miR-146b-5p downregulates IRAK1 and ADAM19 to suppress trophoblast proliferation, invasion, and migration in miscarriage†.

A large proportion of miscarriages are classified as unexplained miscarriages since no cause is identified. No reliable biomarkers or treatments are available for these pregnancy losses. While our transcriptomic sequencing has revealed substantial upregulation of miR-146b-5p in unexplained miscarriage villous tissues, its role and associated molecular processes have yet to be fully characterized. Our work revealed that relative to samples from normal pregnancy, miR-146b-5p was significantly elevated in villous tissues from unexplained miscarriage patients and displayed promising diagnostic potential. Moreover, miR-146b-5p agomir contributed to higher rates of embryonic resorption in ICR mice. When overexpressed in HTR-8/SVneo cells, miR-146b-5p attenuated the proliferative, invasive, and migratory activity of these cells while suppressing the expression of MMP9 and immune inflammation-associated cytokines, including IL1B, IL11, CXCL1, CXCL8, and CXCL12. Conversely, inhibition of its expression enhanced proliferation, migration, and invasion abilities. Mechanistically, IL-1 receptor-associated kinase-1 and a disintegrin and metalloproteinase 19 were identified as miR-146b-5p targets regulating trophoblast function, and silencing IL-1 receptor-associated kinase-1 had similar effects as miR-146b-5p overexpression, while IL-1 receptor-associated kinase-1 overexpression could partially reverse the inhibitory impact of this microRNA on trophoblasts. miR-146b-5p may inhibit trophoblast proliferation, migration, invasion, and implantation-associated inflammation by downregulating IL-1 receptor-associated kinase-1 and a disintegrin and metalloproteinase 19, participating in the pathogenesis of miscarriage and providing a critical biomarker and a promising therapeutic target for unexplained miscarriage.

rJararacin, a recombinant disintegrin from Bothrops jararaca venom: Exploring its effects on hemostasis and thrombosis.

Integrins are a family of heterodimeric transmembrane receptors which link the extracellular matrix to the cell cytoskeleton. These receptors play a role in many cellular processes: adhesion, proliferation, migration, apoptosis, and platelet aggregation, thus modulating a wide range of scenarios in health and disease. Therefore, integrins have been the target of new antithrombotic drugs. Disintegrins from snake venoms are recognized by the ability to modulate the activity of integrins, such as integrin αIIbß3, a fundamental platelet glycoprotein, and αvß3 expressed on tumor cells. For this reason, disintegrins are unique and potential tools for examining integrin-matrix interaction and the development of novel antithrombotic agents. The present study aims to obtain the recombinant form of jararacin and evaluate the secondary structure and its effects on hemostasis and thrombosis. rJararacin was expressed in the Pichia pastoris (P. pastoris) expression system and purified the recombinant protein with a yield of 40 mg/L of culture. The molecular mass (7722 Da) and internal sequence were confirmed by mass spectrometry. Structure and folding analysis were obtained by Circular Dichroism and 1H Nuclear Magnetic Resonance spectra. Disintegrin structure reveals properly folded with the presence of ß-sheet structure. rJararacin significantly demonstrated inhibition of the adhesion of B16F10 cells and platelets to the fibronectin matrix under static conditions. rJararacin inhibited platelet aggregation induced by ADP (IC50 95 nM), collagen (IC50 57 nM), and thrombin (IC50 22 nM) in a dose-dependent manner. This disintegrin also inhibited 81% and 94% of the adhesion of platelets to fibrinogen and collagen under continuous flow, respectively. In addition, rjararacin efficaciously prevents platelet aggregation in vitro and ex vivo with rat platelets and thrombus occlusion at an effective dose (5 mg/kg). The data here provides evidence that rjararacin possesses the potential as an αIIbß3 antagonist, capable of preventing arterial thrombosis.

Disintegrin-like Protein Strategy to Inhibit Aggressive Triple-Negative Breast Cancer.

