diabetic wounds

may provide the basis for the development of new therapeutic approaches to treat this condition and also reduce mortality, morbidity and disability associated with chronic lungs. as most of you probably know, the united states spends more

than 25 billion, make that billion with a b, dollars, annually on wound care. improved techniques for promoting healing of wounds have great impact on health care costs as well. the title of dr. marjana tomic-canic's presentation is

wizz add reof tissue repair and regeneration of skin cells when the manage sick all about gone. please welcome me in joining dr. marjana tomic-canic. (applause) >> good afternoon, everyone. can you hear me well. it is really a great pleasure to

be here today and i want to thank the nursing research for nominating me for this lecture and organizers for giving me the opportunity to share today with you our research and the approaches to heal wounds. and if it wasn't for poetry and influence of my daughter who is

a big harry potter fan and my son who loves graphic design, my title would have been slightly simpler and and would read cellular and molecular wound healing in pathogenesis. that's what i will be talking about today. so the review of the lecture

will be a brief introduction of the skin biology and basic cells and wound healing and li divide the talk into two parts, the first will be lessons learned from patients where we took the approach of taking the biopsies from chronic patients and trying to analyze both cellular and

molecular aspects of inhibition of healing from the many lessons from basic and molecular cellular mechanisms. then the second part of the talk will be flipping the decide trying to go from bench-to-bedside and apply what we learned in practical

settings. and so, the primary function of skin, i have been fascinated with this organ ever since i started my graduate school, is that obviously the goal of skin exist sense barrier formation and its maintenance. and that is achieved through

really a remarkable perpetual process if you want, where basal keratin ocytes where are the -- which are the most proliferative potential compartments of skin, divide and then one cell will turn on and start differentiating completing this sort of process of terminal

differentiation in the barrier formation. so this process, the keratin sights will lose their nuclei, crosslink their proteins and die. and together with lipids will form the barrier as we seat fortified layer.

how is this really achieved? obviously we have to thank epidermal stem cells for it. they reside, there are two compartments, one is basal layer of ep demis where they symmetrically divide and start differentiating terminally and then the second compartment that

is equally important for wound healing, resides in the area of the hair follicle. normally, in a steady state, these cells don't participate much in biology of epidermis unless the wound happens. they are responsible for formation of hair follicle and

the cycling of hair and biology of the hair follicle. but, if the wound happens, the stem cells will start migrating in the epidermis and age to the wound healing process and that becomes very important for what i'm going talk about today. so, clearly skin has this

enormous regenitive potential and proliferative potential because it perpetually differentiates throughout lifetime and serves us well. so, a small biopsy of human skin can generate enough primary cells in cultures to generate sheets that can cover several

basal fields in surface and that is currently being used in burn units where we actually grow otol gus cells and apply them to patients to actually aid in their wound healing. so if you think about the potential, it's really remarkable of what skin as an

organ does. in addition to that, many of you have heard how this was a landmark year of 2007, when a skin cell scientist independently took different parts of the world, and have come up with a protocol where they can use adult skin cells

and really turn it into induced pluripotent stem cells or embyronic live stem cells and now become a really potential to generate different tissues. so from an adult skin cell with four different genes, slightly different from two different cocktails in two different labs,

but an adult somatic skin cell can really be changed into embyronic stem cell and then give rise to different tissues and differentiate into different things. so if you think about this conceptual potential of regenitive medicine and tissue

repair that comes from skin, it is really is remarkable of how the wound healing process occurs throughout the lifetime. and it is really a coordinated cellular process that aims at nothing else but barrier restoration. what i was fatinated about is

you can take human skin off the body, put it in the incubator, maintain it in the early interface and then if you wound that skin, that skin will heal. so this is right out. so the cells do know how to close the wounds and how to enter this evolutionary process

of barrier restoration. you contain this process one step away and then take the skin pieces apart with individual cellular compartments and grow keratinocytes fibroblasts in culture. then reconstruct 3 dimension alley that tissue and make a

skin in the pee tree dish. if you wound that skin, that skin will heal as the slides are showing. so you could see this leading edge of the epithelium crawling over the dermal side and really closing the gap. so, the cell really do know what

to do on their own without any of our help. and how is this process achieved? the basics are clearly it's not a simple process. and that is both exciting and difficult when you have to deal with treatment development

because obviously, when you have multiple cells and multiple factors, it becomes a lot more complicated story. so, a wound, when a wound happens, keratinocytes release prestored interleukin 1 and that is important because this is sort of a big signal to the

surrounding tissue that the barrier has been broken and the pathogens were o. coming in. and i think that if you think about these cells, they are equipped to really very profoundly robustly alert their neighbors, if you want, that barrier had been broken.

