Hyaluronidases are endoglycosidases that catabolize hyaluronan, an abundant component of the extracellular matrix surrounding vertebrate cells. We characterized one of the hyaluronidases, HYAL1, an enzyme deficient in the lysosomal storage disorder Mucopolysaccharidosis IX. HYAL1 stably expressed in BHK cells resulted in several intracellular forms, but only one secreted form. Secretion was not increased by weak bases, and no phosphate was incorporated in metabolic labeling, suggesting this enzyme is not targeted to the lysosome by the mannose 6-phosphate dependent pathway. Further analysis revealed the various forms of HYAL1 differ only in glycosylation, and are all active at pH 3.8. The forms migrated in a Percol density gradient similarly to an endosomal marker, and with partial overlap with the lysosomal marker LPG120 (Lamp1). / May 2006

Hyaluronan (HA) is a glycosaminoglycan that is abundant in the extracellular matrix of vertebrate cells. Under physiological conditions HA exists in a high-molecular-weight form, whereas HA fragments accumulate at sites of tissue injury and inflammation. Hyaluronidases are a group of enzymes that initiate the breakdown of HA. In humans, six hyaluronidase-like sequences have been identified in two locations, 3p21.3 (HYAL1, HYAL2 and HYAL3) and 7q31.3 (HYAL4, SPAM1 and HYALP1). Deficiency of one of these enzymes, HYAL1, was identified in a patient with Mucopolysaccharidosis IX, a disorder characterized by peri-articular soft masses containing HA-filled lysosomes. Given the broad distribution of HA and the mild phenotype of the patient, it is likely that other hyaluronidases or possibly the exoglycosidases, β-hexosaminidase and β-glucuronidase, are playing a major role in HA degradation. To address the potential role of HYAL3 in HA degradation in health and disease, a Hyal3-deficient mouse model was generated. Hyal3-deficient mice were viable, fertile and appeared to have no gross phenotype. The only difference observed was a subtle change in the cellularity and tissue structure of lungs from aged Hyal3-deficient mice. Further studies focused on analysis of HA homeostasis revealed a significant increase in HA in the airways of Hyal3-deficient lungs. Altered HA homeostasis is observed in rodent models of several lung conditions. In order to further study the role of Hyal3 in lungs, an ovalbumin-challenged inflammation model was generated in Hyal3-deficient mice. A significant increase in lung HA levels and altered distribution of HA in the airways of lungs was detected in ovalbumin-challenged Hyal3-deficient mice. Moreover, lung inflammation and airway resistance were increased in Hyal3-deficient mice after ovalbumin-challenge compared to similarly treated Hyal3-control mice. This suggests HA homeostasis that is altered during Hyal3-deficiency might be directly or indirectly promoting inflammation and airway resistance. Because the reported level of HA accumulation was very low in Hyal1-deficient and Hyal2-deficient mice, and in our studies of Hyal3-deficient mice, we performed preliminary studies to assess a role for an exoglycosidase, β-hexosaminidase, in HA turnover. Our preliminary studies indicate there is no or little HA accumulation in β-hexosaminidase-deficient mouse tissues. To conclude, our study of Hyal3- and β-hexosaminidase-deficient mice suggests that these are not the major enzymes involved in HA degradation

Hyaluronidases are endoglycosidases that catabolize hyaluronan, an abundant component of the extracellular matrix surrounding vertebrate cells. We characterized one of the hyaluronidases, HYAL1, an enzyme deficient in the lysosomal storage disorder Mucopolysaccharidosis IX. HYAL1 stably expressed in BHK cells resulted in several intracellular forms, but only one secreted form. Secretion was not increased by weak bases, and no phosphate was incorporated in metabolic labeling, suggesting this enzyme is not targeted to the lysosome by the mannose 6-phosphate dependent pathway. Further analysis revealed the various forms of HYAL1 differ only in glycosylation, and are all active at pH 3.8. The forms migrated in a Percol density gradient similarly to an endosomal marker, and with partial overlap with the lysosomal marker LPG120 (Lamp1).

Hyaluronidases are endoglycosidases that catabolize hyaluronan, an abundant component of the extracellular matrix surrounding vertebrate cells. We characterized one of the hyaluronidases, HYAL1, an enzyme deficient in the lysosomal storage disorder Mucopolysaccharidosis IX. HYAL1 stably expressed in BHK cells resulted in several intracellular forms, but only one secreted form. Secretion was not increased by weak bases, and no phosphate was incorporated in metabolic labeling, suggesting this enzyme is not targeted to the lysosome by the mannose 6-phosphate dependent pathway. Further analysis revealed the various forms of HYAL1 differ only in glycosylation, and are all active at pH 3.8. The forms migrated in a Percol density gradient similarly to an endosomal marker, and with partial overlap with the lysosomal marker LPG120 (Lamp1).

