Learning and memory changes in rats following exogenous human hepatocyte growth factor gene injection into cerebral ischemic penumbra

Human hepatocyte growth factor can be used to treat cerebral infarction, administered by lateral ventricular, cerebellomedullary cistern or subarachnoid injections. However, the target gene ex-pression product is scarcely found in the ischemic penumbra, but extensively distributes in other regions, increasing the risks of gene therapy. The present study directly transfected hepatocyte growth factor gene into the ischemic penumbra of rats with transient middle cerebral artery occlusion. Immunohistochemical analysis revealed that infarct volume was significantly decreased, hepatocyte growth factor protein expression level and vessel quantity in the ischemic penumbra were significantly increased, and learning and memory were significantly improved.

Phosphatase and tensin homology deleted in chromosome 10,hypoxia-inducible factor-1 alpha gene expression in colorectal adenoma and adenocarcinoma and their relation to vascular endothelial growth factor protein expression

To examine phosphatase and tensin homology deleted in chromosome 10 (PTEN),hypoxia-inducible factor-1 alpha (HIF-1 alpha) gene expressions and their relation to vascular endothelial growth factor(VEGF) protein expression in the patients with human colorectal adenomas and adenocarcinomas.Methods The expression of PTEN,HIF-1 alpha gene was detected by using in situ hybridization,and the VEGF expression levels by immunohistochemistry in colorectal adenomas and primary colorectal adenocarcinoma.Results Strong expression of HIF-1 alpha was detectable in the majority of colorectal dadenocarcinoma,particularly surrounding areas of necrosis in adenocarcinoma.PTEN,HIF-1 alpha mRNA and VEGF protein were positive in 51.6%,67.7% and 59.7% respectively in 62 cases of adenocarcinomas,and 77.8%,44.4% and 33.3% respectively in 18 cases of adenomas.The positive rate of VEGF was higher in the patients with colorectal adenocarcinomas than that in those with adenomas,whereas that of PTEN mRNA was contrary.HIF-1 mRNA expression was correlated significantly with lymph node metastasis,liver metastasis,Duke’s stage and recurrence.During colorectal tumor progression,the expression of HIF-1 alpha mRNA was positively correlated with the VEGF protein expression (χ2= 4.751 ,P<0.05),but negatively with the PTEN mRNA expression(χ2=21.84,P<0.01).Conclusion The absence or low expression of PTEN and the increased levels of HIF-1α and VEGF may paly an important role in carcinogenesis and progression of colorectal carcinoma.These results suggest that VEGF upregulated by HIF-1 alpha gene may be involved in angiogenesis of colorectal adenocarcinoma.4 refs,1 tab.

Expression of serine protease SNC19/matriptase and its inhibitor hepatocyte growth factor activator inhibitor type 1 in normal and malignant tissues of gastrointestinal tract

