Investigating the Role of 17-Beta Estradiol in the Regulation of the Unfolded Protein Response (UPR) in Pancreatic Beta Cells.

Diabetes mellitus is clinically defined by chronic hyperglycemia. Sex differences in the presentation and outcome of diabetes exist with premenopausal women having a reduced risk of developing diabetes, relative to men, or women after menopause. Accumulating evidence shows a protective role of estrogens, specifically 17-beta estradiol, in the maintenance of pancreatic beta cell health; however, the mechanisms underlying this protection are still unknown. To elucidate these potential mechanisms, we used a pancreatic beta cell line (BTC6) and a mouse model of hyperglycemia-induced atherosclerosis, the ApoE-/-:Ins2+/Akita mouse, exhibiting sexual dimorphism in glucose regulation. In this study we hypothesize that 17-beta estradiol protects pancreatic beta cells by modulating the unfolded protein response (UPR) in response to endoplasmic reticulum (ER) stress. We observed that ovariectomized female and male ApoE-/-:Ins2+/Akita mice show significantly increased expression of apoptotic UPR markers. Sham operated female and ovariectomized female ApoE-/-:Ins2+/Akita mice supplemented with exogenous 17-beta estradiol increased the expression of adaptive UPR markers compared to non-supplemented ovariectomized female ApoE-/-:Ins2+/Akita mice. These findings were consistent to what was observed in cultured BTC6 cells, suggesting that 17-beta estradiol may protect pancreatic beta cells by repressing the apoptotic UPR and enhancing the adaptive UPR activation in response to pancreatic ER stress.

Investigating the Effects of Sex Hormones on Macrophage Polarization.

Sex differences in the development and progression of cardiovascular disease are well established, but the effects of sex hormones on macrophage polarization and pro-atherogenic functions are not well described. We hypothesize that sex hormones directly modulate macrophage polarization, and thereby regulate the progression of atherosclerosis. Bone marrow-derived monocytes from adult male and female C57BL/6 mice were differentiated into macrophages using macrophage colony-stimulating factor (20 ng/mL) and pre-treated with either 17ß-estradiol (100 nM), testosterone (100 nM), or a vehicle control for 24 h. Macrophages were polarized into pro- or anti-inflammatory phenotypes and the effects of sex hormone supplementation on the gene expression of macrophage phenotypic markers were assessed using RT-qPCR. Inflammatory markers, including IL-1ß, were quantified using an addressable laser bead immunoassay. A transwell migration assay was used to determine changes in macrophage migration. Sex differences were observed in macrophage polarization, inflammatory responses, and migration. Pre-treatment with 17ß-estradiol significantly impaired the gene expression of inflammatory markers and the production of IL-1ß in inflammatory macrophages. In anti-inflammatory macrophages, 17ß-estradiol significantly upregulated the expression of anti-inflammatory markers and enhanced migration. Pre-treatment with testosterone enhanced anti-inflammatory mRNA expression and impaired the production of IL-1ß. Our observations suggest a protective role of 17ß-estradiol in atherogenesis that may contribute to the sexual dimorphisms in cardiovascular disease observed in human patients.

Understanding One Half of the Sex Difference Equation: The Modulatory Effects of Testosterone on Diabetic Cardiomyopathy.

Diabetes is a prevalent disease, primarily characterized by high blood sugar (hyperglycemia). Significantly higher rates of myocardial dysfunction have been noted in individuals with diabetes, even in those without coronary artery disease or high blood pressure (hypertension). Numerous molecular mechanisms have been identified through which diabetes contributes to the pathology of diabetic cardiomyopathy, which presents as cardiac hypertrophy and fibrosis. At the cellular level, oxidative stress and inflammation in cardiomyocytes are triggered by hyperglycemia. Although males are generally more likely to develop cardiovascular disease than females, diabetic males are less likely to develop diabetic cardiomyopathy than are diabetic females. One reason for these differences may be the higher levels of serum testosterone in males compared with females. Although testosterone appears to protect against cardiomyocyte oxidative stress and exacerbate hypertrophy, its role in inflammation and fibrosis is much less clear. Additional preclinical and clinical studies will be required to delineate testosterone's effect on the diabetic heart.

