Serum opacity factor normalizes erythrocyte morphology in Scarb1-/- mice in an HDL-free cholesterol-dependent way.

Compared with WT mice, HDL receptor-deficient (Scarb1-/-) mice have higher plasma levels of free cholesterol (FC)-rich HDL and exhibit multiple pathologies associated with a high mol% FC in ovaries, platelets, and erythrocytes, which are reversed by lowering HDL. Bacterial serum opacity factor (SOF) catalyzes the opacification of plasma by targeting and quantitatively converting HDL to neo HDL (HDL remnant), a cholesterol ester-rich microemulsion, and lipid-free APOA1. SOF delivery with an adeno-associated virus (AAVSOF) constitutively lowers plasma HDL-FC and reverses female infertility in Scarb1-/- mice in an HDL-dependent way. We tested whether AAVSOF delivery to Scarb1-/- mice will normalize erythrocyte morphology in an HDL-FC-dependent way. We determined erythrocyte morphology and FC content (mol%) in three groups-WT, untreated Scarb1-/- (control), and Scarb1-/- mice receiving AAVSOF-and correlated these with their respective HDL-mol% FC. Plasma-, HDL-, and tissue-lipid compositions were also determined. Plasma- and HDL-mol% FC positively correlated across all groups. Among Scarb1-/- mice, AAVSOF treatment normalized reticulocyte number, erythrocyte morphology, and erythrocyte-mol% FC. Erythrocyte-mol% FC positively correlated with HDL-mol% FC and with both the number of reticulocytes and abnormal erythrocytes. AAVSOF treatment also reduced FC of extravascular tissues to a lesser extent. HDL-FC spontaneously transfers from plasma HDL to cell membranes. AAVSOF treatment lowers erythrocyte-FC and normalizes erythrocyte morphology and lipid composition by reducing HDL-mol% FC.

Serum opacity factor rescues fertility among female Scarb1-/- mice by reducing HDL-free cholesterol bioavailability.

Human female infertility, 20% of which is idiopathic, is a public health problem for which better diagnostics and therapeutics are needed. A novel cause of infertility emerged from studies of female mice deficient in the HDL receptor gene (Scarb1). These mice are infertile and have high plasma HDL cholesterol (C) concentrations, due to elevated HDL-free cholesterol (FC), which transfers from HDL to all tissues. Previous studies have indicated that oral delivery of probucol, an HDL-lowering drug, to female Scarb1-/- mice reduces plasma HDL-C concentrations and rescues fertility. Additionally, serum opacity factor (SOF), a bacterial virulence factor, disrupts HDL structure, and bolus SOF injection into mice reduces plasma HDL-C concentrations. Here, we discovered that delivering SOF to female Scarb1-/- mice with an adeno-associated virus (AAVSOF) induces constitutive SOF expression, reduces HDL-FC concentrations, and rescues fertility while normalizing ovary morphology. Although AAVSOF did not alter ovary-FC content, the ovary-mol% FC correlated with plasma HDL-mol% FC in a fertility-dependent way. Therefore, reversing the abnormal plasma microenvironment of high plasma HDL-mol% FC in female Scarb1-/- mice rescues fertility. These data provide the rationale to search for similar mechanistic links between HDL-mol% FC and infertility and the rescue of fertility in women by reducing plasma HDL-mol% FC.

High Free Cholesterol Bioavailability Drives the Tissue Pathologies in Scarb1-/- Mice.

Objective: Overall and atherosclerosis-associated mortality is elevated in humans with very high HDL (high-density lipoprotein) cholesterol concentrations. Mice with a deficiency of the HDL receptor, Scarb1 (scavenger receptor class B type 1), are a robust model of this phenotype and exhibit several additional pathologies. We hypothesized that the previously reported high plasma concentration of free cholesterol (FC)-rich HDL in Scarb1-/- mice produces a state of high HDL-FC bioavailability that increases whole-body FC and dysfunction in multiple tissue sites. Approach and Results: The higher mol% FC in Scarb1-/- versus WT (wild type) HDL (41.1 versus 16.0 mol%) affords greater FC bioavailability for transfer to multiple sites. Plasma clearance of autologous HDL-FC mass was faster in WT versus Scarb1-/- mice. FC influx from Scarb1-/- HDL to LDL (low-density lipoprotein) and J774 macrophages was greater ([almost equal to]4x) than that from WT HDL, whereas FC efflux capacity was similar. The higher mol% FC of ovaries, erythrocytes, heart, and macrophages of Scarb1-/- versus WT mice is associated with previously reported female infertility, impaired cell maturation, cardiac dysfunction, and atherosclerosis. The FC contents of other tissues were similar in the two genotypes, and these tissues were not associated with any overt pathology. In addition to the differences between WT versus Scarb1-/- mice, there were many sex-dependent differences in tissue-lipid composition and plasma FC clearance rates. Conclusions: Higher HDL-FC bioavailability among Scarb1-/- versus WT mice drives increased FC content of multiple cell sites and is a potential biomarker that is mechanistically linked to multiple pathologies.

