Impaired Plakophilin-2 in obesity breaks cell cycle dynamics to breed adipocyte senescence.

Plakophilin-2 (PKP2) is a key component of desmosomes, which, when defective, is known to promote the fibro-fatty infiltration of heart muscle. Less attention has been given to its role in adipose tissue. We report here that levels of PKP2 steadily increase during fat cell differentiation, and are compromised if adipocytes are exposed to a pro-inflammatory milieu. Accordingly, expression of PKP2 in subcutaneous adipose tissue diminishes in patients with obesity, and normalizes upon mild-to-intense weight loss. We further show defective PKP2 in adipocytes to break cell cycle dynamics and yield premature senescence, a key rheostat for stress-induced adipose tissue dysfunction. Conversely, restoring PKP2 in inflamed adipocytes rewires E2F signaling towards the re-activation of cell cycle and decreased senescence. Our findings connect the expression of PKP2 in fat cells to the physiopathology of obesity, as well as uncover a previously unknown defect in cell cycle and adipocyte senescence due to impaired PKP2.

Menstrual blood-derived mesenchymal stromal cells: impact of preconditioning on the cargo of extracellular vesicles as potential therapeutics.

BACKGROUND: Mesenchymal stromal cells (MSCs) have been shown to exert their therapeutic effects through the secretion of broad spectrum of paracrine factors, including extracellular vesicles (EVs). Accordingly, EVs are being pursued as a promising alternative to cell-based therapies. Menstrual blood-derived stromal cells (MenSCs) are a type of MSC that, due to their immunomodulatory and regenerative properties, have emerged as an innovative source. Additionally, new strategies of cell priming may potentially alter the concentration and cargo of released EVs, leading to modification of their biological properties. In this study, we aimed to characterize the EVs released by MenSCs and compare their therapeutic potential under three different preconditioning conditions (proinflammatory stimuli, physioxia, and acute hypoxia). METHODS: MenSCs were isolated from five healthy women. Following culturing to 80% confluence, MenSCs were exposed to different priming conditions: basal (21% O2), proinflammatory stimuli (IFNγ and TNFα, 21% O2), physioxia (1-2% O2), and acute hypoxia (< 1% O2) for 48-72 h. Conditioned media from MenSCs was collected after 48 h and EVs were isolated by a combination of ultra-filtration and differential centrifugation. An extensive characterization ranging from nano-flow cytometry (nFC) to quantitative high-throughput shotgun proteomics was performed. Bioinformatics analyses were used to derive hypotheses on their biological properties. RESULTS: No differences in the morphology, size, or number of EVs released were detected between priming conditions. The proteome analysis associated with basal MenSC-EVs prominently revealed their immunomodulatory and regenerative capabilities. Furthermore, quantitative proteomic analysis of differentially produced MenSC-EVs provided sufficient evidence for the utility of the differential preconditioning in purpose-tailoring EVs for their therapeutic application: proinflammatory priming enhanced the anti-inflammatory, regenerative and immunomodulatory capacity in the innate response of EVs, physioxia priming also improves tissue regeneration, angiogenesis and their immunomodulatory capacity targeting on the adaptive response, while acute hypoxia priming, increased hemostasis and apoptotic processes regulation in MenSC-EVs, also by stimulating immunomodulation mainly through the adaptive response. CONCLUSIONS: Priming of MenSCs under proinflammatory and hypoxic conditions affected the cargo proteome of EVs released, resulting in different therapeutic potential, and thus warrants experimental exploration with the aim to generate better-defined MSC-derived bioproducts.

Unbiased plasma proteomics discovery of biomarkers for improved detection of subclinical atherosclerosis.

