Periapical bacterial disinfection is critical for dental pulp regenerative cell therapy in apical periodontitis in dogs.

BACKGROUND: Application of pulp regenerative cell therapy for mature teeth with periapical lesions is a critical clinical challenge. The bacterial infection in inaccessible location within the root canal system and in the periapical lesions could cause resistance and impediment, leading to limitations in successful therapy. Thus, the aim of this study was to examine the effect of residual bacteria on the outcome of pulp regeneration in mature teeth with apical periodontitis in dogs. METHODS: Periapical lesions were induced in 32 root canals of 4 dogs in two different models in severities, model A and model B. Model A (moderate infection): the canal exposed to the oral cavity for 2 weeks and then closed for 2 weeks. Model B (severe infection): the canal exposed to the oral cavity for 2 months and then closed for 5 months. All root canals were irrigated with 6% sodium hypochlorite, and 3% EDTA and further with 0.015% levofloxacin-containing nanobubbles, which was also used as an intracanal medicament. The aseptic conditions were examined by bacterial anaerobic culture and/or PCR analyses. The root canal treatment was repeated several times, and allogeneic dental pulp stem cells were transplanted into the root canals. The radiographic evaluation of periapical lesions was performed by cone-beam computed tomography before the first treatment, just after cell transplantation, and after 2 months and 6 months in both model A, model B, respectively. The animals were then sacrificed and the jaw blocks were harvested for histological and histobacteriological evaluations of pulp regeneration and periapical tissue healing. Furthermore, the DiI-labelled DPSCs were transplanted into the root canals after complete disinfection (n = 4) or without root canal treatment (n = 4) in the apical periodontitis model (model A) in one dog, and cell localization was compared 72 h after transplantation. RESULTS: In 8 out of 12 canals from model A, and 10 out of 15 canals from model B, pulp regeneration with good vascularization, innervation, and a significant reduction in the radiolucent area of the periapical lesions were observed. However, in the other 4 canals and 5 canals from model A and model B, respectively, no pulp tissue was regenerated, and inflammation in the periapical tissue, and external resorption or healed external resorption were detected. The presence of residual bacteria in the periapical tissues and severe inflammation were significantly associated with inhibition of regenerated pulp tissue in these 9 unsuccessful canals (P < 0.05, each) (OR = 0.075, each) analyzed by multiple logistic regression analysis. For cellular kinetics, transplanted cells remained in the disinfected root canals, while they were not detected in the infected root canals, suggesting their migration through the apical foramen under the influence of inflammation. CONCLUSIONS: A true pulp-dentin complex was regenerated in the root canal by the pulp regenerative therapy in mature teeth with apical lesions. The successful pulp regeneration was negatively associated both with residual bacteria and inflammation in the periapical tissue.

Versican provides the provisional matrix for uterine spiral artery dilation and fetal growth.

The extracellular matrix (ECM) in the endometrium plays a crucial role in mammalian pregnancy. We have shown that versican secreted from the endometrial epithelium promotes embryo implantation. Versican is a proteoglycan, a major player in the provisional matrix, and versikine, its N-terminal fragment cleaved by ADAMTS proteinases, serves as a bioactive molecule. Here, since versican expression in the placenta was dynamically altered in humans and mice, we investigated the role of versican in pregnancy using uterine-specific Vcan deletion mice (uKO mice) and ADAMTS-resistant versican expressing mice (V1R mice). uKO mice exhibited insufficient spiral artery dilation, followed by fetal growth restriction and maternal hypertension. Further analysis revealed impaired proliferation of tissue-resident natural killer cells required for spiral artery dilation. V1R mice showed the same results as the control, eliminating the involvement of versikine. Our results provide a new concept that versican, one factor of ECM, contributes to placentation and following fetal growth.

Biological characteristics and pulp regeneration potential of stem cells from canine deciduous teeth compared with those of permanent teeth.

