Extracellular matrix Flashcards
Which celebrities have an ECM disease?
Lena Dunham, Sia, and Jameela Jamil all have Ehlers-Danlos syndrome. This is a group of disorders affecting the joints and skin. The syndrome is rare, but
combined, its forms affect at least 1 in 5,000 across
the world. The syndrome is often overlooked by many who may have it and mistake it for chronic pain
What 3 things make the ECM clinically relevant?
- Tumor microenvironment
- Stem cell therapy
- SynVisc one
Tumor microenvironment
The immediate environment of the tumor. This contributes to the heterogeneity of cells in the tumor, which influences the behavior of the tumor cells and cancer stem cells. Cancer cells also influence the behavior of immune cells and fibroblasts in the microenvironment.
SynVisc One
Synvisc-One is an injection that supplements the fluid in your knee to help lubricate and cushion the joint, and can provide up to six months of osteoarthritis knee pain relief. The injection contains a gel-like mixture made from an ECM protein called hyaluronan that comes from chicken combs. Categorized as a medical device by the FDA.
Extracellular matrix (ECM)
Diverse collection of molecules that are not passive but rather very dynamic
ECM functions
- Cell adhesion
- Cell to cell specificity
- Filter matrix or barrier like the basal lamina- basement membrane
- Promote/maintain cell differentiation. Ex- stem cell differentiation
- Tendon/bone strength
- Charged surface
- Receptors- integrin receptors
ECM and tissue engineering
The ECM is critically important to products as as Epiderm (MatTek corporation). It is a tissue model consisting of normal, human-derived epidermal keratinocytes (NHEK)
What is a challenge to growing human embryonic stem cells?
Mouse “half dead” (irradiated)feeder layers are typically required for growing hESCs. However, this introduces animal proteins and frowned upon by the FDA due to the possibility of viruses, animal proteins. New feeder cell-free plates and media have been developed
Magnetic Levitation Approach (stem cells)
Magnetic cell levitation is a recent method which has been developed, which offers an alternative method for spheroid generation. During magnetic levitation, cells are pre-loaded with magnetic nanoparticles and encouraged to form multicellular spheroids within several hours via an external magnetic field. The resultant spheroids are easily manipulated by virtue of their magnetic qualities and also confer benefits including cell tracking and imaging. This is a method that does not require ECM or a feeder layer.
Type 1 collagen
Easy to isolate. Forms microfibrils and collagen fibrils, which can aggregate into collagen fibers. They connect muscle to bone and therefore have a lot of tensile strength.
Osteogenesis imperfecta
Caused by autosomal dominant mutations in genes encoding type 1 collagen. Mutations in glycine result in unstable alpha helices.
Type 2 collagen
More mesh-like structure. Found in cartilage along with type 9 collagen. Type 2 fibers are smaller in diameter than type 1 fibrils and oriented randomly in a proteoglycan matrix
What is one example for why ECM is critical to stem cells?
Three women suffered severe, permanent eye damage after stem cells
were injected into their eyes, in an unproven treatment at a clinic in Florida. One, 72, went completely blind from the injections, and the others, 78 and 88, lost much of their eyesight. Before the procedure, all had some
visual impairment but could see well enough to drive. Cells were fat derived mesenchymal stem cells (autologous transplant) but the stem cells were in a different niche or ECM environment
US stem cell/Bioheart
A loosely regulated clinic selling unproven stem cell treatments. Several women suffered vision loss after stem cells were injected into their eyes. Another patient had a catastrophic reaction after visiting a South Miami clinic. The 59-year-old
woman felt faint and started vomiting two hours after receiving
injections for arthritis pain. The FDA’s slow response has permitted U.S. Stem Cell to continue operating years after those first reports of blindness. Although the company stopped injecting its fat-derived
treatments into eyes after the patients sued, it continues to sell the therapy to people with spinal injuries, Parkinson’s disease, MS, and other serious chronic conditions
Kristin Comella
Former CSO of US Stem Cell. She stated in the Washington Post about their procedures:
“Of course, we don’t understand the mechanisms of action, I suppose you would have to talk directly to God to get that.” “Dr.” Comella has a PhD from a non-accredited virtual university out of Panama and resigned in 2019.
Age related macular degeneration
Age-related macular degeneration of the eye is
the most common cause of blindness in the elderly. This loss of vision is caused by the death of the photoreceptors (the rods and cones) resulting from the degeneration and
death of the underlying retinal pigment epithelial (RPE) cells
Stem cell therapy to treat age related macular degeneration
One problem has been immune rejection of the
transplanted stem cells. But, scientists in Sweden say they have discovered a way to refine the production of retinal cells from embryonic stem cells
for treating blindness in the elderly. Using
the CRISPR/Cas9 (Nobel Prize 2020) gene
editing, they have also managed to modify
the cells so that they can hide from the immune system to prevent rejection
Classic experiment of cell to cell specificity
Used a living sponge (animal). Cells were stained and dissociated, then the cell reorganized. The stained cell would be in the same place when they reorganized, suggesting that cells could recognize their neighbors and know where they were.
