Ad-LacZ Groups Research Articles

Adenoviral vector expressing hepatocyte growth factor promotes liver regeneration by preoperative injection: The advantages of performing selective injection to the remnant lobe

In a cirrhotic liver, the regenerative ability is impaired and liver failure may occur after a hepatectomy. Hepatocyte growth factor (HGF) stimulates liver regeneration and adenoviral vector expressing hepatocyte growth factor (AdHGF) allows hepatocyte growth factor (HGF) to be persistently expressed. The aim of this study is to evaluate the benefits of the selective and preoperative injection of AdHGF to the remnant lobes to regenerate the liver. A 70% partial hepatectomy was performed in dimethylnitrosamine-induced cirrhotic rats with a preoperative injection of AdHGF, adenoviral vector carrying beta-galactosidase (AdLacZ), or phosphate-buffered saline (PBS). The morphologic, histologic, and biochemical changes in the remnant liver and survival rates were then assessed. 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. On day 7 after hepatectomy, the survival rates were 87% in the AdHGF group, 53% in the AdLacZ group, and 40% in the PBS group (P < .05). The ratio of the remnant liver weight/body weight (%) was 2.0 +/- 0.1 in the AdHGF group, 1.5 +/- 0.3 in the AdLacZ group, and 1.6 +/- 0.04 in the PBS group (P < .01). The 5-bromo-2'-deoxyuridine labeling index significantly increased in the AdHGF group on day 1, and the fibrous status significantly decreased in the AdHGF group on day 7 after hepatectomy. Preoperatively, the selective injection of AdHGF into the remnant lobes may be an effective treatment prior to a major hepatectomy in a cirrhotic liver.

Suppression of human colon tumor growth by adenoviral vector-mediated NK4 expression in an athymic mouse model

To investigate the suppressive effects of adenoviral vector-mediated expression of NK4, an antagonist of hepatocyte growth factor (HGF), on human colon cancer in an athymic mouse model to explore the possibility of applying NK4 to cancer gene therapy. A human colon tumor model was developed by subcutaneous implantation of tumor tissue formed by LS174T cells grown in athymic mice. Fifteen tumor-bearing mice were randomized into three groups (n = 5 in each group) at d 3 after tumor implantation and mice were injected intratumorally with phosphate-buffered saline (PBS) or with recombinant adenovirus expressing beta-galactosidase (Ad-LacZ) or NK4 (rvAdCMV/NK4) at a 6-d interval for total 5 injections in each mouse. Tumor sizes were measured during treatment to draw a tumor growth curve. At d 26 after the first treatment, all animals were sacrificed and the tumors were removed to immunohistochemically examine proliferating cell nuclear antigen (PCNA), microvessel density (represented by CD31), and apoptotic cells. In a separate experiment, 15 additional athymic mice were employed to develop a tumor metastasis model by intraperitoneal injection (ip) of LS174T cells. These mice were randomized into 3 groups (n = 5 in each group) at d 1 after injection and were treated by ip injection of PBS, or Ad-LacZ, or rvAdCMV/NK4 at a 6-d interval for total two injections in each mouse. All animals were sacrificed at d 14 and the numbers and weights of disseminated tumors within the abdominal cavity were measured. Growth of human colon tumors were significantly suppressed in the athymic mice treated with rvAdCMV/NK4 (2537.4 +/- 892.3 mm(3)) compared to those treated by either PBS (5175.2 +/- 1228.6 mm(3)) or Ad-LacZ (5578.8 +/- 1955.7 mm(3)) (P < 0.05). The tumor growth inhibition rate was as high as 51%. Immunohistochemical staining revealed a similar PCNA labeling index (75.1% +/- 11.2% in PBS group vs 72.8% +/- 7.6% in Ad-LacZ group vs 69.3% +/- 9.4% in rvAdCMV/NK4 group) in all groups, but significantly lower microvessel density (10.7 +/- 2.4 in rvAdCMV/NK4 group vs 25.6 +/- 3.8 in PBS group or 21.3 +/- 3.5 in Ad-LacZ group, P < 0.05), and a markedly higher apoptotic index (7.3% +/- 2.4% in rvAdCMV/NK4 group vs 2.6 +/- 1.1% in PBS group or 2.1% +/- 1.5% in Ad-LacZ group, P < 0.05) in the rvAdCMV/NK4 group compared to the two control groups. In the tumor metastasis model, the number and weight of disseminated tumors of mice treated with rvAdCMV/NK4 were much lower than those of the control groups (tumor number: 6.2 +/- 3.3 in rvAdCMV/NK4 group vs 22.9 +/- 7.6 in PBS group or 19.8 +/- 8.5 in Ad-LacZ group, P < 0.05; tumor weight: 324 +/- 176 mg in rvAdCMV/NK4 group vs 962 +/- 382 mg in PBS group or 1116 +/- 484 mg in Ad-LacZ group, P < 0.05). The recombinant adenovirus, rvAdCMV/NK4, can attenuate the growth of colon cancer in vivo, probably by suppressing angiogenesis and inducing tumor cell apoptosis, but not by direct suppression of tumor cell proliferation. Moreover, rvAdCMV/NK4 may inhibit peritoneal dissemination of colon cancer cells in a murine tumor metastasis model. These findings indicate that NK4 gene transfer may be an effective tool for the treatment of colon cancer.

