N-acetylglucosamine-bearing polymers mimicking O-GlcNAc-modified proteins elicit anti-fibrotic activities in myofibroblasts and activated stellate cells.

O-linked ß-N-acetylglucosamine (O-GlcNAc)-modified proteins are post-translationally modified with GlcNAc conjugated to serine and threonine residues. This modification is associated with various physiological functions such as serine and threonine phosphorylation and Notch signaling. Here, we demonstrated that O-GlcNAc-modified proteins leaked from dead cells and GlcNAc-bearing polymers mimicking the multivalent GlcNAc moiety of these proteins induced anti-fibrotic activities, such as the suppression of α-smooth muscle actin and collagen and the induction of matrix metalloprotease 1 in myofibroblasts. We have previously reported that O-GlcNAc-modified proteins and GlcNAc-bearing polymers could interact with cell surface vimentin and desmin. In the current study, it was demonstrated that a multivalent GlcNAc moiety structure of these molecules activated PI3K/Akt and p38MAPK pathway and elicited these anti-fibrotic activities in myofibroblasts by interacting with cell surface vimentin. Since the interaction of O-GlcNAc-modified proteins with desmin was observed in the fibrotic liver of carbon tetrachloride-treated mice via an in situ proximity ligation assay, it was assumed that the activated stellate cells could bind to the O-GlcNAc-modified proteins from the damaged hepatocytes. In addition, the administration of anti-O-GlcNAc antibody to inhibit the interaction exacerbated liver fibrosis in the mice. Moreover, administration of the GlcNAc-bearing polymers into carbon tetrachloride-treated mice could ameliorate liver fibrosis. Thus, O-GlcNAc-modified proteins leaked from dead cells can interact with myofibroblasts and activated stellate cells and function as fibrosis suppressors. Moreover, we anticipate that GlcNAc-bearing polymers mimicking O-GlcNAc-modified proteins will be applied as novel therapeutic tools for fibrosis.

Interactions of N-acetyl-D-glucosamine-conjugated silk fibroin with lectins, cytoskeletal proteins and cardiomyocytes.

We have reported that cytoskeletal proteins such as desmin and vimentin are expressed on the surface of muscle, mesenchymal and cancer cells, and possess N-acetyl-ß-D-glucosamine (ß-GlcNAc) residue-binding properties. As cell-recognizable ß-GlcNAc residue-bearing biopolymer, we prepared glycoconjugates (SF-GlcNAc) composed of silk fibroin (SF) and monosaccharide N-acetyl-D-glucosamine (GlcNAc) by chemical modification using cyanuric chloride. The covalent immobilization of GlcNAc into SF was assessed by 1H-NMR measurements. The 1H-NMR spectrum of SF-GlcNAc conjugates showed new peaks attributed to the methyl protons of the N-acetyl group in GlcNAc, and the integration of these peaks revealed that the GlcNAc content in the conjugates was 9 wt%. The existence of ß-GlcNAc residues in SF-GlcNAc was examined by the criteria using lectins such as wheat germ agglutinin (WGA). Addition of WGA to SF-GlcNAc solution caused an increase in the turbidity of the solution due to lectin-mediated aggregation. Solid-phase lectin binding assay based on the biotin-avidin interaction showed that biotinylated succinylated WGA bound more strongly onto SF-GlcNAc conjugate-coated wells compared to SF-coated well. Following the establishment of the existence of ß-GlcNAc residues in SF-GlcNAc, the interaction of SF-GlcNAc with desmin was examined by enzyme-linked immunosorbent assay using anti-desmin antibody. The stronger binding of desmin was observed for SF-GlcNAc conjugate-coated wells compared to SF-coated wells. The use of SF-GlcNAc conjugates as a substrate for culturing desmin-expressing human cardiac myocytes demonstrated an increase in the numbers of attached cells and proliferating cells on the conjugate-coated wells compared to SF-coated wells. These results suggest that the immobilization of monosaccharide GlcNAc is a useful method for the versatile functionalization of SF as an application in tissue engineering.

Development of a Gene Delivery System of Oligonucleotides for Fibroses by Targeting Cell-Surface Vimentin-Expressing Cells with N-Acetylglucosamine-Bearing Polymer-Conjugated Polyethyleneimine.

