Combined cord blood stem cells and gene therapy enhances angiogenesis and improves cardiac performance in mouse after acute myocardial infarction

BACKGROUND: Gene and stem cell therapies hold promise for the treatment of ischaemic cardiovascular disease. However, combined stem cell and angiogenic growth factor gene therapy for acute ischaemic myocardium has not been previously reported. This study hypothesized that combined stem cell and gene therapy would not only augment new vessels formation but also improve myocardial function in acute ischaemic myocardium. METHODS: Human angiopoietin-1 (Ang1) cDNA and VEGF(165) cDNA were ligated into AAV vector. The purified CD34(+) cells were obtained from human umbilical cord blood samples. Cord blood CD34(+) cells were transduced with AAV vector encoding either the human Ang1 (AAV-Ang1) or VEGF(165) (AAV-VEGF) cDNA alone, or both (AAV-Ang1 plus VEGF). Immediately after ligation of the left anterior descending coronary artery in male SCID mice, culture-expanded CD34(+) cells transduced with AAV-Ang1, AAV-VEGF or AAV-Ang1 plus VEGF were injected intramyocardially at the left anterior free wall. RESULTS: Western blot showed that Ang1 and VEGF protein expressions were enhanced in the CD34(+)cells transduced with AAV-Ang1 and AAV-VEGF, respectively. Infarct size significantly decreased and capillary density significantly increased after treatment with CD34(+)/AAV-Ang1 plus VEGF when compared with treatment by CD34(+) only. Combined therapy with CD34(+) and AAV-Ang1, CD34(+) and AAV-VEGF, CD34(+) and AAV-Ang1 plus VEGF, all showed significantly higher cardiac performance in echocardiography than the therapy with CD34(+) alone 4 weeks after myocardial infarction. CONCLUSIONS: Combined therapy with human umbilical cord blood CD34(+) cells and both Ang1 and VEGF genes reduced infarct size, attenuated the progression of cardiac dysfunction and increased capillary density in acute myocardial infarction in mice.     

Formulation and characterization of poly (D,L-lactide-co-glycolide) nanoparticle containing vascular endothelial growth factor for gene delivery

OBJECTIVE: The stability, in vitro release, in vitro cell transfection efficiency and in vivo gene transfer of vascular endothelial growth factor (VEGF(165)) plasmid DNA-loaded poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles were investigated. METHODS: DNA-loaded nanoparticles were prepared with PLGA bearing VEGF(165) plasmid DNA and characterized with respect to morphology, size and encapsulation efficiency. The gene transfer efficiency of nanoparticles was tested in vitro on the cultured myocardial cells. And then the suspension of VEGF-loaded nanoparticles (VEGF-NPs) was injected into myocardial tissues in vivo to observe the process of nanoparticles as vectors for gene transfer to cardiac myocytes and to detect its biological effect and evaluate angiogenesis. RESULTS: The encapsulation efficiency of the VEGF-NPs was 58.06 +/- 2.8% and their diameter ranged 100-300 nm. VEGF gene could be successfully transfected into myocardial cells by nanoparticles, which significantly enhanced gene transfection efficiency. A great number of nanoparticles were seen in the myocardial cytoplasm and nucleus. Furthermore, the nanoparticles began to dissolve and degrade. There was a significant increase in the number of capillaries in the VEGF-NPs group when compared with the plasmid DNA group. CONCLUSION: The PLGA nanoparticles are capable of DNA delivery to cardiac myocytes for treating ischaemic myocardium. The direct gene transfer of VEGF-NPs into ischaemic rabbit myocardium can improve cardiac function and increase the number of capillaries.     

