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ZO-1 controls endothelial adherens junctions, cell-cell tension, angiogenesis, and barrier formation.

Tornavaca O, Chia M, Dufton N, Almagro LO, Conway DE, Randi AM, Schwartz MA, Matter K, Balda MS - J. Cell Biol. (2015)

Bottom Line: ZO-1 depletion led to tight junction disruption, redistribution of active myosin II from junctions to stress fibers, reduced tension on VE-cadherin and loss of junctional mechanotransducers such as vinculin and PAK2, and induced vinculin dissociation from the α-catenin-VE-cadherin complex.Claudin-5 depletion only mimicked ZO-1 effects on barrier formation, whereas the effects on mechanotransducers were rescued by inhibition of ROCK and phenocopied by JAM-A, JACOP, or p114RhoGEF down-regulation.ZO-1 was required for junctional recruitment of JACOP, which, in turn, recruited p114RhoGEF.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology, UCL Institute of Ophthalmology, University College London, London EC1V 9EL, England, UK.

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ZO-1 down-regulation reduces endothelial cell migration and angiogenic potential. (A) HDMEC were transfected with the indicated siRNAs for 48 h. Scratch wounds were then inflicted to induce cell migration. Representative images of wounds are shown that were taken at 0 and 16 h after scratching. (B) Percentages of the areas of closure of three independent experiments; shown are means ± 1 SD (error bars). (C–G) The effect of the depletion of ZO-1 on endothelial angiogenic potential was tested in vitro using an MC-based fibrin gel angiogenesis assay (C–E) or Matrigel plugs in vivo (F and G). For in vitro assays, HDMEC were seeded onto beads 24 h after siRNA transfection and were then embedded in a 3D fibrin gel after another 24 h. (C) Sprouting was then analyzed after 4 d by phase-contrast microscopy. (D) The cells were then fixed, permeabilized, and stained for nuclei (blue) and F-actin (red) to monitor coverage of beads with cells. (D and E) The number of sprouts per bead (D) and the mean length (E) were then determined (shown are means ± 1 SD [error bars] of four experiments). (F and G) For in vivo assays, mice were injected with Matrigel-containing FGF and siRNAs as indicted to induce angiogenesis. After 7 d, the plugs were harvested and fixed, embedded in paraffin, and sectioned. Sections in E were stained by hematoxylin and eosin, and were used to quantify the number of vessels shown in F (shown are means ± 1 SD [error bars]; control and FGF, n = 6; siRNA samples, n = 12). Bars, 250 µm.
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fig3: ZO-1 down-regulation reduces endothelial cell migration and angiogenic potential. (A) HDMEC were transfected with the indicated siRNAs for 48 h. Scratch wounds were then inflicted to induce cell migration. Representative images of wounds are shown that were taken at 0 and 16 h after scratching. (B) Percentages of the areas of closure of three independent experiments; shown are means ± 1 SD (error bars). (C–G) The effect of the depletion of ZO-1 on endothelial angiogenic potential was tested in vitro using an MC-based fibrin gel angiogenesis assay (C–E) or Matrigel plugs in vivo (F and G). For in vitro assays, HDMEC were seeded onto beads 24 h after siRNA transfection and were then embedded in a 3D fibrin gel after another 24 h. (C) Sprouting was then analyzed after 4 d by phase-contrast microscopy. (D) The cells were then fixed, permeabilized, and stained for nuclei (blue) and F-actin (red) to monitor coverage of beads with cells. (D and E) The number of sprouts per bead (D) and the mean length (E) were then determined (shown are means ± 1 SD [error bars] of four experiments). (F and G) For in vivo assays, mice were injected with Matrigel-containing FGF and siRNAs as indicted to induce angiogenesis. After 7 d, the plugs were harvested and fixed, embedded in paraffin, and sectioned. Sections in E were stained by hematoxylin and eosin, and were used to quantify the number of vessels shown in F (shown are means ± 1 SD [error bars]; control and FGF, n = 6; siRNA samples, n = 12). Bars, 250 µm.

Mentions: Angiogenesis requires complex actin reorganization and migration of EC to generate functional new vessels. Hence, we asked whether ZO-1 regulates the angiogenic potential of EC. We first used a migration assay to determine if ZO-1 affected collective cell migration. Fig. 3 (A and B) shows that cells lacking normal ZO-1 expression migrated less. An in vitro Matrigel tubulogenesis assay further revealed that ZO-1 depletion led to reduced network formation in vitro (Fig. S1, A and B). ZO-1 depletion was also found to reduce endothelial sprouting in a 3D microcarrier (MC)-based fibrin gel angiogenesis assay (Fig. 3, C–E). ZO-1 thus regulates the angiogenic potential of HDMEC primary cultures.


