NO-Induced Endothelial Barrier Protection: PKG-Mediated Activation of MLCP and Modulation of Cell Adhesion

Background and Aims: The precise regulation of nitric oxide (NO) production in endothelial cells is crucial for maintaining endothelial function. While excessive NO production due to inflammatory stimuli (e.g., histamine, VEGF) disrupts endothelial adherens junctions (AJs) and impairs barrier integrity, we have previously shown that insulin can protect the endothelial barrier through NO. In this study, we investigated the effects of exogenous NO on endothelial barrier function and the role of the cGMP/PKG signaling pathway in mediating these effects.

Methods: Human umbilical vein endothelial cells (HUVECs) were cultured and used in an in vitro model to assess endothelial barrier function. Exogenous NO was delivered using two structurally distinct NO donors, Spermine-NONOate and NOC-18 (10 µM). Barrier function was assessed through permeability (albumin flux) and isometric tension (IT) assays, while intracellular signaling was analyzed by immunoblotting and immunoprecipitation.

Results: Exogenous NO significantly reduced basal endothelial permeability by 35±7% and IT by 80±9%, and also decreased contractile activation by 31±6% (P<0.05, n=5). These effects were associated with a two-fold increase in vasodilator-stimulated phosphoprotein (VASP) phosphorylation. Pre-treatment with KT5823 (1 µM), a selective PKG inhibitor, abolished the NO-induced effects, indicating a PKG-dependent mechanism. In response to thrombin, which increased permeability by 210±12%, IT by 130±8%, and myosin light chain phosphorylation (MLC~P) by 90±8%, NO pre-incubation significantly attenuated these effects. NO also induced the assembly and activation of the myosin light chain phosphatase (MLCP) holoenzyme, increasing the translocation of PP1 catalytic subunit to the myosin phosphatase targeting subunit (MYPT1) by 3-fold and enhancing PP1 activity by 1.5-fold. Additionally, NO triggered a 3-fold increase in PKG translocation to MYPT1, suggesting PKG involvement in MLCP activation. NO also promoted the translocation of VE-cadherin, β-catenin, and γ-catenin to cell-cell junctions, which protected against thrombin-induced junctional disassembly. Furthermore, NO enhanced focal adhesion kinase (FAK) phosphorylation and increased focal adhesion formation.

Conclusion: These findings demonstrate that exogenous NO stabilizes the endothelial barrier by activating MLCP, which deactivates the contractile machinery and enhances both cell-cell and cell-matrix adhesion. The interaction between PKG and MYPT1 suggests that PKG mediates NO’s effects on MLCP, further enhancing endothelial integrity. These results provide insight into the mechanisms through which NO regulates endothelial barrier function and may have therapeutic implications for inflammatory vascular diseases.