Functional structure of a high resolution thyroid nodule unit. Economic impact and results of its implementation in an Endocrinology department.

BACKGROUND AND OBJECTIVE: The high incidence of nodular thyroid pathology has led to growing concern about the economic impact that this pathology represents on the healthcare system. There are conclusive data about the cost-effectiveness of high-resolution units for nodular thyroid pathology; however, their implementation is not homogeneous in the Endocrinology and Nutrition services of our country. The objective of the present study was to evaluate the economic impact of the implementation of the high-resolution thyroid nodule unit (HRTNU) in our center. PATIENTS AND METHODS: The present work is a prospective, observational and descriptive study carried out in 1314 patients (82% women, mean age 58 years ds = 11) evaluated at the HRTNU during the period of August 2022-August 2023. Demographic data (age and sex) were analyzed, referral center and consultation type, number of total consultations and neck ultrasound performed, number of fine needle aspiration (FNAB) performed, and cytology results were analyzed. RESULTS: In the period from August 2022 to August 2023, a total of 1314 patients were evaluated (neck ultrasound and clinical consultation) and a total of 133 FNAB were performed, of which only 2.26% were non-diagnostic. Compared to the percentage of unsatisfactory FNAB from the previous year August 2021-July 2022 of 25%, a mean saving of 9931.43 euros was estimated. 84.47% of the patients evaluated for the first time by the HRTNU were discharged, estimating a mean saving of 133,200 euros. CONCLUSIONS: The implementation of a HRTNU at the Endocrinology and Nutrition departments, coordinated with primary and specialized care, is a cost-effectiveness alternative, as it reduces the number of medical consultations and is accompanied by a higher rate of diagnostic FNAB.

Machine Learning Model in Obesity to Predict Weight Loss One Year after Bariatric Surgery: A Pilot Study.

Roux-en-Y gastric bypass (RYGB) is a treatment for severe obesity. However, many patients have insufficient total weight loss (TWL) after RYGB. Although multiple factors have been involved, their influence is incompletely known. The aim of this exploratory study was to evaluate the feasibility and reliability of the use of machine learning (ML) techniques to estimate the success in weight loss after RYGP, based on clinical, anthropometric and biochemical data, in order to identify morbidly obese patients with poor weight responses. We retrospectively analyzed 118 patients, who underwent RYGB at the Hospital Clínico Universitario of Valencia (Spain) between 2013 and 2017. We applied a ML approach using local linear embedding (LLE) as a tool for the evaluation and classification of the main parameters in conjunction with evolutionary algorithms for the optimization and adjustment of the parameter model. The variables associated with one-year postoperative %TWL were obstructive sleep apnea, osteoarthritis, insulin treatment, preoperative weight, insulin resistance index, apolipoprotein A, uric acid, complement component 3, and vitamin B12. The model correctly classified 71.4% of subjects with TWL < 30% although 36.4% with TWL ≥ 30% were incorrectly classified as "unsuccessful procedures". The ML-model processed moderate discriminatory precision in the validation set. Thus, in severe obesity, ML-models can be useful to assist in the selection of patients before bariatric surgery.

Improved lipid-lowering treatment and reduction in cardiovascular disease burden in homozygous familial hypercholesterolemia: The SAFEHEART follow-up study.

AIM: We aimed to describe clinical and genetic characteristics, lipid-lowering treatment and atherosclerotic cardiovascular disease (ASCVD) outcomes over a long-term follow-up in homozygous familial hypercholesterolemia (HoFH). METHODS: SAFEHEART (Spanish Familial Hypercholesterolaemia Cohort Study) is a long-term study in molecularly diagnosed FH. Data analyzed in HoFH were prospectively obtained from 2004 until 2022. ASCVD events, lipid profile and lipid-lowering treatment were determined. RESULTS: Thirty-nine HoFH patients were analyzed. The mean age was 42 ± 20 years and nineteen (49%) were women. Median follow-up was 11 years (IQR 6,18). Median age at genetic diagnosis was 24 years (IQR 8,42). At enrolment, 33% had ASCVD and 18% had aortic valve disease. Patients with new ASCVD events and aortic valve disease at follow-up were six (15%), and one (3%), respectively. Median untreated LDL-C levels were 555 mg/dL (IQ 413,800), and median LDL-C levels at last follow-up was 122 mg/dL (IQR 91,172). Most patients (92%) were on high intensity statins and ezetimibe, 28% with PCSK9i, 26% with lomitapide, and 23% with lipoprotein-apheresis. Fourteen patients (36%) attained an LDL-C level below 100 mg/dL, and 10% attained an LDL-C below 70 mg/dL in secondary prevention. Patients with null/null variants were youngers, had higher untreated LDL-C and had the first ASCVD event earlier. Free-event survival is longer in patients with defective variant compared with those patients with at least one null variant (p=0.02). CONCLUSIONS: HoFH is a severe life threating disease with a high genetic and phenotypic variability. The improvement in lipid-lowering treatment and LDL-C levels have contributed to reduce ASCVD events.

