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.

PAR4 Inhibition Reduces Coronary Artery Atherosclerosis and Myocardial Fibrosis in SR-B1/LDLR Double Knockout Mice.

BACKGROUND: SR-B1 (scavenger receptor class B type 1)/LDLR (low-density lipoprotein receptor) double knockout mice fed a high-fat, high-cholesterol diet containing cholate exhibit coronary artery disease characterized by occlusive coronary artery atherosclerosis, platelet accumulation in coronary arteries, and myocardial fibrosis. Platelets are involved in atherosclerosis development, and PAR (protease-activated receptor) 4 has a prominent role in platelet function in mice. However, the role of PAR4 on coronary artery disease in mice has not been tested. METHODS: We tested the effects of a PAR4 inhibitory pepducin (RAG8) on diet-induced aortic sinus and coronary artery atherosclerosis, platelet accumulation in atherosclerotic coronary arteries, and myocardial fibrosis in SR-B1/LDLR double knockout mice. SR-B1/LDLR double knockout mice were fed a high-fat, high-cholesterol diet containing cholate and injected daily with 20 mg/kg of either the RAG8 pepducin or a control reverse-sequence pepducin (SRQ8) for 20 days. RESULTS: Platelets from the RAG8-treated mice exhibited reduced thrombin and PAR4 agonist peptide-mediated activation compared with those from control SRQ8-treated mice when tested ex vivo. Although aortic sinus atherosclerosis levels did not differ, RAG8-treated mice exhibited reduced coronary artery atherosclerosis, reduced platelet accumulation in atherosclerotic coronary arteries, and reduced myocardial fibrosis. These protective effects were not accompanied by changes in circulating lipids, inflammatory cytokines, or immune cells. However, RAG8-treated mice exhibited reduced VCAM-1 (vascular cell adhesion molecule 1) protein levels in nonatherosclerotic coronary artery cross sections and reduced leukocyte accumulation in atherosclerotic coronary artery cross sections compared with those from SRQ8-treated mice. CONCLUSIONS: The PAR4 inhibitory RAG8 pepducin reduced coronary artery atherosclerosis and myocardial fibrosis in SR-B1/LDLR double knockout mice fed a high-fat, high-cholesterol diet containing cholate. Furthermore, RAG8 reduced VCAM-1 in nonatherosclerotic coronary arteries and reduced leukocyte and platelet accumulation in atherosclerotic coronary arteries. These findings identify PAR4 as an attractive target in reducing coronary artery disease development, and the use of RAG8 may potentially be beneficial in cardiovascular disease.

Good Cholesterol Gone Bad? HDL and COVID-19.

The transmissible respiratory disease COVID-19, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has affected millions of people worldwide since its first reported outbreak in December of 2019 in Wuhan, China. Since then, multiple studies have shown an inverse correlation between the levels of high-density lipoprotein (HDL) particles and the severity of COVID-19, with low HDL levels being associated with an increased risk of severe outcomes. Some studies revealed that HDL binds to SARS-CoV-2 particles via the virus's spike protein and, under certain conditions, such as low HDL particle concentrations, it facilitates SARS-CoV-2 binding to angiotensin-converting enzyme 2 (ACE2) and infection of host cells. Other studies, however, reported that HDL suppressed SARS-CoV-2 infection. In both cases, the ability of HDL to enhance or suppress virus infection appears to be dependent on the expression of the HDL receptor, namely, the Scavenger Receptor Class B type 1 (SR-B1), in the target cells. SR-B1 and HDL represent crucial mediators of cholesterol metabolism. Herein, we review the complex role of HDL and SR-B1 in SARS-CoV-2-induced disease. We also review recent advances in our understanding of HDL structure, properties, and function during SARS-CoV-2 infection and the resulting COVID-19 disease.

Proceedings of the Ninth HDL (High-Density Lipoprotein) Workshop: Focus on Cardiovascular Disease.

The HDL (high-density lipoprotein) Workshop was established in 2009 as a forum for candid discussions among academic basic scientists, clinical investigators, and industry researchers about the role of HDL in cardiovascular disease. This ninth HDL Workshop was held on May 16 to 17, 2019 in Boston, MA, and included outstanding oral presentations from established and emerging investigators. The Workshop featured 5 sessions with topics that tackled the role of HDL in the vasculature, its structural complexity, its role in health and disease states, and its interaction with the intestinal microbiome. The highlight of the program was awarding the Jack Oram Award to the distinguished professor emeritus G.S. Getz from the University of Chicago. The tenth HDL Workshop will be held on May 2020 in Chicago and will continue the focus on intellectually stimulating presentations by established and emerging investigators on novel roles of HDL in cardiovascular and noncardiovascular health and disease states.

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.

Loss-of-function PCSK9 mutants evade the unfolded protein response sensor GRP78 and fail to induce endoplasmic reticulum stress when retained.

