ABSTRACT
AIMS: Cardiovascular pathology is the main cause of death in chronic kidney disease (CKD) patients. CKD is associated with the accumulation of uremic toxins in the bloodstream, and indoxyl sulfate (IS) is one of the most abundant uremic toxins found in the blood of CKD patients. We conducted an in vitro study to assess the mechanisms underlying the IS-induced endothelial dysfunction that could lead to cardiovascular diseases. We also studied their extracellular vesicles (EVs) owing to their capacity to act as messengers that transmit signals through their cargo. MAIN METHODS: EVs were characterized by nanoparticle tracking analysis, transmission electron microscopy, flow cytometry, and tetraspanin expression. Cell lysates and isolated EVs were analyzed using liquid chromatography coupled with mass spectrometry, followed by Gene Set Enrichment Analysis to identify the altered pathways. KEY FINDINGS: Proteomic analysis of endothelial cells revealed that IS causes an increase in proteins related to adipogenesis, inflammation, and xenobiotic metabolism and a decrease in proliferation. Extracellular matrix elements, as well as proteins associated with myogenesis, response to UV irradiation, and inflammation, were found to be downregulated in IS-treated EVs. Fatty acid metabolism was also found to be increased along with adipogenesis and inflammation observed in cells. SIGNIFICANCE: The treatment of endothelial cells with IS increased the expression of proteins related to adipogenesis, inflammation, and xenobiotic metabolism and was less associated with proliferation. Furthermore, EVs from cells treated with IS may mediate endothelial dysfunction, since they present fewer extracellular matrix elements, myogenesis, inflammatory factors, and proteins downregulated in response to UV radiation.
Subject(s)
Endothelial Cells , Extracellular Vesicles , Indican , Proteomics , Renal Insufficiency, Chronic , Indican/metabolism , Extracellular Vesicles/metabolism , Humans , Renal Insufficiency, Chronic/metabolism , Proteomics/methods , Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , Proteome/metabolismABSTRACT
BACKGROUND: Information on the microbiome's human pathways and active members that can affect SARS-CoV-2 susceptibility and pathogenesis in the salivary proteome is very scarce. Here, we studied a unique collection of samples harvested from April to June 2020 from unvaccinated patients. METHODS: We compared 10 infected and hospitalized patients with severe (n = 5) and moderate (n = 5) coronavirus disease (COVID-19) with 10 uninfected individuals, including non-COVID-19 but susceptible individuals (n = 5) and non-COVID-19 and nonsusceptible healthcare workers with repeated high-risk exposures (n = 5). RESULTS: By performing high-throughput proteomic profiling in saliva samples, we detected 226 unique differentially expressed (DE) human proteins between groups (q-value ≤ 0.05) out of 3376 unambiguously identified proteins (false discovery rate ≤ 1%). Major differences were observed between the non-COVID-19 and nonsusceptible groups. Bioinformatics analysis of DE proteins revealed human proteomic signatures related to inflammatory responses, central cellular processes, and antiviral activity associated with the saliva of SARS-CoV-2-infected patients (p-value ≤ 0.0004). Discriminatory biomarker signatures from human saliva include cystatins, protective molecules present in the oral cavity, calprotectins, involved in cell cycle progression, and histones, related to nucleosome functions. The expression levels of two human proteins related to protein transport in the cytoplasm, DYNC1 (p-value, 0.0021) and MAPRE1 (p-value, 0.047), correlated with angiotensin-converting enzyme 2 (ACE2) plasma activity. Finally, the proteomes of microorganisms present in the saliva samples showed 4 main microbial functional features related to ribosome functioning that were overrepresented in the infected group. CONCLUSION: Our study explores potential candidates involved in pathways implicated in SARS-CoV-2 susceptibility, although further studies in larger cohorts will be necessary.
