Nucleoporin93 (Nup93) Promotes Vascular Health by Limiting Endothelial Cell Aging and Dysfunction

Endothelial cells (ECs) serve as a selective monolayer to mediate blood vessel integrity for vascular function, and EC health is in turn increasingly acknowledged as a crucial factor in limiting age-induced vessel dysfunction. Conversely, chronic inflammation and natural aging drive pro-inflammatory endothelial changes, resulting in EC activation and vessel leakage to precipitate vascular disease. Recent studies report increased nuclear leakiness with aging, displaying the significance of proper nucleocytoplasmic transport for cellular health. As the major regulator of molecular transport across the nuclear envelope, the nuclear pore complex (NPC) in ECs may also be compromised with age to drive vascular disease. Our exciting studies implicate nucleoporin93 (Nup93), a crucial structural NPC protein, as an indispensable player for EC health and vascular protection. We observe a significant reduction in endothelial Nup93 expression in the coronary vasculature of aged mice. Moreover, we find that endothelial loss of Nup93 induces cellular senescence and promotes the surface expression of pro-inflammatory adhesion molecules for enhanced EC-monocyte interaction. Mechanistically, endothelial Nup93 deficiency leads to nuclear leakiness for increased nuclear localization of Yap, a transcription co-factor known to activate EC inflammation. Pharmacological inhibition of Yap activity in Nup93-null ECs reverses the senescence and inflammatory phenotypes, indicating Yap hyperactivation as a major consequence of Nup93 deficiency in ECs. Furthermore, Nup93 loss in human ECs leads to actin stress fiber formation, impaired eNOS-dependent nitric oxide (NO) bioavailability, and enhanced in vitro paracellular permeability. Mechanistically, endothelial Nup93 depletion significantly decreases Sun1 levels, a component of the linker of the nucleoskeleton and cytoskeleton (LINC) complex necessary to connect the nucleus with the cytoskeleton. Endothelial loss of Nup93 also results in a concomitant increase in RhoA activity, where the re-introduction of Sun1 in Nup93-deficient ECs mitigates RhoA activation to restore endothelial barrier function and NO production. Our studies not only show Sun1 as a negative regulator of endothelial RhoA activity, but also implicate Nup93 as a necessary component in preventing aberrant RhoA signaling. Overall, the dissertation herein highlights the previously unrecognized role of NPC proteins in limiting endothelial senescence and dysfunction, thus providing novel insight into the molecular mechanisms underlying vascular aging and disease progression

Registered T2 overlapped over T1.

<p><b>A</b>) Mandibular Body mask showing superimposition over T1; <b>B</b>) Modified Björk mask displayed a vertical shift of the mandibular canal in relation to T1.</p

3D Mandibular Superimposition: Comparison of Regions of Reference for Voxel-Based Registration - Fig 2

<p><b>A</b>) Representation of the measurements obtained independently from T1 and T2 and their difference; <b>B</b>) Representation of the T2-T1 differences based on mandibular registration; <b>C</b>) Partial 3D surface model of the mandible and representation of the 8 landmarks used for the measurements.</p

Measurement of the agreement of changes between T2 and T1 from registrations using Mandibular Body and Modified Bjork masks with changes between T2 and T1 obtained from independent measurements.

<p>Bland-Altman means (in mm), standard deviation and 95% limits of agreement (LoA, in mm) for comparison between corresponding measurements of the changes between T2 and T1.</p

Landmarks identified on patient’s 3D models surface and their definition.

<p>Landmarks identified on patient’s 3D models surface and their definition.</p

Figures that represent the masks (regions of reference) used for mandibular voxel-based registrations.

<p><b>A</b>) Mask1, Björk; <b>B</b>) Mask2, Modified Björk; and <b>C</b>) Mask3, Mandibular Body. The yellow part corresponds to the mask and the red part was erased.</p

3D surface models overlays: red model, T1; white model, T2 from mandibular body mask registration; yellow model, T2 from modified Björk mask registration.

<p><b>A</b>) overlay of registered T2 (mandibular body mask) and T1; <b>B</b>) overlay of registered T2 (modified Björk mask) and T1; <b>C</b>) overlay of registered T2 (mandibular body mask) and registered T2 (modified Björk mask).</p

Figures illustrating the head orientation procedure used to obtain the same 3D coordinate system for all patients.

<p><b>A</b>) superior view; <b>B</b>) lateral view; and <b>C</b>) frontal view.</p

Intraclass correlation coefficient (ICC) comparing changes between T2 and T1 from registrations using Mandibular Body and Modified Bjork masks and changes between T2 and T1 obtained from independent measurements.

<p>ICC with 95% confidence interval (CI).</p

Induction of anti IgY Abs and IgY treatment in mice with pre-existing anti IgY.

<p>Anti IgY in the sera of mice immunized with normal IgY (IgY immunized), treated once intranasally with PR8-specific IgY 8 hours before (anti PR8 IgY −8 h) or three times after infection (anti PR8 IgY +8, 32, 56 h) (Fig. 4A). Endpoint titers (log₂) were determined by ELISA. Morbidity and mortality of IgY-immunized mice treated with PR8-specific IgY (anti PR8 IgY) before (−6 hr) or after (+6 hr) infection with mouse-adapted PR8 (Fig. 4B). The values are the mean of 5–10 mice in each group.</p