Endothelial Protein C Receptor and Its Impact on Rheumatic Disease.

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.

Lipid-binding antiphospholipid antibodies: significance for pathophysiology and diagnosis of the antiphospholipid syndrome.

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.

EPCR deficiency ameliorates inflammatory arthritis in mice by suppressing the activation and migration of T cells and dendritic cells.

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.

The association between EPCR gene p.Ser219Gly polymorphism and venous thromboembolism risk: a case-control study, meta-analysis, and a reproducibility study.

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.

Natural immunity to malaria preferentially targets the endothelial protein C receptor-binding regions of PfEMP1s.

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.

Common Autoantibody among Takayasu Arteritis and Ulcerative Colitis: A Possible Pathophysiology That Includes Gut-Vessel Connection in Vascular Inflammation.

Takayasu arteritis (TAK) is a type of large-vessel vasculitis that predominantly affects young females. The precise pathomechanism of TAK is still under investigation. In TAK, the vasa vasorum is considered to be the initial inflammatory site. Disruption of the vasa vasorum induces the entry of inflammatory cells into the vascular wall of large vessels between the media and adventitia, and infiltrated cells damage the vascular components, eventually leading to stenosis or dilatation of the affected arteries. In addition, T cells are considered key players in TAK, and myeloid cells function as effector cells. Although the roles of B cells in TAK are poorly understood, recent evidence supports their contribution to the pathogenicity of TAK. Particularly, two autoantibodies have been identified in TAK through investigation of anti-endothelial cell antibodies, and they could be involved in the maintenance of vascular inflammation. Furthermore, one of the autoantibodies, anti-endothelial protein C receptor, was shown to be present in ulcerative colitis (UC), which is genetically and clinically associated with TAK. Similar autoantibodies in inflammatory diseases with different target organs indicate a common underlying pathophysiology of these diseases, which could be characterized by the aberrant activation of B cells. This review discusses recent understanding of the pathomechanisms of TAK and UC, with a focus on the involvement of B cells and autoantibodies. The close association of UC with TAK further suggests a common etiology, and the importance of the intestinal microbiota, including dysbiosis, is also becoming known in TAK. Investigation of such common factors among TAK and UC would improve understanding of the interplay between gut and vascular inflammation, which is a new concept for developing vascular inflammation through the gut-vessel connection.

The APC-EPCR-PAR1 axis in sickle cell disease.

Sickle Cell Disease (SCD) is a group of inherited hemoglobinopathies. Sickle cell anemia (SCA) is caused by a homozygous mutation in the ß-globin generating sickle hemoglobin (HbS). Deoxygenation leads to pathologic polymerization of HbS and sickling of erythrocytes. The two predominant pathologies of SCD are hemolytic anemia and vaso-occlusive episodes (VOE), along with sequelae of complications including acute chest syndrome, hepatopathy, nephropathy, pulmonary hypertension, venous thromboembolism, and stroke. SCD is associated with endothelial activation due to the release of danger-associated molecular patterns (DAMPs) such as heme, recurrent ischemia-reperfusion injury, and chronic thrombin generation and inflammation. Endothelial cell activation is mediated, in part, by thrombin-dependent activation of protease-activated receptor 1 (PAR1), a G protein coupled receptor that plays a role in platelet activation, endothelial permeability, inflammation, and cytotoxicity. PAR1 can also be activated by activated protein C (APC), which promotes endothelial barrier protection and cytoprotective signaling. Notably, the APC system is dysregulated in SCD. This mini-review will discuss activation of PAR1 by APC and thrombin, the APC-EPCR-PAR1 axis, and their potential roles in SCD.

Microvascular Environment Influences Brain Microvascular Heterogeneity: Relative Roles of Astrocytes and Oligodendrocytes for the EPCR Expression in the Brain Endothelium.

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.

Differential expression of endothelial protein C receptor (EPCR) in hematopoietic stem and multipotent progenitor cells in young and old mice.

Endothelial protein C receptor (EPCR) has emerged as one of the most conserved and reliable surface markers for the prospective identification and isolation of hematopoietic stem cells (HSCs). Prior studies have consistently demonstrated that EPCR expression enriches HSCs capable of long-term multilineage repopulation in both mouse and human across different hematopoietic tissues, including bone marrow (BM), fetal liver and ex vivo HSC expansion cultures. However, little is known about the expression profiles of EPCR in multipotent progenitor (MPP) populations located immediately downstream of HSCs in the hematopoietic hierarchy and which play a major role in sustaining lifelong blood cell production. Here, we incorporate EPCR antibody detection into a multi-parameter flow cytometric panel, which allows accurate identification of HSCs and five MPP subsets (MPP1-5) in mouse BM. Our data reveal that all MPP populations contain EPCR-expressing cells. Multipotent MPP1 and MPP5 contain higher proportion of EPCR+ cells compared to the more lineage-biased MPP2-4. Notably, high expression of EPCR enriches phenotypic HSC and MPP5, but not MPP1. Comparison of EPCR expression profiles between young and old BM reveals ageing mediated expansion of EPCR-expressing cells only in HSCs, but not in any of the MPP populations. Collectively, our study provides a comprehensive characterization of the surface expression pattern of EPCR in mouse HSC and MPP1-5 cells during normal and aged hematopoiesis.