Venoms are a rich source of bioactive compounds, and among them is leberagin-C (Leb-C), a disintegrin-like protein derived from the venom of Macrovipera lebetina transmediterrannea snakes. Leb-C has shown promising inhibitory effects on platelet aggregation. Previous studies have demonstrated that this SECD protein specifically targets α5ß1, αvß3, and αvß6 integrins through a mimic mechanism of RGD disintegrins. In our current study, we focused on exploring the potential effects of Leb-C on metastatic breast cancer. Our findings revealed that Leb-C disrupted the adhesion, migration, and invasion capabilities of MDA-MB-231 breast cancer cells and its highly metastatic D3H2LN sub-population. Additionally, we observed significant suppression of adhesion, migration, and invasion of human umbilical vein endothelial cells (HUVECs). Furthermore, Leb-C demonstrated a strong inhibitory effect on fibroblast-growth-factor-2-induced proliferation of HUVEC. We conducted in vivo experiments using nude mice and found that treatment with 2 µM of Leb-C resulted in a remarkable 73% reduction in D3H2LN xenograft tumor size. Additionally, quantification of intratumor microvessels revealed a 50% reduction in tumor angiogenesis in xenograft after 21 days of twice-weekly treatment with 2 µM of Leb-C. Collectively, these findings suggest the potential utility of this disintegrin-like protein for inhibiting aggressive and resistant metastatic breast cancer.

CC5 and CC8, Two Disintegrin Isoforms from Cerastes cerastes Snake Venom Decreased Inflammation Response In Vitro and In Vivo.

Inflammation is associated with many pathology disorders and the malignant progression of most cancers. Therefore, targeting inflammatory pathways could provide a promising strategy for disease prevention and treatment. In this study, we experimentally investigated the anti-inflammatory effect of CC5 and CC8, two disintegrin isoforms isolated from Cerastes cerastes snake venom, on LPS-stimulated macrophages, both on human THP-1 and mouse RAW264.7 cell adherence and their underlying mechanisms by measuring cytokine release levels and Western blot assay. Equally, both molecules were evaluated on a carrageenan-induced edema rat model. Our findings suggest that CC5 and CC8 were able to reduce adhesion of LPS-stimulated macrophages both on human THP-1 and mouse RAW264.7 cells to fibrinogen and vitronectin through the interaction with the αvß3 integrin receptor. Moreover, CC5 and CC8 reduced the levels of reactive oxygen species (ROS) mediated by the NF-κB, MAPK and AKT signaling pathways that lead to decreased production of the pro-inflammatory cytokines TNF-α, IL-6 and IL-8 and increased secretion of IL-10 in LPS-stimulated THP-1 and RAW264.7 cells. Interestingly, both molecules potently exhibited an anti-inflammatory effect in vivo by reducing paw swelling in rats. In light of these results, we can propose the CC5 and CC8 disintegrins as interesting tools to design potential candidates against inflammatory-related diseases.

A Disintegrin and Metalloproteinase with Thrombospondin Motifs 4 Regulates Pulmonary Vascular Hyperpermeability through Destruction of Glycocalyx in Acute Respiratory Distress Syndrome.

Acute respiratory distress syndrome (ARDS) has no specific and effective treatment, and there is an urgent need to understand its pathogenesis. Therefore, based on the hypothesis that molecules whose expression is upregulated in injured pulmonary vascular endothelial cells (VECs) are involved in the pathogenesis of ARDS, we conducted a study to elucidate the molecular mechanisms and identify target factors for treatment. Primary human lung microvascular endothelial cells (HMVEC-Ls) were stimulated with lipopolysaccharide (LPS) or poly (I:C) and analyzed via a microarray to identify target genes for ARDS. We found that a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) was induced in murine lung VECs in an LPS-mediated ARDS model. Elevated ADAMTS4 was also observed by the immunostaining of lung samples from ARDS patients. The suppression of ADAMTS4 by siRNA in VECs ameliorated LPS-stimulated vascular permeability. The impairment of the cell surface expression of syndecan-1, a marker of the glycocalyx that is an extracellular matrix involved in vascular permeability, was dramatically inhibited by ADAMTS4 suppression. In addition, the suppression of ADAMTS4 protected against LPS-induced reductions in syndecan-1 and the adherens junction protein vascular endothelial cadherin. These results suggest that ADAMTS4 regulates VEC permeability in ARDS and may be a predictive marker and therapeutic target for ARDS.

Nuclear Paraspeckle Assembly Transcript 1 Enhances Hydrogen Peroxide-Induced Human Vascular Smooth Muscle Cell Injury by Regulating miR-30d-5p/A Disintegrin and Metalloprotease 10.

