超声微泡介导hVEGF165 基因靶向转染糖尿病鼠缺血骨骼肌

目的：探讨超声介导微泡破裂法促进血管内皮生长因子（VEGF）基因在糖尿病鼠缺血骨骼肌内转染的作用,评估其转染效 率和安全性。方法：建立糖尿病鼠缺血骨骼肌动物模型,以绿色荧光蛋白基因为报告基因, 观察接受超声及微泡治疗组hVEGF165 基因在糖尿病鼠缺血骨骼肌内表达,并与对照组相比。同时取糖尿病鼠缺血骨骼肌进行HE染色行组织学检查。结果：在超声介导 微泡破裂组内,hVEGF165 基因表达明显增强(42.87± 5.12),与单纯接受质粒治疗组(5.02± 1.21)和接受质粒和超声治疗组（8.16± 2.43）相比,差异具有统计学意义（P<0.001）,HE 切片未发现肌组织结构的改变。结论：超声介导微泡破裂法能有效促进外源基因 在糖尿病鼠缺血骨骼肌中表达, 为糖尿病周围血管疾病的基因治疗提供了实验依据。     

缺氧诱导血管新生机制的研究新进展

缺氧是机体在缺血、肿瘤等病理因素或高原环境下局部或整体出现的一种内环境状态。大量的研究表明,缺氧可以诱导相应组织血管新生。近年来,我们已逐渐认识到,缺氧可以通过调节几种生长因子的表达以及炎症介质的直接或间接作用促进血管的新生,其中又以血管内皮生长因子(VEGF)及其受体的研究较为深人。对于这些机制的研究,为临床治疗缺血或肿瘤性疾病以及研究高原习服等问题提供了有价值的参考。     

小干扰RNA靶向VEGF基因体内外抑制乳腺癌细胞MCF-7的增殖

 血管生成与肿瘤生长、侵袭、转移密切相关.血管内皮生长因子能特异地促进内皮细胞分裂、增殖及迁移,在肿瘤新生血管生成过程中起着至关重要的作用.通过RNAi抑制VEGF表达的抗血管生成疗法可有效应用于肿瘤治疗.本研究采用化学修饰的siRNA在体内外抑制VEGF基因表达,探讨化学修饰的siRNA介导的RNA干扰技术在乳腺癌基因治疗的可行性和特异性.选用阳离子脂质体Lipofectamine^TM2000作为转染试剂,将针对人VEGF基因的小干扰RNA(small interfering RNA,siRNA)转染人类乳腺细胞株MCF-7和裸鼠移植瘤,在体内外诱导RNAi.采用四甲基偶氮唑蓝（MTT）法,逆转录聚合酶链反应(RT-PCR),蛋白印迹实验等检测siRNA治疗组和对照组VEGF基因表达及细胞增殖变化.体外实验结果显示：靶向VEGF基因siRNA转染乳腺癌MCF-7细胞后,细胞生长抑制率达52.5%;VEGF的mRNA和蛋白表达水平显著降低（P<0.01）;裸鼠体内实验结果显示：siRNA治疗组瘤组织的增长受到明显抑制;RT-PCR结果同时表明治疗组VEGF表达下调.体内外对照组各指标无显著变化.化学修饰的siRNA介导的RNAi在体内外均能成功下调靶基因VEGF的表达,抑制MCF-7细胞增殖,是潜在的肿瘤治疗新方法.     

