Treatment of acute myocardial infarction by hepatocyte growth factor gene transfer: the first demonstration of myocardial transfer of a "functional" gene using ultrasonic microbubble destruction

OBJECTIVES: We examined whether ultrasonic microbubble destruction (US/MB) enables therapeutic myocardial gene transfer of hepatocyte growth factor (HGF) for acute myocardial infarction (MI). BACKGROUND: Hepatocyte growth factor gene transfer provides cardioprotective effects in MI, which requires direct intramyocardial injection or special vectors. Although US/MB was used in myocardial gene transfer, its feasibility in transfer of a therapeutic gene with non-viral vector remains unknown. METHODS: In a rat model of acute MI, naked plasmid (pVaxl) encoding human HGF (1,500 microg) was infused into the left ventricular (LV) chamber during US/MB (HGF-US/MB) or insonation only (HGF-US) or alone (HGF-alone), while control MI rats received empty pVaxl during US/MB (pVaxl-US/MB). For US/MB, transthoracic intermittent insonation with a diagnostic transducer (1.3 MHz) was performed for 2 min at a peak negative pressure of -2,160 kPa during intravenous 20% Optison. RESULTS: Baseline risk area was comparable among the groups. Immunohistology seven days after treatment revealed significant myocardial expression of HGF protein only in HGF-US/MB. At three weeks, LV weight in HGF-US/MB (0.89 +/- 0.03 g) was significantly lower than those in HGF-alone (1.09 +/- 0.08 g), HGF-US (1.04 +/- 0.07 g), and pVaxl-US/MB (1.04 +/- 0.05 g). Moreover, scar size was significantly smaller (16 +/- 6% vs. 39 +/- 5%, 41 +/- 6%, and 40 +/- 4% of total myocardial circumferential length, respectively), while capillary density (49 +/- 8 vs. 34 +/- 5, 37 +/- 6, and 36 +/- 4 capillaries/high-power field, respectively) and arterial density (37 +/- 7 vs. 15 +/- 9, 18 +/- 4, and 14 +/- 11 arterioles/high-power field, respectively) in the risk area were higher in HGF-US/MB than the other groups. CONCLUSIONS: Ultrasound-mediated microbubble destruction may enable myocardial HGF gene transfer with systemic administration of naked plasmid, which enhances angiogenesis, limits infarction size, and prevents LV remodeling after MI.     

Catheter-based plasmid-mediated transfer of genes into ischemic myocardium using the pCOR plasmid

BACKGROUND: Direct transfer of genes holds promise for the sustained delivery of therapeutic proteins to treat cardiovascular diseases. This can be accomplished by several approaches, including use of adenoviral vectors and naked plasmid DNA vectors. We previously demonstrated achieval of effective delivery of genes into the myocardium with a left ventricular-guided catheter-based approach using an adenoviral vector. OBJECTIVE: To evaluate the levels and duration of expression of genes induced after injection of a specific plasmid vector, using the same delivery platform as that in our previous work. METHODS: The pCOR plasmids are narrow-host-range plasmid vectors designed for nonviral gene therapy. We tested the ability of the pCOR plasmid vector to express its transgene after injection into the myocardium of pigs with chronic experimental ischemia using a catheter-based transendocardial delivery system. Four animals were subjected to transendocardial injections of the luciferase reporter pCOR gene into ischemic and nonischemic zones using the Biosense intramyocardial injection catheter. Injections (1 mg per animal, 50 micrograms per injection site) were performed at 20 sites in ischemic and nonischemic zones. Measurements of luciferase activity were performed 3 and 7 days thereafter. RESULTS: We observed high levels of expression of luciferase gene in ischemic and nonischemic regions (on days 3 and 7, respectively, in ischemic zone 58,237 and 33,709 pg; in nonischemic zone 39,928 and 46,036 pg). Control noninjected samples from the left and right ventricles contained no detectable luciferase activity. CONCLUSIONS: With a catheter-based approach, the pCOR plasmid was successfully used to deliver genes into designated myocardial regions, and provides sustained expression of protein for at least 7 days, of roughly similar magnitudes in ischemic and nonischemic myocardium.     

