Combination of VEGF(165)/Angiopoietin-1 gene and endothelial progenitor cells for therapeutic neovascularization

Previous studies have established that vascular endothelial growth factor (VEGF), Angiopoietin-1 (Ang1) and endothelial progenitor cells (EPCs) play important roles in neovascularization, suggesting that combination of them would be a promising therapy for ischemic diseases. So we constructed the adeno-associated virus-2 (AAV-2) vectors simultaneously encoding human VEGF(165) and Ang1 (AAV-Ang1/VEGF), and investigated the combination therapeutic effect of AAV-Ang1/VEGF with EPCs in a rabbit ischemic hindlimb model. In the present study we found that AAV-Ang1/VEGF could successfully and efficiently transfer VEGF(165) and Ang1 gene into bone marrow derived EPCs for gene therapy. Combined administration of AAV-Ang1/VEGF with EPCs had higher blood flow recovery, cellularity, capillary density and smooth muscle alpha-actin positive vessel density than administration of either of them alone. Furthermore, the strategy of pre-intramuscular injection of AAV-Ang1/VEGF followed by EPCs transplantation had a higher therapeutic effect than the strategy of transplantation of AAV-Ang1/VEGF transduced EPCs. It seemed that the former strategy may be a promising therapy for ischemic diseases.     

Angiopoiesis and bone regeneration via co expression of the hVEGF and hBMP genes from an adeno-associated viral vector in vitro and in vivo

Aim:

To investigate the therapeutic potential of adeno-associated virus (AAV)-mediated expression of vascular endothelial growth factor (VEGF) and bone morphogenetic protein (BMP).

Methods:

Four experimental groups were administered the following AAV vector constructs: rAAV-hVEGF₁₆₅-internal ribosome entry site (IRES)-hBMP-7 (AAV-VEGF/BMP), rAAV-hVEGF₁₆₅-GFP (AAV-VEGF), rAAV-hBMP-7-GFP (AAV-BMP), and rAAV-IRES-GFP (AAV-GFP). VEGF₁₆₅ and BMP-7 gene expression was detected using RT-PCR. The VEGF₁₆₅ and BMP-7 protein expression was determined by Western blotting and ELISA. The rabbit ischemic hind limb model was adopted and rAAV was administered intramuscularly into the ischemic limb.

Results:

Rabbit bone marrow-derived mesenchymal stem cells (BMSCs) were cultured and infected with the four viral vectors. The expression of GFP increased from the 7th day of infection and could be detected on the 28th day post-infection. In the AAV-VEGF/BMP group, the levels of VEGF165 and BMP-7 increased with prolonged infection time. The VEGF₁₆₅ and BMP-7 secreted from BMSCs in the AAV-VEGF/BMP group enhanced HUVEC tube formation and resulted in a stronger osteogenic ability, respectively. In rabbit ischemic hind limb model, GFP expression increased from the 4th week and could be detected at 8 weeks post-injection. The rAAV vector had superior gene expressing activity. Eight weeks after gene transfer, the mean blood flow was significantly higher in the AAV-VEGF/BMP group. Orthotopic ossification was radiographically evident, and capillary growth and calcium deposits were obvious in this group.

Conclusion:

AAV-mediated VEGF and BMP gene transfer stimulates angiogenesis and bone regeneration and may be a new therapeutic technique for the treatment of avascular necrosis of the femoral head (ANFH).     

Adeno-associated virus-mediated vascular endothelial growth factor gene transfer into cardiac myocytes

Vascular endothelial growth factor (VEGF) is an angiogenic growth factor that stimulates endothelial cell proliferation, increases endothelial permeability, and promotes collateral vessel formation. We transferred human VEGF gene into rat cardiac myocytes using adeno-associated virus (AAV) vectors and investigated whether VEGF secreted from the transduced cardiac myocytes promoted proliferation of endothelial cells. We produced VEGF-expressing AAV vectors (AAV-VEGF) by the adenovirus-free method. Immunoblotting revealed VEGF protein expression in AAV-VEGF-transduced rat cardiac myocytes. More than 60% of cardiac myocytes were stained positively on immunohistochemical staining using anti-VEGF antibody. Concentration of VEGF in the culture medium of AAV-VEGF-transduced myocytes was increased in a vector dose-dependent manner, and VEGF secretion from the transduced myocytes persisted for > or = 14 days. Thymidine incorporation into human vascular endothelial cells was significantly increased by incubation with the conditioned medium from AAV-VEGF-transduced myocytes. This increased thymidine uptake was significantly inhibited by anti-VEGF antibody. We demonstrated here that AAV-mediated VEGF gene transfer into cardiac myocytes induces the secretion of functional VEGF.     