Highly conserved amino acid residues in apolipoprotein A1 discordantly induce high density lipoprotein assembly in vitro and in vivo.

OBJECTIVE: Apolipoprotein A1 (APOA1) is essential to reverse cholesterol transport, a physiologically important process that protects against atherosclerotic cardiovascular disease. APOA1 is a 28 kDa protein comprising multiple lipid-binding amphiphatic helices initialized by proline residues, which are conserved across multiple species. We tested the hypothesis that the evolutionarily conserved residues are essential to high density lipoprotein (HDL) function. APPROACH: We used biophysical and physiological assays of the function of APOA1P➔A variants, i.e., rHDL formation via dimyristoylphosphatidylcholine (DMPC) microsolubilization, activation of lecithin: cholesterol acyltransferase, cholesterol efflux from human monocyte-derived macrophages (THP-1) to each variant, and comparison of the size and composition of HDL from APOA1-/- mice receiving adeno-associated virus delivery of each human variant. RESULTS: Differences in microsolubilization were profound and showed that conserved prolines, especially those in the C-terminus of APOA1, are essential to efficient rHDL formation. In contrast, P➔A substitutions produced small changes (-25 to +25%) in rates of cholesterol efflux and no differences in the rates of LCAT activation. The HDL particles formed following ectopic expression of each variant in APOA1-/- mice were smaller and more heterogeneous than those from control animals. CONCLUSION: Studies of DMPC microsolubilization show that proline residues are essential to the optimal interaction of APOA1 with membranes, the initial step in cholesterol efflux and HDL production. In contrast, P➔A substitutions modestly reduce the cholesterol efflux capacity of APOA1, have no effect on LCAT activation, but according to the profound reduction in the size of HDL formed in vivo, P➔A substitutions alter HDL biogenesis, thereby implicating other cellular and in vivo processes as determinants of HDL metabolism and function.

Revisiting Reverse Cholesterol Transport in the Context of High-Density Lipoprotein Free Cholesterol Bioavailability.

Dysregulated free cholesterol (FC) metabolism has been implicated in nearly all stages of atherosclerosis, the underlying cause of most cardiovascular disease. According to a widely cited model, the burden of macrophage FC in the arterial wall is relieved by transhepatic reverse cholesterol transport (RCT), which comprises three successive steps: (1) macrophage FC efflux to high-density lipoprotein (HDL) and/or its major protein, apolipoprotein AI; (2) FC esterification by lecithin:cholesterol acyltransferase (LCAT); and (3) HDL-cholesteryl ester (CE) uptake via the hepatic HDL-receptor, scavenger receptor class B type 1 (SR-B1). Recent studies have challenged the validity of this model, most notably the role of LCAT, which appears to be of minor importance. In mice, most macrophage-derived FC is rapidly cleared from plasma (t1/2 < 5 min) without esterification by hepatic uptake; the remainder is taken up by multiple tissue and cell types, especially erythrocytes. Further, some FC is cleared by the nonhepatic transintestinal pathway. Lastly, FC movement among lipid surfaces is reversible, so that a higher-than-normal level of HDL-FC bioavailability-defined by high plasma HDL levels concurrent with a high mol% HDL-FC-leads to the transfer of excess FC to cells in vivo. SR-B1-/- mice provide an animal model to study the mechanistic consequences of high HDL-FC bioavailability that provokes atherosclerosis and other metabolic abnormalities. Future efforts should aim to reduce HDL-FC bioavailability, thereby reducing FC accretion by tissues and the attendant atherosclerosis.

