In vitro effects of mycophenolic acid on the nucleotide pool and on the expression of adhesion molecules of human umbilical vein endothelial cells

The immunosuppressive drug mycophenolate mofetil (MMF) and its active metab olite mycophenolic acid (MPA) selectively inhibit inosine 5'-monophosphate dehydrogenase (IMPDH), and therefore interfere with cellular guanine nucleo tide biosynthesis. IMPDH is additionally involved in the synthesis of membr ane glycoproteins, some of which are adhesion receptors known to play an ac tive part in the regulation of cell-cell contacts, which are crucial in the process of recruitment and transendothelial infiltration of activated leuc ocytes in the transplanted organ. As a consequence, MPA leads to a reductio n of cellular infiltrates in the course of transplant rejection. In the present study, the effects of MPA on human umbilical vein endothelia l cells (HUVEC) are investigated at both molecular and cellular levels. In our experiments, HUVECs are treated with tumor necrosis factor-alpha (TNF-a lpha; 10 ng/ml) in order to mimic activation occurring at a rejection crisi s. The dose-dependent influence of concomitant incubation with MPA (5-20 mu mol/l; 48 h, 37 degreesC, 5% CO2) on their intracellular nucleotide profil e is observed by determining the concentrations of purine and pyrimidine, n ucleotides, using a HPLC method based on solvent generated ion-exchange. Th e possibility of synergistic effects is investigated by incubating endothel ial cells with mixtures of three different immunosuppressants (mycophenolic acid; cyclosporin A, 100 ng/ml; prednisolone, 1 mu mol/l)-a combination co mmonly used after transplantation-varying the amount of MPA (5-20 mu mol/l) . Stimulation with TNF alpha does not significantly modulate the intracellula r levels of nucleotides quantitated. In the presence of MPA concentrations of at least 5 mu mol/l, GTP levels (68 +/- 12%) are significantly decreased compared to controls (100%). At a concentration of 20 mu mol/l MPA, the GT P amount is reduced to 58 +/-7%. In contrast to these observations, the lev els of UDP and UTP are increasing significantly under coincubation with MPA concentrations greater than 5 mu mol/l. At 20 mu mol/l MPA, UDP and UTP ar e increased to 147 +/- 19% and 114 +/- 11%, respectively. All other nucleot ides (CTP, ADP, ATP) reveal no significant alterations in their intracellul ar concentrations under the conditions applied. Incubation of TNF alpha -tr eated. HUVEC monolayers, with a mixture of three immunosuppressive drugs va rying the amount of MPA, show no significant differences compared with the data observed after incubation with MPA alone. In addition, the influence of MPA (10 mu mol/l) on a cellular level is obse rved by measuring the cell surface expression of adhesion molecules on cyto kine-stimulated HUVECs, using TNF alpha (10 ng/ml), interferon-gamma (100 n g/ml), interleukin-1 beta (10 ng/ml) and interleukin-8 (20 ng/ml). Expressi on of the intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesio n molecule-1 (VCAM-1), endothelial leucocyte adhesion molecule-1 (ELAM-1) a nd platelet endothelial cell adhesion molecule-1 (PECAM-1) was assessed by flow cytometry. Activation of endothelial cell monolayers with TNF alpha significantly incr eases the mean fluorescence intensity of VCAM-1 (361 +/- 14%) and ICAM-1 (4 29 +/- 47%) surface expression, compared to controls, and additionally indu ces E-selectin expression (2919 +/- 134%) The same tendencies, but in a les ser degree, are observed under stimulation of cells with either IFN gamma o r IL-1 beta. Incubation with a combination of TNF alpha and MPA leads to a significant reduction in VCAM-1 (329 +/- 13%) and E-selectin (2613 +/- 167% ) expression, compared to the values obtained for HUVEC incubated with the cytokine alone. Treatment of the cells with IL-1 beta /MPA also reduces the expression of VCAM-1 to a level significantly lower than the level observe d after stimulation with IL-1 beta. Incubation with MPA alone reveals no si gnificant modulation in the expression of all surface molecules tested comp ared to the values of unstimulated HUVECs. The experiments show that the im munosuppressive action of MPA not only inhibits lymphocyte proliferation bu t also decreases the expression of adhesion molecules on endothelial cells, which are the first target of the cellular rejection process. (C) 2001 Els evier Science B.V. All rights reserved.