Platelet Activation Signaling Research Articles

P-791 The 4-step ASCOT technique modifies the plasma proteome of patients with premature ovarian insufficiency to resemble that of normoresponder women

Abstract Study question Does the 4-step ASCOT technique induce any modification in the plasma proteomic profile of women with premature ovarian insufficiency (POI)? Summary answer The 4-step ASCOT technique elicits substantial changes in plasma proteome of POI patients, leading to a more similar composition to that observed in normoresponder women. What is known already Nowadays, treatment of POI women is still challenging and different approaches have been proposed to improve their reproductive potential, including stem cells (SC) and platelet-rich plasma (PRP). Previous studies showed that plasma treatments containing a combination of both SC-secreted and platelet-enclosed factors are the most promising regenerative treatment in mouse and human ovarian tissues. Therefore, we designed the 4-step ASCOT technique, consisting in: 1) SC mobilization from bone marrow to peripheral blood to enrich plasma with SC secreted factors, 2) Platelet concentration, 3) platelet activation to deliver additional growth factors and 4) direct ovarian injection. Study design, size, duration Experimental study with plasma samples of 10 POI women undergoing the 4-step ASCOT reactivation technique (NCT04475744) and 5 normoresponder fertile women. Peripheral plasma samples from POI women were collected at recruitment (Pre) and three months after SC-PRP ovarian injection (Post). Proteomic profiles of Pre and Post plasma samples were compared between them and with the normoresponder samples. Participants/materials, setting, methods Proteins were isolated from all plasmas and quantified by the data-independent acquisition parallel accumulation serial fragmentation (diaPASEF) approach. Principal component analysis was performed to compare the plasma proteomic profile of pre- and post-treatment samples, applying the vip function to determine which proteins explained the differences between both time-points (vip>1.5). Then, ElasticNet and Limma methods were employed to determine the statistically significant changes and functional analysis performed to establish the enriched KEGG pathways (false discovery rate<0.05). Main results and the role of chance Plasma proteome of POI patients at recruitment exhibited a distinctly different proteomic pattern from that of normoresponder women. However, principal component analysis revealed a clear distinction between plasma samples obtained from POI patients at recruitment and three months after the 4-step ASCOT technique (principal component 1 (PC1) accounting for 20% of explained variance, and PC2 for a 15.4%). Differences between both time-points were mainly determined by 56 proteins involved in processes with a key role in ovarian function, such as complement and coagulation cascades, platelet activation, Hippo pathway, cell cycle, oocyte meiosis and PI3K-Akt signaling pathway. Of these, fifteen proteins exhibited significantly differential expression between both time points, including the age-related APOF protein and the GC vitamin D binding protein, which was the most prominently changed protein after the treatment. Interestingly, the 4-step ASCOT technique allowed the plasma proteome of patients with POI to reach a similar profile to that observed in normoresponder women. In fact, a detailed analysis uncovered 72 differentially expressed proteins between pre-treatment and normoresponder samples, with only 28 in post-treatment vs. normoresponders. Moreover, the magnitude of expression changes, when compared to normoresponder women, was more significant in pre-treatment than in post-treatment samples. Limitations, reasons for caution Further research will be necessary to determine the extent of the regenerative effects of the identified differentially expressed proteins within the ovaries, and their direct implications in ovarian aging and function. Wider implications of the findings Our findings highlight that the 4-step ASCOT technique induces changes in plasma proteome of POI patients, reaching similar values to normoresponders. These changes, that remain for few months, might contribute to ovarian reactivation, being the stepping stone to design more effective and personalized treatments for patients with impaired ovarian reserves. Trial registration number NCT04475744

Identification of differentially expressed genes to predict the risk of heart failure in older patients with hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a common heart disease. Old people with HCM are at high risk of heart failure (HF). This study aimed to identify differentially expressed genes (DEGs) to evaluate the risk of HF in older patients with HCM. GSE89714 and GSE116250 were downloaded from Gene Expression Omnibus (GEO) database, and DEGs were identified by using limma R package with P < 0.05 and logFC> 1 as cut off. Protein-protein interaction (PPI) network, Genome Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed for the identified DEGs. NetworkAnalyst online tool was applied for Gene Set Enrichment Analysis (GSEA) analysis. We identified 124 overlap DEGs from the 2 datasets. PPI network showed that COL1A1, COL3A1, COL1A2, BGN, COL5A1, LUM, TGFB2, FMOD, ASPN, and COL14A1 were the top ten genes related to HCM and HF compared with control. Functional and pathway analyses showed that the overlap genes were mainly related to ECM-receptor interaction, ECM organization, Focal adhesion, PI3K-Akt signaling, TGF-beta signaling, and Platelet activation signaling and aggregation. Among the overlap genes, COL5A1 and LUM were significantly upregulated, while TGFB2, FMOD, ASPN, and COL14A1 were significantly downregulated in HF dataset compared with HCM dataset. Bioinformatics-based analysis revealed potential genes associated with HCM and HF, which could be utilized to evaluate the risk of HF in older patients with HCM.

