Ad-LacZ Groups Research Articles

Adenoviral VEGF-DΔN ΔC gene therapy for myocardial ischemia.

Background: Cardiovascular diseases are the leading cause of death globally. In spite of the availability of improved treatments, there is still a large group of chronic ischemia patients who suffer from significant symptoms and disability. Thus, there is a clear need to develop new treatment strategies for these patients. Therapeutic angiogenesis is a novel therapy method which has shown promising results in preclinical studies. In this study, we evaluated safety and efficacy of adenoviral (Ad) VEGF-DΔNΔC gene transfer for the treatment of myocardial ischemia in a pig model. Methods: Adenoviral VEGF-DΔNΔC gene transfer was given to pigs (n = 26) via intramyocardial injections using an electromechanical injection catheter. Angiogenic effects were evaluated in an acute myocardial infarction model (n = 18) and functionality of the lymphatic vessels were tested in healthy porcine myocardium (n = 8). AdLacZ was used as a control. Results: AdVEGF-DΔNΔC induced safe and effective myocardial angiogenesis by inducing a four-fold increase in mean capillary area at the edge of the myocardial infarct six days after the gene transfer relative to the control AdLacZ group. The effect was sustained over 21 days after the gene transfer, and there were no signs of vessels regression. AdVEGF-DΔNΔC also increased perfusion 3.4-fold near the infarct border zone relative to the control as measured by fluorescent microspheres. Ejection fraction was 8.7% higher in the AdVEGF-DΔNΔC treated group 21days after the gene transfer relative to the AdLacZ control group. Modified Miles assay detected a transient increase in plasma protein extravasation after the AdVEGF-DΔNΔC treatment and a mild accumulation of pericardial effusate was observed at d6. However, AdVEGF-DΔNΔC also induced the growth of functional lymphatic vasculature, and the amount of pericardial fluid and level of vascular permeability had returned to normal by d21. Conclusion: Endovascular intramyocardial AdVEGF-DΔNΔC gene therapy proved to be safe and effective in the acute porcine myocardial infarction model and provides a new potential treatment option for patients with severe coronary heart disease.

Effect of adenovirus-mediated overexpression of PTEN on brain oxidative damage and neuroinflammation in a rat kindling model of epilepsy.

Background:Epilepsy is a chronic and severe neurological disorder. Phosphatase and tensin homolog deleted on chromosome ten (PTEN)-deficient mice exhibit learning and memory deficits and spontaneous epilepsy. The aim of this study was to investigate the role of PTEN in brain oxidative damage and neuroinflammation in a rat model of epilepsy.Methods:An adenovirus (Ad)-PTEN vector was constructed, and status epilepticus (SE) was induced in 41 model rats using lithium chloride-pilocarpine. Thirty-six SE rats were then allocated into the Ad-PTEN, Ad-LacZ, and SE groups, those were administered intracerebroventricular injections of Ad-PTEN, Ad-enhanced green fluorescent protein, and phosphate buffer saline, respectively. The normal group was comprised of healthy Sprague-Dawley rats. Nissl staining was conducted to evaluate neuronal damage, and immunohistochemistry was conducted to observe the morphology of cells in the hippocampal CA1 region and the distribution of ionized calcium-binding adaptor molecule 1 (Iba1) and ED1 (rat homologue of human CD68). Levels of apoptosis-related proteins, inflammatory-related factors, and oxidative stress-related markers (reactive oxygen species [ROS], glutathione [GSH], superoxide dismutase [SOD], and malondialdehyde [MDA]) were measured. Comparisons between multiple groups were conducted using one-way analysis of variance (ANOVA), and pairwise comparisons after ANOVA were conducted using the Tukey multiple comparisons test.Results:After SE induction, PTEN expression in the rat brain exhibited a four-fold decrease (P = 0.000) and the expression of both Iba1 and ED1 increased. Furthermore, significant neuronal loss, oxidative damage, and neuroinflammation were observed in the SE rat brain. After intracerebroventricular injection of Ad-PTEN, PTEN expression exhibited a three-fold increase (P = 0.003), and the expression of both Iba1 and ED1 decreased. Additionally, neurons were restored and neuronal apoptosis was inhibited. Furthermore, ROS and MDA levels decreased, GSH level and SOD activity increased, and neuroinflammation was reduced.Conclusion:Our study demonstrated that brain oxidative damage and neuroinflammation in SE rats were ameliorated by intracerebroventricular injection of Ad-PTEN.

