Fork head box M1 regulates vascular endothelial growth factor-A expression to promote the angiogenesis and tumor cell growth of gallbladder cancer

BACKGROUND Gallbladder cancer(GBC)is an aggressive type of biliary tract cancer that lacks effective therapeutic targets.Fork head box M1(FoxM1)is an emerging molecular target associated with tumor progression in GBC,and accumulating evidence suggests that vascular endothelial growth factor(VEGF)promotes various tumors by inducing neoangiogenesis.AIM To investigate the role of FoxM1 and the angiogenesis effects of VEGF-A in primary GBC.METHODS Using immunohistochemistry,we investigated FoxM1 and VEGF-A expression in GBC tissues,paracarcinoma tissues and cholecystitis tissues.Soft agar,cell invasion,migration and apoptosis assays were used to analyze the malignant phenotype influenced by FoxM1 in GBC.Kaplan-Meier survival analysis was performed to evaluate the impact of FoxM1 and VEGF-A expression in GBC patients.We investigated the relationship between FoxM1 and VEGF-A by regulating the level of FoxM1.Next,we performed MTT assays and Transwell invasion assays by knocking out or overexpressing VEGF-A to evaluate its function in GBC cells.The luciferase assay was used to reveal the relationship between FoxM1 and VEGF-A.BALB/c nude mice were used to establish the xenograft tumor model.RESULTS FoxM1 expression was higher in GBC tissues than in paracarcinoma tissues.Furthermore,the high expression of Foxm1 in GBC was significantly correlated with a malignant phenotype and worse overall survival.Meanwhile,high expression of FoxM1 influenced angiogenesis;high expression of FoxM1 combined with high expression of VEGF-A was related to poor prognosis.Attenuated FoxM1 significantly suppressed cell proliferation,transfer and invasion in vitro.Knockdown of FoxM1 in GBC cells reduced the expression of VEGF-A.Luciferase assay showed that FoxM1 was the transcription factor of VEGF-A,and knockdown VEGF-A in FoxM1 overexpressed cells could partly reverse the malignancy phenotype of GBC cells.In this study,we found that FoxM1 was involved in regulation of VEGF-A expression.CONCLUSION FoxM1 and VEGF-A overexpression were associated with the prognosis of GBC patients.FoxM1 regulated VEGF-A expression,which played an important role in the progression of GBC.

Vascular endothelial growth factor A, secreted in response to transforming growth factor-β1 under hypoxic conditions, induces autocrine effects on migration of prostate cancer cells

Hypoxia and transforming growth factor-β1 （TGF-β1） increase vascular endothelial growth factor A （VEGFA） expression in a number of malignancies. This effect of hypoxia and TGF-β1 might be responsible for tumor progression and metastasis of advanced prostate cancer. In the present study, TGF-β1 was shown to induce VEGFA165 secretion from both normal cell lines （HPV7 and RWPE1） and prostate cancer cell lines （DU 145 and PC3）. Conversely, hypoxia-stimulated VEGFA165 secretion was observed only in prostate cancer cell lines. Hypoxia induced TGF-β1 expression in PC3 prostate cancer cells, and the TGF-β1 type I receptor （ALK5） kinase inhibitor partially blocked hypoxia-mediated VEGFA16s secretion. This effect of hypoxia provides a novel mechanism to increase VEGFA expression in prostate cancer cells. Although autocrine signaling of VEGFA has been implicated in prostate cancer progression and metastasis, the associated mechanism is poorly characterized. VEGFA activity is mediated via VEGF receptor （VEGFR） 1 （Fit-l） and 2 （FIk-I/KDR）. Whereas VEGFR-1 mRNA was detected in normal prostate epithelial cells, VEGFR-2 mRNA and VEGFR protein were expressed only in PC3 cells. VEGFA165 treatment induced phosphorylation of extracellular signal-regulated kinase 1/2 （ERKI/2） in PC3 cells but not in HPV7 cells, suggesting that the autocrine function of VEGFA may be uniquely associated with prostate cancer. Activation of VEGFR-2 by VEGFA165 was shown to enhance migration of PC3 cells. A similar effect was also observed with endogenous VEGFA induced by TGF-β1 and hypoxia. These findings illustrate that an autocrine loop of VEGFA via VEGFR-2 is critical for the tumorigenic effects of TGF-β1 and hypoxia on metastatic prostate cancers.

