A novel role for thioredoxin-related transmembrane protein TMX4 in platelet activation and thrombus formation.

BACKGROUND: The functions of critical platelet proteins are controlled by thiol-disulfide exchanges, which are mediated by the protein disulfide isomerase (PDI) family. It has been shown that some PDI family members are important in platelet activation and thrombosis with distinct functions. TMX4, a membrane-type PDI family member, is expressed in platelets, whether it has a role in platelet activation remains unknown. OBJECTIVES: To determine the role of TMX4 in platelet activation and thrombosis. METHODS: The phenotypes of TMX4-deficient mice were evaluated in tail bleeding time assay and laser-induced and FeCl3-induced arterial injury models. The functions of TMX4 in platelets were assessed in vitro using TMX4-null platelets, recombinant TMX4 protein and anti-TMX4 antibody. RESULTS: Compared with the control mice, Tie2-Cre/TMX4fl/fl mice deficient of hematopoietic and endothelial TMX4 exhibited prolonged tail bleeding times and reduced platelet thrombus formation. Pf4-Cre/TMX4fl/fl mice deficient of platelet TMX4 also had prolonged tail bleeding times and decreased thrombus formation, which was rescued by injection of recombinant TMX4 protein. Consistently, TMX4 deficiency inhibited platelet aggregation, integrin αIIbß3 activation, P-selectin expression, phosphatidylserine exposure and thrombin generation, without affecting tyrosine phosphorylation of intracellular signaling molecules Syk, LAT and PLCγ2 and calcium mobilization. Recombinant TMX4 protein enhanced platelet aggregation and reduced integrin αIIbß3 disulfide bond, and TMX4 deficiency decreased free thiols of integrin αIIbß3, consistent with a potent reductase activity of TMX4. In contrast, an inactive TMX4 protein and a specific anti-TMX4 antibody inhibited platelet aggregation. CONCLUSIONS: TMX4 is a novel PDI family member that enhances platelet activation and thrombosis.

Lymph node-targeted STING agonist nanovaccine against chronic HBV infection.

Chronic hepatitis B virus (HBV) infection is a global health problem that substantially increases the risk of developing liver disease. The development of a novel strategy to induce anti-HB seroconversion and achieve a long-lasting immune response against chronic HBV infection remains challenging. Here, we found that chronic HBV infection affected the signaling pathway involved in STING-mediated induction of host immune responses in dendritic cells (DCs) and then generated a lymph node-targeted nanovaccine that co-delivered hepatitis B surface antigen (HBsAg) and cyclic diguanylate monophosphate (c-di-GMP) (named the PP-SG nanovaccine). The feasibility and efficiency of the PP-SG nanovaccine for CHB treatment were evaluated in HBV-carrier mice. Serum samples were analyzed for HBsAg, anti-HBs, HBV DNA, and alanine aminotransferase levels, and liver samples were evaluated for HBV DNA and RNA and HBcAg, accompanied by an analysis of HBV-specific cellular and humoral immune responses during PP-SG nanovaccine treatment. The PP-SG nanovaccine increased antigen phagocytosis and DC maturation, efficiently and safely eliminated HBV, achieved a long-lasting immune response against HBV reinjection, and disrupted chronic HBV infection-induced immune tolerance, as characterized by the generation and multifunctionality of HBV-specific CD8+ T and CD4+ T cells and the downregulation of immune checkpoint molecules. HBV-carrier mice immunized with the PP-SG nanovaccine achieved partial anti-HBs seroconversion. The PP-SG nanovaccine can induce sufficient and persistent viral suppression and achieve anti-HBs seroconversion, rendering it a promising vaccine candidate for clinical chronic hepatitis B therapy.

Low-threshold random lasers based on the DCM-DEG gain system with graphene nanosheets.

In this article, low-threshold random lasers based on DCM-DEG (DD) gain system with graphene nanosheets are studied. The experiment results show that the threshold of random lasers reduces rapidly when an appropriate amount of graphene nanosheets is added in DD solution. Meanwhile, the quantity and quality of random lasing modes raise significantly. We discussed the potential reasons why the graphene nanosheets can strengthen the sample's random lasing. And, the influence of the graphene nanosheet concentration on the radiation characteristics of random lasers is further studied. When the concentration of graphene nanosheets is 0.088wt%, the lasing threshold of DD samples with graphene nanosheets (GDD) is only about 31.8% of the lasing threshold of DD samples, and the quality of random lasing modes is five times higher than that of the DD sample. To further reduce the lasing threshold, the gold (Au) nanoparticles are added in the mixed solution to form the GDD solution with Au nanoparticles (GGDD). The results show that the lasing threshold of the GGDD sample is about 7.73 µJ/pulse, which is 5.2% of the lasing threshold of the DD sample. This experiment provides a new method to study low-threshold and high-quality random lasers based on graphene.