ABCA1-Derived Nascent High-Density Lipoprotein-Apolipoprotein AI and Lipids Metabolically Segregate.

OBJECTIVE: Reverse cholesterol transport comprises cholesterol efflux from ABCA1-expressing macrophages to apolipoprotein (apo) AI, giving nascent high-density lipoprotein (nHDL), esterification of nHDL-free cholesterol (FC), selective hepatic extraction of HDL lipids, and hepatic conversion of HDL cholesterol to bile salts, which are excreted. We tested this model by identifying the fates of nHDL-[3H]FC, [14C] phospholipid (PL), and [125I]apo AI in serum in vitro and in vivo. APPROACH AND RESULTS: During in vitro incubation of human serum, nHDL-[3H]FC and [14C]PL rapidly transfer to HDL and low-density lipoproteins (t1/2=2-7 minutes), whereas nHDL-[125I]apo AI transfers solely to HDL (t1/2<10 minutes) and to the lipid-free form (t1/2>480 minutes). After injection into mice, nHDL-[3H]FC and [14C]PL rapidly transfer to liver (t1/2=≈2-3 minutes), whereas apo AI clears with t1/2=≈460 minutes. The plasma nHDL-[3H]FC esterification rate is slow (0.46%/h) compared with hepatic uptake. PL transfer protein enhances nHDL-[14C]PL but not nHDL-[3H]FC transfer to cultured Huh7 hepatocytes. CONCLUSIONS: nHDL-FC, PL, and apo AI enter different pathways in vivo. Most nHDL-[3H]FC and [14C]PL are rapidly extracted by the liver via SR-B1 (scavenger receptor class B member 1) and spontaneous transfer; hepatic PL uptake is promoted by PL transfer protein. nHDL-[125I]apo AI transfers to HDL and to the lipid-free form that can be recycled to nHDL formation. Cholesterol esterification by lecithin:cholesterol acyltransferase is a minor process in nHDL metabolism. These findings could guide the design of therapies that better mobilize peripheral tissue-FC to hepatic disposal.

Neo High-Density Lipoprotein Produced by the Streptococcal Serum Opacity Factor Activity against Human High-Density Lipoproteins Is Hepatically Removed via Dual Mechanisms.

Injection of streptococcal serum opacity factor (SOF) into mice reduces the plasma cholesterol level by ∼40%. In vitro, SOF converts high-density lipoproteins (HDLs) into multiple products, including a small HDL, neo HDL. In vitro, neo HDL accounts for ∼60% of the protein mass of the SOF reaction products; in vivo, the accumulated mass of neo HDL is <1% of that observed in vitro. To identify the underlying cause of this difference, we determined the fate of neo HDL in plasma in vitro and in vivo. Following incubation with HDL, neo HDL-PC rapidly transfers to HDL, giving a small remnant, which fuses with HDL. An increased level of SR-B1 expression in Huh7 hepatoma cells and a reduced level of LDLR expression in CHO cells had little effect on neo HDL-[3H]CE uptake. Thus, the dominant receptors for neo HDL uptake are not LDLR or SR-B1. The in vivo metabolic fates of neo HDL-[3H]CE and HDL-[3H]CE were different. Thirty minutes after the injection of neo HDL-[3H]CE and HDL-[3H]CE into mice, plasma [3H]CE counts were 40 and 53%, respectively, of injected counts, with 10 times more [3H]CE appearing in the livers of neo HDL-[3H]CE-injected than in those of HDL-[3H]CE-injected mice. These data support a model of neo HDL-[3H]CE clearance by two parallel pathways. At early post-neo HDL-[3H]CE injection times, some neo HDL is directly removed by the liver; the remainder transfers its PC to HDL, leaving a remnant that fuses with HDL, which is also hepatically removed more slowly. Given that SR-B1 and SOF both remove CE from HDL, this novel mechanism may also underlie the metabolism of remnants released by hepatocytes following selective SR-B1-mediated uptake of HDL-CE.

Streptococcal serum opacity factor promotes cholesterol ester metabolism and bile acid secretion in vitro and in vivo.