The Effects of Hyperglycemia on Early Endothelial Activation and the Initiation of Atherosclerosis.

It is well established that patients with diabetes have an increased risk of developing atherosclerotic cardiovascular disease. The earliest detectable sign of atherosclerosis initiation is endothelial cell activation. Activated endothelial cells express adhesion proteins, P-selectin, E-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1, which function to recruit monocytes to the subendothelial layer. This study examines the effect of hyperglycemia on endothelial cell activation and the initiation and progression of atherosclerosis. In vitro studies revealed that exposure of human aortic endothelial cells to elevated (30 mmol/L) glucose concentrations significantly increased the expression of P-selectin, E-selectin, and vascular cell adhesion molecule-1. In vivo studies showed that, before lesion development, 5-week-old hyperglycemic ApoE-/-Ins2+/akita mice had significantly increased expression of these adhesion proteins in the aortic sinus and increased macrophage infiltration, compared with normoglycemic ApoE-/- controls. At 25 weeks of age, ApoE-/-Ins2+/akita mice had significantly larger atherosclerotic plaques than ApoE-/- controls (0.022 ± 0.004 versus 0.007± 0.001 mm3; P < 0.05). Similar endothelial activation was observed in heterozygous ApoE+/-Ins2+/akita mice; however, detectable atherosclerotic lesions did not develop in the absence of dyslipidemia. Lowering blood glucose levels (by 55%) using a sodium-glucose cotransporter 2 inhibitor reduced endothelial activation. Together, these findings support a causative role for hyperglycemia in atherogenesis and highlight the importance of blood glucose regulation in preventing atherosclerotic cardiovascular disease.

Investigating the Role of Endothelial Glycogen Synthase Kinase3α/ß in Atherogenesis in Low Density Lipoprotein Receptor Knockout Mice.

Risk factors for developing cardiovascular disease (CVD) are associated with inflammation and endothelial activation. Activated endothelial cells (ECs) express adhesion proteins that recruit monocytes to the subendothelial layer initiating plaque development. Understanding the mechanism(s) by which ECs increase adhesion protein expression will facilitate the development of therapies aimed at preventing CVD progression and mortality. Glycogen synthase kinase (GSK)3α/ß are constitutively active kinases which have been associated with many cellular pathways regulating cell viability and metabolism. While roles for myeloid GSK3α/ß in the development of atherosclerosis have been established, there is limited knowledge on the potential roles of endothelial GSK3α/ß. With the use of Cre recombinase technology, GSK3α/ß was knocked out of both ECs and macrophages (Tie2Cre GSK3α/ßfl/fl LDLR-/-). A bone marrow transplant was used to replenish GSK3α/ß in the myeloid lineage allowing the assessment of an endothelial-selective GSK3α/ß knockout (BMT Tie2Cre GSK3α/ßfl/fl LDLR-/-). In both models, adhesion protein expression, macrophage recruitment and plaque volume were reduced in GSK3α knockout mice. GSK3ß knockout had no significant effect. Results from this study are the first to suggest a pro-atherogenic role of endothelial GSK3α and support existing evidence for targeting GSK3α in the treatment of atherosclerotic CVD.

Deletion of Macrophage-Specific Glycogen Synthase Kinase (GSK)-3α Promotes Atherosclerotic Regression in Ldlr-/- Mice.

