Targeting the transient receptor potential (TRP) channels for restoration of microvascular barrier function in diabetic macular oedema

Here, functional expression of TRPV2 and TRPV4 was uncovered. Subsequently, a panel of endogenous TRP channel activators, upregulated in the eye during diabetes, was screened for ability to mobilise Ca2+ in HRMECs, using fura2-QBT Ca2+ signalling assays. Based on potency and robustness of Ca2+ response, upregulated activators histamine, thrombin, 4-oxo-2-nonenal (4-ONE), and lysophosphatidylcholine (LPC) were selected for further study.

To varying degrees, Ca2+ responses elicited by these compounds could be significantly attenuated pharmacologically, with the TRPV2 antagonist tranilast (100µM), TRPV4 antagonist HC-067047 (10µM), and TRPM7 antagonist NS8593 (20µM). Next, it was investigated whether these channel antagonists can attenuate barrier dysfunction elicited by the upregulated compounds of interest, using xCELLigence electrical impedance assays.

Here, barrier dysfunction elicited by thrombin was significantly attenuated in the presence of TRPV2 antagonist tranilast (100µM), whereas barrier disruption elicited by LPC was significantly attenuated by both TRPV2 antagonist tranilast (100µM) and TRPV4 antagonist HC-067047 (10µM). Following this, mechanism underlying barrier dysfunction were investigated. Here, HRMECs were shown to contract in response to thrombin, an effect that could be attenuated in the presence of TRPV2 antagonist tranilast (100µM) or ROCK1 inhibitor Y27632 (10µM).

Pharmacological inhibition of ROCK1 was also found to significantly attenuate barrier dysfunction elicited by thrombin, directly tying contraction to barrier integrity.<br><br>These findings strongly suggest that TRPV2 mediates endothelial barrier integrity within the retinal microvasculature, and overactivation of the channel by endogenous agonists may be a potential disease mechanism in DMO. <br><br><i>Thesis is embargoed until 31 July 2027.</i><br>