Marked inhibition of retinal neovascularization in rats following soluble-flt-1 gene transfer

BACKGROUND: In mouse models of retinopathy of prematurity (ROP) inhibitors of vascular endothelial growth factor (VEGF) functions administered systemically completely block retinal neovascularization. In contrast, selective ocular VEGF depletion has achieved an approx. 50% inhibition of retinal neovascular growth. It is unclear whether a more complete inhibition of new blood vessel development can be obtained with an anti-VEGF therapy localized to the eye. Therefore, the objective of the present study was to determine the effect of local anti-VEGF therapy in a different animal model which closely mimics human ROP. METHODS: Rats were exposed to alternating cycles of high and low levels of oxygen for 14 days immediately after birth; thereafter, they were intravitreally injected with an adenoviral vector expressing a secreted form of the VEGF receptor flt-1 (Ad.sflt), which acts by sequestering VEGF. Contralateral eyes were injected with the control vector carrying the reporter gene expressing beta-galactosidase (Ad.betaGal). RESULTS: At the peak of retinal neovascular growth, i.e. post-natal day 21 (P21), we observed up to 97.5% decrease in retinal neovascularization in animals injected with Ad.sflt. At the end of observation (P28), no significant difference in retinal vessel number was detected in both oxygen-injured and normoxic Ad.sflt-treated retinas compared with untreated or Ad.betaGal-treated retinas. CONCLUSION: Adenoviral-mediated sflt-1 gene transfer induces a near-complete inhibition of ischemia-induced retinal neovascularization in rats without affecting pre-existing retinal vessels.     

Combination of angiopoietin-1 and vascular endothelial growth factor gene therapy enhances arteriogenesis in the ischemic myocardium

We hypothesised that angiopoietin-1 (Ang-1), in conjunction with vascular endothelial growth factor (VEGF) gene therapy, can enhance arteriogenesis and angiogenesis during myocardial ischemia. Mice were given a single intramyocardial injection of saline, phVEGF-A(165) and phAng-1 or a combination thereof into the non-ischemic normal heart or into the ischemic border zone of the infarcted heart. In the normal and the ischemic myocardium, gene transfer of phVEGF-A(165) alone increased the myocardial capillary density by 16% and 36%, respectively, and phAng-1 had a similar effect. In the normal heart, the ratio of arteriolar to capillary densities increased with phVEGF-A(165) and more so in the ischemic myocardium where phAng-1 also had an effect. Furthermore, the combination of plasmids induced an up to 7.5-fold increase. Transient overexpression of VEGF-A(165) boosts endogenous arteriogenesis in addition to capillary angiogenesis. Ang-1 further boosts this effect at the arteriolar level.     

MicroRNA signature and function in retinal neovascularization Agrawal S,Chaqour B简

Ischemic retinopathies are clinically well-defined chronic microvascular complications characterized by gradually progressive alterations in the retinal microvasculature and a compensatory aberrant neovascularization of the eye. The subsequent metabolic deficiencies result in structural and functional alterations in the retina which is highly susceptible to injurious stimuli such as diabetes, trauma, hyperoxia, inflammation, aging and dysplipidemia. Emerging evidence indicates that an effective therapy may require targeting multiple components of the angiogenic pathway. Conceptually, mircoRNA (miRNA)-based therapy provides the rationale basis for an effective antiangiogenic treatment. miRNAs are an evolutionarily conserved family of short RNAs, each regulating the expression of multiple protein-coding genes. The activity of specific miRNAs is important for vascular cell signaling and blood vessel formation and function. Recently, important progress has been made in mapping the miRNA-gene target network and miRNA-mediated gene expression control. Here we highlight the latest findings on angiogenic and antiangiogenic miRNAs and their targets as well as potential implications in ocular neovascular diseases. Emphasis is placed on how specific vascular-enriched miRNAs regulate cell responses to various cues by targeting several factors, receptors and/or signaling molecules in order to maintain either vascular function or dysfunction. Further improvement of our knowledge in not only miRNA specificity, turnover, and transport but also how miRNA sequences and functions can be altered will enhance the therapeutic utility of such molecules.     

