Why do you think your paper is highly cited?

This review challenges a number of prevailing views on matrix metalloproteinases (MMPs), brings together an enormous amount of information in a critical way, and has an important positive message for the future development of matrix metalloproteinase inhibitors (MMPIs). For instance, we challenge the views that MMPIs' primary disease targets should be invasive or metastatic cancer or that broad-spectrum MMPIs will have any benefit for long-term therapy (of cancer and chronic diseases). By now, it is clear that MMPs are key players in inflammatory and vascular diseases, which constitute much better targets than cancer.

Cancers are genetically unstable, and thus less predictable, whereas, in inflammatory and vascular diseases, the genetics of all tissues are normal, the predictions will be simpler and the readouts will be clearer for chronic inflammation and vascular disease models.

Another mistake from the past, namely oversimplification, should be avoided. By now, we know that MMPs have many substrates in physiology and pathology and that specific MMPs are constitutive, whereas others are induced, for instance, during infections or inflammation. The latter ones are ideal targets for the development of highly specific inhibitors, whereas the inhibition of constitutive MMPs will certainly lead to side effects.

Another point of view is that we are in favor of and contemplate the short-term use of MMPIs for acute and life-threatening diseases, rather than long-term use. Maybe this is less attractive for commercial developments, but we give sufficient examples in the review that should stimulate this research.

Finally, we have invested an enormous amount of energy and critical thinking to summarize the literature on MMP knockout mice and MMPI studies. Stimulating the thinking that many currently available knockout mice are in fact leaky (thus not really knockout), that the essence of science is in confirmation, comparison, and complementation, that not the factual results but the interpretations which we give to in vivo results may often be wrong; these are all essential contributions of this article.

 Does it describe a new discovery, methodology, or synthesis of knowledge?

The article has several layers of information. It gives a synthesis of knowledge on MMPIs and MMP knockout studies. It describes a number of new views and insights as to why previous MMPI studies failed and, at the same time, outlines many opportunities for how the money invested in excellent academic and pharmaceutical research on MMPIs may bring enormous returns to patients, medical professionals, and society.

The synthesis of the present knowledge of MMPIs was considerably enhanced thanks to the insights, critical appraisal, and eye for details by the group of medicinal chemists of Professor Qing-Xiang Amy Sang at Florida State University, USA. In this way, the work is a truly transcontinental effort. This was further enhanced with the help by the staff of Nature Publishing Group.

One trigger to write this review was to further enhance the work of our postdoctoral fellows. Dr. Jialiang Hu developed a new high throughput methodology for screening MMPIs and, by using this, was able to simulate evolution in the test tube and to discover a novel peptide inhibitor for MMP-9 with which we could block the acute lethality of endotoxin shock in mice.

These findings are quite stimulating, particularly in view of the fact that currently, about half of the patients with severe sepsis or shock syndromes, even in our modern hospitals, may not survive their stay in the intensive care unit. The use of MMPIs in sepsis syndromes really needs to be carefully examined.

More than a decade ago, Dr. Liesbet Paemen developed another method for screening and discovered that tetracyclines are MMPIs. In addition, we used her method with success to make the first inhibitory monoclonal antibodies against MMP-9. These antibodies are still today the most selective inhibitors against human MMP-9.

Last but not least, to define MMPIs one needs the enzyme—lots of the enzyme. In fact, together with Dr. Stefan Masure and Dr. Philippe Van den Steen, our group has always invested a lot in the production of the enzyme MMP-9. For years, we used buffy coat neutrophils from thousands of blood donations to produce natural MMP-9. Afterwards, we added a recombinant production line. This gave us the additional opportunities to study domain and point mutants of the protein.

In conclusion, scientists from three continents have integrated their work and ideas in the present scientific literature with the hope being of stimulating future developments.

 Would you summarize the significance of your paper in layman's terms?

Drugs that have been developed and still can be improved for the treatment of cancer, may be much better developed for the treatment of acute inflammatory (for instance meningitis, sepsis, acute exacerbations of arthritis, and multiple sclerosis) and acute vascular—for instance, stroke—diseases.

 How did you become involved in this research, and were there any problems along the way?

In 1954, Piet De Somer founded The Rega Institute for Medical Research, which has established a great tradition in translational research, in particular, in virology and immunology. De Somer's younger mentors, Alfons Billiau, Erik De Clercq, Hendrik Eyssen, and Michel Vandeputte, each maintained an open mind towards innovation and excellence and are quite inspiring personalities.

My doctoral work was already on a proteinase: the well-known serine proteinase t-PA. During my postdoc at Yale University with Peter Lengyel, I refined my knowledge and practical skills on the interactions of cytokines and proteinases. After my return to the University of Leuven in 1987, I started working on MMPs and quickly chose to concentrate on MMP-9, alias gelatinase B, because this enzyme was exquisitely induced and regulated by inflammatory cytokines and chemokines.

