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.

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.

Impact of conducting a genetic study on the management of familial hypercholesterolemia.

BACKGROUND: Clinically diagnosed familial hypercholesterolemia (FH) may require a genetic test (GT) to confirm diagnosis. GT availability/accessibility is resource-dependent and usually restricted to specialized clinics. While GT has a diagnostic value, it has not yet defined its impact on long-term management and prognosis of FH. OBJECTIVE: The aim was to identify the clinical characteristics associated with the request for a GT in suspected heterozygous FH. METHODS: Retrospective study including adult patients with clinically suspected to be FH. Positive GT (GT+) was defined as having a pathogenic/likely pathogenic variant. Patients were stratified based on whether they had a genetic study conducted, and among those with a genetic study, according to those who did or did not have a GT+. RESULTS: From 4854 patients included, 3090 were performed a GT (GT+: 2113). Median follow-up: 6.2 years. A younger age, FH-related physical signs, premature coronary disease, higher low-density lipoprotein cholesterol (LDLc) and lower body mass index and triglycerides, associated higher odds of being conducted a genetic study. These patients had higher baseline LDLc (252 mg/dL vs. 211 mg/dL among clinically diagnosed patients) and experienced larger reductions over the follow-up (157.7 mg/dL vs. 113.5 mg/dL, respectively). A similar pattern was observed among patients with GT+ (vs. negative GT). LDLc target attainment was low but increased to 66-95% when a triple combination with statin/ezetimibe/proprotein convertase subtilisin kexin type 9-inhibitor was used. Cardiovascular events occurred in 3.2% and 3.1% of patients who conducted/not conducted a genetic study. Patients conducted a genetic analysis and those with GT+ tended to present the events earlier. CONCLUSIONS: Genetic study, vs. having a clinical-only diagnosis, impacts the management of FH. Cardiovascular prognosis was similar in both groups, perhaps as a result of the more intensive management of patients with a genetic study.

Metabolomic signature and molecular profile of normal and degenerated human intervertebral disc cells.

BACKGROUND CONTEXT: Intervertebral disc degeneration (IVDD) is an incurable, specific treatment-orphan disease with an increasing burden worldwide. Although great efforts have been made to develop new regenerative therapies, their clinical success is limited. PURPOSE: Characterize the metabolomic and gene expression changes underpinning human disc degeneration. This study also aimed to disclose new molecular targets for developing and optimizing novel biological approaches for IVDD. STUDY DESIGN: Intervertebral disc cells were obtained from IVDD patients undergoing circumferential arthrodesis surgery or from healthy subjects. Mimicking the harmful microenvironment of degenerated discs, cells isolated from the nucleus pulposus (NP) and annulus fibrosus (AF) were exposed to the proinflammatory cytokine IL-1ß and the adipokine leptin. The metabolomic signature and molecular profile of human disc cells were unraveled for the first time. METHODS: The metabolomic and lipidomic profiles of IVDD and healthy disc cells were analyzed by high-performance liquid chromatography-mass spectrometry (UHPLC-MS). Gene expression was investigated by SYBR green-based quantitative real-time RT-PCR. Altered metabolites and gene expression were documented. RESULTS: Lipidomic analysis revealed decreased levels of triacylglycerols (TG), diacylglycerol (DG), fatty acids (FA), phosphatidylcholine (PC), lysophosphatidylinositols (LPI) and sphingomyelin (SM), and increased levels of bile acids (BA) and ceramides, likely promoting disc cell metabolism changing from glycolysis to fatty acid oxidation and following cell death. The gene expression profile of disc cells suggests LCN2 and LEAP2/GHRL as promising molecular therapeutic targets for disc degeneration and demonstrates the expression of genes related to inflammation (NOS2, COX2, IL-6, IL-8, IL-1ß, and TNF-α) or encoding adipokines (PGRN, NAMPT, NUCB2, SERPINE2, and RARRES2), matrix metalloproteinases (MMP9 and MMP13), and vascular adhesion molecules (VCAM1). CONCLUSIONS: Altogether, the presented results disclose the NP and AF cell biology changes from healthy to degenerated discs, allowing the identification of promising molecular therapeutic targets for intervertebral disc degeneration. CLINICAL SIGNIFICANCE: Our results are relevant to improving current biological-based strategies aiming to repair IVD by restoring cellular lipid metabolites as well as adipokines homeostasis. Ultimately, our results will be valuable for successful, long-lasting relief of painful IVDD.