SEA 2024 Standards for Global Control of Vascular Risk. / Estándares de la Sociedad Española de Arteriosclerosis 2024 para el control global del riesgo vascular.

One of the objectives of the Spanish Society of Arteriosclerosis is to contribute to the knowledge, prevention and treatment of vascular diseases, which are the leading cause of death in Spain and entail a high degree of disability and health expenditure. Atherosclerosis is a multifactorial disease and its prevention requires a global approach that takes into account the associated risk factors. This document summarises the current evidence and includes recommendations for patients with established vascular disease or at high vascular risk: it reviews the symptoms and signs to evaluate, the laboratory and imaging procedures to request routinely or in special situations, and includes the estimation of vascular risk, diagnostic criteria for entities that are vascular risk factors, and general and specific recommendations for their treatment. Finally, it presents aspects that are not usually referenced in the literature, such as the organisation of a vascular risk consultation.

Vascular smooth muscle cell phenotype is modulated by ligands of the lymphotoxin ß receptor and the tumor necrosis factor receptor.

OBJECTIVE: Vascular smooth muscle cells (VSMCs) undergo a phenotypic-switching process during the generation of unstable atheroma plaques. In this investigation, the potential implication of the tumor necrosis factor superfamily (TNFSF) ligands, in the gene expression signature associated with VSMC plasticity was studied. MATERIAL AND METHODS: Human aortic (ha)VSMCs were obtained commercially and treated with the cytokine TNFSF14, also called LIGHT, the lymphotoxin alpha (LTα), the heterotrimer LTα1ß2 or with vehicle for 72h. The effect of the different treatments on gene expression was analyzed by quantitative PCR and included the study of genes associated with myofibroblast-like cell function, osteochondrogenesis, pluripotency, lymphorganogenesis and macrophage-like cell function. RESULTS: HaVSMCs displayed a change in myofibroblast-like cell genes which consisted in reduced COL1A1 and TGFB1 mRNA levels when treated with LTα or LIGHT and with augmented MMP9 expression levels when treated with LTα. LTα and LIGHT treatments also diminished the expression of genes associated with osteochondrogenesis and pluripotency SOX9, CKIT, and KLF4. By contrary, all the above genes were no affected by the treatment with the trimer LTα1ß2. In addition, haVSMC treatment with LTα, LTα1ß2 and LIGHT altered lymphorganogenic cytokine gene expression which consisted of augmented CCL20 and CCL21 mRNA levels by LTα and a reduction in the gene expression of CCL21 and CXCL13 by LIGHT and LTα1ß2 respectively. Neither, LTα or LIGHT or LTα1ß2 treatments affected the expression of macrophage-like cell markers in haVSMC. CONCLUSIONS: Altogether, indicates that the TNFSF ligands through their interconnected network of signaling, are important in the preservation of VSMC identity against the acquisition of a genetic expression signature compatible with functional cellular plasticity.

Corticotroph cell hyperplasia as a rare cause of ACTH-dependent Cushing syndrome.

