Generation and characterization of a spontaneously immortalized endothelial cell line from mice microcirculation

Author(s)

Loiola, Rodrigo A. ^[1] ; Torres, Tathiany C. ^[1] ; Aburaya, Carla M. ^[1] ; Landgraf, Maristella A. ^{[2, 1]} ; Landgraf, Richardt G. ^[1] ; Pesquero, Joao Bosco ^[3] ; Fernandes, Liliam ^[1]

Affiliation

^[1] Univ Fed Sao Paulo, Inst Ciencias Ambientais Quim & Farmaceut, BR-09913030 Sao Paulo - Brazil

^[2] Univ Sao Paulo, Inst Ciencias Biomed, Dept Imunol, BR-05508900 Sao Paulo - Brazil

^[3] Univ Fed Sao Paulo, Dept Biofis, BR-04039032 Sao Paulo - Brazil

• Document type: Journal article
• Source: Experimental Cell Research; v. 319, n. 8, p. 1102-1110, MAY 1 2013.
• Web of Science Citations: 6

Abstract

Endothelial cells from microvasculature are directly involved in a large number of vascular diseases; however, culture of these cells is problematic, since most methodologies employ proteolytic enzymes or mechanical techniques, leading to cell damage and contamination of endothelial cultures with other cellular types. Besides, primary cultured cells have a short life span in vitro and undergo replicative senescence after 3-4 passages, limiting long-term studies. In the present work we report the generation of a spontaneously immortalized endothelial culture obtained from mice pulmonary capillaries. Firstly, primary (third passage) and immortalized (100th) cultures were established. Further, monoclonal populations were obtained by serial dilutions from immortalized cultures. Cells were analyzed according to: (1) morphological appearance, (2) expression of specific endothelial markers by fluorescent staining {[}von Willebrand Factor (vWF), endothelial nitric oxide synthase (eNOS), angiotensin converting enzyme (ACE) and Ulex europaeus (UEA-1)] and by flow cytometry (endoglin, VE-cadherin and VCAM-1), and (3) release of nitric oxide (NO), assessed by the specific fluorescent dye DAF-2 DA, and prostacyclin (PGI(2)), quantified by enzyme immune assay. In both cultures cells grew in monolayers and presented cobblestone appearance at confluence. Positive staining for vWF, eNOS, ACE and UEA-1 was detected in cloned as well as in early-passage cultured cells. Similarly, cultures presented equal expressions of endoglin, VE-cadherin and VCAM-1. Values of NO and PGI(2) levels did not differ between cultures. From these results we confirm that the described spontaneously immortalized endothelial cell line is capable of unlimited growth and retains typical morphological and functional properties exhibited by primary cultured cells. Therefore, the endothelial cell line described in the present study can become a suitable tool in the field of endothelium research and can be useful for the investigation of production of endothelial mediators, angiogenesis and inflammation. (c) 2013 Elsevier Inc. All rights reserved. (AU)

• FAPESP's process: 08/06676-8 - Cellular and molecular biology of the kallikrein-kinin and renin-angiotensin systems
• Grantee: João Bosco Pesquero
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 10/01404-0 - In vivo and in vitro studies of the leptin role in different models of lung inflammation: inflammatory mediators and signaling airways participation
• Grantee: Richardt Gama Landgraf
• Support Opportunities: Research Grants - Young Investigators Grants