VEGFR-3 and Tie pathways in vascular network formation

The blood and lymphatic vascular systems are essential for life, but they may become harnessed for sinister purposes in pathological conditions. For example, tumors learn to grow a network of blood vessels (angiogenesis), securing a source of oxygen and nutrients for sustained growth. On the other hand, damage to the lymph nodes and the collecting lymphatic vessels may lead to lymphedema, a debilitating condition characterized by peripheral edema and susceptibility to infections. Promoting the growth of new lymphatic vessels (lymphangiogenesis) is an attractive approach to treat lymphedema patients. Angiopoietin-1 (Ang1), a ligand for the endothelial receptor tyrosine kinases Tie1 and Tie2. The Ang1/Tie2 pathway has previously been implicated in promoting endothelial stability and integrity of EC monolayers. The studies presented here elucidate a novel function for Ang1 as a lymphangiogenic factor. Ang1 is known to decrease the permeability of blood vessels, and could thus act as a more global antagonist of plasma leakage and tissue edema by promoting growth of lymphatic vessels and thereby facilitating removal of excess fluid and other plasma components from the interstitium. These findings reinforce the idea that Ang1 may have therapeutic value in conditions of tissue edema. VEGFR-3 is present on all endothelia during development, but in the adult its expression becomes restricted to the lymphatic endothelium. VEGF-C and VEGF-D are ligands for VEGFR-3, and potently promote lymphangiogenesis in adult tissues, with direct and remarkably specific effects on the lymphatic endothelium in adult tissues. The data presented here show that VEGF-C and VEGF-D therapy can restore collecting lymphatic vessels in a novel orthotopic model of breast cancer-related lymphedema. Furthermore, the study introduces a novel approach to improve VEGF-C/VEGF-D therapy by using engineered heparin-binding forms of VEGF-C, which induced the rapid formation of organized lymphatic vessels. Importantly, VEGF-C therapy also greatly improved the survival and integration of lymph node transplants. The combination of lymph node transplantation and VEGF-C therapy provides a basis for future therapy of lymphedema. In adults, VEGFR-3 expression is restricted to the lymphatic endothelium and the fenestrated endothelia of certain endocrine organs. These results show that VEGFR-3 is induced at the onset of angiogenesis in the tip cells that lead the formation of new vessel sprouts, providing a tumor-specific vascular target. VEGFR-3 acts downstream of VEGF/VEGFR-2 signals, but, once induced, can sustain angiogenesis when VEGFR-2 signaling is inhibited. The data presented here implicate VEGFR-3 as a novel regulator of sprouting angiogenesis along with its role in regulating lymphatic vessel growth. Targeting VEGFR-3 may provide added efficacy to currently available anti-angiogenic therapeutics, which typically target the VEGF/VEGFR-2 pathway.

Cellular and network mechanisms generating spontaneous population events in the immature rat hippocampus

