Marina Cardellini

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.

G972R IRS-1 Variant Impairs Insulin Regulation of Endothelial Nitric Oxide Synthase in Cultured Human Endothelial Cells

Background—Impaired insulin-mediated vasodilation might contribute to vascular damage in insulin-resistant states. Little is known about insulin regulation of nitric oxide (NO) synthesis in insulin-resistant cells. The aim of this work was to investigate insulin regulation of NO synthesis in human umbilical vein endothelial cells (HUVECs) carrying the IRS-1 gene G972R variant, known to be associated with impaired insulin activation of the PI3-kinase (PI3-K) pathway in transfected cells. Methods and Results—HUVECs were screened for the presence of the G972R-IRS-1 (HUVEC-G972R) variant by restriction fragment length polymorphisms. After 24-hour exposure to 10−7 mol/L insulin, endothelial NO synthase (eNOS) mRNA (reverse transcription–polymerase chain reaction), eNOS protein levels (Western blotting), and NOS activity (conversion of [3H]arginine into [3H]citrulline) were increased in wild-type HUVECs (HUVEC-WT), whereas they did not change from baseline in HUVEC-G972R. Compared with HUVEC-WT, in HUVEC-G972R after 2 and 10 minutes of insulin stimulation, IRS-1–associated PI3-K activity was reduced by 47% and 32%, respectively; Akt phosphorylation was decreased by 40% at both time points; and eNOS-Ser1177 phosphorylation was reduced by 38% and 51%, respectively. In HUVEC-WT, eNOS-Thr495 phosphorylation decreased after insulin stimulation. In contrast, in HUVEC-G972R, eNOS-Thr495 phosphorylation increased after insulin stimulation and was 40% greater than in HUVEC-WT. Conclusions—Our data demonstrate that genetic impairment of the (IRS)-1/PI3-K/PDK-1/Akt insulin signaling cascade determines impaired insulin-stimulated NO release and suggest that the G972R-IRS-1 polymorphism, through a direct impairment of Akt/eNOS activation in endothelial cells, may contribute to the genetic predisposition to develop endothelial dysfunction and cardiovascular disease.

TIMP3 Is Reduced in Atherosclerotic Plaques From Subjects With Type 2 Diabetes and Increased by SirT1

OBJECTIVE Atherosclerosis is accelerated in subjects with type 2 diabetes by unknown mechanisms. We identified tissue inhibitor of metalloproteinase 3 (TIMP3), the endogenous inhibitor of A disintegrin and metalloprotease domain 17 (ADAM17) and other matrix metalloproteinases (MMPs), as a gene modifier for insulin resistance and vascular inflammation in mice. We tested its association with atherosclerosis in subjects with type 2 diabetes and identified Sirtuin 1 (SirT1) as a major regulator of TIMP3 expression. RESEARCH DESIGN AND METHODS We investigated ADAM10, ADAM17, MMP9, TIMP1, TIMP2, TIMP3, and TIMP4 expression levels in human carotid atherosclerotic plaques (n = 60) from subjects with and without diabetes. Human vascular smooth muscle cells exposed to several metabolic stimuli were used to identify regulators of TIMP3 expression. SirT1 small interference RNA, cDNA, and TIMP3 promoter gene reporter were used to study SirT1-dependent regulation of TIMP3. RESULTS Here, we show that in human carotid atherosclerotic plaques, TIMP3 was significantly reduced in subjects with type 2 diabetes, leading to ADAM17 and MMP9 overactivity. Reduced expression of TIMP3 was associated in vivo with SirT1 levels. In smooth muscle cells, inhibition of SirT1 activity and levels reduced TIMP3 expression, whereas SirT1 overexpression increased TIMP3 promoter activity. CONCLUSIONS In atherosclerotic plaques from subjects with type 2 diabetes, the deregulation of ADAM17 and MMP9 activities is related to inadequate expression of TIMP3 via SirT1. Studies in vascular cells confirmed the role of SirT1 in tuning TIMP3 expression.

Heterogeneous Effect of Peroxisome Proliferator-activated Receptor γ2 Ala12 Variant on Type 2 Diabetes Risk

Conflicting results have been reported regarding whether the PPARγ2 Pro12Ala polymorphism plays a role in the risk of type 2 diabetes (T2D), suggesting genetic heterogeneity. To investigate this issue, a meta‐analysis of 41 published and 2 unpublished studies (a total of 42,910 subjects) was conducted. Ala12 carriers had a 19% T2D risk reduction, but this association was highly heterogeneous (p = 0.005). A great proportion (48%) of heterogeneity was explained by the controls’ BMI, with risk reduction being greater when BMI was lower. Risk reduction of Ala12 carriers in Asia (35%) was higher than in Europe (15%, p = 0.02) and tended to be higher than in North America (18%, p = 0.10). Difference between Asians and Europeans was no longer significant (p = 0.15) after adjusting for the controls’ BMI. Studies from Europe were still heterogeneous (p = 0.02) with risk reduction in Ala12 carriers being progressively smaller (test for trend in the odds ratios, p = 0.02) from Northern (26% reduction, p < 0.0001) to Central (10%, p = 0.04) and Southern (0%, p = 0.94) Europe. In conclusion, in our meta‐analysis, the reduced risk of T2D in Ala12 carriers is not homogeneous. It is greater in Asia than in Europe and, among Europeans, it is higher in Northern Europe, barely significant in Central Europe, and nonexistent in Southern Europe.

