Sandra Abramowicz

ApoE regulates hematopoietic stem cell proliferation, monocytosis, and monocyte accumulation in atherosclerotic lesions in mice

Leukocytosis is associated with increased cardiovascular disease risk in humans and develops in hypercholesterolemic atherosclerotic animal models. Leukocytosis is associated with the proliferation of hematopoietic stem and multipotential progenitor cells (HSPCs) in mice with deficiencies of the cholesterol efflux-promoting ABC transporters ABCA1 and ABCG1 in BM cells. Here, we have determined the role of endogenous apolipoprotein-mediated cholesterol efflux pathways in these processes. In Apoe⁻/⁻ mice fed a chow or Western- type diet, monocytosis and neutrophilia developed in association with the proliferation and expansion of HSPCs in the BM. In contrast, Apoa1⁻/⁻ mice showed no monocytosis compared with controls. ApoE was found on the surface of HSPCs, in a proteoglycan-bound pool, where it acted in an ABCA1- and ABCG1-dependent fashion to decrease cell proliferation. Accordingly, competitive BM transplantation experiments showed that ApoE acted cell autonomously to control HSPC proliferation, monocytosis, neutrophilia, and monocyte accumulation in atherosclerotic lesions. Infusion of reconstituted HDL and LXR activator treatment each reduced HSPC proliferation and monocytosis in Apoe⁻/⁻ mice. These studies suggest a specific role for proteoglycanbound ApoE at the surface of HSPCs to promote cholesterol efflux via ABCA1/ABCG1 and decrease cell proliferation, monocytosis, and atherosclerosis. Although endogenous apoA-I was ineffective, pharmacologic approaches to increasing cholesterol efflux suppressed stem cell proliferative responses.

Deficiency of ATP-Binding Cassette Transporters A1 and G1 in Macrophages Increases Inflammation and Accelerates Atherosclerosis in Mice

Rationale: Plasma high-density lipoprotein levels are inversely correlated with atherosclerosis. Although it is widely assumed that this is attributable to the ability of high-density lipoprotein to promote cholesterol efflux from macrophage foam cells, direct experimental support for this hypothesis is lacking. Objective: To assess the role of macrophage cholesterol efflux pathways in atherogenesis. Methods and Results: We developed mice with efficient deletion of the ATP-binding cassette transporters A1 and G1 (ABCA1 and ABCG1) in macrophages (MAC-ABCDKO mice) but not in hematopoietic stem or progenitor populations. MAC-ABCDKO bone marrow (BM) was transplanted into Ldlr−/− recipients. On the chow diet, these mice had similar plasma cholesterol and blood monocyte levels but increased atherosclerosis compared with controls. On the Western-type diet, MAC-ABCDKO BM–transplanted Ldlr−/− mice had disproportionate atherosclerosis, considering they also had lower very low-density lipoprotein/low-density lipoprotein cholesterol levels than controls. ABCA1/G1-deficient macrophages in lesions showed increased inflammatory gene expression. Unexpectedly, Western-type diet–fed MAC-ABCDKO BM–transplanted Ldlr−/− mice displayed monocytosis and neutrophilia in the absence of hematopoietic stem and multipotential progenitor cells proliferation. Mechanistic studies revealed increased expressions of machrophage colony stimulating factor and granulocyte colony stimulating factor in splenic macrophage foam cells, driving BM monocyte and neutrophil production. Conclusions: These studies show that macrophage deficiency of ABCA1/G1 is proatherogenic likely by promoting plaque inflammation and uncover a novel positive feedback loop in which cholesterol-laden splenic macrophages signal BM progenitors to produce monocytes, with suppression by macrophage cholesterol efflux pathways.

