Edith Tzeng

Carbon monoxide suppresses arteriosclerotic lesions associated with chronic graft rejection and with balloon injury

Carbon monoxide (CO), one of the products of heme oxygenase action on heme, prevents arteriosclerotic lesions that occur following aorta transplantation; pre-exposure to 250 parts per million of CO for 1 hour before injury suppresses stenosis after carotid balloon injury in rats as well as in mice. The protective effect of CO is associated with a profound inhibition of graft leukocyte infiltration/activation as well as with inhibition of smooth muscle cell proliferation. The anti-proliferative effect of CO in vitro requires the activation of guanylate cyclase, the generation of cGMP, the activation of p38 mitogen-activated protein kinases and the expression of the cell cycle inhibitor p21Cip1. These findings demonstrate a protective role for CO in vascular injury and support its use as a therapeutic agent.

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.

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.

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.

Does hostile neck anatomy preclude successful endovascular aortic aneurysm repair

OBJECTIVES Poor outcomes have been reported with endovascular aneurysm repair (EVAR) in patients with hostile neck anatomy. Unsupported endografts with active fixation may offer certain advantages in this situation. We compared EVAR results using the Ancure (Guidant) endograft in patients with and without hostile neck anatomy. METHODS Records of EVAR patients from October 1999 to July 2002 at a tertiary care hospital were retrospectively reviewed from a division database. Patients with elective open abdominal aortic aneurysm (AAA) repair during the same period were reviewed to determine those unsuitable for EVAR. Hostile neck anatomy, assessed by computer tomography (CT) scans and angiograms, was defined as one or more of the following: (1) neck length </=10 mm, (2) focal bulge in the neck >3 mm, (3) >2-mm reverse taper within 1 cm below the renal arteries, (4) neck thrombus > or =50% of circumference, and (5) angulation > or =60 degrees within 3 cm below renals. RESULTS Three hundred and twenty-two patients underwent EVAR with an average follow-up of 18 months. Patients in Phase II trials (n = 41), repaired with other graft types (n = 48), or without complete anatomic records (n = 27) were excluded. Demographics and co-morbidities were similar in the 115 good-neck (GN) and 91 bad-neck (BN) patients except for age (mean, 72.9 years GN vs 75.7 BN; P = 0.13), gender (11% female GN vs 22% BN; P =.04); neck length (mean, 21.8 mm GN vs 14.4 mm BN: P <.001), and angulation (mean, 22 degrees GN vs 40 degrees BN; (P <.001). Perioperative mortality (0 GN vs 1.1% BN), late mortality (5.2% GN vs 4.4% BN), all endoleaks (19.1% GN vs 17.6% BN), proximal endoleaks (0.8% GN vs 2.1% BN), and graft migration (0 for both groups) did not reach statistical significance. Neck anatomy precluded EVAR in 106 of 165 (64%) patients with open AAA. CONCLUSIONS Unsupported endografts with active fixation can yield excellent results in treating many medically compromised patients with hostile neck anatomy. Nonetheless, an unsuitable neck remains the most frequent cause for open abdominal AAA.

Inducible nitric oxide synthase and vascular injury.

The role nitric oxide (NO) plays in the cardiovascular system is complex and diverse. Even more controversial is the role that the inducible NO synthase enzyme (iNOS) serves in mediating different aspects of cardiovascular pathophysiology. Following arterial injury, NO has been shown to serve many vasoprotective roles, including inhibition of platelet aggregation and adherence to the site of injury, inhibition of leukocyte adherence, inhibition of vascular smooth muscle cell (VSMC) proliferation and migration, and stimulation of endothelial cell (EC) growth. These properties function together to preserve a normal vascular environment following injury. In this review, we discuss what is known about the involvement of iNOS in the vascular injury response. Additionally, we discuss the beneficial role of iNOS gene transfer to the vasculature in preventing the development of neointimal thickening. Lastly, the pathophysiology of transplant vasculopathy is discussed as well as the role of iNOS in this setting.