AIM： To provide the expression profile of serine protease SNC19/matriptase and its inhibitor hepatocyte growth factor activator inhibitor type 1 （HAI-1） in normal and malignant tissues of gastrointestinal tract at mRNA level for further study on their correlations with tumor progression and metastasis. METHODS： Total RNAs were prepared from 37 samples of colorectal cancer tissues, 40 samples of gastric cancer tissues, and their adjacent normal tissues. The expression of SNC19/matriptase and HAI-1 in these samples was detected by real-time fluorescent quantitative PCR using glyceraldehyde-3-phosphate dehydrogenase as internal standard, and the clinical significance for the correlation with clinicopathological parameters was evaluated. RESULTS： In gastric cancer tissues the expression of HAI-1 and SNC19/matriptase was significantly lower than that in the corresponding adjacent normal tissues （Z = -3.280, P= 0.006; Z= -4.651, P= 0.000）. HAI-1：SNC19/matriptase ratio showed no difference between normal and malignant tissues （P〉0.05）. Analysis of clinicopathological parameters showed decreased expression of HAI-1 and HAI-1：SNC19/ matriptase ratio associated with stage Ⅲ/Ⅳ gastric tumors as compared to stage Ⅰ/Ⅱ ones （Z= -2.140, P= 0.031; Z = -2.155, P = 0.031）, and with lymph node-positive gastric cancer tissues as compared to lymph node-negative ones （Z = -2.081, P = 0.036; Z= -2.686, P = 0.006）. The expression of SNC19/matriptase had no relationship with stages and lymph node metastasis （P〉0.05）. The expression of HAI-1 and HAI-1：SNC19/matriptase ratio increased in well-differentiated gastric cancer tissues, but there was no statistical significance （P〉0.05）. The difference of SNC19/matriptase expression was not significant in gastric cancer tissues of different histological differentiation status （P〉0.05）. In colorectal cancer tissues, the expression of HAI-1 and SNC19/matriptase was also markedly lower than that in their adjacent normal tissues （Z= -3.100, P = 0.002; Z= -2.731, P = 0.006）, whereas HAI-1：SNC19/matriptase ratio showed no difference. Decreased expression of HAI-1 was associated with increased invasive depth and lymph node metastasis, but there was no statistical significance （P〉0.05）. The difference of SNC19/matriptase expression and HAI-1： SNC19/matriptase ratio was not significant in different stages and different lymph node metastasis status （P〉0.05）. The expression of SNC19/matriptase, HAI-1 or HAI-1： SNC19/matriptase ratio showed no difference in colorectal cancer tissues of different histological differentiation status （P〉0.05）. CONCLUSION： The expressions of SNC19/matriptase and its inhibitor HAI-1 are decreased in gastrointestinal cancer tissues compared to their normal counterparts, and the decreased expression of HAI-1 may correlate with invasion and lymph node metastasis. The possible mechanisms involved need to be further investigated.

Secretory expression and characterization of a recombinant-deleted variant of human hepatocyte growth factor in Pichia pastoris

AIM： To study the secretory expression of human hepatocyte growth factor （hdHGF） gene in Pichia pastoris. METHODS： The full-length gene of human cDNA encoding the deleted variant of hdHGF was cloned by RT-PCR and overlapping-fragment PCR technique using mRNA of human placenta as a template. The cloned hdHGF cDNA was inserted into the Escherichia coilyeast shuttle vector of pPIC9. The constructed plasmid, pPIC9-hdHGF, was transformed into the GSl15 cells of the methylotrophic yeast, P pastoris, using a chemical method. The Hut ＋ transformants were screened to obtain high-expression strains by the test and analysis of expressed products of shake-flask culture. A secretory form of rhdHGF was made with the aid of the leader peptide sequence of Saccharomyces cerevisiae α-factor. RESULTS： The expressed products, which showed a band of molecular mass of about 80 ku, were observed on 15% SDS-PAGE and identified by Western blotting and N-terminal amino acid sequencing. In the high cell density culture of 5 L fermentor by fed-batch culture protocol, the cell biomass was reached at approximately 135 g （DCW）/L. The productivity of secreted total supernant protein concentration attained a high-level expression of more than 8.0 g/L and the ratio of rhdHGF band area was about 12.3% of the total band area scanned by SDS-PAGE analysis, which estimated that the product of rhdHGF was 500-900 mg/L.CONCLUSION： The P pastoris system represents an attractive tool of generating large quantities of hdHGF for both research and industrial purposes.

