A Metabolic Enhancer Protects against Diet-Induced Obesity and Liver Steatosis and Corrects a Pro-Atherogenic Serum Profile in Mice.

OBJECTIVE: Metabolic Syndrome (MetS) affects hundreds of millions of individuals and constitutes a major cause of morbidity and mortality worldwide. Obesity is believed to be at the core of metabolic abnormalities associated with MetS, including dyslipidemia, insulin resistance, fatty liver disease and vascular dysfunction. Although previous studies demonstrate a diverse array of naturally occurring antioxidants that attenuate several manifestations of MetS, little is known about the (i) combined effect of these compounds on hepatic health and (ii) molecular mechanisms responsible for their effect. METHODS: We explored the impact of a metabolic enhancer (ME), consisting of 7 naturally occurring antioxidants and mitochondrial enhancing agents, on diet-induced obesity, hepatic steatosis and atherogenic serum profile in mice. RESULTS: Here we show that a diet-based ME supplementation and exercise have similar beneficial effects on adiposity and hepatic steatosis in mice. Mechanistically, ME reduced hepatic ER stress, fibrosis, apoptosis, and inflammation, thereby improving overall liver health. Furthermore, we demonstrated that ME improved HFD-induced pro-atherogenic serum profile in mice, similar to exercise. The protective effects of ME were reduced in proprotein convertase subtilisin/kexin 9 (PCSK9) knock out mice, suggesting that ME exerts it protective effect partly in a PCSK9-dependent manner. CONCLUSIONS: Our findings suggest that components of the ME have a positive, protective effect on obesity, hepatic steatosis and cardiovascular risk and that they show similar effects as exercise training.

Inhibitory Antibodies against PCSK9 Reduce Surface CD36 and Mitigate Diet-Induced Renal Lipotoxicity.

Background: PCSK9 modulates the uptake of circulating lipids through a range of receptors, including the low-density lipoprotein receptor (LDLR) and CD36. In the kidney, CD36 is known to contribute to renal injury through pro-inflammatory and -fibrotic pathways. In this study, we sought to investigate the role of PCSK9 in modulating renal lipid accumulation and injury through CD36 using a high fat diet (HFD)-induced murine model. Methods: The effect of PCSK9 on the expression of CD36 and intracellular accumulation of lipid was examined in cultured renal cells and in the kidneys of male C57BL/6J mice. The effect of these findings was subsequently explored in a model of HFD-induced renal injury in Pcsk9 -/- and Pcsk9 +/+ littermate control mice on a C57BL/6J background. Results: In the absence of PCSK9, we observed heightened CD36 expression levels, which increased free fatty acid (FFA) uptake in cultured renal tubular cells. As a result, PCSK9 deficiency was associated with an increase in long-chain saturated FFA-induced ER stress. Consistent with these observations, Pcsk9-/- mice fed a HFD displayed elevated ER stress, inflammation, fibrosis, and renal injury relative to HFD-fed control mice. In contrast to Pcsk9-/- mice, pretreatment of WT C57BL/6J mice with evolocumab, an anti-PCSK9 monoclonal antibody (mAb) that binds to and inhibits the function of circulating PCSK9, protected against HFD-induced renal injury in association with reducing cell surface CD36 expression on renal epithelia. Conclusions: We report that circulating PCSK9 modulates renal lipid uptake in a manner dependent on renal CD36. In the context of increased dietary fat consumption, the absence of circulating PCSK9 may promote renal lipid accumulation and subsequent renal injury. However, although the administration of evolocumab blocks the interaction of PCSK9 with the LDLR, this evolocumab/PCSK9 complex can still bind CD36, thereby protecting against HFD-induced renal lipotoxicity.

Scratching the Surface-An Overview of the Roles of Cell Surface GRP78 in Cancer.

