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Increased shear stress inhibits angiogenesis in veins and not arteries during vascular development

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Abstract

Vascular development is believed to occur first by vasculogenesis followed by angiogenesis. Though angiogenesis is the formation of new vessels, we found that vascular density actually decreases during this second stage. The onset of the decrease coincided with the entry of erythroblasts into circulation. We therefore measured the level of shear stress at various developmental stages and found that it was inversely proportional to vascular density. To investigate whether shear stress was inhibitory to angiogenesis, we altered shear stress levels either by preventing erythroblasts from entering circulation (“low” shear stress) or by injection of a starch solution to increase the blood plasma viscosity (“high” shear stress). By time-lapse microscopy, we show that reverse intussusception (merging of two vessels) is inversely proportional to the level of shear stress. We also found that angiogenesis (both sprouting and splitting) was inversely proportional to shear stress levels. These effects were specific to the arterial or venous plexus however, such that the effect on reverse intussusception was present only in the arterial plexus and the effect on sprouting only in the venous plexus. We cultured embryos under altered shear stress in the presence of either DAPT, a Notch inhibitor, or DMH1, an inhibitor of the bone morphogenetic protein (BMP) pathway. DAPT treatment phenocopied the inhibition of erythroblast circulation (“low” shear stress) and the effect of DAPT treatment could be partially rescued by injection of starch. Inhibition of the BMP signaling prevented the reduction in vascular density that was observed when starch was injected to increase shear stress levels.

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Abbreviations

AF488:

Alexa-Fluor 488

AcLDL:

Acetylated low density lipoprotein

Alk2/3:

Activin receptor-like kinase 2/3

BMP:

Bone morphogenetic protein

CAM:

Chorio-allantoic membrane

DAPT:

Notch inhibitor

N-[N-(3,5-Difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester

DMEM/F-12:

Dulbecco’s modified eagle medium/nutrient mixture F-12

DMH1:

R-Smad inhibitor

4-[6-(4-Isopropoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline, 4-[6-[4-(1-Methylethoxy)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline

HEPES:

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

HUAEC:

Human umbilical artery endothelial cell

HUVEC:

Human umbilical vein endothelial cell

μPIV:

Micro particle image velocimetry

VEGF:

Vascular endothelial growth factor

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Acknowledgments

This work was supported by grants from the Natural Science and Engineering Research Council’s Discovery Program (342134), the Fonds de recherche du Québec—Nature et technologies’ Nouveau Chercheur Program (131373) and the Canadian Foundation for Innovation. The Eugenie Lamothe Fellowship supported GCP and EDJ.

Author information

Correspondence to Elizabeth A. V. Jones.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Movie 1 – Time-lapse of Vascular Remodeling in Avian Embryos. Chicken embryos were followed by ex ovo time-lapse microscopy. Eggs were incubated until the embryo reached 12–14 somites. Embryos were then injected with AF488-AcLDL, which labels endothelial cells and macrophages. The embryos were transferred to a petri dish in which a Teflon covered window was made into the lid (see Methods). Embryos were transferred to a heated fluorescent microscope and an image was taken every 6 minutes for 10 hours using a 5x objective lens. The movies are played at 8 frames per second. The arrows highlight the presence of sprouting angiogenesis (red arrow) and reverse intussusception (yellow arrow). Scale bar: 200 μm. (AVI 11790 kb)

Movie 2 – Time-lapse of Vascular Remodeling Under Low Shear Stress in Avian Embryos. Eggs were incubated until the embryo reached 12–14 somites and then injected with AF488-AcLDL, which labels endothelial cells and macrophages. The erythroblasts at the caudal end of the yolk sac were prevented from entering circulation by injection of Acrylimide/APS followed by injection of TEMED (See Methods). This reduced the effective hematocrit of the blood flow. The embryos were then transferred to a petri dish in which a Teflon covered window was made into the lid (see Methods). An image was taken every 10 minutes for 10 hours using a 10x objective lens. The movies are played at 8 frames per second. Under low shear stress, significant amounts of reverse intussusception are present (white, yellow and red arrows). Scale bar: 100 μm. (AVI 3516 kb)

Movie 1 – Time-lapse of Vascular Remodeling in Avian Embryos. Chicken embryos were followed by ex ovo time-lapse microscopy. Eggs were incubated until the embryo reached 12–14 somites. Embryos were then injected with AF488-AcLDL, which labels endothelial cells and macrophages. The embryos were transferred to a petri dish in which a Teflon covered window was made into the lid (see Methods). Embryos were transferred to a heated fluorescent microscope and an image was taken every 6 minutes for 10 hours using a 5x objective lens. The movies are played at 8 frames per second. The arrows highlight the presence of sprouting angiogenesis (red arrow) and reverse intussusception (yellow arrow). Scale bar: 200 μm. (AVI 11790 kb)

Movie 2 – Time-lapse of Vascular Remodeling Under Low Shear Stress in Avian Embryos. Eggs were incubated until the embryo reached 12–14 somites and then injected with AF488-AcLDL, which labels endothelial cells and macrophages. The erythroblasts at the caudal end of the yolk sac were prevented from entering circulation by injection of Acrylimide/APS followed by injection of TEMED (See Methods). This reduced the effective hematocrit of the blood flow. The embryos were then transferred to a petri dish in which a Teflon covered window was made into the lid (see Methods). An image was taken every 10 minutes for 10 hours using a 10x objective lens. The movies are played at 8 frames per second. Under low shear stress, significant amounts of reverse intussusception are present (white, yellow and red arrows). Scale bar: 100 μm. (AVI 3516 kb)

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Chouinard-Pelletier, G., Jahnsen, E.D. & Jones, E.A.V. Increased shear stress inhibits angiogenesis in veins and not arteries during vascular development. Angiogenesis 16, 71–83 (2013). https://doi.org/10.1007/s10456-012-9300-2

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Keywords

  • Vascular development
  • Angiogenesis
  • Shear stress
  • Vascular remodeling
  • Particle image velocimetry