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IntroductionImmune checkpoint blockade (ICB) immunotherapy has revolutionized cancer treatment, demonstrating exceptional clinical responses in a wide range of cancers. Despite the success, a significant proportion of patients still fail to respond, highlighting the existence of unappreciated mechanisms of immunotherapy resistance. Delineating such mechanisms is paramount to minimize immunotherapy failures and optimize the clinical benefit.MethodsIn this study, we treated tumour-bearing mice with PD-L1 blockage antibody (aPD-L1) immunotherapy, to investigate its effects on cancer-induced emergency myelopoiesis, focusing on bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs). We examined the impact of aPD-L1 treatment on HSPC quiescence, proliferation, transcriptomic profile, and functionality.ResultsHerein, we reveal that aPD-L1 in tumour-bearing mice targets the HSPCs in the BM, mediating their exit from quiescence and promoting their proliferation. Notably, disruption of the PDL1/PD1 axis induces transcriptomic reprogramming in HSPCs, observed in both individuals with Hodgkin lymphoma (HL) and tumour-bearing mice, shifting towards an inflammatory state. Furthermore, HSPCs from aPDL1-treated mice demonstrated resistance to cancer-induced emergency myelopoiesis, evidenced by a lower generation of MDSCs compared to control-treated mice.DiscussionOur findings shed light on unrecognized mechanisms of action of ICB immunotherapy in cancer, which involves targeting of BM-driven HSPCs and reprogramming of cancer-induced emergency myelopoiesis.

Abstract Background It is now realized that Parkinson’s disease (PD) pathology extends beyond the substantia nigra, affecting both central and peripheral nervous systems, and exhibits a variety of non-motor symptoms often preceding motor features. Neuroinflammation induced by activated microglia and astrocytes is thought to underlie these manifestations. α-Synuclein aggregation has been linked with sustained neuroinflammation in PD, aggravating neuronal degeneration; however, there is still a lack of critical information about the structural identity of the α-synuclein conformers that activate microglia and/or astrocytes and the molecular pathways involved. Methods To investigate the role of α-synuclein conformers in the development and maintenance of neuroinflammation, we used primary quiescent microglia and astrocytes, post-mortem brain tissues from PD patients and A53T α-synuclein transgenic mice that recapitulate key features of PD-related inflammatory responses in the absence of cell death, i.e., increased levels of pro-inflammatory cytokines and complement proteins. Biochemical and -omics techniques including RNAseq and secretomic analyses, combined with 3D reconstruction of individual astrocytes and live calcium imaging, were used to uncover the molecular mechanisms underlying glial responses in the presence of α-synuclein oligomers in vivo and in vitro. Results We found that the presence of SDS-resistant hyper-phosphorylated α-synuclein oligomers, but not monomers, was correlated with sustained inflammatory responses, such as elevated levels of endogenous antibodies and cytokines and microglial activation. Similar oligomeric α-synuclein species were found in post-mortem human brain samples of PD patients but not control individuals. Detailed analysis revealed a decrease in Iba1Low/CD68Low microglia and robust alterations in astrocyte number and morphology including process retraction. Our data indicated an activation of the p38/ATF2 signaling pathway mostly in microglia and a sustained induction of the NF-κB pathway in astrocytes of A53T mice. The sustained NF-κB activity triggered the upregulation of astrocytic T-type Cav3.2 Ca2+ channels, altering the astrocytic secretome and promoting the secretion of IGFBPL1, an IGF-1 binding protein with anti-inflammatory and neuroprotective potential. Conclusions Our work supports a causative link between the neuron-produced α-synuclein oligomers and sustained neuroinflammation in vivo and maps the signaling pathways that are stimulated in microglia and astrocytes. It also highlights the recruitment of astrocytic Cav3.2 channels as a potential neuroprotective mediator against the α-synuclein-induced neuroinflammation. Graphical Abstract

Podosphaera xanthii is the main causal agent of powdery mildew (PM) disease for Cucurbita pepo. Disease control is attained principally by applications of chemical fungicides, along with parallel use of tolerant crop varieties and alternate application of elicitors to control development of disease resistance. To get insight into C. pepo molecular responses to P. xanthii infection and elicitor treatment we studied the proteomic profile differences at the phyllosphere of a zucchini cultivar susceptible to PM, at the onset of P. xanthii (PX) infection and after application of Reynoutria sachalinensis (RS) plant extract, respectively, using a nano-LC-HRMS/MS, Q-Exactive-Orbitrap approach. Analysis of peptide sequences regarding four treatment groups (Control; PX; RS; and RSPX (PX-infected priorly treated with RS)) resulted in 2070 CuGenDB annotations. Three comparisons (treatments vs. Control) encompassed most of the Differentially Expressed Proteins (DEPs). In these three comparisons, KEGG and Gene Ontology functional analyses highlighted unique differentially enriched pathways—some of which included highly expressed proteins—in PX-related (proteasome, pentose phosphate pathway, and carbon fixation), RS-related (biosynthesis of secondary metabolites, flavonoids, and starch and sucrose metabolism), and RSPX-related (pyruvate metabolism and polycomb repressive complex) comparisons, respectively, suggesting distinct mechanisms of early plant responses modulated by PX and RS. Furthermore, in four out of six comparisons the thiamine metabolism pathway was found to be enriched, suggesting a pivotal role in PX-induced responses.

