**Meet the editor**

Dr Timothy Radstake, MD, PhD, attended medical school at the Radboud University in Nijmegen, the Netherlands, where he graduated in 2000 with the judicium Cum Laude. In 2003, he obtained his PhD (Cum Laude) on the genetic factors that determine the susceptibility to Rheumatoid arthritis, and the role of dendritic cells in this condition. He then started with his trainee

ship rheumatology at the department of Rheumatology of the Radboud University Nijmegen Medical Centre. He became a staff rheumatologist in July 2011. From 2007 – 2008, he performed a post-doctoral fellowship at the Scleroderma center of the Boston University School of Medicine in Boston, MA, USA. Since then, he holds a faculty position, and together, with Prof. R. Lafyatis and Prof. M. Trojanowska, runs the immunology research line at this center. Currently, he is principal investigator of the research group translational immunology at the Nijmegen Center of Molecular Life Sciences (NCMLS) and Nijmegen Institute of Infection, Inflammation and Immunity (N4i).

Contents

**Preface IX** 

Chapter 1 **Pathogenesis of the** 

**Part 1 The Immune System in SSc 1** 

Chapter 3 **Using Proteomic Analysis for** 

Chapter 4 **Apoptosis of T Lymphocytes in Systemic Sclerosis 69** 

**Endothelial Damage and Related Factors 3**  Paola Cipriani, Vasiliki Liakouli, Alessandra Marrelli,

Sébastien Lepreux, Anne Solanilla, Julien Villeneuve, Joël Constans, Alexis Desmoulière and Jean Ripoche

P. Coral-Alvarado, G. Quintana, C. Cardozo, J. Iriarte, Y. Sanchez, S. Bravo, J. Castano, M.F. Garces, L. Cepeda,

M. Szymanek, G. Chodorowska, A. Pietrzak and D. Krasowska

J.C.A. Broen, L. McGlynn, T.R.D.J. Radstake and P.G. Shiels

Roberto Perricone and Roberto Giacomelli

**Studying the Skin Fibroblast Protein Profile in Systemic Sclerosis 53** 

A. Iglesias-Gamarra and J.E. Caminos

Chapter 5 **Cytokines in Systemic Sclerosis: Focus on IL-17 93**  Julie Baraut, Dominique Farge, Elena Ivan-Grigore,

Franck Verrecchia and Laurence Michel

Chapter 6 **Biological Ageing Research in Systemic Sclerosis:** 

Chapter 7 **Fibrocytes in Scleroderma Lung Fibrosis 117** 

Ronald Reilkoff, Aditi Mathur and Erica Herzog

**Time to Grow up? 103** 

Chapter 2 **Blood Platelets and Systemic Sclerosis 29** 

### Contents

#### **Preface XI**

	- Y. Sanchez, S. Bravo, J. Castano, M.F. Garces, L. Cepeda,
	- A. Iglesias-Gamarra and J.E. Caminos

Chapter 8 **Inhibition of Thrombin as a Novel Strategy in the Treatment of Scleroderma-Associated Interstitial Lung Disease 131**  Galina S. Bogatkevich, Kristin B. Highland, Tanjina Akter, Paul J. Nietert, Ilia Atanelishvili, Joanne van Ryn and Richard M. Silver

#### **Part 2 Clinical Features of SSc 149**


### Preface

Scleroderma, or systemic sclerosis (Ssc), means "tight skin" and is caused by the fibrosis of skin and disappearance of subcutaneous fat. There is much more involved, however. Although the fibrosis of the skin is a disabling and often painful sign of SSc, the involvement of the kidneys (see chapter on renal failure), lungs, and heart mostly lead to severe co-morbidity and premature death. The exact cause of SSc is unknown but there is accumulating evidence that the immune systems drives the three hallmarks that are observed in SSc patients: endothelial cell dysfunction, presence of autoantibodies, and fibroblast activation. In order to update the readers in the multiple facets of Ssc, this book covers two issues. In the first eight chapters, multiple key authors will provide better insights into the role of endothelial damage (chapter 1), platelets (chapter 2), and the observations that came forth from proteomic studies so far (chapter 3). T lymphocytes (chapter 4) and IL-17; a cytokine more recently shown to be increased in the circulation, and T cells isolated from SSc patients (chapter 5) are then discussed. In chapter 6, Broen and colleagues dive deeper into the potential role of the telomere and its dysfunction in SSc. Chapter 7 and 8 will deal with fibroblasts and thrombin as a potential therapeutic target of this clinical syndrome.

Besides the immunopathology of this disease, various clinical issues will be dealt with in the next issues. As such, novel issues with regard to diagnosis and pathogenesis will be discussed (chapter 9), as well as the potential role of capillary nail fold lesions in the diagnosis and follow-up (chapter 10). Scleroderma renal crisis is a feared complication of SSc and will be further discussed in chapter 11. Finally, the hurdles and pitfalls with regard pregnancy in a patient with SSc is widely discussed.

Altogether, although significant advances have occurred in our understanding SSc, the exact cause and most optimal way of treatment remains a medical conundrum. This book is meant to provide an up-to-date in SSc pathogenesis and clinical features. We believe that this book will be able to provide both researchers and clinicians dealing with SSc with the most modern information that will aid them in their work. However, the research into its cause and most efficient treatment is

#### X Preface

rapidly expanding, hopefully resulting in highly needed updates in the not to distant future.

#### **Dr Timothy Radstake, MD, PhD**

Department of Rheumatology, Radboud University Medical Center, Nijmegen, The Netherlands

The Scleroderma Center, Boston University School of Medicine, Massachusetts, U.S.A.

X Preface

distant future.

rapidly expanding, hopefully resulting in highly needed updates in the not to

Department of Rheumatology, Radboud University Medical Center, Nijmegen,

The Scleroderma Center, Boston University School of Medicine, Massachusetts,

**Dr Timothy Radstake, MD, PhD** 

The Netherlands

U.S.A.

**Part 1** 

**The Immune System in SSc** 

**Part 1** 

**The Immune System in SSc** 

**1** 

*Italy* 

**Pathogenesis of the Endothelial Damage** 

Systemic sclerosis (SSc) is an autoimmune connective tissue disorder characterized by a widespread microangiopathy, autoimmunity and fibrosis of the skin and of various internal organs. Vascular damage occurs early in the course of the disease as showed by the presence of Raynaud's phenomenon (RP) that can precede the fibrotic process of months or years. A complex interaction between endothelial cells (ECs), smooth muscle cells (SMCs), pericytes, extracellular matrix (ECM), and intravascular circulating factors is now recognized to contribute to the vascular reactivity, remodeling, and occlusive disease of SSc (Gabrielli et al., 2009). Chronic platelet activation and enhanced coagulation with reduced fibrinolysis, secondary to EC activation that leads to fibrin deposits and contribute to the intimal proliferation and luminal narrowing are also found. The identity of the initial trigger of EC damage remains unknown. Current hypotheses suggest a possible infectious or chemical trigger(s) that activates both cellular and humoral immunity. Products of immune activation may lead to vascular injury possibly through the production of autoantibodies and the release of products of activated T cells that can directly damage the endothelium (Kahaleh, 2008). Microangiopathy is characterized by a reduced capillary density and an irregular chaotic architecture that leads to chronic tissue hypoxia and organ dysfunction with eventual organ failure. Vascular complications, including pulmonary arterial hypertension (PAH) and scleroderma renal crisis (SRC) have emerged as leading causes of disability and mortality in SSc (Guiducci et al., 2007). Despite the hypoxic conditions, there is no evidence for a sufficient compensative angiogenesis in SSc (Distler O et al., 2002). Furthermore, vasculogenesis, the *de novo* formation of blood vessels, is also impaired. An imbalance between angiogenic and angiostatic factors as well as functional alterations of the cellular players, involved in the angiogenic and vasculogenic program, might explain the pathogenetic mechanisms of SSc vasculopathy (Liakouli, 2011; Cipriani, 2011). Either angiogenic or vasculogenic mechanisms may potentially become in the future the target of

novel therapeutic strategies to promote vascular regeneration in SSc.

Microvascular endothelial cell (MVEC) injury and apoptosis is an early and central event in the pathogenesis of SSc vasculopathy that leads to microcirculatory dysfunction and loss of

**2. Vascular endothelial cell damage** 

**1. Introduction** 

Paola Cipriani, Vasiliki Liakouli, Alessandra Marrelli,

Roberto Perricone and Roberto Giacomelli

**and Related Factors** 

*University of L'Aquila* 

## **Pathogenesis of the Endothelial Damage and Related Factors**

Paola Cipriani, Vasiliki Liakouli, Alessandra Marrelli, Roberto Perricone and Roberto Giacomelli *University of L'Aquila Italy* 

#### **1. Introduction**

Systemic sclerosis (SSc) is an autoimmune connective tissue disorder characterized by a widespread microangiopathy, autoimmunity and fibrosis of the skin and of various internal organs. Vascular damage occurs early in the course of the disease as showed by the presence of Raynaud's phenomenon (RP) that can precede the fibrotic process of months or years. A complex interaction between endothelial cells (ECs), smooth muscle cells (SMCs), pericytes, extracellular matrix (ECM), and intravascular circulating factors is now recognized to contribute to the vascular reactivity, remodeling, and occlusive disease of SSc (Gabrielli et al., 2009). Chronic platelet activation and enhanced coagulation with reduced fibrinolysis, secondary to EC activation that leads to fibrin deposits and contribute to the intimal proliferation and luminal narrowing are also found. The identity of the initial trigger of EC damage remains unknown. Current hypotheses suggest a possible infectious or chemical trigger(s) that activates both cellular and humoral immunity. Products of immune activation may lead to vascular injury possibly through the production of autoantibodies and the release of products of activated T cells that can directly damage the endothelium (Kahaleh, 2008). Microangiopathy is characterized by a reduced capillary density and an irregular chaotic architecture that leads to chronic tissue hypoxia and organ dysfunction with eventual organ failure. Vascular complications, including pulmonary arterial hypertension (PAH) and scleroderma renal crisis (SRC) have emerged as leading causes of disability and mortality in SSc (Guiducci et al., 2007). Despite the hypoxic conditions, there is no evidence for a sufficient compensative angiogenesis in SSc (Distler O et al., 2002). Furthermore, vasculogenesis, the *de novo* formation of blood vessels, is also impaired. An imbalance between angiogenic and angiostatic factors as well as functional alterations of the cellular players, involved in the angiogenic and vasculogenic program, might explain the pathogenetic mechanisms of SSc vasculopathy (Liakouli, 2011; Cipriani, 2011). Either angiogenic or vasculogenic mechanisms may potentially become in the future the target of novel therapeutic strategies to promote vascular regeneration in SSc.

#### **2. Vascular endothelial cell damage**

Microvascular endothelial cell (MVEC) injury and apoptosis is an early and central event in the pathogenesis of SSc vasculopathy that leads to microcirculatory dysfunction and loss of

Pathogenesis of the Endothelial Damage and Related Factors 5

and coagulation cascades leading to microvascular thrombosis and further vessel compromise. MVEC apoptosis can result from their interaction with cytotoxic T cells either by Fas or granzymes/perforin-related mechanisms. For example, CD4+ T cells can mediate MVEC apoptosis by a Fas-related mechanism as seen in cytolytic T cells killing of vascular endothelium in the rejection reaction, whereas the granzyme/perforin system mediates apoptosis by the major cytotoxic cells, the CD8+ T cells, NK and LAK cells. Involvement of cytotoxic T cells in SSc is suggested by the presence of a 60 kDa protein in SSc sera that was described as an endothelial cytotoxic factor. This factor was characterized as the granular enzyme, and was detected in the perivascular spaces in SSc skin biopsies. Cytotoxic T lymphocytes (in particular CD8+ T cells, NK cells, LAK cells) granule-specific products such as granzyme B and perforin are able to induce apoptosis in cultured ECs. Granzymes gain access to the cells following cellular membrane damage by perforin (Kahaleh, 1997). The majority of autoantigens targeted in SSc can be cleaved by granzyme B and are recognized preferentially by patients antibodies (Schachna et al., 2002). Furthermore, the activation of cytotoxic cell-mediated pathways is plausible and may be involved in early vascular injury thus initiating and propagating the autoimmune response in SSc. Antibody-dependent cellular cytotoxicity of vascular endothelium is reported in up to 40% of the SSc patients. The effector cells express Fc receptors and are both non-T cells and non adherent T lymphocytes, while the antibody is an IgG with MVEC specificity that mediate MVEC cytotoxicity via the Fas pathway (Sgonc et al., 2000). EC apoptosis may also confer a state of resistance to apoptosis by the surrounding fibroblasts that may lead to myofibroblast differentiation and tissue fibrotic changes that follow (Laplante et al., 2005). However, EC damage and apoptosis is an early event in the course of the disease with progressive loss of capillaries, responsible on one hand of the typical clinical manifestations of vasculopathy and on the other hand the chronic tissue ischemia that leads to organ dysfunction and

**3. Vascular endothelial cell alterations and fibroproliferative vasculopathy** 

Vascular disease in scleroderma patients is both functional and structural with reversible vasospasm as well as a reduction in the capillary density followed by obliterative vasculopathy. These vascular changes involving capillaries, arterioles and small arteries

Important features of the tissue lesions in various stages of scleroderma are early microvascular damage, mononuclear-cell infiltrates, and slowly developing fibrosis. In later stages of scleroderma, the main findings are very densely packed collagen in the dermis, loss of cells, and atrophy. In particular, in the early phase of the disease, endothelial damage is characterized by collapse of vimentin's filaments in the perinuclear region, vacuolization, granular degeneration of the nucleus, cellular necrosis, gaps between endothelial cells, reduplication of basal membranes, followed by vascular lumen obstruction, altered permeability of vessel wall that induce increased passage of both plasma and mononuclear cells with perivascular infiltrates formation in which T lymphocytes and monocytes bearing macrophage markers predominate (Fleischmajer, 1980, 1977; Ishikawa, 1992) with more CD4+ than CD8+ cells (Roumm, 1984). In fact, T cells in skin lesions are predominantly CD4+ cells, display markers of activation, exhibit oligoclonal expansion (Sakkas et al., 2002) and are predominantly type 2 helper T (Th2) cells (Mavalia et al., 1997). Moreover, an

eventual organ failure.

may be observed by nail-fold capillaroscopy.

capillaries with consequent vascular desertification, tissue chronic ischemia and eventual organ failure. The initiating factors or cause of the vascular insult in scleroderma remain unknown. Current hypotheses in SSc vascular disease pathogenesis suggest a possible infectious or chemical trigger(s) that activates both cellular and humoral immunity. Products of immune activation may lead to vascular injury possibly through the production of autoantibodies and the release of products of activated T cells that can directly damage the endothelium. In particular, primary activation of ECs in SSc include autoantibodies showing cross-reactivity between Cytomegalovirus (CMV) epitopes and specific surface molecules of ECs, inducing apoptosis. However, this is unlikely to be the only aetiological factor, since CMV is ubiquitous in the normal population (Lunardi et al., 2000). Antiendothelial cells antibodies (AECAs) present in the SSc sera are reported to activate EC and, induce EC apoptosis *in vivo* independent of the Fas–Fas ligand pathway. This is clearly shown in the chicken model of SSc (UCD-200), where serum transfer into normal chicken embryos results in binding of antibodies to the microvasculature in the chorioallantoic membrane in association with endothelial apoptosis (Worda et al., 2003). The exact identity of the endothelial antigen is not known; (Worda M et al., 2003). Moreover, SSc sera containing distinct AECA subsets (ACAs for limited cutaneous SSc or anti-topoisomerase I antibodies for diffuse cutaneous SSc) can induce EC apoptosis in association with increased gene expression of caspase 3 and the reexpression of EC SSc autoantigen fibrillin 1 (Ahmed et al., 2006). Anti-endothelial cells antibodies (AECAs) are present in 40–50% of the SSc sera and are mostly of the IgG1 isotype. The antibody titres correlate negatively with pulmonary diffusion capacity and positively with pulmonary hypertension and with digital ischemic ulcers, suggesting a pathological role in the development of the vascular disease. The only published proteomic analysis of endothelial antigen(s) recognized by AECA identified 53 proteins consisting of cytoskeleton proteins, proteins involved in cellular mobility, regulation of apoptosis and senescence as well as proteins implicated in clotting and antigen presentation (Bordron et al., 1998). Thus, vascular cell apoptosis could, in turn, expose autoantigens to immune surveillance, evoking an autoimmune response and perpetuating autoimmunity to blood vessels in SSc (Ahmed et al., 2006). However, AECAs are detected in a variety of vascular diseases and specific epitopes and mechanisms have not been clarified in SSc. High levels of reactive oxygen species and oxidative stress have been directly or indirectly implicated in scleroderma (Sambo et al., 2001, 1999; Servettazet al., 2007). The source of reactive oxygen species is the membrane NADPH oxidase system, which is stimulated in all cell types within or surrounding the vessel wall in response to injury (Lassegue, 2001; Sturroch, 2005; Holland, 1998). In scleroderma, the high levels of reactive oxygen species in mesenchymal cells (MSCs) are relatively independent of the inflammatory status; they persist *in vitro* in the absence of growth factors and cytokines, render cells sensitive to stress, and induce DNA damage (Svegliati et al., 2006). MSCs become progressively hypersensitive to cytokines induced by local reactive oxygen species (Sullivan et al., 2008). Cytokines activate mesenchymal precursor cells and lead to the transformation of fibroblasts to myofibroblasts with consequent abnormal collagen synthesis. Furthermore, free radicals contribute to the release of mediators implicated in fibrosis (Bellocq et al., 1999; Barcellos-Hoff et al., 1996). EC apoptosis may also activate the immune-inflammatory system by dendritic cells and macrophage presentation of self-antigen present in the apoptotic debris to CD8+ T cells, and by the direct activation of the alternate complement

capillaries with consequent vascular desertification, tissue chronic ischemia and eventual organ failure. The initiating factors or cause of the vascular insult in scleroderma remain unknown. Current hypotheses in SSc vascular disease pathogenesis suggest a possible infectious or chemical trigger(s) that activates both cellular and humoral immunity. Products of immune activation may lead to vascular injury possibly through the production of autoantibodies and the release of products of activated T cells that can directly damage the endothelium. In particular, primary activation of ECs in SSc include autoantibodies showing cross-reactivity between Cytomegalovirus (CMV) epitopes and specific surface molecules of ECs, inducing apoptosis. However, this is unlikely to be the only aetiological factor, since CMV is ubiquitous in the normal population (Lunardi et al., 2000). Antiendothelial cells antibodies (AECAs) present in the SSc sera are reported to activate EC and, induce EC apoptosis *in vivo* independent of the Fas–Fas ligand pathway. This is clearly shown in the chicken model of SSc (UCD-200), where serum transfer into normal chicken embryos results in binding of antibodies to the microvasculature in the chorioallantoic membrane in association with endothelial apoptosis (Worda et al., 2003). The exact identity of the endothelial antigen is not known; (Worda M et al., 2003). Moreover, SSc sera containing distinct AECA subsets (ACAs for limited cutaneous SSc or anti-topoisomerase I antibodies for diffuse cutaneous SSc) can induce EC apoptosis in association with increased gene expression of caspase 3 and the reexpression of EC SSc autoantigen fibrillin 1 (Ahmed et al., 2006). Anti-endothelial cells antibodies (AECAs) are present in 40–50% of the SSc sera and are mostly of the IgG1 isotype. The antibody titres correlate negatively with pulmonary diffusion capacity and positively with pulmonary hypertension and with digital ischemic ulcers, suggesting a pathological role in the development of the vascular disease. The only published proteomic analysis of endothelial antigen(s) recognized by AECA identified 53 proteins consisting of cytoskeleton proteins, proteins involved in cellular mobility, regulation of apoptosis and senescence as well as proteins implicated in clotting and antigen presentation (Bordron et al., 1998). Thus, vascular cell apoptosis could, in turn, expose autoantigens to immune surveillance, evoking an autoimmune response and perpetuating autoimmunity to blood vessels in SSc (Ahmed et al., 2006). However, AECAs are detected in a variety of vascular diseases and specific epitopes and mechanisms have not been clarified in SSc. High levels of reactive oxygen species and oxidative stress have been directly or indirectly implicated in scleroderma (Sambo et al., 2001, 1999; Servettazet al., 2007). The source of reactive oxygen species is the membrane NADPH oxidase system, which is stimulated in all cell types within or surrounding the vessel wall in response to injury (Lassegue, 2001; Sturroch, 2005; Holland, 1998). In scleroderma, the high levels of reactive oxygen species in mesenchymal cells (MSCs) are relatively independent of the inflammatory status; they persist *in vitro* in the absence of growth factors and cytokines, render cells sensitive to stress, and induce DNA damage (Svegliati et al., 2006). MSCs become progressively hypersensitive to cytokines induced by local reactive oxygen species (Sullivan et al., 2008). Cytokines activate mesenchymal precursor cells and lead to the transformation of fibroblasts to myofibroblasts with consequent abnormal collagen synthesis. Furthermore, free radicals contribute to the release of mediators implicated in fibrosis (Bellocq et al., 1999; Barcellos-Hoff et al., 1996). EC apoptosis may also activate the immune-inflammatory system by dendritic cells and macrophage presentation of self-antigen present in the apoptotic debris to CD8+ T cells, and by the direct activation of the alternate complement and coagulation cascades leading to microvascular thrombosis and further vessel compromise. MVEC apoptosis can result from their interaction with cytotoxic T cells either by Fas or granzymes/perforin-related mechanisms. For example, CD4+ T cells can mediate MVEC apoptosis by a Fas-related mechanism as seen in cytolytic T cells killing of vascular endothelium in the rejection reaction, whereas the granzyme/perforin system mediates apoptosis by the major cytotoxic cells, the CD8+ T cells, NK and LAK cells. Involvement of cytotoxic T cells in SSc is suggested by the presence of a 60 kDa protein in SSc sera that was described as an endothelial cytotoxic factor. This factor was characterized as the granular enzyme, and was detected in the perivascular spaces in SSc skin biopsies. Cytotoxic T lymphocytes (in particular CD8+ T cells, NK cells, LAK cells) granule-specific products such as granzyme B and perforin are able to induce apoptosis in cultured ECs. Granzymes gain access to the cells following cellular membrane damage by perforin (Kahaleh, 1997). The majority of autoantigens targeted in SSc can be cleaved by granzyme B and are recognized preferentially by patients antibodies (Schachna et al., 2002). Furthermore, the activation of cytotoxic cell-mediated pathways is plausible and may be involved in early vascular injury thus initiating and propagating the autoimmune response in SSc. Antibody-dependent cellular cytotoxicity of vascular endothelium is reported in up to 40% of the SSc patients. The effector cells express Fc receptors and are both non-T cells and non adherent T lymphocytes, while the antibody is an IgG with MVEC specificity that mediate MVEC cytotoxicity via the Fas pathway (Sgonc et al., 2000). EC apoptosis may also confer a state of resistance to apoptosis by the surrounding fibroblasts that may lead to myofibroblast differentiation and tissue fibrotic changes that follow (Laplante et al., 2005). However, EC damage and apoptosis is an early event in the course of the disease with progressive loss of capillaries, responsible on one hand of the typical clinical manifestations of vasculopathy and on the other hand the chronic tissue ischemia that leads to organ dysfunction and eventual organ failure.

#### **3. Vascular endothelial cell alterations and fibroproliferative vasculopathy**

Vascular disease in scleroderma patients is both functional and structural with reversible vasospasm as well as a reduction in the capillary density followed by obliterative vasculopathy. These vascular changes involving capillaries, arterioles and small arteries may be observed by nail-fold capillaroscopy.

