Hyaluronan Staining Research Articles

Interleukin-10 regulates fetal extracellular matrix hyaluronan production

Background/PurposeMid-gestational (E14.5) fetal wounds heal regeneratively with attenuated inflammation and high levels of hyaluronan (HA) in their extracellular matrix (ECM), whereas late-gestational (E18.5) fetal wounds heal with scarring. IL-10 plays an essential role in the fetal regenerative phenotype and is shown to recapitulate scarless wound healing postnatally. We hypothesize a novel role of IL-10 as a regulator of HA in the ECM. MethodsMurine fetal fibroblasts (FFb) from C57Bl/6 and IL-10−/− mice were evaluated in vitro. Pericellular matrix (PCM) and HA synthesis were quantified using a particle exclusion assay and ELISA. The effects of hyaluronidase and hyaluronan synthase (HAS) inhibitor (4-methylumbelliferone[4-MU]) were evaluated. An ex vivo fetal forearm culture incisional wound model comparing mid-gestation and late-gestation fetuses was used to evaluate IL-10's effect on HA-rich ECM production with pentachrome and immunohistochemistry. ResultsFFb produce a robust HA-rich PCM which is IL-10 dependent and attenuated with hyaluronidase and HAS inhibition. Mid-gestation fetal wounds produce more ground substance and HA than late-gestation fetal wounds. IL-10 in late-gestation fetal wounds results in elevated ground substance levels and HA staining. ConclusionsOur data demonstrate that IL-10 regulates an HA-rich ECM deposition, suggesting a novel non-immunoregulatory mechanism of IL-10 in mediating regenerative wound healing.

Molecular composition of extracellular matrix in the vestibular nuclei of the rat

Previous studies have demonstrated that the molecular and structural composition of the extracellular matrix (ECM) shows regional differences in the central nervous system. By using histochemical and immunohistochemical methods, we provide here a detailed map of the distribution of ECM molecules in the vestibular nuclear complex (VNC) of the rat. We have observed common characteristics of the ECM staining pattern in the VNC and a number of differences among the individual vestibular nuclei and their subdivisions. The perineuronal net (PNN), which is the pericellular condensation of ECM, showed the most intense staining for hyaluronan, aggrecan, brevican and tenascin-R in the superior, lateral and medial vestibular nuclei, whereas the HAPLN1 link protein and the neurocan exhibited moderate staining intensity. The rostral part of the descending vestibular nucleus (DVN) presented a similar staining pattern in the PNN, with the exception of brevican, which was negative. The caudal part of the DVN had the weakest staining for all ECM molecules in the PNN. Throughout the VNC, versican staining in the PNN, when present, was distinctive due to its punctuate appearance. The neuropil also exhibited heterogeneity among the individual vestibular nuclei in ECM staining pattern and intensity. We find that the heterogeneous distribution of ECM molecules is associated in many cases with the variable cytoarchitecture and hodological organization of the vestibular nuclei, and propose that differences in the ECM composition may be related to specific neuronal functions associated with gaze and posture control and vestibular compensation.

Abstract 3375: Phase 1 pharmacodyamic activity of multiple-dose PEGylated hyaluronidase PH20 (PEGPH20) in patients with solid tumors.

