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N-cadherin acts upstream of VE-cadherin in controlling vascular morphogenesis.

Luo Y, Radice GL - J. Cell Biol. (2005)

Bottom Line: Contrary to previous studies, we found that N-cadherin localizes to endothelial cell-cell junctions in addition to its well-known diffusive membrane expression.Loss of N-cadherin in endothelial cells results in embryonic lethality at mid-gestation due to severe vascular defects.Intriguingly, loss of N-cadherin caused a significant decrease in VE-cadherin and its cytoplasmic binding partner, p120ctn.

View Article: PubMed Central - PubMed

Affiliation: Center for Research on Reproduction and Women's Health, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

ABSTRACT
Endothelial cells express two classic cadherins, VE-cadherin and N-cadherin. The importance of VE-cadherin in vascular development is well known; however, the function of N-cadherin in endothelial cells remains poorly understood. Contrary to previous studies, we found that N-cadherin localizes to endothelial cell-cell junctions in addition to its well-known diffusive membrane expression. To investigate the role of N-cadherin in vascular development, N-cadherin was specifically deleted from endothelial cells in mice. Loss of N-cadherin in endothelial cells results in embryonic lethality at mid-gestation due to severe vascular defects. Intriguingly, loss of N-cadherin caused a significant decrease in VE-cadherin and its cytoplasmic binding partner, p120ctn. The down-regulation of both VE-cadherin and p120ctn was confirmed in cultured endothelial cells using small interfering RNA to knockdown N-cadherin. We also show that N-cadherin is important for endothelial cell proliferation and motility. These findings provide a novel paradigm by which N-cadherin regulates angiogenesis, in part, by controlling VE-cadherin expression at the cell membrane.

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Cadherin/catenin expression in endothelial-restricted N-cadherin CKO embryos. Immunofluorescence was performed on E9.5 embryos for N-cadherin (A and B), VE-cadherin (C and D), PECAM (E), and p120ctn (F and G). In addition to its diffuse cell surface localization, N-cadherin is also observed at cell–cell contacts (arrows) between endocardial cells (A, inset). Western analysis of VE-cadherin in yolk sacs from wild-type (wt) and mutant (cko) embryos (H). m, myocardium; e, endocardium. Bars, 50 μm.
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fig3: Cadherin/catenin expression in endothelial-restricted N-cadherin CKO embryos. Immunofluorescence was performed on E9.5 embryos for N-cadherin (A and B), VE-cadherin (C and D), PECAM (E), and p120ctn (F and G). In addition to its diffuse cell surface localization, N-cadherin is also observed at cell–cell contacts (arrows) between endocardial cells (A, inset). Western analysis of VE-cadherin in yolk sacs from wild-type (wt) and mutant (cko) embryos (H). m, myocardium; e, endocardium. Bars, 50 μm.

Mentions: To confirm the efficiency of the Tie2-Cre–mediated deletion, we examined N-cadherin expression in the endocardium by immunofluorescence. Compared with myocardium, N-cadherin is weakly expressed in endocardium of the wild-type embryo (Fig. 3 A, inset); however, it is both present at cell–cell contacts and found diffusely distributed on the cell surface, similar to HUVEC (Fig. 1). In contrast, N-cadherin was absent from the endocardium in the mutant embryo (Fig. 3 B), but present in the myocardium. Although direct comparison of tissue-specific and constitutive knockout phenotypes can be difficult due to the timing of the deletion, the similarity between the N-cadherin CKO and VE-cadherin KO phenotypes (Carmeliet et al., 1999; Gory-Faure et al., 1999) led us to examine VE-cadherin expression in our CKO embryos using immunofluorescence microscopy. Surprisingly, the VE-cadherin signal was significantly decreased in the endocardium and dorsal aorta (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200411127/DC1) of the N-cadherin CKO embryo compared with the strong immunostaining seen in the wild-type (Fig. 3, C and D). The endothelial identity of the CKO tissue was confirmed by positive staining of an adjacent section with PECAM antibody (Fig. 3 E). Furthermore, Western blot analysis of yolk sac lysates derived from CKO embryos showed a dramatic reduction of VE-cadherin compared with wild-type (Fig. 3 H). Thus, our results provide the first evidence that N-cadherin can regulate the cellular level of VE-cadherin.


