Gene Transfer Of Nitric Oxide Synthase Research Articles

Optimizing endothelial cell functionalization for cell therapy of vascular proliferative disease using a direct contact co-culture system.

Increased susceptibility to thrombosis, neoatherosclerosis, and restenosis due to incomplete regrowth of the protective endothelial layer remains a critical limitation of the interventional strategies currently used clinically to relieve atherosclerotic obstruction. Rapid recovery of endothelium holds promise for both preventing the thrombotic events and reducing post-angioplasty restenosis, providing the rationale for developing cell delivery strategies for accelerating arterial reendothelialization. The successful translation of experimental cell therapies into clinically viable treatment modalities for restoring vascular endothelium critically depends on identifying strategies for enhancing the functionality of endothelial cells (EC) derived from high cardiovascular risk patients, the target group for the majority of angioplasty procedures. Enhancing EC-associated nitric oxide (NO) synthesis by inducing overexpression of NO synthase (NOS) has shown promise as a way of increasing paracrine activity and restoring function of EC. In the present study, we developed a direct contact co-culture approach compatible with highly labile effectors, such as NO, and applied it for determining the effect of EC functionalization via NOS gene transfer on the growth of co-cultured arterial smooth muscle cells (A10 cell line) exhibiting the defining characteristics of neointimal cells. Bovine aortic endothelial cells magnetically transduced with inducible NOS-encoding adenovirus (Ad) formulated in zinc oleate-based magnetic nanoparticles (MNP[iNOSAd]) strongly suppressed growth of proliferating A10 and attenuated the stimulatory effect of a potent mitogen, platelet-derived growth factor (PDGF-BB), whereas EC functionalization with free iNOSAd or MNP formulated with a different isoform of the enzyme, endothelial NOS, was associated with lower levels of NO synthesis and less pronounced antiproliferative activity toward co-cultured A10 cells. These results show feasibility of applying magnetically facilitated gene transfer to potentiate therapeutically relevant effects of EC for targeted cell therapy of restenosis. The direct contact co-culture methodology provides a sensitive and reliable tool with potential utility for a variety of biomedical applications.

Minicircle DNA-mediated endothelial nitric oxide synthase gene transfer enhances angiogenic responses of bone marrow-derived mesenchymal stem cells.

BackgroundNon-viral-based gene modification of adult stem cells with endothelial nitric oxide synthase (eNOS) may enhance production of nitric oxide and promote angiogenesis. Nitric oxide (NO) derived from endothelial cells is a pleiotropic diffusible gas with positive effects on maintaining vascular tone and promoting wound healing and angiogenesis. Adult stem cells may enhance angiogenesis through expression of bioactive molecules, and their genetic modification to express eNOS may promote NO production and subsequent cellular responses.MethodsRat bone marrow-derived mesenchymal stem cells (rBMSCs) were transfected with a minicircle DNA vector expressing either green fluorescent protein (GFP) or eNOS. Transfected cells were analysed for eNOS expression and NO production and for their ability to form in vitro capillary tubules and cell migration. Transcriptional activity of angiogenesis-associated genes, CD31, VEGF-A, PDGFRα, FGF2, and FGFR2, were analysed by quantitative polymerase chain reaction.ResultsMinicircle vectors expressing GFP (MC-GFP) were used to transfect HEK293T cells and rBMSCs, and were compared to a larger parental vector (P-GFP). MC-GFP showed significantly higher transfection in HEK293T cells (55.51 ± 3.3 %) and in rBMSC (18.65 ± 1.05 %) compared to P-GFP in HEK293T cells (43.4 ± 4.9 %) and rBMSC (15.21 ± 0.22 %). MC-eNOS vectors showed higher transfection efficiency (21 ± 3 %) compared to P-eNOS (9 ± 1 %) and also generated higher NO levels. In vitro capillary tubule formation assays showed both MC-eNOS and P-eNOS gene-modified rBMSCs formed longer (14.66 ± 0.55 mm and 13.58 ± 0.68 mm, respectively) and a greater number of tubules (56.33 ± 3.51 and 51 ± 4, respectively) compared to controls, which was reduced with the NOS inhibitor L-NAME. In an in vitro wound healing assay, MC-eNOS transfected cells showed greater migration which was also reversed by L-NAME treatment. Finally, gene expression analysis in MC-eNOS transfected cells showed significant upregulation of the endothelial-specific marker CD31 and enhanced expression of VEGFA and FGF-2 and their corresponding receptors PDGFRα and FGFR2, respectively.ConclusionsA novel eNOS-expressing minicircle vector can efficiently transfect rBMSCs and produce sufficient NO to enhance in vitro models of capillary formation and cell migration with an accompanying upregulation of CD31, angiogenic growth factor, and receptor gene expression.

