Mechanisms of Nifedipine-Downregulated CD40L/sCD40L Signaling in Collagen Stimulated Human Platelets

Abstract

The platelet-derived soluble CD40L (sCD40L) release plays a critical role in the development of atherosclerosis. Nifedipine, a dihydropyridine-based L-type calcium channel blocker (CCB), has been reported to have an anti-atherosclerotic effect beyond its blood pressure-lowering effect, but the molecular mechanisms remain unclear. The present study was designed to investigate whether nifedipine affects sCD40L release from collagen-stimulated human platelets and to determine the potential role of peroxisome proliferator-activated receptor-β/-γ (PPAR-β/-γ). We found that treatment with nifedipine significantly inhibited the platelet surface CD40L expression and sCD40L release in response to collagen, while the inhibition was markedly reversed by blocking PPAR-β/-γ activity with specific antagonist such as GSK0660 and GW9662. Meanwhile, nifedipine also enhanced nitric oxide (NO) and cyclic GMP formation in a PPAR-β/-γ-dependent manner. When the NO/cyclic GMP pathway was suppressed, nifedipine-mediated inhibition of sCD40L release was abolished significantly. Collagen-induced phosphorylation of p38MAPK, ERK1/2 and HSP27, matrix metalloproteinase-2 (MMP-2) expression/activity and reactive oxygen species (ROS) formation were significantly inhibited by nifedipine, whereas these alterations were all attenuated by co-treatment with PPAR-β/-γ antagonists. Collectively, these results demonstrate that PPAR-β/-γ-dependent pathways contribute to nifedipine-mediated downregulation of CD40L/sCD40L signaling in activated platelets through regulation of NO/ p38MAPK/ERK1/2/HSP27/MMP-2 signalings and provide a novel mechanism regarding the anti-atherosclerotic effect of nifedipine.

Fig 1. Effects of nifedipine on the membrane CD40L expression and sCD40L release.

Washed platelets were preincubated with various doses of nifedipine or nifedipine combined with GSK0660 (5 μ M) or GW9662 (5 μ M) for 3 min, followed by addition of collagen (10 μ g/ml) for 6 min. The platelet surface CD40L expression (A) and the levels of sCD40L (B) were determined. Data were presented as means ± SEM (n = 5). ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001 compared with the collagen alone group; ⁺ p < 0.05, ⁺⁺ p < 0.01, ⁺⁺⁺ p < 0.001 compared with respective collagen+nifedipine-treated group.

Fig 2. Effects of PPAR-β/-γ antagonists on nifedipine-mediated NO/cyclic-GMP formation and CD40L/sCD40L cascade.

Platelets were incubated with various drugs, followed by addition of collagen. The levels of NOx (A) cyclic GMP formation (B) platelet surface CD40L expression (C) and sCD40L concentrations (D) were determined. Date were expressed as means ± SEM (n = 5). ^** p < 0.01, ^*** p < 0.001 compared with collagen alone group; ⁺ p < 0.05, ⁺⁺ p < 0.01, ⁺⁺⁺ p < 0.001 compared with respective collagen+nifedipine-treated group.

Fig 3. Effects of PPAR-β/-γ antagonists on nifedipine-mediated phosphorylation of P38MAPK, ERK1/2 and HSP27.

Platelets were pretreated with 5 μ M nifedipine or nifedipine combined with various drugs for 3 min followed by addition of collagen for 6 min. The expression of phospho-P38MAPK and total-P38MAPK (A), phospho-ERK1/2 and total ERK1/2 (B), phospho-HSP27 and total-HSP27 (C) were determined. Date were expressed as means ± SEM (n = 5). ^*** p < 0.001 compared with collagen alone group; ⁺⁺ p < 0.01, ⁺⁺⁺ p < 0.001 compared with respective collagen+nifedipine-treated group.

Fig 4. Effects of PPAR-β/-γ antagonists on nifedipine-mediated MMP2 activity.

Platelets were treated with nifedipine (5 μ M), or nifedipine combined with various drugs for 3 min, followed by addition of collagen for 6 min. The MMP-2 expression (A) and MMP-2 activity (B) were determined. Date were expressed as means ± SEM (n = 5). ^*** p < 0.001 compared with collagen alone group; ⁺⁺ p < 0.01, ⁺⁺⁺ p < 0.001 compared with respective collagen+nifedipine-treated group.

Fig 5. Effects of PPAR-β/-γ antagonists on nifedipine-mediated ROS production.

Platelets were preincubated with nifedipine or nifedipine combined with GSK0660 or GW9662 for 3 min; then NBT or DCFH-DA was added, followed by collagen to measure superoxide (A) and hydrogen peroxide (B) formation respectively. Data were expressed as means ± SEM (n = 5). ^*** p < 0.001 compared with collagen alone group; ⁺⁺ p < 0.01 compared with respective collagen+nifedipine treated group.

Fig 6. Hypothetical model of the signaling pathways of nifedipine-mediated reduction of sCD40L release from collagen-stimulated platelets.

Nifedipine initially activates PPAR-β/-γ followed by increased formation of NO/cyclic GMP and down-regulation of p38MAPK/ERK1/2/HSP27 signaling as well as ROS generation, which then attenuates MMP-2 activity and ultimately inhibits sCD40L release from activated platelets.