C-type lectin-like receptor-2 in platelets mediates ferric chloride–induced platelet activation and attenuates ferroptosis of endothelial cells

BackgroundAn iron overload status induces ferroptosis, an iron-dependent nonapoptotic cell death, in various pathological conditions. We previously reported that hemin (heme), protoporphyrin-IX with ferric iron, activates platelets via C-type lectin-like receptor-2 (CLEC-2) and glycoprotein VI/FcRγ, but protoporphyrin-IX alone blocks CLEC-2-dependent platelet activation. Therefore, we hypothesized that free iron has the ability to activate platelets. ObjectivesThis study aimed to elucidate platelet activation mechanisms of iron (ferric chloride), including the identification of signaling pathways and receptors, and to examine whether platelets regulate ferroptosis. MethodsPlatelet aggregometry, platelet activation marker expression, and protein phosphorylation were examined in ferric chloride–stimulated human and murine platelets. Inhibitors of platelet activation signaling pathways and receptor-deleted platelets were utilized to identify the responsible signaling pathway and receptor. The effect of platelets on ferroptosis of endothelial cells was investigated in vitro. ResultsFerric chloride induced platelet activation dependent on Src family kinase pathways in humans and mice. Ferric chloride–induced platelet aggregation was almost lost in CLEC-2–depleted murine platelets and wild-type platelets preincubated with recombinant CLEC-2 proteins. Furthermore, coculture of wild-type platelets, but not CLEC-2–deficient platelets, attenuated ferroptosis of endothelial cells in vitro. ConclusionFerric chloride activates platelets via CLEC-2 and Src family kinase pathways, and platelets have a protective role in the ferroptosis of endothelial cells dependent on CLEC-2.

Skin proteomic screening and functional analysis of differential proteins associated with coat color in sheep (Ovis aries).

Coat color is an important characteristic and economic trait in domestic sheep. In this study, we explored the potential mechanisms and the signaling pathways involved in coat color regulation for sheep. Isobaric tags for relative and absolute quantification (iTRAQ) technology was used to catalog global protein expression profiles in skin of sheep with black versus white coat color. Immunofluorescence was used to observe the expression localization of differential protein. Western blot and quantitative real time polymerase chain reaction (qRT-PCR) were used to evaluate their role in the coat color formation of sheep. A total of 136 differential proteins were obtained in different coat colors, including 101 up-regulated and 35 down-regulated. Pigmentation function entries were enriched through GO annotation. Tyrosine metabolism and platelet activation signaling pathway were extracted by KEGG analysis. APOA1 (Apolipoprotein A-1) and FGA (Fibrinogen alpha chain) were found to be critical differential proteins by the interaction of differential proteins in the direct-interaction network diagram. Strikingly, twenty candidate differential proteins were screened, from which ACTB (Beta-actin) protein showed higher expression in white sheep skin, while ALB (albumin), APOA1 MAOA (Amine oxidase) and FGA proteins showed higher expression in black sheep skin, which validated by immunofluorescence, western blot and qRT-PCR. Our studies identified several novel proteins that may involved in the coat color formation of sheep. The white and black sheep skin proteome profiles obtained provide a valuable resource for future research to understand the network of protein expression controlling skin physiology and melanogenesis in sheep.

Former smoking associated with epigenetic modifications in human granulosa cells among women undergoing assisted reproduction

Smoking exposure during adulthood can disrupt oocyte development in women, contributing to infertility and possibly adverse birth outcomes. Some of these effects may be reflected in epigenome profiles in granulosa cells (GCs) in human follicular fluid. We compared the epigenetic modifications throughout the genome in GCs from women who were former (N = 15) versus never smokers (N = 44) undergoing assisted reproductive technologies (ART). This study included 59 women undergoing ART. Smoking history including time since quitting was determined by questionnaire. GCs were collected during oocyte retrieval and DNA methylation (DNAm) levels were profiled using the Infinium MethylationEPIC BeadChip. We performed an epigenome-wide association study with robust linear models, regressing DNAm level at individual loci on smoking status, adjusting for age, ovarian stimulation protocol, and three surrogate variables. We performed differentially methylated regions (DMRs) analysis and over-representation analysis of the identified CpGs and corresponding gene set. 81 CpGs were differentially methylated among former smokers compared to never smokers (FDR < 0.05). We identified 2 significant DMRs (KCNQ1 and RHBDD2). The former smoking-associated genes were enriched in oxytocin signaling, adrenergic signaling in cardiomyocytes, platelet activation, axon guidance, and chemokine signaling pathway. These epigenetic variations have been associated with inflammatory responses, reproductive outcomes, cancer development, neurodevelopmental disorder, and cardiometabolic health. Secondarily, we examined the relationships between time since quitting and DNAm at significant CpGs. We observed three CpGs in negative associations with the length of quitting smoking (p < 0.05), which were cg04254052 (KCNIP1), cg22875371 (OGDHL), and cg27289628 (LOC148145), while one in positive association, which was cg13487862 (PLXNB1). As a pilot study, we demonstrated epigenetic modifications associated with former smoking in GCs. The study is informative to potential biological pathways underlying the documented association between smoking and female infertility and biomarker discovery for smoking-associated reproductive outcomes.

