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) with α-amylase and α-glucosidase compared to their synthetic inhibitors, acarbose (-0.21 kcal mol) and miglitol (-2.78 kcal mol), respectively. In vitro α-amylase and α-glucosidase inhibition assays revealed that Lxn had IC values of 435.5 μg mL and 1.84 mg mL, but acarbose has values of 2.5 and 16.19 μg mL. 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) in diabetic rats fed Lxn compared to the diabetic group (572.2 ± 30.5 mg dL, 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.

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.

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 IL-1β in restricted venous flow conditions. Our recent studies showed a signaling adapter protein, Gab2 (Grb2-associated binder2), plays a crucial role in propagating inflammatory signaling triggered by IL-1β and other inflammatory mediators in endothelial cells. The present study shows that Gab2 facilitates the assembly of the CBM (CARD recruited membrane-associated protein 3 [CARMA3]- B cell lymphoma 10 [BCL-10] - mucosa-associated lymphoid tissue lymphoma translocation protein 1 [MALT1]) signalosome, which mediates the activation of Rho and NF-kB 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-1β-induced Rho-dependent exocytosis of P-selectin and VWF and the subsequent adhesion of neutrophils to endothelial cells. MALT1 inhibition also reduced IL-1β-induced NF-kB-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.

Objective: In response to inflammatory insult, endothelial cells express cell adhesion molecules and TF (tissue factor), leading to increased adhesion of leukocytes to the endothelium and activation of coagulation. Enhanced coagulation could further exacerbate inflammation. Identifying key signaling molecule(s) that drive both inflammation and coagulation may help devise effective therapeutic strategies to treat inflammatory and thrombotic disorders. The aim of the current study to determine the role of Gab2 (Grb2-associated binder2), which is known to play a crucial role in the signaling evoked by growth factors and antigen receptors, in inflammatory signaling pathways and contributing to vascular dysfunction. Approach and Results: WT (wild type) and Gab2-silenced endothelial cells were treated with TNFα (tumor necrosis factor alpha), IL (interleukin)-1β, or lipopolysaccharide (LPS). Activation of key signaling proteins in the inflammatory signaling pathways and expression of cell adhesion molecules, TF, and inflammatory cytokines were analyzed. Gab2 −/ − and WT littermate mice were challenged with LPS or S pneumoniae , and parameters of inflammation and activation of coagulation were assessed. Gab2 silencing in endothelial cells markedly attenuated TNFα-induced, IL-1β–induced, and LPS-induced expression of TF, cell adhesion molecules, and inflammatory cytokines/chemokines. Gab2 silencing suppressed TNFα-induced, IL-1β–induced, and LPS-induced phosphorylation and ubiquitination of TAK1 and activation of MAPKs (mitogen-activated protein kinases) and NF-κB (nuclear factor kappa B). Immunoprecipitation studies revealed that the Src kinase Fyn phosphorylates Gab2. Gab2 −/− mice are protected from LPS or S pneumoniae –induced vascular permeability, neutrophil infiltration, thrombin generation, NET formation, cytokine production, and lung injury. Conclusions: Our studies identify, for the first time, that Gab2 integrates signaling from multiple inflammatory receptors and regulates vascular inflammation and thrombosis.

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