and in response to that, a lot of things happen. keratinocytes start moving and proliferating so obviously tow close the gap. fibroblasts are proliferating and lay down the matrix and epithelial cells start sprouting blood vessels and sort of the

form of angiogenesis, macrophages are moving in to clear up the infection. all of these cells are very, very important and they are very, very well coordinated in timely fashion in order to close this wound. and of course, how does that

happen? well, through the cross talk among these cells with wonderful ehills and cell phones and all the gadgets they have, growth factors, and chemokines. they talk to each other and simulate each other in a timed response in the well coordinated

manner. so how, if we deal with this extraordinarily tissue that can heal on perpetual does he that through our lifetime, how can we then face this in our clinical side? so, we are now in the midst of epidemic chronic wounds.

and so, worldwide, there is or there are data out there that show that there are every 30 seconds part of the lense, lower lense, amputated due to diabetes and i think as this develops the data through 2005 and as we are coming towards this age now, i think the time is shorter.

so, with all of this ability that we have and technology and knowledge and time and everything that we have in our toolbox, how can we use amputation as a clinical approach? that is simply not acceptable. so, the focus of mislearns to

try to understand why won't the woundinally in a clinical arrangement, we're faced with a lot of products that are safe to use but they do not reach the threshold of clinical efficacy. and very few products have reached that. and they actually don't have all

that great successful rate. so clearly we are facing with a tremendous clinical burden that really did not come up with solutions or treatment approaches in this big fashion. so, we are trying to do everything as possible we can for our patients but i think in

general, there is a lot more that can be done. so the question really is, why won't wound heal, and why do cells want their magic to be in line with the title? so we decided to use the approach and i teamed up with extraordinary team of clinical

scientists and one of which is harold bream and winthrop university hospital and one is robert at the university of miami. and you know, these are the incredible physicians who treat these station. and has been working in my lab

for a long time, first as a fellow and now as a junior faculty, who actually participated in majority of the studies that i will show you today. so what we decided to do is collect the biopsies in one study with dr. bren to collect

the biopsies from wound healing edge of the wound and standardize these biopsies and do microarrays. and the study with dr. cechner, what we have done is we selected patients who are nonhealers and healing cellars and then compared the two.

we implemented this clinical outcome in our design before we did my crow arrays. so li show you the summary but before that i have to make a slight axe aggression towards the students that i really love and fellow whose are yet to face the study section world and the

grant application world. and it is from grant that actually, national institute of nursing research funded but there was before the one submission before the one that got funded. and basically what i proposed to do global transcriptional

analysis of wound healing, one reviewer said it's simply not possible. so, this is really very important sort of dense and you will receive a lot of reviews in your life from grants and papers. and every single one of them

does influence if you many ways. so once you set that aside and look into the message, to me the message was really clear. he said, you know what? i believe it when i see it. and so, what i am excide said to real doe this and research the grant after i done the initial

studies and the grant got funded and here i am. because everything i have to say started with this original grant and the original study. so before the microarrays are beautiful you show them and essentially the left three patterns are gene patterns and

heat maps of nonhealing edges of the venus ulcers. we compared this to an identifiable pattern of genes that really show and resemble nonheavying. when you zoom in, you understand there is about 1500 of them that are statistically significant,

deregulated in the chronic non-healing wound edge. and of course, i will not go into doe tails but many of them relate to three important keratinocyte profiles. swone care 10o site differentiation, one is migration ability and

proliferation. and i will today talk about proliferation and migration primarily. but when you sort of -- when you look at that profile, and then you go back and look at the patient samples and histology, you what actually see is that

everything we saw in the microarrays is really true. first you actually find tremendously hyperpolice officerrence in epidermis. when you zoom in, you could actually appreciate there are divisions super basally, differentiated as if the dermis

never happens. if you look at this quantified layer, which is supposed to be without nuclei and that cell layer that really needs to protect us, you appreciate that there is a lot of nuclei there. so, what this means is that the cells really do not execute

properly the differentiation process. but they also don't completely execute this activation process of wound healing where they are proliferating but they kind of -- they cannot move so there is an inhibition of migration and loss of differentiation if

you will at this nonhealing edge. so, there is this risk that happens and almost like a grand canyon. a big pile of cells that are disoriented in space and time and they cannot do anything what they are so well programsed to

do. that prosfascinates me and we really tried explain why. so as i said, one of the characteristics of keratinocytes is hyperproliferation. the first question we wanted to ask is what is the fate of progenitor cells in chronic

bounds we start to look at the epidermal stem cells. li no got into details of the best parts of biology but important to know obviously if any stem cells are kept in mostly quiescent times but whether a cycling needs to happen, then there are a couple

of events and signaling molecules that need play a role. one is the bmpr1 alpha needs to be down regulated. data 3 needs to be down regulated and bait cateen in needs to be induced in order for stem cells to go from quiescence to cycling.