BACKGROUND: Nuclear paraspeckle assembly transcript 1 (NEAT1) has been reported to be involved in the progression of many cancers; however, the role and mechanisms underlying NEAT1 in abdominal aortic aneurysm (AAA) remain unclear.Methods and Results: The expression of NEAT1, miR-30d-5p and A disintegrin and metalloprotease 10 (ADAM10) was measured by qRT-PCR and western blot. Functional experiments were conducted by using a CCK-8 assay, EDU assay, flow cytometry, western blot, ELISA, and commercial kits. The target relation was confirmed by dual-luciferase reporter assay and the RIP assay. It was then found that NEAT1 was upregulated in peripheral blood of AAA patients ~3.46-fold, smooth muscle cells (SMCs) isolated from AAA tissues ~2.6-fold and in a hydrogen peroxide (H2O2)-induced injury model of human vascular SMC (HVSMCs) ~2.0- and 3.9-fold at 50 µmol/L and 200 µmol/L H2O2treatment, respectively. NEAT1 deletion attenuated H2O2-induced cell proliferation promotion (40.0% vs. 74.3%), apoptosis inhibition (25.0% vs. 13.5%), and reduction of inflammatory response and oxidative stress in HVSMCs. Mechanistically, NEAT1 targeted miR-30d-5p to prevent the degradation of its target, ADAM10, in HVSMCs. Further rescue experiments suggested miR-30d-5p inhibition mitigated the effects of NEAT1 deletion on H2O2-induced HVSMCs. Moreover, ADAM10 overexpression counteracted the inhibitory functions of miR-30d-5p on H2O2-evoked HVSMC injury. CONCLUSIONS: NEAT1 promoted H2O2-induced HVSMC injury by inducing cell apoptosis, inflammation and oxidative stress through miR-30d-5p/ADAM10 axis, indicating the possible involvement of NEAT1 in the pathogenesis of AAA.

Novel disintegrin-like peptides derived from an amphibian skin cDNA sequence of Hypsiboas punctatus.

Disintegrins comprise a family of small proteins that bind to and alter the physiological function of integrins, especially integrins that mediate platelet aggregation in blood. Here, we report a lysine-glycine-aspartic acid (KGD) disintegrin-like motif present in a 15-amino acid residue peptide identified in a cDNA library of the amphibian Hypsiboas punctatus skin. The original peptide sequence was used as a template from which five new analogs were designed, chemically synthesized by solid phase, and tested for disintegrin activity and tridimensional structural studies using NMR spectroscopy. The original amphibian peptide had no effect on integrin-mediated responses. Nevertheless, derived peptide analogs inhibited integrin-mediated platelet function, including platelet spreading on fibrinogen.

The Effects of αvß3 Integrin Blockage in Breast Tumor and Endothelial Cells under Hypoxia In Vitro.

Breast cancer is characterized by a hypoxic microenvironment inside the tumor mass, contributing to cell metastatic behavior. Hypoxia induces the expression of hypoxia-inducible factor (HIF-1α), a transcription factor for genes involved in angiogenesis and metastatic behavior, including the vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMPs), and integrins. Integrin receptors play a key role in cell adhesion and migration, being considered targets for metastasis prevention. We investigated the migratory behavior of hypoxia-cultured triple-negative breast cancer cells (TNBC) and endothelial cells (HUVEC) upon αvß3 integrin blocking with DisBa-01, an RGD disintegrin with high affinity to this integrin. Boyden chamber, HUVEC transmigration, and wound healing assays in the presence of DisBa-01 were performed in hypoxic conditions. DisBa-01 produced similar effects in the two oxygen conditions in the Boyden chamber and transmigration assays. In the wound healing assay, hypoxia abolished DisBa-01's inhibitory effect on cell motility and decreased the MMP-9 activity of conditioned media. These results indicate that αvß3 integrin function in cell motility depends on the assay and oxygen levels, and higher inhibitor concentrations may be necessary to achieve the same inhibitory effect as in normoxia. These versatile responses add more complexity to the role of the αvß3 integrin during tumor progression.

Crotalus atrox disintegrin reduces hemorrhagic transformation by attenuating matrix metalloproteinase-9 activity after middle cerebral artery occlusion in hyperglycemic male rats.

