M6A-mediated-upregulation of lncRNA BLACAT3 promotes bladder cancer angiogenesis and hematogenous metastasis through YBX3 nuclear shuttling and enhancing NCF2 transcription.

Lymphatic metastasis is recognized as the leading manner of metastasis in bladder cancer (BLCa), but hematogenous metastasis accounts for a majority of cancer-associated deaths. The past two decades have witnessed tremendous attention in long non-coding RNAs (lncRNAs), which are a new hope for the development of targeted drug therapy for metastatic cancers; however, the underlying mechanism of lncRNAs involved in BLCa hematogenous metastasis remains to be elucidated. Here, we identified BLCa-associated transcript 3 (BLACAT3), a lncRNA, which was aberrantly upregulated in BLCa and corelated with poor prognosis of patients with muscle-invasive bladder cancer. Methodologically, m6A epitranscriptomic microarray, RNA sequencing and mass spectrometry (MS) were used to screen the key molecules of the regulatory axis. Functional assays, animal models and clinical samples were used to explore the roles of BLACAT3 in BLCa in vitro and in vivo. Mechanistically, m6A modification contributes to BLACAT3 upregulation by stabilizing RNA structure. BLACAT3 recruits YBX3 to shuttle into the nucleus, synergistically enhances NCF2 transcription, and promotes BLCa angiogenesis and hematogenous metastasis by activating downstream NF-κB signaling. Our findings will develop prognosis prediction tools for BLCa patients and discover novel therapeutic biological targets for metastatic BLCa.

Thymic stromal lymphopoietin induces platelet mitophagy and promotes thrombosis in Kawasaki disease.

Kawasaki disease (KD) is an acute systemic vasculitis primarily affecting infants and children. Activated platelets predispose patients to coronary artery structural lesions that may lead to thrombotic cardiovascular events. To discover potential proteins underlying platelet activation in KD, we conducted a protein chip assay of 34 cytokines and discovered thymic stromal lymphopoietin (TSLP) was aberrantly expressed, which remained elevated after intravenous immunoglobulin G (IVIG) treatment and during convalescence in KD patients in comparison to healthy controls. Enzyme-linked immunosorbent assay (ELISA) corroborated the upregulation of TSLP in KD patients, which was exacerbated in convalescent patients complicated with thrombosis. TSLP receptors on platelets were also significantly upregulated in KD patients complicated with thrombosis. Platelet activation, apoptosis, and mitochondrial autophagy (mitophagy) were increased in convalescence KD patients complicated with thrombosis. In vitro, TSLP induced platelet activation and platelet mitophagy in healthy blood donors, as observed in KD patients. TSLP, similar to mitophagy agonist carbonyl cyanide 3-chlorophenyl hydrazone (CCCP), promoted thrombosis, which was attenuated by the mitophagy inhibitor Mdivi-1. Co-immunoprecipitation in TSLP-treated platelets revealed TSLP receptor (TSLPR) bound to mitophagy regulators, Parkin and Voltage Dependent Anion Channel Protein 1 (VDAC1).Thus, our results demonstrated that TSLP induced platelet mitophagy via a novel TSLPR/Parkin/VDAC1 pathway that promoted thrombosis in KD. These results suggest TSLP as a novel therapeutic target against KD-associated thrombosis.

Hypoxia and low temperature upregulate transferrin to induce hypercoagulability at high altitude.

Studies have shown significantly increased thromboembolic events at high altitude. We recently reported that transferrin could potentiate blood coagulation, but the underlying mechanism for high altitude-related thromboembolism is still poorly understood. Here, we examined the activity and concentration of plasma coagulation factors and transferrin in plasma collected from long-term human residents and short-stay mice exposed to varying altitudes. We found that the activities of thrombin and factor XIIa (FXIIa) along with the concentrations of transferrin were significantly increased in the plasma of humans and mice at high altitudes. Furthermore, both hypoxia (6% O2) and low temperature (0°C), 2 critical high-altitude factors, enhanced hypoxia-inducible factor 1α (HIF-1α) levels to promote the expression of the transferrin gene, whose enhancer region contains HIF-1α binding site, and consequently, to induce hypercoagulability by potentiating thrombin and FXIIa. Importantly, thromboembolic disorders and pathological insults in mouse models induced by both hypoxia and low temperature were ameliorated by transferrin interferences, including transferrin antibody treatment, transferrin downregulation, and the administration of our designed peptides that inhibit the potentiation of transferrin on thrombin and FXIIa. Thus, low temperature and hypoxia upregulated transferrin expression-promoted hypercoagulability. Our data suggest that targeting the transferrin-coagulation pathway is a novel and potentially powerful strategy against thromboembolic events caused by harmful environmental factors under high-altitude conditions.

In vitro assessment and phase I randomized clinical trial of anfibatide a snake venom derived anti-thrombotic agent targeting human platelet GPIbα.

The interaction of platelet GPIbα with von Willebrand factor (VWF) is essential to initiate platelet adhesion and thrombosis, particularly under high shear stress conditions. However, no drug targeting GPIbα has been developed for clinical practice. Here we characterized anfibatide, a GPIbα antagonist purified from snake (Deinagkistrodon acutus) venom, and evaluated its interaction with GPIbα by surface plasmon resonance and in silico modeling. We demonstrated that anfibatide interferds with both VWF and thrombin binding, inhibited ristocetin/botrocetin- and low-dose thrombin-induced human platelet aggregation, and decreased thrombus volume and stability in blood flowing over collagen. In a single-center, randomized, and open-label phase I clinical trial, anfibatide was administered intravenously to 94 healthy volunteers either as a single dose bolus, or a bolus followed by a constant rate infusion of anfibatide for 24 h. Anfibatide inhibited VWF-mediated platelet aggregation without significantly altering bleeding time or coagulation. The inhibitory effects disappeared within 8 h after drug withdrawal. No thrombocytopenia or anti-anfibatide antibodies were detected, and no serious adverse events or allergic reactions were observed during the studies. Therefore, anfibatide was well-tolerated among healthy subjects. Interestingly, anfibatide exhibited pharmacologic effects in vivo at concentrations thousand-fold lower than in vitro, a phenomenon which deserves further investigation.Trial registration: Clinicaltrials.gov NCT01588132.