Targeting myofibroblasts and activated stellate cells in lesion sites of fibrotic tissues is an important approach to treat fibroses. Herein, we focused on targeting the cytoskeletal proteins vimentin, which are reportedly highly expressed on the surface of these cells and have N-acetylglucosamine (GlcNAc)-binding activity. A GlcNAc-bearing polymer synthesized via radical polymerization with a reversible addition-fragmentation chain transfer reagent has been previously found to interact with cell-surface vimentin-expressing cells. We designed a GlcNAc-bearing polymer-conjugated polyethyleneimine (PEI), as the gene carrier to target cell-surface vimentin-expressing cells and specifically deliver nuclear factor-κB decoy oligonucleotides (ODNs) and heat shock protein 47 (HSP47)-small interfering RNA (siRNA) to normal human dermal fibroblasts (NHDFs) that express cell-surface vimentin. The results showed that the expression of tumor necrosis factor-α in lipopolysaccharide-stimulated NHDFs and HSP47 in transforming growth factor-ß1-stimulated NHDFs was suppressed by cellular uptake of the GlcNAc-bearing polymer-conjugated PEI/nuclear factor (NF)-κB decoy ODNs and HSP47-siRNA complexes through cell-surface vimentin, respectively. These findings suggest that the effective and specific delivery of ODNs and siRNA for cell-surface vimentin-expressing cells such as myofibroblasts and activated stellate cells can be achieved using GlcNAc-bearing polymer-conjugated PEI. This therapeutic approach could prove advantageous to prevent the promotion of various fibroses.

Multimeric conformation of type III intermediate filaments but not the filamentous conformation exhibits high affinity to lipid bilayers.

Vimentin, desmin, glial fibrillary acidic protein (GFAP) and peripherin, classified as the type III intermediate filament family, maintain the integrity and architecture of various cell types. Recently, we reported their cell surface expression and binding to multivalent N-acetylglucosamine-conjugated polymers. Furthermore, the presence of vimentin on the surface of various cell types including malignant tumor cells and fibroblasts has been demonstrated. Type III intermediate filament proteins are traditionally considered intracellular proteins and do not possess signal peptides for cell membrane recruitment. Therefore, the mechanism of their transport to the cell surface is unclear. In the current study, we aimed to elucidate this mechanism by focusing on the relationship between their multimeric structure and lipid bilayer affinity. Blue native polyacrylamide gel electrophoresis demonstrated that cell surface-expressed type III intermediate filament proteins formed a multimeric mostly including 4-12-mers but not filamentous structure. Moreover, surface plasmon resonance analysis revealed that the multimeric structure of these recombinant proteins had high affinity to lipid bilayers, whereas their filament-like large multimeric structure did not. Our results suggest that type III intermediate filaments are incorporated into the cell membrane through alteration from a filamentous to a multimeric structure.

Improved Isolation of Mesenchymal Stem Cells Based on Interactions between N-Acetylglucosamine-Bearing Polymers and Cell-Surface Vimentin.

Mesenchymal stem cells (MSCs) in bone marrow and adipose tissues are expected to be effective tools for regenerative medicine to treat various diseases. To obtain MSCs that possess both high differentiation and tissue regenerative potential, it is necessary to establish an isolation system that does not require long-term culture. It has previously been reported that the cytoskeletal protein vimentin, expressed on the surfaces of multiple cell types, possesses N-acetylglucosamine- (GlcNAc-) binding activity. Therefore, we tried to exploit this interaction to efficiently isolate MSCs from rat bone marrow cells using GlcNAc-bearing polymer-coated dishes. Cells isolated by this method were identified as MSCs because they were CD34-, CD45-, and CD11b/c-negative and CD90-, CD29-, CD44-, CD54-, CD73-, and CD105-positive. Osteoblast, adipocyte, and chondrocyte differentiation was observed in these cells. In total, yields of rat MSCs were threefold to fourfold higher using GlcNAc-bearing polymer-coated dishes than yields using conventional tissue-culture dishes. Interestingly, MSCs isolated with GlcNAc-bearing polymer-coated dishes strongly expressed CD106, whereas those isolated with conventional tissue-culture dishes had low CD106 expression. Moreover, senescence-associated ß-galactosidase activity in MSCs from GlcNAc-bearing polymer-coated dishes was lower than that in MSCs from tissue-culture dishes. These results establish an improved isolation method for high-quality MSCs.

