Inhibition of angiogenesis by tissue inhibitor of metalloproteinase-3.
OBJECTIVE
It has been established that Sorsby's fundus dystrophy, a dominantly inherited form of blindness, is caused by mutations in the tissue inhibitor of metalloproteinase-3 (TIMP-3) gene. Because choroidal neovascularization is a prominent feature of Sorsby's fundus dystrophy, the authors have examined whether TIMP-3 protein plays a role in the regulation of angiogenesis.


METHODS
Chemotaxis of endothelial cells toward vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) was examined using a modified Boyden chamber assay. Endothelial cells placed in the upper chamber were allowed to migrate through a polycarbonate membrane with 8 microns pores toward VEGF or bFGF present in the lower chamber. Next, the ability of TIMP-3 to inhibit chemotaxis was studied by incubating the cells with varying amounts of TIMP-3 during the assay. Finally, an in vitro angiogenesis assay was performed on collagen gels. Endothelial cells were seeded onto three-dimensional collagen gels. Treatment with bFGF and VEGF induced invasion of the gel and the formation of tube-like structures. TIMPs (1, 2, and 3) were added to the cultures to determine their effect on invasion. An in vivo chorioallantoic membrane (CAM) assay was performed using methylcellulose discs containing bFGF with or without TIMP-3. Induction of new blood vessels was observed with a stereomicroscope.


RESULTS
TIMP-3 inhibits chemotaxis of vascular endothelial cells toward VEGF and bFGF, inhibits collagen gel invasion and capillary morphogenesis in vitro, and inhibits bFGF-induced angiogenesis in the CAM assay in vivo.


CONCLUSIONS
TIMP-3 has the potential to inhibit angiogenesis. These results allow us to speculate on a possible mechanism by which mutant TIMP-3 protein might contribute to the Sorsby fundus dystrophy phenotype.
Anand-Apte
B
B
Department of Surgical Research, Children's Hospital, Boston, Massachusetts, USA.
Pepper
M S
MS
Voest
E
E
Montesano
R
R
Olsen
B
B
Murphy
G
G
Apte
S S
SS
Zetter
B
B
D000482:Allantois / Q000098:blood supply* / Q000187:drug effects; D000818:Animals; D002455:Cell Division; D002633:Chemotaxis / Q000187:drug effects; D002642:Chick Embryo; D002823:Chorion / Q000098:blood supply* / Q000187:drug effects; D015194:Diffusion Chambers, Culture; D016228:Endothelial Growth Factors / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008222:Lymphokines / Q000378:metabolism; D009389:Neovascularization, Pathologic / Q000139:chemically induced / Q000517:prevention & control*; D011480:Protease Inhibitors / Q000494:pharmacology*; D011506:Proteins / Q000494:pharmacology*; D019717:Tissue Inhibitor of Metalloproteinase-3; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D008222:Lymphokines; D011480:Protease Inhibitors; D011506:Proteins; D019717:Tissue Inhibitor of Metalloproteinase-3; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D016222:Fibroblast Growth Factor 2