Abstract:
Endothelial dysfunction is a hallmark of diabetic vasculopathies. Although hyperglycemia is believed to be the culprit causing endothelial damage, the mechanism underlying early endothelial insult in prediabetes remains obscure. The majority of studies attributed early endothelial damage to an impairment in nitric oxide (NO)-mediated vasodilation in isolation of other endothelial mechanisms. Nevertheless, the low-grade inflammation process and the associated imbalances in lipid assimilation implicated in the pathogenesis of metabolic disease are known to affect the molecular effectors of endothelium-dependent hyperpolarization (EDH). Indeed, such an early deterioration in the interplay between EDH and NO pathways, culminating in endothelial dysfunction, might be triggered by a lipid mediator. Yet, whether the contribution of EDH-type relaxation to the integrative endothelial response is altered in the prediabetic stage remains unclear. To address this issue, we used a non-obese high-calorie-fed rat model with hyperinsulinemia, hyperlipidemia, and delayed development of hyperglycemia. Compared with aortic rings from control rats, HC-fed rat rings displayed attenuated acetylcholine-mediated relaxation. While sensitive to nitric oxide synthase (NOS) inhibition, aortic relaxation in HC-rat tissues was not affected by blocking the inward-rectifier potassium (Kir) channels using BaCl2. Although Kir channel expression was reduced in HC-rat aorta, Kir expression, endothelium-dependent relaxation, and the BaCl2-sensitive component improved in HC rats treated with atorvastatin to reduce serum lipid. Remarkably, HC tissues demonstrated increased reactive species (ROS) in smooth muscle cells, which was reversed in rats receiving atorvastatin. In vitro ROS reduction, with superoxide dismutase, improved endothelium-dependent relaxation in HC-rat tissues. Significantly, connexin-43 expression increased in HC aortic tissues, possibly allowing ROS movement into the endothelium and reduction of eNOS activity. In this context, gap junction blockade with 18-β-glycyrrhetinic acid reduced vascular tone in HC rat tissues but not in controls. This reduction was sensitive to NOS inhibition and SOD treatment, possibly as an outcome of reduced ROS influence, and emerged in BaCl2-treated control tissues. Such vascular alterations in HC-fed rats were accompanied by a localized perivascular adipose tissue (PVAT) inflammation, as manifested by an increase in macrophage infiltration and inflammatory markers exclusively in PVAT depot. In this context, custom-designed novel multi-targeting drug ligands (MTDLs), targeting COX-2, 15-LOX, and/or PPARγ, were developed in an attempt to halt inflammation. In vitro, in silico and biological screening of these compounds demonstrated selective but moderate COX-2/15-LOX inhibitory activity as well as partial PPARγ agonistic actions. Given that the pro-inflammatory phenotype of PVAT is thought to drive FFA spill-over possibly underlying the observed endothelial derangements, we tested whether the modulation of different inflammatory targets will ameliorate the observed endothelial insults at this stage. Therefore, endothelial dysfunction was studied in rat thoracic aortic rings resulting from ex vivo exposure to increased concentrations of saturated fatty acids. Only treatment with anti-inflammatory triple targeting MTDLs affecting COX-2, 15-LOX, and PPAR restored endothelium-dependent relaxation. Whereas the impact of the triple-targeting MTDLs appeared to be similar to the single targeting pioglitazone in acute ex vivo exposure, it was more effective on aortic tissues isolated from prediabetic rats with chronic vascular inflammation. Importantly, treatment with the triple targeting MTDL restored Kir channel expression in tissues exposed to palmitic acid. Altogether, these results suggest that early metabolic challenge, associated with local PVAT inflammation, leads to reduced Kir-mediated EDH, increased vascular ROS potentially impairing NO synthesis and highlight these channels as a possible target for early intervention with vascular dysfunction in metabolic disease. Also, our results provide insights on the potential efficacy of using novel anti-inflammatory agents to alleviate vascular dysfunction in metabolic disease.