Effects of mycophenolate mofetil on key pattern of coronary restenosis: a cascade of in vitro and ex vivo models

Abstract

Background: Mycophenolate mofetil (MMF), the prodrug of mycophenolic acid (MPA), is a rationally designed immunosuppressive drug. The current study investigates the effect of MMF on key pattern of restenosis in a cascade of in vitro and ex vivo models.

Methods: Part I of the study investigated in northern blot and cytoflow studies the effect of MMF (50, 100, 150, 200, 250, and 300 microg/mL) on TNF-alpha induced expression of intercellular adhesion molecule 1 (ICAM-1) in human coronary endothelial cells (HCAEC) and human coronary medial smooth muscle cells (HCMSMC). Part II of the study applied a human coronary 3D model of leukocyte attack, the 3DLA-model. HCAEC and HCMSMC were cultured on both sides of a polycarbonate filters, mimicking the internal elastic membrane. Leukocyte attack (LA) was carried out by adding human monocytes (MC) on the endothelial side. The effect of MMF (50 microg/mL) on adhesion and chemotaxis (0.5, 1, 2, 3, 4, 6, and 24 h after LA) and the effect on proliferation of co-cultured HCMSMC (24 h after LA) was studied. In part III of the study a porcine coronary organ culture model of restenosis (POC-model) was used. After ex vivo ballooning MMF (50 microg/mL) was added to the cultures for a period of 1, 2, 3, 4, 5, 6, and 7 days. The effect on reactive cell proliferation and neointimal thickening was studied at day 7 and day 28 after ballooning.

Results: Expression of ICAM-1 in northern blot and cytoflow studies was neither clearly inhibited nor stimulated after administration of MMF in the clinical relevant concentration of 50 microg/mL. In the 3DLA-model 50 microg/mL of MMF caused a significant antiproliferative effect (p < 0.001) in co-cultured HCMSMC but had no effect on MC-adhesion and MC-chemotaxis. In the ex vivo POC-model neighter reactive cell proliferation at day 7 nor neointimal hyperplasia at day 28 were significantly inhibited by MMF (50 microg/mL).

Conclusion: Thus, the data demonstrate a significant antiproliferative effect of clinical relevant levels of MMF (50 microg/mL) in the 3DLA-model. The antiproliferative effect was a direct antiproliferative effect that was not triggered via reduced expression of ICAM-1 or via an inhibition of MC-adhesion and chemotaxis. Probably due to technical limitations (as e.g. the missing of perfusion) the antiproliferative effect of MMF (50 microg/mL) could not be reproduced in the coronary organ culture model. A cascade of focused in vitro and ex vivo models may help to gather informations on drug effects before large experimental studies are initiated.

Figure 1

Northern blots and relative band densities of TNF-α-induced expression of ICAM-1 mRNA after incubation of HCAECs and HCMSMCs with 50, 100, 150, 200, 250, and 300 μg/mL of MMF.

Figure 2

Graphics and histograms (cytoflow data) of the effect of MMF (50, 100, 150, 200, 250, and 300 μg/mL) on TNF-α-induced expression of ICAM-1 in HCAECs (A, B) and HCMSMCs (C, D) after 18 h. Negative control (dotted line) and ICAM-1 expression in untreated cells (grey line) are included.

Figure 3

(A) Effect of MMF (50 μg/mL) on monocyte adhesion after leukocyte attack on the endothelial side of the human coronary transfilter co-culture model (3DLA units). (B) Effect of MMF (50 μg/mL) on monocyte chemotaxis after leukocyte attack on the endothelial side of the human coronary transfilter co-culture model (3DLA units). (C) Identification of monocytes by positive staining against CD68 (arrow).

Figure 4

(A) Effect of MMF (50 μg/mL) on proliferative activity of co-cultured HCMSMC after leukocyte attack with monocytes on the endothelial side of TNF-α-stimulated 3DLA units. (B) Proliferation of smooth muscle cell demonstrated by positive staining of against BrdU (arrow). F = Filter, *** = p < 0.001.

Figure 5

Effect of a 1–7 days treatment with MMF (50 μg/mL) on reactive cell proliferation in balloon-injured porcine coronary organ cultures at day 7 (A) and day 28 (B). C = control, CB = control ballooning. Positive staining against BrdU (arrow) in the ballooning control group (C).

Figure 6

Effect of a 1–7 days treatment with MMF (50 μg/mL) on reactive neointimal hyperplasia in balloon-injured porcine coronary organ cultures at day 7 and day day 28. C = control, CB = control ballooning. Elastica van Gieson-staining in the ballooning control group (C).