HIF-2α、VEGF、COX-2、MMP-9表达及其与肾细胞癌血管生成的关系分析

目的 探讨HIF-2α(缺氧诱导因子-20α)、VEGF(血管内皮生长因子)、COX-2(环氧化酶-2)、MMP-9(金属基质酶-9)表达及其与肾细胞癌血管生成的关系.方法 应用免疫组化方法,检测79例肾细胞癌手术切除标本的HIF-2α、VEGF、COX-2、MMP-9表达情况,并与MVD(微血管密度)表达进行关联性分析.结果 肾癌组织MMP-9、VEGF的表达均显著高于正常肾组织(P＜0.05);不同临床分期肾癌组织中的MMP-9、VEGF表达有明显差异(P＜0.05).MVD与VEGF、MMP-9蛋白表达呈显著正相关(r=0.381、0.375,P＜0.05);MVD与COX-2表达有关,COX-Z表达0级和4级MVD值最低,与1、2、3级之间比较差异有统计学意义(P＜0.05),0级与4级之间比较差异无统计学意义(P＞0.05),1、2、3级之间差别无统计学意义(P＞0.05).肾细胞癌中HIF-2α阳性组的MVD值高于HIF-2α阴性组中MVD值,差异有统计学意义(t=4.374,P＜0.05);Spearman等级相关分析发现,HIF-2α的表达与MVD间存在正相关关系(r=0.545,P＜0.01).结论 HIF-2α、VEGF、COX-2、MMP-9表达及其与肾细胞癌血管生成之间有明显相关性,在临床上可以根据相关基因表达情况而采取相应的干预措施.     

mTOR activates hypoxia-inducible factor-1α and inhibits neuronal apoptosis in the developing rat brain during the early phase after hypoxia-ischemia

The mammalian target of rapamycin (mTOR) exerts neuroprotective effects under hypoxic or ischemic conditions. To explore whether mTOR participates in neuroprotective signaling through regulation of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) and neuronal apoptosis in developing rat brain with hypoxia-ischemia (HI), we operated on postnatal day 10 rats by ligating the common carotid artery followed by exposure to systemic hypoxia. Brains were collected at various intervals to detect the expression of mTOR, phosphorylated mTOR (p-mTOR), HIF-1α, VEGF and cleaved caspase 3 (CC3), using immunohistochemistry and Western blot analysis. We also used terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) to detect neuronal apoptosis. The p-mTOR protein expression increased at 2 h after HI, peaked at 8 h, lasted 24 h, and then dropped to the basal level. Also, the expression of HIF-1α and VEGF was significantly enhanced and peaked at 8 h after HI. Up-regulated expression of CC3 was observed at 2 h, peaked at 24 h, and lasted 72 h after HI. Increased neuronal apoptosis is associated with reduced HIF-1α and VEGF expression. Furthermore, pretreatment with rapamycin, a mTOR specific inhibitor, significantly inhibited HIF-1α and VEGF protein after HI. The expression of CC3 and the number of TUNEL-positive cells were up-regulated at 8 h and down-regulated at 24 h after HI in the rapamycin-treated group. Our findings suggest that mTOR may participate in the regulation of HIF-1α, VEGF and neuronal apoptosis, serving neuroprotective functions after HI in developing rat brain.     

