Endothelial GATA-6 deficiency promotes pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a chronic and progressive disease characterized by pulmonary vasculopathy with elevation of pulmonary artery pressure, often culminating in right ventricular failure. GATA-6, a member of the GATA family of zinc-finger transcription factors, is highly expressed in quiescent vasculature and is frequently lost during vascular injury. We hypothesized that endothelial GATA-6 may play a critical role in the molecular mechanisms underlying endothelial cell (EC) dysfunction in PAH. Here we report that GATA-6 is markedly reduced in pulmonary ECs lining both occluded and nonoccluded vessels in patients with idiopathic and systemic sclerosis-associated PAH. GATA-6 transcripts are also rapidly decreased in rodent PAH models. Endothelial GATA-6 is a direct transcriptional regulator of genes controlling vascular tone [endothelin-1, endothelin-1 receptor type A, and endothelial nitric oxide synthase (eNOS)], pro-inflammatory genes, CX3CL1 (fractalkine), 5-lipoxygenease-activating protein, and markers of vascular remodeling, including PAI-1 and RhoB. Mice with the genetic deletion of GATA-6 in ECs (Gata6-KO) spontaneously develop elevated pulmonary artery pressure and increased vessel muscularization, and these features are further exacerbated in response to hypoxia. Furthermore, innate immune cells including macrophages (CD11b(+)/F4/80(+)), granulocytes (Ly6G(+)/CD45(+)), and dendritic cells (CD11b(+)/CD11c(+)) are significantly increased in normoxic Gata6-KO mice. Together, our findings suggest a critical role of endothelial GATA-6 deficiency in development and disease progression in PAH.

Mef2c regulates transcription of the extracellular matrix protein cartilage link protein 1 in the developing murine heart.

Cartilage Link Protein 1 (Crtl1) is an extracellular matrix (ECM) protein that stabilizes the interaction between hyaluronan and versican and is expressed in endocardial and endocardially-derived cells in the developing heart, including cells in the atrioventricular (AV) and outflow tract (OFT) cushions. Previous investigations into the transcriptional regulation of the Crtl1 gene have shown that Sox9 regulates Crtl1 expression in both cartilage and the AV valves. The cardiac transcription factor Mef2c is involved in the regulation of gene expression in cardiac and skeletal muscle cell lineages. In this study we have investigated the potential role of Mef2c in the regulation of ECM production in the endocardial and mesenchymal cell lineages of the developing heart. We demonstrate that the Crtl1 5' flanking region contains two highly conserved Mef2 binding sites and that Mef2c is able to bind to these sites in vivo during cardiovascular development. Additionally, we show that Crtl1 transcription is dependent on Mef2c expression in fetal mitral valve interstitial cells (VICs). Combined, these findings highlight a new role for Mef2c in cardiac development and the regulation of cardiac extracellular matrix protein expression.

A role for estrogen receptor-α and estrogen receptor-ß in collagen biosynthesis in mouse skin.

Hormonal regulation of the dermal collagenous extracellular matrix has a key role in maintaining proper tissue homeostasis. However, the factors and pathways involved in this process are not fully defined. This study investigated the role of estrogen receptors (ERs) in the regulation of collagen biosynthesis in mice lacking either ERα or ERß. Collagen content was significantly increased in the skin of ERα(-/-) mice, as measured by acetic acid extraction and the hydroxyproline assay, and correlated with the decreased levels of matrix metalloproteinase (MMP)-15 and elevated collagen production by ERα(-/-) fibroblasts. In contrast, collagen content was decreased in the skin of ERß(-/-) mice, despite markedly increased collagen production by ERß(-/-) fibroblasts. However, expression of several MMPs, including MMP-8 and -15, was significantly elevated, suggesting increased degradation of dermal collagen. Furthermore, ERß(-/-) mice were characterized by significantly reduced levels of small leucine proteoglycans, lumican (Lum), and decorin (Dcn), leading to defects in collagen fibrillogenesis and possibly less stable collagen fibrils. ERα(-/-) mice also exhibited fibrils with irregular structure and size, which correlated with increased levels of Lum and Dcn. Together, these results demonstrate distinct functions of ERs in the regulation of collagen biosynthesis in mouse skin in vivo.

Diazoxide inhibits aortic endothelial cell apoptosis in diabetic rats via activation of ERK.

