Expression Levels Of Vascular Endothelial Growth Factor A Research Articles

NF‑κB‑miR15a‑bFGF/VEGFA axis contributes to the impaired angiogenic capacity of BM‑MSCs in high fat diet‑fed mice.

Potent paracrine properties, such as secretion of angiogenic cytokines and growth factors, have been considered essential for the function of mesenchymal stem cells (MSCs) in tissue regeneration and repair. The present study determined that bone marrow‑derived mesenchymal stem cells from mice fed a high fat diet (HFD) had reduced pro‑angiogenic capacity, as evident from the reduced expression of vascular endothelial growth factor A (VEGFA) and basic fibroblast growth factor (bFGF); therefore, a reduced number of branches was induced in the angiogenesis assay. Additionally, the present study determined that miR‑15a, a putative microRNA targeting both VEGFA and bFGF, may simultaneously downregulate bFGF and VEGFA expression levels through the 3'‑untranslated region. Inhibition of miR‑15a using an antagonist restored the expression of VEGFA and bFGF under fatty acid treatment and thus the angiogenic capacity. Furthermore, the HFD and fatty acids treatments transcriptionally activated the expression of miR‑15a via nuclear factor‑κB. In conclusion, the findings of the present study revealed that inhibition of miR‑15a may restore the therapeutic efficacy of mesenchymal stem cells in patients suffering from obesity.

Beneficial effects of extracts from Luciliasericata maggots on burn wounds in rats.

Lucilia sericata maggots have beneficial properties; however, their protective effects on burn wounds have yet to be fully elucidated. In the present study, a deep second-degree burn rat model was used to investigate the burn wound healing properties of aqueous extract of maggots (MAE). The anti-inflammatory, antioxidative and antibacterial activities were examined. In addition, the protein expression levels of Akt, vascular endothelial growth factor A (VEGFA) and nuclear factor-κB (NF-κB) were detected by western blotting. The findings of the present study revealed that MAE treatment increased burn wound healing and hydroxyproline content in the burn-treated rats. A total of seven compounds (MAE-P1-P7) were separated from MAE and a comparative study was performed to identify the major active component. The results demonstrated that MAE-P6 exerted greater antibacterial activity compared with the other compounds. MAE-P6 treatment reduced tissue levels of malondialdehyde, tumor necrosis factor-α and interleukin-6, and increased superoxide dismutase activity. Furthermore, MAE-P6 increased the expression levels of VEGFA and reduced NF-κB expression through Akt, which was verified by treatment with the Akt-specific inhibitor, LY294002. In conclusion, the present study demonstrated that the beneficial effects of MAE on burn wound healing were due to its antibacterial, antioxidative and anti-inflammatory activities. MAE-P6 reduced the release of inflammatory cytokines via the Akt/NF-κB signaling pathway, and regulated angiogenesis and vasopermeability via the Akt/VEGFA pathway.

Relationship between RSUME and HIF-1α/VEGF-A with invasion of pituitary adenoma

The RWD-containing sumoylation enhancer (RSUME) can stabilize hypoxia-inducible factor-1α (HIF-1α) which promotes vascular endothelial growth factor-A (VEGF-A) expression. RSUME plays an important role in promoting the invasion of pituitary adenoma. In this study, we compared the mRNA and protein levels of RSUME, HIF-1α, and VEGF-A in pituitary adenoma tissue and analyzed the correlation. We found that the expression levels of RSUME, HIF-1α, and VEGF-A in invasive pituitary adenoma were significantly higher than in noninvasive pituitary adenoma. Moreover, a positive correlation was found between RSUME and HIF-1α/VEGF pathways. RSUME and HIF-1α were treated with hypoxia-mimicking CoCl2 and transfected into AtT-20 and GT1.1 cell lines to determine the relationship between them. It was found that RSUME effects post-transcriptional expression of HIF-1α regulated VEGF-A secretion. Reducing RSUME expression using siRNA transfection resulted in a decrease of the invasion inhibition rate of AtT-20 cells, as determined using Transwell and MTT assays. Together, we found that RSUME silencing can inhibit the invasion of pituitary adenoma cells.