SaRNA Guided iNOS Up-Regulation Improves Erectile Function of Diabetic Rats

Promoter targeted saRNAs mediate sequence specific up-regulation of gene expression. We explored the therapeutic effect of RNA activation mediated iNOS gene activation on improving erectile function in a rat model of diabetes mellitus. An optimal saRNA sequence specific for iNOS promoter was cloned into an adenoviral vector, resulting in AdU6/shiNOS and AdU6/shControl. The corresponding viruses were used to transduce cultured rat cavernous smooth muscle cells. Streptozotocin induced diabetes models were established in rats and used to test the effects of intracavernous delivery of iNOS saRNA viruses on erectile function. iNOS expression in the cavernous smooth muscle cells or penile tissue of treated rats was assessed by reverse transcriptase-polymerase chain reaction and Western blot. Cyclic guanosine monophosphate was analyzed by enzyme-linked immunosorbent assay. Intracavernous pressure in response to cavernous nerve stimulation was measured using a data acquisition system on post-injection days 1, 3, 5, 7, 10 and 14. Adenovirus mediated expression of iNOS saRNA caused sustained up-regulation of iNOS in cavernous smooth muscle cells. Intracavernous injection of AdU6/shiNOS activated iNOS expression in vivo and significantly increased peak intracavernous pressure in streptozotocin induced diabetic rats via nitric oxide/intracellular cyclic guanosine monophosphate activation. Results show that saRNA mediated iNOS over expression in the penis can restore erectile function in streptozocin diabetic rats via the nitric oxide-cyclic guanosine monophosphate pathway.

Nitric Oxide Synthase Gene Transfer Overcomes the Inhibition of Wound Healing by Sulfur Mustard in a Human Keratinocyte In Vitro Model

Sulfur mustard (SM) is a chemical warfare agent that causes extensive skin injury. Previously we reported that SM exposure resulted in suppression of inducible nitric oxide synthase (iNOS) expression to inhibit the healing of scratch wounds in a cultured normal human epidermal keratinocyte (NHEK) model. Based on this finding, the present study was to use adenovirus-mediated gene transfer of iNOS to restore the nitric oxide (NO) supply depleted by exposure to SM and to evaluate the effect of NO on wound healing inhibited by SM in NHEKs. The effect of the iNOS gene transfer on iNOS protein expression and NO generation were monitored by Western blot and flow cytometry, respectively. Wound healing with or without the iNOS gene transfer after SM exposure was assessed by light and confocal microscopy. The iNOS gene transfer via adenovirus resulted in overexpression of the iNOS and an increase in NO production regardless of SM exposure in the NHEK model. The gene transfer was also effective in overcoming the inhibition of wound healing due to SM exposure leading to the promotion of wound closure. The findings in this study suggest that the iNOS gene transfer is a promising therapeutic strategy for SM-induced skin injury.

Ex Vivo Adenoviral Vector Gene Delivery Results in Decreased Vector-associated Inflammation Pre- and Post–lung Transplantation in the Pig

Acellular normothermic ex vivo lung perfusion (EVLP) is a novel method of donor lung preservation for transplantation. As cellular metabolism is preserved during perfusion, it represents a potential platform for effective gene transduction in donor lungs. We hypothesized that vector-associated inflammation would be reduced during ex vivo delivery due to isolation from the host immune system response. We compared ex vivo with in vivo intratracheal delivery of an E1-, E3-deleted adenoviral vector encoding either green fluorescent protein (GFP) or interleukin-10 (IL-10) to porcine lungs. Twelve hours after delivery, the lung was transplanted and the post-transplant function assessed. We identified significant transgene expression by 12 hours in both in vivo and ex vivo delivered groups. Lung function remained excellent in all ex vivo groups after viral vector delivery; however, as expected, lung function decreased in the in vivo delivered adenovirus vector encoding GFP (AdGFP) group with corresponding increases in IL-1β levels. Transplanted lung function was excellent in the ex vivo transduced lungs and inferior lung function was seen in the in vivo group after transplantation. In summary, ex vivo delivery of adenoviral gene therapy to the donor lung is superior to in vivo delivery in that it leads to less vector-associated inflammation and provides superior post-transplant lung function.

