Electroporation-mediated gene delivery of cleavage-resistant pro-nerve growth factor causes retinal neuro- and vascular degeneration

Suraporn Matragoon, Mohammed M.H. Al-Gayyar, Barbara A Mysona, Mohammed A. Abdelsaid, Bindu A. Pillai, Kenneth E. Neet, Susan C. Fagan, Azza B. El-Remessy

Research output: Contribution to journalArticle

Abstract

Purpose: Neurotrophins, including nerve growth factor (NGF), are secreted by glia as a pro-form (proNGF) that is normally cleaved into the mature ligand. Increases of proNGF has been well documented in retinal neurodegenerative diseases. Since systemic overexpression of proNGF exhibits embryonic lethality, we aimed to establish a model that specifically and stably overexpresses a cleavage-resistant mutant of proNGF (proNGF123) plasmid in the retina using electroporation. Methods: Male Sprague-Dawley rats were injected intravitreally with pGFP or pGFP-proNGF123 plasmids, then elec- troporated with various settings for optimization. Retinal cell death and ganglion cell count were assessed by TUNEL and immunostaining with anti-Brn3. Expression of proNGF, NGF, and their receptors was examined by western blot. Retinal vascular permeability was assessed by extravasation of bovine serum albumin-fluorescein. Development of acellular capillaries was assessed by periodic acid-Schiff and hematoxylin staining. Results: Successful pGFP-proNGF123 gene delivery and expression of proNGF was demonstrated by western blot and extensive proNGF immunostaining in retina sections. Overexpression of proNGF reduced NGF expression while inducing the expression of neurotrophin receptors, including p75NTRand tyrosine receptor kinase A, but not sortilin. Overexpression of proNGF resulted in ~50% reduction in ganglion cell count and fivefold increase in TUNEL-positive cells in rat retina. In addition, overexpression of proNGF induced breakdown of the blood-retina barrier evident by twofold increase in extravasation of bovine serum albumin-fluorescein after 1 week and induced the development of acellular capillaries after 4 weeks. Conclusions: Electroporation can successfully incorporate and express biologically active cleavage-resistant proNGF locally in rat retinas. Overexpression of cleavage-resistant proNGF can be a useful tool to investigate specific molecular mechanisms by which proNGF causes neurodegeneration and vascular injury in the retina.

Original languageEnglish (US)
Pages (from-to)2993-3003
Number of pages11
JournalMolecular Vision
Volume18
StatePublished - Dec 14 2012

Fingerprint

Retinal Vessels
Electroporation
Nerve Growth Factor
Retina
Genes
Nerve Growth Factor Receptors
In Situ Nick-End Labeling
Bovine Serum Albumin
Fluorescein
Plasmids
Cell Count
Western Blotting
Retinal Diseases
Periodic Acid
Retinal Ganglion Cells
Vascular System Injuries
Nerve Growth Factors
Capillary Permeability
Receptor Protein-Tyrosine Kinases
Hematoxylin

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Matragoon, S., Al-Gayyar, M. M. H., Mysona, B. A., Abdelsaid, M. A., Pillai, B. A., Neet, K. E., ... El-Remessy, A. B. (2012). Electroporation-mediated gene delivery of cleavage-resistant pro-nerve growth factor causes retinal neuro- and vascular degeneration. Molecular Vision, 18, 2993-3003.

Electroporation-mediated gene delivery of cleavage-resistant pro-nerve growth factor causes retinal neuro- and vascular degeneration. / Matragoon, Suraporn; Al-Gayyar, Mohammed M.H.; Mysona, Barbara A; Abdelsaid, Mohammed A.; Pillai, Bindu A.; Neet, Kenneth E.; Fagan, Susan C.; El-Remessy, Azza B.

In: Molecular Vision, Vol. 18, 14.12.2012, p. 2993-3003.