Distinct endogenous network events, generated independently of sensory input, are a general feature of various structures of the immature central nervous system. In the immature hippocampus, these type of events are seen as "giant depolarizing potentials" (GDPs) in intracellular recordings in vitro. GABA, the major inhibitory neurotransmitter of the adult brain, has a depolarizing action in immature neurons, and GDPs have been proposed to be driven by GABAergic transmission. Moreover, GDPs have been thought to reflect an early pattern that disappears during development in parallel with the maturation of hyperpolarizing GABAergic inhibition. However, the adult hippocampus in vivo also generates endogenous network events known as sharp (positive) waves (SPWs), which reflect synchronous discharges of CA3 pyramidal neurons and are thought to be involved in cognitive functions. In this thesis, mechanisms of GDP generation were studied with intra- and extracellular recordings in the neonatal rat hippocampus in vitro and in vivo. Immature CA3 pyramidal neurons were found to generate intrinsic bursts of spikes and to act as cellular pacemakers for GDP activity whereas depolarizing GABAergic signalling was found to have a temporally non-patterned facilitatory role in the generation of the network events. Furthermore, the data indicate that the intrinsic bursts of neonatal CA3 pyramidal neurons and, consequently, GDPs are driven by a persistent Na+ current and terminated by a slow Ca2+-dependent K+ current. Gramicidin-perforated patch recordings showed that the depolarizing driving force for GABAA receptor-mediated actions is provided by Cl- uptake via the Na-K-C1 cotransporter, NKCC1, in the immature CA3 pyramids. A specific blocker of NKCC1, bumetanide, inhibited SPWs and GDPs in the neonatal rat hippocampus in vivo and in vitro, respectively. Finally, pharmacological blockade of the GABA transporter-1 prolonged the decay of the large GDP-associated GABA transients but not of single postsynaptic GABAA receptor-mediated currents. As a whole the data in this thesis indicate that the mechanism of GDP generation, based on the interconnected network of bursting CA3 pyramidal neurons, is similar to that involved in adult SPW activity. Hence, GDPs do not reflect a network pattern that disappears during development but they are the in vitro counterpart of neonatal SPWs.

Latent TGF-β binding proteins -3 and -4 : transcriptional control and extracellular matrix targeting

Extracellular matrix (ECM) is a complex network of various proteins and proteoglycans which provides tissues with structural strength and resilience. By harvesting signaling molecules like growth factors ECM has the capacity to control cellular functions including proliferation, differentiation and cell survival. Latent transforming growth factor β (TGF-β) binding proteins (LTBPs) associate fibrillar structures of the ECM and mediate the efficient secretion and ECM deposition of latent TGF-β. The current work was conducted to determine the regulatory regions of LTBP-3 and -4 genes to gain insight into their tissue-specific expression which also has impact on TGF-β biology. Furthermore, the current research aimed at defining the ECM targeting of the N-terminal variants of LTBP-4 (LTBP-4S and -4L), which is required to understand their functions in tissues and to gain insight into conditions in which TGF-β is activated. To characterize the regulatory regions of LTBP-3 and -4 genes in silico and functional promoter analysis techniques were employed. It was found that the expression of LTBP-4S and -4L are under control of two independent promoters. This finding was in accordance with the observed expression patterns of LTBP-4S and -4L in human tissues. All promoter regions characterized in this study were TATAless, GC-rich and highly conserved between human and mouse species. Putative binding sites for Sp1 and GATA family of transcription factors were recognized in all of these regulatory regions. It is possible that these transcription factors control the basal expression of LTBP-3 and -4 genes. Smad binding element was found within the LTBP-3 and -4S promoter regions, but it was not present in LTBP-4L promoter. Although this element important for TGF-β signaling was present in LTBP-4S promoter, TGF-β did not induce its transcriptional activity. LTBP-3 promoter activity and mRNA expression instead were stimulated by TGF-β1 in osteosarcoma cells. It was found that the stimulatory effect of TGF-β was mediated by Smad and Erk MAPK signaling pathways. The current work explored the ECM targeting of LTBP-4S and identified binding partners of this protein. It was found that the N-terminal end of LTBP-4S possesses fibronectin (FN) binding sites which are critical for its ECM targeting. FN deficient fibroblasts incorporated LTBP-4S into their ECM only after addition of exogenous FN. Furthermore, LTBP-4S was found to have heparin binding regions, of which the C-terminal binding site mediated fibroblast adhesion. Soluble heparin prevented the ECM association of LTBP-4S in fibroblast cultures. In the current work it was observed that there are significant differences in the secretion, processing and ECM targeting of LTBP-4S and -4L. Interestingly, it was observed that most of the secreted LTBP-4L was associated with latent TGF-β1, whereas LTBP-4S was mainly secreted as a free form from CHO cells. This thesis provides information on transcriptional regulation of LTBP-3 and -4 genes, which is required for the deeper understanding of their tissue-specific functions. Further, the current work elucidates the structural variability of LTBPs, which appears to have impact on secretion and ECM targeting of TGF-β. These findings may advance understanding the abnormal activation of TGF-β which is associated with connective tissue disorders and cancer.

