---start---- djd/arthritis question: describe the components of articular cartilage matrix, their role in function of articular cartilage, and how they are affected by at least 2 cytokines or drugs. djd is organ failure what can fail? cartilage bone synovium supporting structures ligements joint capsule note to those who care: i'm not transcribing this lecture. I'm only writing down stuff relevant to the question at hand. my wrist hurts. Joint = machine in machines, bearings wear out - parts that move against each other in joint, cartilage is the stuff that moves against itself. it's the stuff that wears out. DJD/osteoarthritis- traumatic, mechanical wear/tear - all is lumped into this category. slightly different from rheumatoid arthritis (immune mediated) or septic arthritis, but end result is the same. Normal joint function requires normal articular cartilage (bearing surfaces) Articular cartilage function is dependent on an intact structure structure of articular cartilage depends on its matrix components. Synthesis and degradation of matrix of articular cartilage must be balanced! joints are biological structures, in flux,not static - cartilage is always being made and destroyed (as are other tissues). "From Hippocrates to the present age, it is universally allowed that ulcerated cartilage is a troublesome thing and that when once destroyed, it is not repaired." William Hunter, 1743!! We've known for a while - once cartilage loses its framework or overall structure, we can't return it to normal. Now, you can lose a piece of joint cartilage and have a reasonably healthy scar form in that place,and if it is reasonably small,the horse can still run. But it isn't totally normal. It can not be made normal. Ever. What is cartilage? -65-80% water -type II collagen -matrix (ECM) Think about how cartilage works. You walk on joints constantly. It has to last. Matrix components are maily type II collagen and many proteoglycans (PGs) microscopically- recall histology- articular cartilage is relatively hypocellular tissue, that is on a per unit volume, there are few chondrocytes. they are spread out quite a bit and near the surface they get flatter and closer together. beneath it is subchondral bone. So, what's between the cells? you have chondrocytes sort of irregularly dispersed, sitting in matrix. closer to bone, they are sort of stacked up radially. at surface, they are sort of flat. this is similar to the pattern of collagen fibers. this has to do with how collagen wears. How do joints move? back and forth - surfaces rub together- if you have collagen fibers sticking perpendicular to the surface, that is not so good. you have the vertical fibers sticking into subchondral bone, and the deeper parts of the cartilage have vertical fibers. at the surface, the fibers run more parallel or tangential to the surface - so it wears better. Collagen - the key structural molecule of the body in all mammals and pretty much all vertebrates. Many specialized forms of collagen exist. In articular cartilage, we use type II.It's a fibrillar collagen, with stacked fibrils forming the actual fiber. IX, X, and XI are also seen in cartilages but we won't discuss them. type II is the key element for structural integrity of the meshwork forming the articular cartilage. type II -key element for structural integrity of articular cartilage -has specific interactions with other matrix proteins (can't just replace it with some other collagens; will lose these interactions) -provides tensile strength -can be cleaved by various proteinases, either in helical region by collagenase or telopeptide region by stromelysin) Note: aging horses were shown to produce less collagen - the chondrocytes removed from an older horse made less type II collagen than ones from younger horses. So, when you do surgery in younger animals you can expect a superior quality of healing. THis is absolutely the case. If you remove an OCD lesion from a foal or a 6 yr old, the result is just far superior in the foal even if technique is the same. TNF and IL1beta - both cytokines important in joint diseases - these are inflammatory cytokines - are induced during inflammation and cause induction of more inflammatory processes - if you take chondrocytes and treat with these cytokines, the gene that makes type II collagen turns off and the cartilage isn't able to replenish, heal, or make collagen. balance tips to the degradation side b/c you're turning off the synthetic side. [yada yada...description of what northern blot does, and how DNA is used to make a protein] So, if you look at cartilage and type II collagen - that's a lot of it. but there are also orange squiggly things on the slide. what are they? well, they are supramolecular aggregates (SAs) - the other key