Establishment of a chemical tongue injury-recovery mouse model.

Tongue epithelium is one of the most proliferative and regenerative epithelia in our body. However, tongue stem cell research is hampered partly by the lack of optimal animal models to study tongue injury, repair, and regeneration. Here, we establish a novel chemically induced tongue injury-recovery mouse model. Focal application of sodium hydroxide for a limited time led to the denudation of suprabasal layers, leaving the basal layer. Time course study revealed that tongue epithelial cells robustly proliferate over one week after the tongue injury. Importantly, we demonstrated that our novel mouse model could be employed in the lineage tracing of the tongue stem cells under the injury and repair process and further showed that tongue stem cells proliferate faster and generate larger clones in the injury condition than in the steady state condition. Our data indicate the development of a novel chemically induced tongue injury-recovery mouse model for tongue stem cell research, which will significantly facilitate the preclinical study for the pathogenesis and treatment of caustic ingestion.

Effects of photobiomodulation on bone remodeling in an osteoblast-osteoclast co-culture system.

The general bone anabolic effect of photobiomodulation (PBM) is largely accepted. As a result, PBM therapy is expected to be beneficial in the medical fields of dentistry and bone healing. However, most of the previous in vitro studies on PBM and bone metabolism were performed with single-cell cultures of osteoclast-lineage cells or osteoblast-lineage cells. In the present study, the bone-modulating effects of PBM were evaluated in an in vitro osteoblast/osteoclast co-culture system. Mouse bone marrow-derived macrophages (BMMs) and mouse calvarial pre-osteoblasts cells were purified and used as precursor cells for osteoclasts and osteoblasts, respectively. The PBM effects on single-cell culture of osteoclasts or osteoblasts as well as co-culture were examined by 1.2 J/cm2 low-level Ga-Al-As laser (λ = 808 ± 3 nm, 80 mW, and 80 mA; spot size, 1cm2; NDLux, Seoul, Korea) irradiation for 30 s at daily intervals throughout culture period. At the end of culture, the osteoclast differentiation and osteoblast differentiation were assessed by TRAP staining and ALP staining, respectively. The expressions of osteoclastogenic cytokines were evaluated by RT-PCR and Western blot analyses. Under the single-cell culture condition, PBM enhanced osteoblast differentiation but had minor effects on osteoclast differentiation. However, in the co-culture condition, its osteoblastogenic effect was maintained, and osteoclast differentiation was substantially reduced. Subsequent RT-PCR analyses and western blot results revealed marked reduction in receptor activator of NF-κB ligand (RANKL) expression and elevation in osteoprotegerin (OPG) expression by PBM in co-cultured cells. More importantly, these alterations in RANKL/OPG levels were not observed under the single-cell culture conditions. Our results highlight the different effects of PBM on bone cells based on culture conditions. Further, our findings suggest the indirect anti-osteoclastogenic effect of PBM, which is accompanied by a decrease in RANKL expression and an increase in OPG expression.

Ischemia-induced Netrin-4 promotes neovascularization through endothelial progenitor cell activation via Unc-5 Netrin receptor B.

Endothelial progenitor cells (EPCs) promote neovascularization and tissue repair by migrating to vascular injury sites; therefore, factors that enhance EPC homing to damaged tissues are of interest. Here, we provide evidence of the prominent role of the Netrin-4 (NTN4)-Unc-5 Netrin receptor B (UNC5B) axis in EPC-specific promotion of ischemic neovascularization. Our results showed that NTN4 promoted the proliferation, chemotactic migration, and paracrine effects of small EPCs (SEPCs) and significantly increased the incorporation of large EPCs (LEPCs) into tubule networks. Additionally, NTN4 prominently augmented neovascularization in mice with hindlimb ischemia by increasing the homing of exogenously transplanted EPCs to the ischemic limb and incorporating EPCs into vessels. Moreover, silencing of UNC5B, an NTN4 receptor, abrogated the NTN4-induced cellular activities of SEPCs in vitro and blood-flow recovery and neovascularization in vivo in ischemic muscle by reducing EPC homing and incorporation. These findings suggest NTN4 as an EPC-based therapy for treating angiogenesis-dependent diseases.

