Hyaluronan Content Research Articles

Asthmatic bronchial epithelial cells promote the establishment of a Hyaluronan-enriched, leukocyte-adhesive extracellular matrix by lung fibroblasts

BackgroundAirway inflammation is a hallmark of asthma. Alterations in extracellular matrix (ECM) hyaluronan (HA) content have been shown to modulate the recruitment and retention of inflammatory cells. Bronchial epithelial cells (BECs) regulate the activity of human lung fibroblasts (HLFs); however, their contribution in regulating HLF production of HA in asthma is unknown. In this study, we tested the hypothesis that BECs from asthmatic children promote the generation of a pro-inflammatory, HA-enriched ECM by HLFs, which promotes the retention of leukocytes.MethodsBECs were obtained from well-characterized asthmatic and healthy children ages 6–18 years. HLFs were co-cultured with BECs for 96 h and samples were harvested for analysis of gene expression, synthesis and accumulation of HA, and subjected to a leukocyte adhesion assay with U937 monocytes.ResultsWe observed increased expression of HA synthases HAS2 and HAS3 in HLFs co-cultured with asthmatic BECs. Furthermore, we demonstrated greater total accumulation and increased synthesis of HA by HLFs co-cultured with asthmatic BECs compared to healthy BEC/HLF co-cultures. ECM generated by HLFs co-cultured with asthmatic BECs displayed increased HA-dependent adhesion of leukocytes in a separate in vitro binding assay.ConclusionsOur findings demonstrate that BEC regulation of HA production by HLFs is altered in asthma, which may in turn promote the establishment of a more leukocyte-permissive ECM promoting airway inflammation in this disease.

Accumulation of hyaluronan in reared Japanese eel Anguilla japonica during early ontogeny

Hyaluronan (HA) is a main component of the highly hydrated extracellular body matrix of the larval stage of eels (leptocephalus). To determine the physiological and nutritional significance of HA in artificially reared larvae of the Japanese eel Anguilla japonica, HA and water content were measured at various larval stages in captivity. HA accumulated in the larval body with growth and water content also increased in parallel with HA. Almost all the accumulated HA was consumed during metamorphosis, suggesting HA plays a significant role in eel growth and metamorphosis. However, the amounts of HA and water were significantly lower in reared than in wild-caught larvae at the young stage, indicating a poorer nutritional condition of reared larvae, probably due to the deficient performance of currently used artificial diets. HA accumulation in reared larvae, however, eventually attained a level sufficient for metamorphosis by 250 days or more, and the glass eels of reared larvae had the same nutritional condition as that of wild glass eels. These findings are discussed with respect to the development of more effective artificial diets for rearing eel larvae.

Quantification of hyaluronan in human fasciae: variations with function and anatomical site.

Recently, alterations in fascial gliding-like movement have been invoked as critical in the etiology of myofascial pain. Various methods have been attempted for the relief of this major and debilitating clinical problem. Paramount have been attempts to restore correct gliding between fascial layers and the movement over bone, joint, and muscular structures. One of the key elements that underlies such fascial movement is hyaluronan. However, until now, the precise content of hyaluronan within fasciae has been unknown. This study quantifies for the first time the hyaluronan content of human fascial samples obtained from a variety of anatomic sites. Here, we demonstrate that the average amount varies according to anatomic site, and according to the different kinds of sliding properties of the particular fascia. For example, the fascia lata has 35μg of hyaluronan per gram of tissue, similar to that of the rectus sheath (29μgg-1 ). However, the types of fascia adherent to muscle contain far less hyaluronan: 6μgg-1 in the fascia overlying the trapezius and deltoid muscles. In the fascia that surrounds joints, the hyaluronan increases to 90μgg-1 , such as in the retinacula of the ankle, where greater degrees of movement occur. Surprisingly, no significant differences were detected at any site as a function of age or sex (P-value>0.05, t-test) with the sole exception of the plantar fascia. This work can provide a better understanding of the role of hyaluronan in fascia. It will facilitate a better comprehension of the modulation of the hyaluronan-rich layer that occurs in relation to the various conditions that affect fascia, and the diverse factors that underlie the attendant pathologies.

