Slide 1

Doctor Bianchi, thank you very much and also I would like to thank the organisers for inviting me here. I have been given a rather difficult task because the evidence for a role of AT2 receptors in humans is extremely limited and so I’m afraid that I will also have to show you some animal data to further support this story.

Slide 2

So, here you see for those of you who are not familiar with the renin-angiotensin system again a brief overview of the system. There are 2 enzymes, renin and ACE which together form a substrate, angiotensinogen generates the active end product angiotensin II. This as we have heard already, activates two types of receptors, in man at least. In rodents like in mice and rats there are in fact, 3 because mice and rats have 2 types of the AT1 receptor, AT1a and AT1b but in humans there are only 2. As we’ve heard the AT1 receptor in fact, mediates all of the known, well known and well accepted effects of angiotensin II including constriction but also sodium and water retention, growth and remodelling. It is, of course, this receptor which we block when patients are treated with angiotensin receptor antagonist. But now on the AT2 receptor. A lot of data have been obtained in animals but as I said in humans the date are very scarce. It seems on the basis of the data in animals that this receptor opposes some or perhaps all of the effects of the AT1 receptor. So, in fact, it appears to induce relaxation and it maybe anti-growth and anti-remodelling.

Slide 3

So, when we heard about this, when we became interested in this, of course, we studied this using in this case human coronary arteries to see what we could confirm in this case in human vessels. What you see here, we’re looking at as I said a human coronary artery where we treat the artery or expose the artery to angiotensin II and of course, as expected we see a nice constriction occurring in a dose- dependent manner. This constriction can be completely blocked by using an AT1 receptor antagonist, irbesartan in this case but when we simultaneously use the AT2 receptor antagonist PD123319 and we would expect perhaps then if indeed there is AT2 receptor mediated vasorelaxation we would have expected a clear increase in the effects and the constriction of angiotensin II because now we would remove this vasorelaxation. As you can see here, perhaps there is a small increase but it’s very modest and in fact, it was not significant. So, in these vessels at least, there seems to be no clear evidence for a role of AT2 receptors, all effective angiotensin II could be explained on the basis of AT1 receptor activation.

Slide 4

So, yes I have to turn to animal data then to further support the story here and what I’m showing you here are data obtained in rats in vivo. What we have done here we have infused these rats with angiotensin II and we looked at regional flow, for instance, in the heart and other organs by using radio labelled microspheres and what is shown on this slide here is the myocardial vascular conductance which is regional coronary blood flow corrected for mean arterial pressure. If it increases, this is an indication of regional vasodilation. If it decreases, this is an indication of regional vasoconstriction. Again, when we infuse angiotensin in the presence of just vehicle, you see a tendency for vasorelaxation it was not significant, so an increase in conductance. Doses are not affected by irbesartan, the AT1 receptor antagonist, the line in green but importantly when we pre-treated the animals now with the AT2 receptor antagonist, the PD compound, certainly we do see this decrease in conductance, so suggestive for regional constriction.

Slide 5

So, this is indeed the first evidence obtained in our lab at least and confirming, of course, data by others that there is indeed AT2 receptor mediated vasodilation and which we are only able to unmask by simultaneously blocking this receptor and adding angiotensin II. Now, of course, we wanted to know what is the mechanism of this effect, so what we did in the subsequent studies, we treated these rats either with a cyclooxygenase inhibitor, indomethacin and you see here this is again the control situation and it’s a similar increase in conductance in the presence of indomethacin. So, this is not affecting this effect of angiotensin II but when we pre-treat the animals with an NO synthase inhibitor, L-NAME now we see a similar decrease as I’ve shown you before with the AT2 receptor antagonist, PD. So clearly therefore, this effect of the AT2 receptor which is similar now to the effect of an NO synthase inhibitor is probably related to nitricoxide release and this is again in line with the literature. So we have an AT2 receptor which probably causes vasodilation through activation of NO synthase by releasing nitricoxide. Another concept which has been described in the literature is the upregulation of AT2 receptors under pathological conditions.