Elevated Expression of Cathepsin K in Periodontal Ligament Fibroblast by Inflammatory Cytokines Accelerates Osteoclastogenesis via Paracrine Mechanism in Periodontal Disease.

Cathepsin K (CTSK) is a cysteine protease that is mainly produced from mature osteoclasts and contributes to the destruction of connective tissues and mineralized matrix as a consequence of periodontal disease (PD). However, few studies have reported its regulatory role in osteoclastogenesis-supporting cells in inflammatory conditions. Here, we investigated the role of CTSK in osteoclastogenesis-supporting cells, focusing on the modulation of paracrine function. Microarray data showed that CTSK was upregulated in PD patients compared with healthy individuals, which was further supported by immunohistochemistry and qPCR analyses performed with human gingival tissues. The expression of CTSK in the osteoclastogenesis-supporting cells, including dental pulp stem cells, gingival fibroblasts, and periodontal ligament fibroblasts (PDLFs) was significantly elevated by treatment with inflammatory cytokines such as TNFα and IL-1ß. Moreover, TNFα stimulation potentiated the PDLF-mediated osteoclastogenesis of bone marrow-derived macrophages. Interestingly, small interfering RNA-mediated silencing of CTSK in PDLF noticeably attenuated the TNFα-triggered upregulation of receptor activator of nuclear factor kappa-B ligand (RANKL), macrophage colony-stimulating factor, and RANKL/osteoprotegerin ratio, thereby abrogating the enhanced osteoclastogenesis-supporting activity of PDLF. Collectively, these results suggest a novel role of CTSK in the paracrine function of osteoclastogenesis-supporting cells in periodontal disease.

Letrozole Suppresses the Fusion of Osteoclast Precursors through Inhibition of p38-Mediated DC-STAMP Pathway.

Letrozole is a reversible nonsteroidal aromatase inhibitor that is widely used in postmenopausal breast cancer patients. It is well established that letrozole decreases bone density owing to estrogen depletion; however, few studies have reported its direct effect on bone cells in vitro. Therefore, we investigated the effect of letrozole on bone metabolism, focusing on osteoclastogenesis. Letrozole did not affect the viability, proliferation, or migration of bone marrow-derived macrophages (BMMs); however, it reduced the multinucleation of immature osteoclasts and subsequent bone resorption in vitro. Overall, letrozole inhibited the expression of dendritic cell-specific transmembrane protein (DC-STAMP), tartrate-resistant acid phosphatase, calcitonin receptor, and cathepsin K. Among them, the reduced expression of DC-STAMP was the most prominent. However, this downregulation of DC-STAMP expression following letrozole treatment was not related to the inhibition of major osteoclastogenesis pathways, such as the nuclear factor-κB (NF-κB), c-Fos, and nuclear factor of activated T cell c1 (NFATc1) pathways, but was attributed to the inhibition of p38, which is known to reside upstream of DC-STAMP expression. Notably, the anti-osteoclastogenic effect of letrozole was abolished following treatment with the p38 activator anisomycin. Contrary to our expectations, these results strongly suggest a previously unknown anti-osteoclastogenic activity of letrozole, mediated by the downregulation of the p38/DC-STAMP pathway.

Formyl Peptide Receptor 2 Is Involved in Cardiac Repair After Myocardial Infarction Through Mobilization of Circulating Angiogenic Cells.

