Cyclosporin A inhibits PDGF-BB induced hyaluronan synthesis in orbital fibroblasts.

Orbital connective tissue changes are contributors to the pathogenesis in thyroid eye disease (TED). Activated fibroblasts respond to immune stimuli with proliferation and increased hyaluronan (HA) production. Cyclosporin A (CsA) was reported to be beneficial in the treatment of TED. PDGF isoforms are increased in orbital tissue of TED patients and enhance HA production. We aimed to study the effect of CsA on HA production and hyaluronan synthase (HAS1, 2 and 3) and hyaluronidase (HYAL1 and 2) mRNA expressions in orbital fibroblasts (OFs). Measurements were performed in the presence or absence of CsA (10 µM) in unstimulated or PDGF-BB (10 ng/ml) stimulated OFs. The HA production of TED OFs (n = 7) and NON-TED OFs (n = 6) were measured by ELISA. The levels of mRNA expressions were examined using RT-PCR. The proliferation rate and metabolic activity were measured by BrdU incorporation and MTT assays, respectively. Treatment with CsA resulted in an average 42% decrease in HA production of OFs (p < 0.0001). CsA decreased the expression levels of HAS2, HAS3 and HYAL2 (p = 0.005, p = 0.005 and p = 0.002, respectively.) PDGF-BB increased HA production (p < 0.001) and HAS2 expression (p = 0.004). CsA could reduce the PDGF-BB-stimulated HA production (p < 0.001) and HAS2 expression (p = 0.005) below the untreated level. In addition, CsA treatment caused a decrease in proliferation potential (p = 0.002) and metabolic activity (p < 0.0001). These findings point to the fact that CsA affects HA metabolism via HAS2, HAS3 and HYAL2 inhibition in OFs. In addition to its well characterized immunosuppressant properties, CsA's beneficial effect in TED may be related to its direct inhibitory effect on basal and growth factor stimulated HA production.

AßPP-tau-HAS1 axis trigger HAS1-related nuclear speckles and gene transcription in Alzheimer's disease.

As the backbone of the extracellular matrix (ECM) and the perineuronal nets (PNNs), hyaluronic acid (HA) provides binding sites for proteoglycans and other ECM components. Although the pivotal of HA has been recognized in Alzheimer's disease (AD), few studies have addressed the relationship between AD pathology and HA synthases (HASs). Here, HASs in different regions of AD brains were screened in transcriptomic database and validated in AßPP/PS1 mice. We found that HAS1 was distributed along the axon and nucleus. Its transcripts were reduced in AD patients and AßPP/PS1 mice. Phosphorylated tau (p-tau) mediates AßPP-induced cytosolic-nuclear translocation of HAS1, and negatively regulated the stability, monoubiquitination, and oligomerization of HAS1, thus reduced the synthesis and release of HA. Furthermore, non-ubiquitinated HAS1 mutant lost its enzyme activity, and translocated from the cytosol into the nucleus, forming nuclear speckles (NS). Unlike the splicing-related NS, less than 1 % of the non-ubiquitinated HAS1 co-localized with SRRM2, proving the regulatory role of HAS1 in gene transcription, indirectly. Thus, differentially expressed genes (DEGs) related to both non-ubiquitinated HAS1 mutant and AD were screened using transcriptomic datasets. Thirty-nine DEGs were identified, with 64.1 % (25/39) showing consistent results in both datasets. Together, we unearthed an important function of the AßPP-p-tau-HAS1 axis in microenvironment remodeling and gene transcription during AD progression, involving the ubiquitin-proteasome, lysosome, and NS systems.

The hyaluronan-related genes HAS2, HYAL1-4, PH20 and HYALP1 are associated with prognosis, cell viability and spheroid formation capacity in ovarian cancer.