Objective: The interplay of gut microbiota with the kidney system in chronic kidney disease (CKD), is characterized by increased concentrations of uric acid in the gut, which in turn, may increase bacterial uricase activity and may lead to the generation of uremic toxins. Nevertheless, knowledge on these underlying bidirectional molecular mechanisms is still limited. Methods: In this exploratory study, proteomic analysis was performed on fecal samples, targeting to investigate this largely unexplored biological material as a source of information reflecting the gut-kidney axis. Specifically, fecal suspension samples from patients with CKD1 (n = 12) and CKD4 (n = 17) were analysed by LC-MS/MS, using both the Human and Bacterial UniProt RefSeq reviewed databases. Results: This fecal proteomic analysis collectively identified 701 human and 1011 bacterial proteins of high confidence. Differential expression analysis (CKD4/CKD1) revealed significant changes in human proteins (n = 8, including proteins such as galectin-3-binding protein and prolactin-inducible protein), that were found to be associated with inflammation and CKD. The differential protein expression of pancreatic alpha-amylase further suggested plausible reduced saccharolytic fermentation in CKD4/CKD1. Significant changes in bacterial proteins (n = 9, such as glyceraldehyde-3-phosphate dehydrogenase and enolase), participating in various carbohydrate and metabolic pathways important for the synthesis of butyrate, in turn suggested differential butyrate synthesis in CKD4/CKD1. Further, targeted quantification of fecal pancreatic alpha-amylase and butyrate in the same fecal suspension samples, supported these hypotheses. Conclusion: Collectively, this exploratory fecal proteomic analysis highlighted changes in human and bacterial proteins reflecting inflammation and reduced saccharolytic fermentation in CKD4/CKD1, plausibly affecting the butyrate synthesis pathways in advanced stage kidney disease. Integrative multi-omics validation is planned.

Summary: Quiescence is a common cellular state, required for stem cell maintenance and microorganismal survival under stress conditions or starvation. However, the mechanisms promoting quiescence maintenance remain poorly known. Plasma membrane components segregate into distinct microdomains, yet the role of this compartmentalization in quiescence remains unexplored. Here, we show that flavodoxin-like proteins (FLPs), ubiquinone reductases of the yeast eisosome membrane compartment, protect quiescent cells from lipid peroxidation and ferroptosis. Eisosomes and FLPs expand specifically in respiratory-active quiescent cells, and mutants lacking either show accelerated aging and defective quiescence maintenance and accumulate peroxidized phospholipids with monounsaturated or polyunsaturated fatty acids (PUFAs). FLPs are essential for the extramitochondrial regeneration of the lipophilic antioxidant ubiquinol. FLPs, alongside the Gpx1/2/3 glutathione peroxidases, prevent iron-driven, PUFA-dependent ferroptotic cell death. Our work describes ferroptosis-protective mechanisms in yeast and introduces plasma membrane compartmentalization as an important factor in the long-term survival of quiescent cells.

Summary: Liver transplantation is the gold-standard therapy for acute hepatic failure (AHF) with limitations related to organ shortage and life-long immunosuppressive therapy. Cell therapy emerges as a promising alternative to transplantation. We have previously shown that IL-10 and Annexin-A1 released by amniotic fluid human mesenchymal stromal cells (AF-MSCs) and their hepatocyte progenitor-like (HPL) or hepatocyte-like (HPL) cells induce liver repair and downregulate systemic inflammation in a CCl4-AHF mouse model. Herein, we demonstrate that exosomes (EXO) derived from these cells improve liver phenotype in CCl4-induced mice and promote oval cell proliferation. LC-MS/MS proteomic analysis identified MEFG-8 in EXO cargo that facilitates rescue of AHF by suppressing PI3K signaling. Administration of recombinant MFGE-8 protein also reduced liver damage in CCl4-induced mice. Clinically, MEFG-8 expression was decreased in liver biopsies from AHF patients. Collectively, our study provides proof-of-concept for an innovative, cell-free, less immunogenic, and non-toxic alternative strategy for AHF.