This educated choice was a really good one: from the present approximately 20,000 PubMed entries on all MMPs together, about half are on MMP-9. Together with a great colleague and friend, Jo Van Damme, I had the opportunity to create an environment that is challenging and complementary in terms of technology (molecular versus cellular versus in vivo) and in topics (cytokines, chemokines, and proteinases). In doing so, we kept a focus on inflammation research.

It would be incorrect to say that I have never encountered problems in my research, but these problems are sublimated by the friendship of collaborators, both locally and in top institutes abroad, from Oxford to Heidelberg, and from Dublin to Rehovot.

 Where do you see your research leading in the future?

MMP and MMPI research is not different from any other topic; we try to dig further and deeper and, to achieve this, one needs to bring together expertise from many disciplines. One dream is to one day have the crystallographic picture of full-size human MMP-9. This project must be doable in collaboration with other experts and the future picture will tell us more than a thousand words.

Another project is the definition of the spectrum of intracellular MMP-9 substrates. We know a number of important secreted extracellular substrates quite well and have recently listed all known membrane-associated substrates, whereas colleagues abroad are using state-of-the-art proteomics technology to define the repertoire of all extracellular substrates. However, MMP-9 as an inflammatory enzyme must have many substrates from inside cells. Whether and how this enzyme contributes to cleavage of molecules that are released when cells die and how this might contribute to the origin or development of diseases are key questions which keep us busy. In return, solving these questions by using a multidisciplinary approach might give new momentum to MMPI research.

Using recombinant MMP-9 domain mutants, we hope to be able to understand how monomers and oligomers interact with substrates, in other words to understand molecular complex formation, also because this knowledge will give insights on how to develop new types of MMPIs for specific pathologies. Posttranslational modifications of MMPs are also insufficiently understood in terms of functions and potentials for applications.

Thanks to the support by Prof. Raymond Dwek and Prof. Pauline Rudd, MMP-9 has become a model molecule for glycobiology research, a discipline with a great future. In a collaboration with Prof. Bernd Arnold from the German Cancer Research Center, we developed, 10 years ago, two lines of MMP-9 knockout mice with brown fur: one leaky MMP-9-deficient line, and one nonleaky real knockout line.

Because of subfertility problems in the latter mice, it took us 10 years of patience and a bit of good luck to backcross the knockouts to C57BL/7 for 13 generations in order to obtain, in the end, black MMP-9 knockout mice. Only now can we think of animal model experiments that will hopefully show the real role of MMP-9 in health and diseases.

Meanwhile, we remain convinced that perhaps the simplest way to generate a highly selective MMP-9 inhibitor is by hybridoma technology. We have successfully made such inhibitory monoclonal antibodies against human and mouse MMP-9. If, one day, patients will be helped with such monoclonals or derivatives, all our efforts will have been worthwhile.

 Do you foresee any social or political implications for your research?

In the 1980s, we started molecular biology research at the University of Leuven and transferred technology to many Belgian scientists in this discipline. In the 1990s, research on MMPs was superimposed on our cytokine and chemokine gene cloning work and the MMPI project was started. In addition to the start of an amplification effect, I can now count 10 postdocs as Rega Institute alumni. They each successfully continue to work in inflammation research at universities or governmental and pharmaceutical laboratories.

In 1995, we discovered that tetracyclines (e.g., minocycline) inhibit MMP-9 and might be useful for the treatment of inflammatory and vascular diseases. Tetracyclines are inexpensive drugs with a high therapeutic index. It is gratifying to see that, on the basis of our studies, several clinical trials have shown the beneficial effects of tetracyclines for the treatment of multiple sclerosis (MS). In 2003, Dr. Inge Nelissen from our laboratory described that recombinant beta-interferon (used for the treatment of multiple sclerosis) is degraded by MMP-9, which is exactly the enzyme that is induced in the central nervous system of the patients suffering from MS.

As a medical professional, and being concerned about the health of individual patients and also about the societal costs of healthcare, I have been trying on many occasions to advocate the combined use of tetracyclines and beta-interferon for treatment of MS: most probably this will reduce many observed side effects of monotherapy with interferon and, intrinsically, this will be much less expensive.

I foresee that one day our science policymakers will have enough vision and courage to sponsor double-blind clinical trials of such combination therapies of beta-interferon with tetracyclines or other MMPIs versus monotherapy and that the industrial managers will have the insight that it is possible to reduce therapy costs with presently available drug combinations, while improving the quality of life of the patients.

Ghislain Opdenakker, M.D., Ph.D.
Professor of Immunology
Head of the Department of Microbiology and Immunology
Leuven University Medical School
Rega Institute for Medical Research
Leuven, Belgium
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Keywords: matrix metalloproteinases, inflammatory and vascular diseases, sepsis syndromes, inhibitory monoclonal antibodies, interactions of cytokines and proteinases, inflammatory cytokines and chemokines, extracellular substratesO.