Inhibitory effect of wild-type P53 gene transfer on graft coronary artery disease

ObjectiveThe aim of the present study was to investigate the inhibitory effect of wild-type P53 gene transfer on graft coronary artery disease (GCAD) after heart transplantation and the underlying mechanisms. MethodsA rat model of heterotopic heart transplantation was established using Wistar rats as donors and Sprague-Dawley (SD) rats as recipients. The donor hearts were collected and perfused, via the coronary artery, with 800 μl of recombinant adenovirus carrying the P53 gene (Ad-P53). Thirty minutes later, heart transplant was performed. At 5 d after the transplant surgery, the expression of the exogenous P53 gene and protein in the coronary artery tissues of the donor hearts was examined. At 28 d after the transplant surgery, tissues were collected from the transplanted hearts. The degree of coronary artery stenosis was examined, and apoptosis of the coronary artery smooth muscle cells in the donor hearts was analysed. In addition, histological changes in the vital organs of the recipient rats and the levels of serum biochemical indicators in the rats were also examined. ResultsThe exogenous gene was successfully transferred into donor heart tissues and the coronary artery and was highly expressed. At 28 d after the transplant surgery, the ratio of tunica intima thickness to tunica media thickness (I/M) and the ratio of wall thickness to the lumen diameter of the coronary artery were decreased in the Ad-P53 group compared to those in the Ad-LacZ group and the control group (P < 0.05). A terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay revealed that the percentage of apoptotic coronary artery smooth muscle cells in the donor hearts was significantly increased in the Ad-P53 group compared to that in the Ad-LacZ group and the control group (P < 0.01). The wild-type P53 gene had no effect on the morphology and functions of the vital organs of the recipient rats. ConclusionsP53 gene transfer inhibits coronary artery intimal hyperplasia and reduces the degree of luminal stenosis in transplanted hearts. The inhibitory effect may be related to the wild-type P53 gene-induced apoptosis of vascular smooth muscle cells and inhibition of vascular smooth muscle cell proliferation. This approach is effective and safe and may have good prospects for clinical application.

Abstract 640: Overexpression of Vasohibin-2 Exacerbates Development of Angiotensin II-Induced Thoracic Aortic Aneurysms Independent of VeEGF

Objective: Chronic angiotensin II (AngII) infusion promotes both thoracic (TAAs) and abdominal aortic aneurysms (AAAs) in mice. Vasohibin-2 (VASH2) is known to cause angiogenesis at the sprouting front of neovascularization. The purpose of this study was to examine whether VASH2 influenced AngII-induced TAAs. Methods: Male C57BL/6J mice (10-week-old) were injected with VASH2 or LacZ expressing adenovirus (Ad; 7.5 x 10 9 vp/100 μL) via tail vein at 2 week intervals. One week after the first injection, subcutaneous infusion of either AngII (1,000 ng/kg/min) or saline by mini osmotic pumps was started for 3 weeks. Consequently, mice were divided into 4 groups: AngII + Ad VASH2 (n=22), AngII + Ad LacZ (n=21), saline + Ad VASH2 (n=10), saline + Ad LacZ (n=8). Next, in order to examine whether VASH2 affected TAAs via VEGF regulation, bevacizumab was intraperitoneally administrated into mice; AngII + Ad VASH2 + saline (n=15), AngII + Ad VASH2 + bevacizumab (n=15). TAAs were evaluated in all mice by en face method. Third, human aortic smooth muscle cells (hSMCs) were infected with Ad VASH2 or Ad LacZ, stimulated with or without AngII to evaluate further mechanism. Result: Intima area of aortic arch was significantly larger in AngII + Ad VASH2 group than in AngII + Ad LacZ group (19.78 ± 0.40 mm 3 vs 17.74 ± 0.44 mm 3 , P &lt; 0.001). Gelatin zymography demonstrated that AngII upregulated latent MMP-2 expression, and activated MMP-2 most prominently in AngII + VASH2 group. Protein expression of p21 and p53 in thoracic aortas was enhanced in AngII + VASH2 group. Positive TUNEL staining was observed in thoracic aortic wall of AngII + VASH2 group. No significant difference in intima area of aortic arch between AngII + Ad VASH2 + saline group and AngII + Ad VASH2 + bevacizumab group. In vitro, the same results were observed regarding protein expression of p21 and p53, and TUNEL staining. In addition, Annexin-V staining was detected only in AngII + VASH2 group. Conclusion: Overexpression of VASH2 accelerated development of AngII-induced TAAs in vivo. VASH2-induced cell apoptosis may influence AngII-induced TAA formation independent of VEGF.

Ultrasound molecular imaging of the angiogenic response induced by triple-point mutations of hypoxia-inducible factor-1.ALPHA. in aged mice with hind-limb ischemia

