Bisphenol A disrupts granulosa cell function

Because of its widespread use and potential adverse biological effects, bisphenol A (BPA) represents one of the most studied endocrine-disrupting compounds. Within the reproductive system, ovarian granulosa cells have been documented as a target of BPA action, but no consensus has been reached about functional modifications induced by BPA. On these bases, we studied the potential disrupting effects of BPA on the main granulosa cell functional activities, also taking into account a potential interference with the ovarian angiogenic process. Ovarian granulosa cells were isolated from porcine follicles and cultured in the presence or absence of BPA at different concentrations for 48 h. Cell proliferation was studied by measuring adenosine triphosphate content. Progesterone (P4) and estradiol 17β (E2) production was determined by radioimmunoassay. Vascular endothelial growth factor (VEGF) output was quantified by an enzyme-linked immunosorbent assay. Redox status was monitored by measuring superoxide anion and hydrogen peroxide, and by determining the activities of the scavenging enzymes superoxide dismutase, catalase, and peroxidase by colorimetric methods. Granulosa cell proliferation as well as redox status resulted unaffected by BPA. Concentrations of E2 were stimulated by the lower BPA concentration, whereas they were inhibited by the larger doses tested. P4 output was decreased by all BPA concentrations. To the contrary, VEGF production was stimulated. Data indicate that BPA can interfere with reproductive activity by affecting granulosa cell steroidogenesis in vitro; furthermore, BPA can exert a promoting effect on the ovarian angiogenic process by increasing VEGF output in pigs. A disruption of this finely tuned process seems particularly relevant because of the risk of uncontrolled neovascularization.     

Lentivirus-mediated calcitonin gene-related peptide transfection enhances endothelial differentiation of rat bone marrow mesenchymal stem cells

AIM：To study the effect of calcitonin gene-related peptide (CGRP) gene transfection mediated by lentivirus on the differentiation of rat bone marrow mesenchymal stem cells (MSCs) to endothelial cells. METHODS：Rat bone marrow MSCs were isolated by density gradient centrifugation combined with adherence method. Recombinant lentivirus vector carrying CGRP gene (Lenti-CGRP) was transfected into the MSCs. The secretion of CGRP in culture supernatants of the transfected MSCs was detected using ELISA method. The cells at passage 3 were divided into three groups: CGRP group (MSCs transfected with Lenti-CGRP), CGRP+CGRP8-37 (an antagonist of CGRP receptor) group and control group (MSCs transfected with PBS). The differentiation of the MSCs was detected by immunocytochemical staining for CD31 and factor Ⅷ-related antigen. The proliferation of the cells was measured by cell counting, and the angiogenic ability of the cells was analyzed using Matrigel assay. RESULTS：The proportion of CD31-and factor Ⅷ-related antigen-positive cells in CGRP and CGRP+CGRP8-37 groups was larger than that in control group (P<0.05). The numbers of the cells in CGRP and CGRP+CGRP8-37 groups were significantly increased compared with control group (P<0.05). Lumen-like structures were observed in CGRP and CGRP+CGRP8-37 groups. The above indexes in CGRP+CGRP8-37 group were reduced compared with CGRP group. CONCLUSION: Transfection with CGRP gene induces rat bone marrow MSCs to differentiate into endothelial cells and enhances their proliferation, suggesting that CGRP may play a role in the regulation of angiogenesis.     

MCP-1/CCR2 signaling pathway mediates alcohol-induced angiogenesis in breast cancer

AIM To investigate the role of monocyte chemoattractant protein-1 (MCP-1) and its receptor CC chemokine receptor 2 (CCR2) in ethanol-promoted breast cancer angiogenesis and the underlying mechanism. METH?ODS: A mouse model of transplanted breast tumor with moderate alcohol consumption was established. The correlations between the expression of MCP-1/CCR2 and the expression of angiogenesis markers [platelet endothelial cell adhesion molecule-1 (PECAM-1) and vascular endothelial growth factor (VEGF)] in tumor tissues were examined by immunohistochemistry. In vitro, a 3D tumor-endothelial co-culture system was established to observe tumor angiogenesis and the role of MCP-1/CCR2 signaling pathway in alcohol-mediated angiogenesis. The cell migration ability was detected to clarify whether MCP-1/CCR2 enhanced cell mobility to form new vessels. RESULTS MCP-1 and CCR2 were both highly expressed in the breast tumor tissues of tumor-bearing mice consuming alcohol, and their expression levels were consistent with the angiogenic markers PECAM-1 and VEGF (P<0.05). The interaction between mouse breast cancer E0771 cells and endothelial cells was observed to promote angiogenesis in the 3D tumor-endothelial co-culture system with or without alcohol stimulation. MCP-1 promoted this kind of tumor angiogenesis, while CCR2 antagonist effectively inhibited the tumor angiogenesis and especially blocked alcohol-induced angiogenesis. Activation of MCP-1/CCR2 signaling pathway enhanced the migration ability of endothelial cells. CONCLUSION The MCP-1/CCR2 signaling pathway plays an important role in promoting the angiogenesis of breast cancer stimulated by alcohol. The mechanism might be that MCP-1 improves the migration of endothelial cells and then promotes angiogenesis.     