Deficiency of ABCA1 and ABCG1 in Macrophages Increases Inflammation and Accelerates Atherosclerosis in Mice

Rationale: Plasma high-density lipoprotein levels are inversely correlated with atherosclerosis. Although it is widely assumed that this is attributable to the ability of high-density lipoprotein to promote cholesterol efflux from macrophage foam cells, direct experimental support for this hypothesis is lacking. Objective: To assess the role of macrophage cholesterol efflux pathways in atherogenesis. Methods and Results: We developed mice with efficient deletion of the ATP-binding cassette transporters A1 and G1 (ABCA1 and ABCG1) in macrophages (MAC-ABCDKO mice) but not in hematopoietic stem or progenitor populations. MAC-ABCDKO bone marrow (BM) was transplanted into Ldlr−/− recipients. On the chow diet, these mice had similar plasma cholesterol and blood monocyte levels but increased atherosclerosis compared with controls. On the Western-type diet, MAC-ABCDKO BM–transplanted Ldlr−/− mice had disproportionate atherosclerosis, considering they also had lower very low-density lipoprotein/low-density lipoprotein cholesterol levels than controls. ABCA1/G1-deficient macrophages in lesions showed increased inflammatory gene expression. Unexpectedly, Western-type diet–fed MAC-ABCDKO BM–transplanted Ldlr−/− mice displayed monocytosis and neutrophilia in the absence of hematopoietic stem and multipotential progenitor cells proliferation. Mechanistic studies revealed increased expressions of machrophage colony stimulating factor and granulocyte colony stimulating factor in splenic macrophage foam cells, driving BM monocyte and neutrophil production. Conclusions: These studies show that macrophage deficiency of ABCA1/G1 is proatherogenic likely by promoting plaque inflammation and uncover a novel positive feedback loop in which cholesterol-laden splenic macrophages signal BM progenitors to produce monocytes, with suppression by macrophage cholesterol efflux pathways.

Activation of Liver X Receptor Decreases Atherosclerosis in Ldlr−/− Mice in the Absence of ATP-Binding Cassette Transporters A1 and G1 in Myeloid Cells

Objective—Liver X receptor (LXR) activators decrease atherosclerosis in mice. LXR activators (1) directly upregulate genes involved in reverse cholesterol transport and (2) exert anti-inflammatory effects mediated by transrepression of nuclear factor-&kgr;B target genes. We investigated whether myeloid cell deficiency of ATP-binding cassette transporters A1 and G1 (ABCA1/G1), principal targets of LXR that promote macrophage cholesterol efflux and initiate reverse cholesterol transport, would abolish the beneficial effects of LXR activation on atherosclerosis. Approach and Results—LXR activator T0901317 substantially reduced inflammatory gene expression in macrophages lacking ABCA1/G1. Ldlr−/− mice were transplanted with Abca1−/−Abcg1−/− or wild-type bone marrow (BM) and fed a Western-type diet for 6 weeks with or without T0901317 supplementation. Abca1/g1 BM deficiency increased atherosclerotic lesion complexity and inflammatory cell infiltration into the adventitia and myocardium. T0901317 markedly decreased lesion area, complexity, and inflammatory cell infiltration in the Abca1−/−Abcg1−/− BM–transplanted mice. To investigate whether this was because of macrophage Abca1/g1 deficiency, Ldlr−/− mice were transplanted with LysmCreAbca1fl/flAbcg1fl/fl or Abca1fl/flAbcg1fl/fl BM and fed Western-type diet with or without the more specific LXR agonist GW3965 for 12 weeks. GW3965 decreased lesion size in both groups, and the decrease was more prominent in the LysmCreAbca1fl/flAbcg1fl/fl group. Conclusions—The results suggest that anti-inflammatory effects of LXR activators are of key importance to their antiatherosclerotic effects in vivo independent of cholesterol efflux pathways mediated by macrophage ABCA1/G1. This has implications for the development of LXR activators that lack adverse effects on lipogenic genes while maintaining the ability to transrepress inflammatory genes.

