Downregulation of low-density lipoprotein receptor mRNA in lymphatic endothelial cells impairs lymphatic function through changes in intracellular lipids.

Rationale: Impairment in lymphatic transport is associated with the onset and progression of atherosclerosis in animal models. The downregulation of low-density-lipoprotein receptor (LDLR) expression, rather than increased circulating cholesterol level per se, is involved in early atherosclerosis-related lymphatic dysfunction. Enhancing lymphatic function in Ldlr-/- mice with a mutant form of VEGF-C (VEGF-C 152s), a selective VEGFR-3 agonist, successfully delayed atherosclerotic plaque onset when mice were subsequently fed a high-fat diet. However, the specific mechanisms by which LDLR protects against lymphatic function impairment is unknown. Methods and results: We have thus injected wild-type and Pcsk9-/- mice with an adeno-associated virus type 1 expressing a shRNA for silencing Ldlr in vivo. We herein report that lymphatic contractility is reduced upon Ldlr dowregulation in wild-type mice only. Our in vitro experiments reveal that a decrease in LDLR expression at the mRNA level reduces the chromosome duplication phase and the protein expression of VEGFR-3, a membrane-bound key lymphatic marker. Furthermore, it also significantly reduced the levels of 18 lipid subclasses, including key constituents of lipid rafts as well as the transcription of several genes involved in cholesterol biosynthesis and cellular and metabolic processes. Exogenous PCSK9 only reduces lymphatic endothelial-LDLR at the protein level and does not affect lymphatic endothelial cell integrity. This puts forward that PCSK9 may act upon lymphatic muscle cells to mediate its effect on lymphatic contraction capacity in vivo. Conclusion: Our results suggest that treatments that specifically palliate the down regulation of LDLR mRNA in lymphatic endothelial cells preserve the integrity of the lymphatic endothelium and sustain lymphatic function, a prerequisite player in atherosclerosis.

Apolipoprotein A-I carboxy-terminal domain residues 187-243 are required for adiponectin-induced cholesterol efflux.

Adiponectin exerts its atheroprotection by stimulating adenosine triphosphate binding cassette transporter A1 (ABCA1)-mediated cholesterol efflux to apolipoprotein A-I (apoA-I). However, involvement of the apoA-I residues in this process have not been studied. In Tamm-Horsfall 1 (THP-1) macrophages and baby hamster kidney (BHK) cells we assessed adiponectin's potential to restore cholesterol efflux in the presence of apoA-I and ABCA1 mutants, respectively. Adiponectin was unable to restore efflux from THP-1 macrophages in the presence of apoA-I carboxy-terminal domain (CTD) successive mutants from residues 187-243 versus apoA-I mutants alone. Furthermore, adiponectin did not significantly influence cholesterol efflux to apoA-I from BHK-ABCA1 mutant cells. Adiponectin appears to require functional apoA-I CTD residues 187-243 and wild-type ABCA1 to mediate efficient cholesterol efflux from THP-1 macrophages and BHK cells, respectively. Therefore, adiponectin cannot rescue defective cholesterol efflux in apoA-I- or ABCA1-mutant conditions, but rather increases cholesterol efflux in wild-type apoA-I conditions compared to apoA-I exposure alone.

Current models of apolipoprotein A-I lipidation by adenosine triphosphate binding cassette transporter A1.

PURPOSE OF REVIEW: The primary cardioprotective function of high-density lipoprotein (HDL) is to remove excess cellular free cholesterol (FC) from peripheral tissues and deliver it to the liver. Here, we summarize recent research that examines apolipoprotein A-I (apoA-I) lipidation models by adenosine triphosphate binding cassette transporter A1 (ABCA1) and discuss its relevance in atherosclerotic cardiovascular disease (ASCVD). RECENT FINDINGS: The first step in HDL formation involves the interaction between apoA-I and ABCA1, where ABCA1 mediates the removal of FC and phospholipids from lipid-laden macrophages to form discoidal nascent HDL (nHDL). However, there are currently no clear-cut systematic models that characterize HDL formation. A number of recent studies have investigated the importance of apoA-I C- and N-terminal domains required for optimal cholesterol efflux and nHDL production. Furthermore, functional ABCA1 is required for direct or indirect binding to apoA-I where ABCA1 dimer-monomer interconversion facilitates apoA-I lipidation from plasma membrane microdomains. Microparticles are also another lipid source for apoA-I solubilization into nHDL. SUMMARY: ApoA-I and ABCA1 are key factors in macrophage-mediated cholesterol efflux and nHDL production. Understanding of the key steps in HDL formation may unlock the therapeutic potential of HDL and improve clinical management of ASCVD.