OBJECTIVE: Our aim was to characterise a cohort of patients with Cushing's disease (CD) who did not present pituitary adenoma in magnetic resonance imaging (MRI), needing a catheterisation of the inferior petrosal sinus (CIPS), and to study the pathological findings of the pituitary gland in these subjects after transsphenoidal surgery in order to establish the aetiology of CD. Furthermore, we evaluated possible differences in the features of the diagnosis between hyperplasia and adenoma. SUBJECTS AND METHODS: We included 16 subjects. 17 CIPS were done. Hormonal parameters were measured using standard methods. A microscopic histochemical study following standard procedures and immunohistochemical analysis was performed. The diagnostic criteria for adenoma and hyperplasia were based on the WHO classification. RESULTS: One patient was excluded for presenting an ACTH-producing bronchial neuroendocrine tumour. The 15 subjects with CD have a positive CIPS test indicating hypophyseal ACTH production. After transsphenoidal surgery, 12 patients showed a microadenoma and three (20%) a corticotroph cell hyperplasia. We found four recurrences after the transsphenoidal surgery (26%), with a mean time for recurrence of 105 months. We found that recurrence was more frequent in subjects with hyperplasia, and in those subjects with lower right/left ACTH ratio. CONCLUSION: Our study, which was focused on patients with CD with no pituitary adenoma detected by MRI and a positive CRH test after CIPS, has found that 20% showed corticotroph cell hyperplasia as the cause of CD. Right/left ACTH ratio after CIPS was useful to differentiate adenoma from hyperplasia. This finding may have important prognostic and treatment implications. More studies are necessary to confirm our result.

Novel Therapies for Cardiometabolic Disease: Recent Findings in Studies with Hormone Peptide-Derived G Protein Coupled Receptor Agonists.

The increasing prevalence of obesity and type 2 diabetes (T2DM) is provoking an important socioeconomic burden mainly in the form of cardiovascular disease (CVD). One successful strategy is the so-called metabolic surgery whose beneficial effects are beyond dietary restrictions and weight loss. One key underlying mechanism behind this surgery is the cooperative improved action of the preproglucagon-derived hormones, glucagon, glucagon-like peptide-1 (GLP-1), and glucose-dependent insulinotropic polypeptide (GIP) which exert their functions through G protein-coupled receptors (GPCR). Great success has been reached with therapies based on the GLP-1 receptor monoagonism; therefore, a logical and rational approach is the use of the dual and triagonism of GCPC to achieve complete metabolic homeostasis. The present review describes novel findings regarding the complex biology of the preproglucagon-derived hormones, their signaling, and the drug development of their analogues, especially those acting as dual and triagonists. Moreover, the main investigations into animal models and ongoing clinical trials using these unimolecular dual and triagonists are included which have demonstrated their safety, efficacy, and beneficial effects on the CV system. These therapeutic strategies could greatly impact the treatment of CVD with unprecedented benefits which will be revealed in the next years.

Beneficial effects of PCSK9 inhibition with alirocumab in familial hypercholesterolemia involve modulation of new immune players.

Familial hypercholesterolemia (FH) is associated with low-grade systemic inflammation, a key driver of premature atherosclerosis. We investigated the effects of inhibiting proprotein convertase subtilisin/kexin type 9 (PCSK9) function on inflammatory state, endothelial dysfunction and cardiovascular outcomes in patients with FH. Fourteen patients with FH were evaluated before and 8 weeks after administration of a PCSK9 blocking monoclonal antibody (alirocumab, 150 mg/subcutaneous/14 days). In vivo and ex vivo analysis revealed that alirocumab blunted the attachment of leukocytes to TNFα-stimulated human umbilical arterial endothelial cells (HUAEC) and suppressed the activation of platelets and most leukocyte subsets, which was accompanied by the diminished expression of CX3CR1, CXCR6 and CCR2 on several leukocyte subpopulations. By contrast, T-regulatory cell activation was enhanced by alirocumab treatment, which also elevated anti-inflammatory IL-10 plasma levels and lowered circulating pro-inflammatory cytokines. Plasma levels of IFNγ positively correlated with levels of total and LDL-cholesterol, whereas circulating IL-10 levels negatively correlated with these key lipid parameters. In vitro analysis revealed that TNFα stimulation of HUAEC increased the expression of PCSK9, whereas endothelial PCSK9 silencing reduced TNFα-induced mononuclear cell adhesion mediated by Nox5 up-regulation and p38-MAPK/NFκB activation, concomitant with reduced SREBP2 expression. PCSK9 silencing also decreased endothelial CX3CL1 and CXCL16 expression and chemokine generation. In conclusion, PCSK9 inhibition impairs systemic inflammation and endothelial dysfunction by constraining leukocyte-endothelium interactions. PCSK9 blockade may constitute a new therapeutic approach to control the inflammatory state associated with FH, preventing further cardiovascular events in this cardiometabolic disorder.