Endothelial cell (EC) survival is critical in the maintenance of endothelial function as well as in the regulation of angiogenesis and vessel integrity since endothelial dysfunction is the initial lesion of atherosclerosis. The goal of this study was to examine the effect of diazoxide, a mitochondrial ATP-sensitive K(+)(mito K(ATP)) channel opener, on aorta ECs apoptosis and its potential mechanism in Otsuka Long-Evans Tokushima Fatty (OLETF) rats at prediabetic stage. Diazoxide (25 mg kg(-1) day(-1)) was administered intraperitoneally from age 8 weeks to age 30 weeks. Thoracic aorta and cultured thoracic aortic ECs were used. The thickening of thoracic aortic wall and apoptosis of ECs were markedly increased in OLETF rats early from the age of 16 weeks, at the impaired glucose tolerance stage, compared with Long-Evans Tokushima Otsuka rats, in conjunction with intimal hyperplasia and perivascular fibrosis. In contrast, diazoxide treatment inhibited these changes. Further study strongly demonstrated that extracellular signal-regulated kinases (ERKs) are key regulatory proteins in protecting ECs from apoptosis. Diazoxide could significantly enhance phosphorylation of ERK via opening mito K(ATP) channels. This role was reversed by both 5-hydroxydecanoate, selectively closing mito K(ATP) channels, and PD-98509, MEK inhibitors. The present studies demonstrate that diazoxide prevents the onset and development of macrovascular disease in OLETF rats by inhibiting apoptosis directly via phosphorylated ERK increase in aorta ECs. Our findings establish the basis for the therapeutic potential of diazoxide in atherosclerotic disease.

2-Methoxyestradiol induced Bax phosphorylation and apoptosis in human retinoblastoma cells via p38 MAPK activation.

Retinoblastoma (Rb) is a common childhood intraocular cancer that affects approximately 300 children each year in the United States alone. 2-Methoxyestradiol (2ME), an endogenous metabolite of 17-ß-estradiol that dose not bind to nuclear estrogen receptor, exhibits potent apoptotic activity against rapidly growing tumor cells. Here, we report that 2ME induction of apoptosis was demonstrated by early fragmented DNA after 48 h of incubation with 10 µM 2ME in Rb cell lines. Subsequently, a decrease of proliferation was observed in a time- and dose-dependent manner. Further analysis of the mechanism indicates that p38 kinase plays a critical role in 2ME-induced apoptosis in Y79 cells, even though ERK was also activated by 2ME under the same conditions. Activation of p38 kinase also mediates 2ME induced Bax phosphorylated at Thr(167) after a 6 h treatment of 2ME, which in turn prevents formation of the Bcl-2-Bax heterodimer. Both p38 specific inhibitor, SB 203580, or p38 knockdown by specific siRNA, blocked 2ME induction of Bax phosphorylation. Furthermore, only transiently transfected mutant BaxT167A, but not Bax S163A, inhibited 2ME-induced apoptosis. In summary, our data suggest that 2ME induces apoptosis in human Rb cells by causing phosphorylation of p38 Mitogen-activated protein kinase (MAPK), which appears to be correlated with phosphorlation of Bax. This understanding of 2ME's ability may help develop it as a promising therapeutic candidate by inducing apoptosis in a Rb.

Connective tissue growth factor (CTGF/CCN2) mediates angiogenic effect of S1P in human dermal microvascular endothelial cells.

OBJECTIVE: The primary objective of this study was to examine the potential interaction between S1P, a pleiotropic lipid mediator, and CTGF/CCN2, a secreted multimodular protein, in the process of endothelial cell migration. The secondary objective was to determine whether C- and N-terminal domains of CTGF/CCN2 have a specific function in cell migration. MATERIALS AND METHODS: Migration of HDMECs was examined in monolayer wound healing "scratch" assay, whereas capillary-like tube formation was examined in three-dimensional collagen co-culture assays. RESULTS: We observed that S1P stimulates migration of HDMECs concomitant with upregulation of CTGF/CCN2 expression. Furthermore, the blockade of endogenous CTGF/CCN2 via siRNA abrogated S1P-induced HDMEC migration and capillary-like tube formation. Full-length CTGF induced cell migration and capillary-like tube formation with a potency similar to that of S1P, while C-terminal domain of CTGF was slightly less effective. However, N-terminal domain had only a residual activity in inducing capillary-like tube formation. CONCLUSIONS: This study revealed that CTGF/CCN2 is required for the S1P-induced endothelial cell migration, which suggests that CTGF/CCN2 may be an important mediator of S1P-induced physiological and pathological angiogenesis. Moreover, this study shows that the pro-migratory activity of CTGF/CCN2 is located in the C-terminal domain.

Novel wound healing powder formulation for the treatment of venous leg ulcers.

Chronic venous disorders are common in the Western world. The current treatment of venous leg ulcers is unsatisfactory despite the availability of well-documented standards of care. Patients today are interested in alternative approaches to modern medicine. We have developed a wound-healing powder containing natural ingredients with absorptive, aromatic, antiseptic, and anti-inflammatory synergistic properties. This report describes 3 cases that were successfully treated with the powder, demonstrating the potential of herbal remedies in the clinical treatment of venous leg ulcers.

GATA-6 is a novel transcriptional repressor of the human Tenascin-C gene expression in fibroblasts.

In this study we show that GATA-6 is a novel repressor of TN-C gene expression. We demonstrated that overexpression of GATA-6 in fibroblasts inhibited basal levels, as well as markedly decreased IL-4- and TGF-beta-induced TN-C mRNA and protein levels. A GATA-6 response element was mapped to position -467 to -460 of the TN-C promoter. In addition, we showed that GATA-6 binds this site both in vitro and in vivo.