Expression of vascular endothelial growth factors A and C in human pancreatic cancer

AIM： To study the expression of vascular endothelial growth factor A （VEGF-A） and VEGF-C and to determine whether the presence of VEGF-A and VEGF-C was associated with the clinicopathologic characteristics of pancreatic cancer. METHODS： VEGF-A and VEGF-C mRNA transcripts were examined by Northern blot in 6 human pancreatic cancer cell lines and 8 normal pancreatic tissues and 8 pancreatic carcinoma specimens. The expression of VEGF-A and VEGF-C proteins was examined by Western blot in the tested cell lines and by immunohistochemical stain in 50 pancreatic carcinoma samples. RESULTS： VEGF-A and VEGF-C mRNA transcripts were present in all the 6 human pancreatic cancer cell lines. Immunoblotting revealed the presence of VEGF-A and VEGF-C proteins in all the cell lines. Northern blot analysis of total RNA revealed 3.0-fold and 3.6-fold increase in VEGF-A and VEGF-C mRNA transcript in the cancer samples, respectively. Immunohistochemical analysis confirmed the expression of VEGF-A and VEGF-C in cancer cells within the tumor mass. Immunohistochemical analysis of 50 pancreatic cancer tissue samples revealed the presence of VEGF-A and VEGF-C immunoreactivity in 50% and 80% of the cancer tissue samples, respectively. The presence of VEGF-A in these cells was associated with larger tumor size and enhanced local spread （x^2= 6.690, P= 0.035〈0.05） but was not associated with decreased patient survival. However, the presence of VEGF-C in the cancer cells was associated with increased lymph node metastasis （x^2= 5.710, P= 0.017〈0.05）,but was not associated with decreased patient survival. There was no correlation between the expression of VEGF-A and VEGF-C in the same cancer cells. CONCLUSION： VEGF-A and VEGF-C are commonly overexpressed in human pancreatic cancer and may contribute to tumor growth and lymph node metastasis, There is no relationship between the expression of VEGF-A and VEGF-C in pancreatic cancer.

Elevated Vascular Endothelia Growth Factor-A in the Serum and Peritoneal Fluid of Patients with Endometriosis

There has been emergence of evidence suggesting that specific variants of the vascular endothelia growth factor （VEGF） family, based on their ability to regulate angiogenesis, would be pivotal in the pathogenesis of endometriosis. This study was aimed at determining whether high levels of VEGF-A could be found in the serum and peritoneal fluid （PF） of patients with endometriosis. VEGF-A levels were measured by enzyme-linked immunosorbent assay （ELISA） in serum and PF from 46 patients with surgically confirmed endometriosis, and 40 controls with no clinical evidence of the disease or detectable endometriotic lesions at the time of surgical examination. The results showed the mean VEGF-A levels were significantly higher in the serum and PF of patients with endometriosis than in the controls. The VEGF-A levels in the serum and PF of patients with severe endometriosis （stages Ⅲ-Ⅳ） were significantly higher than in those with minimal endometriosis （P〈0.001）. It was concluded that endometriosis was associated with significant modulation in the levels of circulating VEGF-A.

Vascular endothelial growth factor 165b expression in stromal cells and colorectal cancer

AIM:To characterize the implications of vascular endothelial growth factor(VEGF)-A in stromal cells and colorectal cancer and the expression of VEGF-A splice variants.METHODS:VEGF-A expression in tumor and stromal cells from 165 consecutive patients with colorectal cancer was examined by immunohistochemistry.The association between VEGF-A expression status and clinicopathological factors was investigated.Twenty freshfrozen samples were obtained for laser capture microdissection to analyze the splice variants of VEGF-A.RESULTS:VEGF-A was expressed in 53.9% and 42.4% of tumor and stromal cells,respectively.VEGF-A expression in tumor cells(t-VEGF-A) was associated with advanced clinical stage(stage 0,1/9;stage 1,2/16;stage 2,32/55;stage 3,38/66;stage 4,16/19,P < 0.0001).VEGF-A expression in stromal cells(s-VEGF-A) increased in the earlier clinical stage(stage 0,7/9;stage 1,6/16;stage 2,33/55;stage 3,22/66;stage 4,5/19;P = 0.004).Multivariate analyses for risk factors of recurrence showed that only s-VEGF-A expression was an independent risk factor for recurrence(relative risk 0.309,95% confidence interval 0.141-0.676,P = 0.0033).The five-year disease-free survival(DFS) rates of t-VEGF-A-positive and-negative cases were 51.4% and 62.9%,respectively.There was no significant difference in t-VEGF-A expression status.The five-year DFS rates of s-VEGF-A-positive and-negative cases were 73.8% and 39.9%,respectively.s-VEGFA-positive cases had significantly better survival than s-VEGF-A-negative cases(P = 0.0005).Splice variant analysis revealed that t-VEGF-A was mainly composed of VEGF165 and that s-VEGF-A included both VEGF165 and VEGF165b.In cases with no venous invasion(v0),the level of VEGF165b mRNA was significantly higher(v0 204.5 ± 122.7,v1 32.5 ± 36.7,v2 2.1 ± 1.7,P = 0.03).The microvessel density tended to be lower in cases with higher VEGF165b mRNA levels.CONCLUSION:s-VEGF-A appears be a good prognostic factor for colorectal cancer and includes VEGF165 and VEGF165b.

