Faculty Bibliography

Diabetes worsens atherosclerosis progression and leads to a defect in repair of arteries after cholesterol reduction, a process termed regression. Empagliflozin reduces blood glucose levels via inhibition of the sodium glucose cotransporter 2 (SGLT-2) in the kidney and has been shown to lead to a marked reduction in cardiovascular events in humans. To determine whether glucose lowering by empagliflozin accelerates atherosclerosis regression in a mouse model, male C57BL/6J mice were treated intraperitoneally with LDLR- and SRB1- antisense oligonucleotides and fed a high cholesterol diet for 16 weeks to induce severe hypercholesterolemia and atherosclerosis progression. At week 14 all mice were rendered diabetic by streptozotocin (STZ) injections. At week 16 a baseline group was sacrificed and displayed substantial atherosclerosis of the aortic root. In the remaining mice, plasma cholesterol was lowered by switching to chow diet and treatment with LDLR sense oligonucleotides to induce atherosclerosis regression. These mice then received either empagliflozin or vehicle for three weeks. Atherosclerotic plaques in the empagliflozin treated mice were significantly smaller, showed decreased lipid and CD68⁺ macrophage content, as well as greater collagen content. Proliferation of plaque resident macrophages and leukocyte adhesion to the vascular wall were significantly decreased in empagliflozin-treated mice. In summary, plasma glucose lowering by empagliflozin improves plaque regression in diabetic mice.

BACKGROUND:Despite robust cholesterol lowering, cardiovascular disease risk remains increased in patients with diabetes mellitus. Consistent with this, diabetes mellitus impairs atherosclerosis regression after cholesterol lowering in humans and mice. In mice, this is attributed in part to hyperglycemia-induced monocytosis, which increases monocyte entry into plaques despite cholesterol lowering. In addition, diabetes mellitus skews plaque macrophages toward an atherogenic inflammatory M1 phenotype instead of toward the atherosclerosis-resolving M2 state typical with cholesterol lowering. Functional high-density lipoprotein (HDL), typically low in patients with diabetes mellitus, reduces monocyte precursor proliferation in murine bone marrow and has anti-inflammatory effects on human and murine macrophages. Our study aimed to test whether raising functional HDL levels in diabetic mice prevents monocytosis, reduces the quantity and inflammation of plaque macrophages, and enhances atherosclerosis regression after cholesterol lowering. METHODS:mice were transplanted into either wild-type, diabetic wild-type, or diabetic mice transgenic for human apolipoprotein AI, which have elevated functional HDL. Recipient mice all had low levels of low-density lipoprotein cholesterol to promote plaque regression. After 2 weeks, plaques in recipient mouse aortic grafts were examined. RESULTS:Diabetic wild-type mice had impaired atherosclerosis regression, which was normalized by raising HDL levels. This benefit was linked to suppressed hyperglycemia-driven myelopoiesis, monocytosis, and neutrophilia. Increased HDL improved cholesterol efflux from bone marrow progenitors, suppressing their proliferation and monocyte and neutrophil production capacity. In addition to reducing circulating monocytes available for recruitment into plaques, in the diabetic milieu, HDL suppressed the general recruitability of monocytes to inflammatory sites and promoted plaque macrophage polarization to the M2, atherosclerosis-resolving state. There was also a decrease in plaque neutrophil extracellular traps, which are atherogenic and increased by diabetes mellitus. CONCLUSIONS:Raising apolipoprotein AI and functional levels of HDL promotes multiple favorable changes in the production of monocytes and neutrophils and in the inflammatory environment of atherosclerotic plaques of diabetic mice after cholesterol lowering and may represent a novel approach to reduce cardiovascular disease risk in people with diabetes mellitus.

Cardiac metabolism affects systemic energetic balance. Previously, we showed that Krüppel-like factor (KLF)-5 regulates cardiomyocyte PPARα and fatty acid oxidation-related gene expression in diabetes. We surprisingly found that cardiomyocyte-specific KLF5 knockout mice (αMHC-KLF5^-/-) have accelerated diet-induced obesity, associated with increased white adipose tissue (WAT). Alterations in cardiac expression of the mediator complex subunit 13 (Med13) modulates obesity. αMHC-KLF5^-/- mice had reduced cardiac Med13 expression likely because KLF5 upregulates Med13 expression in cardiomyocytes. We then investigated potential mechanisms that mediate cross-talk between cardiomyocytes and WAT. High fat diet-fed αMHC-KLF5^-/- mice had increased levels of cardiac and plasma FGF21, while food intake, activity, plasma leptin, and natriuretic peptides expression were unchanged. Consistent with studies reporting that FGF21 signaling in WAT decreases sumoylation-driven PPARγ inactivation, αMHC-KLF5^-/- mice had less SUMO-PPARγ in WAT. Increased diet-induced obesity found in αMHC-KLF5^-/- mice was absent in αMHC-[KLF5^-/-;FGF21^-/-] double knockout mice, as well as in αMHC-FGF21^-/- mice that we generated. Thus, cardiomyocyte-derived FGF21 is a component of pro-adipogenic crosstalk between heart and WAT.

