Chronic UVR Causes Increased Immunostaining of CD44 and Accumulation of Hyaluronan in Mouse Epidermis

Chronic intense UV radiation is the main cause of epidermal tumors. Because hyaluronan (HA), a large extracellular polysaccharide, is known to promote malignant growth, hyaluronan expression was studied in a model in which long-term UV radiation (UVR) induces epidermal tumors. Mouse back skin was exposed three times a week for 10.5 months to UVR corresponding to one minimal erythema dose, processed for histology, and stained for hyaluronan and the hyaluronan receptor CD44. This exposure protocol caused epidermal hyperplasia in most of the animals; tumors, mainly squamous cell carcinomas (SCCs), were found in ~20% of the animals. Specimens exposed to UVR showed increased hyaluronan and CD44 staining throughout the epidermal tissue. In hyperplastic areas, hyaluronan and CD44 stainings correlated positively with the degree of hyperplasia. Well-differentiated SCCs showed increased hyaluronan and CD44 staining intensities, whereas poorly differentiated tumors and dysplastic epidermis showed areas where HA and CD44 were locally reduced. The findings indicate that HA and CD44 increase in epidermal keratinocytes in the premalignant hyperplasia induced by UV irradiation and stay elevated in dysplasia and SCC, suggesting that the accumulation of hyaluronan and CD44 is an early marker for malignant transformation and may be a prerequisite for tumor formation.     

Accessible hyaluronan receptors identical to ICAM-1 in mouse mast-cell tumours

Immunohistochemical studies of the hyaluronan (HA)-receptor (R), originally found on liver endothelial cells (LEC) and related to the intercellular adhesion molecule 1 (ICAM-1), showed that polyclonal antibodies against HARLEC (HA receptor on LEC) also stain structures in mouse mastocytomas, mainly vessels. To test if intravenously administered HA might target the tumour receptorsin vivo, mice carrying an inoculated mastocytoma in one hind leg muscle were injected in the tail vein with¹²⁵I-tyrosine (T)-labelled HA and killed 75 min after injection when organs and tissues were checked for radioactivity. When doses exceeding the binding capacity of the liver were injected, a significant increase in radioactivity (up to five-fold) within the tumour tissue was found. The weight adjusted difference between control and tumour tissue was greater for smaller tumours, probably due to necrosis in the larger. HA-staining of tumours from animals receiving¹²⁵I-T-HA, showed HA in areas that also stained weakly for ICAM-1 using monoclonal antibodies. ICAM-1 staining was dramatically increased after hyaluronidase treatment of the sections, indicating that the HA is bound to these receptors and thereby blocks antibody recognition.Abbreviations ICAM-1 intercellular adhesion molecule 1 - HA hyaluronan - HARLEC hyaluronan receptor on liver endothelial cells - MW molecular weight     

BMP-2 Induces Versican and Hyaluronan That Contribute to Post-EMT AV Cushion Cell Migration

Distal outgrowth and maturation of mesenchymalized endocardial cushions are critical morphogenetic events during post-EMT atrioventricular (AV) valvuloseptal morphogenesis. We explored the role of BMP-2 in the regulation of valvulogenic extracellular matrix (ECM) components, versican and hyaluronan (HA), and cell migration during post-EMT AV cushion distal outgrowth/expansion. We observed intense staining of versican and HA in AV cushion mesenchyme from the early cushion expansion stage, Hamburger and Hamilton (HH) stage-17 to the cushion maturation stage, HH stage-29 in the chick. Based on this expression pattern we examined the role of BMP-2 in regulating versican and HA using 3D AV cushion mesenchymal cell (CMC) aggregate cultures on hydrated collagen gels. BMP-2 induced versican expression and HA deposition as well as mRNA expression of versican and Has2 by CMCs in a dose dependent manner. Noggin, an antagonist of BMP, abolished BMP-2-induced versican and HA as well as mRNA expression of versican and Has2. We further examined whether BMP-2-promoted cell migration was associated with expression of versican and HA. BMP-2- promoted cell migration was significantly impaired by treatments with versican siRNA and HA oligomer. In conclusion, we provide evidence that BMP-2 induces expression of versican and HA by AV CMCs and that these ECM components contribute to BMP-2-induced CMC migration, indicating critical roles for BMP-2 in distal outgrowth/expansion of mesenchymalized AV cushions.     

