**2.1. Pulmonary endothelial cell heterogeneity**

ECs from various organs have distinctive vascular responses to chemokines, cytokines, and adhesions molecules and exhibit unique functional properties [22, 23]. Phenotypic heterogeneity among ECs may account for important organ-specific behaviors [24]. The unique features of pulmonary ECs allow them to function at multiple basic levels such as function as a dynamic barrier critical for lung gas exchange and the regulation of fluid and solute passage between the blood and interstitial compartments in the lung [25].

Pulmonary endothelium exhibits a high expression of adhesion molecules which contribute to the margination of the large intravascular pool of leucocytes in the lung [26]. Adhesion molecules are expressed by activated ECs in a sequential manner. Cytokines activate E-selectin promoter, which induces E-selectin and a prolonged contact among leukocytes, causing them to roll along the endothelium [27]. Other selectins such as platelet endothelial 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 not comparable to study of the pulmonary microvasculature in vivo or in vitro [27].

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,

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].

M-CSF (macrophage CSF), PDGF, and VEGF [12].

between the blood and interstitial compartments in the lung [25].

**2.1. Pulmonary endothelial cell heterogeneity**

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),

ECs from various organs have distinctive vascular responses to chemokines, cytokines, and adhesions molecules and exhibit unique functional properties [22, 23]. Phenotypic heterogeneity among ECs may account for important organ-specific behaviors [24]. The unique features of pulmonary ECs allow them to function at multiple basic levels such as function as a dynamic barrier critical for lung gas exchange and the regulation of fluid and solute passage

Pulmonary endothelium exhibits a high expression of adhesion molecules which contribute to the margination of the large intravascular pool of leucocytes in the lung [26]. Adhesion molecules are expressed by activated ECs in a sequential manner. Cytokines activate E-selectin promoter, which induces E-selectin and a prolonged contact among leukocytes, causing them to roll along the endothelium [27]. Other selectins such as platelet endothelial

ET-1 and IL-1 and IL-8 [16].

290 Endothelial Dysfunction - Old Concepts and New Challenges

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

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 distinction of macro versus microvascular cells could be important.

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].

In another in vitro study comparing MVEC and PAECs, metabolic requirements for growth were studied in rat pulmonary cells. It was found that PMVEC populations had a higher metabolic function and grow faster than PAEC. PMVEC consumed threefold more glucose in cell culture over comparable time frame than PAECs. PMVECs but not PAECs generated a lactic acidosis, higher ATP concentrations, and lower oxygen consumption than PAECs [34]. The hydraulic conductance of rat PMVEC and PAEC were compared to study lung EC permeability, such as may occur in pulmonary edema to hydraulic stress. The results of these studies indicated dramatic differences in the baseline hydraulic responses to hydrostatic pressure between the two phenotypes with PAEC values averaged 22 times higher than PMVEC in new monolayers. It was speculated that the dramatic differences in PMVEC and RPAEC may be due to different embryologic origins, being derived respectively by vasculogenesis and angiogenesis [35]. The same group investigated the role of cytosolic calcium (Ca2+) in rat PMVECs and PAECs; they found that Thapsigargin (a non-competitive inhibitor of the sarco/ endoplasmic reticulum Ca2+ ATPase) produced higher Ca2+ levels in PAECs than in PMVECs and increased permeability in PAEC but not in PMVEC monolayers. The significance is that whereas increased Ca2+ promotes permeability in PAECs it is not sufficient in PMVECs which show an apparent uncoupling of Ca2+ signaling pathways or dominant Ca2+ − independent

Pulmonary Vascular Endothelial Cells http://dx.doi.org/10.5772/intechopen.76995 293

mechanisms for controlling cellular gap formation and permeability [25].

suggesting that differences between ECs populations may be substantial [24].

**2.2. Pulmonary microvascular endothelial cells**

availability.

