NQO1 promotes osteogenesis and suppresses angiogenesis in DPSCs via MAPK pathway modulation.

BACKGROUND: Influence on stem cells' angiogenesis and osteogenesis of NAD(P)H Quinone Dehydrogenase 1(NQO1) has been established, but its impact on dental pulp stem cells (DPSCs) is unexplored. An important strategy for the treatment of arteriosclerosis is to inhibit calcium deposition and to promote vascular repair and angiogenesis. This study investigated the function and mechanism of NQO1 on angiogenesis and osteogenesis of DPSCs, so as to provide a new ideal for the treatment of arteriosclerosis. METHODS: Co-culture of human DPSCs and human umbilical vein endothelial cells (HUVECs) was used to detect the angiogenesis ability. Alkaline phosphatase (ALP) activity, alizarin red staining (ARS), and transplantation of HA/tricalcium phosphate with DPSCs were used to detect osteogenesis. RESULTS: NQO1 suppressed in vitro tubule formation, migration, chemotaxis, and in vivo angiogenesis, as evidenced by reduced CD31 expression. It also enhanced ALP activity, ARS, DSPP expression and osteogenesis and boosted mitochondrial function in DPSCs. CoQ10, an electron transport chain activator, counteracted the effects of NQO1 knockdown on these processes. Additionally, NQO1 downregulated MAPK signaling, which was reversed by CoQ10 supplementation in DPSCs-NQO1sh. CONCLUSIONS: NQO1 inhibited angiogenesis and promoted the osteogenesis of DPSCs by suppressing MAPK signaling pathways and enhancing mitochondrial respiration.

STL Inhibited Angiogenesis of DPSCs Through Depressing Mitochondrial Respiration by Enhancing RNF217.

Angiogenesis is the determining factor during dental pulp regeneration. Six-twelve leukemia (STL) is identified as a key regulatory factor on the biological function of dental pulp stem cells (DPSCs) under hypoxic conditions, but its effect on angiogenesis is unclear. Co-culture of DPSCs and human umbilical vein endothelial cells (HUVECs) is used to detect tubule formation ability in vitro and the angiogenesis ability in vivo. RNA-seq and bioinformatic analyses are performed to screen differentially expressed genes. Seahorse Cell Mito Stress Test is proceeded to exam mitochondrial respiration. STL decreased tubule formation and mitochondrial respiration of DPSCs in vitro and restrained the number of blood vessels and the expression of VEGF in new formed tissue in vivo. Furthermore, pretreating STL-depleted DPSCs with rotenone, a mitochondrial respiration inhibitor, counteracted the promoting effect of STL knockdown on tubule formation. Then, RNA-seq and bioinformatic analyses identified some angiogenesis relevant genes and pathways in STL-depleted DPSCs. And STL enhanced expression of mRNA-ring finger protein 217 (RNF217), which inhibited the tubule formation and mitochondrial respiration of DPSCs. STL inhibited the angiogenesis of DPSCs through depressing mitochondrial respiration by enhancing RNF217, indicating that STL is a potential target for angiogenesis of DPSCs.

KDM4D enhances osteo/dentinogenic differentiation and migration of SCAPs via binding to RPS5.

OBJECTIVES: Stem cells of the apical papilla (SCAPs) provide promising candidates for dental pulp regeneration. Despite great advances in the transcriptional controls of the SCAPs fate, little is known about the regulation of SCAP differentiation. MATERIALS AND METHODS: Short hairpin RNAs and full-length RNA were used to deplete or overexpress lysine demethylase 4D (KDM4D) gene expression. Western blotting, real-time RT-PCR, alizarin red staining, and scratch migration assays were used to study the role of KDM4D and the ribosomal protein encoded by RPS5 in SCAPs. RNA microarray, chromatin Immunoprecipitation (ChIP), and co-immunoprecipitation (Co-IP) assays were performed to explore the underlying molecular mechanisms. RESULTS: KDM4D enhanced the osteo/dentinogenic differentiation, migration, and chemotaxis of SCAPs. The microarray results revealed that 88 mRNAs were differentially expressed in KDM4D-overexpressed SCAPs. ChIP results showed knock-down of KDM4D increased the level of H3K9me2 and H3K9me3 in CNR1 promoter region. There were 37 possible binding partners of KDM4D. KDM4D was found to combine with RPS5, which also promoted the osteo/dentinogenic differentiation, migration, and chemotaxis of SCAPs. CONCLUSIONS: KDM4D promoted the osteo/dentinogenic differentiation and migration potential of SCAPs in combination with RPS5, which provides a therapeutic clue for improving SCAPs-based dental tissue regeneration.

