Pulmonary arterial hypertension (PAH) is a lethal disease characterized by elevations in pulmonary arterial pressure, in part due to formation of occlusive lesions in the distal arterioles of the lung. These complex lesions may comprise multiple cell types, including endothelial cells (ECs). To better understand the molecular mechanisms underlying EC dysfunction in PAH, lung microvascular endothelial cells (MVECs) were isolated from normoxic rats (N-MVECs) and rats subjected to SU5416 plus hypoxia (SuHx), an experimental model of PAH. Compared with N-MVECs, MVECs isolated from SuHx rats (SuHx-MVECs) appeared larger and more spindle shaped morphologically and expressed canonical smooth muscle cell markers smooth muscle-specific α-actin and myosin heavy chain in addition to endothelial markers such as Griffonia simplicifolia and von Willebrand factor. SuHx-MVEC mitochondria were dysfunctional, as evidenced by increased fragmentation/fission, decreased oxidative phosphorylation, and increased reactive oxygen species (ROS) production. Functionally, SuHx-MVECs exhibited increased basal levels of intracellular calcium concentration ([Ca]) and enhanced migratory and proliferative capacity. Treatment with global (TEMPOL) or mitochondria-specific (MitoQ) antioxidants decreased ROS levels and basal [Ca] in SuHx-MVECs. TEMPOL and MitoQ also decreased migration and proliferation in SuHx-MVECs. Additionally, inhibition of ROS-induced Ca entry via pharmacologic blockade of transient receptor potential vanilloid-4 (TRPV4) attenuated [Ca], migration, and proliferation. These findings suggest a role for mitochondrial ROS-induced Ca influx via TRPV4 in promoting abnormal migration and proliferation in MVECs in this PAH model.
In acute respiratory distress syndrome, both reactive oxygen species (ROS) and increased intracellular calcium ([Ca 2ϩ ]i) are thought to play important roles in promoting endothelial paracellular permeability, but the mechanisms linking ROS and [Ca 2ϩ ]i in microvascular endothelial cells are not known. In this study, we assessed the effect of hydrogen peroxide (H2O2) on [Ca 2ϩ ]i in mouse and human lung microvascular endothelial cells (MLMVEC and HLMVEC, respectively). We found that in both MLMVECs and HLMVECs, exogenously applied H2O2 increased [Ca 2ϩ ]i through Ca 2ϩ influx and that pharmacologic inhibition of the calcium channel transient receptor potential vanilloid 4 (TRPV4) attenuated the H2O2-induced Ca 2ϩ influx. Additionally, knockdown of TRPV4 in HLMVEC also attenuated calcium influx following H2O2 challenge. Administration of H2O2 or TRPV4 agonists decreased transmembrane electrical resistance (TER), suggesting increased barrier permeability. To explore the regulatory mechanisms underlying TRPV4 activation by ROS, we examined H2O2-induced Ca 2ϩ influx in MLMVECs and HLMVECs with either genetic deletion, silencing, or pharmacologic inhibition of Fyn, a Src family kinase. In both MLMVECs derived from mice deficient for Fyn and HLMVECs treated with either siRNA targeted to Fyn or the Src family kinase inhibitor SU-6656 for 24 or 48 h, the H 2O2-induced Ca 2ϩ influx was attenuated. Treatment with SU-6656 decreased the levels of phosphorylated, but not total, TRPV4 protein and had no effect on TRPV4 response to the external agonist, GSK1016790A. In conclusion, our data suggest that application of exogenous H2O2 increases [Ca 2ϩ ]i and decreases TER in microvascular endothelial cells via activation of TRPV4 through a mechanism that requires the Src kinase Fyn.
