Effects of TGFβ Stimulation in Aortic Valvular Interstitial Cells Are Altered in Hypoxia

Objectives: Local hypoxia due to leaflet thickening during calcific aortic valve disease leads to upregulation of hypoxia-inducible factor-1α (Hif-1α). Signaling of pro-fibrotic transforming growth factor β (TGFβ) is known to be altered in hypoxic conditions and to interact with Hif-1α signaling in several tissues, but interrelation with hypoxia in aortic valve is still unknown.

Methods: Ovine aortic valvular interstitial cells (VIC) were cultivated under normoxic (N) or hypoxic (H) conditions (1% O₂) using standard culture conditions (std) or TGFβ stimulation (TGFβ). After 6 days of cultivation gene and protein expression levels were analyzed.

Results: Hypoxic cultivation of VIC leads to significantly higher Hif-1α protein expression (p < 0.01) and higher expression of Hif-1α target gene vascular endothelial growth factor (VEGF) in all cell culture conditions (p < 0.05), while no morphological changes were observed compared to normoxia. Stimulation with TGFβ leads to a positive feedback loop with significantly elevated TGFβ gene expression in both hypoxic and normoxic conditions (p < 0.05). In prolonged hypoxia (12 days) gene expression of TGFβ is significantly lower in H-TGFβ vs. N-TGFβ condition (p = 0.004) but insignificantly altered compared to H-std, indicating loss of positive feedback loop in hypoxic but not in normoxic condition. After 6 days, expression of TGFβ effector Smad3 is significantly lower in H-TGFβ vs. N-TGFβ (p = 0.04), suggesting an altered regulation of TGFβ signaling in hypoxia. Vice versa TGFβ stimulation significantly increases protein expression of factor inhibiting Hif-1α (FIH) in TGF- H-TGFβ vs. N-TGFβ (p = 0.007) and compared to N-std (p < 0.001). Prolonged hypoxia leads to loss of hypoxia-induced VEGF upregulation after TGF stimulation (p = 0.77; std: p = 0.0043). However, TGF does not only impact on Hif-1α signaling: NFkB expression is enhanced after TGFβ stimulation compared to N-std (p = 0.006) and therefore significantly higher than in H-TGFβ (p = 0.003). Nevertheless, pNFkB expression is not altered between conditions, leading to a significantly lower pNFkB/NFkB ratio in N-TGFβ than in H-TGFβ (p = 0.038).

Conclusions: Hypoxia alters TGFβ signaling in aortic VIC with a relevant impact on NFkB signaling and modification of TGFβ positive feedback loop. On the other hand TGFβ leads to upregulation of FIH and loss of hypoxia induced VEGF upregulation indicating a complex crosslink between TGFβ and Hif-1α signaling.