Vascular Endothelial Growth Factor Synthesis Research Articles

Interleukin-6 trans-signalling induces vascular endothelial growth factor synthesis partly via Janus kinases-STAT3 pathway in human mesothelial cells.

Interleukin-6 (IL-6) is a vital inflammatory factor in the peritoneal cavity of peritoneal dialysis (PD) patients. Because intraperitoneal inflammation is closely associated with angiogenesis, we sought to explore the effect of IL-6 on vascular endothelial growth factor (VEGF) synthesis and its transduction pathway in mesothelial cells. Human mesothelial cells (Met-5A) were incubated with different concentrations of glucose and mannitol, and the effect of glucose and mannitol on the expression of IL-6 was determined. Then, the cells were stimulated by IL-6 with or without two soluble receptors of IL-6 (sIL-6R or sgp130), and VEGF synthesis was detected. Finally, the cells were incubated with IL-6/sIL-6R combined with or without the inhibitor of Janus kinases (JAK) AG490. The phosphorylation of the signal transducer and activator of transcription 3 (STAT3) and its intracellular translocation were examined. 1. High glucose and mannitol could upregulate IL-6 mRNA expression and IL-6 secretion in mesothelial cells significantly, and there was no difference of its effect between high glucose and mannitol. 2. Met-5A was a cell line with a single IL-6 receptor. The IL-6/sIL-6R complex induced VEGF synthesis of mesothelial cells, which was alleviated by sgp130 or AG490. IL-6 trans-signalling could induce the phosphorylation of STAT3, which is recruited to the cellular nucleus of Met-5A cells. The present study might provide evidence that high glucose upregulates IL-6 synthesis in Met-5A cells, to some extent, depending on its osmolality and that IL-6 trans-signalling could induce VEGF synthesis partly dependent on the JAK/STAT3 pathway.

Heat Shock Protein 70 Negatively Regulates TGF-β-Stimulated VEGF Synthesis via p38 MAP Kinase in Osteoblasts

Background/Aims: We previously demonstrated that transforming growth factor-β (TGF-β) stimulates the synthesis of vascular endothelial growth factor (VEGF) through the activation of p38 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells. Heat shock protein70 (HSP70) is a ubiquitously expressed molecular chaperone. In the present study, we investigated the involvement of HSP70 in the TGF-β-stimulated VEGF synthesis and the underlying mechanism in these cells. Methods: Culture MC3T3-E1 cells were stimulated by TGF-β. Released VEGF was measured using an ELISA assay. VEGF mRNA level was quantified by RT-PCR. Phosphorylation of each protein kinase was analyzed by Western blotting. Results: VER-155008 and YM-08, both of HSP70 inhibitors, significantly amplified the TGF-β-stimulated VEGF release. In addition, the expression level of VEGF mRNA induced by TGF-β was enhanced by VER-155008. These inhibitors markedly strengthened the TGF-β-induced phosphorylation of p38 MAP kinase. The TGF-β-induced phosphorylation of p38 MAP kinase was amplified in HSP70-knockdown cells. SB203580, an inhibitor of p38 MAP kinase, significantly suppressed the amplification by these inhibitors of the TGF-β-induced VEGF release. Conclusion: These results strongly suggest that HSP70 acts as a negative regulator in the TGF-β-stimulated VEGF synthesis in osteoblasts, and that the inhibitory effect of HSP70 is exerted at a point upstream of p38 MAP kinase.

Designer Leptin Receptor Antagonist Allo-aca Inhibits VEGF Effects in Ophthalmic Neoangiogenesis Models.

