Figure 2.

Lack of BMPR1A in endothelial cells induces endothelial to mesenchymal transition. (A) Immunostaining showing CD31, αSMA, and DAPI in lung tissues from Bmpr1a^iECKO (bottom) mice compared with control (top). White arrowheads indicate CD31 and αSMA double-positive endothelial cells (scale bar = 30 μm). (B)Quantification of CD31 and αSMA double-positive nucleus in the lung of control and Bmpr1a^iECKO mice [**P ≤ 0.005 (unpaired t-test), n = 7]. (C) Snapshot of 3D reconstructed video (see Supplementary material online, Videos S1 and 2) with lung section of control (left) and Bmpr1a^iECKO (right) mice (αSMA; CD31; DAPI). White arrowheads indicate αSMA/CD31 double-positive cells in Bmpr1a^iECKO (right) mice (scale bar = 50 μm). (D) Representative images showing the morphology of control (left) or BMPR1A (right) siRNA-treated PAECs (scale bar = 50 μm). (E) Quantification of elongation index in control or BMPR1A siRNA-treated PAECs [**P ≤ 0.005 (unpaired t-test), n = 11]. (F) Representative images showing the morphology of control (left) and Bmpr1a-deleted (Bmpr1a^KO, right) mouse lung ECs (scale bar = 50 μm). (G) Quantification of elongation index in control and Bmpr1a-deleted mouse lung ECs [**P ≤ 0.005 (unpaired t-test), n = 18]. (H) Western blot showing significantly increased expression of αSMA and SM22α in BMPR1A siRNA-treated PAECs compared with control siRNA-treated PAECs. (I) Quantification of expression of SM22α and αSMA in control or BMPR1A siRNA-treated PAECs [*P ≤ 0.05, **P ≤ 0.005 (unpaired t-test), n = 3]. (J) qRT–PCR showing the RNA expression of EndoMT and EC markers. Expression of transcripts associated with EndoMT (SLUG, SNAIL, ZEB2, and NCAD) is elevated with concomitant downregulation of endothelial transcripts (CDH5, VEGFR2, and PECAM) in BMPR1A siRNA-treated PAECs [**P ≤ 0.005, *P ≤ 0.05 (unpaired t-test), n = 3]. (K) Lineage tracing using ROSA^mT/mG reporter mice showed that a subset of excessive αSMA expressing cells are descendants of endothelial cells. Arrowheads indicate αSMA and mGFP double-positive cells. The inset shows the area in white-dotted rectangle (scale bar = 20 μm). (L) Flow cytometry showing an increased number of αSMA/GFP double-positive cells in Cdh5(PAC)Cre^ERT2; Bmpr1a^fl/fl; ROSA26^mT/mG compared with Cdh5(PAC)Cre^ERT; ROSA26^mT/mG mice. (M) Quantification of the number of αSMA and GFP expressing cells (Q2 area) from control and Bmpr1a^iECKO mice [**P ≤ 0.005 (unpaired t-test), n = 3].