Vascular Endothelial Growth Factor A Protein Levels Research Articles

FUS-stabilized USP35 promotes growth, invasion and angiogenesis in NSCLC through deubiquitinating VEGFA.

Vascular endothelial growth factor A (VEGFA) is an important regulator for non-small cell lung cancer (NSCLC). Our study aimed to reveal its upstream pathway to provide new ideas for developing the therapeutic targets of NSCLC. The mRNA and protein levels of VEGFA, ubiquitin-specific peptidase 35 (USP35), and FUS were determined by quantitative real-time PCR and Western blot. Cell proliferation, apoptosis, invasion and angiogenesis were detected using CCK8 assay, EdU assay, flow cytometry, transwell assay and tube formation assay. The interaction between USP35 and VEGFA was assessed by Co-IP assay and ubiquitination assay. Animal experiments were performed to assess USP35 and VEGFA roles in vivo. VEGFA had elevated expression in NSCLC tissues and cells. Interferences of VEGFA inhibited NSCLC cell proliferation, invasion, angiogenesis, and increased apoptosis. USP35 could stabilize VEGFA protein level by deubiquitination, and USP35 knockdown suppressed NSCLC cell growth, invasion and angiogenesis via reducing VEGFA expression. FUS interacted with USP35 to promote its mRNA stability, thereby positively regulating VEGFA expression. Also, USP35 silencing could reduce NSCLC tumorigenesis by downregulating VEGFA. FUS-stabilized USP35 facilitated NSCLC cell growth, invasion and angiogenesis through deubiquitinating VEGFA, providing a novel idea for NSCLC treatment.

Overexpression of hypoxia-inducible factor 1α and excessive vascularization in the peri-implantation endometrium of infertile women with chronic endometritis.

Chronic endometritis (CE) contributes to impaired endometrial receptivity and is closely associated with poor in vitro fertilization (IVF) outcomes. However, the mechanisms underlying CE are unclear. Here, we investigated the role of the hypoxic microenvironment and endometrial vascularization in the peri-implantation endometrium of infertile women with CE. This retrospective study involved 15 fertile women and 77 infertile patients diagnosed with CE based on CD138+ ≥1/10 high-power fields (HPFs). The CE patients were divided into Group 1 (CD138+ 1-4/10 HPFs, 53 cases) and Group 2 (CD138+ ≥5/10 HPFs, 24 cases). The expression levels of hypoxia-inducible factor 1α (HIF1α), vascular endothelial growth factor A (VEGFA), and vascular endothelial growth factor receptor 2 (VEGFR2) in peri-implantation endometrium were assessed by qRT-PCR and western blot analyses. Spatial levels of HIF1α, VEGFA, and VEGFR2 in various endometrial compartments was determined using immunohistochemistry and H-score analysis. Microvascular density (MVD) was determined using CD34 staining and scored using Image J. Finally, we used qRT-PCR to assess changes in the expression of HIF1α, VEGFA, and VEGFR2 in CE patients after treatment with first-line antibiotics. Relative to Group 1 and control group, during the implantation window, protein and mRNA levels of HIF1α, VEGFA, and VEGFR2 were markedly high in Group 2 (P<0.05). H-score analysis showed that HIF1α, VEGFA, and VEGFR2 in the luminal, glandular epithelium, and stromal compartments were markedly elevated in Group 2, comparing to control group and Group 1 (P<0.05). Moreover, markedly elevated MVD levels were observed in Group 2. Notably, the above indexes did not differ significantly in the control group versus Group 1. Treatment with antibiotics significantly suppressed the endometrial HIF1α and VEGFA levels in CE-cured patients. Here, we for the first time report the upregulation of HIF1α, VEGFA, and VEGFR2, as well as excessive endometrial vascularization in the peri-implantation endometrium of CE patients. Our findings offer new insights into reduced endometrial receptivity in CE-associated infertility.

The effect of pre-analytical variables on downstream application and data analysis of human endometrial biopsies.

