Gregory Shultz, PhD defines the concept of dynamic reciprocity and gives examples. Dr Shultz explains the molecular and cellular differences between healing wounds and chronic non-healing skin wounds and reveals the pathways to promote healing by altering the components of the extracellular matrix.
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Gregory Shultz, PhD
UF Research Foundation Professor
Obstetrics & Gynecology
Institute for Wound Research
University of Florida
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[Dr Frykberg] The next speaker I�ve been trying to get to the meeting for a number of years now. Dr Greg Shultz, you�ve seen referred to in a number of articles, including the one that Tom just referred to. I�ve never met Dr Shultz until today, but for the last at least decade I�ve been reading many of his papers in Wound Repair and Regeneration and other papers. He�s been a long-time advocate for wound bed preparation, for proper approaches to wound care; numerous research topics and papers over the last at least ten, 15 years. He comes to us from the University of Florida in Gainesville. I just mention him � my brother�s in the University of Florida in Jacksonville, also at the Department of Surgery. And one of the topics that was of particular fascination for me, that I know he really, I believe he coined the term and really expounded upon, and that�s this idea of dynamic reciprocity of the extracellular matrix interaction with the cells, and vice-versa. And I really found this to be fascinating for those of us who trained or went to school many, many years ago, we thought of the ECM as just a static substance, as a substrate, and we know now that it�s much more so. Let�s welcome Dr Gregory Shultz to the podium.
[Dr Shultz] Well thank you Dr Frykberg. It is indeed a pleasure to have the opportunity to participate in the Desert Foot conference with you. This is my first time to attend this meeting, and I�m very impressed with the breadth and quality of the information that you all hear, that you�ve heard. And hopefully what I�m going to do is try to give you some biochemical background. Now before that immediately puts you to sleep, let me try to say I�m going to try to take all this biochemistry and molecular biology and distil it down to make it very clinician-friendly. And I also want to acknowledge Ira Herman, who�s a colleague and one of the co-authors of this paper that was published in 2011 in Wound Repair and Regeneration with this topic titled dynamic reciprocity.
So what am I going to do? Well first of all, I�m going to help you try to understand what this concept of dynamic reciprocity really is. I mean, where does this term come from and what does it mean? And as Dr Frykberg indicated, really this is just this concept to try to emphasize that in wound healing there is continuous communication between both the cells and the matrix and the other components that are crucial for wound healing, including growth factors and proteases. So really, when you are doing wound bed preparation, as Dr Allios indicated before, a part of that wound bed preparation and time acronym is really restoring dynamic reciprocity signaling within the wound. And I�ll try to show you how that fits in, because I�m going to give you some examples of how dynamic reciprocity occurs in acute wound healing, and we�ll look at a couple of these key examples, of the multi-domain adhesion proteins. This class that are exemplified by fibronectin and laminin. We�ll look a little bit at some of the growth factors, the matrikines and matricryptins. We�ll look at the integrin receptors, so how do cells actually recognize what matrix proteins they�re encountering, and how does that influence the gene expression within those cells? And obviously one of the best and most important components within wound healing and regulation, molecular regulation in wound healing, are the growth factors. And I�ll show you how many of the growth factors interacting with the receptors require an extracellular matrix component, the proteoglycans, to stabilize that interaction. An example of that is the heparansulfat proteoglycan, that�s really required for the fibroblast growth factor action. And another example, both in a positive and negative, a negative aspect, of regulation of transforming growth factor beta action by decrin.
But really, what I want to do besides just try to give you some background and understanding of what dynamic reciprocity really is explaining, is to show you the differences between the molecular and cellular regulation in normal acute wounds, where
dynamic reciprocity is functioning, and how that gets disrupted in chronic wounds, and I�ll show you, as has been indicated a little bit today, that growth factors, the receptors, and extracellular matrix proteins become destroyed by the excessive inflammation and the downstream effects of proteases interactive oxygen species. And those are the factors that are really disrupting this dynamic reciprocity, this communication between the cells and the matrix. And so really when you�re taking a chronic wound and converting it back into a healing wound, you�re preparing the wound bed, but that�s resulting in restoration of the normal dynamic reciprocity, the normal signaling that�s occurring between the cells and the matrix. And I�ll show you just example because the subsequent two speakers, I think, are going to give you really good examples of how to use acellular matrices or other advanced extracellular matrix products, because they in one sense are helping to restore very rapidly this dynamic reciprocity that�s occurring that needs to occur within chronic wounds.
