Will cause some changes in and the function that eventually leading to a forms or development froma this sometimes goes on to become vulnerable plaques and sometimes it breaks open causing from Moses and causing heart attack or stroke. And some of these plaques can sometimes here become a stabilized plaques However we are still really at the process of understanding the detailed mechanisms for some of these processes in specially horrible plaques. So why apply American engineers such as me interested in questions the reason is shown in this lied if you look at real crosses of course pros in humans and animal models it's been well known that it occurs in specific places those branching point of the carotid artery. And branching points in the coronary arteries and your approach is the same occurs in both animal models as well especially in the area Garge in the last two regions of the oligarchs these are all the regions that are associated with dynamic disturbances so it has been proved it has been proposed that human dynamic disturbance in these places might be associated with that for us crosses but whether a flow disturbance could say could actually cause arthrosclerosis on out was unknown. So this show for the shows. This photo shows the relationship between bad flow or distort flow and after a screw system element so this is a human created. Tree and as you would see here is the plaque. And you will see the plaque in this area. It's I guess it doesn't like me to use this one. So the plaque occurs in this corner area and that corresponds to exactly this area where you will see these whirling patterns of flow and this is really showing the good correlation between flow disturbance and potentially arthrosclerosis. So if I go for that I'll just give you one more introduction to the again and the thing this shows. And within the larger world there are two major forces one is blood pressure caused by the bottom of the blood and the other force that is well known is sure stress this is a viscous fluid going across and that William dragging across the surface and that shear stress so one of the bottom line message is that if you have you need to action or high level Sure that's good that protects and the killer layer that protects against the disease but if you have a few stress that change the direction at a low speed then that usually is also it also shared it with disease development. And again I will focus today on how in the field. That is directly regulated by sheer stress plays a role in this process. So again when you see when you have your stress and the till you recognize that force using a various McKenna sensors there are almost twenty different be kind of sensors and they are. Some of the surface including Calix some of the junction including you can one theory and. The other is. Exist at the bottom of the surface such as into greens and skeleton also. Potential new kind of sensors. And once and that is recognize this different types of stress then they react depending upon what kind of shoes first they saw So this is really a summary of a lot of work done by a lot of people including Larry McIntyre and narrow in on our campus where this is most of these lessons were actually learned from in vitro studies where if the theaters are exposed to a stable unidirectional flow sensor is turned recognize it and then that cause. Signaling pathways including I three kinase A.K.P. pathway protein kinase a pathway and pathways these then lead to a subsequent change in our circulation to produce nitric oxide production but in more chronic tones they will then lead to changes in June expression so in Christie expression of care to know if two or and the failure of. US is known to occur or these changes then lead to subsequently changes in many anti Etheridge and responses so for example if your stress. Unidirectional I'm not sure stress inhibits a pup ptosis proliferation inflammation through Moses In contrast when the theory or cells are exposed to. Flow disturbance or oscillatory such as in these areas the branching point and the theory or sense that change then it activates. Different signalling pathways including P.K. C. Zeta one and June kinase these then lead to subsequently activation of many stress related responses that lead to activation of. Reactive oxygen species and nitrogen species increase these then for there are lead to changes in June expression including And of copper B. and more for the new proteins. These changes does eventually lead to changes that are consistent to pro responses including activation of a pump hoses inflammation proliferation and through Moses So these were really lessons learned from in vitro studies however whether these changes are really responsible for atherosclerosis development was really on clear So my question was really does flow disturbance or disturb flow really in the process in be able our hypothesis has been that disturbed flow characterized by low an oscillating flow regulates and the killer gene expression priming and the theory for atherosclerosis in the presence of or the risk factors such as hypercholesterolemia So to test this hypothesis. We developed a any more moderate this is a partial quite a large Asian model well that those courses using mouse so mouse as as well as humans we have to crack it out to its left and right and one of the reasons that we chose this area is because most common crowded our two regions right come a crowded artery left come across adultery they are straight they are always exposed