miR-196b-5p inhibits proliferation of Wharton's jelly umbilical cord stem cells.

Human umbilical cord mesenchymal stem cells can be obtained from different parts of the umbilical cord, including Wharton's jelly. Transplantation of Wharton's jelly umbilical cord stem cells (WJCMSCs) is a promising strategy for the treatment of various diseases. However, the molecular mechanisms underlying the proliferation of WJCMSCs are incompletely understood. Here, we report that overexpression of miR-196b-5p in WJCMSCs suppresses proliferation and arrests the cell cycle in G0/G1 phase, whereas knockdown of miR-196b-5p promotes WJCMSC proliferation and cell-cycle progression. Moreover, miR-196b-5p overexpression resulted in decreased levels of Cyclin A, Cyclin D, Cyclin E and cyclin-dependent kinases 2 and increased levels of p15INK4b , whereas miR-196b-5p knockdown had the opposite effects. In conclusion, our data suggests that miR-196b-5p inhibits WJCMSC proliferation by enhancing G0/G1-phase arrest.

Depletion of SNRNP200 inhibits the osteo-/dentinogenic differentiation and cell proliferation potential of stem cells from the apical papilla.

BACKGROUND: Tissue regeneration mediated by mesenchymal stem cells (MSCs) is deemed a desirable way to repair teeth and craniomaxillofacial tissue defects. Nevertheless, the molecular mechanisms about cell proliferation and committed differentiation of MSCs remain obscure. Previous researches have proved that lysine demethylase 2A (KDM2A) performed significant function in the regulation of MSC proliferation and differentiation. SNRNP200, as a co-binding factor of KDM2A, its potential effect in regulating MSCs' function is still unclear. Therefore, stem cells from the apical papilla (SCAPs) were used to investigate the function of SNRNP200 in this research. METHODS: The alkaline phosphatase (ALP) activity assay, Alizarin Red staining, and osteogenesis-related gene expressions were used to examine osteo-/dentinogenic differentiation potential. Carboxyfluorescein diacetate, succinimidyl ester (CFSE) and cell cycle analysis were applied to detect the cell proliferation. Western blot analysis was used to evaluate the expressions of cell cycle-related proteins. RESULTS: Depletion of SNRNP200 caused an obvious decrease of ALP activity, mineralization formation and the expressions of osteo-/dentinogenic genes including RUNX2, DSPP, DMP1 and BSP. Meanwhile, CFSE and cell cycle assays revealed that knock-down of SNRNP200 inhibited the cell proliferation and blocked cell cycle at the G2/M and S phase in SCAPs. In addition, it was found that depletion of SNRNP200 up-regulated p21 and p53, and down-regulated the CDK1, CyclinB, CyclinE and CDK2. CONCLUSIONS: Depletion of SNRNP200 repressed osteo-/dentinogenic differentiation potentials and restrained cell proliferation through blocking cell cycle progression at the G2/M and S phase, further revealing that SNRNP200 has crucial effects on preserving the proliferation and differentiation potentials of dental tissue-derived MSCs.

miR-4651 inhibits cell proliferation of gingival mesenchymal stem cells by inhibiting HMGA2 under nifedipine treatment.

