Endothelial Follistatin‐like 1 Regulates The Maturation of The Pulmonary Vasculature by Modulating BMP/Smad Signaling

Abstract

Pulmonary arterial hypertension (PAH) is a progressive disease that is characterized by vascular remodeling and sustained vasoconstriction which consequently lead to high blood pressure in the pulmonary vasculature and right ventricle remodeling. Altered bone morphogenetic protein (BMP) signaling has been implicated in the pathogenesis of PAH as well as in pulmonary vascular maturation. The endogenous BMP antagonist follistatin‐like 1 (Fstl1) is highly expressed in pulmonary vascular endothelium of the developing lung in mice, suggesting a role in pulmonary vessel formation. In this study, the role of Fstl1 in the maturation of the pulmonary vasculature was investigated using Tie2‐cre mediated endothelial‐specific Fstl1 knockout mice. To this end, Fstl1fl/fl mice were bred with Fstl1WT/KO Tie2‐cre mice to generate Fstl1KO/fl Tie2‐cre mice and control littermates Fstl1WT/fl Tie2‐cre, Fstl1KO/fl, and Fstl1WT/fl. Heart and lung tissue was collected from pups at 1 and 3 weeks after birth. The percentage of actin‐positive small vessels per total number of vessels was determined. The expression level of Fstl1 and BMP/Smad‐regulated genes was determined in total RNA isolated from lungs using qPCR. Activation of BMP signaling was investigated using western blot. In wildtype mice, the mRNA expression of Fstl1 was higher in lung tissue at 1 week than 3 weeks (p<0.005). In line with the idea that Fstl1 is a BMP inhibitor, BMP signaling was more active at 3 weeks than 1 week, reflected by the higher level of pSmad1/5/8 at 3 weeks than 1 week. Moreover, the mRNA levels of the BMP/Smad‐regulated genes JAG1 (p<0.05) and endothelin‐1 (p<0.005) were increased at 3 weeks compared to 1 week after birth. Endothelial‐specific deletion of Fstl1 was postnatally lethal, approx. 70% of Fstl KO/fl Tie2‐cre mice died at 3 weeks after birth. Though most were found dead, some were found in respiratory distress prior to dying. In these mice, Fstl1 mRNA expression was reduced (p<0.01) compared to wildtype mice at 1 week. Endothelial deletion of Fstl1 resulted in activation of Smad1/5/8 signaling and increased BMP/Smad‐regulated gene expression of JAG1 (p<0.05), endoglin (p<0.05), and endothelin‐1 (p<0.05) at 1 week. At 3 weeks, JAG1 was reduced (p<0.01) whereas endoglin and endothelin‐1 were unchanged. Furthermore, the percentage of actin‐positive small pulmonary vessels was increased at 3 weeks old of Fstl1KO/fl Tie2‐cre mice compare to wildtype (p<0.005) whereas it was unaltered at 1 week. Loss of endothelial Fstl1 induces activation of BMP‐mediated gene expression and microvasculature remodeling, which presumably due to alteration in endothelin‐1 expression.Support or Funding InformationThis study was supported by a grant from the Netherlands Lung Foundation (grant number: 3.2.12.083)