Ephrin-B2促进大鼠局灶脑缺血再灌注后VEGF表达及血管新生

目的:探讨Ephrin-B2对大鼠脑缺血再灌注后脑组织中血管新生的调节作用及其可能的机制。方法:雄性SD大鼠随机分为正常组及、缺血再灌注组及Ephrin-B2干预组,后两组再分为4天、7天、14天、28天亚组;线栓法制备局灶性大脑中动脉缺血再灌注模型;改良神经功能评分(modified neurological severity scores mNSS)评分法对各时间点模型进行评分;Western blot及荧光定量PCR检测缺血脑组织中血管内皮生长因子(Vascular Endothelial Growth Factor VEGF)的表达;以免疫荧光双标法定位VEGF表达的细胞类型;以CD31+BrdU计数缺血半暗带中新生微血管密度(microvessel densityMVD)。结果:Ephrin-B2干预组与缺血再灌注组各时间点亚组比较,新生微血管密度测定计数较缺血再灌注组均显著增加(P0.05),神经功能评分均显著降低(P0.05),VEGF mRNA水平及蛋白表达水平均显著增加(P0.05),VEGF主要表达于CD31阳性的血管内皮细胞。结论:Ephrin-B2通过上调VEGF的表达促进脑缺血再灌注后缺血半暗带血管新生,从而促进神经功能缺失的修复。     

shRNA下调MTDH基因抑制人乳腺癌MCF-7细胞HIF-1α及VEGF表达

目的：探讨转移粘附基因（metadherin,MTDH）的表达对人乳腺癌细胞中肿瘤血管生成相关分子标志物缺氧诱导因子-1α（HIF-1α）及血管内皮生长因子（VEGF）表达的影响。方法：将针对MTDH基因的干扰质粒MTDH-shRNA转染乳腺癌MCF-7细胞,RT-PCR及Western blot验证其对MTDH基因的沉默效果;应用Western blot检测转染前后MCF-7细胞中缺氧诱导因子-1α（HIF-1α）及血管内皮生长因子（VEGF）在蛋白水平上的表达变化;MTT实验检测下调MTDH对MCF-7细胞增殖情况的影响。结果：MCF-7细胞转染48小时后,MTDH-shRNA转染组和MTDH-shRNA-neg转染组转染效率约70%。MTDH-shRNA转染组中MTDH在mRNA及蛋白水平上表达明显下调,此外HIF-1α及VEGF蛋白表达明显降低,与对照组比较差异有统计学意义（P〈0.05）。MTDH-shRNA转染组MCF-7细胞增殖明显受到抑制,与对照组比较差异有统计学意义（P〈0.05）。结论：在乳腺癌MCF-7细胞中下调MTDH基因可以抑制HIF-1α、VEGF表达及细胞增殖,提示MTDH基因可能对乳腺癌肿瘤血管生成有促进作用。     

超声破裂载基因微泡增强心肌细胞报告基因的转染与表达

目的:通过超声破裂载基因微泡介导报告基因心肌细胞转染,探讨其能否增强心肌细胞外源基因转染与表达.方法:以β-galactosidase质粒为报告基因,将其与自制氟碳气体微泡粘附,制备载基因微泡.利用诊断性超声破裂微泡进行体外心肌细胞基因转染;以磷酸钙共沉淀转染为阳性对照并将其以不同方式与超声破裂微泡技术联合应用,以期进一步增强基因转染效果.分别采用原位染色及酶学定量检测β-galactosidase表达水平,同时进行细胞活性检测.结果:超声破裂载基因氟碳气体微泡(PESDA)转染组心肌细胞β-galactosidase表达水平可达单纯质粒转染组60倍(P＜0.01).磷酸钙共沉淀转染3.67倍(P＜0.01)超声强度、微泡浓度对超声破裂介导基因转染效果有明显影响.超声破裂微泡技术与磷酸钙共沉淀联合应用可进一步提高报告基因的表达(P＜0.05),即使在磷酸钙转染后6 h,超声破裂微泡仍能明显增强报告的基因的表达(P＜0 05).结论:超声破裂微泡技术是一种高效基因转染方法,其不但能增加DNA转染,而且增强入胞后基因的表达.超声破裂微泡与其它基因转染技术联合应用能进一步增加基因转染效率.     

