Activation of Peripheral Blood Mononuclear Cells and Leptin Secretion: New Potential Role of Interleukin-2 and High Mobility Group Box (HMGB)1.

Activation of innate immunity and low-grade inflammation contributes to hyperglycemia and an onset of Type 2 Diabetes Mellitus (T2DM). Interleukin-2 (IL-2), leptin, High Mobility Group Box-1 (HMGB-1), and increased glucose concentrations are mediators of these processes also by modulating peripheral blood mononuclear cells (PBMCs) response. The aim of this study was to investigate if HMGB-1 and IL-2 turn on PBMCs and their leptin secretion. In isolated human PBMCs and their subpopulations from healthy individuals and naïve T2DM patients, leptin release, pro-inflammatory response and Toll-like Receptors (TLRs) activation was measured. After treatment with IL-2 and HMGB1, NK (Natural Killer) have the highest amount of leptin secretion, whilst NK-T have the maximal release in basal conditions. TLR4 (TAK242) and/or TLR2 (TLR2-IgA) inhibitors decreased leptin secretion after IL-2 and HMGB1 treatment. A further non-significant increase in leptin secretion was reported in PBMCs of naive T2DM patients in response to IL-2 and HMGB-1 stimulation. Finally, hyperglycemia or hyperinsulinemia might stimulate leptin secretion from PBMCs. The amount of leptin released from PBMCs after the different treatments was enough to stimulate the secretion of IL-1ß from monocytes. Targeting leptin sera levels and secretion from PBMCs could represent a new therapeutic strategy to counteract metabolic diseases such as T2DM.

Tissue inhibitor of metalloproteinase-3 regulates inflammation in human and mouse intestine.

BACKGROUND & AIMS: Tissue inhibitor of metalloproteinases (TIMP)-3 is an inhibitor of matrix metalloproteinases, which regulates tissue inflammation, damage, and repair. We investigated the role of TIMP-3 in intestinal inflammation in human beings and mice. METHODS: We used real-time polymerase chain reaction and flow cytometry to measure levels of TIMP-3 in intestine samples from patients with Crohn's disease (CD) and those without (controls). We also analyzed TIMP-3 levels in lamina propria mononuclear cells (LPMCs) collected from biopsy samples of individuals with or without CD (controls) and then stimulated with transforming growth factor (TGF)-ß1, as well as in biopsy samples collected from patients with CD and then incubated with a Smad7 anti-sense oligonucleotide (knock down). LPMCs and biopsy samples from patients with CD were cultured with exogenous TIMP-3 and levels of inflammatory cytokines were measured. We evaluated the susceptibility of wild-type, TIMP-3-knockout (TIMP-3-KO), and transgenic (TIMP-3-Tg) mice to induction of colitis with 2, 4, 6-trinitrobenzene-sulfonic-acid (TNBS), and the course of colitis in recombinase-activating gene-1-null mice after transfer of wild-type or TIMP-3-KO T cells. RESULTS: Levels of TIMP-3 were reduced in intestine samples from patients with CD compared with controls. Incubation of control LPMCs with TGF-ß1 up-regulated TIMP-3; knockdown of Smad7, an inhibitor of TGF-ß1, in biopsy samples from patients with CD increased levels of TIMP-3. Exogenous TIMP-3 reduced levels of inflammatory cytokines in CD LPMCs and biopsy samples. TIMP-3-KO mice developed severe colitis after administration of TNBS, whereas TIMP-3-Tg mice were resistant to TNBS-induced colitis. Reconstitution of recombinase-activating gene-1-null mice with T cells from TIMP-3-KO mice increased the severity of colitis, compared with reconstitution with wild-type T cells. CONCLUSIONS: TIMP-3 is down-regulated in inflamed intestine of patients with CD. Its expression is regulated by TGF-ß1, and knock-down of Smad7 in intestinal tissues from patient with CD up-regulates TIMP-3. Loss or reduction of TIMP-3 in mice promotes development of colitis.

