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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

Functional response of iMAECs to shear stress. (A) Endothelial cell tube formation; (B) endothelial cell sprouting; and (C) endothelial cell scratch wound migration assay performed using iMAECs exposed to LS or OS for 24 h. Dotted black line in B denotes the periphery of the Matrigel bead for sprouting assay. Black scale bar = 200 μm. Cells under static condition were used a controls. Data shown as means ± standard error; n = 3; * p < 0.05.
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Figure 6: Functional response of iMAECs to shear stress. (A) Endothelial cell tube formation; (B) endothelial cell sprouting; and (C) endothelial cell scratch wound migration assay performed using iMAECs exposed to LS or OS for 24 h. Dotted black line in B denotes the periphery of the Matrigel bead for sprouting assay. Black scale bar = 200 μm. Cells under static condition were used a controls. Data shown as means ± standard error; n = 3; * p < 0.05.

Mentions: Additionally, we have shown that OS induces endothelial tube formation and migration in various cultured ECs, including HUVEC [34,41,42]. Thus, we tested whether iMAECs respond to shear in a similar manner. As shown in Figure 6A-C, exposure of iMAECs to OS for 24 h induced endothelial tube formation, sprouting capability, and migration in the scratch wound-healing study, in comparison to LS and static conditions. These results suggest that iMAECs respond to shear stress in a manner similar to other cultured primary ECs.


Development of immortalized mouse aortic endothelial cell lines.

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

Functional response of iMAECs to shear stress. (A) Endothelial cell tube formation; (B) endothelial cell sprouting; and (C) endothelial cell scratch wound migration assay performed using iMAECs exposed to LS or OS for 24 h. Dotted black line in B denotes the periphery of the Matrigel bead for sprouting assay. Black scale bar = 200 μm. Cells under static condition were used a controls. Data shown as means ± standard error; n = 3; * p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Functional response of iMAECs to shear stress. (A) Endothelial cell tube formation; (B) endothelial cell sprouting; and (C) endothelial cell scratch wound migration assay performed using iMAECs exposed to LS or OS for 24 h. Dotted black line in B denotes the periphery of the Matrigel bead for sprouting assay. Black scale bar = 200 μm. Cells under static condition were used a controls. Data shown as means ± standard error; n = 3; * p < 0.05.
Mentions: Additionally, we have shown that OS induces endothelial tube formation and migration in various cultured ECs, including HUVEC [34,41,42]. Thus, we tested whether iMAECs respond to shear in a similar manner. As shown in Figure 6A-C, exposure of iMAECs to OS for 24 h induced endothelial tube formation, sprouting capability, and migration in the scratch wound-healing study, in comparison to LS and static conditions. These results suggest that iMAECs respond to shear stress in a manner similar to other cultured primary ECs.

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