Healing and Repair

Extracellular Matrix and Cell-Matrix Interactions
Tissue repair and regeneration depend not only on the activity of soluble factors, but also on interactions between cells and the components of the extracellular matrix (ECM).
The ECM regulates the growth, proliferation, movement, and differentiation of the cells living within it.
It is constantly remodeling, and its synthesis and degradation accompanies morphogenesis, regeneration, wound healing, chronic fibrotic processes, tumor invasion, and metastasis.
The ECM sequesters water, providing turgor to soft tissues, and minerals that give rigidity to bone, but it does much more than just fill the spaces around cells to maintain tissue structure.
Functions of ECM include:
1. Mechanical support for cell anchorage and cell migration, and maintenance of cell polarity.
2. Control of cell growth. ECM components can regulate cell proliferation by signaling through cellular receptors of the integrin family.
3. Maintenance of cell differentiation. The type of ECM proteins can affect the degree of differentiation of the cells in the tissue, also acting largely via cell surface integrins.
4. Scaffolding for tissue renewal.
The maintenance of normal tissue structure requires a basement membrane or stromal scaffold. The integrity of the basement membrane or the stroma of the parenchymal cells is critical for the organized regeneration of tissues. It is particularly noteworthy that although labile and stable cells are capable of regeneration, injury to these tissues results in restitution of the normal structure only if the ECM is not damaged. Disruption of these structures leads to collagen deposition and scar formation.
5. Establishment of tissue microenvironments. Basement membrane acts as a boundary between epithelium and underlying connective tissue and also forms part of the filtration apparatus in the kidney.
6. Storage and presentation of regulatory molecules. For example, growth factors like FGF and HGF are secreted and stored in the ECM in some tissues. This allows the rapid deployment of growth factors after local injury, or during regeneration.
The ECM is composed of three groups of macromolecules:
1. Fibrous structural proteins, such as collagens and elastins that provide tensile strength and recoil.
2. Adhesive glycoproteins that connect the matrix elements to one another and to cells.
3. Proteoglycans and hyaluronan that provide resilience and lubrication.

These molecules assemble to form two basic forms of ECM: interstitial matrix and basement membranes. The interstitial matrix is found in spaces between epithelial, endothelial, and smooth muscle cells, as well as in connective tissue. It consists mostly of fibrillar and nonfibrillar collagen, elastin, fibronectin, proteoglycans, and hyaluronan. Basement membranes are closely associated with cell surfaces, and consist of nonfibrillar collagen (mostly type IV), laminin, heparin sulfate, and proteoglycans.



The main components of the ECM.

COLLAGEN
Collagen is the most common protein in the animal world, providing the extracellular framework for all multicellular organisms.
Currently, 27 different types of collagens encoded by 41 genes dispersed on at least 14 chromosomes are known.
Each collagen is composed of three chains that form a trimer in the shape of a triple helix.
The polypeptide is characterized by a repeating sequence in which glycine is in every third position (Gly-X-Y, in which X and Y can be any amino acid other than cysteine or tryptophan), and it contains the specialized amino acids 4-hydroxyproline and hydroxylysine. Prolyl residues in the Y-position are characteristically hydroxylated to produce hydroxyproline, which serves to stabilize the triple helix.
Types I, II, III and V, and XI are the fibrillar collagens, in which the triple-helical domain is uninterrupted for more than 1000 residues; these proteins are found in extracellular fibrillar structures.
Type IV collagens have long but interrupted triple-helical domains and form sheets instead of fibrils; they are the main components of the basement membrane, together with laminin.
Another collagen with a long interrupted triple-helical domain (type VII) forms the anchoring fibrils between some epithelial and mesenchymal structures, such as epidermis and dermis. Still other collagens are transmembrane and may also help to anchor epidermal and dermal structures.
The messenger RNAs transcribed from fibrillar collagen genes are translated into pre-pro-? chains that assemble in a type-specific manner into trimers.
Hydroxylation of proline and lysine residues and lysine glycosylation occur during translation.
Three chains of a particular collagen type assemble to form the triple helix.