Association of chemokines IP-10/CXCL10 and I-TAC/CXCL11 with insulin resistance and enhance leukocyte endothelial arrest in obesity.

BACKGROUND AND AIMS: Obesity is a key contributing factor to incidental type 2 diabetes and cardiovascular disease. CXCR3 receptor and its ligands CXCL 10 and 11 are associated with atherosclerosis and cardiovascular disease. The aim of our study was to analyse the role of the CXCR3 ligands on insulin resistance (IR) and endothelial dysfunction in human obesity. METHODS AND RESULTS: We have studied 45 obese patients (mean age 44 ± 6 years, body mass index 45 ± 9 kg/m2) who were selected for Roux-Y-gastric bypass surgery and 21 non obese control subjects with similar age and gender distribution. We measured by ELISA the circulating levels of the CXCR3 ligands interferon-γ inducible protein 10 (IP-10/CXCL10) and interferon-γ-inducible T-cell alpha chemoattractant (I-TAC/CXCL11). Using an ex vivo procedure with the flow chamber assay, we have investigated the effect of such chemokines on endothelial leukocytes arrest under dynamic conditions. Peripheral blood levels of CXCL10 and CXCL11 were significantly higher in obese subjects than in controls (p < 0.001) and significantly correlated with BMI, waist circunference and HOMA-IR. Obese patients with HOMA-IR index above 75th percentile showed highest increase of circulating CXCL10 and CXCL11 values. Under dynamic flow conditions, the enhanced adhesion of patient leukocytes to TNFα-induced human arterial endothelial cells was partly dependent on CXCR3. CONCLUSIONS: The study demonstrates that CXCL10 and CXCL11 are associated with IR and enhance leukocyte endothelial arrest in obese subjects. Blockade of CXCR3 signaling might be a new therapeutic approach for the prevention of obesity-associated cardiovascular co-morbidities.

Anthropometric Indicators as a Tool for Diagnosis of Obesity and Other Health Risk Factors: A Literature Review.

Obesity is characterized by the accumulation of an excessive amount of fat mass (FM) in the adipose tissue, subcutaneous, or inside certain organs. The risk does not lie so much in the amount of fat accumulated as in its distribution. Abdominal obesity (central or visceral) is an important risk factor for cardiovascular diseases, diabetes, and cancer, having an important role in the so-called metabolic syndrome. Therefore, it is necessary to prevent, detect, and appropriately treat obesity. The diagnosis is based on anthropometric indices that have been associated with adiposity and its distribution. Indices themselves, or a combination of some of them, conform to a big picture with different values to establish risk. Anthropometric indices can be used for risk identification, intervention, or impact evaluation on nutritional status or health; therefore, they will be called anthropometric health indicators (AHIs). We have found 17 AHIs that can be obtained or estimated from 3D human shapes, being a noninvasive alternative compared to X-ray-based systems, and more accessible than high-cost equipment. A literature review has been conducted to analyze the following information for each indicator: definition; main calculation or obtaining methods used; health aspects associated with the indicator (among others, obesity, metabolic syndrome, or diabetes); criteria to classify the population by means of percentiles or cutoff points, and based on variables such as sex, age, ethnicity, or geographic area, and limitations.

Oral Unsaturated Fat Load Impairs Postprandial Systemic Inflammation in Primary Hypercholesterolemia Patients.

