Thrombin induces endocytosis of endoglin and type-II TGF-beta receptor and down-regulation of TGF-beta signaling in endothelial cells

Thrombin activates protease-activated receptor 1 (PAR1) on endothelial cells (ECs) and is critical for angiogenesis and vascular development. However, the mechanism underlying the proangiogenic effect of thrombin has not been elucidated yet. Here, we report the discovery of a novel functional link between thrombin-PAR1 and transforming growth factor-beta (TGF-beta) signaling pathways. We showed that thrombin via PAR1 induced the internalization of endoglin and type-II TGF-beta receptor (TbetaRII) but not type-I receptors in human ECs. This effect was mediated by protein kinase C-zeta (PKC-zeta) since specific inhibition of PKC-zeta caused an aggregation of endoglin or TbetaRII on cell surface and blocked their internalization by thrombin. Furthermore, acute and long-term pretreatment of ECs with thrombin or PAR1 peptide agonist suppressed the TGF-beta-induced serine phosphorylation of Smad2, a critical mediator of TGF-beta signaling. Moreover, activation of PAR1 led to a profound and spread cytosolic clustering formation of Smad2/3 and markedly prevented Smad2/3 nuclear translocation evoked by TGF-beta1. Since TGF-beta plays a crucial role in the resolution phase of angiogenesis, the down-regulation of TGF-beta signaling by thrombin-PAR1 pathway may provide a new insight into the mechanism of the proangiogenic effect of thrombin.     

Endoglin is overexpressed after arterial injury and is required for transforming growth factor-beta-induced inhibition of smooth muscle cell migration

Endoglin is a homodimeric membrane glycoprotein primarily expressed on endothelial cells. In association with transforming growth factor (TGF)-ss receptors I and II, it can bind TGF-beta1 and -beta3 and form a functional receptor complex. There is increasing evidence that endoglin can modulate the cellular response to TGF-beta, a factor implicated in vascular lesion formation in human and experimental models. The purpose of this study was to analyze the expression of endoglin in normal and balloon-injured porcine coronary arteries and in normal and atherosclerotic human coronary arteries and to determine its ability to mediate the effects of TGF-beta on the migration of vascular smooth muscle cells (SMCs). In normal porcine coronary arteries, endoglin was of low abundance and was found primarily on endothelial cells and adventitial fibroblasts, as well as on a minority of medial SMCs. On days 3, 7, and 14 after angioplasty, endoglin was present not only on endothelial cells but also on adventitial myofibroblasts and medial SMCs of porcine coronary arteries. By day 28, few or no cells expressed endoglin. In situ hybridization revealed that endoglin mRNA expression appeared to be highest in endothelial cells on days 3, 7, and 14 days after injury and absent thereafter. With a second balloon injury, a similar pattern of endoglin protein and mRNA expression was observed. In human vascular tissue, endoglin immunolabeling was higher in endarterectomy specimens removed from diseased coronary arteries than in normal internal mammary arteries. In vitro, antisense oligonucleotides to endoglin decreased its expression and antagonized the TGF-beta-mediated inhibition of human and porcine SMC migration. In summary, upregulation of endoglin occurs during arterial repair and in established atherosclerotic plaques and may be required for modulation of SMC migration by TGF-beta.     

S-endoglin expression is induced in senescent endothelial cells and contributes to vascular pathology

Senescence of endothelial cells (ECs) may contribute to age-associated cardiovascular diseases, including atherosclerosis and hypertension. The functional and gene expression changes associated with cellular senescence are poorly understood. Here, we have analyzed the expression, during EC senescence, of 2 different isoforms (L, long; S, short) of endoglin, an auxiliary transforming growth factor (TGF)-beta receptor involved in vascular remodeling and angiogenesis. As evidenced by RT-PCR, the S/L ratio of endoglin isoforms was increased during senescence of human ECs in vitro, as well as during aging of mice in vascularized tissues. Next, the effect of S-endoglin protein on the TGF-beta receptor complex was studied. As revealed by coimmunoprecipitation assays, S-endoglin was able to interact with both TGF-beta type I receptors, ALK5 and ALK1, although the interaction with ALK5 was stronger than with ALK1. S-endoglin conferred a lower proliferation rate to ECs and behaved differently from L-endoglin in relation to TGF-beta-responsive reporters with ALK1 or ALK5 specificities, mimicking the behavior of the endothelial senescence markers Id1 and plasminogen activator inhibitor-1. In situ hybridization studies demonstrated the expression of S-endoglin in the endothelium from human arteries. Transgenic mice overexpressing S-endoglin in ECs showed hypertension, decreased hypertensive response to NO inhibition, decreased vasodilatory response to TGF-beta(1) administration, and decreased endothelial nitric oxide synthase expression in lungs and kidneys, supporting the involvement of S-endoglin in the NO-dependent vascular homeostasis. Taken together, these results suggest that S-endoglin is induced during endothelial senescence and may contribute to age-dependent vascular pathology.     

