CME Wound Care

Placental Membranes: A Molecular Review

Jonathan Brantley, DPM

Jonathan Brantley, DPM gives an overview of how placental membranes work on a molecular level giving particular attention to TGF-beta3, SDF-1, and N-Gal 3 factors that he feels don't get as much attention as the more common factors that are more known such as VEGF.

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Goals and Objectives
  1. Identify the benefits of transforming growth factor beta 3 as it relates to wound healing
  2. Draw an accurate reproduction of the mechanism of action of neutraphil gelatinase associated lipocalin
  3. Identify the benefits of stromal cell derived factor 1 as it relates to wound healing
  4. Describe the type of cells that mesenchymal stem cells are capable of differentiating into
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    Release Date: 03/16/2018 Expiration Date: 12/31/2018

  • Author
  • Jonathan Brantley, DPM

    Chief, Podiatric Medicine and Surgery
    McGuire VAMC

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  • Lecture Transcript
  • TAPE STARTS – [00:00]

    Male Speaker 1: If you look at the posters or you look at the program, lots and lots of talks about placental tissue and the role or placental tissue in wound healing. So Jonathan Brantley is going to talk to us from a little different perspective and that’s molecular review of placental membranes. Jonathan, there you're.


    Male Speaker 2: So placental membranes -- this is going to be a molecular review and really what I wanted to do by structuring this particular lecture is to get you think outside of the box in regards to how these particular structures work. So of course placental membranes have been around for a number of years, approximately 100 years and the benefits are basically very, very well known. So the issue has always been the acquisition, the availability and then that possible risk of the transmission of some type of an infectious disease when you're using fresh placental membranes. The target has always been if you're going to preserve a fresh placental membrane, then the ultimate usage really was trying to maintain the structural integrity as well as maintaining that native property of those particular placental membranes, so that it literally mimics what a fresh placental membrane does in regards to a prepared or preserved placental membrane. So as you know, there is a huge push right now where there are many, many companies that are actually doing different types of preservation techniques of placental membranes and this is for the purpose of acute and chronic wound healing. So this list that you see here is not an exhaustive list, but it is a relatively comprehensive list of a lot of different products that are in placental membranes. And there are three here that I really want to outline because I don't think there is enough discussion about these three particular products within the mesenchymal stem cell -- I'm sorry --within the placental membranes. So the first one obviously is TGF beta 3, which is for its anti-scarring properties. The second one is going to be stromal cell-derived factor 1, which is for its chemoattractive activity with respect to mesenchymal stem cells and NGAL or neutrophil gelatinase-associated lipocalin for its very, very unique properties in regards to its anti-microbial effect on wounds. So let’s take the TGF beta factor family. So you actually have three isoforms and the isoforms are beta 1, beta 2 and beta 3. They play a very, very critical role in both stimulation of collagen synthesis as well as angiogenesis and they are all potent chemoattractants or anti-inflammatory mediators for different immune cells. So let’s focus on TGF beta 3. Now, no discussion about this would really be complete unless we really look at the pathway of how they can actually cause this functions that I alluded to in the previous slide. So when you look at the TGF beta 3 ligand, essentially what it does is it attaches to the receptor site for the particular ligand, which in this case may be transforming growth factor beta 3 receptor or transforming growth factor beta 2. The affinity is slightly higher in transforming growth factor beta 2, but eventually what happens is once that ligand is actually attached to that particular receptor site, it actually forms what's called a tetramer with the receptor site for 1. When that occurs, now what you're going to see is this transformational conformational change that occurs in a receptor itself where you have something called SMADs. And SMADs essentially just stands for mothers against decapentaplegic homolog, and essentially that’s a specific type of a protein that will attach to that receptor site. So after it becomes phosphorylated and that is SMAD2 and SMAD3, then SMAD4 eventually will be able to attach to that complex. And then that is what actually enters into the nucleus. Now, once that complex enters into the nucleus, everything that we talked about as far as the functions of what happens when TGF beta is actually expressed, this is where it's going to occur.