MicroRNA signature and function in retinal neovascularization

Ischemic retinopathies are clinically well-defined chronic microvascular complications characterized by gradually progressive alterations in the retinal microvasculature and a compensatory aberrant neovascularization of the eye. The subsequent metabolic deficiencies result in structural and functional alterations in the retina which is highly susceptible to injurious stimuli such as diabe-tes, trauma, hyperoxia, inflammation, aging and dys-plipidemia. Emerging evidence indicates that an effec-tive therapy may require targeting multiple components of the angiogenic pathway. Conceptually, mircoRNA(miRNA)-based therapy provides the rationale basis for an effective antiangiogenic treatment. miRNAs are an evolutionarily conserved family of short RNAs, each regulating the expression of multiple protein-coding genes. The activity of specific miRNAs is important for vascular cell signaling and blood vessel formation and function. Recently, important progress has been made in mapping the miRNA-gene target network andmiRNA-mediated gene expression control. Here wehighlight the latest findings on angiogenic and antian-giogenic miRNAs and their targets as well as potentiaimplications in ocular neovascular diseases. Emphasis isplaced on how specific vascular-enriched miRNAs regu-late cell responses to various cues by targeting severafactors, receptors and/or signaling molecules in orderto maintain either vascular function or dysfunction. Fur-ther improvement of our knowledge in not only miRNAspecificity, turnover, and transport but also how miRNAsequences and functions can be altered will enhancethe therapeutic utility of such molecules.     

Regulation of limb development by the sphingosine 1-phosphate receptor S1p1/EDG-1 occurs via the hypoxia/VEGF axis

Angiogenesis, also known as new blood vessel formation, is regulated coordinately with other tissue differentiation events during limb development. Although vascular endothelial cell growth factor (VEGF) is important in the regulation of angiogenesis, chondrogenesis and osteogenesis during limb development, the role of other angiogenic factors is not well understood. Sphingosine 1-phosphate, a platelet-derived lipid mediator, regulates angiogenesis and vascular maturation via its action on the G-protein-coupled receptor S1P(1) (also known as EDG-1). In addition to vascular defects, abnormal limb development was also observed in S1p(1)(-/-) mice. Here we show that strong induction of S1P(1) expression is observed in the blood vessels and the interdigital mesenchymal cells during limb development. Deletion of S1P(1) results in aberrant chondrocyte condensation and defective digit morphogenesis. Interestingly, the vasculature in the S1p(1)(-/-) limbs was hyperplastic and morphologically altered. In addition, the hypoxia inducible factor (HIF)-1 alpha and its response gene VEGF were induced in S1p(1)(-/-) limbs. However, aberrant regulation of HIF-1 alpha and VEGF were not observed in embryonic fibroblasts derived from S1p(1)(-/-) mice, suggesting a non-cell autonomous effect of S1P(1) on VEGF expression. Indeed, similar limb defects were observed in endothelium-specific S1P(1) null mice in vivo. These data suggest that the function of S1P(1) in the developing vasculature is essential for proper limb development.     

黄芪红花配伍对大鼠脑缺血后血管新生及Cav-1/VEGF信号通路的影响

目的:黄芪红花配伍是否通过调节小凹蛋白1(Caveolin-1,Cav-1)/血管内皮生长因子(vascular endothelial growth factor,VEGF)通路促进血管新生保护大鼠脑缺血损伤。方法:60只雄性SD大鼠随机分为5个组:对照组(Sham组,n=12),模型组(MACO组,n=12),黄芪红花40:1组(n=12),20:1组(n=12),5:1(n=12)。大鼠脑缺血再灌注损伤模型采用尼龙线栓法制作,连续给药21d后,评价神经功能学评分,计算脑梗死体积,采用免疫组化法测定皮质区的微血管密度,采用RT-PCR法检测皮质区VEGF m RNA和Cav-1 m RNA表达,采用Western-blotting法测定皮质区VEGF和Cav-1的蛋白表达。结果:连续给药21d后,各组大鼠的神经功能学评分均有所降低,3个不同比例的黄芪红花组的神经功能学评分降低最为明显(P0.01),脑梗死体积较模型组显著减少(P0.05~P0.01),微血管密度、VEGF和Cav-1 m RNA和蛋白表达水平均较模型组明显升高(P0.05~P0.01)。结论:黄芪红花配伍可能通过调节Cav-1/VEGF信号通路促进脑缺血再灌注损伤大鼠脑内的血管新生,从而减轻脑缺血损伤,且最佳的配伍比例为黄芪红花5:1。     