Hyaluronate and CD44 expression patterns in the human placenta throughout pregnancy

Hyaluronan (HA) and CD44 are involved in several processes such as cell migration and differentiation. In the present study, we examined the expression and distribution of both hyaluronan and its cell surface receptor (CD44) in the human placenta, which is a rapidly growing and differentiating organ that plays a fundamental role in fetal life. Hyaluronan was detected by a specific biotinylated binding probe, termed b-PG. In the first half of gestation, HA was strongly expressed in the stroma of the mesenchymal villi which have been previously identified as responsible for the growth and differentation of the villous trees. The other villous types showed an intense staining only in the fetal vessel walls and in the connective tissue closely underlying the trophoblastic cover. In addition, hyaluronan positive staining was also apparent in a restricted rim of villous stroma directly apposed to extravillous cytotrophoblastic cell islands and cell columns. In full term placentas, all villi expressed HA in their stromal tissue with a more homogenous staining than in the first half of gestation. In contrast to hyaluronan, in the first trimester CD44 was restricted to some of the Hofbauer cells which may be able to internalize hyaluronan, thus playing a significant role in its removal in early pregnancy. CD44 was primarily expressed starting from the 16th week of gestation. At the end of pregnancy it was expressed in the various villous types, especially in stem villi. Moreover, the plasma membrane of some extravillous cytotrophoblastic cells in the basal plate and the large majority of the decidual cells showed a positive immunostaining for this receptor. Taken together, these data suggest that HA is strongly involved in early villous morphogenesis, whereas CD44 seem to be play an important role in tissue remodelling later in gestation.     

微波快速免疫荧光组化染色方法的研究

本文应用微波辐射方法加速免疫荧光组化染色(间接和直接法),分别定位15种不同组织抗原,并应用连续切片同时用两种不同的孵育方法即微波辐射和常规孵育方法进行比较。结果证明,经微波辐射后免疫荧光组化染色时间大大缩短,背景染色明显好于常规法,阳性率和阳性强度与常规法基本一致。     

Inhibition of Hyaluronan Synthesis Protects against Central Nervous System (CNS) Autoimmunity and Increases CXCL12 Expression in the Inflamed CNS

Hyaluronan (HA) may have proinflammatory roles in the context of CNS autoimmunity. It accumulates in demyelinated multiple sclerosis (MS) lesions, promotes antigen presentation, and enhances T-cell activation and proliferation. HA facilitates lymphocyte binding to vessels and CNS infiltration at the CNS vascular endothelium. Furthermore, HA signals through Toll-like receptors 2 and 4 to stimulate inflammatory gene expression. We assessed the role of HA in experimental autoimmune encephalomyelitis (EAE), an animal model of MS by administration of 4-methylumbelliferone (4MU), a well established inhibitor of HA synthesis. 4MU decreased hyaluronan synthesis in vitro and in vivo. It was protective in active EAE of C57Bl/6 mice, decreased spinal inflammatory infiltrates and spinal infiltration of Th1 cells, and increased differentiation of regulatory T-cells. In adoptive transfer EAE, feeding of 4MU to donor mice significantly decreased the encephalitogenicity of lymph node cells. The transfer of proteolipid protein (PLP)-stimulated lymph node cells to 4MU-fed mice resulted in a delayed EAE onset and delayed spinal T-cell infiltration. Expression of CXCL12, an anti-inflammatory chemokine, is reduced in MS patients in CSF cells and in spinal cord tissue during EAE. Hyaluronan suppressed production of CXCL12, whereas 4MU increased spinal CXCL12 in naive animals and during neuroinflammation. Neutralization of CXCR4, the most prominent receptor of CXCL12, by administration of AMD3100 diminished the protective impact of 4MU in adoptive transfer EAE. In conclusion, hyaluronan exacerbates CNS autoimmunity, enhances encephalitogenic T-cell responses, and suppresses the protective chemokine CXCL12 in CNS tissue. Inhibition of hyaluronan synthesis with 4MU protects against an animal model of MS and may represent an important therapeutic option in MS and other neuroinflammatory diseases.     

Hyaluronan fragments induce endothelial cell differentiation in a CD44- and CXCL1/GRO1-dependent manner

Hyaluronan is a glycosaminoglycan of the extracellular matrix. In tumors and during chronic inflammatory diseases, hyaluronan is degraded to smaller fragments, which are known to stimulate endothelial cell differentiation. In this study, we have compared the molecular mechanisms through which hyaluronan dodecasaccharides (HA12), and the known angiogenic factor, fibroblast growth factor 2 (FGF-2), induce capillary endothelial cell sprouting in a three-dimensional collagen gel. The gene expression profiles of unstimulated and HA12- or FGF-2-stimulated endothelial cells were compared using a microarray analysis approach. The data revealed that both FGF-2 and HA12 promoted endothelial cell morphogenesis in a process depending on the expression of ornithine decarboxylase (Odc) and ornithine decarboxylase antizyme inhibitor (Oazi) genes. Among the genes selectively up-regulated in response to HA12 was the chemokine CXCL1/GRO1 gene. The notion that the induction of CXCL1/GRO1 is of importance for HA12-induced endothelial cell sprouting was supported by the fact that morphogenesis was inhibited by antibodies specifically neutralizing the CXCL1/GRO1 protein product. HA12-stimulated endothelial cell differentiation was exerted via binding to CD44 since it was inhibited by antibodies blocking CD44 function. Our data show that hyaluronan fragments and FGF-2 affect endothelial cell morphogenesis by the induction of overlapping but also by distinct sets of genes.     

