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.The receptor for advanced glycation end products (RAGE) is a multiligand cell surface molecule belonging to the immunoglobulin superfamily (1). It is expressed as full-length, N-truncated, and C-truncated isoforms, generated in humans by alternative splicing (2). Activation of the full-length RAGE receptor has been implicated in a range of chronic diseases, including various diabetic complications and atherosclerosis (1). In particular, studies in RAGE^−/− mice that carry the dominant-negative form of the receptor (2–6) and in RAGE-overexpressing mice (7) have confirmed an important role of RAGE activation in the development of diabetic nephropathy, neuropathy, and impaired angiogenesis. RAGE activation has also been implicated in the acceleration of atherosclerotic lesion formation as well as in the maintenance of proinflammatory and prothrombotic mechanisms, characteristic of diabetes-accelerated atherosclerosis (8,9). RAGE also represents an important mediator of oxidative stress in diabetes. Activation of RAGE in vitro leads to increased NADPH oxidase expression, mitochondrial oxidase activity, and downregulation of endogenous antioxidant activity (10,11). RAGE^−/− mice have a suppression of neointimal proliferation after externally induced arterial injury in the absence of diabetes (12). Moreover, blockade of RAGE-dependent signaling by soluble RAGE (sRAGE) has been shown to inhibit the progression of atherosclerotic changes (8,9) and kidney disease (3) in diabetic mice, possibly by suppressing the activation of nuclear factor-κB (NF-κB) activation and inflammatory cytokine expression. The present study examined the role of RAGE in the development of diabetes-accelerated atherosclerosis in a model of insulin deficiency, the streptozotocin-induced diabetic RAGE^−/−/apoE^−/− mouse. Our aim was to determine the effect of global RAGE deficiency, which includes absence of both the full-length receptor and endogenous sRAGE on the development of vascular lesions in the presence and absence of diabetes. Furthermore, key mediators of the atherosclerotic process in diabetes were examined, and the effects on these pathways were assessed in these RAGE-deficient mice.     

Diabetes adversely affects macrophages during atherosclerotic plaque regression in mice

OBJECTIVE

Patients with diabetes have increased cardiovascular risk. Atherosclerosis in these patients is often associated with increased plaque macrophages and dyslipidemia. We hypothesized that diabetic atherosclerosis involves processes that impair favorable effects of lipid reduction on plaque macrophages.

RESEARCH DESIGN AND METHODS

Reversa mice are LDL receptor–deficient mice that develop atherosclerosis. Their elevated plasma LDL levels are lowered after conditional knockout of the gene encoding microsomal triglyceride transfer protein. We examined the morphologic and molecular changes in atherosclerotic plaques in control and streptozotocin-induced diabetic Reversa mice after LDL lowering. Bone marrow–derived macrophages were also used to study changes mediated by hyperglycemia.

RESULTS

Reversa mice were fed a western diet for 16 weeks to develop plaques (baseline). Four weeks after lipid normalization, control (nondiabetic) mice had reduced plasma cholesterol (−77%), plaque cholesterol (−53%), and plaque cells positive for macrophage marker CD68+ (−73%), but increased plaque collagen (+116%) compared with baseline mice. Diabetic mice had similarly reduced plasma cholesterol, but collagen content increased by only 34% compared with baseline; compared with control mice, there were lower reductions in plaque cholesterol (−30%) and CD68+ cells (−41%). Diabetic (vs. control) plaque CD68+ cells also exhibited more oxidant stress and inflammatory gene expression and less polarization toward the anti-inflammatory M2 macrophage state. Many of the findings in vivo were recapitulated by hyperglycemia in mouse bone marrow–derived macrophages.