Important features of the tissue lesions in various stages of scleroderma are early microvascular damage, mononuclear-cell infiltrates, and slowly developing fibrosis. In later stages of scleroderma, the main findings are very densely packed collagen in the dermis, loss of cells, and atrophy. In particular, in the early phase of the disease, endothelial damage is characterized by collapse of vimentin's filaments in the perinuclear region, vacuolization, granular degeneration of the nucleus, cellular necrosis, gaps between endothelial cells, reduplication of basal membranes, followed by vascular lumen obstruction, altered permeability of vessel wall that induce increased passage of both plasma and mononuclear cells with perivascular infiltrates formation in which T lymphocytes and monocytes bearing macrophage markers predominate (Fleischmajer, 1980, 1977; Ishikawa, 1992) with more CD4+ than CD8+ cells (Roumm, 1984). In fact, T cells in skin lesions are predominantly CD4+ cells, display markers of activation, exhibit oligoclonal expansion (Sakkas et al., 2002) and are predominantly type 2 helper T (Th2) cells (Mavalia et al., 1997). Moreover, an

Pathogenesis of the Endothelial Damage and Related Factors 7

However, after endothelial cell injury, and in response to appropriate stimuli, mature and progenitor ECs they can form new blood vessels through a combination of two separate processes: angiogenesis and vasculogenesis. The term angiogenesis describes the formation of new capillaries and larger vessels by sprouting of differentiated EC from pre-existing vessels. Angiogenesis is a highly complex and requires a dynamic, temporally and spatially interaction among ECs, ECM molecules, adhesion molecules, proteolytic enzymes and the subtle balance between proangiogenic and angiostatic factors (Distler et al., 2003). In particular, proangiogenic stimuli activate EC, which degrade the basal membrane and the perivascular extracellular matrix, proliferate and migrate into the site of new vessel formation. Stabilisation of vessel wall by pericytes is the final process of sprouting angiogenesis and leads to a functional network of new capillary. In contrast to angiogenesis, vasculogenesis describes the formation of new vessels by circulating EPC, independent from pre-existing vessels. Vasculogenesis was regarded to be restricted to embryogenesis but the discovery of EPC in adult bone marrow and peripheral blood has challenged this theory (Asahara et al., 1997; Shi et al., 1998). After birth, postnatal vasculogenesis contributes to vascular healing in response to endothelial injury through the processes of rapid reendothelialization of denuded vessels and collateral vessel formation in ischemic tissues. In particular, following tissue ischemia, EPC are mobilized from their bone marrow niches into the circulation in response to stress- and/or damage related signals, migrate through the bloodstream and home to the sites of vascular injury, where they contribute to the formation as well of neovessels as to the repair of damaged vessels, collaborating with preexisting mature EC (Urbich et al., 2004). Several studies showed that EPCs promotes structural and functional repair in several organs such as the heart, liver, kidney or brain. However, as mentioned above, progenitor cells can migrate to sites of vascular injury and differentiate not only into an endothelial phenotype (vascular repair), but also into vascular smooth muscle cells contributing to neointimal hyperplasia and eventually fibroproliferative

Both angiogenic and vasculogenic processes are impaired in SSc. The progressive loss of capillaries on one hand, and the vascular remodeling of arteriolar vessels on the other result in insufficient blood flow, causing severe and chronic hypoxia. Tissue hypoxia usually initiates the formation of new blood vessels from the pre-existing microvasculature leading to the expression of pro-angiogenic molecules, mainly of Vascular Endothelial Growth Factor (VEGF), which triggers the angiogenic process. Despite the hypoxic conditions and the increased levels of VEGF in skin and serum of SSc patients, there is, paradoxically, no evidence for a sufficient angiogenesis, thus perpetuating the vicious circle leading to tissue ischemia (Distler JH et al., 2006; Distler O et al., 2004, 2002). Vasculogenesis, is also impaired in SSc patients with a decreased number and several functional defects of endothelial progenitor cells (EPCs) (Kuwana et al., 2004; Del Papa et al., 2006) and mesenchymal stem cells (MSCs), the latter deriving from the bone marrow population and the tissue resident

The aetiological factors involved in the pathogenesis of SSc-associated vascular defects determine a complicate network of EC alterations which account for the lost ability of these

Angiogenic process is an invasive event in which proteolytic activities by EC are required. Specific proteases are needed for the degradation of the membrane basement, for cell

cells, was observed in SSc patients (Cipriani et al., 2007).

vasculopathy.

**5. Endothelial cells** 

cells to perform *in vitro* angiogenesis.

increase in the Vdelta1 + T cells subset that express both adhesion molecules and activation markers suggests a selective V gene subset expansion (Giacomelli et al., 1998). In advanced phases, intimal thickening, delamination, vessel narrowing or obliteration and perivascular fibrosis are present (Rodnan et al., 1980).

At the cellular level, the changes that characterized the early lesions are: loss of endothelial cells, proliferating pericytes and vascular smooth muscle cells, and immune cells in the perivascular space. Endothelial cells are the only mesenchymal cell type that undergo apoptosis in the early phase of scleroderma, whereas vascular smooth-muscle cells and pericytes proliferate vigorously thus leading to the characteristic fibroproliferative SSc vasculopathy. The activation of vascular smooth muscle leads to migration of these cells into the intimal layer of the vessel where they differentiate into a myofibroblast. Fibroblasts and pericytes may also transform into myofibroblasts in scleroderma disease (Rajkumar et al., 2005). It is also suggested that, following vascular injury, bone marrow-derived circulating mesenchymal progenitor cells (e.g., fibrocytes), and epithelial cells via epithelial to mesenchymal transition (EMT) can become myofibroblasts. Recently, a study provided evidence that abnormal fibrillin-1 expression and chronic oxidative stress mediate endothelial-mesenchymal transition (EndoMT) in the tight skin murine model of SSc (Xu et al, 2010) and more recently evidence indicates that the c-Abl tyrosine kinase and the protein kinase C δ (PKC-δ), are crucial for TGFβ induction of EndoMT *in vitro*, and that imatinib mesylate and rottlerin, or similar kinase inhibitor molecules, may be effective therapeutic agents for SSc and other fibroproliferative vasculopathies in which EndoMT is involved (Li & Jimenez, 2011). The exact mediator of cell activation in scleroderma is unknown, but speculation includes the release of mediators from the activated endothelium (e.g., endothelin- 1 (ET-1)) and platelets (e.g., thromboxane or platelet derived growth factor. In fact, endothelial damage cause an imbalance in endothelial vascular signals with increased endothelin production and impaired nitric oxide and prostacyclin release that mediates the vasospasm and contribute to intimal proliferation and vascular fibrosis and stiffness of the vessel wall. Recently, Interleukin 33 (IL-33), a novel member of the IL1 family that promotes Th2 responses and inflammation through the ST2 receptor, was found to be abnormally expressed in the SSc tissue and sera. In particular, in the early phase of the disease, upon EC activation/damage IL-33 may be mobilised from ECs into the circulation to signal through ST2 in key profibrotic players such as inflammatory/immune cells and fibroblasts/myofibroblasts. (Manetti, 2010, 2011). This step probably corresponds to the first symptom of scleroderma. Recurrent Raynaud's phenomenon could be the direct consequence of the structural changes of the vessel and the perturbed control of vascular tone due to an imbalance between vasodilatory and vasoconstrictive mediators. At this stage, the patient may have early signs of skin and visceral fibrosis. Platelet activation and enhanced coagulation with reduced fibrinolysis lead to fibrin deposits and contribute to the intimal proliferation and luminal narrowing.

Besides the skin, fibroproliferative vasculopathy is present in the lungs, kidneys and other organs (Dorfmuller et al., 2007; Nagai et al., 2007; Guiducci et al., 2007). However, the mechanisms underlying the pathological vascular changes in SSc still remain unclear.

#### **4. Impaired angiogenesis and vasculogenesis in SSc**

In the adult mammalian organism, the vasculature is normally quiescent and the ECs have an extremely low turn-over rate with the exception of the reproductive cycle (ovulation, implanation, pregnancy) and wound healing or tissue regeneration (Carmeliet, 2003).

increase in the Vdelta1 + T cells subset that express both adhesion molecules and activation markers suggests a selective V gene subset expansion (Giacomelli et al., 1998). In advanced phases, intimal thickening, delamination, vessel narrowing or obliteration and perivascular

At the cellular level, the changes that characterized the early lesions are: loss of endothelial cells, proliferating pericytes and vascular smooth muscle cells, and immune cells in the perivascular space. Endothelial cells are the only mesenchymal cell type that undergo apoptosis in the early phase of scleroderma, whereas vascular smooth-muscle cells and pericytes proliferate vigorously thus leading to the characteristic fibroproliferative SSc vasculopathy. The activation of vascular smooth muscle leads to migration of these cells into the intimal layer of the vessel where they differentiate into a myofibroblast. Fibroblasts and pericytes may also transform into myofibroblasts in scleroderma disease (Rajkumar et al., 2005). It is also suggested that, following vascular injury, bone marrow-derived circulating mesenchymal progenitor cells (e.g., fibrocytes), and epithelial cells via epithelial to mesenchymal transition (EMT) can become myofibroblasts. Recently, a study provided evidence that abnormal fibrillin-1 expression and chronic oxidative stress mediate endothelial-mesenchymal transition (EndoMT) in the tight skin murine model of SSc (Xu et al, 2010) and more recently evidence indicates that the c-Abl tyrosine kinase and the protein kinase C δ (PKC-δ), are crucial for TGFβ induction of EndoMT *in vitro*, and that imatinib mesylate and rottlerin, or similar kinase inhibitor molecules, may be effective therapeutic agents for SSc and other fibroproliferative vasculopathies in which EndoMT is involved (Li & Jimenez, 2011). The exact mediator of cell activation in scleroderma is unknown, but speculation includes the release of mediators from the activated endothelium (e.g., endothelin- 1 (ET-1)) and platelets (e.g., thromboxane or platelet derived growth factor. In fact, endothelial damage cause an imbalance in endothelial vascular signals with increased endothelin production and impaired nitric oxide and prostacyclin release that mediates the vasospasm and contribute to intimal proliferation and vascular fibrosis and stiffness of the vessel wall. Recently, Interleukin 33 (IL-33), a novel member of the IL1 family that promotes Th2 responses and inflammation through the ST2 receptor, was found to be abnormally expressed in the SSc tissue and sera. In particular, in the early phase of the disease, upon EC activation/damage IL-33 may be mobilised from ECs into the circulation to signal through ST2 in key profibrotic players such as inflammatory/immune cells and fibroblasts/myofibroblasts. (Manetti, 2010, 2011). This step probably corresponds to the first symptom of scleroderma. Recurrent Raynaud's phenomenon could be the direct consequence of the structural changes of the vessel and the perturbed control of vascular tone due to an imbalance between vasodilatory and vasoconstrictive mediators. At this stage, the patient may have early signs of skin and visceral fibrosis. Platelet activation and enhanced coagulation with reduced fibrinolysis lead to fibrin deposits and contribute to the

Besides the skin, fibroproliferative vasculopathy is present in the lungs, kidneys and other organs (Dorfmuller et al., 2007; Nagai et al., 2007; Guiducci et al., 2007). However, the mechanisms underlying the pathological vascular changes in SSc still remain unclear.

In the adult mammalian organism, the vasculature is normally quiescent and the ECs have an extremely low turn-over rate with the exception of the reproductive cycle (ovulation, implanation, pregnancy) and wound healing or tissue regeneration (Carmeliet, 2003).

fibrosis are present (Rodnan et al., 1980).

intimal proliferation and luminal narrowing.

**4. Impaired angiogenesis and vasculogenesis in SSc** 

However, after endothelial cell injury, and in response to appropriate stimuli, mature and progenitor ECs they can form new blood vessels through a combination of two separate processes: angiogenesis and vasculogenesis. The term angiogenesis describes the formation of new capillaries and larger vessels by sprouting of differentiated EC from pre-existing vessels. Angiogenesis is a highly complex and requires a dynamic, temporally and spatially interaction among ECs, ECM molecules, adhesion molecules, proteolytic enzymes and the subtle balance between proangiogenic and angiostatic factors (Distler et al., 2003). In particular, proangiogenic stimuli activate EC, which degrade the basal membrane and the perivascular extracellular matrix, proliferate and migrate into the site of new vessel formation. Stabilisation of vessel wall by pericytes is the final process of sprouting angiogenesis and leads to a functional network of new capillary. In contrast to angiogenesis, vasculogenesis describes the formation of new vessels by circulating EPC, independent from pre-existing vessels. Vasculogenesis was regarded to be restricted to embryogenesis but the discovery of EPC in adult bone marrow and peripheral blood has challenged this theory (Asahara et al., 1997; Shi et al., 1998). After birth, postnatal vasculogenesis contributes to vascular healing in response to endothelial injury through the processes of rapid reendothelialization of denuded vessels and collateral vessel formation in ischemic tissues. In particular, following tissue ischemia, EPC are mobilized from their bone marrow niches into the circulation in response to stress- and/or damage related signals, migrate through the bloodstream and home to the sites of vascular injury, where they contribute to the formation as well of neovessels as to the repair of damaged vessels, collaborating with preexisting mature EC (Urbich et al., 2004). Several studies showed that EPCs promotes structural and functional repair in several organs such as the heart, liver, kidney or brain. However, as mentioned above, progenitor cells can migrate to sites of vascular injury and differentiate not only into an endothelial phenotype (vascular repair), but also into vascular smooth muscle cells contributing to neointimal hyperplasia and eventually fibroproliferative vasculopathy.

Both angiogenic and vasculogenic processes are impaired in SSc. The progressive loss of capillaries on one hand, and the vascular remodeling of arteriolar vessels on the other result in insufficient blood flow, causing severe and chronic hypoxia. Tissue hypoxia usually initiates the formation of new blood vessels from the pre-existing microvasculature leading to the expression of pro-angiogenic molecules, mainly of Vascular Endothelial Growth Factor (VEGF), which triggers the angiogenic process. Despite the hypoxic conditions and the increased levels of VEGF in skin and serum of SSc patients, there is, paradoxically, no evidence for a sufficient angiogenesis, thus perpetuating the vicious circle leading to tissue ischemia (Distler JH et al., 2006; Distler O et al., 2004, 2002). Vasculogenesis, is also impaired in SSc patients with a decreased number and several functional defects of endothelial progenitor cells (EPCs) (Kuwana et al., 2004; Del Papa et al., 2006) and mesenchymal stem cells (MSCs), the latter deriving from the bone marrow population and the tissue resident cells, was observed in SSc patients (Cipriani et al., 2007).

#### **5. Endothelial cells**

The aetiological factors involved in the pathogenesis of SSc-associated vascular defects determine a complicate network of EC alterations which account for the lost ability of these cells to perform *in vitro* angiogenesis.

Angiogenic process is an invasive event in which proteolytic activities by EC are required. Specific proteases are needed for the degradation of the membrane basement, for cell

Pathogenesis of the Endothelial Damage and Related Factors 9

functions of these cells have been found to be involved in the pathogenesis of cardiovascular

At least two different types of circulating progenitors appear able to become mature endothelium (Smadja et al., 2007). One type of progenitor cells displays the markers CD133, CD34, and vascular endothelial growth factor receptor 2 (VEGFR2) (Hristov et al., 2004). These are harvested from late-outgrowth cultures, possess a high proliferation capacity and are sometimes referred as "true EPC "; most of studies focus on this population. A second type of progenitor population is a subset of CD14+ monocytes distinguishable from the conventional endothelial progenitor cells by the fact that they are CD34− (Hristov et al., 2004; Zhao et al., 2003), arise from short-term cultures and show little proliferative capacity. Both circulating progenitor cell types can differentiate into mature endothelium in culture. It has been precisely characterized using, genome-wide transcriptional study, the molecular fingerprint of two distinct EPCs, showing that early-outhgrowth EPC are haematopoietic cells with a molecular phenotype linked to monocytes; whereas late-outgrowth EPC exhibit commitment to the endothelial lineage (Medina et al., 2010). Interestingly both populations can form capillary tubes in vitro, mediate reendothelialization after injury and improve neovascularization (Urbich et al., 2004). It has been previously demonstrated that both subsets contribute to angiogenesis, but through different mechanisms, CD14+ EPC supporting vasculogenic process by paracrine production of growth factors, while lateoutgrowth CD14- EPC directly incorporating into vessel wall (Sieveking et al., 2008; Mukai et al., 2008). Neovascularization and reendothelialization event are complex multistep processes, requiring EPC chemoattraction, adhesion, and finally differentiation into mature EC. Although the signaling cascades that regulate these steps are still incompletely understood, it is well known that VEGF and SDF-1, induced by hypoxia, play a pivotal role in the EPC mobilization from bone marrow, differentiation and attraction to site of ischemia. Recently several studies have demonstrated a role of EPC in the pathogenesis of SSc, suggesting that alteration in the vasculogenic process might contribute to the vasculopathy, distinctive features of the disease. Apparently conflicting results have raised on quantitative and functional characteristics of EPC from SSc patients, probably due to unclear distinctive

disease (Adams et al., 2004, Dimmeler et al., 2001; Hill et al., 2003).

markers of the several cell subsets belonging to the EPC population.

Willebrand factor.

A lower number of circulating EPC, defined as CD34+CD133+VEGFR-2+ mononuclear cells in patients with SSc than in patients with RA or in healthy subjects was seen (Kuwana et al., 2006). In SSc patients, EPC counts did not correlate with the disease subset, the disease duration, or the modified Rodnan skin thickness score. However, the numbers of these cells were lower in SSc patients with pitting scars and active fingertip ulcers. Furthermore, EPC, obtained from the peripheral blood of SSc patients demonstrated an impaired differentiation capacity into mature EC, as shown by a reduced expression of the EC marker von

In contrast to these findings, in another study was found a significantly increase in the number of circulating EPC, identified via the same cell surface markers CD34+ CD133+VEGFR-2+, in SSc patients. Further subgroup analysis revealed a negative correlation between EPC count and disease duration (Del Papa et al., 2008). Based on this finding, the authors suggested that differences in disease duration might account for the discrepancy between their results and the findings reported by the other authors. Apart from disease duration, no correlations between EPC counts and clinical parameters, including digital ulcers, were observed. In this study bone marrow EPC were also evaluated. The number of bone marrow CD133+ cells was significantly decreased in SSc patients compared to healthy controls. Their ability to differentiate into EC *in vitro* was

migration and for creating space in the matrix, to allow the formation of new tubules. Besides their substrate specific properties, proteases exert more complex pro- or antiangiogenic activities, including the activation and modification of growth factors, cytokines and receptors and the generation of matrix fragments which inhibit angiogenesis (Van Hinsberg et al., 2008). Evidence for a mechanism of dysregulated angiogenesis involving these proteases in SSc has emerged from recent experimental studies. A decreased urokinase plasminogen activator (uPA) dependent invasion, proliferation, and capillary morphogenesis, was showed in SSc EC. Urokinase plasminogen activator receptor (uPAR) undergoes truncation between domains 1 and 2, and this modification prevents EC from entering in an angiogenic program (D'Alessio et al., 2004). Furthermore, SSc MVECs produce large amounts of antiangiogenic molecules such as matrix metalloproteinase 12 (MMP-12), involved in the cleavage of the domain of the uPAR, and pentraxin 3 (PTX3). Silencing these two molecules in SSc-MVEC, restore their ability to produce capillaries in vitro (Margheri et al., 2010). Recent work has provided the evidence for the association between a uPAR gene variant, *UPAR* rs344781, and vascular complications, such as digital ulcerations, suggesting a role of this gene in the vascular pathophysiology of SSc (Manetti et al., 2011).

Endothelial cells are directly involved also in vasculogenic process, through the expression of SDF-1. In fact, SDF-1 is a pivotal molecule in the recruitment and retention of CXCR4+ EPC into neo-angiogenic niches (Petit et al., 2007). This molecule, expressed and presented by EC at the site of injury triggers cell arrest and emigration of circulating cells, facilitating the formation of stable vasculature and supporting organ repair (Yao et al., 2003). Additionally, SDF-1 has an angiogenic effect on endothelial cells by inducing cell proliferation, differentiation, sprouting and tube formation in vitro (Salvucci et al., 2002; Yamaguchi et al., 2011). It has been reported that SDF-1 and CXCR4 are clearly up-regulated in the skin and in microvascular endothelial cells during the early edematous phases of SSc. The production of these two molecules progressively decreased, with the lowest levels in the latest phases of the disease. These data strongly suggest that an impairment of EC ability to promote a sustained response to chronic ischemic stress through SDF-1 expression could compromise an adaptive angiogenesis and vasculogenesis in the disease, contributing to the disappearance of the vessels (Cipriani et al., 2006).

Finally, it has been demonstrated that in SSc skin, along with the loss of capillaries, there is a dramatic change in the endothelial phenotype of residual microvessels, characterized by loss of vascular endothelial cadherin (VEcadherin), supposed to be an universal endothelial marker required for tube formation, as well as the over-expression of the anti-angiogenic interferon-α (IFN-α) and over-expression of RGS5, a signaling molecule whose expression coincides with the end of new vessel formation during embryo development and tumour angiogenesis (Fleming et al., 2008).

#### **5.1 Endothelial progenitors**

#### **5.1.1 Hematopoietic endothelial progenitor cells**

Endothelial progenitor cells are known to be a key cellular effectors of vascular regeneration. Growing evidence shows that EPC play an important role in the homeostasis of physiologic vascular network and are involved both in new vessel formation after ischemic insult and in the repair mechanisms of existing vessels (Shi et al., 1998; Urbich et al., 2004; Zammaretti & Zisch, 2005; Adams et al., 2004). Endothelial progenitor cells have emerged as crucial regulators of cardiovascular integrity. Reduced numbers and altered

migration and for creating space in the matrix, to allow the formation of new tubules. Besides their substrate specific properties, proteases exert more complex pro- or antiangiogenic activities, including the activation and modification of growth factors, cytokines and receptors and the generation of matrix fragments which inhibit angiogenesis (Van Hinsberg et al., 2008). Evidence for a mechanism of dysregulated angiogenesis involving these proteases in SSc has emerged from recent experimental studies. A decreased urokinase plasminogen activator (uPA) dependent invasion, proliferation, and capillary morphogenesis, was showed in SSc EC. Urokinase plasminogen activator receptor (uPAR) undergoes truncation between domains 1 and 2, and this modification prevents EC from entering in an angiogenic program (D'Alessio et al., 2004). Furthermore, SSc MVECs produce large amounts of antiangiogenic molecules such as matrix metalloproteinase 12 (MMP-12), involved in the cleavage of the domain of the uPAR, and pentraxin 3 (PTX3). Silencing these two molecules in SSc-MVEC, restore their ability to produce capillaries in vitro (Margheri et al., 2010). Recent work has provided the evidence for the association between a uPAR gene variant, *UPAR* rs344781, and vascular complications, such as digital ulcerations, suggesting a role of this gene in the vascular pathophysiology of SSc (Manetti

Endothelial cells are directly involved also in vasculogenic process, through the expression of SDF-1. In fact, SDF-1 is a pivotal molecule in the recruitment and retention of CXCR4+ EPC into neo-angiogenic niches (Petit et al., 2007). This molecule, expressed and presented by EC at the site of injury triggers cell arrest and emigration of circulating cells, facilitating the formation of stable vasculature and supporting organ repair (Yao et al., 2003). Additionally, SDF-1 has an angiogenic effect on endothelial cells by inducing cell proliferation, differentiation, sprouting and tube formation in vitro (Salvucci et al., 2002; Yamaguchi et al., 2011). It has been reported that SDF-1 and CXCR4 are clearly up-regulated in the skin and in microvascular endothelial cells during the early edematous phases of SSc. The production of these two molecules progressively decreased, with the lowest levels in the latest phases of the disease. These data strongly suggest that an impairment of EC ability to promote a sustained response to chronic ischemic stress through SDF-1 expression could compromise an adaptive angiogenesis and vasculogenesis in the disease, contributing to the

Finally, it has been demonstrated that in SSc skin, along with the loss of capillaries, there is a dramatic change in the endothelial phenotype of residual microvessels, characterized by loss of vascular endothelial cadherin (VEcadherin), supposed to be an universal endothelial marker required for tube formation, as well as the over-expression of the anti-angiogenic interferon-α (IFN-α) and over-expression of RGS5, a signaling molecule whose expression coincides with the end of new vessel formation during embryo development and tumour

Endothelial progenitor cells are known to be a key cellular effectors of vascular regeneration. Growing evidence shows that EPC play an important role in the homeostasis of physiologic vascular network and are involved both in new vessel formation after ischemic insult and in the repair mechanisms of existing vessels (Shi et al., 1998; Urbich et al., 2004; Zammaretti & Zisch, 2005; Adams et al., 2004). Endothelial progenitor cells have emerged as crucial regulators of cardiovascular integrity. Reduced numbers and altered

et al., 2011).

disappearance of the vessels (Cipriani et al., 2006).

**5.1.1 Hematopoietic endothelial progenitor cells** 

angiogenesis (Fleming et al., 2008).

**5.1 Endothelial progenitors** 

functions of these cells have been found to be involved in the pathogenesis of cardiovascular disease (Adams et al., 2004, Dimmeler et al., 2001; Hill et al., 2003).

At least two different types of circulating progenitors appear able to become mature endothelium (Smadja et al., 2007). One type of progenitor cells displays the markers CD133, CD34, and vascular endothelial growth factor receptor 2 (VEGFR2) (Hristov et al., 2004). These are harvested from late-outgrowth cultures, possess a high proliferation capacity and are sometimes referred as "true EPC "; most of studies focus on this population. A second type of progenitor population is a subset of CD14+ monocytes distinguishable from the conventional endothelial progenitor cells by the fact that they are CD34− (Hristov et al., 2004; Zhao et al., 2003), arise from short-term cultures and show little proliferative capacity. Both circulating progenitor cell types can differentiate into mature endothelium in culture. It has been precisely characterized using, genome-wide transcriptional study, the molecular fingerprint of two distinct EPCs, showing that early-outhgrowth EPC are haematopoietic cells with a molecular phenotype linked to monocytes; whereas late-outgrowth EPC exhibit commitment to the endothelial lineage (Medina et al., 2010). Interestingly both populations can form capillary tubes in vitro, mediate reendothelialization after injury and improve neovascularization (Urbich et al., 2004). It has been previously demonstrated that both subsets contribute to angiogenesis, but through different mechanisms, CD14+ EPC supporting vasculogenic process by paracrine production of growth factors, while lateoutgrowth CD14- EPC directly incorporating into vessel wall (Sieveking et al., 2008; Mukai et al., 2008). Neovascularization and reendothelialization event are complex multistep processes, requiring EPC chemoattraction, adhesion, and finally differentiation into mature EC. Although the signaling cascades that regulate these steps are still incompletely understood, it is well known that VEGF and SDF-1, induced by hypoxia, play a pivotal role in the EPC mobilization from bone marrow, differentiation and attraction to site of ischemia. Recently several studies have demonstrated a role of EPC in the pathogenesis of SSc, suggesting that alteration in the vasculogenic process might contribute to the vasculopathy, distinctive features of the disease. Apparently conflicting results have raised on quantitative and functional characteristics of EPC from SSc patients, probably due to unclear distinctive markers of the several cell subsets belonging to the EPC population.

A lower number of circulating EPC, defined as CD34+CD133+VEGFR-2+ mononuclear cells in patients with SSc than in patients with RA or in healthy subjects was seen (Kuwana et al., 2006). In SSc patients, EPC counts did not correlate with the disease subset, the disease duration, or the modified Rodnan skin thickness score. However, the numbers of these cells were lower in SSc patients with pitting scars and active fingertip ulcers. Furthermore, EPC, obtained from the peripheral blood of SSc patients demonstrated an impaired differentiation capacity into mature EC, as shown by a reduced expression of the EC marker von Willebrand factor.

