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Objectives To investigate whether circulating endothelial protein C receptor (EPCR) is associated with disease activity and inflammatory markers in rheumatoid arthritis. Methods Thirty-eight RA patients and 21 healthy controls (HC) were recruited via the A3BC biobank. Peripheral blood mononuclear cells and plasma were isolated from the blood of these participants. Plasma soluble (s)EPCR, IL-6, IL-17 and sCD14 were measured by enzyme-linked immunosorbent assay, cell membrane-associated (m)EPCR by flow cytometry; EPCR gene H3 single nucleotide polymorphism (SNP), which contributes to high plasma sEPCR levels, by PCR and DNA sequencing. Data were analysed using FlowJo10 and GraphPad Prism 10. Results RA patients had higher levels of mEPCR on T cells and plasma sEPCR compared with HC. No difference in the EPCR gene H3 SNP G genotype frequency was found between RA and HC. This SNP was significantly correlated with higher sEPCR levels in HC but not in RA patients. In RA, plasma sEPCR levels were positively correlated with IL-6, IL-17, sCD14, anti-CCP and rheumatoid factor. In contrast, mEPCR levels on T cells and natural killer cells (NK) were inversely associated with disease activity measures including 28/66 swollen joint count, 28/68 tender joint count and/or DAS28-CRP/ESR scores, and positively correlated with EPCR gene H3 SNP, which was also correlated with lower disease activity measures in RA. Conclusion Our findings suggest that EPCR may play an important role in RA, with plasma sEPCR being potentially associated with inflammatory markers and mEPCR and the EPCR gene H3 SNP possibly related to disease activity measures. [ABSTRACT FROM AUTHOR]

The antiphospholipid syndrome (APS) is an autoimmune disease characterized by the presence of pathogenic antiphospholipid antibodies (aPL). Since approximately 30 years ago, lipid-binding aPL, which do not require a protein cofactor, have been regarded as irrelevant for APS pathogenesis even though anticardiolipin are a diagnostic criterion of APS. In this review, we will summarize the available evidence from in vitro studies, animal models, and epidemiologic studies, which suggest that this concept is no longer tenable. Accordingly, we will only briefly touch on the role of other aPL in APS. This topic has been amply reviewed in detail elsewhere. We will discuss the consequences for laboratory diagnostics and future research required to resolve open questions related to the pathogenic role of different aPL specificities. [ABSTRACT FROM AUTHOR]

Même si ces dernières années la définition du syndrome des antiphospholipides (SAPL) n'a pas subi de changements majeurs, certaines connaissances ont évolué au cours du temps. Dans cet article, nous présenterons les nouveautés en termes de critères de classification, physiopathologie, évaluation du risque prothrombotique, cibles antigéniques et enfin traitements potentiels. Even if in recent years the definition of anti-phospholipid syndrome (APS) has not undergone major changes, some knowledges have evolved over time. In this article, we will present the new developments in terms of classification criteria, pathophysiology, assessment of prothrombotic risk, antigenic targets and finally potential treatments. [ABSTRACT FROM AUTHOR]

Endothelial Protein C Receptor (EPCR) is a key regulator of the activated protein C anti-coagulation pathway due to its role in the binding and activation of this protein. EPCR also binds to other ligands such as Factor VII and X, γδ T-cells, plasmodium falciparum erythrocyte membrane protein 1, and Secretory group V Phospholipases A2, facilitating ligand-specific functions. The functions of EPCR can also be regulated by soluble (s)EPCR that competes for the binding sites of membrane-bound (m)EPCR. sEPCR is created when mEPCR is shed from the cell surface. The propensity of shedding alters depending on the genetic haplotype of the EPCR gene that an individual may possess. EPCR plays an active role in normal homeostasis, anti-coagulation pathways, inflammation, and cell stemness. Due to these properties, EPCR is considered a potential effector/mediator of inflammatory diseases. Rheumatic diseases such as rheumatoid arthritis and systemic lupus erythematosus are autoimmune/inflammatory conditions that are associated with elevated EPCR levels and disease activity, potentially driven by EPCR. This review highlights the functions of EPCR and its contribution to rheumatic diseases. [ABSTRACT FROM AUTHOR]

Objectives Endothelial protein C receptor (EPCR) is highly expressed in synovial tissues of patients with RA, but the function of this receptor remains unknown in RA. This study investigated the effect of EPCR on the onset and development of inflammatory arthritis and its underlying mechanisms. Methods CIA was induced in EPCR gene knockout (KO) and matched wild-type (WT) mice. The onset and development of arthritis was monitored clinically and histologically. T cells, dendritic cells (DCs), EPCR and cytokines from EPCR KO and WT mice, RA patients and healthy controls (HCs) were detected by flow cytometry and ELISA. Results EPCR KO mice displayed >40% lower arthritis incidence and 50% less disease severity than WT mice. EPCR KO mice also had significantly fewer Th1/Th17 cells in synovial tissues with more DCs in circulation. Lymph nodes and synovial CD4 T cells from EPCR KO mice expressed fewer chemokine receptors CXCR3, CXCR5 and CCR6 than WT mice. In vitro , EPCR KO spleen cells contained fewer Th1 and more Th2 and Th17 cells than WT and, in concordance, blocking EPCR in WT cells stimulated Th2 and Th17 cells. DCs generated from EPCR KO bone marrow were less mature and produced less MMP-9. Circulating T cells from RA patients expressed higher levels of EPCR than HC cells; blocking EPCR stimulated Th2 and Treg cells in vitro. Conclusion Deficiency of EPCR ameliorates arthritis in CIA via inhibition of the activation and migration of pathogenic Th cells and DCs. Targeting EPCR may constitute a novel strategy for future RA treatment. [ABSTRACT FROM AUTHOR]

Many individual genetic risk loci have been associated with multiple common human diseases. However, the molecular basis of this pleiotropy often remains unclear. We present an integrative approach to reveal the molecular mechanism underlying the PROCR locus, associated with lower coronary artery disease (CAD) risk but higher venous thromboembolism (VTE) risk. We identify PROCR-p.Ser219Gly as the likely causal variant at the locus and protein C as a causal factor. Using genetic analyses, human recall-by-genotype and in vitro experimentation, we demonstrate that PROCR-219Gly increases plasma levels of (activated) protein C through endothelial protein C receptor (EPCR) ectodomain shedding in endothelial cells, attenuating leukocyte-endothelial cell adhesion and vascular inflammation. We also associate PROCR-219Gly with an increased pro-thrombotic state via coagulation factor VII, a ligand of EPCR. Our study, which links PROCR-219Gly to CAD through anti-inflammatory mechanisms and to VTE through pro-thrombotic mechanisms, provides a framework to reveal the mechanisms underlying similar cross-phenotype associations.