Adenovirus mediated vascular endothelial growth factor gene transfer protects dopaminergic neurons in Parkinson's disease: experiment with rats

To evaluate the protective effect of adenovirus mediated vascular endothelial growth factor 165 (VEGF165) gene transfer on dopaminergic neurons in Parkinson disease (PD). Adenovirus vector coding VEGF165 (Ad-VEGF165) was injected into the striate bodies of 16 SD rats, and adenovirus Ad-LacZ was injected into 25 rats and phosphate-buffered saline (PBS) was injected into 16 rats as controls. Then 6-hydroxydopamine (6-OHDA) was injected to establish PD model. X-gal staining was used to detect the expression of the report gene LacZ in the brain of the Ad-LacZ group 3 d, 2 w, and 6 w after injection, 3 rats in each time point. RT-PCR was used to detect the VEGF165 mRNA expression in the striate body of the rats of the 3 groups 2 weeks after injection, 3 rats for each group. Western blotting was performed to check the protein expression of VEGF165 in the striate bodies of the rats of the 3 groups 2 weeks after injection, 3 rats for each group. A certain numbers of rats in each group underwent rotational behavior analysis 1, 2, and 6 weeks after the 6-OHDA injection. Immunohistochemistry was used to examine the number of tyrosine hydroxylase (TH) positive neuron, density of TH-positive fiber in striate body and black substance, laminin-positive vessel density, and glial fibrillary acidic protein (GFAP) positive glial cells. High performance liquid chromatography-electric-chemical discharge (HPLC-ECD) was performed to detect the contents of dopamine (DA) and its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the striatum. Beta-gal was expressed in the striate bodies of all Ad-LacZ transfected rats, showing the successful transfection of LacZ gene. The mRNA expression and protein expression of VEGF165 in the striate body were significantly higher in the Ad-VEGF165 group than in the other 2 groups. The apomorphine-induced rotation number in the Ad-VEGF165 group was 8.3 turns/min +/- 8.7 turns/min 1 week after the transfection, then gradually decreased, and became 5.0 turns/min +/- 4.4 turns/min 6 weeks after. The rotation numbers of the Ad-LacZ and PBS group were 14.7 turns/min +/- 11.2 turns/min and 13.9 turns/min +/- 8.3 turns/min respectively 1 week after the injection, then increased gradually, and became 20.2 turns/min +/- 13.6 turns/min and 21.8 turns/min +/- 11.8 turns/min respectively 6 weeks later, all significantly higher than those of the Ad-LacZ group (all P < 0.01). The ratios of TH-positive cells in the black substance in the transfected side was 0.42 +/- 0.11, and the density of fibers in the striate body of the transfected side to that of the contralateral side was 0.56 +/- 0.10 in the Ad-VEGF165 group, both significantly higher than those of the Ad-LacZ group (0.20 +/- 0.10 and 0.28 +/- 0.09) and PBS group (0.22 +/- 0.13 and 0.24 +/- 0.08), (all P < 0.01). The ratio of laminin-positive vessel density of the transfected side to that of the contralateral side in the Ad-VEGF165 group was 2.09 +/- 0.42, and the ratio of GFAP-positive glial cells of the striate body of the transfected side to that of the contralateral side was 2.77 +/- 1.21 in the Ad-VEGF165 group, both significantly higher than those in the Ad-LacZ group (1.01 +/- 0.16 and 1.64 +/- 0.28) and the PBS group (1.04 +/- 0.09 and 1.56 +/- 0.62) (P < 0.01 and 0.05). HPLC-ECD showed that the contents of DA, HAV, and DOPAC of the striate body at the destroyed side in the Ad-VEGF165 group were all significantly higher than those in the other 2 groups (all P < 0.01). The ratios to the DA, DOPAC, and HVA of the destroyed side striate body to those of the contralateral side in the Ad-VEGF165 group was 0.35 +/- 0.11, 0.46 +/- 0.09, and 0.38 +/- 0.09 respectively, all significantly higher than those in the Ad-LacZ group (0.17 +/- 0.15, 0.21 +/- 0.07, and 0.16 +/- 0.05) and PBS group (0.19 +/- 0.06, 0.20 +/- 0.09, 0.14 +/- 0.03) (all P < 0.01). Gene transfer of Ad-VEGF165 has a protective effect on the dopaminergic neurons of PD. The proliferation of vessels and glial cells induced by VEGF may involve in the process of neuroprotection to the dopaminergic neurons of PD.

