Alena Lizonova

Inducible nitric oxide synthase suppresses the development of allograft arteriosclerosis.

In cardiac transplantation, chronic rejection takes the form of an occlusive vasculopathy. The mechanism underlying this disorder remains unclear. The purpose of this study was to investigate the role nitric oxide (NO) may play in the development of allograft arteriosclerosis. Rat aortic allografts from ACI donors to Wistar Furth recipients with a strong genetic disparity in both major and minor histocompatibility antigens were used for transplantation. Allografts collected at 28 d were found to have significant increases in both inducible NO synthase (iNOS) mRNA and protein as well as in intimal thickness when compared with isografts. Inhibiting NO production with an iNOS inhibitor increased the intimal thickening by 57.2%, indicating that NO suppresses the development of allograft arteriosclerosis. Next, we evaluated the effect of cyclosporine (CsA) on iNOS expression and allograft arteriosclerosis. CsA (10 mg/kg/d) suppressed the expression of iNOS in response to balloon-induced aortic injury. Similarly, CsA inhibited iNOS expression in the aortic allografts, associated with a 65% increase in intimal thickening. Finally, we investigated the effect of adenoviral-mediated iNOS gene transfer on allograft arteriosclerosis. Transduction with iNOS using an adenoviral vector suppressed completely the development of allograft arteriosclerosis in both untreated recipients and recipients treated with CsA. These results suggest that the early immune-mediated upregulation in iNOS expression partially protects aortic allografts from the development of allograft arteriosclerosis, and that iNOS gene transfer strategies may prove useful in preventing the development of this otherwise untreatable disease process.

Efficient inhibition of intimal hyperplasia by adenovirus-mediated inducible nitric oxide synthase gene transfer to rats and pigs in vivo

BACKGROUND Inadequate nitric oxide (NO) availability may underlie vascular smooth muscle overgrowth that contributes to vascular occlusive diseases including atherosclerosis and restenosis. NO possesses a number of properties that should inhibit this hyperplastic healing response, such as promoting reendothelialization, preventing platelet and leukocyte adherence, and inhibiting cellular proliferation. STUDY DESIGN We proposed that shortterm but sustained increases in NO synthesis achieved with inducible NO synthase (iNOS) gene transfer at sites of vascular injury would prevent intimal hyperplasia. We constructed an adenoviral vector, AdiNOS, carrying the human iNOS cDNA and used it to express iNOS at sites of arterial injury in vivo. RESULTS AdiNOS-treated cultured vascular smooth muscle cells produced up to 100-fold more NO than control cells. In vivo iNOS gene transfer, using low concentrations of AdiNOS (2 x 10(6) plaque forming units [PFU]/rat) to injured rat carotid arteries, resulted in a near complete (>95%) reduction in neointima formation even when followed longterm out to 6 weeks post-injury. This protective effect was reversed by the continuous administration of an iNOS selective inhibitor L-N6-(1-iminoethyl)-lysine. However, iNOS gene transfer did not lead to regression of preestablished neointimal lesions. In an animal model more relevant to human vascular healing, iNOS gene transfer (5 x 10(8) PFU/pig) to injured porcine iliac arteries in vivo was also efficacious, reducing intimal hyperplasia by 51.8%. CONCLUSIONS These results indicate that shortterm overexpression of the iNOS gene initiated at the time of vascular injury is an effective method of locally increasing NO levels to prevent intimal hyperplasia.

Reducing the Native Tropism of Adenovirus Vectors Requires Removal of both CAR and Integrin Interactions

