Hyaluronan modulates TRPV1 channel opening, reducing peripheral nociceptor activity and pain.

Hyaluronan (HA) is present in the extracellular matrix of all body tissues, including synovial fluid in joints, in which it behaves as a filter that buffers transmission of mechanical forces to nociceptor nerve endings thereby reducing pain. Using recombinant systems, mouse-cultured dorsal root ganglia (DRG) neurons and in vivo experiments, we found that HA also modulates polymodal transient receptor potential vanilloid subtype 1 (TRPV1) channels. HA diminishes heat, pH and capsaicin (CAP) responses, thus reducing the opening probability of the channel by stabilizing its closed state. Accordingly, in DRG neurons, HA decreases TRPV1-mediated impulse firing and channel sensitization by bradykinin. Moreover, subcutaneous HA injection in mice reduces heat and capsaicin nocifensive responses, whereas the intra-articular injection of HA in rats decreases capsaicin joint nociceptor fibres discharge. Collectively, these results indicate that extracellular HA reduces the excitability of the ubiquitous TRPV1 channel, thereby lowering impulse activity in the peripheral nociceptor endings underlying pain.

Genome integrity, stem cells and hyaluronan.

Faithful preservation of genome integrity is the critical mission of stem cells as well as of germ cells. Reviewed are the following mechanisms involved in protecting DNA in these cells: (a) The efflux machinery that can pump out variety of genotoxins in ATP-dependent manner; (b) the mechanisms maintaining minimal metabolic activity which reduces generation of reactive oxidants, by-products of aerobic respiration; (c) the role of hypoxic niche of stem cells providing a gradient of variable oxygen tension; (d)(e) the presence of hyaluronan (HA) and HA receptors on stem cells and in the niche; (f) the role of role of HA in protecting DNA from oxidative damage; (g) the specific role of HA that may play a role protecting DNA in stem cells; (h) the interactions of HA with sperm cells and oocytes that also may shield their DNA from oxidative damage, and (e) mechanisms by which HA exerts the anti-oxidant activity. While HA has multitude of functions its anti-oxidant capabilities are often overlooked but may be of significance in preservation of integrity of stem and germ cells genome.

Attenuation of the oxidative burst-induced DNA damage in human leukocytes by hyaluronan.

Oxidative burst provides the mechanism for specialized phagocytes, such as granulocytes or monocytes, to kill invading microorganisms through generation of superoxide anions. However, the oxidants generated during the burst damage DNA of the phagocytes and neighboring cells. Human blood leukocytes treated with phorbol myristate acetate (PMA) are considered to represent the experimental model of induction of oxidative burst. We recently reported that DNA damage in PMA-treated leukocytes is assessed by cytometric analysis of the induction of histone H2AX phosphorylation and Ataxia Telangiectasia Mutated (ATM) activation. In the present study we observed that hyaluronic acid (HA) of average molecular weight (MW) 5.4x10(6) and 2x10(6) at 0.1% (w/v) concentration significantly attenuated H2AX phosphorylation and ATM activation induced in leukocytes during oxidative burst. HA also reduced the intracellular level of PMA-induced reactive oxidants as measured by the ability of cells to oxidize 2',7'-dihydro-dichlorofluorescein-diacetate. No such effect was seen with HA of 6x10(4) MW. The data are consistent with earlier observations that HA of high MW protects DNA from oxidative damage induced by endo- or exogenous oxidants. The anti-oxidant effect of HA seen during oxidative burst also explains its anti-inflammatory effect when used to treat arthritic joints.

Hyaluronan as an ophthalmic viscoelastic device.

Hyaluronan solutions known as ophthalmic viscoelastic devices (OVDs) are used in surgical procedures within the eye and on the surface of the eye to prevent dryness and to facilitate wound healing. HA and a variety of derivatives facilitate procedures including vitreoretinal surgery, anterior segment surgery, glaucoma surgery, and corneal transplantation.

