Adeno-associated viral vector-mediated gene transfer of VEGF normalizes skeletal muscle oxygen tension and induces arteriogenesis in ischemic rat hindlimb

Critical limb ischemia is an important clinical problem that often leads to disability and limb loss. Vascular endothelial growth factor (VEGF), delivered either as recombinant protein or as gene therapy, has been shown to promote both collateral artery formation (arteriogenesis) and capillary angiogenesis in animal models of hindlimb ischemia. However, none of the previous studies has demonstrated an improvement in tissue hypoxia, the condition that drives the molecular response to ischemia. Furthermore, the optimal vector and route of gene delivery have not been determined. Recently, adeno-associated viral (AAV) vectors, which efficiently transduce skeletal muscle and produce sustained transgene expression, have been used as gene therapy vectors. We asked whether an intra-arterial injection of AAV-VEGF₁₆₅ normalizes muscle oxygen tension by increasing skeletal muscle oxygen tension, and promotes arteriogenesis and angiogenesis in a rat model of severe hindlimb ischemia. We found that AAV-VEGF treatment normalized muscle oxygen tension in the ischemic limb. In contrast, vehicle and AAV-lacZ-treated limbs remained ischemic. Collateral arteries were more numerous in AAV-VEGF-treated rats, but, surprisingly, capillaries were not. We conclude that intra-arterial AAV-mediated gene transfer of AAV-VEGF₁₆₅ normalizes muscle oxygen tension and leads to arteriogenesis in rats with severe hindlimb ischemia.     

Adeno-associated virus vector-mediated interleukin-10 gene transfer inhibits atherosclerosis in apolipoprotein E-deficient mice

Inflammation is a major contributor to atherosclerosis by its effects on arterial wall biology and lipoprotein metabolism. Interleukin-10 (IL-10) is an anti-inflammatory cytokine that may modulate the atherosclerotic disease process. We investigated the effects of adeno-associated virus (AAV) vector-mediated gene transfer of IL-10 on atherogenesis in apolipoprotein E (ApoE)-deficient mice. A murine myoblast cell line, C2C12, transduced with AAV encoding murine IL-10 (AAV2-mIL10) secreted substantial amounts of IL-10 into conditioned medium. The production of monocyte chemoattractant protein-1 (MCP-1) by the murine macrophage cell line, J774, was significantly inhibited by conditioned medium from AAV2-mIL10-transduced C2C12 cells. ApoE-deficient mice were injected with AAV5-mIL10 into their anterior tibial muscle at 8 weeks of age. The expression of MCP-1 in the vascular wall of the ascending aorta and serum MCP-1 concentration were decreased in AAV5-mIL10-transduced mice compared with AAV5-LacZ-transduced mice. Oil red-O staining of the ascending aorta revealed that IL-10 gene transfer resulted in a 31% reduction in plaque surface area. Serum cholesterol concentrations were also significantly reduced in AAV5-mIL10-transduced mice. To understand the cholesterol-lowering mechanism of IL-10, we measured the cellular cholesterol level in HepG2 cells, resulting in its significant decrease by the addition of IL-10 in a dose-dependent manner. Furthermore, IL-10 suppressed HMG-CoA reductase expression in the HepG2 cells. These observations suggest that intramuscular injection of AAV5-mIL10 into ApoE-deficient mice inhibits atherogenesis through anti-inflammatory and cholesterol-lowering effects.     

Adeno-associated virus-mediated delivery of BCL-w gene improves outcome after transient focal cerebral ischemia

A recombinant adeno-associated virus (rAAV) vector was used to overexpress the anti-apoptotic Bcl-2-family protein, BCL-w, in rat brain. Three weeks after injecting the vector into cerebral cortex and striatum on one side, temporary focal ischemia was induced by occlusion of the ipsilateral middle cerebral artery for 90 min, followed by reperfusion for 24 h. BCL-w expression was increased in cerebral cortex and striatum--and in neurons, astroglia and endothelial cells--in the brains of rats that received the rAAV-BCL-w vector, compared to rats given phosphate-buffered saline or a control vector containing the gene for green fluorescent protein. Recipients of the rAAV-BCL-w vector also showed a 30% reduction in infarct size and a 33-40% improvement in neurological function, compared to the control groups. These results provide evidence for a role of BCL-w in regulating histological and functional outcome after focal cerebral ischemia.     