So let me just go back and give you just this one piece of background. So I would love to claim that I coined the term dynamic reciprocity, but I didn�t. It actually was a term that Dr Mina Bissell, a developmental biologist, and her colleagues, really in the early 1980s, almost 30 years ago. They used this term to describe how the components of the extracellular matrix regulated and influenced gene expression, which in turn regulated cellular differentiation and tissue development. And one of the best examples that she used in this was the development, the embryological development of the mammary gland. And she showed in her work that only when the developing cells were placed on the correct matrix would they differentiate into the terminal differentiated cells that compose the mammary gland acinar complex.
But it has also been enlarged within recent years to recognize that it�s not just the matrix regulating the cells but the cells are also regulating the matrix because of what they�re synthesizing and how they�re turning that matrix over, how they have proteolytic activity that is modifying that. So in one sense, one of the other places where dynamic reciprocity becomes disregulated is in cancer, and one of the primary examples of that is in metastatic spread of cancer.
So for us, in terms of dynamic reciprocity and wound healing, we really want to keep this concept that it is this ongoing, evolving, that is, the dynamic term of this, where we�re looking at the interactions between wound cells and wound microenvironment, and it�s going to include both components of the matrix as well as the growth factors that are secreted by the cells and influence themselves and adjacent cells, as well as the proteases that are influencing and regulating the matrix production which in turn is going to, again, signal the composition and structure of the matrix through interactions with the integrin receptors.
So, as Dr Frykberg said, I think when most of us were taking classes and going through our histology classes, the classical view of the extracellular matrix, that it was really a passive component of the tissue, and particularly in wounds, it was considered to be really an inner scaffolding. And it provided important functions, that is, it helped to form the support scaffold for migration of cells, ingrowth of blood vessels and so forth, and of course to replace tissue that was lost during an injury, but this is really a far too narrow concept about what role the extracellular matrix plays in regulating wound cells, because in addition to just supporting that migration, it, as I�ll show you, directly coordinates wound healing through regulation of gene expression within the wound cells. And this is because the matrix will regulate the gene expression through activation of integrin receptors, synthesis of
specialized extracellular matrix components, and the matrix metalloproteases, or MMP secretion. And that the extracellular matrix plays active roles in both activating and inhibiting the roles of growth factors.
So really, what we need to begin thinking about from a larger conceptual perspective, is that when we have a wound, we have cells that are coming into the wound, and they are synthesizing, degrading or remodeling the matrix, but that matrix is also providing important feedback information that regulates the gene expression and the functioning of these cells within the wound through both the tension, the polarity, migration, proliferation and survival of those cells.
So if we look at a normal wound, and we can recognize that normal wound healing proceeds through these four phases: hemostasis, inflammation, repair and remodeling � at each of these four phases of healing, there are components of dynamic reciprocity signaling that is occurring, certainly during the early phases of the clotting, the early provisional wound matrix, which is composed predominantly of fibrin, is providing that initial scaffolding, but it�s also releasing growth factors that are stored in the platelets. And that�s stimulating the next phase � they�re chemotactically drawing in the inflammatory cells, and they are reciprocally interacting with the matrix because they�re particularly synthesizing and secreting proteases that are doing the initial wound debridement, removing those damaged extracellular matrix proteins that must be removed before new collagen molecules, for example, can be properly inserted.
During the repair phase, as cells migrate in, they�re replacing that early fibrin scaffold with the more mature collagen-containing scaffold. But in addition, as I�ll show you, they�re producing molecules like those multi-domain adhesion proteins, the fibronectin. As I�ll show you those are crucial in helping epithelial cells migrate. They�re also synthesizing and secreting the specific and unique components of the basement membrane that are really important for the epithelial cells to recognize and attach to. And then finally, the last phase of wound healing, the remodeling phase, is also of reciprocal communication between the stresses on the cells and their remodeling of that irregular collagen matrix that was produced during the early stage of wound healing.
