Benefits of testosterone therapy on the cardiovascular system

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T. Hugh Jones, MD, MB, ChB, BSc, MRCP, FRCP, Centre for Diabetes and Endocrinology,
Barnsley District General Hospital, United Kingdom

What I am going to talk about is the benefits of testosterone on the cardiovascular system. Now, for many years, testosterone was thought to have adverse effects on the cardiovascular system and you will see in most drug information leaflets that coronary heart disease is a relative contraindication and chronic heart failure is a contraindication, as well. But I want to try and dispel some of the myths, if you like, and let us go forward and start treating people with heart disease with testosterone where indicated.

You can see from this slide from the British Heart Foundation which looks at the distribution of male on the left here in red and black female on this side, and you can see there are a lot of men dying from coronary heart disease throughout the world. I think the UK is here in orange and the Czech Republic just a couple above, but up in Estonia, there is a very high incidence of death and Japan is quite low. Now, if you correct for the differences between risk factors between sexes, such as smoking differences between the male and female, or obesity, you are still getting a greater than two-to-one ratio, so what is it about males that make them more likely to get heart disease?

Over the years, there have been a number of cross-sectional studies, which have measured various androgens, mainly testosterone, but also DHEA and DHEAS, and they have used endpoints, such as myocardial infarction, coronary artery disease, and some have used angiography. But there has been no evidence except in one right down the bottom here which is Zumuf from 1982, which suggests that testosterone, high testosterone levels, are linked with coronary heart disease.

Associations of testosterone and DHEA with coronary heart data from prospective studies, and there are eight of those in the literature, and cross-sectional studies, (36), suggest that six out of the eight prospective studies show a neutral effect of testosterone and two, an antiatherogenic effect, but those are both of the DHEA. Of the cross-sectional studies, 12 neutral effect and 24 show an antiatherogenic effect. But really we have had no data from interventional studies.

This is just an information slide. When I talk about bioavailable testosterone, I am talking about both bioavailable testosterone as being the free testosterone plus the albumen-bound testosterone and we have assayed this directly using the modified method of Tremblay and Dube, using ammonium sulphate precipitation of the SHBG-bound testosterone. So you find out the percentage of testosterone, which does not bind to SHBG, and then you calculate that from measuring the total testosterone. There is a direct method.

We published this paper in the European Heart Journal in 2000. Nobody in any of the studies before had compared testosterone levels in men with positive angiograms with normal angiography. 

We found 60 men who had greater than 75% stenosis in at least one artery and we compared them with 30 males with normal coronaries, and these were males who were going to have angiography, they may have had chest pain or valvular heart disease but at angiography there was not evidence of atheroma. We found that, of these men with coronary heart disease, they had a significantly lower level of testosterone, with about 50% having hypogonadal levels of bioavailable testosterone.

These are the results from that study. If you measure the total testosterone, you can see the cases of 13.3 and the control is slightly higher at 15.3, and this did not approach a P value of less than 0.05, but it was getting there. But when you look at the bioavailable testosterone, the lower limit of normal, of which 2.5 is equivalent to a total testosterone round about 7.5 to give you an equivalent, you will see that there are significantly lower levels in patients with coronary heart disease compared with normal arteries. The free testosterone, this is used in the analogue assay, again showed significant lower change. We also interestingly found that men with heart disease had a slightly lower oestradial level than the normal controls.

So we went on and said, what is the size of the problem? How big is this problem? In South Yorkshire, an area that serves about almost 2.5 million people, there is one centre for coronary angiography, so there are a lot of people going through that centre. We decided to screen patients and we screened 1,146 men undergoing angiography in the morning and of those patients, 831 eligible patients had significant coronary disease. We defined hypogonadism, quite profound hypogonadism, with a total testosterone of less than 7.5 and/or a bioavailable testosterone of less than 2.5 nanomoles per litre. 

