Inhibition of HDAC1 alleviates monocrotaline-induced pulmonary arterial remodeling through up-regulation of miR-34a

Figures

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  Fig. 1. Protocol for generation of rat pulmonary arterial hypertension (PAH) and reagent intervention models. The rat PAH model was induced by intraperitoneally injection of monocrotaline (MCT) (60 mg/kg) on day 1. MS-275 (3 mg/kg) was administrated to rats by intraperitoneally injection every other day for 28 days after MCT injection. miR-34a agomiR (25 μM) was given to rats via tail vein injection on days 1, 7, 14, 21 and 28 respectively after MCT injection

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  Fig. 2. MS-275 alleviates monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) in rats. a Right ventricular systolic pressure (RVSP) waveform. b Changes of RVSP in each group (n = 6–8). c Changes of right ventricular hypertrophy index (RVHI) in each group, RVHI = RV/(LV + S) (n = 6–8). *P < 0.05 versus control group; #P < 0.05 versus MCT group

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  Fig. 3. Histone deacetylases 1 (HDAC1) is up-regulated in monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) rats. a HDAC1 in the lung tissues from different groups was detected using immunofluorescence staining. b Protein levels of HDAC1/2/3 in the lung tissues from different groups were determined using immunoblotting (n = 5). *P < 0.05 versus control group; #P < 0.05 versus MCT group

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  Fig. 4. Inhibition of histone deacetylases 1 (HDAC1) restores miR-34a level in monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) rats and pulmonary arterial smooth muscle cells (PASMCs). a miR-34a level in lung tissues from different groups was determined using quantitative real-time polymerase chain reaction (qRT-PCR) (n = 3). b HDAC activity in lung tissues from different groups was measured with a fluorometric HDAC activity assay kit (n = 3). c Protein level of HDAC1 in PASMCs transfected with HDAC1-specific siRNA or control siRNA was examined using immunoblotting (n = 3). d miR-34a level in PASMCs transfected with HDAC1-specific siRNA or control siRNA was tested using qRT-PCR (n = 3). *P < 0.05 versus control group; #P < 0.05 versus MCT group

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  Fig. 5. Inhibition of histone deacetylases 1 (HDAC1) reduces the ratio of matrix metalloproteinase 9 (MMP-9)/tissue inhibitor of metalloproteinase 1 (TIMP-1) and MMP-2/TIMP-2 and the expression of collagen I through restoring miR-34a level in monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) rats. a miR-34a level in lung tissues from different groups was determined using quantitative real-time polymerase chain reaction (qRT-PCR) (n = 3). b MMP-2/9 activity in lung tissues from different groups was measured using gelatin zymography (n = 3). c Protein level of TIMP-1 and TIMP-2 in lung tissues from different groups was examined using immunoblotting (n = 3). d Ratio of MMP-9/TIMP-1 and MMP2/TIMP-2 was analyzed in lung tissues from different groups (n = 3). e Collagen I production in lung tissues from different groups was determined using enzyme-linked immunosorbent assay (ELISA) (n = 3). *P < 0.05 versus control group; #P < 0.05 versus MCT group

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  Fig. 6. MS-275 suppresses pulmonary arterial remodeling, excessive extracellular matrix (ECM) accumulation and the increase of right ventricular systolic pressure (RVSP) and right ventricular hypertrophy index (RVHI) through restoring miR-34a level in monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) rats. a Hematoxylin and eosin staining of pulmonary arteries (×400 magnification). b Quantitative morphometric analysis of the medial wall thickness of small pulmonary arteries. c Masson’s trichrome staining of pulmonary arteries (×400 magnification). d Changes of RVSP and RVHI in each group, RVHI = RV/(LV + S) (n = 6–8). *P < 0.05 versus control group; #P < 0.05 versus MCT group

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  Fig. 7. Proposed mechanism for inhibition of histone deacetylases 1 (HDAC1) alleviating monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH). Injection of MCT up-regulates HDAC1 expression, reduces miR-34a level, and subsequently increases the activity of matrix metalloproteinase 2/9 (MMP-2/9). The elevated activity of MMP-2/9 initiates degradation of extracellular matrix (ECM) and finally leads to excessive ECM accumulation through a vicious circle. In addition, tissue inhibitor of metalloproteinase 1/2 (TIMP-1/2) expression is also up-regulated to inhibit excessive MMP activity through a balancing mechanism. However, since the balancing effect is limited, the ratio of MMP-9/TIMP-1 and MMP-2/TIMP-2 is elevated. Inhibition of HDAC1 reverses the process in MCT-induced PAH through restoring miR-34a level