In contrast to these findings, in another study was found a significantly increase in the number of circulating EPC, identified via the same cell surface markers CD34+ CD133+VEGFR-2+, in SSc patients. Further subgroup analysis revealed a negative correlation between EPC count and disease duration (Del Papa et al., 2008). Based on this finding, the authors suggested that differences in disease duration might account for the discrepancy between their results and the findings reported by the other authors. Apart from disease duration, no correlations between EPC counts and clinical parameters, including digital ulcers, were observed. In this study bone marrow EPC were also evaluated. The number of bone marrow CD133+ cells was significantly decreased in SSc patients compared to healthy controls. Their ability to differentiate into EC *in vitro* was

Pathogenesis of the Endothelial Damage and Related Factors 11

A recent study demonstrated that circulating monocytic EPCs were increased in the peripheral blood of SSc patients. *In vitro* and *in vivo* functional analyses revealed that monocytic EPCs derived from SSc patients had an enhanced ability to promote blood vessel formation, when co-cultured with HUVEC. In contrast, the EPC ability to be incorporated into vessels and differentiate into mature endothelial cells was rather impaired in SSc patients. This characteristic was primarily attributable to an enhanced angiogenic property through production of angiogenic factors (Yamaguchi et al., 2010). Because EPCs may critically contribute to the homeostasis of the physiological vascular network, these progenitor cells might be considered interesting candidates for novel cell therapies for the

Endothelial cells could also originate from non- hematopoietic stem cells of the bone marrow (Drake et al., 2003). Mesenchymal stem cells are multipotent cells that are present in the bone marrow and in some tissues as resident stem cells. They retain the capacity to differentiate into several cell lineages of mesenchymal tissues, i.e. bone, cartilage, muscle, tendon or adipose tissue and serve for the preservation and repair of tissues and organs (Tuan et al., 2003; Arnhold et al., 2007). Multipotent mesenchymal progenitor cells have been isolated from bone marrow. Under exposition to angiogenic factors, these cells differentiated to angioblasts *in vitro* (CD34-positive, flk-1- positive, vascular endothelial cadherin-positive) and finally to mature endothelial cells, expressing specific endothelial markers, showing characteristics endothelial functional features and participating in neovascularization of tumours or wound healing *in vitro* (Reyes et al., 2001, 2002). Transplanted MSCs were able to enhance angiogenesis and contribute to remodelling of the vasculature *in vitro* (Ladage et al., 2007; Choi et al., 2008; Copland et al., 2008). Taken together, these data suggest that human MSCs may be considered an alternative source of

There are several evidences that in SSc there is a complex impairment in the BM microenvironment, involving not only the endothelial compartment but also the MSC, thus hypothesizing that alteration in this cell population may also contribute to the defective vasculogenesis in SSc (Del Papa et al., 2006; Cipriani et al., 2007). In particular, it has been reported that the number of colonies formed by MSC obtained from bone marrow of SSc patients were reduced in comparison with healthy controls. The colonies were small and did not expand, and the cells rapidly showed aging and signs of stress (Del Papa et al., 2006). Furthermore, nother study provided evidence of a reduced differentiative ability *in vitro* toward an endothelial phenotype of bone marrow MSC from SSc patients. These cells displayed both an early senescence and decreased capacity to perform specific endothelial activities, such as capillary morphogenesis and chemoinvasion, after VEGF and SDF-1 stimulation (Cipriani et al., 2007). All above suggests that a functional impairment of this population of endothelial progenitors cells may affect endothelial repair machinery,

In the last few years bone marrow-derived MSCs have shown great promise for tissue repair. In experimental models of acute myocardial infarction, intramyocardial injection of mesenchymal stem cells restored cardiac function through the formation of a new vascular network and arteriogenesis (Torensma et al., 2004). Autologous implantation of bone marrow–derived mesenchymal stem cells into chronic nonhealing ulcers has been shown to accelerate the healing process and significantly improve clinical parameters (Martens et al.,

contributing to the defective angiogenesis and vasculogenesis in the disease.

treatment of various ischemic diseases.

**5.1.2 Mesenchymal stem cells (MSCs)** 

EPCs (Oswald et al., 2004).

found reduced in SSc patients. Finally, the number and the size of colonies were reduced, and cells from patients showed morphologic signs of cellular senescence.

Another study assessed EPC counts in the whole blood of patients with SSc, osteoarthritis (OA), and RA (Allanore et al., 2007). Circulating CD34+CD133+ cells were increased in patients with SSc as compared with patients with OA, but the same cells were lower than those in RA patients. The analysis of potential correlations with clinical parameters showed that CD34+CD133+ counts increased in parallel with the European League Against Rheumatism Scleroderma Trial and Research (EUSTAR) group disease activity score. Of note, the authors did not analyse the expression of VEGFR-2.

In another study, the same authors (Avouac et al., 2008) assessed EPC, evaluating VEGFR-2 and lineage (Lin) markers as additional markers, and using 7-aminoactinomycin D (7-AAD) as viability marker. Again, patients with SSc displayed higher numbers of circulating Lin-7AAD-CD34+CD133+VEGFR-2+ EPC than did healthy subjects. Lower EPC counts in SSc patients were associated with higher Medsger severity scores for SSc and with digital ulcers. A decreased CD34+CD133+VEGFR-2+ EPC counts was found in both limited and diffuse subsets of recent-onset, and late-stage SSc, as compared with healthy individuals. The authors showed an increased rate of apoptosis in freshly isolated EPC from SSc patients. Addition of sera from the same patients to cultured EPC from healthy volunteers was able to induce apoptosis of EPC. The proapoptotic effects of SSc sera were abolished by depletion of the IgG fraction, suggesting the presence of anti-EPC autoantibodies in the SSc patient sera (Zhu et al., 2008).

It has been also found a raise of circulating EPC in early stage SSc, in response to tissue ischemia, but they dropped with disease progression. EPC reduction was linked with endothelial dysfunction and capillary loss, as well as the development of severe cardiac involvement and pulmonary arterial hypertension (Nevsakaya et al., 2008).

Whether EPC counts are altered in the peripheral blood of SSc patients is still a matter of controversy. Apparent contradiction in EPC counts between the different studies might be explained by use of different combinations of surface markers, resulting in the analysis of different cell subsets, or by differences in the mean disease duration and severity. Nevertheless, functional defects of EPC in the peripheral blood as well as in the bone marrow have consistently been reported (Del papa et al., 2006; Kuwana et al., 2004) indicating a critical role of EPC in the pathogenesis of SSc. Further studies on the molecular mechanisms underlying these defects is needed in order to develop specific treatment options and restore functional vasculogenesis in patients with SSc.

Several lines of evidence indicate that EPC obtained by short term culture of peripheral blood mononuclear cells (PBMCs) in media favouring endothelial differentiation, are composed predominantly of endothelial-like cells derived from circulating monocytes (Rehman et al., 2003; Urbich et al., 2003). Moreover, it has been identified a monocytederived multipotent cells positive for CD14, CD45, CD34, which contain progenitors able to differentiate into several distinct mesenchymal cell types, including bone, cartilage, fat, and skeletal and cardiac muscle cells, as well as neurons (Kuwana et al., 2003; Kodama et al., 2005, 2006). It has been recently shown that this population of multipotent cells, is able to differentiate into endothelium of a mature phenotype with typical morphologic and functional characteristics (Kuwana et al., 2006). These findings indicate a potential developmental relationship between monocytes and endothelial cells and suggest that the monocyte population could be recruited for vasculogenesis and represent an endothelial precursor population.

found reduced in SSc patients. Finally, the number and the size of colonies were reduced,

Another study assessed EPC counts in the whole blood of patients with SSc, osteoarthritis (OA), and RA (Allanore et al., 2007). Circulating CD34+CD133+ cells were increased in patients with SSc as compared with patients with OA, but the same cells were lower than those in RA patients. The analysis of potential correlations with clinical parameters showed that CD34+CD133+ counts increased in parallel with the European League Against Rheumatism Scleroderma Trial and Research (EUSTAR) group disease activity score. Of

In another study, the same authors (Avouac et al., 2008) assessed EPC, evaluating VEGFR-2 and lineage (Lin) markers as additional markers, and using 7-aminoactinomycin D (7-AAD) as viability marker. Again, patients with SSc displayed higher numbers of circulating Lin-7AAD-CD34+CD133+VEGFR-2+ EPC than did healthy subjects. Lower EPC counts in SSc patients were associated with higher Medsger severity scores for SSc and with digital ulcers. A decreased CD34+CD133+VEGFR-2+ EPC counts was found in both limited and diffuse subsets of recent-onset, and late-stage SSc, as compared with healthy individuals. The authors showed an increased rate of apoptosis in freshly isolated EPC from SSc patients. Addition of sera from the same patients to cultured EPC from healthy volunteers was able to induce apoptosis of EPC. The proapoptotic effects of SSc sera were abolished by depletion of the IgG fraction, suggesting the presence of anti-EPC autoantibodies in the SSc patient

It has been also found a raise of circulating EPC in early stage SSc, in response to tissue ischemia, but they dropped with disease progression. EPC reduction was linked with endothelial dysfunction and capillary loss, as well as the development of severe cardiac

Whether EPC counts are altered in the peripheral blood of SSc patients is still a matter of controversy. Apparent contradiction in EPC counts between the different studies might be explained by use of different combinations of surface markers, resulting in the analysis of different cell subsets, or by differences in the mean disease duration and severity. Nevertheless, functional defects of EPC in the peripheral blood as well as in the bone marrow have consistently been reported (Del papa et al., 2006; Kuwana et al., 2004) indicating a critical role of EPC in the pathogenesis of SSc. Further studies on the molecular mechanisms underlying these defects is needed in order to develop specific treatment

Several lines of evidence indicate that EPC obtained by short term culture of peripheral blood mononuclear cells (PBMCs) in media favouring endothelial differentiation, are composed predominantly of endothelial-like cells derived from circulating monocytes (Rehman et al., 2003; Urbich et al., 2003). Moreover, it has been identified a monocytederived multipotent cells positive for CD14, CD45, CD34, which contain progenitors able to differentiate into several distinct mesenchymal cell types, including bone, cartilage, fat, and skeletal and cardiac muscle cells, as well as neurons (Kuwana et al., 2003; Kodama et al., 2005, 2006). It has been recently shown that this population of multipotent cells, is able to differentiate into endothelium of a mature phenotype with typical morphologic and functional characteristics (Kuwana et al., 2006). These findings indicate a potential developmental relationship between monocytes and endothelial cells and suggest that the monocyte population could be recruited for vasculogenesis and represent an endothelial

involvement and pulmonary arterial hypertension (Nevsakaya et al., 2008).

options and restore functional vasculogenesis in patients with SSc.

and cells from patients showed morphologic signs of cellular senescence.

note, the authors did not analyse the expression of VEGFR-2.

sera (Zhu et al., 2008).

precursor population.

A recent study demonstrated that circulating monocytic EPCs were increased in the peripheral blood of SSc patients. *In vitro* and *in vivo* functional analyses revealed that monocytic EPCs derived from SSc patients had an enhanced ability to promote blood vessel formation, when co-cultured with HUVEC. In contrast, the EPC ability to be incorporated into vessels and differentiate into mature endothelial cells was rather impaired in SSc patients. This characteristic was primarily attributable to an enhanced angiogenic property through production of angiogenic factors (Yamaguchi et al., 2010). Because EPCs may critically contribute to the homeostasis of the physiological vascular network, these progenitor cells might be considered interesting candidates for novel cell therapies for the treatment of various ischemic diseases.

#### **5.1.2 Mesenchymal stem cells (MSCs)**

Endothelial cells could also originate from non- hematopoietic stem cells of the bone marrow (Drake et al., 2003). Mesenchymal stem cells are multipotent cells that are present in the bone marrow and in some tissues as resident stem cells. They retain the capacity to differentiate into several cell lineages of mesenchymal tissues, i.e. bone, cartilage, muscle, tendon or adipose tissue and serve for the preservation and repair of tissues and organs (Tuan et al., 2003; Arnhold et al., 2007). Multipotent mesenchymal progenitor cells have been isolated from bone marrow. Under exposition to angiogenic factors, these cells differentiated to angioblasts *in vitro* (CD34-positive, flk-1- positive, vascular endothelial cadherin-positive) and finally to mature endothelial cells, expressing specific endothelial markers, showing characteristics endothelial functional features and participating in neovascularization of tumours or wound healing *in vitro* (Reyes et al., 2001, 2002). Transplanted MSCs were able to enhance angiogenesis and contribute to remodelling of the vasculature *in vitro* (Ladage et al., 2007; Choi et al., 2008; Copland et al., 2008). Taken together, these data suggest that human MSCs may be considered an alternative source of EPCs (Oswald et al., 2004).

There are several evidences that in SSc there is a complex impairment in the BM microenvironment, involving not only the endothelial compartment but also the MSC, thus hypothesizing that alteration in this cell population may also contribute to the defective vasculogenesis in SSc (Del Papa et al., 2006; Cipriani et al., 2007). In particular, it has been reported that the number of colonies formed by MSC obtained from bone marrow of SSc patients were reduced in comparison with healthy controls. The colonies were small and did not expand, and the cells rapidly showed aging and signs of stress (Del Papa et al., 2006). Furthermore, nother study provided evidence of a reduced differentiative ability *in vitro* toward an endothelial phenotype of bone marrow MSC from SSc patients. These cells displayed both an early senescence and decreased capacity to perform specific endothelial activities, such as capillary morphogenesis and chemoinvasion, after VEGF and SDF-1 stimulation (Cipriani et al., 2007). All above suggests that a functional impairment of this population of endothelial progenitors cells may affect endothelial repair machinery, contributing to the defective angiogenesis and vasculogenesis in the disease.

In the last few years bone marrow-derived MSCs have shown great promise for tissue repair. In experimental models of acute myocardial infarction, intramyocardial injection of mesenchymal stem cells restored cardiac function through the formation of a new vascular network and arteriogenesis (Torensma et al., 2004). Autologous implantation of bone marrow–derived mesenchymal stem cells into chronic nonhealing ulcers has been shown to accelerate the healing process and significantly improve clinical parameters (Martens et al.,

Pathogenesis of the Endothelial Damage and Related Factors 13

deficiency not only impairs TGF-β/ALK-5 signaling but also reduces TGF-β/ALK-1 responses, suggesting that ALK-5 is essential for efficient ALK-1 activation and recruitment into a TGF-β/receptor complex (Bobik et al., 2006). These effects are mainly mediated by ENG (CD105), a coreceptor of TGF-β, predominantly expressed on cell surfaces of ECs. ENG plays a role in vascular integrity and endothelium functioning, whereas soluble ENG (sENG) acts as an antiangiogenic protein interfering with the binding of TGF-β to its receptors. Conflicting results have been reported in the available literature concerning the relationship between ENG and PAH. A previous paper reported an association between a 6 base insertion in intron 7 (6bINS) polymorphism of ENG gene and SSc-related PAH (Wipff et al., 2007). More recently, increased sENG levels were found in SSc patients, both with and without PAH, suggesting a role for ENG in SSc vasculopathy, independent of PAH presence (Coral-Alvarado et al., 2010). Furthermore, ENG might act on fibroblasts to modulate TGF-β signaling by acting as a molecular link regulating or reducing the total pool of TGF-β

The PDGF family consists of four different PDGF strands (A-D), establishing functional homodimers (PDGF-AA, PDGF-BB, PDGF-CC, and PDGF-DD) or an heterodimer PDGF-AB. They exert their biological activities by activating 2 structurally related tyrosine kinase receptors, PDGFRα and PDGFRβ. Ligand-induced receptor homo- or hetero-dimerization leads to autophosphorylation of specific tyrosine residues within the cytoplasmic domain. PDGF-A activates PDGFRα exclusively, while PDGF-B is capable of activating PDGFRαα, PDGFRαβ and PDGFRββ. PDGF-AB and PDGF-C activate PDGFRαα and PDGFRαβ, whereas PDGF-D preferentially activates PDGFRββ. PDGF-B and the PDGFRβ are primarily required for the development of the vasculature both under physiological and pathological angiogenic conditions e.g. during myocardial infarction (Zymek et al., 2006) and tumor vascularization (Vrekoussis et al., 2007) while, they are almost undetectable in healthy tissues. PDGFBB/PDGFRβ signaling-axis is also involved in recruiting pericytes and smooth muscle cells thus contributing to the maturation of the vessel wall (Lindblom et al., 2003). PDGF plays an important role in the pathogenesis of scleroderma and elevated expression of PDGF and its receptors has been found in SSc skin and lung diseases. In particular, expression of PDGF-B was detected in endothelial cell lining of small capillaries and in the infiltrating cells (Gay et al., 1989; Klareskoog et al., 1990). Likewise, elevated levels of PDGF-A and PDGF-B were found in bronchoalveolar lavage (BAL) fluid obtained from SSc patients (Ludwicka et al., 1995). Moreover, elevated plasma levels of PDFG-BB were reported in patients with SSc (Hummers et al., 2009). Indeed, recent studies suggest that the reciprocal induction of pro-angiogenic factors could promote vascularization and improve vessel maturation compared with the release of a single factor, and the interplay between PDGF and VEGF-mediated signalling pathways is just emerging (Bianco et al., 2007; Reinmuth et al., 2007)**.** In fact, activated platelets carry in their alpha granules a set of angiogenesis stimulators such as VEGF, PDGF and SDF-1. Moreover, EPCs trigger EC thus inducing a pro-angiogenic phenotype including the up-regulation of PDGFRβ, thereby turning the PDGFBB/PDGFRβ signaling-axis into a critical element of EPC-induced endothelial angiogenesis (Wyler von Ballmoos et al., 2010). This finding may be utilized to enhance EPC-based therapy of ischemic tissue in future. Furthermore, the recent discovery of novel agonistic antibodies targeting the PDGF receptor that represents a pathogenetic link between immune system and fibrosis might lead to perform intense investigations to clarify the possible role that PDGF might exert on the SSc vasculopathy (Baroni et al., 2006). Finally,

available for activating signal-transducing receptors.

**6.3 Platelet derived growth factor (PDGF)** 

2006). Recently, it has been showed that in one patient with systemic sclerosis who have severe peripheral ischemia, intravenous infusion of expanded autologous mesenchymal stem cells may promote the recovery of the vascular network, restore blood flow, and reduce skin necrosis (Guiducci et al., 2010). Further studies on a larger number of patients with systemic sclerosis are needed to confirm the short- and long-term efficacy and safety of mesenchymal stem-cell infusion as treatment of severe digital ulcers and gangrene of the extremities that are resistant to conventional therapies.

#### **6. Angiogenic factors**

#### **6.1 Vascular endothelial growth factor (VEGF)**

VEGF, one of the strongest angiogenic factors known in biology, is involved in several steps of physiological and pathological angiogenesis. VEGF increases the vascular permeability, stimulates the migration and proliferation of ECs and induces tube formation. The biological effects of VEGF are extremely dose dependent. Loss of even a single allele results in lethal vascular defects in the embryo, and postnatal inhibition of VEGF leads to impaired organ development and growth arrest in mice. Application of VEGF as a recombinant protein or by gene transfer augmented perfusion and development of collateral vessels in animal models of hindlimb ischemia, thereby making VEGF an interesting target for therapeutic angiogenesis (Takeshita et al., 1994, 1996). During SSc, VEGF is strongly overexpressed in the skin and sera (Distle O et al., 2002) of these patients. VEGF exerts its biological functions by binding to 2 different tyrosine kinase receptors: VEGFR-1 and VEGFR-2, which are both upregulated in the affected skin of SSc patients (Distler O et al., 2004), although non compensative new vessel formation is observed. The serum levels of VEGF significantly correlate with the development of fingertip ulcers in these patients. Although elevated levels of VEGF are consistent with active angiogenesis, an uncontrolled chronic overexpression, as seen in SSc patients, throughout various disease stages, might contribute to disturbed vessel morphology and endothelial disturbances rather than to promote new vessel formation. On the other hand, a brief upregulation of VEGF results in instability of newly formed vessels ((Dor et al., 2002). The mechanisms that lead to an over-expression of VEGF in SSc are unclear. Isolated microvascular endothelial cells (MVECs) from SSc patients show an impaired response to VEGF and other growth factors in the Matrigel capillary morphogenesis assay, suggesting that VEGF receptor signaling might be impaired in these cells (D'Alessio et al., 2004). Hypoxia-induced expression of hypoxia inducible factor-1α (HIF1-α) does not appear to play a major role in the induction of VEGF in SSc (Distler O et al., 2004), whereas induction by cytokines such as platelet derived growth factor (PDGF) and transforming growth factor beta (TGF-β) appear to be more important. The function of TGFβ in angiogenesis is strongly context dependent. A weak TGF-β stimulation may cause induction of several angiogenic regulators such as VEGF and matrix proteins to promote angiogenesis, whereas at high TGF-β concentrations, growth-inhibitory effect dominate (Bobik et al., 2006). Functionally important gene polymorphisms that lead to an impairment in biological properties of VEGF have not still been shown in SSc patients (Allanore et al., 2007).

#### **6.2 Transforming growth factor beta (TGF-β) and Endoglin (ENG)**

The vascular effect of TGF-β in angiogenesis results in activation of ECs and vascular smooth muscle cells (VSMCs). It regulates the activation state of ECs, via 2 different types of I receptors, ALK-5, and ALK-1. The TGF-β/ALK-1 pathway stimulates ECs proliferation and migration, whereas the TGF-β/ALK-5 pathway inhibits these processes. ALK-5

2006). Recently, it has been showed that in one patient with systemic sclerosis who have severe peripheral ischemia, intravenous infusion of expanded autologous mesenchymal stem cells may promote the recovery of the vascular network, restore blood flow, and reduce skin necrosis (Guiducci et al., 2010). Further studies on a larger number of patients with systemic sclerosis are needed to confirm the short- and long-term efficacy and safety of mesenchymal stem-cell infusion as treatment of severe digital ulcers and gangrene of the

VEGF, one of the strongest angiogenic factors known in biology, is involved in several steps of physiological and pathological angiogenesis. VEGF increases the vascular permeability, stimulates the migration and proliferation of ECs and induces tube formation. The biological effects of VEGF are extremely dose dependent. Loss of even a single allele results in lethal vascular defects in the embryo, and postnatal inhibition of VEGF leads to impaired organ development and growth arrest in mice. Application of VEGF as a recombinant protein or by gene transfer augmented perfusion and development of collateral vessels in animal models of hindlimb ischemia, thereby making VEGF an interesting target for therapeutic angiogenesis (Takeshita et al., 1994, 1996). During SSc, VEGF is strongly overexpressed in the skin and sera (Distle O et al., 2002) of these patients. VEGF exerts its biological functions by binding to 2 different tyrosine kinase receptors: VEGFR-1 and VEGFR-2, which are both upregulated in the affected skin of SSc patients (Distler O et al., 2004), although non compensative new vessel formation is observed. The serum levels of VEGF significantly correlate with the development of fingertip ulcers in these patients. Although elevated levels of VEGF are consistent with active angiogenesis, an uncontrolled chronic overexpression, as seen in SSc patients, throughout various disease stages, might contribute to disturbed vessel morphology and endothelial disturbances rather than to promote new vessel formation. On the other hand, a brief upregulation of VEGF results in instability of newly formed vessels ((Dor et al., 2002). The mechanisms that lead to an over-expression of VEGF in SSc are unclear. Isolated microvascular endothelial cells (MVECs) from SSc patients show an impaired response to VEGF and other growth factors in the Matrigel capillary morphogenesis assay, suggesting that VEGF receptor signaling might be impaired in these cells (D'Alessio et al., 2004). Hypoxia-induced expression of hypoxia inducible factor-1α (HIF1-α) does not appear to play a major role in the induction of VEGF in SSc (Distler O et al., 2004), whereas induction by cytokines such as platelet derived growth factor (PDGF) and transforming growth factor beta (TGF-β) appear to be more important. The function of TGFβ in angiogenesis is strongly context dependent. A weak TGF-β stimulation may cause induction of several angiogenic regulators such as VEGF and matrix proteins to promote angiogenesis, whereas at high TGF-β concentrations, growth-inhibitory effect dominate (Bobik et al., 2006). Functionally important gene polymorphisms that lead to an impairment in biological properties of VEGF have not still been shown in SSc patients (Allanore et al., 2007).

extremities that are resistant to conventional therapies.

**6.2 Transforming growth factor beta (TGF-β) and Endoglin (ENG)** 

The vascular effect of TGF-β in angiogenesis results in activation of ECs and vascular smooth muscle cells (VSMCs). It regulates the activation state of ECs, via 2 different types of I receptors, ALK-5, and ALK-1. The TGF-β/ALK-1 pathway stimulates ECs proliferation and migration, whereas the TGF-β/ALK-5 pathway inhibits these processes. ALK-5

**6.1 Vascular endothelial growth factor (VEGF)** 

**6. Angiogenic factors** 

deficiency not only impairs TGF-β/ALK-5 signaling but also reduces TGF-β/ALK-1 responses, suggesting that ALK-5 is essential for efficient ALK-1 activation and recruitment into a TGF-β/receptor complex (Bobik et al., 2006). These effects are mainly mediated by ENG (CD105), a coreceptor of TGF-β, predominantly expressed on cell surfaces of ECs. ENG plays a role in vascular integrity and endothelium functioning, whereas soluble ENG (sENG) acts as an antiangiogenic protein interfering with the binding of TGF-β to its receptors. Conflicting results have been reported in the available literature concerning the relationship between ENG and PAH. A previous paper reported an association between a 6 base insertion in intron 7 (6bINS) polymorphism of ENG gene and SSc-related PAH (Wipff et al., 2007). More recently, increased sENG levels were found in SSc patients, both with and without PAH, suggesting a role for ENG in SSc vasculopathy, independent of PAH presence (Coral-Alvarado et al., 2010). Furthermore, ENG might act on fibroblasts to modulate TGF-β signaling by acting as a molecular link regulating or reducing the total pool of TGF-β available for activating signal-transducing receptors.

