Inflammation-dependent mechanism of calcification: a new role for macrophage matrix vesicles
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and distribution of collagen, and GAGs were observed. HA accumulated in regions of medial elastin fragmentation, and of early neointimal remodeling by α-SMA+cells, though it was replaced by collagen and non-lamellar elastin in the neointima of longer-term AAAs. Though fibrillar, neointimal elastic matrix was deficient in fibrillin. Conclusions: Our study indicates significant de novo elastin deposition in the neointima of AAAs, but not in the elastin- disrupted media, and that neointimal elastic fiber formation is deficient, likely due to poor fibrillin production by neointimal SMCs. Future work will investigate specific deficiencies/aberrations in elastic matrix assembly by these cells and investigate feasibility of elastogenic induction to overcome these defects towards reinstating AAA-wall elasticity. http://dx.doi.org/10.1016/j.carpath.2013.01.060 Inflammation-dependent mechanism of calcification: a new role for macrophage matrix vesicles Sophie New, Claudia Goettsch, Masanori Aikawa, Julio Meirelles Marchini, Catherine Shanahan, Kevin Croce, Elena Aikawa Brigham and Women's Hospital, Harvard Medical School, King's College, London, UK Purpose: We previously linked macrophages and early calcification/ microcalcification, using molecular imaging. Here we propose a novel mechanism for calcification via macrophage-derived matrix vesicles (MV), as an alternative pathway to a commonly accepted mechanism of osteogenic cell transition. Methods and Results: Near-infrared fluorescence microscopy with a hydroxyapatite- binding imaging agent detected microcalcifications, containing vesicular structures, in human atherosclerotic plaques. Immunogold electron microscopy with CD68 labeling demonstrated the release of macrophage-derived MV (Figure 1 , left panel). S100A9, a novel inducer of inflammation, was co-localized with plaque macrophages and microcalcifications. Annexins were previously implicated in smooth muscle cell-derived MV. Immunogold localized S100A9 and annexin-V in macrophage-MV of human plaques (Figure, right panel). Western blot showed increased S100A9 and annexin-V in stimulated macrophage-MV. Co-IP indicated the S100A9-annexin-V interaction. S100A9 siRNA reduced the number and calcification potential of MV derived from human primary macrophages (pb0.05). ApoE -/- S100A9 -/- mice had less MV in the plasma, and their macrophage-derived MV had reduced calcification potential compared to apoE -/- mice (n=5). Stimulation with 2 mM calcium/3 mM phosphate increased calcification potential of MV from RAW 264.7 macrophage cell line (Pb0.01; n=3). These conditions did not induce apoptosis and thus this effect is not attributed to apoptotic bodies. Conclusions: This study provides the first direct evidence for the role of macrophage- derived MV in microcalcification. S100A9 may play a key role in this process. http://dx.doi.org/10.1016/j.carpath.2013.01.061 Polyethyieneimine siRNA complexes released from coated electro- spun polyethylene terephthalate bypass graft materials facilitate gene silencing in infiltrating primary human aortic smooth muscle cells Christoph S. Nabzdyk a,b , Maggie Chun b , Hunter Oliver-Allen b , Matthew D. Phaneuf c , Saif G. Pathan c , Jin-Oh You d , Leena Pradhan b , Frank W. LoGerfo b a Tufts Medical Center, Department of Surgery, Tufts University School of Medicine, Boston, MA b Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA c Biosurfaces Inc., Ashland, MA d Harvard University, School of Engineering and Applied Sciences, Boston, MA Purpose: Anastomotic intimal hyperplasia (AIH) remains the leading cause of prosthetic arterial graft failure. Use of electrospun PET (ePET) can be an alternative to conventional PET grafts. RNAi is a promising tool to silence genes contributing to AIH, including thrombospondin-2 (TSP-2) previously shown by us to be up- regulated in AIH. Delivery of silencing RNA (siRNA) from ePET graft to the anastomotic sites could potentially mitigate AIH. Methods: ePET was dip-coated with control siRNA alone or complexed with either, cationic polymer polyethyleneimine (PEI) or the lipophilic transfection reagent Lipofectamine RNAiMax for 45 minutes. UV-spectrophotometry was used to quantify siRNA coating on ePET. Control and TSP-2 siRNA-PEI complexes coated ePET samples (5×5 mm 2 ) were placed onto a monolayer of human aortic smooth muscle cells (HAoSMCs) for 24 hours. Confocal microscopy and Q- RTPCR were performed to evaluate cellular infiltration into ePET and gene silencing of TSP-2. Results: Significant amount of siRNA from the coating solution was adsorbed to ePET if complexed with PEI compared to RNAiMax or no transfection reagent (62%±3% vs. 1%±0.075% or 1.0%±0.01%), HAoSMCs migrated into and proliferated on ePET coated with siRNA-PEI complexes. Compared to control siRNA, TSP-2 siRNA uptake in HAoSMC resulted in significant gene silencing of TSP-2 from ePET coated with siRNA-PEI. Conclusions: Coating of ePET with siRNA-PEI complexes is feasible, does not impair HAoSMCs in-growth and facilitates TSP-2 gene silencing in HAoSMC in vitro. This clinically feasible approach for delivery of target gene siRNAs may help prevent AIH and subsequent graft failure. http://dx.doi.org/10.1016/j.carpath.2013.01.062 Abstracts / Cardiovascular Pathology 22 (2013) e29–e52 e46
Transcript of Inflammation-dependent mechanism of calcification: a new role for macrophage matrix vesicles
anddistribution of collagen, andGAGswere observed. HA accumulatedin regions of medial elastin fragmentation, and of early neointimalremodeling by α-SMA+cells, though it was replaced by collagen andnon-lamellar elastin in the neointima of longer-term AAAs. Thoughfibrillar, neointimal elastic matrix was deficient in fibrillin.Conclusions: Our study indicates significant de novo elastindeposition in the neointima of AAAs, but not in the elastin-disrupted media, and that neointimal elastic fiber formation isdeficient, likely due to poor fibrillin production by neointimal SMCs.Future work will investigate specific deficiencies/aberrations inelastic matrix assembly by these cells and investigate feasibility ofelastogenic induction to overcome these defects towards reinstatingAAA-wall elasticity.
