Simone Kennard

NOTCH3 Expression Is Induced in Mural Cells Through an Autoregulatory Loop That Requires Endothelial-Expressed JAGGED1

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 &ggr;-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.

Notch3 Is Critical for Proper Angiogenesis and Mural Cell Investment

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&agr;. 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.

Transforming Growth Factor-β (TGF-β1) Down-regulates Notch3 in Fibroblasts to Promote Smooth Muscle Gene Expression

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-β (TGF-β1) 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-β1 to induce the differentiation of 10T1/2 fibroblasts, we investigated the specific function of Notch3. Overexpression of activated Notch3 caused repression of TGF-β1-induced smooth muscle-specific genes, whereas knockdown of Notch3 by small interfering RNA did not convincingly alter their expression. Surprisingly, the addition of TGF-β1 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 SM22α promoter. These results offer a novel mechanism by which TGF-β1 promotes the expression of smooth muscle differentiation genes through the inhibition of Notch3 and activation of Hes1.

Differential gene expression in a coculture model of angiogenesis reveals modulation of select pathways and a role for Notch signaling

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 others 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.

Notch2 and Notch3 function together to regulate vascular smooth muscle development.

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.

Calcineurin-mediated dephosphorylation of eNOS at serine 116 affects eNOS enzymatic activity indirectly by facilitating c-Src binding and tyrosine 83 phosphorylation.

It has been shown previously that phosphorylation of the endothelial nitric oxide synthase (eNOS) at serine 116 (S116) under basal conditions suppresses eNOS enzymatic activity in endothelial cells. It has also been shown that vascular endothelial growth factor (VEGF) treatment of endothelial cells produces a rapid S116 dephosphorylation, which is blocked by the calcineurin inhibitor, cyclosporin A (CsA). In this study, we show that activation of eNOS in response to a variety of other eNOS-activating agonists and the cytosolic calcium-elevating agent, thapsigargin also involves CsA-inhibitable S116 dephosphorylation. Studies with the purified eNOS enzyme also demonstrate that neither mimicking phosphorylation at S116 nor phosphorylation of the purified enzyme at S116 in vitro has any effect on enzymatic activity. Phospho-mimicking, however, does interfere with the interaction of eNOS with c-Src, an interaction which is known to activate eNOS by phosphorylation at tyrosine 83 (Y83). Agonist-stimulated eNOS-Src complex formation, as well as agonist-stimulated Y83 phosphorylation, are blocked by calcineurin inhibition by CsA and by a cell-permeable calcineurin inhibitory peptide. Taken together, these data suggest a mechanism of eNOS regulation whereby calcineurin-mediated dephosphorylation of eNOS at S116 affects eNOS enzymatic activity indirectly, rather than directly, by facilitating c-Src binding and Y83 phosphorylation.

Endothelial cells downregulate apolipoprotein D expression in mural cells through paracrine secretion and Notch signaling.

Endothelial and mural cell interactions are vitally important for proper formation and function of blood vessels. These two cell types communicate to regulate multiple aspects of vessel function. In studying genes regulated by this interaction, we identified apolipoprotein D (APOD) as one gene that is downregulated in mural cells by coculture with endothelial cells. APOD is a secreted glycoprotein that has been implicated in governing stress response, lipid metabolism, and aging. Moreover, APOD is known to regulate smooth muscle cells and is found in abundance within atherosclerotic lesions. Our data show that the regulation of APOD in mural cells is bimodal. Paracrine secretion by endothelial cells causes partial downregulation of APOD expression. Additionally, cell contact-dependent Notch signaling plays a role. NOTCH3 on mural cells promotes the downregulation of APOD, possibly through interaction with the JAGGED-1 ligand on endothelial cells. Our results show that NOTCH3 contributes to the downregulation of APOD and by itself is sufficient to attenuate APOD transcript expression. In examining the consequence of decreased APOD expression in mural cells, we show that APOD negatively regulates cell adhesion. APOD attenuates adhesion by reducing focal contacts; however, it has no effect on stress fiber formation. These data reveal a novel mechanism in which endothelial cells control neighboring mural cells through the downregulation of APOD, which, in turn, influences mural cell function by modulating adhesion.

