Timothy O’Brien

H2O2 is required for UVB-induced EGF receptor and downstream signaling pathway activation

Ultraviolet radiation (UVR)-induced receptor phosphorylation is increasingly recognized as a widely occurring phenomenon. However, the mechanisms, mediators, and sequence of events involved in this process remain ill-defined. We have recently shown that exposure of human keratinocytes to physiologic doses of ultraviolet B radiation (UVB) activates epidermal growth factor receptor (EGFR)/extracellular-regulated kinase 1 and 2 (ERK1/2), and p38 signaling pathways via reactive oxygen species. Here we demonstrate that UVB exposure increased intra- and extracellular H2O2 production rapidly in a time-dependent manner. An EGFR-specific monoclonal antibody abrogated EGFR autophosphorylation and markedly decreased the phosphorylation of ERK1/2 whereas p38 activation was unaffected. Overexpression of catalase strongly inhibited UVB-induced EGFR/ERK1/2 pathway activation. These findings establish the sequence of events after UVB irradiation: (i) H2O2 generation, (ii) EGFR phosphorylation, and (iii) ERK activation. Our results identify UVB-induced H2O2 as a second messenger that is required for EGFR and dependent downstream signaling pathways activation.

Gene transfer of endothelial nitric oxide synthase improves nitric oxide-dependent endothelial function in a hypertensive rat model.

OBJECTIVE We have shown previously that there is a relative nitric oxide deficiency at the level of vascular endothelium in the stroke-prone spontaneously hypertensive rat (SHRSP), a model of human essential hypertension, as compared to its normotensive reference strain Wistar Kyoto (WKY) rat. The aim of the current study was to investigate whether adenoviral-mediated gene transfer of an endothelial nitric oxide synthase (eNOS) cDNA (AdCMVeNOS) into carotid arteries of the SHRSP may improve endothelial function. METHODS Enzyme activity of the recombinant eNOS protein encoded by AdCMVeNOS was tested using a Griess assay in endothelial cells in culture. Left carotid arteries of SHRSP were surgically isolated and exposed to either the AdCMVeNOS or control beta-galactosidase-containing virus, (2 x 10(9) pfu/ml) ex vivo and in vivo. The vessels were harvested 24 h after surgery and analysed by Western blotting, immunohistochemistry and by examining endothelial function ex vivo. RESULTS Cultured endothelial cells showed almost 100% transduction with both viruses and a dose response of eNOS expression showed a five-fold increase in nitrite production for AdCMVeNOS with no change for beta-galactosidase-containing virus. Western blotting demonstrated a significant increase of eNOS expression in vessels infused with AdCMVeNOS when compared to controls. Immunohistochemistry showed highly positive staining with monoclonal antibodies against eNOS in the intact endothelial cells of the AdCMVeNOS infused vessels. The areas under the curve of the concentration responses to phenylephrine (10(-9) to 3 x 10(-6) M) in the absence and presence of NG-nitroarginine methyl ester (100 microM) showed increased basal nitric oxide bioavailability in the carotid arteries infused with AdCMVeNOS compared to the control (n = 6 for each; P = 0.0069; 95% CI, 0.864 to 3.277). CONCLUSIONS Our results show that AdCMVeNOS is an effective tool for vascular gene transfer and that it can improve endothelial NO availability in the SHRSP, a genetic model of essential hypertension and endothelial dysfunction.

In Vivo Gene Transfer of Endothelial Nitric Oxide Synthase to Carotid Arteries From Hypercholesterolemic Rabbits Enhances Endothelium-Dependent Relaxations

Background and Purpose —Hypercholesterolemia is associated with abnormal endothelium-dependent vasorelaxation due to decreased nitric oxide bioavailability. Our aim was to examine the effect of adenovirus-mediated gene transfer of endothelial nitric oxide synthase (eNOS) to the hypercholesterolemic rabbit carotid artery in vivo. In addition, we examined whether adenovirus-mediated gene transfer was associated with vascular dysfunction. Methods —Rabbits were fed a 1% cholesterol diet for 4 weeks followed by a 0.5% cholesterol diet for 6 weeks. Vascular reactivity was assessed in nontransduced carotid arteries from chow- and cholesterol-fed animals. In addition, carotid arteries were surgically isolated, and 2 separate doses of adenoviral vectors encoding eNOS or β-galactosidase (AdβGal) on the contralateral side were delivered to the lumen (1×1010 and 5×1010 pfu/mL). Results —Abnormal acetylcholine-mediated endothelium-dependent vasorelaxation was detected in the carotid artery from cholesterol-fed animals, whereas responses to calcium ionophore A23187 and diethylamine NONOate were normal. Vascular reactivity was similar in nontransduced and AdβGal-transduced hypercholesterolemic vessels. In vessels transduced with eNOS, transgene expression was demonstrated by immunostaining in both the endothelium and the adventitia and by Western blot analysis. High-dose but not low-dose eNOS gene transfer enhanced endothelium-dependent relaxation in vessels from cholesterol-fed rabbits. Conclusions —Adenovirus-mediated gene transfer of eNOS to carotid arteries of cholesterol-fed animals improves endothelium-dependent relaxation when an optimal viral titer is administered.

