Hypothesis / aims of study
Lysophosphatidic acid (LPA) is a lipid mediator exerting actions including wound healing, cell proliferation, motility and DNA repair through G protein coupled receptors LPAR1-6. LPAR1 and 2 are studied the most, both activating Gi/o, Gq and G12/13 protein signaling. Tissue damage including irradiation increases LPA secretion activating pro-inflammatory and pro-fibrotic processes. However, it has been shown that LPAR2 activation decreases radiation-induced apoptosis of stem cells in the intestinal crypt decreasing gastrointestinal acute radiation syndrome-associated death [1]. In the bladder, LPA/LPAR1 has been reported to increase voiding frequency and being involved in oncogenesis. In these studies, we have examined the effect of LPAR2 activation and inhibition on urothelial cell survival and bladder function in mice following irradiation.
Study design, materials and methods
Cultured UROtsa (immortalized human uroepithelial) cells were plated in 96 well plates at 500 cells/well and subjected to 0 or 8 Gy irradiation. One hour later, cells were treated with LPAR2 agonist (radioprotectin-1, RP-1, 10 µM), LPAR2 antagonist (H2L5186303, 10 µM ) or vehicle (0.01% DMSO). Cell survival was monitored using the incubation monitoring system CM30.
Adult male C57Bl6 mice were used for assessing bladder function. Their bladders were irradiated with 0 or 20 Gy and RP-1 (0.2 mg/kg/day), H2L5186303 (5 mg/kg/day) or vehicle were delivered by osmotic pumps for a month after irradiation. Bladder function and fibrosis were assessed using 2-hour spot tests and histology (Van Gieson’s stain).
Results
Neither activation nor inhibition of LPAR2 affected non-irradiated UROtsa cells which grew up to 300% of the original number during a week of monitoring. About 50% of the irradiated cells treated with vehicle survived during this period. Treatment with LPAR2 agonist, RP-1, did not improve cell survival, on the contrary, only about 35% cells survived in this group. LPAR2 inhibitor did increase cell survival and growth following irradiation. These cells grew to about 150% of the original number.
Spot tests performed up to one month following irradiation demonstrated multiple void spots smaller in volume in comparison to the tests performed before irradiation (Figure 1). Spot tests of mice treated with LPAR2 agonist exhibited more voids and higher numbers of droppings in comparison to irradiated vehicle-treated animals. LPAR2 antagonist-treated mice had spot tests similar to non-irradiated peers. Tests repeated 6 months later still demonstrated very dramatic bladder dysfunction in both irradiated vehicle- and LPAR2 agonist-treated mice in comparison to non-irradiated and irradiated antagonist-treated animals.
Histology demonstrated very high collagen infiltration in irradiated bladders 6 months post insult (Figure 2). The bladders of irradiated animals treated with RP-1 were still fibrotic, while the bladders of the animals treated with LPAR2 antagonist had normal collagen content.
Interpretation of results
Our results suggest LPAR2 activation is detrimental to irradiated urothelial cells and the bladder. Urothelial cells survival following irradiation worsened with LPAR2 activation. Spot tests demonstrated exacerbated voiding disfunction in irradiated animals treated with RP-1. There was also a high collagen content suggestive of severe fibrosis in the bladders of these animals. Conversely, blocking LPAR2 have improved cell survival, bladder function and decreased the development of fibrosis following irradiation.
Concluding message
LPA/LPARs are important in regulating multiple cellular processes, however, their exact roles remain unclear. There are controversial reports on the role of LPARs in irradiation damage demonstrating that the blockage of LPAR1 and 3 decreases lung fibrosis [2], while activation of LPAR2 improves mucosal barrier function in the colon. In this study, we demonstrated activation of LPAR2 exacerbates urothelial cell survival, bladder function and fibrosis following irradiation while its blockade is beneficial.