Increasing evidence suggests that circulating angiogenic cells (CACs) promote repair of ischemic tissues. Activation of formyl peptide receptor 2 (Fpr2) has been reported to stimulate repair of ischemic heart. This study was conducted to investigate the role of Fpr2 on CAC mobilization and cardiac protection in myocardial infarction (MI). WKYMVm, a strong agonist for Fpr2, was administered in a murine model of acute MI, and mobilization of CACs including endothelial progenitor cells (CD34+ Flk1+ or Sca1+ Flk1+ cells) in peripheral blood was monitored. CAC mobilization by daily injection of WKYMVm for the first 4 days after MI was as efficient as granulocyte colony-stimulating factor and provided myocardial protection from apoptosis with increased vascular density and preservation of cardiac function. Transplantation of bone marrow (BM) from green fluorescent protein mice showed that BM-derived cells homed to ischemic heart after WKYMVm treatment and contributed to tissue protection. Transplantation of BM from Fpr2 knockout mice showed that Fpr2 in BM cells is critical in mediation of WKYMVm-stimulated myocardial protection and neovascularization after MI. These results suggest that activation of Fpr2 in BM after WKYMVm treatment provides cardiac protection through mobilization of CACs after MI, which may lead to the development of a new clinical protocol for treating patients with ischemic heart conditions. Stem Cells 2017;35:654-665.

Quantification and application of a liquid chromatography-tandem mass spectrometric method for the determination of WKYMVm peptide in rat using solid-phase extraction.

A liquid chromatographic-electrospray ionization-time-of-flight/mass spectrometric (LC-ESI-TOF/MS) method was developed and applied for the determination of WKYMVm peptide in rat plasma to support preclinical pharmacokinetics studies. The method consisted of micro-elution solid-phase extraction (SPE) for sample preparation and LC-ESI-TOF/MS in the positive ion mode for analysis. Phenanthroline (10 mg/mL) was added to rat blood immediately for plasma preparation followed by addition of trace amount of 2 m hydrogen chloride to plasma before SPE for stability of WKYMVm peptide. Then sample preparation using micro-elution SPE was performed with verapamil as an internal standard. A quadratic regression (weighted 1/concentration2 ), with the equation y = ax2 + bx + c was used to fit calibration curves over the concentration range of 3.02-2200 ng/mL for WKYMVm peptide. The quantification run met the acceptance criteria of ±25% accuracy and precision values. For quality control samples at 15, 165 and 1820 ng/mL from the quantification experiment, the within-run and the between-run accuracy ranged from 92.5 to 123.4% with precision values ≤15.1% for WKYMVm peptide from the nominal values. This novel LC-ESI-TOF/MS method was successfully applied to evaluate the pharmacokinetics of WKYMVm peptide in rat plasma.

Therapeutic angiogenesis in a murine model of limb ischemia by recombinant periostin and its fasciclin I domain.

Periostin, an extracellular matrix protein, is expressed in injured tissues, such as the heart with myocardial infarction, and promotes angiogenesis and tissue repair. However, the molecular mechanism associated with periostin-stimulated angiogenesis and tissue repair is still unclear. In order to clarify the role of periostin in neovascularization, we examined the effect of periostin in angiogenic potentials of human endothelial colony forming cells (ECFCs) in vitro and in an ischemic limb animal model. Recombinant periostin protein stimulated the migration and tube formation of ECFCs. To identify the functional domains of periostin implicated in angiogenesis, five fragments of periostin, including four repeating FAS-1 domains and a carboxyl terminal domain, were expressed in Escherichia coli and purified to homogeneity. Of the five different domains, the first FAS-1 domain stimulated the migration and tube formation of human ECFCs as potent as the whole periostin. Chemotactic migration of ECFCs induced by the full length and the first FAS-1 domain of periostin was abrogated by blocking antibodies against ß3 and ß5 integrins. Intramuscular injection of the full length and the first FAS-1 domain of periostin into the ischemic hindlimb of mice attenuated severe limb loss and promoted blood perfusion and homing of intravenously administered ECFCs to the ischemic limb. These results suggest that the first FAS-1 domain is responsible for periostin-induced migration and angiogenesis and it can be used as a therapeutic tool for treatment of peripheral artery occlusive disease by stimulating homing of ECFCs.

Reptin regulates pluripotency of embryonic stem cells and somatic cell reprogramming through Oct4-dependent mechanism.

