We are going to actually discuss some of the basic cellular molecular mechanisms of erectile function and the effect of androgen on these mechanisms.
Erectile function requires normal endocrine milieu. We have to be cognizant that endocrinology is an important aspect of this endeavour. Also, we have to really realise that intact innervation of the corpora cavernosa is important, so neurogenic components are important. Also we have to understand this question that just came out, that there should be adequate arterial blood flow. Any vascular disease that can alter this will be of consideration, and I think this is an important point that is missed many times, is that there has to be adequate compliance of the sinusoidal tissue. I want to make one important point here from the outset. A lot of people think that “how come I took my testosterone and I still don’t have an erection”? It is not the PDE-5. It works differently. How does it work differently? The penis is an electro-mechanical pump. And this pump, like any other hydraulic pump needs maintenance. You have to maintain this pump. Testosterone is one of those hormones that is required for maintaining this pump therefore, it is not a light switch but rather something that functions as maintenance in maintaining the cellularity, the cellular function. Therefore, we have to look at it from this point of view. I think this is one of the issues that confounds the literature, that we do not really know if androgen is important for a human erectile function. In animal models, yes, it has many times been shown but in a human we do not know. Why because we expect that I’m going to pop a pill at 6:30 and by 7:00 I’m doing, it doesn’t work that way.
Let’s look at the cross-section of a penis; you can see the two cavernosal bodies, corpus spongiosum, the cavernosal artery and this spongy tissue, which we call the cavernosal body. If we take a microscopic part of it, and we look we see the trabecular smooth muscle, we see the sinusoid lined by a single layer of endothelial, and during the flaccid state, right now all of you sitting here none of you have an erection, unless I am not aware of it, your penis is flaccid. In the flaccid state you are not trapping the blood because the smooth muscle is contracted, and therefore, very minimal blood inflow. There is a maximum blood outflow and therefore the penis is flaccid. Upon sexual stimulation something happens, which vasodilator cavernosal artery, which allows blood inflow to increase. This increase in blood inflow stimulates the endothelial to produce more nitric oxide, which increases the vasodilation of the trabecular
smooth muscle, which relaxes, which allows the entrapment of blood and therefore the penis engorges, and you get an erection. Really, if we have to look at it we have to look at this as a global mechanism, which we call veno-occlusive function, which means during erection the whole pump has to comply by trapping blood under pressure. And this is really the key issue, how the penis traps blood under pressure. And this brings me to the issues that I am going to discuss with you. Androgen effect is not only on one aspect, but on multiple aspects, including the trabecular smooth muscle, the endothelial, the nervous, the cavernosal artery, the connective tissue and the metabolic aspect, which I will discuss in a few minutes.
To have a good erection you must have reasonable arterial dilation the trabecular smooth muscle must relax, venous compression must take place, and it is only when this happens you will have then a blood filling of the sinusoid and you prevent blood outflow by compressing the sub-tunical venules against the tunical albuginea to impede blood outflow, and therefore you have engorgement and you have erection.
So let’s put it in a cartoon fashion. In the flaccid state you have a poor inflow, but you have unimpeded outflow, the smooth muscle is in contractile fashion, and therefore you have no trapping, and therefore you are still in the flaccid state. Upon sexual stimulation vasodilation of the cavernosal arteries and the (inaudible) will increase blood inflow. This allows the smooth muscle to relax. You begin to trap blood then you compress the sub-tunical venules against the tunical albuginea. You impede blood outflow and therefore you get an erection.
Testosterone actually regulates almost every aspect of this process in terms of maintenance. The only thing I want to focus here is hormonal because I am interested in the role of androgen in regulating erectile function
I don’t think anyone in this audience disagrees with me that we know since 1950 that androgens are important for development, maintenance and growth of erectile tissue. That is common knowledge. What the issue at hand is that is testosterone really important in human erectile function? And I think that is what the clinical research, and the best research, has to come together and find out the answer to this question.
When you go from 9 years old to 18 years old we notice that as your androgen levels or as your testosterone levels go up, your penis grows. So there is no question that androgen is important for erectile growth, and for maintaining of erectile tissue.
