Vascular Endothelial Growth Factor Protein Expression Research Articles

All-trans retinoic acid and vascular endothelial growth factor induced the directional osteogenic differentiation of mouse embryonic fibroblasts

To investigate the effect of all-trans retinoic acid (ATRA) and vascular endothelial growth factor (VEGF) on the osteogenic differentiation of mouse embryonic fibroblasts (MEFs). The fetal mice in the uterus of NIH pregnant mice (pregnancy 12-15 days) were collected, and the heads and hearts etc. were removed. Then MEFs were separated from the rest tissues of the fetal mice and cultured by trypsin digestion and adherent culture. HEK-293 cells were used to obtain recombinant adenovirus-red fluorescent protein (Ad-RFP) and Ad-VEGF by repeatedly freezing and thawing. Alkaline phosphatase (ALP) staining and quantitative detection were used to detect the changes of ALP activity in MEFs applied with ATRA or VEGF alone or combined use of ATRA and VEGF on the 3rd and 5th days. The cultured 3rd to 4th generation MEFs were divided into groups A, B, C, and D, and were cultured with DMSO plus Ad-RFP, ATRA, Ad-VEGF, ATRA plus Ad-VEGF, respectively. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the mRNA expressions of osteogenic markers including ALP, collagen type Ⅰ, osteopontin (OPN), osteocalcin (OCN), and angiogenic markers including VEGF, angiopoietin 1 (ANGPT1), and endomucin (EMCN) on the 3rd and 7th days. Immunohistochemical staining was used to detect the protein expressions of OPN and VEGF on the 3rd, 5th, and 7th days in each group. Alizarin red staining was used to detect calcium salt deposition levels in each group at 14 and 21 days after osteogenic induction. Fifteen athymic female nude mice aged 4 to 6 weeks were randomly divided into 3 groups and 5 mice in each group. Then MEFs treated with ATRA, Ad-VEGF, and ATRA plus Ad-VEGF were injected subcutaneously into the dorsal and ventral sides, respectively. X-ray observation, gross observation, and histological staining (Masson, HE, and Safranin O-fast green stainings) were performed at 5 weeks after implantation to observe the ectopic bone formation in nude mice in each group. MEFs were successfully isolated and cultured. The acquired Ad-RFP and Ad-VEGF were successfully transfected into MEFs with approximately 50% and 20% transfection rates. ALP activity tests showed that ATRA or Ad-VEGF could enhance ALP activity in MEFs ( P<0.05), and ATRA had a stronger effect than Ad-VEGF; and the combined use of ATRA and Ad-VEGF significantly enhanced the ALP activity in MEFs ( P<0.05). qRT-PCR test showed that the combined use of ATRA and Ad-VEGF also increased the relative mRNA expressions of early-stage osteogenesis-related markers ALP, OPN, and collagen type I ( P<0.05); the relative mRNA expressions of angiogenesis-related markers VEGF, EMCN, and ANGPT1 increased at 7 days ( P<0.05). Immunohistochemical staining showed that ATRA combined with Ad-VEGF not only enhanced OPN protein expression, but also increased VEGF protein expression on 7th day. Alizarin red staining showed that the application of ATRA or Ad-VEGF induced weak calcium salt deposition, and the combined use of ATRA and Ad-VEGF significantly enhanced the effect of calcium salt deposition in MEFs. The results of implantation experiments in nude mice showed that X-ray films observation revealed obvious bone mass in the ATRA plus Ad-VEGF group, and the bone was larger than that in other groups. Histological staining showed a large amount of collagen and mature bone trabeculae, bone matrix formation, and gray-green collagen bone tissue, indicating that the combined use of ATRA and Ad-VEGF significantly enhanced the osteogenic effect of MEFs in vivo. The combined use of ATRA and VEGF can induce the osteogenic differentiation of MEFs.

Effect of curcumin-lipiodol emulsion combined with absorbable anti-adhesion film through double embolic pathway on angiogenesis and overall survival (OS) time of VX2 liver cancer rabbits

BackgroundCurcumin is a promising anti-tumor drug in recent years. The main function of absorbable anti-adhesion film is to prevent adhesions after surgical operation. The effects of curcumin-lipiodol emulsion injected via the hepatic artery and portal vein catheter combined with using absorbable anti-adhesion film around the liver angiogenesis are unknown. This article mainly discusses the effects of these combined strategies on the inhibition or reduction of postoperative angiogenesis and prolonging survival time of rabbit model.MethodsForty model rabbits with VX2 liver cancer were divided into four groups. Group A is the control group and its intervention is a simple lipiodol emulsion group. The intervention in group B was a simple lipiodol emulsion combined with an anti-adhesion membrane. The intervention in group C was a mixture of curcumin and lipiodol. The intervention in group D was a mixed emulsion of curcumin and lipiodol combined with an anti-adhesion membrane. The levels of aspartate aminotransferase (ALT) and alanine aminotransferase (AST) in the model rabbits were compared to analyze the damage of liver cells 1 day before embolization and 3, 7, 14 days after the embolization. In addition, the levels of vascular endothelial growth factor (VEGF) and microvessel density (MVD) in residual cancer tissues were determined by immunohistochemistry and Western blot. Finally, overall survival time (OS) of each group was recorded.ResultsThe results showed that the ALT and AST levels of venous blood before and after embolization in each group of rabbits were not changed significantly (P>0.05). The levels of VEGF and MVD in the group C and group D were lower than those in the group A (P<0.05). However, there was no significant difference between group B and group A (P>0.05). The decrease of VEGF and MVD in group D was most obvious, and group D had a significant difference compared with group C (P<0.05). Western blot analysis showed that the protein expression of VEGF was consistent with immunohistochemical staining. The overall survival (OS) time of group B showed no significant difference compared with group A (P>0.05). The OS time of group C and group D was longer than that of group A (P<0.05). Group D showed significantly difference on prolongation of the survival time than group C (P<0.05).ConclusionsOverall, the curcumin-lipiodol emulsion injected into the portal vein and hepatic artery supplying the tumor can significantly inhibit angiogenesis and play a role in prolonging the OS time of model rabbits. The application of anti-adhesion film alone cannot inhibit tumor angiogenesis and play a role in prolonging the OS time of model rabbits. However, curcumin-lipiodol emulsion combined with anti-adhesion film can obviously promote these effects.

