Prolyl 4‐Hydroxylase Domain Protein 3 Overexpression Improved Obstructive Sleep Apnea—Induced Cardiac Perivascular Fibrosis Partially by Suppressing Endothelial‐to‐Mesenchymal Transition

Abstract

BackgroundIntermittent hypoxia (IH) induced by obstructive sleep apnea is the key factor involved in cardiovascular fibrosis. Under persistent hypoxia condition, endothelial cells respond by endothelial‐to‐mesenchymal transition (EndMT), which is associated with cardiovascular fibrosis. Prolyl 4‐hydroxylase domain protein 3 (PHD3) is a cellular oxygen sensor and its expression increased in hypoxia. However, its role in obstructive sleep apnea–induced EndMT and cardiovascular fibrosis is still uncertain. We investigated the potential mechanism of obstructive sleep apnea–induced cardiac perivascular fibrosis and the role of PHD3 in it.Methods and ResultsIn vivo, C56BL/6 mice were exposed to IH for 12 weeks. PHD3 expression was changed by lentivirus‐mediated short‐hairpin PHD3 and lentivirus carrying PHD3 cDNA. EndMT related protein levels, histological and functional parameters were detected after 12 weeks. In vitro, human umbilical vein endothelial cells were treated with IH/short‐hairpin PHD3/lentivirus carrying PHD3 cDNA to explore the mechanism of PHD3 in altered function of human umbilical vein endothelial cells. We found that chronic intermittent hypoxia increase PHD3 expression and EndMT. In vivo, IH accelerate cardiac dysfunction and aggravate collagen deposition via the process of EndMT. And, when PHD3 were overexpressed, cardiac dysfunction and collagen excessive deposition were improved. In vitro, IH induced EndMT, which endow human umbilical vein endothelial cells spindle morphology and an enhanced ability to migration and collagen secretion. PHD3 overexpression in cultured human umbilical vein endothelial cells ameliorated IH–induced EndMT through inactivating hypoxia‐inducible factor 1 alpha and small mothers against decapentaplegic 2 and 3.ConclusionsObstructive sleep apnea–induced cardiac perivascular fibrosis is associated with EndMT, and PHD3 overexpression might be beneficial in the prevention of it by inhibiting EndMT. PHD3 overexpression might have therapeutic potential in the treatment of the disease.

Prolyl 4-Hydroxylase Domain Protein 3-Inhibited Smooth-Muscle-Cell Dedifferentiation Improves Cardiac Perivascular Fibrosis Induced by Obstructive Sleep Apnea

“…The obvious modifications in cardiovascular remodelling are characterized by the disruption of normal myocardial structure through excessive collagen deposition. Our previous study confirms that OSA can induce cardiac perivascular fibrosis [12]. However, the detailed mechanisms remain unclear.…”

“…Our previous study had shown that IH can induce PHD3 expression in vitro and vivo [12]. To ascertain whether PHD3 could improve cardiac fibrosis induced by IH, in this study, lentivirus was applied to intervene the expression of PHD3 in vivo and vitro.…”

“…For 3 months, mice, which need to IH exposure, received 60 hypoxic events/h (20 s at 5% O 2 followed by 40 s of room air) during 8 h/d, corresponding to severe OSA. Control mice with normoxic breathing were placed in an identical device, but the hypoxic gas was replaced by room air [12, 26, 27] (we followed the method of Zhang et al 2017).…”

“…The preparation of section and immunohistochemistry analysis was performed as described [12]. For immunohistochemistry, sections were incubated with primary antibody against collagen I (1:1000 Abcam), collagen III (1:1000 Abcam) and α -SMA (1:200 Abcam), OPN (1:200 Abcam) overnight at 4°C and incubated with secondary antibody at 37°C for 30 min.…”

“…Cells were cultured in a humidified 5% CO 2 and 95% air incubator at 37°C and 3-5 passages were used. Cells were treated with IH for 72 h. Air-phase set-point consisted of a 35-minute hypoxic period, followed by 25 minutes of reoxygenation (21% O 2 and 5%CO 2 ) [12, 28, 29] (we followed the method of Zhang et al 2017). Before IH treatment, the VSMCs were infected with lentivirus at a multiplicity of infection of 15 for 24 h. Cells were treated with siRNA (GenePharma Shanghai, China) in order to inhibit HIF-1 α expression.…”