Effect of nifedipine on myocardial ischemia: analysis of collateral flow, pulsatile heat and regional muscle shortening.

Effects of intravenous nifedipine on the ischemic myocardium of dogs subjected to coronary ligation were evaluated with a newly developed myothermal technique. Sensitive, fast-reacting miniature thermistors implanted within the ventricular wall were used in conjunction with low noise thermistor bridges to monitor beat-to-beat intramyocardial heat pulses and to detect intramyocardial cold transients after mechanized injections of cold saline into the left atrium. Myocardial ischemia reduced the amplitude of the pulse signals and decreased mean intramyocardial temperature. Ischemia also reduced the amplitude of the intramyocardial cold transients detected after injection of cold saline solution. All thermal changes due to ischemia were partly reversed by nifedipine. The amplitude of the cold signals correlated positively (r = 0.85) with local myocardial perfusion estimated by the radioactive microsphere technique. Thus, estimates of local perfusion by two independent techniques confirmed that nifedipine increased collateral perfusion. In addition, simultaneous measurements of peripheral coronary pressure indicated that the drug decreased resistance in the coronary bed distal to the occlusion. Ultrasonic measurements of myocardial segment length revealed that nifedipine improved myocardial shortening in ischemic zones, a response that accounts for the nifedipine-induced increases in local heat. Thus, the results show that nifedipine increases collateral flow to the acutely ischemic myocardium and that improved perfusion is accompanied by enhanced contractile performance. These findings also indicate that the protective action of nifedipine on ischemic myocardium is not predominantly mediated by a cardioplegic effect limiting the metabolic needs of the myocardium at risk.     

超声介导微泡造影剂携带肝细胞生长因子基因靶向治疗大鼠急性心肌梗死

目的 研究急性心肌梗死时超声介导微泡造影剂携带肝细胞生长因子(HGF)基因靶向治疗的可行性.方法 将54只雄性SD大鼠建立心肌梗死模型并随机分成3组(每组18只):US/MB-HGF组,予超声照射及尾静脉注射携带HGF质粒基因(pcDNA3.1-HGF)的微泡造影剂;US-HGF组,予超声照射及尾静脉注射PCDNA3.1-HGF,无微泡造影剂;US/MB-P组:即对照组,予超声照射及尾静脉注射携带空质粒的微泡造影剂.并按大鼠被处死的时间顺序,将每组分为3个亚组(每组6只):24 h组、7 d组和14 d组,分别取心肌组织做石蜡切片并做免疫组织化学染色,包括抗凋亡蛋白(bcl-2)的表达、HGF蛋白表达和CD34标记的毛细血管计数情况.结果 (1)心肌组织HGF蛋白表达:US/MB.HGF7 d亚组有明显的HGF蛋白表达,24 h组未见有明显的HGF的表达颗粒.US-HGF7d和14 d亚组可见少量HGF表达.US/MB-P组各亚组均未见HGF表达.(2)CDM标记的毛细血管计数:US/MB-HGF 14 d亚组为(367.6±17.6)个/高倍视野,明显高于US-HGF组[(268.9±0.8)个/高倍视野]和US/MB-P组[(186.8±11.8)个/高倍视野,均P＜0.05],各组24 h和7 d亚组的毛细血管计数均低于其14 d亚组.(3)梗死区bcl-2的表达:US/MB-HGF 7 d亚组为9.9%±0.5%,明显高于US-HGF组(6.3%±1.0%)和uS/MB-P组(3.5%±0.8%,均P＜0.05);US/MB-HGF 14 d亚组为6.7%±0.9%,明显高于US-HGF组(4.5%±0.8%)和US/MB-P组(2.1%±0.9%,均P＜0.05);3组24 h亚组均未见明显表达.结论 超声介导造影剂微泡破裂可以使静脉注射的HGF基因在心肌局部浓度增高,增强其转染和表达,且可明显促进心肌新生血管的生长,减少急性梗死区心肌细胞的凋亡.     