Latent TGF-beta Binding Proteins : Adhesive functions and matrix association of LTBP-2 and potential functions of LTBP-1 and LTBP-3 in mesothelioma

Latent transforming growth factor-beta (TGF-beta) binding proteins (LTBPs) -1, -3 and -4 are ECM components whose major function is to augment the secretion and matrix targeting of TGF-beta, a multipotent cytokine. LTBP-2 does not bind small latent TGF-beta but has suggested functions as a structural protein in ECM microfibrils. In the current work we focused on analyzing possible adhesive functions of LTBP-2 as well as on characterizing the kinetics and regulation of LTBP-2 secretion and ECM deposition. We also explored the role of TGF-beta binding LTBPs in endothelial cells activated to mimic angiogenesis as well as in malignant mesothelioma. We found that, unlike most adherent cells, several melanoma cell lines efficiently adhered to purified recombinant LTBP-2. Further characterization revealed that the adhesion was mediated by alpha3beta1 and alpha6beta1 integrins. Heparin also inhibited the melanoma cell adhesion suggesting a role for heparan sulphate proteoglycans. LTBP-2 was also identified as a haptotactic substrate for melanoma cell migration. We used cultured human embryonic lung fibroblasts to analyze the temporal and spatial association of LTBP-2 into ECM. By We found that LTBP-2 was efficiently assembled to the ECM only in confluent cultures following the deposition of fibronectin (FN) and fibrillin-1. In early, subconfluent cultures it remained primarily in soluble form after secretion. LTBP-2 colocalized transiently with FN and fibrillin-1. Silencing of fibrillin-1 expression by lentiviral shRNAs profoundly disrupted the deposition of LTBP-2 indicating that the ECM association of LTBP-2 depends on a pre-formed fibrillin-1 network. Considering the established role of TGF-beta as a regulator of angiogenesis we induced morphological activation of endothelial cells by phorbol 12-myristate 13-acetate (PMA) and followed the fate of LTBP-1 in the endothelial ECM. This resulted in profound proteolytic processing of LTBP-1 and release of latent TGF-beta complexes from the ECM. The processing was coupled with increased activation of MT-MMPs and specific upregulation of MT1-MMP. The major role of MT1-MMP in the proteolysis of LTBP-1 was confirmed by suppressing the expression with lentivirally induced short-hairpin RNAs as well as by various metalloproteinases inhibitors. TGF-beta can promote tumorigenesis of malignant mesothelioma (MM), which is an aggressive tumor of the pleura with poor prognosis. TGF-beta activity was analyzed in a panel of MM tumors by immunohistochemical staining of phosphorylated Smad-2 (P-Smad2). The tumor cells were strongly positive for P-Smad2 whereas LTBP-1 immunoreactivity was abundant in the stroma, and there was a negative correlation between LTBP-1 and P-Smad2 staining. In addition, the high P-Smad2 immunoreactivity correlated with shorter survival of patients. mRNA analysis revealed that TGF-beta1 was the most highly expressed isoform in both normal human pleura and MM tissue. LTBP-1 and LTBP-3 were both abundantly expressed. LTBP-1 was the predominant isoform in established MM cell lines whereas the expression of LTBP-3 was high in control cells. Suppression of LTBP-3 expression by siRNAs resulted in increased TGF-beta activity in MM cell lines accompanied by decreased proliferation. Our results suggest that decreased expression of LTBP-3 in MM could alter the targeting of TGF-beta to the ECM and lead to its increased activation. The current work emphasizes the coordinated process of the assembly and appropriate targeting of LTBPs with distinct adhesive or cytokine harboring properties into the ECM. The hierarchical assembly may have implications in the modulation of signaling events during morphogenesis and tissue remodeling.