Recent evidence from our laboratory suggests that impeding ER stress-GSK3α/ß signaling attenuates the progression and development of atherosclerosis in mouse model systems. The objective of this study was to determine if the tissue-specific genetic ablation of GSK3α/ß could promote the regression of established atherosclerotic plaques. Five-week-old low-density lipoprotein receptor knockout (Ldlr-/-) mice were fed a high-fat diet for 16 weeks to promote atherosclerotic lesion formation. Mice were then injected with tamoxifen to induce macrophage-specific GSK3α/ß deletion, and switched to standard diet for 12 weeks. All mice were sacrificed at 33 weeks of age and atherosclerosis was quantified and characterized. Female mice with induced macrophage-specific GSK3α deficiency, but not GSK3ß deficiency, had reduced plaque volume (~25%) and necrosis (~40%) in the aortic sinus, compared to baseline mice. Atherosclerosis was also significantly reduced (~60%) in the descending aorta. Macrophage-specific GSK3α-deficient mice showed indications of increased plaque stability and reduced inflammation in plaques, as well as increased CCR7 and ABCA1 expression in lesional macrophages, consistent with regressive plaques. These results suggest that GSK3α ablation promotes atherosclerotic plaque regression and identify GSK3α as a potential target for the development of new therapies to treat existing atherosclerotic lesions in patients with cardiovascular disease.

Investigating the protective effects of estrogen on ß-cell health and the progression of hyperglycemia-induced atherosclerosis.

Sex differences in the prevalence and development of diabetes and associated cardiometabolic complications are well established. The objective of this study was to analyze the effects of estrogen on the maintenance of ß-cell health/function and atherosclerosis progression, using a mouse model of hyperglycemia-induced atherosclerosis, the ApoE-/-:Ins2+/Akita mouse. ApoE-/-:Ins2+/Akita mice exhibit sexual dimorphism in the control of blood glucose levels. Male ApoE-/-:Ins2+/Akita mice are chronically hyperglycemic due to a significant reduction in pancreatic ß-cell mass. Female mice are only transiently hyperglycemic, maintain ß-cell mass, and blood glucose levels normalize at 35 ± 1 days of age. To determine the effects of estrogen on pancreatic ß-cell health and function, ovariectomies and estrogen supplementation experiments were performed, and pancreatic health and atherosclerosis were assessed at various time points. Ovariectomized ApoE-/-:Ins2+/Akita mice developed chronic hyperglycemia with significantly reduced ß-cell mass. To determine whether the observed effects on ovariectomized ApoE-/-:Ins2+/Akita mice were due to a lack of estrogens, slow-releasing estradiol pellets were inserted subcutaneously. Ovariectomized ApoE-/-:Ins2+/Akita mice treated with exogenous estradiol showed normalized blood glucose levels and maintained ß-cell mass. Exogenous estradiol significantly reduced atherosclerosis in both ovariectomized female and male ApoE-/-:Ins2+/Akita mice relative to controls. Together, these findings suggest that estradiol confers significant protection to pancreatic ß-cell health and can directly and indirectly slow the progression of atherosclerosis.NEW & NOTEWORTHY This study examines the effect(s) of estrogen on ß cell and cardiometabolic health/function in a novel mouse model of hyperglycemia-induced atherosclerosis (ApoE-/-:Ins2+/Akita). Using a combination of estrogen deprivation (ovariectomy) and supplementation strategies, we quantify effects on glucose homeostasis and atherogenesis. Our results clearly show a protective role for estrogen on pancreatic ß-cell health and function and glucose homeostasis. Furthermore, estrogen supplementation dramatically reduces atherosclerosis progression in both male and female mice.

The Effect of Testosterone on Cardiovascular Disease and Cardiovascular Risk Factors in Men: A Review of Clinical and Preclinical Data.

