Extracellular matrix

Glycosaminoglycans (GAGs) are carbohydrate polymers and mostly attached to extracellular matrix proteins to form proteoglycans (hyaluronic acid is a notable exception; see below). Proteoglycans have a net negative charge that attracts positively charged sodium ions (Na⁺), which attracts water molecules via osmosis, keeping the ECM and resident cells hydrated. Proteoglycans may also help to trap and store growth factors within the ECM.

Described below are the different types of proteoglycan found within the extracellular matrix.

Heparan sulfate (HS) is a linear polysaccharide found in all animal tissues. It occurs as a proteoglycan (PG) in which two or three HS chains are attached in close proximity to cell surface or ECM proteins.[12][13] It is in this form that HS binds to a variety of protein ligands and regulates a wide variety of biological activities, including developmental processes, angiogenesis, blood coagulation, and tumour metastasis.

In the extracellular matrix, especially basement membranes, the multi-domain proteins perlecan, agrin, and collagen XVIII are the main proteins to which heparan sulfate is attached.

Chondroitin sulfates contribute to the tensile strength of cartilage, tendons, ligaments, and walls of the aorta. They have also been known to affect neuroplasticity.[14]

Keratan sulfates have a variable sulfate content and, unlike many other GAGs, do not contain uronic acid. They are present in the cornea, cartilage, bones, and the horns of animals.

Hyaluronic acid (or "hyaluronan") is a polysaccharide consisting of alternating residues of D-glucuronic acid and N-acetylglucosamine, and unlike other GAGs, is not found as a proteoglycan. Hyaluronic acid in the extracellular space confers upon tissues the ability to resist compression by providing a counteracting turgor (swelling) force by absorbing significant amounts of water. Hyaluronic acid is thus found in abundance in the ECM of load-bearing joints. It is also a chief component of the interstitial gel. Hyaluronic acid is found on the inner surface of the cell membrane and is translocated out of the cell during biosynthesis.[15]

Hyaluronic acid acts as an environmental cue that regulates cell behavior during embryonic development, healing processes, inflammation, and tumor development. It interacts with a specific transmembrane receptor, CD44.[16]

Collagens are the most abundant protein in the ECM. In fact, collagen is the most abundant protein in the human body[17][18] and accounts for 90% of bone matrix protein content.[19] Collagens are present in the ECM as fibrillar proteins and give structural support to resident cells. Collagen is exocytosed in precursor form (procollagen), which is then cleaved by procollagen proteases to allow extracellular assembly. Disorders such as Ehlers Danlos Syndrome, osteogenesis imperfecta, and epidermolysis bullosa are linked with genetic defects in collagen-encoding genes.[11] The collagen can be divided into several families according to the types of structure they form:

1. Fibrillar (Type I, II, III, V, XI)
2. Facit (Type IX, XII, XIV)
3. Short chain (Type VIII, X)
4. Basement membrane (Type IV)
5. Other (Type VI, VII, XIII)

Elastins, in contrast to collagens, give elasticity to tissues, allowing them to stretch when needed and then return to their original state. This is useful in blood vessels, the lungs, in skin, and the ligamentum nuchae, and these tissues contain high amounts of elastins. Elastins are synthesized by fibroblasts and smooth muscle cells. Elastins are highly insoluble, and tropoelastins are secreted inside a chaperone molecule, which releases the precursor molecule upon contact with a fiber of mature elastin. Tropoelastins are then deaminated to become incorporated into the elastin strand. Disorders such as cutis laxa and Williams syndrome are associated with deficient or absent elastin fibers in the ECM.[11]

In 2016, Huleihel et al., reported the presence of DNA, RNA, and Matrix-bound nanovesicles (MBVs) within ECM bioscaffolds.[20] MBVs shape and size were found to be consistent with previously described exosomes. MBVs cargo includes different protein molecules, lipids, DNA, fragments, and miRNAs. Similar to ECM bioscaffolds, MBVs can modify the activation state of macrophages and alter different cellular properties such as; proliferation, migration and cell cycle. MBVs are now believed to be an integral and functional key component of ECM bioscaffolds.

