Lysyl oxidase-mediated VEGF-induced differentiation and angiogenesis in human dental pulp cells.

AIM: To investigate the effects of recombinant human vascular endothelial growth factor (rhVEGF) on odontoblastic differentiation, in vitro angiogenesis, and expression and activity of lysyl oxidase (LOX) in human dental pulp cells (HDPCs), compared with rhFGF-2. To identify the underlying molecular mechanisms, the study focused on whether LOX was responsible for the actions of rhVEGF. METHODOLOGY: Recombinant human vascular endothelial growth factor (rhVEGF) was constructed using the pBAD-HisA plasmid in Escherichia coli. HDPCs were treated with 1-50 µg mL-1 rhVEGF for 14 days. Alkaline phosphatase (ALP) activity was measured, and the formation of calcified nodules was assessed using alizarin red staining after the induction of odontogenic differentiation of HDPCs. The expression level of the odontogenic differentiation markers was detected by reverse transcription polymerase chain reaction. Signal pathways were assessed by Western blot and immunocytochemistry. The data were analysed by anova with Bonferroni's test (α = 0.05). RESULTS: Recombinant human vascular endothelial growth factor significantly increased cell growth (P < 0.05), ALP activity (P < 0.05) and mineralization nodule formation and upregulated the mRNA expression levels of the osteogenic/odontogenic markers that were lower with rhFGF-2. rhVEGF significantly increased amine oxidase activity (P < 0.05) and upregulated LOX and LOXL mRNA expression in HDPCs. Additionally, rhVEGF dose-dependently upregulated angiogenic gene mRNAs and capillary tube formation to a greater degree than rhFGF-2. Inhibition of LOX using ß-aminopropionitrile (BAPN) and LOX or LOXL gene silencing by RNA interference attenuated rhVEGF-induced growth, ALP activity, mineralization, the expression of marker mRNAs and in vitro angiogenesis. Furthermore, treatment with rhVEGF resulted in phosphorylation of Akt, ERK, JNK and p38, and activation of NF-κB, which was inhibited by LOX or LOXL silencing and BAPN. CONCLUSION: Recombinant human vascular endothelial growth factor promoted cell growth, odontogenic potential and in vitro angiogenesis via modulation of LOX expression. These results support the concept that rhVEGF may offer therapeutic benefits in regenerative endodontics.

Role of PIN1 on in vivo periodontal tissue and in vitro cells.

BACKGROUND: Although expression of peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (PIN1) was reported in bone tissue, the precise role of PIN1 in periodontal tissue and cells remain unclear. MATERIAL & METHODS: To elucidate the roles of PIN1 in periodontal tissue, its expression in periodontal tissue and cells, and effects on in vitro 4 osteoblast differentiation and the underlying signaling mechanisms were evaluated. RESULTS: PIN1 was expressed in mouse periodontal tissues including periodontal ligament cells (PDLCs), cementoblasts and osteoblasts at the developing root formation stage (postnatal, PN14) and functional stage of tooth (PN28). Treatment of PIN1 inhibitor juglone, and gene silencing by RNA interference promoted osteoblast differentiation in PDLCs and cementoblasts, whereas the overexpression of PIN1 inhibited. Moreover, osteogenic medium-induced activation of AMPK, mTOR, Akt, ERK, p38 and NF-jB pathways were enhanced by PIN1 siRNA, but attenuated by PIN1 overexpression. Runx2 expressions were induced by PIN1 siRNA, but downregulated by PIN1 overexpression. CONCLUSION: In summary, this study is the first to demonstrate that PIN1 is expressed in developing periodontal tissue, and in vitro PDLCs and cementoblasts. PIN1 inhibition stimulates osteoblast differentiation, and thus may play an important role in periodontal regeneration.

Ureteral Complications in Kidney Transplantation: Analysis and Management of 853 Consecutive Laparoscopic Living-Donor Nephrectomies in a Single Center.