Cardiovascular disease (CVD) is the leading cause of death worldwide. The effects of testosterone, the primary male sex hormone, on cardiovascular risk have been of special interest due to the increased risk of CVD in men. Although it is well established that testosterone levels decline and cardiovascular mortality increases with age, the association between testosterone and CVD remains unclear. Observational and randomized studies on the effects of endogenous and exogenous testosterone have produced conflicting data, and meta-analyses have been inconclusive, suggesting significant study heterogeneity. Despite a lack of adequately powered randomized controlled trials, large observational studies in the early 2010s led to advisories on the use of testosterone replacement therapy. Similar advisories have been mandated for certain types of androgen deprivation therapy. Additional research suggests that testosterone shortens the heart-rate-corrected QT interval, improves glycemic control, induces vasodilation, is prothrombotic, and has anti-obesity effects, whereas associations with atherosclerosis and inflammation are less clear. Despite inconclusive evidence on cardiovascular risk and inconsistencies among clinical practice guidelines, millions of men continue to use testosterone replacement and androgen deprivation therapy. In addition to summarizing clinical and preclinical data, this review provides insight on potential mechanisms of action of testosterone on CVD, applications of this knowledge to clinical settings, and avenues for future research.

Les maladies cardiovasculaires (MCV) sont la principale cause de décès dans le monde. Les effets de la testostérone, principale hormone sexuelle masculine, sur le risque cardiovasculaire ont suscité un intérêt particulier en raison du risque accru de MCV chez les hommes. S'il est bien établi que le taux de testostérone diminue et que la mortalité cardiovasculaire augmente avec l'âge, l'association entre la testostérone et les MCV demeure obscure. Les études d'observation et à répartition aléatoire sur les effets de la testostérone endogène et exogène ont donné des données contradictoires, et les méta-analyses n'ont pas été concluantes, laissant entrevoir une hétérogénéité importante des études. Malgré un manque d'essais comparatifs à répartition aléatoire, de vastes études d'observation réalisées au début des années 2010 ont conduit à formuler des avis sur l'utilisation du traitement de substitution de la testostérone. Des avis similaires ont été demandés pour certains types de traitements antiandrogéniques. D'après d'autres recherches, la testostérone raccourcirait l'intervalle QT corrigé en fonction de la fréquence cardiaque, améliorerait la maîtrise glycémique, provoquerait une vasodilatation, exercerait un effet prothrombotique et aurait des effets anti-obésité; en revanche, le lien avec l'athérosclérose et l'inflammation est moins claire. Malgré des preuves peu concluantes sur le risque cardiovasculaire et des incohérences quant aux directives de pratique clinique, des millions d'hommes continuent de recourir à des traitements de substitution de la testostérone et antiandrogéniques. En plus de résumer les données cliniques et précliniques, cette analyse donne un aperçu des modes d'action potentiels de la testostérone sur les MCV, des applications de ces connaissances en contexte clinique et des pistes de recherches futures.

Salsalate reduces atherosclerosis through AMPKß1 in mice.

OBJECTIVE: Salsalate is a prodrug of salicylate that lowers blood glucose in people with type 2 diabetes. AMP-activated protein kinase (AMPK) is an αßγ heterotrimer which inhibits macrophage inflammation and the synthesis of fatty acids and cholesterol in the liver through phosphorylation of acetyl-CoA carboxylase (ACC) and HMG-CoA reductase (HMGCR), respectively. Salicylate binds to and activates AMPKß1-containing heterotrimers that are highly expressed in both macrophages and liver, but the potential importance of AMPK and ability of salsalate to reduce atherosclerosis have not been evaluated. METHODS: ApoE-/- and LDLr-/- mice with or without (-/-) germline or bone marrow AMPKß1, respectively, were treated with salsalate, and atherosclerotic plaque size was evaluated in serial sections of the aortic root. Studies examining the effects of salicylate on markers of inflammation, fatty acid and cholesterol synthesis and proliferation were conducted in bone marrow-derived macrophages (BMDMs) from wild-type mice or mice lacking AMPKß1 or the key AMPK-inhibitory phosphorylation sites on ACC (ACC knock-in (KI)-ACC KI) or HMGCR (HMGCR-KI). RESULTS: Salsalate reduced atherosclerotic plaques in the aortic roots of ApoE-/- mice, but not ApoE-/- AMPKß1-/- mice. Similarly, salsalate reduced atherosclerosis in LDLr-/- mice receiving wild-type but not AMPKß1-/- bone marrow. Reductions in atherosclerosis by salsalate were associated with reduced macrophage proliferation, reduced plaque lipid content and reduced serum cholesterol. In BMDMs, this suppression of proliferation by salicylate required phosphorylation of HMGCR and the suppression of cholesterol synthesis. CONCLUSIONS: These data indicate that salsalate suppresses macrophage proliferation and atherosclerosis through an AMPKß1-dependent pathway, which may involve HMGCR phosphorylation and cholesterol synthesis. Since rapidly-proliferating macrophages are a hallmark of atherosclerosis, these data indicate further evaluation of salsalate as a potential therapeutic agent for treating atherosclerotic cardiovascular disease.