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.

Abstract 1743: Rationale for evaluating PEGylated recombinant human hyaluronidase PH20 (pegvorhyaluronidase alfa; PEGPH20) in patients with hyaluronan (HA)-accumulating colorectal cancer

Abstract Colorectal cancer is the third most common cancer, and the third most common cause of cancer-related death, among both men and women in the United States. To address this unmet clinical need, ongoing efforts have focused on classifying different subtypes of colorectal cancer, with the goal of identifying therapies with the highest likelihood of success for a given tumor subtype. One marker that has been associated with poor clinical outcome in multiple solid tumor types, including colorectal cancer, is the accumulation of the glycosaminoglycan hyaluronan (HA), which, in preclinical tumor models, increases interstitial fluid pressure, compresses tumor vasculature, and restricts the access of therapeutics. PEGylated recombinant human hyaluronidase PH20 (pegvorhyaluronidase alfa; PEGPH20), which enzymatically degrades HA, is currently being evaluated in combination with existing therapies in clinical trials for HA-accumulating pancreatic, gastric, non-small cell lung, and bile duct cancers. Here, we review the available data for PEGPH20 as published by the authors to evaluate the potential utility of PEGPH20 in treating colorectal cancer. We first studied PEGPH20 in two HA-accumulating murine colon tumor models: CT26 transduced with hyaluronan synthase-3 (HAS3) and MC38 (Charles River Laboratories). In CT26-HAS3 tumors, PEGPH20 monotherapy induced 43% tumor growth inhibition and was associated with tumor blood vessel decompression. In combination with anti-CTLA4 treatment, PEGPH20 further enhanced tumor growth inhibition (79%, p≤0.002 vs anti-CTLA4 alone and PEGPH20 alone). In MC38 tumors, combination of PEGPH20 with anti-PD-L1 treatment significantly increased infiltration of CD8+ T cells (2.8-fold, p<0.05) and NK cells (3-fold, p<0.0001) compared with anti-PD-L1 alone. We then investigated the prevalence of HA accumulation in human colorectal cancer. To do this, we histologically analyzed 138 patient tumor specimens for stromal content and presence of HA. In 38 samples (28%), ≥50% of the tumor stromal area stained positive for HA. Additionally, Phase 1 studies with single agent PEGPH20 included several subjects with colorectal cancer. Pharmacodynamic measurements before and after treatment demonstrated increased tumor perfusion as measured by median volume transfer constant (Ktrans), decreased tumor metabolic activity as measured by 18F-FDG-PET/CT in one patient, and decreased tumor HA following PEGPH20 administration. Collectively, these data warrant further investigation of the potential for PEGPH20 to benefit patients with HA-high colorectal cancer. Citation Format: Renee Clift, Benjamin J. Thompson, Darin Taverna, Barbara Blouw, Jisook Lee, Curtis B. Thompson, Daniel C. Maneval. Rationale for evaluating PEGylated recombinant human hyaluronidase PH20 (pegvorhyaluronidase alfa; PEGPH20) in patients with hyaluronan (HA)-accumulating colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1743.

Defective lung function following influenza virus is due to prolonged, reversible hyaluronan synthesis

Little is known about the impact of viral infections on lung matrix despite its important contribution to mechanical stability and structural support. The composition of matrix also indirectly controls inflammation by influencing cell adhesion, migration, survival, proliferation and differentiation. Hyaluronan is a significant component of the lung extracellular matrix and production and degradation must be carefully balanced. We have discovered an imbalance in hyaluronan production following resolution of a severe lung influenza virus infection, driven by hyaluronan synthase 2 from epithelial cells, endothelial cells and fibroblasts. Furthermore hyaluronan is complexed with inter-α-inhibitor heavy chains due to elevated TNF-stimulated gene 6 expression and sequesters CD44-expressing macrophages. We show that intranasal administration of exogenous hyaluronidase is sufficient to release inter-α-inhibitor heavy chains, reduce lung hyaluronan content and restore lung function. Hyaluronidase is already used to facilitate dispersion of co-injected materials in the clinic. It is therefore feasible that fibrotic changes following severe lung infection and inflammation could be overcome by targeting abnormal matrix production.