Protective effect of hyaluronate on oxidative DNA damage in WI-38 and A549 cells.

Progressive DNA damage in live cells by oxidants is the key factor contributing to cell aging and preconditioning to neoplastic transformation. The strategies to slow aging or prevent cancer rely on protection of DNA from the damage. Since cells reside within intercellular matrix it is of interest to know whether matrix constituents possess properties of modulating oxidative DNA damage. We explored, therefore, the effect of hyaluronate (HA), the ubiquitous component of the matrix, on extent of DNA damage induced by exogenous and endogenously generated oxidants. WI-38 and A549 cells were exposed to 200 microM H2O2 in the absence or presence of HA and induction of histone H2AX phosphorylation and activation of ATM, the reporters of DNA damage, was assessed by multiparameter cytometry. Also explored was effect of HA on constitutive H2AX phosphorylation that reflects DNA damage caused by endogenous oxidants generated during aerobic metabolism. HA of average MW 5.4 million (high MW) and 2 million (medium MW) at 0.1% (w/v) in culture medium totally prevented the H2O2-induced H2AX phosphorylation in both cell types whereas effect of 60,000 average MW (low MW) HA was somewhat less pronounced. Constitutive H2AX phosphorylation in WI-38 cells growing in the presence of 0.1% HA of low MW and medium MW was reduced by about 35 and 30%, respectively; no reduction was observed in A549 cells. The data indicate that HA protected DNA from damage caused by the exogenous oxidant H2O2. In WI-38 fibroblasts, the cells that express the HA-receptor CD44, HA also protected DNA from damage caused by endogenous oxidants. We postulate that expression of CD44 in some cell types such as stem cells may provide the means to internalize HA by endocytosis and one of the functions of the internalized HA may be protection of DNA from oxidants. The mechanism of protective effect of HA may either: i) involve entrapment of iron ions thereby inhibiting the Fenton's reaction that produces secondary oxidative species, and/or: ii) directly scavenging of primary and secondary ROIs, as an antioxidant, resulting in HA degradation. Since no significant degradation of HA upon its exposure in tissue culture medium to H2O2 was detected the scavenging may occur intracellularly.

Hyaluronan conformations on surfaces: effect of surface charge and hydrophobicity.

Extended, relaxed, condensed, and interacting forms of the polysaccharide hyaluronan have been observed by atomic force microscopy (AFM). The types of images obtained depend on the properties of the surfaces used. We have investigated several different surface conditions for HA imaging, including unmodified mica, mica chemically modified with two different kinds of amino-terminated silanes (3-aminopropyltriethoxysilane and N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride), and highly oriented pyrolytic graphite. We found the degree of HA molecular extension or condensation to be variable, and the number of bound chains per unit area was low, for all of the mica-based surfaces. HA was more easily imaged on graphite, a hydrophobic surface. Chains were frequently observed in high degrees of extension, maintained by favorable interaction with the surface after molecular combing. This observation suggests that the HA macromolecule interacts with graphite through hydrophobic patches along its surface. AFM studies of HA behavior on differing surfaces under well-controlled environmental conditions provides useful insight into the variety of conformations and interactions likely to be found under differing physiological conditions.

Extended, relaxed, and condensed conformations of hyaluronan observed by atomic force microscopy.

The conformation of the polysaccharide hyaluronan (HA) has been investigated by tapping mode atomic force microscopy in air. HA deposited on a prehydrated mica surface favored an extended conformation, attributed to molecular combing and inhibition of subsequent chain recoil by adhesion to the structured water layer covering the surface. HA deposited on freshly cleaved mica served as a defect in a partially structured water layer, and favored relaxed, weakly helical, coiled conformations. Intramolecularly condensed forms of HA were also observed, ranging from pearl necklace forms to thick rods. The condensation is attributed to weak adhesion to the mica surface, counterion-mediated attractive electrostatic interactions between polyelectrolytes, and hydration effects. Intermolecular association of both extended and condensed forms of HA was observed to result in the formation of networks and twisted fibers, in which the chain direction is not necessarily parallel to the fiber direction. Whereas the relaxed coil and partially condensed conformations of HA are relevant to the native structure of liquid connective tissues, fully condensed rods may be more relevant for HA tethered to a cell surface or intracellular HA, and fibrous forms may be relevant for HA subjected to shear flow in tight intercellular spaces or in protein-HA complexes.