Context: Primary hypercholesterolemia (PH) is a lipid disorder characterized by elevated levels of cholesterol and low-density lipoprotein (LDL). Low-grade systemic inflammation is associated with PH, which might explain the higher incidence of cardiovascular diseases in this setting. Objective: To evaluate the effect of an oral unsaturated fat load (OUFL) on different immune parameters and functional consequences in patients with PH in postprandial state. Design: A commercial liquid preparation of long-chain triglycerides (Supracal®; ω6/ω3 ratio >20/1, OUFL) was administered to 20 patients and 10 age-matched controls. Whole blood was collected before (fasting state) and 4 h after administration (postprandial state). Flow cytometry was employed to determine platelet and leukocyte activation, and the levels of circulating platelet-leukocyte aggregates. Soluble markers were determined by ELISA, and the parallel-plate flow chamber was employed to study leukocyte adhesion to the dysfunctional arterial endothelium. Results: The PH group had a lower percentage of activated platelets and circulating type 1 monocytes, and blunted neutrophil activation after the OUFL, accompanied by a significant increase in the percentage of regulatory T lymphocytes. In this group, the OUFL led to a significant impairment of leukocyte adhesion to the dysfunctional [tumor necrosis factor α (TNFα)-stimulated] endothelium and reduced the plasma levels of soluble P-selectin, platelet factor-4 (PF-4)/CXCL4, CXCL8, CCL2, CCL5, and TNFα. Conclusion: The OUFL has a beneficial impact on the pro-thrombotic and pro-inflammatory state of PH patients and might be a promising macronutrient approach to dampen the systemic inflammation associated with PH and the development of further cardiovascular events.

Dapagliflozin Does Not Modulate Atherosclerosis in Mice with Insulin Resistance.

Type 2 diabetes mellitus (T2DM) increases morbimortality in humans via enhanced susceptibility to cardiovascular disease (CVD). Sodium-glucose co-transporter 2 inhibitors (SGLT2i) are drugs designed for T2DM treatment to diminish hyperglycaemia by reducing up to 90% of renal tube glucose reabsorption. Clinical studies also suggest a beneficial action of SGLT2i in heart failure and CVD independent of its hypoglycaemiant effect. In the present study, we explored the effect of SGLT2i dapagliflozin (DAPA) in the metabolism and atherosclerosis in Apoe-/-Irs2+/- mice, which display accelerated atherosclerosis induced by insulin resistance. DAPA treatment of Apoe-/-Irs2+/- mice, which were fed a high-fat, high-cholesterol diet, failed to modify body weight, plasma glucose or lipid. Carbohydrate metabolism characterisation showed no effect of DAPA in the glucose tolerance test (GTT) despite augmented insulin levels during the test. In fact, decreased C-peptide levels in DAPA-treated mice during the GTT suggested impaired insulin release. Consistent with this, DAPA treatment of Apoe-/-Irs2+/- isolated islets displayed lower glucose-stimulated insulin secretion compared with vehicle-treated islets. Moreover, insulin-signalling experiments showed decreased pAKT activation in DAPA-treated adipose tissue indicating impaired insulin signalling in this tissue. No changes were seen in lesion size, vulnerability or content of macrophages, vascular smooth muscle cells, T cells or collagen. DAPA did not affect circulating inflammatory cells or cytokine levels. Hence, this study indicates that DAPA does not protect against atherosclerosis in insulin-resistant mice in hypercholesterolemic conditions.

Impact of Cholesterol Metabolism in Immune Cell Function and Atherosclerosis.

Cholesterol, the most important sterol in mammals, helps maintain plasma membrane fluidity and is a precursor of bile acids, oxysterols, and steroid hormones. Cholesterol in the body is obtained from the diet or can be de novo synthetized. Cholesterol homeostasis is mainly regulated by the liver, where cholesterol is packed in lipoproteins for transport through a tightly regulated process. Changes in circulating lipoprotein cholesterol levels lead to atherosclerosis development, which is initiated by an accumulation of modified lipoproteins in the subendothelial space; this induces significant changes in immune cell differentiation and function. Beyond lesions, cholesterol levels also play important roles in immune cells such as monocyte priming, neutrophil activation, hematopoietic stem cell mobilization, and enhanced T cell production. In addition, changes in cholesterol intracellular metabolic enzymes or transporters in immune cells affect their signaling and phenotype differentiation, which can impact on atherosclerosis development. In this review, we describe the main regulatory pathways and mechanisms of cholesterol metabolism and how these affect immune cell generation, proliferation, activation, and signaling in the context of atherosclerosis.

Gender differences on oxidative stress markers and complement component C3 plasma values after an oral unsaturated fat load test.

