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Dynamic endothelial cell rearrangements drive developmental vessel regression.

Franco CA, Jones ML, Bernabeu MO, Geudens I, Mathivet T, Rosa A, Lopes FM, Lima AP, Ragab A, Collins RT, Phng LK, Coveney PV, Gerhardt H - PLoS Biol. (2015)

Bottom Line: Patterning of functional blood vessel networks is achieved by pruning of superfluous connections.The cellular and molecular principles of vessel regression are poorly understood.Establishing and analyzing the first axial polarity map of all endothelial cells in a remodeling vascular network, we propose that balanced movement of cells maintains the primitive plexus under low shear conditions in a metastable dynamic state.

View Article: PubMed Central - PubMed

Affiliation: Vascular Biology Laboratory, London Research Institute-Cancer Research UK, Lincoln's Inn Laboratories, London, United Kingdom; Instituto de Medicina Molecular, Faculdade de Medicina Universidade de Lisboa, Lisboa, Portugal.

ABSTRACT
Patterning of functional blood vessel networks is achieved by pruning of superfluous connections. The cellular and molecular principles of vessel regression are poorly understood. Here we show that regression is mediated by dynamic and polarized migration of endothelial cells, representing anastomosis in reverse. Establishing and analyzing the first axial polarity map of all endothelial cells in a remodeling vascular network, we propose that balanced movement of cells maintains the primitive plexus under low shear conditions in a metastable dynamic state. We predict that flow-induced polarized migration of endothelial cells breaks symmetry and leads to stabilization of high flow/shear segments and regression of adjacent low flow/shear segments.

No MeSH data available.


Related in: MedlinePlus

Developmental vessel regression resembles anastomosis in reverse.A–C, Immunostaining for lumen (ICAM2), junctions (ZO1 and Cdh5), blood vessels (IB4), and basement membrane (Col.IV) shows that lumen breakage (arrows in A) and junction disconnection (arrows in B and C) is an early step in vessel regression. D and E, Single-cell labeling using Cre-induced expression of membrane-bound GFP (mGFP) shows polarized morphology of activated endothelial cells with filopodia projections (yellow arrows in in D and E). Endothelial cells (green dotted-lines) bridge two or more vessel segments in the regressing vessel, showing rings or points of junctional connection (blue arrows in in D and E). Scale bars (A–E: 10 μm).
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pbio.1002125.g002: Developmental vessel regression resembles anastomosis in reverse.A–C, Immunostaining for lumen (ICAM2), junctions (ZO1 and Cdh5), blood vessels (IB4), and basement membrane (Col.IV) shows that lumen breakage (arrows in A) and junction disconnection (arrows in B and C) is an early step in vessel regression. D and E, Single-cell labeling using Cre-induced expression of membrane-bound GFP (mGFP) shows polarized morphology of activated endothelial cells with filopodia projections (yellow arrows in in D and E). Endothelial cells (green dotted-lines) bridge two or more vessel segments in the regressing vessel, showing rings or points of junctional connection (blue arrows in in D and E). Scale bars (A–E: 10 μm).

Mentions: Analyzing endothelial cell configurations in regressing vessels by co-staining for intercellular adhesion molecule 2 (ICAM2) (P35330) (marking the apical/luminal endothelial cell membrane [11]), Col.IV, and isolectin B4 (IB4), we identified disrupted lumen as the first visible sign of vessel regression (Fig 2A). It was demonstrated in zebrafish that vessel regression in the brain vasculature was influenced by vessel perfusion [6]. Indeed, following perfusion of rhodamin-conjugated concanavilin-A in mouse pups, we observed that lumen disconnections were preferentially observed in rhodamin-negative vessel segments (S2A Fig). Co-labeling with vascular endothelial (VE)-cadherin (P55284) or zona occludens protein 1 (ZO1) (P39447) illustrated that the usual continuous junctions lining stable vessels as parallel lines are disrupted in branches with interrupted lumen. Instead, the junctions form isolated ring structures, often surrounding a patch of apical endothelial membrane without contact to the lumen in neighboring vessels (Fig 2B and 2C; S1 and S2 Movies). Such junctional arrangements surrounding apical membrane patches have been previously reported in early stages of lumen formation during anastomosis in the dorsolateral anastomotic vessel (DLAV) of zebrafish embryos [12]. At regression sites, they are additionally surrounded by a continuous Col.IV basement membrane (Fig 2B), suggesting that this configuration of lumen and cell junctions represents an intermediate step common to both anastomosis and regression. Indeed, by mosaic single-cell labeling using low-dose tamoxifen-induced Cre-mediated activation of membrane enhanced green fluorescent protein (eGFP) expression, we observed that endothelial cells in regressing branches extend numerous filopodia, similar to fusing endothelial tip cells, representative of activated endothelium (Fig 2D and 2E and S2 Movie). This suggests that endothelial cells could be actively migrating within the remodeling vascular plexus, and not only at the vascular sprouting front. In agreement, looking at the patterns of endothelial cell distribution in chimeric mouse retinas, we observed a lack of cohesive clonal expansion of proliferating endothelial cells (S2B Fig) [13]. These observations, although derived from static images in the retina, are nevertheless consistent with the idea that rearrangements of endothelial cells contribute to remodeling and possibly drive regression.


