Tetramethylpyrazine inhibits hypoxia-induced pulmonary vascular leakage in rats via the ROS-HIF-VEGF pathway

Tetramethylpyrazine (TMP) is a reactive oxygen species (ROS) antagonist that has potent properties for the treatment of a variety of vascular diseases, such as ischemic stroke and pulmonary hypertension secondary to chronic obstructive pulmonary diseases. However, there are few data about the role of TMP in hypoxia-induced pulmonary vascular leakage. This study examined the effect of TMP on hypoxia-induced pulmonary vascular leakage and the underlying mechanisms. Rat pulmonary microvascular endothelial cells (RPMVECs) treated with TMP or not were subjected to hypoxic or normoxic conditions for 24 h, and the monolayer permeability, intracellular ROS, hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) proteins levels were determined. Additionally, rats administrated TMP were exposed to hypobaric hypoxia to evaluate the effect of TMP in vivo by measuring lung water content, pulmonary vascular leakage into the lungs and immunohistochemistry for HIF-1α and VEGF. Hypoxia was found to cause a significant increase in RPMVEC monolayer permeability and intracellular ROS, HIF-1α and VEGF protein levels. Treatment with TMP decreased the hypoxia-induced RPMVEC monolayer permeability and attenuated the elevation of ROS, HIF-1α and VEGF protein levels. TMP-treated animals showed less pulmonary vascular leakage and HIF-1α and VEGF expression compared with those exposed to hypoxia alone. These observations supported that TMP inhibited the increase in pulmonary vascular permeability induced by hypoxia. The underlying mechanisms may be related to the scavenging of intracellular ROS and the suppression of hypoxia-induced upregulation of HIF-1α and VEGF proteins.     

Rhapontigenin inhibited hypoxia inducible factor 1 alpha accumulation and angiogenesis in hypoxic PC-3 prostate cancer cells

Hypoxia inducible factor 1 alpha (HIF-1α) is frequently over-expressed in the numerous types of cancer and plays an important role in angiogenesis. In the present study, the inhibitory mechanism of rhapontigenin isolated from Vitis coignetiae was investigated on HIF-1α stability and angiogenesis in human prostate cancer PC-3 cells. Rhapontigenin significantly suppressed HIF-1α accumulation at protein level but not at mRNA level in PC-3 cells under hypoxia. Also, rhapontigenin suppressed hypoxia-induced HIF-1α activation in various cancer cells, such as colorectal adenocarcinoma (SW620), breast adenocarcinoma (MCF-7), fibrosarcoma (HT-1080) and prostate carcinoma (LNCaP). Interestingly, rhapontigenin had more potency in inhibition of hypoxia-induced HIF-1α expression than that of resveratrol, a known HIF-1α inhibitor. In addition, rhapontigenin promoted hypoxia-induced HIF-1α degradation and cycloheximide (CHX) blocked protein synthesis. A prolyl hydroxylase (PHD) inhibitor dimethyloxalylglycine (DMOG) is usually utilized to examine whether prolyl hydroxylation is involved in inhibition of HIF-1α accumulation. Here, DMOG recovered HIF-1α accumulation inhibited by rhapontigenin. Immunoprecipitation assay also revealed that rhapotigenin enhanced the binding of hydroxylated HIF-1α to von Hippel-Lindau (VHL) tumor suppressor protein. Furthermore, rhapontigenin reduced vascular endothelial growth factor (VEGF) secretion in hypoxic PC-3 cells as well as suppressed tube formation in human umbilical vein endothelial cells (HUVECs) treated by the conditioned media of hypoxic PC-3 cells. However, anti-angiogenic effect of rhapontigenin in hypoxic PC-3 cells was reversed by DMOG. Taken together, these findings suggest that rhapontigenin inhibits HIF-1α accumulation and angiogenesis in PC-3 prostate cancer cells.     

