A Computational Tool for Quantitative Analysis of Vascular Networks

Zudaire, Enrique; Gambardella, Laure; Kurcz, Christopher; Vermeren, Sonja

doi:10.1371/journal.pone.0027385

Cited by 857 publications

(683 citation statements)

References 30 publications

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“…For quantification of the vascular branching and density, confocal images obtained with a ϫ20 objective, a ϫ0.7 scanning digital zoom, 512 ϫ 512 pixels were analyzed using AngioTool 0.5a (21). For quantification of branching points, vessels were outlined in Adobe Photoshop CS4 software prior to analysis.…”

Section: Methodsmentioning

confidence: 99%

Phosphorylation Regulates FOXC2-Mediated Transcription in Lymphatic Endothelial Cells

Ivanov

Agalarov

Valmu

et al. 2013

Molecular and Cellular Biology

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One of the key mechanisms linking cell signaling and control of gene expression is reversible phosphorylation of transcription factors. FOXC2 is a forkhead transcription factor that is mutated in the human vascular disease lymphedema-distichiasis and plays an essential role in lymphatic vascular development. However, the mechanisms regulating FOXC2 transcriptional activity are not well understood. We report here that FOXC2 is phosphorylated on eight evolutionarily conserved proline-directed serine/threonine residues. Loss of phosphorylation at these sites triggers substantial changes in the FOXC2 transcriptional program. Through genome-wide location analysis in lymphatic endothelial cells, we demonstrate that the changes are due to selective inhibition of FOXC2 recruitment to chromatin. The extent of the inhibition varied between individual binding sites, suggesting a novel rheostat-like mechanism by which expression of specific genes can be differentially regulated by FOXC2 phosphorylation. Furthermore, unlike the wild-type protein, the phosphorylation-deficient mutant of FOXC2 failed to induce vascular remodeling in vivo. Collectively, our results point to the pivotal role of phosphorylation in the regulation of FOXC2-mediated transcription in lymphatic endothelial cells and underscore the importance of FOXC2 phosphorylation in vascular development. Forkhead box (Fox) proteins are a family of transcription factors (TFs) that play an important role in development, cell cycle regulation, and other key biological processes (1). In mammals, more than 40 forkhead family members have been identified, all sharing the evolutionarily conserved forkhead DNA binding domain. Despite the similarity in their DNA-binding domains, different members of the forkhead family have evolved distinct functional roles. The forkhead transcription factor FOXC2 was first demonstrated to play a role in the morphogenesis of the cardiovascular system during embryonic development (2, 3). Subsequent studies revealed that FOXC2 is also implicated in lymphatic vascular development and disease. Mutations in FOXC2 cause lymphedema-distichiasis (LD; OMIM 153400) characterized by lymphedema and double rows of eyelashes (4). In both humans and mice, FOXC2 is highly expressed in the developing lymphatic vessels, as well as in the adult lymphatic valves (5, 6). The critical role of FOXC2 in lymphatic vascular development has been underscored by the demonstration of abnormal lymphatic patterning and failure to form lymphatic valves in Foxc2-deficient mice (5,7,8). LD patients develop similar defects characterized by lymph and venous reflux, indicating failure or absence of lymphatic and venous valves (9, 10). On a mechanistic level, FOXC2 genomic binding sites are enriched in NFATC1 consensus sequences, and the two transcription factors cooperate in vivo during lymphatic vascular morphogenesis (8).Phosphorylation of transcription factors represents a rapid and reversible mechanism for dynamic regulation of transcriptional networks (11). Most of the Fox...

