 Good morning everybody. Welcome to this VPH summer school. It's the second time we organize this. I'm back Benance working here in the UBF. We organized this together with Jerome Noi and Mathieu de Caen from Philips because it's a collaboration with a European research project cardio function that we do this. Now as you have seen from the program what we try to do is try to bring you some information on the use of VPH technologies, VPH approaches. This year's in kind of vascular approaches and fluid dynamics and things like that. What we think is quite important is that since VPH is intrinsically so interdisciplinary also when we start to do research when we try to explain some of these things we have to try to cover many of these aspects and that means that before you can do any modeling or any analysis you have to understand what it's going about and that's why today we will start with some aspects of the basic pathophysiology behind everything that's related to flows and vessels in the body. You will see in the next days we will gradually build this up by starting to talk a little bit about acquisition of data because without any data you cannot do any modeling. Next we will talk a little bit about the models themselves at organ levels, at cell levels, cell levels and these kind of things and not to forget also the technology behind it because there's a lot of computational problems, implementation problems that's why we will cover more on Thursday and then we will end up by looking at an overview application where we try to combine everything. You will see we will get a little bit more information later on in the afternoon we do hands-on sessions which are related to this. We will do the explanation just after lunch and so you can see which one you can participate. But today we're going to start first with talking about really the basic aspects, the biology, the pathophysiology behind it and it's my pleasure to invite the first speaker, Gemma Bilaour, she is like from training a veterinarian so you see already how very interdisciplinary this will be and we want to talk about the basic pathophysiology of heterosclerosis and vascular remodeling because when you start to talk about flows in the body this is one of the basic things and Gemma is working here in Barcelona in the Institute for cardiovascular sciences which is quite an important institute doing a lot of research on thrombosis for example and other things so Welcome and we're looking forward to your lecture. Thank you. Thank you so much indeed for such a nice introduction and for me It's a real pleasure being here and contributing to this course So I want to deeply thank the organizers and coordinators for giving me the chance to just Introduce yourselves in the cardiovascular field from a basic point of view So we'll go through we'll start with atherosclerosis It's impacting vascular remodeling and then move towards the clinical events, which is thrombosis so the Wait a second. Where should I point? Or perhaps there no perhaps there Yeah I'm not pressing the right button Now, thank you. Thank you so much. So let's just start by the most important What's what when we're talking about cardiovascular disease and atherosclerosis? What are we talking about and to understand this we have to talk about numbers We have to talk that it's the leading cause of death worldwide in 2012 The World Heart Organization said that 31% of the causes of death were related to cardiovascular disease And if we take a closer look to Europe, we can see that here is in red here all these Triangles that in both men and women. It's almost 50% of the causes of death Indeed, it's a bit higher in women than men and there are not only differences across genders, but also Around Europe so you can see the incidence of cardiovascular diseases much higher in the Eastern Europe as compared to the Western Europe And what about Spain since we're here today Though it has become a worrisome problem because it has increased during the last three years six percent The incidence of death for cardiovascular disease So we're really talking about a disease that we have to invest on and keep on working And what is the main in the line cause of cardiovascular disease is atherosclerosis? Atherosclerosis has reached epidemic proportions. However, it again has a different distribution of around the countries So if there are all these Countries which are more developed and you know where a style lifestyle Well fast food, tobacco is present cardiovascular these factors and the incidence of atherosclerosis is much much higher than those countries They're still under development and When we talk about atherosclerosis, we are talking about it's very important to have in mind that we're talking about a chronic Disease so it really develops over decades and it's related to both components two important components one the inflammatory mediated Another one is a lipid driven Process so you can see here lipids will go in detail further on but lipids get into the vessel wall which Induces that the lumen of the artery gets narrower So that influences the blood flow and the formation of these atherosclerotic plague which is lipid rich may eventually break and expose Substrates that are not usually exposed to the flowing blood in using the appearance of thrombus formation and the clinical presentation of the disease So depending on where these occurs we can talk about ischemic heart disease certain death myocardial infarction So one we can talk about therovascular disease in which strokes the main participant here It happens in the carotid arteries It can happen in the server arteries and also when it happens distantly we're talking about peripheral artery disease There's the three feet three different manifestations that both of them emerge and compose what's called the cardiovascular disease So just it's important to have in mind how is the vascular structure and these are basic concepts It's composed of three layers that intimate the media of them in Tisha the intimate the first one It's composed of the endothelium which is this mono layer of cells Which are in contact with the lumen with the vessel lumen and these are recovered by an internal lactic lamina The second one the media is the most thick one and it's composed of vascular smooth muscle cells which are exposed contentrically one to each other and it's also recovered by this elastic external in this case elastic lamina and Finally the third layer the most exterior one is the adventitia The dentitia as you can see here is mainly formed by extracellular matrix components Collagen and all that but most important is the one that has vessels and this is important Because the vessel has to be nourished somehow and in healthy conditions vessels are only presented and ventitia and they are called the vasavasodil This is this is diagram in which it illustrates here the endothelium layer and Here on the internal Elastic lamina, so this is the intimate and what happens with this layer that into indirect contact with the blood and the healthy conditions First it has anti platelet properties The first property is that this is surfaces negatively charge and platelets present a Phospholipid layer which is also negative negatively charts So in the healthy conditions there is a repulsion effect and they don't interact in Second term this endothelial layer also express proteins which are called ectodipase And what they do is that through a conversion process of conversion They convert ATP in adenosine and adenosine is known to inhibit platelet aggregation sorry third Most importantly it releases these two compounds Proscycling and nitric oxide and I say this importantly because there are main Components involved in inhibiting platelet reactivity and if these two the compounds are not synthesized There is a tendency throw is a thrombotic risk So in the in the healthy conditions this endothelium exerts anti platelet properties But not only anti platelet properties. It also expresses in its surface Proteins that are in charge of blocking the activation of a coagulation cascade But even in the case that some coagulation occurs the endothelium also is an important source of tissue plasmidogen Activator which is in charge of dissolving the form coagulation of the forming clot So the health endothelium exerts anti platelet anti coagulant and the fiber-analytic properties and therefore prevents thrombosis to occur However, what happens and the presence of this chronic disease in which you are a continuously exposed to cardiovascular risk factors That the endothelium is damaged and all these properties disappear And when we're talking about cardiovascular risk factors, we are talking about some factors that cannot be modified They are like they are dependent age Gender ethnicity genetics, but there are many others of them that we can modify then and we can't Work on them. You can see here blood pleasural cholesterol diabetes sedentarism Bluecoast tobacco all that can be modified And I would like to strengthen this point because most of them are just depending on the lifestyle we carry So if we follow High fat sugar rich diet all these cardiovascular risk factors get increased But if you follow the Mediterranean lifestyle, which the Mediterranean diet you can moderate all these cardiovascular risk factors Just lessen the risk to develop atherosclerosis. I Wanted to especially point out the cholesterol because everybody has the concept that high cholesterol is bad But it's not always like that because cholesterol is the sum of two components of the cholesterol the HDL cholesterol which is the good cholesterol