同时携带人血管内皮生长因子和血管生成素基因的内皮祖细胞血管再生研究

目的用腺相关病毒（adeno-associated virus,AAV）载体将人血管内皮生长因子165 （vascular endothelial growth factors,VEGF165）和血管生成素-1（angiopoietin-1,Angl）基因同时转入骨髓源性内皮祖细胞（endothelial progenitor cells,EPCs）中,观察外源基因的表达并评价其促血管再生的能力。方法用同时携带人VEGF165和Angl基因的AAV载体AAV2-hAngl/VEGF165转染体外培养的兔骨髓源EPCs,用RT-PCR和细胞免疫化学法检测外源基因的表达。实验用新西兰大白兔24只,制作成右下肢缺血模型,随机分成3组：PBS、EPC和EPC/AV组。造模后第10天,向缺血肌组织中分别注射PBS、EPCs和AAV2-hAngl/VEGF165转染的EPCs,用免疫组织化学法评价其血管再生效应。结果EPCs可高效地被AAV2-hAngl/VEGF165转染而表达Angl和VEGF165。细胞移植4周后,EPC/AV组存活的EPCs密度（283±137）/mm^-2明显高于EPC组（191±88）/mm^-2;EPC/AV组的毛细血管密度（856±212）/mm^-2明显高于EPC组（564±177/mm^-2）和PBS组（308±112）/mm^-2;α-SMA阳性血管密度（6.9±3.0）/mm^-2明显高于PBS组（3.6±1.8）/mm^-2,P〈0.05。结论AAV载体可同时将人VEGF165和Angl基因转入EPCs中,转染后的EPCs具有更强的促血管再生能力。     

血管内皮细胞生长因子基因转染血管内皮祖细胞移植提高移植脂肪存活率的实验研究

目的探讨血管内皮细胞生长因子165(VEGF165)基因转染血管内皮祖细胞(EPCs)移植促进游离移植的脂肪组织的血管新生,提高移植脂肪组织存活率。方法体外分离、培养人脐血中EPCs,利用脂质体介导VEGF165基因体外转染EPCs,然后与来自人体的脂肪组织混合移植于裸鼠背部,裸鼠随机分为3组:VEGF165基因转染组、EPCs组及M199培养基对照组。结果脐血中分离培养的EPCs表达CD34、血管内皮细胞生长因子受体及CD133;VEGF165基因转染EPCs体外及体内检测均有VEGF165蛋白的表达。VEGF165基因转染组、EPCs组中,EPCs整合到缺血部位新生血管中,与对照组的脂肪组织存活率分别为(96.2±8.6)%、(75.3±6.8)%和(40.2±2.5)%(P<0.05),VEGF165基因转染组与EPCs组脂肪组织周边区毛细血管密度有显著差异(P<0.05),均高于对照组(P<0.05)。术后3个月时3组脂肪组织周边区的EPCs密度分别为(196±16)个/mm2、(95±11)个/mm2、0个/mm2(P<0.05)。结论脐血中的EPCs体外培养后移植体内可促进游离移植的脂肪组织的血管新生,提高存活率,而转染VEGF165基因的EPCs具有更强的促血管新生的作用。     

血管内皮生长因子转染内皮祖细胞治疗大鼠缺血后肢的研究

目的 使用血管内皮生长因子(VEGF)转染内皮祖细胞(EPC)治疗大鼠缺血后肢,观察EPC、VEGF转染EPC对大鼠缺血后肢的新生血管和肢体成活的影响.方法 制作SD大鼠后肢缺血模型,将动物随机分为3组,每组6只.将构建的VEGF基因真核表达载体转染入骨髓来源的EPCs后通过尾静脉注射人大鼠体内,并与使用磷酸盐缓冲液(PBS)或EPC的动物进行比较,观察转染VEGF的EPCs在缺血部位的聚集和形成新生血管的情况.结果 (1)动物总残肢率比较,CELL组、VEGF组较PBS组明显增加的肢体恢复率(P<0.05),CELL组肢体恢复率较VEGF组差(P<0.05).(2)毛细血管密度与PBS组比较,各时间点中CELL、VEGF组MVD均明显增多(P<0.05).(3)缺血肢体VEGFa的表达:VEGF组的VEGF蛋白表达较PBS组、CELL组、明显增多(P<0.05);(4)手术后7、14、28 d,与PBS对照组比较,CELL、VEGF组细胞的血流灌注有较大程度的恢复(P<0.01).结论 VEGFa基因转染EPCs对缺血部位的血管新生有重要影响,联合应用VEGFa基因和EPCs治疗缺血后肢有较好的协同作用.     

