Anna C. Calkin

Transcriptional integration of metabolism by the nuclear sterol-activated receptors LXR and FXR

Nuclear receptors are integrators of hormonal and nutritional signals, mediating changes to metabolic pathways within the body. Given that modulation of lipid and glucose metabolism has been linked to diseases including type 2 diabetes, obesity and atherosclerosis, a greater understanding of pathways that regulate metabolism in physiology and disease is crucial. The liver X receptors (LXRs) and the farnesoid X receptors (FXRs) are activated by oxysterols and bile acids, respectively. Mounting evidence indicates that these nuclear receptors have essential roles, not only in the regulation of cholesterol and bile acid metabolism but also in the integration of sterol, fatty acid and glucose metabolism.

Hyperglycemia Induces a Dynamic Cooperativity of Histone Methylase and Demethylase Enzymes Associated With Gene-Activating Epigenetic Marks That Coexist on the Lysine Tail

OBJECTIVE Results from the Diabetes Control Complications Trial (DCCT) and the subsequent Epidemiology of Diabetes Interventions and Complications (EDIC) Study and more recently from the U.K. Prospective Diabetes Study (UKPDS) have revealed that the deleterious end-organ effects that occurred in both conventional and more aggressively treated subjects continued to operate >5 years after the patients had returned to usual glycemic control and is interpreted as a legacy of past glycemia known as “hyperglycemic memory.” We have hypothesized that transient hyperglycemia mediates persistent gene-activating events attributed to changes in epigenetic information. RESEARCH DESIGN AND METHODS Models of transient hyperglycemia were used to link NFκB-p65 gene expression with H3K4 and H3K9 modifications mediated by the histone methyltransferases (Set7 and SuV39h1) and the lysine-specific demethylase (LSD1) by the immunopurification of soluble NFκB-p65 chromatin. RESULTS The sustained upregulation of the NFκB-p65 gene as a result of ambient or prior hyperglycemia was associated with increased H3K4m1 but not H3K4m2 or H3K4m3. Furthermore, glucose was shown to have other epigenetic effects, including the suppression of H3K9m2 and H3K9m3 methylation on the p65 promoter. Finally, there was increased recruitment of the recently identified histone demethylase LSD1 to the p65 promoter as a result of prior hyperglycemia. CONCLUSIONS These studies indicate that the active transcriptional state of the NFκB-p65 gene is linked with persisting epigenetic marks such as enhanced H3K4 and reduced H3K9 methylation, which appear to occur as a result of effects of the methyl-writing and methyl-erasing histone enzymes.

Receptor for Advanced Glycation End Products (RAGE) Deficiency Attenuates the Development of Atherosclerosis in Diabetes

OBJECTIVE—Activation of the receptor for advanced glycation end products (RAGE) in diabetic vasculature is considered to be a key mediator of atherogenesis. This study examines the effects of deletion of RAGE on the development of atherosclerosis in the diabetic apoE−/− model of accelerated atherosclerosis. RESEARCH DESIGN AND METHODS—ApoE−/− and RAGE−/−/apoE−/− double knockout mice were rendered diabetic with streptozotocin and followed for 20 weeks, at which time plaque accumulation was assessed by en face analysis. RESULTS—Although diabetic apoE−/− mice showed increased plaque accumulation (14.9 ± 1.7%), diabetic RAGE−/−/apoE−/− mice had significantly reduced atherosclerotic plaque area (4.9 ± 0.4%) to levels not significantly different from control apoE−/− mice (4.3 ± 0.4%). These beneficial effects on the vasculature were associated with attenuation of leukocyte recruitment; decreased expression of proinflammatory mediators, including the nuclear factor-κB subunit p65, VCAM-1, and MCP-1; and reduced oxidative stress, as reflected by staining for nitrotyrosine and reduced expression of various NADPH oxidase subunits, gp91phox, p47phox, and rac-1. Both RAGE and RAGE ligands, including S100A8/A9, high mobility group box 1 (HMGB1), and the advanced glycation end product (AGE) carboxymethyllysine were increased in plaques from diabetic apoE−/− mice. Furthermore, the accumulation of AGEs and other ligands to RAGE was reduced in diabetic RAGE−/−/apoE−/− mice. CONCLUSIONS—This study provides evidence for RAGE playing a central role in the development of accelerated atherosclerosis associated with diabetes. These findings emphasize the potential utility of strategies targeting RAGE activation in the prevention and treatment of diabetic macrovascular complications.