Objective To evaluate the angiogenic response of the HIF-1α with triple-point mutations in aged mice with hind-limb ischemia by ultrasound molecular imaging of αv-integrin expression. Methods Twenty four aged mice under unilateral artery ligation were equally divided into 2 test groups and adenovirus (Ad) vector particles containing the gene for LacZ (Ad-LacZ) or HIF-1a 402/564/803(triple-point mutant, Ad-TM) were injected intramuscularly into the ischemic hind limb.Serial contrast-enhanced ultrasound perfusion imaging of the proximal adductor muscles in the ischemic and contralateral non-ischemic hind limbs was performed (n=6 per group at each time). Ultrasound molecular imaging of the endothelial αv-integrin expression was carried out in mice(n=6 per group) on day 7 after surgery at 8 minutes after intravenous injection of either αv-integrin targeting microbubbles or isotype control microbubbles in random with 30 min interval and the video intensity (VI) was measured.Following ultrasound imaging, the hind-limb was harvested for histological examination of αv-integrin expression. Results Immediately after femoral artery ligation, blood flow (as a percentage of that in the non-ischemia hind limb) was reduced to 15% in the 2 test groups (P>0.05). Although blood flow gradually recovered over time, recovery was persistently better in the Ad-TM group as compared with the Ad-LacZ group. On postoperative day 28, the blood flow ratio was significantly increased in the Ad-TM group (65.6±8.0)% as compared with the Ad-LacZ group (30.7±4.5)% (P<0.01). Minimal ultrasound signals were obtained with both MBαv [VI, (5.2±1.5)U] and MBiso [VI, (5.8±2.2)U] in the non-ischemic hind-limb.Progressive signal enhancement was noted in the ischemic hind limbs with MBαv in the order of the Ad-lacZ group [VI, (15.9±2.8)U], Ad-TM group [VI, (30.8±5.1)U], whereas no significant change of signals was noted in the Ad-TM group[VI, (7.9±1.6)U] and Ad-LacZ group[VI, (8.1±2.0)U] with MBiso. No αv-integrin expression was observed in the non-ischemic hind-limb, a marked endothelial αv-integrin expression in ischemic hind-limb was confirmed by immunofluorescence, especially in the Ad-TM group. Conclusions The angiogenic response is enhanced in the aged mice with hind-limb ischemia by using HIF-1α with triple-point mutations. Key words: Molecular probes; Ultrasound molecular imaging; Hind-limb ischemia; Angiogenesis; αv-integrin

Effects of adenovirus-mediated phosphatase and tensin homolog deleted on chromosome ten gene on the prostate cancer PC-3 cells' metastasis and expression of matrix metalloproteinase-2 and matrix metalloproteinase-9

Objective To investigate the effects of phosphatase and tensin homolog deleted on chromosome ten (PTEN) gene expression on the prostate cancer PC-3 cells' metastasis and the expression of matrix metalloproteinase (MMP)-2 and MMP-9.Methods Recombinant adenovirus vectors were infected into prostate cancer cell line PC-3.The protein expression levels of PTEN,MMP-2 and MMP-9 in the three groups were detected by Western blotting and indirect immunofluorescence assay respectively.The enzymatic activities of MMP-2 and MMP-9 in the three groups were determined by gelatin zymography.Wound scratch assay was used to determine the effect of PTEN on migratory ability of PC-3 cells.Invasion ability of PC-3 cells was measured by Transwell chamber assay.Results After infection by adenovirus,the protein expression of PTEN was significantly up-regulated in PC-3 cells,and the expression and enzymatic activities of MMP-2 and MMP-9 were significantly down-regulated.Wound scratch assay showed that migration distance in Ad-PTEN group at 12,24 and 48 h was (112.36 ± 18.38),(214.75 ± 35.62) and (436.61 ±52.69) μm respectively,which was significantly shorter than in Ad-LacZ group [(166.52 ± 19.28) μm,t=4.55,P＜0.01 at 12 h; (361.87 ±46.15) μm,t=5.64,P＜0.01 at 24 h,and (732.56 ±78.30) μm,t =7.01,P ＜0.01 at 48 h correspondingly].Transwell showed that the number of PC-3 cells that invaded the lower chamber in the Ad-PTEN group (22.40 ± 2.41) was significantly decreased as compared with the control group (38.20 ± 4.44,t =6.99,P ＜ 0.01) and the Ad-LacZ group (37.20 4.97,t =5.98,P ＜ 0.01).Conclusion PTEN gene can significantly inhibit metastasis of the PC-3 cells and expression of MMP-2 and MMP-9,suggesting it may paly an important role in metastasis of prostate cancer. Key words: Prostate cancer; Phosphatase and tensin homolog deleted on chromosome ten; Matrix metalloproteinases-2 ; Matrix metalloproteinases-9 ; Metastasis

Adenovirus-Mediated Anti-sense Extracellular Signal-regulated Kinase 2 Gene Therapy Inhibits Activation of Vascular Smooth Muscle Cells and Angiogenesis, and Ameliorates Transplant Arteriosclerosis

The aim of this study was to explore underlying mechanisms of transplant arteriosclerosis (TA) based on intimal thickening that involve activation of vascular smooth muscle cells (VSMCs) and angiogenesis. We also examined the effects of adenovirus-mediated anti-sense extracellular signal-regulated kinase 2 (ERK2) (Adanti-ERK2) gene therapy on TA. MethodsWe employed a rat aorta transplantation model (Brown-Norway → Lewis). The animals were divided into: (1) an isograft group (n = 6), (2) an empty control group (n = 6), (3) the Ad-LacZ group (n = 6), and (4) the adanti-ERK2 group (n = 6). At 60 days after transplantation, we documented the ratio of intima/(intima + media) the isografts pathologically. Staining for α-actin and platelet-derived growth factor (PDGF)-BB was performed to analyze the migration and secretion of VSMCs. We evaluated angiogenesis and COX-2 staining. ResultIsografts showed normal histology; allografts from the empty control group and the Ad-LacZ group displayed typical TA lesions, while the pathology was significantly improved among the adanti-ERK2 group. The ratios of intima/(intima + media) were 7.6 ± 2.1%, 81.4% ± 6.7%, 85.9% ± 9.4%, and 15.9% ± 4.1% among the isograft group, the empty control, the Ad-LacZ, and the adanti-ERK2 groups respectively. The α-actin+ cells in the intima per field (×400) were 2.1 ± 1.1, 71.3 ± 9.2, 76.4 ± 11.3, and 34.8 ± 5.3, PDGF-BB+ cells, 0.9 ± 0.5, 28.4 ± 3.4, 29.1 ± 3.2, and 8.6 ± 1.7; COX-2+ cells in new capillaries were none, 36.3 ± 8.3, 40.9 ± 9.2, and 10.4 ± 3.9 respectively (P < .05). ConclusionIntimal thickening a key feature of TA, involves activation of VSMC (proliferation, migration and secretion), and the accompanying angiogenesis. Adanti-ERK2 gene therapy modulates the mechanisms, protecting allografts against TA.