CCL17 and CCL22 chemokines are upregulated in human obesity and play a role in vascular dysfunction.

Background/Aims: Chemokines are known to play critical roles mediating inflammation in many pathophysiological processes. The aim of this study was to investigate the role of chemokine receptor CCR4 and its ligands CCL17 and CCL22 in human morbid obesity. Methods: Circulating levels of CCL17 and CCL22 were measured in 60 morbidly obese patients (mean age, 45 ± 1 years; body mass index/BMI, 44 ± 1 kg/m2) who had undergone bariatric bypass surgery, and 20 control subjects. Paired subcutaneous (SCAT) and visceral adipose tissue (VCAT) from patients were analysed to measure expression of CCR4 and its ligands by RT-PCR, western blot and immunohistochemical analysis. The effects of CCR4 neutralization ex vivo on leukocyte-endothelial cells were also evaluated. Results: Compared with controls, morbidly obese patients presented higher circulating levels of CCL17 (p=0.029) and CCL22 (p<0.001) and this increase was positively correlated with BMI (p=0.013 and p=0.0016), and HOMA-IR Index (p=0.042 and p< 0.001). Upregulation of CCR4, CCL17 and CCL22 expression was detected in VCAT in comparison with SCAT (p<0.05). Using the parallel-plate flow chamber model, blockade of endothelial CCR4 function with the neutralizing antibody anti-CCR4 in morbidly obese patients significantly reduced leucocyte adhesiveness to dysfunctional endothelium, a key event in atherogenesis. Additionally, CCL17 and CCL22 increased activation of the ERK1/2 mitogen-activated protein kinase signalling pathway in human aortic endothelial cells, which was significantly reduced by CCR4 inhibition (p=0.016 and p<0.05). Conclusion: Based on these findings, pharmacological modulation of the CCR4 axis could represent a new therapeutic approach to prevent adipose tissue dysfunction in obesity.

The Controversial Role of Vitamin D in Thyroid Cancer Prevention.

Thyroid cancer is the most common endocrine malignancy and exhibits rising incidence. Annual incidence varies by sex, age, and geographical location. It has been reported that impairment of vitamin D signalling promotes thyroid cancer progression. Recent studies have shown that vitamin D, a fat-soluble vitamin that acts as both a nutrient and a hormone, may have utility in the prevention of autoimmune thyroid-related diseases. However, the precise role of vitamin D in the pathobiology of thyroid cancer is controversial. Previous studies have suggested that elevated serum vitamin D levels have a protective role in thyroid cancer. However, there is also evidence demonstrating no inverse relationship between vitamin D levels and the occurrence of thyroid cancer. Furthermore, recent data provide evidence that circulating vitamin D concentration is inversely correlated with disease aggressiveness and poor prognosis, while evidence of an association with tumour initiation remains weak. Nevertheless, a variety of data support an anti-tumorigenic role of vitamin D and its potential utility as a secondary chemopreventive agent. In this review, we highlighted recent findings regarding the association of vitamin D status with the risk of thyroid cancer, prognosis, potential mechanisms, and possible utility as a chemopreventive agent.

SEA 2022 Standards for Global Control of Cardiovascular Risk. / Estándares SEA 2022 para el control global del riesgo cardiovascular.

One of the objectives of the Spanish Society of Arteriosclerosis is to contribute to better knowledge of vascular disease, its prevention and treatment. It is well known that cardiovascular diseases are the leading cause of death in our country and entail a high degree of disability and health care costs. Arteriosclerosis is a multifactorial disease and therefore its prevention requires a global approach that takes into account the different risk factors with which it is associated. Therefore, this document summarizes the current level of knowledge and includes recommendations and procedures to be followed in patients with established cardiovascular disease or at high vascular risk. Specifically, this document reviews the main symptoms and signs to be evaluated during the clinical visit, the laboratory and imaging procedures to be routinely requested or requested for those in special situations. It also includes vascular risk estimation, the diagnostic criteria of the different entities that are cardiovascular risk factors, and makes general and specific recommendations for the treatment of the different cardiovascular risk factors and their final objectives. Finally, the document includes aspects that are not usually referenced in the literature, such as the organization of a vascular risk consultation.