转基因的骨髓间充质干细胞治疗大鼠缺血皮瓣

目的探讨转染血管内皮细胞生长因子(VEGF)基因的大鼠骨髓间充质干细胞(MSCs)同种异体移植促进缺血皮瓣的血管新生,从而提高皮瓣存活率的可能性。方法体外分离、培养、鉴定SD大鼠MSCs,PcDNA3.1(-)/VEGF165质粒转染MSCs,免疫荧光方法检测MSCs体外表达VEGF的情况,CM-DiI标记MSCs。SD大鼠随机分3组:A组[PcDNA3.1(-)/VEGF165质粒转染的MSCs移植]、B组(单纯MSCs移植)、C组(DMEM-F12培养基)。每只大鼠背侧皮下按组分别注射细胞悬液和培养基,注射后ELISA法连续检测大鼠血浆VEGF浓度,注射后第4天掀起1个蒂在尾侧的9 cm×2 cm的随意皮瓣。在术后第14天分别观察皮瓣的存活率、激光多普勒血液监测仪监测血流灌注、CD34免疫组织化学检测皮瓣毛细血管密度、荧光显微镜检测MSCs在皮瓣内的分布和存活状况。结果转染VEGF165基因的MSCs体外和体内检测均高表达VEGF165蛋白。A、B、C三组的皮瓣存活率分别为(83.1±2.6)%、(66.4±6.1)%、(51.5±7.5)%(P< 0.05);A、B、C三组的毛细血管密度(条/mm2)分别为:89.2±6.1、57.1±4.7、28.7±2.8(P< 0.05);血流灌注比值A组高于B、C两组,B组高于C组(P<0.05);转染VEGF165基因的MSCs移植SD大鼠皮瓣后,MSCs存活并参与血管新生。结论转染VEGF基因的大鼠MSCs体外培养后异体移植可促进缺血皮瓣的血管新生,提高存活率。     

人VEGF165基因转染人外周血内皮祖细胞的实验研究

目的 探讨人血管内皮细胞生长因子165(hVEGF165)基因转染对人外周血内皮祖细胞(EPCs)的影响.方法 体外分离、培养、鉴定人外周血EPCs.实验分脂质体介导pcDNA3.1-hVEGF165质粒转染组,pcDNA3.1空质粒转染组,窄白对照组.ELISA法和硝酸还原酶法分别测定各组上清液中VEGF和一氧化氮(NO)的含量;噻唑蓝(MTT)检测它们对EPCs增殖的影响.结果 FITC-UEA-I和DiI-ac-LDL双染色阳性细胞为正在分化的EPCs,脂质体介导pcDNA3.1-hVEGF165质粒转染组EPCs培养基上清液中VEGF和NO表达量明显高于其他两组(P＜0.01);VEGF质粒转染对EPCs的增殖无明显影响.结论 人外周血EPCs可以成功转染hVEGF165基因.同时能表达一定浓度的VEGF蛋白,并可促进NO的分泌,而对EPCs的活性无明显影响.该研究为进一步研究VEGF165基因和EPCs结合治疗缺血性疾病提供了实验依据.     

