Methylglyoxal-derived advanced glycation end products contribute to negative cardiac remodeling and dysfunction post-myocardial infarction.

Advanced glycation end-products (AGEs) have been associated with poorer outcomes after myocardial infarction (MI), and linked with heart failure. Methylglyoxal (MG) is considered the most important AGE precursor, but its role in MI is unknown. In this study, we investigated the involvement of MG-derived AGEs (MG-AGEs) in MI using transgenic mice that over-express the MG-metabolizing enzyme glyoxalase-1 (GLO1). MI was induced in GLO1 mice and wild-type (WT) littermates. At 6 h post-MI, mass spectrometry revealed that MG-H1 (a principal MG-AGE) was increased in the hearts of WT mice, and immunohistochemistry demonstrated that this persisted for 4 weeks. GLO1 over-expression reduced MG-AGE levels at 6 h and 4 weeks, and GLO1 mice exhibited superior cardiac function at 4 weeks post-MI compared to WT mice. Immunohistochemistry revealed greater vascular density and reduced cardiomyocyte apoptosis in GLO1 vs. WT mice. The recruitment of c-kit+ cells and their incorporation into the vasculature (c-kit+CD31+ cells) was higher in the infarcted myocardium of GLO1 mice. MG-AGEs appeared to accumulate in type I collagen surrounding arterioles, prompting investigation in vitro. In culture, the interaction of angiogenic bone marrow cells with MG-modified collagen resulted in reduced cell adhesion, increased susceptibility to apoptosis, fewer progenitor cells, and reduced angiogenic potential. This study reveals that MG-AGEs are produced post-MI and identifies a causative role for their accumulation in the cellular changes, adverse remodeling and functional loss of the heart after MI. MG may represent a novel target for preventing damage and improving function of the infarcted heart.

Reducing methylglyoxal as a therapeutic target for diabetic heart disease.

Diabetes is a well-known risk factor for the development of cardiovascular diseases. Diabetes affects cardiac tissue through several different, yet interconnected, pathways. Damage to endothelial cells from direct exposure to high blood glucose is a primary cause of deregulated heart function. Toxic by-products of non-enzymatic glycolysis, mainly methylglyoxal, have been shown to contribute to the endothelial cell damage. Methylglyoxal is a precursor for advanced glycation end-products, and, although it is detoxified by the glyoxalase system, this protection mechanism fails in diabetes. Recent work has identified methylglyoxal as a therapeutic target for the prevention of cardiovascular complications in diabetes. A better understanding of the glyoxalase system and the effects of methylglyoxal may lead to more advanced strategies for treating cardiovascular complications associated with diabetes.

Role of renin-angiotensin system in activation of macrophages by modified lipoproteins.

Angiotensin II favors the development of atherosclerosis. Our goal was to determine if foam cell formation increases angiotensin II generation by the endogenous renin-angiotensin system (RAS) and if endogenously produced angiotensin II promotes lipid accumulation in macrophages. Differentiated THP-1 cells were treated with acetylated low-density lipoproteins (ac-LDL), native LDL (n-LDL), or no LDL. Expression of RAS genes was assessed and angiotensin I/II levels were quantified in media and cell lysate. Ac-LDL increased angiotensin I/II levels and the angiotensin II/I ratio in cells and media after foam cell formation. Renin mRNA or activity did not change, but renin blockade completely inhibited the increase in angiotensin II. Angiotensinogen mRNA but not protein level was increased. Angiotensin-converting enzyme (ACE) and cathepsin G mRNA and activities were enhanced by ac-LDL. Inhibition of renin, ACE, or the angiotensin II receptor 1 (AT1-receptor) largely abolished cholesteryl ester formation in cells exposed to ac-LDL and decreased scavenger receptor A (SR-A) and acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT-1) protein levels. Inhibition of renin or the AT1-receptor in cells treated with oxidized LDL also decreased SR-A and ACAT-1 protein and foam cell formation. ac-LDL also increased angiotensin II by human peripheral blood monocyte-derived macrophages, whereas blockade of renin decreased cholesterol ester formation in these macrophages. These findings indicate that, during foam cell formation, angiotensin II generation by the endogenous RAS is stimulated and that endogenously generated angiotensin II is crucial for cholesterol ester accumulation in macrophages exposed to modified LDL.

