Monthly Archives: August 2010

Heart failure breakthrough: animal research paved the way!

Heart failure, where the heart is unable to maintain a sufficient blood flow to supply the body’s needs, is a leading cause of death, especially among the over 65’s. Half of all chronic heart failure patients die within four years of diagnosis. It can have a number of causes, for example damage to heart tissue after a heart attack, and leads to a variety of problems in patients. Fatigue and muscle weakness are common as the muscles receive insufficient oxygen, and because waste products cannot be removed from tissues quickly enough fluid can build up in the lungs and other parts of the body, often the legs and abdomen. The extra strain placed on the heart as it tries to maintain adequate blood pressure can lead to further damage to the heart and ultimately cardiac arrest.

Ivabradine can lower the heart rate while maintaining a normal blood pressure - good news for heart failure patients. Image courtesy of the CDC Public Health Image Library.

In heart failure the rate at which the heart beats is often increased, and group of scientists led by Karl Svedberg and Michael Komajda set up the SHIfT study, to evaluate whether a drug called Ivabradine, which lowers the heart rate, could reduce risk of death or hospitalization in a group of patients who had heart failure accompanied by an elevated resting heart rate.  Significantly fewer patients taking Ivabradine in addition to their existing treatments required hospital admission during the course of the study, compared to a control group who were given a placebo in addition to their existing treatment. The most striking outcome was that Ivabradine cut the risk of death by 26%.

So what is Ivabradine, and where does it come from?

Ivabradine slows the heart rate by inhibiting an electrical current known as the If current* which is a major regulator of the activity of the sinoatrial node – better known as the pacemaker. Inhibiting the If current slows the generation of the electrical impulses by the sinoatrial node that trigger heart contraction, and therefore slows the heart rate itself. Ivabradine, then known as S16257, was first developed in the early 1990’s when it was found to be able to block the If current in-vitro in sinoatrial node tissue from rabbits and guinea pigs, and slowed the generation of electrical impulses in a manner that was safer than other bradycardic drugs (1). Ivabradine was then evaluated in live rats and dogs, where it safely reduced the heart rate, and moreover did so without reducing the blood pressure (2,3). While beta-blockers such as Propranolol can reduce the heart rate they also lower the blood pressure – indeed they are used to treat hypertension – and hence are not suitable for many patients, so the development of a drug that could reduce heart rate without affecting blood pressure was very welcome.

Following the successful animal studies Ivabradine entered human clinical trials and in 2005 was approved for the treatment of angina pectoris. In angina pectoris the heart muscle receives too little oxygen, a problem exacerbated by a fast heart beat that increases the need for oxygen, so lowering of the heart rate by Ivabradine reduced oxygen demand and prevents angina attacks. The success of Ivabradine in the treatment of angina pectoris in turn led to its evaluation in heart failure.

The successful outcome of SHIfT study is a major boost to the development of better treatment regimes for heart failure, and if it is confirmed by further clinical trials will improve and prolong the lives of many heart failure patients.

* Hence the name of the SHIfT study – Systolic Heart failure treatment with the If inhibitor ivabradine Trial

Paul Browne

1) Thollon C. et al. “Electrophysiological effects of S 16257, a novel sino-atrial node modulator, on rabbit and guinea-pig cardiac preparations: comparison with UL-FS 49.” Br J Pharmacol. Volume 112(1), Pages 37-42 (1994) PubMedCentral:PMC1910295

2) Gardiner S.M. et al. “Acute and chronic cardiac and regional haemodynamic effects of the novel bradycardic agent, S16257, in conscious rats.”  Br J Pharmacol. Volume 115(4):579-586 (1995) PubMedCentral:PMC1908496

3) Simon L. et al. “Coronary and hemodynamic effects of S 16257, a new bradycardic agent, in resting and exercising conscious dogs.”  J Pharmacol Exp Ther. Volume 275(2), Pages 659-666 (1995) PubMed:7473152

Mice, rats, and the secrets of the genome.

It’s just over a decade since the completion of the first working draft of the human genome was announced, and seven years since the publication of the complete sequence, but in that short time the impact of this new knowledge on all areas medical research has been immense. Sequencing the human genome was a huge achievement, but having got the sequence an even greater task confronts scientists – working out what it all means.  To do this scientists have studied the natural variations that exist between individuals, and have also sequenced the genomes of a wide variety of species, some closely related to us, others separated from us by hundreds of millions of years of evolution. Scientists can analyze the similarities and differences between the genes of different species, and examine how changes to the structure or regulation of these genes are reflected in physiology.  In many cases it is also possible to use genetic modification to study the function of conserved genes in other species in ways that are just not possible, for technical and/or ethical reasons, in humans. A study published a couple of weeks ago in the scientific journal Nature provides an excellent example of how animal research contributes to our understanding of the human genome.