to unidirectional flow unless we do something and they never develop crosses so we wanted to change flow in one side. And we can use the other side as a control side so to change flow in one side without religion. Touchingly cereal we did a surgery called Partial by lying gating three out of four small branches and we left one branch open. To a this then lead to following changes if you look at this sound Doppler measurement before ligation in the left side you can see the signal always goes in each cardiac cycle one way that meaning there is a unidirectional flow however after the ligation then you can see the signal is actually reporting so that means flow is flipped in each cardiac cycle and so we did a competition on modeling study done Giddens group and this is what we found. We found that flow. Goes always unidirectional early in a person or in the right side but after the surgery in the left side flow goes only briefly forward but then it goes backward throughout the cardiac cycle so we were able to generate a low end aspirating flow that mimics the what happens in the naturally natural areas. So using this method Our question was can this flow disturbance cause that was closest to testers we took a mouse called a poor knockout mice they actually increased cholesterol level it's very high upon high cholesterol diet so we did the parts of our usual surgery using a point out on mice and then we gave them a. Diet really high fat diet it's really a super diet for two weeks and this is what happens in the left side in the entire length of the carotid artery developes arthrosclerosis while the right side the control side where they were exposed to good flow they are prevented from atherosclerosis So in the same animal this is what's happening and if you look at the details in. The cross-section here in a time dependent manner this is what you see in in the left side where flow is bad one week to week you can already see this red staining meaning you have crosses already developing in two weeks by three weeks it becomes bigger by four weeks it's almost closely closed off and again this also shows additional signs of advanced lesions in trouble. And all our cholesterol craft. At the same time if you look at the control side they are clean no Afros courses so we have been using this model to really understand how those courses of course and for the first time we were able to demonstrate that yes flow disturbance causes crosses. And this is a using the same model in two weeks by ultrasound in the right side you can see in the entire length of the right come across that artery the flow is very clean there is no disease in the left side again you can see plaque occurring in the entire war you're with very narrow Lumen. And so using this model we have been studying how low disturbance cause that was crosses over time so this is a summary of many papers. So as soon as we do the project ideation flow disturbance occurs and this is sensed by the till your this then lead to changes in coupling and activation of any D.P.A. Chuck States is to produce We have our two X. and spaces and by one week this is the need to a dysfunction by two weeks it induces after a more formation and by one months we can see when sneezing and forming and by about four days and on we can also see immune cells infiltrating. Into larger war so the next question that we really wanted to test was that what are the mechanisms that a simple flow change could lead to all these. Responses So our hypothesis has been that it must be changes in genes we cannot sensitive genes at the level of messenger R.N.A. Micron and and epigenetic changes and metabolite changes so to test this hypothesis. This is what we did and actually we were very lucky that we chose this a straight part of the carotid artery because it's like a long pipe so after we do surgery we can cut it out and we can actually simply flush it and believe it or not this was such a simple technique that gives us almost pure and the theologian's and this technique a very simple technique but very effective technique or allowed us to do many experienced studies so first study that we did was using and the theory in which our genes. We were able to do transcript from studies by doing messenger messenger R.N.A. or a Studies. And then we did a micro out in a already studies to figure out which Micron any is go up or down in a genome wide manner and then we then control. The paint and the theory or genomic D.N.A. from these animals and we were able to do again a genome wide appeal genetic D.N.A. method that is using a technique called Reduced representation by itself right sequencing and deep sequencing This allowed us to look at the entire genomic picture of what happens by different flow conditions. And we recently have completed the same make studies where we can correct fifty micro leader blood and then. We gave it to one of our colleagues at Emory enjoins and he said that can do but. Studies by mass spec which then tells us more than ten thousand changes in metabolites all at once and this allows us to look at the entire metabolism exchanges as well so what we have so far is a we have all mix data coming from messenger R.N.A. Micro are in a D.N.A. method loam and my problem exchange again these are huge amount of data and we are now relying on systems biological approaches to get better to get a better handle on our own data or to get better ideas of how we go about. Using these data. So at the end of the day we look at the pathways we look at