Encapsulated three-dimensional bioprinted structure seeded with urothelial cells: a new construction technique for tissue-engineered urinary tract patch.

Background:Traditional tissue engineering methods to fabricate urinary tract patch have some drawbacks such as compromised cell viability and uneven cell distribution within scaffold. In this study, we combined three-dimensional (3D) bioprinting and tissue engineering method to form a tissue-engineered urinary tract patch, which could be employed for the application on Beagles urinary tract defect mode to verify its effectiveness on urinary tract reconstruction.Methods:Human adipose-derived stem cells (hADSCs) were dropped into smooth muscle differentiation medium to generate induced microtissues (ID-MTs), flow cytometry was utilized to detect the positive percentage for CD44, CD105, CD45, and CD34 of hADSCs. Expression of vascular endothelial growth factor A (VEGFA) and tumor necrosis factor-stimulated gene-6 (TSG-6) in hADSCs and MTs were identified by Western blotting. Then the ID-MTs were employed for 3D bioprinting. The bioprinted structure was encapsulated by transplantation into the subcutaneous tissue of nude mice for 1 week. After retrieval of the encapsulated structure, hematoxylin and eosin and Masson's trichrome staining were performed to demonstrate the morphology and reveal collagen and smooth muscle fibers, integral optical density (IOD) and area of interest were calculated for further semi-quantitative analysis. Immunofluorescent double staining of CD31 and α-smooth muscle actin (α-SMA) were used to reveal vascularization of the encapsulated structure. Immunohistochemistry was performed to evaluate the expression of interleukin-2 (IL-2), α-SMA, and smoothelin of the MTs in the implanted structure. Afterward, the encapsulated structure was seeded with human urothelial cells. Immunofluorescent staining of cytokeratins AE1/AE3 was applied to inspect the morphology of seeded encapsulated structure.Results:The semi-quantitative assay showed that the relative protein expression of VEGFA was 0.355 ± 0.038 in the hADSCs vs. 0.649 ± 0.150 in the MTs (t = 3.291, P = 0.030), while TSG-6 expression was 0.492 ± 0.092 in the hADSCs vs. 1.256 ± 0.401 in the MTs (t = 3.216, P = 0.032). The semi-quantitative analysis showed that the mean IOD of IL-2 in the MT group was 7.67 ± 1.26, while 12.6 ± 4.79 in the hADSCs group, but semi-quantitative analysis showed that there was no statistical significance in the difference between the two groups (t = 1.724, P = 0.16). The semi-quantitative analysis showed that IOD was 71.7 ± 14.2 in non-induced MTs (NI-MTs) vs. 35.7 ± 11.4 in ID-MTs for collagen fibers (t = 3.428, P = 0.027) and 12.8 ± 1.9 in NI-MTs vs. 30.6 ± 8.9 in ID-MTs for smooth muscle fibers (t = 3.369, P = 0.028); furthermore, the mean IOD was 0.0613 ± 0.0172 in ID-MTs vs. 0.0017 ± 0.0009 in NI-MTs for α-SMA (t = 5.994, P = 0.027), while 0.0355 ± 0.0128 in ID-MTs vs. 0.0035 ± 0.0022 in NI-MTs for smoothelin (t = 4.268, P = 0.013), which indicate that 3D bioprinted structure containing ID-MTs could mimic the smooth muscle layer of native urinary tract. After encapsulation of the urinary tract patch for additional cell adhesion, urothelial cells were seeded onto the encapsulated structures, and a monolayer urothelial cell was observed.Conclusion:Through 3D bioprinting and tissue engineering methods, we provided a promising way to fabricate tissue-engineered urinary tract patch for further investigation.

Mechanism of the hypoxia inducible factor 1/hypoxic response element pathway in rat myocardial ischemia/diazoxide post‑conditioning.

Ischemic post-conditioning (IPO) and diazoxide post-conditioning (DPO) has been proven to reduce myocardial ischemia reperfusion injury (MIRI); however, the mechanisms of IPO/DPO are still not clear. The present study aimed to investigate whether mitochondrial ATP-sensitive potassium channels (mitoKATP) channels are activated by IPO/DPO, which may further activate the hypoxia inducible factor 1/hypoxic response element (HIF-1/HRE) pathway to mitigate MIRI. Using a Langendorff perfusion device, healthy male (250–300 g) Sprague Dawley rat hearts were randomly divided into the following groups. Group N was aerobically perfused with K-H solution for 120 min. Group ischaemia/reperfusion (I/R) was aerobically perfused for 20 min, then subjected to 40 min hypoxia plus 60 min reperfusion. Group IPO was treated like the I/R group, but with 10 sec of hypoxia plus 10 sec of reperfusion for six rounds before reperfusion. Group DPO was exposed to 50 µM diazoxide for 5 min before reperfusion and otherwise treated the same as group I/R. In groups IPO+5-hydroxydecanoic acid (5HD), DPO+5HD and I/R+5HD, exposure to 100 µM 5HD (a mitoKATP channel specific blocker) for 5 min before reperfusion as described for groups IPO, DPO and I/R, respectively. In groups IPO+2-methoxyestradiol (2ME2), DPO+2ME2 and I/R+2ME2, exposure to 2 µM 2ME2 (a HIF-1α specific blocker) for 10 min before reperfusion as described for groups IPO, DPO and I/R respectively. Cardiac hemodynamics, myocardial injury and the expression of HIF-1/HRE pathway [HIF-1α, heme oxygenase (HO-1), inducible nitric oxide synthase (iNOS) and vascular endothelial growth factor (VEGF)] were detected in each group. The infarct size and mitochondrial Flameng scores of groups IPO/DPO were significantly decreased compared with the I/R group (P<0.05), but the myocardial protective effects of IPO/DPO could be eliminated by 5HD or 2ME2 (P<0.05). In addition, IPO/DPO could increase the mRNA expression of HIF-1α and the downstream factors of the HIF-1/HRE pathway (the mRNA and protein expression of HO-1, iNOS and VEGF; P<0.05). However, the myocardial protective effects and the activation the HIF-1/HRE pathway mediated by IPO/DPO could be eliminated by 5HD or 2ME2 (P<0.05). Therefore, the activation of the HIF-1/HRE pathway by opening mitoKATP channels may work with the mechanism of IPO/DPO in reducing MIRI.