血管内皮生长因子基因转染血管内皮祖细胞治疗肢体缺血的实验研究

目的利用血管内皮生长因子（vascular endothelial growth factor,VEGF-165）基因转染体外诱导的血管内皮祖细胞（endothelial progenitor cells,EPCs）,并移植到下肢缺血的新西兰兔体内,观测其促进血管新生,改善肢体缺血的效果。方法（1）梯度离心法分离兔骨髓单个核细胞,然后用含有VEGF、bFGF、IGF-1的M199培养液诱导培养EPCs。并以免疫荧光、透射电镜等方法进行鉴定。（2）用携带VEGF165基因的腺病毒质粒（Adv-GFP-VEGF165）转染所培养的细胞,ELISA法检测上清液中VEGF蛋白的表达。（3）制作兔下肢缺血模型,并将其随机分为A、B、C 3组,分别移植EPCs、VEGF165基因转染后的EPCs、M199培养基,多种方法检测移植效果及局部整合情况。结果（1）自兔骨髓诱导出梭形贴壁细胞,免疫荧光及电镜检测证实为EPCs。（2）Adv-GFP-VEGF165成功转染EPCs,ELISA法检测转染VEGF165后的EPCs其上清液中VEGF蛋白浓度明显升高。（3）Brdu示踪显示移植细胞整合到缺血局部,CTA及免疫组化检查显示VEGF-165基因转染后的EPCs移植后其改善肢体缺血效果优于其他两组。结论VEGF基因转染EPCs后能改进EPCs质量,移植后促血管新生能力增强,其效果优于未转染组。     

血管内皮细胞生长因子体外转染血管内皮祖细胞的研究

目的 探讨脂质体介导血管内皮细胞生长因子(VEGF)基因转染血管内皮祖细胞(EPC)应用于基因治疗的可行性、安全性。方法 体外分离、培养、免疫组织化学及流式细胞仪鉴定EPC,转染VEGF后用免疫组织化学和ELISA检测EPC表达VEGF蛋白,噻唑蓝(MTT)检测EPC对VEGF质粒转染的敏感性。结果 EPC表达CD_(34)、CD_(31)、KDR及CD_(133)细胞表面标志,转染后EPC胞内表达VEGF。脂质体介导PcDNA3.1(-)/VEGF_(165)质粒转染、PcDNA3.1(-)/VEGF_(165)空质粒转染、不转染任何质粒的3组EPC培养基上清中表达的VEGF分别为(352±35)ng/L、(45±5)ng/L、0 ng/L(P<0.05),VEGF质粒转染对EPC增殖无影响。结论 EPC可作为VEGF转染的靶细胞,用于基因治疗。     

Ang-1基因转染的内皮祖细胞移植对大鼠肾脏缺血再灌注后的保护作用

目的 评估Ang-1基因转染的内源性内皮祖细胞(endothelial progenitor cells,EPCs)移植治疗肾脏缺血再灌注损伤的作用,进一步完善EPCs对缺血肾脏产生保护作用的旁分泌机制.方法利用重组腺病毒载体Ad-Ang-1感染EPCs,并进行转染效率鉴定,进一步移植治疗大鼠缺血肾脏,术后72 h给予肾功能评估,并检测大鼠缺血肾脏中VEGF、Ang-1以及Ang-2表达情况.结果EPCs移植治疗后,大鼠肾脏功能明显改善,术后72 h检测大鼠患肾,其内VEGF、Ang-1以及Ang-2表达均明显提升.结论 Ang-1基因转染的EPCs移植治疗可以有效地治疗大鼠肾脏IRI,其作用机制可能与EPC归巢后旁分泌过量Ang-1相关,并且促使肾脏血管新生.     

缺氧诱导因子转染内皮祖细胞治疗大鼠缺血后肢

目的 使用缺氧诱导因子-1α(HIF-1α)转染内皮祖细胞(EPC)治疗大鼠后肢缺血,观察EPC、HIF-1α转染EPC对大鼠缺血后肢血管新生和肢体成活的影响.方法 制作SD大鼠后肢缺血模型,将动物随机分为3组,每组6只.将构建的HIF-1α基因真核表达载体转染入EPCs后通过尾静脉注射入大鼠体内,并与注射磷酸盐缓冲液(PBS)或EPC的大鼠进行比较,观察转染HIF-1α对新生血管形成的影响.结果 (1)EPC组、HIF组较PBS组肢体恢复率明显增加(P<0.05),EPC组肢体恢复率较HIF组差(P<0.05).(2)与PBS组比较,各时间点中EPC、HIF组微血管密度(MVD)均明显增多(P<0.05),HIF组较EPC组明显增高(P<0.05).(3)HIF组的HIF与血管内皮生长因子(VEGF)蛋白表达较PBS组、EPC组明显增多(P<0.05).PBS组Capase-3的表达较EPC组、HIF组明显增多(P<0.05).(4)术后7 d,各组的大鼠肢体灌注均明显降低,但EPC、HIF组细胞的血流灌注较PBS组多(P<0.01).术后14、21 d,与PBS对照组比较,HIF组的血流灌注恢复明显(P<0.01),EPC组血流灌注较HIF组少(P<0.05).结论 EPCs对大鼠缺血后肢的局部血管新生有明显促进作用,联合应用HIF-1α和EPCs有更优的治疗效果.     

