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Amino-terminal fragments of laminin γ2 chain retract vascular endothelial cells and increase vascular permeability.

Sato H, Oyanagi J, Komiya E, Ogawa T, Higashi S, Miyazaki K - Cancer Sci. (2014)

Bottom Line: Such effects of γ2pf led to prominent actin stress fiber formation in HUVECs, which was blocked by a ROCK inhibitor.As possible receptors, γ2pf interacted with heparan sulfate proteoglycans on the surface of HUVECs.Moreover, we localized the active site of γ2pf to the N-terminal epidermal growth factor-like repeat.

View Article: PubMed Central - PubMed

Affiliation: Department of Genome Science, Graduate School of Integrated Science and Nanobioscience, Yokohama City University, Yokohama, Japan; Division of Cell Biology, Kihara Institute for Biological Research, Yokohama City University, Yokohama, Japan.

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Vascular permeability-inducing activity of laminin γ2 chain in vivo. Miles assay was carried out by injecting 5 μg each of γ2 proteolytic fragment (γ2pf) (a), γ2 N-terminal domain V (γ2dV) (b), or domain III (γ2dIII) (c) into the back skin of BALB/c mice. Each mouse was injected with PBS (Control) and a test sample on the left and right sides of the skin, respectively. Evans blue dye leaked from blood vessels was extracted and quantified. Lower panels show representative examples from the respective experiments. Similar results were reproduced in two additional experiments. Closed circles indicate the mean ± SD (n = 6); *P < 0.05. N.S., not significant.
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fig05: Vascular permeability-inducing activity of laminin γ2 chain in vivo. Miles assay was carried out by injecting 5 μg each of γ2 proteolytic fragment (γ2pf) (a), γ2 N-terminal domain V (γ2dV) (b), or domain III (γ2dIII) (c) into the back skin of BALB/c mice. Each mouse was injected with PBS (Control) and a test sample on the left and right sides of the skin, respectively. Evans blue dye leaked from blood vessels was extracted and quantified. Lower panels show representative examples from the respective experiments. Similar results were reproduced in two additional experiments. Closed circles indicate the mean ± SD (n = 6); *P < 0.05. N.S., not significant.

Mentions: The γ2pf-induced retraction of endothelial cells seemed to lead to their loss of barrier integrity. To confirm this possibility, we examined the activity of γ2pf on endothelial permeability in vitro (Fig. 4a). When the monolayers of HUVECs on the culture inserts were treated with purified γ2pf, the endothelial cell permeability, as measured by the diffusion of FITC-dextran through the HUVEC sheet, significantly increased compared to the untreated control cultures. In addition, enhanced permeability was observed with the full-length γ2 chain and its N-terminal domain V (γ2dV) (Fig. 4b, see also Fig. 1a). The order of the permeability activity was γ2dV > γ2pf > full-length γ2. Furthermore, we examined the effect of Lmγ2 on vascular permeability in vivo by Miles permeability assay with mice (Fig. 5). The intradermal injection of γ2pf increased the leakage of Evans blue dye two-fold compared to the PBS injection as control (Fig. 5a). Purified γ2dV also increased vascular permeability two-fold (Fig. 5b), but domain III of Lmγ2 did not show any significant activity (Figs 5c, S2, see also Fig. 1a). These results suggest that the N-terminal fragments of Lmγ2 chain function as vascular permeability-promoting factors in pathological conditions.


Amino-terminal fragments of laminin γ2 chain retract vascular endothelial cells and increase vascular permeability.

Sato H, Oyanagi J, Komiya E, Ogawa T, Higashi S, Miyazaki K - Cancer Sci. (2014)

Vascular permeability-inducing activity of laminin γ2 chain in vivo. Miles assay was carried out by injecting 5 μg each of γ2 proteolytic fragment (γ2pf) (a), γ2 N-terminal domain V (γ2dV) (b), or domain III (γ2dIII) (c) into the back skin of BALB/c mice. Each mouse was injected with PBS (Control) and a test sample on the left and right sides of the skin, respectively. Evans blue dye leaked from blood vessels was extracted and quantified. Lower panels show representative examples from the respective experiments. Similar results were reproduced in two additional experiments. Closed circles indicate the mean ± SD (n = 6); *P < 0.05. N.S., not significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4317827&req=5

fig05: Vascular permeability-inducing activity of laminin γ2 chain in vivo. Miles assay was carried out by injecting 5 μg each of γ2 proteolytic fragment (γ2pf) (a), γ2 N-terminal domain V (γ2dV) (b), or domain III (γ2dIII) (c) into the back skin of BALB/c mice. Each mouse was injected with PBS (Control) and a test sample on the left and right sides of the skin, respectively. Evans blue dye leaked from blood vessels was extracted and quantified. Lower panels show representative examples from the respective experiments. Similar results were reproduced in two additional experiments. Closed circles indicate the mean ± SD (n = 6); *P < 0.05. N.S., not significant.
Mentions: The γ2pf-induced retraction of endothelial cells seemed to lead to their loss of barrier integrity. To confirm this possibility, we examined the activity of γ2pf on endothelial permeability in vitro (Fig. 4a). When the monolayers of HUVECs on the culture inserts were treated with purified γ2pf, the endothelial cell permeability, as measured by the diffusion of FITC-dextran through the HUVEC sheet, significantly increased compared to the untreated control cultures. In addition, enhanced permeability was observed with the full-length γ2 chain and its N-terminal domain V (γ2dV) (Fig. 4b, see also Fig. 1a). The order of the permeability activity was γ2dV > γ2pf > full-length γ2. Furthermore, we examined the effect of Lmγ2 on vascular permeability in vivo by Miles permeability assay with mice (Fig. 5). The intradermal injection of γ2pf increased the leakage of Evans blue dye two-fold compared to the PBS injection as control (Fig. 5a). Purified γ2dV also increased vascular permeability two-fold (Fig. 5b), but domain III of Lmγ2 did not show any significant activity (Figs 5c, S2, see also Fig. 1a). These results suggest that the N-terminal fragments of Lmγ2 chain function as vascular permeability-promoting factors in pathological conditions.

Bottom Line: Such effects of γ2pf led to prominent actin stress fiber formation in HUVECs, which was blocked by a ROCK inhibitor.As possible receptors, γ2pf interacted with heparan sulfate proteoglycans on the surface of HUVECs.Moreover, we localized the active site of γ2pf to the N-terminal epidermal growth factor-like repeat.

View Article: PubMed Central - PubMed

Affiliation: Department of Genome Science, Graduate School of Integrated Science and Nanobioscience, Yokohama City University, Yokohama, Japan; Division of Cell Biology, Kihara Institute for Biological Research, Yokohama City University, Yokohama, Japan.

Show MeSH
Related in: MedlinePlus