Highly efficient ex vivo gene transfer to the transplanted heart by means of hypothermic perfusion with a low dose of adenoviral vector

BACKGROUND Hypothermic conditions required for donor heart preservation may reduce gene-transfer efficiency. Experiments were designed to determine whether a perfusion technique could improve the efficiency of gene transfer to donor hearts. METHODS An adenoviral vector encoding beta-galactosidase (3.5 x 10(8) plaque-forming units) was infused into explanted rat hearts under 4 conditions (each n = 6): (1) the virus was diluted in 350 microL of University of Wisconsin solution and infused as a high-pressure bolus into the coronary arteries of donor hearts through the aortic root; (2) the virus was diluted in 5 mL of University of Wisconsin solution and circulated by means of a peristaltic pump (flow, 0.75 mL/min) through the vasculature of the donor heart for 30 minutes; (3) 5 mL of viral solution was circulated as for group 2 for 15 minutes; and (4) 5 mL of viral solution was circulated for 5 minutes at a flow rate of 2.4 mL/min. Transduced hearts were transplanted into the abdomen of syngeneic rats, and transgene expression was assessed by means of immunoassay 4 days later. RESULTS The median beta-galactosidase content was (1) 45.0 ng/mg protein (25th-75th percentile, 33-73 ng/mg), (2) 640 ng/mg protein (25th-75th percentile, 614-878 ng/mg), (3) 493.8 ng/mg protein (25th-75th percentile, 456-527 ng/mg), and (4) 503.3 ng/mg protein (25th-75th percentile, 475-562 ng/mg; P <.01 for group 2 vs group 1, and P <.05 for groups 3 and 4 vs group 1). Transgene expression was predominantly in myocytes and favored the subepicardial region of the right ventricle. CONCLUSION Hypothermic perfusion of the donor heart with an adenoviral vector resulted in efficient transgene expression compared with that induced by a single bolus injection.

Transbronchial gene transfer of endothelial nitric oxide synthase to transplanted lungs

BACKGROUND Experiments were designed to study the efficiency, distribution, and toxicity of transbronchial adenoviral-mediated transfer of endothelial constitutive nitric oxide synthase (ecNOS) gene to transplanted lungs. METHODS Syngeneic orthotopic single-lung transplantation in the rat was performed after airway administration (300 microL, 1 x 10(9) pfu/mL) of either the ecNOS gene or the marker gene beta-Gal (control group) to donor lungs (n=4 each). After 4 days, transgene expression, inflammation, and the presence of apoptosis in the transplanted lungs were assessed by molecular, immunohistochemical, and histologic techniques. RESULTS Gene transfer was confirmed by a positive polymerase chain reaction signal for the recombinant ecNOS gene, and recombinant messenger RNA by reverse transcription polymerase chain reaction. Positive immunohistochemical staining for ecNOS was present in more than 75% of pneumocytes only in ecNOS transduced lungs. Calcium-dependent nitric oxide synthase activity was increased in ecNOS- compared with betaGal-transduced lungs (2,139+/-756 versus 47+/-28 pmol x mg protein(-1) x h(-1); p < 0.05). Minimal to mild inflammation was observed in all transplanted lungs; fewer than 0.5% of cells in both groups were apoptotic. CONCLUSIONS Transbronchial transfer of ecNOS gene to the transplanted lung results in transduction of pneumocytes with expression of a functionally active transgene product.

Gene transfer to coronary artery bypass conduits

BACKGROUND Gene therapy is a rational approach to prevention of stenosis in saphenous vein grafts used as conduits for coronary artery bypass grafting. To explore this possibility we developed methods for adenoviral-mediated gene transfer to canine saphenous veins. METHODS During a single procedure, autogenous canine saphenous vein segments were transduced ex vivo and used as coronary artery bypass grafts. The proximal end of each vein was ligated, adenovirus containing the Escherichia coli beta-galactosidase gene (Ad.CMVLacZ) was delivered at titers of 2.5 x 10(9) or 5 x 10(9) plaque-forming units (pfu)/mL to the lumen through a distal heparin lock, and the segment was immersed in the viral solution for 1 hour at 37 degrees C. Control segments were exposed to diluent alone in an identical manner. Aortocoronary anastomoses were made using cardiopulmonary bypass. Transgene expression was assessed qualitatively and quantitatively after 3 days. RESULTS Beta-galactosidase levels showed a dose-dependent increase: 0.00 +/- 0.00 ng/mg total protein for controls; 5.60 +/- 2.27 ng/mg total protein for a viral titer of 2.5 x 10(9) pfu/mL and 11.97 +/- 6.14 ng/mg for 5 x 10(9) pfu/mL. The two dosage groups differed significantly from each other (p = 0.035) and from controls (p = 0.003). X-gal staining demonstrated mostly endothelial and scattered adventitial transgene expression. CONCLUSIONS Transgene expression after ex vivo gene transfer into saphenous vein grafts in a canine coronary artery bypass model indicates that this method may be useful for delivery of therapeutic genes to prevent or retard vein graft arteriosclerosis.

