Calvin P.H. Vary

Soluble Transforming Growth Factor-β Type II Receptor Inhibits Negative Remodeling, Fibroblast Transdifferentiation, and Intimal Lesion Formation But Not Endothelial Growth

Using the rat balloon catheter denudation model, we examined the role of transforming growth factor-beta (TGF-beta) isoforms in vascular repair processes. By en face in situ hybridization, proliferating and quiescent smooth muscle cells in denuded vessels expressed high levels of mRNA for TGF-beta1, TGF-beta2, TGF-beta3, and lower levels of TGF-beta receptor II (TGF-betaRII) mRNA. Compared with normal endothelium, TGF-beta1 and TGF-beta2, as well as TGF-betaRII, mRNA were upregulated in endothelium at the wound edge. Injected recombinant soluble TGF-betaRII (TGF-betaR:Fc) localized preferentially to the adventitia and developing neointima in the injured carotid artery, causing a reduction in intimal lesion formation (up to 65%) and an increase in lumen area (up to 88%). The gain in lumen area was largely due to inhibition of negative remodeling, which coincided with reduced adventitial fibrosis and collagen deposition. Four days after injury, TGF-betaR:Fc treatment almost completely inhibited the induction of smooth muscle alpha-actin expression in adventitial cells. In the vessel wall, TGF-betaR:Fc caused a marked reduction in mRNA levels for collagens type I and III. TGF-betaR:Fc had no effect on endothelial proliferation as determined by reendothelialization of the denuded rat aorta. Together, these findings identify the TGF-beta isoforms as major factors mediating adventitial fibrosis and negative remodeling after vascular injury, a major cause of restenosis after angioplasty.

Interaction and functional interplay between endoglin and ALK‐1, two components of the endothelial transforming growth factor‐β receptor complex

Transforming growth factor‐β (TGF‐β) signaling in endothelial cells is able to modulate angiogenesis and vascular remodeling, although the underlying molecular mechanisms remain poorly understood. Endoglin and ALK‐1 are components of the TGF‐β receptor complex, predominantly expressed in endothelial cells, and mutations in either endoglin or ALK‐1 genes are responsible for the vascular dysplasia known as hereditary hemorrhagic telangiectasia. Here we find that the extracellular and cytoplasmic domains of the auxiliary TGF‐β receptor endoglin interact with ALK‐1 (a type I TGF‐β receptor). In addition, endoglin potentiates TGF‐β/ALK1 signaling, with the extracellular domain of endoglin contributing to this functional cooperation between endoglin and ALK‐1. By contrast, endoglin appears to interfere with TGF‐β/ALK‐5 signaling. These results suggest that the functional association of endoglin with ALK‐1 is critical for the endothelial responses to TGF‐β.

Vascular Injury Induces Expression of Periostin Implications for Vascular Cell Differentiation and Migration

Objective— Periostin mRNA is among the most strongly upregulated transcripts in rat carotid arteries after balloon injury. The goal of the present study was to gain insight into the significance of periostin in the vasculature. Methods and Results— Periostin expression after injury was localized to smooth muscle cells of the neointima and the adventitia. The expression of periostin in smooth muscle cells in vitro was not regulated by cytokines such as fibroblast growth factor-2 (FGF-2). In contrast, stimulation of MC3T3-E1 osteoblastic cells, NIH3T3 fibroblasts, or mesenchymal C3H10T1/2 cells with FGF-2 reduced periostin mRNA levels to <5% of controls, whereas conversely bone morphogenetic protein-2 (BMP-2) increased periostin mRNA levels. Periostin expression was induced and maintained during retinoic acid-induced smooth muscle cell differentiation in A404 cells. In addition, overexpression of periostin in C3H10T1/2 cells caused an increase in cell migration that could be blocked with an anti-periostin antibody. Conclusions— Periostin expression is associated with smooth muscle cell differentiation in vitro and promotes cell migration. Unlike other mesenchymally derived cell lines, periostin expression is not regulated by FGF-2 in smooth muscle cells. This distinction may be useful in discriminating smooth muscle and fibroblast lineages.

