Coagulation proteased-induced extracellular vesicles: Their potential effects on coagulation and inflammation.

Coagulation proteases, in addition to playing an essential role in blood coagulation, often influence diverse cellular functions by inducing specific signaling pathways via the activation of protease-activated receptors (PARs). PAR activation-induced cellular effects are known to be cell-specific as PARs are expressed selectively in specific cell types. However, a growing body of evidence indicates that coagulation protease-induced PAR activation in a specific cell type could affect cellular responses in other cell types via communicating through extracellular vesicles (EVs) as coagulation proteases induced-PAR signaling could promote the release of EVs in various cell types. EVs are membrane-enclosed nanosized vesicles that facilitate intercellular communication by transferring bioactive molecules, such as proteins, lipids, mRNAs, and microRNAs, from donor cells to recipient cells. Our recent findings established that factor VIIa (FVIIa) promotes the release of EVs from vascular endothelium via endothelial cell protein C receptor-dependent activation of PAR1-mediated biased signaling. FVIIa-released EVs exhibit procoagulant activity and cytoprotective responses both in vitro and in vivo model systems. This review discusses how factor VIIa and other coagulation proteases activate PAR-mediated cell signaling and trigger the release of EVs. The review specifically discusses how FVIIa-released EVs are enriched with phosphatidylserine and anti-inflammatory miRNAs and the impact of FVIIa-released EVs on hemostasis in therapeutic settings. The review also briefly highlights the therapeutic potential of FVIIa-released EVs in treating bleeding and inflammatory disorders, such as hemophilic arthropathy and sepsis.

Synthesis of Novel Benzopril-Derived Trizole Compounds Assisted by Ultrasound: In Vitro and In Silico Analysis for Potential Anticancer Properties.

Benzepril-based novel trizole derivatives are being explored as potential anticancer agents, designed with an N-substituted 1,2,3-triazole moiety linked to benzepril's N-1 position via a methylene bridge. An ultrasound irradiated CuAAC method was used to prepare all these compounds and evaluated their anti-proliferative activities against cancer and drug-resistant cell lines. While some of these compounds demonstrated anti-proliferative activity towards leukemic cancer cell line K562, two of them displayed complete inhibitory activity.  Interestingly, the compounds 5n and 5o showed potent activity against imatinib-resistant cell lines suggesting their promise to overcome cancer drug resistance. Furthermore, molecular docking analysis revealed that compounds 5n and 5o have higher predicted sensitivity towards ACE protein when compared to benazepril and lisinopril indicating their value as potential drug lead molecules. This research introduces a distinctive approach by employing ultrasound to facilitate CuAAC reactions in medicinal chemistry.

Multiomics-assisted characterization of rice-Yellow Stem Borer interaction provides genomic and mechanistic insights into stem borer resistance in rice.

KEY MESSAGE: By deploying a multi-omics approach, we unraveled the mechanisms that might help rice to combat Yellow Stem Borer infestation, thus providing insights and scope for developing YSB resistant rice varieties. Yellow Stem Borer (YSB), Scirpophaga incertulas (Walker) (Lepidoptera: Crambidae), is a major pest of rice, that can lead to 20-60% loss in rice production. Effective management of YSB infestation is challenged by the non-availability of adequate sources of resistance and poor understanding of resistance mechanisms, thus necessitating studies for generating resources to breed YSB resistant rice and to understand rice-YSB interaction. In this study, by using bulk-segregant analysis in combination with next-generation sequencing, Quantitative Trait Loci (QTL) intervals in five rice chromosomes were mapped that could be associated with YSB resistance at the vegetative phase in a resistant rice line named SM92. Further, multiple SNP markers that showed significant association with YSB resistance in rice chromosomes 1, 5, 10, and 12 were developed. RNA-sequencing of the susceptible and resistant lines revealed several genes present in the candidate QTL intervals to be differentially regulated upon YSB infestation. Comparative transcriptome analysis revealed a putative candidate gene that was predicted to encode an alpha-amylase inhibitor. Analysis of the transcriptome and metabolite profiles further revealed a possible link between phenylpropanoid metabolism and YSB resistance. Taken together, our study provides deeper insights into rice-YSB interaction and enhances the understanding of YSB resistance mechanism. Importantly, a promising breeding line and markers for YSB resistance have been developed that can potentially aid in marker-assisted breeding of YSB resistance among elite rice cultivars.

