Fig 1: Endothelial PHD2 deficiency suppressed BMP signaling in lung endothelial cells. (a) RNA‐seq analysis demonstrated suppression of BMP signaling by PHD2 deficiency in HIF‐2α dependent manner in PH mice. Egln1 f/f (WT), Egln1 Tie2Cre (CKO), and Egln1/Hif2a Tie2Cre (EH2) mice. (b) mRNA expression of Bmpr2, Bmpr1b, Acrvl1, and Grem1 were dysregulated in the lung of CKO mice and normalized in EH2 mice. (c) Downregulated protein expression of BMPR2 and upregulation of GREM1 in the CKO lungs were normalized in EH2 lungs. (d) Decreased BMPR2 and increased GREM1 in the lung ECs of CKO lungs via immunostaining. V, vessel. (e) RNA‐sequencing analysis showed that PHD2 deficiency reduced the expression of BMP signaling molecules. HLMVECs were transfected with control siRNA (siCtl) or PHD2 siRNA for 48 h. Three replicates were pooled in an equal amount for RNA‐seq analysis. (f) qRT‐PCR analysis confirmed the reduction of BMP signaling, including upregulation of GREM1 and downregulation of BMPR1A and ID1 in PHD2‐deficient HLMVECs. N = 3. (g) BMPR2 expression and activities were reduced by PHD2 knockdown, but not affected by HIF‐2α knockdown in IPAH patient‐derived PAECs. IPAH PAECs were transfected with siCtl or PHD2 or HIF‐2α siRNA for 48 h, followed by treatment of BMP9 (5 ng/ml) for 16 h. (h) GREM1 is upregulated by HIF‐2α secondary to PHD2 deficiency. HLMVECs were transfected with siCtl or PHD2 siRNA or PHD2 plus HIF2A siRNA for 48 h. N = 3. (i) A diagram showing three putative hypoxia responsible element (HRE) sites in the promoter region of the human GREM1 promoter. Red highlighted texts indicate mutated HRE sequences. (j) Luciferase assay demonstrates that human GREM1 promoter activities were induced by PHD2 deficiency in an HIF‐2α‐dependent manner. HLMVECs were cotransfected with human GREM1 promoter firefly luciferase plasmids, control Renilla luciferase plasmids, and siCtl or PHD2 siRNA for 48 h, followed by treatment with dimethyl sulfoxide or HIF‐2α translational inhibitor C76 (20 μm) for 16 h. N = 3. (k) Mutagenesis studies and luciferase assays demonstrated that HRE sites in the GREM1 promoter mediate GREM1 activation in PHD2‐deficient HLMVECs. N = 3 to 4. (l) FK506 treatment reduced right ventricular (RV) systolic pressure in CKO mice. (m) RV hypertrophy was inhibited in FK506‐treated CKO mice compared to PBS. One‐way analysis of variance with Tukey posthoc analysis for multiple group comparisons (b, h, j, k). Student t test (f, l, m). CKO, conditional knockout; DAPI, 4′,6‐diamidino‐2‐phenylindole; HIF, hypoxia‐inducible factor; HLMVEC, human lung microvascular endothelial cell; IPAH, idiopathic pulmonary arterial hypertension; mRNA, messenger RNA; PAEC, pulmonary arterial endothelial cell; PBS, phosphate‐buffered saline; PH, pulmonary hypertension; PHD2, prolyl hydroxylase 2; qRT‐PCR, quantitative reverse‐transcription polymerase chain reaction; RVSP, right ventricular systolic pressure; siRNA, small interfering RNA; WT, wild‐type. *p < 0.05; **p < 0.01, and ***p < 0.001
Fig 2: Wound-healing assay in NIBD patient-derived colonic epithelial cell monolayers. (A) Representative images of wound healing in the presence or absence of BMP9. (B) The wounded areas covered with migrated cells were measured at 8 and 24 hours after BMP9 stimulation (N = 7 per group). The red dashed lines show the edge of the cells.