Ad-Ang1-IRES-VEGF165腺病毒载体的构建及其促血管形成作用

目的:构建人血管生成素1(Ang1)和血管内皮生长因子VEGF165 (VEGF165)的共表达腺病毒载体Ad-Ang1-IRES-VEGF165(简称Ad-AV),为研究Ad-AV转基因细胞表达产物血管诱生活性提供实验依据。方法:采用IRES介导的Ang1和VEGF165双基因腺病毒共表达模式,通过常规的基因克隆和重组技术,构建Ad-AV双基因共表达腺病毒载体,经感染人胚肾QBI-293A细胞(293A)进行扩增和效价测定后,再感染WI-38人胚肺成纤维细胞,均用ELISA法检测VEGF、Ang1目的基因的表达,并采用鸡胚尿囊膜血管形成实验(CAM)法分析其对血管形成的影响。结果: 扩增的Ad-AV腺病毒效价可达4×10¹⁰pfu/ml;Ad-AV不仅能在293A细胞中成功表达目的基因Ang1、VEGF,而且在WI-38成纤维细胞也能成功表达,其表达产物具有显著的促进CAM上血管生成的活性。结论:成功构建并获得了Ad-Ang1-IRES-VEGF165重组病毒子, 目的基因均能在人胚肾和人胚肺成纤维细胞中表达,其表达产物具有诱导血管形成的功能。     

IRES/polyA-promoter介导的Ang-1与VEGF165双基因表达效率的对比分析

目的:构建IRES及polyA-promoter介导人血管生成素-1(Angiogenin-1,Ang-1)和人血管内皮生长因子165(vascular endothelial growth factor165,VEGF165)双基因共表达的腺病毒载体,比较IRES与polyA-promoter不同表达模式及其对位于二者前后基因的表达效率和诱导兔角膜新生血管的形成功能,为今后构建双基因或多基因高效共表达载体提供实验依据。方法:以pAdTrack-CMV-Ang-1-IRES-VEGF165质粒为模板,PCR扩增人VEGF165及Ang-1基因片段,分别将其亚克隆至改建的 pAdTrack-CMV-PolyA-promoter及pAdTrack-CMV-IRES转移质粒中,构建pTrack-CMV-Ang-1-polyA-promoter-VEGF165、pTrack-CMV- VEGF165-polyA-promoter-Ang-1、pTrack-CMV-VEGF165-IRES-Ang-1基因重组转移质粒,再与腺病毒骨架质粒pAdeasy-1在BJ5183细菌中同源重组,然后经PacI线性化后转染QBI-293A人胚肾成纤维细胞(简称293A细胞),收获腺病毒重组病毒子Ad-Ang-1-polyA-promoter-VEGF165及Ad-VEGF165-polyA-promoter-Ang-1,Ad-VEGF165-IRES-Ang-1,RT-PCR检测Ang-1和VEGF165在QBI-293A细胞中的转录,ELISA法分别检测不同腺病毒载体目的基因的表达量,比较分析Ang-1与VEGF165基因在IRES和polyA-promoter介导的不同腺病毒表达载体中的表达能力,及在同一腺病毒表达载体中前后不同位置的表达效率。并进一步于兔角膜缘注射Ad-Ang-1-polyA-promoter-VEGF165,Ad-VEGF165-polyA-promoter-Ang-1,Ad-VEGF165-IRES-Ang-1,Ad-Ang-1-IRES-VEGF165,检测角膜新生血管的面积,并比较其诱导血管形成能力的差异。结果:测序显示Ang-1和VEGF165序列正确,不同重组腺病毒载体均获得成功包装,病毒效价可达2~5×10¹⁰pfu/ml,RT-PCR检测Ang-1和VEGF165均能有效转录,ELISA法检测结果表明Ang-1、VEGF165基因不仅均能在细胞中有效表达,而且IRES介导的Ang-1及VEGF165基因,无论在IRES上游或下游,其表达量均低于polyA-promoter相同位置的Ang-1及VEGF165基因表达量,大约降低60%~70%左右,同时Ang-1/VEGF165在同一载体上、下游不同位置,其下游基因的表达量均明显低于上游基因表达量,大约降低30%~40%左右。角膜血管形成动物实验的结果表明Ad-VEGF165-PolyA-promoter-Ang-1及Ad-VEGF165-IRES-Ang-1诱导角膜新生血管形成面积和血管密度的能力相对较强,且前者比后者效果更为显著。结论:在腺病毒表达载体中,由IRES/polyA-promoter介导的Ang-1与VEGF165双基因均能在细胞中成功表达,并具血管诱导性,但polyA-promoter比IRES介导的双基因表达效率高,诱导血管形成能力强;同时两者下游基因的表达量及血管诱导性能均明显低于上游基因。     

