Endothelial Nucleoporin93 (Nup93) Maintains Vascular Homeostasis via Sun1-Dependent Regulation of RhoA/ROCK Activity

BACKGROUND: As the innermost lining of blood vessels, endothelial cells regulate barrier function, maintain vascular tone, and limit inflammation for vessel health. Involved in various fundamental endothelial processes, RhoA (Ras homolog family member A)/ROCK (Rho-associated coiled-coil containing protein kinase) signaling has been identified as a major contributor to age-associated hypertension and vascular disease; RhoA/ROCK hyperactivation promotes vessel permeability and impairs nitric oxide (NO) production. Recent studies identify Sun1 (Sad1 and UNC84 domain-containing protein 1), a key component of the linker of nucleoskeleton and cytoskeleton complex, as a major repressor of RhoA/ROCK activity. Our latest studies identify endothelial loss of Nup93 (nucleoporin93), a component of the nuclear pore complex, as a hallmark of vascular aging. Insinuating a role for nuclear envelope components in vessel homeostasis, the role of Nup93 in RhoA/ROCK signal regulation, however, remains entirely unknown. METHODS: Targeted Nup93 knockdown approaches were used in primary human endothelial cells to assess the role of Nup93 in endothelial RhoA/ROCK signaling and downstream readouts, including endothelial barrier function, cellular stiffness, and eNOS (endothelial NO synthase) function. Rescue studies include the use of pharmacological ROCK inhibitors and lentiviral-mediated Sun1 restoration methods. We additionally measure barrier function and vessel reactivity in our novel inducible endothelial-specific Nup93 mouse model. RESULTS: Targeted loss of endothelial Nup93 significantly increases RhoA/ROCK activity for consequent endothelial permeability and decreased NO bioavailability both in vitro and in vivo. Mechanistically, we find that loss of Nup93 drastically reduces endothelial Sun1 levels for a concomitant increase in RhoA activity. Indeed, restoring Sun1 protein levels in Nup93-deficient endothelial cells mitigates RhoA activity, thereby rescuing both endothelial barrier function and eNOS expression. CONCLUSIONS: Taken together, we demonstrate endothelial Nup93 as a novel regulator of vascular permeability and NO-dependent vessel reactivity, contributing to the growing importance of nuclear membrane components in endothelial cell and vascular biology.