#### **6.3 Platelet derived growth factor (PDGF)**

The PDGF family consists of four different PDGF strands (A-D), establishing functional homodimers (PDGF-AA, PDGF-BB, PDGF-CC, and PDGF-DD) or an heterodimer PDGF-AB. They exert their biological activities by activating 2 structurally related tyrosine kinase receptors, PDGFRα and PDGFRβ. Ligand-induced receptor homo- or hetero-dimerization leads to autophosphorylation of specific tyrosine residues within the cytoplasmic domain. PDGF-A activates PDGFRα exclusively, while PDGF-B is capable of activating PDGFRαα, PDGFRαβ and PDGFRββ. PDGF-AB and PDGF-C activate PDGFRαα and PDGFRαβ, whereas PDGF-D preferentially activates PDGFRββ. PDGF-B and the PDGFRβ are primarily required for the development of the vasculature both under physiological and pathological angiogenic conditions e.g. during myocardial infarction (Zymek et al., 2006) and tumor vascularization (Vrekoussis et al., 2007) while, they are almost undetectable in healthy tissues. PDGFBB/PDGFRβ signaling-axis is also involved in recruiting pericytes and smooth muscle cells thus contributing to the maturation of the vessel wall (Lindblom et al., 2003). PDGF plays an important role in the pathogenesis of scleroderma and elevated expression of PDGF and its receptors has been found in SSc skin and lung diseases. In particular, expression of PDGF-B was detected in endothelial cell lining of small capillaries and in the infiltrating cells (Gay et al., 1989; Klareskoog et al., 1990). Likewise, elevated levels of PDGF-A and PDGF-B were found in bronchoalveolar lavage (BAL) fluid obtained from SSc patients (Ludwicka et al., 1995). Moreover, elevated plasma levels of PDFG-BB were reported in patients with SSc (Hummers et al., 2009). Indeed, recent studies suggest that the reciprocal induction of pro-angiogenic factors could promote vascularization and improve vessel maturation compared with the release of a single factor, and the interplay between PDGF and VEGF-mediated signalling pathways is just emerging (Bianco et al., 2007; Reinmuth et al., 2007)**.** In fact, activated platelets carry in their alpha granules a set of angiogenesis stimulators such as VEGF, PDGF and SDF-1. Moreover, EPCs trigger EC thus inducing a pro-angiogenic phenotype including the up-regulation of PDGFRβ, thereby turning the PDGFBB/PDGFRβ signaling-axis into a critical element of EPC-induced endothelial angiogenesis (Wyler von Ballmoos et al., 2010). This finding may be utilized to enhance EPC-based therapy of ischemic tissue in future. Furthermore, the recent discovery of novel agonistic antibodies targeting the PDGF receptor that represents a pathogenetic link between immune system and fibrosis might lead to perform intense investigations to clarify the possible role that PDGF might exert on the SSc vasculopathy (Baroni et al., 2006). Finally,

Pathogenesis of the Endothelial Damage and Related Factors 15

vessel maturation and maintains vessel integrity by the recruitment of periendothelial cells. On the contrary, Ang-2 acts as a vessel-destabilizing cytokine, playing an essential role in vascular remodeling. Recently, has been demonstrated that Ang-1 and -2 are differentially expressed in the sera of patients with SSc. Ang-1, was significantly decreased in SSc patients contributing to the development of SSc-related vasculopathy through promoting activation and apoptosis of ECs and destabilization of blood vessels. On the other hand, Ang-2, was significantly increased in SSc patients (Michalska et al., 2010)). Moreover, high Ang-2 levels are associated with greater severity and higher activity of the disease. Thus, the Ang-1/ Ang-2 imbalance might contribute to the development of the disease, and represent a new

As already mentioned above, MVECS can perform angiogenesis only when provided with a proper enzymatic machinery, enabling them to lyse the extracellular matrix and invade the surrounding tissue. In this regard, the serine protease urokinase- type plasminogen activator uPA-uPAR system is known to play a crucial role in angiogenesis by modulating the adhesive properties of ECs in their interactions with the extracellular matrix and in the degradation of matrix components (Van Hinsberg et al., 2008); D'Alessio et al., 2004;

Another angiogenesis-associated serine protease family, potentially involved in the angiogenic program of SSc are some members of kallikreins. Kallikreins hydrolyze kininogen to kinin. Kinins promote angiogenesis, since they play a role that leads to endothelial cell migration, proliferation and differentiation. Kallikreins 9, 11, and 12, which are associated with proangiogenesis, were downregulated in SSc patients, whereas antiangiogenic kallikrein 3 was upregulated. Further experiments using healthy MVECs treated with antibodies against the relevant kallikreins revealed that while kallikreins 9, 11, and 12 induced cell growth, only kallikrein 12 regulated invasion and capillary morphogenesis. Buffering of kallikrein 12 with antibodies resulted in the acquisition of an SSc-like pattern by

Another pro-angiogenic marker of SSc vasculopathy is the presence of adhesion proteins involved in cell-cell interaction and cell-extracellular matrix interactions that are found increased in SSc skin (Gruscwitz et al., 1992). In particular, increased expression of the adhesion proteins such as intercellular adhesion molecule 1 (ICAM-1), endothelial leukocyte adhesion molecule 1 (ELAM-1), vascular adhesion molecule (VCAM-1), E-selectin, P-selectin in endothelial cells in the skin of patients with a rapidly progressive systemic sclerosis was observed (Sollberg et al., 1992; Gruschwitz et al., 1995; Ihn et al., 1997; Kiener et al., 1994; Ihn et al., 1998). To evaluate the relationship between systemic manifestations and immunological markers of endothelial cell activation, soluble VCAM-1 (sVCAM-1), soluble E selectin (sE-selectin), VEGF, and ET-1 were determined. Interestingly, the injury to the pulmonary and renal vascular trees might have distinct pathogenic mechanisms (Stratton et al., 1998). In particular, in patients with SRC, the level of E-selectin, sVCAM-1, and soluble ICAM-1 (sICAM-1) were elevated, but they were not consistently elevated in patients with

promising therapeutic target in SSc.

Margheri et al., 2006; Manetti et al., 2011).

normal cells *in vitro* angiogenesis (Giusti et al., 2005).

**6.7 uPAR and kallikreins** 

**6.8 Adhesion molecules** 

pulmonary hypertension.

PDGF receptor antagonist STI571 (imatinib) reversed advanced pulmonary vascular disease in 2 animal models of pulmonary hypertension (Schermuly et al., 2005).

#### **6.4 Stromal cell-derived factor 1 (SDF-1/CXCL12)**

The CXC chemokine SDF-1, the most important chemokine induced by ischemia, and its receptor, CXCR4, regulates specific steps in new vessel formation (Salcedo et al., 2003). Experimental deficiency in *SDF-1* or *CXCR4* gene in the embryo results in a lethal phenotype characterised by defective development of cardiovascular system (Kucia et al., 2004). As already mentioned above, SDF-1 is a potent chemoattractant for mature ECs, hematopoietic stem cells (HSCs) and EPCs expressing CXCR4 on their surface, thus influencing both angiogenesis and vasculogenesis (De Falco et al., 2004). SDF-1–CXCR4 interaction further amplifies angiogenesis by increasing VEGF release by ECs. VEGF elevated levels, in turn, promote enhanced expression of CXCR4 on endothelial cells, which can then respond to SDF-1. SDF-1, also prevents the apoptosis of EPCs. Also, SDF-1 contributes to the stabilization of neo-vessel formation by recruiting CXCR4+PDGFR+cKit+ smooth muscle progenitor cells during recovery for vascular injury. In SSc, due to the transient nature of SDF-1 expression, its modulation could be considered a future therapeutic target for inducing new vessel formation in this disease (Cipriani et al., 2006). Finally, SDF-1 polymorphism may modulate SSc vascular phenotype, further arguing for a critical role of SDF-1/CXCR4 axis in the vascular component of SSc pathogenesis (Manetti et al., 2009).

#### **6.5 Endothelin 1 (ET-1)**

ET-1, a highly vasoconstrictor molecule produced from endothelial cells and mesodermal cells such as fibroblasts and smooth muscle cells, promotes leukocyte adhesion to the endothelium as well as vascular smooth muscle cell proliferation and fibroblast activation (Abraham & Distler, 2006)**.** ET-1 expression levels are increased in blood vessels, lung, kidney and skin of patients with SSc. Plasma ET-1 levels are also increased in SSc patients in both early and late stage (Kahaleh, 1991; Kadono et al., 1995). ET-1 mediates its biological effects via the ETA and ETB receptors. ETA receptors are expressed by vascular smooth muscle cells and can mediate vasoconstriction, smooth muscle cell proliferation, fibrosis and inflammation. ETB receptors are predominantly expressed on endothelial cells mediating vasodilation via the release of nitric oxide or potassium channel activation, and removing ET-1 from the circulation. In SSc vasculopathy, ETB receptors are down regulated on endothelial cells which may diminish their vasodilatory role while are up-regulated on smooth muscle cells and can contribute to cell proliferation, hypertrophy, inflammation, fibrosis and vasoconstriction (Abraham et al., 1997; Bauer et al., 20029). ETA/B receptor antagonists including bosentan are now commonly used for the treatment of PAH (Channick et al., 2001; Rubin et al., 2002) and of the prevention of new digital ulcers related to SSc (Matucci-Cerinic et al., 2011). ET-1 represents a potent molecular target for intervention in the management of patients with SSc.

#### **6.6 Angiopoietins**

Angiopoietins are known to be involved in the development, remodeling and stability of blood vessels. Recently has been clarified as they might act alongside VEGF (Ashara et al., 1998). However, Ang-1 and -2 have opposing functions. Ang-1 under physiological conditions has vasoprotective and anti-inflammatory actions (Kim et al., 2001), mediates vessel maturation and maintains vessel integrity by the recruitment of periendothelial cells. On the contrary, Ang-2 acts as a vessel-destabilizing cytokine, playing an essential role in vascular remodeling. Recently, has been demonstrated that Ang-1 and -2 are differentially expressed in the sera of patients with SSc. Ang-1, was significantly decreased in SSc patients contributing to the development of SSc-related vasculopathy through promoting activation and apoptosis of ECs and destabilization of blood vessels. On the other hand, Ang-2, was significantly increased in SSc patients (Michalska et al., 2010)). Moreover, high Ang-2 levels are associated with greater severity and higher activity of the disease. Thus, the Ang-1/ Ang-2 imbalance might contribute to the development of the disease, and represent a new promising therapeutic target in SSc.

#### **6.7 uPAR and kallikreins**

14 Systemic Sclerosis – An Update on the Aberrant Immune System and Clinical Features

PDGF receptor antagonist STI571 (imatinib) reversed advanced pulmonary vascular disease

The CXC chemokine SDF-1, the most important chemokine induced by ischemia, and its receptor, CXCR4, regulates specific steps in new vessel formation (Salcedo et al., 2003). Experimental deficiency in *SDF-1* or *CXCR4* gene in the embryo results in a lethal phenotype characterised by defective development of cardiovascular system (Kucia et al., 2004). As already mentioned above, SDF-1 is a potent chemoattractant for mature ECs, hematopoietic stem cells (HSCs) and EPCs expressing CXCR4 on their surface, thus influencing both angiogenesis and vasculogenesis (De Falco et al., 2004). SDF-1–CXCR4 interaction further amplifies angiogenesis by increasing VEGF release by ECs. VEGF elevated levels, in turn, promote enhanced expression of CXCR4 on endothelial cells, which can then respond to SDF-1. SDF-1, also prevents the apoptosis of EPCs. Also, SDF-1 contributes to the stabilization of neo-vessel formation by recruiting CXCR4+PDGFR+cKit+ smooth muscle progenitor cells during recovery for vascular injury. In SSc, due to the transient nature of SDF-1 expression, its modulation could be considered a future therapeutic target for inducing new vessel formation in this disease (Cipriani et al., 2006). Finally, SDF-1 polymorphism may modulate SSc vascular phenotype, further arguing for a critical role of SDF-1/CXCR4 axis in the vascular component of SSc pathogenesis

ET-1, a highly vasoconstrictor molecule produced from endothelial cells and mesodermal cells such as fibroblasts and smooth muscle cells, promotes leukocyte adhesion to the endothelium as well as vascular smooth muscle cell proliferation and fibroblast activation (Abraham & Distler, 2006)**.** ET-1 expression levels are increased in blood vessels, lung, kidney and skin of patients with SSc. Plasma ET-1 levels are also increased in SSc patients in both early and late stage (Kahaleh, 1991; Kadono et al., 1995). ET-1 mediates its biological effects via the ETA and ETB receptors. ETA receptors are expressed by vascular smooth muscle cells and can mediate vasoconstriction, smooth muscle cell proliferation, fibrosis and inflammation. ETB receptors are predominantly expressed on endothelial cells mediating vasodilation via the release of nitric oxide or potassium channel activation, and removing ET-1 from the circulation. In SSc vasculopathy, ETB receptors are down regulated on endothelial cells which may diminish their vasodilatory role while are up-regulated on smooth muscle cells and can contribute to cell proliferation, hypertrophy, inflammation, fibrosis and vasoconstriction (Abraham et al., 1997; Bauer et al., 20029). ETA/B receptor antagonists including bosentan are now commonly used for the treatment of PAH (Channick et al., 2001; Rubin et al., 2002) and of the prevention of new digital ulcers related to SSc (Matucci-Cerinic et al., 2011). ET-1 represents a potent molecular target for

Angiopoietins are known to be involved in the development, remodeling and stability of blood vessels. Recently has been clarified as they might act alongside VEGF (Ashara et al., 1998). However, Ang-1 and -2 have opposing functions. Ang-1 under physiological conditions has vasoprotective and anti-inflammatory actions (Kim et al., 2001), mediates

in 2 animal models of pulmonary hypertension (Schermuly et al., 2005).

**6.4 Stromal cell-derived factor 1 (SDF-1/CXCL12)** 

intervention in the management of patients with SSc.

(Manetti et al., 2009).

**6.6 Angiopoietins** 

**6.5 Endothelin 1 (ET-1)** 

As already mentioned above, MVECS can perform angiogenesis only when provided with a proper enzymatic machinery, enabling them to lyse the extracellular matrix and invade the surrounding tissue. In this regard, the serine protease urokinase- type plasminogen activator uPA-uPAR system is known to play a crucial role in angiogenesis by modulating the adhesive properties of ECs in their interactions with the extracellular matrix and in the degradation of matrix components (Van Hinsberg et al., 2008); D'Alessio et al., 2004; Margheri et al., 2006; Manetti et al., 2011).

Another angiogenesis-associated serine protease family, potentially involved in the angiogenic program of SSc are some members of kallikreins. Kallikreins hydrolyze kininogen to kinin. Kinins promote angiogenesis, since they play a role that leads to endothelial cell migration, proliferation and differentiation. Kallikreins 9, 11, and 12, which are associated with proangiogenesis, were downregulated in SSc patients, whereas antiangiogenic kallikrein 3 was upregulated. Further experiments using healthy MVECs treated with antibodies against the relevant kallikreins revealed that while kallikreins 9, 11, and 12 induced cell growth, only kallikrein 12 regulated invasion and capillary morphogenesis. Buffering of kallikrein 12 with antibodies resulted in the acquisition of an SSc-like pattern by normal cells *in vitro* angiogenesis (Giusti et al., 2005).

#### **6.8 Adhesion molecules**

Another pro-angiogenic marker of SSc vasculopathy is the presence of adhesion proteins involved in cell-cell interaction and cell-extracellular matrix interactions that are found increased in SSc skin (Gruscwitz et al., 1992). In particular, increased expression of the adhesion proteins such as intercellular adhesion molecule 1 (ICAM-1), endothelial leukocyte adhesion molecule 1 (ELAM-1), vascular adhesion molecule (VCAM-1), E-selectin, P-selectin in endothelial cells in the skin of patients with a rapidly progressive systemic sclerosis was observed (Sollberg et al., 1992; Gruschwitz et al., 1995; Ihn et al., 1997; Kiener et al., 1994; Ihn et al., 1998). To evaluate the relationship between systemic manifestations and immunological markers of endothelial cell activation, soluble VCAM-1 (sVCAM-1), soluble E selectin (sE-selectin), VEGF, and ET-1 were determined. Interestingly, the injury to the pulmonary and renal vascular trees might have distinct pathogenic mechanisms (Stratton et al., 1998). In particular, in patients with SRC, the level of E-selectin, sVCAM-1, and soluble ICAM-1 (sICAM-1) were elevated, but they were not consistently elevated in patients with pulmonary hypertension.

Pathogenesis of the Endothelial Damage and Related Factors 17

For the specific function of involved molecules and cells see the text. EPC: circulating endothelial progenitors; MSC: mesenchymal stem cells; HSC: hematopoietic stem cells; ROS: reactive oxygen species; TGFβ: transforming growth factor beta; HIF1α: hypoxia inducible factor 1 alfa; PDGF: platelet derived factor; PDGFR: platelet derived factor receptor; VEGF: vascular endothelial growth factor; VEGFR: vascular endothelial growth factor receptor; SDF-1: stromal cell-derived factor 1; CXCR4: SDF-1 receptor; ENG: endoglin; sENG: soluble endoglin; ALK-1 and ALK-5: activin receptor-like kinase-1 and -5; Ang-1 and Ang-2: angiopoietin 1 and 2; Tie-2: tyrosine kinase receptor; ET1: endothelin-1; ETAR and ETBR: endothelin receptor A and B; MMP12: matrix metalloproteinase 12; uPAR: urokinase-type

Vascular endothelial cell damage is and early an probably initiating event in the pathogenesis of the SSc resulting in endothelial dysfunction and loss of capillaries. The dysregulation of the angiogenic homeostasis seen in SSc, leads to failure in replacing damaged vessels, thus contributing to the vascular desertification and the chronic ischemia characteristic of the disease. Vasculogenesis is also impaired in SSc. Failure of the angiogenic process in SSc largely depends on alteration in the balance between angiogenic and angiostatic factors. At present, data describing the process of dysregulation in angiogenic homeostasis are incomplete and need further research. On the other hand functional alterations of the cellular players involved in the angiogenic and vasculogenic program are also demonstared. The possibility of reversing this impairment opens new perspectives for

regenerative cellular therapy for the vascular damage of this disease.

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Schuler G & Hambrecht R. (2004). Increase of circulating endothelial progenitor cells in patients with coronary artery disease after exercise-induced ischemia*.* 

expression in human dermal endothelial cells by scleroderma sera containing

Caramaschi P, Majdan M, Krasowska D, Friedl E, Lemarechal H, Ananieva LP, Nievskaya T, Ekindjian OG, Matucci-Cerinic M & Kahan A. (2007). Lack of

plasminogen activator.

**9. Acknowledgment** 

**10. References** 

**8. Conclusions** 

### **7. Angiostatic factors**

#### **7.1 Endostatin and Angiostatin**

Breakdown of the extracellular matrix by granzyme B and other proteases contained in T cell granule content, may contribute to defective wound healing and vascular repair in SSc patients. Among these extracellular matrix derived angiostatic growth factors, endostatin has been characterized as a potent inhibitor of VEGF-induced angiogenesis. Endostatin is a C-terminal, 20 kDa fragment of the basement protein collagen type XVIII that inhibits angiogenesis and tumor growth strongly by reducing endothelial cell proliferation and migration. Circulating endostatin concentrations are significantly increased in patients with SSc and this increase is associated with the presence of more severe clinical involvement (Distler O et al., 2002). Angiostatin is another antiangiogenic factor derived from the cleavage of the plasminogen and proangiogenic plasmin. Recent data suggest that there is a decreased presence and activity of proangiogenic plasmin, and increased production of antiangiogenic angiostatin in SSc plasma. This increase in angiostatin production may account for some of the vascular defects observed in patients with SSc (Mullighan-Kehoe et al., 2007). Finally, circulating thrombospondin-1 (TSP-1), a counteradhesive protein with angiostatic and apoptotic properties was found increased in patients with SSc (Macko et al., 2002).

Fig. 1. Angiogenic endothelial response following ischemic injury.

For the specific function of involved molecules and cells see the text. EPC: circulating endothelial progenitors; MSC: mesenchymal stem cells; HSC: hematopoietic stem cells; ROS: reactive oxygen species; TGFβ: transforming growth factor beta; HIF1α: hypoxia inducible factor 1 alfa; PDGF: platelet derived factor; PDGFR: platelet derived factor receptor; VEGF: vascular endothelial growth factor; VEGFR: vascular endothelial growth factor receptor; SDF-1: stromal cell-derived factor 1; CXCR4: SDF-1 receptor; ENG: endoglin; sENG: soluble endoglin; ALK-1 and ALK-5: activin receptor-like kinase-1 and -5; Ang-1 and Ang-2: angiopoietin 1 and 2; Tie-2: tyrosine kinase receptor; ET1: endothelin-1; ETAR and ETBR: endothelin receptor A and B; MMP12: matrix metalloproteinase 12; uPAR: urokinase-type plasminogen activator.

#### **8. Conclusions**

16 Systemic Sclerosis – An Update on the Aberrant Immune System and Clinical Features

Breakdown of the extracellular matrix by granzyme B and other proteases contained in T cell granule content, may contribute to defective wound healing and vascular repair in SSc patients. Among these extracellular matrix derived angiostatic growth factors, endostatin has been characterized as a potent inhibitor of VEGF-induced angiogenesis. Endostatin is a C-terminal, 20 kDa fragment of the basement protein collagen type XVIII that inhibits angiogenesis and tumor growth strongly by reducing endothelial cell proliferation and migration. Circulating endostatin concentrations are significantly increased in patients with SSc and this increase is associated with the presence of more severe clinical involvement (Distler O et al., 2002). Angiostatin is another antiangiogenic factor derived from the cleavage of the plasminogen and proangiogenic plasmin. Recent data suggest that there is a decreased presence and activity of proangiogenic plasmin, and increased production of antiangiogenic angiostatin in SSc plasma. This increase in angiostatin production may account for some of the vascular defects observed in patients with SSc (Mullighan-Kehoe et al., 2007). Finally, circulating thrombospondin-1 (TSP-1), a counteradhesive protein with angiostatic and apoptotic properties was found increased in patients with SSc (Macko et al.,

Fig. 1. Angiogenic endothelial response following ischemic injury.

**7. Angiostatic factors** 

2002).

**7.1 Endostatin and Angiostatin** 

Vascular endothelial cell damage is and early an probably initiating event in the pathogenesis of the SSc resulting in endothelial dysfunction and loss of capillaries. The dysregulation of the angiogenic homeostasis seen in SSc, leads to failure in replacing damaged vessels, thus contributing to the vascular desertification and the chronic ischemia characteristic of the disease. Vasculogenesis is also impaired in SSc. Failure of the angiogenic process in SSc largely depends on alteration in the balance between angiogenic and angiostatic factors. At present, data describing the process of dysregulation in angiogenic homeostasis are incomplete and need further research. On the other hand functional alterations of the cellular players involved in the angiogenic and vasculogenic program are also demonstared. The possibility of reversing this impairment opens new perspectives for regenerative cellular therapy for the vascular damage of this disease.

#### **9. Acknowledgment**

This work was supported in part by PRIN 2008:200884K784 and FIRA ONLUS 2009.

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**2** 

*2CRG, Barcelona* 

*1,3,4France 2Spain* 

*3Service de Médecine Vasculaire,* 

*Bordeaux University Hospital, Bordeaux 4Department of Physiology and EA 3842,* 

**Blood Platelets and Systemic Sclerosis** 

Systemic sclerosis (SSc) is characterized by a progressive fibrosis of the perivascular and interstitial connective tissues which can involve the skin, heart, lungs, kidneys, and the gastrointestinal tract. SSc is an uncommon, debilitating condition, associated to a vital risk linked to visceral extensions and has a high case-fatality rate among connectivitis. SSc begins in the vast majority of cases with a Raynaud's phenomenon, may have a limited or diffuse skin extension, and is often associated to arterial occlusions, digital ulcerations or necrosis. SSc clinical manifestations are heterogeneous and classifications distinguish limited to diffuse disease, depending on the distribution of the skin lesions and organ involvement [1-3]. There is today no curative treatment. Disease susceptibility differs according to sex, age and race, there is a notable familial clustering, and SSc incidence may be rising [4, 5]. The clinical management of the patients still remains a difficult challenge and the pharmacopeia offers limited choices to the clinician to bring relief to patients. Pulmonary, renal and myocardial complications have benefited from the introduction of angiotensin converting enzyme inhibitors, calcium pump inhibitors, prostacyclin analogs and endothelin antagonists. Based on recent pathophysiological insights, a number of novel

SSc pathogenesis remains obscure. Central features are an inflammatory vasculopathy, perivascular and interstitial sclerosis, altered angiogenesis and autoimmunity. Pathologic changes depend on whether they are observed at an early or late disease stage. The progression of the disease is typified by the accumulation of extra-cellular matrix (ECM) components in connective tissues. There are clinical and pathologic arguments to support the hypothesis that the vasculature is involved at an early step during disease progression.

**1. Introduction** 

agents are being developed [6-8].