Elevated expression of hyaluronan synthase 2 associates with decreased survival in diffusely infiltrating astrocytomas

BackgroundDiffusely infiltrating astrocytomas originate from astrocytic glial cells or their precursor cells and are the most common type of brain tumors in adults. In this retrospective study, we investigated the content of hyaluronan, its cell surface receptor, CD44 and the expression of hyaluronan metabolizing enzymes, in these aggressive tumors. Hyaluronan is the main component of extracellular matrix in the brain. In many tumors, aberrant hyaluronan metabolism implicates aggressive disease progression and metastatic potential.MethodsOur material consisted of 163 diffusely infiltrating astrocytomas (WHO grades II-IV). Tumor samples were processed into tissue microarray (TMA) blocks. The TMA sections were stained for hyaluronan, CD44, hyaluronan synthases 1–3 (HAS1–3) and hyaluronidase 2 (HYAL2). The immunostaining results were compared with χ2 –test or with Kruskal-Wallis test for correlation with clinicopathological parameters and survival analyses were done with Kaplan-Meier log rank test and Cox regression.ResultsHyaluronan and CD44 were strongly expressed in astrocytic gliomas but their expression did not correlate with WHO grade or any other clinicopathological parameters whereas high HAS2 staining intensity was observed in IDH1 negative tumors (p = 0.003). In addition, in non-parametric tests increased HAS2 staining intensity correlated with increased cell proliferation (p = 0.013) and in log rank test with decreased overall survival of patients (p = 0.001). In the Cox regression analysis HAS2 expression turned out to be a significant independent prognostic factor (p = 0.008).ConclusionsThis study indicates that elevated expression of HAS2 is associated with glioma progression and suggests that HAS2 has a prognostic significance in diffusely infiltrating astrocytomas.

P-183 - A pilot trial of PEGPH20 (Pegvorhyaluronidase alfa) in combination with avelumab (anti-PD-L1 MSB0010718C) in chemotherapy resistant pancreatic cancer (PDAC) - Trial in progress

Introduction: PDAC is characterized by excessive hyaluronan (HA) accumulation in the tumor microenvironment, elevating interstitial pressure, resulting in tumor vascular collapse, hypoxia, blocking chemotherapeutic agent perfusion and immune cells. Consequently, checkpoint inhibitors and immunotherapy strategies have failed in PDAC. PEGPH20 targets tumors that accumulate HA. Enzymatically depleting HA from the extracellular matrix (ECM), resulting in decompression of intratumoral blood vessels and increased penetration of antitumor agents. In preclinical models, depletion of HA in the tumor microenvironment has been shown to inhibit the growth of tumors characterized by accumulation of HA. PEGPH20 has been evaluated in a phase II trial in combination with gemcitabine and nab–paclitaxel (PAG) versus gemcitabine plus nab–paclitaxel (AG). For patients with high HA expression on baseline biopsies, the combination arm with PEGPH20 increased progression-free survival (PFS) by 4 months (9.2 vs. 5.2 months; HR 0.51; p = 0.048). Our study tests the hypothesis that elimination of HA in tumor microenvironment by PEGPH20 will result in stromal remodeling and may facilitate the activity of checkpoint inhibitors like avelumab, by at least two mechanisms including increase in drug delivery and increasing immune infiltrate. Methods: A pilot, open label, multicenter, pharmacodynamics, safety, and efficacy study of PEGPH20 in combination with avelumab in chemotherapy resistant advanced or locally advanced PDAC. PEGPH20 3 microg/Kg dose will be administered on days 1, 4, 8, 11, 15, 18 during the first cycle (28 days) and days 1 and 15 thereafter. Avelumab at dose of 10 mg/Kg will start on day 15 after 4 doses of PEGPH20 and continued to be administered every 2 weeks during the study. Enoxaparin will be administered to all subjects to minimize the risk of thromboembolic events (TE) associated to PEGPH20 administration. Pharmacokinetics (Pk) samples for PEGPH20 will be collected. Other assessments: serum CA 19-9 and tissue biomarkers (including collagen content, cancer associated fibroblasts [CAF], and immune infiltrate). Objectives: Primary: To determine the objective response rate (ORR) as per RECIST v1.1 criteria. Secondary: To determine the overall survival (OS), progression free survival (PFS) and CA19-9 tumor marker response. Exploratory: To determine the effect of PEGPH20 in HA content in plasma and paired tumor biopsies. Key inclusion criteria: progression to first line treatment for locally advanced or advanced disease, life expectancy ≥ 3 months and no clinical evidence of prior TE within 12 months or other known TE during the screening.