Prenatal adenovirus vascular endothelial growth factor gene therapy: a promising new treatment for fetal growth restriction

BackgroundFetal growth restriction (FGR) is an important obstetric condition that is commonly associated with reduced uterine blood flow. In normal sheep pregnancies, adenovirus (Ad) mediated overexpression of vascular endothelial growth factor (VEGF) in the uterine arteries increases uterine blood flow. We hypothesised that enhancing uterine blood flow would improve fetal substrate delivery in a sheep model of FGR that is characterised by reduced uterine blood flow from mid-gestation. MethodsSingleton pregnancies were established with embryo transfer in 90 adolescent ewes subsequently overnourished to generate FGR (n=78) or control fed (n=12). In study 1, at a mean gestational age of 89 days (SE 0·2), 45 overnourished ewes were randomised to receive Ad-VEGF (n=18), Ad-LacZ (control vector, n=14), or saline (n=13) injected into each uterine artery. Control-fed ewes (n=12) received saline. Uterine blood flow was monitored with indwelling flowprobes until necropsy at 131 days’ gestation (0·6). Placental mRNA expression of VEGF and its receptors, Flt1/KDR, was assessed in the maternal and fetal placental compartments by quantitative reverse transcription PCR. In study 2, at 88 days’ gestation (0·7), 33 overnourished ewes received Ad-VEGF (n=17) or saline (n=16) and were allowed to deliver spontaneously at 141 days’ gestation (0·4). In both studies fetal growth and wellbeing were evaluated (blind) using serial ultrasound. FindingsIn both studies ultrasound measurements of abdominal circumference (AC) were greater in Ad-VEGF-treated FGR fetuses than in control-treated FGR fetuses at 21 days (0·3) and 28 days (0·5) post injection (p<0·001–0·047). In study 1, fewer fetuses were markedly growth-restricted (weight >2 SD below control mean) in Ad-VEGF compared with Ad-LacZ plus saline groups (5/18 vs 18/27, p=0·038). Indices of head sparing (ultrasound biparietal diameter:AC ratios, relative brain weight, and brain:liver weight ratios) were lower (p=0·001–0·046) and maternal placental Flt1/KDR mRNA expression higher (p=0·028/0·034) in Ad-VEGF than in Ad-LacZ plus saline groups, but no measurable differences in uterine blood flow were detected. In study 2, Ad-VEGF-treated lambs tended to be heavier (p=0·081) with increased placental efficiency (p=0·074). There were no maternal or fetal complications or significant differences in the level of neonatal care required to ensure lamb survival. InterpretationAd-VEGF safely increases fetal growth in this ovine model of FGR. FundingWellbeing of Women.

Adenovirus-Mediated Anti-Sense ERK2 Gene Therapy Ameliorates Transplant Arteriosclerosis through Inhibiting Activation of Vascular Smooth Muscle Cells and Angiogenesis

The aim of this study is to explore the underlying mechanisms of transplant arteriosclerosis (TA) based on intimal thickening, involving activation of vascular smooth muscle cells (VSMCs) and angiogenesis. The effect of Adenovirus mediated anti-sense ERK2 (Adanti-ERK2) gene therapy on TA is also to be examined. Methods: A rat aorta transplantation model (BN→Lewis) was employed. The animals were divided into: (1) the isograft group (n=6), (2) the empty control group (n=6), (3) the Ad-LacZ group (n=6), and (4) the adanti-ERK2 group (n=6). At 60 days after transplantation, the grafts were collected for pathology. The ratio of intima/(intima□media) was documented. The staining of α-actin and PDGF-BB was performed to analyze migration and secretion of VSMCs. The angiogenesis was evaluated and the COX-2 staining was detected. Result: The isografts showed a normal histology, the allografts from the empty control group and the Ad-LacZ group displayed a typical TA lesion, while the pathology was significant improved in the adanti-ERK2 group. The ratio of intima/(intima+media) were 7.6±2.1%, 81.4±6.7%, 85.9±9.4%, and 15.9±4.1% in the isograft group, the empty control group, the Ad-LacZ group, and the adanti-ERK2 group, respectively. The α-actin+ cells in the intima per vision filed (x400) were 2.1±1.1, 71.3±9.2, 76.4±11.3, and 34.8±5.3, the PDGF-BB+ cells were 0.9±0.5, 28.4±3.4, 29.1±3.2, and 8.6±1.7, the COX-2+ cells in the new capillaries were none, 36.3±8.3, 40.9±9.2, and 10.4±3.9. P< 0.05 was observed between the isograft group, the empty control and the Ad-LacZ group, and the Adanti-ERK2 group. Conclusion: Intimal thickening is one key feature of TA. The mechanisms involve the activation of VSMC (proliferation, migration and secretion), and the accompanying angiogenesis. Adanti-ERK2 gene therapy modulates the mechanisms, and protects allograft against TA.