Slide 6

So, we also studied this and in fact, we used again the rats in this case. Rats underwent a myocardial infarction and after about 3 or 4 weeks we again exposed them to angiotensin II. So this is the control situation which I’ve just shown you before where we unmasked this effect, this constrictive effect of angiotensin by adding simultaneously the PD compound but now when we look in this case here on the right to the animals which underwent a myocardial infarction, again we see that with vehicle there is a modest tendency but not significant for an increase in conductance. This is now no longer affected by the PD compound, the line in purple. Here we get to see this decrease in conductance but now we do see that after pre-treatment with an AT1 antagonist, irbesartan, now we suddenly observe an increase in conductance, so unmasking an AT2 receptor mediated vasodilation. Therefore, supporting because it was not shown here in the control situation that apparently there is an upregulation of AT2 receptors after myocardial infarction.

Slide 7

Of course, the data I’ve just shown you, they were obtained in rats but studies in mice are equally important particularly because in such animals we might also study, for instance, the consequences of a knockout of 1 or more of the various angiotensin receptors. So, first I’m showing you here and these data were very recently published, I’m showing you here the studies we recently did in the isolated mouse Langendorff heart, technically quite a difficult preparation. What we did here we exposed Langendorff hearts to increasing concentrations of angiotensin II and here we’re looking at coronary flow and as expected of course, angiotensin II is decreasing coronary flow up to a certain concentration, after that due to tachyphilaxia the effect is disappearing.

Slide 8

When we repeat the study after pre-treating the animals with an NO synthase inhibitor, now we see that its enhanced effect in open circles here is no longer present and so again supporting that this AT2 receptor mediated effect like in the rat is NO mediated, it is absent after removing nitricoxide with an NO synthase inhibitor.

Slide 9

Now as I said, using mice has the advantage that now perhaps also we can study mice which lack certain angiotensin receptors and the mouse we obtained in this case was a mouse lacking AT1a receptors. As I said in rodents there are 2 types of AT1 receptors and it appears to be the AT1a receptor which is the most important vasoconstrictor receptor in mice.
Here you see the wild type, it’s just a typical tracing of one study and of course, angiotensin again is causing a decrease in coronary flow in line with what I’ve shown you before. But surprisingly and to our disappointment in these AT1a knockout mice we see, of course, no constriction but also as we had hoped, of course, we would have hoped to see now a vasodilation , a real direct AT2 receptor mediated effect and you can see here there was no such effect at all.
We also repeated these studies not shown here after pre-constricting the coronary vessels with phenylephrine, for instance, but again no evidence for vasodilation.

Slide 10

The next slide summarises all the findings in the wild type animals, a nice vasoconstriction of course after adding angiotensin II but over a whole dose range in the AT1a knockout animals absolutely no effect on either flow or left ventricular pressure. So, it seems that to see an AT2 receptor mediated effect one apparently also somehow needs the presence of AT1 receptors, in this case in fact AT1a receptors. So, so far a limited amount of human and particularly animal data. What have we seen? AT2 receptor mediated vasodilation, it can be demonstrated in the rat and the mouse heart but unfortunately, not in these large human coronary arteries. So, one explanation could be that in fact we’re looking at the wrong arteries and we should have looked at the smaller ones, the coronary microarteries. What I’ve shown you also is that this effect depends both in the rat and the mouse on nitricoxide and particularly, in the mouse it may require simultaneously AT1 receptor activation. Furthermore, this effect of the AT2 receptor is enhanced in the pathological conditions after myocardial infarction.

Slide 11

So, the question we might now have, does this AT2 receptor mediated vasorelaxation occur in human coronary microarteries? We didn’t see it in large ones, maybe we should look in the smaller arteries and if so what are the mediators? Is it again nitricoxide, and subsequently cGMP and according to various animal data there’s also a role for bradikinin type 2 receptors.

Slide 12

So, could we perhaps study this in further detail in these coronary microarteries? We’ve heard about this already, we used also a myograph setup while we used these microcoronary arteries with a diameter of around 160-500μm. The vessels and it is also important to mention they were obtained from heart valve donors from the Rotterdam Heart Valve Bank and these are usually healthy people who have died, for instance, from a car accident and for instance, who also display an excellent endothelial function. So, these are in general healthy vessels.