Adenoviral Vectors and Gene Transfer to the Blood Vessel Wall

Gene therapy may be defined as the treatment of human disease via the delivery of genes to human tissues. The transferred gene may replace a defective gene or may introduce a new function to a cell. Alternatively, the transgene may enhance an existing cellular function or manifest this function at a time it would not normally be exhibited. Local delivery of genes to the vascular wall is a promising approach to a number of vascular disorders. Overexpression of therapeutic genes within the vessel wall may avoid side effects associated with systemic delivery of the product and result in higher concentrations locally at the site of disease. Inhibition of restenosis after vascular injury1 2 3 4 5 and enhancement of new vessel formation after gene transfer has been demonstrated in numerous animal models.6 7 8 9 Phase 1 clinical trials of vascular gene therapy have also recently been reported.10 11 Two major obstacles to the clinical application of this technology include limitations of the currently available vectors and difficulties delivering genes to the vessel wall in vivo by percutaneous methods. Although direct gene delivery to a surgically isolated segment of the blood vessel has been demonstrated, efficient percutaneous catheter-mediated delivery to the coronary vasculature is more challenging.12 A number of vector systems have been used to deliver genes to the blood vessel wall. The simplest involves the use of plasmid alone or plasmid complexed with liposomes. These methods result in relatively inefficient gene transfer. However, if the therapeutic product is a potent secreted protein, biological effects may be observed after plasmid-based gene delivery. Viral vectors have been used extensively in vascular gene transfer, adenovirus vectors being the most commonly used system. Other vector systems …

Distribution and function of recombinant endothelial nitric oxide synthase in transplanted hearts

Introducing recombinant genes into donor hearts may offer a therapeutic intervention that could potentially attenuate the complications of heart transplantation, including rejection, infection and accelerated atherosclerosis. In the cardiovascular system, reduced bioactivity of endothelial nitric oxide is a feature of atherosclerosis and vascular injury. Nitric oxide is an arterial vasodilator that also inhibits proliferation of vascular smooth muscle cells and platelet aggregation. Experiments were designed to determine the distribution of adenoviral-mediated transfer of recombinant endothelial nitric oxide synthase gene (eNOS) and the effect of recombinant gene expression on the function of transplanted hearts. Adenoviral vectors for (a) bovine eNOS (AdeNOS) or (b) beta-galactosidase (AdLacZ; control) were infused into two groups (n = 12, per group) of explanted rat hearts. The transduced hearts were then implanted heterotopically into the abdomen of syngeneic recipient rats. After four days, the hearts were excised and examined for distribution and function of the recombinant genes. Polymerase chain reaction (PCR) verified the presence of the recombinant eNOS gene in eNOS-transduced but not in beta-galactosidase-transduced hearts; reverse transcriptase-PCR identified mRNA for eNOS in AdeNOS-transduced hearts. NOS activity (conversion of tritiated L-arginine to citrulline) was greater in homogenates of AdeNOS-compared to AdLacZ-transduced hearts. Positive immunoreactivity for eNOS was present in cardiomyocytes predominantly in eNOS-transduced hearts. Myocardial contractility and coronary blood flow, as determined using a Langendorff preparation, were not different between hearts transduced with AdeNOS or AdLacZ. These results suggest that, up to four days post transplantation, adenoviral-mediated transfer of eNOS into transplanted hearts is possible. However, expression of the recombinant protein did not result in measurable changes in myocardial contractility or coronary perfusion.

Absence of toxicity associated with adenoviral-mediated transfer of the β-galactosidase reporter gene to neonatal rat islets in vitro

Transfer of genes with potential therapeutic utility to the pancreatic islets of Langerhans may enhance graft survival after islet transplantation. The aim of this study was to determine the optimal conditions for adenoviral-mediated gene transfer to the islets of Langerhans in the absence of vector-induced toxicity. Neonatal rat islets were transduced in groups of 25 with an adenoviral vector encoding beta-galactosidase (AdbetaGal) at doses of MOI 0, 10, 100 and 1000 pfu per islet cell. All experiments were performed in triplicate. Efficiency of gene transfer was determined by gross inspection and estimation of the percentage of beta-galactosidase positive cells after islet dispersion at 1, 4, 7 and 10 days post-transduction. Islet toxicity was assessed by measuring accumulated insulin levels at each time-point and by assessing static incubation insulin release at 3 and 10 days. Efficient dose-dependent gene transfer to the islets was documented at 1, 4, 7 and 10 days post-transduction. Transgene expression was relatively stable for the duration of the experiment. Insulin accumulation did not differ between transduced and non-transduced islets at each timepoint. Likewise, the insulin secretory response to glucose, obtained by dividing the insulin response to high glucose incubation by the insulin response to low glucose incubation was similar in transduced and non-transduced islets at 3 and 10 days at all doses studied. In summary, adenoviral-mediated transduction of islets results in dose dependent efficient gene transfer with relatively stable transgene expression in the absence of toxicity. This technology may be useful in the study of islet biology and also in the future in gene therapy approaches to the treatment of diabetes mellitus.