Hepatocyte Growth Factor Gene Transfection Research Articles

Suppression of Peritoneal Fibrosis by Sonoporation of Hepatocyte Growth Factor Gene-Encoding Plasmid DNA in Mice.

Gene therapy is expected to be used for the treatment of peritoneal fibrosis, which is a serious problem associated with long-term peritoneal dialysis. Hepatocyte growth factor (HGF) is a well-known anti-fibrotic gene. We developed an ultrasound and nanobubble-mediated (sonoporation) gene transfection system, which selectively targets peritoneal tissues. Thus, we attempted to treat peritoneal fibrosis by sonoporation-based human HGF (hHGF) gene transfection in mice. To prepare a model of peritoneal fibrosis, mice were intraperitoneally injected with chlorhexidine digluconate. We evaluated the preventive and curative effects of sonoporation-based hHGF transfection by analyzing the following factors: hydroxyproline level, peritoneum thickness, and the peritoneal equilibration test. The transgene expression characteristics of sonoporation were also evaluated using multicolor deep imaging. In early-stage fibrosis in mice, transgene expression by sonoporation was observed in the submesothelial layer. Sonoporation-based hHGF transfection showed not only a preventive effect but also a curative effect for early-stage peritoneal fibrosis. Sonoporation-based hHGF transfection may be suitable for the treatment of peritoneal fibrosis regarding the transfection characteristics of transgene expression in the peritoneum under fibrosis.

Early treatment with hepatocyte growth factor improves pulmonary artery and right ventricular remodeling in rats with pulmonary artery hypertension by modulating cytokines expression

To investigate the effect of early treatment with hepatocyte growth factor (HGF) on the cytokine expression and pulmonary artery, right ventricular (RV) remodeling in the rat model of pulmonary artery hypertension (PAH). The rat model of PAH was produced by injecting monocrotaline, and the model rats were randomly divided into empty adenovirus transfection group (MCT group, n = 10) and HGF gene transfection group(HGF group, n = 10). Another group of rats served as the Sham operation group (Sham group n = 10). After 4 weeks of HGF gene transfection, the histological sections of the lungs and right ventricular (RV) were stained with hematoxylin-eosin (HE) and the pulmonary artery and RV remodeling were examined. The rat lung sections were also immunostained with antibodies against factor VIII, and the capillary density were calculated. Meanwhile, the mRNA expression of TNF-α, IL-6, IL-1β and TGF-β1 in pulmonary arteries and RV were detected by RT-PCR. HGF gene treatment significantly decreased the ratio of vessel wall thickness to pulmonary artery diameter and the ratio of vessel wall area to total area, and increased the capillary density of lung in PAH rats. HGF gene treatment also significantly decreased cross-sectional area of cardiomyocytes and collagen deposited in RV. Moreover, HGF gene treatment significantly decreased the expression of cytokines in pulmonary artery (TNF-α: 0.82 ± 0.07 vs 0.49 ± 0.09, IL-6: 1.13 ± 0.19 vs 0.68 ± 0.09, IL-1β: 0.86 ± 0.11 vs 0.51 ± 0.07, TGF-β1: 1.18 ± 0.12 vs 0.59 ± 0.10) and RV (TNF-α: 0.79 ± 0.11 vs 0.48 ± 0.08, IL-6: 1.03 ± 0.11 vs 0.63 ± 0.09, IL-1β: 0.81 ± 0.11 vs 0.52 ± 0.07, TGF-β1: 0.94 ± 0.12 vs 0.53 ± 0.10) in MCT induced PAH rats (P < 0.05) . Early treatment with HGF improves pulmonary artery and RV remodeling, possibly by decreasing the expression of TNF-α, IL-6, IL-1β and TGF-β1 in pulmonary arteries and RV.