Interleukin-3/Granulocyte Macrophage Colony–Stimulating Factor Receptor Promotes Stem Cell Expansion, Monocytosis, and Atheroma Macrophage Burden in Mice With Hematopoietic ApoE Deficiency

Objective—Coronary heart disease is associated with monocytosis. Studies using animal models of monocytosis and atherosclerosis such as ApoE−/− mice have shown bone marrow (BM) hematopoietic stem and multipotential progenitor cell (HSPC) expansion, associated with increased cell surface expression of the common &bgr; subunit of the granulocyte macrophage colony–stimulating factor/interleukin-3 receptor (CBS) on HSPCs. ApoE−/− mice also display increased granulocyte macrophage colony–stimulating factor–dependent monocyte production in the spleen. We investigated the role of the CBS in cholesterol-driven HSPC expansion, monocytosis, and atherosclerosis. Approach and Results—Ldlr−/− mice were transplanted with ApoE−/−Cbs−/− or ApoE−/− BM followed by Western-type diet feeding. Compared with ApoE−/− BM–transplanted controls, ApoE−/−Cbs−/− BM–transplanted mice had reduced BM and splenic HSPC proliferation, fewer blood monocytes and neutrophils, and reduced macrophage content and area of early atherosclerotic lesions. More advanced lesions showed diminished macrophage and collagen content; however, lesion size was unchanged, reflecting an increase in necrotic core area, associated with a marked decrease in Abcg1 expression and increased macrophage apoptosis. Compared with wild-type mice, Western-type diet–fed ApoE−/− mice showed increased CBS expression on granulocyte macrophage colony–stimulating factor–producing innate response activator B cells and expansion of this population. ApoE−/−Cbs−/− BM–transplanted Ldlr−/− mice showed a marked decrease in innate response activator B cells compared with ApoE−/− BM–transplanted Ldlr−/− controls. Conclusions—Increased levels of CBS on HSPCs and splenic innate response activator B cells lead to expansion of these populations in ApoE−/− BM–transplanted Ldlr−/− mice, contributing to monocytosis and increased lesional macrophage content. However, in more advanced lesions, the CBS also has a role in atherosclerotic plaque stabilization.

Deficiency of ATP-Binding Cassette Transporters A1 and G1 in Endothelial Cells Accelerates Atherosclerosis in Mice

Objective— Plasma high-density lipoproteins have several putative antiatherogenic effects, including preservation of endothelial functions. This is thought to be mediated, in part, by the ability of high-density lipoproteins to promote cholesterol efflux from endothelial cells (ECs). The ATP-binding cassette transporters A1 and G1 (ABCA1 and ABCG1) interact with high-density lipoproteins to promote cholesterol efflux from ECs. To determine the impact of endothelial cholesterol efflux pathways on atherogenesis, we prepared mice with endothelium-specific knockout of Abca1 and Abcg1. Approach and Results— Generation of mice with EC-ABCA1 and ABCG1 deficiency required crossbreeding Abca1 fl/fl Abcg1 fl/fl Ldlr −/− mice with the Tie2Cre strain, followed by irradiation and transplantation of Abca1 fl/fl Abcg1 fl/fl bone marrow to abrogate the effects of macrophage ABCA1 and ABCG1 deficiency induced by Tie2Cre. After 20 to 22 weeks of Western-type diet, both single EC-Abca1 and Abcg1 deficiency increased atherosclerosis in the aortic root and whole aorta. Combined EC-Abca1/g1 deficiency caused a significant further increase in lesion area at both sites. EC-Abca1/g1 deficiency dramatically enhanced macrophage lipid accumulation in the branches of the aorta that are exposed to disturbed blood flow, decreased aortic endothelial NO synthase activity, and increased monocyte infiltration into the atherosclerotic plaque. Abca1/g1 deficiency enhanced lipopolysaccharide-induced inflammatory gene expression in mouse aortic ECs, which was recapitulated by ABCG1 deficiency in human aortic ECs. Conclusions— These studies provide direct evidence that endothelial cholesterol efflux pathways mediated by ABCA1 and ABCG1 are nonredundant and atheroprotective, reflecting preservation of endothelial NO synthase activity and suppression of endothelial inflammation, especially in regions of disturbed arterial blood flow.