Nitric Oxide Synthase Gene Transfer Restores Activity of Circulating Angiogenic Cells From Patients With Coronary Artery Disease

Circulating angiogenic cells (CACs), represent a potential new therapeutic tool for the treatment of cardiovascular diseases, but their regenerative function is impaired in patients with coronary artery disease (CAD) and cardiac risk factors. The objective of this study is to assess the effect of lentiviral overexpression of endothelial nitric oxide synthase (eNOS) on the activity of CACs from patients with CAD and cardiac risk factors. In vitro and in vivo assays were employed to evaluate the regenerative capacity of the cells compared to CACs derived from healthy volunteers. Lentiviral eNOS transduction of cells from CAD patients significantly improved chemotactic migration compared with sham transduction, and increased the ability of CACs to induce angiogenic tube formation when cocultured with human umbilical vein endothelial cells (HUVECs) on Matrigel. In addition, eNOS transduction restored the ability of patient-derived CACs to enhance neovascularization and improve ischemic hind limb perfusion, approaching the efficacy of cells from healthy donors. These data indicate that CAC dysfunction seen in high-risk patients can be partially reversed by eNOS overexpression, suggesting that ex vivo gene delivery may improve the efficacy of autologous cell therapy for cardiovascular disease.

Gene transfer of human endothelial nitric oxide synthase inhibits proliferation of human vascular smooth muscle cells and the mechanism

Objectiye To invesigate the effect of human endothelial nitric oxide synthase (heNOS ) gene transfer on the proliferation of in vitro cultured human vascular smooth muscle cells (HVSMCs)and the mechanism. Methods The HVSMCs were transfeced with multiplicity of infection (MOI) of 50,150,250, 300,450. cGMP was measured by radioimmunoassay in HVSMCs. The expression levels of p21 and p27 were detected by Western blotting. Cell cycle and apoptosis were assayed by flow cytometry. Results (1) At 120th h, the A570values were respectively 1.410±0.081, 1.357 ±-0. 150, 1.303±0.311,0. 995 ±0. 248 and 0. 731 ±0. 101 at MOI of 50, 150,250,300,450. The proliferation of HVSMCs was significantly and stably inhibited with MOI 300 of AdCMV-heNOS; (2) Af72nd h after the gene transfer,cGMP levels were increased in heNOS-transduced ( 12. 89 ±2. 06) compared to LacZ- (8.36 ±0. 34) and non-tranduced (7. 91 ± 0. 39) cells ( P ＜ 0. 01 ); (3) At 48th h after the gene transfer, the expression of heNOS in HVSMCs up-regulated p21 and p27 (P＜0. 05); (4) After 48 h tronsfected with sersuln-depriveol and for 24 h serum stimulation, the cell cycle was significantly arrested in G0/G1 phase in heNOStransduced group, and G0/G1 was respectively ( 64. 23 ± 1.58 ) %, ( 64. 96 ± 1.36 ) %, ( 76. 03 ±2. 27 ) % in non-, LacZ- and heNOS-transduced cells; ( 5 ) At first and third day afer the gene transfer,there was no increase in apoptosis at the first day in all transduced cells ( P ＞ 0. 05 ). Conclusion Adenovirus-mediated heNOS gene transfer to HVSMCs inhibits cell proliferation via up-regulation of p21 and p27 resulting in a delay in cell progression not apoptosis. Key words: Endothelial nitric oxide synthase; Cell cycle; p27

Transfection of Endothelial Nitric Oxide Synthase Gene Improves Angiogenic Efficacy of Endothelial Progenitor Cells in Rabbits with Hindlimb Ischemia

Background: The present study explored the effect of endothelial nitric oxide synthase (eNOS) gene transfer on the angiogenic potential of ex vivo expanded endothelial progenitor cells (EPCs) in a rabbit model of hindlimb ischemia. Methods: Rabbit peripheral blood EPCs were cultured and transfected with mammalian expression vector pcDNA3.1-eNOS containing full-length human eNOS gene. Ischemia was induced in the right hind limb of three groups of rabbits by ligation of the distal external iliac artery and excision of the common and superficial femoral arteries. In one group of animals, ten days after the surgery, autologous eNOS-EPCs were transplanted intramuscularly in the ischemic limb. Two other groups received an equivalent number of unmodified EPCs or phosphate buffered saline (PBS) respectively. Results: Two weeks after cell transplantation, the in vivo expression of eNOS was detected in limb tissue sections of eNOS-EPCs treated animals. Animals treated with eNOS-EPCs had a significant reduction in ischemic muscle necrosis and inflammation, augmentation in the capillary density (P< 0.05) and angiographic scores demonstrating distal arterial reconstitution and enhanced angiogenesis in comparison to animals transplanted with EPCs or PBS (P< 0.05). Conclusion: We conclude that modification of EPCs by eNOS constitutes an effective strategy to improve the efficacy of EPCs for therapeutic angiogenesis.