BACKGROUND: We report the incidence and nature of ureteral and surgical complications in our series of 853 consecutive living-donor renal transplants after laparoscopic living-donor nephrectomy. The aim of this study was to analyze the therapeutic approaches to ureteral complications in kidney transplantations and their relationship with recipient outcome. METHODS: The medical records of patients who underwent kidney transplantation from 2000 to 2014 were reviewed retrospectively. After the donor nephrectomies were performed with the use of laparoscopic, hand-assisted laparoscopic, and vesico-ureteral anastomosis, the recipient's ureteral complications were classified according to the mechanism and site of urinary tract involvement: anastomosis stricture, anastomosis leakage, vesico-ureteral reflux, and urolithiasis. RESULTS: Among the 853 cases of kidney transplantation, ureteral complications occurred in 66 patients (7.73%). The most common complication was urinary tract infection caused by vesico-ureteral reflux (n = 24, 2.81%), which was managed with by means of sub-ureteral polydimethylsiloxane injection. The second most common complication was the anastomosis site stricture (n = 23, 2.69%), which was treated by means of ureteral re-implantation or percutaneous nephrostomy. Anastomosis site leakage occurred in 11 patients (1.28%) and was managed by percutaneous nephrostomy with double-J stenting and drainage or ureteral re-implantation. Urolithiasis occurred in 8 patients (0.93%). CONCLUSIONS: There was an 8% rate of recipient ureteral complications at our institution. Of the 66 patients, 46 (5.4%) required surgical repair. The remaining 20 patients with ureteral complications were treated with conservative care or minimally invasive procedures. The keys to successful management of these problems are early diagnosis and prompt reconstruction whenever possible. Most ureteral complications are easily managed with a successful outcome with early intervention.

PIN1 Suppresses the Hepatic Differentiation of Pulp Stem Cells via Wnt3a.

This study aimed to investigate the role of PIN1 on the hepatic differentiation of human dental pulp stem cells (hDPSCs) and its signaling pathway, as well as the potential therapeutic effects of hDPSC transplantation and PIN1 inhibition on CCl4 (carbon tetrachloride)-induced liver fibrosis in mice. The in vitro results showed that hepatic differentiation was suppressed by infection with adenovirus-PIN1 and promoted by PIN1 inhibitor juglone via the downregulation of Wnt3a and ß-catenin. Compared with treatment with either hDPSC transplantation or juglone alone, the combination of hDPSCs and juglone into CCl4-injured mice significantly suppressed liver fibrosis and restored serum levels of alanine transaminase, aspartate transaminase, and ammonia. Collectively, the present study shows for the first time that PIN1 inhibition promotes hepatic differentiation of hDPSCs through the Wnt/ß-catenin pathway. Furthermore, juglone in combination with hDPSC transplantation effectively treats liver fibrosis, suggesting that hDPSC transplantation with PIN1 inhibition may be a novel therapeutic candidate for the treatment of liver injury.

Anti-inflammatory and anti-osteoclastogenic effects of zinc finger protein A20 overexpression in human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Although overexpression of the nuclear factor κB inhibitory and ubiquitin-editing enzyme A20 is thought to be involved in the pathogenesis of inflammatory diseases, its function in periodontal disease remains unknown. The aims of the present study were to evaluate A20 expression in patients with periodontitis and to study the effects of A20 overexpression, using a recombinant adenovirus encoding A20 (Ad-A20), on the inflammatory response and on osteoclastic differentiation in lipopolysaccharide (LPS)- and nicotine-stimulated human periodontal ligament cells (hPDLCs). MATERIAL AND METHODS: The concentration of prostaglandin E2 was measured by radioimmunoassay. Reverse transcription-polymerase chain reactions and western blot analyses were used to measure mRNA and protein levels, respectively. Osteoclastic differentiation was assessed in mouse bone marrow-derived macrophages using conditioned medium from LPS- and nicotine-treated hPDLCs. RESULTS: A20 was upregulated in the gingival tissues and neutrophils from patients with periodontitis and in LPS- and nicotine-exposed hPDLCs. Pretreatment with A20 overexpression by Ad-A20 markedly attenuated LPS- and nicotine-induced production of prostaglandin E2 , as well as expression of cyclooxygenase-2 and proinflammatory cytokines. Moreover, A20 overexpression inhibited the number and size of tartrate-resistant acid phosphatase-stained osteoclasts, and downregulated osteoclast-specific gene expression. LPS- and nicotine-induced p38 phosphorylation and nuclear factor κB activation were blocked by Ad-A20. Ad-A20 inhibited the effects of nicotine and LPS on the activation of pan-protein kinase C, Akt, GSK-3ß and protein kinase Cα. CONCLUSIONS: This study is the first to demonstrate that A20 overexpression has anti-inflammatory effects and blocks osteoclastic differentiation in a nicotine- and LPS-stimulated hPDLC model. Thus, A20 overexpression may be a potential therapeutic target in inflammatory bone loss diseases, such as periodontal disease.

PIN1 inhibition suppresses osteoclast differentiation and inflammatory responses.

