Journal: Cardiovascular Research

Article Title: BMPR1A promotes ID2–ZEB1 interaction to suppress excessive endothelial to mesenchymal transition

doi: 10.1093/cvr/cvac159

Figure Lengend Snippet: Nanoparticle-mediated inhibition of TGFBR2 attenuates EndoMT and progression of PAH in vivo. (A) Representative images showing the morphology of control, BMPR1A, TGFBR2, or BMPR1A/TGFBR2 siRNA-treated PAECs. Morphological alteration in BMPR1A siRNA-treated PAECs was restored by concomitant inhibition of TGFBR2 (scale bar = 50 μm). (B) Quantification of elongation index in control, BMPR1A, TGFBR2, or BMPR1A/TGFBR2 siRNA-treated PAECs [**P ≤ 0.005 (unpaired t-test), n = 11]. (C) Western blots showing the expression of EndoMT markers (αSMA, SM22α, and TGFBR2) and BMPR1A in control, BMPR1A, TGFBR2, or BMPR1A/TGFBR2 siRNA-treated PAECs. Inhibiting TGFBR2 effectively suppressed the ectopic expression of EndoMT markers induced by BMPR1A siRNA treatment. (D) Quantification of αSMA (left) and SM22α (right) protein expression in control, BMPR1A, TGFBR2, or BMPR1A/TGFBR2 siRNA-treated PAECs [**P ≤ 0.005 (unpaired t-test), n = 3]. (E) Schematic illustration on the treatment regimen for 7C1 nanoparticles coated with either control or TGFBR2 siRNA. 7C1 nanoparticles were injected into the tail vein of Bmpr1aiECKO mice after tamoxifen administration. Three rounds of 7C1 nanoparticle injections were performed with a 1-week interval. (F) Western blot with whole lung lysate showed that treatment with TGFBR2 siRNA-coated 7C1 nanoparticle attenuated the ectopic expression of TGFBR2, pSMAD2, and pSMAD3 in Bmpr1aiECKO mice. (G) Quantification of TGFBR2, pSMAD2, and pSMAD3 protein level in control or TGFBR2 siRNA-coated 7C1 nanoparticle-injected Bmpr1aiECKO mice [**P ≤ 0.005 (unpaired t-test), n = 3]. (H) Immunostaining showed αSMA expression was significantly reduced in the lung of TGFBR2 siRNA-coated 7C1 nanoparticle-injected Bmpr1aiECKO mice (right) compared with control (left) (scale bar = 2 mm). (I) Quantification of αSMA-positive EC area in the lung of control or TGFBR2 siRNA-coated 7C1 nanoparticle-injected Bmpr1aiECKO mice [**P ≤ 0.005 (unpaired t-test), n = 14]. (J) Elevated right ventricle systolic pressure was restored to normal range in TGFBR2 siRNA-coated 7C1 nanoparticles-injected Bmpr1aiECKO mice. (K) Quantification of the right ventricle systolic pressure in control or TGFBR2 siRNA-coated 7C1 nanoparticles-injected Bmpr1aiECKO mice [**P ≤ 0.005 (unpaired t-test), n = 7]. (L) Quantification of the right ventricle heart wall thickness in control or TGFBR2 siRNA-coated 7C1 nanoparticles-injected Bmpr1aiECKO mice [**P ≤ 0.005 (unpaired t-test), n = 8].

Article Snippet: Cell culture and siRNA treatment Pulmonary artery ECs were purchased from ATCC (PCS-100-022).

Techniques: Inhibition, In Vivo, Control, Western Blot, Expressing, Injection, Immunostaining

Journal: Cardiovascular Research

Article Title: BMPR1A promotes ID2–ZEB1 interaction to suppress excessive endothelial to mesenchymal transition

doi: 10.1093/cvr/cvac159

Figure Lengend Snippet: Lack of BMPR1A in endothelial cells induces endothelial to mesenchymal transition. (A) Immunostaining showing CD31, αSMA, and DAPI in lung tissues from Bmpr1aiECKO (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 Bmpr1aiECKO 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 Bmpr1aiECKO (right) mice (αSMA; CD31; DAPI). White arrowheads indicate αSMA/CD31 double-positive cells in Bmpr1aiECKO (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 (Bmpr1aKO, 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 ROSAmT/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)CreERT2; Bmpr1afl/fl; ROSA26mT/mG compared with Cdh5(PAC)CreERT; ROSA26mT/mG mice. (M) Quantification of the number of αSMA and GFP expressing cells (Q2 area) from control and Bmpr1aiECKO mice [**P ≤ 0.005 (unpaired t-test), n = 3].

Article Snippet: Cell culture and siRNA treatment Pulmonary artery ECs were purchased from ATCC (PCS-100-022).