Aerobic Endurance Exercise Ameliorates Renal Vascular Sclerosis in Aged Mice by Regulating PI3K/AKT/mTOR Signaling Pathway.

Renal vascular sclerosis caused by aging plays an important role in the occurrence and development of chronic kidney disease. Clinical studies have confirmed that endurance exercise is able to delay the aging of skeletal muscle and brain tissue. However, to date, few studies have assessed whether endurance exercise is able to improve the occurrence of renal vascular sclerosis caused by natural aging and its related mechanisms. In this study, we investigated the protective effect of aerobic endurance exercise on renal vascular sclerosis in aged mice and its effect on the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway. The results suggested that aerobic endurance exercise preserved kidney morphology and renal function. Glomerular basement membrane thickness was evidently increased, podocyte foot processes were effaced in aged mice, and aerobic endurance exercise significantly ameliorated the overall lesion range. The protein expression of vascular endothelial growth factor (VEGF) and JG12 was lower in the senile control group (OC group). The protein expression of VEGF and JG12 was significantly increased after aerobic endurance exercise. Furthermore, aerobic endurance exercise resulted in downregulation of Bax, Caspase 3, IL-6, and senescent cells and upregulation of Bcl-2. The upregulation of PI3K and its downstream signal molecules AKT and mTOR after aerobic endurance exercise was further observed. Our observations indicated that aerobic endurance exercise may inhibit renal vascular sclerosis in aged mice by regulating the PI3K/AKT/mTOR signaling pathway.

Antitumor effect of hypoxia response element chimeric tumor specific promoter regulated oncolytic adenovirus on renal cell carcinoma

Objective To investigate the effect of chimeric promoter composed of hypoxia response element (HRE) and tumor specific cell proliferation-associated nuclear antigen (Ki-67) promoter on the replication ability of oncolytic adenovirus and its cytotoxic activity under hypoxic conditions. Methods The HRE sequence, as an enhancer, was inserted upstream of the Ki-67 promoter and, at the same time carried the reporter gene enhanced green fluorescent protein (EGFP) to construct oncolytic adenovirus Ad-HRE-Ki-67-EGFP, and the titer of adenoviruses was measured by the tissue culture infective dose (TCID50). Normal renal epithelial cells HK-2 and renal cancer cells OSRC-2 and ACHN were infected with two kinds of viruses under normoxic (21% O2) or hypoxic (1% O2) conditions respectively for 24 h, and the virus tumor-targeting was detected by fluorescence microscopy and flow cytometry. Western blotting was performed to detect the expression of hypoxia-related protein hypoxia-inducible factor-1 alpha (HIF-1α), vascular endothelial growth factor (VEGF) and the expression of E1A protein. Cell counting kit-8 (CCK-8) assay was used to detect the cytotoxity of virus on cells. Results The recombinant oncolytic virus Ad-HRE-Ki67-EGFP was successfully constructed. Under normoxic conditions, the efficiency of ACHN and OSRC-2 infection by chimeric HRE oncolytic adenovirus in renal cancer cells was (47.5±2.6)% and (35.2±3.4)%, while that in hypoxic conditions was (86.4±3.4)% and (73.5±2.3)%, indicating that the infection efficiency was higher under hypoxic conditions (ACHN: t=15.760; OSRC-2: t=16.190; P<0.01); Normal renal tubular epithelial cells HK-2 were infected with Ad-Ki-67-EGFP or Ad-HRE-Ki-67-EGFP under normoxic conditions. The infection efficiency was (22.4±3.1)% and (22.9±2.2)% respectively, compared with that of ACHN and OSRC-2 cells [ACHN: (68.4±3.3)% and (72.1±2.9)%; OSRC-2: (56.1±3.1)% and (61.4±2.3)%], the difference was statistically significant (Ad-Ki-67-EGFP: t=17.650, 13.390; Ad-HRE-Ki-67-EGFP: t=23.680, 21.430, P<0.01), indicating that the two adenoviruses have a targeting effect on renal cancer cells. Western blotting data showed that the expression of HIF-1α, VEGF and p53 protein were increased under hypoxic conditions and was time-dependent, indicating that hypoxia can regulate the transcription and translation of HIF-1α, VEGF and p53 proteins. Under hypoxic conditions, the expression level of E1A protein in OSRC-2 and ACHN infected with Ad-HRE-Ki67-EGFP was significantly higher than that after Ad-Ki67-EGFP infection. It indicated that HRE can enhance the replication ability of virus under hypoxic conditions. CCK-8 results showed that when the multiple infection (MOI) values were 20, 100, the survival rate of the Ad-HRE-Ki-67-EGFP-normoxia virus group in ACHN and OSRC-2 cells was (73.4±2.0)%, (56.4±1.5)% and (79.9±1.8)%, (61.3±2.7)%, the Ad-HRE-Ki-67-EGFP-hypoxia virus group was (63.1±2.0)%, (31.6±2.1)% and (68.1±2.6)%, (35.8±3.1)%, the Ad-HRE-Ki-67-EGFP-hypoxia virus group showed a stronger proliferation inhibitory effect [MOI (20): t=6.328, 6.441; MOI (100): t=16.410, 10.790, P<0.01]. Conclusion Under hypoxic conditions, chimeric HRE sequence recombinant oncolytic adenovirus has stronger replication ability and cell killing effect in renal carcinoma cells. Key words: Renal cell carcinoma; Oncolytic adenovirus; Hypoxia response element; Hypoxia