Synergistic effects of ultrasound-targeted microbubble destruction and TAT peptide on gene transfection: An experimental study in vitro and in vivo

Cell-permeable peptides (CPPs) and ultrasound-targeted microbubble destruction (UTMD) have tremendous potential for gene delivery. However, their applications are limited due to nonspecificity of CPPs and low transfection efficiency of UTMD. Here, we developed a ‘smart’ gene delivery system by encapsulating TAT peptide (TATp) and hepatocyte growth factor (HGF) gene within lipid microbubbles, in which TATp was protected from being enzymatically cleaved and HGF gene was protected from degradation. This new strategy had synergistic effects of UTMD and TATp on gene transfection. We investigated the efficacy and safety of HGF gene transfection mediated by the combination of UTMD and TATp in vitro and in vivo. The results from MTT assay and flow cytometry analyses indicated that the combination of UTMD and TATp could enhance HGF gene expression in HUVECs without any significant side effect on cell viability. In rat myocardial infarction models, we demonstrated that the protein and mRNA expressions of HGF in myocardium caused by the combination of UTMD and TATp were the highest.Histopathological findings demonstrated that the combination of UTMD and TATp enhanced myocardial microvasculature and ameliorated myocardial fibrosis. In conclusion, the combination of UTMD and TATp might be a safe and efficient technique for gene delivery.

Recombinant human hepatocyte growth factor transfection alleviates hyperkinetic pulmonary artery hypertension in rabbit models

The study objective was to investigate the effect of recombinant human hepatocyte growth factor gene transfection via an endotracheal approach on hyperkinetic pulmonary artery hypertension rabbit models. The rabbits with established pulmonary artery hypertension were separated into a gene transfection group (rabbits treated with intratracheal instillation of human hepatocyte growth factor 2×10(9) plaque-forming units coded by replication-defective recombinant adenovirus), an empty vector group, and a control group. Two weeks after endotracheal gene transfection, immunohistochemistry examination and Western blot were used to detect the protein expression of human hepatocyte growth factor. The hemodynamic data were measured, and pulmonary angiography was performed to investigate the pulmonary collateral vessels. The vascular density in lung also was analyzed. Two weeks after gene transfection, human hepatocyte growth factor was expressed in the gene transfection group. The mean pulmonary artery pressure in the gene transfection group was lower than in the control and empty vector groups (P<.05 for both). The arteriolar density in the lung tissues of the gene transfection group was higher than in the other groups (P<.05), which was confirmed by immunohistochemistry, double-labeling immunofluorescence, and pulmonary angiography. Human hepatocyte growth factor was expressed in rabbit lung after gene transfection via an airway approach. Recombinant human hepatocyte growth factor transfection ameliorates the pulmonary artery hypertension induced by shunt flow by promoting angiogenesis in lung tissues.

Cellular Injury of Cardiomyocytes during Hepatocyte Growth Factor Gene Transfection with Ultrasound-Triggered Bubble Liposome Destruction

We transfected naked HGF plasmid DNA into cultured cardiomyocytes using a sonoporation method consisting of ultrasound-triggered bubble liposome destruction. We examined the effects on transfection efficiency of three concentrations of bubble liposome (1 × 106, 1 × 107, 1 × 108/mL), three concentrations of HGF DNA (60, 120, 180 μg/mL), two insonification times (30, 60 sec), and three incubation times (15, 60, 120 min). We found that low concentrations of bubble liposome and low concentrations of DNA provided the largest amount of the HGF protein expression by the sonoporated cardiomyocytes. Variation of insonification and incubation times did not affect the amount of product. Following insonification, cardiomyocytes showed cellular injury, as determined by a dye exclusion test. The extent of injury was most severe with the highest concentration of bubble liposome. In conclusion, there are some trade-offs between gene transfection efficiency and cellular injury using ultrasound-triggered bubble liposome destruction as a method for gene transfection.

Effect of hepatocyte growth factor gene transfection on biological features of lymphoma cells

To investigate the biological effect of hepatocyte growth factor (HGF) on HGF gene-transfected Raji cells. Total RNA was extracted from human hepatic tissue, HGF gene cDNA was amplified by RT-PCR, and then cloned into vector pVITRO2-mcs to construct recombinant eukaryotic expression vector pVITRO2-mcs-HGF. The recombinant vector was transfected to Raji cells, and the stably transfected cells were selected by homomycin B in serial passages, and confirmed by real-time fluorescent quantitative PCR, ELISA, immunocytohistochemistry. The biological features of transfected Raji cells were evaluated by semisolid culture. RT-PCR results showed that Raji cells were transfected successfully with recombinant eukaryotic expression vector pVITRO2-mcs-HGF. HGF mRNA and protein were expressed successfully in Raji cells. Expression of HGF gene enhanced proliferation, metastasis and invasion of Raji cells. HGF gene has been cloned and recombined to construct recombinant eukaryotic expression vector pVITRO2-mcs-HGF successfully. Transfected HGF may change the biological features of Raji cells.