CONCLUSIONS

Diabetes hindered plaque regression in atherosclerotic mice (based on CD68+ plaque content) and favorable changes in plaque macrophage characteristics after the reduction of elevated plasma LDL.Type 1 and 2 diabetic patients have earlier onset and more extensive atherosclerosis than similar aged nondiabetic patients. Moreover, the presence of diabetes is associated with more coronary events and worse clinical outcomes (1,2). This might reflect the presence of plaques that are more vulnerable to rupture. Indeed, careful pathologic assessment of atherosclerotic plaques shows that diabetes is associated with more macrophages and lipid-rich areas (3), features of unstable plaques. Although studies in type 1 diabetes have established that more intensive glucose management reduces cardiovascular events (4), clinical trial data in type 2 diabetes remain controversial. The reasons for this are debated and might reflect differences in pathobiology, therapy, or extent of disease when the trials were initiated.Current clinical practice to reduce cardiovascular risk in diabetes includes plasma LDL lowering, which should not only retard the progression of atherosclerosis, but also promote its regression. Given the importance of macrophages in plaque development and pathology (5), the effects of plasma lipid changes on these cells have been an active area of research. In our own studies, we have shown in a surgical transplant model that the content of monocyte-derived CD68+ cells (mostly macrophages and macrophage foam cells) in mouse atherosclerotic plaques declined quickly in normolipidemic conditions. Furthermore, some plaque CD68+ cells were shown to emigrate to regional and systemic lymph nodes in a process dependent on the chemokine (C-C) motif receptor-7 (CCR7) (6).It is now recognized that macrophage phenotypes vary (7). Inflammatory macrophages, often referred to as M1 macrophages, are involved in pathogen recognition and inflammatory cytokine secretion. Tissue repair is thought to be mediated by M2, or alternatively activated, macrophages (8). Both M1 and M2 macrophages, as well as monocyte-derived dendritic cells, are found in human and mouse atherosclerotic plaques (9,10), with the M1 type thought to play a critical role in the progression of atherosclerosis.In the current report, we have focused on whether the diabetic state negatively influences the ability of plasma lipid reduction to regress atherosclerotic plaques. Given the importance of both the amount and the characteristics of plaque macrophages to the disease process, we examined whether diabetes interfered with a reduction in the content of macrophages or in their inflammatory state after the repression of hyperlipidemia. To accomplish this, we turned to the Reversa mouse, in which the hyperlipidemia of the LDL receptor knockout (LdLr^−/−) mouse can be reduced by the conditional inactivation of the microsomal triglyceride transfer protein gene (Mttp) (11). Atherosclerosis was allowed to develop in Reversa mice before they were made diabetic by streptozotocin (STZ) injection to avoid effects of diabetes on plaque progression. Hyperlipidemia then was repressed by Mttp ablation in both normoglycemic and diabetic mice. The morphometric and histologic changes in the plaques, as well as the molecular changes in CD68+ cells laser-captured from the plaques, were then determined. Studies of the effects of hyperglycemia on cultured bone marrow–derived macrophages (BMDMs) were performed to extend the results.     

Thioredoxin-Interacting Protein Deficiency Protects against Diabetic Nephropathy

Expression of thioredoxin-interacting protein (TxNIP), an endogenous inhibitor of the thiol oxidoreductase thioredoxin, is augmented by high glucose (HG) and promotes oxidative stress. We previously reported that TxNIP-deficient mesangial cells showed protection from HG-induced reactive oxygen species, mitogen-activated protein kinase phosphorylation, and collagen expression. Here, we investigated the potential role of TxNIP in the pathogenesis of diabetic nephropathy (DN) in vivo. Wild-type (WT) control, TxNIP^−/−, and TxNIP^+/− mice were rendered equally diabetic with low-dose streptozotocin. In contrast to effects in WT mice, diabetes did not increase albuminuria, proteinuria, serum cystatin C, or serum creatinine levels in TxNIP^−/− mice. Whereas morphometric studies of kidneys revealed a thickened glomerular basement membrane and effaced podocytes in the diabetic WT mice, these changes were absent in the diabetic TxNIP^−/− mice. Immunohistochemical analysis revealed significant increases in the levels of glomerular TGF-β1, collagen IV, and fibrosis only in WT diabetic mice. Additionally, only WT diabetic mice showed significant increases in oxidative stress (nitrotyrosine, urinary 8-hydroxy-2-deoxy-guanosine) and inflammation (IL-1β mRNA, F4/80 immunohistochemistry). Expression levels of Nox4-encoded mRNA and protein increased only in the diabetic WT animals. A significant loss of podocytes, assessed by Wilms’ tumor 1 and nephrin staining and urinary nephrin concentration, was found in diabetic WT but not TxNIP^−/− mice. Furthermore, in cultured human podocytes exposed to HG, TxNIP knockdown with siRNA abolished the increased mitochondrial O₂⁻ generation and apoptosis. These data indicate that TxNIP has a critical role in the progression of DN and may be a promising therapeutic target.     