去铁胺对新生大鼠缺氧缺血性脑的保护作用

目的： 研究去铁胺对新生大鼠缺氧缺血性脑病(HIE)模型新生血管形成的影响。方法：新生7 d SD大鼠建立HIE模型，模型组和治疗组建模前24 h分别用去铁胺或生理盐水注射。第1 d、3 d、7 d和14 d处死大鼠，检测缺氧缺血（左）侧脑组织毛细血管密度(BCDI)、增生毛细血管数、脑含水量、脑萎缩程度及其血管内皮生长因子(VEGF)和低氧诱导因子-1α (HIF-1α) mRNA表达。结果：治疗组左脑含水量和左脑萎缩比显著低于模型组(P<0.01)；左侧脑组织增生毛细血管数目显著高于模型组［(2.01±0.31)条/HPF vs (0.90±0.25)条/HPF ,P<0.01］。去铁胺显著上调左侧脑组织VEGF 和HIF-1α mRNA表达［12 h时VEGF：(1.41±0.07) vs (1.10±0.15)，P<0.05；HIF-1α：(1.49±0.12) vs (1.11±0.16)，P<0.05］。结论:去铁胺可能通过上调脑组织HIF-1α和VEGF表达，促进缺氧缺血脑组织新生血管形成，减轻缺氧缺血性脑损伤。     

Qiliqiangxin improves cardiac function and attenuates cardiac remodeling in rats with experimental myocardial infarction

Objective: It has been reported that Qiliqiangxin (QL), a traditional Chinese medicine compound, could inhibit cardiac hypertrophy and remodeling, and improve cardiac function. However, whether and how it reverses cardiac remodeling in rats post myocardial infarction (MI) remains unknown. This study aims to explore related mechanisms linked with cardiac function improvement and attenuation of cardiac remodeling by QL in rats with experimental MI. Methods: MI was induced by ligation of left anterior descending coronary artery (LAD) in male Sprague-Dawley rats. Rats with LVEF < 50% at four weeks after procedure were treated for another 6 weeks with placebo, QL and captopril. Echocardiography and plasma NT-proBNP were measured at the end of study, and histological studies were performed. Protein expressions of Neuregulin-1 (NRG-1), total-Akt, phospho-Akt (Ser473), hydroxy-HIF-1α (Pro564), VEGF, Bax, Bcl-2 and Caspase 3 were examined by Western blot. mRNA expression of NRG-1 and p53 was detected by real-time PCR. Results: Compared with the placebo group, QL improved cardiac function, reduced left ventricular dimension, inhibited interstitial inflammation and fibrosis, increased neovascularization, and attenuated cardiomyocyte apoptosis. Meanwhile QL significantly upregulated the expression of HIF-1α, VEGF, enhanced phosphorylation of Akt, decreased the ratio of Bax/Bcl-2 and Caspase 3 expression. Furthermore, we observed upregulation of NRG-1 and downregulation of p53 after QL treatment. Conclusion: Our data suggest that the beneficial effects of QL on improving cardiac function and attenuating cardiac remodeling post MI are associated with angiogenesis enhancement and apoptosis inhibition, which may be mediated via activation of NRG-1/Akt signaling and suppression of p53 pathway.     

Blood vessel formation in the tissue-engineered bone with the constitutively active form of HIF-1α mediated BMSCs

The successful clinical outcome of the implanted tissue-engineered bone is dependent on the establishment of a functional vascular network. A gene-enhanced tissue engineering represents a promising approach for vascularization. Our previous study indicated that hypoxia-inducible factor-1α (HIF-1α) can up-regulate the expression of vascular endothelial growth factor (VEGF) and stromal-derived factor 1 (SDF-1) in bone mesenchymal stem cells (BMSCs). The angiogenesis is a co-ordinated process that requires the participation of multiple angiogenic factors. To further explore the angiogenic effect of HIF-1α mediated stem cells, in this study, we systematically evaluated the function of HIF-1α in enhancing BMSCs angiogenesis in vitro and in vivo. A constitutively active form of HIF-1α (CA5) was inserted into a lentivirus vector and transduced into BMSCs, and its effect on vascularization and vascular remodeling was further evaluated in a rat critical-sized calvarial defects model with a gelatin sponge (GS) scaffold. The expression of the key angiogenic factors including VEGF, SDF-1, basic fibroblast growth factor (bFGF), placental growth factor (PLGF), angiopoietin 1 (ANGPT1), and stem cell factor (SCF) at both mRNAs and proteins levels in BMSCs were significantly enhanced by HIF-1α overexpression compared to the in vitro control group. In addition, HIF-1α-over expressing BMSCs showed dramatically improved blood vessel formation in the tissue-engineered bone as analyzed by photography of specimen, micro-CT, and histology. These data confirm the important role of HIF-1α in angiogenesis in tissue-engineered bone. Improved understanding of the mechanisms of angiogenesis may offer exciting therapeutic opportunities for vascularization, vascular remodeling, and bone defect repair using tissue engineering strategies in the future.     

