Retinoid X Receptor (RXR) agonists inhibits mononuclear leukocyte-endothelial cell interactions through peroxisome proliferator-activated receptor-γ (PPARγ) activation

MJ Sanz1,2, F Albertos1,2, L Crespo1, EJ Morcillo1,2, L Piqueras1. 1University Clinic Hospital Research Foundation-INCLIVA, INCLIVA, Spain, 2University of Valencia, Department of Pharmacology, Spain

Introduction: Mononuclear cell migration into the vascular subendothelium constitutes an early event of the atherogenic process.

Objective: The effect of retinoid X receptor-α (RXRα) on leukocyte recruitment is poorly understood. Therefore, this study investigated whether RXR agonists can affect this response and the underlying mechanisms involved.

Methods: Human umbilical arterial endothelial cells (HUAECs) were used for these studies. Mononuclear leukocyte-endothelial cell interactions were evaluated in vitro using the dynamic flow chamber assay. For this purpose, freshly isolated human mononuclear cells were perfused across HUAEC monolayers stimulated or not with TNFα (10 ng/ml) for 4 h. The RXR agonist, bexarotene, (0.1-3 μM) was added to some plates 20 h prior to TNF-α stimulation. Confluent HUAEC were transfected with either control, RXRα, PPARα, PPARβ or PPARγ specifics small interference RNA.

Results: Forty-eight hours postransfection with a specific siRNA, HUAECs showed a reduction in RXRα (80±19% compared to the levels detected in the control siRNA-transfected cells) (p<0.05, students t-test, n=3-4). Preincubation of the HUAEC with bexarotene, at the concentration of 1 μM, inhibited TNFα-induced mononuclear leukocyte endothelial adhesion (vehicle, 127.2±21.8 cells/mm2; bexarotene,47.1±13.8 cells/mm2) (p<0.05, students t-test , n=4-6). Suppression of RXRα expression with a siRNA abrogated these responses. In addition, knockdown of PPARγ reversed the inhibitory effects of bexatotene in TNFα-induced mononuclear cell adhesion (vehicle, 84.5 ±8.2 cells/mm2; bexarotene, 96.5±4.8 cells/mm2)(p<0.05, students t-test, n=4-5).However, gene silencing of PPARα or PPARβ did not block the antiinflammatory effects elicited by RXR agonists. Furthermore, while suboptimal concentrations of bexarotene (0.3 μM) was unable to inhibit TNFα-induced mononuclear cell-adhesion (vehicle, 127.2±21.8 cells/mm2; bexarotene, 102.5±22.8 cells/mm2)(p>0.05, students t-test, n=4-5), significant reductions in this parameter were achieved when they were combined with a suboptimal concentration of the PPARγ agonist rosiglitazone (1 μM), being mononuclear cell adhesion reduced by 63.2±2.1% (p<0.05, students t-test, n=4-7)

Conclusions: The inhibitory effects elicited by bexarotene in mononuclear endothelial cell interactions seem to be mediated via RXRα/PPARγ interaction. Therefore, these results reveal that RXR agonists can inhibit the initial inflammatory response that precedes the atherogenic process by targeting the mononuclear recruitment. Thus, RXR agonists may constitute a new therapeutical tool in the control of the inflammatory process associated to cardiovascular disease.

This work was supported by grants CP07/00179, PI081875, SAF2009-08913 and SAF2011-23777, Spanish Ministry of Science and Innovation, RIER RD08/0075/0016, Carlos III Health Institute, Spanish Ministry of Health Innovation, co-financed by FEDER (European Funds for Regional Development) and different grants from Generalitat Valenciana.