is called high-density lipoprotein cholesterol and there is a bad cholesterol the LDL Cholesterol so it's called the low-density lipoprotein cholesterol and when they give you your cholesterol is 300 It comes from the sum of both of them So perhaps this number results from because there is an increase on the good one and what does the good one do? while the bad one Just devil the deposits in the vessel and forms the atherosclerotic plaque The good cholesterol the HDL cholesterol what it does is that remove this cholesterol from the vessels take it to the liver To get secreted so this is very important to take in mind that the worst scenario is to have a high LDL Cholesterol, but never HDL cholesterol So set this because these are important play in the atherosclerotic plaque They just give a brief overview how the atherosclerotic process takes place We see here that the chronic exposure to tobacco infections Changes in shear stress what it does is that damages this in the filial layer and since it damage All these LDL particles that are present in the bloodstream can enter easily into the intimate Once they enter the problem is that they get in contact with these solar matrix components and they suffer minimum Minimal modifications firstly and afterwards once they're retained get oxidized and this is that this is dangerous and injurious Also, because induces the endothelium to secrete Chemotactin substances in order to recruit inflammatory cells just a protective mechanism inflammatory cells have to get into the vessel and engulf all this lipid and just disappear So atherosclerosis in its early stages is a protective response against accumulation of LDL within the vessel But what happens if you keep LDL cholesterol high and you keep having bad cholesterol levels? When it happens then is that all the system of engulfing But the back monocytes have entered have transformed into macrophage and they engulf all the lipids But this gets overwhelmed and so the lip the macrophage is not macrophage anymore And it becomes a foam cell which is a cell that's very rich in lipids and loses all the function and eventually dies And when it dies not only it releases growing factors that induce the migration of this smooth muscle cells that were in the intima and they the change they phenotype Towards a contractile phenotype to a synthetic phenotype in which they release Proteolytic enzymes they release collagen so forth But also then to migrate into the lesion and so the lesion starts growing The lesion starts growing vascular muscle cells go there at the beginning they synthesize collagen But they also release proteolytic enzymes that break down all the matrix components and lipids get Deposited because all the cell dies and release the cholesterol crystals over there So over time what happens is that you have here a protrusion in which there has been an atherosclerotic plaque development and in our group we demonstrated that not only these macrophages are capable of engulfing this lipid and becoming foam cells But also vascular smooth muscle cells and we see here the vascular smooth cells are there have accumulation of lipids and it and We also explained that this mechanism spoke by which vascular smooth muscle cells engulf this lipids It's very receptacle LLP1. So not only the inflammatory cells get lipid rich, but also the vascular smooth muscle cells So this is an example of two very nice images to human coronary arteries Here we have an initial lesion in which is is rich in vascular smooth muscle muscle cells They have start migrating since they have migrated and they have changed their phenotype They synthesize collagen and still lipid lipid core is not that rich because cells have not died I have not released all the lipid content and also inflammatory cells are starting to come as a protective Mechanisms but have not seen how dangerous is going to get at the lesion itself But over time in advanced lesion what happens is that these vascular smooth muscle cells have get too much rich in lipids And they have lost their function and they have died So vascular smooth muscle cells disappear Therefore their synthesis of collagen disappears and there is an enrichment Have you some more richer and richer of this lipid core rich in cholesterol crystals and in more inflammatory cells that go to the lesion So this is our main features of an after vulnerable atherosclerotic plaque as you can see here We can see some foam cells here rich in lipid most of them have already died I haven't released all the cholesterol crystals We see only few vascular smooth muscle cells and mainly all the inflammatory cells are recruiting in what's called the shoulder of the Lesion that's the region that's more prone to rupture and the region in which thrombus formation Majorly occurs another characteristic as I told you before is that as you can see here that the adventita here in this brownish Is rich in vasomassorum to nourish all the all the cellular layers or the vascular layer? Sorry, but as this layer becomes thicker there is a need to provide more Nutrients there is a need to the vessels to grow However, the growth of the vessels under these conditions in which is inflammatory milieu Only leads to the formation of leaky vessels immature vessels that they end up doing Emerages and emeritus isn't the feature what makes blacks more vulnerable to rupture So this is a representative image and we can see a healthy vessel in which in the vasomassorum here You can see the very nicely in that dentition. There is no vessels in the intimate the media However, in advanced atherosclerotic lesions You only not only see the vessels here vasomassorum Dentition, but you can see many vessels enter in the media and even some of them are hemorrhagic down here And this is what makes the lesions prone to rupture So but this atherosclerosis occurs in all the vascular tree and the answer is no it mainly occurs Where there is a turbulence of the flow if flow follows a light has been demonstrated that way in the zones where flow Follows a laminar pattern in the field cells are functional do not change the gene expression and You know everything's physiological However, it has been demonstrated also in counterpart that in the branches What happens is that there is a turbulent of the flow which induces this Indithelial cells to change the pattern of gene expression and also to become dysfunctional And this is very important because then they give rise to the possibility of Enter the lipids and form here as you can see all the deposits of atherosclerotic plaque So it usually occurs in the zones where there are branches and verification and where the flow has lost is laminar pattern So I wanted to give a hint of because when you talk about changes in flow You usually talk about changes in share rate and what share rate blood flow goes on different layers and we can as they are the advanced in different layers and share rate could be Considered the rate of the different velocities between the different layers of the blood So it could be considered the difference between all the layers and blood flow follows a Here you can see a parabolic pattern and share rate So depending what in the blow velocity the first and also in the diameter of the channel and this is very important because As much you increase the blood velocity or as much you decrease the diameter That means there is an stenosis and blood has to go quicker through a narrow place Share rate increases and what is an what's the impact of that the impact is that erythrocytes get deformed So they they keep and they get they migrate towards the axis of the vessel Just because of the size and what does this induce is that platelets get displaced towards the endothelial layer And this facilitates the interaction between the endothelial layer and platelets And this is very important to have in mind when we're talking about share rates and the impact that share rate may have in the formation of thrombus so when we're talking about a Share rate in the physiological conditions We've talking about hundred and twelve seconds minus one and this occurs in those vessels that are healthy or patent Or also in those vessels that have been stented a stent has been placed just to open the artery And so here the vessel recovers its diameter when we talk about the share rate of around 800 We're talking those vessels in which the arterial vessels in which it has been a moderate Astonotic Legion that there's a still some flow, but you can see here this starting a stenotic procedure of the vessel and Finally the most dangerous one the most representative of those culprits for a clinical vent are the high Severe stenotic or high share rates We're talking about 1730 for 100 seconds minus one and this occurs really when there is almost no space for the blood to occur And they usually end up Rupturing the plaque and forming a thrombi which includes all the presenting the clinical event So now let's talk about the association of the link between share rate and vascular remodeling Because the fact that we'll see here we see here the atheroma and the first of all what happens is that When share rate increases the indyothelial layer just receives a message that the luminous narrowing I have to do I have to Compensate for this error so I have to expand outward at a response as a healthy response So the first response is what's called the the incomplete outward of the outward perfect at work in which you do not lose the lumen size However, then there is some situations Especially has been detected and smokers and it's not know the reasons still not known that The endothelium is not