Protein signatures of unstable and stable atherosclerosis are defined by quantitative proteomics using a preclinical mouse model of plaque instability/rupture.

Carfilzomib is an irreversible proteasome inhibitor indicated for relapsed/refractory multiple myeloma. Carfilzomib toxicity includes renal adverse effects (RAEs) of obscure pathobiology. Therefore, we investigated the mechanisms of nephrotoxicity developed by Carfilzomib. In a first experimental series, we used our previously established in vivo mouse models of Carfilzomib cardiotoxicity, that incorporated 2 and 4 doses of Carfilzomib, to identify whether Carfilzomib affects renal pathways. Hematology and biochemical analyses were performed, while kidneys underwent histological and molecular analyses. In a second and third experimental series, the 4 doses protocol was repeated for 24 hours urine collection and proteomic/metabolomic analyses. To test an experimental intervention, primary murine collecting duct tubular epithelial cells were treated with Carfilzomib and/or Eplerenone and Metformin. Finally, Eplerenone was orally co-administered with Carfilzomib daily (165 mg/kg) in the 4 doses protocol. We additionally used material from 7 patients to validate our findings and patients underwent biochemical analysis and assessment of renal mineralocorticoid receptor (MR) axis activation. In vivo screening showed that Carfilzomib-induced renal histological deficits and increased serum creatinine, urea, NGAL levels, and proteinuria only in the 4 doses protocol. Carfilzomib decreased diuresis, altered renal metabolism, and activated MR axis. This was consistent with the cytotoxicity found in primary murine collecting duct tubular epithelial cells, whereas Carfilzomib + Eplerenone co-administration abrogated Carfilzomib-related nephrotoxic effects in vitro and in vivo. Renal SGK-1, a marker of MR activation, increased in patients with Carfilzomib-related RAEs. Conclusively, Carfilzomib-induced renal MR/SGK-1 activation orchestrates RAEs and water retention both in vivo and in the clinical setting. MR blockade emerges as a potential therapeutic approach against Carfilzomib-related nephrotoxicity.

Abstract Processing of N-terminally elongated antigenic peptide precursors by Endoplasmic Reticulum Aminopeptidase 1 (ERAP1) is a key step in antigen presentation and the adaptive immune response. Although ERAP1 can efficiently process long peptides in solution, it has been proposed that it can also process peptides bound onto Major Histocompatibility Complex I molecules (MHCI). In a previous study, we suggested that the occasionally observed “ontο MHCI” trimming by ERAP1 is likely due to fast peptide dissociation followed by solution trimming, rather than direct action of ERAP1 onto the MHCI complex. However, other groups have proposed that ERAP1 can trim peptides covalently bound onto MHCI, which would preclude peptide dissociation. To explore this interaction, we constructed disulfide-linked MHCI-peptide complexes using HLA-B*08 and a 12mer kinetically labile peptide, or a 16mer carrying a phosphinic transition-state analogue N-terminus with high-affinity for ERAP1. Kinetic and biochemical analyses suggested that while both peptides could access the ERAP1 active site when free in solution, they were unable to do so when tethered in the MHCI binding groove. Our results suggest that MHCI binding protects, rather than promotes, antigenic peptide precursor trimming by ERAP1 and thus solution trimming is the more likely model of antigenic peptide processing.

Hypertensive nephropathy is the second most frequent cause of end-stage renal disease in western societies. In previous experiments in our laboratory with proteomic analysis of renal parenchyma of SHR hypertensive animals, we identified two molecules, namely SGLT2 and CLIC4, associated with the development of hypertension. Here, we apply the methodology of targeted proteomic analysis in kidney biopsies from patients with hypertensive nephropathy to study the role of SGLT2 and CLIC4 in the pathogenesis of the disease. Relative quantification data of SGLT2 and CLIC4 via means of targeted proteomic analysis in human kidney biopsies from hypertensive patients and normotensive controls are reported. In addition, validation data of the proteomic results via immunofluorescence are presented. Renal tissue biopsies (N = 17) from archival material of patients with hypertensive nephropathy and normotensive controls were used. Targeted proteomic analysis was performed using the method: ``Parallel Reaction Monitoring'' (PRM) in renal parenchyma of hypertensive and normotensive patients for the selective identification of SGLT2 and CLIC4 and the relative quantification of their expression using proteotypic peptides for each protein. The expression of SGLT2 molecule was also confirmed by immunofluorescence followed by quantification of fluorescence intensity. According to PRM, the SGLT2 protein was found with reduced and the CLIC4 protein with increased expression levels in hypertensive patients compared to normotensive controls. Comparison of representative immunofluorescence images confirmed a decrease in the expression of SGLT2 in the brush border of proximal tubular epithelial cells in hypertensive patients. Our data show changes in the tubular compartment of the kidney and especially in the proximal tubules associated with the hypertensive nephropathy. The clinical significance of these findings should be further explored for the discovery and/or confirmation of novel therapeutic approaches and biomarkers in the development of hypertensive kidney disease.