Blood flowing through the heart, but the cholesterol building up in an artery can cause a heart attack. Image courtesy of the British Heart Foundation

As the cost of technology such as DNA microarrays has fallen genome-wide association studies (GWAS) have become an increasingly popular way of examining the relationship between genetic differences between individuals and particular diseases. In a GWAS the whole genome of many individuals is screened for variations, and then any association between those variations and particular phenotypes or diseases is determined.  Tanya M. Teslovich and colleagues (1)  analysed the genomes of over 100,000 people who had been enrolled in 46 separate clinical studies, and identified 95 genes that have variants associated with increases in blood lipid (fat and cholesterol) levels.  One of the problems with GWAS studies is that while they are often good at identifying genes that are associated with a disease, they are not so good at identifying which genetic variations actually cause disease, or explaining how the genetic variations contribute to disease.  This is where Tanya Teslovich and colleagues scored highly; they were able to show that 14 of the 95 lipid-associated genes were also associated with the development of coronary artery disease, supporting the proposition that elevated blood lipids contribute to coronary artery disease. They also found that overall the effect of the variants was additive, the more risk variants of these 95 genes you have the greater your chance of having elevated blood lipids.

So that established that the gene variants were associated with elevated blood lipids, but to use that information to develop new treatments you need to know how the particular gene affects lipid levels. As you might expect many of the 95 genes identified were already known from previous studies to be involved in the regulation of blood lipids, and in several cases their precise role has been thoroughly studied. However, several of the genes had not been implicated in regulating blood lipids before, and the team decided to use genetically modified mice to investigate their function.  They injected viral vectors into the liver of the mice that contained either an extra copy of the gene being studied, to increase expression of the gene, or a short-hairpin RNA, to target the gene for knockdown via RNAi. This allowed them to discover that one gene, GALNT2, decreases levels of high-density lipoprotein cholesterol (HDLC), the so-called “good cholesterol”, while two other genes, Ttc39b and Ppp1r3b, increase  HDLC.

Another associated paper (2) in the same issue of Nature takes the analysis even further. Several studies, including the GWAS performed by Tanya M. Teslovich and colleagues, had demonstrated that variations in a particular region of chromosome 1 known as 1p13 were associated with high levels of Low-density lipoprotein “bad” cholesterol (LDLC) in the blood and heart disease, but that these variations were not within the coding sequence of any genes, so they would not affect the structure of any proteins. They first show through genetic studies of human subjects and human liver tissue culture that variations at 1p13 affect the expression of several genes –  and hence the amount of protein produced by those genes – and that one particular variation creates a binding site for the transcription factor C/EBP. Transcription factors are proteins that regulate the expression of genes, and this particular site altered the levels of a gene named SORT1. But what does SORT1 do? To answer this they again turned to GM mice, using virus vectors that specifically reduced or increased the levels of SORT1 in the mouse liver. Reducing or eliminating SORT1 expression in the mouse liver led to a reduction in the levels of LDLC in the blood, and that this was found to be due to SORT1 regulating the production of very-low-density lipoprotein (VLDL), a precursor to low-density lipoprotein, in the liver. As a result of this work a whole new pathway for the regulation of blood lipids has been uncovered, one that may offer new opportunities to scientists developing treatments for hypercholesterolemia.

As a BBC news report indicates, the identification of these genes and the elucidation of their function may aid the development of better diagnostic tools to identify those at risk of heart disease, and ultimately the development of better treatments.

These studies illustrate how important animal models, particularly GM mice, are to efforts to decode the human genome. As the biosciences move towards a more systems based approach to biology, one where knowledge of how networks of genes interact to produce a particular physiological or clinical outcome is applied to areas such as toxicology, the information that studies of GM animals can yield will become increasingly important. This importance has not gone unrecognized by the wider scientific community, the 2007 Nobel Prize in Medicine was awarded to Mario Capecchi, Sir Martin  Evans, and Oliver Smithies for their discoveries of  “ principles for introducing specific gene modifications in mice by the use of embryonic stem cells”.

With this in mind let’s turn briefly to another GM animal that’s been in the news lately – the rat.  While GM mice have become a mainstay of modern scientific research the rat has lagged behind, which is a shame since the larger size, longer lifespan, and more complex behavior of rats make them more effective animal models than mice for studying many human diseases, particularly neurological conditions. The lack of GM rats was due to the difficulty in growing rat embryonic stem cells (ESCs) in culture, a necessary first step in the most common methods of producing GM animals. Last year Matthew Evans wrote an article for the Pro-Test blog discussing how scientists at the University of Cambridge and the University of Southern California had developed a method for growing rat ESCs in culture, and how this achievement paved the way for the production of transgenic rats. Last week the same group of scientists announced that they had employed this method to produce GM rats whose p53 gene, a key tumor suppressor that is defective in several cancers, was deleted.