what we wanted to really look into is what happens with all of these regulatory molecules in the context of the chronic wound patient? and then what we found out is if you look at the left side of these two important molecules that are real -- they are down

regulated, they are down regulated in ulcer and, and they need to be down regulated if the stem cell is entering the cycling mode and leaving the quiescent mode. so this kind of props that may be is there a specialist quiescent to cycling in the

context of the chronic wound environment. and then when you look at the second part of the equation, whether there is activation of beta catenin, we found a profound induction of nuclear bait cateen in in the nonhealing wound and a consequence of

stimmic, which leads to hyperproliferation of the epidermis. and what is interesting about it is that long before we started looking at this, we have shown that if you overexpress c myc in epidermis of mouse skin. these mouse will spontaneously

develop chronic wounds. so clearly this pathway does play a role in the context of it and then so many years later when we start looking at patients, we kind of get into the same story. and which is always nice to get a confirmation of this.

so we have a hyperproliferative now, as a side part of that, so you have a hyperproliferative epidermis but then okay, what was striking to us is the fact that there is actually a complete loss of epidermal stem cells in this hyperproliferative one of the markers is k15,

keratin 15. and this is the normal skin and you can appreciate the basal layer staining that is completely gone in a nonhealing edge of the chronic wound. so this frequent cycling might lead to the deprivation of the source of the stem cells in the

environment of a non-healing wound. thankfully all went on and became transient to find cells. yet the wound doesn't heal and it's not healed because there is no migration. so these cells are piling but they are not moving and this

obviously happens because of all of these signals that are really not coming. so not only are they not migrating in local but they values a loss of homing and loss of different chemokines and cytoicancy where they cannot call the progenitors who come to

the site of the wound. so in addition to not being able to migrate, they also cannot call the cells, other cells such as circulating progenitors, bone marrow progenitors, epithelial projen tors or local epidermal stem cells to come and aid in wound healing.

so none of that happens. when we start looking into signaling that control migration and this whole process, we actually looked at for example, the egf receptor and we found really that interestingly, there is almost a slight decrease - auto profound

decrease of epidermal growth factor receptor in the non-healing wound. what is interesting about it is that that a little bit of -- i'm not sure whether you can see, it's cytoplasmic. so if you think about what egf receptor normally does, it sits

on the membrane and receives a signal. here in the nonhealing wound, you really don't have that receptor in the membrane where it should be. so, if you apply egf receptor externally to the patient, there are no cell that is can respond

to it. so the clinical trial of testing egf receptor would not make any sense because there is simply no way of really transmitting that signal even if it's externally applied. and i think that is one aspect that is very important to

understand. the transition for preclinical testing to human situation and clinical testing, really calls for looking into patient samples much more carefully to make sure that they will be able to respond to whatever we are offering to them.

the same thing, and i will not go into details, the take home message of this complicated slide, that a nonhealing edge of a chronic ulcer, we get the samization none of the receptors are on the membrane but they are signaling molecules are not in place they are impaired in their

function. so, at the end of the day, when you look at this, we did a microarray study where we compared, this is mickey bloomberg who collaborated with us and the lead editor on this paper. what happened here is that genes

that tgf beta normally regulates in healthy skin or healthy wounds, actually are regulated in a non-healing of the chronic ulcer simply because there is no way of proper signaling of the tgf beta in the context of a non-healing edge. so another thing, so we are

still searching of why this signaling in paris and how the growth factors and all of that really influences migration. and when you do microarrays, we all kind of have a set of our choices that we are expecting to see and are very excited to see that it will confirm, but then

what we essentially do it for the surprise is and i think that is the big part of genomics approach is where you really don't know what you're going to find. and one candidate that we real did he not expect was adam 12 to find in a nonhealing edge.

and this is a molecule that really is very interesting and it has a lot of parts and functions and a lot of things but it's actually different grin. so it also regulates the intergrins and regulates a lot of signaling.

so, when we started looking, we have been a lot surprised, not only they it is uniquely induced in chronic wounds but also that it's not regulated in acute wound healing process of human skin. so it becomes only present and induced normally doesn't

function and doesn't do a lot during the regular wound healing but in a chronic wound, it gets turned on tremendously and you can appreciate that here on the protein level every panel of these round ones is a different ulcer and different patients. so you can appreciate this.