链霉素及H7对机械牵张离体心肌HIF-1α、VEGF的影响及机制探讨

目的研究链霉素及H7对机械牵张大鼠心肌组织低氧诱导分子-1α和血管内皮细胞生长因子表达的影响,并探讨二者在其中的作用机制。方法采用大鼠离体灌流心脏模型,膨胀左心室30min,RT-PCR法检测左室心肌细胞HIF-1α、VEGF mRNA的表达,免疫组化观察二者在心肌细胞中定位,Western blot检测HIF-1α蛋白的表达,利用链霉素作为牵张敏感离子通道(SACs)阻断剂研究SACs和PKC抑制剂H7在其中的可能作用。结果与不牵张组HIF-1α和VEGF mRNA无表达的比较,牵张可以明显增加HIF-1α和VEGF mRNA的表达(P<0·05或P<0·01);而链霉素、H7可以明显减少HIF-1α和VEGF mRNA的表达(P<0·05);但是二者不能完全抑制急性牵张刺激激活的HIF-1α和VEGF mRNA水平升高(P<0·05),HIF-1α和VEGF在胞质和胞核中均有表达,并检测到HIF-1α蛋白表达。结论链霉素、H7对膨胀左室致HIF-1α、VEGF表达有明显抑制作用,提示心室膨胀经SACs-PKC-激活胞内信号诱导HIF-1α、VEGF表达。同时链霉素并不能完全抑制HIF-1α、VEGF表达,提示膨胀心室致HIF-1α、VEGF表达进而引起心室肥厚尚有其他传导途径,仍需进一步研究。     

缺氧诱导因子-1α在胶质瘤中的表达及意义

目的探讨缺氧诱导因子-1α（HIF-1α）在人脑不同级别胶质瘤中的表达,分析HIF-1α、血管内皮生长因子（VEGF）和微血管密度（MVD）之间的相互关系及意义。方法采用免疫组化法检测54例胶质瘤组织中HIF-1α及VEGF的表达情况,并以VIII-R-Ag作为标记计数微血管密度（MVD）。结果58例胶质瘤组织HIF-1α的表达阳性率为65.5%（33/58）,HIF-1α的表达强度和胶质瘤的病理分级呈正相关（χ2=14.24,P〈0.05）。HIF-1α的表达和VEGF的表达呈正相关（χ2=4.02,P〈0.05）,HIF-1α和MVD呈正相关（t=3.22,P〈0.05）。结论HIF-1α与人脑胶质瘤组织中血管生成及病理分级密切相关,为治疗胶质瘤的提供一种可能的途径。     

A positive circuit of VEGF increases Glut-1 expression by increasing HIF-1α gene expression in human retinal endothelial cells

Treatment of human retinal microvascular endothelial cells (HRMECs) with vascular endothelial growth factor 165 (VEGF₁₆₅) increased hypoxia-inducible factor 1α (HIF-1α), VEGF, and glucose transporter 1 (Glut-1) mRNA expression and Glut-1 protein localization to the membrane. In contrast, treatment of human retinal pigment epithelium cells with VEGF₁₆₅ did not induce HIF-1α, VEGF, and Glut-1 gene expression. Microvascular endothelial cells are surrounded by astrocytic end feet in the retina. Astrocyte-derived A-kinase anchor protein 12 overexpression during hypoxia downregulated VEGF secretion, and this conditioned medium reduced VEGF and Glut-1 expression in HRMECs, suggesting that communications between astrocytes and endothelial cells may be the determinants of the blood vessel network. In HRMECs, HIF-1α small interfering RNA transfection blocked the VEGF₁₆₅-mediated increase in VEGF and Glut-1 gene expression. Inhibition of protein kinase C (PKC) with inhibitor GF109203X or with a small interfering RNA targeting PKCζ attenuated the VEGF₁₆₅-induced Glut-1 protein expression and VEGF and Glut-1 mRNA expression. In addition, results of an immunoprecipitation assay imply an interaction between VEGF receptor 2 (VEGFR2) and PKCζ in HRMECs. Therefore, VEGF secretion by hypoxic astrocytes may upregulate HIF-1α gene expression, inducing VEGF and Glut-1 expression via the VEGFR2–PKCζ axis in HRMECs.     

通心络对兔缺氧耐受性的影响

目的观察通心络对兔急性缺氧耐受性的作用。方法 24只新西兰大耳白家兔随机分为对照组、缺氧组和通心络组,先在11.4%氧环境下低氧实验,60min后进行密闭缺氧,观察兔密闭缺氧耐受时间,ELISA法检测血清缺氧诱导因子1-α(HIF-1α)的含量、Western blot法检测主动脉组织HIF-1α和血管内皮生长因子(VEGF)的蛋白表达。结果与缺氧前比较,缺氧组与通心络组血清HIF-1α含量明显增加(P<0.01或P<0.05)。与对照组比较,缺氧组和通心络组主动脉组织HIF-1α和VEGF表达明显增强(P<0.01或P<0.05)。与缺氧组比较,通心络组家兔密闭缺氧耐受时间明显增加(P<0.05),血清HIF-1α含量明显增加(P<0.01),主动脉组织HIF-1α表达增强,VEGF表达明显增强(P<0.05)。结论 HIF-1α和VEGF的表达增高可能是缺氧适应的一种机制,通心络能提高兔的缺氧耐受性,其机制可能与上调血清及组织的HIF-1α和VEGF有关。     

