Angiogenesis was barely a blip on the horizon when you started your research in the 1980s. What was it that motivated you?

I simply wanted to better understand how blood vessels grow. This field was launched by Judah Folkman and his colleagues in the early 1970s, but what had been learned was mostly descriptive. People were interested in the cellular mechanisms of the process, but very little was known about the molecules involved. At that time, VEGF, which stands for vascular endothelial growth factor, was one of the most specific growth factors affecting the cells that actually line the blood vessels, so that was a very promising candidate for being a major player in angiogenesis. There was no really formal proof in vivo, however, that this was an important molecule. One way to achieve that at the time, and still today, was to make a knockout mouse for VEGF.

Yes. When Napoleon Ferrara and my group made knockouts of VEGF those were actually the first knockouts ever made in the angiogenesis field. For that matter, the field of knockout mice was very young, too. The first one made was in 1989. I happened to be at the Whitehead Institute at the time, where there was a lot of interest in this knockout technology. It wasn’t that easy at the time. And for the VEGF knockout in particular, there was something very funny and strange about it.

Well, there are two copies of every gene—two alleles. And if you knock out one allele, you have one inactive allele; usually the mouse is sterile. In mice missing both alleles, that’s when you usually see the interesting phenotypes, some of which are lethal. With VEGF, even the mice lacking a single allele died very early on. That was unprecedented. And that made it a real challenge. Originally we thought it wasn’t working. Napoleon Ferrara thought the same.

We realized that the embryos in these heterozygously deficient mice were actually showing the phenotype expected. We characterized the vascular defect. We also used special technology, pioneered by Andre Nagy in Toronto, to actually generate homozygously-deficient embryos in a single step. Usually you breed heterozygously deficient embryos, and then cross-breed those. But since we couldn’t do that, this was the only way to obtain mice that were homozygously deficient for VEGF. They showed an even worse phenotype. Basically that study was something of a landmark seminal study because it showed that VEGF was really very critical for embryonic vascular development. That got a lot of interest from the entire field. That was the first sign. Later on, people started studying the role of VEGF in pathological conditions. Now it has become clear that VEGF is the major player in angiogenesis.

Yes. And it was published back-to-back with the similar study by Napoleon Ferrara.

The VEGF gene was cloned in 1989. I started working on it in 1991 and then it was submitted for publication in December 1995. So it was quite a battle. And the gene was very difficult to clone initially, as well.

Yes, because the phenotype was so spectacular. So people immediately started to believe that VEGF was an important molecule. This was really making a true difference. It was an important player. We showed also that blood vessels are the first organs formed in the embryo. With the VEGF knockout mice, we realized these embryos died very early on, which demonstrated that angiogenesis was very critical for the embryo. So this was a breakthrough on several fronts.

Absolutely, everyone was. Especially this fact, this first example, of what is called a haplo-insufficient phenotype—meaning the lack of a single allele already results in such a severe phenotypes.

As I said, this was a very descriptive field through the early 1990s, and what was lacking was the identification of which molecules were involved. This has now changed dramatically. Now we know a lot of the molecules and whether they’re important or not. And so now we also know how to inhibit these molecules—using what are called rational inhibitors such as antagonists and antibodies—and we now have data from three clinical trials showing that anti-VEGF antibodies, for instance, are actually very effective in fighting cancer. They’re now approved by the FDA in the US. So the field is still young, but it has evolved with enormous rapidity, and we’ve gone from a point where we knew almost nothing about the molecules involved to having developed drugs that fight cancer.

At that time, and it’s still the case, the science was so exciting, so young, and there were so many obvious questions to ask that the entire field was very social and friendly. Very collegial. This is quite different from fields that have been long established and it actually gets more and more difficult to do top science. It becomes more competitive, and then the industry gets involved, and people don’t talk about their results and so on and so forth. But there’s been none of that in this field. There’s always been plenty of new science to step up and grab.

We’re still doing quite a bit, but we actually have also identified a novel function for VEGF. It’s not just about blood vessels now. By making more subtle genetic deviations in the VEGF genes, we actually made a mouse model of the motor neuron degenerative disease, amyotrophic lateral sclerosis, or ALS, which in the States, of course, is known as Lou Gehrig’s disease. It’s a very dramatic incurable paralysis that’s caused by a slow degeneration of the neurons that innervate the muscle. By chance we found out that VEGF is also playing an important role in this disease. These mice with a genetic mutation in the VEGF gene actually develop all the symptoms and signs of ALS. We just published another paper in Nature where we used VEGF gene therapy for treatment of ALS in this mouse model. We’re still evaluating whether we can use the VEGF protein for treatment of the disease.

The disease is so dramatic that no single treatment has shown that you can reverse it. What you can do, at least in rat and mouse models, is slow it down. You delay the disease onset and prolong survival. It seems VEGF is not only having an effect on the blood vessels directly but also on the neurons, particularly large motor neurons.

We have been working for the last couple of years on better understanding the role of the other members of the VEGF family. In particular, what’s called PLGF, which stands for placental growth factor. That molecule differs from VEGF in many different aspects, and it also seems to be a player in angiogenesis, but only in cancer and inflammation—not in the embryo. That makes it an attractive molecule, because if you block it with an antibody you are not going to affect the normal vasculature. This is different than anti-VEGF antibodies, where they also affect normal vessels. In the long run, those sorts of side effects will become a concern for anti-angiogenesis gene treatments. So we need additional molecules besides VEGF, and that’s why we’re exploring PLGF inhibition, and why this will be part of our research for the coming years. We’re also further exploring whether we can use PLGF—either gene delivery or protein treatment—to stimulate vascularization in tissues. That is still a major line of research. And now quite recently, we’re also focusing on understanding how vessels are guided to their target. You know nerves have to face enormous challenges in finding their way from the brain and the spinal cord to their final targets, and this new information about VEGF and neurons tells us that there may be similar and common mechanisms and molecules and guiding tools used by blood vessels, axons, and nerve endings. That’s a very exciting development. So we are studying that in much more detail.

Peter Carmeliet, M.D., Ph.D.
Center for Transgene Technology & Gene Therapy
Flanders Interuniversity Institute for Biotechnology
University of Leuven, Campus Gasthuisberg
Leuven, Belgium