Abstract Enzymatic degradation of hyaluronan (HA), a key component of the extracellular matrix (ECM), can enhance tumor perfusion, improve delivery of chemotherapeutics, and enhance the effects of anti-tumor agents. HA accumulation has been correlated with local invasion, the presence of distal metastasis, higher tumor grade, and poorer overall survival. PEGPH20, a pegylated form of recombinant human hyaluronidase PH20, is an investigational therapeutic agent under clinical development for the treatment of patients with solid tumors that may accumulate HA. Preclinical studies have demonstrated that sustained HA removal, accomplished with PEGPH20, inhibits tumor growth and enhances chemotherapeutic activity in HA-rich xenografts and genetically engineered mouse tumor models. Twenty-six patients with advanced solid tumors were enrolled in a Phase 1 multi-center trial. Patients received weekly or twice weekly doses of IV PEGPH20 (0.5 - 5 μg/kg) for the first 4-week cycle, followed by once-weekly dosing for the subsequent 4-week cycles. Serial blood samples were drawn from each patient, and plasma concentrations were measured by an ultrasensitive hyaluronidase activity assay to assess PEGPH20 pharmacokinetics (PK). Plasma levels of HA catabolites were measured using a quantitative HPLC method to characterize PEGPH20 pharmacodynamics (PD). Other measures of PD activity were evaluated in selected patients, including DCE-MRI, DW-MRI, 18FDG-PET, textural analysis on CT, and histochemical staining of HA in tumor biopsies collected both before and after PEGPH20 treatment. Peak plasma PEGPH20 concentrations increased with dose, and plasma pharmacokinetics (PK) were well described by a linear PK model. Pre-treatment plasma HA levels were typically <1 μg/mL and increased in a dose-dependent fashion after PEGPH20 treatment. Increased plasma HA corresponded with decreased HA staining in patients with available tumor biopsies. DCE-MRI analysis indicated early (24-48 hr) and rapid increase in tumor perfusion compared to baseline in 4 patients with serial MRI assessments. Pharmacodynamic activity was also demonstrated when pre-treatment 18FDG-PET scans were compared with images collected at scheduled times after PEGPH20 dosing. Reduction in FDG uptake exceeding 25% was demonstrated in 3 of 4 patients after one cycle of treatment, consistent with a partial metabolic response. These data are consistent with the mechanism of action reported in preclinical models, and results support continued evaluation of PEGPH20 in combination with anti-tumor agents via modification of the ECM. Citation Format: Ping Jiang, Daniel C. Maneval, Ramesh K. Ramanathan, Jeffrey R. Infante, Mitesh Borad, Alberto Bessudo, Patricia LoRusso, Barry J. Sugarman, Deborah Carson, Marie A. Printz, Curtis B. Thompson, Paneer Selvam, Joy Zhu, Ronald Korn, H Michael Shepard, Gregory I. Frost. Phase 1 pharmacodyamic activity of multiple-dose PEGylated hyaluronidase PH20 (PEGPH20) in patients with solid tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3375. doi:10.1158/1538-7445.AM2013-3375

The Importance of Hyaluronan As a Contributor to Asthma Progression

Lung extracellular matrix (ECM) hyaluronan produced by fibroblasts is pro-inflammatory, inducing cytokine secretion and leukocyte retention. TGFβ-1 induces differentiation of lung fibroblasts to myofibroblasts which secrete more hyaluronan, contributing to airway inflammation and remodeling. In an animal model of asthma, CD44, the hyaluronan receptor, was required for hyaluronan turnover and clearance of eosinophils. We further evaluated the role of CD44 in hyaluranon-related lung inflammatory processes. Lung fibroblasts were treated with +/-TGFβ-1. A scratch-wound culture was used. In previous studies, injury induced hyaluronan production and hyaluronan addition increased cell migration. Cultures were incubated with fluorescent (hyaluronan (fHA), hyaluronan-binding-protein or antibodies to α-smooth muscle actin (αSMA) or CD44. Eosinophils were fluorescently labeled. fHA binding sites on migrating myofibroblasts were primarily along cell margins and the ruffling membrane, and did not correspond to CD44 staining. Hyaluronan staining at wound edge and on migrating cells was upregulated. Hyaluronan-rich cell coats were larger and matrix more cable-like on migrating myofibroblasts. CD44 staining, uniform across the luminal surface, was not altered by scratch-wounding. αSMA staining in response to TGFβ-1, was not changed by CD44 knock-out, despite increased hyaluronan accumulation by myofibroblasts. Hyaluronan-dependent eosinophil retention was increased in ECM induced by TGFβ-1. Hyaluronan binding and accumulation increased on the forward surface of migrating cells. There was increased hyaluronan staining at the wound edge and TGFβ-1 stimulation increased hyaluronan-dependent eosinophil retention. These processes did not correspond to CD44 distribution. Hyaluronan appears to be important in pulmonary inflammation and may be regulated in a CD44-independent manner.