Improvement of cardiac function by hepatocyte growth factor via intracoronary transfection in the swine myocardial infarction model

Objective： To study the amelioration effect of AdenovirusS-mediated human hepatocyte growth factor （AdsHGF） on postinfarction heart failure in the swine myocardial infarction model. Methods： Twelve SuZhong young swine were randomly divided into 2 groups with 6 swine in each group： Ad5-HGF-treated group and null-Ad5 group. Four weeks after ligation at left anterior descending coronary artery in swine hearts, Ad5-HGF was transferred to the swine myocardium. Simultaneously, Gated myocardial perfusion imaging was performed to evaluate cardiac perfusion and heart function. After three weeks, Gated myocardial perfusion imaging was performed again, then the hearts were harvested and sectioned to examine the expression of HGF through ELISA. Results： High expression of human HGF was observed in the myocardium of Ad5-HGF-treated group. From 4 weeks to 7 weeks after operation, Left ventricular ejection fraction was increased in Ad5-HGF-treated group. The improvement in LVEF was greater in Ad5-HGF-treated group than that in null-Ad5 group at 7 weeks after operation. Cardiac perfusion was significantly improved in the Ad5-HGF-treated group. Conclusion： High expression of human HGF was observed in the myocardium through intracoronary transfection, which suggests that HGF can ameliorate heart function in swine with postinfarction heart failure.

Role of TGF-β1 and its Receptors in Breast Carcinogenesis:Evaluation of Gene Expression Patterns and Clinical Implications

OBJECTIVE Transforming growth factor β1 （TGF-β1） is a multifunc- tional cytokine that may play an important role in tumor development and progression. METHODS We evaluated gene expression patterns of TGF-β1 and its receptors [transforming growth factor β type Ⅰ receptor （TβR- Ⅰ ） and transforming growth factor β type Ⅱ receptor （TβR- Ⅱ ）] in tumor tissue from patients with breast cancer or with benign breast diseases （BBD） and adjacent normal tissue from the patients with breast cancer. Included in the study were 527 breast cancer patients and 213 BBD patients who participated in the Shanghai Breast Cancer Study. RESULTS The expression levels of the TGF-β1, TβR- Ⅰ and TβR-Ⅱ genes in breast tissue were quantified using real-time PCR. TIER- Ⅱ expression in cancer tissue was decreased by over 50% as compared to either adjacent normal tissue from the same patients or benign tumor tissue from BBD patients （p〈0.001）. TGF-β1 expression was lower by approximately 20% in cancer tissue compared to adjacent normal tissue （p=0.14） or to benign tumor tissue （p=0.002）. Although TβR-Ⅰ expression was also reduced in cancer tissue compared to adjacent normal tissue, or benign tumor tissue, the magnitude of the reduction was less apparent than that for TβR- Ⅱ. Compared to patients with the lowest tertile value for TβR- Ⅱ, patients with median tertile value for TβR- Ⅱ had more favorable overall survival （HR 0.47, 95% CI 0.27-0.85） and disease-free survival （HR 0.65, 95% CI 0.39-1.06）. No apparent associations, however, were observed between TGF-β1 or TβR- Ⅰ expression and overall or disease-free survival. CONCLUSION The results from this study support the hypothesis that a decreased level of TβR-Ⅱ gene expression, and thus reduced TGF-β1 sensitivity, is related to breast tumor progression.

Gene expression of hepatocyte growth factor and its receptor in HCC and nontumorous liver tissues

AIM To study the changes of gene expression of hepatocyte growth factor (HGF) and hepatocyte growth factor receptor (HGFr) in hepatocellular carcinoma (HCC) tissue and nontumorous liver tissue and the relationship between these changes and the biological behavior of the tumor.METHODS Gene expression of HGF and HGFr in 26 cases of HCC tissue and their adjacent nontumorous liver tissues was determined with digoxigenin-labeled DNA probes.RESULTS Positive expression of HGF in HCC tissue was similar to that in the adjacent nontumorous liver tissue, but positive rate of HGF expression was lower than HGFr gene expression. However, HGFr expression was higher in the metastatic cases than in those without metastasis. It was found that HGFr was overexpressed in HCC tissue as well as in the adjacent nontumorous liver tissue.CONCLUSION There seems to be a close relationship between overexpression of HGFr gene and tumor metastasis, and the HGF and HGFr system plays an important role in regulating tumor growth and metastasis.