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Section: Methodsmentioning

confidence: 99%

Phosphorylation Regulates FOXC2-Mediated Transcription in Lymphatic Endothelial Cells

Ivanov

Agalarov

Valmu

et al. 2013

Molecular and Cellular Biology

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“…Images were tiled to create a picture of the entire retina. The density of the capillary network was analyzed using the AngioTool image analysis sofware 10 having fixed settings for vessel diameter (1-5) and intensity (15)(16)(17)(18)(19)(20)(21)(22).…”

Section: Fluorescein Angiographymentioning

confidence: 99%

Diabetes enhances the efficacy of AAV2 vectors in the retina: therapeutic effect of AAV2 encoding vasoinhibin and soluble VEGF receptor 1

Díaz‐Lezama

Adán‐Castro

et al. 2016

Laboratory Investigation

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Adeno-associated virus (AAV) vector-mediated delivery of inhibitors of blood-retinal barrier breakdown (BRBB) offers promise for the treatment of diabetic macular edema. Here, we demonstrated a reversal of blood-retinal barrier pathology mediated by AAV type 2 (AAV2) vectors encoding vasoinhibin or soluble VEGF receptor 1 (sFlt-1) when administered intravitreally to diabetic rats. Efficacy and safety of the AAV2 vasoinhibin vector were tested by monitoring its effect on diabetes-induced changes in the retinal vascular bed and thickness, and in the electroretinogram (ERG). Also, the transduction of AAV2 vectors and expression of AAV2 receptors and co-receptors were compared between the diabetic and the non-diabetic rat retinas. AAV2 vasoinhibin or AAV2 sFlt-1 vectors were injected intravitreally before or after enhanced BRBB due to diabetes induced by streptozotocin. The BRBB was examined by the Evans blue method, the vascular bed by fluorescein angiography, expression of the AAV2 EGFP reporter vector by confocal microscopy, and the AAV2 genome, expression of transgenes, receptors, and co-receptors by quantitative PCR. AAV2 vasoinhibin and sFlt-1 vectors inhibited the diabetes-mediated increase in BRBB when injected after, but not before, diabetes was induced. The AAV2 vasoinhibin vector decreased retinal microvascular abnormalities and the diabetes-induced reduction of the B-wave of the ERG, but it had no effect in non-diabetic controls. Also, retinal thickness was not altered by diabetes or by the AAV2 vasoinhibin vector. The AAV2 genome, vasoinhibin and sFlt-1 transgenes, and EGFP levels were higher in the retinas from diabetic rats and were associated with an elevated expression of AAV2 receptors (syndecan, glypican, and perlecan) and co-receptors (fibroblast growth factor receptor 1, αvβ5 integrin, and hepatocyte growth factor receptor). We conclude that retinal transduction and efficacy of AAV2 vectors are enhanced in diabetes, possibly due to their elevated cell entry. AAV2 vectors encoding vasoinhibin and sFlt-1 may be desirable gene therapeutics to target diabetic retinopathy and macular edema.

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“…Vascular parameters were measured using the AngioTool software (Zudaire et al, 2011). For each quantification, at least four fluorescent images/retina were taken from 4-5 mice.…”

Section: Quantification Of Retinal Vessel Density Migration and Lacumentioning

confidence: 99%

The matricellular protein CCN1 controls retinal angiogenesis by targeting VEGF, Src homology 2 domain phosphatase-1 and Notch signaling

et al. 2015

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Physiological angiogenesis depends on the highly coordinated actions of multiple angiogenic regulators. CCN1 is a secreted cysteine-rich and integrin-binding matricellular protein required for proper cardiovascular development. However, our understanding of the cellular origins and activities of this molecule is incomplete. Here, we show that CCN1 is predominantly expressed in angiogenic endothelial cells (ECs) at the leading front of actively growing vessels in the mouse retina. Endothelial deletion of CCN1 in mice using a Cre-Lox system is associated with EC hyperplasia, loss of pericyte coverage and formation of dense retinal vascular networks lacking the normal hierarchical arrangement of arterioles, capillaries and venules.

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A Computational Tool for Quantitative Analysis of Vascular Networks

Cited by 857 publications

References 30 publications

Phosphorylation Regulates FOXC2-Mediated Transcription in Lymphatic Endothelial Cells

Phosphorylation Regulates FOXC2-Mediated Transcription in Lymphatic Endothelial Cells

Diabetes enhances the efficacy of AAV2 vectors in the retina: therapeutic effect of AAV2 encoding vasoinhibin and soluble VEGF receptor 1

The matricellular protein CCN1 controls retinal angiogenesis by targeting VEGF, Src homology 2 domain phosphatase-1 and Notch signaling

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