Prognostic significance of vascular endothelial growth factor polymorphisms in colorectal cancer patients

AIM To investigate the associations of the genetic polymor-phisms of vascular endothelial growth factor A(VEGF-A)-1498C>T and-634G>C, with the survival of patients with colorectal cancer(CRC). METHODS A prospective cohort consisting of 131 Brazilians patients consecutively operated on with a curative intention as a result of sporadic colorectal carcinoma was studied. DNA was extracted from peripheral blood and its amplification and allelic discrimination for each genetic polymorphism was performed using the technique of polymerase chain reaction(PCR) in real-time. The real-time PCR technique was used to identify the VEGF-A-1498C>T(rs833031) and-634G>C(rs2010963) polymorphisms. Genotyping was validated for VEGF-A-1498C>T polymorphism in 129 patients and for VEGF-A-634G>C polymorphism in 118 patients. The analysis of association between categorical variables was performed using logistic regression, survival by Kaplan-Meier method and multivariate analysis by the Cox regression method. RESULTS In the univariate analysis there was a significant association(OR = 0.32; P = 0.048) between genotype CC of the VEGF-A-1498C>T polymorphism and the presence of CRC liver metastasis. There was no association between VEGF-A-1498C>T polymorphism and VEGF-A-634G>C polymorphism with further clinical or anatomopathologic variables. The genotype CC of the VEGF-A-1498C>T polymorphism was significantly correlated with the 5-year survival(P = 0.032), but not significant difference(P = 0.27) was obtained with the VEGF-A-634G>C polymorphism with the 5-year survival in the univariate analysis. The genotype CT(HR = 2.79) and CC(HR = 4.67) of the polymorphism VEGF-A-1498C>T and the genotype CC(HR = 3.76) of the polymorphism VEGF-A-634C>G acted as an independent prognostic factor for the risk of death in CRC patients. CONCLUSION The CT and CC genotypes of the VEGF-A-1498C>T and the CC genotype of the VEGF-A-634C>G polymorphisms are prognostic factors of survival in Brazilians patients with sporadic colorectal carcinoma.

腰椎间盘突出伴坐骨神经痛患者血清HIF-1α、VEGFA水平与痛阈值关系及交互作用

目的探讨腰椎间盘突出伴坐骨神经痛(LDHS)患者血清缺氧诱导因子-1α(HIF-1α)、血管内皮生长因子A(VEGFA)水平与痛阈值的关系及交互作用。方法选取2020年5月至2023年2月在本院就诊的126例LDHS患者作为病例组,另选取同期126例体检健康者作为对照组。比较两组血清HIF-1α、VEGFA水平、痛阈值,Pearson相关系数分析血清HIF-1α、VEGFA水平与痛阈值的关系,多元线性回归分析血清HIF-1α、VEGFA水平与LDHS患者痛阈值的关系及交互作用。结果病例组血清HIF-1α、VEGFA水平高于对照组,痛阈值低于对照组(P<0.05)。病例组血清HIF-1α(r=0.777,P<0.001)、VEGFA(r=0.822,P<0.001)水平与痛阈值呈中度正相关,对照组血清HIF-1α(r=0.383,P<0.001)、VEGFA(r=0.453,P<0.001)水平与痛阈值无明显相关性。不考虑交互作用,血清HIF-1α、VEGFA水平与痛阈值存在线性相关(P<0.05),拟合方程为Y=-0.409+0.004×HIF-1α+0.019×VEGFA,所有预测变量可解释因变量的74.3%的方差。考虑交互作用,痛阈值的拟合方程为Y=19.276+0.062×HIF-1α+0.047×VEGFA-0×HIF-1α×VEGFA,所有预测变量可解释因变量的74.7%的方差,VEGFA低表达时,HIF-1α表达与痛阈值的关系较强,当VEGFA高表达时,HIF-1α与痛阈值的关系减弱,直线趋于平缓。结论血清HIF-1α、VEGFA水平与LDHS患者痛阈值关系密切,在对痛阈值影响中具有交互作用。

血清TLR4和VEGFA表达对糖尿病视网膜病变的临床诊断及预后价值评估

目的:探讨Toll样受体4(TLR4)、血管内皮生长因子A(VEGFA)在糖尿病视网膜病变(DR)患者血清中的表达情况及临床意义。方法:选取2021-01/2022-01本院收治的183例2型糖尿病(T2DM)患者为研究对象,分为非糖尿病视网膜病变(NDR)组(54例),增殖性糖尿病视网膜病变(PDR)组(68例)和非增殖性糖尿病视网膜病变(NPDR)组(61例)。同期按照年龄、性别分层随机选择70例于本院进行健康体检志愿者作为对照组。出院后随访1a,根据DR患者是否发生视力残疾,分为预后不良组(40例)与预后良好组(89例)。采用酶联免疫吸附法(ELISA)检测血清中TLR4、VEGFA水平;通过Logistic回归分析DR发生的影响因素;利用受试者工作特征曲线(ROC)分析血清TLR4、VEGFA水平诊断DR及预测预后的临床价值。结果:对照组、NDR组、PDR组和NPDR组间TLR4、VEGFA水平比较均具有差异(F=935.753、516.936,均P<0.05),各组两两比较均具有差异(P<0.05)。预后不良组患者血清中TLR4、VEGFA表达水平均高于预后良好组(P<0.01)。Logistic回归分析结果显示,TLR4、VEGFA、病程、HbA1c均是DR发生的危险因素(P<0.05);ROC结果显示,血清TLR4、VEGFA水平及二者联合预测DR的AUC分别为0.869、0.862、0.931,血清TLR4、VEGFA水平及二者联合预测DR患者视力残疾的AUC分别为0.864、0.863、0.938。结论:DR患者血清中TLR4、VEGFA表达均上调,二者联合检测可作为评估DR发生及预后不良的潜在指标。