4 families of cell adhesion molecules
- Cadherins
- Immunoglobulin (Ig) superfamily
- Integrins
- Lectins
Cell-adhesion molecules
The interactions between cells of the body are achieved via cell adhesion molecules. They have diverse structures and expression levels depending on where they are found in the body. Adhesion receptors can mediate binding through homophilic interactions (molecules of the same type) or heterophilic interactions.
Cadherins
Characteristic of the adherens junction. E-cadherins mediate cell-cell adhesion by forming homophilic cross bridges with identical E-cadherins on adjacent cells. They can also bind to E-cadherins on the same cell. Ubiquitous in all known substrate dependent cells, require calcium to function. Have a strong cis-trans configuration.
Immunoglobulins
Can form homophilic or heterophilic linkages and can serve as cell-cell and cell-matrix adhesion receptors
Cell junctions
CAMs can be clustered in separate regions on the plasma membrane. Cell junctions and be tight and long lasting or weak and transient.
Homophilic binding
When a CAM on one cell binds directly to the same kind of CAM on an adjacent cell.
Heterophilic binding
A CAM on one cell binds to a different class of CAM
Cis interactions
Also called lateral (in the same cell) interactions. Monomeric CAMs on one cell bind to one or more CAMs on the same cell’s plasma membrane.
Trans interactions
Also called intercellular or adhesive interactions. CAMs on one cell bind to CAMs on an adjacent cell.
E-cadherin and the cytoskeleton
The cadherin adhesion complex bridges neighboring cells and the actin-myosin cytoskeleton, and thereby contributes to mechanical coupling between cells which drives many morphogenetic events and tissue repair.
What ion does E-cadherin require to function?
Calcium. Cadherins depend on calcium for their function: removal of calcium abolishes adhesive activity, renders cadherins vulnerable to proteases and, in E-cadherin, induces a dramatic reversible conformational change in the entire extracellular region.
E-cadherin and MDCK cells
Experiments have been conducted using a green fluorescent protein labeled form of E-cadherin. It has shown that clusters of E-cadherin mediate the initial attachment of cells and the subsequent zippering of cells into sheets.
L cell experiments
Used cultured mouse fibroblasts. L cells don’t have cadherins and adhere poorly to other cells, but when an E-cadherin gene was introduced, the cells preferentially adhered to other cells expressing E-cadherin.
Extracellular matrix
A combination of protein and polysaccharides that is secreted and assembled by cells into a network where the components are bound to each other. It’s often involved in holding cells and tissues together. Components include proteoglycans, glycoproteins, collagen, and soluble matrix proteins
Interstitium
May be a new human organ that is larger than skin. Interstitium consists of all the fluid filled spaces between cells like muscle, skin, etc. that have been regarded as merely fluid filled spaces
Interstitium characteristics (6)
- Layer of interconnected, fluid-filled compartments supported by collagen bundles and elastin.
- Compressible and distensible – shock absorber around organs
- May explain how cancers metastisize
- Source of lymph
- Standard fixation procedures drain away the fluid and superficially
looks like tears in tissue - First seen with a probe-based confocal laser endomicroscope
examining metastasis in a patient’s bile duct
The interstitium is lined by
Lined by very unusual, flat cells – look like fibroblasts but function unknown and have little cytoplasm with an oblong nucleus
Basal lamina
Forms the basement membrane along with anchoring collagen fibrils- a sheet-like mesh of ECM components. It plays a role in regeneration after tissue damage and in embryonic development. For example, the basal lamina forms the BBB in some animals, and neurons migrate along it during development.
Adhesion receptors bind to 3 types of molecules that are abundant in the ECM of all tissues
- Proteoglycans- a group of glycoproteins that cushion cells and bind extracellular and cell surface molecules
- Collagen fibers- structural integrity, strength, resilience
- Multi-adhesive matrix proteins- laminin and fibronectin
4 proteins found in the basal lamina
- Type 4 collagen
- Laminins
- Perlecan
- Nidogen
Connective tissue
Includes tendons and cartilage. It differs from other solid tissues because most of its volume is made up of ECM and insoluble protein fibers. Includes elastin and glycosaminoglycans, but collagen is the most abundant fibrous protein in connective tissue. The ECM components are mostly secreted by fibroblasts
Loose connective tissue contains (3)
Fibroblasts, collagen, and elastin
Skin is made of
Stratified squamous epithelia. The dermis and epidermis are linked by fibrils of type 7 collagen
Epidermolysis bullosa
Loss of function mutations in the gene enchoring type 7 collagen (collagen anchoring dermis and epidermis) results in a debilitating blistering disease- dermis and epidermis can separate as the result of any pressure.