Viscosupplementation for treatment of osteoarthritis: from initial discovery to current status and results.

Viscosupplementation is a therapeutic modality based on the replacement of synovial fluid or exudates with an elastoviscous hyaluronan solution. The first clinical trials were carried out on race horses with painful osteoarthritis of traumatic origin. In the early 1970s, the clinical trials were extended to painful osteoarthritis in humans. Analgesic effects lasting longer than the residence time of the injected hyaluronan in joints were reported both in horses and humans. The hyaluronan used was a noninflammatory fraction of the molecule with an average molecular weight of 2-3 million at a 1% concentration. The analgesic effect of this elastoviscous hyaluronan solution was demonstrated in behavioral animal pain models. Later it was shown that the elastoviscous properties of hyaluronan solutions are the determining factors in reducing pain-elicited nerve activity in both normal and inflamed cat and rat joints. It also was demonstrated in animal arthritis models that elastoviscous hyaluronan solutions promote the healing of traumatic intra-articular wounds. From the mid-1980s, several hyaluronan preparations of greatly varying average molecular weight but with the same concentration were introduced as viscosupplementation-based therapeutic agents. The elastoviscous properties of these solutions varied also, because of the greatly varying average molecular weights (0.5-6.0 million), imitating the rheological properties of either healthy or pathological synovial fluid. Currently, viscosupplementation products available worldwide vary greatly in their elastoviscous properties, and their dosage is not standardized in terms of frequency of injections required or in regard to the removal of exudates before injection. The question of which patient at what stage of the disease responds best with long-lasting pain relief to the many therapeutic products marketed with greatly varying elastoviscous properties has not yet been answered. At the same time, viscosupplementation was introduced, the same highly elastoviscous hyaluronan solutions also were applied in ophthalmic surgery as viscosurgical tools to protect sensitive tissues in the eye during surgery and to be used as soft instruments for tissue manipulation. Modified hyaluronan products (gels) also were introduced for augmentation of the intercellular matrix in tissues (viscoaugmentation) and for separating tissues to prevent adhesions and excessive scar formation (viscoseparation). Hyaluronan and its derivatives (gels) also have been used for drug delivery. The therapeutic use of highly elastoviscous solutions and gels of hyaluronan and its derivatives to build intercellular matrices for supplementation, regeneration, and developing new tissues introduced the concept of matrix engineering into medical practice.

Effects of different molecular weight elastoviscous hyaluronan solutions on articular nociceptive afferents.

OBJECTIVE: To compare 3 different hyaluronan (HA) preparations used as therapeutic agents for osteoarthritis pain in humans in order to establish the degree to which a single application affects the sensitivity of nociceptors in both the normal and the acutely inflamed rat joint. METHODS: In anesthetized rats, single-unit recordings were performed from the medial articular nerve of the right knee joint under normal conditions and during an acute experimental arthritis. Fifty fine afferent units (conduction velocities 0.8-15.3 meters/second) responded to passive movements of the knee joint. They were exposed to a torque meter-controlled, standardized stimulus protocol consisting of innocuous and noxious inward and outward rotations of the joint. This stimulus protocol of 50 seconds' duration was repeated every 5 minutes for 2-3 hours. Three commercially available HA preparations and a buffer solution, the solvent of these preparations, were injected intraarticularly after discharges resulting from 6 stimulus protocols were averaged and used as controls. RESULTS: Both in normal and in inflamed joints, the injection of Hyalgan did not reduce nerve impulse frequency of the evoked discharges. The injections of Orthovisc had no effect in normal joints, but produced a transient frequency reduction of the evoked discharge in inflamed joints. Synvisc significantly reduced (by an average of 50%) the impulse discharge in both normal and inflamed joints 50 minutes after injection, and this level of impulse discharge continued until the end of the recording period (120-130 minutes after injection). The buffer, which had elastoviscous properties substantially different from those of Hyalgan, Orthovisc, and Synvisc, had no such effect. CONCLUSION: We conclude that the elastoviscous properties of HA solutions are determining factors in reducing pain-eliciting nerve activity both in normal and in inflamed rat joints.

