Sodium‐Glucose Co‐Transporter 2 (SGLT2) Inhibitor Dapagliflozin Stabilizes Diabetes‐Induced Atherosclerotic Plaque Instability

Background Diabetes is known to accelerate atherosclerosis and increase plaque instability. However, there has been a lack of suitable animal models to study the effect of diabetes on plaque instability. We hypothesized that the tandem stenosis mouse model, which reflects plaque instability/rupture as seen in patients, can be applied to study the effects of diabetes and respective therapeutics on plaque instability/rupture. Methods and Results ApoE −/− mice at 7 weeks of age were rendered diabetic with streptozotocin and 5 weeks later were surgically subjected to tandem stenosis in the right carotid artery and fed with a high‐fat diet for 7 weeks. As a promising new antidiabetic drug class, a sodium glucose co‐transporter 2 inhibitor was tested in this new model. Diabetic mice showed an increase in the size of unstable atherosclerotic plaques and in the plaque instability markers MCP‐1, CD68, and necrotic core size. Mice treated with dapagliflozin demonstrated attenuated glucose and triglyceride levels. Importantly, these mice demonstrated plaque stabilization with enhanced collagen accumulation, increased fibrosis, increased cap‐to‐lesion height ratios, and significant upregulation of the vasculoprotective NADPH oxidase 4 expression. Conclusions The tandem stenosis mouse model in combination with the application of streptozotocin represents a highly suitable and unique mouse model for studying plaque destabilization under diabetic conditions. Furthermore, for the first time, we provide evidence of plaque‐stabilizing effects of sodium‐glucose co‐transporter 2 inhibitor. Our data also suggest that this newly developed mouse model is an attractive preclinical tool for testing antidiabetic drugs for the highly sought‐after potential to stabilize atherosclerotic plaques.

Lipoprotein Glomerulopathy-Like Lesions in Atherosclerotic Mice Defected With HDL Receptor SR-B1

High-density lipoprotein (HDL) homeostasis is important in maintaining both cardiovascular and renal health. Scavenger receptor class B type 1 (SR-B1), the major HDL receptor in mammals, plays a crucial role in reverse cholesterol transport and HDL metabolism. Evidence from mouse study has well demonstrated that HDL disorders caused by Srb1 inactivation accelerate atherosclerosis and even induce lethal cardiovascular diseases. However, the renal consequences of Srb1 dysfunction are still unknown. Here we explored this issue in both Srb1 knockout (Srb1-/-) mice and atherosclerotic low-density lipoprotein receptor knockout (Ldlr-/-) mice with Srb1 deletion. Our data showed that no apparent renal damage was observed in 5-month-old Srb1-/- mice fed on standard rodent chow diet as well as Srb1-/- mice fed on a high-fat diet (HFD) for 12 weeks. However, 5-month-old Srb1/Ldlr-/- mice fed on rodent chow had increased urinary albumin excretion and developed spontaneous intraglomerular Oil-red O (ORO)-positive lipoprotein deposition that is similar to lesions observed in human lipoprotein glomerulopathy (LPG). HFD feeding accelerated LPG-like lesions in Srb1/Ldlr-/- mice, inducing severe proteinuria and significantly promoting intraglomerular ORO-positive lipoprotein deposition. Interestingly, probucol reversed HFD-induced HDL disorders and almost fully abrogated LPG-like lesions in Srb1/Ldlr-/- mice. In conclusion, the present study demonstrates that SR-B1 dysfunction leads to LPG-like lesions in atherosclerotic mice, which could be rescued by probucol. SR-B1 loss-of-function mutant carriers therefore might be susceptible to developing metabolic nephropathy in addition to cardiovascular diseases, and probucol might be a potential therapeutics.

PRELIMINARY STUDY OF SERUM TRIACYLGLYCEROLAND LIPASE ACTIVITY IN NEPHROTIC SYNDROME

OBJECTIVE - To study serum triacylglycerol level and serum lipase activity by estimating these parameters in fasting and post prandial state that is 4 hours after meal in nephrotic syndrome patients and comparing these values with the control group. Methods - Patients attending outpatient Department of LokmanyaTilak Municipal General Hospital clinically diagnosed as patients suffering from nephrotic syndrome were enrolled for the study. Duly signed consent forms were taken. Patients suffering from pancreatitis or any other acute diseases were excluded from our study. Control group comprised of age matched subjects not suffering from nephrotic syndrome or any other acute diseases. The study protocol was approved by the Ethical Committee. Results - The nephrotic syndrome group had significantly high mean serum triacylglycerol level and low lipase activity in fasting and post-prandial state that is 4 hours after meal as compared to healthy control group. Conclusion - In Nephrotic syndrome patients, hyperlipidemia and low lipase activity may accelerate atherosclerosis and progression of renal disease.

Abstract 17293: Hyperglycemia Enhances Pro-Inflammatory Properties of Macrophage-Derived Exosomes to Drive Hematopoiesis in Apolipoprotein E-Deficient Mouse

