Plasma proteins associate with carotid plaques and predict incident atherosclerotic cardiovascular events.

PURPOSE: Performing non-invasive carotid imaging is challenging, owing inter-operator variability and organizational barriers, but plasma proteomics can offer an alternative. We sought plasma proteins that associate with the presence of carotid plaques, their number and predict the incidence of clinically overt atherosclerotic cardiovascular events (ASCVD) above currently recognized risk factors in "apparently healthy" subjects. METHODS: We studied the plasma levels of 368 proteins in 664 subjects from the PLIC study, who underwent an ultrasound imaging screening of the carotids to check for the presence of plaques. We clustered, by artificial intelligence (A.I.), the proteins that associate with the presence, the number of plaques and that predict incident ASCVDs over 22 years (198 events were registered). FINDINGS: 299/664 subjects had at least 1 carotid plaque (1+) (77 with only one plaque, 101 with 2 plaques, 121 with ≥3 plaques (3+)). The remaining 365 subjects with no plaques acted as controls. 106 proteins were associated with 1+ plaques, but 97 proteins significantly predicted 3+ plaques only (AUC = 0.683 (0.601-0.785), p < 0.001), when considered alone. A.I. underscored 87 proteins that improved the performance of the classical risk factors both in detecting 3+ plaques (AUC = 0.918 (0.887-0.943) versus risk factors alone, AUC = 0.760 (0.716-0.801), p < 0.001) and in predicting the incident ASCVD (AUC = 0.739 (0.704-0.773) vs risk factors alone AUC = 0.559 (0.521-0.598), p < 0.001). The chemotaxis/migration of leukocytes and interleukins/cytokines signaling were biological pathways mostly represented by these proteins. DISCUSSION AND CONCLUSIONS: Plasma proteomics marks the number of carotid plaques and improve the prediction of incidence ASCVDs in apparently healthy subjects.

The Molecular Detection of Germline Mutations in the BRCA1 and BRCA2 Genes Associated with Breast and Ovarian Cancer in a Romanian Cohort of 616 Patients.

The objective of this study was to identify and classify the spectrum of mutations found in the BRCA1 and BRCA2 genes associated with breast and ovarian cancer in female patients in Romania. Germline BRCA1 and BRCA2 mutations were investigated in a cohort of 616 female patients using NGS and/or MLPA methods followed by software-based data analysis and classification according to international guidelines. Out of the 616 female patients included in this study, we found that 482 patients (78.2%) did not have any mutation present in the two genes investigated; 69 patients (11.2%) had a BRCA1 mutation, 34 (5.5%) had a BRCA2 mutation, and 31 (5%) presented different type of mutations with uncertain clinical significance, moderate risk or a large mutation in the BRCA1 gene. Our investigation indicates the most common mutations in the BRCA1 and BRCA2 genes, associated with breast and ovarian cancer in the Romanian population. Our results also bring more data in support of the frequency of the c.5266 mutation in the BRCA1 gene, acknowledged in the literature as a founder mutation in Eastern Europe. We consider that the results of our study will provide necessary data regarding BRCA1 and BRCA2 mutations that would help to create a genetic database for the Romanian population.

High dietary inflammatory index associates with inflammatory proteins in plasma.