STUDY QUESTIONWhat are the effects of pre-analytical variables on the downstream analysis of patient-derived endometrial biopsies?SUMMARY ANSWERThere are distinct differences in the protein levels of the master regulator of oxygen homeostasis, hypoxia-inducible factor-1-alpha (HIF1α), and the protein and mRNA levels of three related genes, carbonic anhydrase 9 (CA9), vascular endothelial growth factor A (VEGFA) and progesterone receptor (PR) in human endometrial biopsies, depending on the pre-analytical variables: disease status (cancer vs benign), timing of biopsy (pre- vs post-hysterectomy) and type of biopsy (pipelle vs full-thickness).WHAT IS KNOWN ALREADYPatient-derived biopsies are vital to endometrial research, but pre-analytical variables relating to their collection may affect downstream analysis, as is evident in other tissues.STUDY DESIGN, SIZE, DURATIONA prospective observational study including patients undergoing hysterectomy for endometrial cancer (EC) or benign indications was conducted at a large tertiary gynaecological unit in the UK. Endometrial biopsies were obtained at different time points (pre- or post-hysterectomy) using either a pipelle endometrial sampler or as a full-thickness wedge biopsy.PARTICIPANTS/MATERIALS, SETTING, METHODSThe changes in HIF1α, CA9, VEGFA and PR protein levels were measured by semi-quantitative analysis of immunostaining, and the expression levels of three genes (CA9, VEGFA and PR) were investigated by quantitative real-time PCR, in endometrial biopsies from 43 patients undergoing hysterectomy for EC (n = 22) or benign gynaecological indications (n = 21).MAIN RESULTS AND THE ROLE OF CHANCEAn increase in HIF1α immunostaining was observed in EC versus benign endometrium (functionalis glands) obtained pre-hysterectomy (P < 0.001). An increase in CA9 immunostaining was observed in EC versus benign endometrial functionalis glands at both pre- and post-hysterectomy time points (P = 0.03 and P = 0.003, respectively). Compared with benign endometrial pipelle samples, EC samples demonstrated increased mRNA expression of CA9 (pre-hysterectomy P < 0.001, post-hysterectomy P = 0.008) and VEGFA (pre-hysterectomy P = 0.004, post-hysterectomy P = 0.002). In benign uteri, HIF1α immunoscores (functionalis glands, P = 0.03 and stroma, P = 0.009), VEGFA immunoscores (functionalis glands, P = 0.03 and stroma, P = 0.01) and VEGFA mRNA levels (P = 0.008) were increased in matched post-hysterectomy versus pre-hysterectomy samples. Similarly, in EC, an increase in VEGFA immunoscores (epithelial and stromal) and VEGFA mRNA expression was observed in the matched post-hysterectomy versus pre-hysterectomy biopsies (P = 0.008, P = 0.004 and P = 0.018, respectively). Full-thickness benign post-hysterectomy endometrial biopsies displayed increased VEGFA (P = 0.011) and PR (P = 0.006) mRNA expression compared with time-matched pipelle biopsies.LARGE SCALE DATAN/A.LIMITATIONS, REASONS FOR CAUTIONThis descriptive study explores the effect of pre-analytical variables on the expression of four proteins and three hypoxia-related genes in a limited number of endometrial biopsies from patients with EC and benign controls. Due to the small number, it was not possible to investigate other potential variables such as menstrual cycle phase, region-specific differences within the endometrium, grade and stage of cancer, and surgical technicalities.WIDER IMPLICATIONS OF THE FINDINGSCareful consideration of the effects of these pre-analytical variables is essential when interpreting data relating to human endometrial biopsies. A standardized approach to endometrial tissue collection is essential to ensure accurate and clinically transferrable data.STUDY FUNDING/COMPETING INTEREST(S)The authors have no conflicts of interest to declare. The work included in this manuscript was funded by Wellbeing of Women project grants RG1073 and RG2137 (D.K.H.), Wellbeing of Women Entry-Level Scholarship ELS706 and Medical Research Council MR/V007238/1 (A.M./D.K.H.), Liverpool Women’s Hospital Cancer Charity (M.A.) and University of Liverpool (L.B., L.R. and E.N.).

Differential Responses of Human Dental Pulp Stromal Cells to Bioceramic Materials: A Comparative In Vitro Study

The aim of this study was to compare the effects of white MTA-Angelus (wMTA), Biodentine® (Biodentine) and TotalFill® BC Root Repair MaterialTM putty (TotalFill) on human dental pulp stromal cells (hDPSCs) in vitro. hDPSCs were isolated from third molars of healthy young adults. Material elutes at different concentrations were prepared. Cells were exposed to the eluates for 1, 3, and 7 days. Cell proliferation was evaluated using 3-(4,5-dimethyl-thiazoyl)-2, 5-diphenyl-tetrazolium bromide assay. The expression of alkaline phosphatase (ALP), osteoprotegerin (OPG), osteocalcin (OC), collagen1A (Col1A), runt-related transcription factor 2 (RUNX2), vascular endothelial growth factor-A (VEGF-A), fibroblast growth factor-1 (FGF-1), interleukin 6 (IL6), tumor necrosis factor alpha (TNFα), and interleukin-1-beta (IL1β) was determined by reverse transcription-polymerase chain reaction (RT-PCR). VEGF-A protein levels and ALP activity were quantified in the culture supernatant. Data were analyzed by two-way analysis of variance (ANOVA). p values <0.05 were considered statistically significant. hDPSC proliferation was decreased in a dose-related manner for all materials on day 3. The same effect was observed with wMTA and TotalFill on day 7. RT-PCR showed that Biodentine increased the expression of the osteogenic markers ALP, OPG, and OC. TotalFill decreased the ALP expression and activity, enhanced the production of angiogenic VEGF-A, and downregulated the inflammatory IL6 on day 7. Although the tested materials are used interchangeably in vital pulp therapy, the findings showed varied hDPSC responses. Biodentine did not affect cell proliferation and increased the expression of osteo-/odontogenic markers compared to wMTA and TotalFill, whereas TotalFill decreased cell proliferation and exhibited enhanced angiogenic and anti-inflammatory effects over time. The clinical significance of the results needs further investigation in an attempt to provide recommendations on the selection of bioceramic pulp capping material under different scenarios of pulpal pathosis.

Circular RNA circLMF1 regulates PDGF-BB-induced proliferation and migration of human aortic smooth muscle cells by regulating the miR-125a-3p/VEGFA or FGF1 axis.

