ADAM8 silencing suppresses the migration and invasion of fibroblast-like synoviocytes via FSCN1/MAPK cascade in osteoarthritis.

OBJECTIVE: Osteoarthritis (OA) is a degenerative joint disease that affects elderly populations worldwide, causing pain and disability. Alteration of the fibroblast-like synoviocytes (FLSs) phenotype leads to an imbalance in the synovial inflammatory microenvironment, which accelerates the progression of OA. Despite this knowledge, the specific molecular mechanisms of the synovium that affect OA are still unclear. METHODS: Both in vitro and in vivo experiments were undertaken to explore the role of ADAM8 playing in the synovial inflammatory of OA. A small interfering RNA (siRNA) was targeting ADAM8 to intervene. High-throughput sequencing was also used. RESULTS: Our sequencing analysis revealed significant upregulation of the MAPK signaling cascade and ADAM8 gene expression in IL-1ß-induced FLSs. The in vitro results demonstrated that ADAM8 blockade inhibited the invasion and migration of IL-1ß-induced FLSs, while also suppressing the expression of related matrix metallomatrix proteinases (MMPs). Furthermore, our study revealed that inhibiting ADAM8 weakened the inflammatory protein secretion and MAPK signaling networks in FLSs. Mechanically, it revealed that inhibiting ADAM8 had a significant effect on the expression of migration-related signaling proteins, specifically FSCN1. When siADAM8 was combined with BDP-13176, a FSCN1 inhibitor, the migration and invasion of FLSs was further inhibited. These results suggest that FSCN1 is a crucial downstream factor of ADAM8 in regulating the biological phenotypes of FLSs. The in vivo experiments demonstrated that ADAM8 inhibition effectively reduced synoviocytes inflammation and alleviated the progression of OA in rats. CONCLUSIONS: ADAM8 could be a promising therapeutic target for treating OA by targeting synovial inflammation.

Model for predicting prognosis and immunotherapy based on CD+8 T cells infiltration in neuroblastoma.

BACKGROUND: Neuroblastoma (NBL) is an extracranial malignant tumor in children deriving from the neural crest in the sympathetic nervous system. Although various immunotherapy interventions have made significant breakthroughs in many adult cancers, the efficacy of these immunotherapies was still limited in NBL. NBL has low immunogenicity which results in a lack of tumor-infiltrating T lymphocytes in the tumor microenvironment (TME). Moreover, tumor cells can wield many immune evasion strategies both in the TME and systemically to impede lymphocyte infiltration and activation. All these factors hamper the anti-tumor effects of CD8+ T cells during immunotherapy and the levels of infiltrating CD8+ T cells correlate with therapy response. MATERIALS AND METHODS: In this study, we utilized multidimensional bioinformatic methods to establish a risk model based on CD8+ T cells -related genes (CD8+ TRGs). RESULTS: We obtained 33 CD8+ TRGs with well-predictive ability for prognosis in both GSE49711 and E-MTAB-8248 cohorts. Then, 12 CD8+ TRGs including HK2, RP2, HPSE, ELL2, GFI1, SLC22A16, FCGR3A, CTSS, SH2D1A, RBP5, ATF5, and ADAM9 were finally identified for risk model construction and validation. This model revealed a stable performance in prognostic prediction of the overall survival (OS) and event-free survival (EFS) in patients with NBL. Additionally, our research indicated that the immune and stromal scores, immune-related pathways, immune cell infiltration, the expression of major histocompatibility complex (MHC) and immune checkpoint molecules, immunotherapy response, and drug susceptibility revealed significant differences between high and low-risk groups. CONCLUSIONS: According to our analyses, the constructed CD8+ TRGs-based risk model may be promising for the clinical prediction of anti-tumor therapy responses and prognoses in NBL.

Extracellular vesicle­mediated miR­126­3p transfer contributes to inter­cellular communication in the liver tumor microenvironment.

