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 PurposeThrombin 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 ApproachCrypts 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 ResultsThrombin 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 ImplicationsOverall, 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.

Characterization of Human Colon Organoids From Inflammatory Bowel Disease Patients

“…Organoids were plated in 96-wells opaque plates after 6 or 9 days of culture. CellTiter-Glo R 3D reagent (Promega, France) was added to each well (volume 1:1), and incubated for 30-min at room temperature (Sébert et al, 2018). Triplicate measures were performed for each condition.…”

“…In the presence of appropriate growth factors, intestinal stem cells present in the isolated crypts proliferate and enter into differentiation processes, recreating a complex epithelium, which contains all cell types that compose the intestinal epithelium (paneth cells, enteroendocrine cells, goblet cells, enterocytes, tuft cells, etc.). The epithelium generated by three-dimension cultures of isolated crypts closes on itself, forming a sphere, in which epithelial cells are orientated with their apical side toward the lumen (Sébert et al, 2018). While a number of studies have employed culture organoids from intestinal crypts (Sugimoto et al, 2018;Yip et al, 2018;Ramesh et al, 2019), only very few studies have investigated the possibility to culture organoids from IBD patient-isolated intestinal crypts (Dotti et al, 2017;Noben et al, 2017;Howell et al, 2018).…”

Sexual dimorphism in PAR2-dependent regulation of primitive colonic cells

“…On the other hand, PAR 1 activation triggered Akt activation and colonoid growth [7]. Furthermore, we have shown that PAR 1 is implicated in the maturation and apoptotic behavior of primary colonoids treated by thrombin [8].…”

Protease-Activated Receptors in the Intestine: Focus on Inflammation and Cancer

“…More recently, in human colon organoid cultures, we reported that thrombin significantly reduces the size of budding structures, metabolic activity and proliferation, while increasing apoptosis. In the same study, we reported that both PAR1 and PAR4 antagonists inhibited apoptosis regardless of thrombin doses (91).…”