PURPOSE: Hyaluronan modulates tumour progression, including cell adhesion, cohesion, proliferation and invasion, and the cancer stem cell phenotype. In ovarian cancer, high levels of stromal hyaluronan are associated with poor prognosis. In this work, hyaluronan synthases (HAS1-3) and hyaluronidases (HYAL1-4, PH-20, HYALP1) were examined with regard to different levels of gene expression and its influence on ovarian cancer patients' survival. The impact of a siRNA depletion of HAS2 was investigated in vitro. METHODS: Using the Kaplan-Meier Plotter tool, we investigated the influence of hyaluronic synthases and hyaluronidases on the survival of a collective of 1435 ovarian cancer patients. Differences in gene expression between normal (n = 46) and cancerous (n = 744) ovarian tissue were examined using the TNMplot database. Following an evaluation of hyaluronan-related gene expression in the ATCC ovarian cancer panel, we studied SKOV3 and SW 626 ovarian cancer cells subjected to HAS2 siRNA or control siRNA treatment in terms of HAS1-3, HYAL2 and HYAL3 mRNA expression. We investigated the ability to form spheroids using the Hanging Drop method and the response to chemotherapy at different concentrations using the MTT Assay. By STRING analysis, interactions within the enzymes of the hyaluronic acid system and with binding partners were visualized. RESULTS: HAS1, HYAL1 and HYAL4 mRNA expression is significantly upregulated, whereas HAS2, HYAL2 and HYAL3 mRNA expression is significantly downregulated in ovarian cancer tissue compared to controls. HAS2 improves cell viability, the capability to form tumour spheroids and has a negative prognostic value regarding overall survival. Lower HAS2 expression and high expression of HYAL2 and HYAL3 favours the survival of ovarian cancer patients. HAS2 knockdown cells and control cells showed a moderate response to combinatorial in vitro chemotherapy with taxol and cisplatin. CONCLUSION: In conclusion, our study shows that the hyaluronic acid system has a relevant influence on the survival of ovarian cancer patients and could therefore be considered as a possible prognostic factor.

Hyaluronan Synthase 1: A Novel Candidate Gene Associated With Late-Onset Non-syndromic Hereditary Hearing Loss.

OBJECTIVES: Hyaluronan synthase 1 (HAS1) is a membrane-bound protein that is abundant in the epidermis and dermis, and it is important for skin function. However, its association with hearing loss has not yet been studied. Herein, we sought to evaluate the potential contribution of HAS1: c.1082G>A to genetic hearing loss. METHODS: We used whole-exome sequencing to analyze blood DNA samples of six patients of a family with autosomal dominant familial late-onset progressive hearing loss, which was revealed to be related to a variant of the HAS1 gene. Confirmatory Sanger sequencing was performed with samples from 10 members. A missense variant was detected in HAS1 (c.1082 G>A, p.Cys361Tyr). In silico analyses predicted this variant to result in the functional loss of HAS1. Immunostaining was conducted using wild-type mouse samples to verify HAS1 expression. RESULTS: Has1 was detected in an otocyst at E10.5. In the pup, Has1 expression was localized in the stria vascularis (SV), hair cells, supporting cells of the organ of Corti, and some spiral ganglion neurons. SV marginal cells markedly expressed Has1 in the adult stage. The hearing threshold in the Has1-depleted condition was investigated by accessing the International Mouse Phenotyping Consortium's Auditory Brainstem Response (ABR) data. ABR of Has1 knock-out mice showed threshold elevations at 6, 12, and 18 kHz in young male adults. CONCLUSION: HAS1 may have a close relationship with auditory function and genetic hearing loss. Further investigation is needed to reveal the precise role of HAS1 in the auditory system. HAS1 is a candidate gene for future hereditary hearing loss genetic testing.

Hyaluronan as "Agent Smith" in cancer extracellular matrix pathobiology: Regulatory roles in immune response, cancer progression and targeting.

Extracellular matrix (ECM) critically regulates cancer cell behavior by governing cell signaling and properties. Hyaluronan (HA) acts as a structural and functional ECM component that mediates critical properties of cancer cells in a molecular size-dependent manner. HA fragments secreted by cancer-associated fibroblasts (CAFs) reveal the correlation of HA to CAF-mediated matrix remodeling, a key step for the initiation of metastasis. The main goal of this article is to highlight the vital functions of HA in cancer cell initiation and progression as well as HA-mediated paracrine interactions among cancer and stromal cells. Furthermore, the HA implication in mediating immune responses to cancer progression is also discussed. Novel data on the role of HA in the formation of pre-metastatic niche may contribute towards the improvement of current theranostic approaches that benefit cancer management.