BACKGROUND: Imaging of subclinical atherosclerosis improves cardiovascular risk prediction on top of traditional risk factors. However, cardiovascular imaging is not universally available. This work aims to identify circulating proteins that could predict subclinical atherosclerosis. METHODS: Hypothesis-free proteomics was used to analyze plasma from 444 subjects from PESA cohort study (222 with extensive atherosclerosis on imaging, and 222 matched controls) at two timepoints (three years apart) for discovery, and from 350 subjects from AWHS cohort study (175 subjects with extensive atherosclerosis on imaging and 175 matched controls) for external validation. A selected three-protein panel was further validated by immunoturbidimetry in the AWHS population and in 2999 subjects from ILERVAS cohort study. FINDINGS: PIGR, IGHA2, APOA, HPT and HEP2 were associated with subclinical atherosclerosis independently from traditional risk factors at both timepoints in the discovery and validation cohorts. Multivariate analysis rendered a potential three-protein biomarker panel, including IGHA2, APOA and HPT. Immunoturbidimetry confirmed the independent associations of these three proteins with subclinical atherosclerosis in AWHS and ILERVAS. A machine-learning model with these three proteins was able to predict subclinical atherosclerosis in ILERVAS (AUC [95%CI]:0.73 [0.70-0.74], p < 1 × 10-99), and also in the subpopulation of individuals with low cardiovascular risk according to FHS 10-year score (0.71 [0.69-0.73], p < 1 × 10-69). INTERPRETATION: Plasma levels of IGHA2, APOA and HPT are associated with subclinical atherosclerosis independently of traditional risk factors and offers potential to predict this disease. The panel could improve primary prevention strategies in areas where imaging is not available. FUNDING: This study was supported by competitive grants from the Spanish Ministry of Science, Innovation and Universities (BIO2015-67580-P, PGC2018-097019-B-I00, PID2019-106814RB-I00 and SAF2016-80843-R), through the Carlos III Institute of Health-Fondo de Investigacion Sanitaria grant PRB3 (IPT17/0019 - ISCIII-SGEFI / ERDF, ProteoRed), CIBERCV and CIBERDEM, the Fundacio MaratoTV3 (grant 122/C/2015) and "la Caixa" Banking Foundation (project HR17-00247). The PESA study is co-funded equally by the Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain, and Banco Santander, Madrid, Spain. The ILERVAS study was funded by the Diputacio de Lleida. The study also receives funding from the Instituto de Salud Carlos III (PI15/02019; PI18/00610; RD16/0009) and the FEDER funds. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia, Innovacion y Universidades (MCNU) and the Pro CNIC Foundation.

IFN-Gamma and TNF-Alpha as a Priming Strategy to Enhance the Immunomodulatory Capacity of Secretomes from Menstrual Blood-Derived Stromal Cells.

Mesenchymal stromal cells isolated from menstrual blood (MenSCs) exhibit a potent pro-angiogenic and immunomodulatory capacity. Their therapeutic effect is mediated by paracrine mediators released by their secretomes. In this work, we aimed to evaluate the effect of a specific priming condition on the phenotype and secretome content of MenSCs. Our results revealed that the optimal condition for priming MenSCs was the combination of interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) that produced a synergistic and additive effect on IDO1 release and immune-related molecule expression. The analyses of MenSC-derived secretomes after IFNγ and TNFα priming also revealed an increase in EV release and in the differentially expressed miRNAs involved in the immune response and inflammation. Proliferation assays on lymphocyte subsets demonstrated a decrease in CD4+ T cells and CD8+ T cells co-cultured with secretomes, especially in the lymphocytes co-cultured with secretomes from primed cells. Additionally, the expression of immune checkpoints (PD-1 and CTLA-4) was increased in the CD4+ T cells co-cultured with MenSC-derived secretomes. These findings demonstrate that the combination of IFNγ and TNFα represents an excellent priming strategy to enhance the immunomodulatory capacity of MenSCs. Moreover, the secretome derived from primed MenSCs may be postulated as a therapeutic option for the regulation of adverse inflammatory reactions.

Early renal and vascular damage within the normoalbuminuria condition.