Blood was taken between 8:30 and 10:00 am, the age range was between 20 and 85 years, with a mean age of 59.9, and the BMI ranged from 12 to 64, mean 28.1. Patients were excluded from the original cohort if they had a c-reactive protein of greater than 10mg per litre or if they had malignancy, inflammatory disease, inter-current infection, uncontrolled heart failure and myocardial infarction three months before and hormonal therapy.

Of the 831 who had significant stenosis of greater than 70% in one artery, 14.1% had a total testosterone of less than 7.5, 19.9% had a bioavailable testosterone of less than 2.5. When you put these groups together, 23.4% had either, both or one of the above. So about one-quarter of these patients, these 831 patients, we are getting on to 250 patients who had hypogonadism, which is quite significant. We went on to examine these and 84% were symptomatic of hypogonadism. When you take the less strict criteria of a total testosterone of less than 12 or a bioavailable testosterone of less than 4.5, 52.6% of patients are hypogonadal, so there is a very high prevalence of hypogonadism in men with heart disease. 

We also found a positive correlation between hypogonadism and a history of hypertension and BMI, very positive. So this is a huge number of patients who potentially may require testosterone-replacement therapy, but should we be giving this to men with heart disease? We need to prove the point.

This is the age-stratified prevalence of hypogonadism. The pale grey bar here is total testosterone; the darker bar bioavailable testosterone and the white bar is a combination of both. You can see as you get older the instance, as you would expect, would get higher but, particularly in these age groups from 60 to 90, you can see that the bioavailable testosterone is more sensitive at picking up hypogonadism and this is because obviously the SHBG level rises with age.

I am not going to go into this in too much detail because other speakers have done so, but there is a lot of work to show that there are several associations between testosterone deficiency and cardiovascular risk factors. The lipid story is contradictory, but when you look at the elderly population, there are publications, which suggest that testosterone replacement is beneficial. There is the relationship with visceral obesity, insulin resistance, hypertension and also prothrombotic status.

From a treatment point of view, I am going to talk on three aspects. (1) angina, including vascular reactivity; (2) chronic heart failure; and (3) atherosclerosis.

So first of all, angina and vascular reactivity. 

History is interesting and if you look back in the literature, in 1939, the first published report on the effect of testosterone on vascular flow was in the New England Journal of Medicine, and this reported that testosterone increased the blood flow in the skin of castrated men and also benefited men with intermittent claudication. This was followed in 1941, Bonnell et al, who reported beneficial effects of androgenic and oestrogenic substances in 23 cases of angina where the high proportion of these patients felt better and had less chest pain.

Then there was a profusion of publications in the 1940s, between 1942 and 1946 over the war years, where several people looked at giving testosterone proprionate to men with angina and also it was given to some women. 

If you accumulate the data from these studies, you see that the numbers treated were small and they were not well controlled and there was an attempt in one and two to try placebo control, so these are not statistically proven. But of the 190 patients reported, 174 showed benefit. 

So there must have been something there but, if you look through the literature, there is nothing further for another thirty years when, in the British Heart Journal, Jaffe did a proper randomised double-blind, placebo-controlled study looking at post-exercise, ST segment depression and it showed that 32% of patients at a month improved their time to ST depression and at three months, 51%. Now the dose went to super physiological ranges, but there was a definite benefit. Then, again a gap in the literature until 1993, where this study by Wu and Weng, which was published in the Chinese Medical Journal and which is the paper I picked up on and which really led to the profusion of research that we have managed in our unit that was based on this study, where he gave oral testosterone to 62 elderly men with coronary heart disease, improved the symptoms in 77.4% and ischemia on an ECG by 68.8%. This was not a treadmill test this was standard ECGs. So there is something there, but it needed to be taken further forward.

We started our study using testosterone patches in 1997 but before we had completed the studies, two publications came out from London and Italy where intravenous testosterone was given in high supraphysiological doses, it was an acute administration, and it showed that treadmill testing after improved ischemia threshold.

Then, as Peter Collins told you yesterday, if you infuse acutely testosterone into the coronary arteries at angiography, this increases the coronary blood flow and diameter and this is his paper from then. He looked at 13 men with proven coronary heart disease at angiography and used intra-coronary infusion doses of 10 to -10 to 10 to -7, so the mid-part of this range was in the normal physiological range. He measured coronary artery diameter and blood flow, as I have said.