Novel biochemical pathways of endoglin in vascular cell physiology

The broad role of the transforming growth factor beta (TGFβ) signaling pathway in vascular development, homeostasis, and repair is well appreciated. Endoglin is emerging as a novel, complex, and poorly understood regulatory component of the TGFβ receptor complex, whose importance is underscored by its recognition as the site of mutations causing hereditary hemorrhagic telangiectasia (HHT) [McAllister et al., 1994 ]. Extensive analyses of endoglin function in normal developmental mouse models [Bourdeau et al., 1999 ; Li et al., 1999 ; Arthur et al., 2000 ] and in HHT animal models [Bourdeau et al., 2000 ; Torsney et al., 2003 ] exemplify the importance of understanding endoglins biochemical functions. However, novel mechanisms underlying the regulation of these pathways continue to emerge. These mechanisms include modification of TGFβ receptor signaling at the ligand and receptor activation level, direct effects of endoglin on cell adhesion and migration, and emerging roles for endoglin in the determination of stem cell fate and tissue patterning. The purpose of this review is to highlight the cellular and molecular studies that underscore the central role of endoglin in vascular development and disease. J. Cell. Biochem. 102: 1375–1388, 2007.

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.

Notch and Transforming Growth Factor-β (TGFβ) Signaling Pathways Cooperatively Regulate Vascular Smooth Muscle Cell Differentiation

Notch and transforming growth factor-β (TGFβ) play pivotal roles during vascular development and the pathogenesis of vascular disease. The interaction of these two pathways is not fully understood. The present study utilized primary human smooth muscle cells (SMC) to examine molecular cross-talk between TGFβ1 and Notch signaling on contractile gene expression. Activation of Notch signaling using Notch intracellular domain or Jagged1 ligand induced smooth muscle α-actin (SM actin), smooth muscle myosin heavy chain, and calponin1, and the expression of Notch downstream effectors hairy-related transcription factors. Similarly, TGFβ1 treatment of human aortic smooth muscle cells induced SM actin, calponin1, and smooth muscle protein 22-α (SM22α) in a dose- and time-dependent manner. Hairy-related transcription factor proteins, which antagonize Notch activity, also suppressed the TGFβ1-induced increase in SMC markers, suggesting a general mechanism of inhibition. We found that Notch and TGFβ1 cooperatively activate SMC marker transcripts and protein through parallel signaling axes. Although the intracellular domain of Notch4 interacted with phosphoSmad2/3 in SMC, this interaction was not observed with Notch1 or Notch2. However, we found that CBF1 co-immunoprecipitated with phosphoSmad2/3, suggesting a mechanism to link canonical Notch signaling to phosphoSmad activity. Indeed, the combination of Notch activation and TGFβ1 treatment led to synergistic activation of a TGFβ-responsive promoter. This increase corresponded to increased levels of phosphoSmad2/3 interaction at Smad consensus binding sites within the SM actin, calponin1, and SM22α promoters. Thus, Notch and TGFβ coordinately induce a molecular and functional contractile phenotype by co-regulation of Smad activity at SMC promoters.

Endoglin increases eNOS expression by modulating Smad2 protein levels and Smad2‐dependent TGF‐β signaling

The endothelial nitric oxide synthase (eNOS) is a critical regulator of cardiovascular homeostasis, whose dysregulation leads to different vascular pathologies. Endoglin is a component of the transforming growth factor beta (TGF‐β) receptor complex present in endothelial cells that is involved in angiogenesis, cardiovascular development, and vascular homeostasis. Haploinsufficient expression of endoglin has been shown to downregulate endothelium‐derived nitric oxide in endoglin+/− (Eng+/−) mice and cultured endothelial cells. Here, we find that TGF‐β1 leads to an increased vasodilatation in Eng+/+ mice that is severely impaired in Eng+/− mice, suggesting the involvement of endoglin in the TGF‐β regulated vascular homeostasis. The endoglin‐dependent induction of eNOS occurs at the transcriptional level and is mediated by the type I TGF‐β receptor ALK5 and its downstream substrate Smad2. In addition, Smad2‐specific signaling is upregulated in endoglin‐induced endothelial cells, whereas it is downregulated upon endoglin gene suppression with small interference RNA (siRNA). The endoglin‐dependent upregulation of Smad2 was confirmed using eNOS and pARE promoters, whose activities are known to be Smad2 dependent, as well as with the interference of Smad2 with siRNA, Smurf2, or a dominant negative form of Smad2. Furthermore, increased expression of endoglin in endoglin‐inducible endothelial cells or in transfectants resulted in increased levels of Smad2 protein without affecting the levels of Smad2 mRNA. The increased levels of Smad2 appear to be due to a decreased ubiquitination and proteasome‐dependent degradation leading to stabilization of Smad2. These results suggest that endoglin enhances Smad2 protein levels potentiating TGF‐β signaling, and leading to an increased eNOS expression in endothelial cells. J. Cell. Physiol. 210: 456–468, 2007.

