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Development of immortalized mouse aortic endothelial cell lines.

Ni CW, Kumar S, Ankeny CJ, Jo H - (2014)

Bottom Line: Here, 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.Using this method, we have generated iMAEC lines from wild-type and various genetically modified mice such as p47phox-/-, eNOS-/-, and caveolin-1-/-.

View Article: PubMed Central - HTML - PubMed

Affiliation: Wallace H, Coulter Department of Biomedical Engineering Georgia Institute of Technology and Emory University, 1760 Haygood Drive, Health Science Research Building, E-170, Atlanta, GA 30322, USA. hanjoong.jo@bme.gatech.edu.

ABSTRACT

Background: The 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.

Methods: Here, 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.

Results: iMAECs 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-/-.

Conclusion: In summary, generation of iMAEC lines from various genetically modified mouse lines provides an invaluable tool to study vascular biology and pathophysiology.

No MeSH data available.


Related in: MedlinePlus

VCAM-1 expression is elevated in iMAEC-eNOS while superoxide production is diminished in iMAEC-p47. iMAEC-WT and iMAEC-p47 were stained with DHE (2 μM) for 30 min and images were acquired using fluorescence microscopy.
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Figure 7: VCAM-1 expression is elevated in iMAEC-eNOS while superoxide production is diminished in iMAEC-p47. iMAEC-WT and iMAEC-p47 were stained with DHE (2 μM) for 30 min and images were acquired using fluorescence microscopy.

Mentions: Next, we compared superoxide production between iMAEC-WT and iMAEC-p47phox Figure 7. Given that p47phox is an important component of NADPH oxidases, which produce superoxide, lack of p47 phox in EC is expected to reduce the production of superoxide [21]. Indeed, superoxide production, as demonstrated by dihydroethidium (DHE) staining, was significantly lower in iMAEC-p47 cells as compared to iMAEC-WT, thus both confirming the knockout efficiency in these cells as well as the role of p47phox in superoxide production.


Development of immortalized mouse aortic endothelial cell lines.

Ni CW, Kumar S, Ankeny CJ, Jo H - (2014)

VCAM-1 expression is elevated in iMAEC-eNOS while superoxide production is diminished in iMAEC-p47. iMAEC-WT and iMAEC-p47 were stained with DHE (2 μM) for 30 min and images were acquired using fluorescence microscopy.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4230636&req=5

Figure 7: VCAM-1 expression is elevated in iMAEC-eNOS while superoxide production is diminished in iMAEC-p47. iMAEC-WT and iMAEC-p47 were stained with DHE (2 μM) for 30 min and images were acquired using fluorescence microscopy.
Mentions: Next, we compared superoxide production between iMAEC-WT and iMAEC-p47phox Figure 7. Given that p47phox is an important component of NADPH oxidases, which produce superoxide, lack of p47 phox in EC is expected to reduce the production of superoxide [21]. Indeed, superoxide production, as demonstrated by dihydroethidium (DHE) staining, was significantly lower in iMAEC-p47 cells as compared to iMAEC-WT, thus both confirming the knockout efficiency in these cells as well as the role of p47phox in superoxide production.

Bottom Line: Here, 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.Using this method, we have generated iMAEC lines from wild-type and various genetically modified mice such as p47phox-/-, eNOS-/-, and caveolin-1-/-.

View Article: PubMed Central - HTML - PubMed

Affiliation: Wallace H, Coulter Department of Biomedical Engineering Georgia Institute of Technology and Emory University, 1760 Haygood Drive, Health Science Research Building, E-170, Atlanta, GA 30322, USA. hanjoong.jo@bme.gatech.edu.

ABSTRACT

Background: The 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.

Methods: Here, 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.

Results: iMAECs 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-/-.

Conclusion: In summary, generation of iMAEC lines from various genetically modified mouse lines provides an invaluable tool to study vascular biology and pathophysiology.

No MeSH data available.


Related in: MedlinePlus