Antioxidant N-acetyl cysteine reverses cigarette smoke-induced myocardial infarction by inhibiting inflammation and oxidative stress in a rat model.

The contribution of chronic tobacco exposure in determining post-myocardial infarction (MI) left ventricular (LV) remodeling and possible therapeutic strategies has not been investigated systematically. In this small animal investigation, we demonstrate that chronic tobacco smoke exposure leading up to acute MI in rats is associated with greater histological extent of myocardial necrosis and consequent worse LV function. These findings are associated with increased transcriptomic expression of pro-inflammatory cytokines, tissue repair molecules and markers of oxidative stress in the myocardium. The results demonstrate that an N-acetyl cysteine (NAC) treatment significantly reduced tobacco-exposed induced infarct size and percent fractional shortening. A significantly increased LV end-systolic diameter was observed in tobacco-exposed sham compared to tobacco-naïve sham (4.92±0.41 vs 3.45±0.33; P<0.05), and tobacco-exposed MI compared to tobacco-naïve MI (8.24±0.3 vs 6.1±0.49; P<0.01) rats. Decreased intracardiac mRNA expression of the markers of inflammation, tissue repair and oxidative stress and circulating levels of pro-inflammatory cytokines accompanied these positive effects of NAC. The treatment of tobacco-exposed MI rats with NAC resulted in significantly increased levels of intracardiac mRNA expression of antioxidants, including superoxide dismutase, thioredoxin and nuclear factor-E2-related factor 2, as well as circulating levels of glutathione (7±0.12 vs 10±0.18; P≤0.001), where the levels were almost identical to the tobacco-naïve sham rats. These findings identify a novel post-infarction therapy for amelioration of the adverse effects of tobacco exposure on the infracted myocardium and advocate the use of dietary supplement antioxidants for habitual smokers to prevent and reverse cardiovascular adverse effects in the absence of successful achievement of cessation of smoking.

Cardiac myocyte-specific overexpression of human GTP cyclohydrolase I protects against acute cardiac allograft rejection.

GTP cyclohydrolase I (GTPCH) is the rate-limiting enzyme for tetrahydrobiopterin (BH(4)) synthesis. Decreases in GTPCH activity and expression have been shown in late stages of acute cardiac rejection, suggesting a deficit in BH(4). We hypothesized that increasing intracellular levels of BH(4) by cardiac myocyte-targeted overexpression of GTPCH would diminish acute cardiac allograft rejection. Transgenic mice overexpressing GTPCH in the heart were generated and crossed on C57BL6 background. Wild-type and transgenic mouse donor hearts were transplanted into BALB/c recipient mice. Left ventricular (LV) function, histological rejection, BH(4) levels, and inflammatory cytokine gene expression (mRNA) were examined. Expression of human GTPCH was documented by PCR, Western analysis, and function by a significant (P < 0.001) increase in cardiac BH(4) levels. GTPCH transgene decreased histological rejection (46%; P < 0.003) and cardiac myocyte injury (eosin autofluorescence; 56%; P < 0.0001) independent of changes in inflammatory cytokine expression or nitric oxide content. GTPCH transgene decreased IL-2 (88%; P < 0.002), IL-1R2 (42%; P < 0.0001), and programmed cell death-1 (67%; P < 0.0001) expression, whereas it increased fms-like tyrosine kinase 3 (156%; P < 0.0001) and stromal-derived factor-1 (2; 190%; P < 0.0001) expression. There was no difference in ejection fraction or fractional shortening; however, LV mass was significantly increased (P < 0.05) only in wild-type grafts. The decreases in LV mass, cardiac injury, and histological rejection support a protective role of cardiac GTPCH overexpression and increased BH(4) synthesis in cardiac allografts. The mechanism of the decreased rejection appears related to decreased T cell proliferation and modulation of immune function by higher expression of genes involved in hematopoietic/stromal cell development and recruitment.

Tobacco smoke exposure in either the donor or recipient before transplantation accelerates cardiac allograft rejection, vascular inflammation, and graft loss.