i have to say it's a bearer of bad news in general and if you look at the bladder cancer for example, you could find a. adam 12 secreted in urine of a patient whose tumor is going to go. so in a way, it kind of alerts to the bad situations and

clinical outcomes we still don't know exactly how and why in the context of a chronic wound. and study was done with carl from cornel and hss. so what we have done is generated a transgenic mouse with adam 12 where we actually know how adam 12 and obviously

when you think about it, so if a lot of adam 12 inhibits healing and knock it out, you expect the cells will mike rate and that's what we get. and this is more visual way of testing the migration on its own when you actually take the biopsy off of in and you put and

measure the output. so it's an inverse version of wound healing where you are expecting to close you're now measuring the cells and how far and how wide they can actually migrate. so and the staining here is showing that these are actually

keratinocytes because this is specific antibody. so in addition to what we already knew about chronic wounds that have increased pro taze activity, we found increase of adam 12 and that may be leading to increased processing and growth factors and change

interactions with extracellular matrix and signaling all of which would possibly lead to inhibition of wound healing. so the obvious question is so what regulates all of this what i told you? it's what are the regulators and what are the main switchers?

so we start looking into microarray. mark row rnas and i will not really go into details what have they do but for the take home message, the important thing is when you have a message obviously in the protein, when you have a microrna present,

that doesn't happen. so especially you can have a full transcriptional regulation and you do have expression of the message being made but then mirna comes it never athroughs to turn into protein. and so, we thought that maybe if we approach this genomics

profile that we have from our patients, and if we use the reverse genomics with the help of christina from sloan-kettering who is in computational biology, but if you take these genes, these 1500 regulated genes and just say, okay, if micrornas are target

genes which micrornas would be regulated in that tissue if these are their targets and that's how they are excessed. so that is sort of reversed computational analysis lead to a list of micrornas that might be regulated in the chronic wound and so, again, those who

we collaborated with in this study in miami, we start looking in the different levels and we found they are all initudes and you this is one of them, this is the microrna21 ask, and this is the biopsy and you can appreciate this is in situ hybridization showing

microrna21. so i will not go into details. there are a lot of data there. but in order to shift to the patience and talk about translational aspects, i will not spend a lot of time on this subject. but did is important that we

have identified and documented and proved that these micrornas that we found induced in patients with chronic wounds target signaling molecules they do regulate their expression. or their protein levels if you want.

and that is one of the profound and important issues with a chronic wound and something that we really clinically need to know. so essentially, if you look at the diabetic food ulcer, for example, what happens to these cells, unlike in acute wound

when these cells start migrating, that doesn't happen. then the fibroblast really stop proliferating and they do not lay properly, the matrix and epithelial cells are not migrating and decrease of angiogenesis. macrophages don't come in the

site of the wound in the number of ready to fight infection and you have in addition all of this, is formation of colinnization and unfortunately microbial bioheld and this wound turns in chronic and infected and is there a really bad outcome fe at the end for your

patients. so how is this achieved? the decrease in growth factor coo cytokines and chemokines is one of the reasons why none of this really happens. and so, turning from healing to chronic wound and trying to wrap up with what i have said so far,

is that there is increase of microrna molecules in the nonhealing wound and that reads to deregulation of the signaling molecules that, as an outcome on the cellular process has migration and hyperproliferation and loss of local and ever stem cell cycling and their loss in

the area of the nonhealing wound and then obviously loss of differentiation. all of this really inhibits wound closure and epithelial evasion. and wound is not healed unless it is closed. and that means until

keratinocytes do their job, the wound is not closed. and therefore there is no healing. and keep in mind obviously that i haven't talked about other components. so we are just an epidermis. this is one aspect of the whole

process and i could not list all the participants but you know, we didn't discuss today granalation tissue formation in the context of chronic wounds, nor angiogenesis or this cross talk of the microbiofilm and chronic wound. so clearly there is -- we have

our science worldcom completely cut out for us. now the biggest question that many labs packing this and trying to understand it is why? and what is upstream of all of this? what regulates this microrna and not only micrornas?

there are no magic molecules. i don't want you to go home and think it's easy. we can just reverse niece we are done. that's not going to happen, right? so i think that understanding what comes upstream of all of

this that i have told you will be really important and that is where the excitement right now is and there are a lot of work being done on that. and essentially and conceptual frethis healing to a nonhealing. so you can really think about it in two ways.

so either the cell transitioned and crossed over to the world and it cannot go back, so this is sort of terminal, it's nonhealing and there is no way back. sore this cell has to undergo and underwent really a bad set of decisions and signals and

then god know what is else and end up nonhealing but is there a hope and switches can be reversed and they can really be transitioned over. and actually, both of these are clinically valid approaches and then so when i talk about the first part at clinical meetings,

they all shrug shoulders and say what can we do today for our patients? so, the current approaches are two. obviously one is remove the nonhealing cells and the other approach is reverse. it's very simple if you think

about it. so either you can just cut them out and keep those that are actually do have healing phenotypes or you can try to really convert them. this might be maybe more difficult but then maybe more permanent solution.

