Gene therapy for vascular smooth muscle cell proliferation after arterial injury.

Accumulation of vascular smooth muscle cells as a consequence of arterial injury is a major feature of vascular proliferative disorders. Molecular approaches to the inhibition of smooth muscle cell proliferation in these settings could potentially limit intimal expansion. This problem was approached by introducing adenoviral vectors encoding the herpesvirus thymidine kinase (tk) into porcine arteries that had been injured by a balloon on a catheter. These smooth muscle cells were shown to be infectable with adenoviral vectors, and introduction of the tk gene rendered them sensitive to the nucleoside analog ganciclovir. When this vector was introduced into porcine arteries immediately after a balloon injury, intimal hyperplasia decreased after a course of ganciclovir treatment. No major local or systemic toxicities were observed. These data suggest that transient expression of an enzyme that catalyzes the formation of a cytotoxic drug locally may limit smooth muscle cell proliferation in response to balloon injury.

Gene transfer and vascular disease.

Major advances in molecular biology over the past decade have increased our understanding of how genes are expressed and regulated in mammalian cells in vivo. This knowledge has been translated to investigations of the molecular and cellular biology of the cardiovascular system. Gene transfer, the introduction of a recombinant gene into host somatic cells, has become a useful tool for studying gene structure and function. It can be used to identify regulatory sequences that control gene expression. Transfer of a gene into a new cellular environment provides a means to determine gene function. These methods also allow investigators to introduce new genes into intact animals, permitting the development of animal models of human disease and investigations of gene function in vivo. The field of gene transfer and vascular disease is emerging as a new approach for studying the pathophysiology of vascular disease and for developing potential new genetic treatments for these disorders. In this review, we discuss the methods for gene transfer, how these methods can be employed to investigate the pathophysiology of cardiovascular diseases, and the potential for the development of molecular genetic treatments for human vascular diseases. The focus will be on cardiovascular diseases, although the principles may be applied to other disorders. In summary, gene transfer represents a new approach to applying molecular biology to the study and treatment of human disease.

Expression and function of a recombinant endothelial nitric oxide synthase gene in porcine coronary arteries.

OBJECTIVES: Direct gene transfer of exogenous nitric oxide synthase, with the subsequent increase in nitric oxide production, could represent a potential therapeutic strategy in the treatment of vascular proliferative disorders. The goal of the present study was to determine if porcine coronary arteries could be transduced with an adenoviral vector encoding endothelial nitric oxide synthase (Ad.CMVeNOS) resulting in functional expression. METHODS AND RESULTS: Segments of porcine right coronary artery were exposed for 1 h at 37 degrees C to either replication-deficient adenovirus encoding bovine endothelial nitric oxide synthase (Ad.CMVeNOS, 5 x 10(9) pfu/ml) or control adenovirus encoding Escherichia coli beta-galactosidase (Ad.CMVLacZ, 5 x 10(9) pfu/ml). Immunohistochemistry with a monoclonal antibody specific for nitric oxide synthase (NOS) demonstrated recombinant gene expression in both the endothelial and adventitial layers of Ad.CMVeNOS transduced coronaries with only endogenous NOS confirmed in the endothelium of Ad.CMVLacZ arteries. Coronary arteries transduced with Ad.CMVeNOS yielded 517 +/- 110 (mean +/- S.E.M.) nM/ng nitrite while vessels transduced with Ad.CMVLacZ yielded 126 +/- 84 nM/ng (P < 0.05, n = 6). Isometric tension recording, following prostaglandin F2 alpha constriction, documented a reduced tension in Ad.CMVeNOS transduced coronaries, compared to matched Ad.CMVLacZ coronaries (6.10 +/- 1.08 g vs. 8.45 +/- 1.19 g, respectively, P = 0.05, n = 8). This tension differential was eliminated with prior incubation in NG-monomethyl-L-arginine (L-NMMA, 10(-4) M). The EC50 for calcium ionophore relaxation of Ad.CMVeNOS coronary arteries was reduced compared to Ad.CMVLacZ (-7.90 +/- 0.03 logM vs. -7.26 +/- 0.11 logM, respectively, P < 0.05, n = 8). CONCLUSIONS: These studies demonstrate successful transfer of endothelial nitric oxide synthase into porcine coronary arteries as verified by histochemical localization of recombinant protein with an increase of nitric oxide release as demonstrated by enhanced nitrite production and an alteration in vasomotor function.

