Pharmacological aspects of hyaluronic acid metabolism: A review

The covalent transfer of heavy chains (HCs) from inter-α-inhibitor (IαI) to hyaluronan (HA) via the protein product of tumor necrosis factor-stimulated gene-6 (TSG-6) forms the HC-HA complex, a pathological form of HA that promotes the adhesion of leukocytes to HA matrices. The transfer of HCs to high molecular weight (HMW) HA is a reversible event whereby TSG-6 can shuffle HCs from one HA molecule to another. Therefore, HMW HA can serve as both an HC acceptor and an HC donor. In the present study, we show that transfer of HCs to low molecular weight HA oligosaccharides is an irreversible event where subsequent shuffling does not occur, i.e. HA oligosaccharides from 8 to 21 monosaccharide units in length can serve as HC acceptors, but are unable to function as HC donors. We show that the HC-HA complex is present in the synovial fluid of mice subjected to systemic and monoarticular mouse models of rheumatoid arthritis. Furthermore, we demonstrate that HA oligosaccharides can be used, with TSG-6, to irreversibly shuffle HCs from pathological, HMW HC-HA to HA oligosaccharides, thereby restoring HC-HA matrices from the inflamed joint to their normal state, unmodified with HCs. This process was also effective for HC-HA in the synovial fluid of human rheumatoid arthritis patients (in vitro).

Hyaluronic acid (HA) is a component of the extracellular matrix that has been shown to play an important role in bone formation, resorption, and mineralization both in vivo and in vitro. We examined the effects of HA at several molecular weights on osteoclast formation and function induced by RANKL (receptor activator of NF-kappa B ligand) in a mouse monocyte cell line (RAW 264.7). HA at M(r) < 8,000 (low molecular weight HA (LMW-HA)) enhanced tartrate-resistant acid phosphatase-positive multinucleated cell formation and tartrate-resistant acid phosphatase activity induced by RANKL in a dose-dependent manner, whereas HA at M(r) > 900,000 (high molecular weight HA (HMW-HA)) showed no effect on osteoclast differentiation. LMW-HA enhanced pit formation induced by RAW 264.7 cells, whereas HMW-HA did not, and LMW-HA stimulated the expression of RANK (receptor activator of NF-kappa B) protein in RAW 264.7 cells. In addition, we found that LMW-HA enhanced the levels of c-Src protein and phosphorylation of ERKs and p38 MAPK in RAW 264.7 cells stimulated with RANKL, whereas the p38 MAPK inhibitor SB203580 inhibited RANKL-induced osteoclast differentiation. This enhancement of c-Src and RANK proteins induced by LMW-HA was inhibited by CD44 function-blocking monoclonal antibody. These results indicate that LMW-HA plays an important role in osteoclast differentiation and function through the interaction of RANKL and RANK.

Lung endothelial permeability is a key pathological feature of acute respiratory distress syndrome. Hyaluronic acid (HA), a major component of the glycocalyx layer on the endothelium, is generated by HA synthase (HAS) during inflammation and injury and is critical for repair. We hypothesized that administration of exogenous high molecular weight (HMW) HA would restore protein permeability across human lung microvascular endothelial cells (HLMVEC) injured by an inflammatory insult via upregulation of HAS by binding to CD44. A transwell coculture system was used to study the effects of HA on protein permeability across HLMVEC injured by cytomix, a mixture of IL‐1β, TNFα, and IFNγ, with or without HMW or low molecular weight (LMW) HA. Coincubation with HMW HA, but not LMW HA, improved protein permeability following injury at 24 h. Fluorescence microscopy demonstrated that exogenous HMW HA partially prevented the increase in “actin stress fiber” formation. HMW HA also increased the synthesis of HAS2 mRNA expression and intracellular HMW HA levels in HLMVEC following injury. Pretreatment with an anti‐CD44 antibody or 4‐methylumbelliferone, a HAS inhibitor, blocked the therapeutic effects. In conclusion, exogenous HMW HA restored protein permeability across HLMVEC injured by an inflammatory insult in part through upregulation of HAS2.

Low, medium, and high molecular weight hyaluronic acid effects on human dental pulp stem cells in vitro