Hyaluronan Synthases' Expression and Activity Are Induced by Fluid Shear Stress in Bone Marrow-Derived Mesenchymal Stem Cells.

During biomineralization, the cells generating the biominerals must be able to sense the external physical stimuli exerted by the growing mineralized tissue and change their intracellular protein composition according to these stimuli. In molluscan shell, the myosin-chitin synthases have been suggested to be the link for this communication between cells and the biomaterial. Hyaluronan synthases (HAS) belong to the same enzyme family as chitin synthases. Their product hyaluronan (HA) occurs in the bone and is supposed to have a regulatory function during bone regeneration. We hypothesize that HASes' expression and activity are controlled by fluid-induced mechanotransduction as it is known for molluscan chitin synthases. In this study, bone marrow-derived human mesenchymal stem cells (hMSCs) were exposed to fluid shear stress of 10 Pa. The RNA transcriptome was analyzed by RNA sequencing (RNAseq). HA concentrations in the supernatants were measured by ELISA. The cellular structure of hMSCs and HAS2-overexpressing hMSCs was investigated after treatment with shear stress using confocal microscopy. Fluid shear stress upregulated the expression of genes that encode proteins belonging to the HA biosynthesis and bone mineralization pathways. The HAS activity appeared to be induced. Knowledge about the regulation mechanism governing HAS expression, trafficking, enzymatic activation and quality of the HA product in hMSCs is essential to understand the biological role of HA in the bone microenvironment.

Modulation of hyaluronan synthases and involvement of T cell-derived hyaluronan in autoimmune responses to transplanted islets.

The extracellular matrix glycosaminoglycan hyaluronan (HA) accumulates in human and mouse islets during the onset of autoimmune type 1 diabetes (T1D). HA plays a critical role in T1D pathogenesis, as spontaneous disease is blocked in mice fed the HA synthesis inhibitor 4-methylumbelliferone (4MU). The present study demonstrates the involvement of HA in T cell-mediated autoimmune responses to transplanted islets and in in vivo and in vitro T cell activation. Scaffolded islet implants (SIs) loaded with RIP-mOVA mouse islets expressing chicken ovalbumin (OVA) on their ß cells were grafted into T and B cell-deficient RIP-mOVA mice, which subsequently received CD4+ T cells from DO11.10 transgenic mice bearing OVA peptide-specific T cell receptors (TcRs), followed by injection of OVA peptide to induce an immune response to the OVA-expressing islets. By affinity histochemistry (AHC), HA was greatly increased in grafted islets with T cell infiltrates (compared to islets grafted into mice lacking T cells) and a portion of this HA co-localized with the infiltrating T cells. Transferred T cells underwent HA synthase (HAS) isoform switching - T cells isolated from the SI grafts strongly upregulated HAS1 and HAS2 mRNAs and downregulated HAS3 mRNA, in contrast to T cells from graft-draining mesenteric lymph nodes, which expressed HAS3 mRNA only. Expression of HAS1 and HAS2 proteins by T cells in SI infiltrates was confirmed by immunohistochemistry (IHC). DO11.10 mice fed 4MU had suppressed in vivo T cell immune priming (measured as a reduced recall response to OVA peptide) compared to T cells from control mice fed a normal diet. In co-cultures of naïve DO11.10 T cells and OVA peptide-loaded antigen-presenting cells (APCs), pre-exposure of the T cells (but not pre-exposure of APCs) to 4MU inhibited early T cell activation (CD69 expression). In addition, T cells exposed to 4MU during activation in vitro with anti-CD3/CD28 antibodies had inhibited phosphorylation of the CD3ζ subunit of the TcR, a very early event in TcR signaling. Collectively, our results demonstrate that T cell-derived HA plays a significant role in T cell immune responses, and that expression of T cell HAS isoforms changes in a locale-specific manner during in vivo priming and functional phases of the T cell response.

