Statins reduce endothelial cell apoptosis via inhibition of TRAIL expression on activated CD4 T cells in acute coronary syndrome

Abstract

Objective: Statins reduce cardiovascular-related morbidity and mortality, but their effects on inflammation in atherosclerosis are not fully understood. We investigated whether statins can modulate cytotoxic functions of CD4 T cells in acute coronary syndrome (ACS).

Methods and results: Fresh CD4 T cells were isolated from 55 patients with ACS without statin treatment on admission and from 34 age-matched controls. CD4 T cells collected from ACS patients induced endothelial cell apoptosis significantly more than control T cells. The TNF-related apoptosis-inducing ligand (TRAIL) receptor DR5 was strongly upregulated on endothelial cells, and TRAIL-specific antibodies effectively blocked CD4 T cell-mediated apoptosis, indicating that T cell-mediated endothelial death was dependent on the TRAIL pathway. Expression of both the activating antigen CD69 and TRAIL was enhanced on ACS T cells. In in-vitro assays rosuvastatin, fluvastatin, and pitavastatin directly blocked CD4 T cell-mediated endothelial cell apoptosis and reduced T cell-expression of CD69 and TRAIL through TCR-induced Extracellar signal-Regulated Kinases (ERK) activation.

Conclusions: Activated CD4 T cells expressing TRAIL are enriched in the blood of ACS patients and induce endothelial injury, which may contribute to the destabilization of the plaque. Early statin therapy may suppress T cell-mediated endothelial cell damage in atherosclerotic plaques and thus prevent cardiovascular events.

Figure 1. Cytotoxic CD4 T cells from ACS patients

A, CD4 T cells derived from peripheral blood of ACS and controls (NC) were incubated on human umbilical vein endothelial cells (HUVECs) monolayers and frequencies of apoptotic HUVECs were assessed after 3h. B, Representative apoptotic nuclear changes of HUVECs induced with ACS T cells. Scale bar = 100 µm.

Figure 2. CD4 T cell-induce EC apoptosis through the TRAIL pathway

A, mRNA was isolated from human coronary artery smooth muscle cells (CASMCs), human aortic smooth muscle cells (AoSMCs), and HUVECs and amplified by real-time PCR for transcripts of the TRAIL receptors DR4 and DR5. B, Expression levels for DR4 and DR5 protein were analyzed by Western blots. C, CD4 T cells from patients with ACS and NC were pretreated with anti-TRAIL antibody or isotype control IgG2a antibody before the T cell apoptosis assay. Apoptosis rates were determined after 3 h. All Data are representative of four experiments.

Figure 3. TRAIL expression is increased on activated CD4 T cells in ACS

A, Peripheral blood mononuclear cells (PBMCs) were isolated and CD69 expression was analyzed by flow cytometry. B, PBMCs were stimulated in anti-human CD3-coated plates (After TCR triggering). Control cells were seeded onto uncoated plates (Control). TRAIL expression on CD4 T cells was analyzed by flow cytometry.

Figure 4. Statins inhibit CD4 T cell-induced EC apoptosis and ERK activation

A, Fresh CD4 T cells were isolated from 16 ACS patients and tested for their ability to induce HUVEC apoptosis pretreated with different statins (rosuvastatin, ROS, fluvastatin, FLV, or pitavastatin, PIT) or without statin (control). B, Fresh PBMCs from ACS were pretreated with (dotted line) or without (solid line) statins. Then, PBMCs were stimulated with (After TCR triggering) or without (Control) anti-CD3 mAb, followed by anti-mouse IgG for cross-linking. Intracellular ERK1/2 phospholiration in CD4 T cells was analyzed by flow cytometry. Isotype control (shaded histogram). Data are representative of five experiments.

Figure 5. Statins inhibit CD4 T cell activation and TRAIL expression

A, CD69 expression on CD4 T cells were pretreated with or without statins (15 ACS) and analyzed. B, PBMCs were pretreated with or without statins and then stimulated in anti-human CD3-coated plates (11 ACS). Expression of TRAIL was assessed by flow cytometry.