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.

Activation of the Constitutive Androstane Receptor Inhibits Leukocyte Adhesiveness to Dysfunctional Endothelium.

Leukocyte cell recruitment into the vascular subendothelium constitutes an early event in the atherogenic process. As the effect of the constitutive androstane receptor (CAR) on leukocyte recruitment and endothelial dysfunction is poorly understood, this study investigated whether the role of CAR activation can affect this response and the underlying mechanisms involved. Under physiological flow conditions, TNFα-induced endothelial adhesion of human leukocyte cells was concentration-dependently inhibited by preincubation of human umbilical arterial endothelial cells with the selective human CAR ligand CITCO. CAR agonism also prevented TNFα induced VCAM-1 expression, as well as MCP-1/CCL-2 and RANTES/CCL-5 release in endothelial cells. Suppression of CAR expression with a small interfering RNA abrogated the inhibitory effects of CITCO on these responses. Furthermore, CITCO increased interaction of CAR with Retinoid X Receptor (RXR) and reduced TNFα-induced p38-MAPK/NF-κB activation. In vivo, using intravital microscopy in the mouse cremasteric microcirculation treatment with the selective mouse CAR ligand TCPOBOP inhibited TNFα-induced leukocyte rolling flux, adhesion, and emigration and decreased VCAM-1 in endothelium. These results reveal that CAR agonists can inhibit the initial inflammatory response that precedes the atherogenic process by targeting different steps in the leukocyte recruitment cascade. Therefore, CAR agonists may constitute a new therapeutic tool in controlling cardiovascular disease-associated inflammatory processes.

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.

The nuclear retinoid-related orphan receptor RORα controls adipose tissue inflammation in patients with morbid obesity and diabetes.

BACKGROUND/AIMS: Inflammation governs adipose tissue (AT) dysfunction in obesity. Retinoic acid receptor-related orphan receptor alpha (RORα) is associated with inflammation and insulin resistance in animal studies, but its role in human obesity remains elusive. We investigated the expression and function of RORα on AT inflammation in patients with morbid obesity with/without diabetes. SUBJECTS/METHODS: We assessed RORα expression in paired biopsies of subcutaneous and omental AT from 41 patients (body mass index (BMI) 43.3 ± 0.8 kg/m2) during Roux-en-Y-gastric surgery and explored the functional consequences of pharmacological RORα blockade in AT ex vivo. RESULTS: RORα expression was significantly higher in omental AT than in subcutaneous AT (p = 0.03) and was positively associated with BMI (r = 0.344, p = 0.027) and homeostasis model assessment of insulin resistance (r = 0.319, p = 0.041). In ex vivo assays, IL-8/CXCL8 and MCP-1/CCL2 chemokine release was significantly higher in omental fat explants from diabetic patients than from non-diabetics and was significantly diminished by RORα blockade (p < 0.05). Inhibition of RORα improved protein kinase B signaling and decreased NF-κB activity in omental AT from patients with diabetes (p < 0.05). Under dynamic flow conditions, RORα blockade prevented mononuclear cell attachment to human dysfunctional endothelial cells. CONCLUSIONS: RORα blockade represents a potential therapy to prevent AT dysfunction and inflammation associated with insulin resistance in human obesity.

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.

