Das Basalzellkarzinom (BZK) der Haut gilt als einer der häufigsten semimalignen Tumoren. Trotz der niedrigen Metastasierungsrate von < 0,1 % können BZK schwerwiegende Infiltrationen und Destruktionen knorpeliger sowie knöcherner Strukturen verursachen. Für die Tumorentstehung ist vor allem die kumulative UVB-Dosis in Kindheit und Adoleszenz bedeutsam. Aber auch die Embryonalentwicklung scheint eine Rolle in der Pathogenese von Tumoren zu spielen. Die so genannte embryonale Fusionszone (eFZ) entsteht zwischen der 5-10 Entwicklungswoche durch die Verschmelzung der fünf Gesichtswülste. Es konnte gezeigt werden, dass BZK innerhalb dieses Kompartiments nicht nur gehäuft auftreten sondern auch durch ein ausgeprägtes Tiefenwachstum charakterisiert sind. Als mögliche Ursache für das verstärkt invasive Wachstum von BZK innerhalb der eFZ wurden Änderungen im Hyaluronsäure (HA)-Stoffwechsel der Stromazellen angenommen. Neben der Bedeutung in der Embryonalentwicklung und bei der Geweberegeneration zeigten verschiedene Studien zudem die essentielle Rolle von HA im Rahmen von malignen Zelltransformationen. Vermehrte HA-Ablagerungen in der Tumorumgebung oder erhöhte HA-Serumkonzentrationen wurden bei einer Vielzahl von Tumoren als Zeichen einer fortschreitenden Tumorprogression beschrieben. Um den HA-Stoffwechsel von Stromafibroblasten um BZK gezielt zu untersuchen, wurden der HA-Gehalt, die HA-Größe und die exprimierten Enzyme des HA-Stoffwechsels in Abhängigkeit von ihrer Lokalisationen miteinander verglichen. Dabei zeigte sich, dass sowohl innerhalb als auch außerhalb der eFZ vergleichbare Mengen und Polymergrößen von HA sezerniert werden. Molekularbiologische Untersuchungen an expandierten Fibroblasten aus Biopsien verschiedener Lokalisationen zeigten ebenfalls keine Unterschiede in der Expression von mRNA der HA Stoffwechselenzyme nach Herkunft der Fibroblasten. Somit wird geschlussfolgert, dass HA zwar auch im Stroma von BZK gebildet wird, der HA-Stoffwechsel von Stromafibroblasten jedoch kein Merkmal ist, das mit dem vermehrten Auftreten und invasiven Wachstum von BZK im Bereich der embryonalen Fusionszone des Mittelgesichts korreliert.

