hyaluronic acid

Hyaluronan (also called hyaluronic acid or hyaluronate) is an anionic, non-sulfated glycosaminoglycan distributed widely throughout connective, epithelial, and neural tissues. It is unique among glycosaminoglycans in that it is unsulfated, forms in the plasma membrane instead of the Golgi and can be very large with its molecular weight often reaching the millions.^[1] One of the chief components of the extracellular matrix, hyaluronan contributes significantly to cell proliferation and migration, and may also be involved in the progression of some malignant tumors. The average 70 kg (154 lbs) person has roughly 15 grams of hyaluronan in their body, one-third of which is turned over (degraded and synthesised) every day.^[2] Hyaluronic acid is also a component of the group A streptococcal extracellular capsule,^[3] and is believed to play a role in virulence.^[4][5]

Contents 1 Functions 2 Structure 3 Biological synthesis 4 Cell receptors for hyaluronan 5 Degradation 6 Role of hyaluronan on wound repair process 7 Role of hyaluronan in cancer metastasis 8 Medical applications 9 Cosmetic applications 10 Equine applications 11 Etymology 12 References 13 External links

Functions

Until the late 1970s, hyaluronan was described as a "goo" molecule, a ubiquitous carbohydrate polymer that is part of the extracellular matrix.^[6] For example, hyaluronan is a major component of the synovial fluid and was found to increase the viscosity of the fluid. Along with lubricin, it is one of the fluid's main lubricating components.

Hyaluronan is an important component of articular cartilage, where it is present as a coat around each cell (chondrocyte). When aggrecan monomers bind to hyaluronan in the presence of link protein, large highly negatively-charged aggregates form. These aggregates imbibe water and are responsible for the resilience of cartilage (its resistance to compression). The molecular weight (size) of hyaluronan in cartilage decreases with age, but the amount increases.^[7]

Hyaluronan is also a major component of skin, where it is involved in tissue repair. When skin is excessively exposed to UVB rays, it becomes inflamed (sunburn) and the cells in the dermis stop producing as much hyaluronan, and increase the rate of its degradation. Hyaluronan degradation products also accumulate in the skin after UV exposure.^[8]

While it is abundant in extracellular matrices, hyaluronan also contributes to tissue hydrodynamics, movement and proliferation of cells, and participates in a number of cell surface receptor interactions, notably those including its primary receptors, CD44 and RHAMM. Upregulation of CD44 itself is widely accepted as a marker of cell activation in lymphocytes. Hyaluronan's contribution to tumor growth may be due to its interaction with CD44. Receptor CD44 participates in cell adhesion interactions required by tumor cells.

Although hyaluronan binds to receptor CD44, there is evidence that hyaluronan degradation products transduce their inflammatory signal through Toll-like receptor 2 (TLR2), TLR4 or both TLR2, and TLR4 in macrophages and dendritic cells. TLR and hyaluronan play a role in innate immunity.

High concentrations of hyaluronan in the brains of young rats, and reduced concentrations in the brains of adult rats suggest that hyaluronan plays an important role in brain development.^[9]

Structure

The chemical structure of hyaluronan was determined in the 1950s in the laboratory of Karl Meyer^{[citation needed]}. Hyaluronan is a polymer of disaccharides, themselves composed of D-glucuronic acid and D-N-acetylglucosamine, linked together via alternating β-1,4 and β-1,3 glycosidic bonds. Hyaluronan can be 25,000 disaccharide repeats in length. Polymers of hyaluronan can range in size from 5,000 to 20,000,000 Da in vivo. The average molecular weight in human synovial fluid is 3−4 million Da, and hyaluronan purified from human umbilical cord is 3,140,000 Da.^[10]

Hyaluronan is energetically stable in part because of the stereochemistry of its component disaccharides. Bulky groups on each sugar molecule are in sterically favored positions, whereas the smaller hydrogens assume the less-favorable axial positions.

Biological synthesis

Hyaluronan is synthesized by a class of integral membrane proteins called hyaluronan synthases, of which vertebrates have three types: HAS1, HAS2, and HAS3. These enzymes lengthen hyaluronan by repeatedly adding glucuronic acid and N-acetylglucosamine to the nascent polysaccharide as it is extruded via ABC-transporter through the cell membrane into the extracellular space ^[11].

