We employed shotgun proteomics to test the hypothesis that HDL might carry proteins that make a previously unsuspected contribution to its cardioprotective and antiinflammatory activities. Forty-eight proteins were identified in HDL isolated by ultracentrifugation from healthy controls and/or CAD subjects. Twenty-two proteins with well-characterized roles in lipid metabolism and the antioxidant properties of HDL were detected, which validates our experimental approach. Importantly, we found 13 proteins not previously known to reside in HDL.
We used annotations by the GO Consortium to connect the complex array of proteins we identified in HDL to biological processes. Twenty-two of the proteins were linked to cholesterol and lipoprotein metabolism. Remarkably, there were more acute-phase-response proteins (23 of 48), whose plasma concentrations are altered markedly by acute and chronic inflammation. A number of proteins not previously known to reside in HDL, including C4A/C4B, C9, and vitronectin, were linked to the complement pathway and its regulation. Our detection of the extracellular matrix protein vitronectin and the lysosomal protein prenylcysteine oxidase suggests that HDL components can be derived from cells other than those that synthesize the lipoprotein in the liver and intestine. The detection of multiple proteins with roles in complement activation, together with the identification of specific apoA-I complexes that help kill pathogens (44
), is consistent with the suggestion that HDL serves as a platform for the assembly of proteins involved in the innate immune response.
Proteolysis of artery wall proteins is thought to play a critical role in plaque rupture, the major cause of myocardial infarction and sudden death in subjects with CAD (45
). It is noteworthy that we found α-2-antiplasmin, α-2-HS-glycoprotein, angiotensinogen, inter-α-trypsin inhibitor, α-2-macroglobulin, and serpin peptidase inhibitor (clade F, member 1) in HDL. All of these proteins have serine proteinase inhibitor domains. Serine proteinase inhibitors, termed serpins, are key regulators of numerous biological pathways that initiate inflammation, coagulation, apoptosis, angiogenesis, and matrix degradation (46
). Moreover, we also detected the thiol proteinase inhibitor kininogen-1 and haptoglobin-related protein. The latter contains a trypsin-like serine proteinase domain with a substitution in 1 of its catalytic triad residues that may allow it to act as a decoy substrate to prevent proteolysis. Collectively, these observations raise the possibility that HDL plays a previously unsuspected role in preventing plaque rupture, perhaps by protecting tissue from promiscuous proteolysis.
Complement activation is implicated in tissue damage during acute myocardial infarction (47
). It may therefore be significant that we found multiple complement components and complement-regulatory proteins in HDL. Moreover, in vitro studies indicate that HDL blocks the assembly of the terminal complement attack complex on endothelial cells (49
). Inhibition of complement deposition may limit injury to cardiac cells and prevent activation of the procoagulant response in endothelium, macrophages, and platelets, 2 critical events in acute thrombosis.
When we extended our analysis to HDL3
from men with established CAD, we found the lipoprotein to be selectively enriched in 5 proteins. One protein was C3, suggesting that this HDL-associated immune system protein may contribute to vascular disease. It is noteworthy that C3 is produced by human monocyte-derived macrophages (50
) and that 3 of the other enriched proteins — apoE, apoC-IV, and apoA-IV — are involved in lipid metabolism and cholesterol export from macrophages. apoE and apoC-IV are part of a gene cluster that is induced in macrophages by cholesterol-sensing nuclear receptors that protect against atherosclerosis in mice (51
). apoA-IV, along with apoA-I and apoC-III, belongs to another gene cluster that also protects against atherosclerosis when expressed in mice (52
). Taken together, our observations suggest that HDL3
from CAD patients is selectively enriched in proteins that play critical roles in macrophage biology, lipid metabolism, and the inflammatory response. Several of these proteins are produced by cholesterol-loaded macrophages, suggesting that a fraction of the proteins that are overproduced in CAD patients may be derived from macrophages in atherosclerotic lesions. Alternatively, conditions associated with CAD may enhance their production by the liver, promote their association with circulating HDL, or remodel HDL particles so that they redistribute to denser HDL subfractions (3
Our proteomics analyses suggested that apoE was the most significantly enriched protein in CAD HDL3. To confirm that this approach can estimate protein abundance semiquantitatively, we measured apoE levels immunochemically in HDL3 isolated from a second set of 64 subjects: 32 with established CAD and 32 age-matched controls. Levels of apoE were significantly higher in HDL3 isolated from the CAD subjects. In striking contrast, the 2 groups had similar levels of HDL-cholesterol, apoA-I, apoA-II, and plasma triglycerides. Importantly, relative protein abundance, as assessed by MS and biochemical assays, was similar, supporting the proposal that peptide counts and the peptide index can assess relative protein abundance in biological material.
Many lines of evidence indicate that apoE plays a role in modulating atherogenesis. apoE is produced by cholesterol-loaded macrophages, where it can promote cholesterol efflux during its secretion. Circulating and macrophage-produced apoE removes macrophage cholesterol through its interaction with the ATP-binding cassette transporter (ABC) A1. apoE-rich HDL2
particles are efficient acceptors for macrophage cholesterol exported by another ABC transporter termed ABCG1 (53
). apoE may be important for the expansion of HDL particles required for driving net cholesterol transport from macrophage foam cells (54
). Epidemiological and clinical studies suggest that elevated levels of HDL2
are cardioprotective. Moreover, apoE levels are lower in HDL2
(the less dense subfraction of HDL) isolated from subjects with CAD (55
), and apoE-enriched HDL2
isolated from CETP-deficient subjects removes potentially toxic cholesterol from macrophages much more efficiently than control HDL2
). Our demonstration of elevated levels of apoE in HDL3
isolated from subjects with established CAD raises the possibility that redistribution of apoE from HDL2
impairs cholesterol efflux and promotes the formation of macrophage foam cells in vivo.
In summary, our results indicate that HDL carries a population of proteins that function in lipid metabolism, proteinase inhibition, complement activation, and the acute-phase response. It will be of interest to study a larger number of subjects to determine whether levels of the proteins we detected in HDL3
from CAD subjects might be useful indicators of cardiovascular risk and to investigate the proposal that chronic inflammation alters the protein composition of HDL, making it atherogenic (3