Kenji Matsushita

Nitric Oxide Regulates Exocytosis by S-Nitrosylation of N-ethylmaleimide-Sensitive Factor

Nitric oxide (NO) inhibits vascular inflammation, but the molecular basis for its anti-inflammatory properties is unknown. We show that NO inhibits exocytosis of Weibel-Palade bodies, endothelial granules that mediate vascular inflammation and thrombosis, by regulating the activity of N-ethylmaleimide-sensitive factor (NSF). NO inhibits NSF disassembly of soluble NSF attachment protein receptor (SNARE) complexes by nitrosylating critical cysteine residues of NSF. NO may regulate exocytosis in a variety of physiological processes, including vascular inflammation, neurotransmission, thrombosis, and cytotoxic T lymphocyte cell killing.

Side Population Cells Isolated from Porcine Dental Pulp Tissue with Self‐Renewal and Multipotency for Dentinogenesis, Chondrogenesis, Adipogenesis, and Neurogenesis

Dental pulp has the potential to form dentin as a regenerative response to caries. This regeneration is mediated by stem/progenitor cells. Thus, stem cell therapy might be of potential utility in induction of reparative dentin. We isolated side population (SP) cells from dental pulp based on the exclusion of the DNA binding dye Hoechst 33342 by flow cytometry and compared its self‐renewal capacities and multipotency with non‐SP cells and primary pulp cells. The cumulative cell number of the SP cells was greater than the non‐SP cells and primary pulp cells. Bmi1 was continuously expressed in SP cells, suggesting longer proliferative lifespan and self‐renewal capacity of SP cells. Next, the maintenance of the multilineage differentiation potential of pulp SP cells was investigated. Expression of type II collagen and aggrecan confirmed chondrogenic conversion (30%) of SP cells. SP cells expressed peroxisome proliferator‐activated receptor γ and adaptor protein 2, showing adipogenic conversion. Expression of mRNA and proteins of neurofilament and neuromodulin confirmed neurogenic conversion (90%). These results demonstrate that pulp SP cells maintain multilineage differentiation potential. We further examined whether bone morphogenetic protein 2 (BMP2) could induce differentiation of pulp SP cells into odontoblasts. BMP2 stimulated the expression of dentin sialophosphoprotein (Dspp) and enamelysin in three‐dimensional pellet cultures. Autogenous transplantation of the Bmp2‐supplemented SP cells on the amputated pulp stimulated the reparative dentin formation. Thus, adult pulp contains SP cells, which are enriched for stem cell properties and useful for cell therapy with BMP2 for dentin regeneration.

A Novel Stem Cell Source for Vasculogenesis in Ischemia: Subfraction of Side Population Cells from Dental Pulp

Cell therapy with stem cells and endothelial progenitor cells (EPCs) to stimulate vasculogenesis as a potential treatment for ischemic disease is an exciting area of research in regenerative medicine. EPCs are present in bone marrow, peripheral blood, and adipose tissue. Autologous EPCs, however, are obtained by invasive biopsy, a potentially painful procedure. An alternative approach is proposed in this investigation. Permanent and deciduous pulp tissue is easily available from teeth after extraction without ethical issues and has potential for clinical use. We isolated a highly vasculogenic subfraction of side population (SP) cells based on CD31 and CD146, from dental pulp. The CD31−;CD146− SP cells, demonstrating CD34+ and vascular endothelial growth factor‐2 (VEGFR2)/Flk1+, were similar to EPCs. These cells were distinct from the hematopoietic lineage as CD11b, CD14, and CD45 mRNA were not expressed. They showed high proliferation and migration activities and multilineage differentiation potential including vasculogenic potential. In models of mouse hind limb ischemia, local transplantation of this subfraction of SP cells resulted in successful engraftment and an increase in the blood flow including high density of capillary formation. The transplanted cells were in proximity of the newly formed vasculature and expressed several proangiogenic factors, such as VEGF‐A, G‐CSF, GM‐CSF, and MMP3. Conditioned medium from this subfraction showed the mitogenic and antiapoptotic activity on human umbilical vein endothelial cells. In conclusion, subfraction of SP cells from dental pulp is a new stem cell source for cell‐based therapy to stimulate angiogenesis/vasculogenesis during tissue regeneration.

Regeneration of dental pulp after pulpotomy by transplantation of CD31-/CD146- side population cells from a canine tooth

AIM To achieve complete regeneration of dental pulp in vivo by stem/progenitor cells obtained from a fraction of side population (SP) cells from canine pulp. MATERIALS & METHODS A subfraction of SP cells, CD31(-)/CD146(-) SP cells, were isolated by flow cytometry from canine dental pulp. The efficiency of this subfraction of SP cells was evaluated in an experimental model of pulp injury in the dog. RESULTS The fractionated SP cells formed extensive networks of tube-like structures in vitro. Transplantation of the SP cells into an in vivo model of amputated pulp resulted in complete regeneration of pulp tissue with capillaries and neuronal cells within 14 days. Gene-expression studies demonstrated the expression of pro-angiogenic factors, implying trophic action on endothelial cells. CONCLUSIONS This investigation demonstrates the potential utility of fractionated SP cells as a source of cells for total pulp regeneration complete with angiogenesis and vasculogenesis.