capable to activate this compensatory mechanism And what happens them is the vascular does not response and the atherosclerotic plaque grows inward as you can see here And you keep losing you your lumen However, it's important to have in mind that having outward or inward the modeling does not determine the clinical event In other words, you can have this inward remodeling and have a Presence of angina stable angina in which the plaque is a stable There has been no rupture and there's no clinical event Just the flow is low and you have chest pain and you may have this situation here It has been an algorithm modeling so the lumen was not affected at all But the plaque was soft and the plaque ruptured and induce the protrusion of this thrombi and the Consequent appearance of thrombotic event as you can see here So there are many questions that remain here in this field So it's which are the mechanisms leads to inward or outward modeling But nevertheless the most important as whatever what happens in the vessel you cannot predict the clinical event I wanted to put this images because I think they're very representative So which are the culprit lesions for the corporate events here? You can as I told you this is a stable angina. You have a very low flow You have chest pain that's all but what happens if this plaque just erodes as you can see here And it exposes just a little piece of the inner layers which are thrombogenic and Induce this thrombi that do not get firmly attached and they embolize they appear So you have chest pain episodes and that's what's called a stable angina in which sometimes you have the relief sometimes you have pain The worst these scenarios come here These two scenarios are what's called acute coronary syndrome And what happens is that here the plaque fissures that ruptures and there is a thrombi and when this thrombi Just partially occludes the lumen so there is still flow going through You talk about an acute coronary syndrome with non-ST elevation myocardial infarction I don't know you have heard about non-stemmy or stemmy patients In the other can do the case when you have a thrombi that has completely occluded the artery and there is no way that Blood grows through the distal organ you're talking about stemmy situation ST elevation myocardial infarction and this is important because when a patient arrives to to the Emergency department you first do an AKG and you see you read the pattern This is an electrocardiogram. This is a bit of a heart and here it's divided in different Waves and the thing is that this wave here that's depressed. It's the ST wave So if it's depressed you get this non-ST Because it's not elevated but here when it's up you really know that's you are he's having a complete Coronary occlusion and you have to go rapidly to the PCI department to place an extent So these are the clinical events that may take place So and does the plaque has to rupture or with erosion that's enough Must be said that 70 80% of the cases in which thrombi have been detected in the coronary artist postmortem Have been because the the rupture there has been the rupture of the atherosclerotic plaque However, only although only 20 or 30% of the cases there has been plaque erosion The most interestingly is that among this 20 30% 80% do fight fatal events So although erosion is much much likely less much less likely to happen The thing is when it happens, it's more severe and this happens particularly women Here I wanted to I know a video which is not works The thing is that this is my atherosclerotic plaque and when it gets rupture exposes all this material that that then just Facilitates the platelets to adhere I will try the thing is that when we and wanted to put this video because it was the link between The atherosclerotic lesions and the main culprit of the thrombotic events, which is which are the platelets So you can see here doesn't matter we're talking for partial or total occlusion of non-stem in stemmy But all ultimately when you go and you zoom in you can see that the last player is the platelet We're talking about platelet We're talking about the blood cell that it's synthesized from the bone marrow and it has a very short heart like only seven ten days And you know why because it doesn't have a nucleus and since it doesn't have a nucleus That's it just in size and then dies and the thing is that if you take a look at the electron microscopy studies You can see here that its structure is that's plenty of granules And this is really important because the granules what are released from ill the all these platelets not only Condition how big will be the thrombi but also if there will be other responses Which is inflammatory reactions so forth as we can see in the next slide Look, I wanted to put this live because take into account that platelets only represent 1% of the components in plasma Only 1% and it can do all that So you do you do have to take them into account and look at the size is 4 times 5 times as more than the retro science And when they were get activated look, they release all this amount of components from their granules They have dense granules in which the release ATP ADP will see all these are just Challengers of platelet activation They release other gran other agonists such as serotonin and histamine and look alpha granules all these chemokines Garofractors 5-analytic coagulation factors So when a platelet gets activated and release all its granules in not not only will condition This thrombotic response But also will condition the growth of the atherosclerotic plague and inflammatory reaction that will take place of the site abrupture And it also releases in the last years has been taken into account micro anase and micro anase I'm our RNAs that cannot be have not been encoded to become proteins But they can modulate other RNAs and so they modulate gene expression So platelets as you can see here when they get activated They change their shape and they express or protrude lamellipodia would Allow them to get attached to the vessel and also get it at attached to the platelets As we can see here when they get activated again Not only they contribute to hemostasis and thrombosis, but also to inflammation and atherogenic processes This is this is the platelet and I wanted to highlight that it's full of receptors its surface plenty of receptors Some receptors are involved in allowing its attachment to the vessel wall as you can see here We can have all these glycoproteins because mainly the receptors are rich in glucose So that's why they called like proteins and what they do is that most of them interact with all the components of the extracellular matrix because when when the the vessel erodes or ruptures It exposed components of the inner vessels mostly extracellular material components elastic laminae which express all this a Compound over here and which thanks to the platelet that express all the receptors They can accord in the interact one each other and prevent What and prevent bleeding so I wanted to put this slide because this is really important to have in mind There is a huge difference between hemostasis and thrombosis Because hemostasis is the human response just to stop bleeding if you cut yourself You could your finger you just stop bleeding in 20 seconds Hopefully and this is because platelets have recognized through all these receptors Substrates lambicollagens, which are in the inner Layers of the vessel and they just get attached and stop hemorrhage But that's not the case. This is a physiological response in order to stop hemorrhage But this is not the case in thrombosis Thrombosis is there it has been a culprit. That's not a cut that has induced thrombus to occur So there is a pathological condition that stimulates from a formation and it Platelets go there not to stop bleeding just because they recognize surfaces that are foreigners for them And so usually that's pathological It's always pathological and we are talking about clinical presentations. So hemostasis is physiological Thrombosis is pathological and in both platelets and coagulation come at play. So both are important So here the platelets. I wanted to this is a diagram in which what the first Step that occurs during platelet adhesion to the vessel wall is through bomb-villain factor Bomb-villain factor is a protein that's circulating in the blood regularly But when it detects a surface that is not the endothelium it gets habitably attached to collagen and the Collagen attached to the bomb-villain factor. It's very appealing for the glycoproteins of the platelets to get attached to So here's the first point in which platelets get anchor to the vessel But this is a very weak attachment. So this with just flow platelet would go away So they have to be other interactions in which other Components or other platelet receptors then when when this one has occurred They get attached to the different components and this is forms then more tight attached of the platelet to the vessel wall And this activates what's caught an outside in signaling towards a platelet activation state And as I told you when platelets get activated several things happen first they change their shape and they start expressing lamidipodia Second they release their granules creation all the granule content as you can remember you may recall There were multiple agonists ADP serotonin capable of challenging platelets to activate So all these factors that are released interact with the surface of a platelet nearby Stimulating their activation But in addition because the rupture here the plaque has rupture This has induced the high mollusks of multiple