Safety and short-term toxicity of a novel cationic lipid formulation for human gene therapy.

Among the potential nonviral vectors for human gene therapy are DNA-liposome complexes. In a recent clinical study, this delivery system has been utilized. In this report, a novel cationic lipid, dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium (DMRIE), has been substituted into the DNA-liposome complex with dioleoyl phosphatidylethanolamine (DOPE), which both improves transfection efficiencies and allows increased doses of DNA to be delivered in vivo. The safety and toxicity of this DNA-liposome complex has been evaluated in two species, mice and pigs. The efficacy of DMRIE/DOPE in inducing an antitumor response in mice after transfer of a foreign MHC has been confirmed. No abnormalities were detected after administration of up to 1,000-fold higher concentrations of DNA and lipid than could be tolerated in vivo previously. Examination of serum biochemical enzymes, pathologic examination of tissue, and analysis of cardiac function in mice and pigs revealed no toxicities related to this treatment. This improved cationic lipid formulation is well-tolerated in vivo and could therefore allow higher dose administration and potentially greater efficiency of gene transfer for gene therapy.

Oral antibiotic treatment of right-sided staphylococcal endocarditis in injection drug users: prospective randomized comparison with parenteral therapy.

PURPOSE: To compare the efficacy and safety of inpatient oral antibiotic treatment (oral) versus standard parenteral antibiotic treatment (intravenous) for right-sided staphylococcal endocarditis in injection drug users. PATIENTS AND METHODS: In a prospective, randomized, non-blinded trial, febrile injection drug users were assigned to begin oral or intravenous (IV) treatment on admission, before blood culture results were available. Oral therapy consisted of ciprofloxacin and rifampin. Parenteral therapy was oxacillin or vancomycin, plus gentamicin for the first 5 days. Antibiotic dosing was adjusted for renal dysfunction. Administration of other antibacterial drugs was not permitted during the treatment or follow-up periods. Bacteremic subjects having right-sided staphylococcal endocarditis received 28 days of inpatient therapy with the assigned antibiotics. Test-of-cure blood cultures were obtained during inpatient observation 6 and 7 days after the completion of antibiotic therapy, and again at outpatient follow-up 1 month later. Criteria for treatment failure and for drug toxicity were prospectively defined. RESULTS: Of 573 injection drug users who were hospitalized because of a febrile illness and suspected right-sided staphylococcal endocarditis, 93 subjects (16.2%) had two or more sets of blood cultures positive for staphylococci; 85 of these bacteremic subjects (14.8%) satisfied diagnostic criteria for at least possible right-sided staphylococcal endocarditis (no other source of bacteremia was apparent) and entered the trial. Forty-four (oral, 19; IV, 25) of these 85 subjects completed inpatient treatment and evaluation including test-of-cure blood cultures. There were four treatment failures (oral, 1 [5.2%]; IV, 3 [12.0%]; not significant, Fisher's exact test). Drug toxicity was significantly more common in the parenterally treated group (oral, 3%; IV, 62%; P < 0.0001), consisting largely of oxacillin-associated increases in liver enzymes. CONCLUSIONS: For selected patients with right-sided staphylococcal endocarditis, oral ciprofloxacin plus rifampin is effective and is associated with less drug toxicity than is intravenous therapy.

Arteriography of the left internal mammary artery graft utilizing a balloon-tipped floatation catheter: an alternative approach.

In this report, we describe an alternative method to the conventional arteriographic techniques of the left internal mammary artery (LIMA) graft using a balloon-tipped floatation catheter placed within the left subclavian artery. The floatation catheter will serve as both an occluder of the subclavian artery as well as a port for contrast injection. It may be effectively employed in the rare instances where direct cannulation of the LIMA graft is not possible.

Saphenous vein graft ectasia: an unusual late complication of coronary artery bypass surgery. A case report.

Aneurysms and ectasias of saphenous vein grafts are infrequent complications of coronary artery bypass surgery. They usually present as an expanding asymptomatic mediastinal mass on chest x-ray film or computed tomography scan. Though rare, they must be excluded from the differential diagnosis of mediastinal masses to avoid potentially dangerous needle biopsy. The authors describe ectasia of a saphenous vein graft in a 62-year-old man 14 years after coronary artery bypass surgery. The relevant literature is also discussed.

