Endothelial cells require related transcription enhancer factor-1 for cell-cell connections through the induction of gap junction proteins.

OBJECTIVE: Capillary network formation represents a specialized endothelial cell function and is a prerequisite to establish a continuous vessel lumen. Formation of endothelial cell connections that form the vascular structure is regulated, at least in part, at the transcriptional level. We report here that related transcription enhancer factor-1 (RTEF-1) plays an important role in vascular structure formation. METHODS AND RESULTS: Knockdown of RTEF-1 by small interfering RNA or blockage of RTEF-1 function by the transcription enhancer activators domain decreased endothelial connections in a Matrigel assay, whereas overexpression of RTEF-1 in endothelial cells resulted in a significant increase in cell connections and aggregation. In a model of oxygen-induced retinopathy, endothelial-specific RTEF-1 overexpressing mice had enhanced angiogenic sprouting and vascular structure remodeling, resulting in the formation of a denser and more highly interconnected superficial capillary plexus. Mechanistic studies revealed that RTEF-1 induced the expression of functional gap junction proteins including connexin 43, connexin 40, and connexin 37. Blocking connexin 43 function inhibited RTEF-1-induced endothelial cell connections and aggregation. CONCLUSIONS: These findings provide novel insights into the transcriptional control of endothelial function in the coordination of cell-cell connections.

Response gene to complement 32, a novel hypoxia-regulated angiogenic inhibitor.

BACKGROUND: Response gene to complement 32 (RGC-32) is induced by activation of complement and regulates cell proliferation. To determine the mechanism of RGC-32 in angiogenesis, we examined the role of RGC-32 in hypoxia-related endothelial cell function. METHODS AND RESULTS: Hypoxia/ischemia is able to stimulate both angiogenesis and apoptosis. Hypoxia-inducible factor-1/vascular endothelial growth factor is a key transcriptional regulatory pathway for angiogenesis during hypoxia. We demonstrated that the increased RGC-32 expression by hypoxia was via hypoxia-inducible factor-1/vascular endothelial growth factor induction in cultured endothelial cells. However, overexpression of RGC-32 reduced the proliferation and migration and destabilized vascular structure formation in vitro and inhibited angiogenesis in Matrigel assays in vivo. Silencing RGC-32 had an opposing, stimulatory effect. RGC-32 also stimulated apoptosis as shown by the increased apoptotic cells and caspase-3 cleavage. Mechanistic studies revealed that the effect of RGC-32 on the antiangiogenic response was via attenuating fibroblast growth factor 2 expression and further inhibiting expression of cyclin E without affecting vascular endothelial growth factor and fibroblast growth factor 2 signaling in endothelial cells. In the mouse hind-limb ischemia model, RGC-32 inhibited capillary density with a significant attenuation in blood flow. Additionally, treatment with RGC-32 in the xenograft tumor model resulted in reduced growth of blood vessels that is consistent with reduced colon tumor size. CONCLUSIONS: We provide the first direct evidence for RGC-32 as a hypoxia-inducible gene and antiangiogenic factor in endothelial cells. These data suggest that RGC-32 plays an important homeostatic role in that it contributes to differentiating the pathways for vascular endothelial growth factor and fibroblast growth factor 2 in angiogenesis and provides a new target for ischemic disorder and tumor therapies.

Hyperglycemia attenuates angiogenic capability of survivin in endothelial cells.

Survivin, an anti-apoptotic protein, can be induced by hypoxia and contributes to angiogenic activity in endothelial cells. To determine the potential mechanism of survivin in endothelial dysfunction caused by hyperglycemia in diabetes, we evaluated the role of survivin in hyperglycemia and its effect on endothelial homeostasis. We demonstrated that an increase of D-glucose was sufficient to down-regulate survivin expression, impacting survivin's angiogenic role in endothelial cells. We additionally showed that survivin expression was increased in response to hypoxia yet this reaction was mitigated when the endothelial cells were in hyperglycemic conditions prior to hypoxia. Hyperglycemia also affected survivin-related proliferation and migration of endothelial cells and increased the number of apoptotic cells. In the ischemic porcine myocardium, the expression of survivin was induced. Moreover, survivin expression in the aorta, myocardium, and isolated endothelial cells was attenuated in a porcine model of diabetes in comparison to non-diabetes, which correlated negatively with the levels of fasting blood sugars and positively with territory perfusion. These results demonstrate that hyperglycemia critically alters survivin expression in vitro and in vivo, which leads to attenuation of angiogenic activity and impacts endothelial metabolism.