ABSTRACT
Epithelial intercellular adhesion molecule (ICAM)-1 is apically polarized, interacts with, and guides leukocytes across epithelial barriers. Polarized hepatic epithelia organize their apical membrane domain into bile canaliculi and ducts, which are not accessible to circulating immune cells but that nevertheless confine most of ICAM-1. Here, by analyzing ICAM-1_KO human hepatic cells, liver organoids from ICAM-1_KO mice and rescue-of-function experiments, we show that ICAM-1 regulates epithelial apicobasal polarity in a leukocyte adhesion-independent manner. ICAM-1 signals to an actomyosin network at the base of canalicular microvilli, thereby controlling the dynamics and size of bile canalicular-like structures. We identified the scaffolding protein EBP50/NHERF1/SLC9A3R1, which connects membrane proteins with the underlying actin cytoskeleton, in the proximity interactome of ICAM-1. EBP50 and ICAM-1 form nano-scale domains that overlap in microvilli, from which ICAM-1 regulates EBP50 nano-organization. Indeed, EBP50 expression is required for ICAM-1-mediated control of BC morphogenesis and actomyosin. Our findings indicate that ICAM-1 regulates the dynamics of epithelial apical membrane domains beyond its role as a heterotypic cell-cell adhesion molecule and reveal potential therapeutic strategies for preserving epithelial architecture during inflammatory stress.
Subject(s)
Actomyosin , Intercellular Adhesion Molecule-1 , Animals , Mice , Humans , Actomyosin/metabolism , Intercellular Adhesion Molecule-1/genetics , Intercellular Adhesion Molecule-1/metabolism , Epithelial Cells/metabolism , Hepatocytes/metabolism , Liver/metabolism , Actin Cytoskeleton/metabolism , Leukocytes/metabolism , Cell PolarityABSTRACT
Gonadal hormone deprivation (GHD) and decline such as menopause and bilateral oophorectomy are associated with an increased risk of neurodegeneration. Yet, hormone therapies (HTs) show varying efficacy, influenced by factors such as sex, drug type, and timing of treatment relative to hormone decline. We hypothesize that the molecular environment of the brain undergoes a transition following GHD, impacting the effectiveness of HTs. Using a GHD model in mice treated with Tibolone, we conducted proteomic analysis and identified a reprogrammed response to Tibolone, a compound that stimulates estrogenic, progestogenic, and androgenic pathways. Through a comprehensive network pharmacological workflow, we identified a reprogrammed response to Tibolone, particularly within "Pathways of Neurodegeneration", as well as interconnected pathways including "cellular respiration", "carbon metabolism", and "cellular homeostasis". Analysis revealed 23 proteins whose Tibolone response depended on GHD and/or sex, implicating critical processes like oxidative phosphorylation and calcium signalling. Our findings suggest the therapeutic efficacy of HTs may depend on these variables, suggesting a need for greater precision medicine considerations whilst highlighting the need to uncover underlying mechanisms.
Subject(s)
Norpregnenes , Animals , Norpregnenes/pharmacology , Female , Mice , Proteomics/methods , Estrogen Receptor Modulators/pharmacology , Neurodegenerative Diseases/metabolism , Neurodegenerative Diseases/drug therapy , Mice, Inbred C57BL , Male , Ovariectomy , Gonadal Hormones/metabolism , Brain/metabolism , Brain/drug effects , Brain/pathologyABSTRACT
Despite technological advances in the proteomics field, sample preparation still represents the main bottleneck in mass spectrometry (MS) analysis. Bead-based protein aggregation techniques have recently emerged as an efficient, reproducible, and high-throughput alternative for protein extraction and digestion. Here, a refined paramagnetic bead-based digestion protocol is described for Opentrons® OT-2 platform (OT-2) as a versatile, reproducible, and affordable alternative for the automatic sample preparation for MS analysis. For this purpose, an artificial neural network (ANN) was applied to maximize the number of peptides without missed cleavages identified in HeLa extract by combining factors such as the quantity (µg) of trypsin/Lys-C and beads (MagReSyn® Amine), % (w/v) SDS, % (v/v) acetonitrile, and time of digestion (h). ANN model predicted the optimal conditions for the digestion of 50 µg of HeLa extract, pointing to the use of 2.5% (w/v) SDS and 300 µg of beads for sample preparation and long-term digestion (16h) with 0.15 µg Lys-C and 2.5 µg trypsin (≈1:17 ratio). Based on the results of the ANN model, the manual protocol was automated in OT-2. The performance of the automatic protocol was evaluated with different sample types, including human plasma, Arabidopsis thaliana leaves, Escherichia coli cells, and mouse tissue cortex, showing great reproducibility and low sample-to-sample variability in all cases. In addition, we tested the performance of this method in the preparation of a challenging biological fluid such as rat bile, a proximal fluid that is rich in bile salts, bilirubin, cholesterol, and fatty acids, among other MS interferents. Compared to other protocols described in the literature for the extraction and digestion of bile proteins, the method described here allowed identify 385 unique proteins, thus contributing to improving the coverage of the bile proteome.