Coronary angioplasty and stenting in orthotopic heart transplants: a fruitful act or a futile attempt?

Accelerated allograft coronary artery disease remains the major cause of mortality after the first year of transplantation. Despite the extensive use of stents and angioplasty in coronary artery disease, there is a paucity of data about the efficacy of such interventions in orthotopic heart transplants. The authors herein report the outcome of those patients in their institution who had undergone percutaneous coronary artery angioplasty and stenting at a late stage of their transplantation. Within a 12-year period, 106 adult patients underwent orthotopic heart transplantation at their institution. Eight of these patients with 17 lesions underwent deployment of nine stents and eight angioplasties 8.1 +/- 3.2 years posttransplantation. There were 15 denovo and two restenotic lesions. The indications for intervention were presence of symptoms in five patients and severity of lesions in three asymptomatic patients detected on their follow-up angiogram. All patients had angiographic worsening of lesions at their follow-up angiogram. The initial procedural success for both stented and angioplastied lesions was 100%. Within a mean angiographic follow-up of 261 days, all balloon angioplastied lesions had developed restenosis, whereas within a mean period of 67 days, 50% of stented lesions had developed restenosis. On the follow-up angiogram, deterioration of the nontreated segments were noted throughout the coronary arterial tree; however, the immediate proximal and distal parts of the target segments demonstrated an exaggerated hyperproliferative response as compared to other sites. The overall median time to the detection of restenosis for both stented and angioplastied lesions was 5.2 months (inner quartile 2.5-6.2 months). The authors conclude that angioplasty and stenting late in the course of transplantation is associated with a significant restenosis rate and in such patients earlier or alternative catheter-based interventions must be considered.

Enhanced angiogenesis and unfavorable remodeling in injured porcine coronary artery lesions: effects of local basic fibroblast growth factor delivery.

There is interest in the role of growth factors in the genesis of arterial remodeling. We studied local administration of basic fibroblast growth factor (bFGF) to coronary lesions to determine whether there is a difference in remodeling and whether neovascularization could be induced in such stenoses and distal myocardium. Pigs were randomized to balloon infusion of either saline or bFGF at each thermally injured arterial site. After the animals were killed, their internal elastic lamina, neointima, and lumen areas were measured. Capillaries were counted in the arteries and myocardium. There was a greater loss of lumen and internal elastic in the bFGF group. The neointima, media, and myocardium in the bFGF treated arteries had statistically more capillaries. This study showed that local intracoronary bFGF, at a dose that results in arterial luminal revascularization in injured segments, adversely affects arterial remodeling. Thus, the angiogenic response to exogenous bFGF may be offset by concomitant shrinkage of injured arterial segments.

Hematopoietic stem cells: transcriptional regulation, ex vivo expansion and clinical application.

Maintenance of ex vivo hematopoietic stem cells (HSC) pool and its differentiated progeny is regulated by complex network of transcriptional factors, cell cycle proteins, extracellular matrix, and their microenvironment through an orchestrated fashion. Strides have been made to understand the mechanisms regulating in vivo quiescence and proliferation of HSCs to develop strategies for ex vivo expansion. Ex vivo expansion of HSCs is important to procure sufficient number of stem cells and as easily available source for HSC transplants for patients suffering from hematological disorders and malignancies. Our lab has established a nanofiber-based ex vivo expansion strategy for HSCs, while preserving their stem cell characteristics. Ex vivo expanded cells were also found biologically functional in various disease models. However, the therapeutic potential of expanded stem cells at clinical level still needs to be verified. This review outlines transcriptional factors that regulate development of HSCs and their commitment, genes that regulate cell cycle status, studies that attempt to develop an effective and efficient protocol for ex vivo expansion of HSCs and application of HSC in various non-malignant and malignant disorders. Overall the goal of the current review is to deliver an understanding of factors that are critical in resolving the challenges that limit the expansion of HSCs in vivo and ex vivo.

Kruppel-like factor 2 (KLF2) regulates monocyte differentiation and functions in mBSA and IL-1ß-induced arthritis.