BACKGROUND AND AIM: Unhealthy dietary habits and highly caloric foods induce metabolic alterations and promote the development of the inflammatory consequences of obesity, insulin resistance, diabetes and cardiovascular diseases. Describing an inflammatory effect of diet is difficult to pursue, owing lacks of standardized quali-quantitative dietary assessments. The Dietary Inflammatory Index (DII) has been proposed as an estimator of the pro- or anti-inflammatory effect of nutrients and higher DII values, which indicate an increased intake of nutrients with pro-inflammatory effects, relate to an increased risk of metabolic and cardiovascular diseases and we here assessed whether they reflect biologically relevant plasmatic variations of inflammatory proteins. METHODS: In this cross-sectional study, seven days dietary records from 663 subjects in primary prevention for cardiovascular diseases were analyzed to derive the intake of nutrients, foods and to calculate DII. To associate DII with the Normalized Protein eXpression (NPX), an index of abundance, of a targeted panel of 368 inflammatory biomarkers (Olink™) measured in the plasma, we divided the population by the median value of DII (1.60 (0.83-2.30)). RESULTS: 332 subjects with estimated DII over the median value reported a higher intake of saturated fats but lower intakes of poly-unsaturated fats, including omega-3 and omega-6 fats, versus subjects with estimated dietary DII below the median value (N = 331). The NPX of 61 proteins was increased in the plasma of subjects with DII > median vs. subjects with DII < median. By contrast, in the latter group, we underscored only 3 proteins with increased NPX. Only 23, out of these 64 proteins, accurately identified subjects with DII > median (Area Under the Curve = 0.601 (0.519-0.668), p = 0.035). CONCLUSION: This large-scale proteomic study supports that higher DII reflects changes in the plasmatic abundance of inflammatory proteins. Larger studies are warranted to validate.

Adherence to Mediterranean Diet: A Population-Based Longitudinal Cohort Study.

Adherence to the Mediterranean diet (MedDiet) is recommended for cardiovascular disease prevention. However, recent epidemiological studies report a shift toward lower adherence to MedDiet. We have conducted a prospective cohort study to evaluate changes in individual determinants of MedDiet adherence over time. Clinical information and MedDiet adherence score (MEDAS) were collected in 711 subjects (mean age 68 ± 10 years; 42% males), enrolled in the PLIC study (Progression of Intimal Atherosclerotic Lesions in Carotid arteries), during two visits conducted, on average, 4.5 years apart. MEDAS score worsening and improvements (absolute change, ΔMEDAS) and the variation in the proportion of subjects reporting to meet each MEDAS criteria were assessed. Overall, 34% of the subjects improved their MedDiet adherence (ΔMEDAS: +1.87 ± 1.13), by consuming more olive oil, legumes and fish and use of dishes seasoned with sofrito and 48% subjects worsened their MedDiet adherence (ΔMEDAS: -2.02 ± 1.14) by consuming less fruit, legumes, fish and nuts, with higher rates of worsening in women and subjects aged 50-65 years. Subjects who improved the score were more obese, had higher plasma glucose levels, and metabolic syndrome at the basal visit. In summary, we report an overall decrease in MedDiet adherence, evaluated during a timeframe heavily affected by the COVID-19 pandemic, underlining the need for better dietary interventions.

Targeted Plasma Proteomics to Predict the Development of Carotid Plaques.

BACKGROUND: Cardiovascular risk stratification in primary prevention is a clinical challenge. We recently identified a large set of circulating proteins improving the risk prediction for cardiovascular events. We now evaluate which of these proteins predicts the development of subclinical carotid atherosclerosis (SCA) in primary cardiovascular prevention. METHODS: Three hundred sixty-eight proteins were quantified, by proximity extension assay, from the plasma collected at basal visit from 586 subjects without previous cardiovascular events and without preclinical atherosclerosis. These subjects were reevaluated 11 years after median follow-up (10-12) in a longitudinal observational analysis, to assess the development of SCA, defined as the formation of focal lesion in any carotid tract and detected by carotid ultrasound at basal visit and after follow-up. Common carotid (intima-media thickness [IMT]) was also measured by ultrasound during the same follow-up to identify subjects with faster common carotid intima-media thickness (IMT) progression (increase IMT)>1.3 mm in the common carotid tract). RESULTS: The variation of 68 proteins predicted SCA development and, among them, higher levels of PIgR2 (polymeric immunoglobulin receptor), chemokine (C-C motif) ligand 18, CA1 (carbonic anhydrase 1), Fc gamma receptor IIa and reduced MMP10 (matrix metallopeptidase 10), GT (gastrotropin), IL7R (interleukin 7 receptor) were the most predictive for SCA development. These 7 proteins improved the sensitivity and the specificity for SCA development versus risk factors (age, sex, overweight, hypertension, low HDL-cholesterol, high triglyceride); area under the curve: 0.747 ([0.707-0.784] versus 0.620 [0.577-0.663]; P<0.001). Vice versa, 25 proteins (not in common with the previous 68) predicted faster common carotid IMT progression. Among them, increased IL7D (interleukin 7), chemokine (C-X-C motif) ligand 1, and reduced TNFS13B (TNF superfamily member 13b) significantly increased the sensitivity and the specificity to predict faster common carotid IMT progression as compared with same risk factors (area under the curve: 0.719 [0.680-0.756] versus 0.569 [0.527-0.610]; P<0.001). CONCLUSIONS: A new set of circulating proteins have been identified that may be considered as markers of preclinical atherosclerosis development. The difference of the protein identified to predict SCA versus IMT progression may reflect different etiological factors.