Evaluation of the mechanism of cordyceps polysaccharide action on rat acute liver failure.

IntroductionThis study aimed to investigate the mechanism of action of cordyceps polysaccharide on rat acute liver failure (ALF).Material and methodsSixty rats were randomly divided into five groups: a normal group, a model group without cordyceps polysaccharide and groups with cordyceps polysaccharide in three different doses (5, 10 and 20 mg/ml). Serum alanine aminotransferase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), and total bilirubin (TBIL) contents were measured for assessing liver function. Hematoxylin and eosin (HE) staining was used for observing liver pathology. Apoptosis was detected through the method of terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) staining. Protein expression levels of caspase-1, interleukin-18 (IL-18), IL-10, vascular endothelial growth factor (VEGF), and stromal cell-derived factor-1α (SDF-1α) in liver tissue were detected by Western blot. Proliferating cell nuclear antigen (PCNA) and signal regulatory protein-α1 (SIRPα1) contents were measured by PCR.ResultsThe rat ALF model was established with D-galactosamine induced by lipopolysaccharide (LPS). After modelling, tissue HE staining showed typical manifestation of acute liver injury that emerged in the rat ALF model. The liver failure group showed higher levels of serum ALT and AST, as well as hepatocyte apoptosis, than the groups treated with cordyceps polysaccharide. Cordyceps polysaccharide can effectively suppress the protein expression of caspase-1, IL-18, and IL-10, while simultaneously increasing the protein expression of VEGF and SDF-1α, as well as the mRNA expression of PCNA and SIRPα1.ConclusionsCordyceps polysaccharide can alleviate the immune response and inflammatory injury in ALF by regulating the balance of pro-inflammatory and anti-inflammatory factors and reducing the apoptosis.

氨基酮戊酸-光动力疗法对尖锐湿疣组织中血管内皮生长因子和增殖细胞核抗原表达的影响

Objective To evaluate the effect of aminolevulinic acid-based photodynamic therapy (ALA-PDT) on the expression of vascular endothelial growth factor (VEGF) and proliferating cell nuclear antigen (PCNA) in condyloma acuminatum (CA) tissues. Methods A total of 56 patients with CA were enrolled from Department of Dermatology, The First Affiliated Hospital of Zhengzhou University from October 2016 to September 2017, and skin lesions were obtained before and 1 week after the first ALA-PDT treatment. Immunohistochemical SP method was used to determine the expression of VEGF and PCNA in keratinocytes in the CA tissues. Chi-square test and rank sum test were carried out to analyze differences between pre- and post-treatment expression rate and intensity of VEGF and PCNA, and Spearman correlation analysis was conducted to analyze the correlations between the protein expression of VEGF and PCNA. Results The expression rates of VEGF and PCNA in keratinocytes in the CA tissues were 71.43% (40/56) and 73.21% (41/56) respectively before ALA-PDT, and 44.64% (25/56) and 41.07% (23/46) respectively after ALA-PDT. There were significant differences between pre- and post-treatment expression rate and intensity of VEGF and PCNA (expression rate: χ2 = 8.25, 11.81 respectively, both P < 0.05; expression intensity: H = 11.29, 12.22 respectively, both P < 0.05) . The expression of VEGF was positively correlated with the expression of PCNA in the CA tissues before and after the ALA-PDT treatment (rs = 0.202, 0.273, respectively, both P < 0.05) . Conclusion The expression of VEGF and PCNA decreased in CA tissues after ALA-PDT treatment, which may be one of the mechanisms underlying the treatment of CA with ALA-PDT. Key words: Condylomata acuminata; Photochemotherapy; Aminolevulinic acid; Vascular endothelial growth factors; Proliferating cell nuclear antigen

MicroRNA‑15a‑5p induces pulmonary artery smooth muscle cell apoptosis in a pulmonary arterial hypertension model via the VEGF/p38/MMP‑2 signaling pathway.