Slide 13

Again we’re looking now here at angiotensin II responsiveness and here the angiotensin II concentration and as expected, of course, we see a nice vasoconstriction occurring in a concentration dependent manner and this can be blocked by irbesartan, the AT1 antagonist.

Slide 14

But now we did see that after pre-treatment of the vessels with the PD compound the AT2 receptor antagonist we did see a clear and significant increase in constriction, so now indeed confirming the idea that also in human vessels, although indirectly here, there is evidence of AT2 receptor mediated vasodilation. There was no change in the EC50, so in the potency of angiotensin II, the only change we observed was an increase in the maximum constricting effect of angiotensin II.

Slide 15

Interestingly, when we compared the increase in the maximum effect with age, we observed that its increase in maximum effect was larger in older subjects. So, that would support the idea that contribution of AT2 receptors increases with age and of course, with age also there will be a relatively endothelial dysfunction and vascular dysfunction, so this would be in agreement with the concept I mentioned earlier and I showed you data to support it that there’s an upregulation of AT2 receptors under pathological conditions.

Slide 16

Now, what about the mechanism? We removed the endothelium in these vessels and what we then saw is that no longer the PD compound was capable of increasing the effect of angiotensin if anything there was a decrease but it was not significant, so clearly it is endothelium dependent. This effect of AT2 receptors doesn’t mean, of course, that the AT2 receptors are located on the endothelium, it only means that their effect is mediated somehow involving an endothelial factor.

Slide 17

It might be the AT2 receptor but perhaps it might also be, for instance, the bradikinin type 2 receptor which is known to be present on endothelial cells because here too we do see when we treat, pre-treat the vessels with the Hoe140 compound which blocks these B2 receptors, we no longer see this increase in constriction after the PD compound. So, clearly it needs the endothelium and the B2 receptors are required.

Slide 18

Furthermore, we repeated the studies of course in the presence of L-NAME and here too we see that the enhanced effect of PD is no longer seen after pre-treatment with L-NAME, so supporting the idea that also like in rats and mice nitricoxide is required for this effect.

Slide 19

Finally, of course, nitricoxide will stimulate the generation of cGMP and we did see indeed that when angiotensin was added to vessel segments, there was an increase in cGMP which was not affected by irbesartan and it was brought back by the PD compound, the AT2 antagonist to baseline levels. So supporting also a role for cGMP.

Slide 20

Well, the final proof we needed, of course, was to show actually that angiotensin, not indirectly but now directly, that angiotensin was capable of causing a relaxation. We did this in the following manner. We pre-constricted the vessels with a compound, a thromboxane A2 analogue which is called U46619, so there was already a certain degree of pre-constriction.

Slide 21

On top of that we added angiotensin and now, of course, there’s not much more effect to be expected, no further increase in constriction because there’s already a large degree of constriction.

Slide 22

Also when we pre-treated the vessels with the PD compound there is no further enhancement of the effect of angiotensin II because it was already near maximal.

Slide 23

But importantly now, when we pre-treat these vessels with the AT1 antagonist, irbesartan so to block AT1 receptors, then, of course, angiotensin II is capable of only stimulating AT2 receptors under conditions where there is a pre-constriction. Then you see that clearly angiotensin II and I think this is the first time it was shown that angiotensin II is a vasodilator in human vessels, so mediated via AT2 receptors and the evidence, of course, was obtained by also repeating the study by adding both antagonists and then this effect was gone.

Slide 24

So yes indeed angiotensin II is causing vasorelaxation in coronary microarteries obtained from humans in a manner which involves endothelium, B2 receptors and nitricoxide and cGMP. Of course, we had to show that the receptors are locally expressed. So what you are looking at here at RT-PCR data not only in coronary microarteries but interestingly enough also in the larger coronary arteries which I showed you before where we were unable to see this clear increase of angiotensin II responsiveness in the presence of the PD compound. So apparently the receptors are there, so either the expression is too low or perhaps these receptors are exerting other effects, for instance, related to growth.