Adenovirus-mediated tissue factor pathway inhibitor gene transfer induces apoptosis by blocking the phosphorylation of JAK-2/STAT-3 pathway in vascular smooth muscle cells

ObjectiveIn our previous study, we have demonstrated that tissue factor pathway inhibitor (TFPI) gene could induce vascular smooth muscle cell (VSMC) apoptosis. This study was conducted to investigate whether the overexpression of the TFPI gene can induce VSMC apoptosis by inhibiting JAK-2/STAT-3 pathway phosphorylation and thereby inhibiting the expression of such downstream targets as the apoptotic protein Bcl-2 and cell cycle protein cyclin D1. The effect of TFPI on the expression of survivin, a central molecule in cell survival, was also investigated. MethodsRat VSMCs were infected with recombinant adenovirus containing either the TFPI (Ad-TFPI) or LacZ (Ad-LacZ) gene or DMEM in vitro. TFPI expression was detected by ELISA. TUNEL staining and electron microscope were carried out to determine the apoptosis of VSMCs. The expression levels of JAK-2, p-JAK-2, STAT-3, p-STAT-3, cyclin D1, Bcl-2 and survivin were examined by western blot analysis. ResultsTFPI protein was detected in the TFPI group after gene transfer and the peak expression was at the 3rd day. At the 3rd, 5th and 7th days after gene transfer, the apoptotic rates by TUNEL assay in the TFPI group were 10.91±1.66%, 13.46±1.28% and 17.04±1.95%, respectively, whereas those in the LacZ group were 3.28±0.89%, 4.01±0.72% and 4.89±1.17%, respectively. We observed cell contraction, slight mitochondrial swelling, nuclear pyknosis and apoptotic body formation in TFPI-treated VSMCs using electron microscopy. JAK-2, p-JAK-2, STAT-3, p-STAT-3, cyclin D1 and Bcl-2, which are all involved in the JAK-2/STAT-3 pathway, were detected in the VSMCs on the 3rd, 5th and 7th days after gene transfer, which is consistent with previously demonstrated time points when VSMCs apoptosis occurred. The expression levels of p-JAK-2, p-STAT-3, cyclin D1 and Bcl-2 were significantly decreased over time in the TFPI group (each P<0.05) but not in the Ad-LacZ and DMEM groups. However, this attenuation of expression was not observed for JAK-2 and STAT-3 in any of the groups at any time points after gene transfer (each P>0.05). The expression level of survivin in the TFPI group also weakened significantly over time compared with the levels in the Ad-LacZ and DMEM groups (each P<0.05) at the 3rd, 5th and 7th days after gene transfer. ConclusionThe results demonstrated that TFPI played an apoptosis-inducing role in VSMCs in a manner that involves both the suppression of JAK-2/STAT-3 pathway phosphorylation and the down-regulation of survivin. Our data show for the first time that targeting the JAK-2/STAT-3 pathway and survivin by overexpressing TFPI may be a new avenue for the treatment of restenosis.

WoLiver Regeneration Effect of Oncostatin M Following Hepatectomy for the Rat Cirrhotic Liver Model

Background/Aims: Liver resection represents the treatment of choice for hepatocellular carcinoma (HCC) arising in well-compensated cirrhosis. Gene expression of the multifunctional cytokine, Oncostatin M (OSM), stimulates liver regeneration and adenoviral vector expressing OSM (AdOSM) allows a persistent expression of the gene. The aim of this study is to evaluate the benefits of the preoperative injection of AdOSM to the remnant lobes to regenerate the liver. Methods: A 70% partial hepatectomy was performed in dimethylnitrosamine-administrated cirrhotic rats with a preoperative injection of AdOSM, adenoviral vector carrying β-galactosidase (AdLacZ), or phosphate-buffered saline (PBS). The morphologic, histologic, and biochemical changes in the remnant liver and survival rates were then assessed. Results: Portal injection with clamping the portal branches of the resected lobes for 5 min made it possible to effectively transduce the adenoviral vector into the remnant lobes. The ratio of the remnant liver weight/body weight (%) was 2.3 ± 0.5 in the AdOSM group, 1.1 ± 0.3 in the AdLacZ group (p < 0.001), and 1.6 ± 0.4 in the PBS group (p = 0.02). The fibrous ratio (%) was 21.3 ± 4.6 in the AdOSM group and 35.2 ± 4.5 in the AdLacZ group on day 4 after hepatectomy and fibrous status was significantly decreased in the AdOSM group (p = 0.02). Serum hyaluronic acid which is the indicator of liver fibrosis was 215 ± 141 ng/mL in the AdOSM group and 1963 ± 1225 ng/mL in the AdLacZ group (p = 0.03). Conclusions: The OSM gene therapy may increase the possibility of hepatectomy in a cirrhotic liver by improving fibrosis, hepatic function, and hepatocyte regeneration.

