**2.3. Pulmonary endothelial cells in ARDS and pneumonia**

inter-endothelial junctions consist of adherens, tight and gap junctional complexes, and promote adhesion of opposing cells in the monolayer of microvascular ECs [38]. Microvascular EC gene

**Figure 4.** Scanning micrograph of mouse alveoli (a) and vessel with pericyte (P) surrounding the alveolar lining. Source is mouse alveoli from authors (JG) collection of images processed in the vascular biology laboratory, Augusta University

**Figure 3.** Transmission electron micrograph of a mouse alveolar capillary (Cp) with microvascular endothelial (EC) lining (arrows). Source is mouse alveoli from authors (JG) collection of images processed in the vascular biology laboratory, Augusta University health, electron microscope Core Laboratory (Libby Perry and Brendan Marshall PhD).

294 Endothelial Dysfunction - Old Concepts and New Challenges

clusters include genes related to lipid transport and metabolism [3].

health, electron microscope Core Laboratory (Libby Perry and Brendan Marshall PhD).

ARDS is a severe lung inflammatory disorder with a declining but still unacceptably high.

Mortality (25–46%) [42, 43]. The healthy alveolar-capillary barrier is formed by the microvascular endothelium, the alveolar epithelium, and the basement membrane. The homogeneous pulmonary microvasculature layer of ECs lining the pulmonary circulation forms a tight barrier [44]. The EC barrier dysfunction that occurs in acute lung injury is tightly linked to agonist-induced cytoskeletal remodeling resulting in the disruption of cell–cell contacts, paracellular gap formation, and EC barrier compromise [45, 46]. Tight junctions are formed by the fusion of the outer layers of the plasma membranes and are comprised of occludins, claudins, and junctional adhesion molecules that in turn bind to other protein partners in the actin cytoskeleton [8, 36]. Integrity of adherens junctions (AJs) is critical in regulating paracellular permeability and disruption of VE-cadherin homophilic adhesions leads to excessive accumulation of fluid in the interstitial space and is associated with inflammation, atherogenesis, and acute lung injury [38]. AJs are composed of VE-cadherin and its cytoplasmic binding partners: α-, β- γ-, p120 catenins, which link AJs to the actin cytoskeleton. The assembly of the VE-cadherin-catenin complex is regulated by phosphorylation, and their dissociation leads to cytoskeletal changes and loss of cohesive structure required for an intact EC barrier [36]. Therefore, the complex network of cytoskeletons is critical in the EC barrier regulation.

On the cellular level, in ARDS, there is increased pulmonary capillary EC permeability and fluid leakage into the pulmonary parenchyma that is followed by neutrophils, cytokines, and an acute inflammatory response [47]. Adhesion molecule upregulation on the vascular endothelium of the lung results from the systemic inflammatory cascade that occurs in EC activation. In fact, the expression of molecules that mediate adhesion and signaling of leukocytes is nearly synonymous with endothelial activation [48]. When ECs are activated by toxins such as LPS, other bacterial toxins, viral infections, thrombin or hypoxia, the ECs release cytokines such as TNF-α, IL-1ß or IL-8, and a shift toward a pro-inflammatory phenotype occurs [36]. There is a consecutive expression of adhesion molecules, including PECAM (CD-31), ICAM–1, VCAM, and E selectin, that plays a central role in the leukocyte endothelial adhesion. These adhesion molecules are responsible for recruiting and directing leukocytes to the sites of inflammation.

(Ang-2), an endothelial growth factor. Ang-2, a mediator of pulmonary vascular permeability, binds to the tyrosine kinase receptor and plays a key role in endothelial junctional integrity [57]. Ang–2 levels have been shown to be higher in ARDS patients than in patients with hydrostatic pulmonary edema [58]. Increased levels have also been linked with the severity and mortality of ARDS [58]. Stimulation of PMVEC with IL–8 leads to cytoskeletal reorganization and cell retraction which in turn leads to gap formation between cells and IL-8 levels that are higher in non-survivors of ARDS [59]. Studies for biomarkers are ongoing with the

Pulmonary Vascular Endothelial Cells http://dx.doi.org/10.5772/intechopen.76995 297