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 (GATA2S401A) acts similarly to wild-type GATA2. In contrast, a GATA2 mutant that mimics Akt phosphorylation (GATA2S401D) is restrained in the cytoplasm. Cultured preadipocytes bearing GATA2S401A do not convert to adipocytes and express high levels of inflammatory cytokines like monocyte chemotactic protein-1 (MCP-1). On the contrary, GATA2S401D preadipocytes differentiate to adipocytes. When GATA2S401A 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 GATA2S401D 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.

Increased O-glycosylation of insulin signaling proteins results in their impaired activation and enhanced susceptibility to apoptosis in pancreatic beta-cells

Because adverse effects of glucose were attributed to its increased routing through the hexosamine pathway (HBP), we inquired whether HBP activation affects pancreatic β‐cell survival. Exposure of human islets to high glucose resulted in increased apoptosis of β‐cells upon serum deprivation that was reversed by azaserine. Also, glucosamine, a direct precursor of the downstream product of the HBP, increased human β‐cells apoptosis upon serum deprivation, which was reversed by benzyl‐2‐acetamido‐2‐deoxy‐α‐D‐galactopyranoside (BADGP), an inhibitor of protein O‐glycosylation. These results were reproduced in RIN rat β‐cells. Glucosamine treatment resulted in inhibition of tyrosine‐phosphorylation of the insulin receptor (IR), IRS‐1, and IRS‐2, which was associated with increased O‐glycosylation. These changes caused impaired activation of the PI 3‐kinase/Akt survival signaling that resulted in reduced GSK‐3 and FOXO1a inactivation. BADGP reversed the glucosamine‐induced reduction in insulin‐stimulated phosphorylation of IR, IRS‐1, IRS‐2, Akt, GSK‐3, and FOXO1a. Impaired FOXO1a inactivation sustained expression of the pro‐apoptotic protein Bim, without affecting Bad, Bcl‐XL, or Bcl‐2 expression. These results indicate that hyperglycemia may increase susceptibility to apoptosis of human and rat β‐cell through activation of the HBP. Increased routing of glucose through this metabolic pathway results in impaired activation of the IR/IRSs/PI3‐kinase/Akt survival pathway by induction of O‐glycosylation of signaling molecules.

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 (r2 = 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.

MiR-216a: a link between endothelial dysfunction and autophagy

Endothelial dysfunction and impaired autophagic activity have a crucial role in aging-related diseases such as cardiovascular dysfunction and atherosclerosis. We have identified miR-216a as a microRNA that is induced during endothelial aging and, according to the computational analysis, among its targets includes two autophagy-related genes, Beclin1 (BECN1) and ATG5. Therefore, we have evaluated the role of miR-216a as a molecular component involved in the loss of autophagic function during endothelial aging. The inverse correlation between miR-216a and autophagic genes was conserved during human umbilical vein endothelial cells (HUVECs) aging and in vivo models of human atherosclerosis and heart failure. Luciferase experiments indicated BECN1, but not ATG5 as a direct target of miR-216a. HUVECs were transfected in order to modulate miR-216a expression and stimulated with 100 μg/ml oxidized low-density lipoprotein (ox-LDL) to induce a stress repairing autophagic process. We found that in young HUVECs, miR-216a overexpression repressed BECN1 and ATG5 expression and the ox-LDL induced autophagy, as evaluated by microtubule-associated protein 1 light chain 3 (LC3B) analysis and cytofluorimetric assay. Moreover, miR-216a stimulated ox-LDL accumulation and monocyte adhesion in HUVECs. Conversely, inhibition of miR-216a in old HUVECs rescued the ability to induce a protective autophagy in response to ox-LDL stimulus. In conclusion, mir-216a controls ox-LDL induced autophagy in HUVECs by regulating intracellular levels of BECN1 and may have a relevant role in the pathogenesis of cardiovascular disorders and atherosclerosis.

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.

Plasma interleukin-6 levels are increased in subjects with impaired glucose tolerance but not in those with impaired fasting glucose in a cohort of Italian Caucasians.

While the relationship between impaired glucose tolerance (IGT) and circulating interleukin‐6 (IL‐6) is well established, there is no information whether IL‐6 levels are elevated in impaired fasting glucose (IFG).