Ligand-dependent interactions between SR-B1 and S1PR1 in macrophages and atherosclerotic plaques.

HDLs carry sphingosine-1-phosphate (S1P) and stimulate signaling pathways in different cells including macrophages and endothelial cells, involved in atherosclerotic plaque development. HDL signaling via S1P relies on the HDL receptor scavenger receptor class B, type I (SR-B1) and the sphingosine-1-phosphate receptor 1 (S1PR1), which interact when both are heterologously overexpressed in the HEK293 cell line. In this study, we set out to test if SR-B1 and S1PR1 interacted in primary murine macrophages in culture and atherosclerotic plaques. We used knock-in mice that endogenously expressed S1PR1 tagged with eGFP-(S1pr1eGFP/eGFP mice), combined with proximity ligation analysis to demonstrate that HDL stimulates the physical interaction between SR-B1 and S1PR1 in primary macrophages, that this is dependent on HDL-associated S1P and can be blocked by an inhibitor of SR-B1's lipid transfer activity or an antagonist of S1PR1. We also demonstrate that a synthetic S1PR1-selective agonist, SEW2871, stimulates the interaction between SR-B1 and S1PR1 and that this was also blocked by an inhibitor of SR-B1's lipid transport activity. Furthermore, we detected abundant SR-B1/S1PR1 complexes in atherosclerotic plaques of S1pr1eGFP/eGFP mice that also lacked apolipoprotein E. Treatment of mice with the S1PR1 antagonist, Ex26, for 12 h disrupted the SR-B1-S1PR1 interaction in atherosclerotic plaques. These findings demonstrate that SR-B1 and S1PR1 form ligand-dependent complexes both in cultured primary macrophages and within atherosclerotic plaques in mice and provide mechanistic insight into how SR-B1 and S1PR1 participate in mediating HDL signaling to activate atheroprotective responses in macrophages.

Apolipoprotein A1 Protects Against Necrotic Core Development in Atherosclerotic Plaques: PDZK1-Dependent High-Density Lipoprotein Suppression of Necroptosis in Macrophages.

BACKGROUND: Atherosclerosis is a chronic disease affecting artery wall and a major contributor to cardiovascular diseases. Large necrotic cores increase risk of plaque rupture leading to thrombus formation. Necrotic cores are rich in debris from dead macrophages. Programmed necrosis (necroptosis) contributes to necrotic core formation. HDL (high-density lipoprotein) exerts direct atheroprotective effects on different cells within atherosclerotic plaques. Some of these depend on the SR-B1 (scavenger receptor class B type I) and the adapter protein PDZK1 (postsynaptic density protein/Drosophila disc-large protein/Zonula occludens protein containing 1). However, a role for HDL in protecting against necroptosis and necrotic core formation in atherosclerosis is not completely understood. METHODS: Low-density lipoprotein receptor-deficient mice engineered to express different amounts of ApoA1 (apolipoprotein A1), or to lack PDZK1 were fed a high fat diet for 10 weeks. Atherosclerotic plaque areas, necrotic cores, and key necroptosis mediators, RIPK3 (receptor interacting protein kinase 3), and MLKL (mixed lineage kinase domain-like protein) were characterized. Cultured macrophages were treated with HDL to determine its effects, as well as the roles of SR-B1, PDZK1, and the PI3K (phosphoinositide 3-kinase) signaling pathway on necroptotic cell death. RESULTS: Genetic overexpression reduced, and ApoA1 knockout increased necrotic core formation and RIPK3 and MLKL within atherosclerotic plaques. Macrophages were protected against necroptosis by HDL and this protection required SR-B1, PDZK1, and PI3K/Akt pathway. PDZK1 knockout increased atherosclerosis in LDLRKO mice, increasing necrotic cores and phospho-MLKL; both of which were reversed by restoring PDZK1 in BM-derived cells. CONCLUSIONS: Our findings demonstrate that HDL in vitro and ApoA1, in vivo, protect against necroptosis in macrophages and necrotic core formation in atherosclerosis, suggesting a pathway that could be a target for the treatment of atherosclerosis.

Extensive diet-induced atherosclerosis in scavenger receptor class B type 1-deficient mice is associated with substantial leukocytosis and elevated vascular cell adhesion molecule-1 expression in coronary artery endothelium.