Expression of vascular endothelial growth factor and its role in oncogenesis of human gastric carcinoma

AIM: To establish the role of vascular endothelial growth factor (VEGF) in the oncogenesis of human gastric carcinoma more directly. METHODS: The expression of VEGF and its receptor kinase-domain insert containing receptor (KDR) in human gastric cancer tissue were observed by immunohistochemical staining. VEGF levels were manipulated in human gastric cancer cell using eukaryotic expression constructs designed to express the complete VEGF(165) complimentary DNA in either the sense or antisense orientation. The biological changes of the cells were observed in which VEGF was up-regulated or down-regulated. RESULTS: VEGF-positive rate was 50%, and VEGF was mainly localized in the cytoplasm and membrane of the tumor cells, while KDR was mainly located in the membrane of vascular endothelial cells in gastric cancer tissues and peri-cancerous tissue. In 2 cases of 50 specimens, the gastric cancer cells expressed KDR, localized in both the cytoplasm and membrane. Introduction of VEGF(165) antisense into human gastric cancer cells (SGC-7901, immunofluorescence intensity, 31.6%)) resulted in a significant reduction in VEGF-specific messenger RNA and total and cell surface VEGF protein (immunofluorescence intensity, 8.9%) (P【0.05). Conversely, stable integration of VEGF(165) in the sense orientation resulted in an increase in cellular and cell surface VEGF (immunofluorescence intensity, 75.4%) (P【0.05). Lowered VEGF levels were associated with a marked decrease in the growth of nude mouse xenografted tumor (at 33 days postimplantation, tumor volume: 345.40 +/- 136.31 mm3)(P【0.05 vs control SGC-7901 group: 1534.40 +/- 362.88 mm3), whereas up-regulation of VEGF resulted in increased xenografted tumor size (at 33 days postimplantation, tumor volume: 2350.50 +/- 637.70 mm3) (P【0.05 vs control SGC-7901 group). CONCLUSION: This study provides direct evidence that VEGF plays an important role in the oncogenesis of human gastric cancer.

Enhanced expression of epidermal growth factor receptor gene in gastric mucosal cells by the serum derived from rats treated with electroacupuncture at stomach meridian acupoints

AIM: To investigate the effect of serum derived from rats treated with electroacupuncture at stomach meridian acupoints on the expression of epidermal growth factor receptor (EGFR) gene in gastric mucosal cells. METHODS: The stress-induced gastric mucosal injury in rat model was established by water-immersion and restrained stress methods. 52 rats were randomly divided into: normal group (n = 8), model group (n = 8), model serum group (n = 12), stomach serum group (n = 12), and gallbladder serum group (n = 12). The gastric mucosal cells were separated by pronase-EDTA digestion method and incubated with serum. The EGFR gene expression in gastric mucosal cells was detected by reverse transcription-polymerase chain reaction (RT-PCR) method. RESULTS: Compared with normal group (0.6860 ± 0.0594), the serum derived from rats of the stomach group (1.2272 ± 0.0813, P = 0.00 < 0.01) and gallbladder group (0.9640 ± 0.0387, P = 0.00 < 0.01) had a tendency to enhance the EGFR gene expression in gastric mucosal cells. Such tendency existed in the model group (0.7104 ± 0.0457) but with no signifi cant difference (P = 0.495 > 0.05) and in model serum group (0.8516 ± 0.0409) with an extremely obvious difference (P = 0.001 < 0.01). Furthermore, the EGFR gene expression in stomach serum group was significantly higher than that in gallbladder serum group (P = 0.00 < 0.01). CONCLUSION: The present study shows that serumderived from rats treated with electroacupuncture at stomach meridian acupoints can distinctly increase the EGFR gene expression of gastric mucosal cells. Therefore, there is certain meridian specificity in the serum, which could provide a proof for the TCM theory “particular relation between meridian and internal organ”.

