Crosstalk of endothelin-1 and platelet-derived growth factor in cardiac allograft arteriosclerosis
Roope K. Sihvola, MD*,*,
Ville P. Pulkkinen, MSc*,
Petri K. Koskinen, MD, PhD* and
Karl B. Lemström, MD, PhD*
* Cardiopulmonary Research Group, Transplantation Laboratory, University of Helsinki, and Helsinki University Central Hospital, Helsinki, Finland
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Figure 1 Effect of Bosentan (Ro 47-0203), an ET-1 receptor antagonist, on (A) incidence and (B) mean score of intimal thickness in epicardial arteries and intramyocardial arterioles of rat cardiac allografts. Allograft recipients were given Bosentan, 100 mg/kg per day (n = 9), or vehicle (n = 7), and, as background immunosuppression, CsA, 2 mg/kg per day for the first week and 1 mg/kg per day thereafter. The grafts were removed 60 days after transplantation. Data are given as the mean value ± SEM; data were analyzed by using the Mann-Whitney U test. Grade 0 = normal artery with intact internal elastic lamina; grade 1 = <10% occlusion of the lumen by arterial intimal thickening and proliferation, disruption of the internal elastic lamina and the presence of some foam or vacuolated ECs; grade 2 = >10% but <50% occlusion of the lumen; grade 3 = >50% but <100% occlusion of the lumen; and grade 4 = 100% vessel occlusion of the lumen. Photomicrographs of the coronary arteries of vehicle-treated (C) and Ro 47-0203–treated (D) rat cardiac allografts. Original magnification x200. Hematoxylin-eosin and resorcin-fuchsin staining for internal elastic lamina.
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Figure 2 To analyze the correlation between the development of intimal thickening and ET-1 ligand and receptor expression, allograft recipients were given CsA at a dose of either 1.0, 1.5 or 2 mg/kg per day for two weeks, followed by one week of CsA at 1 mg/kg per day. The correlation of intimal thickening with ET-1 (left column), ETA (middle column) and ETB (right column) protein expression in the arterial wall of chronically rejecting allografts, analyzed by linear regeression analysis. Correlation coefficients (r2) are given. Immunohistochemistry findings were scored from 0 to 3 (0 = no visible staining; 1 = few cells with faint staining; 2 = moderate intensity with multifocal staining; and 3 = intense diffuse staining of the cells).
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Figure 3 (A) Effects of ET-1 and PDGF on 3H-TdR incorporation into coronary artery SMCs in vitro. After 96- or 72-h serum starvation, quiescent SMCs were either incubated with ET-1, PDGF-AA, PDGF-BB or PDGF-AB for 48 h, or prestimulated with ET-1 for 24 h; thereafter, PDGF-AA, -AB or -BB was added to cultures for 48 h, respectively. Incorporation of 3H-TdR (1 µCi/ml) was measured during last 24 h. Six parallel wells were used for the determinations, and the experiment was repeated twice. (B) Effects of ET-1 and PDGF into coronary artery SMC migration in vitro. The cells were seeded in porous membranes separating the upper and lower chambers. After 2 h, ET-1, PDGF-AA, PDGF-BB and PDGF-AB were added to the lower chamber and incubated for 24 h at 37°C. Migrated cells on the lower side of the filter were quantitated by counting the cells on the lower side of the filter. Three parallel wells were used for the determinations, and the experiment was repeated twice. Data are expressed as the mean value ± SEM. NIL = no treatment.
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