Induction of regulatory T cells by physiological level estrogen.

Naturally occurring CD4+CD25+ regulatory T cells (Treg) exert an important role in mediating maternal tolerance to the fetus during pregnancy, and this effect might be regulated via maternal estrogen secretion. Although estrogen concentration in the pharmaceutical range has been shown to drive expansion of CD4+CD25+ Treg cells, little is known about how and through what mechanisms E2 within the physiological concentration range of pregnancy affects this expansion. Using in vivo and in vitro mouse models in these experiments, we observed that E2 at physiological doses not only expanded Treg cell in different tissues but also increased expression of the Foxp3 gene, a hallmark for CD4+CD25+ Treg cell function, and the IL-10 gene as well. Importantly, our results demonstrate that E2, at physiological doses, stimulated the conversion of CD4+CD25- T cells into CD4+CD25+ T cells which exhibited enhanced Foxp3 and IL-10 expression in vitro. Such converted CD4+CD25+ T cells had similar regulatory function as naturally occurring Treg cells, as demonstrated by their ability to suppress naïve T cell proliferation in a mixed lymphocyte reaction. We also found that the estrogen receptor (ER) exist in the CD4+CD25- T cells and the conversion of CD4+CD25- T cells into CD4+CD25+ T cells stimulated by E2 could be inhibited by ICI182,780, a specific inhibitor of ER(s). This supports that E2 may directly act on CD4+CD25- T cells via ER(s). We conclude that E2 is a potential physiological regulatory factor for the peripheral development of CD4+CD25+ Treg cells during the implantation period in mice.

Endothelial differentiation gene-1, a new downstream gene is involved in RTEF-1 induced angiogenesis in endothelial cells.

Related Transcriptional Enhancer Factor-1 (RTEF-1) has been suggested to induce angiogenesis through regulating target genes. Whether RTEF-1 has a direct role in angiogenesis and what specific genes are involved in RTEF-1 driven angiogenisis have not been elucidated. We found that over-expressing RTEF-1 in Human dermal microvascular endothelial cells-1 (HMEC-1) significantly increased endothelial cell aggregation, growth and migration while the processes were inhibited by siRNA of RTEF-1. In addition, we observed that Endothelial differentiation gene-1 (Edg-1) expression was up-regulated by RTEF-1 at the transcriptional level. RTEF-1 could bind to Edg-1 promoter and subsequently induce its activity. Edg-1 siRNA significantly blocked RTEF-1-driven increases in endothelial cell aggregation in a Matrigel assay and retarded RTEF-1-induced endothelial cell growth and migration. Pertussis Toxin (PTX), a Gi/Go protein sensitive inhibitor, was found to inhibit RTEF-1 driven endothelial cell aggregation and migration. Our data demonstrates that Edg-1 is a potential target gene of RTEF-1 and is involved in RTEF-1-induced angiogenesis in endothelial cells. Gi/Go protein coupled receptor pathway plays a role in RTEF-1 driven angiogenesis in endothelial cells.

Panax notoginseng and its components decreased hypertension via stimulation of endothelial-dependent vessel dilatation.

UNLABELLED: Ginsenoside Rb1 and Rg1 are major components of Panax notoginseng (P.N.), an herb with known clinical efficacy in hypertension and myocardial ischemia in Eastern countries. This investigation is to elicit the mechanism of these components in hypertension via their effect on vascular reaction. To assess the ability of P.N. in hypertension, P.N. extracts were injected in spontaneously hypertensive rats (SHR) via the vena caudalis; Low dosages of P.N. extracts significantly lowered blood pressure in SHR. Examination with Rb1 and Rg1 revealed significant vasodilatation using mouse coronary arteries in a dose-dependent manner. Rb1- and Rg1-induced vasodilatation was blocked by pre-incubation with eNOS and PI3K inhibitors. Coronaries of eNOS-/- mice showed attenuated vasodilatation with Rb1 and Rg1. In addition, both Rb1 and Rg1 induce nitric oxide (NO) generation through increasing the phosphorylation of eNOS, activating Na+-independent l-arginine transport, and stimulating cationic amino acid transport (CAT)-1 mRNA expression in cultured endothelial cells. CONCLUSION: Ginsenoside Rb1 and Rg1 increased endothelial-dependent vessel dilatation through the activation of NO by modulating the PI3K/Akt/eNOS pathway and l-arginine transport in endothelial cells. These findings may have important implications for understanding the mechanisms of clinical efficacy of the herb P.N. when used in the regulation of blood pressure.

