Michele Geoffrion

The intracellular redox state is a core determinant of mitochondrial fusion

Mitochondrial hyperfusion has recently been shown to function as a cellular stress response, providing transient protection against apoptosis and mitophagy. However, the mechanisms that mediate this response remain poorly understood. In this study, we demonstrate that oxidized glutathione (GSSG), the core cellular stress indicator, strongly induces mitochondrial fusion. Biochemical and functional experiments show that GSSG induces the generation of disulphide‐mediated mitofusin oligomers, in a process that also requires GTP hydrolysis. Our data outline the molecular events that prime the fusion machinery, providing new insights into the coupling of mitochondrial fusion with the cellular stress response.

Glycation of LDL by Methylglyoxal Increases Arterial Atherogenicity: A Possible Contributor to Increased Risk of Cardiovascular Disease in Diabetes

OBJECTIVE To study whether modification of LDL by methylglyoxal (MG), a potent arginine-directed glycating agent that is increased in diabetes, is associated with increased atherogenicity. RESEARCH DESIGN AND METHODS Human LDL was isolated and modified by MG in vitro to minimal extent (MGmin-LDL) as occurs in vivo. Atherogenic characteristics of MGmin-LDL were characterized: particle size, proteoglycan-binding, susceptibility to aggregation, LDL and non-LDL receptor–binding, and aortal deposition. The major site of modification of apolipoprotein B100 (apoB100) modification was investigated by mass spectrometric peptide mapping. RESULTS MGmin-LDL contained 1.6 molar equivalents of MG modification—mostly hydroimidazolone—as found in vivo. MGmin-LDL had decreased particle size, increased binding to proteoglycans, and increased aggregation in vitro. Cell culture studies showed that MGmin-LDL was bound by the LDL receptor but not by the scavenger receptor and had increased binding affinity for cell surface heparan sulfate–containing proteoglycan. Radiotracer studies in rats showed that MGmin-LDL had a similar fractional clearance rate in plasma to unmodified LDL but increased partitioning onto the aortal wall. Mass spectrometry peptide mapping identified arginine-18 as the hotspot site of apoB100 modification in MGmin-LDL. A computed structural model predicted that MG modification of apoB100 induces distortion, increasing exposure of the N-terminal proteoglycan–binding domain on the surface of LDL. This likely mediates particle remodeling and increases proteoglycan binding. CONCLUSIONS MG modification of LDL forms small, dense LDL with increased atherogenicity that provides a new route to atherogenic LDL and may explain the escalation of cardiovascular risk in diabetes and the cardioprotective effect of metformin.

Netrin-1 promotes adipose tissue macrophage retention and insulin resistance in obesity

During obesity, macrophage accumulation in adipose tissue propagates the chronic inflammation and insulin resistance associated with type 2 diabetes. The factors, however, that regulate the accrual of macrophages in adipose tissue are not well understood. Here we show that the neuroimmune guidance cue netrin-1 is highly expressed in obese but not lean adipose tissue of humans and mice, where it directs the retention of macrophages. Netrin-1, whose expression is induced in macrophages by the saturated fatty acid palmitate, acts via its receptor Unc5b to block their migration. In a mouse model of diet-induced obesity, we show that adipose tissue macrophages exhibit reduced migratory capacity, which can be restored by blocking netrin-1. Furthermore, hematopoietic deletion of Ntn1 facilitates adipose tissue macrophage emigration, reduces inflammation and improves insulin sensitivity. Collectively, these findings identify netrin-1 as a macrophage retention signal in adipose tissue during obesity that promotes chronic inflammation and insulin resistance.