Endothelial Rap1 (Ras-Association Proximate 1) Restricts Inflammatory Signaling to Protect From the Progression of Atherosclerosis.

OBJECTIVE: Small GTPase Rap1 (Ras-association proximate 1) is a novel, positive regulator of NO release and endothelial function with a potentially key role in mechanosensing of atheroprotective, laminar flow. Our objective was to delineate the role of Rap1 in the progression of atherosclerosis and its specific functions in the presence and absence of laminar flow, to better define its role in endothelial mechanisms contributing to plaque formation and atherogenesis. Approach and Results: In a mouse atherosclerosis model, endothelial Rap1B deletion exacerbates atherosclerotic plaque formation. In the thoracic aorta, where laminar shear stress-induced NO is otherwise atheroprotective, plaque area is increased in Athero-Rap1BiΔEC (atherogenic endothelial cell-specific, tamoxifen-inducible Rap1A+Rap1B knockout) mice. Endothelial Rap1 deficiency also leads to increased plaque size, leukocyte accumulation, and increased CAM (cell adhesion molecule) expression in atheroprone areas, whereas vascular permeability is unchanged. In endothelial cells, in the absence of protective laminar flow, Rap1 deficiency leads to an increased proinflammatory TNF-α (tumor necrosis factor alpha) signaling and increased NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation and elevated inflammatory receptor expression. Interestingly, this increased signaling to NF-κB activation is corrected by AKTVIII-an inhibitor of Akt (protein kinase B) translocation to the membrane. Together, these data implicate Rap1 in restricting Akt-dependent signaling, preventing excessive cytokine receptor signaling and proinflammatory NF-κB activation. CONCLUSIONS: Via 2 distinct mechanisms, endothelial Rap1 protects from the atherosclerosis progression in the presence and absence of laminar flow; Rap1-stimulated NO release predominates in laminar flow, and restriction of proinflammatory signaling predominates in the absence of laminar flow. Our studies provide novel insights into the mechanisms underlying endothelial homeostasis and reveal the importance of Rap1 signaling in cardiovascular disease.

High-Density Lipoprotein Biogenesis: Defining the Domains Involved in Human Apolipoprotein A-I Lipidation.

The first step in removing cholesterol from a cell is the ATP-binding cassette transporter 1 (ABCA1)-driven transfer of cholesterol to lipid-free or lipid-poor apolipoprotein A-I (apoA-I), which yields cholesterol-rich nascent high-density lipoprotein (nHDL) that then matures in plasma to spherical, cholesteryl ester-rich HDL. However, lipid-free apoA-I has a three-dimensional (3D) conformation that is significantly different from that of lipidated apoA-I on nHDL. By comparing the lipid-free apoA-I 3D conformation of apoA-I to that of 9-14 nm diameter nHDL, we formulated the hypothetical helical domain transitions that might drive particle formation. To test the hypothesis, ten apoA-I mutants were prepared that contained two strategically placed cysteines several of which could form intramolecular disulfide bonds and others that could not form these bonds. Mass spectrometry was used to identify amino acid sequence and intramolecular disulfide bond formation. Recombinant HDL (rHDL) formation was assessed with this group of apoA-I mutants. ABCA1-driven nHDL formation was measured in four mutants and wild-type apoA-I. The mutants contained cysteine substitutions in one of three regions: the N-terminus, amino acids 34 and 55 (E34C to S55C), central domain amino acids 104 and 162 (F104C to H162C), and the C-terminus, amino acids 200 and 233 (L200C to L233C). Mutants were studied in the locked form, with an intramolecular disulfide bond present, or unlocked form, with the cysteine thiol blocked by alkylation. Only small amounts of rHDL or nHDL were formed upon locking the central domain. We conclude that both the N- and C-terminal ends assist in the initial steps in lipid acquisition, but that opening of the central domain was essential for particle formation.