Oct4 has been implicated in regulation of pluripotency in embryonic stem cells (ESCs) and reprogramming of somatic cells into induced pluripotent stem cells. However, the molecular mechanisms involved in Oct4-dependent regulation of pluripotency and reprogramming have not been clear. To gain insight into the mechanism of regulation of Oct4-mediated self-renewal of ESCs and reprogramming of somatic cells, we attempted to identify Oct4-binding proteins using affinity purification and mass spectrometry. We identified Reptin, a key component of ATP-dependent chromatin remodeling complexes, as an Oct4-binding protein. Depletion of endogenous Reptin using lentiviral short hairpin RNA (shRNA) led to a decrease in the number and size of alkaline phosphatase-positive colonies of mouse ESCs. In addition, shRNA-mediated silencing of Reptin resulted in decreased expression of pluripotency-specific marker genes, including Oct4, Sox2, Nanog, and SSEA-1. Results of the Oct4 reporter assay showed synergism between Oct4 and Reptin, and depletion of endogenous Reptin abolished Oct4 transcriptional activity. Results of a chromatin immunoprecipitation assay showed the overlapping interaction of Reptin and Oct4 to CR4 in the Oct4 enhancer in ESCs. Knockdown of Reptin using shRNA suppressed the reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells, whereas overexpression of Reptin resulted in enhanced efficiency of induced pluripotent stem cell generation. These results strongly suggest that Reptin plays a key role in maintaining the pluripotency of ESCs and in establishing the pluripotency during reprogramming of somatic cells by regulation of Oct4-mediated gene regulation.

WKYMVm-induced activation of formyl peptide receptor 2 stimulates ischemic neovasculogenesis by promoting homing of endothelial colony-forming cells.

Endothelial colony-forming cells (ECFCs) are recruited to the sites of ischemic injury in order to contribute to neovascularization and repair of injured tissues. However, therapeutic potential of ECFCs is limited due to low homing and engraftment efficiency of transplanted ECFCs. The G-protein-coupled formyl peptide receptor (FPR) 2 has been implicated in regulation of inflammation and angiogenesis, while the role of FPR2 in homing and engraftment of ECFCs and neovascularization in ischemic tissues has not been fully defined. This study was undertaken to investigate the effects of WKYMVm, a selective FPR2 agonist isolated by screening synthetic peptide libraries, on homing ability of ECFCs and vascular regeneration of ischemic tissues. WKYMVm stimulated chemotactic migration, angiogenesis, and proliferation ability of human ECFCs in vitro. Small interfering RNA-mediated silencing of FPR2, but not FPR3, abrogated WKYMVm-induced migration and angiogenesis of ECFCs. Intramuscular injection of WKYMVm resulted in attenuation of severe hind limb ischemia and promoted neovascularization in ischemic limb. ECFCs transplanted via tail vein into nude mice were incorporated into capillary vessels in the ischemic hind limb, resulting in augmented neovascularization and improved ischemic limb salvage. Intramuscular injection of WKYMVm promoted homing of exogenously administered ECFCs to the ischemic limb and ECFC-mediated vascular regeneration. Silencing of FPR2 expression in ECFCs resulted in abrogation of WKYMVm-induced in vivo homing of exogenously transplanted ECFCs to the ischemic limb, neovascularization, and ischemic limb salvage. These results suggest that WKYMVm promotes repair of ischemic tissues by stimulating homing of ECFCs and neovascularization via a FPR2-dependent mechanism.

Stimulation of cutaneous wound healing by an FPR2-specific peptide agonist WKYMVm.

Diabetes is one of the most common human diseases and 15% of the 200 million diabetics worldwide suffer from diabetic wounds. Development of new therapeutic agents is needed for treatment of diabetic wounds. Wound healing is mediated by multiple steps, including inflammation, epithelialization, neoangiogenesis, and granulation. Formyl peptide receptor 2 has been known to stimulate angiogenesis, which is essential for tissue repair and cutaneous wound healing. In this study, we explored the therapeutic effects of WKYMVm (Trp-Lys-Tyr-Met-Val-D-Met-NH2), a synthetic peptide agonist of formyl peptide receptor 2, on cutaneous wounds in streptozotocin-induced diabetic rats. Topical application of WKYMVm onto cutaneous wounds stimulated formation of von Willebrand factor-positive capillary and α-smooth muscle actin-positive arteriole with a maximal stimulation on day 6, suggesting WKYMVm-stimulated angiogenesis. Infiltration of immune cells could be detected on early phase during wound healing and WKYMVm treatment acutely augmented infiltration of CD68-positive macrophages. In addition, reepithelialization and granulation tissue formation were accelerated by treatment with WKYMVm. These results suggest that WKYMVm has therapeutic effects on diabetic wounds by stimulating angiogenesis and infiltration of immune cells.