OBJECTIVE:. In adipose, less macrophage lipid accumulation was found with global but not myeloid-specific LpL deficiency. Neither deletion affected the expression of inflammatory genes. Global LpL deficiency also reduced the numbers of elicited peritoneal macrophages. Finally, we assessed gene expression in macrophages from atherosclerotic lesions during regression; LpL deficiency did not affect the polarity of plaque macrophages. CONCLUSIONS:The phenotypic changes observed in macrophages upon deletion of Lpl in vitro is not mimicked in tissue macrophages.

Dual peroxisome proliferator-activated receptor (PPAR)α/γ agonists that were developed to target hyperlipidemia and hyperglycemia in type 2 diabetes patients, caused cardiac dysfunction or other adverse effects. We studied the mechanisms that underlie the cardiotoxic effects of a dual PPARα/γ agonist, tesaglitazar, in wild type and diabetic (leptin receptor deficient - db/db) mice. Mice treated with tesaglitazar-containing chow or high fat diet developed cardiac dysfunction despite lower plasma triglycerides and glucose levels. Expression of cardiac peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), which promotes mitochondrial biogenesis, had the most profound reduction among various fatty acid metabolism genes. Furthermore, we observed increased acetylation of PGC1α, which suggests PGC1α inhibition and lowered sirtuin 1 (SIRT1) expression. This change was associated with lower mitochondrial abundance. Combined pharmacological activation of PPARα and PPARγ in C57BL/6 mice reproduced the reduction of PGC1α expression and mitochondrial abundance. Resveratrol-mediated SIRT1 activation attenuated tesaglitazar-induced cardiac dysfunction and corrected myocardial mitochondrial respiration in C57BL/6 and diabetic mice but not in cardiomyocyte-specific Sirt1-/- mice. Our data shows that drugs, which activate both PPARα and PPARγ lead to cardiac dysfunction associated with PGC1α suppression and lower mitochondrial abundance likely due to competition between these two transcription factors.

Lipins play important roles in adipogenesis, insulin sensitivity, and gene regulation, and mutations in these genes cause lipodystrophy, myoglobinuria, and inflammatory disorders. While all lipins (lipin 1, 2, and 3) act as phosphatidic acid phosphatase (PAP) enzymes, which are required for triacylglycerol (TAG) synthesis from glycerol 3-phosphate, lipin 1 has been the focus of most of the lipin-related research. In the current issue of the JCI, Zhang et al. show that while lipin 2 and 3 are expendable for the incorporation of dietary fatty acids into triglycerides, lipin 2/3 PAP activity has a critical role in phospholipid homeostasis and chylomicron assembly in enterocytes.

We assessed the importance of triglyceride (TG) lipolysis and circulating HDL levels in the resolution of atherosclerosis and the phenotype of vascular macrophages. We hypothesized that hyperTG impairs atherosclerosis regression due to decreased HDL particle numbers (HDL-P) and/or HDL function assessed as Cholesterol Efflux Capacity (CEC). To study hyperTG, we performed atherosclerosis regression studies in control Lipoprotein lipase (LpL) flox (LpLfl/fl) and tamoxifen inducible LpL KO (iLpL-/-) mice; the latter showing plasma TG of ~500mg/dL after tamoxifen treatment. We used two different atherosclerosis regression models – the aortic transplant and inhibition of the LDL receptor (LDLR) using antisense oligonucleotides followed by re-expression after ASO discontinuation. We analyzed atherosclerosis regression (lesion size and CD68+ macrophages) in aortic arches, roots and branchiocephalic …

Although LDL is thought to be predominantly produced by lipoprotein lipase (LpL) mediated hydrolysis of VLDL, we show that another pathway for LDL production exists and is evident with deletion of the LDL receptor (LDLR). Plasma LDL levels in humans and animals with LpL loss have been interpreted as demonstrating the obligate requirement for lipolysis in LDL production. However, studies in isolated hepatocytes and kinetic analyses in humans have indicated that in addition to decreased catabolism, reductions in the LDL receptor lead to increased direct LDL production. We first quantified lipids in plasma fractions in mice with inducible deletion of LpL (iLpl-/-) on chow diet. As expected, loss of LpL led to reduced plasma LDL cholesterol levels of 3±0.5 mg/dl compared to 20±6 mg/dl in control Lpl floxed mice (Lplfl/fl), consistent with the importance of the VLDL to LDL pathway in LDL production. HDL was also …