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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

Characterization of iMAEC lines by Dil-Ac-LDL staining. iMAEC lines including wild-type (iMAEC-WT), Caveolin-1 knockout (iMAEC-cav1), eNOS knockout (iMAEC-eNOS), and p47phox knockout (iMAEC-p47), were cultured. (A) Total cell lysates were collected from iMAEC lines or control cells including HUVEC, 3T3, and RASMC. Western blotting was performed using specific antibodies against Flk-1, eNOS and Cav-1. Actin serves as an internal loading control. (B) iMAECs were incubated with Dil-Ac-LDL (10 μg/mL) for 4 h, and images were taken by fluorescence microscopy. HUVEC served as positive control while 3T3 and RASMC served as negative controls. Scale bar: 50 μm. (C) Graph shows the cell shape index of HUVECs, iMAECs and primary MAECs. For cell shape index calculation, 25 cells were chosen randomly from each group and analyzed by ImageJ software. Data represent means ± standard error.
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Figure 3: Characterization of iMAEC lines by Dil-Ac-LDL staining. iMAEC lines including wild-type (iMAEC-WT), Caveolin-1 knockout (iMAEC-cav1), eNOS knockout (iMAEC-eNOS), and p47phox knockout (iMAEC-p47), were cultured. (A) Total cell lysates were collected from iMAEC lines or control cells including HUVEC, 3T3, and RASMC. Western blotting was performed using specific antibodies against Flk-1, eNOS and Cav-1. Actin serves as an internal loading control. (B) iMAECs were incubated with Dil-Ac-LDL (10 μg/mL) for 4 h, and images were taken by fluorescence microscopy. HUVEC served as positive control while 3T3 and RASMC served as negative controls. Scale bar: 50 μm. (C) Graph shows the cell shape index of HUVECs, iMAECs and primary MAECs. For cell shape index calculation, 25 cells were chosen randomly from each group and analyzed by ImageJ software. Data represent means ± standard error.

Mentions: Using the described method, we developed several iMAEC lines, including wild-type (iMAEC-WT), Caveolin-1 knockout (iMAEC-cav1), eNOS knockout (iMAEC-eNOS), and p47phox knockout (iMAEC-p47). The phenotype of these immortalized cells was then examined. First, we performed Western blotting to confirm the lack of protein expression in knockout cell lines. As expected, all iMAEC and HUVEC expressed Flk1, while 3T3 and RASMC did not. Next, caveolin-1 in iMAEC-cav1 or eNOS in iMAEC-eNOS did not express cavleolin-1 or eNOS protein, respectively (Figure 3A). To further confirm the identity of these cell lines, we studied Dil-Ac-LDL uptake, as endothelial cells internalize and degrade Ac-LDL 7-15 times more efficiently than smooth muscle cells [38]. As shown in Figure 3B, iMAEC showed homogeneous Dil-Ac-LDL uptake similar to primary HUVEC. As expected, 3T3 fibroblasts and rat aorta smooth muscle cells (RASMC) failed to uptake Dil-Ac-LDL, demonstrating the staining specificity of Dil-Ac-LDL to endothelial cells. We also determined the shape-index of iMAECs to compare the morphology of these cells to HUVECs and primary MAECs. We found that iMAECs are more spindle-shaped compared to the other ECs such as HUVECs (Figure 3C), which may reflect maintenance of the cytoskeletal organization under high shear stress environment of mouse aortic ECs in vivo as compared to the venous origin of HUVECs. Next, several endothelial-specific protein markers, PECAM-1, VE-Cadherin, and von Willebrand factor (vWF), were examined by immunocytochemistry. PECAM-1 and VE-Cadherin expression was localized as anticipated at the cell borders in iMAEC and HUVEC, as expected, but not in negative controls cells 3T3 and RASMC (Figure 4). Interestingly, iMAEC-cav1 cells showed a reduced junctional staining pattern for VE-cadherin. vWF was also observed in the cytosol of iMAECs and HUVECs, but not in the respective negative controls. In contrast, the smooth muscle cell specific maker, α-SMA, was positive only in RASMCs and negative in all ECs and the 3T3 cells, further confirming the specificity of iMAEC lines. These results demonstrate that iMAECs generated from wild-type and knockout mouse aortas retain key endothelial cell markers.