Adeno-associated virus vector-mediated minidystrophin gene therapy improves dystrophic muscle contractile function in mdx mice

Duchenne muscular dystrophy (DMD) is the most common disabling and lethal genetic muscle disorder, afflicting 1 of every 3500 males. Patients with DMD experience progressive muscle degeneration and weakness and succumb to respiratory or cardiac failure by their early twenties. No treatment is currently available for DMD. Mutations in the dystrophin gene result in lack of a functional dystrophin protein in striated muscle, which induces instability in the muscle cell membrane leading to persistent muscle injury after contraction. We have previously created novel minidystrophin genes and demonstrated that adeno-associated virus (AAV)-mediated intramuscular delivery of the minigenes effectively ameliorated mdx dystrophic histopathology and led to normal cell membrane integrity for more than 1 year. In this paper, we investigated whether AAV-minidystrophin could also improve mdx muscle contractile function. Two-month-old adult male mdx mice, with established muscular dystrophy, were given a single-dose injection of an AAV-minidystrophin vector in the tibialis anterior (TA) muscle of one leg, with the untreated contralateral leg used as a control. The treated TA muscle showed both (1) a significant increase in isometric force generation and (2) a significant increase in resistance to lengthening activation-induced muscle force decrements. We conclude that AAV-minidystrophin gene treatment is effective in improving mdx muscle contractile function.     

Therapeutic effects of viral vector-mediated antiangiogenic gene transfer in malignant ascites

Malignant ascites is a common complication of advanced intraabdominal neoplasms for which standard treatments are suboptimal. Evidence suggests that tumor-mediated angiogenesis and enhanced vascular permeability in the peritoneal wall due to high levels of vascular endothelial growth factor play a fundamental role in the pathogenesis of malignant ascites. To explore the advantage of viral vector-mediated "targeted antiangiogenic therapy" in ascites formation, we constructed and administered adenoviral vectors encoding several different antiangiogenic proteins (angiostatin, endostatin, platelet factor 4, and a fusion protein between angiostatin and endostatin) alone or in combination intraperitoneally in mice with peritoneal carcinomatosis from breast cancer (TA3 cells) and ovarian cancer (SKOV-3 i.p. and ES-2 cell lines) to explore the potential of additive or synergistic activity. Our data demonstrated statistically significant downregulation of ascites formation, tumor growth, vascularity, and prolongation of animal survival after intraperitoneal treatment with antiangiogenic adenoviral vectors in three different ascites tumor models. Combined treatment proved to be more effective than treatment with one vector alone. Reduced ascites formation was accompanied by decreased microvascular density in the peritoneal wall and increased apoptosis of tumor cells after administration of antiangiogenic vectors in vivo. Of interest was the observation that AdPF4 caused a significant decrease in the level of VEGF secreted by tumor cells both in vitro and in TA3 ascites tumor-bearing animals in vivo. These data suggest that adenoviral vector-mediated delivery of genes encoding antiangiogenic proteins may represent a potentially new treatment modality for malignant ascites.     

