Shedding of soluble platelet-derived growth factor receptor-β from human brain pericytes - PubMed (original) (raw)

Shedding of soluble platelet-derived growth factor receptor-β from human brain pericytes

Abhay P Sagare et al. Neurosci Lett. 2015.

Abstract

Platelet-derived growth factor receptor-β (PDGFRβ) is expressed in the brain by vascular mural cells-brain capillary pericytes and arterial vascular smooth muscle cells (VSMCs). Recent evidence shows that blood-brain barrier (BBB) disruption and increased permeability, especially in the hippocampus, positively correlates with elevated levels of soluble PDGFRβ (sPDGFRβ) in cerebrospinal fluid (CSF) in patients with mild dementia. To determine which vascular cell type(s) contributes to increased sPDGFRβ in CSF, we compared PDGFRβ expression and sPDGFRβ shedding in response to injury in early passage primary cultures of human brain pericytes, brain arterial VSMCs, and brain endothelial cells. PDGFRβ protein was undetectable in endothelial cells, but was found both in pericytes and VSMCs. PDGFRβ relative protein abundance was by 4.2-fold (p<0.05) higher in pericytes compared to VSMCs. Hypoxia (1% O2) or amyloid-β peptide (25 μM) compared to normoxia (21% O2) both increased over 48 h shedding of sPDGFRβ and its levels in the culture medium from pericytes cultures, but not from VSMCs cultures, by 4.3-fold and 4.6-fold, respectively, compared to the basal sPDGFRβ levels in the medium (1.43±0.15 ng/ml). This was associated with the corresponding loss of cell-associated PDGFRβ from pericytes and no change in cellular levels of PDGFRβ in VSMCs. Thus, sPDGFRβ is a biomarker of pericyte injury, and elevated sPDGFRβ levels in biofluids in patients with dementia and/or other neurodegenerative disorders likely reflects pericyte injury, which supports the potential for sPDGFRβ to be developed and validated as a biomarker of brain pericyte injury and BBB dysfunction.

Keywords: Hypoxia; Pericytes; Soluble platelet-derived growth factor receptor-β; Vascular smooth muscle cells.

Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

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Conflict of interest statement

Conflict of interest

The authors declare no financial conflict of interest.

Figures

Fig. 1

Fig. 1. Abundant PDGFRβ expression in cultured primary human brain pericytes compared to other vascular cell types

(A and B) Immunoblotting for PDGFRβ and β-actin (A) and relative abundance of PDGFRβ protein levels compared to β-actin (B) in cultured primary human brain endothelial cells, brain arterial vascular smooth muscle cells (VSMCs), and brain pericytes by Western blot analysis. (C) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of PDGFRβ mRNA levels in VSMCs and pericytes. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as an internal control. (C) Representative images of PDGFRβ immunostaining in cultured primary human brain endothelial cells, arterial VSMCs, and brain pericytes; scale bar, 20 μm. In B and C, means ± s.e.m. from 6 independent cultures from 6 donors in triplicate. P<0.05, significance by Student’s _t-_test (B and C).

Fig. 2

Fig. 2. Hypoxia and amyloid-β peptide (Aβ) lead to shedding of soluble platelet-derived growth factor receptor-β (sPDGFRβ) from cultured primary human brain pericytes, but not from brain arterial vascular smooth muscle cells (VSMCs)

(A and B) Immunoblotting for sPDGFRβ (A) and quantification of sPDGFRβ levels (ng/ml) by quantitative Western blot analysis (B) in the culture medium from primary human VSMCs and pericytes cultured under normoxic (21% O2) or hypoxic (1% O2) conditions, or incubated with human synthetic Aβ40 (25 μM) for 48 h. (C and D) Immunoblottting for cell-associated PDGFRβ (C) and relative abundance of cellular PDGFRβ levels (D) in primary human VSMCs and pericytes cultured under normoxic (21% O2) or hypoxic (1% O2) conditions, or incubated with human synthetic Aβ40 (25 μM) for 48 h. β-Actin was used as an internal loading control. Means ± s.e.m., from 3 independent cultures from 3 donors in triplicates. P<0.05, by Student’s _t-_test.

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