PMC

Cyclic stretch increased expression of MMPs. qPCR showing AVICs exposed to 14% cyclic stretch have increases in MMP-1, MMP-14, MMP-16-1, and MMP-16-2 mRNA levels. ^#P < 0.005; n = 3.

Cyclic stretch increases interleukin expression. A) qPCR showing increased mRNA levels for IL-1α, IL-1β, IL-8, and IL-33. B, C) ELISA demonstrating that stretched AVICs secrete increased amounts of IL-1β and IL-8. *P < 0.05, ^#P < 0.005; n = 3.

Model of how stretch activates NF-κB signaling and inflammatory pathways. Black summarizes stretch responsive pathway based on our data. Gray indicates data in context of proposed mechanism by which BAVs develop AVC as the result of mechanical stretch due to their abnormal valve morphology.

Conditioned medium from stretched AVICs activates THP-1 macrophages. A) Differentiation of THP-1 monocytes into adherent macrophages, quantified by total lactate dehydrogenase assay performed on THP-1 cells treated with conditioned medium from stretched or static AVICs for 24 h. B) THP-1 cells treated with conditioned medium have increased expression of IL-1α, IL-1β, and IL-33 compared to controls. **P < 0.01, ^#P < 0.005; n = 3.

miR-148a represses expression of inflammatory genes. AVICs were transfected with miR-148a mimic for 72 hours. A) The expression levels of ILs after transfection with miR-148a-3p mimic; n = 6. B) Expression of MMPs after transfection with miR-148a-3p mimic; n = 6. C) miR-148a mimic blocks/attenuates the stretch activation of IKBKB, IL-1β, IL-8, MMP-1, and MMP-14. AVICs were transfected with the scramble control or miR-148a-3p mimic 24 hours before being exposed to stretch or static conditions for 24 hours; n = 3. D) Transfection with miR-148a-3p inhibitor results in increased expression of ILs; n = 4. E) miR-148a inhibition results in increased expression of MMP-1 and MMP-16-1. Of note, the expression levels of MMP-14 trended toward being increased by miR-148a-3p inhibition (P = 0.06); n = 4. *P < 0.05, ^#P < 0.005, ^P < 0.001.

miR-148a-3p is repressed by stretch and represses both IKBKB and NF-κB signaling. A) qPCR demonstrates that AVICs exposed to cyclic stretch have decreased miR-148a-3p levels. B) Human diseased BAVs leaflets have decreased miR-148a-3p levels, determined by qPCR, compared to tricuspid aortic valves. C) IKBKB mRNA levels are increased in stretched AVICs compared to static controls. *P < 0.05; n = 4. D) IKBKB mRNA levels were decreased by qPCR in miR-148a-3p mimic-treated AVICs compared to scramble controls. AVICs transfected with miR-148a-3p inhibitor did not have increased expression of IKBKB. E) Western blot analysis showing protein levels of IKKβ, IκB, and phospho-p-65 after transfection with miR-148a-3p mimic. F) Targetscan miRNA target prediction algorithm predicts 2 miR-148a-3p binding sites in the 3′ UTR of IKBKB. G) Luciferase reporters bearing putative miR-148a binding sites were transfected into HEK293 cells. Reporter for the 132–154 site of the IKBKB 3′ UTR demonstrated a 35% reduction (P < 0.02) of luciferase when cotransfected with miR-148a-3p. The miR-148a-3p-mediated repression of this site was abrogated when 3 bases in the seed site was mutated. The 1281–1303 site was not significantly repressed by miR-148a-3p mimic, yielding a 12% reduction (P = 0.06); n = 3.