Extracellular vesicles (EVs) and their contents are gaining recognition as important mediators of intercellular communication through the transfer of bioactive molecules, such as non­coding RNA. The present study comprehensively assessed the microRNA (miRNA/miR) content within EVs released from HepG2 liver cancer (LC) cells and LX2 hepatic stellate cells (HSCs) and determined the contribution of EV miRNA to intercellular communication. Using both transwell and spheroid co­cultures of LC cells and HSCs, miR­126­3p within EV was established as a mediator of HSC to LC cell communication that influenced tumor cell migration and invasion, as well as the growth of multicellular LC/HSC spheroids. Manipulation of miR­126­3p either by enforced expression using pre­miR­126­3p or by inhibition using antimiR­126­3p did not alter tumor cell viability, proliferation or sensitivity to either sorafenib or regorafenib. By contrast, enforced expression of miR­126­3p decreased tumor­cell migration. Knockdown of miR­126­3p in tumor cells increased disintegrin and metalloproteinase domain­containing protein 9 (ADAM9) expression and in HSCs increased collagen­1A1 accumulation with an increase in compactness of multicellular spheroids. Within LC/HSC spheroids, ADAM9 and vascular endothelial growth factor expression was increased by silencing of miR­126­3p but diminished with the restoration of miR­126­3p. These studies implicate miR­126­3p in functional effects on migration, invasion and spheroid growth of tumor cells in the presence of HSCs, and thereby demonstrate functional EV­RNA­based intercellular signaling between HSCs and LC cells that is directly relevant to tumor­cell behavior.

The role of YAP1 target gene CTGF in the anoikis resistance of rheumatoid arthritis synovial fibroblasts.

OBJECTIVE: To analyse pro-survival mechanisms elicited in RA synovial fibroblasts (RASFs) upon detachment from their extracellular matrix dependent on the disintegrin metalloproteinase ADAM15 and Yes-associated protein kinase 1 (YAP1). METHODS: Detachment-induced apoptosis was determined by caspase 3/7 assays. Immunofluorescent stainings, cell surface biotinylation and immunoblotting were applied to analyse phosphorylated kinases and subcellular localization of YAP1 and connective tissue growth factor (CTGF). Caspase and transwell transmigration assays served to study CTGF function. RESULTS: Silencing of ADAM15 or YAP1 in RASFs leads to significantly increased levels of detachment-induced caspase activity. In non-silenced RASFs detachment causes simultaneous ADAM15-enhanced phosphorylation of YAP1 at S127, known for promoting its cytoplasmic localization, and Src-dependent phosphorylation at tyrosine Y357. The majority of nuclear YAP1 leaves the nucleus shortly after cell detachment, but prolonged detachment causes a marked nuclear re-entry of YAP1, resulting in significantly increased synthesis of CTGF. The newly synthesized CTGF, however, is not detectable in the supernatant, but is bound to the outside of the plasma membrane. In vitro studies demonstrated autocrine binding of CTGF to the EGF receptor and ß1 integrin, with concomitant triggering of survival kinases, AKT1, ERK1/2, Src and focal adhesion kinase. Functional studies revealed anti-apoptotic effects of CTGF on detached RASFs and an enhancement of their potential for endothelial transmigration using HUVEC-coated transwells. CONCLUSION: The elucidation of a new molecular mechanism that protects RASFs in the highly pro-apoptotic environment of inflamed RA joints by promoting anoikis-resistance and transendothelial migration via ADAM15/YAP1-mediated CTGF upregulation uncovers potentially new targets for future therapeutic intervention.

Haemorrhagic snake venom metalloproteases and human ADAMs cleave LRP5/6, which disrupts cell-cell adhesions in vitro and induces haemorrhage in vivo.

