After centuries of playing second fiddle to the blood system, our lymphatic circulation is coming into its own as a key player in diseases ranging from cancer to asthma. Phyllida Brown reports.
Sometimes it's hard to be a scientist. Just five years ago, Mihaela Skobe, a molecular biologist now at Mount Sinai School of Medicine in New York, and her team were struggling to publish their work on the lymphatic system. Editors and reviewers thought it boring. The polite but yawning rejection letters came. “They would say that everything in the paper was fine but that lymphatics were unimportant,” says Skobe. “There was a complete lack of interest.”
Not any more. Once dismissed as a mere drainage network, the body's ‘second circulation’ system (see Graphic, below) is emerging as a crucial player in numerous diseases from cancer to asthma, and as a vital part of the normal immune system. As a result of these discoveries, researchers are trying to intervene in its activities, for example to reduce the spread of tumours, to boost the efficiency of vaccines, or to treat the painful and disfiguring swelling known as lymphoedema.
“It's a hot field now,” says Michael Detmar, a dermatologist and lymphatic system researcher at Harvard Medical School in Boston, Massachusetts. These days, he says, many laboratories are switching from the study of blood vessels to the lymphatic system. Detmar is one of several researchers showing how important the lymphatic system is for tumour spread. Other groups are finding it plays a key role in inflammatory diseases such as asthma or transplant rejection. “We are in the pioneering phase,” says Detmar. “There is a feeling that there is still so much to discover.”
It might seem strange that one of the body's major organs is terra incognita. Lymphatic vessels may be drains, but they are pretty sophisticated ones. Not only do they collect fluid that has leaked into tissues from the bloodstream and return it to the blood; they also process that fluid, sending it through lymph nodes where key cells of the immune system, called dendritic cells, present fragments of foreign molecules to other cells to mount an immune response.
But the fact is that the lymphatic system has played second fiddle to the blood system for centuries. Only properly discovered in the 1600s (see A brief history of our second circulation), it faded into obscurity and, apart from a brief flurry of interest around 1900, was largely neglected until about ten years ago.
The turning point came in the early 1990s when Kari Alitalo, a cancer researcher at the University of Helsinki, began studying a family of proteins involved in generating new blood vessels. These proteins, called vascular endothelial growth factors, or VEGFs, stimulate the growth of cells lining blood vessels and enable new vessels to sprout. Tumours often subvert these signals to build a blood supply that nourishes their invading mass. Like other scientists, Alitalo reasoned that if these signals, or the receptor proteins that enable cells to receive them were blocked, blood vessels could be prevented from growing, and tumours could be starved to death.
But what Alitalo and his team discovered next was to lead their focus away from blood-vessel growth signals and into the backwater of the lymphatic system. He and his team happened upon a new VEGF receptor that was present mainly on the cells that line the insides of lymphatic vessels1. The receptor was similar to a known receptor for VEGFs but neither of the two members of this family, VEGF-A or VEGF-B, activated it. So the hunt for the signal was on.
When they eventually found this signal, they discovered that it was an endothelial growth factor similar to the known VEGFs. They named it VEGF-C and the receptor VEGF receptor 3 (ref. 2). In mice that had been genetically engineered to express excessive amounts of VEGF-C, the lymphatic vessels proliferated but — crucially — the blood vessels did not. This was the first signal known to act specifically on the lymphatic system3. Meanwhile, on the other side of the world, Marc Achen and Steven Stacker at the Ludwig Institute for Cancer Research, Melbourne, were hunting for further VEGFs. Faxes flew from Melbourne to Helsinki and back. Together, the researchers soon identified another signal that also acted on VEGF receptor 3. This one was dubbed, not surprisingly, VEGF-D (ref. 4).
“It was very exciting,” says Alitalo. He and the Australians hoped that their discoveries could eventually lead to treatments to help build new lymphatic vessels in people suffering from lymphoedema — for example after breast cancer surgery.
But progress was hampered by the fact that there were no ‘markers’ for the lymphatic system. These are proteins characteristic of the tissue being studied that scientists use to specifically target dyes, and so to see the tissue more easily. It was not until 1999, when David Jackson, a biochemist at the Weatherall Institute of Molecular Medicine in Oxford, discovered such a marker, a protein called LYVE-1 (ref. 5), that lymphatic research really went into orbit.
For one thing, researchers could now probe the role of the lymphatic system in the spread of cancerous tumours. Many tumours, such as breast cancer and melanoma, spread from the original tumour via the lymphatic system to other organs, and a person's prognosis is worse if lymph nodes are involved. Yet, although the role of blood vessels in tumour spread had been well studied, researchers knew almost nothing about whether tumour cells actively persuaded lymphatic vessels to grow and assist their spread or whether lymphatics were just passive ducts.
Alitalo's group, together with Michael Pepper at the University of Geneva Medical Centre, and Gerhard Christofori at the Research Institute of Molecular Pathology in Vienna, created genetically engineered mice that developed tumours in the pancreas and had abnormally high levels of VEGF-C in the same organ. The team found lymphatic vessels sprouting in the animals' tumours. What is more, the animals' lymphatic vessels often contained tumour cells that had originated in the pancreas6.