Fibronectins are glycoproteins that connect cells with collagen fibers in the ECM, allowing cells to move through the ECM. Fibronectins bind collagen and cell-surface integrins, causing a reorganization of the cell's cytoskeleton to facilitate cell movement. Fibronectins are secreted by cells in an unfolded, inactive form. Binding to integrins unfolds fibronectin molecules, allowing them to form dimers so that they can function properly. Fibronectins also help at the site of tissue injury by binding to platelets during blood clotting and facilitating cell movement to the affected area during wound healing.[11]

Laminins are proteins found in the basal laminae of virtually all animals. Rather than forming collagen-like fibers, laminins form networks of web-like structures that resist tensile forces in the basal lamina. They also assist in cell adhesion. Laminins bind other ECM components such as collagens and nidogens.[11]

The ECM can exist in varying degrees of stiffness and elasticity, from soft brain tissues to hard bone tissues. The elasticity of the ECM can differ by several orders of magnitude. This property is primarily dependent on collagen and elastin concentrations,[4] and it has recently been shown to play an influential role in regulating numerous cell functions.

Cells can sense the mechanical properties of their environment by applying forces and measuring the resulting backlash.[21] This plays an important role because it helps regulate many important cellular processes including cellular contraction,[22] cell migration,[23] cell proliferation,[24] differentiation[25] and cell death (apoptosis).[26] Inhibition of nonmuscle myosin II blocks most of these effects,[25][23][22] indicating that they are indeed tied to sensing the mechanical properties of the ECM, which has become a new focus in research during the past decade.

Differing mechanical properties in ECM exert effects on both cell behaviour and gene expression.[27] Although the mechanism by which this is done has not been thoroughly explained, adhesion complexes and the actin-myosin cytoskeleton, whose contractile forces are transmitted through transcellular structures are thought to play key roles in the yet to be discovered molecular pathways.[22]

ECM elasticity can direct cellular differentiation, the process by which a cell changes from one cell type to another. In particular, naive mesenchymal stem cells (MSCs) have been shown to specify lineage and commit to phenotypes with extreme sensitivity to tissue-level elasticity. MSCs placed on soft matrices that mimic brain differentiate into neuron-like cells, showing similar shape, RNAi profiles, cytoskeletal markers, and transcription factor levels. Similarly stiffer matrices that mimic muscle are myogenic, and matrices with stiffnesses that mimic collagenous bone are osteogenic.[25]

Stiffness and elasticity also guide cell migration, this process is called durotaxis. The term was coined by Lo CM and colleagues when they discovered the tendency of single cells to migrate up rigidity gradients (towards more stiff substrates)[23] and has been extensively studied since. The molecular mechanisms behind durotaxis are thought to exist primarily in the focal adhesion, a large protein complex that acts as the primary site of contact between the cell and the ECM.[28] This complex contains many proteins that are essential to durotaxis including structural anchoring proteins (integrins) and signaling proteins (adhesion kinase (FAK), talin, vinculin, paxillin, α-actinin, GTPases etc.) which cause changes in cell shape and actomyosin contractility.[29] These changes are thought to cause cytoskeletal rearrangements in order to facilitate directional migration.

Many cells bind to components of the extracellular matrix. Cell adhesion can occur in two ways; by focal adhesions, connecting the ECM to actin filaments of the cell, and hemidesmosomes, connecting the ECM to intermediate filaments such as keratin. This cell-to-ECM adhesion is regulated by specific cell-surface cellular adhesion molecules (CAM) known as integrins. Integrins are cell-surface proteins that bind cells to ECM structures, such as fibronectin and laminin, and also to integrin proteins on the surface of other cells.

Fibronectins bind to ECM macromolecules and facilitate their binding to transmembrane integrins. The attachment of fibronectin to the extracellular domain initiates intracellular signalling pathways as well as association with the cellular cytoskeleton via a set of adaptor molecules such as actin.[8]