Katsutoshi Miyosawa

New CETP Inhibitor K-312 Reduces PCSK9 Expression: A Potential Effect on LDL Cholesterol Metabolism

Despite significant reduction of cardiovascular events by statin treatment, substantial residual risk persists, driving emerging needs for the development of new therapies. We identified a novel cholesteryl ester transfer protein (CETP) inhibitor, K-312, that raises HDL and lowers LDL cholesterol levels in animals. K-312 also suppresses hepatocyte expression of proprotein convertase subtilisin/kexin 9 (PCSK9), a molecule that increases LDL cholesterol. We explored the underlying mechanism for the reduction of PCSK9 expression by K-312. K-312 inhibited in vitro human plasma CETP activity (IC50; 0.06 μM). Administration of K-312 to cholesterol-fed New Zealand White rabbits for 18 wk raised HDL cholesterol, decreased LDL cholesterol, and attenuated aortic atherosclerosis. Our search for additional beneficial characteristics of this compound revealed that K-312 decreases PCSK9 expression in human primary hepatocytes and in the human hepatoma cell line HepG2. siRNA silencing of CETP in HepG2 did not compromise the suppression of PCSK9 by K-312, suggesting a mechanism independent of CETP. In HepG2 cells, K-312 treatment decreased the active forms of sterol regulatory element-binding proteins (SREBP-1 and -2) that regulate promoter activity of PCSK9. Chromatin immunoprecipitation assays demonstrated that K-312 decreased the occupancy of SREBP-1 and SREBP-2 on the sterol regulatory element of the PCSK9 promoter. PCSK9 protein levels decreased by K-312 treatment in the circulating blood of cholesterol-fed rabbits, as determined by two independent mass spectrometry approaches, including the recently developed, highly sensitive parallel reaction monitoring method. New CETP inhibitor K-312 decreases LDL cholesterol and PCSK9 levels, serving as a new therapy for dyslipidemia and cardiovascular disease.

Mass spectrometry meets the challenge of understanding the complexity of the lipoproteome: recent findings regarding proteins involved in dyslipidemia and cardiovascular disease

Despite the fact that link between dyslipidemia and atherosclerosis was made over 100 years ago, atherosclerosis remains a major cause of morbidity and mortality worldwide. Major efforts focus towards understanding lipid metabolism, particularly by studying its particle compartments in circulation: the lipoproteins. In recent years, mass spectrometry has played an integral role in the deep sequencing of the lipoproteome and in metabolism studies conducted in vivo. This review highlights the path of lipoprotein research towards state-of-the-art mass spectrometry with special emphasis on the method of selected reaction monitoring and its impact on apolipoprotein metabolism studies. Also presented is what is expected for the lipoprotein field with the recent advent of high resolution/accurate mass parallel reaction monitoring mass spectrometry. The benefits of high resolution/accurate mass measurements are demonstrated by example instrument workflows and by detailing a novel method to quantify very low levels of circulating proprotein convertase subtilisin-kexin type 9 in rabbit. It is anticipated that future clinical studies or clinical trials aimed to treat dyslipidemia by manipulating key regulatory proteins will benefit from the new and exciting opportunities afforded by the latest technologies in mass spectrometry.