Subject(s)
Neural Networks, Computer , Animals , Humans , HeLa Cells , Mice , Rats , Proteomics/methods , Trypsin/metabolism , Trypsin/chemistry , AutomationABSTRACT
Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a severe rare liver disease that affects between 1/50,000 and 1/100,000 children. In physiological conditions, bile is produced by the liver and stored in the gallbladder, and then it flows to the small intestine to play its role in fat digestion. To prevent tissue damage, bile acids (BAs) are kept in phospholipid micelles. Mutations in phosphatidyl choline transporter ABCB4 (MDR3) lead to intrahepatic accumulation of free BAs that result in liver damage. PFIC3 onset usually occurs at early ages, progresses rapidly, and the prognosis is poor. Currently, besides the palliative use of ursodeoxycholate, the only available treatment for this disease is liver transplantation, which is really challenging for short-aged patients. To gain insight into the pathogenesis of PFIC3 we have performed an integrated proteomics and phosphoproteomics study in human liver samples to then validate the emerging functional hypotheses in a PFIC3 murine model. We identified 6246 protein groups, 324 proteins among them showing differential expression between control and PFIC3. The phosphoproteomic analysis allowed the identification of 5090 phosphopeptides, from which 215 corresponding to 157 protein groups, were differentially phosphorylated in PFIC3, including MDR3. Regulation of essential cellular processes and structures, such as inflammation, metabolic reprogramming, cytoskeleton and extracellular matrix remodeling, and cell proliferation, were identified as the main drivers of the disease. Our results provide a strong molecular background that significantly contributes to a better understanding of PFIC3 and provides new concepts that might prove useful in the clinical management of patients.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B , Cholestasis, Intrahepatic , Proteomics , Humans , Cholestasis, Intrahepatic/metabolism , Cholestasis, Intrahepatic/genetics , Cholestasis, Intrahepatic/pathology , Animals , Proteomics/methods , Mice , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily B/metabolism , ATP Binding Cassette Transporter, Subfamily B/deficiency , Liver/metabolism , Liver/pathology , Male , Disease Models, Animal , Phosphorylation , Female , Bile Acids and Salts/metabolism , MutationABSTRACT
The lack of sensitive diagnostic methods to detect Mycobacterium avium subsp. paratuberculosis (Map) subclinical infections has hindered the control of paratuberculosis (PTB). The serum proteomic profiles of naturally infected cows presenting focal and diffuse pathological forms of PTB and negative controls (n = 4 per group) were analyzed using TMT-6plex quantitative proteomics. Focal and diffuse are the most frequent pathological forms in subclinical and clinical stages of PTB, respectively. One (focal versus (vs.) control), eight (diffuse vs. control), and four (focal vs. diffuse) differentially abundant (DA) proteins (q-value < 0.05) were identified. Ingenuity pathway analysis of the DA proteins revealed changes in the acute-phase response and lipid metabolism. Six candidate biomarkers were selected for further validation by specific ELISA using serum from animals with focal, multifocal, and diffuse PTB-associated lesions (n = 108) and controls (n = 56). Overall, the trends of the serum expression levels of the selected proteins were consistent with the proteomic results. Alpha-1-acid glycoprotein (ORM1)-based ELISA, insulin-like growth factor-binding protein 2 (IGFBP2)-based ELISA, and the anti-Map ELISA had the best diagnostic performance for detection of animals with focal, multifocal, and diffuse lesions, respectively. Our findings identify potential biomarkers that improve diagnostic sensitivity of PTB and help to elucidate the mechanisms involved in PTB pathogenesis.
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CD137 (4-1BB) is a member of the TNFR family that mediates potent T cell costimulatory signals upon ligation by CD137L or agonist monoclonal antibodies (mAbs). CD137 agonists attain immunotherapeutic antitumor effects in cancer mouse models, and multiple agents of this kind are undergoing clinical trials. We show that cIAP1 and cIAP2 are physically associated with the CD137 signaling complex. Moreover, cIAPs are required for CD137 signaling toward the NF-κB and MAPK pathways and for costimulation of human and mouse T lymphocytes. Functional evidence was substantiated with SMAC mimetics that trigger cIAP degradation and by transfecting cIAP dominant-negative variants. Antitumor effects of agonist anti-CD137 mAbs are critically dependent on the integrity of cIAPs in cancer mouse models, and cIAPs are also required for signaling from CARs encompassing CD137's cytoplasmic tail.