Cholesterol accumulation on dendritic cells reverses chronic hepatitis B virus infection-induced dysfunction.

Chronic hepatitis B (CHB) infection remains a serious public health problem worldwide; however, the relationship between cholesterol levels and CHB remains unclear. We isolated peripheral blood mononuclear cells from healthy blood donors and CHB patients to analyze free cholesterol levels, lipid raft formation, and cholesterol metabolism-related pathways. Hepatitis B virus (HBV)-carrier mice were generated and used to confirm changes in cholesterol metabolism and cell-surface lipid raft formation in dendritic cells (DCs) in the context of CHB. Additionally, HBV-carrier mice were immunized with a recombinant HBV vaccine (rHBVvac) combined with lipophilic statins and evaluated for vaccine efficacy against HBV. Serum samples were analyzed for HBsAg, anti-HBs, and alanine aminotransferase levels, and liver samples were evaluated for HBV DNA and RNA and HBcAg. CHB reduced free cholesterol levels and suppressed lipid raft formation on DCs in patients with CHB and HBV-carrier mice, whereas administration of lipophilic statins promoted free cholesterol accumulation and restored lipid rafts on DCs accompanied by an enhanced antigen-presentation ability in vitro and in vivo. Cholesterol accumulation on DCs improved the rHBVvac-mediated elimination of serum HBV DNA and intrahepatic HBV DNA, HBV RNA, and HBcAg and promoted the rHBVvac-mediated generation and polyfunctionality of HBV-specific CD11ahi CD8αlo cells, induction of the development of memory responses against HBV reinfection, and seroconversion from HBsAg to anti-HBs. The results demonstrated the important role of cholesterol levels in DC dysfunction during CHB, suggesting that strategies to increase cholesterol accumulation on DCs might enhance therapeutic vaccine efficacy against HBV and support development toward clinical CHB treatment.

CpG-C ODN M362 as an immunoadjuvant for HBV therapeutic vaccine reverses the systemic tolerance against HBV.

Chronic Hepatitis B virus (CHB) infection is a global public health problem. Oligodeoxynucleotides (ODNs) containing class C unmethylated cytosine-guanine dinucleotide (CpG-C) motifs may provide potential adjuvants for the immunotherapeutic strategy against CHB, since CpG-C ODNs stimulate both B cell and dendritic cell (DC) activation. However, the efficacy of CpG-C ODN as an anti-HBV vaccine adjuvant remains unclear. In this study, we demonstrated that CpG M362 (CpG-C ODN) as an adjuvant in anti-HBV vaccine (cHBV-vaccine) successfully and safely eliminated the virus in HBV-carrier mice. The cHBV-vaccine enhanced DC maturation both in vivo and in vitro, overcame immune tolerance, and recovered exhausted T cells in HBV-carrier mice. Furthermore, the cHBV-vaccine elicited robust hepatic HBV-specific CD8+ and CD4+ T cell responses, with increased cellular proliferation and IFN-γ secretion. Additionally, the cHBV-vaccine invoked a long-lasting follicular CXCR5+ CD8+ T cell response following HBV re-challenge. Taken together, CpG M362 in combination with rHBVvac cleared persistent HBV and achieved long-term virological control, making it a promising candidate for treating CHB.

Tyro3, Axl, and Mertk receptors differentially participate in platelet activation and thrombus formation.