Tumor Necrosis Factor-stimulated Gene-6 (TSG-6) Amplifies Hyaluronan Synthesis by Airway Smooth Muscle Cells

We tested the hypothesis that the artificial addition of heavy chains from inter-α-inhibitor to hyaluronan (HA), by adding recombinant TSG-6 (TNF-stimulated gene-6) to the culture medium of murine airway smooth muscle (MASM) cells, would enhance leukocyte binding to HA cables produced in response to poly(I:C). As predicted, the addition of heavy chains to HA cables enhanced leukocyte adhesion to these cables, but it also had several unexpected effects. (i) It produced thicker, more pronounced HA cables. (ii) It increased the accumulation of HA in the cell-associated matrix. (iii) It decreased the amount of HA in the conditioned medium. Importantly, these effects were observed only when TSG-6 was administered in the presence of poly(I:C), and TSG-6 did not exert any effect on its own. Increased HA synthesis occurred during active, poly(I:C)-induced HA synthesis and did not occur when TSG-6 was added after poly(I:C)-induced HA synthesis was complete. MASM cells derived from TSG-6(-/-), HAS1/3(-/-), and CD44(-/-) mice amplified HA synthesis in response to poly(I:C) + TSG-6 in a manner similar to WT MASM cells, demonstrating that they are expendable in this process. We conclude that TSG-6 increases the accumulation of HA in the cell-associated matrix, partially by preventing its dissolution from the cell-associated matrix into the conditioned medium, but primarily by inducing HA synthesis.

Increased hyaluronan content and stromal cell CD44 associate with HER2 positivity and poor prognosis in human breast cancer

Previous in vitro studies have suggested interactions between hyaluronan (HA), CD44 and HER2. We have studied the expression of HA and CD44 in a material of 278 breast cancer cases, half of which were HER2-positive. Intense stromal HA staining was associated with HER2 positivity, large tumor size, lymph node positivity, hormone receptor negativity, poor differentiation, a high body mass index, increased relapse rate and shortened overall survival. Among the 139 HER2-positive cases, the relapse rate was associated with the intensity of stromal HA staining as most of the relapses occurred in the cases with intense stromal HA staining. The presence of HA in the carcinoma cells was related to the frequency of relapses as none of the patients without HA in carcinoma cells experienced a relapse, whereas 33.3% of those with a high percentage of HA-positive carcinoma cells suffered a relapse. CD44 positivity in carcinoma cells was related to poor differentiation, postmenopausal status and triple negative breast carcinoma. CD44 positivity in stromal cells was associated with HER2 positivity, large tumor size, hormone receptor negativity, poor differentiation, increased relapse rate and shortened overall survival. The association between HER2 positivity and intense stromal HA staining indicates that HA could be one of the factors involved in the unfavorable outcome of HER2-positive patients. This study also suggests that HA in breast carcinoma cells and CD44 in stromal cells may have clinical significance.

Antioxidant Protects against Increases in Low Molecular Weight Hyaluronan and Inflammation in Asphyxiated Newborn Pigs Resuscitated with 100% Oxygen

BackgroundNewborn resuscitation with 100% oxygen is associated with oxidative-nitrative stresses and inflammation. The mechanisms are unclear. Hyaluronan (HA) is fragmented to low molecular weight (LMW) by oxidative-nitrative stresses and can promote inflammation. We examined the effects of 100% oxygen resuscitation and treatment with the antioxidant, N-acetylcysteine (NAC), on lung 3-nitrotyrosine (3-NT), LMW HA, inflammation, TNFα and IL1ß in a newborn pig model of resuscitation.Methods & Principal FindingsNewborn pigs (n = 40) were subjected to severe asphyxia, followed by 30 min ventilation with either 21% or 100% oxygen, and were observed for the subsequent 150 minutes in 21% oxygen. One 100% oxygen group was treated with NAC. Serum, bronchoalveolar lavage (BAL), lung sections, and lung tissue were obtained. Asphyxia resulted in profound hypoxia, hypercarbia and metabolic acidosis. In controls, HA staining was in airway subepithelial matrix and no 3-NT staining was seen. At the end of asphyxia, lavage HA decreased, whereas serum HA increased. At 150 minutes after resuscitation, exposure to 100% oxygen was associated with significantly higher BAL HA, increased 3NT staining, and increased fragmentation of lung HA. Lung neutrophil and macrophage contents, and serum TNFα and IL1ß were higher in animals with LMW than those with HMW HA in the lung. Treatment of 100% oxygen animals with NAC blocked nitrative stress, preserved HMW HA, and decreased inflammation. In vitro, peroxynitrite was able to fragment HA, and macrophages stimulated with LMW HA increased TNFα and IL1ß expression.Conclusions & SignificanceCompared to 21%, resuscitation with 100% oxygen resulted in increased peroxynitrite, fragmentation of HA, inflammation, as well as TNFα and IL1ß expression. Antioxidant treatment prevented the expression of peroxynitrite, the degradation of HA, and also blocked increases in inflammation and inflammatory cytokines. These findings provide insight into potential mechanisms by which exposure to hyperoxia results in systemic inflammation.