Serum IL-18 levels in mice with collagen-induced arthritis treated by recombinant adenovirus containing mIL-18BP and mIL-4 fusion gene

To investigate serum IL-18 levels in mice with collagen-induced arthritis treated by recombinant adenoviral vector containing mIL-18BP and mIL-4 fusion gene (AdmIL-18BP/mIL-4). Arthritis was induced by injection of collagen in male DBA-1/BOM mice. Mice with collagen-induced arthritis (CIA) were intra-articularly injected with 10(7)pfu/6μL of AdmIL-18BP/mIL-4; and in mice of control groups AdLacZ or PBS were used. The animals were sacrificed at week 1, 2 and 4 after treatment. Serum IL-18 levels were determined by ELISA at the different time points. The mean serum levels of IL-18 at weeks 1, 2, and 4 after injection of AdmIL-18BP/mIL-4 were (36.5±5.4)ng/L, (32.5 ± 3.2) ng/L and (28.7 ±2.9)ng/L, respectively, which were significantly lower than those at the same time point of AdLacZ group [(66.2 ±5.1)ng/L, (69.2 ±4.2)ng/L and (77.7 ±3.9)ng/L] and PBS group [(67.3 ±7.1)ng/L, (71.9 ±1.8)ng/L and (78.7±4.1)ng/L] (P<0.01 at all time points). In the therapy group, there were no significant differences in the mean serum concentrations of IL-18 at all time points. The serum IL-18 levels in CIA mice are down-regulated by treatment of recombinant adenovirus containing mIL-18BP and mIL-4 fuse gene, which might be a promising therapeutic strategy for rheumatoid arthritis.

Effects of Ad-p27mt gene transfer on the expression of Bax, Bcl-2, VEGF and MMP-9 in the transplanted liver tumors in nude mice

In this study, the mechanism by which Ad-p27mt inhibits the growth, invasion and metastasis of transplanted liver tumor was studied by examining the effects of Ad-27mt gene transfer on the expression of Bax, Bcl-2, VEGF and MMP-9 in the transplanted liver tumors in nude mice. The model of transplanted hepatic tumor was established in nude mice. The mice were then divided into three groups, which were injected with PBS, Ad-LacZ and Ad-p27mt and the growth of the transplanted liver tumor was observed. The expressions of P27, Bax and Bcl-2 proteins were detected by Western blotting and the expressions of VEGF and MMP-9 were immunohistochemically determined. Our result showed that the tumor size, expressions of Bax, Bcl-2 proteins, VEGF and MMP-9 were all lower than those in PBS and Ad-LacZ groups and the differences were statistically significant (P<0.05). Our study suggested that Ad-p27mt could inhibit the growth, invasion and metastasis of hepatic cancer by lowering the expressions of VEGF and MMP-9.

Impact of p27mt gene on transplantation model of human colorectal cancer in nude mice

To investigate the inhibitory and anti-metastatic effect of mutant p27 gene (p27mt) on the growth of colorectal cancer xenografts in nude mice and its underlying mechanism. Inhibitory effect of p27mt gene on the growth of colorectal cancer xenografts was determined by measurement of tumor size before and after direct intra-tumoral injection of Ad-p27mt in a pre-established transplantation model of human colorectal cancer in nude mice. Cell cycle and apoptosis were detected by flow cytometry performed on single-cell suspension from an isolated tumor. Expression of MMP-9 in tumor tissue was detected by immunohistochemistry. The average sizes of transplantation tumors were 1.94 +/- 0.67 cm(3), 2.75 +/- 0.83 cm(3) and 3.01 +/- 0.76 cm(3) in the Ad-p27mt, Ad-LacZ and control groups, respectively (P < 0.05). The average proliferation rates were 37.34% +/- 1.45%, 53.16% +/- 3.27% and 54.48% +/- 2.43%, in the Ad-p27mt, Ad-LacZ and control groups, respectively (P < 0.05). The average apoptosis rates were 19.79% +/- 3.32%, 6.38% +/- 4.91% and 7.25% +/- 5.20% in the Ad-p27mt, Ad-LacZ and control groups, respectively (P < 0.01). The average MMP-9 expression rates were 20%, 75% and 66.7% in the Ad-p27mt, Ad-LacZ and control groups, respectively (P < 0.01). p27mt inhibits the growth of transplanted tumor by blocking the proliferation of cancer xenografts and by promoting apoptosis of transplantated tumor cells, as well as decrease transpl-anted tumor metastasis.