Tumor necrosis factor-α-activated mesenchymal stem cells promote endothelial progenitor cell homing and angiogenesis.

Mesenchymal stem cells (MSCs) accelerate regeneration of ischemic or injured tissues by stimulation of angiogenesis through a paracrine mechanism. Tumor necrosis factor-α (TNF-α)-activated MSCs secrete pro-angiogenic cytokines, including IL-6 and IL-8. In the present study, using an ischemic hindlimb animal model, we explored the role of IL-6 and IL-8 in the paracrine stimulation of angiogenesis and tissue regeneration by TNF-α-activated MSCs. Intramuscular injection of conditioned medium derived from TNF-α-treated MSCs (TNF-α CM) into the ischemic hindlimb resulted in attenuated severe limb loss and stimulated blood perfusion and angiogenesis in the ischemic limb. Immunodepletion of IL-6 and IL-8 resulted in attenuated TNF-α CM-stimulated tissue repair, blood perfusion, and angiogenesis. In addition, TNF-α CM induced migration of human cord blood-derived endothelial progenitor cells (EPCs) through IL-6- and IL-8-dependent mechanisms in vitro. Intramuscular injection of TNF-α CM into the ischemic limb led to augmented homing of tail vein-injected EPCs into the ischemic limb in vivo and immunodepletion of IL-6 or IL-8 from TNF-α CM attenuated TNF-α CM-stimulated homing of EPCs. In addition, intramuscular injection of recombinant IL-6 and IL-8 proteins resulted in increased homing of intravenously transplanted EPCs into the ischemic limb and improved blood perfusion in vivo. These results suggest that TNF-α CM stimulates angiogenesis and tissue repair through an increase in homing of EPCs through paracrine mechanisms involving IL-6 and IL-8.

Proteomic identification of betaig-h3 as a lysophosphatidic acid-induced secreted protein of human mesenchymal stem cells: paracrine activation of A549 lung adenocarcinoma cells by betaig-h3.

Lysophosphatidic acid (LPA) is enriched in the serum and malignant effusion of cancer patients and plays a key role in tumorigenesis and metastasis. LPA-activated mesenchymal stem cells promote tumorigenic potentials of cancer cells through a paracrine mechanism. LPA-conditioned medium (LPA CM) from human adipose tissue-derived mesenchymal stem cells (hASCs) elicited adhesion and proliferation of A549 human lung adenocarcinoma cells. To identify proteins involved in the LPA-stimulated paracrine functions of hASCs, we analyzed the LPA CM using liquid-chromatography tandem mass spectrometry-based shotgun proteomics. We identified ßig-h3, an extracellular matrix protein that is implicated in tumorigenesis and metastasis, as an LPA-induced secreted protein in hASCs. LPA-induced ßig-h3 expression was abrogated by pretreating hASCs with the LPA receptor(1/3) inhibitor Ki16425 or small interfering RNA-mediated silencing of endogenous LPA(1). LPA-induced ßig-h3 expression was blocked by treating the cells with the Rho kinase inhibitor Y27632, implying that LPA-induced ßig-h3 expression is mediated by the LPA(1)- Rho kinase pathway. Immunodepletion or siRNA-mediated silencing of ßig-h3 abrogated LPA CM-stimulated adhesion and proliferation of A549 cells, whereas retroviral overexpression of ßig-h3 in hASCs potentiated it. Furthermore, recombinant ßig-h3 protein stimulated the proliferation and adhesion of A549 human lung adenocarcinoma cells. These results suggest that hASC-derived ßig-h3 plays a key role in tumorigenesis by stimulating the adhesion and proliferation of cancer cells and it can be applicable as a biomarker and therapeutic target for lung cancer.