Drug-induced gingival overgrowth (DIGO) is recognized as a side effect of nifedipine (NIF); however, the underlying molecular mechanisms remain unknown. In this study, we found that overexpressed miR-4651 inhibits cell proliferation and induces G0/G1-phase arrest in gingival mesenchymal stem cells (GMSCs) with or without NIF treatment. Furthermore, sequential window acquisition of all theoretical mass spectra (SWATH-MS) analysis, bioinformatics analysis, and dual-luciferase report assay results confirmed that high-mobility group AT-hook 2 (HMGA2) is the downstream target gene of miR-4651. Overexpression of HMGA2 enhanced GMSC proliferation and accelerated the cell cycle with or without NIF treatment. The present study demonstrates that miR-4651 inhibits the proliferation of GMSCs and arrests the cell cycle at the G0/G1 phase by upregulating cyclin D and CDK2 while downregulating cyclin E through inhibition of HMGA2 under NIF stimulation. These findings reveal a novel mechanism regulating DIGO progression and suggest the potential of miR-4651 and HMGA2 as therapeutic targets.

FAM96B inhibits the senescence of dental pulp stem cells.

Dental pulp stem cells (DPSCs) are considered a remarkable source for the regeneration of dental pulp tissues, but their therapeutic effectiveness remains limited, especially in elderly people. Previous studies found that senescence has a negative effect on the proliferation and differentiation potential of DPSCs. Moreover, numerous long non-coding RNA (lncRNA) and messenger RNA were significantly differentially regulated in DPSCs from young and elderly donors. However, the changes in DPSCs protein during senescence have not been addressed. In this study, differences in DPSC protein expression profiles and coexpression of protein and lncRNA were analyzed using proteomics and bioinformatics. The results showed 75 upregulated proteins and 69 downregulated proteins in DPSCs from elderly donors. Vasopressin-regulated water reabsorption, Parkinson's disease, Alzheimer's disease, and protein export were the top four functional pathways associated with DPSCs. High mobility group N1 (HMGN1), HMGN2, UCHL1, and the family with sequence similarity 96 member B homeobox gene (FAM96B) were associated with DPSCs senescence. Then, we investigated FAM96B function in DPSCs. After FAM96B depletion, telomerase reverse transcriptase (TERT) activity decreased, but the number of senescence-associated ß-galactosidase (SA-ß-gal) positive cells and the protein levels of p16, p53 were significantly increased. Gain-of-function assays suggested that FAM96B overexpression was positively correlated with TERT activity, but negatively correlated with the number of SA-ß-gal positive cells and the protein levels of P16 and P53. Moreover, after FAM96B overexpression, the results showed a significant increase in alkaline phosphatase activity and an enhanced mineralization ability of DPSCs. The reverse-transcription polymerase chain reaction results also showed that dentin sialophosphoprotein and osteocalcin were expressed at greater levels. The carboxyfluorescein succinimidyl ester (CFSE) results displayed that FAM96B increased the proliferation potential of DPSCs. Our study revealed candidate proteins that might be related to DPSCs senescence and provided information to elucidate the mechanism of the biological changes in DPSCs' aging. Moreover, FAM96B was demonstrated to play an important role in suppressing DPSCs senescence and promoting osteogenic differentiation and proliferation.

Epiregulin promotes osteogenic differentiation and inhibits neurogenic trans-differentiation of adipose-derived mesenchymal stem cells via MAPKs pathway.