component to maintain the biochemical and mechanical function. long backbone of hyaluronate, and backbones of aggrecans sticking off the side attached by "link protein". on the aggrecan are many branches of glycosaminoglycans (GAGs) (chondroitin sulfate and keratan sulfate). what is the function of this SA? to hold water. it is very hydrophilic, esp the GAG sidechains. now, think about this. you have these SAs holding big volumes of water. you hve a firm network of strong collagen fibers, and inside that are the SAs holding a lot of water, so now you have a really resilient mechanical structure - it's resilient, wear resistant,a nd well lubricated. type II collagen is specific inn its ability to interact with these SAs. this interaction is critical. you want to avoid articular degeneration. if you lose GAGs or aggrecans or any part of the SA, the water isn't bound into the matrix. Then you lose resilience. If you lose resilience, you're vulnerable - can't withstand normal stress - wears out. so how do you keep this from happening? collagen and other stuff in balance b/w synthesis and degradation. how do you keep synthesis and degradation in balance? prosynthetic compounds: TGFb, insulin like growth factor, IFNg degradation compounds: IL1, TNFa, bradykinin, retinoids but it isn't that simple. primary elements involved in degradation of matrix are: Matrix metalloproteinases: MMPs these are very big in the literature right now. anyone know what other people are interested in them? oncologists. the big interest in these enzymes is their importance in cancer metastasis. to spread to other tissue, tumor has to degrade normal tissues ... MMPs are: major elements in tissue remodeling highly conserved across species found in reproductive system, too found where there is a lot of tissue turnover involved in articular cartilage turnover MMPs hydrolyze at least one componenet of ECM. most exist in a latent form prior to activation the conserved molecular structure includes activation site, Zn binding region, etc. How do MMPs get involved in arthritis? some are capable of cleaving aggrecan - some are able to cut the aggrecan near the attachment to the hyaluronan backbone. "cysteine switch" there is a list of all kinds of different MMPs he's showing us...the cysteine switch region is important for enzyme activation. in the literature it says this PRC motif is consistent across all spp, but in the horse there is a serine instead of a proline. MMP cascade: prostromelysin is inactive MMP. kallikrein, plasmin can activate it to stromelysin, which then can self activate more of itself. ----break----- moving right along.... footloose and fancy free... the MMP collgenases cleave fibrillary collagens - of which Type II is one MMP 1 and 13 are inn cartilage, and 13 is probably more important in articular cartilage.. stromelysin MMPs degrade aggrecan, laminin, fibronectin, and smoe collagens (not II). are important in inflamed joints. MMP 2 and 9 are gelatinases; are players in articular matrix degradation, break down collagen fibers once the degradation has started. don't know #s and names, just know MMPs exist and break stuff down. MMP 12 rather specifically breaks down elastin. mmp 15, 16, 17 are membranetype MMPs. his interest is cartilage so we're looking at it. TNFa and IL1b are imprtant cytokines. when you treat with these cytokines, small amounts of them cause huge upregulation of genes for these MMPs. why are certain genes turned on and others are not? TIMP1 - tissue inhibitor of metalloproteinase -constitutively expressed in articular cartilage -a major regulator of ECM degradation - binds to and inactivates MMPs this is a degradation inhibitor. like so many other things in the body,there is a balance b/w agonists and antagonists. Think about MMP being important to break down the tissue. if TIMP1 is important to turn off those enzymes, you should find out how to turn it on or off. remember TNFa and IL1b turned on MMP genes. well, TIMP1 is already there, and doesn't increase that much after treatment with those cytokines. in fact, high doses of TNFa downregulate TIMP1 production. net effect: increase in MMPs, causing enhanced degradation of ECM. TNFa and IL1 b- downregulates link protein, mildly downregulates aggrecan, definitely downregulates type II collagen can you treat these with drugs? phenylbutazone, ketoprofen, flunixin are all NSAIDs used in horses. TIMP1 is basically unaffected by these drugs MMP3, decorin, biglycan, and aggrecan are also unaffected by normal doses of these drugs. the literature hotly debates the effect of NSAIDs on articular cartilage. some studies say asyou use increased doses you end up with decreased synthesis of these proteins, but it's not at the gene regulated level,may be at