Proteomic analysis of endothelial cells and extracellular vesicles in response to indoxyl sulfate: Mechanisms of endothelial dysfunction in chronic kidney disease.

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.

Proteomic analysis of STEAP1 knockdown in human LNCaP prostate cancer cells.

Prostate cancer (PCa) continues to be one of the most common cancers in men worldwide. The six transmembrane epithelial antigen of the prostate 1 (STEAP1) protein is overexpressed in several types of human tumors, particularly in PCa. Our research group has demonstrated that STEAP1 overexpression is associated with PCa progression and aggressiveness. Therefore, understanding the cellular and molecular mechanisms triggered by STEAP1 overexpression will provide important insights to delineate new strategies for PCa treatment. In the present work, a proteomic strategy was used to characterize the intracellular signaling pathways and the molecular targets downstream of STEAP1 in PCa cells. A label-free approach was applied using an Orbitrap LC-MS/MS system to characterize the proteome of STEAP1-knockdown PCa cells. More than 6700 proteins were identified, of which a total of 526 proteins were found differentially expressed in scramble siRNA versus STEAP1 siRNA (234 proteins up-regulated and 292 proteins down-regulated). Bioinformatics analysis allowed us to explore the mechanism through which STEAP1 exerts influence on PCa, revealing that endocytosis, RNA transport, apoptosis, aminoacyl-tRNA biosynthesis, and metabolic pathways are the main biological processes where STEAP1 is involved. By immunoblotting, it was confirmed that STEAP1 silencing induced the up-regulation of cathepsin B, intersectin-1, and syntaxin 4, and the down-regulation of HRas, PIK3C2A, and DIS3. These findings suggested that blocking STEAP1 might be a suitable strategy to activate apoptosis and endocytosis, and diminish cellular metabolism and intercellular communication, leading to inhibition of PCa progression.

Proteomics profiling of vitreous humor reveals complement and coagulation components, adhesion factors, and neurodegeneration markers as discriminatory biomarkers of vitreoretinal eye diseases.

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).

Refinement of two-dimensional electrophoresis for vitreous proteome profiling using an artificial neural network.

Despite technological advances, two-dimensional electrophoresis (2DE) of biological fluids, such as vitreous, remains a major challenge. In this study, artificial neural network was applied to optimize the recovery of vitreous proteins and its detection by 2DE analysis through the combination of several solubilizing agents (CHAPS, Genapol, DTT, IPG buffer), temperature, and total voltage. The highest protein recovery (94.9% ± 4.5) was achieved using 4% (w/v) CHAPS, 0.1% (v/v) Genapol, 20 mM DTT, and 2% (v/v) IPG buffer. Two iterations were required to achieve an optimized response (580 spots) using 4% (w/v) CHAPS, 0.2% (v/v) Genapol, 60 mM DTT, and 0.5% (v/v) IPG buffer at 35 kVh and 25 °C, representing a 2.4-fold improvement over the standard initial conditions of the experimental design. The analysis of depleted vitreous using the optimized protocol resulted in an additional 1.3-fold increment in protein detection over the optimal output, with an average of 761 spots detected in vitreous from different vitreoretinopathies. Our results clearly indicate the importance of combining the appropriate amount of solubilizing agents with a suitable control of the temperature and voltage to obtain high-quality gels. The high-throughput of this model provides an effective starting point for the optimization of 2DE protocols. This experimental design can be adapted to other types of matrices. Graphical abstract.

Proteome analysis of vitreous humor in retinal detachment using two different flow-charts for protein fractionation.

The deeper understanding of retinal detachment (RD) pathogenesis may improve the visual outcome after surgery. Given the main role of the vitreous in retinal eye diseases, two strategies were explored to identify its proteome in RD. Fractionation techniques such as anion exchange chromatography (IEX) and SDS-PAGE combined with MALDI-TOF/TOF analysis allowed to identify 127 proteins in vitreous of RD patients. From these proteins, 19 were identified using only the IEX fractionation strategy, and 117 using a bidimensional (IEX and SDS-PAGE) fractionation. Of these proteins, 68 had not yet been found in other vitreous proteomic studies. The fractionation with IEX and SDS-PAGE largely improved the number of identified proteins proving that it is crucial to combine several methodologies to cover vitreous proteome.

Vitreous humor in the pathologic scope: insights from proteomic approaches.

The vitreous humor (VH) is the largest component of the eye. It is a colorless, gelatinous, highly hydrated matrix that fills the posterior segment of the eye between the lens and retina in vertebrates. In VH, a diversity of proteins that can influence retinal physiology is present, including growth factors, hormones, proteins with transporter activity, and enzymes. More importantly, the protein composition of VH has been described as being altered in a number of disease states. Therefore, attempts aiming at establishing a map of VH proteins and detecting putative biomarkers for ocular illness or protein fluctuations with putative physiologic significance were conducted over the last two decades, using proteomic approaches. Proteomic strategies often involve gel-based or LC techniques as sample fractioning approaches, subsequently coupled with MS procedures. This set of studies resulted in the proteomic characterization of a range of ocular disease samples, with particular incidence on diabetic retinopathy. However, practical therapeutic applications arising from these studies are scarce at the moment. A pertinent example of therapeutic targets arising from VH proteomics has emerged concerning vasoproliferative factors present in the vitreous, which should be involved in neovascularization and subsequent fibrovascular proliferation of the retina, in ocular disease context. Therefore, this review attempts to sum up the information acquired from the proteomic approaches to ocular disease conducted in VH samples, highlighting its clinical potential for disclosing ocular disease mechanisms and engendering pharmacological therapeutic treatments.

Trends in protein-based biosensor assemblies for drug screening and pharmaceutical kinetic studies.

The selection of natural and chemical compounds for potential applications in new pharmaceutical formulations constitutes a time-consuming procedure in drug screening. To overcome this issue, new devices called biosensors, have already demonstrated their versatility and capacity for routine clinical diagnosis. Designed to perform analytical analysis for the detection of a particular analyte, biosensors based on the coupling of proteins to amperometric and optical devices have shown the appropriate selectivity, sensibility and accuracy. During the last years, the exponential demand for pharmacokinetic studies in the early phases of drug development, along with the need of lower molecular weight detection, have led to new biosensor structure materials with innovative immobilization strategies. The result has been the development of smaller, more reproducible biosensors with lower detection limits, and with a drastic reduction in the required sample volumes. Therefore in order to describe the main achievements in biosensor fields, the present review has the main aim of summarizing the essential strategies used to generate these specific devices, that can provide, under physiological conditions, a credible molecule profile and assess specific pharmacokinetic parameters.

Trends in proteomic analysis of human vitreous humor samples.

Proteomic analysis of human vitreous humor (VH) may elucidate the pathogenesis of retinal ocular diseases and may provide information for the development of potential therapeutic targets due to its pivotal location near lens and retina. The discovery of whole VH proteome involves a complex analysis of thousands of proteins simultaneously. Therefore, in proteomic studies the protein fractionation is important for reducing sample complexity, facilitating the access to the low-abundant proteins, and recognizing them as biotargets for clinical research. Although several separation methods have been used, gel-based proteomics are the most popular and versatile ones applied for global protein separation. However, chromatographic methods and its combination with other separation techniques are now beginning to be used as promising set-ups for VH protein identification. This review attempts to offer an overview of the techniques currently used with VH, exploring its methodological demands, exposing its advantages, and helping the reader to plan future experiences. Moreover, this review shows the relevance of VH proteomic analysis as a tool for the study of the mechanisms underlying some ocular diseases and for the development of new therapeutic approaches.