But in chronic wounds, instead of wounds progressing through these phases of healing � remember all chronic wounds started as an acute wound, that you�ve heard before, and I�ll just re-emphasize, indicates that most of these wounds get stuck in a chronic self-amplifying and inflammation stage. And it�s the disruption of the dynamic reciprocity signaling caused by reactive oxygen and protease species, that really blocks the cells from moving out of this inflammatory phase into the repair phase. So in one sense, from the wound bed preparation, what we�re trying to do is re-establish this correct reciprocal communication between the cells and the matrix, and the matrix and the cells.
So just to remind you, the structure of normal skin, and of course you�re very familiar with this, but will have an epithelium sitting on a basal lamina � a basement membrane. And here you can see the epithelial cells and this light grey line which is the basement membrane that they�re sitting on. And this impacts very importantly how the basal cells of the epithelium behave. In addition, there are all of the normal matrix components that you�re aware of � large amounts of collagen arranged into collagen fibrils and larger fibers. But importantly, to recognize that when we look at an electron micrograph like this, we see a lot of space, right? We see things that just look like gaps in the matrix. Well, that�s an
artefact. It�s just because the dyes and the stains that we use to try to provide electron-dense material don�t stain this component, which is predominantly the proteoglycans. And these are molecules that are providing the water-holding capacity of the tissue and cushioning, whereas the collage and elastin fibers provide the tensile strength and the flexibility.
Another very important component of dynamic reciprocity signaling is the structure of the basement membrane or basal lamina. And this drawing just shows you that this is a very different structure than what is seen within the normal dermis, because it contains unique proteins like laminin and other multi-domain adhesion protein. And instead of the fibrillar-type collagens, Type 1 and 2 and 3, it predominantly contains Type 4 collagen. It can form this matrix in essentially two dimensions, and then there are other components that provide important recognition for the integrin receptors that are coming through the plasma membrane.
So what does a basement membrane or a basal lamina actually look like? Well, this is a scanning EM showing a chick cornea, so here�s the corneal epithelial cells, the equivalent of our skin, basal epithelial cells, and here�s the basal lamina. And underneath that is the corneal stroma, or in our case the dermis. So look at this amazing structure and how intimately associated the epithelial cells are with this, and how much it separates the epithelial component from the dermis. Epithelial cells will migrate across a dermal matrix composed mainly of collagen, but they must resynthesize a basal lamina to have their proper orientation and proper barrier function for an epithelium. So when a wound really is healing and maturing, one of the key things that has to happen is the basement lamina or basement membrane has to be reassociated.
So now I�m just going to give you a couple of real quick examples of some of these multi-domain adhesion proteins, and why these are important for us in normal healing, and how chronic wounds need to be able to synthesize these proteins and keep them around long enough for them to do what they need. And we�re going to look at one of these key ones, the fibronectin molecule. So fibronectin is a multi-domain adhesion protein. So bla, bla, bla � what do biochemists mean when we say that? Well, this is just a diagram, a drawing, that shows within a fibronectin molecule, that there are all of these domains formed by these amino acid clusters. And this particular domain contains a unit that binds to heparansulfat and fibrin. This domain binds to collagen. This domain contains the integrin or cell binding domain. And here�s another heparansulfat and a fibrin domain. So what this is doing is, this particular molecule, the fibronectin protein, is really key in helping cells attach to the matrix. It�s kind of the liaison between cells and matrix. And to show you how important this molecule is, if we take normal cells like fibroblasts or epithelial cells, and we plate them on just glass without any other proteins, these cells are so confused because they don�t have glass receptors, and they just sit as little rounded-up balls on the surface of the glass and they do not spread, as you see in this photomicrograph when they are plated on glass coated with fibronectin. So these cells are able to recognize that fibronectin through their specific integrin receptors in their membrane. It changes their gene expression and their behavior, and in fact it will also greatly enhance migration.
Now, as another important component to recognize is that fibrin, and its precursor fibrinogen, are not recognized by epithelial cells. Epithelial cells, when they�re plated on fibrinogen or fibrin, are unable to bind to that. So that�s why epithelial cells in an acute wound don�t move across the top of a fibrin clot. They don�t have fibrin integrin receptors.
They only have integrin receptors for fibronectin or collagen. And so this shows you that the matrix is determining what cells can do and how they can perform. And the way they do that is they have these things called integrin receptors � these molecules that are synthesized and they�re in the plasma membrane, and they have binding domains that are specifically able to recognize structures on the surface.