人血管生成素-1和血管内皮生长因子VEGF_(165)腺病毒载体构建

目的:克隆人血管内皮生长因子(vascularendothelialgrowthfactor165,VEGF165)和血管生成素-1(angiopoietin-1)的全长编码基因,构建表达该基因的复制缺陷型腺病毒载体Ad-Ang1和Ad-VEGF165。方法:通过RT-PCR方法克隆人VEGF165和Ang1全长编码基因。Ad-VEGF165和Ad-Ang1通过同源重组方法构建。将Ad-VEGF165和/或Ad-Ang1转染大鼠胚胎心脏成肌细胞(H9C2)24h后,Westernblot方法分析VEGF165和Ang1蛋白表达量;核酸电泳分析细胞基因组降解检测凋亡水平。结果:测序显示VEGF165序列与基因库序列相同;Ang1序列与基因库序列存在一个碱基的差异,但编码氨基酸无改变。VEGF165和Ang1蛋白表达分别为对照组的11.65倍和3.53倍。Ad-VEGF165和/或Ad-Ang1转染后的H9C2细胞对过氧化氢诱导的细胞凋亡具有明显的抵抗能力。结论:所构建的Ad-Ang1和Ad-VEGF165能有效转染心脏成肌细胞,表达出功能性目的蛋白,具有抗细胞凋亡作用。     

同种异体骨髓间质干细胞移植联合VEGF基因转染治疗心肌梗死的实验研究

目的：探讨同种异体骨髓间质千细胞（BMSCs）移植联合血管内皮生长因子（VEGF）基因转染对大鼠急性心肌梗死血管再生和心功能的影响：方法：体外分离、纯化、培养大鼠BMSCs，以BrdU标记细胞；制备、抽提、纯化质粒PAdTrack／VEGF165。结扎冠状动脉建立急性心肌梗死模型2周后，随机将其分为4组（n=12），进行心肌内BMSCs移植和／或VEGF165转染，组Ⅰ：BMSCs移植＋VEGF165转染；组Ⅱ：BMSCs移值；组Ⅲ：VEGF165转染；组Ⅳ：DMEM注射作为对照组。4周后行免疫组化和超声心动图检查：结果：组Ⅰ和组Ⅱ梗死及缺血心肌处可见大量BrdU标记的移植细胞，其中缺血心肌处部分移植细胞分化为血管内皮细胞并形成新生毛细血管。Ⅷ因子染色阳性的新生血管密度分布为组Ⅰ〉组Ⅲ〉组Ⅱ〉组Ⅳ（P均〈0．01）。细胞移植和基因转染治疗后室壁厚度和室壁运动幅度改善，EF值增加幅度（△EF）组Ⅰ〉组Ⅱ〉组Ⅲ〉组Ⅳ（P均〈0．01）：结论：同种异体骨髓间质干细胞移植联合VEGF基因转染能进一步增强大鼠梗死缺血区血管再生、改善室壁厚度和心功能，有利于心肌梗死后的康复。     

Periadventitial angiopoietin-1 gene transfer induces angiogenesis in rabbit carotid arteries

This study was performed to evaluate angiogenic responses of angiopoietin-1 (Ang1) in vivo after adenovirus-mediated gene transfer in the periadventitial space of the rabbit carotid arteries using a collar technique. Adenoviruses encoding LacZ and vascular endothelial growth factor (VEGF) receptor-1-Ig fusion protein (VEGF-R1-Ig) adenoviruses were used as controls. Increased neovessel formation was seen in adventitia of the Ang1 transduced arteries 7 days after the gene transfer. Neovessels in the Ang1 transduced arteries were large in size and well perfused. Ang1 binds to Tie2 (tyrosine kinase with immunoglobulin and epidermal growth factor homology domain) receptors, which were expressed in the endothelium of the neovessels. When VEGF-R1-Ig was used with Ang1, it resulted in a decrease in the number of neovessels, which implies that VEGF-A or some other VEGF-R1 ligand(s) play a crucial role in angiogenesis occurring in response to Ang1. There were no significant differences in the total number of capillaries in the adventitia of the VEGF-R1-Ig transduced arteries as compared to LacZ controls. Neointima formation was not increased in the Ang1 transduced arteries as compared to the controls. We conclude that in the periadventitial space Ang1 shows angiogenic activity and is a potentially useful factor for the induction of therapeutic vascular growth in vivo.     