Donor graft adenoviral iNOS gene transfer improve transplant survival and ischemia / reperfusion injury

Background: Stress response genes, such as inducible nitric oxide synthase (iNOS) and heme oxygenase (HO)-1, are known to be upregulated by ischemia/reperfusion (I/R) injury. We have previously shown that the L-arginine/NOS pathway has a protective effect on hepatic cold I/R injury. In this study, to examine whether hepatic iNOS gene transfer could improve transplant survival following severe I/R injury, donor grafts underwent prolonged cold-storage for 24 hrs prior to transplant. Methods: Adenovirus encoding iNOS (AdiNOS) or control gene vector (AdLacZ) was delivered to the liver by donor intravenous pretreatment 4 days before harvesting. Orthotopic syngeneic LEW rat liver transplantation (OLT) was performed after 18 or 24 hrs preservation in cold UW. Serum LFTs, histopathology, hepatic ICAM-1 mRNA expression, and neutrophil infiltration were assessed after reperfusion. L-NIL and tin-protoporphyrin IX (SnPP) were used as selective inhibitors for iNOS and HO-1, respectively. Survival data (24 hrs preservation) was analyzed by Kaplan-Meier and log-rank x2 tests. Results: AdiNOS, but not AdLacZ, treatment at a relatively low titer (2 × 109 pfu) produced abundant iNOS protein expression in the donor liver before harvesting and in the recipient isograft as early as 1 hr post-reperfusion, indicating iNOS expression was due to the transduced AdiNOS and not induction of endogenous iNOS. Using the prolonged cold storage (24 hrs) model of severe I/R injury, animals treated with AdiNOS showed significantly improved survival from 30% to 70% (table, # indicates p <0.05 vs. saline or AdLacZ groups). AdiNOS delivery also markedly decreased serum AST/ALT levels and tissue necrosis. Hepatic ICAM-1 mRNA expression and neutrophil infiltration were also reduced. (mean±SE, n=3-6 animals per group, p < 0.05). HO-1 protein expression was induced in the AdiNOS-treated, but not AdLacZ-treated, donor liver grafts. Administration of L-NIL (30 mg/kg iv) or SnPP (10 μM/kg iv) partially abrogated the protection afforded by AdiNOS (data not shown). Conclusion: These results demonstrate that AdiNOS pretreatment decreases hepatic transplant preservation injury. With severe I/R injury due to prolonged cold storage, AdiNOS delivery also improves transplant survival, indicating the clinical relevance of this treatment modality. Hepatic HO-1 induction by iNOS transduction may account in part for the protective effect of donor pre-treatment with AdiNOS. Tabled 1 Two-week animal survival after liver transplantation. Treatment n Survival (days) 14-days survival Saline 10 1, 1, 1, 2, 3, 3, 4, >14 ×3 3/10 (30.0%) AdiNOS 10 2, 4, 5, >14 × 7 7/10 (70.0%)# AdLacZ 8 1, 1, 2, 2, 3, 5, >14 × 2 2/8 (25.0%) Open table in a new tab Tabled 1 Serum AST (18 hr CIT) (IU/L) Necrotic area (%) ICAM-1 mRNA (fold increase) Neutrophil (/mm2) HO-1 band density (/normal liver) Group 6h 24h 48h 48h 3h 48h Donor liver 3h Ad-LacZ 2314 ±324 3434 ±814 1064 ±405 10.3 ±6.8 13.3 ±4.7 61.9 ±17.7 1.1 ±0.5 1.8 ±0.6 Ad-iNOS 1367 ±163* 1402 ±276* 296 ±50 0.6 ±0.9* 7.3 ±3.6* 13.9 ±8.8* 5.7 ±1.0* 3.1 ±0.3 Open table in a new tab