Neuronal Nitric Oxide Synthase Gene Transfer into the Rat Prostate Using In Vivo Electroporation.

It has been reported that nitric oxide (NO) mainly contributes to prostate or urethral smooth muscles relaxation, and that nitrergic innervation and neuronal NO synthase (nNOS) levels are decreased in benign prostatic hyperplasia. The purpose of the present study was to evaluate the feasibility to gene therapy for benign prostatic hyperplasia by transferring nNOS gene into the rat prostate with in vivo electroporation (EP) procedure. Male Sprague-Dawley rats were divided into four groups (sham, only EP, only nNOS injection, and nNOS gene injection with EP groups). Fifty micrograms of luciferase gene and nNOS expression vectors in 50 µL of K-PBS (potassium-phosphate buffered saline) were injected into the prostate. Immediately after the injection of these vectors, the vector injection points were electroporated by the two-square parallel electrodes. Two days after gene transfer, luciferase analysis and an immunohistochemical staining for nNOS were performed, and NO2 (-) /NO3 (-) (NOX ) release was measured using high-performance liquid chromatography coupled with the microdialysis procedure. The optimal electric pulse conditions were 50 V, 1 Hz and 10 msec. In vivo EP with these conditions showed the increase in the luciferase gene expression approximately 300-fold of the control group. In the nNOS gene injection with EP group, the marked nNOS immunoreactivity was observed, and NOX release was significantly higher, as compared to other groups. The results suggest that EP is a feasible technique for in vivo gene transfer into the rat prostate, and that the transferred nNOS gene functionally expresses and contributes to NO production.

Oxidative Stress and Compartment of Gene Expression Determine Proatherosclerotic Effects of Inducible Nitric Oxide Synthase

Genetic and pharmacological inhibition of inducible nitric oxide synthase (iNOS) decreases atherosclerosis development. Potential proatherogenic effects of iNOS include iNOS mediated oxidative stress and iNOS expression in different cellular compartments. Lesional iNOS can potentially produce nitric oxide radicals (NO), superoxide radicals (O2(-)), or both; these radicals may then react to form peroxynitrite. Alternatively, O2(-) radicals from oxidases co-expressed with iNOS could react with NO to produce peroxynitrite. Therefore, the expression profiles of the genes that modulate the redox system in different iNOS-expressing cell compartments may determine the role of iNOS in atherosclerosis. We used apoE (apoE(-/-)) and apoE/iNOS double knockout (apoE(-/-)/ iNOS(-/-)) mice to assess vascular NO, O2(-), and peroxynitrite formation by electron spin resonance spectroscopy, high performance liquid chromatography, and 3-nitrotyrosine staining. The relevance of the iNOS expressing cell compartment was tested by bone marrow transplantation. We show that iNOS significantly contributes to vascular NO production and itself produces O2(-), leading to peroxynitrite formation in atherosclerotic lesions. Our bone marrow transplantation experiments show that bone marrow derived cells exclusively mediate the proatherosclerotic effects of iNOS in males, while both parenchymal and bone marrow derived iNOS equally contribute to atherosclerosis in females. Moreover, iNOS expression affects vascular remodeling. These findings establish for the first time that the proatherosclerotic effects of iNOS vary with sex in addition to the compartment of its expression.