Sickle cell disease (SCD) is an inherited red blood cell disorder that affects millions of people throughout the world and in most common among those whose ancestors came from sub-Saharan Africa, Spanish-speaking regions in the Western hemisphere, Saudi Arabia, India, and Mediterranean countries [60]. SCD is caused by a mutant β-globin gene that substitutes valine for glutamic acid at position 6 in the β-globin chain of hemoglobin A. The resultant hemoglobin is called hemoglobin S and is characterized by red blood cells (RBCs) that are crescent or sickle shaped rather than the normal rounded disc shape [61]. One of the most common forms of acute pulmonary disease associated with morbidity and mortality in SCD is acute chest syndrome (ACS). Hypoxia induces abnormal hemoglobin S polymerization and RBC sickling, and the abnormal cells are rigid and unable to pass through narrow capillaries leading to vessel occlusion and ischemia [62]. ACS is the most common form of acute pulmonary disease associated with SCD. ACS is diagnosed by a new infiltrate on chest x-ray that is consistent with alveolar consolidation triggered by infection, fat embolization, or pulmonary sequestration of sickled erythrocytes [61, 62]. The patient experiences chest pain, fever, tachypnea, wheezing, or cough. Under hypoxic and infectious conditions, cell–cell junctions can be destabilized causing the passage of systemic inflammatory mediators into the lungs, producing pulmonary edema; in this sense, ACS is similar to ARDS [61, 62]. Other pathologies include alterations in activated EC metabolic functions that may contribute to the vaso-occlusive events in ACS [63]. The balance between vasoconstriction and vasodilation in ACS may be altered. The ET-1 gene is upregulated in the lung and is released by activated lung ECs in response to hypoxia and reduced NO bioavailability [64, 65]. In a transgenic mouse model consistent with chronic organ lesions, tissue lesions, and acute vaso-occlusive events analogous to human SCD, SAD mice [S(β6val) Antilles (β23lle) D-Punjab(β121Gln)] [66], it was found that ET-1 is produced at a higher level in the pulmonary MVEC of SAD mice than wild type (WT) mice. Further, in the SAD mice, bosentan, an ET receptor antagonist, was shown to prevent death of SAD mice exposed to a severe hypoxic challenge [64].

Painful vaso-occlusive crisis (VOC), one of the major and specific manifestations of SCD, is the most debilitating manifestation of SCD [67]. In VOC, the circulation of blood vessels is obstructed by sickled red blood cells causing ischemic injury and severe pain. Sickling and/ or hypoxia associated with VOC in SCD may shift the balance of endothelial vasodilator and vasoconstrictor response in favor of vasoconstriction [68]. The study of Hammerman et al. [63] measured NO products from cultured pulmonary ECs exposed to red blood cells

potential that the EC biomarkers will aid in the diagnosis of acute lung injury.

**2.4. Endothelial cells in acute chest syndrome in sickle cell disease**

Muller et al. analyzed the autopsy lung specimen ECs for PECAM (CD-31), ICAM–1 in patients with Gram-negative sepsis-induced ARDS and found these adhesion molecules strongly expressed compared to normal lung autopsy specimen [49]. Another study demonstrated that blockade of the VCAM-1 receptor on the pulmonary vascular endothelium diminishes lung injury in established pancreatitis-induced ARDS [50]. In our own unpublished data, mice with lung injury induced by LPS and Gram-positive toxin, pneumolysin, an attenuation of ICAM-1 by a low-anticoagulant heparin, has been shown to attenuate neutrophils and acute lung injury (data not published). Neutrophil recruitment into the lung is the hallmark of acute lung injury (ALI) [51]. Neutrophils enter the interstitial spaces by rolling on the endothelium, and this is mediated by the selectins. The neutrophils adhere to the endothelium and affect the endothelial cytoskeleton inducing remodeling of the tight junctions and further facilitating the transmigration of neutrophils [51].

Once activated, ECs display recruited neutrophils in ARDS; there is considerable evidence that pro-and anti-inflammatory cytokines and chemokines play a major role in the pathogenesis of acute lung injury from sepsis and pneumonia [51]. There is a complex network of inflammatory cytokines and chemokines that play a major role in mediating, amplifying, and perpetuating the lung injury process. The pro-inflammatory cytokines IL–1 beta and TNF-α have been located in bronchoalveolar lavage fluid (BALF) from ARDS patients [52]. In influenza, early induction of the cytokines IFN-α, TNF-α, IL-1α, and IL-6 and the chemokines CCL2, CCL3, CXCL2 (IL-8), and CXCL10 are associated with clinical symptoms and morbidity in humans [14, 53, 54]. Simultaneous production of anti-inflammatory cytokines can counteract pro-inflammatory cytokine effects and modify the intensity of the inflammatory process in ARDS [52].