Why is it difficult sometimes to make an argument that androgen ise important for maintaining the erectile tissue health? Because I showed in the very first slide that unless you have erectile tissue health, maintenance of innervation network, and maintenance of the smooth muscle and the cavernosal arteries, all in a healthy way you are not going to have a good erection. So our framework works as follows – testosterone can be metabolised into 5- a DHT by the 5- a reductase, and either 5- a they had with testosterone or testosterone would bind to the androgen receptor. And binding to the androgen receptor in the corpus cavernosa will illicit metabolic function including activation of the neural nitric oxide synthase (nNos), endothelial nitric oxide synthase (eNos), expression of PDE-5 and many others. Also, the androgen receptor will regulate changes in the nerve fibres, smooth muscle content, connective tissue deposition, and, an interesting observation we made recently, is the regulation of the adipogenesis, or the pathway of the lineage of the fibrostroma progenitor cells going either into smooth muscle cells or into adipocytes. When you have healthy erectile tissue you have a functional veno-occlusion. So therefore our endpoint, or our outcome measure as you will put it in your clinical setting, is the veno-occlusion dysfunction. So, if everything is going smoothly, veno-occlusive function is working. If any alteration in the metabolic or structural component of the tissue is altered due to androgen deficiency or androgen insufficiency or androgen deprivation, the veno-occlusive function will malfunction, and if that is the case, there will be no erection.
All the studies I am going to show you are animal model studies. That is why I am taking you from your clinic into the laboratory. Let’s look at the veno-occlusive function. Here is a group of male rabbits, which are intact, and if we look at the intracavernosal pressure in response to electric field stimulation of the cavernosal nerve you can see that there is a nice increase in the intracavernosal pressure. If you castrate these animals two weeks later, if you repeat the experiment, if you stimulate the pelvic nerve you can see that there is no trapping of blood because due to androgen deficiency, due to castration, something happened, both metabolically and structurally. Therefore the veno-occlusive function malfunctions or failed, and therefore no trapping and no erection. If you take a group of animals, which were castrated and now you treat them back with androgen, you actually recover. So this is a reversal process within that frame of time. If you give them oestrodial instead of testosterone, you do not recover the veno-occlusive function. Therefore androgen is absolutely critical, at least from this point of view, for maintaining the veno-occlusive function. Why? Because we believe that it maintains many aspects of the metabolic and structural components, as I will show you in a few minutes.
This is a very drastic measurewhen you take an animal and you do orchiectomy, you completely remove the source of testosterone because in our rodents and in other animals, the adrenal does not contribute much. So we also repeated this study using LHRH agonist, Lupron, and as you can see, we compared: this is the control, this is the orchiectomised, the surgical model. But also if we do the medical castration 2 weeks, 4 weeks and 8 weeks, 8 weeks after Lupron it almost mimics what we do with surgery, although the testosterone level is not completely as though in the castrated model. But you can see the intracavernosal pressure is significantly reduced by medical castration almost similar to what we see in the surgical castration. It does not matter which mechanism that leads to androgen deprivation, or androgen deficiency, both mechanisms will lead to a functional dysfunction of veno-occlusive mechanism, and erectile dysfunction.
This is really an important point for many of you who see patients and you want to prescribe PDE-5 to them. If they are androgen deficient, PDE-5 will not work. Here is the experiment and this is supported by work from the group of Dr. Maggi from Italy. This is the control, this is a group of animals, which were castrated but now if you give them vardenafil you can see that you do not return back to the normal state, in fact you stay exactly as in the castrated state. In other words PDE-5 alone, if the animal is androgen deprived or androgen deficient, it does not work. You can do that also in the medically castrated animal and again you see that vardenafil does not bring back the erectile response to the normal. So we concluded based on this data and those from Dr. Maggi’s group is that PDE-5 alone does not work. Why, because the pump has failed. Why, because this electromechanical pump has not been well-maintained, it is not going to work just by giving PDE-5.
Is there a threshold value for testosterone in erectile function? It took us a few years to address this question.
If you look here, these are different doses of testosterone. If you look at the body weight there is not much difference, definitely there is as you decrease the testosterone level to the castrated, you can see that there is a decrease in the penis size. Of course, this is not magic, this is data which existed for many years but just to show that there is a dose-dependent for reduction in prostate weight and seminal vesicles.
In the animal model, there is a range from 1.4 to about 0.2 ng/mL where the erectile function is maintained. So erectile function is maintained over a wide range of testosterone.
