Atherosclerosis is a complex arterial disease characterized by vascular wall inflammation and atherosclerotic plaque accumulation. Endothelial cells play a critical role in the development of atherosclerosis, so damage to the vascular endothelium might increase the risk of triggering the pathogenesis of atherosclerosis. Our study demonstrated that reduced UGP2 expression is involved in the progression of atherosclerosis via stimulation of endothelial apoptosis in vivo and in vitro. Downregulation of UGP2 was associated with increased endothelial cells apoptosis and severe atherosclerotic plaque. With these in mind, UGP2 might be a novel molecular target for cell apoptosis and atherosclerosis.
UGP2 is an enzyme involved in the biosynthesis of glycogen by catalyzing the reaction of uridine triphosphate (UTP) and glucose-1-P to generate UDP-glucose, UDP-glucose is the activated glucose required for glycogen synthesis. UGP2 has previously been identified as a marker protein in various types of malignancies where its upregulation is correlated with a poor disease outcome[11–15]. A recent cohort study on Developmental and/or epileptic encephalopathies (DEEs) revealed that the reduction of UGP2 expression in brain cells leads to global transcriptome changes, a reduced ability to produce glycogen, alterations in glycosylation and increased sensitivity to ER stress. Changes in glycosylation can modulate inflammatory responses, promote cell metastasis or regulate apoptosis. ER stress can react in various ways, including the induction of the unfolded protein response (UPR) and apoptosis. The endothelial cells as an effective mediator to regulate the vascular system, with roles in processes such as vascular homeostasis, cell cholesterol, and signal transduction. Convincing evidence indicates that dysfunction of the endothelial cells is associated with various vascular diseases, including diabetes mellitus, arterial thrombosis and hypercholesterolemia. Endothelial cells apoptosis can destroy the vascular structure, results in macrophages infiltration and lipids deposition, and finally contributes to atherogenesis[39, 40]. Our study is the first to show a role of UGP2 in the development of atherosclerosis, and confirmed that the deletion of UGP2 promotes atherosclerosis by increasing Cleaved caspase-3 expressions and apoptosis levels in HUVECs.
As an important biological modulator, ROS not only play a role in maintaining normal physiological functions but also participate in cell damage and cell death because of their highly intensive activities. As one of the adverse stimuli, ROS mediates activation of Cleaved caspase-3 and cell apoptosis via multiple mechanisms such as impairing membrane integrity and causing mitochondrial damage[42, 43]. During atherogenesis, the primary process of endothelial cell dysfunction and apoptosis is accompanied by a secondary event of oxidative stress, high levels of ROS can induce oxidative stress that is closely associated with the pathogenesis of atherosclerosis. Low density lipoprotein (LDL) oxidation by ROS is an early event in the development of atherosclerotic lesions. In addition, ROS can lead to NF-κB activation, protein modification, and oxidative damages to other biomolecules. ROS also disrupt redox-dependent signaling in the vessel wall to promote progress of atherosclerosis. In the present study, the HUVECs were pre-treated by UGP2 siRNA before incubation with H2O2, elevation of apoptosis was found, accompanied by augmentation of ROS generation and upregulation of Cleaved caspase-3 expressions. This result indicated that ROS are indispensable for UGP2 knockdown induced apoptosis in HUVECs.
It is well-known that TP53 is a tumor-suppressor gene, playing key roles in cell cycle control and induction of apoptosis through the regulation of a battery of target genes. In response to death signals, activated TP53 regulates various genes of pro-apoptotic proteins, which are transcription-dependent or independent, leading to final cell death[20–23]. Otherwise, it has recently been shown that activated TP53 promotes cell survival and capable of anti-apoptosis ability under DNA damage by implicating in the regulation of carbohydrate metabolisms. Numerous studies revealed that TP53 is also a redox-regulating transcription factor via regulation of ROS production and the expression of various metabolizing enzymes, like TIGAR and GLS2[26, 27]. TP53 also plays a central role in atherosclerosis. Convincing evidence has showed that TP53 deters the development of atherosclerosis by inhibiting DNA damage signaling in VSMCs. In addition, TP53 protects the genome from oxidation by reactive oxygen species (ROS), a major cause of DNA damage in atherosclerosis. In the present study, we found that UGP2 was directly transactivated by TP53, and UGP2 knockdown increased ROS generation and Cleaved caspase-3 activation through a TP53-dependent manner in vivo. Therefore, it is highly likely that UGP2 is transactivated by TP53 in response to survival stress, contributes to ROS reduction, Cleaved caspase-3 downregulation and reduction of apoptosis in HUVECs, finally results in the inhibition of atherosclerosis.
In ldlr-knockout mice fed a Western high-fat diet, we found that targeted deletion of the UGP2 gene resulted in substantially increased atherosclerotic lesion areas, indicating a protective role of UGP2 against atherogenesis. Furthermore, we observed that the atherosclerotic lesions in ugp2-deficient mice had higher lipid content, reduced cap thickness, lower collagen contents and less calcification, all of which are hallmarks of vulnerable atherosclerotic plaques. In line with the findings from HUVECs, the levels of ROS and Cleaved caspase-3 expressions was upregulated and endothelial cells apoptosis was enhanced in atherosclerotic lesions of ugp2-deficient mice. As it is well established that endothelial dysregulation and apoptosis is considered as the initiating factors in the development and pathogenesis of atherosclerosis. Thus, endothelial cells apoptosis induced by UGP2 deficiency results in increased atherosclerotic lesions area and more vulnerable atherosclerotic plaques in mice.
In summary, our present study not only identified the critical glycogen metabolism enzyme UGP2 as a novel direct target of TP53, but also demonstrated a novel role of UGP2 in the regulation of apoptosis through the reduction of ROS and anti-activation of Cleaved caspase-3. Based on our current findings, further extensive studies on the function of UGP2 and glycogen metabolism might provide a clue to develop a promising strategy for atherosclerosis therapy.