血管内皮细胞生长因子-165和血管形成素-1促进干细胞动员

目的 研究缺血肌肉转染腺病毒(Ad-)携带人血管内皮细胞生长因子-165(VEGF-165)和人血管形成素-1(Ang-1)基因后引起的细胞动员效应。方法 制作大鼠下肢血管闭塞模型,肌肉内注射Ads-VEGF-165和／或Ad5-Ang-1,以Western法检测生长因子蛋白表达、免疫组化检测基因导入后在缺血肌肉引起的效应。结果(1)局部转基因肌肉组织高表达人VEGF-165蛋白和人Ang-1蛋白;(2)与对照组相比,接收两因子转染后的骨骼肌纤维间溴化脱氧尿嘧啶(BrdU)阳性的浸润细胞显著增多,呈协同效应。有细胞同时表达内皮细胞、骨骼肌细胞、平滑肌细胞标志性抗原。部分细胞表达CD117^ 并分化为新生微血管的内皮细胞。结论(1)两因子能促进CD117^ 骨髓干细胞动员到缺血组织部位并分化为内皮细胞参与血管新生;(2)两因子可能协同动员召集能在缺血部位聚集并分化为其它类型细胞的干／祖细胞;(3)两因子可能同时作为血管生成因子和细胞动员剂用于缺血性疾病的干细胞移植治疗中,以增强缺血损伤修复效果和节省干细胞用量。     

Effect of Erythropoietin on Angiogenesis With the Increased Adhesion of Platelets to the Microvessels in the Hind-Limb Ischemia Model in Mice

Erythropoietin (EPO) has been shown to enhance angiogenesis, but its precise mechanisms of enhancement during ischemia are not fully elucidated. We examined the effect of EPO on blood flow recovery from acute hind-limb ischemia induced by ligation of the femoral artery in male C57Bl/6 mice. The density of microvessels with platelet adhesion in ischemic tissues was assessed by intravital microscopy. Treatment with EPO (100 and 1000 IU/kg, i.p.) restored blood flow in a dose-dependent manner and increased plasma levels of soluble-P-selectin, vascular endothelial growth factor (VEGF), and stromal cell–derived factor (SDF-1). Flow cytometric analysis revealed increased P-selectin expression on platelets in EPO-treated mice compared to PBS-treated mice. Intravital microscopic studies showed that EPO increased density of microvessels with platelet adhesion selectively in the ischemic tissues. Neutralizing antibody against P-selectin reduced the density of microvessels with platelet adhesion enhanced with EPO and impaired blood flow recovery with reductions in VEGF and SDF-1 levels. These results suggest that EPO administration enhances recovery from hind-limb ischemia, and platelet adhesion to the microvessels is a key event to enhance the angiogenesis in the ischemic tissues.     

当归补血汤对脑缺血再灌注损伤大鼠血管新生及Ang-1、Ang-2、VEGF和Tie-2的影响

目的 探究当归补血汤对脑缺血再灌注损伤大鼠血管新生及Ang-1、Ang-2、VEGF和Tie-2的影响.方法 采用线栓法复制脑缺血再灌注损伤大鼠模型,模型复制后随机分为假手术组、模型组、当归补血汤低剂量组和当归补血汤高剂量组.连续给药14 d,评价术后第1天和第14天的神经功能,免疫组化检测脑缺血区域及周围的微血管密度和Tie-2表达水平,酶联免疫吸附法检测血清中Ang-1和Ang-2浓度,Weatern Blotting法检测脑组织中VEGF表达水平.结果 连续给药14 d后当归补血汤高剂量组和低剂量组的神经功能评分、MVD、Ang-1和Ang-2浓度、Tie-2和VEGF表达水平均显著高于模型组,且随着当归补血汤剂量的增加,各指标亦显著性增加.结论 当归补血汤促进脑缺血再灌注损伤大鼠的神经功能的恢复,而其作用机制可能为通过促进Ang-1、Ang-2、Tie-2和VEGF的表达而使脑缺血部位的血管新生.     

