Abstract-Endothelial cells and mural cells (smooth muscle cells, pericytes, or fibroblasts) are known to communicate with one another. Their interactions not only serve to support fully functional blood vessels but also can regulate vessel assembly and differentiation or maturation. In an effort to better understand the molecular components of this heterotypic interaction, we used a 3D model of angiogenesis and screened for genes, which were modulated by coculturing of these 2 different cell types.In doing so, we discovered that NOTCH3 is one gene whose expression is robustly induced in mural cells by coculturing with endothelial cells. Knockdown by small interfering RNA revealed that NOTCH3 is necessary for endothelial-dependent mural cell differentiation, whereas overexpression of NOTCH3 is sufficient to promote smooth muscle gene expression. Moreover, NOTCH3 contributes to the proangiogenic abilities of mural cells cocultured with endothelial cells. Interestingly, we found that the expression of NOTCH3 is dependent on Notch signaling, because the ␥-secretase inhibitor DAPT blocked its upregulation. Furthermore, in mural cells, a dominant-negative Mastermind-like1 construct inhibited NOTCH3 expression, and endothelial-expressed JAGGED1 was required for its induction. Additionally, we demonstrated that NOTCH3 could promote its own expression and that of JAGGED1 in mural cells. Taken together, these data provide a mechanism by which endothelial cells induce the differentiation of mural cells through activation and induction of NOTCH3. These findings also suggest that NOTCH3 has the capacity to maintain a differentiated phenotype through a positive-feedback loop that includes both autoregulation and JAGGED1 expression. W ithin the vasculature, endothelial cells and mural cells (defined here as vascular support cells that include smooth muscle cells, pericytes, and fibroblasts) are closely associated and can regulate the activity of each other throughout development and into adulthood. [1][2][3] Several groups have shown that mural cells influence blood vessel assembly by controlling such events as endothelial cell proliferation, migration, sprouting, and regression. 4 -10 Later, in intact vessels, these cells influence how endothelial cells respond to humoral and hemodynamic cues. 11 Likewise, endothelial cells are known to modulate mural cell phenotype and function. In addition to proliferation and migration, endothelial cells can promote smooth muscle differentiation and influence contractile activity. 12,13 Despite their intimate association and obvious abilities to respond to one another in intact vessels, there is still much to be learned about the nature of their interactions, particularly during blood vessel formation.A handful of signaling mediators form the basis of our understanding about how these 2 cell types communicate during vasculogenesis and angiogenesis. Growth factor/receptor families, including platelet-derived growth factor (PDGF)-B/PDGF receptor (PDGFR)-␤, transforming growth factor-␤, and ...

The nickel-containing enzyme urease is an essential colonization factor of the human gastric pathogen Helicobacter pylori which enables the bacteria to survive the low-pH conditions of the stomach. Transcription of the urease genes is positively controlled in response to increasing concentrations of nickel ions and acidic pH. Here we demonstrate that acid-induced transcription of the urease genes is mediated directly by the ArsRS two-component system. Footprint analyses identify binding sites of the phosphorylated ArsR response regulator within the ureA and ureI promoters. Furthermore, deletion of a distal upstream ArsR binding site of the ureA promoter demonstrates its role in acid-dependent activation of the promoter. In addition, acid-induced transcription of the ureA gene is unaltered in a nikR mutant, providing evidence that pH-responsive regulation and nickel-responsive regulation of the ureA promoter are mediated by independent mechanisms involving the ArsR response regulator and the NikR protein.