Research output: Contribution to journalArticle

Matragoon, S, Al-Gayyar, MMH, Mysona, BA, Abdelsaid, MA, Pillai, BA, Neet, KE, Fagan, SC & El-Remessy, AB 2012, 'Electroporation-mediated gene delivery of cleavage-resistant pro-nerve growth factor causes retinal neuro- and vascular degeneration', Molecular Vision, vol. 18, pp. 2993-3003.
Matragoon, Suraporn ; Al-Gayyar, Mohammed M.H. ; Mysona, Barbara A ; Abdelsaid, Mohammed A. ; Pillai, Bindu A. ; Neet, Kenneth E. ; Fagan, Susan C. ; El-Remessy, Azza B. / Electroporation-mediated gene delivery of cleavage-resistant pro-nerve growth factor causes retinal neuro- and vascular degeneration. In: Molecular Vision. 2012 ; Vol. 18. pp. 2993-3003.
@article{20bef2704a084182a6c3a8eb49c81cc1,
title = "Electroporation-mediated gene delivery of cleavage-resistant pro-nerve growth factor causes retinal neuro- and vascular degeneration",
abstract = "Purpose: Neurotrophins, including nerve growth factor (NGF), are secreted by glia as a pro-form (proNGF) that is normally cleaved into the mature ligand. Increases of proNGF has been well documented in retinal neurodegenerative diseases. Since systemic overexpression of proNGF exhibits embryonic lethality, we aimed to establish a model that specifically and stably overexpresses a cleavage-resistant mutant of proNGF (proNGF123) plasmid in the retina using electroporation. Methods: Male Sprague-Dawley rats were injected intravitreally with pGFP or pGFP-proNGF123 plasmids, then elec- troporated with various settings for optimization. Retinal cell death and ganglion cell count were assessed by TUNEL and immunostaining with anti-Brn3. Expression of proNGF, NGF, and their receptors was examined by western blot. Retinal vascular permeability was assessed by extravasation of bovine serum albumin-fluorescein. Development of acellular capillaries was assessed by periodic acid-Schiff and hematoxylin staining. Results: Successful pGFP-proNGF123 gene delivery and expression of proNGF was demonstrated by western blot and extensive proNGF immunostaining in retina sections. Overexpression of proNGF reduced NGF expression while inducing the expression of neurotrophin receptors, including p75NTRand tyrosine receptor kinase A, but not sortilin. Overexpression of proNGF resulted in ~50{\%} reduction in ganglion cell count and fivefold increase in TUNEL-positive cells in rat retina. In addition, overexpression of proNGF induced breakdown of the blood-retina barrier evident by twofold increase in extravasation of bovine serum albumin-fluorescein after 1 week and induced the development of acellular capillaries after 4 weeks. Conclusions: Electroporation can successfully incorporate and express biologically active cleavage-resistant proNGF locally in rat retinas. Overexpression of cleavage-resistant proNGF can be a useful tool to investigate specific molecular mechanisms by which proNGF causes neurodegeneration and vascular injury in the retina.",
author = "Suraporn Matragoon and Al-Gayyar, {Mohammed M.H.} and Mysona, {Barbara A} and Abdelsaid, {Mohammed A.} and Pillai, {Bindu A.} and Neet, {Kenneth E.} and Fagan, {Susan C.} and El-Remessy, {Azza B.}",
year = "2012",
month = "12",
day = "14",
language = "English (US)",
volume = "18",
pages = "2993--3003",
journal = "Molecular Vision",
issn = "1090-0535",

}

TY - JOUR

T1 - Electroporation-mediated gene delivery of cleavage-resistant pro-nerve growth factor causes retinal neuro- and vascular degeneration

AU - Matragoon, Suraporn

AU - Al-Gayyar, Mohammed M.H.

AU - Mysona, Barbara A

AU - Abdelsaid, Mohammed A.

AU - Pillai, Bindu A.

AU - Neet, Kenneth E.

AU - Fagan, Susan C.

AU - El-Remessy, Azza B.