Advances on studies of miR-21 in vascular endothelial function and angiogenesis

MicroRNAs (miRNAs)are a class of non-coding, endogenous, single-stranded small RNA molecules composed of 19~25 nucleotides. miRNAs are widely involved in the process of human life activities. Recent studies have shown that part of miRNAs regulate the vascular endothelial function and angiogenesis. High expression of miRNA-21 is found to play important roles in the cell proliferation, cell apoptosis, cell growth and death of vascular endothelial cells. This review will focus on the recent progress related to miRNAs in vascular endothelial function and angiogenesis, providing a new insight in cardiovascular disease prevention, clinical diagnosis, prognosis and target therapeutics.     

Regulatory effect of astragaloside IV on SDF-1α/CXCR4 in EPCs

AIM: To investigate the effects of astragaloside IV (AS-IV) on chemokine receptor 4 (CXCR4) and stromal cell-derived factor 1α (SDF-1α) in endothelial progenitor cells (EPCs) and its mechanism. METHODS: Rat bone marrow-derived EPCs were cultured in vitro. The proliferation, adhesion, migration, apoptosis and tube formation capacity of EPCs treated with AS-IV and AMD3100, a specific blocker of CXCR4, were observed. The effects of AS-IV on the expression of SDF-1α/CXCR4 at mRNA and protein levels and the protein level of p-CXCR4 in the EPCs were determined. RESULTS: AS-IV significantly enhanced the proliferation, adhesion, migration and tube formation abilities of EPCs, reduced the apoptosis of EPCs, and up-regulated the mRNA and protein expression of SDF-1α and CXCR4 and the p-CXCR4 protein level in the EPCs. On the other hand, AMD3100 blocked the up-regulating effect of AS-IV on the mRNA and protein expression of CXCR4 and the p-CXCR4 protein level in the EPCs, but did not affect the effect of AS-IV on the expression of SDF-1α. CONCLUSION: AS-IV might enhance the biological function of EPCs by regulating the expression of SDF-1α/CXCR in EPCs.     

Primary culture,identification and in vitro angiogenesis of mouse pulmonary microvascular endothelial cells

AIM: To establish a simple and reliable method for primary culture of mouse pulmonary microvascular endothelial cells (PMVECs), and to investigate the ability of angiogenesis of the cells in vitro. METHODS: PMVECs from C57BL/6 new born mice were cultured with peripheral pulmonary tissue pasted method. The cells were observed under inverted phase-contrast microscope and identified by immunocytochemical and immunofluorescent methods. PMVECs were seeded at the density of 20 000 cells per well in 96-well plate on top of growth factor-reduced matrigel and incubated for 24 h at 37 ℃ in 5% CO₂. The plates were examined every 2 h for tube formation under an inverted microscope. RESULTS: The primary PMVECs showed fusiform shape or polygonal, regular cobblestone morphology after fusion to monolayer, and expressed factor Ⅷ -related antigen (ⅧF-Ag). Under fluorescence microscope, those cells demonstrated positive staining for uptaking acetylated low-density lipoprotein (DiI-Ac-LDL) and binding Bandeiraea simpli-cifolia lectin I (FITC-BSI). The purity of PMVECs was above 90%. PMVECs formed capillary-like structures within 4 h to 6 h after plated on matrigel and fully developed a "honeycomb" appearance at 10~12 h, and then the capillary tubes began to break apart. CONCLUSION: The tissue block pasted method for primary cultivation of PMVECs is easy to handle, with high yield and purity, and will further facilitate related studies of well-established tube-formation assay in vitro.     

Cloning and expression of mouse canstatin cDNA in E.coli

AIM: To clone and express mouse canstatin (m canstatin) cDNA and provide a basis for the further research on its anti-angiogenic activity and potential application for cancer therapy. METHODS: Total RNA was extracted from mouse liver tissue by Trizol Reagent, and mouse canstatin cDNA was amplified by RT- PCR, then cloned into vector pMD18-T for sequencing. pET30a(+)-m canstatin recombinant plasmid was constructed and expressed in E.coli BL21 with induction of IPTG. RESULTS: Mouse canstatin cDNA is 684 bp coding 227 amino acids. The sequences of both cDNA and amino acid share high homology with human canstatin, with cDNA identity at 89% and amino acids identity at 96% to human canstatin. In the present study, pET30a(+)-m canstatin recombinant plasmid was expressed in E.coli BL21. CONCLUSION: Mouse canstatin cDNA has been cloned for the first time. Constructed pET30a(+)-m canstatin recombinant plasmid is highly expressed in E.coli BL21.     

Progresses on antitumor effect of snake venom active components

Many snake venoms contain complex mixtures of pharmacologically important molecules, some of which show potential therapeutic value in the treatment of cancer and other human disorders. In this review, we mainly reports the effects of snake venom active components, such as disintegrins and lectins in paralyzing cancer cells, blocking on cell migration, interaction with integrins, inhibition of tumor dissemination and angiogenesis. The advanced researches on the snake venom's apoptosis-inducing components on tumors are also introduced.