Accuracy of Pancreatic Stone Protein for diagnosis of sepsis in children admitted to pediatric intensive care or high-dependency care: a pilot study.

BACKGROUND: Pancreatic Stone Protein (PSP) is one of the most promising diagnostic and prognostic markers. The aim of the study was to assess the accuracy of PSP, compared to C-Reactive Protein (CRP), and Procalcitonin (PCT) for sepsis diagnosis in pediatric patients. Furthermore, we explored the correlation of PSP levels with sepsis severity and organ failure measured with PELOD-2 score. METHODS: Forty pediatric patients were enrolled following admission to pediatric intensive care, high dependency care or pediatric ward. PSP blood levels were measured in Emergency Department (nanofluidic point-of-care immunoassay; abioSCOPE, Abionic SA, Switzerland) on day 1, 2, 3, 5 and 7 from the onset of the clinical signs and symptoms of sepsis or SIRS. Inclusion criteria were: 1) patient age (1 month to 18 years old), 2) signs and symptoms of SIRS, irrespective of association with organ dysfunction. Exclusion criteria were: 1) hemato-oncological diseases and/or immunodeficiencies, 2) pancreatic diseases. RESULTS: Septic patients showed higher PSP levels than those with non-infectious systemic inflammation. The optimal cut-off in diagnosis of sepsis for PSP at day 1 was 167 ng/ml resulted in a sensitivity of 59% (95% IC 36%-79%) and a specificity of 83% (95% IC 58%-96%) with an AUC of 0.636 for PSP in comparison to AUC of 0.722 for PCT and 0.503 for C-RP. ROC analysis for outcome (survival versus no survival) has showed AUC 0.814 for PSP; AUC 0.814 for PCT; AUC of 0.657 for C-RP. CONCLUSIONS: PSP could distinguish sepsis from non-infectious systemic inflammation; however, our results need to be confirmed in larger pediatric population.

Contribution of vitamin D3 and thiols status to the outcome of COVID-19 disease in Italian pediatric and adult patients.

The coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARSCoV-2), was declared a global pandemic by the World Health Organization (WHO) on March 2020, causing unprecedented disease with million deaths across the globe, mostly adults. Indeed, children accounted for only a few percent of cases. Italy was the first Western country struck by the COVID-19 epidemic. Increasing age, which is one of the principal risk factors for COVID-19 mortality, is associated with declined glutathione (GSH) levels. Over the last decade, several studies demonstrated that both vitamin D (VD) and GSH have immunomodulatory properties. To verify the association between VD, GSH and the outcome of COVID-19 disease, we conducted a multicenter retrospective study in 35 children and 128 adult patients with COVID-19. Our study demonstrated a hypovitaminosis D in COVID-19 patients, suggesting a possible role of low VD status in increasing the risk of COVID-19 infection and subsequent hospitalization. In addition, we find a thiol disturbance with a GSH depletion associated to the disease severity. In children, who fortunately survived, both VD and GSH levels at admission were higher than in adults, suggesting that lower VD and thiols levels upon admission may be a modifiable risk factor for adverse outcomes and mortality in hospitalized patients with COVID-19.

Study of the Association between Thiols and Oxidative Stress Markers in Children with Obesity.

Obesity has reached epidemic proportions, and the World Health Organization defined childhood overweight and obesity as a noncommunicable disease that represents the most serious public health challenges of the twenty-first century. Oxidative stress, defined as an imbalance between oxidants and antioxidants causing an impairment of the redox signals, is linked to the development of metabolic diseases. In addition, reactive oxygen species generated during metabolic disorder could increase inflammation, causing the development of insulin resistance, diabetes, and cardiovascular disease. We analyze serum levels of cysteine (Cys), cysteinyl-glycine (Cys-Gly), homocysteine (Hcy), and glutathione (GSH), and other markers of oxidative stress, such as thiobarbituric acid reactive substances (T-BARS), 8-isoprostane, and protein carbonyl in our children with obesity. Total antioxidant status was also determined. We found lower GSH and Cys-Gly levels, and higher Hcy and oxidative stress markers levels. We also found a positive correlation between Body Mass Index (BMI), Cys, GSH, and Hcy levels, between insulin and Cys levels, and between BMI and the homeostasis model assessment-estimated insulin resistance (HOMA-IR) with 8-isoprostane levels. Finally, we found a correlation between age and GSH and Cys levels. The deficiency of GSH could be restored by dietary supplementation with GSH precursors, supplying an inexpensive approach to oppose oxidative stress, thus avoiding obesity complications.