Expression of developmentally regulated endothelial cell locus 1 was induced by tumor-derived factors including VEGF

Developmentally regulated endothelial cell locus 1 (Del1) is a new angiogenic molecules expressed specifically in early embryonic endothelial cells. We investigated the relationship between Del1 and tumor cell-derived vascular endothelial growth factor (VEGF). Dunn osteosarcoma cells and high- and low-metastatic murine sarcoma cells did not express Del1. However, the expression of Del1 was observed in these primary tumor tissues and the pulmonary metastatic tissues after subcutaneous inoculation in vivo. Every tumor cell-conditioned medium containing VEGF induced the expression of Del1 in murine lung microvascular endothelial (MLE) cells, although control MLE cells did not express Del1. The anti-mouse VEGF monoclonal antibody inhibited the induction of the Del1 expression. In addition, mouse recombinant interleukin-1alpha and tumor necrosis factor-alpha also induced Del1 in MLE cells. Del1 may play an important role in tumor angiogenesis through the effects of tumor-derived factors including VEGF.     

VEGF 164 gene transfer by electroporation improves diabetic sensory neuropathy in mice

BACKGROUND: Diabetic neuropathy is the most common cause of peripheral neuropathy and a serious complication of diabetes. Vascular endothelial growth factor (VEGF) stimulates angiogenesis and has neurotrophic and neuroprotective activities. To examine the efficiency of VEGF 164 electro-gene therapy for neuropathy, intramuscular VEGF 164 gene transfer by electroporation was performed to treat sensory neuropathy in diabetic mice. METHODS: VEGF 164 was overexpressed in the tibial anterior (TA) muscles of streptozotocin-induced diabetic mice with hypoalgesia, using a VEGF 164 plasmid injection with electroporation. From 2 weeks after electro-gene transfer, the nociceptive threshold was measured weekly using the paw-pressure test. The TA muscles, sciatic nerve, liver and spleen were histochemically examined at 4 weeks after electro-gene transfer. RESULTS: Two weeks after electro-gene transfer into the bilateral TA muscles, the elevated nociceptive threshold was decreased to a normal level in all treated mice. Improvement of the hypoalgesia continued for 14 weeks. When the VEGF 164 plasmid was injected with electroporation into a unilateral TA muscle, recovery from hypoalgesia was observed in not only the ipsilateral hindpaw, but also the contralateral one, suggesting that VEGF circulates in the blood. No increase in the number of endoneurial vessels in the sciatic nerve was found in the VEGF 164 plasmid-electroporated mice. CONCLUSIONS: These findings suggest that VEGF 164 electro-gene therapy completely recovered the sensory deficits, i.e. hypoalgesia, in the diabetic mice through mechanisms other than angiogenesis in the endoneurium of the peripheral nerve, and may be useful for treatment for diabetic sensory neuropathy in human subjects.     