Nitric oxide synthase gene therapy enhances the toxicity of cisplatin in cancer cells

Nitric oxide (NO.) derived from donor drugs has been shown to be an effective chemosensitizer in vitro. We investigated the combination of inducible nitric oxide synthase (iNOS) gene transfer, driven by a strong constitutive promoter (cytomegalovirus; CMV) with the DNA cross-linking agent cisplatin in mouse and human tumour cell lines. Proof of principal experiments were performed in the radiation-induced fibrosarcoma-1 (RIF-1) murine cell line. Cells were transfected with constitutively expressed CMV/iNOS plasmid DNA using a cationic lipid vector, before exposure to cisplatin. In vivo efficacy was determined in an intradermal RIF-1 tumour model, with intraperitoneal administration of cisplatin. Additionally, treatment potential was investigated in various human tumour cell lines including human prostate (DU145 and PC3) and human colon (HT29 and HCT116) cancer cell lines. Experimental endpoints were established using western blot, Greiss test, clonogenic assay and tumour growth delay. Transfection of RIF-1 tumour cells in vitro with the CMV/iNOS significantly enhanced the cytotoxicity of cisplatin (0.2-1.0 microM). In vivo transfer of CMV/iNOS by direct injection into established RIF-1 tumours caused a significant (p = 0.0027) delay in tumour growth. CMV/iNOS gene transfer in vitro resulted in the strong expression of iNOS DNA in all cell lines, and significantly increased levels of NO. in all cell lines except HCT116. Significant chemosensitization of cisplatin cytotoxicity was observed in the presence of NO. derived from the overexpression iNOS. We conclude that p53 status of the various cell lines was unlikely to be responsible for cisplatin-induced apoptosis.

Invited Commentary

Pulmonary hypertension, regardless of the cause, represents a significant challenge to cardiothoracic surgeons with regard to preoperative risk assessment, perioperative management, and postoperative outcomes. Despite multiple pharmacologic strategies, including prostanoids, endothelin-receptor antagonists, and phosphodiesterase inhibitors, pulmonary hypertension remains a source of significant morbidity and mortality. One of the most recognized regulators of pulmonary vascular vasomotor tone is nitric oxide (NO). Otherwise known as endothelial-derived relaxation factor, NO is biosynthesized from L-arginine in the vasculature by two forms of nitric oxide synthase: constitutive nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS). Upregulation of endogenous NO or exogenous delivery of NO has been well studied in a variety of cardiovascular models as well as humans. Indeed adenoviral gene transfer of eNOS has been previously reported to ameliorate monocrotaline and hypoxia-induced pulmonary hypertension. Zhang and colleagues [1Zhang F. Wu S. Lu X. Wang M. Liu M. Gene transfer of endothelial nitric oxide synthase attenuates flow-induced pulmonary hypertension in rabbits.Ann Thorac Surg. 2008; 85: 581-586Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar] report a therapeutically-aimed study on the effects of tracheal-delivered adenoviral eNOS gene transfer in a rabbit model of pulmonary overcirculation. They establish a background associated with congenital heart disease by performing pre-tricuspid shunts (carotid-jugular bypass). They demonstrate successful delivery of the gene and achieve a functional response by significantly decreasing pulmonary vascular resistance. The model seems challenging as only 18 of 40 animals survived or developed significant pulmonary hypertension. Although their biochemical data is firm, some of the kinetics associated with gene transfer remains nebulous. Quite frankly, the effects of only 10 minutes of adenoviral exposure are impressive. Notably missing are the data related to transfection efficiency and time course of activity. They surveyed tissues after 4 days for gene expression, but hemodynamic testing was done at 3 months. Adenoviral gene expression is usually lost by 3 to 4 weeks after transduction. It remains unknown if pulmonary hypertension returns when transfected eNOS gene expression ceases. Furthermore, the effects of endogenous NO compared with the transgene are unable to be deciphered, and thus, the effect of exogenous eNOS on their physiologic outcomes comes into question. Although they claim that eNOS was mostly expressed in the endothelial cells of resistance size vessels, it would have been nice to see immunohistochemical evidence of uptake of eNOS in other locations in the tracheobronchial tree or cell-type (ie, vascular smooth muscle). This is especially important as many other reports have demonstrated tracheal delivery of transgenes to be located in airway epithelial cells. Finally, others have shown that this model of flow-induced pulmonary hypertension causes an increased expression of native eNOS. Why would delivery of even more eNOS help? Because there is no true control group in their study, they are unable to comment on this paradox. They speculate that although eNOS is elevated, its biologic activity is decreased because the endothelial function is impaired. As such, it would have been nice to see some assessment of endothelial function in their model. Despite some of the specific limitations just outlined, their results offer additional evidence particularly related to NO for adopting translational motivated therapies for cardiopulmonary disease. The pulmonary vasculature is easily accessible and offers a clinically relevant avenue for using such innovative technology. Indeed, inhaled NO has proven effective for short-term treatment of adult and newborn acute pulmonary hypertension. Although L-arginine has gained popularity for treating patients with chronic pulmonary hypertension, no randomized study has documented its long-term effects, particularly in light of some of its pro-proliferative metabolites. As such, direct manipulation of the nitric oxide and nitric oxide synthase axis with locally delivered eNOS could offer an effective strategy for this difficult patient population. Gene Transfer of Endothelial Nitric Oxide Synthase Attenuates Flow-Induced Pulmonary Hypertension in RabbitsThe Annals of Thoracic SurgeryVol. 85Issue 2PreviewNitric oxide, a potent vasodilator with an important role in the regulation of pulmonary vascular tone, is synthesized by a family of nitric oxide synthases. To determine whether endothelial nitric oxide synthase (eNOS) gene transfer may prevent pulmonary hypertension, the effects of transfer of the eNOS gene to the lung were studied in rabbits with pulmonary hypertension induced by high pulmonary blood flow. Full-Text PDF