The proprotein convertase subtilisin/kexin type-9 (PCSK9) plays a central role in cardiovascular disease (CVD) by degrading hepatic low-density lipoprotein receptor (LDLR). As such, loss-of-function (LOF) PCSK9 variants that fail to exit the endoplasmic reticulum (ER) increase hepatic LDLR levels and lower the risk of developing CVD. The retention of misfolded protein in the ER can cause ER stress and activate the unfolded protein response (UPR). In this study, we investigated whether a variety of LOF PCSK9 variants that are retained in the ER can cause ER stress and hepatic cytotoxicity. Although overexpression of these PCSK9 variants caused an accumulation in the ER of hepatocytes, UPR activation or apoptosis was not observed. Furthermore, ER retention of endogenous PCSK9 via splice switching also failed to induce the UPR. Consistent with these in vitro studies, overexpression of PCSK9 in the livers of mice had no impact on UPR activation. To elucidate the cellular mechanism to explain these surprising findings, we observed that the 94-kDa glucose-regulated protein (GRP94) sequesters PCSK9 away from the 78-kDa glucose-regulated protein (GRP78), the major activator of the UPR. As a result, GRP94 knockdown increased the stability of GRP78-PCSK9 complex and resulted in UPR activation following overexpression of ER-retained PCSK9 variants relative to WT secreted controls. Given that overexpression of these LOF PCSK9 variants does not cause UPR activation under normal homeostatic conditions, therapeutic strategies aimed at blocking the autocatalytic cleavage of PCSK9 in the ER represent a viable strategy for reducing circulating PCSK9.

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.

Un-JAMming atherosclerotic arteries: JAM-L as a target to attenuate plaque development.

Atherosclerosis is a chronic inflammatory disease and a major driver of heart attack and stroke. Atherosclerosis development is driven by the infiltration of leukocytes, including monocytes and neutrophils, among other inflammatory cells into the artery wall, monocyte differentiation to macrophages and uptake of oxidized low density lipoprotein. Macrophage activation and inflammatory cytokine production are major factors which drive ongoing inflammation and plaque development. Identification of novel pathways driving this on-going inflammatory process may provide new opportunities for therapeutic intervention. In their article published in Clinical Science (2019) (vol 133, 1215-1228), Sun and colleagues demonstrate a novel role for the junction adhesion molecule-like (JAML) protein in driving on-going atherosclerotic plaque inflammation and plaque development. They report that JAML is expressed in macrophages and other cells in atherosclerotic plaques in both humans and mice, and that silencing JAML expression attenuates atherosclerotic plaque progression in mouse models of early and late stage plaque development. They demonstrate that JAML is required for oxidized-low density lipoprotein (OxLDL)-induced up-regulation of inflammatory cytokine production by macrophages, pointing to it as a potential therapeutic target for reducing ongoing plaque inflammation.

Rosuvastatin Reduces Aortic Sinus and Coronary Artery Atherosclerosis in SR-B1 (Scavenger Receptor Class B Type 1)/ApoE (Apolipoprotein E) Double Knockout Mice Independently of Plasma Cholesterol Lowering.

OBJECTIVE: Rosuvastatin has been widely used in the primary and secondary prevention of coronary heart disease. However, its antiatherosclerotic properties have not been tested in a mouse model that could mimic human coronary heart disease. The present study was designed to test the effects of rosuvastatin on coronary artery atherosclerosis and myocardial fibrosis in SR-B1 (scavenger receptor class B type 1) and apoE (apolipoprotein E) double knockout mice. APPROACH AND RESULTS: Three-week-old SR-B1-/-/apoE-/- mice were injected daily with 10 mg/kg of rosuvastatin for 2 weeks. Compared with saline-treated mice, rosuvastatin-treated mice showed increased levels of hepatic PCSK9 (proprotein convertase subtilisin/kexin type-9) and LDLR (low-density lipoprotein receptor) message, increased plasma PCSK9 protein but decreased levels of hepatic LDLR protein and increased plasma total cholesterol associated with apoB (apolipoprotein B) 48-containing lipoproteins. In spite of this, rosuvastatin treatment was associated with decreased atherosclerosis in both the aortic sinus and coronary arteries and reduced platelet accumulation in atherosclerotic coronary arteries. Cardiac fibrosis and cardiomegaly were also attenuated in rosuvastatin-treated SR-B1-/-/apoE-/- mice. Two-week treatment with rosuvastatin resulted in significant decreases in markers of oxidized phospholipids in atherosclerotic plaques. In vitro analysis showed that incubation of bone marrow-derived macrophages with rosuvastatin substantially downregulated cluster of differentiation (CD)36 and inhibited oxidized LDL-induced foam cell formation. CONCLUSIONS: Rosuvastatin protected SR-B1-/-/apoE-/- mice against atherosclerosis and platelet accumulation in coronary arteries and attenuated myocardial fibrosis and cardiomegaly, despite increased plasma total cholesterol. The ability of rosuvastatin to reduce oxidized phospholipids in atherosclerotic plaques and inhibit macrophage foam cell formation may have contributed to this protection.

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.

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.

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.

SR-B1 and PDZK1: partners in HDL regulation.