Procollagen is secreted from the cell and cleaved by proteases to form the basic unit of the fibrils. Collagen fibril formation is associated with the oxidation of lysine and hydroxylysine residues by the extracellular enzyme lysyl oxidase. This results in cross-linking between the chains of adjacent molecules, which stabilizes the array, and is a major contributor to the tensile strength of collagen. Vitamin C is required for the hydroxylation of procollagen, a requirement that explains the inadequate wound healing in scurvy.
Genetic defects in collagen production cause many inherited syndromes, including various forms of the Ehlers-Danlos syndrome and osteogenesis imperfect.
ELASTIN, FIBRILLIN, AND ELASTIC FIBERS
Tissues such as blood vessels, skin, uterus, and lung require elasticity for their function. Proteins of the collagen family provide tensile strength, but the ability of these tissues to expand and recoil (compliance) depends on the elastic fibers. These fibers can stretch and then return to their original size after release of the tension.
Morphologically, elastic fibers consist of a central core made of elastin, surrounded by a peripheral network of microfibrils.
Substantial amounts of elastin are found in the walls of large blood vessels, such as the aorta, and in the uterus, skin, and ligaments.
The peripheral microfibrillar network that surrounds the core consists largely of fibrillin, a 350-kD secreted glycoprotein, which associates either with itself or with other components of the ECM. The microfibrils serve, in part, as scaffolding for deposition of elastin and the assembly of elastic fibers. They also influence the availability of active TGF? in the ECM. As already mentioned, inherited defects in fibrillin result in formation of abnormal elastic fibers in Marfan syndrome, manifested by changes in the cardiovascular system (aortic dissection) and the skeleton.
CELL ADHESION PROTEINS
Most adhesion proteins, also called CAMs (cell adhesion molecules), can be classified into four main families:
1. Immunoglobulin family CAMs.
2. Cadherins.
3. Integrins
4. Selectins.
These proteins function as transmembrane receptors but are sometimes stored in the cytoplasm. As receptors, CAMs can bind to similar or different molecules in other cells, providing for interaction between the same cells (homotypic interaction) or different cell types (heterotypic interaction).
Selectins play an important role in leukocyte/endothelial cells interactions. Integrins bind to ECM proteins such as fibronectin, laminin, and osteopontin providing a connection between cells and ECM, and also to adhesive proteins in other cells, establishing cell-to-cell contact. Fibronectin is a large protein that binds to many molecules, such as collagen, fibrin, proteoglycans, and cell surface receptors.
It consists of two glycoprotein chains, held together by disulfide bonds. Fibronectin messenger RNA has two splice forms, giving rise to tissue fibronectin and plasma fibronectin.
The plasma form binds to fibrin, helping to stabilize the blood clot that fills the gaps created by wounds, and serves as a substratum for ECM deposition and formation of the provisional matrix during wound healing. Laminin is the most abundant glycoprotein in the basement membrane and has binding domains for both ECM and cell surface receptors. In the basement membrane, polymers of laminin and collagen type IV form tightly bound networks. Laminin can also mediate the attachment of cells to connective tissue substrates.
Cadherins and integrins link the cell surface with the cytoskeleton through binding to actin and intermediate filaments. These linkages, particularly for the integrins, provide a mechanism for the transmission of mechanical force and the activation of intracellular signal transduction pathways that respond to these forces. Ligand binding to integrins causes clustering of the receptors in the cell membrane and formation of focal adhesion complexes. The cytoskeletal proteins that co-localize with integrins at the cell focal adhesion complex include talin, vinculin, and paxillin. The integrin-cytoskeleton complexes function as activated receptors and trigger a number of signal transduction pathways, including the MAP kinase, PKC, and PI3K pathways, which are also activated by growth factors. Not only is there a functional overlap between integrin and growth factor receptors, but integrins and growth factor receptors interact (“cross-talk”) to transmit environmental signals to the cell that regulate proliferation, apoptosis, and differentiation.
The name cadherin is derived from the term “calcium-dependent adherence protein.” This family contains almost 90 members, which participate in interactions between cells of the same type. These interactions connect the plasma membrane of adjacent cells, forming two types of cell junctions called:
(1) zonula adherens, small, spotlike junctions located near the apical surface of epithelial cells.