Gene Transfer of Endothelial Nitric Oxide Synthase Attenuates Flow-Induced Pulmonary Hypertension in Rabbits

Nitric oxide, a potent vasodilator with an important role in the regulation of pulmonary vascular tone, is synthesized by a family of nitric oxide synthases. To determine whether endothelial nitric oxide synthase (eNOS) gene transfer may prevent pulmonary hypertension, the effects of transfer of the eNOS gene to the lung were studied in rabbits with pulmonary hypertension induced by high pulmonary blood flow. Adenoviral vector encoding the eNOS gene was intratracheally transfected into the lung of rabbits with flow-induced pulmonary hypertension. Rabbits instilled intratracheally with adenoviral vector without encoding the eNOS gene served as a control group. Hemodynamic data were recorded before and after transfection, and transgene expression was investigated. Pulmonary hypertension was significantly attenuated in eNOS gene-transfected rabbits compared with control animals (mean pulmonary arterial pressure, 22.3 +/- 5.5 versus 41.0 +/- 6.9 mm Hg; pulmonary vascular resistance, 326 +/- 42 versus 618 +/- 66 dynes x s x cm(-5); p < 0.01). Systemic arterial pressure and systemic vascular resistance were unaffected. There was an increase in calcium-dependent conversion of L-arginine to L-citrulline in the lung (16.81 +/- 0.72 versus 4.11 +/- 0.41 pmol x mg protein(-1) x h(-1)) and cyclic guanosine monophosphate levels (0.138 +/- 0.015 versus 0.065 +/- 0.003 pmol/mg protein). Immunohistochemical staining showed expression of the eNOS gene was detected mainly in endothelial cells of small pulmonary vessels. Transgene expression was confirmed using Western blot analysis. These data suggest that intratracheal adenoviral-mediated eNOS gene transfer can attenuate flow-induced pulmonary hypertension in rabbits and may represent a new form of therapy for the treatment of flow-induced pulmonary hypertension.

Neuronal nitric oxide synthase gene transfer decreases [Ca 2+] i in cardiac sympathetic neurons

Gene transfer of neuronal nitric oxide synthase (nNOS) can decrease cardiac sympathetic outflow and facilitate parasympathetic neurotransmission. The precise pathway responsible for nitric oxide (NO) mediated inhibition of sympathetic neurotransmission is not known, but may be related to NO–cGMP activation of cGMP-stimulated phosphodiesterase (PDE2) that enhances the breakdown of cAMP to deactivate protein kinase A (PKA), resulting in a decrease in Ca 2+ influx mediated exocytosis of the neurotransmitter. We investigated depolarization evoked Ca 2+ influx in nNOS gene transduced sympathetic neurons from stellate ganglia with a noradrenergic cell specific vector (Ad.PRS-nNOS or empty vector), and examined how nNOS gene transfer affected cAMP and cGMP levels in these neurons. We found that targeting nNOS into these sympathetic neurons reduced amplitudes of voltage activated Ca 2+ transients by 44%. nNOS specific inhibition by N-[(4 S)-4-Amino-5-[(2-aminoetyl](amino] pentyl]- N′-nitroguanidine (AAAN) reversed this response. nNOS gene transfer also increased intracellular cGMP (47%) and decreased cAMP (29%). A PDE2 specific inhibitor Bay60-7557 reversed the reduction in cAMP caused by Ad.PRS-nNOS. These results suggest that neuronal NO modulates cGMP and PDE2 to regulate voltage gated intracellular Ca 2+ transients in sympathetic neurons. Therefore, we propose this as a possible key step involved in NO decreasing cardiac sympathetic neurotransmission.

