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. 2015 Aug 15;404(2):49-60.
doi: 10.1016/j.ydbio.2015.05.011. Epub 2015 May 22.

Polo-like kinase 2 regulates angiogenic sprouting and blood vessel development

Hongbo Yang  1 , Longhou Fang  1 , Rui Zhan  1 , Jeffrey M Hegarty  1 , Jie Ren  1 , Tzung K Hsiai  2 , Joseph G Gleeson  3 , Yury I Miller  1 , JoAnn Trejo  4 , Neil C Chi  5
Affiliations

Polo-like kinase 2 regulates angiogenic sprouting and blood vessel development

Hongbo Yang et al. Dev Biol. .

Abstract

Angiogenesis relies on specialized endothelial tip cells to extend toward guidance cues in order to direct growing blood vessels. Although many of the signaling pathways that control this directional endothelial sprouting are well known, the specific cellular mechanisms that mediate this process remain to be fully elucidated. Here, we show that Polo-like kinase 2 (PLK2) regulates Rap1 activity to guide endothelial tip cell lamellipodia formation and subsequent angiogenic sprouting. Using a combination of high-resolution in vivo imaging of zebrafish vascular development and a human umbilical vein endothelial cell (HUVEC) in vitro cell culture system, we observed that loss of PLK2 function resulted in a reduction in endothelial cell sprouting and migration, whereas overexpression of PLK2 promoted angiogenesis. Furthermore, we discovered that PLK2 may control angiogenic sprouting by binding to PDZ-GEF to regulate RAP1 activity during endothelial cell lamellipodia formation and extracellular matrix attachment. Consistent with these findings, constitutively active RAP1 could rescue the endothelial cell sprouting defects observed in zebrafish and HUVEC PLK2 knockdowns. Overall, these findings reveal a conserved PLK2-RAP1 pathway that is crucial to regulate endothelial tip cell behavior in order to ensure proper vascular development and patterning in vertebrates.

Keywords: Angiogenesis; Human umbilical vein endothelial cells; Polo-like kinase 2; Vascular development; Zebrafish.

