We previously reported identification of two subgroups of healthy women at high-risk for incident CVD, one with low HDL-C and high CRP levels and the other with high HDL-C and high CRP levels 
. We believe the high CRP in each case to be indicative of an underlying potentiating relationship between inflammation and HDL particles in the establishment of risk. A major finding of the current work was that high levels of HDL particles with both apoA-I and apoA-II (LpA-I:A-II) associated with incident CVD risk in the high-risk group of women with high levels of HDL-C and CRP as determined by multivariable models adjusted for relevant clinical covariates and blood markers. In the same high-risk group, apoA-II levels were found not to be associated with risk which was suggestive that apoA-II associated risk derived not from the absolute amount of apoA-II but rather from the number of particles of apoA-II-carrying HDL. An additional important finding of the current study was that LpA-I:A-II associated risk was found to interact positively with the increased risk previously demonstrated for high levels of apoE in these women 
. Regarding HDL particles with apoA-I but not apoA-II (LpA-I), there was no association of risk with LpA-I in the high HDL-C/high CRP women. Also, it should be noted that for women with low levels of HDL-C and high levels of CRP, neither LpA-I:A-II nor LpA-I associated with risk.
As stated above, we found risk in the female high HDL-C/high CRP subgroup associated with high levels of LpA-I:A-II particles but not with apoA-II levels. Regarding CVD risk and apoA-II in general, the situation is not clear in that although it is generally thought that there is an inverse relationship of risk with apoA-II levels, other studies indicate that apoA-II may be pro-atherogenic 
. Our lack of demonstrating apoA-II risk may thus be a reflection of sensitivity of potential apoA-II associated risk to salient features of specific populations. Regarding LpA-I:A-II and risk, there are few human studies directly assessing potential associations. One of particular relevance to the current study was an investigation of LpA-I and LpA-I:A-II regarding atherosclerotic lesions in hyperalphalipoproteinemic subjects. Results indicated less cardioprotective effects for higher LpA-I:A-II levels 
. Additionally, a report from the Framingham Offspring Study in an investigation involving participants with and without coronary heart disease revealed slightly but significantly higher levels of LpA-I:A-II in cases versus HDL-C-matched controls 
. On the other hand, earlier studies revealed for LpA-I:A-II levels in individuals with coronary artery disease as compared to controls, in one case lower levels 
and in another case no difference 
With regard to apoA-II and lipoprotein metabolism, there have been substantial efforts directed at elucidation of possible links. Reported actions of apoA-II involve: primarily inhibitory effects on remodeling of HDL through modulation of activities of lipid transfer proteins (CETP, PLTP), enzymes (LCAT, LPL, HL, endothelial lipase), and receptors (SR-B1, cubilin, heat shock protein); efflux of cholesterol and phospholipids; and triglyceride metabolism 
. Recent work potentially relevant to findings of the current study involved an investigation in human subjects of apoA-II metabolism in the setting of raised levels of HDL-C brought about by torcetrapib inhibition of CETP 
. Results indicated delayed catabolism of and alterations in remodeling of apoA-II containing HDL particles presumably stemming from the large size of resultant particles and apoA-II mediated inhibition of phospholipases (endothelial lipase and HL). In addition to the previously mentioned effects of apoA-II on HDL remodeling, lipid efflux, and triglyceride metabolism; human apoA-II may play a role in the anti-oxidant properties of HDL although the issue remains unclear. One study reported the efficient transport by apoA-II rich HDL of two enzymes, paraoxonase 1 (PON1) and platelet activating factor acetylhydrolase (PAF-AH), that are thought to play a leading role in anti-oxidant functionality of HDL 
. However, another study reported for human apoA-II enriched HDL, impaired protection against oxidative modification of apoB-containing lipoproteins and displacement of PON1 by apoA-II. This was thought to explain why PON1 is found mostly on LpA-I particles and why, at least in part, apoA-II-rich HDL demonstrated lack of anti-atherogenic properties 
. It should also be noted that studies in mice involving apoA-II-enriched HDL have demonstrated pro-inflammatory transformation of HDL 
. In addition to these findings, a recent study reports novel apoA-II associated pro-inflammatory activity in the suppression of lipopolysaccharide (LPS) inhibition by LPS binding proteins 
. The finding in the current study of LpA-I:A-II associated risk would thus be consistent with pro-inflammatory and pro-atherogenic characteristics of apoA-II as noted above.