Inhibition of TGF-β1 signaling by eNOS gene transfer improves ventricular remodeling after myocardial infarction through angiogenesis and reduction of apoptosis

Endothelial nitric oxide synthase and nitric oxide have been implicated in protection against myocardial ischemia injury. However, the angiogenic effect of endothelial nitric oxide synthase on infarcted myocardium and the role of tumor growth factor beta1 signaling in cardiac remodeling mediated by endothelial nitric oxide synthase/nitric oxide have not yet been elucidated. Human endothelial nitric oxide synthase gene in an adenovirus vector was delivered locally into rat heart 4 days prior to the induction of myocardial infarction by left anterior descending coronary artery ligation. Cardiomyocyte apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, and neovascularization was identified immunohistochemically. Endothelial nitric oxide synthase gene transfer significantly reduced infarct size and improved cardiac contractility and left ventricular diastolic function at 24 h after myocardial infarction. In addition, endothelial nitric oxide synthase significantly reduced myocardial-infarction-induced cardiomyocyte apoptosis. Activation of tumor growth factor beta1 and Smad-2 after myocardial infarction was also dramatically reduced by endothelial nitric oxide synthase. Moreover, the deterioration of both systolic and diastolic functions, in conjunction with thin left ventricular remodeling at 7 days after myocardial infarction, was prevented by endothelial nitric oxide synthase. Capillary density, as identified by alpha-smooth muscle actin immunostaining, was significantly increased in the infarcted myocardium after endothelial nitric oxide synthase transfer compared with myocardial infarction control. All cardioprotective effects of endothelial nitric oxide synthase were blocked by N(omega)-nitro-l-arginine methyl ester administration, indicating a nitric-oxide-mediated event. These results demonstrate that the endothelial nitric oxide synthase/nitric oxide system provides cardiac protection after myocardial infarction injury through inhibition of cardiac apoptosis, stimulation of neovascularization, and suppression of tumor growth factor beta1/Smad-2 signaling.

Effect of endothelial cell-based iNOS gene transfer on cavernosal eNOS expression and mouse erectile responses

Inducible nitric oxide synthase (iNOS) gene transfer is reported to augment erectile responses in rats, although it is also shown to impair vasorelaxation in cerebral arteries. We investigated the effect of endothelial cell-based iNOS gene transfer on endothelial NOS (eNOS) expression and mouse erectile responses. Human coronary artery endothelial cells (EC) transduced with empty vector (control) or iNOS were grown in culture and transplanted into the corpus cavernosum of severe combined immunodeficient mice. Endothelial NOS expression was compared in control and iNOS-transduced cells grown in the presence or absence of a selective iNOS inhibitor, L-N6- (1-iminoethyl) lysine hydrochloride (L-NIL). At 3-5 days after cell transplantation, we recorded intracorporal pressure (ICP) responses to cavernosal nerve stimulation and measured cavernosal total NO and eNOS protein expression. In this study, EC transduced with iNOS produced significantly more NO than controls but exhibited a twofold downregulation of eNOS protein and mRNA. This effect was reversed by L-NIL. In vivo, the cell-based gene transfer of iNOS led to significantly increased ICP responses, compared to mice transplanted with control ECs. Consistent with the in vitro data, cavernosal lysates had significantly reduced eNOS expression. In conclusion, EC gene transfer of iNOS downregulates EC expression of eNOS by an NOS-dependent mechanism. In the cavernosum of mice transplanted with Inos-transduced EC, nerve-stimulated erectile responses were augmented by the short-term gene transfer. However, our findings suggest that iNOS gene transfer may have deleterious effects on endothelial function if used as a treatment for erectile dysfunction.

Nitric oxide synthase gene transfer and treatment of restenosis: from bench to bedside

The concept of nitric oxide synthase (NOS) gene therapy as a therapeutic tool for inhibiting smooth muscle cell proliferation after vascular injury has been described extensively. This overview summarizes research work that moved NOS gene transfer from the first proof of concept in a rodent model to preclinical development activities, which eventually enabled early clinical testing of an inducible NOS lipoplex gene therapy approach in the setting of coronary stent implantation after balloon angioplasty. The nonviral character of this gene therapy approach may have important impact on the transition of NOS-based gene therapy to clinical practice.

