Phosphorylation of ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) by Akt promotes stability and mitogenic function of S-phase kinase-associated protein-2 (Skp2).

The regulation of the cell cycle by the ubiquitin-proteasome system is dependent on the activity of E3 ligases. Skp2 (S-phase kinase associated protein-2) is the substrate recognition subunit of the E3 ligase that ubiquitylates the cell cycle inhibitors p21(cip1) and p27(kip1) thus promoting cell cycle progression. Increased expression of Skp2 is frequently observed in diseases characterized by excessive cell proliferation, such as cancer and neointima hyperplasia.

Recombinant human interferon alpha 2b prevents and reverses experimental pulmonary hypertension.

Pulmonary hypertension (PH) is a progressive and fatal disease with no cure. Vascular remodeling in PH involves intraluminal growth of endothelial and smooth muscle cells, leading to obliterative vascular lesions. Cell growth in these lesions is quasi-neoplastic, with evidence of monoclonality, apoptosis resistance and cancer-like metabolic derangements.

Genetic deletion of toll-like receptor 4 on platelets attenuates experimental pulmonary hypertension.

Rationale: Recent studies demonstrate a role for toll-like receptor 4 (TLR4) in the pathogenesis of pulmonary hypertension (PH); however, the cell types involved in mediating the effects of TLR4 remain unknown.

Objectives: The objective of this study was to determine the contribution of TLR4 expressed on nonparenchymal cells to the pathogenesis of PH.

Methods And Results: TLR4 bone marrow chimeric mice revealed an equal contribution of TLR4 on nonparenchymal and parenchymal cells in the pathogenesis of PH as determined by measuring right ventricular (RV) systolic pressure and RV hypertrophy.

Caveolae-dependent and -independent uptake of albumin in cultured rodent pulmonary endothelial cells.

Although a critical role for caveolae-mediated albumin transcytosis in pulmonary endothelium is well established, considerably less is known about caveolae-independent pathways. In this current study, we confirmed that cultured rat pulmonary microvascular (RPMEC) and pulmonary artery (RPAEC) endothelium endocytosed Alexa488-labeled albumin in a saturable, temperature-sensitive mode and internalization resulted in co-localization by fluorescence microscopy with cholera B toxin and caveolin-1. Although siRNA to caveolin-1 (cav-1) in RPAEC significantly inhibited albumin uptake, a remnant portion of albumin uptake was cav-1-independent, suggesting alternative pathways for albumin uptake.

Ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) and nuclear factor-κB (NF-κB): a feed-forward loop for systemic and vascular inflammation.

The interaction between vascular cells and macrophages is critical during vascular remodeling. Here we report that the scaffolding protein, ezrin-binding phosphoprotein 50 (EBP50), is a central regulator of macrophage and vascular smooth muscle cells (VSMC) function. EBP50 is up-regulated in intimal VSMC following endoluminal injury and promotes neointima formation.

High mobility group box 1 contributes to the pathogenesis of experimental pulmonary hypertension via activation of Toll-like receptor 4.

Survival rates for patients with pulmonary hypertension (PH) remain low, and our understanding of the mechanisms involved are incomplete. Here we show in a mouse model of chronic hypoxia (CH)-induced PH that the nuclear protein and damage-associate molecular pattern molecule (DAMP) high mobility group box 1 (HMGB1) contributes to PH via a Toll-like receptor 4 (TLR4)-dependent mechanism. We demonstrate extranuclear HMGB1 in pulmonary vascular lesions and increased serum HMGB1 in patients with idiopathic pulmonary arterial hypertension.

High mobility group Box 1 inhibits human pulmonary artery endothelial cell migration via a Toll-like receptor 4- and interferon response factor 3-dependent mechanism(s).

In pulmonary hypertension the loss of precapillary arterioles results from vascular injury causing endothelial dysfunction. Endothelial cell migration and proliferation are critical for vascular regeneration. This study focused on the effect of high mobility group box 1 protein (HMGB1) on these critical processes.

Chronic hypoxia induces right heart failure in caveolin-1-/- mice.