BAMBI Elimination Enhances Alternative TGF-β Signaling and Glomerular Dysfunction in Diabetic Mice

BMP, activin, membrane-bound inhibitor (BAMBI) acts as a pseudo-receptor for the transforming growth factor (TGF)-β type I receptor family and a negative modulator of TGF-β kinase signaling, and BAMBI^−/− mice show mild endothelial dysfunction. Because diabetic glomerular disease is associated with TGF-β overexpression and microvascular alterations, we examined the effect of diabetes on glomerular BAMBI mRNA levels. In isolated glomeruli from biopsies of patients with diabetic nephropathy and in glomeruli from mice with type 2 diabetes, BAMBI was downregulated. We then examined the effects of BAMBI deletion on streptozotocin-induced diabetic glomerulopathy in mice. BAMBI^−/− mice developed more albuminuria, with a widening of foot processes, than BAMBI^+/+ mice, along with increased activation of alternative TGF-β pathways such as extracellular signal–related kinase (ERK)1/2 and Smad1/5 in glomeruli and cortices of BAMBI^−/− mice. Vegfr2 and Angpt1, genes controlling glomerular endothelial stability, were downmodulated in glomeruli from BAMBI^−/− mice with diabetes. Incubation of glomeruli from nondiabetic BAMBI^+/+ or BAMBI^−/− mice with TGF-β resulted in the downregulation of Vegfr2 and Angpt1, effects that were more pronounced in BAMBI^−/− mice and were prevented by a MEK inhibitor. The downregulation of Vegfr2 in diabetes was localized to glomerular endothelial cells using a histone yellow reporter under the Vegfr2 promoter. Thus, BAMBI modulates the effects of diabetes on glomerular permselectivity in association with altered ERK1/2 and Smad1/5 signaling. Future therapeutic interventions with inhibitors of alternative TGF-β signaling may therefore be of interest in diabetic nephropathy.     

Genetic Disruption of Myostatin Reduces the Development of Proatherogenic Dyslipidemia and Atherogenic Lesions In Ldlr Null Mice

OBJECTIVE

Insulin-resistant states, such as obesity and type 2 diabetes, contribute substantially to accelerated atherogenesis. Null mutations of myostatin (Mstn) are associated with increased muscle mass and decreased fat mass. In this study, we determined whether Mstn disruption could prevent the development of insulin resistance, proatherogenic dyslipidemia, and atherogenesis.

RESEARCH DESIGN AND METHODS

C57BL/6 Ldlr^−/− mice were cross-bred with C57BL/6 Mstn^−/− mice for >10 generations to generate Mstn^−/−/Ldlr^−/− double-knockout mice. The effects of high-fat/high-cholesterol diet on body composition, plasma lipids, systemic and tissue-specific insulin sensitivity, hepatic steatosis, as well as aortic atheromatous lesion were characterized in Mstn^−/−/Ldlr^−/− mice in comparison with control Mstn^+/+/Ldlr^−/− mice.

RESULTS

Compared with Mstn^+/+/Ldlr^−/− controls, Mstn^−/−/ Ldlr^−/− mice were resistant to diet-induced obesity, and had greatly improved insulin sensitivity, as indicated by 42% higher glucose infusion rate and 90% greater muscle [³H]-2-deoxyglucose uptake during hyperinsulinemic-euglycemic clamp. Mstn^−/−/Ldlr^−/− mice were protected against diet-induced hepatic steatosis and had 56% higher rate of hepatic fatty acid β-oxidation than controls. Mstn^−/−/Ldlr^−/− mice also had 36% lower VLDL secretion rate and were protected against diet-induced dyslipidemia, as indicated by 30–60% lower VLDL and LDL cholesterol, free fatty acids, and triglycerides. Magnetic resonance angiography and en face analyses demonstrated 41% reduction in aortic atheromatous lesions in Ldlr^−/− mice with Mstn deletion.

