Expression and activity of thrombomodulin in human gingival epithelium: in vivo and in vitro studies

Abstract

Epidermal keratinocytes thrombomodulin (TM) has been shown to regulate thrombin at sites of cutaneous injury in addition to a role for epidermal differentiation. TM, a major anticoagulant proteoglycan of the endothelial cell membrane, is a thrombin receptor that acts as a co-factor for protein C activation. Thrombin has pro-inflammatory effects for periodontitis. However, little is known about TM in gingival tissue with periodontitis. We used immunohistochemistry to examine expression of TM in gingival epithelium from patients with periodontitis. In vitro, we observed TM expression at varying Ca2+ concentrations by confocal laser scanning microscopy, examined the expression of TM mRNA and tested TM co-factor activity. Furthermore, we measured TM concentration in gingival crevicular fluid (GCF) from 11 severe adult cases of periodontitis using enzyme-linked immunosorbent assay. Immunoreactive TM was present in gingival epithelium and junctional epithelium, and was reduced in inflamed gingival epithelium compared to healthy gingival epithelium. Ultrastructurally, TM, including microvilli, was observed on the cell membrane. TM localization in cells cultured in 0.09 mM Ca2+ differed from that in cells exposed to 1.2 mM Ca2+. Northern analysis demonstrated TM mRNA in gingival keratinocytes more than in human umbilical vein endothelial cells (HUVEC). Gingival keratinocytes also facilitated protein C activation by thrombin, although less strongly than HUVEC. TM in GCF at sites with bleeding on probing in patients was significantly elevated (p < 0.001, Student's t-test). TM in gingival epithelium may regulate thrombin activity at sites of coagulation and inflammation with periodontal disease, although inflammation may impair this regulation of thrombin.

Significance of thrombomodulin release from gingival epithelial cells in periodontitis patients

“…Gingival epithelial cells were obtained from healthy gingival tissues using an explant culture method (18). Cells were maintained in a serum‐free keratinocyte medium (Gibco, Grand Island, NY, USA) supplemented with epidermal growth factor (5 ng/mL) and bovine pituitary extracts (35–50 µg/mL) containing 0.09 m m Ca 2+ .…”

“…We previously showed that gingival epithelial cells expressed thrombomodulin, and gingival crevicular fluid at bleeding sites in periodontitis patients without systemic disease contained high levels of soluble thrombomodulin (18), suggesting that the thrombomodulin content in gingival crevicular fluid was associated with local inflammation. However, the source of the soluble form of thrombomodulin in gingival crevicular fluid remains unknown in periodontitis.…”

Novel Role of the Human Alveolar Epithelium in Regulating Intra-Alveolar Coagulation

“…APC suppresses thrombin formation by proteolytically inactivating coagulation factors Va and VIIIa. TM and EPCR were originally described in endothelial cells (10,11), but subsequently were also detected in several other cell types (12)(13)(14), including epithelial cells. The alveolar epithelium has a large surface area, and thus is uniquely situated to actively modulate the intraalveolar environment.…”

“…Cells were washed with serum free medium then incubated with 0.1 U/ml ␣-thrombin and 12 ug/ml protein C for 2h at 37ЊC. The reaction was stopped with 40ul of hirudin (50 U/ml) (13). Supernatants were transferred to a new microplate and APC was assayed against an APC standard curve after incubation with the chromogenic substrate S2238.…”

The Antimetastatic Role of Thrombomodulin Expression in Islet Cell-Derived Tumors and Its Diagnostic Value

“…Activated protein C exerts additional anticoagulant effects by inactivating coagulant cofactors Va and VIIIa (7,8,12). Thrombomodulin is also expressed at extravascular sites, such as in syncytiotrophoblasts in the placenta, in the epithelial tissues of gingiva, in the skin, lungs and digestive organs, and in the synovial lining cells (15)(16)(17)(18)(19). However, the functional role of thrombomodulin in the extra-vascular space, apart from the possible limitation of thrombin generation at these sites, remains uncertain.…”