Slide 25

So to summarise this part, we have seen that the PD compound enhances angiotensin II induced constriction and this effect was particularly large in older subjects. This potentiation was absent in the presence of L-NAME, the Hoe compound and also after removal of the endothelium. Angiotensin, in fact, relaxed vessels that are pre-constricted in the presence of irbesartan and this could be blocked by the PD compound. Finally, radioligand binding studies and RT-PCR confirmed the presence of these AT2 receptors in human coronary microarteries.

Slide 26

So what about evidence for AT2 receptor mediated effects in humans in vivo? As I said the data are very, very limited in fact there’s almost nothing. The only study I’m aware of is a study published a few years ago where there was intrabrachial infusion of the PD compound and this was found to increase forearm vascular resistance in women during AT1 receptor blockade, so supporting again that AT2 receptors might normally exert vasodilatory effects. In our own hands when we infused angiotensin II in very high levels in the forearm vascular bed of healthy volunteers during AT1 receptor blockade, there was a tendency for vasodilation but it was not significant.

Slide 27

So very limited data in vivo, largely related to the fact that this compound cannot normally be given to human subjects. But what evidence is available and several studies have been done relating polymorphisms in the angiotensin type II receptor gene to, for instance, in this case cardiac hypertrophy. This is a study we published a few years ago in women with hypertrophic cardiomyopathy and what you can see here that indeed there is a significant correlation or an association with this polymorphism, the women in this case with the CC genotype having less cardiac hypertrophy than those with the AA or AC genotype. This has been confirmed looking usually at other polymorphisms also in the AT2 receptor gene, the various polymorphisms and so supporting that perhaps the AT2 receptor is not only related to vasodilation but perhaps also affects growth.

Slide 28

Now, I have to turn again to animal data. These are data published by us almost 10 years ago already where we look here at the growth effects of angiotensin II in cardiomyocytes. We looked at protein synthesis and DNA synthesis and as you can see here, when we add angiotensin without any blocker, there is virtually no effect on protein synthesis, there is a clear effect on DNA synthesis. When we block the AT1 receptor, nothing is left of this effect and here still we see no effect. But when we block the AT2 receptor, now we see a clear increase in protein synthesis and even further enhanced DNA synthesis. So supporting again the idea indirectly that AT2 receptors are mediating effects that are opposite the AT1 receptors, in this case suppressing protein synthesis and suppressing DNA synthesis. For that reason there is no effect at all when both receptors are stimulated and only we do see an AT1 receptor mediated effect, if we block AT2 receptor. So this could underline the previously described association with the various polymorphisms in the AT2 receptor gene and cardiac hypertrophy.

Slide 29

The final issue then, when will AT2 receptor stimulation ever occur in humans? Well, this is in fact what will occur all the time when subjects are treated with an AT1 receptor antagonist. For instance, when we block ACE, of course there will be a reduced amount of angiotensin II and then neither the AT1 nor the AT2 receptor will be stimulated or at least, the stimulation will be at a lower degree. But when we block the AT1 receptors now, of course AT1 receptors are, well cannot be stimulated anymore but due to the feedback mechanisms within the renin-angiotensin system and this, of course, will also happen during ACE inhibition, we get a rise in renin, a rise in angiotensin I here not a rise in angiotensin II because ACE is blocked but here we will see a rise in angiotensin II,. This cannot stimulate the AT1 receptors which are blocked but it can stimulate AT2 receptors.

Slide 30

So in fact, it could very well be that part at least of the effect of AT1 receptor blockade is not so much due to the blockade of this AT1 receptor but perhaps to some degree to simultaneously stimulation of AT2 receptor. There is indeed evidence supporting this, also I go back to animal data. One of the first studies and more recently various other studies have confirmed this concept. What you’re looking at here is again a matter of post myocardial infarction in mice. We looked at cardiac hypertrophy, this is reduced by AT1 receptor antagonist treatment which is not very surprising of course, and also there is during such treatment an increase in capillary density. But importantly, when we simultaneously treat these animals with an AT2 antagonist, this effect is partially gone. Now, we no longer get such a decrease in cardiac hypertrophy nor is the increase in capillary density still significant. So, supporting the idea that indeed simultaneously AT2 receptor stimulation during AT1 blockade is important for the beneficial effect of such drugs.

