The SphK1/S1P axis is a novel pathway in PAH that promotes PASMC proliferation, a major contributor to pulmonary vascular remodeling. Our results suggest that this pathway is a potential therapeutic target in PAH.

Hyperoxia increases reactive oxygen species (ROS) production in vascular endothelium; however, the mechanisms involved in ROS generation are not well characterized. We determined the role and regulation of NAD(P)H oxidase in hyperoxia-induced ROS formation in human pulmonary artery endothelial cells (HPAECs). Exposure of HPAECs to hyperoxia for 1, 3, and 12 h increased the generation of superoxide anion, which was blocked by diphenyleneiodonium but not by rotenone or oxypurinol. Furthermore, hyperoxia enhanced NADPH- and NADH-dependent and superoxide dismutase- or diphenyleneiodonium-inhibitable ROS production in HPAECs. Immunohistocytochemistry and Western blotting revealed the presence of gp91, p67 phox, p22 phox, and p47 phox subcomponents of NADPH oxidase in HPAECs. Transfection of HPAECs with p22 phox antisense plasmid inhibited hyperoxia-induced ROS production. Exposure of HPAECs to hyperoxia activated p38 MAPK and ERK, and inhibition of p38 MAPK and MEK1/2 attenuated the hyperoxia-induced ROS generation. These results suggest a role for MAPK in regulating hyperoxia-induced NAD(P)H oxidase activation in HPAECs.

Barrier dysfunction of pulmonary endothelial monolayer is associated with dramatic cytoskeletal reorganization, activation of actomyosin contractility, and gap formation. The linkage between the microtubule (MT) network and the contractile cytoskeleton has not been fully explored, however, clinical observations suggest that intravenous administration of anti-cancer drugs and MT inhibitors (such as the vinca alkaloids) can lead to the sudden development of pulmonary edema in breast cancer patients. In this study, we investigated the crosstalk between MT and actomyosin cytoskeleton and characterized specific molecular mechanisms of endothelial cells (EC) barrier dysfunction induced by MT inhibitor nocodazole (ND). Our results demonstrate that MT disassembly by ND induced rapid decreases in transendothelial electrical resistance (TER) and actin cytoskeletal remodeling, indicating EC barrier dysfunction. These effects involved ND-induced activation of Rho GTPase. Rho-mediated activation of its downstream target, Rho-kinase, induced phosphorylation of Rho-kinase effector EC MLC phosphatase (MYPT1) at Thr(696) and Thr(850) resulting in MYPT1 inactivation. Phosphatase inhibition leaded to accumulation of diphospho-MLC, which induced acto-myosin polymerization, stress fiber formation and gap formation. Inhibition of Rho-kinase by Y27632 abolished ND-induced MYPT1 phosphorylation, MLC phosphorylation, and stress fiber formation. In addition, MT preservation via the MT stabilizer paclitaxel, Rho inhibition (via C3 exotoxin, or dominant negative (DN)-Rho, or DN-Rho-kinase) attenuated ND-induced TER decreases, stress fiber formation and MLC phosphorylation. Collectively, our results demonstrate a leading role for Rho-dependent mechanisms in crosstalk between the MT and actomyosin cytoskeleton, and suggest Rho-kinase and MYPT1 as major Rho effectors mediating pulmonary EC barrier disruption in response to ND-induced MT disassembly.

In vascular endothelium, the major research focus has been on reactive oxygen species (ROS) derived from Nox2. The role of Nox4 in endothelial signal transduction, ROS production, and cytoskeletal reorganization is not well defined. In this study, we show that human pulmonary artery endothelial cells (HPAECs) and human lung microvascular endothelial cells (HLMVECs) express higher levels of Nox4 and p22 phox compared to Nox1, Nox2, Nox3, or Nox5. Immunofluorescence microscopy and Western blot analysis revealed that Nox4 and p22 phox , but not Nox2 or p47 phox , are localized in nuclei of HPAECs. Further, knockdown of Nox4 with siRNA decreased Nox4 nuclear expression significantly. Exposure of HPAECs to hyperoxia (3-24 h) enhanced mRNA and protein expression of Nox4, and Nox4 siRNA decreased hyperoxia-induced ROS production. Interestingly, Nox4 or Nox2 knockdown with siRNA upregulated the mRNA and protein expression of the other, suggesting activation of compensatory mechanisms. A similar upregulation of Nox4 mRNA was observed in Nox2 Ϫ/Ϫ ko mice. Downregulation of Nox4, or pretreatment with N-acetylcysteine, attenuated hyperoxia-induced cell migration and capillary tube formation, suggesting that ROS generated by Nox4 regulate endothelial cell motility. These results indicate that Nox4 and Nox2 play a physiological role in hyperoxia-induced ROS production and migration of ECs. Antioxid. Redox Signal. 11,[747][748][749][750][751][752][753][754][755][756][757][758][759][760][761][762][763][764]