Knockdown of Glyoxalase 1 Mimics Diabetic Nephropathy in Nondiabetic Mice

Differences in susceptibility to diabetic nephropathy (DN) between mouse strains with identical levels of hyperglycemia correlate with renal levels of oxidative stress, shown previously to play a central role in the pathogenesis of DN. Susceptibility to DN appears to be genetically determined, but the critical genes have not yet been identified. Overexpression of the enzyme glyoxalase 1 (Glo1), which prevents posttranslational modification of proteins by the glycolysis-derived α-oxoaldehyde, methylglyoxal (MG), prevents hyperglycemia-induced oxidative stress in cultured cells and model organisms. In this study, we show that in nondiabetic mice, knockdown of Glo1 increases to diabetic levels both MG modification of glomerular proteins and oxidative stress, causing alterations in kidney morphology indistinguishable from those caused by diabetes. We also show that in diabetic mice, Glo1 overexpression completely prevents diabetes-induced increases in MG modification of glomerular proteins, increased oxidative stress, and the development of diabetic kidney pathology, despite unchanged levels of diabetic hyperglycemia. Together, these data indicate that Glo1 activity regulates the sensitivity of the kidney to hyperglycemic-induced renal pathology and that alterations in the rate of MG detoxification are sufficient to determine the glycemic set point at which DN occurs.

Macrophage Mitochondrial Energy Status Regulates Cholesterol Efflux and Is Enhanced by Anti-miR33 in Atherosclerosis

RATIONALE Therapeutically targeting macrophage reverse cholesterol transport is a promising approach to treat atherosclerosis. Macrophage energy metabolism can significantly influence macrophage phenotype, but how this is controlled in foam cells is not known. Bioinformatic pathway analysis predicts that miR-33 represses a cluster of genes controlling cellular energy metabolism that may be important in macrophage cholesterol efflux. OBJECTIVE We hypothesized that cellular energy status can influence cholesterol efflux from macrophages, and that miR-33 reduces cholesterol efflux via repression of mitochondrial energy metabolism pathways. METHODS AND RESULTS In this study, we demonstrated that macrophage cholesterol efflux is regulated by mitochondrial ATP production, and that miR-33 controls a network of genes that synchronize mitochondrial function. Inhibition of mitochondrial ATP synthase markedly reduces macrophage cholesterol efflux capacity, and anti-miR33 required fully functional mitochondria to enhance ABCA1-mediated cholesterol efflux. Specifically, anti-miR33 derepressed the novel target genes PGC-1α, PDK4, and SLC25A25 and boosted mitochondrial respiration and production of ATP. Treatment of atherosclerotic Apoe(-/-) mice with anti-miR33 oligonucleotides reduced aortic sinus lesion area compared with controls, despite no changes in high-density lipoprotein cholesterol or other circulating lipids. Expression of miR-33a/b was markedly increased in human carotid atherosclerotic plaques compared with normal arteries, and there was a concomitant decrease in mitochondrial regulatory genes PGC-1α, SLC25A25, NRF1, and TFAM, suggesting these genes are associated with advanced atherosclerosis in humans. CONCLUSIONS This study demonstrates that anti-miR33 therapy derepresses genes that enhance mitochondrial respiration and ATP production, which in conjunction with increased ABCA1 expression, works to promote macrophage cholesterol efflux and reduce atherosclerosis.

Targeting macrophage necroptosis for therapeutic and diagnostic interventions in atherosclerosis

Necroptosis promotes necrotic core and vulnerable atherosclerosis in humans and mice and is a prospective therapeutic and diagnostic tool. Atherosclerosis results from maladaptive inflammation driven primarily by macrophages, whose recruitment and proliferation drive plaque progression. In advanced plaques, macrophage death contributes centrally to the formation of plaque necrosis, which underlies the instability that promotes plaque rupture and myocardial infarction. Hence, targeting macrophage cell death pathways may offer promise for the stabilization of vulnerable plaques. Necroptosis is a recently discovered pathway of programmed cell necrosis regulated by RIP3 and MLKL kinases that, in contrast to apoptosis, induces a proinflammatory state. We show herein that necroptotic cell death is activated in human advanced atherosclerotic plaques and can be targeted in experimental atherosclerosis for both therapeutic and diagnostic interventions. In humans with unstable carotid atherosclerosis, expression of RIP3 and MLKL is increased, and MLKL phosphorylation, a key step in the commitment to necroptosis, is detected in advanced atheromas. Investigation of the molecular mechanisms underlying necroptosis showed that atherogenic forms of low-density lipoprotein increase RIP3 and MLKL transcription and phosphorylation—two critical steps in the execution of necroptosis. Using a radiotracer developed with the necroptosis inhibitor necrostatin-1 (Nec-1), we show that 123I-Nec-1 localizes specifically to atherosclerotic plaques in Apoe−/− mice, and its uptake is tightly correlated to lesion areas by ex vivo nuclear imaging. Furthermore, treatment of Apoe−/− mice with established atherosclerosis with Nec-1 reduced lesion size and markers of plaque instability, including necrotic core formation. Collectively, our findings offer molecular insight into the mechanisms of macrophage cell death that drive necrotic core formation in atherosclerosis and suggest that this pathway can be used as both a diagnostic and therapeutic tool for the treatment of unstable atherosclerosis.