Inflammatory responses and osteoclast differentiation play pivotal roles in the pathogenesis of osteolytic bone diseases such as periodontitis. Although overexpression or inhibition of peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (PIN1) offers a possible therapeutic strategy for chronic inflammatory diseases, the role of PIN1 in periodontal disease is unclear. The aim of the present study was to evaluate PIN1 expression in periodontitis patients as well as the effects of PIN1 inhibition by juglone or PIN1 small-interfering RNA (siRNA) and of PIN1 overexpression using a recombinant adenovirus encoding PIN1 (Ad-PIN1) on the inflammatory response and osteoclastic differentiation in lipopolysaccharide (LPS)- and nicotine-stimulated human periodontal ligament cells (PDLCs). PIN1 was up-regulated in chronically inflamed PDLCs from periodontitis patients and in LPS- and nicotine-exposed PDLCs. Inhibition of PIN1 by juglone or knockdown of PIN1 gene expression by siRNA markedly attenuated LPS- and nicotine-stimulated prostaglandin E2 (PGE2) and nitric oxide (NO) production, as well as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression, whereas PIN1 overexpression by Ad-PIN1 increased it. LPS- and nicotine-induced nuclear factor (NF)-κB activation was blocked by juglone and PIN1 siRNA but increased by Ad-PIN1. Conditioned medium prepared from LPS- and nicotine-treated PDLCs increased the number of tartrate-resistant acid phosphatase-stained osteoclasts and osteoclast-specific gene expression. These responses were blocked by PIN1 inhibition and silencing but stimulated by Ad-PIN1. Furthermore, juglone and PIN1 siRNA inhibited LPS- and nicotine-induced osteoclastogenic cytokine expression in PDLCs. This study is the first to demonstrate that PIN1 inhibition exhibits anti-inflammatory effects and blocks osteoclastic differentiation in LPS- and nicotine-treated PDLCs. PIN1 inhibition may be a therapeutic strategy for inflammatory osteolysis in periodontal disease.

Oct4 is a critical regulator of stemness in head and neck squamous carcinoma cells.

Cancer stem cells (CSCs) have been suggested as responsible for the initiation and progression of cancers. Octamer-binding transcription factor 4 (Oct4) is an important regulator of embryonic stem cell fate. Here, we investigated whether Oct4 regulates stemness of head and neck squamous carcinoma (HNSC) CSCs. Our study showed that ectopic expression of Oct4 promotes tumor growth through cyclin E activation, increases chemoresistance through ABCC6 expression and enhances tumor invasion through slug expression. Also, Oct4 dedifferentiates differentiated HNSC cells to CSC-like cells. Furthermore, Oct4(high) HNSC CSCs have more stem cell-like traits compared with Oct4(low) cells, such as self-renewal, stem cell markers' expression, chemoresistance, invasion capacity and xenograft tumorigeneity in vitro and in vivo. In addition, knockdown of Oct4 led to markedly lower HNSC CSC stemness. Finally, there was a significant correlation between Oct4 expression and survival of 119 HNSC patients. Collectively, these data suggest that Oct4 may be a critical regulator of HNSC CSCs and its targeting may be potentially valuable in the treatment of HNSC CSCs.

Anti-inflammatory effects of glutamine on LPS-stimulated human dental pulp cells correlate with activation of MKP-1 and attenuation of the MAPK and NF-κB pathways.

AIM: To evaluate the anti-inflammatory effects of glutamine and the underlying signal pathway mechanisms in lipopolysaccharide (LPS)-stimulated human dental pulp cells (HDPCs). METHODS: Human dental pulp cells were exposed to 10 µg mL(-1) LPS and various concentrations of glutamine for 24 h. The production of PGE2 and nitric oxide was determined by enzyme-linked immunosorbent assay (ELISA) and Griess reagent kit, respectively. Cytokines were examined by ELISA, reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time PCR. iNOS and COX protein expression as well as signal pathways were accessed by Western blot. The data were analysed by anova with Bonferroni's test (α = 0.05). RESULTS: Glutamine reduced LPS-induced iNOS and COX-2 protein expression as well as production of NO and PGE2 in a dose-dependent fashion. Additionally, glutamine suppressed the production and mRNA expression of inflammatory cytokines including interleukin-1ß (IL-1ß), TNF-α, and IL-8. Furthermore, glutamine attenuated phosphorylation of extracellular signal-regulated kinase (ERK), p38, c-Jun N-terminal kinase (JNK) and IκB-α, and nuclear translocation of NF-κB p65, but enhanced mitogen-activated protein kinase phosphatase-1 (MKP-1) expression in LPS-treated HDPCs. CONCLUSION: Glutamine exerted an anti-inflammatory effect via activation of MKP-1 and inhibition of the NF-κB and MAPK pathways in LPS-treated HDPCs.