Caveolin-1 (Cav-1)-/- mice develop mild pulmonary hypertension as they age. In this study, we sought to determine the effect of chronic hypoxia, an established model of pulmonary hypertension, on young Cav-1-/- mice with no measurable signs of pulmonary hypertension. Exposure of Cav-1-/- mice to chronic hypoxia resulted in an initial rise in right ventricular (RV) systolic pressure (RVSP) similar to wild-type (WT) mice.

Endothelium derived nitric oxide synthase negatively regulates the PDGF-survivin pathway during flow-dependent vascular remodeling.

Chronic alterations in blood flow initiate structural changes in vessel lumen caliber to normalize shear stress. The loss of endothelial derived nitric oxide synthase (eNOS) in mice promotes abnormal flow dependent vascular remodeling, thus uncoupling mechanotransduction from adaptive vascular remodeling. However, the mechanisms of how the loss of eNOS promotes abnormal remodeling are not known.

Complement C3 deficiency attenuates chronic hypoxia-induced pulmonary hypertension in mice.

Background: Evidence suggests a role of both innate and adaptive immunity in the development of pulmonary arterial hypertension. The complement system is a key sentry of the innate immune system and bridges innate and adaptive immunity. To date there are no studies addressing a role for the complement system in pulmonary arterial hypertension.

The scaffolding protein EBP50 promotes vascular smooth muscle cell proliferation and neointima formation by regulating Skp2 and p21(cip1).

Objective: The Ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is a scaffolding protein known to regulate ion homeostasis in the kidney and intestine. Previous work showed that EBP50 expression increases after balloon injury in rat carotids. This study was designed to determine the role of EBP50 on vascular smooth muscle cells (VSMC) proliferation and the development of neointimal hyperplasia.

Bone morphogenetic protein receptor II is a novel mediator of endothelial nitric-oxide synthase activation.

Activation of bone morphogenetic protein (BMP) receptor II (BMPRII) promotes pulmonary artery endothelial cell (PAEC) survival, proliferation, and migration. Mutations to BMPRII are associated with the development of pulmonary arterial hypertension (PAH). Endothelial dysfunction, including decreased endothelial nitric-oxide synthase (eNOS) activity and loss of bioactive nitric oxide (NO), plays a prominent role in the development of PAH.

A noninhibitory mutant of the caveolin-1 scaffolding domain enhances eNOS-derived NO synthesis and vasodilation in mice.

Aberrant regulation of eNOS and associated NO release are directly linked with various vascular diseases. Caveolin-1 (Cav-1), the main coat protein of caveolae, is highly expressed in endothelial cells. Its scaffolding domain serves as an endogenous negative regulator of eNOS function.

Thrombospondin-1 supports blood pressure by limiting eNOS activation and endothelial-dependent vasorelaxation.

Aims: Thrombospondin-1 (TSP1), via its necessary receptor CD47, inhibits nitric oxide (NO)-stimulated soluble guanylate cyclase activation in vascular smooth muscle cells, and TSP1-null mice have increased shear-dependent blood flow compared with wild-type mice. Yet, the endothelial basement membrane should in theory function as a barrier to diffusion of soluble TSP1 into the arterial smooth muscle cell layer. These findings suggested that endothelial-dependent differences in blood flow in TSP1-null mice may be the result of direct modulation of endothelial NO synthase (eNOS) activation by circulating TSP1.

Nitrite potently inhibits hypoxic and inflammatory pulmonary arterial hypertension and smooth muscle proliferation via xanthine oxidoreductase-dependent nitric oxide generation.

Background: Pulmonary arterial hypertension is a progressive proliferative vasculopathy of the small pulmonary arteries that is characterized by a primary failure of the endothelial nitric oxide and prostacyclin vasodilator pathways, coupled with dysregulated cellular proliferation. We have recently discovered that the endogenous anion salt nitrite is converted to nitric oxide in the setting of physiological and pathological hypoxia. Considering the fact that nitric oxide exhibits vasoprotective properties, we examined the effects of nitrite on experimental pulmonary arterial hypertension.