早期糖尿病大鼠视网膜中血管生成素-1、血管内皮生长因子的表达

目的:研究血管生成素-1(angiopoietin-1,Ang-1)和血管内皮生长因子(vascular endothelial growth factor,VEGF)在早期糖尿病大鼠视网膜中的表达及其意义。方法:制备类似人类Ⅰ型糖尿病大鼠动物模型,取40只SD雄性大鼠分糖尿病组28只及正常对照组12只,糖尿病组28只SD雄性大鼠腹腔注射STZ65mg.Kg-1,建模成功后分别在1周、2周、3周、4周各取糖尿病组7只及正常对照组3只摘取大鼠眼球,应用免疫组化法检测Ang-1、VEGF在视网膜中的表达。结果:Ang-1、VEGF在正常大鼠视网膜的染色均为阴性,Ang-1在1、2、3周糖尿病大鼠视网膜内界膜、内核层及散在血管旁周细胞及Mǔller细胞中呈阳性表达,4周时呈阴性染色反应,Ang-1在糖尿病大鼠视网膜中表达1周阳性率约为39.5%,2周阳性率约为59.7%,3周阳性率约为78.9%,4周阳性率约为19.0%。VEGF在糖尿病大鼠视网膜中表达1周阳性率约为38.6%,2周阳性率约为54.3%,3周阳性率为78.9%,4周阳性率约为42%。结论:Ang-1、VEGF可能在糖尿病视网膜病变早期形成中起到重要...     

VEGF promotes tumorigenesis and angiogenesis of human glioblastoma stem cells

There is increasing evidence for the presence of cancer stem cells (CSCs) in malignant brain tumors, and these CSCs may play a pivotal role in tumor initiation, growth, and recurrence. Vascular endothelial growth factor (VEGF) promotes the proliferation of vascular endothelial cells (VECs) and the neurogenesis of neural stem cells. Using CSCs derived from human glioblastomas and a retrovirus expressing VEGF, we examined the effects of VEGF on the properties of CSCs in vitro and in vivo. Although VEGF did not affect the property of CSCs in vitro, the injection of mouse brains with VEGF-expressing CSCs led to the massive expansion of vascular-rich GBM, tumor-associated hemorrhage, and high morbidity, suggesting that VEGF promoted tumorigenesis via angiogenesis. These results revealed that VEGF induced the proliferation of VEC in the vascular-rich tumor environment, the so-called stem cell niche.     

Human FGF-1 gene transfer promotes the formation of collateral vessels and arterioles in ischemic muscles of hypercholesterolemic hamsters

BACKGROUND: Acidic fibroblast growth factor (FGF-1) has been identified as a potent mitogen for vascular cells, inducing formation of mature blood vessels in vitro and in vivo and represents one of the most promising approaches for the treatment of ischemic cardiovascular diseases by gene therapy. Nevertheless, and most probably due to the few experimental models able to address the issue, no study has described the therapeutic effects of FGF-1 gene transfer in subjects with peripheral arterial disease (PAD) exhibiting a clinically relevant cardiovascular pathology. METHODS: In order to assess the potency of FGF-1 gene transfer for therapeutic angiogenesis in ischemic skeletal muscles displaying decreased gene expression levels and sustained impaired formation of collateral vessels and arterioles, we developed a model of PAD in hamsters with a background of hypercholesterolemia. Hamsters fed a cholesterol-rich diet and subjected to hindlimb ischemia exhibit a sustained impaired angiogenic response, as evidenced by decreased angiographic score and histological quantification of arterioles in the ischemic muscles. RESULTS: In this model, we demonstrate that NV1FGF (a human FGF-1 expression plasmid), given intramuscularly 14 days after induction of hindlimb ischemia, promoted the formation of both collateral vessels and arterioles 14 days after treatment (i.e. 28 days post-ischemia). CONCLUSIONS: Our data provide evidence that NV1FGF can reverse the cholesterol-induced impairment of revascularization in a hamster model of hindlimb ischemia by promoting the growth of both collateral vessels and arterioles in ischemic muscles exhibiting significantly decreased levels of gene expression compared with control muscles. Therefore, this study underscores the relevance of NV1FGF gene therapy to overcome perfusion defects in patients with PAD.     

