In the present study, we tested the hypothesis that chronic inflammation and oxidative/nitrosative stress induce caveolin 1 (Cav-1) degradation, providing an underlying mechanism of endothelial cell activation/dysfunction and pulmonary vascular remodeling in patients with idiopathic pulmonary arterial hypertension (IPAH). We observed reduced Cav-1 protein despite increased Cav-1 messenger RNA expression and also endothelial nitric oxide synthase (eNOS) hyperphosphorylation in human pulmonary artery endothelial cells (PAECs) from patients with IPAH. In control human lung endothelial cell cultures, tumor necrosis factor α-induced nitric oxide (NO) production and S-nitrosation (SNO) of Cav-1 Cys-156 were associated with Src displacement and activation, Cav-1 Tyr-14 phosphorylation, and destabilization of Cav-1 oligomers within 5 minutes that could be blocked by eNOS or Src inhibition. Prolonged stimulation (72 hours) with NO donor DETANONOate reduced oligomerized and total Cav-1 levels by 40%-80%, similar to that observed in IPAH patient-derived PAECs. NO donor stimulation of endothelial cells for >72 hours, which was associated with sustained Src activation and Cav-1 phosphorylation, ubiquitination, and degradation, was blocked by NOS inhibitor L-NAME, Src inhibitor PP2, and proteosomal inhibitor MG132. Thus, chronic inflammation, sustained eNOS and Src signaling, and Cav-1 degradation may be important causal factors in the development of IPAH by promoting PAEC dysfunction/activation via sustained oxidative/nitrosative stress.

Regulation of gene transcription in vascular smooth muscle cells (VSMCs) by serum response factor (SRF) plays a crucial role in vascular development and in the pathophysiology of vascular diseases. Nevertheless, the regulation of specific genes by SRF in vascular diseases is poorly understood. Therefore, we investigated the regulation of smooth muscle myosin light chain kinase (smMLCK) by using spontaneously hypertensive rats (SHR) as an experimental model. We found that smMLCK expression in blood vessels increases during the development of hypertension and is always greater in blood vessels from SHR compared with normotensive rats. Analysis of the DNA sequences of the promoters isolated from SHR and normotensive rats revealed that SHR contain a 12-base pair insertion adjacent to the CArG box. This insertion increases SRF binding to the CArG box and positively regulates SRF-dependent promoter activity. The increase in smMLCK expression was blocked by dominant-negative SRF, dominant-negative Ras, or antisense oligonucleotides to ERK. In vivo, inhibiting MEK decreased smMLCK expression and blood pressure in SHR partly by decreasing SRF binding to the smMLCK promoter. These data provide novel insight into the regulation of smMLCK expression at the molecular level and demonstrate the importance of SRF in regulating smMLCK promoter activity in SHR.

Key Points Gα12 interaction with α-SNAP regulates basal EC vWF secretion. PAR-1 activation-dependent signaling via Gα12/RhoA and Gαq/11 enhances vWF secretion.

The primary function of the mammalian lung is to facilitate diffusion of oxygen to venous blood and to ventilate carbon dioxide produced by catabolic reactions within cells. However, it is also responsible for a variety of other important functions, including host defense and production of vasoactive agents to regulate not only systemic blood pressure, but also water, electrolyte and acid-base balance. Caveolin-1 is highly expressed in the majority of cell types in the lung, including epithelial, endothelial, smooth muscle, connective tissue cells, and alveolar macrophages. Deletion of caveolin-1 in these cells results in major functional aberrations, suggesting that caveolin-1 may be crucial to lung homeostasis and development. Furthermore, generation of mutant mice that under-express caveolin-1 results in severe functional distortion with phenotypes covering practically the entire spectrum of known lung diseases, including pulmonary hypertension, fibrosis, increased endothelial permeability, and immune defects. In this Chapter, we outline the current state of knowledge regarding caveolin-1-dependent regulation of pulmonary cell functions and discuss recent research findings on the role of caveolin-1 in various pulmonary disease states, including obstructive and fibrotic pulmonary vascular and inflammatory diseases.

Nitrate tolerance developed after persistent nitroglycerin (GTN) exposure limits its clinical utility. Previously, we have shown that the vasodilatory action of GTN is dependent on endothelial nitric oxide synthase (eNOS/NOS3) activity. Caveolin-1 (Cav-1) is known to interact with NOS3 on the cytoplasmic side of cholesterol-enriched plasma membrane microdomains (caveolae) and to inhibit NOS3 activity. Loss of Cav-1 expression results in NOS3 hyperactivation and uncoupling, converting NOS3 into a source of superoxide radicals, peroxynitrite, and oxidative stress. Therefore, we hypothesized that nitrate tolerance induced by persistent GTN treatment results from NOS3 dysfunction and vascular toxicity. Exposure to GTN for 48–72 h resulted in nitrosation and depletion (>50%) of Cav-1, NOS3 uncoupling as measured by an increase in peroxynitrite production (>100%), and endothelial toxicity in cultured cells. In the Cav-1 deficient mice, NOS3 dysfunction was accompanied by GTN tolerance (>50% dilation inhibition at low GTN concentrations). In conclusion, GTN tolerance results from Cav-1 modification and depletion by GTN that causes persistent NOS3 activation and uncoupling, preventing it from participating in GTN-medicated vasodilation.