Soluble Isoform of Suppression of Tumorigenicity 2 (ST2) Biomarker in a Large Cohort of Healthy Pediatric Population: Determination of Reference Intervals.

Introduction: Only little data exists on ST2 reference intervals in healthy pediatric populations despite the high importance of this biomarker in adults with heart failure. The aim of the study was to assess the reference intervals of ST2 in a wide healthy pediatric cohort. Methods: We evaluated the serum concentrations of ST2 biomarker in 415 healthy pediatric subjects referred to our analysis laboratory. Subjects were categorized according to age (i.e., 0−6 (n = 79), 7−11 (n = 142) and 12−18 years (n = 191)) and sex. They were not suffering from any cardiac disorders, metabolic disorders, lung diseases, autoimmune disorders or malignancies. A written consent was obtained for each individual. No duplicate patients were included in the analysis and the presence of outliers was investigated. Reference intervals (Mean and central 95% confidence intervals) were determined. Results: Three outliers have been identified and removed from the analysis (60.0, 64.0 and 150.2 ng/mL). A total of 412 subjects were therefore included. The mean value for the whole population was 15.8 ng/mL (2.4−36.4 ng/mL). Males present a significantly higher mean concentration compared to females (17.2 versus 14.4 ng/mL, p = 0.001). A significant trend toward higher ST2 values with age was also observed, but for males only (r = 0.43, p < 0.0001). If considering age partitions, only males of 12−18 years (mean = 21.7 ng/mL) had significantly higher ST2 values compared to the other groups (ranging from 11.9 for males 0−6 years to 15.2 for females 12−18 years; p < 0.0001). Conclusions: We described age and sex-specific reference intervals for ST2 in a large healthy pediatric population. We found that ST2 values differ between sexes if considering all participants. A significant increase in ST2 with age was also observed, but only for males of 12−18 years.

ITCH E3 ubiquitin ligase downregulation compromises hepatic degradation of branched-chain amino acids.

OBJECTIVE: Metabolic syndrome, obesity, and steatosis are characterized by a range of dysregulations including defects in ubiquitin ligase tagging proteins for degradation. The identification of novel hepatic genes associated with fatty liver disease and metabolic dysregulation may be relevant to unravelling new mechanisms involved in liver disease progression METHODS: Through integrative analysis of liver transcriptomic and metabolomic obtained from obese subjects with steatosis, we identified itchy E ubiquitin protein ligase (ITCH) as a gene downregulated in human hepatic tissue in relation to steatosis grade. Wild-type or ITCH knockout mouse models of non-alcoholic fatty liver disease (NAFLD) and obesity-related hepatocellular carcinoma were analyzed to dissect the causal role of ITCH in steatosis RESULTS: We show that ITCH regulation of branched-chain amino acids (BCAAs) degradation enzymes is impaired in obese women with grade 3 compared with grade 0 steatosis, and that ITCH acts as a gatekeeper whose loss results in elevation of circulating BCAAs associated with hepatic steatosis. When ITCH expression was specifically restored in the liver of ITCH knockout mice, ACADSB mRNA and protein are restored, and BCAA levels are normalized both in liver and plasma CONCLUSIONS: Our data support a novel functional role for ITCH in the hepatic regulation of BCAA metabolism and suggest that targeting ITCH in a liver-specific manner might help delay the progression of metabolic hepatic diseases and insulin resistance.