Hyaluronan involvement in the changes of mouse interpubic tissue during late pregnancy and post-partum

The present work quantifies hyaluronan (HA) during the late pregnancy and post-partum in order to provide a better understanding of the role of HA in the adaptations that occur in the pubic symphysis during this period. HA was quantified in situ (histochemically) and in interpubic tissue extracts by fluorimetric assay. Samples were taken from virgin mice and from pregnant animals at various stages of pregnancy: 12th-18th days into pregnancy, the day of delivery (D19) and the 3rd and 5th day post-partum. The quantitative fluorimetric analysis indicated a gradual increase of HA in the interpubic tissue throughout late pregnancy (2.4-14.6 microg/mg dry weight). This was followed by a decrease beginning on D19 (12.4 microg/mg), reaching close to virgin levels (2.2 microg/mg) on the 5th day post-partum. The same optical density changes could be seen in the HA staining. Furthermore, the histochemical analysis demonstrated the presence of HA both in the extracellular matrix of the tissue and within its cells. Such results indicate that the extracellular presence of HA may contribute to the transformation of the symphysis into a flexible structure. In addition, HA's intracellular presence (until the 18th day of pregnancy) may contribute to cellular proliferation. Finally, during parturition and on the 5th day post-partum, HA may contribute to the maintenance of the myofibroblastic phenotype of ligament cells, aiding the ligament involution after parturition.     

Structural and conformational differences of acylated hyaluronan modified in protic and aprotic solvent system

Acylated hyaluronan (HA) in aqueous (DMSO/H₂O) and nonaqueous (DMSO) solutions was studied by means of nuclear magnetic resonance, differential scanning calorimetry (DSC), mass spectrometry and UV/vis spectroscopy. It has been demonstrated that structural and conformational properties of the acylated hyaluronan derivates are strongly dependent on the nature of reaction solvent. Acylation in DMSO was more selective than that carried out in DMSO/H₂O, though in both cases in average a maximum of one acyl chain was detected per HA dimer. The hydrophobic functionalization of hyaluronan induced its interaction with hydrophobic dye as a consequence of acyl chain aggregation. The higher the degree of acylation the more hydrophobic dye was interacting with HA. For concentrated samples, aggregation was more evident in case of acylated HA in aqueous solution. This phenomenon was explained by its different conformational arrangement in solution which was further supported by DSC data indicating an existence of hydrophobic cavities. The formation of self-aggregated assemblies indicates potential applications of this type of HA derivate as drug delivery system.     

Hyaluronan synthesis in cultured tobacco cells (BY‐2) expressing a chlorovirus enzyme: Cytological studies

Extraction of hyaluronan from animals or microbial fermentation has risks including contamination with pathogens and microbial toxins. In this work, tobacco cultured‐cells (BY‐2) were successfully transformed with a chloroviral hyaluronan synthase (cvHAS) gene to produce hyaluronan. Cytological studies revealed accumulation of HA on the cells, and also in subcellular fractions (protoplasts, miniplasts, vacuoplasts, and vacuoles). Transgenic BY‐2 cells harboring a vSPO‐cvHAS construct containing the vacuolar targeting signal of sporamin connected to the N‐terminus of cvHAS accumulated significant amounts of HA in vacuoles. These results suggested that cvHAS successfully functions on the vacuolar membrane and synthesizes/transports HA into vacuoles. Efficient synthesis of HA using this system provides a new method for practical production of HA. Biotechnol. Bioeng. 2013; 110: 1174–1179. © 2012 Wiley Periodicals, Inc.     