But what is interesting is if you go below that 0.2 nmol, 0.2 ng/mL of testosterone, this is what you see. This is really a key piece of data. And this data will very shortly in the Journal of Andrology. Below 0.2 ng/mL or roughly 250 mcg/ml you can see that there is a dose-dependent decrease in erectile function as measured by the area under the care of the intracavernosal pressure. And you can see beyond this value of testosterone, there is really rapid diminishing of erectile function. So we believe there is a threshold for maintaining erectile function by testosterone. But what we also found was that this threshold is not the same for maintaining the other androgen-dependent organs, such as the prostate or the seminal vesicles. It is not the same. And therefore, sexual function seems to be maintained by even much lower dose of testosterone. In our study we calculated in two different groups of animals. It is roughly 10 to 12% of the normal level. So, if you take the normal level when you reach about 10% of that that is when the erectile function begins to go. Somewhere between 100% and 80% it is maintained. It is when you go to about the lowest 10% that is when the erectile function begins to go. I think this is a very interesting finding. What would be nice are studies of this sort, but of course it is not that simple, can be executed in the clinical research and demonstrated as the question was raised this morning. Is there a threshold value for androgen for the various health issues, whether it is bone resorption, or erectile function and other concerns?
To summarize this, erectile function is maintained by a wide range of systemic testosterone levels that are as low as 10 – 12% of normal physiologic plasma concentration. Below this concentration, erectile function significantly and positively correlated with testosterone plasma level in a dose-dependent manner. This correlation was consistent at all three stimulation frequencies irrespective of whether the data was normalised based on intracavernosal pressure, systemic endothelial pressure or area under the care. In other words, the data is solid.
I promised you that we are going to go and discuss the molecular and cellular mechanism. So here is what we are going to do. I already showed you that androgen deprivation alters the veno-occlusive function, and results in erectile dysfunction. What are the mechanisms? We are asking the question does androgen regulate nitric oxide synthases, does androgen regulate PDE-5, this is the metabolic component. Does androgen regulate adrenergic receptor expression? Does androgen regulate smooth muscle growth and function? Does androgen regulate neural structure and function because we said that was important from the outset? And, does androgen regulate the differentiation of progenitor cells into an adipogenic lineage, and contribute to devolving of a fat mass within the penis which actually will interfere with the veno-occlusive function.
What are the effects of androgen on expression of nitro oxide synthases?
Since nitric oxide synthase is considered a key modulator of erectile function and you have two forms: neural form and you have the endothelial form, and both forms are important for producing relaxation in the trabecular smooth muscle.
What would be important is to ask, is this key enzyme, which takes arginine and oxygen, to make nitric oxide and citrulline, is this enzyme regulated by androgen?
The answer is yes, not just from our work, it’s from (inaudible) work, from Dr. Maggi’s work, from Dr. Tom Miller’s work, and from many, many others. I just want to point out that this is a control. If you castrate the animal, which is (inaudible) for the vehicle nitric oxide synthase is gone. If now you give varying doses of androgen you can bring it back to varying levels.
This is work from Marin in Spain showing that this is the intact animal, this is the castrated, this is the castrated plus testosterone you can see that the endothelial matter for endothelial nitric synthase is also regulated by androgen.
Does androgen also regulate the other enzyme, which the world talks about all the time, the PDE-5 and the answer is yes. As you can see here that this is the control, PDE-5 is the band androgen, so in the castrated animal, PDE-5 expression is diminished. If you give back androgen at different doses you can recover PDE-5 activity, and this is also confirmed by studies from Dr. Maggi’s group.
Interestingly enough, also if you look at the activity of PDE-5, not just a (inaudible) expression, you can see that the activity is increased by testosterone, not by oestrodial, not in the castrate. But it is increased much more over that of the control.
So clearly we can say that androgen regulates the key neurotransmitter, which is the nitric oxide synthase, brought from endothelial and urine sources. But it also regulates the regulating mechanism, which involves PDE-5. So therefore androgen regulates the metabolic aspect of the erectile function. This showed up on the cover of Journal of Andrology some years ago from a review I wrote. All I want to draw your attention to is that the a 1 and a 2 adrenergic receptors on the trabecular smooth muscle are also regulated by many factors, including androgen.
In the study we have done in 1999 we show that a 1 adrenergic receptor is down regulated by androgen, and it is up regulated by testosterone. This might be controversial, no it is not. When the smooth muscle is deprived of androgen, the smooth muscle changes its phenotype; maybe from contractile to a different phenotype and therefore the expression of alpha adrenergic receptor will change. And this actually is evidence that there are changes in the trabecular smooth muscle due to androgen deprivation.
What are the effects of androgen on the smooth muscle itself in terms of content and function?