TNFα reduces eNOS activity in endothelial cells through serine 116 phosphorylation and Pin1 binding: Confirmation of a direct, inhibitory interaction of Pin1 with eNOS

Production of NO by the endothelial nitric oxide synthase (eNOS) has a major role in blood pressure control and suppression of atherosclerosis. In a previous study, we presented evidence implicating the Pin1 prolyl isomerase in negative modulation of eNOS activity in bovine aortic endothelial cells (BAECs). Pin1 recognizes phosphoserine/phosphothreonine-proline motifs in target proteins and catalyzes prolyl isomerization at the peptide bond. In the present study, we show, first, with purified proteins, that Pin1 binds to eNOS directly via the Pin1 WW domain. Binding is enhanced by mimicking phosphorylation of eNOS at S116. Interaction of Pin1 with eNOS markedly reduces eNOS enzymatic activity. Second, in BAECs, we show that TNFα induces ERK 1/2-mediated S116 phosphorylation of eNOS, accompanied by Pin1 binding. TNFα treatment of BAECs results in a reduction in NO release from the cells in a manner that depends on the activities of both Pin1 and ERK 1/2. Evidence is also presented that this mechanism of eNOS regulation cannot occur in rat and mouse cells because there is no proline residue in the mouse and rat amino acid sequences adjacent to the putative phosphorylation site. Moreover, we find that phosphorylation of this site is not detectable in mouse eNOS.

Ptp1b deletion in pro-opiomelanocortin neurons increases energy expenditure and impairs endothelial function via TNF-α dependent mechanisms

Protein tyrosine phosphatase 1b (Ptp1b) is a negative regulator of leptin and insulin-signalling pathways. Its targeted deletion in proopiomelanocortin (POMC) neurons protects mice from obesity and diabetes by increasing energy expenditure. Inflammation accompanies increased energy expenditure. Therefore, the present study aimed to determine whether POMC-Ptp1b deletion increases energy expenditure via an inflammatory process, which would impair endothelial function. We characterized the metabolic and cardiovascular phenotypes of Ptp1b+/+ and POMC-Ptp1b-/- mice. Clamp studies revealed that POMC-Ptp1b deletion reduced body fat and increased energy expenditure as evidenced by a decrease in feed efficiency and an increase in oxygen consumption and respiratory exchange ratio. POMC-Ptp1b deletion induced a 2.5-fold increase in plasma tumour necrosis factor α (TNF-α) levels and elevated body temperature. Vascular studies revealed an endothelial dysfunction in POMC-Ptp1b-/- mice. Nitric oxide synthase inhibition [N-nitro-L-arginine methyl ester (L-NAME)] reduced relaxation to a similar extent in Ptp1b+/+ and POMC-Ptp1b-/- mice. POMC-Ptp1b deletion decreased ROS-scavenging enzymes [superoxide dismutases (SODs)] whereas it increased ROS-generating enzymes [NADPH oxidases (NOXs)] and cyclooxygenase-2 (COX-1) expression, in aorta. ROS scavenging or NADPH oxidase inhibition only partially improved relaxation whereas COX-2 inhibition and thromboxane-A2 (TXA2) antagonism fully restored relaxation in POMC-Ptp1b-/- mice Chronic treatment with the soluble TNF-α receptor etanercept decreased body temperature, restored endothelial function and reestablished aortic COX-2, NOXs and SOD expression to their baseline levels in POMC-Ptp1b-/- mice. However, etanercept promoted body weight gain and decreased energy expenditure in POMC-Ptp1b-/- mice. POMC-Ptp1b deletion increases plasma TNF-α levels, which contribute to body weight regulation via increased energy expenditure and impair endothelial function via COX-2 and ROS-dependent mechanisms.