Periodontitis is a multifactorial disease that progresses via dynamic interaction between bacterial and host-derived genetic factors. The recent trend of omics analyses has discovered many ...periodontitis-related risk factors. However, how much the individual factor affects the pathogenesis of periodontitis is still unknown. This article aims to identify multiple key factors related to the pathogenesis of periodontitis and quantitatively predict the influence of each factor on alveolar bone resorption by omics analysis and mathematical modeling. First, we induced periodontitis in mice (n = 3 or 4 at each time point) by tooth ligation. Next, we assessed alveolar bone resorption by micro–computed tomography, alterations in the gene expression by RNA sequencing, and the microbiome of the gingivae by 16S ribosomal RNA sequencing during disease pathogenesis. Omics data analysis identified key players (bacteria and molecules) involved in the pathogenesis of periodontitis. We then constructed a mathematical model of the pathogenesis of periodontitis by employing ordinary differential equations that described the dynamic regulatory interplay between the key players and predicted the alveolar bone integrity as output. Finally, we estimated the model parameters using our dynamic experimental data and validated the model prediction of influence on alveolar bone resorption by in vivo experiments. The model predictions and experimental results revealed that monocyte recruitment induced by bacteria-mediated Toll-like receptor activation was the principal reaction regulating alveolar bone resorption in a periodontitis condition. On the other hand, osteoblast-mediated osteoclast differentiation had less impact on bone integrity in a periodontitis condition.
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CMK, NUK, OILJ, SAZU, UKNU, UL, UM, UPUK
Periodontal ligament–associated protein 1 (PLAP-1)/asporin is an extracellular matrix protein preferentially expressed in periodontal ligaments. PLAP-1/asporin inhibits the cytodifferentiation and ...mineralization of periodontal ligament cells and has important roles in the maintenance of periodontal tissue homeostasis. However, the involvement of PLAP-1/asporin in inflammatory responses during periodontitis is poorly understood. This study hypothesized that PLAP-1/asporin might affect the pathogenesis of periodontitis by regulating periodontopathic bacteria-induced inflammatory responses. Proinflammatory cytokine expression induced by Toll-like receptor 2 (TLR2) and TLR4 was significantly downregulated when PLAP-1/asporin was overexpressed in periodontal ligament cells. Similarly, recombinant PLAP-1/asporin inhibited TLR2- and TLR4-induced proinflammatory cytokine expression in macrophages. We also confirmed that NF-κB activity induced by TLR2 and TLR4 signaling was suppressed by the addition of recombinant PLAP-1/asporin. Furthermore, IκB kinase α degradation induced by TLR4 was reduced by PLAP-1/asporin. Immunoprecipitation assays demonstrated the binding abilities of PLAP-1/asporin to both TLR2 and TLR4. Taken together, PLAP-1/asporin negatively regulates TLR2- and TLR4-induced inflammatory responses through direct molecular interactions. These findings indicate that PLAP-1/asporin has a defensive role in periodontitis lesions by suppressing pathophysiologic TLR signaling and that the modulating effects of PLAP-1/asporin might be useful for periodontal treatments.
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CMK, NUK, OILJ, SAZU, UKNU, UL, UM, UPUK
PLAP-1/asporin is an extracellular matrix protein that is predominantly expressed in the human periodontal ligament (PDL) and has an aspartic acid (D) repeat polymorphism in its N-terminal region. In ...this study, we hypothesized that the D repeat polymorphism of PLAP-1/asporin may affect the physiological functions of periodontal ligaments. We established periodontal ligament cell lines transfected with the D13- or D14-PLAP-1 gene. Alkaline phosphatase staining and alizarin red staining revealed that the cytodifferentiation of the D14-PLAP-1-expressing PDL cells was more repressed compared with that of the D13-PLAP-1-expressing cells. Furthermore, the D14-PLAP-1-expressing cells inhibited BMP-2-induced cytodifferentiation more strongly than did the D13-PLAP-1-expressing cells. Western blotting analysis and luciferase assay revealed that D14-PLAP-1 suppressed BMP-2 signal transduction more efficiently than did D13-PLAP-1, and co-immunoprecipitation demonstrated the stronger affinity of the D14-PLAP-1 protein to BMP-2 compared with the D13-PLAP-1 protein. Analysis of these data suggests that the D repeat polymorphism of PLAP-1/asporin has a significant influence on the functions of PDL cells.
