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A ROLE FOR FLK-1 (VASCULAR ENDOTHELIAL GROWTH FACTOR-RECEPTOR 2) IN INTESTINAL BARRIER MAINTENANCE
Ivan Pacheco, Panida Lungchukiet, Craig Spencer, R John MacLeod
GIDRU/Dept of Physiology, Queen’s University, Kingston, Ontario
AIM: The current experiments sought to determine a role of Vascular endothelial growth factor (VEGF) secretion from colonic myofibroblasts after activation of the extracellular calcium-sensing receptor (CaSR).
Material and METHODS: 18Co, a colonic myofibroblast cell line and primary isolates of human colonic myofibroblasts were used to model the sub-epithelia while T84 cells grown on Transwells to a resistance of ~1800 Ohms/cm2 were used to model the epithelia.
RESULTS: Activation of the CaSR by incubation for 12h with 3 or 5 mM Ca2+ stimulated VEGF secretion into the medium 5 and 11 fold (p<0.05), respectively. Transient transfection with siRNA against the CaSR reduced both 3 and 5 mM Ca2+ stimulated VEGF to ~160% of basal levels of secretion (p<0.05, n=8). High Ca2+ (3 mM) challenge of high resistance T84 cells to activate the CaSR increased the expression of both Flt-1 and Flk-1 (VEGF receptors 1 and 2), respectively. We used the Ca2+-switch protocol to break the barrier and measured recovery from 200 to 1800 Ohms/cm2 over 24 h. T84 cells treated with 3 mM Ca2+ recovered resistance faster 2h, but by 6 h there was no difference between the two groups. Apical secretion of VEGF remained constant (100-200 pg/mg) as TER increased from 200 to 1200 0hms/cm2, however secretion into the basolateral compartment increased from 100 to 800 pg/mg, following the increases in TER. To assess whether the VEGF produced by the epithelia was involved in the maintenance of barrier, we incubated high resistance T84 cells overnight with SU5614 (10 µM), a selective inhibitor of Flk-1. The Flk-1 inhibitor did not alter TER of the cells. We then performed Ca2+ switch in the presence and absence of SU5614. Within 1h the untreated cells had recovered 67% while the SU5614 treated cells had recovered 41% (1220 vs 753 Ohms/cm2). Untreated cells recovered completely within 4h, while it took 24h for those treated with SU5614 to return to starting TERs.
CONCLUSIONS: Our current results suggest that VEGF secretion is polarized in intact epithelia and works through Flk-1 to maintain high resistance. Unlike its effect on vascular endothelia, VEGF/Flk-1 may decrease intestinal barrier permeability.