Contraindications of VEGF-based therapeutic angiogenesis: effects on macrophage density and histology of normal and ischemic brains

Therapeutic angiogenesis by vascular endothelial growth factor (VEGF) is advocated as a promising treatment strategy for brain ischemic stroke. However, data in the literature demonstrating the benefit of therapeutic angiogenesis are contradictory. In this paper, we describe the effects of non-angiogenic and angiogenic doses of VEGF165 on macrophage density and histology of normal and ischemic brains of adult rats. VEGF165 was administered intra-arterially for 7 days following temporary occlusion of the middle cerebral artery. In contrast to ischemic brains treated with non-angiogenic doses of VEGF165 which showed preserved neuropil and reduced numbers of macrophages, ischemic brains treated by an angiogenic dose showed phagocytized neuropil and high macrophage density. Though neither non-angiogenic nor angiogenic doses caused macrophage infiltration in normal brains, damage of the brain matrix occurred with the angiogenic dose. These results suggest an angiogenic dose of VEGF165 injures the nervous tissue rather than promote recovery. Angiogenesis by VEGF monotherapy for ischemic stroke should be viewed with caution, or avoided. Since our data show intravascular administration of VEGF165 does not cause macrophage inflammation, in contrast to reports in the literature whereby VEGF165 was applied directly to the brain, our findings also indicate the relationships between VEGF, angiogenesis, and macrophage inflammation are governed by the route VEGF is administered to the brain.     

重组VEGF165基因腺相关病毒的包装和鉴定

目的包装重组VEGF165基因的无致病性腺相关病毒,以其作为基因载体感染HUVEC细胞并检测其表达,并在体外检测病毒生物学活性。方法从含有VEGF165基因的pET-32a（＋）-VEGF165原核表达载体中扩增出VEGF165片段,构建重组骨架质粒pAAV-VEGF165。将此质粒和对照质粒pAAV-GFP分别与腺相关病毒包装质粒pAAV-RC、辅助质粒pAAV-Helper用钙-磷共转法转染HEK293细胞,通过同源重组分别产生rAAV-VEGF165和rAAV-GFP重组腺相关病毒。通过real-timePCR法测定病毒滴度后,转染HUVEC细胞并检测其表达,并通过感染HUVEC来检测其促增殖作用。结果扩增出的VEGF165片段成功构建至重组骨架质粒中,pAAV-VEGF165经双酶切和测序鉴定正确。病毒包装效率达90%以上,收获纯化rAAV-VEGF165病毒滴度达6.0×1010pfu/mL。重组腺病毒rAAV-VEGF165能够感染HUVEC细胞并得到显著性表达;与未转染组和GFP组相比,能够显著促进HUVEC细胞的增殖。结论我们成功包装了重组腺相关病毒rAAV-VEGF165,它能够感染HUVEC细胞并高表达VEGF165蛋白,并具有促增殖作用,这为进一步开展VEGF165基因的基因靶向治疗奠定了基础。     

Adeno-associated virus serotype 8 ApoA-I gene transfer reduces progression of atherosclerosis in ApoE-KO mice: comparison of intramuscular and intravenous administration

Apolipoprotein A-I (ApoA-I)/high-density lipoprotein (HDL)-raising treatments are effective antiatherosclerotic strategies. We have compared the antiatherogenic effects of human ApoA-I (hApoA-I) overexpression by intraportal and intramuscular gene transfer in atherosclerotic ApoE-knockout mice. Atherosclerotic lesions were induced by atherogenic diet. After atherosclerosis induction, a group of animals was killed and served as atherosclerosis baseline-control group. The remaining animals were randomized into the following groups: (1) atherosclerosis-progression-control, (2) intraportal/vector administration, and (3) intramuscular/vector administration. Aortas and hearts were processed for atherosclerotic quantification by en face Sudan IV and Oil Red-O, respectively. Liver and muscle specimens were processed for protein/gene expression analysis. A sustained increase in hApoA-I/HDL plasma levels was observed in both transduced groups. hApoA-I overexpression abolished plaque progression versus progression-control group. hApoA-I overexpression significantly reduced lesion macrophage, feature indicative of plaque stabilization. Scavenger receptor class-B type I (SR-BI), but not ATP-binding cassette, sub-family A (ABCA), member 1 (ABCA-1), was significantly upregulated in treated groups versus progression-controls. The results of this study show a similar effect of hApoA-I/HDL overexpression on plaque progression/stabilization by 2 different routes of administration. Our results showing similar effects using either intramuscular administration and intraportal route of administration may have significant clinical implications, given the reduced medical risk to patient and cost of intramuscular injections.     