Background: The current epidemic of opioid overdoses in the United States necessitates a robust public health and clinical response. We described patterns of non-fatal opioid overdoses (NFOODs) in a small western region using data from the 9-1-1 Computer Assisted Dispatch (CAD) record and electronic Patient Clinical Records (ePCR) completed by EMS responders. We determined whether CAD and ePCR variables could identify NFOOD cases in 9-1-1 data for intervention and surveillance efforts. Methods: We conducted a retrospective analysis of 1 year of 9-1-1 emergency medical CAD and ePCR (including naloxone administration) data from the sole EMS provider in the response area. Cases were identified based on clinician review of the ePCR, and categorized as definitive NFOOD, probable NFOOD, or non-OOD. Sensitivity, specificity, positive and negative predictive values (PPV and NPV) of the most prevalent CAD and ePCR variables were calculated. We used a machine learning technique-Random-Forests (RF) modeling-to optimize our ability to accurately predict NFOOD cases within census blocks. Results: Of 37,960 9-1-1 calls, clinical review identified 158 NFOOD cases (0.4%), of which 123 (77.8%) were definitive and 35 (22.2%) were probable cases. Overall, 106 (67.1%) received naloxone from the EMS responder at the scene. As a predictor of NFOOD, naloxone administration by paramedics had 67.1% sensitivity, 99.6% specificity, 44% PPV, and 99.9% NPV. Using CAD variables alone achieved a sensitivity of 36.7% and specificity of 99.7%. Combining ePCR variables with CAD variables increased the diagnostic accuracy with the best RF model yielding 75.9% sensitivity, 99.9% specificity, 71.4% PPV, and 99.9% NPV. Conclusion: CAD problem type variables and naloxone administration, used alone or in combination, had sub-optimal predictive accuracy. However, a Random Forests modeling approach improved accuracy of identification, which could foster improved surveillance and intervention efforts. We identified the set of NFOODs that EMS encountered in a year and may be useful for future surveillance efforts.

BackgroundThe rs867186 single-nucleotide polymorphism in the PROCR gene (g.6936A > G, c.4600A > G) results in a serine-to-glycine substitution at codon 219 of endothelial protein C receptor (EPCR). We performed a case–control study followed by an updated meta-analysis of the association between this polymorphism and the risk of venous thromboembolism (VTE).Objective and methodsWe enrolled 263 VTE patients and 320 unrelated healthy controls for the case–control study. The total number of cases and controls for the meta-analysis were 5,768 and 30,017, respectively. A new online MetaGenyo Statistical Analysis System software was used to perform the current meta-analysis. Furthermore, a reproducibility study was conducted to validate our results.ResultsAmong well-defined thrombosis risk factors, Factor V Leiden was more frequent in the VTE group (p A polymorphism between the two groups. There was no difference in the mutation frequency of Factor V Leiden and prothrombin 20210G>A between cases with and without provoking factors and cases with and without VTE recurrence. The rs867186 “G” carriership did not influence the risk of VTE [odds ratio (OR) 1.339; 95% confidence interval (CI): 0.904–1.984] in our study. No significant differences could be demonstrated among the rs867186 genotype frequencies between VTE cases with and without provoking factors (p = 0.430). PROCR rs867186 was associated with an OR of 1.72 (95% CI: 0.95–3.13, p = 0.075) in terms of VTE recurrence. In the meta-analysis, a significant association was found between EPCR Ser219Gly polymorphism and VTE under the dominant model (OR = 1.27, 95% CI: 1.11–1.46, p = 0.0006), the recessive model (OR = 1.60, 95% CI: 1.26–2.04, p = 0.0001), the GG vs. AA contrast model (OR = 1.64, 95% CI: 1.28–2.09, p = 0.0001), and the GA vs. AA contrast model (OR = 1.24, 95% CI: 1.08–1.43, p = 0.002).ConclusionThe rs867186 was not associated with the first VTE risk in our case–control study; however, a tendency to VTE recurrence was observed. Based on the results of our reproducibility study, MetaGenyo is acceptable for meta-analysis in case of genetic epidemiology studies. Although the risk conferred by the rs867186 is mild in all meta-analyses, including ours, identifying patients carrying the minor allele might have an impact on personalized VTE risk assessment, risk-score calculation, and patient management.

ABSTRACT Antibody responses to variant surface antigens (VSAs) produced by the malaria parasite Plasmodium falciparum may contribute to age-related natural immunity to severe malaria. One VSA family, P. falciparum erythrocyte membrane protein-1 (PfEMP1), includes a subset of proteins that binds endothelial protein C receptor (EPCR) in human hosts and potentially disrupts the regulation of inflammatory responses, which may lead to the development of severe malaria. We probed peptide microarrays containing segments spanning five PfEMP1 EPCR-binding domain variants with sera from 10 Malian adults and 10 children to determine the differences between adult and pediatric immune responses. We defined serorecognized peptides and amino acid residues as those that elicited a significantly higher antibody response than malaria-naïve controls. We aimed to identify regions consistently serorecognized among adults but not among children across PfEMP1 variants, potentially indicating regions that drive the development of immunity to severe malaria. Adult sera consistently demonstrated broader and more intense serologic responses to constitutive PfEMP1 peptides than pediatric sera, including peptides in EPCR-binding domains. Both adults and children serorecognized a significantly higher proportion of EPCR-binding peptides than peptides that do not directly participate in receptor binding, indicating a preferential development of serologic responses at functional residues. Over the course of a single malaria transmission season, pediatric serological responses increased between the start and the peak of the season, but waned as the transmission season ended. IMPORTANCE Severe malaria and death related to malaria disproportionately affect sub-Saharan children under 5 years of age, commonly manifesting as cerebral malaria and/or severe malarial anemia. In contrast, adults in malaria-endemic regions tend to experience asymptomatic or mild disease. Our findings indicate that natural immunity to malaria targets specific regions within the EPCR-binding domain, particularly peptides containing EPCR-binding residues. Epitopes containing these residues may be promising targets for vaccines or therapeutics directed against severe malaria. Our approach provides insight into the development of natural immunity to a binding target linked to severe malaria by characterizing an “adult-like” response as recognizing a proportion of epitopes within the PfEMP1 protein, particularly regions that mediate EPCR binding. This “adult-like” response likely requires multiple years of malaria exposure, as increases in pediatric serologic response over a single malaria transmission season do not appear significant.

Postmortem neuropathology shows clear regional differences in many brain diseases. For example, brains from cerebral malaria (CM) patients show more hemorrhagic punctae in the brain's white matter (WM) than grey matter (GM). The underlying reason for these differential pathologies is unknown. Here, we assessed the effect of the vascular microenvironment on brain endothelial phenotype, focusing endothelial protein C receptor (EPCR). We demonstrate that the basal level of EPCR expression in cerebral microvessels is heterogeneous in the WM compared to the GM. We used in vitro brain endothelial cell cultures and showed that the upregulation of EPCR expression was associated with exposure to oligodendrocyte conditioned media (OCM) compared to astrocyte conditioned media (ACM). Our findings shed light on the origin of the heterogeneity of molecular phenotypes at the microvascular level and might help better understand the variation in pathology seen in CM and other neuropathologies associated with vasculature in various brain regions. [ABSTRACT FROM AUTHOR]

Hematopoietic stem cells (HSCs) are the self-renewing multipotent progenitors to all blood cell types. Identification and isolation of HSCs for study has depended on the expression of combinations of surface markers on HSCs that reliably distinguish them from other cell types. However, the increasing number of markers required to isolate HSCs has made it tedious, expensive, and difficult for newcomers, suggesting the need for a simpler panel of HSC markers. We previously showed that phenotypic HSCs could be separated based on expression of CD11a and that only the CD11a negative fraction contained true HSCs. Here, we show that CD11a and another HSC marker, endothelial protein C receptor (EPCR), can be used to effectively identify and purify HSCs. We introduce a new two-color HSC sorting method that can highly enrich for HSCs with efficiencies comparable to the gold standard combination of CD150 and CD48. Our results demonstrate that adding CD11a and EPCR to the HSC biologist's toolkit improves the purity of and simplifies isolation of HSCs. Stem Cells Translational Medicine 2018;7:468-476.