某些肿瘤组织中血管内皮生长因子基因的表达

血管内皮生长因子(vascular endothelial growth factors,VEGF)是新发现的生长因子,特异作用于血管内皮细胞,促进其增殖及新生血管的生成.已确认,它和实体肿瘤的生长有着十分密切的关系.文章报道,利用力子杂交技术分析了胃癌、肾癌、结肠癌和膀胱癌及其癌旁相应组织中VEGF mRNA的表达,结果发现,癌组织较其癌旁组织中vEGF mRNA的表达增高.SGC-7901细胞加TPA 4h后则明显促进VEGF mRNA的表达.转染含人反义N-ras₁ DNA片段重组质粒的人膀胱癌BIU-87细胞系可抑制VEGFmRNA表达.     

血管内皮细胞生长因子在非血管新生方面的重要功能

血管内皮细胞生长因子(vascular endothelial growth factor,VEGF或VEGF-A),又称为血管通透因子(vascular permeable factor,VPF)是一种具有多种功能的生物大分子,它是分泌性糖蛋白生长因子超家族中的一员.VEGF主要通过两个高亲和力的酪氨酸激酶受体来传递各种信号:VEGF受体1和2(VEGFR1,VEGFR2),从而引起细胞的多种生理反应.在胚胎时期,VEGF可以促进血管内皮细胞的增殖、迁移、管状形成和提高内皮细胞的存活率,对于血管新生和发育十分关键;而在成体时期,VEGF则主要参与正常血管结构的维持,并调节生理和病理性血管新生.近几年来的临床试验表明,使用多种阻断VEGF作用的抑制剂能有效促进肿瘤血管的退化和减小肿瘤的体积,但是同时在部分病人中也观察到了多方面的副作用.这些结果显示,VEGF也具有非血管新生方面的重要功能.因此,在研制基于拮抗VEGF作用的抗癌药物时,这些功能更不容忽视.研究表明,在成体的小肠、胰岛、甲状腺、肾脏和肝脏等器官组织中,VEGF都发挥着十分重要的作用,如果VEGF水平降低,这些器官组织的毛细血管网状结构将部分退化.VEGF还可以促进骨髓形成、组织修复与再生、促进卵巢囊泡成熟,并且参与血栓、炎症反应和缺氧缺血的病理过程.本文主要对VEGF在血管新生之外的功能及其分子机制进行了简要探讨.     

阻断VEGF旁分泌通路抑制乳腺癌血管生成与肿瘤生长

以人乳腺癌细胞株MCF 7为研究对象 ,通过构建有义与反义血管内皮生长因子 (VEGF)基因表达质粒 ,并转染MCF 7细胞 ,建立了高与低水平表达VEGF的细胞克隆。稳定转染反义VEGF表达质粒的细胞产生和分泌VEGF的能力明显下降 ,尽管在体外培养条件下细胞的增殖速度与未经转染的对照相比不是减慢而是略有增快 ,但在体内的成瘤能力、生长速度和转移能力等却明显低于未经转染的对照细胞或稳定转染有义VEGF表达质粒高水平表达VEGF的细胞克隆。通过体内电穿孔技术介导反义VEGF12 1及可溶性VEGF受体sFlk 1表达质粒转移至荷瘤鼠肿瘤组织内 ,反义VEGF12 1及sFlk 1的表达能显著抑制肿瘤的生长。研究结果证实了VEGF旁分泌通路在诱导乳腺癌肿瘤血管生成、促进肿瘤生长和转移方面起重要作用 ,阻断VEGF旁分泌通路能有效抑制乳腺癌的生长     

血管内皮生长因子家族及其受体与肿瘤血管生成研究进展

血管内皮生长因子(vascular endothelial growth factor,VEGF),又名血管通透性因子(vascular permeability factor,VPF)是重要的血管生成正性调节因子,是目前抗癌治疗的研究靶点之一。现已发现的VEGF家族成员包括VEGF—A、VEGF—B、VEGF—C、VEGF—D、VEGF—E和胎盘生长因子(placenta growth factor,PLGF)。VEGF的受体有VEGFR—1(fit—1)、VEGFR-2(flk-1／KDR)、VEGFR-3(fit-4)、neuropilin(NPR1／NPR2)。该家族的成员可以选择性地增强血管和／或淋巴管内皮细胞的有丝分裂,刺激内皮细胞增殖并促进血管生成,提高血管特别是微小血管的通透性,使血浆大分子外渗沉积在血管外的基质中,促进新生毛细血管网的建立,为肿瘤细胞的生长提供营养等。作者对VEGF家族成员及其受体的理化特征、VEGF与肿瘤的关系、VEGF抑制剂的研制作一综述。     