IL-33, a nuclear alarmin released during cell death, exerts context-specific effects on adaptive and innate immune cells, eliciting potent inflammatory responses. We screened blood, skin, and kidney tissues from patients with systemic lupus erythematosus (SLE), a systemic autoimmune disease driven by unabated type I IFN production, and found increased amounts of extracellular IL-33 complexed with neutrophil extracellular traps (NETs), correlating with severe, active disease. Using a combination of molecular, imaging, and proteomic approaches, we show that SLE neutrophils, activated by disease immunocomplexes, release IL-33–decorated NETs that stimulate robust IFN-α synthesis by plasmacytoid DCs in a manner dependent on the IL-33 receptor ST2L. IL33-silenced neutrophil-like cells cultured under lupus-inducing conditions generated NETs with diminished interferogenic effect. Importantly, NETs derived from patients with SLE are enriched in mature bioactive isoforms of IL-33 processed by the neutrophil proteases elastase and cathepsin G. Pharmacological inhibition of these proteases neutralized IL-33–dependent IFN-α production elicited by NETs. We believe these data demonstrate a novel role for cleaved IL-33 alarmin decorating NETs in human SLE, linking neutrophil activation, type I IFN production, and end-organ inflammation, with skin pathology mirroring that observed in the kidneys.

Background: Human mesenchymal stem/stromal cells (MSCs) and their secreted molecules exert beneficial effects in injured tissues by promoting tissue regeneration and angiogenesis and by inhibiting inflammation and fibrosis. We have previously demonstrated that the therapeutic activity of fetal MSCs derived from amniotic fluid (AF-MSCs) and their hepatic progenitor-like cells (HPL) is mediated by paracrine effects in a mouse model of acute hepatic failure (AHF). Methods: Herein, we have combined proteomic profiling of the AF-MSCs and HPL cell secretome with ex vivo and in vivo functional studies to identify specific soluble factors, which underpin tissue regeneration in AHF. Findings: The anti-inflammatory molecule Annexin-A1 (ANXA1) was detected at high levels in both AF-MSC and HPL cell secretome. Further functional analyses revealed that the shRNA-mediated knock-down of ANXA1 in MSCs (shANXA1-MSCs) decreased their proliferative, clonogenic and migratory potential, as well as their ability to differentiate into HPL cells. Liver progenitors (oval cells) from AHF mice displayed reduced proliferation when cultured ex vivo in the presence of conditioned media from shANXA1-MSCs compared to control MSCs secretome. Intra-hepatic delivery of conditioned media from control MSCs but not shANXA1-MSCs reduced liver damage and circulating levels of pro-inflammatory cytokines in AHF. Interpretation: Collectively, our study uncovers secreted Annexin-A1 as a novel effector of MSCs in liver regeneration and further underscores the potential of cell-free therapeutic strategies for liver diseases. Fund: Fondation Santé, GILEAD Asklipeios Grant, Fellowships of Excellence – Siemens, IKY, Reinforcement of Postdoctoral Researchers, IKY. Keywords: MSCs, Secretome, Annexin-A1, Hepatic progenitors, Acute hepatic failure

Background: The increased prevalence of cardiovascular disease (CVD) indicates a demand for novel therapeutic approaches. Proteome analysis of vascular tissues from animal models and humans with CVD could lead to the identification of novel druggable targets. Methods: LC-MS/MS analysis of thoracic aortas from three mouse models of non-diabetic and diabetic (streptozotocin (STZ)-induced) atherosclerosis followed by bioinformatics/pathway analysis was performed. Selected findings were confirmed by proteomics analysis of human vessels from patients with CVD as well as in vitro studies (migration, proliferation, angiogenesis assays) using endothelial (HUVEC) cells. Findings: Comparative tissue proteomics of low density lipoprotein receptor deficient (Ldlr−/−) and diabetic Ldlr−/− (Ldlr−/−STZ) with wild type (WT) animals led to the identification of 284 differentially expressed proteins in both models. Among them, 177 proteins were also differentially expressed in diabetic apolipoprotein E deficient (ApoE−/−STZ) mice, suggesting expression changes associated with atherosclerosis independent of the model used. These proteins recapitulated the hallmarks of atherosclerosis. Comparison of these findings with differentially expressed proteins in human vessels with CVD enabled shortlisting of six commonly dysregulated proteins. Among them, lysine-specific demethylase 5D (KDM5D) exhibited pronounced overexpression accompanied by a reduction in the protein levels of its substrate, the trimethylated lysine 4 of histone H3 (H3K4me3), in patients with CVD. Functional interference studies applying a KDM5 inhibitor on HUVEC reduced cell proliferation, migration and tube-forming ability in vitro. Interpretation: This high-throughput proteomics strategy identified KDM5 histone demethylases being potentially involved in CVD, possibly by affecting H3K4 methylation. Fund: [SysVasc, HEALTH-2013 603288], [ERA-CVD PROACT: ANR-17-ECVD-0006, 01KL1805], [FRM, DEQ20170336759]. Keywords: Cardiovascular disease, Atherosclerosis, Diabetes, Proteomics, KDM5, H3K4