Lack of the Antioxidant Enzyme Glutathione Peroxidase-1 Accelerates Atherosclerosis in Diabetic Apolipoprotein E–Deficient Mice

Background— Recent clinical studies have suggested a major protective role for the antioxidant enzyme glutathione peroxidase-1 (GPx1) in diabetes-associated atherosclerosis. We induced diabetes in mice deficient for both GPx1 and apolipoprotein E (ApoE) to determine whether this is merely an association or whether GPx1 has a direct effect on diabetes-associated atherosclerosis. Methods and Results— ApoE-deficient (ApoE−/−) and ApoE/GPx1 double-knockout (ApoE−/−GPx1−/−) mice were made diabetic with streptozotocin and aortic lesion formation, and atherogenic pathways were assessed after 10 and 20 weeks of diabetes. Aortic proinflammatory and profibrotic markers were determined by both quantitative reverse-transcription polymerase chain reaction analysis after 10 weeks of diabetes and immunohistochemical analysis after 10 and 20 weeks of diabetes. Sham-injected nondiabetic counterparts served as controls. Atherosclerotic lesions within the aortic sinus region, as well as arch, thoracic, and abdominal lesions, were significantly increased in diabetic ApoE−/−GPx1−/− aortas compared with diabetic ApoE−/− aortas. This increase was accompanied by increased macrophages, &agr;-smooth muscle actin, receptors for advanced glycation end products, and various proinflammatory (vascular cell adhesion molecule-1) and profibrotic (vascular endothelial growth factor and connective tissue growth factor) markers. Quantitative reverse-transcription polymerase chain reaction analysis showed increased expression of receptors for advanced glycation end products (RAGE), vascular cell adhesion molecule-1, vascular endothelial growth factor, and connective tissue growth factor. Nitrotyrosine levels were significantly increased in diabetic ApoE−/−GPx1−/− mouse aortas. These findings were observed despite upregulation of other antioxidants. Conclusions— Lack of functional GPx1 accelerates diabetes-associated atherosclerosis via upregulation of proinflammatory and profibrotic pathways in ApoE−/− mice. Our study provides evidence of a protective role for GPx1 and establishes GPx1 as an important antiatherogenic therapeutic target in patients with or at risk of diabetic macrovascular disease.

Reconstituted High-Density Lipoprotein Attenuates Platelet Function in Individuals With Type 2 Diabetes Mellitus by Promoting Cholesterol Efflux

Background— Individuals with diabetes mellitus have an increased risk of cardiovascular disease and exhibit platelet hyperreactivity, increasing their resistance to antithrombotic therapies such as aspirin and clopidogrel. Reconstituted high-density lipoprotein (rHDL) has short-term beneficial effects on atherosclerotic plaques, but whether it can effectively reduce the reactivity of diabetic platelets is not known. Methods and Results— Individuals with type 2 diabetes mellitus were infused with placebo or rHDL (CSL-111; 20 mg · kg−1 · h−1) for 4 hours, resulting in an ≈1.4-fold increase in plasma HDL cholesterol levels. rHDL infusion was associated with a >50% reduction in the ex vivo platelet aggregation response to multiple agonists, an effect that persisted in washed platelets. In vitro studies in platelets from healthy individuals revealed that the inhibitory effects of rHDL on platelet function were time and dose dependent and resulted in a widespread attenuation of platelet function and a 50% reduction in thrombus formation under flow. These effects could be recapitulated, in part, by the isolated phospholipid component of rHDL, which enhanced efflux of cholesterol from platelets and reduced lipid raft assembly. In contrast, the apolipoprotein AI component of rHDL had minimal effect on platelet function, cholesterol efflux, or lipid raft assembly. Conclusion— These findings suggest that rHDL therapy is highly effective at inhibiting the heightened reactivity of diabetic platelets, partly through reducing the cholesterol content of platelet membranes. These properties, combined with the known short-term beneficial effects of rHDL on atherosclerotic lesions, suggest that rHDL infusions may be an effective approach to reduce atherothrombotic complications in diabetic individuals. Clinical Trial Registration Information— URL: http://www.clinicaltrials.gov. Unique identifier: NCT00395148.