Plasma high density lipoprotein-cholesterol (HDL-C) concentrations negatively correlate with atherosclerotic cardiovascular disease. HDL is thought to have several atheroprotective functions, which are likely distinct from the epidemiological inverse relationship between HDL-C levels and risk. Specifically, strategies that reduce HDL-C while promoting reverse cholesterol transport (RCT) may have therapeutic value. The major product of the serum opacity factor (SOF) reaction versus HDL is a cholesteryl ester (CE)-rich microemulsion (CERM), which contains apo E and the CE of ~400,000 HDL particles. Huh7 hepatocytes take up CE faster when delivered as CERM than as HDL, in part via the LDL-receptor (LDLR). Here we compared the final RCT step, hepatic uptake and subsequent intracellular processing to cholesterol and bile salts for radiolabeled HDL-, CERM- and LDL-CE by Huh7 cells and in vivo in C57BL/6J mice. In Huh7 cells, uptake from LDL was greater than from CERM (2-4X) and HDL (5-10X). Halftimes for [(14)C]CE hydrolysis were 3.0±0.2, 4.4±0.6 and 5.4±0.7h respectively for HDL, CERM and LDL-CE. The fraction of sterols secreted as bile acids was ~50% by 8h for all three particles. HDL, CERM and LDL-CE metabolism in mice showed efficient plasma clearance of CERM-CE, liver uptake and metabolism, and secretion as bile acids into the gall bladder. This work supports the therapeutic potential of the SOF reaction, which diverts HDL-CE to the LDLR, thereby increasing hepatic CE uptake, and sterol disposal as bile acids.

High-Density Lipoprotein Processing and Premature Cardiovascular Disease.

High plasma concentrations of low-density lipoprotein-cholesterol (LDL-C) are a well-accepted risk factor for cardiovascular disease (CVD), and the statin class of hypolipidemic drugs has emerged as an effective means of lowering LDL-C and reducing CVD risk. In contrast, the role of plasma high-density lipoproteins (HDL) in protection against atherosclerotic vascular disease is the subject of considerable controversy. Although the inverse correlation between plasma HDL-C and CVD is widely acknowledged, reduction of CVD risk by interventions that increase HDL-C have not been uniformly successful. Several studies of large populations have shown that the first step in reverse cholesterol transport (RCT), the transfer of cholesterol from the subendothelial space of the arterial wall via the plasma compartment to the liver for disposal, is impaired in patients with CVD. Here we review HDL function, the mechanisms by which HDL supports RCT, and the role of RCT in preventing CVD.

CD1d serves as a surface receptor for oxidized cholesterol induction of peroxisome proliferator-activated receptor-γ.

OBJECTIVE: The cluster of differentiation-1d (CD1d) recognizes and presents the lipid antigens to NK-T lymphocytes. Atherosclerotic lesions contain atherogenic lipids, mainly cholesterol and its oxides. Peroxisome proliferator-activated receptor-γ (PPARγ) is also known to exist in atherosclerotic lesions, participating in regulation of lipid metabolism. The current study tested whether CD1d acts as a surface receptor that mediates induction and activation of PPARγ by oxysterols commonly found in atherosclerotic lesions. METHODS AND RESULTS: CD1d overexpression in HEK 293 cells transfected with CD1d cDNA was confirmed by fluorescence, flow cytometry, Western blotting and mRNA expression. Tritiated ((3)H) 7-ketocholesterol (7K) was used for lipid binding assays. Radioactive assessment demonstrated an increased 7K-binding activity HEK 293 cells with CD1d overexpression. The 7K binding could be blocked by another oxysterol, 25-hydroxycholesterol, but not by native free cholesterol. Addition of CD1d:IgG dimer protein or an anti-CD1d antibody, but not control IgG, significantly diminished 7K binding to CD1d-expressing HEK 293 cells. CD1d deficiency markedly diminished the 7K-binding in macrophages and smooth muscle cells. Western blot and gel shift assays demonstrated that CD1d-mediated 7K binding induced expression and activation of PPARγ. The PPARγ agonist PGJ2 enhances the 7K stimulatory effect on PPARγ expression and activity but the antagonist GW9662 inhibits the 7K effect on the CD1d-expressing cells. CONCLUSIONS: CD1d acts as a cell surface receptor that recognizes and binds oxysterols and initializes a pathway connecting oxysterol binding to PPARγ activation.

Streptococcal serum opacity factor increases the rate of hepatocyte uptake of human plasma high-density lipoprotein cholesterol.