Transfection of HGF gene enhances endothelial progenitor cell (EPC) function and improves EPC transplant efficiency for balloon-induced arterial injury in hypercholesterolemic rats

Risk factors for coronary heart disease can reduce the number of endothelial progenitor cells (EPCs) and impair EPC function, thus hindering their utility in the treatment of cardiovascular diseases. In the present study, we began exploring the feasibility of genetic modification of EPCs with hepatocyte growth factor (HGF) to counter the effects of these risk factors and enhance the biological functions of EPCs. The effects of HGF transfection on proliferation, migration and angiogenesis of EPCs were investigated. Additionally, the role of ERK1/2 in this process was evaluated through the observation of ERK1/2 and ERK1/2 phosphorylation as well as by pharmacological analysis. Finally, we evaluated the effect of HGF-transfected EPCs (HGF-EPCs) on neointima formation after balloon-induced arterial injury in hypercholesterolemic rats. Our data showed that EPCs transfected with the HGF gene released high levels of soluble HGF protein, which were maintained for at least nine days. Transfection with HGF also enhanced the proliferative, migratory and angiogenic capabilities of EPCs, and promoted the activation of ERK1/2 without affecting its expression. ERK1/2 blockade by the chemical inhibitor PD98059 partially inhibited these effects. In hypercholesterolemic rats, HGF-EPCs homed to the site of vascular injury at a significantly higher rate than did EPCs without the exogenous HGF gene. Furthermore, systemically applied HGF-EPCs were more effective in decreasing neointima formation and increasing re-endothelialization. These data suggest that gene delivery combined with EPC transplant may be a practical and promising therapy for the prevention of neointimal formation after vascular injury.

Effects of local release of hepatocyte growth factor on peripheral nerve regeneration in acellular nerve grafts

Options for reconstructing peripheral nerve gaps after trauma are limited. The acellular nerve is a new kind of biomaterial used to reconstruct the peripheral nerve defect, but its use could be improved upon. We aimed to investigate the effect of adenoviral transfection with hepatocyte growth factor (HGF) on the functional recovery of transected sciatic nerves repaired by acellular nerve grafting. 30 Rats were divided into three groups (10/group) for autografting and acellular grafting, as well as acellular grafting with adenovirus transfection of HGF (1 × 10 8 pfu) injected in muscles around the proximal and distal allograft coapation. Sciatic functional index (SFI) was evaluated every 4 weeks to week 16 by measuring rat footprints on walking-track testing. The three groups presented initial complete functional loss, followed by slow but steady recovery, with final similar SFIs. Weight of the gastrocnemius and soleus muscles, histologic and morphometric study and neovascularization in the nerve grafts were evaluated at week 16. Autografting gave the best functional recovery, but HGF-treated acellular grafting gave better recovery than acellular grafting alone. Neovascularization was greater with HGF-treated acellular grafting than with autografting and acellular grafting alone. Axonal regeneration distance of autografting on the 20th postoperative day was the longest in the three groups,while that of acellular grafting alone was the smallest. Acellular nerve grafting may be useful for functional peripheral nerve regeneration, and with human HGF gene transfection may improve on acellular grafting alone in functional recovery.

Combined Strategy Using Myoblasts and Hepatocyte Growth Factor in Dilated Cardiomyopathic Hamsters