red blood cells and red blood cells are a very important Source a very rich source of ADP. So there is a huge amount of release of ADP Which is attached to the platelet receptor and again further enhances the platelet activation process But what's the third thing that occurs which is very important is that there is an enzyme cause called phospholipase which is in the surface of the platelet that gets activated and what it does is that gets this acid Fatty acid from the membrane which is arachidonic acid and Converts it to tromboxan which is a potent inducer of platelet activation And I wanted to highlight this because there are different Differences strengths of agonists that can induce platelet agonation and although large amounts of ADP are released This is considered a weak platelet agonist and as you go down the line here You can see that tromboxane is more powerful and finally the most powerful one is from bin And they say finally because from bin is the end product is the end product of the coagulation gasket I would come will come further on so because of the importance of all this Receptors in platelets and interacting with different agonists in activating platelets The last event what occurs is that there is a conformational change in this receptor over here Which is called the glycoprotein 2B3A receptor I'm a specifying its name because it's really important when platelets get activated They induce a conformational change of this Receptor from a closed form to an open form and when it gets open the receptor it allows That the attachment of fibrinogen of envelope and factor both of which are regularly in circulation And what happens when fibrinogen gets attacked? That's rapidly looking for another glycoprotein 2B3A. So if the platelet nearby it's also activated It favors platelets to get interacted and form what's called the platelet aggregates And when platelets are aggregates, they are then firmly stuck to the vessel and it's very different to get dislodged So because of the importance of these receptors all the albumin terium nowadays in the clinical arena have focused on blocking all these pathways And just for curiosity As I told you later when arachidonic gets activated from strongbox hand, which is a bot antagonist and here when aspirin come at play It just blocks this pathway and prevents platelet activation to occur But despite the usefulness and the success of plate aspirin there are still 20% of events that occur being an aspirin And that's why there are many other platelet activation pathways that need to be blocked So we can be find the Fidia inhibitors from boxane part that blocks from being ADPs and 2B3As The most widely used in the clinical arena as you can see here the other ones are aspirin of course obviously and then This blockers of ADP and blockers of 2B3A Because why because ADPs released in large amounts So let's try to block the receptors although a weak receptor We have a lot a lot of ADP going around the site of rupture And why the 2B3A inhibitor because it's the last step if you prevent platelets from attach one to another They will never aggregate and they will never form a thrombus And why do not block all all the platelet receptors at the same time? Because then you have the risk of bleeding and you have to find the sweet point in which you prevent thrombosis But in case there is this physiological hemostasis this can take place and when You have been combining from being inhibitors having inhibitors with ADPs and what has happened Okay, the person does not have any event because of thrombosis, but it dies because of hemorrhage So this is why you have when you use the combination you have to use the right combination to still have on top working hemostasis So this is just a brief summary which has been presented here What happens is the the surface of the plaque at the road to the rupture allows attachment of a villain factor which the first initial step of Adhesion of the platelets this the plate that gets further firmly adhered it gets activated It changed the shape it releases all the granule content and activates arachidonic acid to form thrombocyan And finally it changes the conformational change of the 2B3A allowing platelet platelet interaction and thrombiformation Platelets also within the granules you can see that you have seen they have inflammatory mediasis And this is really important because then that recruit is inflammatory mid inflammatory cells to the growing thrombi So the thrombi not only has platelet and inflammatory and red blood cells that have been trapped, but also inflammatory mediasis But so far we have talked about one of the components in thrombosis which are platelets But there are two components of thrombosis, which is the coagulation cascade because despite platelets get aggregated They kept they have to get firmly Attached one to another in in the net of fibrin which it surrounds the thrombi and Allows it to do not dissolute and here's with the coagulation cascade comes at play The coagulation cascade is a process coagulation factors are simorgens That means that are proteins that are inactive in blood and they circulate in the regular conditions But when either one or the other Pathway thrombotic of the coagulation cascade get activated What happens that these these simorgens get cleaved and they get activated and there is a cascade and one activates the other and Activates the other so it's then you end up having frombin here and finally which converts pharyngeal into fibrin No, so first we have the extrinsic pathway in which the most important component is tissue factor Tissue factor is present in all this lipid rich foam cells I talked to you about monocytes that have been engulfing the lipids vascular smooth muscle cells that have been Capturing the lipids and in addition to the lipids all this lipids what induced to the cells is to express tissue factor And this is very dangerous because when there's the rupture of the plaque this tissue factor gets exposed and Triggers the extrinsic coagulation Pathway this there is another pathway which called intrinsic pathway or the contact pathway in which the activation of platelets and the breakdown of several cells Release negatively charged particles and this negatively charged particles are capable of activating factor 11 in factor 11a But nevertheless if there is a breakdown of the atherosclerotic plaque with exposure of tissue factor Which is able to cleave factor 7 in factor 7a This come this this complex can also Activate as you can see here the intrinsic pathway and I start cleaving all the factors one to another one to another Until eventual deformation of thrombin. Do you remember I told you was a very potent platelet agonist But thrombin not only activates platelets But converts the fibrinogen that is synthesized in the liver and it converters to fibrin forming this fibrin measures You can see here But I want to draw your attention to factor 8a in factor 5 Which are key steps in the coagulation cascade because factor 8 just allows the conversion of the web both converge both coagulation pathways converge in activation of 10a This is very important and also factor 5 because allows the conversion of prothrombin to thrombin And why did I want to pay the draw your attention to these two because these two only get activated in the surface of activated platelets in In other words if platelets do not get activated this this coagulation pathway does not occur And what platelets get activated what did happens is that they change their phospholipid bilayer? So they exposed in this external surface negatively charge Ions which attract the factor 5 and factor 8 to get it here So there is a retrosclotic plaque tissue factories released and activates the extrinsic pathway And then all the coagulation pathway takes place Thank you that platelets have been activated and have exposed the ability to get a factor 5 and factor 8 attach So what what is first is it coagulation or is activation the coagulation cascade? So several studies have been trying to just find out with which come first and the things that we perform several Studies with the perfusion chamber differential rate with the use of a human coronary substrates And with what we did see is that first when you when you trigger a from boost first What you see is firing here in red that is the resultant of the coagulation cascade and Afterwards on top of five in platelets start to get adhered But it's not that coagulation first take place, but takes place more rapidly So when the coagulation cascade gates activated rapidly trombine converts fibers into fibering Meanwhile platelets have get slightly adhered tightly adhered Activated and aggregated so that that's why the reason why firing gets firstly the positions in the vessels So because of the the important role of this fact to 8 and 5 to 5 here the conversion There have been many drugs that have tried to stop the coagulation cascade and most of them have focused mainly in two factors One is factor 10 because if you block factor 10 you block the conversion of the two Extrinsic and in strength the pathway is a conversion point and Another one is it's factor 2 or from bin because once you stop from bin Formation to occur not only you inhibit platelets, but also you prevent fiber information And nowadays there is a fight against what should we do should we block factor 10 or shot with to block factor 2? so but in Parallel to the coagulation cascade there is also running what's called the fiber in a latex system because I told you later Later on there in the physiological conditions in the thelium is in charge of of releasing