l‐Mimosine and Dimethyloxaloylglycine Decrease Plasminogen Activation in Periodontal Fibroblasts

Background: The use of prolyl hydroxylase inhibitors such as l ‐mimosine (L‐MIM) and dimethyloxaloylglycine (DMOG) to improve angiogenesis is a new approach for periodontal regeneration. In addition to exhibiting pro‐angiogenic effects, prolyl hydroxylase inhibitors can modulate the plasminogen activator system in cells from non‐oral tissues. This study assesses the effect of prolyl hydroxylase inhibitors on plasminogen activation by fibroblasts from the periodontium. Methods: Gingival and periodontal ligament fibroblasts were incubated with L‐MIM and DMOG. To investigate whether prolyl hydroxylase inhibitors modulate the net plasminogen activation, kinetic assays were performed with and without interleukin (IL)‐1. Moreover, plasminogen activators and the respective inhibitors were analyzed by casein zymography, immune assays, and quantitative polymerase chain reaction. Results: The kinetic assay showed that L‐MIM and DMOG reduced plasminogen activation under basal and IL‐1–stimulated conditions. Casein zymography revealed that the effect of L‐MIM involves a decrease in urokinase‐type plasminogen activator activity. In agreement with these findings, reduced levels of urokinase‐type plasminogen activator and elevated levels of plasminogen activator inhibitor 1 were observed. Conclusion: L‐MIM and DMOG can reduce plasminogen activation by fibroblasts from the gingiva and the periodontal ligament under basal conditions and in the presence of an inflammatory cytokine.     

Platelet-derived growth factor-B gene delivery sustains gingival fibroblast signal transduction

Background and Objective: Platelet‐derived growth factor‐BB is a potent mediator of tooth‐supporting periodontal tissue repair and regeneration. A limitation of the effects of topical platelet‐derived growth factor‐BB application is its short half‐life in vivo. Gene therapy has shown strong promise for the long‐term delivery of platelet‐derived growth factor in both skin ulcer healing and periodontal tissue engineering. However, little is known regarding the extended effects of platelet‐derived growth factor‐B on cell signaling via gene delivery, especially at the level of phosphorylation of intracellular kinases. This study sought to evaluate the effect of gene transfer by Ad‐PDGF‐B on human gingival fibroblasts (HGFs) and the subsequent regulation of genes and cell‐surface proteins associated with cellular signaling. Material and Methods: HGFs from human subjects were treated by adenoviral PDGF‐B, PDGF‐1308 (a dominant negative mutant of PDGF) and recombinant human platelet‐derived growth factor‐BB, and then incubated in serum‐free conditions for various time points and harvested at 1, 6, 12, 24, 48, 72 and 96 h. Exogenous PDGF‐B was measured by RT‐PCR and Western blot. Cell proliferation was evaluated by [methyl‐³H]thymidine incorporation assay. We used proteomic arrays to explore phosphorylation patterns of 23 different intracellular kinases after PDGF‐B gene transfer. The expression of α and β PDGFR and Akt were measured by Western blot analysis. Results: Sustained in vitro expression of PDGF‐B in HGFs by Ad‐PDGF‐B transduction was seen at both the mRNA and protein levels. Compared to rhPDGF‐BB and Ad‐PDGF‐1308, Ad‐PDGF‐B maintained cell growth in serum‐free conditions, with robust increases in DNA synthesis. Gene delivery of PDGF‐B also prolonged downregulation of the growth arrest specific gene (gas) PDGFαR. Of the 23 intracellular kinases that we tested in proteomic arrays, Akt revealed the most notable long‐term cell signaling effect as a result of the over‐expression of Ad‐PDGF‐B, compared with pulse recombinant human platelet‐derived growth factor BB. Prolonged Akt phosphorylation was induced by treatment with Ad‐PDGF‐B, for at least up to 96 h. Conclusion: These findings further demonstrate that gene delivery of PDGF‐B displays sustained signal transduction effects in human gingival fibroblasts that are higher than those conveyed by treatment with recombinant human platelet‐derived growth factor‐BB protein. These data on platelet‐derived growth factor gene delivery contribute to an improved understanding of these pathways that are likely to play a role in the control of clinical outcomes of periodontal regenerative therapy.     