Heme-induced loss of renovascular endothelial protein C receptor promotes chronic kidney disease in sickle mice.

ABSTRACT: Chronic kidney disease (CKD) is a major contributor to morbidity and mortality in sickle cell disease (SCD). Anemia, induced by chronic persistent hemolysis, is associated with the progressive deterioration of renal health, resulting in CKD. Moreover, patients with SCD experience acute kidney injury (AKI), a risk factor for CKD, often during vaso-occlusive crisis associated with acute intravascular hemolysis. However, the mechanisms of hemolysis-driven pathogenesis of the AKI-to-CKD transition in SCD remain elusive. Here, we investigated the role of increased renovascular rarefaction and the resulting substantial loss of the vascular endothelial protein C receptor (EPCR) in the progressive deterioration of renal function in transgenic SCD mice. Multiple hemolytic events raised circulating levels of soluble EPCR (sEPCR), indicating loss of EPCR from the cell surface. Using bone marrow transplantation and super-resolution ultrasound imaging, we demonstrated that SCD mice overexpressing EPCR were protective against heme-induced CKD development. In a cohort of patients with SCD, plasma sEPCR was significantly higher in individuals with CKD than in those without CKD. This study concludes that multiple hemolytic events may trigger CKD in SCD through the gradual loss of renovascular EPCR. Thus, the restoration of EPCR may be a therapeutic target, and plasma sEPCR can be developed as a prognostic marker for sickle CKD.

A potential mechanism for the cytoprotective effects of activated protein C-released endothelial extracellular vesicles.

ABSTRACT: Activated protein C (APC) was shown to release extracellular vesicles (EVs). APC bound to the EVs was thought to be responsible for cytoprotection. Our study demonstrates that the cytoprotective effects of APC-released EVs are independent of APC. APC-released EVs carry anti-inflammatory microRNAs in their cargo.

Unraveling Links between Chronic Inflammation and Long COVID: Workshop Report.

As COVID-19 continues, an increasing number of patients develop long COVID symptoms varying in severity that last for weeks, months, or longer. Symptoms commonly include lingering loss of smell and taste, hearing loss, extreme fatigue, and "brain fog." Still, persistent cardiovascular and respiratory problems, muscle weakness, and neurologic issues have also been documented. A major problem is the lack of clear guidelines for diagnosing long COVID. Although some studies suggest that long COVID is due to prolonged inflammation after SARS-CoV-2 infection, the underlying mechanisms remain unclear. The broad range of COVID-19's bodily effects and responses after initial viral infection are also poorly understood. This workshop brought together multidisciplinary experts to showcase and discuss the latest research on long COVID and chronic inflammation that might be associated with the persistent sequelae following COVID-19 infection.

MicroRNA-10a enrichment in factor VIIa-released endothelial extracellular vesicles: potential mechanisms.

BACKGROUND: Factor VIIa induces the release of extracellular vesicles (EVs) from endothelial cells (EEVs). Factor VIIa-released EEVs are enriched with microRNA-10a (miR10a) and elicit miR10a-dependent cytoprotective responses. OBJECTIVES: To investigate mechanisms by which FVIIa induces miR10a expression in endothelial cells and sorts miR10a into the EVs. METHODS: Activation of Elk-1 and TWIST1 expression was analyzed by immunofluorescence microscopy and immunoblot analysis. Small interfering RNA silencing approach was used to knock down the expression of specific genes in endothelial cells. EVs secreted from endothelial cells or released into circulation in mice were isolated by centrifugation and quantified by nanoparticle tracking analysis. Factor VIIa or EVs were injected into mice; mice were challenged with lipopolysaccharides to assess the cytoprotective effects of FVIIa or EVs. RESULTS: FVIIa activation of ERK1/2 triggered the activation of Elk-1, which led to the induction of TWIST1, a key transcription factor involved in miR10a expression. Factor VIIa also induced the expression of La, a small RNA-binding protein. Factor VIIa-driven acid sphingomyelinase (ASM) activation and the subsequent activation of the S1P receptor pathway were responsible for the induction of La. Silencing of ASM or La significantly reduced miR10a levels in FVIIa-released EEVs without affecting the cellular expression of miR10a. Factor VIIa-EEVs from ASM knocked-down cells failed to provide cytoprotective responses in cell and murine model systems. Administration of FVIIa protected wild-type but not ASM-/- mice against lipopolysaccharide-induced inflammation and vascular leakage. CONCLUSION: Our data suggest that enhanced cellular expression of miR10a coupled with La-dependent sorting of miR10a is responsible for enriching FVIIa-released EVs with miR10a.