Cathepsin G deficiency decreases complexity of atherosclerotic lesions in apolipoprotein E-deficient mice.

Cathepsin G is a serine protease with a broad range of catalytic activities, including production of angiotensin II, degradation of extracellular matrix and cell-cell junctions, modulation of chemotactic responses, and induction of apoptosis. Cathepsin G mRNA expression is increased in human coronary atheroma vs. the normal vessel. To assess whether cathepsin G modulates atherosclerosis, cathepsin G knockout (Cstg(-/-)) mice were bred with apolipoprotein E knockout (Apoe(-/-)) mice to obtain Ctsg(+/-)Apoe(-/-) and Ctsg(+/+)Apoe(-/-) mice. Heterozygous cathepsin G deficiency led to a 70% decrease in cathepsin G activity in bone marrow cells, but this reduced activity did not impair generation of angiotensin II in bone marrow-derived macrophages (BMDM). Atherosclerotic lesions were compared in male Cstg(+/-)Apoe(-/-) and Cstg(+/+)Apoe(-/-) mice after 8 wk on a high-fat diet. Plasma cholesterol levels and cholesterol distribution within serum lipoprotein fractions did not differ between genotypes nor did the atherosclerotic lesion areas in either the aortic root or aortic arch. Cstg(+/-)Apoe(-/-) mice, however, showed a lower percentage of complex lesions within the aortic root and a smaller number of apoptotic cells compared with Cstg(+/+)Apoe(-/-) littermates. Furthermore, apoptotic Cstg(-/-) BMDM were more efficiently engulfed by phagocytic BMDM than were apoptotic Ctsg(+/+) BMDM. Thus cathepsin G activity may impair efferocytosis, which could lead to an accumulation of lesion-associated apoptotic cells and the accelerated progression of early atherosclerotic lesions to more complex lesions in Apoe(-/-) mice.

Immunochemical analysis of the electronegative LDL subfraction shows that abnormal N-terminal apolipoprotein B conformation is involved in increased binding to proteoglycans.

Electronegative LDL (LDL(-)) is a minor subfraction of modified LDL present in plasma. Among its atherogenic characteristics, low affinity to the LDL receptor and high binding to arterial proteoglycans (PGs) could be related to abnormalities in the conformation of its main protein, apolipoprotein B-100 (apoB-100). In the current study, we have performed an immunochemical analysis using monoclonal antibody (mAb) probes to analyze the conformation of apoB-100 in LDL(-). The study, performed with 28 anti-apoB-100 mAbs, showed that major differences of apoB-100 immunoreactivity between native LDL and LDL(-) concentrate in both terminal extremes. The mAbs Bsol 10, Bsol 14 (which recognize the amino-terminal region), Bsol 2, and Bsol 7 (carboxyl-terminal region) showed increased immunoreactivity in LDL(-), suggesting that both terminal extremes are more accessible in LDL(-) than in native LDL. The analysis of in vitro-modified LDLs, including LDL lipolyzed with sphingomyelinase (SMase-LDL) or phospholipase A(2) (PLA(2)-LDL) and oxidized LDL (oxLDL), suggested that increased amino-terminal immunoreactivity was related to altered conformation due to aggregation. This was confirmed when the aggregated subfractions of LDL(-) (agLDL(-)) and oxLDL (ag-oxLDL) were isolated and analyzed. Thus, Bsol 10 and Bsol 14 immunoreactivity was high in SMase-LDL, ag-oxLDL, and agLDL(-). The altered amino-terminal apoB-100 conformation was involved in the increased PG binding affinity of agLDL(-) because Bsol 10 and Bsol 14 blocked its high PG-binding. These observations suggest that an abnormal conformation of the amino-terminal region of apoB-100 is responsible for the increased PG binding affinity of agLDL(-).

