Abstract-During blood vessel assembly, endothelial cells recruit mesenchymal progenitors and induce their differentiation into mural cells via contact-dependent transforming growth factor-␤ (TGF-␤) activation. We investigated whether gap junction channels are formed between endothelial cells and recruited mesenchymal progenitors and whether intercellular communication is necessary for endothelial-induced mural cell differentiation. Key Words: blood vessel formation Ⅲ gap junctions Ⅲ endothelial cells Ⅲ mural cell differentiation Ⅲ transforming growth factor-␤ E mbryonic blood vessel formation begins with the coalescence of mesodermal progenitors and their differentiation into endothelial and blood cells, forming blood islands 1 that fuse and define a primitive circulatory plexus. 2 During branching and remodeling of the initial plexus that leads to a well-defined vascular network, 3 endothelial tubes acquire a surrounding vessel wall of mural cells (pericytes or smooth muscle cells) via the secretion of platelet-derived growth factor-B that acts as a chemoattractant and mitogen for mural cell precursors. 4 -6 On contact with endothelial cells, the recruited progenitors are induced toward a mural cell fate, 7 in a process mediated by the activation of transforming growth factor-beta (TGF-␤).Although TGF-␤ activation is not completely understood, 8,9 it is clear that activated TGF-␤ plays a critical role in the induction of mural cell differentiation. Via TGF-␤ control elements 10 or upregulation of serum response factor (SRF), 11 TGF-␤ promotes coordinated transcriptional activation of cytoskeletal and contractile genes, including smooth muscle (SM)-␣-actin, SM-␥-actin, SM22␣, calponin, and SM-myosin heavy chain, needed for differentiated function. 12 Mural cells not only modulate blood flow but also stabilize vessels 6,13 and may sustain endothelial survival through the production of Ang-1 14 or VEGF-A. 15 The exact interactions between endothelial and mesenchymal cells during vessel assembly that lead to TGF-␤ activation and mural cell differentiation are not well defined but are presumably complex given the number of genes implicated by mutation studies to regulate these processes during vascular development. Our studies aim to elucidate the role of one component of endothelial-mesenchymal cell interactions in vessel assembly-heterocellular communication via gap junctions.Gap junctions are aggregates of intercellular channels 16 that connect the cytoplasms of adjoining cells and allow the passage of second messengers, ions, and metabolites. 17 Gap junction channels are composed of connexin (Cx) proteins, of which there are at least 20. 18,19 The presence of gap junctions has been documented among and between vascular cells. 20 -22 Conflicting reports of their exact Cx composition suggest that Cx distribution varies among vascular beds and vessel types and is differentially regulated in response to injury and growth.In adults, quiescent endothelial cells predominantly express Cx37 and Cx40 [23][24][25][26][2...

Background-We studied the growth-promoting effects of 2 sodium pump-selective cardiotonic steroids, ouabain and marinobufagenin, on cultured cells from vascular smooth muscle (VSMCs) from human umbilical vein and a rat VSMC line, A7r5. Methods and Results-Both ouabain and marinobufagenin activated proliferation of these cells in a concentrationdependent manner, reflecting the cardiotonic steroid sensitivity of the specific ␣ 1 subunit contained within each cell source. The observed effective concentration ranges of both compounds was below that necessary to induce cytoplasmic ion alterations by sodium pump inhibition. Conclusions-These data indicate that the ouabain-activated proliferative effect previously observed in canine VSMCs occurs in other VSMC sources. This growth effect seems to be initiated by drug interaction with the sodium pump, reflected by the affinity of the steroid for the pump, and is independent of altered transmembrane ionic gradients. Key Words: cells Ⅲ drugs Ⅲ muscle, smooth Ⅲ vasculature T he role of the sodium pump, or Na ϩ ,K ϩ -ATPase, in the catalysis of Na ϩ and K ϩ active transport across the plasma membrane of animal cells is well documented. Recent research in this area has shown that this enzyme complex may have functions distinct from those involved in active ion transport. Significant evidence has emerged indicating that the sodium pump can also function as a transmembrane signal transducing complex (for reviews, see Xie and Askari 1 and Schoner 2 ). Studies by Kometiani et al, 3 Haas et al, 4 and Liu et al 5 have shown that low concentrations of the cardiotonic steroid ouabain activated rat cardiac myocyte hypertrophy and ERK1/2 phosphorylation and that these actions are totally independent of changes in intracellular Na ϩ and Ca 2ϩ concentrations. In addition, we have demonstrated that 0.1 to 1.0 nmol/L ouabain induced proliferation of cultured canine vascular smooth muscle cells (VSMCs) via a signaling cascade involving Src, the epidermal growth factor receptor, and ERK1/2. 6 These low concentrations of ouabain have also been shown to stimulate proliferation of human prostate smooth muscle cells. 7 Together with the recent observations of endogenous production of these species of cardiotonic steroids, 8,9 these studies suggest that they may function as autocoidal mediators of growth.VSMC proliferation has been linked to a variety of adaptive and pathophysiological responses, and a potential endogenous regulator of this function would be of considerable interest. In addition, it has been suggested that these endogenous compounds could well be a part of the cardiovascular remodeling that occurs in hypertension. 10 Thus, this study extends our previous work, because it addresses 3 critical functional issues: (1) Does the response occur in human VSMCs? (2) Do other autocoidal steroids evoke the same response? and (3) Does the response involve ligand interaction with the Na ϩ ,K ϩ -ATPase? To accomplish these goals, we have elected to assess 2 compounds, ouabain and marinobufagen...

Animals deficient for connexin 45 (Cx45), Cx43, or Cx40 and Cx37 all suffer embryonic or post-natal lethal vascular phenotypes. We developed an in vitro model of blood vessel assembly to dissect the specific roles of these connexins in this process. Previously, we showed that heterocellular gap junction channel formation between endothelial and mesenchymal cells is required for TGF-β activation and endothelial-induced mural cell differentiation, and that Cx43-containing channels support these processes. Developmental studies suggest that Cx45 is required for mural cell development during embryogenesis, although its exact role was not delineated. OBJECTIVE The focus of this study was to investigate the role of Cx45 in endothelial-induced mural cell differentiation. METHODS AND RESULTS We created mural cell precursors that stably express only Cx45 in Cx43-deficient mesenchymal cells (ReCx45), and used our in vitro model of blood vessel assembly to assess the capacity of this Cx to support endothelial-induced mural cell differentiation. Lucifer Yellow dye injection and dual whole-cell patch clamping revealed that functional gap junctions exhibiting properties of Cx45-containing channels formed amongst ReCx45 transfectants, and between ReCx45 and endothelial cells. Heterocellular Cx45-containing gap junction channels enabled TGF-β activation, and promoted the upregulation of mural cell-specific proteins in the mesenchymal precursors. CONCLUSION These studies reveal a critical role for Cx45 in the regulation of endothelial-induced mural cell differentiation, which is consistent with the phenotype of Cx45-deficient embryos that exhibit dysregulated TGF-β and lack mural cell development.

No abstract