intermediate metabolism level. you will read a lot of articles about NSAIDs which have no effect on articular cartilage and that is supposed to be a big selling point - carprofen, for one. Does it make a difference wrt joint function? maybe. How do NSAIDs work in arthritis? they do make you feel better. do they protect your joints? no. they do not slow joint deterioration, we know that much. to date, most of the evidence suggests that in fact NSAIDs will increase progression of DJD. people in study felt better on NSAIDs but had more rapid joint deterioration (because they were using the joints more); people on placebo had less joint deterioration. another group of drugs we looked at - glucocorticoids, corticosteroids - these also are hotly contested in literature. Horses get joint injected with steroids, runs, falls down,kills someone, owner is sued...vet is sued...steroids are used b/c they WORK. they have potent antiinflammatory properties. but also have deleterious effects. molecular evidence suggests - if you take chondrocytes and treat with TNF, there is little effect on aggrecan synthesis, but then adding dexamethasone turns off aggrecan production. at the same time, you turn off MMP3 production (which is turned on by TNF). this is good. you decrease degradation (but you've also decreased synthesis). what about TIMP1? thereis decreased TIMP production under influence of dexamethasone. that's bad. this work helps us to recognize dose/response effects. if you work with lame horses, steroids are an important step. used to be, you got a bottle of depomedrol and filled the joint with the contents of the bottle. you were way overdosing. and there are dose related effects - you can show that at certain doses you getmore beneficial effects, and fewer deleterious effects. so we have tried to lower our doses to effective level. link protein gene- notice that when you treat with IL1b it is slightly downregulated, then it is heavily upregulated when you add steroids. a few things about some drugs used -- arthritis is related to wear and tear wear and tear is going to affect weight bearing surfaces articular cartilage function depends on structure structure depends on molecular composition, which depends on balance b/w synthesis and degradation. STeroids are greatly effective at decreasing the degrading processes in terms of MMPs, but they also have deleterious effects on synthesis. the way to mitigate the problem is to use smallest effective dose for clinical effect. choice of steroid depends - duration of action is greatest variable.potency isn't that important,you just adjust your dose. Adequan - polysulfated glycosaminoglycans PSGAG. been around a long time and still debated. two reasonably well proven mechanisms of action- can inhibit MMPs and some other proteinases, and also when you give it it stimulates hyaluronan synthesis by synovial cells. hyaluronan is found in synovial fluid - it's the stuff that lends the viscosity to the joint fluid. this drug also can be given intraarticular or intramuscular. when used IM, in dog/rat/horse it DOES reach the joint. how? well, it is small molecule. probably works better given ia, but it inhibits complement and greatly lessens resistance to sepsis. Hyaluronic acid HA (sodium hyaluronate) given intraarticular, to replenish supply in synovial fluid. this is an important component of synovial fluid, but not the sole component. the mechanism of joint lubrication is greatly dependent on the inherent elasticity of the cartilage. some of these products are available for IV administration (LEGEND). how does that work? cellular mechanisms...hyaluronan can bind CD44 receptors in articular cells, turn on more synthesis, etc. Glucosamine: aminomonosaccharide: excellent absorption, mild "anti reactive" agent, substrate for cartilage formation, directly stimulates PG synthesis,possibly HA. it's very safe, and somewhat expensive esp in horse long term. very small molecule, readily absorbed Chondroitin sulfate - large molecule- not sure if it is well absorbed. not as well as glucosamine. good absorption by enteral pinocytosis(?). positive feedback on PG synthesis experimentally. it' a repeating disaccharide. it's safe. won't hurt to try. data is sort of marginal about whether or not it does anything useful. where do you go with arthritis from here? people are looking at new ways to interrupt degradative pathways. not many good ways to increase synthesis. also, the surgical problem of dealing with lost cartilage - how to resurface a joint. people are looking at gene therapy too - we propose using gene transfer - add in a gene to crank up production of a product short term - for joint resurfacing, fracture healing, whatever. this may become commonplace during our careers. ----end----