Lymphatic mispatterning

Natasha Harvey will head to ComBio this year with a few years’ worth of results and an exciting new story of a protein called Nedd4 and how it could hold a key to vascular networks and their problems.

Derived from the Latin word for ‘fresh water’, lymph and its transport through a complex network of vessels and nodes helps the body maintain its homeostasis.

The lymphatic vessels do this by returning tissue fluid and protein to the bloodstream. They also have a role in absorbing dietary lipids from the digestive tract and shuttling cells of the immune system to where they are needed.

The lymphatic circulation has long been the neglected cousin in cardiovascular research and we still know surprisingly little about how the whole system is constructed and regulated. But, according to Associate Professor Natasha Harvey, head of the Lymphatic Development Laboratory at the Centre for Cancer Biology, SA Pathology, all that is changing as lymphatic research is going through a ‘boom’ time.

A developmental biologist, Harvey’s research seeks to pinpoint key signalling pathways and molecules used in forming the lymphatic vascular system (lymphangiogenesis) during development.

“The cardiovascular system, comprising the heart, blood vessels and lymphatic vessels, is the first organ network to develop in the vertebrate embryo,” she said.

The growth, development and function of lymphatic vessels also tends to ‘go wrong’ in several disease settings in humans. Abnormal vessel formation manifests in conditions such as lymphoedema and vascular malformations, whereas excessive lymphangiogenesis is associated with inflammatory diseases and cancer.

By identifying and characterising healthy lymphatic vessels, Harvey is aiming to find new therapeutic targets that block or promote the growth of lymphatic vessels. That is the plan anyway.

Revisiting a successful partnership

At ComBio, Harvey will present one of the major projects ongoing in her group.

“For the last 4 years or so, we have been looking at the role of a ubiquitin ligase called Nedd4 in the development of lymphatic and blood vessels,” she explained. “Several years ago, we found that embryos lacking Nedd4 expression (knockout mice) have striking defects in cardiovascular development and since then have been trying to understand what Nedd4 is doing during that process.

“This is being undertaken in collaboration with Nedd4 guru Professor Sharad Kumar, who I actually did my PhD with, so it is also great to be working with him again on this.”

Nedd4 belongs to a family of functionally diverse enzymes involved in ubiquitination, a process of protein modification that is essential for many aspects of cellular regulation. The attachment of ubiquitin to target proteins marks them for degradation, regulates receptor internalisation into cells and directs protein trafficking within cells.

Proteins such as Nedd4 thus have the potential to control cell function at multiple levels. For instance, Nedd4 regulates cell growth through the insulin-like growth factor 1 and insulin signalling pathways, and functions in the stability and trafficking of a range of other plasma membrane proteins. However, evidence from many sources suggests that Nedd4 has additional and important cellular targets.

“Our current work on Nedd4 started when Sharad obtained the knockout mice and noticed that the embryos had a cardiovascular defect, so he asked us to take a look at them,” said Harvey.

On first inspection, the embryos showed obvious oedema and haemorrhage, a clear indicator of problems with the development of their blood and lymph vessels. “We then looked further at the patterning and formation of the lymphatic vessels and could see abnormalities, particularly in the skin. The vessels looked sparse, spindly and unhealthy, and were formed into island-type structures. Basically, they were strikingly mispatterned,” Harvey reported.

Because Nedd4 is a ubiquitin ligase, Harvey hypothesised that without Nedd4 being expressed, proteins within vascular cells were not being degraded normally or were signalling abnormally and impacting on vascular development.

Pictures tell the story

“This work has involved a lot of confocal microscopy imaging - often using whole-mount stained embryos as we are really trying to get a 3-dimensional view of the vessel patterns. We have spent a lot of time working on techniques to visualise the vessels with as much detail as possible.”

This approach allows Harvey’s team to really examine how the lymphatic vasculature is built during development - what pathways it takes, how it gets connected together as development proceeds and where the signals that drive that patterning come from. The result is some quite stunning imaging that has been made possible by recent upgrades in imaging capabilities at the Centre for Cancer Biology.

“We also work with primary endothelial cells, which we purify from embryonic skin and manipulate in vitro to look at cellular functions such as proliferation, migration and tube formation. Then, by exposing the cells in culture to different signals or by modifying their gene expression using siRNA, we can assess what genes regulate the processes we want to look at in these cells.”

Indeed, Harvey’s group pioneered methods to purify, culture and transfect primary blood and lymphatic vascular endothelial cells, which have traditionally proven difficult to culture. Their work has paved the ways for new and improved assays to dissect gene function and signal transduction pathways in these cells ex vivo.

The ideal endpoint

Harvey’s talk will represent a large body of work that she described as “just coming together at the moment - we are trying to get that last piece of the puzzle to publish a really strong and interesting story”.

Their work has basically confirmed that Nedd4 is crucial for morphogenesis of the blood and lymphatic vascular networks during development.

“We can see that Nedd4 is regulating pathways important for vascular development, although we don’t know exactly how yet and that is what we are now aiming to define. But, because Nedd4 is important for both blood vessel and lymphatic development, it really takes us further in understanding the signalling pathway required for construction of those vascular networks.

“We are also certain that these pathways are in turn involved in disease settings, and by understanding the developmental growth mechanisms we hope to identify targets that could be modified to stop abnormal vessel growth such as in tumour metastasis or to promote the growth and repair of lymphatic vessels in conditions such as lymphoedema. It is a long way down the track, but that is the Holy Grail for our type of work.”

Associate Professor Natasha Harvey currently heads the Lymphatic Development Laboratory at the Centre for Cancer Biology, SA Pathology. She completed a PhD and postdoc in the Division of Haematology at SA Pathology in Adelaide before a postdoc stint in the US at St Jude Children’s Research Hospital, where she focused on vascular development.