SOX2 regulates self-renewal and tumorigenicity of stem-like cells of head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinomas (HNSCCs) display cellular heterogeneity and contain cancer stem cells (CSCs). Sex-determining region Y [SRY]-box (SOX)2 is an important regulator of embryonic stem cell fate and is aberrantly expressed in several types of human tumours. Nonetheless, the role of SOX2 in HNSCC remains unclear. METHODS: We created cells ectopically expressing SOX2 from previously established HNSCC cells and examined the cell proliferation, self-renewal capacity, and chemoresistance of these cells compared with control cells. In addition, we knocked down SOX2 in primary spheres obtained from HNSCC tumour tissue and assessed the attenuation of stemness-associated traits in these cells in vitro and in vivo. Furthermore, we examined the clinical relevance of SOX2 expression in HNSCC patients. RESULTS: SOX2 is aberrantly expressed in primary tissue of HNSCC patients but not in healthy tissue. SOX2 expression correlated with tumour recurrence and poor prognosis of HNSCC patients. Ectopic expression of SOX2 induced cell proliferation via cyclin B1 expression and stemness-associated features, such as self-renewal and chemoresistance. In addition, a knockdown of SOX2 in HNSCC CSCs attenuated their self-renewal capacity, chemoresistance (through ABCG2 suppression), invasion capacity (via snail downregulation), and in vivo tumorigenicity. CONCLUSIONS: These results suggest that SOX2 may have important roles in the 'stemness' and progression of HNSCC. Targeting SOX2-positive tumour cells (CSCs) could be a new therapeutic strategy in HNSCCs.

The role of SDF-1 and CXCR4 on odontoblastic differentiation in human dental pulp cells.

AIM: To examine the role of stromal cell-derived factor 1 (SDF-1) signalling during odontogenic differentiation in human dental pulp cells (HDPCs). METHODOLOGY: Human dental pulp cells were treated with differentiation medium, recombinant human SDF-1, neutralizing antibody for SDF-1 or CXCR4, pertussis toxin (PTX) and AMD3100. The expression of SDF-1 and its receptor chemokine receptor type 4 (CXCR4) was measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Odontoblastic differentiation was determined using alkaline phosphatase (ALP) activity assay, mineralized nodule formation and marker mRNAs by RT-PCR. RESULTS: Marked upregulation of SDF-1 and CXCR4 mRNA and protein was observed in cells grown 7 days in osteogenic induction medium. The addition of recombinant human SDF-1 to HDPCs significantly (P < 0.05) increased ALP activity, mineralized nodule formation and odontoblast marker mRNAs in a dose-dependent manner. Blocking SDF-1 signalling using antibodies against SDF-1 or CXCR4, or the G-protein-coupled receptor inhibitor PTX, and CXCR4 inhibitor AMD3100, strongly suppressed induction of odontogenic differentiation in HDPCs. CONCLUSIONS: Odontoblastic differentiation was stimulated by SDF-1 activation and repressed by SDF-1/CXCR4 inhibition. Thus, SDF-1/CXCR4 signalling may be a new therapeutic target and strategy to promote repair and regeneration in endodontics.

Nicotine and lipopolysaccharide stimulate the production of MMPs and prostaglandin E2 by hypoxia-inducible factor-1α up-regulation in human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Although hypoxia-inducible factor 1α (HIF-1α) is up-regulated in the periodontal pockets of periodontitis patients, the expression and precise molecular mechanisms of HIF-1α remain unknown in human periodontal ligament cells (PDLCs). The aim of this study was to explore the effects, as well as the signaling pathway, of nicotine and lipopolysaccharide (LPS) on the expression of HIF-1α and on the production of its target genes, including cyclooxygenase-2 (COX-2)-derived prostaglandin E(2) (PGE(2) ), MMP-2 and MMP-9 in PDLCs. MATERIAL AND METHODS: The expression of COX-2 and HIF-1α proteins was evaluated using western blotting. The production of PGE(2) and MMPs was evaluated using enzyme immunoassays and zymography, respectively. RESULTS: LPS and nicotine synergistically induced the production of PGE(2) , MMP-2 and MMP-9, and increased the expression of MMP-2, MMP-9, COX-2 and HIF-1α proteins. Inhibition of HIF-1α activity by chetomin or knockdown of HIF1α gene expression by small interfering RNA markedly attenuated the production of LPS- and nicotine-stimulated PGE(2) and MMPs, as well as the expression of COX-2 and HIF-1α. Furthermore, pretreatment with inhibitors of COX-2, p38, extracellular signal-regulated kinase, Jun N-terminal kinase, protein kinase C, phosphatidylinositol 3-kinase and nuclear factor-kappaB decreased the expression of nicotine- and LPS-induced HIF-1α and COX-2, as well as the activity of PGE(2) and MMPs. CONCLUSION: These data demonstrate novel mechanisms by which nicotine and LPS promote periodontal tissue destruction, and provide further evidence that HIF-1α is a potential target in periodontal disease associated with smoking and dental plaque.