The Attenuated Secretion of Hyaluronan by UVA-Exposed Human Fibroblasts Is Associated with Up- and Downregulation of HYBID and HAS2 Expression via Activated and Inactivated Signaling of the p38/ATF2 and JAK2/STAT3 Cascades.

Little is known about the effects on hyaluronan (HA) metabolism of UVA radiation. This study demonstrates that the secretion of HA by human dermal fibroblasts (HDFs) is downregulated by UVA, accompanied by the down- and upregulation of mRNA and protein levels of the HA-synthesizing enzyme (HAS2) and the HA-degrading protein, HYaluronan Binding protein Involved in HA Depolymerization(HYBID), respectively. Signaling analysis revealed that the exposure distinctly elicits activation of the p38/MSK1/CREB/c-Fos/AP-1 axis, the JNK/c-Jun axis, and the p38/ATF-2 axis, but downregulates the phosphorylation of NF-kB and JAK/STAT3. A signal inhibition study demonstrated that the inhibition of p38 significantly abrogates the UVA-accentuated mRNA level of HYBID. Furthermore, the inhibition of STAT3 significantly downregulates the level of HAS2 mRNA in non-UVA exposed HDFs. Analysis using siRNAs demonstrated that transfection of ATF-2 siRNA but not c-Fos siRNA abrogates the increased protein level of HYBID in UVA-exposed HDFs. An inhibitor of protein tyrosine phosphatase but not of protein serine/threonine phosphatase restored the diminished phosphorylation level of STAT3 at Tyr 705, accompanied by a significant abolishing effect on the decreased mRNA expression level of HAS2. Silencing with a protein tyrosine phosphatase PTP-Meg2 siRNA revealed that it abrogates the decreased phosphorylation of STAT3 at Tyr 705 in UVA-exposed HDFs. These findings suggest that the UVA-induced decrease in HA secretion by HDFs is attributable to the down- and upregulation of HAS2 and HYBID expression, respectively, changes that are mainly ascribed to the inactivated signaling of the STAT3 axis due to the activated tyrosine protein phosphatase PTP-Meg2 and the activated signaling of the p38/ATF2 axis, respectively.

C1q-HA Matrix Regulates the Local Synthesis of Hyaluronan in Malignant Pleural Mesothelioma by Modulating HAS3 Expression.

Increased hyaluronic acid (HA) production is often associated with cancer progression. In malignant pleural mesothelioma (MPM), HA is found at elevated levels in pleural effusions and sera of patients, and it has been widely debated whether MPM cells are able to produce HA by themselves or through the release of growth factors stimulating other cells. Another key component of the MPM microenvironment is C1q, which can act as a pro-tumorigenic factor favoring cell adhesion, migration and proliferation. The aim of the current study was to prove that MPM primary cells are able to synthesize HA and to inquire the stimulus given by C1q-HA matrix to HA synthesis. We confirmed the presence of a HA coat and cable-like structures around MPM primary cells, as well as an intracellular pool, mainly localized in the cytoplasmic and perinuclear region. After evaluating HA synthase (HAS) enzymes' basal expression in MPM primary cells, we found that C1q bound to HA was able to impinge upon HA homeostasis by upregulating HAS3 both at the mRNA and the protein levels. High expression of HAS3 has been correlated with a shorter life expectancy in MPM by bioinformatical analysis. These data confirmed that C1q bound to HA may exert pro-tumorigenic activity and identified HAS3 as a potential target in MPM.

Adhesive Properties of the Hyaluronan Pericellular Coat in Hyaluronan Synthases Overexpressing Mesenchymal Stem Cells.