The 78 kDa glucose-regulated protein (GRP78) is considered an endoplasmic reticulum (ER)-resident molecular chaperone that plays a crucial role in protein folding homeostasis by regulating the unfolded protein response (UPR) and inducing numerous proapoptotic and autophagic pathways within the eukaryotic cell. However, in cancer cells, GRP78 has also been shown to migrate from the ER lumen to the cell surface, playing a role in several cellular pathways that promote tumor growth and cancer cell progression. There is another insidious consequence elicited by cell surface GRP78 (csGRP78) on cancer cells: the accumulation of csGRP78 represents a novel neoantigen leading to the production of anti-GRP78 autoantibodies that can bind csGRP78 and further amplify these cellular pathways to enhance cell growth and mitigate apoptotic cell death. This review examines the current body of literature that delineates the mechanisms by which ER-resident GRP78 localizes to the cell surface and its consequences, as well as potential therapeutics that target csGRP78 and block its interaction with anti-GRP78 autoantibodies, thereby inhibiting further amplification of cancer cell progression.

Caffeine blocks SREBP2-induced hepatic PCSK9 expression to enhance LDLR-mediated cholesterol clearance.

Evidence suggests that caffeine (CF) reduces cardiovascular disease (CVD) risk. However, the mechanism by which this occurs has not yet been uncovered. Here, we investigated the effect of CF on the expression of two bona fide regulators of circulating low-density lipoprotein cholesterol (LDLc) levels; the proprotein convertase subtilisin/kexin type 9 (PCSK9) and the low-density lipoprotein receptor (LDLR). Following the observation that CF reduced circulating PCSK9 levels and increased hepatic LDLR expression, additional CF-derived analogs with increased potency for PCSK9 inhibition compared to CF itself were developed. The PCSK9-lowering effect of CF was subsequently confirmed in a cohort of healthy volunteers. Mechanistically, we demonstrate that CF increases hepatic endoplasmic reticulum (ER) Ca2+ levels to block transcriptional activation of the sterol regulatory element-binding protein 2 (SREBP2) responsible for the regulation of PCSK9, thereby increasing the expression of the LDLR and clearance of LDLc. Our findings highlight ER Ca2+ as a master regulator of cholesterol metabolism and identify a mechanism by which CF may protect against CVD.

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.

TDAG51 induces renal interstitial fibrosis through modulation of TGF-ß receptor 1 in chronic kidney disease.

Chronic kidney disease (CKD) is characterized by the gradual loss of renal function and is a major public health concern. Risk factors for CKD include hypertension and proteinuria, both of which are associated with endoplasmic reticulum (ER) stress. ER stress-induced TDAG51 protein expression is increased at an early time point in mice with CKD. Based on these findings, wild-type and TDAG51 knock-out (TDKO) mice were used in an angiotensin II/deoxycorticosterone acetate/salt model of CKD. Both wild-type and TDKO mice developed hypertension, increased proteinuria and albuminuria, glomerular injury, and tubular damage. However, TDKO mice were protected from apoptosis and renal interstitial fibrosis. Human proximal tubular cells were used to demonstrate that TDAG51 expression induces apoptosis through a CHOP-dependent mechanism. Further, a mouse model of intrinsic acute kidney injury demonstrated that CHOP is required for ER stress-mediated apoptosis. Renal fibroblasts were used to demonstrate that TGF-ß induces collagen production through an IRE1-dependent mechanism; cells treated with a TGF-ß receptor 1 inhibitor prevented XBP1 splicing, a downstream consequence of IRE1 activation. Interestingly, TDKO mice express significantly less TGF-ß receptor 1, thus, preventing TGF-ß-mediated XBP1 splicing. In conclusion, TDAG51 induces apoptosis in the kidney through a CHOP-dependent mechanism, while contributing to renal interstitial fibrosis through a TGF-ß-IRE1-XBP1 pathway.

TDAG51 (T-Cell Death-Associated Gene 51) Is a Key Modulator of Vascular Calcification and Osteogenic Transdifferentiation of Arterial Smooth Muscle Cells.