Experimental and clinical data suggest that pro-angiogenic, pro-inflammatory and mitogenic cytokine leptin can be implicated in ocular neovascularization and other eye pathologies. At least in part, leptin action appears to be mediated through functional interplay with vascular endothelial growth factor (VEGF). VEGF is a potent regulator of neoangiogenesis and vascular leakage with a proven role in conditions such as proliferative diabetic retinopathy, age-related macular degeneration and diabetic macular edema. Accordingly, drugs targeting VEGF are becoming mainstream treatments for these diseases. The crosstalk between leptin and VEGF has been noted in different tissues, but its involvement in the development of eye pathologies is unclear. Leptin is coexpressed with VEGF during ocular neovascularization and can potentiate VEGF synthesis and angiogenic function. However, whether or not VEGF regulates leptin expression or signaling has never been studied. Consequently, we addressed this aspect of leptin/VEGF crosstalk in ocular models, focusing on therapeutic exploration of underlying mechanisms. Here we show, for the first time, that in retinal (RF/6A) and corneal (BCE) endothelial cells, VEGF (100 ng/mL, 24 h) stimulated leptin mRNA synthesis by 70 and 30%, respectively, and protein expression by 56 and 28%, respectively. In parallel, VEGF induced RF/6A and BCE cell growth by 33 and 20%, respectively. In addition, VEGF upregulated chemotaxis and chemokinesis in retinal cells by ~40%. VEGF-dependent proliferation and migration were significantly reduced in the presence of the leptin receptor antagonist, Allo-aca, at 100–250 nmol/L concentrations. Furthermore, Allo-aca suppressed VEGF-dependent long-term (24 h), but not acute (15 min) stimulation of the Akt and ERK1/2 signaling pathways. The efficacy of Allo-aca was validated in the rat laser-induced choroidal neovascularization model where the compound (5 μg/eye) significantly reduced pathological vascularization with the efficacy similar to that of a standard treatment (anti-VEGF antibody, 1 μg/eye). Cumulatively, our results suggest that chronic exposure to VEGF upregulates leptin expression and function. As leptin can in turn activate VEGF, the increased abundance of both cytokines could amplify pro-angiogenic and pro-inflammatory environement in the eye. Thus, combined therapies targeting ObR and VEGF should be considered in the treatment of ocular diseases.

Effects of low-level laser therapy and epidermal growth factor on the activities of gingival fibroblasts obtained from young or elderly individuals.

This study evaluated the effects of low-level laser therapy (LLLT) and epidermal growth factor (EGF) on fibroblasts obtained from young and elderly individuals. Gingival fibroblasts from young (Y) and elderly (E) individuals were seeded in wells of 24-well plates with Dulbecco's modified Eagle's medium (DMEM) containing 10% of fetal bovine serum (FBS). After 24h, the cells were irradiated (LASERTable-InGaAsP-780 ± 3nm, 25mW, 3J/cm2) or exposed to EGF (100μM). After 72h, cells were evaluated for viability, migration, collagen and vascular endothelial growth factor (VEGF) synthesis, and gene expression of growth factors. Data were analyzed by Kruskal-Wallis and Mann-Whitney tests (α = 5%). Y and E fibroblasts irradiated with laser or exposed to EGF showed increased viability and collagen synthesis. Enhanced cell migration was observed for Y fibroblasts after both treatments, whereas only the LLLT stimulated migration of E cells. VEGF synthesis was higher for Y and E cells exposed to EGF, while this synthesis was reduced when E fibroblasts were irradiated. Increased gene expression of VEGF was observed only for Y and E fibroblasts treated with LLLT. Regardless of a patient's age, the LLLT and EGF applications can biostimulate gingival fibroblast functions involved in tissue repair.

Leukotriene B4 Receptor 2 Is Critical for the Synthesis of Vascular Endothelial Growth Factor in Allergen-Stimulated Mast Cells.

Mast cells are among the principal effector cells in the pathogenesis of allergic asthma. In allergic reactions, allergen (Ag)-induced cross-linking of IgE bound to FcεRI on mast cells results in the production of vascular endothelial growth factor (VEGF), which is essential for the initiation and development of the allergic response. Despite the central role of VEGF in allergic asthma, the signaling events responsible for the production of VEGF remain unclear, particularly in Ag-stimulated mast cells. In the present study, we observed that blocking leukotriene B4 receptor 2 (BLT2) completely abrogated the production of VEGF in Ag-stimulated bone marrow-derived mast cells (BMMCs). The synthesis of BLT2 ligands (leukotriene B4 and 12(S)-hydroxyeicosatetraenoic acid) was also required for VEGF production, suggesting a mediating role of an autocrine BLT2 ligands-BLT2 axis in the production of VEGF in mast cells. The NADPH oxidase 1-reactive oxygen species-NF-κB cascade is downstream of BLT2 during Ag signaling to VEGF synthesis in mast cells. Furthermore, the level of VEGF synthesis in genetically mast cell-deficient Kit(W/Wv) mice was significantly lower than that in wild-type mice in the OVA-induced asthma model, suggesting that mast cells play a critical role in the synthesis of VEGF in OVA-induced allergic asthma. Importantly, VEGF production was restored to the levels observed in wild-type mice after adoptive transfer of normal BMMCs into Kit(W/Wv) mice but was not restored in BLT2(-/-) BMMC-reconstituted Kit(W/Wv) mice in the OVA-induced asthma model. Taken together, our results suggest that BLT2 expression in mast cells is essential for the production of VEGF in OVA-induced allergic asthma.