血管内皮生长因子治疗下肢缺血性疾病的研究进展

单独使用血管内皮生长因子（VEGF）基因治疗下肢缺血性疾病的研究由来已久，目前尚无满意疗效。目前已证实血管内皮前体细胞（EPC）可定向分化血管内皮细胞并能形成新生血管，其中VEGF在EPC定向分化、体内动员、体外扩增及转染EPC后定向移植治疗周围缺血性疾病等过程中发挥重要作用。笔者对VEGF在基因治疗、细胞治疗和基因转染细胞治疗下肢缺血性疾病的研究进展综述如下。     

Autologous endothelial progenitor cells transplantation promoting endothelial recovery in mice

Transplantation of endothelial progenitor cells (EPCs) restores endothelial function. The present study was designed to determine the effect of autologous EPCs transplantation on the regeneration of endothelium in mice. Mice splenectomy was performed 14 days before carotid artery injury, and mononuclear cells were isolated and cultured in endothelial growth media for 7 days. EPCs were confirmed by immunostaining (CD31, endothelial nitric oxide synthase (eNOS) and double positive for 1,1'dioctadecyl-3,3,3',3-tetramethylindocarbocyanine (DiI)-low-density lipoprotein and ulex europaeus agglutinin (UEA)). Cell counts and fluorescence-activated cell sorting for stem cell marker were performed. 1 x 10(6) 4-,6-Diamidino-2-phenylindole- labeled EPCs or saline were injected through tail vein after wire injury. Two weeks after transplantation, cell tracking and immunohistochemical staining showed homing and incorporation of labeled EPCs in injury artery. Administration of EPCs enhanced reendothelialization (P < 0.05) after 1 week and inhibition of neointima formation at 3 weeks compared with that of saline (P < 0.05, n = 6). These data demonstrate that delivery of autologous EPCs is associated with accelerated reendothelialization and reduced neointimal formation. Thus, delivery of autologous EPCs represents an important vasculoprotective approach to attenuate the response to acute vascular injury.     

Quantitative endothelial biomicroscopy

Twenty patients were evaluated by quantitative endothelial biomicroscopy and the resulting endothelial cell densities compared with those from a specular microscope. Estimated cell densities by this technique, when compared with the specular microscope, demonstrated a Pearson correlation coefficient of +0.977, an average error of -7% and an absolute error of 12%. There were no errors greater than 26% except for one extremely low cell density of 318 cells/mm2 for which the estimate was 476 cells/mm2. This rapid, inexpensive technique requires counting the number of endothelial cells seen across the horizontal diameter of the 0.2-mm projected spot beam of a standard biomicroscope. From this count and the known spot size the endothelial cell density may be accurately calculated. A simplified four-step technique for performing specular microscopy using only the standard biomicroscope and a method for accurately measuring the size of the projected slit lamp spot beam are explained.     