Improvement of bacterial blight resistance of the high yielding, fine-grain, rice variety, Gangavati sona through marker-assisted backcross breeding.

Gangavati sona (GS) is a high-yielding, fine-grain rice variety widely grown in the Tungabhadra command area in Karnataka, India; however, it is susceptible to bacterial blight (BB). Therefore, the present study was conducted to improve the GS variety for BB resistance. Three BB-resistant genes (xa5, xa13, and Xa21) were introgressed into the genetic background of susceptible cultivar GS through marker-assisted backcrossing (MABB) by using Improved samba Mahsuri (ISM), a popular, high-yielding, bacterial blight resistant rice variety as a donor parent. Foreground selection was carried out using gene-specific markers, viz., xa5FM (xa5), xa13prom (xa13), and pTA248 (Xa21), while background selection was carried out using well-distributed 64 polymorphic microsatellite markers. The true heterozygote F1 was used as the male parent for backcrossing with GS to obtain BC1F1. The process was repeated in BC1F1 generation, and a BC2F1 plant (IGS-5-11-5) possessing all three target genes along with maximum recurrent parent genome (RPG) recovery (86.7%) was selfed to obtain BC2F2s. At BC2F2, a single triple gene homozygote plant (IGS-5-11-5-33) with 92.6% RPG recovery was identified and advanced to BC2F5 by a pedigree method. At BC2F5, the seven best entries were selected, possessing all three resistance genes with high resistance levels against bacterial blight, yield level, and grain quality features equivalent to better than GS. The improved versions of GS will immensely benefit the farmers whose fields are endemic to BB.

Factor VIIa releases phosphatidylserine-enriched extracellular vesicles from endothelial cells by activating acid sphingomyelinase.

BACKGROUND: Our recent studies showed that activated factor (F) VII (FVIIa) releases extracellular vesicles (EVs) from the endothelium. FVIIa-released EVs were found to be enriched with phosphatidylserine (PS) and contribute to the hemostatic effect of FVIIa in thrombocytopenia and hemophilia. OBJECTIVE: To investigate mechanisms by which FVIIa induces EV biogenesis and enriches EVs with PS. METHODS: FVIIa activation of acid sphingomyelinase (aSMase) was evaluated by its translocation to the cell surface. The role of aSMase in the biogenesis of FVIIa-induced EVs and their enrichment with PS was investigated using specific siRNAs and inhibitors of aSMase and its downstream metabolites. Wild-type and aSMase-/- mice were injected with a control vehicle or FVIIa. EVs released into circulation were quantified by nanoparticle tracking analysis. EVs hemostatic potential was assessed in a murine thrombocytopenia model. RESULTS: FVIIa activation of aSMase is responsible for both the externalization of PS and the release of EVs in endothelial cells. FVIIa-induced aSMase activation led to ceramide generation and de novo expression of transmembrane protein 16F. Inhibitors of ceramidases, sphingosine kinase, or sphingosine-1-phosphate receptor modulator blocked FVIIa-induced expression of transmembrane protein 16F and PS externalization without interfering with FVIIa release of EVs. In vivo, FVIIa release of EVs was markedly impaired in aSMase-/- mice compared with wild-type mice. Administration of a low dose of FVIIa, sufficient to induce EVs release, corrected bleeding associated with thrombocytopenia in wild-type mice but not in aSMase-/- mice. CONCLUSION: Our study identifies a novel mechanism by which FVIIa induces PS externalization and releases PS-enriched EVs.

[Spatial-temporal characteristics and influencing factors of pulmonary tuberculosis cases in Shanghai from 2013 to 2020].