The impact of different levels of wheat diets on hepatic oxidative stress, immune response, and lipid metabolism in Tibetan sheep (Ovis aries)

BackgroundCompared with corn, wheat contains higher crude protein, amino acids concentration. However, wheat contains a mass of anti-nutritional factors, resulting in increased of the digesta viscosity and impaired the intestinal function in ruminant.ObjectiveThis study aimed to investigate the effects of substitution of different amounts of wheat for corn on hepatic metabolism in the Tibetan lamb.MethodsNinety Tibetan lambs (Body weight = 12.37 ± 0.92 kg) were randomly assigned to three groups: 0% wheat diet (Control), 10% wheat diet (Low group), and 15% wheat diet (High group). The feeding trial lasted for 130 d, including a 10 d adaption period. Hepatic gene expression profiling was performed via RNA sequencing after the conclusion of the feeding trials.ResultsResults showed that greater level of glutathione peroxidase levels in L group compared with those of the C and H groups (P < 0.05). The immune indexes, including interleukin-1β (IL-1β), immunoglobulin A (IgA), and IgM were also elevated in L group compared with the other groups (P < 0.05). Compared with H group, the hepatocytes were arranged radially, and hepatic plates anastomosed with each other to form a labyrinth-like structure in L group. Transcriptomic analysis showed 872 differentially expressed genes (DEG) between H and L group, of which 755 were down-regulated and 117 were up-regulated. Through Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, 32 pathways were significantly enriched (Q-value < 0.05), such as the cAMP signaling pathway, Th1 and Th2 cell differentiation, leukocyte transendothelial migration, platelet activation and adipocytokine signaling pathway. Additionally, the expression of comment DEGs were verified via quantitative reverse-transcription polymerase chain reaction.ConclusionIn summary, our findings suggest that wheat can be supplemented up to 10% in Tibetan sheep, contributing to improve the hepatic oxidative stress, immune response and lipid metabolism through regulating the expression of related genes.

Integration of metabolomics and network pharmacology technology to explain the effect mechanisms of Danggui Buxue decoction in vascular dementia.

Danggui Buxue decoction (DBD) is a traditional Chinese medicine herbal decoction that has a good therapeutic effect on vascular dementia (VaD). However, its pharmacodynamic substances and underlying mechanisms are ambiguous. The work aimed to decipher the pharmacodynamic substances and molecular mechanisms of DBD against VaD rats based on gas chromatography-mass spectrometry metabonomics, network pharmacology, molecular docking, and experimental verification. The results indicated that DBD significantly improved the learning abilities and cognitive impairment in the VaD rat model. Integration analysis of the metabolomics and network pharmacology approach revealed that DBD might primarily affect arachidonic acid (AA) and inositol phosphate metabolic pathways by regulating the platelet activation signaling pathways. Six core targets (TNF [tumor necrosis factor], IL-6 [interleukin 6], PTGS2 [prostaglandin-endoperoxide synthase 2], MAPK1, MAPK3, and TP53) in the platelet activation signaling pathways also had a good affinity to seven main active components (saponins, organic acids, flavonoids, and phthalides) of DBD through the verification of molecular docking. Enzyme-linked immunosorbent assay results (ELISA) showed that the levels of TNF, IL-6, PTGS2, thromboxane B2, and caspase-3 in the platelet activation signaling pathway can be regulated by DBD. Our results indicated that DBD treated VaD mainly by modulating the platelet activation signaling pathway, and AA and inositol phosphate metabolism.

Qihuang Zhuyu formula alleviates coronary microthrombosis by inhibiting PI3K/Akt/αIIbβ3-mediated platelet activation

BackgroundCoronary microembolism (CME) is commonly seen in the peri-procedural period of Percutaneous Coronary Intervention (PCI), where local platelet activation and endothelial cell inflammation crosstalk may lead to micro thrombus erosion and rupture, with serious consequences. Qihuang Zhuyu Formula (QHZYF) is a Chinese herbal compound with high efficacy against coronary artery disease, but its antiplatelet mechanism is unclear. Hypothesis/PurposeThis study aimed to elucidate the effects and mechanisms of QHZYF on sodium laurate-induced CME using network pharmacology and in vitro and in vivo experiments. MethodsWe employed high-performance liquid chromatography mass spectrometry to identify the main components of QHZYF. Network pharmacology analysis, molecular docking and surface plasmon resonance (SPR) were utilized to predict the primary active components, potential therapeutic targets, and intervention pathways mediating the effects of QHZYF on platelet activation. Next, we pretreated a sodium laurate-induced minimally invasive CME rat model with QHZYF. In vivo experiments were performed to examine cardiac function in rats, to locate coronary arteries on heart sections to observe internal microthrombi, to extract rat Platelet-rich plasma (PRP) for adhesion assays and CD62p and PAC-1 (ITGB3/ITGA2B) flow assays, and to measure platelet-associated protein expression in PRP. In vitro clot retraction and Co-culture of HUVECs with PRP were performed and the gene pathway was validated through flow cytometry and immunofluorescence. ResultsCombining UPLC-Q-TOF/MS technology and database mining, 78 compounds were finally screened as the putative and representative compounds of QHZYF, with 75 crossover genes associated with CME. QHZYF prevents CME mainly by regulating key pathways of the inflammation and platelets, including Lipid and atherosclerosis, Fluid shear stress, platelet activation, and PI3K-Akt signaling pathways. Five molecules including Calyson, Oroxin A, Protosappanin A,Kaempferol and Geniposide were screened and subjected to molecular docking and SPR validation in combination with Lipinski rules (Rule of 5, Ro5). In vivo experiments showed that QHZYF not only improved myocardial injury but also inhibited formation of coronary microthrombi. QHZYF inhibited platelet activation by downregulating expression of CD62p receptor and platelet membrane protein αIIbβ3 and reduced the release of von Willebrand Factor (vWF), Ca2+ particles and inflammatory factor IL-6. Further analysis revealed that QHZYF inhibited the activation of integrin αIIbβ3, via modulating the PI3K/Akt pathways. In in vitro experiments, QHZYF independently inhibited platelet clot retraction. Upon LPS induction, the activation of platelet membrane protein ITGB3 was inhibited via the PI3K/Akt pathway, revealing an important mechanism for attenuating coronary microthrombosis. We performed mechanistic validation using PI3K inhibitor LY294002 and Akt inhibitor MK-2206 to show that QHZYF inhibited platelet membrane protein activation and inflammation to improved coronary microvessel embolism by regulating PI3K/Akt/αIIbβ3 pathways, mainly by inhibiting PI3K and Akt phosphorylation. ConclusionQHZYF interferes with coronary microthrombosis through inhibition of platelet adhesion, activation and inflammatory crosstalk, thus has potential in clinical anti-platelet applications. Calyson, Oroxin A, Protosappanin A, Kaempferol and Geniposide may be the major active ingredient groups of QHZYF that alleviate coronary microthrombosis.