High levels of low density lipoprotein (LDL) cholesterol and low levels of high density lipoprotein (HDL) cholesterol are risk factors for cardiovascular disease. Mice that lack genes involved in the clearance of LDL from the bloodstream, such as the LDL receptor and apolipoprotein E, are widely used models of experimental atherosclerosis. Conversely, mice that lack the HDL receptor, scavenger receptor class B type I, and therefore have disrupted HDL functionality, also develop diet-inducible atherosclerosis but are a seldom-used disease model. In this study, we compared atherosclerosis and associated phenotypes in scavenger receptor class B type I knockout mice with those of wild type, LDL receptor knockout, and apolipoprotein E knockout mice after 20 weeks of being fed an atherogenic diet containing sodium cholate. We found that while scavenger receptor class B type I knockout mice had substantially lower plasma cholesterol than LDL receptor and apolipoprotein E knockout mice, they developed atherosclerotic plaques with similar sizes and compositions in their aortic sinuses, and more extensive atherosclerosis in their descending aortas and coronary arteries. This was associated with elevated tumor necrosis factor alpha levels in scavenger receptor class B type I knockout mice compared to wild type and LDL receptor knockout mice, and lymphocytosis, monocytosis, and elevated vascular cell adhesion molecule expression in coronary artery endothelial cells compared to the other mice examined. We conclude that extensive atherosclerosis in arteries that are not generally susceptible to atherosclerosis in scavenger receptor class B type I knockout mice is driven by factors in addition to hypercholesterolemia, including inflammation, dysregulation of the immune system and increased sensitivity of endothelial cells in arteries that are normally resistant to atherosclerosis. Scavenger receptor class B type I knockout mice fed a cholate containing atherogenic diet may prove to be a useful model to study mechanisms of atherosclerosis and evaluate treatments that rely on intact LDL clearance pathways.

High Density Lipoprotein and Its Precursor Protein Apolipoprotein A1 as Potential Therapeutics to Prevent Anthracycline Associated Cardiotoxicity.

Cardiovascular disease and cancer are the leading causes of death in developed societies. Despite their effectiveness, many cancer therapies exhibit deleterious cardiovascular side effects such as cardiotoxicity and heart failure. The cardiotoxic effects of anthracyclines such as doxorubicin are the most well-characterized of cardiotoxic anti-cancer therapies. While other anti-neoplastic drugs also induce cardiotoxicity, often leading to heart failure, they are beyond the scope of this review. This review first summarizes the mechanisms of doxorubicin-induced cardiotoxicity. It then reviews emerging preclinical evidence that high density lipoprotein and its precursor protein apolipoprotein A1, which are known for their protective effects against ischemic cardiovascular disease, may also protect against doxorubicin-induced cardiotoxicity both directly and indirectly, when used therapeutically.

Treatment with apolipoprotein A1 protects mice against doxorubicin-induced cardiotoxicity in a scavenger receptor class B, type I-dependent manner.

Doxorubicin, an agent used to treat a variety of cancers, is cardiotoxic by triggering cardiomyocyte apoptosis. We previously showed that treating cultured cardiomyocytes with human high-density lipoprotein in vitro or transgenic overexpression of human apolipoprotein A1, its main structural protein, protects against doxorubicin-induced cardiomyocyte apoptosis in a manner dependent on the scavenger receptor class B type I [Durham KK, Chathely KM, Mak KC, Momen A, Thomas CT, Zhao YY, MacDonald ME, Curtis JM, Husain M, Trigatti BL. HDL protects against doxorubicin-induced cardiotoxicity in a scavenger receptor class B type 1-, phosphatidylinositol 3-kinase-, and Akt-dependent manner. Am J Physiol Heart Circ Physiol 314: H31-H44, 2018]. This was due to high-density lipoprotein-induced activation of Akt signaling in cardiomyocytes. We now demonstrate that mice lacking the scavenger receptor class B, type I exhibit increased sensitivity to doxorubicin-induced cardiomyocyte apoptosis in vivo. Cardiomyocytes expressing scavenger receptor class B, type I are protected from doxorubicin-induced apoptosis by preincubation with high-density lipoprotein isolated from wild-type mice, whereas high-density lipoprotein from scavenger receptor class B, type 1 knockout mice is less effective. Cardiomyocytes from scavenger receptor class B, type I knockout mice, however, are not protected by high-density lipoprotein in vitro, and hearts from knockout mice are more sensitive to doxorubicin in vivo. Pharmacological administration of purified apolipoprotein A1 dramatically protected wild-type mice from doxorubicin-induced cardiotoxicity and left ventricular dysfunction, whereas this protection was lost in scavenger receptor class B, type I-deficient mice. This demonstrates, at least in mice, that high-density lipoprotein therapy can confer protection against doxorubicin-induced cardiomyocyte apoptosis in a manner mediated by the scavenger receptor class B, type I. NEW & NOTEWORTHY We show that scavenger receptor class B, type I (SR-B1) mediates HDL-dependent protection against doxorubicin-induced cardiomyocyte apoptosis and that this is a property of SR-B1 in cardiomyocytes in vitro and in hearts in vivo. We also demonstrate that pharmacological treatment with apolipoprotein A1, the major HDL structural protein, protects mice against doxorubicin-induced cardiomyocyte apoptosis and left ventricular dysfunction in an SR-B1-dependent manner. This suggests that HDL-targeted pharmacological therapy may hold promise for protecting against the deleterious, cardiotoxic side effects of this commonly used chemotherapeutic drug.