Where and how to conserve: Extending the scope of spatial reserve network design

Ongoing habitat loss and fragmentation threaten much of the biodiversity that we know today. As such, conservation efforts are required if we want to protect biodiversity. Conservation budgets are typically tight, making the cost-effective selection of protected areas difficult. Therefore, reserve design methods have been developed to identify sets of sites, that together represent the species of conservation interest in a cost-effective manner. To be able to select reserve networks, data on species distributions is needed. Such data is often incomplete, but species habitat distribution models (SHDMs) can be used to link the occurrence of the species at the surveyed sites to the environmental conditions at these locations (e.g. climatic, vegetation and soil conditions). The probability of the species occurring at unvisited location is next predicted by the model, based on the environmental conditions of those sites. The spatial configuration of reserve networks is important, because habitat loss around reserves can influence the persistence of species inside the network. Since species differ in their requirements for network configuration, the spatial cohesion of networks needs to be species-specific. A way to account for species-specific requirements is to use spatial variables in SHDMs. Spatial SHDMs allow the evaluation of the effect of reserve network configuration on the probability of occurrence of the species inside the network. Even though reserves are important for conservation, they are not the only option available to conservation planners. To enhance or maintain habitat quality, restoration or maintenance measures are sometimes required. As a result, the number of conservation options per site increases. Currently available reserve selection tools do however not offer the ability to handle multiple, alternative options per site. This thesis extends the existing methodology for reserve design, by offering methods to identify cost-effective conservation planning solutions when multiple, alternative conservation options are available per site. Although restoration and maintenance measures are beneficial to certain species, they can be harmful to other species with different requirements. This introduces trade-offs between species when identifying which conservation action is best applied to which site. The thesis describes how the strength of such trade-offs can be identified, which is useful for assessing consequences of conservation decisions regarding species priorities and budget. Furthermore, the results of the thesis indicate that spatial SHDMs can be successfully used to account for species-specific requirements for spatial cohesion - in the reserve selection (single-option) context as well as in the multi-option context. Accounting for the spatial requirements of multiple species and allowing for several conservation options is however complicated, due to trade-offs in species requirements. It is also shown that spatial SHDMs can be successfully used for gaining information on factors that drive a species spatial distribution. Such information is valuable to conservation planning, as better knowledge on species requirements facilitates the design of networks for species persistence. This methods and results described in this thesis aim to improve species probabilities of persistence, by taking better account of species habitat and spatial requirements. Many real-world conservation planning problems are characterised by a variety of conservation options related to protection, restoration and maintenance of habitat. Planning tools therefore need to be able to incorporate multiple conservation options per site, in order to continue the search for cost-effective conservation planning solutions. Simultaneously, the spatial requirements of species need to be considered. The methods described in this thesis offer a starting point for combining these two relevant aspects of conservation planning.

Fine-tuning of the signalling network controlling morphogenesis and stem cell development in teeth