A Mouse Model of the PAR4 Sequence Variant P310L Shows Decreased Platelet Reactivity and Thrombosis

Protease activated receptors 1 and 4 (PAR1, PAR4) on platelets mediate thrombin signaling and are vital for thrombus development. Hypercoagulation and thrombin generation are major risk factors for thrombosis. Sustained thrombin signaling through PAR4 promotes platelet activation, phosphatidylserine exposure, and subsequent thrombin generation. A single-nucleotide polymorphism (SNP, rs2227376) leads to an amino acid change in extracellular loop 3 (ECL3) of PAR4, which decreases PAR4 activation (PAR4-P310L). This SNP is also associated with a significantly lower risk for venous thromboembolism (VTE) in a GWAS meta-analysis. We aim to determine the mechanism by which PAR4-P310L (rs2227376) impacts platelet function and reduces the risk of VTE using mice with a homologous mutation (PAR4-P322L). Ex vivo platelet function was determined using flow cytometry and aggregometry. Arterial and venous thrombosis was examined using the ferric-chloride induced carotid artery injury and inferior vena cava (IVC) stasis models, respectively. Platelets from heterozygous (PAR4-322P/L) and homozygous (PAR4-322L/L) mice had decreased thrombin-mediated aggregation and decreased PAR4 reactivity compared to wild types. These mice also had a prolonged time to occlusion in arterial thrombosis, with 55% of PAR4-322P/L mice unable to develop stable thrombi within 30 minutes. In venous thrombosis, wild-type mice developed clotted IVCs that averaged 31 mg after 48 hours, while global PAR4-knockout mice averaged 21 mg (p&amp;lt;0.01), PAR4-322P/L 23 mg (p&amp;lt;0.01), and PAR4-322L/L 28 mg. Since platelet procoagulant activity is known to drive venous thrombosis, we used lactadherin to show that PAR4-P322L platelets express less phosphatidylserine in response to thrombin (5 nM) and convulxin (1 nM). Platelet-rich plasma from PAR4-P322L mice also generated less thrombin compared to wild-types, and whole blood from mice lacking PAR4 showed delayed clot formation in rotational thromboelastometry. Altogether, this suggests a direct impact of PAR4 signaling on platelet-mediated thrombin generation. Our new mouse model shows that the polymorphism in ECL3, PAR4-322L, decreases platelet reactivity and thrombosis. We also show PAR4-322L leads to less procoagulant platelets and decreases the endogenous thrombin potential. This decreased ability to generate thrombin offers a mechanism for PAR4's role in VTE. Our findings highlight a key role of PAR4 signaling in platelet activation and procoagulant activity in thrombosis.

Single Cell Transcriptomics Reveal That HMGA1 Chromatin Regulators Drive Transcriptional Networks Involved in Megakaryocyte Expansion, Platelet Activation, and Interferon Signaling in JAK2 V617F Myeloproliferative Neoplasms