Fig 3: Decreased ALK1 expression is associated with reduced NOTCH activity and NOTCH target gene expression in the colonic epithelial cells of CD patients. (A) Representative blot (left) and the difference of JAG1 and NOTCH intracellular domain (NICD) protein expression between NIBD and CD patients (right). (B) BMP9 concentration in the serum of NIBD controls (N = 17) and CD patients (N = 23). (C) NOTCH target gene expression in colonic epithelial cells from CD patients (N = 15) and NIBD controls (N = 12). (D) NOTCH target gene expression in NIBD patient-derived colonic epithelial cell monolayers. Expanded cells were cultured in expansion media in the presence or absence of BMP9 and ALK1–Fc chimera protein. N = 6 per group. Each gene expression was normalized to (C) GAPDH or (D) RPLP0. ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001. P values were determined by the (A–C) Mann–Whitney test or the (D) Friedman test followed by the Dunn multiple comparison test.
Fig 4: Loss of TGFβRI leads to elevated basal BMP activity and BMP9 responsiveness in chondrocytes. Cells were transfected with 12×SBE-Luc reporter (77) and Renilla luciferase control reporter. Relative 12×SBE-Luc activity shows the normalized level of 12×SBE-Luc/Renilla Luc. Assays were repeated 2 times. n = 3/technical repeats in each assay. Error bars show SE. Significance was established using 2-way ANOVA and t test. *P < 0.05/n (n = number of groups). (A) Elevated basal BMP activity and BMP9 responsiveness in chondrocytes. The basal BMP activity in Tgfbr1Δ/Δ cells is more than 2-fold higher than in control cells (columns 1 and 5). TGFβ1 treatment (2 ng/mL) increases BMP activity in control cells but does not affect Tgfbr1Δ/Δ cells (columns 2 and 6). BMP2 (100 ng/mL) and BMP9 (100 ng/mL) have similar activities in control cells at the tested concentrations (columns 3 and 4). BMP9 induces higher BMP activity than BMP2 in Tgfbr1Δ/Δ cells (columns 7 and 8). (B) NOGGIN does not block elevated basal BMP activity in Tgfbr1Δ/Δ cells. Elevated BMP activity in Tgfbr1Δ/Δ cells compared with control cells (columns 1 and 5). BMP2 activity in control and Tgfbr1Δ/Δ cells (columns 2 and 6). NOGGIN inhibits BMP2 activity in both control and Tgfbr1Δ/Δ cells (columns 3 and 4, and columns 7 and 8). NOGGIN does not block elevated basal BMP activity in Tgfbr1Δ/Δ cells (columns 5 and 8). (C) BMP9 antibody does not block elevated basal BMP activity in Tgfbr1Δ/Δ cells. BMP reporter activity is activated by BMP9 in control ATDC5 cells (columns 1 and 2). α-BMP9 antibody blocks exogenous BMP9 action in control and Tgfbr1Δ/Δ cells (columns 3 and 7). Tgfbr1Δ/Δ cells exhibit elevated basal BMP activity (columns 1 and 5). Tgfbr1Δ/Δ cells respond to BMP9 (columns 5 and 6). α-BMP9 antibody has no effect on elevated basal BMP activity in Tgfbr1Δ/Δ cells (columns 5 and 8). (D) ACVRL1-Fc does not block elevated basal BMP activity in Tgfbr1Δ/Δ cells. BMP reporter activity is activated by BMP9 in control ATDC5 cells (columns 1 and 2). ACVRL1-Fc blocks exogenous BMP9 action in control and Tgfbr1Δ/Δ cells (columns 3 and 7). Tgfbr1Δ/Δ cells exhibit elevated basal BMP activity (columns 1 and 5). Tgfbr1Δ/Δ cells respond to BMP9 (columns 5 and 6). ACVRL1-Fc has no effect on elevated basal BMP activity in Tgfbr1Δ/Δ cells (columns 5 and 8). (E) ACTRIIB-Fc