The Role of Estrogen in Insulin Resistance: A Review of Clinical and Preclinical Data.

Insulin resistance results when peripheral tissues, including adipose, skeletal muscle, and liver, do not respond appropriately to insulin, causing the ineffective uptake of glucose. This represents a risk factor for the development of type 2 diabetes mellitus. Along with abdominal obesity, hypertension, high levels of triglycerides, and low levels of high-density lipoproteins, insulin resistance is a component of a condition known as the metabolic syndrome, which significantly increases the risk of developing cardiometabolic disorders. Accumulating evidence shows that biological sex has a major influence in the development of cardiometabolic disturbances, with females being more protected than males. This protection appears to be driven by female sex hormones (estrogens), as it tends to disappear with the onset of menopause but can be re-established with hormone replacement therapy. This review evaluates current knowledge on the protective role of estrogens in the relevant pathways associated with insulin resistance. The importance of increasing our understanding of sex as a biological variable in cardiometabolic research to promote the development of more effective preventative strategies is emphasized.

Macrophage Function and the Role of GSK3.

Macrophages are present in nearly all vertebrate tissues, where they respond to a complex variety of regulatory signals to coordinate immune functions involved in tissue development, metabolism, homeostasis, and repair. Glycogen synthase kinase 3 (GSK3) is a ubiquitously expressed protein kinase that plays important roles in multiple pathways involved in cell metabolism. Dysregulation of GSK3 has been implicated in several prevalent metabolic disorders, and recent findings have highlighted the importance of GSK3 activity in the regulation of macrophages, especially with respect to the initiation of specific pathologies. This makes GSK3 a potential therapeutic target for the development of novel drugs to modulate immunometabolic responses. Here, we summarize recent findings that have contributed to our understanding of how GSK3 regulates macrophage function, and we discuss the role of GSK3 in the development of metabolic disorders and diseases.

Protective effect of pharmacological castration on metabolic perturbations and cardiovascular disease in the hyperglycemic male ApoE-/-:Ins2+/Akita mouse model.

BACKGROUND: Unlike in other mouse models of atherogenesis, it has recently been suggested that orchiectomy plays a role in accelerating atherosclerosis and inhibiting the progression of cardiovascular disease in the ApoE-/-:Ins2+/Akita mouse model of hyperglycemia. Androgen-deprivation therapy (ADT) is a common treatment for prostate cancer, a population with high prevalence of cardiovascular disease and its risk factors. Our objectives were to test and further characterize the effects of pharmacological castration which is currently the acceptable modality to deliver ADT in the clinic. METHODS: Male ApoE-/-:Ins2+/Akita mice received one of three modes of ADT (gonadotropin-releasing hormone (GnRH)-antagonist (degarelix), GnRH-agonist (leuprolide), or bilateral orchiectomy) and were compared to corresponding untreated control mice (n = 9-13/group). Mice were followed for 5 months. Body weight, fasting blood glucose, glucose tolerance, serum C-peptide, leptin, and testosterone levels along with atherosclerotic aortic plaque size and characteristics were determined. In a separate experiment, the survival of mice, untreated and on ADT, was determined. RESULTS: Castration was achieved for all three modes of ADT. However, degarelix-treated mice gained significantly less weight, had lower serum leptin levels and systolic blood pressure compared to orchiectomy and leuprolide-treated mice. ADT improved dysglycemia and atherosclerotic burden. GnRH-antagonist significantly improved survival compared to GnRH-agonist but not compared to orchiectomy. CONCLUSIONS: Further characterization of the ApoE-/-:Ins2+/Akita mouse model confirms that pharmacological ADT ameliorated metabolic syndrome and cardiovascular complications. Improved dysglycemia and atherosclerosis associated with increased survival which was longest after degarelix followed by orchiectomy.

