Extracellular Matrix

Functions of Extracellular Matrix

The extracellular matrix (ECM) is a collection of proteins, carbohydrates and fluid that perform a variety of functions in tissues. First, the ECM provides structural support to cells by allowing cells to attach to a common substrate. By connecting to the ECM, cells become integrated into a structural unit that has more robust mechanical properties than if cells were only attached to each other.

In this image nuclei are stained in blue and the ECM is stained in green. Note how the ECM forms a network that surrounds the cells.

ECM also regulates different activities within cells in two different ways. First, the mechanical properties of ECM appear to influence the behavior of cells. An ECM that is stiffer or more resistant to tension elicits different responses in cells compared to an ECM that is more deformable. For example, a stiff ECM can induce morphological changes in cells, trigger cell division or differentiation, and alter gene expression.

Cells sense the stiffness of the ECM through contacts they make with components of the ECM. These contacts often aggregate in structures called focal adhesions. Focal adhesions associate with actin filaments within the cytosol of cells.

Cells can generate tension on focal adhesions through actin and myosin filaments. Bipolar myosin filaments crosslink actin filaments that are attached to different focal adhesions. Focal adhesions are linked to the ECM via a set of integral membrane proteins in the cell membrane called integrins. When the myosin filaments are active, they pull on the actin filaments which in turn pull on the focal adhesions. If the focal adhesions are attached to an ECM that is stiff, the cell will be able to generate more tension in its actin and myosin filaments as the components in the ECM will resist the tension generated by actin and myosin. The higher tension initiates a signaling pathway which changes cell behavior. If attached to a less stiff ECM, then the cell will not be able to generate as much internal tension and the signaling pathways will not be activated.

A second way that ECM controls cell behavior is by controlling the availability of signaling molecules. Hormones and other signaling molecules must diffuse through ECM to reach receptors on the surfaces of cells. Some of the components of the ECM bind to signaling molecules which slows their diffuse and in some cases, prevents them from binding to receptors on the surfaces of cells. Thus, ECM can regulate the concentration of a signaling molecule that reaches cells and determine which cells respond to that signaling molecule.

Components

There are three classes of protein components in extracellular matrix that determine its mechanical properties: collagen fibers, elastic fibers and glycoproteins. In general, these molecules either resist tensile and stretching forces or compressing forces. Collagen is the main component that resist tension. Elastin also resist tension but behaves similar to rubber in that it can be stretched and will recoil after the force is removed. On the other side are glycosaminoglycans that resist compressive forces.

Collagen

Collagen is the most abundant class of proteins and pound for pound some are as strong as steel. There are several different types of collagens and their locations within the body varies. Most collagens, about 80-90% of total collagen, form fibers that provide the most mechanical strength. Aggregation and lateral interactions between the individual fibers increase the mechanical strength.

There are over twenty different types of collagen but the most abundant and medically significant are types I - IV. Types I - III all form a structure called a fibril which gives them greater mechanical strength and ability to resist tension. We’ll discuss these collagens in more detail when we cover connective tissue. Type IV forms a branched network which allows it to form a sheet-like structure. We’ll discuss type IV collagen in more detail when we cover epithelia.

All collagens regardless of type are composed of three polypeptide chains. Each of these polypeptides can be over 1000 amino acids giving the trimer an overall length of 300 nm and a width of 1.5 nm. A collagen trimer can be composed of polypeptides encoded by different genes (Type I and IV) or polypeptides from the same gene (Type II and III). The polypeptides form alpha-helices that wrap around each other to form coiled coil interactions. The sequence for most of the length of a collagen polypeptide is a repeat of 3 amino acids: glycine and usually proline and lysine. Glycine which is the smallest amino acid allows for tight packing of the polypeptides in the trimer. Note the extensive lateral interactions that give the structure its mechanical strength.

Many different types of cells can synthesize collagen but one of the most prominent in extracellular matrix is the fibroblast shown here in an electron micrograph.

The synthesis of collagen polypeptides in fibroblasts involves several key steps that allow them to assembly into collagen fibrils. Because collagen is a secreted protein, it is synthesized on ER-bound ribosomes and translocated across the ER membrane during translation. One important feature of collagen is that it contains extra sequence at its N and C-termini called prodomains. These prevent collagen trimers from assembling into fibrils inside the cell which would be catastrophic for the cell.

