Abstract
Purpose :
Retinal vascular leakage resulting from blood-retinal barrier (BRB) dysfunction is a significant factor in retinal eye diseases such as diabetic macular edema (DME). Plasmalemma vesicle-associated protein (PLVAP) is highly upregulated in DME patients. It is involved in caveolae-mediated transcellular transport, a process involved in BRB breakdown, but its exact function remains elusive. Our goal is to identify novel PLVAP-associated proteins, to investigate PLVAP regulation during breakdown of the BRB in more detail.
Methods :
We used various methods to enrich the expression of functionally relevant PLVAP protein. These methods include stimulation with phorbol 12-myristate 13-acetate (PMA) and vascular endothelial growth factor (VEGF), as well as the introduction of lentiviral overexpression constructs of PLVAP. Correct membrane-associated localization was verified by confocal microscopy and transmission electron microscopy (TEM). Isolated and fractionated protein samples were analyzed by mass spectrometry.
Results :
PLVAP upregulation following stimulation with PMA, VEGFA, and overexpression vectors, was confirmed, and membrane localization was verified. Immunofluorescence was used to demonstrate that PMA induction alone or in combination with PLVAP overexpression yields the most precise anticipated localization of PLVAP at caveolar vesicles. This was later confirmed with TEM. Mass spectrometry results are currently analyzed.
Conclusions :
We show that overexpression of PLVAP, in combination with PMA treatment, is the most effective way to upregulate PLVAP localized in caveolae. This methodology enables us to identify proteins that are directly associated with PLVAP. Ultimately our results will lead to new insights into the molecular mechanisms of BRB breakdown in DME, which may lead to better treatment options for patients.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.