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MOLECULAR BIOLOGY
Matrix-buster Inhibitor Has Second Way to Throttle AngiogenesisThe molecular world abounds in yin-yang pairs in which one protein turns off the very process the other initiates. Among the most closely watched of these partnerships is that of the matrix metalloproteinases (MMPs) and their regulators, the tissue inhibitors of metalloproteinases (TIMPs)--and for good reason. MMPs work by breaking down the dense matrix surrounding cells, freeing them to wander the body during processes like metastasis and angiogenesis. TIMPs rein in the MMPs, essentially cutting off the supply of migrating cancer or endothelial cells. TIMP-2's two distinct anti-angiogenic activities are associated with two different regions of the protein. The N-terminal (T2N), which blocks MMP activity, inhibits angiogenesis during embryonic development in vivo. The C-terminal (T2C), which stops endothelial-cell proliferation, inhibits both embryonic and pathologic angiogenesis in vivo. T2C's anti-angiogenic power resides in a small, easily synthesized region, Loop 6.
TIMP-2's N- and C-terminal regions inhibit embryonic angiogenesis, but in a very different fashion. T2N blots out blood vessels (left) while T2C causes vessels to retract, creating a tendril effect (right). (Images courtesy of Marsha Moses) Given the TIMPs' anti-angiogenic action, it is no wonder that pharmaceutical companies have been rushing to develop synthetic MMP-inhibiting agents. Yet in clinical trials, these manmade versions have often performed poorly, producing serious side effects while failing to stop angiogenesis in cancer patients. It now appears that inhibiting the MMPs is not enough to arrest the new blood vessel growth that accompanies tumors. Working in a mouse model, Marsha Moses, Cecilia Fernandez, and their colleagues found that TIMP-2, a variant known to stifle angiogenesis in vivo, owes its power to stop cancer-related angiogenesis to its unique ability to inhibit endothelial cells from proliferating, rather than its ability to bind MMP. What is more, TIMP-2's anti-proliferative power appears to be restricted to a small, easily synthesized region of the molecule, Loop 6. "It is a new angiogenesis inhibitor, it is small, and it is bioavailable," said Moses, HMS associate professor of surgery at Children's Hospital. The study appears in the Oct. 17 Journal of Biological Chemistry.
"One reason synthetic MMP-inhibitors have not met everybody's expectations is that we are only focusing on the MMP-inhibitory activity. And it may not be enough against tumors that induce robust angiogenesis," said Marsha Moses (left), shown with Catherine Butterfield (center) and Cecilia Fernandez. (Photo by Phil Farnsworth) Not that TIMP-2's MMP-binding region is entirely lacking in anti-angiogenic ability. Moses; Fernandez, an MIT graduate student; and their colleagues found that this region is capable of stopping the new blood vessel growth that occurs during embryonic development. Loop 6 was also found to inhibit embryonic angiogenesis, but in a somewhat different fashion. TIMP-2's MMP-binding region appeared to dissolve blood vessels while Loop 6 produced a kind of mass retraction. The findings represent the latest insight into a motley tribe of proteins. The TIMPs consist of four members, each with a reputation for idiosyncratic behavior. All of them bind MMPs and in this regard have the potential to disrupt angiogenesis. But each also performs other unique functions. TIMP-3, for example, appears to induce apoptosis while TIMP-1 inhibits it. In the 1990s, Moses showed that TIMP-2's claim to uniqueness lay in its ability to stop the most critical step in angiogenesis, the proliferation of endothelial cells. But the source of this power remained a mystery. Cell StopperA clue in the search came when scientists discovered that the TIMPs' anti-MMP activity lay in their N-terminal region. Reasoning that the inhibitors' anti-proliferative powers must lie near the opposite, or C-, terminal, Fernandez began a series of experiments. First she separated the N- and C-terminal domains and mixed each with rapidly growing cultured endothelial cells. Sure enough, only those cells exposed to the C-terminal portion stopped dividing. Cutting the C-terminal portion into ever smaller pieces, she was able to home in on Loop 6 as the critical inhibitor."Of course, inhibiting endothelial-cell proliferation in vitro is not the whole ball of wax," said Moses. To further test Loop 6's mettle, Fernandez decided to expose it, along with the larger C- and also N-terminal portions, to two hotbeds of angiogenic activity--the developing embryo and a mouse model of cancer. Working with Geraldine Jackson, a technician in the lab of Judah Folkman, the Julia Dyckman Andrus professor of pediatric surgery, she impregnated chick embryos with methylcellulose disks containing either the N- or C-terminals. "It turned out, and we were somewhat surprised, that both the N- and C-terminals inhibited angiogenesis, though when we looked at the vessels, they were very, very different," said Moses (see images, page 6). Molecular MarvelWorking with lab technician Catherine Butterfield, Fernandez next introduced N- or C-terminal-containing pellets into the corneas of mice that had been primed with growth factor to undergo rapid endothelial cell proliferation. Only those mice receiving the C-terminal pellets experienced relief from the aberrant angiogenesis. Again, Loop 6 alone was sufficient to arrest the process.It is not clear how Loop 6 works its anti-angiogenic magic. "We do not know if it has its own novel receptor or is competing for another receptor," said Moses. "There is also some data suggesting it is interfering with cell cycle progression." Whatever its mode of operation, the molecule could provide an appealing candidate to those looking for anti-angiogenic cancer therapies. "What is very cool about Loop 6 is that it is small, 2.9 kilodaltons," she said. "Translationally speaking, you do not have to go through any elaborate expression system and worry about all those things. And we found, at the end of the day, that it doesn't even have to be in loop form." --Misia Landau |