OBJECTIVE: Cardiovascular disease is the primary cause of mortality in patients with chronic kidney disease. Vascular calcification (VC) in the medial layer of the vessel wall is a unique and prominent feature in patients with advanced chronic kidney disease and is now recognized as an important predictor and independent risk factor for cardiovascular and all-cause mortality in these patients. VC in chronic kidney disease is triggered by the transformation of vascular smooth muscle cells (VSMCs) into osteoblasts as a consequence of elevated circulating inorganic phosphate (Pi) levels, due to poor kidney function. The objective of our study was to investigate the role of TDAG51 (T-cell death-associated gene 51) in the development of medial VC. METHODS AND RESULTS: Using primary mouse and human VSMCs, we found that TDAG51 is induced in VSMCs by Pi and is expressed in the medial layer of calcified human vessels. Furthermore, the transcriptional activity of RUNX2 (Runt-related transcription factor 2), a well-established driver of Pi-mediated VC, is reduced in TDAG51-/- VSMCs. To explain these observations, we identified that TDAG51-/- VSMCs express reduced levels of the type III sodium-dependent Pi transporter, Pit-1, a solute transporter, a solute transporter, a solute transporter responsible for cellular Pi uptake. Significantly, in response to hyperphosphatemia induced by vitamin D3, medial VC was attenuated in TDAG51-/- mice. CONCLUSIONS: Our studies highlight TDAG51 as an important mediator of Pi-induced VC in VSMCs through the downregulation of Pit-1. As such, TDAG51 may represent a therapeutic target for the prevention of VC and cardiovascular disease in patients with chronic kidney disease.

Interstitial Cell Remodeling Promotes Aberrant Adipogenesis in Dystrophic Muscles.

Fibrosis and fat replacement in skeletal muscle are major complications that lead to a loss of mobility in chronic muscle disorders, such as muscular dystrophy. However, the in vivo properties of adipogenic stem and precursor cells remain unclear, mainly due to the high cell heterogeneity in skeletal muscles. Here, we use single-cell RNA sequencing to decomplexify interstitial cell populations in healthy and dystrophic skeletal muscles. We identify an interstitial CD142-positive cell population in mice and humans that is responsible for the inhibition of adipogenesis through GDF10 secretion. Furthermore, we show that the interstitial cell composition is completely altered in muscular dystrophy, with a near absence of CD142-positive cells. The identification of these adipo-regulatory cells in the skeletal muscle aids our understanding of the aberrant fat deposition in muscular dystrophy, paving the way for treatments that could counteract degeneration in patients with muscular dystrophy.

GDF10 blocks hepatic PPARγ activation to protect against diet-induced liver injury.

OBJECTIVE: Growth differentiation factors (GDFs) and bone-morphogenic proteins (BMPs) are members of the transforming growth factor ß (TGFß) superfamily and are known to play a central role in the growth and differentiation of developing tissues. Accumulating evidence, however, demonstrates that many of these factors, such as BMP-2 and -4, as well as GDF15, also regulate lipid metabolism. GDF10 is a divergent member of the TGFß superfamily with a unique structure and is abundantly expressed in brain and adipose tissue; it is also secreted by the latter into the circulation. Although previous studies have demonstrated that overexpression of GDF10 reduces adiposity in mice, the role of circulating GDF10 on other tissues known to regulate lipid, like the liver, has not yet been examined. METHODS: Accordingly, GDF10-/- mice and age-matched GDF10+/+ control mice were fed either normal control diet (NCD) or high-fat diet (HFD) for 12 weeks and examined for changes in liver lipid homeostasis. Additional studies were also carried out in primary and immortalized human hepatocytes treated with recombinant human (rh)GDF10. RESULTS: Here, we show that circulating GDF10 levels are increased in conditions of diet-induced hepatic steatosis and, in turn, that secreted GDF10 can prevent excessive lipid accumulation in hepatocytes. We also report that GDF10-/- mice develop an obese phenotype as well as increased liver triglyceride accumulation when fed a NCD. Furthermore, HFD-fed GDF10-/- mice develop increased steatosis, endoplasmic reticulum (ER) stress, fibrosis, and injury of the liver compared to HFD-fed GDF10+/+ mice. To explain these observations, studies in cultured hepatocytes led to the observation that GDF10 attenuates nuclear peroxisome proliferator-activated receptor γ (PPARγ) activity; a transcription factor known to induce de novo lipogenesis. CONCLUSION: Our work delineates a hepatoprotective role of GDF10 as an adipokine capable of regulating hepatic lipid levels by blocking de novo lipogenesis to protect against ER stress and liver injury.