OBJECTIVE: A continuous association between albuminuria and cardiorenal risk exists further below moderately increased albuminuria ranges. If only based in albumin to creatinine ratio (ACR) higher than 30 mg/g, a significant percentage of individuals may be out of the scope for therapeutic management. Despite epidemiological outcomes, the identification of biochemical changes linked to early albuminuria is underexplored, and normoalbuminuric individuals are usually considered at no risk in clinical practice. Here, we aimed to identify early molecular alterations behind albuminuria development. METHODS: Hypertensive patients under renin-angiotensin system (RAS) suppression were classified as control, (ACR < 10 mg/g) or high-normal (ACR = 10-30 mg/g). Urinary protein alterations were quantified and confirmed by untargeted and targeted mass spectrometry. Coordinated protein responses with biological significance in albuminuria development were investigated. Immunohistochemistry assays were performed in human kidney and arterial tissue to in situ evaluate the associated damage. RESULTS: A total of 2663 identified proteins reflect inflammation, immune response, ion transport and lipids metabolism (P value ≤ 0.01). A1AT, VTDB and KNG1 varied in high-normal individuals (P value < 0.05), correlated with ACR and associated with the high-normal condition (odds ratio of 20.76, 6.00 and 7.04 were found, respectively (P value < 0.001)). After 12 months, protein variations persist and aggravate in progressors to moderately increased albuminuria. At tissue level, differential protein expression was found in kidney from individuals with moderately increased albuminuria and atherosclerotic aortas for the three proteins, confirming their capacity to reflect subclinical organ damage. CONCLUSION: Early renal and vascular damage is molecularly evidenced within the normoalbuminuria condition.

Beyond the Extracellular Vesicles: Technical Hurdles, Achieved Goals and Current Challenges When Working on Adipose Cells.

Adipose tissue and its crosstalk with other organs plays an essential role in the metabolic homeostasis of the entire body. Alteration of this communication (i.e., due to obesity) is related to the development of several comorbidities including type 2 diabetes, cardiovascular diseases, or cancer. Within the adipose depot, adipocytes are the main cell type and thus the main source of secreted molecules, which exert modulating effects not only at a local but also at a systemic level. Extracellular vesicles (EVs) have recently emerged as important mediators in cell-cell communication and account for part of the cellular secretome. In recent years, there has been a growing body of research on adipocyte-derived extracellular vesicles (Ad-EVs). However, there is still a lack of standardized methodological approaches, especially regarding primary adipocytes. In this review, we will provide an outline of crucial aspects when working on adipose-derived material, with a special focus on primary adipocytes. In parallel, we will point out current methodological challenges in the EV field and how they impact the transcriptomic, proteomic and functional evaluations of Ad-EVs.

Phosphoproteomics identify arachidonic-acid-regulated signal transduction pathways modulating macrophage functions with implications for ovarian cancer.

Arachidonic acid (AA) is a polyunsaturated fatty acid present at high concentrations in the ovarian cancer (OC) microenvironment and associated with a poor clinical outcome. In the present study, we have unraveled a potential link between AA and macrophage functions. Methods: AA-triggered signal transduction was studied in primary monocyte-derived macrophages (MDMs) by phosphoproteomics, transcriptional profiling, measurement of intracellular Ca2+ accumulation and reactive oxygen species production in conjunction with bioinformatic analyses. Functional effects were investigated by actin filament staining, quantification of macropinocytosis and analysis of extracellular vesicle release. Results: We identified the ASK1 - p38δ/α (MAPK13/14) axis as a central constituent of signal transduction pathways triggered by non-metabolized AA. This pathway was induced by the Ca2+-triggered activation of calmodulin kinase II, and to a minor extent by ROS generation in a subset of donors. Activated p38 in turn was linked to a transcriptional stress response associated with a poor relapse-free survival. Consistent with the phosphorylation of the p38 substrate HSP27 and the (de)phosphorylation of multiple regulators of Rho family GTPases, AA impaired actin filament organization and inhibited actin-driven macropinocytosis. AA also affected the phosphorylation of proteins regulating vesicle biogenesis, and consistently, AA enhanced the release of tetraspanin-containing exosome-like vesicles. Finally, we identified phospholipase A2 group 2A (PLA2G2A) as the clinically most relevant enzyme producing extracellular AA, providing further potentially theranostic options. Conclusion: Our results suggest that AA contributes to an unfavorable clinical outcome of OC by impacting the phenotype of tumor-associated macrophages. Besides critical AA-regulated signal transduction proteins identified in the present study, PLA2G2A might represent a potential prognostic tool and therapeutic target to interfere with OC progression.

