Pulmonary Vascular Endothelial Cells

Pulmonary vascular endothelial cells (ECs) line the surface of the lung vasculature and accommodate the various levels of blood flow. Pulmonary endothelium is a critical regulator of vascular homeostasis by inhibiting coagulation of the blood. The ECs bind tissue factor pathway inhibitors (TFPI), modulate hemostasis with opposing effects such as antiplatelet, anticoagulant and fibrinolytic properties. Lung endothelium regulates synthesis and metabolism of vasoactive compounds such as nitric oxide and endothelin-1, both potent regulators of vascular tone. Cytokines, chemokines, interleukins, adhesion molecules, and growth factors can be secreted by pulmonary ECs with positive and adverse effects. Pulmonary endothelium exhibits heterogeneity with diverse expression of molecules and specific differences in signaling induced by various infections such as Gram-positive bacteria. The distinction of macro or microvascular endothelium occurs from the larger vessels to small capillaries in the lung alveoli system. Lectin-binding patterns discriminate between pulmonary artery and pulmonary microvascular capillary endothelium. The lung is one of the body’s organs with the highest expression of vascular endothelial growth factor that stimulates small vessel formation of the microvascular endothelium. Acute respiratory distress syndrome and acute chest syndrome in sickle cell disease are two prototypes of devastating diseases caused by pulmonary EC dysfunction

Interplay of myosin phosphatase and protein phosphatase-2A in the regulation of endothelial nitric-oxide synthase phosphorylation and nitric oxide production

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Adenosine A1 Receptors Promote Vasa Vasorum Endothelial Cell Barrier Integrity via Gi_{i} and Akt-Dependent Actin Cytoskeleton Remodeling

Background: In a neonatal model of hypoxic pulmonary hypertension, a dramatic pulmonary artery adventitial thickening, accumulation of inflammatory cells in the adventitial compartment, and angiogenic expansion of the vasa vasorum microcirculatory network are observed. These pathophysiological responses suggest that rapidly proliferating vasa vasorum endothelial cells (VVEC) may exhibit increased permeability for circulating blood cells and macromolecules. However, the molecular mechanisms underlying these observations remain unexplored. Some reports implicated extracellular adenosine in the regulation of vascular permeability under hypoxic and inflammatory conditions. Thus, we aimed to determine the role of adenosine in barrier regulation of VVEC isolated from the pulmonary arteries of normoxic (VVEC-Co) or chronically hypoxic (VVEC-Hyp) neonatal calves. Principal Findings We demonstrate via a transendothelial electrical resistance measurement that exogenous adenosine significantly enhanced the barrier function in VVEC-Co and, to a lesser extent, in VVEC-Hyp. Our data from a quantitative reverse transcription polymerase chain reaction show that both VVEC-Co and VVEC-Hyp express all four adenosine receptors (A1, A2A, A2B, and A3), with the highest expression level of A1 receptors (A1Rs). However, A1R expression was significantly lower in VVEC-Hyp compared to VVEC-Co. By using an A1R-specific agonist/antagonist and siRNA, we demonstrate that A1Rs are mostly responsible for adenosine-induced enhancement in barrier function. Adenosine-induced barrier integrity enhancement was attenuated by pretreatment of VVEC with pertussis toxin and GSK690693 or LY294002, suggesting the involvement of G$_{i}$ proteins and the PI3K-Akt pathway. Moreover, we reveal a critical role of actin cytoskeleton in VVEC barrier regulation by using specific inhibitors of actin and microtubule polymerization. Further, we show that adenosine pretreatment blocked the tumor necrosis factor alpha (TNF-α)-induced permeability in VVEC-Co, validating its anti-inflammatory effects. Conclusions: We demonstrate for the first time that stimulation of A1Rs enhances the barrier function in VVEC by activation of the G$_{i}$/PI3K/Akt pathway and remodeling of actin microfilament

Ezrin/radixin/moesin proteins differentially regulate endothelial hyperpermeability after thrombin

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The Protective Role of MLCP-Mediated ERM Dephosphorylation in Endotoxin-Induced Lung Injury in Vitro and in Vivo