There are few reports on treating dilated cardiomyopathy (DCM) with myoblast transplantation, and these show limited efficacy. Hepatocyte growth factor has cardioprotective effects on failed myocardium. Here, we combined these two treatments and analyzed cardiac function in DCM hamsters. Twenty-seven-week-old BIO TO-2 hamsters, which show moderate cardiac remodeling, were divided into four treatment groups: myoblast transplantation (T group, n = 24), human hepatocyte growth factor gene transfection (H group, n = 29), combined treatment (T+H group, n = 21), and medium alone (C group, n = 26). Significantly better fractional shortening was observed in the T+H group compared with the others (14.9% +/- 1.0%, 11.7% +/- 1.5%, 11.3% +/- 1.3%, and 8.6% +/- 1.1 %, in the T+H, H, T, and C groups, respectively). Immunohistochemical analysis showed alpha- and beta-sarcoglycan expression in the hearts of the H and T+H groups but not in the other groups. There was less myocardial fibrosis in the H and T+H groups than in the other two, and neovascularization in the T+H group was significantly greater than in the other groups (266 +/- 24, 209 +/- 27, 199 +/- 36, and 96 +/- 17 vessels/mm2, in the T+H, H, T, and C groups, respectively). Survival was significantly prolonged in the H and T+H groups compared with the other groups. Hepatocyte growth factor gene transfection and myoblast transplantation preserved the cardiac function of DCM hamsters, probably through different mechanisms, and the combined treatments preserved cardiac performance better than either treatment alone. The combined therapy is a promising strategy for treating DCM.

Biological effect of hepatocyte growth factor gene transfection to rat cerebral ischemic model: the experimental study

目的 探讨肝细胞生长因子(HGF)基因转染大鼠急性脑卒中模型后的表达情况及其生物学效应.方法 39只实验用大鼠,线栓法制备成可控性急性大鼠脑卒中模型.利用基因重组技术将HGF基因与增强型绿色荧光蛋白(EGFP)基因标记的真核表达载体PIRES2-EGFP进行融合,构建真核细胞表达质粒.通过脂质体介导法,立体定向下多点注射到大鼠急性脑卒中模型的缺血半暗带区域行基因转染,转染7 d后断头取大鼠脑组织,切片观察HGF基因在大鼠脑内的表达情况;并采用兔抗人Ⅷ因子相关抗原多克隆抗体对大鼠脑组织进行免疫组化染色,显微镜下随机检测5个高倍视野下缺血半暗带区血管数量;同时在基因转染前及转染后7 d,应用CT灌注扫描对实验鼠脑血流量进行监测,观察基因转染对脑血流量的影响.将大鼠脑卒中模型随机分成3组(每组13只):(1)注射脂质体/pIRES2-EGFP-HGF质粒的实验组;(2)注射脂质体/PIRES2-EGFP空载质粒的空载体对照组;(3)空白对照组(未进行注射).结果 酶切鉴定及基因测序证实,HGF基因片段已克隆到PIRES2-EGFP的BamH I和Sal I位点之间.HGF基因转染大鼠缺血半暗带区7 d后,利用荧光显微镜观察可见到绿色荧光蛋白的表达,用免疫组化方法进一步证实了HGF的表达.实验组大鼠转染局部血管数量(46.71±7.11)条/5个高倍视野,明显多于对照组的(20.43±3.21)条/5个高倍视野和空白对照组的(18.00±3.27)条/5个高倍视野(F=74.447;P＜0.01);CT灌注显示其梗死侧大脑半球的脑血流量(63.82±6.08)%也高于对照组(53.58±4.19)%和空白对照组(52.46±4.93)%(F=10.445;P＜0.01).结论 脂质体转染HGF基因能够在缺血半暗带区表达,表达产物能够发挥生物学效应,促进局部血管侧支循环形成。

Hepatocyte growth factor suppresses acute renal inflammation by inhibition of endothelial E-selectin

Vascular endothelial activation, marked by de novo expression of E-selectin, is an early and essential event in the process of leukocyte extravasation and inflammation. Evidence suggests that hepatocyte growth factor (HGF) ameliorates inflammation in animal models of renal disease, implying that HGF might inhibit specific components of the inflammatory response. This study examined the effect of HGF on endothelial E-selectin expression in acute inflammation induced by tumor necrosis factor (TNF)-α. In vitro, HGF suppressed TNF-α-induced cell surface expression of E-selectin in human umbilical vein endothelial cells (HUVEC) and inhibited E-selectin mediated monocytic adhesion to endothelial monolayers. HGF activated phosphatidylinositol 3-kinase (PI3K)–Akt that in turn inhibited its downstream transducer glycogen synthase kinase (GSK)3. Blockade of the PI3K–Akt pathway with specific inhibitors abrogated HGF induced inhibitory phosphorylation of GSK3 and suppression of E-selectin. In addition, selective inhibition of GSK3 activity by lithium suppressed TNF-α-induced E-selectin expression and monocytic adhesion, reminiscent of the action of HGF. Moreover, ectopic expression of an uninhibitable mutant GSK3β, in which the regulatory serine-9 is replaced by alanine, abolished HGF's suppressive effect on endothelial E-selectin. In vivo, administration of exogenous HGF reduced endothelial expression of E-selectin induced by bolus injection of TNF-α. This was associated with less sequestration of circulating fluorescence-labeled macrophages in the kidney. These findings suggest that HGF ameliorates acute renal inflammation in part by downregulating E-selectin mediated macrophage adhesion to the inflamed endothelium.