Dense connective tissue
Includes bone and tendon. Dense connective tissue, is one of the types of connective tissue also referred to as dense fibrous tissue due to relative abundance of the collagen fibers. It also contains fewer cells and less ground substance in comparison with the other type, loose connective tissue.
Human cornea layers (3)
Example of highly ordered collagen:
1. Corneal epithelial cells- can regenerate
2. Stroma- made of collagen, strong, optically clear
3. Corneal endothelial cells
Corneal endothelial cells
Have Descener’s membrane. Maintain osmotic balance in the stroma and don’t divide. If disease targets the endothelial layer, you get a corneal cataract
What causes corneal opacities?
Loss of corneal endothelial cells/function
When was the first corneal transplant?
First cornea transplant was in 1905 – one of the first successful transplants of all tissue/organs
Types of corneal transplants (3)
- Penetrating keratoplasty – full thickness cornea in its entirety
- Lamellar keratoplasty – limited to diseased areas only
- Endothelial keratoplasty- Descemet membrane and endothelial cell layer only
Dr. Geoffrey Tabin
Known for the Himalayan Cataract Project
NIH (National Eye Institute) project between CPSI Biotech and
Schepens Eye Research Institute
The two companies are working on hypothermic storage and cryopreservation of corneas.
Bioengineered cornea implants
Only 1 in 70 patients receive a cornea transplant. However, two clinical trials in Iran and India have shown that an
implanted engineered cornea plug using pig collagen directly into the center of the cataractous cornea can restore vision from 0% (totally blind) to 100% (20/20 vision) in many patients. Can be stored up to 2 years unlike a transplanted cornea, which can only be stored for 2 weeks.
Collagen
Collagens are trimeric molecules that are often bundled together into fibers. It is the most abundant fibrous protein in connective tissue. Most collagen consists of fibrillar collagens (type 1-3) which are located in connective tissue. They have a triple helix structure.
Synthesis of fibrillar collagens
Synthesized in the endoplasmic reticulum. The trimers are linked by disulfide bonds, and selected residues in the Gly-X-Y triplet repeats are covalently modified. The modifications facilitate zipper-like formation and stabilization of the alpha helices. They are bound by chaperone protein Hsp47 to stabilize the helices. They are packaged in vesicles and brought to the golgi to be sorted. In the last step, the fibrils can assembles into larger and larger bundles. Some will eventually form tendons
Lysyl oxidase
Enzyme that plays a critical role in the biogenesis of connective tissue matrices by crosslinking the extracellular matrix proteins, collagen and elastin. Therefore, it helps to stabilize the matrix
Scurvy
Scurvy is the name for vitamin C deficiency. It can lead to anemia, exhaustion, spontaneous bleeding, limb pain, swelling in some parts of the body. Vitamin C is a cofactor that is responsible for adding hydroxyl groups to pro-alpha chains in collagen. Without hydroxylation, the chains are unstable and can’t assemble into normal fibrils. Blood vessels, skin, and tendons will become fragile without the support of collagen.
Glycosaminoglycans
Negatively-charged polysaccharide compounds (long and linear polymers). They are composed of repeating disaccharide units that are expressed on the cell surface and in the ECM. Their functions within the body are widespread and determined by their molecular structure. Historically, the function of GAGs was thought to be limited to cell hydration and structural scaffolding. However, evidence now suggests that GAGs play a key role in cell signaling, which serves to modulate a vast amount of biochemical processes
Where are GAGs synthesized?
GAGs naturally occur as a component of proteoglycans. Proteoglycan core proteins are synthesized in the ER, and the GAGs are attached to the core proteins in the Golgi complex
Hyaluronan
A nonsulfated glycosaminoglycan. It is made by a plasma membrane enzyme called HA synthase and is secreted into the extracellular space as it’s synthesized.
Functions of hyaluronan
It’s a major component of the ECM that surrounds migrating and proliferating cells, especially in embryonic tissues. It forms a backbone of proteoglycan aggregates in the ECM, especially cartilage. Therefore, it provides stiffness and resilience as well as a lubricating quality to many connective tissues, like in joints
CD44
An adhesion receptor that contains hyaluronan binding domains. Hyaluronan coating has a loose, porous nature that keeps cells apart from each other, giving them the freedom to move and proliferate.