Snake venom metalloproteases (SVMPs) are members of the a disintegrin and metalloprotease (ADAM) family of proteins, as they possess similar domains. SVMPs are known to elicit snake venom-induced haemorrhage; however, the target proteins and cleavage sites are not known. In this work, we identified a target protein of vascular apoptosis-inducing protein 1 (VAP1), an SVMP, relevant to its ability to induce haemorrhage. VAP1 disrupted cell-cell adhesions by relocating VE-cadherin and γ-catenin from the cell-cell junction to the cytosol, without inducing proteolysis of VE-cadherin. The Wnt receptors low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) are known to promote catenin relocation, and are rendered constitutively active in Wnt signalling by truncation. Thus, we examined whether VAP1 cleaves LRP5/6 to induce catenin relocation. Indeed, we found that VAP1 cleaved the extracellular region of LRP6 and LRP5. This cleavage removes four inhibitory ß-propeller structures, resulting in activation of LRP5/6. Recombinant human ADAM8 and ADAM12 also cleaved LRP6 at the same site. An antibody against a peptide including the LRP6-cleavage site inhibited VAP1-induced VE-cadherin relocation and disruption of cell-cell adhesions in cultured cells, and blocked haemorrhage in mice in vivo. Intriguingly, animals resistant to the effects of haemorrhagic snake venom express variants of LRP5/6 that lack the VAP1-cleavage site, or low-density lipoprotein receptor domain class A domains involved in formation of the constitutively active form. The results validate LRP5/6 as physiological targets of ADAMs. Furthermore, they indicate that SVMP-induced cleavage of LRP5/6 causes disruption of cell-cell adhesion and haemorrhage, potentially opening new avenues for the treatment of snake bites.

Potential for Recombinant ADAMTS13 as an Effective Therapy for Acquired Thrombotic Thrombocytopenic Purpura.

OBJECTIVE: The metalloprotease ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) regulates the size of von Willebrand factor multimers. A deficiency in ADAMTS13 activity is associated with the life-threatening disease thrombotic thrombocytopenic purpura (TTP). The vast majority of patients have acquired TTP, where circulating anti-ADAMTS13 autoantibodies are causative for the decreased ADAMTS13 activity. Current treatment consists of plasma exchange, but improved therapies are highly warranted. APPROACH AND RESULTS: We have developed a new rat model mimicking various aspects of acquired TTP to investigate the therapeutic efficacy of human recombinant ADAMTS13. A polyclonal antibody against ADAMTS13 completely blocked endogenous rat ADAMTS13 activity in Sprague-Dawley rats. When TTP was triggered using recombinant von Willebrand factor, the animals displayed severe TTP-like symptoms, such as thrombocytopenia, hemolytic anemia, and von Willebrand factor-rich thrombi in the kidneys and brain. Subsequent injection of 400, 800, or 1600 U/kg recombinant ADAMTS13 prevented full development of these symptoms. Analysis of plasma samples confirmed that recombinant ADAMTS13 was able to override circulating anti-ADAMTS13 inhibitory antibodies, resulting in restoration of ADAMTS13 activity and degradation of ultralarge von Willebrand factor multimers. CONCLUSIONS: Recombinant ADAMTS13 was shown to be effective in averting severe acquired TTP-like symptoms in rats and holds promising value for the treatment of this severe and life-threatening disease in humans.

[Inhibitory effect of von Willebrand factor-cleaving protease on angiogenesis].