Secoisolariciresinol diglucoside: relevance to angiogenesis and cardioprotection against ischemia-reperfusion injury

Therapeutic angiogenesis represents a novel approach for the prevention and treatment of ischemic heart disease. This study examined a novel method of stimulating myocardial angiogenesis using secoisolariciresinol diglucoside (SDG), a plant lignan isolated from flaxseed. SDG has been shown to decrease serum cholesterol and reduce the extent of atherosclerosis. In the present study, the angiogenic properties of SDG were investigated in three different models. First, in the in vitro model, human coronary arteriolar endothelial cells (HCAEC) treated with SDG (50 and 100 microM) showed a significant increase in tubular morphogenesis compared with control. Western blot analysis indicated an increased expression of vascular endothelial growth factor (VEGF), kinase insert domain-containing receptor (KDR), Flt-1, angiopoietin-1 (Ang-1), Tie-1, and phosphorylated endothelial nitric oxide synthase (p-eNOS) in the SDG-treated cells. Second, in the ex vivo ischemia/reperfusion model, SDG-treated rats (20 mg/kg b.wt./day for 2 weeks orally) showed an increased level of aortic flow and functional recovery after 2 h of reperfusion following 30 min of ischemia compared with the control group [dP/dt (mm Hg/s) of 2110 +/- 35 versus 1752 +/- 62]. SDG reduced infarct size compared with the control group by 32% (38 versus 26%) and also decreased cardiomyocyte apoptosis. Increased protein expression of VEGF, Ang-1, and p-eNOS was also observed in the SDG-treated group. Third, in the in vivo myocardial infarction model, SDG increased capillary density and myocardial function as evidenced by increased fractional shortening and ejection fraction. In conclusion, these results suggest that SDG has potent angiogenic and antiapoptotic properties that may contribute to its cardioprotective effect in ischemic models.     

Silk fibroin scaffolds loaded with angiogenic genes in adenovirus vectors for tissue regeneration

Vascularization remains a critical challenge in dermal tissue regeneration. In this study, a vascular endothelial growth factor (VEGF165) and angiopoietin‐1 (Ang‐1) dual gene coexpression vector that encoded green fluorescent protein (GFP) was constructed from an arginine–glycine–aspartic acid‐modified adenovirus. Silk fibroin (SF) scaffolds loaded with adenovirus vectors were fabricated by freeze‐drying method. In vitro, the human endothelial‐derived cell line EA.hy926 was infected with adenovirus vectors and then expressed GFP, secreted VEGF165 and Ang‐1, and promoted cell proliferation effectively. The VEGF165 and Ang‐1 genes loaded in the SF scaffolds significantly promoted the formation of abundant microvascular networks in the chick embryo chorioallantoic membrane. In vivo, angiogenic genes loaded in the scaffolds promoted vascularization and collagen deposition in scaffolds, thus effectively accelerating dermal tissue regeneration in a dorsal full‐thickness skin defect wound model in Sprague–Dawley rats. In conclusion, SF scaffolds loaded with arginine–glycine–aspartic acid‐modified adenovirus vectors encoding VEGF165 and Ang‐1 could stimulate the formation of vascular networks through the effective expression of target genes in vascular endothelial cells, thereby accelerating the regeneration of dermal tissue.     