Sialidase down-regulation reduces non-HDL cholesterol, inhibits leukocyte transmigration, and attenuates atherosclerosis in ApoE knockout mice.

Atherosclerosis is a complex disease that involves alterations in lipoprotein metabolism and inflammation. Protein and lipid glycosylation events, such as sialylation, contribute to the development of atherosclerosis and are regulated by specific glycosidases, including sialidases. To evaluate the effect of the sialidase neuraminidase 1 (NEU1) on atherogenesis, here we generated apolipoprotein E (ApoE)-deficient mice that express hypomorphic levels of NEU1 (Neu1hypoApoe-/-). We found that the hypomorphic NEU1 expression in male Apoe-/- mice reduces serum levels of very-low-density lipoprotein (VLDL) and LDL cholesterol, diminishes infiltration of inflammatory cells into lesions, and decreases aortic sinus atherosclerosis. Transplantation of Apoe-/- bone marrow (BM) into Neu1hypoApoe-/- mice significantly increased atherosclerotic lesion development and had no effect on serum lipoprotein levels. Moreover, Neu1hypoApoe-/- mice exhibited a reduction in circulating monocyte and neutrophil levels and had reduced hyaluronic acid and P-selectin adhesion capability on monocytes/neutrophils and T cells. Consistent with these findings, administration of a sialidase inhibitor, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, had a significant anti-atherogenic effect in the Apoe-/- mice. In summary, the reduction in NEU1 expression or function decreases atherosclerosis in mice via its significant effects on lipid metabolism and inflammatory processes. We conclude that NEU1 may represent a promising target for managing atherosclerosis.

Low-density lipoprotein (LDL)-dependent uptake of Gram-positive lipoteichoic acid and Gram-negative lipopolysaccharide occurs through LDL receptor.

Lipoteichoic acid (LTA) and lipopolysaccharide (LPS) are bacterial lipids that stimulate pro-inflammatory cytokine production, thereby exacerbating sepsis pathophysiology. Proprotein convertase subtilisin/kexin type 9 (PCSK9) negatively regulates uptake of cholesterol by downregulating hepatic lipoprotein receptors, including low-density lipoprotein (LDL) receptor (LDLR) and possibly LDLR-related protein-1 (LRP1). PCSK9 also negatively regulates Gram-negative LPS uptake by hepatocytes, however this mechanism is not completely characterized and mechanisms of Gram-positive LTA uptake are unknown. Therefore, our objective was to elucidate the mechanisms through which PCSK9 regulates uptake of LTA and LPS by investigating the roles of lipoproteins and lipoprotein receptors. Here we show that plasma PCSK9 concentrations increase transiently over time in septic and non-septic critically ill patients, with highly similar profiles over 14 days. Using flow cytometry, we demonstrate that PCSK9 negatively regulates LDLR-mediated uptake of LTA and LPS by HepG2 hepatocytes through an LDL-dependent mechanism, whereas LRP1 and high-density lipoprotein do not contribute to this uptake pathway. Bacterial lipid uptake by hepatocytes was not associated with cytokine production or hepatocellular injury. In conclusion, our study characterizes an LDL-dependent and LDLR-mediated bacterial lipid uptake pathway regulated by PCSK9, and provides evidence in support of PCSK9 inhibition as a potential therapeutic strategy for sepsis.

PDZK1 in leukocytes protects against cellular apoptosis and necrotic core development in atherosclerotic plaques in high fat diet fed ldl receptor deficient mice.