Genetically determined hypercholesterolaemia results into premature leucocyte telomere length shortening and reduced haematopoietic precursors.

AIMS: Leucocyte telomere length (LTL) shortening is a marker of cellular senescence and associates with increased risk of cardiovascular disease (CVD). A number of cardiovascular risk factors affect LTL, but the correlation between elevated LDL cholesterol (LDL-C) and shorter LTL is debated: in small cohorts including subjects with a clinical diagnosis of familial hypercholesterolaemia (FH). We assessed the relationship between LDL-C and LTL in subjects with genetic familial hypercholesterolaemia (HeFH) compared to those with clinically diagnosed, but not genetically confirmed FH (CD-FH), and normocholesterolaemic subjects. METHODS AND RESULTS: LTL was measured in mononuclear cells-derived genomic DNA from 206 hypercholesterolaemic subjects (135 HeFH and 71 CD-FH) and 272 controls. HeFH presented shorter LTL vs. controls (1.27 ± 0.07 vs. 1.59 ± 0.04, P = 0.045). In particular, we found shorter LTL in young HeFH as compared to young controls (<35 y) (1.34 ± 0.08 vs. 1.64 ± 0.08, P = 0.019); moreover, LTL was shorter in statin-naïve HeFH subjects as compared to controls (1.23 ± 0.08 vs. 1.58 ± 0.04, P = 0.001). HeFH subjects presented shorter LTL compared to LDL-C matched CD-FH (1.33 ± 0.05 vs. 1.55 ± 0.08, P = 0.029). Shorter LTL was confirmed in leucocytes of LDLR-KO vs. wild-type mice and associated with lower abundance of long-term haematopoietic stem and progenitor cells (LT-HSPCs) in the bone marrow. Accordingly, HeFH subjects presented lower circulating haematopoietic precursors (CD34 + CD45dim cells) vs. CD-FH and controls. CONCLUSIONS: We found (i) shorter LTL in genetically determined hypercholesterolaemia, (ii) lower circulating haematopoietic precursors in HeFH subjects, and reduced bone marrow resident LT-HSPCs in LDLR-KO mice. We support early cellular senescence and haematopoietic alterations in subjects with FH.

The Association of Proprotein Convertase Subtilisin/Kexin Type 9 to Plasma Low-Density Lipoproteins: An Evaluation of Different Methods.

Proprotein convertase subtilisin/kexin type-9 (PCSK9) is key regulator of low-density lipoprotein (LDL) metabolism. A significant proportion of PCSK9 is believed to be associated with LDL in plasma as it circulates, although this finding is still a matter of debate. The purpose of this study was to establish an experimental method to investigate the presence of such an interaction in the bloodstream. We compared a number of well-established methods for lipoprotein (LP) isolation to clarify whether PCSK9 associates differently to circulating lipoproteins, such as KBr gradient ultracentrifugation, physical precipitation of ApoB-LPs, fast protein liquid chromatography (FPLC) and iodixanol gradient ultracentrifugation. Our data show heterogeneity in PCSK9 association to lipoproteins according to the method used. Two methods, iodixanol ultracentrifugation and column chromatography, which did not involve precipitation or high salt concentration, consistently showed an interaction of PCSK9 with a subfraction of LDL that appeared to be more buoyant and have a lower size than average LDL. The percent of PCSK9 association ranged from 2 to 30% and did not appear to correlate to plasma or LDL cholesterol levels. The association of PCSK9 to LDL appeared to be sensitive to high salt concentrations. FPLC and iodixanol gradient ultracentrifugation appeared to be the most suitable methods for the study of this association.