Therapeutic Inhibition of miR-33 Promotes Fatty Acid Oxidation but Does Not Ameliorate Metabolic Dysfunction in Diet-Induced Obesity

Objective—miR-33 has emerged as an important regulator of lipid homeostasis. Inhibition of miR-33 has been demonstrated as protective against atherosclerosis; however, recent studies in mice suggest that miR-33 inhibition may have adverse effects on lipid and insulin metabolism. Given the therapeutic interest in miR-33 inhibitors for treating atherosclerosis, we sought to test whether pharmacologically inhibiting miR-33 at atheroprotective doses affected metabolic parameters in a mouse model of diet-induced obesity. Approach and Results—High-fat diet (HFD) feeding in conjunction with treatment of male mice with 10 mg/kg control anti-miR or anti-miR33 inhibitors for 20 weeks promoted equivalent weight gain in all groups. miR-33 inhibitors increased plasma total cholesterol and decreased serum triglycerides compared with control anti-miR, but not compared with PBS-treated mice. Metrics of insulin resistance were not altered in anti-miR33–treated mice compared with controls; however, respiratory exchange ratio was decreased in anti-miR33–treated mice. Hepatic expression of miR-33 targets Abca1 and Hadhb were derepressed on miR-33 inhibition. In contrast, protein levels of putative miR-33 target gene SREBP-1 or its downstream targets genes Fasn and Acc were not altered in anti-miR33–treated mice, and hepatic lipid accumulation did not differ between groups. In the adipose tissue, anti-miR33 treatment increased Ampk gene expression and markers of M2 macrophage polarization. Conclusions—We demonstrate in a mouse model of diet-induced obesity that therapeutic silencing of miR-33 may promote whole-body oxidative metabolism but does not affect metabolic dysregulation. This suggests that pharmacological inhibition of miR-33 at doses known to reduce atherosclerosis may be a safe future therapeutic.

Glyoxalase-1 overexpression in bone marrow cells reverses defective neovascularization in STZ-induced diabetic mice

AIMS Methylglyoxal (MG) accumulates in diabetes and impairs neovascularization. This study assessed whether overexpressing the MG-metabolizing enzyme glyoxalase-1 (GLO1) in only bone marrow cells (BMCs) could restore neovascularization in ischaemic tissue of streptozotocin-induced diabetic mice. METHODS AND RESULTS After 24 h of hyperglycaemic and hypoxic culture, BMCs from GLO1 overexpressing and wild-type (WT) diabetic mice were compared for migratory potential, viability, and mRNA expression of anti-apoptotic genes (Bcl-2 and Bcl-XL). In vivo, BMCs from enhanced green fluorescent protein (eGFP) mice that overexpress GLO1 were used to reconstitute the BM of diabetic mice (GLO1-diabetics). Diabetic and non-diabetic recipients of WT GFP(+) BM served as controls (WT-diabetics and non-diabetics, respectively). Following hindlimb ischaemia, the mobilization of BMCs was measured by flow cytometry. In hindlimbs, the presence of BM-derived angiogenic (GFP(+)CXCR4(+)) and endothelial (GFP(+)vWF(+)) cells and also arteriole density were determined by immunohistochemistry. Hindlimb perfusion was measured using laser Doppler. GLO1-BMCs had superior migratory potential, increased viability, and greater Bcl-2 and Bcl-XL expression, compared with WT BMCs. In vivo, the mobilization of pro-angiogenic BMCs (CXCR4(+), c-kit(+), and Flk(+)) was enhanced post-ischaemia in GLO1-diabetics compared to WT-diabetics. A greater number of GFP(+)CXCR4(+) and GFP(+)vWF(+) BMCs incorporated into the hindlimb tissue of GLO1-diabetics and non-diabetics than in WT-diabetics. Arteriole and capillary density and perfusion were also greater in GLO1-diabetics and non-diabetics. CONCLUSION This study demonstrates that protection from MG uniquely in BM is sufficient to restore BMC function and neovascularization of ischaemic tissue in diabetes and identifies GLO1 as a potential therapeutic target.

