**2. Pulmonary endothelial cell dysfunction**

Vascular endothelium is a critical regulator of vascular homeostasis. All EC inhibit coagulation of the blood. ECs bind tissue factor pathway inhibitors (TFPIs) that prevent the initiation of coagulation by blocking the actions of the factor (f) VIIa tissue factor (TF) complex [2]. Like other ECs, lung ECs modulate hemostasis with sometimes opposing effects such as antiplatelet, anticoagulant, and fibrinolytic properties; yet after injury or activation ECs are capable of exerting procoagulant functions. The balance between endothelial anti and prothrombotic activities determines whether thrombus formation, propagation, or dissolution occurs [3]. An intact endothelium in a healthy vessel inhibits the adhesion of platelets, platelet activation, and aggregation and adhesion of platelets and leucocytes to vessel wall through the release of nitric oxide (NO) [4]. On the other hand, injury or activation of ECs results in a procoagulant phenotype that contributes to localized clot formation.

Activation of the coagulation cascade is one of the early occurring events in lung injury, and it is initiated via the extrinsic pathway [5]. Endothelium activated by inflammation and/or injury releases the procoagulant molecule TF which binds with circulating coagulation fVII to form a TF/fVIIa complex that cleaves fIX and thrombin. (**Figure 1**). Thrombin further activates platelets

**Figure 1.** Schematic representation of pulmonary microvascular endothelial cells (MVEC) activated by inflammation and/or injury following release of the pro-coagulant molecule TF which binds with circulating coagulation factor VII (fVII) to form a TF/fVIIa complex that cleaves fIX, and generates thrombin.

and coagulation factors in the intrinsic coagulation pathway generating more thrombin and formation of a fibrin mesh. The adhesion of platelets is facilitated by von Willebrand factor (vWF). vWF is a product of normal EC and is not synthesized after endothelial injury. The clotting pathway is also induced by cytokines such as tumor necrosis factor alpha (TNF-α) or interleukin 1 (IL-1) or bacterial endotoxin such as lipopolysaccharide (LPS) to secrete TF which activates the extrinsic clotting pathway [3]. In a tightly regulated system, the proteases and molecules of the coagulation cascade can be inhibited by circulating protease inhibitors, such as antithrombin, heparin cofactor II, TF pathway inhibitor and C1 inhibitor. These bind with the active sites of proteases, thereby inactivating them. In addition, coagulation factors can be degraded through activation of the protein C and protein S complex, synthesized by ECs as a cofactor that is then catalyzed by the presence of thrombomodulin and endothelial protein C receptor (EPCR). Other pathways of coagulation factor degradation are disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13). ADAMTS13 cleaves the multimeric strands of vWF, thereby disrupting platelet adhesion. ECs synthesize tissue plasminogen activator (t-PA), promoting fibrinolytic activity to clear fibrin deposits from endothelial surfaces [3, 6]. Thrombin also binds to its protease activated receptor-1 (PAR-1) and induces a signaling cascade resulting in EC junctional gaps that lead to increased endothelial permeability [7].

to the smallest capillaries, thereby accommodating various levels of blood flow from the turbulent high pressures from large vessels entering and leaving the heart as well as small vessels such as that of the minute capillaries of the lungs, liver, kidneys, and the moderate vessels throughout the body. ECs from different blood vessels and microvascular ECs from different tissues have distinct and characteristic gene expression profiles. Pervasive differences in gene expression pat-

Vascular endothelium is a critical regulator of vascular homeostasis. All EC inhibit coagulation of the blood. ECs bind tissue factor pathway inhibitors (TFPIs) that prevent the initiation of coagulation by blocking the actions of the factor (f) VIIa tissue factor (TF) complex [2]. Like other ECs, lung ECs modulate hemostasis with sometimes opposing effects such as antiplatelet, anticoagulant, and fibrinolytic properties; yet after injury or activation ECs are capable of exerting procoagulant functions. The balance between endothelial anti and prothrombotic activities determines whether thrombus formation, propagation, or dissolution occurs [3]. An intact endothelium in a healthy vessel inhibits the adhesion of platelets, platelet activation, and aggregation and adhesion of platelets and leucocytes to vessel wall through the release of nitric oxide (NO) [4]. On the other hand, injury or activation of ECs results in a procoagulant

Activation of the coagulation cascade is one of the early occurring events in lung injury, and it is initiated via the extrinsic pathway [5]. Endothelium activated by inflammation and/or injury releases the procoagulant molecule TF which binds with circulating coagulation fVII to form a TF/fVIIa complex that cleaves fIX and thrombin. (**Figure 1**). Thrombin further activates platelets

**Figure 1.** Schematic representation of pulmonary microvascular endothelial cells (MVEC) activated by inflammation and/or injury following release of the pro-coagulant molecule TF which binds with circulating coagulation factor VII

terns distinguish the EC of large vessels from microvascular ECs [1].