BACKGROUND: Clinical studies have demonstrated that dental pulp stem cells isolated from permanent teeth (PT-DPSCs) are safe and efficacious for complete pulp regeneration in mature pulpectomized permanent teeth with complete apical closure. Moreover, dental pulp stem cells from deciduous teeth (DT-DPSCs) have also been shown to be useful for pulp regenerative cell therapy of injured immature permanent teeth. However, direct comparisons of the pulp regenerative potential of DT-DPSCs and PT-DPSCs from the same individual have not been performed. This study aimed to compare the differences in stem cell properties and pulp regenerative potential of DT-DPSCs and PT-DPSCs of identical origin. METHODS: DT-DPSCs and PT-DPSCs were isolated from the same individual dogs at 4 months and 9 months of age, respectively. The expression of cell surface antigen markers, proliferation and migration activities, and gene expression of stem cell markers, angiogenic/neurotrophic factors and senescence markers were compared. The effects of conditioned medium (CM) derived from these cells on cellular proliferation, migration, angiogenesis, neurite outgrowth and immunosuppression were also compared. Autologous transplantation of DT-DPSCs or PT-DPSCs together with G-CSF was performed to treat pulpectomized teeth in individual dogs. The vascularization and reinnervation of the regenerated pulp tissues were qualitatively and quantitatively compared between groups by histomorphometric analyses. RESULTS: The rates of positive CXCR4 and G-CSFR expression in DT-DPSCs were significantly higher than those in PT-DPSCs. DT-DPSCs migrated at a higher rate with/without G-CSF and exhibited increased expression of the stem cell markers Oct3/4 and CXCR4 and the angiogenic factor VEGF and decreased expression of the senescence marker p16 than PT-DPSCs. DT-DPSC-derived CM promoted increased cell proliferation, migration with G-CSF, and angiogenesis compared with PT-DPSC-derived CM; however, no difference was observed in neurite outgrowth or immunosuppression. The regenerated pulp tissues in the pulpectomized teeth were quantitatively and qualitatively similar between the DT-DPSCs and PT-DPSCs transplant groups. CONCLUSIONS: These results demonstrated that DT-DPSCs could be a potential clinical alternative to PT-DPSCs for pulp regenerative therapy. DT-DPSCs can be preserved in an individual cell bank and used for potential future pulp regenerative therapy before the supply of an individual's own sound discarded teeth has been exhausted.

Bone marrow endothelial dysfunction promotes myeloid cell expansion in cardiovascular disease.

Abnormal hematopoiesis advances cardiovascular disease by generating excess inflammatory leukocytes that attack the arteries and the heart. The bone marrow niche regulates hematopoietic stem cell proliferation and hence the systemic leukocyte pool, but whether cardiovascular disease affects the hematopoietic organ's microvasculature is unknown. Here we show that hypertension, atherosclerosis and myocardial infarction (MI) instigate endothelial dysfunction, leakage, vascular fibrosis and angiogenesis in the bone marrow, altogether leading to overproduction of inflammatory myeloid cells and systemic leukocytosis. Limiting angiogenesis with endothelial deletion of Vegfr2 (encoding vascular endothelial growth factor (VEGF) receptor 2) curbed emergency hematopoiesis after MI. We noted that bone marrow endothelial cells assumed inflammatory transcriptional phenotypes in all examined stages of cardiovascular disease. Endothelial deletion of Il6 or Vcan (encoding versican), genes shown to be highly expressed in mice with atherosclerosis or MI, reduced hematopoiesis and systemic myeloid cell numbers in these conditions. Our findings establish that cardiovascular disease remodels the vascular bone marrow niche, stimulating hematopoiesis and production of inflammatory leukocytes.

Frontotemporal EEG as potential biomarker for early MCI: a case-control study.

BACKGROUND: Previous studies using EEG (electroencephalography) as biomarker for dementia have attempted to research, but results have been inconsistent. Most of the studies have extremely small number of samples (average N = 15) and studies with large number of data do not have control group. We identified EEG features that may be biomarkers for dementia with 120 subjects (dementia 10, MCI 33, against control 77). METHODS: We recorded EEG from 120 patients with dementia as they stayed in relaxed state using a single-channel EEG device while conducting real-time noise reduction and compared them to healthy subjects. Differences in EEG between patients and controls, as well as differences in patients' severity, were examined using the ratio of power spectrum at each frequency. RESULTS: In comparing healthy controls and dementia patients, significant power spectrum differences were observed at 3 Hz, 4 Hz, and 10 Hz and higher frequencies. In patient group, differences in the power spectrum were observed between asymptomatic patients and healthy individuals, and between patients of each respective severity level and healthy individuals. CONCLUSIONS: A study with a larger sample size should be conducted to gauge reproducibility, but the results implied the effectiveness of EEG in clinical practice as a biomarker of MCI (mild cognitive impairment) and/or dementia.