Dynamic endothelial cell rearrangements drive developmental vessel regression.

Franco CA, Jones ML, Bernabeu MO, Geudens I, Mathivet T, Rosa A, Lopes FM, Lima AP, Ragab A, Collins RT, Phng LK, Coveney PV, Gerhardt H - PLoS Biol. (2015)

Developmental vessel regression resembles anastomosis in reverse.A–C, Immunostaining for lumen (ICAM2), junctions (ZO1 and Cdh5), blood vessels (IB4), and basement membrane (Col.IV) shows that lumen breakage (arrows in A) and junction disconnection (arrows in B and C) is an early step in vessel regression. D and E, Single-cell labeling using Cre-induced expression of membrane-bound GFP (mGFP) shows polarized morphology of activated endothelial cells with filopodia projections (yellow arrows in in D and E). Endothelial cells (green dotted-lines) bridge two or more vessel segments in the regressing vessel, showing rings or points of junctional connection (blue arrows in in D and E). Scale bars (A–E: 10 μm).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4401640&req=5

pbio.1002125.g002: Developmental vessel regression resembles anastomosis in reverse.A–C, Immunostaining for lumen (ICAM2), junctions (ZO1 and Cdh5), blood vessels (IB4), and basement membrane (Col.IV) shows that lumen breakage (arrows in A) and junction disconnection (arrows in B and C) is an early step in vessel regression. D and E, Single-cell labeling using Cre-induced expression of membrane-bound GFP (mGFP) shows polarized morphology of activated endothelial cells with filopodia projections (yellow arrows in in D and E). Endothelial cells (green dotted-lines) bridge two or more vessel segments in the regressing vessel, showing rings or points of junctional connection (blue arrows in in D and E). Scale bars (A–E: 10 μm).
Mentions: Analyzing endothelial cell configurations in regressing vessels by co-staining for intercellular adhesion molecule 2 (ICAM2) (P35330) (marking the apical/luminal endothelial cell membrane [11]), Col.IV, and isolectin B4 (IB4), we identified disrupted lumen as the first visible sign of vessel regression (Fig 2A). It was demonstrated in zebrafish that vessel regression in the brain vasculature was influenced by vessel perfusion [6]. Indeed, following perfusion of rhodamin-conjugated concanavilin-A in mouse pups, we observed that lumen disconnections were preferentially observed in rhodamin-negative vessel segments (S2A Fig). Co-labeling with vascular endothelial (VE)-cadherin (P55284) or zona occludens protein 1 (ZO1) (P39447) illustrated that the usual continuous junctions lining stable vessels as parallel lines are disrupted in branches with interrupted lumen. Instead, the junctions form isolated ring structures, often surrounding a patch of apical endothelial membrane without contact to the lumen in neighboring vessels (Fig 2B and 2C; S1 and S2 Movies). Such junctional arrangements surrounding apical membrane patches have been previously reported in early stages of lumen formation during anastomosis in the dorsolateral anastomotic vessel (DLAV) of zebrafish embryos [12]. At regression sites, they are additionally surrounded by a continuous Col.IV basement membrane (Fig 2B), suggesting that this configuration of lumen and cell junctions represents an intermediate step common to both anastomosis and regression. Indeed, by mosaic single-cell labeling using low-dose tamoxifen-induced Cre-mediated activation of membrane enhanced green fluorescent protein (eGFP) expression, we observed that endothelial cells in regressing branches extend numerous filopodia, similar to fusing endothelial tip cells, representative of activated endothelium (Fig 2D and 2E and S2 Movie). This suggests that endothelial cells could be actively migrating within the remodeling vascular plexus, and not only at the vascular sprouting front. In agreement, looking at the patterns of endothelial cell distribution in chimeric mouse retinas, we observed a lack of cohesive clonal expansion of proliferating endothelial cells (S2B Fig) [13]. These observations, although derived from static images in the retina, are nevertheless consistent with the idea that rearrangements of endothelial cells contribute to remodeling and possibly drive regression.

Bottom Line: Patterning of functional blood vessel networks is achieved by pruning of superfluous connections.The cellular and molecular principles of vessel regression are poorly understood.Establishing and analyzing the first axial polarity map of all endothelial cells in a remodeling vascular network, we propose that balanced movement of cells maintains the primitive plexus under low shear conditions in a metastable dynamic state.

View Article: PubMed Central - PubMed

Affiliation: Vascular Biology Laboratory, London Research Institute-Cancer Research UK, Lincoln's Inn Laboratories, London, United Kingdom; Instituto de Medicina Molecular, Faculdade de Medicina Universidade de Lisboa, Lisboa, Portugal.

ABSTRACT
Patterning of functional blood vessel networks is achieved by pruning of superfluous connections. The cellular and molecular principles of vessel regression are poorly understood. Here we show that regression is mediated by dynamic and polarized migration of endothelial cells, representing anastomosis in reverse. Establishing and analyzing the first axial polarity map of all endothelial cells in a remodeling vascular network, we propose that balanced movement of cells maintains the primitive plexus under low shear conditions in a metastable dynamic state. We predict that flow-induced polarized migration of endothelial cells breaks symmetry and leads to stabilization of high flow/shear segments and regression of adjacent low flow/shear segments.

No MeSH data available.


Related in: MedlinePlus