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Experimental Branch Retinal Vein Occlusion Induces Upstream Pericyte Loss and Vascular Destabilization.

Dominguez E, Raoul W, Calippe B, Sahel JA, Guillonneau X, Paques M, Sennlaub F - PLoS ONE (2015)

Bottom Line: We here show that laser-induced experimental BRVO in mice leads to a wave of TUNEL-positive endothelial cell (EC) apoptosis in the upstream vascular network associated with a transient edema and hemorrhages.The vascular remodeling was associated with increased expression of TGFβ, TSP-1, but also FGF2 expression.These early changes might pave the way for capillary loss and subsequent chronic ischemia and edema that characterize the late stage disease.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U968, Paris, F-75012, France; Sorbonne Universités, UPMC Univ Paris 06, UMR_S 968, Institut de la Vision, Paris, F-75012, France; CNRS, UMR_7210, Paris, F-75012, France.

ABSTRACT

Aims: Branch retinal vein occlusion (BRVO) leads to extensive vascular remodeling and is important cause of visual impairment. Although the vascular morphological changes following experimental vein occlusion have been described in a variety of models using angiography, the underlying cellular events are ill defined.

Methods and results: We here show that laser-induced experimental BRVO in mice leads to a wave of TUNEL-positive endothelial cell (EC) apoptosis in the upstream vascular network associated with a transient edema and hemorrhages. Subsequently, we observe an induction of EC proliferation within the dilated vein and capillaries, detected by EdU incorporation, and the edema resolves. However, the pericytes of the upstream capillaries are severely reduced, which was associated with continuing EC apoptosis and proliferation. The vascular remodeling was associated with increased expression of TGFβ, TSP-1, but also FGF2 expression. Exposure of the experimental animals to hypoxia, when pericyte (PC) dropout had occurred, led to a dramatic increase in endothelial cell proliferation, confirming the vascular instability induced by the experimental BRVO.

Conclusion: Experimental BRVO leads to acute endothelial cells apoptosis and increased permeability. Subsequently the upstream vascular network remains destabilized, characterized by pericyte dropout, un-physiologically high endothelial cells turnover and sensitivity to hypoxia. These early changes might pave the way for capillary loss and subsequent chronic ischemia and edema that characterize the late stage disease.

No MeSH data available.


Related in: MedlinePlus

Hypoxia induced endothelial cell proliferation in control and BRVO retinas.(A and B) Representative micrographs of EdU (green)—CollIV (red) double-labeled retinal flatmounts at 7d after BRVO raised in normoxia (A) or hypoxia from d3 to d7 (B). All mice were daily injected with EdU from d3 to d7 before sacrifice (please not that retinas in Fig 2 were injected daily with EdU and are therefor not comparable in terms of numbers of EdU+cells). (C) Quantification of EdU positive ECs of the superior vein and upstream capillary bed at d7 of control retinas (without BRVO) and BRVO retinas, raised in normoxia and hypoxia. All mice were daily injected with EdU from d3 to d7 before sacrifice. Values in histograms are mean ± SEM of mRNA expression of occluded area from 9–10 retinas per group. Mann-Whitney non parametric test, ** p<0.01, normoxic BRVO versus hypoxic BRVO. Scale bars AB = 200μm.
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pone.0132644.g006: Hypoxia induced endothelial cell proliferation in control and BRVO retinas.(A and B) Representative micrographs of EdU (green)—CollIV (red) double-labeled retinal flatmounts at 7d after BRVO raised in normoxia (A) or hypoxia from d3 to d7 (B). All mice were daily injected with EdU from d3 to d7 before sacrifice (please not that retinas in Fig 2 were injected daily with EdU and are therefor not comparable in terms of numbers of EdU+cells). (C) Quantification of EdU positive ECs of the superior vein and upstream capillary bed at d7 of control retinas (without BRVO) and BRVO retinas, raised in normoxia and hypoxia. All mice were daily injected with EdU from d3 to d7 before sacrifice. Values in histograms are mean ± SEM of mRNA expression of occluded area from 9–10 retinas per group. Mann-Whitney non parametric test, ** p<0.01, normoxic BRVO versus hypoxic BRVO. Scale bars AB = 200μm.

Mentions: PC dropout leads to a destabilization of the vascular network [17]. To test whether the loss of PCs we observe after BRVO leads to vascular destabilization, we exposed control and BRVO animals to hypoxia of 10% O2 (from d3 to d7) or normal atmosphere (20,9% O2) and evaluated EC proliferation using EdU incorporation at d7. EdU (green)- CollIV (red) double stained retinal flatmounts of 7d BRVO retina that were kept under normoxic conditions (Fig 6A) and exposed to hypoxia (Fig 6B) showed a dramatic increase in EdU+ECs in the hypoxic condition. Quantification of EdU+ECs showed that hypoxia did not significantly increase cell proliferation in non-occluded control retina (Fig 6C; 5.25 EdU+ECs /mm² for normoxia versus 7.25 EdU+ECs /mm² for hypoxia). In contrast, hypoxic conditions nearly doubled EC proliferation in the occluded retina with BRVO (Fig 6C; 85.46 EdU+ECs /mm² for normoxia versus 152.2 EdU+ECs /mm² for hypoxia). Please note that the values of Edu+cells in normoxic BRVO retina are lower in this set of experiments compared to the results presented in Fig 2, as EdU was only injected from d3 to d5 (d0 to d7 in Fig 2F).


