Human Aldose Reductase Expression Prevents Atherosclerosis Regression in Diabetic Mice.

Guidelines to reduce cardiovascular risk in diabetes include aggressive LDL lowering, but benefits are attenuated compared with those in patients without diabetes. Consistent with this, we have reported in mice that hyperglycemia impaired atherosclerosis regression. Aldose reductase (AR) is thought to contribute to clinical complications of diabetes by directing glucose into pathways producing inflammatory metabolites. Mice have low levels of AR, thus raising them to human levels would be a more clinically relevant model to study changes in diabetes under atherosclerosis regression conditions. Donor aortae from Western diet-fed Ldlr-/- mice were transplanted into normolipidemic wild-type, Ins2Akita (Akita+/- , insulin deficient), human AR (hAR) transgenic, or Akita+/- /hAR mice. Akita+/- mice had impaired plaque regression as measured by changes in plaque size and the contents of CD68+ cells (macrophages), lipids, and collagen. Supporting synergy between hyperglycemia and hAR were the even more pronounced changes in these parameters in Akita+/- /hAR mice, which had atherosclerosis progression in spite of normolipidemia. Plaque CD68+ cells from the Akita+/- /hAR mice had increased oxidant stress and expression of inflammation-associated genes but decreased expression of anti-inflammatory genes. In summary, hAR expression amplifies impaired atherosclerosis regression in diabetic mice, likely by interfering with the expected reduction in plaque macrophage inflammation.

miR33 inhibition overcomes deleterious effects of diabetes mellitus on atherosclerosis plaque regression in mice.

RATIONALE: Diabetes mellitus increases cardiovascular disease risk in humans and remains elevated despite cholesterol-lowering therapy with statins. Consistent with this, in mouse models, diabetes mellitus impairs atherosclerosis plaque regression after aggressive cholesterol lowering. MicroRNA 33 (miR33) is a key negative regulator of the reverse cholesterol transport factors, ATP-binding cassette transporter A1 and high-density lipoprotein, which suggested that its inhibition may overcome this impairment. OBJECTIVE: To assess the effects of miR33 inhibition on atherosclerosis regression in diabetic mice. METHODS AND RESULTS: Reversa mice, which are deficient in the low-density lipoprotein receptor and in which hypercholesterolemia is reversed by conditional inactivation of the microsomal triglyceride transfer protein gene, were placed on an atherogenic diet for 16 weeks, then either made diabetic by streptozotocin injection or kept normoglycemic. Lipid-lowering was induced by microsomal triglyceride transfer protein gene inactivation, and mice were treated with anti-miR33 or control oligonucleotides. Although regression was impaired in diabetic mice treated with control oligonucleotides, anti-miR33 treatment decreased plaque macrophage content and inflammatory gene expression in these mice. The decreased macrophage content in anti-miR33 treated diabetic mice was associated with a blunting of hyperglycemia-induced monocytosis and reduced monocyte recruitment to the plaque, which was traced to an inhibition of the proliferation of bone marrow monocyte precursors associated with the upregulation of their Abca1. CONCLUSIONS: miR33 inhibition overcomes deleterious effects of diabetes mellitus in atherosclerosis regression in mice, which suggests a therapeutic strategy in diabetic patients, who remain at elevated cardiovascular disease risk, despite plasma cholesterol lowering.

Effects of native and myeloperoxidase-modified apolipoprotein a-I on reverse cholesterol transport and atherosclerosis in mice.

OBJECTIVE: Preclinical and clinical studies have shown beneficial effects of infusions of apolipoprotein A-I (ApoA-I) on atherosclerosis. ApoA-I is also a target for myeloperoxidase-mediated oxidation, leading in vitro to a loss of its ability to promote ATP-binding cassette transporter A1-dependent macrophage cholesterol efflux. Therefore, we hypothesized that myeloperoxidase-mediated ApoA-I oxidation would impair its promotion of reverse cholesterol transport in vivo and the beneficial effects on atherosclerotic plaques. APPROACH AND RESULTS: ApoA-I(-/-) or apolipoprotein E-deficient mice were subcutaneously injected with native human ApoA-I, oxidized human ApoA-I (myeloperoxidase/hydrogen peroxide/chloride treated), or carrier. Although early postinjection (8 hours) levels of total ApoA-I in plasma were similar for native versus oxidized human ApoA-I, native ApoA-I primarily resided within the high-density lipoprotein fraction, whereas the majority of oxidized human ApoA-I was highly cross-linked and not high-density lipoprotein particle associated, consistent with impaired ATP-binding cassette transporter A1 interaction. In ApoA-I(-/-) mice, ApoA-I oxidation significantly impaired reverse cholesterol transport in vivo. In advanced aortic root atherosclerotic plaques of apolipoprotein E-deficient mice, native ApoA-I injections led to significant decreases in lipid content, macrophage number, and an increase in collagen content; in contrast, oxidized human ApoA-I failed to mediate these changes. The decrease in plaque macrophages with native ApoA-I was accompanied by significant induction of their chemokine receptor CCR7. Furthermore, only native ApoA-I injections led to a significant reduction of inflammatory M1 and increase in anti-inflammatory M2 macrophage markers in the plaques. CONCLUSIONS: Myeloperoxidase-mediated oxidation renders ApoA-I dysfunctional and unable to (1) promote reverse cholesterol transport, (2) mediate beneficial changes in the composition of atherosclerotic plaques, and (3) pacify the inflammatory status of plaque macrophages.

Hypoxia in murine atherosclerotic plaques and its adverse effects on macrophages.

Hypoxia has been found in the atherosclerotic plaques of larger mammals, including humans. Whether hypoxia occurs in the plaques of standard mouse models with atherosclerosis has been controversial, given their small size. In this review, we summarize the findings of a recent report demonstrating that direct evidence of hypoxia can indeed be found in the plaques of mice deficient in apolipoprotein E (apoE-/-mice). Furthermore, studies in vitro showed that hypoxia promoted lipid synthesis and reduced cholesterol efflux through the ABCA1 pathway, and that the transcription factor HIF-1α mediated many, but not all, of the effects. These results are discussed in the context of the literature and clinical practice.

Rapid regression of atherosclerosis with MTP inhibitor treatment.

OBJECTIVE: Regression of atherosclerosis is a vital treatment goal of atherosclerotic vascular disease. Inhibitors of the microsomal triglyceride transfer protein (MTP) have been shown to reduce apolipoprotein B (apoB)-containing lipoproteins in animals and humans effectively. Therefore, the major aim of our study is to evaluate the effect of MTP inhibition on atherosclerotic plaque regression. METHODS: LDL-receptor-deficient (LDLr(-/-)) mice were fed a Western diet for 16 weeks and then harvested for baseline (n = 8), switched to chow diet (n = 8) or chow diet containing MTP inhibitor (BMS 212122; n = 8) for 2 weeks before harvesting. RESULTS: Treatment with MTP inhibitor led to rapid reduction in plasma lipid levels, which were accompanied by a significant decrease in lipid content and monocyte-derived (CD68+) cells in atherosclerotic plaques compared to baseline and chow diet control groups. MTP inhibitor-treated mice had increased collagen content, a marker associated with increased stability in human plaques. Furthermore, plaques of these mice showed a significant decrease in tissue factor and pro-inflammatory M1 macrophage marker monocyte chemoattractant protein-1 (MCP-I) and an increase in anti-inflammatory M2 macrophage markers arginase-I and mannose receptor 1 compared to mice in the baseline group. CONCLUSION: Reversal of hyperlipidemia in atherosclerotic mice by inhibition of MTP leads to rapid and beneficial changes in the composition and inflammatory state of the plaque.

Transient infantile hypertriglyceridemia, fatty liver, and hepatic fibrosis caused by mutated GPD1, encoding glycerol-3-phosphate dehydrogenase 1.

The molecular basis for primary hereditary hypertriglyceridemia has been identified in fewer than 5% of cases. Investigation of monogenic dyslipidemias has the potential to expose key metabolic pathways. We describe a hitherto unreported disease in ten individuals manifesting as moderate to severe transient childhood hypertriglyceridemia and fatty liver followed by hepatic fibrosis and the identification of the mutated gene responsible for this condition. We performed SNP array-based homozygosity mapping and found a single large continuous segment of homozygosity on chromosomal region 12q13.12. The candidate region contained 35 genes that are listed in Online Mendelian Inheritance in Man (OMIM) and 27 other genes. We performed candidate gene sequencing and screened both clinically affected individuals (children and adults with hypertriglyceridemia) and also a healthy cohort for mutations in GPD1, which encodes glycerol-3-phosphate dehydrogenase 1. Mutation analysis revealed a homozygous splicing mutation, c.361-1G>C, which resulted in an aberrantly spliced mRNA in the ten affected individuals. This mutation is predicted to result in a truncated protein lacking essential conserved residues, including a functional site responsible for initial substrate recognition. Functional consequences of the mutation were evaluated by measuring intracellular concentrations of cholesterol and triglyceride as well as triglyceride secretion in HepG2 (hepatocellular carcinoma) human cells lines overexpressing normal and mutant GPD1 cDNA. Overexpression of mutant GPD1 in HepG2 cells, in comparison to overexpression of wild-type GPD1, resulted in increased secretion of triglycerides (p = 0.01). This finding supports the pathogenicity of the identified mutation.

Cholesterol 27-hydroxylase but not apolipoprotein apoE contributes to A2A adenosine receptor stimulated reverse cholesterol transport.

Movement of free cholesterol between the cellular compartment and acceptor is governed by cholesterol gradients that are determined by several enzymes and reverse cholesterol transport proteins. We have previously demonstrated that adenosine A(2A) receptors inhibit foam cell formation and stimulate production of cholesterol 27-hydroxylase (CYP27A1), an enzyme involved in the conversion of cholesterol to oxysterols. We therefore asked whether the effect of adenosine A(2A) receptors on foam cell formation in vitro is mediated by CYP27A1 or apoE, a carrier for cholesterol in the serum. We found that specific lentiviral siRNA infection markedly reduced apoE or 27-hydroxylase mRNA in THP-1 cells. Despite diminished apoE expression (p < 0.0002, interferon-gamma (IFNγ) CGS vs. IFNγ alone, n=4), CGS-21680, an adenosine A(2A) receptor agonist, inhibits foam cell formation. In contrast, CGS-21680 had no effect on reducing foam cell formation in CYP27A1 KD cells (4 ± 2%; p<0.5113, inhibition vs. IFNγ alone, n=4). Previously, we reported the A(2A) agonist CGS-21680 increases apoAI-mediated cholesterol efflux nearly twofold in wild-type macrophages. Adenosine receptor activation had no effect on cholesterol efflux in CYP27A1 KD cells but reduced efflux in apoE KD cells. These results demonstrate that adenosine A(2A) receptor occupancy diminishes foam cell formation by increasing expression and function of CYP27A1.

Rat carboxylesterase ES-4 enzyme functions as a major hepatic neutral cholesteryl ester hydrolase.

Although esterification of free cholesterol to cholesteryl ester in the liver is known to be catalyzed by the enzyme acyl-coenzyme A:cholesterol acyltransferase, ACAT, the neutral cholesteryl ester hydrolase (nCEH) that catalyzes the reverse reaction has remained elusive. Because cholesterol undergoes continuous cycling between free and esterified forms, the steady-state concentrations in the liver of the two species and their metabolic availability for pathways, such as lipoprotein assembly and bile acid synthesis, depend upon nCEH activity. On the basis of the general characteristics of the family of rat carboxylesterases, we hypothesized that one member, ES-4, was a promising candidate as a hepatic nCEH. Using under- and overexpression approaches, we provide multiple lines of evidence that establish ES-4 as a bona fide endogenous nCEH that can account for the majority of cholesteryl ester hydrolysis in transformed rat hepatic cells and primary rat hepatocytes.

Antagonism of miR-33 in mice promotes reverse cholesterol transport and regression of atherosclerosis.

Plasma HDL levels have a protective role in atherosclerosis, yet clinical therapies to raise HDL levels have remained elusive. Recent advances in the understanding of lipid metabolism have revealed that miR-33, an intronic microRNA located within the SREBF2 gene, suppresses expression of the cholesterol transporter ABC transporter A1 (ABCA1) and lowers HDL levels. Conversely, mechanisms that inhibit miR-33 increase ABCA1 and circulating HDL levels, suggesting that antagonism of miR-33 may be atheroprotective. As the regression of atherosclerosis is clinically desirable, we assessed the impact of miR-33 inhibition in mice deficient for the LDL receptor (Ldlr-/- mice), with established atherosclerotic plaques. Mice treated with anti-miR33 for 4 weeks showed an increase in circulating HDL levels and enhanced reverse cholesterol transport to the plasma, liver, and feces. Consistent with this, anti-miR33-treated mice showed reductions in plaque size and lipid content, increased markers of plaque stability, and decreased inflammatory gene expression. Notably, in addition to raising ABCA1 levels in the liver, anti-miR33 oligonucleotides directly targeted the plaque macrophages, in which they enhanced ABCA1 expression and cholesterol removal. These studies establish that raising HDL levels by anti-miR33 oligonucleotide treatment promotes reverse cholesterol transport and atherosclerosis regression and suggest that it may be a promising strategy to treat atherosclerotic vascular disease.

Diabetes adversely affects macrophages during atherosclerotic plaque regression in mice.

OBJECTIVE: Patients with diabetes have increased cardiovascular risk. Atherosclerosis in these patients is often associated with increased plaque macrophages and dyslipidemia. We hypothesized that diabetic atherosclerosis involves processes that impair favorable effects of lipid reduction on plaque macrophages. RESEARCH DESIGN AND METHODS: Reversa mice are LDL receptor-deficient mice that develop atherosclerosis. Their elevated plasma LDL levels are lowered after conditional knockout of the gene encoding microsomal triglyceride transfer protein. We examined the morphologic and molecular changes in atherosclerotic plaques in control and streptozotocin-induced diabetic Reversa mice after LDL lowering. Bone marrow-derived macrophages were also used to study changes mediated by hyperglycemia. RESULTS: Reversa mice were fed a western diet for 16 weeks to develop plaques (baseline). Four weeks after lipid normalization, control (nondiabetic) mice had reduced plasma cholesterol (-77%), plaque cholesterol (-53%), and plaque cells positive for macrophage marker CD68+ (-73%), but increased plaque collagen (+116%) compared with baseline mice. Diabetic mice had similarly reduced plasma cholesterol, but collagen content increased by only 34% compared with baseline; compared with control mice, there were lower reductions in plaque cholesterol (-30%) and CD68+ cells (-41%). Diabetic (vs. control) plaque CD68+ cells also exhibited more oxidant stress and inflammatory gene expression and less polarization toward the anti-inflammatory M2 macrophage state. Many of the findings in vivo were recapitulated by hyperglycemia in mouse bone marrow-derived macrophages. CONCLUSIONS: Diabetes hindered plaque regression in atherosclerotic mice (based on CD68+ plaque content) and favorable changes in plaque macrophage characteristics after the reduction of elevated plasma LDL.

MiR-33 contributes to the regulation of cholesterol homeostasis.

Cholesterol metabolism is tightly regulated at the cellular level. Here we show that miR-33, an intronic microRNA (miRNA) located within the gene encoding sterol-regulatory element-binding factor-2 (SREBF-2), a transcriptional regulator of cholesterol synthesis, modulates the expression of genes involved in cellular cholesterol transport. In mouse and human cells, miR-33 inhibits the expression of the adenosine triphosphate-binding cassette (ABC) transporter, ABCA1, thereby attenuating cholesterol efflux to apolipoprotein A1. In mouse macrophages, miR-33 also targets ABCG1, reducing cholesterol efflux to nascent high-density lipoprotein (HDL). Lentiviral delivery of miR-33 to mice represses ABCA1 expression in the liver, reducing circulating HDL levels. Conversely, silencing of miR-33 in vivo increases hepatic expression of ABCA1 and plasma HDL levels. Thus, miR-33 appears to regulate both HDL biogenesis in the liver and cellular cholesterol efflux.

A2A adenosine receptor stimulation decreases foam cell formation by enhancing ABCA1-dependent cholesterol efflux.

Immune and inflammatory cells play a critical role in the pathogenesis of atherosclerotic plaques. We have demonstrated that A2ARs inhibit foam cell formation and stimulate production of ABCA1, the primary transporter of lipoproteins. We asked whether the effects of A2ARs on foam cell formation in vitro are mediated by transporters involved in reverse cholesterol transport, ABCA1 and ABCG1. Foam cells were generated from THP-1 cells by incubation with 100 nM PMA for 2 days and incubated with acLDL (50 microg/mL) plus IFN-gamma (500 U/mL) +/- A2AR agonist CGS-21680 (1 microM). Radiolabeled cholesterol (0.2 microCi/ml) was added to cells, and efflux was measured using a liquid scintillation counter. Lentiviral siRNA infection markedly reduces ABCA1 or ABCG1 mRNA in THP-1 cells. Despite diminished ABCG1 expression (KD), CGS-21680 inhibits foam cell formation (81+5% inhibition; P<0.0001 vs. IFN-gamma alone; n=3) but has no effect on foam cell formation in ABCA1 KD cells (5+3% inhibition; P<0.85 vs. IFN-gamma alone; n=3). The A2A agonist increases apoA-I-mediated cholesterol efflux nearly twofold in THP-1-derived macrophages (from 9.5% to 17.5+2.5% [3H]-cholesterol efflux; P<0.0090 vs. control; n=3) but not in ABCA1 KD cells. Activation of Epac, a signaling molecule downstream of the A2AR, increased ABCA1 (23+5%; P<0.0007 vs. control; n=3) and phospho-ABCA1 (13+5%; P<0.0003 vs. control; n=3) protein. These results demonstrate that A2AR occupancy diminishes foam cell formation by stimulating increased reverse cholesterol transport via ABCA1.

Effects of amino acid substitutions at glycine 420 on SR-BI cholesterol transport function.

Scavenger receptor class B type I (SR-BI) facilitates the uptake of HDL cholesteryl esters (CEs) in a two-step process involving binding of HDL to its extracellular domain and transfer of HDL core CEs to a metabolically active membrane pool, where they are subsequently hydrolyzed by a neutral CE hydrolase. Recently, we characterized a mutant, G420H, which replaced glycine 420 in the extracellular domain of SR-BI with a histidine residue and had a profound effect on SR-BI function. The G420H mutant receptor exhibited a reduced ability to mediate selective HDL CE uptake and was unable to deliver HDL CE for hydrolysis, despite the fact that it retained the ability to bind HDL. This did not hold true if glycine 420 was replaced with an alanine residue; G420A maintained wild-type HDL binding and cholesterol transport activity. To further understand the role that glycine 420 plays in SR-BI function and why there was a disparity between replacing glycine 420 with a histidine versus an alanine, we generated a battery of point mutants by substituting glycine 420 with amino acids possessing side chains that were charged, hydrophobic, polar, or bulky and tested the resulting mutants for their ability to support HDL binding, HDL cholesterol transport, and delivery for hydrolysis. The results indicated that substitution with a negatively charged residue or a proline impaired cell surface expression of SR-BI or its interaction with HDL, respectively. Furthermore, substitution of glycine 420 with a positively charged residue reduced HDL CE uptake as well as its subsequent hydrolysis.

Nitric oxide mediates neurodegeneration and breakdown of the blood-brain barrier in tPA-dependent excitotoxic injury in mice.

Stroke and many neurodegenerative diseases culminate in neuronal death through a mechanism known as excitotoxicity. Excitotoxicity proceeds through a complex signaling pathway that includes the participation of the serine protease tissue plasminogen activator (tPA). tPA mediates neurotoxic effects on resident central nervous system cells as well alters blood-brain barrier (BBB) permeability, which further promotes neurodegeneration. Another signaling molecule that promotes neurodegeneration and BBB dysfunction is nitric oxide (NO), although its precise role in pathological progression remains unclear. We examine here the potentially interrelated roles of tPA, NO and peroxynitrite (ONOO-), which is the toxic metabolite of NO, in BBB breakdown and neurodegeneration following intrahippocampal injection of the glutamate analog kainite (KA). We find that NO and ONOO- production are linked to tPA-mediated excitotoxic injury, and demonstrate that NO provision suffices to restore the toxic effects of KA in tPA-deficient mice that are normally resistant to excitotoxicity. NO also promotes BBB breakdown and excitotoxicity. Interestingly, BBB breakdown in itself does not suffice to elicit neurodegeneration; a subsequent ONOO(-)-mediated event is required. In conclusion, NO and ONOO- function as downstream effectors of tPA-mediated excitotoxicity.

Changes in plasma membrane properties and phosphatidylcholine subspecies of insect Sf9 cells due to expression of scavenger receptor class B, type I, and CD36.

In mammalian cells scavenger receptor class B, type I (SR-BI), mediates the selective uptake of high density lipoprotein (HDL) cholesteryl ester into hepatic and steroidogenic cells. In addition, SR-BI has a variety of effects on plasma membrane properties including stimulation of the bidirectional flux of free cholesterol (FC) between cells and HDL and changes in the organization of plasma membrane FC as indicated by increased susceptibility to exogenous cholesterol oxidase. Recent studies in SR-BI-deficient mice and in SR-BI-expressing Sf9 insect cells showed that SR-BI has significant effects on plasma membrane ultrastructure. The present study was designed to test the range of SR-BI effects in Sf9 insect cells that typically have very low cholesterol content and a different phospholipid profile compared with mammalian cells. The results showed that, as in mammalian cells, SR-BI expression increased HDL cholesteryl ester selective uptake, cellular cholesterol mass, FC efflux to HDL, and the sensitivity of membrane FC to cholesterol oxidase. These activities were diminished or absent upon expression of the related scavenger receptor CD36. Thus, SR-BI has fundamental effects on cholesterol flux and membrane properties that occur in cells of evolutionarily divergent origins. Profiling of phospholipid species by electrospray ionization mass spectrometry showed that scavenger receptor expression led to the accumulation of phosphatidylcholine species with longer mono- or polyunsaturated acyl chains. These changes would be expected to decrease phosphatidylcholine/cholesterol interactions and thereby enhance cholesterol desorption from the membrane. Scavenger receptor-mediated changes in membrane phosphatidylcholine may contribute to the increased flux of cholesterol and other lipids elicited by these receptors.

Glycine 420 near the C-terminal transmembrane domain of SR-BI is critical for proper delivery and metabolism of high density lipoprotein cholesteryl ester.

Scavenger receptor BI, SR-BI, is a physiologically relevant receptor for high density lipoprotein (HDL) that mediates the uptake of cholesteryl esters and delivers them to a metabolically active membrane pool where they are subsequently hydrolyzed. A previously characterized SR-BI mutant, A-VI, with an epitope tag inserted into the extracellular domain near the C-terminal transmembrane segment, revealed a separation-of-function between SR-BI-mediated HDL cholesteryl ester uptake and cholesterol efflux to HDL, on one hand, and cholesterol release to small unilamellar phospholipid vesicle acceptors and an increased cholesterol oxidase-sensitive pool of membrane free cholesterol on the other. To further elucidate amino acid residues responsible for this separation-of-function phenotype, we engineered alanine substitutions and point mutations in and around the site of epitope tag insertion, and tested these for various cholesterol transport functions. We found that changing amino acid 420 from glycine to histidine had a profound effect on SR-BI function. Despite the ability to mediate selective HDL cholesteryl ester uptake, the G420H receptor had a greatly reduced ability to: 1) enlarge the cholesterol oxidase-sensitive pool of membrane free cholesterol, 2) mediate cholesterol efflux to HDL, even at low concentrations of HDL acceptor where binding-dependent cholesterol efflux predominates, and 3) accumulate cholesterol mass within the cell. Most importantly, the G420H mutant was unable to deliver the HDL cholesteryl ester to a metabolically active membrane compartment for efficient hydrolysis. These observations have important implications regarding SR-BI function as related to its structure near the C-terminal transmembrane domain.