Postmortem neuropathology shows clear regional differences in many brain diseases. For example, brains from cerebral malaria (CM) patients show more hemorrhagic punctae in the brain’s white matter (WM) than grey matter (GM). The underlying reason for these differential pathologies is unknown. Here, we assessed the effect of the vascular microenvironment on brain endothelial phenotype, focusing endothelial protein C receptor (EPCR). We demonstrate that the basal level of EPCR expression in cerebral microvessels is heterogeneous in the WM compared to the GM. We used in vitro brain endothelial cell cultures and showed that the upregulation of EPCR expression was associated with exposure to oligodendrocyte conditioned media (OCM) compared to astrocyte conditioned media (ACM). Our findings shed light on the origin of the heterogeneity of molecular phenotypes at the microvascular level and might help better understand the variation in pathology seen in CM and other neuropathologies associated with vasculature in various brain regions.

Background: Hematopoietic stem cell (HSC) transplantation is considered a possible treatment option capable of curing various diseases. The aim of this study was the co-culturing of mesenchymal stem cell (MSC) spheres with HSCs under hypoxic condition to enhance the proliferation, self-renewal, stemness, and homing capacities of HSCs. Methods and results: HSCs were expanded after being subjected to different conditions including cytokines without feeder (Cyto), co-culturing with adherent MSCs (MSC), co-culturing with adherent MSCs + hypoxia (MSC + Hyp), co-culturing with MSCs spheres (Sph-MSC), co-culturing with MSCs spheres + hypoxia (Sph-MSC + Hyp), co-culturing with MSC spheres + cytokines (Sph-MSC + Cyto). After 10 days, total nucleated cell (TNC) and CD34+/CD38− cell counts, colony-forming unit assay (CFU), long-term culture initiating cell (LTC-IC), the expression of endothelial protein C receptor (EPCR), nucleostemin (NS), nuclear factor I/X (Nfix) CXCR4, and VLA-4 were evaluated. The TNC, CD34+/CD38− cell count, CFU, and LTC-IC were higher in the Sph-MSC + Hyp and Sph-MSC + Cyto groups as compared with those of the MSC + Hyp group (P < 0.001). The expanded HSCs co-cultured with MSC spheres in combination with hypoxia expressed more EPCR, CXCR4, VLA-4, NS, and Nfix mRNA. The protein expression was also more up-regulated in the Sph-MSC + Cyto and Sph-MSC + Hyp groups. Conclusion: Co-culturing HSCs with MSC spheres under hypoxic condition not only leads to higher cellular yield but also increases the expression of self-renewal and homing genes. Therefore, we suggest this approach as a simple and non-expensive strategy that might improve the transplantation efficiency of HSCs. [ABSTRACT FROM AUTHOR]

Cytoadhesion of Plasmodium falciparum -infected red blood cells is a virulence determinant associated with microvascular obstruction and organ complications. The gastrointestinal tract is a major site of sequestration in fatal cerebral malaria cases and kidney complications are common in severe malaria, but parasite interactions with these microvascular sites are poorly characterized. To study parasite tropism for different microvascular sites, we investigated binding of parasite lines to primary human microvascular endothelial cells from intestine (HIMEC) and peritubular kidney (HKMEC) sites. Of the three major host receptors for P. falciparum , CD36 had low or negligible expression; endothelial protein C receptor (EPCR) had the broadest constitutive expression; and intercellular adhesion molecule 1 (ICAM-1) was weakly expressed on resting cells and was strongly upregulated by TNF-α on primary endothelial cells from the brain, intestine, and peritubular kidney sites. By studying parasite lines expressing var genes linked to severe malaria, we provide evidence that both the DC8 and Group A EPCR-binding subsets of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family encodes binding affinity for brain, intestinal, and peritubular kidney endothelial cells, and that DC8 parasite adhesion was partially dependent on EPCR. Collectively, these findings raise the possibility of a brain-gut-kidney binding axis contributing to multi-organ complications in severe malaria. [ABSTRACT FROM AUTHOR]

Cytoadhesion of Plasmodium falciparum-infected red blood cells is a virulence determinant associated with microvascular obstruction and organ complications. The gastrointestinal tract is a major site of sequestration in fatal cerebral malaria cases and kidney complications are common in severe malaria, but parasite interactions with these microvascular sites are poorly characterized. To study parasite tropism for different microvascular sites, we investigated binding of parasite lines to primary human microvascular endothelial cells from intestine (HIMEC) and peritubular kidney (HKMEC) sites. Of the three major host receptors for P. falciparum, CD36 had low or negligible expression; endothelial protein C receptor (EPCR) had the broadest constitutive expression; and intercellular adhesion molecule 1 (ICAM-1) was weakly expressed on resting cells and was strongly upregulated by TNF-α on primary endothelial cells from the brain, intestine, and peritubular kidney sites. By studying parasite lines expressing var genes linked to severe malaria, we provide evidence that both the DC8 and Group A EPCR-binding subsets of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family encodes binding affinity for brain, intestinal, and peritubular kidney endothelial cells, and that DC8 parasite adhesion was partially dependent on EPCR. Collectively, these findings raise the possibility of a brain-gut-kidney binding axis contributing to multi-organ complications in severe malaria.

Identification and isolation of hematopoietic stem cells (HSCs) in mice is most commonly based on the expression of surface molecules Kit and Sca-1 and the absence of markers of mature lineages. However, Sca-1 is absent or weakly expressed in hematopoietic progenitors in many strains, including nonobese diabetic (NOD), BALB/c, C3H, and CBA mice. In addition, both Kit and Sca-1 levels are modulated following bone marrow injury. In these cases, other markers and dye exclusion methods have been employed to identify HSCs, yet there is no antibody-based stain that enables identification of HSCs and early progenitors when Kit and Sca-1 are inadequate. CD201 is a marker that is highly restricted to HSCs and progenitors, and CD27 is expressed at moderate-to-high levels on HSCs. We show here that combining CD201 and CD27 enables highly efficient isolation of long-term HSCs in NOD mice as well as in other strains, including SJL, FVB, AKR, BALB/c, C3H, and CBA. We also find that HSCs appear to maintain expression of CD201 and CD27 after hematopoietic injury when Kit expression is downregulated. These results suggest a widely applicable yet simple alternative for HSC isolation in settings where Kit and Sca-1 expression are insufficient.

It is not yet clear whether cellular junctions between splenic sinus endothelial cells are open or closed. In order to clarify this, immunolocalization of thrombomodulin (TM), endothelial protein C receptor (EPCR), protease-activated receptor 1 (PAR1), sphingosine 1-phosphate receptor 1 (S1P1), β-catenin phosphorylated at Try142 (β-catenin Y142) and β-catenin phosphorylated at Try654 (β-catenin Y654), which are related proteins that regulate dissociation and association of the adherens junctions of endothelial cells, are examined in rats using laser microscopy and electron microscopy. TM, EPCR, PAR1 and S1P1 were colocalized in the entire circumference of the endothelial cells, as well as in the caveolar membranes and junctional membranes of adjacent endothelial cells. These molecules may protect the adherens junctions of the endothelial cells. On the other hand, β-catenin Y142 and β-catenin Y 654 colocalized with α-catenin and β-catenin, respectively and in addition, β-catenin Y142 and β-catenin Y 654 were localized in the vicinity of the adherens junctions of the endothelial cells from immunogold electron microcopy. The adherens junctions are considered to be partially dissociated at the site where β-catenin Y142 and β-catenin Y 654 are localized. Thus, the system that protects the adherens junctions and the system that dissociates them may concurrently coexist in the endothelial cells and dissociation and association of the adherens junctions may be constantly repeated at the cell boundary of the endothelial cells. [ABSTRACT FROM AUTHOR]