CONCLUSIONS

Inactivation of Mstn protects against the development of insulin resistance, proatherogenic dyslipidemia, and aortic atherogenesis in Ldlr^−/− mice. Myostatin may be a useful target for drug development for prevention and treatment of obesity and its associated type 2 diabetes and atherosclerosis.Heart disease and diabetes rank among the most prevalent disorders in most Western countries, and their incidence rates within the elderly population are particularly high (1). Aging is associated with decrease in muscle mass, increase in fat mass (2), insulin resistance, and athersclerosis progression, all conditions that predispose individuals to cardiometabolic diseases. Therefore, adiposity, sarcopenia, and heart disease are interrelated consequences of aging that contribute substantially to morbidity and mortality among older humans.Currently, most available therapies for heart disease, such as statins, are based on lowering of plasma cholesterol. These interventions have little effect on adiposity and sarcopenia. We surmised that unlike available pharmacological therapies that are mostly targeted at cholesterol synthesis or metabolism, novel therapeutic strategies, such as myostatin inactivation, that directly target muscle and fat mass accumulation may be effective in protection against obesity and its metabolic ramifications.Genetic disruption of myostatin, a transforming growth factor-β (TGF-β) family member that functions as an endogenous inhibitor of muscle growth (3–5), leads to increased skeletal muscle mass and decreased fat mass in humans, cattle, mice, and other species (6,7). Disruption of myostatin gene has also been shown to prevent the development of obesity (6). Inhibition of myostatin, either directly or through overexpression of myostatin propeptide, an endogenous myostatin inhibitor, has also been shown to prevent the development of obesity and insulin resistance (6).In this study, we show that the loss of myostatin attenuates the development of atherogenic lipid profile and the progression of atheromatous lesion growth in LDL receptor–null (Ldlr^−/−) mice, a widely used experimental model of atherogenesis. When Ldlr^−/− mice, lacking the cell-surface transmembrane receptor that recognizes apolipoprotein B100 (apoB100), are fed Western-type diet, they display hypercholesterolemia and atheromatous lesions resembling those observed in patients with familial hypercholesterolemia (8). Our data show that Mstn disruption in Ldlr^−/− mice not only attenuates diet-induced fat accumulation and improves whole body insulin sensitivity, as has been reported recently (9,10), but it also prevents the hepatic hypersecretion of proatherogenic lipoprotein, protects against the development of proatherogenic dyslipidemia, and reduces atherogenesis progression.     

Loss-of-Function Mutations in ABCA1 and Enhanced β-Cell Secretory Capacity in Young Adults

Loss-of-function mutations affecting the cholesterol transporter ATP-binding cassette transporter subfamily A member 1 (ABCA1) impair cellular cholesterol efflux and are associated with reduced HDL-cholesterol (HDL-C) levels. ABCA1 may also be important in regulating β-cell cholesterol homeostasis and insulin secretion. We sought to determine whether loss-of-function ABCA1 mutations affect β-cell secretory capacity in humans by performing glucose-potentiated arginine tests in three subjects homozygous for ABCA1 mutations (age 25 ± 11 years), eight heterozygous subjects (28 ± 7 years), and eight normal control subjects pair-matched to the heterozygous carriers. To account for any effect of low HDL-C on insulin secretion, we studied nine subjects with isolated low HDL-C with no ABCA1 mutations (age 26 ± 6 years) and nine pair-matched control subjects. Homozygotes for ABCA1 mutations exhibited enhanced oral glucose tolerance and dramatically increased β-cell secretory capacity that was also greater in ABCA1 heterozygous subjects than in control subjects, with no differences in insulin sensitivity. Isolated low HDL-C subjects also demonstrated an increase in β-cell secretory capacity but in contrast to those with ABCA1 mutations, exhibited impaired insulin sensitivity, supporting β-cell compensation for increased insulin demand. These data indicate that loss-of-function mutations in ABCA1 in young adults may be associated with enhanced β-cell secretory capacity and normal insulin sensitivity and support the importance of cellular cholesterol homeostasis in regulating β-cell insulin secretion.     

Apolipoprotein A-I Mimetic Peptides Prevent Atherosclerosis Development and Reduce Plaque Inflammation in a Murine Model of Diabetes

OBJECTIVE

To determine the effect of the apolipoprotein A-I (ApoA-I) mimetic peptide, D-4F, on atherosclerosis development in a pre-existing diabetic condition.