BACKGROUND: Tobacco exposure in cardiac transplant recipients, before and after transplantation, may increase the risk of cardiac allograft vasculopathy and allograft loss, but no direct evidence for this phenomenon is forthcoming. In this experimental study, we investigated early consequences of tobacco smoke exposure in cardiac transplant donors and recipients with an emphasis on alloinflammatory mediators of graft outcome. METHODS AND RESULTS: Using heterotopic rat cardiac transplantation, we tested the effects of donor or recipient tobacco smoke exposure in 6 groups of animals (rat heterotopic cardiac transplantation) as follows: tobacco-naïve allogeneic rejecting controls (n=6), tobacco-naïve nonrejecting controls (n=3; killed on day 5 to simulate survival times of tobacco-treated animals), isografts (n=3), both donor and recipient rats exposed to tobacco smoke (n=4), only donor rats exposed to tobacco smoke (n=7), and only recipient rats exposed to tobacco smoke (n=6). Polymerase chain reaction studies of tissue and peripheral (systemic) protein expression were performed to evaluate inflammatory (tumor necrosis factor-alpha, interferon-gamma, interleukin-6) and alloimmune (interleukin-1 receptor 2, programmed cell death-1, and stromal cell-derived factor-1) pathways, as was histological analysis of the cardiac allografts. Our experiments reveal that pretransplantation tobacco exposure in donors and/or recipients results in heightened systemic inflammation and increased oxidative stress, reduces posttransplantation cardiac allograft survival by 33% to 57%, and increases intragraft inflammation (tumor necrosis factor-alpha, interferon-gamma, interleukin-6) and alloimmune activation (CD3, interleukin-1 receptor 2, programmed cell death-1, and stromal cell-derived factor-1) with consequent myocardial and vascular destruction. CONCLUSIONS: These sentinel findings confirm that tobacco smoke exposure in either donors or recipients leads to accelerated allograft rejection, vascular inflammation, and graft loss. Molecular pathways that intersect as arbiters in this phenomenon include instigation of alloimmune activation associated with tobacco smoke-induced inflammation.

Enhanced susceptibility of cyclin kinase inhibitor p21 knockout mice to high fat diet induced atherosclerosis.

Cyclin kinase inhibitor p21 is one of the most potent inhibitors of aortic smooth muscle cell proliferation, a key mediator of atherosclerosis. This study tests if p2l deficiency will result in severe atherosclerosis in a mouse model. p21-/- and strain matched wild type mice were fed with high fat diet for 21 weeks. Analysis for biochemical parameters (cholesterol, triglycerides) in serum and mRNA expression of CD36, HO-1, TGF-beta, IFN-gamma, TNF-alpha, PPAR-gamma and NADPH oxidase components (p22phox, NOX-1 and Rac-1) was performed in aortic tissues by Real Time PCR. p21-/- mice gained significantly (p < 0.01) more weight than wild type mice, triglycerides (p < 0.05) and cholesterol levels (p < 0.01) were more pronounced in the sera of p21-/- compared to wild type mice fed with high fat diet. High fat diet resulted in significantly decreased TGF-beta (p < 0.02), HO-l (p < 0.02) and increased CD36 (p < 0.03) mRNA expression in aortic tissues of p21-/- mice compared to animal fed with regular diet. IFN-gamma mRNA expression (235 +/- 11 folds) increased significantly in high fat diet fed p21-/- mice and a multifold modulation of PPAR-gamma(136 +/- 7), p22phox, NOX-1 and Rac-1 (15-35-folds) mRNA in aortic tissues from p21-/- mice compared to the wild type mice. Severity of atherosclerotic lesions was significantly higher in p21-/- compared to wild type mice. The results demonstrate that the deficiency of p21 leads to altered expression of pro-atherogenic genes, and severe atherosclerosis in mice fed with high fat diet. This opens the possibility of p21 protein as a therapeutic tool to control progression of atherosclerosis.

Adverse effects of nicotine and immunosuppression on proximal tubular epithelial cell viability, tissue repair and oxidative stress gene expression.

BACKGROUND: Renal dysfunction in non-renal transplantation is a major arbiter of poor late allograft outcomes. Tobacco recidivism is an important modifiable risk marker for cardiac allograft loss, but its effects on renal dysfunction remain poorly studied. METHODS: In a 96-well plate, 10(-5) proximal tubular epithelial (PTE) cells (HK-2, American Type Culture Collection) were cultured overnight and treated with sirolimus (SRL; 100 nmol/liter), nicotine (N; 10(-7) mol/liter) and mycophenolate mofetil (MMF; 10 micromol/liter), alone or in combination for 24 hours. Cell viability was quantified by treatment with tetrazolium salt WST-1 and calculated as the difference in percent inhibition with respect to the optical densitometry (OD) of treated and untreated cells. Gene and protein expression was analyzed using real-time polymerase chain reaction and Western blot techniques. RESULTS: OD decreased with SRL (-52.7 +/- 2.85%), N (-47.3 +/- 3.84%) and MMF (-53.3 +/- 2.4%) in isolation. Further reduction in OD occurred when N was combined with SRL (-63 +/- 2.3%, p < 0.04), MMF (-64.3 +/- 1.45%, p < 0.02) or the combination of SRL and MMF (-78.2%, p < 0.007). Compared with control, treatment of PTE cells with N increased mRNA expression of transforming growth factor-beta (TGF-beta; 10-fold), connective tissue growth factor (CTGF; 25-fold), osteopontin (OPN; 10-fold) and NADPH oxidase components (p22(phox), NOX-1 and Rac-1 at 18-, 16- and 12-fold, respectively). The pre-treatment of cells with inhibitor of superoxide generator diphenylene iodonium (DPI) reversed these effects. CONCLUSIONS: Nicotine adversely amplified the effects of SRL and MMF on tissue repair and oxidative stress markers, subsequently modulating PTE viability. However, caution is advised in extrapolating these in vitro findings to the human model.

