Papel do receptor TLR4 no efeito anticontrátil do PVAT

Abstract

Previous studies have demonstrated that genetic deletion of Toll-like receptor 4 (TLR4) significantly impairs vascular function. In this context, the present study aimed to investigate the modulatory role of TLR4 on the anticontractile effect of perivascular adipose tissue (PVAT) and its interaction with endothelial cells under physiological conditions, specifically evaluating the effects of acute TLR4 agonism induced by lipopolysaccharide (LPS). In human endothelial cells (EA.hy926), stimulation with LPS (1 μg/mL) reduced basal nitric oxide (NO) production during the first 30 minutes, followed, after 1 hour, by increased inducible nitric oxide synthase (iNOS) expression and elevated NO levels. This temporal profile indicates a transition from an initial eNOS-dependent response to a late iNOS-mediated response. Blockade of the TLR4 receptor with a specific antibody reduced nitric oxide (NO) production, and LPS stimulation did not exert any additional effect on this reduction, suggesting that the observed response is dependent on TLR4 activation. In vascular reactivity experiments, LPS suppressed the anticontractile effect of PVAT in aortas from C57BL/6J mice. In contrast, aortas from TLR4⁻/⁻ mice exhibited no PVAT anticontractile effect under basal conditions and showed no functional alterations in response to LPS stimulation, highlighting the crucial role of PVAT TLR4 in the modulation of vascular tone. Non-selective inhibition of nitric oxide synthases with L-NAME increased contractile responses; however, this increase was less pronounced in aortas treated with L-NAME in combination with LPS compared with those treated with L-NAME alone. This finding suggests reduced NO bioavailability in the presence of LPS and further supports the involvement of the NO pathway in the modulation of vascular reactivity. Activation of the ERK1/2 pathway was identified as a central event in PVAT dysfunction induced by acute TLR4 agonism. The selective MEK1/2 inhibitor PD98059 restored the anticontractile effect of PVAT, normalized NO production, and reversed the increase in inhibitory phosphorylation of eNOS at the Thr495 residue. In addition, LPS increased the production of reactive oxygen species (ROS) in both the endothelium and PVAT, an effect prevented by PD98059, indicating that ERK1/2 signaling also contributes to enhanced oxidative stress. Taken together, these findings identify the TLR4–ERK1/2–eNOS axis as a critical mediator of PVAT dysfunction induced by acute TLR4 agonism, providing a mechanistic basis for understanding how TLR4 activation suppresses eNOS function, impairs PVAT-mediated anticontractile modulation, and compromises vascular homeostasis.