PY - 2012/12/14

Y1 - 2012/12/14

N2 - Purpose: Neurotrophins, including nerve growth factor (NGF), are secreted by glia as a pro-form (proNGF) that is normally cleaved into the mature ligand. Increases of proNGF has been well documented in retinal neurodegenerative diseases. Since systemic overexpression of proNGF exhibits embryonic lethality, we aimed to establish a model that specifically and stably overexpresses a cleavage-resistant mutant of proNGF (proNGF123) plasmid in the retina using electroporation. Methods: Male Sprague-Dawley rats were injected intravitreally with pGFP or pGFP-proNGF123 plasmids, then elec- troporated with various settings for optimization. Retinal cell death and ganglion cell count were assessed by TUNEL and immunostaining with anti-Brn3. Expression of proNGF, NGF, and their receptors was examined by western blot. Retinal vascular permeability was assessed by extravasation of bovine serum albumin-fluorescein. Development of acellular capillaries was assessed by periodic acid-Schiff and hematoxylin staining. Results: Successful pGFP-proNGF123 gene delivery and expression of proNGF was demonstrated by western blot and extensive proNGF immunostaining in retina sections. Overexpression of proNGF reduced NGF expression while inducing the expression of neurotrophin receptors, including p75NTRand tyrosine receptor kinase A, but not sortilin. Overexpression of proNGF resulted in ~50% reduction in ganglion cell count and fivefold increase in TUNEL-positive cells in rat retina. In addition, overexpression of proNGF induced breakdown of the blood-retina barrier evident by twofold increase in extravasation of bovine serum albumin-fluorescein after 1 week and induced the development of acellular capillaries after 4 weeks. Conclusions: Electroporation can successfully incorporate and express biologically active cleavage-resistant proNGF locally in rat retinas. Overexpression of cleavage-resistant proNGF can be a useful tool to investigate specific molecular mechanisms by which proNGF causes neurodegeneration and vascular injury in the retina.

AB - Purpose: Neurotrophins, including nerve growth factor (NGF), are secreted by glia as a pro-form (proNGF) that is normally cleaved into the mature ligand. Increases of proNGF has been well documented in retinal neurodegenerative diseases. Since systemic overexpression of proNGF exhibits embryonic lethality, we aimed to establish a model that specifically and stably overexpresses a cleavage-resistant mutant of proNGF (proNGF123) plasmid in the retina using electroporation. Methods: Male Sprague-Dawley rats were injected intravitreally with pGFP or pGFP-proNGF123 plasmids, then elec- troporated with various settings for optimization. Retinal cell death and ganglion cell count were assessed by TUNEL and immunostaining with anti-Brn3. Expression of proNGF, NGF, and their receptors was examined by western blot. Retinal vascular permeability was assessed by extravasation of bovine serum albumin-fluorescein. Development of acellular capillaries was assessed by periodic acid-Schiff and hematoxylin staining. Results: Successful pGFP-proNGF123 gene delivery and expression of proNGF was demonstrated by western blot and extensive proNGF immunostaining in retina sections. Overexpression of proNGF reduced NGF expression while inducing the expression of neurotrophin receptors, including p75NTRand tyrosine receptor kinase A, but not sortilin. Overexpression of proNGF resulted in ~50% reduction in ganglion cell count and fivefold increase in TUNEL-positive cells in rat retina. In addition, overexpression of proNGF induced breakdown of the blood-retina barrier evident by twofold increase in extravasation of bovine serum albumin-fluorescein after 1 week and induced the development of acellular capillaries after 4 weeks. Conclusions: Electroporation can successfully incorporate and express biologically active cleavage-resistant proNGF locally in rat retinas. Overexpression of cleavage-resistant proNGF can be a useful tool to investigate specific molecular mechanisms by which proNGF causes neurodegeneration and vascular injury in the retina.

UR - http://www.scopus.com/inward/record.url?scp=84871697833&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84871697833&partnerID=8YFLogxK

M3 - Article

VL - 18

SP - 2993

EP - 3003

JO - Molecular Vision

JF - Molecular Vision

SN - 1090-0535

ER -