Stromal Matrix and Angiogenesis in Healing Wounds

00:00 In the next series of talks, we're going to talk a little bit more about the biology of neoplasia, we'll talk specifically about tumor angiogenesis and stroma because these are important targets for therapies as we get smarter and smarter about combating cancer.

00:17 We'll talk about invasion and metastasis and how that happens, and we'll finish up with the effect of tumor on host. How does cancer kill you or how does cancer cause morbidity? So first up, in this talk today right here we're going to do the biology of neoplasia. Okay, let's start with some basic concepts again. I want to tell you that cancer is not making new stuff up. It's basically using the same pathways that normal tissues do. It's just using them in a different way or in an uncontrolled way. So there's nothing magical about tumors, they're just doing things that other tissues do normally but in a controlled way, but cancers do it in an uncontrolled way. So we're looking here, this is not cancer, this is just looking at the concept of parenchyma and stroma. So parenchyma are going to be the cells, in this case the glandular epithelium lining a lumen that are going to be responsible for the business end of the tissue. This happens to be, I believe, endometrium, and that endometrial tissue will proliferate under the influence of hormones that have various secretory products and will get prepared on a monthly basis to accept perhaps a fur-like zygote. Okay, parenchyma and any tissue at all is the business end. It's what gives it its function. So in skin, it's the epidermis. In the heart, it's the cardiac myocytes. In the liver, it's the hepatocytes. In the kidney, it's the nephron and the tubules, so the business end.

01:55 But the parenchyma, the business end can't do its job if it doesn't have a good supporting framework around it, that's the stroma. So the supporting connective tissue is not only just extracellular matrix, but it's got the blood vessels and lymphatics. It's got nerves, it's got fibroblast, and myofibroblast and a variety of inflammatory cells that are all part of the support network that allow the epithelial cells or the parenchyma to do their job. Stroma, the supporting connective tissue, can be incredibly variable in amount and in type of stroma between different tissues as we'll show you on the next slide. And very importantly the kind of stroma, how it looks, how it's organized is going to drive structure and function correlations. The first one up here is bone and bone is primarily extracellular matrix with rather sparse contribution of the cells that are actually making or maintaining the bone.

02:58 So there are little lacunae in there with osteoblast, but around all the osteoblast are these dig sheets of collagen and hydroxyapatite and other bone-forming proteins that make a matrix that is mostly stroma. In the middle, we have colon where we have a greater number of cells in the parenchyma. So you can see all the glands and in between the glands is going to be our stroma. So it's not quite as stroma rich as say bone. And then finally in liver, there is relatively minimal amounts of stroma, there are lots of parenchymal cells, the hepatocytes as you see radiating here away from the portal triad. Okay, same thing can be said about cancers. So, in some cancers there's a very dense stromal component that is called desmoplasia. The first panel here is adenocarcinoma of the breast and some breast cancers can be quite cellular. This one tends not to be. This is why breast cancers frequently feel very rock hard and firm because they elicit a lot of stroma of the connective tissue extracellular matrix around them. Notably in there, the tumor cells are relatively sparse, there are little islands up near the top and a little island at the right lower corner, but the majority that this is stroma that is not malignant. So I just grind up this tissue and don’t pay attention to where my DNA and RNA is coming from, it will look like scar. That's the signature of this tissue because the majority of the cells and the majority of what's in there is stroma. There are other tumors that have, maybe 50/50, this example here is adenocarcinoma of the lung. There is still a bit of a desmoplastic response, there's still some fibrosis but there is much greater cellularity. And if I were to grind this one up and then look at the DNA signature, I might be able to see that there were some tumor genes that were being expressed, but keep in mind all the stroma around there is going to be normal non-malignant tissue, but it's being elicited by things that the tumor is putting out. Interesting, right? And then finally, we have small cell lung carcinoma which is basically back to back, ___ cells. I mean there's very little matrix here, there's very little stroma here. Which one do you think is going to be a hard tumor? Well, yeah the one on the left. Which one's going to be a soft squishy tumor? The one on the right. Okay, and so the amount of stroma will also give us some indication of the physical properties of the tumor itself. Okay, so why am I harping on this? Well in fact, all the stroma production is really just the tumor recapitulating normal wound healing. There was actually a really interesting paper back in the 1970s from a colleague in Boston who is still alive, Dr. Hal Dvorak, who basically described cancers as wounds that do not heal. They continue to elicit the wounding response, the normal inflammatory and healing response, and they have just laid down more and more matrix. So in order to understand what the tumors are doing, let's look at what normal wound healing is like. And we have covered this before, but it doesn't hurt to review that and it will be important as we go forward. So here we have a normal tissue with a blood vessel there in the middle. In red cell is going by, everybody's happy. There is a certain amount of epithelium at the top and some stroma underneath and we're good. Now, life intervenes. We have a cut. We have some injury, some trauma to this and we're going to get a disruption. We've talked about coagulation and other things, but what is all going to be happening here initially is some vasodilation as a result of that initial injury. Leakage of some of the plasma proteins including things like fibrinogen and we're going to lay down now a clot that will have inflammatory cells within it. It will have various clotting proteins and fibrinogen turning into fibrin. We'll also have elements of the basement membrane, fibronectin and the extracellular matrix, all in there. As we go along, in order to do the healing, we will have angiogenesis. So the inflammatory cells, the epithelial cells, the endothelial cells, and the various protein components that are in this wound will elicit the new ingrowth of blood vessels for the nearest intact blood vessel. And they will provide a conduit, a railroad truck upon which we can bring in fibroblast that will then heal the wound. So the goal here is to actually now make more matrix that require the ingrowth of fibroblast coming in along these blood vessels, healing a wound, very metabolically active so we're going to need a good blood supply so that we have necessary nutrition and oxygen. And we will have growth factors that are giving you the same area as part of that matrix deposition. And eventually, we restore integrity to the tissue. We may have lost some of our epithelium, but we now have a matrix that's collagen rich and it has lot of the other extracellular matrix proteins being maintained by fibroblast and macrophages of course kind of supervising the entire process. Important step in here and that we will come back to when we talk about tumors in particular is angiogenesis. We definitely have to have the new ingrowth of vessels to provide the adequate nutrition and the necessary conduit in which fibroblast can grow in.