Expression of Vascular Endothelial Growth Factor in Bone Morphogenetic Protein-2 Induced Osteogenesis

An experimental model of femoral muscular pouch in 20 mice was adopted. The expression of VEGF was examined by in situ hybridization method and immunohistochemical method in bone morphogenetic protein- 2 induced osteogenesis . The experimental results demonstrated that the expression signals of VEGF mRNA and VEGF appeared in cytoplasm during condensation of mesenehymal cell. As the mesenchymal cells differentiated into precartilage, the expression signals decreased in mesenehymal cells, but increased in chondrocytes and kept getting denser in the process of cartilage maturity. The peak expression of VEGF mRNA and VEGF in the experimental group appeared on the 14 th day, accompanied by numerons hypertrophic chondrocytes. When mature cartilage calcified and neu, bone trabecula formed, the expression of VEGF mRNA and VEGF decreased in chondrocytes, but still expressed moderately in the osteoblasts and osteocytes. Signals of VEGF mRNA and VEGF can not be detected in the control groups.

Identification and expression of epidermal growth factor gene in mouse testis

Epidermal growth factor (EGF) is produced primarily by Leydig cells of human testis. Expression of the EGF gene was assessed in mouse testis during the course of sexual maturation by the application of the RT-PCR method and the use of specific oligonucleotide primers. Testis EGF mRNA content increased with the developmental age of the mice, i.e., day 15 < day 30 < day 45 postnatal. The expression of the EGF gene appears to correlate with maturation of the testis and proliferation of Leydig cells.

Relationship between microsatellite instability and hepatocyte growth factor expression and their prognostic significance in colorectal cancer

Objective To investigate the expression of hepatocyte growth factor(HGF)and its relationship with microsatellite instability(MSI)and their influence on survival in patients with colorectal cancer.Methods Immunohistochemistry(IHC)was used to detect the expression of HGF and MSI in 98 specimens of colorectal cancer.Tumors lacking protein expression of any of the our mis-

Gene therapy for pathological scar with hepatocyte growth factor mediated by recombinant adenovirus vector

A complementary DNA (cDNA) encoding human hepatocyte growth factor was intro-duced into a replication-defective type 5 adenovirus (lacking E1, E3 domains) vector by homolo-gous recombination of intracellular plasmid DNA, thus a recombinant vector containing HGF (Ad-HGF) was obtained. Ad-HGF and Ad-GFP (adenovirus vector carrying green fluorescence protein gene) were expanded in 293 cells and purified by cesium chloride gradient centrifugation for large-scale preparation, then were infected to the primarily cultured scar fibroblast of rabbit ear to observe the transfer efficiency and expression level of HGF in vitro. To evaluate the effect of Ad-HGF on established scar Ad-HGF solution was injected into excessively formed scar, which bears some clinical and histologic similarities to human hypertrophic scars. The results showed that: (i) the transfer efficiency was 36.8%±14.1% on day 3 in primarily cultured scar fibroblasts treated with Ad-GFP and lasted more than 20 d; (ii) high-level expression of HGF protein was de-tected by means of ELISA in supernatant of scar fibroblasts treated with Ad-HGF, the amount of expression was 76 ng/4.0×105 cells on day 3; (iii) on day 32 after a single intradermal injection of Ad-HGF at different doses (8.6×109 pfu, 8.6×108 pfu, 8.6×107 pfu, 8.6×106 pfu) per scar, most of the scars in the former two dose groups were dramatically flattened, some were even similar to that of the normal skin. The value of HI (hypertrophic index) showed that there was a therapeutic effect of Ad-HGF on scars at the dose of 109 pfu and 108 pfu. Whereas no therapeutic effects were seen at lower dose (107 pfu and 106 pfu of Ad-HGF) groups. In addition, clusters of hair were ob-served to different extent on healed wound treated with Ad-HGF. Histopathologic examination re-vealed that in most healed wounds of Ad-HGF treated group, the dermal layer was thinner, the amount of fibrous tissue was much fewer, and hair follicles growth and sebaceous glands were observed. In Sirius red-stained sections the amount of type I collagen in the Ad-HGF-treated scars was diminished markedly, compared to that in Ad-GFP group, in which a huge amount of type I collagen was still observed; (iv) immune response against HGF was absent. Antibody against HGF was not detectable by ELISA in serum from rabbit treated with Ad-HGF; (v) no local or systemic side-effects and toxicity associated with the gene transfer were found. These results demonstrated the potential use of treating pathologic scar by Ad-HGF, an alterative strategy of gene therapy for scar in clinical practice.