Introduction: Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell (HSC) disorders characterized by hyperactive JAK/STAT signaling, thromboses, abnormal bleeding, and increased risk of transformation to myelofibrosis (MF) or acute myeloid leukemia (AML). Importantly, clinical outcomes are poor after transformation and actionable mechanisms that drive progression remain elusive. We discovered that High Mobility Group A1 ( HMGA1) chromatin regulators are required for transformation to AML in preclinical models of MPN by up-regulating transcriptional networks involved in GATA2 and proliferation (Li et al, Blood, 2022). Unexpectedly, loss of just a single Hmga1 allele within HSC prevents progression to MF in JAK2 V617F mice. Intriguingly, Hmga1 also drives fibrosis and tumor progression in pancreatic tumor models (Chia et al, JCI 2023). We therefore sought to elucidate mechanisms underlying HMGA1 early in progression that could be targeted in therapy to prevent MF. Methods : To elucidate Hmga1 function early in MPN, we performed single cell RNA sequencing (scRNAseq) of bone marrow (BM)-derived Lin -, Sca +, c-kit + (LSK) cells from JAK2 V617F mice at 16 weeks old before the development of MF with both Hmga1 alleles intact ( Hmga1+/+) compared to those with haploinsufficiency ( Hmga1+/-). We used gene set enrichment analysis (GSEA) to identify Hmga1-dependent transcriptional networks, CellChat to define cell interactions, and trajectory analysis to assess differentiation. To identify relevant networks in patients, we assessed HMGA1 pathways in transcriptomes from platelets, peripheral blood mononuclear cells, or CD34 + stem and progenitor cells from MPN patients and healthy, age-matched controls via bulk or scRNAseq. Results: We discovered that loss of just a single Hmga1 allele in HSPC is sufficient to dampen expansion of long-term, quiescent HSC (qHSC), megakaryocyte-biased HSC (Mk-HSC), and megakaryocyte-erythroid-biased HSC (MEP-HSC) while expanding HSC with a lymphoid bias (Ly-HSC) in JAK2 V617Fmice by scRNAseq. Pathway analysis in the most quiescent HSC clusters reveal that Hmga1 induces pathways involved in interferon α (IFNα) and interferon γ (IFNγ) signaling. Trajectory analysis show that JAK2 V617F qHSC with intact Hmga1 have diminished differentiation capacity, leading to decreased Ly-HSC. Because Hmga1 haploinsufficiency dramatically decreased megakaryocyte hyperplasia and thrombocytosis in JAK2 V617Fmice, we focused on HMGA1 transcriptional networks in Mk-HSC and MEP-HSC. Similar to qHSC, Hmga1 up-regulates networks involved in IFNα and IFNγ signaling in Mk- and MEP-biased HSC. In contrast to the qHSC, Hmga1 activates transcriptional networks involved in cell cycle progression in HSC poised to differentiate into Mk and MEP, including: 1) MYC Target pathways, 2) E2F Targets, and, 3) Mitotic Spindle genes. In addition, gene networks involved in oxidative phosphorylation are activated, which associate with proliferative states. Unexpectedly, we also discovered that Hmga1 up-regulates genes involved in coagulation and platelet activation, including genes encoding Platelet factor 4, von Willebrand factor, glycoprotein 2b, glycoprotein 3a, and P-selectin. Because megakaryocytes are associated with inflammatory signals in MPN, we queried inflammatory gene networks, which revealed that Hmga1 induces genes encoding IL6, IL1-β, and gene networks involved in signaling through TNFα and NF-κB. CellChat shows that Hmga1 increases MIF receptor-ligand signaling. Moreover, HMGA1 and similar networks are up-regulated in transcriptomes of platelets, PBMC, and CD34 + cells from patients with JAK2 V617F MPN. Conclusions: We uncovered a previously unknown epigenetic program in JAK2 V617FMPN whereby Hmga1 induces transcriptional networks that drive expansion in mutant HSC with greatest impact on HSC biased to differentiate into megakaryocytes (Mk-HSC, MEP-HSC). Mechanistically, Hmga1 up-regulates genes involved in cell cycle progression, platelet activation, and inflammatory signaling in Mk-biased HSC. HMGA1 also induces genes involved in IFNα and IFNγ signaling. Together, our studies reveal a new paradigm whereby HMGA1 drives progression early in MPN by activating transcriptional networks required for megakaryocyte expansion, inflammatory signaling, and IFN networks, the latter of which may sensitize JAK2 mutant HSC to IFN therapy.

Identification of novel gene signatures and immune cell infiltration in intervertebral disc degeneration using bioinformatics analysis.

Background: Intervertebral disc degeneration (IDD) is the leading cause of lower back pain, and an overall understanding of the molecular mechanisms related to IDD is still lacking. The purpose of this study was to explore gene signatures and immune cell infiltration related to IDD via bioinformatics analysis. Methods: A total of five expression profiles of mRNA and non-coding RNA were downloaded from the Gene Expression Omnibus (GEO) database. The potentially involved lncRNA/circRNA-miRNA-mRNA networks and protein-protein interaction networks were constructed by miRNet, circBank, STRING, and the Cytoscape database. Gene ontology, Kyoto Encyclopaedia of Genes and Genomes Analysis, Gene Set Enrichment Analysis, Gene Set Variation Analysis, Immune Infiltration Analysis, and Drug-Gene Interaction were used to analyse the top 20 hub genes. RT-qPCR was conducted to confirm the 12 differential expressions of genes both in the nucleus pulposus and annulus fibrosus tissues Results: There were 346 differentially expressed mRNAs, 12 differentially expressed miRNAs, 883 differentially expressed lncRNAs, and 916 differentially expressed circRNAs in the GEO database. Functional and enrichment analyses revealed hub genes associated with platelet activation, immune responses, focal adhesion, and PI3K-Akt signalling. The apoptotic pathway, the reactive oxygen species pathway, and oxidative phosphorylation play an essential role in IDD. Immune infiltration analysis demonstrated that the Treg cells had significant infiltration, and three levels of immune cells, including dendritic cells, Th2 cells, and tumour-infiltrating lymphocytes, were inhibited in IDD. Drug-gene interaction analysis showed that COL1A1 and COL1A2 were targeted by collagenase clostridium histolyticum, ocriplasmin, and PDGFRA was targeted by 66 drugs or molecular compounds. Finally, 24 cases of IDD tissues and 12 cases of normal disc tissues were collected, and the results of RT-qPCR were consistent with the bioinformatics results. Conclusion: Our data indicated that the 20 hub genes and immune cell infiltration were involved in the pathological process of IDD. In addition, the PDGFRA and two potential drugs were found to be significant in IDD development.