Lipin-1 contributes to modified low-density lipoprotein-elicited macrophage pro-inflammatory responses.

Atherosclerosis is a chronic inflammatory disease of large and medium-sized arteries and the underlying cause of cardiovascular disease, a major cause of mortality worldwide. The over-accumulation of modified cholesterol-containing low-density lipoproteins (e.g. oxLDL) in the artery wall and the subsequent recruitment and activation of macrophages contributes to the development of atherosclerosis. The excessive uptake of modified-LDL by macrophages leads to a lipid-laden "foamy" phenotype and pro-inflammatory cytokine production. Modified-LDLs promote foam cell formation in part by stimulating de novo lipid biosynthesis. However, it is unknown if lipid biosynthesis directly regulates foam cell pro-inflammatory mediator production. Lipin-1, a phosphatidate phosphohydrolase required for the generation of diacylglycerol during glycerolipid synthesis has recently been demonstrated to contribute to bacterial-induced pro-inflammatory responses by macrophages. In this study we present evidence demonstrating the presence of lipin-1 within macrophages in human atherosclerotic plaques. Additionally, reducing lipin-1 levels in macrophages significantly inhibits both modified-LDL-induced foam cell formation in vitro, as observed by smaller/fewer intracellular lipid inclusions, and ablates modified-LDL-elicited production of the pro-atherogenic mediators tumor necrosis factor-α, interleukin-6, and prostaglandin E2. These findings demonstrate a critical role for lipin-1 in the regulation of macrophage inflammatory responses to modified-LDL. These data begin to link the processes of foam cell formation and pro-inflammatory cytokine production within macrophages.

Procollagen C-endopeptidase Enhancer Protein 2 (PCPE2) Reduces Atherosclerosis in Mice by Enhancing Scavenger Receptor Class B1 (SR-BI)-mediated High-density Lipoprotein (HDL)-Cholesteryl Ester Uptake.

Studies in human populations have shown a significant correlation between procollagen C-endopeptidase enhancer protein 2 (PCPE2) single nucleotide polymorphisms and plasma HDL cholesterol concentrations. PCPE2, a 52-kDa glycoprotein located in the extracellular matrix, enhances the cleavage of C-terminal procollagen by bone morphogenetic protein 1 (BMP1). Our studies here focused on investigating the basis for the elevated concentration of enlarged plasma HDL in PCPE2-deficient mice to determine whether they protected against diet-induced atherosclerosis. PCPE2-deficient mice were crossed with LDL receptor-deficient mice to obtain LDLr(-/-), PCPE2(-/-) mice, which had elevated HDL levels compared with LDLr(-/-) mice with similar LDL concentrations. We found that LDLr(-/-), PCPE2(-/-) mice had significantly more neutral lipid and CD68+ infiltration in the aortic root than LDLr(-/-) mice. Surprisingly, in light of their elevated HDL levels, the extent of aortic lipid deposition in LDLr(-/-), PCPE2(-/-) mice was similar to that reported for LDLr(-/-), apoA-I(-/-) mice, which lack any apoA-I/HDL. Furthermore, LDLr(-/-), PCPE2(-/-) mice had reduced HDL apoA-I fractional clearance and macrophage to fecal reverse cholesterol transport rates compared with LDLr(-/-) mice, despite a 2-fold increase in liver SR-BI expression. PCPE2 was shown to enhance SR-BI function by increasing the rate of HDL-associated cholesteryl ester uptake, possibly by optimizing SR-BI localization and/or conformation. We conclude that PCPE2 is atheroprotective and an important component of the reverse cholesterol transport HDL system.

Acrolein impairs the cholesterol transport functions of high density lipoproteins.