Effect of p27mt gene on apoptosis of the colorectal cancer cell line Lovo

To construct p27mt recombinant adenovirus, transfect the colorectal cell line Lovo and observe the effects of p27mt on Lovo cell apoptosis and cell cycle inhibition. We constructed recombinant adenovirus containing p27mt by homologous recombination in bacteria. The colorectal cancer cell line Lovo was infected with recombinant replication-defective adenovirus Ad-p27mt, and expression of p27mt was determined by Western blotting; the inhibitory effect of p27mt on Lovo cells was detected by cytometry. Cell cycle was determined by flow cytometry. DNA fragment analysis identified the occurrence of apoptosis. The recombinant adenovirus which already contained p27mt target gene was successfully constructed. When multiplicity of infection was >or= 50, the infection efficiency was 100%. After transfection of Lovo cells with Ad-p27mt the cells had high p27 expression which was identified by immunoblotting assay. PI staining and flow cytometry showed that 77.96% of colorectal cancer cells were inhibited in phase G(0)/G(1), while in the Ad-LacZ group and blank control group, 27.57% and 25.29% cells were inhibited in the same phase, respectively. DNA fragment analysis, flow cytometry and TUNEL assay demonstrated that p27mt is able to induce apoptosis in colorectal cancer cells. p27mt has an obvious blocking effect on colorectal cancer cell cycle, and most cells were inhibited in phase G(0)/G(1). Therefore, p27mt can induce apoptosis in colorectal cells.

Adenovirus mediated angiostatin gene therapy for ovarian cancer: experiment with nude mice

To built an expression vector of angiostatin (AG) gene with recombinated replication defective adenovirus and investigate the therapeutic effect of human AG gene on ovarian cancer. (1) Human AG K (1 - 3) cDNA was inserted into the vector pShuttle to build the recombinant plasmid pShttle-AG (K1-3). pAdeno-X-AG (K1-3) was built by double-cut and recombinated pShttle-AG (K1-3) to vector pAdeno-X, and then recombinant adenovirus was finally prepared by transfection of pAdeno-X-AG (K1-3) into to the human embryo kidney cells of the line 293. (2) Human ovarian cancer cells of the line SKOV3 were inoculated subcutaneously into nude mice of the line BALB/c nu/nu to establish model of human ovarian cancer. Then the mice were randomly divided into 3 groups to be injected with Ad = AG (K1-3), Ad-LacZ, or phosphate buffered saline (PBS) around the cancer every 5 days. The tumor size was measured every 5 days to calculate the tumor volume and tumor inhibition rate. Three days after the last injection the mice were killed. The tumor tissues, livers, and kidneys of the mice underwent immunohistochemistry to calculate the microvessel density (MVD) and expression of vessel endothelial growth factor (VEGF) and AG. The tumor volume and weight of the Ad-AG (K1-3) group were significantly less than those of the PBS and Ad-LacZ groups (all P < 0.01), however, there were not significant difference between the latter two groups (both P > 0.05). The expression levels of CD34 and VEGF of the Ad-AG (K1-3) group were both significantly lower than those of the PBS and Ad-LacZ groups (all P < 0.01), however (both P > 0.05). Human angiostatin mediated by adenovirus suppresses the angiogenesis and the growth of human ovarian cancer in the nude mice model, which suggests that it is promising in clinical application.

Adenovirus-mediated gene transfer of tissue factor pathway inhibitor induces apoptosis in vascular smooth muscle cells

To investigate the pro-apoptotic effect of tissue factor pathway inhibitor (TFPI) gene transfer mediated by adenovirus on vascular smooth muscle cells (VSMCs). Rat VSMCs were infected with recombinant adenovirus containing either the TFPI (Ad-TFPI) or LacZ (Ad-LacZ) gene or DMEM in vitro. TFPI expression was detected by ELISA. Apoptosis of VSMCs was determined by electron microscopy and flow cytometry. The expression of cytochrome c, procaspase-3, cleaved caspase-3, cleaved caspase-9 and inhibitor of apoptosis protein-1(IAP-1) were examined by western blot and RT-PCR. TFPI protein was detected in the TFPI group after gene transfer and the peak expression was at the 3rd day. At the 3rd, 5th and 7th day after gene transfer, the apoptotic rates in the TFPI group were 11.95%, 71.96% and 37.83%, respectively, whereas those in the LacZ group were 1.34%, 1.83% and 6.37%, respectively. We observed cell contraction, slight mitochondrial swelling, nuclear pyknosis and apoptotic body formation in TFPI-treated VSMCs using electron microscopy. Cytochrome c, cleaved caspase-3 and cleaved caspase-9, which are all involved in mitochondrial pathway, were detected in the cytoplasm on the 3rd, 5th and 7th day after TFPI gene transfer. Procaspase-3 expression was significantly decreased over time in the TFPI group (each P < 0.05), which were not seen in the Ad-LacZ and DMEM groups. The expression of IAP-1 mRNA in the TFPI group was also decreased compared with the Ad-LacZ and DMEM groups (each P < 0.05) at the 3rd and 7th day after gene transfer. The results demonstrated that overexpression of TFPI gene might induce VSMC apoptosis in vitro through the mitochondrial pathway; meanwhile, IAP-1 expression is decreased. These findings indicated that TFPI might inhibit restenosis by inducing apoptosis in VSMCs.

