**2.2. Pulmonary microvascular endothelial cells**

**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.

292 Endothelial Dysfunction - Old Concepts and New Challenges

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

The lung is one of the body's organs with the highest expression of VEGF [39]. VEGF stimulates small vessel formation of MVEC and is an essential component of the angiogenic process and MVECs survival. VEGF signaling orchestrates capillary development along the basement membrane of airway epithelium [23]. Excessive VEGF expression has a broad impact on ECs including vascular permeability increase [39]. VEGF can be induced by hypoxia-inducible factor (HIF) in cells under hypoxic conditions. Gene studies evaluating VEGF have shown a higher level of expression of actin binding proteins—Lin11, Isl-1 and Mec-3 (LIM) proteins 1, actinin-associated LIM protein, Arginase (Arg) binding protein 2, Slingshot, vav3, myosin IB, myosin 5C, myosin7A, and myosin light chain kinase in the microvascular ECs [3, 40]. These are proteins that play important roles in basic biological processes including cytoskeleton organization. This increase in cytoskeletal protein expression may be related to the ability of MVECs to undergo extensive cytoskeletal remodeling and migration during angiogenesis [3]. VEGF increases permeability by at least two different pathways: one, involving proto-oncogene (Raf-1), mitogen-activated protein kinase/ERK kinase (MEK), and extracellular signalregulated kinases 1,2 (ERK); and the other involving endothelial nitric oxide synthase (eNOS). Protein kinase C (PKC) is a mediator of VEGF-induced ERK-1/2 phosphorylation and hyperpermeability which increases permeability via increased NO production [33]. Endothelin-1 (ET-1) is known to play a pathogenic role in pulmonary arterial hypertension (PAH). VEGF may have beneficial effects by decreasing ET-1 production in HLMVEC thereby modulating

Pulmonary Vascular Endothelial Cells http://dx.doi.org/10.5772/intechopen.76995 295

Microvascular ECs are known to produce macrophage inflammatory protein-1beta (MIP-1β) and MIP-2 (the mouse equivalent to human interleukin-8) in the lung which act as major chemotactic factors responsible for the recruitment of neutrophils into the alveolar spaces during

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

endothelin production in PAH [41].

inflammation or infection [22, 41].

critical in the EC barrier regulation.

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

**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).

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

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

The lung is one of the body's organs with the highest expression of VEGF [39]. VEGF stimulates small vessel formation of MVEC and is an essential component of the angiogenic process and MVECs survival. VEGF signaling orchestrates capillary development along the basement membrane of airway epithelium [23]. Excessive VEGF expression has a broad impact on ECs including vascular permeability increase [39]. VEGF can be induced by hypoxia-inducible factor (HIF) in cells under hypoxic conditions. Gene studies evaluating VEGF have shown a higher level of expression of actin binding proteins—Lin11, Isl-1 and Mec-3 (LIM) proteins 1, actinin-associated LIM protein, Arginase (Arg) binding protein 2, Slingshot, vav3, myosin IB, myosin 5C, myosin7A, and myosin light chain kinase in the microvascular ECs [3, 40]. These are proteins that play important roles in basic biological processes including cytoskeleton organization. This increase in cytoskeletal protein expression may be related to the ability of MVECs to undergo extensive cytoskeletal remodeling and migration during angiogenesis [3]. VEGF increases permeability by at least two different pathways: one, involving proto-oncogene (Raf-1), mitogen-activated protein kinase/ERK kinase (MEK), and extracellular signalregulated kinases 1,2 (ERK); and the other involving endothelial nitric oxide synthase (eNOS). Protein kinase C (PKC) is a mediator of VEGF-induced ERK-1/2 phosphorylation and hyperpermeability which increases permeability via increased NO production [33]. Endothelin-1 (ET-1) is known to play a pathogenic role in pulmonary arterial hypertension (PAH). VEGF may have beneficial effects by decreasing ET-1 production in HLMVEC thereby modulating endothelin production in PAH [41].

Microvascular ECs are known to produce macrophage inflammatory protein-1beta (MIP-1β) and MIP-2 (the mouse equivalent to human interleukin-8) in the lung which act as major chemotactic factors responsible for the recruitment of neutrophils into the alveolar spaces during inflammation or infection [22, 41].