Plasmid-mediated VEGF gene transfer induces cardiomyogenesis and reduces myocardial infarct size in sheep

We have recently reported that in pigs with chronic myocardial ischemia heart transfection with a plasmid encoding the 165 isoform of human vascular endothelial growth factor (pVEGF165) induces an increase in the mitotic index of adult cardiomyocytes and cardiomyocyte hyperplasia. On these bases we hypothesized that VEGF gene transfer could also modify the evolution of experimental myocardial infarct. In adult sheep pVEGF165 (3.8 mg, n=7) or empty plasmid (n=7) was injected intramyocardially 1 h after coronary artery ligation. After 15 days infarct area was 11.3+/-1.3% of the left ventricle in the VEGF group and 18.2+/-2.1% in the empty plasmid group (P<0.02). The mechanisms involved in infarct size reduction (assessed in additional sheep at 7 and 10 days after infarction) included an increase in early angiogenesis and arteriogenesis, a decrease in peri-infarct fibrosis, a decrease in myofibroblast proliferation, enhanced cardiomyoblast proliferation and mitosis of adult cardiomyocytes with occasional cytokinesis. Resting myocardial perfusion (99mTc-sestamibi SPECT) was higher in VEGF-treated group than in empty plasmid group 15 days after myocardial infarction. We conclude that plasmid-mediated VEGF gene transfer reduces myocardial infarct size by a combination of effects including neovascular proliferation, modification of fibrosis and cardiomyocyte regeneration.     

Vascular endothelial growth factor and angiopoietin-1 stimulate postnatal hematopoiesis by recruitment of vasculogenic and hematopoietic stem cells

Tyrosine kinase receptors for angiogenic factors vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) are expressed not only by endothelial cells but also by subsets of hematopoietic stem cells (HSCs). To further define their role in the regulation of postnatal hematopoiesis and vasculogenesis, VEGF and Ang-1 plasma levels were elevated by injecting recombinant protein or adenoviral vectors expressing soluble VEGF(165), matrix-bound VEGF(189), or Ang-1 into mice. VEGF(165), but not VEGF(189), induced a rapid mobilization of HSCs and VEGF receptor (VEGFR)2(+) circulating endothelial precursor cells (CEPs). In contrast, Ang-1 induced delayed mobilization of CEPs and HSCs. Combined sustained elevation of Ang-1 and VEGF(165) was associated with an induction of hematopoiesis and increased marrow cellularity followed by proliferation of capillaries and expansion of sinusoidal space. Concomitant to this vascular remodeling, there was a transient depletion of hematopoietic activity in the marrow, which was compensated by an increase in mobilization and recruitment of HSCs and CEPs to the spleen resulting in splenomegaly. Neutralizing monoclonal antibody to VEGFR2 completely inhibited VEGF(165), but not Ang-1-induced mobilization and splenomegaly. These data suggest that temporal and regional activation of VEGF/VEGFR2 and Ang-1/Tie-2 signaling pathways are critical for mobilization and recruitment of HSCs and CEPs and may play a role in the physiology of postnatal angiogenesis and hematopoiesis.     

Reducible poly(amido ethylenediamine) for hypoxia-inducible VEGF delivery.

Delivery of the hypoxia-inducible vascular endothelial growth factor (RTP-VEGF) plasmid using a novel reducible disulfide poly(amido ethylenediamine) (SS-PAED) polymer carrier was studied in vitro and in vivo. In vitro transfection of primary rat cardiomyoblasts (H9C2) showed SS-PAED at a weighted ratio of 12:1 (polymer/DNA) mediates 16 fold higher expression of luciferase compared to an optimized bPEI control. FACS analysis revealed up to 57+/-2% GFP positive H9C2s. The efficiency of plasmid delivery to H9C2 using SS-PAED was found to depend upon glutathione (GSH) levels inside the cell. SS-PAED mediated delivery of RTP-VEGF plasmid produced significantly higher levels of VEGF expression (up to 76 fold) under hypoxic conditions compared to normoxic conditions in both H9C2 and rat aortic smooth muscle cells (A7R5). Using SS-PAED, delivery of RTP-VEGF was investigated in a rabbit myocardial infarct model using 100 mug RTP-VEGF. Results showed up to 4 fold increase in VEGF protein expression in the region of the infarct compared to injections of SS-PAED/RTP-Luc. In conclusion, SS-PAED mediated therapeutic delivery improves the efficacy of ischemia-inducible VEGF gene therapy both in vitro and in vivo and therefore, has potential for the promotion of neo-vascular formation and improvement of tissue function in ischemic myocardium.     

