Abstract
Endothelial dysfunction, referring to a disturbance in the vascular homeostasis, has been implicated in many disease conditions including ischemic/reperfusion injury and atherosclerosis. Endothelial mitochondria have been increasingly recognized as a regulator of calcium homeostasis which has implications in the execution of diverse cellular events and energy production. The mitochondrial calcium uniporter complex through which calcium enters the mitochondria is composed of several proteins, including the pore-forming subunit MCU and its regulators MCUR1, MICU1, and MICU2. Mitochondrial calcium overload leads to opening of MPTP (mitochondrial permeability transition pore) and results in apoptotic cell death. Whereas, blockage of calcium entry into the mitochondria results in reduced ATP production thereby activates AMPK-mediated pro-survival autophagy. Here, we investigated the expression of mitochondrial calcium uniporter complex components (MCU, MCUR1, MICU1, and MICU2), induction of autophagy and apoptotic cell death in endothelial cells in response to oxygen-glucose deprivation. Human pulmonary microvascular endothelial cells (HPMVECs) were subjected to oxygen-glucose deprivation (OGD) at 3-h timepoints up to 12 h. Interestingly, except MCUR1 which was significantly downregulated, all other components of the uniporter (MCU, MICU1, and MICU2) remained unchanged. MCUR1 downregulation has been shown to activate AMPK mediated pro-survival autophagy. Similarly, MCUR1 downregulation in response to OGD resulted in AMPK phosphorylation and LC3 processing indicating the activation of pro-survival autophagy. Despite the activation of autophagy, OGD induced Caspase-mediated apoptotic cell death. Blockade of autophagy did not reduce OGD-induced apoptotic cell death whereas serum starvation conferred enough cellular and functional protection. In conclusion, the autophagic flux induced by MCUR1 downregulation in response to OGD is insufficient in protecting endothelial cells from undergoing apoptotic cell death and requires enhancement of autophagic flux by additional means such as serum starvation.
Highlights
Endothelial cells are essential regulators of vascular function
To determine the expression pattern of the mitochondrial calcium uniporter complex components in Human pulmonary microvascular endothelial cells (HPMVECs) in normal and oxygen-glucose deprivation (OGD) conditions, protein levels of MCU, MCUR1, MICU1, and MICU2 were assessed by western blot analysis (Figure 1A)
These findings suggest that OGD results in downregulation of MCUR1, the positive regulator of the mitochondrial calcium uniporter complex
Summary
Endothelial cells are essential regulators of vascular function. Endothelial dysfunction is widely implicated in the development and progression of many vascular diseases (Deedwania, 2003; Gutierrez et al, 2013). Mitochondrial matrix calcium regulates important cofactors for enzymes involved in the Krebs cycle – namely pyruvate dehydrogenase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase (Mallilankaraman et al, 2012a; Tarasov et al, 2012; Vatrinet et al, 2017). These enzymes are essential players of Krebs cycle which provides reducing equivalents to the electron transport chain (ETC), thereby contributing to the majority of mitochondrial ATP production (Quijano et al, 2016). Mitochondrial Ca2+ signaling has been shown to regulate NO production in endothelial cells (Williams et al, 2013; Park and Park, 2015)
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