Platelet Glycoprotein-Ib (GPIb) May Serve as a Bridge between Type 2 Diabetes Mellitus (T2DM) and Atherosclerosis, Making It a Potential Target for Antiplatelet Agents in T2DM Patients.

Type 2 diabetes mellitus (T2DM) is a persistent metabolic condition that contributes to the development of cardiovascular diseases. Numerous studies have provided evidence that individuals with T2DM are at a greater risk of developing cardiovascular diseases, typically two to four times more likely than those without T2DM, mainly due to an increased risk of atherosclerosis. The rupture of an atherosclerotic plaque leading to pathological thrombosis is commonly recognized as a significant factor in advancing cardiovascular diseases caused by TD2M, with platelets inducing the impact of plaque rupture in established atherosclerosis and predisposing to the primary expansion of atherosclerosis. Studies suggest that individuals with T2DM have platelets that display higher baseline activation and reactivity than those without the condition. The expression enhancement of several platelet receptors is known to regulate platelet activation signaling, including platelet glycoprotein-Ib (GPIb). Furthermore, the high expression of platelet GP1b has been reported to increase the risk of platelet adhesion, platelet-leucocyte interaction, and thrombo-inflammatory pathology. However, the study exploring the role of GP1b in promoting platelet activation-induced cardiovascular diseases in T2DM patients is still limited. Therefore, we summarize the important findings regarding pathophysiological continuity between T2DM, platelet GPIb, and atherosclerosis and highlight the potential therapy targeting GPIb as a novel antiplatelet agent for preventing further cardiovascular incidents in TD2M patients.

Screening core genes and signaling pathways after SFTSV infection by integrated transcriptome profiling analysis

A newly discovered tick-borne virus called the severe fever with thrombocytopenia syndrome virus (SFTSV) can cause the severe fever with thrombocytopenia syndrome (SFTS). The mortality and incidence rate of SFTS patients remain extremely high due to the fast global dissemination of its arthropod vectors, and the mechanism of viral pathogenesis remains largely unknown. In this study, high-throughput RNA sequencing (RNA-Seq) was used to sequence HEK 293 cells treated with SFTSV at four time points. 115, 191, 259, and 660 differentially expressed genes (DEGs) were identified at 6, 12, 24, and 48 h post-infection, respectively. We found that SFTSV infection induced the expression of genes responsible for numerous cytokine-related pathways, including TNF, CXCL1, CXCL2, CXCL3, CXCL8, CXCL10, and CCL20. With the extension of infection time, the expression of most genes involved in these pathways increased significantly, indicating the host's inflammatory response to SFTSV. Moreover, the expression levels of GNA13, ARHGEF12, RHOA, ROCK1, and MYL12A, elements of the platelet activation signaling pathway, were downregulated during SFTSV infection, suggesting that the SFTSV infection may cause thrombocytopenia by inhibiting platelet activation. Our results contribute to further understanding the interaction between SFTSV and the host.

Metabolomics-based Approach to Analyze the Therapeutic Targets and Metabolites of a Synovitis Ointment for Knee Osteoarthritis

Background: Knee osteoarthritis (KOA) is a clinically common degenerative joint disease that is not fatal but has a high prevalence. Synovitis ointment (HMYG) is a traditional Chinese medicine formula that has been clinically proven to treat KOA; however, its therapeutic targets remain unknown. Objective: This study aimed to identify metabolites and potential targets of synovitis ointment alleviation in rats with KOA using ultra-high-performance liquid-chromatography–mass spectrometry (UHPLC-MS) metabolomics. Methods: The meniscus on each side of the knee was removed to model KOA in rats. The synovitis ointment treatment was provided for 4 weeks. The lateral diameter of the knee was measured once a week, and after 4 weeks, serum was collected to observe changes in the knee through a metabolomic analysis. Results: Synovitis ointment reduced the lateral diameter of the knee joint, relieved knee swelling, and improved knee volume. In total, 28 differential metabolites, which were mainly involved in arginine and proline metabolism and apoptosis, were identified in the Con and HMYG groups. 15-Deoxy-d-12, 14-PGJ2 and fomepizole were found to be the key metabolites after the HMYG treatment of KOA. Compared with known drugs (diclofenac diethylamine emulsion and Jin Huang San), 2-(SGlutathionyl) acetyl glutathione, daidzein, pelargonic acid, and sulfamethoxazole increased in the HMYG, and the metabolic pathways included the oxytocin signaling pathway, platelet activation, olfactory transduction, phototransduction, and cGMP-PKG signaling pathway. The expression levels of cleaved-caspase-3, Bcl-2, PIK3a, TP53, TGFB1, and NFKB1 were reversed after HMYG treatment. Conclusion: It has been observed that synovitis ointment relieves KOA. UHPLC-MS can analyze the potential mechanism of action of the herbal compound of the synovitis ointment.