Progress and challenges in viral vector-mediated gene transfer to the brain

Gene transfer into the brain allows the manipulation of transgene expression in both time and space. Recently developed gene transfer technologies allow transgenes to be expressed in any anatomically, biochemically or functionally distinct group of brain cells. Gene transfer has been used to alter the expression of neurotransmitter receptors, ion channels, signaling proteins, neuronal growth, differentiation and survival factors, and thus to modify brain anatomy, neuron physiology, behavior and pathology. However, challenges remain in making gene therapy a more widespread tool for the treatment of neurological disease. We have identified the following as areas needing development: access and delivery of viral vectors to the brain; diffusion of viral vectors and transgenes throughout large areas of brain tissue; viral vector side effects and toxicity, inflammatory and immune responses to vectors; long-term stable transgene expression; cell type-specific expression of transgenes; and the ability of the experimenter or physician to switch transgene expression 'on' and 'off' at will. In the last year, neuro-gene therapy has shown that brain defects in experimental disease models can be prevented and corrected, and that viral vectors and encoded transgenes can be made to diffuse over larger brain areas. In addition, the cause of vector-induced inflammation and immune responses have begun to be elucidated, so that rational approaches can be developed to avoid these complications. Further improvements in viral vectors will facilitate clinical trials in the near future.     

ADAMTS13 reduces VWF‐mediated acute inflammation following focal cerebral ischemia in mice

Summary. Background: ADAMTS13 cleaves hyperactive ultra‐large von Willebrand factor (ULVWF) multimers into smaller and less active forms. It remains unknown whether VWF‐mediated inflammatory processes play a role in the enhanced brain injury due to ADAMTS13 deficiency. Objective: We tested the hypothesis that the deleterious effect of ADAMTS13 deficiency on ischemic brain injury is mediated through VWF‐dependent enhanced vascular inflammation. Methods: Transient focal cerebral ischemia was induced by 60 min of occlusion of the right middle cerebral artery. Myeloperoxidase (MPO) activity and inflammatory cytokines in the infarcted region were evaluated 23 h after reperfusion injury. Neutrophil infiltration within the infarct and surrounding areas was quantitated by immunohistochemistry. Results: We report that ADAMTS13‐deficient mice exhibited significantly enlarged infarct size, concordant with increased myeloperoxidase (MPO) activity, neutrophil infiltration and expression of the pro‐inflammatory cytokines interleukin‐6 (IL‐6) and tumor necrosis factor‐α (TNF‐α). In contrast, VWF‐deficient mice exhibited significantly reduced MPO activity, neutrophil infiltration and inflammatory cytokine induction, demonstrating a role of VWF in these inflammatory processes. Mice deficient for both ADAMTS13 and VWF exhibited an identical reduction of the same inflammatory parameters, demonstrating that the increased inflammation observed in ADAMTS13‐deficient mice is VWF dependent. Finally, the increased infarct size observed in ADAMTS13‐deficient mice was completely abrogated by prior immunodepletion of neutrophils, demonstrating a causal role for acute inflammation in the enhanced brain injury that occurs in the setting of ADAMTS13 deficiency. Conclusion: These findings provide new evidence for ADAMTS13 in reducing VWF‐mediated acute cerebral inflammation following ischemic stroke.     

VEGF-induced neuroprotection,neurogenesis, and angiogenesis after focal cerebral ischemia

Vascular endothelial growth factor (VEGF) is an angiogenic protein with therapeutic potential in ischemic disorders, including stroke. VEGF confers neuroprotection and promotes neurogenesis and cerebral angiogenesis, but the manner in which these effects may interact in the ischemic brain is poorly understood. We produced focal cerebral ischemia by middle cerebral artery occlusion for 90 minutes in the adult rat brain and measured infarct size, neurological function, BrdU labeling of neuroproliferative zones, and vWF-immunoreactive vascular profiles, without and with intracerebroventricular administration of VEGF on days 1-3 of reperfusion. VEGF reduced infarct size, improved neurological performance, enhanced the delayed survival of newborn neurons in the dentate gyrus and subventricular zone, and stimulated angiogenesis in the striatal ischemic penumbra, but not the dentate gyrus. We conclude that in the ischemic brain VEGF exerts an acute neuroprotective effect, as well as longer latency effects on survival of new neurons and on angiogenesis, and that these effects appear to operate independently. VEGF may, therefore, improve histological and functional outcome from stroke through multiple mechanisms.     

