Twilight years for blood vessels

All blood vessels are lined by a monolayer of endothelial cells, which forms both a barrier and a gateway between the blood and the tissues.

Endothelial cells thus have highly regulated and precise functions to allow the correct passage of nutrients and molecules back and forth, and in processes such as inflammation to fight infection or tissue damage. However, they also contribute to and are essential for tumour growth and metastasis.

Professor Jenny Gamble, who heads the vascular biology program at the Centenary Institute in Sydney, has a long-standing interest in how blood vessels, and particularly endothelia, function at the cellular level under normal conditions and in disease – from vessel formation through to endothelial cell death.

At the Hunter Cell Biology meeting recently, Gamble spoke about the phenomenon of cell senescence, and more specifically, how it happens in the vascular system. She feels that for a mechanism causing such a major alteration in the phenotype of cells, senescence has been somewhat neglected by the cell biology field in recent years, with much more emphasis placed on other major mechanisms such as differentiation, apoptosis and the cell cycle.

And with their discovery of a potentially key gene in the senescence process, it is a rather exciting topic for Gamble’s group at the moment.

Cellular senescence is basically an irreversible halt in the cell cycle that causes the cell to stop proliferating and go into growth arrest.

Unlike in apoptosis, the senescent cell does not die and break up, but instead just sits there: not dividing, but still alive and metabolically active.

Senescent cells show typical changes in morphology, gene expression and function, and can be readily identified. However, it is the relevance of senescence to disease processes, and particularly for tumour formation, that is really capturing recent attention.

“Studying cell senescence has increased in popularity in the last five years or so because people now realise that together with apoptosis, senescence is a mechanism to halt uncontrolled cell proliferation, and thus has a potentially major role in inhibiting cellular transformation,” Gamble says.

Researchers showed a few years back that when cells become transformed – that is, they undergo an oncogenic change to become cancerous – but prior to the fully tumourigenic state, many undergo senescence.

“It seems that this is one way of the organism shutting down those proliferating cells and trying to control the cell transformation,” Gamble says.

Many premalignant tumours, including lymphoma and prostate cancer, contain numerous senescent cells, and it is only when the cell accumulates further oncogenic or cancerous changes that it can bypass the senescence mechanism to become fully transformed and progress to the metastatic state.

In fact, senescence is a rare event in these same tumours that have become metastatic. These findings, many of which were made in a string of studies published four years ago in Nature, have important implications for both tumour development and tumour therapy.

A particularly interesting study in this collection, by a group in the Netherlands, also implicated senescence as a physiologically normal and important protective process in humans. These scientists was studying moles or naevi, which are basically a bunch of benign senescent cells sitting around on top of the skin, sometimes for a whole lifetime.

However, some of these moles will have the potential to become malignant melanomas. Their results showed that senescence in the skin cells was acting to keep a potentially oncogenic state in check, rather than simply representing a loss of replicative potential.

---PB--- Senescence versus apoptosis

This idea of senescence having an important role in normal physiology was also raised by Gamble’s group around the same time with an unexpected finding that made her interest in cell senescence suddenly more than academic.

A screen for genes involved in angiogenesis yielded one candidate that causes endothelial cells to become senescent when overexpressed.

Fittingly, they called this gene SEN1 and began to look more closely at senescence in the vascular system.

“We wanted to know whether senescence commonly operates there, what are the signals that generate it and what are the repercussions of increased senescence for vascular function,” she says.

They went on to establish SEN1 as a sort of cell sensor – it was upregulated when endothelial cells were hit with conditions of stress, such as hydrogen peroxide (oxidative stress) or radiation, and these cells become senescent, a known possible outcome of stress signalling.

Gamble next asked whether SEN1 function was important under conditions in the vascular system when senescence is known to occur, such as in atherosclerosis.

On looking at atherosclerotic plaques in animal models, her group found that although there was still an endothelial covering, SEN1 gene expression was abnormally upregulated in the plaque region.

Although not proving that SEN1 is mediating the vascular senescence, it is tantalising to suggest this possibility.