Disruption of Mammalian Target of Rapamycin Complex 1 in Macrophages Decreases Chemokine Gene Expression and Atherosclerosis

Rationale: The mammalian target of rapamycin complex 1 inhibitor, rapamycin, has been shown to decrease atherosclerosis, even while increasing plasma low-density lipoprotein levels. This suggests an antiatherogenic effect possibly mediated by the modulation of inflammatory responses in atherosclerotic plaques. Objective: Our aim was to assess the role of macrophage mammalian target of rapamycin complex 1 in atherogenesis. Methods and Results: We transplanted bone marrow from mice in which a key mammalian target of rapamycin complex 1 adaptor, regulatory-associated protein of mTOR, was deleted in macrophages by Cre/loxP recombination (Mac-RapKO mice) into Ldlr−/− mice and then fed them the Western-type diet. Atherosclerotic lesions from Mac-RapKO mice showed decreased infiltration of macrophages, lesion size, and chemokine gene expression compared with control mice. Treatment of macrophages with minimally modified low-density lipoprotein resulted in increased levels of chemokine mRNAs and signal transducer and activator of transcription (STAT) 3 phosphorylation; these effects were reduced in Mac-RapKO macrophages. Although wild-type and Mac-RapKO macrophages showed similar STAT3 phosphorylation on Tyr705, Mac-RapKO macrophages showed decreased STAT3Ser727 phosphorylation in response to minimally modified low-density lipoprotein treatment and decreased Ccl2 promoter binding of STAT3. Conclusions: The results demonstrate cross-talk between nutritionally induced mammalian target of rapamycin complex 1 signaling and minimally modified low-density lipoprotein–mediated inflammatory signaling via combinatorial phosphorylation of STAT3 in macrophages, leading to increased STAT3 activity on the chemokine (C-C motif) ligand 2 (monocyte chemoattractant protein 1) promoter with proatherogenic consequences.

Deficiency of ATP-Binding Cassette Transporter B6 in Megakaryocyte Progenitors Accelerates Atherosclerosis in Mice

Objective—The ATP-binding cassette (ABC) transporter B6 (ABCB6) is highly expressed in megakaryocyte progenitors, but its role in platelet production and disease has not been elucidated. Approach and Results—Among various ABC transporters, ABCB6 was highly expressed in megakaryocyte progenitors, exhibiting the same pattern of expression of genes involved in heme synthesis pathway. Transplantation of Abcb6 deficient (Abcb6−/−) bone marrow into low density lipoprotein receptor deficient recipient mice resulted in expansion and proliferation of megakaryocyte progenitors, attributable to increased reactive oxygen species production in response to porphyrin loading. The enhanced megakaryopoiesis in Abcb6−/− bone marrow–transplanted mice was further illustrated by increased platelet counts, mean platelet volume, and platelet activity. Platelets from Abcb6−/− bone marrow–transplanted mice had higher levels of chemokine (C-C motif) ligand 5, which was associated with increased plasma chemokine (C-C motif) ligand 5 levels. There were also increased platelet–leukocyte aggregates, which resulted in leukocyte activation. Abcb6−/− bone marrow–transplanted mice had accelerated atherosclerosis which was associated with deposition of the chemotactic agent, chemokine (C-C motif) ligand 5 in atherosclerotic plaques, resulting in increased macrophage accumulation. Conclusions—Our findings identify a new role of ABCB6 in preventing atherosclerosis development by dampening platelet production, reactivity, and chemokine (C-C motif) ligand 5 deposition in atherosclerotic lesions.