Probably you have seen this slide a number of times but I think it is an important slide. If you look at the red or pink stain it represents the trabecular smooth muscle, and that is the control. Look what happens when you deprive the animal from androgen, there is considerable reduction in the trabecular smooth muscle, and there is increased in the blue stain, which represents the connective tissue. What does that mean? It means you are losing tissue compliance. The fewer smooth muscles the fewer smooth muscle that can expand, relax and expand, therefore, you actually are altering tissue compliance. If you are altering tissue compliance you are not going to impede blood outflow and if you cannot impede blood outflow you are going to have what we call venogenic leak or vasculogenic impotence as shown by Dr. Tom Lue and Dr. Rogers. I think that is what we believe is happening here. There is a reduction in trabecular smooth muscle and there is an increase in conductive tissue deposition. What happens when you give back testosterone? So you give this group testosterone and you go back here and now you can see that almost they are similar. So this is a reversible process. I think that is good news in a sense that maybe within this frame of time, that this loss of smooth muscle can be recovered by giving back androgen, but not by giving back oestrogen.
If we quantify the data from the slide I showed you before, you can see the control does not lose any muscle. If you castrate the trabecular smooth muscle there is roughly 23/24% loss of trabecular smooth muscle. If you give oestrodial you do not recover, but if you give testosterone you recover back almost 20 some percent of the smooth muscle that is lost. That is why here you recover erectile function, here and here you do not recover erectile function.
When we look at some of these tissues from the intact and castrated animal under the electro microscopy we see something interesting. This is the control, you can see this is the smooth muscle; this is the nucleus, very nice organisational structure. When you look at the two-week post-orchiectomy you can see the first thing that goes is the canthus the cells are beginning to die. There is vacuoles there is a accumulation of flocculent material. If you look carefully there is a swollen mitochondria, there is increased number.
Therefore, there is considerable effect of androgen on the trabecular smooth muscle.
Just to summarise, what we notice is the reduction in contractile elements, there is an increase in mitochondria number and swelling, there is smooth muscle degeneration, there is cytoplasmic vacuolisation with the flocculent material. And, which was very, very obvious, you do not need an expert to see that, is very disorganised contours and irregularity in the smooth muscle. So clearly androgen deficiency is not good for the trabecular smooth muscle.
Does that really mean anything at all? Yes, it does. This is a study done by Dr. Ajay Nehra, Irwin Goldstein and their colleagues in our group in Boston. What you see here is a prospective study. They took patients who are going for penile implantation and they took tissue during surgery and they looked at the smooth muscle content and since before that they have done dynamic infusion, cavernosonography and cavernosonometry, they were able to measure the flow to maintain, and then they correlated with the percent of smooth muscle. If you have less than 30% smooth muscle, you need roughly 120 mL to maintain erection. This is a very, very high flow, it is almost impossible. If you have somewhere between 38 and 30 you need roughly somewhere around 20 to 25 mL but if you have more than 38% you need about 10. So there really is a correlation between the number of trabecular smooth muscle cells, and the flow to maintain, and this correlation clearly says that you need considerable amount of trabecular smooth muscle to maintain veno-occlusive mechanism to maintain erectile function. Therefore, androgen deprivation, which results in loss of smooth muscle will bring you in this direction, which will have venogenic impotence or venous leak, which is very difficult to manage and PDE-5 will not allow you to manage this part here.
So now I am going to ask the question, what about the effect of androgen deprivation on the cavernosal dorsal and nerve arteries?
I want to show you first, this is recent data, this is not published we are still working on that; if you look at the very left banner here, at least for me it is the left, as you can see this is the myelin sheets of (inaudible). You can see that it is very nice it is quite a few. After castration you can see the diameter is smaller, there is lots of myelin there is some disorganisation. Now if you give androgen back you can see that it looks pretty much like what we see in the control. This is very encouraging that androgen are affecting not only the trabecular smooth muscle, but also affecting the cavernosal nerve, which will make sense because when we electrically stimulate the nerve to get erection, we don’t get a good erection in this animal suggesting that androgen has multi-point of action. They affect the cavernosal nerve, they affect the trabecular smooth muscle, they affect the nitric oxide synthase they affect the PDE-5.
This is data from Dr. Rogers and Tom Lui group, and they also show that here in the controlled, this is the dorsal nerve. This is not the cavernosal nerve. You can see the myelin here you see a larger diameter compared to the castrated. You see smaller diameter and loss of myelin. So clearly androgen has effect on the innervation processes and maintaining of nerve function. I think this is an important thing. This is an area for us of interest and we hope to pursue this further as we go along.
Probably the last question I want to ask, and I would like to show you some data, and this is very exciting because it converges with the metabolic syndrome issue, and it converges with the diabetes issue, is that when there is low androgen or androgen deficiency or androgen deprivation, there is disregulation of the smooth muscle function, but also increase of accumulation of adipocytes in the erectile tissue.
