Atherosclerosis is a chronic inflammatory arterial disease characterized by build-up of atheromatous plaque, which narrows the lumen of arteries. Hypercholesterolemia and excessive oxidative stress in arterial walls are among the main causative factors of atherosclerosis. Transient receptor potential channel M2 (TRPM2) is a Ca²⁺-permeable cation channel activated by oxidative stress. However, the role of TRPM2 in atherosclerosis in animal models is not well studied. In the present study, with the use of adeno-associated virus (AAV)-PCSK9 and TRPM2 knockout (TRPM2^−/−) mice, we determined the role of TRPM2 in hypercholesterolemia-induced atherosclerosis. Our results demonstrated that TRPM2 knockout reduced atherosclerotic plaque area in analysis of En face Oil Red O staining of both whole aortas and aortic-root thin sections. Furthermore, TRPM2 knockout reduced the expression of CD68, α-SMA, and PCNA in the plaque region, suggesting a role of TRPM2 in promoting macrophage infiltration and smooth-muscle cell migration into the lesion area. Moreover, TRPM2 knockout reduced the expression of ICAM-1, MCP-1, and TNFα and decreased the ROS level in the plaque region, suggesting a role of TRPM2 in enhancing monocyte adhesion and promoting vascular inflammation. In bone-marrow-derived macrophages and primary cultured arterial endothelial cells, TRPM2 knockout reduced the production of inflammatory cytokines/factors and decreased ROS production. In addition, a TRPM2 antagonist N-(p-amylcinnamoyl) anthranilic acid (ACA) was able to inhibit atherosclerotic development in an ApoE^−/− mouse model of atherosclerosis. Taken together, the findings of our study demonstrated that TRPM2 contributes to the progression of hypercholesterolemia-induced atherosclerosis. Mechanistically, TRPM2 channels may provide an essential link that can connect ROS to Ca²⁺ and inflammation, consequently promoting atherosclerotic progression.

Cardiac fatty acid metabolism is essential for maintaining normal cardiac function at baseline and in response to various disease stress, like diabetes. EP4 is widely expressed in cardiomyocytes and has been demonstrated to play a role in cardio function. However, its function in regulating cardiac fatty acid metabolism is remained unknown. Mice were fed with standard chow or high-fat for eight weeks. The effects of EP4 deficiency on cardiac function, cardiomyocytes hypertrophy and myocardial fibrosis were studied. The possible regulatory mechanisms were further investigated. EP4-/- mice exhibited concentric hypertrophy and myocardial fibrosis with cardiac energy deprivation due to reduction of fatty acid uptake and inhibition of ATP generation mediated by FOXO1/CD36 signalling. Moreover, pharmacologically activated EP4 alleviated impaired fatty acid transport and insufficient ATP generation in cardiomyocytes. EP4 tightly coordinates the rates of cardiac fatty acid uptake and ATP generation via FOXO1/CD36 signalling axis. Our study provides evidences for the link between EP4 and cardiac fatty acid transport and further pointed out that EP4 could be a potential target for modulating fatty acid metabolism and curbing cardiac tissue-specific impairment of function following diabetes.

Thioredoxin is one of the most important redox regulating proteins. Although thioredoxin has been shown to protect cells against different kinds of oxidative stress, the role of thioredoxin in myocardial ischemia and reperfusion injury has not been fully understood. This study was conducted to explore the protective role of human thioredoxin on myocardial ischemia and reperfusion injury and its potential mechanisms. Purified human thioredoxin was injected into adult Wister rats, which were subjected to 30 minutes of myocardial ischemia followed by 2 or 24 hours of reperfusion. We detected 1) the infarct size; 2) the level of malondisldehyde (MDA) in serum; 3) the expression of caspase-9, and cytochrome c in/out of mitochondia by Western blotting; 4) apoptosis by terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) assay and caspase-3 and its protein by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting; 5) the expression of bcl-2 and bax in cardium by immunohistochemical (IHC) assay. Human thioredoxin reduced myocardial ischemia/reperfusion injury as evidenced by significant decrease of myocardial infarct size (P<0.01), notable reduction of myocyte apoptosis (P <0.01), lower systemic oxidative stress level (P <0.01) after reperfusion for 2 hours, and few inflammatory cell infiltration after reperfusion for 24 hours in rats. Furthermore, treatment with human thioredoxin significantly reduced the release of mitochondrial cytochrome C (P<0.05), and inhibited the activity of caspase-9 (P <0.05) and caspase-3 (P <0.01 in mRNA and P <0.05 at protein level). Meanwhile, human thioredoxin markedly increased bcl-2 expression (P<0.05). These results strongly suggest that human thioredoxin has cardioprotective effects on myocardial ischemia/reperfusion and its anti-apoptotic role may be mediated by modulating bcl-2 and the mitochondria-dependent apoptotic signaling pathway.