RESEARCH DESIGN AND METHODS

We induced hyperglycemia in 6-week-old apoE^−/− female mice using streptozotocin. Half of the diabetic apoE^−/− mice received D-4F in drinking water. Ten weeks later, plasma lipids, glucose, insulin levels, atherosclerotic lesions, and lesion macrophage content were measured.

RESULTS

Diabetic apoE^−/− mice developed ∼300% more lesion area, marked dyslipidemia, increased glucose levels, and reduced plasma insulin levels when compared with nondiabetic apoE^−/− mice. Atherosclerotic lesions were significantly reduced in the D-4F–treated diabetic apoE^−/− mice in whole aorta (1.11 ± 0.73 vs. 0.58 ± 0.44, percentage of whole aorta, P < 0.01) and in aortic roots (36,038 ± 18,467 μm²/section vs. 17,998 ± 12,491 μm²/section, P < 0.01) when compared with diabetic apoE^−/− mice that did not receive D-4F. Macrophage content in atherosclerotic lesions from D-4F–treated diabetic apoE^−/− mice was significantly reduced when compared with nontreated animals (78.03 ± 26.1 vs. 29.6 ± 15.2 P < 0.001, percentage of whole plaque). There were no differences in glucose, insulin, total cholesterol, HDL cholesterol, and triglyceride levels between the two groups. Arachidonic acid, PGE₂, PGD₂, 15-HETE, 12-HETE, and 13-HODE concentrations were significantly increased in the liver tissue of diabetic apoE^−/− mice compared with nondiabetic apoE^−/− mice and significantly reduced by D-4F treatment.

CONCLUSIONS

Our results suggest that oral D-4F can prevent atherosclerosis development in pre-existing diabetic mice and this is associated with a reduction in hepatic arachidonic acid and oxidized fatty acid levels.Type 1 diabetes is associated with two- to fourfold higher risk of coronary artery disease (CAD) and macrovascular disease (1,2). The excess cardiovascular risk in this population is not entirely explained by traditional risk factors, including hyperglycemia.Oxidative modification of LDL and the subsequent formation of foam cells are thought to be an initial step in atherogenesis (3). Multiple animal and in vitro studies have supported a role for oxidative processes in all phases of CAD, from foam cell formation to plaque rupture and thrombosis (4–6). Initiation of lipid peroxidation and formation of an array of bioactive fatty acid oxidation products are widely held as critical steps in the atherosclerotic process. It has been suggested that the inflammatory properties of lipoproteins may also be important for the development of the atherosclerotic process in diabetes (7). However, the mechanism by which diabetic dyslipidemia contributes to the development of CAD in type 1 diabetes is not clear.HDL and apolipoprotein A-I (apoA-I), its major protein, have been efficacious in the treatment of atherosclerosis (8). ApoA-I mimetic peptides 4F (L-4F and D-4F), that form a class A amphipathic helix similar to those found in apoA-I, were found to be efficacious in murine models of atherosclerosis (9) by a mechanism that is independent of plasma cholesterol levels and in part related to its ability to remove oxidized lipids from lipoproteins (9). Moreover, recent studies have shown that apoA-I mimetic peptides increase antioxidants, confer robust vascular protection and improve insulin sensitivity in rodent models of diabetes and obesity (10–12).In this study, we examined whether oral administration of D-4F can inhibit atherosclerosis development in a pre-existing diabetic condition. Our results show that D-4F is able to decrease atherosclerotic lesion development in diabetic mice, and this is associated with a reduction of hepatic arachidonic acid and hepatic oxidized fatty acids levels.     

Sphingosine 1-Phosphate Receptor 3–Deficient Dendritic Cells Modulate Splenic Responses to Ischemia-Reperfusion Injury

