Thrombus generation after adenovirus-mediated gene transfer into atherosclerotic arteries

Thrombosis represents a major issue during arterial local delivery. We evaluated the occurrence of thrombosis after adenovirus (Ad)-mediated gene transfer into normal and atherosclerotic arteries. A replication-deficient Ad vector expressing the beta-galactosidase reporter gene (Ad.RSV betagal; 4 x 10(9) PFU) was injected into normal and atherosclerotic arteries (n = 11 in both groups). The contralateral artery received either an Ad vector carrying no transgene (Ad.MLPnull) (n = 7 in both groups, 4 x 10(9) PFU) or vehicle buffer (n = 4 in normal group, n = 8 in atherosclerotic group). Animals were sacrificed 3 days following gene transfer for thrombus detection and assessment of beta-galactosidase activity. Thrombus was absent in normal arteries and in atherosclerotic arteries injected with vehicle buffer only. In contrast, nonocclusive thrombus was present in atherosclerotic arteries injected with either Ad.RSV betagal (5 of 11) or Ad.MLPnull (3 of 7). Beta-galactosidase activity was predominantly found in the endothelial layer of the transfected arteries. Gene transfer and expression occurred despite the presence of the thrombus (4 of 5), and its efficiency did not significantly differ regardless of the thrombus. We conclude that thrombus frequently occurred in atherosclerotic arteries after Ad-mediated gene transfer. Further studies are warranted to identify the mechanisms of thrombus generation after Ad-mediated gene transfer into atherosclerotic arteries.     

Direct gene transfer into the mouse heart

Direct injection of plasmid DNA into the myocardium of several species has been shown to be useful for studying cardiac gene expression. However, despite a better understanding of mouse genetics and the availability of several disease models in mice, gene injection with plasmid DNA into the mouse heart has not been reported. In this study, we demonstrate a simple and reproducible method for gene transfer into the mouse heart via direct injection of plasmid DNA. A firefly luciferase gene, driven by the RSV promoter, was used to quantitatively determine the spatial and temporal characteristics of gene transfer. Luciferase gene expression was stable for 8 weeks and showed a dose-dependent response over a range of 0.3-3 micrograms of input DNA. Inter-animal variability was low and gene expression was restricted to the left ventricle, near the site of injection. This method was also demonstrated to be suitable for detecting the expression of structural genes under the control of cellular promoters. Immunohistochemistry was used to detect the expression of an epitope-tagged myosin heavy chain driven by a rat alpha-myosin heavy chain promoter. Thus, naked DNA injection into the mouse heart results in a highly reproducible expression of constructs with either viral or cellular promoters. It is a relatively inexpensive and efficient means of studying cardiac gene regulation in vivo and a useful tool for screening the potential transgenes before generating transgenic mice.     

Enhanced in vivo adenovirus-mediated gene transfer to rat hepatocarcinomas by selective administration into the hepatic artery

Adenovirus-mediated gene therapy of experimental hepatocarcinoma is hindered by low transduction efficacy in vivo. We evaluated the extent of gene expression following various routes of administration of recombinant adenovirus AdCMVlacZ in diethylnitrosamine-induced rat hepatocarcinoma. We first characterized the vascularization of diethylnitrosamine-induced hepatocarcinomas using a computerized tomography scanner approach. The efficacy of gene transfer was then evaluated by three routes of administration: intraportal, selective injection through the hepatic artery and direct injection into the tumor. Diethylnitrosamine-induced hepatocarcinomas had predominantly an arterial blood supply, 67% of the total liver blood supply. Compared with intraportal administration, arterial injection improved gene transfer into tumors whereas that to the non-tumor areas was diminished. In addition, this route of injection allowed the efficient transduction of dysplastic nodules. Diethylnitrosamine-induced hepatocarcinoma in rats is a relevant model for the study of human hepatocarcinoma due to its vascularization. Arterial infusion improved the ratio of transduced tumorous to nontumorous cells and allowed targeting of gene transfer to dysplastic nodules. This will be useful in the design of gene therapy for hepatocarcinoma.     