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CMK, NUK, OILJ, SAZU, UKNU, UL, UM, UPUK
Sphingomyelin phosphodiesterase 3 (Smpd3), which encodes neutral sphingomyelinase 2 (nSMase2), is a key molecule for skeletal development as well as for the cytodifferentiation of odontoblasts and ...alveolar bone. However, the effects of nSMase2 on the cytodifferentiation of periodontal ligament (PDL) cells are still unclear. In this study, the authors analyzed the effects of Smpd3 on the cytodifferentiation of human PDL (HPDL) cells. The authors found that Smpd3 increases the mRNA expression of calcification-related genes, such as alkaline phosphatase (ALPase), type I collagen, osteopontin, Osterix (Osx), and runt-related transcription factor (Runx)-2 in HPDL cells. In contrast, GW4869, an inhibitor of nSMase2, clearly decreased the mRNA expression of ALPase, type I collagen, and osteocalcin in HPDL cells, suggesting that Smpd3 enhances HPDL cytodifferentiation. Next, the authors used exome sequencing to evaluate the genetic variants of Smpd3 in a Japanese population with aggressive periodontitis (AgP). Among 44 unrelated subjects, the authors identified a single nucleotide polymorphism (SNP), rs145616324, in Smpd3 as a putative genetic variant for AgP among Japanese people. Moreover, Smpd3 harboring this SNP did not increase the sphingomyelinase activity or mRNA expression of ALPase, type I collagen, osteopontin, Osx, or Runx2, suggesting that this SNP inhibits Smpd3 such that it has no effect on the cytodifferentiation of HPDL cells. These data suggest that Smpd3 plays a crucial role in maintaining the homeostasis of PDL tissue.
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CMK, NUK, OILJ, SAZU, UKNU, UL, UM, UPUK
Background and Objective
Single nucleotide polymorphisms (SNPs) of paraoxonase 1 (PON1) are known to be associated with the pathogenesis of osteoporosis and periodontitis. However, the effects of ...PON1 on the osteoblastic differentiation of periodontal ligament (PDL) cells are unclear. In this study, we examined the effects of PON1 on the osteoblastic differentiation of PDL cells, and analysed the role of PON1 SNPs on the pathogenesis of aggressive periodontitis (AgP) in the Japanese population.
Material and Methods
Human PDL (HPDL) cells were exposed to the PON1 plasmid and PON1 inhibitor, 2‐hydroxyquinoline, and cultured in mineralization medium. Expression of calcification‐related genes and calcified nodule formation were assessed by real‐time PCR, an alkaline phosphatase (ALPase) activity assay and Alizarin red staining. Sanger sequencing was performed to evaluate whether PON1 SNPs are associated with the pathogenesis of AgP in Japanese people.
Results
During osteoblastic differentiation of HPDL cells, expression of PON1 mRNA increased in a time‐dependent manner. PON1 stimulated an increase in expression of mRNA for calcification‐related genes, as well as ALPase activity. In contrast, 2‐hydroxyquinoline clearly inhibited the expression of calcification‐related genes, ALPase activity and calcified nodule formation in HPDL cells. Moreover, there was a statistically significant difference in the minor allele frequency of PON1 SNP rs854560 between the Japanese control database and patients with AgP in the Japanese population (P = .0190).
Conclusion
PON1 induced cytodifferentiation and mineralization of HPDL cells, and PON1 SNP rs854560 may be associated with the pathogenesis of AgP in the Japanese population.
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CMK, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Periodontal ligaments (PDLs) play an important role in remodeling the alveolar bond and cementum. Characterization of the periodontal tissue transcriptome remains incomplete, and an improved ...understanding of PDL features could aid in developing new regenerative therapies. Here, we aimed to generate and analyze a large human PDL transcriptome. We obtained PDLs from orthodontic treatment patients, isolated the RNA, and used a vector-capping method to make a complementary DNA library from >20,000 clones. Our results revealed that 58% of the sequences were full length. Furthermore, our analysis showed that genes expressed at the highest frequencies included those for collagen type I, collagen type III, and proteases. We also found 5 genes whose expressions have not been previously reported in human PDL. To access which of the highly expressed genes might be important for PDL cell differentiation, we used real-time polymerase chain reaction to measure their expression in differentiating cells. Among the genes tested, the cysteine protease cathepsin K had the highest upregulation, so we measured its relative expression in several tissues, as well as in osteoclasts, which are known to express high levels of cathepsin K. Our results revealed that PDL cells express cathepsin K at similar levels as osteoclasts, which are both expressed at higher levels than those of the other tissues tested. We also measured cathepsin K protein expression and enzyme activity during cell differentiation and found that both increased during this process. Immunocytochemistry experiments revealed that cathepsin K localizes to the interior of lysosomes. Last, we examined the effect of inhibiting cathepsin K during cell differentiation and found that cathepsin K inhibition stimulated calcified nodule formation and increased the levels of collagen type I and osteocalcin gene expression. Based on these results, cathepsin K seems to regulate collagen fiber accumulation during human PDL cell differentiation into hard tissue-forming cells.
