Abstract
Purpose :
Transient receptor potential (TRP) channels mediate endothelial permeability responses outside of the retina. Here, we have determined the role of TRP channel-mediated Ca2+ signalling in diabetes-associated blood-retinal-barrier dysfunction (BRB) in vitro and evaluated the utility of pharmacologically targeting TRP channels as a novel therapeutic strategy to prevent excessive retinal vasopermeability responses.
Methods :
In vitro studies were performed using primary human retinal microvascular endothelial cells (HRMECs). The ability of molecules upregulated in the diabetic eye (histamine (Hist), thrombin (Thr), and lysophosphatidylcholine (LPC)) to elicit TRP channel-mediated Ca2+ responses was examined in Flexstation3 Fura2-QBT assays (n=6 wells). HRMEC barrier properties were investigated in XCELLigence electrical impedance assays (n=3 wells). Endothelial contractility responses were investigated in calcein-AM assays (n=2 wells) and isolated rat retinal microvessels (n=3 vessels). At least 3 biological replicates were carried out for each experiment.
Results :
Hist, Thr, and LPC were found to mobilise Ca2+ in HRMECs at physiologically relevant concentrations (< 10µM). Ca2+ mobilisation occurred via TRPV2, TRPV4, and TRPM7 channels, based on attenuated response magnitudes when pharmacological antagonists were present. The TRPV2 antagonist tranilast (100µM), decreased the degree of barrier dysfunction in response to Hist, Thr, and LPC (p<0.0005) (Fig. 1) and the TRPV4 antagonist, HC-067047 (10µM), reduced the magnitude of responses to LPC (p<0.0001). Addition of Hist, Thr, and LPC to HRMECs resulted in significant endothelial contraction in HRMECs (p<0.05).
Conclusions :
Molecules upregulated in the diabetic eye mobilise HRMEC Ca2+ via TRPV2, TRPV4, and TRPM7 channels leading to disruption of barrier properties and contractility responses. The ability of TRP channel antagonists to attenuate diabetes associated BRB disruption in vivo will be investigated in future studies. To our knowledge, this is the first time TRPV2 has been implicated in mediating endothelial permeability responses in any vascular bed.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.