骨髓干细胞介导的人肝细胞生长因子基因转染在兔心肌梗死组织中的表达

目的:观察以骨髓干细胞(BMSCs)介导的人肝细胞生长因子(hHGF)基因转染在兔心肌梗死组织中能否稳定表达.方法:家兔16只,复制急性心肌梗死(AMI)模型.随机分为2组:移植组:BMSCs加hHGF移植,对照组.另外随机选取8只兔作为假手术组.AMI术后3 d,抽取股骨骨髓1.5 ml,分离BMSCs培养.于对数生长期加入5-溴脱氧尿苷孵育24 h,以标记移植细胞.构建pcDNA3.1-hHGF重组表达载体,移植前用脂质体包裹表达载体转染BMSCs,孵育24 h.AMI术后14 d,行自体细胞移植,对照组接受等量的无血清培养基注射,假手术组只开胸,不注射.细胞移植后28 d,检测左心室血流动力学指标;取心肌组织,RT-PCR检测hHGFmRNA的表达;酶联免疫吸附法(ELISA)测定心肌组织中HGF的含量.结果:与假手术组比较,心肌梗死后28d,对照组左心室功能降低,但BMSCs加hHGF组左心心室功能有改善;RT-PCR结果显示移植组有hHGFmRNA表达,ELISA测定HGF含量移植组高于对照组.结论:AMI组织中HGF含量降低,转染HGF的BMSCs能在AMI组织中生存并能表达分泌HGF,其左心室功能有改善.     

Treatment of ischemic limbs based on local recruitment of vascular endothelial growth factor-producing inflammatory cells with ultrasonic microbubble destruction

OBJECTIVES: We sought to clarify the mechanism for neovascularization by ultrasonic microbubble destruction (US/MB) and its ability to improve the function of ischemic limbs. BACKGROUND: In tissue, US/MB can cause capillary rupture, leading to angiogenesis and arteriogenesis. METHODS: Seven days after removal of the femoral artery (day 0) in mice, microbubble/ultrasound treatment was performed by intermittent insonation (1.6 MHz, mechanical index 1.1) to the ischemic limbs after intravenous infusion of phospholipid-stabilized microbubbles BR14 (US/MB group). Effects were compared with those in untreated mice with ischemic limbs (control group). RESULTS: Immunostaining of the treated muscles revealed a greater leukocyte (CD45-positive cell) count in the US/MB group on days 3 and 7. These cells included F4/80-positive cells (macrophages) and CD3-positive cells (T-lymphocytes), both of which were immunoreactive to vascular endothelial growth factor (VEGF) antibody. Muscular VEGF content by Western blotting was elevated in the US/MB group on day 3, which declined but remained greater until day 21. The US/MB group showed a greater capillary density by alkaline phosphatase stain on day 7 without further increase at day 21. Surface vascularity of the muscles and blood flow were greater in the US/MB group on day 7, which further increased by day 21. Moreover, the US/MB group showed a two-fold longer treadmill time compared with the untreated control group on day 21. None of these favorable effects were observed in mice treated with ultrasound only or microbubbles only. CONCLUSIONS: Ultrasonic destruction of microbubbles delivered to the ischemic limbs can recruit inflammatory cells producing VEGF, which is followed by neovascularization and functional improvement, and thus has a therapeutic potential.     

Could plasmid‐mediated gene transfer into the myocardium be augmented by left ventricular guided laser myocardial injury?

