Takeshi Into

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.

Stimulation of human Toll-like receptor (TLR) 2 and TLR6 with membrane lipoproteins of Mycoplasma fermentans induces apoptotic cell death after NF-κB activation

Mycoplasmal membrane diacylated lipoproteins not only initiate proinflammatory responses through Toll‐like receptor (TLR) 2 and TLR6 via the activation of the transcriptional factor NF‐κB, but also initiate apoptotic responses. The aim of this study was to clarify the apoptotic machineries. Mycoplasma fermentans lipoproteins and a synthetic lipopeptide, MALP‐2, showed cytocidal activity towards HEK293 cells transfected with a TLR2‐encoding plasmid. The activity was synergically augmented by co‐expression of TLR6, but not by co‐expression of other TLRs. Under the condition of co‐expression of TLR2 and TLR6, the lipoproteins could induce maximum NF‐κB activation and apoptotic cell death in the cells 6 h and 24 h after stimulation respectively. Dominant‐negative forms of MyD88 and FADD, but not IRAK‐4, reduced the cytocidal activity of the lipoproteins. In addition, both dominant‐negative forms also downregulated the activation of both NF‐κB and caspase‐8 in the cells. Additionally, the cytocidal activity was sufficiently attenuated by a selective inhibitor of p38 MAPK. These findings suggest that mycoplasmal lipoproteins can trigger TLR2‐ and TLR6‐mediated sequential bifurcate responses: NF‐κB activation as an early event, which is partially mediated by MyD88 and FADD; and apoptosis as a later event, which is regulated by p38 MAPK as well as by MyD88 and FADD.

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.

Involvement of Leucine Residues at Positions 107, 112, and 115 in a Leucine-Rich Repeat Motif of Human Toll-Like Receptor 2 in the Recognition of Diacylated Lipoproteins and Lipopeptides and Staphylococcus aureus Peptidoglycans

S-(2,3-bispalmitoyloxypropyl)Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe (FSL-1) derived from Mycoplasma salivarium stimulated NF-κB reporter activity in human embryonic kidney 293 (HEK293) cells transfected with Toll-like receptor 2 (TLR2) or cotransfected with TLR2 and TLR6, but not in HEK293 cells transfected with TLR6, in a dose-dependent manner. The activity was significantly higher in HEK293 cells transfected with both TLR2 and TLR6 than in HEK293 cells transfected with only TLR2. The deletion mutant TLR2ΔS40-I64 (a TLR2 mutant with a deletion of the region of Ser40 to Ile64) failed to activate NF-κB in response to FSL-1. The deletion mutant TLR2ΔC30-S39 induced NF-κB reporter activity, but the level of activity was significantly reduced compared with that induced by wild-type TLR2. A TLR2 point mutant with a substitution of Glu178 to Ala (TLR2E178A), TLR2E180A, TLR2E190A, and TLR2L132E induced NF-κB activation when stimulated with FSL-1, M. salivarium lipoproteins, and Staphylococcus aureus peptidoglycans, but TLR2L107E, TLR2L112E (a TLR2 point mutant with a substitution of Leu112 to Glu), and TLR2L115E failed to induce NF-κB activation, suggesting that these residues are essential for their signaling. Flow cytometric analysis demonstrated that TLR2L115E, TLR2L112E, and TLR2ΔS40-I64 were expressed on the cell surface of the transfectants as wild-type TLR2 and TLR2E190A were. In addition, these mutants, except for TLR2E180A, functioned as dominant negative form of TLR2. This study strongly suggested that the extracellular region of Ser40-Ile64 and leucine residues at positions 107, 112, and 115 in a leucine-rich repeat motif of TLR2 are involved in the recognition of mycoplasmal diacylated lipoproteins and lipopeptides and in the recognition of S. aureus peptidoglycans.

Autophagy in regulation of Toll-like receptor signaling.