Effects and mechanisms of docosahexaenoic acid on the generation of angiopoietin-2 by rat brain microvascular endothelial cells under an oxygen- and glucose-deprivation environment.

ObjectiveThe aim of this study was to investigate the effects of docosahexaenoic acid (DHA) on the generation of angiopoietin-2 (Ang-2) by rat brain microvascular endothelial cells under an oxygen- and glucose-deprivation environment (OGD), and its relationship, if any, with cyclooxygenase 2 (COX-2) expression.MethodsAnnexin V and propidium iodide apoptosis assay was used to detect apoptosis. Enzyme linked immunosorbent assay was used to detect Ang-2, vascular endothelial growth factor (VEGF), prostaglandin E2 (PGE2), and prostaglandin I2 (PGI2) content. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect Ang-2 and VEGF mRNA expression. Western blot was used to detect expression of COX-2 protein.ResultsDHA reduced the apoptosis rate (P = 0.026) and decreased the secretion of Ang-2, VEGF, PGE2, and PGI2 (P = 0.006, P = 0.000, P = 0.002, P = 0.004 respectively). The relative expression of Ang2 and Vegf mRNA, as well as COX-2 expression, also decreased (P = 0.000, P = 0.005, P = 0.007 respectively). These effects were antagonized by GW9662 (peroxisome proliferator-activated receptor-γ antagonist). COX-2 protein expression levels were positively correlated with Ang2 and Vegf mRNA expression levels (γ = 0.69, P = 0.038 and γ = 0.76, P = 0.032, respectively). Ang-2 and VEGF mRNA levels were positively correlated with Ang-2 (γ = 0.84, P = 0.012) and VEGF (γ = 0.71, P = 0.036) secretion levels respectively.ConclusionDHA reduced apoptosis induced by an OGD environment, thus decreasing Ang-2 and VEGF synthesis. This phenomenon was associated with a decrease in COX-2 protein expression, PGE2 and PGI2 secretion, and generation regulation via intracellular transcriptional pathways.

Regulation of vascular endothelial growth factor production in mouse thymic epithelial cell lines

Vascular endothelial growth factor (VEGF) is synthesized in small amounts by thymus epithelial cells in adults and plays a role in supporting vascular homeostasis. However its role becomes dramatically important during the process of thymus reparation after involution caused by chemo-, x-ray or hormonal therapy. The aim of the study was to evaluate the influence of different factors on VEGF production by mouse thymic epithelial cells in vitro. As a model two cell lines were used: cortical cTEC1-2 and medullar mTEC3-10 cells. These cells were characterized by their ability to synthesize VEGF mRNA and protein as well as by their expression of VEGF receptors. VEGFR1 and VEGFR2 mRNA expression in these cells were absent while NRP-1 mRNA revealed low level of expression. It was shown by ELISA that cTEC1-2 cells produced VEGF about 30 times more than mTEC3-10. When cultivated in the presence of cytokines, hormonal factors or thymocytes, both cell lines responded differently. Introduction of keratinocyte growth factor (KGF) induced VEGF mRNA expression as well as VEGF production in medullar cells but simultaneously down-regulated VEGF mRNA expression in cortical cells. Dexamethasone suppressed mRNA VEGF expression and VEGF production in cortical cells while in medullar cells only VEGF production was reduced. Introduction of IL-7, IL-1b or murine thymocytes increased while addition of Semaphorin 3A, SDF-1a or ACTH decreased VEGF production by cortical epithelial cells with no influence on medullar cells. We suggest that our data obtained in vitro can be used for further development of special programs for directed regulation of VEGF synthesis in the thymus epithelial cells in the vivo.