Angiotensin converting enzyme inhibition blocks interstitial hyaluronan dissipation in the neonatal rat kidney via hyaluronan synthase 2 and hyaluronidase 1

A functional renin-angiotensin system (RAS) is required for normal kidney development. Neonatal inhibition of the RAS in rats results in long-term pathological renal phenotype and causes hyaluronan (HA), which is involved in morphogenesis and inflammation, to accumulate. To elucidate the mechanisms, intrarenal HA content was followed during neonatal completion of nephrogenesis with or without angiotensin converting enzyme inhibition (ACEI) together with mRNA expression of hyaluronan synthases (HAS), hyaluronidases (Hyal), urinary hyaluronidase activity and cortical lymphatic vessels, which facilitate the drainage of HA from the tissue. In 6-8 days old control rats cortical HA content was high and reduced by 93% on days 10-21, reaching adult low levels. Medullary HA content was high on days 6-8 and then reduced by 85% to 12-fold above cortical levels at day 21. In neonatally ACEI-treated rats the reduction in HA was abolished. Temporal expression of HAS2 corresponded with the reduction in HA content in the normal kidney. In ACEI-treated animals cortical HAS2 remained twice the expression of controls. Medullary Hyal1 increased in controls but decreased in ACEI-treated animals. Urine hyaluronidase activity decreased with time in control animals while in ACEI-treated animals it was initially 50% lower and did not change over time. Cells expressing the lymphatic endothelial mucoprotein podoplanin in ACEI-treated animals were increased 18-fold compared to controls suggesting compensation. In conclusion, the high renal HA content is rapidly reduced due to reduced HAS2 and increased Hyal1 mRNA expressions. Normal angiotensin II function is crucial for inducing these changes. Due to the extreme water-attracting and pro-inflammatory properties of HA, accumulation in the neonatally ACEI-treated kidneys may partly explain the pathological renal phenotype of the adult kidney, which include reduced urinary concentration ability and tubulointerstitial inflammation.     

A pericellular hyaluronan matrix is required for the morphological maturation of cortical neurons.

BackgroundHyaluronan (HA) is a major component of the extracellular matrix (ECM) and is involved in many cellular functions. In the adult brain, HA forms macromolecular aggregates around synapses and plays important roles in neural plasticity. In contrast to the well-characterized function of HA in the adult brain, its roles in the developing brain remain largely unknown.MethodsBiochemical and histochemical analyses were performed to analyze the amount, solubility, and localization of HA in the developing mouse brain. By combining in utero labeling, cell isolation, and in vitro cultures, we examined the expression of hyaluronan synthase (HAS) and morphological maturation of cortical neurons.ResultsThe amount of HA increased during perinatal development and decreased in the adult. HA existed as a soluble form in the early stages; however, its solubility markedly decreased during postnatal development. HA localized in cell-sparse regions in the embryonic stages, but was broadly distributed during the postnatal development of the cerebral cortex. Developing cortical neurons expressed both Has2 and Has3, but not Has1, suggesting the autonomous production of HA by neurons themselves. HA formed a pericellular matrix around the cell bodies and neurites of developing cortical neurons, and the inhibition of HA synthesis reduced neurite outgrowth.ConclusionThe formation of the pericellular HA matrix is essential for the proper morphological maturation of developing neurons.General significanceThis study provides new insights into the roles of hyaluronan in the brain.development.     

Mammalian hyaluronan synthases

Three mammalian hyaluronan (HA) synthase genes, HAS1, HAS2, and HAS3, have been cloned and expressed, allowing the mechanisms for regulation of HA biosynthesis and function to be studied. The hyaluronan synthase (HAS) isoforms differ in kinetic characteristics and product size. The expression of each HAS isoform is controlled in a different fashion when mammalian cells are stimulated by various cytokines and the expression patterns are both spatially and temporally regulated during embryonic development. The existence of three different HAS isoforms with different characteristics implies that the broad range of biological and physiological roles performed by HA are regulated by controlling the activities and expression of the HAS isoforms. This review focuses on recent findings on the regulatory mechanisms for controlling HA biosynthesis and provides new insights into the enzymic basis for the functional regulation of HA.     

Collagen fragments inhibit hyaluronan synthesis in skin fibroblasts in response to ultraviolet B (UVB): new insights into mechanisms of matrix remodeling