tissue plasminogen activator here which just Converts the plasminogen that has been released to the liver to plasmin and plasmin has very very high affinity To 5 5 in polymers so although the fibrin has already the fiber network has formed if there is a healthy and the thelium There is no problem because we'll have plasmin and plasmin will break down all the polymers of fibrin and then have the different time is here So that's why in the physiological conditions hemostasis you make a cut it stops bleeding And then you have no more issues because they have renalytic system has has completely fantastic But what happens in the thrombosis and the thrombosis there is a huge increase in the release of one inhibitor Which is called pi plasminogen activator inhibitor that what it does is that prevents the formation of plasminogen to plasmin So if there is no plasmin, there is no breakdown in case there is some plasmin when you have an acute event What also rapidly rises in the blood is alpha 2 anti-plasmin. So just in case some plasmin has been formed There's an rise a tremendous rise in this protein which blocks definitely the breakdown of polymers of fibrin so You can see here that the vulnerable plaque not only is rich in tissue factor which induces the coagulation cascade But also is very rich in this inhibitor on the fibrinolytic system So if you have thrombosis if you have plated activation you have coagulation and you have impaired fibrinolytic system That's what's happening here. You have the formation of this thrombi Which is rich in red platelets in firin that recovers all the measure of platelets and because these firin Does not allow cells to pass through red blood cells have get attached and also inflammatory cells have get attached And if this occurs in the coronary artery here, you have an acute coronary event So just to end on my talk I would like to to give a hint and when you when you take into account I'm going to Model vessel and see what happens in thrombosis and coagulation Because it has had acute event or I want to model patients and anti-platelet treatment There is another factor that you have to have surely mine, which is the presence of comorbidities in other words if this patient is of ease if this patient has diabetes and if this patient has hypertension and This is because all these conditions Per se independently of the presence of acute coronary event Worse than the progression of the thrombotic process and I wanted to draw special attention to one that has been lately most epidemi that has had a Pandemia in the last 30 years and which is quietly perfectly now I would say all the mechanisms that drive an increased thrombus formation because diabetes They're still under no relation between having high glucose levels and the risk of thrombosis But it's definitely sure that when you are obese your risk of thrombosis is increased on your five in a little system is impaired We're talking about the disease Look at look at these numbers here that not only is present in the United States with 46 percent of total population But just the south of Europe these numbers are really alarming So which is really it should take into consideration that the likelihood that you have to perform in a study of an obese patient Is really high 40% So What do you understand by obesity and this is a term that needs to be clarified when we talk about obesity you regularly Are you we talk about body mass index? This is the best known among peers The term and when we have BMI of above 25 You're overweight if it's above 30 you have obesity type 1 and so on obesity type 2 and type 3 So this is the most part of the population are within here of overweight obesity class 1 But sometimes this this may lead to to mistake or to me misleading Perception because you just have a very high body mass index because you are all muscle So you must have a body maxing of 29 and it just because you are so fit this one over here So in the clinical arena it has gained every time more and more the concept that BMI should not be used But should be used intra-abdominal adipose tissue Adiposity because here it's clear that the waist circumference is clear that you are not fitted So if it's above 102 centimeters in men that means you are overweight And if it's a both 88 in female that means you're a weight And it's been important studying what is there has been a very nice correlation About the centimeters of this waist circumference and the intra-abdominal play presence of adipose tissue There's a nice study here the hope study in which the presence of this abdominal tissue was clearly Associated with an increased death Increase risk of cardiovascular death of myocardial infarction and overall causes of death So we were talking about the problem that you will really routinely see in your in your in your patients or your studies So but obesity not know look at all the deleterious effect that has in the lungs in the language and ecology in the bones and Do not forget the ones related to cardiovascular disease because there is a clear Association between gaining weight and the risk to suffer Cardiovascular death But not only cardiovascular death because if you're lucky enough and you don't suffer an event It's clear that your life expense expectancy can be shortened it Look here If we take a body mass index of around 30 that means you're just overweight obesity type 1 and you are around 20 30 years old here you have lost already two years of your life just because you have that way and What are the what are the problems with gaining weight and having more adipose tissue because But I say adipose tissue is a source of energy So there is all the equipment of adipose adipocytes just in case You don't have food and they get released to give you energy or you cannot eat for 12 hours You have them being released and give you energy But in addition during the last 15 years it has become more evident that there is there an important source of look at this of leptins resistant which are proteins involved in Enhancing the thrombotic process it also again. It has by one to remember by one the inhibitor of the five in a latex system It also releases all these inflammatory mediators So again, it perpetuates the inflammatory milieu already present with patients with cardiovascular risk factors in the first Sclerosis and all these leads to an increase in the thrombotic risk as you can see here and the physiological Conditions there is this fatty tissue is not inflammatory tissue. This is a healthy tissue and just a source of energy There is not enough. I have to keep it over there. There is no release of inflammatory mediators There is no release of these proteins that induce thrombosis. So it's no inflammation of thrombosis keep lean But what happens if you are obese then all these adipocytes are a source of inflammatory mediators And they look at more inflammatory cells over there that not only induce further endothelial damage and allows Endothelial not to cover and protect against the presence of events But also secrete all these amounts of proteins which induce platelet activation They also release tissue factor and different factors of the coagulation cascade Inducing and hypercroglabital state and finally remember another important source of pi 1 So the final it is still impaired. So we're in your new month to model Situation in where in which there will be a thrombotic event You have to take into account will it be of ease will not be of ease will have a potential Because all these parameters will certainly change your modulation Just so just to summarize and stand I would like to just take home messages First one related to atherosclerosis It's very important to have in mind that it's mainly associated to vascular region with bifurcations So it makes no sense to evaluate thrombosis in the aorta better to do it in the branch In the in the carotid in the coronary arteries So where there is disturbances of this flow that induces Damage endothelial layers and this damage of the endothelial layer allows lipids to entry under development of the nascarotic lesion Second conclusion would be atherosclerosis is due to this lipid accumulation In the intimate of the vessels and the recruitment of these inflammatory cells that in the first stage is protective In order to prevent the development of atherosclerosis But the continuous exposure of a decade of too high levels of LDL just overwhelm this protective system and becomes Deleterious and third plaque or atherosclerotic plaque indeed may may rupture usually they rupture But do not forget that if they erode the likelihood to be a total occlusion of there is really high was almost 80 percent And what about thrombosis? Thrombosis results from the interaction of platelets with coagulation cascade This is very important both have to be combined and both have to be considered You cannot omit platelets if you're looking to thrombosis and you cannot omit coagulation second that Platelets not only are involved in thrombosformation, but the their activation also released a hundred of mediators inflammatory mediators That will potentiate the growth of the plaque and also the recruitment of further inflammatory cells And that in the pathological conditions the fibrinitic system you want to take it into account Is not working properly in fact is not working at all and that is why well thrombosis do not dissolve And finally that the presence of copper mitzis and hands all these risk of thrombosis because first induces endothelial dysfunction Activates platelets in they're more reactive more angrier third they impairs fibrinolysis and finally enhances