eNOS Overexpression Exacerbates Vascular Closure in the Obliterative Phase of OIR and Increases Angiogenic Drive in the Subsequent Proliferative Stage

Edgar, Kevin; Gardiner, Tom; Haperen, Rien van; Crom, Rini de; McDonald, Denise

doi:10.1167/iovs.12-9797

Cited by 19 publications

(30 citation statements)

References 48 publications

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“…An important advantage of our study was that we used mice previously backcrossed onto a C57 backgrounddvascular development is well characterized in this strain, as is their responsiveness to the OIR model. 19,33 Moreover, whenever possible, all experiments were performed with littermate WT controls, further reducing the likelihood of variations in genetic background confounding our results.…”

Section: Discussionmentioning

confidence: 99%

“…19,33 On P7, mouse pups and their nursing dams were placed in a chamber in which the oxygen level was maintained at 75% by a PROOX 110 oxygen regulator (Reming Bioinstruments, Redfield, NY). At P12, the mice were returned to room air and tissue was collected or placed with room airemaintained surrogate dams until P17; because nursing mice are adversely affected by hyperoxia, the use of room air surrogates ensures consistent nutrition between litters.…”

Section: Mouse Model Of Oirmentioning

confidence: 99%

“…After washing with phosphate-buffered saline (PBS), the eyes were prepared for retinal flat mount in a Maltese cross conformation. 19 Specimens were permeabilized for 4 hours at 37 C in permeabilization buffer (PBS with 0.5% Triton X-100, 1% normal goat serum, 0.1 mmol/L CaCl 2 , and 0.05% NaN 3 ). Then, they were reacted with biotinylated isolectin B4 (Sigma-Aldrich, Gillingham, UK; 20 mg/mL) in permeabilization buffer overnight at 4 C and washed for 4 hours at 37 C before reaction with Alexa Fluor-488 streptavidin conjugate (Molecular Probes, Paisley, UK; dilution 1:500) in the dark for 3 hours at 37 C. After several further washes, the slides were mounted onto glass coverslips with Vectashield medium (Vector Laboratories, Peterborough, UK).…”

Section: Assessment Of Normal Retinal Development and Vascular Coveramentioning

confidence: 99%

“…We have previously shown that these hyperfluorescent areas correspond to NV tufts extending into the vitreal compartment of the eye. 19 The quantified areas were expressed as a percentage of the total retinal area.…”

Section: Assessment Of Normal Retinal Development and Vascular Coveramentioning

confidence: 99%

“…7,9,17,18 Previously, we and others have shown a pivotal role for eNOS-derived free radicals in the vasoobliterative phase of ROP. 7,19 Currently, the mechanisms that lead to disturbed eNOS function and the nitroso-reduction oxidation imbalance are poorly defined. Further definition will aid in the development of therapeutic strategies for the treatment of this sight-threatening disease.…”

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confidence: 99%

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Hyperoxia Depletes (6R)-5,6,7,8-Tetrahydrobiopterin Levels in the Neonatal Retina

Edgar

Matesanz

Gardiner

et al. 2015

The American Journal of Pathology

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Retinopathy of prematurity is a sight-threatening complication of premature birth caused by nitro-oxidative insult to the developing retinal vasculature during therapeutic hyperoxia exposure and later ischemia-induced neovascularization on supplemental oxygen withdrawal. In the vasodegenerative phase, during hyperoxia, defective endothelial nitric oxide synthase (NOS) produces reactive oxygen and nitrogen free radicals rather than vasoprotective nitric oxide for unclear reasons. Crucially, normal NOS function depends on availability of the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4). Because BH4 synthesis is controlled enzymatically by GTP cyclohydrolase (GTPCH), we used GTPCH-depleted mice [hyperphenylalaninemia strain (hph1)] to investigate the impact of hyperoxia on BH4 bioavailability and retinal vascular pathology in the neonate. Hyperoxia decreased BH4 in retinas, lungs, and aortas in all experimental groups, resulting in a dose-dependent decrease in NOS activity and, in the wild-type group, elevated NOS-derived superoxide. Retinal dopamine levels were similarly diminished, consistent with the dependence of tyrosine hydroxylase on BH4. Despite greater depletion of BH4, the hph(+/-) and hph1(-/-) groups did not show exacerbated hyperoxia-induced vessel closure, but exhibited greater vascular protection and reduced progression to neovascular disease. This vasoprotective effect was independent of enhanced circulating vascular endothelial growth factor (VEGF), which was reduced by hyperoxia, but to local retinal ganglion cell layer-derived VEGF. In conclusion, a constitutively higher level of VEGF expression associated with retinal development protects GTPCH-deficient neonates from oxygen-induced vascular damage.

