Gingival epithelial cell-derived microvesicles activate mineralization in gingival fibroblasts

Abstract

Soft tissue calcification occurs in many parts of the body, including the gingival tissue. Epithelial cell-derived MVs can control many functions in fibroblasts but their role in regulating mineralization has not been explored. We hypothesized that microvesicles (MVs) derived from gingival epithelial cells could regulate calcification of gingival fibroblast cultures in osteogenic environment. Human gingival fibroblasts (HGFs) were cultured in osteogenic differentiation medium with or without human gingival epithelial cell-derived MV stimulation. Mineralization of the cultures, localization of the MVs and mineral deposits in the HGF cultures were assessed. Gene expression changes associated with MV exposure were analyzed using gene expression profiling and real-time qPCR. Within a week of exposure, epithelial MVs stimulated robust mineralization of HGF cultures that was further enhanced by four weeks. The MVs taken up by the HGF's did not calcify themselves but induced intracellular accumulation of minerals. HGF gene expression profiling after short exposure to MVs demonstrated relative dominance of inflammation-related genes that showed increases in gene expression. In later cultures, OSX, BSP and MMPs were significantly upregulated by the MVs. These results suggest for the first time that epithelial cells maybe associated with the ectopic mineralization process often observed in the soft tissues.

Figure 1

Mineralization of HGF cultures exposed to epithelial MVs. (a) Representative images of 7- and 28-day-old HGF cultures stained with von Kossa. Mineralized nodules were detected only in cells treated with MVs in osteogenic medium (OMMV) after one week. After a 28-day treatment, MVs promoted progressive mineralization (OMMV) compared to the control medium (OM). There was minimal mineralization in the basic medium (BM). Scale bars: 1000 µm. (b) IncuCyte real-time assessment of mineralization of MV-treated HGF cultures using calcein uptake for 8 and 17 days. Cells in BM and in control OM media showed only weak background fluorescence during the 17-day long experiment. In contrast, cells exposed to OMMV showed progressively increasing fluorescence from day 3 onward. Scale bars: 200 µm. (c) Real-time quantification of mineralization of the calcein fluorescence using the IncuCyte System. Mineralization was quantified as total calcein integrated intensity units per image (N = 3). ***p < 0.001.

Figure 2

Accumulation of intracellular minerals in 28-day-old HGF cultures treated with epithelial MVs. (a) Representative scanning electron microscope (SEM) images of HGF cultures treated in basic medium (BM), osteogenic control medium (OM) and in OM with MVs (OMMV). Note the accumulation of electron dense material (white arrows) in the cytoplasm around the nucleus of cells in OMMV. Scale bars: 100 µm. (b), (c) focused ion beam (FIB) cross section images (cross-sectional view of cells on culture plastic) of HGF cultures in OMMV. Intracellular vesicles with high density nodules (black arrows) can be detected. Bar = 10 µm in lower magnification image and 1 µm in higher magnification image. (d), (e), The energy-dispersive X-ray spectroscopy (EDS) analysis. EDS analysis showed that both calcium and phosphorus were present in the high-density nodules in the cell interior (e) while control areas contained carbon only (d). The Ca/P ratio of these nodules was determined to be 2:1.

Figure 3

Double immunofluorescence staining of epithelial MVs and mineralized nodules in HGF cultures. Membranes of the MV were labeled with Vybrant DiI cell labeling solution (red) and minerals with calcein green. After 3-day treatment with MVs, numerous red MVs were clearly detected as dots and diffuse patches on the cell layer regardless whether calcein was present (Cal +) or not (Cal−). At this time point, only a few mineralized green dots were detected. After 7-day treatment with MVs, the number of mineral nodules significantly increased around nuclei. However, no co-location of the MVs and the mineralized nodules were detected. HGFs without MVs showed no green calcein positive nodules. Scale bars: 50 µm.

Figure 4

Profiling of HGF genes differentially expressed after 1- and 3-day treatment with epithelial MVs in osteogenic medium. (a), (b) Gene ontology enriched annotations of biological process (GOTERM_BP), cellular component (GOTERM_CC) and molecular function (GOTREM_MF) associated with differentially expressed genes (DEGs) on HGFs in response to MV stimulation after 1 day (a) and 3 days (b) using The Database for Annotation, Visualization, and Integrated Discovery (DAVID). After 1-day stimulation, annotation about cell cycle, cell component and protein binding were enriched. On the other hand, genes related extracellular matrix (ECM) component were enriched after 3-days stimulation. Three biological replicates were included in the analysis.

Figure 5

Profiling of HGF genes related to extracellular matrix (ECM), Inflammatory response (INF) and Ossification (OSS) differentially expressed after 1- and 3-day treatment with epithelial MVs in osteogenic medium. (a), (b) differentially expressed genes (DEGs) were analyzed in groups related to ECM, INF and OSS after (a) 1 day and (b) 3 days stimulation using BaseSpace Correlation Engine. Genes related Inflammatory response were increased after 1- and 3-day stimulation. Genes related ECM were downregulated on 1 and 3 days. Ossification-related genes were mainly down-regulated. Three biological replicates were included in the analysis.

Figure 6

The effect of epithelial MVs on HGFs gene expression after 7- and 28-day stimulation. Gene expressions were analyzed by RT-qPCR. Cells without MVs (OM) were used as control. Results show mean ± standard error of the mean (SEM) from triplicate experiments (*p < 0.05, **p < 0.01, ***p < 0.001). The genes are grouped by function; (a), (c) MMPs, and (b), (d) osteogenic differentiation-related genes after 7-days (a), (b) and 28-day (c), (d) stimulation. Expression of MMP-3 and OSX was highly up-regulated (a) while expression of ALP and COL I was downregulated after 7-days stimulation (b). After 28-day culture, MMP1 and MMP3 were significantly up-regulated with MV stimulation (c). Among osteogenic differentiation-related genes, OSX and BSP were up-regulated with MV stimulation. On the other hand, expression of ALP, RUNX2 and COL I was downregulated (d).