Accumulation of mechanical forces in tumors is related to hyaluronan content and tissue stiffness

Hyaluronan is abundant in the extracellular matrix of many desmoplastic tumors and determines in large part the tumor biochemical and mechanical microenvironment. Additionally, it has been identified as one of the major physiological barriers to the effective delivery of drugs to solid tumors and its targeting with the use of pharmaceutical agents has shown to decompress tumor blood vessels, and thus improve tumor perfusion and efficacy of cytotoxic drugs. In this study, we investigated the contribution of hyaluronan to the accumulation of mechanical forces in tumors. Using experimental data from two orthotopic breast tumor models and treating tumors with two clinically approved anti-fibrotic drugs (tranilast and pirfenidone), we found that accumulation of growth-induced, residual forces in tumors are associated with hyaluronan content. Furthermore, mechanical characterization of the tumors revealed a good correlation of the accumulated forces with the elastic modulus of the tissue. Our results provide important insights on the mechano-pathology of solid tumors and can be used for the design of therapeutic strategies that target hyaluronan.

Hyaluronan content and distribution in the rat ventral prostate after castration.

Hyaluronan (HA) has been implicated in tissue remodeling, healing, and tumor growth. This study investigated the variation in hyaluronan content, distribution, and metabolism in the rat ventral prostate (VP) in response to androgen deprivation after castration. The mRNA abundance of hyaluronan synthases (Has1-3) and hyaluronidases (Hyal 1-3) were assessed by reverse transcription (RT)-PCR and immunohistochemistry, respectively. The results demonstrated an increased concentration, but an overall reduction in HA content. HA was located in both epithelium and stroma of the prostate of both the noncastrated and castrated animals. Quantitative RT-PCR (qRT-PCR) showed that Has1 and Has2 are major synthases, and that Hyal 1 was the predominant hydrolase expressed in the VP. qRT-PCR also showed that Has1 and Has2 mRNA increased transiently after castration, whereas Has3 mRNA declined markedly. While Hyal 1 mRNA increased slowly up to day 21 after castration, Hyal 2 and Hyal 3 mRNA dropped significantly. CD44 was found in the epithelial cells and in some stromal cells in both hormonal conditions. In conclusion, castration results in increased abundance of Has1 and Has2 mRNA, but is associated with a decrease in the total content of HA, with an increased concentration, and a predominance of short-chain HA molecules.

Review: Limb regeneration in humans: Dream or reality?