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CMK, NUK, OILJ, SAZU, UKNU, UL, UM, UPUK
Background and Objective
The periodontal ligament (PDL) is vital to maintaining the homeostasis of the tooth and periodontal tissue. The influence of iron levels on the cytodifferentiation of PDL ...cells has not been studied, despite evidence that iron overload or deficiency can have adverse effects on alveolar bone density. The purpose of this study was to examine the effects of altered iron levels on cytodifferentiation in human PDL cells.
Material and Methods
Human PDL cells were incubated with culture media supplemented with 10–50 μm ammonium ferric citrate or 5 μm deferoxamine (an iron chelator) during differentiation. Intracellular iron status was assessed by measuring changes in the expression of ferritin RNA and protein. PDL cell differentiation and function were evaluated by measuring osteoblast differentiation gene markers and the capacity of cultures to form mineralized nodules.
Results
Iron accumulation resulted in upregulation of light and heavy chain ferritin proteins. Concurrently, osteoblast differentiation gene markers and mineralized nodule formation were suppressed. Iron deficiency resulted in downregulation of light and heavy chain ferritin proteins, suppression of alkaline phosphatase activity and formation of mineralized nodules during PDL cell differentiation.
Conclusion
We conclude that iron is critical for normal cell differentiation of human PDL cells.
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CMK, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Mycophenolate mofetil attenuates renal injury in the rat remnant kidney.
Mycophenolate mofetil (MMF), an inhibitor of lymphocyte proliferation, has been used to prevent allograft rejection. We ...investigated whether MMF also limits progressive renal injury in rats with 5/6 renal ablation, a model not primarily related to immunologic mechanisms.
Eighty-eight adult male Munich-Wistar rats underwent ablation and received either vehicle (N = 42) or oral MMF (N = 46), 10mg/kg/day. Forty-seven sham-operated rats were also studied.
Thirty days after surgery, remnant kidneys exhibited glomerular hypertension and hypertrophy. MMF treatment did not correct these abnormalities. Immunohistochemistry revealed interstitial lymphocyte infiltration 7 and 30days after ablation. Proliferating cells abounded seven days after ablation, especially in tubules, declining in number along the following weeks. By contrast, the number of macrophages was moderately increased in the first weeks, attaining values eightfold as high as control 60days after ablation. MMF attenuated these cellular events at all phases of the study. Sixty days after ablation, marked albuminuria, glomerulosclerosis and interstitial expansion were prominent in untreated rats. MMF treatment largely attenuated glomerular and interstitial injury without changing proteinuria.
This is the first evidence that MMF may impact favorably on progressive renal diseases of “nonimmunologic” origin.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
We recently standardized a model (L(Lact)) of severe chronic kidney disease based on impaired nephrogenesis by suppression of angiotensin II activity during lactation (Machado FG, Poppi EP, Fanelli ...C, Malheiros DM, Zatz R, Fujihara CK. Am J Physiol Renal Physiol 294: F1345-F1353, 2008). In this new study of the L(Lact) model, we sought to gain further insight into renal injury mechanisms associated with this model and to verify whether the renoprotection obtained with the association of the angiotensin II receptor blocker losartan (L) and hydrochlorothiazide (H), which arrested renal injury in the remnant kidney model, would provide similar renoprotection. Twenty Munich-Wistar dams, each nursing six pups, were divided into control, untreated, and L(Lact) groups, given losartan (L; 250 mg·kg(-1)·day(-1)) until weaning. The male L(Lact) offspring remained untreated until 7 mo of age, when renal functional and structural parameters were studied in 17 of them, used as pretreatment control (L(Lact)Pre), and followed no further. The remaining rats were then divided among groups L(Lact)+V, untreated; L(Lact)+L, given L (50 mg·kg(-1)·day(-1)) now as a therapy; L(Lact)+H, given H (6 mg·kg(-1)·day(-1)); and L(Lact)+LH, given L and H. All parameters were reassessed 3 mo later in these groups and in age-matched controls. At this time, L(Lact) rats exhibited hypertension, severe albuminuria, glomerular damage, marked interstitial expansion/inflammation, enhanced cell proliferation, myofibroblast infiltration, and creatinine retention. L monotherapy normalized albuminuria and prevented hypertension and the progression of renal injury, inflammation, and myofibroblast infiltration. In contrast to the remnant model, the LH combination promoted only slight additional renoprotection, perhaps because of a limited tendency to retain sodium in L(Lact) rats.
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