Pigment epithelium derived factor upregulates expression of vascular endothelial growth factor by human mesenchymal stem cells: Possible role in PEDF regulated matrix mineralization

Pigment epithelium-derived factor (PEDF) encoded by serpinf1 is a potent antiangiogenic factor found in a wide variety of fetal and adult tissues. Several reports have shown that lack of PEDF leads to osteogenesis imperfecta (OI) type VI whose hallmark is a defect in mineralization that leads to excessive osteoid build up that fails to mineralize. Because PEDF is antiangiogenic factor it would pose serious consequences on bone development and healing of fractures. To understand possible mechanisms by which PEDF plays a role in bone development and regulation of matrix mineralization, we determined the effects of exogenous PEDF on vascular endothelial growth factor (VEGF) expression by human mesenchymal stem cells (hMSCs) and mechanisms of its regulation by PEDF. Human MSCs incubated in normal medium supplemented with exogenous PEDF increased VEGF expression; this increase was also seen when PEDF was added to hMSCs undergoing osteogenic differentiation. MSCs maintained in osteogenic medium increased synthesis of both VEGF and PEDF but both factors were maintained relatively in balance during differentiation. To understand mechanisms by which exogenous PEDF regulated VEGF expression, hMSCs exposed to PEDF activated Erk signaling pathway in MSCs; inhibition of Erk signaling reduced VEGF mRNA expression as well as protein production suggesting that PEDF regulates VEGF expression in MSCs via Erk signaling pathway. In conclusion, PEDF increases VEGF expression by MSCs suggesting that regulation of VEGF by PEDF may be part of the mechanisms by which PEDF regulates osteoblastic mineralization.

Downregulation of LH and FSH receptors after hCG and eCG treatments in the porcine oviduct

The influence of induction of ovulation and superovulation with eCG and hCG on LH and FSH receptor levels in porcine oviducts on day 3 postcoitum was studied. In experiment I, gilts were assigned into cyclic (control; n = 5) and inseminated (n = 5) groups. In experiment II, there were 3 groups of animals: inseminated (n = 5), induced ovulation/inseminated (750 IU eCG, 500 IU hCG; n = 5) and superovulated/inseminated (1500 IU eCG, 1000 IU hCG; n = 5) gilts. Oviduct tissues were collected 3 d after insemination or PBS infusion. The messenger RNA (mRNA) expression of FSH receptor (FSHR) and luteinizing hormone/chorionic gonadotropin receptor (LH/CGR) was measured by real-time reverse transcription PCR and protein levels using Western blots. Localization of LH/CGR and FSHR-positive cells was studied by immunohistochemical staining. Insemination by itself did not influence mRNA and protein levels of LH/CGR. However, FSHR mRNA expression in the isthmus and ampulla of the oviduct was affected by insemination (P < 0.05). Similarly, insemination decreased FSHR protein level in the isthmus (P < 0.05). Stimulation with hCG and eCG did not affect LH/CGR and FSHR mRNA expression, either in the isthmus or in the ampulla. Nevertheless, superovulation decreased LH/CGR protein level in the oviductal ampulla (P < 0.05) in comparison with inseminated gilts. Similarly, protein levels of FSHR in the oviductal ampulla decreased after superovulation (P < 0.05). LH/CGR-positive cells were observed in the mucosa as well as in smooth muscle cells of both parts of the oviduct. Follicle-stimulating hormone receptor–positive cells were observed in smooth muscle cells and blood vessels of the isthmus. In the ampulla, FSHR-positive cells were observed in the smooth muscle as well as in the mucosa. Summarizing, the present study revealed for the first time that stimulation with eCG and hCG, especially in high doses, can change LH/CGR and FSHR levels in porcine oviducts. This may in turn alter many signaling pathways, eg, PGs or vascular endothelial growth factor synthesis, and consequently disturb the oviductal environment, with possible detrimental effects on fertilization and/or embryonic development.