Augmented adenovirus-mediated gene transfer to atherosclerotic vessels

Vascular endothelium is an important target for gene transfer in atherosclerosis. In this study, we examined gene transfer to normal and atherosclerotic blood vessels from two species, using an organ culture method. Using normal aorta, we determined optimal dose, duration of exposure to adenovirus, and duration of incubation of vessels in tissue culture. Aortas from normal and atherosclerotic monkeys were cut into rings and incubated for 2 hours with a recombinant adenovirus, carrying the reporter gene for beta-galactosidase driven by a cytomegalovirus (CMV) promoter. After 20 hours of incubation, transgene expression was assessed with a morphometric method after histochemical staining and a chemiluminescent assay of enzyme activity. Expression of beta-galactosidase after histochemical staining, expressed as percentage of total cells, was similar in adventitial cells of normal monkeys (21 +/- 4%, mean +/- SE%) and atherosclerotic monkeys (25 +/- 12%). Transgene expression in endothelium was higher in atherosclerotic than in normal vessel (53 +/- 3% versus 27 +/- 7%, P < .05). Chemiluminescent assay indicated greater beta-galactosidase activity (2.5 +/- 0.6 mU/mg of protein) in the intima and media of atherosclerotic than normal vessels (0.6 +/- 0.2 mU/mg of protein, P < .05). Aortas from normal (n = 6) and atherosclerotic (n = 5) rabbits also were examined. Transgene expression (after histochemical staining) in endothelium was much greater in atherosclerotic than normal rabbits (39 +/- 3% versus 9 +/- 2%, P < .05) and expression in adventitial cells was similar (normal 23 +/- 2%, atherosclerotic 24 +/- 4%). Chemiluminescent assay indicated greater beta-galactosidase activity (1.2 +/- 0.4 mU/mg of protein) in the intima and media of atherosclerotic than normal vessels (0.2 +/- 0.1 mU/mg protein, P < .05). These findings suggest that an adenoviral vector with a CMV promoter provides similar transgene expression in adventitia of both normal and atherosclerotic vessels. Gene transfer to the endothelium was much more effective in atherosclerotic than in normal vessels. Thus it may be possible to achieve greater transgene expression in atherosclerotic than in normal arteries.     

Interferons impair early transgene expression by adenovirus-mediated gene transfer in muscle cells

Sarcosine reductase is the only reductase system present in Tissierella creatinophila when grown on creatinine plus formate. The acetyl-phosphate-forming component protein C was purified to homogeneity. SDS-PAGE of the purified protein revealed two protein bands with apparent mol. masses of 62 and 50 kDa. The N-terminal amino acid sequence of the two subunits was determined. Antibodies raised against each of the subunits of protein C from Eubacterium acidaminophilum cross-reacted with the corresponding protein present in T. creatinophila, Clostridium litorale and Clostridium sporogenes. The arsenate-dependent hydrolysis of acetyl phosphate catalyzed by protein C was partly inhibited by antibodies directed against the large subunit. Antibodies raised against the small subunit were twice as effective, which indicates that this subunit is the primary site of acetyl transfer from acetyl phosphate. The protein A component of the sarcosine reductase of T. creatinophila was purified to homogeneity by cochromatography with thioredoxin reductase on DEAE-Sephacel, hydroxylapatite, Q-Sepharose, and Sephacryl 100-HR. Protein A had an apparent mol. mass of 21 kDa. Its N-terminal amino acid sequence showed high similarities to that of other proteins A. Initial steps for the purification and preliminary characterization of the sarcosine-specific, substrate-binding protein Bsarcosine component of T. creatinophila indicated the involvement of a 50-kDa protein.     

Penetration of zona-free mouse ova

We have studied atrio-ventricular conduction in 9 patients with atrial fibrillation and flutter and ventricular response below 90 per minute, by means of His bundle electrograms. Despite the finding of intraventricular conduction defects in 6 cases, the block of the atrial impulses occurred at the high atrio-ventricular junction in every case. In four patients the arrhythmia was an incidental finding and there were no other signs of cardiac disease. Several patients tolerated low ventricular rates for years without symptoms, but in two cases it was necessary to insert a pacemaker to treat cerebral ischemic symptoms. The problems of interpretation of the His bundle electrogram, in the presence of atrial fibrillation, are discussed in detail.     

