P-Rex2 Regulates Angiogenesis and Endothelial Barrier Dysfunction through Rac1-Dependent ROS Production

Background: Rac1, a member of the Rho family of GTPases, plays a key role in several cellular processes, including the maintenance of endothelial cell-cell junction integrity and NADPH oxidase-dependent ROS production. The activity of Rac1 is regulated by guanine nucleotide exchange factors (GEFs), which convert Rac1 from its inactive GDP-bound form to its active GTP-bound form. P-Rex2, a GEF highly expressed in the cardiovascular system, has not been fully investigated in terms of its role in regulating angiogenesis and endothelial barrier integrity. The aim of this study was to explore the role of P-Rex2 in the regulation of angiogenesis and endothelial barrier function under inflammatory conditions.

Methods: The study was carried out on cultured human umbilical vein endothelial cells (HUVEC). EC barrier function was analysed by measuring flux of albumen through HUVEC monolayers cultured on filter membranes. P-Rex2 and Rac1 was overexpressed using lentiviral vector system and down regulated using CRISPR/Cas system. Changes in EC cytoskeleton and cell-cell junctions were studied by immuno-cyto-chemistry (ICC) followed by fluorescent microscopy. Statistical analysis was performed using Graph-pad (Version 6) software.

Results: Lentiviral-mediated overexpression of P-Rex2 resulted in enhanced basal endothelial cell (EC) sprouting and an amplification of VEGF-induced sprouting, indicating increased angiogenic potential in vitro. This was associated with elevated basal EC permeability, as well as an exacerbation of TNF-α-induced hyperpermeability, loss of cell-cell junction integrity, and disruption of the peripheral actin cytoskeleton. In contrast, knockdown of P-Rex2 abolished these effects, both on angiogenesis and TNF-α-mediated hyperpermeability, suggesting a crucial role for P-Rex2 in regulating these processes.
P-Rex2 overexpression also led to the activation of Rac1 and enhanced reactive oxygen species (ROS) production. Pharmacological inhibition of Rac1 activation in P-Rex2 overexpressing cells effectively protected the EC barrier against TNF-α-induced disruption. Additionally, overexpression of constitutively active Rac1 (Rac1-CA) improved EC barrier function under basal conditions but led to the formation of excessive actin stress fibers in P-Rex2 overexpressing ECs, further supporting the involvement of Rac1 in P-Rex2-mediated endothelial barrier dysfunction.
Treatment with the ROS scavenger N-acetyl-L-cysteine (NAC) abrogated EC sprouting and protected the EC barrier from P-Rex2-induced loss of VE-cadherin from cell-cell junctions and the increased accumulation of actin at the cell periphery. Similarly, the NADPH oxidase inhibitor VAS2870 protected against EC barrier disruption in P-Rex2 overexpressing cells, both under basal conditions and following TNF-α treatment. These findings suggest that P-Rex2 mediates NADPH oxidase-dependent ROS production in ECs, contributing EC sprouting and the disruption of endothelial barrier integrity.

Conclusion: In conclusion, our data demonstrate that P-Rex2 regulates endothelial cell (EC) sprouting and endothelial barrier dysfunction downstream of TNF-α signaling through a Rac1-dependent mechanism involving NADPH oxidase activation. P-Rex2 may represent a valuable therapeutic target to prevent inflammation-induced ROS production and therapeutic angiogenesis.