Structural and genomic analysis of single nucleotide polymorphisms in human host factor endothelial protein C receptor (EPCR) reveals complex interplay with malaria parasites.

Plasmodium parasites responsible for malaria follow a complex life cycle of which half takes place inside the human host. Parasites present diverse antigens at different stages of their life cycle and interact with many surface molecules to attach to and enter host cells. The CIDRα1 domain of Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) in infected erythrocytes adheres to one such vascular receptor endothelial protein C receptor (EPCR). EPCR is implicated in the pathogenesis of severe malaria as preferential binding of CIDRα1 to endothelium results in widespread sequestration of infected erythrocytes leading to endothelium inflammation and severe disease. A single EPCR variant S219G is clinically reported to provide protection from severe malaria. In this work, we have collated all single nucleotide polymorphisms (SNPs) in EPCR from dbSNP. We structurally mapped the SNPs on the three-dimensional complex of EPCR and PfEMP1 CIDRα1. Analysis shows that most EPCR mutations lie on the receptor surface and are non-conservative. Of the 11 mutations in the CIDRα1-interaction region of EPCR, S88P, L96V/I, and R98L/H/P/C are seen with comparably higher occurrences in diverse populations. Our structural analysis details a framework of the interactions between the parasite ligand and host factor EPCR. These structural glimpses provide a blueprint for designing both field-based variant sequencing studies and vaccine development.

Plasmodium falciparum Parasite Lines Expressing DC8 and Group A PfEMP1 Bind to Brain, Intestinal, and Kidney Endothelial Cells.

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.

Usefulness of soluble endothelial protein C receptor combined with left ventricular global longitudinal strain for predicting slow coronary flow: A case-control study.

BACKGROUND: Slow coronary flow (SCF) is an angiographic entity characterized by delayed coronary opacification without an evident obstructive lesion in the epicardial coronary artery. However, patients with SCF have decreased left ventricular (LV) global longitudinal strain (GLS). SCF is associated with inflammation, and soluble endothelial protein C receptor (sEPCR) is a potential biomarker of inflammation. Therefore, under evaluation herein, was the relationship between SCF and sEPCR and the predictive value of sEPCR and LV GLS for SCF was investigated. METHODS: Twenty-eight patients with SCF and 34 controls were enrolled. SCF was diagnosed by the thrombolysis in myocardial infarction frame count (TFC). The plasma level of sEPCR was quantified using enzyme-linked immunosorbent assay. LV GLS was measured by two-dimensional speckle-tracking echocardiography. RESULTS: Plasma sEPCR was significantly higher in patients with SCF than in controls and was positively correlated with the mean TFC (r = 0.67, p < 0.001) and number of involved vessels (r = 0.61, p < 0.001). LV GLS was decreased in patients with SCF compared to that in controls. sEPCR level (OR = 3.14, 95% CI 1.55-6.36, p = 0.001) and LV GLS (OR = 1.44, 95% CI 1.02-2.04, p = 0.04) were independent predictors of SCF. sEPCR predicted SCF (area under curve [AUC]: 0.83); however, sEPCR > 9.63 ng/mL combined with LV GLS > -14.36% demonstrated better predictive power (AUC: 0.89; sensitivity: 75%; specificity: 91%). CONCLUSIONS: Patients with SCF have increased plasma sEPCR and decreased LV GLS. sEPCR may be a useful potential biomarker for SCF, and sEPCR combined with LV GLS can better predict SCF.

Co-culture of mesenchymal stem cell spheres with hematopoietic stem cells under hypoxia: a cost-effective method to maintain self-renewal and homing marker expression.

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.

Pathogenic lipid-binding antiphospholipid antibodies are associated with severity of COVID-19.

