Thrombosis and Atherosclerosis Research Institute (S.K.L., R.A.M., J.Z., W.W., P.L.G., J.I.W., B.L.T.), McMaster University, Hamilton, Ontario, Canada.
Hamilton Health Sciences, Ontario, Canada (S.K.L., R.A.M., J.Z., W.W., P.L.G., J.I.W., B.L.T.).
Arterioscler Thromb Vasc Biol. 2023 Nov;43(11):2165-2178. doi: 10.1161/ATVBAHA.123.319767. Epub 2023 Sep 7.
SR-B1 (scavenger receptor class B type 1)/LDLR (low-density lipoprotein receptor) double knockout mice fed a high-fat, high-cholesterol diet containing cholate exhibit coronary artery disease characterized by occlusive coronary artery atherosclerosis, platelet accumulation in coronary arteries, and myocardial fibrosis. Platelets are involved in atherosclerosis development, and PAR (protease-activated receptor) 4 has a prominent role in platelet function in mice. However, the role of PAR4 on coronary artery disease in mice has not been tested.
We tested the effects of a PAR4 inhibitory pepducin (RAG8) on diet-induced aortic sinus and coronary artery atherosclerosis, platelet accumulation in atherosclerotic coronary arteries, and myocardial fibrosis in SR-B1/LDLR double knockout mice. SR-B1/LDLR double knockout mice were fed a high-fat, high-cholesterol diet containing cholate and injected daily with 20 mg/kg of either the RAG8 pepducin or a control reverse-sequence pepducin (SRQ8) for 20 days.
Platelets from the RAG8-treated mice exhibited reduced thrombin and PAR4 agonist peptide-mediated activation compared with those from control SRQ8-treated mice when tested ex vivo. Although aortic sinus atherosclerosis levels did not differ, RAG8-treated mice exhibited reduced coronary artery atherosclerosis, reduced platelet accumulation in atherosclerotic coronary arteries, and reduced myocardial fibrosis. These protective effects were not accompanied by changes in circulating lipids, inflammatory cytokines, or immune cells. However, RAG8-treated mice exhibited reduced VCAM-1 (vascular cell adhesion molecule 1) protein levels in nonatherosclerotic coronary artery cross sections and reduced leukocyte accumulation in atherosclerotic coronary artery cross sections compared with those from SRQ8-treated mice.
The PAR4 inhibitory RAG8 pepducin reduced coronary artery atherosclerosis and myocardial fibrosis in SR-B1/LDLR double knockout mice fed a high-fat, high-cholesterol diet containing cholate. Furthermore, RAG8 reduced VCAM-1 in nonatherosclerotic coronary arteries and reduced leukocyte and platelet accumulation in atherosclerotic coronary arteries. These findings identify PAR4 as an attractive target in reducing coronary artery disease development, and the use of RAG8 may potentially be beneficial in cardiovascular disease.
喂食高脂肪、高胆固醇饮食并含有胆酸盐的 SR-B1（清道夫受体 B 类 1 型）/LDLR（低密度脂蛋白受体）双敲除小鼠会出现阻塞性冠状动脉粥样硬化为特征的冠状动脉疾病，伴有冠状动脉血小板积聚和心肌纤维化。血小板参与动脉粥样硬化的发展，PAR（蛋白酶激活受体）4 在小鼠血小板功能中起重要作用。然而，PAR4 在小鼠冠状动脉疾病中的作用尚未得到检验。
我们测试了 PAR4 抑制肽聚糖（RAG8）对饮食诱导的主动脉窦和冠状动脉粥样硬化、粥样硬化冠状动脉中的血小板积聚以及 SR-B1/LDLR 双敲除小鼠心肌纤维化的影响。喂食高脂肪、高胆固醇饮食并含有胆酸盐的 SR-B1/LDLR 双敲除小鼠，每天注射 20mg/kg 的 RAG8 肽聚糖或对照反向序列肽聚糖（SRQ8），共 20 天。
与对照 SRQ8 处理的小鼠相比，RAG8 处理的小鼠的血小板在体外测试时显示出降低的凝血酶和 PAR4 激动肽介导的激活。尽管主动脉窦粥样硬化程度没有差异，但 RAG8 处理的小鼠表现出降低的冠状动脉粥样硬化、降低的粥样硬化冠状动脉中的血小板积聚和降低的心肌纤维化。这些保护作用没有伴随着循环脂质、炎症细胞因子或免疫细胞的变化。然而，与 SRQ8 处理的小鼠相比，RAG8 处理的小鼠在非粥样硬化冠状动脉横切面上的 VCAM-1（血管细胞黏附分子 1）蛋白水平降低，在粥样硬化冠状动脉横切面上的白细胞积聚减少。
喂食高脂肪、高胆固醇饮食并含有胆酸盐的 SR-B1/LDLR 双敲除小鼠中，PAR4 抑制性 RAG8 肽聚糖减少了冠状动脉粥样硬化和心肌纤维化。此外，RAG8 减少了非粥样硬化冠状动脉中的 VCAM-1，并减少了粥样硬化冠状动脉中的白细胞和血小板积聚。这些发现表明 PAR4 是减少冠状动脉疾病发展的一个有吸引力的靶点，RAG8 的使用可能对心血管疾病有益。