Cerebrospinal Fluid Levels of AFP and hCG: Validation of the Analytical Method and Application in the Diagnosis of Central Nervous System Germ Cell Tumors.

The determination of Human Chorionic Gonadotropin (hCG) and Alpha Fetoprotein (AFP) levels on serum and amniotic fluid plays a fundamental role in the diagnosis and follow-up of specific physiological or pathological conditions (e.g., pregnancy, threat of abortion or germ cell tumors). Recently, the quantification of hCG and AFP in other biological fluids has gained great attention to support the diagnosis, prognosis and follow-up of neoplastic diseases deriving from trophoblastic cells, such as germinomas. Most of the commercial kits for hCG and AFP assays are developed to be used on biological fluids such as serum/plasma and/or urine by manufacturing companies. The aim of this work was to evaluate the suitability of the analytical method certified for the use on serum, and/or amniotic fluid for the quantification of hCG and AFP in cerebrospinal fluid, carrying out an internal validation protocol. The data reported here show that the automated immunochemical method is fit for quantification of hCG and AFP in cerebrospinal fluid (CSF), allowing selective and specific diagnosis of secreting germ cell tumors. This is confirmed by the positive correlation between elevated levels of hCG or AFP and the diagnosis of brain tumors.

Dysregulated miRNAs in bone cells of patients with Gorham-Stout disease.

Gorham-Stout disease (GSD) is a very rare disease characterized by increased bone erosion with angiomatous proliferation. The mechanisms underlying this disorder have not been deeply investigated. Due to its rarity, no guidelines are currently available for treatment and management of GSD. We recently evaluated the cellular alterations of the bone remodeling in patients showing that osteoclast precursors displayed increased ability to differentiate into osteoclasts and that affected osteoclasts resorb bone more actively than control cells. Moreover, osteoblasts isolated from a patient showed a defective ability to form mineralized nodules. In this paper, we investigated the molecular pathways involved in the cellular defects of GSD bone cells. For this study, we recruited nine patients and performed miRNome analysis of bone cells. Between the 178 miRNAs robustly expressed in GSD osteoclasts, significant modulation of three miRNAs (miR-1246, miR-1-3p, and miR-137-3p) involved in the regulation of osteoclast formation and activity or in the angiomatous proliferation was found in patients' cells. Interestingly, miR-1246 was also up-regulated in serum exosomes from patients. Analysis of miRNAs from patient osteoblasts suggested alteration of miR-204a-5p, miR-615-3p and miR-378a-3p regulating osteoblast function and differentiation. The resulting miRNA pattern may help to understand better the mechanisms involved in GSD and to identify new potential therapeutic targets for this rare disease.

Unusual Presentation of Denys-Drash Syndrome in a Girl with Undisclosed Consumption of Biotin

We describe a 46,XX girl with Denys-Drash syndrome, showing both kidney disease and genital abnormalities, in whom a misdiagnosis of hyperandrogenism was made. A 15 year-old girl was affected by neonatal nephrotic syndrome, progressing to end stage kidney failure. Hair loss and voice deepening were noted during puberty. Pelvic ultrasound and magnetic resonance imaging showed utero-tubaric agenesis, vaginal atresia and urogenital sinus, with inguinal gonads. Gonadotrophin and estradiol levels were normal, but testosterone was increased up to 285 ng/dL at Tanner stage 3. She underwent prophylactic gonadectomy. Histopathology reported fibrotic ovarian cortex containing numerous follicles in different maturation stages and rudimental remnants of Fallopian tubes. No features of gonadoblastoma were detected. Unexpectedly, testosterone levels were elevated four months after gonadectomy (157 ng/dL). Recent medical history revealed chronic daily comsumption of high dose biotin, as a therapeutic support for hair loss. Laboratory immunoassay instruments used streptavidin-biotin interaction to detect hormones and, in competitive immunoassays, high concentrations of biotin can result in false high results. Total testosterone, measured using liquid chromatography tandem mass spectrometry, was within reference intervals. Similar testosterone levels were detected on repeat immunoassay two weeks after biotin uptake interruption. Discordance between clinical presentation and biochemical results in patients taking biotin, should raise the suspicion of erroneous results. Improved communication among patients, health care providers, and laboratory professionals is required concerning the likelihood of biotin interference with immunoassays.