Vergetures et laser

Scar tissues were collected from patients with keloids, hypertrophic scars and mature scars. Normal skin was obtained from healthy individuals. Clinical attributes were used to select which tissue to obtain but the distribution of the specific hyaluronan (HA) staining was then used for the definite classification of the various scar types. Light microscopic and ultrastructural analyses were performed with an HA-binding probe, antibodies for collagen I and III and staining for mast cells.Ultrastructural studies of keloids revealed an altered collagen structure in the dermal layers, with an abundance of collagen fibres of similar diameter in both the reticular dermis (RD) and the papillary dermis (PD) compared to normal skin. Furthermore, the keloids displayed epidermal changes, which involved the basement membrane (BM), with fewer hemidesmosomes and an altered shape of desmosomes in the entire enlarged spinous layer. The frequency of mast cells found in keloids was lower than in other scar tissues and normal skin.These alterations in epidermis could influence the hydrodynamic and cell regulatory properties of the wounded skin with impaired function and insufficient regulative capacity to hinder the ever-growing collagen tissue that is characteristic for keloids.

Abstract 2458: The expression of hyaluronan and hyaluronidases in cutaneous melanoma

Abstract Aberrant hyaluronan expression implicates aggressive disease progression and metastatic potential in many cancers. In tumors originating from simple epithelium, hyaluronan amount is increased both in the epithelial and stromal component and it correlates with patient's poor overall prognosis. The role of hyaluronan degrading enzymes (hyaluronidases, HYAL1-2) in cancer progression is controversial. Hyaluronidases are proposed to be both tumor suppressors and tumor promoters. In this retrospective study, we characterized the expression levels of HYAL1 and HYAL2 and correlated their expression to hyaluronan in the different stages of primary skin melanoma as well as in metastatic melanoma. Superfically (pT1) and deeply (pT4) invasive melanoma (Breslow < 1mm (n=18) and Breslow > 4mm (n=18)), in situ melanoma (n=17), lymph node metastasis (n=19), dysplastic nevi (n=28) and benign nevi samples (n=29) were analyzed for hyaluronan staining and HYAL1 and HYAL2 immunoreactivity (ABCAM antibodies) in paraffin tissue sections. Stainings were evaluated for their intensity and coverage. The tissue area for hyaluronan and HYAL1/HYAL2 positive staining was estimated with a five-level scoring from 0-4 and the staining intensity of melanocytic cells was evaluated with a four-level scoring (0-3; no color, weak, moderate and strong) by two independent observers. Compared to benign nevi and in situ melanoma, the cell-associated hyaluronan staining coverage and intensity were clearly reduced in the invasive part of melanoma with advanced Breslow depth. Strong hyaluronan staining intensity was detected in 36% of in situ melanoma cases and in 21% of superficial melanoma (Breslow < 1mm) cases, while in the deeply invasive melanoma group (Breslow > 4mm), only 6% of cases showed strong hyaluronan staining intensity. In addition, in lymph node metastases the melanoma cells showed low hyaluronan staining intensity or were totally hyaluronan negative. However, the tumor stroma was highly hyaluronan positive in all groups. HYAL1 immunoreactivity was found in melanocytic cells both in benign nevi and melanoma samples while the tumor stromal cells were negative. The coverage of HYAL2 immunoreactivity was parallel to HYAL1, although its staining intensity was lower than that of HYAL1. Our results suggest that hyaluronan content is clearly decreased in invasive primary and metastatic melanoma compared to benign nevi. The results from hyaluronan stainings will be compared with clinical follow-up data to study the prognostic value of hyaluronan in melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2458. doi:1538-7445.AM2012-2458