Sepiapterin decreases acute rejection and apoptosis in cardiac transplants independently of changes in nitric oxide and inducible nitric-oxide synthase dimerization.

Tetrahydrobiopterin (BH(4)), a cofactor of inducible nitric-oxide synthase (iNOS), is an important post-translational regulator of NO bioactivity. We examined whether treatment of cardiac allograft recipients with sepiapterin [S-(-)-2-amino-7,8-dihydro-6-(2-hydroxy-1-oxopropyl)-4-(1H)-pteridinone], a precursor of BH(4), inhibited acute rejection and apoptosis in cardiac transplants. Heterotopic cardiac transplantation was performed in Wistar-Furth donor to Lewis recipient strain rats. Recipients were treated daily after transplantation with 10 mg/kg sepiapterin. Grafts were harvested on post-transplant day 6 for analysis of BH(4) (high-performance liquid chromatography), expression of inflammatory cytokines (reverse transcription- and real-time polymerase chain reaction), iNOS (Western blots), and NO (Griess reaction and NO analyzer). Histological rejection grade was scored, and graft function was determined by echocardiography. Apoptosis, protein nitration, and oxidative stress were determined by immunohistochemistry. Treatment of allografts with sepiapterin increased cardiac BH(4) levels by 3-fold without changing protein levels of GTP cyclohydrolase, the enzyme that regulates de novo BH(4) synthesis. Sepiapterin decreased inflammatory cell infiltrate and significantly inhibited histological rejection scores and apoptosis similar in magnitude to cyclosporine. Sepiapterin also decreased nitrative and oxidative stress. Sepiapterin caused a smaller increase in left ventricular mass versus untreated allografts but without improving fractional shortening. Sepiapterin did not alter tumor necrosis factor-alpha and interferon-gamma expression, whereas it decreased interleukin (IL)-2 expression. Sepiapterin did not change total iNOS protein or monomer levels, or plasma and tissue NO metabolites levels. It is concluded that the mechanism(s) of antirejection are due in part to decreased apoptosis, protein nitration, and oxidation of cardiomyocytes, which seems to be mediated at the immune level by limiting inflammatory cell infiltration via decreased IL-2-mediated T-lymphocyte expansion.

NADPH oxidase subunits (NOX-1, p22phox, Rac-1) and tacrolimus-induced nephrotoxicity in a rat renal transplant model.

BACKGROUND: TGF-beta and oxidative stress are known mediators of renal injury. However, the precise mechanisms by which TGF-beta and oxidative stress may be involved in the development of nephrotoxicity are not known. We examined whether anti-TGF-beta antibody limits nephrotoxicity produced by tacrolimus (TAC) and whether this altered genes that regulate oxidative stress. METHODS: Renal transplants were performed in Wistar-Furth and Lewis rat strains. Groups included: isograft controls; untreated allografts; allografts treated with 0.25 mg/kg TAC till 90 days with or without 1.0 mg/kg anti-TGF-beta antibody or control antibody. Serum creatinine and BUN levels and renal histology were determined. Real time PCR and western analysis were used to quantify mRNA and protein expression. RESULTS: BUN and creatinine were elevated in TAC-treated rats. TAC increased expression of TGF-beta (37-fold) and NADPH oxidase subunits, NOX-1 (18-fold), p22(phox) (31-fold) and Rac-1 mRNA (20-fold), respectively. Contrariwise, expression of antioxidant genes, superoxide dismutase (SOD) and thioredoxin (TRX) was decreased. Anti-TGF-beta antibody but not control antibody reversed the TAC-induced changes in gene expression, renal histology and function. CONCLUSIONS: Our findings suggest a potential for anti-TGF-beta antibody as a novel adjunct therapeutic tool to prevent TAC-induced nephrotoxicity in transplant recipients. The mechanism of protection involves suppression of TGF-beta and the expression of genes that regulate oxidative stress. Moreover, the specific up-regulation of NOX-1, a non-phagocytic NADPH oxidase subunit and its reversal by anti-TGF-beta antibody strongly implicates for the first time the up-regulation of renal parenchymal cell NADPH oxidase in the aetiology of immunosuppression-induced nephrotoxicity.