Collagen undergoes two important modifications in the ER. First, certain prolines and lysines are hydroxylated. These modifications will allow for assembly into trimers and covalent crosslinks between collagen trimers outside the cell. Second, disulfide bonds between collagen polypeptides mediates their assembly into trimers by facilitating interaction between correct collagen proteins.

Once secreted, the prodomain are removed from the trimer by proteases that reside outside fibroblasts to produce the mature collagen trimer. Collagen trimers then self-assemble into fibrils through an entropy-driven process (does not require input of energy).

The interactions between collagen trimers allow them to assemble into fibrils but the interactions are insufficient to account for collagen’s ability to resist tension. Covalent crosslinks between adjacent trimers generates stronger interaction between trimers. Lysyl oxidase catalyzes a reaction that generates covalent bonds between hydroxlysines in adjacent trimers. Mutations that affect hydroxylation of lysines generate weaker collagen.

Some collagen fibrils (type I) then aggregate into large bundles called fibers. Note in the image below collagen fibers are shown in cross-section (green circle) and longitudinally (green arrow). In cross-section each circle is a collagen fibril and the collection of fibrils composes a fiber.

This gives type I collagen three levels of lateral interactions:

• Trimer - assembles inside fibroblasts.
• Fibrils - aggregation of trimers outside of fibroblasts and crosslinked by lysyl oxidase..
• Fibers - aggregation of fibrils.

The extensive interactions are in part what make type I so effective at resisting tension.

The proper assembly of collagen is dependent upon two enzymes that act immediately after collagen is synthesized in the ER. Proline and lysyl hydroxylases convert proline and lysine into hydroxylated versions. Hydroxylated proline facilitates packing of individual collagen polypeptides into a trimer and hydroxylated lysine is required to generate covalent attachments between trimers.. Both enzymes require vitamin C as a cofactor and people who don’t consume enough vitamin C will produce collagen that lacks hydroxylated lysines and prolines. Because these trimers cannot be crosslinked, the collagen fibrils will be weaker leading to tissues that are more prone to damage.

Although often considered a disease of the 17th and 18th century that affected sailors, scurvy can still be seen in patients today, especially in the homeless population. This population often has poor nutrition due to inconsistent sources of food and will rarely have access to fresh fruits and vegetables, leading to deficiencies in vitamin C.

Elastic Fibers

Next, we will examine elastic fibers that are often found enmeshed with collagen fibers as shown in this cross section of an artery. The elastic fibers stain dark blue and appear as a wavy line whereas the collagen stains light blue. Elastic fibers have different mechanical properties from collagen. They allow for stretching of tissues under external force, but generate a recoil force when the external force is removed. Elastic fibers are prominent in the walls of arteries especially the aorta. The elastic fibers stretch to allow the aorta to accommodate a large volume of blood during systole. When the pressure drops during diastole, the fibers recoil pushing blood into the circulatory system. Because of elastic fibers a constant blood pressure is maintained throughout the circulatory system, even though the pumping of the heart delivers blood in a pulsatile fashion.

Elastic fibers are a composite material composed of two primary components: elastin and fibrillin. Both are synthesized by fibroblasts and other cells and are secreted into the surrounding tissue where they assemble into elastic fibers. Fibrillin fibers are thin and arranged in more or less parallel arrays. They are required for correct assembly of elastic fibers. Elastin appears as a amorphous substance. Elastin is what gives elastic fibers its characteristic mechanical properties of stretching and recoiling.

Elastin is the main structural component of elastic fibers. Compared to collagen, elastin is largely unstructured which gives it that amorphous appearance in electron micrographs. Elastin has a hydrophobic domain which mostly lacks structure and an alpha-helical domain. Lysyl oxidase generates covalent attachments between lysines in the alpha-helical domain of adjacent elastin polypeptides.

The unstructured nature of elastin accounts for its ability to stretch and recoil. Relaxed fibers largely unstructured and disordered. The hydrophobic domains cluster to avoid water. Tension stretches elastin generating order in hydrophobic domains. When tension is removed, elastin returns to its lower energy state of disordered fibers, causing the elastin network to recoil. This is another example of entropy having a role in the structure of a component of the ECM. The relaxation of elastic fibers does not require input of energy but is due to the transition from high energy state to low energy state.

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Glycosaminoglycans

Extracellular matrix must not only resist tensile or pulling forces but they must also resist compression. The primary component in ECM that responds to compression are a family of molecules called glycosaminoglycans (GAGs). GAGs resist compression by occupying a large volume and retaining water within that space. This is similar to how a plastic bottle filled with water resists compression. An air-filled bottle collapses under applied force because the force expels the air from the bottle. In contrast, a bottle filled with water that is sealed so that it retains water, resists compression from an outside force.

