Peeking into the Stingers: A Comprehensive SWATH-MS Study of the European Hornet Vespa crabro (Linnaeus, 1758) (Hymenoptera: Vespidae) Venom Sac Extracts.

This study aimed to investigate the venom sac extracts (VSEs) of the European hornet (EH) Vespa crabro (Linnaeus, 1758) (Hymenoptera: Vespidae), focusing on the differences between stinging females, gynes (G), and workers (W), at the protein level. Using a quantitative "Sequential Window Acquisition of all Theoretical Fragment Ion Mass Spectra" (SWATH-MS) analysis, we identified and quantified a total of 240 proteins. Notably, within the group, 45.8% (n = 110) showed significant differential expression between VSE-G and VSE-W. In this set, 57.3% (n = 63) were upregulated and 42.7% (n = 47) downregulated in the G. Additionally, the two-hundred quantified proteins from the class Insecta belong to sixteen different species, six of them to the Hymenoptera/Apidae lineage, comprising seven proteins with known potential allergenicity. Thus, phospholipase A1 (Vesp v 1), phospholipase A1 verutoxin 2b (VT-2b), hyaluronidase A (Vesp v 2A), hyaluronidase B (Vesp v 2B), and venom allergen 5 (Vesp v 5) were significantly downregulated in the G, and vitellogenin (Vesp v 6) was upregulated. Overall, 46% of the VSE proteins showed differential expression, with a majority being upregulated in G. Data are available via ProteomeXchange with identifier PXD047955. These findings shed light on the proteomic differences in VSE between EH castes, potentially contributing to our understanding of their behavior and offering insights for allergy research.

The Hepatokine RBP4 Links Metabolic Diseases to Articular Inflammation.

OBJECTIVES: This study investigates the role of retinol binding protein 4 (RBP4) in an articular context. RBP4, a vitamin A transporter, is linked to various metabolic diseases. METHODS: Synovial fluid RBP4 levels were assessed in crystalline arthritis (CA) patients using ELISA. RBP4's impact on articular cell types was analysed in vitro through RT-PCR and flow cytometry. Proteomic analysis was conducted on primary human osteoarthritis chondrocytes (hOACs). RESULTS: Synovial fluid RBP4 concentrations in CA patients correlated positively with glucose levels and negatively with synovial leukocyte count and were elevated in hypertensive patients. In vitro, these RBP4 concentrations activated neutrophils, induced the expression of inflammatory factors in hOACs as well as synoviocytes, and triggered proteomic changes consistent with inflammation. Moreover, they increased catabolism and decreased anabolism, mitochondrial dysfunction, and glycolysis promotion. Both in silico and in vitro experiments suggested that RBP4 acts through TLR4. CONCLUSIONS: This study identifies relevant RBP4 concentrations in CA patients' synovial fluids, linking them to hypertensive patients with a metabolic disruption. Evidence is provided that RBP4 acts as a DAMP at these concentrations, inducing robust inflammatory, catabolic, chemotactic, and metabolic responses in chondrocytes, synoviocytes, and neutrophils. These effects may explain RBP4-related metabolic diseases' contribution to joint destruction in various rheumatic conditions like CA.

Controlling the protein corona of polymeric nanocapsules: effect of polymer shell on protein adsorption.

Understanding the interactions between nanocarriers and plasma proteins is essential for controlling their biological fate. Based on the reported potential of polymeric nanocapsules (NCs) for the targeted delivery of oncological drugs, the main objective of this work has been to investigate how the surface chemical composition influences their protein corona fingerprint. Thus, we developed six NC prototypes with different polymer shells and physicochemical properties and quantified the amount of protein adsorbed upon incubation in human plasma. Using sequential window acquisition of all theoretical mass spectra (SWATH-MS) and following the Minimum Information about Nanomaterial Biocorona Experiments (MINBE) guidelines, we identified different protein corona patterns. As expected, the presence of polyethylene glycol (PEG) in the polymer shell reduced the protein corona, particularly the adsorption of immunoglobulins. However, by comparing the different prototypes, we concluded that the protein adsorption pattern was not exclusively driven by PEG. In fact, a highly PEGylated prototype exhibited intense apolipoprotein IV adsorption. On the other hand, we also observed that polymeric NCs containing 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) exhibited high adsorption of vitronectin, a protein that is known for enhancing the uptake of nanosystems by lung epithelium and several cancer cells. Overall, the gathered information allowed us to identify promising polymeric NCs with an expected prolonged circulation time, enhanced tumor targeting, liver accumulation, and preferential uptake by the immune system. In this sense, the analyses of the protein corona performed along this work will hopefully contribute to advancing a new generation of rationally designed nanometric drug delivery systems.