Streptococcus pneumoniae is the leading cause of hospital community-acquired pneumonia. Patients with pneumococcal pneumonia may develop complicated parapneumonic effusions or empyema that can lead to pleural organization and subsequent fibrosis. The pathogenesis of pleural organization and scarification involves complex interactions between the components of the immune system, coagulation, and fibrinolysis. EPCR (endothelial protein C receptor) is a critical component of the protein C anticoagulant pathway. The present study was performed to evaluate the role of EPCR in the pathogenesis of S. pneumoniae infection–induced pleural thickening and fibrosis. Our studies show that the pleural mesothelium expresses EPCR. Intrapleural instillation of S. pneumoniae impairs lung compliance and lung volume in wild-type and EPCR-overexpressing mice but not in EPCR-deficient mice. Intrapleural S. pneumoniae infection induces pleural thickening in wild-type mice. Pleural thickening is more pronounced in EPCR-overexpressing mice, whereas it is reduced in EPCR-deficient mice. Markers of mesomesenchymal transition are increased in the visceral pleura of S. pneumoniae–infected wild-type and EPCR-overexpressing mice but not in EPCR-deficient mice. The lungs of wild-type and EPCR-overexpressing mice administered intrapleural S. pneumoniae showed increased infiltration of macrophages and neutrophils, which was significantly reduced in EPCR-deficient mice. An analysis of bacterial burden in the pleural lavage, the lungs, and blood revealed a significantly lower bacterial burden in EPCR-deficient mice compared with wild-type and EPCR-overexpressing mice. Overall, our data provide strong evidence that EPCR deficiency protects against S. pneumoniae infection–induced impairment of lung function and pleural remodeling. [ABSTRACT FROM AUTHOR]

Heterotopic ossification (HO) comprises the abnormal formation of ectopic bone in extraskeletal soft tissue. The factors that initiate HO remain elusive. Herein, we found that calcified apoptotic vesicles (apoVs) led to increased calcification and stiffness of tendon extracellular matrix (ECM), which initiated M2 macrophage polarization and HO progression. Specifically, single-cell transcriptome analyses of different stages of HO revealed that calcified apoVs were primarily secreted by a PROCR + fibroblast population. In addition, calcified apoVs enriched calcium by annexin channels, absorbed to collagen I via electrostatic interaction, and aggregated to produce calcifying nodules in the ECM, leading to tendon calcification and stiffening. More importantly, apoV-releasing inhibition or macrophage deletion both successfully reversed HO development. Thus, we are the first to identify calcified apoVs from PROCR + fibroblasts as the initiating factor of HO, and might serve as the therapeutic target for inhibiting pathological calcification., (© 2024 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

Background: Previous studies show that spinal cord ischemia and hypoxia is an important cause of spinal cord necrosis and neurological loss. Therefore, the study aimed to identify genes related to ischemia and hypoxia after spinal cord injury (SCI) and analyze their functions, regulatory mechanism, and potential in regulating immune infiltration., Methods: The expression profiles of GSE5296, GSE47681, and GSE217797 were downloaded from the Gene Expression Omnibus database. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed to determine the function and pathway enrichment of ischemia- and hypoxia-related differentially expressed genes (IAHRDEGs) in SCI. LASSO model was constructed, and support vector machine analysis was used to identify key genes. The diagnostic values of key genes were evaluated using decision curve analysis and receiver operating characteristic curve analysis. The interaction networks of miRNAs-IAHRDEGs and IAHRDEGs-transcription factors were predicted and constructed with the ENCORI database and Cytoscape software. CIBERSORT algorithm was utilized to analyze the correlation between key gene expression and immune cell infiltration., Results: There were 27 IAHRDEGs identified to be significantly expressed in SCI at first. These genes were mostly significantly enriched in wound healing function and the pathway associated with lipid and atherosclerosis. Next, five key IAHRDEGs (Abca1, Casp1, Lpl, Procr, Tnfrsf1a) were identified and predicted to have diagnostic value. Moreover, the five key genes are closely related to immune cell infiltration., Conclusion: Abca1, Casp1, Lpl, Procr, and Tnfrsf1a may promote the pathogenesis of ischemic or hypoxic SCI by regulating vascular damage, inflammation, and immune infiltration.

Diabetes mellitus is a major risk factor for cardiovascular disease. Platelets from diabetic patients are hyperreactive and release microparticles that carry activated cysteine proteases or calpains. Whether platelet-derived calpains contribute to the development of vascular complications in diabetes is unknown. Here we report that platelet-derived calpain1 (CAPN1) cleaves the protease-activated receptor 1 (PAR-1) on the surface of endothelial cells, which then initiates a signaling cascade that includes the activation of the tumor necrosis factor (TNF)-α converting enzyme (TACE). The latter elicits the shedding of the endothelial protein C receptor and the generation of TNF-α, which in turn, induces intracellular adhesion molecule (ICAM)-1 expression to promote monocyte adhesion. All of the effects of CAPN1 were mimicked by platelet-derived microparticles from diabetic patients or from wild-type mice but not from CAPN1−/− mice, and were not observed in PAR-1-deficient endothelial cells. Importantly, aortae from diabetic mice expressed less PAR-1 but more ICAM-1 than non-diabetic mice, effects that were prevented by treating diabetic mice with a calpain inhibitor as well as by the platelet specific deletion of CAPN1. Thus, platelet-derived CAPN1 contributes to the initiation of the sterile vascular inflammation associated with diabetes via the cleavage of PAR-1 and the release of TNF-α from the endothelial cell surface. [ABSTRACT FROM AUTHOR]

BackgroundCerebral malaria (CM) is characterized by the sequestration of Plasmodium-infected erythrocytes (pRBCs) to host brain microvasculature beds via P. falciparum erythrocyte membrane protein 1 (PfEMP1). Under normal conditions, activated protein C (APC) bound to endothelial protein C receptor (EPCR) has cytoprotective properties via the activation of protease-activated receptor 1 (PAR1). During malaria infection, pRBCs transports PfEMP1 to the membranes to bind EPCR in the same region as APC. As a result, APC is less capable of inducing cytoprotective effects via PAR1. Two studies involving adult malaria patients revealed that EPCR rs867186-GG allele is associated with protection against severe malaria, while three other studies involving child malaria patients could not show association between EPCR rs867186-GG genotype and severe malaria or increased mortality among children with CM.MethodsWe examined the association between the EPCR rs867186-GG genotype and the protection against cerebral malaria. Peripheral blood samples were collected from 47 malaria patients and 34 healthy individuals from a study conducted from 2004 to 2007 at the NSCB Medical College Hospital in India. CM and malaria-associated complications were defined based on WHO criteria. Genomic DNA was isolated from the peripheral blood mononuclear cells. Primer sequences were designed to contain rs867186 of the PROCR gene (NM 006404) and were used to amplify a 660 bp product as described before. PCR products were purified, and DNA sequences were determined by Sanger Sequencing (Genewiz, NJ). Nonparametric tests were used to compare the groups. To analyze differences in allele frequencies, we used chi-squared or Fisher's exact tests for categorical variables if the expected values were less than 5. P-value