Hepatocyte growth factor prevents lupus nephritis in a murine lupus model of chronic graft-versus-host disease

Chronic graft-versus-host disease (GVHD) induced in (C57BL/6 × DBA/2) F1 (BDF1) mice by the injection of DBA/2 mouse spleen cells represents histopathological changes associated with systemic lupus erythematosus (SLE), primary biliary cirrhosis (PBC) and Sjogren's syndrome (SS), as indicated by glomerulonephritis, lymphocyte infiltration into the periportal area of the liver and salivary glands. We determined the therapeutic effect of hepatocyte growth factor (HGF) gene transfection on lupus using this chronic GVHD model. Chronic GVHD mice were injected in the gluteal muscle with either HVJ liposomes containing 8 μg of the human HGF expression vector (HGF-HVJ liposomes) or mock vector (untreated control). Gene transfer was repeated at 2-week intervals during 12 weeks. HGF gene transfection effectively prevented the proteinuria and histopathological changes associated with glomerulonephritis. While liver and salivary gland sections from untreated GVHD mice showed prominent PBC- and SS-like changes, HGF gene transfection reduced these histopathological changes. HGF gene transfection greatly reduced the number of splenic B cells, host B cell major histocompatibility complex class II expression, and serum levels of IgG and anti-DNA antibodies. IL-4 mRNA expression in the spleen, liver, and kidneys was significantly decreased by HGF gene transfection. CD28 expression on DBA/2 CD4+ T cells was decreased by the addition of recombinant HGF in vitro. Furthermore, IL-4 production by DBA/2 CD4+ T cells stimulated by irradiated BDF1 dendritic cells was significantly inhibited by the addition of recombinant HGF in vitro. These results suggest that HGF gene transfection inhibited T helper 2 immune responses and reduced lupus nephritis, autoimmune sialoadenitis, and cholangitis in chronic GVHD mice. HGF may represent a novel strategy for the treatment of SLE, SS and PBC.

Hepatocyte growth factor ameliorates dermal sclerosis in the tight-skin mouse model of scleroderma

The tight-skin (TSK/+) mouse, a genetic model of systemic sclerosis (SSc), develops cutaneous fibrosis and defects in pulmonary architecture. Because hepatocyte growth factor (HGF) is an important mitogen and morphogen that contributes to the repair process after tissue injury, we investigated the role of HGF in cutaneous fibrosis and pulmonary architecture defects in SSc using TSK/+ mice. TSK/+ mice were injected in the gluteal muscle with either hemagglutinating virus of Japan (HVJ) liposomes containing 8 μg of a human HGF expression vector (HGF-HVJ liposomes) or a mock vector (untreated control). Gene transfer was repeated once weekly for 8 weeks. The effects of HGF gene transfection on the histopathology and expression of tumor growth factor (TGF)-β and IL-4 mRNA in TSK/+ mice were examined. The effect of recombinant HGF on IL-4 production by TSK/+ CD4+ T cells stimulated by allogeneic dendritic cells (DCs) in vitro was also examined. Histologic analysis revealed that HGF gene transfection in TSK/+ mice resulted in a marked reduction of hypodermal thickness, including the subcutaneous connective tissue layer. The hypodermal thickness of HGF-treated TSK/+ mice was decreased two-fold to three-fold compared with untreated TSK/+ mice. However, TSK/+ associated defects in pulmonary architecture were unaffected by HGF gene transfection. HGF gene transfection significantly inhibited the expression of IL-4 and TGF-β1 mRNA in the spleen and skin but not in the lung. We also performed a mixed lymphocyte culture and examined the effect of recombinant HGF on the generation of IL-4. Recombinant HGF significantly inhibited IL-4 production in TSK/+ CD4+ T cells stimulated by allogeneic DCs. HGF gene transfection inhibited IL-4 and TGF-β mRNA expression, which has been postulated to have a major role in fibrinogenesis and reduced hypodermal thickness, including the subcutaneous connective tissue layer of TSK/+ mice. HGF might represent a novel strategy for the treatment of SSc.