**2. SSc pathogenesis in 2011: An elusive mechanism** 

Sébastien Lepreux1, Anne Solanilla1, Julien Villeneuve2, Joël Constans3, Alexis Desmoulière4 and Jean Ripoche1

*1INSERM U 1026 and Université de Bordeaux, Bordeaux* 

*Faculty of Pharmacy, University of Limoges, Limoges*


### **Blood Platelets and Systemic Sclerosis**

Sébastien Lepreux1, Anne Solanilla1, Julien Villeneuve2, Joël Constans3, Alexis Desmoulière4 and Jean Ripoche1 *1INSERM U 1026 and Université de Bordeaux, Bordeaux 2CRG, Barcelona 3Service de Médecine Vasculaire, Bordeaux University Hospital, Bordeaux 4Department of Physiology and EA 3842, Faculty of Pharmacy, University of Limoges, Limoges 1,3,4France 2Spain* 

#### **1. Introduction**

28 Systemic Sclerosis – An Update on the Aberrant Immune System and Clinical Features

Wipff J, Kahan A, Hachulla E, Sibilia J, Cabane J, Meyer O, Mouthon L, Guillevin L, Junien

Worda M, Sgonc R, Dietrich H, Niederegger H, Sundick RS, Gershwin ME & Wick G. (2003).

Wyler von Ballmoos M, Yang Z, Völzmann J, Baumgartner I, Kalka C & Di Santo S. (2010).

*Rheumatology* 46:622

48:2605–14.

C, Boileau C & Allanore Y. (2007). Association between an endoglin gene polymorphism and systemic sclerosis related pulmonary arterial hypertension.

In vivo analysis of the apoptosis-inducing effect of anti endothelial cell antibodies in systemic sclerosis by the chorionallantoic membrane assay. *Arthritis Rheum*.

Endothelial progenitor cells induce a phenotype shift in differentiated endothelial cells towards PDGF/PDGFRβ axis-mediated angiogenesis. *PLoS One*. 5(11):e14107.

> Systemic sclerosis (SSc) is characterized by a progressive fibrosis of the perivascular and interstitial connective tissues which can involve the skin, heart, lungs, kidneys, and the gastrointestinal tract. SSc is an uncommon, debilitating condition, associated to a vital risk linked to visceral extensions and has a high case-fatality rate among connectivitis. SSc begins in the vast majority of cases with a Raynaud's phenomenon, may have a limited or diffuse skin extension, and is often associated to arterial occlusions, digital ulcerations or necrosis. SSc clinical manifestations are heterogeneous and classifications distinguish limited to diffuse disease, depending on the distribution of the skin lesions and organ involvement [1-3]. There is today no curative treatment. Disease susceptibility differs according to sex, age and race, there is a notable familial clustering, and SSc incidence may be rising [4, 5]. The clinical management of the patients still remains a difficult challenge and the pharmacopeia offers limited choices to the clinician to bring relief to patients. Pulmonary, renal and myocardial complications have benefited from the introduction of angiotensin converting enzyme inhibitors, calcium pump inhibitors, prostacyclin analogs and endothelin antagonists. Based on recent pathophysiological insights, a number of novel agents are being developed [6-8].

#### **2. SSc pathogenesis in 2011: An elusive mechanism**

SSc pathogenesis remains obscure. Central features are an inflammatory vasculopathy, perivascular and interstitial sclerosis, altered angiogenesis and autoimmunity. Pathologic changes depend on whether they are observed at an early or late disease stage. The progression of the disease is typified by the accumulation of extra-cellular matrix (ECM) components in connective tissues. There are clinical and pathologic arguments to support the hypothesis that the vasculature is involved at an early step during disease progression.

Blood Platelets and Systemic Sclerosis 31

Platelets are cytoplasmic fragments that derive from the fragmentation of megakaryocytes (MKs) in the bone marrow sinuses. They harbor a unique store of secretory organelles having a distinct content in bioactive peptides. Alpha granules contain both soluble proteins destined to be secreted and membrane-bound proteins [31, 32]. Dense granules mostly contain an adenine nucleotide pool of ATP and ADP, bivalent cations calcium and magnesium and amines, including serotonin and histamine [33]. Platelet lysosomes contain a complex set of enzymes including acid proteases, such as cathepsin, carboxypeptidases, collagenases and various glycohydrolases [34]. Following platelet activation, the organelle content is released in a process termed secretion. The bulk of proteins secreted by platelets are remarkably large and diverse, as shown by proteomic studies on either platelet releasates or isolated granules. Classifications of the platelet secretome content with reference to biological effects pinpoint relations that platelets may encompass in various biological functions, including inflammation, tissue repair and angiogenesis [34-37]. Such a complexity has opened many new challenges with reference to the platelet role in human

In addition to MK biosynthesis, platelets also carry mediators that are endocytosed from plasma and possibly concentrated and/or modified within platelets. Such a plasma uptake has, for example, been demonstrated for fibrinogen, albumin, immunoglobulins, amino acids, and several inflammatory mediators including vascular endothelial growth factor (VEGF), histamine or serotonin [38, 39]. Passive and/or active mechanisms are responsible for the platelet uptake of plasma material. Platelets may be considered as mobile nodes, gathering (through endocytosis) and imparting information to target cells [40]. Apart from the rapid mobilization and release of granule content, platelets also express biomediators, including IL-1, tissue factor, fibrinogen, thrombospondin, von Willebrand factor (vWF), GPIIb and GPIIIa, through a time-, translational-dependent pathway that is triggered upon platelet activation [41-45]. Finally, the traditional concept of platelet loss of function following activation is debated, as activated platelets circulate or persist in clots while

**4.2 Platelet-derived microparticles recapitulate essential platelet functions** 

Activated platelets shed microparticles (MPs). Platelets are the major source of circulating MPs. MP biological roles recapitulate essential platelet functions as MPs represent a transport and delivery system of mediators participating to hemostasis, thrombosis, vascular repair and inflammation, acting both locally and systemically. MPs may transfer information to EC through adhesion and/or fusion, an event thought to contribute to the

One of the primary roles of platelets is to maintain vascular integrity [49]. At sites of vascular damage, platelets promptly adhere to exposed ECM [50], but also to activated EC (below), a first step in a sequence of events that result in platelet activation, initiation and propagation of hemostasis and thrombosis and in the release of key material contributing to wound repair and tissue regeneration, including ECM components and ECM remodeling proteins, matrix metalloproteinases (MMPs) and their inhibitors, the tissue inhibitors of

**4. Platelets: Central actors of inflammation** 

diseases.

keeping functional properties.

control of EC phenotype in inflammation [46-48].

**4.3.1 Platelets maintain vascular integrity** 

**4.3 Platelet-endothelium: A friend and foe relationship** 

**4.1 Platelets transport bioactive mediators in the bloodstream** 

Indeed, endothelium damage, perivascular edema and mononuclear cell infiltration are among earliest pathological changes that may precede the development of fibrosis [9-11]. Deregulated vascular tone, as evidenced by the Raynaud's phenomenon and morphological abnormalities of nail-fold capillary loops, as evidenced by capillaroscopy, also witness the underlying vasculopathy. In such a hypothetical vascular perspective, endothelial cell (EC) injury fuels other manifestations of the disease by the way of a deregulated inflammatory reaction. Whatever the primary target, a putative sequence of events leading to progression of the disease may follows microvascular injury leading in turn to perivascular and interstitial inflammation, autoimmunity leading to further endothelium injury, chronic progression of the inflammatory reaction, activation of interstitial fibroblasts, pericytes and other fibrocompetent cells leading to myofibroblast generation and subsequent persistent fibrogenic response [6, 12-18]. There are arguments to incriminate the genetic background in the disease susceptibility and progression. Genetic studies have underlined the inherent complexity of the disease, highlighting the involvement of the vasculature, the immune system and the ECM. Several polymorphisms in potential candidate genes have been identified [19-24]. The number of genes involved further underlines the disease spectrum heterogeneity. Environmental factors are linked to the disease, this being the case for exposure to silica, organic solvents or vinyl chloride. Infectious agents have been proposed to be involved, through endothelium damage, molecular mimicry-mediated autoimmune responses or other mechanisms [25, 26]. But how exposure to environmental risk factors combined to a permissive genetic background translates into initiation and progression of the disease is unclear. Therefore, fundamental questions remain currently unanswered with regard to key SSc features such as vascular, autoimmune and fibrotic events. The nature of the EC injury remains unknown. Several potential mechanisms have been explored, such as EC-specific T cells or auto-antibodies, vasculotropic viruses or environmental factors, oxidative stress, profibrotic and/or inflammatory cytokines. Altered angiogenesis is also a characteristic feature of SSc. Prevalent alterations of the capillary network in SSc include chaotic malformations with giant and bushy capillaries and reduced capillary density. There is, however, an insufficient angiogenic response, a defective vasculogenesis and a capillary loss in SSc [27]. Recent progresses have allowed progressive uncovering of the molecular mechanisms of tissue fibrosis. However, the widespread accumulation of ECM in SSc remain largely misunderstood, and therapeutic attempts to control fibrosis unfortunately remained unsuccessful [17, 18, 28-30].

#### **3. SSc: A role for platelets?**

A recent expansion of knowledge from basic research has illuminated the role of platelets in the inflammatory signalization network and underlined a hitherto unsuspected role for platelets in inflammatory diseases. Moreover, platelets are linked to endothelium homeostasis. They have been implicated in several vascular and fibrotic disorders. Hence, platelets stand as foreseeable contributors to SSc natural history. Indeed, there are signs of ongoing platelet activation in SSc but the underlying mechanisms remain ill-defined. First, it is not known how platelets could be activated during the course of the disease, through endothelium injury, immunological mechanisms or other reasons; second, it is not clear which platelet-derived mediators may specifically influence the progression of the disease. In the following sections are summarized some of the mechanisms linking platelets with inflammation, angiogenesis, vascular tone and fibrosis.

Indeed, endothelium damage, perivascular edema and mononuclear cell infiltration are among earliest pathological changes that may precede the development of fibrosis [9-11]. Deregulated vascular tone, as evidenced by the Raynaud's phenomenon and morphological abnormalities of nail-fold capillary loops, as evidenced by capillaroscopy, also witness the underlying vasculopathy. In such a hypothetical vascular perspective, endothelial cell (EC) injury fuels other manifestations of the disease by the way of a deregulated inflammatory reaction. Whatever the primary target, a putative sequence of events leading to progression of the disease may follows microvascular injury leading in turn to perivascular and interstitial inflammation, autoimmunity leading to further endothelium injury, chronic progression of the inflammatory reaction, activation of interstitial fibroblasts, pericytes and other fibrocompetent cells leading to myofibroblast generation and subsequent persistent fibrogenic response [6, 12-18]. There are arguments to incriminate the genetic background in the disease susceptibility and progression. Genetic studies have underlined the inherent complexity of the disease, highlighting the involvement of the vasculature, the immune system and the ECM. Several polymorphisms in potential candidate genes have been identified [19-24]. The number of genes involved further underlines the disease spectrum heterogeneity. Environmental factors are linked to the disease, this being the case for exposure to silica, organic solvents or vinyl chloride. Infectious agents have been proposed to be involved, through endothelium damage, molecular mimicry-mediated autoimmune responses or other mechanisms [25, 26]. But how exposure to environmental risk factors combined to a permissive genetic background translates into initiation and progression of the disease is unclear. Therefore, fundamental questions remain currently unanswered with regard to key SSc features such as vascular, autoimmune and fibrotic events. The nature of the EC injury remains unknown. Several potential mechanisms have been explored, such as EC-specific T cells or auto-antibodies, vasculotropic viruses or environmental factors, oxidative stress, profibrotic and/or inflammatory cytokines. Altered angiogenesis is also a characteristic feature of SSc. Prevalent alterations of the capillary network in SSc include chaotic malformations with giant and bushy capillaries and reduced capillary density. There is, however, an insufficient angiogenic response, a defective vasculogenesis and a capillary loss in SSc [27]. Recent progresses have allowed progressive uncovering of the molecular mechanisms of tissue fibrosis. However, the widespread accumulation of ECM in SSc remain largely misunderstood, and therapeutic attempts to control fibrosis unfortunately

A recent expansion of knowledge from basic research has illuminated the role of platelets in the inflammatory signalization network and underlined a hitherto unsuspected role for platelets in inflammatory diseases. Moreover, platelets are linked to endothelium homeostasis. They have been implicated in several vascular and fibrotic disorders. Hence, platelets stand as foreseeable contributors to SSc natural history. Indeed, there are signs of ongoing platelet activation in SSc but the underlying mechanisms remain ill-defined. First, it is not known how platelets could be activated during the course of the disease, through endothelium injury, immunological mechanisms or other reasons; second, it is not clear which platelet-derived mediators may specifically influence the progression of the disease. In the following sections are summarized some of the mechanisms linking platelets with

remained unsuccessful [17, 18, 28-30].

inflammation, angiogenesis, vascular tone and fibrosis.

**3. SSc: A role for platelets?** 

#### **4. Platelets: Central actors of inflammation**

#### **4.1 Platelets transport bioactive mediators in the bloodstream**

Platelets are cytoplasmic fragments that derive from the fragmentation of megakaryocytes (MKs) in the bone marrow sinuses. They harbor a unique store of secretory organelles having a distinct content in bioactive peptides. Alpha granules contain both soluble proteins destined to be secreted and membrane-bound proteins [31, 32]. Dense granules mostly contain an adenine nucleotide pool of ATP and ADP, bivalent cations calcium and magnesium and amines, including serotonin and histamine [33]. Platelet lysosomes contain a complex set of enzymes including acid proteases, such as cathepsin, carboxypeptidases, collagenases and various glycohydrolases [34]. Following platelet activation, the organelle content is released in a process termed secretion. The bulk of proteins secreted by platelets are remarkably large and diverse, as shown by proteomic studies on either platelet releasates or isolated granules. Classifications of the platelet secretome content with reference to biological effects pinpoint relations that platelets may encompass in various biological functions, including inflammation, tissue repair and angiogenesis [34-37]. Such a complexity has opened many new challenges with reference to the platelet role in human diseases.

In addition to MK biosynthesis, platelets also carry mediators that are endocytosed from plasma and possibly concentrated and/or modified within platelets. Such a plasma uptake has, for example, been demonstrated for fibrinogen, albumin, immunoglobulins, amino acids, and several inflammatory mediators including vascular endothelial growth factor (VEGF), histamine or serotonin [38, 39]. Passive and/or active mechanisms are responsible for the platelet uptake of plasma material. Platelets may be considered as mobile nodes, gathering (through endocytosis) and imparting information to target cells [40]. Apart from the rapid mobilization and release of granule content, platelets also express biomediators, including IL-1, tissue factor, fibrinogen, thrombospondin, von Willebrand factor (vWF), GPIIb and GPIIIa, through a time-, translational-dependent pathway that is triggered upon platelet activation [41-45]. Finally, the traditional concept of platelet loss of function following activation is debated, as activated platelets circulate or persist in clots while keeping functional properties.

#### **4.2 Platelet-derived microparticles recapitulate essential platelet functions**

Activated platelets shed microparticles (MPs). Platelets are the major source of circulating MPs. MP biological roles recapitulate essential platelet functions as MPs represent a transport and delivery system of mediators participating to hemostasis, thrombosis, vascular repair and inflammation, acting both locally and systemically. MPs may transfer information to EC through adhesion and/or fusion, an event thought to contribute to the control of EC phenotype in inflammation [46-48].

#### **4.3 Platelet-endothelium: A friend and foe relationship 4.3.1 Platelets maintain vascular integrity**

One of the primary roles of platelets is to maintain vascular integrity [49]. At sites of vascular damage, platelets promptly adhere to exposed ECM [50], but also to activated EC (below), a first step in a sequence of events that result in platelet activation, initiation and propagation of hemostasis and thrombosis and in the release of key material contributing to wound repair and tissue regeneration, including ECM components and ECM remodeling proteins, matrix metalloproteinases (MMPs) and their inhibitors, the tissue inhibitors of

Blood Platelets and Systemic Sclerosis 33

78]. Platelets also link coagulation to angiogenesis, through the proteolytic release of several cryptic angiogenic regulators [79]. VEGF is a critical angiogenic mediator in SSc (below) and its connections with platelets have been extensively studied. VEGF is transported by platelets [80]. Following binding to its receptor, VEGF induces EC survival, growth, permeability and migration. Other key angiogenic regulators secreted by platelets are platelet-derived growth factor (PDGF), transforming growth factor, (TGF)- and angiopoietins. The angiogenic response is dependent on a complex regulated balance between the generation of pro- and anti-angiogenic molecules, with reference to magnitude and temporal production sequence [81]. The angiogenic activity of VEGF can only be appreciated by integrating the action of other mediators present in the EC environment [82, 83]. PDGF, TGF- and angiopoietins are such mediators, acting in concert with VEGF for the stabilization of the vascular wall; their imbalanced expression has indeed been implicated in aberrant angiogenesis [82, 84]. Interestingly, there is a regulated differential secretion of proand anti-angiogenic mediators by platelets with reference to the nature of the agonist [85].

The blood flow is dependent on the constriction/dilation of resistance arteries. Endothelium and periarterial autonomic nervous plexus provide essential controls of the vascular tone [86, 87]. ECs are a source of vasoactive mediators that regulate blood flow, including the relaxing factors nitric oxid (NO), prostacyclin (PGI2) or endothelium-derived hyperpolarizing factor and vasoconstrictive factors including thromboxane A2 or endothelin-1 [88, 89]. Recent progress has highlighted the involvement of platelets and platelet-derived MPs in the control of the vascular tone. The integrity of the endothelium appears central to the vasomotor response to platelets; on intact endothelium, platelet derived mediators, including serotonin and ADP, cause the release of relaxing factors by the endothelium. If endothelium dysfunction has occurred, the absence of its protective role allows platelet-derived mediators, including serotonin and thromboxan A2 to induce vasoconstriction by directly acting on smooth muscle cells [90, 91]. Interestingly, VEGF may prove to have an increasing importance in vascular flow regulation, apart from its role in angiogenesis. Recent evidences indicate that it preserves the structure and function of

Platelets are a rich source of pro-fibrotic molecules. Tissue fibrosis results from a deregulated wound-healing response to chronic injury. It is typically associated to a perpetuating inflammatory response, resulting for example from the persistence or repeated release of a tissue irritant, in contrast to the regulated acute inflammatory response that ends in a resolution step, repair of tissue damage and tissue homeostasis [92]. A central feature of tissue fibrosis is a quantitatively and qualitatively altered production of ECM. The increased biosynthesis of ECM components, particularly fibrillar collagens, by fibrocompetent cells contributes to generate a permanent and destructive tissue scarring that impairs organ function. Fibrocompetent cell activation is therefore at the root of the natural history of tissue fibrosis, and one of its decisive manifestations is transdifferentiation towards myofibroblasts, expressing -smooth muscle actin (SMA), and harboring characteristic stress fibers and contractile phenotype [93]. It is generally accepted that the major source of myofibroblasts are local connective tissue fibroblasts. However, they can also be recruited

**4.5 Platelet-derived mediators participate to vascular tone regulation** 

neuro-effectors junctions [87].

**4.6 Platelets are a source of fibrogenic mediators** 

metalloproteinases (TIMPs). Platelets also provide essential material for the angiogenic process, as summarized below. The role of platelets in tissue repair goes beyond vascular integrity. Indeed, platelets are essential for organ repair and regeneration as remarkably exemplified in the liver [37, 51-53].

#### **4.3.2 The platelet/endothelium dialogue in inflammation**

The dialogue between platelets and EC is a representative paradigm that has been extensively studied and reviewed because of its relevance in atherosclerosis [54]. Platelet-ECs interactions are critical in the initiation and progression of vascular inflammation. Platelet-derived inflammatory mediators turn EC phenotype to proinflammatory and procoagulant, and platelets facilitate leukocyte recruitment through the endothelium by providing chemotactic signals and platelet-bound ligands [49, 54]. Platelets are brought to inflammatory sites through vascular leakage, attachment to leukocytes but they also respond to chemotactic signals [55]. Coagulation and inflammation are intricately linked, and platelets represent an integrating platform for coagulation and inflammation cascades. For example, they anchor the procoagulant complex leading to the generation of thrombin a potent proinflammatory mediator. Platelets also contribute to other inflammatory cascades; for example, they propagate complement system activation [56]. Further readings may be found in [49, 54, 57-59].

The inflammatory reaction leads to platelet activation. ECM components, chemokines, triggering of platelet receptors with ligands on inflammatory cells activate platelets. Importantly, activated ECs bind and activate platelets, and the underlying mechanisms have been recently précised [50]. In resting conditions, the endothelium is not adhesive for platelets and prevents platelet activation through multiple mechanisms [60]. Activated ECs support platelet adhesion and activation. Endothelium denudation, resulting in ECM exposure, is not a prerequisite for platelet activation. Indeed, platelets roll and adhere on activated ECs, as shown, for example, after stimulation with Tumor Necrosis Factor-, or following ischemia/reperfusion injury [61, 62]. EC activation (which can occurs through multiple mechanisms, inflammatory mediators, hypoxia, complement activation products, infectious agents...) results in the upregulation of adhesion molecules, including E- and Pselectin, v3, intercellular adhesion molecule (ICAM)-1 or vWF, all ligands that mediate platelet rolling and firm adhesion in a process presenting profound analogies with the multistep-adhesion mechanisms of leukocytes on ECs [50, 54, 63-68]. Throughout the adhesion process platelets become activated. Activated platelets are a rich source of inflammatory mediators. They secrete a host of cytokines, chemokines, and lipid inflammatory mediators, deliver free acid arachidonic to bystander polymorphonuclear cells, allowing them to generate leukotriens. Activated platelets therefore contribute to generate a complex inflammatory milieu in their vicinity [37, 69, 70]. Platelet-derived inflammatory mediators deliver in turn activating signals to target cells including EC and leukocytes, resulting in the amplification of inflammation.

#### **4.4 Platelets are a source of angiogenesis mediators**

An important mechanism through which platelets control wound healing is linked to angiogenesis, as exemplified in tumor angiogenesis [71]. Platelets and platelet-derived MPs provide critical material for the generation and stabilization of the neo-angiogenic vessels; they secrete positive regulators and inhibitors of angiogenesis, including chemokines [72-

metalloproteinases (TIMPs). Platelets also provide essential material for the angiogenic process, as summarized below. The role of platelets in tissue repair goes beyond vascular integrity. Indeed, platelets are essential for organ repair and regeneration as remarkably

The dialogue between platelets and EC is a representative paradigm that has been extensively studied and reviewed because of its relevance in atherosclerosis [54]. Platelet-ECs interactions are critical in the initiation and progression of vascular inflammation. Platelet-derived inflammatory mediators turn EC phenotype to proinflammatory and procoagulant, and platelets facilitate leukocyte recruitment through the endothelium by providing chemotactic signals and platelet-bound ligands [49, 54]. Platelets are brought to inflammatory sites through vascular leakage, attachment to leukocytes but they also respond to chemotactic signals [55]. Coagulation and inflammation are intricately linked, and platelets represent an integrating platform for coagulation and inflammation cascades. For example, they anchor the procoagulant complex leading to the generation of thrombin a potent proinflammatory mediator. Platelets also contribute to other inflammatory cascades; for example, they propagate complement system activation [56]. Further readings may be

The inflammatory reaction leads to platelet activation. ECM components, chemokines, triggering of platelet receptors with ligands on inflammatory cells activate platelets. Importantly, activated ECs bind and activate platelets, and the underlying mechanisms have been recently précised [50]. In resting conditions, the endothelium is not adhesive for platelets and prevents platelet activation through multiple mechanisms [60]. Activated ECs support platelet adhesion and activation. Endothelium denudation, resulting in ECM exposure, is not a prerequisite for platelet activation. Indeed, platelets roll and adhere on activated ECs, as shown, for example, after stimulation with Tumor Necrosis Factor-, or following ischemia/reperfusion injury [61, 62]. EC activation (which can occurs through multiple mechanisms, inflammatory mediators, hypoxia, complement activation products, infectious agents...) results in the upregulation of adhesion molecules, including E- and Pselectin, v3, intercellular adhesion molecule (ICAM)-1 or vWF, all ligands that mediate platelet rolling and firm adhesion in a process presenting profound analogies with the multistep-adhesion mechanisms of leukocytes on ECs [50, 54, 63-68]. Throughout the adhesion process platelets become activated. Activated platelets are a rich source of inflammatory mediators. They secrete a host of cytokines, chemokines, and lipid inflammatory mediators, deliver free acid arachidonic to bystander polymorphonuclear cells, allowing them to generate leukotriens. Activated platelets therefore contribute to generate a complex inflammatory milieu in their vicinity [37, 69, 70]. Platelet-derived inflammatory mediators deliver in turn activating signals to target cells including EC and

An important mechanism through which platelets control wound healing is linked to angiogenesis, as exemplified in tumor angiogenesis [71]. Platelets and platelet-derived MPs provide critical material for the generation and stabilization of the neo-angiogenic vessels; they secrete positive regulators and inhibitors of angiogenesis, including chemokines [72-

exemplified in the liver [37, 51-53].

found in [49, 54, 57-59].

**4.3.2 The platelet/endothelium dialogue in inflammation** 

leukocytes, resulting in the amplification of inflammation.

**4.4 Platelets are a source of angiogenesis mediators** 

78]. Platelets also link coagulation to angiogenesis, through the proteolytic release of several cryptic angiogenic regulators [79]. VEGF is a critical angiogenic mediator in SSc (below) and its connections with platelets have been extensively studied. VEGF is transported by platelets [80]. Following binding to its receptor, VEGF induces EC survival, growth, permeability and migration. Other key angiogenic regulators secreted by platelets are platelet-derived growth factor (PDGF), transforming growth factor, (TGF)- and angiopoietins. The angiogenic response is dependent on a complex regulated balance between the generation of pro- and anti-angiogenic molecules, with reference to magnitude and temporal production sequence [81]. The angiogenic activity of VEGF can only be appreciated by integrating the action of other mediators present in the EC environment [82, 83]. PDGF, TGF- and angiopoietins are such mediators, acting in concert with VEGF for the stabilization of the vascular wall; their imbalanced expression has indeed been implicated in aberrant angiogenesis [82, 84]. Interestingly, there is a regulated differential secretion of proand anti-angiogenic mediators by platelets with reference to the nature of the agonist [85].