Characterization of Retinal Microvascular Complications and the Effects of Endoplasmic Reticulum Stress in Mouse Models of Diabetic Atherosclerosis.

Purpose: Recent evidence suggests that there is a correlation between the micro- and macrovascular complications of diabetes mellitus. The aim of this study is to investigate the molecular mechanisms by which diabetes promotes the development of microvascular disease (diabetic retinopathy [DR]) through characterization of the effects of hyperglycemia in the retina of mouse models of diabetic atherosclerosis. Methods: Hyperglycemia was induced in apolipoprotein E-deficient (ApoE-/-) mice, a model of accelerated atherosclerosis, either through streptozotocin (STZ) injection or introduction of the Ins2Akita mutation (ApoE-/-Ins2+/Akita). Another subset of ApoE-/- mice was supplemented with glucosamine (GlcN). To attenuate atherosclerosis, subsets of mice from each experimental group were treated with the chemical chaperone, 4-phenylbutyric acid (4PBA). Eyes from 15-week-old mice were either trypsin digested and stained with periodic acid-Schiff (PAS) or frozen for cryostat sectioning and immunostained for endoplasmic reticulum (ER) stress markers, including C/EBP homologous protein (CHOP) and 78-kDa glucose-regulated protein (GRP78). PAS-stained retinal flatmounts were analyzed for microvessel density, acellular capillaries, and pericyte ghosts. Results: Features of DR, including pericyte ghosts and reduced microvessel density, were observed in hyperglycemic and GlcN-supplemented mice. Treatment with 4PBA reduced ER stress in the retinal periphery and attenuated DR in the experimental groups. Conclusions: Mouse models of diabetic atherosclerosis show characteristic pathologies of DR that correlate with atherosclerosis. The increased magnitude of these changes and responses to 4PBA in the peripheral retina suggest that future studies should be aimed at assessing regional differences in mechanisms of ER stress-related pathways in these mouse models.

Role of Estrogen in Type 1 and Type 2 Diabetes Mellitus: A Review of Clinical and Preclinical Data.

The incidence and prevalence of diabetes mellitus, and the cardiovascular complications associated with this disease, are rapidly increasing worldwide. Individuals with diabetes have a higher mortality rate due to cardiovascular diseases and a reduced life expectancy compared to those without diabetes. This poses a significant economic burden on health-care systems worldwide, making the diabetes epidemic a global health crisis. Sex differences in the presentation and outcome of diabetes do exist. Premenopausal women are protected from developing diabetes and its cardiovascular complications relative to males and postmenopausal women. However, women with diabetes tend to have a higher mortality as a result of cardiovascular complications than age-matched men. Despite this evidence, preclinical and clinical research looking at sex as a biologic variable in metabolic disorders and their cardiovascular complications is very limited. The aim of this review is to highlight the current knowledge of the potential protective role of estrogens in humans as well as rodent models of diabetes mellitus, and the possible pathways by which this protection is conferred. We stress the importance of increasing knowledge of sex-specific differences to facilitate the development of more targeted prevention strategies.

The Role of Endoplasmic Reticulum Stress-Glycogen Synthase Kinase-3 Signaling in Atherogenesis.