Subject(s)
Neoplasms , Signal Transduction , Humans , Animals , Mice , NF-kappa B , Antibodies, Monoclonal/pharmacology , Spectrum Analysis, Raman , Neoplasms/drug therapyABSTRACT
BACKGROUND & AIMS: Hepatoblastoma (HB) is the most frequent childhood liver cancer. Patients with aggressive tumors have limited therapeutic options; therefore, a better understanding of HB pathogenesis is needed to improve treatment. HBs have a very low mutational burden; however, epigenetic alterations are increasingly recognized. We aimed to identify epigenetic regulators consistently dysregulated in HB and to evaluate the therapeutic efficacy of their targeting in clinically relevant models. METHODS: We performed a comprehensive transcriptomic analysis of 180 epigenetic genes. Data from fetal, pediatric, adult, peritumoral (n = 72) and tumoral (n = 91) tissues were integrated. Selected epigenetic drugs were tested in HB cells. The most relevant epigenetic target identified was validated in primary HB cells, HB organoids, a patient-derived xenograft model, and a genetic mouse model. Transcriptomic, proteomic and metabolomic mechanistic analyses were performed. RESULTS: Altered expression of genes regulating DNA methylation and histone modifications was consistently observed in association with molecular and clinical features of poor prognosis. The histone methyltransferase G9a was markedly upregulated in tumors with epigenetic and transcriptomic traits of increased malignancy. Pharmacological targeting of G9a significantly inhibited growth of HB cells, organoids and patient-derived xenografts. Development of HB induced by oncogenic forms of ß-catenin and YAP1 was ablated in mice with hepatocyte-specific deletion of G9a. We observed that HBs undergo significant transcriptional rewiring in genes involved in amino acid metabolism and ribosomal biogenesis. G9a inhibition counteracted these pro-tumorigenic adaptations. Mechanistically, G9a targeting potently repressed the expression of c-MYC and ATF4, master regulators of HB metabolic reprogramming. CONCLUSIONS: HBs display a profound dysregulation of the epigenetic machinery. Pharmacological targeting of key epigenetic effectors exposes metabolic vulnerabilities that can be leveraged to improve the treatment of these patients. IMPACT AND IMPLICATIONS: In spite of recent advances in the management of hepatoblastoma (HB), treatment resistance and drug toxicity are still major concerns. This systematic study reveals the remarkable dysregulation in the expression of epigenetic genes in HB tissues. Through pharmacological and genetic experimental approaches, we demonstrate that the histone-lysine-methyltransferase G9a is an excellent drug target in HB, which can also be harnessed to enhance the efficacy of chemotherapy. Furthermore, our study highlights the profound pro-tumorigenic metabolic rewiring of HB cells orchestrated by G9a in coordination with the c-MYC oncogene. From a broader perspective, our findings suggest that anti-G9a therapies may also be effective in other c-MYC-dependent tumors.
Subject(s)
Hepatoblastoma , Liver Neoplasms , Humans , Animals , Mice , Hepatoblastoma/drug therapy , Hepatoblastoma/genetics , Hepatoblastoma/metabolism , Proteomics , Epigenesis, Genetic , Liver Neoplasms/drug therapy , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , DNA Methylation , Carcinogenesis/geneticsABSTRACT
The liver is the only solid organ capable of regenerating itself to regain 100% of its mass and function after liver injury and/or partial hepatectomy (PH). This exceptional property represents a therapeutic opportunity for severe liver disease patients. However, liver regeneration (LR) might fail due to poorly understood causes. Here, we have investigated the regulation of liver proteome and phosphoproteome at a short time after PH (9 h), to depict a detailed mechanistic background of the early LR phase. Furthermore, we analyzed the dynamic changes of the serum proteome and metabolome of healthy living donor liver transplant (LDLT) donors at different time points after surgery. The molecular profiles from both analyses were then correlated. Insulin and FXR-FGF15/19 signaling were stimulated in mouse liver after PH, leading to the activation of the main intermediary kinases (AKT and ERK). Besides, inhibition of the hippo pathway led to an increased expression of its target genes and of one of its intermediary proteins (14-3-3 protein), contributing to cell proliferation. In association with these processes, metabolic reprogramming coupled to enhanced mitochondrial activity cope for the energy and biosynthetic requirements of LR. In human serum of LDLT donors, we identified 56 proteins and 13 metabolites statistically differential which recapitulate some of the main cellular processes orchestrating LR in its early phase. These results provide mechanisms and protein mediators of LR that might prove useful for the follow-up of the regenerative process in the liver after PH as well as preventing the occurrence of complications associated with liver resection.