MAPK/ERK signal pathway involved expression of COX-2 and VEGF by IL-1β induced in human endometriosis stromal cells in vitro

Objective: Now there are more and more evidences that Cyclooxygenase-2 (COX-2) plays an important role in angiogenesis of endometriosis (EMs). Vascular endothelial growth factor (VEGF) has a potent angiogenic activity. However, it is worth studying about the regulating mechanism of COX-2/COX-1 and VEGF in the development of human endometriosis in vitro. The current study was designed to investigate the effect of 4 cytokines on COX-2/COX-1 expression and the effect of IL-1β on VEGF release in human endometriosis stromal cells (ESC), and to explore the related signaling pathways involved in vitro. Methods: Isolation, culture and identification of ESC. Cells were treated with 4 cytokines, and the inhibitor mitogen-activated protein-Erk (MEK) and the inhibitor p38 mitogen-activated protein kinase (MAPK) prior to adding cytokine IL-1β. COX-2 protein expression was measured by western blot and VEGF secretion was determined by ELISA. Results: Among four kinds of cytokines, IL-1β treatment increased COX-2 protein expression and VEGF release in three ESC, and TNF-α had the same effect on COX-2 protein level as IL-1β only in ectopic and eutopic ESC, and MCSF had only slight effect on ectopic ESC. In contrast, cytokines had no effect on COX-1 expression. We also demonstrated that MAPK reduced the synthesis of COX-2 by IL-1β induced. COX-2 inhibitor reduced VEGF release by IL-1β induced. Conclusions: i) In human ESC in vitro, IL-1β up-regulated the COX-2 expression through the activation of p38 MAPK pathway, and not to COX-1. ii) Up-regulation of VEGF level by IL-1β treatment was found in human endometriosis stromal cell and COX-2 inhibitor was involved in this process.     

TRPV3 promotes the angiogenesis through HIF-1α-VEGF signaling pathway in A549 cells

BackgroundAngiogenesis is an essential physiological process in the growth and metastasis of primary tumors. Ca²⁺ signaling is crucial for tumor angiogenesis. The purpose of this study was to detect the potential role of Ca²⁺ permeable transient receptor potential vanilloid-3 (TRPV3) in the angiogenesis of non-small cell lung cancer (NSCLC).MethodsSmall interfering RNA was used to down-regulate TRPV3 expression in A549 cells. A laser scanning confocal microscope was used to examine intracellular calcium concentration ([Ca²⁺]i). Human umbilical vein endothelial cells (HUVECs) tube formation and migration assay, Western blot, MTT and ELISA were performed to detect the potential mechanisms of TRPV3 in tumor angiogenesis. A mouse tumor xenograft model was performed to expound the effects of TRPV3 on tumor cell growth.ResultsInhibition of TRPV3 reduced [Ca²⁺]i and protein expressions of VEGF and HIF-1α in A549 cells. Moreover, HIF-1α depletion decreased the secretion and expression of VEGF. Depletion of TRPV3 inhibited HUVECs proliferation, tube formation and migration induced by conditioned medium. And TRPV3 inhibition could decrease the volume of xenograft tumors and MVD of CD34⁺ cells. The expression levels of HIF-1α, VEGF and p-CaMKП in the xenograft tumors in RuR and siTRPV3 groups was reduced.ConclusionsTRPV3 calcium channel protein may play a key role in NSCLC angiogenesis. TRPV3 could promote the angiogenesis through HIF-1α-VEGF signaling pathway. Targeting TRPV3 channel protein by novel approaches would be useful for reversing NSCLC angiogenesis.     