Hyaluronan synthesis (HAS) has been shown to be inhibited by 4-Methylumbelliferone (hymecromone, heparvit), a 7-Hydroxy-4-methylcoumarin derivative.^[12] This selective inhibition (without inhibiting other Glycosaminoglycans) may prove useful in preventing metastasis of malignant tumor cells.^[13]

Cell receptors for hyaluronan

So far, cell receptors that have been identified for HA fall into three main groups: CD44, RHAMM (Receptor for HA Mediated Motility) and ICAM-1 (Intracellular adhesion molecule-1). CD44 and ICAM-1 were already known as cell adhesion molecules with other recognized ligands before their HA binding was discovered.^[14]

CD44 is widely distributed through out the body, and the formal demonstration of HA-CD44 binding was proposed by Aruffo et al. ^[15] in 1990. To date it is recognized as the main cell surface receptor for HA. CD44 mediates cell interaction with HA and the binging of the two functions as an important part in various physiologic events,^[16][17] such as cell aggregation, migration, proliferation and activation; cell-cell and cell-substrate adhesion; endocytosis of HA which leads to HA catabolism in macrophages; and assembly of petircellular matrices from HA and proteoglycan. Two significant roles of CD44 in skin were proposed by Kaya et al.^[18] The first one is regulation of keratinocyte proliferation in response to extracellular stimuli, and the second one is the maintenance of local HA homeostasis.^[19]

ICAM-1 is mainly known as a metabolic cell surface receptor for HA, and this protein may mainly responsible for the clearance of HA from lymph and blood plasma, which accounts for perhaps most of its whole-body turnover ^[20][21] Ligand binding of this receptor thus triggers a highly co-ordinated cascade of events that includes the formation of an endocytotic vesicle, its fusion with primary lysosomes, enzymatic digestion to monosccharides, active transmembrane transport of these sugars to cell sap, phosphorylation of GlcNAc and enzymatic deacetylation.^[22][23][24] Like its name, ICAM-1 may also serve as a cell adhesion molecule and the binding of HA to ICAM-1 may contribute to the control of ICAM-1-mediated inflammatory activation.^[25]

Degradation

Hyaluronan is degraded by a family of enzymes called hyaluronidases. In humans, there are at least seven types of hyaluronidase-like enzymes, several of which are tumor suppressors. The degradation products of hyaluronan, the oligosaccharides and very low-molecular-weight hyaluronan, exhibit pro-angiogenic properties. In addition, recent studies showed that hyaluronan fragments, not the native high-molecular mass of hyaluronan, can induce inflammatory responses in macrophages and dendritic cells in tissue injury and in skin transplant rejection.

Role of hyaluronan on wound repair process

Skin provides a mechanical barrier to the external environment and acts to prevent the ingress of infectious agents.^[26] Once injured the beneath tissues are exposed to infection therefore rapid and effective healing is of crucial significance to re-construct a barrier function. Skin wound healing is a complex process includes many interacting processes initiated by haemostasis and the release of platelet derived factors.^[27] Then the following stages are inflammation, granulation tissue formation, reepithelization and remodeling. HA is likely to play a multifaceted role in mediation of these cellular and matrix events. The proposed roles of HA in this sequence of skin wound healing events are elucidated in details below.

Inflammation

Many biological factors, such as growth factors, cytokines and eicosanoids and so on, are generated in the inflammation process. These factors are necessary for the subsequent steps of wound healing due to their roles in promoting migration of inflammatory cells, fibroblasts and endothelial cells into the wound site.^[28]

The wound tissue in the early inflammatory phase of wound repair is abounding with HA, probably a reflection of increased synthesis.^[29] HA acts as a promoter of early inflammation which is crucial in the whole skin wound healing process. In a murine air pouch model of carrageenan/IL-1-induced inflammation, HA was observed to enhance cellular infiltration.^[30][31] Kobayashi and colleagues ^[32][33] showed a dose-dependent increase of the proinflammatory cytokines TNF-α and IL-8 production by human uterine fibroblasts at HA concentrations of 10μg/ml to 1 mg/ml via a CD44 mediated mechanism. Endothelial cells, in response to inflammatory cytokines such as TNF-α, and bacterial lipopolysaccharide, also synthesize HA and this has been shown to facilitate primary adhesion of cytokine-activated lymphocytes expressing the HA-binding variants of CD44 under laminar and static flow conditions.^[34][35] It is interesting that HA has contradictory duo-functions in the inflammatory process. It not only can promote the inflammation as stated above, while it also can moderate the inflammatory response, which may contribute to the stabilization of granulation tissue matrix, as described in the following part.

