Development of immortalized mouse aortic endothelial cell lines

Abstract

BackgroundThe understanding of endothelial cell biology has been facilitated by the availability of primary endothelial cell cultures from a variety of sites and species; however, the isolation and maintenance of primary mouse aortic endothelial cells (MAECs) remain a formidable challenge. Culturing MAECs is difficult as they are prone to phenotypic drift during culture. Therefore, there is a need to have a dependable in vitro culture system, wherein the primary endothelial cells retain their properties and phenotypes.MethodsHere, we developed an effective method to prepare immortalized MAEC (iMAEC) lines. Primary MAECs, initially isolated from aortic explants, were immortalized using a retrovirus expressing polyoma middle T-antigen. Immortalized cells were then incubated with DiI-acetylated-low density lipoprotein and sorted via flow cytometry to isolate iMAECs.ResultsiMAECs expressed common markers of endothelial cells, including PECAM1, eNOS, VE-cadherin, and von Willebrand Factor. iMAECs aligned in the direction of imposed laminar shear and retained the ability to form tubes. Using this method, we have generated iMAEC lines from wild-type and various genetically modified mice such as p47phox-/-, eNOS-/-, and caveolin-1-/-.ConclusionIn summary, generation of iMAEC lines from various genetically modified mouse lines provides an invaluable tool to study vascular biology and pathophysiology.

A Direct Comparison of in Vitro and in Vivo Nucleic Acid Delivery Mediated by Hundreds of Nanoparticles Reveals a Weak Correlation

“…We administered the LNPs to immortalized mouse aortic endothelial cells (iMAECs), and mouse macrophages (RAWs). We chose iMAECs since they are isolated directly from the murine heart, and have been shown to recapitulate endothelial cell signaling and function 34 . We chose RAWs since they are a commonly used macrophage cell line.…”

Isolation and In Vitro Culture of Vascular Endothelial Cells from Mice

“…Thus, numerous studies have been attempted to establish immortalized cell lines from mouse EC [ 5 , 6 , 20 ], and several EC lines originating from EC of mouse vessels have been established. Immortalized MAEC express common markers of EC, including PECAM1, eNOS, VE-cadherin, and vWF, and proliferate to form tube-like structures, suggesting that the MAEC retain typical endothelial properties and phenotypes [ 5 ]. However, phenotype changes have been reported in the EC lines [ 21 ].…”

“…Mice have been widely used as animal models to investigate the underlying mechanisms of human diseases. Since primary EC are highly useful for in vitro studies in order to investigate the mechanisms underlying EC function or dysfunction, EC culture from mouse vessels has been attempted by investigators over the last few years; however, few reports on the isolation and characterization of EC from mouse vessels appear in the literature [ 5 , 6 , 7 , 8 , 9 , 10 , 11 ]. Dong et al [ 11 ] report a strategy for isolation of EC from murine lung selected with CD-31 and magnetic beads.…”

Functional screening of mammalian mechanosensitive genes using Drosophila RNAi library– Smarcd3/Bap60 is a mechanosensitive pro-inflammatory gene

“…Human umbilical vein endothelial cells (HUVECs), human aortic endothelial cells (HAECs) and immortalized mouse aortic endothelial cells (iMAECs) were maintained in their respective culture medium as we previously described22232425. Shear stress was applied to the cells in 10-cm culture plates for 24 hours using a cone-and-plate viscometer that exerts 15 dyn/cm 2 of unidirectional flow (laminar shear or LS) and ±5 dyn/cm 2 of oscillatory shear stress (OS), respectively, as we previously described2627.…”