specific. Candidate McCain a sensitive genes and then we validate them and we do functional studies and from these type of studies we not only understand the disease mechanisms but also we can actually develop. And one of the example is shown in this slide where here is a process and if one became a sensitive Gene this is what happens we can prevent atherosclerosis as an example so I'll give you a little more examples of some of these approaches. So also another thing we just completed that we are still working on is we have completed the R.N.A. sequencing from our data and this is to understand a new area called a long non-coding our D.N.A. is. So this was our forest a transcript from data where we did a. Messy R.N.A. or a studies using a little your genes and we found that flow disturbance changes gene expression for more than five hundred genes and all of these we followed some of the genes that we identified from here and one example is D.N.A. Matthew transferees one or D.N.A. he one now. The reason that we chose to look at the empty one is because this D.N.A. method transfer It's one is an epic genetic master regulator what it does is that it puts a method a group on site was in when it is in C. sequence. And. What would the importance of this gene is that if this gene is involved in many diseases including cancer such as. Leukemia or. Dysplastic syndrome and there has been a specific drug that inhibits the empty and that drug is called Five Days or two D.X. is cited in and so because of this type of work we actually studied father whether this D.M.P. one place or an important role in a process and at the time there was nothing known about the D.M.P. one in in atherosclerosis or and within your dysfunction. So the first was to really can from our finding whether D.M.P. one gene goes up at the gene and protein levels and here is staining showing that the Q one protein level goes up in the flow this. Compared to the control side and. Showing no signs of D.N.A. If you want protein expression and this is not only in the flow of the story very year by caused by surgery but in naturally flow disturbed areas such as the oligarchs you can see in the last will flow is bad you will see a lot of red staining that means D.M.T. one is very high in flow disturbed areas but not in the good flow region called. Such as here. So next it was that we tested whether blocking D.M.P. one has any effect on atherosclerosis can we prevent those crosses so to. Tested we treat it any more with five days a drug you know those depend on manner and this is our control or in the left side versus right side you will see a lot about those courses and if you treat them with five days a drug you know those depend on man or you can see why ways are prevent significantly. And moderate We also did studies where we didn't do any surgery and we just gave them Western diet for two months and if we do that then they develop crosses naturally into these branching point regions of erotic art and that also describe development of that for us courses can also be prevented if we treat them with the five A's or. So we now have idea that five ways are or D.M.P. plays a very important role so then we wanted to understand understand what is really the mechanism what how does it occur so to get that what we did was we used this a lot of years D.N.A. methyl on data and compare that to a. Transcript on data because we got both D.N.A. methylation data and gene expression data from the same animals so we didn't compare it to see whether a D.N.A. methylation parent get anything matches any gene expression patterns the basic idea is this when genes is specially at the promoter of regions is D.N.A. methylated then it tends to prevent its gene expression so if I come by comparing high. Methylation of the promoter regions of each gene and it's gene expression we can actually figure out which gene is behaving in a consistent manner and we also used five days of data as well you know to to understand it so by going through this systematic bias. Systems biological approaches we'd then were able to find some genes actually we were surprised that we only found eleven flow sensitive genes there are regulated in this manner by D.N.A. methylation and expression Penner pattern you know five ways are dependent manner. And these are shown in here so these were the eleven genes that we identified from a genome I completely unbiased approaches. These were not so another important thing was that out of eleven genes half of them including Hoxsey five and care of three contains a cyclic M.P. response element so from our completely unbiased approach we found that cycling a response element genes are somehow regulated by this D.N.A. met alone dependent manner. And that's something that we are actually actively following to figure out what this means and. How we can actually get a better understanding. So the next topic that I'm going to cover is my crowd and my crowd in a way is a noncoding more and when it's more true or it's about twenty two nucleotides and I'm sure many of you have heard about it if you'd have it the think of it as a cousin of S I out in a small or in a that behaves like. This is how it works so it is made in the nucleus like any other genes. Through a transcription and this is initially made into pry my quote any so long longer sequence and this primary quaternary can be processed by enzyme towards Russia and did you see a complex and this is then made into a premium once you have smaller premier it goes out into