Vasohibin-1、VEGF 及 HIF-1α在子宫内膜腺癌中的表达及意义

目的：通过研究血管生成抑制蛋白 Vasohibin-1、缺氧诱导因子1α（hypoxia-inducible factor-1α,HIF-1α）在正常增殖期子宫内膜（normal endometrium,NE）、子宫内膜非典型增生（endometrial atypical hyperplasia,EAH）和子宫内膜腺癌（endometrioid adenocarcinoma,EA）组织中的表达,探讨上述指标与血管内皮生长因子（ vascular endometrial growth factor,VEGF）及 EA 临床病理特性的关系。方法采用免疫组织化学方法检测 Vasohibin-1、VEGF 及 HIF-1α在15例 NE、30例 EAH 和50例 EA 组织中的表达,分析各指标之间及与 EA 临床病理特性的关系。结果在 EA 组织中,Vasohibin-1、VEGF 和 HIF-1α的阳性表达率明显高于 NE 组（ P <0.05）;VEGF 和 HIF-1α的表达阳性率与 EA 临床手术分期有关且随期别升高而升高（ P <0.05）,而 Vasohibin-1与临床手术分期无关（ P >0.05）;在 EA 组织中, Vasohibin-1、HIF-1α与 VEGF 的表达呈正相关（ P <0.01）,而 Vasohibin-1与 HIF-1α无相关性（ P >0.05）。结论Vasohibin-1、VEGF 及 HIF-1α均在 EA 组织中高表达,Vasohibin-1在高水平的 VEGF 诱导下表达升高,但并不足以抑制EA 组织中新生血管形成,可能存在其他旁路调节途径来调节两者之间的平衡。     

Inhibition of angiogenesis and HIF-1alpha activity by antimycin A1

We identified antimycin A1 as an inhibitor of the hypoxia-response element (HRE) from screening using a reporter under the control of HRE under hypoxic conditions. Antimycin A1 was effective at 20 pg/ml in inhibiting the reporter activity. The expression of vascular endothelial growth factor (VEGF) mRNA during hypoxia was also inhibited by antimycin A1. Angiogenesis induced by implantation of mouse sarcoma-180 cells was significantly inhibited by non-toxic doses of antimycin A1. Hypoxia inducible factor (HIF)-1alpha protein levels were significantly decreased by antimycin A1, but its mRNA level was not affected. Antimycin A1 is known to be an inhibitor of mitochondrial electron transport system, and depletion of mitochondria abolished antimycin A1-effect, at least in part. Inhibitors of proteasome or protein synthesis did not affect the decrease in HIF-1alpha level induced by antimycin A1. These results indicate that antimycin A1 inhibited angiogenesis through decrease in VEGF production caused by inhibition of HIF-1alpha activation.     

Molecular biomarkers of cantharidin-induced cardiotoxicity in Sprague-Dawley rats: Troponin T,vascular endothelial growth factor and hypoxia inducible factor-1α

Early diagnosis of cantharidin-induced myocardial injury is the key to reduce the fatality rate in clinical practice. The purpose of the present study was to explore biomarkers that can be used for the prediction and diagnosis of cantharidin-induced myocardial injury. Of 65 male Sprague-Dawley rats weighing 200-230 g, 25 rats were divided into five groups according to the administration dose of cantharidin (0, 1.34, 2.67, 4 and 5.34 mg/kg; n = 5 per group) and the other 40 rats were treated with 2.67 mg/kg cantharidin and divided into nine groups according to the administration time (0, 1, 2, 4, 6, 8, 12, 24, 48 and 72 hours; n = 4 per group). Pathological changes of hypoxia, necrosis and inflammation were confirmed in heart samples that were exposed to cantharidin by hematoxylin-eosin staining and overall scores of pathological changes among heart samples in cantharidin exposure groups showed an increasing trend compared with in the control group. Coexpression of vascular endothelial growth factor (VEGF), hypoxia inducible factor-1α (HIF-1α) and caspase9 was shown in the myocardium by immunofluorescence staining. Western blotting results showed that expression of VEGF, HIF-1α and caspase9 in cantharidin-treated rat hearts showed an increasing trend compared with in the control group. Results of enzyme-linked immunosorbent assay suggested that plasma levels of troponin T (TN-T), VEGF and HIF-1α were elevated at different intervals after cantharidin administration, and VEGF and HIF-1α had a significant linear relationship with TN-T that was verified by multiple linear regression analysis. Preliminary results serve to illustrate that TN-T, VEGF and HIF-1α might be valuable molecular markers in cantharidin-induced myocardial injury and that diagnostic accuracy needs to be studied further.