银杏叶提取物(EGB)对局灶性脑缺血大鼠脑片HIF-1 VEGF表达及新生血管的影响

目的 探讨银杏叶提取物(EGB)对大鼠局灶性脑缺血/再灌注中脑组织内血管内皮细胞生长因子(VEGF)及血管通透因子(HIF-1)表达的影响.方法 采用Zea Longa线栓法制备大鼠局灶性脑缺血/再灌注模型(即MCAO模型),54只大鼠随机分为三组,即对照组(n=6)、EGB组(n=24)及缺血/再灌注组(n=24),分别在缺血/再灌注4、6、24、72 h取材.应用免疫组化法(SABC法),利用计算机图像分析系统软件,计数脑梗死后不同时间点阳性细胞的数量.检测VEGF及HIF-1蛋白的表达变化情况.结果 对照组:脑组织VEGF和HIF表达均较低;EGB组:VEGF及HIF阳性细胞密度均相对较高;缺血/再灌注组:VEGF在4 h开始升高,24 h达到高峰,72 h下降到接近正常水平,HIF-1在缺血后4 h开始降低,8 h开始升高,24 h达到高峰,72 h下降到接近正常水平.结论 EGB对于缺血性脑损伤有保护作用.     

亚低温对局灶性脑缺血大鼠血管新生的影响

目的探讨亚低温对局灶性脑缺血大鼠血管新生的影响。方法采用改良的线栓法制作大鼠永久性大脑中动脉闭塞模型，将造模成功的大鼠分为常温组和亚低温组。造模后14d通过股静脉注射异硫氰酸荧光素右旋糖酐（FITC-dextran）标记微血管，结合共聚焦显微镜和3DDoctor3．5版软件分析梗死灶周围区微血管的直径、面积及血管分支数目，并采用TTC染色观察脑梗死灶体积的变化。结果脑梗死后14d，梗死侧微血管直径明显小于对侧，但是血管分支数目及面积较对侧增加，且亚低温组梗死侧微血管直径、面积及分支数目明显大于常温组梗死侧；亚低温组梗死体积也明显小于常温组，差异均具有统计学意义（P〈0.05）。结论亚低温治疗能减小脑梗死灶体积并促进脑梗死后血管新生：     

Adeno-associated virus vector-mediated gene transfer into dystrophin-deficient skeletal muscles evokes enhanced immune response against the transgene product

Duchenne muscular dystrophy (DMD) is an X-linked, lethal muscular disorder caused by a defect in the DMD gene. AAV vector-mediated micro-dystrophin cDNA transfer is an attractive approach to treatment of DMD. To establish effective gene transfer into skeletal muscle, we examined the transduction efficiency of an AAV vector in skeletal muscles of dystrophin-deficient mdx mice. When an AAV vector encoding the LacZ gene driven by a CMV promoter (AAV-CMVLacZ) was introduced, beta-galactosidase expression markedly decreased in mdx muscle 4 weeks after injection due to immune responses against the transgene product. We also injected AAV-CMVLacZ into skeletal muscles of mini-dystrophin-transgenic mdx mice (CVBA3'), which show ameliorated phenotypes without overt signs of muscle degeneration. AAV vector administration, however, evoked substantial immune responses in CVBA3' muscle. Importantly, AAV vector using muscle-specific MCK promoter also elicited responses in mdx muscle, but at a considerably later period. These results suggested that neo-antigens introduced by AAV vectors could evoke immune reactions in mdx muscle, since increased permeability allowed a leakage of neo-antigens from the dystrophin-deficient sarcolemma of muscle fibers. However, resident antigen-presenting cells, such as myoblasts, myotubes and regenerating immature myofibers, might also play a role in the immune response.     