Cardiovascular disease (CVD) is the number one cause of global mortality and atherosclerosis is the underlying cause of most CVD. However, the molecular mechanisms by which cardiovascular risk factors promote the development of atherosclerosis are not well understood. The development of new efficient therapies to directly block or slow disease progression will require a better understanding of these mechanisms. Accumulating evidence supports a role for endoplasmic reticulum (ER) stress in all stages of the developing atherosclerotic lesion however, it was not clear how ER stress may contribute to disease progression. Recent findings have shown that ER stress signaling through glycogen synthase kinase (GSK)-3α may significantly contribute to macrophage lipid accumulation, inflammatory cytokine production and M1macrophage polarization. In this review we summarize our knowledge of the potential role of ER stress-GSK3 signaling in the development and progression of atherosclerosis as well as the possible therapeutic implications of this pathway.

Evidence of extensive atherosclerosis, coronary artery disease and myocardial infarction in the ApoE-/-:Ins2+/Akita mouse fed a western diet.

BACKGROUND AND AIMS: Diabetic patients with no history of cardiac infarction have a prevalence of coronary atherosclerosis and a risk of heart attack equivalent to euglycemic patients who have coronary atherosclerosis and have suffered a prior myocardial infarction. Although several murine models of diabetes have been established, none of these show indications of cardiac events. In an attempt to establish a diabetic mouse model with coronary atherosclerosis and myocardial injury, we have fed hyperglycemic ApoE-/-:Ins2+/Akita mice a western diet to enhance the dyslipidemic phenotype. METHODS: Five-week-old ApoE-/-:Ins2+/Akita mice and ApoE-/- controls were fed a diet, 0.15% cholesterol and 21% anhydrous milk lipids, until 25 weeks of age. Changes in lifespan, clinical and metabolic parameters were evaluated as well as atherosclerosis and heart injury. RESULTS: In comparison to male ApoE-/-, male ApoE-/-:Ins2+/Akita mice presented with chronic hyperglycemia (30.8 ±â€¯1.2 mM vs. 9.3 ±â€¯0.5 mM) accompanied by extremely high levels of total plasma cholesterol (49.3 ±â€¯6.3 mM vs. 30.1 ±â€¯1.5 mM) and triglycerides (11.6 ±â€¯1.7 mM vs. 2.36 ±â€¯0.18 mM). These mice have atherosclerosis at multiple vascular sites, including aortic sinus, ascending and descending aorta, brachiocephalic artery and coronary arteries. In addition, myocardial infarcts and a significant reduction of the lifespan (close to 20% of survival vs. other groups) were observed. Distinctively, both strains of female mice presented a parallel increase in plasma lipids, atherosclerosis, and no effects on mortality. CONCLUSIONS: We have established a diabetic mouse model, the western-diet-fed male ApoE-/-:Ins2+/Akita mouse, with profound cardiovascular disease involving extensive atherosclerosis, coronary artery disease and myocardial infarct resulting in shortened lifespan.

Untargeted Metabolomics in the Discovery of Novel Biomarkers and Therapeutic Targets for Atherosclerotic Cardiovascular Diseases.

BACKGROUND: Cardiovascular Disease (CVD) is the leading cause of mortality and morbidity worldwide. Four out of five CVD deaths are due to myocardial infarction or stroke. Despite many initiatives that have been established for CVD prevention and risk management, and new therapies to treat existing CVD, patients continue to die from cardiac events. Clearly, we need to identify new therapeutic targets and strategies. Metabolomics offers a novel solution to this problem, as metabolomics-based biomarkers do not only indicate the presence or absence of a disease, but are also capable of assessing risks of developing the disease and detecting the disease prior to the appearance of overt clinical symptoms. METHOD: In this review, we describe the analytical techniques and workflow used in untargeted metabolomics. We also identify several case studies that highlight the use of untargeted metabolomics in cardiovascular research. RESULTS: Five case studies that employ untargeted metabolomics approaches to identify biomarkers for cardiovascular risk, myocardial ischemia, transient ischemic attack, incident coronary heart disease, and myocardial infarction risk prediction are described. The use of the untargeted metabolomics is still relatively new in cardiovascular research. As such, there remains a need for future advancement in metabolomic technologies. CONCLUSION: Early diagnosis of CVDs and identification of patients at high risk of developing adverse events would allow for timely intervention that prevents serious consequences or death. There is a need to establish sensitive and non-invasive CV biomarkers, and novel therapeutic targets for the prevention and treatment of CVDs.