OBJECTIVE: To investigate the inhibitory effect of von Willebrand factor-cleaving protease, a disintegrin-like and metalloprotease with thrombospondin-1 repeats (ADAMTS13)on angiogenesis induced by vascular endothelial growth factor (VEGF)in vitro and in vivo. METHODS: Cell proliferation assay, differentiation (tube formation)assay and wound migration assay were performed by using human umbilical vein endothelial cells (HUVECs)to explore the effect of ADAMTS13 on angiogenesis in vitro. Cells were treated with different concentrations of ADAMTS13 (1, 5, 25, 50 and 100 nmol/L)and the number of cells was counted via MTT assay. In addition, effect of ADAMTS13 on differentiation was assessed by measuring the length of capillary-like tube structures formed by HUVECS in matrigel. Effect of ADAMTS13 on HUVEC migration was assessed via calculation of wound healing distance after 8 hrs culture with VEGF or ADAMTS13. Chick embryo chorioallantoic membrane (CAM) assay and Matrigel plug assay were performed to investigate the effect of ADAMTS13 on angiogenesis in vivo. RESULTS: ADAMTS13 significantly inhibited the proliferation of HUVECs induced by VEGF in a dose-dependent manner. Migration distance of HUVECs was (79 ± 22) µm in control group, (250 ± 8)µm in VEGF-treated group and (170 ± 23)µm in VEGF and ADAMTS13 cotreated-group after 8 hrs, respectively. The tube length is (450.6 ± 16.6)% in VEGF-treated group and (235.3 ± 19.0)% in VEGF and ADAMTS13 cotreated-group of that of control group after HUVECs cultured in matrigel for 16 hrs. The number of blood vessels decreased after treatment with ADAMTS13 in CAM assay. The number of blood vessels was (228.2 ± 10.8)%, (69.2 ± 21.1)%, (184.6 ± 15.2)% in VEGF treated-group, ADAMTS13 treated-group and VEGF and ADAMTS13 cotreated-group of that of control group, respectively. Formation of capillary-like network in matrigel plugs containing VEGF was reduced to 43.5% by ADAMTS13 in matrigel plug assay in mouse model. CONCLUSION: ADAMTS13 inhibits the HUVECs proliferation, differentiation and migration in vitro. ADAMTS13 inhibits chick embryos vascularization and formation of capillary-like network in vivo.

Pathological von Willebrand factor fibers resist tissue plasminogen activator and ADAMTS13 while promoting the contact pathway and shear-induced platelet activation.

BACKGROUND: Under severe stenotic conditions, von Willebrand factor (VWF) multimerizes into large insoluble fibers at pathological shear rates. OBJECTIVE: Evaluate the mechanics and biology of VWF fibers without the confounding effects of endothelium or collagen. METHODS: Within a micropost-impingement microfluidic device, > 100-µm long VWF fibers multimerized on the post within 10 min using EDTA-treated platelet-free plasma (PFP) perfused at wall shear rates > 5000 s(-1) . RESULTS: von Willebrand factor fiber thickness increased to > 10 µm as a result of increasing the shear rate to 10,000 s(-1) . In a stress-strain test, fibrous VWF had an elastic modulus of ~50 MPa. The insoluble VWF fibers were non-amyloid because they rapidly dissolved in trypsin, plasmin or 2% SDS, but were resistant to 50 nm ADAMTS13 or 100 nm tissue plasminogen activator in plasma. Following fiber formation, perfusion of low corn trypsin inhibitor (CTI)-treated (4 µg mL(-1) ), recalcified citrated plasma at 1500 s(-1) caused fibrin formation on the VWF fibers, a result not observed with purified type 1 collagen or a naked micropost. During VWF fiber formation, contact pathway factors accumulated on VWF because the use of EDTA/D-Phe-Pro-Arg chloromethylketone (PPACK)/apixaban/high CTI-treated PFP during VWF fiber formation prevented the subsequent fibrin production from low-CTI, recalcified citrated PFP. VWF fibers displayed FXIIa-immunostaining. When PPACK-inhibited whole blood was perfused over VWF fibers, platelets rolled and arrested on the surface of VWF, but only displayed P-selectin if prevailing shear rates were pathological. Platelet arrest on VWF fibers was blocked with αIIb ß3 antagonist GR144053. CONCLUSIONS: We report VWF fiber-contact pathway crosstalk and mechanisms of thrombolytic resistance in hemodynamic settings of myocardial infarction.

The Protective Roles of IL-6 Trans-Signaling Regulated by ADAM9 on the Liver in Carbon Tetrachloride-Induced Liver Injury in Mice.