Hyaluronan (HA), a natural component of the extracellular matrix, is supposed to have a regulatory function in the stem cell niche. Bone marrow-derived human mesenchymal stem cells (hMSCs) are known to express all three hyaluronan synthases (HASes), which are responsible for HA production. HA is extruded into the extracellular matrix, but also stays bound to the plasma membrane forming a pericellular coat, which plays a key role during early cell adhesion. Since HAS isoenzymes, HAS1, HAS2 and HAS3, produce HA with different molecular weights, a difference in their role for cell adhesion is expected. Here, we transduced the immortalized hMSC cell line SCP1 to constitutively express eGFP-tagged HASes (SCP1-HAS-eGFP) by lentiviral gene transfer. The overexpression of the HAS-eGFP was shown on RNA and protein levels, HA was determined by ELISA and the stained HA-coat was analyzed using confocal microscopy. Time-lapse microscopy, spreading assay and single cell force spectroscopy using atomic force microscopy were applied to characterize adhesion of the different HAS transduced SCP1 cells. We showed in this study that HAS3 overexpressing cells formed the thickest pericellular coat compared with control or HAS1 and HAS2 transduced cells. Furthermore, SCP1-HAS3-eGFP displayed faster and stronger adhesion compared to cells overexpressing the other synthases or control cells. We conclude that overexpression of HASes in hMSCs differentially modulates their initial adhesive interactions with the substrate. This observation might be helpful in regenerative medicine goals.

Hyaluronan in the Tumor Microenvironment.

The extracellular matrix is part of the microenvironment and its functions are associated with the physical and chemical properties of the tissue. Among the extracellular components, the glycosaminoglycan hyaluronan is a key component, defining both the physical and biochemical characteristics of the healthy matrices. The hyaluronan metabolism is strictly regulated in physiological conditions, but in the tumoral tissues, its expression, size and binding proteins interaction are dysregulated. Hyaluronan from the tumor microenvironment promotes tumor cell proliferation, invasion, immune evasion, stemness alterations as well as drug resistance. This chapter describes data regarding novel concepts of hyaluronan functions in the tumor. Additionally, we discuss potential clinical applications of targeting HA metabolism in cancer therapy.

Contact allergen (PPD and DNCB)-induced keratinocyte sensitization is partly mediated through a low molecular weight hyaluronan (LMWHA)/TLR4/NF-κB signaling axis.

Allergic contact dermatitis (ACD) is caused by topical exposure to chemical allergens. Keratinocytes play a key role in innate immunity, as well as in ACD progression. The transmembrane Toll-like receptor 4 (TLR4), strongly implicated in skin inflammation, has the ability to bind Damage Associated Molecular Patterns (DAMPs), like Low Molecular Weight Hyaluronan (LMWHA). Previously, we had determined that p-phenylenediamine (PPD) and 2,4-dinitrochlorobenzene (DNCB) modulate keratinocyte HA deposition in a manner correlated to their sensitization. In the present study, we aimed to investigate putative co-operation of HA and TLR4 in the process of PPD and DNCB-induced keratinocyte activation. Contact sensitizers were shown to significantly increase the expression of Hyaluronan Synthases (HAS) and TLR4 in NCTC2544 human keratinocytes, as demonstrated by western blot and Real-Time PCR. These data, in correlation to earlier shown enhanced HA degradation suggest that the contact sensitizers facilitate HA turnover of keratinocytes and increase the release of pro-inflammatory, LMWHA fragments. Treatment with exogenous LMWHA enhanced TLR4, HAS levels and Nuclear factor-kappa beta (NF-κΒ) activation. PPD, DNCB and LMWHA-effects were shown to be partly executed through TLR4 downstream signaling as shown by Real-Time, western blot, siRNA and confocal microscopy approaches. Specifically, PPD and DNCB stimulated the activation of the TLR4 downstream mediator NF-κB. Therefore, the shown upregulation of TLR4 expression is suggested to further facilitate the release of endogenous, bioactive HA fragments and sustain keratinocyte activation. In conclusion, keratinocyte contact allergen-dependent sensitization is partly mediated through a LMWHA/TLR4/ NF-κB signaling axis.

Adenosine and hyaluronan promote lung fibrosis and pulmonary hypertension in combined pulmonary fibrosis and emphysema.