BACKGROUND AND AIMS: PDZK1 (Post-synaptic density protein/Drosophila disc-large protein/Zonula occludens protein containing 1) stabilizes the HDL receptor protein, SR-B1, in the liver, and mediates SR-B1 signaling outside of the liver. Complete knockout of pdzk1 increases atherosclerosis in apoE-deficient mice, but the effect of PDZK1 in leukocytes is not known. In this study, we tested the role of leukocyte PDZK1 in atherosclerosis development by using bone marrow transplantation to generate ldlr deficient mice lacking PDZK1 in leukocytes. METHODS: Ldlr-/- mice were transplanted with either pdzk1-/- or pdzk1+/+ bone marrow and fed a high-fat diet to induce atherosclerosis. RESULTS: Bone marrow specific pdzk1 knockout slightly increased atherosclerotic plaque sizes but strikingly increased sizes of necrotic cores and cellular apoptosis in within plaques. PDZK1 deficiency prevented HDL dependent protection of macrophages from apoptosis in vitro and sensitized peritoneal macrophages to apoptosis in situ. PDZK1 deficiency in macrophages also impaired their ability to engulf apoptotic cells, and attenuated the IL-4 dependent induction of mannose receptor in vitro and mannose receptor protein levels in macrophages in atherosclerotic plaques. CONCLUSIONS: PDZK1 is required for anti-atherogenic responses in macrophages including HDL dependent protection against apoptosis and macrophage mediated efferocytosis and limits the accumulation of apoptotic cells within atherosclerotic plaques protecting against necrotic core development.

High-density lipoprotein protects cardiomyocytes against necrosis induced by oxygen and glucose deprivation through SR-B1, PI3K, and AKT1 and 2.

The cardioprotective lipoprotein HDL (high-density lipoprotein) prevents myocardial infarction and cardiomyocyte death due to ischemia/reperfusion injury. The scavenger receptor class B, type 1 (SR-B1) is a high-affinity HDL receptor and has been shown to mediate HDL-dependent lipid transport as well as signaling in a variety of different cell types. The contribution of SR-B1 in cardiomyocytes to the protective effects of HDL on cardiomyocyte survival following ischemia has not yet been studied. Here, we use a model of simulated ischemia (oxygen and glucose deprivation, OGD) to assess the mechanistic involvement of SR-B1, PI3K (phosphatidylinositol-3-kinase), and AKT in HDL-mediated protection of cardiomyocytes from cell death. Neonatal mouse cardiomyocytes and immortalized human ventricular cardiomyocytes, subjected to OGD for 4 h, underwent substantial cell death due to necrosis but not necroptosis or apoptosis. Pretreatment of cells with HDL, but not low-density lipoprotein, protected them against OGD-induced necrosis. HDL-mediated protection was lost in cardiomyocytes from SR-B1-/- mice or when SR-B1 was knocked down in human immortalized ventricular cardiomyocytes. HDL treatment induced the phosphorylation of AKT in cardiomyocytes in an SR-B1-dependent manner. Finally, chemical inhibition of PI3K or AKT or silencing of either AKT1 or AKT2 gene expression abolished HDL-mediated protection against OGD-induced necrosis of cardiomyocytes. These results are the first to identify a role of SR-B1 in mediating the protective effects of HDL against necrosis in cardiomyocytes, and to identify AKT activation downstream of SR-B1 in cardiomyocytes.

HDL protects against doxorubicin-induced cardiotoxicity in a scavenger receptor class B type 1-, PI3K-, and Akt-dependent manner.