Effect of human hepatocyte growth factor on promoting wound healing and preventing scar formation by adenovirus-mediated gene transfer

Objective To evaluate the effects of hepatocyte growth factor (HGF) on the prevention of scar formation and the promotion of wound healing by gene transfer Methods A total of 12 female New Zealand rabbits were used in this study Rabbits were anesthetized with an intravenous injection of sodium pentobarbital, and identical wounds were made over the ventral surface of each ear Five circular wounds, 7 mm in diameter, were created in each ear by excision through the skin to the underlying cartilage using sterile technique After the surgical procedures, 10 of the rabbits were randomly allocated to five groups, with 2 rabbits in each group: Ad HGF group 1, Ad HGF group 2, Ad HGF group 3, Ad GFP (a reporter gene) group and the solvent group Immediately after surgery, 6×10 7 pfu Ad HGF, 6×10 8 pfu Ad HGF, 6×10 9 pfu of Ad HGF, 6×10 9 pfu of Ad GFP, or same volume of solvent (PBS, pH 7.2) was applied once to each wound in groups 1 to 5, respectively One additional rabbit was used to evaluate the transfer efficiency of the adenovirus vector by transferring Ad GFP (6×10 9 pfu) into its wounds Ice slides of wounds from this animal were observed under fluorescence microscopy Another additional rabbit was used to evaluate the expression of HGF and TGFβ1 after transferring Ad HGF (6×10 9 pfu) into each of its wound Immunohistochemistry was used for detection Results The effect of HGF on reducing excessive dermal scarring was observed by adenovirus mediated gene transfer Transfection of the human HGF cDNA into skin wounds through an adenoviral vector suppressed the over expression of TGFβ1, which plays an essential role in the progression of dermal fibrogenesis Application of HGF to the wounds significantly enhanced wound healing and inhibited over scarring Conclusion HGF gene therapy could be a new approach for preventing excessive dermal scarring in wound healing

Hepatocyte Growth Factor Gene-modified Bone Marrow-derived Mesenchymal Stem Cells Transplantation Promotes Angiogenesis in a Rat Model of Hindlimb Ischemia

Summary： Angiogenic gene therapy and cell-based therapy for peripheral arterial disease （PAD） have been studied intensively currently. This study aimed to investigate whether combining mesenchymal stem cells （MSCs）transplantation with ex vivo human hepatocyte growth factor （HGF） gene transfer was more therapeutically efficient than the MSCs therapy alone in a rat model of hindlimb ischemia. One week after establishing hindlimb ischemia models, Sprague-Dawley （SD） rats were randomized to receive HGF gene-modified MSCs transplantation （HGF-MSC group）, untreated MSCs transplantation （MSC group）, or PBS injection （PBS group）, respectively. Three weeks after injection, angiogenesis was significantly induced by both MSCs and HGF-MSCs transplantation, and capillary density was the highest in the HGF-MSC group. The number of transplanted cell-derived endothelial cells was greater in HGF-MSC group than in MSC group after one week treatment. The expression of angiogenic cytokines such as HGF and VEGF in local ischemic muscles was more abundant in HGF-MSC group than in the other two groups. In vitro, the conditioned media obtained from HGF-MSCs cultures exerted proproliferative and promigratory effects on endothelial cells. It is concluded that HGF gene-modified MSCs transplantation therapy may induce more potent angiogenesis than the MSCs therapy alone. Engraftment of MSCs combined with angiogenic gene delivery may be a promising therapeutic strategy for the treatment of severe PAD.