Combined pulmonary fibrosis and emphysema (CPFE) is a syndrome that predominantly affects male smokers or ex-smokers and it has a mortality rate of 55% and a median survival of 5 years. Pulmonary hypertension (PH) is a frequently fatal complication of CPFE. Despite this dismal prognosis, no curative therapies exist for patients with CPFE outside of lung transplantation and no therapies are recommended to treat PH. This highlights the need to develop novel treatment approaches for CPFE. Studies from our group have demonstrated that both adenosine and its receptor ADORA2B are elevated in chronic lung diseases. Activation of ADORA2B leads to elevated levels of hyaluronan synthases (HAS) and increased hyaluronan, a glycosaminoglycan that contributes to chronic lung injury. We hypothesize that ADORA2B and hyaluronan contribute to CPFE. Using isolated CPFE lung tissue, we characterized expression levels of ADORA2B and HAS. Next, using a unique mouse model of experimental lung injury that replicates features of CPFE, namely airspace enlargement, PH and fibrotic deposition, we investigated whether 4MU, a HAS inhibitor, was able to inhibit features of CPFE. Increased protein levels of ADORA2B and HAS3 were detected in CPFE and in our experimental model of CPFE. Treatment with 4MU was able to attenuate PH and fibrosis but not airspace enlargement. This was accompanied by a reduction of HAS3-positive macrophages. We have generated pre-clinical data demonstrating the capacity of 4MU, an FDA-approved drug, to attenuate features of CPFE in an experimental model of chronic lung injury.This article has an associated First Person interview with the first author of the paper.

Dissecting the role of hyaluronan synthases in the tumor microenvironment.

The tumor microenvironment is becoming a crucial factor in determining the aggressiveness of neoplastic cells. The glycosaminoglycan hyaluronan is one of the principal constituents of both the tumor stroma and the cancer cell surfaces, and its accumulation can dramatically influence patient survival. Hyaluronan functions are dictated by its ability to interact with several signaling receptors that often activate pro-angiogenic and pro-tumorigenic intracellular pathways. Although hyaluronan is a linear, non-sulfated polysaccharide, and thus lacks the ability of the other sulfated glycosaminoglycans to bind and modulate growth factors, it compensates for this by the ability to form hyaluronan fragments characterized by a remarkable variability in length. Here, we will focus on the role of both high and low molecular weight hyaluronan in controlling the hallmarks of cancer cells, including cell proliferation, migration, metabolism, inflammation, and angiogenesis. We will critically assess the multilayered regulation of HAS2, the most critical hyaluronan synthase, and its role in cancer growth, metabolism, and therapy.

TGF-ß1 upregulates the expression of hyaluronan synthase 2 and hyaluronan synthesis in culture models of equine articular chondrocytes.

We investigated the effect of transforming growth factor beta 1 (TGF-ß1) on equine hyaluronan synthase 2 (HAS2) gene expression and hyaluronan (HA) synthesis in culture models of articular chondrocytes. Equine chondrocytes were treated with TGF-ß1 at different concentrations and times in monolayer cultures. In three-dimensional cultures, chondrocyte-seeded gelatin scaffolds were cultured in chondrogenic media containing 10 ng/mL of TGF-ß1. The amounts of HA in conditioned media and in scaffolds were determined by enzyme-linked immunosorbent assays. HAS2 mRNA expression was analyzed by semi-quantitative reverse transcription polymerase chain reaction. The uronic acid content and DNA content of the scaffolds were measured by using colorimetric and Hoechst 33258 assays, respectively. Cell proliferation was evaluated by using the alamarBlue assay. Scanning electron microscopy (SEM), histology, and immunohistochemistry were used for microscopic analysis of the samples. The upregulation of HAS2 mRNA levels by TGF-ß1 stimulation was dose and time dependent. TGF-ß1 was shown to enhance HA and uronic acid content in the scaffolds. Cell proliferation and DNA content were significantly lower in TGF-ß1 treatments. SEM and histological results revealed the formation of a cartilaginous-like extracellular matrix in the TGF-ß1-treated scaffolds. Together, our results suggest that TGF-ß1 has a stimulatory effect on equine chondrocytes, enhancing HA synthesis and promoting cartilage matrix generation.

Knockout of hyaluronan synthase 1, but not 3, impairs formation of the retrocalcaneal bursa.