09:04 So again, physiologic angiogenesis, not tumor angiogenesis although that will becoming physiologic angiogenesis involves an initial process by which we take blood vessel precursors, endothelial cell precursors. And either developmentally generate vessels.

09:25 That's vasculogenesis. So blood vessels form during fetal development is called vasculogenesis. But once you have an adult form, any new blood vessel ingrowth is going to be angiogenesis. That's the distinction there. With this initial collection of precursor cells, then we get cell-cell signaling that says you need to grow, you need to grow in a particular vectorial direction and you need to mature and form tubes. So there has to be recognition of cell-cell and cell matrix interactions, signaling, as well as mechanical forces to get good angiogenesis. So we're going to bring those cells in, they're going to form a tube, forming that tube requires cellular proliferation, migration along a concentration gradient. As we prune back, we will have components of apoptosis. And depending on the vascular bed, depending on the cells that are doing the calling, depending on the needs, there will be some specialization. So for example in the glomerulus of the kidney, we will have fenestrated endothelium in the hepatocyte sinusoids will have fenestrated endothelium with holes in it. In the brain, we're going to have incredibly tight junctions maintained by the endothelial and astrocyte interactions. So there will be subsequent specialization. And that allows us to have a very organized artery, brain-blood branching, branching, branching into capillaries and then re-coalescing all those branches into a post-capillary venial and into a vein. So that there is an organization of the normal vasculature. Now most of what I'm showing you here is vasculogenesis happening during development, but similar remodelling and processes have to happen when we have angiogenesis in the place of wound healing. So, the mature network should be stable and the structure and function of the wall in the network is appropriate to the needs of the tissue. What are the environmental cues that drive angiogenesis? Now we're going to talk about angiogenesis. Hypoxia. Well that's actually makes a lot of sense. If a tissue is hypoxic, it's not giving enough blood supply so we need to have new blood vessel ingrowth to improve the oxygen delivery to that tissue. Acidosis, and that's kind of the corollary. If a tissue is not getting adequate oxygen, it's not going to be using oxygen as a recipient for electrons in the electron transport chain so it's not going to be making ATP very efficiently.

12:12 Instead of just calling it a day and quitting, what that cell will do is use anaerobic glycolysis.

12:19 It will just breakdown whatever glucose it is to generate just 2 ATPs, but in doing so we'll also generate a lot of lactic acid. So acidosis in a tissue from lactic acid is also going to be an environmental cue for new ingrowth of vessels. Growth factors during development are going to be very important in vasculogenesis. I mean as we're building the initial blood vessel system and getting arteries coming off the aorta and having that go into a capillary bed and then into veins and returning through the inferior vena cava and going through a pump, all of those required gradients of growth factors. Interestingly enough, sex hormones, testosterone, estrogen can influence the angiogenesis and certainly inflammatory cytokines can do that as well. So keep all those in mind in this normal physiologic angiogenesis and then we'll revisit this when we talk about tumor angiogenesis. And with that, we've finished this first stage.