Adenovirus-mediated VEGF(165) gene transfer enhances wound healing by promoting angiogenesis in CD1 diabetic mice

It has been previously shown that vascular endothelial growth factor (VEGF) plays a central role in promoting angiogenesis during wound repair and that healing-impaired diabetic mice show decreased VEGF expression levels. In order to investigate the potential benefits of gene therapy with growth factors on wound repair, a replication-deficient recombinant adenovirus vector carrying the human VEGF(165) gene (AdCMV.VEGF(165)) was topically applied on excisional wounds of streptozotocin-induced diabetic mice. Treatment with AdCMV.VEGF(165) significantly accelerated wound closure when compared with AdCMV.LacZ-treated, as well as saline-treated control mice, by promoting angiogenesis at the site of injury. Our findings suggest that AdCMV.VEGF(165) may be regarded as a therapeutic tool for the treatment of diabetic ulcers.     

自体骨髓细胞移植对大鼠局灶性脑缺血区新生血管形成的作用

背景骨髓细胞移植是一种简单而有效的促进新生血管生长的治疗手段.同样新生血管的形成和血液循环的再建立,对于脑缺血区域受损而未死亡神经元的修复以及为植入的组织与细胞的成活和分化提供必要的环境条件均具有重要意义.而自体骨髓细胞移植是否会促进脑缺血区新生血管的形成进行血运重建目前尚不清楚.目的探讨经颈动脉移植自体骨髓细胞对脑缺血区新生血管形成的影响.设计以实验动物为研究对象,随机对照的验证性研究.单位一所大学医院的神经外科和泌尿外科研究所.材料实验于2002-09/2003-04在江西医学院第二附属医院神经外科实验室,江西医学院泌尿外科研究所完成.选择雄性SD大鼠10只,体质量250～300 g,清洁级.干预复制局灶性脑缺血大鼠模型,经颈动脉注入自体骨髓细胞.动物随机分为实验组5只,经颈动脉注入自体骨髓细胞,对照组5只注入生理盐水.通过免疫组织化学染色方法进行微血管计数,观察脑缺血区血管增生状况.主要观察指标微血管密度.F8因子免疫组化染色.结果自体骨髓细胞移植后大鼠在皮质缺血区的微血管密度为(159.15±40.4)血管数/mm2,明显较对照组大鼠(81.70±32.18)血管数/mm2多,差异有显著性意义(P＜0.05).骨髓细胞移植组皮质缺血区可见很多散在的单个内皮细胞.而相应的脑缺血对照组少见散在的单个内皮细胞.结论经颈动脉注入的自体骨髓细胞能够促进脑缺血区新生血管的形成.     

Adeno-associated virus vector-mediated bcl-2 gene transfer into post-ischemic gerbil brain in vivo: prospects for gene therapy of ischemia-induced neuronal death

The proto-oncogene bcl-2 is known as an anti-apoptotic gene that confers the ability to block neuronal cell death after transient ischemia. In order to examine whether the bcl-2 gene can be used for protection of ischemic brain injury, we generated adeno-associated virus (AAV) vectors capable of expressing human bcl-2. Replication-defective AAV vectors were found effectively to transfer and express bcl-2 gene in the gerbil hippocampal neurons. Transduction with AAV bcl-2 5 days before forebrain ischemia prevented the DNA fragmentation in the CA1 neurons that is commonly associated with ischemia-induced cell death. Furthermore, the application of AAV bcl-2 as late as 1 h following an ischemic insult also prevented DNA fragmentation in CA1 neurons. These results suggest that the bcl-2 protein has neuroprotective functions that inhibit ischemic cell death and demonstrate the potential of AAV bcl-2 for use in post-ischemic gene therapy in the brain. Gene Therapy (2000) 7, 1244-1249.     