The aim of the present study was to investigate the role of microRNA-15a-5p (miR-15a-5p) in pulmonary arterial hypertension (PAH) and elucidate the underlying pro-apoptotic mechanism. Reverse transcription-quantitative PCR analysis and gene microarray hybridization were used to measure the expression of miR-15a-5p in the lung tissues of rats with monocrotaline (MCT)-induced PAH. Flow cytometry and caspase-3/9 activity assays were adopted to measure the apoptosis of pulmonary artery smooth muscle cells (PASMCs). The expression of apoptosis-related proteins was analyzed using western blotting. The results demonstrated that the expression of miR-15a-5p was significantly increased in the lung tissues of rats with MCT-induced PAH. In addition, the overexpression of miR-15a-5p reduced PASMC proliferation, induced apoptosis, promoted the activity of caspase-3/9, induced the protein expression of B-cell lymphoma 2-associated X protein (Bax), decreased the expression of B-cell lymphoma 2 (Bcl-2), increased inflammation, as indicated by the expression of tumor necrosis factor-α (TNF)-α and interleukin (IL)-1β, IL-6 and IL-18, suppressed the protein expression of vascular endothelial growth factor (VEGF), and promoted the protein expression levels of phosphorylated (p)-p38 mitogen-activated protein kinase (p38) and matrix metalloproteinase (MMP)-2 in the PASMCs of rats with MCT-induced PAH. By contrast, the downregulation of miR-15a-5p increased cell proliferation, decreased apoptosis, reduced the activity of caspase-3/9 and the protein expression of Bax, increased the expression of Bcl-2, inhibited inflammation (as suggested by the expression of TNF-α, IL-1β, IL-6 and IL-18), induced the protein expression of VEGF, and suppressed the protein expression of p-p38 and MMP-2 in the PASMCs of rats with MCT-induced PAH. The inhibition of VEGF attenuated the effects of the overexpression of miR-15a-5p on the inhibition of cell proliferation, apoptotic rate, caspase-3/9 activity and protein expression of Bax, and it attenuated the increased inflammation, as indicated by the protein expression of p38 and MMP-2 in the PASMCs. In conclusion, the data of the present study demonstrated that miR-15a-5p induced the apoptosis of PASMCs in an animal model of PAH via the VEGF/p38/MMP-2 signaling pathway. However, further research is required to fully elucidate the role of miR-15a-5p in the development of PAH.

Effect of ovarian steroids on vascular endothelial growth factor a expression in bovine uterine endothelial cells during adenomyosis

BackgroundAdenomyosis is a uterine dysfunction defined as the presence of endometrial glands within the myometrium. There is evidence that proangiogenic factors may play a role during the development of adenomyosis; however, exact mechanism remains unknown. The aim of the study was to determine the action of vascular endothelial growth factor A (VEGFA) in uterine tissue and uterine vascular endothelial cells during adenomyosis.ResultsUterine tissues were collected and examined for the presence and extent of adenomyosis. Gene and protein expression of VEGFA and its two receptors (VEGFR1 and VEGFR2) was evaluated with quantitative polymerase chain reaction and Western blotting, respectively, in endometrium and myometrium during adenomyosis. Immunolocalization of VEGFA and its receptors within uterine tissues during adenomyosis was also determined. In an in vitro experiment, endothelial cells from non-adenomyotic bovine uteri were treated with media conditioned by non-adenomyotic or adenomyotic uterine slices treated with 17-beta-oestradiol (E2) or progesterone (P4). Both gene and protein expression of VEGFR2 were elevated in endometrium in stages 3–4 of adenomyosis. Protein expression of VEGFA and VEGFR2 as well as VEGFA secretion were increased in endothelial cells treated with media conditioned by adenomyotic uterine slices after E2 treatment.ConclusionsResults suggest that VEGFA signalling is an important component, next to E2, that enhances VEGFA action and participates in adenomyosis development in cows.

Influence of miR-199a on rats with non-small cell lung cancer via regulating the HIF-1α/VEGF signaling pathway.

Micro-ribonucleic acids (miRNAs) are involved in the occurrence of various cancers, and the hypoxia-inducible factor 1-α (HIF-1α) is the main regulator of cell proliferation induced by hypoxia. The relationships of miR-199a and HIF-1α expressions with non-small cell lung cancer (NSCLC) remain unclear, so they were explored in this work. On the basis of establishing the rat model of NSCLC, the messenger RNA (mRNA) expressions of miR-199a, HIF-1α and the vascular endothelial growth factor (VEGF) were analyzed in NSCLC rats, and the correlations of miR-199a with the mRNAs of HIF-1α and VEGF and cancer staging were investigated. To further study the role of miR-199a in NSCLC cell proliferation via the HIF-1α/VEGF signaling pathway, NSCLC cells were treated with the signaling pathway inhibitor and transfected with miR-199a mimics, respectively. Also, the roles of the inhibitor PX-478 and miR-199a mimics in the expressions of miR-199a, HIF-1α, and VEGF proteins, as well as their influences on cell proliferation ability were detected. In NSCLC rats, the expression of miR-199a was substantially decreased (p<0.01), but the expressions of HIF-1α and VEGF were notably raised (p<0.01). MiR-199a was negatively correlated with the expression of VEGF. As cancer developed, the expression of miR-199a was gradually lowered, but the expressions of HIF-1α and VEGF were gradually increased. Both HIF-1α/VEGF signaling pathway inhibitor PX-478 and miR-199a mimics significantly reduced the expressions of HIF-1α and VEGF proteins (p<0.01) and suppressed the cell proliferation activity. MiR-199a prevents the proliferation of NSCLC cells via the targeted down-regulation of the HIF-1α/VEGF signaling pathway.

Vascular Endothelial Growth Factor (VEGF-C) Protein Expression Related with Lymphangiogensis in Iraqi Cervical Cancer Patients

