[Frontiers in Bioscience, Landmark, 25, 536-548, Jan 1, 2020]

Blocking PERK resuces vascular smooth muscle cells from homocysteine-induced ER stress and apoptosis

Wei Li1, Fujun Shang1, Xiaoli Li1, Shaoping Lu1, Xiaolin Niu1, Zhimin Zhang1, Jing Liu1, Xue Li1, Lianyou Zhao1

1Department of Cardiology, Tangdu Hospital, Fourth Military Medical University, Xi’an 710038, China


1. Abstract
2. Introduction
3. Materials and methods
    3.1. Reagents
    3.2. Cell culture and treatments
    3.3. Cell viability and injury detection
    3.4. Immunofluorescence staining
    3.5. Western blotting analysis
    3.6. Real-time quantitative RT-PCR
    3.7. Evaluation of apoptosis
    3.8. Statistical analysis
4. Results
    4.1. Hcy induces ERS in VSMCs
    4.2. Hcy activates PERK signaling pathway in VSMCs
    4.3. Hcy is cytotoxic and induces apoptosis in VSMCs
    4.4. Inhibition of PERK pathway protects the VSMCs against Hyc induced cell damage
5. Discussion
6. References


Hyperhomocysteinemia induces stress response in endoplasmic reticulum (ERS). Here, we tested whether blockage of homocysteine (Hcy) induced ERS and subsequent apoptosis in vascular smooth muscle cells can be inhibited by blockage of PERK/eIF2α/ATF4/CHOP signaling. Short-term exposure of vascular smooth muscle cells to Hcy led to the phosphorylation of PERK (pPERK), which in turn, phosphorylated eIF2 alpha (peIF2α) and inhibited the unfolded protein response. Long-term Hcy exposure, however, increased the expression of ATF-4 and CHOP and led to apoptosis. Treatment of cells with salubrinal, a specific inhibitor for eIF2α decreased the expression of ATF-4 and CHOP, and prevented apoptosis. Together, the results show that PERK pathway is involved in Hcy-induced vascular smooth muscle cell apoptosis and that blocking the PERK pathway protects against this injury.


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Abbreviations: Hyperhomocysteinemia (HHcy), homocysteine (Hcy), extracellular matrix (ECM), endoplasmic reticulum (ER), ER stress (ERS), protein kinase-like ER kinase (PERK), inositol-requiring kinase 1 (IRE1), transcription factor-activating transcription factor 6 (ATF6), unfolded protein response (UPR), eukaryotic initiation factor 2 (eIF2α), transcription factor 4 (ATF4), C/EBP homologous protein (CHOP), vascular smooth muscle cells (VSMCs), Dulbecco’s modified Eagle’s medium (DMEM), Cell Counting Kit-8 (CCK-8), Mouse aortic VSMCs (MOVAS cells), bicinchoninic acid (BCA), transferred to a polyvinylidene fluoride (PVDF), Tris-buffered saline containing Tween 20 (TBST), transferase-mediated dUTP nick end-labeling (TUNEL), Social Sciences (SPSS), Lactate dehydrogenase (LDH).

Key Words: Homocysteine, VSMCs, salubrinal, Endoplasmic reticulum stress, Apoptosis

Send correspondence to: Lianyou Zhao, Department of Cardiology, Tangdu Hospital, Fourth Military Medical University, Xi’an 710038, China, Tel: 86 18091863768, Fax:86-029-84777422, E-mail: zhaolianyoutd@163.com