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. 2008 Jan;172(1):37-49.
doi: 10.2353/ajpath.2008.070130. Epub 2007 Dec 21.

Regulation of Endothelial Cell Cytoskeletal Reorganization by a Secreted Frizzled-Related protein-1 and Frizzled 4- And Frizzled 7-dependent Pathway: Role in Neovessel Formation

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Regulation of Endothelial Cell Cytoskeletal Reorganization by a Secreted Frizzled-Related protein-1 and Frizzled 4- And Frizzled 7-dependent Pathway: Role in Neovessel Formation

Pascale Dufourcq et al. Am J Pathol. .
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Abstract

Consistent with findings of Wnt pathway members involved in vascular cells, a role for Wnt/Frizzled signaling has recently emerged in vascular cell development. Among the few Wnt family members implicated in vessel formation in adult, Wnt7b and Frizzled 4 have been shown as involved in vessel formation in the lung and in the retina, respectively. Our previous work has shown a role for secreted Frizzled-related protein-1 (sFRP-1), a proposed Wnt signaling inhibitor, in neovascularization after an ischemic event and demonstrated its role as a potent angiogenic factor. However the mechanisms involved have not been investigated. Here, we show that sFRP-1 treatment increases endothelial cell spreading on extracellular matrix as revealed by actin stress fiber reorganization in an integrin-dependent manner. We demonstrate that sFRP-1 can interact with Wnt receptors Frizzled 4 and 7 on endothelial cells to transduce downstream to cellular machineries requiring Rac-1 activity in cooperation with GSK-3beta. sFRP-1 overexpression in endothelium specifically reversed the inactivation of GSK-3 beta and increased neovascularization in ischemia-induced angiogenesis in mouse hindlimb. This study illustrates a regulated pathway by sFRP-1 involving GSK-3beta and Rac-1 in endothelial cell cytoskeletal reorganization and in neovessel formation.