Enhancement of adenovirus-mediated gene transfer to human bone marrow cells

Adenovirus infection of CD34+ hematopoietic stem/progenitor cells is dependent on the multiplicity of infection (MOI), time of incubation, the volume in which the co-incubation occurs and the presence or absence of growth factors. Studies revealed that a brief co-incubation (1-8 hours), resulted in low levels of transgene expression, suggesting that adenovirus infection of CD34+ cells occurs slowly, and optimal transduction requires a 24 hour exposure to adenovirus. Infection by Ad/beta-gal or Ad/p53 at a MOI of 500:1 provided a high transduction efficiency but inhibited hematopoietic function. However, treatment at a MOI of 50-100 resulted in efficient transduction (10.7-15.7% positive) without detectable toxicity. Secondary proof of adenovirus transgene expression was demonstrated by detection of mRNA for p53 in Ad/p53 infected stem cells. We conclude that a 24 hour exposure to recombinant adenovirus encoding p53 or beta-gal, at a MOI of 50-100 is optimal for in vitro gene transfer to BM cells and has no significant effect on hematopoietic function. Adenovirus-mediated transduction of BM cells can also be modulated by growth factors (IL-3, GM-CSF and G-CSF) with improved gene delivery and maintenance of hematopoietic function. In summary, adenovirus vectors can be used to transiently transduce stem cells, and conditions have been defined to maximize expression and limit inhibitory effects on CD34+ cells. These data support continued investigation of this vector for local cytokine delivery and purging of stem cell products.     

Apoptosis by retrovirus- and adenovirus-mediated gene transfer of Fas ligand to glioma cells: implications for gene therapy

Astrocytic tumors frequently express Fas/APO-1 (Fas), in sharp contrast to surrounding normal brain cells, providing a potential window through which selective killing of tumor cells could be pursued. To assess this possibility, we transduced Fas into U251, a glioma cell line resistant to anti-Fas antibody-mediated apoptosis, and obtained transfectants with high levels of Fas expression. Anti-Fas antibody showed significantly enhanced cytotoxicity for the transfectants, suggesting that U251 cells maintained an intercellular cascade of Fas-mediated apoptosis. When U251 transfectants with high-level Fas expression were transduced with Fas ligand-encoding gene via retrovirus, they were unaffected by exposure to anti-Fas antibody or Fas ligand adenovirus (Adeno-FL). Thus, retroviral induction of Fas ligand into the glioma cells with high levels of Fas led to the selection of cells that were resistant to Fas-dependent apoptosis. These resistant U251 transfectants were susceptible to FADD adenovirus (Adeno-FADD)-induced apoptosis, indicating that a cascade of death signals was blocked at the steps between Fas ligand and FADD. As for adenoviral transduction of Fas ligand into gliomas, gliomas with a relatively high level of expression of Fas were remarkably sensitive to Adeno-FL-induced apoptosis. Besides, Adeno-FADD induced pronounced apoptosis in all glioma cells. Our data suggest the possibility of using adenovirus-mediated transduction of Fas ligand and FADD genes as a therapeutic approach to target gliomas.     

Inhibition of phosphatidylinositol 3-kinase activity by adenovirus-mediated gene transfer and its effect on insulin action

Phosphatidylinositol 3-kinase (PI 3-K) is implicated in cellular events including glucose transport, glycogen synthesis, and protein synthesis. It is activated in insulin-stimulated cells by binding of the Src homology 2 (SH2) domains in its 85-kDa regulatory subunit to insulin receptor substrate-1 (IRS-1), and, others. We have previously shown that IRS-1-associated PI 3-kinase activity is not essential for insulin-stimulated glucose transport in 3T3-L1 adipocytes, and that alternate pathways exist in these cells. We now show that adenovirus-mediated overexpression of the p85N-SH2 domain in these cells behaves in a dominant-negative manner, interfering with complex formation between endogenous PI 3-K and its SH2 binding targets. This not only inhibited insulin-stimulated IRS-1-associated PI 3-kinase activity, but also completely blocked anti-phosphotyrosine-associated PI 3-kinase activity, which would include the non-IRS-1-associated activity. This resulted in inhibition of insulin-stimulated glucose transport, glycogen synthase activity and DNA synthesis. Further, Ser/Thr phosphorylation of downstream molecules Akt and p70 S6 kinase was inhibited. However, co-expression of a membrane-targeted p110(C) with the p85N-SH2 protein rescued glucose transport, supporting our argument that the p85N-SH2 protein specifically blocks insulin-mediated PI 3-kinase activity, and, that the signaling pathways downstream of PI 3-kinase are intact. Unexpectedly, GTP-bound Ras was elevated in the basal state. Since p85 is known to interact with GTPase-activating protein in 3T3-L1 adipocytes, the overexpressed p85N-SH2 peptide could titrate out cellular GTPase-activating protein by direct association, such that it is unavailable to hydrolyze GTP-bound Ras. However, insulin-induced mitogen-activated protein kinase phosphorylation was inhibited. Thus, PI 3-kinase may be required for this action at a step independent of and downstream of Ras. We conclude that, in 3T3-L1 adipocytes, non-IRS-1-associated PI 3-kinase activity is crucial for insulin's metabolic signaling, and that overexpressed p85N-SH2 protein inhibits a variety of insulin's ultimate biological effects.     

