ECM for Ligament & Tendon (and other soft tissues) Collagen, Elastin, MMPs, TIMPs BME 615

Ligaments and tendons are similar - but not the same tissues

Extracellular matrix components contributing to ligament composition, organization, and function ComponentFunction Collagen 1Most abundant protein; dominantly contributes to stiffness and strength of tissue; forms fibrils and fibers; highly organized parallel fibers Collagen 3Fiber forming protein; forms smaller diameter fibers that are less organized. Associated with healing and damage Elastin Forms fibers, but a minor component and doesn ’ t contribute much to structural stiffness or strength BiglycanSLRP contributes to thick fibrilogenesis and organization of fibrils DecorinSLRP contributes to thin fibrilogenesis and organization of fibrils. Decorates outside of fibril to stop appositional growth. FibronectinContributes to cell adhesion and matrix organization TenascinContributes to cell migration, Contributes to enzyme expression MMP-2Gelatinase responsible for degradation of collagen matrix TIMP-1Inhibitor for MMP enzyme activity TGF  1 Regulates release of ECM proteins, promotes cell proliferation and migration

Ligament/Tendon Composition Amiel, et al. “Ligament Structure, Chemistry, and Physiology” in Knee Ligaments: Structure, Function, Injury, and Repair, Daniel, et al., eds. Raven Press, New York

Collagen Collagen is the duct tape of the natural world. Adapted from Sir David Attenborough

Collagen Tropocollagen or "collagen molecule" is a subunit of larger collagen aggregates such as fibrils. It is approximately 300 nm long and 1.5 nm in diameter, made up of 3 polypeptide strands called alpha chains. Each chain possesses the conformation of a left-handed helix. These 3 left-handed helices twist together into a right-handed triple helix. Tropocollagen forms in a "super helix“ that aggregates as a quaternary structure stabilized by numerous hydrogen bonds. With type I collagen and possibly all fibrillar collagens, each triple-helix associates into a right-handed super-super-helix referred to as a microfibril. Each microfibril interdigitates with neighboring microfibrils so they are well ordered and crystalline.

Fibrillogenesis  Collagen I formation Most collagen forms in a similar manner, but the following process is typical for type I:  Inside the cell Two types of peptide chains are formed during translation on ribosomes along the RER:  -1 and  -2 chains. These peptide chains (known as preprocollagen) have registration peptides (telopeptides) on each end and a signal peptide. Polypeptide chains are released into the lumen of the RER. Signal peptides are cleaved inside the RER and the chains are now known as pro-alpha chains. Hydroxylation of lysine and proline occurs. This process is dependent on ascorbic acid (Vitamin C) as cofactor. Glycosylation of specific hydroxylysine residues occurs. Triple helical structure is formed inside the endoplasmic reticulum from each two  -1 chains and one  -2 chain. Procollagen is shipped to the golgi apparatus, where it is packaged and secreted by exocytosis.  Outside the cell Registration peptides are cleaved and tropocollagen is formed by procollagen peptidase. Multiple tropocollagen molecules form collagen fibrils, via covalent cross-linking by lysyl oxidase which links hydroxylysine and lysine residues. Multiple collagen fibrils form into collagen fibers. Collagen may be attached to cell membranes via several types of proteins, including fibronectin or integrins.

Fibrillogenesis & Collagen Hierarchy Amiel, et al. “Ligament Structure, Chemistry, and Physiology” in Knee Ligaments: Structure, Function, Injury, and Repair, Daniel, et al., eds. Raven Press, New York

Fibrillogenesis & Collagen Hierarchy Amiel, et al. “Ligament Structure, Chemistry, and Physiology” in Knee Ligaments: Structure, Function, Injury, and Repair, Daniel, et al., eds. Raven Press, New York

Fibrillogenesis Electrostatic alignment produces quarter stagger Hydrogen bonds Nimni M, ed. Collagen Vol 1, CRC Press, Boca Raton, FL 1988

Fibrillogenesis Nimni M, ed. Collagen Vol 1, CRC Press, Boca Raton, FL 1988

Periodicity of Collagen Fibril Nimni M, ed. Collagen Vol 1, CRC Press, Boca Raton, FL 1988

