Inhibition of BMPER Mitigates Pulmonary Hypertension by Modulating LRP1-YAP Interaction in Smooth Muscle Cells

Abstract

Background: BMPER (bone morphogenetic protein-binding endothelial regulator) is a secreted protein that is highly expressed in endothelial cells. It regulates the BMP (bone morphogenetic protein) pathway during vascular development and adulthood. Mutations in the BMP pathway are recognized as risk factors for pulmonary arterial hypertension group 1 pulmonary hypertension (PH). However, the roles of BMPER in pulmonary arterial hypertension remain unknown.

Methods: We assessed BMPER expression in Group 1 pulmonary arterial hypertension patient samples and examined its role in vascular remodeling using in vivo and in vitro approaches.

Results: BMPER level was elevated in pulmonary arterial hypertension lungs and significantly associated with pulmonary vascular resistance, but was not increased in patient plasma. Global and endothelial cell-specific depletion of BMPER in a mouse model of hypoxia-induced PH displayed attenuation in pulmonary artery smooth muscle cell proliferation, a hallmark of pulmonary vascular remodeling, and reduced right ventricular pressures. Conversely, adeno-associated virus-assisted BMPER overexpression targeted to the pulmonary endothelium led to the spontaneous development of PH. Mechanistically, BMPER promoted YAP (yes-associated protein 1) activation through the release of YAP sequestration by LRP1 (low-density lipoprotein receptor-related protein 1), a BMPER endocytic receptor, in the membrane of pulmonary artery smooth muscle cells. Moreover, the protective effect of BMPER depletion can be reversed by simultaneous depletion of LRP1 in mice with hypoxia-induced PH.

Conclusions: Collectively, these results implicate secreted BMPER as a discrete regulator for pulmonary vascular remodeling and suggest its inhibition as a new potential therapeutic strategy against PH.

Keywords: cell proliferation; endothelial cells; hypertension, pulmonary; hypoxia; vascular remodeling.

Figure 1.. BMPER level is increased in human PAH patient lungs.

(A-B) BMPER protein level was elevated in lung lysates, but not plasma of PAH patients compared to non-PH (ctrl) donors. Western blots for BMPER were performed. n=11 (Ctrl), 11 (PAH) for lung and n=3 for plasma samples in each group. In human plasma, BMPER dominant forms are BMPER N-terminal domain fragment (NTD, 40 kDa), processed from the full-length form that exists dominantly inside of the cells. (C) BMPER level was associated with PVR of PAH patients. n=11. (D) Immunohistochemistry of BMPER, showing BMPER was mainly enriched in intima of pulmonary vessels from PAH patients or non-PH donors. (E) Immunofluorescence of non-PH and PAH lung sections, stained with CD31 (green), BMPER (red) and DAPI (blue) or IgG controls. V, vessel. Statistical analysis was performed using unpaired two-tailed Student’s t-test (B) or simple linear regression analysis (C).

Figure 2.. BMPER iKO mice display attenuated pulmonary hypertension in response to hypoxia.

(A) Western blotting demonstrated the diminished BMPER expression in iKO lungs compared with wild-type (WT) mice. (B-C) BMPER depletion decreased right ventricle (RV) systolic pressure (RVSP, b) and RV hypertrophy (Fulton index: RV/(LV+septum) expressed as mg/mg, c) in mice exposed to 3-weeks of 10% hypoxia. (D) Echocardiography demonstrated BMPER depletion decreased RV wall thickness at diastole (RVWTD) in BMPER iKO mice. Representative echocardiography images (M-mode) were shown. (E) Echocardiography demonstrated BMPER depletion recovered hypoxia-reduced the pulmonary artery acceleration time/ejection time (PA AT/ET) ratio in BMPER iKO mice. Representative echocardiography images (PW-mode) were shown. Nx, normoxia. Hx, hypoxia. WT, BMPER^flox/flox;CAG-CreER^−/−. iKO, BMPER^flox/flox;CAG-CreER^+/−. n=6 (A), 7 (B, C), 5~11 (D, E). Statistics were analyzed with 2-way ANOVA followed by a Fisher’s LSD test (A-E).

Figure 3.. BMPER iKO mice display attenuated pulmonary arterial remodeling responses.

(A) Immunostaining demonstrated vascular remodeling responses in BMPER iKO and WT mice following 3-weeks of 10% hypoxia or normoxia exposure. Lung sections were subjected for H&E and EVG staining or immunostained with anti-SMA (red). V, vessel. Br, bronchiole. Quantification of pulmonary vascular remodeling was performed, including medial wall thickness, vessel number and distal artery number. FM, fully muscularized. PM, partially muscularized. NM, non-muscularized. (B-C) Smooth muscle cell (SMC) content was increased by hypoxia and this increase was inhibited by BMPER depletion. Lung lysates were blotted for α-SMA (B) or isolated lung SMC-enriched lysates were subjected for Western blots with PCNA antibody (C). n=8 mice per group and ≥39 vessels per group for the analysis of medial wall thickness, muscularized vessel number and distal arterioles per 100 alveoli (A), 5 (B, C). Statistics were analyzed with 2-way ANOVA followed by a Fisher’s LSD test (A-C).