Hyaluronan and Collagen in Human Hypertrophic Cardiomyopathy: A Morphological Analysis

Introduction. The hypertrophic cardiomyopathy (HCM) disease process is not only limited to cardiomyocyte abnormalities but also engages the extracellular matrix. Hyaluronan (HA) and its receptor CD44 are involved in cellular growth and tissue proliferation but have so far been less studied in myocardial hypertrophy. In HCM, collagens are abundant but their histological distribution and relation to hyaluronan have not been described. Material and Methods. Myocardial specimens from 5 patients with symptomatic left ventricular tract obstruction undergoing myectomy due to HCM were processed for histochemistry and immunohistochemistry. Results. HA staining was more intense in HCM patients. The histological distribution of HA was the same in patients and controls, that is, interstitial staining including the space between cardiomyocytes, in fibrous septa, and in the adventitia of intramyocardial blood vessels. CD44 was not detected in the myocardium of patients or controls. Collagen I showed the same general localisation as HA but detailed distribution differed. Conclusions. This is the first study that describes the distribution of hyaluronan in human HCM. HA staining is more intense in HCM patients but without coexpression of its receptor CD44, at least not in the chronic phase of HCM. HA and collagen I have the same localisation.

Abstract B35: Hyaluronan (HA) accumulation in tumors predicts response to pegylated rHuPH20 hyaluronidase (PEGPH20) in human tumors: A biomarker strategy.

Abstract Increasing evidence suggests that the tumor microenvironment (TME) is a potential source of novel anti-tumor targets. Extracellular matrix components in the TME include the matrix glycosaminoglycan, hyaluronan (HA), which accumulates in 20–30% of solid tumors. HA functions to increase water content of HA-rich stroma, thereby creating high tumor interstitial fluid pressure (IFP) characteristic of many tumors. Cells engineered to overexpress hyaluronan synthase form tumors more readily in mouse models, and HA accumulation has been associated with disease progression and poor prognosis in human cancers. Enzymatic degradation of HA with hyaluronidase can reduce IFP and facilitate delivery of anti-tumor agents. We have previously presented data that demonstrate the therapeutic potential of PEGPH20 in tumor xenografts derived from a spectrum of human malignancies. Here we highlight the utility of HA detection methods that use aggrecan-derived HA binding region (HABR). Accumulation of HA was measured in tumor tissues from 14 different xenografts and compared with anti-tumor activity of PEGPH20. When cell line-derived tumors were classified according to HABR staining intensity as HA+1,2,3 (low to high staining), the anti-tumor activity of PEGPH20 correlated with higher accumulation of HA in tumor specimens (r=0.8). To more rigorously test the hypothesis that HA-depletion is an effective candidate therapeutic approach for cancer, we utilized non-small cell lung cancer (SCC) patient tumor explants which were characterized for HA accumulation in advance of implantation into immunodeficient mice. Three SCC explants (HA+1,2,3) were treated with PEGPH20 and tumor growth was measured over a 2-week interval. Tumor growth inhibition corresponded to HA accumulation status, with the most dramatic reduction (97%; 4/10 regressions) observed in the HA+3 SCC explants. We have extended this work and detected tumor HA in pre- and post-treatment tumor biopsies collected from two patients enrolled in a Phase 1 study with PEGPH20. In both sets of specimens, a reduction in HA accumulation was demonstrated following PEGPH20 treatment. These results provide a rationale to assess tumor HA staining as a promising biomarker for clinical investigation with PEGPH20 for the treatment of solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B35.