Aggrecan and versican: two brothers close or apart.

Aggrecan (Acan) and versican (Vcan) are large chondroitin sulfate proteoglycans of the extracellular matrix. They share the same structural domains at both N- and C-termini. The N-terminal G1 domain binds hyaluronan (HA), forms an HA-rich matrix, and regulates HA-mediated signaling. The C-terminal G3 domain binds other extracellular matrix molecules and forms a supramolecular structure that stores transforming growth factor ß (TGFß) and bone morphogenetic proteins (BMPs) and regulates their signaling. EGF-like motifs in the G3 domain may directly act like an EGF ligand. Both Acan and Vcan are present in cartilage, intervertebral disc, brain, heart, and aorta. Their localizations are essentially reciprocal. This review describes their structural domains, expression patterns and functions, and regulation of their expression.

Accumulation of versican and lack of versikine ameliorate acute colitis.

Versican is a large chondroitin sulfate/dermatan sulfate proteoglycan that plays a key role in the formation of the provisional matrix. Here, we generated dextran sulfate sodium-induced colitis in knockin-mice, R/R, expressing ADAMTS-resistant versican, and investigated the impact of accumulating versican and its turnover in the inflammatory colon mucosa. Histologically, R/R colon showed decreased levels of tissue destruction and an increased number of myofibroblasts and macrophages. Characterization of inflammatory cells revealed an increase in F4/80+ macrophages in R/R colon, compared with wildtype, without a clear shift between M1 and M2 populations. Intestinal stroma exhibited a higher number of myofibroblasts in R/R, suggesting increased levels of tissue regeneration. Coculture of macrophages and stromal fibroblasts obtained from inflammatory colon showed that wild-type macrophages inhibited myofibroblastic differentiation of R/R fibroblasts but not wild-type. This inhibitory effect was due to an increased level of versikine, a cleaved fragment of versican by ADAMTS proteinases. Taken together, our results demonstrate versikine as the direct regulator that inhibits repair of inflamed tissue.

Characterization of stable hypoxia-preconditioned dental pulp stem cells compared with mobilized dental pulp stem cells for application for pulp regenerative therapy.

BACKGROUND: Dental pulp stem cells (DPSCs) have been developed as a potential source of mesenchymal stem cells (MSCs) for regeneration of dental pulp and other tissues. However, further strategies to isolate highly functional DPSCs beyond the colony-forming methods are required. We have demonstrated the safety and efficacy of DPSCs isolated by G-CSF-induced mobilization and cultured under normoxia (mobilized DPSCs, MDPSCs) for pulp regeneration. The device for isolation of MDPSCs, however, is not cost-effective and requires a prolonged cell culture period. It is well known that MSCs cultured under hypoxic-preconditions improved MSC proliferation activity and stemness. Therefore, in this investigation, we attempted to improve the clinical utility of DPSCs by hypoxia-preconditioned DPSCs (hpDPSCs) compared with MDPSCs to improve the potential clinical utility for pulp regeneration in endodontic dentistry. METHODS: Colony-forming DPSCs were isolated and preconditioned with hypoxia in a stable closed cultured system and compared with MDPSCs isolated from the individual dog teeth. We examined the proliferation rate, migration potential, anti-apoptotic activity, and gene expression of the stem cell markers and angiogenic/neurotrophic factors. Trophic effects of the conditioned medium (CM) were also evaluated. In addition, the expression of immunomodulatory molecules upon stimulation with IFN-γ was investigated. The pulp regenerative potential and transplantation safety of hpDPSCs were further assessed in pulpectomized teeth in dogs by histological and immunohistochemical analyses and by chemistry of the blood and urine tests. RESULTS: hpDPSCs demonstrated higher proliferation rate and expression of a major regulator of oxygen homeostasis, HIF-1α, and a stem cell marker, CXCR-4. The direct migratory activity of hpDPSCs in response to G-CSF was significantly higher than MDPSCs. The CM of hpDPSCs stimulated neurite extension. However, there were no changes in angiogenic, migration, and anti-apoptotic activities compared with the CM of MDPSCs. The expression of immunomodulatory gene, PTGE was significantly upregulated by IFN gamma in hpDPSCs compared with MDPSCs. However, no difference in nitric oxide was observed. The regenerated pulp tissue was quantitatively and qualitatively similar in hpDPSC transplants compared with MDPSC transplants in dog teeth. There was no evidence of toxicity or adverse events of the hpDPSC transplantation. CONCLUSIONS: These results demonstrated that the efficacy of hpDPSCs for pulp regeneration was identical, although hpDPSCs improved stem cell properties compared to MDPSCs, suggesting their potential clinical utility for pulp regeneration.