Atherosclerosis is a major cause of cardiovascular disease, in which vascular smooth muscle cells (VSMCs) proliferation and migration play a vital role. Circular RNAs (circRNAs) have been reported to be correlated with the VSMCs function. Therefore, this study is designed to explore the role and mechanism of circRNA lipase maturation factor 1 (circLMF1) in Human aortic VSMCs (HASMCs). The microarray was used for detecting the expression of circLMF1 in proliferative and quiescent HASMCs. Levels of circLMF1, microRNA-125a-3p (miR-125a-3p), vascular endothelial growth factor A (VEGFA), and fibroblast growth factor 1 (FGF1) were determined by real-time quantitative polymerase chain reaction (RT-qPCR). Cell viability, cell cycle progression, and migration were assessed by Cell Counting Kit-8 (CCK-8), flow cytometry, wound healing, and transwell assays, respectively. Western blot assay determined proliferating cell nuclear antigen (PCNA), Cyclin D1, matrix metalloproteinase (MMP2), osteopontin (OPN), VEGFA, and FGF1 protein levels. The possible interactions between miR-125a-3p and circLMF1, and miR-125a-3p and VEGFA or FGF1 were predicted by circbank or targetscan, and then verified by a dual-luciferase reporter, RNA Immunoprecipitation (RIP), RNA pull-down assays. CircLMF1, VEGFA, and FGF1 were increased, and miR-125a-3p was decreased in platelet-derived growth factor-BB (PDGF-BB)-inducted HASMCs. Functionally, circLMF1 knockdown hindered cell viability, cell cycle progression, and migration in PDGF-BB-treated HASMCs. Mechanically, circLMF1 could regulate VEGFA or FGF1 expression through sponging miR-125a-3p. Our findings revealed that circLMF1 deficiency could inhibit cell viability, cell cycle progression, and migration of PDGF-BB stimulated atherosclerosis model partly through the miR-125a-3p/VEGFA or FGF1 axis, suggesting that targeting circLMF1 can be a feasible therapeutic strategy for atherosclerosis.

Expression and prognostic significance of vascular endothelial growth factor-A (VEGF-A) and its receptor in canine prostate cancer.

Vascular endothelial growth factor-A (VEGF-A) and its receptor, VEGF receptor-2 (VEGFR-2), represent a complex family of angiogenic molecules consisting of different ligands and receptors. Due to the importance of VEGF-A/VEGFR-2 signaling in tumor proliferation and angiogenesis, this study aimed to evaluate the protein and gene expression levels of VEGF-A and VEGFR-2 in canine prostate cancer (PC). We analyzed VEGF-A and VEGFR-2 expression in 87 PC samples by immunohistochemistry and quantitative-polymerase chain reaction. PC samples were graded according to the Gleason score and the immunohistochemical staining for VEGF-A and VEGFR-2 was quantified using a selected threshold from the ImageJ Software. Microvascular density was assessed by cluster of differentiation 31 staining and counting the number of positive vessels. Additionally, the homology of VEGF-A and VEGFR-2 between humans and dogs was assessed, followed by the construction of a protein structure homology model to compare the tertiary structures of these proteins in both species. Negative to weakly positive expression levels of VEGF-A and VEGFR-2 were observed in the epithelial cells of the normal prostate (NP) and prostatic hyperplasia samples. In contrast, the canine proliferative atrophy and PC samples exhibited higher VEGF-A (p < .0001) and VEGFR-2 (p < .0001) compared to NP. Moreover, positive correlations between the expression levels of VEGF-A and VEGFR-2 (Spearman's coefficient (r) = .68, p = .013) and the expression levels of VEGF-A and VEGFR-2 proteins (r = .8, p < .0001) were also observed in the NP samples. Additionally, the patients with PC exhibiting higher VEGFR-2 expression levels experienced a shorter survival period (p = .0372). Furthermore, we found an association between the microvascular density and overall survival. Dogs with a higher number of vessels showed a shorter survival time. We further demonstrated that the VEGF-A and VEGFR-2 exhibited high homology between humans and dogs, and identified their protein structures in both species. In conclusion, VEGFR-2 appears to be an independent prognostic factor in animals with PC. VEGF-A and VEGFR-2 are highly conserved between humans and dogs, which can be investigated further in future cross-species studies to explore their therapeutic applications.

Intravitreal connective tissue growth factor neutralizing antibody or bevacizumab alone or in combination for prevention of proliferative vitreoretinopathy in an experimental model