Hyaluronan (HA) is a glycosaminoglycan that is abundant in the extracellular matrix of vertebrate cells. Under physiological conditions HA exists in a high-molecular-weight form, whereas HA fragments accumulate at sites of tissue injury and inflammation. Hyaluronidases are a group of enzymes that initiate the breakdown of HA. In humans, six hyaluronidase-like sequences have been identified in two locations, 3p21.3 (HYAL1, HYAL2 and HYAL3) and 7q31.3 (HYAL4, SPAM1 and HYALP1). Deficiency of one of these enzymes, HYAL1, was identified in a patient with Mucopolysaccharidosis IX, a disorder characterized by peri-articular soft masses containing HA-filled lysosomes. Given the broad distribution of HA and the mild phenotype of the patient, it is likely that other hyaluronidases or possibly the exoglycosidases, β-hexosaminidase and β-glucuronidase, are playing a major role in HA degradation. To address the potential role of HYAL3 in HA degradation in health and disease, a Hyal3-deficient mouse model was generated. Hyal3-deficient mice were viable, fertile and appeared to have no gross phenotype. The only difference observed was a subtle change in the cellularity and tissue structure of lungs from aged Hyal3-deficient mice. Further studies focused on analysis of HA homeostasis revealed a significant increase in HA in the airways of Hyal3-deficient lungs. Altered HA homeostasis is observed in rodent models of several lung conditions. In order to further study the role of Hyal3 in lungs, an ovalbumin-challenged inflammation model was generated in Hyal3-deficient mice. A significant increase in lung HA levels and altered distribution of HA in the airways of lungs was detected in ovalbumin-challenged Hyal3-deficient mice. Moreover, lung inflammation and airway resistance were increased in Hyal3-deficient mice after ovalbumin-challenge compared to similarly treated Hyal3-control mice. This suggests HA homeostasis that is altered during Hyal3-deficiency might be directly or indirectly promoting inflammation and airway resistance. Because the reported level of HA accumulation was very low in Hyal1-deficient and Hyal2-deficient mice, and in our studies of Hyal3-deficient mice, we performed preliminary studies to assess a role for an exoglycosidase, β-hexosaminidase, in HA turnover. Our preliminary studies indicate there is no or little HA accumulation in β-hexosaminidase-deficient mouse tissues. To conclude, our study of Hyal3- and β-hexosaminidase-deficient mice suggests that these are not the major enzymes involved in HA degradation

Progression to metastatic disease is the leading cause of deaths resulting from breast cancer. Understanding the mechanisms underlying a cell's ability to move away from its site of origin and populate a distant site is important for the future development of therapies. The interactions between a tumor cell and the microenvironment can modulate a cell's ability to invade through tissues and access distant organs. In this study we present evidence indicating the differential modulation of invasive and proliferative phenotypes by hyaluronan present in the cellular microenvironment.We establish the role of CD44, the primary receptor for hyaluronan, in breast cancer progression and metastasis through the use of transgenic mouse models of breast cancer. While no differences were seen in the onset of primary breast tumors, mice expressing CD44 had a reduced rate of pulmonary metastasis compared to mice that lacked CD44. This establishes an anti-invasive role for CD44 in breast tumor progression. We also identify a decreased population of alveolar macrophages in CD44 negative mice that could affect metastatic breast cancer cell colonization of the lungs.We then focused our study in vitro, where we assessed the invasive properties of breast cancer cells as they move through three dimensional (3D) matrices containing or lacking hyaluronan. We show that in 3D type I collagen gels, breast cancer cells invade more readily in the absence of hyaluronan compared to when hyaluronan (HA) is embedded within the gel. HA mediated inhibition of invasion is dependent on CD44 binding as demonstrated through the use of a CD44 functional blocking antibody.We also show that HA promotes differential phenotypes of breast cancer cell. HA promotes filopodia formation and invasion when soluble in the cell microenvironment. Alternatively, matrix-embedded HA inhibits invasion and promotes migration through the formation of lamellipodia. The differential HA invasive and proliferative phenotypes are mediated by differential activation of ERK or γPAK. Activation of γPAK is mediated by CD44 while ERK activation by HA occurs by CD44 independent mechanisms.We also demonstrate an inhibition of MMP9 mediated invasion by HA when embedded within a type IV collagen matrix, but not a type I collagen matrix. This differential activity indicates that it is not only the immobilization of HA in a matrix that determines its activity, but also the context in which it is present within the matrix.These data underscore the importance of studying matrix components in an environment that closely resembles in vivo conditions. HA is a prime example as it has the capability of both promoting and inhibiting invasion depending on how it is presented to a cell. Differential HA activity also underlies the importance of understanding extracelluar matrix degradation and the release of matrix components as these can adversely affect disease progression.