Overexpression of tissue inhibitor of metalloproteinase 3 in macrophages reduces atherosclerosis in low-density lipoprotein receptor knockout mice.

OBJECTIVE: Tissue inhibitor of metalloproteinase 3 (TIMP3) is a stromal protein that inhibits the activity of proteases and receptors. TIMP3 is downregulated in metabolic and inflammatory disorders, such as type 2 diabetes mellitus and atherosclerosis, particularly in regions enriched with monocyte/macrophage cells. To investigate the role of TIMP3 in atherosclerosis, we generated a new mouse model in which Timp3 was overexpressed in the atherosclerotic plaque via a macrophage-specific promoter (MacT3). We elucidated any potential antiatherosclerotic effects of TIMP3, including regulation of monocyte/macrophage recruitment within atherosclerotic plaques, in MacT3 mice crossbred with low-density lipoprotein receptor knockout (LDLR(-/-)) mice. METHODS AND RESULTS: MacT3/LDLR(-/-) mice had an improvement of atherosclerosis and metabolic parameters compared with LDLR(-/-). En face aorta and aortic root examination of MacT3/LDLR(-/-) mice revealed smaller atherosclerotic plaques with features of stability, such as increased collagen content and decreased necrotic core formation. Atherosclerotic plaques in MacT3/LDLR(-/-) mice contained fewer T cells and macrophages. Furthermore, TIMP3 overexpression in macrophages resulted in reduced oxidative stress signals, as evidenced by lower lipid peroxidation, protein carbonylation, and nitration in atheromas. CONCLUSIONS: Our study confirmed that macrophage-specific overexpression of TIMP3 decreases the inflammatory content and the amplitude of atherosclerotic plaques in mice.

The molecular link between oxidative stress, insulin resistance, and type 2 diabetes: A target for new therapies against cardiovascular diseases.

Type 2 Diabetes Mellitus (T2D) is a chronic disease with a pandemic incidence whose pathogenesis has not yet been clarified. Raising evidence highlighted the role of oxidative stress in inducing insulin resistance, pancreatic beta-cell dysfunction, and leading to cardiovascular disease (CVD). Therefore, understanding the link between oxidative stress, T2D and CVD may help to further understand the pathological processes beyond this association, to personalize the algorithm of the cure, and to find new therapeutic targets. Here, we discussed the role of oxidative stress and the decrease of antioxidant defenses in the pathogenesis of T2D. Furthermore, some aspects of hypoglycemic therapies and their potential role as antioxidant agents were examined, which might be pivotal in preventing CVD in T2D patients.

Increased tumor necrosis factor alpha-converting enzyme activity induces insulin resistance and hepatosteatosis in mice.

UNLABELLED: Tumor necrosis factor alpha-converting enzyme (TACE, also known as ADAM17) was recently involved in the pathogenesis of insulin resistance. We observed that TACE activity was significantly higher in livers of mice fed a high-fat diet (HFD) for 1 month, and this activity was increased in liver > white adipose tissue > muscle after 5 months compared with chow control. In mouse hepatocytes, C(2)C(12) myocytes, and 3T3F442A adipocytes, TACE activity was triggered by palmitic acid, lipolysaccharide, high glucose, and high insulin. TACE overexpression significantly impaired insulin-dependent phosphorylation of AKT, GSK3, and FoxO1 in mouse hepatocytes. To test the role of TACE activation in vivo, we used tissue inhibitor of metalloproteinase 3 (Timp3) null mice, because Timp3 is the specific inhibitor of TACE and Timp3(-/-) mice have higher TACE activity compared with wild-type (WT) mice. Timp3(-/-) mice fed a HFD for 5 months are glucose-intolerant and insulin-resistant; they showed macrovesicular steatosis and ballooning degeneration compared with WT mice, which presented only microvesicular steatosis. Shotgun proteomics analysis revealed that Timp3(-/-) liver showed a significant differential expression of 38 proteins, including lower levels of adenosine kinase, methionine adenosysltransferase I/III, and glycine N-methyltransferase and higher levels of liver fatty acid-binding protein 1. These changes in protein levels were also observed in hepatocytes infected with adenovirus encoding TACE. All these proteins play a role in fatty acid uptake, triglyceride synthesis, and methionine metabolism, providing a molecular explanation for the increased hepatosteatosis observed in Timp3(-/-) compared with WT mice. CONCLUSION: We have identified novel mechanisms, governed by the TACE-Timp3 interaction, involved in the determination of insulin resistance and liver steatosis during overfeeding in mice.