Other teams also found evidence pointing the finger at the lymphatic system as an active agent in spreading tumours. Detmar and Skobe transplanted human breast cancers, engineered to make a lot of VEGF-C, into mice, and found lymphatic vessels sprouting within the transplanted tumours. Indeed, the greater the degree of lymph-vessel growth, the more the tumours spread in the animals' lymph nodes and lungs7.
“There is now little doubt that the interplay between tumours and the lymphatic system is the main route used by solid cancers to spread.”
There is now little doubt that this interplay between tumours and the lymphatic system is the main route used by solid cancers to spread. But could clinicians one day intervene to stop it? Quite possibly. Stacker and Achen and colleagues from Melbourne showed that VEGF-D was, like VEGF-C, capable of triggering lymphatic vessels to grow inside transplanted tumours in mice. It also increased the spread of tumours to lymph nodes. But when VEGF-D was blocked, this increased spread could be checked8.
Alitalo's group meanwhile, showed that mice genetically engineered to have VEGF-C and VEGF-D signalling blocked could not make new lymphatic vessels; their existing vessels even shrank9. The team then blocked VEGF-C and VEGF-D signalling in mice with human-breast-tumour transplants, and found it could reduce the amount of tumour spread by two-thirds10. The implications were clear: if VEGF-C and VEGF-D could be blocked in a mouse, then perhaps they could also be blocked in people with cancer to help prevent tumour spread.
Mice to men
Evidence shows that tumours in people behave like those in mice. Across a range of different human tumours, those that contain high levels of VEGF-C or VEGF-D are more likely to spread11. As a general rule, the more lymphatic vessels there are in the tumour, the greater the risk. Stacker and Achen are hopeful that trials testing agents that block VEGF-C and VEGF-D can begin soon. After all, points out Achen, an antibody called bevacizumab, or Avastin, that slows the growth of blood vessels in spreading colon cancer by blocking VEGF-A, has already prolonged some patients' lives.
But Alitalo warns that such trials may be a long time coming. One problem, he says, is that tumour spread can be a slow process, and pharmaceutical companies are wary of embarking on costly trials that take as long as three years to reveal results.
As if all this were not news enough, it seems that the lymphatic system has been hiding an even bigger surprise up its sleeve. Over the past year, several research teams have begun to uncover evidence that the lymphatic system could be a major culprit in unwanted inflammation. Inflammation plays a key role in asthma, which affects an estimated five million people in Britain alone. It is also associated with other common conditions including psoriasis and rheumatoid arthritis, and with some medical problems such as transplant rejection.
Dontscho Kerjaschki, a pathologist at the Medical University of Vienna, has been studying what happens when kidney transplants are rejected. Normally, the kidney cortex, the part that filters blood, has hardly any lymphatic vessels. But in about a third of biopsies from transplanted kidneys, Kerjaschki found a 50-fold increase in the number of lymphatic vessels12. In most cases, such biopsies came from patients with chronic rejection, a condition in which the graft continues to be attacked by the host's immune system after initial immunosuppression treatment, until the transplant breaks down. Kerjaschki thinks the unusually extensive lymphatics could be involved, speculating that they may bring a continual supply of immune cells into the graft. “Maybe the lymphatics are organizing the whole catastrophe,” he says.
Hints that the lymphatics can mastermind an immune response come from the discovery of a protein called podoplanin by Kerjaschki's team. Podoplanin sticks to a signalling mol-ecule called CCL21, which is found mainly in lymphatic vessels. CCL21 is a powerful attractant to immune cells such as dendritic cells and macrophages13. In kidney grafts, where some inflammatory cells are already present, the complex of CCL21 and podoplanin breaks down, releasing CCL21 into the vessel, and so attracting further inflammatory cells.
Another piece of the jigsaw implicating lymphatic vessels in harmful immune responses comes from Donald McDonald, a vascular biologist at the University of California, San Francisco, and his colleagues. They have studied a mouse model of asthma. The animals' lungs are chronically infected with Mycoplasma pulmonis, a bacterial infection that produces swelling of the mucous membranes, alterations to vessel linings and scarring — all symptoms that resemble those of human asthma. McDonald's group found that infected mice grew additional lymphatic vessels in their tracheas, and their airway blood vessels also proliferated. When the team treated the mice with antibiotics, the blood vessels shrank but the extra lymphatic vessels persisted14.
“This was a surprise,” says McDonald. He speculates that the new lymphatic vessels help to set the lung up for more rapid and accentuated immune responses to subsequent infections, exacerbating inflammation. His team is now looking at the signalling molecules involved, hoping that it may eventually be possible to manipulate the inflammatory immune responses to help control asthma.
Back in Helsinki, where it all began, Alitalo is upbeat. He is happy that there are still fundamental questions to answer, including how cancer cells move beyond the lymph nodes as they travel through the vessels to distant organs, and how exactly tumour cells enter lymphatic vessels in the first place. And Skobe? She and her team are getting to grips with the molecules in lymphatic vessels that are involved in tumour spread. Skobe is not giving away details of her latest work yet. But when she does have new results to report, it seems unlikely, this time around, that the journal editors will need convincing.
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