**5. Microvesicles: their nature, release and pathophysiological relevance**

A chronic inflammatory state has been widely documented since the early stages of CKD and becomes more pronounced in those with CKD stage V undergoing HD. Oxidant stress (Wratten et al., 2000, Morena et al. 2011), endothelial dysfunction (Recio-Mayoral et al., 2011), high circulating cytokine-producing monocyte subpopulation (Ramirez et al., 2006), reduced number and/or impaired function of endothelial progenitor cells (Krenning et al., 2009), are today considered as hallmarks of vascular damage and defective repair. Uremia also causes telomere shortening and premature cellular senescence of immunocompetent cells (Jimenez et al, 2005). In recent years, increasing attention has been drawn by the awareness of the pathophysiologic role of small, circular membrane fragments named as Microvesiscles (MVs) (Ratajczak et al., 2006) (**Figure 6**).

Fig. 6.

102 Progress in Hemodialysis – From Emergent Biotechnology to Clinical Practice

The proposed mechanisms include blood interaction with endotoxins from the contaminated dialysate through HD membranes. A large number of studies have greatly contributed to increasing our knowledge in the mechanisms of endotoxin transfer across the membrane In fact, when using high permeability membranes, backfiltration and backdiffusion occur and have ebeen extensively described (Fiore & Ronco, 2007, Ronco, 2007). Thus, the transmembrane passage of endotoxins or other cytokine stimulating substances (CIS) occurs during HD (Schindler et al., 2004, Tetta et al., 2006). The reduction of backfiltration of standard dialysate may reduce the plasma concentration of IL-1ra, a sensitive indicator of inflammation in HD patients (*Dinarello personal communication, 2004*), and IL-1 (Panichi et al., 1998). Studies on large groups of patients have shown that highvolume exchange HDF, a treatment in which dialysate backfiltration is minimal, if any, is associated with significantly lower CRP plasma values (Panichi et. 1998). Comparing in a double cross-over study patients treated with high-flux and on line HDF using ultrapure dialysate and infusate, it was shown that a significant reduction of pro-inflammatory CD14+/CD16+ mononuclear subset (Carracedo et al., 2006) occurs in on line HDF. These studies emphasize that the convective component has an additional anti-inflammatory

The new technology of pyrogen-adsorbing, non-complement activating, high-permeability synthetic membrane and dedicated machines (Tetta et al., 2011), as well as the awareness of the deleterious effects derived from contamination of dialysis fluids has reduced the clinical impact to a periodic microinflammatory stimulus. Undoubtedly, the availability of monitors for on-line HDF and its increased popularity have spurred more restrictive measures on safety issues and monitoring. Water quality is a mandatory issue. The safety of online HDF has been shown repeatedly in several monocenter (Canaud et al., 1998, Pizzarelli et al., 1998

Nowadays, the philosophy of ''ultrapure dialysate''is in common practice (Kessler et al., 2002). The clinical, consolidated experience on line HDF warrants well-defined procedures and leaves no space for ''experiments'' in what is now routine (Canaud et al, 2011). The ''hemocompatibility network'' should eventually prevent the periodic microinflammation induction through the implementation of rigid protocols of disinfection and maintenance of water-treatment systems and HD monitors (Cappelli et al., 2006; Kessler

**5. Microvesicles: their nature, release and pathophysiological relevance** 

A chronic inflammatory state has been widely documented since the early stages of CKD and becomes more pronounced in those with CKD stage V undergoing HD. Oxidant stress (Wratten et al., 2000, Morena et al. 2011), endothelial dysfunction (Recio-Mayoral et al., 2011), high circulating cytokine-producing monocyte subpopulation (Ramirez et al., 2006), reduced number and/or impaired function of endothelial progenitor cells (Krenning et al., 2009), are today considered as hallmarks of vascular damage and defective repair. Uremia also causes telomere shortening and premature cellular senescence of immunocompetent cells (Jimenez et al, 2005). In recent years, increasing attention has been drawn by the awareness of the pathophysiologic role of small, circular membrane fragments named as

and multicenter studies (Canaud et al., 2001,Vaslaki et al., 2000).

