Brain and retinal vascular endothelial cells with extended life span established by ectopic expression of telomerase

Xiaolin Gu, Jing Zhang, Darrell W Brann, Fu Shin X. Yu

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

PURPOSE. To study blood-retina barrier (BRB) regulation, we sought to establish neuronal microvascular endothelial cells (ECs) with expanded life span by ectopic expression of the human telomerase gene (hTERT). METHODS. Primary cultures of human brain and bovine retinal microvascular endothelial cells (HBECs and BRECs, respectively) were transfected with the catalytic component of human telomerase human telomerase reverse transcriptase (hTERT), and colonies were selected with puromycin. The endothelial origin of these cells was confirmed by immunocytochemistry. Reconstituted basement membrane matrix and three-dimensional collagen gel were used to induce the formation of tubulelike structures. To assess endothelial permeability, ECs were cultured on the upper chamber of migration assay membrane filters, with or without astrocyte coculture. Transepithelial electrical resistance (TER) was measured using a voltohmmeter. RESULTS. Both bovine retinal and human brain microvascular ECs expressing hTERT resembled young primary ECs in their morphology and growth response after more than 100 population doublings. Both bovine and human hTERT cells expressed von Willebrand factor, a key marker distinguishing ECs from other cell types; formed angiogenic webs in reconstituted basement membrane matrix; and, in a VEGF-dependent manner, formed tubule-like structures in three-dimensional collagen gel. Coculture of both types of cells with astrocytes resulted in a decrease in EC permeability, as assessed by TER. VEGF induced the breakdown of the HBEC monolayer barrier, and astrocytes in coculture appeared to attenuate the effects of VEGF. CONCLUSIONS. Ectopic expression of hTERT enables adult HBECs to bypass the first mortality checkpoint but not the second mortality checkpoint, allowing generation of neuronal ECs with extended, but not indefinite life span.

Original languageEnglish (US)
Pages (from-to)3219-3225
Number of pages7
JournalInvestigative Ophthalmology and Visual Science
Volume44
Issue number7
DOIs
StatePublished - Jul 1 2003

Fingerprint

Retinal Vessels
Telomerase
Endothelial Cells
Brain
Coculture Techniques
Astrocytes
Vascular Endothelial Growth Factor A
Electric Impedance
Basement Membrane
Permeability
Collagen
Gels
Ectopic Gene Expression
Puromycin
Mortality
von Willebrand Factor
Retina
Immunohistochemistry
human TERT protein
Membranes

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Brain and retinal vascular endothelial cells with extended life span established by ectopic expression of telomerase. / Gu, Xiaolin; Zhang, Jing; Brann, Darrell W; Yu, Fu Shin X.

In: Investigative Ophthalmology and Visual Science, Vol. 44, No. 7, 01.07.2003, p. 3219-3225.

Research output: Contribution to journalArticle

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N2 - PURPOSE. To study blood-retina barrier (BRB) regulation, we sought to establish neuronal microvascular endothelial cells (ECs) with expanded life span by ectopic expression of the human telomerase gene (hTERT). METHODS. Primary cultures of human brain and bovine retinal microvascular endothelial cells (HBECs and BRECs, respectively) were transfected with the catalytic component of human telomerase human telomerase reverse transcriptase (hTERT), and colonies were selected with puromycin. The endothelial origin of these cells was confirmed by immunocytochemistry. Reconstituted basement membrane matrix and three-dimensional collagen gel were used to induce the formation of tubulelike structures. To assess endothelial permeability, ECs were cultured on the upper chamber of migration assay membrane filters, with or without astrocyte coculture. Transepithelial electrical resistance (TER) was measured using a voltohmmeter. RESULTS. Both bovine retinal and human brain microvascular ECs expressing hTERT resembled young primary ECs in their morphology and growth response after more than 100 population doublings. Both bovine and human hTERT cells expressed von Willebrand factor, a key marker distinguishing ECs from other cell types; formed angiogenic webs in reconstituted basement membrane matrix; and, in a VEGF-dependent manner, formed tubule-like structures in three-dimensional collagen gel. Coculture of both types of cells with astrocytes resulted in a decrease in EC permeability, as assessed by TER. VEGF induced the breakdown of the HBEC monolayer barrier, and astrocytes in coculture appeared to attenuate the effects of VEGF. CONCLUSIONS. Ectopic expression of hTERT enables adult HBECs to bypass the first mortality checkpoint but not the second mortality checkpoint, allowing generation of neuronal ECs with extended, but not indefinite life span.

AB - PURPOSE. To study blood-retina barrier (BRB) regulation, we sought to establish neuronal microvascular endothelial cells (ECs) with expanded life span by ectopic expression of the human telomerase gene (hTERT). METHODS. Primary cultures of human brain and bovine retinal microvascular endothelial cells (HBECs and BRECs, respectively) were transfected with the catalytic component of human telomerase human telomerase reverse transcriptase (hTERT), and colonies were selected with puromycin. The endothelial origin of these cells was confirmed by immunocytochemistry. Reconstituted basement membrane matrix and three-dimensional collagen gel were used to induce the formation of tubulelike structures. To assess endothelial permeability, ECs were cultured on the upper chamber of migration assay membrane filters, with or without astrocyte coculture. Transepithelial electrical resistance (TER) was measured using a voltohmmeter. RESULTS. Both bovine retinal and human brain microvascular ECs expressing hTERT resembled young primary ECs in their morphology and growth response after more than 100 population doublings. Both bovine and human hTERT cells expressed von Willebrand factor, a key marker distinguishing ECs from other cell types; formed angiogenic webs in reconstituted basement membrane matrix; and, in a VEGF-dependent manner, formed tubule-like structures in three-dimensional collagen gel. Coculture of both types of cells with astrocytes resulted in a decrease in EC permeability, as assessed by TER. VEGF induced the breakdown of the HBEC monolayer barrier, and astrocytes in coculture appeared to attenuate the effects of VEGF. CONCLUSIONS. Ectopic expression of hTERT enables adult HBECs to bypass the first mortality checkpoint but not the second mortality checkpoint, allowing generation of neuronal ECs with extended, but not indefinite life span.

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