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First published online June 6, 2008
doi: 10.1242/10.1242/dev.017038

Development 135, 2331-2338 (2008)
Published by The Company of Biologists 2008
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LKB1 in endothelial cells is required for angiogenesis and TGFβ-mediated vascular smooth muscle cell recruitment

Anou Londesborough, Kari Vaahtomeri*, Marianne Tiainen*,{dagger}, Pekka Katajisto, Niklas Ekman{ddagger}, Tea Vallenius and Tomi P. Mäkelä§

Genome-Scale Biology Program and Institute of Biomedicine, Biomedicum Helsinki, 00014 University of Helsinki, Finland.


Figure 1
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  		Fig. 1. Abnormal development of Lkb1lox/-, Mox2-Cre conceptuses at E9.5. (A-H) Gross morphology of wild-type (A,E), Lkb1lox/-, Mox2-Cre (B,C,F,G) and Lkb1-/- (D,H) embryos and yolk sacs (E-H). (I-L) Higher magnification of E-H, respectively. Defects observed in Lkb1lox/-, Mox2-Cre embryos vary from mild abnormalities and reduced size (B) to severe developmental retardation (C) resembling the Lkb1-/- phenotype (D). Similar to Lkb1-/- yolk sacs (H,L), the yolk sacs of Lkb1lox/-, Mox2-Cre embryos (F,G,J,K) fail to remodel the primary vascular plexus into large vitelline vessels, as seen in E9.5 wild-type yolk sacs (E,I).

 

Figure 2
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  		Fig. 2. Vascular disruption in embryos after Tie1-Cre induced inactivation of endothelial Lkb1. (A) Whole-mount immunofluorescence staining of Cre (red) and VE-cadherin (green) in E11.5 Tie1-Cre-positive yolk sac. (B,C) E11.5 wild-type (B) and Lkb1lox/-, Tie1-Cre (C) yolk sacs. The Lkb1lox/-, Tie1-Cre yolk sac is pale owing to a lack of blood in the vitelline vessels (arrow). (D,E) E11.5 wild-type (D) and Lkb1lox/-, Tie1-Cre (E) embryos. Pericardial swelling (arrow) and dilated embryonic vessels (arrowheads) are apparent in the Lkb1lox/-, Tie1-Cre embryo. (F,G) E12.5 wild-type (F) and Lkb1lox/-, Tie1-Cre (G) embryos, demonstrating paleness and severe hemorrhaging in the mutant (arrowheads).

 

Figure 3
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  		Fig. 3. Blood vessel structure of Lkb1lox/-, Tie1-Cre conceptuses at E11.5. (A,B) Whole-mount VE-cadherin staining of wild-type (A) and Lkb1lox/-, Tie1-Cre (B) yolk sacs. (C,D) Whole-mount PECAM1 staining of wild-type (C) and Lkb1lox/-, Tie1-Cre (D) yolk sacs. Insets show higher magnification of single vessels. (E,F) Hematoxylin and Eosin-stained sagittal sections from wild-type (E) and Lkb1lox/-, Tie1-Cre (F) embryos. Arrows indicate the aortas; asterisks, the livers. Arrowhead in F indicates congested blood in the Lkb1lox/-, Tie1-Cre aorta.

 

Figure 4
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  		Fig. 4. SMA and PDGFRβ staining demonstrates a reduction of vSMCs in Lkb1lox/-, Tie1-Cre conceptuses at E11.5 (A-F) Whole-mount immunofluorescence staining of PECAM1 (green) and SMA (red) in wild-type (A,C,E) and Lkb1lox/-, Tie1-Cre (B,D,F) yolk sacs, demonstrating loss of SMA staining in the mutant. (G,H) Whole-mount immunofluorescence staining of PDGFRβ in wild-type (G) and Lkb1lox/-, Tie1-Cre (H) yolk sacs, showing loss of PDGFRβ expression in Lkb1lox/-, Tie1-Cre yolk sacs. (I,J) SMA staining of cross sections of the aorta at the thoracic level of wild-type (I) and Lkb1lox/-, Tie1-Cre (J) embryos demonstrates reduced SMA staining in the mutant.

 

Figure 5
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  		Fig. 5. Suppressed TGFβ signaling in Lkb1-deficient ECs. (A) Quantitative RT-PCR analysis of LKB1, TGFβ1 and PAI1 mRNA levels in immortalized HUVECs, or wild-type and Lkb1lox/-, Tie1-Cre yolk sacs, as indicated. HUVECs were transfected with a control-siRNA or two independent siRNAs targeting LKB1 (LKB1-si1 and LKB1-si2). Measurements were normalized according to GAPDH mRNA levels and are presented relative to control samples (control-siRNA transfected HUVECs or wild-type yolk sac). Bars represent the mean (±s.d.) of two samples (siRNA treated), four samples (control-siRNA) or three samples (yolk sacs; *P<0.05, **P<0.01). (B,C) pSMAD2 (red) and PECAM1 (brown) staining of E12 wild-type (B) and Lkb1lox/-, Tie1-Cre (C) yolk sac sections. (D,E) Higher magnifications of boxed areas in B and C. (F) Percentage of pSMAD2-positive ECs and mesenchymal cells in wild-type and Lkb1lox/-, Tie1-Cre yolk sacs, with means (±s.d.) of three embryos indicated (*P<0.05). e, endoderm; EC, endothelial cell; mes, mesenchymal cell.

 

Figure 6
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  		Fig. 6. Partial rescue of LKB1 deficiency by exogenous TGFβ. (A) Relative TGFβ1 and PAI1 mRNA levels in LKB1-siRNA transfected HUVECS with (+) or without (-) TGFβ1 treatment (1 ng/ml, 4 hours). Measurements were normalized according to GAPDH mRNA levels and the ratio (±s.d.) of LKB1-siRNA HUVECs to control HUVECs, which were normalized to one, is presented. Bars represent means of LKB1-si1 and LKB1-si2 duplicate samples compared with four controls (*P<0.05, **P<0.01). The data in non-treated samples (-TGFβ1) is summarized from Fig. 5A and is shown for reference. (B-D) pSMAD2 staining of E12.5 wild-type (B), Lkb1lox/-, Tie1-Cre (C) and TGFβ-treated Lkb1lox/-, Tie1-Cre (D) yolk sac sections. (E) Percentage of pSMAD2-positive cells in E12.5 wild-type and Lkb1lox/-, Tie1-Cre yolk sacs with or without exogenous TGFβ. Means±s.d. of three embryos are indicated (*P<0.05). (F-H) SMA staining of E12.5 wild-type (F), Lkb1lox/-, Tie1-Cre (G) and TGFβ-treated Lkb1lox/-, Tie1-Cre (H) yolk sac sections, demonstrating loss of SMA staining in Lkb1lox/-, Tie1-Cre embryos and partial recovery following TGFβ1 treatment. e, endoderm; EC, endothelial cell; mes, mesenchymal cell.

 

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© The Company of Biologists Ltd 2008