There is a search for biomarkers in ARDS to assess the activation and dysfunction of ECs. One marker may be endothelial progenitor cells (EPCs) as a marker of EC dysfunction and damage. It has been reported that there is an association between the EPC count and survival in ARDS [55]. The cells are present at very low levels in normal patients, but the number of EPCs increased significantly in conditions associated with vascular damage such as ARDS. In the study of Moussa et al. [56], EPC counts were increased in patients with moderate and severe ARDS compared with non-ARDS patients [56]. Higher EPC counts were also found in nonsurvivors of ARDS in this same study [56]. Other promising biomarkers are angiopoietin–2 (Ang-2), an endothelial growth factor. Ang-2, a mediator of pulmonary vascular permeability, binds to the tyrosine kinase receptor and plays a key role in endothelial junctional integrity [57]. Ang–2 levels have been shown to be higher in ARDS patients than in patients with hydrostatic pulmonary edema [58]. Increased levels have also been linked with the severity and mortality of ARDS [58]. Stimulation of PMVEC with IL–8 leads to cytoskeletal reorganization and cell retraction which in turn leads to gap formation between cells and IL-8 levels that are higher in non-survivors of ARDS [59]. Studies for biomarkers are ongoing with the potential that the EC biomarkers will aid in the diagnosis of acute lung injury.

#### **2.4. Endothelial cells in acute chest syndrome in sickle cell disease**

On the cellular level, in ARDS, there is increased pulmonary capillary EC permeability and fluid leakage into the pulmonary parenchyma that is followed by neutrophils, cytokines, and an acute inflammatory response [47]. Adhesion molecule upregulation on the vascular endothelium of the lung results from the systemic inflammatory cascade that occurs in EC activation. In fact, the expression of molecules that mediate adhesion and signaling of leukocytes is nearly synonymous with endothelial activation [48]. When ECs are activated by toxins such as LPS, other bacterial toxins, viral infections, thrombin or hypoxia, the ECs release cytokines such as TNF-α, IL-1ß or IL-8, and a shift toward a pro-inflammatory phenotype occurs [36]. There is a consecutive expression of adhesion molecules, including PECAM (CD-31), ICAM–1, VCAM, and E selectin, that plays a central role in the leukocyte endothelial adhesion. These adhesion molecules are responsible for recruiting and directing leukocytes to the

Muller et al. analyzed the autopsy lung specimen ECs for PECAM (CD-31), ICAM–1 in patients with Gram-negative sepsis-induced ARDS and found these adhesion molecules strongly expressed compared to normal lung autopsy specimen [49]. Another study demonstrated that blockade of the VCAM-1 receptor on the pulmonary vascular endothelium diminishes lung injury in established pancreatitis-induced ARDS [50]. In our own unpublished data, mice with lung injury induced by LPS and Gram-positive toxin, pneumolysin, an attenuation of ICAM-1 by a low-anticoagulant heparin, has been shown to attenuate neutrophils and acute lung injury (data not published). Neutrophil recruitment into the lung is the hallmark of acute lung injury (ALI) [51]. Neutrophils enter the interstitial spaces by rolling on the endothelium, and this is mediated by the selectins. The neutrophils adhere to the endothelium and affect the endothelial cytoskeleton inducing remodeling of the tight junctions and

Once activated, ECs display recruited neutrophils in ARDS; there is considerable evidence that pro-and anti-inflammatory cytokines and chemokines play a major role in the pathogenesis of acute lung injury from sepsis and pneumonia [51]. There is a complex network of inflammatory cytokines and chemokines that play a major role in mediating, amplifying, and perpetuating the lung injury process. The pro-inflammatory cytokines IL–1 beta and TNF-α have been located in bronchoalveolar lavage fluid (BALF) from ARDS patients [52]. In influenza, early induction of the cytokines IFN-α, TNF-α, IL-1α, and IL-6 and the chemokines CCL2, CCL3, CXCL2 (IL-8), and CXCL10 are associated with clinical symptoms and morbidity in humans [14, 53, 54]. Simultaneous production of anti-inflammatory cytokines can counteract pro-inflammatory

cytokine effects and modify the intensity of the inflammatory process in ARDS [52].