Targeted in vitro and in vivo gene transfer into T lymphocytes: potential of direct inhibition of allo-immune activation.

BACKGROUND: Successful inhibition of alloimmune activation in organ transplantation remains one of the key events in achieving a long-term graft survival. Since T lymphocytes are largely responsible for alloimmune activation, targeted gene transfer of gene of cyclin kinase inhibitor p21 into T cells might inhibit their aberrant proliferation. A number of strategies using either adenoviral or lentiviral vectors linked to mono or bispecific antibodies directed against T cell surface markers/cytokines did not yield the desired results. Therefore, this study was designed to test if a CD3promoter-p21 chimeric construct would in vitro and in vivo transfer p21 gene to T lymphocytes and result in inhibition of proliferation. CD3 promoter-p21 chimeric constructs were prepared with p21 in the sense and antisense orientation. For in vitro studies EL4-IL-2 thyoma cells were used and for in vivo studies CD3p21 sense and antisense plasmid DNA was injected intramuscularly in mice. Lymphocyte proliferation was quantified by 3H-thymidine uptake assay; IL-2 mRNA expression was studied by RT-PCR and using Real Time PCR assay, we monitored the CD3, p21, TNF-alpha and IFN-gamma mRNA expression. RESULTS: Transfection of CD3p21 sense and antisense in mouse thyoma cell line (EL4-IL-2) resulted in modulation of mitogen-induced proliferation. The intramuscular injection of CD3p21 sense and antisense plasmid DNA into mice also modulated lymphocyte proliferation and mRNA expression of pro-inflammatory cytokines. CONCLUSION: These results demonstrate a novel strategy of in vitro and in vivo transfer of p21 gene to T cells using CD3-promoter to achieve targeted inhibition of lymphocyte proliferation and immune activation.

Beneficial effect of alpha-tocopheryl succinate in rat cardiac transplants.

BACKGROUND: Anti-oxidant vitamins have increasingly been used to supplement traditional post-surgical treatment in cardiac transplant recipients. However, the mechanism(s) of action have not been determined. In this study we examined the effects of a novel vitamin E analog, alpha-tocopheryl polyethylene glycol-100 succinate (alpha-TPGS), and low-dose cyclosporine (CsA) in the treatment of acute and delayed cardiac rejection. METHODS: In situ sonomicrometry, histologic rejection and graft survival were determined in untreated rat cardiac allograft recipients and recipients receiving CsA, alpha-TPGS or CsA plus alpha-TPGS. DNA binding of nuclear factor (NF)-kappaB and AP-1, inducible nitric oxide synthase (iNOS) protein, caspase-3 activity and lymphocyte proliferation were determined. RESULTS: alpha-TPGS significantly (p < 0.05) prolonged graft survival equipotent to low-dose CsA. Treatment with CsA plus alpha-TPGS further enhanced graft survival (p < 0.001). CsA or alpha-TPGS alone decreased rejection, with the greatest decrease seen using combination therapy. Graft fractional shortening was improved by CsA or alpha-TPGS alone (p < 0.01), whereas distention in systolic and diastolic lengths in untreated allografts was prevented by CsA, alpha-TPGS and combination therapy. Nitrosylation of heme protein was inhibited by alpha-TPGS and abolished by CsA or CsA plus alpha-TPGS. Expression of iNOS was decreased 50% by alpha-TPGS equipotent to CsA, but apparently via an NF-kappaB- and AP-1-independent pathway. Caspase-3 activity, an index of apoptosis, was increased only in untreated allografts. In addition, alpha-TPGS markedly inhibited mitogen-stimulated proliferation by both rat and human lymphocytes. CONCLUSIONS: alpha-TPGS has a significant effect in limiting lymphocyte proliferation and activation. This might explain the equipotent action of alpha-TPGS vs low-dose CsA and its action to potentiate graft survival and limit graft rejection and dysfunction.

Antagonizing reactive oxygen by treatment with a manganese (III) metalloporphyrin-based superoxide dismutase mimetic in cardiac transplants.

