1.1 Mechanisms behind increased ROS production in ECs
At moderate concentrations, ROS play an important role in maintaining the proliferation and survival of ECs, but excessive ROS have detrimental effects on the vascular system (Forstermann et al. , 2017). In patients with DM, hyperglycaemia accelerates protein glycation and forms advanced glycation end products (AGEs) (Kay et al. , 2016). These AGEs bind with cell surface receptors for AGEs (RAGE) to activate downstream signaling pathways, such as extracellular signal-regulated kinase (ERK), and subsequently increase ROS production (Yuan et al. , 2019). Excessive ROS also trigger nuclear poly ADP-ribose polymerase, which inhibits activity of glyceraldehydes-3-phosphate dehydrogenase (GADPH) and activates the polyol pathway of glucose (Giri et al. , 2018). Activated polyol pathway produces ROS via depleting nicotinamide adenine dinucleotide phosphate and glutathione, as well increasing the oxidation of nicotinamide adenine dinucleotide hydrogen during the conversion of sorbitol to fructose. Inhibition of GADPH also accelerates the generation of diacylglycerol, subsequently activating protein kinase C (PKC) and stimulating nicotinamide adenine dinucleotide phosphate oxidases (NOXs) to generate additional ROS (Yuan et al. , 2019). Mitochondria are the central regulators for aerobic energy generation, and ROS are the essential by-product during this process. Diabetes and hyperglycemia disrupt the mitochondrial respiratory chain and alter mitochondrial ultrastructure (e.g. mitochondrial fission and fusion), thereby increasing the ROS production within mitochondria (Forresteret al. , 2018; Brownlee, 2001). Hyperglycemia upregulates both abundance and activity of the sodium-hydrogen exchanger (NHE) within ECs (Klug et al. , 2021). Activated NHE promotes the influx of sodium influx and enhances intracellular calcium (Ca2+) via triggering sodium-calcium exchanger (Baartscheer et al. , 2017). Increased intracellular sodium (Na+) triggers the sodium-calcium exchanger (NCX) and enhances calcium (Ca2+) influx into the cytosol. The increased cytosolic Ca2+ then stimulates the PKC-NOXs pathway, which further increases ROS production (Rastogi et al. , 2016). Recently, Uthman et al have directly proven the causal link between NHE activity and oxidative stress in ECs: tumor necrosis factor-α (TNF-α) enhanced NHE activity and intracellular sodium (Na+), as well ROS production, and the increased ROS generation was mitigated by cariporide, a potent inhibitor for NHE. The crucial role of NHE/Na+-axis in inflammatory related oxidative stress was further supported by the fact that sodium pump inhibitor ouabain increased intracellular Na+ and ROS production in human ECs (Uthman et al. , 2022).
Excessive ROS increase vascular tone and undermine cardiac inotropic function, contributing to cardiomyopathy (Ritchie et al. , 2020). Oxidative stress causes endothelial nitric oxide synthase (eNOS) uncoupling and impairs NO production, the key vasodilator. ROS produced by NOXs also oxidize the sarcoendoplasmic reticulum calcium transport ATPase and limit the sensitivity of SMC to NO (Griendling et al. , 2021). Besides, ROS induce vascular stiffness via upregulating the expression of vasoactive factors like vascular endothelial growth factor (VEGF) and extracellular proteins like matrix metalloproteinases (Griendling et al. , 2021). Vascular remodeling elevates blood pressure and increases the intensity of cyclic stretch caused by vasoconstriction-dilation circles (Ohishi, 2018). Enhanced stretch might exacerbate oxidative stress via upregulating expression of NOXs in ECs, further increasing ROS production within in ECs (Li et al. , 2021). Besides, oscillatory shear stress created by disturbed blood flow also induces oxidative stress in ECs via activating NOXs (Siu et al. , 2016).
Depletion of nitric oxide (NO) is a crucial mediator for ROS related cardiac dysfunction (Shah et al. , 2021). EC-derived NO triggers soluble guanylate cyclase (cGMP) and protein kinase G (PKG) of adjacent CMs and leads to the phosphorylation of troponin I and reduction of myofilament Ca2+ sensitivity, thus enhancing myocardial relaxation in both isolated CMs and whole hearts (Królet al. , 2021; Feil et al. ). Activation of the NO/cGMP/PKG signaling pathway also maintains phosphorylation of titin within CMs and prevents the development of cardiac hypertrophy (Shah et al. , 2021). Moreover, oxidative stress leads to increased secretion of pro-inflammatory cytokines and chemokines from ECs, as well as upregulated expression of adhesion molecules and enhanced monocyte-endothelial attachment (Yuan et al. , 2019). Intensified ROS production activates src family kinase (SFK) to phosphorylate vascular endothelial-cadherin (VE-cadherin), leading to VE-cadherin internalisation and adherens junction disruption. Activated SFK also promotes the transformation from G actin to F actin to generate stress fibres under the cellular membrane, increasing the intracellular tension (Zhang et al. , 2017). ROS are also involved in the endothelial dysfunction induced by stretch and oscillatory shear stress through intracellular cascades, such as mitogen-activated protein kinase p38 (p38 MAPK), ERK, c-Jun N-terminal kinase (JNK) and nuclear factor kappa B (NF-κB) (Lehoux, 2006). The pivotal role of EC-derived ROS in development of cardiovascular disease is summarised in Figure 1.