    So this is when you're going to get the gene expression for collagen synthesis, for angiogenesis formation and such. That gives you an idea of where and what this is doing as far as how it's trying to express these particular molecules. This is a relatively busy slide, but when you look at the extra role of TGF beta 1, beta 2 and beta 3, beta 1 and 2 actually work as an inversely proportional factor to TGF beta 3. So if you look here at what an elevated level of TGF beta 1 and 2 will do in regards to the migration and activation of inflammatory cells, that cause an increase where if you have an increase in beta 3 that’s actually limiting that. So you don't really have that same type of increase. And the second step that you would see here is the proliferative phase where TGF beta 1 and 2 when they are elevated as well as 3 in this particular case, you're going to see an increase in re-epithelialization, more angiogenesis production. But if 3 is not elevated, then obviously you're going to see kind of a decrease in that extracellular matrix deposition. I'm going to explain that a little later in some further slides. Now, the big thing about scar formation is scar formation traditionally occurs when you have an increase in fibroblast to myofibroblast conversion. So TGF beta 1 and 2 when they are elevated, you're having a greater differentiation of fibroblast to myofibroblast conversion, which means that you're going to have more scar formation. And if the converse is true and you have less of beta 1 and beta 2, but more beta 3, now you have less scar formation because you have less transition from your fibroblast to your myofibroblast. So the TGF beta 3 actually plays a very, very important role on keratinocytes. And it's not always what you think or suspect would actually occur. So the absence of beta 3 actually causes keratinocytes to express an increased proliferation rate in those keratinocytes. So this is when you look at a wound and you start to see those margins really kind of stacking up on each other, but there is also a reduced migratory factor that’s associated with this. So now you're not saying that epithelial tongue that you desire to occur. Now, when you add TGF beta 3 to these particular wounds, then yes, you do see a reduce in the amount of proliferation of those keratinocytes, but there really is no affect on the migratory capacity of these keratinocytes. And all this really comes about because you kind of scratch your head and it's like, well, that doesn't really make sense. If I had them, then I really should see an increase, but the reason or the rationale behind that is because the transforming growth factor beta receptor 2, which I said has a very, very high affinity for TGF beta 3 or it's really not that -- it's not highly expressed in the keratinocytes. So if it's really not highly expressed, it doesn't really matter how much you actually put in to that particular wound for the purpose of them attaching to the keratinocytes. Then if there is few of them that are actually being an expressed by that cell type, then those cells are actually somewhat insensitive to the addition of TGF beta 3. So how does it really affect scarring? As I had alluded to a little earlier where you have more fibroblast to myofibroblast conversion, which is really kind of what is causing that retention of the tissue and then further scarring of that particular tissue or what is manifested as scarring in that tissue. Well, transforming growth factor beta 3 actually exhibits decreased expression of alpha smooth muscle actin in granulation tissue. So what's so important about that? Well, the alpha smooth muscle actin really is the marker for myofibroblast. So if it's the marker for myofibroblast and you know that if you have more fibroblast to myofibroblast conversion, that’s going to lead to more scar tissue.


    So if you have something that is going to decrease the alpha smooth muscle actin, then that’s telling you that you really don't have a whole lot of myofibroblast in that particular area and with that being the case, then your scarring is going to be a lot less once you see the end result. So, here is something that I thought would kind of throw a fly into the ointment because it's also – it's a little rabbit holes that we are jumping down all the time, we are looking for this and looking for that. But a lot of the drugs that our patients are on are actually inhibiting TGF beta. So if TGF beta is necessary for this re-epithelization, for granular tissue formation, I mean we will just take the first one. You're looking at Losartan, which is a very, very common antihypertensive drug that actually blocks angiotensin too. Now, that might be great for the cardiac condition, for the vascular condition, but what happens if you actually have a wound and you have a drug that particular patent is taking that’s actually inhibiting the very growth factor that’s necessary to cause that increase in fibrosis to occur. That’s just a whole new different quandary of problems and it's a pathway or rabbit hole that you may not want to venture down because most of our patients are on this really, really high pharmacy list.