Molar incisor hypomineralization is a complex developmental enamel defect that affects the permanent dentition of children with significant functional and aesthetic implications. Saliva is an ideal diagnostic tool and ensures patients’ compliance by diminishing the discomfort especially in pediatric population. Lately, salivary proteome analysis has progressively evolved in various biomedical disciplines. As changes in saliva composition are associated with oral diseases, it is reasonable to assume that the saliva proteome of MIH-affected children might be altered compared to healthy children. This study analyzed the human and microbial salivary proteome in children with MIH in order to identify salivary markers indicative of the pathology. The conducted proteomic analysis generated a comprehensive dataset comprising a total of 1515 high confidence identifications and revealed a clear discrimination between the two groups. Statistical comparison identified 142 differentially expressed proteins, while the pathway analysis indicated deregulation of inflammation, immune response mechanisms, and defense response to bacteria in MIH patients. Bacterial proteome analysis showed a lower diversity for the microbial species, which highlights the dysbiotic environment established in the MIH pathology.

Hepatitis C virus is the major cause of chronic liver diseases and the only cytoplasmic RNA virus known to be oncogenic in humans. The viral genome gives rise to ten mature proteins and to additional proteins, which are the products of alternative translation initiation mechanisms. A protein—known as ARFP (alternative reading frame protein) or Core+1 protein—is synthesized by an open reading frame overlapping the HCV Core coding region in the (+1) frame of genotype 1a. Almost 20 years after its discovery, we still know little of the biological role of the ARFP/Core+1 protein. Here, our differential proteomic analysis of stable hepatoma cell lines expressing the Core+1/Long isoform of HCV-1a relates the expression of the Core+1/Long isoform with the progression of the pathology of HCV liver disease to cancer.

TIA1 is a broadly expressed DNA/RNA binding protein that regulates multiple aspects of RNA metabolism. It is best known for its role in stress granule assembly during the cellular stress response. Three RNA recognition motifs mediate TIA1 functions along with a prion-like domain that supports multivalent protein-protein interactions that are yet poorly characterized. Here, by fusing the enhanced ascorbate peroxidase 2 (APEX2) biotin-labeling enzyme to TIA1 combined with mass spectrometry, the proteins in the immediate vicinity of TIA1 were defined in situ. Eighty-six and 203 protein partners, mostly associated with ribonucleoprotein complexes, were identified in unstressed control and acute stress conditions, respectively. Remarkably, the repertoire of TIA1 protein partners was highly dissimilar between the two cellular states. Under unstressed control conditions, the biological processes associated with the TIA1 interactome were enriched for cytosolic ontologies related to mRNA metabolism, such as translation initiation, nucleocytoplasmic transport, and RNA catabolism, while the protein identities were primarily represented by RNA binding proteins, ribosomal subunits, and eicosanoid regulators. Under acute stress, TIA1-labeled partners displayed a broader subcellular distribution that included the chromosomes and mitochondria. The enriched biological processes included splicing, translation, and protein synthesis regulation, while the molecular function of the proteins was enriched for RNA binding activity, ribosomal subunits, DNA double-strand break repair, and amide metabolism. Altogether, these data highlight the TIA1 spatial environment with its different partners in diverse cellular states and pave the way to dissect TIA1 role in these processes.