Analgesic effect of elastoviscous hyaluronan solutions and the treatment of arthritic pain.

Elastoviscous hyaluronan solutions have an analgesic effect when injected intra-articularly in animal and human joints. This was first discovered using animal behavioral models and later confirmed in neurophysiological studies in cat and rat joints. These studies on both normal and experimentally produced arthritis in joints confirmed that only elastoviscous solutions of hyaluronan or certain of its derivatives (hylans) have a desensitizing effect on nociceptive sensory receptors. Recently, this desensitizing effect of elastoviscous hyaluronan solutions was also demonstrated on intact or on isolated patches of oocyte cell membranes. Viscosupplementation, the exchange of pathological synovial fluid in arthritic joints with pure elastoviscous solutions of hyaluronan or hylans, is a widely accepted therapeutic modality used to provide long-lasting analgesia in human knee joints. The clinical studies performed on human and animal temporomandibular joints since the mid-1970s are reviewed. These trials used three distinctly different preparations made from hyaluronan of different average molecular weight, polydispersity and, consequently, different elastoviscous properties. These differences are demonstrated and the consequences on the potential efficacy of the preparations are discussed.

In vitro model of "wound healing" analyzed by laser scanning cytometry: accelerated healing of epithelial cell monolayers in the presence of hyaluronate.

BACKGROUND: In vitro models of "wound healing" rely on analysis of confluent cell cultures that are mechanically wounded, e.g., by scratching the cell monolayer. Damage and removal of cells during wounding provides mitogenic signals to the adjacent cells and induces their migration to close the wound. The progress of healing is generally estimated by microscopy or time-lapse cinematography by assessing cell proliferation and/or migration that leads to the wound closure. METHODS: The aim of the present study was to adapt laser scanning cytometry (LSC) to measure cellular changes related to damage and recovery of a monolayer of primary epithelial cells from rat kidneys growing with and without hyaluronate ( approximately 6 x 10(6) average molecular weight). Because x-y coordinates of the cell position on the slide were recorded by LSC, the apoptotic and proliferative changes in individual cells induced by wounding and wound closure could be correlated, by multiparameter analysis, with the cell location with respect to the wound. RESULTS: The initial change, observed as soon as 4 h after scratching and seen among the cells at the wound edge, was the appearance of apoptotic cells, characterized by cell shrinkage, typically condensed chromatin, and activation of caspases, the latter detected by binding of fluorochrome-labeled inhibitor of caspases. Their frequency was reduced to up to sixfold in the presence of hyaluronate. Cell proliferation, measured by frequency of cells incorporating bromodeoxyuridine, also reflected by percentage of cells in S, G(2), and mitosis, was higher in proximity of the wound but was not significantly affected by hyaluronate. However, the monolayer gap closure was accelerated in the presence of hyaluronate. CONCLUSIONS: By offering the means to measure apoptosis and proliferation in relation to the cell position (distance) with respect to the wound in cell monolayer and to relocate them for visual inspection, LSC is uniquely suited to quantitatively analyze in vitro the process of wound healing. Hyaluronate, the ubiquitous component of intercellular matrix, preparations of which are being used in the clinic to suppress inflammatory reactions in tissues and promote healing, accelerated the healing process by protecting cells from apoptosis and stimulating cell migration to close the gap in the cell monolayer.