Decreased small arterial compliance with increased serum vascular endothelial growth factor-A and circulating endothelial progenitor cell in dilated cardiomyopathy

Background Evidence showed that both myocardium and blood vessels were damaged in dilated cardiomyopathy （DCM）. However, the changes in arterial compliance, serum cytokines and circulating endothelial progenitor cells （EPC）, and their correlations remain unknown. Methods Sixty-five DCM patients and 49 healthy volunteers were studied. Both large artery compliance （C1） and small artery compliance （C2） were measured with the CVProfUor DO-2020. Quantitative enzyme-linked immunosorbent assays （ELISAs） were used to measure the levels of vascular endothelial growth factor-A （VEGF-A） and VEGF receptor 2 （VEGF-R2）. Circulating EPC was assessed by EPC colony-forming assays and flow cytometry （CD133^＋/CD34^＋cells）. Phagocytized Dil-acLDL and binded FITC-UEA-I were used to analyze endothelial lineage marker expression by immunofluorescence. Results Although C2 was markedly lower in DCM patients than in control group （（3.8±1.8） ml/mmHg × 100 vs （5.0±2.2） ml/mmHg × 100, P〈0.0001）, there was no statistically significant difference in C1 between the two groups （P〉0.05）. Levels of VEGF-A, the numbers of colony-forming units （CFU） and the fractions of EPC were obviously higher in DCM patients than in control group （（127.6±139.5） pg/ml vs （58.8±42.9） pg/ml, P〈0.0001; （2.5±1.5）% vs （0.5±0.3）%, P〈0.05; 23.5±12.8 vs 10.8±7.4, P〈0.01, respectively） and however, there was no significant difference in VEGF-R2 between two groups （P〉0.05）. LgVEGF-A was positively correlated with the number of EPC-CFU （r=-0.435; P〈0.05） and inversely correlated with C2 （r=-0.543; P〈0.001） in DCM patients. Conclusions The reduction of C2, a sensitive marker reflecting endothelial dysfunction, was observed in DCM patients and closely related to the increase in serum VEGF-A.

lncRNA SBF2-AS1通过调控miR-140-5p/VEGFA分子轴促进宫颈癌HeLa细胞上皮间质转化

目的:探讨1ncRNASBF2-AS1通过调控miR-140-5p/.血管内皮生长因子A(VEGFA)分子轴对宫颈癌HeLa细胞上皮间质转化(EMT)的影响。方法:细胞培养和转染后分为NC、miR-140-5p mimic、miR-140-5p mimic+pcDNA-VEGFA、si-lncRNA SBF2-AS1+pcDNA-VEGFA及si-lncRNA SBF2-AS 1+miR-140-5p mimic组5组。采用qPCR检测1ncRNA SBF2-AS1在宫颈癌组织及细胞系中的表达水平,双荧光素酶报告基因验让1ncRNASBF2-AS1、miR-140-5p与VEGFA的靶向关系,WB检测HeLa细胞中VEGFA及EMT标志物N-cadherin、Vimentin和E-cadherin的表达水平,Transwell实验检测HeLa细胞侵袭和迁移能力。结果:1ncRNA SBF2-AS 1在宫颈癌组织及细胞系中高表达(P<0.05或P<0.01),1ncRNA SBF2-AS 1靶向结合miR-140-5p,且VEGFA是miR-140-5p的靶基因(P<0.05)。敲降lncRNA SBF2-AS1抑制HeLa细胞侵袭、迁移及EMT。进一步实验证实,1ncRNA SBF2-AS1通过miR-140-5p上调VEGFA的表达水平,从而促进HeLa细胞侵袭、迁移及EMT(P<0.05或P<0.01)。结论:1ncRNA SBF2-AS1通过miR-140-5p/VEGFA分子轴促进HeLa细胞EMT。

Impact of umbelliprenin-containing niosome nanoparticles on VEGF-A and CTGF genes expression in retinal pigment epithelium cells