Functional expression of smooth muscle-specific ion channels in TGF-ß(1)-treated human adipose-derived mesenchymal stem cells.

Human adipose tissue-derived mesenchymal stem cells (hASCs) have the power to differentiate into various cell types including chondrocytes, osteocytes, adipocytes, neurons, cardiomyocytes, and smooth muscle cells. We characterized the functional expression of ion channels after transforming growth factor-ß1 (TGF-ß1)-induced differentiation of hASCs, providing insights into the differentiation of vascular smooth muscle cells. The treatment of hASCs with TGF-ß1 dramatically increased the contraction of a collagen-gel lattice and the expression levels of specific genes for smooth muscle including α-smooth muscle actin, calponin, smooth mucle-myosin heavy chain, smoothelin-B, myocardin, and h-caldesmon. We observed Ca(2+), big-conductance Ca(2+)-activated K(+) (BKCa), and voltage-dependent K(+) (Kv) currents in TGF-ß1-induced, differentiated hASCs and not in undifferentiated hASCs. The currents share the characteristics of vascular smooth muscle cells (SMCs). RT-PCR and Western blotting revealed that the L-type (Cav1.2) and T-type (Cav3.1, 3.2, and 3.3), known to be expressed in vascular SMCs, dramatically increased along with the Cavß1 and Cavß3 subtypes in TGF-ß1-induced, differentiated hASCs. Although the expression-level changes of the ß-subtype BKCa channels varied, the major α-subtype BKCa channel (KCa1.1) clearly increased in the TGF-ß1-induced, differentiated hASCs. Most of the Kv subtypes, also known to be expressed in vascular SMCs, dramatically increased in the TGF-ß1-induced, differentiated hASCs. Our results suggest that TGF-ß1 induces the increased expression of vascular SMC-like ion channels and the differentiation of hASCs into contractile vascular SMCs.

TAZ mediates lysophosphatidic acid-induced migration and proliferation of epithelial ovarian cancer cells.

BACKGROUND: Transcriptional co-activator with PDZ-binding motif (TAZ), a downstream effector of the Hippo pathway, has been reported to regulate organ size, tissue homeostasis, and tumorigenesis by acting as a transcriptional co-activator. Lysophosphatidic acid (LPA) is a bioactive lipid implicated in tumorigenesis and metastasis of ovarian cancer through activation of G protein-coupled receptors. However, the involvement of TAZ in LPA-induced tumorigenesis of ovarian cancer has not been elucidated. METHODS: In order to demonstrate the role of TAZ in LPA-stimulated tumorigenesis, the effects of LPA on TAZ expression and cell migration were determined by Western blotting and chemotaxis analyses in R182 human epithelial ovarian cancer cells. RESULTS AND CONCLUSION: Treatment of R182 cells with the LPA receptor inhibitor Ki16425 blocked LPA-induced cell migration. In addition, transfection of R182 cells with small interfering RNA specific for LPA receptor 1 resulted in abrogation of LPA-stimulated cell migration. LPA induced phosphorylation of ERK and p38 MAP kinase in R182 cells and pretreatment of cells with the MEK-ERK pathway inhibitor U0126, but not the p38 MAPK inhibitor SB202190, resulted in abrogation of LPA-induced cell migration. Pretreatment of R182 cells with U0126 attenuated LPA-induced mRNA levels of TAZ and its transcriptional target genes, such as CTGF and CYR61, without affecting phosphorylation level of YAP. These results suggest that MEK-ERK pathway plays a key role in LPA-induced cell migration and mRNA expression of TAZ in R182 cells, without affecting stability of TAZ protein. In addition, small interfering RNA-mediated silencing of TAZ expression attenuated LPA-stimulated migration of R182 cells. These results suggest that TAZ plays a key role in LPA-stimulated migration of epithelial ovarian cancer cells.

Macrophages regulate smooth muscle differentiation of mesenchymal stem cells via a prostaglandin F2α-mediated paracrine mechanism.