Our study was undertaken to evaluate the important role that a disintegrin and metalloproteinase 9 (ADAM9) regulates IL-6 trans-signaling in carbon tetrachloride (CCl4)-induced liver injury in mice. Mice were divided into four groups. Each group respectively received mineral oil injection, CCl4 injection, anti-ADAM9 monoclonal antibody (mAb) pretreatment and CCl4 injection, anti-ADAM9 mAb and recombinant mouse ADAM9 molecules pretreatment with CCl4 injection. Our results showed that anti-ADAM9 mAb pretreatment significantly aggravated liver injury, inhibited IL-6 trans-signaling, which led to downregulation of proliferating cell nuclear antigen (PCNA), vascular endothelial growth factor (VEGF), upregulation of Caspase3, cytochrome P450 2E1 (CYP2E1), and hepatocytes apoptosis at 24 h after CCl4 injection. Recombinant ADAM9 molecules pretreatment reversed the impact of anti-ADAM9 mAb pretreatment in mice. In conclusion, our study suggested that ADAM9 could regulate the hepatocytes proliferation, apoptosis, angiogenesis, and CYP2E1 expression by activating IL-6 trans-signaling and play important protective roles during CCl4-induced liver injury in mice.

Antitumor and anti-angiogenic activity of the recombinant human disintegrin domain of A disintegrin and metalloproteinase 15.

A disintegrin and metalloproteinases (ADAMs), a family of transmembrane glycoproteins, are expressed in numerous tissues and organs, and have been implicated in a variety of physiological and pathological processes. ADAM15 is unique among the ADAMs in having an Arg-Gly-Asp motif in its disintegrin domain. In the present study, the antitumor and anti-angiogenic effects of the recombinant human disintegrin domain (rhdd) ADAM15, expressed by Escherichia coli, were evaluated. rhddADAM15 inhibited the proliferation and migration of several tumor cells, with a half maximal inhibitory concentration of 1.0-6.0 µM. In addition, rhddADAM15 inhibited the proliferation of Bel-7402 cells via the mitogen-activated protein kinase pathway and reduced the activation of Src. rhddADAM15 (1-10 µM) inhibited the proliferation, migration and tube formation of vascular endothelial EA.hy926 cells. G0/G1 arrest (10.96 ± 1.40%) and apoptotic cells (55.85 ± 1.06%) were observed in the EA.hy926 cells treated with 4 µM and 6 µM rhddADAM15, respectively. In vivo, rhddADAM15 significantly inhibited angiogenesis in zebrafish. rhddADAM15 at concentrations of 20 nmol/fish or 5 nmol/fish inhibited the angiogenesis of subintestinal and intersegmental vessels in the zebrafish by 72 ± 1.26 and 48 ± 2.92%, respectively. In conclusion, the results of the present study identified rhddADAM15 as a potent inhibitor of tumor formation and angiogenesis, rendering it a promising tool for use in anticancer treatment.

Effect of recombinant ADAMTS-13 on microthrombosis and brain injury after experimental subarachnoid hemorrhage.