ABSTRACT The development of tissue-selective virus-based vectors requires a better understanding of the role of receptors in gene transfer in vivo, both to rid the vectors of their native tropism and to introduce new specificity. CAR and αv integrins have been identified as the primary cell surface components that interact with adenovirus type 5 (Ad5)-based vectors during in vitro transduction. We have constructed a set of four vectors, which individually retain the wild-type cell interactions, lack CAR binding, lack αv integrin binding, or lack both CAR and αv integrin binding. These vectors have been used to examine the roles of CAR and αv integrin in determining the tropism of Ad vectors in a mouse model following intrajugular or intramuscular injection. CAR was found to play a significant role in liver transduction. The absence of CAR binding alone, however, had little effect on the low level of expression from Ad in other tissues. Binding of αv integrins appeared to have more influence than did binding of CAR in promoting the expression in these tissues and was also found to be important in liver transduction by Ad vectors. An effect of the penton base modification was a reduction in the number of vector genomes that could be detected in several tissues. In the liver, where CAR binding is important, combining defects in CAR and αv integrin binding was essential to effectively reduce the high level of expression from Ad vectors. While there may be differences in Ad vector tropism among species, our results indicate that both CAR and αv integrins can impact vector distribution in vivo. Disruption of both CAR and αv integrin interactions may be critical for effectively reducing native tropism and enhancing the efficacy of specific targeting ligands in redirecting Ad vectors to target tissues.

Adenoviral transfer of the inducible nitric oxide synthase gene blocks endothelial cell apoptosis

BACKGROUND We have previously reported that vascular inducible nitric oxide synthase (iNOS) gene transfer inhibits injury-induced intimal hyperplasia in vitro and in vivo. One mechanism by which NO may prevent intimal hyperplasia is by preserving the endothelium or promoting its regeneration. To study this possibility we examined the effect of iNOS gene transfer on endothelial cell (EC) proliferation and viability. METHODS An adenoviral vector (AdiNOS) containing the human iNOS cDNA was constructed and used to infect cultured sheep arterial ECs. NO production was measured, and the effects of continuous NO exposure on EC proliferation, viability, and apoptosis were evaluated. RESULTS AdiNOS-infected ECs produced 25- to 100-fold more NO than control (AdlacZ) infected cells as measured by nitrite accumulation. This increased NO synthesis did not inhibit EC proliferation as reflected by tritiated thymidine incorporation. Chromium 51 release assay revealed that EC viability was also unaffected by AdiNOS infection and NO synthesis. In addition, prolonged exposure to NO synthesis did not induce EC apoptosis. Instead, NO inhibited lipopolysaccharide-induced apoptosis in these cells by reducing caspase-3-like protease activity. CONCLUSIONS Vascular iNOS gene transfer, while inhibiting smooth muscle cell proliferation, does not impair EC mitogenesis or viability. Augmented NO synthesis may also protect ECs against apogenic stimuli such as lipopolysaccharide. Therefore iNOS gene transfer may promote endothelial regeneration and can perhaps accelerate vascular healing.

Adenovirus-mediated inducible nitric oxide synthase gene transfer inhibits hepatocyte apoptosis ☆ ☆☆

BACKGROUND Apoptosis limits hepatocyte viability in bioartificial livers in vitro and may contribute to liver dysfunction in vivo. Nitric oxide (NO) inhibits hepatocyte apoptosis; however, methods to deliver NO in a sustained manner to hepatocytes are limited. Here, we tested the feasibility of inducible NO synthase (iNOS) gene transfer as an approach to deliver an intracellular source of NO to inhibit spontaneous and tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis in cultured hepatocytes. METHODS An adenoviral vector carrying the human iNOS gene (AdiNOS) was used to overexpress iNOS in cultured rat hepatocytes. Spontaneous apoptosis was induced by prolonged culture (4 days), and stimulated apoptosis was induced by exposure to TNF-alpha + actinomycin D (TNF-alpha ActD). Nitrite (NO2-), cell viability, and cellular caspase-3-like protease activity were measured. RESULTS AdiNOS gene transfer resulted in sustained NO production and protected hepatocytes from spontaneous and TNF-alpha + ActD-induced apoptosis. Apoptosis was associated with increases in caspase-3-like protease activity, which was suppressed by iNOS gene transfer in an NO-dependent manner. Dithiothreitol partially reversed the NO-induced suppression of caspase-3-like activity, which is consistent with S-nitrosylation of caspase-3. CONCLUSIONS Adenovirus-mediated iNOS gene transfer effectively blocks spontaneous and TNF-alpha + ActD-induced cell killing in hepatocytes. iNOS gene transfer could be used to suppress apoptotic hepatocyte death in vitro and possibly in vivo.

