Abstract
Objective:Mitochondrial Ca2+ overload is implicated in hyperglycaemia-induced endothelial cell dysfunction, but the key molecular events responsible remain unclear. We examined the involvement of mitochondrial calcium uniporter, which mediates mitochondrial Ca2+ uptake, in endothelial cell dysfunction resulting from high-glucose treatment.Methods:Human umbilical vein endothelial cells were exposed to various glucose concentrations and to high glucose (30 mM) following mitochondrial calcium uniporter inhibition or activation with ruthenium red and spermine, respectively. Subsequently, mitochondrial calcium uniporter and mitochondrial calcium uniporter regulator 1 messenger RNA and protein expression was measured by real-time polymerase chain reaction and western blotting. Ca2+ concentrations were analysed by laser confocal microscopy, and cytoplasmic and mitochondrial oxidative stress was detected using 2′,7′-dichlorofluorescein diacetate and MitoSOX Red, respectively. Apoptosis was assessed by annexin V-fluorescein isothiocyanate/propidium iodide staining, and a wound-healing assay was performed using an in vitro model.Results:High glucose markedly upregulated mitochondrial calcium uniporter and mitochondrial calcium uniporter regulator 1 messenger RNA expression, as well as protein production, in a dose- and time-dependent manner with a maximum effect demonstrated at 72 h and 30 mM glucose concentration. Moreover, high-glucose treatment significantly raised both mitochondrial and cytoplasmic Ca2+ and reactive oxygen species levels, increased apoptosis and compromised wound healing (all p < 0.05). These effects were enhanced by spermine and completely negated by ruthenium red, which are known to activate and inhibit mitochondrial calcium uniporter, respectively.Conclusion:Mitochondrial calcium uniporter plays an important role in hyperglycaemia-induced endothelial cell dysfunction and may constitute a therapeutic target to reduce vascular complications in diabetes.
Highlights
Endothelial cells (ECs) play a crucial role in maintaining vascular homeostasis,[1] and dysfunction of the vascular endothelium is associated with the pathogenesis of cardiovascular diseases.[2]
Total Reactive oxygen species (ROS) and mitochondrial O2− levels in human umbilical vein endothelial cells (HUVECs) treated with high glucose (HG) were remarkably higher than in cells exposed to the NG and Mnt condition (p < 0.05) (Figure 2(b))
There was no significant difference in [Ca2+]mito, [Ca2+]cyt, total ROS and mitochondrial O2− levels between NG and Mnt group (p > 0.05) (Figure 2).HG increased the expression of Mitochondrial calcium uniporter (MCU) and Mitochondrial calcium uniporter regulator 1 (MCUR1) and resulted in heightened calcium and oxidative stress levels in the cytoplasm and mitochondria of HUVECs
Summary
Endothelial cells (ECs) play a crucial role in maintaining vascular homeostasis,[1] and dysfunction of the vascular endothelium is associated with the pathogenesis of cardiovascular diseases.[2] compelling evidence has implicated dysfunctional ECs in the development of diabetic vascular diseases,[3,4] the significance of which is underscored by the World Health Organization’s estimate that there are currently 422 million patients with diabetes worldwide.[5] The classical in vitro model of diabetes development comprises the induction of endothelial dysfunction by high glucose (HG) levels. Understanding the molecular mechanism by which vascular EC dysfunction arises under HG conditions may be useful for both the prevention and treatment of diabetic vascular complications.
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