MicroRNA 217 modulates endothelial cell senescence via silent information regulator 1.

BACKGROUND: Aging is a major risk factor for the development of atherosclerosis and coronary artery disease. Through a microarray approach, we have identified a microRNA (miR-217) that is progressively expressed in endothelial cells with aging. miR-217 regulates the expression of silent information regulator 1 (SirT1), a major regulator of longevity and metabolic disorders that is progressively reduced in multiple tissues during aging. METHODS AND RESULTS: miR-217 inhibits SirT1 expression through a miR-217-binding site within the 3'-UTR of SirT1. In young human umbilical vein endothelial cells, human aortic endothelial cells, and human coronary artery endothelial cells, miR-217 induces a premature senescence-like phenotype and leads to an impairment in angiogenesis via inhibition of SirT1 and modulation of FoxO1 (forkhead box O1) and endothelial nitric oxide synthase acetylation. Conversely, inhibition of miR-217 in old endothelial cells ultimately reduces senescence and increases angiogenic activity via an increase in SirT1. miR-217 is expressed in human atherosclerotic lesions and is negatively correlated with SirT1 expression and with FoxO1 acetylation status. CONCLUSIONS: Our data pinpoint miR-217 as an endogenous inhibitor of SirT1, which promotes endothelial senescence and is potentially amenable to therapeutic manipulation for prevention of endothelial dysfunction in metabolic disorders.

Tissue inhibitor of metalloproteinase 3 deficiency causes hepatic steatosis and adipose tissue inflammation in mice.

BACKGROUND & AIMS: Obesity-driven, low-grade inflammation affects systemic metabolic function and can lead to insulin resistance, hepatic steatosis, and atherosclerosis. Decreased expression of tissue inhibitor of metalloproteinase 3 (Timp3) is a catalyst for insulin resistance and inflammation. Timp3 is a natural inhibitor of matrix metalloproteinases, tumor necrosis factor-alpha-converting enzyme (TACE), and vascular endothelial growth factor receptor 2, and therefore could affect signaling processes involved in inflammation and angiogenesis. METHODS: We assessed the effects of Timp3 on inflammation, tissue remodeling, and intermediary metabolism in mice, under conditions of environmental stress (high-fat diet), genetic predisposition to insulin resistance (insulin receptor [Insr] haploinsufficiency), and varying levels of inflammation (Timp3 or Tace deficiencies). Metabolic tests, immunohistochemistry, real-time polymerase chain reaction, and immunoblotting were used to compare data from wild-type, Insr(+/-), Timp3(-/-), Insr(+/-)Timp3(-/-), and Insr(+/-)Tace(+/-) mice placed on high-fat diets for 10 weeks. RESULTS: Insr(+/-)Timp3(-/-) mice showed a higher degree of adipose and hepatic inflammation compared with wild-type, Insr(+/-), Timp3(-/-), and Insr(+/-)Tace(+/-) mice. In particular, the Insr(+/-)Timp3(-/-) mice developed macrovesicular steatosis and features of severe nonalcoholic fatty liver disease, including lobular and periportal inflammation, hepatocellular ballooning, and perisinusoidal fibrosis. These were associated with increased expression of inflammatory and steatosis markers, including suppressor of cytokine signaling 3 and stearoyl CoA desaturase 1, in both liver and adipose tissue. Interestingly, Insr(+/-)Tace(+/-) mice had a nearly opposite phenotype. CONCLUSIONS: Timp3, possibly through its regulation of TACE, appears to have a role in the pathogenesis of fatty liver disease associated with obesity.