稽留流产患者血清和绒毛中HIF-1alpha和VEGF表达水平及其相关性分析

目的：探讨缺氧诱导因子-1alpha（HIF-1alpha）和血管内皮生长因子（VEGF）在稽留流产患者血清和绒毛中的表达水平及其相关性 分析。方法：选择2014 年8 月至2015 年8 月我院妇产科76 例稽留流产患者为观察组及行人工流产的60 例正常早产孕妇为对 照组;根据患者稽留流产时间将稽留流产患者分为稽留时间<2 周组（12 例）,2~4周组（33 例）,>4周组（31 例）;采用酶联免疫吸 附（ELISA）和免疫组化SP 法检测并分析患者血清与绒毛中HIF-1琢与VEGF的表达水平。结果：观察组血清中HIF-1alpha与VEGF 表达水平均显著低于对照组,差异有统计学意义（P<0.05）;观察组和对照组绒毛VEGF 和HIF-1-alpha均表达,其中VEGF 主要表达 于滋养层细胞细胞质和细胞间质,HIF-1琢主要表达于滋养层细胞的细胞核与细胞质,且观察组患者绒毛HIF-1-alpha与VEGF 表达水 平均显著低于对照组,差异有统计学意义（P<0.05）;不同稽留时间患者绒毛HIF--alpha与VEGF表达水平间比较,差异均无统计学意 义（P>0.05）观察组患者血清HIF-1alpha与VEGF的表达水平呈现正相关关系（r=0.601;P<0.05）;且绒毛HIF-1琢与VEGF的表达水平 也呈现正相关关系（r=0.401;P<0.05）。结论：HIF-1琢和VEGF在血清和绒毛中的低表达可能是稽留流产的发生的重要原因,临床 上可以通过检测患者血清和绒毛组织中HIF-1琢和VEGF的水平,有针对性的对患者进行监护和治疗,预防稽留流产的发生。     

Smad3 mediates TGF-beta1 induction of VEGF production in lung fibroblasts

Transforming growth factor-beta1 (TGF-beta1) is a key factor in a variety of physiological and pathological processes. Vascular endothelial growth factor (VEGF) is a key angiogenic factor, and vascular change is one of the features of airway remodeling. We examined the effect of TGF-beta1 on VEGF production by fibroblasts from mice lacking expression of Smad2 or Smad3 as well as human lung fibroblasts treated with or without Smad2 or Smad3 siRNA. TGF-beta1 stimulated VEGF production by fibroblasts from Smad2 deficient animals and wildtype animals. In contrast, TGF-beta1 did not affect VEGF production by fibroblasts from Samd3 deficient mice. Similarly, TGF-beta1 failed to stimulate VEGF production by HFL-1 cells treated with Samd3 siRNA but significantly increased VEGF production by the cells treated with Smad2 siRNA. These result suggest that TGF-beta1 stimulation of VEGF production by fibroblasts is regulated by Smad3 but not by Smad2 signaling.     

内蒙古白绒山羊VEGF164基因cDNA克隆及组织表达特异性分析

旨在克隆内蒙古白绒山羊血管内皮生长因子(vascular endothelial growth factor,VEGF164)基因并分析其基本表达模式。采用RT-PCR技术克隆基因,将得到的基因cDNA序列及其编码的氨基酸序列进行生物信息学分析。利用半定量RT-PCR方法进行组织表达检测。获得了内蒙古白绒山羊VEGF164基因编码区cDNA全长序列,扩增片段全长573 bp,包含了完整的ORF,编码190个氨基酸残基。核苷酸序列与绵羊的VEGF164(EU857623.1)基因同源性为99%,相应的氨基酸序列同源性为99%。SMART程序分析表明,ORF编码的蛋白质具有信号肽序列及血小板衍生和血管内皮生长因子家族(PDGF,VEGF)结构域。Psite程序分析表明,有1个蛋白激酶C磷酸化位点,4个酪蛋白激酶磷酸化位点。ProtComp Version 9.0程序分析将其定位于细胞外。RT-PCR检测表明,VEGF164基因在绒山羊脑、心脏、睾丸、胰腺、脾、肾和肺组织中均有表达。     

Whole-body hyperthermia induces up-regulation of vascular endothelial growth factor accompanied by neovascularization in cardiac tissue