does not block elevated basal BMP activity in Tgfbr1Δ/Δ cells. BMP reporter activity is activated by BMP9 in control ATDC5 cells (columns 1 and 2). ACTRIIB-Fc blocks exogenous BMP9 action in control and Tgfbr1Δ/Δ cells (columns 3 and 7). Tgfbr1Δ/Δ cells exhibit elevated basal BMP activity (columns 1 and 5). Tgfbr1Δ/Δ cells respond to BMP9 (columns 5 and 6). ACVRL1-Fc has no effect on elevated basal BMP activity in Tgfbr1Δ/Δ cells (columns 5 and 8). (F) ACVRL1 antibody blocks elevated basal BMP activity in Tgfbr1Δ/Δ cells. BMP reporter activity is activated by BMP9 in control ATDC5 cells (columns 1 and 2). Anti-ACVRL1 antibody does not block BMP9 action in control cells (columns 2 and 4). Tgfbr1Δ/Δ cells exhibit elevated basal BMP activity (columns 1 and 5). Anti-ACVRL1 antibody blocks elevated basal BMP activity in Tgfbr1Δ/Δ cells (columns 5 and 7). Anti-ACVRL1 antibody restores BMP9 activity to control level in Tgfbr1Δ/Δ cells (columns 2, 4, and 8). (G) Loss of ACVRL1 kinase activity restores BMP signaling to normal level in the Tgfbr1Δ/Δ cells. Elevated BMP activity in Tgfbr1Δ/Δ cells compared with control cells (columns 1 and 3). Mutation of threonine-196 to valine near the canonical start of the ACVRL1 kinase domain (Acvrl1KD) using Crispr-Cas9 system in the Tgfbr1Δ/Δ cells (column 5); BMP activity in Tgfbr1/Acvrl1 double-mutant cells is restored to normal (columns 1 and 5). BMP9 activity in control and Tgfbr1Δ/Δ cells (columns 2 and 4). BMP9 activity in Tgfbr1/Acvrl1 double-mutant cells is lower than Tgfbr1Δ/Δ cells and restored to control levels (columns 2, 4, and 6). (H) Anti-ACTRIIB antibody blocks elevated basal BMP activity in Tgfbr1Δ/Δ cells. BMP reporter activity is activated by BMP9 in control ATDC5 cells (columns 1 and 2). Anti-ACTRIIB antibody deceases BMP9 action in control cells (columns 2 and 4). Tgfbr1Δ/Δ cells exhibit elevated basal BMP activity (columns 1 and 5). Anti-ACTRIIB antibody blocks elevated basal BMP activity in Tgfbr1Δ/Δ cells (columns 5 and 7). Anti-ACTRIIB antibody decreases BMP9 activity by 2-fold in Tgfbr1Δ/Δ cells (columns 6 and 8) but does not completely restore BMP9 activity to control levels (columns 4 and 8).
Fig 5: BMP9–ALK1 signaling enhances human colonic IEC barrier integrity. (A) Gene expression of junctional proteins in NIBD patient-derived colonic epithelial cell monolayers. Expanded cells were cultured in expansion media in the presence or absence of BMP9 and ALK1–Fc chimera protein. Each gene expression was normalized to RPLP0 (N = 6 per group). (B) Gene expression of tight junction proteins in colonic epithelial cells isolated from CD patients (N = 15) and NIBD controls (N = 12). Each gene expression was normalized to GAPDH. (C) Epithelial permeability assay in NIBD patient-derived colonic epithelial cells cultured on a collagen scaffold. TEER was measured over time (N = 3 per group). (D) Cells were stimulated with BMP9 in EM in the presence or absence of ALK1–Fc chimera protein or cultured in DM on day 4. The changes in TEER between days 4 and 6 are shown as ΔTEER% (N = 4–8 per group). ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001. P values were determined by the (A and D) Kruskal–Wallis test followed by the Dunn multiple comparison test, and the (B) Mann–Whitney test.
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