Endoglin structure and function - Determinants of endoglin phosphorylation by transforming growth factor-beta receptors

Determination of the functional relationship between the transforming growth factor-β (TGFβ) receptor proteins endoglin and ALK1 is essential to the understanding of the human vascular disease, hereditary hemorrhagic telangiectasia. TGFβ1 caused recruitment of ALK1 into a complex with endoglin in human umbilical vein endothelial cells (HUVECs). Therefore, we examined TGFβ receptor-dependent phosphorylation of endoglin by the constitutively active forms of the TGFβ type I receptors ALK1, ALK5, and the TGFβ type II receptor, TβRII. Of these receptors, TβRII preferentially phosphorylated endoglin on cytosolic domain serine residues Ser634 and Ser635. Removal of the carboxyl-terminal tripeptide of endoglin, which comprises a putative PDZ-liganding motif, dramatically increased endoglin serine phosphorylation by all three receptors, suggesting that the PDZ-liganding motif is important for the regulation of endoglin phosphorylation. Constitutively active (ca)ALK1, but not caALK5, phosphorylated endoglin on cytosolic domain threonine residues. caALK1-mediated threonine phosphorylation required prior serine phosphorylation, suggesting a sequential mechanism of endoglin phosphorylation. Wild-type, but not a threonine phosphorylation-defective endoglin mutant blocked cell detachment and the antiproliferative effects of caALK1 expressed in HUVECs. These results suggest that ALK1 is a preferred TGFβ receptor kinase for endoglin threonine phosphorylation in HUVECs and indicate a role for endoglin phosphorylation in the regulation of endothelial cell adhesion and growth by ALK1.

Mutation and expression analysis of the endoglin gene in hereditary hemorrhagic telangiectasia reveals null alleles

Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant disorder characterized by multisystemic vascular dysplasia and recurrent hemorrhage from the sites of vascular lesions. Two genes have been identified for HHT. Endoglin, a TGF‐β binding protein which maps to chromosome 9q3, is the gene for HHT1. The type and location of most of the previously described mutations in the endoglin (ENG) gene suggested a dominant‐negative model of receptor–complex dysfunction for the molecular basis of this disorder. In this article we describe 11 novel ENG mutations in HHT kindreds, which include missense and splice‐site mutations. Two identical missense mutations in unrelated families disrupt the start codon of the gene. In addition, some frameshift and nonsense mutations lead to very low or undetectable levels of transcript from the mutant allele. These combined data suggest that the nature of most ENG mutations is to create a null (nonfunctional) allele, and that there is no requirement for the synthesis of a truncated endoglin protein in the pathogenesis of HHT. Hum Mutat 11:286–294, 1998.

Endoglin structure and function: Determinants of endoglin phosphorylation by TGFβ receptors

Determination of the functional relationship between the transforming growth factor-β (TGFβ) receptor proteins endoglin and ALK1 is essential to the understanding of the human vascular disease, hereditary hemorrhagic telangiectasia. TGFβ1 caused recruitment of ALK1 into a complex with endoglin in human umbilical vein endothelial cells (HUVECs). Therefore, we examined TGFβ receptor-dependent phosphorylation of endoglin by the constitutively active forms of the TGFβ type I receptors ALK1, ALK5, and the TGFβ type II receptor, TβRII. Of these receptors, TβRII preferentially phosphorylated endoglin on cytosolic domain serine residues Ser634 and Ser635. Removal of the carboxyl-terminal tripeptide of endoglin, which comprises a putative PDZ-liganding motif, dramatically increased endoglin serine phosphorylation by all three receptors, suggesting that the PDZ-liganding motif is important for the regulation of endoglin phosphorylation. Constitutively active (ca)ALK1, but not caALK5, phosphorylated endoglin on cytosolic domain threonine residues. caALK1-mediated threonine phosphorylation required prior serine phosphorylation, suggesting a sequential mechanism of endoglin phosphorylation. Wild-type, but not a threonine phosphorylation-defective endoglin mutant blocked cell detachment and the antiproliferative effects of caALK1 expressed in HUVECs. These results suggest that ALK1 is a preferred TGFβ receptor kinase for endoglin threonine phosphorylation in HUVECs and indicate a role for endoglin phosphorylation in the regulation of endothelial cell adhesion and growth by ALK1.