activation the coagulation cascade So said that I would like to just thank you all for your attention And I will be more than glad to take any questions and have a wonderful stay in Barcelona Thank you very much for this comprehensive overview So I think it's already clear and you nicely showed it that when you want to study vascular function You need to combine everything it goes from structure Damage to structure related to flows know everything about the biology see at the Factors that will enhance thrombosis or will take it away and things like that So it's a very delicate balance as you also say it's like between physiology and Pathology and these are the things which are quite important And so things are always not as simple as you would try to hope them to be as an engineer for example in order to do the modeling They're not only three four factors. No, no exactly exactly Maybe just first a general question also for this type of public is like when you look at this research field What's the thing which is now important? Do we need more molecular biology? Do we need to know more about the physics behind it? Or what is missing in order to solve some of the problems? So do we just need to get better drugs? I think that you cannot get better drugs if you don't get a better knowledge of what's happening And all all this history it's known from experimental models which are far away from reality Be honest and also from post mortem analysis because that's that imagine what has happened post mortem So I think that it's really important to combine The modeling within vivo scenario because I think it could be the most closest To to the discoverer at least to the testing of anti-platelet drugs Because when we when we're testing to platelet we have tested men anti-platelet drugs now And we have used ex vivo perfusion chambers and they can give you an scenario what's happening But it's it's not reality. It's just the vessel within the chamber and exposed to flowing blood Perhaps with with the modeling you have the chance to really take into account for the for the components like hematocritic red blood cells Like how the the flow can disturb How can the black cat rupture because I have exposed you that the black kid rupture and the black kids erode But you know what it remains unknown how this happens. Nobody knows how this breaks and how this erodes And perhaps if here we combine the the modeling processes and just know look if you reach this Force if you reach this strength then the plaque cannot resist anymore And this only I think modeling can can reach it be honest When you when you look at it it's like is this a Research field or when you go to the clinic what's important to the cardiologist just need to know what's the size of the vessel and depending on that treats or How do you link these things how you do translate not only from the basics to the engineering but to the clinic and how do you All these concepts are important just to have in mind what you what you have to address So when you go to the clinics What the the clinicians really want to know is is the vulnerability of the patient Is how are they played in it? Will they get activated soon other Coagulation system. How is it the fibrolytic system because they don't do not know all these all these basic components. They just measured Different factors on the line and if they have pi levels they have so pi levels no fibrolysis They have fiber in products that's going from analysis and so forth And they measure the function of the platelets and they say oh, they need more anti platelet agents But perhaps it's not a matter of anti platelet agents Is because they have not taken into account that the patient is obese and perhaps if they treat first obesity They will not need to enhance anti platelet agents and they perhaps they have to Put combined all these concepts together in us just to find the perfect target. In fact There are many every every year you go to European science of cardiology and different guidelines go out So there is no consensus. Which is the right drug to use and all drugs have Drawbacks because I told you there was a war between the factor 2 and factor 10 a inhibitors for the correlation cascade And it's just because once you give them orally, but give more bleeding The other ones you give less continuously. So every every single day the person has to Put it itself, but it does not keep bleeding So there are so many parameters to take into account that this perhaps Thanks to this is what gives research going on And but there is when you look at the pipeline of anti platelet anticoagulant agents, it's empty. It's completely empty So unless we keep on doing research and we find new targets within the following five years All drugs will become generic Which is a disaster from most of the industry and also disaster for the researchers because they they invest a lot of money in doing research Let's be let's be honest But we need to find new targets and to find new targets. We need to know how things happen And to come back to modeling Where would you start or what would be for example that you say like you have a student coming to you interested in doing modeling Do you want to go to the basic the kind of the the molecular part or more to the cellular part? Or do you say like no, no, let's go to the patient because in the end It's the patient which is the most I would go to the to the cellular part Not to the molecular part the molecular molecular part is more for the discovery Not rather than for treating and I think that from a modeling point of view is important to to treat properly the patient So I would go to the cellular part and take into account This cottage of the blood is dependent of red blood cells of matocrates Of the share rate of the platelet numbers. What happens if it's slow? There is the rest numbers than being high and One is this and the other one will be also Integrated in the modeling all the coagulation cascade Is really important if you can model what happens if you increase the concentration of different factors What happens to the resulting modeling in the susceptibility form of thrombi And one of the things you also mentioned on forehand is like when you look at vascular problems You see it's cardiovascular neuro vascular peripheral vascular to Are there separate entities? Are they combined? Do they come together? When do you study which one the truth is that they're not as combined as we Would wish so because when you talk about stroke, it's true that you have ischemic stroke But it's not most often you have the hemorrhagic stroke. It has nothing to do with this. There is a breakdown of vessel So usually when you dedicate yourself to serobascular disease Then you just focus on what's happening there that if the heart is difficult serobascular It's it's even worse. So it's really tough to get in there You don't have the images vessels are very small and you do not happens It's not exactly clear what is the function of the risk factors in the presence Of stroke and neither the impact of different anti-platelet agents because most of them have failed In demonstrated benefit. So why because probably ischemic stroke or stroke due to thrombosis occlusion is not that often And usually goes hemorrhagic stroke and as for peripheral artery disease Nowadays, it's it's easily treatable. So you can with a flow Doppler or whatever you can just know where the thrombi is And just perform a surgery and take it out without other secondary effect that having had pains in the leg So and and when you want to look at vessel properties, for example an individual patient For example, if you would study cardiovascular diseases to get coronary Properties, it's rather difficult if you would do peripheral arteries if you would study their properties How much do you think you can translate measurements that you would do peripherally to? Not much not much because Just just to put in an example when you the share rate that occurs in the coronary arteries, which makes them break The same share rate in the carotid arteries do nothing So when you use when you study a special location, you have to study the special conditions Which make the plaque prone to rupture. So if you study this 700 1700 30 500 seconds minus one in the coronary arteries Don't apply that for the carotid artery because it will not work even less to peripheral disease So you have to be and we have any idea why that is because as an engineer you would say you have Sheer stresses in a certain vessel. No, you do the same thing every time. No, what are we missing? It's it's there are two potential hypotheses One is that the structure of the carotid artery since it receives the pulse Of of the body if you take into account is not the same the coronary artery that just just nourishing the heart That the carotid artery that's you're receiving you can notice yourself So this has made all the vessel structure more more more More harder. Yes with the different composition. It's the celeromatic components This is the first reason why they say it's not that easy to break because it's it's Able to challenge all the differences in the share rates and blood velocities compared to the coronary artery This is the first one and and the second one is just that The composition or the inflammatory recruitment of the cells to the different zones of our eyes And it's just a matter of debate and a matter of investigation of what it happens So it's a target for Molly's target Okay, any questions? Thank you for the presentation And What is the role of personalized medicine in this? so the genetic background of Very good question