the service or through. Expert in five and then it it binds two and then it uses ties of which for the process is prima micron in a in too much of a form and then it puts into a risk complex once it goes into risk complex with the help of adult two proteins or agro proteins then it does following things once it binds to specific genes. And then it it degrades this is one of the best things that happens or it prevents translation or sometimes it also induce fear than elation So all in all these. Regional teen a decreased. A degradation of specific gene targets. And there have been many fewer sensitive my crowd is reported but at the time when we first reported there was no report regarding whether the any of these. Micro R.N.A.'s play a role in atherosclerosis. So this was our initial or a data showing a lot of A. Sensitive. And we have validated many of them and another thing that we did was that in each of our studies actually we do not only in vivo studies but concomitant in vitro studies where we correct and that we called to them on the either lab and I should stress conditions that simulate good stable unidirectional flow. That simulates. A distance flow disturbance. So this is how it looks like when you when you culture and the theory is on the lam you know sure they are lying to the direction of the flow in oscillatory Shearman or they show a cover stone no particular directions and then we can actually confirm what we saw in vivo you know in in vitro conditions as well so from this type of work we have selected. My crowd and. Is that we wanted to study and one of them is called My crowd and a seventh so this goes up by bad flow. Long story short what we identified was that this crowd in a target this one court came through tissue inhibitor of. His or M M P's and one of the the reason that this may be very important is because Camp Three is a indigenous inhibitor of M.P.'s and Adams and these three is well known as well as M.P.'s are well known in in Afros crosses and one of the critical thing is that camp three is lost in many on it is these conditions including Afros crosses a domino theory any reason and cancer metastasis and heart failure so however why three is lost in many of these chronic conditions was on known until we actually provided this for us the evidence that this my crowd anyway is actually one of the upstream regulator of the temp. So I'm going to skip some of the details. So then what we did was we studied how what this. Is is doing in the theatres so first thing that we identified was that my crowd and it's I want to induce inflammation such as in the in vitro experiment where we can see if we incubate. With premium year seven. As opposed to the control or inflammation is is occurring as measured by the side at the center in the theory research one other thing that happens is that. The probability goes up so if you treat and the filler serves with. Probability to be you mean goes up and this is mediated by temp three week. Expressed temporary we can actually bring back the increased probability so temp three plays a an important role in this new seven pathway. So another thing was that we did look at Invisible what happens what it how does. Work you know an invisible condition so again this is our hypothesis fro disturbance increases in your servant and if you have increased in your seventh then this down regulates temporary and so this is a protein staining showing in in our control right side versus left side as you can see the red staining showing three protein level which is highly expressed in the healthy arteries but in response to bad flow they lose complete this last three can be rescued if we treat my eyes with. Seven twelve. So this again shows that this is our hypothesis is correct. And once you three as I told you just previously three is the individual inhibitor of M.P.'s or atoms if you lose ten three and then P. goes haywire so we expect that once you lose three we will see very high levels of M.M.P. is an atom activities and I will show you some data using this gelatin is active here say as well as standing for a tip here say. So again this is an M.P. activity measured in vivo and this is again used in our. Samples so to measure M.M.P. activity we used a recently labeled. Gelatin and if you see a lot of greens that means it is high in CA low disturbed side compared to the control side but if we inject mice with. We can actually prevent an ampere creation again suggesting the important role of M.P.'s. So we measured add on activity by using four second fragmentation as is and you can see in the control there is none much signal going on but in the way you will see a lot of red staining I don't know if you can see it that means shared its activity has increased and this increased activity is again prevented if we treat mice with and. So finally then we wanted to see whether descent actually can prevent atherosclerosis development so again here is ours control you can see in the left side by flow disturbance the entire length of the last left come across a day or two it develops process and this process can be prevented if we treat mice with and to be a servant so we gave to mirror twice a week at five milligrams per kilogram those through subcutaneous injection and we were able to demonstrate that. Is a very potent. Therapy. And again we also treated a chronic animals without doing any surgery just giving them hyper tired so in two months they develop crosses so that you specially in the early guard cheeriest without