ZO-1 controls endothelial adherens junctions, cell-cell tension, angiogenesis, and barrier formation.

Tornavaca O, Chia M, Dufton N, Almagro LO, Conway DE, Randi AM, Schwartz MA, Matter K, Balda MS - J. Cell Biol. (2015)

ZO-1 down-regulation reduces endothelial cell migration and angiogenic potential. (A) HDMEC were transfected with the indicated siRNAs for 48 h. Scratch wounds were then inflicted to induce cell migration. Representative images of wounds are shown that were taken at 0 and 16 h after scratching. (B) Percentages of the areas of closure of three independent experiments; shown are means ± 1 SD (error bars). (C–G) The effect of the depletion of ZO-1 on endothelial angiogenic potential was tested in vitro using an MC-based fibrin gel angiogenesis assay (C–E) or Matrigel plugs in vivo (F and G). For in vitro assays, HDMEC were seeded onto beads 24 h after siRNA transfection and were then embedded in a 3D fibrin gel after another 24 h. (C) Sprouting was then analyzed after 4 d by phase-contrast microscopy. (D) The cells were then fixed, permeabilized, and stained for nuclei (blue) and F-actin (red) to monitor coverage of beads with cells. (D and E) The number of sprouts per bead (D) and the mean length (E) were then determined (shown are means ± 1 SD [error bars] of four experiments). (F and G) For in vivo assays, mice were injected with Matrigel-containing FGF and siRNAs as indicted to induce angiogenesis. After 7 d, the plugs were harvested and fixed, embedded in paraffin, and sectioned. Sections in E were stained by hematoxylin and eosin, and were used to quantify the number of vessels shown in F (shown are means ± 1 SD [error bars]; control and FGF, n = 6; siRNA samples, n = 12). Bars, 250 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4362456&req=5

fig3: ZO-1 down-regulation reduces endothelial cell migration and angiogenic potential. (A) HDMEC were transfected with the indicated siRNAs for 48 h. Scratch wounds were then inflicted to induce cell migration. Representative images of wounds are shown that were taken at 0 and 16 h after scratching. (B) Percentages of the areas of closure of three independent experiments; shown are means ± 1 SD (error bars). (C–G) The effect of the depletion of ZO-1 on endothelial angiogenic potential was tested in vitro using an MC-based fibrin gel angiogenesis assay (C–E) or Matrigel plugs in vivo (F and G). For in vitro assays, HDMEC were seeded onto beads 24 h after siRNA transfection and were then embedded in a 3D fibrin gel after another 24 h. (C) Sprouting was then analyzed after 4 d by phase-contrast microscopy. (D) The cells were then fixed, permeabilized, and stained for nuclei (blue) and F-actin (red) to monitor coverage of beads with cells. (D and E) The number of sprouts per bead (D) and the mean length (E) were then determined (shown are means ± 1 SD [error bars] of four experiments). (F and G) For in vivo assays, mice were injected with Matrigel-containing FGF and siRNAs as indicted to induce angiogenesis. After 7 d, the plugs were harvested and fixed, embedded in paraffin, and sectioned. Sections in E were stained by hematoxylin and eosin, and were used to quantify the number of vessels shown in F (shown are means ± 1 SD [error bars]; control and FGF, n = 6; siRNA samples, n = 12). Bars, 250 µm.
Mentions: Angiogenesis requires complex actin reorganization and migration of EC to generate functional new vessels. Hence, we asked whether ZO-1 regulates the angiogenic potential of EC. We first used a migration assay to determine if ZO-1 affected collective cell migration. Fig. 3 (A and B) shows that cells lacking normal ZO-1 expression migrated less. An in vitro Matrigel tubulogenesis assay further revealed that ZO-1 depletion led to reduced network formation in vitro (Fig. S1, A and B). ZO-1 depletion was also found to reduce endothelial sprouting in a 3D microcarrier (MC)-based fibrin gel angiogenesis assay (Fig. 3, C–E). ZO-1 thus regulates the angiogenic potential of HDMEC primary cultures.

Bottom Line: ZO-1 depletion led to tight junction disruption, redistribution of active myosin II from junctions to stress fibers, reduced tension on VE-cadherin and loss of junctional mechanotransducers such as vinculin and PAK2, and induced vinculin dissociation from the α-catenin-VE-cadherin complex.Claudin-5 depletion only mimicked ZO-1 effects on barrier formation, whereas the effects on mechanotransducers were rescued by inhibition of ROCK and phenocopied by JAM-A, JACOP, or p114RhoGEF down-regulation.ZO-1 was required for junctional recruitment of JACOP, which, in turn, recruited p114RhoGEF.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology, UCL Institute of Ophthalmology, University College London, London EC1V 9EL, England, UK.

Show MeSH
Related in: MedlinePlus