Connective tissue growth factor (CTGF) is released by retinal pigment epithelial (RPE) cells and detectable in proliferative membranes (PrMs). This experimental study was performed to investigate the mRNA and protein levels of both CTGF and vascular endothelial growth factor A (VEGF-A) in a rabbit model of proliferative vitreoretinopathy (PVR). In addition, the effects of a single intravitreal injection of the safe dose of anti-CTGF or bevacizumab as monotherapy and in combination were evaluated. PVR was induced in the right eye of albino rabbits by intravitreal injection of cultured adult human RPE cells. Quantitative real-time reverse transcription PCR (qRT-PCR) and Western blot analysis of CTGF and VEGF-A were performed on whole eye tissue in the PVR model versus controls at different time points. In the next step, the PVR models were assigned to five groups. The monotherapy groups received a single intravitreal injection of 0.1 ml of anti-CTGF 100 μg/ml (final concentration of 6.6 μg/ml in the vitreous) or 0.03 ml of 25 mg/ml bevacizumab. In the combined group, the abovementioned amounts of anti-CTGF and bevacizumab were injected intravitreally from separate sites in one session. No antibody injection was performed in the control group. Intravitreal injection of 0.1 ml of control IgG (1 mg/ml of isotype matched) antibody was performed in the placebo group. After 2 weeks, histologic evaluation including, trichrome staining for collagen, immunostaining by anti-alpha-smooth muscle actin for myofibroblasts, and anti-collagen type-1 antibody on paraffin embedded anterior-posterior sections was done. In addition, fundus photography was performed for clinically equivalent PVR staging. Twenty-four hours following PVR induction, CTGF mRNA and protein levels increased five- and- three-fold compared to controls, respectively (P < 0.001). VEGF-A mRNA and protein levels decreased significantly after 72 h of PVR induction compared to controls (P < 0.05). Means of PrM thickness and myofibroblast cell counts significantly decreased in the anti-CTGF group (P < 0.001 and P < 0.05, respectively). The mean area of collagen type-1 fibers of PrM in the mono- and combination therapy groups that received intravitreal anti-CTGF was significantly reduced (P < 0.001); in addition, mild PVR (stage-1 and 2) formation occurred in comparison with moderate to severe PVR (stage-4 and higher) in other groups. In conclusion, we found that intravitreal injection of CTGF neutralizing antibody resulted in a reduction in PrM thickness, collagen fibers and myofibroblast density in the PVR model. CTGF inhibition may represent a potential therapeutic target for PVR.

Protective effects of piperine on the retina of mice with streptozotocin-induced diabetes by suppressing HIF-1/VEGFA pathway and promoting PEDF expression.

To evaluate the protective mechanisms of piperine in the retina of mice with streptozotocin-induced diabetes. In experiments in vitro, stimulation by chemical hypoxia was established in ARPE-19 cells. Then, the expression of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor A (VEGFA), and pigment epithelium-derived factor (PEDF) was assessed at the mRNA and protein levels. In experiments in vivo, diabetes mellitus was established by intraperitoneally injecting 150 mg/kg streptozotocin once. After 3wk of the onset of diabetes, 15 mg/kg piperine was intraperitoneally injected once daily for 1 or 3wk. Then, the retinal morphology and mRNA and protein expression were assessed. In hypoxia, 1-100 µmol/L piperine significantly decreased the expression of VEGFA mRNA and increased the expression of PEDF mRNA without affecting HIF-1α mRNA. Meanwhile, 100 µmol/L piperine substantially decreased the protein level of VEGFA and increased the protein level of PEDF. The HIF-1α protein level was also hampered by piperine. In the diabetic retina of mice, the morphological damage was alleviated by piperine. Likewise, the retinal vascular leakage was substantially decreased by piperine. Further, the protein levels of HIF-1α and VEGFA were significantly reduced by piperine. Moreover, the level of the antiangiogenic factor of PEDF dramatically increased by piperine. Piperine may exert protective effects on the retina of mice with diabetes via regulating the pro-antiangiogenic homeostasis composed of HIF-1/VEGFA and PEDF.

Deficiency of Mettl3 in Bladder Cancer Stem Cells Inhibits Bladder Cancer Progression and Angiogenesis

RNA N6-methyladenosine is a key step of posttranscriptional modulation that is involved in governing gene expression. The m6A modification catalyzed by Mettl3 has been widely recognized as a critical epigenetic regulation process for tumorigenic properties in various cancer cell lines, including bladder cancer. However, the in vivo function of Mettl3 in bladder cancer remains largely unknown. In our study, we found that ablation of Mettl3 in bladder urothelial attenuates the oncogenesis and tumor angiogenesis of bladder cancer using transgenic mouse model. In addition, conditional knockout of Mettl3 in K14+ bladder cancer stem cell population leads to inhibition of bladder cancer progression. Coupled with the global transcriptome sequencing and methylated RNA immunoprecipitation sequencing results, we showed that deletion of Mettl3 leads to the suppression of tyrosine kinase endothelial (TEK) and vascular endothelial growth factor A (VEGF-A) through reduced abundance of m6A peaks on a specific region. In addition, the depletion of Mettl3 results in the decrease in both messenger RNA (mRNA) and protein levels of TEK and VEGF-A in vitro. Taken together, Mettl3-mediated m6A modification is required for the activation of TEK–VEGF-A-mediated tumor progression and angiogenesis. Our findings may provide theoretical basis for bladder cancer treatment targeting Mettl3.

TPTEP1 suppresses high glucose-induced dysfunction in retinal vascular endothelial cells by interacting with STAT3 and targeting VEGFA.