BACKGROUND: A common complication after aneurysmal subarachnoid hemorrhage (SAH) is delayed cerebral ischemia (DCI), which is associated with vasospasm and other mechanisms such as microthrombosis. ADAMTS-13 activity plays a role in the prevention of thrombus formation in the cerebral microvasculature. Previously, we observed that patients with DCI have lower levels of ADAMTS-13. OBJECTIVES: To examine whether recombinant human ADAMTS-13 (rADAMTS-13) reduces cerebral microthrombus formation and brain injury in an experimental mouse model of SAH including wild-type and ADAMTS-13(-/-) mice. METHODS: Experimental SAH was induced with the prechiasmatic blood injection model. The following experimental groups were investigated: (i) C57BL/6J mice (n = 10); (ii) C57BL/6J mice (n = 10) treated with rADAMTS-13 20 min after SAH; (iii) ADAMTS-13(-/-) mice (n = 10); and (iv) ADAMTS-13(-/-) mice (n = 10) treated with rADAMTS-13 20 min after SAH. Mice were killed at 48 h. Results are presented as means with standard errors of the mean. RESULTS: Infusion with rADAMTS-13 reduced the extent of microthrombosis by ~ 50% in both wild-type mice (mean fibrinogen area: 0.28% ± 0.09% vs. 0.15% ± 0.04%; P = 0.20) and ADAMTS-13(-/-) mice (mean fibrinogen area: 0.32% ± 0.05% vs. 0.16% ± 0.03%; P = 0.016). In addition, rADAMTS-13 reduced brain injury by > 60% in both wild-type mice (mean microglia area: 0.65% ± 0.18% vs. 0.18% ± 0.04%; P = 0.013) and ADAMTS-13(-/-) mice (mean microglia area: 1.24% ± 0.36% vs. 0.42% ± 0.13%; P = 0.077). CONCLUSIONS: Our results support the further study of rADAMTS-13 as a treatment option for the prevention of microthrombosis and brain injury after SAH.

Electroconvulsive seizure induces thrombospondin-1 in the adult rat hippocampus.

Synaptic dysfunction has recently gained attention for its involvement in mood disorders. Electroconvulsive therapy (ECT) possibly plays a role in synaptic repair. However, the underlying mechanisms remain uncertain. Thrombospondin-1 (TSP-1), a member of the TSP family, is reported to be secreted by astrocytes and to regulate synaptogenesis. We investigated the effects of electroconvulsive seizure (ECS) on the expression of TSPs in the adult rat hippocampus. Single and repeated ECS significantly increased TSP-1 mRNA expression after 2h and returned to sham levels at 24h. Conversely, the TSP-2 and -4 mRNA levels did not change. Only repeated ECS induced TSP-1 proteins. ECS also induced glial fibrillary acidic protein (GFAP) expression. The GFAP expression occurred later than the TSP-1 mRNA expression following single ECS; however, it occurred earlier and was more persistent following repeated ECS. ECS had no effect on the α2δ-1 or neuroligin-1 expressions, both of which are TSP-1 receptors. Furthermore, chronic treatment with antidepressants did not induce the expression of TSP-1 or GFAP. These findings suggest that repeated ECS, but not chronic treatment with antidepressants, induces TSP-1 expression partially via the activation of astrocytes. Therefore, TSP-1 is possibly involved in the synaptogenic effects of ECS.

Assessment of the matrix degenerative effects of MMP-3, ADAMTS-4, and HTRA1, injected into a bovine intervertebral disc organ culture model.

STUDY DESIGN: In vitro study to develop an intervertebral disc degeneration organ culture model, using coccygeal bovine intervertebral discs (IVDs) and injection of proteolytic enzymes MMP-3, ADAMTS-4, and HTRA1. OBJECTIVE: This study aimed to develop an in vitro model of enzyme-mediated intervertebral disc degeneration to mimic the clinical outcome in humans for investigation of therapeutic treatment options. SUMMARY OF BACKGROUND DATA: Bovine IVDs are comparable with human IVDs in terms of cell composition and biomechanical behavior. Researchers injected papain and trypsin into them to create an intervertebral disc degeneration model with a degenerated nucleus pulposus (NP) area. They achieved macroscopic cavities as well as a loss of glycosaminoglycans (GAGs). However, none of these enzymes are clinically relevant. METHODS: Bovine IVDs were harvested maintaining the endplates. Active forms of MMP-3, ADAMTS-4, and HTRA1 were injected at a dose of 10 µg/mL each. Phosphate-buffered saline was injected as a control. Discs were cultured for 8 days and loaded diurnally (days 1-4 with ≈0.4 MPa for 16 hr) and left under free swelling condition from days 4 to 8 to avoid expected artifacts because of dehydration of the NP. Outcome parameters included disc height, metabolic cell activity, DNA content, GAG content, total collagen content, relative gene expression, and histological investigation. RESULTS: The mean metabolic cell activity was significantly lower in the NP area of discs injected with ADAMTS-4 than the day 0 control discs. Disc height was decreased after injection with HTRA1 and was significantly correlated with changes in GAG/DNA of the NP tissue. Total collagen content tended to be lower in groups injected with ADAMTS4 and MMP-3. CONCLUSION: MMP-3, ADAMTS-4, and HTRA1 provoked neither visible matrix degradation nor major shifts in gene expression. However, cell activity was significantly reduced and HTRA1 induced loss of disc height that positively correlated with changes in GAG/DNA content. The use of higher doses of these enzymes or a combination thereof may, therefore, be necessary to induce disc degeneration.

