Reversal of impaired wound repair in iNOS-deficient mice by topical adenoviral-mediated iNOS gene transfer.

Most evidence indicates that nitric oxide plays a role in normal wound repair; however, involvement of inducible nitric oxide synthase (iNOS) has not been established. Experiments were carried out to determine the requirement for iNOS in closing excisional wounds. Wound closure was delayed by 31% in iNOS knockout mice compared with wild-type animals. An identical delay in wound closure was observed in wild-type mice given a continuous infusion of the partially selective iNOS inhibitor N6-(iminoethyl)-L-lysine. Delayed wound healing in iNOS-deficient mice was completely reversed by a single application of an adenoviral vector containing human iNOS cDNA (AdiNOS) at the time of wounding. Reverse transcription PCR identified iNOS mRNA expression in wild-type mice peaking 4-6 d after wounding, and confirmed expression of human iNOS in the adenoviral vector containing human iNOS cDNA-treated animals. These results establish the key role of iNOS in wound closure, and suggest a gene therapy strategy to improve wound healing in iNOS-deficient states such as diabetes, and during steroid treatment.

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.

Clonal variation in the production of tumor-associated alpha 2-macroglobulin in a malignant human melanoma and association with growth stimulation.

alpha 2-Macroglobulin (alpha 2-M) is known as a wide-spectrum proteinase inhibitor and to bind covalently certain growth factors. We have previously characterized tumor-associated alpha 2-M synthesized and secreted by human tumor cell lines. Of the cell lines studied, the melanoma cell line HMB-2 produced the largest amount of this glycoprotein. Immunofluorescence staining of cultured HMB-2 cells suggested that the cell population is heterogeneous with respect to alpha 2-M production. We have now isolated clones from the parental HMB-2 cells and characterized eight representative ones in detail. They varied considerably in the quantity of alpha 2-M secreted, from 4.2 to 46.5% of total protein. No relationship between the production of alpha 2-M by these clones and their pigmentation or tumorigenicity in nude mice was found. However, statistically there was a strong correlation between the modal chromosome number and population doubling time (r2 = 0.88, P less than 0.001) and also between the modal chromosome number and alpha 2-M production (r2 = 0.73, P less than 0.01). The growth rate of the clones correlated with the level of alpha 2-M in culture medium (r2 = 0.69, P less than 0.01). Clones with lower alpha 2-M production had a proportionally shorter population doubling time than the clones with intermediate or high production. Northern hybridization indicated quantitative variation in the alpha 2-M mRNA expressed by the parental cells and the clones, that was comparable but not identical with the quantity of alpha 2-M in the respective culture media; both parental cells and clones expressed platelet-derived growth factor A-chain mRNAs with little difference in levels. Serum-free medium from low alpha 2-M producer clones stimulated normal stationary fibroblasts significantly more than clones producing intermediate or high amounts of alpha 2-M. alpha 2-M decreased and anti-alpha 2-M IgG increased the stimulation. These results suggest that production of tumor-associated alpha 2-M is related to both autocrine and paracrine growth-stimulating activity of the tumor cells.

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.

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.

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.

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.

Optimizing cardiovascular gene therapy: increased vascular gene transfer with modified adenoviral vectors.