HO-1 and CO decrease platelet-derived growth factor-induced vascular smooth muscle cell migration via inhibition of Nox1.

Objective: Heme oxygenase-1 (HO-1), via its enzymatic degradation products, exhibits cell and tissue protective effects in models of vascular injury and disease. The migration of vascular smooth muscle cells (VSMC) from the medial to the intimal layer of blood vessels plays an integral role in the development of a neointima in these models. Despite this, there are no studies addressing the effect of increased HO-1 expression on VSMC migration.

Caveolin-1 regulates BMPRII localization and signaling in vascular smooth muscle cells.

Recent studies demonstrate the interaction of BMPRII and caveolin-1 in various cell types. In this study we test the hypothesis that caveolin-1 interacts with and regulates BMPRII-dependent signaling in vascular smooth muscle cells. We demonstrate that BMPRII localizes to caveolae and directly interacts with caveolin-1 in mouse aortic smooth muscle cells.

Variant estrogen receptor-c-Src molecular interdependence and c-Src structural requirements for endothelial NO synthase activation.

Little is known about the tyrosine kinase c-Src's function in the systemic circulation, in particular its role in arterial responses to hormonal stimuli. In human aortic and venous endothelial cells, c-Src is indispensable for 17beta-estradiol (E2)-stimulated phosphatidylinositol 3-kinase/Akt/endothelial NO synthase (eNOS) pathway activation, a possible mechanism in E2-mediated vascular protection. Here we show that c-Src supports basal and E2-stimulated NO production and is required for E2-induced vasorelaxation in murine aortas.

Dissecting the molecular control of endothelial NO synthase by caveolin-1 using cell-permeable peptides.

In endothelia, NO is synthesized by endothelial NO synthase (eNOS), which is negatively regulated by caveolin-1 (Cav-1), the primary coat protein of caveolae. We show that delivery of Cav-1 amino acids 82-101 (Cav) fused to an internalization sequence from Antennapedia (AP) blocks NO release in vitro and inflammation and tumor angiogenesis in vivo. To characterize the molecular mechanism by which the AP-Cav peptide and Cav-1 mediate eNOS inhibition, we subdivided the Cav portion of AP-Cav into three domains (Cav-A, -B, and -C), synthesized five overlapping peptides (AP-Cav-A, -AB, -B, -BC, and -C), and tested their effects on eNOS-dependent activities.

Endothelial-specific expression of caveolin-1 impairs microvascular permeability and angiogenesis.

The functions of caveolae and/or caveolins in intact animals are beginning to be explored. Here, by using endothelial cell-specific transgenesis of the caveolin-1 (Cav-1) gene in mice, we show the critical role of Cav-1 in several postnatal vascular paradigms. First, increasing levels of Cav-1 do not increase caveolae number in the endothelium in vivo.

Endothelial NO synthase phosphorylated at SER635 produces NO without requiring intracellular calcium increase.

Shear stress stimulates NO production involving the Ca2+-independent mechanisms in endothelial cells. We have shown that exposure of bovine aortic endothelial cells (BAEC) to shear stress stimulates phosphorylation of eNOS at S635 and S1179 by the protein kinase A- (PKA-) dependent mechanisms. We examined whether phosphorylation of S635 of eNOS induced by PKA stimulates NO production in a calcium-independent manner.

Compensatory phosphorylation and protein-protein interactions revealed by loss of function and gain of function mutants of multiple serine phosphorylation sites in endothelial nitric-oxide synthase.

We examined the influence of individual serine phosphorylation sites in endothelial nitric-oxide synthase (eNOS) on basal and stimulated NO release, cooperative phosphorylation, and co-association with hsp90 and Akt. Mutation of the serine phosphorylation sites 116, 617, and 1179 to alanines affected the phospho-state of at least one other site, demonstrating cooperation between multiple phosphorylation events, whereas mutation of serine 635 to alanine did not cause compensation. Mutation of serines 116 and 617 to alanine promoted a greater protein-protein interaction with hsp90 and Akt and greater phosphorylation on serine 1179, the major site for Akt phosphorylation.