Hyaluronan (HA), a high molecular weight non-sulfated glycosaminoglycan, is an integral component of the extracellular matrix of developing and mature connective tissues including tendon. There are few published reports quantifying HA content during tendon growth and maturation, or detailing its effects on the mechanical properties of the tendon extracellular matrix. Therefore, the goal of the current study was to examine the role of HA synthesis during post-natal skeletal growth and maturation, and its influence on tendon structure and biomechanical function. For this purpose, the morphological, biochemical, and mechanical properties of Achilles tendons from wild type (WT) and hyaluronan synthase 1 and 3 deficient mouse strains (Has1-/- (Has1KO), Has3-/- (Has3KO), and Has1-/- 3-/- (Has1/3KO)) were determined at 4, 8, and 12 weeks of age. Overall, HAS-deficient mice did not show any marked differences from WT mice in Achilles tendon morphology or in the HA and chondroitin/dermatan sulfate (CS/DS) contents. However, HAS1-deficiency (in the single or Has1/3 double KO) impeded post-natal formation of the retrocalcaneal bursa, implicating HAS1 in regulating HA metabolism by cells lining the bursal cavity. Together, these data suggest that HA metabolism via HAS1 and HAS3 does not markedly influence the extracellular matrix structure or function of the tendon body, but plays a role in the formation/maintenance of peritendinous bursa. Additional studies are warranted to elucidate the relationship of HA and CS/DS metabolism to tendon healing and repair in vivo. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2622-2632, 2018.

Hyaluronic Acid in the Third Millennium.

Since its first isolation in 1934, hyaluronic acid (HA) has been studied across a variety of research areas. This unbranched glycosaminoglycan consisting of repeating disaccharide units of N-acetyl-d-glucosamine and d-glucuronic acid is almost ubiquitous in humans and in other vertebrates. HA is involved in many key processes, including cell signaling, wound reparation, tissue regeneration, morphogenesis, matrix organization and pathobiology, and has unique physico-chemical properties, such as biocompatibility, biodegradability, mucoadhesivity, hygroscopicity and viscoelasticity. For these reasons, exogenous HA has been investigated as a drug delivery system and treatment in cancer, ophthalmology, arthrology, pneumology, rhinology, urology, aesthetic medicine and cosmetics. To improve and customize its properties and applications, HA can be subjected to chemical modifications: conjugation and crosslinking. The present review gives an overview regarding HA, describing its history, physico-chemical, structural and hydrodynamic properties and biology (occurrence, biosynthesis (by hyaluronan synthases), degradation (by hyaluronidases and oxidative stress), roles, mechanisms of action and receptors). Furthermore, both conventional and recently emerging methods developed for the industrial production of HA and its chemical derivatization are presented. Finally, the medical, pharmaceutical and cosmetic applications of HA and its derivatives are reviewed, reporting examples of HA-based products that currently are on the market or are undergoing further investigations.

TGF-β1 upregulates the expression of hyaluronan synthase 2 and hyaluronan synthesis in culture models of equine articular chondrocytes

We investigated the effect of transforming growth factor beta 1 (TGF-β1) on equine hyaluronan synthase 2 (HAS2) gene expression and hyaluronan (HA) synthesis in culture models of articular chondrocytes. Equine chondrocytes were treated with TGF-β1 at different concentrations and times in monolayer cultures. In three-dimensional cultures, chondrocyte-seeded gelatin scaffolds were cultured in chondrogenic media containing 10 ng/mL of TGF-β1. The amounts of HA in conditioned media and in scaffolds were determined by enzyme-linked immunosorbent assays. HAS2 mRNA expression was analyzed by semi-quantitative reverse transcription polymerase chain reaction. The uronic acid content and DNA content of the scaffolds were measured by using colorimetric and Hoechst 33258 assays, respectively. Cell proliferation was evaluated by using the alamarBlue assay. Scanning electron microscopy (SEM), histology, and immunohistochemistry were used for microscopic analysis of the samples. The upregulation of HAS2 mRNA levels by TGF-β1 stimulation was dose and time dependent. TGF-β1 was shown to enhance HA and uronic acid content in the scaffolds. Cell proliferation and DNA content were significantly lower in TGF-β1 treatments. SEM and histological results revealed the formation of a cartilaginous-like extracellular matrix in the TGF-β1-treated scaffolds. Together, our results suggest that TGF-β1 has a stimulatory effect on equine chondrocytes, enhancing HA synthesis and promoting cartilage matrix generation.

