Figure 5. Inhibition of TGF-β receptor–mediated signaling prevents phenotypic changes caused by RA deficiency in cultured human ASM.

hASM cells from 3 donors were cultured with control medium (CTR), pan-RA receptor antagonist (BMS), RA synthesis inhibitor (DEAB), and TGF-β1 with or without the TGF-β type I receptor kinase inhibitor SB. (A) Phospho-SMAD2 (p-SMAD2) to total SMAD2 (tSMAD2) ratio in BMS- and DEAB-treated hASM is reduced when hASM is also treated with SB, suggesting a decrease in TGF-β activity in RA-deficient hASM with TGF-β receptor blockade (n = 3). (B and C) Expression of TGF-β target COL1A2 (B) and smooth muscle–specific marker ACTA2 (C) in BMS- and DEAB-treated hASM is lowered by adding SB to the culture medium, showing that RA-deficiency-induced changes in hASM are ameliorated with inhibition of TGF-β signaling (n = 3). (D) Hypercontractility resulting from BMS and DEAB treatment is diminished by cotreatment with SB (n = 3). hASM cells cultured with TGF-β1 with or without SB are included as additional controls and for assessing the efficacy of SB in downregulating TGF-β signaling. All measured parameters are graphed relative to non–SB-treated CTR hASM (CTR/–SB). Data represent the mean ± SEM. Two-way ANOVA was used for statistical analysis. Bonferroni’s correction for multiple comparisons was applied to adjust P values. Means with different letters are significantly different with P < 0.05.