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postgraduate thesis: Role of tissue hypoxia in periodontitis

TitleRole of tissue hypoxia in periodontitis
Authors
Advisors
Issue Date2011
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
Citation
Li, J. [李京平]. (2011). Role of tissue hypoxia in periodontitis. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b4784956
AbstractIn periodontitis, local oxygen supply and consumption in gingival tissues may be significantly altered due to the inflammatory process. The etiology agent of periodontal disease i.e. anaerobic bacterial biofilm is known to confer a low oxygen tension in the vicinity of periodontitis lesion. The oxygen shortage will lead to the stabilization of HIF-1α, the regulatory subunit of hypoxia-inducible factor (HIF)-1, which through controlling specific downstream genes transcription may modulate multiple cellular functions and hence shape the process of periodontitis. Lipopolysaccharide (LPS), a cell wall component of anaerobic bacteria, has been considered to be involved in the pathogenesis of periodontitis. Its interaction with host peptides including LPS-binding protein (LBP), CD14, MD-2 and Toll-like receptor (TLR) 4 may trigger the production of inflammatory cytokines. In this project we hypothesize that hypoxia and bacterial components may induce HIF-1α activity, which in turn impacts upon on the pathological process of periodontitis. This project aimed to detect in vivo expression of HIF-1α and TLR4 in human gingivae; to examine whether LPS could induce HIF-1α activity through pattern recognition receptor like TLR4 on human primary gingival fibroblasts (HGF); and to investigate the combined effect of hypoxia and LPS on type I collagen metabolism in HGF. Human gingival biopsies were collected from advanced periodontitis or clinically healthy sites. By immunohistochemistry, both HIF-1α and TLR4 peptides appeared to express in gingival epithelium. In periodontal pockets, there appeared a marked increase in HIF-1α and TLR4 expression in fibroblast-like and leukocyte-like cells. Human primary gingival keratinocytes (HGK) and fibroblasts (HGF) were cultured. Transcripts of TLR4, MD-2 and CD14 were identified in HGK, HGF and periodontal tissue using RT-PCR. Their protein products were identified in both cell types in vitro using immunoblotting. LBP transcript was only found in gingival biopsies but not in HGK and HGF culture. HGF treated by Escherichia coli LPS ranging from 0.2 μg/mL to 200 μg/mL showed nuclear accumulation of HIF-1α peptide, detectable by immunocytofluorescence and immunoblotting. This accumulation could be attenuated by treatment with TLR4-neutralizing antibody. Under hypoxia, LPS further increased HIF-1α accumulation. Using quantitative real-time PCR (qPCR), hypoxia and/or LPS appeared to enhance the transcription of certain enzymes or enzyme subunits that are related to collagen assembly and crosslink, including prolyl 4-hydroxylases, lysyl hydroxylases, lysyl oxidase and lysyl oxidase-like enzymes. These increased transcription could be downregulated by pretreatment with TLR4-neutralizing antibody or an HIF-α inhibitor, 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1). Finally, preliminary experiments showed KN-93 [Ca2+/Calmodulin-dependent protein kinase (CaMK) II inhibitor] or cyclosporine-A (calcineurin inhibitor) appeared able to attenuate the LPS-induced HIF-1α accumulation, indicating a possible role for intracellular calcium signal in regulating HIF-1α. In conclusions, human periodontitis is associated with increased expression of TLR4 and HIF-1α in gingivae; hypoxia causes and LPS/TLR4 signal associate with HIF-1α accumulation and activity in human gingival fibroblasts, and subsequently modulate in a certain extend collagen metabolism through upregulating the transcript expression of several collagen-related proteins. All these implicate possibility of an adaptive physiological or pathological response of human gingival fibroblasts towards gram-negative bacterial biofilm challenge in human periodontium.
DegreeDoctor of Philosophy
SubjectPeriodontitis.
Anoxemia.
Dept/ProgramDentistry

 