BACKGROUND: Coronavirus disease 19 (COVID-19)-associated coagulopathy is a hallmark of disease severity and poor prognosis. The key manifestations of this prothrombotic syndrome-microvascular thrombosis, stroke, and venous and pulmonary clots-are also observed in severe and catastrophic antiphospholipid syndrome. Antiphospholipid antibodies (aPL) are detectable in COVID-19 patients, but their association with the clinical course of COVID-19 remains unproven. OBJECTIVES: To analyze the presence and relevance of lipid-binding aPL in hospitalized COVID-19 patients. METHODS: Two cohorts of 53 and 121 patients from a single center hospitalized for PCR-proven severe acute respiratory syndrome-coronavirus 2 infection were analyzed for the presence of aPL and clinical severity of COVID-19. RESULTS: We here demonstrate that lipid-binding aPL are common in COVID-19. COVID-19 patients with lipid-binding aPL have higher median concentrations of C-reactive protein and D-dimer, and are more likely to have a critical clinical course and fatal outcome. Lipid-binding aPL isolated from COVID-19 patients target the recently described cell surface complex of lysobisphosphatidic acid (LBPA) with the protein C receptor (EPCR) to induce prothrombotic and inflammatory responses in monocytes and endothelial cells. We show that B1a cells producing lipid-reactive aPL of the IgG isotype circulate in the blood of COVID-19 patients. In vivo, COVID-19 aPL accelerate thrombus formation in an experimental mouse model dependent on the recently delineated signaling pathway involving EPCR-LBPA. CONCLUSIONS: COVID-19 patients rapidly expand B1a cells secreting pathogenic lipid-binding aPL with broad thrombotic and inflammatory effects. The association with markers of inflammation and coagulation, clinical severity, and mortality suggests a causal role of aPL in COVID-19-associated coagulopathy.

Immunohistochemical study of dissociation and association of adherens junctions in splenic sinus endothelial cells.

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), ß-catenin phosphorylated at Try142 (ß-catenin Y142) and ß-catenin phosphorylated at Try654 (ß-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, ß-catenin Y142 and ß-catenin Y 654 colocalized with α-catenin and ß-catenin, respectively and in addition, ß-catenin Y142 and ß-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 ß-catenin Y142 and ß-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.

Endothelial Cell Protein C Receptor Deficiency Attenuates Streptococcus pneumoniae-induced Pleural Fibrosis.

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.

Aging Reprograms the Hematopoietic-Vascular Niche to Impede Regeneration and Promote Fibrosis.

Regenerative capacity is frequently impaired in aged organs. Stress to aged organs often causes scar formation (fibrosis) at the expense of regeneration. It remains to be defined how hematopoietic and vascular cells contribute to aging-induced regeneration to fibrotic transition. Here, we find that aging aberrantly reprograms the crosstalk between hematopoietic and vascular cells to impede the regenerative capacity and enhance fibrosis. In aged lung, liver, and kidney, induction of Neuropilin-1/hypoxia-inducible-factor 2α (HIF2α) suppresses anti-thrombotic and anti-inflammatory endothelial protein C receptor (EPCR) pathway, leading to formation of pro-fibrotic platelet-macrophage rosette. Activated platelets via supplying interleukin 1α synergize with endothelial-produced angiocrine chemokine to recruit fibrogenic TIMP1high macrophages. In mouse models, genetic targeting of endothelial Neuropilin-1-HIF2α, platelet interleukin 1α, or macrophage TIMP1 normalized the pro-fibrotic hematopoietic-vascular niche and restored the regenerative capacity of old organs. Targeting of aberrant endothelial node molecules might help propel "regeneration without scarring" in the repair of multiple organs.

Platelet-derived calpain cleaves the endothelial protease-activated receptor 1 to induce vascular inflammation in diabetes.

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.

Genetic risk factors for venous thromboembolism among infertile men with Klinefelter syndrome.

BACKGROUND: Klinefelter syndrome (KS) is one of the commonest sex chromosome disorders. Affected males become infertile and highly susceptible to several health problems, including vascular thromboembolism (VTE). The risk of VTE may be exacerbated by an underlying genetically inherited thrombophilia. In this study, we aimed to investigate the genotype and allele frequencies of common gene polymorphisms related to hereditary thrombophilia in infertile males with KS compared to normal, fertile men. METHODS: Eighty-five infertile males with KS and 75 healthy control males were included in this case-control study. Genetic testing was done using an extended thrombophilia gene panel by Multiplex PCR reverse hybridization method. RESULTS: There was an increased frequency of mutant alleles and heterozygous genotypes of FV Leiden, FV H 1299R, Pro G20210A, MTHFR C677T and PAI-1 4G/5G thrombophilic gene polymorphisms in KS patients compared to the control group. It was shown that 10.7% of KS patients had the A3 haplotype of the EPCR gene in comparison to 5.3% of control patients. The A3/A3 genotype was found only in KS patients (7.1%). Carriers of more than one mutant allele in KS patients exceeded the control (p < 0.001). CONCLUSION: A high prevalence of thrombophilic gene polymorphisms and the coexistence of different mutant alleles were evident in infertile KS males. These data highlight the importance of conducting further studies to understand the role of hereditary thrombophilia in predicting venous thrombosis in patients with Klinefelter syndrome.