Objective: To use the spatiotemporal distribution model and INLA algorithm to study the spatiotemporal characteristics and influencing factors of tuberculosis in Shanghai and to provide a theoretical basis for formulating regional tuberculosis epidemic prevention and control measures. Methods: Based on the data of registered pulmonary tuberculosis cases in Shanghai during 2013-2020 derived from the tuberculosis management information system of China Disease Control and Prevention Information System, the hierarchical Bayesian model was adopted to fit the tuberculosis case data, identify the spatiotemporal variation characteristics of tuberculosis, and explore the potential socioeconomic characteristics and other factors related to health services and spatiotemporal characteristics. Results: From 2013 to 2020, 29 281 registered tuberculosis cases were reported in Shanghai, with an average annual incidence of 25.224/100 000. From 2013 to 2020, the incidence trend increased first and then decreased, the highest incidence was reported in 2014 (27.991/100 000). The incidence of tuberculosis in Shanghai is characterized by spatial clustering. Through the spatial characteristics and risk analysis of the reported incidence of tuberculosis, it is found that the high-risk area of tuberculosis in Shanghai is the suburban communities, whereas downtown communities are the low-risk areas. The incidence risk of pulmonary tuberculosis is associated with the gross domestic product per capita (RR=0.48), the number of beds per 10 000 persons (RR=0.56), the normalized vegetation index (RR=0.50), and the night light index (RR=0.80). Conclusions: With the steady progress of tuberculosis prevention and control in the central urban area of Shanghai, special attention should be paid to the prevention and control in the suburbs further to improve the social and economic level in the suburbs and increase the coverage rate of urban green space, to reduce the incidence of tuberculosis and reduce the disease burden of tuberculosis in Shanghai.

Toxoplasmosis lymphadenopathy-A series of 8 cases diagnosed in a 2-year timeframe: Is it a needle in a haystack?

Toxoplasmosis is a zoonosis caused by Toxoplasma gondii, an obligate intracellular parasite. Clinical presentation of infection depends on the age and immune status of the patient. In immunocompetent patient, it may present as a non-specific lymphadenopathy and self- limiting illness. In contrast, in immunocompromised patients it can be a life-threatening infection. We present a series of 8 cases of toxoplasma lymphadenitis diagnosed in our institute in last two years. Lymphadenopathy raises a suspicion of malignancy; however, diagnosis of reactive lymphadenitis often marks the end of diagnostic evaluation. Careful morphological evaluation, the classic triad, often can direct at a conclusive diagnosis. We are presenting a case series to draw attention to this entity as majority of the published literature is in the form of case report and serological surveys and very occasional study showing both the histopathology and serology in tandem.

Mg-Containing Zn3O3 Structures for Detection of CO2: A DFT Study on CHEM Effects of SERS and Electronic Properties.

Surface-enhanced Raman spectra (SERS) and electronic-structure-based properties are important tools for investigation of the molecular sensing ability of nanoparticles. The present computational study is intended to explore the sensing ability of Zn3O3 and Mg-containing Zn3O3 structures for CO2 molecules by CHEM effects of the SERS technique. Geometries of CO2-adsorbed Zn3O3, Zn2MgO3 (Mg as a substitutional impurity), and Zn3O3Mg (Mg as an interstitial impurity) structures are modeled using the B3LYP/6-31G(d,p) level of density functional theory. The Mg site of the Zn2MgO3 and Zn3O3Mg structures is preferential for the adsorption of CO2. The observed energy trends are supported by geometrical analysis, molecular orbital interactions, redshifts in CO2 vibrational modes, and topological properties. Raman activity enhancement of the CO2 symmetric vibrational mode is significant when the molecule is adsorbed at the Mg site of Zn3O3Mg. The observed Raman activity enhancement is supported by SERS spectra obtained from anharmonic calculations carried out on B3LYP/6-31G(d,p) geometries and substantiated by a larger change in the polarizability with energy corresponding to the symmetric vibrational mode of CO2. The TDDFT calculations, frequency-dependent polarizabilities, and charge transfer interactions show that Zn3O3Mg is a good substrate for sensing of CO2, with visible wavelengths, by resonance Raman effect. The trends with adsorption energy, Raman activity, and excited state properties are also substantiated by B3LYP/6-311+G(d,p) calculations.