Gut Microbiota Functional Dysbiosis Relates to Individual Diet in Subclinical Carotid Atherosclerosis.

Gut Microbiota (GM) dysbiosis associates with Atherosclerotic Cardiovascular Diseases (ACVD), but whether this also holds true in subjects without clinically manifest ACVD represents a challenge of personalized prevention. We connected exposure to diet (self-reported by food diaries) and markers of Subclinical Carotid Atherosclerosis (SCA) with individual taxonomic and functional GM profiles (from fecal metagenomic DNA) of 345 subjects without previous clinically manifest ACVD. Subjects without SCA reported consuming higher amounts of cereals, starchy vegetables, milky products, yoghurts and bakery products versus those with SCA (who reported to consume more mechanically separated meats). The variety of dietary sources significantly overlapped with the separations in GM composition between subjects without SCA and those with SCA (RV coefficient between nutrients quantities and microbial relative abundances at genus level = 0.65, p-value = 0.047). Additionally, specific bacterial species (Faecalibacterium prausnitzii in the absence of SCA and Escherichia coli in the presence of SCA) are directly related to over-representation of metagenomic pathways linked to different dietary sources (sulfur oxidation and starch degradation in absence of SCA, and metabolism of amino acids, syntheses of palmitate, choline, carnitines and Trimethylamine n-oxide in presence of SCA). These findings might contribute to hypothesize future strategies of personalized dietary intervention for primary CVD prevention setting.

Low Plasma Lecithin: Cholesterol Acyltransferase (LCAT) Concentration Predicts Chronic Kidney Disease.

Low high-density lipoprotein-cholesterol (HDL-c) is the most remarkable lipid trait both in mild-to-moderate chronic kidney disease (CKD) patients as well as in advanced renal disease stages, and we have previously shown that reduced lecithin:cholesterol acyltransferase (LCAT) concentration is a major determinant of the low HDL phenotype. In the present study, we test the hypothesis that reduced LCAT concentration in CKD contributes to the progression of renal damage. The study includes two cohorts of subjects selected from the PLIC study: a cohort of 164 patients with CKD (NefroPLIC cohort) and a cohort of 164 subjects selected from the PLIC participants with a basal estimated glomerular filtration rate (eGFR) > 60 mL/min/1.73 m2 (PLIC cohort). When the NefroPLIC patients were categorized according to the LCAT concentration, patients in the 1st tertile showed the highest event rate at follow-up with an event hazard ratio significantly higher compared to the 3rd LCAT tertile. Moreover, in the PLIC cohort, subjects in the 1st LCAT tertile showed a significantly faster impairment of kidney function compared to subjects in the 3rd LCAT tertile. Serum from subjects in the 1st LCAT tertile promoted a higher reactive oxygen species (ROS) production in renal cells compared to serum from subjects in the third LCAT tertile, and this effect was contrasted by pre-incubation with recombinant human LCAT (rhLCAT). The present study shows that reduced plasma LCAT concentration predicts CKD progression over time in patients with renal dysfunction, and, even more striking, it predicts the impairment of kidney function in the general population.

LIPA gene mutations affect the composition of lipoproteins: Enrichment in ACAT-derived cholesteryl esters.

