Jiaping Wu

PR39 Inhibits Apoptosis in Hypoxic Endothelial Cells Role of Inhibitor Apoptosis Protein-2

Background—PR39 is a proline- and arginine-rich peptide implicated in wound healing and myocardial ischemia protection. To determine the potential mechanisms of PR39 in ischemia, we examined the role of PR39 in hypoxia-induced apoptosis in vascular endothelial cells. Methods and Results—Hypoxia results in an increase of apoptosis in bovine aortic endothelial cells (BAECs), as determined by terminal deoxynucleotidyl transferase–mediated dUTP biotin nick-end labeling (TUNEL) analysis and caspase-3 activity. Hypoxia induced 66.2±2.7% TUNEL-positive cells, whereas in the presence of synthesized PR39 peptide, TUNEL-positive cells were reduced to 29.6±1.9% (P <0.05). After 24 hours of hypoxia, the addition of PR39 reduced caspase-3 activity to 3.17±0.47 pMol/min from 10.52±0.55 pMol/min in hypoxic BAECs. Moreover, PR39 increased inhibitor of apoptosis protein-2 (IAP-2) gene and protein expression by 3-fold in a time- and dose-dependent manner. The induction of IAP-2 by PR39 conferred an increase in IAP-2 gene transcription and IAP-2 mRNA stability. Furthermore, inhibiting IAP-2 with second mitochondria-derived activator of caspase (Smac) and with small interfering RNA targeting IAP-2 abrogated the ability of PR39 to reduce caspase-3 activity. Conclusions—We provide the first direct evidence for PR39 as an antiapoptotic factor in endothelial cells during hypoxia. These data suggest that PR39 inhibits hypoxia-induced apoptosis and decreases caspase-3 activity in endothelial cells through an increase of IAP-2 expression.

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-1α (HIF-1α), whereas hypoxia-stimulated RGS5 promoter activity was absent in HIF-1β−/− 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.

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.

RTEF-1, an Upstream Gene of Hypoxia-inducible Factor-1α, Accelerates Recovery from Ischemia

The amount of available hypoxia-inducible factor (HIF)-1α has been considered to be largely a consequence of post-translational modification by multiple ubiquitin-proteasome pathways. However, the role of transcriptional regulation of HIF-1α is less certain, and the mechanisms of transcriptional regulation of HIF-1α require further investigation. Here we report that related transcriptional enhancer factor-1 (RTEF-1), a member of the TEF transcriptional factor family, transcriptionally regulates the HIF-1α gene under normoxic and hypoxic conditions. The expression of HIF-1α mRNA was decreased in endothelial cells in which RTEF-1 was knocked down with siRNA. Sequential deletional analysis of the HIF-1α promoter revealed that the MCAT-like element in the HIF-1α promoter was essential for HIF-1α transcription. Binding of RTEF-1 to the MCAT-like element was confirmed by ChIP. Treatment of endothelial cells with a HIF-1 inhibitor resulted in retardation of RTEF-1-induced proliferation and tube formation. Moreover, increased HIF-1α expression was observed in transgenic mice expressing RTEF-1 under the VE-cadherin promoter (VE-Cad/RTEF-1). VE-Cad/RTEF-1 mice subjected to hindlimb ischemia demonstrated increased levels of HIF-1α, accelerated recovery of blood flow, and increased capillary density compared with littermate controls. These results identify RTEF-1 as a regulator of HIF-1α transcription, which results in up-regulation of HIF-1α and acceleration of recovery from ischemia.

Role of A20 in cIAP-2 Protection against Tumor Necrosis Factor α (TNF-α)-Mediated Apoptosis in Endothelial Cells

Tumor necrosis factor α (TNF-α) influences endothelial cell viability by altering the regulatory molecules involved in induction or suppression of apoptosis. However, the underlying mechanisms are still not completely understood. In this study, we demonstrated that A20 (also known as TNFAIP3, tumor necrosis factor α-induced protein 3, and an anti-apoptotic protein) regulates the inhibitor of apoptosis protein-2 (cIAP-2) expression upon TNF-α induction in endothelial cells. Inhibition of A20 expression by its siRNA resulted in attenuating expression of TNF-α-induced cIAP-2, yet not cIAP-1 or XIAP. A20-induced cIAP-2 expression can be blocked by the inhibition of phosphatidyl inositol-3 kinase (PI3-K), but not nuclear factor (NF)-κB, while concomitantly increasing the number of endothelial apoptotic cells and caspase 3 activation. Moreover, TNF-α-mediated induction of apoptosis was enhanced by A20 inhibition, which could be rescued by cIAP-2. Taken together, these results identify A20 as a cytoprotective factor involved in cIAP-2 inhibitory pathway of TNF-α-induced apoptosis. This is consistent with the idea that endothelial cell viability is dependent on interactions between inducers and suppressors of apoptosis, susceptible to modulation by TNF-α.