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Figures

Figure 1
Figure 1. PLK2 is expressed in endothelial cells
(A–B”’) Immunostaining of HUVECs reveals that PLK2 is expressed in ECs. (B-B”’) Enlarged image of the boxed area in A-A”’ shows that PLK2 can aggregate at the leading edge of extending ECs (arrowhead). (A, B) - merge; (A’, B’) - anti-PLK2 immunostaining (green); (A”, B”) - phalloidin actin staining (red). DAPI nuclear staining (blue). Scale bar, 40 µm. (C) Quantitative measurements of PLK2 localized at the leading edge and in the cytoplasm of HUVECs (Mean +/− s.e.m. *p = 0.0056 by Student's t-test). (D–F) in situ hybridization shows that plk2b is expressed in the zebrafish vasculature at 16 hpf (n = 31/31), 24 hpf (n = 46/47), and 48 hpf (n = 28/30). (D’–F’) Enlarged image of the boxed area of D–F shows that plk2b is expressed in the cardinal vein, aorta, and intersomitic vessels of the zebrafish body and tail.
Figure 2
Figure 2. plk2b regulates endothelial cell sprouting
(A–D) Fluorescence micrographs show that loss of plk2b function in Tg(kdrl:mcherry-ras) zebrafish embryos results in underdeveloped intersomitic vessel sprouting (open arrowheads) at 36 hpf. (A) control MO (n = 0/149); (B) plk2b ATG MO (MO1) (n = 125/171); (C) plk2b splice MO (MO2) (n = 89/137); (D) dn-plk2b RNA – dominant negative plk2b (n = 49/85). Injecting (E) 80 pg of plk2b (n = 31/103) or (F) 80 pg of hPLK2 (n = 24/71) can rescue the plk2b MO1 vascular sprouting defect. (G) Injecting 80 pg of plk2b RNA into wild-type Tg(kdrl:mcherry-ras) embryos did not cause any significant vascular phenotypes (n = 0/105). However, (H) injecting 160 pg of plk2b resulted in increased ISV spouting and branches (arrowheads) (n = 91/145). Top, dorsal longitudinal anastomotic vessel (yellow asterisk); bottom, dorsal aorta/cardinal vein. Scale bar, 80 µm. Quantitative measurements of (I) intersomitic vessel length and (J) the number of ISV branches for each corresponding condition. Mean +/− s.e.m. *p<0.05 by ANOVA.
Figure 3
Figure 3. PLK2 regulates endothelial cell migration
(A, B) EC tube formation assays, (C, D) transwell EC migration assays, and (E–F’) wound healing EC assays show that when compared to control siRNA knockdown, PLK2 siRNA knockdown in HUVECs results in (B and G) decreased EC network/tube formation (p = 0.0028), (D and H) reduced EC migration formation (p = 0.0185), and (F, F’ and I) slower EC wound healing/closure, respectively (p = 0.0114). (J–K) Quantitative measurements of the percentage of (J) BrdU positive cells and (K) TUNEL staining positive cells in control and PLK2 siRNA transfected HUVECs show that PLK2 knockdown does not affect HUVEC cell proliferation (p = 0.6007) and apoptosis (p = 0.7970). (A, B, E–F’) Scale bar, 1.6 mm. (C, D) Scale bar, 400 µm. hpw – hours post wounding. Mean +/− s.e.m. *p<0.05, **p<0.01 by Student's t-test.
Figure 4
Figure 4. PLK2 regulates endothelial cell lamellipodia and cell adhesion formation
(A-A’) 26 hpf plk2b MO1 injected Tg(fli1a:eGFP) fish (n = 31/40) exhibit fewer lamellipodia, and extending intersomitic vessels compared to age-matched control MO injected Tg(fli1a:eGFP) fish (n = 0/48). Scale bar, 40 µm. Top, dorsal longitudinal anastomotic vessel; bottom, dorsal aorta/cardinal vein. (B-B’) Phalloidin staining reveals that PLK2 siRNA transfected HUVECs display reduced lamellipodia when compared to control siRNA transfected HUVECs. Scale bar, 20 µm. Immunostaining of (C-C”’, D-D”’) pFAK and (E-E”’, F-F”’) integrin αVβ3 shows that focal adhesions and integrins, respectively, are localized to the lamellipodia of migrating/extending (C, E) control siRNA transfected HUVECs, but they fail to organize and aggregate in (D, F) PLK2 siRNA transfected HUVECs. Scale bar, 10 µm. (G) Quantitative measurements of the number of lamellipodia and filopodia reveal that zebrafish plk2b knockdowns have reduced lamellipodia but relatively the same number of filopodia (finger-like protrusions crossing the cell edge) when compared to controls. Conversely, zebrafish RNA injection of 160 pg plk2b resulted in more lamellipodia only. (H) Quantitative measurements of the number of lamellipodia and filopodia reveal that human PLK2 knockdowns have reduced lamellipodia (p = 0.0023) but relatively the same number of filopodia (p = 0.2615) when compared to controls. (I) Quantitative measurements of the number of pFAK (p = 0.0143) and integrin αVβ3 plaques (p = 0.0224) reveals that knockdown of PLK2 reduced the number of cell adhesions in HUVECs. (J) EC adhesion assays show that PLK2 siRNA HUVECs adhered less to type I collagen (p = 0.0017) and fibronectin-coated coverslips (p = 0.0061) compared to control siRNA HUVECs. Arrowheads and arrows point to lamellipodia and filopodia, respectively. Red – phalloidin/actin staining; Blue – DAPI staining; Green – (A) GFP, (C–D) pFAK, or (E, F) integrin αVβ3. Mean +/− s.e.m. *p<0.05, **p<0.01 by ANOVA for G and Student's t-test for H–J.
Figure 5
Figure 5. PLK2 regulates HUVEC migration in vitro via RAP1 activity
(A) RAP1 immunoblot of RAP1-GTP binding assays shows that there is less RAP1-GTP in PLK2 siRNA HUVECs than in control siRNA HUVECs (22 kDa). PLK2 immunoblot shows the efficiency of PLK2 siRNA knockdown in HUVECs. (B) Quantitative measurements shows that the ratio of RAP1-GTP/Total RAP1 is less in PLK2 siRNA (Mean +/− s.e.m. *p = 0.015 by Student's t-test). (C) Immunoprecipitation (IP) of HUVEC lysates with anti-PLK2 antibody reveals that PLK2 interacts in a complex with PDZ-GEF1 (180 kDa). Western analyses of immunoprecipitations were performed with anti-PDZ-GEF (WB: PDZ-GEF) and anti-PLK2 antibodies (WB: PLK2). IP IgG – control antibody; α-PLK2 – anti-PLK2 antibody. (D) EC tube formation assays, (E) Transwell EC migration assays, and (F, G) wound healing EC assays show that ca-RAP1 virus can rescue the PLK2 siRNA knockdown HUVEC (compare D” to D”’) EC network/tube formation, (compare E” to E”’) cell migration, and (compare F”, G” to F”’, G”’) wound healing defects. (D-D”’, F-F”’, G-G”’) Scale bar, 1.6 mm. (E-E”’) Scale bar, 400 µm. (H–J) Quantitative measurements were performed on (H) EC tube formation assays, (I) transwell EC migration assays, and (J) wound healing EC assays. hpw - hours post wounding. Mean +/− s.e.m. *p<0.05, **p<0.01 by ANOVA for H–J.
Figure 6
Figure 6. PLK2 regulates focal adhesion formation and integrin localization through RAP1 activity
(A–H) Immunofluorescence studies show that ca-RAP1 virus can rescue the reduced EC sprouting and lamellipodia defects observed in PLK2 siRNA HUVECs through restoring focal adhesion formation (compare C to D) and integrin αVβ3 organization (compare G to H). Arrowheads point to lamellipodia extension as detected by the organization of F-actin (phalloidin) at the leading edge of extending HUVEC membranes. Green – (A’–−D’) anti-pFAK; or (E’–H’) anti-integrin αVβ3; Red – phalloidin; Blue – DAPI. Scale bar, 20 µm.
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References

    1. Adams RH, Alitalo K. Molecular regulation of angiogenesis and lymphangiogenesis. Nature reviews. Molecular cell biology. 2007;8:464–478. - PubMed
    1. Bedell VM, Yeo SY, Park KW, Chung J, Seth P, Shivalingappa V, Zhao J, Obara T, Sukhatme VP, Drummond IA, Li DY, Ramchandran R. roundabout4 is essential for angiogenesis in vivo. Proceedings of the National Academy of Sciences of the United States of America. 2005;102:6373–6378. - PMC - PubMed
    1. Bentley D, Toroian-Raymond A. Disoriented pathfinding by pioneer neurone growth cones deprived of filopodia by cytochalasin treatment. Nature. 1986;323:712–715. - PubMed
    1. Bertrand JY, Chi NC, Santoso B, Teng S, Stainier DY, Traver D. Haematopoietic stem cells derive directly from aortic endothelium during development. Nature. 2010;464:108–111. - PMC - PubMed
    1. Bivona TG, Quatela S, Philips MR. Analysis of Ras activation in living cells with GFP-RBD. Methods in enzymology. 2006;407:128–143. - PubMed

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