Risk prediction of second primary malignancies in primary colorectal neuroendocrine neoplasms patients: a population-based study.

PURPOSE: In this study, we aimed to identify risk factors for developing second primary malignancies (SPMs) in colorectal neuroendocrine neoplasms (NENs) patients and develop a competing-risk nomogram to predict SPMs' probabilities quantitatively. METHODS: Patients with colorectal NENs were retrospectively collected from the Surveillance, Epidemiology, and End Results (SEER) database during 2000-2013. Potential risk factors for SPMs' occurrence in colorectal NENs' patients were identified by the Fine and Gray's proportional sub-distribution hazards model. Then, a competing-risk nomogram was constructed to quantify SPMs' probabilities. The discriminative abilities and calibrations of this competing-risk nomogram were assessed by the area under the receiver-operating characteristic (ROC) curves (AUC) and calibration curves. RESULTS: We identified 11,017 colorectal NENs' patients, and randomly divided them into training (n = 7711 patients) and validation (n = 3306 patients) cohorts. In the whole cohort, 12.4% patients (n = 1369) had developed SPMs during the maximum follow-up of approximately 19 years (median 8.9 years). Sex, age, race, primary tumor location, and chemotherapy were identified as risk factors for SPMs' occurrence in colorectal NENs' patients. Such factors were selected to develop a competing-risk nomogram and showed excellent predictive ability for SPMs' occurrence (the 3-, 5-, and 10-year AUC values were 0.631, 0.632, and 0.629 in the training cohort and 0.665, 0.639, 0.624 in the validation cohort, respectively). CONCLUSIONS: This research identified risk factors for SPMs' occurrence in colorectal NENs' patients. Competing-risk nomogram was constructed and proved to have good performance.

Alterations to Sphingomyelin Metabolism Affect Hemostasis and Thrombosis.

BACKGROUND: Our recent studies suggest that sphingomyelin levels in the plasma membrane influence TF (tissue factor) procoagulant activity. The current study was performed to investigate how alterations to sphingomyelin metabolic pathway would affect TF procoagulant activity and thereby affect hemostatic and thrombotic processes. METHODS: Macrophages and endothelial cells were transfected with specific siRNAs or infected with adenoviral vectors to alter sphingomyelin levels in the membrane. TF activity was measured in factor X activation assay. Saphenous vein incision-induced bleeding and the inferior vena cava ligation-induced flow restriction mouse models were used to evaluate hemostasis and thrombosis, respectively. RESULTS: Overexpression of SMS (sphingomyelin synthase) 1 or SMS2 in human monocyte-derived macrophages suppresses ATP-stimulated TF procoagulant activity, whereas silencing SMS1 or SMS2 increases the basal cell surface TF activity to the same level as of ATP-decrypted TF activity. Consistent with the concept that sphingomyelin metabolism influences TF procoagulant activity, silencing of acid sphingomyelinase or neutral sphingomyelinase 2 or 3 attenuates ATP-induced enhanced TF procoagulant activity in macrophages and endothelial cells. Niemann-Pick disease fibroblasts with a higher concentration of sphingomyelin exhibited lower TF activity compared with wild-type fibroblasts. In vivo studies revealed that LPS+ATP-induced TF activity and thrombin generation were attenuated in ASMase-/- mice, while their levels were increased in SMS2-/- mice. Further studies revealed that acid sphingomyelinase deficiency leads to impaired hemostasis, whereas SMS2 deficiency increases thrombotic risk. CONCLUSIONS: Overall, our data indicate that alterations in sphingomyelin metabolism would influence TF procoagulant activity and affect hemostatic and thrombotic processes.

Mucoepidermoid Carcinoma of the Salivary Gland: Long Term Outcomes from a Tertiary Cancer Center in India.