VEGF regulation of endothelial nitric oxide synthase in glomerular endothelial cells

BACKGROUND: Vascular endothelial growth factor (VEGF) regulation of endothelial nitric oxide synthase (eNOS) and signaling pathways involved have not been well studied in glomerular endothelial cells (GENCs). METHODS: GENCs grown from tsA58 Immortomice were used. Immunoblotting and in-cell Western blot analysis were employed to assess changes in VEGF receptor signaling pathway and eNOS phosphorylation of ser1177. Immunokinase assay and immunoblotting with phosphospecific antibodies were performed to assess activity of kinases. RESULTS: VEGF rapidly induced tyrosine phosphorylation of type 1 and type 2 VEGF receptors. Physical association between VEGF-receptor 2 (VEGF-R2) and insulin receptor substrate (IRS-1) and phosphatidylinositol 3'-kinase (PI3K) was induced by VEGF, which augmented PI3K activity in VEGF-R2 immunoprecipitates. VEGF stimulated Akt phosphorylation in a PI3K-dependent manner. VEGF increased eNOS phosphorylation on Ser1177. Activation of eNOS was associated with nitric oxide generation as measured by medium nitrite content. Signaling mechanisms involved in VEGF stimulation of eNOS were explored. VEGF-induced eNOS phosphorylation was abolished by SU1498, a VEGF-R2 inhibitor, LY294002, a PI3K inhibitor, and infection of cells with an adenovirus carrying a dominant negative-mutant of Akt, demonstrating the requirement of the VEGF-R2/IRS-1/PI3K/Akt axis for activation of eNOS. VEGF also activated extracellular signal-regulated protein kinase (ERK) in a time-dependent manner; and VEGF-stimulated eNOS phosphorylation on Ser1177 was prevented by PD098059, an upstream inhibitor of ERK, demonstrating that ERK was involved in VEGF regulation of eNOS. ERK phosphorylation was abolished by LY294002, suggesting ERK was downstream of PI3K in VEGF-treated GENC. CONCLUSIONS: Our data demonstrate that in GENC, VEGF stimulates VEGF-R2/IRS-1/PI3K/Akt axis to regulate eNOS phosphorylation on Ser1177 in conjunction with the ERK signaling pathway.     

Measurement of endothelial permeability

Conclusions  Endothelial permeability is now well characterized as a closely regulated process of reversible structural modification which controls passage of a wide range of circulating elements. The variety of experimental methodologies presented in this review mirrors the complexity of this essential function. These recent advances in experimental technique have already greatly contributed to our understanding of basic pathophysiological processes such as sepsis, atherosclerosis, and ischemia/reperfusion injury. In the future, such methodologies will doubtless illuminate other important clinical problems such as the mechanisms of metastasis, immune deposition, and autoimmunity.     

Glomerular endothelial cell differentiation

BACKGROUND: Glomerular endothelial cells differ from most other endothelial cells in that they are extraordinarily flattened and highly fenestrated. In this differentiated form, they allow formation of glomerular ultrafiltrate at a prodigious rate. METHODS: Molecular processes that dictate the development and differentiation of glomerular endothelium are reviewed. RESULTS: During glomerular development, angioblasts already present in the metanephric blastema well before any organized angiogenic sprouts invade the capillary cleft of developing nephrons at the comma and S-shape stages in response to chemotactic and guiding cues from primitive podocytes. The angioblasts then undergo homotypic aggregation into precapillary cords as yet devoid of a lumen. Lumen development then proceeds through the loss of superfluous endothelial cells by apoptosis as well as flattening of the remaining viable endothelial cells. The final step, fenestration, is critically dependent on appropriate stimuli, most notably vascular endothelial growth factor A (VEGF-A), from differentiated podocytes. Current evidence suggests that the fenestrae of fully differentiated glomerular endothelium can be lost within hours if the VEGF-A stimulus is removed, and that the glomerular endotheliosis, loss of glomerular filtration rate (GFR) and proteinuria observed in preeclampsia are due to the circulating inhibitor of VEGF-A, soluble VEGF receptor 1 (VEGFR-1). CONCLUSION: Differentiation of the glomerular endothelium is highly dependent on podocyte-derived stimuli and their loss leads to the derangements of glomerular function in preeclampsia.     