Recombinant Antisense C-myc Adenovirus Increase in vitro Sensitivity of Osteosarcoma MG-63 Cells to Cisplatin

C-myc is an oncogene with the important role of cell proliferation controller. It has been found to be amplified and overexpressed in osteosarcoma. Moreover, it can promote cell transformation and induce metastatic features. Some studies showed that overexpression of c-myc could induce resistance in response to antineoplastic agents. Currently, we constructed the recombinant adenovirus (Ad-Asc-myc) encoding antisense c-myc fragment and investigated its effect on the in vitro sensitivity of osteosarcoma MG-63 cells to cisplatin(CDDP). The osteosarcoma MG-63 cells were transfected by the Ad-Asc-myc in vitro, and Western Blot, MTT assay, RT-PCR, flow cytometry (FCM), and transmission electron microscopy (TEM) were used to study expression of c-myc and caspase-3 protein, tumor cell proliferation in vitro, cell apoptotic morphology and cell cycle change. Ad-Asc-myc encoding antisense c-myc fragment was obtained with the titer of 2.0 × 109 pfu/ml. Ad-Asc-myc downregulated the expression of c-myc protein after transfected MG-63 cells for 48 hours, combined with the treatment of 2.0, 5.0 μg/ml cisplatin for 2 hours can inhibited tumor cells proliferation in vitro by 33.4 and 54.2 percent, respectively, which had significant difference compared with control recombinant adenovirus (Ad-LacZ) groups (P < 0.05). RT-PCR revealed that Ad-Asc-myc downregulated expression of bcl-2 and upregulated expression of Bax, and no appreciable changes were observed in the expression of E2F-1. Detection of caspase-3 protein TEM, and FCM analysis showed that Ad-Asc-myc could induce apoptosis of transfected cells, which was enhanced by the treatment of cisplatin. Cell cycle analysis showed that obvious G2/M phase arrested in transfected cells. In conclusion, Ad-Asc-myc increased the in vitro sensitivity of osteosarcoma MG-63 cells to cisplatin as well as induced apoptosis.

Adenovirus mediated antisense c-myc gene on the chemotherapy sensitivity of osteosarcoma cells to cisplatin

To construct the recombinant adenovirus encoding antisense c-myc fragment and to investigate its effect on the chemotherapy sensitivity of osteosarcoma MG-63 cells to cisplatin. The recombinant adenovirus (Ad-Asc-myc) encoding antisense c-myc fragment was constructed by cloning c-myc cDNA of about 720 base pairs in a reverse direction into adenovirus vector, then undergoing recombination, amplifying and being complemented in vivo. The osteosarcoma MG-63 cells were transfected by the Ad-Asc-myc in vitro, and Wright staining, Acridine Orange staining, Western Blot, MTT, Flow Cytometry (FCM) were used to study cell morphology, expression of c-myc protein, tumor cell proliferation in vitro, apoptosis and cell cycle change. Ad-Asc-myc encoding antisense c-myc fragment was obtained with the titer of 2 x 10(9) pfu/ml. Ad-Asc-myc down-regulated the expression of c-myc protein after transfected MG-63 cells for 48 h, combined with the treatment of 2.0, 5.0 microg/ml cisplatin for 2 h could inhibit tumor cells proliferation in vitro by 33.4% and 54.2% respectively, which were significantly difference compared with control recombinant adenovirus (Ad-LacZ) groups (P < 0.05). Acridine Orange staining and FCM analysis showed that Ad-Asc-myc could induce apoptosis of transfected cells, which was enhanced by the treatment of cisplatin cell. Cycle analysis showed that obvious G2/M phase arrested in transfected cells. Ad-Asc-myc increases the chemotherapy sensitivity of osteosarcoma MG-63 cells to cisplatin as well as induced apoptosis.