Disruption of Mammalian Target of Rapamycin Complex 1 in Macrophages Decreases Chemokine Gene Expression and AtherosclerosisNovelty and Significance

Rationale: The mammalian target of rapamycin complex 1 inhibitor, rapamycin, has been shown to decrease atherosclerosis, even while increasing plasma low-density lipoprotein levels. This suggests an antiatherogenic effect possibly mediated by the modulation of inflammatory responses in atherosclerotic plaques. Objective: Our aim was to assess the role of macrophage mammalian target of rapamycin complex 1 in atherogenesis. Methods and Results: We transplanted bone marrow from mice in which a key mammalian target of rapamycin complex 1 adaptor, regulatory-associated protein of mTOR, was deleted in macrophages by Cre/loxP recombination (Mac-RapKO mice) into Ldlr−/− mice and then fed them the Western-type diet. Atherosclerotic lesions from Mac-RapKO mice showed decreased infiltration of macrophages, lesion size, and chemokine gene expression compared with control mice. Treatment of macrophages with minimally modified low-density lipoprotein resulted in increased levels of chemokine mRNAs and signal transducer and activator of transcription (STAT) 3 phosphorylation; these effects were reduced in Mac-RapKO macrophages. Although wild-type and Mac-RapKO macrophages showed similar STAT3 phosphorylation on Tyr705, Mac-RapKO macrophages showed decreased STAT3Ser727 phosphorylation in response to minimally modified low-density lipoprotein treatment and decreased Ccl2 promoter binding of STAT3. Conclusions: The results demonstrate cross-talk between nutritionally induced mammalian target of rapamycin complex 1 signaling and minimally modified low-density lipoprotein–mediated inflammatory signaling via combinatorial phosphorylation of STAT3 in macrophages, leading to increased STAT3 activity on the chemokine (C-C motif) ligand 2 (monocyte chemoattractant protein 1) promoter with proatherogenic consequences.

TTC39B deficiency stabilizes LXR reducing both atherosclerosis and steatohepatitis

Cellular mechanisms that mediate steatohepatitis, an increasingly prevalent condition in the Western world for which no therapies are available, are poorly understood. Despite the fact that its synthetic agonists induce fatty liver, the liver X receptor (LXR) transcription factor remains a target of interest because of its anti-atherogenic, cholesterol removal, and anti-inflammatory activities. Here we show that tetratricopeptide repeat domain protein 39B (Ttc39b, C9orf52) (T39), a high-density lipoprotein gene discovered in human genome-wide association studies, promotes the ubiquitination and degradation of LXR. Chow-fed mice lacking T39 (T39−/−) display increased high-density lipoprotein cholesterol levels associated with increased enterocyte ATP-binding cassette transporter A1 (Abca1) expression and increased LXR protein without change in LXR messenger RNA. When challenged with a high fat/high cholesterol/bile salt diet, T39−/− mice or mice with hepatocyte-specific T39 deficiency show increased hepatic LXR protein and target gene expression, and unexpectedly protection from steatohepatitis and death. Mice fed a Western-type diet and lacking low-density lipoprotein receptor (Ldlr−/−T39−/−) show decreased fatty liver, increased high-density lipoprotein, decreased low-density lipoprotein, and reduced atherosclerosis. In addition to increasing hepatic Abcg5/8 expression and limiting dietary cholesterol absorption, T39 deficiency inhibits hepatic sterol regulatory element-binding protein 1 (SREBP-1, ADD1) processing. This is explained by an increase in microsomal phospholipids containing polyunsaturated fatty acids, linked to an LXRα-dependent increase in expression of enzymes mediating phosphatidylcholine biosynthesis and incorporation of polyunsaturated fatty acids into phospholipids. The preservation of endogenous LXR protein activates a beneficial profile of gene expression that promotes cholesterol removal and inhibits lipogenesis. T39 inhibition could be an effective strategy for reducing both steatohepatitis and atherosclerosis.