Objective: Metastatic spread of tumor cells to distant organs is the leading cause of mortality from cancer. Although metastatic tumor spread can occur via a different mechanism. lymphangiogenic factors recognized were vascular endothelial growth factor (VEGF)–C and –D, which bind to a tyrosine kinase&#x0D; &#x0D; receptor, VEGF receptor (R)–3. Binding affinities to VEGFR-2 receptor increase on the lymphatic and blood endothelium therefore enables both growth factors to also exert lymphangiogenic and angiogenic effects and increased incidence of lymph node metastasis. The aim of this study is to evaluate the VEGF-C protein expression in cervical cancer cells and lymph vessels and found the relationship of this marker with lymphangiogensis of Iraqi cervical cancer samples.&#x0D; Method: In this study, expression of VEGF-C was noticed in 55 cervical samples by Immuno-&#x0D; histochemistry. 35 cases diagnosed as invasive cervical cancer in addition to 20 normal samples consider as control. Immunohistochemistry was performed and the cytoplasm level of VEGF-C was scored by the percentage of positive cells and intensity.&#x0D; Results:&#x0D; The present data evaluated the prognostic significance of VEGF-C to cervical cancer, cytoplasm staining was seen in 29 cases (82.9 %) in cervical cancer tissues. Only 4 out of 35 cases (11.4 %) displayed cytoplasmic and nuclear tissue. There is significant difference of VEGF-C staining in lymphatic vessels and cancer cells (χ2= 5.04, p = 0.023*) regarding to positive expression (20/ 57.1%), (25/ 71.4 %) respectively and negative VEGF-C staining 15 (42.9%), 10 (28.6 %) respectively. High positive percentage of VEGF-C expression in cytoplasm of malignant cases in score 2&amp; 3 (25.7%, 45.7 %, P-value= 0.0392 *, 0.029* respectively) as compared to normal cases (15%, 30% respectively). Demographic criteria of patients revealed association with VEGF-C expression patterns. Differentiation Well + moderately and histologic type Squamous carcinoma showed significantly associated with VEGF-C (P=0.0071** &amp; 0.0071** respectively). Positive VEGF-C staining in cancer cells had more lymphatic vessel (17/68 %) as compared to negative cases (3/30 %) with Chi-Square 8.263, p value= 0.0061**. Also, positive VEGF-C staining had more lymph node associated (9/36%) compared to negative cases (1/10 %) with Chi-Square 13.503, p value= 0.0001**.&#x0D; &#x0D; &#x0D; Conclusion:&#x0D; In conclusion, high expression of vascular endothelial growth factor was noticed in cervical cancer cells and lymph vascular invasion indicating the important role of this marker as prognostic factor for Iraqi cervical cancer. Additionally, these results suggested that VEGF-C promoted cervical cancer metastasis using immunohistochemistry technique. Our findings offer new vision into the role of VEGF-C in cervical cancer development and give potential target for study the lymphangiogensis of tumor in Iraqi women.

Expression of EphrinA1, vascular endothelial growth factor and microRNA-210 after hydronephrosis

Objective To investigate the difference and correlations of the EphrinA1, vascular endothelial growth factor (VEGF) expression and microRNA (miRNA)-210 in the kidney tissue of rats in the unilateral ureteral obstruction (UUO) model. And to understand the regulation mechanism of the three factors in the occurrence of renal tissue injury in rats. Methods Using unilateral ureteral ligation to establish a model of hydronephrosis in mice (except for the NC group), and they wrere randomly divided into an acute hydronephrosis group (UUO group) and a false surgical control group (NC group). The UUO group was divided into four groups (UUO 2 d group, UUO 5 d group, UUO 9 d group, and UUO 14 d group), and eight mice in each group. The pathological changes of renal tissue were observed by hematoxylin-eosin (HE) staining. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the mRNA expression of EphrinA1, VEGF and miRNA-210; Western blot was used to detect the protein expression of EphrinA1 and VEGF. Resulsts Hydronephrosis was not shown in the NC group, and hydronephrosis and inflammatory cell infiltration were observed in the UUO group. Compared with NC group, the mRNA expression of EphrinA1 , VEGF and miRNA-210 were up-regulated in UUO 2 d group (P<0.05). With the extension of the time of hydronephrosis, the expressions of all three factors were down-regulated in 9 d and 14 d groups compared with 2 d and 5 d groups, and reached the lowest point at 14 d (P<0.05); Western blot result indicated that the expression of EphrinA1 and VEGF in renal tissue of UUO 2 d and 5 d group was increased compared with NC group (P<0.05). Conclusions The UUO mouse model verified that miRNR-210 participated in hydronephrosis. The consistency of EphrinA1, VEGF, and miRNA-210 indicated that the three factors played an important role in the occurrence and development of UUO. Key words: Hydronephrosis; Ephrin-A1; Vascular endothelial growth factor; MicroRNAs; Mice

Inhibition of β-elemene on the expressions of HIF-lα, VEGF and iNOS in diabetic rats model.

To evaluate the effect of β-elemene on the expressions of hypoxia-inducible factor (HIF)-lα, vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS) in a streptozotocin (STZ) induced diabetic Sprague-Dawley (SD) rat model. SD rats were administered an abdominal injection of STZ and induced to a diabetic model. After 6wk course of diabetes, the treatment groups were given β-elemene through periocular and intravitreous injection separately and the control groups were given blank emulsion injection. HE staining was used to observe the morphology of retina. The mRNA expressions of HIF-1α, VEGF and iNOS was assayed by real-time polymerase chain reaction (PCR) and the protein expression was measured by Western blot and immunocytochemistry methods. The results indicated that the protein and mRNA expressions of HIF-1α, VEGF and iNOS after treated by β-elemene periocularly and intravitreally injections were all found to be reduced compared with the levels in the diabetic rats group (P<0.05). The inhibitory effect of intravitreal injection was more remarkable. The results show β-elemene protect the retina of diabetic rats from high glucose damage by downregulating the expression of HIF-1α, VEGF and iNOS.

Down-regulation of mir-27b promotes angiogenesis and fibroblast activation through activating PI3K/AKT signaling pathway.