To analyze the patterns of failure and factors affecting recurrence and overall survival in mucoepidermoid carcinoma of the salivary gland. The hospital records were retrospectively analyzed from October 2010 to January 2016. Patients diagnosed as mucoepidermoid carcinoma of the salivary gland were eligible for analysis. All patients received surgery as the primary treatment modality with or without post operative radiotherapy. Statistical analysis for factors affecting recurrence was done by cox regression analysis and p value less than 0.05 was considered significant. A total of 116 patients were diagnosed to have malignant salivary gland tumors of which 69 were mucoepidermoid carcinomas (69.5%). The median age was 43 years (8-75 years). Majority of the tumors occurred in major salivary glands gland (77%). 51% patients were females. Most common stage was stage II (36%) followed by stage I (27.5%), stage IV (20.3%) and stage III (16%). High grade carcinomas comprised 34.8%, intermediate grade 30.4% and low grade 34.8%. 36 patients (52.2%) received adjuvant radiotherapy (60 Gy in 30 fractions). At a median follow up of 42 months (8-70 months), 8 (11.6%) patients died (7 cancer related and 1 noncancer related). The locoregional recurrence rate was 4.3% whereas the distant metastasis rate was 11.6%. Most common site of distant metastasis was lung. The mean disease free survival time was 61.4 months and the mean overall survival was 62 months. On univariate analysis, age < 50 years, node positive, presence of PNI, high grade, presence of LVI and local recurrence were significant factors for distant metastasis (p < 0.05). On multivariate analysis, high grade, presence of LVI and local recurrence were significant factors for distant metastasis (p < 0.05). Mucoepidermoid carcinomas of salivary gland have good long term local control and overall survival. Majority of the recurrences are distant metastasis. High grade, LVI and local recurrence are significant risk factors for distant relapse.

The Role of microRNAs in Inflammation.

Inflammation is a biological response of the immune system to various insults, such as pathogens, toxic compounds, damaged cells, and radiation. The complex network of pro- and anti-inflammatory factors and their direction towards inflammation often leads to the development and progression of various inflammation-associated diseases. The role of small non-coding RNAs (small ncRNAs) in inflammation has gained much attention in the past two decades for their regulation of inflammatory gene expression at multiple levels and their potential to serve as biomarkers and therapeutic targets in various diseases. One group of small ncRNAs, microRNAs (miRNAs), has become a key regulator in various inflammatory disease conditions. Their fine-tuning of target gene regulation often turns out to be an important factor in controlling aberrant inflammatory reactions in the system. This review summarizes the biogenesis of miRNA and the mechanisms of miRNA-mediated gene regulation. The review also briefly discusses various pro- and anti-inflammatory miRNAs, their targets and functions, and provides a detailed discussion on the role of miR-10a in inflammation.

DRR Dhan 58, a Seedling Stage Salinity Tolerant NIL of Improved Samba Mahsuri Shows Superior Performance in Multi-location Trials.

BACKGROUND: Improved Samba Mahsuri (ISM) is an elite, high-yielding, bacterial blight resistant, fine-grained rice variety with low glycaemic index. It is highly sensitive to salt stress, particularly at seedling stage, which significantly reduces its yield potential in coastal areas. A salinity tolerant QTL, Saltol, associated with seedling stage tolerance was previously mapped on chromosome 1 (10.6-11.5 Mb) from the Indian landrace, Pokkali and is effective in different genetic backgrounds. The objective of this study was to enhance salinity tolerance of ISM by incorporating the Saltol QTL through marker-assisted backcross breeding using the breeding line, FL478 (Pokkali/IR29). RESULTS: Foreground selection was carried out at each generation using five Saltol-specific markers and three bacterial blight resistance genes, Xa21, xa13 and xa5. Background selection was conducted using 66 well distributed polymorphic SSR markers and at the BC3F2 generation, a single plant with maximum recurrent parent genome recovery (95.3%) was identified and advanced to the BC3F4 generation. Based on bacterial blight resistance, seedling stage salinity tolerance and resemblance to ISM, four advanced breeding lines were selected for testing in replicated experiments near Hyderabad, India. A promising near-isogenic line, DRR Dhan 58, was evaluated in multi-location trials-coastal salinity and it showed significant salinity tolerance, resistance to bacterial blight disease, high yield and excellent grain quality during the 2019 and 2020 trials. DRR Dhan 58 was 95.1% similar to ISM based on genotyping with the 90 K SNP chip. Whole genome resequencing analysis of Pokkali and FL478 which were salinity tolerant checks, ISM and DRR Dhan 58 showed a high degree of relatedness with respect to the candidate gene loci for Saltol and OsSKC1 (Shoot K+ Concentration 1). CONCLUSION: DRR Dhan 58, possessing Saltol and three bacterial blight resistance genes (Xa21, xa13 and xa5) in the genetic background of the Indian mega-variety of rice, Samba Mahsuri, was developed for potential cultivation in areas prone to seedling stage salinity, as well as areas with endemic bacterial blight disease. This entry had a 24% yield advantage over the recurrent parent ISM under coastal saline conditions in multi-location trials and was recently released for commercial cultivation in India.