Microvesiscles (MVs) (Ratajczak et al., 2006) (**Figure 6**).

effects (Ramirez et al., 2007).

et al. 2002).

For long time MVs were considered to be inert cellular debris. The frequently observed vesicles by electron microscopy in the interstitial space of tissues or in blood were considered as the consequence of cell damage or the result of dynamic plasma membrane turnover (Siekevitz et al., 1972). As the vesicle population detectable both *in vitro* and *in vivo*  is a mixed population of exosomes and shedding vesicles, we will refer to them collectively as MVs. Released MVs may remain in the extracellular space in proximity of the place of origin or may enter into the biological fluids reaching distant sites. This may explain the presence of MVs in plasma, urine, milk and cerebrospinal fluid. The bulk of MVs present in the circulation is derived from platelets (George, 1982), and in less extent from other blood cells and endothelial cells (Martinez et al., 2005). The MVs derived from platelets are also designed as microparticles while those derived from polymorphonuclear leukocytes are also named ectosomes (Hess et al., 1999). Finally, MVs released during morphogenesis of multicellular organisms are indicated as argosomes (Greco et al., 2001). Besides normal cells, also tumor cells may release MVs and in patients suffering for neoplastic diseases tumorderived MVs may be detected within the biological fluids (Kim et al, 2003, Iero et al., 2008). Therefore, MVs are an assorted population, differing in cellular origin, number, size and antigenic composition (Diamant et al., 2004) shed by various cell types in physiological and pathological conditions. The release of MVs may be constitutive or consequent to cell activation by soluble agonists, by physical or chemical stress such as the oxidative stress and hypoxia, and by shear stress (Ratajczak et al., 2006). Exosomes have an endosome origin and are a rather homogenous population with a size ranging from 30 to 120nm (7). They are stored as intraluminal vesicles within multivesicular bodies of the late-endosome and are released when these multivesicular bodies fuse with the cell membrane. Our knowledge on

The Evolution of Biocompatibility: From Microinflammation to Microvesiscles 105

**5.1.4 MVs may mediate a horizontal transfer of genetic information.** The occurrence of epigenetic changes has been frequently reported in co-culture conditions. An explanation of this phenomenon is the transfer of genetic information between cells. We demonstrated that MVs derived from human endothelial progenitors (EPC) can also act as a vehicle for mRNA transport among cells (Deregibus et al., 2007). MVs generated from EPC were incorporated in normal endothelial cells by interaction with 4- and 1 integrins expressed on their surface and activated an angiogenic program. Besides mRNA, MVs may transfer microRNAs (miRNA) to target cells (Yuan et al., 2009). Since miRNAs are naturally occurring regulators of protein translation, this observation opens the possibility that stem cells can alter the expression of genes in neighbouring cells by transferring microRNAs contained in MVs. We recently characterized miRNA shuttled by MVs released by human adult mesenchymal stem cells (MSCs) (Collino et al., 2010). Hierarchical clustering and similarity analysis of microRNAs showed that microRNA compartmentalization and secretion by MVs are both highly regulated

The biologic role of MVs and their implication in pathophysiology depends on the several factors namely the cell of origin, their phenotype, the genetic material (mRNA and microRNA) and the target cells. In CKD, several studies have accrued evidence that MVs or MPs could participate to the vascular damage and the evolution of the atherosclerotic lesion. **5.2.1 Circulating platelet-derived microparticles (PMPs)** with procoagulant activity are considered a potential cause of thrombosis in uremic patients undergoing HD (Ando et al., 2002). Elevated counts of circulating PMPs have been reported in association with thrombotic disorders, such as cerebrovascular accidents (Katopodis et al., 1997), unstable angina (Katopodis et al., 1997), and acute myocardial infarction (Gawaz et al., 1996). In addition, PMPs that adhered to vascular endothelium and leukocytes activate such cells and transport their chemical mediators to those cells, potentially leading to the development of thrombosis and atherosclerosis (Mallat et al., 1999, Barry et al.,