Hemorrhagic transformation after ischemic stroke is an independent predictor for poor outcome and is characterized by blood vessel rupture leading to brain edema. To date, no therapies for preventing hemorrhagic transformation exist. Disintegrins from the venom of Crotalus atrox have targets within the coagulation cascade, including receptors on platelets. We hypothesized that disintegrins from C. atrox venom can attenuate hemorrhagic transformation by preventing activation of matrix metalloproteinase after middle cerebral artery occlusion (MCAO) in hyperglycemic rats. We subjected 48 male Sprague-Dawley rats weighing 240-260 g to MCAO and hyperglycemia to induce hemorrhagic transformation of the infarction. At reperfusion, we administered either saline (vehicle), whole C. atrox venom (two doses were used), or fractionated C. atrox venom (HPLC Fraction 2). Rats were euthanized 24 hr post-ictus for measurement of infarction and hemoglobin volume. Reversed-phase HPLC was performed to fractionate the whole venom and peaks were combined to form Fraction 2, which contained the disintegrin Crotatroxin. Fraction 2 protected against hemorrhagic transformation after MCAO, and attenuated activation of matrix metalloproteinase-9. Administering matrix metalloproteinase antagonists prevented the protection by Fraction 2. The results of this study indicate that disintegrins found in C. atrox venom may have therapeutic potential for reducing hemorrhagic transformation after ischemic stroke. Moreover, the RP-HPLC fractions retained sufficient protein activity to suggest that gentler and less efficient orthogonal chromatographic methods may be unnecessary to isolate proteins and explore their function.

Fc-saxatilin inhibits VEGF-induced permeability by regulating claudin-5 expression in human brain microvascular endothelial cells.

The disruption of the blood-brain barrier influences the degree of brain damage and prognosis in cerebral ischemia or other brain diseases accompanied by inflammation. Vascular endothelial growth factor (VEGF) released during brain ischemia or inflammation has been implicated in the breakdown of the blood-brain barrier by increasing endothelial permeability. Saxatilin, a disintegrin-containing RGD motif, has been reported to disaggregate platelets via interactions with platelet integrins and to have a thrombolysis effect. Additionally, the Fc-saxatilin fusion protein reduces vascular leakage in cerebral ischemia in mice. In this study, we show that Fc-saxatilin prevents VEGF-induced permeability in human brain microvascular endothelial cells (HBMECs). The activation of Src and Fak, downstream signaling proteins of VEGF in the induction of endothelial permeability, was inhibited by Fc-saxatilin in HBMECs. The downregulation of a tight junction protein, claudin-5, at the protein and mRNA levels by VEGF was recovered by Fc-saxatilin. Our findings suggest that Fc-saxatilin attenuates VEGF-induced endothelial permeability via the regulation of downstream signaling, and this may contribute to its protective effect against vascular leakage in the ischemic brain.

Recombinant RGD-disintegrin DisBa-01 blocks integrin αvß3 and impairs VEGF signaling in endothelial cells.

BACKGROUND: Integrins mediate cell adhesion, migration, and survival by connecting the intracellular machinery with the surrounding extracellular matrix. Previous studies demonstrated the interaction between αvß3 integrin and VEGF type 2 receptor (VEGFR2) in VEGF-induced angiogenesis. DisBa-01, a recombinant His-tag fusion, RGD-disintegrin from Bothrops alternatus snake venom, binds to αvß3 integrin with nanomolar affinity blocking cell adhesion to the extracellular matrix. Here we present in vitro evidence of a direct interference of DisBa-01 with αvß3/VEGFR2 cross-talk and its downstream pathways. METHODS: Human umbilical vein (HUVECs) were cultured in plates coated with fibronectin (FN) or vitronectin (VN) and tested for migration, invasion and proliferation assays in the presence of VEGF, DisBa-01 (1000 nM) or VEGF and DisBa-01 simultaneously. Phosphorylation of αvß3/VEGFR2 receptors and the activation of intracellular signaling pathways were analyzed by western blotting. Morphological alterations were observed and quantified by fluorescence confocal microscopy. RESULTS: DisBa-01 treatment of endothelial cells inhibited critical steps of VEGF-mediated angiogenesis such as migration, invasion and tubulogenesis. The blockage of αvß3/VEGFR2 cross-talk by this disintegrin decreases protein expression and phosphorylation of VEGFR2 and ß3 integrin subunit, regulates FAK/SrC/Paxillin downstream signals, and inhibits ERK1/2 and PI3K pathways. These events result in actin re-organization and inhibition of HUVEC migration and adhesion. Labelled-DisBa-01 colocalizes with αvß3 integrin and VEGFR2 in treated cells. CONCLUSIONS: Disintegrin inhibition of αvß3 integrin blocks VEGFR2 signalling, even in the presence of VEGF, which impairs the angiogenic mechanism. These results improve our understanding concerning the mechanisms of pharmacological inhibition of angiogenesis.