UVB irradiation causes characteristic features of skin aging including remodeling of the dermal extracellular matrix. A key feature during this process is the up-regulation of matrix metalloproteinases and cleavage of collagen. Hyaluronic acid (HA), a major component of the dermal matrix, decreases after chronic UVB exposure. However, the factors that govern the decline of HA synthesis during the course of actinic aging are largely unknown. The aim of the present study was to explore whether collagen degradation causes inhibition of HA synthesis in human skin fibroblasts. After treatment of fibroblasts with collagen fragments (CF) in vitro, resolution of the actin cytoskeleton and inhibition of HA secretion occurred because of specific down-regulation of hyaluronan synthase 2 (HAS2) expression. The α(v)β(3)-agonist, RGDS, latrunculin A, and an inhibitor of Rho-activated kinase inhibited HAS2 expression. Conversely, blocking antibodies to α(v)β(3) abolished the down-regulation of HAS2 and the cytoskeletal effects. Furthermore, inhibition of cofilin phosphorylation in response to CF was prevented by α(v)β(3)-blocking antibodies. The key role of ERK signaling was shown by reduced nuclear accumulation of phosphoERK and of ELK-1 phosphorylation in response to CF. In addition, the ERK inhibitor PD98059 reduced HAS2 expression. Also, UVB irradiation of fibroblasts caused down-regulation of HAS2, which was sensitive to matrix metalloproteinase inhibitors and to α(v)β(3)-blocking antibodies. In conclusion, these data suggest that CF activate α(v)β(3)-integrins and in turn inhibit Rho kinase (ROCK) signaling and nuclear translocation of phosphoERK, resulting in reduced HAS2 expression. Therefore, a novel mechanism is presented how proteolytic collagen cleavage may inhibit HA synthesis in dermal fibroblasts during extrinsic skin aging.     

Hyaluronan affects protein and collagen synthesis by in vitro human skin fibroblasts

Given the importance of hyaluronan (HA) for the homeostasis of connective tissues during embryogenesis and aging and its role in tissue repair, the aim of the present study was to examine the effect of exogenous HA on the synthesis of total protein, collagen and HA by in vitro human dermal fibroblasts. With differences between different cell strains, HA, at concentrations between 0.5 and 1 microM, induced a significant decrease in total protein synthesised and secreted into the medium compared to controls (P < 0.05), and particularly in collagen (-40%; P < 0.05). The ratios between collagen types I and III and between collagen types V and I were normal. Pulse and chase experiments showed that protein degradation was normal. The presence of exogenous HA did not affect HA synthesis. Data strongly indicate that a relatively high concentration of HA in the extracellular space, such as during development and in the first phases of tissue repair, would partially limit the deposition of the extracellular matrix, and of collagen in particular. This would suggest a role for HA in delaying tissue differentiation during embryogenesis and in preventing fibrosis and scar formation in fetus and in the early phases of wound healing.     

Microwave irradiation improvements in the silver staining of the nucleolar organizer (Ag-NOR) technique

The well-know technique of silver staining of the nucleolar organizer (Ag-NOR) is improved in contrast, selectivity and speed when performed with microwave irradiation. The Ag-NOR technique is a very useful tool for studies on the functional morphology and molecular architecture of the nucleolus, and is reputed to be one of the best techniques for diagnosis and prognosis of cancer lesions. To test the generality of the enhancing effects. our study has involved the use of both mammalian and plant cells. Two steps in the process are improved quantitatively by microwave irradiation: fixation and staining itself. Fixation with the ethanol-based reagent, Kryofix, for 3 min in the microwave oven, resulted in good structural preservation at the optical level, and enhanced the contrast and selectivity of silver staining. On the contrary, we found that neither glutaraldehyde fixation, nor a treatment of sections with Carnoy's solution, improved Ag-NOR staining. After an analysis of the effects of the different substances involved in sample preparation, we conclude that ethanol is an essential factor for fixation for nucleolar staining, particularly if aldehydes are eliminated from fixative solutions. The process of staining was performed with a drop of staining solution on a semithin section of plastic-embedded tissue intthe microwave oven for 1 min. Staining under these conditions always improved the visualization of nucleoli, regardless of the fixation procedure. Therefore, microwave irradiation at both steps is recommended for giving the best results. Microwave irradiation probably enhances fixation by controlled heat, whereas the increase in reactivity of the staining solution is a direct effect by the microwaves on the silver ions themselves. We used this method to study nucleolar materials during mitosis in proliferating plant cells. Current applications of Ag-NOR staining can be improved with this technical modification.     

Matrix hyaluronan alters epidermal growth factor receptor-dependent cell morphology

EGFR, a critical regulator of oncogenic signaling during cancer progression, is capable of integrating multireceptor signaling pathways that promote metastasis. EGFR is subject to regulatory cues from the extracellular matrix (ECM), of which hyaluronan (HA) is a major component. In mammary tumors, HA is deposited in the ECM where it functions in biomechanical support and modulates intracellular signaling. We utilized a 3D collagen system in which HA is either polymerized in collagen matrix or provided soluble in the media (sHA). Here we report that collagen-embedded HA (eHA) inhibits EGFR activation, filopodia formation, and cell spreading on a collagen matrix. These findings demonstrate a novel role for eHA as a protective molecule when encountered in the collagen matrix during cancer progression.