The synthesis of VEGF in allergen-stimulated mast cells is through a leukotriene B4 receptor-2-dependent signaling pathway

Abstract Mast cells are among the principal effector cells in the pathogenesis of allergic asthma. In allergic reactions, allergen(Ag)-induced cross-linking of IgE bound to FcɛRI on mast cells results in the production of vascular endothelial growth factor(VEGF), which is essential for the initiation and development of the allergic response. Despite the central role of VEGF in allergic asthma, the signaling events responsible for the production of VEGF remain unclear, particularly in Ag-stimulated mast cells. In the present study, we observed that blocking leukotriene B4 receptor-2(BLT2) completely abrogated the production of VEGF in Ag-stimulated bone marrow-derived mast cells(BMMCs). The synthesis of BLT2 ligands (leukotriene B4 [LTB4] and 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE]) was also required for VEGF production, suggesting a mediating role of an autocrine BLT2 ligands-BLT2 axis in the production of VEGF in mast cells. The Nox1-reactive oxygen species(ROS)-NF-kB cascade is downstream of BLT2 during Ag signaling to VEGF synthesis in mast cells. Furthermore, the level of VEGF synthesis in genetically mast cell-deficient Kit(W/Wv) mice was significantly lower than that in wild-type(WT) mice in the ovalbumin(OVA)-induced asthma model, suggesting that mast cells play a critical role in the synthesis of VEGF in OVA-induced allergic asthma. Importantly, VEGF production was restored to the levels observed in WT mice after adoptive transfer of normal BMMCs into Kit(W/Wv) mice but was not restored in BLT2−/− BMMC-reconstituted Kit(W/Wv) mice in the OVA-induced asthma model. Taken together, our results suggest that BLT2 expression in mast cells is essential for the production of VEGF in OVA-induced allergic asthma.

MiR-18b suppresses high-glucose-induced proliferation in HRECs by targeting IGF-1/IGF1R signaling pathways

MicroRNAs (miRNAs) are important for the proliferation of endothelial cells and have been shown to be involved in diabetic retinopathy (DR). In previous study, we found that miRNAs might play a critical role in hyperglycemia-induced endothelial cell proliferation based on miRNA expression profiling. Here, the roles of microRNA-18b (miR-18b) in the proliferation of human retinal endothelial cells (HRECs) were investigated in an in vitro model of HRECs grown in high glucose. We identified that levels of miR-18b were decreased in high-glucose-induced HRECs, compared with those in cells incubated in normal glucose. However, the reduction of miR-18b up-regulated vascular endothelial growth factor (VEGF) secretion and promoted effects on in vitro proliferation of HRECs. Mechanistically, insulin growth factor-1 (IGF-1) was identified as a target of miR-18b. IGF-1 simulation could antagonize the effect induced by miR-18b up-regulation, promoting cell proliferation and increasing VEGF production. In contrast, the opposite results were observed with silencing IGF-1, which was consistent with the effects of miR-18b overexpression. MiR-18b exerted its function on VEGF synthesis and cell proliferation by suppressing the IGF-1/insulin growth factor-1 receptor (IGF1R) pathway, consequently inhibiting the downstream phosphorylation of Akt, MEK, and ERK. Hence, this may provide a new insight into understanding the mechanism of DR pathogenesis, as well as a potential therapeutic target for proliferative DR.

Regenerative effect of the polydeoxyribonucleotide after sciatic nerve transection in mouse

Factors that have effects on postoperative nerve regeneration are surgery period, maneuver and the use of material that promotes nerve regeneration. Of various materials that promote nerve regeneration, vascular endothelial growth factor (VEGF) is known as cytokine that plays an important role in regenerating not only vessels but also nerves. Polydeoxyribonucleotide (PDRN) promotes the synthesis of VEGF. Thus, assuming that PDRN has the effect of peripheral nerve regeneration, mice were classified into a group with PDRN being administered, a group with VEGF being administered, and a group with nothing administered. Placentex® (PDRN) and VEGF were used, and 40 male mice were used. Then, the mice were classified into two test groups with Placentex® and VEGF injection, and the control group with NaCl 0.9%. After 2 and 4 weeks, the mice were killed and their histological examination was conducted through electron microscope. It revealed that each group was insignificantly different in terms of all comparative items, but the finding after 4 weeks revealed that the test group with PDRN and VEGF showed a statistically significant effect of promoting nerve regeneration. Further, all histological examinations found that the group with PDRN and VEGF promoted nerve regeneration. Regarding the period, the group with VEGF, compared to the PDRN group, showed a better result, but the difference proved to be statistically insignificant. It was confirmed that PDRN had the effect of promoting nerve regeneration; thus PDRN can help restore peripheral nerve damage, caused by trauma and surgery, and promote the prognosis.

