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

Abstract

The immunosuppressive drug mycophenolate mofetil (MMF) and its active metabolite mycophenolic acid (MPA) selectively inhibit inosine 5′-monophosphate dehydrogenase (IMPDH), and therefore interfere with cellular guanine nucleotide biosynthesis. IMPDH is additionally involved in the synthesis of membrane glycoproteins, some of which are adhesion receptors known to play an active part in the regulation of cell–cell contacts, which are crucial in the process of recruitment and transendothelial infiltration of activated leucocytes in the transplanted organ. As a consequence, MPA leads to a reduction of cellular infiltrates in the course of transplant rejection. In the present study, the effects of MPA on human umbilical vein endothelial cells (HUVEC) are investigated at both molecular and cellular levels. In our experiments, HUVECs are treated with tumor necrosis factor-α (TNF-α; 10 ng/ml) in order to mimic activation occurring at a rejection crisis. The dose-dependent influence of concomitant incubation with MPA (5–20 μmol/l; 48 h, 37°C, 5% CO 2) on their intracellular nucleotide profile is observed by determining the concentrations of purine and pyrimidine nucleotides, using a HPLC method based on solvent generated ion-exchange. The possibility of synergistic effects is investigated by incubating endothelial cells with mixtures of three different immunosuppressants (mycophenolic acid; cyclosporin A, 100 ng/ml; prednisolone, 1 μmol/l)—a combination commonly used after transplantation—varying the amount of MPA (5–20 μmol/l). Stimulation with TNFα does not significantly modulate the intracellular levels of nucleotides quantitated. In the presence of MPA concentrations of at least 5 μmol/l, GTP levels (68±12%) are significantly decreased compared to controls (100%). At a concentration of 20 μmol/l MPA, the GTP amount is reduced to 58±7%. In contrast to these observations, the levels of UDP and UTP are increasing significantly under coincubation with MPA concentrations greater than 5 μmol/l. At 20 μmol/l MPA, UDP and UTP are increased to 147±19% and 114±11%, respectively. All other nucleotides (CTP, ADP, ATP) reveal no significant alterations in their intracellular concentrations under the conditions applied. Incubation of TNFα-treated HUVEC monolayers, with a mixture of three immunosuppressive drugs varying 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 μmol/l) on a cellular level is observed by measuring the cell surface expression of adhesion molecules on cytokine-stimulated HUVECs, using TNFα (10 ng/ml), interferon-γ (100 ng/ml), interleukin-1β (10 ng/ml) and interleukin-8 (20 ng/ml). Expression of the intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), endothelial leucocyte adhesion molecule-1 (ELAM-1) and platelet endothelial cell adhesion molecule-1 (PECAM-1) was assessed by flow cytometry. Activation of endothelial cell monolayers with TNFα significantly increases the mean fluorescence intensity of VCAM-1 (361±14%) and ICAM-1 (429±47%) surface expression, compared to controls, and additionally induces E-selectin expression (2919±134%). The same tendencies, but in a lesser degree, are observed under stimulation of cells with either IFNγ or IL-1β. Incubation with a combination of TNFα 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-1β/MPA also reduces the expression of VCAM-1 to a level significantly lower than the level observed after stimulation with IL-1β. Incubation with MPA alone reveals no significant modulation in the expression of all surface molecules tested compared to the values of unstimulated HUVECs. The experiments show that the immunosuppressive action of MPA not only inhibits lymphocyte proliferation but also decreases the expression of adhesion molecules on endothelial cells, which are the first target of the cellular rejection process.