Crosslink Formation Nimni M, ed. Collagen Vol 1, CRC Press, Boca Raton, FL 1988

Collagen Fibril Microstructure Amiel, et al. “Ligament Structure, Chemistry, and Physiology” in Knee Ligaments: Structure, Function, Injury, and Repair, Daniel, et al., eds. Raven Press, New York

Collagen Types Amiel, et al. “Ligament Structure, Chemistry, and Physiology” in Knee Ligaments: Structure, Function, Injury, and Repair, Daniel, et al., eds. Raven Press, New York

Composition Nimni M, ed. Collagen Vol 1, CRC Press, Boca Raton, FL 1988

Ligament & Tendon Histology Amiel, et al. “Ligament Structure, Chemistry, and Physiology” in Knee Ligaments: Structure, Function, Injury, and Repair, Daniel, et al., eds. Raven Press, New York

Collagen Fibrillogenesis Enzymatic Stages in Collagen Maturation Amiel, et al. “Ligament Structure, Chemistry, and Physiology” in Knee Ligaments: Structure, Function, Injury, and Repair, Daniel, et al., eds. Raven Press, New York

Fiber & Fibril Stretching Nimni M, ed. Collagen Vol 1, CRC Press, Boca Raton, FL 1988

Collagen’s Amino Acids  A distinctive feature of collagen is the regular arrangement of amino acids in each of the three chains of these collagen subunits.amino acids  The sequence often follows the pattern Gly-Pro-X or Gly-X-Hyp, where X may be any of various other amino acid residues.GlyProHyp  Pro or Hyp constitute about 1/6 of the total sequence. With Gly accounting for the 1/3 of the sequence, this means that approximately half of the collagen sequence is not Gly, Pro, or Hyp.  Due to its key role in the determination of cell phenotype, cell adhesion, tissue regulation and infrastructure, many sections of its non-proline rich regions have cell or matrix association / regulation roles.  The relatively high content of Pro and Hyp rings, with their geometrically constrained carboxyl and (secondary) amino groups, along with the rich abundance of Gly, accounts for the tendency of the individual polypeptide strands to form left-handed helices spontaneously.carboxylamino  Because Gly is the smallest amino acid with no side chain, it plays a unique role in fibrous structural proteins. In collagen, Gly is required at every third position because the assembly of the triple helix puts this residue at the interior (axis) of the helix, where there is no space for a larger side group than glycine’s single hydrogen atom.  For the same reason, the rings of the Pro and Hyp must point outward. These two amino acids help stabilize the triple helix.

Collagen Pathologies  Vitamin C deficiency causes scurvy, a disease in which defective collagen prevents the formation of strong connective tissue.  Gums deteriorate and bleed, with loss of teeth  skin discolors  wounds do not heal  Prior to the eighteenth century, scurvy was notorious among long duration military, particularly naval, expeditions without foods containing Vitamin C.  An autoimmune disease such as lupus erythematosus or rheumatoid arthritis may attack healthy collagen fibers.  Many bacteria and viruses have virulence factors which destroy collagen or interfere with its production.  Latherism inhibits collagen cross-linking  Genetic defects prevent proper formation of collagen molecules (Ehlers-Danlos or Marfan syndromes or OI defects)

Collagen Degradation  Type I collagen together with other ECM molecules is degraded in normal remodeling processes, e.g. growth, wound healing, bone turnover, etc.  ECM is degraded during pathological processes, such as arthritis, osteolysis or spreading of tumor cells.  Four major enzyme classes are aspartate, cysteine, serine and matrix metalloproteinases.  Serine and cysteine proteases use their HO- and HS-side chains, aspartate proteases use aspartate residues and metalloproteases use heavy metals, to immobilize and polarize water molecules so the oxygen atom attacks the carbonyl-carbon of an amide bond, cleaving the type I collagen molecule.

Collagen Degradation Nimni M, ed. Collagen Vol 1, CRC Press, Boca Raton, FL 1988

Matrix Metalloproteinases (MMPs)

Function of MMPs  Proteins of the MMP family (at least 28) are involved in the breakdown of ECM in normal physiological processes, such as embryonic development, reproduction, angiogenesis, morphogenesis, and tissue remodeling, as well as in disease processes, such as arthritis, cirrhosis, and metastasis.  Most MMPs are secreted as inactive proproteins which are activated when cleaved by extracellular proteinases.  In addition, mechanical forces may increase or decrease the expression of MMPs in ligament cells depending on the force.