Serum opacity factor (SOF), a virulence determinant of Streptococcus pyogenes, converts plasma high-density lipoproteins (HDL) to three distinct species: lipid-free apolipoprotein (apo) A-I, neo HDL, a small discoidal HDL-like particle, and a large cholesteryl ester-rich microemulsion (CERM) that contains the cholesterol esters (CE) of up to ∼400000 HDL particles and apo E as its major protein. Similar SOF reaction products are obtained with HDL, total plasma lipoproteins, and whole plasma. We hypothesized that hepatic uptake of CERM-CE via multiple apo E-dependent receptors would be faster than that of HDL-CE. We tested our hypothesis using human hepatoma cells and lipoprotein receptor-specific Chinese hamster ovary (CHO) cells. The uptake of [(3)H]CE by HepG2 and Huh7 cells from HDL after SOF treatment, which transfers >90% of HDL-CE to CERM, was 2.4 and 4.5 times faster, respectively, than from control HDL. CERM-[(3)H]CE uptake was inhibited by LDL and HDL, suggestive of uptake by both the LDL receptor (LDL-R) and scavenger receptor class B type I (SR-BI). Studies in CHO cells specifically expressing LDL-R and SR-BI confirmed CERM-[(3)H]CE uptake by both receptors. RAP and heparin inhibit CERM-[(3)H]CE but not HDL-[(3)H]CE uptake, thereby implicating LRP-1 and cell surface proteoglycans in this process. These data demonstrate that SOF treatment of HDL increases the rate of CE uptake via multiple hepatic apo E receptors. In so doing, SOF might increase the level of hepatic disposal of plasma cholesterol in a way that is therapeutically useful.

Serum opacity factor enhances HDL-mediated cholesterol efflux, esterification and anti inflammatory effects.

Serum opacity factor (SOF) is a streptococcal protein that disrupts the structure of human high density lipoproteins (HDL) releasing lipid-free apo A-I while forming a large cholesteryl ester-rich particle and a small neo HDL. Given its low cholesterol and high phospholipid contents, we tested the hypotheses that neo HDL is a better substrate for cholesterol esterification via lecithin:cholesterol acyltransferase (LCAT), better than HDL as an acceptor of THP-1 macrophage cholesterol efflux, and improves reduction of oxidized LDL-induced production of inflammatory markers. We observed that both cholesterol efflux and esterification were improved by recombinant (r)SOF treatment of whole plasma and that the underlying cause of the improved cholesterol esterification in plasma and macrophage cholesterol efflux to rSOF-treated plasma was due to the rSOF-mediated conversion of HDL to neo HDL. Moreover, the reduction of secretion of TNF-α and IL-6 by THP-1 cells by neo HDL was twice that of HDL. Studies in BHK cells overexpressing cholesterol transporters showed that efflux to neo HDL occurred primarily via ABCA1 not ABCG1. Thus, rSOF improves two steps in reverse cholesterol transport with a concomitant reduction in the release of macrophage markers of inflammation. We conclude that rSOF catalyzes a novel reaction that might be developed as a new therapy that prevents or reverses atherosclerosis via improved reverse cholesterol transport.

Role for the BRCA1 C-terminal repeats (BRCT) protein 53BP1 in maintaining genomic stability.

p53-binding protein-1 (53BP1) is phosphorylated in response to DNA damage and rapidly relocalizes to presumptive sites of DNA damage along with Mre11 and the phosphorylated histone 2A variant, gamma-H2AX. 53BP1 associates with the BRCA1 tumor suppressor, and knock-down experiments with small interfering RNA have revealed a role for the protein in the checkpoint response to DNA damage. By generating mice defective in m53BP1 (m53BP1(tr/tr)), we have created an animal model to further explore its biochemical and genetic roles in vivo. We find that m53BP1(tr/tr) animals are growth-retarded and show various immune deficiencies including a specific reduction in thymus size and T cell count. Consistent with a role in responding to DNA damage, we find that m53BP1(tr/tr) mice are sensitive to ionizing radiation (gamma-IR), and cells from these animals exhibit chromosomal abnormalities consistent with defects in DNA repair. Thus, 53BP1 is a critical element in the DNA damage response and plays an integral role in maintaining genomic stability.