Degeneration in vivo of rat hippocampal neurons by wild-type Alzheimer amyloid precursor protein overexpressed by adenovirus-mediated gene transfer

In an attempt to elucidate the pathological implications of intracellular accumulation of the amyloid precursor protein (APP) in postmitotic neurons in vivo, we transferred APP695 cDNA into rat hippocampal neurons by using a replication-defective adenovirus vector. We first improved the efficiency of adenovirus-mediated gene transfer into neurons in vivo by using hypertonic mannitol. When a beta-galactosidase-expressing recombinant adenovirus suspended in 1 M mannitol was injected into a dorsal hippocampal region, a number of neurons in remote areas were positively stained, presumably owing to increased retrograde transport of the virus. When an APP695-expressing adenovirus was injected into the same site, part of the infected neurons in the hippocampal formation underwent severe degeneration in a few days, whereas astrocytes near the injection site showed no apparent degeneration. These degenerating neurons accumulated different epitopes of APP, and beta/A4 protein (Abeta)-immunoreactive materials were undetected in the extracellular space. A small number of degenerating neurons showed nuclear DNA fragmentation. Electron microscopic examinations demonstrated that degenerating neurons had shrunken perikarya along with synaptic abnormalities. Microglial cells/macrophages were often found in close proximity to degenerating neurons, and in some cases they phagocytosed these neurons. These results suggest that intracellular accumulation of wild-type APP695 causes a specific type of neuronal degeneration in vivo in the absence of extracellular Abeta deposition.     

Salvage angiogenesis induced by adenovirus-mediated gene transfer of vascular endothelial growth factor protects against ischemic vascular occlusion

PURPOSE: Vascular endothelial growth factor (VEGF) is a potent stimulator of angiogenesis, and transgene expression from adenovirus vectors can provide in vivo delivery of proteins. On the basis of this knowledge, we hypothesized that local administration of a replication-deficient adenovirus vector expressing complementary DNA for VEGF (AdVEGF) would induce collateral vessel formation in the setting of ischemia that could protect against subsequent acute vascular occlusion. METHODS: Hindlimb ischemia was induced in Sprague-Dawley rats by means of unilateral ligation of the common iliac artery immediately followed by administration of 4 x 10(9)-plaque-forming units VEGF, the control vector AdNull, or phosphate-buffered saline solution into the iliofemoral adipose tissue and thigh muscles. Untreated rats with common iliac ligation were used as an additional control group. RESULTS: Local VEGF expression was observed for 5 days in AdVEGF-treated rats but not in controls. Three weeks after ligation and vector administration, the ipsilateral femoral artery was ligated for a model of an acute vascular occlusion in the setting of preexisting ischemia. Blood flow to the ischemic hindlimb relative to the contralateral hindlimb evaluated with color microspheres demonstrated significantly increased blood flow in the AdVEGF-treated rats compared with each control group (p < 0.0001). Relative blood flow assessed by means of 99mTc-sestamibi radionuclide scans also demonstrated increased blood flow to the ligated hindlimb of AdVEGF-treated rats compared with each control group (p < 0.002). AdVEGF-treated rats also demonstrated increased vascularity in the ligated limb compared with each control group as assessed by means of angiography (p < 0.0001) and histologic quantification of blood vessels less than 80 microm diameter in local adipose tissue and capillaries per muscle fiber (p < 0.0002). AdVEGF treatment prevented a rise in femoral venous lactate femoral venous concentrations 1 hour after femoral artery ligation in control rats (p < 0.04). CONCLUSIONS: An adenovirus vector expressing VEGF complementary DNA is capable of stimulating an angiogenic response that protects against acute vascular occlusion in the setting of preexisting ischemia, suggesting that in vivo gene transfer of VEGF complementary DNA might be useful in prophylaxis of advancing arterial occlusive disease.     

Cytoplasmic gene expression system enhances the efficiency of cationic liposome-mediated in vivo gene transfer into mouse brain

The use of in vivo electroporation in combination with a chemotherapeutic agent (electrochemotherapy) for the treatment of liver tumors was examined. Induced rat hepatomas were treated with a 0.5 unit intratumor bleomycin dose followed by rectangular direct current pulses. Six pulses were administered during treatment using a needle array electrode that rotated the applied electric field around the tumors. A 84.6% objective response rate resulted for tumors that received both bleomycin and electric pulses. Control groups that received partial or no treatment showed less than 10% objective response rates. These results indicate that electrochemotherapy can be translated from the treatment of cutaneous cancers to the treatment of liver tumors and other internal tumors.