AIM:To investigate the impact of niosome nanoparticles carrying umbelliprenin(UMB),an anti-angiogenic and anti-inflammatory plant compound,on the expression of vascular endothelial growth factor(VEGF-A)and connective tissue growth factor(CTGF)genes in a human retinal pigment epithelium(RPE)-like retina-derived cell line.METHODS:UMB-containing niosomes were created,optimized,and characterized.RPE-like cells were treated with free UMB and UMB-containing niosomes.The IC_(50)values of the treatments were determined using an MTT assay.Gene expression of VEGF-A and CTGF was evaluated using real-time polymerase chain reaction after RNA extraction and cDNA synthesis.Niosomes’characteristics,including drug entrapment efficiency,size,dispersion index,and zeta potential were assessed.Free UMB had an IC_(50)of 96.2μg/mL,while UMB-containing niosomes had an IC_(50)of 25μg/mL.RESULTS:Treatment with UMB-containing niosomes and free UMB resulted in a significant reduction in VEGF-A expression compared to control cells(P=0.001).Additionally,UMB-containing niosomes demonstrated a significant reduction in CTGF expression compared to control cells(P=0.05).However,there was no significant reduction in the expression of both genes in cells treated with free UMB.CONCLUSION:Both free UMB and niosome-encapsulated UMB inhibits VEGF-A and CTGF genes expression.However,the latter demonstrates significantly greater efficacy,potentially due to the lower UMB dosage and gradual delivery.These findings have implications for anti-angiogenesis therapeutic approaches targeting age-related macular degeneration.

肝泡状棘球蚴组织中HIF-1α、VEGFA的表达及对血管生成的作用

目的探讨HIF-1α、VEGFA在沙鼠肝泡状棘球蚴组织的表达及血管生成过程中的作用。方法126只沙鼠随机分成空白组(6只)、假手术组(60只)、模型组(60只),采用开腹直视肝脏穿刺法建立泡状棘球蚴动物模型,假手术组接种同体积的PBS,术后第3d、7d、14d、28d、42d、56d、70d、84d、98d、112d随机取6只沙鼠,取临近病变的边缘区组织及正常肝组织。采用HE观察病理改变;qRT-PCR、原位杂交、免疫组化检测HIF-1α、VEGFA表达,CD34标记微血管进行MVD计数。结果HE染色根据病理特点将病程分为早期(14d内)、中期(14d^56d)、晚期(56d后)。模型组泡状棘球蚴组织随感染时间不同,其HIF-1αmRNA随感染时间呈动态改变,术后第14d其表达量高于空白组、低于假手术组(F=82.732,P<0.001);术后第42d、112d其相对表达量均高于空白组与假手术组(χ^2=11.536,χ^2=15.189,P<0.01);模型组术后第14dVEGFAmRNA相对表达量低于空白组及假手术组(χ^2=15.174,P<0.01);术后第42d、112d,VEGFAmRNA表达量均高于空白组与假手术组(χ^2=15.158,χ^2=15.158,P<0.01)。模型组术后第14d、42d、112dHIF-1α表达均高于空白组、假手术组(χ^2=8.627,χ^2=9.000,F=15.690,P<0.01);模型组术后第14d、42dVEGFA表达高于空白组、假手术组(F=11.250,F=70.059,P<0.001);模型组术后第14d、42d、112d,其MVD-CD34均高于空白组、假手术组(χ^2=12.517,P<0.01,χ^2=13.157,P<0.01;χ^2=13.220,P<0.01)。结论沙鼠感染肝泡状棘球蚴中期,病变边缘区HIF-1α、VEGFA表达均升高,同时伴有大量微血管生成,可能存在HIF-1α转录因子激活并上调VEGFA的表达,促进肝泡状棘球蚴组织边缘区的血管新生。