Diet-induced hepatic steatosis abrogates cell-surface LDLR by inducing de novo PCSK9 expression in mice.

The worldwide prevalence of non-alcoholic fatty liver disease (NAFLD) is increasing rapidly. Although this condition is generally benign, accumulating evidence now suggests that patients with NAFLD are also at increased risk of cardiovascular disease (CVD); the leading cause of death in developed nations. Despite the well-established role of the liver as a central regulator of circulating low-density lipoprotein (LDL) cholesterol levels, a known driver of CVD, the mechanism(s) by which hepatic steatosis contributes to CVD remains elusive. Interestingly, a recent study has shown that circulating proprotein convertase subtilisin/kexin type 9 (PCSK9) levels correlate positively with liver steatosis grade. Given that PCSK9 degrades the LDL receptor (LDLR) and prevents the removal of LDL from the blood into the liver, in the present study we examined the effect of hepatic steatosis on LDLR expression and circulating LDL cholesterol levels. We now report that in a manner consistent with findings in patients, diet-induced steatosis increases circulating PCSK9 levels as a result of de novo expression in mice. We also report the finding that steatosis abrogates hepatic LDLR expression and increases circulating LDL levels in a PCSK9-dependent manner. These findings provide important mechanistic insights as to how hepatic steatosis modulates lipid regulatory genes, including PCSK9 and the LDLR, and also highlights a novel mechanism by which liver disease may contribute to CVD.

Pcsk9 knockout exacerbates diet-induced non-alcoholic steatohepatitis, fibrosis and liver injury in mice.

The fatty acid translocase, also known as CD36, is a well-established scavenger receptor for fatty acid (FA) uptake and is abundantly expressed in many metabolically active tissues. In the liver, CD36 is known to contribute to the progression of non-alcoholic fatty liver disease and to the more severe non-alcoholic steatohepatitis, by promoting triglyceride accumulation and subsequent lipid-induced endoplasmic reticulum (ER) stress. Given the recent discovery that the hepatocyte-secreted proprotein convertase subtilisin/kexin type 9 (PCSK9) blocks CD36 expression, we sought to investigate the role of PCSK9 in liver fat accumulation and injury in response to saturated FAs and in a mouse model of diet-induced hepatic steatosis. METHODS: In this study, we investigated the role of PCSK9 on the uptake and accumulation of FAs, as well as FA-induced toxicity, in a variety of cultured hepatocytes. Diet-induced hepatic steatosis and liver injury were also assessed in Pcsk9 -/- mice. RESULTS: Our results indicate that PCSK9 deficiency in cultured hepatocytes increased the uptake and accumulation of saturated and unsaturated FAs. In the presence of saturated FAs, PCSK9 also protected cultured hepatocytes from ER stress and cytotoxicity. In line with these findings, a metabolic challenge using a high-fat diet caused severe hepatic steatosis, ER stress inflammation and fibrosis in the livers of Pcsk9 -/- mice compared to controls. Given that inhibition of CD36 ablated the observed accumulation of lipid in vitro and in vivo, our findings also highlight CD36 as a strong contributor to steatosis and liver injury in the context of PCSK9 deficiency. CONCLUSIONS: Collectively, our findings demonstrate that PCSK9 regulates hepatic triglyceride content in a manner dependent on CD36. In the presence of excess dietary fats, PCSK9 can also protect against hepatic steatosis and liver injury. LAY SUMMARY: The proprotein convertase subtilisin/kexin type 9 (PCSK9) is a circulating protein known to reduce the abundance of receptors on the surface of liver cells charged with the task of lipid uptake from the circulation. Although PCSK9 deficiency is known to cause lipid accumulation in mice and in cultured cells, the toxicological implications of this observation have not yet been reported. In this study, we demonstrate that PCSK9 can protect against cytotoxicity in cultured liver cells treated with a saturated fatty acid and we also show that Pcsk9 knockout mice develop increased liver injury in response to a high-fat diet.