any surgery and this kind of chronic process development can also be inhibited if you treat my eyes with and to me a seventh. So in summary what we showed in that study was that. When they are exposed to flow disturbance this then lead to activation of. Now a very novel pathway that we also described in this paper but due to time I'm going to skip that part anyways through. X.R. in one dependent pathway we were able to show that music goes up in flow disturb and the tedium This goes some of the some of them remains in there and some of them goes out into circulation as well as some of them into goes into a media as with most or with a really have impact as well you know in the theory or serves mere seventy decreases temp three once you lose ten three M M P's and Adams go up and this lead to changes in many many changes including extracellular matrix of a mentation and. Probability increases and some of these are also this mere seventh going out to circulation X. on circulating leukocytes at to beating them and in increasing the inflammation so they bring leukocytes such as monocytes into the subway and the filler layer which then becomes forms and subsequent grossest about the point. Now after we did this we also looked at another disease called AAA or abdominally or again use them in my eyes and this is our controlled experiment where you can see here are the kidneys for for your reference this is AAA developing in mice and this type of AAA can also be prevented if we treat them with seventy or so these type of studies demonstrate the importance of and commune seventy seven activating and peace leading to atherosclerosis and AAA development. So now so far I have been using and seven by Cajuns three injecting it a naked form of and we just dissolve it in P.B.'s we just stick it in on the nice the skin and it goes everywhere. And it affects every surface actually and so not only. It affected and the theory micron in a seven but it also affected many of the servers so some of which may not be actually so good so we wanted to develop an additional power mechanism whereby we can introduce new servant to all need to areas where they are inflamed if we could achieve it I think this could actually really improve our therapeutic potential So this study in what we call nanomedicine study was done in collaboration with my colleagues for our at U.C. Davis and this was just published in Asia's nano So here we are targeting V. can one marker of the inflamed and the failure. And then and then we then hope to put our There are pieces inside the Nano particle. So this is how our leap in a party can look like. Through a really a elaborate. Sequence or pathways synthetic pathways we develop this leap in a particle so within this slip in a particle and communities inside and is surrounded by positively charged care and it lifted so on that by a neutral lip it outside and then it has pegs to increase the circulation time and some pegs also have this peptide moiety this peptide is became one targeting peptide. And so then what we did was then we injected this particle containing we came on targeting with as well as seven again before before I showed that there are here's our control so we wanted to see we're we can when all is expressed on and the three are surface so most in CA. Where flows to store we can see a lot of red staining meaning we can one is high compared to CA and also in Eric arch without any surgery in a naturally flow disturbed areas. We can see a lot of we can one expressed introduced the various but not in the greater. Flow is good so we know we come on is expressed in through this third regions. And if we inject. My quote in a containing a particle it goes and binds to only L.C.A. area will flow is. In the showing that now and then a particle only goes to places where money is high and if you do if you don't put the targeting peptide it does not bind so it shows us that our targeting strategy is very specific. And it binds to in the field surface and we also were actually surprised that this near a particle biased in the theory M It gets inside and the theory but it goes no further so we don't know why but it it gets stuck in the and the Thalia layer. And we are going to skip the in-vitro data but we were able to show that in the in the in vitro condition it goes into the series and it works really specifically to block. This is invisible data. So what we did was we then we did a partial ideations surgery to induce a new servant for expression in there. And then we injected mice with this new. Particle with. The victim one targeting peptide and if it works this is what what should happen if it goes into in the field and it should prevent it should prevent the increase in music and that's what we really saw. And not only new seven twelve level changed its downstream Gene target cord came through he actually gets rescued as I told you Kemp three level drops in flow disturbed their areas and. This can be rescued by treating animals with a cam one targeting. So this was our final experiment using our no particle So again this is a controlled experiment showing a lot of crosses in the CA by partial following partial ideation and this atherosclerosis can be prevented if we treat my eyes with the. Victim when targeting and. And this does not work if we don't put the target in peptide in the front. So again from this experiment we were able to demonstrate that our. Deliberate in targeting then a particle goes specifically to in the field and prevents arthrosclerosis and