Inhibition of Vascular Endothelial Growth Factor Synthesis in Human Retinal Pigment Epithelial Cells by Pazopanib

Age-related macular degeneration (AMD) is a leading cause of central vision blindness in most developed countries. Several new drugs are now available to attempt to prevent progression or treat AMD, however, they have limited effectiveness and therefore are of limited use. Human retinal pigment epithelial (hRPE) cells have been implicated in pathogenesis of AMD via the synthesis of vascular endothelial growth factor (VEGF), which can result in pathological proliferation of vascular endothelial cells and hRPE cells via tyrosine-kinase signaling pathways. Therefore, tyrosine-kinase inhibitors should be considered as potential pharmaco-therapeutic agents in the prevention and treatment of AMD. Since pazopanib (PZB) is a tyrosine-kinase inhibitor of VEGF-R1/VEGF-R2, we investigated its effect on hRPE cell proliferation and VEGF synthesis. We showed that PZB inhibited hRPE cell proliferation and VEGF synthesis in hRPE cells, and therefore it may be of therapeutic value in AMD.

Computational Model of MicroRNA Control of HIF-VEGF Pathway: Insights into the Pathophysiology of Ischemic Vascular Disease and Cancer.

HRMs (hypoxia-responsive miRNAs) are a specific group of microRNAs that are regulated by hypoxia. Recent studies revealed that several HRMs including let-7 family miRNAs were highly induced in response to HIF (hypoxia-inducible factor) stabilization in hypoxia, and they potently participated in angiogenesis by targeting AGO1 (argonaute 1) and upregulating VEGF (vascular endothelial growth factor). Here we constructed a novel computational model of microRNA control of HIF-VEGF pathway in endothelial cells to quantitatively investigate the role of HRMs in modulating the cellular adaptation to hypoxia. The model parameters were optimized and the simulations based on these parameters were validated against several published in vitro experimental data. To advance the mechanistic understanding of oxygen sensing in hypoxia, we demonstrated that the rate of HIF-1α nuclear import substantially influences its stabilization and the formation of HIF-1 transcription factor complex. We described the biological feedback loops involving let-7 and AGO1 in which the impact of external perturbations were minimized; as a pair of master regulators when low oxygen tension was sensed, they coordinated the critical process of VEGF desuppression in a controlled manner. Prompted by the model-motivated discoveries, we proposed and assessed novel pathway-specific therapeutics that modulate angiogenesis by adjusting VEGF synthesis in tumor and ischemic cardiovascular disease. Through simulations that capture the complex interactions between miRNAs and miRNA-processing molecules, this model explores an innovative perspective about the distinctive yet integrated roles of different miRNAs in angiogenesis, and it will help future research to elucidate the dysregulated miRNA profiles found in cancer and various cardiovascular diseases.

Blockade of vascular endothelial growth factor-A/receptor 2 exhibits a protective effect on angiotensin-II stimulated podocytes.

Vascular endothelial growth factor (VEGF) and Angiotensin II (Ang-II) are important in glomerulosclerosis, which is one of the main causes of chronic kidney disease. Previous studies have demonstrated that angiotensin type 1 receptor blocker (ARB) can inhibit the synthesis of VEGF mediated by Ang‑II and can effectively treat diabetic nephropathy. In the present study, the expression of VEGF and its receptors (VEGFR1/VEGFR2) was examined in Ang‑II stimulated podocytes, which were treated with SU5416, a specific VEGFR2 inhibitor. The protein expression of synaptopodin, VEGFR1/2, phosphorylated VEGFR2 and extracellular signal‑regulated kinases (ERK) was assessed by western blot analysis. The mRNA expression of transforming growth factor (TGF)‑β1 was examined by reverse transcription‑quantitative polymerase chain reaction. It was observed that Ang‑II increased the expression of VEGF‑A and VEGFR2. Simultaneously, the increased expression of phosphorylated (p‑)VEGFR2 and p‑ERK induced by Ang‑II was downregulated by SU5416. SU5416 can decrease the expression of synaptopodin and increase the expression of TGF‑β1 induced by Ang‑II as well as ARB treatment. The expression of VEGFR1 remained unchanged by either Ang‑II or SU5416 treatment. However, the normal podocytes administered SU5416 alone showed low levels of synaptopodin and high expression of TGF‑β1 compared with the control. In conclusion, VEGF‑A/VEGFR2 may be essential for podocytes in a normal state. It is suggested that blockade of VEGF‑A/VEGFR2 may exhibit a protective effect on Ang-II stimulated podocytes.