http://dx.doi.org/10.1016/j.carpath.2013.01.060
Inflammation-dependent mechanism of calcification: a new rolefor macrophage matrix vesiclesSophieNew, ClaudiaGoettsch,Masanori Aikawa, JulioMeirellesMarchini,Catherine Shanahan, Kevin Croce, Elena AikawaBrigham and Women's Hospital, Harvard Medical School, King's College,London, UK
Purpose: We previously linked macrophages and early calcification/microcalcification, using molecular imaging. Here we propose a novelmechanism for calcification via macrophage-derived matrix vesicles(MV), as an alternative pathway to a commonly accepted mechanismof osteogenic cell transition.Methods and Results: Near-infrared fluorescence microscopy with ahydroxyapatite- binding imaging agent detected microcalcifications,containing vesicular structures, in human atherosclerotic plaques.Immunogold electron microscopy with CD68 labeling demonstratedthe release of macrophage-derived MV (Figure 1, left panel). S100A9,a novel inducer of inflammation, was co-localized with plaquemacrophages and microcalcifications. Annexins were previouslyimplicated in smooth muscle cell-derived MV. Immunogold localizedS100A9 and annexin-V in macrophage-MV of human plaques(Figure, right panel). Western blot showed increased S100A9 andannexin-V in stimulated macrophage-MV. Co-IP indicated theS100A9-annexin-V interaction. S100A9 siRNA reduced the numberand calcification potential of MV derived from human primarymacrophages (pb0.05). ApoE−/− S100A9−/− mice had less MV in theplasma, and their macrophage-derived MV had reduced calcificationpotential compared to apoE−/− mice (n=5). Stimulation with 2 mMcalcium/3 mM phosphate increased calcification potential of MVfrom RAW 264.7 macrophage cell line (Pb0.01; n=3). Theseconditions did not induce apoptosis and thus this effect is notattributed to apoptotic bodies.
Conclusions: This study provides the first direct evidence for therole of macrophage- derived MV in microcalcification. S100A9 mayplay a key role in this process.
http://dx.doi.org/10.1016/j.carpath.2013.01.061
Polyethyieneimine siRNA complexes released from coated electro-spun polyethylene terephthalate bypass graft materials facilitategene silencing in infiltrating primary human aortic smoothmuscle cellsChristoph S. Nabzdyka,b, Maggie Chunb, Hunter Oliver-Allenb,Matthew D. Phaneufc, Saif G. Pathanc, Jin-Oh Youd, Leena Pradhanb,Frank W. LoGerfobaTufts Medical Center, Department of Surgery, Tufts University School ofMedicine, Boston, MAbBeth Israel DeaconessMedical Center, HarvardMedical School, Boston,MAcBiosurfaces Inc., Ashland, MAdHarvard University, School of Engineering and Applied Sciences, Boston, MA
Purpose: Anastomotic intimal hyperplasia (AIH) remains the leadingcause of prosthetic arterial graft failure. Use of electrospun PET(ePET) can be an alternative to conventional PET grafts. RNAi is apromising tool to silence genes contributing to AIH, includingthrombospondin-2 (TSP-2) previously shown by us to be up-regulated in AIH. Delivery of silencing RNA (siRNA) from ePET graftto the anastomotic sites could potentially mitigate AIH.Methods: ePET was dip-coated with control siRNA alone orcomplexed with either, cationic polymer polyethyleneimine (PEI) orthe lipophilic transfection reagent Lipofectamine RNAiMax for 45minutes. UV-spectrophotometry was used to quantify siRNA coatingon ePET. Control and TSP-2 siRNA-PEI complexes coated ePET samples(5×5 mm2) were placed onto a monolayer of human aortic smoothmuscle cells (HAoSMCs) for 24 hours. Confocal microscopy and Q-RTPCR were performed to evaluate cellular infiltration into ePET andgene silencing of TSP-2.Results: Significant amount of siRNA from the coating solution wasadsorbed to ePET if complexed with PEI compared to RNAiMax or notransfection reagent (62%±3% vs. 1%±0.075% or 1.0%±0.01%),HAoSMCs migrated into and proliferated on ePET coated withsiRNA-PEI complexes. Compared to control siRNA, TSP-2 siRNAuptake in HAoSMC resulted in significant gene silencing of TSP-2from ePET coated with siRNA-PEI.Conclusions: Coating of ePET with siRNA-PEI complexes is feasible,doesnot impairHAoSMCs in-growthand facilitates TSP-2 gene silencinginHAoSMC invitro. This clinically feasible approach fordeliveryof targetgene siRNAs may help prevent AIH and subsequent graft failure.
http://dx.doi.org/10.1016/j.carpath.2013.01.062
Abstracts / Cardiovascular Pathology 22 (2013) e29–e52e46
