Prodrug BMP-7 attenuates pulmonary fibrosis through downregulation of bone marrow derived Apo E+ alveolar macrophage

Abstract

Background Bone morphogenetic protein-7 (BMP-7) antagonizes transforming growth factor-β (TGF-β), which is critically involved in pulmonary fibrosis. This study investigated the ability of prodrug BMP-7, designed as a micelle formulation nanoparticle of BMP-7, to ameliorate pulmonary fibrosis through nasal inhalation in a preclinical bleomycin (BLM)-induced fibrosis mouse model. In addition, the effect on alveolar macrophages (AM) in BALF was examined using single-cell RNA sequencing. Materials and Methods After successfully demonstrating the delivery of fluorescently labeled prodrug BMP-7 into murine lungs in vivo, animal experiments were conducted on 38 mice (C57BL/6J) divided into three groups: control, BLM, and BLM with prodrug BMP-7. The prodrug BMP-7 and vehicle were inhaled nasally at intervals of 72 h for 21 days, respectively. Subsequently, a single-cell RNA sequence was performed on BALF by dividing it into four groups to analyze the role of prodrug BMP-7 on AM: control, prodrug BMP-7 alone, BLM, and BLM with prodrug BMP-7. Results Compared with the BLM group, BALF inflammatory cells were significantly reduced in the prodrug BMP-7 group, and the inhibition of fibrosis was confirmed by Masson’s trichrome staining and the modified Ashcroft lung fibrosis scoring system. In addition, the downregulation of collagen 1, α-SMA, fibronectin, and TGF-β/SMAD signals and a significant decrease in CXCL10 and CXCL2, which are chemokines secreted by AM, were observed. In BALF, the single-cell RNA sequence proportion of Apo E+ macrophages significantly increased in the BLM group and decreased in the prodrug BMP-7 group. In addition, Apo E+ expression increased in the lung tissues of patients with idiopathic pulmonary fibrosis. Conclusions These findings indicate that prodrug BMP-7 could be an effective therapeutic agent for pulmonary fibrosis through modulation of Apo E+ AM.

Bone morphogenetic Protein-7 (BMP-7)은 폐섬유화에 결정적으로 관여하는 Transforming Growth Factor-β (TGF-β)를 길항 한다. 본 연구에서는 나노입자로 설계된 prodrug BMP-7이 Bleomycin (BLM) 폐섬유증 마우스 모델에서 항섬유화 효과를 나타내는지 분석하고, 폐포 대식세포에 대한 prodrug BMP-7의 역할을 규명하기 위해 bronchoalveolar lavage fluid (BALF) 단일 세포 RNA 시퀀싱을 수행했다. 38마리의 마우스 (C57BL/6)를 대조군, BLM 그룹, prodrug BMP-7 그룹으로 나누었으며, prodrug BMP-7과 vehicle은 각각 21일 동안 72시간 간격으로 흡입되었다. BLM 그룹에 비해 prodrug BMP-7 흡입 그룹에서 유의미한 섬유화 감소가 관찰되었으며, 마손삼색 염색 및 변형된 Ashcroft 폐 섬유증 채점 시스템을 사용하여 이를 증명하였다. Western blot에서 prodrug BMP-7 그룹의 collagen 1, α-SMA, fibronectin의 mRNA 감소, TGF-β/SMAD 하향조절이 관찰되었고, 마우스 폐 조직과 BALF에서의 CXCL10, CXCL2와 등의 케모카인 감소를 ELISA를 통해 확인했다. 다음으로, BALF 단일 세포 RNA 시퀀싱을 통해 Apo E+ 대식세포의 비율이 BLM 군에서 유의하게 증가하고 prodrug BMP-7에서는 유의하게 감소함을 관찰했으며, 특발성폐섬유증 환자의 폐 조직에서 ApoE 발현 증가와 BLM 폐섬유화증 마우스 모델에서 prodrug BMP-7 흡입 시 Apo E 발현의 감소를 확인했다. 이는 prodrug BMP-7이 Apo E+ 폐포 대식세포의 조절을 통해 폐섬유화에 대한 효과적인 치료제가 될 수 있음을 시사한다.