Protective Effects of Vitamin D Against Cardiovascular Disease in Diabetes

Abstract

Chronic hyperglycemia is the major initiator of diabetic vascular complications that are likely mediated by advanced glycation end-products (AGEs). The aim of this study was to assess the protective action of calcitriol (vitamin D3) in an in-vitro model of hyperglycaemic injury. Vascular impairment was induced by treating isolated rat macrovasculature with methylglyoxal (MG), a precursor of AGEs. Concurrently, the role of the phosphoinositide 3-kinase (PI3K)/ mammalian target of rapamycin (mTOR) signaling pathway and its impact on the autophagic process were investigated. The pulmonary artery and aortic rings were isolated from adult male Wistar rats (250-300g). Functional reactivity was measured as vasodilator response to acetylcholine (ACh) in vascular rings pre-constricted with the α1-adrenoceptor agonist phenylephrine (PE). To determine optimal experimental conditions, vascular reactivity was studied at 1-3 h (short-term) and after 20 h incubation in culture medium containing MG (100-400 µM). Consequently, the effects of 5 nM calcitriol and 200 nM rapamycin (an mTOR inhibitor) on vascular dysfunction induced by MG were determined. Alongside functional vascular reactivity, Western blot analysis of phosphorylated mTOR complex 1 (mTORC1) expression and a downstream biomarker of autophagy, Unc-51-like autophagy activating kinase 1 (ULK-1) was conducted in rat aortic lysates. The expression of microtubule-associated protein light chain 3 (LC3, an intracellular marker of autophagosome formation) was assessed in isolated rat aortic smooth muscle cells (SMCs), using confocal immunofluorescent microscopy. The effect on endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS) gene expressions were also assessed in rat aortic lysates using quantitative polymerase chain reaction (qPCR). Finally, the role of the PI3K signaling upstream of mTOR activation was studied by measuring phosphorylation of protein kinase B (Akt at T308 and S473) using time-resolved Fӧrster resonance energy transfer (TR-FRET) in 4 µm thick rat aortic slices. MG induced a concentration- and time-dependent decrease in ACh-mediated relaxation in both vessels, but the pulmonary artery was more sensitive. The MG effect was mainly due to endothelial dysfunction. Notably, intact eNOS function exerted additional protection against MG damage as evident from the experiments with eNOS inhibitor and denudation of the endothelium. The MG-induced vasculature dysfunction, and associated increased phosphorylation of mTOR, were reversed by calcitriol and rapamycin. Calcitriol and rapamycin reversed the MG-mediated: i) phosphorylation of ULK-1; ii) the increased levels of LC3 in isolated aortic SMCs, and iii) the decreased eNOS, but not iNOS, gene expression, implicating the role of reduced autophagy and eNOS in MG-induced injury. Finally, calcitriol and rapamycin inhibited the MG-mediated phosphorylation and activation of Akt at T308 suggesting the involvement of PI3K/Akt pathway acting upstream of mTORC1 activation. In conclusion, vitamin D3 plays an important role in protecting macrovessels against AGEs-induced injury via inhibition of mTOR-dependent inhibition of autophagy and reduced eNOS expression that involves multiple pathways including inhibition of the PI3K/Akt signaling. These mechanisms suggest vitamin D3 could have a protective effect in AGE-induced vascular injuries in diabetes.

Date of Award	24 Jan 2025
Original language	English
Awarding Institution	University of Bath
Sponsors	The Egyptian Government
Supervisor	Sergey Smirnov (Supervisor), Malcolm Watson (Supervisor) & Sarah Bailey (Supervisor)