To study the effects of mir-27b on angiogenesis and fibroblast activation and to explore its further mechanism. Humanmicrovascular endothelial cell (HMEC)-1 and humannormal skin fibroblast (BJ) cells were treated with mir-27b inhibitor negative control reagent, mir-27b inhibitor, LY294002, and mir-27b inhibitor + LY294002, respectively. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used to detect the T-cell proliferation. The migration ability was detected by Scratch assays. The angiogenesis of HMEC-1 cells was observed by in vitro tube formation assay. The mRNA and protein expression of vascular endothelial growth factor (VEGF) in HMEC-1 cells and the mRNA and protein expression of collagen I, collagen III, α-SMA, and MMP1 in BJ cells were detected by quantitativereal-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. Meanwhile, the PI3K/protein kinase B (AKT) pathway-related proteins were also detected by Western blot. The proliferation, migration, angiogenesis, the mRNA and protein expression of VEGF and the protein expression of p-PI3K and p-AKT in HMEC-1 cells were increased after treated with mir-27b inhibitor. Meanwhile, the proliferation, migration, and the protein expression of collagen I, collagen III, α-SMA, MMP1, p-PI3K, and p-AKT in BJ cells were increased after treated with mir-27b inhibitor. However, the angiogenesis and fibroblast activation of mir-27b inhibitor was reversed by LY294002, and the activate effect to PI3K/AKT pathway was also inhibited. Down-regulation of mir-27b could promote angiogenesis and fibroblast activation, and its mechanism is related to activate PI3K/AKT signaling pathway.

Novel HDGF/HIF-1\u03b1/VEGF axis in oral cancer impacts disease prognosis

BackgroundHepatoma-derived growth factor (HDGF) participates in angiogenesis and represents a negative prognostic factor in oral cancer. The current study was designed to elucidate the regulatory mechanism between HDGF and vascular endothelial growth factor (VEGF) and the clinical impact of oral cancer.MethodsTCGA data and surgical samples from oral cancer patients were used for the clinicopathological parameter and survival analysis. Human oral cancer SCC4 and SAS cells were treated with recombinant HDGF protein. VEGF gene expression and protein level were analyzed by RT-PCR, Western blotting, and enzyme-linked immunosorbent assay. The signaling pathways for regulating VEGF expression were investigated. The nucleolin neutralizing antibody and HIF-1α inhibitor were applied to SCC4 cells to investigate their effects on the HDGF-stimulated VEGF pathways.ResultsTCGA and immunohistochemical analysis revealed a positive correlation between HDGF and VEGF expression in oral cancer tissues. Recombinant HDGF significantly increased VEGF gene and protein expression in oral cancer SCC4 cells in a dose-dependent manner. HDGF enhanced the phosphorylation levels of AKT and IkB and the protein level of HIF-1α and NF-κB. The nucleolin-neutralizing antibody abolished HDGF-stimulated HIF-1α, NF-κB and VEGF protein expression in SCC4 cells. The HIF-1α inhibitor antagonized the HDGF-induced VEGF gene expression. High VEGF expression was strongly correlated with HDGF expression, advanced disease, and poor survival.ConclusionThis study postulated a new pathway in which HDGF activated HIF-1α and then induced VEGF expression through binding to membrane nucleolin under normoxic conditions, leading to poor disease control. The HDGF/HIF-1α/VEGF axis is important for developing future therapeutic strategies.

Vascular Endothelial Growth Factor (VEGF-C) Protein Expression Related with Lymphangiogensis in Iraqi Cervical Cancer Patients

Objective: Metastatic spread of tumor cells to distant organs is the leading cause of mortality from cancer. Although metastatic tumor spread can occur via a different mechanism. lymphangiogenic factors recognized were vascular endothelial growth factor (VEGF)–C and –D, which bind to a tyrosine kinase receptor, VEGF receptor (R)–3. Binding affinities to VEGFR-2 receptor increase on the lymphatic and blood endothelium therefore enables both growth factors to also exert lymphangiogenic and angiogenic effects and increased incidence of lymph node metastasis. The aim of this study is to evaluate the VEGF-C protein expression in cervical cancer cells and lymph vessels and found the relationship of this marker with lymphangiogensis of Iraqi cervical cancer samples. Method: In this study, expression of VEGF-C was noticed in 55 cervical samples by Immuno- histochemistry. 35 cases diagnosed as invasive cervical cancer in addition to 20 normal samples consider as control. Immunohistochemistry was performed and the cytoplasm level of VEGF-C was scored by the percentage of positive cells and intensity. Results: The present data evaluated the prognostic significance of VEGF-C to cervical cancer, cytoplasm staining was seen in 29 cases (82.9 %) in cervical cancer tissues. Only 4 out of 35 cases (11.4 %) displayed cytoplasmic and nuclear tissue. There is significant difference of VEGF-C staining in lymphatic vessels and cancer cells (χ2= 5.04, p = 0.023*) regarding to positive expression (20/ 57.1%), (25/ 71.4 %) respectively and negative VEGF-C staining 15 (42.9%), 10 (28.6 %) respectively. High positive percentage of VEGF-C expression in cytoplasm of malignant cases in score 2&amp; 3 (25.7%, 45.7 %, P-value= 0.0392 *, 0.029* respectively) as compared to normal cases (15%, 30% respectively). Demographic criteria of patients revealed association with VEGF-C expression patterns. Differentiation Well + moderately and histologic type Squamous carcinoma showed significantly associated with VEGF-C (P=0.0071** &amp; 0.0071** respectively). Positive VEGF-C staining in cancer cells had more lymphatic vessel (17/68 %) as compared to negative cases (3/30 %) with Chi-Square 8.263, p value= 0.0061**. Also, positive VEGF-C staining had more lymph node associated (9/36%) compared to negative cases (1/10 %) with Chi-Square 13.503, p value= 0.0001**. Conclusion: In conclusion, high expression of vascular endothelial growth factor was noticed in cervical cancer cells and lymph vascular invasion indicating the important role of this marker as prognostic factor for Iraqi cervical cancer. Additionally, these results suggested that VEGF-C promoted cervical cancer metastasis using immunohistochemistry technique. Our findings offer new vision into the role of VEGF-C in cervical cancer development and give potential target for study the lymphangiogensis of tumor in Iraqi women.