Clinical benefit of prostaglandin E1-treatment of patients with ischemic heart disease: stimulation of therapeutic angiogenesis in vital and infarcted myocardium.

New evidence suggests that Prostaglandin E1 (PGE-1) stimulates myocardial angiogenesis in human chronic ischemic myocardium. We sought to investigate whether PGE-1 may participate in the process of neoangiogenesis within the myocardial infarct scar. Neovascularization was investigated in 14 explanted hearts from patients with ischemic cardiomyopathy, who had been bridged to heart transplantation (HTX) with PGE-1 and compared with 14 hearts from patients who did not receive PGE-1 prior to HTX. In transmural sections obtained from the left ventricular wall and containing myocardial scar tissue, CD34 and vascular endothelial growth factor (VEGF) were quantified immunohistochemically to estimate capillary density and amount of angiogenesis. Additionally, to assess the hypoxic state of myocardium of the infarct border zone, hypoxia inducible factor 1-alpha (HIF-1alpha) was determined by immunohistochemistry and quantified by means of planimetric analysis. PGE-1-treated patients had significantly more CD34-and VEGF-positive cells in infarct areas as compared to nonPGE-1 group, respectively (CD34: 116.7 +/- 5.9 vs. 45.1 +/- 5.2 capillary profiles/mm(2), P < 0.001, and VEGF: 48.3 +/- 4.9 vs. 22.9 +/- 4.7 capillary profiles/mm(2)). HIF-1alpha enrichment (in %) as well as staining intensity (in estimated units (eU)) was significantly decreased in PGE-1-treated as compared to non-treated controls (enrichment: 11.3 +/- 2.5% vs. 19.4 +/- 4.36%; staining intensity: 0.95 +/- 0.3 vs. 1.97 +/- 0.44 eU). Our data demonstrate that PGE-1 stimulates neoangiogenesis in infarct areas adjacent to viable myocardium, via upregulation of VEGF expression. The induction of therapeutic angiogenesis along with the improved hypoxic state of chronic ischemic myocardial tissue might explain the favorable clinical outcome in PGE-1 treated patients.     

Cardioprotective c-kit+ cells are from the bone marrow and regulate the myocardial balance of angiogenic cytokines

Clinical trials of bone marrow stem/progenitor cell therapy after myocardial infarction (MI) have shown promising results, but the mechanism of benefit is unclear. We examined the nature of endogenous myocardial repair that is dependent on the function of the c-kit receptor, which is expressed on bone marrow stem/progenitor cells and on recently identified cardiac stem cells. MI increased the number of c-kit+ cells in the heart. These cells were traced back to a bone marrow origin, using genetic tagging in bone marrow chimeric mice. The recruited c-kit+ cells established a proangiogenic milieu in the infarct border zone by increasing VEGF and by reversing the cardiac ratio of angiopoietin-1 to angiopoietin-2. These oscillations potentiated endothelial mitogenesis and were associated with the establishment of an extensive myofibroblast-rich repair tissue. Mutations in the c-kit receptor interfered with the mobilization of the cells to the heart, prevented angiogenesis, diminished myofibroblast-rich repair tissue formation, and led to precipitous cardiac failure and death. Replacement of the mutant bone marrow with wild-type cells rescued the cardiomyopathic phenotype. We conclude that, consistent with their documented role in tumorigenesis, bone marrow c-kit+ cells act as key regulators of the angiogenic switch in infarcted myocardium, thereby driving efficient cardiac repair.     