Intramuscular delivery of rAAV-mediated kallikrein gene reduces hypertension and prevents cardiovascular injuries in model rats

Aim： The overexpression of the human tissue kallikrein （HK） gene can reduce blood pressure and ameliorate the secondary syndromes associated with hyper- tension in animal models. The current study was designed to investigate hy- potensive effect of intramuscular delivery of HK gene. Methods： We generated an recombinant adeno-associated virus （rAAV） vector expressing human tissue kallikrein under the control of a cytomegalovirus promoter and administered the rAAV-HK vector to a spontaneously hypertensive rat model at a dose of lx 10^10 virons/rat through intramuscular injection. Results： A persistent, high-level ex- pression of HK post-gene delivery was confirmed by ELISA. The systolic blood pressure in the rats receiving rAAV-LacZ and saline increased from 171.3 mmHg to 182.3 mmHg 28 weeks＇ post injection. In contrast, the delivery of the HK gene by AAV vectors attenuated the increase of the systolic blood pressure in the treated group. The systolic blood pressure was only slightly lowered （from a level of 174 mmHg to 170.5 mmHg） post-vector administration. The difference in blood pres- sure between the treated group and the control groups is statistically significant at 12.6 mmHg. The hypotensive effect of rAAV-HK persisted until the end of the testing period. In addition, a significant amelioration of cardiovascular hypertrophy, renal injury, and collagen depositions in the rAAV-HK-treated ani- mals were also observed. Conclusion： All the effects are comparable with those of intravenous delivery. Therefore, the intramuscular administration of rAAV-HK may be used in gene therapy for hypertension.     

冠状静脉窦逆行灌注VEGF蛋白的实验研究

目的观察经冠状静脉窦逆行灌注血管内皮生长因子(VEGF)对其分布的影响。方法20只新西兰大白兔随机分为实验组(VEGF组)和对照组,2组动物常规全麻,气管插管,胸骨正中切开,暴露心脏,结扎左室支建立心肌梗死模型,冠状静脉窦插管,实验组逆行灌注VEGF蛋白,对照组以生理盐水对照。全程记录心电,逆行灌注后30min取梗死区(左室心尖部)及非梗死区(高侧壁)心肌检测VEGF蛋白分布情况。结果结扎左室支即刻所有动物出现ST段抬高并与T波融合,免疫组织化学显示实验组梗死区有大量的VEGF蛋白,Western blot分析显示实验组梗死区在22kd处有明显的阳性条带,而非梗死区及对照组则为阴性。结论冠状静脉窦逆行灌注技术能很好地将VEGF蛋白靶向导入梗死区。     

丹参多酚酸对脑梗死小鼠血管微循环的影响

目的 探究丹参多酚酸对脑梗死大鼠神经血管微循环及缺血侧血流的影响。方法 将大鼠随机分为假手术组、模型组、尼莫地平10 mg/kg组和丹参多酚酸低、高剂量组（10、25 mg/kg）。制备大鼠中动脉缺血模型,通过神经功能评分、2,3,5-三苯基氯化四氮唑染色以及采用激光散斑血流成像检测脑血流量,鉴定模型构建是否成功。各组大鼠按照分组进行给药,尼莫地平组ig尼莫地平10 mg/kg,丹参多酚酸组ip 10、25 mg/kg注射用丹参多酚酸,连续治疗7 d,1次/d。取脑组织分别进行2,3,5-三苯基氯化四氮唑染色染色评估脑梗死体积,伊文思蓝含量测定评估血、脑、脊液屏障结构完整性,免疫荧光染色测量血小板内皮细胞黏附分子（CD31）表达。采用Western blotting实验检测血管内皮生长因子（VEGF）、血管生成素-1(Ang-1)、磷酸甘油醛脱氢酶（GAPDH）蛋白表达。结果 与模型组相比,丹参多酚酸25 mg/kg组神经功能评分、脑梗死体积占比明显减低,脑血流量明显增加（P<0.05）;且缺血侧CD31相对表达明显减少;伊文思蓝含量以及VEGF与Ang1蛋白表达明显增加（P&l...     