There is a search for biomarkers in ARDS to assess the activation and dysfunction of ECs. One marker may be endothelial progenitor cells (EPCs) as a marker of EC dysfunction and damage. It has been reported that there is an association between the EPC count and survival in ARDS [55]. The cells are present at very low levels in normal patients, but the number of EPCs increased significantly in conditions associated with vascular damage such as ARDS. In the study of Moussa et al. [56], EPC counts were increased in patients with moderate and severe ARDS compared with non-ARDS patients [56]. Higher EPC counts were also found in nonsurvivors of ARDS in this same study [56]. Other promising biomarkers are angiopoietin–2

further facilitating the transmigration of neutrophils [51].

sites of inflammation.

296 Endothelial Dysfunction - Old Concepts and New Challenges

Sickle cell disease (SCD) is an inherited red blood cell disorder that affects millions of people throughout the world and in most common among those whose ancestors came from sub-Saharan Africa, Spanish-speaking regions in the Western hemisphere, Saudi Arabia, India, and Mediterranean countries [60]. SCD is caused by a mutant β-globin gene that substitutes valine for glutamic acid at position 6 in the β-globin chain of hemoglobin A. The resultant hemoglobin is called hemoglobin S and is characterized by red blood cells (RBCs) that are crescent or sickle shaped rather than the normal rounded disc shape [61]. One of the most common forms of acute pulmonary disease associated with morbidity and mortality in SCD is acute chest syndrome (ACS). Hypoxia induces abnormal hemoglobin S polymerization and RBC sickling, and the abnormal cells are rigid and unable to pass through narrow capillaries leading to vessel occlusion and ischemia [62]. ACS is the most common form of acute pulmonary disease associated with SCD. ACS is diagnosed by a new infiltrate on chest x-ray that is consistent with alveolar consolidation triggered by infection, fat embolization, or pulmonary sequestration of sickled erythrocytes [61, 62]. The patient experiences chest pain, fever, tachypnea, wheezing, or cough. Under hypoxic and infectious conditions, cell–cell junctions can be destabilized causing the passage of systemic inflammatory mediators into the lungs, producing pulmonary edema; in this sense, ACS is similar to ARDS [61, 62]. Other pathologies include alterations in activated EC metabolic functions that may contribute to the vaso-occlusive events in ACS [63]. The balance between vasoconstriction and vasodilation in ACS may be altered. The ET-1 gene is upregulated in the lung and is released by activated lung ECs in response to hypoxia and reduced NO bioavailability [64, 65]. In a transgenic mouse model consistent with chronic organ lesions, tissue lesions, and acute vaso-occlusive events analogous to human SCD, SAD mice [S(β6val) Antilles (β23lle) D-Punjab(β121Gln)] [66], it was found that ET-1 is produced at a higher level in the pulmonary MVEC of SAD mice than wild type (WT) mice. Further, in the SAD mice, bosentan, an ET receptor antagonist, was shown to prevent death of SAD mice exposed to a severe hypoxic challenge [64].

Painful vaso-occlusive crisis (VOC), one of the major and specific manifestations of SCD, is the most debilitating manifestation of SCD [67]. In VOC, the circulation of blood vessels is obstructed by sickled red blood cells causing ischemic injury and severe pain. Sickling and/ or hypoxia associated with VOC in SCD may shift the balance of endothelial vasodilator and vasoconstrictor response in favor of vasoconstriction [68]. The study of Hammerman et al. [63] measured NO products from cultured pulmonary ECs exposed to red blood cells and/or plasma from SCD patients during VOC [63]. Exposure to the plasma from SCD patients during VOC increased total NO production by both macro and microvascular lung ECs [63]. However, these increases were not accompanied by changes in eNOS or iNOS expression. Based on their findings, the authors suggested that altered NO production might contribute to the pathogenesis of ACS [63].

**3. Conclusion**

nary microvasculature.

**Acknowledgements**

**Conflict of interest**

**Research support**

**Author details**

manuscript.

Dr. Brendan Marshall and Libby Perry.