Elucidation of GlcNAc-binding properties of type III intermediate filament proteins, using GlcNAc-bearing polymers.

Vimentin, desmin, glial fibrillary acidic protein (GFAP) and peripherin belong to type III intermediate filament family and are expressed in mesenchymal cells, skeletal muscle cells, astrocytes and peripheral neurons, respectively. Vimentin and desmin possess N-acetyl-d-glucosamine (GlcNAc)-binding properties on cell surfaces. The rod II domain of these proteins is a GlcNAc-binding site, which also exists in GFAP and peripherin. However, the GlcNAc-binding activities and behaviors of these proteins remain unclear. Here, we characterized the interaction and binding behaviors of these proteins, using various well-defined GlcNAc-bearing polymers synthesized by radical polymerization with a reversible addition-fragmentation chain transfer reagent. The small GlcNAc-bearing polymers strongly interacted with HeLa cells through vimentin expressed on the cell surface and interacted with vimentin-, desmin-, GFAP- and peripherin-transfected vimentin-deficient HeLa cells. These proteins present high affinity to GlcNAc-bearing polymers, as shown by surface plasmon resonance. These results show that type III intermediate filament proteins possess GlcNAc-binding activities on cell surfaces. These findings provide important insights into novel cellular functions and physiological significance of type III intermediate filaments.

Imaging and therapy of liver fibrosis using bioreducible polyethylenimine/siRNA complexes conjugated with N-acetylglucosamine as a targeting moiety.

Diagnosis and therapy of early stage liver fibrosis is very important for the treatment of fatal liver diseases. Here, we report on the targeted imaging and therapy of activated hepatic stellate cells (HSCs) and fibrotic liver tissue using N-acetylglucosamine (GlcNAc)- and indocyanine green (ICG)-conjugated PEI/siRNA complexes. The conjugation of a disulfide bond to PEI (PEI-D) was achieved by Michael addition. We modified PEI with N-acetylglucosamine (PEI-D-GlcNAc), which can specifically interact with desmin on activated HSCs, using the EDC coupling method. Confocal microscopic analysis showed that the PEI-D-GlcNAc/siRNA was internalized by HSCs upon interaction with surface desmin. In vitro western blot analysis confirmed that PEI-D-GlcNAc provided strong protein knock-down after transfection with TGFß1siRNA into HSCs. After a tail vein injection of ICG-conjugated complexes, the PEI-D-GlcNAc-ICG/siRNA complex accumulated to a greater extent in the livers of fibrotic mice than in normal mice over an extended duration. Moreover, immunohistofluorescence analysis confirmed that the PEI-D-GlcNAc-ICG/siRNA complex specifically colocalized with HSCs, which are desmin-positive cells, in fibrotic liver tissues. In vivo TGFß1siRNA delivery also resulted in superior protein knock-down when using the PEI-D-GlcNAc complex. These results demonstrate that the PEI-D-GlcNAc-ICG/TGFß1siRNA complex is a useful tool for imaging and treatment of liver fibrosis.

Dynamic behaviors of vimentin induced by interaction with GlcNAc molecules.

The cytoskeleton protein vimentin is dramatically altered following pathological events such as fibrosis and tumorigenesis. Vimentin binds to multivalent N-acetylglucosamine (GlcNAc) molecules at the cell surface and interacts with O-linked ß-GlcNAc proteins. Moreover, dying cells can be engulfed by neighboring cells through surface interactions between vimentin and many O-GlcNAc proteins in cell debris. Here, we show that vimentin was altered by its interaction with GlcNAc-bearing molecules such as GlcNAc-bearing polymers. The interaction with GlcNAc-bearing polymers promoted the cell surface recruitment of vimentin followed by the phosphorylation of vimentin serine 71 and the increase in tetrameric vimentin disassembled from vimentin filaments in HeLa cells. Moreover, it was found that GlcNAc-bearing polymers and O-GlcNAc proteins from dying cells promoted vimentin expression and cell migration in the Madin-Darby canine kidney and Michigan Cancer Foundation-7 cells. These results suggest that interactions between surface vimentin and GlcNAc molecules, including the O-GlcNAc proteins from dying cells, may play a pivotal role in vimentin expression and the migration of cancer cells. We propose new mechanisms of vimentin expression in cancer cells.