白藜芦醇对小鼠局灶性脑缺血的保护作用及其机制

目的:探讨不同剂量白藜芦醇对局灶性脑缺血后的神经保护作用及对脑内基质金属蛋白酶(matrixmetallopoteinases,MMPs)表达的影响。方法:实验于2003-11/2004-02在解放军第四军医大学西京医院神经外科研究所实验室分两部分进行。60只健康雄性昆明白小鼠,体质量18～22g。第1部分中随机分为4组,分别为白藜芦醇10,30,60mg/kg强饲预治疗组及单纯缺血组,第2部分随机分为白藜芦醇30mg/kg及单纯缺血组。各组均为10只。采用线段血管内栓塞大脑中动脉(MCA0)获得小鼠脑缺血模型,2,3,5-氯化三苯基四氮唑(TTC)染色显示脑梗死范围,应用酶谱印记技术检测缺血后脑组织中MMPs活性。结果:各白藜芦醇强饲预治疗组的脑梗死体积和脑水肿程度均明显小于单纯缺血组犤(120±17)mm,(21.5±2.5)%犦,差异均有非常显著3性意义(P<0.01)。30mg/kg白藜芦醇强饲预治疗组的脑梗死体积和脑水肿程度犤(87±15)mm,(12.9±1.3)%犦明显小于10mg/kg组3犤(102±11)mm,(17.8±2.3)%,P<0.01犦,但与60mg/kg相比,无3明显差别犤(89±13)mm,(12.5±1.6)%,P>0.05犦。30mg/kg剂量3的白藜芦醇明显抑制了缺血后脑组织MMP-9的活性。结论:白藜芦醇具有脑保护作用,机制与对MMP-9的抑制有关。     

VEGF对兔局灶性脑缺血再灌注损伤治疗作用的剂量-效应关系及MR影像学评价

目的:探讨血管内皮细胞生长因子(VEGF)对家兔局灶性脑缺血再灌注损伤的治疗作用及MR影像学评价。方法:采用兔大脑中动脉阻断(MCAO)局灶性脑缺血再灌注模型,在缺血2h再灌注损伤后即刻应用微量进样器将不同剂量的VEGF立体定向导入梗死灶周,于再灌注72h,观察梗死体积、灶周缺血半暗带caspase-3蛋白表达、凋亡率、ADC值比率(ADCR),评价神经功能缺损。结果:缺血再灌注即刻灶周给予VEGF,1.25ng/μl VEGF的应用对脑水肿、神经功能恢复和血管形成影响不大,而2.5ng/μl和5.0ng/μl的VEGF应用明显降低脑含水量、灶周皮层凋亡率及caspase-3活性表达,ADCR升高,脑组织表面血管增粗、增加,2.5ng/μl组的变化较5.0ng/μl组更明显,同时也能明显降低梗死体积(P<0.05)。结论:2.5ng/μl VEGF应用治疗家兔局灶性脑缺血再灌注损伤可诱导血管发生和神经保护作用,是进一步研究和评价VEGF治疗效应的最佳剂量。     

抗炎治疗促进脑梗死后脑内神经发生和功能恢复的实验研究

目的探讨米诺环素对大脑中动脉闭塞(MCAO)大鼠的神经保护作用及潜在的促神经发生作用。方法电凝法制备MCAO模型。实验大鼠随机分为假手术组、MCAO模型组及抗炎组后分别给予相应处理,于2周行转子杆、爬梯实验以及水迷宫测试评价运动及认知功能,并通过病理学处理及免疫组化检测对比各组各时间点梗死灶周5-溴脱氧核苷尿嘧啶(BrdU)、神经上皮干细胞蛋白(Nestin)、胶原纤维酸性蛋白(GFAP)阳性细胞计数。组间比较用单因素方差分析,两两比较用t检验。结果与模型组相比,急性期米诺环素短期治疗并不能明显减小梗死面积,但运动功能、空间学习及记忆能力均有所恢复(P<0.05)。抗炎组BrdU、Nestin以及GFAP阳性细胞数较对照组增加(P<0.05),BrdU、GFAP阳性细胞呈增加趋势,2周时增加最为明显;Nestin阳性细胞1周达高峰,之后逐渐下降。结论脑梗死急性期米诺环素治疗可能通过促进神经发生过程补偿、修复局部病灶的功能损伤与缺失,从而促进脑梗死大鼠神经功能恢复。     