RTEF-1 Attenuates Blood Glucose Levels by Regulating Insulin-Like Growth Factor Binding Protein-1 in the Endothelium

Rationale: Related transcriptional enhancer factor-1 (RTEF-1) plays an important role in endothelial cell function by regulating angiogenesis; however, the mechanism underlying the role of RTEF-1 in the endothelium in vivo is not well defined. Objective: We investigated the biological functions of RTEF-1 by disrupting the gene that encodes it in mice endothelium -specific RTEF-1–deficient transgenic mice (RTEF-1–/–). Methods and Results: RTEF-1−/− mice showed significantly increased blood glucose levels and insulin resistance, accompanied by decreased levels of insulin-like growth factor binding protein-1 (IGFBP-1) mRNA in the endothelium and decreased serum IGFBP-1 levels. Additionally, the RTEF-1−/− phenotype was exacerbated when the mice were fed a high-fat diet, which correlated with decreased IGFBP-1 levels. In contrast, vascular endothelial cadherin/RTEF-1–overexpressing1 transgenic mice (VE-Cad/RTEF1) demonstrated improved glucose clearance and insulin sensitivity in response to a high-fat diet. Furthermore, we demonstrated that RTEF-1 upregulates IGFBP-1 through selective binding and promotion of transcription from the insulin response element site. Insulin prevented RTEF-1 expression and significantly inhibited IGFBP-1 transcription in endothelial cells in a dose-dependent fashion. Conclusions: To the best of our knowledge, this is the first report demonstrating that RTEF-1 stimulates promoter activity through an insulin response element and also mediates the effects of insulin on gene expression. These results show that RTEF-1–stimulated IGFBP-1 expression may be central to the mechanism by which RTEF-1 attenuates blood glucose levels. These findings provide the basis for novel insights into the transcriptional regulation of IGFBP-1 and contribute to our understanding of the role of vascular endothelial cells in metabolism.

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.

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. Conclusion—These findings provide novel insights into the transcriptional control of endothelial function in the coordination of cell–cell connections.

Structural and functional analysis of the related transcriptional enhancer factor-1 and NF-κB interaction

The related transcriptional enhancer factor-1 (RTEF-1) increases gene transcription of hypoxia-inducible factor 1α (HIF-1α) and enhances angiogenesis in endothelium. Both hypoxia and inflammatory factor TNF-α regulate gene expression of HIF-1α, but how RTEF-1 and TNF-α coordinately regulate HIF-1α gene transcription is unclear. Here, we found that RTEF-1 interacts with p65 subunit of NF-κB, a primary mediator of TNF-α. RTEF-1 increased HIF-1α promoter activity, whereas expression of p65 subunit inhibited the stimulatory effect. By contrast, knockdown of p65 markedly enhanced RTEF-1 stimulation on the HIF-1α promoter activity (7-fold). A physical interaction between RTEF-1 and p65 was confirmed by coimmunoprecipitation experiments in cells and glutathione S-transferase (GST)-pull-down assays. A computational analysis of RTEF-1 crystal structures revealed that a conserved surface of RTEF-1 potentially interacts with p65 via four amino acid residues located at T347, Y349, R351, and Y352. We performed site-directed mutagenesis and GST-pull-down assays and demonstrated that Tyr352 (Y352) in RTEF-1 is a key site for the formation of RTEF-1 and p65-NF-κB complex. An alanine mutation at Y352 of RTEF-1 disrupted the interaction of RTEF-1 with p65. Moreover, expression of RTEF-1 decreased TNF-α-induced HIF-1α promoter activity, IL-1β, and IL-6 mRNA levels in cells; however, the effect of RTEF-1 was largely lost when Y352 was mutated to alanine. These results indicate that RTEF-1 interacts with p65-NF-κB through Y352 and that they antagonize each other for HIF-1α transcriptional activation, suggesting a novel mechanism by which RTEF-1 regulates gene expression, linking hypoxia to inflammation.