Preparation and characterization of a VEGF-Fc fusion protein matrix for enhancing HUVEC growth.

To enhance vascularization of hydrophobic implants in vivo, a VEGF-Fc fusion protein consisting of vascular endothelial growth factor (VEGF) fused to the immunoglobulin G Fc domain was prepared as an artificial extracellular matrix (ECM). VEGF-Fc was stably immobilized on a polystyrene plate due to the hydrophobicity of the Fc domain, and significantly enhanced the adhesion of human umbilical vein endothelial cells (HUVECs). Additionally, the use of VEGF-Fc as an ECM markedly promoted the proliferation of HUVECs longer than 72 h and induced the reorganization of actin filaments into larger stress fibers within these cells. The VEGF-Fc fusion protein may be a promising artificial ECM for enhancing endothelial cell growth.

Engulfment and clearance of apoptotic cells based on a GlcNAc-binding lectin-like property of surface vimentin.

The clearance of apoptotic cells is important to maintain tissue homeostasis. The engulfment of apoptotic cells is performed by professional phagocytes, such as macrophages, and also by non-professional phagocytes, such as mesenchymal cells. Here, we show that vimentin, a cytoskeletal protein, functions as an engulfment receptor on neighboring phagocytes, which recognize O-linked ß-N-acetylglucosamine (O-GlcNAc)-modified proteins from apoptotic cells as "eat me" ligands. Previously, we reported that vimentin possesses a GlcNAc-binding lectin-like property on cell surface. However, the physiological relevance of the surface localization and GlcNAc-binding property of vimentin remained unclear. In the present study, we observed that O-GlcNAc proteins from apoptotic cells interacted with the surface vimentin of neighboring phagocytes and that this interaction induced serine 71-phosphorylation and recruitment of vimentin to the cell surface of the neighboring phagocytes. Moreover, tetrameric vimentin that was disassembled by serine 71-phosphorylation possessed a GlcNAc-binding activity and was localized to the cell surface. We demonstrated our findings in vimentin-expressing common cell lines such as HeLa cells. Furthermore, during normal developmental processes, the phagocytic engulfment and clearance of apoptotic footplate cells in mouse embryos was mediated by the interaction of surface vimentin with O-GlcNAc proteins. Our results suggest a common mechanism for the clearance of apoptotic cells, through the interaction of surface vimentin with O-GlcNAc-modified proteins.

Interactions of vimentin- or desmin-expressing liver cells with N-acetylglucosamine-bearing polymers.

It is necessary to develop highly functionalized liver cell culture systems for liver tissue engineering such as bioartificial livers and liver cell chips. To maintain a high level of hepatocyte function, well-organized patterning culture systems of hepatocytes and nonparenchymal cells would be advantageous. To design the patterning culture system using these cells, cell-recognizable polymers should be useful to regulate not only the hepatocytes, but also the nonparenchymal cells. Here, we report that N-acetylglucosamine (GlcNAc)-bearing polymers are useful as nonparenchymal cell-recognizable polymers. It has previously been reported that mesenchymal cells adhered to GlcNAc-bearing polymer-coated dishes through surface vimentin. It was also observed that nonparenchymal cells expressing vimentin or desmin specifically adhered to GlcNAc-bearing polymer-coated dishes. Especially, in hepatic stellate cells (HSCs) cultured on GlcNAc-bearing polymer-coated dishes, the expression of α-smooth muscle actin as an activated HSCs marker was suppressed in long-term. Therefore, HSCs were shown to maintain a quiescent state on PVGlcNAc-coated dishes during a long-term culture. These results demonstrated that GlcNAc-bearing polymers could be beneficial to culture nonparenchymal cells such as HSCs. Our findings suggest that galactose- and GlcNAc-bearing polymers can regulate the culture of all liver cells and may be useful tools for the establishment of liver tissue engineering.

Cardiac differentiation of embryonic stem cells by substrate immobilization of insulin-like growth factor binding protein 4 with elastin-like polypeptides.

