J Nutr Health.  2020 Dec;53(6):563-569. 10.4163/jnh.2020.53.6.563.

The function of zinc in the primary vascular smooth muscle cell proliferation in rats

• Cho YE
• Kwun IS

• KMID: 2510201
• DOI: http://doi.org/10.4163/jnh.2020.53.6.563

Abstract

Purpose
The vascular smooth muscle cells (VSMCs) in mature animals have implicated to play a major role in the progression of cardiovascular diseases such as atherosclerosis. This study aimed at optimizing the protocol in culturing primary VSMCs (pVSMCs) from rat thoracic aorta and investigating the effect of cellular zinc (Zn) deficiency on cell proliferation of the isolated pVSMCs.
Methods
The thoracic aorta from 7-month-old Sprague Dawley rats was isolated, minced and digested by the enzymatic process of collagenase I and elastase, and then inoculated with the culture Dulbecco Modified Eagle Medium (DMEM) at 37°C in an incubator. The primary cell culture morphology was observed using phase-contrast microscopy and cellular Zn was depleted using Chelex-100 resin (extracellular zinc depletion only) or 3 µM N,N,N′,N′-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) (extracellular and intracellular zinc depletion). Western blot analysis was used for the detection of SM22α and calponin as smooth muscle cell marker proteins and von Willebrand factor as endothelial cell marker protein to detect the culture purity. Cell proliferation by Zn depletion (1 day) was measured by MTT assay.
Results
A primary culture protocol for pVSMCs from rat thoracic aorta was developed and optimized. Isolated cultures exhibited hill and valley morphology as the major characteristics of pVSMCs and expressed the smooth muscle cell protein markers, SM22α and calponin, while the endothelial marker von Willebrand factor was hardly detected. Zn deprivation for 1 day culture decreased rat primary vascular smooth muscle cell proliferation and this pattern was more prominent under severe Zn depletion (3 µM TPEN), while less prominent under mild Zn depletion (Chelexing).
Conclusion
Our results suggest that cellular Zn deprivation decreased pVSMC proliferation and this may be involved in phenotypic modulation of pVSMC in the aorta.

Keyword

primary vascular smooth muscle cells (pVSMCs); primary cell culture; vascular calcification; zinc; atherosclerosis