Granulation and organization of the granulation tissue matrix

Granulation tissue is the perfused, fibrous connective tissue that replaces a fibrin clot in healing wounds. It typically grows from the base of a wound and is able to fill wounds of almost any size it heals.^[36] HA is abundant in granulation tissue matrix. A variety of cell functions that are essential for tissue repair may attribute to this HA-rich network. These functions include facilitation of cell migration into the provisional wound matrix, cell proliferation and organization of the granulation tissue matrix.^[37] Absolutely, initiation of inflammation is extremely crucial for the formation of granulation tissue, therefore the pro-inflammatory role of HA as discussed above also contribute to this stage of wound healing.^[38]

HA and cell migration

Cell migration is essential for the formation of granulation tissue.^[39] The early stage of granulation tissue is dominated by a HA-rich extracellular matrix which is regarded as a conductive environment for migration of cells into this temporary wound matrix. Contributions of HA to cell migration may attribute to its physicochemical properties as stated above, as well as its direct interactions with cells. For the former scenario, HA provides an open hydrated matrix that facilitates cell migration;^[40] whereas in the latter scenario, directed migration and control of the cell locomotory mechanisms are mediated via the specific cell interaction between HA and cell surface HA receptors. As discussed before, the three principal cell surface receptors for HA are CD44, RHAMM and ICAM-1. RHAMM is more related to cell migration. Basically, it forms links with several protein kinases associated with cell locomotion, for example, extracellular signal-regulated protein kinase (ERK), p125fak, and pp60c-src.^[41][42][43] During fetal development, the migration path through which neural crest cells migrate is rich in HA.^[44] HA is closely associated with the cell migration process in granulation tissue matrix, and studies show that cell movement can be inhibited, at least partially, by HA degradation or blocking HA receptor occupancy.^[45]

By providing the dynamic force to the cell, HA synthesis has also been shown to associate with cell migration.^[46] Basically, HA is synthesized at the plasma membrane and released directly into the extracellular environment.^[47] This may contribute to the hydrated microenvironment at sites of synthesis, and is essential for cell migration by facilitating cell detachment.

Role of HA in moderation of the inflammatory response

Although inflammation is an integral part of granulation tissue formation, for normal tissue repair to proceed, inflammation needs to be moderated. The initial granulation tissue formed is highly inflammatory with a high rate of tissue turnover mediated by matrix degrading enzymes and reactive oxygen metabolites that are products of inflammatory cells.^[48] Stabilization of granulation tissue matrix can be achieved by moderating inflammation. HA functions as an important moderator in this moderation process, which contradicts to its role in inflammatory stimulation as described above. HA can protect against free-radical damage to cells.^[49] This may attribute to its free-radical scavenging property, a physicochemical characteristic shared by large polyionic polymers. In a rat model of free-radical scavenging property investigated by Foschi D. and colleagues, HA has been shown to reduce damage to the granulation tissue.^[50]

In addition to the free-radical scavenging role, HA may also function in the negative feedback loop of inflammatory activation through its specific biological interactions with the biological constituents of inflammation.^[51] TNF-α, an important cytokine generated in inflammation, stimulates the expression of TSG-6 (TNF-stimulated gene 6) in fibroblasts and inflammatory cells. TSG-6, a HA-binding protein, also forms a stable complex with the serum proteinase inhibitor IαI (Inter-α-inhibitor) with a synergistic effect on the latter’s plasmin-inhibitory activity. Plasmin is involved in activation of the proteolytic cascade of matrix metalloproteinases and other proteinases leading to inflammatory tissue damage. Therefore, the action of TSG-6/ IαI complex, which may be additionally organized by binding to HA in the extracellular matrix, may serve as a potent negative feedback loop to moderate inflammation and stabilize the granulation tissue as healing progresses.^[52][53] In the murine air pouch model of carragenan/IL-1 (Interleukin-1β) induced inflammation where HA has been shown to have a proinflammatory property, reduction of inflammation can be achieved by administrating TSG-6, and the result is comparable with systemic dexamethasone treatment.