High density lipoproteins (HDL) are considered athero-protective, primarily due to their role in reverse cholesterol transport, where they transport cholesterol from peripheral tissues to the liver for excretion. The current study was designed to determine the impact of HDL modification by acrolein, a highly reactive aldehyde found in high abundance in cigarette smoke, on the cholesterol transport functions of HDL. HDL was chemically-modified with acrolein and immunoblot and mass spectrometry analyses confirmed apolipoprotein crosslinking, as well as acrolein adducts on apolipoproteins A-I and A-II. The ability of acrolein-modified HDL (acro-HDL) to serve as an acceptor of free cholesterol (FC) from COS-7 cells transiently expressing SR-BI was significantly decreased. Further, in contrast to native HDL, acro-HDL promotes higher neutral lipid accumulation in murine macrophages as judged by Oil Red O staining. The ability of acro-HDL to mediate efficient selective uptake of HDL-cholesteryl esters (CE) into SR-BI-expressing cells was reduced compared to native HDL. Together, the findings from our studies suggest that acrolein modification of HDL produces a dysfunctional particle that may ultimately promote atherogenesis by impairing functions that are critical in the reverse cholesterol transport pathway.

Macrophage apoAI protects against dyslipidemia-induced dermatitis and atherosclerosis without affecting HDL.

Tissue cholesterol accumulation, macrophage infiltration, and inflammation are features of atherosclerosis and some forms of dermatitis. HDL and its main protein, apoAI, are acceptors of excess cholesterol from macrophages; this process inhibits tissue inflammation. Recent epidemiologic and clinical trial evidence questions the role of HDL and its manipulation in cardiovascular disease. We investigated the effect of ectopic macrophage apoAI expression on atherosclerosis and dermatitis induced by the combination of hypercholesterolemia and absence of HDL in mice. Hematopoietic progenitor cells were transduced to express human apoAI and transplanted into lethally irradiated LDL receptor(-/-)/apoAI(-/-) mice, which were then placed on a high-fat diet for 16 weeks. Macrophage apoAI expression reduced aortic CD4(+) T-cell levels (-39.8%), lesion size (-25%), and necrotic core area (-31.6%), without affecting serum HDL or aortic macrophage levels. Macrophage apoAI reduced skin cholesterol by 39.8%, restored skin morphology, and reduced skin CD4(+) T-cell levels. Macrophage apoAI also reduced CD4(+) T-cell levels (-32.9%) in skin-draining lymph nodes but had no effect on other T cells, B cells, dendritic cells, or macrophages compared with control transplanted mice. Thus, macrophage apoAI expression protects against atherosclerosis and dermatitis by reducing cholesterol accumulation and regulating CD4(+) T-cell levels, without affecting serum HDL or tissue macrophage levels.

Regulation of high-density lipoprotein on hematopoietic stem/progenitor cells in atherosclerosis requires scavenger receptor type BI expression.

OBJECTIVE: Recently, we demonstrated that scavenger receptor type BI (SR-BI), a high-density lipoprotein (HDL) receptor, was expressed on murine hematopoietic stem/progenitor cells (HSPC) and infusion of reconstituted HDL and purified human apolipoprotein A-I (apoA-I) suppressed HSPC proliferation. We hypothesized that SR-B1 expression is required for the observed antiproliferative effects of HDL on HSPC. APPROACH AND RESULTS: SR-BI-deficient (SR-BI(-/-)) mice and wild-type controls were fed on chow or high-fat diet (HFD) for 8 to 10 weeks. Under chow diet, a significant increase in Lin(-) Sca1(+) cKit(+) cells (LSK cells, so-called HSPC) was found in the bone marrow of SR-BI(-/-) mice when compared with wild-type mice. HFD induced a further expansion of CD150(+)CD48(-) LSK cells (HSC), HSPC, and granulocyte monocyte progenitors in SR-BI(-/-) mice. Injection of reactive oxygen species inhibitor N-acetylcysteine attenuated HFD-induced HSPC expansion, leukocytosis, and atherosclerosis in SR-BI(-/-) mice. ApoA-I infusion inhibited HSPC cell proliferation, Akt phosphorylation and reactive oxygen species production in HSPC and plaque progression in low-density lipoprotein receptor knockout (LDLr(-/-)) apoA-I(-/-) mice on HFD but had no effect on SR-BI(-/-) mice on HFD. Transplantation of SR-BI(-/-) bone marrow cells into irradiated LDLr(-/-) recipients resulted in enhanced white blood cells reconstitution, inflammatory cell production, and plaque development. In patients with coronary heart disease, HDL levels were negatively correlated with white blood cells count and HSPC frequency in the peripheral blood. By flow cytometry, SR-BI expression was detected on human HSPC. CONCLUSIONS: SR-BI plays a critical role in the HDL-mediated regulation HSPC proliferation and differentiation, which is associated with atherosclerosis progression.