Dimeric Disintegrins from the Steppe Viper V. ursinii Venom.

Four dimeric disintegrins were isolated from the venom of the steppe viper V. ursinii using liquid chromatography. Disintegrins prevented adhesion of MCF7 cells to fibronectin, which indicates their interaction with integrin receptors of the αVß1 type. According to mass spectrometry data, the molar masses of disintegrins are about 14 kDa. The method of peptide mapping established the structure of a new heterodimeric disintegrin weighing 13 995.5 Da and shows that it belongs to the class of RGD/KGD-containing disintegrins.

Isolation and characterization of an anti-leishmanial disintegrin from Cerastes cerastes venom.

Investigating new antimicrobial and antiparasitic components from Viperidae venoms represents an alternative therapeutic strategy. In this study, we report the characterization of a disintegrin isolated from Cerastes cerastes venom, exhibiting antiparasitic activity on Leishmania infantum promastigotes. Indeed, isolated disintegrin, referred to Disintegrin_Cc, induced 84.75% of parasiticidal activity and deep morphological alterations on the parasites. SDS-PAGE analysis indicated that this disintegrin was homogenous. This dimeric disintegrin of 14,193.97 Da contains an RGD domain and four intramolecular disulfide bridges. It presents a high percentage of identity with other related snake disintegrins. Predicted 3D structure indicated that this peptide shares partial homology with well-known active antimicrobial peptides. Disintegrin_Cc inhibited 80% of arachidonic acid-induced platelet aggregation. The obtained results suggest that the isolated molecule plays a dual role as a disintegrin and as an anti-leishmanial compound. This component could be useful as a drug in the treatment of leishmaniasis.

Comprehensive Snake Venomics of the Okinawa Habu Pit Viper, Protobothrops flavoviridis, by Complementary Mass Spectrometry-Guided Approaches.

The Asian world is home to a multitude of venomous and dangerous snakes, which are used to induce various medical effects in the preparation of traditional snake tinctures and alcoholics, like the Japanese snake wine, named Habushu. The aim of this work was to perform the first quantitative proteomic analysis of the Protobothrops flavoviridis pit viper venom. Accordingly, the venom was analyzed by complimentary bottom-up and top-down mass spectrometry techniques. The mass spectrometry-based snake venomics approach revealed that more than half of the venom is composed of different phospholipases A2 (PLA2). The combination of this approach and an intact mass profiling led to the identification of the three main Habu PLA2s. Furthermore, nearly one-third of the total venom consists of snake venom metalloproteinases and disintegrins, and several minor represented toxin families were detected: C-type lectin-like proteins (CTL), cysteine-rich secretory proteins (CRISP), snake venom serine proteases (svSP), l-amino acid oxidases (LAAO), phosphodiesterase (PDE) and 5'-nucleotidase. Finally, the venom of P. flavoviridis contains certain bradykinin-potentiating peptides and related peptides, like the svMP inhibitors, pEKW, pEQW, pEEW and pENW. In preliminary MTT cytotoxicity assays, the highest cancerous-cytotoxicity of crude venom was measured against human neuroblastoma SH-SY5Y cells and shows disintegrin-like effects in some fractions.

Synergistic suppression of a disintegrin acurhagin-C in combination with AZD4547 and reparixin on terminating development for human osteosarcoma MG-63 cell.

