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1.
Cell Physiol Biochem ; 41(6): 2319-2332, 2017.
Article in English | MEDLINE | ID: mdl-28463834

ABSTRACT

BACKGROUND/AIMS: Platelet microvesicles (PMVs) contribute to angiogenesis and vasculogenesis, but the mechanisms underlying these contributions have not been fully elucidated. In the present study, we investigated whether PMVs regulate the angiogenic properties of endothelial cells (ECs) via mechanisms extending beyond the transport of angiogenic regulators from platelets. METHODS: In vitro Matrigel tube formation assay and in vivo Matrigel plug assay were used to evaluate the pro-angiogenic activity of PMVs. The effects of PMVs on the migration of human umbilical vein endothelial cells (HUVECs) were detected by transwell assay and wound-healing assay. Real-time PCR and western blot were conducted to examine mRNA and protein expression of pro-angiogenic factors in HUVECs. Matrix metalloproteinase (MMP) activity was assayed by gelatin zymography. Moreover, the effects of specific MMP inhibitors were tested. RESULTS: PMVs promoted HUVEC capillary-like network formation in a dose-dependent manner. Meanwhile, PMVs dose-dependently facilitated HUVEC migration. Levels of MMP-2 and MMP-9 expression and activity were up-regulated in HUVECs stimulated with PMVs. Inhibition of MMPs decreased their pro-angiogenic and pro-migratory effects on HUVECs. Moreover, we confirmed the pro-angiogenic activity of PMVs in vivo in mice with subcutaneous implantation of Matrigel, and demonstrated that blockade of MMPs attenuated PMV-induced angiogenesis. CONCLUSION: The findings of our study indicate that PMVs promote angiogenesis by up-regulating MMP expression in ECs via mechanism extending beyond the direct delivery of angiogenic factors.


Subject(s)
Human Umbilical Vein Endothelial Cells/enzymology , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase 9/metabolism , Neovascularization, Physiologic/physiology , Up-Regulation/physiology , Angiogenesis Inhibitors/pharmacology , Blood Platelets/metabolism , Cell Movement/drug effects , Cell Movement/physiology , Cell Proliferation/drug effects , Cell Proliferation/physiology , Dipeptides/pharmacology , Humans , Matrix Metalloproteinase 2/chemistry , Matrix Metalloproteinase 9/chemistry , Matrix Metalloproteinase Inhibitors/pharmacology , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/genetics , Mitogen-Activated Protein Kinase 3/metabolism , Neovascularization, Physiologic/drug effects , Up-Regulation/drug effects
2.
PLoS One ; 11(5): e0156686, 2016.
Article in English | MEDLINE | ID: mdl-27243975

ABSTRACT

Numerous clinical conditions have been linked to ectopic mineralization (EM). This process of pathological biomineralization is complex and not fully elucidated, but thought to be started within matrix vesicles (MVs). We hypothesized that high mobility group box 1 (HMGB1), a cytokine associated with biomineralizing process under physiological and pathological conditions, induces EM via promoting MVs secretion from macrophages. In this study, we found that HMGB1 significantly promoted secretion of MVs from macrophages and subsequently led to mineral deposition in elevated Ca/Pi medium in vitro. Transmission electron microscopy of calcifying MVs showed formation of hydroxyapatite crystals in the vesicle interior. Subcutaneous injection into mice with MVs derived from HMGB1-treated cells showed a greater potential to initiate regional mineralization. Mechanistic experiments revealed that HMGB1 activated neutral sphingomyelinase2 (nSMase2) that involved the receptor for advanced glycation end products (RAGE) and p38 MAPK (upstream of nSMase2). Inhibition of nSMase2 with GW4869 or p38 MAPK with SB-239063 prevented MVs secretion and mineral deposition. Collectively, HMGB1 induces MVs secretion from macrophages at least in part, via the RAGE/p38 MAPK/nSMase2 signaling pathway. Our findings thus reveal a novel mechanism by which HMGB1 induces ectopic mineralization.


Subject(s)
Calcification, Physiologic/physiology , Extracellular Vesicles/metabolism , HMGB1 Protein/metabolism , Macrophages/metabolism , Receptor for Advanced Glycation End Products/metabolism , Sphingomyelin Phosphodiesterase/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Calcification, Physiologic/genetics , Cell Line , Durapatite/chemistry , Enzyme Activation , Imidazoles/pharmacology , Male , Mice , Mice, Inbred C57BL , Microscopy, Electron, Transmission , Pyrimidines/pharmacology , RNA Interference , RNA, Small Interfering/genetics , Receptor for Advanced Glycation End Products/genetics , Sphingomyelin Phosphodiesterase/antagonists & inhibitors , Toll-Like Receptor 2/genetics , Toll-Like Receptor 4/genetics , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors
3.
Thromb Haemost ; 115(3): 632-45, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26632487

ABSTRACT

Pathogens-induced platelet activation contributes to inflammation in cardiovascular diseases, but underlying mechanisms remain elusive. Staphylococcal superantigen-like protein 5 (SSL5) is a known activator of platelets. Here we examined whether SSL5 is implicated in Staphylococcus aureus (S. aureus)-induced inflammation and potential mechanisms involved. As expected, we show that SSL5 activates human platelets and induces generation of platelet microparticles (PMPs). Flow cytometry and scanning electron microscopy studies demonstrate that SSL5-induced PMPs (SSL5-PMPs) bind to monocytes, causing aggregate formation. In addition, SSL5-PMPs provoke monocyte expression and release of inflammatory mediators, including interleukin-1ß (IL-1ß), tumour necrosis factor-α (TNFα), monocyte chemoattractant protein-1 (MCP-1) and matrix metalloproteinase-9 (MMP-9) in a dose- and time-dependent manner. SSL5-PMPs also enhance MCP-1-induced monocyte migration. Blockade of CD40 and CD40 ligand (CD40L) interactions with neutralising antibodies significantly reduce monocyte release of inflammatory mediators and migration induced by SSL5-PMPs. SiRNA-mediated silencing of CD40 or TNF receptor (TNFR)-associated factor 6 (TRAF6) gene largely abrogates phosphorylation and nuclear translocation of NFκB (p65). In conclusion, SSL5 provokes the release of inflammatory mediators in monocytes, at least in part, via PMPs-mediated activation of the CD40/TRAF6/NFκB signalling pathway, though it normally inhibits leukocyte function. Our findings thus reveal a novel mechanism by which S. aureus induces inflammation.


Subject(s)
Bacterial Proteins/metabolism , CD40 Antigens/metabolism , Monocytes/cytology , NF-kappa B p50 Subunit/metabolism , TNF Receptor-Associated Factor 6/metabolism , Active Transport, Cell Nucleus , Blood Platelets/metabolism , Cell Movement , Cell-Derived Microparticles , Chemokine CCL2/metabolism , Flow Cytometry , Gene Silencing , Humans , Inflammation , Interleukin-1beta/metabolism , Intracellular Signaling Peptides and Proteins , Matrix Metalloproteinase 9/metabolism , RNA Interference , RNA, Small Interfering/metabolism , Receptors, Tumor Necrosis Factor/metabolism , Recombinant Proteins/metabolism , Signal Transduction , Staphylococcus aureus , Tumor Necrosis Factor-alpha/metabolism
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