Differences in cardiovascular risk profile based on relationship between post-load plasma glucose and fasting plasma levels.

BACKGROUND: It has been shown that subjects with normal glucose tolerance (NGT), whose plasma glucose (PG) levels do not return to their fasting PG level within 2 h during an oral glucose tolerance test (OGTT) (Group I), have a significantly higher risk to develop type 2 diabetes than NGT subjects whose 2-h glucose returns to, or drops below, the fasting level (Group I). However, it is still unsettled whether individuals in Group II have a more atherogenic profile than Group I subjects. METHODS: To address this issue, we examined 266 non-diabetic offspring of type 2 diabetic patients, recruited in the context of EUGENE2 cross-sectional study. All subjects underwent an euglycaemic-hyperinsulinemic clamp to assess glucose tolerance and insulin sensitivity. Furthermore, cardiovascular risk factors and ultrasound measurement of carotid intima-media thickness (IMT) were evaluated. RESULTS: Individuals in Group II exhibited significantly higher waist circumference, blood pressure, triglycerides, 2-h post-load PG, hsC-reactive protein, interleukin-6, insulin-like growth factor-1 (IGF-1), IMT, and lower insulin sensitivity than subjects in Group I. CONCLUSIONS: Subjects with NGT, whose PG concentration does not return to their fasting PG level within 2 h during OGTT, have an atherogenic profile, suggesting that performing OGTT with measurement of PG every 30 min may be useful to assess the risk for cardiovascular disease in glucose-tolerant subjects.

Adiponectin isoforms are not associated with the severity of coronary atherosclerosis but with undiagnosed diabetes in patients affected by stable CAD.

BACKGROUND AND AIMS: Total adiponectin is emerging as an independent risk factor for cardiovascular diseases, but the role of adiponectin isoforms in coronary artery disease (CAD) is still unknown. We investigated the role of adiponectin isoforms with respect to the severity of coronary disease and to the presence of undiagnosed diabetes in patients with CAD. METHODS AND RESULTS: We recruited 205 CAD patients, all living in the central area of Italy, with a history of a previous myocardial infarction but apparently not affected by type 2 diabetes (DM2). We compared the CAD patients to a control population (n=100) matched for age, sex, BMI and cardiovascular risk factors, but without overt diabetes and cardiovascular disease. In all patients we measured Total Adiponectin (Tot-Ad) and its isoforms, metabolic, pro- and anti-inflammatory markers and we performed an oral glucose tolerance test (OGTT). CAD patients underwent a coronary angiography and/or coronary multi-slice computed tomography. Based on the severity of CAD they were divided into mono-vessel versus multi-vessel patients. Tot-Ad levels and its isoforms were comparable in patients with mono-vessel versus multi-vessel CAD. After the OGTT, in CAD patients, the results showed that 19% of patients were affected by unknown DM2, 36.1% by unknown impaired glucose tolerance (IGT), and only 43.9% were truly normoglycemic (NGT). Low levels of high molecular weight-adiponectin (HMW-Ad) were significantly associated with undiagnosed IGT or DM2 status (p<0.01). CONCLUSIONS: In our cohort of CAD patients, Tot-Ad and its isoforms do not correlate with severity of CAD, but with undiagnosed defects of glucose metabolism.

Peroxiredoxin6, a Multitask Antioxidant Enzyme Involved in the Pathophysiology of Chronic Noncommunicable Diseases.