Early studies have indicated no correlation between the amount of mechanical injury and the level of myocardial gene expression following direct plasmid vector injection. Recently, however, evidence suggests that combined laser myocardial injury and plasmid-based gene delivery exert synergistic effects on gene expression and activity. The purpose of the study was to determine whether laser-induced myocardial injury followed by transendocardial gene transfer increases gene expression compared to gene transfer alone. We assessed the ability of a plasmid vector to express its transgene after injection into porcine ischemic myocardium with and without preceding laser myocardial injury. Thirteen animals had transendocardial injections of the luciferase reporter gene in a plamid vector using a catheter-based injection system. Injections (0.5 mg per animal, 50 microg per injection site) were divided into 10 sites in the ischemic territory. Eight animals underwent transendocardial laser injury of the ischemic region (2 Joule per pulse x 10 sites) prior to gene delivery. In five animals, gene injection sites were dispersed between laser channels, and in three animals laser and gene delivery were applied in close proximity (< 5 mm) or at the same location. Luciferase activity was measured at 3 and 7 days. Luciferase expression in ischemic zones was markedly elevated at day 3 and 7, and similar whether animals were pretreated using laser injury followed by gene transfer compared to gene transfer alone. Neither same-spot injection nor dispersed gene delivery were associated with augmented gene expression compared to gene transfer alone. Using the above-described catheter-based approach to combine localized laser injury and injection of naked DNA into ischemic myocardium, laser injury did not augment gene expression above levels present with gene transfer alone.     

Electromagnetic guidance for catheter-based transendocardial injection: a platform for intramyocardial angiogenesis therapy. Results in normal and ischemic porcine models

OBJECTIVES: To test the feasibility of myocardial angiogenic gene expression using a novel catheter-based transendocardial injection system. BACKGROUND: Angiogenesis has been induced by direct injection of growth factors into ischemic myocardium during open-heart surgery. Catheter-based transendocardial injection of angiogenic factors may provide equivalent benefit without need of surgery. METHODS: A new guidance system for intramyocardial therapy utilizes magnetic fields and catheter-tip sensors to locate a position in space and reconstruct three-dimensional left ventricular (LV) electromechanical maps without using fluoroscopy. A retractable 27G needle was coupled with the guidance system for LV transendocardial injection. In 12 pigs, the catheter was used to inject 0.1 ml of methylene-blue (MB) dye and 8 pigs had myocardial injections of adenoviral vector (1 x 10(10) particles per site) containing the LacZ transgene. Ten pigs underwent catheter-based transendocardial injection and six pigs were injected using transepicardial approach with the gene encoding adenovirus vascular endothelial growth factor-121 (Ad.VEGF121; 1 x 10(10) viral particles x 6 sites) and sacrificed at 24 h. Injection sites were identified with ultraviolet light by coinjection of fluorescent beads. RESULTS: Overall, 138 of 152 attempted injection MB tracks (91%) were found after sacrifice. Tissue staining was 7.1+/-2.1 mm in depth and 2.3+/-1.8 mm in width. No animal had pericardial effusion or tamponade. In Ad.LacZ injected animals, gross pathology showed positive staining in injected zones, and histology confirmed positive myocyte staining. Adenovirus vascular endothelial growth factor-121 injected sites showed high levels of VEGF121 production that was of similar magnitude whether injected using the transendocardial (880.4+/-412.2 pg VEGF121/mg protein) or transepicardial (838.3+/-270 pg VEGF121/mg protein) delivery approach (p = 0.62). CONCLUSIONS: Using this magnetic guidance catheter-based navigational system, transgenes can effectively be transfected into designated myocardial sites. Thus, if it is determined that direct intramyocardial injection of angiogenic factors enhances collateral function in patients, this less invasive catheter-based system offers a similar gene delivery efficiency and, thus, may have clear advantages compared with the surgically-based transepicardial injection approach.     