Endothelial nitric oxide synthase gene transfer restores endothelium–dependent relaxations and attenuates lesion formation in carotid arteries in apolipoprotein E–deficient mice

Nitric oxide (NO) and monocyte chemoattractant protein-1 (MCP-1) exert partly opposing effects in vascular biology. NO plays pleiotropic vasoprotective roles including vasodilation and inhibition of platelet aggregation, smooth muscle cell proliferation, and endothelial monocyte adhesion, the last effect being mediated by MCP-1 downregulation. Early stages of arteriosclerosis are associated with reduced NO bioactivity and enhanced MCP-1 expression. We have evaluated adenovirus-mediated gene transfer of human endothelial NO synthase (eNOS) and of a N-terminal deletion (8ND) mutant of the MCP-1 gene that acts as a MCP-1 inhibitor in arteriosclerosis-prone, apolipoprotein E-deficient (ApoE(-/-)) mice. Endothelium-dependent relaxations were impaired in carotid arteries instilled with a noncoding adenoviral vector but were restored by eNOS gene transfer (p < 0.01). A perivascular collar was placed around the common carotid artery to accelerate lesion formation. eNOS gene transfer reduced lesion surface areas, intima/media ratios, and macrophage contents in the media at 5-week follow-up (p < 0.05). In contrast, 8ND-MCP-1 gene transfer did not prevent lesion formation. In conclusion, eNOS gene transfer restores endothelium-dependent vasodilation and inhibits lesion formation in ApoE(-/-) mouse carotids. Further studies are needed to assess whether vasoprotection is maintained at later disease stages and to evaluate the long-term efficacy of eNOS gene therapy for primary arteriosclerosis.

Nitric oxide facilitates cardiomyogenesis in mouse embryonic stem cells.

Stem cell therapy holds great promise for the replacement of damaged or dysfunctional myocardium. Nitric oxide (NO) has been shown to promote embryonic stem (ES) cell differentiation in other systems. We hypothesized that NO, through NO synthase gene transfer or exogenous NO exposure, would promote the differentiation of mouse ES cells into cardiomyocytes (CM). In our study, NO treatment increased both the number and the size of beating foci in embryoid body (EB) outgrowths. Within 2 weeks, 69% of the inducible NO synthase-transduced EB displayed spontaneously beating foci, as did 45% of the NO donor-treated EB, compared with only approximately 15% in controls. Cardiac-specific genes and protein expression were significantly increased in NO-treated ES. Electron microscopy and immunocytochemistry revealed that these NO-induced contracting cells exhibited characteristics consistent with CM. At day 7 in culture, troponin T was expressed in 45.6 +/- 20.6% of the NO-treated ES cells but in only 9.25 +/- 1.77% of control cells. Interestingly, 50.4 +/- 18.4% of NO-treated ES cells were troponin T-negative and annexin V-positive. This apoptotic phenotype was seen in <1% of the control ES cells. These data strongly support our hypothesis that mouse ES cells can be accelerated to differentiate into CM by NO treatment. NO may influence cardiac differentiation by both inducing a switch toward a cardiac phenotype and inducing apoptosis in cells not committed to cardiac differentiation.

Adenovirus mediated endothelial nitric oxide synthase gene transfer prevents restenosis of vein grafts.

The possibility of endothelial nitric oxide synthase (eNOS) gene transfer, which prevents restenosis of vein grafts, was explored in 16 goats. The recombinant adenoviral vector coding endothelial nitric oxide synthase (AdCMVeNOS) and adenoviral vector (AdCMV) were constructed. A total of 6 cm jugular vein was removed, cut into two equal lengths for vein grafts, and infected with AdCMVeNOS or AdCMV in vitro. One segment (2 cm) of each carotid artery was removed. The vein graft that had been infected with AdCMVeNOS was anastomosed to the right carotid artery, and the vein graft that had been infected with AdCMV was anastomosed to the left. The functional expression of eNOS in vein grafts was assessed by the immunohistochemical staining and measurement of NO concentration. The inhibition of intimal hyperplasia in vein grafts was evaluated by the assay of 3H-TDR incorporation, histologic analysis, measurement of intimal thickness, and percent area stenosis. Adenovirus mediated eNOS gene transfer to goat vein grafts resulted in functional transgene expression with increased NO release. Increased local NO production could inhibit intimal hyperplasia and increase the patent rate of vein grafts.