Granzyme B deficiency promotes osteoblastic differentiation and calcification of vascular smooth muscle cells in hypoxic pulmonary hypertension

Min Mao,Yao Fu,Yang Liu,Xiaoying Wang,Anqi Ge,Chen Zhang,Min Zhang,Xin Ge,Rui Gu,Jiayin Gao,Daling Zhu

doi:10.1038/s41419-018-0315-5

Abstract

Calcification is a major risk factor for vascular integrity. This pathological symptom and the underlying mechanisms in hypoxic pulmonary artery hypertension remain elusive. Here we report that pulmonary vascular medial calcification is elevated in pulmonary artery hypertension models as a result of an osteoblastic phenotype change of pulmonary arterial smooth muscle cells induced by hypoxia. Notably, inhibiting store-operated calcium channels significantly decreased osteoblastic differentiation and calcification of pulmonary arterial smooth muscle cells under hypoxia. We identified granzyme B, a major constituent of cytotoxic T lymphocytes/natural killer cell granules involved in apoptosis, as the main regulator of pulmonary arterial calcification. Overexpression of granzyme B blocked the mineralization through its effect on store-operated calcium channels in cultured pulmonary arterial smooth muscle cells under hypoxic conditions. Mice with overexpression of granzyme B exposed to hypoxia for 3 weeks showed attenuated vascular calcification and pathological progression of hypoxic pulmonary arterial hypertension. Our findings emphasize the central function of granzyme B in coordinating vascular calcification in hypoxic pulmonary arterial hypertension.

Highlights

Hypoxic pulmonary arterial hypertension (HPAH) is characterized by functional and structural changes in the pulmonary vasculature, leading to increased pulmonary vascular resistance and remodeling and right ventricular hypertrophy[1,2]
Scale bars = 50 μm; n = 10. d Western blotting of Runx[2], MSX2, BMP2, SOX9, and SM22α expression in lung tissues from normoxia, chronic hypoxia, and MCTtreated rats. β-Actin served as the standard; n = 10. e Pulmonary arterial SMC (PASMC) were exposed to hypoxia for 24 h and expression of Runx[2], MSX2, BMP2, SOX9, and SM22α was evaluated with western blotting and real-time PCR. β-Actin served as the standard for western blotting; 18 s served as the standard for real-time PCR; n = 6. f PASMCs were cultured under hypoxia for 7 days upon treatment with procalcifying media containing 2.5 mM inorganic phosphate
Increased vascular medial calcification in PAH The rat models of pulmonary hypertension induced by chronic hypoxia and monocrotalin (MCT) were constructed to characterize the behavior and main localization of pulmonary vascular calcification

Summary

Introduction

Hypoxic pulmonary arterial hypertension (HPAH) is characterized by functional and structural changes in the pulmonary vasculature, leading to increased pulmonary vascular resistance and remodeling and right ventricular hypertrophy[1,2]. A Von Kossa staining revealed obvious deposits of calcium mineral in the medial vessels from the PAH rat model induced by chronic hypoxia or MCT. C Immunohistochemical analysis of cellular expression of Runx[2] at the medial layer in lung tissues from normoxia, chronic hypoxia, and MCT-treated rats. D Western blotting of Runx[2], MSX2, BMP2, SOX9, and SM22α expression in lung tissues from normoxia, chronic hypoxia, and MCTtreated rats. E PASMCs were exposed to hypoxia for 24 h and expression of Runx[2], MSX2, BMP2, SOX9, and SM22α was evaluated with western blotting and real-time PCR. HYP, hypoxia, MCT, monocrotaline; NOR, normoxia of the vessel, which differentiate from a contractile to osteoblastic phenotype in response to pathological signals, such as inflammatory cytokines or mineral imbalance[8,10]. Definitive studies on the regulation of osteoblastic differentiation and calcification of SMCs in HPAH are needed

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Conclusion