Identification of Proteomic Biomarkers in Proliferative Verrucous Leukoplakia through Liquid Chromatography With Tandem Mass Spectrometry.

Proliferative verrucous leukoplakia (PVL) is an oral potentially malignant disorder associated with high risk of malignant transformation. Currently, there is no treatment available, and restrictive follow-up of patients is crucial for a better prognosis. Oral leukoplakia (OL) shares some clinical and microscopic features with PVL but exhibits different clinical manifestations and a lower rate of malignant transformation. This study aimed to investigate the proteomic profile of PVL in tissue and saliva samples to identify potential diagnostic biomarkers with therapeutic implications. Tissue and saliva samples obtained from patients with PVL were compared with those from patients with oral OL and controls. Label-free liquid chromatography with tandem mass spectrometry was employed, followed by qualitative and quantitative analyses, to identify differentially expressed proteins. Potential biomarkers were identified and further validated using immunohistochemistry. Staining intensity scan analyses were performed on tissue samples from patients with PVL, patients with OL, and controls from Brazil, Spain, and Finland. The study revealed differences in the immune system, cell cycle, DNA regulation, apoptosis pathways, and the whole proteome of PVL samples. In addition, liquid chromatography with tandem mass spectrometry analyses showed that calreticulin (CALR), receptor of activated protein C kinase 1 (RACK1), and 14-3-3 Tau-protein (YWHAQ) were highly expressed in PVL samples. Immunohistochemistry validation confirmed increased CARL expression in PVL compared with OL. Conversely, RACK1 and YWHA were highly expressed in oral potentially malignant disorder compared to the control group. Furthermore, significant differences in CALR and RACK1 expression were observed in the OL group when comparing samples with and without oral epithelial dysplasia, unlike the PVL. This research provides insights into the molecular mechanisms underlying these conditions and highlights potential targets for future diagnostic and therapeutic approaches.

Circulating extracellular vesicles promote recovery in a preclinical model of intracerebral hemorrhage.

Circulating extracellular vesicles (EVs) are proposed to participate in enhancing pathways of recovery after stroke through paracrine signaling. To verify this hypothesis in a proof-of-concept study, blood-derived allogenic EVs from rats and xenogenic EVs from humans who experienced spontaneous good recovery after an intracerebral hemorrhage (ICH) were administered intravenously to rats at 24 h after a subcortical ICH. At 28 days, both treatments improved the motor function assessment scales score, showed greater fiber preservation in the perilesional zone (diffusion tensor-fractional anisotropy MRI), increased immunofluorescence markers of myelin (MOG), and decreased astrocyte markers (GFAP) compared with controls. Comparison of the protein cargo of circulating EVs at 28 days from animals with good vs. poor recovery showed down-expression of immune system activation pathways (CO4, KLKB1, PROC, FA9, and C1QA) and of restorative processes such as axon guidance (RAC1), myelination (MBP), and synaptic vesicle trafficking (SYN1), which is in line with better tissue preservation. Up-expression of PCSK9 (neuron differentiation) in xenogenic EVs-treated animals suggests enhancement of repair pathways. In conclusion, the administration of blood-derived EVs improved recovery after ICH. These findings open a new and promising opportunity for further development of restorative therapies to improve the outcomes after an ICH.

Proteomics of Vespa velutina nigrithorax Venom Sac Queens and Workers: A Quantitative SWATH-MS Analysis.

Health risks caused by stings from Vespa velutina nigrithorax (VV), also known as the yellow-legged Asian hornet, have become a public concern, but little is known about its venom composition. This study presents the proteome profile of the VV's venom sac (VS) based on Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS). The study also performed proteomic quantitative analysis and examined the biological pathways and molecular functions of the proteins in the VS of VV gynes (i.e., future queens [SQ]) and workers [SW]. The total protein content per VS was significantly higher in the SW than in the SQ (274 ± 54 µg/sac vs. 175 ± 22 µg/sac; p = 0.02). We quantified a total of 228 proteins in the VS, belonging to 7 different classes: Insecta (n = 191); Amphibia and Reptilia (n = 20); Bacilli, γ-Proteobacteria and Pisoniviricetes (n = 12); and Arachnida (n = 5). Among the 228 identified proteins, 66 showed significant differential expression between SQ and SW. The potential allergens hyaluronidase A, venom antigen 5 and phospholipase A1 were significantly downregulated in the SQ venom.