Hepatic apolipoprotein M (apoM) overexpression stimulates formation of larger apoM/sphingosine 1-phosphate-enriched plasma high density lipoprotein.

Apolipoprotein M (apoM), a lipocalin family member, preferentially associates with plasma HDL and binds plasma sphingosine 1-phosphate (S1P), a signaling molecule active in immune homeostasis and endothelial barrier function. ApoM overexpression in ABCA1-expressing HEK293 cells stimulated larger nascent HDL formation, compared with cells that did not express apoM; however, the in vivo role of apoM in HDL metabolism remains poorly understood. To test whether hepatic apoM overexpression increases plasma HDL size, we generated hepatocyte-specific apoM transgenic (APOM Tg) mice, which had an ∼3-5-fold increase in plasma apoM levels compared with wild-type mice. Although HDL cholesterol concentrations were similar to wild-type mice, APOM Tg mice had larger plasma HDLs enriched in apoM, cholesteryl ester, lecithin:cholesterol acyltransferase, and S1P. Despite the presence of larger plasma HDLs in APOM Tg mice, in vivo macrophage reverse cholesterol transport capacity was similar to that in wild-type mice. APOM Tg mice had an ∼5-fold increase in plasma S1P, which was predominantly associated with larger plasma HDLs. Primary hepatocytes from APOM Tg mice generated larger nascent HDLs and displayed increased sphingolipid synthesis and S1P secretion. Inhibition of ceramide synthases in hepatocytes increased cellular S1P levels but not S1P secretion, suggesting that apoM is rate-limiting in the export of hepatocyte S1P. Our data indicate that hepatocyte-specific apoM overexpression generates larger nascent HDLs and larger plasma HDLs, which preferentially bind apoM and S1P, and stimulates S1P biosynthesis for secretion. The unique apoM/S1P-enriched plasma HDL may serve to deliver S1P to extrahepatic tissues for atheroprotection and may have other as yet unidentified functions.

The conformation of lipid-free human apolipoprotein A-I in solution.

Apolipoprotein AI (apoA-I) is the principal acceptor of lipids from ATP-binding cassette transporter A1, a process that yields nascent high density lipoproteins. Analysis of lipidated apoA-I conformation yields a belt or twisted belt in which two strands of apoA-I lie antiparallel to one another. In contrast, biophysical studies have suggested that a part of lipid-free apoA-I was arranged in a four-helix bundle. To understand how lipid-free apoA-I opens from a bundle to a belt while accepting lipid it was necessary to have a more refined model for the conformation of lipid-free apoA-I. This study reports the conformation of lipid-free human apoA-I using lysine-to-lysine chemical cross-linking in conjunction with disulfide cross-linking achieved using selective cysteine mutations. After proteolysis, cross-linked peptides were verified by sequencing using tandem mass spectrometry. The resulting structure is compact with roughly four helical regions, amino acids 44-186, bundled together. C- and N-terminal ends, amino acids 1-43 and 187-243, respectively, are folded such that they lie close to one another. An unusual feature of the molecule is the high degree of connectivity of lysine40 with six other lysines, lysines that are close, for example, lysine59, to distant lysines, for example, lysine239, that are at the opposite end of the primary sequence. These results are compared and contrasted with other reported conformations for lipid-free human apoA-I and an NMR study of mouse apoA-I.

Lymphatic vasculature mediates macrophage reverse cholesterol transport in mice.