BACKGROUND: Previously, several studies have shown that Tyro3, Axl, and Mertk (TAM) receptors participate in platelet activation and thrombosis. However, the role of individual receptors is not fully understood. METHODS: Using single receptor-deficient platelets from TAM knockout mice in the C57BL/6 J strain, we performed a knockout study using single TAM-deficient mice. We treated platelets isolated from TAM knockout mice with the Glycoprotein VI (GPVI) agonists convulxin, poly(PHG), and collagen-related triple-helical peptide (CRP), as well as thrombin for in-vitro experiments. We used a laser-induced cremaster arterial injury model for thrombosis experiments in vivo. RESULTS: Deficiency of the tyrosine kinase receptors, Axl or Tyro3, but not Mertk, inhibited aggregation, spreading, JON/A binding, and P-selectin expression of platelets in vitro. In vivo, platelet thrombus formation was significantly decreased in Axl-/- and Tyro3-/- mice, but not in Mertk-/- mice. Upon stimulation with glycoprotein VI (GPVI) agonists, tyrosine phosphorylation of signaling molecules, including spleen tyrosine kinase (Syk) and phospholipase C-γ2 (PLCγ2), was decreased in Axl-/- and Tyro3-/- platelets, but not in Mertk-/- platelets. While platelet aggregation induced by agonists did not differ in the presence or absence of the Gas6 neutralizing antibody, the platelet aggregation was inhibited by anti-Axl or anti-Tyro3 neutralizing antibodies antibody, but not the anti-Mertk antibody. Additionally, the recombinant extracellular domain of Axl or Tyro3, but not that of Mertk, also inhibited platelet aggregation. CONCLUSIONS: These data suggest that Axl and Tyro3, but not Mertk, have an important role in platelet activation and thrombus formation, and mechanistically may do so by a pathway that regulates inside to outside signaling and heterotypic interactions via the extracellular domains of TAMs.

SM22α inhibits lamellipodium formation and migration via Ras-Arp2/3 signaling in synthetic VSMCs.

We previously demonstrated that smooth muscle (SM) 22α promotes the migration activity in contractile vascular smooth muscle cells (VSMCs). Based on the varied functions exhibited by SM22α in different VSMC phenotypes, we investigated the effect of SM22α on VSMC migration under pathological conditions. The results demonstrated that SM22α overexpression in synthetic VSMCs inhibited platelet-derived growth factor (PDGF)-BB-induced cell lamellipodium formation and migration, which was different from its action in contractile cells. The results indicated two distinct mechanisms underlying inhibition of lamellipodium formation by SM22α, increased actin dynamic stability and decreased Ras activity via interference with interactions between Ras and guanine nucleotide exchange factor. The former inhibited actin cytoskeleton rearrangement in the cell cortex, while the latter significantly disrupted actin nucleation activation of the Arp2/3 complex. Baicalin, a herb-derived flavonoid compound, inhibited VSMC migration via upregulation of SM22α expression in vitro and in vivo. These data suggest that SM22α regulates lamellipodium formation and cell migration in a phenotype-dependent manner in VSMCs, which may be a new therapeutic target for vascular lesion formation.

Phosphorylation of smooth muscle 22α facilitates angiotensin II-induced ROS production via activation of the PKCδ-P47phox axis through release of PKCδ and actin dynamics and is associated with hypertrophy and hyperplasia of vascular smooth muscle cells in vitro and in vivo.

RATIONALE: We have demonstrated that smooth muscle (SM) 22α inhibits cell proliferation via blocking Ras-ERK1/2 signaling in vascular smooth muscle cells (VSMCs) and in injured arteries. The recent study indicates that SM22α disruption can independently promote arterial inflammation through activation of reactive oxygen species (ROS)-mediated NF-κB pathways. However, the mechanisms by which SM22α controls ROS production have not been characterized. OBJECTIVE: To investigate how SM22α disruption promotes ROS production and to characterize the underlying mechanisms. METHODS AND RESULTS: ROS level was measured by dihydroethidium staining for superoxide and TBA assay for malondialdehyde, respectively. We showed that downregulation and phosphorylation of SM22α were associated with angiotensin (Ang) II-induced increase in ROS production in VSMCs of rats and human. Ang II induced the phosphorylation of SM22α at Serine 181 in an Ang II type 1 receptor-PKCδ pathway-dependent manner. Phosphorylated SM22α activated the protein kinase C (PKC)δ-p47phox axis via 2 distinct pathways: (1) disassociation of PKCδ from SM22α, and in turn binding to p47phox, in the early stage of Ang II stimulation; and (2) acceleration of SM22α degradation through ubiquitin-proteasome, enhancing PKCδ membrane translocation via induction of actin cytoskeletal dynamics in later oxidative stress. Inhibition of SM22α phosphorylation abolished the Ang II-activated PKCδ-p47phox axis and inhibited the hypertrophy and hyperplasia of VSMCs in vitro and in vivo, accompanied with reduction of ROS generation. CONCLUSIONS: These findings indicate that the disruption of SM22α plays pivotal roles in vascular oxidative stress. PKCδ-mediated SM22α phosphorylation is a novel link between actin cytoskeletal remodeling and oxidative stress and may be a potential target for the development of new therapeutics for cardiovascular diseases.