Toll-like receptors (TLRs) serve as the major innate immune sensors for detection of specific molecular patterns on various pathogens. TLRs activate signaling events mainly by utilizing ubiquitin-dependent mechanisms. Recent research advances have provided evidence that TLR signaling is linked to induction of autophagy. Autophagy is currently known to affect both of the immune defense and suppression of inflammatory responses. In TLR-associated immune responses, autophagic lysis of intracellular microbes (called xenophagy) contributes to the former mechanism, while the latter seems to be mediated by the control of the mitochondrial integrity or selective autophagic clearance of aggregated signaling proteins (called aggrephagy). Several autophagy-related ubiquitin-binding proteins, such as SQSTM1/p62 and NDP52, mediate xenophagy and aggrephagy. In this review, we summarize the expanded knowledge regarding TLR signaling and autophagy signaling. After that, we will focus on autophagy-associated signaling downstream of TLRs and the effect of autophagy on TLR signaling, thus highlighting the signaling crosstalk between the TLR-associated innate immune responses and the regulation of innate immunity by xenophagy and aggrephagy.

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.

Regulation of MyD88 Aggregation and the MyD88-dependent Signaling Pathway by Sequestosome 1 and Histone Deacetylase 6

MyD88 is an essential adaptor molecule for Toll-like receptors (TLRs) and interleukin (IL)-1 receptor. MyD88 is thought to be present as condensed forms or aggregated structures in the cytoplasm, although the reason has not yet been clear. Here, we show that endogenous MyD88 is present as small speckle-like condensed structures, formation of which depends on MyD88 dimerization. In addition, formation of large aggregated structures is related to cytoplasmic accumulation of sequestosome 1 (SQSTM1; also known as p62) and histone deacetylase 6 (HDAC6), which are involved in accumulation of polyubiquitinated proteins. A gene knockdown study revealed that SQSTM1 and HDAC6 were required for MyD88 aggregation and exhibited a suppressive effect on TLR ligand-induced expression of IL-6 and NOS2 in RAW264.7 cells. SQSTM1 and HDAC6 were partially involved in suppression of several TLR4-mediated signaling events, including activation of p38 and JNK, but they hardly affected degradation of IκBα (inhibitor of nuclear factor κB). Biochemical induction of MyD88 oligomerization induced recruitment of SQSTM1 and HDAC6 to the MyD88-TRAF6 signaling complex. Repression of SQSTM1 and HDAC6 enhanced formation of the MyD88-TRAF6 complex and conversely decreased interaction of the ubiquitin-specific negative regulator CYLD with the complex. Furthermore, ubiquitin-binding regions on SQSTM1 and HDAC6 were essential for MyD88 aggregation but were not required for interaction with the MyD88 complex. Thus, our study reveals not only that SQSTM1 and HDAC6 are important determinants of aggregated localization of MyD88 but also that MyD88 activates a machinery of polyubiquitinated protein accumulation that has a modulatory effect on MyD88-dependent signal transduction.

Regulation of Toll-like receptor signaling by NDP52-mediated selective autophagy is normally inactivated by A20.

Toll-like receptor (TLR) signaling is linked to autophagy that facilitates elimination of intracellular pathogens. However, it is largely unknown whether autophagy controls TLR signaling. Here, we report that poly(I:C) stimulation induces selective autophagic degradation of the TLR adaptor molecule TRIF and the signaling molecule TRAF6, which is revealed by gene silencing of the ubiquitin-editing enzyme A20. This type of autophagy induced formation of autophagosomes and could be suppressed by an autophagy inhibitor and lysosomal inhibitors. However, this autophagy was not associated with canonical autophagic processes, including involvement of Beclin-1 and conversion of LC3-I to LC3-II. Through screening of TRIF-interacting ‘autophagy receptors’ in human cells, we identified that NDP52 mediated the selective autophagic degradation of TRIF and TRAF6 but not TRAF3. NDP52 was polyubiquitinated by TRAF6 and was involved in aggregation of TRAF6, which may result in the selective degradation. Intriguingly, only under the condition of A20 silencing, NDP52 could effectively suppress poly(I:C)-induced proinflammatory gene expression. Thus, this study clarifies a selective autophagic mechanism mediated by NDP52 that works downstream of TRIF–TRAF6. Furthermore, although A20 is known as a signaling fine-tuner to prevent excess TLR signaling, it paradoxically downregulates the fine-tuning effect of NDP52 on TLR signaling.

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.