Current therapies available for the treatment of human osteosarcoma, an aggressive bone tumor, are insufficient. To examine an alternative approach of integrin-based anti-osteosacoma strategy, acurhagin-C, a Glu-Cys-Asp (ECD)-disintegrin, was isolated and evaluated for its application in combination with two potent inhibitors of basic fibroblast growth factor (bFGF) and interleukin-8 (IL-8). The investigation of human osteosarcoma MG-63 cells pre-incubated with a FGF receptor-1 (FGFR-1) blocker AZD4547, a CXC-chemokine receptor-1/-2 (CXCR1/2) antagonist reparixin, and acurhagin-C via two given modes of separation and combination was executed. Detected by flow cytometry, integrins-α2/-α5/-αv/-ß1, FGFR-1, CXCR1 and CXCR2 constitutively express on the resting membrane. However, bFGF/IL-8-activated MG-63 cells only statistically enhanced the surface exposure of integrins-α5/-ß1, FGFR-1 and CXCR2. In activated MG-63 cells, acurhagin-C targeting integrin-α5 not only might potentiate the inhibitory effect of AZD4547 and reparixin on the surface expression of integrin-α5, FGFR-1 and CXCR2, but also acurhagin-C used alone remained effectively to diminish the surface exposure of those targeted receptors. Hence, a complicated crosstalk mechanism should be involved in the membrane interactions. Furthermore, co-administration of acurhagin-C with AZD4547 and reparixin also showed to have the synergistic suppression toward cell proliferation and the gene expression of matrix metalloproteinase-2. Also, the administration of three-in-one mode could nearly abrogate the cellular attachment onto collagen-IV- and fibronectin-coated wells, as well as penetration into Matrigel-barrier. These data supported an ECD-disintegrin acurhagin-C targeting integrin-α5 upon combined used with AZD4547 and reparixin may become a promising therapeutic approach for attenuating osteosarcoma development.

Disintegrins from the Venom of Vipera ammodytes ammodytes Efficiently Inhibit Migration of Breast Cancer Cells.

Integrins are plasma membrane proteins, whose dysfunction frequently results in cancer pathology, and therefore they represent important targets of anti-tumour therapy. Snake venoms are a rich source of disintegrins (Dis), proteins that specifically bind integrins and thus interfere with their functions. In an attempt to discover new molecules for treatment of breast cancer, the major type of cancer in women, we isolated a dimeric Dis (Vaa-Dis) from the venom of the nosehorned viper. By cell viability testing we demonstrated that 50 nM and higher concentrations of Vaa-Dis were toxic to highly invasive human breast adenocarcinoma cell line MDA-MB-231. Wound-healing assay revealed that already at one order of magnitude lower concentrations Vaa-Dis efficiently inhibited MDA-MB-231 cell migration. This exposed a promising anti-metastatic potential of Vaa-Dis and a good perspective of these natural snake venom proteins for further research and development towards the application in breast cancer treatment.

Hepatocytes contribute to immune regulation in the liver by activation of the Notch signaling pathway in T cells.

The "liver tolerance effect" has been attributed to a unique potential of liver-resident nonprofessional APCs including hepatocytes (HCs) to suppress T cell responses. The exact molecular mechanism of T cell suppression by liver APCs is still largely unknown. In mice, IL-10-dependent T cell suppression is observed after Th1-mediated hepatitis induced by Con A. In this study, we show that HCs, particularly those from regenerating livers of Con A-pretreated mice, induced a regulatory phenotype in naive CD4(+) T cells in vitro. Using reporter mice, we observed that these T regulatory cells released substantial amounts of IL-10, produced IFN-γ, failed to express Foxp3, but suppressed proliferation of responder T cells upon restimulation with anti-CD3 mAb. Hence, these regulatory cells feature a similar phenotype as the recently described IL-10-producing Th1 cells, which are generated upon activation of Notch signaling. Indeed, inhibition of γ-secretase and a disintegrin and metalloproteinase 17 but not a disintegrin and metalloproteinase 10, respectively, which blocked Notch activation, prevented IL-10 secretion. HCs from Con A-pretreated mice showed enhanced expression of the Notch ligand Jagged1 and significantly increased receptor density of Notch1 on CD4(+) T cells. However, HCs from Con A-pretreated IFN regulatory factor 1(-/-) mice, which cannot respond to IFN-γ, as well as those from IFN-γ(-/-) mice failed to augment IL-10 production by CD4(+) T cells. In conclusion, it seems that HCs fine-tune liver inflammation by upregulation of Jagged1 and activation of Notch signaling in Th1 cells. This mechanism might be of particular importance in the regenerating liver subsequent to Th1-mediated hepatitis.

IL-1ß-induced matrix metalloproteinase-13 is activated by a disintegrin and metalloprotease-28-regulated proliferation of human osteoblast-like cells.