Cigarette smoke condensate stimulates urokinase production through the generation of reactive oxygen species and activation of the mitogen activated protein kinase pathways in human gingival fibroblasts

Background and Objective:  Tobacco smoking is a significant risk factor for periodontal disease. It has been suggested that smoking may alter connective tissue remodeling in the periodontium. In the present study, we investigated whether cigarette smoke condensate modulates the production of the serine protease urokinase in human gingival fibroblasts.
Material and Methods:  Primary cultures of human gingival fibroblasts were stimulated with cigarette smoke condensate. Urokinase production was evaluated through casein zymography and western blotting. Plasmin activation was assessed by means of a radial diffusion assay. The roles of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and reactive oxygen species in cigarette smoke condensate-stimulated urokinase production were studied using distinct selective inhibitors (SP600125, PD98059, N -acetyl cysteine). Reactive oxygen species production was determined using a fluorometric assay. Activation of ERK and JNK pathways were evaluated using western blots.
Results:  In gingival fibroblasts, cigarette smoke condensate potently stimulated urokinase production and plasmin activation. Cigarette smoke condensate-stimulated urokinase production was dependent on the activity of ERK/JNK pathways and was inhibited by the reactive oxygen species scavenger, N -acetyl cysteine. Cigarette smoke condensate strongly stimulated ERK and JNK phosphorylation and the generation of reactive oxygen species.
Conclusion:  Cigarette smoke condensate stimulates urokinase production and plasmin activation in gingival fibroblasts. Moreover, cigarette smoke condensate-stimulated urokinase production depends on both the activation of ERK/JNK pathways and on the generation of intracellular reactive oxygen species. These results show that cigarette smoke may alter connective tissue remodeling by inducing production of the urokinase-type plasminogen activator through specific signaling pathways.     

Differential uPA expression by TGF-beta1 in gingival fibroblasts

Transforming Growth Factor-beta1 (TGF-beta1) plays a key role in connective tissue remodeling and inflammation. Under pathological conditions, like periodontal disease, fibroblasts may display an altered response to this growth factor. To investigate this question, we have studied whether TGF-beta1 may differentially regulate the expression of urokinase at the protein level in primary cultures of fibroblasts derived from healthy gingiva, granulation tissue from gingival wounds, and chronic periodontal disease. We observed that TGF-beta1 may repress urokinase expression in healthy gingival fibroblasts and promote its production in granulation-tissue fibroblasts. A significant correlation was found between expression of the myofibroblast marker alpha-smooth-muscle actin and stimulation of urokinase production by TGF-beta1. Immunostaining of gingival wounds showed that myofibroblasts were involved in urokinase production. TGF-beta1-stimulated urokinase expression was blocked after inhibition of the c-jun-NH2 terminal kinase signaling pathway. We propose that stimulation of urokinase production by TGF-beta1 is involved in the responses of activated fibroblasts to tissue injury.     

Enamel matrix derivative induces production of vascular endothelial cell growth factor in human gingival fibroblasts

Enamel matrix derivative (EMD) may enhance periodontal wound healing by inducing angiogenesis. We sought to investigate the effect and the mechanism of action of EMD on vascular endothelial growth factor (VEGF) production by human gingival fibroblasts. Cells were stimulated with EMD, transforming growth factor‐β1 (TGF‐β1), or fibroblast growth factor 2 (FGF‐2), with or without antibodies to TGF‐β1 or FGF‐2. The levels of VEGF in the culture media were measured using an ELISA. We examined the effects of SB203580 [a p38 mitogen‐activated protein kinase (MAPK) inhibitor], U0126 [an extracellular signal‐regulated kinase (ERK) inhibitor], SP600125 [a c‐Jun N‐terminal kinase (JNK) inhibitor], and LY294002 [a phosphatidylinositol 3‐kinase (PI3K)/Akt inhibitor] on EMD‐induced VEGF production. Enamel matrix derivative stimulated the production of VEGF in a dose‐ and time‐dependent manner. Treatment of human gingival fibroblasts with antibodies to TGF‐β1 or FGF‐2 significantly decreased EMD‐induced VEGF production, whereas the addition of exogenous TGF‐β1 and FGF‐2 stimulated VEGF production. Enamel matrix derivative‐induced VEGF production was significantly attenuated by SB203580, U0126, and LY294002. Our results suggest that EMD stimulates VEGF production partially via TGF‐β1 and FGF‐2 in human gingival fibroblasts and that EMD‐induced VEGF production is regulated by ERK, p38 MAPK, and PI3K/Akt pathways. Enamel matrix derivative‐induced production of VEGF by human gingival fibroblasts may be involved in the enhancement of periodontal wound healing by inducing angiogenesis.     