OBJECTIVE: Post-prandial lipaemia (PL), oxidative stress (OS), and complement component C3 (C3) values are related to the atherosclerosis process. The post-prandial response of C3 after an oral fat load test (OFLT) using unsaturated fat is poorly addressed. The aim of this study was to analyze and compare the post-prandial response of OS markers and C3 values in men and women after an OFLT using unsaturated fat. METHODS: The study included a total of 22 healthy subjects with normal lipids and normal blood glucose (11 men and 11 pre-menopausal women). An oral unsaturated fat load test (OFLT: 50g fat per m2 body surface) was performed using a commercial liquid preparation of long chain triglycerides (Supracal®). OS markers and C3 were measured using standardized methods at fasting state and every 2h up to 8h after the OFLT. RESULTS: Men showed statistically significant higher C3, oxidized glutathione (GSSG), and oxidized-reduced glutathione (GSSG/GSH) ratio values at fasting state compared to that obtained in women. In addition, post-prandial C3 values and GSSG/GSH ratios were significantly higher in men compared to women. The GSSG value and GSSG/GSH ratio significantly decreased in men after the OFLT compared to fasting values. In contrast, the post-prandial OS markers decrease observed in women was not statistically significant. CONCLUSIONS: In fasting state, men showed higher statistically significant C3 values and OS markers than women. The post-prandial OS markers (GSSG and GSSG/GSH ratio) significantly decrease after the OFLT with unsaturated fat in men compared to women.

Easy One-Step Amplification and Labeling Procedure for Copy Number Variation Detection.

BACKGROUND: The specific characteristics of copy number variations (CNVs) require specific methods of detection and characterization. We developed the Easy One-Step Amplification and Labeling procedure for CNV detection (EOSAL-CNV), a new method based on proportional amplification and labeling of amplicons in 1 PCR. METHODS: We used tailed primers for specific amplification and a pair of labeling probes (only 1 labeled) for amplification and labeling of all amplicons in just 1 reaction. Products were loaded directly onto a capillary DNA sequencer for fragment sizing and quantification. Data obtained could be analyzed by Microsoft Excel spreadsheet or EOSAL-CNV analysis software. We developed the protocol using the LDLR (low density lipoprotein receptor) gene including 23 samples with 8 different CNVs. After optimizing the protocol, it was used for genes in the following multiplexes: BRCA1 (BRCA1 DNA repair associated), BRCA2 (BRCA2 DNA repair associated), CHEK2 (checkpoint kinase 2), MLH1 (mutL homolog 1) plus MSH6 (mutS homolog 6), MSH2 (mutS homolog 2) plus EPCAM (epithelial cell adhesion molecule) and chromosome 17 (especially the TP53 [tumor protein 53] gene). We compared our procedure with multiplex ligation-dependent probe amplification (MLPA). RESULTS: The simple procedure for CNV detection required 150 min, with <10 min of handwork. After analyzing >240 samples, EOSAL-CNV excluded the presence of CNVs in all controls, and in all cases, results were identical using MLPA and EOSAL-CNV. Analysis of the 17p region in tumor samples showed 100% similarity between fluorescent in situ hybridization and EOSAL-CNV. CONCLUSIONS: EOSAL-CNV allowed reliable, fast, easy detection and characterization of CNVs. It provides an alternative to targeted analysis methods such as MLPA.

Type 1 diabetic mellitus patients with increased atherosclerosis risk display decreased CDKN2A/2B/2BAS gene expression in leukocytes.