OBJECTIVE: Oxidative stress might be an important factor contributing to injury during alloimmune activation. Herein, we evaluated the efficacy of a superoxide dismutase mimetic, manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride (MnTmPyP), on cytokine gene expression and apoptotic signaling in a rat model of cardiac transplantation. METHODS: Lewis-->Lewis (isografts) or Wistar-Furth-->Lewis (allografts) heterotopic rat transplants without and with treatment with MnTmPyP were used. Reactive oxygen formation was determined on the basis of dihydroethidine fluorescence and lucigenin-enhanced chemiluminescence. In situ graft function was determined by means of sonomicrometry. Inflammatory cytokine, proapoptotic, and antiapoptotic gene expression at either postoperative day 4 (early rejection) or postoperative day 6 (late rejection) was determined by means of reverse transcriptase polymerase chain reaction. RESULTS: An increased production of reactive oxygen in allografts was inhibited to isograft control levels by MnTmPyP. MnTmPyP restored either the percentage of fractional shortening, the distended diastolic and systolic myocardial segment lengths, or both in allografts. Of the increases in cytokine and proapoptotic gene expression in allografts, only interleukin 6 was decreased by MnTmPyP. MnTmPyP inhibited antiapoptotic gene expression (Bcl-2 and Bcl-xL) during early rejection but restored expression at later stages. The increase in activated caspase-3 levels in allografts was inhibited by MnTmPyP. CONCLUSIONS: The mechanism of the beneficial effect of MnTmPyP on graft function appear related, in part, to scavenging O2*- and by decreasing apoptotic signaling rather than an effect on inflammatory cytokine gene expression.

Post-translational modification of manganese superoxide dismutase in acutely rejecting cardiac transplants: role of inducible nitric oxide synthase.

BACKGROUND: Nitration of a critical tyrosine residue in the active site of manganese superoxide dismutase (MnSOD) can lead to enzyme inactivation. In this study, we examined the effect of inducible nitric oxide synthase (iNOS) on MnSOD expression, activity and nitration in acutely rejecting cardiac transplants. METHODS: Lewis (isograft) or Wistar-Furth (allograft) donor hearts were transplanted into Lewis recipient rats. Some rats received L-N6-(1-iminoethyl) lysine (l-NIL), a specific iNOS inhibitor. Protein nitration was determined by immunohistochemical, Western blot and slot-blot analyses. MnSOD enzyme activity and gene expression were determined using Western, reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoprecipitation techniques. RESULTS: MnSOD protein levels were decreased 50% by post-operative day 6 (POD 6), which was prevented by L-NIL. RT-PCR analysis indicated that this decrease could not be explained by any changes in MnSOD mRNA. MnSOD enzyme activity but not protein was decreased at POD 5 in untreated allografts. The loss of MnSOD activity at POD 5 was also prevented by L-NIL. Immunoreactive nitrotyrosine was apparent in untreated allografts at POD 6. Slot-blot analysis indicated that nitrotyrosine formation in allografts could be blocked by L-NIL. Nitration of MnSOD was evident upon immunoprecipitation of MnSOD followed by Western blotting for nitrotyrosine. CONCLUSIONS: These results suggest that the decreased MnSOD enzyme activity in acutely rejecting cardiac allografts can be attributed to a post-translational modification related to nitration arising via an iNOS-dependent pathway. This could be a potential major source of amplified oxidative stress in acute graft rejection.

Reciprocal role of cyclins and cyclin kinase inhibitor p21WAF1/CIP1 on lymphocyte proliferation, allo-immune activation and inflammation.

BACKGROUND: Immune activation that results due to the aberrant proliferation of lymphocytes leads to inflammation and graft rejection in organ transplant recipients. We hypothesize that the cell cycle control and inflammation are parallel events, inhibition of cellular proliferation by cyclin kinase inhibitor specifically p21 will limit inflammation and prevent allograft rejection. METHODS: We performed in vitro and in vivo studies using lymphocytes, and rat heart transplant model to understand the role of cyclins and p21 on mitogen and allo-induced lymphocyte activation and inflammation. Lymphocyte proliferation was studied by 3H-thymidine uptake assay and mRNA expression was studied RT-PCR. RESULTS: Activation of allo- and mitogen stimulated lymphocytes resulted in increased expression of cyclins, IL-2 and pro-inflammatory cytokines, which was inhibited by cyclosporine. The over-expression of p21 prolonged graft survival in a completely mismatched rat heart transplant model resulted by inhibiting circulating and intra-graft expression of proinflammatory cytokines. CONCLUSION: Cyclins play a significant role in transplant-induced immune activation and p21 over-expression has potential to inhibit T cell activation and inflammation. The results from this study will permit the design of alternate strategies by controlling cell cycle progression to achieve immunosuppression in transplantation.

Nitric oxide formation in acutely rejecting cardiac allografts correlates with GTP cyclohydrolase I activity.