and so both of them are clinically val in the clinic. but it's not that easy when you have removed non-healing cells cells. so what does that mean? how do you know which one is really nonhealing cell and the tissue part and how you're going

to cut it out if you don't know? so, we clearly need something more of the sophisticated approach to create this molecular -- we can know and guide the extent of surgical debriefment in the chronic boundary moving this tissue. so if you look at the concept of

most surgery in skin tumor cells, what happens is when a tumor is in the skin of most surjohnses and cut out a tumor and then they analyze it under the microscope while the patient still on the table. they look to see histology whether they are efficient in

removing the bad tissue. so is it all tumor tissue gone? and if it's not, they go back and then repack and then until it is clear and histology shows the normal skin tissue, this patient is still in the room and is being treated and debreeded. if we borrow that approach, and

we use that strategy to really try to debrief chronic wounds, we clearly need more than a microscope and histology. then we can analyze this tissue and try to come up with should go that will tell us how far to and therefore development of the biomarkers and any possible way

imaginable. li show you the approaches where we are at this stage. but the bottom line is, so if you can tell what is the nonhealing tissue, even though it might not happen because we still don't have a really quick massive to-do list, but if you

actually know that you have the ability of patients far enough then you might go and redo that more efficiently as quickly as you can to get that patient going. and so, is there any biological basis to what i am telling you? the other important question in

the clinical arena of wound healing is, can we tell when a patient comes in on that first visit whether if we arapply standards of care, this patient will go on to heal or do we have -- or will not. so the question predicted biomarkers whether we can

predict clinical outcomes early on, will clearly defined how we are going to treat the patient. because i have to spend more time explaining to you why it's important today. you what do is you apply standards of treatment protocol for four weeks and then you

measure the size of the wound in every visit and dressing and you're looking whether that wound has healing trajectory. and if it heals later than 50% that patient will heal with the standards of care. but if it, by weak four, this does not happen, then biological

applications -- then different treatment pro protocol gets implemented and more biological treatments are being offered to the patient in order for them to heal. so, and this is basically based on work from david who has shown this four week outcome is a

surrogate endpoint and really important to really know that. so if we don't have to wait for four weeks, until we actually help the patient who is not healing, if t. will be tremendous clinically if we can tell that from the first visit and really start very aggressive

treatment plans from the get go. and of course, a very decisive biological treatment on patient who don't need it. so there are two different benefits of this approach. and so, what we have set up to do is we decided to collect specimens before and after

surgical debriefment and really rin all the possible analysis. so in addition, we have grown primary cells out of these wounds and studied them in culture and also look at different voracity ofinalis and histology and immunohistochemistry.

one thing had becomes apparent. on this nonhealing edge of wound healing, i told you what happens in epidermis. but there this say dermal site and you can see there is a profound level of fibrosis us that resolves partially in this positive reason and biopsy and

this is how normal skin looks like. but one thing was striking because granalation tissue is always a big problem in the chronic wounds and i always thought that there is not enough collagen. but when you look at the

collagen staining, society cell makes the collagen analysis and lays it out as a matrix. so if you look at, it it will be made in cell and could appreciate there is a lot more cell making collagen a nonhealing wound than the positive biopsy.

but yet when you look at the collagen orientation look at the polarized right and how this collagen matrix is laid out, then you can appreciate it really this in a nonhealing edge, this really does not -- this obviously contributes to the lack of migration in the

epithelium. and again, positive biopsy looks a lot better. and this is how the normal skin looks like. so, obviously, striking differences that you would expect in gene profiles, so again this is a nonhealing

profile and tremendously different in positive biopsy. and so if you think about these are all the cells as a part of that wound, and so there is clearly a set of cells that have not only different profiles of different healing potential, within that wound and being able

to really reach out to them and find them and simulate them to really come and do what they are so well-known to do, would be really tremendous. and if you think about this, it's another aspect of it. we can actually use the gene profile to tell us whether we

are far enough and i will show you a little bit later of how we can achieve that. now when you look at the primary cell ground from the wound that a nonhealing edge, you can again, i'm not sure how far you can appreciate resolution but these cells are really they look

sick, not normal, they are big and large and don't move at all. when you look at the cells from positive biopsy, clearly they will come up light to normal when you try to test their migration cells, i have to say that i was a big police officer when you grow primary cells out

of nonhuman wound and put all the cytokine of the growth media that these cells will reverse on them and there will be no difference when you grow them long in culture. but interestingly enough, not only don't grow well but they also do not migrate and don't