Anti-Monocyte Chemoattractant Protein-1 Gene Therapy Protects Against Focal Brain Ischemia in Hypertensive Rats

Monocyte chemoattractant protein-1 (MCP-1) is expressed in the ischemic cortex after focal brain ischemia and appears to exacerbate ischemic damage. The authors examined the effect of gene transfer of dominant negative MCP-1, called 7ND, 90 minutes after induction of focal brain ischemia in hypertensive rats. Adenoviral vectors encoding mutant MCP-1 (Ad7ND; n = 11), or Escherichia coli β-galactosidase (AdlacZ; n = 17) as control were injected into the lateral ventricle of male spontaneously hypertensive rats. Both AdlacZ (n = 12) and Ad7ND (n = 6) administration provided transgene expression as early as 6 hours after injection and the expression further increased on day 1, followed by a sustained detection on day 5. Five days after ischemia, infarct volume (75 ± 13 mm3, n = 5, mean ± SD) significantly reduced to 72% of control (104 ± 22 mm3, n = 5, P < 0.05) by 7ND gene transfer. Numbers of leukocytes in the vessels (48.3 ± 32.9/cm2) and macrophage/monocyte infiltration (475.2 ± 125.5 /mm2) of the infarct area in the Ad7ND group were significantly less than those measured in the AdlacZ group (143.8 ± 72.1/cm2 and 671.8 ± 125.5/mm2, P < 0.05, respectively). In summary, the postischemic gene transfer of dominant negative MCP-1 attenuated the infarct volume and infiltration of inflammatory cells, suggesting potential usefulness of the anti–MCP-1 gene therapy.

Anti—Monocyte Chemoattractant Protein-1 Gene Therapy Protects against Focal Brain Ischemia in Hypertensive Rats

Monocyte chemoattractant protein-1 (MCP-1) is expressed in the ischemic cortex after focal brain ischemia and appears to exacerbate ischemic damage. The authors examined the effect of gene transfer of dominant negative MCP-1, called 7ND, 90 minutes after induction of focal brain ischemia in hypertensive rats. Adenoviral vectors encoding mutant MCP-1 (Ad7ND; n = 11), or Escherichia coli beta-galactosidase (AdlacZ; n = 17) as control were injected into the lateral ventricle of male spontaneously hypertensive rats. Both AdlacZ (n = 12) and Ad7ND (n = 6) administration provided transgene expression as early as 6 hours after injection and the expression further increased on day 1, followed by a sustained detection on day 5. Five days after ischemia, infarct volume (75 +/- 13 mm, n = 5, mean +/- SD) significantly reduced to 72% of control (104 +/- 22 mm3, n = 5, P < 0.05) by 7ND gene transfer. Numbers of leukocytes in the vessels (48.3 +/- 32.9/cm2) and macrophage/monocyte infiltration (475.2 +/- 125.5/mm2) of the infarct area in the Ad7ND group were significantly less than those measured in the AdlacZ group (143.8 +/- 72.1/cm2 and 671.8 +/- 125.5/mm2, P < 0.05, respectively). In summary, the postischemic gene transfer of dominant negative MCP-1 attenuated the infarct volume and infiltration of inflammatory cells, suggesting potential usefulness of the anti-MCP-1 gene therapy.

Vascular endothelial growth factor induces abnormal microvasculature in the endoglin heterozygous mouse brain.

Hereditary hemorrhagic telangiectasia (HHT), associated with brain arteriovenous malformations, is caused by a loss of function mutation in either the endoglin (HHT1) or activin receptor-like kinase 1 gene (ALK-1, HHT2). Endoglin heterozygous (Eng+/-)mice have been proposed as a disease model. To better understand the role of endoglin in vascular malformation development, we examined the effect of vascular endothelial growth factor (VEGF) hyperstimulation on microvessels in adult endoglin heterozygous (Eng+/-) mice using an adenoviral vector to deliver recombinant human VEGF165 cDNA (AdhVEGF) into basal ganglia. VEGF expression was increased in AdhVEGF mice compared with the AdlacZ and saline group (P < 0.05) and localized to multiple cell types (neurons, astrocytes, endothelial cells, and smooth muscle cells) by double-labeled immunostaining. VEGF overexpression increased microvessel count for up to 4 weeks in both the Eng+/+ and Eng+/- groups (Eng+/+ 185 +/- 14 vs. Eng+/- 201 +/- 10 microvessels/mm2). Confocal microscopic examination revealed grossly abnormal microvessels in eight of nine Eng+/- mouse brains compared with zero of nine in Eng+/+ mice (P < 0.05). Abnormal microvessels featured enlargement, clustering, twist, or spirals. VEGF receptor Flk-1 and TGF-beta receptor 1 (T beta R1) expression were reduced in the Eng+/- mouse brain compared with control. Excessive VEGF stimulation may play a pivotal role in the initiation and development of brain vessel malformations in states of relative endoglin insufficiency in adulthood. These observations are relevant to our general understanding of the maintenance of vascular integrity.

