SUMMARY (Project 1) Our long-term goal is to identify the structural and functional features responsible for HDL’s cardioprotective functions in humans, which may have important implications for predicting cardiovascular disease (CVD) risk and developing therapeutics targeted to HDL. The main goal of Project 1 is to determine the impact of specific subspecies of HDL on cardiovascular risk in humans and to determine the factors that govern the sizes of HDL in vivo. The central hypothesis is that different sizes of HDL have very different abilities to promote cholesterol efflux from macrophages by the ABCA1 pathway. We recently showed that smaller HDLs promote cholesterol efflux by the ABCA1 pathway much more strongly than larger forms of HDL. Moreover, in a study of over 550 heart-healthy patients with type 1 diabetes (T1D), we found that a low level of extra-small HDL was the strongest predictor of an increased risk of cardiac death, revascularization, and MI. Using chemical crosslinking, proteolysis, and MS/MS analysis, we demonstrated that apolipoprotein A-I (APOA1) forms two isomers (LL5/5 and LL5/4), which we termed rotamers, in human HDL. Expression of the LL5/4 rotamer in mice selectively elevated levels of extra-small HDL with enhanced cholesterol efflux capacity. Based on these observations, we propose two specific aims. Aim 1 will extend our observations in patients with T1D to patients with type 2 diabetes (T2D). Using state-of-the art methods we developed to measure the concentration of total HDL (HDL-P) and the sizes and concentrations of four HDL subspecies (extra-small, small-, medium- and large-HDL), we will determine if specific sizes of HDL predict CVD risk more strongly than total HDL-P and independently of traditional lipid-risk factors in 500 patients with T2D in Look AHEAD, a prospective study of incident CVD risk. We will complement these studies by determining whether low levels of extra-small HDL predict incident CVD risk in a validation cohort of T1D patients. We will also determine if cholesterol efflux capacity, HDL oxidation, and HDL’s anti-inflammatory properties predict CVD risk. Aim 2 will determine the impact of the two major APOA1 rotamers on the sizes and concentrations of HDL, cholesterol efflux capacity of HDL, and atherosclerosis in humanized mouse models. Because we previously showed that low levels of extra-small HDL strongly predict incident CVD in patients with T1D, we will study both nondiabetic and diabetic mice, using a validated mouse model of T1D. We will complement our mouse mechanistic studies by analyzing the association between rotamer distribution and CVD risk in patients with T1D and T2D. The demonstration that HDL’s structural features associate with its size, function, and CVD risk will provide mechanistic and translational insights into HDL’s cardioprotective functions.