Arteriogenesis induced by intramyocardial vascular endothelial growth factor 165 gene transfer in chronically ischemic pigs

Exogenous vascular endothelial growth factor (VEGF) improves tissue perfusion in large animals and humans with chronic myocardial ischemia. Because tissue perfusion is mainly dependent on the arteriolar tree, we hypothesized that the neovascularizing effect of VEGF should include arteriogenesis, an effect not as yet described in large mammalian models of myocardial ischemia. In the present study we investigated the effect of intramyocardial plasmid-mediated human VEGF(165) gene transfer (pVEGF(165)) on the proliferation of vessels with smooth muscle in a pig model of myocardial ischemia. In addition, we assessed the effect of treatment on capillary growth, myocardial perfusion, myocardial function and collateralization. Three weeks after positioning of an Ameroid constrictor (Research Instruments SW, Escondido, CA) in the left circumflex artery, pigs underwent basal perfusion (single-photon emission computed tomography [SPECT] with (99m)Tc-sestamibi) and regional function (echocardiography) studies at rest and under dobutamine stress, and were then randomly assigned to receive transepicardial injection of pVEGF(165) 3.8 mg (n = 8) or placebo (empty plasmid, n = 8). All experimental steps and data analysis were done in a blinded fashion. Five weeks later, pVEGF(165)-treated pigs showed a significantly higher density of small (8-50 microm in diameter) vessels with smooth muscle, higher density of capillaries, and improved myocardial perfusion. These results indicate an arteriogenic effect of VEGF in a large mammalian model of myocardial ischemia and encourage the use of VEGF to promote arteriolar growth in patients with severe coronary artery disease.     

In vivo imaging shows abnormal function of vascular endothelial growth factor-induced vasculature

Although the angiogenic effect of vascular endothelial growth factor (VEGF) is widely recognized, a central question concerns whether the vessels formed on its overexpression effectively increase tissue perfusion in vivo. To explore this issue, here we exploit AAV vectors to obtain the prolonged expression of VEGF and angiopoietin-1 (Ang1) in rat skeletal muscle. Over a period of 6 months, muscle blood flow (MBF) and vascular permeability were measured by positron emission tomography and single-photon emission computed tomography, respectively. All measurements were performed under resting conditions and after electrically induced muscle exercise. Despite the potent angiogenic effect of VEGF, documented by vessel counting and intravascular volume assessment, the expression of this factor did not improve resting MBF, and it even decreased perfusion after exercise. This deleterious effect was related to the formation of leaky vascular lacunae, which accounted for the occurrence of arteriovenous shunts that excluded the downstream microcirculation. These effects were significantly counteracted by the coinjection of VEGF and Ang1, which determined a marked increase in resting MBF and, most notably, a significant improvement after exercise that persisted over time. Taken together, these results challenge the effectiveness of VEGF as a sole factor to induce angiogenesis and suggest the use of factor combinations to achieve competent vessel formation.     

病毒性心肌炎小鼠血管紧张素的改变与心肌胶原的关系

【目的】观察病毒性心肌炎(VM)不同阶段血管紧张素(Ang)的改变与胶原含量的关系,探讨VM的病理机制。【方法】建立VM小鼠模型,放免法检测不同时点血浆及心肌组织的AngⅠ和AngⅡ浓度、含量,氯氨T法测定心肌的胶原含量。【结果】VM小鼠在感染21d、30d血浆及心肌组织的AngⅠ和AngⅡ浓度、含量以及胶原含量均较感染10d及正常组显著增高。VM小鼠AngⅠ、AngⅡ的浓度在血浆与心肌中均呈高度正相关;血浆和心肌中AngⅠ、AngⅡ的浓度均与心肌胶原含量呈正相关。【结论】VM小鼠恢复期血浆和心肌中AngⅠ、AngⅡ的浓度以及心肌胶原含量明显增高,而AngⅠ、AngⅡ的浓度与心肌胶原含量的改变密切相关。     

Reduction of infarct size in the rat heart by LPS preconditioning is associated with expression of angiogenic growth factors and increased capillary density.