MiR‑140‑5p regulates angiogenesis following ischemic stroke by targeting VEGFA.

MicroRNA (miRNA or miR) expression profiles are altered in tissues under hypoxic-ischemic conditions. The expression of miR‑140 is downregulated >2-fold following hypoxic-ischemic brain damage, however, its role in angiogenesis subsequent to cerebral ischemia is not fully understood. The present study aimed to investigate the role of miR-140-5p in angiogenesis and the molecular mechanism mediated by vascular endothelial growth factorA (VEGFA) in an invitro model for brain ischemia. A rat middle cerebral artery occlusion (MCAO) model was constructed, and the results from reverse transcription-quantitative polymerase chain reaction and western blot analysis demonstrated that the expression levels of miR-140‑5p were significantly decreased, while the expression levels of VEGFA were significantly increased between12and48h in the rat cerebral following MCAO. Furthermore, human umbilical vein endothelial cells (HUVECs) were exposed to low oxygen conditions and it was demonstrated that hypoxia downregulated miR-140-5p and upregulated VEGFA expression levels. The miR-140-5p mimic was transfected into the normoxic and hypoxic HUVECs and3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Transwell migration and tube formation assays were performed. The results indicated that miR‑140‑5p inhibited angiogenesis by decreasing cell proliferation, migration and tube formation. Additionally, in human embryonic kidney293cells, results from the luciferase reporter assay revealed that miR‑140‑5p directly targeted the3'untranslated region of VEGFA and that miR‑140‑5p regulated the protein expression of VEGFA. To further analyze this effect, a VEGFA‑pEGFP‑C1 plasmid was transfected into the normoxic and hypoxic HUVECs, and it was revealed that the inhibitory effect of miR‑140‑5p on angiogenesis was attenuated by the overexpression of VEGFA. In conclusion, to the best of our knowledge, the present study is the first to suggest that miR‑140‑5p exerts an inhibitory effect on angiogenesis in an invitro model of ischemia, and this effect is achieved partially by targeting VEGFA. The present study provided a novel biomarker for the treatment of cerebral ischemia.

PI3K/Akt/mTOR signaling & its regulator tumour suppressor genes PTEN & LKB1 in human uterine leiomyomas

Background & objectives:Despite their high occurrence and associated significant level of morbidity manifesting as spectrum of clinical symptoms, the pathogenesis of uterine leiomyomas (ULs) remains unclear. We investigated expression profile of tumour suppressor genes PTEN (phosphatase and tensin homolog deleted on chromosome ten) and LKB1 (liver kinase B1), and key signaling components of P13K (phosphatidylinositol 3-kinase)/Akt (protein kinase B)/mTOR (mammalian target of rapamycin) pathway in leiomyomas and adjacent normal myometrium in women of reproductive age, to explore the possibility of targeting this pathway for future therapeutic implications.Methods:Real time PCR (qPCR) was used to quantify relative gene expression levels of PTEN, Akt1, Akt2, mTOR, LKB1 and VEGFA (vascular endothelial growth factor A) in leiomyoma as compared to adjacent normal myometrium. Immunohistochemistry was subsequently performed to analyze expression of PTEN, phospho-Akt, phospho-mTOR, phospho-S6, LKB1 and VEGFA in leiomyoma and adjacent normal myometrium.Results:Significant upregulation of PTEN (2.52 fold; P=0.03) and LKB1 (3.93 fold; P=0.01), and downregulation of VEGFA (2.95 fold; P=0.01) genes were observed in leiomyoma as compared to normal myometrium. Transcript levels of Akt1, Akt2 and mTOR did not vary significantly between leiomyoma and myometrium. An increased immunoexpression of PTEN (P=0.015) and LKB1 (P<0.001) and decreased expression of VEGFA (P=0.01) was observed in leiomyoma as compared to myometrium. Immunostaining for activated (phosphorylated) Akt, mTOR and S6 was absent or low in majority of leiomyoma and myometrium.Interpretation & conclusions:Upregulation of PTEN and LKB1 in concert with negative or low levels of activated Akt, mTOR and S6 indicates that PI3K/Akt/mTOR pathway may not play a significant role in pathogenesis of leiomyoma.