Intrinsic Bone Defects in Cystinotic Mice.

Cystinosis is a rare lysosomal storage disorder caused by loss-of-function mutations of the CTNS gene, encoding cystinosin, a symporter that mediates cystine efflux from lysosomes. Approximately 95% of patients with cystinosis display renal Fanconi syndrome, short stature, osteopenia, and rickets. In this study, we investigated whether the absence of cystinosin primarily affects bone remodeling activity, apart from the influences of the Fanconi syndrome on bone mineral metabolism. Using micro-computed tomography and histomorphometric and bone serum biomarker analysis, we evaluated the bone phenotype of 1-month-old Ctns-/- knockout (KO) male mice without tubulopathy. An in vitro study was performed to characterize the effects of cystinosin deficiency on osteoblasts and osteoclasts. Micro-computed tomography analysis showed a reduction of trabecular bone volume, bone mineral density, and number and thickness in KO mice compared with wild-type animals; histomorphometric analysis revealed a reduction of osteoblast and osteoclast parameters in tibiae of cystinotic mice. Decreased levels of serum procollagen type 1 amino-terminal propeptide and tartrate-resistant acid phosphatase in KO mice confirmed reduced bone remodeling activity. In vitro experiments showed an impairment of Ctns-/- osteoblasts and osteoclasts. In conclusion, cystinosin deficiency primarily affects bone cells, leading to a bone loss phenotype of KO mice, independent from renal failure.

Soluble ST2 is a biomarker for cardiovascular mortality related to abnormal glucose metabolism in high-risk subjects.

AIMS: Inflammation plays a role in the development and progression of type 2 diabetes macroangiopathy. Interleukin 33 (IL-33) drives production of Th2-associated cytokines. The soluble form of suppression of tumorigenicity 2 (sST2) acting as a decoy receptor blocks IL-33 and tones down Th2 inflammatory response. We investigated the role of sST2 as a predictor of CV and all-cause mortality in a cohort of patients affected by established atherosclerotic disease. METHODS: 399 patients with atherosclerotic disease from the Tor Vergata Atherosclerosis Registry performed follow-up every year by phone interview. The primary endpoint was cardiovascular death and the secondary endpoint was death for any other disease. RESULTS: sST2 plasma levels were significantly increased from normal glucose-tolerant patients to patients with history of type 2 diabetes (p < 0.00001). Levels of sST2 were significantly correlated with fasting plasma glucose (R = 0.16, p = 0.002), HbA1c (R = 0.17, p = 0.002), and HOMA (R = 0.16, p = 0.004). Dividing patients in tertiles of sST2 levels, those belonging to the highest tertile showed an increased rate of all-cause and cardiovascular mortality, (all-cause mortality p = 0.045 and CVD mortality p = 0.02). A multivariate Cox analysis revealed that sST2 increased the risk in cardiovascular mortality per SD by hazard ratio 1.050 (95% CI 1.006-1.097, p = 0.025) after adjustment for age and hs-CRP while it did not significantly change the risk for all-cause mortality. CONCLUSIONS: High circulating level of sST2 is associated to increased CVD mortality and markers of metabolic dysfunction in subjects with atherosclerotic disease.

Calibrated automated thrombogram values in infants with cardiac surgery before and after cardiopulmonary bypass.