Versican contributes to ligament formation of knee joints.

Versican is a large proteoglycan in the extracellular matrix. During embryonic stages, it plays a crucial role in the development of cartilage, heart, and dermis. Previously, we reported that Prx1-Vcan conditional knockout mice, lacking Vcan expression in mesenchymal condensation areas of the limb bud, show the impaired joint formation and delayed cartilage development. Here, we investigated their phenotype in adults and found that they develop swelling of the knee joint. Histologically, their newborn joint exhibited impaired formation of both anterior and posterior cruciate ligaments. Immunostaining revealed a decrease in scleraxis-positive cells in both articular cartilage and ligament of Prx1-Vcan knee joint, spotty patterns of type I collagen, and the presence of type II collagen concomitant with the absence of versican expression. These results suggest that versican expression during the perinatal period is required for cruciate ligaments' formation and that its depletion affects joint function in later ages.

Versican: A Dynamic Regulator of the Extracellular Matrix.

Versican is a large chondroitin sulfate/dermatan sulfate proteoglycan belonging to the aggrecan/lectican family. In adults, this proteoglycan serves as a structural macromolecule of the extracellular matrix in the brain and large blood vessels. In contrast, versican is transiently expressed at high levels during development and under pathological conditions when the extracellular matrix dramatically changes, including in the inflammation and repair process. There are many reports showing the upregulation of versican in cancer, which correlates with cancer aggressiveness. Versican has four classical splice variants, and all the variants contain G1 and G3 domains at N- and C-termini, respectively. There are two glycosaminoglycan attachment domains CSα and CSß. The largest V0 variant contains both CSα and CSß, V1 contains CSß, V2 contains CSα, and the shortest G3 variant has neither of them. Versican degradation is initiated by cleavage at a site in the CSß domain by ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) proteinases. The N-terminal fragment containing the G1 domain has been reported to exert various biological functions, although its mechanisms of action have not yet been elucidated. In this review, we describe the role of versican in inflammation and cancer and also address the biological function of versikine.

Treatment of Pulpectomized Teeth With Trypsin Prior to Transplantation of Mobilized Dental Pulp Stem Cells Enhances Pulp Regeneration in Aged Dogs.

There is an age-dependent decline of pulp regeneration, due to the decline of migration, proliferation, and cell survival of resident stem cells. Trypsin is a proteolytic enzyme clinically used for tissue repair. Here, we investigated the effects of trypsin pretreatment of pulpectomized teeth prior to cell transplantation on pulp regeneration in aged dogs. The amount of regenerated pulp was significantly higher in trypsin-pretreated teeth compared to untreated teeth. Trypsin pretreatment increased the number of cells attached to the dentinal wall that differentiated into odontoblast-like cells. The trypsin receptor, PAR2, was higher in vitro expression in the periodontal ligament cells (PDLCs) from aged dogs compared to those from young. The direct effects of trypsin on aged PDLCs were increased expression of genes related to immunomodulation, cell survival, and extracellular matrix degradation. To examine the indirect effects on microenvironment, highly extracted proteins from aged cementum were identified by proteomic analyses. Western blotting demonstrated that significantly increased fibronectin was released by the trypsin treatment of aged cementum compared to young cementum. The aged cementum extract (CE) and dentin extract (DE) by trypsin treatment increased angiogenesis, neurite extension and migration activities as elicited by fibronectin. Furthermore, the DE significantly increased the mRNA expression of immunomodulatory factors and pulp markers in the aged DPSCs. These results demonstrated the effects of trypsin on the microenvironment in addition to the resident cells including PDLCs in the aged teeth. In conclusion, the potential utility of trypsin pretreatment to stimulate pulp regeneration in aged teeth and the underlying mechanisms were demonstrated.

