Autocrine Activity of Exosomes in Glucocorticoid-Induced Injury of Bone Microvascular Endothelial Cells Identified by Protein Array

Background: Glucocorticoid could induce injury and apoptosis of bone microvascular endothelial cells (BMECs) in the femoral head and the application of icariin showed a protective effect. However, the impact of autocrine exosomes during these processes is still to be confirmed.Methods: Exosomes were extracted from BMECs treated with hydrocortisone or hydrocortisone plus icariin by super-speed centrifugation; exosome-carried proteins were evaluated via BCA assay, Western blotting, protein array assay and Elisa test, while miRNA expression profile was assessed via high-throughput sequencing and confirmed by quantitative polymerase chain reaction (qPCR) to screen candidate molecules responsible for BMEC-Exo function. BMECs were incubated with and without exosomes before glucocorticoid intervention and then the impact of BMECs-derived exosomes on BMECs viability, apoptosis, migration, angiogenesis, and protein expression was further assessed by a series of functional assays. Results: Exosomes secreted by BMECs could ameliorate glucocorticoid-induced endothelial cellular injury, improve cell viability, decrease cell apoptosis, and promote cell migration and angiogenesis compared with the blank control. These effects of secreted exosomes could be reinforced by icariin intervention. Meanwhile, mechanism studies showed that expression level of eNOS, COX-2, and pERK were significantly increased while the cleaved caspase-3 level was decreased in BMECs after coculture with exosomes. Although icariin treatment would not significantly change the size and total protein content of BMECs-derived exosomes, expression of exosome-carried vascular endothelial growth factor (VEGF) and transforming growth factor β1 (TGF-β1) was enhanced and numerous miRNAs involved in cell proliferation and apoptosis were up-regulated (e.g., hsa-miR-1469 and hsa-miR-133a-5p) or down-regulated (e.g., hsa-miR-10b-5p) (p < 0.05). 29 differentially expressed inflammatory factors were detected between the exosomes secreted by the Icariin-treated and the Model groups.Conclusion: To sum up, the present study indicates that autocrine exosomes could significantly improve glucocorticoid-induced injury of BMECs, partially mediated by activation of MAPK/ERK pathway and regulation of several inflammation/apoptosis/proliferation-associated proteins. Icariin intervention could reinforce these effects and may act as a promising drug for improving glucocorticoid-induced injury of BMECs. In vivo or animal studies are still required to better understand the function of BMEC-derived exosomes.

A Serological Snapshot of COVID-19 Initial Stages in Israel by a 6-Plex Antigen Array

A 6-plex protein array presenting the whole inactivated virus and five nucleocapsid and spike-derived SARS-CoV-2 antigens was used to generate a serological snapshot of SARS-CoV-2 seroprevalence and seroconversion in Israel in the early months of the pandemic. Our findings confirm a very low seroprevalence of anti-SARS-CoV-2 antibodies in the Israeli adult population.

Evaluation of Signaling Pathways Profiling in Human Dermal Endothelial Cells Treated by Snake Venom Cysteine-Rich Secretory Proteins (svCRiSPs) from North American Snakes Using Reverse Phase Protein Array (RPPA)

Cysteine-Rich Secretory Proteins (CRiSPs) are typically found in many snake venoms; however, the role that these toxins play in the pathophysiology of snakebites is still unclear. Herein, we compared the effects of snake venom CRiSPs (svCRiSPs) from the most medically important species of North American snakes on endothelial cell permeability and vascular permeability. We used reverse phase protein array (RPPA) to identify key signaling molecules on human dermal lymphatic (HDLECs) and blood (HDBECs) endothelial cells treated with svCRiSPs. The results showed that Css-CRiSP isolated from Crotalus scutulatus scutulatus and App-CRiSP from Agkistrodon piscivorus piscivorus are the most potent causes of increase vascular and endothelial permeability in comparison with other svCRiSPs used in this study. We examined the protein expression levels and their activated phosphorylation states in HDLECs and HDBECs induced by App-CRiSP and Css-CRiSP using RPPA. Interestingly, both App-CRiSP and Css-CRiSP induced caveolin-1 expression in HDBECs. We also found that stimulating HDBECs with Css-CRiSP and App-CRiSP significantly induced the phosphorylation of mTOR and Src, respectively. In HDLECs, Css-CRiSP significantly downregulated the expression of N-Cadherin and phospholipase C-gamma, while App-CRiSP significantly enhanced Akt and JNK phosphorylation. These results suggest that the increased endothelial permeability in HDLECs and HDBECs by Css-CRiSP and App-CRiSP may occur through different pathways.