4-Hydroxy-2-nonenal (4-HNE), one of the major biologically active aldehydes formed during inflammation and oxidative stress, has been implicated in a number of cardiovascular and pulmonary disorders. 4-HNE has been shown to increase vascular endothelial permeability; however, the underlying mechanisms are unclear. Hence, in the current study, we tested our hypothesis that 4-HNE-induced changes in cellular thiol redox status may contribute to modulation of cell signaling pathways that lead to endothelial barrier dysfunction. Exposure of bovine lung microvascular endothelial cells (BLMVECs) to 4-HNE induced reactive oxygen species generation, depleted intracellular glutathione, and altered cell-cell adhesion as measured by transendothelial electrical resistance. Pretreatment of BLMVECs with thiol protectants, N-acetylcysteine and mercaptopropionyl glycine, attenuated 4-HNE-induced decrease in transendothelial electrical resistance, reactive oxygen species generation, Michael protein adduct formation, protein tyrosine phosphorylation, activation of ERK, JNK, and p38 MAPK, and actin cytoskeletal rearrangement. Treatment of BLMVECs with 4-HNE resulted in the redistribution of FAK, paxillin, VE-cadherin, ␤-catenin, and ZO-1, and intercellular gap formation. Western blot analyses confirmed the formation of 4-HNE-derived Michael adducts with the focal adhesion and adherens junction proteins. Also, 4-HNE decreased tyrosine phosphorylation of FAK without affecting total cellular FAK contents, suggesting the modification of integrins, which are natural FAK receptors. 4-HNE caused a decrease in the surface integrin in a time-dependent manner without altering total ␣5 and ␤3 integrins. These results, for the first time, revealed that 4-HNE in redox-dependent fashion affected endothelial cell permeability by modulating cell-cell adhesion through focal adhesion, adherens, and tight junction proteins as well as integrin signal transduction that may lead dramatic alteration in endothelial cell barrier dysfunction during heart infarction, brain stroke, and lung diseases.

Oxygen therapy often rescues and reduces the mortality resulting from acute respiratory distress syndrome, chronic obstructive pulmonary diseases, exposure to toxic fumes, and drowning (1). However, prolonged exposure to supra-physiological concentrations of oxygen, referred to as hyperoxia, causes extensive damage to the alveolar-capillary barrier resulting in increased permeability and decreased lung function (2). Although the molecular mechanisms of hyperoxia-induced lung injury and cell death are complex, recent studies suggest that the generation of excessive reactive oxygen species (ROS), 1 loss of antioxidant defense pathways, cytokine-mediated inflammation, and modulation of signal transduction may regulate pulmonary edema and apoptosis/necrosis of endothelial and epithelial cells (3). The vascular endothelium has long been recognized to generate superoxide (O 2 . ), hydrogen peroxide (H 2 O 2 ), hydroxyl radical ( ⅐ OH), and nitric oxide (NO) via enzymatic and nonenzymatic reactions. In endothelial cells (ECs), in addition to the mitochondrial electron transport, other potential enzymatic pathways of ROS production include cyclooxygenase/lipoxygenase, cytochrome P450, xanthine oxidase, NADPH oxidase, NO synthase, and peroxidase. In the lung, the vascular NADPH oxidase seems to play an important role in excessive production of O 2 . in atherosclerosis, ischemic lung, pulmonary hypertension, and ventilator-associated lung injury (4 -9). NADPH oxidase catalyzes the one-electron reduction of molecular oxygen to O 2 . by using NADPH or NADH as an electron donor (9). Activated NADPH oxidase is a multimeric protein complex consisting of at least three cytosolic subunits of p47 phox , p67 phox , and p40 phox ; a regulatory small molecular weight G-protein of either Rac1 or Rac2 and a membraneassociated cytochrome b 558 reductase made up of p22 phox and gp91 phox . We and others (10, 11) have shown that most of the subcomponents of phagocytic NADPH oxidase are expressed in vascular ECs. ECs exhibit a low output in of O 2. production under basal conditions, and stimulation by TNF-␣, pulsatile stretch, hypoxia reoxygenation, and phorbol ester enhanced the