**2. Pulmonary endothelial cell dysfunction**

288 Endothelial Dysfunction - Old Concepts and New Challenges

phenotype that contributes to localized clot formation.

(fVII) to form a TF/fVIIa complex that cleaves fIX, and generates thrombin.

Lung ECs also regulate the synthesis and metabolism of vasoactive compounds such as nitric oxide (NO) and endothelin-1 (ET-1), potent regulators of pulmonary vascular tone [8]. EC-derived NO, synthesized by the endothelial nitric oxide synthase (eNOS) from the precursor L-arginine, regulates the healthy endothelium. Antithrombotic effects of EC-derived NO are likely related to release of prostaglandin I<sup>2</sup> and inhibition of plasminogen activator inhibitor-1 (PAI-1), a prothrombotic protein [6, 9].

The enzyme eNOS depends on intracellular calcium (Ca2+) level. In response to a rise in EC intracellular Ca2+ eNOS catalyzes the production of NO. The Ca2+-dependent eNOS synthesizes small amounts of NO until the Ca2+ levels decrease. This Ca2+-dependent eNOS provides the basal release of NO and is sufficient to inhibit the adhesion and activation of platelets providing homeostasis in unstimulated ECs [10].

Cytokines are small soluble proteins that are important in cell signaling and can change the behavior or properties of cells. Cytokines can be secreted by many cells including pulmonary ECs [11]. Cytokines can be grouped into families including the interferons, the chemoattractants (chemokines), the tumor necrosis factors (TNFs), the interleukins (IL-2, IL-3, IL-4 etc.), the epidermal growth factor family (EGF) and transforming growth factors-alpha and beta (TGF-α and β), the growth factors include vascular endothelial growth factor (VEGF and others) that are important in vasculogenesis and angiogenesis. The VEGF family of growth factors restores the oxygen supply to tissues in hypoxic conditions [12].

Pulmonary EC express adhesion molecules and pro and anti-inflammatory cytokines and are intricately involved in inflammatory processes [12, 13]. It was shown that there is a central role via the sphingosine-1-phosphate (S1P) receptor in pulmonary endothelium for regulating an excessive pro-inflammatory cytokine and chemokine production in an influenza virusinduced cytokine storm [14]. A deficiency of alpha 1-antitrypsin (A1AT), a protein that has been shown to trigger an inflammatory response leading to increased circulating concentrations of pro-inflammatory cytokines such as TNF-α from ECs. In A1AT, cytokines activate their receptors and stimulate nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) activity and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor (IkBα), a regulatory protein that inhibits NF-kB degradation. The translocation of NF-kB increases the transcription of inflammatory genes, including an increased secretion of TNF-α from ECs [15]. The role of pulmonary EC cytokines has also been shown in the pathology of lung fibrosis where numerous cytokines have been implicated in pathogenesis including TGF-β, TNF-α, ET-1 and IL-1 and IL-8 [16].

cell adhesion molecule (PECAM, CD31), ICAM-1, and the inducible VCAM-1 are all essential to the subsequent firm attachment of leukocytes to and migration through the endothelium. Many studies of lung EC expression of molecules, including adhesion molecules, were initially accomplished on human pulmonary artery EC (HPAEC) or even human umbilical vein EC (HUVEC) due to the availability of these cells for culture. However, more recently there is a recognition that there are differences between the expression of pulmonary macrovascular and microvascular EC. For example, the study of adhesion molecules of macrovascular large vessel cells cannot be extrapolated to microvascular capillary cells. In vitro study of HPAEC is

Pulmonary Vascular Endothelial Cells http://dx.doi.org/10.5772/intechopen.76995 291

not comparable to study of the pulmonary microvasculature in vivo or in vitro [27].

different patterns of crosstalk between Rho, p38 MAPK, and NFκB signaling [28].

tion of macro versus microvascular cells could be important.