HGF gene transfer increases kidney graft survival

The introduction of cyclosporine (CsA) into clinical practice has resulted in marked improvement in the short-term outcome of organ transplantation, and 1-year survival of renal allografts has improved significantly. However, the annual rate of kidney graft loss caused by chronic allograft nephropathy (CAN) has remained stable over last decade. CAN may result from perioperative ischemia at the time of transplantation. Furthermore, chronic CsA nephrotoxicity may progress to an irreversible renal lesion characterized by tubular atrophy, striped interstitial fibrosis, hyalinosis of the afferent arteriole, and progressive renal impairment. Recent report demonstrated the therapeutic effects of hepatocyte growth factor (HGF) in preventing CAN using a well-established rat CAN model. They showed that HGF treatment during the initial 4 weeks after engraftment prevented onset of CAN and associated death and provided longlasting benefit in terms of graft survival. Exogenous HGF is very unstable in the blood circulation due to the rapid clearance by the liver. To circumvent this problem, we developed a new gene transfer system by electroporation in vivo. Therefore, this gene transfer approach represents a useful technique to investigate the therapeutic potential of HGF gene transfer for long-term survival of kidney allograft. The goal of the present study was to assess the renoprotective potential and safety of HGF gene transfer using a porcine kidney transplant warm ischemia injury model or CsA nephrotoxicity model. In the first set of experiments, following left porcine kidney removal, 10 minutes of warm ischemic injury was intentionally induced. Next, the HGF expression vector or vehicle was infused into the renal artery with the renal vein clamped ex vivo, and electric pulses were discharged using bathtub-type electrodes. Kidney grafts were then transplanted after removing the right kidney. Histopathologic examination of vehicle-transfected kidney transplant revealed initial tubular injury followed by tubulointerstitial fibrosis. In contrast, HGF-transfected kidneys showed no initial tubular damage and no interstitial fibrosis at 6 months posttransplant. In the next set of experiments, CsA was subcutaneously administered daily under low sodium diet, and HGF gene was transferred into skeletal muscle by electroporation on days 7 and 14. We also examined the antiapoptotic mechanism of HGF using human proximal tubular epithelial cells. HGF gene transfer rescued CsA-induced initial tubular injury, and suppressed interstitial infiltration of endothelium-1 (ED-1)-positive macrophages in CsA nephrotoxicity. In addition, HGF significantly inhibited tubular cell apoptosis, and increased the number of proliferating tubular epithelial cells. In vitro studies suggest that HGF executes the antiapoptotic function by enhancing the phosphorylation of Akt and Bcl-2. Northern blot analysis demonstrated that HGF gene transfer suppressed cortical mRNA levels of transforming growth factor-β (TGF-β). Consequently, HGF gene transfer significantly reduced a striped interstitial phenotypic alteration and fibrosis. We conclude that electroporation-mediated HGF gene transfection protects the kidney against graft injury.

Treatment of experimental hepatic fibrosis by combinational delivery of urokinase‐type plasminogen activator and hepatocyte growth factor genes