Endothelial nitric oxide synthase protects aortic allografts from the development of transplant arteriosclerosis

BACKGROUND Inducible nitric oxide synthase (iNOS) is up-regulated in rejecting allografts and is protective against allograft arteriosclerosis; it suppresses neointimal smooth muscle cell accumulation and inhibits adhesion of platelets and leukocytes to the endothelium. However, the functional importance of endothelial NOS (eNOS) in the rejecting allografts remains unclear. METHODS We examined the effects of selective eNOS deficiency in aortic allografts in a murine chronic rejection model using grafts from eNOS knockout (KO) mice (C57BL/6 background; H2b) and normal C3H (H2K) as recipients. Grafts from wild-type C57BL/6 mice served as controls. Grafts from iNOS KO mice served as a second group of controls where the contribution from iNOS was eliminated but eNOS was preserved. Aortic grafts were harvested and analyzed at days 10-14, 18-22, and 26-30 after transplantation. RESULTS Endothelial NOS-deficient grafts showed significantly increased intima/media ratios at days 26-30 compared to controls. Immunostaining demonstrated that in eNOS KO grafts, eNOS was not detectable whereas iNOS was expressed prominently in infiltrating recipient mononuclear cells. In control grafts, eNOS expression was preserved in the endothelium even by day 30, and associated with a decrease in intimal thickening. We further demonstrated that early overexpression of iNOS by ex vivo gene transfer completely prevented the development of arteriosclerosis associated with eNOS deficiency. CONCLUSIONS We found that eNOS plays a protective role in allografts, and that in eNOS-deficient allografts, early overexpression of iNOS is capable of preventing the development of allograft arteriosclerosis. In allografts with dysfunctional vascular endothelium and impaired eNOS activity as a result of ischemia or native arteriosclerotic disease, iNOS gene therapy may serve to improve their long-term survival and function.

Optimization of ex vivo inducible nitric oxide synthase gene transfer to vein grafts.

BACKGROUND Vein graft failure as the result of intimal hyperplasia (IH) remains a significant clinical problem. Ex vivo modification of vein grafts using gene therapy is an attractive approach to attenuate IH. Gene transfer of the inducible nitric oxide synthase (iNOS) gene effectively reduces IH. However, iNOS activity after gene transfer may be impaired by the availability of cofactor, such as tetrahydrobiopterin (BH4). The purpose of this study is to determine the optimal conditions for ex vivo adenoviral-mediated iNOS gene transfer into arterial and venous vessels. METHODS Porcine internal jugular veins and carotid arteries were infected ex vivo with the adenoviral iNOS vector (AdiNOS) and with an adenovirus carrying the cDNA encoding guanosine triphosphate cyclohydrolase I (AdGTPCH), the rate-limiting enzyme for BH4 synthesis. The production of nitrite, cyclic guanosine monophosphate (cGMP), and biopterin were assessed daily. RESULTS Nitric oxide (NO) production after iNOS gene transfer was maximal when vessels were cotransduced with AdGTPCH. NO production in these vessels persisted for more than 10 days. Vein segments generated approximately 2-fold more nitrite, cGMP, and biopterin than arterial segments infected with AdiNOS/AdGTPCH. Submerging vein segments into adenoviral solution resulted in improved gene transfer with greater nitrite and cGMP release compared with infections carried out under pressure intraluminally. Similarly, injury to the vein segments before infection with AdiNOS resulted in less nitrite production. CONCLUSIONS These data demonstrate that AdiNOS can efficiently transduce vein segments ex vivo and that the cotransfer of GTPCH can optimize iNOS enzymatic activity. This cotransfer technique may be used to engineer vein grafts before coronary artery bypass to prevent IH.

Nitric oxide prevents p21 degradation with the ubiquitin-proteasome pathway in vascular smooth muscle cells.