低氧诱导因子-1α参与低氧预处理诱导的心肌血管生成

目的：研究低氧诱导因子-1α（HIF-1α）在低氧预处理（HPC）诱导心肌血管生成中的作用,并探讨其细胞信号转导的机制。方法：雄性SD大鼠随机分为对照组和HPC组。动物置于低氧仓内，持续通入10% O2和90% N2 4 h复制低氧预处理模型。用Ⅷ因子免疫组化染色检测HPC后7 d和21 d心肌组织微血管密度。制备心肌组织蛋白提取物，分别以磷酸化的细胞外信号调节激酶（ERK1/2）抗体和HIF-1α抗体检测HPC后1 d、7 d和21 d p-ERK1/2活性及HIF-1α表达。结果：HPC后7 d和21 d心肌组织微血管密度分别较对照组高36.99%和37.76%（均P<0.01）；p-ERK1/2活性在HPC后1d 较对照组高18.67%；HPC诱导HIF-1α的表达，其表达高峰出现在HPC后1 d。结论：HPC可以促进心肌微血管生成，其机制涉及ERKs活化和HIF-1α表达上调。     

Upregulation of vascular endothelial growth factor by cobalt chloride-simulated hypoxia is mediated by persistent induction of cyclooxygenase-2 in a metastatic human prostate cancer cell line

Upregulation of vascular endothelial growth factor (VEGF) expression induced by hypoxia is crucial event leading to neovascularization. Cyclooxygenase-2, an inducible enzyme that catalyzes the formation of prostaglandins (PGs) from arachidonic acid, has been demonstrated to be induced by hypoxia and play role in angiogenesis and metastasis. To investigate the potential effect of COX-2 on hypoxia-induced VEGF expression in prostate cancer. We examined the relationship between COX-2 expression and VEGF induction in response to cobalt chloride (CoCl₂)-simulated hypoxia in three human prostate cancer cell lines with differing biological phenotypes. Northern blotting and ELISA revealed that all three tested cell lines constitutively expressed VEGF mRNA, and secreted VEGF protein to different degrees (LNCaP > PC-3 > PC3ML). However, these cell lines differed in the ability to produce VEGF in the presence of CoCl₂-simulated hypoxia. CoCl₂ treatment resulted in 40% and 75% increases in VEGF mRNA, and 50% and 95% in protein secretion by LNCaP and PC-3 cell lines, respectively. In contrast, PC-3ML cell line, a PC-3 subline with highly invasive, metastatic phenotype, exhibits a dramatic upregulation of VEGF, 5.6-fold in mRNA and 6.3-fold in protein secretion after treatment with CoCl₂. The upregulation of VEGF in PC-3ML cells is accompanied by a persistent induction of COX-2 mRNA (6.5-fold) and protein (5-fold). Whereas COX-2 expression is only transiently induced in PC-3 cells and not affected by CoCl₂ in LNCaP cells. Moreover, the increases in VEGF mRNA and protein secretion induced by CoCl₂ in PC-3ML cells were significantly suppressed following exposure to NS398, a selective COX-2 inhibitor. Finally, the effect of COX-2 inhibition on CoCl₂-induced VEGF production was reversed by the treatment with exogenous PGE₂. Our data demonstrate that VEGF induction by cobalt chloride-simulated hypoxia is maintained by a concomitant, persistent induction of COX-2 expression and sustained elevation of PGE₂ synthesis in a human metastatic prostate cancer cell line, and suggest that COX-2 activity, reflected by PGE₂ production, is involved in hypoxia-induced VEGF expression, and thus, modulates prostatic tumor angiogenesis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Hypoxia-inducible factor-1alpha expression in experimental cirrhosis: correlation with vascular endothelial growth factor expression and angiogenesis

