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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

iMAEC-WT maintains shear-sensitive endothelial phenotype. iMAEC-WT were exposed to LS or OS or kept for static for 24 h. (A) iMAEC-WT aligned in the direction of the flow when exposed to LS but not OS or static control. Arrows indicate the imposed flow direction. (B) Total cell lysates were collected and protein level of KLF2 and eNOS was measured by Western blot. Data were shown as means ± standard error, n = 3. (C-F) Expression of shear-sensitive genes and microRNA under LS and OS for 24 h using early and late passages of iMAEC cells. iMAECs from early and late passages were subjected to either LS or OS for 24 h and expression of VCAM1, KLF2 and Interleukin-8(C, D, F) was determined by qPCR and normalized to 18S. (E) Graph shows the expression of shear-sensitive miRNA, miR-712 as determined by qPCR and normalized to RNU6B. Cells under static condition served as control. * p < 0.05 compared to respective LS controls.
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Figure 5: iMAEC-WT maintains shear-sensitive endothelial phenotype. iMAEC-WT were exposed to LS or OS or kept for static for 24 h. (A) iMAEC-WT aligned in the direction of the flow when exposed to LS but not OS or static control. Arrows indicate the imposed flow direction. (B) Total cell lysates were collected and protein level of KLF2 and eNOS was measured by Western blot. Data were shown as means ± standard error, n = 3. (C-F) Expression of shear-sensitive genes and microRNA under LS and OS for 24 h using early and late passages of iMAEC cells. iMAECs from early and late passages were subjected to either LS or OS for 24 h and expression of VCAM1, KLF2 and Interleukin-8(C, D, F) was determined by qPCR and normalized to 18S. (E) Graph shows the expression of shear-sensitive miRNA, miR-712 as determined by qPCR and normalized to RNU6B. Cells under static condition served as control. * p < 0.05 compared to respective LS controls.

Mentions: To further test whether iMAECs behave similarly to primary human EC cultures, we tested morphological and gene expression changes of these cells in response to shear stress. Like other cultured ECs, iMAECs exposed to unidirectional laminar shear stress (LS) aligned in the direction of the imposed flow, while those cultured under the oscillatory shear (OS) or static conditions did not. Further, it is well-known that LS induces expression of KLF2 and eNOS as compared to static culture or OS [39,40]. Consistent with previous reports [21], iMAEC-WT aligned to the direction of the flow in LS conditions but not in OS conditions after 24 h (Figure 5A). Further, LS increased KLF2 and eNOS protein expression (Figure 5B). These results demonstrate that iMAECs respond to shear stress in a manner consistent with other cultured ECs. We also followed the effect of shear stress on early passages of iMAECs (passage # 6-10) and compared to higher passage numbers of iMAECs (passage # 69-81) so as to confirm that the late passages of iMAECs respond to shear stress in the same fashion. We tested the expression of well-known shear sensitive genes, such as VCAM1, KLF2, KLF4, and Interleukin-8, as well as a shear-sensitive microRNA, miR-712 in response to either LS or OS for 24 h. We found that the expression of OS-induced VCAM1, Interleukin-8 and miR-712 showed a similar magnitude of fold-change in the early and late passages of iMAECs (Figure 5C-E). Also, the difference in expression of KLF2 between LS and OS was comparable to early passage cells (Figure 5E). These results indicate that iMAECs retain a functional endothelial phenotype and do not change significantly with multiple passages.


Development of immortalized mouse aortic endothelial cell lines.

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

iMAEC-WT maintains shear-sensitive endothelial phenotype. iMAEC-WT were exposed to LS or OS or kept for static for 24 h. (A) iMAEC-WT aligned in the direction of the flow when exposed to LS but not OS or static control. Arrows indicate the imposed flow direction. (B) Total cell lysates were collected and protein level of KLF2 and eNOS was measured by Western blot. Data were shown as means ± standard error, n = 3. (C-F) Expression of shear-sensitive genes and microRNA under LS and OS for 24 h using early and late passages of iMAEC cells. iMAECs from early and late passages were subjected to either LS or OS for 24 h and expression of VCAM1, KLF2 and Interleukin-8(C, D, F) was determined by qPCR and normalized to 18S. (E) Graph shows the expression of shear-sensitive miRNA, miR-712 as determined by qPCR and normalized to RNU6B. Cells under static condition served as control. * p < 0.05 compared to respective LS controls.
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Figure 5: iMAEC-WT maintains shear-sensitive endothelial phenotype. iMAEC-WT were exposed to LS or OS or kept for static for 24 h. (A) iMAEC-WT aligned in the direction of the flow when exposed to LS but not OS or static control. Arrows indicate the imposed flow direction. (B) Total cell lysates were collected and protein level of KLF2 and eNOS was measured by Western blot. Data were shown as means ± standard error, n = 3. (C-F) Expression of shear-sensitive genes and microRNA under LS and OS for 24 h using early and late passages of iMAEC cells. iMAECs from early and late passages were subjected to either LS or OS for 24 h and expression of VCAM1, KLF2 and Interleukin-8(C, D, F) was determined by qPCR and normalized to 18S. (E) Graph shows the expression of shear-sensitive miRNA, miR-712 as determined by qPCR and normalized to RNU6B. Cells under static condition served as control. * p < 0.05 compared to respective LS controls.
Mentions: To further test whether iMAECs behave similarly to primary human EC cultures, we tested morphological and gene expression changes of these cells in response to shear stress. Like other cultured ECs, iMAECs exposed to unidirectional laminar shear stress (LS) aligned in the direction of the imposed flow, while those cultured under the oscillatory shear (OS) or static conditions did not. Further, it is well-known that LS induces expression of KLF2 and eNOS as compared to static culture or OS [39,40]. Consistent with previous reports [21], iMAEC-WT aligned to the direction of the flow in LS conditions but not in OS conditions after 24 h (Figure 5A). Further, LS increased KLF2 and eNOS protein expression (Figure 5B). These results demonstrate that iMAECs respond to shear stress in a manner consistent with other cultured ECs. We also followed the effect of shear stress on early passages of iMAECs (passage # 6-10) and compared to higher passage numbers of iMAECs (passage # 69-81) so as to confirm that the late passages of iMAECs respond to shear stress in the same fashion. We tested the expression of well-known shear sensitive genes, such as VCAM1, KLF2, KLF4, and Interleukin-8, as well as a shear-sensitive microRNA, miR-712 in response to either LS or OS for 24 h. We found that the expression of OS-induced VCAM1, Interleukin-8 and miR-712 showed a similar magnitude of fold-change in the early and late passages of iMAECs (Figure 5C-E). Also, the difference in expression of KLF2 between LS and OS was comparable to early passage cells (Figure 5E). These results indicate that iMAECs retain a functional endothelial phenotype and do not change significantly with multiple passages.

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