(2) desmosomes, stronger and more extensive junctions, present in epithelial and muscle cells.
Migration of keratinocytes in the re-epithelialization of skin wounds is dependent on the formation of dermosomal junctions. Linkage of cadherins with the cytoskeleton occurs through two classes of catenins. ?-catenin links cadherins with ?-catenin, which, in turn, connects to actin, thus completing the connection with the cytoskeleton. Cell-to-cell interactions mediated by cadherins and catenins play a major role in regulating cell motility, proliferation, and differentiation and account for the inhibition of cell proliferation that occurs when cultured normal cells contact each other (“contact inhibition”). Diminished function of E-cadherin contributes to certain forms of breast and gastric cancer. As already mentioned, free ?-catenin acts independently of cadherins in the Wnt signaling pathway, which participates in stem cell homeostasis and regeneration. Mutation and altered expression of the Wnt/?-catenin pathway is implicated in cancer development, particularly in gastrointestinal and liver cancers.
In addition to the main families of adhesive proteins, some other secreted adhesion molecules are mentioned because of their potential role in disease processes:
(1) SPARC (secreted protein acidic and rich in cysteine), also known as osteonectin, contributes to tissue remodeling in response to injury and functions as an angiogenesis inhibitor.
(2) The thrombospondins, a family of large multifunctional proteins, some of which, similar to SPARC, also inhibit angiogenesis.
(3) Osteopontin (OPN) is a glycoprotein that regulates calcification is a mediator of leukocyte migration involved in inflammation, vascular remodeling, and fibrosis in various organs.
(4) the tenascin family, which consist of large multimeric proteins involved in morphogenesis and cell adhesion.
GLYCOSAMINOGLYCANS (GAGS) AND PROTEOGLYCANS
GAGs make up the third type of component in the ECM, besides the fibrous structural proteins and cell adhesion proteins.
GAGs consist of long repeating polymers of specific disaccharides. With the exception of hyaluronan , GAGs are linked to a core protein, forming molecules called proteoglycans. Proteoglycans are remarkable in their diversity. At most sites, ECM may contain several different core proteins, each containing different GAGs. Proteoglycans were originally described as ground substances or mucopolysaccharides, whose main function was to organize the ECM, but it is now recognized that these molecules have diverse roles in regulating connective tissue structure and permeability. Proteoglycans can be integral membrane proteins and, through their binding to other proteins and the activation of growth factors and chemokines, act as modulators of inflammation, immune responses, and cell growth and differentiation.
There are four structurally distinct families of GAGs: heparan sulfate, chondroitin/dermatan sulfate, keratan sulfate, and hyaluronan (HA).
The first three of these families are synthesized and assembled in the Golgi apparatus and rough endoplasmic reticulum as proteoglycans. By contrast, HA is produced at the plasma membrane by enzymes called hyaluronan synthases and is not linked to a protein backbone.
HA is a polysaccharide of the GAG family found in the ECM of many tissues and is abundant in heart valves, skin and skeletal tissues, synovial fluid, the vitreous of the eye, and the umbilical cord.
It is a huge molecule that consists of many repeats of a simple disaccharide stretched end-to-end. It binds a large amount of water (about 1000-fold its own weight), forming a viscous hydrated gel that gives connective tissue the ability to resist compression forces.
HA helps provide resilience and lubrication to many types of connective tissue, notably for the cartilage in joints.
Its concentration increases in inflammatory diseases such as rheumatoid arthritis, scleroderma, psoriasis, and osteoarthritis.
Enzymes called hyaluronidases fragment HA into lower molecular weight molecules (LMW HA) that have different functions than the parent molecule. LMW HA produced by endothelial cells binds to the CD44 receptor on leukocytes, promoting the recruitment of leukocytes to the sites of inflammation.
In addition, LMW HA molecules stimulate the production of inflammatory cytokines and chemokines by white cells recruited to the sites of injury. The leukocyte recruitment process and the production of pro-inflammatory molecules by LMW HA are strictly regulated processes; these activities are beneficial if short-lived, but their persistence may lead to prolonged inflammation.