Intravascular transfer contributes to postprandial increase in numbers of very-low-density hepatitis C virus particles.

BACKGROUND & AIMS: The physical association of hepatitis C virus (HCV) particles with lipoproteins in plasma results in distribution of HCV in a broad range of buoyant densities. This association is thought to increase virion infectivity by mediating cell entry via lipoprotein receptors. We sought to determine if factors that affect triglyceride-rich lipoprotein (TRL) metabolism alter the density and dynamics of HCV particles in the plasma of patients with chronic HCV infection. METHODS: Fasting patients (n = 10) consumed a high-fat milkshake; plasma was collected and fractionated by density gradients. HCV- RNA was measured in the very-low-density fraction (VLDF, d < 1.025 g/mL) before and at 7 serial time points postprandially. RESULTS: The amount of HCV RNA in the VLDF (HCV(VLDF)) increased a mean of 26-fold, peaking 180 minutes after the meal (P < .01). Quantification of HCV RNA throughout the density gradient fractions revealed that HCV(VLDF) rapidly disappeared, rather than migrating into the adjacent density fraction. Immuno-affinity separation of the VLDF, using antibodies that recognize apolipoprotein B-100 and not apolipoprotein B-48, showed that HCV(VLDF) is composed of chylomicron- and VLDL-associated HCV particles; peaking 120 and 180 minutes after the meal, respectively. Plasma from fasting HCV-infected patients mixed with uninfected plasma increased the quantity of HCV(VLDF), compared with that mixed with phosphate-buffered saline, showing extracellular assembly of HCV(VLDF). CONCLUSIONS: Dietary triglyceride alters the density and dynamics of HCV in plasma. The rapid clearance rate of HCV(VLDF) indicates that association with TRL is important for HCV infectivity. HCV particles, such as exchangeable apolipoproteins, appear to reassociate with TRLs in the vascular compartment.

Cloning of apoB intrabodies: specific knockdown of apoB in HepG2 cells.

Apolipoprotein (apo) B is essential for the assembly and secretion of triglyceride-rich lipoproteins made by the liver. As the sole protein component in LDL, apoB is an important determinant of atherosclerosis susceptibility and a potential pharmaceutical target. Single-chain antibodies (sFvs) are the smallest fragment of an IgG molecule capable of maintaining the antigen binding specificity of the parental antibody. In the present study, we describe the cloning and construction of two intracellular antibodies (intrabodies) to human apoB. We targeted these intrabodies to the endoplasmic reticulum for the purpose of retaining nascent apoB within the ER, thereby preventing its secretion. Expression of the 1D1 intrabody in the apoB-secreting human hepatoma cell line HepG2 resulted in marked reduction of apoB secretion. This study demonstrates the utility of an intrabody to specifically block the secretion of a protein determinant of plasma LDL as a therapeutic strategy for the treatment of hyperlipidemia.

Genetic etiology of isolated low HDL syndrome: incidence and heterogeneity of efflux defects.

OBJECTIVE: We have used a multitiered approach to identify genetic and cellular contributors to high-density lipoprotein (HDL) deficiency in 124 human subjects. METHODS AND RESULTS: We resequenced 4 candidate genes for HDL regulation and identified several functional nonsynonymous mutations including 2 in apolipoprotein A-I (APOA1), 4 in lecithin:cholesterol acyltransferase (LCAT), 1 in phospholipid transfer protein (PLTP), and 7 in the ATP-binding cassette transporter ABCA1, leaving 88% (110/124) of HDL deficient subjects without a genetic diagnosis. Cholesterol efflux assays performed using cholesterol-loaded monocyte-derived macrophages from the 124 low HDL subjects and 48 control subjects revealed that 33% (41/124) of low HDL subjects had low efflux, despite the fact that the majority of these subjects (34/41) were not carriers of dysfunctional ABCA1 alleles. In contrast, only 2% of control subjects presented with low efflux (1/48). In 3 families without ABCA1 mutations, efflux defects were found to cosegregate with low HDL. CONCLUSIONS: Efflux defects are frequent in low HDL syndromes, but the majority of HDL deficient subjects with cellular cholesterol efflux defects do not harbor ABCA1 mutations, suggesting that novel pathways contribute to this phenotype.