Macrovascular ECs more abundantly express eNOS and generate more NO than do microvascular ECs [23, 30]. NOS and inducible NOS (iNOS) production in rat pulmonary macrovascular EC was shown to be greater than rat pulmonary microvascular ECs when cell cultures were stimulated with various combinations of TNF-α, interferon gamma (IFN-), and LPS,

HPAEC are also useful to investigate the effects of various compounds and drugs. Other EC types such as human umbilical artery (HUVEC) or bovine pulmonary artery cells (BPAECs) have also been used previously due to their dependable, more robust nature and commercial

Pulmonary microvascular ECs (MVECs) are an active and dynamic layer of cells in the most delicate portion of the lung at the alveolar level (**Figure 3**) where they function to exert both specific and general endothelial function. In the microvascular circulation, the arteries are less than 70 μm in diameter, are nonmuscular arterioles, and extend into the alveolar capillaries. The walls of capillaries are composed of a single layer of MVECs. The general function of lung EC include regulation of systemic blood flow, tissue perfusion thorough changes in vessel diameter and vascular tone, performed in conjunction with underlying smooth muscle cells and pericytes [6] (**Figure 4**). The pulmonary microcirculation is less permeable to protein and water flux as compared to large pulmonary vessels [26]. Experiments have shown that the MVECs form a tighter barrier compared to the macrovascular barrier while showing less permeability to sucrose and albumin compared to macrovascular EC [36]. Lung injury or inflammation are associated with activation of mediators or secretion of cytokines that induce a prolonged increase in paracellular permeability and vessel wall leakiness. Endothelial barrier properties are known to be strictly dependent on the integrity of endothelial adherens and tight junctions [37]. The

**Figure 2.** Lectin immunocytochemistry is used to identify pulmonary microvascular cells (top, Griffonia) compared to pulmonary macrovascular cells bottom, (*Helix pomatia* Lectin). ECs were cultured at 37°C in complete endothelial growth basal medium-2 until confluent. Endothelial cells were isolated from murine lungs or human pulmonary artery EC were used and stained with either *Top*-*Griffonia* to identify microvascular EC and compared to HPAEC or *Bottom*-*Helix pomatia* stain to identify human pulmonary macrovascular EC.

In another in vitro study comparing MVEC and PAECs, metabolic requirements for growth were studied in rat pulmonary cells. It was found that PMVEC populations had a higher metabolic function and grow faster than PAEC. PMVEC consumed threefold more glucose in cell culture over comparable time frame than PAECs. PMVECs but not PAECs generated a lactic acidosis, higher ATP concentrations, and lower oxygen consumption than PAECs [34]. The hydraulic conductance of rat PMVEC and PAEC were compared to study lung EC permeability, such as may occur in pulmonary edema to hydraulic stress. The results of these studies indicated dramatic differences in the baseline hydraulic responses to hydrostatic pressure between the two phenotypes with PAEC values averaged 22 times higher than PMVEC in new monolayers. It was speculated that the dramatic differences in PMVEC and RPAEC may be due to different embryologic origins, being derived respectively by vasculogenesis and angiogenesis [35]. The same group investigated the role of cytosolic calcium (Ca2+) in rat PMVECs and PAECs; they found that Thapsigargin (a non-competitive inhibitor of the sarco/ endoplasmic reticulum Ca2+ ATPase) produced higher Ca2+ levels in PAECs than in PMVECs and increased permeability in PAEC but not in PMVEC monolayers. The significance is that whereas increased Ca2+ promotes permeability in PAECs it is not sufficient in PMVECs which show an apparent uncoupling of Ca2+ signaling pathways or dominant Ca2+ − independent mechanisms for controlling cellular gap formation and permeability [25].

Macrovascular ECs more abundantly express eNOS and generate more NO than do microvascular ECs [23, 30]. NOS and inducible NOS (iNOS) production in rat pulmonary macrovascular EC was shown to be greater than rat pulmonary microvascular ECs when cell cultures were stimulated with various combinations of TNF-α, interferon gamma (IFN-), and LPS, suggesting that differences between ECs populations may be substantial [24].

HPAEC are also useful to investigate the effects of various compounds and drugs. Other EC types such as human umbilical artery (HUVEC) or bovine pulmonary artery cells (BPAECs) have also been used previously due to their dependable, more robust nature and commercial availability.