脳梗塞の遺伝子治療

Topical application of GDNF greatly reduced the infarct size (48%) and brain edema (30%) at 24 hr of continuous MCAO in rats. The reduction of the infarct size was not related to a change of cerebral blood flow (CBF), but was accompanied by marked reduction of positive cells for TUNEL and caspases in the affected area. Thus, GDNF showed a direct protective effect against ischemic brain damage, but not secondary by improving CBF. Based on the strong protective effect of NTF protein against ischemic brain damage and the considerable transfer of a foreign gene into ischemic brain, an adenovirus vector containing the GDNF gene (Ad-GDNF) was prepared, and a possible protective effect of the Ad-GDNF transfer was examined after transient MCAO in rat. Pretreatment of animals with Ad-GDNF 24 hr before the subsequent 90 min of transient MCAO effectively reduced infract volume and area without affecting regional CBF compared to the vehicle or Ad-LacZ animal groups.

Protective effects of intracerebral adenoviral-mediated GDNF gene transfer in a rat model of Parkinson's disease

This study focuses on the potential protective effects of intracerebral adeno-viral mediated glial cell line derived neurotrophic factor (GDNF) gene transfer in a rat model of Parkinson's disease (PD). Thirty-five SD rats were divided into three groups to receive perinigral injections of recombinant adenovirus encoding GDNF (Ad-GDNF), LacZ (Ad-LacZ) or PBS, respectively. One week later, an intrastriatal injection of 6-hydroxydopamine (6-OHDA) was administered to induce the progressive degeneration of dopaminergic neurons. Immunohistochemistry showed that GDNF treatment prior to neuronal damage could promote survival and morphological recovery of tyrosine hydroxylase (TH)-positive neurons in the midbrain. Approximately 70% of nigral TH-positive cells survived in the Ad-GDNF group, compared to approximately 30% for the Ad-LacZ or PBS control group. Histochemical analysis of monoamine levels in the striatum demonstrated that the dopamine content was higher for the Ad-GDNF group than the control groups. Similarly, Ad-GDNF treated animals showed improved apomorphine-induced rotational behavior. The exogenous GDNF gene was efficiently expressed in the brain as detected by ELISA. This work demonstrates that intracerebral adeno-viral mediated GDNF gene transfer can protect dopaminergic neurons in vivo from 6-OHDA-induced injuries. The approach used in this study could potentially be used therapeutically in patients with PD and further work is required to explore this idea in depth.

Peptide-Retargeted Adenovirus Encoding a Tissue Inhibitor of Metalloproteinase-1 Decreases Restenosis after Intravascular Gene Transfer

In this study we have attached cyclic targeting peptides by way of a poly-lysine spacer on the surface of an adenovirus using a transglutaminase enzymatic reaction to enhance transduction efficiency and to modify tissue tropism in vivo. Nuclear targeted lacZ- and TIMP-1-encoding adenoviruses were coupled to a peptide-motif (HWGF) that can bind to matrix metalloproteinase (MMP)-2 and MMP-9. Modified viruses were used to evaluate gene transfer efficiency, biodistribution, and the effect on neointima formation following balloon denudation injury. In vitro, both rabbit aortic smooth muscle cells and human endothelial hybridoma cells demonstrated significantly increased reporter gene expression with HWGF-modified adenoviruses (AdlacZ(HWGF)) compared with control (AdlacZ) or mismatch peptide-modified (AdlacZ(MM)) adenoviruses. However, in human hepatocellular Hep-G2 cells, both AdlacZ(HWGF) and AdlacZ(MM) produced significantly lower transgene expression compared with the respective control viruses. In vivo, local intravascular catheter-mediated gene transfer of a HWGF-targeted TIMP-1-encoding adenovirus (AdTIMP-1(HWGF)) significantly reduced intimal thickening in a rabbit aortic balloon denudation model (P < 0.05) compared with the control adenovirus. X-Gal staining and biodistribution analyses with TaqMan RT-PCR revealed that the cyclic peptides altered vector tropism and, in particular, reduced transduction of the liver. We found that the HWGF peptide modification increased transduction efficiency of the adenovirus-mediated gene transfer in smooth muscle cells and endothelial cells in in vitro and enhanced gene transfer to the arterial wall in vivo; that peptide modification of adenoviruses beneficially modulated tissue tropism in vivo; and that efficient TIMP-1 gene transfer reduced intimal thickening in an established restenosis model in rabbits.

