In vitro prevention of cyclosporin-induced cell contraction by mycophenolic acid.

Nephrotoxicity is a major side-effect of cyclosporin A (CsA), which induces a vasoconstrictive response in vascular smooth muscle and mesangial cells. Mycophenolic acid (MPA) is used in combination with low-dose CsA to reduce nephrotoxicity. We previously demonstrated that MPA affected mesangial cell contractile response to angiotensin II or KCl. Aims of the present study were to evaluate if MPA can prevent CsA-induced contraction of human mesangial and aortic smooth muscle cells (ASMC). Using a morphoquantitative approach, we evidenced that pretreatment with MPA (1 microM) prevented the reduction of cell area induced by CsA within 30 min in both cell types. We then compared the expression of three main cytoskeleton proteins: tubulin, alpha-smooth actin (SMA) and basic calponin, in ASMC and in mesangial cells treated with MPA and/or CsA. CsA alone did not significantly change the expression level of these proteins neither in mesangial cells nor in ASMC. MPA decreased the expression level of tubulin in both mesangial cells and ASMC. Surprisingly, MPA, which stimulated SMA and calponin expression in mesangial cells, exerted an inhibitory effect on both contractile protein expression in ASMC. In conclusion, our results evidenced opposite effects of MPA on calponin and SMA protein expression in ASMC and in mesangial cells, despite similar antiproliferative properties, suggesting that sarcomeric protein expression is controlled by different intracellular mechanisms in mesangial and smooth muscle cells. However, MPA interferes in both cell types with the constrictive properties CsA, which may partially explain the protective effects of MPA against CsA nephrotoxicity.

Cytoskeletal reorganization by mycophenolic acid alters mesangial cell migration and contractility.

Cytoskeleton alterations are a hallmark of mesangial cell activation during glomerulosclerosis. The aim of this study was to investigate whether mycophenolic acid (MPA) affects cytoskeletal organization and motility of human mesangial cells. Using the IP15 cell line, we found that treatment with 1 micromol/L MPA inhibited both receptor-dependent (angiotensin II) and receptor-independent (KCl) contractile responses, as well as serum-induced migration activity, suggesting alterations in the intracellular mechanisms that control mesangial cell motility. Immunofluorescence studies of MPA-treated cells provided evidence for decreased membrane disassembly/reassembly of alpha-smooth muscle actin and F-actin fibers, which was correlated with sustained quantitative and qualitative modifications of actin-associated proteins: calponin was overexpressed and became associated with actin fibers, whereas phosphorylation levels of cofilin and myosin light chain increased, suggesting both an activation of the mechanisms responsible for actin polymerization and an inhibition of actin-depolymerizing processes. These observations support a stabilizing effect of MPA on the mesangial actin cytoskeleton, which constitutes an additive action by which MPA, beyond its anti-inflammatory, antiproliferative and antifibrotic properties, might protect against excessive mesangial activation in the context of various glomerulopathies and kidney transplantation.

The -308 G/A tumor necrosis factor-alpha gene dimorphism: a risk factor for unstable angina.

Since the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) may play a major role in the pathophysiology of acute coronary syndromes, 299 consecutive male patients hospitalized for coronary artery disease (i.e., lumen lost > or = 50%) were genotyped for the functional -308G/A TNF-alpha polymorphism using restriction fragment length polymorphism method, in order to evaluate its potential association with the risk of unstable angina and/or myocardial infarction. A higher frequency of carriers of the A allele was observed in patients with unstable angina (n = 58) when compared to control patients with stable angina (n = 95) (39.66% vs. 23.16% respectively, p = 0.029, odds ratio = 2.2) but not in patients with myocardial infarction (n = 146) (23.97% vs. 23.16%, p = NS). Furthermore, we evidenced an interaction of the polymorphism studied with body mass index in patients with unstable angina. Thus, when stratified analysis was performed, results in patients with a body mass index < or = 27 showed a more striking association between A allele carriage frequency and unstable angina (p = 0.012, odds ratio = 3.0). These results suggest the crucial role of TNF-alpha in the mechanisms responsible for unstable angina in accordance with the concept of vulnerable plaque. On the other hand, mechanisms controlling myocardial infarction appear more complex and heterogeneous.

Mycophenolic acid antagonizes the activation of cultured human mesangial cells.

BACKGROUND: Activation of mesangial cells is observed in several forms of chronic renal disease, and in culture conditions upon stimulation by fetal calf serum (FCS), or agonists such as transforming growth factor beta (TGF-beta). Mycophenolate mofetil (MMF), the precursor of mycophenolic acid (MPA), is currently used in organ transplantation and has been shown to be protective in clinical and experimental glomerulonephritis. This study assessed the effects of MPA on markers of human mesangial cells (HMC) activation. METHODS: Primary cultures of HMC and of an immortalized HMC clone (IP15 cells characterized in this report) were stimulated either by FCS or by TGF-beta, and treated by MPA at clinically relevant concentrations (1 to 10 micromol/L) for 24 hours to 14 days. HMC proliferation, smooth muscle alpha-actin (SMA), collagen type I alpha-1 chain (coll I) and fibronectin synthesis were used as markers of HMC phenotypic activation. RESULTS: Exposure of HMC to MPA inhibited proliferation induced by FCS without cytotoxicity. MPA counteracted the stimulatory effects of FCS and TGF-beta on coll I mRNA and protein and fibronectin protein. SMA expression was increased upon exposure to MPA, without cell hypertrophy. CONCLUSION: Treatment of cultured HMC with MPA inhibited mesangial cell proliferation and matrix production induced by stimulation with either FCS or TGF-beta. Such mechanisms may contribute to the favorable effects of treatment using mycophenolate mofetil in chronic fibrotic kidney diseases, including chronic allograft rejection.