Single-cell analysis of platelets from patients with periodontitis and diabetes

BackgroundBlood platelets are known to play a role in the development of atherosclerotic disease, thrombi, and hemostasis. Investigation of blood platelet transcriptome could provide evidence of disorders that increase vulnerability to cardiovascular disease. However, research on the molecular insights of platelet activation in patients with periodontitis and patients with periodontitis and type 2 diabetes mellitus (DM) is still lacking. ObjectivesIn this study, we analyzed expression in blood platelets from patients with periodontitis and patients with concurrent periodontitis and DM to examine the transcriptomic profile of platelets induced by periodontitis and the modifying effects of DM. MethodsWe obtained the transcriptional profiles of blood platelets from 11 healthy donors, 10 patients with periodontitis, and 6 patients with periodontitis and DM using single-cell RNA sequencing. The biological processes and coexpressed modules of transcriptionally altered genes were further explored. ResultsBoth the patients with periodontitis and DM and those with periodontitis without DM showed higher levels of platelet activation and coagulation signals than the healthy individuals. Platelets from the patients with periodontitis had higher expression levels of genes for RHO GTPase effectors, whereas platelets from the patients with periodontitis and DM demonstrated higher expression of genes involved in oxidative phosphorylation and cellular responses to stress than those from the controls. However, compared with the patients with only periodontitis, those with periodontitis and DM presented a lower expression level of genes for hemostasis and platelet receptors. ConclusionThese results suggest that periodontitis contributes to establishment of blood coagulation via platelet dysregulation, whereas the comorbidities of patients with periodontitis and DM impair the components of platelets, thus preventing normal functions.

Comprehensive analysis of glycoprotein VI-mediated platelet activation signaling pathway for predicting pan-cancer survival and response to anti-PD-1 immunotherapy

Platelets play a vital role in cancer and immunity. However, few comprehensive studies have been conducted on the role of platelet-related signaling pathways in various cancers and their responses to immune checkpoint blockade (ICB) therapy. In the present study, we focused on the glycoprotein VI-mediated platelet activation (GMPA) signaling pathway and comprehensively evaluated its roles in 19 types of cancers listed in The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO). Cox regression and meta-analyses showed that for all 19 types of cancers, patients with high GMPA scores tended to have a good prognosis. Furthermore, the GMPA signature score could serve as an independent prognostic factor for patients with skin cutaneous melanoma (SKCM). The GMPA signature was linked to tumor immunity in all 19 types of cancers, and was correlated with SKCM tumor histology. Compared to other signature scores, the GMPA signature scores for on-treatment samples were more robust predictors of the response to anti-PD-1 blockade in metastatic melanoma. Moreover, the GMPA signature scores were significantly negatively correlated with EMMPRIN (CD147) and positively correlated with CD40LG expression at the transcriptomic level in most cancer patient samples from the TCGA cohort and on-treatment samples from anti-PD1 therapy cohorts. The results of this study provide an important theoretical basis for the use of GMPA signatures, as well as GPVI-EMMPRIN and GPVI-CD40LG pathways, to predict the responses of cancer patients to various types of ICB therapy.

Apolipoprotein A-IV Has Bi-Functional Actions in Alcoholic Hepatitis by Regulating Hepatocyte Injury and Immune Cell Infiltration.

Alcohol abuse can lead to alcoholic hepatitis (AH), a worldwide public health issue with high morbidity and mortality. Here, we identified apolipoprotein A-IV (APOA4) as a biomarker and potential therapeutic target for AH. APOA4 expression was detected by Gene Expression Omnibus (GEO) databases, Immunohistochemistry, and qRT-PCR in AH. Bioinformatics Methods (protein-protein interaction (PPI) network, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Set Enrichment Analysis (GSEA) were used to show down-stream gene and pathways of APOA4 in AH. AML-12 cells were used to evaluate the biological function of APOA4 using an ELISA kit (AST, ALT, and IL-1β) and flow cytometry (ROS activity). Both in vivo and in vitro, APOA4 expression was significantly elevated in the AH model induced by alcohol (ETOH). AML-12 cell damage was specifically repaired by APOA4 deficiency, while AST, ALT, and IL-1β activity that was increased by ETOH (200 µmol, 12 h) were suppressed. APOA4 inhibition increased intracellular ROS induced by ETOH, which was detected by flow cytometry. Functional and PPI network analyses showed Fcgamma receptor (FCGR) and platelet activation signaling were potential downstream pathways. We identified CIDEC as a downstream gene of APOA4. The CIDEC AUC values for the ROC curves were 0.861. At the same time, APOA4 silencing downregulated the expression of CIDEC, whereas the knockdown of CIDEC did not influence the expression of APOA4 in AML-12 cells. Collectively, APOA4 regulates CIDEC expression and immune cell infiltration and may hold great potential as a biomarker and therapeutic target for AH.