INTRODUCTION: Impaired thrombin generation has been associated to increase bleeding after cardiac surgery with cardiopulmonary bypass (CPB), especially in children. The aim of this study was to evaluate standard coagulation assay, thrombin generation by calibrated automated thrombogram (CAT), thromboelastography (TEG) and procoagulant phospholipids (PPL) activity in infants undergoing cardiac surgery with CPB. MATERIALS AND METHODS: Prospective observational study performed in children aged <24months undergoing cardiac surgery with CPB. Exclusion criteria were preoperative coagulopathy or anticoagulant therapy. Coagulation was evaluated by standard coagulation assays (prothrombin time, activated partial thromboplastin time, fibrinogen level, platelet count), TEG, CAT and PPL at anaesthesia induction (T1) and after 12h (T2). Perioperative bleeding management was performed according to the institutional guidelines. RESULTS: Forty-nine children aged <24months were enrolled. At T1 ETP and peak height evaluated by CAT were significantly lower in infants aged <6months. Standard coagulation tests, TEG and PPL did not correlate with age. At T2 platelet count, plasmatic fibrinogen level, all TEG parameters, ETP and peak height by CAT were significantly impaired compared to baseline values (T1), despite allogeneic blood product transfusions. CONCLUSIONS: Thrombin generation is significantly impaired in children affected by congenital heart disease, compared to healthy children and adults. CAT parameters resulted age-dependent, and thrombin generation is lower in infants aged <6months. After cardiac surgery with CPB, a coaugulopathy, revealed by CAT, TEG, but not by PT and aPTT assays, is persistent 12h after surgery despite transfusions of blood products.

A score including ADAM17 substrates correlates to recurring cardiovascular event in subjects with atherosclerosis.

OBJECTIVE: Atherosclerosis disease is a leading cause for mortality and morbidity. The narrowing/rupture of a vulnerable atherosclerotic plaque is accountable for acute cardiovascular events. However, despite of an intensive research, a reliable clinical method which may disclose a vulnerable patient is still unavailable. APPROACH AND RESULTS: We tested the association of ADAM17 (A Disintegrin and Metallo Protease Domain 17) circulating substrates (sICAM-1, sVCAM-1, sIL6R and sTNFR1) with a second major cardiovascular events [MACEs] (cardiovascular death, peripheral artery surgeries, non-fatal myocardial infarction and non-fatal stroke) in 298 patients belonging to the Vascular Diabetes (AVD) study. To evaluate ADAM17 activity we create ADAM17 score through a RECPAM model. Finally we tested the discrimination ability and the reclassification of clinical models. At follow-up (mean 47 months, range 1-118 months), 55 MACEs occurred (14 nonfatal MI, 14 nonfatal strokes, 17 peripheral artery procedures and 10 cardiovascular deaths) (incidence = 7.8% person-years). An increased risk for incident events was observed among the high ADAM17 score individuals both in univariable (HR 19.20, 95% CI 15.82-63.36, p < 0.001) and multivariable analysis (HR 3.42, 95% CI 1.55-7.54, p < 0.001). Finally we found that ADAM17 score significantly increases the prediction accuracy of the Framingham Recurring-Coronary-Heart-Disease-Score, with a significant improvement in discrimination (integrated discrimination improvement = 9%, p = 0.012) and correctly reclassifying 10% of events and 41% of non-events resulting in a cNRI = 0.51 (p = 0.005). CONCLUSION: We demonstrated a positive role of ADAM17 activity to predicting CV events. We think that an approach that targets strategies beyond classic cardiovascular risk factors control is necessary in individuals with an established vascular atherosclerosis.

Recognition of zinc transporter 8 and MAP3865c homologous epitopes by new-onset type 1 diabetes children from continental Italy.

There are several pieces of evidence indicating that Mycobacterium avium subspecies paratuberculosis (MAP) infection is linked to type 1 diabetes (T1D) in Sardinian patients. An association between MAP and T1D was recently observed in an Italian cohort of pediatric T1D individuals, characterized by a different genetic background. It is interesting to confirm the prevalence of anti-MAP antibodies (Abs) in another pediatric population from continental Italy, looking at several markers of MAP presence. New-onset T1D children, compared to age-matched healthy controls (HCs), were tested by indirect enzyme-linked immunosorbent assay for the presence of Abs toward the immunodominant MAP3865c/ZnT8 homologues epitopes, the recently identified C-terminal MAP3865c281-287 epitope and MAP-specific protein MptD. Abs against MAP and ZnT8 epitopes were more prevalent in the sera of new-onset T1D children compared to HCs. These findings support the view that MAP3865c/ZnT8 cross-reactivity is involved in the pathogenesis of T1D, and addition of Abs against these peptides to the panel of existing T1D biomarkers should be considered. It is important now to investigate the timing of MAP infection during prospective follow-up in at-risk children to elucidate whether Ab-titers against these MAP/ZnT8 epitopes are present before T1D onset and if so if they wane after diagnosis.