Doxorubicin is a widely used chemotherapeutic with deleterious cardiotoxic side effects. HDL has been shown to protect cardiomyocytes in vitro against doxorubicin-induced apoptosis. Scavenger receptor class B type 1 (SR-B1), a high-affinity HDL receptor, mediates cytoprotective signaling by HDL through Akt. Here, we assessed whether increased HDL levels protect against doxorubicin-induced cardiotoxicity in vivo and in cardiomyocytes in culture and explored the intracellular signaling mechanisms involved, particularly the role of SR-B1. Transgenic mice with increased HDL levels through overexpression of human apolipoprotein A1 (apoA1Tg/Tg) and wild-type mice (apoA1+/+) with normal HDL levels were treated repeatedly with doxorubicin. After treatment, apoA1+/+ mice displayed cardiac dysfunction, as evidenced by reduced left ventricular end-systolic pressure and +dP/d t, and histological analysis revealed cardiomyocyte atrophy and increased cardiomyocyte apoptosis after doxorubicin treatment. In contrast, apoA1Tg/Tg mice were protected against doxorubicin-induced cardiac dysfunction and cardiomyocyte atrophy and apoptosis. When SR-B1 was knocked out, however, overexpression of apoA1 did not protect against doxorubicin-induced cardiotoxicity. Using primary neonatal mouse cardiomyocytes and human immortalized ventricular cardiomyocytes in combination with genetic knockout, inhibitors, or siRNA-mediated knockdown, we demonstrated that SR-B1 is required for HDL-mediated protection of cardiomyocytes against doxorubicin-induced apoptosis in vitro via a pathway involving phosphatidylinositol 3-kinase and Akt1/2. Our findings provide proof of concept that raising apoA1 to supraphysiological levels can dramatically protect against doxorubicin-induced cardiotoxicity via a pathway that is mediated by SR-B1 and involves Akt1/2 activation in cardiomyocytes. NEW & NOTEWORTHY We have identified an important role for the scavenger receptor class B type 1 in facilitating high-density lipoprotein-mediated protection of cardiomyocytes against stress-induced apoptosis and shown that increasing plasma high-density lipoprotein protects against the deleterious side effects of the chemotherapeutic and cardiotoxic drug doxorubicin.

Sphingosine-1-Phosphate Receptor 1, Expressed in Myeloid Cells, Slows Diet-Induced Atherosclerosis and Protects against Macrophage Apoptosis in Ldlr KO Mice.

We generated myeloid specific sphingosine-1-phosphate receptor 1 (S1pr1) deficient mice by crossing mice that had myeloid specific expression of Cre recombinase (lyzMCre) with mice having the S1pr1 gene flanked by loxP recombination sites. We transplanted bone marrow from these mice and control lyzMCre mice with intact macrophage S1pr1 gene expression into low-density lipoprotein (LDL) receptor gene (Ldlr) deficient mice. The resulting chimeras were fed a high fat atherogenic diet for nine or twelve weeks and evaluated for atherosclerosis development in the aortic sinus. Selective S1pr1 deficiency in bone marrow-derived myeloid cells resulted in accelerated development of atherosclerosis, necrotic core formation and the appearance of apoptotic cells within atherosclerotic plaques of Ldlr knockout mice in response to a high fat diet. Examination of macrophages in culture revealed that the sphingosine-1-phosphate receptor 1 selective agonist, SEW2871 or high density lipoprotein (HDL), protected macrophages against apoptosis induced by endoplasmic reticulum (ER) stress or oxidized LDL, through activation of phosphatidylinositol-3-kinase/Akt signaling. Targeted S1pr1-deletion prevented Akt activation and protection against apoptosis by either SEW2871 or HDL. Our data suggests that sphingosine-1-phosphate receptor 1 in macrophages plays an important role in protecting them against apoptosis in vitro and in atherosclerotic plaques in vivo, and delays diet induced atherosclerosis development in Ldlr deficient mice.

Macrophage Apoptosis and Necrotic Core Development in Atherosclerosis: A Rapidly Advancing Field with Clinical Relevance to Imaging and Therapy.

Cardiovascular diseases represent 1 of the main causes of death worldwide, and atherosclerosis is 1 of the major contributors leading to ischemic heart disease. Macrophages actively participate in all stages of atherosclerosis development, from plaque initiation to the transition to vulnerable plaques. Macrophage apoptosis, in particular, has been recognized as a critical step in the formation of the necrotic core, a key characteristic of unstable lesions. In this review, we discuss the role of macrophage apoptosis and clearance of apoptotic cells by efferocytosis in the development of atherosclerosis, with particular emphasis on their contribution to the development of the necrotic core and the clinical implications of this process for plaque stabilization. We consider the molecular triggers of macrophage apoptosis during atherogenesis, the role of endoplasmic reticulum (ER) stress, the roles of key cellular mediators of apoptosis and efferocytosis, and mechanisms of defective efferocytosis in the progression of atherosclerotic plaques. Finally, we discuss the important clinical implications of rapidly evolving macrophage science, such as novel approaches to imaging vulnerable atherosclerotic plaques with macrophage-sensitive positron emission tomography and magnetic resonance imaging, the role of macrophages in mediating beneficial pleiotropic actions of lipid-lowering therapies, and novel therapeutic modalities targeting ER stress, autophagy, and deficient efferocytosis. Advances in understanding the critical role of macrophages in the progression and destabilization of atherosclerosis have the potential to greatly improve the prevention and management of atherosclerotic diseases over the next decade.