The distinction of macro or microvascular endothelium is between those from the larger vessels to the small capillaries that feed the entire alveolar system in the lung [29]. Lectin-binding pattern discriminates between PAEC and Pulmonary microvascular EC (MVEC), and lectin protein agglutinins isolated from plant or animal sources are often used for distinguishing between cell phenotypes [23, 30]. It was identified that *Helix pomatia* (an agglutinin from the *Helix pomatia* snail) (HPA) and *Griffonia* lectins (a lectin from Bandeiraea simplicifolia (BS I), these lectins are isolated from a variety of natural sources including plants, mollusks, fish eggs) differentially bind to macro and microvascular EC. HPA preferentially binds macrovascular endothelium whereas BS I preferentially binds to microvascular endothelium (**Figure 2**) [31]. In cell culture experiments investigating diseases such as acute respiratory distress syndrome (ARDS), pulmonary edema, or acute chest syndrome in sickle cell disease (SCD), the distinc-

Majority of studies in the lung have been performed on macrovascular ECs from the HPAEC, bovine PAEC or human umbilical AEC. HPAEC are used to study various diseases of the lung involved in endothelial dysfunction such as hypoxia, inflammation, and environmental stresses. It was shown in one study in macrovascular HPAEC that thrombin induces protein kinase C (PKC)-dependent ezrin, moesin, radixin (ERM) phosphorylation on critical threonine residues ERM and translocation of phosphorylated ERM to the EC periphery and that the ERM proteins play differential roles in thrombin-induced modulation of EC permeability [32]. The results in this study are important to the knowledge of the EC barrier in the lung diseases; however, the critical EC in ARDS, pulmonary edema, and ACS are the pulmonary microvascular endothelial cells (PMVEC), and it is unclear if the results would be similar as our understanding of the molecular regulation of PMVEC permeability is incomplete [33].

Gram-positive bacterial pathogens cause lung inflammation and alterations in lung ECs. In macrovascular ECs, pharmacological inhibition of Rho kinase with the Rho kinase (ROCK) inhibitor Y27632 significantly suppressed p38 mitogen-activated protein kinase (MAPK) cascade activation, while inhibition of p38 MAPK with specific inhibitor p38α and β, SB203580 had no effect on Rho activation [28]. In contrast, inhibition of p38 MAPK in microvascular ECs suppressed lipoteichoic acid and peptidoglycan (LTA/PepG), found on the cell wall of Grampositive bacteria induced activation of Rho, while Rho inhibitor suppressed activation of p38 MAPK [28]. These results demonstrate cell type-specific differences in signaling induced by *Staphylococcus aureus* derived pathogens in pulmonary endothelium. Thus, although Grampositive bacterial compounds caused barrier dysfunction in both ECs types, it was induced by

ECs participate in the control of the adhesion and migration of inflammatory cells and the exchange of fluid from the vasculature into damaged tissue. Resting ECs do not interact with leukocytes; however, they store proteins such as P-selectin and chemokines in specialized secretory vesicles called Weibel-Palade bodies (WPBs) in microvascular ECs for interaction with leukocytes when needed [17]. ICAM-1 and E-selectin mediate the firm adhesion to ECs and are an obligatory step in neutrophil migration as neutrophils initially adhere to ECs, then migrate through the EC barrier [18]. Resting ECs also suppress the transcription of other adhesion molecules such as E-selectin, vascular cell-adhesion molecule 1 (VCAM) and intercellular adhesion molecule 1 (ICAM-1) [2]. Upon EC activation, the resulting inflammation is characterized by tissue infiltration of neutrophils, followed by macrophages [19, 20].

EC activation can be induced by endotoxin, cytokines and chemokines, and viruses and bacterial pathogens, all of which can activate NF-kB resulting in modulation of EC synthesis of pro-inflammatory cytokines and chemokines. When stimulated, the endothelium displays increased adhesiveness for monocytes, lymphocytes, and granulocytes mediated by endothelial leukocyte adhesion molecules such as ICAM-1, VCAM-1 and E-selectin. The secretion of inflammatory cytokines and of leukocyte specific chemo attractants such al IL-8 and MCP-1 also contributes to leukocyte recruitment during inflammatory responses [21]. In general, human endothelium can express a broad spectrum of pro-and anti-inflammatory cytokines, including IL-1, IL-5, IL-6 and IL-8, MCP-1 (monocyte chemotactic protein-1), CSFs (colonystimulating factors), GM-CSF (granulocyte/macrophage CSF), G-CSF (granulocyte CSF), M-CSF (macrophage CSF), PDGF, and VEGF [12].