Atherosclerosis severity but not undiagnosed diabetes predicts new cardiovascular events of subjects in secondary cardiovascular prevention.

OBJECTIVE: Undiagnosed diabetes (DM2), especially in individuals that have experienced a major atherosclerotic vascular event, could increase the risk of a second major cardiovascular (CV) event. The aim of this study was to evaluate the impact of type 2 diabetes (DM2), diagnosed after a major cardiovascular event, on subsequent CV disease in high risk individuals. METHODS: 411 subjects without known DM2 and with a history of a prior major CV event were followed for a second major CV event (fatal and nonfatal MI, fatal and nonfatal stroke or any arterial revascularization procedure). At baseline, each individual underwent a physical, biochemical examination, an OGTT and dosed A1c. In addition, patients were classified as having monovascular or polyvascular disease. The average follow-up duration was 31 months. RESULTS: The incidence of second CV events was 10.70 per 100 person-years (114 events/1066 person-years). The diagnosis of occult DM2 was not associated with major CV events, either using A1c values ≥6.5%, fasting glucose ≥126 mg/dL or 2 h post-load glucose ≥200 mg/dL. Polyvascular disease was the only significant predictor of a second major CV event (HR 2.60, 95% CI 1.72-3.95) after adjustment for age, BMI, smoking status, systolic blood pressure, high-density and low-density lipoprotein cholesterol and high sensitivity C-reactive protein. CONCLUSION: DM2 that was newly diagnosed after established vascular atherosclerotic disease did not increase the risk of new major CV events. In our population only the polyvascular disease was able to identify the subjects at high risk for a second major cardiovascular event.

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.

Generalized impairment of vasodilator reactivity during hyperinsulinemia in patients with obesity-related metabolic syndrome.

Defective insulin-dependent vasodilation might contribute importantly to metabolic and vascular abnormalities of the metabolic syndrome (MetS). However, despite extensive investigation, the precise mechanisms involved in insulin's vasoactive effects have not been fully elucidated. Therefore, this study sought to better characterize insulin's physiological actions on vascular reactivity and their potential derangement in the MetS. Forearm blood flow responses to graded doses of acetylcholine, sodium nitroprusside, and verapamil were assessed by strain-gauge plethysmography in patients with obesity-related MetS (n = 20) and in matched controls (n = 18) before and after intra-arterial infusion of insulin (0.2 mU·kg(-1)·min(-1)). Possible involvement of increased oxidative stress in the impaired insulin-stimulated vasodilator responsiveness of patients with MetS (n = 12) was also investigated using vitamin C (25 mg/min). In control subjects, significant potentiation of the vasodilator responses to acetylcholine, nitroprusside, and verapamil was observed after insulin infusion (all P < 0.05). However, no significant change in vasodilator reactivity to either of these drugs was observed following hyperinsulinemia in patients with MetS (all P > 0.05). Interestingly, administration of vitamin C to patients with MetS during hyperinsulinemia significantly enhanced the vasodilator responsiveness to acetylcholine, nitroprusside, and verapamil (all P < 0.05 vs. hyperinsulinemia alone). In conclusion, insulin exerts a generalized facilitatory action on vasodilator reactivity, and this effect is impaired in patients with MetS likely because of increased oxidative stress. Given the importance of vasodilator reactivity in affecting glucose disposal and vascular homeostasis, this defect may then contribute to the development of metabolic and vascular complications in insulin-resistant states.

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.

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.

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.