Hepatocyte growth factor secreted by periodontal ligament and gingival fibroblasts is a major chemoattractant for gingival epithelial cells

To ascertain whether periodontal fibroblasts could be involved in the pathogenesis of periodontal pocket formation, the chemotactic activity of periodontal ligament fibroblast-conditioned medium (PLF-CM) and gingival fibroblast-conditioned medium (GF-CM) for gingival epithelial cells was examined using a modified Boyden chamber assay. Both PLF-CM and GF-CM possessed significant chemotactic activity, which was decreased markedly by treatment with anti-human hepatocyte growth factor (HGF) neutralizing antibody. Furthermore, the chemotactic activity of PLF-CM and GF-CM was well correlated with HGF content. These results show that PLF and GF secrete an HGF-like factor, and suggest that such a factor derived from periodontal fibroblasts might play a role in epithelial apical migration in periodontitis.     

Plasminogen activation by fibroblasts from periodontal ligament and gingiva is not directly affected by chemokines in vitro

Objective

Chronic inflammation in periodontal disease is associated with increased plasminogen activation and elevated levels of chemokines. It is unknown whether chemokines can regulate the activation of plasminogen via modulation of plasminogen activators (PA) and the corresponding plasminogen activator inhibitors (PAI) in periodontal tissue.

Design

To establish a link between chemokines and activation of plasminogen, human periodontal ligament fibroblasts (PDL) and gingival fibroblasts (GF) were incubated with IL-8, monocyte chemoattractant protein-1, macrophage inflammatory protein-1alpha, and platelet factor-4, either alone or in the presence of the inflammatory mediators TGF-β and IL-1. The potential of the cell lysates to activate plasminogen was based on kinetic studies with the substrate casein. Casein zymography was performed to determine the molecular sizes of the PA. Total PAI-1 in the cell-conditioned medium was quantified by immunoassay.

Results

We report that the chemokines did not affect activation of plasminogen by PDL and GF. Even in the presence of TGF-β which suppressed, and IL-1 which stimulated plasminogen activation, the chemokines had no direct effect. Inhibition of PA and plasmin, but not of matrix metalloproteinases and cysteine proteinases prevented caseinolysis. The plasminogen activation capacity of the cell lysates was represented by a single band with features of uPA. The immunoassay showed that the release of PAI-1 in PDL and GF remained unaffected by the chemokines, also when stimulated with TGF-β.

Conclusions

These results suggest that plasminogen activation by PDL and GF is not directly affected by the chemokines even in the presence of the inflammatory mediators TGF-β and IL-1.     

Association of gene polymorphisms for plasminogen activators with alveolar bone loss

BACKGROUND AND OBJECTIVE: The plasminogen activating system is a protease/inhibitor system central to extracellular matrix remodeling with a suggested role in periodontal disease pathology. A few studies have reported polymorphisms in the genes of plasminogen activator inhibitors to be associated with periodontal disease severity. Two gene polymorphisms - a BamHI restriction fragment length polymorphism in the urokinase plasminogen activator gene (uPA) and a HindIII restriction fragment length polymorphism in the plasminogen activator inhibitor type 1 gene (PAI-1) - have been associated with conditions having a vascular component, and our objective was to assess the association of these gene polymorphisms with alveolar bone loss in chronic periodontal disease of adults. MATERIAL AND METHODS: Genotype was determined by polymerase chain reaction amplification of whole blood, pertinent histories were obtained by interview, and alveolar bone loss was assessed from current radiographs. RESULTS: In 77 elderly patients with a normal distribution of alveolar bone loss, we demonstrated a significant association between levels of alveolar bone loss and these polymorphisms in the uPA and PAI-1 genes. Controlling for the contributions of smoking or diabetes to periodontal bone loss, estimated odds ratios for predicting lower levels of alveolar bone loss, associated with a greater degree of periodontal health, were strongest when defined by the concurrent presence of a homozygous urokinase plasminogen activator genotype and the nuclease-sensitive plasminogen activator inhibitor type 1 (HindIII) allele (odds ratio = 2.6; 95% confidence interval: 5.8-1.3). CONCLUSION: The urokinase plasminogen activator (BamHI) and plasminogen activator inhibitor type 1 (HindIII) genotypes may serve as useful markers for heritability of bone loss associated with periodontal disease.     