BACKGROUND: Adenovirus is widely used as a vector for gene transfer to the vasculature. However, the efficiency of these vectors can be limited by ineffective viral-target cell interactions. Viral attachment, which largely determines adenoviral tropism, is mediated through binding of the adenoviral fiber coat protein to the Coxsackievirus and adenovirus receptor, while internalization follows binding of the adenoviral RGD motif to alpha(v)-integrin receptors. Modifications of the fiber coat protein sequence have been successful for targeting the adenovirus to more prevalent receptors in the vasculature, including heparan sulfate-containing receptors and alpha(v)-integrin receptors. HYPOTHESIS: Modified adenoviral vectors targeted to receptors more prevalent in the vasculature result in an increased transfer efficiency of the virus in vitro and in vivo even in the presence of clinically relevant doses of heparin. DESIGN: We tested 2 modified E1- and E3-deleted Ad5 type adenoviral vectors containing the beta-galactosidase gene. AdZ.F(pK7) contains multiple positively charged lysines in the fiber coat protein that target the adenovirus to heparan sulfate receptors, while AdZ.F(RGD) contains an RGD integrin-binding sequence in the fiber coat protein that allows binding to alpha(v)-integrin receptors. The gene transfer efficiency of these modified viruses was compared in rat aortic smooth muscle cells in vitro and in an in vivo porcine model of balloon-induced arterial injury. Because of the use of heparin during most vascular surgical procedures and the concern that heparin might interfere with the binding of AdZ.F(pK7) to heparan sulfate receptors, the effect of heparin on the in vitro and in vivo transfer efficiency of these 2 modified adenoviruses was evaluated. RESULTS: In vitro infection of rat aortic smooth muscle cells with AdZ.F(pK7) and AdZ.F(RGD) resulted in significantly higher levels of beta-galactosidase expression compared with the unmodified adenovirus (mean +/- SEM, 1766.3 +/- 89.1 and 44.8 +/- 3.4 vs 10.1 +/- 0.7 mU per milligram of protein; P<.001). Following heparin administration, the gene transfer efficiency achieved with AdZ.F(pK7) diminished slightly in a concentration-dependent manner. However, the transfer efficiency was still greater than with the unmodified virus (mean +/- SEM, 1342.3 +/- 101.8 vs 4.8 +/- 0.4 mU per milligram of protein; P<.001). In vivo, following injury to the pig iliac artery with a 4F Fogarty balloon catheter, we found that AdZ.F(pK7) transduced the artery approximately 35-fold more efficiently than AdZ.F and 3-fold more efficiently than AdZ.F(RGD) following the administration of intravenous heparin, 100 U/kg body weight, and heparinized saline irrigation. CONCLUSIONS: Modifications of the adenovirus that lead to receptor targeting resulted in significantly improved gene transfer efficiencies. These improvements in transfer efficiencies observed with the modified vectors decreased slightly in the presence of heparin. However, AdZ.F(pK7) was still superior to AdZ.F(RGD) and AdZ.F despite heparin administration. These data demonstrate that modifications of adenoviral vectors that enhance binding to heparan sulfate receptors significantly improve gene transfer efficiency even in the presence of heparin and suggest an approach to optimize gene transfer into blood vessels.

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.

Circulating antibodies to the feline leukemia virus and FOCMA in cats.

Presence of antibodies to the structural proteins of the feline leukemia virus and the feline oncornavirus-associated cell membrane antigen FOCMA was analyzed in sera of 120 cats from household environments. In our experiments 35% of the sera tested were positive with the viable cells of FL-74 feline lymphoma cell line. On the other hand, only 5% of the feline sera reacted with FeLV from FL-74 cells in the solid phase RIA. Selected positive sera were analyzed for the presence of antibodies to the viral structural proteins of FeLV and FOCMA in lysates of FL-74 cells by radioimmunoprecipitation. Absorption of the selected sera (reacting with viable FL-74 cells) with disrupted FeLV had a negligible effect on the precipitating activity of the 70000 protein.

Immunoprecipitation of membrane proteins of cultured human sarcoma cells.

Human sarcoma associated antigens (HSAA) have previously been identified by indirect immune fluorescence in human sarcoma cells in culture using sera from patients bearing different types of sarcoma. To further characterize these HSAA, surface proteins of cultured cells were labeled with 125Iodine, [3H]-glucosamine and [35S]-methionine and solubilized. After immunoprecipitation labeled proteins were detected in immune complexes by SDS polyacrylamide gel electrophoresis and autoradiography, which allowed comparison with antigens described by other groups. A surface protein (Mr 96 000) was precipitated with sera from sarcoma bearing patients, and two glycoproteins (Mr 115 000 and 85 000) were preferentially precipitated with antisera from rabbits immunized with membranes from two human sarcoma cell lines. At least two of these proteins were found in each of five human sarcoma cell lines studied (U-4SS, U-3930S, U-20S, B-5GT and B-6FS). None of the proteins were precipitated with three human control sera, and only occasionally a faint band was observed in immunoprecipitates from control cells (B-25F, B-41B, B-42FC, U-2S, and U-393S with the immune sera. These proteins are probably some of the antigens responsible for the immune fluorescence observed in determination of HSAA. However, purification of the proteins and competition experiments are needed before this can be finally established.

Alpha-2-macroglobulin is not synthesized by human urothelial cell lines in vitro.

