Thrombin modifies growth, proliferation and apoptosis of human colon organoids: a protease-activated receptor 1- and protease-activated receptor 4-dependent mechanism

Abstract

Background and purpose: Thrombin is massively released upon tissue damage associated with bleeding or chronic inflammation. The effects of this thrombin on tissue regrowth and repair has been scarcely addressed and only in cancer cell lines. Hence, the purpose of the present study was to determine thrombin's pharmacological effects on human intestinal epithelium growth, proliferation and apoptosis, using three-dimensional cultures of human colon organoids.

Experimental approach: Crypts were isolated from human colonic resections and cultured for 6 days, forming human colon organoids. Cultured organoids were exposed to 10 and 50 mU·mL^-1 of thrombin, in the presence or not of protease-activated receptor (PAR) antagonists. Organoid morphology, metabolism, proliferation and apoptosis were followed.

Key results: Thrombin favoured organoid maturation leading to a decreased number of immature cystic structures and a concomitant increased number of larger structures releasing cell debris and apoptotic cells. The size of budding structures, metabolic activity and proliferation were significantly reduced in organoid cultures exposed to thrombin, while apoptosis was dramatically increased. Both PAR1 and PAR4 antagonists inhibited apoptosis regardless of thrombin doses. Thrombin-induced inhibition of proliferation and metabolic activity were reversed by PAR4 antagonist for thrombin's lowest dose and by PAR1 antagonist for thrombin's highest dose.

Conclusions and implications: Overall, our data suggest that the presence of thrombin in the vicinity of human colon epithelial cells favours their maturation at the expense of their regenerative capacities. Our data point to thrombin and its two receptors PAR1 and PAR4 as potential molecular targets for epithelial repair therapies.

Figure 1

Organoid characterization. (A) Microphotographs of isolated crypts at day 0 of culture, representative cystic, columnar and releasing organoids at days 3 and 6 of culture and representative budding structure at day 6 of organoid culture. Scale bar is 100 μm. (B) Organoid diameter (μm), at day 6 of culture. Data are means ± SEM, organoids n = 39 for cystic, n = 85 for columnar, n = 23 for releasing and n = 3 for budding. These data are representative of organoid cultures originating from five individuals.

Figure 2

Effects of thrombin (THRB) on organoid characteristics. (A) Diameter (μm) of cystic, columnar, releasing and budding organoids at 6 days of culture and after 3 days of exposure to thrombin at 10 or 50 mU·mL⁻¹ or to vehicle. (B) The percentage of the different types of structures, at 6 days of culture and after 3 days of exposure to thrombin at 10 or 50 mU·mL⁻¹ or to vehicle. Data are means ± SEM with n = 5 different individuals from which tissues were harvested, and 70 organoids were analysed per condition. *P < 0.05 (one‐way ANOVA, Bonferroni multiple comparisons test).

Figure 3

Effects of thrombin (THRB) on organoid metabolic activity. Concentration of ATP released per total surface of organoids as a measure of metabolic activity/cell survival in 6 day organoid cultures exposed to thrombin (10 or 50 mU·mL⁻¹) or to vehicle. Data are means ± SEM with n = 5 different individuals from which tissues were harvested. Individual sign shape represents data generated from the culture of the same patient tissue. *P < 0.05 (one‐way ANOVA, Bonferroni multiple comparisons test).

Figure 4

Effects of thrombin (THRB) on organoid cell apoptosis. The % of apoptotic cells (red staining) present in epithelial lining of organoid structures over total cells (cyan DAPI nucleus staining) per organoid cultured for 6 days and exposed for the last 3 days of culture to thrombin (10 or 50 mU·mL⁻¹) or vehicle. Data are means ± SEM with n = 5 different individuals from which tissues were harvested. Individual sign shape represents data generated from the culture of the same patient tissue. *P < 0.05 (one‐way ANOVA, Bonferroni multiple comparisons test). Photomicrographs are representative pictures of caspase‐3 staining in control condition (vehicle exposure) (left panel), thrombin 10 mU·mL⁻¹ incubation (middle panel) or thrombin 50 mU·mL⁻¹ (right panel) incubation. Scale bar is 50 μm.

Figure 5

Effects of thrombin (THRB) on organoid cell proliferation. The % of proliferative cells (Ki67 red staining) over total cells (cyan DAPI nucleus staining) per organoid cultured for 6 days and exposed for the last 3 days of culture to thrombin (10 or 50 mU·mL⁻¹) or vehicle. Data are means ± SEM with n = 5 different individuals from which tissues were harvested. Individual sign shape represents data generated from the culture of the same patient tissue. *P < 0.05 (one‐way ANOVA, Bonferroni multiple comparisons test). Photomicrographs are representative pictures of Ki67 staining in control condition (vehicle exposure) (left panel), thrombin 10 mU·mL⁻¹ incubation (middle panel) or thrombin 50 mU·mL⁻¹ (right panel) incubation. Scale bar is 50 μm.

Figure 6

PAR1 and PAR4 expression in human colon organoid cultures. (A) Relative mRNA expression of PAR1 and PAR4 in 6 day organoid cultures exposed to vehicle (control) or to thrombin (10 and 50 mU·mL⁻¹). (B) Photomicrographs representative of PAR1 immunostaining (red staining) and nucleus staining (blue staining with DAPI) in 6 day cultures of human colon organoids (two different z stack). Data in (A) are means ± SEM of n = 5 different individuals from which tissues were harvested. *P < 0.05 (one‐way ANOVA, Bonferroni multiple comparisons test). Scale bar is 50 μm in (B).

Figure 7

Effects of PAR1 and PAR4 antagonists on thrombin‐induced changes in organoid composition, metabolic activity, apoptosis and cell proliferation. The % of (A) cystic organoids, (B) releasing organoids and (C) budding organoids, (D) ATP concentration, (E) % of apoptotic cells per organoid and (F) % of proliferative cells per organoid in response to thrombin (50 mU·mL⁻¹ for A–C and 10 or 50 mU·mL⁻¹ for D–F) added on the last 3 days of culture in the presence of PAR1 antagonist F16357 (10 μM), PAR4 antagonist ML354 (140 nM) or vehicle, in human organoid cultures for 6 days. Data are means ± SEM of n = 5 different individuals from which tissues were harvested. (F) The ratio of proliferative cells to total cells per organoid was calculated by use of Ki67 as a marker. *P < 0.05 (one‐way ANOVA, Bonferroni multiple comparisons test).