Efficiency of Intramyocardial Injections of Autologous Bone Marrow Mononuclear Cells in Patients with Ischemic Heart Failure: A Randomized Study

Intramyocardial transplantation of autologous bone marrow mononuclear cells (BMMC) is believed to be a promising method for the treatment of patients with chronic ischemic heart disease. The aim of this study was to evaluate long-term results of intramyocardial bone marrow cell transplantation in patients with severe ischemic heart failure. One hundred nine patients with chronic myocardial infarction and end-stage chronic heart failure were randomized into two groups: 55 patients received intramyocardial BMMC injection and 54 received optimal medical therapy. The NOGA system (Biosense-Webster) was used to administer 41?±?16?×?106 BMMC into the border zone of myocardial infarction. None of the patients developed periprocedural complications following BMMC injections. The injections led to improvement of CCS class (3.1?±?0.4 to 1.6?±?0.6 after 6 months and 1.6?±?0.4 after 12 months; p?=?0.001) and NYHA functional class (3.3?±?0.2 to 2.3?±?0.2 after 6 months and 2.5?±?0.1 after 12 months; p?=?0.006). Left ventricular ejection fraction increased significantly in the BMMC group (27.8?±?3.4% vs 32.3?±?4.1%; p?=?0.04) while it tended to decrease in the control group (26.8?±?3.8% to 25.2?±?4.1%; p?=?0.61). Summed rest score improved in the BMMC group after 12 months (30.2?±?5.6 to 27.8?±?5.1; p?=?0.032). The improvement of stress score was more noticeable (34.5?±?5.4 to 28.1?±?5.2; p?=?0.016). Neither stress nor rest score changed in patients numbers on medical therapy. In BMMC group 6 (10.9%) patients died at 12-month follow-up compared with 21 (38.9%) in control group (log-rank test, p?=?0.0007). Intramyocardial bone marrow cell transplantation to patients with ischemic heart failure is safe and improved survival, clinical symptoms, and has beneficial effect on LV function     

直接心肌植入VEGF_(165)基因的实验研究

目的 :评估VEGF1 6 5基因直接心肌内注射促进心肌血运重建的疗效。方法 :中国实验小型猪 11只 ,体重 (2 9 3± 4 3)kg。在左冠状动脉回旋支 (LCX)近心端放置Ameroid收缩环。术后 6周行冠脉造影示LCX狭窄 >95 %为慢性心肌缺血模型形成。将选用模型随机分为治疗组 (n =6 )和对照组 (n =5 )。治疗组将VEGF1 6 5质粒注射到左室侧壁 10处有标记的心肌中层。对照组只注射空白质粒。治疗前和治疗 6周后 ,采用体表超声心动图观察静息与小剂量多巴酚丁胺负荷 (LDDSE)状态下左心室壁运动及功能 (LVEF、RT、MVCF、WMSI) ,通过2 0 1 TI SPECT观察心肌灌注改变。处死动物后进行心肌血管密度和VEGF基因mRNA表达测定。结果 :治疗 6周后 ,在静息与LDDSE(10 μg min kg)状态下 ,A组RT和WMSI较治疗前显著改善 ,侧壁灌注缺损明显缩小 ,B组改善不明显。A组每个视野的血管面积、血管周长及血管数目均较B组多。A组 2 3例局部心肌VEGF基因mRNA的表达明显 ,B组 3 3例均无表达。结论 :直接心肌内注射VEGF1 6 5基因可促进慢性缺血心肌的血管再生 ,改善局部心肌灌注和室壁运动。     

Lack of Cardiac Nerve Sprouting after Intramyocardial Transplantation of Bone Marrow-Derived Stem Cells in a Swine Model of Chronic Ischemic Myocardium