Juvederm and Restylan
Two different types of ECM fillers that are just two different forms of hyaluronan. The goal is to make patients look younger. Includes Zyderm, which is bovine collagen
Proteoglycans
Glycoproteins that contain one or more covalently bound glycosaminoglycan chains. They are bound to type 1 collagen fibrils and may bind the fibrils together to form thicker collagen fibers in many connective tissues, especially skeletal muscle. Type 2 collagen is randomly oriented in a proteoglycan matrix and is cross linked to the proteoglycans. Proteoglycans play a role in cell-matrix adhesion.
Syndecans
One class of heparan sulfate proteoglycans. Syndecans are integral membrane proteins. They interact with a large number of ligands, such as growth factors, adhesion receptors, soluble small molecules, proteinases, and other extracellular matrix proteins to initiate downstream signaling pathways. They can bind to fibroblast growth factor (FGF) which regulates developmental processes.
Fibronectin
A family of multi-adhesive matrix proteins form their own distinct fibrils in the ECM of most connective tissues. It functions as an adhesion molecule (attaches cells to the ECM) as well as playing a role in migration and differentiation of various cell types in embryogenesis. Also promote blood clotting and immune function in wound healing
How was it discovered that fibrin is an adhesion molecule?
Promoted the discovery that fibronectin functions as an adhesion molecule. It was observed that fibronectin is found on the surface of normal fibroblasts which adhere tightly to petri dishes, but not on cancer cells, which adhere weakly.
Laminin
A multi-adhesive protein in the basal lamina. They are heterotrimeric and are usually large and cross shaped. They interact with the receptors anchored in the plasma membrane of cells adjacent to basement membranes. In doing so laminins regulate multiple cellular activities and signaling pathways.
Integrins
Integrins connect epithelial cells to the basal lamina. They also connect epithelial cells to the cytoskeleton through adapter proteins.
Integrin conformations
- Inactive- low affinity for extracellular ligands- bent closed
- Partially active- intermediate affinity- extended closed
- Fully active- high affinity- extended open
Integrins recognize
RGDs, but not their similar scrambled relatives
Anoikis
Anoikis is the name given to the induction of apoptosis in cells upon loss of attachment to the extracellular matrix (ECM) and neighboring cells. Anoikis plays a fundamental role in preventing inappropriate translocation and attachment of cells, which can lead to abnormal growth in an ectopic environment. Anoikis is largely regulated by interaction with the ECM and nearby cells
Integrins and focal adhesion
Focal adhesions anchor an epithelium to the underlying components of the ECM. Integrins are located within the focal adhesion and connect actin bundles to the ECM.
Extravasation
Movement into tissue by WBCs- they must move from the blood due to infection or injury. It requires successive formation and breakage between leukocytes and endothelial cells lining the blood vessels. Mediated by CAMs called selectins, and the WBCs bind to integrins on the endothelial cells
Leukocyte Adhesion Deficiency Disease
An immunodeficiency disorder involving both B and T cells and is characterized by an inability of leukocytes to migrate to the site of infection to kill offending microbes. Type 1- steady adhesion of leukocyte to endothelial surfaces is defective by mutations in CD18 gene resulting in defective or deficient beta-2 integrin. Symptoms- delayed falling off of the umbilical cord stump, neutrophils are unable to aggregate and counts are always high, frequent bacterial and fungal infections, periodontitis,
Duchenne Muscular Dystrophy
Mutations in ECM genes that encode proteins and their transmembrane receptors can result in diverse SM diseases, a large proportion of which are types of fibrosis and muscular dystrophy. It is a genetic disorder characterized by progressive muscle degeneration and weakness due to the alterations of a protein called dystrophin that helps keep muscle cells intact.
Ehlers-Danlos Syndrome
A group of inherited disorders that affect the collagen in your connective tissues — primarily your skin, joints and blood vessel walls. Symptoms- overly flexible joints, fragile skin, skin that is stretchy, soft, and velvety
Artificial ECM trial
A new injectable artificial ECM reverses paralysis in mice with severed spinal cords with the ability to walk regained in 4 weeks. Currently there is no therapy to trigger spinal cord regeneration but this artificial ECM nanofiber scaffold may be the first. It is a liquid gel that mimics the spinal cord ECM and has “dancing synthetic ligands” that constantly move and can jump off the scaffold so that the moving receptors on the target cells can be better stimulated
Artificial ECM trial results
Severed axons regenerate and more motor neurons survive. Scar tissue is minimized. Myelin forms around the cells, functional blood vessels are formed. Nanofiber artificial ECM disappears after 12 weeks