κ阿片受体激动剂U50488H对血管紧张素Ⅱ诱导内皮细胞产生白介素-6和白介素-8的影响

目的探讨κ阿片受体在血管紧张素Ⅱ诱导内皮细胞产生白介素-6和白介素-8中的作用。方法整体实验观察大鼠静脉注射U50488H(1.5 mg.kg-1)后血中AngⅡ水平的变化;体外培养人脐静脉内皮细胞,分为对照组、AngⅡ组、AngⅡ+U50488H组,AngⅡ+U50488H+nor-BNI(κ阿片受体阻断剂)组。分别用磷酸盐缓冲溶液、AngⅡ(10-9~10-5mol.L-1)或提前给予κ阿片受体激动剂或(和)阻断剂诱导0~24 h,收集0、3、6、12、24 h等时间点的细胞培养液,采用酶联免疫吸附法分别检测细胞培养液IL-6和IL-8水平。结果静脉注射U50488H可明显降低血中AngⅡ的水平,该作用可被nor-BNI(2 mg.kg-1)所阻断。AngⅡ可浓度和时间依赖性诱导内皮细胞产生释放IL-6和IL-8,提前给予U50488H(10-5mol.L-1)可明显抑制AngⅡ诱导内皮细胞产生释放IL-6和IL-8。而nor-BNI(10-5mol.L-1)本身没有任何作用但可完全阻断U50488H的上述作用。结论 U50488H可通过下调AngⅡ水平和抑制AngⅡ的作用来减少内皮细胞产生IL-6和IL-8,表明κ阿片受体可能在抗炎中具有一定调节作用。     

Transferability of cefodizime to cerebrospinal fluid of rabbits with meningitis caused by Staphylococcus aureus

The transferability of cefodizime (THR-221, CDZM) to cerebrospinal fluid (CSF) was studied employing rabbits with experimental meningitis caused by Staphylococcus aureus. The mean blood concentration was 195 +/- 18.3 micrograms/ml using phosphate buffer solution (PBS) standard and 474 +/- 22.0 micrograms/ml using rabbit serum standard, respectively, at 15 minutes after intravenous administration of the drug at a dose level of 100 mg/kg. The mean concentration in CSF vs. PBS standard was maximum at 60 minutes after administration, and the mean maximum concentration was 8.74 +/- 2.16 micrograms/ml. Pharmacokinetic parameters calculated from those values were as follows, respectively, for PBS standard and rabbit serum standard; Cmax (CSF/serum): 4.48% and 1.84%. AUC (CSF/serum): 6.15% and 2.02% between 15 and 60 minutes, 10.6% and 3.00% between 15 and 120 minutes and 13.4% and 3.48% between 15 and 180 minutes. T 1/2 for CDZM in CSF: 141 minutes in both cases. T 1/2 (CSF/serum): 3.27 and 2.11. Concentrations in CSF determined using an high performance liquid chromatography method in another rabbits were similar to those determined using the bioassay vs. rabbit serum standard. The bioassayed concentration of this drug (AUC (CSF/serum] vs. PBS standard ranked 9th among 23 other beta-lactam antibiotics tested. That is, the drug distributed favorably as compared to other antibiotics, and it may be worthwhile of running clinical trials on this drug in meningitis when antimicrobial potential against main pathogens of meningitis are considered.     

Involvement of chymase in angiogenesis in hamster sponge granulomas

We investigated the angiogenic effect of chymase, an alternative angiotensin II-generating enzyme, on angiogenesis using hamster sponge implant model. Exogenous administration of angiotensin II (Ang II) or angiotensin I (Ang I) directly into the sponges enhanced angiogenesis, as determined from the hemoglobin contents in the sponge granuloma tissues. Chymostatin, an inhibitor of chymase, inhibited angiogenesis induced by Ang I but not by Ang II, suggesting the presence of a chymase-like Ang II-generating activity in the sponge granuloma. TCV-116 (5 mg/kg p.o.), an antagonist of Ang II type 1 receptor, and chymostatin suppressed bFGF-induced angiogenesis, suggesting the significance of the endogenous angiotensin system. Chymase activity in the sponge granuloma increased in parallel with the rise in hemoglobin contents induced by bFGF. We also examined the effects of direct administration of human pro-chymase gene or purified hamster chymase, and demonstrated that in vivo human pro-chymase gene transfection and direct injection of purified chymase enhanced angiogenesis, which was 50% inhibited by TCV-116. Sponge granulomas treated with Ang II was supressed by vascular endothelial growth factor (VEGF) antisense. Our results suggest that chymase enhanced angiogenesis partly through the local production of Ang II, followed by up-regulation of VEGF.     