This is a cross-section of a rabbit penis, and this is the tunical albuginea here. This is the cavernosal body and you can see the interface here, very nice interface with no problems.
Take a look at the castrated animal and you can see now there is accumulation of empty white cells, which at the time we did not really understand, why is that?
Then doing this study more rigorously we found out that these are actually fat cells or adipocytes. This is the control, see this is cavernosal body here, this is the tunical albuginea, you can see the contrast, this is the interface. That is the intact animal. Look at the castrated animal. This is the tunical albuginea; this is the cavernosa body, look where the fat cells are nicely nesting like ostrich eggs. What is the significance of this observation? The significance: if compression of the cavernosal body against the tunical albuginea to impede blood outflow to compress the subclinical venue is important for veno-occlusion having these fat cells here may not permit that. And may allow blood outflow and therefore you have what we call venogenic or vasculogenic impotence of venous leak.
This is just another stain looking at these cells. This is trichrome, this is the control you can see the smooth muscle is pink, the connective tissue is blue, this is the tunical albuginea. Again here the castrated animal shows these empty fat cells.
You always have someone in the audience who says how do you know they are fat cells? Well this is the problem, when we do total staining you can see this is the castrated animal, you can see the osmium tetroxide is picked up by the fat cells here in the castrated animal. There are very few intact, and there is actually many more.
However to go further we did electro microscopy and you can see these fat cells have a nucleus, have a nice cytoplasm and have a large lipid droplet. This convinces us that these are really not microphages accumulating fat but actually these are adipocytes.
Is this reversible? I think it is. If you look this is an intact animal, there are no fat cells. This is castrated animal fat cells, it is just beginning to accumulate, and if we give them back androgen these fat cells disappear. One interesting observation and we do not have an answer for it, is that the corpus spongiosum does not accumulate any fat cells although there is smooth muscle cells there. Why? We do not know.
This is an interesting observation and this is maybe relevant to those who discussed the metabolic syndrome, as well as diabetes. What you are looking at on the left panel is a cavernosal tissue from a non-diabetic dog, a control dog. You see how nicely organised. The one on the right is a cavernosal tissue from a diabetic dog, and you can see accumulation of these fat cells in the cavernosal tissue from the diabetic. This message has two things that diabetes reduces testosterone levels, and therefore this can happen or, there is more than one pathway for these adipocytes to accumulate.
So, what is the mechanism? Dr. Shalendar Bhasin, who is now the Chief of Endocrinology at Boston University School of Medicine, has brought forward a nice couple of papers. The last one is published in the February issue of this year suggesting that if the fibrostroma cells, or the stem cells, the bending on the endocrine milieu. So if the endocrine milieu is androgen sufficient, these progenitor cells will follow this lineage going to a smooth muscle cell lineage, and therefore in the presence of androgen, we end up having trabecular smooth muscle. In the event of androgen deprivation of androgen insufficiency, this progenitor or this fibrostroma progenitor cells will go toward a different lineage, which is preadipocyte, and these preadipocytes become adipocytes. I think this is what is really happening in androgen deprivation in the studies I showed you.
I want to summarize, we believe based on the data from the laboratory, not from the clinic, that androgen does regulate nitric oxide synthase activity, and since nitric oxide synthase is a key enzyme, and produces a key neurotransmitter in erectile function, therefore androgen are important for erection. Androgen also regulates PDE-5 expression and activity. Androgen regulates smooth muscle cell function, and this leads to changes in expression of alpha adrenogenic receptors. I hope I convinced you that androgen is important for maintaining smooth muscle growth and function. And I hope that I also convince you that there are some changes in the innervation due to androgen deficiency.
As far as the issue of androgen and differentiation of progenitor cells to smooth muscle cells, or to adipogenic lineage, we have to continue this research but I think the preliminary data I showed you is absolutely interesting and hopefully we will be able to substantiate that as we go along. I hope I also at least brought the issue to discussion regarding the threshold value, and we believe there is a threshold value for testosterone in maintaining erectile function.
To conclude, testosterone regulates composition, organisation and fibro-elastic property of penile cavernosal tissue, which are critical for veno-occlusive and erectile function.
And androgen deficiency produces metabolic structure and functional alteration in the corpus cavernosa, resulting in veno-occlusive dysfunction.
I just want to thank my colleagues, specifically Dr. (inaudible) who is instrumental in many of these studies, and also Dr. Goldstein for continuous collaboration and interaction.
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