Reepithelization

HA plays an important role in the normal epidermis. HA also has crucial functions in the reepithelization process due to several of its properties: it serves as an integral part of the extracellular matrix of basal keratinocytes, which are major constitutes of epidermis; its free-radical scavenging function and its role in keratinocyte proliferation and migration.^[54]

In normal skin, HA is found in relative high concentrations in the basal layer of the epidermis where proliferating keratinocytes are found.^[55] CD44 is collocated with HA in the basal layer of epidermis where additionally it has been shown to be preferentially expressed on plasma membrane facing the HA-rich matrix pouches.^[56][57] Maintaining the extracellular space and providing an open as well as hydrated structure for the passage of nutrients are the main functions of HA in epidermis. Tammi R. and other colleagues ^[58] found that HA content increases at the presence of retinoic acid (vitamin A). The proposed effects of retinoic acid that are against skin photo-damage and aging may be correlated at least in part with an increase of skin HA content, giving rise to increase of tissue hydration. It has been suggested that the free-radical scavenging property of HA contributes to protection against solar radiation, supporting the role of CD44 acting as a HA receptor in the epidermis.^[59]

Epidermal HA also functions as a manipulator in the process of keratinocyte proliferation, which is essential in normal epidermal function as well as during reepithelization in tissue repair. In the wound healing process, HA is expressed in the wound margin, in the connective tissue matrix, and collocating with CD44 expression in migrating keratinocytes.^[60][61] Kaya et al. found that suppression of CD44 expression by an epidermis specific antisense transgene resulted in animals with defective HA accumulation in the superficial dermis, accompanied by distinct morphologic alterations of basal keratinocytes and defective keratinocyte proliferation in response to mitogen and growth factors. Decrease in skin elasticity, impaired local inflammatory response and impaired tissue repair were also observed.^[62] There observations are strongly supportive of the important roles HA and CD44 have in skin physiology and tissue repair.^[63]

Fetal wound healing and scarring

Lack of fibrous scarring is the primary feature of fetal wound healing. Even for longer periods, HA content in fetal wounds is still higher than that in adult wounds, which suggests that HA may, at least in part, reduce collagen deposition and therefore leading to reduced scarring.^[64] This suggestion is in agreement with the research of West et al., who showed that in adult and late gestation fetal wound healing, removal of HA results in fibrotic scarring.^[65] Though the exact role of HA in skin scarring is still under investigation, based on all the facts that have been observed, it must be a great contributor to the less fibrous scarring.

Role of hyaluronan in cancer metastasis

Figure 1. The process of cancer metastasis and which HA-associated molecules play a role in the steps. Abbreviations: hyaluronic acid (HA), hyaluronic acid synthase (HAS), hyaluronic acid receptor (HAR), hyaluronidase (HAase/HYAL)

As shown in Figure 1, the various types of molecules that interact with hyaluronan can contribute to many of the stages of cancer metastasis.

Hyaluronan synthases (HAS) play roles in all of the stages of cancer metastasis. By producing anti-adhesive HA, HAS can allow tumor cells to release from the primary tumor mass and if HA associates with receptors such as CD44, the activation of Rho GTPases can promote epithelial-mesenchymal transition (EMT) of the cancer cells. During the processes of intravasation or extravasation, the interaction of HAS produced HA with receptors such as CD44 or RHAMM promote the cell changes that allow for the cancer cells to infiltrate the vascular or lymphatic systems. While traveling in these systems, HA produced by HAS protects the cancer cell from physical damage. Finally, in the formation of a metastatic lesion, HAS produces HA to allow the cancer cell to interact with native cells at the secondary site and to produce a tumor for itself.^[66]