Gene Therapy for Ischemic Heart Disease

Gene therapy is evolving as an alternative mode to pharmacological intervention in the treatment of cardiovascular diseases. Experimental observations indicating that introduction of genes encoding for angiogenic peptide growth factors could result in improvement in perfusion to ischemic myocardium have led to the initiation of a number of preliminary clinical trials to evaluate this therapeutic modality. Sustained expression of the growth factor product from somatic cells transfected with the DNA for that protein has proven to be one of the major advantages of a gene therapy—based approach over administration of the recombinant protein. A number of gene therapy vectors have been developed, prominent among these being adenoviral vectors and naked plasmid DNA. Whereas plasmid DNA results in less efficient transfection, its tolerability profile may be superior to adenoviral vectors. Plasmid DNA is particularly suitable when the gene product to be produced is capable of being secreted by the cell which is producing it. Vascular endothelial growth factor (VEGF) is not only essential to the process of angiogenesis, but, because it can be secreted from intact cells, appears to be ideal for gene transfer therapy aimed at improving perfusion to ischemic myocardium. The DNA can be delivered to the myocardium by intra-arterial or intramuscular injection. At present, direct injection into the muscle either via a small thoracotomy incision or by use of a recently developed percutaneous catheter technique appears to be superior to arterial administration. Several clinical trials based on intramyocardial injection of VEGF DNA in patients with otherwise inoperable coronary artery disease and intractable angina pectoris have recently been completed. These phase I trials have documented the tolerability of gene transfer using plasmid DNA and show promise of being able to improve myocardial perfusion and reduce anginal symptoms in the majority of patients treated thus far. While the trials involving gene transfer via a thoracotomy did not allow for randomization to a placebo group, the recent advent of a percutaneous delivery modality has allowed for randomization which should enhance our ability to determine whether angiogenic gene therapy will prove to be as effective as initial results suggest. In the future, results from such randomized placebo-controlled trials, improvement in vectors utilized for gene transfer and innovative new delivery techniques will undoubtedly enhance the potential of this novel approach to myocardial revascularization.     

AngⅡ受体阻断剂与ACEI对肾性高血压大鼠血以及血浆和组织AngⅡ含量变化的影响

目的明确血管紧张素转换酶抑制剂(ACEI)卡托普利与血管紧张素Ⅱ(AngⅡ)受体阻断剂洛沙坦二类药物的药效和作用特点。方法本实验采用①离体血管环微量生物反应测定法检测AngⅡ引起的血管收缩反应;②建立两肾一夹型高血压大鼠模型;利用颈动脉插管法和鼠尾测压计检测血压,观测急性与慢性血压的变化;③用放射免疫法检测高血压大鼠血浆与肾组织中的AngⅡ含量。结果①离体血管环实验:AngⅡ能引起剂量依赖性的血管收缩反应,卡托普利(0.1 mg/kg)对低剂量AngⅡ收缩反应有轻度的抑制效应;随着外源性AngⅡ量增多,其抑制血管收缩作用明显减弱。同样条件下洛沙坦却能完全抑制AngⅡ所引起的血管收缩反应。②在体急性降压实验:最大有效剂量的卡托普利使模型鼠的平均动脉压由(18.4±3.9)kPa降为(8.7±1.2)kPa,降压幅度达到9.6 kPa,之后给洛沙坦最大降压有效剂量(2 mg/kg),血压未再下降;改变给药顺序,平均动脉压由(16.8±1.1)kPa降为(11.4±2.4)kPa,降压幅度为5.30 kPa,然后给最大有效降压剂量的卡托普利,血压持续降为(9.3±1.8)kPa,幅度达2.12 kPa,差异具有明显的统计学意义(P<0.05)。③慢性降压实验:高血压大鼠模型给予实验因素干预后,卡托普利组平均动脉压由(18.9±2.5)kPa降为(11.8±1.6)kPa,洛沙坦平均动脉压由(19.7±2.4)kPa降为(11.7±2.0)kPa,降压幅度分别为7.19 kPa和7.93 kPa,与对照组比较差异无统计学意义。④放射免疫实验:血浆中AngⅡ含量卡托普利组为(376±72)ng/L,明显低于对照组的(526±77)ng/L,而洛沙坦组为(1 036±159)ng/L,明显高于对照组(P<0.01),二者差异具有统计学意义。肾组织中AngⅡ含量,卡托普利组(392±81)pg/g,较对照组的(431±80)pg/g降低8.9%,洛沙坦组为(294±86)pg/g,较对照组降低32.4%(P<0.05)。结论①洛沙坦是通过阻断AngⅡ受体而发挥作用,而卡托普利只能够减少内源性AngⅡ的生成,离体状态下药效弱于洛沙坦。②急性在体实验卡托普利的最大降压效应强于洛沙坦;慢性在体实验二者药效差异无统计学意义。③长期作用下,肾组织的AngⅡ水平对调节血管张力起主要的作用,血浆中AngⅡ辅助起作用。     