Reverse cholesterol transport (RCT) refers to the mobilization of cholesterol on HDL particles (HDL-C) from extravascular tissues to plasma, ultimately for fecal excretion. Little is known about how HDL-C leaves peripheral tissues to reach plasma. We first used 2 models of disrupted lymphatic drainage from skin--1 surgical and the other genetic--to quantitatively track RCT following injection of [3H]-cholesterol-loaded macrophages upstream of blocked or absent lymphatic vessels. Macrophage RCT was markedly impaired in both models, even at sites with a leaky vasculature. Inhibited RCT was downstream of cholesterol efflux from macrophages, since macrophage efflux of a fluorescent cholesterol analog (BODIPY-cholesterol) was not altered by impaired lymphatic drainage. We next addressed whether RCT was mediated by lymphatic vessels from the aortic wall by loading the aortae of donor atherosclerotic Apoe-deficient mice with [2H]6-labeled cholesterol and surgically transplanting these aortae into recipient Apoe-deficient mice that were treated with anti-VEGFR3 antibody to block lymphatic regrowth or with control antibody to allow such regrowth. [2H]-Cholesterol was retained in aortae of anti-VEGFR3-treated mice. Thus, the lymphatic vessel route is critical for RCT from multiple tissues, including the aortic wall. These results suggest that supporting lymphatic transport function may facilitate cholesterol clearance in therapies aimed at reversing atherosclerosis.

Suppressed monocyte recruitment drives macrophage removal from atherosclerotic plaques of Apoe-/- mice during disease regression.

Experimental models of atherosclerosis suggest that recruitment of monocytes into plaques drives the progression of this chronic inflammatory condition. Cholesterol-lowering therapy leads to plaque stabilization or regression in human atherosclerosis, characterized by reduced macrophage content, but the mechanisms that underlie this reduction are incompletely understood. Mice lacking the gene Apoe (Apoe-/- mice) have high levels of cholesterol and spontaneously develop atherosclerotic lesions. Here, we treated Apoe-/- mice with apoE-encoding adenoviral vectors that induce plaque regression, and investigated whether macrophage removal from plaques during this regression resulted from quantitative alterations in the ability of monocytes to either enter or exit plaques. Within 2 days after apoE complementation, plasma cholesterol was normalized to wild-type levels, and HDL levels were increased 4-fold. Oil red O staining and quantitative mass spectroscopy revealed that esterified cholesterol content was markedly reduced. Plaque macrophage content decreased gradually and was 72% lower than baseline 4 weeks after apoE complementation. Importantly, this reduction in macrophages did not involve migratory egress from plaques or CCR7, a mediator of leukocyte emigration. Instead, marked suppression of monocyte recruitment coupled with a stable rate of apoptosis accounted for loss of plaque macrophages. These data suggest that therapies to inhibit monocyte recruitment to plaques may constitute a more viable strategy to reduce plaque macrophage burden than attempts to promote migratory egress.

Apolipoprotein A-I modulates regulatory T cells in autoimmune LDLr-/-, ApoA-I-/- mice.

The immune system is complex, with multiple layers of regulation that serve to prevent the production of self-antigens. One layer of regulation involves regulatory T cells (Tregs) that play an essential role in maintaining peripheral self-tolerance. Patients with autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis have decreased levels of HDL, suggesting that apoA-I concentrations may be important in preventing autoimmunity and the loss of self-tolerance. In published studies, hypercholesterolemic mice lacking HDL apoA-I or LDLr(-/-), apoA-I(-/-) (DKO), exhibit characteristics of autoimmunity in response to an atherogenic diet. This phenotype is characterized by enlarged cholesterol-enriched lymph nodes (LNs), as well as increased T cell activation, proliferation, and the production of autoantibodies in plasma. In this study, we investigated whether treatment of mice with lipid-free apoA-I could attenuate the autoimmune phenotype. To do this, DKO mice were first fed an atherogenic diet containing 0.1% cholesterol, 10% fat for 6 weeks, after which treatment with apoA-I was begun. Subcutaneous injections of 500 µg of lipid-free apoA-I was administered every 48 h during the treatment phase. These and control mice were maintained for an additional 6 weeks on the diet. At the end of the 12-week study, DKO mice showed decreased numbers of LN immune cells, whereas Tregs were proportionately increased. Accompanying this increase in Tregs was a decrease in the percentage of effector/effector memory T cells. Furthermore, lipid accumulation in LN and skin was reduced. These results suggest that treatment with apoA-I reduces inflammation in DKO mice by augmenting the effectiveness of the LN Treg response.