indeed because this this is where modeling has to has play because when you went to The the goal of personalized medicine is just taking into consideration all the factors that makes that person different And in the clinics we cannot we can just say it has a platelet function below three It has an event no further events and that's it But we're not tricky improperly because we are not taking into account how the coagulation system is working How is it risk because you you can image all the atherosclerosis plagues and just make a picture of the burden Of how so acceptable susceptible is this patient to suffer an event And with modeling you can include all these conditions and just make like a score Of what is the risk of this patient to suffer another event And this only can may be made through modeling. So Only through modeling we will definitely reach personalized medicine if no, there is no way because it's Too cost effective It will be too expensive to take one by one and make all the battery of a sage We need to measure the five unit the system the coagulation factors the platelet All the presence factors factors if the intercellular is working all that will be okay. This patient is 100 000 euros Okay, give it away. We'll leave it personalized medicine With computer modeling you can say well, it has all we can make a score With all these factors the likelihood to happen is this I would treat it with this And this I think that in fact arise on 2020 is very interested in reaches this goal And which it should be the aim of all of you, which is mine also Maybe just to pick in on this one. There's more and more debate in general in medicine about the link of genotype phenotype Well in the past people would say like or if we can sequence the whole genome of an individual patient We know everything that's going on now in different fields. It's clear that maybe it's more subtle How is it in this field? No, no, it's the same. I mean the genome the wide genome at the general Hohemian genome has discovered that it was a real breakthrough and everybody had you know, they had discovered The entire solution to cover a carnival to solve cardiovascular disease And that's very far from that because it's it's nothing to do There have been many many genetic studies to study genome and the association of the risk and And that's it all all results are no and they have spent thousands and thousands of billions of dollars doing that And the phenotype things you're exposed to it doesn't matter how you're born But how you've developed is the other main features and in suffering from an event So what do we do with genetic information? Just genetic information get it? No, no because genetic information is good for this that kind of pathologies for instance Hypercustid familial hypercustidolemia in which there are families that already When they are born they have 300 and 350 milligrams. There's a liter of cholesterol and here genetics too has a paper Because genetics there is the one that's determining that there is a receptor of lipids. That's not working well That's why lipids are so high. What can how can we target this gene? This this isn't a scenario other scenarios which we have hemophilias We have the absence of factors coagulation factors or we have the absence of of platelet Very low quantum number of platelets. What do we do? Can it die by hemorrhage? So all the inherited disorders do have an important row in genetics But not as we look from bosses as a whole as a pathophilic pathological problem That's really threatening all the population And how do you think these genetic abnormalities can be used for example also to start the research or start the modeling? Is that a place? It is a place. It's definitely a place because not only what happens if this factor in factor five laden There's a disease that some factors are missing coagulation factors are missing But by modeling you can say what happens if we put the factor in what pattern we take the factor out But we cover with other factors So this can only be done modeling Because not only when doing experimental model models with animals I worry where this is expensive But it all you also have many ethical boundaries that you cannot overcome So with modeling you don't have this this issue Thank you for your presentation. I have two questions. The first one is related to the turbulence of the flow in the vessels You said that the pathway activates more when you have separation of vessels When you have rigidity Rigidification of the vessels due to I don't know age So even in in straight vessels you could have and the second question is related to the difference in The size of the vessels For example, when you have very small vessels the red blood cells change shape and you have different behaviors Does the pathway change here or is it the same? No matter For for modeling point of view, could you model the same? The parameters are different for example for the shear rates, but the pathway is the same. Yeah Let's start in the first one that was at the turbulence and the rigidity of the vessel The thing is that what happens with the rigidity of the vessel is that it is cannot compensate for changes And this is very important since it cannot compensate for changes There is a likelihood that Cellular elements of the blood get stuck there And since they get stuck there more easily formed from by So indeed, if you have a very high rigidity Not only in the thylial layer For sure is this functional because because it has disappeared all the capacity to compensate But also it cannot adapt to flow conditions and there And then cellular elements that should not be interacting one to each other's do so So we do it is an increased risk of suffering from boses And as for the red blood cells you made a very important point which is under low flow conditions What happens to the red blood cells is that they get deformed And when they deform themselves They are more prone to get Attach one to another to change the viscosity of the blood and to challenge platelets To get one close to the other one another clear example Is the case for vein thrombosis because we have talking all the time I've been talking about arterial thrombosis In which platelets are the main component But if you go to to venous thrombosis The the major component is the red blood cell Why because it has become you have the vessel blood vessels that The the the bulbs do not work properly All the and they work slower that makes that the flow rate goes down And all the red blood cells get deformed and help platelets to get in contact With the covalent cascade and activate the covalent cascade. So while in the arterial tree High share rate platelets get activated thrombosis forms In the venous tree is the opposite is the lower the share rate The more prone to form thermosformation and I'm very glad you've brought this question out because you also have to distinguish If you want to emulate venous thrombosis in world in which estasis and the coagulation Cascade are the most important factors or arterial thrombosis in which a high flow rate and platelets are the most important factors Partially related to this also something you just Briefly mentioned with nitric oxide and things like that is like vasoreactivity How important is vasoreactivity in all this story? It's it's crucial the vasoreactivity is just it's true I just went through it very fast and Just and didn't point out how important it is because while you have vasoreactivity And the vessel is able to adapt to any situation that works fine the system works So it's something you have to take into account. I don't know if in modeling It's you're able to to take into account how you can compensate for these of movements or not But it seems theoretically if we know what to model we could model it's a combination of first knowing and then Being able to but that that would be wonderful because then if you can put this Taking into account this when you model that that then it would be very very close to reality Which is the most important of the modeling part But partially already mentioned like when it's normal and vasoreactivity is normal Then things are compensated but very often you have like a lack of vasoreactivity So the question is then also like maybe we can simplify the models by just leaving it out if we want to model disease Yes, or do you still no, no, you're right if you would just want to model disease Then you just leave it after the equation Okay, thank you. There was a question here first very nice presentation and another thing it's that the Opposite numbers are quite scary But I would like to question to one. Yes to ask if do you think stem cell therapies Would play a role in treatment of other sclerosis. Okay. You said stem cell therapies. Yes. Yeah Stem cell therapy is very very hot nowadays and we are studying cell therapies over there in the lab But we are dressing more it's used to to once the heart has infected in order to recover the heart because stem cells Numbers in the in the stream in the bloodstream. It's extremely low So when you just find one out of One one thousand million you say it doesn't have it doesn't have nothing to do here So numbers are so small in circulation of stem cells That unless you you challenge they release from the bone marrow by secreted by infusing some growth factors or whatever that I induce they release and perhaps then you find more in the flowing blood I would not consider them at all Particularly because if there is they are extremely low and they're a healthy patient When it's diseased it's even lower. So if you're lucky you find one if not you