N-cadherin acts upstream of VE-cadherin in controlling vascular morphogenesis.

Luo Y, Radice GL - J. Cell Biol. (2005)

Cadherin/catenin expression in endothelial-restricted N-cadherin CKO embryos. Immunofluorescence was performed on E9.5 embryos for N-cadherin (A and B), VE-cadherin (C and D), PECAM (E), and p120ctn (F and G). In addition to its diffuse cell surface localization, N-cadherin is also observed at cell–cell contacts (arrows) between endocardial cells (A, inset). Western analysis of VE-cadherin in yolk sacs from wild-type (wt) and mutant (cko) embryos (H). m, myocardium; e, endocardium. Bars, 50 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171890&req=5

fig3: Cadherin/catenin expression in endothelial-restricted N-cadherin CKO embryos. Immunofluorescence was performed on E9.5 embryos for N-cadherin (A and B), VE-cadherin (C and D), PECAM (E), and p120ctn (F and G). In addition to its diffuse cell surface localization, N-cadherin is also observed at cell–cell contacts (arrows) between endocardial cells (A, inset). Western analysis of VE-cadherin in yolk sacs from wild-type (wt) and mutant (cko) embryos (H). m, myocardium; e, endocardium. Bars, 50 μm.
Mentions: To confirm the efficiency of the Tie2-Cre–mediated deletion, we examined N-cadherin expression in the endocardium by immunofluorescence. Compared with myocardium, N-cadherin is weakly expressed in endocardium of the wild-type embryo (Fig. 3 A, inset); however, it is both present at cell–cell contacts and found diffusely distributed on the cell surface, similar to HUVEC (Fig. 1). In contrast, N-cadherin was absent from the endocardium in the mutant embryo (Fig. 3 B), but present in the myocardium. Although direct comparison of tissue-specific and constitutive knockout phenotypes can be difficult due to the timing of the deletion, the similarity between the N-cadherin CKO and VE-cadherin KO phenotypes (Carmeliet et al., 1999; Gory-Faure et al., 1999) led us to examine VE-cadherin expression in our CKO embryos using immunofluorescence microscopy. Surprisingly, the VE-cadherin signal was significantly decreased in the endocardium and dorsal aorta (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200411127/DC1) of the N-cadherin CKO embryo compared with the strong immunostaining seen in the wild-type (Fig. 3, C and D). The endothelial identity of the CKO tissue was confirmed by positive staining of an adjacent section with PECAM antibody (Fig. 3 E). Furthermore, Western blot analysis of yolk sac lysates derived from CKO embryos showed a dramatic reduction of VE-cadherin compared with wild-type (Fig. 3 H). Thus, our results provide the first evidence that N-cadherin can regulate the cellular level of VE-cadherin.

Bottom Line: Contrary to previous studies, we found that N-cadherin localizes to endothelial cell-cell junctions in addition to its well-known diffusive membrane expression.Loss of N-cadherin in endothelial cells results in embryonic lethality at mid-gestation due to severe vascular defects.Intriguingly, loss of N-cadherin caused a significant decrease in VE-cadherin and its cytoplasmic binding partner, p120ctn.

View Article: PubMed Central - PubMed

Affiliation: Center for Research on Reproduction and Women's Health, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

ABSTRACT
Endothelial cells express two classic cadherins, VE-cadherin and N-cadherin. The importance of VE-cadherin in vascular development is well known; however, the function of N-cadherin in endothelial cells remains poorly understood. Contrary to previous studies, we found that N-cadherin localizes to endothelial cell-cell junctions in addition to its well-known diffusive membrane expression. To investigate the role of N-cadherin in vascular development, N-cadherin was specifically deleted from endothelial cells in mice. Loss of N-cadherin in endothelial cells results in embryonic lethality at mid-gestation due to severe vascular defects. Intriguingly, loss of N-cadherin caused a significant decrease in VE-cadherin and its cytoplasmic binding partner, p120ctn. The down-regulation of both VE-cadherin and p120ctn was confirmed in cultured endothelial cells using small interfering RNA to knockdown N-cadherin. We also show that N-cadherin is important for endothelial cell proliferation and motility. These findings provide a novel paradigm by which N-cadherin regulates angiogenesis, in part, by controlling VE-cadherin expression at the cell membrane.

Show MeSH
Related in: MedlinePlus