**5.2.2 Endothelial MVs (EMVs) -** Treatment modalities that reduce the inflammatory potential of the cells originating MVs have interestingly been correlated with a decreased number of endothelial microparticles (Carracedo et al., 2005, Ramirez et al., 2005). Circulating EMPs have recently been reported to correlate with impaired vascular function in HD patients (Faure et al., 2006). A recent study showed an increase in the percentage of CD14*+*CD16*+* monocytes in CKD-NonD and HD patients. In PD patients, regardless of RRF, the percentage of CD14*+*CD16*+* was similar to controls (Merino et al., 2010). It is interesting to note that HD patients displayed significantly higher apoptotic EMPs and VEGF levels than the two PD and CKD-non dialyszed groups. In contrast, there were no differences between CKD-NonD and PD groups. In CKD-non dialyszed and HD patients, the percentage of CD14*+*CD16*+* was correlated with endothelial damage. It appears that PD, compared with HD, reduces but does not fully prevent the endothelial damage induced by uremia, in spite of presenting a microinflammatory status similar to that of the controls. The role of EMVs is still to be elucidated in the complex unbalance observed in CKD patients between circulating

endothelial cells and endothelial progenitor cells.

processes.

1997).

**5.2 Microvesicles in CKD** 

the mechanism of assembly and sorting of the exosomes is only partial, due to the fact that a common sorting signal for all cell types has not so far been identified (Johnstone et al., 2006). Shedding vesicles are usually larger than exosomes with size ranging from 100nm to 1m. Formation of shedding vesicles takes place from the budding of small cytoplasmic protrusions followed by their detachment from the cell surface. This process is dependent on calcium influx, calpain and cytoskeleton reorganization.

### **5.1 MV biological activities**

It is now recognized that MVs are an integral part of the intercellular microenvironment and may act as regulators of cell-to-cell communication. This concept is based on the observation that MVs released from a given cell type may interact through specific receptor-ligands with other cells leading to target cell stimulation directly or by transferring surface receptors (Janowska-Wieczorek et al., 2001, Morel et al., 2004). This interaction may either be limited to a receptor-mediated binding to the surface of target cells forming a platform for assembly of multimolecular complexes or leading to cell signaling, either be followed by internalization as result of direct fusion or endocytic uptake by target cells (Cocucci et al., 2008). Once internalized, MVs can fuse their membranes with those of endosomes, thus leading to a horizontal transfer of their content in the cytosol of target cells. Alternatively, they may remain segregated within endosomes and be transferred to lysosomes or dismissed by the cells following the fusion with the plasmamembrane, thus leading to a process of transcytosis. It was proposed that MV-mediated cell-to-cell communication emerged very early during evolution as a template for the development of further more refined mechanisms of cell communication (Ratajczak et al., 2006). MVs may influence the behavior of target cells in multiple ways.


**5.1.4 MVs may mediate a horizontal transfer of genetic information.** The occurrence of epigenetic changes has been frequently reported in co-culture conditions. An explanation of this phenomenon is the transfer of genetic information between cells. We demonstrated that MVs derived from human endothelial progenitors (EPC) can also act as a vehicle for mRNA transport among cells (Deregibus et al., 2007). MVs generated from EPC were incorporated in normal endothelial cells by interaction with 4- and 1 integrins expressed on their surface and activated an angiogenic program. Besides mRNA, MVs may transfer microRNAs (miRNA) to target cells (Yuan et al., 2009). Since miRNAs are naturally occurring regulators of protein translation, this observation opens the possibility that stem cells can alter the expression of genes in neighbouring cells by transferring microRNAs contained in MVs. We recently characterized miRNA shuttled by MVs released by human adult mesenchymal stem cells (MSCs) (Collino et al., 2010). Hierarchical clustering and similarity analysis of microRNAs showed that microRNA compartmentalization and secretion by MVs are both highly regulated processes.

