Abstract
BackgroundImmunity and inflammation are considered to be central features of pulmonary artery hypertension (PAH), in which macrophages are one of the main components of inflammatory cell infiltration around the pulmonary artery. M2b macrophages, which are different from M1 and M2 macrophages, are believed to have immunomodulatory activities and produce little fibrosis. The purpose of this study was to explore the effect of M2b macrophages on pulmonary artery smooth muscle cells (PASMCs) derived from monocrotaline-induced PAH rats.MethodsPASMCs were cultured in serum-free medium, the supernatant of M0 macrophages, or the supernatant of M2b macrophages for 24 hours. Then cell proliferation was assessed by cell counting kit-8 and cell migration ability was detected by wound healing and transwell assays. The apoptosis rate of cells was determined by TUNEL staining and annexin V-PE/7-ADD staining. Western blot was used to detect the expression of Bcl-2 family proteins, cleaved caspase-9 and PI3K/Akt/FoxO3a pathway. LY294002 (a specific inhibitor of PI3K) was used to investigate its effect on PASMCs and its relationship with M2b macrophages.ResultsConditioned medium from M2b macrophages significantly inhibited the proliferation and migration of PASMCs compared with the control group and M0 macrophage group. Furthermore, conditioned medium from M2b macrophages promote PASMC apoptosis and increased the expression of pro-apoptotic proteins Bax and cleaved caspase-9, inhibited the expression of anti-apoptotic proteins Bcl-2 and Bcl-xl. Finally, conditioned medium from M2b macrophages inhibited the PI3K/Akt/FoxO3a pathway. Inhibition of PI3K/Akt/FoxO3a pathway also significantly inhibit the proliferation, migration, and apoptosis resistance of PASMCs.ConclusionConditioned medium from M2b macrophages can inhibit the proliferation, migration, and apoptosis resistance of PASMCs, which may be at least partially by deregulating the PI3K/Akt/FoxO3a pathway.
Highlights
Pulmonary artery hypertension (PAH) is characterized by a progressive increase in pulmonary vascular resistance, identified by progressive hypoxia and right heart failure, which seriously affects the survival and prognosis of patients with cardiovascular and respiratory diseases (Humbert, Sitbon & Simonneau, 2004)
The results showed that >90% of the cells were LIGHT+ CD45+ after stimulation, indicating most of bone marrow cells were polarized into M2b macrophages (Figs. 1E and 1F). Quantitative real-time PCR (qRT-PCR) was used to identify the phenotypes of the polarized macrophages
The pro-apoptotic protein expression of Bax and cleaved caspase-9 was significantly increased, the anti-apoptotic proteins Bcl-2 and Bcl-xl were significantly decreased, and the ratio of Bax/Bcl-2 was significantly increased in the M2b group compared with the other two groups. These results suggest that Bcl-2 family proteins and cleaved caspase-9 may be involved in the conditioned medium from M2b macrophage induction of pulmonary artery smooth muscle cells (PASMCs) apoptosis
Summary
Pulmonary artery hypertension (PAH) is characterized by a progressive increase in pulmonary vascular resistance, identified by progressive hypoxia and right heart failure, which seriously affects the survival and prognosis of patients with cardiovascular and respiratory diseases (Humbert, Sitbon & Simonneau, 2004). Pulmonary vascular remodeling is mainly due to medial hypertrophy and neointimal lesion formation, and the apoptosis resistance, proliferation, and migration of pulmonary artery smooth muscle cells (PASMCs) play a vital role in this process. Exploring the molecular mechanism of PASMC proliferation, migration, and apoptosis resistance is an essential means to prevent pulmonary vascular remodeling, and to treat PAH. Macrophages are one of the main components of inflammatory cell infiltration around the pulmonary artery during the development of PAH and are an essential source of local factors regulating pulmonary vascular remodeling. Conditioned medium from M2b macrophages significantly inhibited the proliferation and migration of PASMCs compared with the control group and M0 macrophage group. Inhibition of PI3K/Akt/FoxO3a pathway significantly inhibit the proliferation, migration, and apoptosis resistance of PASMCs
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