Role of CD44 in the organization of keratinocyte pericellular hyaluronan

CD44 is a ubiquitous cell surface glycoprotein, involved in important cellular functions including cell adhesion, migration, and modulation of signals from cell surface receptors. While most of these CD44 functions are supposed to involve hyaluronan, relatively little is known about the contribution of CD44 to hyaluronan maintenance and organization on cell surface, and the role of CD44 in hyaluronan synthesis and catabolism. Blocking hyaluronan binding either by CD44 antibodies, CD44-siRNA or hyaluronan decasaccharides (but not hexasaccharides) removed most of the hyaluronan from the surfaces of both human (HaCaT) and mouse keratinocytes, resembling results on cells from CD44⁻/⁻ animals. In vitro, compromising CD44 function led to reduced and increased amounts, respectively, of intracellular and culture medium hyaluronan, and specific accumulation below the cells. In vivo, CD44-deficiency caused no marked differences in hyaluronan staining intensity or localization in the fetal skin or in adult ear skin, while tail epidermis showed a slight reduction in epidermal hyaluronan staining intensity. However, CD44-deficient tail skin challenged with retinoic acid or tape stripping revealed diffuse accumulation of hyaluronan in the superficial epidermal layers, normally negative for hyaluronan. Our data indicate that CD44 retains hyaluronan in the keratinocyte pericellular matrix, a fact that has not been shown unambiguously before, and that hyaluronan abundance in the absence of CD44 can result in hyaluronan trapping in abnormal locations possibly interfering there with normal differentiation and epidermal barrier function.

Impact of γ-Irradiation on Extracellular Matrix of Porcine Pulmonary Valves

The extracellular matrix plays an important role in heart valve function. To improve the processing of porcine pulmonary valves for clinical use, we have studied the influence of cryopreservation, decellularization, and irradiation on extracellular matrix components. Decellularization was carried out followed by DNAseI/RNAseA digestion and isotonic washout. Valves were cryopreserved in 10% DMSO/10% fetal bovine serum, and then subjected to 25-40 kGy γ-radiation. Extracellular matrix constituents were evaluated by histologic staining, immunohistochemistry, transmission electron microscopy, and liquid chromatography/mass spectrometry. Histologic, immunohistochemical, ultrastructural, and biochemical analyses demonstrated a marked reduction in the expression of extracellular matrix components particularly in the valves that had been γ-irradiated following decellularization and cryopreservation. In this group, histology and immunohistochemistry showed an obvious reduction in staining for chondroitin sulphates, versican, hyaluronan, and collagens. Transmission electron microscopy revealed the smallest fibril diameter of collagen, shortest D-period, and loss of compactness of collagen fiber packaging and fragmentation of elastic fibers. Biochemical analysis showed loss of collagen and elastin crosslinks. Decellularization followed by cryopreservation showed some reduction in staining for collagens and versican, smaller diameter, shorter D-period in collagen fibers, and ridges in elastic fibers. Cryopreservation alone showed minimal changes in ECM staining intensity, collagen, and elastin ultrastructure and biochemistry. γ-Irradiated valves that have been decellularized and cryopreserved produces significant changes in the expression of ECM components, thus providing useful information for improving valve preparation for clinical use and also some indication as to why irradiated human heart valves were not clinically successful.

Prostate Cancer Increases Hyaluronan in Surrounding Nonmalignant Stroma, and This Response Is Associated with Tumor Growth and an Unfavorable Outcome

Our objective was to investigate whether the presence of a tumor increases hyaluronan (HA) levels in surrounding prostate tissues and whether this extratumoral HA influences tumor growth and outcome. From a series of 287 men diagnosed with prostate cancer at transurethral resection and followed up with watchful waiting, tissue microarrays were constructed, stained, and scored for HA. A high HA staining score in the tumor stroma or in nonmalignant prostate tissue stroma were both associated positively with higher Gleason score and larger tumor volume, and was associated with a poor outcome. HA staining score was not an independent marker for outcome (multivariate Cox, with Gleason score, tumor volume, stage, and HA variables). In an orthotopic rat prostate cancer model, hyaluronic acid synthase-1 mRNA levels and HA staining were increased in normal prostate tissue surrounding prostate cancer. Orthotopic prostate cancer growth was increased by intraprostatic injection of HA. In conclusion, cancer in the prostate apparently stimulates HA synthesis both in tumor stroma and in the surrounding normal tissue. This promoted tumor growth and was associated with an unfavorable outcome.

Viewing Hyaluronan

Hyaluronan (HA) is an ubiquitous extracellular matrix polymer that plays many roles in health and disease. The ability to view the spatial and temporal expression of HA in tissues and on/in cells has provided researchers with insights into the tremendously diverse biological processes in which HA is involved. Biochemical extraction, quantity, and size measurement of HA can tell part of the story, but these techniques are incomplete in placing HA at the scene of a biological event and determining which other molecules are likely to be cooperating. HA, however, is not immunogenic, so preparing antibodies for histochemistry is problematic. Fortunately, a probe for HA was devised based on the HA binding region of aggrecan, and today this probe is commercially available and very useful for histochemistry. This article discusses the conditions and considerations that the authors' lab and others have developed for optimal HA staining in many tissues and cell types.