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Section: Discussionmentioning

confidence: 99%

Section: Mouse Model Of Oirmentioning

confidence: 99%

Section: Assessment Of Normal Retinal Development and Vascular Coveramentioning

confidence: 99%

Section: Assessment Of Normal Retinal Development and Vascular Coveramentioning

confidence: 99%

mentioning

confidence: 99%

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Hyperoxia Depletes (6R)-5,6,7,8-Tetrahydrobiopterin Levels in the Neonatal Retina

Edgar

Matesanz

Gardiner

et al. 2015

The American Journal of Pathology

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eNOS controls angiogenic sprouting and retinal neovascularization through the regulation of endothelial cell polarity

Smith

Oubaha

Cagnone

et al. 2021

Cell. Mol. Life Sci.

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The roles of nitric oxide (NO) and endothelial NO synthase (eNOS) in the regulation of angiogenesis are well documented. However, the involvement of eNOS in the sprouting of endothelial tip-cells at the vascular front during sprouting angiogenesis remains poorly defined. In this study, we show that downregulation of eNOS markedly inhibits VEGF-stimulated migration of endothelial cells but increases their polarization, as evidenced by the reorientation of the Golgi in migrating monolayers and by the fewer filopodia on tip cells at ends of sprouts in endothelial cell spheroids. The effect of eNOS inhibition on EC polarization was prevented in Par3-depleted cells. Importantly, downregulation of eNOS increased the expression of polarity genes, such as PARD3B, PARD6A, PARD6B, PKCΖ, TJP3, and CRB1 in endothelial cells. In retinas of eNOS knockout mice, vascular development is retarded with decreased vessel density and vascular branching. Furthermore, tip cells at the extremities of the vascular front have a marked reduction in the number of filopodia per cell and are more oriented. In a model of oxygen-induced retinopathy (OIR), eNOS deficient mice are protected during the initial vaso-obliterative phase, have reduced pathological neovascularization, and retinal endothelial tip cells have fewer filopodia. Single-cell RNA sequencing of endothelial cells from OIR retinas revealed enrichment of genes related to cell polarity in the endothelial tip-cell subtype of eNOS deficient mice. These results indicate that inhibition of eNOS alters the polarity program of endothelial cells, which increases cell polarization, regulates sprouting angiogenesis and normalizes pathological neovascularization during retinopathy.

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Effect of maternal pregestational diabetes mellitus on congenital heart diseases

et al. 2022

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eNOS Overexpression Exacerbates Vascular Closure in the Obliterative Phase of OIR and Increases Angiogenic Drive in the Subsequent Proliferative Stage

Abstract: In hyperoxia, reduced BH₄ bioavailability causes overexpressed eNOS to become dysfunctional, exacerbating vaso-obliteration. In the proliferative phase, however, eNOS has important prorepair functions enhancing angiogenic growth potential and recovery in ischemia.

Cited by 19 publications

References 48 publications

Hyperoxia Depletes (6R)-5,6,7,8-Tetrahydrobiopterin Levels in the Neonatal Retina

Hyperoxia Depletes (6R)-5,6,7,8-Tetrahydrobiopterin Levels in the Neonatal Retina

eNOS controls angiogenic sprouting and retinal neovascularization through the regulation of endothelial cell polarity

Effect of maternal pregestational diabetes mellitus on congenital heart diseases

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