SIGNIFICANCE: Chronic noncommunicable diseases (NCDs) are the leading causes of disability and death worldwide. NCDs mainly comprise diabetes mellitus, cardiovascular diseases, chronic obstructive pulmonary disease, cancer, and neurological degenerative diseases, which kill more than 80% of population, especially the elderly, worldwide. Recent Advances: Several recent theories established NCDs as multifactorial diseases, where a combination of genetic, epigenetic, and environmental factors contributes to their pathogenesis. Nevertheless, recent findings suggest that the common factor linking all these pathologies is an increase in oxidative stress and the age-related loss of the antioxidant mechanisms of defense against it. Impairment in mitochondrial homeostasis with consequent deregulation in oxidative stress balance has also been suggested. CRITICAL ISSUES: Therefore, antioxidant proteins deserve particular attention for their potential role against NCDs. In particular, peroxiredoxin(Prdx)6 is a unique antioxidant enzyme, belonging to the Prdx family, with double properties, peroxidase and phospholipase activities. Through these activities, Prdx6 has been shown to be a powerful antioxidant enzyme, implicated in the pathogenesis of different NCDs. Recently, we described a phenotype of diabetes mellitus in Prdx6 knockout mice, suggesting a pivotal role of Prdx6 in the pathogenesis of cardiometabolic diseases. FUTURE DIRECTIONS: Increasing awareness on the role of antioxidant defenses in the pathogenesis of NCDs may open novel therapeutic approaches to reduce the burden of this pandemic phenomenon. However, knowledge of the role of Prdx6 in NCD prevention and pathogenesis is still not clarified.

Timp3 deficiency in insulin receptor-haploinsufficient mice promotes diabetes and vascular inflammation via increased TNF-alpha.

Activation of inflammatory pathways may contribute to the beginning and the progression of both atherosclerosis and type 2 diabetes. Here we report a novel interaction between insulin action and control of inflammation, resulting in glucose intolerance and vascular inflammation and amenable to therapeutic modulation. In insulin receptor heterozygous (Insr+/-) mice, we identified the deficiency of tissue inhibitor of metalloproteinase 3 (Timp3, an inhibitor of both TNF-alpha-converting enzyme [TACE] and MMPs) as a common bond between glucose intolerance and vascular inflammation. Among Insr+/- mice, those that develop diabetes have reduced Timp3 and increased TACE activity. Unchecked TACE activity causes an increase in levels of soluble TNF-alpha, which subsequently promotes diabetes and vascular inflammation. Double heterozygous Insr+/-Timp3+/- mice develop mild hyperglycemia and hyperinsulinemia at 3 months and overt glucose intolerance and hyperinsulinemia at 6 months. A therapeutic role for Timp3/TACE modulation is supported by the observation that pharmacological inhibition of TACE led to marked reduction of hyperglycemia and vascular inflammation in Insr+/- diabetic mice, as well as by the observation of increased insulin sensitivity in Tace+/- mice compared with WT mice. Our results suggest that an interplay between reduced insulin action and unchecked TACE activity promotes diabetes and vascular inflammation.

Endothelial function and arterial stiffness in normotensive normoglycemic first-degree relatives of diabetic patients are independent of the metabolic syndrome.

BACKGROUND AND AIM: The aim of the present study was to investigate endothelial function and arterial stiffness in normotensive normoglycemic first-degree relatives (offspring) of diabetic subjects and to explore the relationship with the metabolic syndrome and its components. METHODS AND RESULTS: Forty-five healthy normotensive normoglycemic subjects (aged 18-42 years), 29 first-degree relatives of diabetic subjects (FDR) and 16 with no parental history of type 2 diabetes mellitus were studied. Endothelial function was measured as flow-mediated dilation of the brachial artery (FMD) and arterial stiffness as carotid-femoral pulse wave velocity (PWV). Insulin resistance was calculated by homeostasis model assessment (HOMA). Plasma levels of inflammation markers (hsCRP, TNF-alpha, IL-1beta, CD40L, VCAM, and ICAM) were evaluated. Normotensive normoglycemic FDR presented a 33% lower flow-mediated dilation than the control group (9.8+/-5.2 vs. 16.2+/-7.6%, p<0.01). FMD was reduced in FDR, with or without insulin resistance, whereas arterial stiffness was significantly increased only in FDR with insulin resistance. To investigate the role of FDR status independently of altered components of the metabolic syndrome, subjects with no altered components of the metabolic syndrome were compared according to their FDR status: FDR subjects with no altered components of the metabolic syndrome presented a blunted endothelial function (lower FMD: 11.2+/-1.6 vs. 16.8+/-2.0%, p<0.05) and stiffer large arteries (higher PWV: 9.6+/-0.3 vs. 8.8+/-0.3m/s, p<0.05) than controls. CONCLUSION: Normoglycemic first-degree relatives of diabetic subjects have blunted endothelial function and increased stiffness of the large arteries. These alterations are already present at a very young age, before any alteration in glycemic control or blood pressure values can be detected, and are independent of the presence of the metabolic syndrome and its altered components.

