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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

Coordinated polarity induced by high flow triggers vessel regression.A, Overview of the axial polarization pattern of endothelial cells in a WT P6 retina vascular network labeled for endothelial cell nuclei (Erg), lumen (ICAM2), and Golgi (Golph4), and corresponding image segmentation of the vascular plexus in (a), highlighting the lumen of blood vessels (grey), and the axial polarity of all endothelial cells (red arrows). B, Analysis of the endothelial axial polarity angle in the main vessels, correlated to predicted blood flow direction by the rheology in silico model. Endothelial cells robustly position their Golgi apparatus against the blood flow in all vascular regions analyzed. C, Quantitative analysis of the percentage of endothelial cells polarized at 180°(±45°) compared to the flow direction in the different vascular beds (n = 3 retinas). D, Quantitative analysis of cell density, mean wall shear stress and branching point density in P6 mouse retina vascular plexus (n = 3 retinas). E,S6 Movie still images from time-lapse live imaging of a Tg(fli1a:eGFP) zebrafish embryo (grey) injected with pTol2:fli1ep:mCherry-GM130 (green). F, Quantification of endothelial cell axial polarity in ISVs showing dorsal axial polarization during the sprouting phase (not lumenized). In stable ISVs, endothelial shows significantly enriched dorsal or ventral axial polarity when in venous or arterial ISVs, respectively, corresponding to polarization against the predicted blood flow direction. G, Representative images of axial polarity and color-coded representation of the rheology prediction for velocity and wall shear stress in the corresponding vessel segments. Axial polarity length correlates with higher levels of luminal shear stress. In low shear vessels endothelial cells show decreased polarization and tend to point towards high flow vessel segments (black arrows). Scale bars (A: 50 μm; E: 20 μm). The data used to make this figure can be found in S1 Data.
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pbio.1002125.g005: Coordinated polarity induced by high flow triggers vessel regression.A, Overview of the axial polarization pattern of endothelial cells in a WT P6 retina vascular network labeled for endothelial cell nuclei (Erg), lumen (ICAM2), and Golgi (Golph4), and corresponding image segmentation of the vascular plexus in (a), highlighting the lumen of blood vessels (grey), and the axial polarity of all endothelial cells (red arrows). B, Analysis of the endothelial axial polarity angle in the main vessels, correlated to predicted blood flow direction by the rheology in silico model. Endothelial cells robustly position their Golgi apparatus against the blood flow in all vascular regions analyzed. C, Quantitative analysis of the percentage of endothelial cells polarized at 180°(±45°) compared to the flow direction in the different vascular beds (n = 3 retinas). D, Quantitative analysis of cell density, mean wall shear stress and branching point density in P6 mouse retina vascular plexus (n = 3 retinas). E,S6 Movie still images from time-lapse live imaging of a Tg(fli1a:eGFP) zebrafish embryo (grey) injected with pTol2:fli1ep:mCherry-GM130 (green). F, Quantification of endothelial cell axial polarity in ISVs showing dorsal axial polarization during the sprouting phase (not lumenized). In stable ISVs, endothelial shows significantly enriched dorsal or ventral axial polarity when in venous or arterial ISVs, respectively, corresponding to polarization against the predicted blood flow direction. G, Representative images of axial polarity and color-coded representation of the rheology prediction for velocity and wall shear stress in the corresponding vessel segments. Axial polarity length correlates with higher levels of luminal shear stress. In low shear vessels endothelial cells show decreased polarization and tend to point towards high flow vessel segments (black arrows). Scale bars (A: 50 μm; E: 20 μm). The data used to make this figure can be found in S1 Data.