Background/Aims: Hyperphosphatemia promotes medial vascular calcification, at least partly, by induction of osteo-/chondrogenic transdifferentiation of vascular smooth muscle cells (VSMCs). The complex signaling pathways regulating this process are still incompletely understood. The present study investigated the role of cytosolic serine hydroxymethyl transferase 1 (SHMT1) in phosphate-induced vascular calcification. Methods: Endogenous expression of SHMT1 was suppressed by silencing in primary human aortic smooth muscle cells (HAoSMCs) followed by treatment without and with phosphate or antioxidants. Results: In HAoSMCs, SHMT1 mRNA expression was up-regulated by phosphate. Silencing of SHMT1 alone was sufficient to induce osteo-/chondrogenic transdifferentiation of HAoSMCs, as shown by increased tissue-nonspecific alkaline phosphatase (ALPL) activity and osteogenic markers MSX2, CBFA1 and ALPL mRNA expression. Furthermore, phosphate-induced ALPL mRNA expression and activity as well as calcification were augmented in SHMT1 silenced HAoSMCs as compared to negative control siRNA transfected HAoSMCs. Silencing of SHMT1 decreased total antioxidant capacity and up-regulated NADH/NADPH oxidase system components NOX4 and CYBA mRNA expression in HAoSMCs, effects paralleled by increased mRNA expression of matrix metalloproteinase MMP2 as well as BAX/BCL2 ratio. More importantly, additional treatment with antioxidants TEMPOL or TIRON blunted the increased osteogenic markers mRNA expression in SHMT1 silenced HAoSMCs. Conclusion: Silencing of SHMT1 promotes osteo-/chondrogenic signaling in VSMCs, at least in part, by inducing cellular oxidative stress. It thus aggravates phosphate-induced calcification of VSMCs. The present findings support a regulatory role of SHMT1 in vascular calcification during conditions of hyperphosphatemia such as chronic kidney disease.

Abstract Background Cardiovascular disease (CVD) describes the pathological conditions of the heart and blood vessels. Despite the large number of studies on CVD and its etiology, its key modulators remain largely unknown. To this end, we performed a comprehensive proteomic analysis of blood plasma, with the scope to identify disease-associated changes after placing them in the context of existing knowledge, and generate a well characterized dataset for further use in CVD multi-omics integrative analysis. Methods LC–MS/MS was employed to analyze plasma from 32 subjects (19 cases of various CVD phenotypes and 13 controls) in two steps: discovery (13 cases and 8 controls) and test (6 cases and 5 controls) set analysis. Following label-free quantification, the detected proteins were correlated to existing plasma proteomics datasets (plasma proteome database; PPD) and functionally annotated (Cytoscape, Ingenuity Pathway Analysis). Differential expression was defined based on identification confidence (≥ 2 peptides per protein), statistical significance (Mann–Whitney p value ≤ 0.05) and a minimum of twofold change. Results Peptides detected in at least 50% of samples per group were considered, resulting in a total of 3796 identified proteins (838 proteins based on ≥ 2 peptides). Pathway annotation confirmed the functional relevance of the findings (representation of complement cascade, fibrin clot formation, platelet degranulation, etc.). Correlation of the relative abundance of the proteins identified in the discovery set with their reported concentrations in the PPD was significant, confirming the validity of the quantification method. The discovery set analysis revealed 100 differentially expressed proteins between cases and controls, 39 of which were verified (≥ twofold change) in the test set. These included proteins already studied in the context of CVD (such as apolipoprotein B, alpha-2-macroglobulin), as well as novel findings (such as low density lipoprotein receptor related protein 2 [LRP2], protein SZT2) for which a mechanism of action is suggested. Conclusions This proteomic study provides a comprehensive dataset to be used for integrative and functional studies in the field. The observed protein changes reflect known CVD-related processes (e.g. lipid uptake, inflammation) but also novel hypotheses for further investigation including a potential pleiotropic role of LPR2 but also links of SZT2 to CVD.

Background/Aims: Fibulin-3, an extracellular matrix glycoprotein, inhibits vascular oxidative stress and remodeling in hypertension. Oxidative stress is prevalent in chronic kidney disease (CKD) patients and is an important mediator of osteo-/chondrogenic transdifferentiation and calcification of vascular smooth muscle cells (VSMCs) during hyperphosphatemia. Therefore, the present study explored the effects of Fibulin-3 on phosphate-induced vascular calcification. Methods: Experiments were performed in primary human aortic smooth muscle cells (HAoSMCs) treated with control or with phosphate without or with additional treatment with recombinant human Fibulin-3 protein or with hydrogen peroxide as an exogenous source of oxidative stress. Results: Treatment with calcification medium significantly increased calcium deposition in HAoSMCs, an effect significantly blunted by additional treatment with Fibulin-3. Moreover, phosphate-induced alkaline phosphatase activity and mRNA expression of osteogenic and chondrogenic markers MSX2, CBFA1, SOX9 and ALPL were all significantly reduced by addition of Fibulin-3. These effects were paralleled by similar regulation of oxidative stress in HAoSMCs. Phosphate treatment significantly up-regulated mRNA expression of the oxidative stress markers NOX4 and CYBA, down-regulated total antioxidant capacity and increased the expression of downstream effectors of oxidative stress PAI-1, MMP2 and MMP9 as well as BAX/BLC2 ratio in HAoSMCs, all effects blocked by additional treatment with Fibulin-3. Furthermore, the protective effects of Fibulin-3 on phosphate-induced osteogenic and chondrogenic markers expression in HAoSMCs were reversed by additional treatment with hydrogen peroxide. Conclusions: Fibulin-3 attenuates phosphate-induced osteo-/ chondrogenic transdifferentiation and calcification of VSMCs, effects involving inhibition of oxidative stress. Up-regulation or supplementation of Fibulin-3 may be beneficial in reducing the progression of vascular calcification during hyperphosphatemic conditions such as CKD.