SGLT-2 (Sodium-Glucose Cotransporter 2) Inhibition Reduces Ang II (Angiotensin II)-Induced Dissecting Abdominal Aortic Aneurysm in ApoE (Apolipoprotein E) Knockout Mice.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is a pathological condition of permanent vessel dilatation that predisposes to the potentially fatal consequence of aortic rupture. SGLT-2 (sodium-glucose cotransporter 2) inhibitors have emerged as powerful pharmacological tools for type 2 diabetes mellitus treatment. Beyond their glucose-lowering effects, recent studies have shown that SGLT-2 inhibitors reduce cardiovascular events and have beneficial effects on several vascular diseases such as atherosclerosis; however, the potential effects of SGLT-2 inhibition on AAA remain unknown. This study evaluates the effect of oral chronic treatment with empagliflozin-an SGLT-2 inhibitor-on dissecting AAA induced by Ang II (angiotensin II) infusion in apoE (apolipoprotein E)-/- mice. Approach and Results: Empagliflozin treatment significantly reduced the Ang II-induced increase in maximal suprarenal aortic diameter in apoE-/- mice independently of blood pressure effects. Immunohistochemistry analysis revealed that empagliflozin diminished Ang II-induced elastin degradation, neovessel formation, and macrophage infiltration at the AAA lesion. Furthermore, Ang II infusion resulted in a marked increase in the expression of chemokines (CCL-2 [chemokine (C-C motif) ligand 2] and CCL-5 [chemokine (C-C motif) ligand 5]), VEGF (vascular endothelial growth factor), and MMP (matrix metalloproteinase)-2 and MMP-9 in suprarenal aortic walls of apoE-/- mice, and all were reduced by empagliflozin cotreatment. Western blot analysis revealed that p38 MAPK (p38 mitogen-activated protein kinase) and NF-κB (nuclear factor-κB) activation was also reduced in the suprarenal aortas of apoE-/- mice cotreated with empagliflozin. Finally, in vitro studies in human aortic endothelial cells and macrophages showed that empagliflozin inhibited leukocyte-endothelial cell interactions and release of proinflammatory chemokines. CONCLUSIONS: Pharmacological inhibition of SGLT-2 by empagliflozin inhibits AAA formation. SGLT-2 inhibition might represent a novel promising therapeutic strategy to prevent AAA progression.

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.

Upregulation of angiostatic chemokines IP-10/CXCL10 and I-TAC/CXCL11 in human obesity and their implication for adipose tissue angiogenesis.

BACKGROUND/AIMS: Impaired angiogenesis is linked to adipose tissue (AT) dysfunction, inflammation, and insulin resistance in human obesity. Chemokine (C-X-C motif) receptor. (CXCR3) ligands are important regulators of angiogenesis in different disease contexts such as cancer; however, their role in human morbid obesity is unknown. We investigated the role of the CXCR3 axis in AT angiogenesis in morbidly obese patients. SUBJECTS/METHODS: The study group comprised 50 morbidly obese patients (mean age 44 ± 1 years, body mass index 44 ± 1 kg/m2) who had undergone laparoscopic Roux-Y-gastric bypass surgery, and 25 age-matched non-obese control subjects. We measured the circulating levels of the CXCR3 ligands monokine induced by interferon-γ (MIG/CXCL9), interferon-γ inducible protein 10 (IP-10/CXCL10), and interferon-γ-inducible T-cell alpha chemoattractant (I-TAC/CXCL11) in all studied subjects. Additionally, the expression of CXCR3 ligands was analyzed in paired biopsies of subcutaneous and visceral AT obtained during the laparoscopic procedure in morbidly obese patients. Additionally, we explored the functional role of CXCR3 ligands on angiogenesis in AT from morbidly obese patients using an ex vivo assay. RESULTS: Plasma levels of CXCL10 and CXCL11 were significantly higher in morbidly obese patients than in controls (p < 0.01). In ex vivo assays, angiogenic growth was markedly lower in visceral AT than in subcutaneous AT (p < 0.05), which was related to significant tissue upregulation of CXCL10, CXCL11 and CXCR3 (p < 0.05). CXCL10 or CXCL11 inhibited AT angiogenesis (p < 0.05), and blockade of CXCR3 function significantly increased capillary sprouting in visceral fat deposits (p < 0.05). Western blot analysis showed that the p38 mitogen-activated protein kinase signaling pathway was implicated in the angiostatic effects of CXCR3 in AT. CONCLUSIONS: CXCL10 and CXCL11 may play. deleterious role in obesity as potential inhibitors of AT angiogenesis. Accordingly, pharmacological blockade of CXCR3 could represent. therapy to prevent AT dysfunction in obesity.

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.