Role of tissue hypoxia in periodontitis

Role of tissue hypoxia in periodontitis

李京平(Jingping Li)

香港大學/Doctor of Philosophy/Dentistry/博士(2011年)

並列摘要

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.

並列關鍵字

Periodontitis ； Anoxemia