Cerebral cavernous malformations are driven by ADAMTS5 proteolysis of versican.

Cerebral cavernous malformations (CCMs) form following loss of the CCM protein complex in brain endothelial cells due to increased endothelial MEKK3 signaling and KLF2/4 transcription factor expression, but the downstream events that drive lesion formation remain undefined. Recent studies have revealed that CCM lesions expand by incorporating neighboring wild-type endothelial cells, indicative of a cell nonautonomous mechanism. Here we find that endothelial loss of ADAMTS5 reduced CCM formation in the neonatal mouse model. Conversely, endothelial gain of ADAMTS5 conferred early lesion genesis in the absence of increased KLF2/4 expression and synergized with KRIT1 loss of function to create large malformations. Lowering versican expression reduced CCM burden, indicating that versican is the relevant ADAMTS5 substrate and that lesion formation requires proteolysis but not loss of this extracellular matrix protein. These findings identify endothelial secretion of ADAMTS5 and cleavage of versican as downstream mechanisms of CCM pathogenesis and provide a basis for the participation of wild-type endothelial cells in lesion formation.

Effects of Fibroblast Growth Factor 2 on Burn Injury and Repair Process: Analysis Using a Refined Mouse Model.

BACKGROUND: Burn injury is one of the most debilitating traumas, which induces multiple organ dysfunctions, resulting in high levels of morbidity and mortality. Fibroblast growth factor 2 (FGF2) has been applied to burn injury, whose precise mechanisms underlying facilitating the healing have not been fully understood. Although various animal models have been developed in pigs, rabbits, rats, and mice, no mouse model that creates burns consistent in their extent and depth have not been developed. Here, we developed a mouse burn model, and investigated details of the burn process, and elucidated the mechanisms of FGF2 effects. METHODS: A device with an 8-mm metal probe and a temperature controller was developed, which controls the temperature of the probe. Using the device, 1 or 2 of full-thickness burn injuries were generated on the back under catagen/telogen of 6-month-old C57BL/6 male mice. After 24 hours, FGF2 or phosphate-buffered saline was injected into the injured region, and at days 3, 5, and 7, histological and immunohistochemical analysis was performed to observe the injury and repair process. RESULTS: The device constantly generated a mouse full-thickness burn injury. The repair was initiated on the bottom of the burn as well as the margin. Local treatment with FGF2 displayed higher levels of immunostaining for both CD31+ and alpha-smooth muscle actin. CONCLUSIONS: The device we developed is useful to generate a mouse burn injury model. FGF2 facilitates tissue repair with an increased number of both CD31+ and αSMA+ cells.

CCR3 antagonist protects against induced cellular senescence and promotes rejuvenation in periodontal ligament cells for stimulating pulp regeneration in the aged dog.

Pulp regeneration after transplantation of mobilized dental pulp stem cells (MDPSCs) declines in the aged dogs due in part to the chronic inflammation and/or cellular senescence. Eotaxin-1/C-C motif chemokine 11 (CCL11) is an inflammation marker via chemokine receptor 3 (CCR3). Moreover, CCR3 antagonist (CCR3A) can inhibit CCL11 binding to CCR3 and prevent CCL11/CCR3 signaling. The study aimed to examine the effect of CCR3A on cellular senescence and anti-inflammation/immunomodulation in human periodontal ligament cells (HPDLCs). The rejuvenating effects of CCR3A on neurite extension and migratory activity to promote pulp regeneration in aged dog teeth were also evaluated. In vivo, the amount of regenerated pulp tissues was significantly increased by transplantation of MDPSCs with CCR3A compared to control without CCR3A. In vitro, senescence of HPDLCs was induced after p-Cresol exposure, as indicated by increased cell size, decreased proliferation and increased senescence markers, p21 and IL-1ß. Treatment of HPDLCs with CCR3A prevented the senescence effect of p-Cresol. Furthermore, CCR3A significantly decreased expression of CCL11, increased expression of immunomodulatory factor, IDO, and enhanced neurite extension and migratory activity. In conclusion, CCR3A protects against p-Cresol-induced cellular senescence and enhances rejuvenating effects, suggesting its potential utility to stimulate pulp regeneration in the aged teeth.