Diabetic retinopathy (DR) is a vascular complication of diabetes mellitus that causes visual impairment and blindness. Long noncoding RNAs (lncRNAs) have been revealed to be involved in biological processes of several diseases including DR. We designed this study to investigate the specific role of TPTEP1 in DR. First, we mimicked diabetic conditions with high glucose (HG) stimulation of human retinal vascular endothelial cells (HRVECs) and measured TPTEP1 expression in HG-stimulated HRVECs using RT-qPCR analysis. Then, CCK-8, Transwell, and Matrigel tube formation assays as well as western blot analysis were performed to reveal the biological functions of TPTEP1 in HG-stimulated HRVECs. Subsequently, bioinformatics analysis, RNA pull down, luciferase reporter and ChIP assays as well as western blot analysis evaluated the relationship of TPTEP1, signal transducer and activator of transcription 3 (STAT3) and vascular endothelial growth factor A (VEGFA) in HG-stimulated HRVECs. Finally, to verify the regulation of the TPTEP1/STAT3/VEGFA axis in HG-stimulated HRVECs, rescue experiments were carried out in HG-stimulated HRVECs. TPTEP1 presented a significant downregulation in HG-stimulated HRVECs. Additionally, TPTEP1 overexpression reduced viability, migration, and angiogenesis in HG-stimulated HRVECs. Moreover, TPTEP1 suppressed phosphorylation and nuclear translocation of STAT3, and thereby downregulated VEGFA mRNA and protein levels. Furthermore, TPTEP1 overexpression-mediated suppression of HG-induced dysfunction in HRVECs was countervailed by STAT3 upregulation or VEGFA upregulation. TPTEP1 alleviated HG-induced dysfunction in HRVECs via interacting with STAT3 and targeting VEGFA.

Long Non-Coding RNA JPX Contributes to Tumorigenesis by Regulating miR-5195-3p/VEGFA in Non-Small Cell Lung Cancer

BackgroundLung cancer is the most frequently diagnosed cancer. Of all lung cancers, 80–85% are verified as non-small-cell lung cancer (NSCLC). Just proximal to X-inactive specific transcript (JPX), functions as lncRNA, contributed to tumor progression and suggested a poor prognosis in NSCLC. However, the pathogenesis of JPX involved in NSCLC is still unclear.MethodsThe expressions of JPX, miR-5195-3p, and Vascular endothelial growth factor A (VEGFA) were detected by real-time quantitative polymerase chain reaction (RT-qPCR). Proliferation, colony number, apoptosis, invasion, and migration were analyzed by Cell Counting Kit-8 (CCK-8), colony formation, flow cytometry, transwell, and wound healing assays, severally. The protein levels of VEGFA, E-cadherin, N-cadherin, and Vimentin were detected by Western blot assay. The interaction between JPX, miR-5195-3p and VEGFA was predicted by starBase, and then verified by the dual-luciferase reporter, RNA Immunoprecipitation (RIP) and RNA pull-down assay. The biological role of JPX on NSCLC tumor growth was assessed by the xenograft tumor model in vivo.ResultsJPX and VEGFA were upregulated, and miR-5195-3p was downregulated in NSCLC. JPX induced proliferation, colony number, invasion, migration, epithelial–mesenchymal transition (EMT), and inhibited apoptosis of NSCLC cells. JPX is directly bound to miR-5195-3p. JPX regulated NSCLC cell proliferation, apoptosis and EMT by modulating miR-5195-3p. miR-5195-3p hindered NSCLC cells proliferation, EMT and accelerated apoptosis by directly targeting VEGFA. JPX silencing hindered the cell growth of NSCLC in vivo.ConclusionJPX facilitated proliferation, colony number, invasion, migration, EMT, and repressed apoptosis by miR-5195-3p/VEGFA axis, offering a possible therapeutic strategy for NSCLC.

Sorafenib blocks the activation of the HIF-2α/VEGFA/EphA2 pathway, and inhibits the rapid growth of residual liver cancer following high-intensity focused ultrasound therapy in vivo

BackgroundInsufficient high-intensity focused ultrasound (HIFU) can promote the rapid progression of the residual tumor through the hypoxia inducible factor-2α +(HIF-2α)/vascular endothelial growth factor A (VEGFA)/ephrin type-A receptor 2 (EphA2) pathway. Although sorafenib has been shown to significantly improve the survival of patients with advanced liver cancer, the use of sorafenib in residual tumor tissues following HIFU has rarely been elucidated. Thus, this study aimed to investigate the potential adjuvant therapeutic effects of sorafenib following HIFU in order to reduce the relapse rate following insufficient HIFU. MethodsXenograft tumors were established using nude mice injected with liver cancer cells. At approximately 4 weeks after the inoculation of the tumor cells (tumors reached 1.3–1.5 cm), all mice were randomly divided into 3 groups as follows: i) The control group (no treatment); ii) the HIFU-alone group, and iii) the combination group (HIFU + sorafenib), with 6 mice per group. The residual tumor volume was determined among the different treatment groups. The protein expression levels of HIF-2α, VEGFA and EphA2 were determined by immunohistochemistry and western blotting, and the mRNA levels were detected by RT-qPCR. The microvessel density (MVD) was calculated by CD31 immunohistochemistry staining. ResultsThe results revealed that by comparing the control group, insufficient HIFU promoted HIF-2α, VEGFA and EphA2 expression (P < 0.05). Compared with the HIFU-alone group, the protein and mRNA levels of HIF-2α, VEGFA and EphA2 were markedly decreased in the group that received combined treatment with HIFU and sorafenib (P < 0.05). Similar results were obtained for MVD expression. Synergistic tumor growth inhibitory effects were also observed between the control group and HIFU group (P < 0.05). ConclusionsThe findings of this study demonstrate that the expression of HIF-2α, VEGFA and EphA2 can be inhibited by sorafenib, and that sorafenib is likely to provide an effective adjunct treatment for patients with HCC following HIFU ablation.