Previous experimental studies suggested that mesenchymal stem cell transplantation causes cardiac nerve sprouting; however, whether bone marrow (BM)-derived mononuclear cells (MNC) and endothelial progenitor cells (EPC) can also lead to cardiac nerve sprouting and alter gap junction expression remains unclear. We investigated the effect of electroanatomical mapping-guided direct intramyocardial transplantation of BM-MNC (n = 8) and CD31+EPC (n = 8) compared with saline control (n = 8) on cardiac nerve sprouting and gap junction expression in a swine model of chronic ischemic myocardium. At 12 weeks after transplantation, the distribution and density of cardiac nerve sprouting were determined by staining of tyrosine hydroxylase (TH) and growth associated protein 43(GAP-43) and expression of connexin 43 in the targeted ischemic and remote normal myocardium. After 12 weeks, no animal developed sudden death after the transplantation. There were no significant differences in the number of cells with positive staining of TH and GAP-43 in the ischemic and normal myocardium between three groups. Furthermore, expression of connexin 43 was also similar in the ischemic and normal myocardia in each group of animals (P > 0.05). The results of this study demonstrated that intramyocardial BM-derived MNC or EPC transplantation in a large animal model of chronic myocardial ischemia was not associated with increased cardiac nerve sprouting over the ischemic myocardium.     

激光心肌血运重建术与VEGF基因治疗犬心肌缺血心功能的变化

目的　观察 TML R联合 VEGF1 65c DNA治疗心肌缺血犬后心功能的变化。方法　健康杂种犬 36只 ,随机分为 VEGF1 65基因组、空质粒组 ,激光心肌打孔为对照组 (n=12 ) ,结扎冠状动脉造成急性心肌缺血后 6 0 min分别行CO2 激光心肌打孔和基因转染。采用热稀释法和彩色多谱勒超声心动图检测心排量 (CO)和左心室射血分数(L VEF)。结果　治疗后 6周和 12周 VEGF1 65c DNA组 L VEF和 CO显著高于激光打孔组 (P<0 .0 5 ) ,以治疗后 12周最为显著。结论　犬急性心肌缺血后采用激光心肌打孔联合 VEGF1 65基因治疗可显著改善心功能。     

Intake of fermented beverages protect against acute myocardial injury: target organ cardiac effects and vasculoprotective effects

Mild-to-moderate alcohol consumption has been associated with reduced risk of morbi/mortality from coronary artery disease. However, whether beer intake affords cardioprotection remains unclear. We investigated whether beer intake (alcohol-containing and alcohol-free brew) provides cardioprotection in a pig model of myocardial infarction (MI). Pigs were randomly assigned to: (1) be fed for 10?days a high-cholesterol diet (HC); (2) HC?+?low-dose beer (LB; 12.5?g alcohol/day); (3) HC?+?moderate-dose beer (MB; 25?g alcohol/day); or IV) HC?+?alcohol-free-MB (0.0?g alcohol/day) before MI induction and kept 21?days with the same regime. Scar size, echocardiography, biochemical and oxidative parameters were assessed. Myocardial tissue was obtained for molecular analysis and histology. All beer-fed animals were less prone to arrhythmogenesis during ischemia. At sacrifice, beer intake was associated with lower oxidative stress and higher HDL-antioxidant capacity. Within the ischemic myocardium beer-fed animals showed higher Akt/eNOS and AMPK activation and reduced sirtuin1-related apoptosis. Compared to controls beer intake was associated with lower lipid infiltration, higher TGFβ-related collagen fibril formation and diminished MMP9 activity in the fibrous tissue limiting scar size (HC?+?LB and HC?+?MB P?相似文献   

Adeno-associated viral vector delivers cardiac-specific and hypoxia-inducible VEGF expression in ischemic mouse hearts