The endothelium-dependent effect of RTEF-1 in pressure overload cardiac hypertrophy: role of VEGF-B.

AIMS: Related transcription enhancer factor-1 (RTEF-1) has previously been demonstrated to play an important role in both endothelial cells and cardiomyocytes. However, the function of RTEF-1 in the communication between these two adjacent cell types has not been elucidated. METHODS AND RESULTS: We have found that endothelium-specific RTEF-1 transgenic mice (VE-Cad/RTEF-1) developed significant cardiac hypertrophy after transverse aortic constriction surgery, as evidenced by an increased ratio of heart weight to tibia length, enlarged cardiomyocyte size, thickened left ventricular wall and elevated expression of hypertrophic gene markers, with up-regulation of vascular endothelial growth factor B (VEGF-B). Additionally, VEGF-B was increased in endothelial cells from VE-Cad/RTEF-1 mice, as well as in endothelial cells with forced RTEF-1 expression (HMEC-1/RTEF-1), and coincidentally decreased when RTEF-1 was deficient in HMEC-1. Using chromatin immunoprecipitation and luciferase assays, we found that RTEF-1 increased VEGF-B promoter activity through a direct interaction. Hypertrophy-associated genes and protein synthesis were up-regulated in cardiomyocytes that were incubated with conditioned medium from HMEC-1/RTEF-1 and the endothelial cells of VE-Cad/RTEF-1 mice. This effect could be abrogated by treating the myocytes with VEGF-B small interfering RNA and extracellular signal-regulated kinase 1/2 inhibitor. CONCLUSION: Our data demonstrated that increased RTEF-1 in endothelial cells upregulates VEGF-B, which is able to stimulate hypertrophic genes in cardiomyocytes. These results suggest that the RTEF-1-driven increase of VEGF-B plays an important role in communication between the endothelium and myocardium.

Hypoxia-regulated angiogenic inhibitors.

The regulation of angiogenesis by hypoxia is an essential homeostatic mechanism that depends on a precise balance between positive and negative angiogenic regulatory molecules. Proangiogenic factors are well characterized; however, several in vivo and in vitro studies indicate that there are feedback mechanisms in place to inhibit angiogenesis during hypoxia. Understanding the signaling pathways leading to the negative feedback of angiogenesis will undoubtedly provide important tools to develop novel therapeutic strategies not only to enhance the angiogenic response in coronary artery disease but also to hinder deregulated angiogenesis in tumorigenesis.

RGS5, a hypoxia-inducible apoptotic stimulator in endothelial cells.

Endothelial cells rapidly respond to changes in oxygen homeostasis by regulating gene expression. Regulator of G protein signaling 5 (RGS5) is a negative regulator of G protein-mediated signaling that is strongly expressed in vessels during angiogenesis; however, the role of RGS5 in hypoxia has not been fully understood. Under hypoxic conditions, we found that the expression of RGS5, but not other RGS, was induced in human umbilical vein endothelial cells (HUVEC). RGS5 mRNA was increased when HUVEC were incubated with chemicals that stabilized hypoxia-inducible factor-1alpha (HIF-1alpha), whereas hypoxia-stimulated RGS5 promoter activity was absent in HIF-1beta(-/-) cells. Vascular endothelial growth factor (VEGF), which is regulated by HIF-1, did not appear to be involved in hypoxia-induced RGS5 expression; however, VEGF-mediated activation of p38 but not ERK1/2 was increased by RGS5. Overexpression of RGS5 in HUVEC exhibited a reduced growth rate without affecting the cell proliferation. Annexin V assay revealed that RGS5 induced apoptosis with significantly increased activation of caspase-3 and the Bax/Bcl-2 ratio. Small interfering RNA-specific for RGS5, caspase-3 inhibitor, and p38 inhibitor resulted in an attenuation of RGS5-stimulated apoptosis. Matrigel assay proved that RGS5 significantly impaired the angiogenic effect of VEGF and stimulated apoptosis in vivo. We concluded that RGS5 is a novel HIF-1-dependent, hypoxia-induced gene that is involved in the induction of endothelial apoptosis. Moreover, RGS5 antagonizes the angiogenic effect of VEGF by increasing the activation of p38 signaling, suggesting that RGS5 could be an important target for apoptotic therapy.