The humble lab rat, now available in GM. Image courtesy of Understanding Animal Research.

This is not the first time GM rats have been produced, as for the past few years scientists have been able to use zinc finger nucleases to knock-out rat genes. Zinc fingers, so called because one or more zinc ions stabilizes the finger like structure, are found in many proteins, allowing them to bind specifically to a structure within a cell, such as a particular DNA sequence. Scientists found that they could produce artificial zinc fingers that recognize particular genes, and then join a nuclease to that zinc finger so that it cuts out the target gene. This method, discussed in more detail in this excellent article by Elie Dolgin, allows scientists to knock-out genes in rat embryos. The downside of the zinc finger nuclease technique can only be used to knock-out genes, whereas the ESC method is more flexible – it can also be used to add extra copies of a gene, or to delete genes in specific tissues or stages of development.

It is now clear that the rat is joining the mouse at the forefront of the GM revolution in medicine, and that has to be great news for medical science and the patients that depend on it.

Paul Browne

1)      Teslovich T.M. et al. “Biological, clinical and population relevance of 95 loci for blood lipids” Nature Volume 466, Pages 707-713 (2010) DOI:10.1038/nature09270

2)      Musunuru K. et al. “From noncoding variant to phenotype via SORT1 at the 1p13 cholesterol locus” Nature Volume 466, Pages 714–719 (2010) DOI:10.1038/nature09266

Of mice and mTOR: Can damaged spinal cords be taught to repair themselves?

There’s an interesting story on the BBC website about new research on nerve cell regeneration after spinal cord damage in mice, work undertaken by a team led by Dr. Zhigang He of the F.M. Kirby Neurobiology Center at Children’s Hospital Boston.

Nerve regeneration in mice requires mTOR. Image courtesy of Understanding Animal Research.

Those of you who follow developments on the field of spinal cord repair may find this story familiar. The study published online in Nature Neuroscience this week (1) follows up on work by the same team published two years ago (2) which examined whether regeneration of the optic nerve of mice could be promoted by using an adenovirus-based vector to locally delete several genes known to suppress cell growth. They found that knocking out gene named PTEN in the optic nerve encouraged regeneration of nerve cells following damage. PTEN inhibits the activity of the enzyme mTOR which is an important promoter of cell growth and survival, so removing PTEN increases mTOR activity and promotes regrowth of damaged nerve tissue. In agreement with this that found that mTOR levels are high in nerve cells of the central nervous system (CNS) in mice seven days after birth, when nerve cells are still naturally capable of regeneration and repair, but far lower two months later when such nerve regeneration is no longer observed. mTOR is a key regulator of cell growth throughout the body, and it’s activity is highly conserved throughout the evolutionary tree.

The latest study demonstrates that nerve regeneration through activation of the mTOR pathway does not only happen at the optic nerve but can also be induced in other parts of the CNS, with obvious implications for repairing spinal cord damage.

As the BBC report points out this is still quite preliminary work; the study demonstrated that the regenerated nervous tissue had all the features of normal nervous tissue, including the ability to form synapses that are needed to transmit signals from one nerve cell to another, but they have yet to show that it improves the function of the damaged nerve tissue. Other factors will need to be used alongside PTEN inhibition to encourage the regenerating cells to form a bridge across the damaged section of the spinal cord. The authors point out several promising approaches to achieving this are currently under development, for example growth factors that guide the growth of regenerating nerve cells to the correct location.

The PTEN knock-out approach used in these two studies, while useful in laboratory studies, is not suitable for the clinic because its effects are permanent. It should, however, be possible to develop a drug, perhaps using RNAi or Morphilinos, to temporarily turn off PTEN activity at the site of injury and promote nerve regeneration. Whatever approach is used it will be important to be able to target the increase in mTOR activity to the site of injury. mTOR is involved in the regulation of cell growth throughout the body and indiscriminate activation of it might have adverse consequences, after all several drugs are under development that turn off mTOR activity in cancer cells.

All in all it is an interesting piece of work, even if it only goes part of the way towards developing a therapy that can repair damaged spinal cords and prevent paralysis. This is an exciting time for research on treating paralysis, with a variety of techniques under development. These range from transplantation of stem cells such as Olfactory ensheathing cells or embryonic stem cells, to the development of robotic limbs controlled through brain-machine interfaces. Such a variety of approaches is entirely justified by the complexity of the problem that needs to be solved, not every technology will be appropriate for every patient, and furthermore it is likely that the regeneration-promoting technique described in this week’s report may help to increase the effectiveness of stem cell transplants.