#### **4.5 Platelet-derived mediators participate to vascular tone regulation**

The blood flow is dependent on the constriction/dilation of resistance arteries. Endothelium and periarterial autonomic nervous plexus provide essential controls of the vascular tone [86, 87]. ECs are a source of vasoactive mediators that regulate blood flow, including the relaxing factors nitric oxid (NO), prostacyclin (PGI2) or endothelium-derived hyperpolarizing factor and vasoconstrictive factors including thromboxane A2 or endothelin-1 [88, 89]. Recent progress has highlighted the involvement of platelets and platelet-derived MPs in the control of the vascular tone. The integrity of the endothelium appears central to the vasomotor response to platelets; on intact endothelium, platelet derived mediators, including serotonin and ADP, cause the release of relaxing factors by the endothelium. If endothelium dysfunction has occurred, the absence of its protective role allows platelet-derived mediators, including serotonin and thromboxan A2 to induce vasoconstriction by directly acting on smooth muscle cells [90, 91]. Interestingly, VEGF may prove to have an increasing importance in vascular flow regulation, apart from its role in angiogenesis. Recent evidences indicate that it preserves the structure and function of neuro-effectors junctions [87].

#### **4.6 Platelets are a source of fibrogenic mediators**

Platelets are a rich source of pro-fibrotic molecules. Tissue fibrosis results from a deregulated wound-healing response to chronic injury. It is typically associated to a perpetuating inflammatory response, resulting for example from the persistence or repeated release of a tissue irritant, in contrast to the regulated acute inflammatory response that ends in a resolution step, repair of tissue damage and tissue homeostasis [92]. A central feature of tissue fibrosis is a quantitatively and qualitatively altered production of ECM. The increased biosynthesis of ECM components, particularly fibrillar collagens, by fibrocompetent cells contributes to generate a permanent and destructive tissue scarring that impairs organ function. Fibrocompetent cell activation is therefore at the root of the natural history of tissue fibrosis, and one of its decisive manifestations is transdifferentiation towards myofibroblasts, expressing -smooth muscle actin (SMA), and harboring characteristic stress fibers and contractile phenotype [93]. It is generally accepted that the major source of myofibroblasts are local connective tissue fibroblasts. However, they can also be recruited

Blood Platelets and Systemic Sclerosis 35

selectin (CD62p) is a component of –granules and of intracellular EC storage organelles. There has been debate whether the increase in plasma CD62p truly reflects platelet activation, as it is also expressed on activated ECs. It is however currently clear that most, if not all, measured plasma CD62p has a platelet origin [114, 115]. Activated ECs have also been discussed as potential contributors to soluble CD154 (sCD154). In fact, correlation studies demonstrated that it can be considered as mostly derived from platelets [116], but, recently, other cell sources have been described, that may potentially contribute to elevated circulating sCD154 levels [117]. Soluble P-selectin glycoprotein ligand-1 is elevated in SSc [118], which may also reflect some degree of platelet activation as it is expressed by platelets [119]. Chemokine (C-X-C motif) ligand-4 (CXCL4, Platelet Factor 4) is elevated in SSc; albeit until recently described as exclusively derived from platelets, other sources exist such as T-cells or macrophages that may be contributors to elevated circulating levels [120]. Tissue-plasminogen activator (tPA) has also been described as a marker of EC damage [121]. Studies of the expression of platelet membrane-bound activation markers also show that platelets are not globally activated, but that this activation concerns a fraction of their population [122]. Correlation studies in general do not evidence relations with clinical features of the disease, but this is a matter of debate as correlations were found for some markers such as sCD154, thromboxane B2 or MPs (Table 1). Finally, morphological electron microscopy-based studies also suggest platelet enhanced activability in SSc, and there are signs of platelet granule release [111, 123, 124]. A major point is that platelet activation may be prominently linked to the Raynaud's phenomenon. Indeed, features of platelet activation are also found in the primary

features References

[135] (in the CREST variant); [136] (also in

[126]; [131]; [133]; Radstake Arthritis Rheum 2010;62 Suppl 10 :1210 (Abstract);

platelets)

[134]

[134]; [137-139];

Raynaud's phenomenon (below).

Serotonin Not found

CXCL4 Not found

Thromboxane B2 in plasma or urines (Thromboxane A2

metabolite)

Circulating platelet

Platelet activation markers Correlations with disease

aggregates Not found [125-127] Platelet-leucocyte aggregates Not found [128] [128]

[139]

TSP-1 Not found [133] tPA [132] Membrane CD62P Not found [122]; [128] Soluble CD62P Not found [140]


Positive correlation with dSSc (Herrick 1996)

from circulating fibrocytes and bone marrow-derived mesenchymal precursors, or they can derive from local epithelial and endothelial cells through the epithelial-mesenchymal transition [94-99]. Multiple soluble signals activate fibrocompetent cells. Among the most studied in SSc are the growth factors TGF-1, PDGF, endothelin-1 and connective tissue growth factor. These mediators are potent fibroblast activators. TGF-1 holds a peculiar position in SSc, as its deregulated expression represents a critical step. TGF-1 promotes myofibroblastic transdifferentiation of quiescent fibroblasts, clearly demonstrated by the induction of -SMA [100], stimulates their proliferation and the synthesis of ECM components, including fibrillar collagens. Epithelial-mesenchymal transition is linked to inflammation and fibrosis and several pro-fibrotic mediators induce the epithelialmesenchymal transition [101]. A hierarchical induction of pro-fibrotic mediators in tissue fibrosis has been emphasized [102]. Chemokines, as monocyte chemotactic protein MCP-1, RANTES (regulated upon activation, normal T-cell expressed and secreted), interleukin (IL)-8, activate fibrogenic cells, including skin fibroblasts, stimulating their proliferation, chemotaxis, and ECM biosynthesis. The interaction between keratinocytes and fibroblasts is a critical step during the early phase of wound healing in the skin. Fibroblasts produce keratinocyte growth factor and, conversely, keratinocytes signal to fibroblasts leading to their activation, production of a variety of cytokines/chemokines/angiogenic mediators, induction of ECM component synthesis and -SMA expression [102, 103]. There are important connections between vascular remodeling, angiogenesis and tissue fibrosis. Deregulated vascular remodeling is an important parameter in promoting the development of fibrosis and an imbalance between the production of angiogenic and antiangiogenic factors, such as CXC chemokines, at sites of tissue injury, is relevant to tissue fibrosis [17, 104-106]. Finally, a regulated balance between ECM production and degradation is a *sine qua non* condition for harmonious wound repair and tissue regeneration. Indeed, as shown in humans and in experimental tissue fibrosis models, the increased expression of some of TIMPs, a family of enzymes that control MMP activity through the inhibition of their proteolytic activity and the control of proform cleavage, leads to an altered MMP/TIMP balance, ECM homeostatic degradation/production disequilibrium, and ECM accumulation [107].

Platelets are essential actors in these mechanisms. They transport and secrete most of above –mentioned pro-fibrotic mediators, angiogenic and anti-angiogenic mediators, including CXC chemokines, ECM components and ECM remodeling proteins MMPs and TIMPs [108, 109]. Platelets influence cell growth and differentiation in a variety of situations [110]. They provide key control signals to angiogenesis. Persistent platelet activation in the microvascular bed may therefore contribute to tissue fibrosis [111-113]. However, few studies have considered their potential contribution. In fact, platelet-derived mediators have only recently being acknowledged as being of key importance in fibrosis as observed in liver and pulmonary diseases and, very recently, in SSc (below).

#### **5. Evidence for platelet activation in SSc**

There are signs of platelet activation in SSc, as indicated by measurements of various soluble and membrane-bound markers, circulating MPs and platelet- and plateletleukocyte aggregates (Table 1). Soluble markers have been largely studied. As there are signs of ongoing EC injury in SSc, the specificity of these markers is an important point. P-

from circulating fibrocytes and bone marrow-derived mesenchymal precursors, or they can derive from local epithelial and endothelial cells through the epithelial-mesenchymal transition [94-99]. Multiple soluble signals activate fibrocompetent cells. Among the most studied in SSc are the growth factors TGF-1, PDGF, endothelin-1 and connective tissue growth factor. These mediators are potent fibroblast activators. TGF-1 holds a peculiar position in SSc, as its deregulated expression represents a critical step. TGF-1 promotes myofibroblastic transdifferentiation of quiescent fibroblasts, clearly demonstrated by the induction of -SMA [100], stimulates their proliferation and the synthesis of ECM components, including fibrillar collagens. Epithelial-mesenchymal transition is linked to inflammation and fibrosis and several pro-fibrotic mediators induce the epithelialmesenchymal transition [101]. A hierarchical induction of pro-fibrotic mediators in tissue fibrosis has been emphasized [102]. Chemokines, as monocyte chemotactic protein MCP-1, RANTES (regulated upon activation, normal T-cell expressed and secreted), interleukin (IL)-8, activate fibrogenic cells, including skin fibroblasts, stimulating their proliferation, chemotaxis, and ECM biosynthesis. The interaction between keratinocytes and fibroblasts is a critical step during the early phase of wound healing in the skin. Fibroblasts produce keratinocyte growth factor and, conversely, keratinocytes signal to fibroblasts leading to their activation, production of a variety of cytokines/chemokines/angiogenic mediators, induction of ECM component synthesis and -SMA expression [102, 103]. There are important connections between vascular remodeling, angiogenesis and tissue fibrosis. Deregulated vascular remodeling is an important parameter in promoting the development of fibrosis and an imbalance between the production of angiogenic and antiangiogenic factors, such as CXC chemokines, at sites of tissue injury, is relevant to tissue fibrosis [17, 104-106]. Finally, a regulated balance between ECM production and degradation is a *sine qua non* condition for harmonious wound repair and tissue regeneration. Indeed, as shown in humans and in experimental tissue fibrosis models, the increased expression of some of TIMPs, a family of enzymes that control MMP activity through the inhibition of their proteolytic activity and the control of proform cleavage, leads to an altered MMP/TIMP balance, ECM homeostatic degradation/production

Platelets are essential actors in these mechanisms. They transport and secrete most of above –mentioned pro-fibrotic mediators, angiogenic and anti-angiogenic mediators, including CXC chemokines, ECM components and ECM remodeling proteins MMPs and TIMPs [108, 109]. Platelets influence cell growth and differentiation in a variety of situations [110]. They provide key control signals to angiogenesis. Persistent platelet activation in the microvascular bed may therefore contribute to tissue fibrosis [111-113]. However, few studies have considered their potential contribution. In fact, platelet-derived mediators have only recently being acknowledged as being of key importance in fibrosis as observed in liver

There are signs of platelet activation in SSc, as indicated by measurements of various soluble and membrane-bound markers, circulating MPs and platelet- and plateletleukocyte aggregates (Table 1). Soluble markers have been largely studied. As there are signs of ongoing EC injury in SSc, the specificity of these markers is an important point. P-

disequilibrium, and ECM accumulation [107].

and pulmonary diseases and, very recently, in SSc (below).

**5. Evidence for platelet activation in SSc** 

selectin (CD62p) is a component of –granules and of intracellular EC storage organelles. There has been debate whether the increase in plasma CD62p truly reflects platelet activation, as it is also expressed on activated ECs. It is however currently clear that most, if not all, measured plasma CD62p has a platelet origin [114, 115]. Activated ECs have also been discussed as potential contributors to soluble CD154 (sCD154). In fact, correlation studies demonstrated that it can be considered as mostly derived from platelets [116], but, recently, other cell sources have been described, that may potentially contribute to elevated circulating sCD154 levels [117]. Soluble P-selectin glycoprotein ligand-1 is elevated in SSc [118], which may also reflect some degree of platelet activation as it is expressed by platelets [119]. Chemokine (C-X-C motif) ligand-4 (CXCL4, Platelet Factor 4) is elevated in SSc; albeit until recently described as exclusively derived from platelets, other sources exist such as T-cells or macrophages that may be contributors to elevated circulating levels [120]. Tissue-plasminogen activator (tPA) has also been described as a marker of EC damage [121]. Studies of the expression of platelet membrane-bound activation markers also show that platelets are not globally activated, but that this activation concerns a fraction of their population [122]. Correlation studies in general do not evidence relations with clinical features of the disease, but this is a matter of debate as correlations were found for some markers such as sCD154, thromboxane B2 or MPs (Table 1). Finally, morphological electron microscopy-based studies also suggest platelet enhanced activability in SSc, and there are signs of platelet granule release [111, 123, 124]. A major point is that platelet activation may be prominently linked to the Raynaud's phenomenon. Indeed, features of platelet activation are also found in the primary Raynaud's phenomenon (below).


Blood Platelets and Systemic Sclerosis 37

primary or acquired defect. Proteomic studies on platelet compartments in SSc patients

[126]; [131]; [154-158]

[138]; [157]

[153]

Platelet activation in SSc may first be the consequence of endothelium injury. Indeed, the canonical role of platelets is to react to endothelium damage, a circumstance that activates a bidirectional dialogue between ECs and platelets, initiating and sustaining inflammation. Through soluble and platelet-bound signals, activated platelets confer ECs a proinflammatory and procoagulant phenotype and, reciprocally, EC activation, as met in inflammation, leads to platelet activation resulting in a pro-inflammatory amplification loop. The balance of signals that keep platelet from being activated may be overcome in a variety of conditions. As summarized above, not only platelets react to the endothelial barrier loss of integrity with consequent exposure of the underneath collagen-rich matrix, but they also react to slighter features of EC activation, which do not lead *per se* to severe endothelium damage, as met for example in conditions in which ECs are activated by cytokines including TNF IL-1, VEGF, by oxidative stress or hypoxia [80, 162-165]. These signals induce the expression of a range of molecules promoting platelet adhesion on ECs. Platelets bind to these docking structures, an event that initiates their activation. These conditions also induce ECs to produce platelet agonists, such as multimeric vWF and ADP. Inflammation and coagulation are inextricably linked. Activated ECs express tissue factor, leading to the activation of coagulation, platelet activation, thrombin generation and further amplification of the inflammatory reaction. This is dramatically exemplified in renal involvement in SSc, characterized by thrombotic microangiopathy lesions. On electron microscopy studies, aggregates of platelets admixed with fibrin and fragmented red blood cells are observed within small interstitial vessels or glomerular capillaries. Hypoxia is a noxious stimulus that activates a range of inflammatory pathways, leading to EC activation through multiple mechanisms, including the production of angiogenic factors [166-168]. Hypoxia is thought to play a critical role in SSc. SSc-associated microangiopathy results in a disturbed blood flow in the capillaries with consequent hypoxia, which is likely to be

could help finding a potential primary or acquired platelet defects in this disease.

Platelet function changes References

Enhanced adhesion to collagen [159];

Reduced sensitivity to PGI2 [160]

Hyperaggregability not found [161]

**7.2 Potential mechanisms mediating platelet activation in SSc** 

Increased platelet sensitivity to collagen-

Increased expression of collagen type 1

Table 2. Changes in platelet functions in SSc

Hyperaggregability

induced aggregation

receptor

Spontaneous (in whole blood) In response to agonists


Table 1. Markers of platelet activation in SSc

Platelet activation in SSc is documented by several studies. Markers have been measured in plasma or serum or on platelets (CD62P). The elevation of circulating tPA is not always found [139, 144]. Abbreviations: -TG, -thromboglobulin ; TSP-1, thrombospondin-1 ; dSSc, diffuse cutaneous SSc; lSSc, limited cutaneous SSc ([2]). Other abbreviations are spelled in the text.

#### **6. Lack of platelet morphological abnormalities and count in SSc**

There are no specific recognized abnormalities of the platelet count in SSc. Moderate thrombocytosis or thrombocytopenia are occasionally observed, and have been related to inflammation or microangiopathy, respectively [145, 146]. Platelet morphology is not altered.

#### **7. Potential mechanisms mediating platelet activation in SSc**

Mechanisms underlying platelet activation in SSc remain ill-defined. Questions that have been pursued are: (i) is there a platelet dysfunction in patients that would reduce the threshold level of platelet response to activating signals? (ii) are platelets activated in SSc as the result of EC injury, autoantibodies directed at platelets or other reasons? (iii) what is the role played by the Raynaud's phenomenon?

#### **7.1 Is there a platelet disorder in SSc leading to platelet hyperactivability?**

Several studies have examined the eventuality of platelet dysfunction in SSc (Table 2). Reports suggest augmented responsiveness of platelets to their physiological agonists, either strong agonists, such as thrombin or collagen, or weak agonists, such as serotonin, ADP, epinephrine or arachidonic acid, resulting in a reduced threshold of platelet aggregation to the agonist stimulus. Conversely, there is a reduced response to inhibitors of platelet activation such as prostacyclin. However, there are contradictory reports (Table 2). Platelet response to agonists as measured by the hyperactive phenotype is often difficult to interpret. There is a significant variability between individuals, as several genetic modifiers may influence platelet function and as platelet hyperactivity, as measured by aggregometry, can be detected in healthy individuals [147, 148]. Also, *in vivo*, platelet activation results from the combinational input of agonists that act in a complex synergistic way and vary from donor to donor [149-152]. The application of neural networks approaches for predicting platelet response to a complex milieu may represent a useful tool for studying platelet activation response to agonists in SSc. There are no straightforward interpretations of changes in platelet function in SSc. Interestingly, binding assays of radiolabelled collagen indicate a specific increase in the expression of the platelet collagen type 1 receptor in SSc patients [153], which would explain the enhanced response to collagen and indicate a

Positive correlation with lSSc, with digital ulcers and

Inverse correlation to the modified Rodnan thickness

Platelet activation in SSc is documented by several studies. Markers have been measured in plasma or serum or on platelets (CD62P). The elevation of circulating tPA is not always found [139, 144]. Abbreviations: -TG, -thromboglobulin ; TSP-1, thrombospondin-1 ; dSSc, diffuse cutaneous SSc; lSSc, limited cutaneous SSc ([2]). Other abbreviations are spelled in the text.

There are no specific recognized abnormalities of the platelet count in SSc. Moderate thrombocytosis or thrombocytopenia are occasionally observed, and have been related to inflammation or microangiopathy, respectively [145, 146]. Platelet morphology is not altered.

Mechanisms underlying platelet activation in SSc remain ill-defined. Questions that have been pursued are: (i) is there a platelet dysfunction in patients that would reduce the threshold level of platelet response to activating signals? (ii) are platelets activated in SSc as the result of EC injury, autoantibodies directed at platelets or other reasons? (iii) what is the

Several studies have examined the eventuality of platelet dysfunction in SSc (Table 2). Reports suggest augmented responsiveness of platelets to their physiological agonists, either strong agonists, such as thrombin or collagen, or weak agonists, such as serotonin, ADP, epinephrine or arachidonic acid, resulting in a reduced threshold of platelet aggregation to the agonist stimulus. Conversely, there is a reduced response to inhibitors of platelet activation such as prostacyclin. However, there are contradictory reports (Table 2). Platelet response to agonists as measured by the hyperactive phenotype is often difficult to interpret. There is a significant variability between individuals, as several genetic modifiers may influence platelet function and as platelet hyperactivity, as measured by aggregometry, can be detected in healthy individuals [147, 148]. Also, *in vivo*, platelet activation results from the combinational input of agonists that act in a complex synergistic way and vary from donor to donor [149-152]. The application of neural networks approaches for predicting platelet response to a complex milieu may represent a useful tool for studying platelet activation response to agonists in SSc. There are no straightforward interpretations of changes in platelet function in SSc. Interestingly, binding assays of radiolabelled collagen indicate a specific increase in the expression of the platelet collagen type 1 receptor in SSc patients [153], which would explain the enhanced response to collagen and indicate a

pulmonary arterial hypertension [141]

score [143]

**6. Lack of platelet morphological abnormalities and count in SSc** 

**7. Potential mechanisms mediating platelet activation in SSc** 

**7.1 Is there a platelet disorder in SSc leading to platelet hyperactivability?** 

features References

[141]; [142]

[128]; [143]

Platelet activation markers Correlations with disease

sCD154

Platelet-derived MPs

Table 1. Markers of platelet activation in SSc

role played by the Raynaud's phenomenon?

primary or acquired defect. Proteomic studies on platelet compartments in SSc patients could help finding a potential primary or acquired platelet defects in this disease.


Table 2. Changes in platelet functions in SSc

#### **7.2 Potential mechanisms mediating platelet activation in SSc**

Platelet activation in SSc may first be the consequence of endothelium injury. Indeed, the canonical role of platelets is to react to endothelium damage, a circumstance that activates a bidirectional dialogue between ECs and platelets, initiating and sustaining inflammation. Through soluble and platelet-bound signals, activated platelets confer ECs a proinflammatory and procoagulant phenotype and, reciprocally, EC activation, as met in inflammation, leads to platelet activation resulting in a pro-inflammatory amplification loop. The balance of signals that keep platelet from being activated may be overcome in a variety of conditions. As summarized above, not only platelets react to the endothelial barrier loss of integrity with consequent exposure of the underneath collagen-rich matrix, but they also react to slighter features of EC activation, which do not lead *per se* to severe endothelium damage, as met for example in conditions in which ECs are activated by cytokines including TNF IL-1, VEGF, by oxidative stress or hypoxia [80, 162-165]. These signals induce the expression of a range of molecules promoting platelet adhesion on ECs. Platelets bind to these docking structures, an event that initiates their activation. These conditions also induce ECs to produce platelet agonists, such as multimeric vWF and ADP. Inflammation and coagulation are inextricably linked. Activated ECs express tissue factor, leading to the activation of coagulation, platelet activation, thrombin generation and further amplification of the inflammatory reaction. This is dramatically exemplified in renal involvement in SSc, characterized by thrombotic microangiopathy lesions. On electron microscopy studies, aggregates of platelets admixed with fibrin and fragmented red blood cells are observed within small interstitial vessels or glomerular capillaries. Hypoxia is a noxious stimulus that activates a range of inflammatory pathways, leading to EC activation through multiple mechanisms, including the production of angiogenic factors [166-168]. Hypoxia is thought to play a critical role in SSc. SSc-associated microangiopathy results in a disturbed blood flow in the capillaries with consequent hypoxia, which is likely to be

Blood Platelets and Systemic Sclerosis 39

with the endothelium, making them potential contributors to SSc vasculopathy. The microcirculation is a characteristic target in SSc pathogenesis. Microcirculation hemorheologic conditions result in an intimate platelet/endothelium interface, characterized by a near capillary wall platelet concentration. Clearly, cross-interactions between ECs and platelets are inextricably linked under the form of feed-back activation loops and whether or not being a primary event, any condition leading to an endothelium insult drives platelet activation. Conversely, platelet activation drives to EC activation; the fact that platelet activation is observed in the primary Raynaud's phenomenon, in the absence of EC detectable damage, may signify that subtle early events activating platelets, such as disturbed blood flow or hypoxia, precede the onset of the disease. Following platelet activation in the microcirculation, as described above, a wide array of soluble and plateletbound mediators with a pleiotropic range of actions are released that can contribute to several pathophysiological features of the disease including (i) vascular tone dysregulation, (ii) endothelium activation, (iii) inflammation, (iv) activation of the coagulation system, (v) fibrogenic response, (vi) altered angiogenesis. Apart from the possible deleterious cross-talk between endothelium and platelets, described above, two chief features of SSc progression that may be connected to platelets have been the subject of very recent reports. Altered angiogenesis and progressive perivascular and interstitial fibrosis are hallmarks of SSc [27]. First as summarized above, platelets are essential contributors to angiogenesis during wound healing, and ongoing platelet activation in capillary beds may be involved in the altered angiogenesis associated with SSc. In fact, in line with the major VEGF transporter role of platelets, platelet VEGF is increased in SSc patients. However, this is not the case for other angiogenic regulators, such as TGF-1, PDGF-BB or angiopoietins [122]. VEGF, jointly with other mediators, determines the angiogenic or non-angiogenic status of the EC and an imbalance between the relative concentrations of angiogenic mediators is likely to alter angiogenesis homeostasis [170, 177]. Indeed, the deregulated expression of VEGF is thought to lead to abnormal angiogenesis, as exemplified by the resulting disorganization of the capillary network with large, leaky, fragile capillaries; dynamic parameters, such as the magnitude or the kinetics of VEGF release being critically important [170, 205-207]. Therefore platelet activation in the patient's microvascular beds may result in the release of inappropriate combinations of angiogenic and angiostatic mediators, and such disequilibrium may be relevant to the vascular disease in SSc [122, 208, 209]. Further, as summarized above, there are connections between angiogenesis, vascular remodeling and fibrosis and an unbalance between factors promoting and factors inhibiting angiogenesis may also be relevant to the progression of fibrosis. Second, as summarized above, platelets provide a cornucopia of mediators that may be relevant to the natural history of tissue fibrosis, i.e. through fibrocompetent cell activation. Sustained activation of fibrocompetent cells contribute to excessive ECM production in tissues. Indeed, fibroblasts expanded from the fibrotic skin or lungs from SSc patients have a myofibroblastic phenotype and there is a strong correlation between myofibroblast labeling in the lesional skin and the Rodnan skin score [210]. Crucial platelet links with tissue fibrosis have recently been emphasized in humans. In liver fibrosis the secretion of platelet-derived CXCL4 is instrumental [211]. In SSc, the role of platelet-derived serotonin in skin fibrosis is strongly suggested by the stimulation of the production of ECM by serotonin through binding to dermal fibroblasts serotonin receptor 5-HT2B, increased expression of 5-HT2B in the fibrotic skin of SSc patients and reduction of experimental fibrosis through pharmacological targeting of the 5-HT/5-

HT2B signaling and anti-platelet drugs [212].

aggravated by tissue fibrosis [169-172]. Hypoxia alters vascular endothelium but also directly activates platelets [173, 174] (our unpublished results). Altogether, conditions that turn EC phenotype to proinfammatory/prothrombotic are translated into platelet adhesion and activation, with the consequent release of inflammatory, mitogenic, angiogenic and fibrogenic platelet-derived mediators described above. These mediators further activate ECs, promoting the production of cytokine/chemokines, induction of adhesion and procoagulant molecules, and production and activation of MMPs. Moreover, ECs undergoing apoptosis become proadhesive for platelets [175], and activated platelets can in turn induce EC apoptosis [176].