Other proteins that are important in dynamic reciprocal communication are the glycosaminoglycans and proteoglycans, because the fibroblast growth factor family as one example requires the presence of the heparansulfat positively, negatively-charged strands to fit within this positively-charged canyon formed by the FGF and FGF receptor quartet. Without this heparansulfat, this is too unstable and the receptor doesn�t signal.
So with that background of what should happen in dynamic reciprocity and communication, what happens when these wounds fail to progress through these four phases, and there�s a disruption of that dynamic reciprocity communication? We get chronic wounds, and the reason is that in these chronic wounds as you�ve heard several times today already, there are high levels of bacteria, often present as a biofilm, and it�s the downstream process of inflammation, particularly the proteases, reactive oxygen and nitrogen species, that degrade these matrix proteins and the growth factors and the receptors, and that disrupts the dynamic reciprocity signaling. So really what you�re doing when you are preparing the wound bed is you�re rebalancing this molecular environment to allow the restoration of dynamic communication. And in fact, the levels of proteases are a great indicator of whether a wound bed is prepared well or still so out of balance that it cannot progress out of the inflammatory phase to healing, because if we measure the level of MMPs in chronic wounds or acute wounds, you can see that the levels of proteases in chronic wounds are on average about 50 times higher than they are in an acute wound.
And only when the high levels of proteases begin to decrease will the wound begin to heal. In fact, there are now clinical trials ongoing � Tom Serena�s a key clinical participant in this - in which the levels of proteases are being assessed by a rapid point of care indicator, because what we have shown and what others have shown is that the levels of proteases in the wound at the start of treatment are a great indicator of how long it�s going to take for those wounds to heal, and so the faster and better you can depress those growth factors, the better the wound will convert back to the molecular environment that can restore the dynamic reciprocity. So how do these proteases destroy the components? Well, they have off-target effects, and I�ll show you examples of these off-target effects. You remember that fibronectin, that multi-domain adhesion protein that epithelial cells need to move across the ulcer bed? Well, it turns out, in most chronic wounds such as this chronic venous ulcer, when it�s immuno-stained for intact fibronectin, you can see the intact fibronectin under the adjacent skin, but there�s no intact fibronectin in the ulcer bed. In contrast, when that wound bed is prepared well and dynamic reciprocity and communication is restored, the proteases are low, now the fibronectin synthesized by these cells can persist, and now the epithelial cells begin to move across the wound bed. This has restored the dynamic reciprocity communication that�s necessary for those cells to heal.
Similarly, growth factors are also very sensitive to proteases � normal skin has very little �cause there�s very little mitotic activity, but in a healing wound there�s high amounts of this growth factor, PDGF as an example. But immuno-stain for PDGF in chronic wounds, there�s almost none there � not because it�s not being synthesized, but because it�s not surviving in the high protease environment. When that wound bed is converted into the appropriate
balance of proteases now, the growth factor is present and the wounds begin to heal. And similarly, a similar type of process occurs for this receptor. Now this isn�t the growth factor, this is the receptor that becomes stripped. The transforming growth factor beta receptor is the sensitive component, and only as that wound begins to heal and reciprocal communication is re-established will those wounds begin to heal.
So, how do you rapidly and effectively re-establish dynamic reciprocity in chronic wounds? You do it through wound bed preparation. You reduce inflammation and proteases that are the cause that disrupt this dynamic reciprocity, and then you have multiple approaches that can accelerate and promote that restoration of the dynamic reciprocity. You�re going to hear two presentations following this one on acellular matrices as one way to do that in kind of a jump-start fashion, and particularly those matrices that contain growth factors or other components that are important. And this is just one example that in fact even contains an intact basement membrane that epithelial cells like.
So in summary, what I hope I�ve done is demystify this concept of dynamic reciprocity and bring it back to you in some relevant way to help you understand that when you�re preparing a wound bed to begin to heal, what you�re actually doing at a molecular level is re-establishing this communication between the cells and the matrix, and then the matrix is influencing the behavior of the cells. And your task as wound experts and wound clinicians is to take these wounds out of this chronic inflammatory phase and enhance the restoration of the dynamic reciprocity through multiple treatments and adjuvant therapies. And when you have restored the disrupted dynamic reciprocity, the wounds now are going to begin to heal as an acute wound.