Taming of the wild vessel: promoting vessel stabilization for safe therapeutic angiogenesis

VEGF (vascular endothelial growth factor) is the master regulator of blood vessel growth. However, it displayed substantial limitations when delivered as a single gene to restore blood flow in ischaemic conditions. Indeed, uncontrolled VEGF expression can easily induce aberrant vascular structures, and short-term expression leads to unstable vessels. Targeting the second stage of the angiogenic process, i.e. vascular maturation, is an attractive strategy to induce stable and functional vessels for therapeutic angiogenesis. The present review discusses the limitations of VEGF-based gene therapy, briefly summarizes the current knowledge of the molecular and cellular regulation of vascular maturation, and describes recent pre-clinical evidence on how the maturation stage could be targeted to achieve therapeutic angiogenesis.     

Regulatory molecules for coronary expressions of VEGF and its angiogenic receptor KDR in hypoestrogenic middle-aged female rats

We studied the effects of estrogen deprivation and replacement on the protein and gene expression levels molecules that can be considered to be essential for coronary angiogenesis in middle-aged female rats. The animals were subjected to sham operation, ovariectomy, or ovariectomy with estrogen replacement therapy (ERT). Following ovariectomy, protein and gene expressions of vascular endothelial growth factor (VEGF) and its angiogenic receptor (KDR) showed a marked decline in coronary vessels, as determined by immunohistochemistry and in situ hybridization. ERT resulted in restoration of the ovariectomy-induced changes to intact levels. The coronary expression level of basic fibroblast growth factor was unaffected by estrogen deprivation or treatment. The changes in VEGF and KDR expressions were strongly associated with those in endothelial nitric oxide synthase (eNOS) expression in coronary vessels. Moreover, the age- and gender-dependent accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) protein appeared to be a determinant molecule of VEGF expression in middle-aged female rats. We reached a conclusion that the VEGF-KDR system plays a key role in coronary angiogenesis in hypoestrogenic elderly women and is critically regulated by estrogen, eNOS and HIF-1alpha.     

EphrinB2/EphB4 signaling regulates non‐sprouting angiogenesis by VEGF

Vascular endothelial growth factor (VEGF) is the master regulator of angiogenesis, whose best‐understood mechanism is sprouting. However, therapeutic VEGF delivery to ischemic muscle induces angiogenesis by the alternative process of intussusception, or vascular splitting, whose molecular regulation is essentially unknown. Here, we identify ephrinB2/EphB4 signaling as a key regulator of intussusceptive angiogenesis and its outcome under therapeutically relevant conditions. EphB4 signaling fine‐tunes the degree of endothelial proliferation induced by specific VEGF doses during the initial stage of circumferential enlargement of vessels, thereby limiting their size and subsequently enabling successful splitting into normal capillary networks. Mechanistically, EphB4 neither inhibits VEGF‐R2 activation by VEGF nor its internalization, but it modulates VEGF‐R2 downstream signaling through phospho‐ERK1/2. In vivo inhibitor experiments show that ERK1/2 activity is required for EphB4 regulation of VEGF‐induced intussusceptive angiogenesis. Lastly, after clinically relevant VEGF gene delivery with adenoviral vectors, pharmacological stimulation of EphB4 normalizes dysfunctional vascular growth in both normoxic and ischemic muscle. These results identify EphB4 as a druggable target to modulate the outcome of VEGF gene delivery and support further investigation of its therapeutic potential.     