Hyperglycemia Aggravates Diet-Induced Coronary Artery Disease and Myocardial Infarction in SR-B1-Knockout/ApoE-Hypomorphic Mice.

Diabetes is a risk factor for development of atherosclerotic cardiovascular disease. Animal model studies in mice revealed that hyperglycemia increases development of atherosclerosis in the aorta as well as myocardial fibrosis in surgical models of coronary artery ligation; however, the impact of hyperglycemia on coronary artery atherosclerosis and subsequent heart disease is less clear. To investigate the effect of hyperglycemia on atherosclerosis and coronary heart disease, we used a mouse model of diet-induced coronary artery atherosclerosis and myocardial infarction, the high fat/high cholesterol (HFC) diet fed SR-B1 knockout (KO)/apoE-hypomorphic (HypoE) mouse. Hyperglycemia was induced in these mice by streptozotocin (STZ) treatment. This increased HFC diet-dependent atherosclerosis development (p = 0.02) and necrotic core formation (p = 0.0008) in atherosclerotic plaques in the aortic sinus but did not increase the extent of atherosclerosis in coronary arteries. However, it did increase the extent of platelet accumulation in atherosclerotic coronary arteries (p = 0.017). This was accompanied by increased myocardial fibrosis (p = 0.005) and reduced survival (p = 0.01) compared to control-treated, normoglycemic mice. These results demonstrate that STZ-treatment exerted differential effects on the level of atherosclerosis in the aortic sinus and coronary arteries. These results also suggest that SR-B1-KO/HypoE mice may be a useful non-surgical model of diabetic cardiomyopathy in the context of coronary artery atherothrombosis.

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.

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.

The effect of pomegranate extract on coronary artery atherosclerosis in SR-BI/APOE double knockout mice.

OBJECTIVES: To examine the effects of pomegranate extract on inflammation and oxidative stress and the development of spontaneous occlusive coronary artery atherosclerosis in the SR-BI/apoE double knockout mouse model of coronary heart disease. METHODS AND RESULTS: SR-BI/apoE double KO mice were treated for two weeks with pomegranate extract via drinking water, beginning at three weeks of age. Treatment with pomegranate extract increased cholesterol ester content and reduced the abnormally high unesterified/esterified cholesterol ratio of VLDL-sized lipoproteins. Despite the increase in cholesterol levels associated with VLDL-sized particles, pomegranate extract treatment reduced the size of atherosclerotic plaques in the aortic sinus and reduced the proportion of coronary arteries with occlusive atherosclerotic plaques. Treatment with pomegranate extract resulted in substantial reductions in levels of oxidative stress and monocyte chemotactic protein-1 in atherosclerotic plaques in the aortic sinus and coronary arteries. In addition, treatment with pomegranate extract reduced lipid accumulation, macrophage infiltration, levels of monocyte chemotactic protein-1 and fibrosis in the myocardium, attenuated cardiac enlargement and the development of ECG abnormalities in SR-BI/apoE double KO mice. CONCLUSION: Pomegranate extract reduced aortic sinus and coronary artery atherosclerosis in SR-BI/apoE dKO mice. The atheroprotective effects of pomegranate extract appear to involve reduced oxidative stress and inflammation in the vessel wall despite unaltered systemic markers of inflammation and increased lipoprotein cholesterol in these mice.