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Epidemiological studies have shown an inverse correlation between plasma high density lipoprotein (HDL) concentrations and cardiovascular disease risk. At variance with these observations, clinical trials that significantly raised plasma HDL-C levels did not have improved clinical outcomes, emphasizing the importance of understanding HDL function. Recently, a significant correlation has been reported between human procollagen c-endopeptidase enhancer protein 2 (PCPE2) single nucleotide polymorphisms and HDL. PCPE2, a 52 kDa glycoprotein found in the extracellular matrix enhances cleavage of C-terminal procollagen by bone morphogenetic protein 1. Mice lacking PCPE2 have elevated concentrations of enlarged plasma HDL, a phenomenon associated with defective cholesterol efflux. HDL synthesis depends on ABCA1-mediated lipid efflux to lipid-poor apoA-I balanced by cholesterol uptake through hepatic scavenger receptor class B type I (SR-BI). Our studies focused on investigating if the elevated concentration of enlarged plasma HDL in PCPE2 deficient mice was atheroprotective. PCPE2 deficient mice were crossed with LDLr -/- mice (SKO) giving LDLr -/- , PCPE2 -/- (DKO) mice that had elevated HDL levels compared to SKO mice. Despite elevated HDL levels, we found that DKO mice had significantly more lipid and CD68+ infiltration into the aortic root, similar to that reported for LDLr -/- , apoA-I -/- mice that lack plasma apoA-I/HDL. Furthermore, DKO mice showed reduced HDL apoA-I fractional clearance and reverse cholesterol transport rates compared to SKO mice suggesting PCPE2 plays a significant role in HDL remodeling and/or cholesterol uptake by the liver. To test the effect of PCPE2 on SR-BI function we incubated 3 H-cholesteryl ether ( 3 H-CE) enriched HDL from SKO and DKO mice with CHO cells overexpressing PCPE2. Compared to CHO control cells, overexpression of PCPE2 increased 3 H-CE HDL uptake that was independent of HDL particle origin. Western Blot analysis showed no difference in SR-BI expression between control and PCPE2 transfected cells, suggesting that PCPE2 enhanced SR-BI function and promoted HDL cholesterol ester uptake. We conclude that PCPE2 is atheroprotective and an essential component of the reverse cholesterol transport system.

Cytokines/chemokines and their receptors have been an important consideration when investigating the role of inflammation in atherosclerosis. In humans and animal models, monocyte recruitment and accumulation within the artery wall occurs in response to elevated plasma cholesterol levels. To mimic this process in a mouse model of atherosclerosis, our lab has utilized the LDLr-/-, ApoA-I-/- double knockout mouse. Our studies have shown that LDLr-/-, ApoA-I-/- mice have increased immune cell mobilization driving the progression of atherosclerosis when compared to LDLr-/- mice, and that treating mice with frequent, low doses (200 μg) of subcutaneous administered lipid-free apoA-I or rHDL reverses the atherogenic process. Based on these studies we wanted to investigate which inflammatory pathways were most affected by apoA-I treatment. To do this, we carried out RT-PCR to obtain the relative mRNA abundance on 40 different markers of inflammation in two genotypes of atherogenic diet fed mice. Each genotype of mice was divided into two subsets. One subset was fed an atherogenic diet for 12 weeks while the other subset received 12 weeks of diet and subcutaneous injections of 200 μg of apoA-I for the last 6 weeks of the study. Spleen was isolated from all animals and their inflammatory gene mRNA expression examined. Most significantly both genotypes showed a large decrease in IL-3 mRNA expression but also showed a significant increase in CD163 mRNA expression with apoA-I treatment. Decrease in splenic IL-3 expression is a significant mediator of inflammation regression because of the role IL-3 plays in monocyte production inside the spleen. Other investigators have shown that myeloid cells reserved in the spleen give rise to monocytes in an IL-3 rich environment. IL-3 develops and maintains circulating monocytes, which may accumulate in the arteries during atherosclerosis. This suggests apoA-I treatment affects IL-3 levels in the spleen, and thus the amount of monocytes in circulation. An increase in CD163 mRNA expression in the spleen implies higher accumulation of monocytes inside the spleen, and out of circulation. This suggests that apoA-1 treatment can decrease the amount of monocytes in the arteries, which can be a result of reverse in the atherogenic process.