Atorvastatin improves the proliferation and migration of endothelial progenitor cells via the miR-221/VEGFA axis.

The present study was aimed at investigating the detailed functions of atorvastatin, a lipid-lowering agent, in the pathogenesis of coronary slow flow (CSF), a clinical disease characterized by delayed angiographic coronary opacity without obstructive coronary disease. In the present study, we successfully identified isolated endothelial progenitor cells (EPCs) from the peripheral blood of patients with CSF. Their vascular endothelial growth factor-A (VEGFA) protein levels were determined using immunoblotting analyses. We determined cell viability using MTT assays, cell migration capacity using Transwell assays, and the angiogenic capacity using a tube formation assay. The target association between miR-221 and VEGFA was validated with a luciferase reporter assay. Atorvastatin treatment increased EPC VEGFA protein levels, proliferation, migration, and angiogenesis. miR-221 expression was down-regulated after atorvastatin treatment; miR-221 overexpression exerted an opposing effect to atorvastatin treatment on VEGFA protein, EPC proliferation, migration, and angiogenesis. The protective effects of atorvastatin treatment on VEGFA protein and EPCs could be significantly suppressed by miR-221 overexpression. miR-221 directly bound the VEGFA 3′UTR to inhibit its expression. In conclusion, atorvastatin improves the cell proliferation, migration, and angiogenesis of EPCs via the miR-221/VEGFA axis. Thus, atorvastatin could be a potent agent against CSF, pending further in vivo and clinical investigations.

VEGFA and NFE2L2 Gene Expression and Regulation by MicroRNAs in Thyroid Papillary Cancer and Colloid Goiter.

Deregulation of VEGFA (Vascular Endothelial Growth Factor A) and NFE2L2 (Nuclear Factor (Erythroid-derived 2)-Like 2), involved in angiogenesis and oxidative stress, can lead to thyroid cancer progression. MiR-17-5p and miR-612 are possible regulators of these genes and may promote thyroid disorders. In order to evaluate the involvement of VEGFA, NFE2L2, hsa-miR-17-5p, and hsa-miR-612 in thyroid pathology, we examined tissue samples from colloid goiter, papillary thyroid cancer (PTC), and a normal thyroid. We found higher levels of VEGFA and NFE2L2 transcripts and the VEGFA protein in goiter and PTC samples than in normal tissue. In the goiter, miR-612 and miR-17-5p levels were lower than those in PTC. Tumors, despite showing lower VEGFA mRNA expression, presented higher VEGFA protein levels compared to goiter tissue. In addition, NRF2 (Nuclear Related Transcription Factor 2) protein levels in tumors were higher than those in goiter and normal tissues. Inhibition of miR-17-5p resulted in reduced NFE2L2 expression. Overall, both transcript and protein levels of NFE2L2 and VEGFA were elevated in PTC and colloid goiter. Hsa-miR-612 showed differential expression in PTC and colloid goiter, while hsa-miR-17-5p showed differential expression only in colloid goiter, suggesting that hsa-miR-17-5p may be a positive regulator of NFE2L2 expression in PTC.

Reduced Expression of VEGF-A in Human Retinal Pigment Epithelial Cells and Human Muller Cells Following CRISPR-Cas9 Ribonucleoprotein-Mediated Gene Disruption.

PurposeTo evaluate the effects of vascular endothelial growth factor-A (VEGF-A) gene editing in human retinal pigment epithelial (RPE) cells and human Muller cells, which are the main VEGF-A producing cells in the eye.MethodsCRISPR-Cas9 ribonucleoprotein was used to target exon 1 in VEGF-A gene. Lipofectamine CRISPRMAX was used as a vehicle. In vitro gene editing efficiency was assessed on oligonucleotides and genomic DNAs. Sanger sequencing was performed to detect indels. VEGF-A messenger RNA and protein expressions were assessed using quantitative polymerase chain reaction and enzyme-linked immunosorbent assay.ResultsIn vitro cleavage assay on a 60-nucleotide DNA duplex showed 88% cleavage of the precursor. The cleavage efficiency was 40% in RPE cells and 32% in Muller cells. Sanger sequencing in the CRISPR-Cas9 treated RPE and Muller cells showed indels at the predicted cut site in both cells. After the VEGF-A gene disruption, VEGF-A protein levels decreased 43% in RPE cells (P < 0.0001) and 38% in Muller cells (P < 0.0001).ConclusionsCRISPR-Cas9–mediated gene disruption resulted in a significant decrease in the VEGF-A gene protein expression in human RPE and Muller cells. CRISPR-Cas9 ribonucleoprotein may allow simultaneous targeting of multiple VEGF-A producing cells.Translational RelevanceVEGF-A gene disruption using CRISPR-Cas9 ribonucleoprotein has a potential in treating retinal vascular diseases.