BACKGROUND: Type 1 diabetes mellitus (T1DM) patients display increased risk of cardiovascular disease (CVD) and are characterized by a diminished regulatory T (Treg) cell content or function. Previous studies have shown an association between decreased CDKN2A/2B/2BAS gene expression and enhanced CVD. In the present study the potential relationship between CDKN2A/2B/2BAS gene expression, immune cell dysfunction and increased cardiovascular risk in T1DM patients was explored. METHODS: A cross-sectional study was performed in 90 subjects divided into controls and T1DM patients. Circulating leukocyte subpopulations analysis by flow cytometry, expression studies on peripheral blood mononuclear cell by qPCR and western blot and correlation studies were performed in both groups of subjects. RESULTS: Analysis indicated that, consistent with the described T cell dysfunction, T1DM subjects showed decreased circulating CD4+CD25+CD127- Treg cells. In addition, T1DM subjects had lower mRNA levels of the transcription factors FOXP3 and RORC and lower levels of IL2 and IL6 which are involved in Treg and Th17 cell differentiation, respectively. T1DM patients also exhibited decreased mRNA levels of CDKN2A (variant 1 p16Ink4a), CDKN2A (p14Arf, variant 4), CDKN2B (p15Ink4b) and CDKN2BAS compared with controls. Notably, T1DM patients had augmented pro-atherogenic CD14++CD16+-monocytes, which predict cardiovascular acute events and enhanced common carotid intima-media thickness (CC-IMT). CONCLUSIONS: Decreased expression of CDKN2A/2B/2BAS in leukocytes associates with increased CC-IMT atherosclerosis surrogate marker and proatherogenic CD14++CD16+ monocytes in T1DM patients. These results suggest a potential role of CDKN2A/2B/2BAS genes in CVD risk in T1DM.

Anti-inflammatory Therapies for Cardiovascular Disease: Signaling Pathways and Mechanisms.

Cardiovascular diseases (CVD) are the clinical manifestation of atherosclerosis, a chronic inflammatory disease promoted by several risk factors such as dyslipidemia, type 2 diabetes mellitus, hypertension, and smoking. Acute CVD events are the result of an unresolved inflammatory chronic state that promotes the rupture of unstable plaque lesions. Of note, the existing intensive therapies modify risk factors but do not prevent life-threatening recurrent ischemic events in high-risk patients, who have a residual inflammatory risk displayed by increased C-reactive protein (CRP) levels. Better understanding of the role of innate and adaptive immunity in plaque development and rupture has led to intensive investigation of anti-inflammatory strategies for CVD. Some of them are being tested in specific clinical trials and use lower doses of existing medications originally developed for other inflammatory diseases such as rheumatoid arthritis and psoriasis, which have high CVD risk. Other investigations are retrospective and meta-analyses of existing clinical trials that evaluate the incidence of CVD in these inflammatory diseases. Others are based on preclinical testing such as vaccines. In this article, we summarize the main anti-inflammatory strategies and associated molecular mechanisms that are being evaluated in preclinical or clinical CVD studies.

Systemic Inflammation in Metabolic Syndrome: Increased Platelet and Leukocyte Activation, and Key Role of CX3CL1/CX3CR1 and CCL2/CCR2 Axes in Arterial Platelet-Proinflammatory Monocyte Adhesion.

BACKGROUND: Metabolic syndrome is associated with low-grade systemic inflammation, which is a key driver of premature atherosclerosis. We characterized immune cell behavior in metabolic syndrome, its consequences, and the potential involvement of the CX3CL1/CX3CR1 and CCL2/CCR2 chemokine axes. METHODS: Whole blood from 18 patients with metabolic syndrome and 21 age-matched controls was analyzed by flow cytometry to determine the leukocyte immunophenotypes, activation, platelet-leukocyte aggregates, and CX3CR1 expression. ELISA determined the plasma marker levels. Platelet-leukocyte aggregates adhesion to tumor necrosis factor-α (TNFα)-stimulated arterial endothelium and the role of CX3CL1/CX3CR1 and CCL2/CCR2 axes was investigated with the parallel-plate flow chamber. RESULTS: When compared with the controls, the metabolic syndrome patients presented greater percentages of eosinophils, CD3+ T lymphocytes, Mon2/Mon3 monocytes, platelet-eosinophil and -lymphocyte aggregates, activated platelets, neutrophils, eosinophils, monocytes, and CD8+ T cells, but lower percentages of Mon1 monocytes. Patients had increased circulating interleukin-8 (IL-8) and TNFα levels and decreased IL-4. CX3CR1 up-regulation in platelet-Mon1 monocyte aggregates in metabolic syndrome patients led to increased CX3CR1/CCR2-dependent platelet-Mon1 monocyte adhesion to dysfunctional arterial endothelium. CONCLUSION: We provide evidence of generalized immune activation in metabolic syndrome. Additionally, CX3CL1/CX3CR1 or CCL2/CCR2 axes are potential candidates for therapeutic intervention in cardiovascular disorders in metabolic syndrome patients, as their blockade impairs the augmented arterial platelet-Mon1 monocyte aggregate adhesiveness, which is a key event in atherogenesis.