Rosiglitazone Attenuates Atherosclerosis in a Model of Insulin Insufficiency Independent of Its Metabolic Effects

Objectives—Recent studies have demonstrated a role for thiazolidinediones in attenuating atherosclerosis. However, these studies were performed in insulin-resistant animal models in association with reductions in insulin and glucose levels. To assess the vascular effects of thiazolidinediones, independent of their metabolic effects, we observed the effect of rosiglitazone on diabetes-associated atherosclerosis in a model of insulin insufficiency. Methods and Results—Control and diabetic apolipoprotein E–deficient mice received rosiglitazone or placebo. Diabetic mice demonstrated a 3-fold increase in plaque area, which was attenuated by rosiglitazone. There was no significant difference in glucose, insulin, or cholesterol levels between treated and untreated diabetic animals. Rosiglitazone attenuated the increase in superoxide production observed in diabetic mice. A 4-fold increase in the reverse cholesterol transport marker ABCA1 was observed in treated diabetic mice. Rosiglitazone reduced angiotensin II receptor gene expression in control and diabetic mice, and macrophage accumulation was increased in diabetic mice compared with controls and was attenuated by rosiglitazone. Conclusions—These findings suggest peroxisome proliferator-activated receptor-&ggr; ligands such as rosiglitazone confer vascular protection independent of their effects on metabolic control. These antiatherosclerotic effects may have important clinical ramifications not only in insulin resistance/type 2 diabetes and also in type 1 diabetes.

Accelerated Nephropathy in Diabetic Apolipoprotein E-Knockout Mouse: Role of Advanced Glycation End Products

Hyperlipidemia not only may be relevant to cardiovascular disease in diabetes but may also play a role in the development and progression of diabetic nephropathy. Furthermore, there is increasing evidence that advanced glycation end products (AGE) play an important role in diabetic renal disease. The objectives of this study were first to characterize renal injury in diabetic apolipoprotein E knockout (apo E-KO) mice and second to explore the role of AGE in the development and progression of renal disease in this model. Diabetes was induced by injection of streptozotocin in 6-wk-old apo E-KO mice. Diabetic animals received no treatment or treatment with the inhibitor of AGE formation aminoguanidine (1 g/kg per d) or the cross-link breaker [4,5-dimethyl-3-(2-oxo2-phenylethyl)-thiazolium chloride] ALT-711, which cleaves preformed AGE (20 mg/kg per d) for 20 wk. Nondiabetic apo E-KO mice as well as nondiabetic and diabetic C57BL/6 mice served as controls. Compared with nondiabetic apo E-KO mice, induction of diabetes in apo E-KO mice resulted in accelerated renal injury characterized by albuminuria and glomerular and tubulointerstitial injury. These abnormalities were associated with increased expression of collagen type I and type IV and transforming growth factor-beta1 (TGF-beta1), increased alpha-smooth muscle actin immunostaining and macrophage infiltration, and increased serum and renal AGE. The two treatments, which attenuated renal AGE accumulation in a disparate manner, were associated with less albuminuria, structural injury, macrophage infiltration, TGF-beta1, and collagen expression. The accelerated renal injury that was observed in diabetic apo E-KO mice was attenuated by approaches that inhibit renal AGE accumulation.

Imatinib Attenuates Diabetic Nephropathy in Apolipoprotein E-Knockout Mice

In the diabetic kidney, clinical as well as experimental observations have shown an upregulation of growth factors such as PDGF. These studies, however, were not designed to address whether upregulation of PDGF is merely a manifestation of diabetic renal injury or whether PDGF plays an active role in the pathophysiology of diabetic nephropathy. The objectives of this study were first to assess whether PDGF-dependent pathways are involved in the development of diabetic nephropathy and second to determine the effects of PDGF receptor antagonism on this disorder and associated molecular and cellular processes. This study used the diabetic apolipoprotein E-knockout (apoE-KO) mouse, a recently described model of accelerated diabetic nephropathy. Diabetes was induced by injection of streptozotocin in 6-wk-old apoE-KO mice. Diabetic animals received treatment with a tyrosine kinase inhibitor that inhibits PDGF action, imatinib (STI-571, 10 mg/kg per d orally) or no treatment for 20 wk. Nondiabetic apoE-KO mice served as controls. This model of accelerated renal disease with albuminuria as well as glomerular and tubulointerstitial injury was associated with increased renal expression of PDGF-B, proliferating cells, and alpha-smooth muscle actin-positive cells. Furthermore, there was increased accumulation of type I and type IV collagen as well as macrophage infiltration. Imatinib treatment ameliorated both renal functional and structural parameters of diabetes as well as overexpression of a number of growth factors, collagens, proliferating cells, alpha-smooth muscle actin-positive cells, and macrophage infiltration within the kidney. Tyrosine kinase inhibition with imatinib seems to retard the development of experimental diabetic nephropathy.