Methylglyoxal-Induced Endothelial Cell Loss and Inflammation Contribute to the Development of Diabetic Cardiomyopathy.

The mechanisms for the development of diabetic cardiomyopathy remain largely unknown. Methylglyoxal (MG) can accumulate and promote inflammation and vascular damage in diabetes. We examined if overexpression of the MG-metabolizing enzyme glyoxalase 1 (GLO1) in macrophages and the vasculature could reduce MG-induced inflammation and prevent ventricular dysfunction in diabetes. Hyperglycemia increased circulating inflammatory markers in wild-type (WT) but not in GLO1-overexpressing mice. Endothelial cell number was reduced in WT-diabetic hearts compared with nondiabetic controls, whereas GLO1 overexpression preserved capillary density. Neuregulin production, endothelial nitric oxide synthase dimerization, and Bcl-2 expression in endothelial cells was maintained in the hearts of GLO1-diabetic mice and corresponded to less myocardial cell death compared with the WT-diabetic group. Lower receptor for advanced glycation end products and tumor necrosis factor-α (TNF-α) levels were also observed in GLO1-diabetic versus WT-diabetic mice. Over a period of 8 weeks of hyperglycemia, GLO1 overexpression delayed and limited the loss of cardiac function. In vitro, MG and TNF-α were shown to synergize in promoting endothelial cell death, which was associated with increased angiopoietin 2 expression and reduced Bcl-2 expression. These results suggest that MG in diabetes increases inflammation, leading to endothelial cell loss. This contributes to the development of diabetic cardiomyopathy and identifies MG-induced endothelial inflammation as a target for therapy.

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.

Insulin-degrading enzyme deficiency in bone marrow cells increases atherosclerosis in LDL receptor-deficient mice.

BACKGROUND: Insulin-degrading enzyme (IDE), a protease implicated in several chronic diseases, associates with the cytoplasmic domain of the macrophage Type A scavenger receptor (SR-A). Our goal was to investigate the effect of IDE deficiency (Ide(-/-)) on diet-induced atherosclerosis in low density lipoprotein-deficient (Ldlr(-/-)) mice and on SR-A function. METHODS: Irradiated Ldlr(-/-) or Ide(-/-)Ldlr(-/-) mice were reconstituted with wild-type or Ide(-/-) bone marrow and, 6 weeks later, were placed on a high-fat diet for 8 weeks. RESULTS: After 8 weeks on a high-fat diet, male Ldlr(-/-) recipients of Ide(-/-) bone marrow had more atherosclerosis, higher serum cholesterol and increased lesion-associated ß-amyloid, an IDE substrate, and receptor for advanced glycation end products (RAGE), a proinflammatory receptor for ß-amyloid, compared to male Ldlr(-/-) recipients of wild-type bone marrow. IDE deficiency in male Ldlr(-/-) recipient mice did not affect atherosclerosis or cholesterol levels and moderated the effects of IDE deficiency of bone marrow-derived cells. No differences were seen between Ldlr(-/-) and Ide(-/-)Ldlr(-/-) female mice reconstituted with Ide(-/-) or wild-type bone marrow. IDE deficiency in macrophages did not alter SR-A levels, cell surface SR-A, or foam cell formation. CONCLUSION: IDE deficiency in bone marrow-derived cells results in larger atherosclerotic lesions, increased lesion-associated Aß and RAGE, and higher serum cholesterol in male, Ldlr(-/-) mice.

Dietary fatty acids make a rapid and substantial contribution to VLDL-triacylglycerol in the fed state.