To investigate the effect of combinational delivery of urokinase-type plasminogen activator (uPA) and hepatocyte growth factor (HGF) genes on hepatic fibrosis. Replication-deficient adenoviral vectors expressing either human HGF (AdHGF) or uPA (AduPA) were generated. HGF gene was transferred into primary cultured hepatocytes and uPA gene to hepatic stellate cell (HSC) to investigate the effect on the biological character of cells. Combinational adenoviruses were infused into hepatic fibrosis rats. Serum markers as well as histological and immunohistochemical examination were carried out to test the reversal of hepatic fibrosis. Transfection of exogenous HGF gene induced expression of c-met/HGF receptor and stimulated hepatocyte proliferation. uPA gene delivered into HSC decreased the amount of collagen types I and III accompanied with the increased expression of matrix metalloproteinase-2. In vivo, the area of extracellular matrix in the fibrotic liver decreased to 72% in AdHGF-treated rats (P<0.01), 64% in the AduPA-treated group (P<0.01), and 51% in bi-genes transfection (P<0.01), compared with that of the controls. Moreover, immunohistochemical staining of collagen types I and III revealed that combinational genes delivery exerted more effect on reversal of hepatic fibrosis than mono-gene transfection. Our study indicated that simultaneous delivery of two antifibrotic genes could confer synergistic effect on hepatic fibrosis.

The effect of gene transfer with hepatocyte growth factor for pulmonary vascular hypoplasia in neonatal porcine model

Severe degree of pulmonary vascular hypoplasia remains a major limitation in congenital heart surgery. Considering the potential effect of gene transfer with hepatocyte growth factor to induce the angiogenesis in the lung, we assessed the effects of hepatocyte growth factor gene transfer in neonatal porcine lung with pulmonary vascular hypoplasia to achieve treatment for severe pulmonary vascular hypoplasia. The model of pulmonary vascular hypoplasia was introduced with left pulmonary artery banding in piglet lung. After 7 days of pulmonary artery banding, piglets were transfected selectively to the left lung via the left pulmonary artery with a hemagglutinating virus of Japan E vector bearing the cDNA encoding human hepatocyte growth factor (H group) or control vector (C group). Seven days after the transfection, selective angiography of the left pulmonary artery showed the progression of left pulmonary vascular hypoplasia of the left lung in the C group but a significant attenuation of left pulmonary vascular hypoplasia in the H group. A right pulmonary artery occlusion test showed a marked increase in right ventricular systolic pressure in the C group, but this was significantly attenuated in the H group (C: 22.0 +/- 2.9, H: 13.0 +/- 2.7 mm Hg; P < .05). Histologic examination revealed that hepatocyte growth factor gene transfection increased the pulmonary vasculature in the left lung. Our results demonstrated that gene transfer of hepatocyte growth factor via the pulmonary artery showed the angiogenic effects in porcine model of pulmonary vascular hypoplasia after pulmonary artery banding.

Electroporation-mediated HGF gene transfection protected the kidney against graft injury

The annual rate of kidney graft loss caused by chronic allograft nephropathy (CAN) has not improved over the past decade. Recent reports suggest that acute renal ischemia results in development of CAN. The goal of the present study was to assess the renoprotective potential and safety of hepatocyte growth factor (HGF) gene transfer using a porcine kidney transplant warm ischemia injury model. Following left porcine kidney removal, 10 min of warm ischemic injury was intentionally induced. Next, the HGF expression vector or vehicle was infused into the renal artery with the renal vein clamped ex vivo, and electric pulses were discharged using bathtub-type electrodes. Kidney grafts were then transplanted after removing the right kidney. Histopathological examination of vehicle-transfected kidney transplant revealed initial tubular injury followed by tubulointerstitial fibrosis. In contrast, HGF-transfected kidneys showed no initial tubular damage and no interstitial fibrosis at 6 months post-transplant. We conclude that electroporation-mediated ex vivo HGF gene transfection protects the kidney against graft injury in a porcine model.

Hepatocyte Growth Factor Suppresses Vascular Medial Hyperplasia and Matrix Accumulation in Advanced Pulmonary Hypertension of Rats