The establishment of cardiomyocyte differentiation of embryonic stem cells (ESCs) is a useful strategy for cardiovascular regenerative medicine. Here, we report a strategy for cardiomyocyte differentiation of ESCs using substrate immobilization of insulin-like growth factor binding protein 4 (IGFBP4) with elastin-like polypeptides. Recently, IGFBP4 was reported to promote cardiomyocyte differentiation of ESCs through inhibition of the Wnt/ß-catenin signaling. However, high amounts of IGFBP4 (approximately 1 µg/mL) were required to inhibit the Wnt/ß-catenin signaling and induce differentiation to cardiomyocytes. We report herein induction of cardiomyocyte differentiation using IGFBP4-immobilized substrates. IGFBP4-immobilized substrates were created by fusion with elastin-like polypeptides. IGFBP4 was stably immobilized to polystyrene dishes through fusion of elastin-like polypeptides. Cardiomyocyte differentiation of ESCs was effectively promoted by strong and continuous inhibition of Wnt/ß-catenin signaling with IGFBP4-immobilized substrates. These results demonstrated that IGFBP4 could be immobilized using fusion of elastin-like polypeptides. Our results also demonstrate that substrate immobilization of IGFBP4 is a powerful tool for differentiation of ESCs into cardiomyocytes. These findings suggest that substrate immobilization of soluble factors is a useful technique for differentiation of ESCs in regenerative medicine and tissue engineering.

Targeting N-acetylglucosamine-bearing polymer-coated liposomes to vascular smooth muscle cells.

The targeted delivery of anti-inflammatory agents has great therapeutic potential for treating restenosis following percutaneous coronary intervention. To develop a drug delivery system targeted to injured blood vessels, we examined whether N-acetylglucosamine (GlcNAc)-bearing polymer-coated liposomes (GlcNAc-Ls) are specifically taken up by vascular smooth muscle cells (VSMCs). Flow cytometric analysis revealed that GlcNAc-Ls were taken up by VSMCs in vitro. Furthermore, GlcNAc-Ls were intravenously administered to mice that had undergone wire-mediated vascular injury. GlcNAc-Ls markedly accumulated at the intramural site of the injured vessel walls but not at the contralateral (uninjured) vessel walls. These results demonstrated that GlcNAc-Ls can be specifically taken up by VSMCs both in vitro and in vivo. We propose a novel strategy of using GlcNAc-Ls that has potential for application in drug delivery targeted to injured blood vessels.

Gene delivery system based on highly specific recognition of surface-vimentin with N-acetylglucosamine immobilized polyethylenimine.

Gene and drug-delivery systems that use immobilization of carbohydrates are useful for the specific targeting of lectin-expressing tissues. Here, we report that N-acetylglucosamine (GlcNAc) with polyethylenimine (GlcNAc-PEI) specifically interacted with vimentin-expressing cells such as 293FT and HeLa cells. Recently, the intermediate filaments vimentin and desmin have been reported to have GlcNAc-binding lectin-like properties on the cell surface. Therefore, GlcNAc-conjugated agents can be targeted to vimentin- and desmin-expressing cells and tissues. Vimentin-expressing 293FT and HeLa cells were efficiently transfected with green fluorescent protein and luciferase genes by using GlcNAc-PEI; the expression of these genes in vimentin-knockdown cells were low. Confocal microscopic analysis showed that GlcNAc-PEI complexes interacted with vimentin on the cell surface of HeLa cells. These results demonstrate that GlcNAc-PEI/DNA complexes were specifically taken up by 293FT and HeLa cells via vimentin. We suggest that this gene-delivery system could be used to target various vimentin-expressing cells such as fibroblasts and tumor cells.

Vimentin and desmin possess GlcNAc-binding lectin-like properties on cell surfaces.