Preeclampsia is a severe pregnancy‐related disease that is found in 3%–5% of pregnancies worldwide and is primarily related to the decreased proliferation and invasion of trophoblast cells and abnormal uterine spiral artery remodelling. However, studies on the pathogenesis of placental trophoblasts are insufficient, and the aetiology of PE remains unclear. Here, we report that endothelial protein C receptor (EPCR), a transmembrane glycoprotein, was down‐regulated in placentas from preeclamptic patients. Moreover, lack of EPCR significantly reduced the trophoblast cell proliferation, invasion and tube formation capabilities. Microscale thermophoresis analysis showed that EPCR directly bound to protease‐activated receptor 1 (PAR‐1), a G protein‐coupled receptor. This change resulted in a substantial reduction in active Rac1 and caused excessive actin rearrangement. Our findings reveal a previously unidentified role of EPCR in the regulation of trophoblast proliferation, invasion and tube formation through promotion of actin polymerization, which is required for normal placental development. [ABSTRACT FROM AUTHOR]

Background: Endothelial protein C receptor (EPCR) plays an anticoagulant and anti‐inflammatory role by promoting the activation of protein C by thrombin bound to thrombomodulin (TBM). Incompatibility between pig TBM and human/primate thrombin is thought to contribute to dysregulated coagulation in pig‐to‐primate organ xenografts, and expression of human TBM (hTBM) in pigs has shown benefit in preclinical models. However, it is not known whether there are incompatibilities—or molecular barriers—between endogenous pig EPCR (pEPCR) and transgenically expressed human TBM. Aim: To clone and express pEPCR, and determine its function in the human protein C pathway in vitro. Methods: Pig endothelial protein C receptor cDNA was generated from pig lung RNA by RT‐PCR. Primate COS‐7 transfectants expressing various combinations of human and pig TBM and EPCR were incubated with human thrombin and human protein C, and tested for TBM cofactor activity. Results: The predicted protein sequence of pEPCR shared 72.3% amino acid sequence identity with hEPCR, and residues critical for protein C binding were conserved. COS‐7 cells transfected with hEPCR, pEPCR or vector showed minimal TBM cofactor activity (0.13 ± 0.04, 0.13 ± 0.02 and 0.14 ± 0.06 U, respectively). The cofactor activity of hTBM‐transfected cells (1.18 ± 0.29 U) was 8‐fold higher than vector‐transfected cells (P =.004) and further increased 4‐fold and 3‐fold by co‐transfection with hEPCR (5.01 ± 1.12 U, P =.004) or pEPCR (3.73 ± 0.65 U, P =.003), respectively. Conclusions: Our data show that pEPCR is largely compatible with the human TBM/thrombin complex, when expressed on COS‐7 cells in vitro, promoting the activation of human protein C. These findings suggest that endogenous pEPCR will enhance the activity of transgenic hTBM in the xenograft setting. [ABSTRACT FROM AUTHOR]

Background: Cerebral malaria (CM) is characterized by the sequestration of Plasmodium -infected erythrocytes (pRBCs) to host brain microvasculature beds via P. falciparum erythrocyte membrane protein 1 (PfEMP1). Under normal conditions, activated protein C (APC) bound to endothelial protein C receptor (EPCR) has cytoprotective properties via the activation of protease-activated receptor 1 (PAR1). During malaria infection, pRBCs transports PfEMP1 to the membranes to bind EPCR in the same region as APC. As a result, APC is less capable of inducing cytoprotective effects via PAR1. Two studies involving adult malaria patients revealed that EPCR rs867186-GG allele is associated with protection against severe malaria, while three other studies involving child malaria patients could not show association between EPCR rs867186-GG genotype and severe malaria or increased mortality among children with CM. Methods: We examined the association between the EPCR rs867186-GG genotype and the protection against cerebral malaria. Peripheral blood samples were collected from 47 malaria patients and 34 healthy individuals from a study conducted from 2004 to 2007 at the NSCB Medical College Hospital in India. CM and malaria-associated complications were defined based on WHO criteria. Genomic DNA was isolated from the peripheral blood mononuclear cells. Primer sequences were designed to contain rs867186 of the PROCR gene (NM 006404) and were used to amplify a 660 bp product as described before. PCR products were purified, and DNA sequences were determined by Sanger Sequencing (Genewiz, NJ). Nonparametric tests were used to compare the groups. To analyze differences in allele frequencies, we used chi-squared or Fisher's exact tests for categorical variables if the expected values were less than 5. P-value <0.05 was considered statistically significant. Results: Our results showed significantly higher rates of AG and GG genotypes in CM patients compared to mild malaria (P = 0.0034). Conclusion: Our results indicate that rs867186-GG or rs867186-AG genotypes are not associated with protection against HCM. [ABSTRACT FROM AUTHOR]

Background: Due to low predictive values of obstructive sleep apnea (OSA) screening tools, there is a need for biomarker for screening of OSA patients at an early stage. The aim of the study was to evaluate differentially expressed proteins in blood and urine samples of OSA patients.Methods: In this study, we used isobaric tagging for relative and absolute quantification (iTRAQ) based proteomics approach to identify differentially expressed proteins, which were subsequently verified and validated using enzyme-linked immunosorbent assay (ELISA) technique in adult OSA patients.Results: Seventeen differentially expressed proteins were selected from iTRAQ data for verification, based on their clinical significance and reproducibility among different iTRAQ experiment sets. Five of these proteins (plasma = 2; urine = 3) were further validated in plasma (non-OSA- = 42; OSA = 198) and urine samples (non-OSA = 46; OSA = 197). ROC curve analysis for all OSA vs. non-OSA subjects ensured optimal diagnostic utility of two urinary proteins: Endothelial protein c receptor (EPCR) (AUC = 73%, cut-off: 35 pg/ml) and dermcidin (AUC = 74%, cut-off: 4.6 pg/ml). For severe OSA, diagnostic accuracy significantly improved with AUC as 88% and 82% for EPCR (cut-off: 46 pg/ml) and dermcidin (cut-off: 5.2 pg/ml) respectively. Sensitivity and specificity of combined performance of both urinary proteins for severe OSA were 94% and 91% respectively.Conclusion: In this study, urinary EPCR and dermcidin emerged as novel biomarkers for screening severe OSA patients. [ABSTRACT FROM AUTHOR]

Acute kidney injury is a common and complex complication that has high morality and the risk for chronic kidney disease among survivors. The accuracy of current AKI biomarkers can be affected by water retention and diuretics. Therefore, we aimed to identify a urinary non-recovery marker of acute kidney injury in patients with acute decompensated heart failure. We used the isobaric tag for relative and absolute quantification technology to find a relevant marker protein that could divide patients into control, acute kidney injury with recovery, and acute kidney injury without recovery groups. An enzyme-linked immunosorbent assay of the endothelial cell protein C receptor (EPCR) was used to verify the results. We found that the EPCR was a usable marker for non-recovery renal failure in our setting with the area under the receiver operating characteristics 0.776 ± 0.065; 95%CI: 0.648-0.905, ( p < 0.001). Further validation is needed to explore this possibility in different situations.

