FrzA/sFRP-1, a secreted antagonist of the Wnt-Frizzled pathway, controls vascular cell proliferation in vitro and in vivo.
OBJECTIVE
FrzA, a member of the group of secreted frizzled related proteins (sFRP) that is expressed in the cardiovascular system, has been shown to antagonize the Wnt/frizzled signaling pathway. We have recently demonstrated its role in vascular cell growth control in vitro. In this study, we aimed to examine the mechanisms by which FrzA exerts its antiproliferative effect on vascular cells in vitro and its potential effect in vivo.


RESULTS
On synchronized, growth-arrested endothelial cells (EC) and smooth muscle cells (SMC) treated with the recombinant purified FrzA protein, flow cytometry analysis showed that the recombinant FrzA protein delayed G1 phase and entry into S-phase. Western blot experiments demonstrated that the treatment of EC or SMC with FrzA was associated with a decrease in the level of the cyclins and cyclin-dependent kinases and an increase in cytosolic phospho-beta-catenin levels. The FrzA-induced cell cycle delay was resolved by 24 h. C57BL/6J mice underwent surgery to produce unilateral hindlimb ischemia and empty adenoviruses (AdE) or adenoviruses coding for FrzA (AdFrzA) were injected at the time of the surgery. In AdFrzA-treated mice in the 7 days following surgery, we showed a decrease in cell proliferation, capillary density, and blood flow recovery and a reduced expression of cyclin and cdk activity in the ischemic muscle compared to that in the AdE-treated ischemic muscle. To gain insight into the pathway activated by FrzA overexpression, we showed an increase in the level of cytosolic phospho-beta-catenin, a marker of beta-catenin degradation, in AdFrzA-treated ischemic muscle compared to that in control AdE-treated ischemic muscle.


CONCLUSIONS
We provided the first evidence that an impairment of the Wnt-Frizzled pathway, via FrzA overexpression, controlled proliferation and neovascularization after muscle ischemia.
Ezan
Jérome
J
Inserm U441, Avenue du Haut Lévêque, 33 600 Pessac, France, and Université Victor Segalen Bordeaux 2, rue léo Saignat, 33 000 Bordeaux, France.
Leroux
Lionel
L
Barandon
Laurent
L
Dufourcq
Pascale
P
Jaspard
Béatrice
B
Moreau
Catherine
C
Allières
Cécile
C
Daret
Danièle
D
Couffinhal
Thierry
T
Duplàa
Cécile
C
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002455:Cell Division; D002478:Cells, Cultured; D018844:Cyclin-Dependent Kinases; D016213:Cyclins; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism*; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000008:administration & dosage; D006614:Hindlimb; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D009389:Neovascularization, Pathologic; D015398:Signal Transduction / Q000502:physiology*; D015534:Trans-Activators / Q000378:metabolism; D014161:Transduction, Genetic / Q000379:methods; D051176:beta Catenin; D000094742:Secreted Frizzled-Related Proteins
D018844:Cyclin-Dependent Kinases; D016213:Cyclins; D003598:Cytoskeletal Proteins; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D015534:Trans-Activators; D051176:beta Catenin; C495267:CTNNB1 protein, mouse; C485539:Sfrp1 protein, mouse; D000094742:Secreted Frizzled-Related Proteins