The Gab2-MALT1 axis regulates thromboinflammation and deep vein thrombosis.

Deep vein thrombosis (DVT) is the third most common cause of cardiovascular mortality. Several studies suggest that DVT occurs at the intersection of dysregulated inflammation and coagulation upon activation of inflammasome and secretion of interleukin 1ß (IL-1ß) in restricted venous flow conditions. Our recent studies showed a signaling adapter protein, Gab2 (Grb2-associated binder 2), plays a crucial role in propagating inflammatory signaling triggered by IL-1ß and other inflammatory mediators in endothelial cells. The present study shows that Gab2 facilitates the assembly of the CBM (CARMA3 [CARD recruited membrane-associated guanylate kinase protein 3]-BCL-10 [B-cell lymphoma 10]-MALT1 [mucosa-associated lymphoid tissue lymphoma translocation protein 1]) signalosome, which mediates the activation of Rho and NF-κB in endothelial cells. Gene silencing of Gab2 or MALT1, the effector signaling molecule in the CBM signalosome, or pharmacological inhibition of MALT1 with a specific inhibitor, mepazine, significantly reduced IL-1ß-induced Rho-dependent exocytosis of P-selectin and von Willebrand factor (VWF) and the subsequent adhesion of neutrophils to endothelial cells. MALT1 inhibition also reduced IL-1ß-induced NF-κB-dependent expression of tissue factor and vascular cell adhesion molecule 1. Consistent with the in vitro data, Gab2 deficiency or pharmacological inhibition of MALT1 suppressed the accumulation of monocytes and neutrophils at the injury site and attenuated venous thrombosis induced by the inferior vena cava ligation-induced stenosis or stasis in mice. Overall, our data reveal a previously unrecognized role of the Gab2-MALT1 axis in thromboinflammation. Targeting the Gab2-MALT1 axis with MALT1 inhibitors may become an effective strategy to treat DVT by suppressing thromboinflammation without inducing bleeding complications.

Selective inhibition of activated protein C anticoagulant activity protects against hemophilic arthropathy in mice.

Recurrent spontaneous or trauma-related bleeding into joints in hemophilia leads to hemophilic arthropathy (HA), a debilitating joint disease. Treatment of HA consists of preventing joint bleeding by clotting factor replacement, and in extreme cases, orthopedic surgery. We recently showed that administration of endothelial cell protein C receptor (EPCR) blocking monoclonal antibodies (mAb) markedly reduced the severity of HA in factor VIII (FVIII)-/- mice. EPCR blocking inhibits activated protein C (APC) generation and EPCR-dependent APC signaling. The present study was aimed to define the role of inhibition of APC anticoagulant activity, APC signaling, or both in suppressing HA. FVIII-/- mice were treated with a single dose of isotype control mAb, MPC1609 mAb, that inhibits anticoagulant, and signaling properties of APC, or MAPC1591 mAb that only blocks the anticoagulant activity of APC. Joint bleeding was induced by needle puncture injury. HA was evaluated by monitoring joint bleeding, change in joint diameter, and histopathological analysis of joint tissue sections for synovial hypertrophy, macrophage infiltration, neoangiogenesis, cartilage degeneration, and chondrocyte apoptosis. No significant differences were observed between MPC1609 and MAPC1591 in inhibiting APC anticoagulant activity in vitro and equally effective in correcting acute bleeding induced by the saphenous vein incision in FVIII-/- mice. Administration of MAPC1591, and not MPC1609, markedly reduced the severity of HA. MAPC1591 inhibited joint bleed-induced inflammatory cytokine interleukin-6 expression and vascular leakage in joints, whereas MPC1609 had no significant effect. Our data show that an mAb that selectively inhibits APC's anticoagulant activity without compromising its cytoprotective signaling offers a therapeutic potential alternative to treat HA.