Inducible nitric oxide synthase (iNOS) is a prominent component of the complex array of mediators in acute graft rejection. While NO production is determined by iNOS expression, BH4 (tetrahydrobiopterin), a cofactor of iNOS synthesized by GTP cyclohydrolase I, has been considered critical in sustaining NO production. In the present study, we examined time-dependent changes in iNOS and GTP cyclohydrolase I in rat cardiac allografts. The increase in iNOS protein and mRNA in allografts was similar at POD4 (post-operative day 4) and POD6. However, the peak increase in intragraft NO level at POD4 was not sustained at POD6. This disparity could not be explained by any decrease in iNOS enzyme activity measured ex vivo with optimal amounts of substrate and cofactors. Lower iNOS activity could be explained by changes in total biopterin levels in allografts at POD4 that was decreased to baseline at POD6. Changes in biopterin production correlated with lower GTP cyclohydrolase I protein levels but not by any change in GTP cyclohydrolase I mRNA. Functionally, allografts displayed bradycardia and distended diastolic and systolic dimensions at POD6 but not at POD4. Likewise, histological rejection scores were increased at POD4 but with a secondary increased stage at POD6. It is hypothesized that the dissimilar amounts of NO at early and later stages of rejection is due to uncoupling of iNOS arising from disproportionate synthesis of BH4. These findings provide insight into a potential pathway regulating NO bioactivity in graft rejection. Such knowledge may potentially assist in the design of newer strategies to prevent acute graft rejection.

Recombinant p21 protein inhibits lymphocyte proliferation and transcription factors.

Cellular proliferation determines the events leading to the initiation and development of inflammation, immune activation, cancer, atherogenesis, and other disorders associated with aberrant cell proliferation. Cyclin inhibitor p21 plays a unique role in limiting cell cycle progression. However, its effectiveness can only be demonstrated with direct in vitro and in vivo delivery to control aberrant proliferation. We demonstrate that using a protein-transducing domain p21 protein a) localizes within the nuclear compartments of cells, b) interacts with transcription factors, NF-kappaB, and NFATs (NFATc and NFATp), and c) inhibits lymphocyte proliferation and expression of proinflammatory cytokines. This study using lymphocyte proliferation as a model suggests that the recombinant p21 protein can directly be delivered as a therapeutic protein to provide a novel, viable, and powerful strategy to limit proliferation, inflammation, alloimmune activation, cancer, and vascular proliferative disorders such as atherosclerosis.

Protective mechanisms of a metalloporphyrinic peroxynitrite decomposition catalyst, WW85, in rat cardiac transplants.

Nitric oxide (NO) derived from inducible NO synthase has been implicated in cardiac rejection. However, little is known about the role of the reactive nitrogen species peroxynitrite. We examined the protective actions of a peroxynitrite decomposition catalyst, WW85, in an experimental model of acute cardiac rejection. Heterotopic, abdominal transplantation of rat donor hearts was performed. Groups included isografts, allografts, or allografts treated with WW85, cyclosporine, or cyclosporine + WW85. We determined graft survival, histological rejection, and graft function (by in situ sonomicrometry). Intragraft biochemical analysis of cytokines and proapoptotic and antiapoptotic gene expression using reverse transcriptase-polymerase chain reaction were determined. Treatment with WW85 or cyclosporine alone prolonged graft survival, improved graft function, and decreased histological rejection. Graft survival was further significantly (P < 0.001) enhanced by combination treatment. A decrease was also shown in nitrotyrosine, poly(ADP-ribose) polymerase (PARP) activation, and lipid peroxide formation by WW85 that was potentiated when given in combination with cyclosporine. Benefits could not be ascribed to changes in intragraft myeloperoxidase activity. Only combination therapy produced significant decreases in inflammatory cytokine gene expression, suggesting that WW85 acted primarily downstream of these stimuli. In general, WW85 had no direct action on expression of the proapoptotic gene, Fas ligand; however, WW85 given alone or with cyclosporine enhanced expression of antiapoptotic genes Bcl-2 and Bcl-xL. Collectively, these findings suggest a protective action of the peroxynitrite decomposition catalyst WW85 on graft rejection that is independent of any action on leukocyte sequestration and cytokine gene expression. Rather, effects seem to be downstream on decreased protein nitration, decreased lipid peroxidation, and decreased PARP activation.

Treatment with {alpha}-phenyl-N-tert-butylnitrone, a free radical-trapping agent, abrogates inflammatory cytokine gene expression during alloimmune activation in rat cardiac allografts.

