Abstract

BackgroundThe A10 and A7r5 cell lines derived from the thoracic aorta of embryonic rat are widely used as models of non-differentiated, neonatal and neointimal vascular smooth muscle cells in culture. The recent discovery of resident multipotent vascular stem cells within the vessel wall has necessitated the identity and origin of these vascular cells be revisited. In this context, we examined A10 and A7r5 cell lines to establish the similarities and differences between these cell lines and multipotent vascular stem cells isolated from adult rat aortas by determining their differentiation state, stem cell marker expression and their multipotency potential in vitro.MethodsVascular smooth muscle cell differentiation markers (alpha-actin, myosin heavy chain, calponin) and stem cell marker expression (Sox10, Sox17 and S100β) were assessed using immunocytochemistry, confocal microscopy, FACS analysis and real-time quantitative PCR.ResultsBoth A10 and A7r5 expressed vascular smooth muscle differentiation, markers, smooth muscle alpha - actin, smooth muscle myosin heavy chain and calponin. In parallel analysis, multipotent vascular stem cells isolated from rat aortic explants were immunocytochemically myosin heavy chain negative but positive for the neural stem cell markers Sox10+, a neural crest marker, Sox17+ the endoderm marker, and the glia marker, S100β+. This multipotent vascular stem cell marker profile was detected in both embryonic vascular cell lines in addition to the adventitial progenitor stem cell marker, stem cell antigen-1, Sca1+. Serum deprivation resulted in a significant increase in stem cell and smooth muscle cell differentiation marker expression, when compared to serum treated cells. Both cell types exhibited weak multipotency following adipocyte inductive stimulation. Moreover, Notch signaling blockade following γ-secretase inhibition with DAPT enhanced the expression of both vascular smooth muscle and stem cell markers.ConclusionsWe conclude that A10 and A7r5 cells share similar neural stem cell markers to both multipotent vascular stem cells and adventitial progenitors that are indicative of neointimal stem-derived smooth muscle cells. This may have important implications for their use in examining vascular contractile and proliferative phenotypes in vitro.

Highlights

  • The arterial blood vessel is comprised of three distinct layers; an innermost monolayer of endothelial cells, a medial layer composed primarily of vascular smooth muscle cells [vSMCs] [ 1– 3] and some multipotent vascular stem cells [Multipotent vascular stem cell (MVSC)] [ 1, 4, 5], and an outer adventitial layer of fibroblast cells and some vSMC-related stem cell antigen-1 (Sca1+ ) positive progenitor cells [ 6, 7]

  • Multipotent vascular stem cells isolated from rat aortic explants were immunocytochemically myosin heavy chain negative but positive for the neural stem cell markers Sox10+, a neural crest marker, Sox17+ the endoderm marker, and the glia marker, S100β+

  • Notch signaling blockade following γ-secretase inhibition with DAPT enhanced the expression of both vascular smooth muscle and stem cell markers

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Summary

Introduction

The arterial blood vessel is comprised of three distinct layers; an innermost monolayer of endothelial cells, a medial layer composed primarily of vascular smooth muscle cells [vSMCs] [ 1– 3] and some multipotent vascular stem cells [MVSCs] [ 1, 4, 5], and an outer adventitial layer of fibroblast cells and some vSMC-related stem cell antigen-1 (Sca1+ ) positive progenitor cells [ 6, 7]. VSMCs in culture are thought to be ‘phenotypically modulated’ [ 10, 11, 3] These ‘contractile’ cells initially express markers for SMC differentiation such as smooth muscle α-actin (SMA), smooth muscle myosin heavy chain (SM-MHC), calponin (CNN1) and SM-22α. The recent discovery of resident multipotent vascular stem cells within the vessel wall has necessitated the identity and origin of these vascular cells be revisited. In this context, we examined A10 and A7r5 cell lines to establish the similarities and differences between these cell lines and multipotent vascular stem cells isolated from adult rat aortas by determining their differentiation state, stem cell marker expression and their multipotency potential in vitro

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