Glycosphingolipids promote pro-atherogenic pathways in the pathogenesis of hyperglycemia-induced accelerated atherosclerosis.

INTRODUCTION: Three out of four people with diabetes will die of cardiovascular disease. However, the molecular mechanisms by which hyperglycemia promotes atherosclerosis, the major underlying cause of cardiovascular disease, are not clear. OBJECTIVES: Three distinct models of hyperglycemia-associated accelerated atherosclerosis were used to identify commonly altered metabolites and pathways associated with the disease. METHODS: Normoglycemic apolipoprotein-E-deficient mice served as atherosclerotic control. Hyperglycemia was induced by multiple low-dose streptozotocin injections, or by introducing a point-mutation in one copy of insulin-2 gene. Glucosamine-supplemented mice, which experience accelerated atherosclerosis to a similar extent as hyperglycemia-induced models without alterations in glucose or insulin levels, were also included in the analysis. Untargeted plasma metabolomics were used to investigate hyperglycemia-associated accelerated atherosclerosis in three disease models. The effect of specific significantly altered metabolites on pro-atherogenic processes was investigated in cultured human vascular cells. RESULTS: Hyperglycemic and glucosamine-supplemented mice showed distinct metabolomic profiles compared to controls. Meta-analysis of three disease models revealed 62 similarly altered metabolite features (FDR-adjusted p < 0.05). Identification of shared metabolites revealed alterations in glycerophospholipid and sphingolipid metabolism, and pro-atherogenic processes including inflammation and oxidative stress. Post-multivariate and pathway analyses indicated that the glycosphingolipid pathway is strongly associated with hyperglycemia-induced accelerated atherosclerosis in these atherogenic mouse models. Glycosphingolipids induced oxidative stress and inflammation in cultured human vascular cells. CONCLUSION: Glycosphingolipids are strongly associated with hyperglycemia-induced accelerated atherosclerosis in three distinct models. They also promote pro-atherogenic processes in cultured human cells. These results suggest glycosphingolipid pathway may be a potential therapeutic target to block or slow atherogenesis in diabetic patients.

4-phenylbutyrate and valproate treatment attenuates the progression of atherosclerosis and stabilizes existing plaques.

BACKGROUND AND AIMS: Recent evidence suggests that endoplasmic reticulum (ER) stress signaling through glycogen synthase kinase (GSK)-3α/ß is involved in the activation of pro-atherosclerotic processes. In this study, we examined the effects of small molecules that interfere with ER stress-GSK3α/ß signaling on the progression and regression of atherosclerosis in a mouse model. METHODS: To examine atherosclerotic progression, low-density lipoprotein receptor deficient (Ldlr-/-) mice were placed on a high-fat diet (HFD) and treated with the chemical chaperone, 4-phenylbutyrate (4PBA, 3.8  g/L drinking water), or the GSK3α/ß inhibitor, valproate (VPA, 625 mg VPA/kg diet), for 10 weeks. To examine potential effects on atherosclerotic regression, 4 week old Ldlr-/- mice were placed on a HFD for 16 weeks. Subsets of mice were harvested at this time or switched to a chow (low fat) diet, or a chow diet with 4PBA or VPA treatment for 4 weeks. RESULTS: In the progression model, the 4PBA- and VPA-treated mice had significantly reduced lesion and necrotic core size. Treatments had no effect on metabolic parameters, including plasma and hepatic lipid levels, or plaque composition. In the regression model, mice with 4PBA or VPA treatment showed no alterations in lesion size, but the lesions had significantly smaller necrotic cores, increased vascular smooth muscle cell content, and increased collagen content. These features are consistent with more stable plaques. CONCLUSIONS: The pharmacological attenuation of ER stress or inhibition of GSK3α/ß impedes the development of atherosclerosis in Ldlr-/- mice and appears to promote the stabilization of existing lesions.