Regulation of Macrophage and Dendritic Cell Function by Chondroitin Sulfate in Innate to Antigen-Specific Adaptive Immunity.

Chondroitin sulfate (CS), a type of glycosaminoglycan (GAG), is a linear acidic polysaccharide comprised of repeating disaccharides, modified with sulfate groups at various positions. Except for hyaluronan (HA), GAGs are covalently bound to core proteins, forming proteoglycans (PGs). With highly negative charges, GAGs interact with a variety of physiologically active molecules, including cytokines, chemokines, and growth factors, and control cell behavior during development and in the progression of diseases, including cancer, infections, and inflammation. Heparan sulfate (HS), another type of GAG, and HA are well reported as regulators for leukocyte migration at sites of inflammation. There have been many reports on the regulation of immune cell function by HS and HA; however, regulation of immune cells by CS has not yet been fully understood. This article focuses on the regulatory function of CS in antigen-presenting cells, including macrophages and dendritic cells, and refers to CSPGs, such as versican and biglycan, and the cell surface proteoglycan, syndecan.

Accumulation of versican facilitates wound healing: Implication of its initial ADAMTS-cleavage site.

Versican is a large chondroitin sulfate/dermatan sulfate proteoglycan in the extracellular matrix, and is expressed at high levels in tissues during development and remodeling in pathological conditions. Its core protein is cleaved at a region close to the N-terminal end of CSß domain by several members of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family, i.e., ADAMTS-1, 4, 5, 9, 15, and 20. Here, using a CRISPR/Cas9 system, we generated knock-in mice (V1R), which express an ADAMTS cleavage-resistant versican. Some V1R homozygote mice, termed R/R, exhibit syndactyly and organ hemorrhage. In wound healing experiments, R/R wound shows accumulation of versican and activated TGFß-signaling in the early stage, leading to faster healing than wild type wound. Immunostaining for Ki67, CD31, smooth muscle α-actin, periostin demonstrates higher levels of overall cell proliferation and an increased number of endothelial cells and myofibroblasts. Immunostaining for CD11b and qRT-PCR for macrophage markers revealed increased levels of inflammatory cell infiltration, especially those of M1 macrophages. Cultured R/R dermal fibroblasts revealed increased deposition of versican, type I and III collagens, and hyaluronan, and upregulation of Smad2/3 signaling. Taken together, these results demonstrate that the cleavage site determines versican turnover and that versican plays a central role in the provisional matrix during the wound repair.

Versican is crucial for the initiation of cardiovascular lumen development in medaka (Oryzias latipes).

Versican is an evolutionary conserved extracellular matrix proteoglycan, and versican expression loss in mice results in embryonic lethality owing to cardiovascular defects. However, the in utero development of mammals limits our understanding of the precise role of versican during cardiovascular development. Therefore, the use of evolutionarily distant species that develop ex utero is more suitable for studying the mechanistic basis of versican activity. We performed ENU mutagenesis screening to identify medaka mutants with defects in embryonic cardiovascular development. In this study, we described a recessive point mutation in the versican 3'UTR resulting in reduced versican protein expression. The fully penetrant homozygous mutant showed termination of cardiac development at the linear heart tube stage and exhibited absence of cardiac looping, a constricted outflow tract, and no cardiac jelly. Additionally, progenitor cells did not migrate from the secondary source towards the arterial pole of the linear heart tube, resulting in a constricted outflow tract. Furthermore, mutants lacked blood flow and vascular lumen despite continuous peristaltic heartbeats. These results enhance our understanding of the mechanistic basis of versican in cardiac development, and this mutant represents a novel genetic model to investigate the mechanisms of vascular tubulogenesis.