Vimentin and desmin are intermediate filament proteins found in various mesenchymal and skeletal muscle cells, respectively. These proteins play an important role in the stabilization of the cytoplasmic architecture. Here, we found, using artificial biomimicking glycopolymers, that vimentin and desmin possess N-acetylglucosamine (GlcNAc)-binding lectin-like properties on the cell surfaces of various vimentin- and desmin-expressing cells such as cardiomyocytes and vascular smooth muscle cells. The rod II domain of these proteins was demonstrated to be localized to the cell surface and to directly bind to the artificial biomimicking GlcNAc-bearing polymer, by confocal laser microscopy and surface plasmon resonance analysis. These glycopolymers strongly interact with lectins and are useful tools for the analysis of lectin-carbohydrate interactions, since glycopolymers binding to lectins can induce the clustering of lectins due to multivalent glycoside ligand binding. Moreover, immunocytochemistry and pull-down assay with His-tagged vimentin-rod II domain protein showed that the vimentin-rod II domain interacts with O-GlcNAc proteins. These results suggest that O-GlcNAc proteins might be one candidate for physiological GlcNAc-bearing ligands with which vimentin and desmin interact. These findings demonstrate a novel function of vimentin and desmin that does not involve stabilization of the cytoplasmic architecture by which these proteins interact with physiological GlcNAc-bearing ligands such as O-GlcNAc proteins on the cell surface through their GlcNAc-binding lectin-like properties.

Potential feasibility of early bone marrow cell injection into the spleen for creating functional hepatocytes.

BACKGROUND: Bone marrow cells (BMCs) are believed to have the ability to generate functional hepatocytes and have some merits as a therapeutic modality for metabolic liver diseases. However, the appearance of BMC-derived hepatocytes (BMDHs) is low. We hypothesized that early BMC injection would be feasible for creating BMDHs for two main reasons: (1) the liver is a hematopoietic organ in neonatal rats and (2) it may allow sufficient time to generate more BMDHs before severe liver injury occurs. METHODS: We used Long Evans Cinnamon (LEC) rats as recipients, a model of (1) Wilson disease and (2) liver carcinogenesis. Green fluorescent protein-expressing BMCs were injected into newborn LEC rats through the spleen. The oxidative activity of ceruloplasmin, which is low in LEC rats, was measured to evaluate the treatment. In addition, we performed fluorescence in situ hybridization to clarify the origin of the BMDHs and immunohistochemical analysis to confirm whether these BMDHs had malignant potential. RESULTS: At 18 months after injection, we found some green fluorescent protein-expressing areas macroscopically in the liver of treated LEC rats. The oxidative activity of ceruloplasmin increased in treated LEC rats (n=7) and were much higher than that in untreated LEC rats (P=0.015). Moreover, we confirmed that the BMDHs were generated by cell fusion and was not detected in any of the neoplastic lesions or cholngiofibrotic regions. CONCLUSION: Our results suggest that this novel strategy for creating BMDHs is effective and safe.

Bone marrow CXCR4 induction by cultivation enhances therapeutic angiogenesis.

AIMS: The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor (CXCR4, CXC chemokine receptor 4) play a critical role in the process of post-natal neovascularization. Here, we investigated the role of CXCR4(+) bone marrow cells (BMCs) in neovascularization in a murine hindlimb ischaemia model. METHODS AND RESULTS: We found that the expression of CXCR4 in BMCs was specifically upregulated by cultivation; therefore, we used freshly isolated BMCs and cultivated BMCs, designated as BMC(Fr) and BMC(Cul), respectively. The increased CXCR4 expression corresponded to the migratory capacity in response to SDF-1 alpha. Real-time reverse transcription-polymerase chain reaction and immunohistochemical analyses revealed that SDF-1 alpha expression was significantly increased in the ischaemic limbs of mice. Blood flow perfusion and capillary density were significantly accelerated in mice implanted with BMC(Cul) as compared with those in mice implanted with BMC(Fr). The stimulatory effect of BMC(Cul) on neovascularization was significantly impaired when BMC(Cul) derived from CXCR4(+/-) mice were implanted. The implanted BMC(Cul) showed high retention in the ischaemic limbs. Further, the implantation of BMC(Cul) significantly increased the expression of interleukin (IL)-1 beta and vascular endothelial growth factor-A in the ischaemic limbs. CONCLUSION: The upregulation of CXCR4 expression by cultivation may serve as a useful source of BMCs for accelerating therapeutic angiogenesis in ischaemic cardiovascular diseases.

Surface coating of bone marrow cells with N-acetylglucosamine for bone marrow implantation therapy.