These are his results. Here we have the percentage change from baseline of the diameter of the coronary vessels and you can see that there is a dose-dependent response over the whole dose range and then the flow of the arteries also increased. So this is physiological levels and improvement. 

We published our study in Circulation in 2000 and we used the Andropatch, which is produced by Watson Pharmaceuticals in the United States. We had the idea for the study and it took about 12 to 18 months to get the patches and the placebo because import / export licenses for testosterone are really quite difficult, so we were waiting a long time to do this study.

The study involved was a double blind, randomised placebo-controlled add-on trial. The patients who were used in this study were people with severe angina, the majority of who were waiting for coronary artery bypass graft. They were on at least two drugs and most of them were on three anti-anginal drugs. When you do treadmill testing to see what this improvement of ischemia is, if you start a patient with angina on his first drug, you may get an improvement in exercise time of about 90 seconds on average, but with each drug that is added, that time drops by about 30 seconds. So it is important to realize that these were people with severe angina on multi-drug therapy and with an addition of a third or fourth agent in the form of testosterone, you may not expect to see any benefit. When you use treadmill testing, there is a learning curve for the patient and, in fact, the second test that they do, they can improve on the first. So we had a two-week single-blind placebo running phase and then 12 weeks of active treatment with assessment at one month of placebo treatment or three months at the end of the study. So we used treadmill testing with the primary outcome of ST depression of 1mm. We also got the patients to keep angina diaries and patients are notorious and not very good at doing that, so we also applied the health questionnaire, SF-36.

These were the results. This is the active group and this is the placebo group. You can see the baseline in the active group actually could go a bit further than the baseline, but if you look at the statistics, there was an improvement at one month and further improvement, which is interesting, at three months. 

If you look at it, dissect it down and look at the percentage change, the duration of time the patient, on average one month, was going was 34 seconds further before they got that ST depression of 1mm, and 52 seconds further at three months. These patients were feeling a lot better. They did not statistically show a reduction in angina. Individual patients did and I had one particular patient who kept on the patch afterward and if he came off the patch, his angina came back two or three times a day. When he went on the patch, his angina disappeared. So certain people have different sensitivity. I am not going to go into the questionnaire in great detail, but what essentially was happening was that these patients were feeling physically better, they were doing more, they were actually doing more before they got their angina and they were much happier in themselves, their mood improved, as well.

What the interesting thing was is that there was a correlation between the baseline bioavailable testosterone and the response to ischemia. You can see up here, this is the change in time to 1mm ST depression at week 14 and this is the level of the baseline bioavailable testosterone, so the lower your baseline testosterone, the better your response to ischemia. This is a very important thing to know because you need to assess testosterone and if it is low, then those patients are going to benefit. So someone with a testosterone of 20 may not benefit from this anti-anginal effect.

To prove it further, we had gone on to do the effect of testosterone therapy on people who are quite hypogonadal with angina. We have done a randomised, single-blind placebo-controlled crossover study of intramuscular testosterone and we have only used a dose of Sustanon, which is testosterone esters 100 every two weeks, and this is low dose. Normally, in the United Kingdom, you will try to take them up to this dose every week. What we did was a one-month on placebo or treatment and they had a one-month washout phase and then crossed over to the other phase and 10 men were studied. Their total testosterone was 4.4, so this is very low. All had greater than 70% stenosis of at least one coronary artery and/or a proven myocardial infarction and the time to ST depression was assessed.

What we have here is at a month; these patients were walking 73 seconds further before they got their ST depression, so much more effect in someone who is hypogonadal. 

Just as an aside, there was this paper in JCM in 2001, in men who had induced hypogonadism as a result of GnRH therapy for their prostate cancer and you can see these are the wave forms of the radial and aortic arteries before treatment and after treatment. This delay that comes in here shows that the arteries are getting stiffer. So low testosterone leads to stiffening or reduced vascular reactivity of arteries. 