Hyaluronidases (HAase or HYAL) also play many roles in cancer metastasis. By helping to degrade the ECM surrounding the tumor, hyaluronidases help the cancer cell escape from the primary tumor mass and play a major role in intravasation by allowing degradation of the basement membrane of the lymph or blood vessel. Hyaluronidases again play these roles in establishment of a metastatic lesion by helping with extravasation and clearing the ECM of the secondary site.^[67] Finally, hyaluronidases play a key role in the process of angiogenesis. HA fragments promote angiogenesis and hyaluronidases produce these fragments.^[68] Interestingly, hypoxia also increases production of HA and activity of hyaluronidases.^[69]

The hyaluronan receptors, CD44 and RHAMM, are most thoroughly studied in terms of their roles in cancer metastasis. Increased clinical CD44 expression has been positively correlated to metastasis in a number of tumor types.^[70] Mechanistically, CD44 affects adhesion of cancer cells to each other and to endothelial cells, rearranges the cytoskeleton through the Rho GTPases, and increases the activity of ECM degrading enzymes.^[71] Increased RHAMM expression has also been clinically correlated with cancer metastasis. Mechanistically, RHAMM promotes cancer cell motility through a number of pathways including focal adhesion kinase (FAK), Map Kinase (MAPK), pp60 (c-src), and the downstream targets of Rho Kinase (ROK).^[72] RHAMM can also cooperate with CD44 to promote angiogenesis towards the metastatic lesion.^[73]

Medical applications

Hyaluronan is naturally found in many tissues of the body, such as skin, cartilage, and the vitreous humour. It is therefore well suited to biomedical applications targeting these tissues. The first hyaluronan biomedical product, Healon, was developed in the 1970s and 1980s by Pharmacia, and is approved for use in eye surgery (i.e., corneal transplantation, cataract surgery, glaucoma surgery and surgery to repair retinal detachment). Other biomedical companies also produce brands of hyaluronan for ophthalmic surgery.^[74][75][76]

Hyaluronan is also used to treat osteoarthritis of the knee.^[77] Such treatments, called viscosupplementation, are administered as a course of injections into the knee joint and are believed to supplement the viscosity of the joint fluid, thereby lubricating the joint, cushioning the joint, and producing an analgesic effect. It has also been suggested that hyaluronan has positive biochemical effects on cartilage cells. However, some placebo controlled studies have cast doubt on the efficacy of hyaluronan injections, and hyaluronan is recommended primarily as a last alternative to surgery.^[78][79] Oral use of hyaluronan has been lately suggested, although its effectiveness needs to be demonstrated. At present, there are some preliminary clinical studies that suggest that oral administration of Hyaluronan has a positive effect on osteoarthritis, but it remains to be seen if there is any real benefit to the treatment.

Dry, scaly skin (xerosis) such as that caused by atopic dermatitis (eczema) may be treated with a prescription skin lotion containing sodium hyaluronate as its active ingredient.^[80]

Due to its high biocompatibility and its common presence in the extracellular matrix of tissues, hyaluronan is gaining popularity as a biomaterial scaffold in tissue engineering research.^[81] In particular, a number of research groups have found that hyaluronan's properties for tissue engineering and Regenerative medicine are significantly improved with crosslinking, producing a hydrogel. This added feature allows a researcher to form a desired shape as well as to deliver therapeutic molecules into a host.^[82] Hyaluronan can be crosslinked by attaching thiols (trade names: Extracel, HyStem),^[83] methacrylates,^[84] and tyramines (trade name: Corgel).^[85] Hyaluronan can also be crosslinked directly with formaldehyde (trade name: Hylan-A) or with divinyl sulfone (trade name: Hylan-B).^[86]

In some cancers, hyaluronan levels correlate well with malignancy and poor prognosis. Hyaluronan is thus often used as a tumor marker for prostate and breast cancer. It may also be used to monitor the progression of the disease.

Hyaluronan may also be used postoperatively to induce tissue healing, notably after cataract surgery.^[87] Current models of wound healing propose that larger polymers of hyaluronic acid appear in the early stages of healing to physically make room for white blood cells, which mediate the immune response.

