Fig 2: Long-term oscillating flow decreases Ng expression, suppresses eNOS regulation, and activates adhesion protein. (A) Schematics of long-term laminar flow (18 h) and long-term oscillating flow (18 h) experiments. (B) Long-term oscillating flow significantly decreased Ng expression compared to long-term laminar flow. (C) Both mock and Ng siRNA HAEC were exposed to long-term laminar flow and long-term oscillating flow (n = 3–4). (D) AKT phosphorylation was decreased by both siRNA and oscillating flow. (E) eNOS phosphorylation was decreased by siRNA. Oscillating flow in the mock sample decreased eNOS phosphorylation. (F) eNOS expression is increased by oscillating flow in Ng siRNA. (G) NF-κB phosphorylation is increased by oscillating flow in Ng siRNA. (H and I) both ICAM-1 and VCAM-1 are increased by siRNA and oscillating flow. Data presented as mean ± standard error mean. *p < 0.05 by 2-way ANOVA compared to oscillating flow and Ng siRNA. #p < 0.05 by 2-way ANOVA compared to interaction between oscillating flow and Ng siRNA.

Fig 3: Shear stress regulates Ng expression and suppresses ATK-eNOS phosphorylation in Ng siRNA. (A) HAEC were exposed to short-term laminar flow for 30 min (n = 4). (B) Ng expression is significantly decreased by laminar flow in both mock and Ng siRNA. (C) eNOS expression was significantly decreased by Ng siRNA but eNOS expression in Ng siRNA was not increased by shear stress. (D) Ng siRNA decreases eNOS phosphorylation (S1177) during shear stress. (E) AKT phosphorylation (S473) was suppressed by Ng siRNA, while shear stress activated AKT phosphorylation in a mock sample. (F) pERK did not show any changes between mock and Ng siRNA. (H) NF-κB phosphorylation was suppressed by Ng siRNA, while NF-κB expression did not show any changes. Data presented as mean ± standard error mean. *p < 0.05 by unpaired t-test compared to mock control.

Fig 4: Valsartan treatment rescues phospho‐eNOS expression in the aorta of MFS mice. (a) Representative p‐eNOS staining, and (b) average quantification of aortic roots of WT and MFS mice treated long‐term with valsartan. p‐eNOS expression is plotted against a number of different aortic parameters assessed in this study to determine possible correlations between the two. From the aortic parameters that were compared to p‐eNOS expression, (c) average elastic fiber length was found to be significantly correlated whereas (d) aortic root diameter and (e) aortic wall thickness was not significantly associated with p‐eNOS levels

Fig 5: Lack of Ng suppresses Ca2+-dependent AKT-eNOS regulation and endothelial dysfunction in mice. (A) The effect of pharmacological regulation in the Ca2+-dependent eNOS pathway (A28522: NFAT inhibitor, Cyclosporine A and FK606: CaN inhibitor, LY294002: PI3K inhibitor, DetaNONOate: NO donor). NFAT nuclear localization was decreased by CaN inhibitors (Cs A and FK606) in both mock and Ng siRNA. AKT phosphorylation was decreased by a PI3K inhibitor (LY294002) in both mock and Ng siRNA. CaN inhibitors (Cs A and FK606) significantly decreased AKT phosphorylation in Ng SiNRA compared to mock SiRNA, which implies that a lack of Ng modulates the CaN-AKT pathway (n = 3). (B) Lack of Ng activated Ca2+-dependent CaN expression in the aorta of Ng −/− mice (n = 4). (C) Lack of Ng in mice significantly decreased AKT phosphorylation (S473) in the aorta. (D) Ca2+-dependent CaMKII phosphorylation (T286) was increased in the aorta of Ng −/− mice. (E) There was no change in the eNOS phosphorylation between Ng +/+ mice and Ng −/− mice. (F) Ng −/− mice demonstrated increased adhesion molecule (VCAM-1) in the aorta. (G) Flow-mediated dilation (FMD) was performed in the femoral artery of Ng +/+ mice and Ng −/− mice. (H) Ng +/+ mice demonstrated significant femoral artery diameter changes between 30 s and 300 s after ischemia, while there was no difference in the Ng −/− mice (n = 4). Data presented as mean ± standard error mean. *p < 0.05 by paired t-test.