A Novel Combinatorial Therapy With Pulp Stem Cells and Granulocyte Colony-Stimulating Factor for Total Pulp Regeneration

Treatment of deep caries with pulpitis is a major challenge in dentistry. Stem cell therapy represents a potential strategy to regenerate the dentin‐pulp complex, enabling conservation and restoration of teeth. The objective of this study was to assess the efficacy and safety of pulp stem cell transplantation as a prelude for the impending clinical trials. Clinical‐grade pulp stem cells were isolated and expanded according to good manufacturing practice conditions. The absence of contamination, abnormalities/aberrations in karyotype, and tumor formation after transplantation in an immunodeficient mouse ensured excellent quality control. After autologous transplantation of pulp stem cells with granulocyte‐colony stimulating factor (G‐CSF) in a dog pulpectomized tooth, regenerated pulp tissue including vasculature and innervation completely filled in the root canal, and regenerated dentin was formed in the coronal part and prevented microleakage up to day 180. Transplantation of pulp stem cells with G‐CSF yielded a significantly larger amount of regenerated dentin‐pulp complex compared with transplantation of G‐CSF or stem cells alone. Also noteworthy was the reduction in the number of inflammatory cells and apoptotic cells and the significant increase in neurite outgrowth compared with results without G‐CSF. The transplanted stem cells expressed angiogenic/neurotrophic factors. It is significant that G‐CSF together with conditioned medium of pulp stem cells stimulated cell migration and neurite outgrowth, prevented cell death, and promoted immunosuppression in vitro. Furthermore, there was no evidence of toxicity or adverse events. In conclusion, the combinatorial trophic effects of pulp stem cells and G‐CSF are of immediate utility for pulp/dentin regeneration, demonstrating the prerequisites of safety and efficacy critical for clinical applications.

HMG-CoA Reductase Inhibitors Inhibit Endothelial Exocytosis and Decrease Myocardial Infarct Size

Three-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors protect the vasculature from inflammation and atherosclerosis by cholesterol dependent and cholesterol independent mechanisms. We hypothesized that HMG-CoA reductase inhibitors decrease exocytosis of Weibel-Palade bodies, endothelial cell granules whose contents promote thrombosis and vascular inflammation. We pretreated human aortic endothelial cells with simvastatin for 24 hours, then stimulated the cells with thrombin, and measured the amount of vWF released into the media. We then measured the effect of simvastatin on myocardial infarction in mice. Simvastatin decreased thrombin-stimulated Weibel-Palade body exocytosis by 89%. Simvastatin inhibited exocytosis in part by increasing synthesis of nitric oxide (NO), which S-nitrosylated N-ethylmaleimide sensitive factor (NSF), a critical regulator of exocytosis. Simvastatin treatment attenuated myocardial infarct size by 58% in wild-type but not eNOS knockout mice. Furthermore, simvastatin decreased endothelial exocytosis and neutrophil infiltration into ischemic-reperfused myocardium, which was mediated in part by P-selectin contained in Weibel-Palade bodies. However, simvastatin did not affect exocytosis and inflammation in myocardial infarcts of eNOS knockout mice. Inhibition of endothelial exocytosis is a novel mechanism by which HMG-CoA reductase inhibitors may reduce vascular inflammation, inhibit thrombosis, and protect the ischemic myocardium. These findings may explain part of the pleiotropic effects of statin therapy for patients with cardiovascular disease.

Matrix Metalloproteinase-3 Accelerates Wound Healing following Dental Pulp Injury

Matrix metalloproteinases (MMPs) are implicated in a wide range of physiological and pathological processes, including morphogenesis, wound healing, angiogenesis, inflammation, and cancer. Angiogenesis is essential for reparative dentin formation during pulp wound healing. The mechanism of angiogenesis, however, still remains unclear. We hypothesized that certain MMPs expressed during pulp wound healing may support recovery processes. To address this issue, a rat pulp injury model was established to investigate expression of MMPs during wound healing. Real-time RT-PCR analysis showed that expression MMP-3 and MMP-9 (albeit lower extent) was up-regulated at 24 and 12 hours after pulp injury, respectively, whereas expression of MMP-2 and MMP-14 was not changed. MMP-3 mRNA and protein were localized in endothelial cells and/or endothelial progenitor cells in injured pulp in vivo. In addition, MMP-3 enhanced proliferation, migration, and survival of human umbilical vein endothelial cells in vitro. Furthermore, the topical application of MMP-3 protein on the rat-injured pulp tissue in vivo induced angiogenesis and reparative dentin formation at significantly higher levels compared with controls at 24 and 72 hours after treatment, respectively. Inhibition of endogenous MMP-3 by N-Isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid resulted in untoward wound healing. These results provide suggestive evidence that MMP-3 released from endothelial cells and/or endothelial progenitor cells in injured pulp plays critical roles in angiogenesis and pulp wound healing.