The development of the mature cardiovascular system is one of the most captivating stories in embryonic development. The heart is the first organ to form in embryogenesis, and is functional early in development to perfuse the embryo with blood supplying oxygen and the nutrients required for organogenesis. The structural changes in heart development required for formation of the mature four chambered heart are under tight molecular regulation. Severe defects manifest as gross structural malformations of the valves, septa, or vessels that result in physiological consequences that my include hypertension, arrhythmia, or heart failure and may ultimately lead to lethality. According to the American Heart Association, cardiovascular disease is the leading cause of mortality worldwide. A more detailed understanding of the origin of congenital heart defects is necessary for improving prediction, diagnosis, and treatment of cardiovascular disease. Derived from the epicardium, coronary vessel formation relies on growth factor as well as extracellular matrix (ECM) influences on cells of the epicardium that regulate proliferation, motility, invasion and differentiation. The Transforming Growth Factor β (TGFβ) family of receptors have been well described in regulating cardiovascular development. The Type III TGFβ receptor (TGFβR3) has been shown to be required for development of the coronary vessels. Mouse embryos lacking TGFβR3 exhibit inhibited invasion of epicardially derived cells (EPDCs) into the myocardium. This delay of cell invasion of EPDCs and formation of coronary vessels is lethal at E 14.5. Relative to Tgfbr3+/+ cells, epicardial cells lacking TGFβR3 are hypo-proliferative, deficient in cell invasion, and deficient in executing TGFβ ligand and High-Molecular Weight Hyaluronan (HMWHA) stimulated cell invasion. Hyaluronan (HMWHA) is a glycosaminoglycan unmodified sugar extracellular matrix (ECM) molecule synthesized by the Hyaluronan Synthase (Has) family of enzymes. Mouse embryos lacking Hyaluronan Synthase 2 (Has2) are lethal at E 9.5 as a result of severely blocked cardiogenesis due to insufficient endocardial EMT. HA serves structural and bioactive functions in its capacity to stimulate signal transduction pathways required for EMT. Src kinase is a non-receptor tyrosine kinase well characterized to function in growth factor as well as ECM signal transduction, but its role in epicardial cell biology is unclear. Our hypothesis is that Src kinase is a critical regulator of TGFβ and Hyaluronan induced epicardial cell invasion, differentiation and migration during coronary vessel development. Our studies reveal that Src activity is required for TGFβ2-induced synthesis of HA in epicardial cells. We show Src is required for TGFβ2-induced vascular smooth muscle differentiation as well as TGFβ2-induced EMT, cell invasion, and filamentous actin polymerization. Src activity is sufficient to drive epicardial activation of EMT, but not vascular smooth muscle differentiation. These data show that Src is required in the context of TGFβ2-stimulated invasion and differentiation, and sufficient to drive activation of EMT. Next we demonstrate that TGFβR3 and Src are required for HMWHA induced cell invasion and filamentous actin polymerization in epicardial cells. HMWHA induces activation of Src kinase in Tgfbr3+/+ epicardial cells, but not Tgfbr3-/- epicardial cells. siRNA knockdown of TGFβR3 in Tgfbr3+/+ epicardial cells subsequently stimulated with HMWHA phenocopy this deficit in Src activation. Tgfbr3-/- epicardial cells fail to activate Rac1 or RhoA GTPases in the presence of HMWHA. Finally, we demonstrate stimulus independent activation of TGFβR3 is sufficient to activate Src. Taken together, these constitute novel findings establishing TGFβR3 as an HMWHA responsive receptor that is upstream of Src signal transduction. Migration of the epicardium to cover the looped and functioning heart tube is an early step required for development of the coronary vessels. We demonstrate that Tgfbr3-/- epicardial cells are delayed in cell migration relative to Tgfbr3+/+ cells in a wound healing model of cell migration. Tgfbr3-/- cells lack expression of BMP2 mRNA, we found that exogenous BMP2 is sufficient to drive Tgfbr3-/- (but not Tgfbr3+/+) cell migration to levels comparable to unstimulated Tgfbr3+/+ epicardial cells, without enhancing cell proliferation. We demonstrate that Src is required for this BMP2 induced cell migration and filamentous actin polymerization in Tgfbr3-/- cells. These studies demonstrate mechanisms required for TGFβ ligand as well as HMWHA stimulated epicardial cell behavior changes have a common mediator in Src kinase, and provide novel insights into early events in the development of the cardiovascular system. The adult epicardium has been demonstrated to participate in repair of ischemic myocardium in mouse models of myocardial infarction. Expression of molecules required for coronary vessel development are re-expressed in this regeneration (as discussed in chapter 5). Elucidating these pathways will constitute important future targets in aiding in adult cardiovascular regeneration and cardioprotection in adult heart disease.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Bronchoalveolar lavage (BAL) is an established method providing diagnostic support and evaluation of disease activity in interstitial lung disease (ILD). The aims of the present investigation were 1) to study the inflammatory response in pneumonitis evoked by irradiation. 2) to evaluate how well lung tissue inflammation is reflected in BAL findings. 3) to study the effect of smoking on radiation-induced pneumonitis. BAL was performed in 21 patients (11 smokers, 10 non-smokers) who were treated for breast cancer, stage 1 (TjMaNq) by post-surgery irradiation to an accumulated target dose of 56 Gy. It was founa that irradiation induced an alveolitis in the non-smoking patient group while the smoking patients did not differ from their smoking controls. The alveolitis in non-smokers was characterized by an increase in lymphocytes, mast cells and elevated concentrations of hyaluronan (HA), and fibronectin (FN). Three of the non-smoking patients had chest X-ray infiltrates indicating the presence of pneumonitis. An animal experimental model for radiation-induced pneumonitis and fibrosis was established in rats, allowing comparative analysis of BAL fluid and morphology. In the rat model a divergence was noted between the differential cell counts in BAL and cells observed in the interstitial tissue, which was most notable for neutrophils (PMN) and mast cells whereas there was a good correlation between HA content in BAL and HA deposition in the lung tissue. A marked infiltration of intraseptally-located mast cells occurred during the pneumonitis-phase, and this increase was paralleled by a deposition of HA in the interstitial tissue. Histochemical fixation and staining properties of the mast cells revealed that the majority of these cells were of connective tissue mast cell type (CTMC). Compound 48/80, a mast cell secretagogue, significantly altered the HA content both in BAL and in lung tissue in the irradiated animals. Regular treatment throughout the whole experimental period induced depletion of mast cell granules and a decrease in HA deposition whereas 48/80 treatment during the pneumonitis phase enhanced HA deposition. A rat model with smoke exposure was developed, and the effect of cigarette smoke on radiation-induced inflammation was studied. Rats that smoked 3 weeks prior to irradiation and continued to smoke throughout the observation period (7 weeks) had a significantly reduced inflammatory response compared to irradiated non-smoking rats. The most prominent BAL findings in the smoke-exposed rats were a decrease in PMN, mast cells and a decrease in HA. In conclusion, irradiation induces an alveolitis characterized mainly by mononuclear cells. Mast cells seem to be of importance in the remodelling of the connective tissue in the radiation-induced inflammatory response. Hyaluronan is an important component in the early connective tissue response preceding later collagen deposition, and its interstitial deposition is very well reflected in BAL. Moreover, tobacco-smoke suppresses the radiation-induced inflammation with a decreased recruitment of effector cells including mast cells. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1992, härtill 5 uppsatser.</p> / digitalisering@umu