Mentions: Stimulated by our analysis of the axial endothelial polarity in vessel regression and the importance of haemodynamics in regulating blood vessel pruning in the zebrafish brain [6], we took advantage of the newly developed approach for the computation of haemodynamic forces in mouse retinal vascular networks [16] to investigate the correlation between endothelial axial polarity and blood flow patterns in the mouse retina (Figs 5A and S5). Interestingly, in the retinal plexus, axial polarity vectors were largest in high flow vessels, such as arteries and arterioles, with very little variance and vectors pointing exclusively against the direction of blood flow (Figs 5B, 5C, and S6). Also in veins, polarity was generally directed against the flow, but the vectors were, in general, smaller (Figs 5B, 5C, and S6). Surprisingly, even closer to the retinal sprouting front and distant from the feeding arteries, where flow and shear levels are predicted to be low through our simulations, endothelial axial polarity is still significantly directed against flow (Fig 5A–5C, S1 Data, and S6 Fig). Linear regression analysis identified a strong correlation between increasing wall shear stress and polarization (analyzed as scalar product of polarity and shear vectors, S6 Fig and S1 Data). In order to better understand the relationship between flow-induced shear and vascular parameters across the retina, we performed unbiased combinatorial quantitative analysis of wall shear stress, cell density, and branchpoint density as a function of the distance from the optic nerve (Fig 5D, S1 Data). With increasing distance from the optic nerve, branchpoint density increased, reflecting the transition from the more remodeled to the unremodeled plexus. In parallel, the wall shear stress levels decreased with distance from the optic nerve. However, surprisingly, endothelial cell density, measured as nuclei/μm2, was highest closest to the optic nerve and decreased towards the periphery. Thus, although the increase in branch points signifies a more ramified vascular plexus in the developing periphery, endothelial cell density is lower in this region. Conversely, the highest wall shear stress found in the most central and remodeled area correlated with higher cell density (Fig 5D, S1 Data), indicating that the transition from primitive to remodeled plexus is not driven by cell loss. Given that minimal proliferation is detected in these more central areas (Fig 1E), the increased density of endothelial cells closer to the optic nerve independently argues against endothelial cell apoptosis as a driver of vascular remodeling. Instead it would be consistent with cells incorporating into higher flow segments as low-flow segments regress. To directly observe endothelial axial Golgi-to-nucleus polarity under the influence of blood flow, we analyzed transgenic zebrafish embryos expressing transiently mCherry-GM130, a Golgi-specific protein, during the process of lumenization and blood flow onset in intersegmental vessels. Endothelial cells in ISVs without continuous lumen showed variable axial polarities, with cells positioning their Golgi apparatus in the direction of migration, occasionally directed towards the aorta, however, mostly directed towards the DLAV (Fig 5E and 5F and S6 Movie). Interestingly, as ISVs formed a continuous lumen and flow is established, endothelial cells redirected their axial polarity towards the aorta and against the blood flow direction. Thus endothelial cells respond dynamically to the onset of flow and rapidly redirect their Golgi against the direction of flow (Fig 5E and S6 Movie). Quantification of axial polarization in ISVs demonstrates that during the sprouting phase arterially connected endothelial cells show dorsal polarization, which is reversed in mature arterial ISVs, to point against the flow direction (Fig 5F, S1 Data). In stable venous ISVs, axial polarity points dorsally, i.e., against the flow direction (Fig 5F, S1 Data).


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)