So what is the mechanism of this vasodilatatory action of testosterone? The evidence now from our work and others is that the testosterone effect happens within two minutes so it cannot be a genomic effect. The earliest genomic effect that has been seen in biology is 40 minutes. It is endothelium independent so it is not working through a similar mechanism to oestrogen because oestrogen needs the endothelium. What it is showing is that it acts directly on the smooth muscle cell itself, it is non-genomic and it is independent of the classic androgen receptor. We have done this two ways: we have used mice which have got a frame shift mutation of the androgen receptor making it inactive and the arteries from the mouth, the iliac arteries, still respond to the same extent with testosterone. We have also, if you use flutamide, it does not get rid of the dilatatory effects. It binds directly to the cell membrane and we have shown, in fact, that it acts as a calcium antagonist and we have been taking this work further forward to try and look at which calcium channel is it blocking, what effect did it have. I will talk about that in the next slide.

So just to show you, we use a myograph system where the artery is taken out from the animal or human and the artery is kept under tension with two wires and you constrict the artery with potassium chloride or prostaglandin. So we maximally constrict the artery prior to adding testosterone. The blue line is the control of the whole artery and the red line is with the endothelium removed and you remove it by rotating a human hair which you put inside the artery and twist it round, which is histologically proven to remove the whole endothelium, and you can see you get a similar effect. The problem here is the dosage required. You can see that we are in the micromole range for testosterone action, which is not physiological. But you remember in these parafusion systems or myograph systems you are actually pushing the fluid through the artery and you may not get levels of testosterone at the artery consistent with what is in the fluid and it may be much lower. So that is one of the criticisms, is testosterone a vasodilatation?

So we went on and working with a group in the University of Leeds who specialize in looking at the L-type calcium channels and they have human embryonic kidney cells which express just the Alpha 1-C subunit of human cardiovascular L-type calcium channel that does not express any other calcium channels at all. We can also look at the rat aortic smooth muscle cell line, which is stable. There are no human cell lines, which are easy to look at. What we found here is, using this patch clamping, this is the percentage in addition of calcium into the cell, you will see that the effect, this is 1 nanomole, 10 nanomole, 13 nanomole, so we are getting effects which are quite dramatic over the physiological range.

This is the effect of 1 nanomole of testosterone, so we are getting this inhibition of flux of calcium across the cell and that is similar to the slide I have just shown you.

The effect of testosterone. This L-calcium channel, which is the pore we transected into the cells, is the site at which Nifedipine and other calcium channel blockers act. So is testosterone a natural Nifedipine-type agent? 

By looking at calcium florescence, if you stimulate the cells with high-dose potassium, you get maximum calcium fluorescence. Ethanol has to be used to dissolve the testosterone, so it is not having a significant effect and then we are increasing doses of testosterone again, sensitive within the physiological range, you are getting inhibition of calcium flux into the cell. So it is inhibiting calcium into the cell. This is the effect of Nifedipine, 5 micromole, which you can see is similar to the maximal effect of testosterone. So have we got a natural Nifedipine-type agent? It is a bit more complex than that.

There are various sites, and I am not going to go into this slide in too much detail, but testosterone in the literature has now been shown to have actions on four different mechanisms of controlling calcium release into the cell. If you lose potassium from the cell, that triggers voltage-operated calcium channels like the one we have just described for calcium to come in. There are also store-operated calcium channels and receptor-operated calcium channels and testosterone has been shown, albeit in higher doses than most of these, to inhibit each of these mechanisms.

I am going to talk about chronic heart failure next. 

Looking through the literature, there is only one study on the use of testosterone in heart failure. Now chronic heart failure is a major public health burden and the survival of heart failure is similar to many cancers, so the five-year survival is very poor. It is a disabling, progressive disorder, characterized by reduced exercise capacity and breathlessness, poor quality of life associated from muscle wasting and depression. 