In fact, endothelial injury in the microcirculation and arterioles is a predominant feature of SSc which has extensively been reviewed [13, 30, 177-180]. There are circulating stigmata of endothelial injury, including von Willebrand factor and supranormal (larger) vWF multimers [126, 131, 139, 181-184], soluble adhesion molecules, such as E-selectin or vascular cell adhesion molecule (VCAM)-1 and ICAM-1, thrombomodulin, tPA, or endothelin [185- 188]. Increased nitrate in the plasma or serum of patients has been described, and been related to EC injury, as there are correlations with soluble E-selectin and soluble VCAM-1 [189, 190]. Elevated circulating ECs were also attributed to vascular damage in SSc [191]. EC apoptosis is a common and early feature in SSc [192]; however pathologic evidence for EC apoptosis remains controversial [177]. However there are inherent limitations to the interpretations of such biomarkers [193], these results show that there is an early insult to the vasculature during the course of the disease.

Importantly, the Raynaud's phenomenon itself, one, if not the first manifestation of SSc, preceding the onset of other symptoms of the disease [194-198], is associated with platelet activation [132]. The absence of endothelium abnormalities in the primary Raynaud's phenomenon is generally accepted, although limited morphological abnormalities have been described [199]. However, features of platelet activation are found in primary Raynaud's phenomenon [158, 200, 201] and platelet activation was proposed to play a role in its pathogenesis [135, 202]. Therefore, events responsible for the Raynaud's phenomenon lead to platelet activation in the absence of EC damage, a possible argument to place platelet activation as a very early pathogenic event in the reciprocal activation dialogue between platelets and ECs. Intriguingly, several factors that have been put forward as being potentially causative or susceptible to modify the progression of SSc, as exposure of extremities to cold, mechanical vibrations, exposure to organic solvents or silica, CMV infection are associated to some extent to platelet activation, and for some of them to trigger the Raynaud's phenomenon.

Finally, autoantibodies against platelet gpIIb/IIIa have been described in SSc [203]. However their role in platelet activation remains uncertain.

#### **8. Platelet activation and the progression of SSc**

SSc is a complex disease for which no specific causative mechanism has been identified. The disease may be initiated in the vasculature, as morphological changes are apparent before the onset of the disease; however, it is not clear how endothelium injury begins. Platelets play a large and complex physiological role in health and disease, as they contribute to hemostasis, inflammation, tissue repair, and to the innate and adaptative immunity, standing as essential links [69, 204]. Platelets establish intimate bidirectional relationship

aggravated by tissue fibrosis [169-172]. Hypoxia alters vascular endothelium but also directly activates platelets [173, 174] (our unpublished results). Altogether, conditions that turn EC phenotype to proinfammatory/prothrombotic are translated into platelet adhesion and activation, with the consequent release of inflammatory, mitogenic, angiogenic and fibrogenic platelet-derived mediators described above. These mediators further activate ECs, promoting the production of cytokine/chemokines, induction of adhesion and procoagulant molecules, and production and activation of MMPs. Moreover, ECs undergoing apoptosis become proadhesive for platelets [175], and activated platelets can in

In fact, endothelial injury in the microcirculation and arterioles is a predominant feature of SSc which has extensively been reviewed [13, 30, 177-180]. There are circulating stigmata of endothelial injury, including von Willebrand factor and supranormal (larger) vWF multimers [126, 131, 139, 181-184], soluble adhesion molecules, such as E-selectin or vascular cell adhesion molecule (VCAM)-1 and ICAM-1, thrombomodulin, tPA, or endothelin [185- 188]. Increased nitrate in the plasma or serum of patients has been described, and been related to EC injury, as there are correlations with soluble E-selectin and soluble VCAM-1 [189, 190]. Elevated circulating ECs were also attributed to vascular damage in SSc [191]. EC apoptosis is a common and early feature in SSc [192]; however pathologic evidence for EC apoptosis remains controversial [177]. However there are inherent limitations to the interpretations of such biomarkers [193], these results show that there is an early insult to

Importantly, the Raynaud's phenomenon itself, one, if not the first manifestation of SSc, preceding the onset of other symptoms of the disease [194-198], is associated with platelet activation [132]. The absence of endothelium abnormalities in the primary Raynaud's phenomenon is generally accepted, although limited morphological abnormalities have been described [199]. However, features of platelet activation are found in primary Raynaud's phenomenon [158, 200, 201] and platelet activation was proposed to play a role in its pathogenesis [135, 202]. Therefore, events responsible for the Raynaud's phenomenon lead to platelet activation in the absence of EC damage, a possible argument to place platelet activation as a very early pathogenic event in the reciprocal activation dialogue between platelets and ECs. Intriguingly, several factors that have been put forward as being potentially causative or susceptible to modify the progression of SSc, as exposure of extremities to cold, mechanical vibrations, exposure to organic solvents or silica, CMV infection are associated to some extent to platelet activation, and for some of them to trigger

Finally, autoantibodies against platelet gpIIb/IIIa have been described in SSc [203].

SSc is a complex disease for which no specific causative mechanism has been identified. The disease may be initiated in the vasculature, as morphological changes are apparent before the onset of the disease; however, it is not clear how endothelium injury begins. Platelets play a large and complex physiological role in health and disease, as they contribute to hemostasis, inflammation, tissue repair, and to the innate and adaptative immunity, standing as essential links [69, 204]. Platelets establish intimate bidirectional relationship

turn induce EC apoptosis [176].

the Raynaud's phenomenon.

the vasculature during the course of the disease.

However their role in platelet activation remains uncertain.

**8. Platelet activation and the progression of SSc** 

with the endothelium, making them potential contributors to SSc vasculopathy. The microcirculation is a characteristic target in SSc pathogenesis. Microcirculation hemorheologic conditions result in an intimate platelet/endothelium interface, characterized by a near capillary wall platelet concentration. Clearly, cross-interactions between ECs and platelets are inextricably linked under the form of feed-back activation loops and whether or not being a primary event, any condition leading to an endothelium insult drives platelet activation. Conversely, platelet activation drives to EC activation; the fact that platelet activation is observed in the primary Raynaud's phenomenon, in the absence of EC detectable damage, may signify that subtle early events activating platelets, such as disturbed blood flow or hypoxia, precede the onset of the disease. Following platelet activation in the microcirculation, as described above, a wide array of soluble and plateletbound mediators with a pleiotropic range of actions are released that can contribute to several pathophysiological features of the disease including (i) vascular tone dysregulation, (ii) endothelium activation, (iii) inflammation, (iv) activation of the coagulation system, (v) fibrogenic response, (vi) altered angiogenesis. Apart from the possible deleterious cross-talk between endothelium and platelets, described above, two chief features of SSc progression that may be connected to platelets have been the subject of very recent reports. Altered angiogenesis and progressive perivascular and interstitial fibrosis are hallmarks of SSc [27]. First as summarized above, platelets are essential contributors to angiogenesis during wound healing, and ongoing platelet activation in capillary beds may be involved in the altered angiogenesis associated with SSc. In fact, in line with the major VEGF transporter role of platelets, platelet VEGF is increased in SSc patients. However, this is not the case for other angiogenic regulators, such as TGF-1, PDGF-BB or angiopoietins [122]. VEGF, jointly with other mediators, determines the angiogenic or non-angiogenic status of the EC and an imbalance between the relative concentrations of angiogenic mediators is likely to alter angiogenesis homeostasis [170, 177]. Indeed, the deregulated expression of VEGF is thought to lead to abnormal angiogenesis, as exemplified by the resulting disorganization of the capillary network with large, leaky, fragile capillaries; dynamic parameters, such as the magnitude or the kinetics of VEGF release being critically important [170, 205-207]. Therefore platelet activation in the patient's microvascular beds may result in the release of inappropriate combinations of angiogenic and angiostatic mediators, and such disequilibrium may be relevant to the vascular disease in SSc [122, 208, 209]. Further, as summarized above, there are connections between angiogenesis, vascular remodeling and fibrosis and an unbalance between factors promoting and factors inhibiting angiogenesis may also be relevant to the progression of fibrosis. Second, as summarized above, platelets provide a cornucopia of mediators that may be relevant to the natural history of tissue fibrosis, i.e. through fibrocompetent cell activation. Sustained activation of fibrocompetent cells contribute to excessive ECM production in tissues. Indeed, fibroblasts expanded from the fibrotic skin or lungs from SSc patients have a myofibroblastic phenotype and there is a strong correlation between myofibroblast labeling in the lesional skin and the Rodnan skin score [210]. Crucial platelet links with tissue fibrosis have recently been emphasized in humans. In liver fibrosis the secretion of platelet-derived CXCL4 is instrumental [211]. In SSc, the role of platelet-derived serotonin in skin fibrosis is strongly suggested by the stimulation of the production of ECM by serotonin through binding to dermal fibroblasts serotonin receptor 5-HT2B, increased expression of 5-HT2B in the fibrotic skin of SSc patients and reduction of experimental fibrosis through pharmacological targeting of the 5-HT/5- HT2B signaling and anti-platelet drugs [212].

Blood Platelets and Systemic Sclerosis 41

Work supported by the Groupe Français de Recherche sur la Sclérodermie and the Association des Sclérodermiques de France. Julien Villeneuve is supported by an EMBO

[1] Preliminary criteria for the classification of systemic sclerosis (scleroderma).

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**10. Acknowledgments** 

Rheumatol. 1988;15:202-5.

2008;37:223-35.

Pathol. 1992;166:255-63.

1979;149:1326-35.

long term fellowship.

**11. References** 

#### **9. Conclusion; Platelet activation in SSc: A deleterious loop?**

The primary triggering event in SSc remains unclear. However, inflammation of the vasculature is a common denominator, whether resulting from an autoimmune response to a yet undefined antigen or other mechanisms. Whatever the primary target, inflammation leads to EC activation and, due to the reciprocal activating interplay between platelets and endothelium, EC activation in turn activates platelets and vice-versa. Platelet activation, through the release of cytokines, chemokines, angiogenic and chemotactic mediators...., amplifies the inflammatory reaction by triggering its many facets, including leukocyte recruitment, leading to further endothelium activation and perivascular inflammation, deregulated angiogenesis and, eventually, fibrosis (Figure 1). A chronic inflammatory scenario may contribute to fibrosis by the way of fibrocompetent cell activation, if the initial pathogenic trigger persists, either continuously or repetitively. Self-sustained myofibroblastdependent fibrotic process takes place on the grounds of a chronic inflammation. Platelets may therefore stand at an important place in the ill-understood hierarchy of cell and soluble mediators interplay responsible for the disease. This scenario is evidently highly simplified due to the extreme complexity and heterogeneity of the disease pathophysiology. The lack of suitable animal models [213, 214] that would accurately recapitulate each steps of SSc progression remains a real handicap to understand the natural history of this disease.

#### Fig. 1. Platelet activation in SSc: a deleterious loop?

This schematic model underlines the potential role that platelets may play in the initiation and progression of SSc; see text for details. The role of platelet-derived MPs may also be an important point to consider, as they recapitulate several platelet functions and may transfer platelet deleterious effects at sites distant from their generation.

#### **10. Acknowledgments**

Work supported by the Groupe Français de Recherche sur la Sclérodermie and the Association des Sclérodermiques de France. Julien Villeneuve is supported by an EMBO long term fellowship.

#### **11. References**

40 Systemic Sclerosis – An Update on the Aberrant Immune System and Clinical Features

The primary triggering event in SSc remains unclear. However, inflammation of the vasculature is a common denominator, whether resulting from an autoimmune response to a yet undefined antigen or other mechanisms. Whatever the primary target, inflammation leads to EC activation and, due to the reciprocal activating interplay between platelets and endothelium, EC activation in turn activates platelets and vice-versa. Platelet activation, through the release of cytokines, chemokines, angiogenic and chemotactic mediators...., amplifies the inflammatory reaction by triggering its many facets, including leukocyte recruitment, leading to further endothelium activation and perivascular inflammation, deregulated angiogenesis and, eventually, fibrosis (Figure 1). A chronic inflammatory scenario may contribute to fibrosis by the way of fibrocompetent cell activation, if the initial pathogenic trigger persists, either continuously or repetitively. Self-sustained myofibroblastdependent fibrotic process takes place on the grounds of a chronic inflammation. Platelets may therefore stand at an important place in the ill-understood hierarchy of cell and soluble mediators interplay responsible for the disease. This scenario is evidently highly simplified due to the extreme complexity and heterogeneity of the disease pathophysiology. The lack of suitable animal models [213, 214] that would accurately recapitulate each steps of SSc

progression remains a real handicap to understand the natural history of this disease.

**Myofibroblasts**

**Cytokines/chemokines/ Angiogenic/profibrotic mediators Coagulation activation**

Fig. 1. Platelet activation in SSc: a deleterious loop?

platelet deleterious effects at sites distant from their generation.

**angiogenesis/vascular tone** 

**Pericytes**

**Fibrosis**

**Basal membrane**

**Endothelium**

This schematic model underlines the potential role that platelets may play in the initiation and progression of SSc; see text for details. The role of platelet-derived MPs may also be an important point to consider, as they recapitulate several platelet functions and may transfer

**Platelet activation**

**Inflammation; endothelial cell activation/injury**

**Altered infectious?...)**

**Initial event (autoimmune, oxydative stress, toxic, hypoxic,** 

**Fibroblasts**

**Inflammation**

**9. Conclusion; Platelet activation in SSc: A deleterious loop?** 


Blood Platelets and Systemic Sclerosis 43

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**3** 

*Colombia* 

**Using Proteomic Analysis for Studying the Skin** 

**Fibroblast Protein Profile in Systemic Sclerosis** 

Increased efforts have been made during the last few decades to develop new technologies capable of identifying and quantifying the expression proteome in different cellular systems in physiological and physiopathological conditions for determining illness biomarkers, pharmaceutical targets and/or posttranslational modifications (PTM) by means of proteomic techniques. 2D gel electrophoresis, with immobilized pH gradients, associated with mass spectrometry, is one of the fundamentals steps in studying proteomics. The 2D technique can be used in studying the quantitative expression of protein profiles according to iso-electric point (Ip), molecular weight (Mr), protein solubility and the relative abundance of the above. This methodology provides a protein profile reflecting changes in

Proteins can be classified into those known by their structure and function, those recognized by determined domains and about which there is some knowledge, and those whose function is still not known. Proteomics is defined as the large-scale study of proteins expressed for a specific tissue from a genome, (global proteomics) or differentially expressed proteins (differential proteomics). Determining differentially expressed proteins, or proteins suffering a change in physiological circumstances, is the clue to understanding such pathology's cellular mechanisms. Although an expressed gene in specific tissues (as an answer to biologic alterations) could be analyzed by a mRNA expression study (transcriptomics), these results do not always coincide with the expected expression profiles since the number and activity of proteins associated with the same regulation in different stages could be modified. Genomic data integration is required, as well as transcritomics,

C. Cardozo3, J. Iriarte2, Y. Sanchez4, S. Bravo5, J. Castano6, M.F. Garces7, L. Cepeda7,

*3Biothechnology Department, Universidad Nacional de Colombia, Colombia 4 Pathology Department, Universidad Nacional de Colombia, Colombia* 

*1Rheumatology Section Fundacion Santa Fe de Bogota, Medicine School, Universidad de los Andes, Colombia 2Rheumatology Unit, Medicine School Universidad Nacional de Colombia, Colombia* 

*5Phisiology Department, Universidad de Santiago de Compostela-Espana, Espana 6Cellular biology, physiology and immunology Department, Universidad de Cordoba-Espana, Espana 7Biochemistry Unit, Universidad Nacional de Colombia, Colombia* 

**1. Introduction** 

 \*

protein expression levels, isoforms and PTM.

A. Iglesias-Gamarra2 and J.E. Caminos7.

*2Rheumatology Unit, Medicine School Universidad Nacional de Colombia* 

P. Coral-Alvarado1, G. Quintana1,2 et al.\* *1Rheumatology Section Fundacion Santa Fe de Bogota,* 

*Medicine School, Universidad de los Andes* 


### **Using Proteomic Analysis for Studying the Skin Fibroblast Protein Profile in Systemic Sclerosis**

P. Coral-Alvarado1, G. Quintana1,2 et al.\*

*1Rheumatology Section Fundacion Santa Fe de Bogota, Medicine School, Universidad de los Andes 2Rheumatology Unit, Medicine School Universidad Nacional de Colombia Colombia* 

#### **1. Introduction**

52 Systemic Sclerosis – An Update on the Aberrant Immune System and Clinical Features

[207] Springer ML, Chen AS, Kraft PE, Bednarski M, Blau HM. VEGF gene delivery to muscle: potential role for vasculogenesis in adults. Mol Cell. 1998;2:549-58. [208] Distler JH, Strapatsas T, Huscher D, Dees C, Akhmetshina A, Kiener HP, et al.

[209] Trojanowska M. Cellular and molecular aspects of vascular dysfunction in systemic

[210] Kissin EY, Merkel PA, Lafyatis R. Myofibroblasts and hyalinized collagen as markers of skin disease in systemic sclerosis. Arthritis Rheum. 2006;54:3655-60. [211] Zaldivar MM, Pauels K, von Hundelshausen P, Berres ML, Schmitz P, Bornemann J, et

[212] Dees C, Akhmetshina A, Zerr P, Reich N, Palumbo K, Horn A, et al. Platelet-derived serotonin links vascular disease and tissue fibrosis. J Exp Med. 2011;208:961-72. [213] Artlett CM. Animal models of scleroderma: fresh insights. Curr Opin Rheumatol.

[214] Beyer C, Schett G, Distler O, Distler JH. Animal models of systemic sclerosis: prospects

connective tissue disease. Ann Rheum Dis. 2011;70:1197-202.

sclerosis. Nat Rev Rheumatol. 2010;6:453-60.

liver fibrosis. Hepatology. 2010;51:1345-53.

and limitations. Arthritis Rheum. 2010;62:2831-44.

2010;22:677-82.

Dysbalance of angiogenic and angiostatic mediators in patients with mixed

al. CXC chemokine ligand 4 (Cxcl4) is a platelet-derived mediator of experimental

Increased efforts have been made during the last few decades to develop new technologies capable of identifying and quantifying the expression proteome in different cellular systems in physiological and physiopathological conditions for determining illness biomarkers, pharmaceutical targets and/or posttranslational modifications (PTM) by means of proteomic techniques. 2D gel electrophoresis, with immobilized pH gradients, associated with mass spectrometry, is one of the fundamentals steps in studying proteomics. The 2D technique can be used in studying the quantitative expression of protein profiles according to iso-electric point (Ip), molecular weight (Mr), protein solubility and the relative abundance of the above. This methodology provides a protein profile reflecting changes in protein expression levels, isoforms and PTM.

Proteins can be classified into those known by their structure and function, those recognized by determined domains and about which there is some knowledge, and those whose function is still not known. Proteomics is defined as the large-scale study of proteins expressed for a specific tissue from a genome, (global proteomics) or differentially expressed proteins (differential proteomics). Determining differentially expressed proteins, or proteins suffering a change in physiological circumstances, is the clue to understanding such pathology's cellular mechanisms. Although an expressed gene in specific tissues (as an answer to biologic alterations) could be analyzed by a mRNA expression study (transcriptomics), these results do not always coincide with the expected expression profiles since the number and activity of proteins associated with the same regulation in different stages could be modified. Genomic data integration is required, as well as transcritomics,

A. Iglesias-Gamarra2 and J.E. Caminos7.

<sup>\*</sup> C. Cardozo3, J. Iriarte2, Y. Sanchez4, S. Bravo5, J. Castano6, M.F. Garces7, L. Cepeda7,

*<sup>1</sup>Rheumatology Section Fundacion Santa Fe de Bogota, Medicine School, Universidad de los Andes, Colombia 2Rheumatology Unit, Medicine School Universidad Nacional de Colombia, Colombia* 

*<sup>3</sup>Biothechnology Department, Universidad Nacional de Colombia, Colombia 4 Pathology Department, Universidad Nacional de Colombia, Colombia* 

*<sup>5</sup>Phisiology Department, Universidad de Santiago de Compostela-Espana, Espana 6Cellular biology, physiology and immunology Department, Universidad de Cordoba-Espana, Espana 7Biochemistry Unit, Universidad Nacional de Colombia, Colombia* 

Using Proteomic Analysis for Studying the Skin Fibroblast Protein Profile in Systemic Sclerosis 55

Proteomics in clinical practice offers the technical skill for identifying biomarkers for diagnosis and therapeutic intervention. Potential biomarkers developed from proteomic analysis will have further specificity and sensitivity in clinical trials, since they measure protein alteration involved in an illness (9). A good understanding of data management, correlation, interpretation, and validation is crucial in obtaining precise results contributing towards understanding cellular alterations which could be involved in developing SSc. Only two proteomic studies were found in the current literature. Rottoli has analyzed the type of immune response and protein composition in pulmonary fibrosis patients' LBA medium associated with SSc, sarcoidosis and idiopathic pulmonary fibrosis. Proteomic analysis revealed quantitative differences between the three illnesses, finding increased SSc in plasmatic proteins such as alpha1-beta glycoprotein, C3 complement, alpha 1 antitrypsin, beta- haptoglobin, and prothrombin (10,11). Czubaty has used a commercial cell line (HeLa S3) for proteomic analysis of Topoisomerase I protein patterns by comparing co-immunoprecipitation with mass spectrometry and identified 36 new proteins which were associated with Topoisomerase I and their possible interaction site in the RRM domain (12). However, these studies have been carried out in a not very specific

A two-phase fibroblast proteomic study was thus proposed (pre-treatment and posttreatment) in fibroblasts, these being the cells initially involved in SSc physiopathology in one of its most important expressions: fibrosis. Fibrosis is one of the pathognomonic

Characteristically, there is uncontrollable collagen production and that of other extracellular matrix proteins due to resident fibroblasts in the skin, lungs and other vital organs leading to an excessive accumulation of connective tissue. As the illness progresses, this increased deposit of connective tissue alters the tissues' normal architecture, ending in a functional alteration of the latter and determining a very significant involvement in morbidity-

Protein expression pattern was observed when carrying out a proteomic analysis on SSc patients' fibroblasts during different stages of the illness and comparing them to healthy individuals' fibroblasts. Their appearance was analyzed and thus an increase, decrease, or absence of their profiles was determined, looking for an association of the proteins found with phases and serological and clinical characteristics. Proteins involved in the illness' etiopathology during its different stages were isolated as this could have therapeutic implications in an illness in which current treatment is very limited and not very efficient.

This was a cases and controls study in which 11 patients who fulfilled with American College of Rheumatology SSc criteria were included during different phases of the illness (14,15) and subdivided into two groups: limited SSc and diffuse SSc, according to the

The cutaneous involvement of the skin was evaluated according to the modified Rodnan index (16) which ranges from 0 (normal) to 3 (severe), measured in 17 different body areas

pathological findings for SSc, representing one of the most exemplary phenotypes.

mortality of patients suffering fibrosis-related SSc (13).

medium, such as LBA.

**2. Materials and methods** 

(maximum possible score is 51).

parameters proposed by Le Roy (1). Table 1.

**2.1 Patients** 

proteomics, variome, peptidomics, and metabolome to understand physiological phenomenon in a comprehensive manner.

Systemic sclerosis (SSc) is a chronic illness of the connective tissue having unknown etiology; it has a variable course and severity and is characterized by intercellular matrix alterations and secondary fibrosis of enormous amounts of connective tissue. This results in hardening and thickening of the skin, alterations in the microvasculature and the large vessels, secondary to changes in the endothelial cells together with Raynaud's phenomenon, self-immunity alterations, and musculoskeletal and visceral degenerative fibrotic changes (1).

Despite recent advances having been made in understanding some molecular pathways involved in SSc, its etiopathogenesis remains unknown. Treatment for these patients has had very limited effectiveness, and the natural course of the illness inevitably leads to a fatal outcome. A better understanding of the illness' physiopathology is required to be able to orientate suitable therapeutic measures, carry out effective monitoring of its response, and determine severity criteria indicating a bad prognosis for the illness. This is where genomics, micro-array analysis and proteomics appear as valuable therapeutic and diagnosis tools.

Several groups have reported gene expression profiles for SSc-patient's tissues and cells (2-6).

Zhou found that fibroblasts in SSc patients showed different RNAm expression profiles for fibrilarine autoantigens, B centromere protein, P27 centromeric autoantigen , RNA polymerase I , DNA topoisomerase I, and PMScl (2). Luzina found high chemokine and cytokine levels in bronchoalveolar lavage (LAB) in SSc patients (3). Whitfield examined skin biopsies in four SSc patients, identifying 2,776 genes which expressed themselves in different ways to that of healthy controls (4).Tan (using a fibroblast culture) identified 62 genes which expressed themselves in different ways in SSc (5). Zhou reported fibroblast micro-array analysis results for fibroblasts from 18 sets of discordant twins in SSc (6,7). Protein analysis using two-dimensional electrophoresis electrophoresis on polyacrylamide gel (2D PAG) will contribute to and extend the knowledge produced by analyzing gene expression, especially for proteins undergoing crucial PTM in their function.