Subject(s)
Liver Regeneration , Liver Transplantation , Mice , Animals , Humans , Liver Regeneration/genetics , Liver Transplantation/methods , Proteome/genetics , Proteome/metabolism , Living Donors , Liver/surgery , Liver/metabolismABSTRACT
BACKGROUND: In our continuing search for biologically active natural enemies from North of Africa with special reference to Tunisian fungi, our teamwork screened fungi from different ecological habitats in Tunisia. Our previous study on the comparative effectiveness of filamentous fungi in the biocontrol of Meloidogyne javanica, a taxon (Lecanicillium) showed high potentiality against M. javanica. We undertook the present study to evaluate the ability and understand the mechanism of this fungal parasite as a biological control candidate against the root-knot nematode M. javanica. This study used in vitro bioassays with fungal filtrate cultures, scanning electron microscopy (SEM) observation, and isobaric tag for relative and absolute quantitation (iTRAQ) methodology to characterize the biological and molecular features of this fungus. RESULTS: The microscopic and SEM observation revealed that Lecanicillium sp. exhibited exceptional hyperparasitism against M. javanica eggs. The hyphae of this fungi penetrated the eggs, causing destructive damage to the outer eggshell. The exposure to five concentrations of Lecanicillium sp. filtrate cultures showed high inhibition of egg hatching, which increases depending on the exposure time; the best results are recorded at 50%, 75%, and 100% dilutions after seven days of exposure. The SEM observation of nematode-parasitized eggs and juveniles suggests that the production of lytic enzymes degrades the egg cuticle and fungal hyphae penetrate unhatched M.javanica juveniles. Forty-seven unique proteins were identified from the Lecanicillium sp. isolate. These proteins have signalling and stress response functions, bioenergy, metabolism, and protein synthesis and degradation. CONCLUSION: Collectively, Lecanicillium sp. had ovicidal potentiality proved by SEM and proteomic analysis against root-knot nematode' eggs. This study recommended applying this biological control candidate as a bio-agent on vegetable crops grown in situ.
Subject(s)
Hypocreales , Tylenchoidea , Animals , Proteomics , Pest Control, Biological/methods , Tylenchoidea/microbiology , TunisiaABSTRACT
Introduction: Diabetic retinopathy (DR) and age-related macular degeneration (AMD) are leading causes of visual impairment and blindness in people aged 50 years or older in middle-income and industrialized countries. Anti-VEGF therapies have improved the management of neovascular AMD (nAMD) and proliferative DR (PDR), no treatment options exist for the highly prevalent dry form of AMD. Methods: To unravel the biological processes underlying these pathologies and to find new potential biomarkers, a label-free quantitative (LFQ) method was applied to analyze the vitreous proteome in PDR (n=4), AMD (n=4) compared to idiopathic epiretinal membranes (ERM) (n=4). Results and discussion: Post-hoc tests revealed 96 proteins capable of differentiating among the different groups, whereas 118 proteins were found differentially regulated in PDR compared to ERM and 95 proteins in PDR compared to dry AMD. Pathway analysis indicates that mediators of complement, coagulation cascades and acute phase responses are enriched in PDR vitreous, whilst proteins highly correlated to the extracellular matrix (ECM) organization, platelet degranulation, lysosomal degradation, cell adhesion, and central nervous system development were found underexpressed. According to these results, 35 proteins were selected and monitored by MRM (multiple reaction monitoring) in a larger cohort of patients with ERM (n=21), DR/PDR (n=20), AMD (n=11), and retinal detachment (n=13). Of these, 26 proteins could differentiate between these vitreoretinal diseases. Based on Partial least squares discriminant and multivariate exploratory receiver operating characteristic (ROC) analyses, a panel of 15 discriminatory biomarkers was defined, which includes complement and coagulation components (complement C2 and prothrombin), acute-phase mediators (alpha-1-antichymotrypsin), adhesion molecules (e.g., myocilin, galectin-3-binding protein), ECM components (opticin), and neurodegeneration biomarkers (beta-amyloid, amyloid-like protein 2).