The goal of this study was to investigate the role of MLC phosphatase (MLCP) in a LPS model of acute lung injury (ALI). We demonstrate that ectopic expression of a constitutively-active (C/A) MLCP regulatory subunit (MYPT1) attenuates the ability of LPS to increase endothelial (EC) permeability. Down-regulation of MYPT1 exacerbates LPS-induced expression of ICAM1 suggesting an anti-inflammatory role of MLCP. To determine whether MLCP contributes to LPS-induced ALI in vivo, we utilized a nanoparticle DNA delivery method to specifically target lung EC. Expression of a C/A MYPT1 reduced LPS-induced lung inflammation and vascular permeability. Further, increased expression of the CS1β (MLCP catalytic subunit) also reduced LPS-induced lung inflammation, whereas the inactive CS1β mutant increased vascular leak. We next examined the role of the cytoskeletal targets of MLCP, the ERM proteins (Ezrin/Radixin/Moesin), in mediating barrier dysfunction. LPS-induced increase in EC permeability was accompanied by PKC-mediated increase in ERM phosphorylation, which was more prominent in CS1β depleted cells. Depletion of Moesin and Ezrin, but not Radixin attenuated LPS-induced increases in permeability. Further, delivery of a Moesin phospho-null mutant into murine lung endothelium attenuated LPS-induced lung inflammation and vascular leak suggesting that MLCP opposes LPS-induced ALI by mediating the dephosphorylation of Moesin and Ezrin

PKC-Dependent Phosphorylation of eNOS at T495 Regulates eNOS Coupling and Endothelial Barrier Function in Response to G(+) -Toxins

Gram positive (G(+)) infections make up similar to 50% of all acute lung injury cases which are characterized by extensive permeability edema secondary to disruption of endothelial cell (EC) barrier integrity. A primary cause of increased permeability are cholesterol-dependent cytolysins (CDCs) of G(+)-bacteria, such as pneumolysin (PLY) and listeriolysin-O (LLO) which create plasma membrane pores, promoting Ca2+-influx and activation of PKC alpha. In human lung microvascular endothelial cells (HLMVEC), pretreatment with the nitric oxide synthase (NOS) inhibitor, ETU reduced the ability of LLO to increase microvascular cell permeability suggesting an endothelial nitric oxide synthase (eNOS)-dependent mechanism. LLO stimulated superoxide production from HLMVEC and this was prevented by silencing PKC alpha or NOS inhibition suggesting a link between these pathways. Both LLO and PLY stimulated eNOS T495 phosphorylation in a PKC-dependent manner. Expression of a phosphomimetic T495D eNOS (human isoform) resulted in increased superoxide and diminished nitric oxide (NO) production. Transduction of HLMVEC with an active form of PKC alpha resulted in the robust phosphorylation of T495 and increased peroxynitrite production, indicative of eNOS uncoupling. To determine the mechanisms underlying eNOS uncoupling, HLMVEC were stimulated with LLO and the amount of hsp90 and caveolin-1 bound to eNOS determined. LLO stimulated the dissociation of hsp90, and in particular, caveolin-1 from eNOS. Both hsp90 and caveolin-1 have been shown to influence eNOS uncoupling and a peptide mimicking the scaffolding domain of caveolin-1 blocked the ability of PKC alpha to stimulate eNOS-derived superoxide. Collectively, these results suggest that the G(+) pore-forming toxins promote increased EC permeability via activation of PKC alpha, phosphorylation of eNOS-T495, loss of hsp90 and caveolin-1 binding which collectively promote eNOS uncoupling and the production of barrier disruptive superoxide

Heat Shock Protein 90 Inhibitors Prevent LPS-Induced Endothelial Barrier Dysfunction by Disrupting Rhoa Signaling

Permeability of the endothelial monolayer is increased when exposed to the bacterial endotoxin LPS. Our previous studies have shown that heat shock protein (Hsp) 90 inhibitors protect and restore LPS-mediated hyperpermeability in bovine pulmonary arterial endothelial cells. In this study, we assessed the effect of Hsp90 inhibition against LPS-mediated hyperpermeability in cultured human lung microvascular endothelial cells (HLMVECs) and delineated the underlying molecular mechanisms. We demonstrate that Hsp90 inhibition is critical in the early phase, to prevent LPS-mediated hyperpermeability, and also in the later phase, to restore LPS-mediated hyperpermeability in HLMVECs. Because RhoA is a well known mediator of endothelial hyperpermeability, we investigated the effect of Hsp90 inhibition on LPS-mediated RhoA signaling. RhoA nitration and activity were increased by LPS in HLMVECs and suppressed when pretreated with the Hsp90 inhibitor, 17-allylamino-17 demethoxy-geldanamycin (17-AAG). In addition, inhibition of Rho kinase, a downstream effector of RhoA, protected HLMVECs from LPS-mediated hyperpermeability and abolished LPS-induced myosin light chain (MLC) phosphorylation, a target of Rho kinase. In agreement with these findings, 17-AAG or dominant-negative RhoA attenuated LPS-induced MLC phosphorylation. MLC phosphorylation induced by constitutively active RhoA was also suppressed by 17-AAG, suggesting a role for Hsp90 downstream of RhoA. Inhibition of Src family kinases also suppressed RhoA activity and MLC phosphorylation. Together, these data indicate that Hsp90 inhibition prevents and repairs LPS-induced lung endothelial barrier dysfunction by suppressing Src-mediated RhoA activity and signaling