OBJECTIVE: Mesenchymal stem cells are useful for vascular regeneration of injured tissues. Macrophages are involved in acute or chronic inflammatory diseases, and interleukin-1ß (IL-1ß), a proinflammatory cytokine, plays a key role in the activation of macrophages within injured tissues. To explore the role of macrophages on mesenchymal stem cell-mediated vascular regeneration, we examined the effects of IL-1ß-activated macrophages on differentiation of human adipose tissue-derived mesenchymal stem cells (hASCs) to smooth muscle cells (SMCs) and the vascular regenerative capacity of the differentiated SMCs in a hindlimb ischemia animal model. METHODS AND RESULTS: We demonstrate that IL-1ß-conditioned medium from RAW 264.7 macrophages induces differentiation of human adipose tissue-derived mesenchymal stem cells to α-smooth muscle actin-positive SMCs, and the differentiated SMCs exhibited increased contractility in response to KCl and carbachol treatment. Transplantation of the differentiated SMCs attenuated severe hindlimb ischemia and promoted vascular regeneration. IL-1ß treatment stimulated secretion of prostaglandin F(2α) from RAW 264.7 cells. Small interfering RNA-mediated silencing of the prostaglandin F(2α) receptor completely abrogated IL-1ß conditioned medium-stimulated α-smooth muscle actin expression. Moreover, prostaglandin F(2α) treatment stimulated expression of α-smooth muscle actin in human adipose tissue-derived mesenchymal stem cells. CONCLUSIONS: These results suggest that IL-1ß-activated macrophages promote differentiation of human adipose tissue-derived mesenchymal stem cells to SMCs through a prostaglandin F(2α)-mediated paracrine mechanism.

Vasohibin-1 promotes osteoclast differentiation in periodontal disease by stimulating the expression of RANKL in gingival fibroblasts.

Vasohibin-1 (VASH1) is a key inhibitor of vascular endothelial growth factor-induced angiogenesis. Although the involvement of VASH1 in various pathological processes has been extensively studied, its role in periodontal disease (PD) remains unclear. We aimed to investigate the role of VASH1 in PD by focusing on osteoclastogenesis regulation. We investigated VASH1 expression in PD by analyzing data from the online Gene Expression Omnibus (GEO) database and using a mouse ligature-induced periodontitis model. The effects of VASH1 on osteoclast differentiation and osteoclastogenesis-supporting cells were assessed in mouse bone marrow-derived macrophages (BMMs) and human gingival fibroblasts (GFs). To identify the stimulant of VASH1, we used culture broth from Porphyromonas gingivalis (Pg), a periopathogen. The GEO database and mouse periodontitis model revealed that VASH1 expression was upregulated in periodontitis-affected gingival tissues, which was further supported by immunohistochemistry and qRT-PCR analyses. VASH1 expression was significantly stimulated in GFs after treatment with the Pg broth. Direct treatment with recombinant VASH1 protein did not stimulate osteoclast differentiation in BMMs but did contribute to osteoclast differentiation by inducing RANKL expression in GFs through a paracrine mechanism. Small interfering RNA-mediated silencing of VASH1 in GFs abrogated RANKL-mediated osteoclast differentiation in BMMs. Additionally, VASH1-activated RANKL expression in GFs was significantly suppressed by MK-2206, a selective inhibitor of AKT. These results suggest that Pg-induced VASH1 may be associated with RANKL expression in GFs in a paracrine manner, contributing to osteoclastogenesis via an AKT-dependent mechanism during PD progression.

Lysophosphatidic acid induces proliferation and osteogenic differentiation of human dental pulp stem cell through lysophosphatidic acid receptor 3/extracellular signal-regulated kinase signaling axis.