Pulmonary hypertension (PH) is a progressive disease characterized by raised pulmonary vascular resistance, thought to be curable only through lung transplantation. Pathophysiologically, proliferation of pulmonary artery smooth muscle cells triggers pulmonary arterial stenosis and/or regurgitation, especially in advanced PH. Using a rat model of advanced pulmonary vascular disease produced by injecting monocrotaline, we show that hepatocyte growth factor (HGF) targets pulmonary arterioles and blocks the progression of PH. In these rats, endogenous HGF production was dramatically downregulated during developing experimental PH, but c-Met/HGF receptor was abundant in the medial layers of pulmonary arterioles. HGF gene transfection 2 weeks after the monocrotaline injection resulted in milder medial hyperplasia in lung arterioles and inhibited overgrowth of pulmonary artery smooth muscle cells. Notably, exogenous HGF reduced lung expression levels of endothelin-1 and transforming growth factor-beta, which are critically involved in PH-linked fibrogenic events. Overall, medial wall thickening of pulmonary arteries was almost completely prevented by HGF, and the total collagen deposition in the lung decreased; both effects contributed to the suppression of pulmonary artery hypertension. Our results suggest that the loss of endogenous HGF may be a feature of the pathogenesis of PH and that HGF supplementation may minimize pathological lung conditions, even advanced PH.

191. Improvement of Myocardial Dysfunction by Therapeutic Angiogenesis Induced by Transfection of HGF Gene Using Left Ventricular Electromechanical Mapping in Porcine Chronic Myocardial Infarction Model

Top of pageAbstract Therapeutic angiogenesis using angiogenic growth factors via direct myocardial injection has been reported to improve myocardial blood flow in several animal models and patients with ischemic heart disease. In this study, we evaluated the efficacy of therapeutic angiogenesis in porcine chronic infarction model by the direct injection of hepatocyte growth factor (HGF) into myocardium using a needle injection catheter system, to evaluate the potential utility to treat IHD patients. Ischemia was induced by placing an ameroid constrictor around the proximal left circumflex artery. Four weeks later, endocardial electromechanical mapping was used to identify the area of ischemic myocardium. The area of ischemic myocardium as evidenced by uncoupling of mechanical function and electrical activity was about 10cm2 in each group. Each pig received six injections of 0.5 ml of saline or HGF (total dose 0.4 or 4mg) to ischemic area. No animal had pericardial effusion or tamponade, and no episodes of sustained ventricular arrhythmia were noted immediately after intramyocardial injection. At 1month after injection, the ischemic area (% of pre-ischemic area) was significantly reduced in HGF (0.4 or 4mg) group as compared to control (cont;100%, HGF (0.4mg); 43%lowast, HGF (4mg); 26%**,*P<0.05,**P<0.01 vs. control).Moreover, A ratio of the ischemic territory blood supply to the normal territory evaluated by microsphere beads in each animal was significantly increased in HGF (4mg) group as compared to control (cont;0.82, HGF (0.4mg); 1.02, HGF (4mg); 1.19*,*P<0.01 vs. control). These favorable outcomes provide the potential utility to treat IHD patients using catheter-based, intramyocardial injection of HGF gene Figure 1.

Gene transfer of hepatocyte growth factor improves angiogenesis and function of chronic ischemic myocardium in canine heart

Background Hepatocyte growth factor (HGF) induces angiogenesis in myocardium. In the present study, its effects in chronic ischemic myocardium were tested. Methods Four weeks after left anterior descending coronary artery ligation in canine hearts, HVJ-liposome containing either human HGF gene (160 μg; HGF group, n = 7) or nothing (control group, n = 6) was directly injected into ischemic myocardium. Four weeks after gene transfection, the thickness fraction (TF), an index of regional myocardial contractility (assessed by epicardial pulse-Doppler crystals), the myocardial perfusion flow (assessed by color microspheres), and the capillary density (assessed by immunostaining of vessels) were evaluated in ischemic myocardium. Results Thickness fraction (percent of nonischemic myocardium) was significantly improved in the HGF group (80 ± 15 from 52 ± 16 of pregene; p < 0.05) whereas it was not changed in the control group (52 ± 10 from 50 ± 8 of pregene). The perfusion flow (% of nonischemic myocardium) was significantly improved in the HGF group (98 ± 17 from 51 ± 14 of pregene; p < 0.05) while it was not changed in the Control group (58 ± 13 from 62 ± 18 of pregene). The capillary density was significantly higher in the HGF group (894 ± 211/mm 2; p < 0.05) than that in the control group (511 ± 127/mm 2). Conclusions Gene transfection of HGF improved angiogenesis, thereby improved regional myocardial function and perfusion in chronic ischemic myocardium. It indicates a potent therapeutic value of HGF gene transfection for chronic ischemic heart diseases such as myocardial infarction.