Plasminogen activators and inhibitor type-1 in alveolar osteitis

Alveolar osteitis (AO) is characterized by excess fibrinolysis, leading to early dissociation of the clot that normally follows tooth extraction. Nonetheless, scarce information is available on the fibrinolytic system in AO. In this study, we report on the differential composition of postextraction wound healing tissue and of peri-alveolar gingival epithelium from normal healing and AO patients in terms of plasminogen activators, plasminogen activator inhibitor-type 1, and urokinase-type plasminogen activator receptor. Plasminogen activators were studied by overlay zymography, western blotting, and enzyme-linked immunosorbent assay (ELISA). Plasminogen activator inhibitor-type 1 and urokinase receptor were measured by ELISA. In AO, the fibrinolytic activity of wound healing tissue was accounted for by an increase ( approximately 85%) of urokinase-type plasminogen activator, whereas tissue-type plasminogen activator was unchanged. Plasminogen activator inhibitor-type 1 showed a 6.7-fold increase in AO. These results point to key roles of urokinase in AO hyper-fibrinolysis and of plasminogen activator inhibitor type-1 in slowing down the healing response. Peri-alveolar gingival epithelium in AO showed an overall decrease of all the components of the fibrinolytic system, including the urokinase receptor, which indicates a decrease of the migration properties of epithelial cells.     

人牙龈和牙周韧带成纤维细胞的体外培养及生物学特性研究——细胞形态、生长曲线及碱性磷酸酶活性测定

目的 :建立人牙龈和牙周韧带成纤维细胞体外培养模型并对其生物学特性作初步探讨。方法 :采用组织块法常规条件下分别进行牙龈和牙周韧带成纤维细胞的培养 ,通过光镜、透射电镜、生长曲线及碱性磷酸酶测定等手段对其部分生物学特性进行研究。结果 :两种培养的原代及传代细胞在光镜下细胞排列及结构无明显差别 ,传代培养时牙龈成纤维细胞有接触抑制现象 ,而牙周韧带成纤维细胞则可呈复层生长 ,牙周韧带成纤维细胞排列方向性较明显 ,生长曲线表明牙周韧带成纤维细胞增殖活性高于牙龈成纤维细胞 ;碱性磷酸酶活性测定及染色法显示两者有明显的差别。结论 :体外培养的两种细胞在形态及排列上相似 ,但在细胞亚型的组成上存在差异。     

Subculture affects the phenotypic expression of human periodontal ligament cells and their response to fibroblast growth factor-2 and bone morphogenetic protein-7 in vitro

Background and Objective: Although periodontal ligament cells display several osteoblastic traits, their phenotypic expression is still not well established. It remains a matter of debate whether they resemble a terminally differentiated cell type or an intermediate maturation state that potentially can be directed towards a fibroblastic or an osteoblastic phenotype. Material and Methods: To explore the characteristics of periodontal ligament cells in greater detail, fourth‐passage, sixth‐passage and eighth‐passage human periodontal ligament cells were cultured for up to 3 wk. Ki‐67, alkaline phosphatase, osteocalcin, osteoprotegerin and receptor activator of nuclear factor‐κB ligand (RANKL) mRNA expression was quantified by real‐time polymerase chain reaction. Furthermore, the cellular response to fibroblast growth factor‐2 and bone morphogenetic protein‐7 was examined in first‐passage and fourth‐passage cells. Dermal fibroblasts (1BR.3.G) and osteoblast‐like cells (MG63) served as reference cell lines. Results: Proliferation decreased over time and was highest in fourth‐passage cells. The expression of differentiation parameters, osteoprotegerin and RANKL increased with culture time and was higher in fourth‐passage cells than in cells of later passages. The RANKL/osteoprotegerin ratio increased steadily until day 21. Administration of fibroblast growth factor‐2 enhanced cell numbers in both passages, whereas alkaline phosphatase and osteocalcin production remained unchanged. By contrast, exposure of periodontal ligament cells to bone morphogenetic protein‐7 resulted in a reduction of cell number in the first and fourth passages, whereas the production of alkaline phosphatase and osteocalcin was enhanced. In dermal fibroblasts, differentiation parameters did not respond to both stimuli. MG63 cells behaved similarly to periodontal ligament cells. Conclusion: These results indicate that subculture affects the phenotypic expression of human periodontal ligament cells with respect to the characteristics that these cells share with osteoblasts. Furthermore, the periodontal ligament cell phenotype can be altered by fibroblastic and osteoblastic growth factors.     