Estrogen improves endothelial function

Purpose: To determine the effect of estrogen on endothelium-dependent relaxation in the cutaneous microcirculation of women. Methods: Three groups of women participated in the study. Group 1 (n = 20) was premenopausal and had a mean age of 39 years (range 24-50 years). Group 2 (n = 9) was postmenopausal and had a mean age of 58 years (range 53-65 years). Group 3 (n = 11) was postmenopausal and taking estrogen replacement therapy; the mean age was 53 years (range 43-58 years). Eleven women in group 1 underwent testing twice, once during menstruation (mean serum estradiol level 73 ± 30 pg/ml) and once during midcycle (mean serum estradiol level 268 ± 193 pg/ml; p = 0.003). Single-point laser Doppler ultrasound and laser Doppler imaging with a scanner were used to measure vasodilatation in the forearm skin in response to iontophoresis of 1% acetylcholine (endothelium dependent) and 1% sodium nitroprusside (endothelium-independent smooth muscle relaxant). Results: All three groups were matched for body mass index and fasting glucose, total, high-density lipoprotein, and low-density lipoprotein cholesterol and triglyceride levels. All women had normal blood pressure, and none smoked. Mean serum estradiol levels were 196 ± 170 pg/ml (group 1), 35 ± 12 pg/ml (group 2), and 107 ± 78 pg/ml (group 3) (p = 0.004). Maximum microvascular vasodilatation (percentage increase over baseline) in response to acetylcholine was reduced in group 2 (93% ± 43%) compared with group 1 (187% ± 63%) and group 3 (142% ± 56%) (p = 0.001). The response to sodium nitroprusside also was diminished in group 2 (73% ± 27%) compared with group 1 (126% ± 45%) and group 3 (100% ± 32%) (p = 0.02). Within group 1 the acetylcholine response was higher during the midcycle phase (186% ± 31%) compared with the menstrual phase (147% ± 57%) (p < 0.05). The sodium nitroprusside response also was higher during the midcycle phase (144% ± 31%) compared with the menstrual phase (94% ± 41%) (p < 0.05) Conclusion: The results indicate that estrogens might enhance endothelium-dependent and endothelium-independent vasodilatation in the microcirculation of women.(J Vasc Surg 1998;27:1141-7.)     

Hyperuricemia induces endothelial dysfunction

BACKGROUND: Hyperuricemia has been linked to cardiovascular and renal diseases, possibly through the generation of reactive oxygen species (ROS) and subsequent endothelial dysfunction. The enzymatic effect of xanthine oxidase is the production of ROS and uric acid. Studies have shown that inhibiting xanthine oxidase with allopurinol can reverse endothelial dysfunction. Furthermore, rat studies have shown that hyperuricemia-induced hypertension and vascular disease is at least partially reversed by the supplementation of the nitric oxide synthase (NOS) substrate, L-arginine. Therefore, we hypothesized that uric acid induces endothelial dysfunction by inhibiting nitric oxide production. METHODS: Hyperuricemia was induced in male Sprague-Dawley rats with an uricase inhibitor, oxonic acid, by gavage; control rats received vehicle. Allopurinol was placed in drinking water to block hyperuricemia. Rats were randomly divided into four groups: (1) control, (2) allopurinol only, (3) oxonic acid only, and (4) oxonic acid + allopurinol. Rats were sacrificed at 1 and 7 days, and their serum analyzed for serum uric acid and nitrites/nitrates concentrations. The effect of uric acid on nitric oxide production was also determined in bovine aortic endothelial cells. RESULTS: Oxonic acid induced mild hyperuricemia at both 1 and 7 days (P < 0.05). Allopurinol reversed the hyperuricemia at 7 days (P < .001). Serum nitrites and nitrates (NO(X)) were reduced in hyperuricemic rats at both 1 and 7 days (P < .001). Allopurinol slightly reversed the decrease in NO(X) at 1 day and completely at 7 days (P < .001). There was a direct linear correlation between serum uric acid and NO(X) (R(2)= 0.56) and a trend toward higher systolic blood pressure in hyperuricemic rats (P= NS). Uric acid was also found to inhibit both basal and vascular endothelial growth factor (VEGF)-induced nitric oxide production in bovine aortic endothelial cells. CONCLUSION: Hyperuricemic rats have a decrease in serum nitric oxide which is reversed by lowering uric acid levels. Soluble uric acid also impairs nitric oxide generation in cultured endothelial cells. Thus, hyperuricemia induces endothelial dysfunction; this may provide insight into a pathogenic mechanism by which uric acid may induce hypertension and vascular disease.