Significant inter-individual variation in terms of susceptibility to several stress-related disorders, such as myocardial infarction and Alzheimer’s disease, and therapeutic response has been observed among healthy subjects. The molecular features responsible for this phenomenon have not been fully elucidated. Proteomics, in association with bioinformatics analysis, offer a comprehensive description of molecular phenotypes with clear links to human disease pathophysiology. The aim of this study was to conduct a comparative plasma proteomics analysis of glucocorticoid resistant and glucocorticoid sensitive healthy subjects and provide clues of the underlying physiological differences. For this purpose, 101 healthy volunteers were given a very low dose (0.25 mg) of dexamethasone at midnight, and were stratified into the 10% most glucocorticoid sensitive (S) (n = 11) and 10% most glucocorticoid resistant (R) (n = 11) according to the 08:00 h serum cortisol concentrations determined the following morning. One month following the very-low dose dexamethasone suppression test, DNA and plasma samples were collected from the 22 selected individuals. Sequencing analysis did not reveal any genetic defects in the human glucocorticoid receptor (NR3C1) gene. To investigate the proteomic profile of plasma samples, we used Liquid Chromatography–Mass Spectrometry (LC-MS/MS) and found 110 up-regulated and 66 down-regulated proteins in the S compared to the R group. The majority of the up-regulated proteins in the S group were implicated in platelet activation. To predict response to cortisol prior to administration, a random forest classifier was developed by using the proteomics data in order to distinguish S from R individuals. Apolipoprotein A4 (APOA4) and gelsolin (GSN) were the most important variables in the classification, and warrant further investigation. Our results indicate that a proteomics signature may differentiate the S from the R healthy subjects, and may be useful in clinical practice. In addition, it may provide clues of the underlying molecular mechanisms of the chronic stress-related diseases, including myocardial infarction and Alzheimer’s disease.

Background: The absence of efficient inhibitors for diabetic kidney disease (DKD) progression reflects the gaps in our understanding of DKD molecular pathogenesis. Methods: A comprehensive proteomic analysis was performed on the glomeruli and kidney cortex of diabetic mice with the subsequent validation of findings in human biopsies and omics datasets, aiming to better understand the underlying molecular biology of early DKD development and progression. Results: LC–MS/MS was employed to analyze the kidney proteome of 2 DKD models: Ins2Akita (early and late DKD) and db/db mice (late DKD). The abundance of detected proteins was defined. Pathway analysis of differentially expressed proteins in the early and late DKD versus the respective controls predicted dysregulation in DKD hallmarks (peroxisomal lipid metabolism and β-oxidation), supporting the functional relevance of the findings. Comparing the observed protein changes in early and late DKD, the consistent upregulation of 21 and downregulation of 18 proteins was detected. Among these were downregulated peroxisomal and upregulated mitochondrial proteins. Tissue sections from 16 DKD patients were analyzed by IHC confirming our results. Conclusion: Our study shows an extensive differential expression of peroxisomal proteins in the early stages of DKD that persists regardless of the disease severity, providing new perspectives and potential markers of diabetic kidney dysfunction.

The gold standard vascular substitutes, used in cardiovascular surgery, are the Dacron or expanded polytetrafluoroethylene (ePTFE)-derived grafts. However, major adverse reactions accompany their use. For this purpose, decellularized human umbilical arteries (hUAs) may be proven as a significant source for the development of small diameter conduits. The aim of this study was the evaluation of a decellularization protocol in hUAs. To study the effect of the decellularization to the hUAs, histological analysis was performed. Then, native and decellularized hUAs were biochemically and biomechanically evaluated. Finally, broad proteomic analysis was applied. Histological analysis revealed the successful decellularization of the hUAs. Furthermore, a great amount of DNA was removed from the decellularized hUAs. Biomechanical analysis revealed statistically significant differences in longitudinal direction only in maximum stress (p < 0.013) and strain (p < 0.001). On the contrary, all parameters tested for circumferential direction exhibited significant differences (p < 0.05). Proteomic analysis showed the preservation of the extracellular matrix and cytoskeletal proteins in both groups. Proteomic data are available via ProteomeXchange with identifier PXD020187. The above results indicated that hUAs were efficiently decellularized. The tissue function properties of these conduits were well retained, making them ideal candidates for the development of small diameter vascular grafts.