BACKGROUND AND AIMS: Cholesteryl ester storage disease (CESD) due to LIPA gene mutations is characterized by hepatic steatosis, hypercholesterolemia and hypoalphalipoproteinemia, exposing affected patients to an increased cardiovascular risk. Further insights into the impact of LIPA gene mutations on lipid/lipoprotein metabolism are limited. Aim of the study was to investigate the effect of carrying one or two mutant LIPA alleles on lipoprotein composition and function. METHODS: Lipoproteins were isolated from 6 adult CESD patients, 5 relatives carrying one mutant LIPA allele (carriers) and 12 sex/age matched controls. Lipid profile, lipoprotein mass composition and the fatty acid distribution of cholesteryl esters (CEs) were assessed. HDL function was evaluated as the ability to promote nitric oxide release by endothelial cells. RESULTS: Despite the lipid-lowering therapy, total cholesterol, LDL-cholesterol and triglycerides were increased in CESD patients compared to controls, while HDL-cholesterol was reduced. Carriers also displayed elevated total and LDL-cholesterol. Very low and intermediate density lipoproteins from CESD patients and carriers were enriched in CEs compared to the control ones, with a concomitant reduction of triglycerides. Fatty acid composition of CEs in serum and lipoproteins showed a depletion of linoleate content in CESD patients, due to the reduced LCAT activity. In CESD HDL, fatty acid distribution of CEs was shifted towards saturated ones, if compared to control HDL. The changes in HDL composition did not affect HDL ability to promote nitric oxide release by endothelial cells. CONCLUSIONS: LIPA gene mutations significantly affected plasma levels and lipid composition of lipoproteins, likely contributing to the increased cardiovascular risk of affected patients.

Prevalence and relationship between metabolic syndrome and risk of cardiovascular disease: Evidence from two population-based studies.

BACKGROUND AND AIM: The metabolic syndrome (MetS) has become one of the most important clinical issues in the cardiovascular field for this decade because of the marked increase in cardiovascular (CV) risk associated with a clustering of risk factors. The aim of the current study was to evaluate the relationship between MetS and its components and cardiovascular disease (CVD). METHODS: This population-based cross-sectional study was based on data from two studies carried out in Russia (ESSE-RF) and Italy (PLIC). One sample from each cohort was selected, matching individuals by sex and age. A comparison between samples of MetS components distribution and CV risk, according to SCORE chart, has been conducted. RESULTS: A total of 609 individuals (mean [SD] age 55 [8] years, about 39% males) for each cohort were selected. Almost half of PLIC cohort participants belonged to the moderate CV risk group (47% vs 27%), while in ESSE-RF cohort a relatively higher prevalence of individuals classified in the high and very high risk group was observed (19% vs 11%, 21% vs 6%, respectively). Overall, 43% of ESSE-RF participants were diagnosed with MetS, compared with the 27% of PLIC members (the difference in prevalence becomes 37% vs 21%, considering a more conservative cut-off for waist circumference). Both cohorts showed a trend towards the increase of MetS components moving from the lowest to the highest CV risk class, with a high prevalence of patients with four or five MetS determinants allocated in the high/very high CV risk group. CONCLUSIONS: Developing effective public health strategies for the prevention, detection and treatment of MetS should be an urgent priority to reduce the burden of CVD, not only in subjects at high/very high CV risk, but also in those characterized by a lower risk, as even rare CV events that come from low risk group bring a tangible burden to healthcare systems.

Progression of conventional cardiovascular risk factors and vascular disease risk in individuals: insights from the PROG-IMT consortium.

AIMS: Averaged measurements, but not the progression based on multiple assessments of carotid intima-media thickness, (cIMT) are predictive of cardiovascular disease (CVD) events in individuals. Whether this is true for conventional risk factors is unclear. METHODS AND RESULTS: An individual participant meta-analysis was used to associate the annualised progression of systolic blood pressure, total cholesterol, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol with future cardiovascular disease risk in 13 prospective cohort studies of the PROG-IMT collaboration (n = 34,072). Follow-up data included information on a combined cardiovascular disease endpoint of myocardial infarction, stroke, or vascular death. In secondary analyses, annualised progression was replaced with average. Log hazard ratios per standard deviation difference were pooled across studies by a random effects meta-analysis. In primary analysis, the annualised progression of total cholesterol was marginally related to a higher cardiovascular disease risk (hazard ratio (HR) 1.04, 95% confidence interval (CI) 1.00 to 1.07). The annualised progression of systolic blood pressure, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol was not associated with future cardiovascular disease risk. In secondary analysis, average systolic blood pressure (HR 1.20 95% CI 1.11 to 1.29) and low-density lipoprotein cholesterol (HR 1.09, 95% CI 1.02 to 1.16) were related to a greater, while high-density lipoprotein cholesterol (HR 0.92, 95% CI 0.88 to 0.97) was related to a lower risk of future cardiovascular disease events. CONCLUSION: Averaged measurements of systolic blood pressure, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol displayed significant linear relationships with the risk of future cardiovascular disease events. However, there was no clear association between the annualised progression of these conventional risk factors in individuals with the risk of future clinical endpoints.