Propranolol for proliferating hemangioma: therapeutic efficacy and expressions of vascular endothelial growth factor-A and hypoxia-inducible factor 1α in patients before and after treatment

Objective To evaluate the clinical efficacy of propranolol in treating proliferating infantile haemangiomas,and to measure the expression levels of vascular endothelial growth factor-A (VEGF-A) and hypoxiainducible factor 1α (HIF-1α) in sera and urine of patients during the treatment.Methods Thirty infants with proliferating haemangiomas were treated with propranolol at doses of 0.5-2 mg/kg per day.The radius of haemangiomas was measured,and blood and urine samples were obtained from these patients before,and at 4 and 12 weeks after the beginning of treatment.Clinical efficacy was estimated according to a four-graded scale as well as the feedback from parents of these patients.Enzyme-linked immunosorbent assay (ELISA) was performed to determine the serum and urine concentrations of VEGF-A and HIF-1α.Thirty check-up infants collected from the Department of Child Health Care served as the healthy controls.Statistical analysis was done by two-way analysis of variance followed by the least significant difference (LSD) test.Results After 12 weeks of treatment,clinical response was excellent in 2 patients,good in 11,moderate in 14,and poor in 3.The serum levels of VEGF-A and HIF-1α were (268.174 ± 95.056) μg/L and (10.809 ± 1.686) mg/L respectively in the control group,sequentially decreased in the patients from baseline to 4 and 12 weeks after the beginning of treatment (VEGF-A:(385.692 ± 136.146) vs.(264.853 ± 122.12) vs.(211.345 ± 104.035) μg/L; HIF-1α:(31.462 ± 7.458) vs.(21.454 ± 5.489) vs.(12.052 ± 3.623) mg/L).The trend in expression changes of VEGF-A and HIF-1α in urine samples was similar to that in blood samples in these patients.Positive correlation was observed between the expression level of VEGF-A and HIF-1α in sera (r=0.730,P＜ 0.05) and urine (r=0.667,P＜ 0.05) of these patients.Moreover,the levels of serum VEGF-A,urine VEGF-A,serum HIF-1α and urine HIF-1α were all negatively correlated with the time course following propranolol administration (r =-0.390,-0.689,-0.806,-0.683,P ＜ 0.05,0.01,0.05,0.01 respectively).Conclusion Propranolol is effective for the treatment of proliferating infantile haemangiomas,likely by reducing serum and urinary concentrations of VEGF-A and HIF-lα in children. Key words: Propranolol; Hemangioma; Vascular endothelial growth factor A; Hypoxia-inducible factor 1α

Cyclooxygenase-2 and vascular endothelial growth factor expressions are involved in ultrafiltration failure