We looked at the acute haemodynamic effect of testosterone using a buccal tablet. We took 12 men with stable, chronic heart failure, we had a placebo-controlled crossover study, so patients came in one day, received a placebo, went home, came back the second day and had testosterone or vice versa, and we put canulism tubes in them to measure all aspects of heart and vascular circulation. We used two buccal testosterone tablets, 60mg, and using this dose we got the testosterone level up to about a mean of 18 nanomoles per litre, so quite high, and we measured all of these parameters. In a nutshell, the two things that came out positive were cardiac index. When you actually insert all of these catheters, the cardiac index has a natural fall, so this is the placebo effect. On the testosterone treated, this did not happen, so cardiac index was maintained. 

Then if you look at peripheral vascular resistance, this is the placebo group and this is the treated group, it reduced - sorry, that is the active group and that is the placebo group - it reduced peripheral vascular resistance quite significantly, which was maintained throughout the study time. 

If you look at the baseline testosterone again, again showing this point, this is the treatment effect on cardiac index and that is on peripheral vascular resistance, you will see that the active substance was more effective, more beneficial on cardiac index. What we have here are the median testosterone levels, so if a testosterone level with a bioavailable median of 4.5 compared to a lower testosterone, so the lower the baseline testosterone you get, the better improvement in cardiac index. Here, it is vice versa, you get more systemic vascular dilatation if the baseline testosterone was low. Again, this is a very sensitive response to the initial testosterone level.

So then the question is, does testosterone therapy benefit men with chronic heart failure? 

We then embarked on a study because we thought the background to this, we knew it was contraindicated in heart failure, but people with heart failure are wasted so testosterone is anabolic, it has various effects on hormone immune activation, which may be beneficial. It is a vasodilator as we have shown and we have also shown that men with heart failure have got lower testosterone than DHEA levels. So relative androgen deficiency in men with heart failure contributes to the path of physiology and symptoms. So does replacement improve the outlook?

There is one study in the literature where they use Oxymethalone and 12 men with heart failure were given this drug for three months. It increased left ventricular rejection fraction, reduced LV diameter and mass. Single centre, single author, open-label, no placebo, so it was not a well-done study, but it did show a positive effect. 

We did a randomised double blind, placebo-controlled trial of testosterone therapy in male patients with chronic heart failure with the primary outcome measuring a distance walk on the shuttle-walk test. 

So they had 12 weeks of treatment, we used testosterone esters again, Sustanon 100, so we did not have to wait for import / export licenses and we just gave it fortnightly, so low dose testosterone IM injection and non-parametric tests were used for the statistics.

The shuttle-walk test is strongly and independently predictive of the peak VO2 in chronic heart failure, so it is the best test of functional capacity. Patients walk up and down a 10-meter marked course by two cones, one at each end, and they have to walk around each cone. The speed is controlled by a series of beeps and the speed is increased by increments over 12 levels, as shown here. 

So level one is quite easy, but when you get to level 12, you are really walking quite fast with 2.37 metres per second compared with 0.5, and you are walking more shuttle distances. A shuttle is from one cone round and back to the other cone. So it is very well validated.

We looked at various parameters - heart rate, blood pressure, ejection fraction, fractional shortening and others. 

We had 20 men, ambulant, stable heart failure, mean age 61.5 and ejection fraction of 34.8%. The mean total testosterone was 12.7. All of these patients completed the trial.

So we had half the patients on active treatment and half on placebo. The black lines are the accumulated for each, but these are the individual lines and you can see some patients are much more sensitive and did a lot better. 

Some on placebo did better, but the point is, the statistics show there was a definite improvement in capacity and the capacity improvement was an increase of 95 metres walking, which is the length of an American football pitch I am told, and this for people with severe heart failure is very, very significant. So this is a three-month study.

We also looked at Minnesota Heart Failure Score, which was positive, so these people were positive on a score test. 

Also the Beck Depression Inventory showed that they had improved in their mood. Had they improved in muscle bulk or muscle function? 

We looked at CT scans of thighs and also grip strength, but none of these over the three-month period in this group showed any statistical benefit. Now there may be benefit on improved muscle action at the myofibril level.

There was nothing that was definite on these other markers.