Exaggerated postprandial lipemia is associated with coronary heart disease and type II diabetes, yet few studies have examined the effect of sequential meals on lipoprotein metabolism. We have used 13C-labeled fatty acids to trace the incorporation of fatty acid derived from a meal into apolipoprotein B-100 (apoB-100)-containing lipoproteins and plasma nonesterified fatty acids (NEFA) following two consecutive meals. Healthy volunteers (n=8) were given breakfast labeled with [1-(13)C]palmitic acid, eicosapentaenoic acid, and docosahexaenoic acid, followed 5 h later by lunch containing [1-(13)C]oleic acid. Blood samples were taken over a 9-h period. ApoB-100-containing lipoproteins were isolated by immunoaffinity chromatography. Chylomicron-triacylglycerol (TG) concentrations peaked at 195 min following breakfast but at 75 min following lunch (P<0.001). VLDL-TG concentrations, in contrast, rose to a broad peak after breakfast and then fell steadily after lunch. Breakfast markers followed chylomicron-TG concentrations and appeared in plasma NEFA with a similar profile, whereas [1-(13)C]oleic acid peaked 2 h after lunch in plasma TG and NEFA. Breakfast markers appeared steadily in VLDL, peaking 1-3 h after lunch, whereas [1-(13)C]oleic acid was still accumulating in VLDL at 9 h. Around 17% of VLDL-TG originated from recent dietary fat 5 h after breakfast, and around 40% at the end of the experiment. We conclude that there is rapid flux of fatty acids from the diet into endogenous pools. Further study of these processes may open up new targets for intervention to reduce VLDL-TG concentrations and postprandial lipemia.

Selective partitioning of dietary fatty acids into the VLDL TG pool in the early postprandial period.

Circulating triacylglycerol (TG) arises mainly from dietary fat. However, little is known about the entry of dietary fat into the major TG pool, very low-density lipoprotein (VLDL) TG. We used a novel method to study the specific incorporation of dietary fatty acids into postprandial VLDL TG in humans. Eight healthy volunteers (age 25.4 +/- 2.2 years, body mass index 22.1 +/- 2.3 kg/m2) were fed a mixed meal containing 30 g fish oil and 600 mg [1-13C]palmitic acid. Chylomicrons and VLDL were separated using immunoaffinity against apolipoprotein B-100. The fatty acid composition of lipoproteins was analyzed by gas chromatography/mass spectrometry. [1-13C]palmitic acid started to appear in VLDL TG 3 h after meal intake, and a similar delay was observed for eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Approximately 20% of dietary fatty acids entered the VLDL TG pool 6 h after meal intake. DHA was clearly overincorporated into this pool compared with [1-13C]palmitic acid and EPA. This seemed to depend on a marked elevation of this fatty acid in the nonesterified fatty acid pool. In summary, the contribution of dietary fatty acids to early postprandial VLDL TG is substantial. The role of DHA in VLDL TG production will require further investigation.

Immunochemical evidence that human apoB differs when expressed in rodent versus human cells.

LDL from human apolipoprotein B-100 (apoB-100) transgenic (HuBTg+/+) mice contains more triglyceride than LDL from normolipidemic subjects. To obtain novel monoclonal antibody (MAb) probes of apoB conformation, we generated hybridomas from HuBTg+/+ that had been immunized with LDL isolated from human plasma. One apoE-specific and four anti-apoB-100-specific hybridomas were identified. Two MAbs, 2E1 and 3D11, recognized an epitope in the amino-terminal 689 residues of apoB in native apoB-containing lipoproteins (LpBs) from human plasma or from the supernatant of human hepatoma HepG2 cells, but did not react with LpB from HuBTg+/+ mice or LpB secreted by human apoB-100-transfected rat McArdle 7777 hepatoma cells. 2E1 reacted weakly and 3D11 reacted strongly with apoB from HuBTg+/+ mice after SDS-PAGE. The lack of expression of the 2E1 and 3D11 epitopes on native LpB from HuBTg+/+ mice did not solely reflect the abnormal lipid composition of murine LpB. Both epitopes were detected in all human plasma samples tested and in all human plasma LpB classes. Therefore, human apoB expressed by rodent hepatocytes or hepatoma cells appears to adopt a different conformation or undergoes different posttranslational modification than apoB expressed in human hepatocytes or hepatoma cells.