Reactive Oxygen Species Can Provide Atheroprotection via NOX4-Dependent Inhibition of Inflammation and Vascular Remodeling

Objective— Oxidative stress is considered a hallmark of atherosclerosis. In particular, the superoxide-generating type 1 NADPH oxidase (NOX1) has been shown to be induced and play a pivotal role in early phases of mouse models of atherosclerosis and in the context of diabetes mellitus. Here, we investigated the role of the most abundant type 4 isoform (NOX4) in human and mouse advanced atherosclerosis. Approach and Results— Plaques of patients with cardiovascular events or established diabetes mellitus showed a surprising reduction in expression of the most abundant but hydrogen peroxide (H2O2)-generating type 4 isoform (Nox4), whereas Nox1 mRNA was elevated, when compared with respective controls. As these data suggested that NOX4-derived reactive oxygen species may convey a surprisingly protective effect during plaque progression, we examined a mouse model of accelerated and advanced diabetic atherosclerosis, the streptozotocin-treated ApoE −/− mouse, with (NOX4 −/−) and without genetic deletion of Nox4. Similar to the human data, advanced versus early plaques of wild-type mice showed reduced Nox4 mRNA expression. Consistent with a rather protective role of NOX4-derived reactive oxygen species, NOX4 −/− mice showed increased atherosclerosis when compared with wild-type mice. Deleting NOX4 was associated with reduced H2O2 forming activity and attenuation of the proinflammatory markers, monocyte chemotratic protein-1, interleukin-1&bgr;, and tumor necrosis factor-&agr;, as well as vascular macrophage accumulation. Furthermore, there was a greater accumulation of fibrillar collagen fibres within the vascular wall and plaque in diabetic Nox4 −/− ApoE −/− mice, indicative of plaque remodeling. These data could be replicated in human aortic endothelial cells in vitro, where Nox4 overexpression increased H2O2 and reduced the expression of pro-oxidants and profibrotic markers. Interestingly, Nox4 levels inversely correlated with Nox2 gene and protein levels. Although NOX2 is not constitutively active unlike NOX4 and forms rather superoxide, this opens up the possibility that at least some effects of NOX4 deletion are mediated by NOX2 activation. Conclusions— Thus, the appearance of reactive oxygen species in atherosclerosis is apparently not always a nondesirable oxidative stress, but can also have protective effects. Both in humans and in mouse, the H2O2-forming NOX4, unlike the superoxide-forming NOX1, can act as a negative modulator of inflammation and remodeling and convey atheroprotection. These results have implications on how to judge reactive oxygen species formation in cardiovascular disease and need to be considered in the development of NOX inhibitory drugs.

Gemfibrozil decreases atherosclerosis in experimental diabetes in association with a reduction in oxidative stress and inflammation

Aims/hypothesisIt is postulated that peroxisome proliferator-activated receptor α agonists confer cardiovascular benefits in diabetes, independently of their effects on lipid metabolism. We investigated putative mechanisms responsible for these anti-atherogenic effects in an in vivo model of diabetes-associated atherosclerosis.Materials and methodsControl and streptozotocin-induced diabetic apolipoprotein-deficient mice received gemfibrozil (100xa0mg kg−1 day−1) or no treatment for 20xa0weeks. Aortic plaque deposition was assessed by Sudan IV staining and subsequent en face quantification. Superoxide production was measured using lucigenin-enhanced chemiluminescence. Markers of pathways including inflammation and oxidative stress were measured using real-time RT-PCR.ResultsNo significant effect of gemfibrozil was observed on glycated haemoglobin, cholesterol or insulin in diabetic mice. Diabetes was associated with a three-fold increase in plaque area and a significant increase in NADPH-dependent superoxide compared with control mice. Gemfibrozil significantly attenuated plaque area and superoxide production in diabetic mice. In addition, gemfibrozil reduced the expression of the genes encoding the NADPH oxidase subunits p47phox, gp91phox and Rac-1. In addition, gemfibrozil reduced the expression of the genes encoding nuclear factor kappa B (NF-κB) subunit, p65, the NF-κB-dependent chemokine monocyte chemoattractant protein-1, and tissue factor.Conclusions/interpretationsThis study demonstrates that gemfibrozil exerts anti-atherogenic actions, independently of changes in cholesterol and glucose metabolism. Such findings emphasise the possible usefulness of fibrates such as gemfibrozil in a setting of atherosclerosis even in the absence of dyslipidaemia.