Cell Fate Mapping of Vascular Smooth Muscle cells in Medial Arterial Calcification
 

Medial arterial calcification is a  type of vascular calcification and occurs in the medial part of arteries and mostly associated with aging, diabetes and chronic kidney disease Vascular smooth muscle cells (VSMCs) reside in the medial layer of blood vessels and provide structural and regulatory functions of normal vessels. VSMCs can transdifferentiate from contractile phenotype into osteoblast-like cells in response to disease conditions. This results in downregulation of SM-specific proteins expression (aSMA, SM22a) and upregulation of osteo-/chondrogenic proteins expression (RUNX2, SOX9, osteopontin, osteocalcin, alkaline phosphatase and BMP-2).

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Objective:

To generate Myh11-CreERT;mT/mG double reporter mice for adenine-induced uremia model of medial arterial calcification (MAC) to investigate the fate of VSMCs surrounding calcification after removal of the hydroxyapatite by EDTA/PGG nanoparticle.

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Methods:

Tamoxifen inducible VSMC-specific Myh11-CreERT2 Cre recombinase male mice were crossed with tomato/GFP double reporter female mice. Male mice with Myh11-CreERT2: mTmG transgenes were used for this research. Both experimental and control mice were injected 100 mL of tamoxifen for 5 consecutive days. The experimental group was put on normal phosphate (0.8%) adenine diet for 6 weeks and then on high phosphate (1.8 %) adenine diet for additional 6 weeks. Controls were fed regular chow. Heart, aorta, kidney and blood were collected after the last day of the 12th week. Micro CT was used to assess calcification of heart, aorta and kidney. 10 mm frozen sections were histologically assessed for recombination, calcification and fibrosis using Alizarin red, Alcian Blue, elastic staining.

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Results:

Fluorescent and confocal microscopic investigation showed that tamoxifen induced expression of mGFP was exclusively in Myh11-CreERT2 Cre positive VSMCs. Activity of serum alkaline phosphatase was significantly elevated in adenine diet mice compared to controls indicating active calcification. Micro CT assessment and Alizarin Red staining showed presence of extensive calcification in aorta, heart and kidneys of adenine diet mice. Alcian blue staining of acid mucopolysaccharide around calcified areas may indicate VSMCs differentiation into chondrocytes in aortas of adenine diet mice. Cells in the vicinity of calcification have structural features of chondrocytes with round shaped nucleus. These Cells do not express mGFP and this may be due to downregulated expression of SM-specific gene after transdifferentiation. Disorganized and broken elastic fibers are observed in aortas of adenine diet mice at the calcified and surrounding areas.