Hypoxia Mediates Runt-Related Transcription Factor 2 Expression via Induction of Vascular Endothelial Growth Factor in Periodontal Ligament Stem Cells.

Periodontitis is characterized by the loss of periodontal tissues, especially alveolar bone. Common therapies cannot satisfactorily recover lost alveolar bone. Periodontal ligament stem cells (PDLSCs) possess the capacity of self-renewal and multilineage differentiation and are likely to recover lost alveolar bone. In addition, periodontitis is accompanied by hypoxia, and hypoxia-inducible factor-1α (HIF-1α) is a master transcription factor in the response to hypoxia. Thus, we aimed to ascertain how hypoxia affects runt-related transcription factor 2 (RUNX2), a key osteogenic marker, in the osteogenesis of PDLSCs. In this study, we found that hypoxia enhanced the protein expression of HIF-1α, vascular endothelial growth factor (VEGF), and RUNX2 ex vivo and in situ. VEGF is a target gene of HIF-1α, and the increased expression of VEGF and RUNX2 proteins was enhanced by cobalt chloride (CoCl2, 100 μmol/L), an agonist of HIF-1α, and suppressed by 3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole (YC-1, 10 μmol/L), an antagonist of HIF-1α. In addition, VEGF could regulate the expression of RUNX2, as RUNX2 expression was enhanced by human VEGF (hVEGF165) and suppressed by VEGF siRNA. In addition, knocking down VEGF could decrease the expression of osteogenesis-related genes, i.e., RUNX2, alkaline phosphatase (ALP), and type I collagen (COL1), and hypoxia could enhance the expression of ALP, COL1, and osteocalcin (OCN) in the early stage of osteogenesis of PDLSCs. Taken together, our results showed that hypoxia could mediate the expression of RUNX2 in PDLSCs via HIF-1α-induced VEGF and play a positive role in the early stage of osteogenesis of PDLSCs.

Changes in neurological and pathological outcomes in a modified rat spinal cord injury model with closed canal.

Most animal spinal cord injury models involve a laminectomy, such as the weight drop model or the transection model. However, in clinical practice, many patients undergo spinal cord injury while maintaining a relatively complete spinal canal. Thus, open spinal cord injury models often do not simulate real injuries, and few previous studies have investigated whether having a closed spinal canal after a primary spinal cord injury may influence secondary processes. Therefore, we aimed to assess the differences in neurological dysfunction and pathological changes between rat spinal cord injury models with closed and open spinal canals. Sprague-Dawley rats were randomly divided into three groups. In the sham group, the tunnel was expanded only, without inserting a screw into the spinal canal. In the spinal cord injury with open canal group, a screw was inserted into the spinal canal to cause spinal cord injury for 5 minutes, and then the screw was pulled out, leaving a hole in the vertebral plate. In the spinal cord injury with closed canal group, after inserting a screw into the spinal canal for 5 minutes, the screw was pulled out by approximately 1.5 mm and the flat end of the screw remained in the hole in the vertebral plate so that the spinal canal remained closed; this group was the modified model, which used a screw both to compress the spinal cord and to seal the spinal canal. At 7 days post-operation, the Basso-Beattie-Bresnahan scale was used to measure changes in neurological outcomes. Hematoxylin-eosin staining was used to assess histopathology. To evaluate the degree of local secondary hypoxia, immunohistochemical staining and western blot assays were applied to detect the expression of hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF). Compared with the spinal cord injury with open canal group, in the closed canal group the Basso-Beattie-Bresnahan scores were lower, cell morphology was more irregular, the percentage of morphologically normal neurons was lower, the percentages of HIF-1α- and VEGF-immunoreactive cells were higher, and HIF-1α and VEGF protein expression was also higher. In conclusion, we successfully established a rat spinal cord injury model with closed canal. This model could result in more serious neurological dysfunction and histopathological changes than in open canal models. All experimental procedures were approved by the Institutional Animal Care Committee of Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, China (approval No. HKDL201810) on January 30, 2018.

Hydrogen sulfide improves vessel formation of the ischemic adductor muscle and wound healing in diabetic db/db mice.

Objective(s):It has been demonstrated that hydrogen sulfide plays a vital role in physiological and pathological processes such as regulating inflammation, oxidative stress, and vessel relaxation. The aim of the study was to explore the effect of hydrogen sulfide on angiogenesis in the ischemic adductor muscles of type 2 diabetic db/db mice and ischemic diabetic wound healing.Materials and Methods:The femoral arteries of diabetic db/db mice were isolated and ligated for preparation of ischemic hind limb model. Round incision was made on ischemic and non-ischemic limbs. The wounds were treated with sodium bisulfide (hydrogen sulfide donor). Real-time PCR and Western blotting were used to measure transcription of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), platelet derived growth factor (PDGF), hypoxia inducible factor-1α (HIF-1α) and endothelial nitric oxide synthase (eNOS) and protein expression of VEGF, VEGF receptor (VEGFR) and PDGF, PDGF receptor (PDGFR), respectively. Angiogenesis and morphological changes in adductor muscles were observed.Results:Hydrogen sulfide significantly increased transcription of VEGF, EGF, PDGF, HIF-1α, eNOS and protein expression of VEGF, PDGF, and phosphorylated VEGFR and PDGFR. Treatment with hydrogen sulfide significantly improved ischemic wound healing and formation of granulation tissue, and increased the number of small vessels in the ischemic adductor muscles.Conclusion:Our data suggested that hydrogen sulfide attenuated injury of ischemic adductor muscle, and promoted the ischemic diabetic wound healing via modulating angiogenesis in type 2 diabetic db/db mice.