Sphingosine 1-phosphate (S1P) regulates diverse cellular functions through extracellular ligation to S1P receptors, and it also functions as an intracellular second messenger. Human pulmonary artery endothelial cells (HPAECs) effectively utilized exogenous S1P to generate intracellular S1P. We, therefore, examined the role of lipid phosphate phosphatase (LPP)-1 and sphingosine kinase1 (SphK1) in converting exogenous S1P to intracellular S1P. Exposure of and increased intracellular S1P production by 2-3-fold compared with vector control cells. Down-regulation of LPP-1 by siRNA decreased intracellular S1P production from extracellular S1P but had no effect on the phosphorylation of Sph to S1P. Knockdown of SphK1, but not SphK2, by siRNA attenuated the intracellular generation of S1P. Overexpression of wild type SphK1, but not SphK2 wild type, increased the accumulation of intracellular S1P after exposure to extracellular S1P. These studies provide the first direct evidence for a novel pathway of intracellular S1P generation. This involves the conversion of extracellular S1P to Sph by LPP-1, which facilitates Sph uptake, followed by the intracellular conversion of Sph to S1P by SphK1.Sphingosine 1-phosphate (S1P) 2 is a bioactive lipid mediator that plays an important role in regulating intracellular mobilization of Ca 2ϩ , cytoskeletal reorganization, cell growth, differentiation, motility, angiogenesis, and survival (1-5). In biological fluids such as plasma, S1P is present at 0.2-0.5 M, whereas higher concentrations (1-5 M) in serum are attributed to enhanced release from activated platelets (1, 5). S1P is generated by phosphorylation of free sphingosine (Sph) by two sphingosine kinases (SphKs) 1 and 2, which are highly conserved enzymes present in most of the mammalian cells and tissues (6 -9). Cellular levels of S1P are regulated through its formation via SphKs and by its degradation by S1P lyase (SPL) (10 -12), S1P phosphatases (SPPs) (13-15), and intracellular lipid phosphate phosphatases (LPPs) (16 -18). Platelets lack S1P lyase (19), but in most cells the balance between S1P formation and degradation translates to low basal levels of intracellular S1P. S1P exerts dual actions in cells; it acts as an intracellular second messenger and functions extracellularly as a ligand for a family of five G-protein-coupled receptors formerly known as endothelial differentiation gene (Edg) receptors. To date, five G-protein-coupled receptors, S1P-1 (Edg-1), S1P-2 (Edg-5), S1P-3 (Edg-3), S1P-4 (Edg-6), and S1P-5 (Edg-8), have been identified. All these receptors bind to and are activated by extracellular S1P and dihydro-S1P (1, 5, 20 -22). In the vessel wall extracellular S1P is a potent stimulator of angiogenesis (23, 24) and is a major chemotactic factor for endothelial cells (ECs). Recently, circulating S1P and the immunosuppressive drug FTY720, which is also phosphorylated by SphKs, have been implicated in lymphocyte homing and immunoregulation (25,26). In addition to its extracellular action, S1P functions as an intr...

Transforming growth factor-beta1 (TGF-beta1) is a cytokine critically involved in acute lung injury and endothelial cell (EC) barrier dysfunction. We have studied TGF-beta1-mediated signaling pathways and examined a role of microtubule (MT) dynamics in TGF-beta1-induced actin cytoskeletal remodeling and EC barrier dysfunction. TGF-beta1 (0.1-50 ng/ml) induced dose-dependent decrease in transendothelial electrical resistance (TER) in bovine pulmonary ECs, which was linked to increased actin stress fiber formation, myosin light chain (MLC) phosphorylation, EC retraction, and gap formation. Inhibitor of TGF-beta1 receptor kinase RI (5 microM) abolished TGF-beta1-induced TER decline, whereas inhibitor of caspase-3 zVAD (10 microM) was without effect. TGF-beta1-induced EC barrier dysfunction was linked to partial dissolution of peripheral MT meshwork and decreased levels of stable (acetylated) MT pool, whereas MT stabilization by taxol (5 microM) attenuated TGF-beta1-induced barrier dysfunction and actin remodeling. TGF-beta1 induced sustained activation of small GTPase Rho and its effector Rho-kinase; phosphorylation of myosin binding subunit of myosin specific phosphatase; MLC phosphorylation; EC contraction; and gap formation, which was abolished by inhibition of Rho and Rho-kinase, and by MT stabilization with taxol. Finally, elevation of intracellular cAMP induced by forskolin (50 microM) attenuated TGF-beta1-induced barrier dysfunction, MLC phosphorylation, and protected the MT peripheral network. These results suggest a novel role for MT dynamics in the TGF-beta1-mediated Rho regulation, EC barrier dysfunction, and actin remodeling.