Characterization of hyaluronan and TSG‐6 in skin scarring: differential distribution in keloid scars, normal scars and unscarred skin

Background Hyaluronan (HA) is a major component of the extracellular matrix (ECM) with increased synthesis during tissue repair. Tumour necrosis factor-stimulated gene-6 (TSG-6) is known to catalyze the covalent transfer of heavy chains (HC1 and HC2) from inter-α-inhibitor (IαI) onto HA, and resultant HC•HA complexes have been implicated in physiological and pathological processes related to remodelling and inflammation.Objective The aims of this study were to determine the expression of HA, TSG-6 and the IαI polypeptides in unscarred skin, normal scars and keloid scars.Methods Formalin-fixed paraffin-embedded sections of unscarred skin, normal scars and keloid scars were prepared from patient samples collected during scar revision surgery. Haematoxylin and eosin, as well as immunofluorescent staining for HA, TSG-6 and the three polypeptide chains of IαI (i.e. HC1, HC2 and bikunin) were performed.Results All skin types stained positive for TSG-6, HC1, HC2 and bikunin, associated with keratinocytes, fibroblasts and skin appendages all in close proximity to HA. Keloid lesions showed altered HA organization patterns compared with unscarred skin and normal scars. TSG-6 staining was significantly more intense in the epidermis compared with the dermis of all sample types. There was a significant reduction in TSG-6 levels within keloid lesions compared with the dermis of unscarred skin (P = 0.017).Conclusion TSG-6 is expressed in unscarred skin, where its close association with HA and IαI could give rise to TSG-6-mediated HC•HA formation within this tissue. A reduction in the beneficial effects of TSG-6, caused by diminished protein levels in keloid lesions, could contribute to this abnormal scarring process.

Hyaluronan, TSG-6, and inter-α-inhibitor in periprosthetic breast capsules: reduced levels of free hyaluronan and TSG-6 expression in contracted capsules.

The exact mechanism of capsular contracture (CC) is still unknown. The covalent modification of hyaluronan (HA) with the heavy chains (HC) of inter-α-inhibitor (IαI) has been identified as an important pathway in inflammation and tissue remodeling, where HC·HA formation is catalyzed by TSG-6 (the protein product of tumor necrosis factor stimulated gene-6). The authors quantitatively assess the correlation between severity of CC (measured by Baker grade) and expression of HA, TSG-6, and IαI (ie, the polypeptides HC1, HC2, and bikunin) in periprosthetic breast capsules. Immunofluorescent staining for HA, TSG-6, HC1, HC2, and bikunin was carried out on periprosthetic breast capsules (n = 7) of each Baker grade from four anatomical locations. Quantitative analysis was performed to identify differences in staining intensity. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to determine differences in TSG-6 gene expression levels. Severity of contracture was associated with reduced staining for both free HA (Pearson correlation coefficient, r = -0.645, P < .001) and TSG-6 (r = -0.642, P = .002). RT-qPCR showed a significant negative correlation between severity of contracture and TSG-6 gene expression levels (r = -0.750, P = .001). The negative correlation between TSG-6 expression levels and severity of CC suggests a possible protective role for TSG-6 in the context of CC formation, and this may have a clinically relevant role in prevention of breast CC.

Intra‐articular injection of hyaluronan restores the aberrant expression of matrix metalloproteinase‐13 in osteoarthritic subchondral bone