It has been shown that the adeno-associated virus (AAV) vector can deliver the VEGF gene efficiently into the ischemic mouse myocardium. However, the AAV genomes can be found in extracardiac organs after intramyocardial injection. To limit unwanted VEGF expression in organs other than the heart, we tested the use of the cardiac myosin light chain 2v (MLC-2v) promoter and the hypoxia-response element to mediate cardiac-specific and hypoxia-inducible VEGF expression. An AAV vector, MLCVEGF, with 250 bp of the MLC-2v promoter and nine copies of the hypoxia-response element driving VEGF expression, was constructed. Gene expression was studied in vitro by infection of rat cardiomyocytes, rat skeletal myocytes, and mouse fibroblasts with the vector and in vivo by direct injection of the vector into normal and ischemic mouse hearts. With MLCVEGF infection, VEGF expression was higher in cardiomyocytes than the other two cell lines and was hypoxiainducible. VEGF expression was also higher in ischemic hearts than in normal hearts. No VEGF expression was detectable in organs with detectable MLCVEGF vectors other than the heart. MLCVEGF-injected ischemic hearts had more capillaries and small vessels around the injection site, smaller infarct size, and better cardiac function than the negative controls. Hence, MLCVEGF can mediate cardiac-specific and hypoxia-inducible VEGF expression, neoangiogenesis, infarct-size reduction, and cardiac functional improvement.     

OPG inhibits gene expression of RANK and CAII in mouse osteoclast-like cell

This study was designed to determine the effects of the osteoprotegerin (OPG) on the mRNA expression of carbonic anhydrase II (CAII) and the receptor activator of NF-??B (RANK) in mouse osteoclast-like cells. Marrow cells were harvested from femora and tibiae of mouse and cultured in 6-well chamber slides. After 1?day of incubation, the marrow cells were exposed to M-CSF (25?ng/ml), RANKL (50?ng/ml), and different concentrations of OPG (50, 75, and 100?ng/ml, respectively) for 3?days. Osteoclast-like cells were confirmed by both tartrate-resistant acid phosphatase (TRAP) stain and bone resorption assay. The expression of RANK and CAIImRNA was determined with real-time fluorescent quantitative polymerase chain reaction. The numbers of multinucleated, TRAP-positive osteoclast-like cells, and resorption pits formed were observed. Compared with the M-CSF?+?RANKL group, RANKmRNA expression was statistically decreased in the M-CSF and M-CSF?+?RANKL?+?OPG (100?ng/ml) groups (P?=?0.004, P?=?0.024, respectively); Compared with the M-CSF, M-CSF?+?RANKL, and M-CSF?+?RANKL?+?OPG (100?ng/ml) group, CAIImRNA expression in the M-CSF?+?RANKL?+?OPG (75?ng/ml) groups was statistically decreased (P?=?0.001, P?=?0.008, and P?=?0.036, respectively). These data suggest that OPG could regulate the expression of RANK and CA II mRNA in the marrow culture system.     

重组腺病毒相关病毒携带人血管内皮生长因子165诱导家兔缺血心肌血管生成的研究

目的　将携带人血管内皮生长因子 16 5 (hVEGF 16 5 )cDNA的重组腺病毒相关病毒 (rAAV)载体注入家兔缺血心肌 ,观察血管内皮生长因子 (VEGF)的血管生成效应。方法　制作家兔急性心肌梗死模型 ,将rAAV hVEGF 16 5注入缺血心肌。 4周后取注射部位心肌 ,用逆转录聚合酶链式反应 (RT PCR)和免疫组织化学、Westernblot方法检测目的基因的表达 ,并计数注射部位心肌毛细血管数目 ,评价外源VEGF的生物学活性。结果　注射rAAV VEGF部位心肌VEGFmRNA及其蛋白的表达可持续 8周 ,而且比对照组增加。远隔脏器未见外源VEGF的播散和表达。注射rAAV VEGF部位毛细血管密度比对照组增加。结论　rAAV hVEGF 16 5可以有效地转染成年家兔心肌组织 ,外源基因稳定长期表达 ,同时能够刺激新血管生成 ,并具有良好的安全性。     

