Erk1 and Erk2 regulate endothelial cell proliferation and migration during mouse embryonic angiogenesis

Abstract

Angiogenesis is a complex process orchestrated by both growth factors and cell adhesion and is initiated by focal degradation of the vascular basement membrane with subsequent migration and proliferation of endothelial cells. The Ras/Raf/MEK/ERK pathway is required for EC function during angiogenesis. Although in vitro studies implicate ERK1 and ERK2 in endothelial cell survival, their precise role in angiogenesis in vivo remains poorly defined. Cre/loxP technology was used to inactivate Erk1 and Erk2 in endothelial cells during murine development, resulting in embryonic lethality due to severely reduced angiogenesis. Deletion of Erk1 and Erk2 in primary endothelial cells resulted in decreased cell proliferation and migration, but not in increased apoptosis. Expression of key cell cycle regulators was diminished in the double knockout cells, and decreased DNA synthesis could be observed in endothelial cells during embryogenesis. Interestingly, both Paxillin and Focal Adhesion Kinase were expressed at lower levels in endothelial cells lacking Erk1 and Erk2 both in vivo and in vitro, leading to defects in the organization of the cytoskeleton and in cell motility. The regulation of Paxillin and Focal Adhesion Kinase expression occurred post-transcriptionally. These results demonstrate that ERK1 and ERK2 coordinate endothelial cell proliferation and migration during angiogenesis.

Figure 1. Embryonic Lethality and Defective Angiogenesis in Erk1 ^{− /−} ;Erk2^fl/fl;Tie2-Cre mutant embryos.

(A) E10.5 day (top) and E9.5 (bottom) embryos; control embryo is at the left, DKO at the right. Bars = 0.5 mm. (B) Consecutive paraffin sections of control (left) and Erk1^−/−;Erk2^fl/fl;Tie2-Cre (EC-DKO) (right) embryos were stained with anti-ERK1/2 (top) and anti-CD31 (bottom) antibodies. Representative data for E9.5 embryo are shown; a total of 4 embryos were analyzed. Red dotted lines mark the outer lining of the EC layer and arrowheads point to representative EC. Bars = 20 µm. The graphic panel indicates the ratio of ERK positive EC to total CD31-positive cells, expressed as percent positive EC. (C) E9.5 embryos analyzed by whole mount staining with anti-CD31 antibody. Controls are in the top row and EC-DKO mutants in the bottom row. Arrowheads highlight examples of blood vessel staining and branching in the controls that are reduced in the EC-DKO embryos. Bars = 0.5 mm.

Figure 2. Loss of ERK1/2 in EC affects Cell cycle/proliferation and Cell migration/angiogenesis.

(A) Protein expression of ERK1/2 in lentivirus infected aortic control and DKO EC by Western analysis. Erk1^+/−;Erk2^fl/fl (lane 1) and Erk1^−/−;Erk2^flfl without (lane 2) and with (lane 3) Cre. (B) Principal biological processes affected by the loss of ERK1/2 in aortic EC. Indicated is the % genes differentially expressed in DKO EC. (C and D) Gene expression analysis of cell cycle/proliferation (C) and cell migration/ECM remodeling/angiogenesis (D) regulators by q-PCR in cultured aortic EC with and without ERK1/2, n = 3.

Figure 3. Reduced cell proliferation in ERK1/2 deficient EC.

(A) Representative paraffin sections from E9.5 control (left, n = 4) and EC-DKO (right, n = 4) embryo sections stained for anti-BrDU. The red dotted lines indicate the outer lining of the EC layer and the arrowheads point to representative EC. The bar graph represents the fraction of BrDU positive to total EC. Bars = 20 µm. (B) Anti-cleaved Caspase-3 staining of representative paraffin sections from E9.5 control (left, n = 4) and EC-DKO (right, n = 4) embryo. The red dotted lines indicate the outer lining of the EC layer. The graphic panel indicates the fraction of EC staining for cleaved Caspase-3 to total EC. Bars = 20 µm. (C) Anti-BrDU staining on cultured aortic EC of the indicated genotype, without (top panel) and with (bottom panel) Cre. Red-BrDU and Blue-DaPI. The graph indicates the ratio of BrDU positive to total EC with 3 independent experiments represented. Bars = 20 µm. (D) Western blot analysis on lysates from Erk1^+/−;Erk2^fl/fl-Cre (lane 1), Erk1^−/−;Erk2^fl/fl-Cre (lane 2) and Erk1^−/−;Erk2^fl/fl+Cre for the antibodies indicated.

Figure 4. EC lacking ERK1/2 display reduced migration and invasion potential in vitro.

(A) Confluent monolayer of control (top panel) and DKO (bottom panel) aortic EC were wounded and wound closure was monitored over 24 hrs. Representative results from t = 0 h (left panel) and t = 24 h (right panel) are shown (n = 3). The graph indicates the quantification of the number of cells migrating into the wound over the indicated time points. Bars = 40 µm. (B) In vitro matrigel sandwich assay showing invasion and tube-like formation by control aortic EC (top panel) that is absent in DKO EC (bottom panel). Left panel shows the phase contrast images and the right panel GFP images. Bars = 80 µm.

Figure 5. ERK1/2 deficient EC show altered actin organization and reduced expression of Paxillin and FAK in vitro.

(A) Representative micrographs of phalloidin stained control (top) and DKO (bottom) aortic EC (n = 3). Red = Actin and Green = lentivirus infected cells. The bar graph represents % EC showing the presence of intra-cellular stress fibers. Bars = 20 µm. (B) Representative micrographs of Paxillin-pFAK membrane co-localization on FN-coated dishes in Erk1^−/−;Erk2^fl/fl (top) and Erk1^−/−;Erk2^fl/fl+Cre (bottom) aortic EC (n = 3). Red-Paxillin and Green-pY397 FAK. Boxed areas are enlarged to show membrane staining in EC. Graphic panel represents % EC showing Paxillin-pY397 FAK co-localization. Bars = 20 µm. (C) Western blot with indicated antibodies of lentivirus infected aortic EC. Erk1^+/−;Erk2^fl/fl (lane 1) and Erk1^−/−;Erk2^fl/fl without (lane 2) and with (lane 3) Cre.

Figure 6. ERK1/2 deficient EC show reduced expression of Paxillin and FAK in vivo.

(A) Representative cryosections of E9.5 Erk1^−/−;Erk2^fl/fl control (left) and Erk1^−/−;Erk2^fl/fl ;Tie2-Cre (EC-DKO, right) embryos (n = 4 of each genotype) stained with anti-Paxillin. Graphic panel indicates the ratio of Paxillin positive area to total EC area in %. (B) Anti-FAK staining of representative cryosections from E9.5 Erk1^−/−;Erk2^fl/fl control (left, n = 4) and EC-DKO mutant (right, n = 4) embryo. Graphic panel indicates the ratio of FAK positive area to total EC area in %. Boxed areas are enlarged to show membrane staining in EC. The red dotted lines indicate the outer lining of the EC layer and the arrowheads point to representative EC. Lower magnification: Bars = 25 µm and Higher magnification: Bars = 20 µm.