Spin-trapping nitrones such as alpha-phenyl-N-tert-butylnitrone (PBN) have traditionally been used to trap and stabilize free radicals for detection by electron paramagnetic resonance (EPR) spectroscopy. Unlike classical antioxidants, these agents have never been evaluated therapeutically in allograft transplantation. In the present study, we examined potential mechanisms of action of treatment with PBN in a rat model of acute cardiac allograft transplantation. Graft rejection was determined by histological examination and graft function determined by in situ sonomicrometry. DNA binding for nuclear factor (NF)-kappaB and activator protein (AP-1) were determined by gel shift assays. Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was performed for inducible nitric-oxide synthase (iNOS) and inflammatory cytokines. Histological rejection scores were elevated in untreated allografts and decreased by treatment with PBN. In situ sonomicrometry revealed decreased heart rate and distended end diastolic and end systolic segment lengths with rejection. Although PBN did not alter heart rate, it did normalize the distention of both diastolic and systolic cardiac dimension. EPR spectroscopy revealed nitrosylation of myocardial heme protein in untreated allografts that was decreased by treatment with PBN. PBN also decreased iNOS protein and iNOS mRNA. RT-PCR analysis revealed enhanced cytokine gene expression for interferon-gamma, interleukin-6, and interleukin-10 in untreated allografts. Expression for these genes was potently inhibited or abolished in recipients treated with PBN. PBN treatment also decreased DNA binding of transcription factors, NF-kappaB and AP-1. Thus, PBN retains significant anti-inflammatory properties through its action to down-regulate cytokine gene expression that contribute to protection against acute alloimmune activation in cardiac allografts.

Anti-transforming growth factor antibody at low but not high doses limits cyclosporine-mediated nephrotoxicity without altering rat cardiac allograft survival: potential of therapeutic applications.

BACKGROUND: Long-term treatment of cardiac transplant recipients with cyclosporine results in a progressive decline in kidney function in a large number of patients. This complication is one of the most important prognostic parameters that determine the outcome of cardiac transplantation. Transforming growth factor-beta (TGF-beta) is one of the most potent mediators of the fibrogenic effects of cyclosporine. METHODS AND RESULTS: With the use of an experimental rodent model, heterotopic heart transplantation was performed, creating histocompatibility-disparate allografts. Because TGF-beta in part mediates both the immunosuppressive and nephrotoxic effects of cyclosporine, recipients were treated with cyclosporine with and without anti-TGF-beta antibody to determine whether anti-TGF-beta antibody could reduce the nephrotoxic effects of cyclosporine. Intrarenal expression of TGF-beta, collagen, fibronectin, matrix metalloproteinase-2, and tissue inhibitor of metalloproteinase-2 was studied with the use of reverse transcription-polymerase chain reaction. Intrarenal expression of TGF-beta protein was studied by immunohistochemistry and with the use of ELISA to quantify circulating levels of TGF-beta protein in plasma. Cyclosporine-induced graft survival (immunosuppressive effect) was abrogated with a higher concentration (2.5 mg/kg) of anti-TGF-beta antibody, whereas a lower concentration (1 mg/kg) inhibited both cyclosporine-induced expression of fibrogenic molecules and renal toxicity. CONCLUSIONS: These results provide credence to the pivotal role of TGF-beta in immunosuppression-associated renal toxicity in recipients of cardiac transplantation. Furthermore, these findings support a potentially significant therapeutic use of optimal concentration of anti-TGF-beta antibody to ameliorate cyclosporine-associated nephrotoxicity in cardiac transplant recipients.

Inhibition of nitrosylation, nitration, lymphocyte proliferation, and gene expression in acute and delayed cardiac allograft rejection by an orally active dithiocarbamate.

Dithiocarbamate derivatives sequester metals such as iron and may have benefits in inflammatory diseases. We examined the actions of a new dithiocarbamate-based oral formulation, NOX-700, on protein modification by nitric oxide (NO), gene expression, and lymphocyte proliferation in a model of acute and delayed cardiac rejection. Chronic treatment with NOX-700 prolonged graft survival. In combination with low-dose cyclosporine (CsA), NOX-700 produced a synergistic action to prolong graft survival. NOX-700 decreased myocardial heme nitrosylation. A single bolus injection with NOX-700 in untreated recipients did not decrease heme nitrosylation but normalized NO metabolites and caused the formation of a mononitrosyl iron complex indicating NO scavenging in vivo. NOX-700 alone given with CsA inhibited protein nitration. NOX-700 or CsA each alone decreased intragraft inflammatory cell infiltration. NOX-700 also potentiated the CsA-induced inhibition of splenocyte proliferation ex vivo stimulated by concanavalin A. In splenocytes derived from treated rats but stimulated ex vivo in a mixed lymphocyte response (MLR), interferon-gamma and cyclin D3 gene expression was inhibited by NOX-700 suggesting down-regulation of lymphocyte activation and proliferation by in vivo treatment. These studies suggest that NOX-700 is protective in cardiac rejection, in part, by scavenging of NO and by limiting lymphocyte activation infiltration.