Results of the current study indicated positive interaction of LpA-I:A-II with apoE in terms of CVD risk in the female high HDL-C/high CRP subgroup (HR2). In this regard it may be notable that apoA-II and apoE are known to form a heterodimeric complex that overall accounts for approximately 30% of plasma apoE in normolipidemic subjects; and furthermore, it has been reported that the complex on HDL demonstrates reduced binding affinity for the LDL receptor 
. Thus, it is tempting to speculate that the conditions of high-risk in the HR2 subgroup (high LpA-I:A-II and apoE levels) could result, in part, through formation of the apoE/apoA-II complex subsequently resulting in reduced uptake of HDL-C via the LDL receptor-mediated uptake pathway for apoE-rich HDL 
. This could increase HDL residence time in the circulation which could foster dysfunctional transformation of HDL in the inflammatory setting indicated by the high CRP defining the subgroup.
Limitations in the current study involved a number of issues. Because the central aim of the study was to examine potential risk in the female high HDL-C/high CRP subgroup as related to apoA-II levels, and as preliminary studies revealed no evidence of such risk not only for apoA-II but additionally for apoA-I; we sought instead to extend our studies to LpA-I and LpA-I:A-II particles. This necessitated an approach involving several assumptions for the estimation of these parameters. The first of these was that LpA-I and LpA-I:A-II constitute the major subclasses of HDL. This appears justified in that although HDL particles containing apoA-II without apoA-I have been reported 
, they appear to comprise a small fraction of HDL, and in general, they are not detected in the circulation 
. Another concern is that apoA-II is known to be present in VLDL 
; however, this occurs in the setting of low plasma HDL-C levels which was clearly not the case in the present study of the high HDL-C subgroup. The second assumption was that HDL particle concentration was in direct proportion to measured HDL-C. Although it is known that LpA-I and LpA-I:A-II particles are heterogeneous within each subclass with regard to size and density and consequently cholesterol content; on average, the assumption of direct proportionality seems reasonable. This notion is supported by results of previous studies demonstrating statistically significant linear correlation of HDL particle concentration with HDL-C levels 
. The third assumption was that the search for the number of apoA-I and apoA-II molecules per particle based upon goodness of fit to observed values of the k2/k1 ratio would provide valid estimates. In this regard, our estimates compared well with experimentally determined values from other studies. The value of N (number A-I molecules/LpA-I particle) in the current study ranged from 5.1 to 6.3 as compared to 3.0 to 7.0 over the total size range of HDL particles as reported in a recent study 
. Additionally, the ratio of number of A-I molecules to number of A-II molecules in LpA-I:A-II particles in the current study ranged from 0.62 to 0.94 comparing well with reported values for human HDL of 1.00 
, 1.20 
, 1.30 
, 1.56 
, and 1.89 
. In addition to these points, there were other limitations to the study including no direct data provided relating to the primacy of high levels of HDL-C, CRP, apoE, and LpA-I:A-II as related to dysfunctional transformation of HDL in the establishment of risk. These issues should be addressed in future studies by assessment of multiple facets of HDL functionality as well as physico-chemical characterization of HDL to elucidate the nature of potential dysfunctional transformation in such populations.
In summary, we have studied a potential role for apoA-II in the risk of incident CVD risk in a previously identified subgroup of healthy women defined by high levels of HDL-C and CRP 
and for whom apoE was previously shown to be a risk factor 
. Results of the current study demonstrated that high levels of apoA-II containing HDL particles (LpA-I:A-II) also associated with incident CVD risk using multivariable modeling adjusted for relevant clinical and biochemical covariates. In addition, LpA-I:A-II and apoE were found to interact positively in the establishment of risk. Regarding other related factors, it should be noted that neither HDL-C, nor HDL particles without apoA-II (LpA-I), nor apoA-I, nor apoA-II levels associated with risk in the subgroup. LpA-I:A-II associated risk in the absence of apoA-II associated risk was suggestive that the number of LpA-I:A-II particles and not the quantity of apoA-II was important in the establishment of such risk. We conclude that apoA-II and apoE, major apolipoprotein constituents of HDL, are important in the ongoing characterization of the nature of HDL particles with regard to pathophysiologic mechanisms responsible for high CVD risk in populations with concurrently high levels of HDL-C and CRP.