this shows that actually descent in your seventh is not does not have any side effects that that as far as we can tell so we looked at the potential side effects of this and there are particles we collected samples from many tissues and liver or kidney long spleen bone marrow and circulating P.B.M. says is that we don't see any effect of. Any of the tissues that we looked so we don't have a so far we're not seeing any side effects of this. Nanoparticle we only see its effect in inflamed in the field service. So in summary this is what we have done we are going with the flow to understand atherosclerosis and to develop a new. Therapies So in summary we showed that partial Crotty ligation induces flow disturbance which is sensed by in the field immediately that lead to changes in genes pro and use including. D.N.A. Matthew transfer is one that subsequently lead to changes in temporary and. Fifty's as well as some target genes leading to. The failure dysfunction atherosclerosis formation and advanced lesion formations we have also developed a new and he added an extra piece so. We showed the. Ways are on as and. So so far what we have done is that we have taken this all mix approach using our parts and model from this we have really selected a few genes we still have a lot more to go based on our radar and then we will continue to study. Inside as well as new therapeutic development and we will continue to develop our nano nano medicine side. So so far we have been doing Willy Well in terms of preventing Afros crosses in mice but that's not my and Gore I think Gore is somewhere down the stream. That is this is where we want to go we want to develop a therapy that would be actually useful for humans and at the beginning I told you that we have. Tens and drug eluting stent despite that these are that for of course this is still number one killer in the entire world we need a better therapy and I think what we have done here could actually provide the different therapeutics that are not that the. Drug eluting stent cannot cure So how do we go from here to here I think we are actually we want to take a route where we are actually beginning to work with a bigger animals such as baboons and pigs and continue to develop a targeted therapy there appeared to develop the ability libraries and these will I believe to. Take us to the clinical trial. So again I have to tell you that I have not done anything myself and these are actually the group of my students and fellows who in the lab who have done all the work including Richard here and here are my Actually the most important people who have done the work Sandeep Kumar currently a research instructor in my life don't do so on channel can just sit on the epigenetic studies. And my lead manager don't want and I'm your red man who is now a system professor in cardiology have really contributed a lot to the study and my colleagues. In this department done GIVENS I did you're going to often Kingbird and you really helped a lot and. Been Jones and young we go at Emory is my important colleagues as well as colleagues and U.C. Davis. University. And these are my funding sources thank you very much for point for your attention and if you have any questions I would be happy to answer thanks. Ever so. Heavily with. The nano. And I ask you I mean I would still guess most of the articles goes to this. Very little was. The reason that you. Or. I saw that it was normalized to the scrambled. Type or if you normalize it to be more. Because in those cases a big one is NOT have regulated you would expect to scramble right actually. Right right right so he would not. But if you. Come here and. We have not compared it to untreated so I don't know the answer I mean yes yes very selectively. Very very difficult. Actually yes actually. We were actually very surprised that we didn't see any fact I think. One one was in one potential reason is that because we're actually taking the whole labor issue so maybe there may be some literally fact that is hidden We can't tell but when you look at the entire liver or tissues. When we looked at blood this is actually a and that's where actually we would have seen some in fact one of the things that we find in our most of. Almost all the. Systems. Actually we measured it and we haven't seen. A little there is a little bit of complementary C five a. But in terms of any other three markers right we have measured it and it didn't change. Versus really nice with a nice progression so I guess my question is what is. Out there it would be imagine getting to versus in the middle of the growing or at what point will it. Have because the first question and then a second question is it any. Mere. One with. So. The. The first the second question first so the an empty one has a some controlling site in the three prime it's three prime and that we're actually some my quad is actually control for the empty one expression so that's also became a sensitive. That regulates the empty one so there's that connection for in terms of whether we can use this as either a therapeutics or just a prevention so far we have focused on just preventive studies because that's easier to do. And I think. So that's actually that clinically I think prevention or stopping the disease progression whenever that whatever that is is actually a very important aspect of clinical. Use of this type of work so I think that regardless of we don't know therapeutics in fact we haven't really tried but I think we are at this point we're just focusing on