通心络胶囊对脑梗死大鼠脑皮质血管新生和皮层神经元凋亡的影响

目的:探讨通心络胶囊对大鼠脑梗死皮层血管新生与神经元凋亡的影响。方法:M C A o大鼠分成通心络干预组与对照组,于梗死后第4天处死大鼠,免疫组化检测脑缺血皮质V E G F、V III因子及凋亡蛋白酶caspase-3表达,W estern blot半定量检测V E G F、caspase-3蛋白表达情况,TU N E L法检测梗死皮层神经元凋亡。结果:通心络干预组缺血皮质血管密度和V E G F阳性细胞数量均较对照组为高(P均<0.01)。W estern blot显示脑缺血皮层同侧V E G F121、V E G F165有明显表达,而缺血对侧表达很少;与对照组相比,通心络组缺血侧V E G F121、V E G F165蛋白表达均较对照组显著增高(P<0.01)。caspase-3免疫组化显示通心络组缺血侧阳性细胞数量较对照组为低;两组大鼠缺血侧与对侧皮层W estern blot均可显示出pre-caspase-3和活化的caspase-3两条带,以活化的caspase-3分析,对照组缺血侧caspase-3含量明显增高,而通心络组caspase-3含量明显降低,与对侧等其他各组间没有明显差异;TU...     

Adenoviral gene transfer of basic fibroblast growth factor promotes angiogenesis in rat brain

Cerebral ischemic disease often causes morbidity and mortality, while the induction of new blood vessels is expected to provide a therapeutic effect in this occlusive cerebrovascular disease. In this study, we utilized two replication-deficient adenoviral vectors containing cDNA from basic fibroblast growth factor (bFGF), a well-known angiogenic factor, and examined whether biological angiogenic activity of adenovirally gene-transferred bFGF could be observed in the rat brain. One vector contained native cDNA from bFGF without the secretory signal sequence and the other contained the same cDNA fused with an interleukin-2 secretory signal sequence. After ventricular administration of these viral vectors, gene-transferred cells demonstrated a high immunoreactivity against the anti-bFGF antibody and a remarkably high concentration of bFGF was detected in the cerebrospinal fluid. A semiquantitative analysis of angiogenic activity revealed that bFGF gene transfer induced angiogenesis in normal rat brains, with a more pronounced angiogenic effect seen with the vector of a secreted form than with the vector without a secretory signal sequence. These results suggest that bFGF gene transfer using these adenoviral vectors might be useful for the treatment of ischemic cerebrovascular disease.     

Helper-dependent adenoviral vector-mediated gene transfer in aged rat brain

Transfer of the neurotrophin gene into brain can attenuate age-related deficits such as neuronal atrophy and memory loss, but a suitable vector for this procedure has been lacking. The toxicity and immunogenicity of first-generation adenoviral vectors with E1 deletion (fgAdv) prohibit the application of gene transfer in the majority of central nervous system disorders. Here, we report less toxic and persistent gene expression mediated by helper-dependent adenovirus (hdAdv) in aged rat brain. After intrahippocampal or intraventricular inoculation of the vector, transgene expression was monitored by X-Gal staining and compared with fgAdv-mediated expression. Host inflammatory and immune responses against these vectors were evaluated by immunohistochemical detection of microglia, astrocytes, and infiltrating macrophages, as well as by enzyme-linked immunosorbent assay of cytokines TNF-alpha and IL-1beta. Transgene expression mediated by hdAdv persisted for more than 183 days regardless of inoculation site, as compared with 33 and 66 days for fgAdv-mediated expression after intraventricular and intrahippocampal inoculation, respectively. Inoculation with hdAdv was also associated with reduced numbers of activated microglial cells, astrocytes, and infiltrating macrophages in brain tissue. Secretion of the proinflammatory cytokines TNF-alpha and IL-1beta was minimal after hdAdv but not after fgAdv inoculation. These findings indicate that hdAdv would provide a safe and effective means to transfer therapeutic genes into aged brain.