The plasticity of dendritic cells (DCs) permits phenotypic modulation ex vivo by gene expression or pharmacologic agents, and these modified DCs can exert therapeutic immunosuppressive effects in vivo through direct interactions with T cells, either inducing T regulatory cells (T_REGs) or causing anergy. Sphingosine 1-phosphate (S1P) is a sphingolipid and the natural ligand for five G protein–coupled receptors (S1P1, S1P2, S1P3, S1P4, and S1P5), and S1PR agonists reduce kidney ischemia-reperfusion injury (IRI) in mice. S1pr3^−/− mice are protected from kidney IRI, because DCs do not mature. We tested the therapeutic advantage of S1pr3^−/− bone marrow–derived dendritic cell (BMDC) transfers in kidney IRI. IRI produced a rise in plasma creatinine (PCr) levels in mice receiving no cells (NCs) and mice pretreated with wild-type (WT) BMDCs. However, S1pr3^−/− BMDC–pretreated mice were protected from kidney IRI. S1pr3^−/− BMDC–pretreated mice had significantly higher numbers of splenic T_REGs compared with NC and WT BMDC–pretreated mice. S1pr3^−/− BMDCs did not attenuate IRI in splenectomized, Rag-1^−/−, or CD11c⁺ DC–depleted mice. Additionally, S1pr3^−/− BMDC–dependent protection required CD169⁺ marginal zone macrophages and the macrophage–derived chemokine CCL22 to increase splenic CD4⁺Foxp3⁺ T_REGs. Pretreatment with S1pr3^−/− BMDCs also induced T_REG-dependent protection against IRI in an allogeneic mouse model. In summary, adoptively transferred S1pr3^−/− BMDCs prevent kidney IRI through interactions within the spleen and expansion of splenic CD4⁺Foxp3⁺ T_REGs. We conclude that genetically induced deficiency of S1pr3 in allogenic BMDCs could serve as a therapeutic approach to prevent IRI-induced AKI.     

Advanced glycation end product precursors impair ABCA1-dependent cholesterol removal from cells

Abnormal HDL metabolism may contribute to the increased atherosclerosis associated with diabetes. The ATP-binding cassette transporter A1 (ABCA1) is an atheroprotective cell protein that mediates cholesterol transport from cells to apolipoprotein (apo) A-I, the major protein in HDL. Because formation of advanced glycation end products (AGEs) is associated with diabetic vascular complications, we examined the effects of carbonyls implicated in AGE formation on the ABCA1 pathway in cultured fibroblasts and macrophages. Treating cells with glycolaldehyde (GA) and glyoxal (GO) strongly inhibited ABCA1-dependent transport of cholesterol from cells to apoA-I, while methylglyoxal had little effect. This occurred under conditions where other lipoprotein receptors or lipid metabolic pathways were little affected, indicating that ABCA1 was uniquely sensitive to these carbonyls. GA and GO destabilized ABCA1 and nearly abolished its binding of apoA-I, indicating that these carbonyls directly modified ABCA1. Immunohistology of coronary arteries from hyperlipidemic swine revealed that inducing diabetes with streptozotocin increased atherosclerotic lesion area and dramatically reduced the fraction of macrophages that expressed detectable ABCA1. These results raise the possibility that reactive carbonyl-mediated damage to ABCA1 promotes accumulation of cholesterol in arterial macrophages and thus contribute to the increased cardiovascular disease associated with diabetes, insulin resistance, and other inflammatory conditions.     

Efflux of Creatine Kinase from Isolated Soleus Muscle Depends on Age,Sex and Type of Exercise in Mice

Elevated plasma creatine kinase (CK) activity is often used as an indicator of exercise-induced muscle damage. Our aim was to study effects of contraction type, sex and age on CK efflux from isolated skeletal muscles of mice. The soleus muscle (SOL) of adult (7.5-month old) female C57BL/6J mice was subjected to either 100 passive stretches, isometric contractions or eccentric contractions, and muscle CK efflux was assessed after two-hour incubation in vitro. SOL of young (3-month old) male and female mice was studied after 100 eccentric contractions. For adult females, muscle CK efflux was larger (p < 0.05) after eccentric contractions than after incubation without exercise (698 ± 344 vs. 268 ± 184 mU·h⁻¹, respectively), but smaller (p < 0.05) than for young females after the same type of exercise (1069 ± 341 mU·h⁻¹). Eccentric exercise-induced CK efflux was larger in muscles of young males compared to young females (2046 ± 317 vs 1069 ± 341 mU · h⁻¹, respectively, p < 0.001). Our results show that eccentric contractions induce a significant increase in muscle CK efflux immediately after exercise. Isolated muscle resistance to exercise-induced CK efflux depends on age and sex of mice.

Key points

• Muscle lengthening contractions induce the highest CK efflux in vitro compared with similar protocol of isometric contractions or passive stretches.
• Muscle CK efflux in vitro is applicable in studying changes of sarcolemma permeability/integrity, a proxy of muscle damage, in response to muscle contractile activity.
• Isolated muscle resistance to exercise-induced CK efflux is greater in female compared to male mice of young age and is further increased in adult female mice.