Development of immortalized mouse aortic endothelial cell lines.

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

Characterization of iMAEC lines by Dil-Ac-LDL staining. iMAEC lines including wild-type (iMAEC-WT), Caveolin-1 knockout (iMAEC-cav1), eNOS knockout (iMAEC-eNOS), and p47phox knockout (iMAEC-p47), were cultured. (A) Total cell lysates were collected from iMAEC lines or control cells including HUVEC, 3T3, and RASMC. Western blotting was performed using specific antibodies against Flk-1, eNOS and Cav-1. Actin serves as an internal loading control. (B) iMAECs were incubated with Dil-Ac-LDL (10 μg/mL) for 4 h, and images were taken by fluorescence microscopy. HUVEC served as positive control while 3T3 and RASMC served as negative controls. Scale bar: 50 μm. (C) Graph shows the cell shape index of HUVECs, iMAECs and primary MAECs. For cell shape index calculation, 25 cells were chosen randomly from each group and analyzed by ImageJ software. Data represent means ± standard error.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Characterization of iMAEC lines by Dil-Ac-LDL staining. iMAEC lines including wild-type (iMAEC-WT), Caveolin-1 knockout (iMAEC-cav1), eNOS knockout (iMAEC-eNOS), and p47phox knockout (iMAEC-p47), were cultured. (A) Total cell lysates were collected from iMAEC lines or control cells including HUVEC, 3T3, and RASMC. Western blotting was performed using specific antibodies against Flk-1, eNOS and Cav-1. Actin serves as an internal loading control. (B) iMAECs were incubated with Dil-Ac-LDL (10 μg/mL) for 4 h, and images were taken by fluorescence microscopy. HUVEC served as positive control while 3T3 and RASMC served as negative controls. Scale bar: 50 μm. (C) Graph shows the cell shape index of HUVECs, iMAECs and primary MAECs. For cell shape index calculation, 25 cells were chosen randomly from each group and analyzed by ImageJ software. Data represent means ± standard error.
Mentions: Using the described method, we developed several iMAEC lines, including wild-type (iMAEC-WT), Caveolin-1 knockout (iMAEC-cav1), eNOS knockout (iMAEC-eNOS), and p47phox knockout (iMAEC-p47). The phenotype of these immortalized cells was then examined. First, we performed Western blotting to confirm the lack of protein expression in knockout cell lines. As expected, all iMAEC and HUVEC expressed Flk1, while 3T3 and RASMC did not. Next, caveolin-1 in iMAEC-cav1 or eNOS in iMAEC-eNOS did not express cavleolin-1 or eNOS protein, respectively (Figure 3A). To further confirm the identity of these cell lines, we studied Dil-Ac-LDL uptake, as endothelial cells internalize and degrade Ac-LDL 7-15 times more efficiently than smooth muscle cells [38]. As shown in Figure 3B, iMAEC showed homogeneous Dil-Ac-LDL uptake similar to primary HUVEC. As expected, 3T3 fibroblasts and rat aorta smooth muscle cells (RASMC) failed to uptake Dil-Ac-LDL, demonstrating the staining specificity of Dil-Ac-LDL to endothelial cells. We also determined the shape-index of iMAECs to compare the morphology of these cells to HUVECs and primary MAECs. We found that iMAECs are more spindle-shaped compared to the other ECs such as HUVECs (Figure 3C), which may reflect maintenance of the cytoskeletal organization under high shear stress environment of mouse aortic ECs in vivo as compared to the venous origin of HUVECs. Next, several endothelial-specific protein markers, PECAM-1, VE-Cadherin, and von Willebrand factor (vWF), were examined by immunocytochemistry. PECAM-1 and VE-Cadherin expression was localized as anticipated at the cell borders in iMAEC and HUVEC, as expected, but not in negative controls cells 3T3 and RASMC (Figure 4). Interestingly, iMAEC-cav1 cells showed a reduced junctional staining pattern for VE-cadherin. vWF was also observed in the cytosol of iMAECs and HUVECs, but not in the respective negative controls. In contrast, the smooth muscle cell specific maker, α-SMA, was positive only in RASMCs and negative in all ECs and the 3T3 cells, further confirming the specificity of iMAEC lines. These results demonstrate that iMAECs generated from wild-type and knockout mouse aortas retain key endothelial cell markers.

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