We reported previously that matrix metalloproteinase (MMP)-13 accelerates bone remodeling in oral periradicular lesions, and indicated a potentially unique role for MMP-13 in wound healing and regeneration of alveolar bone. The ADAM (a disintegrin and metalloprotease) family is a set of multifunctional cell surface and secreted glycoproteins, of which ADAM-28 has been localized in bone and bone-like tissues. In this study, we show that interleukin (IL)-1ß induces the expression of MMP-13 and ADAM-28 in homogeneous α7 integrin-positive human skeletal muscle stem cell (α7(+)hSMSC)-derived osteoblast-like (α7(+)hSMSC-OB) cells, and promotes proliferation while inhibiting apoptosis in these cells. At higher concentrations, however, IL-1ß failed to induce the expression of these genes and caused an increase in apoptosis. We further employed ADAM-28 small interfering RNA (siRNA) to investigate whether IL-1ß-induced MMP-13 expression is linked to this IL-1ß-mediated changes in cell proliferation and apoptosis. Silencing ADAM-28 expression potently suppressed IL-1ß-induced MMP-13 expression and activity, decreased cell proliferation and increased apoptosis in α7(+)hSMSC-OB cells. In contrast, MMP-13 siRNA had no effect on ADAM-28 expression, suggesting ADAM-28 regulates MMP-13. Exogenous MMP-13 induced α7(+)hSMSC-OB cell proliferation and could rescue ADAM-28 siRNA-induced apoptosis, and we found that proMMP-13 is partially cleaved into its active form by ADAM-28 in vitro. Overall, our results suggest that IL-1ß-induced MMP-13 expression and changes in cell proliferation and apoptosis in α7(+)hSMSC-OB cells are regulated by ADAM-28.

Multimeric disintegrin protein polymer fusions that target tumor vasculature.

Recombinant protein therapeutics have increased in number and frequency since the introduction of human insulin, 25 years ago. Presently, proteins and peptides are commonly used in the clinic. However, the incorporation of peptides into clinically approved nanomedicines has been limited. Reasons for this include the challenges of decorating pharmaceutical-grade nanoparticles with proteins by a process that is robust, scalable, and cost-effective. As an alternative to covalent bioconjugation between a protein and nanoparticle, we report that biologically active proteins may themselves mediate the formation of small multimers through steric stabilization by large protein polymers. Unlike multistep purification and bioconjugation, this approach is completed during biosynthesis. As proof-of-principle, the disintegrin protein called vicrostatin (VCN) was fused to an elastin-like polypeptide (A192). A significant fraction of fusion proteins self-assembled into multimers with a hydrodynamic radius of 15.9 nm. The A192-VCN fusion proteins compete specifically for cell-surface integrins on human umbilical vein endothelial cells (HUVECs) and two breast cancer cell lines, MDA-MB-231 and MDA-MB-435. Confocal microscopy revealed that, unlike linear RGD-containing protein polymers, the disintegrin fusion protein undergoes rapid cellular internalization. To explore their potential clinical applications, fusion proteins were characterized using small animal positron emission tomography (microPET). Passive tumor accumulation was observed for control protein polymers; however, the tumor accumulation of A192-VCN was saturable, which is consistent with integrin-mediated binding. The fusion of a protein polymer and disintegrin results in a higher intratumoral contrast compared to free VCN or A192 alone. Given the diversity of disintegrin proteins with specificity for various cell-surface integrins, disintegrin fusions are a new source of biomaterials with potential diagnostic and therapeutic applications.

FS145, the first flea-derived disintegrin, inhibits angiogenesis through specifically binding integrin αvß3.

FS145, a protein containing a WGD motif, was previously described from the salivary transcriptome of the flea Xenopsylla cheopis. Nevertheless, its biological function and complete structure are still uncertain. Herein, FS145 was confirmed to adopt a common αßß structure with the WGD motif exposed on its surface and located right at the top of a loop composed of residues 72-81. Furthermore, FS145 dose-dependently inhibited the proliferation, adhesion, migration, and tube formation of HUVECs by not only binding to integrin αvß3 but also by subsequently inactivating the FAK/Src/MAPK pathway along with the reduction of the expression of MMP-2, MMP-9, VEGFA, bFGF, Ang2, Tie2, HIF-1α, and FAK. Moreover, FS145 also inhibited aortic vessel sprout and showed strong anti-angiogenic activities as assessed ex vivo, by employing the rat aortic ring assay, chick embryo chorioallantoic membrane, and zebrafish embryo models. Altogether, our results suggest that FS145 suppresses angiogenesis ex vivo and in vitro by blocking integrin αvß3. The current study reveals the first anti-angiogenesis disintegrin with WGD motif from invertebrates and provides a beneficial pharmacological activity to inhibit abnormal angiogenesis.