these preventive effects first because it's easier to do. What it. Was like. What is. Very. Very much so. So D.N.A. So the empty one which is the one and the empty three are so. Sensitive So although we see a lot more D.M.T. one regulation three seems to be which is involved in the you know over methylation D.N.A. methylation. Is also. Activated but to a lesser degree compared to the maintenance methylation regulated by the end of the one but you. Were right. But five it turns out that you know. The empty one is really the major one that is so why ways are is actually selective more selective toward the empty one. Than the empty three So while we cannot exclude the role for the empty three it I think it does mine or. The major or seems to come from the one I was to first question in my own. Right so it's a while so so far. We required. A receptor knockout but actually we recently have been able to do it in seventy seven. Mice by just raising cholesterol levels using something called A.P.C.'s canine so pieces canine is made from labor and it goes out into circulation and it binds to receptor actually it leads to a down regulation of receptor so if you have high level high level of pieces canine through viral infection it cetera then actually you can effectively cause L.D.L. receptor production and if we do that actually we can use regular mice and we can cause afterwards Gross's. Chick fil A may be needed or other if it is not. Supermarket in maybe diabetic conditions or so so for alone does not lead to all the way to us courses I think Road disturbance is what really sets off a chronic inflammation but you need additional factors such as high cholesterol or diabetic condition. The cetera to really brings it to. Formation I think you have question first. About you know. You know I'm only. One. War. But when I was. So I'm going to answer the first question and then I'm going to ask you the second question so are you asking whether there are different pools of genes there are regulated by. Flo. Actually we have that data we're actually writing a manuscript right now where we treated and in my eyes with either starting drug war without starting drug and we compared which genes in the failure are regulated by cholesterol dependent manner as opposed to flowed the pen and then actually we were surprised to find that almost used to preserve genes are completely segregated. So I think this actually really. Validate some of our hypothesis that these these gene targets that are regulated by high cholesterol is almost independent of the genes that we are regulating with flow disturbance so the second question was. Right so modified change of course really rapidly as soon as we do the surgery and. So as all ears for days after our partial are used in surgery we can see. And measure minor side coming in and infiltrate into the flow disturbed areas by fire. Or staining and so I think probably. The real event of course a lot faster a lot faster how modern society actually gets activated when we only what we only did was slow change is is still completely not understand understood but part of the reason is a factor such as my quad in A is produced from and the. You know very local area that goes out into circulation and hits. Leukocytes And actually we have shown that our. Our my crowd is actually have that specific direct order especially on monocytes. So using all these of. These I know little. It's a central event so what so after of course is is requires several types it's not just in the theory it's not just glucose or smooth muscle cells they all are actually involved in developing and so each still has its own drawers so without monocytes becoming macrophage and engulfing high cholesterol and fat you're not going to have atherosclerosis. But that initial event requires and the theory to become dysfunctional and so these are all related events. Nothing just happens by myself. So there was question. Why not what So good example is that if you exercise so if you go to a doctor's office with a. Problem with the cardiovascular system what do they tell you diet and exercise. And in fact. Exercise especially the aerobic exercise which increases cardiac output actually one of the benefit of the increased cardiac output is is on improving the failure function by actually improving flow patterns and. Decreasing the bad flow and I think that has a direct effect. Three. Or. So so actually there are some studies especially using branches. How your indulgence shape is actually has a genetic defect that's that's genetic defect the. Natural shape of the and branching point so some people are more susceptible because human dynamics will be bad worse in some certain people. You know. So there are a lot of master switches there are many transcription factors as Actually it has as you use a flow change within a few hours you can actually see a dramatic change in gene expression because a lot of transcription factors are changing and these really drive a lot of gene changes very rapidly so there are very rapid change engine expression and some of them are more sustained some of them actually comes on early on that goes away and then at the genetic changes. We presume genetic changes would be slower but I wouldn't actually. Completely assume that is the case because I think we I think actually change may or correct me sooner than. Later. I think somebody has a problem I published a paper but. If you're not sore it changes so many things so it's really hard to interpret. OK thank you very much.