It’s just another example of how important animal research is to progress in one of today’s most exciting areas of medicine.

Paul Browne

1)      Liu K. et al. “PTEN deletion enhances the regenerative ability of adult corticospinal neurons” Nature Neuroscience, published online 8 August 2010; doi:10.1038/nn.2603

2)      Park K.K. et al. “Promoting axon regeneration in the adult CNS by modulation of the PTEN/mTOR pathway.”  Science, Volume 322(5903), Pages 963-966 (2008) doi:10.1126/science.1161566

Leicester – The New British Battleground?

Back across the pond, in Leicester (pronounced “les-ter”), animal rights activists are warming up for a battle against a new £15 million (around $24 million) biomedical facility which the University of Leicester is building. Looking through the local rags, an interesting article came up in “this is Leicestershire” from a reporter who took a look round the current facilities.

So let’s set some context to the story:

The university has never let the media in before. They’re allowing it, they say, to set the public record straight.

Access was accepted with no preconditions and no promise to push the university’s side of the story.

So, here we are, on the threshold, fumbling into surgical scrubs, pulling on polythene overshoes that will stop us contaminating the facility with the outside world.

A security card is swiped, a pin code punched and a pair of heavy orange doors slowly part.


The facility manager says: “Treat this like a royal visit. If you see a door you want us to open, we’ll open it for you.”

Conditions – like similar labs across the country – are second to none:

Cages are clean, relatively roomy and well-stocked with plenty of things to gnaw at, burrow through or make nests in.

Clipboards full of completed job sheets show they’ve been inspected daily and had their cages changed at least weekly.

A big stainless steel cage-washer runs almost constantly in a room down the corridor.

Animals can be monitored every five minutes if the experiment they are undergoing puts them at risk of suffering or stress, says the facility manager.

Home Office inspectors come in every six to eight weeks. They can also make unannounced spot checks.

Every experiment, even on a humble mouse, has to clear a university ethics committee.

These ethics committees are comparable to the IACUC committees that exist in labs across the United States.  In this Leicester Lab (which is looking to improve its facilities with a new £15 million lab) the 3Rs are at the forefront of researchers:

The more you see, the more you realise everything in here is controlled and moderated.

Nothing, not even the wood shavings these rodents use as a bed and toilet, contains a rogue variable.

The shavings are sourced from Finland. They are ground down from white wood aspen trees because red woods contain chemicals that can be harmful to mice.

The chippings are sterilised and irradiated so no bugs or bacteria can influence the results of experiments.

That makes for better science, says Prof Barer.

Fortunately, the author also makes mention of the benefits which animal research brings to society.

The benefits of animal research are there for us all to see, say animal test supporters.

Foods which help to prevent cancer have been identified in this Leicester lab, as have new ways to get oxygen into bodies after lung failure. That’s the kind of science that is helping desperately premature babies to survive and could yet save thousands in a flu pandemic.

Prof Barer works in the field of TB research.

“Tuberculosis kills two million people every year,” he says.

“If I see an opportunity to reduce the suffering caused by that disease through the careful, considered use of animal research, then I will.

“I don’t like it, but I think it is justified. As a diabetic, I’m someone whose life expectancy is directly related to discoveries made in animals.

What is more interesting is the absolute steadfast blindness shown from animal rights activists in the area:

Protestor Chris Williams believes animal experimentation is wrong morally and ethically, and is driven by bad science.

He also believes they are experimenting on dogs and primates in the University of Leicester.


“I’m 110 per cent certain they’ve got dogs in that building and 90 per cent sure they’ve got primates”, he says.

Unless the university is lying to the Home Office and funding the research covertly, he is mistaken.

Chris is a spokesman for the Stop the Leicester Animal Lab protest.

He doesn’t have a job. He’s been campaigning, pretty much full-time, for the best part of two years.

Fortunately the reporter actually gathers his facts, rather than creates them.

Chris accuses the university of being economical with the truth.

The same could be said of the Stop the Leicester Animal Website.

None of the horrific photographs it contains – dogs and rats in desperate states – come from the facility at Leicester.

“It wouldn’t be a very effective website if we didn’t have photos,” says Chris.

But the suffering shown on their website doesn’t come from Leicester. Perhaps people should be told that.

The article is a nice piece which looks at some of the conceptions and misconceptions surrounding animal research. Perhaps animal rights activists need to spend more time being reporters themselves and finding out what actually happens in labs themselves rather than trusting every YouTube video they find.