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 ...

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...

Diabetic retinopathy (DR) is one of the most common complications of diabetes and is a leading cause of blindness in people of the working age in Western countries. A major pathology of DR is microvascular complications such as non-perfused vessels, microaneurysms, dot/blot hemorrhages, cotton-wool spots, venous beading, vascular loops, vascular leakage and neovascularization. Multiple mechanisms are involved in these alternations. This review will focus on the role of inflammation in diabetic retinal microvascular complications and discuss the potential therapies by targeting inflammation.

These data demonstrate that the deletion of NOX2 can reduce I/R-induced cell death and preserve retinal GCL neurons after I/R injury. The neuronal cell injury caused by I/R is associated with the activation of ERK and NF-κB signaling mechanisms.

Diabetic retinopathy (DR) is one of the most common complications of diabetes. This devastating disease is a leading cause of blindness in people of working age in industrialized countries and affects the daily lives of millions of people. Despite tight glycemic control, blood pressure control, and lipid-lowering therapy, the number of DR patients keeps growing and therapeutic approaches are limited. Moreover, there are significant limitations and side-effects for the current therapies. Thus, there is a great need for development of new strategies for prevention and treatment of DR. Studies have shown that DR has prominent features of chronic, subclinical inflammation. This review will focus on the role of inflammation in DR and summarize the progress of studies of anti-inflammatory strategies for DR.

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 ...

Accumulating evidence has shown that diabetes accelerates aging and endothelial cell senescence is involved in the pathogenesis of diabetic vascular complications, including diabetic retinopathy. Oxidative stress is recognized as a key factor in the induction of endothelial senescence and diabetic retinopathy. However, specific mechanisms involved in oxidative stress-induced endothelial senescence have not been elucidated. We hypothesized that Sirt6, which is a nuclear, chromatin-bound protein critically involved in many pathophysiologic processes such as aging and inflammation, may have a role in oxidative stress-induced vascular cell senescence. Measurement of Sirt6 expression in human endothelial cells revealed that H2O2 treatment significantly reduced Sirt6 protein. The loss of Sirt6 was associated with an induction of a senescence phenotype in endothelial cells, including decreased cell growth, proliferation and angiogenic ability, and increased expression of senescence-associated β-galactosidase activity. Additionally, H2O2 treatment reduced eNOS expression, enhanced p21 expression, and dephosphorylated (activated) retinoblastoma (Rb) protein. All of these alternations were attenuated by overexpression of Sirt6, while partial knockdown of Sirt6 expression by siRNA mimicked the effect of H2O2. In conclusion, these results suggest that Sirt6 is a critical regulator of endothelial senescence and oxidative stress-induced downregulation of Sirt6 is likely involved in the pathogenesis of diabetic retinopathy.

During wound repair, new blood vessels form in response to angiogenic signals emanating from injured tissues. Dermal fibroblasts are known to play an important role in wound healing, and have been linked to angiogenesis; therefore, we sought to understand the mechanisms through which these cells control blood vessel formation. Using a three-dimensional angiogenesis assay we demonstrate that dermal fibroblasts enhance the tube-forming potential of endothelial cells, and this augmentation is partially due to secreted factors present in conditioned media. Interestingly, we identified tissue inhibitor of metalloproteinase-1 (TIMP-1) as a factor uniquely secreted by fibroblasts, and addition of exogenous TIMP-1 increased vessel assembly. The enhancing activity of TIMP-1 was matrix metalloproteinase (MMP)-dependent, since a mutant version of TIMP-1 was unable to promote angiogenesis. Consistent with this, chemical inhibition of MMP-2/9 showed a similar increase in angiogenesis, and addition of exogenous MMP-9 blocked the enhancing effect of TIMP-1. We further demonstrated that TIMP-1 inhibits the production of tumstatin, an anti-angiogenic fragment of collagen IV that is produced by MMP-9 cleavage. Our results support the notion that dermal fibroblasts regulate blood vessel formation through multiple mediators, and provide novel evidence that fibroblast-derived TIMP-1 acts on endothelial cells in a pro-angiogenic capacity.