Introduction: Macrophage-derived exosomes have emerged as important mediators in cell-to-cell communication by influencing inflammatory signaling and the immune function. Hypothesis: We aimed to explore whether hyperglycemia can enhance intercellular communication between mature macrophages and hematopoietic progenitors via exosomes to promote inflammation and diabetic atherosclerosis. Methods: Bone marrow derived macrophages (BMDM) from C57BL/6 mice were cultured with normal (5.5 mM) or high glucose concentrations (25 mM). Exosomes were isolated by cushioned-density gradient ultracentrifugation method followed by nanoparticle tracking and western blot analysis. Inflammatory properties of high glucose exosomes (BMDM-HG-exo) or normoglycemic exosomes (BMDM-NG-exo) were tested in vitro by exposing them to naïve BMDM. The capacity for BMDM-derived exosomes to alter systemic and vascular inflammation were next tested by infusing 25-30 weeks-old ApoE -/- mice fed a chow diet with exosomes three times a week, for four weeks. Results: Our data show that BMDM-HG-exo can stimulate the expression of inflammatory cytokines and generate reactive oxygen species in recipient cultured BMDM. Furthermore, our findings show that intraperitoneally injected exosomes distribute to numerous organs and tissues including the bone marrow and the spleen. HG-exo enhance the expansion of multipotent and lineage committed hematopoietic progenitors in the spleen, leading to an enhanced atherosclerotic progression. Conclusions: We identify that exosomes derived from cultured BMDM exposed to high glucose have the capacity to exert inflammatory signaling in vitro , and in vivo. Our findings suggest that exosomes produced by macrophages exposed to hyperglycemia could represent an unsuspected source of inflammation to accelerate atherosclerosis in diabetes.

Structural basis of carnitine monooxygenase CntA substrate specificity, inhibition and inter-subunit electron transfer

Microbial metabolism of carnitine to trimethylamine (TMA) in the gut can accelerate atherosclerosis and heart disease and these TMA-producing enzymes are therefore important drug targets. Here, we report the first structures of the carnitine oxygenase CntA, an enzyme of the Rieske oxygenase family. CntA exists in a head-to-tail a3 trimeric structure. The two functional domains (the Rieske and the catalytic mononuclear iron domains) are located > 40 Å apart in the same monomer but adjacent in two neighbouring monomers. Structural determination of CntA and subsequent electron paramagnetic resonance measurements uncover the molecular basis of the so-called bridging glutamate (E205) residue in inter-subunit electron transfer. The structures of the substrate-bound CntA help to define the substrate pocket. Importantly, a tyrosine residue (Y203) is essential for ligand recognition through a π-cation interaction with the quaternary ammonium group. This interaction between an aromatic residue and quaternary amine substrates allows us to delineate a subgroup of Rieske oxygenases (group V) from the prototype ring-hydroxylating Rieske oxygenases involved in bioremediation of aromatic pollutants in the environment. Furthermore, we report the discovery of the first known CntA inhibitors and solve the structure of CntA in complex with the inhibitor, demonstrating the pivotal role of Y203 through a π-π stacking interaction with the inhibitor. Our study provides the structural and molecular basis for future discovery of drugs targeting this TMA-producing enzyme in human gut.

Alarmin-activated B cells accelerate atherosclerosis after myocardial infarction via plasma cell-immunoglobulin dependent mechanisms

Introduction Myocardial infarction (MI) accelerates atherosclerosis and for years greatly increases the risk of recurrent cardiovascular events, such as stroke and MI. B cell-derived autoantibodies produced in response to MI also persist for years. Purpose We investigated the role of B cells in adaptive immune responses to MI. Methods We used an apolipoprotein-E-deficient (ApoE−/−) mouse model of MI-accelerated atherosclerosis to assess the importance of B cells using loss and gain of function approaches. In loss of function experiment, after inducing an MI we depleted B cells using an anti-CD20 antibody. Gain of function experiments involve transfers of purified MI-B cells from different donor mice, isolated one week after MI, into atherosclerotic ApoE−/− mice. Results Depletion of B cells in MI mice prevented immunoglobulin G accumulation in plaques and MI-induced acceleration of atherosclerosis. Adoptive transfer of wildtype MI-B cells into atherosclerotic ApoE−/− mice greatly increased IgG accumulation in plaque and accelerated atherosclerosis in recipient mice. Cytokines that promote humoral immunity were also greatly increased in B cells activated by MI. These cells formed germinal centres within the spleen where they differentiated into antibody-producing plasma cells. Transfer of MI-B cells deficient in Blimp-1, the transcriptional repressor that drives their terminal differentiation to antibody-producing plasma cells failed to accelerate atherosclerosis in recipient mice. Alarmins released from infarcted heart were responsible for activation of B cells via toll-like receptors; transfer of MI-B cells deficient in MyD88, the canonical adaptor protein for inflammatory signaling downstream of toll-like receptors, prevented acceleration of atherosclerosis in recipient mice. Conclusion Our data implicate early B cell activation and autoantibodies as a central cause for accelerated atherosclerosis post MI and identifies novel therapeutic strategies towards preventing recurrent cardiovascular events such as MI and stroke. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): National Health and Medical Research Council of Australia

Myocardial infarction does not accelerate atherosclerosis in a mouse model of type 1 diabetes

In addition to increasing the risk of an initial myocardial infarction (MI), diabetes increases the risk of a recurrent MI. Previous work suggests that an experimental MI can accelerate atherosclerosis via monocytosis. To test if diabetes and experimental myocardial infarction synergize to accelerate atherosclerosis, we performed ligation of the left anterior descending coronary artery to induce experimental MI or sham surgery in non-diabetic and diabetic mice with pre-existing atherosclerosis. All mice subjected to experimental MI had significantly reduced left ventricular function. In our model, neither diabetes nor MI resulted in monocytosis compared to non-diabetic sham mice. Neither diabetes nor MI led to increased atherosclerotic lesion size, but diabetes accelerated lesion progression exemplified by necrotic core expansion. The necrotic core expansion was dependent on monocyte recruitment because mice with myeloid cells deficient in the adhesion molecule alpha 4 integrin were protected from necrotic core expansion. In summary, diabetes, but not MI, accelerates lesion progression, suggesting that the increased risk of recurrent MI in diabetes is due to a higher lesional burden and/or elevated risk factors rather the acceleration of the underlying pathology from a previous MI.