Subchondral bone is a candidate for treatment of osteoarthritis (OA). We investigated the effects of intra-articular injection of hyaluronan (IAI-HA) on subchondral bone in rabbit OA model. OA was induced by anterior cruciate ligament transection, with some rabbits receiving IAI-HA. OA was graded morphologically, and expression of mRNA was assessed by real-time RT-PCR. Tissue sections were stained with hyaluronan-binding protein, and penetration of fluorescent hyaluronan was assessed. The in vitro inhibitory effect of hyaluronan on MMP-13 was analyzed in human osteoarthritic subchondral bone osteoblasts (OA Ob) by real-time RT-PCR and ELISA. Binding of hyaluronan to OA Ob via CD44 was assessed by immunofluorescence cytochemistry. Expression of MMP-13 and IL-6 mRNA in cartilage and subchondral bone, and morphological OA grade, increased over time. IAI-HA ameliorated the OA grade and selectively suppressed MMP-13 mRNA in subchondral bone. IAI-HA enhanced the hyaluronan staining of subchondral bone marrow cells and osteocyte lacunae. Fluorescence was observed in the subchondral bone marrow space. In OA Ob, hyaluronan reduced the expression and production of MMP-13, and anti-CD44 antibody blocked hyaluronan binding to OA Ob. These findings indicate that regulation of MMP-13 in subchondral bone may be a critical mechanism during IAI-HA.

Hyaluronan synthases (HAS1-3) and hyaluronidases (HYAL1-2) in the accumulation of hyaluronan in endometrioid endometrial carcinoma

BackgroundHyaluronan accumulation correlates with the degree of malignancy in many solid tumor types, including malignant endometrial carcinomas. To elucidate the mechanism of hyaluronan accumulation, we examined the expression levels of the hyaluronan synthases (HAS1, HAS2 and HAS3) and hyaluronidases (HYAL1 and HYAL2), and correlated them with hyaluronan content and HAS1-3 immunoreactivity.MethodsA total of 35 endometrial tissue biopsies from 35 patients, including proliferative and secretory endometrium (n = 10), post-menopausal proliferative endometrium (n = 5), complex atypical hyperplasia (n = 4), grade 1 (n = 8) and grade 2 + 3 (n = 8) endometrioid adenocarcinomas were divided for gene expression by real-time RT-PCR, and paraffin embedded blocks for hyaluronan and HAS1-3 cytochemistry.ResultsThe mRNA levels of HAS1-3 were not consistently changed, while the immunoreactivity of all HAS proteins was increased in the cancer epithelium. Interestingly, HAS3 mRNA, but not HAS3 immunoreactivity, was increased in post-menopausal endometrium compared to normal endometrium (p = 0.003). The median of HYAL1 mRNA was 10-fold and 15-fold lower in both grade 1 and grade 2+3 endometrioid endometrial cancers, as compared to normal endometrium (p = 0.004-0.006), and post-menopausal endometrium (p = 0.002), respectively. HYAL2 mRNA was also reduced in cancer (p = 0.02) and correlated with HYAL1 (r = 0.8, p = 0.0001). There was an inverse correlation between HYAL1 mRNA and the epithelial hyaluronan staining intensity (r = -0.6; P = 0.001).ConclusionThe results indicated that HYAL1 and HYAL2 were coexpressed and significantly downregulated in endometrioid endometrial cancer and correlated with the accumulation of hyaluronan. While immunoreactivity for HASs increased in the cancer cells, tumor mRNA levels for HASs were not changed, suggesting that reduced turnover of HAS protein may also have contributed to the accumulation of hyaluronan.

Evaluation of hyaluronan and calcifications in stenotic and regurgitant aortic valves☆

Hyaluronan (HA) is a major component of the interstitium and has been observed in normal heart valves. The function of HA in heart valves is unknown but contribution to biomechanical function has been proposed. The purpose of this investigation was to study the distribution of HA in relation to calcifications in diseased human aortic valves. Human aortic valves were collected at aortic valve replacement, of whom nine patients had regurgitation and 13 stenotic disease. The valves were decalcified and stained for the visualisation of HA. The specimens were macroscopically evaluated for magnitude of calcification using image analysis. The microscopic amount and distribution of HA and calcifications were semiquantitatively evaluated using histochemistry. The overall HA staining showed an inverse relationship against the magnitude of observed valve calcifications (p<0.001) and type of disease (p=0.014). Multiple-group comparison revealed regionally reduced HA staining in diffuse and heavy calcified regions inside the valve (both p<0.001) compared with normal-structured parts of the valve. HA was concentrated on the ventricular side of the valve (p=0.002). The content of HA was reduced in calcified aortic valves and had a heterogeneous distribution, potentially contributing to poor valve function. HA may also be involved in the pathophysiological process in degenerative aortic stenosis.