Appendage regeneration occurs by a sequence of events resembling those that take place during development in the embryo. This requires embryonic conditions such as hydration and hyaluronate content where Wnt and other signaling pathways, together with non- coding RNAs, can be re-expressed. These conditions among vertebrates are fully met only in amputated limbs of amphibians, likely because they are neotenic and maintain larval characteristics, including immaturity of their immune system and permanence of numerous stem cells. Although some key genes orchestrating limb regeneration are also present in amniotes, including humans, these genes are not expressed after injury. In amniotes a key problem for regeneration derives from the efficient immune system, largely deficient in anamniotes. As a consequence, wounds and appendage loss tend to scar instead of regenerating. Efforts of regenerative medicine in the attempt to induce the regrowth of limbs in humans must produce outgrowths with high hydration and hyaluronate content in order to create the immune-suppressed conditions similar to those present during development. The induced blastema must be manipulated for long periods of time in order to maintain the same regions present during limb development, an apical epidermal ridge and a polarizing region that forms gradients of expression of Wnt, Shh, FGF, BMP and Hox-genes. Pharmacological treatments to direct the regenerating limb into normal growth without risk of inducing abnormal or tumorigenic growth must be monitored during the course of the regeneration process — a medical treatment lasting years to fully regain the size of the lost appendage.

Dermatoporosis: a further step to recognition.

Skin ageing has long been considered only as a cosmetic issue. However, with increasing lifespan, we experience a functional dimension of skin ageing which extends beyond cosmetics and appearance. Ten years ago, we proposed the neologism dermatoporosis to cover different characteristics of a chronic cutaneous insufficiency/fragility syndrome, to understand underlying molecular mechanisms and to develop preventative or therapeutic strategies.

A phase IB/II randomized study of mFOLFIRINOX (mFFOX) + pegylated recombinant human hyaluronidase (PEGPH20) versus mFFOX alone in patients with good performance status metastatic pancreatic adenocarcinoma (mPC): SWOG S1313 (NCT #01959139).

208 Background: PEGPH20 degrades hyaluronan (HA), a major component of the stroma, increases delivery of gemcitabine and prolongs survival in preclinical models. We evaluated the activity of PEGPH20 in combination with mFFOX in mPC , unselected for tumor HA. Methods: Pertinent eligibility: untreated mPC, PS of 0-1 and adequate organ function. Standard FFOX was modified to add prophylactic growth factor support and omit bolus 5FU. Due to increased thromboembolic (TE) events with PEGPH20, an amendment instituted LMWH prophylaxis in the PEGPH20 arm only. Following a dose finding cohort of mFFOX + PEGPH20, the Phase II study randomized patients (1:1) to the combination arm or mFFOX alone (n = 138). The primary endpoint was overall survival (OS), with a null median OS of 10 mo and an alternative of 15 mo (1-sided type 1 error 0.1, 80% power). Results: PEGPH20 at 3 mcg/kg, q2 weeks was more tolerable than twice weekly dosing. The randomized phase II study began May 2015. The planned interim futility analysis when 35 deaths occurred gave a HR of 0.44 favoring the standard arm, thus the study was closed to accrual (March 2017). As of August 22, 2017 (n = 111), the estimated median OS in the mFFOX arm was 15.1 mo (95% CI 10.1-15.7) vs 7.6 mo (95% CI 4.6-9.2) in the PEGPH20 arm. Conclusions: OS in the mFFOX control arm (15.1 mo) is longest yet reported. Addition of PEGPH20 to mFFOX is not recommended for further study and appears to be detrimental (HR = 0.48). The impact of PEGPH20 on OS was unexpected and in contrast to favorable results reported for the combination of gemcitabine/nab-paclitaxel + PEGPH20 especially in the HA high cohort (Hingorani S et al. A 4008, ASCO 2017). PEGPH20 with mFFOX caused increased toxicity (mostly GI and TE events) and decreased treatment duration compared to mFFOX alone. Tumor HA content will be analyzed. Funding: NIH/NCI grants CA180888, CA180819; and in part by Halozyme Inc. Clinical trial information: 01959139. [Table: see text]

