Mesenchymal stem cell-derived exosomes improve vascular remodeling by inhibiting EGFR/ErbB2 heterodimerization in hypoxic pulmonary hypertension

Abstract

A key characteristic of hypoxic pulmonary hypertension (HPH) is pulmonary vascular remodeling, involving abnormal proliferation and migration of pulmonary artery smooth muscle cells (PASMCs). Recent studies indicate that mesenchymal stem cell-derived exosomes (MSC-exo) exhibit therapeutic effects on HPH. MSC-exosomes were isolated from the conditioned medium of bone mesenchymal stem cells using ultracentrifugation, confirmed via Western blotting (WB), transmission electron microscopy (TEM), and nanoparticle tracking analyses (NTA). Platelet-derived growth factor BB (PDGFBB) induced pathological behavior in PASMCs, replicating the conditions observed in HPH. HPH rats were subjected to a low oxygen environment (10 ± 1% oxygen) for 8 h daily over 28 days. Parameters such as right ventricular systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), and pulmonary vascular remodeling were evaluated. MSC-exosomes suppressed PDGFBB-induced proliferation and migration of PASMCs. Additionally, MSC-exosomes protected rats from hypoxia-induced increases in RVSP, right ventricular hypertrophy, and pulmonary vascular remodeling. The expression of epidermal growth factor receptor (EGFR) and Erb-B2 receptor tyrosine kinase 2 (ErbB2) was investigated in both HPH lung tissues and PDGFBB-induced PASMCs. Results indicated significant upregulation of EGFR/ErbB2 expression in HPH and PDGFBB-induced PASMCs, which was suppressed by MSC-exosomes. The study demonstrates that MSC-exosomes inhibit the development of HPH by suppressing excessive proliferation and migration of PASMCs through the inhibition of EGFR/ErbB2 heterodimerization.

Keywords: Epidermal growth factor receptor; Erb-B2 receptor tyrosine kinase 2; Hypoxic pulmonary hypertension; Mesenchymal stem cell-derived exosomes; Pulmonary vascular remodeling.

Fig. 1

Preparation and Characterization of Exosomes Derived from Bone Mesenchymal Stem Cells. A: Schematic diagram of MSC-exo extraction by ultracentrifugation; B: Shape of MSC-exo detected by TEM (Scale bar = 100–200 nm); C: Distribution of MSC-exo diameter and particles detected by NTA. D: Expression of MSC-exo protein markers analyzed by Western blotting; MSC-exo: exosome derived from mesenchymal stem cells; TEM: transmission electron microscopy; NTA: nanoparticle tracking analysis.

Fig. 2

MSC-exo Ameliorated PDGFBB-Induced PASMC Proliferation. A: Cell viability determined by CCK-8 analysis; B: Detection of PCNA protein expression by Western blotting analysis; C: Immunofluorescence staining of Ki67 in PASMC after co-incubation with PDGFBB for 24 h (Scale bar = 50 μm); (n ≥ 3); Data are presented as mean ± SEM; **** indicates P < 0.0001, *** indicates P < 0.001, and ** indicates P < 0.01 for normoxia vs. PDGFBB. ^#### indicates P < 0.0001, ^### indicates P < 0.001, and ^## indicates P < 0.01 for MSC-exo vs. PDGFBB. PDGFBB: platelet-derived growth factor BB.

Fig. 3

MSC-exo Ameliorated PDGFBB-Induced PASMC Migration. A: Cell migration assessed by wound healing assay (Scale bar = 100 μm); B: Percentage of wound closure of PASMCs induced by PDGFBB at 24 h and 48 h; (n ≥ 3); Data are presented as mean ± SEM; **** indicates P < 0.0001, *** indicates P < 0.001, and ** indicates P < 0.01 for normoxia vs. PDGFBB. ^#### indicates P < 0.0001, ^### indicates P < 0.001, and ^## indicates P < 0.01 for MSC-exo vs. PDGFBB.

Fig. 4

EGFR/ErbB2 is Highly Expressed in PDGFBB-Induced PASMCs. A-C: RT-qPCR used to determine the mRNA expression of Hif1α, EGFR, and ErbB2 in PDGFBB-induced PASMCs; D: Immunofluorescence staining labeled EGFR and ErbB2 in PASMCs (Scale bar = 50 μm); E: Western blots used to determine the protein expression of ErbB2 in PASMCs; (n ≥ 3); Data are presented as mean ± SEM; **** indicates P < 0.0001, *** indicates P < 0.001, ** indicates P < 0.01, and * indicates P < 0.05. EGFR: epidermal growth factor receptor; ErbB2: Erb-B2 receptor tyrosine kinase; Hif1α: hypoxia-inducible factor 1 subunit alpha.

Fig. 5

MSC-exo Reversed Pulmonary Vascular Hypertrophy and Thickening in Chronic Hypoxic Pulmonary Hypertension Rats. A-B: Measurement of RVSP and the real-time detected pressure waveform; C: Representative lung sections stained with hematoxylin-eosin (Scale bar = 100 μm–50 μm); D: Measurement of medial arterial wall area and thickness of distal pulmonary vessels; (n ≥ 3); Data are presented as mean ± SEM; **** indicates P < 0.0001, *** indicates P < 0.001, ** indicates P < 0.01, and * indicates P < 0.05. RVSP: right ventricular systolic pressure.

Fig. 6

MSC-exo Ameliorated the Muscularization and Fibrosis of Peripheral Pulmonary Vessels in HPH Rats. A: Immunohistochemical staining for α-SMA in peripheral pulmonary vessels and representative images of terminal pulmonary vessels (Scale bar = 50 μm–20 μm); B: Percentage of muscularization in distal pulmonary vessels; C: Representative lung sections stained with Masson’s stain and the percentage of positive collagen volume (Scale bar = 100 μm–50 μm); D: Percentage of positive collagen volume in distal pulmonary vessels; (n ≥ 3); Data are presented as mean ± SEM; **** indicates P < 0.0001, ** indicates P < 0.01, and * indicates P < 0.05. α-SMA: α-smooth muscle actin.

Fig. 7

MSC-exo Suppressed Right Ventricular Hypertrophy in HPH Rats. A: Representative images of H&E staining of the right ventricular muscle cell (Scale bar = 50 μm–20 μm); B: Percentage of single cell cross-sectional area; (n ≥ 3); C: Ratio of RV/LV + S, indicating the degree of right ventricular hypertrophy; Data are presented as mean ± SEM; *** indicates P < 0.001 and ** indicates P < 0.01. RV/LV + S: right ventricle / left ventricle plus septum.

Fig. 8

EGFR/ErbB2 Pathway is Highly Expressed in HPH Rats. A: Western blots used to determine the protein expression of Hif1α in lung tissue; B: Western blots used to determine the protein expression of EGFR in lung tissue; C: Western blots used to determine the protein expression of ErbB2 in lung tissue; (n ≥ 3); Data are presented as mean ± SEM; ** indicates P < 0.01 and * indicates P < 0.05.