Kruppel-like factor 2 (KLF2) plays an important role in the regulation of a variety of immune cells, including monocytes. We have previously shown that KLF2 inhibits proinflammatory activation of monocytes. However, the role of KLF2 in arthritis is yet to be investigated. In the current study, we show that recruitment of significantly greater numbers of inflammatory subset of CD11b(+)F4/80(+)Ly6C+ monocytes to the inflammatory sites in KLF2 hemizygous mice compared to the wild type littermate controls. In parallel, inflammatory mediators, MCP-1, Cox-2 and PAI-1 were significantly up-regulated in bone marrow-derived monocytes isolated from KLF2 hemizygous mice, in comparison to wild-type controls. Methylated-BSA and IL-1ß-induced arthritis was more severe in KLF2 hemizygous mice as compared to the littermate wild type controls. Consistent with this observation, monocytes isolated from KLF2 hemizygous mice showed an increased number of cells matured and differentiated towards osteoclastic lineage, potentially contributing to the severity of cartilage and bone damage in induced arthritic mice. The severity of arthritis was associated with the higher expression of proteins such as HSP60, HSP90 and MMP13 and attenuated levels of pPTEN, p21, p38 and HSP25/27 molecules in bone marrow cells of arthritic KLF2 hemizygous mice compared to littermate wild type controls. The data provide new insights and evidences of KLF2-mediated transcriptional regulation of arthritis via modulation of monocyte differentiation and function.

Recombinant endothelial nitric oxide synthase-transduced human saphenous veins: gene therapy to augment nitric oxide production in bypass conduits.

BACKGROUND: Nitric oxide is a potent vasodilator that also inhibits platelet aggregation and smooth muscle cell proliferation, properties that may prevent early and late occlusion of saphenous vein coronary bypass conduits. We determined whether human saphenous veins can be transduced with adenovirus vector-encoding bovine endothelial nitric oxide synthase (Ad.CMVeNOS), resulting in functional expression of recombinant nitric oxide synthase. METHODS AND RESULTS: Harvested segments of human saphenous vein were exposed for 1 hour at 37 degrees C to replication-deficient Ad.CMVeNOS (5 x 10(9) PFU/mL) or control adenovirus-encoding Escherichia coli beta-galactosidase (Ad.CMVLacZ; 5 x 10(9) PFU/mL). The vein segments were analyzed for recombinant endothelial nitric oxide synthase expression and activity 48 hours later. Histochemical staining for recombinant beta-galactosidase activity was localized to the luminal endothelium and adventitia of vein segments transduced with Ad.CMVLacZ. Similarly, immunohistochemical staining with a monoclonal antibody for nitric oxide synthase localized recombinant gene expression to endothelial and adventitial cells in Ad.CMVeNOS veins; only endogenous nitric oxide synthase was identified in the endothelium of Ad.CMVLacZ veins. Nitrite generation after stimulation with calcium ionophore increased in Ad.CMVeNOS veins (1420.0+/-298.2 nM/mg versus 130.3+/-19.9 nM/mg; n=3; P<.05). Isometric tension recording demonstrated augmented maximal relaxation to calcium ionophore (32+/-4.5% versus 17.4+/-7.4%; n=6; P<.05) after precontraction with norepinephrine. Bioassay superfusion demonstrated a twofold augmentation of the biodetector ring relaxation during calcium ionophore stimulation of Ad.CMVeNOS veins. CONCLUSIONS: Adenovirus-mediated gene transfer to human saphenous veins resulted in functional transgene expression with increased nitric oxide release. These or similar molecular techniques to increase nitric oxide production may reduce the risk of early thrombosis in saphenous vein grafts.

Recombinant gene transfer of endothelial nitric oxide synthase augments coronary artery relaxations during hypoxia.

