In 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. / published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy
| Identifer | oai:union.ndltd.org:HKU/oai:hub.hku.hk:10722/174514 |
| Date | January 2011 |
| Creators | Li, Jingping, 李京平 |
| Contributors | Leung, WK, Fung, ML |
| Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
| Source Sets | Hong Kong University Theses |
| Language | English |
| Detected Language | English |
| Type | PG_Thesis |
| Source | http://hub.hku.hk/bib/B47849563 |
| Rights | The author retains all proprietary rights, (such as patent rights) and the right to use in future works., Creative Commons: Attribution 3.0 Hong Kong License |
| Relation | HKU Theses Online (HKUTO) |
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