Objectives To investigate whether activated protein C (APC), a physiological anticoagulant can inhibit the inflammatory/invasive properties of immune cells and rheumatoid arthritis synovial fibroblasts (RASFs) in vitro and prevent inflammatory arthritis in murine antigen-induced arthritis (AIA) and CIA models. Methods RASFs isolated from synovial tissues of patients with RA, human peripheral blood mononuclear cells (PBMCs) and mouse thymus cells were treated with APC or TNF-α/IL-17 and the following assays were performed: RASF proliferation and invasion by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and cell invasion assays, respectively; cytokines and signalling molecules using ELISA or western blot; Th1 and Th17 phenotypes in human PBMCs or mouse thymus cells by flow cytometry. The in vivo effect of APC was evaluated in AIA and CIA models. Results In vitro, APC inhibited IL-1β, IL-17 and TNF-α production, IL-17-stimulated cell proliferation and invasion and p21 and nuclear factor κB activation in RASFs. In mouse thymus cells and human PBMCs, APC suppressed Th1 and Th17 phenotypes. In vivo , APC inhibited pannus formation, cartilage destruction and arthritis incidence/severity in both CIA and AIA models. In CIA, serum levels of IL-1β, IL-6, IL-17, TNF-α and soluble endothelial protein C receptor were significantly reduced by APC treatment. Blocking endothelial protein C receptor, the specific receptor for APC, abolished the early or preventative effect of APC in AIA. Conclusion APC prevents the onset and development of arthritis in CIA and AIA models via suppressing inflammation, Th1/Th17 phenotypes and RASF invasion, which is likely mediated via endothelial protein C receptor. [ABSTRACT FROM AUTHOR]

Objective To investigate the influence and mechanism of Danshen Tongluo Jiedu Tang (Salvia Root Collateral-freeing Removing Toxicity Decoction) combined with bone marrow stem cell transplantation (BMSCT) on myocardial fibrosis in rats with acute myocardial infarction (AMI). Methods The bone marrow mesenchymal stem cells (BMSCs) were isolated and cultured by using whole bone marrow adherence method combined with density gradient centrifugation. The model of AMI was established through ligating the left anterior descending branch (LAD) of coronary artery. The rats were randomly divided into sham-operation group, AMI model group, BMSCT group, Danshen Tongluo Jiedu Tang group (Chinese medicinal group) and Danshen Tongluo Jiedu Tang+BMSCT group (combination group). The model was established in all groups except of sham-operation group, and then Chinese medicinal group and combination group were respectively given orally DTJT and other groups were given orally equivalent saline water once a day for 14 days continuously. The content of collagen in myocardial tissue was observed after Masson staining, and concentration of procollagen type III N-terminal peptide (PⅢP), procollagen type IV N-terminal peptide (PIVP), nitric oxide (NO) and endothelin-1 (ET-1) were detected by using ELISA. The expressions of endothelial protein C receptor (EPCR) and thrombomodulin (TM) were detected by using Western blotting assay, and all data was analyzed by using SPSS 19.0 statistical software. Results Compared with sham-operation group, the protein expressions of EPCR and TM decreased, NO level decreased, levels of ET-1, PⅢP and PⅣP increased, and myocardial collagen fiber generation in varying degrees was observed in other groups (P<0.01). The protein expressions of EPCR and TM and NO level increased, and levels of ET-1, PⅢP and PⅣP and myocardial collagen fiber generation decreased in BMSCT group, Chinese medicinal group and combination group compared with AMI group (P<0.01). The protein expressions of EPCR and TM and NO level increased, and levels of ET-1, PⅢP and PⅣP and myocardial collagen fiber generation decreased in Chinese medicinal group compared with BMSCT group (P<0.01). The protein expressions of EPCR and TM and NO level increased, and levels of ET-1, PⅢP and PⅣP and myocardial collagen fiber generation decreased in combination group compared with Chinese medicinal group (P<0.01). Conclusion Danshen Tongluo Jiedu Tang combined with BMSCT can inhibit myocardial fibrosis, and the mechanism may be related to that the protein expressions of myocardial EPCR and TM are up-regulated and vascular endothelial function is protected in AMI rats. [ABSTRACT FROM AUTHOR]

This article is focused on the pathophysiology, prevalence , clinical manifestations, diagnosis, and treatment C deficiency with increased risk of thrombosis. Activated protein C (aPC) exerts its anticoagulant activity through inactivation of coagulation factors Va and VIIIa. Aside its anticoagulant property, aPC demonstrates anti-inflammatory and cytoprotectiveeffects, which are mediated through the endothelial protein C receptor and the protease-activated receptor-I (PAR-1). Studying and understanding the biological function of APS makes it possible to control coagulation and inflammation and to apply protein C preparations as anticoagulant and cytoprotective drugs in a physicians clinicalpractice.

Background: The rs867186 single-nucleotide polymorphism in the PROCR gene (g.6936A > G, c.4600A > G) results in a serine-to-glycine substitution at codon 219 of endothelial protein C receptor (EPCR). We performed a case-control study followed by an updated meta-analysis of the association between this polymorphism and the risk of venous thromboembolism (VTE)., Objective and Methods: We enrolled 263 VTE patients and 320 unrelated healthy controls for the case-control study. The total number of cases and controls for the meta-analysis were 5,768 and 30,017, respectively. A new online MetaGenyo Statistical Analysis System software was used to perform the current meta-analysis. Furthermore, a reproducibility study was conducted to validate our results., Results: Among well-defined thrombosis risk factors, Factor V Leiden was more frequent in the VTE group ( p  < 0.001), while there was no difference in mutation frequency of prothrombin 20210G>A polymorphism between the two groups. There was no difference in the mutation frequency of Factor V Leiden and prothrombin 20210G>A between cases with and without provoking factors and cases with and without VTE recurrence. The rs867186 "G" carriership did not influence the risk of VTE [odds ratio (OR) 1.339; 95% confidence interval (CI): 0.904-1.984] in our study. No significant differences could be demonstrated among the rs867186 genotype frequencies between VTE cases with and without provoking factors ( p  = 0.430). PROCR rs867186 was associated with an OR of 1.72 (95% CI: 0.95-3.13, p  = 0.075) in terms of VTE recurrence. In the meta-analysis, a significant association was found between EPCR Ser219Gly polymorphism and VTE under the dominant model (OR = 1.27, 95% CI: 1.11-1.46, p  = 0.0006), the recessive model (OR = 1.60, 95% CI: 1.26-2.04, p  = 0.0001), the GG vs. AA contrast model (OR = 1.64, 95% CI: 1.28-2.09, p  = 0.0001), and the GA vs. AA contrast model (OR = 1.24, 95% CI: 1.08-1.43, p  = 0.002)., Conclusion: The rs867186 was not associated with the first VTE risk in our case-control study; however, a tendency to VTE recurrence was observed. Based on the results of our reproducibility study, MetaGenyo is acceptable for meta-analysis in case of genetic epidemiology studies. Although the risk conferred by the rs867186 is mild in all meta-analyses, including ours, identifying patients carrying the minor allele might have an impact on personalized VTE risk assessment, risk-score calculation, and patient management., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Pituk, Miklós, Schlammadinger, Rázsó and Bereczky.)