Nitrite-generated NO circumvents dysregulated arginine/NOS signaling to protect against intimal hyperplasia in Sprague-Dawley rats

Vascular disease, a significant cause of morbidity and mortality in the developed world, results from vascular injury. Following vascular injury, damaged or dysfunctional endothelial cells and activated SMCs engage in vasoproliferative remodeling and the formation of flow-limiting intimal hyperplasia (IH). We hypothesized that vascular injury results in decreased bioavailability of NO secondary to dysregulated arginine-dependent NO generation. Furthermore, we postulated that nitrite-dependent NO generation is augmented as an adaptive response to limit vascular injury/proliferation and can be harnessed for its protective effects. Here we report that sodium nitrite (intraperitoneal, inhaled, or oral) limited the development of IH in a rat model of vascular injury. Additionally, nitrite led to the generation of NO in vessels and SMCs, as well as limited SMC proliferation via p21Waf1/Cip1 signaling. These data demonstrate that IH is associated with increased arginase-1 levels, which leads to decreased NO production and bioavailability. Vascular injury also was associated with increased levels of xanthine oxidoreductase (XOR), a known nitrite reductase. Chronic inhibition of XOR and a diet deficient in nitrate/nitrite each exacerbated vascular injury. Moreover, established IH was reversed by dietary supplementation of nitrite. The vasoprotective effects of nitrite were counteracted by inhibition of XOR. These data illustrate the importance of nitrite-generated NO as an endogenous adaptive response and as a pathway that can be harnessed for therapeutic benefit.

Interim analysis results from the RESTORE-CLI, a randomized, double-blind multicenter phase II trial comparing expanded autologous bone marrow-derived tissue repair cells and placebo in patients with critical limb ischemia

OBJECTIVES Cell therapy is a novel experimental treatment modality for patients with critical limb ischemia (CLI) of the lower extremities and no other established treatment options. This study was conducted to assess the safety and clinical efficacy of intramuscular injection of autologous tissue repair cells (TRCs). METHODS A prospective, randomized double-blinded, placebo controlled, multicenter study (RESTORE-CLI) was conducted at 18 centers in the United States in patients with CLI and no option for revascularization. Enrollment of 86 patients began in April 2007 and ended in February 2010. For the prospectively planned interim analysis, conducted in February 2010, 33 patients had the opportunity to complete the trial (12 months of follow-up), and 46 patients had completed at least 6 months of follow-up. The interim analysis included analysis of both patient populations. An independent physician performed the bone marrow or sham control aspiration. The aspirate was processed in a closed, automated cell manufacturing system for approximately 12 days to generate the TRC population of stem and progenitor cells. An average of 136 ± 41 × 10(6) total viable cells or electrolyte (control) solution were injected into 20 sites in the ischemic lower extremity. The primary end point was safety as evaluated by adverse events, and serious adverse events as assessed at multiple follow-up time points. Clinical efficacy end points included major amputation-free survival and time to first occurrence of treatment failure (defined as any of the following: major amputation, death, de novo gangrene, or doubling of wound size), as well as major amputation rate and measures of wound healing. RESULTS There was no difference in adverse or serious adverse events between the two groups. Statistical analysis revealed a significant increase in time to treatment failure (log-rank test, P = .0053) and amputation-free survival in patients receiving TRC treatment, (log-rank test, P = .038). Major amputation occurred in 19% of TRC-treated patients compared to 43% of controls (P = .14, Fisher exact test). There was evidence of improved wound healing in the TRC-treated patients when compared with controls at 12 months. CONCLUSIONS Intramuscular injection of autologous bone marrow-derived TRCs is safe and decreases the occurrence of clinical events associated with disease progression when compared to placebo in patients with lower extremity CLI and no revascularization options.