Inflammation induces the expression of angiogenic growth factors in tissues, which leads to microvascular growth. Bacterial lipopolysaccharide (LPS) provokes a transient inflammatory response in the heart and induces delayed cardiac resistance to post-ischemic contractile dysfunction. In this study, we examined: 1) the effects of LPS on myocardial expression of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), 2) whether an increase in the density of myocardial microvessels follows the expression of angiogenic growth factors, and 3) the effect of LPS on myocardial resistance to infarction and its relationship with microvascular growth. Rats were treated with LPS (from Salmonella typhimurium, 0.5 mg/kg i.p.). The expression of bFGF and VEGF in the myocardium was examined at 6 and 12 h after LPS treatment by immunofluorescent staining. Myocardial capillary and arteriole densities were determined 3 days after LPS treatment by morphometry, using immunofluorescent staining of von Willebrand factor (a marker protein of endothelial cells) and alpha-smooth muscle actin (a marker protein of smooth muscle cells). To examine cardiac resistance to infarction, hearts were subjected to 40 min of regional ischemia and 2 h of reperfusion by reversible occlusion of left coronary artery at 3 days after LPS treatment. LPS induced cardiac bFGF and VEGF at 6 and 12 h after treatment. The expression of these growth factors was followed by an increase in myocardial capillary density (2032 +/- 78/mm2 vs. 1617 +/- 47/mm2 in saline control, P < 0.05), but not arteriole density, at 3 days. Meanwhile, infarct size was significantly reduced by LPS preconditioning (infarct/left ventricle 12.3 +/- 1.04% vs. 21.7 +/- 1.65% in saline control, 43% reduction, P < 0.05). These results suggest that LPS preconditioning induces cardiac bFGF and VEGF, and an increase in myocardial capillary density. This increased myocardial capillary density is associated with a reduced infarct size after in vivo regional ischemia-reperfusion.     

Mitofusin 2 inhibits angiotensin II-induced myocardial hypertrophy

BACKGROUND AND OBJECTIVES: Myocardial hypertrophy is a common clinical finding leading to heart failure and sudden death. Mitofusin 2 (Mfn2), a hyperplasia suppressor protein, is downregulated in hypertrophic heart. This study examined the role of Mfn2 in myocardial hypertrophy and its potential signal pathway. Methods AND RESULTS: In in vitro studies, neonatal cardiac myocytes were isolated and cultured. Incubation of cultured cardiomycytes with angiotensin II (Ang II) inhibited gene expression of Mfn2; induced cell hypertrophy and protein synthesis; and activated protein kinase Akt. Pretreatment of cells with AdMfn2-a replication-deficient adenoviral vector encoding rat Mfn2 gene-upregulated Mfn2 expression and subsequently attenuated Ang II-induced cell hypertrophy; protein synthesis; and Akt activation. In in vivo studies, direct gene delivery of AdMfn2 into myocardium decreased the infusion of Ang II-induced atrial natriuretic factor (ANF, a hypertrophic marker) expression and cardiomyocyte cross-sectional area. Consistently, upregulation of Mfn2 in myocardium decreased the thicknesses of anterior and posterior walls of left ventricle (LV) and the ratio of LV mass/body weight in Ang II-treated rats. Of note, AdGFP (control for AdMfn2) did not affect the effects of Ang II in vitro or in vivo. CONCLUSIONS: Upregulation of Mfn2 inhibits Ang II-induced myocardial hypertrophy. In this process, inhibition of Akt activation seems to play a significant role. These findings indicate Mfn2 is a critical protein in modulating myocyte hypertrophy.     