The Immunomodulatory Signature of Extracellular Vesicles From Cardiosphere-Derived Cells: A Proteomic and miRNA Profiling.

Experimental data demonstrated that the regenerative potential and immunomodulatory capacity of cardiosphere-derived cells (CDCs) is mediated by paracrine mechanisms. In this process, extracellular vesicles derived from CDCs (EV-CDCs) are key mediators of their therapeutic effect. Considering the future applicability of these vesicles in human diseases, an accurate preclinical-to-clinical translation is needed, as well as an exhaustive molecular characterization of animal-derived therapeutic products. Based on that, the main goal of this study was to perform a comprehensive characterization of proteins and miRNAs in extracellular vesicles from porcine CDCs as a clinically relevant animal model. The analysis was performed by identification and quantification of proteins and miRNA expression profiles. Our results revealed the presence of clusters of immune-related and cardiac-related molecular biomarkers in EV-CDCs. Additionally, considering that priming stem cells with inflammatory stimuli may increase the therapeutic potential of released vesicles, here we studied the dynamic changes that occur in the extracellular vesicles from IFNγ-primed CDCs. These analyses detected statistically significant changes in several miRNAs and proteins. Notably, the increase in interleukin 6 (IL6) protein, as well as the increase in mir-125b (that targets IL6 receptor) was especially relevant. These results suggest a potential involvement of EV-CDCs in the regulation of the IL6/IL6R axis, with implications in inflammatory-mediated diseases.

Improved integrative analysis of the thiol redox proteome using filter-aided sample preparation.

Changes in the oxidation state of protein Cys residues are involved in cell signalling and play a key role in a variety of pathophysiological states. We had previously developed GELSILOX, an in-gel method that enables the large-scale, parallel analysis of dynamic alterations to the redox state of Cys sites and protein abundance changes. Here we present FASILOX, a further development of the GELSILOX approach featuring: i) significantly increased peptide recovery, ii) enhanced sensitivity for the detection of Cys oxidative alterations, and iii) streamlined workflow that results in shortened assay duration. In mitochondria isolated from the adipose tissue of obese, diabetic patients, FASILOX revealed a sexually dimorphic trait of Cys oxidation involving mainly mitochondrial oxidative phosphorylation complexes. These results provide the first evidence for a decreased efficiency in the antioxidant response of men as compared to women.

Cytoskeletal transgelin 2 contributes to gender-dependent adipose tissue expandability and immune function.

During adipogenesis, preadipocytes' cytoskeleton reorganizes in parallel with lipid accumulation. Failure to do so may impact the ability of adipose tissue (AT) to shift between lipid storage and mobilization. Here, we identify cytoskeletal transgelin 2 (TAGLN2) as a protein expressed in AT and associated with obesity and inflammation, being normalized upon weight loss. TAGLN2 was primarily found in the adipose stromovascular cell fraction, but inflammation, TGF-ß, and estradiol also prompted increased expression in human adipocytes. Tagln2 knockdown revealed a key functional role, being required for proliferation and differentiation of fat cells, whereas transgenic mice overexpressing Tagln2 using the adipocyte protein 2 promoter disclosed remarkable sex-dependent variations, in which females displayed "healthy" obesity and hypertrophied adipocytes but preserved insulin sensitivity, and males exhibited physiologic changes suggestive of defective AT expandability, including increased number of small adipocytes, activation of immune cells, mitochondrial dysfunction, and impaired metabolism together with decreased insulin sensitivity. The metabolic relevance and sexual dimorphism of TAGLN2 was also outlined by genetic variants that may modulate its expression and are associated with obesity and the risk of ischemic heart disease in men. Collectively, current findings highlight the contribution of cytoskeletal TAGLN2 to the obese phenotype in a gender-dependent manner.-Ortega, F. J., Moreno-Navarrete, J. M., Mercader, J. M., Gómez-Serrano, M., García-Santos, E., Latorre, J., Lluch, A., Sabater, M., Caballano-Infantes, E., Guzmán, R., Macías-González, M., Buxo, M., Gironés, J., Vilallonga, R., Naon, D., Botas, P., Delgado, E., Corella, D., Burcelin, R., Frühbeck, G., Ricart, W., Simó, R., Castrillon-Rodríguez, I., Tinahones, F. J., Bosch, F., Vidal-Puig, A., Malagón, M. M., Peral, B., Zorzano, A., Fernández-Real, J. M. Cytoskeletal transgelin 2 contributes to gender-dependent adipose tissue expandability and immune function.