Endoplasmic Reticulum Stress and Ca2+ Depletion Differentially Modulate the Sterol Regulatory Protein PCSK9 to Control Lipid Metabolism.

Accumulating evidence implicates endoplasmic reticulum (ER) stress as a mediator of impaired lipid metabolism, thereby contributing to fatty liver disease and atherosclerosis. Previous studies demonstrated that ER stress can activate the sterol regulatory element-binding protein-2 (SREBP2), an ER-localized transcription factor that directly up-regulates sterol regulatory genes, including PCSK9 Given that PCSK9 contributes to atherosclerosis by targeting low density lipoprotein (LDL) receptor (LDLR) degradation, this study investigates a novel mechanism by which ER stress plays a role in lipid metabolism by examining its ability to modulate PCSK9 expression. Herein, we demonstrate the existence of two independent effects of ER stress on PCSK9 expression and secretion. In cultured HuH7 and HepG2 cells, agents or conditions that cause ER Ca2+ depletion, including thapsigargin, induced SREBP2-dependent up-regulation of PCSK9 expression. In contrast, a significant reduction in the secreted form of PCSK9 protein was observed in the media from both thapsigargin- and tunicamycin (TM)-treated HuH7 cells, mouse primary hepatocytes, and in the plasma of TM-treated C57BL/6 mice. Furthermore, TM significantly increased hepatic LDLR expression and reduced plasma LDL concentrations in mice. Based on these findings, we propose a model in which ER Ca2+ depletion promotes the activation of SREBP2 and subsequent transcription of PCSK9. However, conditions that cause ER stress regardless of their ability to dysregulate ER Ca2+ inhibit PCSK9 secretion, thereby reducing PCSK9-mediated LDLR degradation and promoting LDLR-dependent hepatic cholesterol uptake. Taken together, our studies provide evidence that the retention of PCSK9 in the ER may serve as a potential strategy for lowering LDL cholesterol levels.

Characterization of Proliferating Lesion-Resident Cells During All Stages of Atherosclerotic Growth.

BACKGROUND: Monocyte recruitment leads to accumulation of macrophage foam cells and contributes to atherosclerotic lesion growth. Recent studies have reported that lesion-resident macrophages can proliferate and represent a major cellular component during lesion development. This study was designed to assess whether the rate of macrophage proliferation changes during well-established stages of lesion growth and to characterize other populations of proliferating cells within these lesions. METHODS AND RESULTS: Using murine models of atherosclerosis (Apoe(-/-) and LDLr(-/-) mice) and human coronary artery lesions, in situ proliferation of lesion-resident cells at different stages of growth was assessed by staining for Ki67 and bromodeoxyuridine (BrdU). In early lesions, close to half of all actively growing macrophages were proliferating in situ. BrdU pulse labeling allowed for accurate identification of in situ proliferating macrophages compared to those derived from monocyte recruitment. Local macrophage proliferation declined as lesions advanced. Interestingly, intimal inflammatory cell infiltrates containing proliferating T lymphocytes were identified during the active phase of lesion growth and correlated with apoptotic cell death. Inflammatory cell infiltrates were completely resolved in advanced lesions and replaced with the necrotic core. CONCLUSIONS: Our findings indicate that atherosclerotic lesions contain locally proliferating macrophages primarily during early and intermediate stages of lesion growth. Furthermore, T-lymphocyte-enriched inflammatory cell infiltrates represent a novel subset of proliferating cells within the atherosclerotic lesion that correlate with apoptosis and precede the necrotic core. These findings have novel implications in understanding the pathogenesis of atherosclerosis and may implicate proliferating T lymphocytes as a contributing factor to lesion progression and stability.

Characterization of mice harboring a variant of EPCR with impaired ability to bind protein C: novel role of EPCR in hematopoiesis.