Differential effects of glyoxalase 1 overexpression on diabetic atherosclerosis and renal dysfunction in streptozotocin-treated, apolipoprotein E-deficient mice

The reactive dicarbonyls, glyoxal and methylglyoxal (MG), increase in diabetes and may participate in the development of diabetic complications. Glyoxal and MG are detoxified by the sequential activities of glyoxalase 1 (GLO1) and glyoxalase 2. To determine the contribution of these dicarbonyls to the etiology of complications, we have genetically manipulated GLO1 levels in apolipoprotein E‐null (Apoe−/−) mice. Male Apoe−/− mice, hemizygous for a human GLO1 transgene (GLO1TGApoe−/− mice) or male nontransgenic Apoe−/− litter mates were injected with streptozotocin or vehicle and 6 or 20 weeks later, aortic atherosclerosis was quantified. The GLO1 transgene lessened streptozotocin (STZ)‐induced increases in immunoreactive hydroimidazolone (MG‐H1). Compared to nondiabetic mice, STZ‐treated GLO1TGApoe−/− and Apoe−/− mice had increased serum cholesterol and triglycerides and increased atherosclerosis at both times after diabetes induction. While the increased GLO1 activity in the GLO1TGApoe−/− mice failed to protect against diabetic atherosclerosis, it lessened glomerular mesangial expansion, prevented albuminuria and lowered renal levels of dicarbonyls and protein glycation adducts. Aortic atherosclerosis was also quantified in 22‐week‐old, male normoglycemic Glo1 knockdown mice on an Apoe−/− background (Glo1KDApoe−/− mice), an age at which Glo1KD mice exhibit albuminuria and renal pathology similar to that of diabetic mice. In spite of ~75% decrease in GLO1 activity and increased aortic MG‐H1, the Glo1KDApoe−/− mice did not show increased atherosclerosis compared to age‐matched Apoe−/− mice. Thus, manipulation of GLO1 activity does not affect the development of early aortic atherosclerosis in Apoe−/− mice but can dictate the onset of kidney disease independently of blood glucose levels.

Paradoxical Suppression of Atherosclerosis in the Absence of microRNA-146a

Rationale: Inflammation is a key contributor to atherosclerosis. MicroRNA-146a (miR-146a) has been identified as a critical brake on proinflammatory nuclear factor &kgr; light chain enhancer of activated B cells signaling in several cell types, including endothelial cells and bone marrow (BM)–derived cells. Importantly, miR-146a expression is elevated in human atherosclerotic plaques, and polymorphisms in the miR-146a precursor have been associated with risk of coronary artery disease. Objective: To define the role of endogenous miR-146a during atherogenesis. Methods and Results: Paradoxically, Ldlr−/− (low-density lipoprotein receptor null) mice deficient in miR-146a develop less atherosclerosis, despite having highly elevated levels of circulating proinflammatory cytokines. In contrast, cytokine levels are normalized in Ldlr−/−;miR-146a−/− mice receiving wild-type BM transplantation, and these mice have enhanced endothelial cell activation and elevated atherosclerotic plaque burden compared with Ldlr−/− mice receiving wild-type BM, demonstrating the atheroprotective role of miR-146a in the endothelium. We find that deficiency of miR-146a in BM-derived cells precipitates defects in hematopoietic stem cell function, contributing to extramedullary hematopoiesis, splenomegaly, BM failure, and decreased levels of circulating proatherogenic cells in mice fed an atherogenic diet. These hematopoietic phenotypes seem to be driven by unrestrained inflammatory signaling that leads to the expansion and eventual exhaustion of hematopoietic cells, and this occurs in the face of lower levels of circulating low-density lipoprotein cholesterol in mice lacking miR-146a in BM-derived cells. Furthermore, we identify sortilin-1(Sort1), a known regulator of circulating low-density lipoprotein levels in humans, as a novel target of miR-146a. Conclusions: Our study reveals that miR-146a regulates cholesterol metabolism and tempers chronic inflammatory responses to atherogenic diet by restraining proinflammatory signaling in endothelial cells and BM-derived cells.