PURPOSE We have shown that gene transfer of the inducible nitric oxide synthase (iNOS) gene to injured arteries inhibits the development of intimal hyperplasia. One mechanism by which nitric oxide (NO) may inhibit this process is through the upregulation of the cyclin-dependent kinase inhibitor p21, which induces a G0/G1 cell cycle arrest, leading to an inhibition of vascular smooth muscle cell (VSMC) proliferation. Because NO induced such a dramatic upregulation of p21 and because p21 is a universal inhibitor of the cell cycle, this study aimed to determine how NO upregulates p21 protein expression in VSMCs. METHODS p21 messenger RNA (mRNA) levels in rat aortic smooth muscle cells (RASMCs) were determined by Northern blot analysis after treatment with S-nitroso-N-acetylpenicillamine (SNAP) or after adenoviral iNOS gene transfer. p21 protein levels in RASMCs in similar conditions were determined by Western blot analysis. Levels of ubiquinated p21 in these same treatment groups were assessed by immunoprecipitation of p21 from RASMCs, followed by western blot analysis for ubiquitin. Protein tyrosine and protein serine/threonine phosphatase activity after treatment with SNAP, plus or minus the phosphatase inhibitors calyculin A or cantharidin, were measured with (32)P-labeled myelin basic protein as a substrate. RESULTS NO exposure by the NO-donor SNAP or iNOS gene transfer induced a dose- and time-dependent increase in p21 protein expression in RASMCs. p21 mRNA levels were significantly increased after SNAP treatment only at the 6-hour point, but were not increased at 24 hours. In contrast, protein levels were increased from 6 to 24 hours, and transcriptional inhibitors did not inhibit this increase in protein synthesis. The increase in p21 protein expression induced by NO was associated with less of the ubiquinated form of p21 at both early and late points. Furthermore, NO induced an increase in both protein tyrosine and protein serine/threonine phosphatase activity. Inhibition of these phosphatases with calyculin A or cantharidin prevented the upregulation of p21 protein expression by NO. CONCLUSION These data indicate that one mechanism by which NO upregulates p21 protein expression is through the prevention of p21 protein degradation by the ubiquitin-proteasome pathway in association with increased protein tyrosine and serine/threonine phosphatase activity.

Improved Production of Adenovirus Vectors Expressing Apoptotic Transgenes

Adenovirus vectors expressing gene products that can induce apoptosis have potential utility in gene therapy applications ranging from the treatment of proliferative diseases to transplantation. However, adenovirus vectors carrying proapoptotic gene products are difficult to produce, as the apoptotic environment is not conducive to adenovirus gene expression and replication. Production of AdFasL/G, an adenovirus vector that expresses high levels of Fas ligand, was severely reduced in the 293 packaging cell line. Increased yields of AdFasL/G were achieved by inclusion of peptide-based caspase inhibitors in the growth medium. However, use of these inhibitors for large-scale production would be difficult and expensive. A screen for gene products that increase the yield of AdFasL/G in 293 cells revealed that the poxvirus serpin CrmA and the adenovirus 14.7K product were able to increase virus yields significantly. Apoptosis induced by AdFasL/G was attenuated in 293CrmA cell lines and virus titers were increased dramatically. However, serial passage of AdFasL/G on 293CrmA cells resulted in the generation of replication-competent adenovirus. To resolve this problem, the CrmA gene was introduced into AE25 cells, an E1-complementing cell line that has limited sequence identity with the vectors. AdFasL/G titers were increased 100-fold on AE25CrmA cells relative to the AE25 cells and RCA contamination was not detectable. In addition, adenovirus vectors that express FADD, caspase 8, and Fas/APO1 were produced efficiently in AE25CrmA and 293CrmA.

Rescue and production of vaccine and therapeutic adenovirus vectors expressing inhibitory transgenes

Expression of certain transgenes from an adenovirus vector can be deleterious to its own replication. This can result in the inhibition of virus rescue, reduced viral yields, or, in the worst case, make it impossible to construct a vector expressing the inhibiting transgene product. A gene regulation system based on the tet operon was used to allow the rescue and efficient growth of adenovectors that express transgenes to high levels. A key advantage to this system is that repression of transgene expression is mediated by the packaging cell line, thus, expression of regulatory products from the adenovector are not required. This provides a simple, broadly applicable system wherein transgene repression is constitutive during vector rescue and growth and there is no effect on adenovector-mediated expression of gene products in transduced cells. Several high-level expression vectors based on first- and second-generation adenovectors were rescued and produced to high titer that otherwise could not be grown. Yields of adenovectors expressing inhibitory transgene products were increased, and the overgrowth of cultures by adenovectors with nonfunctional expression cassettes was prevented. The gene regulation system is a significant advancement for the development of adenovirus vectors for vaccine and other gene transfer applications.