Benfotiamine counteracts glucose toxicity effects on endothelial progenitor cell differentiation via Akt/FoxO signaling.

Dysfunction of mature endothelial cells is thought to play a major role in both micro- and macrovascular complications of diabetes. However, recent advances in biology of endothelial progenitor cells (EPCs) have highlighted their involvement in diabetes complications. To determine the effect of glucotoxicity on EPCs, human EPCs have been isolated from peripheral blood mononuclear cells of healthy donors and cultured in the presence or absence of high glucose (33 mmol/l) or high glucose plus benfotiamine to scavenge glucotoxicity. Morphological analysis revealed that high glucose significantly affected the number of endothelial cell colony forming units, uptake and binding of acLDL and Lectin-1, and the ability to differentiate into CD31- and vascular endothelial growth factor receptor 2-positive cells. Functional analysis outlined a reduced EPC involvement in de novo tube formation, when cocultured with mature endothelial cells (human umbilical vein endothelial cells) on matrigel. To explain the observed phenotypes, we have investigated the signal transduction pathways known to be involved in EPC growth and differentiation. Our results indicate that hyperglycemia impairs EPC differentiation and that the process can be restored by benfotiamine administration, via the modulation of Akt/FoxO1 activity.

Plasma interleukin-6 levels are independently associated with insulin secretion in a cohort of Italian-Caucasian nondiabetic subjects.

We have investigated the relationships between plasma interleukin-6 (IL-6) levels and insulin sensitivity and insulin secretion in a cohort of Italian-Caucasian glucose-tolerant subjects. Insulin sensitivity was assessed by euglycemic-hyperinsulinemic clamp, and first-phase insulin secretion was measured by intravenous glucose tolerance test. Fasting plasma IL-6 concentration was negatively correlated with the rate of insulin-stimulated glucose disposal (M) (P = 0.001). The correlation remained statistically significant, while attenuated, after adjusting for sex, age, and BMI (P < 0.03); after an additional adjustment for free fatty acids (FFAs), a further attenuation was observed, but statistical significance was maintained (P < 0.044). Fasting plasma IL-6 concentration was positively correlated with first-phase insulin secretion assessed as acute insulin response (AIR) (P = 0.001). The correlation remained significant after adjusting for sex, age, and BMI (P = 0.003). To estimate the independent contribution of plasma IL-6 levels to AIR, we carried out forward stepwise linear regression analysis in a model that included sex, age, BMI, waist-to-hip ratio, FFAs, and insulin-stimulated glucose disposal. Only insulin sensitivity and plasma IL-6 concentration were independently associated with AIR, accounting, respectively, for 19.0 and 5.2% of its variation. These data indicate that IL-6 is associated in a reciprocal manner with the two pathophysiological components of type 2 diabetes, i.e., insulin resistance and insulin secretion.

Variants of the interleukin-10 promoter gene are associated with obesity and insulin resistance but not type 2 diabetes in caucasian italian subjects.