Transforming growth factor-betas and vascular disorders

Transforming growth factor-beta (TGF-beta) superfamily members, TGF-beta and bone morphogenetic proteins (BMPs), are potent regulatory cytokines with diverse functions on vascular cells. They signal through heteromeric type I and II receptor complexes activating Smad-dependent and Smad-independent signals, which regulate proliferation, differentiation, and survival. They are potent regulators of vascular development and vessel remodeling and play key roles in atherosclerosis and restenosis, regulating endothelial, smooth muscle cell, macrophage, T cell, and probably vascular calcifying cell responses. In atherosclerosis, TGF-beta regulates lesion phenotype by controlling T-cell responses and stimulating smooth muscle cells to produce collagen. It contributes to restenosis by augmenting neointimal cell proliferation and collagen accumulation. Defective TGF-beta signaling in endothelial cells attributable to mutations in endoglin or the type I receptor ALK-1 leads to hereditary hemorrhagic telangiectasia, whereas defective BMP signaling attributable to mutations in the BMP receptor II has been associated with development of primary pulmonary hypertension. The development of mouse models with either cell type-specific or general inactivation of TGF-beta/BMP signaling has started to reveal the importance of the regulatory network of TGF-beta/BMP pathways in vivo and their significance for atherosclerosis, hereditary hemorrhagic telangiectasia, and primary pulmonary hypertension. This review highlights recent findings that have advanced our understanding of the roles of TGF-beta superfamily members in regulating vascular cell responses and provides likely avenues for future research that may lead to novel pharmacological therapies for the treatment or prevention of vascular disorders.     

Endoglin, a TGF-beta receptor-associated protein, is expressed by smooth muscle cells in human atherosclerotic plaques

Endoglin is a transmembrane protein that is found in association with transforming growth factor-beta (TGF-beta) superfamily receptor complexes and has an expression pattern that appears to be restricted primarily to endothelial cells, activated macrophages, trophoblasts, and fibroblasts. Since mutations in endoglin have been shown to be linked to hereditary hemorrhagic telangiectasia type 1, a disease manifested as vascular malformations characterized by excessive layers of vascular smooth muscle cells (VSMC), the expression of endoglin was investigated in VSMC. In vivo, the majority of SMC in human atherosclerotic plaques expressed high levels of endoglin, while endoglin was not detected in SMC from samples of the normal arterial wall. In vitro studies demonstrate that human aortic smooth muscle cells (HASMC) express the L-isoform of endoglin. Like endothelial cells, HASMC express endoglin protein as a dimer on the cell surface that binds TGF-beta1. In vitro, endoglin expression by HASMC is upregulated in response to TGF-beta1, suggesting that the presence of this factor in the atherosclerotic plaque might be responsible for the increased expression of endoglin. The demonstration of increased levels of endoglin in VSMC in human atherosclerotic plaques suggests a role for SMC endoglin in the maintenance of vascular integrity and in the response of the vessel wall to injury.     

Endoglin-mediated vascular remodeling: mechanisms underlying hereditary hemorrhagic telangiectasia

Endoglin is emerging as a pivotal component of the gateway for signaling by transforming growth factor-beta (TGF-beta) in vascular endothelial cells. Mutations in endoglin cause a rare vascular disorder in humans known as hereditary hemorrhagic telengiectasia (HHT). Although rare, in-depth analysis of mutant mice and mononuclear cells from the blood of patients with HHT have provided novel and exciting insights into how the vasculature is formed, maintained, and repaired during disease. Here, we review recent data on how endoglin is thought to function in endothelial cells and place it in the broader context of signaling by TGF-beta family members in vascular cells in general. We highlight where the controversies on underlying molecular mechanisms currently lie and indicate areas of present research focus.     

Identification of intracellular pathways through which TGF-beta1 upregulates PAI-1 expression in endothelial cells

Upregulation of plasminogen activator inhibitor type 1 (PAI-1) expression is a critical mechanism through which transforming growth factor-beta1 (TGF-beta1) accelerates intimal growth. The aim of this study was to identify signaling pathways through which TGF-beta1 upregulates PAI-1 expression in endothelial cells (EC) and test interventions for blocking these pathways. We transduced cultured bovine EC with an adenoviral vector containing the PAI-1 promoter fused to a beta-galactosidase reporter gene. We used these cells, along with vectors expressing potential modifiers of TGF-beta1 signaling and pharmacologic antagonists of mitogen-activated protein kinase (MAPK) pathways to identify key mediators of basal and TGF-beta1-regulated PAI-1 expression. Basal activity of the PAI-1 promoter was directly correlated with Ras activation and was blocked by a dominant negative (DN) type I TGF-beta receptor. TGF-beta1-stimulated activity of the PAI-1 promoter did not require Ras activation, and was lessened or eliminated by expression of either DN type I or type II TGF-beta receptors and by inhibition of either of two MAPKs: MEK and p38. Our results suggest unanticipated pathways of TGF-beta1 signaling in EC and point to new strategies to limit TGF-beta1-induced vascular disease.     