This thesis presents strategies for constructing multi-functional biomaterials based on hyaluronan (HA) derivatives for various biomedical applications, such as drug delivery, tissue regeneration, and imaging biomaterials. The aim of this study is to improve the functionalities of HA biomaterials as well as simplify the preparation procedures.  Native HA polymer contains D-glucuronic acid residue with a carboxyl group per disaccharide unit that can be easily modified by carbodiimide-mediated amidation reaction. Therefore, we have designed a series of orthogonal groups (hydrazide, carbazate, aldehyde, and thiol) that can be linked to HA under mild conditions using the carbodiimide chemistry. Multiple functionalities can be introduced to the obtained HA derivatives via chemoselective “click”-type transformations.   The modified HA derivatives were used for the preparation of either nanogel particles (NPs) or bulk hydrogels. Due to “click” character of the reactions used, structural HA transformations were performed with high fidelity on different scales including molecular (polymers), nanometer (NPs), and a visible scale (bulk hydrogels). By linking pyrene or camptothecin to hydrophilic HA backbone, amphiphilic polymers were obtained and utilized as drug delivery carriers or prodrugs, respectively. Subsequently, physically loaded drug (doxorubicin) could be released upon degradation of HA carriers, while the chemically linked camptothecin was released intact by a thiol-triggered cleavage reaction. Bisphosphonated HA (HA-BP) polymers were prepared to induce hydrogel scaffold bio-mineralization for bone regeneration application. Moreover, we could recruit strong binding capacity of bisphosphonate (BP) groups to calcium ions for the formation of physically crosslinked HA-BP gel upon simple mixing of the polymer and calcium phosphate nanoparticle components. This gel was more stable in vivo compared to hydrazone crosslinked HA gels. Furthermore, the hydrogel composed of fluorine-19 (19F) linked HA polymer was successfully observed by both 1H and 19F MR imaging.         In conclusion, the presented herein study describes new approaches for building up multi-functional biomaterials from the HA-based blocks. The utilization of carbodiimide and click chemistries along with the enzymatic degradation of HA allowed simple and efficient interconversion between HA macromolecules, nanoparticles and macroscopic hydrogels. These HA-based biomaterials show high potential for use in the fields of drug delivery, bone regeneration, and imaging techniques.

Degenerative disc disease is a common ailment with enormous medical, psychosocial and economic ramifications. This study was designed to investigate the utility of a thiol-modified hyaluronan(TMHA) and elastin-like polypeptide(EP) composite material as a potential tissue engineering scaffold to reconstitute the nucleus pulposus in early degenerative disc disease. TMHA and EP were combined in various concentrations and cross-linked using poly(ethylene glycol)diacrylate. Resulting materials were evaluated biomechanically and biologically. Confined compression testing revealed that the addition of EP to TMHA-based gels resulted in a stiffer construct, but remained an order of magnitude less stiff than native nucleus. The in vitro cell culture experiments with human intervertebral disc cells demonstrated 70% cell viability at three weeks with apparent maintenance of phenotype. The addition of EP did not have a significant biologic effect. An in vivo pilot study demonstrated biocompatibility of the TMHA-based hydrogels; additional power is required to adequately assess treatment effect.

Background: Spinal cord injury (SCI) is heterogeneous in nature and can be complicated by inflammation and scarring in the subarachnoid space (arachnoiditis). The constellation of traumatic injury and arachnoiditis can lead to extensive intraparenchymal cysts or post-traumatic syringomyelia (PTS), due to alterations in fluid flow and pressure dynamics in the subarachnoid space. Hypothesis: Intrathecal injection of a bioengineered hydrogel containing hyaluronan (HA) will improve functional recovery following severe spinal cord injury associated with arachnoiditis. Methods: Acute to subacute pathophysiological events were characterized in non-injured sham rats, rats receiving a clip compression/contusion injury (SCI), rats receiving an intrathecal kaolin injection (Arachnoiditis) and in rats receiving SCI plus kaolin injection (PTS). Next, a HA containing hydrogel (HAMC) or artificial cerbralspinal fluid (aCSF) control was injected into the subarachnoid space 24 hours following PTS injury. To assess treatment efficacy, subacute pathophysiology was assessed as was long-term neurobehavioural and neuroanatomical recovery. Finally, in vitro studies examined the effect of HA on TLR4 activation using lipopolysaccharide in primary rat microglial cultures. Results: PTS animals exhibited a greater parenchymal injury response as compared to the sum of SCI alone or arachnoiditis alone. Injection of HAMC reduced the extent of scarring and inflammation in the subarachnoid space and improved neurobehavioural and neuroanatomical recovery relative to aCSF controls. These improvements were associated with reduced chondroitin sulfate proteoglycan and IL-1α expression and a trend towards and axonal preservation. In vitro studies demonstrated that HA is capable of reducing TLR4 mediated inflammation in microglia. Conclusions: Acute arachnoiditis potentiates the intensity of intraparenchymal inflammatory and scarring events following SCI. When HAMC was injected intrathecally following PTS injury, it mitigated some of the pernicious effects of arachnoiditis. Part of the therapeutic action of HAMC can be attributed to the ability of HA to reduce TLR4 mediated inflammation in microglia, possibly through an extracellular mechanism.