Tooth development is regulated by sequential and reciprocal interactions between epithelium and mesenchyme. The molecular mechanisms underlying this regulation are conserved and most of the participating molecules belong to several signalling families. Research focusing on mouse teeth has uncovered many aspects of tooth development, including molecular and evolutionary specifi cs, and in addition offered a valuable system to analyse the regulation of epithelial stem cells. In mice the spatial and temporal regulation of cell differentiation and the mechanisms of patterning during development can be analysed both in vivo and in vitro. Follistatin (Fst), a negative regulator of TGFβ superfamily signalling, is an important inhibitor during embryonic development. We showed the necessity of modulation of TGFβ signalling by Fst in three different regulatory steps during tooth development. First we showed that tinkering with the level of TGFβ signalling by Fst may cause variation in the molar cusp patterning and crown morphogenesis. Second, our results indicated that in the continuously growing mouse incisors asymmetric expression of Fst is responsible for the labial-lingual patterning of ameloblast differentiation and enamel formation. Two TGFβ superfamily signals, BMP and Activin, are required for proper ameloblast differentiation and Fst modulates their effects. Third, we identifi ed a complex signalling network regulating the maintenance and proliferation of epithelial stem cells in the incisor, and showed that Fst is an essential modulator of this regulation. FGF3 in cooperation with FGF10 stimulates proliferation of epithelial stem cells and transit amplifying cells in the labial cervical loop. BMP4 represses Fgf3 expression whereas Activin inhibits the repressive effect of BMP4 on the labial side. Thus, Fst inhibits Activin rather than BMP4 in the cervical loop area and limits the proliferation of lingual epithelium, thereby causing the asymmetric maintenance and proliferation of epithelial stem cells. In addition, we detected Lgr5, a Wnt target gene and an epithelial stem cell marker in the intestine, in the putative epithelial stem cells of the incisor, suggesting that Lgr5 is a marker of incisor stem cells but is not regulated by Wnt/β-catenin signalling in the incisor. Thus the epithelial stem cells in the incisor may not be directly regulated by Wnt/β-catenin signalling. In conclusion, we showed in the mouse incisors that modulating the balance between inductive and inhibitory signals constitutes a key mechanism regulating the epithelial stem cells and ameloblast differentiation. Furthermore, we found additional support for the location of the putative epithelial stem cells and for the stemness of these cells. In the mouse molar we showed the necessity of fi ne-tuning the signalling in the regulation of the crown morphogenesis, and that altering the levels of an inhibitor can cause variation in the crown patterning.

Water in Action : Encounters among 2- to 3-Year-Old Children, Adults, and Water in Day Care

In line with cultural psychology and developmental theory, a single case approach is applied to construct knowledge on how children s interaction emerge interlinked to historical, social, cultural, and material context. The study focuses on the negotiation of constraints and meaning construction among 2-to 3-year-old children, a preschool teacher, and the researcher in settings with water. Water as an element offers a special case of cultural canalization: adults selectively monitor and guide children s access to it. The work follows the socio-cultural tradition in psychology, particularly the co-constructivist theory of human development and the Network of Meanings perspective developed at the University of São Paulo. Valsiner s concepts of Zone of Free Movement and Zone of Promoted Action are applied together with studies where interactions are seen as spaces of construction where negotiation of constraints for actions, emotions, and conceptions occur. The corpus was derived at a Finnish municipal day care centre. During a seven months period, children s actions were video recorded in small groups twice a month. The teacher and the researcher were present. Four sessions with two children were chosen for qualitative microanalysis; the analysis also addressed the transformations during the months covered by the study. Moreover, the data derivation was analyzed reflectively. The narrowed down arenas for actions were continuously negotiated among the participants both nonverbally and verbally. The adults expectations and intentions were materialized in the arrangements of the setting canalizing the possibilities for actions. Children s co-regulated actions emerged in relation to the adults presence, re-structuring attempts, and the constraints of the setting. Children co-constructed novel movements and meanings in relation to the initiatives and objects offered. Gestures, postures, and verbalizations emerged from the initially random movements and became constructed to have specific meanings and functions; meaning construction became abbreviated. The participants attempted to make sense of the ambiguous (explicit and implicit) intentions and fuzzy boundaries of promoted and possible actions: individualized yet overlapping features were continuously negotiated by all the participants. Throughout the months, children s actions increasingly corresponded adults (re-defined) conceptions of water researchers as an emerging group culture. Water became an instrument and a context for co-regulations. The study contributes to discussions on children as participants in cultural canalization and emphasizes the need for analysis in early childhood education practices on the implicit and explicit constraint structures for actions.