Coordinated polarity induced by high flow triggers vessel regression.A, Overview of the axial polarization pattern of endothelial cells in a WT P6 retina vascular network labeled for endothelial cell nuclei (Erg), lumen (ICAM2), and Golgi (Golph4), and corresponding image segmentation of the vascular plexus in (a), highlighting the lumen of blood vessels (grey), and the axial polarity of all endothelial cells (red arrows). B, Analysis of the endothelial axial polarity angle in the main vessels, correlated to predicted blood flow direction by the rheology in silico model. Endothelial cells robustly position their Golgi apparatus against the blood flow in all vascular regions analyzed. C, Quantitative analysis of the percentage of endothelial cells polarized at 180°(±45°) compared to the flow direction in the different vascular beds (n = 3 retinas). D, Quantitative analysis of cell density, mean wall shear stress and branching point density in P6 mouse retina vascular plexus (n = 3 retinas). E,S6 Movie still images from time-lapse live imaging of a Tg(fli1a:eGFP) zebrafish embryo (grey) injected with pTol2:fli1ep:mCherry-GM130 (green). F, Quantification of endothelial cell axial polarity in ISVs showing dorsal axial polarization during the sprouting phase (not lumenized). In stable ISVs, endothelial shows significantly enriched dorsal or ventral axial polarity when in venous or arterial ISVs, respectively, corresponding to polarization against the predicted blood flow direction. G, Representative images of axial polarity and color-coded representation of the rheology prediction for velocity and wall shear stress in the corresponding vessel segments. Axial polarity length correlates with higher levels of luminal shear stress. In low shear vessels endothelial cells show decreased polarization and tend to point towards high flow vessel segments (black arrows). Scale bars (A: 50 μm; E: 20 μm). The data used to make this figure can be found in S1 Data.
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pbio.1002125.g005: Coordinated polarity induced by high flow triggers vessel regression.A, Overview of the axial polarization pattern of endothelial cells in a WT P6 retina vascular network labeled for endothelial cell nuclei (Erg), lumen (ICAM2), and Golgi (Golph4), and corresponding image segmentation of the vascular plexus in (a), highlighting the lumen of blood vessels (grey), and the axial polarity of all endothelial cells (red arrows). B, Analysis of the endothelial axial polarity angle in the main vessels, correlated to predicted blood flow direction by the rheology in silico model. Endothelial cells robustly position their Golgi apparatus against the blood flow in all vascular regions analyzed. C, Quantitative analysis of the percentage of endothelial cells polarized at 180°(±45°) compared to the flow direction in the different vascular beds (n = 3 retinas). D, Quantitative analysis of cell density, mean wall shear stress and branching point density in P6 mouse retina vascular plexus (n = 3 retinas). E,S6 Movie still images from time-lapse live imaging of a Tg(fli1a:eGFP) zebrafish embryo (grey) injected with pTol2:fli1ep:mCherry-GM130 (green). F, Quantification of endothelial cell axial polarity in ISVs showing dorsal axial polarization during the sprouting phase (not lumenized). In stable ISVs, endothelial shows significantly enriched dorsal or ventral axial polarity when in venous or arterial ISVs, respectively, corresponding to polarization against the predicted blood flow direction. G, Representative images of axial polarity and color-coded representation of the rheology prediction for velocity and wall shear stress in the corresponding vessel segments. Axial polarity length correlates with higher levels of luminal shear stress. In low shear vessels endothelial cells show decreased polarization and tend to point towards high flow vessel segments (black arrows). Scale bars (A: 50 μm; E: 20 μm). The data used to make this figure can be found in S1 Data.
Mentions: Stimulated by our analysis of the axial endothelial polarity in vessel regression and the importance of haemodynamics in regulating blood vessel pruning in the zebrafish brain [6], we took advantage of the newly developed approach for the computation of haemodynamic forces in mouse retinal vascular networks [16] to investigate the correlation between endothelial axial polarity and blood flow patterns in the mouse retina (Figs 5A and S5). Interestingly, in the retinal plexus, axial polarity vectors were largest in high flow vessels, such as arteries and arterioles, with very little variance and vectors pointing exclusively against the direction of blood flow (Figs 5B, 5C, and S6). Also in veins, polarity was generally directed against the flow, but the vectors were, in general, smaller (Figs 5B, 5C, and S6). Surprisingly, even closer to the retinal sprouting front and distant from the feeding arteries, where flow and shear levels are predicted to be low through our simulations, endothelial axial polarity is still significantly directed against flow (Fig 5A–5C, S1 Data, and S6 Fig). Linear regression analysis identified a strong correlation between increasing wall shear stress and polarization (analyzed as scalar product of polarity and shear vectors, S6 Fig and S1 Data). In order to better understand the relationship between flow-induced shear and vascular parameters across the retina, we performed unbiased combinatorial quantitative analysis of wall shear stress, cell density, and branchpoint density as a function of the distance from the optic nerve (Fig 5D, S1 Data). With increasing distance from the optic nerve, branchpoint density increased, reflecting the transition from the more remodeled to the unremodeled plexus. In parallel, the wall shear stress levels decreased with distance from the optic nerve. However, surprisingly, endothelial cell density, measured as nuclei/μm2, was highest closest to the optic nerve and decreased towards the periphery. Thus, although the increase in branch points signifies a more ramified vascular plexus in the developing periphery, endothelial cell density is lower in this region. Conversely, the highest wall shear stress found in the most central and remodeled area correlated with higher cell density (Fig 5D, S1 Data), indicating that the transition from primitive to remodeled plexus is not driven by cell loss. Given that minimal proliferation is detected in these more central areas (Fig 1E), the increased density of endothelial cells closer to the optic nerve independently argues against endothelial cell apoptosis as a driver of vascular remodeling. Instead it would be consistent with cells incorporating into higher flow segments as low-flow segments regress. To directly observe endothelial axial Golgi-to-nucleus polarity under the influence of blood flow, we analyzed transgenic zebrafish embryos expressing transiently mCherry-GM130, a Golgi-specific protein, during the process of lumenization and blood flow onset in intersegmental vessels. Endothelial cells in ISVs without continuous lumen showed variable axial polarities, with cells positioning their Golgi apparatus in the direction of migration, occasionally directed towards the aorta, however, mostly directed towards the DLAV (Fig 5E and 5F and S6 Movie). Interestingly, as ISVs formed a continuous lumen and flow is established, endothelial cells redirected their axial polarity towards the aorta and against the blood flow direction. Thus endothelial cells respond dynamically to the onset of flow and rapidly redirect their Golgi against the direction of flow (Fig 5E and S6 Movie). Quantification of axial polarization in ISVs demonstrates that during the sprouting phase arterially connected endothelial cells show dorsal polarization, which is reversed in mature arterial ISVs, to point against the flow direction (Fig 5F, S1 Data). In stable venous ISVs, axial polarity points dorsally, i.e., against the flow direction (Fig 5F, S1 Data).

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