Amelioration of cyclosporin A effect on microvasculature by endothelin inhibitor

We have previously shown that endothelial injury by cyclosporin A (CyA) is associated with an increased endothelin-1 (ET-1) release. We now sought to determine, in an animal model of angiogenesis, if inhibiting the effect of ET-1 on endothelial cells (ECs) would reverse the CyA-mediated endothelial injury in an animal model of angiogenesis.

An angiogenic mixture of Matrigel (0.5 ml), fibroblast growth factor (1 ng/ml), vascular endothelial growth factor (100 ng/ml), and heparin (64 unit/ml) was injected as a subcutaneous plug in the flank of C3H mice (n = 5). In experimental groups CyA (20 mg/ml), CyA, and BQ 123 (ET-A receptor antagonist), CyA and PD 142893 (ET-A and ET-B receptor antagonist), or CyA and ET-1 antibody were added to the angiogenic mixture. Angiogenesis in the mixture was quantified by modified planimetric point counting method in skin/Matrigel cross-sections stained with factor VIII to highlight endothelial neocapillaries. Mean +/- SD of angiogenic area was analyzed with analysis of variance and Bonferroni test. The survival curves obtained by Kaplan-Meier analysis were compared between the groups, and the statistical significance of survival and mortality rates was computed by log rank's and Fisher's exact test, respectively.

The mean +/- SD of angiogenic area in control animals (without CyA in the angiogenic mixture) was 56.76 +/- 4.2. CyA inhibited angiogenesis in the subcutaneous angiogenic plug. Adding CyA to the angiogenic mixture significantly reduced angiogenic area (5.33 +/- 1.4, P <.001) while vehicle for CyA had no such effect (56.33 +/- 3.8, P =.10). Polyclonal ET-1 antibody or PD 142893 ameliorated the effect of CyA, whereas BQ 123 did not. The mean angiogenic areas in animals with ET-1 antibody, PD 142893, or BQ 123 in the angiogenic mixture were 57.20 +/- 7.5 (P =.06), 46.00 +/- 11.5 (P = 1.0), 8.60 +/- 2.9 (P <.001), respectively.

Our data show that blocking ET-B receptors specifically ameliorates the microvascular injury to the neocapillaries in angiogenesis caused by CyA. Antiendothelin-1 antibody and ETR antagonist (PD 142893) could, therefore, reduce the ill effects of CyA on microvascular endothelium.