Abstract
Purpose :
Dysregulated endothelial barrier function contributes to a wide variety of pathologies including diabetic macular edema and proliferative diabetic retinopathy. While vascular endothelial growth factor (VEGF) promotes barrier opening, other factors, including members of the transforming growth factor b (TGFb) family, maintain or restore barrier integrity. In this project we continued our effort to elucidate the mechanism by which activin A suppresses VEGF-mediated permeability.
Methods :
All experiments were performed with high glucose -treated (10 days 30 mmol/L) primary human retinal endothelial cells. Cell permeability was assessed using electrical cell-substrate impedance sensing. To detect pore formation, we used the gelatin trapping assay (GTA). The organization of adherens junctions (AJ) was assessed by immunofluorescence staining. VEGF-induced signaling and the phosphatase level VE-PTP was investigated by western blot analysis.
Results :
We previously reported that activin attenuates VEGF-induced permeability, and that the mechanism involves increased expression of VE-PTP, which suppresses VEGF-mediated signaling. This VE-PTP-based mechanism does not appear to be relevant after 24 (instead of 48) hours of exposure to activin, which also suppressed VEGF-induced permeability. VEGF-induced signaling was not suppressed and the level of VE-PTP was not increased after 24 h of activin. TNF-induced permeability was also affected (enhanced) in cells that had been treated with activin for 24 h. By examining AJ disorganization and pore formation we found that both VEGF and TNF disorganized the AJ and increased pore formation, and activin suppressed (for VEGF) or enhanced (for TNF) these effects. The observation that activin affected both permeability agonist directed our attention towards explanations of the activin effect that were not unique to the VEGF pathway. One such possibility is receptor trafficking. In cells that had be exposed to activin for 24 h expression of numerous regulators of trafficking, including Rab 5a and Rab 11a, was altered. These data are the basis of our working hypothesis that activin suppresses VEGF-mediated barrier relaxation at the 24 h time point by perturbing trafficking of activated VEGFR2.
Conclusions :
Activin attenuates VEGF-induced permeability by multiple mechanism that are determined by the duration of exposure to activin.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.