Interleukin (IL)-10 is a major anti-inflammatory cytokine that has been associated with obesity and type 2 diabetes. The three polymorphisms -1082G/A, -819C/T, and -592C/A in the IL10 promoter were reported to influence IL10 transcription. We investigated whether these polymorphisms were associated with type 2 diabetes and related traits in a cohort of Italian Caucasians comprising 551 type 2 diabetic and 1,131 control subjects. The -819C/T and -592C/A polymorphisms were in perfect linkage disequilibrium (r(2) = 1.0). The -1082G/A polymorphism was not associated with type 2 diabetes or related traits. Although the -592C/A polymorphism was not associated with type 2 diabetes, nondiabetic homozygous carriers of the A allele showed increased BMI and insulin resistance and lower plasma IL-10 levels compared with the other genotypes. In the nondiabetic group, the ATA haplotype was associated with an increased risk for obesity (odds ratio 1.28 [95% CI 1.02-1.60]; P = 0.02). The ATA/ATA composite genotype was associated with an increased risk for obesity (1.96 [1.16-3.31]; P = 0.01) and insulin resistance (1.99 [1.12-3.53]; P = 0.01). This study suggests that polymorphisms and haplotypes of the IL10 promoter may be associated with obesity and insulin resistance in a large sample of Italian Caucasians.

Vascular, metabolic, and inflammatory abnormalities in normoglycemic offspring of patients with type 2 diabetes mellitus.

Endothelial dysfunction, insulin resistance, and elevated levels of circulating proinflammatory markers are among the earliest detectable abnormalities in people at risk for atherosclerosis. Accelerated atherosclerosis is a leading contributor to morbidity and mortality in type 2 diabetes mellitus, a complex genetic disorder. Therefore, we hypothesized that normoglycemic offspring of patients with type 2 diabetes mellitus (NOPD) may have impaired vascular and metabolic function related to an enhanced proinflammatory state. We compared NOPD (n = 51) with matched healthy control subjects without family history of diabetes (n = 35). Flow- and nitroglycerin-mediated brachial artery vasodilation were assessed by ultrasound to evaluate endothelium-dependent and -independent vascular function. Each subject also underwent an oral glucose tolerance test to evaluate metabolic function. Fasting levels of plasma adiponectin and circulating markers of inflammation (high-sensitivity C-reactive protein, CD40 ligand, interleukin 1beta, tumor necrosis factor alpha, vascular cell adhesion molecule 1, and intracellular adhesion molecule) were measured. Both NOPD and the control group had fasting glucose and insulin levels well within the reference range. However, results from oral glucose tolerance test and quantitative insulin sensitivity check index revealed that NOPD were insulin resistant with significantly impaired flow- and nitroglycerin-mediated dilation compared with the control group. Adiponectin levels were lower, whereas many circulating markers of inflammation were higher, in NOPD compared with the control group. Normoglycemic offspring of patients with type 2 diabetes mellitus have impaired vascular and metabolic function accompanied by an enhanced proinflammatory state that may contribute to their increased risk of diabetes and its vascular complications.

In vivo and in vitro studies support that a new splicing isoform of OLR1 gene is protective against acute myocardial infarction.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), encoded by the OLR1 gene, is a scavenger receptor that plays a fundamental role in the pathogenesis of atherosclerosis. LOX-1 activation is associated with apoptosis of endothelial cells, smooth muscle cells (SMCs), and macrophages. This process is an important underlying mechanism that contributes to plaque instability and subsequent development of acute coronary syndromes. Independent association genetic studies have implicated OLR1 gene variants in myocardial infarction (MI) susceptibility. Because single nucleotide polymorphisms (SNPs) linked to MI are located in intronic sequences of the gene, it remains unclear as to how they determine their biological effects. Using quantitative real-time PCR and minigene approach, we show that intronic SNPs, linked to MI, regulate the expression of a new functional splicing isoform of the OLR1 gene, LOXIN, which lacks exon 5. Macrophages from subjects carrying the "non-risk" disease haplotype at OLR1 gene have an increased expression of LOXIN at mRNA and protein level, which results in a significant reduction of apoptosis in response to oxLDL. Expression of LOXIN in different cell types results in loss of surface staining, indicating that truncation of the C-terminal portion of the protein has a profound effect on its cellular trafficking. Furthermore, the proapoptotic effect of LOX-1 receptor in cell culture is specifically rescued by the coexpression of LOXIN in a dose-dependent manner. The demonstration that increasing levels of LOXIN protect cells from LOX-1 induced apoptosis sets a groundwork for developing therapeutic approaches for prevention of plaque instability.