Blood outgrowth endothelial cells from Hereditary Haemorrhagic Telangiectasia patients reveal abnormalities compatible with vascular lesions

OBJECTIVE: Hereditary haemorrhagic telangiectasia (HHT) is originated by mutations in endoglin (HHT1) and ALK1 (HHT2) genes. The purpose of this work was to isolate and characterize circulating endothelial cells from HHT patients. METHODS: Pure primary cultures of blood outgrowth endothelial cells (BOECs) were obtained from 50 ml of peripheral blood by selection on collagen plates with endothelial medium. RESULTS: The amount of endoglin in HHT1-BOECs is half the controls, but HHT2-BOECs are also endoglin-deficient. Since the TGF-beta/ALK1 pathway activates the endoglin promoter activity, these results suggest the involvement of ALK1 in endoglin gene expression. Endothelial TGF-beta pathways, mediated by ALK1 and ALK5, are impaired in HHT cells. HHT-BOECs show disorganized and depolymerized actin fibers, as compared to the organized stress fibers of healthy-BOECs. Functionally, HHT-BOECs have impaired tube formation, in contrast with the cord-like structures derived from normal donors. CONCLUSIONS: Decreased endoglin expression, impaired TGF-beta signalling, disorganized cytoskeleton, and failure to form cord-like structures are common characteristics of endothelial cells from HHT patients. These features may lead to fragility of small vessels and bleeding characteristic of the HHT vascular dysplasia and to a disrupted and abnormal angiogenesis, which may explain the clinical symptoms associated with this disease.     

Reduced endothelial secretion and plasma levels of transforming growth factor-beta1 in patients with hereditary hemorrhagic telangiectasia type 1

OBJECTIVE: To determine if patients with hereditary hemorrhagic telangiectasia (HHT) show alterations in transforming growth factor (TGF)-beta and its pathways. METHODS: Blood samples were obtained from HHT patients and controls, while endothelial cells were derived from umbilical veins of newborns (HUVEC) from HHT families. TGF-beta1 in plasma, or secreted by HUVEC, and plasma endoglin levels were measured by ELISA. Cellular levels of endoglin and receptor Smad proteins were tested by metabolic labeling and immunoprecipitation, mRNA levels for endoglin and TGF-beta1 by real-time PCR, and receptor Smad phosphorylation by Western blotting. RESULTS: TGF-beta1 and endoglin plasma levels analyzed in 197 individuals showed an inverse correlation with age. Circulating levels of TGF-beta1 were reduced in HHT1 patients (with Endoglin mutations) compared to control, but not in HHT2 patients (with ALK1 mutations). Endoglin levels were unchanged in plasma but decreased in activated monocytes and HUVEC with an HHT1 genotype. These HUVEC also expressed reduced levels of endoglin and TGF-beta1 mRNA, secreted less TGF-beta1, and showed normal receptor Smad expression and phosphorylation. CONCLUSIONS: Decreased plasma TGF-beta1 levels in HHT1 patients correlate with reduced production by endothelial cells. The lower endoglin expression in these cells may alter the regulation of TGF-beta1 via Smad-independent pathways.     

Transforming growth factor beta receptor endoglin is expressed in cardiac fibroblasts and modulates profibrogenic actions of angiotensin II

Angiotensin II (Ang II) is a powerful mediator of adverse cardiac remodeling and fibrosis. However, the mechanisms of Ang II-induced myocardial fibrosis remain to be clarified. We postulated that Ang II alters transforming growth factor beta (TGF-beta) receptor expression, specifically that of endoglin, and thereby modulates cardiac fibroblast (CF) collagen metabolism. Experiments were conducted using CF from adult Sprague Dawley rats to determine the expression of TGF-beta1 receptors including endoglin, and the role of Ang II type 1 (AT1) and type 2 (AT2) receptors, and MAPK p42/44 in this process. The functional role of endoglin in modulating Ang II effects on matrix metalloproteinase-1 (MMP-1) and type I collagen expression was also analyzed. Endoglin gene and protein expression were consistently identified in quiescent CFs. Ang II increased the expression of endoglin mRNA and protein in a concentration and time-dependent manner, with no effect on TGF-beta receptors I and II expression. This effect was AT1 receptor mediated, because AT1 receptor antagonists valsartan, candesartan, and losartan inhibited Ang II-induced endoglin expression, whereas the AT2 receptor antagonist PD123319 had no effect. MAPKp42/44 inhibition attenuated Ang II-induced endoglin expression. Ang II-induced decrease in MMP-1 protein expression and increase in type I collagen protein expression were both blocked by a specific endoglin antibody. Hence, our results indicate that endoglin is upregulated in CFs by Ang II via the AT1 receptor and modulates profibrotic effects of Ang II. These findings provide novel insights into Ang II-induced cardiac remodeling.