Members of the myocyte enhancer binding factor-2 (MEF2) family of MADS (MCM1, agamous, deficiens, and serum response factor) box transcription factors are expressed in the skeletal, cardiac, and smooth muscle lineages of vertebrate and Drosophila embryos. These factors bind an adenine-thymidine-rich DNA sequence associated with muscle-specific genes. The function of MEF2 was determined by generating a loss-of-function of the single mef2 gene in Drosophila (D-mef2). In loss-of-function embryos, somatic, cardiac, and visceral muscle cells did not differentiate, but myoblasts were normally specified and positioned. These results demonstrate that different muscle cell types share a common myogenic differentiation program controlled by MEF2.

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 mature aortic valve is composed of a structured trilaminar extracellular matrix that is interspersed with aortic valve interstitial cells (AVICs) and covered by endothelium. Dysfunction of the valvular endothelium initiates calcification of neighboring AVICs leading to calcific aortic valve disease (CAVD). The molecular mechanism by which endothelial cells communicate with AVICs and cause disease is not well understood. Using a co-culture assay, we show that endothelial cells secrete a signal to inhibit calcification of AVICs. Gain or loss of nitric oxide (NO) prevents or accelerates calcification of AVICs, respectively, suggesting that the endothelial cell-derived signal is NO. Overexpression of Notch1, which is genetically linked to human CAVD, retards the calcification of AVICs that occurs with NO inhibition. In AVICs, NO regulates the expression of Hey1, a downstream target of Notch1, and alters nuclear localization of Notch1 intracellular domain. Finally, Notch1 and NOS3 (endothelial NO synthase) display an in vivo genetic interaction critical for proper valve morphogenesis and the development of aortic valve disease. Our data suggests that endothelial cell-derived NO is a regulator of Notch1 signaling in AVICs in the development of the aortic valve and adult aortic valve disease.

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...

The formation of skeletal muscle during embryogenesis involves the commitment of mesodermal progenitors to the myogenic lineage followed by the expression of muscle structural genes. The muscle-specific basic-helix-loop-helix proteins, MyoD, myogenin, myf5, and MRF4, have been shown to regulate muscle development (reviewed in ref.

Periostin is a fasciclin-containing adhesive glycoprotein that facilitates the migration and differentiation of cells that have undergone epithelial-mesenchymal transformation during embryogenesis and in pathological conditions. Despite the importance of post-transformational differentiation as a general developmental mechanism, little is known how periostin's embryonic expression is regulated. To help resolve this deficiency, a 3.9-kb periostin proximal promoter was isolated and shown to drive tissue-specific expression in the neural crest-derived Schwann cell lineage and in a subpopulation of periostin-expressing cells in the cardiac outflow tract endocardial cushions. In order to identify the enhancer and associated DNA binding factor(s) responsible, in vitro promoter dissection was undertaken in a Schwannoma line. Ultimately a 304-bp(peri) enhancer was identified and shown to be capable of recapitulating 3.9 kb(peri-lacZ)in vivo spatiotemporal patterns. Further mutational and EMSA analysis helped identify a minimal 37-bp region that is bound by the YY1 transcription factor. The 37-bp enhancer was subsequently shown to be essential for in vivo 3.9 kb(peri-lacZ) promoter activity. Taken together, these studies identify an evolutionary-conserved YY1-binding 37-bp region within a 304-bp periostin core enhancer that is capable of regulating simultaneous novel tissue-specific periostin expression in the cardiac outflow-tract cushion mesenchyme and Schwann cell lineages.

Blood vessels are composed of two primary cell types, endothelial cells and smooth muscle cells, each providing a unique contribution to vessel function. Signaling between these two cell types is essential for maintaining tone in mature vessels, and their communication is critical during development, and for repair and remodeling associated with blood vessel growth. This review will highlight the pathways that endothelial cells and smooth muscle cells utilize to communicate during vessel formation and discuss how disruptions in these pathways contribute to disease.

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