Subject(s)
Diabetic Retinopathy , Epiretinal Membrane , Wet Macular Degeneration , Humans , Vitreous Body , Angiogenesis Inhibitors , Proteomics , Vascular Endothelial Growth Factor A , Visual Acuity , Complement System Proteins , BiomarkersABSTRACT
Introduction: Cycling Dof transcription factors (CDFs) have been involved in different aspects of plant growth and development. In Arabidopsis and tomato, one member of this family (CDF1) has recently been associated with the regulation of primary metabolism and abiotic stress responses, but their roles in crop production under open field conditions remain unknown. Methods: In this study, we compared the growth, and tuber yield and composition of plants ectopically expressing the CDF1 gene from Arabidopsis under the control of the 35S promoter with wild-type (WT) potato plants cultured in growth chamber and open field conditions. Results: In growth chambers, the 35S::AtCDF1 plants showed a greater tuber yield than the WT by increasing the biomass partition for tuber development. Under field conditions, the ectopic expression of CDF1 also promoted the sink strength of the tubers, since 35S::AtCDF1 plants exhibited significant increases in tuber size and weight resulting in higher tuber yield. A metabolomic analysis revealed that tubers of 35S::AtCDF1 plants cultured under open field conditions accumulated higher levels of glucose, starch and amino acids than WT tubers. A comparative proteomic analysis of tubers of 35S::AtCDF1 and WT plants cultured under open field conditions revealed that these changes can be accounted for changes in the expression of proteins involved in energy production and different aspects of C and N metabolism. Discussion: The results from this study advance our collective understanding of the role of CDFs and are of great interest for the purposes of improving the yield and breeding of crop plants.
ABSTRACT
The existence of sex differences in disease incidence is attributed, in part, to sex differences in metabolism. Uncovering the precise mechanism driving these differences is an extraordinarily complex process influenced by genetics, endogenous hormones, sex-specific lifetime events, individual differences and external environmental/social factors. In fact, such differences may be subtle, but across a life span, increase susceptibility to a pathology. Whilst research persists in the hope of discovering an elegant biological mechanism to underpin sex differences in disease, here, we show, for the first time, that such a mechanism may be subtle in nature but influenced by multiple sex-specific factors. A proteomic dataset was generated from a gonadectomized mouse model treated with Tibolone, a menopausal hormone therapy. Following functional enrichment analysis, we identified that Alzheimer's disease and the electron transport chain-associated pathways were regulated by sex-hormone interactions. Specifically, we identified that the expression of three respirasome proteins, NDUFA2, NDUFA7 and UQCR10, is significantly altered by compounding factors that contribute to sex differences. These proteins function in bioenergetics and produce reactive oxygen species, which are each dysregulated in many diseases with sex differences in incidence. We show sex-specific reprogrammed responses to Tibolone following gonadectomy, which primarily influence the expression of proteins contributing to metabolic pathways. This further infers that metabolic differences may underpin the observed sex differences in disease, but also that hormone therapy research now has potential in exploring sex-specific interventions to produce an effective method of prevention or treatment.
Subject(s)
Mitochondrial Membranes , Proteomics , Animals , Mice , Female , Male , Mitochondrial Membranes/metabolism , Gonadal Steroid Hormones/metabolism , Brain/metabolism , Proteins/metabolism , Hormones/metabolismABSTRACT
Proliferative diabetic retinopathy (PDR), proliferative vitreoretinopathy (PVR), and neovascular age-related macular degeneration (nAMD) are among the leading causes of blindness. Due to the multifactorial nature of these vitreoretinal diseases, omics approaches are essential for a deeper understanding of the pathophysiologic processes underlying the evolution to a proliferative or neovascular etiology, in which patients suffer from an abrupt loss of vision. For many years, it was thought that the function of the vitreous was merely structural, supporting and protecting the surrounding ocular tissues. Proteomics studies proved that vitreous is more complex and biologically active than initially thought, and its changes reflect the physiological and pathological state of the eye. The vitreous is the scenario of a complex interplay between inflammation, fibrosis, oxidative stress, neurodegeneration, and extracellular matrix remodeling. Vitreous proteome not only reflects the pathological events that occur in the retina, but the changes in the vitreous itself play a central role in the onset and progression of vitreoretinal diseases. Therefore, this review offers an overview of the studies on the vitreous proteome that could help to elucidate some of the pathological mechanisms underlying proliferative and/or neovascular vitreoretinal diseases and to find new potential pharmaceutical targets.