Mesenchymal stem cells (MSCs) exists low efficiency to trans-differentiate into other germinal layer cell types. One key issue is to discover the effect of important factor on MSCs differentiation abiltiy. In this study, we investigated the role and mechanism of epiregulin (EREG) on the osteogenic differentiation and neurogenic trans-differentiation in adipose-derived stem cells (ADSCs). We discovered that the depletion of EREG inhibited the osteogenic differentiation in vitro. And 25 ng/mL recombinant human epiregulin protein (rhEREG) effectively improved the osteogenic differentiation of EREG-depleted-ADSCs. Depletion of EREG promoted the formation of neural spheres, and increased the expressions of nestin, ßIII-tubulin, NeuroD, NCAM, TH, and NEF in ADSCs. Then, 25 ng/mL rhEREG significantly inhibited these neurogenic differentiation indicators. Inhibition of p38 MAPK, JNK, or Erk1/2 signaling pathway separately, blocked the rhEREG-enhanced osteogenic differentiation ability and the rhEREG-inhibited neurogenic trans-differentiation ability of ADSCs. In conclusions, EREG promoted the osteogenic differentiation and inhibited the neurogenic trans-differentiation potentials of ADSCs via MAPK signaling pathways.

SFRP2 promotes stem cells from apical papilla-mediated periodontal tissue regeneration in miniature pig.

Mesenchymal stem cell-based therapy is a reliable treatment for periodontal tissue regeneration, while ideal regeneration rate is still a facing problem. In previous study, we found SFRP2 a promising gene in modulating mesenchymal stem cells potential. We further investigated its role on periodontal tissue regeneration. We created periodontitis model in miniature pigs and locally injected with stem cells from apical papilla (SCAP). The periodontitis models were classed into three groups, SFRP2-SCAP group (injected with SCAP overexpressing with SFRP2), SCAP group (injected with SCAP transduced with vector backbone) and saline group (vehicle group injected with saline). Clinical assignment, CT scanning, histopathological assessment and quantitative analysis were applied to evaluate the regeneration effect. Twelve weeks after the injection, we found healthier gingival status in SFRP2-SCAP group than the other two groups. Clinical assignment results showed values of probing depth, gingival recession and attachment loss were improved in SFRP2-SCAP group than that of SCAP group and saline group. The volume of newborn bone was also enhanced in SFRP2-SCAP group than SCAP group and saline group. The difference of clinical assignments and newborn bone between each group was significant relevant. HE staining demonstrated increased tissue regeneration in SFRP2-SCAP group than SCAP group and saline group. Our findings revealed that SFRP2 could enhance SCAP-mediated periodontal tissue regeneration and provide a potential target for improving the regeneration of periodontal tissue.

Analysis of the characteristics and expression profiles of coding and noncoding RNAs of human dental pulp stem cells in hypoxic conditions.

BACKGROUND: Human dental pulp stem cell (DPSC)-mediated regenerative endodontics is a promising therapy for damaged teeth; however, the hypoxic environment in root canals can affect tissue regeneration. In this study, we investigate the characteristics and possible regulatory mechanisms of DPSC function under hypoxic conditions. METHODS: Human DPSCs were cultured under normoxia (20% O2) and hypoxia (3% O2). DPSC proliferation and osteo/odontogenic differentiation potential were assessed by Cell Counting Kit-8 (CCK8) assay, carboxyfluorescein succinimidyl ester (CFSE) assay, alkaline phosphatase (ALP) activity, Alizarin red staining, real-time RT-PCR assays, and western blot analysis. Microarray and bioinformatic analyses were performed to investigate the differences in the mRNA, lncRNA, and miRNA expression profiles of DPSCs. RESULTS: DPSCs exhibited a more powerful proliferation ability and lower osteo/odontogenic differentiation potential in hypoxic conditions. A total of 60 mRNAs (25 upregulated and 35 downregulated), 47 lncRNAs (20 upregulated and 27 downregulated), and 14 miRNAs (7 upregulated and 7 downregulated) in DPSCs were differentially expressed in the hypoxia group compared with the normoxia group. Bioinformatic analysis identified that 7 mRNAs (GRPR, ERO1L, ANPEP, EPHX1, PGD, ANGPT1, and NQO1) and 5 lncRNAs (AF085958, AX750575, uc002czn.2, RP3-413H6.2, and six-twelve leukemia (STL)) may be associated with DPSCs during hypoxia according to CNC network analysis, while 28 mRNAs (including GYS1, PRKACB, and NQO1) and 13 miRNAs (including hsa-miR-3916 and hsa-miR-192-5p) may be involved according to miRNA target gene network analysis. The depletion of one candidate lncRNA, STL, inhibited the osteo/odontogenic differentiation potentials of DPSCs. CONCLUSIONS: Our results revealed that hypoxia could enhance the proliferation ability and impair the osteo/odontogenic differentiation potential of DPSCs in vitro. Furthermore, our results identified candidate coding and noncoding RNAs that could be potential targets for improving DPSC function in regenerative endodontics and lead to a better understanding of the mechanisms of hypoxia's effects on DPSCs.