A synthetic prostacyclin agonist with thromboxane synthase inhibitory activity, ONO-1301, protects myocardium from ischemia/reperfusion injury

ONO-1301, a synthetic prostacyclin agonist with thromboxane synthase inhibitory activity, promotes the production of hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) by various cell types. Here, we evaluated the therapeutic efficacy of ONO-1301 in rats with ischemia/reperfusion injury. Ligation of the left anterior descending arteries was performed in 10-week-old Wistar rats, and released 30 min later. A slow-release form of ONO-1301 was administered subcutaneously at 3h and 3 weeks after reperfusion injury. Hemodynamic parameters were significantly improved in the ONO-1301 group. Histological analysis revealed that ONO-1301 suppressed ischemic and fibrotic changes in the myocardium (ischemic area, control group: 58.6 ± 8.7% vs. ONO-1301 group: 44.4 ± 5.8%, fibrotic area, 33.5 ± 5.9% vs. 22.3 ± 6.2%, P<0.05, respectively), and enhanced neovascularization in the border zone. HGF expression was up-regulated by ONO-1301. Double-immunostaining revealed that myofibroblasts in the border zone of ischemic myocardium mainly expressed HGF. Our findings suggest that ONO-1301 might have therapeutic potential in treating ischemic heart disease.     

Size Distribution Analysis of the Adeno-Associated Virus Vector by the c(s) Analysis of Band Sedimentation Analytical Ultracentrifugation with Multiwavelength Detection

Adeno-associated virus (AAV) vector is a promising platform for in vivo gene therapy. The accurate assessment of distribution state of particles contained in AAV vector samples is one of the most important and challenging matters and is necessary because the product-related impurities with the capsid structure (empty particles, intermediate particles, and aggregates) could be a possible cause of reducing the therapeutic efficacy and enhancing the unfavorable immune response. In this study, we report an effective approach for size distribution analysis with component identification. A small amount of AAV vectors were used by the analytical zone centrifugation c(s) analysis of band sedimentation analytical ultracentrifugation (BS-AUC) with multiwavelength detection. Using PBS/H₂¹⁸O, the concentration of each component could be determined in BS-AUC with high resolution. Compared with the sedimentation velocity AUC (SV-AUC), which generally requires 2 × 10¹² vg of AAV vectors, BS-AUC could be performed with about 1/25 of the AAV vector amount at 260 nm detection and ideally with about 1/50 of the AAV vector amount at 230 nm detection (4 × 10¹⁰ vg), depending on the extinction coefficient of the AAV sample at each wavelength. According to the limit of quantification of this BS-AUC, 6.3 × 10¹¹ cp mL⁻¹ of empty particle (EP) and 4.4 × 10¹¹ vg mL⁻¹ of full particle (FP) could be quantified for 4 × 10¹⁰ vg in 15 µL of AAV8-CMV-EGFP. These results demonstrated that proposed BS-AUC approach we established here can compensate for the drawback in terms of the sample amount of SV-AUC.