Slide 31

So the final slide, what are the conclusions?

Thank you very much. I hope there will be some questions.

Slide 32

Chairman: Thank you very much for this presentation. Now it’s open for discussion. Well, you have mentioned that angiotensin II receptor type 2 receptor perhaps are more active in microcirculation and there is a polymorphism of the receptor that might be associated with ventricular hypertrophy and also your group has been studying a lot of ACE EIAD polymorphism as a local production of angiotensin II being the carrier of DD genotypes producing more local angiotensin II than the EE genotype. My question is devoted to try to see whether there are some data or hypotheses or speculation that might involve the polymorphism of angiotensin receptor II blockers in the hypertrophic response to DD receptor of ACE because we know that this is one of the most established polymorphisms that affects the amount of cardiovascular remodelling, the ACE D genotypes. Could you speculate some interaction of these genotypes with these angiotensin II receptors?

Prof Danser: Yes, I fully agree and this is in fact what we are doing now by not limiting ourselves to just one polymorphism like the AT2 receptor but by combining to get a complete picture of the various polymorphisms. Unfortunately, in the population I just showed you here, the ones with the hypertrophic cardiomyopathy the numbers are too small to make such subdivisions where you would indeed combine, for instance, DD with some of the various polymorphisms of the AT2 receptor. So you will end up with such low numbers this is I think not feasible, either we have to increase the population which is going to take years but which we will do. Alternatively  we are collaborating with the department of Cardiology within our university and they have access to much larger databases involving up to thousands or ten thousands of patients and which are treated, of course, also with ACE inhibitors and where we have similar data on left ventricular hypertrophy and where we now might establish groups not only looking at ACE and AT1 but also AT2, angiotensinogen and aldosterone polymorphisms, so that we can now make these combinations where you could speculate that indeed with the DD, for instance, there is enhanced ACE and therefore enhanced angiotensin II generation and in combination with the various subtypes or various genotypes of the AT2 receptor there might be more or less AT2 receptor stimulation. This will be possible only in large populations and further support hopefully we will be able to obtain by also measuring in blood then of course, the components of the renin-angiotensin system. But this is going to take well a few years before we can establish all of that.

Chairman: Well, perhaps another question. We might discuss the relationship between ACE inhibitors and blockers of angiotensin II subtype 1 receptor has the ability to reduce organ damage because according to some data these blockers could be superior to a converting enzyme inhibitor because they do have an activation of subtype 2 of receptors but this has not been found in the clinical grounds. This could be due to the interference of bradikinin and so on. Can you speculate on that?

Prof Danser: You mean the combination of an ACE inhibitor with an AT1 antagonist?

Chairman: No, the comparison between ACE inhibitors and angiotensin II blockers should produce some increase according to this data but this does not occur in the clinical to my knowledge.

Prof Danser: No you’re absolutely right that it seems that the ACE inhibitors and the AT1 receptor antagonist are equally effective and then you could speculate that well, there are differences related to, for instance, bradikinin but as I’ve just shown you here the complex issue is that of course, ACE inhibitors will increase bradikinin levels but in fact this might also be happening during AT1 receptor blockade due to the stimulation of AT2 receptor. So purely the idea that they are equally effective could in fact, also be taken as support for this concept that indeed AT2 receptor and therefore, bradikinin stimulation is of importance. But I fully agree that hard evidence for this is lacking and the major problem as I mentioned before is that we are unable to block AT2 receptors in humans. Very scarce data where it has been tried to use this PD compound but this is not normally available for human use.

Chairman: Thank you very much and thank all of you who have been here, not many but at least some group of people that are interested in basic science. Thanks to all of you.