Brown Adipose Tissue Sheds Extracellular Vesicles That Carry Potential Biomarkers of Metabolic and Thermogenesis Activity Which Are Affected by High Fat Diet Intervention.

Brown adipose tissue (BAT) is a key target for the development of new therapies against obesity due to its role in promoting energy expenditure; BAT secretory capacity is emerging as an important contributor to systemic effects, in which BAT extracellular vesicles (EVs) (i.e., batosomes) might be protagonists. EVs have emerged as a relevant cellular communication system and carriers of disease biomarkers. Therefore, characterization of the protein cargo of batosomes might reveal their potential as biomarkers of the metabolic activity of BAT. In this study, we are the first to isolate batosomes from lean and obese Sprague-Dawley rats, and to establish reference proteome maps. An LC-SWATH/MS analysis was also performed for comparisons with EVs secreted by white adipose tissue (subcutaneous and visceral WAT), and it showed that 60% of proteins were exclusive to BAT EVs. Precisely, batosomes of lean animals contain proteins associated with mitochondria, lipid metabolism, the electron transport chain, and the beta-oxidation pathway, and their protein cargo profile is dramatically affected by high fat diet (HFD) intervention. Thus, in obesity, batosomes are enriched with proteins involved in signal transduction, cell communication, the immune response, inflammation, thermogenesis, and potential obesity biomarkers including UCP1, Glut1, MIF, and ceruloplasmin. In conclusion, the protein cargo of BAT EVs is affected by the metabolic status and contains potential biomarkers of thermogenesis activity.

Repurposing drugs to inhibit innate immune responses associated with TLR4, IL1, and NLRP3 signaling in joint cells.

Osteoarthritis (OA) affects more than 300 million people worldwide and it is about to become the first disabling disease. OA is characterized by the progressive degradation of the articular cartilage but is a disease of the whole joint. Articular innate immune responses (IIR) associated with tissue degradation contribute to its progression. However, no treatment is available to block these IIRs. Through data text mining and computational pharmacology, we identified two clinical available drugs, naloxone, and thalidomide, with potential inhibitory properties on toll-like receptor 4 (TLR4), a major activator of these IIR. Proteome analysis confirmed that activation of this receptor or the IL1 receptor generated OA-like and gout-like proteomic changes in human primary chondrocytes. Both compounds were found to block TLR4 complex and inhibit TLR4 and IL1R-mediated IIR in OA chondrocytes, osteoblasts, and synoviocytes. Furthermore, naloxone and thalidomide inhibitory effects involved the downregulation of the NLRP3 inflammasome pathway, which is downstream of TLR4/IL1R signaling. We demonstrated that these compounds, within a therapeutic range of concentrations, exhibited anti-inflammatory and anti-catabolic properties in joint primary OA cells without any toxic effect. This data underpins naloxone & thalidomide repurpose to treat OA-associated inflammatory responses.

Shotgun Proteomics for the Identification and Profiling of the Tear Proteome of Keratoconus Patients.

Purpose: The qualitative approach followed in this study aims to obtain an extensive view of the keratoconus (KC) tear proteome, which could highlight proteins previously undetected and enlarge our knowledge of the disease's pathophysiology. Methods: Twenty-five patients diagnosed with KC and 25 control subjects were studied in a prospective, cross-sectional study. KC screening examinations, including clinical and tomographic examinations, were performed on all participants. Tear samples were collected using Schirmer strips and analyzed by liquid chromatography-tandem mass spectrometry in a data-dependent workflow. A spectral count was used as a semiquantification tool. The tear proteomes of both groups were identified and profiled, and the functional interactions and biological characterization of differential proteins were analyzed using in silico tools. Results: We identified a total of 232 proteins, of whom 133 were expressed in both groups' samples; 41 were observed only in control samples and 58 were identified just in tears of patients with KC. A semiquantitative analysis showed the dysregulation of 17 proteins in the KC samples. An in silico analysis linked proteins only expressed in KC samples to oxidative stress, skin development, and apoptosis. The dysregulation of proteins involved in iron transport, inflammation, oxidative stress, and protease inhibition was observed in the semiquantitative results. Conclusions: A shotgun analysis showed that the tear proteome of patients with KC differed from controls by more than one-third of the total proteins identified, highlighting the relationship of the proteins only expressed in KC tears with processes of cell death, oxidative damage, and inflammation. The underexpression of proteins involved in iron pathways might support the iron imbalance as a contributing factor to cellular damage and death in KC disease.