TNFα reverse signaling promotes sympathetic axon growth and target innervation.

Reverse signaling via members of the tumor necrosis factor (TNF) superfamily controls multiple aspects of immune function. Here we document TNFα reverse signaling in the nervous system to our knowledge for the first time and show that it has a crucial role in establishing sympathetic innervation. During postnatal development, sympathetic axons express TNFα as they grow and branch in their target tissues, which in turn express TNF receptor 1 (TNFR1). In culture, soluble forms of TNFR1 act directly on postnatal sympathetic axons to promote growth and branching by a mechanism that depends on membrane-integrated TNFα and on downstream activation of ERK. Sympathetic innervation density is substantially lower in several tissues in postnatal and adult mice lacking either TNFα or TNFR1. These findings reveal that target-derived TNFR1 acts as a reverse-signaling ligand for membrane-integrated TNFα to promote growth and branching of sympathetic axons.

Recombinant disintegrin domain of ADAM15 inhibits the proliferation and migration of Bel-7402 cells.

ADAM15 (A Disintegrin And Metalloproteinase 15), a transmembrane protein containing seven domains, interacts with some integrins via its disintegrin domain and overexpresses in many solid tumors. In this study, the effect of the recombinant human disintegrin domain (rhddADAM15) on the proliferation and migration of Bel-7402 cells was evaluated in vitro and in vivo in zebrafish xenografts. rhddADAM15 (4 µM) severely inhibited the proliferation and migration of Bel-7402 cells, inducing a partial G2/S arrest and morphological nucleus changes of apoptosis. Moreover, the activity of caspases 8, 9 and 3 in Bel-7402 cells was increased. In addition, the zebrafish was used as a model for apoptosis-induction and tumor-xenograft. rhddADAM15 (1 pM) inhibited the growth and metastasis of Bel-7402 cell xenografts in zebrafish and a lower concentration (0.1 pM) induced severe apoptosis in the somatic cells of zebrafish. In conclusion, our data identified rhddADAM15 as a potent inhibitor of tumor growth and metastasis, making it a promising tool for use in anticancer treatment.

Recombinant ADAMTS13 reduces tissue plasminogen activator-induced hemorrhage after stroke in mice.

OBJECTIVE: Tissue plasminogen activator (tPA) is approved for treatment of acute ischemic stroke, but it increases the risk of cerebral hemorrhage. Accumulating evidence suggests that von Willebrand factor (VWF) plays a pivotal role in thrombus formation and microcirculatory disturbances after ischemic stroke. By cleaving VWF, ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) protects mice from stroke. Therefore, we hypothesized that recombinant ADAMTS13 (rADAMTS13) could increase the safety of tPA thrombolysis in stroke. METHODS: We examined blood-brain barrier (BBB) permeability after intraventricular injection of tPA, VWF, and rADAMTS13 in nonischemic mice. We investigated the role of rADAMTS13 on reducing tPA-induced BBB dysfunction and cerebral hemorrhage in a mouse stroke model. RESULTS: Intraventricular injection of tPA or VWF under nonischemic conditions resulted in a significant increase in BBB permeability. In contrast, rADAMTS13 blocked both tPA- and VWF-induced BBB opening. BBB disruption following stroke was exacerbated by intravenous administration of tPA, but this was attenuated by injection of rADAMTS13. Correspondingly, tPA-associated hemorrhage after stroke was significantly reduced by rADAMTS13. The antihemorrhagic effect of rADAMTS13 was reversed by injection of recombinant VWF. We also showed that rADAMTS13 inhibited tPA-mediated upregulation of vascular endothelial growth factor (VEGF) in vascular endothelium after stroke. The upregulation of VEGF was suppressed by either an Akt inhibitor wortmannin or a Rho kinase inhibitor fasudil. Furthermore, rADAMTS13 downregulated tPA-induced phosphorylation of Akt and activation of RhoA. INTERPRETATION: These findings demonstrate that the VWF-cleaving protease rADAMTS13 reduced tPA-induced hemorrhage by regulating BBB integrity, and suggest that this effect may occur through the Akt/RhoA-mediated VEGF pathways.