Conformation of dimeric apolipoprotein A-I milano on recombinant lipoprotein particles.

Apolipoprotein A-I Milano (apoA-I(Milano)) is a naturally occurring human mutation of wild-type apolipoprotein A-I (apoA-I(WT)) having cystine substituted for arginine(173). Two molecules of apo-I(WT) form disks with phospholipid having a defined relationship between the apoA-I(WT) molecules. ApoA-I(Milano) forms cystine homodimers that would not allow the protein to adopt the conformation reported for apoA-I(WT). The conformational constraints for dimeric apoA-I(Milano) recombinant high-density lipoprotein (rHDL) disks made with phospholipid were deduced from a combination of chemical cross-linking and mass spectrometry. Lysine-selective homobifunctional cross-linkers were reacted with homogeneous rHDL having diameters of 78 and 125 A. After reduction, cross-linked apoA-I(Milano) was separated from monomeric apoprotein by gel electrophoresis and then subjected to in-gel trypsin digest. Cross-linked peptides were confirmed by MS/MS sequencing. The cross-links provided distance constraints that were used to refine models of lipid-bound dimeric apoA-I(Milano). These studies suggest that a single dimeric apoA-I(Milano) on 78 A diameter rHDL girdles the edge of a phospholipid disk assuming a "belt" conformation similar to the "belt" region of apoA-I(WT) on rHDL. However, the C-terminal end of dimeric apoA-I(Milano) wraps around the periphery of the particle to shield the fatty acid chains from water rather than folding back onto the "belt" as does apoA-I(WT). The two apoA-I(Milano) dimers on a 125 A diameter rHDL do not encircle the periphery of a phospholipid disk but appear to reside on the surface of a laminar micelle.

Apolipoprotein A-I and its role in lymphocyte cholesterol homeostasis and autoimmunity.

OBJECTIVE: The purpose of this study was to determine the effects of an atherogenic diet on immune function in LDLr(-/-), ApoA-I(-/-) mice. METHODS AND RESULTS: When LDLr(-/-), ApoA-I(-/-) (DKO), and LDLr(-/-) (SKO) mice were fed an atherogenic diet, DKO had larger peripheral lymph nodes (LNs) and spleens compared to SKO mice. LNs were enriched in cholesterol and contain expanded populations of T, B, dendritic cells, and macrophages. Expansion of all classes of LN cells was accompanied by a approximately 1.5-fold increase in T cell proliferation and activation. Plasma antibodies to dsDNA, beta2-glycoprotein I, and oxidized LDL were increased in DKO, similar to levels in diet-fed Fas(lpr/lpr) mice, suggesting the development of an autoimmune phenotype. Both LN enlargement and cellular cholesterol expansion were "prevented" when diet-fed DKO mice were treated with helper dependent adenovirus expressing apoA-I. Independent of the amount of dietary cholesterol, DKO mice consistently showed lower plasma cholesterol than SKO mice, yet greater aortic cholesterol deposition and inflammation. CONCLUSIONS: ApoA-I prevented cholesterol-associated lymphocyte activation and proliferation in peripheral LN of diet-fed DKO mice. A approximately 1.5-fold increase in T cell activation and proliferation was associated with a approximately 3-fold increase in concentrations of circulating autoantibodies and approximately 2-fold increase in the severity of atherosclerosis suggesting a common link between plasma apoA-I, inflammation, and atherosclerosis.

Inflammation and skin cholesterol in LDLr-/-, apoA-I-/- mice: link between cholesterol homeostasis and self-tolerance?

