(Philadelphia, PA) – Researchers at the University of Pennsylvania School of Medicine have discovered that zebrafish produce enzymes analogous to the two human cyclooxygenase (COX) enzymes – COX-1 and -2 – proteins that have a role in a variety of ailments including cardiovascular disease, some types of cancer, and arthritis. Furthermore, drugs that target the COX enzymes, nonsteroidal anti-inflammatory drugs (NSAIDs) and selective inhibitors of COX-2, seem to act in a manner similar to humans.
"We have learned a great deal about how the COX enzymes and their inhibitors work from mouse models of COX gene inactivation," said Garret A. FitzGerald, MD, Chair of the Department of Pharmacology and director of the Center for Experimental Therapeutics at Penn. " However, these systems have their limitations. The zebrafish promises to play a complementary role in which both biology and the role of drugs can be investigated."
Traditionally zebrafish have been useful in searching for gene mutations induced by exposure to toxic chemicals, which coincide with abnormalities of development. Such an approach is termed "forward genetics."
The Penn paper, detailed in this week's print edition of the Proceedings of the National Academy of Sciences, is an early example of the use of zebrafish for "reverse genetics." A particular set of genes – in this case, the COXs – are sought out and their function is uncovered by manipulating their action in zebrafish. The COX enzymes produce prostaglandins, fatty acids that perform a number of hormone-like tasks.
Prostaglandins alter the activities of the cells near and around where they are made. They also cause inflammation and can regulate blood flow to some organs, transport across cell membranes, and transmissions between neurons.
The COX proteins are encoded by separate genes on separate chromosomes and, likewise, have two separate but interdependent functions. COX-1 is responsible for the "day-to-day" production of prostaglandins, while COX-2 is highly regulated by numerous other cellular signals when needed.
Drugs which selectively target COX-2, such as Pharmacia/Pfizer's Celebrexâ and Bextraâ and Merck's Vioxxâ have attracted much attention. They have been remarkably successful, based on claims that they relieve pain and inflammation while lessening the risk of stomach ulceration and bleeding associated with traditional NSAIDs. One issue of controversy has been whether the benefit of COX – 2 inhibitors in the stomach is offset by an increased risk of heart attack.
"The zebrafish has particular advantages for the study of drug action," says Tilo Grosser, MD, Research Associate in Pharmacology and first author of the study. "The embryos are translucent, so we can study the pattern of gene expression during development, as well as in the adult. The near completion of the zebrafish genome project allows us to hunt for relatives of human genes of interest. Then we can manipulate them and see how they function."
Indeed, this is just the approach that Grosser, FitzGerald and their colleagues took in the present study. They thought the zebrafish might be a particularly useful model of the cardiovascular effects of COX inhibitors. First, they identified the zebrafish versions of the COX genes and showed that they behaved like the human enzymes. Next they studied their distribution. Interestingly, both COX- 1 and COX-2 were extensively expressed in the blood vessels of zebrafish, but with different patterns of distribution.
In humans, prostaglandin products of COX-1 causes blood vessels to constrict and platelets to become sticky – the first step in a heart attack or stroke. COX-2 products, by contrast, are formed in blood vessels where they dilate blood vessels and prevent the activation of platelets. NSAIDs and aspirin block both forms of the enzyme. COX-2 inhibitors leave the hazardous COX-1 products unaltered, thereby perhaps predisposing some individuals to a risk of heart attack.
"We were impressed that COX inhibitors behaved much the same in zebrafish as in humans," said Grosser. An NSAID blocked the stickiness of zebrafish thrombocytes -zebrafish platelets – and prolonged the bleeding time, while a COX-2 inhibitor failed to exhibit these cardioprotective properties.
The researchers also illustrated the value of this model system by "knocking down" the COX genes. This revealed a new role for COX-1 during development, one that may have been masked in mice and humans by maternal formation of prostaglandins.
The high fecundity of zebrafish makes them a particularly attractive species for high thruput screening in the selection of new drugs. "Rapid acquisition of 'proof of principle ' is a particular challenge for the pharmaceutical industry, " said FitzGerald. "It is our hope that the zebrafish will facilitate this effort considerably."
This work was supported by the Alexander von Humboldt Foundation and the National Institutes of Health.