Nitric oxide inhibits lipopolysaccharide-induced apoptosis in pulmonary artery endothelial cells

Our group recently reported that cultured sheep pulmonary artery endothelial cells (SPAECs) became resistant to lipopolysaccharide (LPS)-induced apoptosis several days after constitutive synthesis of nitric oxide (NO) after adenoviral (Ad) transfer of inducible NO synthase (iNOS) or exposure to the NO donor S-nitroso-N-acetylpenicillamine (SNAP) (E. Tzeng, Y.-M. Kim, B. R. Pitt, A. Lizonova, I. Kovesdi, and T. R. Billiar. Surgery 122: 255-263, 1997). In the present study, we confirmed this observation by establishing stable transfectants after retroviral gene transfer [replication-deficient retrovirus (DFG)] of human iNOS (DFG-iNOS) SPAECs and then used all three approaches (Ad, DFG, and SNAP) to determine underlying mechanisms of this phenomenon. Continuous endogenous production of NO in itself did not cause apoptosis as assessed by phase-contrast microscopy, nuclear morphology, and internucleosomal DNA fragmentation. Prolonged (72-96 h) synthesis of NO, however, after DFG- or replication-deficient adenovirus (Ad. CMV)-iNOS or SNAP (100 microM, 96 h) inhibited LPS-induced apoptosis. The kinetics of such protection suggested that NO may be inducing other gene products. Ad-mediated transfer of manganese superoxide dismutase (MnSOD) decreased the sensitivity of wild-type SPAECs to LPS-induced apoptosis. MnSOD, however, was not induced in an NG-monomethyl-L-arginine (L-NMMA)-sensitive time-dependent fashion after Ad.CMV-iNOS. Other inducible genes that may be affected by NO and that may protect against potential oxidant-mediated LPS-induced apoptosis including 70-kDa heat shock protein, heme oxygenase-1, metallothionein, and Bcl-2 also were not elevated in an L-NMMA-sensitive, time-dependent fashion. Although the candidate gene product underlying NO-induced protection remains unclear, we did note that prolonged synthesis of NO inhibited LPS-induced activation of an interleukin-1beta-converting enzyme-like cysteine protease (cysteine protease protein-32-like) in a dithiothreitol-sensitive fashion, suggesting that S-nitrosylation of an important downstream target of convergence of apoptotic signals may contribute to the sensitivity of SPAECs to LPS.Our group recently reported that cultured sheep pulmonary artery endothelial cells (SPAECs) became resistant to lipopolysaccharide (LPS)-induced apoptosis several days after constitutive synthesis of nitric oxide (NO) after adenoviral (Ad) transfer of inducible NO synthase (iNOS) or exposure to the NO donor S-nitroso- N-acetylpenicillamine (SNAP) (E. Tzeng, Y.-M. Kim, B. R. Pitt, A. Lizonova, I. Kovesdi, and T. R. Billiar. Surgery 122: 255-263, 1997). In the present study, we confirmed this observation by establishing stable transfectants after retroviral gene transfer [replication-deficient retrovirus (DFG)] of human iNOS (DFG-iNOS) SPAECs and then used all three approaches (Ad, DFG, and SNAP) to determine underlying mechanisms of this phenomenon. Continuous endogenous production of NO in itself did not cause apoptosis as assessed by phase-contrast microscopy, nuclear morphology, and internucleosomal DNA fragmentation. Prolonged (72-96 h) synthesis of NO, however, after DFG- or replication-deficient adenovirus (Ad.CMV)-iNOS or SNAP (100 μM, 96 h) inhibited LPS-induced apoptosis. The kinetics of such protection suggested that NO may be inducing other gene products. Ad-mediated transfer of manganese superoxide dismutase (MnSOD) decreased the sensitivity of wild-type SPAECs to LPS-induced apoptosis. MnSOD, however, was not induced in an N G-monomethyl-l-arginine (l-NMMA)-sensitive time-dependent fashion after Ad.CMV-iNOS. Other inducible genes that may be affected by NO and that may protect against potential oxidant-mediated LPS-induced apoptosis including 70-kDa heat shock protein, heme oxygenase-1, metallothionein, and Bcl-2 also were not elevated in an l-NMMA-sensitive, time-dependent fashion. Although the candidate gene product underlying NO-induced protection remains unclear, we did note that prolonged synthesis of NO inhibited LPS-induced activation of an interleukin-1β-converting enzyme-like cysteine protease (cysteine protease protein-32-like) in a dithiothreitol-sensitive fashion, suggesting that S-nitrosylation of an important downstream target of convergence of apoptotic signals may contribute to the sensitivity of SPAECs to LPS.