Therapeutic angiogenesis induced by human hepatocyte growth factor gene in hindlimb ischemia of dogs

A preclinical study of treating peripheral ar-tery occlusive disease (PAD) was performed by using ahepatocyte growth factor (HGF) gene-expressing vector,plasmid pUDKH, in a dog model with complete ischemia of one hindlimb. After ligation of femoral artery of onehindlimb, pUDKH was transferred directly into the ischemic limb muscles. The angiogenic activity of the plasmid pUDKH was evaluated. On D 30 after injecting once of pUDKH at different doses into local muscles immediately after opera-tion, the degree of augmentation of collateral vessel forma-tion was significantly greater than that treated by blankvector. In addition, the blood flow rate of femoral artery in dogs treated with pUDKH was recovered on D 90, while the flow rate was only 1/5 to 1/3 in control dogs. The pulse am-plitude of pUDKH-treated dogs was recovered on D 90, but it was hardly detectable in most of the control dogs. The sideeffects of intramuscular transfection of pUDKH were alsoinvestigated, and no significant positive change was found. It is suggested that angiogenesis induced by HGF gene has the potential for clinical use in the treatment of peripheral arte-rial diseases.

Treatment of chronical myocardial ischemia by adenovirus-mediated hepatocyte growth factor gene transfer in minipigs

Growth factor gene transfer-induced therapeutic angiogenesis has become a novel approach for the treatment of myocardial ischemia. In order to provide a basis for the clinical application of an adeno- virus with hepatocyte growth factor gene (Ad-HGF) in the treatment of myocardial ischemia, we estab- lished a minipig model of chronically ischemic myocardium in which an Ameroid constrictor was placed around the left circumflex branch of the coronary artery (LCX). A total of 18 minipigs were ran- domly divided into 3 groups: a surgery control group, a model group and an Ad-HGF treatment group implanted with Ameroid constrictor. Ad-HGF or the control agent was injected directly into the ischemic myocardium, and an improvement in heart function and blood supply were evaluated. The results showed that myocardial perfusion remarkably improved in the Ad-HGF group compared with that in both the control and model groups. Four weeks after the treatment, the density of newly formed blood vessels was higher and the number of collateral blood vessels was greater in the Ad-HGF group than in the model group. The area of myocardial ischemia reduced evidently and the left ventricular ejection fraction improved significantly in the Ad-HGF group. These results suggest that HGF gene therapy may become a novel approach in the treatment of chronically ischemic myocardium.

Therapeutic induction of angiogenesis by direct myocardial administration of an adenovirus vector encoding human hepatocyte growth factor gene and its safety

After the study in vitro and in rats, we assessed further the effects and safety of local angiogen therapy using intramyocardial delivery of an adenovirus carrying hepato-cyte growth factor gene (Ad-HGF) in a canine ischemia model. The angiogenic activity of Ad-HGF was evaluated from three aspects. First, the augmentation of collateral ves-sel development was assessed by angiography 30 d after sur-gery. The results showed that the density of collateral vessels in treated group was higher than that of control group. Sec-ondly, infarct size was evaluated by TTC staining and image analysis. The results showed that the infarct size of treated group was smaller than that of control group. Thirdly, the myocardial regional blood flow was determined by the method of colored microspheres. The results showed that the blood flow recovered to the level before ligation in treated group, but that of the control group was lower than normal level. In addition, during the study of chronic toxicity, we tested the anti-adenovirus antibodies by neutralization method. The antibodies yielded after the fourth injection decreased slowly from peak level and disappeared 12 weeks after drug withdrawal. Overall, Ad-HGF can promote an-giogenesis in ischemic myocardium and reduce infarct size. So this method may be considered as a therapeutic angio-genesis induction strategy for ischemic disease including myocardial infarction and peripheral artery disease. At the same time, Ad-HGF could induce the yield of anti-adenovirus antibodies to neutralize adenovirus, which may be the mechanism of adenovirus clearance.