Endothelial cells are an active component of the lung and line the large and small vessels of the lung. They all engage in forming a barrier separation but also are an active constituent in the healthy and diseased lungs. The pulmonary ECs manifest disruption and breakdown under abnormal conditions such as hypoxia and infection and pathologic conditions such as infection, ARDS, and ACS. The ongoing research in pulmonary ECs has highlighted the significance of pulmonary microvascular and macrovascular EC in health and disease with continuing focus toward improving morbidity and mortality of disease involving the pulmo-

Pulmonary Vascular Endothelial Cells http://dx.doi.org/10.5772/intechopen.76995 299

The authors work is generously supported by the Hemoglobinopathy Translational Research Core (HTRC) support (JG) and Dr. Madaio, and the Augusta University Health Medical Center, Department of Medicine (JG), NIH grant HL101902 (ADV). We also thank Dr. Anita Kovacs-Kasa for her work in pulmonary microvascular cell immunocytochemistry figure and the Augusta University Health Vascular Biology Center Electron Microscope Imaging Center,

We declare no author has any disclosure or conflict of interest for any product or result in this

This work was supported by the generous support of Dr. Madaio, and the Augusta University

Health Department of Medicine (JG), NIH grant HL101902 (ADV).

1 Vascular Biology Center, Augusta University Health, Augusta, GA, USA

2 Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine,

Joyce N. Gonzales1,2\* and Alexander D. Verin1,2

Georgia Regents University, Augusta, GA, USA

\*Address all correspondence to: jgonzales@augusta.edu

The vascular inflammation and increased thrombotic activity known to occur in patients with ACS in SCD may be associated with platelet activation of ECs through CD40, a platelet associated pro-inflammatory molecule that promotes ECs activation and is known to be elevated in the circulation of SCD patients [69]. Cluster of differentiation (CD)40, a protein found on antigen-presenting cells and its ligand (L), a protein receptor, are members of the TNF superfamily of molecules. The binding of CD40 to the endothelial cell induces a variety of downstream effects and initiates a variety of immune and inflammatory responses including the production of reactive oxygen species (ROS), chemokines, and cytokines and the expression of adhesion molecules such as E-selectin, ICAM-1 and VCAM-1. The inflammatory response then fosters recruitment of leukocytes around the EC [69]. Furthermore, the ROS generated by CD40L antagonizes NO synthesis and additionally promotes EC dysfunction [69]. A cohort of SCD patients was evaluated for the association of CD40L and inflammation with SCD clinical complications including ACS [69]. It was found that plasma CD40L was associated with ACS and that SCD patients with a lifetime history of ACS presented with significantly higher plasma CD40L than in SCD patients that had never experienced an episode of ACS [70]. Thrombospondin (TSP-1), also a platelet derived protein that activates ECs was found in the same study to correlate with increased ACS ECs activation of cytokines and chemokines [70].

One of the factors that have been identified in ACS is increased adherence between sickled red blood cells (RBC) and ECs [71]. Some investigators interpret abnormal endothelial adhesion as evidence of a pro-inflammatory state [72]. The pro-inflammatory state in SCD is associated with endothelial damage, increased production of ROS, hemolysis, and increased production of pro-inflammatory cytokines [73]. Transgenic SCD mice have been used to study the inflammatory responses that occur in SCD in many organs including the lung. The transgenic mice models have an active inflammatory response similar to human SCD patients [74]. Adhesion molecules VCAM, ICAM and PECAM have been shown to be upregulated in LPS-treated normal and transgenic-treated lungs [74]. IL-6 and NF-kB expressions were also increased in the lungs of transgenic SCD mice suggesting a vigorous inflammatory response with activated macro and microvascular ECs in the lungs [74]. LPS challenge is associated with increased mortality and increased levels of serum and BALF cytokines TNF-α, IL-1β and VCAM-1 in sickle mice compared with control subjects [72].

The role of the lung ECs and their interactions with sickle RBCs depend on multiple factors including the presence of inflammatory cells, cytokines, reactive oxygen species, hypoxic stress and infection that augment sickle cells' and white blood cells' (WBC) adherence to the endothelium. ACS is associated with infections, pneumonia, and fever, and in this setting, there is activation of pro-inflammatory factors such as cytokines that further activate the ECs and promote changes in vascular tone and permeability, anticoagulant-procoagulant balance, and leukocyte trafficking in the lungs of the SCD patient [74].