Bone marrow implantation (BMI) has been performed clinically for the treatment of ischemic cardiovascular diseases. To achieve BMI effectively, accumulation of many bone marrow cells (BMCs) in an infarcted area of the myocardium is important. Previously, we reported that cardiomyocytes show strong interaction with N-acetylglucosamine (GlcNAc) and they can take up GlcNAc-conjugated liposomes. Thus, we examined whether GlcNAc-coated BMCs exhibit strong interaction with cardiomyocytes. The cell surface of BMCs was coated with GlcNAc without causing cell injury by GlcNAc-lipophilic polymers. It was found that the GlcNAc-coated BMCs exhibited strong interaction with cardiomyocytes. At 7 days of coculturing the GlcNAc-coated BMCs with cardiomyocytes, BMC-derived cardiomyocytes were generated. The number of BMC-derived cardiomyocytes was higher following coculture with GlcNAc-coated BMCs than following coculture with uncoated and maltose (MA)-coated BMCs. In this study, we demonstrated that the surface coating of BMCs with GlcNAc can be performed easily by using GlcNAc-lipophilic polymers and that GlcNAc-coated BMCs exhibited strong interaction with cardiomyocytes. Therefore, we think that cell surface coating with GlcNAc would help promote accumulation of BMCs in the infarcted area of the myocardium and that this accumulation would be helpful in the treatment of ischemic cardiovascular diseases with BMI.

Deficiency of tumour necrosis factor-alpha and interferon-gamma in bone marrow cells synergistically inhibits neointimal formation following vascular injury.

AIMS: Neointimal formation after percutaneous coronary intervention (PCI), termed restenosis, limits therapeutic revascularization. Since it is now known that vascular injury involves an inflammatory response, we examined the role of tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) in the neointimal formation after injury. METHODS AND RESULTS: Control (BALB/c), TNF-alpha-deficient (Tnf(-/-)), IFN-gamma-deficient (Ifng(-/-)), or double-deficient (Tnf(-/-)Ifng(-/-)) mice were subjected to wire-mediated vascular injury of the right femoral artery. Neointimal formation after injury was significantly reduced after the injury in the Tnf(-/-)Ifng(-/-) mice, compared to that in the control, Tnf(-/-), and Ifng(-/-) mice. Immunohistochemical analysis showed that TNF-alpha and IFN-gamma were expressed in neointimal lesions in the control mice, but not in mice with deficiency of the corresponding cytokine. No significant difference in re-endothelialization was observed among these groups. The number of proliferating cell nuclear antigen in the neointimal lesions was significantly decreased in the Tnf(-/-)Ifng(-/-) mice. Bone marrow transplantation experiments revealed that deficiency of TNF-alpha and IFN-gamma specifically in bone marrow cells significantly inhibited neointimal formation after vascular injury. CONCLUSION: The absence of TNF-alpha and IFN-gamma in bone marrow cells synergistically inhibits neointimal formation following vascular injury, and thus, may provide new insights into the mechanisms underlying restenosis after PCI.

Critical role of bone marrow apoptosis-associated speck-like protein, an inflammasome adaptor molecule, in neointimal formation after vascular injury in mice.

BACKGROUND: Inflammatory cytokines such as interleukin (IL)-1 beta and IL-18 play an important role in the development of atherosclerosis and restenosis. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is an adaptor protein that regulates caspase-1-dependent IL-1 beta and IL-18 generation; however, the role of ASC in vascular injury remains undefined. Here, we investigated the contribution of ASC to neointimal formation after vascular injury in ASC-deficient (ASC(-/-)) mice. METHODS AND RESULTS: Wire-mediated vascular injury was produced in the femoral artery of ASC(-/-) and wild-type mice. Immunohistochemical analysis revealed that ASC was markedly expressed at the site of vascular injury. Neointimal formation was significantly attenuated in ASC(-/-) mice after injury. IL-1 beta and IL-18 were expressed in the neointimal lesion in wild-type mice but showed decreased expression in the lesion of ASC(-/-) mice. To investigate the contribution of bone marrow-derived cells, we developed bone marrow-transplanted mice and found that neointimal formation was significantly decreased in wild-type mice in which bone marrow was replaced with ASC(-/-) bone marrow cells. Furthermore, in vitro experiments showed that the proliferation activity of ASC(-/-) vascular smooth muscle cells was not impaired. CONCLUSIONS: These findings suggest that bone marrow-derived ASC is critical for neointimal formation after vascular injury and identify ASC as a novel therapeutic target for atherosclerosis and restenosis.