Plastic surgical perspectives on vascular endothelial growth factor as gene therapy for angiogenesis

The practice of plastic surgery has always remained at the frontier of medical science. Over the past few decades, this frontier has been marked by significant developments in the field of gene therapy. Gene therapy serves to replace, supplement, or manipulate a patient's genetic makeup to restore function that has been lost or to correct function that is aberrant. Recent technology may allow surgeons to augment the processes of wound healing and angiogenesis by transfecting genes encoding desirable proteins, such as vascular endothelial factor (VEGF), into ischemic tissues. VEGF is a vital growth factor in the development of blood vessels. Although its mechanisms of action are numerous, its sole function seems to be the augmentation of angiogenesis. VEGF is active in growth and development, in wound healing, and in various pathologic conditions, such as psoriasis and rheumatoid arthritis. The role of VEGF in the field of plastic surgery is just beginning to be explored; it may someday prove to be very rewarding.     

血管内皮生长因子基因治疗大鼠脑缺血的实验研究

为探讨血管内皮生长因子(VEGF)基因治疗大鼠脑缺血的可行性,构建了pcD2/hVEGF121真核表达质粒,建立持续性大脑中动脉堵塞(MCAO)的局灶性脑梗塞模型,大鼠脑皮质直接注射法转移pcD2/hVEGF121真核表达质粒。应用逆转录聚合酶链反应(RT-PCR)、VEGF免疫组织化学法、脑血管计数及梗塞面积测定等方法检测转移pcD2/hVEGF121真核表达质粒后大鼠脑中VEGF基因表达及生物学效应。结果发现,与转移空载质粒的对照组相比,转移VEGF基因后7d的大鼠脑组织中有VEGFmRNA高表达,VEGF免疫组化染色可见VEGF蛋白表达水平增高,脑血管数增多,梗塞体积缩小。因此,直接注射法转移VEGF基因能够在缺血脑组织中表达,表达产物能够发挥生物学效应,进而起到保护脑组织作用。     

Favorable effect of VEGF gene transfer on ischemic peripheral neuropathy

Ischemic peripheral neuropathy is a frequent, irreversible complication of lower extremity vascular insufficiency. We investigated whether ischemic peripheral neuropathy could be prevented and/or reversed by gene transfer of an endothelial cell mitogen designed to promote therapeutic angiogenesis. Intramuscular gene transfer of naked DNA encoding vascular endothelial growth factor (VEGF) simultaneously with induction of hindlimb ischemia in rabbits abrogated the substantial decrease in motor and sensory nerve parameters, and nerve function recovered promptly. When gene transfer was administered 10 days after induction of ischemia, nerve function was restored earlier and/or recovered faster than in untreated rabbits. These findings are due in part to enhanced hindlimb perfusion. In addition, however, the demonstration of functional VEGF receptor expression by Schwann cells indicates a direct effect of VEGF on neural integrity as well. These findings thus constitute a new paradigm for the treatment of ischemic peripheral neuropathy.     

Therapeutic angiogenesis with vascular endothelial growth factor in peripheral and coronary artery disease: a review

Therapeutic angiogenesis constitutes an alternative treatment for patients with extensive tissue ischaemia in whom primary vascular reconstruction procedures are not feasible or have previously failed. At present vascular endothelial growth factor (VEGF) has been the most widely used angiogenic factor in experimental and human clinical trials. Early clinical data provide evidence that gene transfer of the VEGF gene can achieve beneficial angiogenesis, with minimal side-effects. Ongoing phase III clinical studies will reveal definitive efficacy.     