Subject(s)
Diabetic Retinopathy , Vitreoretinopathy, Proliferative , Humans , Vitreous Body/pathology , Proteome , Vitreoretinopathy, Proliferative/genetics , Vitreoretinopathy, Proliferative/pathology , Retina/pathology , Diabetic Retinopathy/genetics , Diabetic Retinopathy/pathologyABSTRACT
BACKGROUND: Cholangiocarcinoma (CCA) is still a deadly tumour. Histological and molecular aspects of thioacetamide (TAA)-induced intrahepatic CCA (iCCA) in rats mimic those of human iCCA. Carcinogenic changes and therapeutic vulnerabilities in CCA may be captured by molecular investigations in bile, where we performed bile proteomic and metabolomic analyses that help discovery yet unknown pathways relevant to human iCCA. METHODS: Cholangiocarcinogenesis was induced in rats (TAA) and mice (JnkΔhepa + CCl4 + DEN model). We performed proteomic and metabolomic analyses in bile from control and CCA-bearing rats. Differential expression was validated in rat and human CCAs. Mechanisms were addressed in human CCA cells, including Huh28-KRASG12D cells. Cell signaling, growth, gene regulation and [U-13C]-D-glucose-serine fluxomics analyses were performed. In vivo studies were performed in the clinically-relevant iCCA mouse model. RESULTS: Pathways related to inflammation, oxidative stress and glucose metabolism were identified by proteomic analysis. Oxidative stress and high amounts of the oncogenesis-supporting amino acids serine and glycine were discovered by metabolomic studies. Most relevant hits were confirmed in rat and human CCAs (TCGA). Activation of interleukin-6 (IL6) and epidermal growth factor receptor (EGFR) pathways, and key genes in cancer-related glucose metabolic reprogramming, were validated in TAA-CCAs. In TAA-CCAs, G9a, an epigenetic pro-tumorigenic writer, was also increased. We show that EGFR signaling and mutant KRASG12D can both activate IL6 production in CCA cells. Furthermore, phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme in serine-glycine pathway, was upregulated in human iCCA correlating with G9a expression. In a G9a activity-dependent manner, KRASG12D promoted PHGDH expression, glucose flow towards serine synthesis, and increased CCA cell viability. KRASG12D CAA cells were more sensitive to PHGDH and G9a inhibition than controls. In mouse iCCA, G9a pharmacological targeting reduced PHGDH expression. CONCLUSIONS: In CCA, we identified new pro-tumorigenic mechanisms: Activation of EGFR signaling or KRAS mutation drives IL6 expression in tumour cells; Glucose metabolism reprogramming in iCCA includes activation of the serine-glycine pathway; Mutant KRAS drives PHGDH expression in a G9a-dependent manner; PHGDH and G9a emerge as therapeutic targets in iCCA.
Subject(s)
Bile Duct Neoplasms , Cholangiocarcinoma , Animals , Arachnodactyly , Bile Duct Neoplasms/genetics , Bile Duct Neoplasms/pathology , Bile Ducts, Intrahepatic/metabolism , Bile Ducts, Intrahepatic/pathology , Carcinogenesis/genetics , Cholangiocarcinoma/pathology , Contracture , Epigenesis, Genetic , ErbB Receptors/genetics , ErbB Receptors/metabolism , Glucose , Glycine/metabolism , Humans , Interleukin-6/genetics , Interleukin-6/metabolism , Mice , Phosphoglycerate Dehydrogenase/genetics , Proteomics , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Rats , Serine/metabolismABSTRACT
The aim of the study was to explore new markers in serum proteome associated with the response to antiosteoporosis drugs, namely teriparatide and denosumab. We obtained serum samples from 14 patients with osteoporosis, both at baseline and after 6 months of treatment with teriparatide (n = 10) or denosumab (n = 4). Samples were analyzed by nanoliquid chromatography coupled to high-resolution mass spectrometry on a QTOF 5600 (SCIEX) apparatus. The spectrometry data were analyzed with Mascot against the UniProtKB base and then several quality-control filters were applied for the identification of peptides (false discovery rate, FDR q < 0.02) and their quantification (FDR q < 0.05). In the group treated with teriparatide, 28 proteins were identified with significant differences before and after treatment. A pathway analysis by using the Reactome database revealed significant enrichment in the Insulin Like Growth Factor 1 (IGF-I) (FDR q 4 × 10−2) and innate immune system (FDR q 2 × 10−3) pathways. Among patients treated with denosumab, we observed significant differences in the levels of 10 proteins, which were also enriched in the pathways related to the innate immune system (FDR q 3 × 10−2). These results suggest that the innate immune system may be involved in the response to antiosteoporosis drugs.