Background/purpose: Human dental pulp stem cells (hDPSCs) possess excellent proliferative and osteogenic differentiation potentials. This study aimed to elucidate the role of lysophosphatidic acid (LPA) signaling in the proliferation and osteogenic differentiation of hDPSCs. Materials and methods: hDPSCs were treated with LPA and proliferation was measured using the cell counting kit-8 assay. Following the osteogenic differentiation of hDPSCs using osteogenic medium in the presence or absence of LPA, alkaline phosphatase (ALP) staining, ALP activity measurements, and RT-qPCR were performed to analyze the osteoblast differentiation. Small interfering RNA (siRNA)-mediated LPAR3 silencing and extracellular signal-regulated (ERK)/mitogen-activated protein (MAP) kinase inhibitors were used to elucidate the molecular mechanisms underlying LPA-induced proliferation and differentiation of hDPSCs. Results: LPA treatment significantly induced proliferation and osteogenic differentiation of hDPSCs. The depletion of LPAR3 expression by LPAR3-speicifc siRNA in hDPSCs diminished LPA-induced proliferation and osteogenic differentiation. The LPAR3-mediated proliferation and osteogenic differentiation of hDPSCs in response to LPA were significantly suppressed by U0126, a selective inhibitor of ERK. Conclusion: These findings suggest that LPA induces the proliferation and osteogenic differentiation of hDPSCs via LPAR3-ERK-dependent pathways.

Periostin mediates human adipose tissue-derived mesenchymal stem cell-stimulated tumor growth in a xenograft lung adenocarcinoma model.

Mesenchymal stem cells stimulate tumor growth in vivo through a lysophosphatidic acid (LPA)-dependent mechanism. However, the molecular mechanism by which mesenchymal stem cells stimulate tumorigenesis is largely elusive. In the present study, we demonstrate that conditioned medium from A549 human lung adenocarcinoma cells (A549 CM) induces expression of periostin, an extracellular matrix protein, in human adipose tissue-derived mesenchymal stem cells (hASCs). A549 CM-stimulated periostin expression was abrogated by pretreatment of hASCs with the LPA receptor 1 (LPA(1)) inhibitor Ki16425 or short hairpin RNA-mediated silencing of LPA(1), suggesting a key role of the LPA-LPA(1) signaling axis in A549 CM-stimulated periostin expression. Using a xenograft transplantation model of A549 cells, we demonstrated that co-injection of hASCs potentiated tumor growth of A549 cells in vivo and that co-transplanted hASCs expressed not only periostin but also α-smooth muscle actin (α-SMA), a marker of carcinoma-associated fibroblasts. Small interfering RNA- or short hairpin RNA-mediated silencing of periostin resulted in blockade of LPA-induced α-SMA expression in hASCs. In addition, silencing of periostin resulted in blockade of hASC-stimulated growth of A549 xenograft tumors and in vivo differentiation of transplanted hASCs to α-SMA-positive carcinoma-associated fibroblasts. Conditioned medium derived from LPA-treated hASCs (LPA CM) potentiated proliferation and adhesion of A549 cells and short interfering RNA-mediated silencing or immunodepletion of periostin from LPA CM abrogated proliferation and adhesion of A549 cells. These results suggest a pivotal role for hASC-secreted periostin in growth of A549 xenograft tumors within the tumor microenvironment.

Virulence factors released from Porphyromonas gingivalis induce electrophysiological dysfunction in human pluripotent stem cell-derived cardiomyocytes.

Background/purpose: Periodontal disease development correlates with the occurrence of systemic diseases. The present study investigated the association between periodontal disease and the development of cardiac arrhythmia. Materials and methods: Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) were treated with Porphyromonas gingivalis (Pg). Cardiotoxicity and electrophysiological properties of hESC-CMs were measured using the cell counting kit-8 assay and a multi-electrode array, respectively. Reverse-transcription-quantitative polymerase chain reaction (RT-qPCR) revealed the mRNA expression of S100 calcium binding protein A1 (S100A1), calsequestrin 2 (CASQ2), troponin I3 (TNNI3), myosin light chain 2 (MYL2), integrin subunit beta 1 (ITGB1), and cadherin 2 (CDH2) in hESC-CMs. Results: Treatment with Pg broth significantly decreased the beat period, field potential duration, spike amplitude, and conduction velocity without affecting the viability of hESC-CMs. In addition, the mRNA expression of CASQ2, TNNI3, and MYL2, which are all associated with calcium handling, were downregulated by Pg broth treatment. Conclusion: These findings indicate that Pg may induce cardiac arrhythmia mediated by virulence factors.