Elevation of serum big endothelin is associated with endothelial dysfunction in patients with chronic heart failure

The Journal of Heart and Lung Transplantation Volume 20, Number 2 fused for 2, 4, or 8 hours (n⫽4/group) then procured for analysis of bcl-2 mRNA (RTPCR) and protein (Western blot) expression, NF␬B activity (EMSA), and TNF-␣ production (ELISA). Results: Compared to nontransplanted PVG heart controls, bcl-2 mRNA and protein expression decreased over time, becoming significant at 8 hours of reperfusion (bcl-2:G3PDH densitometry ratio ⫽ 1.27⫾0.36 at 2h, 0.67⫾0.57 at 4h, and 0.47⫾0.15 at 8h reperfusion vs. 1.16⫾0.12, normal heart, p⫽0.602, 0.025, 0.003, respectively, ANOVA with LSD post hoc; Western blot gel scan arbitrary densitometry units ⫽ 74.8⫾7.2 at 2h, 78.5⫾6.0 at 4h, and 64.5⫾6.5 at 8h reperfusion vs. 80.75⫾1.3, normal heart, p⫽0.164, 0.589, 0.002, respectively). NF␬B activity at 2 hours of reperfusion indicated a 70% increase compared to controls by densitometry. TNF-␣ production increased at 2 and 4 hours of reperfusion and was decreasing towards baseline at 8 hours of reperfusion (pg TNF-␣/mg total protein ⫽ 492.0⫾234.3 at 2h, 530.4⫾297.8 at 4h, and 352.8⫾70.0 at 8h reperfusion vs. 270.0⫾64.0, normal heart, p⫽0.09, 0.044, 0.651, respectively). Conclusions: Upon reperfusion of cardiac allografts, an early increase in NF␬B activity is associated with increased production of TNF-␣ and transcriptional and translational downregulation of bcl-2 over time. The loss of antioxidant effects of bcl-2 may contribute to worsening myocardial oxidative stress following transplantation. 30 IMPAIRMENT OF CYTOLYTIC ACTIVITY IN ALLOGRAFT ENVIRONMENT AFFECTS EARLY MEDIAL CELL LOSS BUT DOES NOT ALTER OUTCOME OF ALLOGRAFT ARTERIOSCLEROSIS J.L. O’Neill, J.L. Jordan, J. Zhou, T.D. Lee, G.M. Hirsch, Dalhousie University, Halifax, NS, Canada Purpose: We investigated the importance of cytolytic effector mechanisms in the progression of allograft vasculopathy in a mouse aortic interposition model. Methods: Abdominal aortic interposition grafts were performed using C3H/HeJ donors and immunodeficient recipients. To test the role of CD8⫹ CTL we utilized both CD8 knockouts (B6.129S2-Cd8atm1Mak) and animals deficient in B2 microglobulin (which are significantly deficient in CD8⫹ cells; C57BL/6J-B2mtm1Unc). To test the role of the two principle cytolytic effector pathways we employed perforin knockout (C57BL/6J-PfptmlSdz) and FasL deficient mice (B6Smn.C3H-FasLgld). C57BL/6J recipients were used as positive controls. TUNEL assay was used to detect apoptosis in the media at 2 wk post-transplant and sections were digitally analyzed at 8 wk post-transplant to measure intimal area and medial cell number. Results: Medial apoptosis was significantly diminished in all aortic grafts in immunocompromised environments compared to positive control. Intimal lesion formation was similar in all aortic allografts regardless of cytolytic immunodefiency, including FasLgld animals. B2m knockout recipients showed significant preservation of medial cell number over the positive control. Other immunodeficient strains, however, showed no medial cell preservation. Conclusion: Aortic interposition grafts in environments with impaired cytolytic activity show decreased early medial damage but ultimate medial destruction and intimal lesion formation at late time points remains unaltered. Our results contradict previous published work on the protective effects of Fas ligand deficient recipients and suggest that intact CD8⫹ T cell subsets and cytolytic effector pathways are unnecessary for the ultimate development of allograft vasculopathy.

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31 ELEVATION OF SERUM BIG ENDOTHELIN IS ASSOCIATED WITH ENDOTHELIAL DYSFUNCTION IN PATIENTS WITH CHRONIC HEART FAILURE R. Berger, K. Strecker, B. Stanek, M. Huelsmann, R. Pacher, T. Neunteufl, University of Vienna, Vienna, Austria Background: We have previously reported that ETA receptor blockade improves impaired endothelium-dependent flow-mediated vasodilation (FMD) in chronic heart failure (CHF) patients. In the present study, we investigated the relationship between plasma levels of the ‘precursor’ big endothelin (ET) and FMD in this patient population. Methods: 35 CHF patients (New York Heart Association function class I - 6 patients, class II - 6 patients, class III - 23 patients; left ventricular ejection fraction [LVEF] ⬍ 30%) and 10 age, sex and risk factor - matched controls have been studied. Big ET plasma concentrations were measured using radioimmunoassay and FMD of the brachial artery was assessed using high resolution ultrasound. Patients were stratified into those with big ET plasma levels below the upper normal range of 1.8 fmol/ml (Group A), those between 1.8 and 4.3 (a cutpoint which was repeatedly shown to indicate very poor prognosis) fmol/ml (Group B) and those above 4.3 fmol/ml (Group C). Results: Big ET plasma levels were 3.5 ⫹ 2.4 fmol/ml in CHF patients and 2.2 ⫹ 0.6 fmol/ml in controls (P⫽0.01). FMD was 6.0 ⫹ 4.5 % in CHF patients and 10.1 ⫹ 4.9 % in controls. Comparing Group A (n⫽13, LVEF 20 ⫹ 4 %), Group B (n⫽10, LVEF 19 ⫹ 6 %), Group C (n⫽12, LVEF 19 ⫹ 6 %) and controls, FMD differed significantly between group A (10.0 ⫹ 3.4 %) and controls on one side and group B (3.8 ⫹ 4.6 %) and group C (3.6 ⫹ 1.6 %) on the other side (P⫽0.0001), but was similar between group A and controls and between group B and group C. In a multivariate analysis including age, gender, smoking, diabetes, hypercholesterolemia, hypertension and big ET, big ET was the only independent predictor of endothelial dysfunction (r ⫽ -0.59, p ⫽ 0.0002). Conclusion: Patients with normal big ET plasma levels have a similar FMD compared to controls, whereas patients with elevated big ET plasma levels have an impaired FMD independent of the degree of elevation. Big ET is a strong independent predictor of endothelial dysfunction. 32 TUMOUR NECROSIS FACTOR- A A2 POLYMORPHISM; A PREDISPOSING FACTOR TO NON-ISCHAEMIC MYOCARDIAL DYSFUNCTION? C. Densem1, I.V. Hutchinson2, N. Yonan1, N.H. Brooks1, 1 Wythenshawe Hospital, Manchester, UK; 2Manchester University, Manchester, United Kingdom Tumour necrosis factor-a (TNF), a pro-inflammatory cytokine, is implicated in the pathophysiology of heart failure. TNF levels are elevated in patients with myocardial dysfunction; the degree of rise relating to symptoms. Transgenic mice producing high TNF quantities develop systolic dysfunction and ventricular dilatation. Chronic infusion of TNF produces a similar clinical picture. G to A polymorphism in the promoter region of the TNF gene (TNFA2, position -308) is associated with higher TNF production in humans. We hypothesized that this genetic variant would be over represented in cardiac transplant recipients.