The cardiovasculature is the first functional system in the developing embryo and as such, it plays a crucial role in the proper nourishment and formation of all other body regions and organs. A detailed understanding the mechanisms that regulate cardiac morphogenesis is necessary to develop possible strategies for diagnostics and treatment of cardiovascular diseases.One molecule identified as important for the proper formation of the heart is Hyaluronan synthase 2 (Has2), a membrane protein in charge of assembling the glycosaminoglycan hyaluronan (HA). Mouse embryos lacking Has2 do not produce HA, display severe cardiovascular abnormalities and die during early embryogenesis. Thus, Has2 and HA are necessary for the early stages of heart formation, but many questions remain to be answered in regards to their mechanism of action and their role in later events such as the formation of the coronary vessels. Our current study addresses these questions employing two cell lines: NIH-3T3s, as a model of mesenchymal endocardial cushion cells; and epicardial cells, which have an epithelial phenotype.Here we show that HA induces biological activity in embryonic cells in a manner that is dependent on its molecular size. High molecular weight HA (HMW-HA), but not low molecular weight HA (LMW-HA), induces invasion of NIH-3T3 cells while it promotes differentiation and invasion of epicardial cells. We also demonstrate that stimulation of cells with HMW-HA promotes the association of MEKK1 with the HA receptor CD44. This leads to the activation of two distinct pathways, one ERK-dependent and another NFκB-dependent, which are crucial for the induction of cellular responsesFinally, we have demonstrated that the growth factors TGFβ2 and EGF induce Has2 expression and/or phosphorylation. TGFβ2 governs Has2 via MEKK3-dependent mechanisms, while EGF does not require MEKK3. Increased Has2 activity as a result of TGFβ2 and EGF stimulation leads to enhanced HA synthesis. These increased levels of HA are coincident with enhanced cellular differentiation and invasion. Taken together, these findings underscore how EGF, TGFβ2 and HA signals are integrated to form highly complex networks that are crucial for the proper formation of organs and tissues during development.

Hyaluronan, an unbranched glycosaminoglycan of the extracellular matrix, has an amazingly simple structure. Initially thought to fulfil only hydrating and space-filling functions in tissues, evidence generated during the past decades shows that hyaluronan is involved in intriguingly complex signalling events in health and disease. In cancer, increased hyaluronan levels have been correlated with poor patient survival. The research underlying this thesis sheds light on the interplay between hyaluronan, its producing and degrading enzymes as well as the triggered intracellular signalling in the metastatic cascade. Utilising breast cancer and normal mammary cells, paper I and II investigate the initial steps of tumour progression: proliferation, invasion and epithelial-mesenchymal transition. Hyaluronan synthase 2 plays a central role in all these processes. In paper III, the focus is shifted toward growth factor-induced hyaluronan production. Stimulation with PDGF-BB, which can be secreted by tumour cells, increased hyaluronan production via upregulation of HAS2 in fibroblast cultures. Finally, paper IV discusses the involvement of hyaluronidases and CD44 in angiogenesis and intravasation – events that are associated with advanced cancer stages.

Předložená práce se zabývá reologickými vlastnostmi roztoků hyaluronanu. Studuje chování hyaluronanových roztoků v prostředí několika rozpouštědel, dále se zabývá vlivem měnícího se pH a rostoucí teploty na viskozitu roztoků. V neposlední řadě je zkoumána správná příprava roztoků, jejich časová stabilita a tvorba agregátů.Všechny experimenty vycházejí z praktického upotřebení, např. při výrobě hyaluronanu, jeho sterilizaci a následném skladování roztoků.

Předkládaná dizertační práce se zabývá vývojem technologie výroby nového typu biodegradabilních vláken na bázi oxidované kyseliny hyaluronové. V rámci práce je postupně představován vývoj jednotlivých jednotkových operací výroby, jejichž správné porozumění a schopnost jejich řízení jsou klíčové pro žádaný chod celé vícestupňové technologie. V rámci práce je představen nezbytný vývoj technologického zařízení, průběžně konstruovaného pro účely laboratorního testování a následně až po samotnou linku pro finální výrobu vláken, která byla realizována v roce 2015. V rámci dizertační práce jsou dále navrhovány možnosti dodatečné chemické úpravy vláken s ohledem na zvyšování jejich stability ve vlhkém prostředí. S ohledem na cílené aplikace vláken pro vnitřní chirurgické implantace, jsou v práci vlákna též hodnocena z hlediska jejich materiálové biokomaptibility (toxicity).