APOA1 oxidation is associated to dysfunctional high-density lipoproteins in human abdominal aortic aneurysm.

BACKGROUND: High-density lipoproteins (HDL) are a complex mixture of lipids and proteins with vasculoprotective properties. However, HDL components could suffer post-translational modifications (PTMs) under pathological conditions, leading to dysfunctional HDL. We studied whether HDL are modified in abdominal aortic aneurysm (AAA) and the effect on HDL functionality. METHODS: HDL were isolated by ultracentrifugation from AAA tissue (HDL-T) and from plasma of healthy volunteers and then incubated with AAA tissue-conditioned medium (HDL-AAA CM). PTMs from these particles were characterized using Comet-PTM. The ability of HDL-AAA CM for promoting cholesterol efflux was determined ex vivo and in vivo by using J774A.1 [3H]cholesterol-labeled mouse macrophages and after injecting [3H]cholesterol-labeled mouse macrophages and HDL into the peritoneal cavity of wild-type C57BL/6 mice, respectively. Trp50 and Trp108 oxidized forms of APOA1 in HDL incubated with conditioned-medium of activated neutrophils and in plasma of AAA patients and controls were measured by targeted parallel reaction monitoring. FINDINGS: Oxidation was the most prevalent PTM in apolipoproteins, particularly in APOA1. Trp50 and Trp108 in APOA1 were the residues most clearly affected by oxidation in HDL-T and in HDL-AAA CM, when compared to their controls. In addition, cholesterol efflux was decreased in macrophages incubated with HDL-AAA CM in vitro and a decreased macrophage-to-serum reverse cholesterol transport was also observed in mice injected with HDL-AAA CM. Finally, both oxidized Trp50 and Trp108 forms of APOA1 were increased in HDL incubated with conditioned-medium of activated neutrophils and in plasma of AAA patients in relation to controls. INTERPRETATION: Oxidative modifications of HDL present in AAA tissue and plasma were closely associated with the loss of vasculoprotective properties of HDL in AAA. FUND: MINECO, ISCiii-FEDER, CIBERDEM, CIBERCV and LA CAIXA.

Interplay between post-translational cyclooxygenase-2 modifications and the metabolic and proteomic profile in a colorectal cancer cohort.

BACKGROUND: Colorectal cancer (CRC) is the second most common cause of cancer death worldwide. It is broadly described that cyclooxygenase-2 (COX-2) is mainly overexpressed in CRC but less is known regarding post-translational modifications of this enzyme that may regulate its activity, intracellular localization and stability. Since metabolic and proteomic profile analysis is essential for cancer prognosis and diagnosis, our hypothesis is that the analysis of correlations between these specific parameters and COX-2 state in tumors of a high number of CRC patients could be useful for the understanding of the basis of this cancer in humans. AIM: To analyze COX-2 regulation in colorectal cancer and to perform a detailed analysis of their metabolic and proteomic profile. METHODS: Biopsies from both healthy and pathological colorectal tissues were taken under informed consent from patients during standard colonoscopy procedure in the University Hospital of Bellvitge (Barcelona, Spain) and Germans Trias i Pujol University Hospital (Campus Can Ruti) (Barcelona, Spain). Western blot analysis was used to determine COX-2 levels. Deglycosylation assays were performed in both cells and tumor samples incubating each sample with peptide N-glycosidase F (PNGase F). Prostaglandin E2 (PGE2) levels were determined using a specific ELISA. 1H high resolution magic angle spinning (HRMAS) analysis was performed using a Bruker AVIII 500 MHz spectrometer and proteomic analysis was performed in a nano-liquid chromatography-tandem mass spectrometer (nano LC-MS/MS) using a QExactive HF orbitrap MS. RESULTS: Our data show that COX-2 has a differential expression profile in tumor tissue of CRC patients vs the adjacent non-tumor area, which correspond to a glycosylated and less active state of the protein. This fact was associated to a lesser PGE2 production in tumors. These results were corroborated in vitro performing deglycosylation assays in HT29 cell line where COX-2 protein profile was modified after PNGase F incubation, showing higher PGE2 levels. Moreover, HRMAS analysis indicated that tumor tissue has altered metabolic features vs non-tumor counterparts, presenting increased levels of certain metabolites such as taurine and phosphocholine and lower levels of lactate. In proteomic experiments, we detected an enlarged number of proteins in tumors that are mainly implicated in basic biological functions like mitochondrial activity, DNA/RNA processing, vesicular trafficking, metabolism, cytoskeleton and splicing. CONCLUSION: In our colorectal cancer cohort, tumor tissue presents a differential COX-2 expression pattern with lower enzymatic activity that can be related to an altered metabolic and proteomic profile.