Decreased expression of hyaluronan synthase 1 and 2 associates with poor prognosis in cutaneous melanoma.

BACKGROUND: Hyaluronan is a large extracellular matrix molecule involved in several biological processes such as proliferation, migration and invasion. In many cancers, hyaluronan synthesis is altered, which implicates disease progression and metastatic potential. We have previously shown that synthesis of hyaluronan and expression of its synthases 1-2 (HAS1-2) decrease in cutaneous melanoma, compared to benign melanocytic lesions. METHODS: In the present study, we compared immunohistological staining results of HAS1 and HAS2 with clinical and histopathological parameters to investigate whether HAS1 or HAS2 has prognostic value in cutaneous melanoma. The specimens consisted of 129 tissue samples including superficial (Breslow ≤ 1 mm) and deep (Breslow > 4 mm) melanomas and lymph node metastases. The differences in immunostainings were analysed with non-parametric Mann-Whitney U test. Associations between immunohistological staining results and clinical parameters were determined with the χ(2) test. Survival between patient groups was compared by the Kaplan-Meier method using log rank test and Cox's regression model was used for multivariate analyses. RESULTS: The expression of HAS1 and HAS2 was decreased in deep melanomas and metastases compared to superficial melanomas. Decreased immunostaining of HAS2 in melanoma cells was significantly associated with several known unfavourable histopathologic prognostic markers like increased mitotic count, absence of tumor infiltrating lymphocytes and the nodular subtype. Furthermore, reduced HAS1 and HAS2 immunostaining in the melanoma cells was associated with increased recurrence of melanoma (p = 0.041 and p = 0.006, respectively) and shortened disease- specific survival (p = 0.013 and p = 0.001, respectively). CONCLUSIONS: This study indicates that reduced expression of HAS1 and HAS2 is associated with melanoma progression and suggests that HAS1 and HAS2 have a prognostic significance in cutaneous melanoma.

Deficiency of hyaluronan synthase 1 (Has1) results in chronic joint inflammation and widespread intra-articular fibrosis in a murine model of knee joint cartilage damage.

OBJECTIVE: Articular cartilage defects commonly result from traumatic injury and predispose to degenerative joint diseases. To test the hypothesis that aberrant healing responses and chronic inflammation lead to osteoarthritis (OA), we examined spatiotemporal changes in joint tissues after cartilage injury in murine knees. Since intra-articular injection of hyaluronan (HA) can attenuate injury-induced osteoarthritis in wild-type (WT) mice, we investigated a role for HA in the response to cartilage injury in mice lacking HA synthase 1 (Has1(-/-)). DESIGN: Femoral groove cartilage of WT and Has1(-/-) mice was debrided to generate a non-bleeding wound. Macroscopic imaging, histology, and gene expression were used to evaluate naïve, sham-operated, and injured joints. RESULTS: Acute responses (1-2 weeks) in injured joints from WT mice included synovial hyperplasia with HA deposition and joint-wide increases in expression of genes associated with inflammation, fibrosis, and extracellular matrix (ECM) production. By 4 weeks, some resurfacing of damaged cartilage occurred, and early cell responses were normalized. Cartilage damage in Has1(-/-) mice also induced early responses; however, at 4 weeks, inflammation and fibrosis genes remained elevated with widespread cartilage degeneration and fibrotic scarring in the synovium and joint capsule. CONCLUSIONS: We conclude that the ineffective repair of injured cartilage in Has1(-/-) joints can be at least partly explained by the markedly enhanced expression of particular genes in pathways linked to ECM turnover, IL-17/IL-6 cytokine signaling, and apoptosis. Notably, Has1 ablation does not alter gross HA content in the ECM, suggesting that HAS1 has a unique function in the metabolism of inflammatory HA matrices.