外源性肝细胞生长因子抗肝细胞凋亡机制的研究

目的通过小鼠尾静脉高压注射的方法在肝细胞中建立持续、高效表达外源性肝细胞生长因子(HGF)的体系,探讨其在肝细胞凋亡中的作用机制。方法通过尾部静脉注射pCMV-HGF质粒,检测肝组织中HGF表达的高峰。实验动物分为模型组、pcDNA3保护组、pCMV-HGF质粒保护组和生理盐水对照组,Western blot检测tBid、细胞色素C在各组肝细胞质及线粒体中的表达情况。结果在小鼠尾静脉快速大量注射质粒4h后肝脏中即有外源性HGF表达,8h达高峰。D-GalN/LPS可诱导明显的细胞凋亡,pCMV-HGF保护组tBid及细胞色素C的表达明显减少。结论通过小鼠尾静脉高压注射的方法在肝细胞中建立持续表达外源性HGF的体系,通过抑制Bid的活性片段tBid表达和细胞色素C释放来减轻凋亡蛋白的激活。     

Safety evaluation of clinical gene therapy using hepatocyte growth factor to treat peripheral arterial disease

Therapeutic angiogenesis using angiogenic growth factors is expected to be a new treatment for patients with critical limb ischemia (CLI). Because hepatocyte growth factor (HGF) has potent angiogenic activity, we investigated the safety and efficiency of HGF plasmid DNA in patients with CLI as a prospective open-labeled clinical trial. Intramuscular injection of naked HGF plasmid DNA was performed in ischemic limbs of 6 CLI patients with arteriosclerosis obliterans (n=3) or Buerger disease (n=3) graded as Fontaine III or IV. The primary end points were safety and improvement of ischemic symptoms at 12 weeks after transfection. Severe complications and adverse effects caused by gene transfer were not detected in any patients. Of particular importance, no apparent edema was observed in any patient throughout the trial. In addition, serum HGF concentration was not changed throughout the therapy period in all patients. In contrast, a reduction of pain scale of more than 1 cm in visual analog pain scale was observed in 5 of 6 patients. Increase in ankle pressure index more than 0.1 was observed in 5 of 5 patients. The long diameter of 8 of 11 ischemic ulcers in 4 patients was reduced >25%. Intramuscular injection of naked HGF plasmid is safe, feasible, and can achieve successful improvement of ischemic limbs. Although the present data are conducted to demonstrate the safety as phase I/early phase IIa, the initial clinical outcome with HGF gene transfer seems to indicate usefulness as sole therapy for CLI.     

Percutaneous endocardial transfer and expression of genes to the myocardium utilizing fluoroscopic guidance.

Experimental studies indicate that administration of angiogenic proteins or genes by the epicardial or intracoronary route can stimulate development of new collateral vessels and improve myocardial perfusion. An endocardial catheter-based approach to this therapy would obviate the need for surgery, while preserving the effectiveness of direct intramyocardial administration. Fluoroscopic guidance and prototype, preformed, coaxial catheters were used to examine the feasibility of percutaneous catheter-based adenovirus (Ad)-mediated gene transfer and expression in normal swine myocardium. The feasibility of intramyocardial administration (100 microl/injection) of a radiocontrast agent and black tissue dye to all regions of the left ventricle (septum, anterior, lateral, and inferior wall) was confirmed fluoroscopically and on postmortem examination. Injections of replication-deficient adenovirus (10 injections of 10(11) particle units/100 microl each) coding for beta-galactosidase (Adbetagal) or vascular endothelial growth factor (Ad(GV)VEGF121.10) were administered to the left ventricular free wall to examine endocardial based gene transfer and expression. beta-Galactosidase activity was detected by histochemical staining and quantitative assay in targeted regions of the myocardium. Regional VEGF expression was found to be significantly greater in targeted regions (1.3 +/- 0.4 ng/mg protein) as compared with non-targeted regions (0.3 +/- 0.1 ng/mg protein) or regions injected with control (Adbetagal) virus (0.2 +/- 0.03 ng/mg protein, P < 0.001). Catheter-based Ad mediated endocardial gene transfer and expression is feasible using percutaneous, fluoroscopically guided, preformed, coaxial catheters. This approach should be clinically useful to administer angiogenic genes to the ischemic myocardium.