Genomic analysis in the colon tissues of omega-3 fatty acid-treated rats identifies novel gene signatures implicated in ulcerative colitis.

Several long-term intervention trials only studied the ex vivo immunological function to elucidate the beneficial mechanisms of n-3 polyunsaturated fatty acids (PUFA) in the ulcerative colitis (UC). An unbiased whole-transcriptome analysis would be more valuable to obtain a comprehensive understanding of the processes and genes regulated by n-3 PUFA in vivo. In this study, we have performed microarray analysis in the colon tissues of dextran sulfate sodium (DSS)-induced UC in rats supplemented with n-6 PUFA, n-3PUFA and long-chain n-3PUFA (LC-n3PUFA). We have identified the novel gene signatures previously not linked to colitis such as Etv3, Clec4d, CD180, CD72, Megf11, and Angptl4 which are most downregulated in both n-3PUFA and LC-n3PUFA groups compared to the n-6PUFA group. The most upregulated genes were Nr1i3, Nptx2, and Zfp810 in both n-3PUFA and LC-n3PUFA groups. The RT-PCR analysis confirmed similar results. Interestingly, LPS treatment in macrophages upregulated the Megf11, Etv3, CD180, and Angptl4, and correlated with increased secretion of cytokines. Gene silencing of Etv3, Megf11, and CD180 in rats using intravascular delivery of siRNA-lipoparticles attenuated the DSS-induced ulceration and mucosal damage. Thus, our genome-wide microarray analysis identified novel genes regulated by omega-3 PUFA and offers new drug targets that could prevent or reduce UC.

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.

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.

Gab2 (Grb2-Associated Binder2) Plays a Crucial Role in Inflammatory Signaling and Endothelial Dysfunction.

[Figure: see text].

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.

EPCR deficiency or function-blocking antibody protects against joint bleeding-induced pathology in hemophilia mice.

We recently showed that clotting factor VIIa (FVIIa) binding to endothelial cell protein C receptor (EPCR) induces anti-inflammatory signaling and protects vascular barrier integrity. Inflammation and vascular permeability are thought to be major contributors to the development of hemophilic arthropathy following hemarthrosis. The present study was designed to investigate the potential influence of FVIIa interaction with EPCR in the pathogenesis of hemophilic arthropathy and its treatment with recombinant FVIIa (rFVIIa). For this, we first generated hemophilia A (FVIII-/-) mice lacking EPCR (EPCR-/-FVIII-/-) or overexpressing EPCR (EPCR++ FVIII-/-). Joint bleeding was induced in FVIII-/-, EPCR-/-FVIII-/-, and EPCR++FVIII-/- mice by needle puncture injury. Hemophilic synovitis was evaluated by monitoring joint bleeding, change in joint diameter, and histopathological analysis of joint tissue sections. EPCR deficiency in FVIII-/- mice significantly reduced the severity of hemophilic synovitis. EPCR deficiency attenuated the elaboration of interleukin-6, infiltration of macrophages, and neoangiogenesis in the synovium following hemarthrosis. A single dose of rFVIIa was sufficient to fully prevent the development of milder hemophilic synovitis in EPCR-/-FVIII-/- mice. The development of hemophilic arthropathy in EPCR-overexpressing FVIII-/- mice did not significantly differ from that of FVIII-/- mice, and 3 doses of rFVIIa partly protected against hemophilic synovitis in these mice. Consistent with the data that EPCR deficiency protects against developing hemophilic arthropathy, administration of a single dose of EPCR-blocking monoclonal antibodies markedly reduced hemophilic synovitis in FVIII-/- mice subjected to joint bleeding. The present data indicate that EPCR could be an attractive new target to prevent joint damage in hemophilia patients.

Increased Accumulation and Retention of rhFVIIa (eptacog beta) in Knee Joints of Hemophilia A Mice Compared to Wild-Type Mice.

Our earlier studies showed that recombinant human factor VIIa (rhFVIIa) administered intravascularly in mice disappeared rapidly from the circulation. However, a small fraction of rhFVIIa that entered extravascular remained functionally active for an extended period. The present study aims to investigate the dose-dependency of rhFVIIa accumulation and retention in mouse knee joints and test whether the hemophilic condition affects rhFVIIa sequestration in joints. Wild-type and FVIII-/- mice were injected with three doses of rhFVIIa (eptacog beta, 90, 250, and 500 µg/kg) via the tail vein. At varying times following rhFVIIa administration, blood and knee joints were collected to measure FVIIa activity and antigen levels in plasma and joint tissues. Joint tissue sections were analyzed by immunohistochemistry for the presence of rhFVIIa. Vascular permeability was assessed by either Evans Blue dye or fluorescein dextran extravasation. The study showed that rhFVIIa accumulated in knee joints of wild-type and FVIII-/- mice in a dose-dependent manner. rhFVIIa antigen and FVIIa activity could be detectable in joints for at least 7 days. Significantly higher levels of rhFVIIa accumulation were observed in knee joints of FVIII-/- mice compared with that of wild-type mice. Immunohistochemical analyses confirmed higher levels of rhFVIIa retention in FVIII-/- mice compared with wild-type mice. Additional studies showed that FVIII-/- mice were more permissible to vascular leakage. In conclusion, the present data demonstrate a dose-dependent accumulation of rhFVIIa in knee joints, and the hemophilic condition enhances the entry of rhFVIIa from circulation to the extravascular. The present data will be useful in improving rhFVIIa prophylaxis.

Lactucaxanthin - a potential anti-diabetic carotenoid from lettuce (Lactuca sativa) inhibits α-amylase and α-glucosidase activity in vitro and in diabetic rats.

Intestinal and pancreatic α-amylase and α-glucosidase inhibitors offer an approach to lower the levels of post-prandial hyperglycemia through the control of dietary starch breakdown in digestion. This study hypothesized that lactucaxanthin (Lxn) in lettuce (Lactuca sativa) inhibits the activity of α-amylase and α-glucosidase. In this study, the interaction of Lxn with α-amylase and α-glucosidase in silico and its inhibitory effect on these enzymes were studied using in vitro and STZ-induced diabetic rat models. Lxn was isolated from lettuce with 96% purity confirmed by HPLC and LCMS. The in silico analysis showed that Lxn has a lower binding energy (-6.05 and -6.34 kcal mol-1) with α-amylase and α-glucosidase compared to their synthetic inhibitors, acarbose (-0.21 kcal mol-1) and miglitol (-2.78 kcal mol-1), respectively. In vitro α-amylase and α-glucosidase inhibition assays revealed that Lxn had IC50 values of 435.5 µg mL-1 and 1.84 mg mL-1, but acarbose has values of 2.5 and 16.19 µg mL-1. The in vivo results showed an increased activity for α-amylase and α-glucosidase in the intestine (4.7 and 1.30 fold, p < 0.05) and pancreas (1.3 and 1.48 fold, p < 0.05) of STZ induced diabetic rats compared to normal rats. Whereas the activity decreased (p < 0.05) in the Lxn fed diabetic rats, except for the intestinal α-glucosidase activity (1.69 ± 0.12 PNP per min per mg protein). This was confirmed by the low blood glucose level (239.4 ± 18.2 mg dL-1) in diabetic rats fed Lxn compared to the diabetic group (572.2 ± 30.5 mg dL-1, p < 0.05). Lxn significantly inhibited (p < 0.05) the activity of α-amylase and α-glucosidase and could be of medical and nutritional relevance in the treatment of diabetes.