Expression of vascular endothelial growth fac-tor gene in primary cultured rat hepatocytes

BACKGROUND: It is the key point for vascular endothelialgrowth factor (VEGF121) gene related therapy as to how totransfect and express the gene safely, effectively and repeat-edly. This study was designed to investigate the VEGF121transfection and expression in primary cultured rat hepato-cyte.METHODS: After construction of vector internal ribosomeentry site-enhanced yellow fluorescent protein (pIRES-EY-FP)/VEGF121, the transfection and expression of the exoge-nous VEGF121 gene in primary cultured rat hepatocyteswere observed through RT-PCR, Western blot and fluores-cent microscopy.RESULTS: pIRES-EYFP/VEGF121 plasmid was construct-ed and transfected successfully into primary cultured rathepatocytes, the transfection and expression of gene in pri-mary cultured rat hepatocytes were examined by RT-PCRand Western blot, and yellow-green fluorescence was ob-served through a fluorescent microscope.CONCLUSION: The successful transfection and expressionof plasmid pIRES-EYFP/VEGF121 in primary cultured rathepatocytes provides a foundation for hepatocyte transplan-tation and gene therapy after modification of hepatocytesby the gene.

Reduction of tumorigenicity of SMMC-7721 hepatoma cells by vascular endothelial growth factor antisense gene therapy

AIM: To test the hypothesis to block VEGF expression of SMMC-7721 hepatoma cells may inhibit tumor growth using the rat hepatoma model. METHODS: Amplify the 200 VEGF cDNA fragment and insert it into human U6 gene cassette in the reverse orientation transcribing small antisense RNA which could specifically interact with VEGF165, and VEGF121 mRNA. Construct the retroviral vector containing this antisense VEGF U6 cassette and package the replication-deficient recombinant retrovirus. SMMC-7721 cells were transduced with these virus and positive clones were selected with G418. PCR and Southern blot analysis were performed to determine if U6 cassette integrated into the genomic DNA of positive clone. Transfected tumor cells were evaluated for RNA expression by ribonuclease protection assays. The VEGF protein in the supernatant of parental tumor cells and genetically modified tumor cells was determined with ELISA. In vitro and in vivo growth properties of antisense VEGF cell clone in nude mice were analyzed. RESULTS: Restriction enzyme digestion and PCR sequencing verified that the antisense VEGF RNA retroviral vector was successfully constructed.After G418 selection, resistant SMMC-7721 cell clone was picked up. PCR and Southern blot analysis suggested that U6 cassette was integrated into the cell genomic DNA. Stable SMMC-7721 cell clone transduced with U6 antisense RNA cassette could express 200 bp small antisense VEGF RNA and secrete reduced levels of VEGF in culture condition. Production of VEGF by antisense transgene-expressing cells was 65+/-10 ng/L per 10(6) cells, 42045 ng/L per 10(6) cells in sense group and 485+/-30 ng/L per 10(6) cells in the negative control group, (P【 0.05). The antisense-VEGF cell clone appeared phenotypically indistinguishable from SMMC-7721 cells and SMMC-7721 cells transfected sense VEGF. The growth rate of the antisense-VEGF cell clone was the same as the control cells. When S.C. was implanted into nude mice, growth of antisense-VEGF cell lines was greatly inhibited compared with control cells. CONCLUSION: Expression of antisense VEGF RNA in SMMC-7721 cells could decrease the tumorigenicity, and antisense-VEGF gene therapy may be an adjuvant treatment for hepatoma.