Combined - whole blood and skin fibroblasts- transcriptomic analysis in Psoriatic Arthritis reveals molecular signatures of activity, resistance and early response to treatment.

BackgroundAn interplay between immune cells and resident skin and joint stromal cells is implicated in psoriatic arthritis (PsA), yet the mechanisms remain elusive with a paucity of molecular biomarkers for activity and response. Combined transcriptomic and immunophenotypic analysis of whole blood and skin fibroblasts could provide further insights.MethodsWhole blood RNA-seq was performed longitudinally in 30 subjects with PsA at the beginning, one and six months after treatment, with response defined at six months. As control groups, 10 healthy individuals and 10 subjects with rheumatoid arthritis (RA) were recruited combined with public datasets from patients with psoriasis (PsO) and systemic lupus erythematous (SLE). Differential expression analysis and weighted gene co-expression network analysis were performed to identify gene expression signatures, while deconvolution and flow cytometry to characterize the peripheral blood immune cell profile. In a subset of affected and healthy individuals, RNA-seq of skin fibroblasts was performed and subjected to CellChat analysis to identify the blood-skin fibroblast interaction network.ResultsPsA demonstrated a distinct “activity” gene signature in the peripheral blood dominated by TNF- and IFN-driven inflammation, deregulated cholesterol and fatty acid metabolism and expansion of pro-inflammatory non-classical monocytes. Comparison with the blood transcriptome of RA, PsO, and SLE revealed a “PsA-specific signature” enriched in extracellular matrix remodeling. This was further supported by the skin fibroblast gene expression profile, displaying an activated, proliferating phenotype, and by skin-blood interactome analysis revealing interactions with circulating immune cells through WNT, PDGF and immune-related semaphorins. Of note, resistance to treatment was associated with upregulation of genes involved in TGFβ signaling and angiogenesis and persistent increase of non-classical monocytes. Differentially expressed genes related to platelet activation and hippo signaling discriminated responders and non-responders as early as one month after treatment initiation.ConclusionTranscriptome analysis of peripheral blood and skin fibroblasts in PsA reveals a distinct disease activity signature and supports the involvement of skin fibroblasts through their activation and interaction with circulating immune cells. Aberrant TGFβ signaling and persistently increased non-classical monocytes characterize treatment-resistant PsA, with pro-inflammatory pathways related to platelet activation and Hippo signaling predicting early response to treatment.

Innate immune TLR7 signaling mediates platelet activation and platelet-leukocyte aggregate formation in murine bacterial sepsis

Thrombocytopenia is a common complication in sepsis and is associated with higher mortality. Activated platelets express CD62P, which facilitates platelet-leukocyte aggregate (PLA) formation and contributes to thrombocytopenia in sepsis. We have reported that thrombocytopenia in murine sepsis is partly attributable to TLR7 signaling, but the underlying mechanism is unclear. In the current study, we tested the hypothesis that TLR7 mediates platelet activation and PLA formation during sepsis. In vitro, whole blood from WT mice treated with loxoribine, a TLR7 agonist, exhibited a dose-dependent increase in activated platelets compared to the control (PBS with 0.05% DMSO) or loxoribine-treated TLR7−/− whole blood. In a murine model of sepsis, there was a significant increase in platelet activation and PLA formation 24 hours after cecal ligation and puncture (CLP) as evidenced by double positive expression of CD41+/CD62P+ and CD45+/CD62P+, respectively. The sepsis-induced PLA formation was significantly attenuated in TLR7−/− mice. Finally, in ex-vivo experiments, plasma isolated from septic mice induced WT platelet activation, but such effect was significantly attenuated in platelets deficient of TLR7. These findings demonstrate a pivotal role of TLR7 signaling in platelet activation and PLA formation during bacterial sepsis.

B.3 Activated gene pathways in post-infectious hydrocephalus (PIH):: proteogenomics and the PIH expressome

Background: Proteogenomics, the integration of proteomics and RNASeq expands the discovery landscape for candidate expressed gene networks to obtain novel insights into host response in post-infectious hydrocephalus (PIH). We examined the cerebrospinal fluid (CSF) of infants with PIH, and case controlled against age-matched infants with non-postinfectious hydrocephalus (NPIH) to probe the molecular mechanisms of PIH, leveraging molecular identification of bacterial and viral pathogens. Methods: Ventricular CSF samples of 100 infants ≤ 3 months of age with PIH (n=64) and NPIH (n=36) were analyzed with proteomics and RNASeq. 16S rRNA/DNA sequencing and virome capture identified Paenibacillus spp. and cytomegalovirus as dominant pathogenetic bacteria implicated in our PIH cohort. Proteogenomics assessed differential expression, gene set enrichment and activated gene pathways. Results: Of 616 proteins and 11,114 genes, there was enrichment for the immune system, cell-cell junction signaling and response to oxidative stress. Proteogenomics yielded 33 functionally and genetically associated gene sets related to neutrophil activation, platelet activation, and cytokines (interleukins and interferon) signaling. Conclusions: We identified PIH patients with severe disease at time of hydrocephalus surgery, to have differential expression of proteins/genes involved in neuroinflammation, ependymal barrier integrity and reaction to oxidative stress. Further studies are needed to examine those proteins/genes as biomarkers for PIH.