乌头汤通过下调HIF-1α/VEGFA/Ang信号通路抑制AIA风寒湿痹证大鼠血管翳形成

目的:探讨乌头汤对佐剂型关节炎(AIA)风寒湿痹证大鼠血管翳形成的抑制作用及其潜在作用机制。方法:将无特定病原体(SPF)雄性SD大鼠40只,按随机数字表法分为空白组、AIA风寒湿痹证组[完全弗氏佐剂(CFA),200μg]、乌头汤组(15 g·kg^(-1)·d^(-1))、吲哚美辛组(10 mg·kg^(-1)),除空白组外,其余各组注射CFA前给予风寒湿刺激。连续给药30 d,期间观察AIA风寒湿痹证大鼠的基本情况、发病时间、关节炎评分、足容积。最终取外周动脉血、踝关节和滑膜组织。酶联免疫吸附测定法(ELISA)检测血清缺氧诱导因子-1α(HIF-1α)、血管内皮生长因子A(VEGFA)蛋白含量和风湿3项,包括抗O(ASO)、C反应蛋白(CRP)和类风湿因子(RF);苏木素-伊红(HE)染色观察关节组织形态学改变;免疫组化检测踝关节通路关键蛋白HIF-1α和VEGFA;实时荧光定量聚合酶链式反应(Real-time PCR)检测大鼠滑膜组织中HIF-1α、VEGFA、血管生成素-1(Ang-1)、血管生成素-2(Ang-2)mRNA表达。结果:与空白组比较,AIA风寒湿痹证大鼠足肿容积和关节炎评分显著升高(P<0.01);血清CRP、RF、ASO阳性表达显著(P<0.01);HE染色可见踝关节滑膜炎性增生,血管新生、血管翳形成,骨破坏程度加重,提示模型构建成功。乌头汤干预后,发病时间显著延迟(P<0.01);足容积和关节炎评分显著降低(P<0.01);血清CRP、RF、ASO含量显著下降(P<0.01);滑膜组织炎性增生明显缓解,血管新生及血管翳减少,骨破坏减轻;滑膜HIF-1α、VEGFA、Ang-1、Ang-2mRNA明显降低(P<0.05,P<0.01);血清与踝关节HIF-1α和VEGFA蛋白表达显著下降(P<0.01)。在吲哚美辛组中,大鼠发病时间显著延迟(P<0.01);足容积和关节炎评分显著降低(P<0.01);血清CRP、RF、和ASO含量显著下降(P<0.01);HIF-1α/VEGFA/Ang信号通路激活,病理组织损伤改善。结论:乌头汤可延缓AIA风寒湿痹证大鼠发病时间,降低足容积、关节炎评分、风湿活动度,改善关节组织病理情况,对血管翳形成具有抑制作用,其作用机制可能与下调HIF-1α/VEGFA/Ang通路表达有关。

循环miRNA-93在DHEA多囊卵巢综合征大鼠模型中的表达及其意义

目的:通过检测多囊卵巢综合征(PCOS)大鼠血清与卵巢组织中miRNA-93及血管内皮生长因子A(VEGFA)的表达水平,探讨循环miRNA-93及靶基因VEGFA在PCOS发生发展过程中的作用机理与诊断价值。方法:选取3~4周龄雌性(SD)大鼠,随机分为实验组和对照组,每组20只。实验组每日颈背部皮下注射脱氢表雄酮(DHEA),按6mg/100g剂量溶于0.2ml无菌注射用油;对照组每日颈背部皮下注射0.2ml无菌注射用油剂。28天后观察两组大鼠的动情周期、卵巢形态学、性激素、体重、卵巢体积、OGTT血糖、空腹胰岛素、胰岛素抵抗指数(HOMA-IR)及血脂等代谢相关指标的变化。RT-PCR法检测大鼠血清和卵巢组织中miRNA-93表达,ELISA法检测血清中VEGFA表达,IHC、Western blot法分析卵巢组织中VEGFA蛋白表达量。结果:实验组大鼠失去规律的动情周期,卵巢体积增大,呈多囊样变,血清FSH、T、LH升高,糖耐量异常,空腹胰岛素及胰岛素抵抗指数明显高于对照组,血清胆固醇、甘油三酯高于对照组,PCOS大鼠模型构建成功。RT-PCR、ELISA、Western blot、IHC等结果显示,miRNA-93及靶基因VEGFA在PCOS大鼠模型的血清、卵巢组织中表达一致且显著升高(P<0.05)。结论:miRNA-93在PCOS大鼠模型血清和卵巢组织中呈高表达,可能通过调控其靶基因VEGFA在PCOS发生发展中起重要作用,在PCOS诊断中循环miRNA-93可能作为一种新型非侵入性的诊断标记物。

miR-126修饰的间质干细胞来源的外泌体对大鼠早期缺血性股骨头坏死的影响

目的观察微小RNA-126(miR-126)修饰的间质干细胞来源的外泌体(MSC-126-Exos)对类固醇激素诱导的早期缺血性股骨头坏死(SANFH)的治疗作用,并探讨其潜在机制。方法分别以80 mg/L MSC-126-Exos和MSCExos处理人脐静脉内皮细胞(HUVECs)并设PBS处理组为对照,MTT、划痕实验以及成管实验分别观察各组细胞增殖、迁移和毛细血管网的形成,Western blot检测HUVECs中血管内皮生长因子A(VEGFA)的表达。构建大鼠SANFH模型,分别注射等量MSC-126-Exos、MSC-Exos和PBS,6周后,通过对股骨头区域组织进行CD31免疫荧光染色及其Micro-CT扫描,观察股骨头血管数量及骨质情况。结果体外实验中,MSC-126-Exos组的HUVECs的增殖、迁移和成管较MSC-Exos组显著增强,且VEGFA的表达水平也上调,差异有统计学意义(P<0.05)。在体实验中,MSC-126-Exos组大鼠股骨头坏死区CD31免疫荧光标记的血管数量较MSC-Exos组和模型组显著增多(P<0.05),且股骨头坏死区Micro-CT扫描显示MSC-126-Exos组的小梁厚度(Tb.Th)、小梁数(Tb.N)和每组织体积骨量(BV/TV)值也显著高于MSC-Exos组和模型组,而小梁分离度(Tb.Sp)显著降低(P<0.05)。结论 MSC-126-Exos可以促进内皮细胞功能,改善股骨头区的血运从而增强骨质沉积,对大鼠SANFH具有显著的治疗效果。