Regulation of MyD88-dependent signaling events by S nitrosylation retards toll-like receptor signal transduction and initiation of acute-phase immune responses.

ABSTRACT Nitric oxide (NO) has been thought to regulate the immune system through S nitrosylation of the transcriptional factor NF-κB. However, regulatory effects of NO on innate immune responses are unclear. Here, we report that NO has a capability to control Toll-like receptor-mediated signaling through S nitrosylation. We found that the adaptor protein MyD88 was primarily S nitrosylated, depending on the presence of endothelial NO synthase (eNOS). S nitrosylation at a particular cysteine residue within the TIR domain of MyD88 resulted in slight reduction of the NF-κB-activating property. This modification could be restored by the antioxidant glutathione. Through S nitrosylation, NO could negatively regulate the multiple steps of MyD88 functioning, including translocation to the cell membrane after LPS stimulation, interaction with TIRAP, binding to TRAF6, and induction of IκBα phosphorylation. Interestingly, glutathione could reversely neutralize such NO-derived effects. We also found that an acute febrile response to LPS was precipitated in eNOS-deficient mice, indicating that eNOS-derived NO exerts an initial suppressive effect on inflammatory processes. Thus, NO has a potential to retard induction of MyD88-dependent signaling events through the reversible and oxidative modification by NO, by which precipitous signaling reactions are relieved. Such an effect may reflect appropriate regulation of the acute-phase inflammatory responses in living organisms.

Pathogen recognition by Toll-like receptor 2 activates Weibel-Palade body exocytosis in human aortic endothelial cells

The endothelial cell-specific granule Weibel-Palade body releases vasoactive substances capable of modulating vascular inflammation. Although innate recognition of pathogens by Toll-like receptors (TLRs) is thought to play a crucial role in promotion of inflammatory responses, the molecular basis for early-phase responses of endothelial cells to bacterial pathogens has not fully been understood. We here report that human aortic endothelial cells respond to bacterial lipoteichoic acid (LTA) and synthetic bacterial lipopeptides, but not lipopolysaccharide or peptidoglycan, to induce Weibel-Palade body exocytosis, accompanied by release or externalization of the storage components von Willebrand factor and P-selectin. LTA could activate rapid Weibel-Palade body exocytosis through a TLR2- and MyD88-dependent mechanism without de novo protein synthesis. This process was at least mediated through MyD88-dependent phosphorylation and activation of phospholipase Cγ. Moreover, LTA activated interleukin-1 receptor-associated kinase-1-dependent delayed exocytosis with de novo protein synthesis and phospholipase Cγ-dependent activation of the NF-κB pathway. Increased TLR2 expression by transfection or interferon-γ treatment increased TLR2-mediated Weibel-Palade body exocytosis, whereas reduced TLR2 expression under laminar flow decreased the response. Thus, we propose a novel role for TLR2 in induction of a primary proinflammatory event in aortic endothelial cells through Weibel-Palade body exocytosis, which may be an important step for linking innate recognition of bacterial pathogens to vascular inflammation.

Neuropeptide Release from Dental Pulp Cells by RgpB via Proteinase-Activated Receptor-2 Signaling

Dental pulp inflammation often results from dissemination of periodontitis caused mostly by Porphyromonas gingivalis infection. Calcitonin gene-related peptide and substance P are proinflammatory neuropeptides that increase in inflamed pulp tissue. To study an involvement of the periodontitis pathogen and neuropeptides in pulp inflammation, we investigated human dental pulp cell neuropeptide release by arginine-specific cysteine protease (RgpB), a cysteine proteinase of P. gingivalis, and participating signaling pathways. RgpB induced neuropeptide release from cultured human pulp cells (HPCs) in a proteolytic activity-dependent manner at a range of 12.5–200 nM. HPCs expressed both mRNA and the products of calcitonin gene-related peptide, substance P, and proteinase-activated receptor-2 (PAR-2) that were also found in dental pulp fibroblast-like cells. The PAR-2 agonists, SLIGKV and trypsin, also induced neuropeptide release from HPCs, and HPC PAR-2 gene knockout by transfection of PAR-2 antisense oligonucleotides inhibited significantly the RgpB-elicited neuropeptide release. These results indicated that RgpB-induced neuropeptide release was dependent on PAR-2 activation. The kinase inhibitor profile on the RgpB-neuropeptide release from HPC revealed a new PAR-2 signaling pathway that was mediated by p38 MAPK and activated transcription factor-2 activation, in addition to the PAR-2-p44/42 p38MAPK and -AP-1 pathway. This new RgpB activity suggests a possible link between periodontitis and pulp inflammation, which may be modulated by neuropeptides released in the lesion.