Abstract
There is a need to develop new techniques for quantitative measurement of receptors expression on particular vasculature cells types. Here, we describe and demonstrate a novel method to measure quantitatively and simultaneously the expression of endothelin B receptor (ETB) on vascular smooth muscle cells (VSMC). We isolated cells from male rat tissues such as: brain pial, brain intraparenchymal and retina vessels. To analyze solid tissues, a single-cell suspension was prepared by a combined mechanic and enzymatic process. The cells were stained with Fixable Viability Dye, followed by fixation, permeabilization and antibodies staining. The expression of ETB receptors on VSMC was measured by flow-cytometry and visualized by fluorescence microscopy. We obtained a high percentage of viable cells 87.6% ± 1.5% pial; 84.6% ± 4.3% parenchymal and 90.6% ± 4% retina after isolation of single cells. We performed a quantitative measurement of ETB receptor expression on VSMC and we identified two subpopulations of VSMC based on their expression of smooth muscle cells marker SM22α. The results obtained from pial vessels are statistically significant (38.4% ± 4% vs 9.8% ± 3.32%) between the two subpopulations of VSMC. The results obtained from intraparenchymal and retina vessels were not statistically significant. By specific gating on two subpopulations, we were able to quantify the expression of ETB receptors. The two subpopulation expressed the same level of ETB receptor (p = 0.45; p = 0.3; p = 0.42) in pial, parenchymal and retina vessels, respectively. We applied our method to the animals after induction of subarachnoid hemorrhage (SAH). There was statistically significant expression of ETB receptor (p = 0.02) on VSMC between sham 61.4% ± 4% and SAH 77.4% ± 4% rats pial vessels. The presented technique is able to quantitatively and selectively measure the level of protein expression on VSMC. The entire technique is optimized for rat tissue; however the protocol can also be adapted for other species.
Highlights
Cerebral blood flow and metabolism are constantly at high levels and the richly vascularized brain receives about 20% of cardiac output at rest
We established a method for quantitative detection of endothelin B receptor (ETB) receptor on vascular smooth muscle cells from solid rat tissue
We found that two subpopulations of vascular smooth muscle cells (VSMC), SM22αdim and SM22αbright expressing the same percentage of ETB receptor
Summary
Cerebral blood flow and metabolism are constantly at high levels and the richly vascularized brain receives about 20% of cardiac output at rest. The tunica media contains mainly smooth muscle cells, which regulate the vascular tone in the blood vessels [2]. Tissue for quantitative protein analysis with the gold standard western blot method will inevitably contain all three layers. In order to determine pathology of the vascular diseases, it is necessary to develop a method, which will quantitatively measure expressions of the receptors/proteins on cells of interest. We proposed a combined technique of mechanical, enzymatic and density gradient centrifugation to achieve high and reproducible yield levels of viable cells after isolation from CNS vasculature. This study focuses on how to isolate viable single cell populations of smooth muscle cells from different tissue origin and measure quantitatively the endothelin B protein (ETB) expression by flow-cytometry. We used the robust changes seen after experimental subarachnoid hemorrhage (SAH) stroke of the endothelin B receptor (ETB) for validation of our new method
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