Novel Immune Features of the Systemic Inflammation Associated with Primary Hypercholesterolemia: Changes in Cytokine/Chemokine Profile, Increased Platelet and Leukocyte Activation.

Primary hypercholesterolemia (PH) is associated with a low grade systemic inflammation that is likely the main driver of premature atherosclerosis. Accordingly, we characterized the immune cell behaviour in PH and its potential consequences. Whole blood from 22 PH patients and 21 age-matched controls was analysed by flow cytometry to determine the percentage of leukocyte immunophenotypes, activation, and platelet-leukocyte aggregates. Plasma markers were determined by Enzyme-Linked ImmunoSorbent Assay (ELISA). The adhesion of platelet-leukocyte aggregates to tumor necrosis factor-α (TNFα)-stimulated arterial endothelium was investigated using the dynamic model of the parallel-plate flow chamber. PH patients presented greater percentage of Mon 3 monocytes, Th2 and Th17 lymphocytes, activated platelets, and leukocytes than controls. The higher percentages of circulating platelet-neutrophil, monocyte and lymphocyte aggregates in patients caused increased platelet-leukocyte adhesion to dysfunctional arterial endothelium. Circulating CXCL8, CCL2, CX3CL1, and IL-6 levels positively correlated with key lipid features of PH, whereas negative correlations were found for IL-4 and IL-10. We provide the first evidence that increased platelet and leukocyte activation leads to elevated platelet-leukocyte aggregates in PH and augmented arterial leukocyte adhesiveness, a key event in atherogenesis. Accordingly, modulation of immune system behavior might be a powerful target in the control of further cardiovascular disease in PH.

The GLP-1 analogue lixisenatide decreases atherosclerosis in insulin-resistant mice by modulating macrophage phenotype.

AIMS/HYPOTHESIS: Recent clinical studies indicate that glucagon-like peptide-1 (GLP-1) analogues prevent acute cardiovascular events in type 2 diabetes mellitus but their mechanisms remain unknown. In the present study, the impact of GLP-1 analogues and their potential underlying molecular mechanisms in insulin resistance and atherosclerosis are investigated. METHODS: Atherosclerosis development was evaluated in Apoe -/- Irs2 +/- mice, a mouse model of insulin resistance, the metabolic syndrome and atherosclerosis, treated with the GLP-1 analogues lixisenatide or liraglutide. In addition, studies in Apoe -/- Irs2 +/- mice and mouse-derived macrophages treated with lixisenatide were performed to investigate the potential inflammatory intracellular pathways. RESULTS: Treatment of Apoe -/- Irs2 +/- mice with either lixisenatide or liraglutide improved glucose metabolism and blood pressure but this was independent of body weight loss. Both drugs significantly decreased atheroma plaque size. Compared with vehicle-treated control mice, lixisenatide treatment generated more stable atheromas, with fewer inflammatory infiltrates, reduced necrotic cores and thicker fibrous caps. Lixisenatide-treated mice also displayed diminished IL-6 levels, proinflammatory Ly6Chigh monocytes and activated T cells. In vitro analysis showed that, in macrophages from Apoe -/- Irs2 +/- mice, lixisenatide reduced the secretion of the proinflammatory cytokine IL-6 accompanied by enhanced activation of signal transducer and activator of transcription (STAT) 3, which is a determinant for M2 macrophage differentiation. STAT1 activation, which is essential for M1 phenotype, was also diminished. Furthermore, atheromas from lixisenatide-treated mice showed higher arginase I content and decreased expression of inducible nitric oxide synthase, indicating the prevalence of the M2 phenotype within plaques. CONCLUSIONS/INTERPRETATION: Lixisenatide decreases atheroma plaque size and instability in Apoe -/- Irs2 +/- mice by reprogramming macrophages towards an M2 phenotype, which leads to reduced inflammation. This study identifies a critical role for this drug in macrophage polarisation inside plaques and provides experimental evidence supporting a novel mechanism of action for GLP-1 analogues in the reduction of cardiovascular risk associated with insulin resistance.