Human obese white adipose tissue sheds depot-specific extracellular vesicles and reveals candidate biomarkers for monitoring obesity and its comorbidities.

Extracellular vesicles (EVs) have been recently postulated as key players in metabolic disorders emerging as an alternative way of paracrine/endocrine communication. However, the nature of EVs shed by adipose tissue (AT) and their role in obesity is still very limited. Here, we isolated human morbid obese visceral (VAT) and subcutaneous (SAT) whole AT shed EVs from donors submitted to bariatric surgery to characterize their protein cargo by qualitative and quantitative/SWATH mass spectrometry analysis. We identified 574 different proteins shed by morbid obese VAT and 401 proteins in those from SAT, establishing the first obese AT EV proteome reference map. Only 50% of identified proteins in VAT vesicles were common to those in SAT; additionally, EVs shed by obese VAT showed more AT and obesity-related adipokines than SAT. Functional classification shows that obese VAT vesicles exhibit an enrichment of proteins implicated in AT inflammation and insulin resistance such as TGFBI, CAVN1, CD14, mimecan, thrombospondin-1, FABP-4 or AHNAK. Selected candidate biomarkers from the quantitative-SWATH analysis were validated in EVs from independent morbid obese and from moderate obese to lean individuals showing that morbid obese VAT vesicles are characterized by a diminution of syntenin 1 and the elevation of TGFBI and mimecan. Interestingly, TGFBI and mimecan containing vesicles could be detected and quantified at circulating level in plasma. Thus, a significant elevation of -TGFBI-EVs was detected on those obese patients with a history of T2D compared to nondiabetic, and an augmentation of mimecan-EVs in obese plasma compared to those in healthy lean individuals. Thus, we conclude that obese AT release functional EVs carrying AT and obesity candidate biomarkers which vary regarding the AT of origin. Our findings suggest that circulating EV-TGFBI may facilitate monitoring T2D status in obese patients, and EV-mimecan may be useful to track adiposity, and more precisely, visceral obesity.

Protein content of blood-derived extracellular vesicles: An approach to the pathophysiology of cerebral hemorrhage.

Introduction: Extracellular vesicles (EVs) participate in cell-to-cell paracrine signaling and can be biomarkers of the pathophysiological processes underlying disease. In intracerebral hemorrhage, the study of the number and molecular content of circulating EVs may help elucidate the biological mechanisms involved in damage and repair, contributing valuable information to the identification of new therapeutic targets. Methods: The objective of this study was to describe the number and protein content of blood-derived EVs following an intracerebral hemorrhage (ICH). For this purpose, an experimental ICH was induced in the striatum of Sprague-Dawley rats and EVs were isolated and characterized from blood at baseline, 24 h and 28 days. The protein content in the EVs was analyzed by mass spectrometric data-dependent acquisition; protein quantification was obtained by sequential window acquisition of all theoretical mass spectra data and compared at pre-defined time points. Results: Although no differences were found in the number of EVs, the proteomic study revealed that proteins related to the response to cellular damage such as deubiquitination, regulation of MAP kinase activity (UCHL1) and signal transduction (NDGR3), were up-expressed at 24 h compared to baseline; and that at 28 days, the protein expression profile was characterized by a higher content of the proteins involved in healing and repair processes such as cytoskeleton organization and response to growth factors (COR1B) and the regulation of autophagy (PI42B). Discussion: The protein content of circulating EVs at different time points following an ICH may reflect evolutionary changes in the pathophysiology of the disease.

Cardiac Extracellular Matrix Hydrogel Enriched with Polyethylene Glycol Presents Improved Gelation Time and Increased On-Target Site Retention of Extracellular Vesicles.

Stem-cell-derived extracellular vesicles (EVs) have demonstrated multiple beneficial effects in preclinical models of cardiac diseases. However, poor retention at the target site may limit their therapeutic efficacy. Cardiac extracellular matrix hydrogels (cECMH) seem promising as drug-delivery materials and could improve the retention of EVs, but may be limited by their long gelation time and soft mechanical properties. Our objective was to develop and characterize an optimized product combining cECMH, polyethylene glycol (PEG), and EVs (EVs-PEG-cECMH) in an attempt to overcome their individual limitations: long gelation time of the cECMH and poor retention of the EVs. The new combined product presented improved physicochemical properties (60% reduction in half gelation time, p < 0.001, and threefold increase in storage modulus, p < 0.01, vs. cECMH alone), while preserving injectability and biodegradability. It also maintained in vitro bioactivity of its individual components (55% reduction in cellular senescence vs. serum-free medium, p < 0.001, similar to EVs and cECMH alone) and increased on-site retention in vivo (fourfold increase vs. EVs alone, p < 0.05). In conclusion, the combination of EVs-PEG-cECMH is a potential multipronged product with improved gelation time and mechanical properties, increased on-site retention, and maintained bioactivity that, all together, may translate into boosted therapeutic efficacy.