TGF-beta1, FGF-2, and receptor mRNA expression in suture mesenchyme and dura versus underlying brain in fusing and nonfusing mouse cranial sutures.

Recent studies have supported a functional role for the transforming growth factor beta-1 (TGF-beta1) and fibro-blast growth factor 2 (FGF-2) signaling cascades in the process of mouse cranial suture fusion. TGF-beta1 and FGF-2 protein expression have been shown to be elevated in the fusing posterior frontal suture versus the nonfusing sagittal suture. The authors evaluated simultaneous mRNA expression of TGF-beta1 and its R1 receptor and FGF-2 and its R2 receptor during mouse cranial suture fusion. They evaluated the suture mesenchyme-dura complex separately from the underlying brain to determine whether there is tissue-specific biologic activity (i.e., brain versus suture mesenchyme-dura) for each cytokine and receptor. Data were collected from 150 male CD-1 mice studied over five time periods from postnatal days 22 to 45. They utilized reverse-transcriptase polymerase chain reaction as a means to detect TGF-beta1, TGF-beta receptor 1 (TGF-betaR1), FGF-2, and FGF receptor 2 (FGFR2) mRNA expression in mouse cranial tissues, beginning with the period of initiation of posterior frontal cranial suture fusion (postnatal day 22) and extending through completion of posterior frontal suture fusion (postnatal day 45). Expression of FGF-2 was significantly greater in posterior frontal suture mesenchyme and dura compared with sagittal suture mesenchyme and dura during the period of initiation of posterior frontal suture fusion, localizing this cytokine's expression to posterior frontal suture mesenchyme and dura during the process of cranial suture fusion. TGF-beta1 and FGFR2 mRNA expression was found to be up-regulated in posterior frontal suture mesenchyme and dura relative to the underlying brain tissue throughout the study period, whereas TGF-betaR1 and FGF-2 mRNA expression was significantly elevated relative to the underlying brain only at time points corresponding to the initiation of posterior frontal suture fusion (between postnatal days 22 and 31). These results indicate that there is tissue-specific mRNA expression of TGF-beta1, FGF-2, and their receptors between suture mesenchyme and dura and the underlying brain, which correlates with the period of posterior frontal suture fusion in the mouse model. Differences in gene expression between suture mesenchyme and dura relative to the underlying brain may be an important regulator of cranial suture biology. Understanding these differences may eventually help to identify possible targets and time windows by which to most effectively modulate cranial suture fusion.

P2Y purine receptor responses and expression in the pulmonary circulation of juvenile rabbits.

The purine nucleotide ATP mediates pulmonary vasodilation at birth by stimulation of P2Y purine receptors in the pulmonary circulation. The specific P2Y receptors in the pulmonary circulation and the segmental distribution of their responses remain unknown. We investigated the effects of purine nucleotides, ATP, ADP, and AMP, and pyrimidine nucleotides, UTP, UDP, and UMP, in juvenile rabbit pulmonary arteries for functional characterization of P2Y receptors. We also studied the expression of P2Y receptor subtypes in pulmonary arteries and the role of nitric oxide (NO), prostaglandins, and cytochrome P-450 metabolites in the response to ATP. In conduit size arteries, ATP, ADP, and AMP caused greater relaxation responses than UTP, UDP, and UMP. In resistance vessels, ATP and UTP caused comparable vasodilation. The response to ATP was attenuated by the P2Y antagonist cibacron blue, the NO synthase antagonist N(omega)-nitro-l-arginine methyl ester (l-NAME), and the cytochrome P-450 inhibitor 17-octadecynoic acid but not by the P2X antagonist alpha,beta-methylene ATP or the cyclooxygenase inhibitor indomethacin in conduit arteries. In the resistance vessels, l-NAME caused a more complete inhibition of the responses to ATP and UTP. Responses to AMP and UMP were NO and endothelium dependent, whereas responses to ADP and UDP were NO and endothelium independent in the conduit arteries. RT-PCR showed expression of P2Y(1), P2Y(2), and P2Y(4) receptors, but not P2Y(6) receptors, in lung parenchyma, pulmonary arteries, and pulmonary artery endothelial cells. These data suggest that distinct P2Y receptors mediate the vasodilator responses to purine and pyrimidine nucleotides in the juvenile rabbit pulmonary circulation. ATP appears to cause NO-mediated vasodilation predominantly through P2Y2 receptors on endothelium.