Groups of MMPs Groupings based on the substrate specificity and on cellular localization Collagenases degrade triple-helical fibrillar collagens into distinctive 3/4 and 1/4 fragments. These collagens are the major components of bone, cartilage, etc. and MMPs are the only known mammalian enzymes capable of degrading them. Collagenases are MMPs #1, #8, #13, and #18. In addition, #14 and #2 can cleave fibrillar collagen. Gelatinases degrade type IV collagen and gelatin. These enzymes are distinguished by a gelatin-binding region that forms a separate folding unit. The gelatinases are #2 and #9. Stromelysins display a broad ability to cleave extracellular matrix proteins but are unable to cleave the triple-helical fibrillar collagens. The three canonical members of this group are #3, #10, and #11. Membrane type MMPs (#14, #15, #16, #17, #24, and #25) have a furin cleavage site in the pro-peptide, which is a feature also shared by #11.

 Inflammatory diseases  MMP1, MMP3, and MMP9 are implicated in rheumatoid and osteoarthritis.  Cardiovascular disease  Increased levels of MMPs, especially MMP9, are found at sites of atherosclerosis and aneurysm formation.  Secretion and activation of MMPs by macrophages degrades ECM for atherosclerotic plaque and plaque rupture.  Lung disease  Elevated levels of MMPs are implicated in pathophysiology of various lung diseases, including acute respiratory distress syndrome, asthma, bronchiectasis, and cystic fibrosis.  Central Nervous System disease  MMP9 plays a important role in multiple sclerosis and Guillain-Barre’s syndrome  Chronic wounds and inflammation of the skin and oral cavity  Acute and chronic wounds have high levels of MMP2/9.  MMP9 is implicated in blistering skin diseases and contact hypersensitivity.  MMPs are implicated in periodontal disease and inflammatory bowel diseases. MMPs in Diseases

 Four-member family of MMP inhibitors (TIMP1, 2, 3, & 4)  TIMP concentrations generally far exceed the concentrations of MMPs in tissue and extracellular fluids, thereby limiting their proteolytic activity to focal pericellular sites.  In contrast to its usual inhibitory role, a low concentration of TIMP2 enhances MMP14 induced activation of MMP2.  TIMPs have growth promoting activities independent of their MMP inhibitory function  TIMP3 can have apoptosis-inducing properties.  The transcription of TIMPs is regulated by similar cytokines and growth factors that control MMP expression i.e. TGFß, TNFα, IL-1, IL-6. Tissue Inhibitors of MMPs (TIMPs)

Elastin  A protein in tissue that is highly elastic  Allows many tissues to resume their non-deformed shape after stretching or contracting.  Helps skin return to its original position when it is poked or pinched.  An important load-bearing tissue in the bodies of mammals and used in places where mechanical energy is required to be stored.  Encoded by the ELN gene.  Extremely stable and insoluble, lasts a lifetime, little transcribed in adult.

Elastin Stretching Stretch a network of elastin molecules (green). The molecules are joined by covalent bonds (red) to generate a cross-linked network. Each elastin molecule in the network expands and contracts as a coil, so that the entire assembly can stretch and recoil like a rubber band.

Elastin Composition  Tropoelastin is a specialized protein with a Mol. Wt. of 64 to 66 kDa, and an irregular or random coil conformation made up of 830 amino acids.  Tropoelastin made of amino acids such as glycine (over 30%), valine, alanine, & proline (these 4 make up 75%).  Elastin fiber is made by linking many soluble tropoelastin protein molecules, in a reaction catalyzed by lysyl oxidase, to make an insoluble, durable x-linked fiber. The amino acid responsible for these cross-links is lysine.

Elastin Function Elastin serves an important function in: – Arteries for pressure wave propagation to help blood flow, abundant in large elastic vessels such as the aorta. – Lungs – Elastic ligaments (ligamentum nuchae) – Skin – Bladder – Elastic cartilage (outer ear, larynx, epiglottis) – Intervertebral disc

Elastin in Aortic Wall

Elastin in Lung Tissue

Skin Wound Healing