Multicenter reverse-phase protein array data integration

Among the technologies available for protein biomarker discovery and validation, reverse-phase protein array (RPPA) benefits from unequalled sample throughput. Panels of high-quality antibodies enable the quantification by RPPA of protein abundance and posttranslational modifications in biological specimens with high precision and sensitivity. Incorporation of RPPA technology into clinical and drug development pipelines requires robust assays that generate reproducible results across multiple laboratories. We implemented the first international multicenter pilot study to investigate RPPA workflow variability. We characterized the proteomic responses of a series of breast cancer cells to two cancer drugs. This analysis quantified 86,832 sample spots, representing 108 biological samples, arrayed at three independent RPPA platforms. This unique integrated set of data is publicly available as a resource to the proteomic and cancer research communities to catalyse further analysis and investigation. We anticipate that this dataset will form a reference for the comparison of RPPA workflows and reagents, which can be expanded in the future, and will aid the identification of platform-robust treatment-marker antigens in breast cancer cells.

A Reverse Phase Protein Array Based Phospho-antibody Characterization Approach and Its Applicability for Clinical Derived Tissue Specimens

Background: Systematic quantification of phosphoprotein within cell signaling networks in solid tissues remains challenging and precise quantification in large scale samples has great potential for biomarker identification and validation. Methods:We developed a reverse phase protein array (RPPA) based phosphor-antibody characterization workflow by taking advantage of the lysis buffer compatible with alkaline phosphatase (AP) treatment and here termed it as a bottom-up antibody screening that differs from the conventional RPPA antibody validation procedure and applied it onto fresh frozen and formalin-fixed and paraffin-embedded tissue (FFPE) to test its applicability.Results:We tested the feasibility of this method by screening 106 phospho-antibodies with RPPA first followed by western blots on a panel of cell lines and demonstrated that AP treatment could serve as an independent factor that can be adopted for rapid RPPA phospho-antibody selection. We also performed studies on different clinical materials. For fresh frozen (FF) samples, pre-selected highly-specific antibodies showed a desirable data reproducibility and antibody specificity based on AP treatment indicating a potential for fresh tissue-based phospho-protein RPPA profiling. Of further clinical significance, using the same approach, based on two sets of FFPE samples from 63 melanoma and 40 lung cancer patients, we showed great interexperimental reproducibility and significant correlation with pathological markers MelanA for melanoma as well as a panel of lung cancer biomarkers for subtyping (EGFR, Napsin A, p63/p40, TTF1 and CK7) generating meaningful data that match clinical features. Conclusions:Our findings establish a highly efficient approach for phospho-antibody characterization by taking advantage of RPPA whereby the same methodology can be applied for tissue-based proteomics and phosphoproteomics in clinical assay development and application.