Phosphorylation of GATA2 by Akt increases adipose tissue differentiation and reduces adipose tissue-related inflammation: a novel pathway linking obesity to atherosclerosis.

BACKGROUND: Obesity-related inflammation is emerging as a major cause of insulin resistance and cardiovascular diseases. GATA2 transcription factor is an inhibitor of adipogenesis and an activator of vascular cells. We hypothesized that GATA2 activity is controlled by insulin during adipogenesis, linking metabolic homeostasis and inflammation. METHODS AND RESULTS: We show that insulin induces GATA2 phosphorylation on serine 401 in a PI-3K/Akt-dependent manner. Insulin-dependent phosphorylation of serine 401 impairs GATA2 translocation to the nucleus and its DNA binding activity. A GATA2 mutant not phosphorylable by Akt (GATA2(S401A)) acts similarly to wild-type GATA2. In contrast, a GATA2 mutant that mimics Akt phosphorylation (GATA2(S401D)) is restrained in the cytoplasm. Cultured preadipocytes bearing GATA2(S401A) do not convert to adipocytes and express high levels of inflammatory cytokines like monocyte chemotactic protein-1 (MCP-1). On the contrary, GATA2(S401D) preadipocytes differentiate to adipocytes. When GATA2(S401A) preadipocytes are injected in mice fed a high-fat diet, they do not differentiate adequately into adipocytes, maintaining the expression of inflammatory markers like MCP-1. In contrast, injection of GATA2(S401D) preadipocytes in mice fed a high-fat diet results in development of adipocytes and no expression of inflammatory markers. CONCLUSIONS: GATA2 could be a new target in the prevention and treatment of obesity-related inflammation and its complications.

Short-term treatment with atorvastatin reduces platelet CD40 ligand and thrombin generation in hypercholesterolemic patients.

BACKGROUND: Soluble CD40L (sCD40L), a substance that maximally reflects in vivo platelet activation, is increased in patients with hypercholesterolemia. We investigated the relation between sCD40L and platelet CD4OL in hypercholesterolemic patients before and after a short-term treatment with atorvastatin. METHODS AND RESULTS: Collagen-induced platelet CD40L and plasma levels of sCD40L and prothrombin fragment F1+2, a marker of thrombin generation, were investigated in 30 hypercholesterolemic patients and 20 healthy subjects. Hypercholesterolemic patients were then randomized to either diet (n=15; group A) or atorvastatin 10 mg/d (group B); the aforementioned variables were measured at baseline and after 3 days of treatment. Compared with referents, hypercholesterolemic patients showed higher values of platelet CD40L (P<0.005), sCD40L (P<0.005), and F1+2 (P<0.003). Platelet CD40L was significantly correlated with sCD40L (P<0.001), and the latter was significantly correlated with F1+2 (P<0.001). The intervention trial showed no changes in group A but a significant decrease in platelet CD40L (P<0.01), sCD40L (P<0.002), and F1+2 (P<0.03) in group B. In vitro studies demonstrated that cholesterol enhanced platelet CD40L and CD40L-mediated clotting activation by human monocytes; also, atorvastatin dose-dependently inhibited platelet CD40L expression and clotting activation by CD40L-stimulated monocytes. CONCLUSIONS: This study shows that, in hypercholesterolemia, platelet overexpression of CD40L may account for enhanced plasma levels of sCD40L and F1+2. Atorvastatin exerts a direct antithrombotic effect via inhibition of platelet CD40L and CD40L-mediated thrombin generation, independently of its cholesterol-lowering effect.