respond to growth factor signals at all. from the nonhealing edge. the positive briefment biopsy is a different story. these cells are able to migrate and as i said, those of you may be sitting in the first row, you appreciate there is more cells

on this side than here. so clearly there is a proliferative capacity different and better. so again, this is all sort of contributing to the biological basis of the approach and yes, there is -- within the nonhealing wounds, there is a

sub set of cells that are able to respond to growth factors and able to migrate and probably complete the healing. so, again, finding that region and finding that debriefment margin and distinguish the tissue a from tissue b, nonhealing from a healing

potential phenotype is really a big question and that is what we were set to study and of course our wizards looks slightly different and wound healing clinic. this is rob and davis and this is the research and clinical team put together that really is

trying to figure out these biomarkers. every once in a while, we will actually get a slide that looks like this and that is really quite exciting. i'm sorry there is not enough light here maybe for you to see. but the nonhealing edge, this is

the staining for beta catenin, and what we are looking for nuclear presence. nuclear presence is bad. absence ever beta catenin is good in healing outcome. so what we do is we stain the cells and we quantify this in a tissue and really link this to

the clinical outcome and look at the healing, at week 4 or the briefment biopsy. every once in a while you will get a slide like this. you could almost draw the line of the tissue where you actually have a loss of beta catenin in the nuclei which will be healing

potential in the other side where we had all the nuclei present for beta catenin. so again, there is the briefment margin and i think it's a matter of how sophisticated we are in finding it and developing the right tools to really get there. but that's really a great hope.

and of course, transition to the genomics approach we clearly that the day and age might not be clinically feasible and practical for fewer expense -- aspects, but in general, it is clear that can can serve as an excellent guide to determine what is a nonhuman from human

tissue. and if you look at this is the patient or these are three different patients before and after the debriefment and you can see the profiles and then this patient obviously has something different. and it turns out that this

patient was not bee reeved far enough and this is a non-healing profile and whey did all the same things when we went back to check the clinical outcome t turns out that that was a non-healing patient and the surgeon went back and debriefed these patience and redid more

extensive debriefment for this patient in order for him to get to heal. but clearly, imagine this is kind of to oversimplified version, the pregnancy test for a wound this a way. so you could use the types say, this say healer and this is a

nonhealer. and of course we don't need but the big microarray chip to do that. what we are trying to did is really narrow down the list so that we can have a small stack of genes that can always tell when is wounded healing and

where the tissue that does not. and again, this is the -- just to show you the marker that is i have mentioned previously in the talk, this is the egf receptor. it's not -- this is down regulated and this is the biopsy. you can appreciate there is a

restoration of the membrane of staining and the same thing for the stem cell marker where it actually gets restored. notice these slides look a lot different than normal skin. so we are not talking about healthy tissue. but we are talking about tissue

that has a human potential. and so, to summarize this part is that the nonhealing wound, so what we are currently doing is collecting a lot of tests from patients and trying to look at healing of a predictive marker and in nonhealing wounds, a lot of them either do not change

over time or actually change far worse, such as these here. and signal early to beta catenin, when you look at week 0 or week 4 and you can see here the clinical outcome and trajectory of healing. it's not in place. so this patient clearly is a

now, how can we really say that upfront is the really one thing that we are trying to figure out. and then again, this is the profile of the healing ulcer as i showed before and again the complete decrease of adam 12 ask decrease of bmwr1 and a decrease

of beta catenin and look at the trajectory of the wound. it's healing. so again, doing sort of this approach with the clinical outcome hand-in-hand really gives us tremendous potential to search for really appropriate so far we are narrow down the

list and we are in the clinical study where we are testing this and trying to narrow down the list and guide us through this clinical process. but i really hope that we will have a gene chip for wounds relatively soon and hopefully sooner than later.

but clearly, our goals are, which wound will heal upfront and where is the debriefment manager in and again, we are now using all the possible approaches. and i want to point out one other thing. and this is again the wonderful

world of gadgets and all developments and computational medicine if you want, that is happening right now. and i want to point out my collaboration a longstanding collaboration the doctor who developed a wound electronic medical record.

he has a research tool that has or that follows 132 clinical variables for each patient. so imagine how wonderful that could be. so he is following from the wound size that is measured all the time to every single aspect that is important and clinical

timing, and presence of infection and all the treatment and everything that might be important for the wound healing. so what we are now trying to do is build a platform that can now sort of link this clinical data, this is data that can be exploited and it's searchable

table. so manage fin we could now see all of these biomarkers and all these things we are doing for the tissue for this patient and connected with this clinical outcomes and variability he is doing. and really being able to pin up

a subset of patients and really do a more personalized approach rather than this general sort of groups of different nonhealing and of course one of the apps for that would be wonderful and that is sort of our goal would be that one day every clinician who treats wounds will have that

app and it will send a message and say okay, there are biomarkers and nonhealing outcomes coming. please do what your protocol tells to you do. i think this is the reality and i think we are there and that's a really great place to be.