Hyaluronan has also been used in the synthesis of biological scaffolds for wound healing applications. These scaffolds typically have proteins such as fibronectin attached to the hyaluronan to facilitate cell migration into the wound. This is particularly important for individuals with diabetes who suffer from chronic wounds.^[88]

In 2007, the EMEA extended its approval of Hylan GF-20 as a treatment for ankle and shoulder osteoarthritis pain.^[89]

Cosmetic applications

Hyaluronan is a common ingredient in skin care products.

In 2003 the FDA approved hyaluronan injections for filling soft tissue defects such as facial wrinkles. Restylane is a common trade name for the product. Hyaluronan injections temporarily smooth wrinkles by adding volume under the skin, with effects typically lasting for six months.

Juvederm is non-animal-sourced Hyaluronic acid injectable filler, similar to Restylane but differing slightly in terms of effect and longevity. It is used for lip augmentation, reduction of folds and wrinkles as well as removal of scars. The effects of Juvederm treatments are also temporary, and costs are similar to that of Restylane.^[90]

Equine applications

Hyaluronan is used in treatment of articular disorders in horses, particularly those in competition or heavy work. It is indicated for carpal and fetlock joint dysfunctions, but not when joint sepsis or fracture are suspected. It is especially used for synovitis associated with equine osteoarthritis. It can be injected directly into an affected joint, or intravenously for less localized disorders. It may cause mild heating of the joint if directly injected, but this does not affect the clinical outcome. Intra-articularly administered medicine is fully metabolized in less than a week.^[91]

Note that according to Canadian regulation, hyaluronan in HY-50 preparation should not be administered to animals to be slaughtered for horse meat.^[92] However in Europe, the same preparation is not considered to have any such effect, and edibility of the horse meat is not affected.^[93]

Etymology

Hyaluronic acid is derived from hyalos (Greek for vitreous) and uronic acid because it was first isolated from the vitreous humour and possesses a high uronic acid content.

The term hyaluronate refers to the conjugate base of hyaluronic acid. Because the molecule typically exists in vivo in its polyanionic form, it is most commonly referred to as hyaluronan.

References

External links

• ATC codes: D03AX05, M09AX01, R01AX09, S01KA01
• MeSH Hyaluronan

v • d • e Polysaccharides: glycosaminoglycans Unsulfated, extracellular Hyaluronan Sulfated, extracellular Chondroitin (Chondroitin sulfate, Dermatan sulfate) · Heparan sulfate · Keratan sulfate Sulfated, intracellular Heparin see also enzymes, disorders

v • d • e Preparations for treatment of wounds and ulcers (D03) Cicatrizants epichlorohydrin: Cadexomer iodine · Dextranomer · Crilanomer B5: Dexpanthenol · Calcium pantothenate nitrate: Glyceryl trinitrate · Isosorbide dinitrate other: Becaplermin · Hyaluronic acid · Enoxolone Proteolytic enzymes Trypsin · Clostridiopeptidase

v • d • e Decongestants and other nasal preparations (R01) Topical Sympathomimetics, plain Cyclopentamine • Ephedrine • Phenylephrine • Oxymetazoline • Tetryzoline • Xylometazoline • Naphazoline • Tramazoline • Metizoline • Tuaminoheptane • Fenoxazoline • Tymazoline • Epinephrine Antiallergic agents, excluding corticosteroids Spaglumic acid histamine antagonists (Levocabastine, Antazoline, Thonzylamine) mast cell stabilizer (some are also antihistamines) (Cromoglicic acid, Nedocromil, Azelastine, Olopatadine) Corticosteroids Beclometasone • Prednisolone • Dexamethasone • Flunisolide • Budesonide • Betamethasone • Tixocortol • Fluticasone • Mometasone furoate • Triamcinolone • Ciclesonide Other nasal preparations Calcium hexamine thiocyanate • Retinol • Ipratropium bromide • Ritiometan • Mupirocin • Hexamidine • Framycetin • Hyaluronic acid • Eucalyptus oil Systemic use: Sympathomimetics Phenylpropanolamine • Pseudoephedrine • Phenylephrine • Phenylpropylamine