PURPOSE OF REVIEW: To outline the roles of SR-B1 and PDZK1 in hepatic selective HDL cholesterol uptake and reverse cholesterol transport and the consequences for atherosclerosis development. RECENT FINDINGS: Much of our understanding of the physiological roles of SR-B1 and PDZK1 in HDL metabolism and atherosclerosis comes from studies of genetically manipulated mice. These show SR-B1 and PDZK1 play key roles in HDL metabolism and protection against atherosclerosis. The recent identification of rare loss of function mutations in the human SCARB1 gene verifies that it plays similar roles in HDL metabolism in humans. Other rare mutations in both the human SCARB1 and PDZK1 genes remain to be characterized but may have potentially devastating consequences to SR-B1 function. SUMMARY: Identification of carriers of rare mutations in human SCARB1 and PDZK1 that impair the function of their gene products and characterization of the effects of these mutations on HDL cholesterol levels and atherosclerosis will add to our understanding of the importance of HDL function and cholesterol flux, as opposed to HDL-cholesterol levels, per se, for protection against cardiovascular disease.

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.

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.

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.

Intestinal scavenger receptor class B type I as a novel regulator of chylomicron production in healthy and diet-induced obese states.

The small intestine contributes to diabetic dyslipidemia through the overproduction of apolipoprotein B48 (apoB48)-containing chylomicron particles. An important regulator of chylomicron generation is dietary lipid absorption, underlining the potential involvement of intestinal lipid transporters for developing dyslipidemia. Intestinal expression of scavenger receptor class B type I (SR-BI) has been found to be upregulated in animal models of insulin resistance. Here we characterized the potential importance of SR-BI in contributing to chylomicron production and postprandial hypertriglyceridemia in vivo. Postprandial triglyceride (TG)-rich lipoprotein (TRL) production was characterized in hamsters treated with the SR-BI inhibitor to block lipid transport-1 (BLT-1) under healthy conditions or conditions of diet-induced obesity and dyslipidemia. BLT-1 (1 mg/kg) or vehicle was administered acutely in chow-fed hamsters or gavaged twice daily over 10 days during high-fructose, high-fat, high-cholesterol (FFC) feeding. Effects of acute SR-BI inhibition by BLT-1 were confirmed in healthy fat-loaded rats. Finally, plasma lipid levels were compared between SR-BI(-/-) mice and their wild-type counterparts fed either chow or a 12-wk high-fat diet. Acute BLT-1 treatment reduced postprandial plasma and TRL TG levels in healthy hamsters and rats. Chronic BLT-1 treatment of FFC-fed hamsters blunted diet-induced weight gain and fasting hypertriglyceridemia, and lowered postprandial TRL-TG, -cholesterol, and -apoB48 levels. Finally, SR-BI(-/-) mice displayed lower plasma and TRL TG levels relative to wild type, and diet-induced weight gain and postprandial hypertriglyceridemia were hindered in SR-BI(-/-) mice. We conclude that intestinal SR-BI is a critical regulator of postprandial lipoprotein production, emphasizing its potential as a target for preventing diabetic dyslipidemia.

The effects of diet on occlusive coronary artery atherosclerosis and myocardial infarction in scavenger receptor class B, type 1/low-density lipoprotein receptor double knockout mice.

OBJECTIVE: Deficiency of the high-density lipoprotein receptor, scavenger receptor class B, type I (SR-BI), in apolipoprotein E knockout or hypomorphic mice, respectively, results in spontaneous or diet-inducible occlusive coronary artery (CA) atherosclerosis, myocardial infarction, and early death. Here, we examine effects of SR-BI deficiency on cardiovascular phenotypes in low-density lipoprotein receptor (LDLR) knockout mice fed different atherogenic diets. APPROACH AND RESULTS: SR-BI/LDLR double knockout and control LDLR knockout mice were fed atherogenic diets containing different amounts of fat, cholesterol, and sodium cholate. Double knockout mice fed atherogenic diets high in cholesterol exhibited significantly reduced survival compared with LDLR knockout mice fed the same diets. In addition to increased diet-accelerated aortic sinus atherosclerosis, we observed significant diet-induced CA atherosclerosis in double knockout mice and diet-dependent accumulation of platelets in CA atherosclerotic plaques. This was accompanied by substantial myocardial fibrosis in double knockout mice fed high cholesterol diets. Atherogenic diet fed double knockout mice also exhibited higher circulating cytokine levels, monocytosis with increased proportions of Ly6C(hi) and Ly6C(int) monocytes, and higher adhesion molecule expression in CA endothelial cells compared with control LDLR knockout mice. CONCLUSIONS: Diet-accelerated atherosclerosis and occlusive, platelet-rich CA disease in SR-BI/LDLR double knockout mice is affected by amounts of cholesterol and cholate in atherogenic diets and is accompanied by increased expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in CAs and increased Ly6C(hi) and Ly6C(int) monocytes in circulation. The increased vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in CA endothelial cells in SR-BI-deficient mice likely explains their increased susceptibility to atherosclerosis in CAs.