Extracellular vesicles derived from endometrial human mesenchymal stem cells enhance embryo yield and quality in an aged murine model†.

Advanced age is a risk factor undermining women's fertility. Hence, the optimization of assisted reproduction techniques is an interdisciplinary challenge that requires the improvement of in vitro culture systems. Here, we hypothesize that supplementation of embryo culture medium with extracellular vesicles from endometrial-derived mesenchymal stem cells (EV-endMSCs) may have a positive impact on the embryo competence of aged oocytes. In this work, 24 weeks old B6D2 female mice were used as egg donors and in vitro fertilization assays were performed using males from the same strain (8-12 weeks); the presumptive zygotes were incubated in the presence of 0, 10, 20, 40, or 80 µg/ml of EV-endMSCs. The results from the proteomic analysis of EV-endMSCs and the classification by Reactome pathways allowed us to identify proteins closely related with the fertilization process. Moreover, in our aged murine model, the supplementation of the embryo culture medium with EV-endMSCs improved the developmental competence of the embryos as well as the total blastomere count. Finally, gene expression analysis of murine blastocysts showed significant changes on core genes related to cellular response to oxidative stress, metabolism, placentation, and trophectoderm/inner cell mass formation. In summary, we demonstrate that EV-endMSCs increase the quality of the embryos, and according to proteomic and genomic analysis, presumably by modulating the expression of antioxidant enzymes and promoting pluripotent activity. Therefore, EV-endMSCs could be a valuable tool in human assisted reproduction improving the developmental competence of aged oocytes and increasing the odds of implantation and subsequent delivery.

Unraveling the Molecular Signature of Extracellular Vesicles From Endometrial-Derived Mesenchymal Stem Cells: Potential Modulatory Effects and Therapeutic Applications.

Endometrial-derived Mesenchymal Stem Cells (endMSCs) are involved in the regeneration and remodeling of human endometrium, being considered one of the most promising candidates for stem cell-based therapies. Their therapeutic effects have been found to be mediated by extracellular vesicles (EV-endMSCs) with pro-angiogenic, anti-apoptotic, and immunomodulatory effects. Based on that, the main goal of this study was to characterize the proteome and microRNAome of these EV-endMSCs by proteomics and transcriptomics approaches. Additionally, we hypothesized that inflammatory priming of endMSCs may contribute to modify the therapeutic potential of these vesicles. High-throughput proteomics revealed that 617 proteins were functionally annotated as Extracellular exosome (GO:0070062), corresponding to the 70% of the EV-endMSC proteome. Bioinformatics analyses allowed us to identify that these proteins were involved in adaptive/innate immune response, complement activation, antigen processing/presentation, negative regulation of apoptosis, and different signaling pathways, among others. Of note, multiplexed quantitative proteomics and Systems Biology analyses showed that IFNγ priming significantly modulated the protein profile of these vesicles. As expected, proteins involved in antigen processing and presentation were significantly increased. Interestingly, immunomodulatory proteins, such as CSF1, ERAP1, or PYCARD were modified. Regarding miRNAs expression profile in EV-endMSCs, Next-Generation Sequencing (NGS) showed that the preferred site of microRNAome targeting was the nucleus (n = 371 microTargets), significantly affecting signal transduction (GO:0007165), cell proliferation (GO:0008283), and apoptotic processes (GO:0006915), among others. Interestingly, NGS analyses highlighted that several miRNAs, such as hsa-miR-150-5p or hsa-miR-196b-5p, were differentially expressed in IFNγ-primed EV-endMSCs. These miRNAs have a functional involvement in glucocorticoid receptor signaling, IL-6/8/12 signaling, and in the role of macrophages. In summary, these results allowed us to understand the complexity of the molecular networks in EV-endMSCs and their potential effects on target cells. To our knowledge, this is the first comprehensive study based on proteomic and genomic approaches to unravel the therapeutic potential of these extracellular vesicles, that may be used as immunomodulatory effectors in the treatment of inflammatory conditions.