Learning Challenges in Biogas Production for Sustainability : An activity theoretical study of a network from a swine industry chain

This study is about the challenges of learning in the creation and implementation of new sustainable technologies. The system of biogas production in the Programme of Sustainable Swine Production (3S Programme) conducted by the Sadia food processing company in Santa Catarina State, Brazil, is used as a case example for exploring the challenges, possibilities and obstacles of learning in the use of biogas production as a way to increase the environmental sustainability of swine production. The aim is to contribute to the discussion about the possibilities of developing systems of biogas production for sustainability (BPfS). In the study I develop hypotheses concerning the central challenges and possibilities for developing systems of BPfS in three phases. First, I construct a model of the network of activities involved in the BP for sustainability in the case study. Next, I construct a) an idealised model of the historically evolved concepts of BPfS through an analysis of the development of forms of BP and b) a hypothesis of the current central contradictions within and between the activity systems involved in BP for sustainability in the case study. This hypothesis is further developed through two actual empirical analyses: an analysis of the actors senses in taking part in the system, and an analysis of the disturbance processes in the implementation and operation of the BP system in the 3S Programme. The historical analysis shows that BP for sustainability in the 3S Programme emerged as a feasible solution for the contradiction between environmental protection and concentration, intensification and specialisation in swine production. This contradiction created a threat to the supply of swine to the food processing company. In the food production activity, the contradiction was expressed as a contradiction between the desire of the company to become a sustainable company and the situation in the outsourced farms. For the swine producers the contradiction was expressed between the contradictory rules in which the market exerted pressure which pushed for continual increases in scale, specialisation and concentration to keep the production economically viable, while the environmental rules imposed a limit to this expansion. Although the observed disturbances in the biogas system seemed to be merely technical and localised within the farms, the analysis proposed that these disturbances were formed in and between the activity systems involved in the network of BPfS during the implementation. The disturbances observed could be explained by four contradictions: a) contradictions between the new, more expanded activity of sustainable swine production and the old activity, b) a contradiction between the concept of BP for carbon credits and BP for local use in the BPfS that was implemented, c) contradictions between the new UNFCCC1 methodology for applying for carbon credits and the small size of the farms, and d) between the technologies of biogas use and burning available in the market and the small size of the farms. The main finding of this study relates to the zone of proximal development (ZPD) of the BPfS in Sadia food production chain. The model is first developed as a general model of concepts of BPfS and further developed here to the specific case of the BPfS in the 3S Programme. The model is composed of two developmental dimensions: societal and functional integration. The dimension of societal integration refers to the level of integration with other activities outside the farm. At one extreme, biogas production is self-sufficient and highly independent and the products of BP are consumed within the farm, while at the other extreme BP is highly integrated in markets and networks of collaboration, and BP products are exchanged within the markets. The dimension of functional integration refers to the level of integration between products and production processes so that economies of scope can be achieved by combining several functions using the same utility. At one extreme, BP is specialised in only one product, which allows achieving economies of scale, while at the other extreme there is an integrated production in which several biogas products are produced in order to maximise the outcomes from the BP system. The analysis suggests that BP is moving towards a societal integration, towards the market and towards a functional integration in which several biogas products are combined. The model is a hypothesis to be further tested through interventions by collectively constructing the new proposed concept of BPfS. Another important contribution of this study refers to the concept of the learning challenge. Three central learning challenges for developing a sustainable system of BP in the 3S Programme were identified: 1) the development of cheaper and more practical technologies of burning and measuring the gas, as well as the reduction of costs of the process of certification, 2) the development of new ways of using biogas within farms, and 3) the creation of new local markets and networks for selling BP products. One general learning challenge is to find more varied and synergic ways of using BP products than solely for the production of carbon credits. Both the model of the ZPD of BPfS and the identified learning challenges could be used as learning tools to facilitate the development of biogas production systems. The proposed model of the ZPD could be used to analyse different types of agricultural activities that face a similar contradiction. The findings could be used in interventions to help actors to find their own expansive actions and developmental projects for change. Rather than proposing a standardised best concept of BPfS, the idea of these learning tools is to facilitate the analysis of local situations and to help actors to make their activities more sustainable.