Recombinant Adenoviral Gene Transfer Does Not Affect Cardiac Allograft Vasculopathy

Adenovirus serotype 5 has remained the pre-eminent vector in pre-clinical gene therapy applications in cardiac transplantation. Concerns over the potential effects of adenoviral vectors on the later development of cardiac allograft vasculopathy (CAV) are addressed in this study. Hearts (n = 22) harvested from Brown Norway rats were perfused ex vivo with either University of Wisconsin (UW) solution with no virus, Ad-CMV-LacZ or Ad-CMV-Null. Donor hearts were transplanted heterotopically into the abdomen of Lewis rats. All recipients received cyclosporine for the duration of the experiment. Transplanted hearts were recovered for analysis at 120 days. Sections of the heart were stained with elastic-van Gieson stain for morphometric analysis of the vessels to ascertain the degree of vascular luminal occlusion. Hematoxylin-eosin staining facilitated diagnosis of chronic rejection. Seventy-seven percent of transplanted hearts showed signs of chronic rejection with no difference in the proportion of animals between groups (p = 0.797). No difference was noted in the degree of vascular luminal occlusion between the Ad-Null (0.57 +/- 0.22), Ad-LacZ (0.62 +/- 0.19) and UW (0.47 +/- 0.29) groups (p = 0.653). Vascularized cardiac allografts transplanted from Brown Norway to Lewis rats demonstrated cardiac allograft vasculopathy CAV at 120 days. Adenoviral perfusion of the donor heart ex vivo did not affect the development of CAV.

Impact of different drugs and different volumes injected intraspinally on the nerve function: experiment with rats

To investigate the impact of different drugs and different volumes injected intraspinally on the nerve function and find out the safe intraspinally injection volume. Ninety-six adult Sprague-Dawley rats were randomly divided into 12 equal groups to be injected intraspinally with normal saline (NS) 2 microl, 4 microl, 6 microl, or 10 microl, viral buffer 2 microl, 4 microl, 6 microl, or 10 microl, or Ad-LacZ (1 x 10(9) pfu/ml) 2 microl, 4 microl, 6 microl, or 10 microl at the level of T13 respectively immediately after the laminectomy was performed. Another 6 adult SD rats only underwent T13 luminal decompression and used as controls. Basso-Beattie-Bresnahan locomotor rating scale was used to evaluate the motor function of their hind limbs after 1, 2, 3, 4, 5, and 6 days, and 1, 2, 3, and 4 weeks. One and 2 weeks after the injection 2 rats from each group were killed and the remaining 4 rats in each group were killed 4 weeks after the injection. Histological examination was done with HE staining and toluidine blue staining. X-gal staining was used to observe the expression of adenovirus in the spinal cord. The extent of motor deficits and tissue damage increased following the increase of intraspinal injection volume (P = 0.000). The severity of damage of the Ad-LacZ was significantly greater than those of the NS and virus buffer groups (P = 0.044), however, there were no significant differences in the severity of damage among NS groups and virus buffer groups (P = 1.000). X-Gal staining showed that the reporter gene LacZ was expressed in all Ad-LacZ groups. The great the dose the more severe the damage. Four weeks later the effective residual rates f the grey and white matters of the Ad-LacZ groups were all significantly lower than those of the other 2 groups, however, there were no significant differences in the effective residual rates f the grey and white matters among the NS and virus buffer groups. Intraspinal injection results in functional deficit and tissue damage in the spinal cord. The extent of tissue damage increases following the increase of intraspinal injection volume. Different drugs cause different tissue damage. The intraspinal injection of Ad-LacZ results in more tissue damage. The volume of 4 microl is the safe volume for injection, 6 microl is a critical volume.

Percutaneous Cardiac Recirculation-Mediated Gene Transfer of an Inhibitory Phospholamban Peptide Reverses Advanced Heart Failure in Large Animals

The purpose of this study was to develop a clinically applicable high-efficiency percutaneous means of therapeutic gene delivery to the failing heart. Substantial advances in the understanding of the cellular and molecular basis of heart failure (HF) have recently fostered interest in the potential utility of gene and cell therapy as novel therapeutic approaches. However, successful clinical translation is currently limited by the lack of safe, efficient, and selective delivery systems. We developed a novel percutaneous closed-loop recirculatory system that provides homogeneous myocardial delivery for gene transfer in the failing large animal heart. After 4 weeks' rapid pacing in adult sheep to induce HF, the animals were randomly allocated to receive either adenovirus expressing a pseudophosphorylated mutant (AdS16E) of phospholamban (PLN) or Ad-beta-galactosidase (AdLacZ). Two weeks after gene delivery, in the presence of continued pacing, left ventricular (LV) ejection fraction had significantly improved in the AdS16E-treated animals (27 +/- 3% to 50 +/- 4%; p < 0.001), whereas a further decline occurred in the AdLacZ group (34 +/- 4% to 27 +/- 3%; p < 0.05). In conjunction, AdS16E delivery resulted in significant reductions in LV filling pressures and end-diastolic diameter (both p < 0.05). In conjunction, AdS16E-treated animals showed significant improvement in the expression of PLN and Ca2+-adenosine triphosphatase activity. In separate animals, recirculating AdLacZ delivery was shown to achieve superior myocardial gene expression in contrast to intracoronary delivery and was associated with lower systemic expression. We report the development of a novel closed-loop system for cardiac gene therapy. Using this approach delivery of AdS16E reversed HF progression in a large animal HF model.