Aims This study compared the six-month medical/pharmacy costs and healthcare resource utilization (HCRU) for knee OA patients undergoing intra-articular therapy with different classes of hyaluronic acid (HA) (by molecular weight) or corticosteroid (ICS). Materials and methods Patients across high molecular weight (HMW) HA, medium molecular weight (MMW) HA, low molecular weight (LMW) HA, and ICS therapy groups were matched from a U.S. claims database (Optum’s de-identified Clinformatics Data Mart Database), with a final size of 6,234 patients per group. Adjusted six-month medical/prescription costs per patient per month (PPPM), and HCRU rates and costs, were determined. Secondary endpoints included complication rates and adjusted costs, new prescription analgesic use, and adjunctive/supplemental intra-articular treatment. Results Mean adjusted PPPM medical costs were highest for LMW HA ($527.14), followed by HMW HA ($469.35) and MMW HA ($441.97) (p < .001), and lowest for the ICS group ($240.26; p < .001). Office visit, arthrocentesis, and subsequent ICS/arthrocentesis rates and corresponding costs, as well as costs for any complications, decreased from LMW HA to MMW HA to HMW HA. The ICS group had greater arthrocentesis, subsequent ICS/arthrocentesis, and office visit costs versus MMW and HMW HA groups. The ICS group had higher rates of new prescription analgesic use (15.8% versus 11.7%–12.2%) and adjunctive ICS (21.8% vs. 9.4%–11.1%) and HA (14.1% versus 1.6%–5.3%) treatment than the HA groups. HMW HA had the lowest rates of adjunctive non-index HA treatment. Limitations Claims data contains limited clinical data and relied on the accuracy of coding of diagnoses and procedures. Conclusions Among HA products, HMW HA may provide greater short-term clinical and economic benefits. Additionally, intra-articular HA therapy may provide improved short-term clinical and economic results over ICS, in terms of lower rates of adjunctive intra-articular treatments, HCRU, and new prescription analgesic use. Complication rates were low reflecting the safety profiles of HA and ICS.

Intra-abdominal infection is the second most common cause of sepsis, and the mortality rate from abdominal sepsis remains high. High molecular weight (HMW) hyaluronic acid (HA) has been studied in sterile injury models as an anti-inflammatory and anti-permeability agent. This study evaluated the therapeutic effects of intraperitoneal HMW HA administration in mice with peritonitis-induced sepsis. Sepsis was induced in C57BL/6 mice by cecal ligation and puncture (CLP), followed 4 h later by an intraperitoneal injection of HMW HA (20 mg/kg) solution or phosphate buffered saline (PBS). Survival, physiological data, organ injury, bacterial burden, and inflammatory cytokine levels were assessed in the CLP mice. To assess the effect of HA on macrophage phagocytosis activity, RAW264.7 cells, primed with lipopolysaccharide, were exposed with either PBS or HMW HA (500 μg/mL) prior to exposure to 10 CFU of E coli bacteria. HMW HA instillation significantly improved blood oxygenation, lung histology, and survival in CLP mice. Inflammatory cytokine levels in the plasma and bacterial burdens in the lung and spleen were significantly decreased by HA administration at 24 h after CLP. At 6 h after CLP, HA significantly decreased bacterial burden in the peritoneal lavage fluid. HMW HA administration significantly increased E coli bacterial phagocytosis by RAW264.7 cells in part through increased phosphorylation of ezrin/radixin/moesin, a known downstream target of CD44 (a HA receptor); ezrin inhibition abolished the enhanced phagocytosis by RAW264.7 cells induced by HA. Intraperitoneal administration of HMW HA had therapeutic effects against CLP-induced sepsis in terms of suppressing inflammation and increasing antimicrobial activity.

Background:intra-articular (IA) injections of hyaluronic acid (HA) preparations are one of conservative treatment of the knee osteoarthritis (OA). However, the results of this treatment are inconsistent.Objectives:to evaluate the effectiveness of...

Aim: to evaluate the effectiveness of hyaluronic acid (HA) preparations with different molecular weights and in combination with chondroitin sulfate (HS) for intra-articular (IA) injections in the treatment of patients with stage I–III knee OA.Subjects and methods. IA HA injections were performed 160 patients with primary and post-traumatic knee OA of the I–III stages at the department of traumatology-orthopedics, V.A. Nasonova Research Institute of Rheumatology for the period from September 2017 to June 2019. Patients were divided into 4 groups. Group 1 consisted of 80 patients treated with low molecular weight (LMW) HA, group 2–20 patients treated with medium molecular weight (MMW) HA, group 3–30 patients treated with high molecular weight (HMW) HA, and group 4–30 patients who were intraarticular introduced HA with HS. The course of IA injections was 2 for LMW, HMW, and HA with HS, and 3 for MMW HA. Injections were performed with an interval of 1 week. To evaluate the results of treatment, we studied the intensity of pain according to VAS and the total score of KOOS before treatment and on follow-up examinations 1, 3 and 6 months after the course of IA HA injections.Results. The maximum reduction in pain with IA HA injections at stage I of knee OA occurred by 3 months after the course of treatment. Moreover, improvement was detected by 1 month in 84.3% of cases, and remained until the end of the study in 71.1% of patients. All HA preparations used in stage I of knee OA were effective. At stage II of the knee OA after 3 months after the course of IA HA, different efficiencies of HA preparations were revealed. So, in the groups of LMW, MMW and HA with HS, the improvement persisted up to 3 months, and in the group of HMW HA – up to 1 month. After 3 months, the best results were shown by HA with HS, by 6 months the results were comparable. IA HA injections at the II stage of knee OA led to good and excellent results 1 month after the course of treatment in 53.9% of cases, but by the end of the study, improvement remained in only 30.8% of patients. In the case of the use of HA in stage III of the knee OA, the effectiveness of the studied drugs was comparable, and the maximum improvement was achieved by 1 month. The positive effect of IA HA injections in patients with stage III of the knee OA one month after the course of treatment was obtained in 40.6% of cases, by 3 months it decreased to 18.8%, and by 6 months – to 15.7% of patients.Conclusions. IA injections of HA at stage I of the knee OA is a highly effective method of conservative treatment, which allows to relieve pain and improve the condition of the knee joint for a period of 6 months or more. The use of HA preparations at stage II of the knee OA allows reducing pain up to 3 months with IA injections of LMW and MMW HA, as well as HA with HS. HMW HA helps reduce pain intensity for a period of 1 month. The use of HA preparations in stage III of the knee OA leads to a short-term relief of symptoms of OA.