直接心肌内注射血管内皮生长因子基因对慢性缺血心肌侧枝循环生成的影响

目的观察血管内皮生长因子基因(rAAV2-hVEGF165)直接心肌内注射促进慢性缺血心肌侧枝循环生成的作用。方法小型猪左冠状动脉回旋支放置血管缩窄环,建立慢性心肌缺血模型。5周后应用心电图、冠脉造影和心脏核磁共振成像确认左回旋支闭塞或相应心肌的缺血。动物随机分为实验组和对照组,分别在缺血心肌内直接注射rAAV2-VEGF165(1×1012virus genomeml)或同等量的磷酸盐缓冲液或rAAV2-LACZ(1×1012virus genomeml)。治疗后6个月,冠状动脉造影了解有无侧枝循环生成。结果放置血管缩窄环后5周,所有动物均出现左回旋支完全、次全闭塞和旋支供血区域的心肌缺血。治疗后3个月,缺血心肌内可检测到LACZ和VEGF蛋白的阳性表达;治疗后6个月,VEGF组缺血心肌内毛细血管和小动脉密度均高于对照组(P<0.05);结论直接在小型猪慢性缺血心肌内注射rAAV2-VEGF165,VEGF165基因可转染入心肌组织内,并可在缺血部位显著促进侧枝循环形成。     

靶向超声介导VEGF_(165) cDNA基因及抗ICAM-1单克隆抗体治疗大鼠心肌缺血的对比性研究

目的 探讨靶向超声介导VEGF_(165) cDNA、抗ICAM-1单克隆抗体及两者协同作用于受损心肌的有效性及可行性.方法 将80只心肌缺血的雄性SD大鼠随机分为四组:第一组采用超声击破微泡造影剂的方法将VEGF_(165) cDNA基因及抗ICAM-1单克隆抗体混合物定向于受损心肌;第二组采用超声击破微泡造影剂的方法将VEGF_(165) cDNA基因定向于受损心肌;第三组采用超声击破微泡造影剂的方法将抗ICAM-1单克隆抗体定向于受损心肌;第四组为对照组只注射生理盐水1 ml,不采用超声波照射.每组分别于24 h和14 d后处死10只.处死后分别行坏死区中性粒细胞记数,梗死区测定,免疫组织化学检测缺血心肌中的VEGF蛋白表达,缺血坏死心肌区域毛细血管记数.结果 第一组大鼠心肌缺血治疗效果较第二、第三组效果显著,第一组中性粒细胞浸润以及心肌梗死区域均明显低于其他各组,VEGF蛋白表达以及毛细血管记数明显高于其他三组.结论 采用超声触发破坏含靶基因及抗体的微泡造影剂的方式,可明显对抗心肌再损伤,促进缺血心肌的新生血管再生,改善心肌缺血,且两者协同作用比单独作用效果更佳.     

Combined angiopoietin-1 and vascular endothelial growth factor gene transfer restores cavernous angiogenesis and erectile function in a rat model of hypercholesterolemia.

Hypercholesterolemia-related endothelial cell dysfunction and decreased endothelium-derived nitric oxide formation may account for impaired angiogenesis and subsequent erectile dysfunction. Angiopoietin-1 (Ang1) is a critical angiogenic factor for vascular maturation and enhances vascular endothelial growth factor (VEGF)-induced angiogenesis in a complementary manner. We hypothesized that combined adenovirus-delivered human Ang1 (ad-Ang1) and VEGF165 (ad-VEGF165) gene transfer might promote angiogenesis cooperatively in a rat model of hypercholesterolemic erectile dysfunction and result in a recovery of erectile function. Ad-Ang1 and ad-VEGF165 were injected either alone or in combination into the corpus cavernosum of the penis. Combined gene transfer of both ad-Ang1 and ad-VEGF165 significantly increased cavernous angiogenesis, eNOS phosphorylation, and cGMP expression compared with that in the groups treated with either therapy alone. Erectile function, as evaluated by electrical stimulation of the cavernous nerve 2 and 8 weeks after treatment, was completely restored in the combined treatment group, whereas intracavernous injection of either ad-Ang1 or ad-VEGF165 alone elicited partial improvement. The results indicate that combined application of angiogenic factors may enhance cavernous angiogenesis cooperatively by reinforcing the endothelium both structurally and functionally, which results in an additive effect on erectile function in hypercholesterolemic rats.