    The next one is stromal cell derived factor or SDF-1. It also has multiple different functions where number one, it's a potent chemokine for bone marrow-derived mesenchymal stem cells that actually express the receptor site for that SDF-1. So if you don't have the actual receptor site for that, then SDF-1 is kind of rendered ineffective. The receptor site is actually called CXCR4. Now, if you don't have that, all the effects that mesenchymal stem cell or that bone cell will traditionally exhibit, it's not going to happen because you're not having that attachment of SDF-1. So it also plays a very crucial role in mobilization, trafficking and the homing effect of bone mesenchymal stem cells. It stimulates bone mesenchymal stem cells both through autocrine pathways as well as paracrine pathways. And interestingly enough, it actually mobilizes hematopoietic progenitor cells as well as stem cells and bone marrow-derived endothelial cells. So this here is really no more than just a depiction of what these particular cells actually have the ability to differentiate into and this is no secret. We are all very, very familiar with this, but bone marrow-derived mesenchymal stem cells actually and their derivatives can modulate blood production and the immunity at different levels. And currently, they have been known to play a prominent role in the emergence of bone mesenchymal stem cells and how they regulate hematopoietic stem cells. And the secretions that actually come from these bone mesenchymal stem cells are things such as, which you're very familiar with, VEGF, basic fibroblastic growth factor, all of the isoforms of transforming growth factors beta as well as interleukin 6 and others. So how do you actually up regulate the expression for SDF-1? The way that whole pathway begins is first you're going to have some type of an initial ischemic injury to that wounded tissue and this is basically what happens in just about all wounds. When you have that initial ischemic injury, then you're going to release or at least tissues that are surrounding that are going to release hypoxia inducible factor 1. Now, the reason that this is occurring is because you have to establish some type of hemostasis to stop the hemorrhaging from occurring. So during that short period of time, obviously now you're going to have the expression of HIF-1. Well, HIF-1 actually attaches to the promoter region of SDF-1 and when that occurs, now that causes the activation or now the true expression of stromal cell-derived factor 1 and then of course it looks for that receptor site at CXCR4. And then of course the next set of events that’s going to occur is the release or the secretion of all those various types of growth factors and such as well as the trafficking and homing to that particular area.


    So you're going to have some peaks and you're going to experience some troughs of SDF-1. And the peaks that you're generally going to see obviously like I alluded to earlier is that when you have the initial wounding of that tissue, there is an ischemic event that occurs where first you're going to see some very relatively short ischemia or hypoxia and then ischemia, but this is when you will see a huge expression of SDF-1. So at that point, now you wonder, okay so when is the next peak going to be occurring within that particular wound. Well, the next peak actually occurs about a week or so afterwards because now you have that proliferation or at least that migration of epithelial cells, fibroblast and endothelial cells, which also have the ability to express those particular receptor sites for SDF-1. And then of course if you have a peak, you're going to have a trough as well and the trough that you see is right after wounding, you're always going to have this really, really severe inflammatory state. And with this severe inflammatory state, this is the point where you're going to have a tremendous amount of pro-inflammatory cytokines. And with these pro-inflammatory cytokines, you have pro-inflammatory cytokines such as tumor necrosis factor alpha, interleukin 1, which are actually suppressors of the expression of SDF-1. So moving on to neutrophil gelatinase–associated lipocalin, which I have always kind of find as a very, very interesting concept how this works. Essentially with NGAL, it has a bactericidal effect inside the wound and just to give an orderly sequence of the events, bacteria will secrete something called siderophores. And siderophores essentially will sequester free iron inside that host. And when it sequesters this free iron inside that host, the whole purpose behind that is it is trying to basically commandeer that iron because it needs to actually have iron as the element of choice for bacterial cell respiration as well as bacterial cell DNA replication. So if you prevent that iron from being acquired by the bacterial cell or even a fungal cell, then you're able to effectively interrupt those two particular types of pathways. Some of siderophores they have various different names depending on the type of bacteria that they are secreted from where you have some that are called enterochelin or its degradation process, which is called 2,3-dihydroxybenzoic acid. Then you also have some that are expressed from E. coli and bacteroides species and that is enterobactin as well as bacillibactin respectively. So this here is just an artist rendition of how that whole process occurs. So you see your bacterial cells over here on the right side and then you see your fungal cells and they both have the ability to secrete these siderophores. And siderophores enter into that particular host looking for free iron to, for lack of a better term, really sequester [Indecipherable] [0:19:08]. And then you see here at the 6 o’clock region, lipocalin-2, which is just another name for NGAL or neutrophil gelatinase-associated lipocalin, which sequesters or it is able to prevent the re-acquisition of that siderophore with the iron containing in it so that it is not available to the bacteria or that fungi to use there by interrupting bacterial cell respiration and bacterial cell DNA replication. With that being said, I would like to thank you for your attention and your consideration. And if you have any question at this time, I would be more than happy to answer them.