Figures

Figure 1
Figure 1
sFRP-1 induces HUVEC spreading on extracellular matrix and promotes HUVEC cell spreading to collagen type I through α2β1-integrin. a: HUVECs were allowed to spread on polylysine (PL), type I collagen (Coll I), type IV collagen (Coll IV), or laminin (LM) in the absence or presence of rb sFRP-1 in serum-free medium for 2 hours. The percentage of spread cells were scored as described in the Materials and Methods section. Cell surface area was quantified in adherent HUVECs after staining by rhodamine-phalloidin Treatments were performed in triplicate, and results are expressed as mean percent spreading ± SD. *P < 0.05 relative to controls. b: F-actin (red fluorescence) and focal adhesion plaque (green fluorescence) distributions were detected in adherent HUVECs after staining with rhodamine-phalloidin or vinculin labeling in the absence (control) (□) or in the presence (▪) of recombinant bovine sFRP-1 (+rb sFRP-1). c: Spreading assays of HUVECs were performed in the presence of function-blocking antibodies against β1, α2β1, and αvβ3 integrins on wells coated with type I collagen. A mouse IgG served as control. Cells were preincubated for 15 minutes before plating with function-blocking mAbs at a concentration of 4 μg/ml in the absence (□) or presence (▪) of recombinant bovine sFRP-1. Spreading was quantified by counting the number of spread cells per field as described in the Materials and Methods section (×20 objective). Results are expressed as mean spreading as percentage of control ± SD. *P < 0.05 relative to controls.
Figure 2
Figure 2
Involvement of Frizzled 4 and 7 in sFRP-1-induced endothelial cell spreading. Forty-eight hours after transfection with siRNA control or indicated Frizzled siRNA (Fzd2, Fzd4, Fzd5, Fzd6, Fzd7), MS1 cells were submitted to a spreading assay on wells coated with type I collagen. a: The percentage of spread cells were scored as described in the Materials and Methods section. b: F-actin (red fluorescence) distribution in adherent MS1 cells was detected by rhodamine-phalloidin.
Figure 3
Figure 3
Interaction between sFRP-1 and extracellular domain of Frizzled 4 and Frizzled 7 in vitro. a: CHO cells were transfected with expression vectors as indicated. Total cell extract (T) were immunoprecipitated (IP) with anti-His tag antibody. The immunoprecipitates were immunoblotted (WB) with anti-myc tag antibody. Positions of specific bands corresponding to bovine sFRP-1-myc::his, mFz4CRD-myc-GPI, or mFz7CRD-myc-GPI are indicated. b: CHO cells expressing either Fzd4-HA or Fzd7 or none (Ctle) were incubated with rb sFRP-1-myc for 1 hour. Fzd4 and Fzd7 expressed, as determined by using histochemical detection (red staining), localized efficiently at the cell membrane, and induced accumulation of sFRP-1 puncta (green). This recruitment and co-localization of Fzd4 or Fzd7 with sFRP-1 in puncta could highlight sites of frizzled activity.
Figure 4
Figure 4
Role of Rac1 in sFRP-1 induced spreading on endothelial cells. sFRP-1 leads to an activation of Rac1, which is required for sFRP-1-induced EC spreading. a: Immunoprecipitation using a GST-human PAK1-PBD fusion protein that binds to GTP-bound Rac1 (active form) was used to detect, by Western blotting, the magnitude of Rac1 activation in response to sFRP-1 in HUVECs. Serum-starved HUVECs were exposed or not to recombinant bovine sFRP-1 for the indicated times. Cells were also pretreated with SB 216763 (10 μmol/L) before stimulation. The results are expressed as Rac GTP/total Rac ratio and are representative of three independent experiments. b: HUVECs were infected with adenovirus expressing the β-galactosidase or the dominant-negative form of Rac1 (Ad N17Rac) 18 hours before stimulation with the recombinant bovine sFRP-1. The PAE cell line expressing N17 Rac under the control of an IPTG-inducible promoter was also treated by recombinant bovine sFRP-1. After stimulation, spreading assays on type I collagen were performed as described in the Materials and Methods section. Data are expressed in average percent spreading ± SD (n = 3). *P < 0.05 relative to control without recombinant bovine sFRP-1.
Figure 5
Figure 5
GSK-3β/Rac-1 pathway involved in sFRP-1-induced EC spreading. a: HUVECs were either infected with adenovirus expressing β-galactosidase, GSK-3β, GSK-3β-S9A, or GSK-3β-KM or treated with SB216763. After the treatment, a spreading assay was performed. Data are mean percent spreading ± SD. *P < 0.05 relative to control. b: HUVECs infected with adenovirus coding for β-galactosidase or N17Rac or V12Rac and PAE cell line expressing N17Rac under the control of an IPTG-inducible promoter were also treated or not with recombinant bovine sFRP-1 for 30 minutes in serum-free medium. After stimulation, the cellular proteins were analyzed by Western blotting with anti-phospho-GSK-3β (Ser 9) and total GSK-3β. The results shown are representative of three independent experiments. c: HUVECs were infected in a two-step process: first with adenovirus coding for β-galactosidase, N17Rac, or GSK-3β-KM, 6 hours before a second infection with adenovirus producing β-galactosidase, GSK-3β-S9A, GSK-3β-KM, or V12Rac. Eighteen hours later, cells were treated (▪) or not (□) with rb sFRP-1 for 30 minutes in serum-free medium. After stimulation, spreading assays on type I collagen were performed as described in the Materials and Methods section. Data are mean percent spreading ± SD. *P < 0.05 relative to control without recombinant bovine sFRP-1.
Figure 6
Figure 6
sFRP-1 induction of EC spreading is independent of β-catenin and requires GSK-3β activation. Endothelial cell lines expressing β-catenin (+/+) or deleted for β-catenin (−/−) as demonstrated by Western blot, were treated (▪) or not (□) with rb sFRP-1 for 30 minutes in serum-free medium. The role of GSK-3β was investigated in ECs deleted for β-catenin by infection of ECs with adenovirus expressing GSK-3β-KM. After stimulation, spreading assays on type I collagen were performed as described in the Materials and Methods section. Data are mean percent spreading ± SD. *P < 0.05 relative to control without recombinant bovine sFRP-1.
Figure 7
Figure 7
Characterization of the Tie2-tTA/TRE-bov sFRP-1 mice. a: Tie2-tTA transactivator mouse line expressing tTA under the control of the endothelial-specific tie2 promoter was mated with TRE-bov sFRP-1 transgenic mice leading to double-transgenic mice with inducible and endothelium-restricted expression of bovine sFRP-1 and of LacZ as reporter gene. b: LacZ transgene expression was confined to endothelial cells in Tie2-tTA/TRE-bov sFRP-1 compared to littermate embryo (Litt) at day 11.5 dpc. β-galactosidase expression was evaluated by staining with the chromogenic substrate X-Gal on paraformaldehyde 2% fixed embryos in toto. c: Expression by RT-PCR of bov sFRP-1 during the kinetics of ischemia. d: LacZ transgene expression in Tie2-tTA/TRE-bov sFRP-1 in hindlimb after ischemia: at day 11 after ischemia. X-Gal staining of whole mount hindlimb of Litt and Tie2-tTA/TRE-bov sFRP-1 mice and of thin section displayed strong expression of LacZ in neovessels. Section and inset was X-Gal stained for lacZ expression and then double-immunostained with anti-CD31 (in brown) and anti-α-actin (in red) antibodies to localize blood vessel endothelium and pericytes at day 11 in Tie2-tTA/TRE-bov sFRP-1 mice. X-Gal staining demonstrated the selective expression in ECs of neovessels. Scale bar = 50 μm.
Figure 8
Figure 8
Role of sFRP-1 in neovessel formation and on the level of phosphorylated GSK-3β and β-catenin after hindlimb ischemia. a: Quantitative evaluation of capillary density using CD31 immunostaining (number of vessels/mm2) in tissues retrieved from ischemic anterior tibialis muscle of littermates and Tie2-tTA/TRE-bov sFRP-1 mice treated from day 0 to day 6 with doxycycline (Dox, 0 to 6 days) after ischemia. *P < 0.001 and in mice treated with doxycycline after ischemia, from day 0 to day 25 (Dox, 0 to 25 days) after ischemia with doxycycline. *P < 0.001. Treatment through the whole period of ischemia with doxycycline in Tie2-tTA/TRE-bov sFRP-1 animals, switched off bovine sFRP-1 expression and restored a kinetics of capillary density similar to that in littermates, thereby confirming the specific role of endothelial bovine sFRP-1 in the modulation of the angiogenic response after ischemia. The levels of either p-GSK-3β and GSK-3β total (b) or p-β-catenin and total β-catenin (c) was compared by Western blot in Tie2-tTA/TRE-bov sFRP-1 versus littermate (Litt) in hindlimb extracts at day 15 after ischemia. Doxycycline was removed from the drinking water 6 days after ischemia, allowing sFRP-1 induction (−doxycycline). Controls were done with mice treated from day 0 to day 15 after injury with doxycycline (+doxycycline). A quantification of the ratio of phospho-β-catenin:β-catenin is presented.

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