Glucose modulates growth of gingival fibroblasts and periodontal ligament cells: correlation with expression of basic fibroblast growth factor

Diabetes mellitus is a systemic disease with profound effects on oral health and periodontal wound healing. Uncontrolled diabetes adversely affects surgical wound healing and is often associated with abnormal proliferation of fibroblasts, excessive angiogenesis and poor bone regeneration. Human gingival fibroblasts and periodontal ligament cells from both diabetics and non-diabetics were evaluated for growth responses following culture in 20 mM glucose, a concentration compatible with blood glucose levels in uncontrolled diabetics. Gingival fibroblasts derived from 9 non-diabetic patients and 3 insulin-dependent diabetics either proliferated or showed little change of growth in elevated glucose. Enhanced proliferation was observed following 1 wk of culture in glucose. Growth of periodontal ligament cells from 5 non-diabetic patients was inhibited by 20 mM glucose. Fibroblasts that were markedly growth stimulated were probed for expression of basic fibroblast growth factor (bFGF) using a reverse-transcribed polymerase chain reaction (RT-PCR). Results indicate that fibroblasts exhibiting the greatest increase in growth in response to high glucose also exhibited increased expression of bFGF. No changes were observed in mRNA expression for platelet-derived growth factor-AA, platelet-derived growth factor-BB, insulin-like growth factor and transforming growth factor-β₁. Mitogenic effects induced by the cytosol of fibroblasts exhibiting increases of growth in 20 mM glucose were abrogated by neutralizing antibodies to bFGF. In addition, some periodontal ligament cells that were growth inhibited by high glucose had reduced expression of bFGF. These data suggest that bFGF may play a role in the abnormal wound healing associated with periodontal surgery of uncontrolled diabetics.     

Enamel matrix derivative induces the expression of tissue inhibitor of matrix metalloproteinase‐3 in human gingival fibroblasts via extracellular signal‐regulated kinase

Zeldich E, Koren R, Dard M, Weinberg E, Weinreb M, Nemcovsky CE. Enamel matrix derivative induces the expression of tissue inhibitor of matrix metalloproteinase‐3 in human gingival fibroblasts via extracellular signal‐regulated kinase. J Periodont Res 2010; doi: 10.1111/j.1600‐0765.2009.01218.x © 2009 John Wiley & Sons A/S Background and Objective: Periodontal disease is characterized by increased expression and activity of matrix metalloproteinases (MMPs) and insufficient expression/activity of their inhibitors, tissue inhibitors of matrix metalloproteinases (TIMPs). This altered MMP–TIMP balance results in progressive destruction of gingival and periodontal extracellular matrix. Enamel matrix derivative (EMD), clinically used for periodontal regeneration in a device called Emdogain^®, has been suggested to enhance gingival healing following periodontal procedures in humans. We previously showed that EMD increases the proliferation of human and rat gingival fibroblasts and protects them from tumor necrosis factor‐induced apoptosis. In the present study, the modulation of MMP and TIMP expression by EMD was investigated. Material and Methods: Primary human gingival fibroblasts were treated in vitro with tumor necrosis factor, EMD or both in serum‐free conditions, and RNA was analyzed with an extracellular matrix‐focused microarray and quantitative real‐time polymerase chain reaction. Results: Microarray analysis showed detectable expression of MMP‐1, MMP‐2, MMP‐3, MMP‐7 and MMP‐13, as well as TIMP‐1 and TIMP‐3 in untreated cells. There was no apparent regulation of the expression of MMP‐2, MMP‐7, MMP‐13 and TIMP‐1 by either tumor necrosis factor or EMD. In contrast, tumor necrosis factor significantly increased MMP‐1 expression, and EMD reduced it when both agents were present. Also, EMD significantly induced TIMP‐3 expression, an effect which was dependent on activation of extracellular signal‐regulated kinase 1/2, since it was totally abolished by a selective extracellular signal‐regulated kinase pathway inhibitor. Conclusion: These data suggest that EMD may affect gingival health by ways other than cell proliferation/survival, i.e. by stimulation of TIMP‐3 production, which could improve the MMP–TIMP balance in gingival tissue and curb extracellular matrix destruction.