The interaction of protein C (PC) with the endothelial PC receptor (EPCR) enhances activated PC (APC) generation. The physiological importance of EPCR has been demonstrated in EPCR knockout mice which show early embryonic lethality due to placental thrombosis. In order to study the role of EPCR independent of PC interaction, we generated an EPCR point mutation knock-in mouse (EPCR(R84A/R84A)) which lacks the ability to bind PC/APC. EPCR(R84A/R84A) mice are viable and reproduce normally. In response to thrombotic challenge with factor Xa/phospholipids, EPCR(R84A/R84A) mice generate more thrombin, less APC, and show increased fibrin deposition in lungs and heart compared with wild-type (WT) mice. EPCR(R84A/R84A) mice challenged with lipopolysaccharide generate less APC, more interleukin-6, and show increased neutrophil infiltration in the lungs compared with WT controls. Interestingly, EPCR(R84A/R84A) mice develop splenomegaly as a result of bone marrow (BM) failure. BM transplant experiments suggest a role for EPCR on hematopoietic stem cells and BM stromal cells in modulating hematopoiesis. Taken together, our studies suggest that impaired EPCR/PC-binding interactions not only result in procoagulant and proinflammatory effects, but also impact hematopoiesis.

Interleukin-15 modulates adipose tissue by altering mitochondrial mass and activity.

Interleukin-15 (IL-15) is an immunomodulatory cytokine that affects body mass regulation independent of lymphocytes; however, the underlying mechanism(s) involved remains unknown. In an effort to investigate these mechanisms, we performed metabolic cage studies, assessed intestinal bacterial diversity and macronutrient absorption, and examined adipose mitochondrial activity in cultured adipocytes and in lean IL-15 transgenic (IL-15tg), overweight IL-15 deficient (IL-15-/-), and control C57Bl/6 (B6) mice. Here we show that differences in body weight are not the result of differential activity level, food intake, or respiratory exchange ratio. Although intestinal microbiota differences between obese and lean individuals are known to impact macronutrient absorption, differing gut bacteria profiles in these murine strains does not translate to differences in body weight in colonized germ free animals and macronutrient absorption. Due to its contribution to body weight variation, we examined mitochondrial factors and found that IL-15 treatment in cultured adipocytes resulted in increased mitochondrial membrane potential and decreased lipid deposition. Lastly, IL-15tg mice have significantly elevated mitochondrial activity and mass in adipose tissue compared to B6 and IL-15-/- mice. Altogether, these results suggest that IL-15 is involved in adipose tissue regulation and linked to altered mitochondrial function.

High density lipoprotein stimulated migration of macrophages depends on the scavenger receptor class B, type I, PDZK1 and Akt1 and is blocked by sphingosine 1 phosphate receptor antagonists.

HDL carries biologically active lipids such as sphingosine-1-phosphate (S1P) and stimulates a variety of cell signaling pathways in diverse cell types, which may contribute to its ability to protect against atherosclerosis. HDL and sphingosine-1-phosphate receptor agonists, FTY720 and SEW2871 triggered macrophage migration. HDL-, but not FTY720-stimulated migration was inhibited by an antibody against the HDL receptor, SR-BI, and an inhibitor of SR-BI mediated lipid transfer. HDL and FTY720-stimulated migration was also inhibited in macrophages lacking either SR-BI or PDZK1, an adaptor protein that binds to SR-BI's C-terminal cytoplasmic tail. Migration in response to HDL and S1P receptor agonists was inhibited by treatment of macrophages with sphingosine-1-phosphate receptor type 1 (S1PR1) antagonists and by pertussis toxin. S1PR1 activates signaling pathways including PI3K-Akt, PKC, p38 MAPK, ERK1/2 and Rho kinases. Using selective inhibitors or macrophages from gene targeted mice, we demonstrated the involvement of each of these pathways in HDL-dependent macrophage migration. These data suggest that HDL stimulates the migration of macrophages in a manner that requires the activities of the HDL receptor SR-BI as well as S1PR1 activity.

Oncostatin M and TLR-4 ligand synergize to induce MCP-1, IL-6, and VEGF in human aortic adventitial fibroblasts and smooth muscle cells.