### **5.2 Microvesicles in CKD**

104 Progress in Hemodialysis – From Emergent Biotechnology to Clinical Practice

the mechanism of assembly and sorting of the exosomes is only partial, due to the fact that a common sorting signal for all cell types has not so far been identified (Johnstone et al., 2006). Shedding vesicles are usually larger than exosomes with size ranging from 100nm to 1m. Formation of shedding vesicles takes place from the budding of small cytoplasmic protrusions followed by their detachment from the cell surface. This process is dependent

It is now recognized that MVs are an integral part of the intercellular microenvironment and may act as regulators of cell-to-cell communication. This concept is based on the observation that MVs released from a given cell type may interact through specific receptor-ligands with other cells leading to target cell stimulation directly or by transferring surface receptors (Janowska-Wieczorek et al., 2001, Morel et al., 2004). This interaction may either be limited to a receptor-mediated binding to the surface of target cells forming a platform for assembly of multimolecular complexes or leading to cell signaling, either be followed by internalization as result of direct fusion or endocytic uptake by target cells (Cocucci et al., 2008). Once internalized, MVs can fuse their membranes with those of endosomes, thus leading to a horizontal transfer of their content in the cytosol of target cells. Alternatively, they may remain segregated within endosomes and be transferred to lysosomes or dismissed by the cells following the fusion with the plasmamembrane, thus leading to a process of transcytosis. It was proposed that MV-mediated cell-to-cell communication emerged very early during evolution as a template for the development of further more refined mechanisms of cell communication (Ratajczak et al., 2006). MVs may influence the

**5.1.1 MVs may act as signaling complexes by direct stimulation of target cells** (Ratajczak et al., 2006, Cocucci et al., 2008). MVs derived from platelets, for instance, play an important role in coagulation as their phosphatidylserine-enriched membranes provide a surface for assembly of clotting factors (Zwaal et al., 2004). After activation, platelets shed MVs coated with tissue factor which may interact with macrophages, neutrophils and other platelets by ligation with molecules expressed on the surface of these cells such as P-selectin (Polgar et al., 2005). On the other hand, MVs released from neutrophils express activated Mac-1 able to induce platelet activation (Andrews & Berndt, 2004). Moreover, platelet-derived MVs, besides coagulation, trigger various cell responses as they activate endothelial cells (Barry et al., 1997), polymorphonuclear

**5.1.2 MVs may act by transferring receptors between cells.** The transferring of receptors between cells is supported by the observation that bystander B cells rapidly acquire antigen receptors from activated B cells by a membrane transfer (Quah et al., 2008). **5.1.3 MVs may deliver proteins within the target cells.** An example of this mechanism is the recently reported MV-mediated transfer of a cell death message via encapsulated caspase-1 (Sarkar et al., 2009). It has been found that endotoxin stimulated monocytes induce the cell death of vascular smooth muscle cells by releasing MVs containing caspase-1. This trans-cellular apoptosis induction pathway depends on the function of the delivered caspase-1 within the target cells. It has been also suggested that MVs may contribute to dissemination of certain infective agents, such as HIV or prions (Facler &

neutrophils (Miyhamoto et al., 1988) and monocytes (Barry et al., 1999). .

on calcium influx, calpain and cytoskeleton reorganization.

**5.1 MV biological activities** 

behavior of target cells in multiple ways.

Peterlin, 2000, Fevrier et al., 2004).

The biologic role of MVs and their implication in pathophysiology depends on the several factors namely the cell of origin, their phenotype, the genetic material (mRNA and microRNA) and the target cells. In CKD, several studies have accrued evidence that MVs or MPs could participate to the vascular damage and the evolution of the atherosclerotic lesion.


The Evolution of Biocompatibility: From Microinflammation to Microvesiscles 107

together with new concepts in machine technology have already shown their great potential

Adam A, Cugno M, Molinaro G, Perez M, Lepage Y, & Agostoni A. (2002). Aminopeptidase

Ando M, Iwata A, Ozeki Y, Tsuchiya K, Akiba T, & Nihei H (2002). Circulating platelet-

Arenas MD, Niveiro E, Moledous A, Gil MT, Albiach B, & Carretón MA (2006) .Fatal acute

Barret BJ. (2002). Reducing the burden of cardiovascular disease in patients on dialysis. *Dial* 

Barry OP, Pratico D, Lawson JA, & FitzGerald GA (1997). Transcellular activation of

Barry OP, Kazanietz MG, Praticò D, & Fitzgerald GA(1999). Arachidonic acid in platelet