Background/Objective: Atherosclerosis is one of the leading causes of cardiovascular diseases and mortality around the world. One exciting strategy for atherosclerosis treatment is immunotherapy, especially active immunization. Active immunization relies on the delivery of antigens in a vaccine platform to introduce humoral and cellular immunity, alleviating atherosclerotic progression. Transient receptor potential channel isoform M2 (TRPM2) is an ROS-activated Ca²⁺-permeable ion channel that can promote atherosclerosis via stimulating vascular inflammation. In the present study, we developed a strategy of active immunization with the TRPM2 E3 domain peptide in a vaccine platform, aiming to induce the endogenous production of anti-TRPM2 blocking antibody in mice in vivo, consequently inhibiting TRPM2 channel activity to alleviate atherosclerotic progression. Methods: ApoE knockout mice were fed with a high cholesterol diet to develop atherosclerosis. The mice were injected with or without the E3 peptide vaccines, followed by analysis of atherosclerotic lesion by en face Oil Red O staining of the whole aorta and histologic analysis of thin tissue sections from aortic roots. Results: The results show that immunization with a pig TRPM2 E3 region-based peptide (P1) could effectively alleviate high cholesterol diet-induced atherosclerosis in ApoE knockout mice. We worked out the best vaccine formulation for the most effective atheroprotection, namely P1 at the dose of 67.5 µg per mouse (2.5 mg/kg body weight) with aluminum salts as adjuvant. Conclusions: The present study provides a novel target TRPM2 for peptide vaccine-based anti-atherosclerotic strategy and lays the foundation for future preclinical/clinical trials using TRPM2 E3 P1 vaccine for a potential therapeutic option against atherosclerosis.

An abnormality of Ca²⁺ signaling may aggravate lipid accumulation in steatotic hepatocytes, leading to non-alcoholic fatty liver disease. However, the molecular identity of Ca²⁺-permeable channels and the mechanism of involvement of these channels in steatotic hepatocytes are not well-studied. In the present study, we investigated the role of a Ca²⁺-permeable channel TRPM2 in lipid metabolism in steatotic hepatocytes. A mouse model of non-alcoholic fatty liver disease was established by high-fat-diet feeding. Fat accumulation, fibrosis, lipophagic indexes, TFEB and lysosomal acid lipase in the liver tissue and/or hepatocytes were compared between TRPM2-knockout mice and wild-type mice. Knockout of the TRPM2 gene aggravated liver fat accumulation and fibrosis. Mechanistically, the TRPM2 knockout impaired the lipophagic process, decreased lysosomal abundance and attenuated lysosomal/autolysosomal acidification in mouse hepatocytes. Furthermore, the TRPM2 knockout reduced TFEB expression and its nuclear translation and also reduced the expression/activity of lysosomal acid lipase. These data demonstrate that TRPM2 deficiency may reduce lipophagy via its action on TFEB and lysosomal acid lipase, consequently contributing to liver steatosis and NAFLD under high-fat feeding conditions.

Atherosclerosis is one of the leading causes of cardiovascular diseases and mortality around the world. One exciting strategy for atherosclerosis treatment is immunotherapy, especially active immunization. Active immunization relies on the delivery of antigens in a vaccine platform to introduce humoral and cellular immunity alleviating atherosclerotic progression. TRPM2 is an ROS-activated Ca2+-permeable ion channel that can promote atherosclerosis via stimulating vascular inflammation. In the present study, we developed a strategy of active immunization with TRPM2 E3 domain peptide in a vaccine platform, aiming to induce endogenous production of anti-TRPM2 blocking antibody in mice in vivo, consequently inhibiting TRPM2 channel activity to alleviate atherosclerotic progression. The results show that immunization with a pig TRPM2 E3 region-based peptide (P1) could effectively alleviate high cholesterol diet-induced atherosclerosis in ApoE-/- mice. We worked out the best vaccine formulation for most effective atheroprotection, namely P1 at the dose of 67.5 µg per mouse (2.5 mg/kg body weight) with aluminium salts as adjuvant. The present study laid the foundation for future clinical trials using TRPM2 E3 vaccine for potential therapeutic option against atherosclerosis.