UDP-sugar accumulation drives hyaluronan synthesis in breast cancer

Increased uptake of glucose, a general hallmark of malignant tumors, leads to an accumulation of intermediate metabolites of glycolysis. We investigated whether the high supply of these intermediates promotes their flow into UDP-sugars, and consequently into hyaluronan, a tumor-promoting matrix molecule. We quantified UDP-N-Acetylglucosamine (UDP-GlcNAc) and UDP-glucuronic acid (UDP-GlcUA) in human breast cancer biopsies, the levels of enzymes contributing to their synthesis, and their association with the hyaluronan accumulation in the tumor. The content of UDP-GlcUA was 4 times, and that of UDP-GlcNAc 12 times higher in the tumors as compared to normal glandular tissue obtained from breast reductions. The surge of UDP-GlcNAc correlated with an elevated mRNA expression of glutamine-fructose-6-phosphate aminotransferase 2 (GFAT2), one of the key enzymes in the biosynthesis of UDP-GlcNAc, and the expression of GFAT1 was also elevated. The contents of both UDP-sugars strongly correlated with tumor hyaluronan levels. Interestingly, hyaluronan content did not correlate with the mRNA levels of the hyaluronan synthases (HAS1–3), thus emphasizing the role of the UDP-sugar substrates of these enzymes. The UDP-sugars showed a trend to higher levels in ductal vs. lobular cancer subtypes. The results reveal for the first time a dramatic increase of UDP-sugars in breast cancer, and suggest that their high supply drives the accumulation of hyaluronan, a known promoter of breast cancer and other malignancies. In general, the study shows how the disturbed glucose metabolism typical for malignant tumors can influence cancer microenvironment through UDP-sugars and hyaluronan.

Hyaluronan content governs tissue stiffness in pancreatic islet inflammation

We have identified a novel role for hyaluronan (HA), an extracellular matrix polymer, in governing the mechanical properties of inflamed tissues. We recently reported that insulitis in type 1 diabetes of mice and humans is preceded by intraislet accumulation of HA, a highly hygroscopic polymer. Using the double transgenic DO11.10 × RIPmOVA (DORmO) mouse model of type 1 diabetes, we asked whether autoimmune insulitis was associated with changes in the stiffness of islets. To measure islet stiffness, we used atomic force microscopy (AFM) and developed a novel "bed of nails"-like approach that uses quartz glass nanopillars to anchor islets, solving a long-standing problem of keeping tissue-scale objects immobilized while performing AFM. We measured stiffness via AFM nanoindentation with a spherical indenter and found that insulitis made islets mechanically soft compared with controls. Conversely, treatment with 4-methylumbelliferone, a small-molecule inhibitor of HA synthesis, reduced HA accumulation, diminished swelling, and restored basal tissue stiffness. These results indicate that HA content governs the mechanical properties of islets. In hydrogels with variable HA content, we confirmed that increased HA leads to mechanically softer hydrogels, consistent with our model. In light of recent reports that the insulin production of islets is mechanosensitive, these findings open up an exciting new avenue of research into the fundamental mechanisms by which inflammation impacts local cellular responses.

Определение факторов, влияющих на содержание гиалуронана в ротовой жидкости

Определение факторов, влияющих на содержание гиалуронана в ротовой жидкости

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.

Local injection of high-molecular hyaluronan promotes wound healing in old rats by increasing angiogenesis.

Impaired angiogenesis contributes to delayed wound healing in aging. Hyaluronan (HA) has a close relationship with angiogenesis and wound healing. However, HA content decreases with age. In this study, we used high molecular weight HA (HMW-HA) (1650 kDa), and investigated its effects on wound healing in old rats by local injection. We found that HMW-HA significantly increases proliferation, migration and tube formation in endothelial cells, and protects endothelial cells against apoptosis. Local injection of HMW-HA promotes wound healing by increasing angiogenesis in old rats. HMW-HA increases the phosphorylation of Src, ERK and AKT, leading to increased angiogenesis, suggesting that local injection of HMW-HA promotes wound healing in elderly patients.

Hyaluronan Production by Renomedullary Interstitial Cells: Influence of Endothelin, Angiotensin II and Vasopressin.