BackgroundLong-term peritoneal dialysis (PD) is associated with ultrafiltration failure (UFF). The aim of the study was to investigate changes in cyclooxygenase-2 (COX-2), vascular endothelial growth factor A (VEGF-A), and vascular endothelial growth factor C (VEGF-C) expressions in a rat model of UFF induced by PD solution. MethodsSprague–Dawley rats were divided into six groups (n = 8/group): normal untreated control group, sham operation group, uremic group (nephrectomy without PD), uremic 2-wk PD group (PD solution for 2 wk), uremic 4-wk PD group (PD solution for 4 wk), and uremic 4-wk PD + celecoxib group (PD solution plus COX-2 inhibitor celecoxib 20 mg/kg for 4 wk). Peritoneal function was determined by peritoneal equilibration test. Peritoneal morphology was determined by hematoxylin and eosin and Masson staining. Microvessel and lymphatic microvessel formation was determined by immunohistochemistry. COX-2, VEGF-A, and VEGF-C expressions were determined by real-time polymerase chain reaction and immunohistochemistry. ResultsUremic rat model was successfully established. PD-induced peritoneal morphologic changes associated with UFF, characterized by inflammation, edema, and collagen accumulation. PD solution increased the density of microvessels marked by CD31 (microvessel density) and lymphatic microvessels marked by LYVE-1 (lymphatic vessel density) in peritoneum. COX-2, VEGF-A, and VEGF-C expression levels in the uremic 4-wk PD group were higher than those in the uremic group (all P < 0.05). All these changes were partially reversed by celecoxib. VEGF-A and VEGF-C protein expressions were positively correlated with microvessel density and lymphatic vessel density formation. ConclusionsCOX-2 could increase VEGF-A and VEGF-C expressions in peritoneal tissue, resulting in increased formation of peritoneal microvessels and lymphatic microvessels, playing pivotal roles in the development of UFF.

Effect of unfocused extracorporeal shock wave therapy on growth factor gene expression in wounds and intact skin of horses

To compare the effect of extracorporeal shock wave therapy (ESWT) on expression of fibroblast growth factor-7 (FGF-7), transforming growth factor-β1 (TGF-β1), insulin-like growth factor-1 (IGF-1), platelet-derived growth factor-A (PDGF), and vascular endothelial growth factor-A (VEGF) in skin with surgically created skin wounds and intact skin in horses. 14 healthy horses. 8 horses were treated with ESWT at 6 locations along the neck at 36, 24, 12, 6, 2, or 1 hour prior to collection of full-thickness biopsy specimens from each location; a control specimen was collected from a sham-treated location. In 6 horses, 5 full-thickness wounds were created in each forelimb. Wounds in 1 forelimb/horse received ESWT immediately after creation and subsequently on days 7, 14, and 21; wounds in the contralateral forelimb remained untreated. Biopsy specimens were collected from 1 wound on each forelimb on days 7, 14, 21, 28, and 35. Expression levels of FGF-7, TGF-β1, IGF-1, PDGF, and VEGF were assessed in tissue samples from the horses' necks and forelimbs. In surgically created wounds, ESWT treatment was associated with reduced TGF-β1 expression, compared with expression in control wounds, during the entire study period. At 28 days following wound creation, IGF-1 expression was significantly increased for treated and untreated wounds, compared with findings on days 7, 14, 21, and 35. There was no significant effect of treatment on FGF-7, TGF-β1, IGF-1, PDGF, or VEGF expression in intact skin. Intervention with ESWT to suppress TGF-β1 may decrease granulation tissue production, resulting in improved wound healing on the distal portion of horses' limbs.

Rosuvastatin calcium inhibits the vascular development of zebrafish by down regulating the expression levels of vascular endothelial growth factor

Objective: To observe the effect of rosuvastatin calcium on the vascular development of zebrafish,and investigate the inhibition mechanism involved.Methods: The zebrafish embryos incubated with rosuvastatin calcium were identified as the experimental group.Meanwhile,the embryos immersed in 0.5% dimethyl sulfoxide(DMSO) were identified as the control group.The embryos were treated with drugs at 0.5 hour post fertilization(hpf) stage of development.After 24 hpf of exposure to the drugs,the numbers of intersegmental vessels of embryos were inspected and recorded by fluorescence microscope.Total RNAs were extracted from 0,6,12 and 24 hpf zebrafish embryos.The expression levels of vascular endothelial growth factor A(VEGF-A) were examined by real-time quantitative reverse transcription polymerase chain reaction(RT-PCR).Results:The growth of intersegmental vessels of zebrafish was significantly inhibited by rosuvastatin calcium.And the inhibition effect of rosuvastatin calcium was dose-dependent in the concentration range of 0.01-0.08 nmol/L.Compared with that of the control group,the expression levels of VEGF-A was reduced in the experiment group,and it was significantly different at 24 hpf(P0.05).Conclusion:Rosuvastatin calcium could inhibit the vascular development of zebrafish by down regulating the expression levels of VEGF-A.