Key words: Skeletal muscle, eccentric contractions, muscle damage, CK activity     

Loss of both ABCA1 and ABCG1 results in increased disturbances in islet sterol homeostasis, inflammation, and impaired β-cell function

Cellular cholesterol homeostasis is important for normal β-cell function. Disruption of cholesterol transport by decreased function of the ATP-binding cassette (ABC) transporter ABCA1 results in impaired insulin secretion. Mice lacking β-cell ABCA1 have increased islet expression of ABCG1, another cholesterol transporter implicated in β-cell function. To determine whether ABCA1 and ABCG1 have complementary roles in β-cells, mice lacking ABCG1 and β-cell ABCA1 were generated and glucose tolerance, islet sterol levels, and β-cell function were assessed. Lack of both ABCG1 and β-cell ABCA1 resulted in increased fasting glucose levels and a greater impairment in glucose tolerance compared with either ABCG1 deletion or loss of ABCA1 in β-cells alone. In addition, glucose-stimulated insulin secretion was decreased and sterol accumulation increased in islets lacking both transporters compared with those isolated from knockout mice with each gene alone. Combined deficiency of ABCA1 and ABCG1 also resulted in significant islet inflammation as indicated by increased expression of interleukin-1β and macrophage infiltration. Thus, lack of both ABCA1 and ABCG1 induces greater defects in β-cell function than deficiency of either transporter individually. These data suggest that ABCA1 and ABCG1 each make complimentary and important contributions to β-cell function by maintaining islet cholesterol homeostasis in vivo.     

Targeted Reduction of Vascular Msx1 and Msx2 Mitigates Arteriosclerotic Calcification and Aortic Stiffness in LDLR-Deficient Mice Fed Diabetogenic Diets

When fed high-fat diets, male LDLR^−/− mice develop obesity, hyperlipidemia, hyperglycemia, and arteriosclerotic calcification. An osteogenic Msx-Wnt regulatory program is concomitantly upregulated in the vasculature. To better understand the mechanisms of diabetic arteriosclerosis, we generated SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR^−/− mice, assessing the impact of Msx1+Msx2 gene deletion in vascular myofibroblast and smooth muscle cells. Aortic Msx2 and Msx1 were decreased by 95% and 34% in SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR^−/− animals versus Msx1(fl/fl);Msx2(fl/fl);LDLR^−/− controls, respectively. Aortic calcium was reduced by 31%, and pulse wave velocity, an index of stiffness, was decreased in SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR^−/− mice vs. controls. Fasting blood glucose and lipids did not differ, yet SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR^−/− siblings became more obese. Aortic adventitial myofibroblasts from SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR^−/− mice exhibited reduced osteogenic gene expression and mineralizing potential with concomitant reduction in multiple Wnt genes. Sonic hedgehog (Shh) and Sca1, markers of aortic osteogenic progenitors, were also reduced, paralleling a 78% reduction in alkaline phosphatase (TNAP)–positive adventitial myofibroblasts. RNA interference revealed that although Msx1+Msx2 supports TNAP and Wnt7b expression, Msx1 selectively maintains Shh and Msx2 sustains Wnt2, Wnt5a, and Sca1 expression in aortic adventitial myofibroblast cultures. Thus, Msx1 and Msx2 support vascular mineralization by directing the osteogenic programming of aortic progenitors in diabetic arteriosclerosis.     