Experimental Branch Retinal Vein Occlusion Induces Upstream Pericyte Loss and Vascular Destabilization.

Dominguez E, Raoul W, Calippe B, Sahel JA, Guillonneau X, Paques M, Sennlaub F - PLoS ONE (2015)

Hypoxia induced endothelial cell proliferation in control and BRVO retinas.(A and B) Representative micrographs of EdU (green)—CollIV (red) double-labeled retinal flatmounts at 7d after BRVO raised in normoxia (A) or hypoxia from d3 to d7 (B). All mice were daily injected with EdU from d3 to d7 before sacrifice (please not that retinas in Fig 2 were injected daily with EdU and are therefor not comparable in terms of numbers of EdU+cells). (C) Quantification of EdU positive ECs of the superior vein and upstream capillary bed at d7 of control retinas (without BRVO) and BRVO retinas, raised in normoxia and hypoxia. All mice were daily injected with EdU from d3 to d7 before sacrifice. Values in histograms are mean ± SEM of mRNA expression of occluded area from 9–10 retinas per group. Mann-Whitney non parametric test, ** p<0.01, normoxic BRVO versus hypoxic BRVO. Scale bars AB = 200μm.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4514656&req=5

pone.0132644.g006: Hypoxia induced endothelial cell proliferation in control and BRVO retinas.(A and B) Representative micrographs of EdU (green)—CollIV (red) double-labeled retinal flatmounts at 7d after BRVO raised in normoxia (A) or hypoxia from d3 to d7 (B). All mice were daily injected with EdU from d3 to d7 before sacrifice (please not that retinas in Fig 2 were injected daily with EdU and are therefor not comparable in terms of numbers of EdU+cells). (C) Quantification of EdU positive ECs of the superior vein and upstream capillary bed at d7 of control retinas (without BRVO) and BRVO retinas, raised in normoxia and hypoxia. All mice were daily injected with EdU from d3 to d7 before sacrifice. Values in histograms are mean ± SEM of mRNA expression of occluded area from 9–10 retinas per group. Mann-Whitney non parametric test, ** p<0.01, normoxic BRVO versus hypoxic BRVO. Scale bars AB = 200μm.
Mentions: PC dropout leads to a destabilization of the vascular network [17]. To test whether the loss of PCs we observe after BRVO leads to vascular destabilization, we exposed control and BRVO animals to hypoxia of 10% O2 (from d3 to d7) or normal atmosphere (20,9% O2) and evaluated EC proliferation using EdU incorporation at d7. EdU (green)- CollIV (red) double stained retinal flatmounts of 7d BRVO retina that were kept under normoxic conditions (Fig 6A) and exposed to hypoxia (Fig 6B) showed a dramatic increase in EdU+ECs in the hypoxic condition. Quantification of EdU+ECs showed that hypoxia did not significantly increase cell proliferation in non-occluded control retina (Fig 6C; 5.25 EdU+ECs /mm² for normoxia versus 7.25 EdU+ECs /mm² for hypoxia). In contrast, hypoxic conditions nearly doubled EC proliferation in the occluded retina with BRVO (Fig 6C; 85.46 EdU+ECs /mm² for normoxia versus 152.2 EdU+ECs /mm² for hypoxia). Please note that the values of Edu+cells in normoxic BRVO retina are lower in this set of experiments compared to the results presented in Fig 2, as EdU was only injected from d3 to d5 (d0 to d7 in Fig 2F).

Bottom Line: We here show that laser-induced experimental BRVO in mice leads to a wave of TUNEL-positive endothelial cell (EC) apoptosis in the upstream vascular network associated with a transient edema and hemorrhages.The vascular remodeling was associated with increased expression of TGFβ, TSP-1, but also FGF2 expression.These early changes might pave the way for capillary loss and subsequent chronic ischemia and edema that characterize the late stage disease.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U968, Paris, F-75012, France; Sorbonne Universités, UPMC Univ Paris 06, UMR_S 968, Institut de la Vision, Paris, F-75012, France; CNRS, UMR_7210, Paris, F-75012, France.

ABSTRACT

Aims: Branch retinal vein occlusion (BRVO) leads to extensive vascular remodeling and is important cause of visual impairment. Although the vascular morphological changes following experimental vein occlusion have been described in a variety of models using angiography, the underlying cellular events are ill defined.

Methods and results: We here show that laser-induced experimental BRVO in mice leads to a wave of TUNEL-positive endothelial cell (EC) apoptosis in the upstream vascular network associated with a transient edema and hemorrhages. Subsequently, we observe an induction of EC proliferation within the dilated vein and capillaries, detected by EdU incorporation, and the edema resolves. However, the pericytes of the upstream capillaries are severely reduced, which was associated with continuing EC apoptosis and proliferation. The vascular remodeling was associated with increased expression of TGFβ, TSP-1, but also FGF2 expression. Exposure of the experimental animals to hypoxia, when pericyte (PC) dropout had occurred, led to a dramatic increase in endothelial cell proliferation, confirming the vascular instability induced by the experimental BRVO.

Conclusion: Experimental BRVO leads to acute endothelial cells apoptosis and increased permeability. Subsequently the upstream vascular network remains destabilized, characterized by pericyte dropout, un-physiologically high endothelial cells turnover and sensitivity to hypoxia. These early changes might pave the way for capillary loss and subsequent chronic ischemia and edema that characterize the late stage disease.

No MeSH data available.


Related in: MedlinePlus