Bhatt DL, & Topol EJ. (2002). Need to test the arterial Inflammation hypothesis. *Circulation*

Bingel M, Lonnemann G, Shaldon S, Koch KM, & Dinarello CA. (1986). Human interleukin-1

Bradbury BD, Critchlow CV, Weir MR Stewart R, Krishnan M, & Hakim RH (2009). Impact

Camussi G, Deregibus MC, Bruno S, Grange C, Fonsato V, & Tetta C (2011). Exosome /

Canaud B, Bosc JY, Leray H, Stec F, Argiles A, Leblanc M, & Mion C.(1998). On line

Canaud B, Wizemann V, Pizzarelli F, Greenwood R, Schultze G, Weber C, & Falkenhagen D

on-line haemodiafiltration therapy. *Nephrol Dial Transplant.* 16:2181–2187. Canaud B, Chenine L, Renaud S, & Leray H. (2011). Optimal therapeutic conditions for

hemodiafiltration: State of the art. *Nephrol Dial Transplant.* 5:3–11

online hemodiafiltration. *Contrib Nephrol*. 168:28-38.

production during hemodialysis. *Nephron*. 1986;43(3):161-3

P in individuals with a history of angio-oedema on ACE inhibitors. *Lancet*.

derived microparticles with procoagulant activity may be a potential cause of

systemic hypersensitivity reaction during haemodialysis. *Nephrol Dial Transplant.*

platelets and endothelial cells by bioactive lipids in platelet microparticles. *J Clin* 

microparticles up-regulates cyclooxygenase-2-dependent prostaglandin formation via a protein kinase C/mitogen-activated protein kinase-dependent pathway. *J Biol* 

of elevated C-reactive protein levels on Erythropoiesis-stimulating agent (ESA) dose and responsiveness in naemodialysis patients. *Nephrol Dial Transplant* 24: 919-

microvesicle - mediated epigenetic reprogramming of cells *Am J Cancer Res* 1(1):98-

(2001). Cellular interleukin- 1 receptor antagonist production in patients receiving

Ciro Tetta and Emanuele Gatti are full-time employees of Fresenius Medical Care.

The authors thank Dr Sudhir Bowry for critically reviewing the manuscript.

thrombosis in uremic patients *Kidney Int*, 62:1757–1763

to improve survival and cardiovascular stability.

**7. Conflict of interest** 

**8. Acknowledgement** 

359(9323):2088-9.

21(10):2966-70

*Transplant* 31: 155–163

*Invest* 99: 2118-2127.

*Chem* 274: 7545-7556.

106:136–140.

925.

110

**9. References** 

### **5.2.3 MVs in treatment modalities**

Preliminary studies in our laboratory have shown an interesting trend in the reduction of total MVs in a cross-over clinical study when patients shifted from high-flux HD to on-line HDF (**Figure 7**).

Fig. 7. Total MVs count in patients on maintenance HD. In a cross-over design, 8 patients were started on bicarbonate HD (black columns) and 8 patients on on-line HDF (grey columns). MVs were counted by cytofluorimetry. MVs were also characterized (data not shown) by the following specific markers: CD62P, CD41, CD42, CD31, for platelets; CD45, for leukocytes; CD31, CD146, CD144 for the endothelium: CD235 and CD242 (ICAM 4), for erythrocytes.

More studies are needed to better assess the relevance of these observations and to better characterize the type and biological effects of the MVs. It is still to be fully elucidated whether MVs are a consequence or a cause of disease. Increasing evidence for their pathophysiologic role in other human diseases such as sepsis and tumors (Camussi et al., 2011) is rapidly accruing. Many points require further investigation. i. The stimuli and the molecular pathways that regulate the assembly within MVs of the biological active molecules that they shuttle. ii. The stimuli that trigger their release. iii. The surface receptors that may confer selective specificity. iv. The full diagnostic potential of MVs in different pathological conditions. v. The strategy to inhibit formation or to remove from circulation potentially harmful MVs. The recognition of MVs has opened a new era and new perspectives of investigation also in biocompatibility of extracorporeal treatments.