HDL Cholesterol Efflux Predicts Graft Failure in Renal Transplant Recipients

High-density lipoprotein (HDL) particles are involved in the protection against cardiovascular disease by promoting cholesterol efflux, in which accumulated cholesterol is removed from macrophage foam cells. We investigated whether HDL cholesterol efflux capacity is associated with cardiovascular mortality, all-cause mortality, and graft failure in a cohort of renal transplant recipients (n=495, median follow-up 7.0 years). Cholesterol efflux capacity at baseline was quantified using incubation of human macrophage foam cells with apolipoprotein B–depleted plasma. Baseline efflux capacity was not different in deceased patients and survivors (P=0.60 or P=0.50 for cardiovascular or all-cause mortality, respectively), whereas recipients developing graft failure had lower efflux capacity than those with functioning grafts (P<0.001). Kaplan–Meier analysis demonstrated a lower risk for graft failure (P=0.004) but not cardiovascular (P=0.30) or all-cause mortality (P=0.31) with increasing gender-stratified tertiles of efflux capacity. Cox regression analyses adjusted for age and gender showed that efflux capacity was not associated with cardiovascular mortality (hazard ratio [HR], 0.89; 95% confidence interval [95% CI], 0.67 to 1.19; P=0.43). Furthermore, the association between efflux capacity and all-cause mortality (HR, .79; 95% CI, 0.63 to 0.98; P=0.031) disappeared after further adjustment for potential confounders. However, efflux capacity at baseline significantly predicted graft failure (HR, 0.43; 95% CI, 0.29 to 0.64; P<0.001) independent of apolipoprotein A-I, HDL cholesterol, or creatinine clearance. In conclusion, this prospective study shows that cholesterol efflux capacity from macrophage foam cells is not associated with cardiovascular or all-cause mortality but is a strong predictor of graft failure independent of plasma HDL cholesterol levels in renal transplant recipients.     

Deletion of Placental Growth Factor Prevents Diabetic Retinopathy and Is Associated With Akt Activation and HIF1α-VEGF Pathway Inhibition

A new diabetic mouse strain, the Akita.PlGF knockout (^−/−), was generated to study the role of placental growth factor (PlGF) in the pathogenesis of diabetic retinopathy (DR). PlGF deletion did not affect blood glucose but reduced the body weight of Akita.PlGF^−/− mice. Diabetes-induced retinal cell death, capillary degeneration, pericyte loss, and blood-retinal barrier breakdown were prevented in these mice. Protein expression of PlGF was upregulated by diabetes, particularly in vascular cells. Diabetes-induced degradation of ZO-1 and VE-cadherin was reversed due to PlGF deficiency; their expression was correlated with that of sonic hedgehog and angiopoietin-1. PlGF deletion in Akita mice resulted in an increased Akt phosphorylation. Diabetes-activated hypoxia-inducible factor (HIF)1α–vascular endothelial growth factor (VEGF) pathway, including expression of HIF1α, VEGF, VEGFR1–3, and the extent of phospho (p)-VEGFR1, p-VEGFR2, and p–endothelial nitric oxide synthase, was inhibited in the retinas of diabetic PlGF^−/− mice. However, expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, CD11b, and CD18 was not inhibited by PlGF deletion, nor was retinal leukostasis. These results suggest that PlGF is critical for the development of DR, and its genetic deletion protects the retina from diabetic damage. Protective mechanisms are associated with Akt activation and HIF1α-VEGF pathway inhibition, but independent of retinal leukostasis in the retinas of diabetic PlGF^−/− mice.     

Loss of NLRP3 increases bacterial cystitis via IRAKM

BackgroundWe attempted to characterize the molecular mechanisms that underpin urinary tract infections using a mouse model of cystitis induced by bacterial infection in a background of NOD-, LRR- and PYD domains-containing protein (NLRP3) deficiency.MethodsMale NLRP3 knockout (NLRP3^−/−) and control mice (12 weeks old) were intraurethrally inoculated with 2×10⁸ Escherichia coli (E. coli) and euthanized 1, 3, and 7 days later to assess the degree of bladder infection. Immunohistochemical detection of NLRP3 and interleukin-1 receptor-associated kinase M (IRAKM) was performed. Quantitative PCR analysis was performed to analyze the expression of interleukin (IL)-1β and tumor necrosis factor (TNF)-α.ResultsBladder infection was observed in control mice 1 day after inoculation with E. coli. The infection had disappeared by day 7. IL-1β and TNF-α levels were lower 1 day after injection but higher on days 3 and 7 in the NLRP3^−/− group compared with the control mice (P<0.05). Expression of NLRP3 and IRAKM in wild-type (WT) group were significantly decreased 1 day post infection, and by day 7 were increased back to similar level on day 0. On the contrary, in the NLRP3^−/− group, IRAKM was significantly lower than WT mice on day 0 and were significantly decreased by day 7.ConclusionsDeficiency of NLRP3 expression in NLRP3^−/− mice contributes to the pathogenesis of chronic inflammation associated with cystitis through IRAKM.