Impact of genetic testing and family health history of cystic fibrosis in the early prenatal diagnosis and prevention of a new case of genetic disorder.

Cystic fibrosis (CF) is a multi-system autosomal recessive disorder, results of mutations in the CF transmembrane conductance regulator (CFTR) gene, located on the long arm of chromosome 7. We present a special family couple with particular medical history of CF, who comes to our Clinic for genetic tests and a prenatal genetic counseling, to prevent the birth of a new affected CF child. Genetic analysis showed that the first affected child, a daughter, is compound heterozygous for two clinically significant recessive mutations: c.1521_1523delCTT; p.Phe508del, inherited from her mother, who carries the same CFTR mutation, and c.1853_1863delTTTTGCATGAA; p.IIe618Argfs 2, inherited from her father, who is heterozygous, healthy carrier, for the same CFTR mutation. In our case report, early prenatal genetic testing, pre- and post-test genetic counseling was crucial in the management of the present pregnancy, to prevent the birth of a new affected CF child.

Treatment with fibrates is associated with higher LAL activity in dyslipidemic patients.

Lysosomal acid lipase (LAL) is responsible for the hydrolysis of cholesteryl esters (CE) and triglycerides (TG) within the lysosomes; generated cholesterol and free fatty acids (FFA) are released in the cytosol where they can regulate their own synthesis and metabolism. When LAL is not active, as in case of genetic mutations, CE and TG accumulate in the lysosomal compartment, while the lack of release of cholesterol and FFA in the cytosol leads to an upregulation of their synthesis. Thus, LAL plays a central role in the intracellular homeostasis of lipids. Since there are no indications about the effect of different lipid-lowering agents on LAL activity, aim of the study was to address the relationship between LAL activity and the type of lipid-lowering therapy in a cohort of dyslipidemic patients. LAL activity was measured on dried blood spot from 120 patients with hypercholesterolemia or mixed dyslipidemia and was negatively correlated to LDL-cholesterol levels. Among enrolled patients, ninety-one were taking one or more lipid-lowering drugs, as statins, fibrates, ezetimibe and omega-3 polyunsaturated fatty acids. When patients were stratified according to the type of lipid-lowering treatment, i.e. untreated, taking statins or taking fibrates, LAL activity was significantly higher in those with fibrates, even after adjustment for sex, age, BMI, lipid parameters, liver function, metabolic syndrome, diabetes and statin use. In a subset of patients tested after 3 months of treatment with micronized fenofibrate, LAL activity raised by 21%; the increase was negatively correlated with baseline LAL activity. Thus, the use of fibrates is independently associated with higher LAL activity in dyslipidemic patients, suggesting that the positive effects of PPAR-α activation on cellular and systemic lipid homeostasis can also include an improved LAL activity.

Multilevel Models to Estimate Carotid Intima-Media Thickness Curves for Individual Cardiovascular Risk Evaluation.