Activation of the metabolic sensor-AMP activated protein kinase reverses impairment of angiogenesis in aging myocardial microvascular endothelial cells. Implications for the aging heart

Impairment of angiogenesis - new capillary blood vessel formation from pre-existing vessels, is frequent in aging tissues and cells. Reduced angiogenesis in aging individuals is associated with increased incidence of myocardial infarctions and other cardiovascular diseases. Therefore there is a need to develop novel strategies to enhance angiogenesis in aging individuals. Our previous study demonstrated aging-related impairment of angiogenesis in aging (vs. young) rat myocardial microvascular endothelial cells (MMEC), and identified reduced activation of the vascular endothelial growth factor (VEGF, the most potent stimulator of angiogenesis) gene as the main underlying mechanism. In the present study we examined the possibility of increasing angiogenesis and activating VEGF gene expression in aging MMECs using a chemical activator of the metabolic sensor - AMP activated protein kinase (AMPK). We hypothesized that activation of VEGF gene in aging MMECs by AMPK would stimulate angiogenesis and reverse the impairment in angiogenesis seen in these cells. We used MMECs isolated from aging (24 months old) Fisher F-344 rats and treated them with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), a specific pharmacological stimulator of AMPK. We examined: 1) in vitro angiogenesis; and 2) the expression of phosphorylated AMPK, VEGF, and P-MAPK/Erk1/2. Treatment of aging MMECs with AICAR increased in vitro angiogenesis and VEGF mRNA expression by 2.1-fold and 3.7-fold, respectively. Furthermore, AICAR treatment resulted in phosphorylation of MAPK/Erk1/2. This study demonstrated the successful use AICAR to reverse aging-related impairment of angiogenesis in aging MMECs by enhancing VEGF gene expression and also identified phosphorylation of MAPK/Erk1/2 as a likely mechanism of these changes.     

Combined administration of naked DNA vectors encoding VEGF and bFGF enhances tissue perfusion and arteriogenesis in ischemic hindlimb

Few studies have examined in detail the combined effects of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) gene delivery on collateral development. Here, we evaluated the potential synergism of naked DNA vectors encoding VEGF and bFGF using a skeletal-muscle based ex vivo angiogenesis assay and compared tissue perfusion and limb loss in a murine model of hindlimb ischemia. In the ex vivo angiogenesis assay, the VEGF+bFGF combination group had a larger capillary sprouting area than those of the LacZ, VEGF, and bFGF groups. Consistent with these results, regional blood flow recovery on day 14 was also highest in the VEGF+bFGF combination group, followed by the bFGF, VEGF, and LacZ groups. The limb loss frequency was 0% in the combination group, whereas the limb loss frequencies of the other groups were 7-29%. The ischemic muscles of the combination group revealed evidence of increased angiogenesis and arteriogenesis and the upregulated expression of genes that may be associated with arteriogenesis, such as those for cardiac ankyrin repeat protein, early growth response factor-1, and transforming growth factor-beta1. Our study has implications for the development of a combined gene therapy for the vascular occlusive diseases.     

IGFBP‐4 enhances VEGF‐induced angiogenesis in a mouse model of myocardial infarction

Vascular endothelial growth factor (VEGF) is a well‐known angiogenic factor, however its ability in promoting therapeutic angiogenesis following myocardial infarction (MI) is limited. Here, we aimed to investigate whether dual treatment with insulin‐like growth factor binding protein‐4 (IGFBP‐4), an agent that protects against early oxidative damage, can be effective in enhancing the therapeutic effect of VEGF following MI. Combined treatment with IGFBP‐4 enhanced VEGF‐induced angiogenesis and prevented cell damage via enhancing the expression of a key angiogenic factor angiopoietin‐1. Dual treatment with the two agents synergistically decreased cardiac fibrosis markers collagen‐I and collagen‐III following MI. Importantly, while the protective action of IGFBP‐4 occurs at an early stage of ischemic injury, the action of VEGF occurs at a later stage, at the onset angiogenesis. Our findings demonstrate that VEGF treatment alone is often not enough to protect against oxidative stress and promote post‐ischemic angiogenesis, whereas the combined treatment with IGFBP4 and VEGF can utilize the dual roles of these agents to effectively protect against ischemic and oxidative injury, and promote angiogenesis. These findings provide important insights into the roles of these agents in the clinical setting, and suggest new strategies in the treatment of ischemic heart disease.