Person:

Olsen, Bjorn

PURPOSE: 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.

Perlecan plays an important tole in basement membranes and other extracellular matrices. Mice with a knockout gene for perlecan die at or before birth with severe defects in the brain, heart, and skeleton.

The matrix metalloproteinases (MMPs) are a family of zinc-containing matrix degrading endopeptidases. A subfamily of membrane type (MT) -MMPs has been de- scribed recently. We have determined the structure of the gene (Mmp14) encoding the first MT-MMP to be de scribed, MT1-MMP (MMP-14), and mapped it to mouse chromosome 14. The mouse MMP-14 protein is encoded by ten exons. The novel C-terminal peptide domains of MMP-14 are encoded by a single large exon that also encodes the 3'-untranslated region. The structure of the exons encoding the catalytic domain and pro-domain of MMP-14 is distinct from previously described MMP genes, whereas the exons encoding the hemopexin-like domains are similar to those of most other MMP genes. Mmp14 and the gene for tissue inhibitor of metalloproteinases-2 (Timp2) show a temporally and spatially co- regulated expression during mouse development. They are co-expressed during vascular and urogenital development and during the development of osteocartilaginous and musculotendinous structures. The stringent co-expression of these two genes suggests common regulatory pathways that may have important functional implications for the activation of pro-gelatinase A in health and disease.

To analyze the relationship between c-myc gene expression and chondrocyte proliferation and maturation during endochondral ossification, Day 18-19 chick embryo sterna were pulse-labeled with [3H]thymidine, and serial sections were processed for autoradiography and in situ hybridization. Proliferating chondrocytes, located in four distinct areas of the developing sternum, all contained high levels of c-myc transcripts, whereas postmitotic chondrocytes (such as hypertrophic chondrocytes) contained undetectable amounts. These findings were confirmed by Northern blot analysis and by the observation that antisense cmyc oligomer treatment inhibited proliferation in cultured chondrocytes. Constitutive overexpression of cmyc by retroviral vectors in immature chondrocyte cultures (c-myc cultures) maintained the cells in a proliferative state and blocked their maturation into hypertrophic chondrocytes. The lack of maturation in the c-myc cultures was corroborated by analysis of type X collagen gene regulation. Control immature cultures contained strong repressor activity for the type X collagen gene promoter, as revealed by transfection assays; repressor activity was lost upon maturation and activation of type X collagen synthesis. In the c-myc cultures, however, repressor activity persisted. Thus, c-myc participates in the normal changes in proliferation accompanying chondrocyte maturation in vivo and in culture. The decreases in c-myc expression and cell proliferation appear to be required for completion of maturation.

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) play a critical role in extracellular matrix homeostasis. We have previously cloned human and mouse TIMP-3 cDNAs and mapped their chromosomal loci (Apte, S. S., Mattei, M-G., and Olsen, B. R.(1994) Genomics 19, 86-90; Apte, S. S., Hayashi, K., Seldin, M. F., Mattei, M-G., Hayashi, M., and Olsen, B. R.(1994) Dev. Dynam. 200, 177-197); the identification of TIMP3 mutations in Sorsby's fundus dystrophy has underscored the functional importance of TIMP-3. We now report that TIMP-3 is encoded by five exons spanning over 30 kilobase pairs of mouse genomic DNA. In the attribution of protein domains to specific exons, as well as exon structures, the Timp-3 and Timp-1 genes are similar, confirming the common evolutionary origin of the TIMPs and defining a distinct gene family. We have expressed human and mouse TIMP-3 in mouse NSO myeloma cells. In each case, an N-glycosylated 27-kDa protein was generated, that, like TIMP-1 and TIMP-2, inhibited collagenase-1, stromelysin-1, and gelatinases A and B. TIMP-3 and TIMP-1 inhibition were quantitatively similar, implying that all TIMPs are equally efficient in MMP inhibition. Instead, differential regulation of the TIMP genes or divergent C-terminal protein sequences may underlie distinct biological functions for each TIMP.

We have cloned and characterized cDNAs encoding the alpha 1 chain of type XV collagen from a human placenta library. Using primer extension cloning we extended the cDNAs to the 5' end of the mRNA and determined the complete deduced primary structure of the human alpha 1(XV) chain. The polypeptide chain contains nine triple helical domains separated by eight non-triple helical regions and flanked by large amino-terminal (555 amino acid residues) and carboxyl-terminal (256 amino acid residues) non-triple helical domains. Comparison of amino acid sequences of the human alpha 1(XV) chain with those of mouse alpha 1(XVIII) collagen showed remarkable similarity within both amino- and carboxyl-terminal non-triple helical domains. Within the carboxyl third of the amino-terminal domain a tandem repeat structure is found with an about 45-amino acid residue sequence repeated four times. This amino acid sequence has a strikingly high similarity to rat cartilage proteoglycan core protein. Northern blot analysis of human embryonic RNA revealed that alpha 1(XV) mRNA is expressed predominantly in internal organs such as the adrenal gland, kidney, and pancreas.