Atherosclerosis 193 (2007) 283–291 Pro-apoptotic low-density lipoprotein subfractions in type II diabetes Chao-yuh Yang a,b,∗ , Hsin-Hung Chen a , Max T. Huang a , Joe L. Raya a , Jun-Hai Yang a , Chu-Huang Chen a , John W. Gaubatz a , Henry J. Pownall a , Addison A. Taylor a , Christie M. Ballantyne a , Floor A. Jenniskens c , Charles V. Smith c,d a Department of Medicine, Baylor College of Medicine, 6565 Fannin, MS A.601, Houston, TX 77030, United States b Department of Biochemistry, Baylor College of Medicine, 6565 Fannin, MS A.601, Houston, TX 77030, United States c Center for Developmental Pharmacology and Toxicology, Columbus Children’s Research Institute, Ohio State University, Columbus, OH 43205, United States d Center for Developmental Pharmacology and Toxicology, Children’s Hospital & Regional Medical Center, Seattle, WA 98101, United States Received 21 March 2006; received in revised form 27 July 2006; accepted 25 August 2006 Available online 9 October 2006 Abstract Objective: To test the hypothesis that differences in subfractions of circulating lipoproteins between diabetic and non-diabetic subjects exist and might contribute to the increased risk for atherosclerosis in type II diabetics. Methods and results: LDL isolated from diabetic (D) and control subjects (N) were separated by FPLC into five subfractions (L1–L5). The fractional distributions of N- and D-LDL were not different, but the most strongly retained subfractions of D-LDL (D-L5) were markedly more pro-apoptotic to bovine aortic endothelial cells in vitro than were the other subfractions in D- or N-LDL. D-L5 induced time- and concentration-dependent apoptosis that was inhibited by z-VAD-fmk. The most electronegative D-LDL subfractions contained substantial amounts of apoproteins AI, E and CIII, higher concentrations of non-esterified fatty acids and LpPLA2, and lower trinitrobenzenesulfonic acid (TNBSA) reactivities. Electronegative subfractions of D-LDL exhibited longer lag times and lower net increases in absorbance at 234 nm with Cu-catalyzed oxidation in vitro. Conclusions: The toxicities of electronegative subfractions of LDL from diabetic subjects to endothelial cells in vitro may be pivotal to vascular complications of diabetes in vivo, but the specific molecular alterations responsible for the toxicities of these subfractions of diabetic LDL are not known. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Electronegative LDL; Atherosclerosis; Diabetes; Apoptosis; Fatty acids; Lipoprotein phospholipase A2 1. Introduction Diabetes mellitus is characterized by abnormal carbohy- drate, lipid and protein metabolism, resulting from defects in insulin secretion, insulin action or both. Type II diabetes accounts for most diabetes and, of these, 90% are obese [1]. The Multiple Risk Factor Intervention Trial (MRFIT) study ∗ Corresponding author at: Department of Medicine, Baylor College of Medicine, 6565 Fannin, MS A.601, Houston, TX 77030, United States. Tel.: +1 713 798 4210; fax: +1 713 798 4121. E-mail address: cyang@bcm.tmc.edu (C.-y. Yang). reported that the absolute risk for death from cardiovascular disease (CVD) was much higher for diabetic men than for non-diabetic men of every age group, ethnic background and risk factor level [2]. Overall, the risk was three times higher for diabetics than for non-diabetics. Rates of coronary heart disease (CHD) mortality in type II diabetic patients with no history of myocardial infarction are as high as in non-diabetics with a history of myocardial infarction [3]. Diabetes is a strong, independent risk factor for atheroscle- rotic cardiovascular disease (ACVD), which is the major cause of morbidity and mortality in individuals with types I and II diabetes. However, the specific mechanisms by which 0021-9150/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2006.08.059