BACKGROUND: Coronary arteries respond to hypoxia with transient relaxations, which increases coronary blood flow, in part, by release of nitric oxide. We hypothesized that increased expression of nitric oxide synthase might further augment blood vessel relaxation during hypoxia. The present study examined the effect of adenovirus-mediated transfer of bovine endothelial nitric oxide synthase (eNOS) on hypoxia-induced transient relaxations in canine coronary arteries. METHODS AND RESULTS: Paired segments of coronary arteries were exposed to vehicle (phosphate-buffered saline with albumin) or an adenovirus encoding either E coli beta-galactosidase (Ad.CMVLacZ, viral control; 10(10) pfu/mL) or eNOS (Ad.CMVeNOS; 10(10) pfu/mL) for 2 hours at 37 degrees C. Immunohistochemistry with a monoclonal antibody specific for eNOS documented both endothelial and adventitial expression in Ad.CMVeNOS arteries, whereas vehicle and viral controls demonstrated only constitutive expression. Levels of cGMP were increased 5-fold in Ad.CMVeNOS arteries compared with controls. In arteries exposed to Ad.CMVeNOS, maximum contraction to prostaglandin F(2alpha) was reduced compared with viral controls, and this effect was eliminated by pretreatment with a competitive inhibitor of eNOS (N(G)-monomethyl-L-arginine, 10(-3) mol/L). Hypoxia-induced transient relaxation (95% N(2)-5% CO(2)) in Ad.CMVeNOS arteries (45.2+/-8.8%, n=6) was augmented compared with vehicle (26.3+/-6.0%) or viral (27.2+/-7.1%) controls. CONCLUSIONS: Adenovirus-mediated gene transfer of nitric oxide synthase reduces receptor-dependent contractions and augments hypoxia-induced relaxations in canine coronary arteries; this method of augmentation of NO production might be advantageous for reduction of coronary artery vasospasm.

Left internal mammary artery graft perforation due to high-pressure stent deployment.

Perforation of newly placed left internal mammary artery (LIMA) grafts due to stent deployment is an infrequent but potentially dangerous complication of coronary interventions. It may lead to brisk hemorrhage and massive cardiac tamponade requiring emergent pericardiocentesis and surgery. We report a case of a LIMA graft perforation following stent deployment with a high-pressure balloon 12 days after surgery. The patient was treated with emergent pericardiocentesis, rapid autotransfusion of the pericardial aspirate into the systemic circulation, and surgical repair of the ruptured vessel.

Expression and function of a recombinant PDGF B gene in porcine arteries.

Platelet-derived growth factor (PDGF) B is a mitogen and chemoattractant for smooth muscle cells in vitro, and expression of a recombinant PDGF B gene in porcine arteries stimulates intimal thickening. To define the mechanisms by which PDGF B gene expression induces intimal thickening in vivo, we examined its effects on smooth muscle cell proliferation and migration, extracellular matrix synthesis, and inflammatory cell infiltration in intimal lesions of pig arteries after direct gene transfer of a recombinant PDGF B gene. PDGF B gene expression was associated with rapid formation of an intima, including 3- to 10-fold increases in intimal thickness and intima-to-media area ratio 4 to 21 days after gene transfer compared with control transfected arteries. Intimal smooth muscle cell proliferation was detected at 2 days, peaked at 7 days (P < .01), and declined by 14 days, although the total number of intimal nuclei progressively increased to 21 days (P < .01). Calculations of expected-to-observed ratios of intimal cells, based on BrdC proliferation indexes, demonstrated that the increases in intimal cell number on days 2 through 7 could not be accounted for by proliferation alone, suggesting that recombinant PDGF BB acts to stimulate cell proliferation and migration of smooth muscle cells into the intima. Extracellular matrix deposition and procollagen synthesis were observed after 7 days (P < .01) and were associated with a decline in cell density in the intima, suggesting that extracellular matrix synthesis may contribute to progressive intimal thickening in response to PDGF B gene expression. There was minimal accumulation of inflammatory cells, including macrophages and CD3(+) lymphocytes, in transfected arteries. These data suggest that PDGF B gene expression promotes intimal expansion by both proliferation and migration of smooth muscle cells followed by synthesis of extracellular matrix and therefore acts through several mechanisms to play a role in the pathogenesis of intimal lesions in vivo.

Catheter-mediated pulmonary vascular gene transfer and expression.