The content of hyaluronan (HA) in the interstitium of the renal medulla changes in relation to body hydration status. We investigated if hormones of central importance for body fluid homeostasis affect HA production by renomedullary interstitial cells in culture (RMICs). Simultaneous treatment with vasopressin and angiotensin II (Ang II) reduced HA by 69%. No change occurred in the mRNA expressions of hyaluronan synthase 2 (HAS2) or hyaluronidases (Hyals), while Hyal activity in the supernatant increased by 67% and CD44 expression reduced by 42%. The autocoid endothelin (ET-1) at low concentrations (10−10 and 10−8 M) increased HA 3-fold. On the contrary, at a high concentration (10−6 M) ET-1 reduced HA by 47%. The ET-A receptor antagonist BQ123 not only reversed the reducing effect of high ET-1 on HA, but elevated it to the same level as low concentration ET-1, suggesting separate regulating roles for ET-A and ET-B receptors. This was corroborated by the addition of ET-B receptor antagonist BQ788 to low concentration ET-1, which abolished the HA increase. HAS2 and Hyal2 mRNA did not alter, while Hyal1 mRNA was increased at all ET-1 concentrations tested. Hyal activity was elevated the most by high ET-1 concentration, and blockade of ET-A receptors by BQ123 prevented about 30% of this response. The present study demonstrates an important regulatory influence of hormones involved in body fluid balance on HA handling by RMICs, thereby supporting the concept of a dynamic involvement of interstitial HA in renal fluid handling.

Involvements of p38 MAPK and oxidative stress in the ozone-induced enhancement of AHR and pulmonary inflammation in an allergic asthma model

BackgroundExposure to ambient ozone (O3) increases the susceptivity to allergens and triggers exacerbations in patients with asthma. However, the detailed mechanisms of action for O3 to trigger asthma exacerbations are still unclear.MethodsAn ovalbumin (OVA)-established asthmatic mouse model was selected to expose to filtered air (OVA-model) or 1.0 ppm O3 (OVA-O3 model) during the process of OVA challenge. Next, the possible involvements of p38 MAPK and oxidative stress in the ozone actions on the asthma exacerbations were investigated on the mice of OVA-O3 model by treating them with SB239063 (a p38 MAPK inhibitor), and/or the α-tocopherol (antioxidant). Biological measurements were conducted including airway hyperresponsiveness (AHR), airway resistance (Raw), lung compliance (CL), inflammation in the airway lumen and lung parenchyma, the phosphorylation of p38 MAPK and heat shock protein (HSP) 27 in the tracheal tissues, and the malondialdehyde (MDA) content and the glutathione peroxidase (GSH-Px) activity in lung tissues.ResultsIn OVA-allergic mice, O3 exposure deteriorated airway hyperresponsiveness (AHR), airway resistance (Raw), lung compliance (CL) and pulmonary inflammation, accompanied by the increased oxidative stress in lung tissues and promoted p38 MAPK and HSP27 phosphorylation in tracheal tissues. Administration of SB239063 (a p38 MAPK inhibitor) on OVA-O3 model exclusively mitigated the Raw, the CL, and the BAL IL-13 content, while α-tocopherol (antioxidant) differentially reduced the BAL number of eosinophils and macrophages, the content of BAL hyaluronan, the peribronchial inflammation, as well as the mRNA expression of TNF-α and IL-5 in the lung tissues of OVA-O3 model. Administration of these two chemical inhibitors similarly inhibited the AHR, the BAL IFN-γ and IL-6 production, the perivascular lung inflammation and the lung IL-17 mRNA expression of OVA-O3 model. Interestingly, the combined treatment of both compounds together synergistically inhibited neutrophil counts in the BALF and CXCL-1 gene expression in the lung.ConclusionsO3 exposure during the OVA challenge process promoted exacerbation in asthma. Both p38 MAPK and oxidative stress were found to play a critical role in this process and simultaneous inhibition of these two pathways significantly reduced the O3-elicited detrimental effects on the asthma exacerbation.