Tumors develop by invoking a supportive environment characterized by aberrant angiogenesis and infiltration of tumor-associated macrophages (TAMs). In a transgenic model of breast cancer, we found that TAMs localized to the tumor parenchyma and were smaller than mammary tissue macrophages. TAMs had low activity of the metabolic regulator mammalian/mechanistic target of rapamycin complex 1 (mTORC1), and depletion of negative regulator of mTORC1 signaling, tuberous sclerosis complex 1 (TSC1), in TAMs inhibited tumor growth in a manner independent of adaptive lymphocytes. Whereas wild-type TAMs exhibited inflammatory and angiogenic gene expression profiles, TSC1-deficient TAMs had a pro-resolving phenotype. TSC1-deficient TAMs relocated to a perivascular niche, depleted protein C receptor (PROCR)-expressing endovascular endothelial progenitor cells, and rectified the hyperpermeable blood vasculature, causing tumor tissue hypoxia and cancer cell death. TSC1-deficient TAMs were metabolically active and effectively eliminated PROCR-expressing endothelial cells in cell competition experiments. Thus, TAMs exhibit a TSC1-dependent mTORC1-low state, and increasing mTORC1 signaling promotes a pro-resolving state that suppresses tumor growth, defining an innate immune tumor suppression pathway that may be exploited for cancer immunotherapy., Competing Interests: Declaration of interests MSKCC has filed a patent application (number 63/502054) with the US Patent and Trademark Office directed toward targeting the mTORC1 signaling pathway in macrophages for cancer immunotherapy. M.O.L. is a scientific advisory board member of and holds equity or stock options in Amberstone Biosciences and META Pharmaceuticals., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Histone H2A monoubiquitination is associated with transcriptional repression and needs to be removed by deubiquitinases to facilitate gene transcription in eukaryotes. However, the deubiquitinase responsible for genome-wide H2A deubiquitination in plants has yet to be identified. In this study, we found that the previously identified PWWP-EPCR-ARID-TRB (PEAT) complex components interact with both the ubiquitin-specific protease UBP5 and the redundant histone acetyltransferases HAM1 and HAM2 (HAM1/2) to form a larger version of PEAT complex in Arabidopsis thaliana. UBP5 functions as an H2A deubiquitinase in a nucleosome substrate-dependent manner in vitro and mediates H2A deubiquitination at the whole-genome level in vivo. HAM1/2 are shared subunits of the PEAT complex and the conserved NuA4 histone acetyltransferase complex, and are responsible for histone H4K5 acetylation. Within the PEAT complex, the PWWP components (PWWP1, PWWP2, and PWWP3) directly interact with UBP5 and are necessary for UBP5-mediated H2A deubiquitination, while the EPCR components (EPCR1 and EPCR2) directly interact with HAM1/2 and are required for HAM1/2-mediated H4K5 acetylation. Collectively, our study not only identifies dual roles of the PEAT complex in H2A deubiquitination and H4K5 acetylation but also illustrates how these processes collaborate at the whole-genome level to regulate the transcription and development in plants., (Copyright © 2023 The Author. Published by Elsevier Inc. All rights reserved.)

Introduction: Urinary incontinence (UI) is associated with increasing age and is more frequently experienced by women. Despite 40% prevalence in the community, little is known about the prevalence/incidence of UI in older women during hospital admission. UI during hospital admissions, within this group, has also been under-researched in terms of its relationship to specific clinical conditions and mortality rates. Given that UI has serious implications for both patient care and women's general health and well-being on discharge, this protocol describes a planned research project which aims to determine mortality, morbidity, prevalence and incidence of UI in older women (≥55 years) during hospital admission to inform nursing practice. Additionally, it aims to explore the experience of nurses who deliver women's care., Methods and Analysis: This is an explanatory mixed-methods study consisting of two phases: (1) retrospecitive analysis of electronic patient care records (EPCR) to determine prevalence/incidence of UI, clinical conditions most likely associated with UI and any associations between UI and death, (2) nurse interviews to explore views, knowledge and perceptions of performing the nursing assessment and providing care for older women (≥55 years) with UI during admission. EPCR will be gained from a National Health Service (NHS) teaching hospital. Nurse interviews will be conducted with nurses from an alternative but similar-sized NHS hospital., Ethics and Dissemination: Ethical approval is provided by the University of Salford Ethics Committee and regulatory approval by the NHS Health Research Authority (Integrated Research Application System project ID: 303118). Local NHS trust approval to access electronic care records for the purposes of analysis of anonymised data has been provided by one of the two collaborating NHS hospitals. Findings will be disseminated through open-access geriatric or urogynaecology journals and presented to relevant stakeholders at local, national and international meetings including scientific meetings such as the UK Continence Society and International Continence Society., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Antibody responses to variant surface antigens (VSAs) produced by the malaria parasite Plasmodium falciparum may contribute to age-related natural immunity to severe malaria. One VSA family, P. falciparum erythrocyte membrane protein-1 (PfEMP1), includes a subset of proteins that binds endothelial protein C receptor (EPCR) in human hosts and potentially disrupts the regulation of inflammatory responses, which may lead to the development of severe malaria. We probed peptide microarrays containing segments spanning five PfEMP1 EPCR-binding domain variants with sera from 10 Malian adults and 10 children to determine the differences between adult and pediatric immune responses. We defined serorecognized peptides and amino acid residues as those that elicited a significantly higher antibody response than malaria-naïve controls. We aimed to identify regions consistently serorecognized among adults but not among children across PfEMP1 variants, potentially indicating regions that drive the development of immunity to severe malaria. Adult sera consistently demonstrated broader and more intense serologic responses to constitutive PfEMP1 peptides than pediatric sera, including peptides in EPCR-binding domains. Both adults and children serorecognized a significantly higher proportion of EPCR-binding peptides than peptides that do not directly participate in receptor binding, indicating a preferential development of serologic responses at functional residues. Over the course of a single malaria transmission season, pediatric serological responses increased between the start and the peak of the season, but waned as the transmission season ended. IMPORTANCE Severe malaria and death related to malaria disproportionately affect sub-Saharan children under 5 years of age, commonly manifesting as cerebral malaria and/or severe malarial anemia. In contrast, adults in malaria-endemic regions tend to experience asymptomatic or mild disease. Our findings indicate that natural immunity to malaria targets specific regions within the EPCR-binding domain, particularly peptides containing EPCR-binding residues. Epitopes containing these residues may be promising targets for vaccines or therapeutics directed against severe malaria. Our approach provides insight into the development of natural immunity to a binding target linked to severe malaria by characterizing an "adult-like" response as recognizing a proportion of epitopes within the PfEMP1 protein, particularly regions that mediate EPCR binding. This "adult-like" response likely requires multiple years of malaria exposure, as increases in pediatric serologic response over a single malaria transmission season do not appear significant., Competing Interests: The authors declare no conflict of interest.

The biologic basis of genetic ancestry-dependent variability in disease incidence and outcome is just beginning to be explored. We recently reported enrichment of a population of ZEB1-expressing cells located adjacent to ductal epithelial cells in normal breasts of women of African ancestry compared to those of European ancestry. In this study, we demonstrate that these cells have properties of fibroadipogenic/mesenchymal stromal cells that express PROCR and PDGFRα and transdifferentiate into adipogenic and osteogenic lineages. PROCR + /ZEB1 + /PDGFRα+ (PZP) cells are enriched in normal breast tissues of women of African compared to European ancestry. PZP: epithelial cell communication results in luminal epithelial cells acquiring basal cell characteristics and IL-6-dependent increase in STAT3 phosphorylation. Furthermore, level of phospho-STAT3 is higher in normal and cancerous breast tissues of women of African ancestry. PZP cells transformed with HRas G12V  ± SV40-T/t antigens generate metaplastic carcinoma suggesting that these cells are one of the cells-of-origin of metaplastic breast cancers., (© 2023. Springer Nature Limited.)