Rationale:The heterotypic interactions of endothelial cells and mural cells (smooth muscle cells or pericytes) are crucial for assembly, maturation, and subsequent function of blood vessels. Yet, the molecular mechanisms underlying their association have not been fully defined.Objective: Our previous in vitro studies indicated that Notch3, which is expressed in mural cells, mediates these cell-cell interactions. To assess the significance of Notch3 on blood vessel formation in vivo, we investigated its role in retinal angiogenesis. Methods and Results:We show that Notch3-deficient mice exhibit reduced retinal vascularization, with diminished sprouting and vascular branching. Moreover, Notch3 deletion impairs mural cell investment, resulting in progressive loss of vessel coverage. In an oxygen-induced retinopathy model, we demonstrate that Notch3 is induced in hypoxia and interestingly, pathological neovascularization is decreased in retinas of Notch3-null mice. Analysis of oxygen-induced retinopathy mediators revealed that angiopoietin-2 expression is significantly reduced in the absence of Notch3. Furthermore, in vitro experiments showed that Notch3 is sufficient for angiopoietin-2 induction, and this expression is additionally enhanced in the presence of hypoxia-inducible factor 1␣. Conclusions: These results provide compelling evidence that Notch3 is important for the investment of mural cells and is a critical regulator of developmental and pathological blood vessel formation. (Circ Res. 2010;107:860-870.)Key Words: Notch3 Ⅲ retina Ⅲ angiogenesis Ⅲ smooth muscle cell Ⅲ pericytes Ⅲ blood vessel B lood vessel formation is a dynamic and complex process that serves a vital role in both health and disease. At the onset of blood vessel formation, endothelial cells coalesce into tube-like structures, which become stabilized by the recruitment of mural cells such as pericytes and vascular smooth muscle cells (VSMCs) that encase the nascent vessel. 1,2 A host of reports have implied that endothelial cells and mural cells are closely associated, and the interactions between them are required for the regulation of vessel formation, stabilization, remodeling and function in vivo and in vitro. [2][3][4] However, the way in which endothelial and mural cells communicate with each other remains poorly understood. Several different ligand-receptor systems have been implicated in heterotypic cell interactions to regulate the development and maintenance of the vasculature. Endothelial cell-secreted platelet-derived growth factor-B is known to be necessary for the recruitment of pericytes to newly formed vessels through platelet-derived growth factor receptor-␤. [5][6][7] Angiopoietin (Ang)-1 and Tie2 signaling has been shown to be critical for vessel maturation and stabilization, 3,8 whereas the differentiation of VSMCs surrounding blood vessels depends on endothelial-derived transforming growth factor-␤. 3,9 In addition to these signaling mediators, it is believed that additional receptor-ligand pairs regulate vascular cellce...

Select signaling pathways have emerged as key players in regulating smooth muscle gene expression during myofibroblast and smooth muscle differentiation, an event that is important for wound healing and vascular remodeling. These include the transforming growth factor-␤ (TGF-␤1) signaling cascade, which has been assigned multiple roles in these cells, and the Notch pathway. Notch family members have been implicated in governing cell fate in a variety of cells; however, the mechanisms are not well understood. We sought to explore how these prominent signaling mediators regulate differentiation, and in particular, how they might converge to control the transcription of smooth muscle genes. Using TGF-␤1 to induce the differentiation of 10T1/2 fibroblasts, we investigated the specific function of Notch3. Overexpression of activated Notch3 caused repression of TGF-␤1-induced smooth muscle-specific genes, whereas knockdown of Notch3 by small interfering RNA did not convincingly alter their expression. Surprisingly, the addition of TGF-␤1 caused a significant decrease in Notch3 RNA and protein and a reciprocal increase in Hes1 gene transcription. The repression of Notch3 was mediated by SMAD activity and p38 mitogen-activated protein (MAP) kinase, whereas analysis of the Hes1 promoter revealed direct activation by Smad2 but not Smad3. Furthermore, the Hes1 repressor protein augmented Smad3 transactivation of the SM22␣ promoter. These results offer a novel mechanism by which TGF-␤1 promotes the expression of smooth muscle differentiation genes through the inhibition of Notch3 and activation of Hes1.The differentiation of smooth muscle cells and myofibroblasts is characterized by the coordinate up-regulation of smooth muscle genes that include smooth muscle ␣-actin, SM22␣, and h1-calponin (1-3). These proteins serve as indicators of cell function as they are associated with the contractile properties of the cell and signify a state of maturation. A well established modulator of myofibroblast and smooth muscle cells is transforming growth factor-␤1 (TGF-␤1), 2 which has been shown to have multiple roles in vascular remodeling and wound healing (4 -6). In the vasculature, this cytokine can promote smooth muscle differentiation and inhibit proliferation and migration, all of which are compatible with a stable vessel (1, 7). Conversely, TGF-␤1 has also been shown to be robustly expressed in experimental balloon injury models and can cause neointimal hyperplasia (1, 7). During wound healing, TGF-␤1 activates fibroblasts to elicit contraction and the production of critical extracellular matrix components, but it is also associated with fibrosis, leading to an abundance of myofibroblasts that cause scarring (5). These data imply that the activity of TGF-␤1 is largely context-dependent and that its effect on a cell is determined by additional regulators that together establish its mode of function. TGF-␤1 has been shown to directly activate smooth muscle gene expression primarily, but not exclusively, through SMAD proteins that ...