Background and Purpose- The value of carotid intima-media thickness (cIMT)-a marker of subclinical atherosclerosis-in defining the cardiovascular risk is still debated. The aim of this study was to estimate standard cIMT progression, adjusting values over time for the main cardiovascular risk factors, in a sample of low-to-moderate cardiovascular risk subjects, to identify normative cIMT progression values. Methods- From the progression of lesions in the intima of the carotid cohort, we selected subjects who underwent 4 planned serial clinical evaluations and ultrasound cIMT determinations, on average every 4 years. Subject taking glucose-lowering therapies in secondary cardiovascular prevention or with cardiovascular risk score >5 were excluded from the analysis. The growth of cIMT across the study period (12 years) was assessed by use of individual growth curve modeling within multilevel models. Results- A total of 1175 (36% men; mean age, 53±11 years at baseline) participants at low/intermediate cardiovascular risk have been included in this analysis. A significant and marked slope of the mean and maximum cIMT growth curves (ß=0.009 and ß=0.012, respectively) was observed, confirming that it is a function of age. A stratified analysis by decades of age highlighted a nonlinear cIMT progression over time. In addition, different patterns of cIMT development between sex were observed. Finally, different slopes in mean and maximum cIMT curves, with a significant spread since the fifth decade, were observed in the cIMT growth curve models of subjects developing multifocal carotid atherosclerosis compared with the rest of the population. Conclusions- These findings proved that the rate of change in cIMT over time is a sign of the development of atherosclerosis, which cannot be a priori assumed linear. These data, therefore, support the clinical relevance of these growth curve models for cIMT progression to be considered as useful tool to identify subjects with faster atherosclerosis progression and thus at increased cardiovascular risk.

Identification of AnnexinA1 as an Endogenous Regulator of RhoA, and Its Role in the Pathophysiology and Experimental Therapy of Type-2 Diabetes.

Annexin A1 (ANXA1) is an endogenously produced anti-inflammatory protein, which plays an important role in the pathophysiology of diseases associated with chronic inflammation. We demonstrate that patients with type-2 diabetes have increased plasma levels of ANXA1 when compared to normoglycemic subjects. Plasma ANXA1 positively correlated with fatty liver index and elevated plasma cholesterol in patients with type-2 diabetes, suggesting a link between aberrant lipid handling, and ANXA1. Using a murine model of high fat diet (HFD)-induced insulin resistance, we then investigated (a) the role of endogenous ANXA1 in the pathophysiology of HFD-induced insulin resistance using ANXA1-/- mice, and (b) the potential use of hrANXA1 as a new therapeutic approach for experimental diabetes and its microvascular complications. We demonstrate that: (1) ANXA1-/- mice fed a HFD have a more severe diabetic phenotype (e.g., more severe dyslipidemia, insulin resistance, hepatosteatosis, and proteinuria) compared to WT mice fed a HFD; (2) treatment of WT-mice fed a HFD with hrANXA1 attenuated the development of insulin resistance, hepatosteatosis and proteinuria. We demonstrate here for the first time that ANXA1-/- mice have constitutively activated RhoA. Interestingly, diabetic mice, which have reduced tissue expression of ANXA1, also have activated RhoA. Treatment of HFD-mice with hrANXA1 restored tissue levels of ANXA1 and inhibited RhoA activity, which, in turn, resulted in restoration of the activities of Akt, GSK-3ß and endothelial nitric oxide synthase (eNOS) secondary to re-sensitization of IRS-1 signaling. We further demonstrate in human hepatocytes that ANXA1 protects against excessive mitochondrial proton leak by activating FPR2 under hyperglycaemic conditions. In summary, our data suggest that (a) ANXA1 is a key regulator of RhoA activity, which restores IRS-1 signal transduction and (b) recombinant human ANXA1 may represent a novel candidate for the treatment of T2D and/or its complications.

Pentraxin 3 deficiency protects from the metabolic inflammation associated to diet-induced obesity.

AIMS: Low-grade chronic inflammation characterizes obesity and metabolic syndrome. Here, we aim at investigating the impact of the acute-phase protein long pentraxin 3 (PTX3) on the immune-inflammatory response occurring during diet-induced obesity. METHODS AND RESULTS: PTX3 deficiency in mice fed a high-fat diet for 20 weeks protects from weight gain and adipose tissue deposition in visceral and subcutaneous depots. This effect is not related to changes in glucose homeostasis and lipid metabolism but is associated with an improved immune cell phenotype in the adipose tissue of Ptx3 deficient animals, which is characterized by M2-macrophages polarization and increased angiogenesis. These findings are recapitulated in humans where carriers of a PTX3 haplotype (PTX3 h2/h2 haplotype), resulting in lower PTX3 plasma levels, presented with a reduced prevalence of obesity and decreased abdominal adiposity compared with non-carriers. CONCLUSION: Our results support a critical role for PTX3 in the onset of obesity by promoting inflammation and limiting adipose tissue vascularization and delineate PTX3 targeting as a valuable strategy for the treatment of adipose tissue-associated inflammatory response.