The effects of covalently immobilized hyaluronic acid substrates on the adhesion, expansion, and differentiation of embryonic stem cells for in vitro tissue engineering

It is well known that hyaluronic acid and its principal receptor, CD44, are implicated in the regulation of the tissue repair process, but their role in the formation of chronic diabetic ulcers has not been studied. Hyaluronic acid metabolism and CD44 expression are regulated by lactate, where their increased production is considered to affect the properties of fibroblasts in non-insulin-dependent diabetes mellitus. The aim of our work was to investigate the possible role of hyaluronic acid and CD44, and their regulation by lactate, in the abnormal wound healing of diabetes. Fibroblasts were derived from uninjured skin from four non-insulin-dependent diabetic patients with ulcers and four without ulcers; and from four healthy age-matched volunteers. We observed that diabetic fibroblasts of both groups produced more L-lactate ( approximately 30%) and incorporated more (3)H-glucosamine into the medium hyaluronic acid ( approximately 28%) than controls. Fibroblasts of the diabetic group with ulcers, unlike those of the group without ulcers, showed significant increases in the high molecular weight hyaluronic acid accumulation in the pericellular matrix (30.5%, p < 0.01) and CD44 expression (27.0%, p < 0.05). Exogenous L-lactate dose-dependently, and equally for all fibroblasts lines, stimulated the accumulation of medium hyaluronic acid (3.7-fold) and CD44 expression (1.5-fold). However, fibroblasts from diabetic patients with ulcers were more (1.4-fold) sensitive to L-lactate in terms of CD44 expression, and responded to L-lactate by the increased accumulation of high molecular weight hyaluronic acid in the pericellular matrix (32.1%, p < 0.01). We propose that specific properties of fibroblasts from diabetic patients with ulcers may be involved in the increased susceptibility of these patients to chronic ulceration.

A major hurdle for regeneration after spinal cord injury (SCI) is the ability of axons to penetrate and grow through the scar tissue. After SCI, inflammatory cells, astrocytes and meningeal cells all play a role in developing the glial scar. In addition, degradation of native high molecular weight (MW) hyaluronic acid (HA), a component of the extracellular matrix, has been shown to induce activation and proliferation of astrocytes. However, it is not known if the degradation of native HA actually enhances glial scar formation. We hypothesize that the presence of high MW HA (HA with limited degradation) after SCI will decrease glial scarring. Here, we demonstrate that high MW HA decreases cell proliferation and reduces chondroitin sulfate proteoglycan (CSPG) production in cultured neonatal and adult astrocytes. In addition, stiffness-matched high MW HA hydrogels crosslinked to resist degradation were implanted in a rat model of spinal dorsal hemisection injury. The numbers of immune cells (macrophages and microglia) detected at the lesion site in animals with HA hydrogel implants were significantly reduced at acute time points (one, three and ten days post-injury). Lesioned animals with HA implants also exhibited significantly lower CSPG expression at ten days post-injury. At nine weeks post-injury, animals with HA hydrogel implants exhibited a significantly decreased astrocytic response, but did not have significantly altered CSPG expression. Combined, these data suggest that high MW HA, when stabilized against degradation, mitigates astrocyte activation in vitro and in vivo. The presence of HA implants was also associated with a significant decrease in CSPG deposition at ten days after SCI. Therefore, HA-based hydrogel systems hold great potential for minimizing undesired scarring as part of future repair strategies after SCI.