MicroRNA-4316 inhibits gastric cancer proliferation and migration via directly targeting VEGF-A

Background and aimsmicroRNAs (miRNAs) have been reported to regulate proliferation and migration by down-regulating the expression of target genes. The aims of this study were to investigate whether miR-4316 inhibited proliferation and migration by downregulating vascular endothelial growth factor A (VEGF-A) and its clinical significance in gastric cancer (GC).MethodsThe clinical tissues of the GC patients for miR-4316 and VEGF-A were detected by qRT-PCR. The protein levels of VEGF-A and c-Met were determined by western blotting. Cell Proliferation, migration, and colony forming assays were conducted to show whether miR-4316 affects proliferation by CCK-8, migration by transwell, wound healing and colony formation assays. The bioinformatic methods and luciferase reporter assay were applied to detect the relationship between miRNA and VEGF-A on its targeting 3-untranslated regions (3-UTRs). CCK-8, colony formation, wound healing, and transwell assay were performed to explore the function of miR-4316.ResultsThe results of qRT-PCR indicated that miR-4316 expression level was significantly downregulated in human GC tissues and GC cell lines compared with their control. miR-4316 inhibited proliferation, migration and colony formation in GC cell lines by reducing VEGF-A. And western blot results indicated that miR-4316 significantly inhibited GC through repressing VEGF-A and c-Met. The investigation of Luciferase assay indicated that VEGF-A is a direct target gene of miR-4316.ConclusionsmiR-4316 suppressed proliferation and migration of GC through the VEGF-A gene. MiR-4316 acts as a tumor suppressor by targeting VEGF-A and this indicated that MiR-4316 might be a potential therapeutic target for GC.

Distinct Roles of VEGFA and ANGPT2 in Lung Adenocarcinoma and Squamous Cell Carcinoma.

Background: Vascular endothelial growth factor A (VEGFA) and angiopoietin 2 (ANGPT2) are key mediators in angiogenesis. The expression and clinical significance of VEGFA and ANGPT2 have been investigated in lung cancer, but the results are controversial. The specific roles of VEGFA and ANGPT2 in adenocarcinoma (ADC) and squamous cell carcinoma (SQC) are still not fully understood. To characterize it, we conducted the current study.Materials and Methods: The relationships between clinic-pathological characteristics and the protein expressions of VEGFA and ANGPT2 were analyzed on tissue microarrays by immunohistochemistry (IHC) staining. Then public databases were used to evaluate the association of VEGFA and ANGPT2 mRNA expressions with clinic-pathological parameters and prognosis. Cobalt chloride (CoCl2) was adopted to mimic a hypoxic microenvironment and western blot was used to detect the expression of hypoxia inducible factor-1α (HIF-1α), VEGFA and ANGPT2 in lung cancer cell lines.Results: IHC staining revealed that the expressions of VEGFA and ANGPT2 were enriched in lung cancer tissues compared with normal tissues. Additionally, both VEGFA and ANGPT2 protein levels were significantly associated with the tumor size and lymph node metastasis only in ADC, not SQC. More importantly, increased VEGFA and ANGPT2 protein levels were negatively correlated with overall survival (OS) of ADC individuals. Meta-analyses of 22 gene expression omnibus (GEO) databases of lung cancer implicated that patients with higher VEGFA and ANGPT2 mRNA expressions tended to have advanced stage in ADC rather than SQC. Kaplan-Meier plot analyses further verified that high levels of VEGFA and ANGPT2 mRNA were associated with poor survival only in ADC. Moreover, the combination of VEGFA and ANGPT2 could more precisely predict prognosis in ADC. In hypoxia-mimicking conditions, induced expression of HIF-1α unregulated VEGFA and ANGPT2 proteins abundance.Conclusion: Our results showed hypoxia upregulated the protein levels of VEGFA and ANGPT2 in lung cancer cell lines, and the roles of VEGFA and ANGPT2 were distinct in ADC and SQC. Combined detections of VEGFA and ANGPT2 may be valuable prognostic biomarkers for ADC and double block of VEGFA and ANGPT2 may improve therapeutic outcome.

Associations Between Genetic Polymorphisms in the VEGFA, ACE, and SOD2 Genes and Susceptibility to Diabetic Nephropathy in the Han Chinese.