Promotion of Astragaloside IV for EA-hy926 Cell Proliferation and Angiogenic Activity via ERK1/2 Pathway.

The aim of this study was to determine the pro-angiogenic effects of Astragaloside IV (AS-IV) in vitro and reveal the potential mechanisms. A kind of human umbilical vein endothelial cells (HUVECs), named EA-hy926 cells, were treated with various dosages of AS-IV. We then utilized Cell Counting Kit-8 (CCK-8), real-time PCR and Western blot to detect EA-hy926 cells' proliferation and proangiogenic effect from AS-IV. Data showed that AS-IV promoted EA-hy926 cells proliferation, as assessed by CCK-8. The AS-IV was also associated with an increased tube formation and upregulation of vascular endothelial growth factor (VEGF) mRNA and protein in a dose-dependent manner. Interestingly, the influence of AS-IV on cell proliferation and angiogenisis could be abolished by inhibitor PD98059 through suppressed extracellular signal regulated protein kinases1/2 (ERK1/2) phosphorylation. These data demonstrated that the AS-IV activated the ERK1/2 pathway to control VEGF synthesis. Our findings conclude that the AS-IV promotes EA-hy926 cells proliferation and angiogenesis through ERK1/2 pathway, and it is also a regulator of VEGF.

Pretreatment of mesenchymal stem cells with angiotensin II enhances paracrine effects, angiogenesis, gap junction formation and therapeutic efficacy for myocardial infarction

BackgroundPretreatment of mesenchymal stem cells (MSCs) with growth factors is reported to be an effective route for improving cell-based therapy of myocardial infarction (MI). Angiotensin II (Ang II) triggers vascular endothelial growth factor (VEGF) synthesis in MSCs. This study aimed to investigate the effects and mechanisms of Ang II pretreatment in enhancing the therapeutic efficacy of MSCs in MI. MethodsMSCs and endothelial cells (ECs) were isolated from Sprague-Dawley rats. After pretreated with or without 100nM of Ang II for 24h, the MSCs were directly injected into the border zones of the ischemic heart. Cardiac function, fibrosis, infarct size, VEGF expression, angiogenesis, and cell differentiation in the infarcted myocardium were determined after 30days. The cell apoptosis of MSCs post hypoxia was assessed using flow cytometry. The angiogenic activity of MSCs was analyzed using tube formation assay. The gap junction protein connexin-43 (Cx43) expression was detected. ResultsCompared with the MSC group, pretreatment of MSCs with Ang II resulted in better cardiac function, less cardiac fibrosis, smaller infarct size, and higher expression of VEGF and Von Willebrand Factor in ischemic myocardium, but no promotion of cardiomyocyte-like differentiation of MSCs. Ang II pretreatment enhanced the survival of MSCs and the H9c2 cells surrounding MSCs, and augmented the tube formation of ECs and MSCs. Ang II pretreatment up-regulated the Cx43 expression. ConclusionsThe pretreatment of MSCs with Ang II improved the outcome of MSC-based therapy for MI via the mechanisms of enhancing the paracrine production of VEGF, angiogenesis, and gap junction formation.

Resveratrol suppresses TGF-β-induced VEGF synthesis in osteoblasts: Inhibition of the p44/p42 MAPKs and SAPK/JNK pathways.