Relationship between Angiogenic and Inflammatory Biomarkers and Diabetic Retinopathy

Background: Diabetic retinopathy (DR) is a common complication of diabetes mellitus and a major cause of vision loss in the working age population. Its pathogenesis is poorly understood but may involve low grade chronic inflammation and angiogenesis. The aim of this study was to evaluate the relationship between serum levels of one inflammatory (IL-6) and angiogenic cytokine (VEGF-A) with the presence and severity of DR in type 2 diabetic mellitus patients. Methods: From January to June 2019, we conducted a cross-sectional analytical study on 84 patients out of which 31 developed DR and 53 did not. All patients underwent complete ophthalmological examination and laboratory analysis for IL-6 and VEGF-A with ELISA Technique. We studied the relation of IL-6 and VEGF-A with the presence and severity of DR, HBA1c, the duration of diabetes. Results: The group with DR had statistically significant higher levels of VEGF-A compared to the control group (390.5 pg/ml vs. 173.1 pg/ml;p = 0.007). There was no significant difference in IL-6 levels between both groups (42.8 pg/ml vs. 31.7 pg/ml;p = 0.10). Equally there was no association between these 2 cytokines and macular oedema or with the severity of DR. The level of IL-6 was associated to the balance of diabetes (p = 0.006) although VEGF-A was not (p = 0.15). Moreover, Il-6 (p = 0.31) and VEGF-A (p = 0.24) were not linked to the duration of diabetes. Conclusion: Serum concentrations of VEGF-A have an effect on the development of DR. They correlate with the presence of the disease but IL-6 does not. However, IL-6 was associated to the balance of diabetes. These 2 biomarkers may play a role in the pathophysiology of diabetes and the diabetic retinopathy. Findings on Il-6 and VEGF-A may therefore contribute to the development of the diagnosis tools and caretaking of diabetes and diabetic retinopathy.

基于HIF-1α/VEGFA信号通路探讨柴胡桂枝汤对三阴性乳腺癌细胞的影响

目的:基于缺氧诱导因子-1α(HIF-1α)/血管内皮生长因子A(VEGFA)信号通路探讨柴胡桂枝汤对三阴性乳腺癌(TNBC)细胞的影响。方法:建立TNBC移植瘤模型,随机分为模型组,卡培他滨组(0.2 mg·kg^(-1)),柴胡桂枝汤低、中、高剂量组(10.62、21.23、42.46 g·kg^(-1)),每组10只,干预21 d。给药结束后,酶联免疫吸附测定法(ELISA)检测血清中肿瘤坏死因子-α(TNF-α)水平;实时荧光定量聚合酶链式反应(Real-time PCR)检测肿瘤组织中HIF-1αmRNA表达情况;免疫组化法(IHC)检测肿瘤组织中HIF-1α、TNF-α、VEGFA的表达水平及CD34染色检测肿瘤内血管生成情况并计算微血管密度(MVD);蛋白免疫印迹法(Western blot)检测肿瘤组织中HIF-1α、VEGFA、表皮生长因子受体(EGFR)蛋白的表达。结果:与模型组比较,卡培他滨组、柴胡桂枝汤低、中、高剂量组TNF-α水平显著降低(P<0.01),HIF-1αmRNA显著降低(P<0.01),HIF-1α、TNF-α、VEGFA和CD34阳性细胞表达及MVD明显降低(P<0.05,P<0.01),HIF-1α、VEGFA、EGFR蛋白水平显著降低(P<0.01)。与卡培他滨组比较,柴胡桂枝汤中、高剂量组TNF-α水平显著降低(P<0.01),HIF-1αmRNA显著降低(P<0.01),HIF-1α、TNF-α和VEGFA阳性细胞表达显著降低(P<0.01),CD34表达及MVD显著降低(P<0.01),HIF-1α、VEGFA、EGFR蛋白水平均显著降低(P<0.01)。结论:柴胡桂枝汤可能通过调控HIF-1α/VEGFA信号通路,抑制TNBC细胞血管生成,从而发挥抗肿瘤作用。

Elevated VEGF-A & PLGF concentration in aqueous humor of patients with uveal melanoma following Iodine-125 plaque radiotherapy