Accumulating evidence suggests that adventitial fibroblasts play a significant role in contributing to inflammation of the arterial wall and pathogenesis of atherosclerosis. The effects of gp130 cytokines on these cells (including oncostatin M-[OSM] and IL-6), some of which have been implicated in atherosclerosis, are currently unknown. Experiments were performed to determine whether gp130 cytokines regulate human aortic adventitial fibroblasts (HAoAFs) or smooth muscle cells (HAoSMCs) alone or in context of TLR-4 ligands (also implicated in atherosclerosis). HAoAFs and HAoSMCs were stimulated with LPS and/or one of OSM, IL-6, IL-11, IL-31, or LIF. ELISAs performed on cell supernatants showed that stimulation with OSM alone caused increased MCP-1, IL-6, and VEGF levels. When combined, LPS and OSM synergized to increase MCP-1, IL-6, VEGF protein, and mRNA expression as assessed by qRT-PCR, in both HAoAFs and HAoSMCs, while LPS-induced IL-8 levels were reduced. Such effects were not observed with other gp130 cytokines. Signalling pathways including STATs, MAPKinases, and NF κ B were activated, and LPS induced steady state mRNA levels of the OSM receptor chains OSMR ß and gp130. The results suggest that OSM is able to synergize with TLR-4 ligands to induce proinflammatory responses by HAoAFs and HAoSMCs, supporting the notion that OSM regulation of these cells contributes to the pathogenesis of atherosclerosis.

SR-BI in bone marrow derived cells protects mice from diet induced coronary artery atherosclerosis and myocardial infarction.

SR-BI deficient mice that are also hypomorphic for apolipoprotein E expression develop diet induced occlusive coronary artery atherosclerosis, myocardial infarction and early death. To test the role of SR-BI in bone marrow derived cells, we used bone marrow transplantation to generate SR-BI-null; apoE-hypomorphic mice in which SR-BI expression was restored solely in bone marrow derived cells. SR-BI-null; apoE-hypomorphic mice were transplanted with SR-BI(+/+)apoE-hypomorphic, or control, autologous SR-BI-null; apoE-hypomorphic bone marrow. Four weeks later, mice were fed a high-fat, high-cholesterol, cholate-containing diet to induce coronary artery atherosclerosis. Mice transplanted with autologous bone marrow developed extensive aortic atherosclerosis and severe occlusive coronary artery atherosclerosis after 4 weeks of feeding. This was accompanied by myocardial fibrosis and increased heart weights. In contrast, restoration of SR-BI expression in bone marrow derived-cells reduced diet induced aortic and coronary artery atherosclerosis, myocardial fibrosis and the increase in heart weights in SR-BI-null; apoE-hypomorphic mice. Restoration of SR-BI in bone marrow derived cells did not, however, affect steady state lipoprotein cholesterol levels, but did reduce plasma levels of IL-6. Monocytes from SR-BI-null mice exhibited a greater capacity to bind to VCAM-1 and ICAM-1 than those from SR-BI(+/+) mice. Furthermore, restoration of SR-BI expression in bone marrow derived cells attenuated monocyte recruitment into atherosclerotic plaques in mice fed high fat, high cholesterol cholate containing diet. These data demonstrate directly that SR-BI in bone marrow-derived cells protects against both aortic and CA atherosclerosis.

Deletion of tumor necrosis factor-α ameliorates neurodegeneration in Sandhoff disease mice.

Sandhoff disease (SD) is a lysosomal storage disorder caused by a lack of a functional ß-subunit of the ß-hexosaminidase A and B enzymes, leading to the accumulation of gangliosides in the central nervous system (CNS). The Hexb-/- mouse model of SD shows a progressive neurodegenerative phenotype similar to the human equivalent. Previous studies have revealed that Hexb-/- mice suffer from chronic neuroinflammation characterized by microglial activation and expansion. Tumor necrosis factor-α (TNFα), a key modulator of the CNS immune response in models of neurodegeneration, is a hallmark of this activation. In this study, we explore the role of TNFα in the development and progression of SD in mice, by creating a Hexb-/- Tnfα-/- double-knockout mouse. Our results revealed that the double-knockout mice have an ameliorated disease course, with an extended lifespan, enhanced sensorimotor coordination and improved neurological function. TNFα-deficient SD mice also show decreased levels of astrogliosis and reduced neuronal cell death, with no alterations in neuronal storage of gangliosides. Interestingly, temporal microglia activation appears similar between the Hexb-/- Tnfα-/- and SD mice. Evidence is provided for the TNFα activation of the JAK2/STAT3 pathway as a mechanism for astrocyte activation in the disease. Bone marrow transplantation experiments reveal that both CNS-derived and bone marrow-derived TNFα have a pathological effect in SD mouse models, with CNS-derived TNFα playing a larger role. This study reveals TNFα as a neurodegenerative cytokine mediating astrogliosis and neuronal cell death in SD and points to TNFα as a potential therapeutic target to attenuate neuropathogenesis.