ABSTRACT
Oxidative stress is defined as an unbalance between pro-oxidants and antioxidants, as evidenced by an increase in reactive oxygen and reactive nitrogen species production over time. It is important in the pathophysiology of retinal disorders such as diabetic retinopathy, age-related macular degeneration, retinal detachment, and proliferative vitreoretinopathy, which are the focus of this article. Although the human organism's defense mechanisms correct autoxidation caused by endogenous or exogenous factors, this may be insufficient, causing an imbalance in favor of excessive ROS production or a weakening of the endogenous antioxidant system, resulting in molecular and cellular damage. Furthermore, modern lifestyles and environmental factors contribute to increased chemical exposure and stress induction, resulting in oxidative stress. In this review, we discuss the current information about oxidative stress and the vitreous proteome with a special focus on vitreoretinal diseases. Additionally, we explore therapies using antioxidants in an attempt to rescue the body from oxidation, restore balance, and maximize healthy body function, as well as new investigational therapies that have shown significant therapeutic potential in preclinical studies and clinical trial outcomes, along with their goals and strategic approaches to combat oxidative stress.
ABSTRACT
OBJECTIVE: Intratumor heterogeneity drives cancer progression and therapy resistance. However, it has yet to be determined whether and how subpopulations of cancer cells interact and how this interaction affects the tumour. DESIGN: We have studied the spontaneous flow of extracellular vesicles (EVs) between subpopulations of cancer cells: cancer stem cells (CSC) and non-stem cancer cells (NSCC). To determine the biological significance of the most frequent communication route, we used pancreatic ductal adenocarcinoma (PDAC) orthotopic models, patient-derived xenografts (PDXs) and genetically engineered mouse models (GEMMs). RESULTS: We demonstrate that PDAC tumours establish an organised communication network between subpopulations of cancer cells using EVs called the EVNet). The EVNet is plastic and reshapes in response to its environment. Communication within the EVNet occurs preferentially from CSC to NSCC. Inhibition of this communication route by impairing Rab27a function in orthotopic xenographs, GEMMs and PDXs is sufficient to hamper tumour growth and phenocopies the inhibition of communication in the whole tumour. Mechanistically, we provide evidence that CSC EVs use agrin protein to promote Yes1 associated transcriptional regulator (YAP) activation via LDL receptor related protein 4 (LRP-4). Ex vivo treatment of PDXs with antiagrin significantly impairs proliferation and decreases the levels of activated YAP.Patients with high levels of agrin and low inactive YAP show worse disease-free survival. In addition, patients with a higher number of circulating agrin+ EVs show a significant increased risk of disease progression. CONCLUSION: PDAC tumours establish a cooperation network mediated by EVs that is led by CSC and agrin, which allows tumours to adapt and thrive. Targeting agrin could make targeted therapy possible for patients with PDAC and has a significant impact on CSC that feeds the tumour and is at the centre of therapy resistance.
ABSTRACT
One of the critical issues to warrant the success of a proteome-wide analysis is sample preparation. Efficient protein extraction in the absence of interferent material is mandatory to achieve an ample proteome coverage by mass spectrometry. The study of biological fluids is always challenging due to their specific biochemical composition. However, there is increasing interest in their characterization as it will provide proteins that may advice disease setting, state, and progression. In particular, bile is proximal to liver and pancreas, and its study is especially attractive since it might provide valuable information for the clinical management of severe diseases afflicting these organs, which are at an urgent need of new biomarkers. Though previous efforts have been made to optimize protocols to analyze bile proteome, only partial descriptions were achieved due to its complex composition, where proteins represent less than 5% of the organic components. Here we describe a new method that significantly increases the bile proteome coverage while reducing by a factor of six the amount of sample required for the proteomic analysis.