Whole-body hyperthermia (WBH) promotes cardiac protection against ischemia/reperfusion injury, in part by up-regulation of heat shock proteins (HSP). Whether heat stress also promotes up-regulation of angiogenic factors or induces endothelial cell proliferation is unknown. We studied the effects of heat stress on up-regulation of vascular endothelial growth factor (VEGF) and growth of new blood vessels following WBH. Anesthetized rats were subjected to WBH at 42 degrees C for 15 min. The control (n=23) and heated (n=55) groups were allowed to recover for 4, 12, 24, 48, or 72 h prior to harvesting the heart for Western Blot and immunohistochemical assessment of VEGF, HSP70, and platelet endothelial cell adhesion molecular-1 (PECAM-1). A significant increase in VEGF and HSP70 expression was observed as early as 4 h post-heating. The Western Blot analysis revealed a close temporal correlation between up-regulation of HSP70 and VEGF. Maximum VEGF and HSP70 expression occurred at 12 and 24 h post-heating in the left and right ventricles, respectively. The right ventricle showed the greatest expression of both VEGF and HSP70. Immunostaining revealed that VEGF was focally increased in the endothelial cells of capillaries, small arteries, and in interstitium. At 48 and 72 h post-heating, multiple areas of extensive capillary proliferation occurred in the epicardial region of the right ventricle. These observations were verified by quantitative analysis of the density of blood vessels as determined by PECAM-1 staining. Our experiments show that sublethal heat stress can lead to upregulation of both VEGF and HSP70 in cardiac tissue and promote focal endothelial proliferation in the heart.     

康柏西普在糖尿病大鼠早期视网膜病变中的作用及对VEGF、ICAM-1及CRP的影响

摘要 目的：探讨康柏西普在糖尿病大鼠早期视网膜病变中的作用及对血管内皮生长因子 (vascular endothelial growth factor,VEGF)和细胞间粘附分子-1(Intercellular adhesion molecule-1,ICAM-1)及C-反应蛋白(C-reactive protein,CRP)的影响。方法：糖尿病大鼠早期视网膜病变模型(n=27)随机平分为三组-模型组、替米沙坦组与康柏西普组,造模成功后当天三组分别给予注射生理盐水、替米沙坦、康柏西普治疗,1次/w,持续4 w,检测VEGF、ICAM-1及CRP表达情况。结果：大鼠造模成功后均出现食欲增多、饮水、尿量、体重减轻的现象。替米沙坦组与康柏西普组治疗第1 w与第4 w的体重高于模型组(P<0.05),康柏西普组高于替米沙坦组(P<0.05)。替米沙坦组与康柏西普组治疗第1 w与第4 w的空腹血糖低于模型组(P<0.05),康柏西普组低于替米沙坦组(P<0.05)。替米沙坦组与康柏西普组治疗第4 w的视网膜VEGF、ICAM-1、CRP蛋白相对表达水平低于模型组(P<0.05),康柏西普组低于替米沙坦组(P<0.05)。康柏西普组视网膜厚度变薄不明显,内、外核层细胞排列整齐,神经纤维层未见明显空泡样变性。结论：康柏西普在糖尿病大鼠早期视网膜病变中的应用能抑制VEGF、ICAM-1及CRP的表达,能促进降低血糖,增加大鼠体重。     

可溶性VEGF受体Flt-1的基因克隆及其活性产物在原核中的表达

可溶性血管内皮细胞生长因子受体 ( Flt- 1 )具有受体拮抗剂作用 ,可竞争结合血管内皮细胞生长因子 ,( VEGF)并与其膜表面受体 Flt- 1及 KDR形成异源二聚体 ,最终阻断 VEGF的生物学活性 .利用 RT- PCR技术从人脐静脉内皮细胞扩增出 Flt- 1胞外 ～ 区 c DNA片段 ,通过基因重组将该片段克隆于谷胱甘肽转移酶 ( GST)融合蛋白表达载体 PGEX2 -T中 ,连接产物转化大肠杆菌XL1 - blue,经 IPTG诱导可获大量稳定表达的 Flt- 1 - GST融合蛋白 .该表达产物经变性复性处理后 ,可特异性结合 12 5 - VEGF.大量具有活性的可溶性 Flt- 1的获得有助于新的抗肿瘤血管形成方法的探索 .