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Notch signaling has been implicated in the regulation of smooth muscle differentiation, but the precise role of Notch receptors is ill defined. Although Notch3 receptor expression is high in smooth muscle, Notch3 mutant mice are viable and display only mild defects in vascular patterning and smooth muscle differentiation. Notch2 is also expressed in smooth muscle and Notch2 mutant mice show cardiovascular abnormalities indicative of smooth muscle defects. Together, these findings infer that Notch2 and Notch3 act together to govern vascular development and smooth muscle differentiation. To address this hypothesis, we characterized the phenotype of mice with a combined deficiency in Notch2 and Notch3. Our results show that when Notch2 and Notch3 genes are simultaneously disrupted, mice die in utero at mid-gestation due to severe vascular abnormalities. Assembly of the vascular network occurs normally as assessed by Pecam1 expression, however smooth muscle cells surrounding the vessels are grossly deficient leading to vascular collapse. In vitro analysis show that both Notch2 and Notch3 robustly activate smooth muscle differentiation genes, and Notch3, but not Notch2 is a target of Notch signaling. These data highlight the combined actions of the Notch receptors in the regulation of vascular development, and suggest that while these receptors exhibit compensatory roles in smooth muscle, their functions are not entirely overlapping.

The human gastric pathogen Helicobacter pylori exhibits a remarkably small repertoire of transcriptional regulators including three complete two-component systems as well as the orphan response regulators HP1021 and HP1043. Both HP1021 and HP1043 show atypical receiver sequences and are required for the normal cell growth of H. pylori. Recently, we demonstrated that phosphorylation of HP1021 and HP1043 according to the two-component paradigm is not a prerequisite for the cell growth-associated functions of these response regulators, raising the question of how the activity of this regulatory proteins is modulated. Here, we report that strict transcriptional control of its expression is not involved in the cell-growth associated function of HP1021. We show that expression of hp1043 is controlled both on the post-transcriptional or post-translational level and by transcriptional regulation. Furthermore, we provide evidence that hp1043 can be replaced by the orthologous gene cj0355 from Campylobacter jejuni.

Communication between endothelial and mural cells (smooth muscle cells, pericytes, and fibroblasts) can dictate blood vessel size and shape during angiogenesis, and control the functional aspects of mature blood vessels, by determining things such as contractile properties. The ability of these different cell types to regulate each other's activities led us to ask how their interactions directly modulate gene expression. To address this, we utilized a three-dimensional model of angiogenesis and screened for genes whose expression was altered under coculture conditions. Using a BeadChip array, we identified 323 genes that were uniquely regulated when endothelial cells and mural cells (fibroblasts) were cultured together. Data mining tools revealed that differential expression of genes from the integrin, blood coagulation, and angiogenesis pathways were overrepresented in coculture conditions. Scans of the promoters of these differentially modulated genes identified a multitude of conserved C promoter binding factor (CBF)1/CSL elements, implicating Notch signaling in their regulation. Accordingly, inhibition of the Notch pathway with gamma-secretase inhibitor DAPT or NOTCH3-specific small interfering RNA blocked the coculture-induced regulation of several of these genes in fibroblasts. These data show that coculturing of endothelial cells and fibroblasts causes profound changes in gene expression and suggest that Notch signaling is a critical mediator of the resultant transcription.