Epigenetic regulation of vascular endothelial growth factor a dynamic expression in transitional cell carcinoma

Vascular endothelial growth factor A (VEGF-A) is a key mediator in the neovascularization of cancers. We have found that VEGF-A was expressed at significantly higher levels in high-grade transitional cell carcinoma (TCC) cells than low-grade TCC cells in our previous study. In the present study, promoter methylation pattern was assessed and quantified by bisulfite genomic sequencing (BGS) and specific VEGF-A CpG sites in low-grade, but not in high-grade, TCC cells were observed. Reporter assays indicated that hypermethylation of nine CpG sites can inhibit the transcriptional activity of the VEGF-A gene. Subsequent chromatin immunoprecipitation (ChIP) assay revealed down-regulation of transcription activity of VEGF-A with increasing binding of methyl-CpG-binding protein 2 (MBD2) and trimethyl-histone H3 (Lys9) proteins to these CpG sites in low-grade TCC cells during hypermethylation. Furthermore, treatment of low-grade TCC cells with DNA methyltransferase inhibitor and histone deacetylase inhibitor can restore the expression of VEGF-A and promote the invasive ability of low-grade TCC cells. Hypermethylation with lower expression levels of VEGF-A in low-grade TCC tumors than high-grade TCC tumors was also confirmed in clinical specimens by reverse transcriptase-PCR and pyrosequencing analyses. Our findings are the first results indicating that VEGF-A expression is suppressed in low-grade TCC tumors by promoter hypermethylation. This offers a new perspective on the role of VEGF-A in TCC tumor behavior.

VEGF-A Links Angiogenesis and Inflammation in Inflammatory Bowel Disease Pathogenesis

Vascular endothelial growth factor A (VEGF-A) mediates angiogenesis and might also have a role in inflammation and immunity. We examined whether VEGF-A signaling has a role in inflammatory bowel disease (IBD). Expression levels of VEGF-A, and its receptors VEGFR-1 and VEGFR-2, were examined in samples from patients with IBD and compared with those of controls. The capacity of VEGF-A to induce angiogenesis was tested in human intestinal microvascular endothelial cells using cell-migration and matrigel tubule-formation assays. Levels of vascular cellular adhesion molecule-1 and intercellular adhesion molecule were measured by flow cytometry to determine induction of inflammation; neutrophil adhesion was also assayed. Expression patterns were determined in tissues from mice with dextran sulfate sodium (DSS)-induced colitis; the effects of VEGF-A overexpression and blockade were assessed in these mice by adenoviral transfer of VEGF-A and soluble VEGFR-1. Intestinal angiogenesis was measured by quantitative CD31 staining and leukocyte adhesion in vivo by intravital microscopy. Levels of VEGF-A and VEGFR-2 increased in samples from patients with IBD and colitic mice. VEGF-A induced angiogenesis of human intestinal microvascular endothelial cells in vitro as well as an inflammatory phenotype and adherence of neutrophils to intestinal endothelium. Overexpression of VEGF-A in mice with DSS-induced colitis worsened their condition, whereas overexpression of soluble VEGFR-1 had the opposite effect. Furthermore, overexpression of VEGF-A increased mucosal angiogenesis and stimulated leukocyte adhesion in vivo. VEGF-A appears to be a novel mediator of IBD by promoting intestinal angiogenesis and inflammation. Agents that block VEGF-A signaling might reduce intestinal inflammation in patients with IBD.