Resveratrol, which is found in grape and berry skins and red wine, is generally known to be beneficial for human health due to its anti-inflammation and antioxidant effects. We have recently reported that transforming growth factor-β (TGF-β) stimulates vascular endothelial growth factor (VEGF) synthesis through Smad-independent pathways, such as the p38 mitogen-activated protein (MAP) kinase, p44/p42 MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) pathways, in osteoblast-like MC3T3-E1 cells. The aim of the present study was to investigate the effect of resveratrol on the TGF-β-induced VEGF synthesis and the mechanism in osteoblast-like MC3T3-E1 cells. Resveratrol significantly suppressed the TGF-β-stimulated release of VEGF and the VEGF mRNA expression levels. SRT1720, a synthetic sirtuin 1 (SIRT1) activator, also reduced the VEGF release and the mRNA levels. With regard to the intracellular signaling in the TGF-β-stimulated VEGF synthesis, resveratrol and SRT1720 significantly attenuated the phosphorylation of p44/p42 MAP kinase and SAPK/JNK stimulated by TGF-β; however, the TGF-β-induced phosphorylation of Smad2 and p38 MAP kinase was hardly affected by resveratrol or SRT1720. These results strongly suggest that the TGF-β-stimulated VEGF synthesis is suppressed by resveratrol through the inhibition of p44/p42 MAP kinase and SAPK/JNK in osteoblasts, and that the suppressive effect is mediated, at least in part, via SIRT1 activation.

Rac limits TGF-β-induced VEGF synthesis in osteoblasts

We previously showed that transforming growth factor-β (TGF-β) stimulates vascular endothelial growth factor (VEGF) synthesis via p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the involvement of Rac, which is a member of the Rho family of small GTPases, in the TGF-β-stimulated VEGF synthesis in MC3T3-E1 cells. TGF-β markedly increased the levels of GTP-bound Rac. NSC23766, a selective inhibitor of Rac-guanine nucleotide exchange factor interaction, significantly increased both the release of VEGF and the mRNA expression levels induced by TGF-β. In addition, the release of VEGF stimulated by TGF-β was amplified in Rac-knock down cells. Meanwhile, SIS3, a specific inhibitor of TGF-β-dependent Smad3 phosphorylation, significantly reduced the TGF-β-stimulated VEGF release. However, the phosphorylation of Smad2 or Smad3 induced by TGF-β was hardly affected by NSC23766. On the other hand, NSC23766 enhanced the TGF-β-induced phosphorylation of p38 MAP kinase without affecting the phosphorylation of p44/p42 MAP kinase or SAPK/JNK. Furthermore, the phosphorylation of p38 MAP kinase induced by TGF-β was markedly upregulated in the Rac-knock down cells. These results strongly suggest that Rac negatively regulates the TGF-β-stimulated VEGF synthesis via the inhibition of p38 MAP kinase in osteoblasts.

Resveratrol reduces prostaglandin E1-stimulated osteoprotegerin synthesis in osteoblasts: Suppression of stress-activated protein kinase/c-Jun N-terminal kinase

Resveratrol, a natural polyphenol mainly existing in red grapes and berries, possesses beneficial effects on human being. We have previously reported that prostaglandin E1 (PGE1) stimulates vascular endothelial growth factor synthesis via activation of p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) but not p44/p42 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the PGE1-effect on osteoprotegerin (OPG) synthesis and the effect of resveratrol on the synthesis in MC3T3-E1 cells. PGE1 induced the expression levels of OPG mRNA and stimulated the OPG release. Resveratrol significantly reduced the PGE1-induced OPG release and the mRNA expression. SRT1720, an activator of SIRT1, suppressed the release of OPG. The protein levels of SIRT1 were not up-regulated by resveratrol with or without PGE1. Both SB203580 and SP600125, a specific p38 MAP kinase inhibitor and a specific SAPK/JNK inhibitor, respectively, but not PD98059, a specific MEK inhibitor, reduced the PGE1-stimulated OPG release. Resveratrol or SRT1720 failed to affect the phosphorylation of p38 MAP kinase. On the contrary, PGE1-induced phosphorylation of SAPK/JNK was significantly attenuated by both resveratrol and SRT1720. Our results strongly suggest that resveratrol inhibits PGE1-stimulated OPG synthesis via suppressing SAPK/JNK but not p38 MAP kinase in osteoblasts.