Mitoproteomics: Tackling Mitochondrial Dysfunction in Human Disease.

Mitochondria are highly dynamic and regulated organelles that historically have been defined based on their crucial role in cell metabolism. However, they are implicated in a variety of other important functions, making mitochondrial dysfunction an important axis in several pathological contexts. Despite that conventional biochemical and molecular biology approaches have provided significant insight into mitochondrial functionality, innovative techniques that provide a global view of the mitochondrion are still necessary. Proteomics fulfils this need by enabling accurate, systems-wide quantitative analysis of protein abundance. More importantly, redox proteomics approaches offer unique opportunities to tackle oxidative stress, a phenomenon that is intimately linked to aging, cardiovascular disease, and cancer. In addition, cutting-edge proteomics approaches reveal how proteins exert their functions in complex interaction networks where even subtle alterations stemming from early pathological states can be monitored. Here, we describe the proteomics approaches that will help to deepen the role of mitochondria in health and disease by assessing not only changes to mitochondrial protein composition but also alterations to their redox state and how protein interaction networks regulate mitochondrial function and dynamics. This review is aimed at showing the reader how the application of proteomics approaches during the last 20 years has revealed crucial mitochondrial roles in the context of aging, neurodegenerative disorders, metabolic disease, and cancer.

The immunomodulatory activity of extracellular vesicles derived from endometrial mesenchymal stem cells on CD4+ T cells is partially mediated by TGFbeta.

Endometrial mesenchymal stem cells (endMSCs) reside in the basal and functional layer of human endometrium and participate in tissue remodelling, which is required for maintaining the regenerative capacity of the endometrium. The endMSCs are multipotent stem cells and exhibit immunomodulatory effects. This paper aimed to evaluate the regulatory effects of extracellular vesicles derived from endMSCs (EV-endMSCs) in the setting of T cell activation. In vitro stimulations of lymphocytes were performed in the presence of EV-endMSCs. These in vitro-stimulated lymphocytes were functionally and phenotypically characterized to distinguish CD4+ and CD8+ T cell differentiation subsets. Moreover, the inhibition of TGFß was performed with neutralizing antibodies. The phenotype and nanoparticle tracking analysis of the EV-endMSCs demonstrated that they are similar in terms of size distribution to other mesenchymal stem cells-derived exosomes. The in vitro assays showed an immunomodulatory potential of these vesicles to counteract the differentiation of CD4+ T cells. The quantification of active TGFß in EV-endMSCs was found to be very high when compared with extracellular vesicles-free concentrated supernatants. Finally, the neutralization of TGFß significantly attenuated the immunomodulatory activity of EV-endMSCs. In summary, this is the first report demonstrating that EV-endMSCs exhibit a potent inhibitory effect against CD4+ T cell activation, which is partially mediated by TGFß signalling.

Murine embryos exposed to human endometrial MSCs-derived extracellular vesicles exhibit higher VEGF/PDGF AA release, increased blastomere count and hatching rates.