Diet-fed low density lipoprotein receptor-deficient/apolipoprotein A-I-deficient (LDLr-/-, apoA-I-/-) mice accumulate a 10-fold greater mass of cholesterol in their skin despite a 1.5- to 2-fold lower plasma cholesterol concentration compared with diet-fed LDLr-/- mice. The accumulation of cholesterol predominantly in the skin has been shown to occur in a growing number of other hypercholesterolemic double knockout mouse models sharing deficits in genes regulating cellular cholesterol homeostasis. Exploring the relationship between cholesterol balance and inflammation, we have examined the time course of cholesterol accumulation in a number of extrahepatic tissues and correlated with the onset of inflammation in diet-fed LDLr-/-, apoA-I-/- mice. After 4 weeks of diet, LDLr-/-, apoA-I-/- mice showed a significant increase in skin cholesterol mass compared with LDLr-/- mice. In addition, after 4 weeks on the diet, cholesterol accumulation in the skin was also found to be associated with macrophage infiltration and accompanied by increases in tumor necrosis factor-alpha, cyclooxygenase-2, and langerin mRNA, which were not seen in the liver. Overall, these data suggest that as early as 4 weeks after starting the diet, the accumulation of skin cholesterol and the onset of inflammation occur concurrently. In summary, the use of hypercholesterolemic LDLr-/-, apoA-I-/- mice may provide a useful tool to investigate the role that apoA-I plays in maintaining cholesterol homeostasis and its relationship to inflammation.

Intermolecular contact between globular N-terminal fold and C-terminal domain of ApoA-I stabilizes its lipid-bound conformation: studies employing chemical cross-linking and mass spectrometry.

The structure of apoA-I on discoidal high density lipoprotein (HDL) was studied using a combination of chemical cross-linking and mass spectrometry. Recombinant HDL particles containing 145 molecules of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and two molecules of apoA-I with a 96-A diameter were treated with the lysine-specific cross-linker, dithiobis(succinimidylpropionate) at varying molar ratios from 2:1 to 200:1. At low molar ratios of dithiobis(succinimidylpropionate) to apoA-I, two products were obtained corresponding to approximately 53 and approximately 80 kDa. At high molar ratios, these two products merged, yielding a product of approximately 59 kDa, close to the theoretical molecular mass of dimeric apoA-I. To identify the intermolecular cross-links giving rise to the two different sized products, bands were excised from the gel, digested with trypsin, and then analyzed by liquid chromatography-electrospray-tandem mass spectrometry. In addition, tandem mass spectrometry of unique cross-links found in the 53- and 80-kDa products suggested that a distinct conformation exists for lipid-bound apoA-I on 96-A recombinant HDL, emphasizing the inherent flexibility and malleability of the N termini and its interaction with its C-terminal domain.

Quality control in the apoA-I secretory pathway: deletion of apoA-I helix 6 leads to the formation of cytosolic phospholipid inclusions.

From a total of 47 known apolipoprotein A-I (apoA-I) mutations, only 18 are linked to low plasma HDL apoA-I concentrations, and 78% of these map to apoA-I helices 6 and 7 (residues 143-186). Gene transfer and transgenic mouse studies have shown that several helix 6 apoA-I mutations have reduced hepatic HDL production. Our objective was to examine the impact of helix 6 modifications on intracellular biosynthetic processing and secretion of apoA-I. Cells were transfected with wild-type or mutant apoA-I, radiolabeled with [(35)S]Met/Cys, and then placed in unlabeled medium for up to 4 h. Results show that >90% of newly synthesized wild-type apoA-I was secreted by 60 min. Over the same length of time, only 20% of helix 6 deletion mutant (Delta 6 apoA-I) was secreted, whereas 80% remained cell associated. Microscopic and biochemical studies revealed that cell-associated Delta 6 apoA-I was located predominantly within the cytoplasm as lipid-protein inclusions, whereas wild-type apoA-I was localized in the endoplasmic reticulum/Golgi. Results using other helix deletions or helix 6 substitution mutations indicated that only complete removal of helix 6 resulted in massive cytoplasmic accumulation. These data suggest that alterations in native apoA-I conformation can lead to aberrant trafficking and accumulation of apolipoprotein-phospholipid structures. Thus, conformation-dependent alterations in intracellular trafficking and turnover may underlie the reduced plasma HDL concentrations observed in individuals harboring deletion mutations within helix 6.