Angiogenesis progresses together with fibrogenesis during chronic liver injury. Hypoxia-inducible factor-1alpha (HIF-1alpha), a master regulator of homeostasis, plays a pivotal role in hypoxia-induced angiogenesis through its regulation of vascular endothelial growth factor (VEGF). The association between hypoxia, angiogenesis and VEGF expression has been demonstrated in experimental cirrhosis. However, expression of HIF-1alpha has yet to be reported. The aim of this study was to investigate the significance of HIF-1alpha expression during experimental liver fibrosis and the relationships between HIF-1alpha expression, VEGF expression and angiogenesis. Cirrhosis was induced in male Wistar rats by intraperitoneal administration of diethyl nitrosamine (DEN) (100 mg/kg, once a week). The serial sections from liver tissues were stained with anti-HIF-1alpha, anti-VEGF and anti-CD34 antibodies before being measured by light microscopy. Our results showed that HIF-1alpha expression gradually increases according to the severity of fibrosis (p<0.01). Moreover, its expression was found to be correlated with angiogenesis (r=0.916) and VEGF expression (r=0.969). The present study demonstrates that HIF-1alpha might have a role in the development of angiogenesis via regulation of VEGF during experimental liver fibrogenesis and suggests that this factor could be a potential target in the manipulation of angiogenesis in chronic inflammatory diseases of the liver.     

缺氧诱导因子-1α抑制剂对大鼠局部脑缺血再灌注损伤的影响

目的研究大鼠局部脑缺血再灌注损伤时,缺氧诱导因子-1α(HIF-1α)抑制剂2-甲氧雌二醇(2ME2)对损伤脑组织的影响。方法雄性SD大鼠60只随机分为假手术组、大脑中动脉阻塞再灌注组(MCAO组)、假性治疗组(DMSO组)、2ME2治疗组(2ME2组)。治疗组在术后1h腹腔注射相应剂量药物。观察各组大鼠神经行为学缺陷;再灌注7d,TTC染色观察脑梗死体积变化;再灌注24h,Western blotting检测HIF-1α及其下游基因的表达变化;制备脑组织切片分别作Nissl染色、免疫组织化学染色及原位缺口末端标记(TUNEL)。结果2ME2组神经功能较MCAO及DMSO组有明显改善(P<0.05),同时,2ME2组梗死面积明显减小(P<0.05)。Western blotting结果显示,HIF-1α的表达经2ME2治疗后降低,其下游基因VEGF、BNIP3及Caspase-3的表达有同样的变化。Nissl染色可见2ME2治疗组皮质神经元结构较清晰,胞体肿胀、核固缩、核溶解程度较模型组及假性治疗组明显减轻,淡染区域减小;免疫组织化学法观察到2ME2组HIF-1α、Caspase-3、BNIP3、VEGF及TUNEL标记的阳性细胞数明显减少(P<0.05)。结论2ME2可能通过降低HIF-1α水平并下调其下游的BNIP3和VEGF的表达,降低血脑屏障的通透性并减少凋亡因子Caspase-3的作用,发挥神经元的保护作用。     

HIF-1α change in serum and callus during fracture healing in ovariectomized mice

The purpose was to detect the effects of ovariectomy (OVX) on femoral fracture healing through different angiogenesis and HIF-1α expression in mice. Thirty-six young female C57 mice were randomized into two groups: OVX and age-matched intact control (CON). The femoral fracture was generated at 3 weeks after OVX or CON. At 2 or 4 weeks after fracture, the femoral fracture area was evaluated healing status by bone mineral density (BMD), callus formation and mineralization and neovascularization in callus, biomechanical analysis, and HIF-1α tests. OVX mice showed lower BMD as compared with CON mice. Callus geometric microstructural parameters of the femora in OVX mice were significantly lower than CON mice. OVX induced significant changes of biomechanical parameters in the femoral fracture healing area. The callus forming, callus neovascularization and HIF-1α tests in OVX mice were significantly lower than in CON mice. HIF-1α results have the positive proportion with osteoporotic fracture healing.