A Study On The Roles And Effects Of Syndecans On Endothelial Cell Biology During Angiogenesis

PhD (md res)Angiogenesis, the formation of new blood vessels from pre-existing ones, is a key process during development, wound healing and the female reproductive cycle. However, it is also a pathological feature of many diseases such as cancer, age-related macular degeneration and chronic inflammatory pathologies. Given the complexity underlying the fine regulation of this process, despite the great number of studies, our understanding of it is not yet exhaustive. Syndecans are heparan sulphate proteoglycans with roles in cell adhesion, migration and growth factor interactions. The aim of this body of work was to investigate the role of these molecules in the regulation of angiogenesis. For this purpose, in the first part of my project, I investigated the effects of the extracellular core proteins of all four syndecans on endothelial cell functions. These studies identified that all four proteins are able to inhibit angiogenesis and strongly suppress endothelial cell migration, invasion and tube formation. On the basis of these discoveries, I focused on the syndecan-2 extracellular core protein and showed that it is shed during inflammation and can interact with the endothelial cell surface tyrosine phosphatase receptor CD148. Upon binding to CD148, the syndecan-2 extracellular core protein reduces the expression of active β1 integrins on endothelial cell surface and inhibits endothelial cell migration. This provides a mechanism through which shed syndecan-2 can regulate angiogenesis. Critically, this is an entirely novel pathway for the reduction of angiogenesis which is independent to the main angiogenic factor VEGF. Lastly, since previous works suggested that the syndecan-4-null mouse may exhibit angiogenic defects, I set out to determine the role of syndecan-4 in angiogenesis. