Seongah Han

ATP-Binding Cassette Transporters and HDL Suppress Hematopoietic Stem Cell Proliferation

Inhibiting Leukocytosis Leukocytosis—an elevated white blood cell count—contributes by unknown mechanisms to the pathogenesis of atherosclerosis and associated coronary heart disease. Now, Yvan-Charvet et al. (p. 1689, published online 20 May; see the Perspective by Hansson and Björkholm) show that the adenosine triphosphate–binding cassette transporters ABCA1 and ABCG1 are critical suppressors of atherosclerosis-associated leukocytosis. Mice deficient in both transporters in blood-producing hematopoietic cells possessed increased levels of hematopoietic stem and multipotential progenitor cells and accelerated atherosclerosis. ABCA1 and ABGA1 protect against atherosclerosis by promoting cholesterol efflux from cholesterol-laden macrophage foam cells to lipid-poor high-density lipoprotein (HDL) and apolipoprotein A-1. The leukocytosis and atherosclerosis in ABCA1- and ABG1-deficient mice were reversed in the presence of high amounts of HDL. Thus, signaling already known to inhibit atherosclerosis by reducing cholesterol in atherosclerotic plaques also reduces atherosclerosis-associated leukocytosis. Pathways that reduce cholesterol in atherosclerosis also suppress increased immune cell numbers associated with the disease. Elevated leukocyte cell numbers (leukocytosis), and monocytes in particular, promote atherosclerosis; however, how they become increased is poorly understood. Mice deficient in the adenosine triphosphate–binding cassette (ABC) transporters ABCA1 and ABCG1, which promote cholesterol efflux from macrophages and suppress atherosclerosis in hypercholesterolemic mice, displayed leukocytosis, a transplantable myeloproliferative disorder, and a dramatic expansion of the stem and progenitor cell population containing Lin–Sca-1+Kit+ (LSK) in the bone marrow. Transplantation of Abca1–/– Abcg1–/– bone marrow into apolipoprotein A-1 transgenic mice with elevated levels of high-density lipoprotein (HDL) suppressed the LSK population, reduced leukocytosis, reversed the myeloproliferative disorder, and accelerated atherosclerosis. The findings indicate that ABCA1, ABCG1, and HDL inhibit the proliferation of hematopoietic stem and multipotential progenitor cells and connect expansion of these populations with leukocytosis and accelerated atherosclerosis.

Combined deficiency of ABCA1 and ABCG1 promotes foam cell accumulation and accelerates atherosclerosis in mice

HDLs protect against the development of atherosclerosis, but the underlying mechanisms are poorly understood. HDL and its apolipoproteins can promote cholesterol efflux from macrophage foam cells via the ATP-binding cassette transporters ABCA1 and ABCG1. Experiments addressing the individual roles of ABCA1 and ABCG1 in the development of atherosclerosis have produced mixed results, perhaps because of compensatory upregulation in the individual KO models. To clarify the role of transporter-mediated sterol efflux in this disease process, we transplanted BM from Abca1(-/-)Abcg1(-/-) mice into LDL receptor-deficient mice and administered a high-cholesterol diet. Compared with control and single-KO BM recipients, Abca1(-/-)Abcg1(-/-) BM recipients showed accelerated atherosclerosis and extensive infiltration of the myocardium and spleen with macrophage foam cells. In experiments with isolated macrophages, combined ABCA1 and ABCG1 deficiency resulted in impaired cholesterol efflux to HDL or apoA-1, profoundly decreased apoE secretion, and increased secretion of inflammatory cytokines and chemokines. In addition, these cells showed increased apoptosis when challenged with free cholesterol or oxidized LDL loading. These results suggest that the combined effects of ABCA1 and ABCG1 in mediating macrophage sterol efflux are central to the antiatherogenic properties of HDL.

Dual role of transcription factor FoxO1 in controlling hepatic insulin sensitivity and lipid metabolism.

Hepatic insulin resistance affects both carbohydrate and lipid metabolism. It has been proposed that insulin controls these 2 metabolic branches through distinct signaling pathways. FoxO transcription factors are considered effectors of the pathway regulating hepatic glucose production. Here we show that adenoviral delivery of constitutively nuclear forkhead box O1 (FoxO1) to mouse liver results in steatosis arising from increased triglyceride accumulation and decreased fatty acid oxidation. FoxO1 gain of function paradoxically increased insulin sensitivity by promoting Akt phosphorylation, while FoxO1 inhibition via siRNA decreased it. We show that FoxO1 regulation of Akt phosphorylation does not require DNA binding and is associated with repression of the pseudokinase tribble 3 (Trb3), a modulator of Akt activity. This unexpected dual role of FoxO1 in promoting insulin sensitivity and lipid synthesis in addition to glucose production has the potential to explain the peculiar admixture of insulin resistance and sensitivity that is commonly observed in the metabolic syndrome.

Increased inflammatory gene expression in ABC transporter deficient macrophages: free cholesterol accumulation, increased signaling via Toll-like receptors and neutrophil infiltration of atherosclerotic lesions

Background— Two macrophage ABC transporters, ABCA1 and ABCG1, have a major role in promoting cholesterol efflux from macrophages. Peritoneal macrophages deficient in ABCA1, ABCG1, or both show enhanced expression of inflammatory and chemokine genes. This study was undertaken to elucidate the mechanisms and consequences of enhanced inflammatory gene expression in ABC transporter–deficient macrophages. Methods and Results— Basal and lipopolysaccharide-stimulated thioglycollate-elicited peritoneal macrophages showed increased inflammatory gene expression in the order Abca1−/−Abcg1−/−>Abcg1−/−>Abca1−/−>wild-type. The increased inflammatory gene expression was abolished in macrophages deficient in Toll-like receptor 4 (TLR4) or MyD88/TRIF. TLR4 cell surface concentration was increased in Abca1−/−Abcg1−/−>Abcg1−/−> Abca1−/−> wild-type macrophages. Treatment of transporter-deficient cells with cyclodextrin reduced and cholesterol-cyclodextrin loading increased inflammatory gene expression. Abca1−/−Abcg1− bone marrow–derived macrophages showed enhanced inflammatory gene responses to TLR2, TLR3, and TLR4 ligands. To assess in vivo relevance, we injected intraperitoneally thioglycollate in Abcg1−/− bone marrow–transplanted, Western diet–fed, Ldlr-deficient mice. This resulted in a profound inflammatory infiltrate in the adventitia and necrotic core region of atherosclerotic lesions, consisting primarily of neutrophils. Conclusions— The results suggest that high-density lipoprotein and apolipoprotein A-1 exert anti-inflammatory effects by promoting cholesterol efflux via ABCG1 and ABCA1 with consequent attenuation of signaling via Toll-like receptors. In response to a peripheral inflammatory stimulus, atherosclerotic lesions containing Abcg1−/− macrophages experience an inflammatory “echo,” suggesting a possible mechanism of plaque destabilization in subjects with low high-density lipoprotein levels.

Increased CD36 protein as a response to defective insulin signaling in macrophages.

Accelerated atherosclerosis is a major cause of morbidity and death in insulin-resistant states such as obesity and the metabolic syndrome, but the underlying mechanisms are poorly understood. We show that macrophages from obese (ob/ob) mice have increased binding and uptake of oxidized LDL, in part due to a post-transcriptional increase in CD36 protein. Macrophages from ob/ob mice are also insulin resistant, as shown by reduced expression and signaling of insulin receptors. Three lines of evidence indicate that the increase in CD36 is caused by defective insulin signaling: (a) Treatment of wild-type macrophages with LY294002, an inhibitor of insulin signaling via PI3K, results in an increase in CD36; (b) insulin receptor knockout macrophages show a post-transcriptional increase in CD36 protein; and (c) administration of thiazolidinediones to intact ob/ob mice and ob/ob, LDL receptor-deficient mice results in a reversal of macrophage insulin receptor defects and decreases CD36 protein. The last finding contrasts with the increase in CD36 that results from treatment of macrophages with these drugs ex vivo. The results suggest that defective macrophage insulin signaling predisposes to foam cell formation and atherosclerosis in insulin-resistant states and that this is reversed in vivo by treatment with PPAR-gamma activators.

Decreased Atherosclerosis in Low-Density Lipoprotein Receptor Knockout Mice Transplanted With Abcg1−/− Bone Marrow

Objective—Recent studies indicate that the ATP-binding cassette transporter ABCG1 can promote cholesterol efflux from macrophages to high-density lipoprotein. This study was designed to assess the in vivo role of macrophage ABCG1 in atherosclerosis. Methods and Results—Bone marrow from Abcg1−/− mice was transplanted into irradiated Ldlr−/− recipients, and atherosclerosis was evaluated by aortic root assay after 7 or 11 weeks of feeding on a Western diet. After 7 weeks, there was no difference in lesion area in mice receiving either wild-type or Abcg1−/− bone marrow, whereas after 11 weeks, lesion area was moderately but significantly reduced in Abcg1−/− recipients. ABCG1-deficient peritoneal macrophages showed induction of several liver X receptor target genes, such as Abca1 and Srebp1c, and a dramatic increase in apolipoprotein E (apoE) protein both in cell media and lysates, without parallel change in apoE mRNA. Abca1 knockdown prevented the increase in apoE secretion but had minimal effects on apoE accumulation in cell lysates of Abcg1−/− macrophages. Plasma apoE levels were markedly increased in recipients of Abcg1−/− bone marrow. Conclusions—These studies reveal an inverse relationship between Abcg1 expression and apoE accumulation and secretion in macrophages. The reduced atherosclerosis in recipients of Abcg1-deficient bone marrow may be explained by induction of Abca1 and an associated increase in macrophage apoE secretion.

The Macrophage at the Crossroads of Insulin Resistance and Atherosclerosis

The macrophage has emerged as an important player in the pathogenesis of both atherosclerosis and insulin resistance. Cross-talk between inflammatory macrophages and adipocytes may be involved in insulin resistance in peripheral tissues. Defective insulin signaling in cells of the arterial wall including macrophages may promote the development of atherosclerosis. Insulin resistant macrophages are more susceptible to endoplasmic reticulum stress and apoptosis in response to various stimuli such as nutrient deprivation, free cholesterol loading, and oxidized LDL. Increased apoptosis of insulin resistant macrophages and impaired phagocytic clearance of apoptotic cells by insulin resistant macrophages in atherosclerotic lesions may lead to enhanced postapoptotic necrosis, larger lipid-rich cores, increased inflammation, and more complex vulnerable plaques.

Macrophage deficiency of p38α MAPK promotes apoptosis and plaque necrosis in advanced atherosclerotic lesions in mice

ER stress occurs in macrophage-rich areas of advanced atherosclerotic lesions and contributes to macrophage apoptosis and subsequent plaque necrosis. Therefore, signaling pathways that alter ER stress-induced apoptosis may affect advanced atherosclerosis. Here we placed Apoe-/- mice deficient in macrophage p38alpha MAPK on a Western diet and found that they had a marked increase in macrophage apoptosis and plaque necrosis. The macrophage p38alpha-deficient lesions also exhibited a significant reduction in collagen content and a marked thinning of the fibrous cap, which suggests that plaque progression was advanced in these mice. Consistent with our in vivo data, we found that ER stress-induced apoptosis in cultured primary mouse macrophages was markedly accelerated under conditions of p38 inhibition. Pharmacological inhibition or genetic ablation of p38 suppressed activation of Akt in cultured macrophages and in atherosclerotic lesions. In addition, inhibition of Akt enhanced ER stress-induced macrophage apoptosis, and expression of a constitutively active myristoylated Akt blocked the enhancement of ER stress-induced apoptosis that occurred with p38 inhibition in cultured cells. Our results demonstrate that p38alpha MAPK may play a critical role in suppressing ER stress-induced macrophage apoptosis in vitro and advanced lesional macrophage apoptosis in vivo.

Defective Phagocytosis of Apoptotic Cells by Macrophages in Atherosclerotic Lesions of ob/ob Mice and Reversal by a Fish Oil Diet

Rationale: The complications of atherosclerosis are a major cause of death and disability in type 2 diabetes. Defective clearance of apoptotic cells by macrophages (efferocytosis) is thought to lead to increased necrotic core formation and inflammation in atherosclerotic lesions. Objective: To determine whether there is defective efferocytosis in a mouse model of obesity and atherosclerosis. Methods and Results: We quantified efferocytosis in peritoneal macrophages and in atherosclerotic lesions of obese ob/ob or ob/ob;Ldlr−/− mice and littermate controls. Peritoneal macrophages from ob/ob and ob/ob;Ldlr−/− mice showed impaired efferocytosis, reflecting defective phosphatidylinositol 3-kinase activation during uptake of apoptotic cells. Membrane lipid composition of ob/ob and ob/ob;Ldlr−/− macrophages showed an increased content of saturated fatty acids (FAs) and decreased &ohgr;-3 FAs (eicosapentaenoic acid and docosahexaenoic acid) compared to controls. A similar defect in efferocytosis was induced by treating control macrophages with saturated free FA/BSA complexes, whereas the defect in ob/ob macrophages was reversed by treatment with eicosapentaenoic acid/BSA or by feeding ob/ob mice a fish oil diet rich in &ohgr;-3 FAs. There was also defective macrophage efferocytosis in atherosclerotic lesions of ob/ob;Ldlr−/− mice and this was reversed by a fish oil–rich diet. Conclusions: The findings suggest that in obesity and type 2 diabetes elevated levels of saturated FAs and/or decreased levels of &ohgr;-3 FAs contribute to decreased macrophage efferocytosis. Beneficial effects of fish oil diets in atherosclerotic cardiovascular disease may involve improvements in macrophage function related to reversal of defective efferocytosis and could be particularly important in type 2 diabetes and obesity.

Regulation of hepatic LDL receptors by mTORC1 and PCSK9 in mice

Individuals with type 2 diabetes have an increased risk of atherosclerosis. One factor underlying this is dyslipidemia, which in hyperinsulinemic subjects with early type 2 diabetes is typically characterized by increased VLDL secretion but normal LDL cholesterol levels, possibly reflecting enhanced catabolism of LDL via hepatic LDLRs. Recent studies have also suggested that hepatic insulin signaling sustains LDLR levels. We therefore sought to elucidate the mechanisms linking hepatic insulin signaling to regulation of LDLR levels. In WT mice, insulin receptor knockdown by shRNA resulted in decreased hepatic mTORC1 signaling and LDLR protein levels. It also led to increased expression of PCSK9, a known post-transcriptional regulator of LDLR expression. Administration of the mTORC1 inhibitor rapamycin caused increased expression of PCSK9, decreased levels of hepatic LDLR protein, and increased levels of VLDL/LDL cholesterol in WT but not Pcsk9-/- mice. Conversely, mice with increased hepatic mTORC1 activity exhibited decreased expression of PCSK9 and increased levels of hepatic LDLR protein levels. Pcsk9 is regulated by the transcription factor HNF1α, and our further detailed analyses suggest that increased mTORC1 activity leads to activation of PKCδ, reduced activity of HNF4α and HNF1α, decreased PCSK9 expression, and ultimately increased hepatic LDLR protein levels, which result in decreased circulating LDL levels. We therefore suggest that PCSK9 inhibition could be an effective way to reduce the adverse side effect of increased LDL levels that is observed in transplant patients taking rapamycin as immunosuppressive therapy.