(HV T) ventilation causes pulmonary endothelial barrier dysfunction. HV T ventilation also increases lung nitric oxide (NO) and cGMP. NO contributes to HV T lung injury, but the role of cGMP is unknown. In the current study, ventilation of isolated C57BL/6 mouse lungs increased perfusate cGMP as a function of V T. Ventilation with 20 ml/kg V T for 80 min increased the filtration coefficient (K f), an index of vascular permeability. The increased cGMP and Kf caused by HVT were attenuated by nitric oxide synthase (NOS) inhibition and in lungs from endothelial NOS knockout mice. Inhibition of soluble guanylyl cyclase (sGC) in wild-type lungs (10 M ODQ) also blocked cGMP generation and inhibited the increase in K f, suggesting an injurious role for sGC-derived cGMP. sGC inhibition also attenuated lung Evans blue dye albumin extravasation and wetto-dry weight ratio in an anesthetized mouse model of HV T injury. Additional activation of sGC (1.5 M BAY 41-2272) in isolated lungs at 40 min increased cGMP production and K f in lungs ventilated with 15 ml/kg V T. HVT endothelial barrier dysfunction was attenuated with a nonspecific phosphodiesterase (PDE) inhibitor (100 M IBMX) as well as an inhibitor (10 M BAY 60-7550) specific for the cGMPstimulated PDE2A. Concordantly, we found a V T-dependent increase in lung cAMP hydrolytic activity and PDE2A protein expression with a decrease in lung cAMP concentration that was blocked by BAY 60-7550. We conclude that HV T-induced endothelial barrier dysfunction resulted from a simultaneous increase in NO/sGC-derived cGMP and PDE2A expression causing decreased cAMP. nitric oxide; guanosine 3Ј,5Ј-cyclic monophosphate; phosphodiesterase 2A; adenosine 3Ј,5Ј-cyclic monophosphate; atrial natriuretic peptide; endothelial permeability MECHANICAL VENTILATION is necessary to support patients with the acute respiratory distress syndrome. However, mechanical ventilation also contributes to lung injury by a mechanism that directly correlates with tidal volume (V T ) (39). High V T (HV T ) ventilation causes pulmonary endothelial barrier dysfunction and edema, leading to ventilator-induced lung injury (VILI) (42). The mechanisms behind this injury are complex, but it is evident that HV T -induced changes in intracellular signaling play a significant role (12).Recent evidence from animal models of VILI implicates nitric oxide (NO) in the pathogenesis of HV T endothelial barrier dysfunction (4,6,29). NO is synthesized by three isoforms of nitric oxide synthase (NOS): endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS. Ventilatory lung stretch stimulates pulmonary microvascular NO production by an increase in eNOS expression (6), phosphorylation of eNOS by phosphatidylinositol 3-kinase-induced protein kinase B activation (22), or induction of iNOS expression (13,29). Once produced by NOS, NO reacts with reactive oxygen species (ROS) to generate peroxynitrite, a toxic free radical that contributes to VILI (29). NO can also stimulate endothelial soluble guanylyl cyclase (sGC) to generat...
Oxidant injury contributes to acute lung injury (ALI). We previously reported that activation of protein kinase GI (PKGI) posttranscriptionally increased the key antioxidant enzymes catalase and glutathione peroxidase 1 (Gpx-1) and attenuated oxidant-induced cytotoxicity in mouse lung microvascular endothelial cells (MLMVEC). The present studies tested the hypothesis that the antioxidant effect of PKGI is mediated via inhibition of the c-Abl tyrosine kinase. We found that activation of PKGI with the cGMP analog 8pCPT-cGMP inhibited c-Abl activity and decreased c-Abl expression in wild-type but not PKGI(-/-) MLMVEC. Treatment of wild-type MLMVEC with atrial natriuretic peptide also inhibited c-Abl activation. Moreover, treatment of MLMVEC with the c-Abl inhibitor imatinib increased catalase and GPx-1 protein in a posttranscriptional fashion. In imatinib-treated MLMVEC, there was no additional effect of 8pCPT-cGMP on catalase or GPx-1. The imatinib-induced increase in antioxidant proteins was associated with an increase in extracellular H2O2 scavenging by MLMVEC, attenuation of oxidant-induced endothelial barrier dysfunction, and prevention of oxidant-induced endothelial cell death. Finally, in the isolated perfused lung, imatinib prevented oxidant-induced endothelial toxicity. We conclude that cGMP, through activation of PKGI, inhibits c-Abl, leading to increased key antioxidant enzymes and resistance to lung endothelial oxidant injury. Inhibition of c-Abl by active PKGI may be the downstream mechanism underlying PKGI-mediated antioxidant signaling. Tyrosine kinase inhibitors may represent a novel therapeutic approach in oxidant-induced ALI.
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