PET imaging of serotonin transporters with 4-[18F]-ADAM in a Parkinsonian rat model.

This study was undertaken to address the effects of fetal mesencephalic tissue transplantation on the serotonin system in a rat model of Parkinson's disease (PD) while also investigating the usefulness of 4-[18F]-ADAM (a serotonin transporter imaging agent) coupled with micro-PET for imaging serotonin transporters (SERTs). A PD model was induced by unilateral injection of 6-hydroxydopamine (6-OHDA) into the right medial forebrain bundle of the nigrostriatal pathway, while cell transplantation was performed via intrastriatal injection of mesencephalic brain tissue dissected from embryonic (E14) rats. The 4-[18F]-ADAM/micro-PET scanning was performed following both 6-OHDA lesioning and transplantation. Immunohistochemistry (IHC) studies were also performed following the final PET scan, and the results were compared to show a 17-43% decrease in the specific uptake ratio (SUR) and a 23-52% decrease in serotonin transporter immunoreactivity (SERT-ir) within various brain regions on the lesioned side. The number of methamphetamine-induced rotations also decreased significantly at the 4th week postgraft. In addition, striatal SUR and the SERT-ir levels were restored to 77% and 83% 5 weeks postgraft. These results suggest that Parkinson's disease also affects the serotonergic system, while both the dopaminergic and serotonergic systems can be partially restored in a rat model of PD after E14 mesencephalic tissue transplantation. In addition, we have also determined that 4-[18F]-ADAM/micro-PET can be used to detect serotonergic neuron loss, monitor the progress of Parkinson's disease, and oversee the effectiveness of therapy.

Aß leads to Ca²âº signaling alterations and transcriptional changes in glial cells.

The pathogenesis of Alzheimer's disease includes accumulation of toxic amyloid beta (Aß) peptides. A recently developed cell-permeable peptide, termed Tat-Pro, disrupts the complex between synapse-associated protein 97 (SAP97) and the α-secretase a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), thereby leading to an alteration of the trafficking of the enzyme, which is important for nonamyloidogenic processing of amyloid precursor protein (APP). We report that Tat-Pro treatment, as well as the treatment with exogenous Aß, deregulates Ca(2+) homeostasis specifically in astrocytes through increased expression of key components of Ca(2+) signaling, metabotropic glutamate receptor-5 and inositol 1,4,5-trisphosphate receptor-1. This is accompanied by potentiation of (S)-3,5-dihydroxyphenylglycine-induced Ca(2+) transients. Calcineurin inhibition reverts all these effects. Furthermore, our data demonstrate that astrocytes express all the components for the amyloidogenic and nonamyloidogenic processing of APP including APP itself, beta-site APP-cleaving enzyme 1 (BACE1), ADAM10, γ-secretase, and SAP97. Indeed, treatment with Tat-Pro for 48 hours significantly increased the amount of Aß(1-42) in the medium of cultured astrocytes. Taken together, our results suggest that astroglia might be active players in Aß production and indicate that the calcium hypothesis of Alzheimer's disease may recognize glial cells as important intermediates.