A genomic sequence comparison within and flanking the ERRbeta genes of eight species demonstrated that short-form hERRbeta lacks an F domain and is the matched homolog of mouse and rat ERRbeta proteins in humans. However, hERRbeta2-Delta10 and the previously reported hERRbeta2 isoforms are primate specific. RT-PCR analysis showed that short-form hERRbeta has a wide distribution in the 24 of 27 human tissues and cell lines tested, whereas hERRbeta2 and hERRbeta2-Delta10 were only expressed in testis and kidney. The three human ERRbeta-splicing isoforms have different transcriptional activities when measured on an estrogen response element-driven luciferase reporter in transfection assays. The localization of a nuclear localization signal of short-form hERRbeta was also determined. Interestingly, the F domain of hERRbeta2 alters the function of the nuclear localization signal. Therefore, the ERRbeta isoforms are likely to have diverse biological functions in vivo, and characterizing the three isoforms of ERRbeta will lead to an understanding of the multiple levels of gene regulation involved in steroid receptor-signaling pathways in humans and may provide novel therapeutic targets for human diseases.

Objective-We examined the impact of C-reactive protein (CRP) on vascular smooth muscle cell (VSMC) expression of tissue factor (TF) and TF pathway inhibitor (TFPI

BackgroundPlatelets play a fundamental role in maintaining hemostasis and have been shown to participate in hematogenous dissemination of tumor cells. Abundant platelets were detected in the tumor microenvironment outside of the blood vessel, thus, platelet -tumor cell interaction outside of the bloodstream may play a role in regulating primary tumor growth and metastasis initiation. However, it is unclear that platelet depletion affects tumor vessel structure and dynamics.MethodsUsing thrombocytopenia induction in two different tumor-bearing mouse models, tumor tissues were performed by Westernblotting and immunohistochemical staining. Vascular permeability was evaluated by determination of intratumoral Evans blue and Miles vascular permeability assay. Furthermore, microdialysis was used to examining the intratumoral extracellular angiogenic growth factors (VEGF, TGF-β) by ELISA.ResultsPlatelet depletion showed no change in tumor growth and reduced lung metastasis. Platelet depletion led to reduced tumor hypoxia and Met receptor activation and was associated with a decreased release of MMP-2, 9, PAI-1, VEGF, and TGF-β. Tumor vessels in platelet-depleted mice showed impaired vessel density and maturation.ConclusionsOur findings demonstrate that platelets within the primary tumor microenvironment play a critical role in the induction of vascular permeability and initiation of tumor metastasis.

Objective Plasminogen activator inhibitor-1 (PAI-1) regulates angiogenesis via effects on extracellular matrix proteolysis and cell adhesion. However, no previous study has implicated PAI-1 in controlling vascular endothelial growth factor (VEGF) signaling. We tested the hypothesis that PAI-1 down-regulates VEGF receptor-2 (VEGFR-2) activation by inhibiting a vitronectin (VN)-dependent cooperative binding interaction between VEGFR-2 and αVβ3. Approach and Results We studied PAI-1's effects on VEGF signaling in human umbilical vein endothelial cells (HUVECs). PAI-1 inhibited VEGF-induced phosphorylation of VEGFR-2 in HUVECs grown on VN, but not on fibronectin or collagen. PAI-1 inhibited binding of VEGFR-2 to β3 integrin, VEGFR-2 endocytosis, and intracellular signaling pathways downstream of VEGFR-2. The anti-VEGF effect of PAI-1 was mediated by 2 distinct pathways, one requiring binding to VN and another requiring binding to very-low-density-lipoprotein receptor (VLDLR). PAI-1 inhibited VEGF-induced angiogenesis in vitro and in vivo, and pharmacological inhibition of PAI-1 promoted collateral arteriole development and recovery of hindlimb perfusion after femoral artery interruption. Conclusions PAI-1 inhibits activation of VEGFR-2 by VEGF by disrupting a VN-dependent, pro-angiogenic binding interaction involving αVβ3 and VEGFR-2. These results broaden our understanding of the roles of PAI-1, VN, and endocytic receptors in regulating VEGFR-2 activation and suggest novel therapeutic strategies for regulating VEGF signaling.

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Metformin, an anti-diabetic drug commonly used for type 2 diabetes therapy, is associated with anti-angiogenic effects in conditions beyond diabetes. miR-21 has been reported to be involved in the process of angiogenesis. However, the precise regulatory mechanisms by which the metformin-induced endothelial suppression and its effects on miR-21-dependent pathways are still unclear. Bioinformatic analysis and identification of miR-21 and its targets and their effects on metformin-induced antiangiogenic activity were assessed using luciferase assays, quantitative real-time PCR, western blots, scratch assays, CCK-8 assays and tubule formation assays. In this study, miR-21 was strikingly downregulated by metformin in a time- and dose-dependent manner. miR-21 directly targeted the 3′-UTR of PTEN and SMAD7, and negatively regulated their expression. Overexpression of miR-21 abrogated the metformin-mediated inhibition of endothelial cells proliferation, migration, tubule formation and the TGF-β-induced AKT, SMAD- and ERK-dependent phosphorylations, and conversely, down-regulation of miR-21 aggravated metformin’s action and revealed significant promotion effects. Our study broadens our understanding of the regulatory mechanism of miR-21 mediating metformin-induced anti-angiogenic effects, providing important implications regarding the design of novel miRNA-based therapeutic strategies against angiogenesis.

The coordinated activation of both VEGFR-1 and VEGFR-2 represents a more potent arteriogenic stimulus compared to the isolated activation of either one of these two receptors. These data imply that the activation of both monocytes and endothelial cells is necessary to obtain a maximal VEGF-induced activation of arteriogenesis.