The study and treatment of pulmonary diseases may be greatly facilitated by in vivo expression of specific recombinant genes in the pulmonary vasculature and lung parenchyma. To evaluate the feasibility of gene transfer to the pulmonary vasculature, cationic liposomes and adenoviral vectors encoding a human placental alkaline phosphatase (hpAP) gene were delivered into a pulmonary artery of 24 pigs by percutaneous right heart catheterization. Pulmonary tissue was harvested within 20 minutes or 5, 14, or 28 days later and was analyzed for gene transfer and expression. Five days after exposure to liposomes or adenoviral vectors, transfer of DNA and expression of mRNA were demonstrated in transfected lung tissue. Recombinant alkaline phosphatase protein was observed in both the vasculature and in alveolar septa but not in the bronchi. Expression of hpAP protein was observed at 5 days, was diminished at 14 days, and was absent 28 days after gene transfer with both liposome and adenoviral vectors. No major adverse effects of gene expression were detected by histological examination of the transfected lung segments compared with control segments. Gene transfer to the lung by either vector was not associated with significant biochemical abnormalities or histological changes 5, 14, or 28 days later in other organs, including carotid artery, heart, liver, spleen, kidney, skeletal muscle, ovary, and testes. These studies demonstrate that after intravascular gene delivery to the lung, recombinant genes are expressed in the vasculature and alveoli. This approach may provide a useful model for the experimental study of pulmonary vascular diseases, including pulmonary fibrosis and pulmonary thrombosis disorders.

Expression and function of recombinant endothelial nitric oxide synthase gene in canine basilar artery.

Endothelial NO synthase (eNOS) is an enzyme responsible for the production of a potent vasodilator and a key regulator of vascular tone, NO. In peripheral arteries, expression of a recombinant eNOS gene increases production of NO in the blood vessel wall. This approach appears to be a promising strategy for gene therapy of cerebrovascular disease. The major objective of the present study was to determine whether a recombinant eNOS gene (AdCMVNOS) can be functionally expressed in cerebral arteries. Replication-defective recombinant adenovirus vectors encoding bovine eNOS and Escherichia coli beta-galactosidase (AdCMVLacZ) genes, driven by the cytomegalovirus promoter, were used for ex vivo gene transfer. Rings of canine basilar artery were incubated with increasing titers of the vectors in MEM. Twenty-four or forty-eight hours after gene transfer, expression and function of AdCMVNOS were evaluated by (1) immunohistochemical staining, (2) isometric tension recording, and (3) cGMP radioimmunoassay. Transfection with AdCMVNOS resulted in the expression of recombinant eNOS protein in the vascular adventitia and endothelium, associated with significantly reduced contractile responses to UTP and enhanced endothelium-dependent relaxation to calcium ionophore A23187. Basal production of cGMP was significantly increased in the transfected vessels. The reduced contractions to UTP with increased cGMP production were reversed by a NOS inhibitor, N(G)-monomethyl-L-arginine. Contractions to UTP or production of cGMP were not affected in arteries transfected with AdCMVLacZ reporter gene. The results of the present study represent the first successful transfer and functional expression of recombinant eNOS gene in cerebral arteries. Our findings suggest that cerebral arterial tone can be modulated by recombinant eNOS expression in the vessel wall.

Expression and function of recombinant endothelial NO synthase in coronary artery smooth muscle cells.

Smooth muscle cells (SMCs) play a key role in the pathogenesis of vascular diseases. The objectives of this study were to determine whether transfer of recombinant endothelial nitric oxide synthase (eNOS) gene to porcine coronary artery smooth muscle cell (CSMCs) would result in expression of a functional enzyme and to assess the effect of expression of eNOS on cell proliferation. CSMCs were transduced in vitro with adenoviral vectors encoding cDNA for eNOS (AdeNOS) and beta-galactosidase (Ad beta Gal). In contrast to Ad beta Gal- or sham-transduced cells, CSMCs transduced with AdeNOS stained positive with the NADPH-diaphorase stain, acquired calcium-dependent NOS activity (measured by the conversion of [3H]L-arginine to [3H]L-citrulline), had increasing cyclic 3',5' cGMP levels with increasing concentrations of the vector, and produced increased amounts of nitrite. cGMP production by AdeNOS-transduced cells was augmented by increasing intracellular levels of the eNOS cofactor tetrahydrobiopterin. CSMCs transduced with AdeNOS showed diminished serum-stimulated DNA synthesis as measured by thymidine uptake. Cell proliferation was diminished in AdeNOS-transduced CSMCs as assessed by cell counts 3 and 6 days after serum stimulation of quiescent CSMCs. The present study demonstrates that adenovirus-mediated gene transfer of eNOS to CSMCs results in the expression of a functional enzyme whose activity can be augmented by increasing intracellular levels of tetrahydrobiopterin. Expression of recombinant eNOS in CSMCs results in inhibition of serum-stimulated DNA synthesis and cell proliferation. These findings imply that eNOS gene transfer to SMCs may be a unique mode of increasing local NO production in the arterial wall.