DC FieldValueLanguage
dc.contributor.advisorLeung, WK-
dc.contributor.advisorFung, ML-
dc.contributor.authorLi, Jingping-
dc.contributor.author李京平-
dc.date.issued2011-
dc.identifier.citationLi, J. [李京平]. (2011). Role of tissue hypoxia in periodontitis. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b4784956-
dc.description.abstractIn periodontitis, local oxygen supply and consumption in gingival tissues may be significantly altered due to the inflammatory process. The etiology agent of periodontal disease i.e. anaerobic bacterial biofilm is known to confer a low oxygen tension in the vicinity of periodontitis lesion. The oxygen shortage will lead to the stabilization of HIF-1α, the regulatory subunit of hypoxia-inducible factor (HIF)-1, which through controlling specific downstream genes transcription may modulate multiple cellular functions and hence shape the process of periodontitis. Lipopolysaccharide (LPS), a cell wall component of anaerobic bacteria, has been considered to be involved in the pathogenesis of periodontitis. Its interaction with host peptides including LPS-binding protein (LBP), CD14, MD-2 and Toll-like receptor (TLR) 4 may trigger the production of inflammatory cytokines. In this project we hypothesize that hypoxia and bacterial components may induce HIF-1α activity, which in turn impacts upon on the pathological process of periodontitis. This project aimed to detect in vivo expression of HIF-1α and TLR4 in human gingivae; to examine whether LPS could induce HIF-1α activity through pattern recognition receptor like TLR4 on human primary gingival fibroblasts (HGF); and to investigate the combined effect of hypoxia and LPS on type I collagen metabolism in HGF. Human gingival biopsies were collected from advanced periodontitis or clinically healthy sites. By immunohistochemistry, both HIF-1α and TLR4 peptides appeared to express in gingival epithelium. In periodontal pockets, there appeared a marked increase in HIF-1α and TLR4 expression in fibroblast-like and leukocyte-like cells. Human primary gingival keratinocytes (HGK) and fibroblasts (HGF) were cultured. Transcripts of TLR4, MD-2 and CD14 were identified in HGK, HGF and periodontal tissue using RT-PCR. Their protein products were identified in both cell types in vitro using immunoblotting. LBP transcript was only found in gingival biopsies but not in HGK and HGF culture. HGF treated by Escherichia coli LPS ranging from 0.2 μg/mL to 200 μg/mL showed nuclear accumulation of HIF-1α peptide, detectable by immunocytofluorescence and immunoblotting. This accumulation could be attenuated by treatment with TLR4-neutralizing antibody. Under hypoxia, LPS further increased HIF-1α accumulation. Using quantitative real-time PCR (qPCR), hypoxia and/or LPS appeared to enhance the transcription of certain enzymes or enzyme subunits that are related to collagen assembly and crosslink, including prolyl 4-hydroxylases, lysyl hydroxylases, lysyl oxidase and lysyl oxidase-like enzymes. These increased transcription could be downregulated by pretreatment with TLR4-neutralizing antibody or an HIF-α inhibitor, 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1). Finally, preliminary experiments showed KN-93 [Ca2+/Calmodulin-dependent protein kinase (CaMK) II inhibitor] or cyclosporine-A (calcineurin inhibitor) appeared able to attenuate the LPS-induced HIF-1α accumulation, indicating a possible role for intracellular calcium signal in regulating HIF-1α. In conclusions, human periodontitis is associated with increased expression of TLR4 and HIF-1α in gingivae; hypoxia causes and LPS/TLR4 signal associate with HIF-1α accumulation and activity in human gingival fibroblasts, and subsequently modulate in a certain extend collagen metabolism through upregulating the transcript expression of several collagen-related proteins. All these implicate possibility of an adaptive physiological or pathological response of human gingival fibroblasts towards gram-negative bacterial biofilm challenge in human periodontium.-
dc.languageeng-
dc.publisherThe University of Hong Kong (Pokfulam, Hong Kong)-
dc.relation.ispartofHKU Theses Online (HKUTO)-
dc.rightsThe author retains all proprietary rights, (such as patent rights) and the right to use in future works.-
dc.rightsCreative Commons: Attribution 3.0 Hong Kong License-
dc.source.urihttp://hub.hku.hk/bib/B47849563-
dc.subject.lcshPeriodontitis.-
dc.subject.lcshAnoxemia.-
dc.titleRole of tissue hypoxia in periodontitis-
dc.typePG_Thesis-
dc.identifier.hkulb4784956-
dc.description.thesisnameDoctor of Philosophy-
dc.description.thesislevelDoctoral-
dc.description.thesisdisciplineDentistry-
dc.description.naturepublished_or_final_version-
dc.identifier.doi10.5353/th_b4784956-
dc.date.hkucongregation2012-

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