Glycosaminoglycans (GAGs) are the main component that retains water to resist compression. The base component of GAGs is a disaccharide of two different sugars. These disaccharides are joined into polymers that can contain 1000s of disaccharides. The sugars that compose the disaccharides differ between GAGs, but a feature they share is a negative charge on one of the sugars. Thus, GAGs are long, negatively charged polymers. The negative strong charge in GAGs attracts sodium which through osmosis attracts and retains large amounts of water within GAGs.

Proteoglycans

GAGs come in two different types. Proteoglycans are a single polypeptide that contains a few or many GAGs attached as side chains. Addition of GAGs to the protein occurs in the secretory pathway. The disaccharide side-chains are linked to proteins in the ER and Golgi. Proteoglycans have proteins of different lengths and differ in the number and type of GAGs that are attached. Fibroblasts and other cells secrete proteoglycans through the secretory pathway.

Hyaluronan is another glycosaminoglycan but is unique because it is not attached to a proteins but exists as a free polysaccharide. Hyaluronan can contain up to 25000 repeats of a disaccharide and can reach a length of 20 µm, the size of an average cell. Hyaluronan lacks the structure of most proteins and contains many regions of that form random, flexible coils. Remember that the sugars in hyaluronan are negatively charged and repel each other. That generates a lot of space within the hyaluronan and allows it to occupy an incredibly large volume. In addition, hyaluronan like other GAGs bind and retains water which enables it to resist compression.

Digestions of Extracellular Matrix

Like many structures in the body, the components of the extracellular matrix must be replaced over time due to damage to the proteins that compose the ECM. Specific enzymes called matrix metalloproteinases (MMPs) digest the proteins in the ECM, and fibroblasts can synthesize new protein fibers to replace the digested components.

Digestion of the ECM also plays an important role in certain biological processes including during development and immune responses. Some immune cells synthesize both secreted and membrane-bound MMPs to digest the protein components in the ECM.

One reason that immune cells produce MMPs is to release factors that attract other immune cells to sites of infection or damage. Many immune cells travel to infection or damage through the circulatory system and rely on signals from those regions to guide them. Recall that components of the ECM can bind key signaling molecules. Digestion of the ECM releases those signaling molecules which serve as a chemoattractant to immune cells, guiding them to the site of infection or damage.

Fibroblasts limit the extent of digestion of ECM by secreting proteins called tissue inhibitors of MMPs (TIMPs). TIMPs inhibit the activity to MMPs to ensure that the enzymes don’t degrade too much of the ECM which impair the structure and function of cells in the tissue or organ. A balance between the amount of matrix metalloproteinases and inhibitors of matrix metalloproteinases is essential to maintain the integrity of the ECM while facilitating necessary biological process. Disease can arise when the balance between MMP

ECM and Tissue Engineering

Our understanding of the roles the extracellular matrix plays in nurturing cells has not merely been an academic exercise but we have applied what we’ve learned to developing methods to grow human-compatible tissues in vitro that can be transplanted into patients.

As diagrammed here, we can grow skin in vitro by isolating two key cells from a patient. Fibroblasts are isolated and cultured to produce an extracellular matrix. Next, keratinocytes which are the cell that make up the bulk of skin are isolated and added to the ECM. Overtime the keratinocytes and ECM develop into what is called a human skin equivalent which structurally and functionally resembles normal human skin. Human skin equivalents can be transplanted into patients whose skin has been damaged from environmental causes or genetic mutations. Because the cells used to make the human skin equivalent came from the patient, there is a lower risk of the patient’s immune system rejecting the transplant.

Our knowledge of the ECM has also allowed us to grow mini tissues and organs in vitro that resemble structurally and functionally our internal organs. The process involves isolating and growing stem cells. The stems cells are induced to differentiate and then injected into a three-dimensional, ECM structure. After culturing for a few days, the cells develop into an organoid. Analyzing the organoid shows that it has developed a structure similar to an internal organ, in this case, small intestine. Note the nuclei in blue define a tube with a central lumen that is similar to normal small intestine. The cells also express a key protein, cadherin, that mediates adhesion between cells. These organoids are not ready for transplant into humans but are useful for studying diseases processes in vitro and the effect of drugs.