don't find anyone That's why in in cardiovascular research We are using them to repair the heart But what we do is that we obtain them and then we isolate them we expand them We get six thousand we get a million and then we inject them But we have manipulated stem cells We have not allowed them to just flow As physiological would be a pathological would be Talking about Cells and stem cells also It's like what you say a lot of the things start with endothelial damage But from what you say and when you look at the drugs, they don't seem to directly address the endothelium What what is the reason because it's too late The thing is that endothelial damage has already happened and it happened a few decades ago So i'm already late the problem is the the first manifestation of the disease is that the endothelium is not working well So and then all the procedures start and then 20 years later 30 years later you have the vent So you do not address the endothelial damage because of that But if you're if you're doing a follow-up study All of them include the analysis of molecules involved with endothelial function To see if an intervention we do that we do it We do we give dieter dieter interventions to obese people that are healthier not under any drug And what we do is measure endothelial function and see over two years what happens with the diet intervention Is endothelium is still working although they are obese So in this scenario it's it's useful in the in the use of anti-platelet armamentarium with purposes just to Stop the risk of having another infarct. It's too late Yep Thanks for your talk You seems very keen to to modeling But I would like to know your opinion. Okay. What you dislike about modeling and also In your opinion, we should be the the key element That we should take into account as a as a modeling community to integrate that technology into the clinical practice I'm very keen with modeling because we have been trying to get two european grants during the last seven eight years And with at the moment no success. We have two submitted european projects at the moment and the thing is that's why I'm so keen on because Um, I really believe that it could lead us to personalize medicine But what I hate is when you write down the the protocol they say Okay, but you have to give me this detail and I say no No, no, I don't know this detail if I knew this detail or how many Factors are released by the platelets or how many platelets would get attached Definitely, I will not be I would be just looking for new Both to be coming out millionaire because I will knew everything And the problem with modeling is that you need too many parameters which are still not known and you keep answering and I'm running a project of biomarkers And the thing is that by by I will I will provide all the Particles and micro vesicles that are released in an atherosclerotic lesion A prior atherosclerotic lesion and post atherosclerotic lesion And I will provide all these markers and then they will model What's the risk of this plaque to rupture? Combining imaging techniques and see if it has a very liquid rich core or not And they always ask me which which other markers I have to measure If say if I knew the markers I would just set up a kit with Pfizer and sell please measure this marker and you will know the risk So I do not know the markers it just you have to go by chance and Put an array of markers to see which are the ones that may be released from the plaque Because it's not known it's because when you have an event the issue is that you know it's too late So since we're not able to know if this plaque is going or not rupture What I find at the site of the plaque is it from this plaque or another one rupture and had the event Or perhaps it ruptured three days ago and it just the trombosis got dislodged So there may did too many answers that the too many questions that have not been answered yet That cannot provide you as much as detail evaluation This is the only thing that it makes more difficult to To understand to get to get to collaborate just to see I need all these parameters And I cannot give you all that this is the only thing and the second question. Sorry, which was The key elements to integrate modeling today clinical practice the key elements would be To take into account as much Factors as you can that's why they keep asking give me as many factors as you can because I need them all The more reliable you make the model For definitely for sure the most success it will be and we have we need In the in the in the clinics. We definitely need what's called ready to use assays or Betside assays that can provide you with a quick cancer, which is the vulnerability of the patient But to do that you need You need to know To take into account as I said many times all the cardiovascular risk factors How many does it have white is the impacts to a very cardiovascular risk factor in thermal formation How the number of platelets dematocrit red blood cells the viscosity of the blood If it's never in if it's not in ever in was it treated was not treated So all these factors may definitely Take you to the proper treatment of the patient It's interesting that you mentioned also the saying like these biomarkers because it's one of the other To me slightly disappointing fields of research that a huge amount of money went In trying to find like the holy grail one biomarker that for specific disease would tell you everything But I think like it's clear by now that this is not the way that it works No, it's like just one biomarker Would not help too much or what's the role of biomarkers you think the role of biomarkers first of all It's a it's a pretty new story So we still have to have faith and that stem cells have already running for 15 years So we can now have to be more Accepted but with biomarkers it's a newer field and the thing is that they they try to since since they don't take into account Modeling there is not at the moment. We cannot model anything Then let's find the particle that tell us if if something has happened and again With whom with a patient has had 30 years of disease. Is it visa hypertensive? It has been taking statins 20 years That's why it has not been successful because there may too many confounding factors to read a biomarker And the biomarker is just a molecule that is found in blood and can give you diagnostic How the patient is So let's call the biomarker could be the readout of pi one But since pi one is increased in obesity is increased in inflammation It's not a good biomarker because it's increased in everybody So the thing is to find one protein that gives you a hint of what that patient that patient that has suffered from event Is is susceptible to have another vent And so since many of them are redundant and most of them are present in all the pathways pathological pathways That's why it has not been success and probably because we do not need to look for one biomarker But we need to look for a panel of biomarkers Which are indeed only expressed in those situations in which the patient is susceptible to again full of sufferinothermotic So it's integration of information. This is the most important. There was a question there Thank you Question that is not so related to modeling or maybe it's also relevant, but i'm curious You mentioned something about the women are more susceptible to atherosclerosis and also Could you please comment on that and also the effect of contraception because Okay, everybody very good The truth is that I had one hour and a half. I didn't use all my time That's why good questions because you follow the talk the thing is that women die more of cardiovascular disease But are less susceptible to atherosclerosis So the thing is that there is the women reach an age where they reach the metaphors in which there are no estrogens And the lack of estrogens Lead to endothelial dysfunction Lead platelets to aggregate and to a dysfunction in the coagulation pathway So under 50 54 the women is much more protected than men against suffering any Cardiovascular event But then since estrogens disappear not only they lose their protective effect But they lose a complete protective effect and they not get neutral. They get the opposite because The human body reacts as something was missing And if something misses then they increases the risk and that's why they die more than men But if we if we analyze the death of cardiovascular death of women men over over the decade You would see that the 40 years women is much much more protective because of the presence of Of estrogens in line with this the use of contra-conceptives or Is not an issue because what only does is that changes the levels of some of them to make sure you're not in the Fertile moment But the concentrations of When you are reached the menopause estrogens completely disappear Here is that they work with the progesterones levels in order to avoid that you get pregnant But they do not abolish your estrogens, which is quite different If we go to the peri menopausal issue There it's very important to have them because they also Help to stimulate estrogen production And it's seen multiple studies have seen that while the use of contra-conceptives after the peri menopausal Years is dangerous While you have in the peri menopausal It's very beneficial because they allow your estrogens to keep balance and to keep steady for two three years more Okay, thank you very much for this very interesting talk and very stimulating discussion I think thank you. Thank you. Hope it's created a lot of ideas and everybody on what to do next So thanks again and now it's coffee time