Mapping Autoantibodies in Children With Acute Rheumatic Fever

BackgroundAcute rheumatic fever (ARF) is a serious sequela of Group A Streptococcus (GAS) infection associated with significant global mortality. Pathogenesis remains poorly understood, with the current prevailing hypothesis based on molecular mimicry and the notion that antibodies generated in response to GAS infection cross-react with cardiac proteins such as myosin. Contemporary investigations of the broader autoantibody response in ARF are needed to both inform pathogenesis models and identify new biomarkers for the disease.MethodsThis study has utilised a multi-platform approach to profile circulating autoantibodies in ARF. Sera from patients with ARF, matched healthy controls and patients with uncomplicated GAS pharyngitis were initially analysed for autoreactivity using high content protein arrays (Protoarray, 9000 autoantigens), and further explored using a second protein array platform (HuProt Array, 16,000 autoantigens) and 2-D gel electrophoresis of heart tissue combined with mass spectrometry. Selected autoantigens were orthogonally validated using conventional immunoassays with sera from an ARF case-control study (n=79 cases and n=89 matched healthy controls) and a related study of GAS pharyngitis (n=39) conducted in New Zealand.ResultsGlobal analysis of the protein array data showed an increase in total autoantigen reactivity in ARF patients compared with controls, as well as marked heterogeneity in the autoantibody profiles between ARF patients. Autoantigens previously implicated in ARF pathogenesis, such as myosin and collagens were detected, as were novel candidates. Disease pathway analysis revealed several autoantigens within pathways linked to arthritic and myocardial disease. Orthogonal validation of three novel autoantigens (PTPN2, DMD and ANXA6) showed significant elevation of serum antibodies in ARF (p &lt; 0.05), and further highlighted heterogeneity with patients reactive to different combinations of the three antigens.ConclusionsThe broad yet heterogenous elevation of autoantibodies observed suggests epitope spreading, and an expansion of the autoantibody repertoire, likely plays a key role in ARF pathogenesis and disease progression. Multiple autoantigens may be needed as diagnostic biomarkers to capture this heterogeneity.

Serum anti-DIDO1, anti-CPSF2, and anti-FOXJ2 antibodies as predictive risk markers for acute ischemic stroke

Background Acute ischemic stroke (AIS) is a serious cause of mortality and disability. AIS is a serious cause of mortality and disability. Early diagnosis of atherosclerosis, which is the major cause of AIS, allows therapeutic intervention before the onset, leading to prevention of AIS. Methods Serological identification by cDNA expression cDNA libraries and the protein array method were used for the screening of antigens recognized by serum IgG antibodies in patients with atherosclerosis. Recombinant proteins or synthetic peptides derived from candidate antigens were used as antigens to compare serum IgG levels between healthy donors (HDs) and patients with atherosclerosis-related disease using the amplified luminescent proximity homogeneous assay-linked immunosorbent assay. Results The first screening using the protein array method identified death-inducer obliterator 1 (DIDO1), forkhead box J2 (FOXJ2), and cleavage and polyadenylation specificity factor (CPSF2) as the target antigens of serum IgG antibodies in patients with AIS. Then, we prepared various antigens including glutathione S-transferase-fused DIDO1 protein as well as peptides of the amino acids 297–311 of DIDO1, 426–440 of FOXJ2, and 607–621 of CPSF2 to examine serum antibody levels. Compared with HDs, a significant increase in antibody levels of the DIDO1 protein and peptide in patients with AIS, transient ischemic attack (TIA), and chronic kidney disease (CKD) but not in those with acute myocardial infarction and diabetes mellitus (DM). Serum anti-FOXJ2 antibody levels were elevated in most patients with atherosclerosis-related diseases, whereas serum anti-CPSF2 antibody levels were associated with AIS, TIA, and DM. Receiver operating characteristic curves showed that serum DIDO1 antibody levels were highly associated with CKD, and correlation analysis revealed that serum anti-FOXJ2 antibody levels were associated with hypertension. A prospective case–control study on ischemic stroke verified that the serum antibody levels of the DIDO1 protein and DIDO1, FOXJ2, and CPSF2 peptides showed significantly higher odds ratios with a risk of AIS in patients with the highest quartile than in those with the lowest quartile, indicating that these antibody markers are useful as risk factors for AIS. Conclusions Serum antibody levels of DIDO1, FOXJ2, and CPSF2 are useful in predicting the onset of atherosclerosis-related AIS caused by kidney failure, hypertension, and DM, respectively.