Conditionally immortalized human glomerular endothelial cells expressing fenestrations in response to VEGF

Abstract

Glomerular endothelial cells (GEnC) are specialized cells with important roles in physiological filtration and glomerular disease. Despite their unique features, GEnC have been little studied because of difficulty in maintaining them in cell culture. We have addressed this problem by generation of conditionally immortalized (ci) human GEnC using technology with which we have previously produced ci podocytes. Primary culture GEnC were transduced with temperature-sensitive simian virus 40 large tumour antigen and telomerase using retroviral vectors. Cells were selected, cloned, and then characterized by light and electron microscopy (EM), response to vascular endothelial growth factor (VEGF), and tumour necrosis factor (TNF)alpha, expression of endothelial markers by focused gene array, immunofluorescence and Western blotting, and formation and behaviour of monolayers. CiGEnC proliferated at the permissive temperature (33 degrees C) and became growth arrested at the non-permissive temperature (37 degrees C). CiGEnC retained morphological features of early-passage primary culture GEnC up to at least p41, confirming successful immortalization. EM demonstrated fenestrations, increased in number by VEGF. mRNA analysis confirmed expression of the endothelial markers platelet endothelial cell adhesion molecule 1, intercellular adhesion molecule 2, VEGF receptor 2, and von Willebrand factor, validated by immunofluorescence and Western blotting. CiGEnC also expressed Tie2, and TNFalpha upregulated E-selectin. CiGEnC formed monolayers with barrier properties responsive to cyclic adenosine 3',5' monophosphate (cAMP) and thrombin. CiGEnC retain the markers and behaviour of primary culture GEnC. They express fenestrations which are upregulated in response to VEGF. These cells are a unique resource for further study of GEnC and their roles in glomerular filtration, glomerular disease, and response to glomerular injury.

Direct detection and measurement of wall shear stress using a filamentous bio-nanoparticle

“…We have previously shown that ibidi systems are able to orient semi-rigid molecular systems [24]. Human conditionally immortalized glomerular endothelial cells (GEnCs) [25] were grown as described in the ESM. The completed M13 construct was bound to the flow slide coating (collagen IV or endothelial cells) and any unbound M13 was rinsed off.…”

Rosiglitazone enhances glucose uptake in glomerular podocytes using the glucose transporter GLUT1

“…Conditionally immortalised human glomerular endothelial cells were also used. The characteristics of these cells have been reported elsewhere [22]. In addition, murine 3T3-L1 fibroblast clones were obtained from LGC Promochem (Teddington, UK).…”

Adenoviral E4 Gene Stimulates Secretion of Pigmental Epithelium Derived Factor (PEDF) that Maintains Long-term Survival of Human Glomerulus-derived Endothelial Cells

“…Primary endothelial cells maintained in culture have a shortened life-span and require supplementation of growth media with cytokines and serum that result in the loss of their unique endothelial cell features (6). Different methods were previously used to transform endothelial cells to increase their life-span including transformation with human telomerase (17,18), polyoma middle T antigen (19), SV40 large T antigen, adenoviral E4 genes (10) or combination of transforming agents (8).…”

“…Thus the limited life span and dependence on serum and growth factors put a considerable limitation to studies that can be conducted with primary endothelial cells. Human primary glomerulus-derived endothelial cells (PrGECs) either transformed with a simian virus 40 (SV40) (7) or conditionally immortalized with temperature-sensitive SV40 large T antigen and telomerase using retroviral vectors (8) retain the ability to grow up to 60 passages and maintain endothelial cell markers. However, culturing these transformed or immortalized cell lines still requires supplementation with serum and growth factors and their withdrawal leads to quick cell death (9).…”