So we have shown in heart failure that we can increase functional capacity, improve symptoms and mood, but we did not show any particular change after three months in LV function, hemodynamics or muscle bulk. 

So testosterone therapy may be useful as an add-on for improving functional capacity. This week we have completed a one-year study but we have not got the results for that.

Now I am going to talk about atherosclerosis in the last five minutes. 

This is important - in atherosclerosis, the plaque are eccentric, so you have got all this function in artery and this is where you get the vascular reactivity and the response. 

It has been shown in male rabbits that if you remove their testes, they will develop more atheroma on a high cholesterol diet. However, if you replace that testosterone, it inhibits atheroma. Replacement after a period of hypogonadism led to plaque regression. 

So there is animal evidence, and I am going to skip through that.

We were quite interested in is testosterone going to improve the actual plaque or atheroma? 

Now these need long-term outcome studies, but we have looked at inflammatory disease because people with atherosclerosis have higher inflammatory markets, particularly IL-1α, IL-1β, IL-6, tumenacrosus factor and interferon gamma, and the anti-inflammatory is IL-10. If you look at our cohort of the population with coronary heart disease in men, you will find out that we have shown the interleukin-1 levels in patients with one artery disease compared to three are a lot lower, so it is a lot higher to do with the vascular burden with IL-1.

But we have also shown that with IL-1 here, if you are eugonadal with coronary heart disease, your IL-1 levels are lower and as your testosterone level falls, your IL-1 level increases, so there is a suggestion that testosterone has or is linked in some way to immunosuppression. 

We went on to look at 27 hypogonadal male subjects with confirmed cardiovascular disease in a randomised crossover study using the Sustanon 100 again, so it was one month on, washout phase, one month on the placebo or the drug. We measured early-morning fast in blood for all the inflammatory markers.

Essentially, we have shown that, this is with placebo and this is with treatment, testosterone treatment in hypogonadal men lowers IL-1β and also lowers TNF-Alpha. 

Now TNF-Alpha, especially in heart failure, is one of the worst predictive factors that death is going to occur, so maybe testosterone, in addition to its other effect, is having immunological benefit.

In summary from atheroma, epidemiological studies show either a neutral or a positive correlation with low testosterone levels. Recent studies using measurements of atheroma burdens show positive correlations with low testosterone and positive correlations of low testosterone with atherogenic risk factors. Animal work suggests testosterone has anti-atherogenic effects. Testosterone may exert athero-protective effects by suppression of pro-atherogenic cytokines but we are also looking at the effect of testosterone on smooth muscle apoptosis or growth. 

So the pathogenics of atherosclerosis as we know is complex and multi-factorial, there is increasing evidence to suggest that testosterone may play a role in the male, but the major questions to be answered are (1) is testosterone deficiency a cause or consequence, (2) does testosterone replacement reduce vascular events and ameliorate atherogenesis in the future? 

In all of our studies, especially in heart failure where testosterone in the literature is contraindicated, we have not shown any adverse effects of testosterone on the hematocrit, fluid retention or on lipid profiles. Compliance in this group of men is high. Any dropouts from our studies have been mainly due to local skin reactions to the patch.

So, in summary, there is increasing evidence that testosterone has beneficial effects on angina, heart failure and, potentially, atherosclerosis. There is little evidence in the literature that testosterone has adverse effects. Now is the time to take this further with longer-term trials, especially five-year outcome studies, which, obviously, would cost a lot of money.

In conclusion, we have quite a large group here, and I want to thank everyone who has been involved in this because the work obviously would not be possible. Also, the importance of collaboration between two specialties - Endocrinology, myself, and Cardiology, Kevin Channer. Richard Jones is responsible for a lot of the lab data myography. Kate English and Peter Pugh for a lot of the clinical work on the heart disease. Joanne Hall, Katherine Kerry, Joanne Nettleship for a lot of the assays and cytokine work, and we have collaboration with Leeds, with Professor Peers on the patch clamping, and, also, we have looked at arteries from mesentery pulmonary and we needed collaboration from surgeons, both in Sheffield and Hull, to work at this.