A big question in this context, and one that Gamble is currently pursuing is what makes a cell that is in some type of trouble choose senescence over apoptosis.

Apoptosis in fact involves many of the same downstream signalling paths such as the p53 and p16 pathways. Both mechanisms are clearly going on all the time and particularly in the tumourigenic state.

“We know a lot about what senescent cells look like and how they act, and even a lot about what goes on to make senescence happen in a cell. However, we don’t know how the cell makes that decision in the first place.”

What are the upstream signals that shunt the cell down the senescence pathway?

A really interesting finding from Gamble’s work was that if SEN1 is overexpressed in an endothelial cell, that cell becomes senescent, but if you knock out the gene function, the endothelial cell dies by apoptosis.

“We think that the level of SEN1 expression in a given cell must be critically regulated. So, this gene might be a sensor for cellular alteration and depending on changes in the SEN1 expression (such as strength or duration), the cell will undergo apoptosis or senescence.

“So SEN1 functions as a fulcrum to determine how the cell will respond. The other interesting question is what benefit would the vessel have from each choice.” SEN1 will be a valuable tool to allow us to start dissecting this decision pathway for the cell, she says.

“Our working hypothesis is that SEN1 is a marker of senescence, defining regions of blood vessels where there is uncontrolled cell proliferation and inflammation.

“The major function of this gene is to get the vascular system back under control. We are looking now to see whether it also works more generically as one of the key genes acting in cellular senescence in all cell types.”

---PB--- Senescence limits the damage

Gamble’s group is also interested in the connection between ageing of the vascular system, senescence and cardiovascular disease.

Both genetic and environmental factors contribute to ageing, such as changes in gene expression that affect repair and defence mechanisms and oxidative damage to tissues and cells by free radicals.

“The interesting thing is that some of those biochemical pathways involved in senescence are the same ones involved in ageing – you get the p16 pathway activated for instance. We also know that both senescence in the vascular system and the incidence of cardiovascular disease increase with age.”

It has been proposed (and shown in some animal models) that vascular disease and ageing share many common mechanisms.

Endothelial cell senescence in both vascular disease and aged vessels is thought to be one such common mechanism that leads to vascular dysfunction, although the molecular basis of endothelial cell ageing has not been established.

Gamble proposes that as the vascular system is hit continuously over many years with stresses from diet and oxidation, as well as genetic and molecular mistakes that have to be defended, the vasculature continues to build up senescent cells as a way to limit the damage.

“In the end, there is an excess or multiple regions of inappropriately functioning senescent cells. However, is cell senescence with ageing the cause of the disease or a consequence of the disease that then contributes to disease progression?”

Gamble’s hope is that SEN1 provides them with a new tool to address these questions in vitro, in animal models and in a range of clinical material. It could be used both as a marker of senescence or vascular dysfunction, and as a way to turn the process of senescence on or off, initially in cultured cell and model systems.

To start with, Gamble’s group is profiling those endothelial cells that become senescent. They want to define the senescent phenotype and signalling pathways involved.

“This is a cell that is acting to control excessive proliferation and inflammation – it is performing its pre-ordained function, but is having some problems. We are trying to determine what those problems are and what are their consequences.”

The cell profiling is concentrating on all those functions that Gamble knows an endothelial cell does using a range of cell biological, molecular and physiological techniques.

Experiments will focus on the gene and signalling pathways an endothelial cell goes through to reach senescence, and of course incorporate SEN1 as a valuable tool in this part of the work.

In an all-out effort, they are even trying to crystallise the protein expressed by SEN1. Endothelial cells control vessel strength and permeability by maintaining adhesion at cell junctions and other cell-cell interactions. They also control the thrombotic nature of the vessel inner surface and the proliferative response.

“So, we are trying to build up a picture of what a senescent endothelial cell looks and acts like, and how that might impact on the total function of the vascular system, particularly with ageing and disease.

“It seems that there is an early-type response when it is advantageous for the vasculature to have senescent cells, but then if they fail to be cleared, the function of the system becomes compromised and then you don’t want senescent cells – and that is when the real damage to the vascular system happens.”