Endometrial Mesenchymal Stromal Cells (endMSCs) are multipotent cells with immunomodulatory and pro-regenerative activity which is mainly mediated by a paracrine effect. The exosomes released by MSCs have become a promising therapeutic tool for the treatment of immune-mediated diseases. More specifically, extracellular vesicles derived from endMSCs (EV-endMSCs) have demonstrated a cardioprotective effect through the release of anti-apoptotic and pro-angiogenic factors. Here we hypothesize that EV-endMSCs may be used as a co-adjuvant to improve in vitro fertilization outcomes and embryo quality. Firstly, endMSCs and EV-endMSCs were isolated and phenotypically characterized for in vitro assays. Then, in vitro studies were performed on murine embryos co-cultured with EV-endMSCs at different concentrations. Our results firstly demonstrated a significant increase on the total blastomere count of expanded murine blastocysts. Moreover, EV-endMSCs triggered the release of pro-angiogenic molecules from embryos demonstrating an EV-endMSCs concentration-dependent increase of VEGF and PDGF-AA. The release of VEGF and PDGF-AA by the embryos may indicate that the beneficial effect of EV-endMSCs could be mediating not only an increase in the blastocyst's total cell number, but also may promote endometrial angiogenesis, vascularization, differentiation and tissue remodeling. In summary, these results could be relevant for assisted reproduction being the first report describing the beneficial effect of human EV-endMSCs on embryo development.

Differential proteomic and oxidative profiles unveil dysfunctional protein import to adipocyte mitochondria in obesity-associated aging and diabetes.

Human age-related diseases, including obesity and type 2 diabetes (T2DM), have long been associated to mitochondrial dysfunction; however, the role for adipose tissue mitochondria in these conditions remains unknown. We have tackled the impact of aging and T2DM on adipocyte mitochondria from obese patients by quantitating not only the corresponding abundance changes of proteins, but also the redox alterations undergone by Cys residues thereof. For that, we have resorted to a high-throughput proteomic approach based on isobaric labeling, liquid chromatography and mass spectrometry. The alterations undergone by the mitochondrial proteome revealed aging- and T2DM-specific hallmarks. Thus, while a global decrease of oxidative phosphorylation (OXPHOS) subunits was found in aging, the diabetic patients exhibited a reduction of specific OXPHOS complexes as well as an up-regulation of the anti-oxidant response. Under both conditions, evidence is shown for the first time of a link between increased thiol protein oxidation and decreased protein abundance in adipose tissue mitochondria. This association was stronger in T2DM, where OXPHOS mitochondrial- vs. nuclear-encoded protein modules were found altered, suggesting impaired mitochondrial protein translocation and complex assembly. The marked down-regulation of OXPHOS oxidized proteins and the alteration of oxidized Cys residues related to protein import through the redox-active MIA (Mitochondrial Intermembrane space Assembly) pathway support that defects in protein translocation to the mitochondria may be an important underlying mechanism for mitochondrial dysfunction in T2DM and physiological aging. The present draft of redox targets together with the quantification of protein and oxidative changes may help to better understand the role of oxidative stress in both a physiological process like aging and a pathological condition like T2DM.

N-acetylcysteine inhibits kinase phosphorylation during 3T3-L1 adipocyte differentiation.

OBJECTIVES: Reports investigating the effects of antioxidants on obesity have provided contradictory results. We have previously demonstrated that treatment with the antioxidant N-acetylcysteine (NAC) inhibits cellular triglyceride (Tg) accumulation as well as total cellular monoamine oxidase A (MAOA) expression in 3T3-L1 mature adipocytes (Calzadilla et al., Redox Rep. 2013;210-218). Here we analyzed the role of NAC on adipogenic differentiation pathway. METHODS: Assays were conducted using 3T3-L1 preadipocytes (undifferentiated cells: CC), which are capable of differentiating into mature adipocytes (differentiated cells: DC). We studied the effects of different doses of NAC (0.01 or 1 mM) on DC, to evaluate cellular expression of phospho-JNK½ (pJNK½), phospho-ERK½ (pERK½) and, mitochondrial expression of citrate synthase, fumarate hydratase and MAOA. RESULTS: Following the differentiation of preadipocytes, an increase in the expression levels of pJNK½ and pERK½ was observed, together with mitotic clonal expansion (MCE). We found that both doses of NAC decreased the expression of pJNK½ and pERK½. Consistent with these results, NAC significantly inhibited MCE and modified the expression of different mitochondrial proteins. DISCUSSION: Our results suggested that NAC could inhibit Tg and mitochondrial protein expression by preventing both MCE and kinase phosphorylation.