Reduction of VEGF-A and CTGF expression in diabetic nephropathy is associated with podocyte loss

Micro-vascular and renal complications in diabetic patients are a considerable clinical challenge. In a previous study, we found a significant decrease in vascular endothelial growth factor A (VEGF-A) mRNA levels in glomeruli from patients with diabetic nephropathy (DN). We now set out to investigate the relationship between reduced VEGF-A and connective tissue growth factor (CTGF) expression levels, the number of podocytes, and the extent of interstitial fibrosis. Laser capture microdissection was applied to obtain glomerular RNA from 28 patients with DN and 22 controls. mRNA levels of VEGF-A, CTGF, nephrin, podocin, and Wilms tumor1 (WT1) were measured using real-time polymerase chain reaction. Protein expression was evaluated using immuno-stainings for VEGF-A and CTGF, as well as markers for podocytes (WT1) and endothelial cells (CD31). We found a significant decrease in glomerular mRNA levels for VEGF-A (2.5 times), CTGF (1.6), nephrin (2.8), podocin (3.3), and WT1 (1.7) in patients with DN. There was a significant correlation between expression of podocyte markers and VEGF-A mRNA levels, and an inverse correlation between podocin message and the extent of interstitial fibrosis. CD31-positive area was significantly decreased (3.2 times) in patients with DN. Reduction of angiogenic factors correlated with the extent of interstitial fibrosis. This downregulation was related to a reduction of podocytes in DN. The results may suggest that downregulation of VEGF-A and CTGF in DN is a result of podocyte loss.

Overexpression of biologically active VEGF121 fusion proteins in Escherichia coli

Vascular endothelial growth factor-A (VEGF) exists as five different isoforms, which exert their growth stimulatory effects through interaction with the FLK and KDR receptors. The VEGF(121) isoform has been employed as a highly selective carrier of therapeutic agents to target tumor endothelial cells resulting in inhibition of tumor growth and metastasis. VEGF(121) and VEGF(121)/rGel fusion toxin containing hexa-histidine tags were expressed in Escherichia coli AD494 (DE3) pLysS. Media containing glycerol as a primary carbon source increased the specific expression levels of soluble VEGF(121) and VEGF(121)/rGel (mg/L/OD10) by more than two-fold over LB media when grown in a batchtype cultivation in a bioreactor. High cell densities over OD 40 were achieved using a fed-batch method and employing feeding medium containing glycerol and yeast extract. The overall production of the target proteins was improved 18-fold for VEGF(121) (59.2mg/L) and 27-fold for VEGF(121)/rGel (42.5mg/L), respectively, compared to the conventional flask cultivation method (3.3 and 1.6mg/L for VEGF(121) and VEGF(121)/rGel, respectively). The purified VEGF(121) and VEGF(121)/rGel fusion proteins were biologically active as assessed by phosphorylation of KDR receptors and cytotoxicity against KDR expressing cells.

Vascular endothelial growth factor A (VEGF-A) mRNA expression levels decrease after menopause in normal breast tissue but not in breast cancer lesions.

We hypothesized that the regulation of microvascular functions and angiogenesis in breast tissue, a well known target of ovarian steroid action, is dependent on the hormonal exposure of the breast. Relative expression levels of VEGF-A (vascular endothelial growth factor A), a putative key regulator of angiogenesis in breast cancer, were analysed in the tumour and the adjacent non-neoplastic breast tissue of 19 breast cancer patients by quantitative reverse transcriptase polymerase chain reaction. In non-neoplastic breast specimens the expression levels of all detected VEGF-A-isoforms (189, 165, 121) were significantly higher in premenopausal compared to post-menopausal women (P = 0.02) and were inversely correlated with the patient's age (P = 0.006). In contrast, in cancerous tissues menopausal status had no influence on VEGF-A-expression levels. Benign and malignant tissues exhibited a similar expression pattern of VEGF-A-isoforms relative to each other. Thus, the regulation of the vasculature in normal breast tissue, as opposed to breast cancer tissue, appears to be hormonally dependent. Endogenous and therapeutically used hormonal steroids might, therefore, cause clinically relevant changes of the angiogenic phenotype of the human breast. © 1999 Cancer Research Campaign