Heme oxygenase-1 protects human periodontal ligament cells against substance P-induced RANKL expression.

BACKGROUND AND OBJECTIVE: Although substance P (SP) stimulates bone resorption activity and this is reported to be correlated with the degree of periodontal inflammation, it is unclear how human periodontal ligament cells regulate neuropeptide-induced osteoclastogenesis or the possible involvement of heme oxygenase-1 (HO-1) might be. This study examines how SP affects osteoprotegerin (OPG) and RANKL expression via HO-1. MATERIAL AND METHODS: Using immortalized human periodontal ligament cells, the effects of SP on the expression of HO-1, RANKL and OPG mRNA and proteins were determined by RT-PCR and western blotting, respectively. Various concentrations of SP (10(-7), 10(-8), 10(-9) and 10(-10) m) were added to the medium, and the cells were treated for 0, 0.25, 0.5, 1, 2 and 3 d. RESULTS: Substance P upregulated RANKL and HO-1 and downregulated OPG mRNA and protein expression in periodontal ligament cells, in a concentration- and time-dependent manner. A HO-1 inducer inhibited both the upregulation of RANKL expression and downregulation of OPG expression by SP in periodontal ligament cells. By contrast, treatment with a HO-1 inhibitor or HO-1 small interferring RNA (siRNA) enhanced SP-stimulated RANKL expression. Inhibitors of ERK and p38 MAP kinases, phosphoinositide 3-kinase and nuclear factor-kappaB blocked the effects of SP on RANKL expression in periodontal ligament cells. CONCLUSION: These results suggest that SP stimulates osteoclastic differentiation by increasing the expression of RANKL vs. OPG via the HO-1 pathway in periodontal ligament cells. The HO-1 pathway may be an effective therapeutic target for inhibiting chronic periodontitis involving alveolar bone resorption.

N(1)-guanyl-1,7,-diamineoheptane, an inhibitor of deoxyhypusine synthase, suppresses differentiation and induces apoptosis via mitochondrial and AMPK pathways in immortalized and malignant human oral keratinocytes.

BACKGROUND: Although N(1)-guanyl-1,7,-diamineoheptane (GC7), an inhibitor of deoxyhypusine synthase, has been shown to inhibit cell growth, the mechanism of its action is not completely understood. In this study, we investigated the mechanisms of the effects of GC7 on cell growth, differentiation and apoptosis in relation to adenosine monophosphate-activated protein kinase (AMPK) activation, as AMPK is known to be a possible target for cancer treatment. METHODS: The effects of GC7 on the growth of immortalized human oral keratinocytes (IHOK) and primary oral cancer cells (HN4), was investigated using MTT assay, Western blotting, cell cycle analysis, DNA fragmentation and expression of apoptotic pathway proteins. RESULTS: N(1)-guanyl-1,7,-diamineoheptane inhibited cell proliferation in a time- and dose-dependent manner in IHOK and HN4 cells. GC7 treatment decreased the expression of differentiation markers, such as involucrin, CK13 and CK19. The major mechanism of growth inhibition by GC7 treatment was induction of apoptosis, which is supported by sub-G(1) phase arrest, annexin V-FITC staining and DNA fragmentation analysis. GC7 treatment increased the cytosolic level of cytochrome c and resulted in caspase-3 activation. GC7 treatment also resulted in a strong activation of AMPK. Furthermore, specific AMPK activator blocked the GC7-induced growth inhibition effect, as well as apoptosis. CONCLUSION: These results demonstrate that GC7 blocks immortalized and malignant keratinocyte cell proliferation and differentiation by inducing apoptosis through the mitochondrial and AMPK pathways. On the basis of these observations, we propose that a strategy combining GC7 and AMPK inhibition could be developed into a novel chemotherapeutic modality in oral cancer.