Qualitative and Quantitative Comparison of Plasma Exosomes from Neonates and Adults.

Exosomes are extracellular vesicles that contain nucleic acids, lipids and metabolites, and play a critical role in health and disease as mediators of intercellular communication. The majority of extracellular vesicles in the blood are platelet-derived. Compared to adults, neonatal platelets are hyporeactive and show impaired granule release, associated with defects in Soluble N-ethylmaleimide-sensitive fusion Attachment protein REceptor (SNARE) proteins. Since these proteins participate in biogenesis of exosomes, we investigated the potential differences between newborn and adult plasma-derived exosomes. Plasma-derived exosomes were isolated by ultracentrifugation of umbilical cord blood from full-term neonates or peripheral blood from adults. Exosome characterization included size determination by transmission electron microscopy and quantitative proteomic analysis. Plasma-derived exosomes from neonates were significantly smaller and contained 65% less protein than those from adults. Remarkably, 131 proteins were found to be differentially expressed, 83 overexpressed and 48 underexpressed in neonatal (vs. adult) exosomes. Whereas the upregulated proteins in plasma exosomes from neonates are associated with platelet activation, coagulation and granule secretion, most of the underexpressed proteins are immunoglobulins. This is the first study showing that exosome size and content change with age. Our findings may contribute to elucidating the potential "developmental hemostatic mismatch risk" associated with transfusions containing plasma exosomes from adults.

Impact of liver-specific GLUT8 silencing on fructose-induced inflammation and omega oxidation.

Excessive consumption of high-fructose diets is associated with insulin resistance, obesity, and non-alcoholic fatty liver disease (NAFLD). However, fructose differentially affects hepatic regulation of lipogenesis in males and females. Hence, additional studies are necessary in order to find strategies taking gender disparities in fructose-induced liver damage into consideration. Although the eighth member of facilitated glucose transporters (GLUT8) has been linked to fructose-induced macrosteatosis in female mice, its contribution to the inflammatory state of NAFLD remains to be elucidated. Combining pharmacological, biochemical, and proteomic approaches, we evaluated the preventive effect of targeted liver GLUT8 silencing on liver injury in a mice female fructose-induced non-alcoholic steatohepatitis female mouse model. Liver GLUT8-knockdown attenuated fructose-induced ER stress, recovered liver inflammation, and dramatically reduced fatty acid content, in part, via the omega oxidation. Therefore, this study links GLUT8 with liver inflammatory response and suggests GLUT8 as a potential target for the prevention of NAFLD.

Beneficial Effects of Bariatric Surgery-Induced by Weight Loss on the Proteome of Abdominal Subcutaneous Adipose Tissue.

Bariatric surgery (BS) is the most effective treatment for obesity and has a positive impact on cardiometabolic risk and in the remission of type 2 diabetes. Following BS, the majority of fat mass is lost from the subcutaneous adipose tissue depot (SAT). However, the changes in this depot and functions and as well as its relative contribution to the beneficial effects of this surgery are still controversial. With the aim of studying altered proteins and molecular pathways in abdominal SAT (aSAT) after body weight normalization induced by BS, we carried out a proteomic approach sequential window acquisition of all theoretical mass spectra (SWATH-MS) analysis. These results were complemented by Western blot, electron microscopy and RT-qPCR. With all of the working tools mentioned, we confirmed that after BS, up-regulated proteins were associated with metabolism, the citric acid cycle and respiratory electron transport, triglyceride catabolism and metabolism, formation of ATP, pyruvate metabolism, glycolysis/gluconeogenesis and thermogenesis among others. In contrast, proteins with decreased values are part of the biological pathways related to the immune system. We also confirmed that obesity caused a significant decrease in mitochondrial density and coverage, which was corrected by BS. Together, these findings reveal specific molecular mechanisms, genes and proteins that improve adipose tissue function after BS characterized by lower inflammation, increased glucose uptake, higher insulin sensitivity, higher de novo lipogenesis, increased mitochondrial function and decreased adipocyte size.