[Increase of saliva nitrate and nitrite level in patients with oral candidiasis].

OBJECTIVE: To observe the alterations of saliva nitrate and nitrite level in patients with oral candidiasis. METHODS: Parotid saliva and whole saliva were collected from 33 patients and 34 healthy volunteers. Concentrations of nitrate and nitrite in saliva were determined by high-performance liquid chromatography. Follow-up observation was performed on 10 patients after treatment. The data were statistically analyzed with independent-samples t test or paired-samples t test at alpha = 0.05. RESULTS: There was significant increase of the concentrations and secretion rate of parotid saliva nitrate in patient group as compared with controls: (49.70 +/- 0.50) vs (21.51 +/- 0.60) mg/L (t = 2.692, P = 0.009) and (27.71 +/- 0.50) vs (12.55 +/- 0.60) microg/min (t = 2.554, P = 0.013), respectively. Significantly increased concentrations and secretion rate of nitrate and nitrite [nitrate: (6.46 +/- 0.94) vs (1.11 +/- 0.70) mg/L (t = 3.792, P = 0.000); nitrite: (8.48 +/- 0.58) vs (3.39 +/- 0.53) mg/L (t = 2.888, P = 0.005); nitrate secretion rate: (10.57 +/- 0.91) vs (2.10 +/- 0.74) microg/min (t = 3.464, P= 0.001); nitrite secretion rate: (13.91 +/- 0.55) vs (6.42 +/- 0.58) microg/min (t = 2.397, P = 0.020)] were revealed in whole saliva of patients group. Significantly decreased nitrate and nitrite levels were also observed in patients after treatment, especially the changes of parotid saliva nitrate secretion rate [(37.50 +/- 0.50) vs (14.34 +/- 0.64) microg/min (t = 3.142, P = 0.012)], whole saliva nitrate [(14.29 +/- 1.01) vs (2.59 +/- 1.03) mg/L (t = 3.475, P = 0.007)] and whole saliva nitrate secretion rate [(25.97 +/- 0.93) vs (4.12 +/- 1.00) microg/min (t = 3.922, P = 0.003)]. CONCLUSION: The present study revealed the significant increase of salivary nitrate and nitrite level in patients with oral candidiasis is considered to be associated with the host defense reaction.

[Clinical and pathological analysis of oral manifestations of 40 patients with secondary syphilis].

OBJECTIVE: To analyze the clinical and pathological features of 40 patients with secondary syphilis. METHODS: A total of 40 cases of secondary syphilis confirmed by serology were collected during 1994-2004 and were first diagnosed on presentation with oral lesions. RESULTS: The white patch in oral mucosa was found in 32 cases with painless or slight pain in most cases. The most common site of the lesion was the tongue. The histological examination on eight cases was initially misdiagnosed as oral candidosis or lichen planus, but confirmed as syphilis after serology revealed nonspecific inflammation with intraepithelial microabscess and dense perivascular infiltration of lymphocytes and plasma cells in connective tissue. The symptoms showed dramatic improvement in 16 cases after benzathine penicillin treatment. CONCLUSIONS: The oral manifestations of syphilis have specific clinical and pathological feature and attention should be paid to the suspicious oral lesions when patients are first presented in a dental office.