We have examined the effect of exogenous linear chain high molecular weight hyaluronic acid (HMW HA) on endogenously synthesized hyaluronic acid (HA) and associated binding proteins in primary cultures of fibroblast-like stromal cells that were obtained by collagenase digestion of the murine peripatellar fat pad. The cultures were expanded in DMEM that was supplemented with fetal bovine serum and basic fibroblast growth factor (bFGF) then exposed to macrophage-colony-stimulating factor (MCSF) to induce macrophage properties, before activation of inflammatory pathways using E. coli lipopolysaccharide (LPS). Under all culture conditions, a significant amount of endogenously synthesized HA localized in LAMP1-positive lysosomal vesicles. However, this intracellular pool was depleted after the addition of exogenous HMW HA and was accompanied by enhanced proteolytic processing and secretion of de novo synthesized versican, much of which was associated with endosomal compartments. No changes were detected in synthesis, secretion, or proteolytic processing of aggrecan or lubricin (PRG4). The addition of HMW HA also modulated a range of LPS-affected genes in the TLR signaling and phagocytosis pathways, as well as endogenous HA metabolism genes, such as Has1, Hyal1, Hyal2, and Tmem2. However, there was no evidence for association of endogenous or exogenous HMW HA with cell surface CD44, TLR2 or TLR4 protein, suggesting that its physiochemical effects on pericelluar pH and/or ionic strength might be the primary modulators of signal transduction and vesicular trafficking by this cell type. We discuss the implications of these findings in terms of a potential in vivo effect of therapeutically applied HMW HA on the modification of osteoarthritis-related joint pathologies, such as pro-inflammatory and degradative responses of multipotent mesenchymal cells residing in the synovial membrane, the underlying adipose tissue, and the articular cartilage surface.

Hyaluronan (HA) is a naturally occurring non-sulfated glycosaminoglycan (GAG) localized to the cell surface and the tissue extracellular matrix (ECM). It is composed of disaccharides containing glucuronic acid and N-acetylglucosamine, is synthesized by the HA synthase (HAS) enzymes and is degraded by hyaluronidase (HYAL) or reactive oxygen and nitrogen species (ROS/RNS) actions. HA is deposited as a high molecular weight (HMW) polymer and degraded to low molecular weight (LMW) fragments and oligosaccharides. HA affects biological functions by interacting with HA-binding proteins (hyaladherins). HMW HA is anti-inflammatory, immunosuppressive, and antiangiogenic, whereas LMW HA has pro-inflammatory, pro-angiogenetic, and oncogenic effects. ROS/RNS naturally degrade HMW HA, albeit at enhanced levels during tissue injury and inflammatory processes. Thus, the degradation of endothelial glycocalyx HA by increased ROS challenges vascular integrity and can initiate several disease progressions. Conversely, HA exerts a vital role in wound healing through ROS-mediated HA modifications, which affect the innate immune system. The normal turnover of HA protects against matrix rigidification. Insufficient turnover leads to increased tissue rigidity, leading to tissue dysfunction. Both endogenous and exogenous HMW HA have a scavenging capacity against ROS. The interactions of ROS/RNS with HA are more complex than presently perceived and present an important research topic.

MP17-05 MYELOID CELLS IN BLADDER CANCER MICROENVIRONMENT DEGRADE HYALURONIC ACID INTO INFLAMMATORY LOW MOLECULAR WEIGHT HYALURON

ObjectiveCosmetic emulsions containing hyaluronic acid are ubiquitous in the cosmetic industry. However, the addition of (different molecular weight) hyaluronic acid can affect the filament stretching properties of concentrated emulsions. This property is often related to the “stringiness” of an emulsion, which can affect the consumer's choice for a product. It is thus very important to investigate and predict the effect of hyaluronic acid on the filament stretching properties of cosmetic emulsions.MethodsModel emulsions and emulsions with low and high molecular weights are prepared and their filament stretching properties are studied by the use of an extensional rheometer. Two different stretching speeds are employed during the stretching of the emulsions, a low speed at 10 µm/s and a high speed at 10 mm/s. The shear rheology of the samples is measured by rotational rheology.ResultsWe find that filament formation only occurs at high stretching speeds when the emulsion contains high molecular weight hyaluronic acid. The formation of this filament, which happens at intermediate states of the break‐up, coincides with an exponential decay in the break‐up dynamics. The beginning and end of the break‐up of high molecular weight hyaluronic acid emulsions show a power law behaviour, where the exponent depends on the initial stretching rate. At a lower stretching speed, no filament is observed for both high molecular weight and low molecular weight hyaluronic acid emulsions and the model emulsion. The emulsions show a power law behaviour over the whole break‐up range, where the exponent also depends on the stretching rate. No significant difference is observed between the shear flow properties of the emulsions containing different molecular weights hyaluronic acid.ConclusionIn this work, we underline the importance of the molecular weight of hyaluronic acid on the elongational properties of concentrated emulsions. The filament formation properties, for example the stringiness, of an emulsion is a key determinant of a product liking and repeat purchase. Here, we find that high molecular weight hyaluronic acid and a high stretching speed are the control parameters affecting the filament formation of an emulsion.