Proteomic identification of putative biomarkers for early detection of sudden cardiac death in a family with a LMNA gene mutation causing dilated cardiomyopathy.

UNLABELLED: Dilated cardiomyopathy (DCM) is a severe heart disease characterized by progressive ventricular dilation and impaired systolic function of the left ventricle. We recently identified a novel pathogenic mutation in the LMNA gene in a family affected by DCM showing sudden death background. We now aimed to identify potential biomarkers of disease status, as well as sudden death predictors, in members of this family. We analysed plasma samples from 14 family members carrying the mutation, four of which (with relevant clinical symptoms) were chosen for the proteomic analysis. Plasma samples from these four patients and from four sex- and age-matched healthy controls were processed for their enrichment in low- and medium-abundance proteins (ProteoMiner™) prior to proteomic analysis by 2D-DIGE and MS. 111 spots were found to be differentially regulated between mutation carriers and control groups, 83 of which were successfully identified by MS, corresponding to 41 different ORFs. Some proteins of interest were validated either by turbidimetry or western blot in family members and healthy controls. Actin, alpha-1-antytripsin, clusterin, vitamin-D binding protein and antithrombin-III showed increased levels in plasma from the diseased group. We suggest following these proteins as putative biomarkers for the evaluation of DCM status in LMNA mutation carriers. BIOLOGICAL SIGNIFICANCE: We developed a proteomic analysis of plasma samples from a family showing history of dilated cardiomyopathy caused by a LMNA mutation, which may lead to premature death or cardiac transplant. We identified a number of proteins augmented in mutation carriers that could be followed as potential biomarkers for dilated cardiomyopathy on these patients.

2D-DIGE-based proteomic analysis of intracoronary versus peripheral arterial blood platelets from acute myocardial infarction patients: Upregulation of platelet activation biomarkers at the culprit site.

PURPOSE: Platelets play a fundamental role in the atherothrombotic events that lead to an acute myocardial infarction. In the present study we compared the proteome of intracoronary and peripheral arterial platelets from ST-elevation myocardial infarction (STEMI) patients in the search for potential platelet biomarkers/drug targets related to what is happening at the culprit site. EXPERIMENTAL DESIGN: Ten STEMI patients were recruited and blood collected from the occluded coronary artery, at the culprit site, in the moment of reperfusion. Systemic blood obtained from the radial artery of the same patients was used as control. Proteome analysis was based on high-resolution 2D-DIGE and mass spectrometry. Validations were by western blotting in a group of 11 patients. RESULTS: Sixteen differentially regulated protein features were identified, corresponding to 15 ORFs, mostly related to cytoskeletal and signaling proteins. We demonstrate the up-regulation of integrin αIIb (ITA2B), the adapter Src kinase-associated phosphoprotein-2 (SKAP2), and thrombospondin-1 isoforms in intracoronary platelets. CONCLUSION AND CLINICAL RELEVANCE: This study constitutes the first analyzing in detail the proteome of arterial intracoronary platelets from STEMI patients. We show variations in the platelet proteome when comparing intracoronary and peripheral platelets. Observed differences might be related to platelet activation events at the culprit site.

CILAIR-Based Secretome Analysis of Obese Visceral and Subcutaneous Adipose Tissues Reveals Distinctive ECM Remodeling and Inflammation Mediators.

In the context of obesity, strong evidences support a distinctive pathological contribution of adipose tissue depending on its anatomical site of accumulation. Therefore, subcutaneous adipose tissue (SAT) has been lately considered metabolically benign compared to visceral fat (VAT), whose location is associated to the risk of developing cardiovascular disease, insulin resistance, and other associated comorbidities. Under the above situation, the chronic local inflammation that characterizes obese adipose tissue, has acquired a major role on the pathogenesis of obesity. In this work, we have analyzed for the first time human obese VAT and SAT secretomes using an improved quantitative proteomic approach for the study of tissue secretomes, Comparison of Isotope-Labeled Amino acid Incorporation Rates (CILAIR). The use of double isotope-labeling-CILAIR approach to analyze VAT and SAT secretomes allowed the identification of location-specific secreted proteins and its differential secretion. Additionally to the very high percentage of identified proteins previously implicated in obesity or in its comorbidities, this approach was revealed as a useful tool for the study of the obese adipose tissue microenvironment including extracellular matrix (ECM) remodeling and inflammatory status. The results herein presented reinforce the fact that VAT and SAT depots have distinct features and contribute differentially to metabolic disease.