now i will just a few more minutes talk about a couple of approaches of the other side. and that cerealy the reverse, how to reverse the fortune of these cells and obviously we can circumvent the missing signals by different ways. one is use think of different

approaches and hi to say, gene deliver and assets today are really quite well developed and ideal subject for that simply because it's topical and especially wounds because for wounds, you don't have to have a long term. you have to have gene deliver

they will stimulate the healing but doesn't have to stay there three years. it can really be temporal. and i think that is really a wonderful place to start real applying gene therapy. obviously, we could apply a lot of different cells to wounds and

that is the different therapy that are at most fda arrived and new ones that are being developed. so they could be obviouslyee - on tol gus or not and obviously different matrix. all of which will simulate the local cells to really get on the

program that they know so well and execute the healing. and this is one of the projects that we have done and this is sort of delivery of the vascular endothelial growth factor and these are experiments done in different types of diabetes, which is type 1 and type 2.

and what we have shown is if you heat the mouse obviously with adinovirus delivering vegf, it stimulates healing and it's, so what. vegf simalates angiogenis and it's expected this would be the outcome. what we were surprised to see is

that we have seen a tremendous effect on epidermis. and not only that wound has healed much faster, but look at how much epidermal layers we have here. so clearly in is not only simalates angiogenesis but epithelialization.

the other thing also the thickness of the granalation tissue which is tremendous compared to the sail lean treatments or just the vector alone. and this is again the cause in stain to show how well the collagen laid out.

so this really targets multiple cell types and that's one of the approach that is is important. if we are using approach that is will hit the single point, it's most likely not going to be very efficient in the complex scheme of things such as chronic wounds.

but there are ways we can stimulate more than one cell type then they all contribute to the wound healing, that has a very, very big value in the chronic wound field. and then, the last beta slide if you want that li show you, is that what we call dr.--

(indiscernible) trial. clinician scientist at the university of mime whoa has nih study where he studies clinical trial of bone marrow progenitors for patients with chronic and we kind of piggyback on that study and we come and collect

the tissue and grow the cells and he does the great stuff which is treating the patients. and one they think he never thought he would get, it turns thought they are university of miami, documentary on stem cell therapy healing force of the future.

ask they won an emo it. it was about a clinical trial and another they do for heart patients in the stem cell institute. you never know when you become a scientist, maybe there is an oscar in the making. what i want to say is when we

collect the biopsies from his patients before and after treatment with bone marrow progenitors, we grow these cells and then we start following them looking at different things and then showing here microrna profiles of them and you could appreciate that after the first

treatment, the response is already good in terms of looking at the expression of microrna21 and 106. but 16 is still there. and you kind of get everything in order after a second treatment. but what this tells me is that

there might be a way of what i call, ordered restoration. so clearly, you don't have a magic bullet with everything goes back to normal. but there is a sequence of events that can actually lead to a healing process, which in a way, what i told but switches,

may be tells us that we can actually revert these nonhealing cells slowly but surely through the process become a healing. and i didn't each touch upon these progenitors because that technology clearly gives a tremendous opportunity also in the chronic wounds as many other

clinical disciplines, but that is again another potential of how we can reverse the nonhuman cells into a healing cell. so in summary, i will not read it, but in general, what i have told you is that genomics approach identified nonhealing wound phenotype and what we

learned about it is that there is inhibition of cellular migration and loss of appropriate signals control growth factors and obviously lack of differentiation in the epidermis of nonhealing wounds and we have also found that there is distinct population

within the chronic wounds that has a healing potential and that is linked to clinical outcomes in patients and this loss of the signals of control and are really not only coming for what we knew already about high proas protease activity but also decrease of receptauthorities

many are signaling molecules that many of the signals in the cell and i'll just increase in microrna molecules. and obviously one approach would be to really debrief the cells and restore healing phenotype or the other son to reverse their fortune.

and i think that this is -- i have to say probably most exciting times in biomedicine because i think technology that we have in our hands are so tremendous that we are yet to learn how to really neutralize the advantage of our patients and i think the good days are

coming in many ways and i'm really, really excited to be a scientist at this time. and with that, i have to say there are a lot of people i have to thank. and it's impossible to list them all. i have been privileged to really

work with extraordinary set of people in every situation. i have been in many others because today collaborations are the key. and i will have to thank obviously, people in my lab. this is fairly accurate picture and yes, this is miami.