PCSK9 deficiency reduces insulin secretion and promotes glucose intolerance: the role of the low-density lipoprotein receptor.

Aims: PCSK9 loss of function genetic variants are associated with lower low-density lipoprotein cholesterol but also with higher plasma glucose levels and increased risk of Type 2 diabetes mellitus. Here, we investigated the molecular mechanisms underlying this association. Methods and results: Pcsk9 KO, WT, Pcsk9/Ldlr double KO (DKO), Ldlr KO, albumin AlbCre+/Pcsk9LoxP/LoxP (liver-selective Pcsk9 knock-out mice), and AlbCre-/Pcsk9LoxP/LoxP mice were used. GTT, ITT, insulin and C-peptide plasma levels, pancreas morphology, and cholesterol accumulation in pancreatic islets were studied in the different animal models. Glucose clearance was significantly impaired in Pcsk9 KO mice fed with a standard or a high-fat diet for 20 weeks compared with WT animals; insulin sensitivity, however, was not affected. A detailed analysis of pancreas morphology of Pcsk9 KO mice vs. controls revealed larger islets with increased accumulation of cholesteryl esters, paralleled by increased insulin intracellular levels and decreased plasma insulin, and C-peptide levels. This phenotype was completely reverted in Pcsk9/Ldlr DKO mice implying the low-density lipoprotein receptor (LDLR) as the proprotein convertase subtilisin/kexin Type 9 (PCSK9) target responsible for the phenotype observed. Further studies in albumin AlbCre+/Pcsk9LoxP/LoxP mice, which lack detectable circulating PCSK9, also showed a complete recovery of the phenotype, thus indicating that circulating, liver-derived PCSK9, the principal target of monoclonal antibodies, does not impact beta-cell function and insulin secretion. Conclusion: PCSK9 critically controls LDLR expression in pancreas perhaps contributing to the maintenance of a proper physiological balance to limit cholesterol overload in beta cells. This effect is independent of circulating PCSK9 and is probably related to locally produced PCSK9.

Correction: Predictive value for cardiovascular events of common carotid intima media thickness and its rate of change in individuals at high cardiovascular risk - Results from the PROG-IMT collaboration.

[This corrects the article DOI: 10.1371/journal.pone.0191172.].

Epidemiology of cardiovascular risk factors in two population-based studies.

We aimed to compare cardiovascular risk factors prevalence in Italy and Russia through cross-sectional database analysis. The study has been based on data from ESSE-RF and from baseline of PLIC study, two population-based epidemiological studies aimed to investigate prevalence of risk factors and evaluating contribution of traditional and new risk factors into morbidity and cardiovascular mortality. A total of 2203 patients with left and right intima-media thickness (IMT) measurements constituted the source population (1205 from PLIC study and 998 from ESSE-RF study). Sample of ESSE-RF study had slightly more diabetic and hypertensive individuals, while the percentage of subjects with high cholesterol value was lower than in the other sample (67.1% vs 79.9%). The median LDL-C value was higher among individuals not treated with statins in the PLIC sample (p < 0.001), while was comparable among subjects receiving statin therapy. On the other hand, the percentage of individuals with positive cardiovascular history was higher in ESSE-RF sample. This could also explain the higher mean IMT value (0.71 ±â€¯0.17 vs 0.63 ±â€¯0.13) in the whole sample, and among patients without past cardiovascular events (regardless of statin treatment), despite some differences in major risk factors. Despite Russian and Italian populations are culturally and geographically different, they are not so different based on characteristics analyzed.