Characterization and functional analysis of novel circulating NK cell sub-populations.

Natural killer (NK) cells are innate lymphoid cells having potent cytolytic function that provide host defense against microbial infections and tumors. Using our generated monoclonal antibody (mAb), named FE-1H10, new NK cell sub-populations in peripheral blood were identified. The molecules recognized by mAb FE-1H10 were expressed on a sub-population of CD3-CD56dim NK cells. The epitope recognized by mAb FE-1H10 was demonstrated to be N-glycan and proven to be different from CD57. Upon K562 stimulation, the CD56dimFE-1H10+ NK cell sub-population exhibited significantly lower cytolytic function with low ability to degranulate and release cytolytic granules compared to the CD56dimFE-1H10- NK cell sub-population. Moreover, the CD56dimFE-1H10+ NK cells produced less IFN-γ and TNF-α than the CD56dimFE-1H10- NK cells. We demonstrated here that mAb FE-1H10 could identify two sub-populations of circulating CD56dim NK cells with different functions. Our discovery of new sub-populations of NK cells improves our understanding of NK cell biology and may lead to the development of new approaches for NK cell therapy.

The plant alkaloid conophylline inhibits matrix formation of fibroblasts.

Conophylline is a Vinca alkaloid from leaves of the tropical plant Ervatamia microphylla and has been shown to mimic the effect of the growth and differentiation factor activin A on pancreatic progenitor cells. However, activin A stimulates fibrosis of pancreatic stellate cells, whereas conophylline inhibits it, suggesting that this compound may serve as an antifibrotic drug. Here we investigated the effects of conophylline on human foreskin fibroblasts, especially focusing on extracellular matrix (ECM) proteins. A gene microarray analysis revealed that conophylline remarkably suppressed expression of the gene for hyaluronan synthase 2 (HAS2) and of its antisense RNA, whereas the expression of collagen genes was unaffected. Of note, immunostaining experiments revealed that conophylline substantially inhibits incorporation of versican and collagens into the ECM in cells treated with transforming growth factor ß (TGFß), which promotes collagen synthesis, but not in cells not treated with TGFß. Moreover, a protein biosynthesis assay disclosed that conophylline decreases collagen biosynthesis, concomitant with a decrease in total protein biosynthesis, indicating that conophylline-mediated inhibition of fibrosis is not specific to collagen synthesis. Conophylline affected neither TGFß-induced nuclear translocation of SMAD family member 2/3 (SMAD2/3) nor phosphorylation of SMAD2. However, conophylline substantially inhibited phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), suggesting that conophylline inhibits HAS2 expression via TGFß-mediated activation of the ERK1/2 pathway. Taken together, our results indicate that conophylline may be a useful inhibitor of ECM formation in fibrosis.

Craniofacial abnormality with skeletal dysplasia in mice lacking chondroitin sulfate N-acetylgalactosaminyltransferase-1.

Chondroitin sulfate (CS) proteoglycan is a major component of the extracellular matrix and plays an important part in organogenesis. To elucidate the roles of CS for craniofacial development, we analyzed the craniofacial morphology in CS N-acetylgalactosaminyltransferase-1 (T1) gene knockout (KO) mice. T1KO mice showed the impaired intramembranous ossification in the skull, and the final skull shape of adult mice included a shorter face, higher and broader calvaria. Some of T1KO mice exhibited severe facial developmental defect, such as eye defects and cleft lip and palate, causing embryonic lethality. At the postnatal stages, T1KO mice with severely reduced CS amounts showed malocclusion, general skeletal dysplasia and skin hyperextension, closely resembling Ehlers-Danlos syndrome-like connective tissue disorders. The production of collagen type 1 was significantly downregulated in T1KO mice, and the deposition of CS-binding molecules, Wnt3a, was decreased with CS in extracellular matrices. The collagen fibers were irregular and aggregated, and connective tissues were dysorganized in the skin and calvaria of T1KO mice. These results suggest that CS regulates the shape of the craniofacial skeleton by modulating connective tissue organization and that the remarkable reduction of CS induces hypoplasia of intramembranous ossification and cartilage anomaly, resulting in skeletal dysplasia.