We have analyzed 14 human urothelial cell lines in two different transformation grades for the production of a tumor-associated-alpha-2-macroglobulin. It has been shown in radioimmunoprecipitation and immunoblotting tests that human urothelial cells in vitro do not synthesize the wide-spectrum proteinase inhibitor alpha-2-macroglobulin. Four of the tested cell lines were additionally tested for the expression of specific mRNA which resulted in negative findings. The significance of the absence of alpha-2-macroglobulin in the human urothelial cells is discussed.

The retrovirus particles in human myeloma cells RPMI8226: morphological, biochemical, immunological and infective transmission studies.

The retrovirus designated RPMI8226V (isolated from human myeloma cells RPMI8226) has been characterized with respect to its morphological, biochemical and immunological properties as well as its propagation in various animal and human cells. The myeloma cells RPMI8226 produce intracytoplasmatic A-type particles and extracellular particles. The extracellular particles have been classified as immature particles with translucent core center, typical mammalian C-type virus particles and C-type particles with intermediate membrane. However, the budded particles in secondarily infected human neoplastic cells contained complete doughnut-shaped nucleoids. This type of budding is rather characteristic for B-type particles. The 3H-uridine labeled RPMI8226 viral particles have a buoyant density 1.17 g/ml in sucrose gradient containing high molecular weight RNA and the distribution of viral structural proteins in SDS-PAGE is characteristic for oncornaviruses. The internal structural proteins according to MW are ranged from 13 000 to 30 000 daltons. The virus contains a magnesium-dependent reverse transcriptase. The cellular homogenate and viral concentrate from RPMI8226 cultures do not react with antibodies against ALSV, MuLV, FeLV, RD114, MP-MV and SiSLV. The only reaction was scored with anti BLV antibodies. However, anti BLV serum inhibiting the reverse transcriptase activity of BLV to 60% does not cross-react with the reverse transcriptase of RPMI8226V. In contrast to BLV concentrates, neither XC nor KC cells show syncytia formation by RPMI8226V. The RPMI8226V replication is restricted to human tumor and normal human glia-like cells. The possible origin of the virus is discussed.

Human neoplastic cells in tissue culture: two established cell lines derived from giant cell tumor and fibrosarcoma.

The establishment and cultivation of two human neoplastic cell lines is described. The cell line B-5GT was derived from bone giant cell tumor and B-6FS from poorly differentiated fibrosarcoma. In comparison to the normal skin fibroblasts both cell lines have a potential for "indefinite" multiplication in vitro and they exhibit growth properties which are associated with malignant transformation. The parameters investigated included cell morphology, chromosome characteristics, terminal cell density, growth pattern, residual DNA synthesis and growth in soft agar. Both cell lines exhibited human karyotype with aneuploidy and differed in their karyotype from each other.

BrdU-induced changes in the provirus of PR-RSV in mammalian cells.

Previously we demonstrated differences in the organization and methylation pattern of integrated Rous sarcoma proviral sequences in helper-dependent virogenic rat TWERC cells. In the present study we attempted to induce changes in the integrated viral genome of TWERC cells using 5-bromodeoxyuridine (BrdU). Four clones (A, B, C, and F) derived from the parental cell line were treated for 10 months with different concentrations of BrdU. Restriction enzyme analysis of the parental cell line and its clones showed that the cells contained in their genomic DNA two copies of deleted provirus. In TWERC cells, the PR-RSV provirus lost the whole env gene and part of the 3' end of the pol gene. The proviral sequences in DNA from the TWERC-derived clones were found to be hypermethylated. A slightly different situation was seen in clone C where demethylation of the provirus in the 3' region and in the 5' end of the genome was found. The level of mRNA expression both in the parental cells and in the clones correlated with the methylation pattern of the PR-RSV provirus. Clone C was less methylated and expressed more virus-specific RNA. The possible role of BrdU in these events is discussed.

[Provirus structure and expression in rat cell lines transformed by the avian sarcoma virus]. / Struktura i ékspressia provirusa v liniiakh kletok krys, transformirovannykh virusom sarkomy ptits.

Two type of exogenous action on transformed cells have been investigated. The first system includes clones of TWERC cells transformed by Rous sarcoma virus and cultivated in the presence of 5-bromodeoxyuridine. The second system consists of spontaneously transformed rat cells Sam IV superinfected with avian sarcoma virus B-77. In these cell lines and clones the structure of the provirus and its expression have been analyzed by different molecular probes.