Abstract
Tetrahydrobiopterin (BH4), 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 BH4, 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 BH4 (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 BH4 levels by 3-fold without changing protein levels of GTP cyclohydrolase, the enzyme that regulates de novo BH4 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-α and interferon-γ 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.
Footnotes
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This work was supported by the National Institutes of Health National Heart, Lung, and Blood Institute [Grants HL078937, HL067244] (to G.M.P. and J.V.V., respectively).
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.108.148569.
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ABBREVIATIONS: iNOS, inducible nitric-oxide synthase; BH4, tetrahydrobiopterin; GTPCH, GTP cyclohydrolase 1; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; 4-HNE, 4-hydroxy-2-nonenal; LV, left ventricular; LVIDd, left ventricular internal diameter in diastole; LVIDs, left ventricular internal diameter in systole; FS, fractional shortening; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PCR, polymerase chain reaction; RT, reverse transcription; BH2, dihydrobiopterin; HPLC, high-performance liquid chromatography; NOx, total sum of nitrite + nitrate; TNF, tumor necrosis factor; IFN, interferon; IL, interleukin.
- Received November 17, 2008.
- Accepted March 18, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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