Cu/Zn-superoxide dismutase gene attenuates ischemia-reperfusion injury in the rat kidney.

Evidence has accumulated for a role of toxic oxygen radicals in the pathogenesis of ischemia-reperfusion injury in the kidney. The aim of this study was to evaluate the hypothesis that reducing postischemic renal injury is possible by delivery of the gene for the antioxidant enzyme superoxide dismutase (SOD). Female Sprague-Dawley rats received intravenous injections of recombinant adenovirus (1 x 10(9) pfu) containing the transgenes for Escherichia coli beta-galactosidase (Ad-LacZ, as control) or human Cu/Zn-SOD (Ad-SOD). Three days later, renal ischemia was produced by cross-clamping the left renal vessels for 60 min. The right kidney was removed before reperfusion and processed for the transgene. Renal SOD protein and activity in rats given Ad-SOD was 2.5-fold higher than from the animals receiving Ad-LACZ: Urinary lactate dehydrogenase concentrations were elevated by ischemia-reperfusion in the Ad-LacZ group (1403 +/- 112 U/L), yet values were 50% lower in Ad-SOD-treated rats. Free radical production was elevated by ischemia-reperfusion but was significantly lower in SOD-treated animals. Importantly, on postischemic day 1, glomerular filtration rates were reduced to 0.21 ml/min per 100 g in the Ad-LacZ group, whereas values remained significantly higher (0.39) in the Ad-SOD group. Two weeks after ischemia-reperfusion, inflammation, interstitial fibrosis, tubular atrophy and tissue levels of tumor necrosis factor alpha and interleukin-1 were significantly higher in the Ad-LacZ-treated than in Ad-SOD-treated rats. In conclusion, these results indicate that SOD expression can be increased by delivery of the sod gene to the kidney by intravenous injection and that sod gene transduction minimized ischemia-reperfusion-induced acute renal failure.

Adenovirus-based overexpression of tissue inhibitor of metalloproteinases 1 reduces tissue damage in the joints of tumor necrosis factor alpha transgenic mice.

Rheumatoid arthritis is a prototype of a destructive inflammatory disease. Inflammation triggered by the overexpression of tumor necrosis factor alpha (TNFalpha) is a driving force of this disorder and mediates tissue destruction. Since matrix metalloproteinases (MMPs) are among the molecules activated by TNFalpha, we hypothesized that overexpression of their natural inhibitor, tissue inhibitor of metalloproteinases 1 (TIMP-1), in TNFalpha transgenic mice could inhibit the development of destructive arthritis. Systemic treatment was carried out by replication-defective adenoviral vectors for TIMP-1, beta-galactosidase, or phosphate buffered saline (PBS), which were applied once at the onset of arthritis. Clinical, serologic, radiologic, and histologic outcomes were assessed 18 days after the treatment. The AdTIMP-1 group showed significantly reduced paw swelling and increased grip strength compared with the 2 control groups, whereas total body weight, TNFalpha, and interleukin-6 levels were similar in all 3 groups. Radiographic assessment revealed a significant reduction of joint destruction in the AdTIMP-1 group; this was confirmed by histologic analyses showing reduced formation of pannus and erosions in the AdTIMP-1 group compared with the AdLacZ and PBS control groups. The formation of arthritis-specific autoantibodies to heterogeneous nuclear RNP A2 was not observed in the AdTIMP-1 group but was present in the 2 control groups. These results indicate a central role of MMPs in TNFalpha-mediated tissue damage in vivo and a promising therapeutic role for TIMP-1.