AIM: To measure the concentration of vascular endothelial growth factor-A(VEGF-A), and placental growth factor(PLGF) in aqueous humor of uveal melanoma patients before and after Iodine-125 plaque therapy(IPT), determine the postoperative fluctuation and evaluate associated factors in vivo.METHODS: Participants were 18 Chinese patients with uveal melanoma who were elected to IPT. Undiluted aqueous humor samples were collected at Iodine plaque implant and removal time, then stored immediately at-80℃ until assayed. The concentration of VEGF-A, PLGF and other 7 cytokines comprising interleukin-2(IL-2), IL-8, IL-10, interferon(IFN)-γ, programmed death(PD)-1, transforming growth factor(TGF)-β1 and insulin-like growth factor(IGF)-1 in aqueous humor was measured using Raybiotech immunoassay kit, a high throughput strategy. The VEGF-A and PLGF levels were compared across preoperation and postoperation subgroups, as well as those of other 7 interleukins. Correlation and grouped analyses were conducted to determine the independent effects of clinical parameters and other cytokines on VEGF-A and PLGF concentration or fluctuation. This study set a self-control design.RESULTS: VEGF-A(P=0.038) and PLGF(P=0.026) were the only two increased cytokines after IPT. Preoperative and postoperative level of VEGF-A and PLGF(r=0.575, P=0.013;r=0.987, P<0.001) correlated with each other significantly. Level of VEGF-A(r=0.626, P=0.005;r=0.588, P=0.01) and PLGF(r=0.616, P=0.007;r=0.588, P=0.01) had positive correlation with tumor thickness consistently. Elevated VEGF-A or PLGF level were strong predictive factors of each other(P=0.007, OR=60.0). The elevated VEGF-A group showed a higher postoperative level of IFN-γ(P=0.005), IL-2(P<0.001) and IL-10(P=0.004) in aqueous humor. When the elevated PLGF group got similar results that a higher postoperative level of IFN-γ(P=0.007), IL-2(P<0.001) and IL-10(P=0.013) in aqueous humor. CONCLUSION: This study reveals that VEGF-A and PLGF in aqueous humor significantly increased with tumor thickness and radiation process in uveal melanoma patients. VEGF-A and PLGF may be crucial in uveal melanoma genesis and radiotherapy reactions. Immune mediators comprised IFN-γ, IL-2 and IL-10 could play roles in the link between inflammation and angiogenesis in uveal melanoma when exposed to radiotherapy.

血管内皮生长因子A调控人脐静脉内皮细胞miRNA的全基因表达谱分析

目的·分析人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)中微小RNA(microRNA,miRNA)在血管内皮生长因子A(vascular endothelial growth factor A,VEGFA)刺激下的变化,探索miRNA在调节VEGFA下游基因表达变化中的作用,并寻找新的血管新生调控miRNA。方法·通过NanoString nCounter分别检测了HUVEC在VEGFA刺激后0、1、4、12 h时miRNA的表达量,将具有相同变化趋势的差异表达miRNA聚类并预测其靶基因。通过基因本体数据库富集分析和京都基因与基因组百科全书通路分析,预测差异表达miRNA靶基因的相关功能,并建立互作网络。结果·VEGFA刺激HUVEC后,有39个miRNA的表达量发生了显著差异性变化。生物信息学分析结果显示差异表达miRNA所对应的靶mRNA有129个,动态表达的miRNA与其靶基因间的调控参与了血管新生中多个生物过程和信号通路。结论·VEGFA刺激诱导HUVEC差异性表达miRNA,miRNA在调节血管新生中具有重要作用;其中,miR-107和miR-21可能是血管新生的调控因子。

莪术油对卵巢癌VEGFA,STAT3,mTOR的调控机制

目的:观察莪术油对卵巢癌血管内皮生长因子A(VEGFA)/信号转导及转录激活因子3(STAT3)/雷帕霉素靶蛋白(mTOR)的影响,探讨莪术油及其活性成分作用于卵巢癌VEGFA,STAT3,mTOR的效应机制。方法:利用网络药理学技术分析莪术作用与卵巢癌的作用机制,生物信息学分析VEGFA,STAT3,mTOR在卵巢癌中的表达情况及对卵巢癌预后的影响,以此探究莪术油干预卵巢癌VEGFA,STAT3,mTOR的可行性。建立裸鼠移植瘤模型,利用苏木素-伊红(HE),实时荧光定量聚合酶链式反应(Real-time PCR),蛋白免疫印迹法(Western blot)及免疫组化检测莪术油及其活性成分对卵巢癌VEGFA,STAT3,mTOR的影响。结果:生物信息学分析和文献研究显示,VEGFA,STAT3,mTOR在卵巢癌的发生发展中起到了特殊的调控作用,并可能是卵巢癌增殖的关键靶点。网络药理学分析显示,莪术能调控卵巢癌的多个疾病靶点,并能通过这多个靶点调控卵巢癌中的VEGFA,STAT3,mTOR。各组卵巢癌瘤体组织HE染色显示,模型组瘤体组织肿瘤细胞分布致密,内部无囊泡。与模型组比较,其他给药组细胞密度缩小,瘤体组织内部也出现了一些较小的空囊泡;其中以莪术油组为最。与模型组比较,莪术油及其活性成分能够抑制卵巢癌瘤体组织中VEGFA,STAT3,mTOR mRNA和蛋白的表达(P<0.05)。结论:莪术油及其活性成分能降低卵巢癌模型裸鼠瘤体组织中VEGFA,STAT3,mTOR的表达,可能是通过VEGFA,STAT3,mTOR来达到抑制卵巢癌增殖的。