i'm sorry, yes, we do have fun occasionally. and then of course the university of -- umass wound center. and obviously, nih, because everything i said today really was funded exclusively by nih, although i do have other

projects that are not, but comes from that initial grant from national institutes of nursing research that really sort of retained me in a wound it was t. wasn't my first rl1. but it was my first in the wound healing and really i have to thank them for really sort of

shedding the light on that path and keeping me in that field. and of course, most importantly, thanks to all of our patients because they are really special and i'm really hoping we might not heal the wounds of the entire humanity but chronic wounds we hope to be very soon.

thank you very much and i'll take questions. >> thank you very much. we do have time for a few questions. if you will take the microphones on the side and say your name. we can step up to the mics. >> congratulations for excellent

coverage and excellent work. so you identified quite a few interesting targets. so how many of them you think you might be able to use in the patient care and also what is the -- part testify that arranged this process of defective wound healing?

>> so there are two different questions in that question. one is right before these 1500 possible targets. so what we focused is mainly trying to identify the potential biomarkers that will predict the outcomes of healing. but apart from that, there are

lots of different potential targets that would be validated as a target -- pharmaceutical target. but the issue with that is really, yes, answering the question very, very shortly, there are a set of targets that can be used, for example these

micrornas. and i mean they are major regulators and they can be targeted because they really regulate a lot of what is happening. so they would be the obvious but i think from the microarrays, it doesn't point

out a singe set of targets. it tells you more about what is going to in molecular and cellular level. and really understanding how the cells or tissue got there would be really important and that's where they are tremendously valuable.

that's where the targets are. because when you take a biopsy of a nonhealing wound, that is if you want an end point, that tissue is not healing. but what we still don't know is how the process really sort of -- if you turn back the time of the origin when those cells

actually progress to the nonhealing. i think that would be really important to understand. and that is something that we are working on, really trying to find out what happened before they actually got to be terminally non-healing so to

speak. does that answer the first part of question? it is really, you know, if you ask me, is there a magic molecule, there isn't. there really isn't. and the chronic wound, i don't think there will be a magic

molecule. a single one definitely -- definitely not. it's the complexity of the disease as such it's not i found like my reviewer. it's very similar and some would be here and will go and prove me wrong and i will have nothing

more than that. but what i wanted to say is that you ask -- another thing about the diabetes. again in diabetics, there are multiple components. metabolic say very important one but the other one that goes with that diabetes in developing

chronic wounds, there are a couple of things. one is peripheral neuropathy. patients lose a sensation in their feet and that really contributes in part -- nobody really knows. we still don't know and pinpoint how much this neuropathy really

plays a scroll how much diabetes they are linked together. i could tell you one thing, there are sets of patients that develop peripheral neuropathy. they don't have debaties but they colored similar conic ulcers in their feet. diabetes predispose system

because of obviously metabolic aspects because of loss of ability to fight infections and there are a lot of other aspects of diabetes that kind of play a role of why the tissue is not but there has to be something linked to the local to the feet and lower extremities.

if you really look at the wounds and surgical wounds on that, they actually heal, maybe slower but they do. so it's not, shi say it's not complete disability of healing, obviously die beat cease a big part testify. we have done experiments to show

that young die beatic mice don't have, that much of impairment and healing compared to the age that this was mind. if you think about, it is always some underlying com pone that he went plays a role in addition to the diabetes that plays a role in diabetics.

>> good luck in the challenging area. >> thank you. >> one more question. thank you for your presentation. i was curious, does high glucose change the differentiation and -- (indiscernible).

>> we have done this in scheduleature and i have to say i haven't seen any effect in culture but obviously that doesn't mean much. but i can tell you the experiments that we have done in the mice that is published, in experimental dermatology.

and if you look at the mice, the young diabetic mouse controlled or uncontrolled diabetes. if you look at normal hyper and hypoglycemic, you really don't see a tremendous difference in wound closure capacity of on these mice. they are all the same.

it doesn't mean they are good but ta doesn't obviously again, we are talking about mice and extrapolating, i would really have tremendous activity doing it. i think it's important to understand that in aging it becomes a problem.

so again it's a component that really -- of aging in combination with diabetes in the context of mice. we absolutely see the difference. but not in the glucose level. i would like to invite you to, we have a reception following

this talk in the nih library so as you exit the auditorium, turn to your left and you'll see it is right there. we can continue the conversation there. and the reception is sponsored.

diabetic wounds 2017-01-30T03:09:00-08:00 Rating: 4.5 Diposkan Oleh: Ramdani Sanghiang Wibawa Tunggal