Degenerative disc disease is a common ailment with enormous medical, psychosocial and economic ramifications. This study was designed to investigate the utility of a thiol-modified hyaluronan(TMHA) and elastin-like polypeptide(EP) composite material as a potential tissue engineering scaffold to reconstitute the nucleus pulposus in early degenerative disc disease. TMHA and EP were combined in various concentrations and cross-linked using poly(ethylene glycol)diacrylate. Resulting materials were evaluated biomechanically and biologically. Confined compression testing revealed that the addition of EP to TMHA-based gels resulted in a stiffer construct, but remained an order of magnitude less stiff than native nucleus. The in vitro cell culture experiments with human intervertebral disc cells demonstrated 70% cell viability at three weeks with apparent maintenance of phenotype. The addition of EP did not have a significant biologic effect. An in vivo pilot study demonstrated biocompatibility of the TMHA-based hydrogels; additional power is required to adequately assess treatment effect.

A cysteine-containing elastin-like polypeptide (ELP2cys) was successfully synthesized and purified, and was shown to behave in a similar fashion to other well-characterized ELPs. Incorporating the ELP2cys as a crosslinking agent into a solution of sulphated hyaluronan (CMHA-S) not only decreased the gelation time of the solution but also increased the crosslinking density of the resultant hydrogel, in turn increasing both the resiliency and stiffness of the construct. Preliminary in vitro work involved culture of human disc cells, followed by their encapsulation within the hydrogel. Unfortunately the results were inconclusive, although it appeared as though the addition of ELP2cys to the matrix did not negatively affect the viability of the cells, as compared to hydrogels with CMHA-S only. This study showed that ELP2cys is a valuable addition to the family of recombinant elastin-like polypeptides, and shows promise as a crosslinking agent in the formation of hyaluronan hydrogels.

Background.The extracellular matrix surrounds all cells within our bodies. The glycosaminoglycan hyaluronan is a major component in the extracellular matrix. Despite its structural simplicity it has been shown to be involved in several important functions. It is a lubricant and shock absorber, as well as an important player in inflammation and tumor invasion. Many of its functions are closely related to its size and concentration in tissues. Therefore methods for measuring these properties are of great importance to properly understand the role that hyaluronan play in different events. Proteins are found both inside and outside cells, and they have a wide variety of functions. The protein structure and function is determined by the properties of their building blocks, the amino acids. Several diseases have been linked to changes in the amino acid sequence of certain proteins by mutations, causing the proteins to form extracellular deposits of structures called amyloid aggregates. The aim of this thesis is to investigate the function of hyaluronan in cell cultures, develop new methods for size determination hyaluronan and to use multivariate methods to provide prediction and better understanding of factors driving protein amyloid aggregation. Methods.Cardiomyocytes and fibroblast were cultured and stimulated by different growth factors. Hyaluronan was purified and its size and concentration were measured. Crosstalk between cardiomyocytes and fibroblast were investigated and gene expression of hyaluronan synthases was determined. A new method for size measurement of hyaluronan was developed. The amyloid aggregation rate of different mutants of acylphosphatase was predicted by multivariate analysis. Results. Cardiomyocytes stimulated by PDGF-BB produced hyaluronan. Cardiomyocytes could induce fibroblast to increase its hyaluronan production, through an unknown soluble factor. The cardiomyocyte gene expression changed when stimulated by hyaluronan. GEMMA was presented as a new method for size determination of hyaluronan. Amyloid aggregation of different acylphosphatase mutants could be predicted using a multivariate regression model of the physicochemical and structural properties of the amino acid sequence. Conclusion. It was shown that cardiomyocytes are not only able to produce hyaluronan, but also induce an increased hyaluronan production in other cells. GEMMA was proven suitable for size determination of hyaluronan at very low concentrations. Multivariate analysis showed that hydrophobic patterns and charge where the most important factors for amyloid aggregation of acylphosphatase.