Objective: To investigate the association between polymorphisms in the rs2010963 and rs69947 loci of the vascular endothelial growth factor A (VEGFA) gene; the rs4646994 locus of the angiotensin I converting enzyme (ACE) gene; and the rs4880 locus of the superoxide dismutase 2 (SOD2) gene with genetic susceptibility to type 2 diabetic nephropathy (T2DN) in the Chinese Han population. Methods: A total of 650 Chinese Han patients with T2DN and 580 non-nephropathy patients with type 2 diabetes were enrolled in this study. Sanger sequencing was used to detect the genotypes of the rs2010963 and rs69947 loci within VEGFA, the rs4646994 locus of for the ACE gene, and the rs4880 locus of the SOD2 gene in all subjects. Enzyme-linked immunosorbent assays were used to detect VEGFA, ACE, and SOD2 levels in serum. Results: The risk of T2DN was significantly increased (odds ratio [OR] = 1.15, confidence interval [95% CI]: 1.03-1.30) in patients with the GC/CC genotypes compared to those with the GG genotype at the rs2010963 locus of the VEGFA gene under the dominant model of inheritance. Similarly, the risk of T2DN was significantly increased (OR = 1.17, 95% CI: 1.05-1.31) in patients with the CA/AA genotypes compared to those with the CC genotype at the rs69947 locus of the VEGFA gene under the dominant model of inheritance. In addition, the risk of T2DN was increased (OR = 1.57, 95% CI: 1.37-1.74) in patients with the AA genotype compared to the CC/CA genotypes under the recessive model of inheritance. The risk of T2DN was also significantly increased (OR = 1.54, 95% CI: 1.24-1.97) in patients with the ID/DD genotypes compared to those with the II genotype at the rs4646994 locus of the ACE gene under the dominant model; and (OR = 1.41, 95% CI: 1.26-1.57) for DD genotype compared to the II/ID genotypes under the recessive model. We also found the risk of T2DN was significantly increased (OR = 1.25, 95% CI: 1.11-1.39) under the dominant model in patients with the TC/CC genotypes compared to the TT genotype, and for the CC genotype (compared to the TT/TC genotype) (OR = 1.45, 95% CI: 1.18-1.66) under the recessive model at the rs4880 locus of the SOD2 gene rs4880. The haplotypes GAC (OR = 1.17, 95% CI: 1.04-1.29), CAT (OR = 1.12, 95% CI: 1.03-1.60), and CAC (OR = 1.13, 95% CI: 1.01-1.24) constructed from VEGFA gene rs2010963 and rs69947 loci, and the SOD2 gene rs4880 locus were associated with a higher risk for T2DN. Finally, we found that VEGFA protein levels from subjects with the rs2010963 GG genotype and the rs69947 CC genotype were higher in both case groups and the control group than in subjects with rs2010963 GA/AA genotypes and rs69947 CA/AA genotypes, respectively (p < 0.05). The ACE protein levels for variants at the rs4646994 locus showed that the case group and control group subjects with the DD genotype had the highest levels, followed by the ID genotype and the II genotype (p < 0.05). Conclusion: Genetic variation in the VEGFA gene at the rs2010963 and rs69947 loci, the ACE gene at the rs4646994 locus, and the SOD2 gene at the rs4880 locus may increase the risk of developing T2DN.

Prognostic significance of VHL, HIF1A, HIF2A, VEGFA and p53 expression in patients with clear‑cell renal cell carcinoma treated with sunitinib as first‑line treatment

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cell cancer, characterized by the highest mortality rate among other RCC subtypes due to the occurrence of metastasis and drug resistance following surgery. The Von Hippel-Lindau tumor suppressor (VHL)-hypoxia-inducible factor 1 subunit α (HIF1A)/hypoxia-inducible factor 2α (HIF2A)-vascular endothelial growth factor A (VEGFA) protein axis is involved in the development and progression of ccRCC, whereas sunitinib, a tyrosine kinase inhibitor, blocks the binding of VEGFA to its receptor. The aim of the present study was to examine the possible association of the gene expression of VHL, HIF1A, HIF2A, VEGFA and tumor protein P53 (P53) in cancer tissue with the outcome of ccRCC patients who were treated with sunitinib as first-line therapy following nephrec-tomy. A total of 36 ccRCC patients were enrolled, 11 of whom were administered sunitinib post-operatively. Tumor and control samples were collected, and mRNA and protein levels were assessed by reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. High mRNA and protein expression levels of HIF2A and VEGFA were found to be associated with shorter overall survival (OS) and progression-free survival (PFS) rates, as well as with unfavorable risk factors of cancer recurrence and mortality. Resistance to sunitinib was also observed; the OS and PFS rates were shorter (median OS and PFS: 12 and 6 months, respectively, vs. undetermined). Sunitinib resistance was associated with high HIF2A and VEGFA protein levels (b=0.57 and b=0.69 for OS and PFS, respectively; P<0.001). Taken together, the findings of this study suggest that the protein levels of HIF2A and VEGFA in tumor tissue may serve as independent prognostic factors in ccRCC. ccRCC patients with increased intratumoral HIF2A and VEGFA protein levels, and unaltered VHL protein levels, are not likely to benefit from sunitinib treatment following nephrectomy; however, this hypothesis requires verification by large-scale replication studies.

Intradermal delivery of modified mRNA encoding VEGF-A in patients with type 2 diabetes

Chemically modified mRNA is an efficient, biocompatible modality for therapeutic protein expression. We report a first-time-in-human study of this modality, aiming to evaluate safety and potential therapeutic effects. Men with type 2 diabetes mellitus (T2DM) received intradermal injections of modified mRNA encoding vascular endothelial growth factor A (VEGF-A) or buffered saline placebo (ethical obligations precluded use of a non-translatable mRNA control) at randomized sites on the forearm. The only causally treatment-related adverse events were mild injection-site reactions. Skin microdialysis revealed elevated VEGF-A protein levels at mRNA-treated sites versus placebo-treated sites from about 4–24 hours post-administration. Enhancements in basal skin blood flow at 4 hours and 7 days post-administration were detected using laser Doppler fluximetry and imaging. Intradermal VEGF-A mRNA was well tolerated and led to local functional VEGF-A protein expression and transient skin blood flow enhancement in men with T2DM. VEGF-A mRNA may have therapeutic potential for regenerative angiogenesis.