(1) Background: Prostate cancer (PCa) is characterized by high heterogeneity. The aim of this study was to investigate molecular alterations underlying PCa development based on proteomics data. (2) Methods: Liquid chromatography coupled to tandem mass spectrometry was conducted for 22 fresh-frozen tissue specimens from patients with benign prostatic hyperplasia (BPH, n = 5) and PCa (n = 17). Mann Whitney test was used to define significant differences between the two groups. Association of protein abundance with PCa progression was evaluated using Spearman correlation, followed by verification through investigating the Prostate Cancer Transcriptome Atlas. Functional enrichment and interactome analysis were carried out using Metascape and String. (3) Results: Proteomics analysis identified 1433 proteins, including 145 proteins as differentially abundant between patients with PCa and BPH. In silico analysis revealed alterations in several pathways and hallmarks implicated in metabolism and signalling, represented by 67 proteins. Among the latter, 21 proteins were correlated with PCa progression at both the protein and mRNA levels. Interactome analysis of these 21 proteins predicted interactions between Myc proto-oncogene (MYC) targets, protein processing in the endoplasmic reticulum, and oxidative phosphorylation, with MYC targets having a central role. (4) Conclusions: Tissue proteomics allowed for characterization of proteins and pathways consistently affected during PCa development. Further validation of these findings is required.

Background: Recent findings have shown that the fibrin gel derived from cord blood units (CBUs) play a significant role in wound healing and tissue regeneration. The aim of this study was to standardize the fibrin gel production process in order to allow for its regular use. Methods: CBUs (n = 200) were assigned to 4 groups according to their initial volume. Then, a two-stage centrifugation protocol was applied in order to obtain platelet rich plasma (PRP). The concentration of platelets (PLTs), white blood cells (WBCs) and red blood cells (RBCs) were determined prior to and after the production process. In addition, targeted proteomic analysis using multiple reaction monitoring was performed. Finally, an appropriate volume of calcium gluconate was used in PRP for the production of fibrin gel. Results: The results of this study showed that high volume CBUs were characterized by greater recovery rates, concentration and number of PLTs compared to the low volume CBUs. Proteomic analysis revealed the presence of key proteins for regenerative medicine. Fibrin gel was successfully produced from CBUs of all groups. Conclusion: In this study, low volume CBUs could be an alternative source for the production of fibrin gel, which can be used in multiple regenerative medicine approaches.

High resolution proteomics approaches have been successfully utilized for the comprehensive characterization of the cell proteome. However, in the case of quantitative proteomics an open question still remains, which quantification strategy is best suited for identification of biologically relevant changes, especially in clinical specimens. In this study, a thorough comparison of a label-free approach (intensity-based) and 8-plex iTRAQ was conducted as applied to the analysis of tumor tissue samples from non-muscle invasive and muscle-invasive bladder cancer. For the latter, two acquisition strategies were tested including analysis of unfractionated and fractioned iTRAQ-labeled peptides. To reduce variability, aliquots of the same protein extract were used as starting material, whereas to obtain representative results per method further sample processing and MS analysis were conducted according to routinely applied protocols. Considering only multiple-peptide identifications, LC-MS/MS analysis resulted in the identification of 910, 1092 and 332 proteins by label-free, fractionated and unfractionated iTRAQ, respectively. The label-free strategy provided higher protein sequence coverage compared to both iTRAQ experiments. Even though pre-fraction of the iTRAQ labeled peptides allowed for a higher number of identifications, this was not accompanied by a respective increase in the number of differentially expressed changes detected. Validity of the proteomics output related to protein identification and differential expression was determined by comparison to existing data in the field (Protein Atlas and published data on the disease). All methods predicted changes which to a large extent agreed with published data, with label-free providing a higher number of significant changes than iTRAQ. Conclusively, both label-free and iTRAQ (when combined to peptide fractionation) provide high proteome coverage and apparently valid predictions in terms of differential expression, nevertheless label-free provides higher sequence coverage and ultimately detects a higher number of differentially expressed proteins. The risk for receiving false associations still exists, particularly when analyzing highly heterogeneous biological samples, raising the need for the analysis of higher sample numbers and/or application of adjustment for multiple testing.