Figure 4.. EC-BMPER depletion attenuates pulmonary hypertension induced by hypoxia or PHD2 depletion in ECs.

(A-B) BMPER depletion normalized RVSP (A) and RV hypertrophy (B) in mice exposed to 3-weeks of 10% hypoxia. Nx, normoxia. Hx, hypoxia. BMPER eWT, BMPER^flox/flox;Cdh5-CreER^−/−. BMPER eKO, BMPER^flox/flox;Cdh5-CreER^+/−. (C) BMPER levels increased in the lung lysates of PHD2 eKO (PHD2^flox/flox;Cdh5-CreER^+/−) mice compared to eWT mice. (D-J) BMPER depletion attenuated the impact of PHD2 depletion on RVSP (D), PA AT/ET ratio (E), medial wall thickness (G, quantified with H&E and EVG images, representative pictures were shown in F), and partially on RV hypertrophy, indicated by RVWTD (H), weight ratios of RV/(LV+S) (I) and RV/BW (body weight; J) in mice. eWT, PHD2^flox/flox;BMPER^flox/flox; Cdh5-CreER^−/−. PHD2 eKO, PHD2^flox/flox;Cdh5-CreER^+/−. PB DKO, PHD2^flox/flox;BMPER^flox/flox; Cdh5-CreER^+/−. n=5 (A, B), Statistics were analyzed with 2-way ANOVA and followed by a Fisher’s LSD test. n=7 (C), 6 (D, E, H-J), and ≥23 vessels per group for the analysis of medial wall thickness (G). Statistical analysis was performed using 2-way ANOVA followed by a Fisher’s LSD test (A-B), unpaired two-tailed Student’s t-test (C), one-way ANOVA followed by a Fisher’s LSD test (D-E, G-J).

Figure 5.. Mice with overexpressed BMPER in lung endothelium develop pulmonary hypertension spontaneously.

(A) BMPER and α-SMA levels increased in the lung lysates, but not in the serum, of AAV-BMPER-injected mice. (B) BMPER levels were significantly higher in ECs than other cells isolated from the AAV-BMPER-injected mouse lungs. (C-F) AAV-BMPER increased RVSP (C), RV hypertrophy (D), RVWTD (E) and decreased PA AT/ET ratio (F) in mice. (G-I) AAV-BMPER increased pulmonary vessel remodeling. Lung sections were subjected for H&E and EVG staining (G) or immunostained with anti-CD31 (green), SMA (red) and DAPI (blue, H). Quantification of pulmonary vascular remodeling in AAV-BMPER-injected mice, including medial wall thickness, vessel number and distal arteries per 100 alveoli (I). FM, fully muscularized. PM, partially muscularized. NM, non-muscularized. n=6 (A), 8 (C-F) mice per group. n ≥26 (AAV-GFP, I) or 22 vessels (AAV-BMPER, I). Statistics were analyzed with unpaired two-tailed Student’s t-test (A, C-F, I).

Figure 6.. BMPER and LRP1 reciprocally regulate YAP activation in PASMCs.

(A) SMC DNA synthesis was inhibited by BMPER depletion in endothelial cells. BrdU incorporation in human pulmonary artery SMCs (HPASMCs) was performed following culturing them with condition media collected from human pulmonary artery endothelial cells (HPAECs), which had been transfected with BMPER (B) or control (Ctrl) siRNAs and incubated at 21% or 2% O₂ for 4 hours. (B) BMPER depletion decreased YAP activation in hypoxic lungs, shown with Western blot assays. (C) BMPER treatment increased YAP activation in MPASMCs, shown with Western blot assays. (D) Mouse PASMCs were transduced with LRP1 or control shRNA lentivirus and then treated with BMPER (20 nM, 8 hr). (E-F) YAP and LRP1 were colocalized in the membrane. BMPER or LRP1 depletion induced YAP translocation into the nucleus in MPASMCs. Arrows indicate the membrane-bound YAP and LRP1. (G) A complex of endogenous YAP and LRP1 was detected in PASMCs and BMPER inhibits their association. n=24 fields per group (A), 4 (B, D), 3 (C, G), 12 (F). Statistics were analyzed with one-way (C) and 2-way ANOVA (B, D) followed by a Fisher’s LSD test or unpaired two-tailed Student’s t-test (F, G).

Figure 7.. LRP1 negates the protective effects of BMPER depletion in mice.

(A-B) LRP1 iKO mice displayed elevated RVSP and RV hypertrophy. LRP1 KO mice (LRP1^flox/flox;CAG-CreER^+/−) and their littermate control (WT, LRP1^flox/flox;CAG-CreER^−/−) mice were incubated for 3 weeks of 10% hypoxia and then subjected for RV catheterization and echocardiography. (C) LRP1 depletion increased YAP activation in normoxic lungs. However, hypoxia-induced YAP activation was not further increased by LRP1 depletion. Western blot assays were performed with mouse lung lysates. (D-E) BMPER;LRP1 DKO mice showed reversed phenotype compared to BMPER iKO mice, displaying elevated RVSP and RV hypertrophy. n=6 (A, B), 3 (C) and 5–7 (D, E). Statistics were analyzed with 2-way ANOVA followed by a Fisher’s LSD test (A-E).