The endothelial cell protein C receptor (EPCR) serves a key role in activated protein C (APC)-mediated cytoprotective effects in endothelial cells, and is involved in the development of certain types of human cancer. To the best of our knowledge, the present study is the first to demonstrate that EPCR may exert effects on gastric cancer angiogenesis in vitro. To detect microvessel density (MVD), the microvascular endothelial cells were stained for cluster of differentiation (CD)31 and CD34 in 61 cases of surgical resection of gastric carcinoma tissues, and the association between the expression of EPCR protein and MVD was analyzed. In addition, to analyze the effect of EPCR expressed by gastric cancer cells on the proliferation, migration and angiogenic abilities of endothelial cells, human umbilical vein endothelial cells (HUVECs) were cultured with tumor-conditioned medium derived from EPCR knockdown or protease-activated receptor 1 (PAR1)-blocked MGC803 gastric cancer cells. A CCK-8 assay was used to assess the proliferation ability of the HUVECs. A Transwell assay was performed to assess the migration ability of the HUVECs and a Matrigel-based tube formation assay was used to assess the angiogenic activity of the HUVECs. The results demonstrated that the expression of EPCR was correlated with the MVD of gastric cancer tissues. When cultured with tumor-conditioned medium derived from EPCR knockdown or PAR1-blocked MGC803 cells, the proliferation, migration and tubules formation abilities of HUVECs were markedly inhibited markedly. The expression of phosphorylated (p)-extracellular signal regulated kinase 1/2, p-protein kinase B (AKT; s473) and p-AKT (T308) in the HUVECs was decreased. In addition, EPCR knockdown inhibited PAR1 activation in the MGC803 cells. These results indicated that the expression of EPCR in gastric cancer cell line MGC803 contributes to tumor angiogenesis in vitro by activating ERK1/2 and AKT, and that this effect of EPCR is dependent on PAR1 activation. [ABSTRACT FROM AUTHOR]

Objective To explore the effects and mechanism of endothelial protein C receptor (EPCR) on the proliferation and migration of human breast cancer cells MDA-MB-231. Methods MDA-MB-231 cells were divided into the EPCR transfection group, unrelated sequence transfection group, and control group which were transfected with EPCR-siR-NA, siRNA-NC, and empty plasmid, respectively. Cell-ELISA was used to detect the activation of PAR-1 in the membranes of MDA-MB-231. CCK-8 assay was performed to observe the proliferation of cells in each group. Transwell chamber was employed to observe the cells migration. After treatment of anti-PAR-1 antibody, the proliferation and migration abilities of MDA-MB-231 cells were detected by CCK-8 and Transwell assay. Western blotting was used to determine the protein expression levels of Erkl/2 and AKT. Results Compared with the control group and unrelated sequence transfection group, the binding rate of un-cleaved activated PAR-1 antibody increased, and the proliferation and migration abilities significantly decreased in the transfection group ( all P < 0. 05 ). Compared with the control group, after being treated with anti-PAR-1 antibody, the proliferation and migration of MDA-MB-231 cells significantly decreased (both P< 0. 05 ). After knockdown of EPCR or the PAR-1 antibody treatment, the expression of p-Akt (S473 ) and p-Akt (T308 ) significantly decreased in the MDA-MB-231 cells (both P < 0. 05) , but the expression of p-Erkl/2 did not change significantly (P > 0.05). Conclusion EPCR contributes to the proliferation and migration of MDA-MB-231 cells by activating AKT, and this effect of EPCR may be dependent on PAR-1. [ABSTRACT FROM AUTHOR]

Introduction Bone remodeling relies on a delicate balance between formation and resorption of bone tissues, processes in which bone-forming osteoblasts and bone-resorbing osteoclasts play central roles. Recently, we reported that anticoagulant activated protein C (APC) promotes osteoblast proliferation, but the role of the blood coagulation system in bone remodeling remains unclear. In this study, to further elucidate the relationship between bone remodeling and blood coagulation, we investigated the effect of APC on osteoclast differentiation. Materials and methods Normal human osteoclast precursor cells were cultured in their growth medium including soluble RANKL, M-CSF, and FBS, and on days 4 and 7, the culture medium was replaced with the same medium containing various concentrations of APC, protein C (PC), sphingosine 1-phosphate (S1P) receptor agonist, FTY720, or APC + various substances without FBS. On day 8, TRAP-positive multinucleated cells (≥ 3 nuclei) were counted manually using a light microscope. The effects of APC on NF-κB and NFATc1 activation were evaluated using specific ELISA. Results APC suppressed RANKL-induced osteoclast differentiation, and this APC-induced suppression of osteoclast differentiation was inhibited by zymogen protein C and aprotinin, a serine protease inhibitor. Immunohistochemistry and RT-PCR analyses suggested that endothelial protein C receptor (EPCR) and protease-activated receptor-1 (PAR-1) were expressed in osteoclast precursor cells and osteoclasts. Both anti-PAR-1 antibody and anti-EPCR antibody (RCR-252), which blocks APC binding to EPCR, inhibited the APC-induced suppression of osteoclast differentiation. FTY720 had no effect on osteoclast differentiation. However, FTY 720 and S1P receptor antagonist, VP 23019, inhibited the APC-induced suppression of osteoclast differentiation. On the other hand, recombinant soluble human ApoER2 and anti-human ApoER2 inhibited the APC-induced suppression of osteoclast differentiation. Further, APC had no effect on NF-κB and NFATc1 activation. Conclusions APC suppresses human osteoclast differentiation mainly by inhibiting the formation of multinucleated cells via EPCR, PAR-1, S1P receptor, and ApoER2 in a manner that depends on APC protease activity. [ABSTRACT FROM AUTHOR]

The present study investigated the influence of cigarette smoke extract (CSE) and nicotine on the expression of thrombomodulin (TM) and endothelial protein C receptor (EPCR) in human umbilical vein endothelial cells (HUVECs). Smoking is associated with intravascular thrombosis. As a vital anticoagulation cofactor, TM is located on the endothelial cell surface and regulates intravascular coagulation by binding to thrombin, hence activating protein C. Activated protein C is a natural anticoagulant that interacts with EPCR to enhance the function of anticoagulation system. The effects of CSE (0.5-5%) and nicotine (10-3-10-9 mol/l) on the expression of TM and EPCR in HUVECs were observed. Reverse transcription-quantitative polymerase chain reaction and flow cytometric analysis techniques were used for detecting TM and EPCR mRNA and protein expression levels, respectively. After 6-h exposure, TM protein and mRNA expression levels decreased in a dose-dependent manner. Stimulation with 5% CSE for 0, 6, 10, 12 and 24 h led to a decrease in the levels of TM mRNA and protein over time, which reached a peak at 12 h. The levels were significantly reduced compared with the control group (P<0.001). However, CSE had no effect on EPCR. Furthermore, nicotine had no influence on TM and EPCR. In conclusion, the present study supports a novel molecular mechanism of cigarette smoking-associated thrombosis by the decreased expression of TM. Further studies are required to identify specific components in CSE responsible for decreasing TM expression and its associated consequences. [ABSTRACT FROM AUTHOR]

Activation of protein C is greatly enhanced by the presence of thrombomodulin (TM) and endothelial protein C receptor (EPCR) on the endothelial surface. Impairment of the anticoagulant protein C system occurs during endotoxemia and contributes to sepsis-associated hypercoagulability. Previous studies have demonstrated that unfractionated heparin (UFH) can attenuate coagulation in endotoxemic mice. However, whether UFH has an effect on the protein C system remains to be elucidated. The current study evaluated the therapeutic effect of UFH on the protein C system in a mouse model of lipopolysaccharide (LPS)-induced sepsis, and further investigated the effect of UFH on the expression of TM and EPCR in vitro using human umbilical vein endothelial cells (HUVECs). The in vivo data indicated that UFH preconditioning attenuated the decline in circulating activated protein C following LPS administration, and also reduced LPS-induced shedding of TM and EPCR. In HUVECs, LPS stimulation led to the downregulation of TM and EPCR expression, and UFH dose-dependently restored the mRNA and protein levels of TM and EPCR. In addition, UFH inhibited the LPS-induced activation of mitogen-activated protein kinase 14, proto-oncogene tyrosine-protein kinase Src and nuclear factor κB signaling in HUVECs. In summary, these results suggest that UFH has a protective effect on the protein C system during sepsis. Thus, UFH may be a candidate therapeutic agent for the treatment of patients with sepsis. [ABSTRACT FROM AUTHOR]