Proteomic analysis involves many methodologies orientated towards identifying and characterizing altered proteins as a result of illness. Thousands of proteins are evaluated in just one trial in such studies, leading to detecting expression profiles as a consequence of abnormality in cell function or interaction. Traditional methods used in proteome analysis have included 2D PAG where proteins are first separated according to their electric charge and then by their mass in the second direction before being stained, thus allowing mixtures of 1,000 to 3,000 proteins to be visualized. The development of special software and the use of Internet have allowed multiple genes and databases to be compared. When being combined with mass spectrometry, a separation appears which allows efficient identification of proteins of interest, including many of their PTM. This analysis can be applied in comparative studies of expression profiles during different stages of the illness or healthy tissue compared to affected tissue, thus being able to identify the different modifications in the protein characteristics of clinical interest in different illnesses (8).

Clinical proteomics is aimed at identifying proteins involved in pathological processes, as well as evaluating changes in their expression during different stages of an illness. Proteomics in clinical practice offers the technical skill for identifying biomarkers for diagnosis and therapeutic intervention. Potential biomarkers developed from proteomic analysis will have further specificity and sensitivity in clinical trials, since they measure protein alteration involved in an illness (9). A good understanding of data management, correlation, interpretation, and validation is crucial in obtaining precise results contributing towards understanding cellular alterations which could be involved in developing SSc.

Only two proteomic studies were found in the current literature. Rottoli has analyzed the type of immune response and protein composition in pulmonary fibrosis patients' LBA medium associated with SSc, sarcoidosis and idiopathic pulmonary fibrosis. Proteomic analysis revealed quantitative differences between the three illnesses, finding increased SSc in plasmatic proteins such as alpha1-beta glycoprotein, C3 complement, alpha 1 antitrypsin, beta- haptoglobin, and prothrombin (10,11). Czubaty has used a commercial cell line (HeLa S3) for proteomic analysis of Topoisomerase I protein patterns by comparing co-immunoprecipitation with mass spectrometry and identified 36 new proteins which were associated with Topoisomerase I and their possible interaction site in the RRM domain (12). However, these studies have been carried out in a not very specific medium, such as LBA.

A two-phase fibroblast proteomic study was thus proposed (pre-treatment and posttreatment) in fibroblasts, these being the cells initially involved in SSc physiopathology in one of its most important expressions: fibrosis. Fibrosis is one of the pathognomonic pathological findings for SSc, representing one of the most exemplary phenotypes.

Characteristically, there is uncontrollable collagen production and that of other extracellular matrix proteins due to resident fibroblasts in the skin, lungs and other vital organs leading to an excessive accumulation of connective tissue. As the illness progresses, this increased deposit of connective tissue alters the tissues' normal architecture, ending in a functional alteration of the latter and determining a very significant involvement in morbiditymortality of patients suffering fibrosis-related SSc (13).

Protein expression pattern was observed when carrying out a proteomic analysis on SSc patients' fibroblasts during different stages of the illness and comparing them to healthy individuals' fibroblasts. Their appearance was analyzed and thus an increase, decrease, or absence of their profiles was determined, looking for an association of the proteins found with phases and serological and clinical characteristics. Proteins involved in the illness' etiopathology during its different stages were isolated as this could have therapeutic implications in an illness in which current treatment is very limited and not very efficient.

#### **2. Materials and methods**

#### **2.1 Patients**

54 Systemic Sclerosis – An Update on the Aberrant Immune System and Clinical Features

proteomics, variome, peptidomics, and metabolome to understand physiological

Systemic sclerosis (SSc) is a chronic illness of the connective tissue having unknown etiology; it has a variable course and severity and is characterized by intercellular matrix alterations and secondary fibrosis of enormous amounts of connective tissue. This results in hardening and thickening of the skin, alterations in the microvasculature and the large vessels, secondary to changes in the endothelial cells together with Raynaud's phenomenon, self-immunity alterations, and musculoskeletal and visceral degenerative

Despite recent advances having been made in understanding some molecular pathways involved in SSc, its etiopathogenesis remains unknown. Treatment for these patients has had very limited effectiveness, and the natural course of the illness inevitably leads to a fatal outcome. A better understanding of the illness' physiopathology is required to be able to orientate suitable therapeutic measures, carry out effective monitoring of its response, and determine severity criteria indicating a bad prognosis for the illness. This is where genomics, micro-array analysis and proteomics appear as valuable therapeutic and

Several groups have reported gene expression profiles for SSc-patient's tissues and cells

Zhou found that fibroblasts in SSc patients showed different RNAm expression profiles for fibrilarine autoantigens, B centromere protein, P27 centromeric autoantigen , RNA polymerase I , DNA topoisomerase I, and PMScl (2). Luzina found high chemokine and cytokine levels in bronchoalveolar lavage (LAB) in SSc patients (3). Whitfield examined skin biopsies in four SSc patients, identifying 2,776 genes which expressed themselves in different ways to that of healthy controls (4).Tan (using a fibroblast culture) identified 62 genes which expressed themselves in different ways in SSc (5). Zhou reported fibroblast micro-array analysis results for fibroblasts from 18 sets of discordant twins in SSc (6,7). Protein analysis using two-dimensional electrophoresis electrophoresis on polyacrylamide gel (2D PAG) will contribute to and extend the knowledge produced by analyzing gene

Proteomic analysis involves many methodologies orientated towards identifying and characterizing altered proteins as a result of illness. Thousands of proteins are evaluated in just one trial in such studies, leading to detecting expression profiles as a consequence of abnormality in cell function or interaction. Traditional methods used in proteome analysis have included 2D PAG where proteins are first separated according to their electric charge and then by their mass in the second direction before being stained, thus allowing mixtures of 1,000 to 3,000 proteins to be visualized. The development of special software and the use of Internet have allowed multiple genes and databases to be compared. When being combined with mass spectrometry, a separation appears which allows efficient identification of proteins of interest, including many of their PTM. This analysis can be applied in comparative studies of expression profiles during different stages of the illness or healthy tissue compared to affected tissue, thus being able to identify the different modifications in the protein characteristics of clinical interest in different illnesses (8).

Clinical proteomics is aimed at identifying proteins involved in pathological processes, as well as evaluating changes in their expression during different stages of an illness.

expression, especially for proteins undergoing crucial PTM in their function.

phenomenon in a comprehensive manner.

fibrotic changes (1).

diagnosis tools.

(2-6).

This was a cases and controls study in which 11 patients who fulfilled with American College of Rheumatology SSc criteria were included during different phases of the illness (14,15) and subdivided into two groups: limited SSc and diffuse SSc, according to the parameters proposed by Le Roy (1). Table 1.

The cutaneous involvement of the skin was evaluated according to the modified Rodnan index (16) which ranges from 0 (normal) to 3 (severe), measured in 17 different body areas (maximum possible score is 51).

Using Proteomic Analysis for Studying the Skin Fibroblast Protein Profile in Systemic Sclerosis 57

The patients were appropriately and conveniently informed about the investigation and

The biopsies would arrive at the lab immediately after samples had been taken to

According to previously-defined protocols, the sample should have arrived at the lab

 Tubes marked with the names of the patients, indicating whether the fragment of skin had been taken from a clinically healthy area or from a clinically sick one; and

 Preparation of the means of transport for the biopsy: A DMEM medium was used with a F-12 medium supplemented with an antibiotic solution (100ug/ml streptomycin,

Preparation of the material and supplies for the culture: a culture medium was

The skin biopsies immersed in the transport medium and the serums were transported at 4°C and taken to a lab specializing in human fibroblast cultures. Once in the lab, the samples were processed in the white zone, cell culture room, in the safety cabin following

 Each sample was washed three times with HANK´s saline solution supplemented at 3% with antibiotic and antimycotic solution (100mg/ml penicillin, 100ug/ml streptomycin,

 The samples were cut by a scalpel into small explants (half millimeter maximum size). The fragments so obtained were planted as explants in six-well culture plates; A total blood sample was taken from each patient in a dry tube to obtain serum by centrifuging at 2,500 rpm for 20 minutes at room temperature, with which the fibroblast

2ml DNEM culture medium with F-12 supplemented at 20% with autologous serum

Each sample was identified with a number for each patient, followed by whether the

The cultures were monitored daily under an inverted microscope at 10X by 40X enlargement:

Cell preparation for obtaining the proteins was carried out, following the following steps:

 Once the washing solution had been removed, 600 ul extraction protein buffer was added to each well as described in the protocol for 2D electrophoresis or Trizol study

 The fibroblasts were incubated for 10 minutes and then homogenized with the help of a rake. Cell separation was confirmed with an inverted microscope and each well's content was placed in a 1.5 ml Eppendorf tube and stored at -80°C until proteins were analyzed.

the lab procedures that would be carried out for analyzing samples;

Serum taken from patients was convenient and suitable for lab procedures.

ensure rapid processing; and

0.25ug/ml B anphotericine);

sample was healthy or unhealthy.

for obtaining NRA; and

Observations and photographs were registered;

Confluence was obtained around the fourth week.

The culture medium was changed every third day; and

Cells were previously washed with 1X PBS solution;

on the day the sample was taken as follows (18):

 Dry tubes to take the blood sample from the same patient. The following procedures had been previously carried out in the lab:

0.25ug/ml B anphotericin) at 3% in sealed sterile glasses; and

prepared to be supplemented with autologous human serum.

culture medium would be autologously supplemented;

management protocols for these areas, according to the Lab Quality Manual.

and 1% antimycotic antibiotic solution was added to each well; and

The first cells began to be observed during the second week after culture.


Table 1. General characteristics of SSc patients

Patients had no treatment or had suspended 4 weeks before taking the biopsy (a treatment scheme was defined as involving any of the following medications, alone or combined: prednisone, D-penicillamine, colchicine, micophenolate mofetil, methotrexate, cyclophosphamide).

Healthy controls were individuals without an autoimmune illness or who had not undergone previous immunodepressor treatment.

Registration forms were completed; they then contained SSc patients' demographic data, clinical characteristics and antibody levels.

All individuals involved in the study signed the participation consent form according to established ethical norms.

#### **2.2 Skin biopsy**

Following the cutaneous biopsy technique's guidelines by means of punch (17), two skin biopsies were taken from each SSc patient: a skin sample with SSc involvement obtained from the body area having the maximum Rodan score and another clinically healthy skin sample having a zero Rodan score. The same technique was used for a skin biopsy of healthy individuals taken from a non-esthetic non-visible area. The material was prepared for cell culture.

#### **2.3 Obtaining fibroblasts from skin biopsies from healthy controls and SSc patients**

This stage of the study, as well as the rest of the procedures, had been previously agreed on by the interdisciplinary team for which the critical route in each process was determined. Clear coordination of activities was needed to guarantee that:


SSc subtype

Table 1. General characteristics of SSc patients

undergone previous immunodepressor treatment.

Clear coordination of activities was needed to guarantee that:

clinical characteristics and antibody levels.

cyclophosphamide).

established ethical norms.

**2.2 Skin biopsy** 

for cell culture.

**Clinical features SSc Patients (n=11)**  Age at onset/yrs 44.75 ±10 Female/male ratio 3:01 Disease duration yrs 9,65 ± 4

lSSc 7 dSSc 3 Morphea 1

Raynaud phenomenon % 90 Raynaud duration, yrs 9.1 ± 4 Rodnan Score 22.1 ± 9 Calcinosis % 45 Telangiectsias % 64 Renal Crisis % 0 Digital ulcers % 0 Gastrointestinal involvement % 20 Pulmonary involvement % 0 Antibodies Anticentromere % 60 Antibodies Anti SL-70 % 30 Antibodies Antinuclear % 90

Patients had no treatment or had suspended 4 weeks before taking the biopsy (a treatment scheme was defined as involving any of the following medications, alone or combined: prednisone, D-penicillamine, colchicine, micophenolate mofetil, methotrexate,

Healthy controls were individuals without an autoimmune illness or who had not

Registration forms were completed; they then contained SSc patients' demographic data,

All individuals involved in the study signed the participation consent form according to

Following the cutaneous biopsy technique's guidelines by means of punch (17), two skin biopsies were taken from each SSc patient: a skin sample with SSc involvement obtained from the body area having the maximum Rodan score and another clinically healthy skin sample having a zero Rodan score. The same technique was used for a skin biopsy of healthy individuals taken from a non-esthetic non-visible area. The material was prepared

**2.3 Obtaining fibroblasts from skin biopsies from healthy controls and SSc patients**  This stage of the study, as well as the rest of the procedures, had been previously agreed on by the interdisciplinary team for which the critical route in each process was determined. The following procedures had been previously carried out in the lab:


The skin biopsies immersed in the transport medium and the serums were transported at 4°C and taken to a lab specializing in human fibroblast cultures. Once in the lab, the samples were processed in the white zone, cell culture room, in the safety cabin following management protocols for these areas, according to the Lab Quality Manual.


The cultures were monitored daily under an inverted microscope at 10X by 40X enlargement:


Confluence was obtained around the fourth week.

Cell preparation for obtaining the proteins was carried out, following the following steps:


Using Proteomic Analysis for Studying the Skin Fibroblast Protein Profile in Systemic Sclerosis 59

removed to avoid reduction reversibility. This step was repeated, but this time the strips

Meanwhile the 2D gel was placed in low fusion point agarose solution which was dissolved in SDS\_PAGE running buffer. The proteins were separated in BIO RAD chambers, whether with Mini-Protean 3 cell (cat165-3301/02) or Mini-Protean Tetra cell (CAT 165-8000/01); the

Once the proteins had been separated in 2D, they were silver stained according to manufacturer's recommendations (Invitrogen, Silver Express staining kit, cat.LC 61000). The gels were documented with Quantity One 1-D Analysis Software and differential

The spots or differential expression points between controls and patients were analyzed with MALDI-TOF/TOF (4700 Proteomics Analyzer, Applied Biosystems). Four points were split and sent to the Córdoba University's central research support service (SCAI)

It should be pointed out that the best results obtained in separating proteins from fibroblasts in patients and controls by means of 2D electrophoresis were on 4-7 pH strips and 12% gels. All 2D electrophoresis trials were carried out from the same human fibroblast culture lysate

The results were presented descriptively with measurements, medians and interquartile ranges expressed according to expected variables. Association measurements having binominal categorical variables were presented in the analysis, depending on population distribution. A Wilcoxon or Mann-Whitney chi square test was used and association was measured by odds ratio (OR). Controls having similar conditions to the chosen patient cases regarding age and gender were sought to avoid differential expression which could have been explained by a physiological condition associated with these two variables and which

Proteins in cells from silver stained fibroblast cultures were observed in representative 2D SD-Page electrophoresis. The trials were carried out on 12% gel and IPG strips having pH4- 7. Standardization studies were carried out on strips having pH 3-10 but most proteins were located in the pH 4-7 range where better resolution appeared. Each fibroblast sample was

2D electrophoresis images of human fibroblast proteins from controls and SSc patients were analyzed by PDQest software allowing the gels to be normalized. Proteins (spots) which were differentially expressed in controls and scleroderma patients (marked with arrows and numbers on each gel in Figure 1) were isolated, digested with trypsin and the peptides so produced were analyzed by mass spectrometry (peptide mass fingerprints) (MALDI-TOF). The analyzed spots from silver stained gels as well as isolated ones stained with Coomasie blue were mainly from different haptoglobin protein isoforms (Table 2),

were placed in the 2D running buffer (tri/glycine/SDS at pH 8.8).

expression points were found with PDQuest 2-D Analysis Software.

proteomics unit in Spain.

**4. Statistical study** 

**5. Results** 

having greater than

for both patients' samples and triplicate controls.

could have increased or decreased potential associations.

analyzed by 2D electrophoresis in triplicate.

procedure began with a 40V voltage and was slowly increased to 60V voltage.

#### **3. 2D electrophoresis for analyzing human fibroblast proteins in SSc patients and controls**

14 fibroblast culture samples were studied by 2D-SD PAGE obtained from skin biopsies from three healthy controls and skin from a healthy and unhealthy region in 11 SSc patients. A recognition code was assigned. All individuals involved in the study signed the participation consent form, according to the ethical standards for such protocol.

The fibroblasts were lysed in a 600 ul protein extraction buffer made up of 7M thiourea, 2M ABS-14 detergent (1%), 40 mM Tris base and 0.001% bromophenol, all of which form part of BIO RAD protein extraction kit (cat. 163-2086). Anpholites (pH 3-10) were added at 200 mM final concentration before starting the lysis for reducing cysteine disulphide links.

The samples suspended in lysis buffer were initially sonified on ice to break up the genomic DNA cells and fragments (10%). The product was spun at 16,000 g for 20 minutes, separating proteins from the remains of cells and other macromolecules. These samples were stored at -80°C until subsequent analysis.

The Lowry method (RC DC, assay protein, Bio-Rad) was used for protein quantification; uni-dimensional electrophoresis was carried out by means of Laemmli's method to obtain an electrophoretic map and thus guarantee the integrity of proteins from fibroblast lysates. Once protein concentration and integrity had been verified, 2D PAG SDS electrophoresis trials were carried out. Electrophoresis was carried out on 10% and 12% acrylamide gels (30%/0.8v/v acrylamide/bisacrylamide), best results being obtained at 12%.

Isoelectrofocusing (IEF) followed BIO RAD's recommended method (cat.163-2105); 7 cm IPG strips, pH 3-10 and pH 4-7 ranks were selected. The latter were placed on trays to hold samples of interest suspended in rehydration buffer (125ul total volume); this buffer contained (m urea, 2% CHAPS, 50 Mm dithiethreitol (DTT), 3-10 anpholites (0.2%) and blue bromophenol traces. The fibroblast lysates were left (for one or two hours) and it was verified that the gel was totally covered by the previous solution, after which mineral oil was placed on the strip to avoid evaporation. The samples were covered and incubated for sixteen hours. Two functions were fulfilled in this step: the strips were hydrated and the samples were absorbed by the pH strip gel (which is why time taken and conditions for this procedure were so important).

Human fibroblast culture protein IEF was carried out on Protean IEF Cell equipment (BIO RAD), initially on a linear gradient until reaching 250V for 30 minutes, then at 4,000V for 2 hours on a linear gradient and finally on a fast ramp until the equipment reached 12,000V when the IEF finished. Small wicks of filter paper were placed before passing the strip from the hydration tray to the IEF equipment; they were moistened with ultrapure water and the strip was then placed. However, everything had to be covered with mineral oil so as to avoid evaporation before starting the process.

The 2D in which the proteins were separated according to weight was developed on 12% gels according to the preliminary analysis. Once the IEF was finished, the strips were separated from the electrode and placed in the equilibrium solution trays again, with 2% of equilibrium buffer I (6M urea, 2% SDS, 0.375M Tris-HCl (pH 8.8), 20% glycerol and 2% DTT). They were incubated for 10 minutes, the disulphur groups thus being reduced. The strips were then incubated for 10 minutes in equilibrium II buffer (6M urea, 2% SDS, 0.375M Tris-HCl (pH 8.8), 20% glycerol and 0.5g iodoacetamide). The sulphidryl groups were removed to avoid reduction reversibility. This step was repeated, but this time the strips were placed in the 2D running buffer (tri/glycine/SDS at pH 8.8).

Meanwhile the 2D gel was placed in low fusion point agarose solution which was dissolved in SDS\_PAGE running buffer. The proteins were separated in BIO RAD chambers, whether with Mini-Protean 3 cell (cat165-3301/02) or Mini-Protean Tetra cell (CAT 165-8000/01); the procedure began with a 40V voltage and was slowly increased to 60V voltage.

Once the proteins had been separated in 2D, they were silver stained according to manufacturer's recommendations (Invitrogen, Silver Express staining kit, cat.LC 61000).

The gels were documented with Quantity One 1-D Analysis Software and differential expression points were found with PDQuest 2-D Analysis Software.

The spots or differential expression points between controls and patients were analyzed with MALDI-TOF/TOF (4700 Proteomics Analyzer, Applied Biosystems). Four points were split and sent to the Córdoba University's central research support service (SCAI) proteomics unit in Spain.

It should be pointed out that the best results obtained in separating proteins from fibroblasts in patients and controls by means of 2D electrophoresis were on 4-7 pH strips and 12% gels. All 2D electrophoresis trials were carried out from the same human fibroblast culture lysate for both patients' samples and triplicate controls.

#### **4. Statistical study**

58 Systemic Sclerosis – An Update on the Aberrant Immune System and Clinical Features

**3. 2D electrophoresis for analyzing human fibroblast proteins in SSc patients** 

14 fibroblast culture samples were studied by 2D-SD PAGE obtained from skin biopsies from three healthy controls and skin from a healthy and unhealthy region in 11 SSc patients. A recognition code was assigned. All individuals involved in the study signed the

The fibroblasts were lysed in a 600 ul protein extraction buffer made up of 7M thiourea, 2M ABS-14 detergent (1%), 40 mM Tris base and 0.001% bromophenol, all of which form part of BIO RAD protein extraction kit (cat. 163-2086). Anpholites (pH 3-10) were added at 200 mM

The samples suspended in lysis buffer were initially sonified on ice to break up the genomic DNA cells and fragments (10%). The product was spun at 16,000 g for 20 minutes, separating proteins from the remains of cells and other macromolecules. These samples

The Lowry method (RC DC, assay protein, Bio-Rad) was used for protein quantification; uni-dimensional electrophoresis was carried out by means of Laemmli's method to obtain an electrophoretic map and thus guarantee the integrity of proteins from fibroblast lysates. Once protein concentration and integrity had been verified, 2D PAG SDS electrophoresis trials were carried out. Electrophoresis was carried out on 10% and 12% acrylamide gels

Isoelectrofocusing (IEF) followed BIO RAD's recommended method (cat.163-2105); 7 cm IPG strips, pH 3-10 and pH 4-7 ranks were selected. The latter were placed on trays to hold samples of interest suspended in rehydration buffer (125ul total volume); this buffer contained (m urea, 2% CHAPS, 50 Mm dithiethreitol (DTT), 3-10 anpholites (0.2%) and blue bromophenol traces. The fibroblast lysates were left (for one or two hours) and it was verified that the gel was totally covered by the previous solution, after which mineral oil was placed on the strip to avoid evaporation. The samples were covered and incubated for sixteen hours. Two functions were fulfilled in this step: the strips were hydrated and the samples were absorbed by the pH strip gel (which is why time taken and conditions for this

Human fibroblast culture protein IEF was carried out on Protean IEF Cell equipment (BIO RAD), initially on a linear gradient until reaching 250V for 30 minutes, then at 4,000V for 2 hours on a linear gradient and finally on a fast ramp until the equipment reached 12,000V when the IEF finished. Small wicks of filter paper were placed before passing the strip from the hydration tray to the IEF equipment; they were moistened with ultrapure water and the strip was then placed. However, everything had to be covered with mineral oil so as to

The 2D in which the proteins were separated according to weight was developed on 12% gels according to the preliminary analysis. Once the IEF was finished, the strips were separated from the electrode and placed in the equilibrium solution trays again, with 2% of equilibrium buffer I (6M urea, 2% SDS, 0.375M Tris-HCl (pH 8.8), 20% glycerol and 2% DTT). They were incubated for 10 minutes, the disulphur groups thus being reduced. The strips were then incubated for 10 minutes in equilibrium II buffer (6M urea, 2% SDS, 0.375M Tris-HCl (pH 8.8), 20% glycerol and 0.5g iodoacetamide). The sulphidryl groups were

participation consent form, according to the ethical standards for such protocol.

final concentration before starting the lysis for reducing cysteine disulphide links.

(30%/0.8v/v acrylamide/bisacrylamide), best results being obtained at 12%.

were stored at -80°C until subsequent analysis.

procedure were so important).

avoid evaporation before starting the process.

**and controls** 

The results were presented descriptively with measurements, medians and interquartile ranges expressed according to expected variables. Association measurements having binominal categorical variables were presented in the analysis, depending on population distribution. A Wilcoxon or Mann-Whitney chi square test was used and association was measured by odds ratio (OR). Controls having similar conditions to the chosen patient cases regarding age and gender were sought to avoid differential expression which could have been explained by a physiological condition associated with these two variables and which could have increased or decreased potential associations.

#### **5. Results**

Proteins in cells from silver stained fibroblast cultures were observed in representative 2D SD-Page electrophoresis. The trials were carried out on 12% gel and IPG strips having pH4- 7. Standardization studies were carried out on strips having pH 3-10 but most proteins were located in the pH 4-7 range where better resolution appeared. Each fibroblast sample was analyzed by 2D electrophoresis in triplicate.

2D electrophoresis images of human fibroblast proteins from controls and SSc patients were analyzed by PDQest software allowing the gels to be normalized. Proteins (spots) which were differentially expressed in controls and scleroderma patients (marked with arrows and numbers on each gel in Figure 1) were isolated, digested with trypsin and the peptides so produced were analyzed by mass spectrometry (peptide mass fingerprints) (MALDI-TOF). The analyzed spots from silver stained gels as well as isolated ones stained with Coomasie blue were mainly from different haptoglobin protein isoforms (Table 2), having greater than




Table 2. Proteins identified by mass spectrometry (MALDI/TOF- TOF) which were

which differ in expression profile between controls subjects and patients.

separated by 2 D electrophoresis and obtained from cultures of human fibroblasts from SSc patients and healthy subjects. The proteins corresponding to isolated spots in Figure 1,


Table 2. Proteins identified by mass spectrometry (MALDI/TOF- TOF) which were separated by 2 D electrophoresis and obtained from cultures of human fibroblasts from SSc patients and healthy subjects. The proteins corresponding to isolated spots in Figure 1, which differ in expression profile between controls subjects and patients.

Using Proteomic Analysis for Studying the Skin Fibroblast Protein Profile in Systemic Sclerosis 65

Table 3. Main clinical and serological variables and intensity of expression of the spots in

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