Techniques: Immunostaining, Control, Western Blot, Expressing, Quantitative RT-PCR, RNA Expression, Flow Cytometry

Journal: Cardiovascular Research

Article Title: BMPR1A promotes ID2–ZEB1 interaction to suppress excessive endothelial to mesenchymal transition

doi: 10.1093/cvr/cvac159

Figure Lengend Snippet: ID2–ZEB1 interactions regulate transcription of TGFBR2. (A) Western blots showing TGFBR2 and SM22α expression in control or BMPR1A siRNA-treated PAEC cells with control, ID1, ID2, or ID3 overexpressing adenovirus infection. Note that ID2-overexpressed PAECs showed dramatic reduction of TGFBR2 expression. (B) Quantification of TGFBR2 and SM22α protein expression in C [**P ≤ 0.005 (unpaired t-test), n = 3]. (C) Western blots showing TGFBR2 expression in control, ID1, ID2, or ID3 siRNA-treated PAECs. Expression of TGFBR2 was significantly increased in ID2 siRNA-treated cells, but not in ID1 or ID3 siRNA. (D) Quantification of TGFBR2 protein expression in control, ID1, ID2, or ID3 siRNA-treated PAECs [***P < 0.0001, **P ≤ 0.005 (unpaired t-test), n = 3]. (E) Representative images showing the morphology of control or ID2 siRNA-treated PAECs (scale bar = 50 μm). Right panel shows quantification of elongation index [**P ≤ 0.005 (unpaired t-test), n = 27]. (F) Representative images showing the morphology of control or BMPR1A siRNA-treated PAECs with control or ID2 adenoviral overexpression (scale bar = 50 μm). Right panel shows quantification of elongation index [**P ≤ 0.005, *P ≤ 0.05 (unpaired t-test), n = 36]. (G) Quantification of TGFBR2 RNA expression in control, ZEB1 or TCF3 siRNA-treated PAECs. [**P ≤ 0.005 (unpaired t-test), n = 3]. (H) Western blots showing TGFBR2 expression in control or ZEB1 overexpressing PAECs. Overexpression of ZEB1 induces robust expression of TGFBR2 in PAECs. (I) Western blots showing the expression of TGFBR2, SM22α, and ZEB1 and BMPR1A in control, BMPR1A, ZEB1, or BMPR1A/ZEB1 siRNA-treated PAECs. Inhibiting ZEB1 effectively suppressed the ectopic expression of TGFBR2 and SM22α induced by BMPR1A siRNA treatment. (J) Luciferase assay using pGL4-luciferase reporter vector containing the TGFBR2 promoter region (−1670/+36) with control or ZEB1 overexpressing PAECs [**P ≤ 0.005 (unpaired t-test), n = 3]. (K) Co-immunoprecipitation using Flag-ZEB1 and HA-ID2-overexpressing PAECs showing that ID2 could physically interact with ZEB1. Flag-VEGFR3 infected cells were used as negative control for flag antibody. (L) Display of genomic sequence corresponding to the 5′-upstream and transcription initiation site (font) of TGFBR2. Three putative E-box motifs located at −496, −386, and −81 positions from the TGFBR2 transcription initiation site were highlighted. The mutated sequence with site-directed mutagenesis in pGL4- TGFBR2 promoter was highlighted in black box. (M) Luciferase assay using pGL4-basic Luc vector containing control, mut1, mut2, or mut3 TGFBR2 promoter region with ZEB1 overexpressing HEK293 cells. Note that an E-box motif at the −81 position within the TGFBR2 promoter confers ZEB1-mediated regulation of TGFBR2 expression [**P ≤ 0.005 (unpaired t-test), n = 3]. (N) Chromatin immunoprecipitation showing the interaction between ZEB1 and the E-box motif at the −81 position/−496 position within the TGFBR2 promoter or Lif (negative control). Note that only the E-box motif at the −81 position was pulled down with Flag-ZEB1-binding antibody [**P ≤ 0.005 (unpaired t-test), n = 3]. (O) Chromatin immunoprecipitation showing that ID2 overexpression inhibits the binding of ZEB1 to the TGFBR2 promoter (E-box motif at the −81 position) [**P ≤ 0.005 (unpaired t-test), n = 3]. (P) Our working model: In the presence of endothelial BMPR1A, ID2–ZEB1 interaction increases. We speculate that ID2 could prevent ZEB1 from binding to the promoter of TGFBR2, and inducing transcription by sequestering ZEB1, which helps effectively maintain endothelial fate (left). In Bmpr1aiECKO mice, lack of BMPR1A and ID2 allows ZEB1 to bind to the promoter of TGFBR2, and derepresses its expression, which leads to the loss of endothelial identity and EndoMT.

Article Snippet: Cell culture and siRNA treatment Pulmonary artery ECs were purchased from ATCC (PCS-100-022).

Techniques: Western Blot, Expressing, Control, Infection, Over Expression, RNA Expression, Luciferase, Plasmid Preparation, Immunoprecipitation, Negative Control, Sequencing, Mutagenesis, Chromatin Immunoprecipitation, Binding Assay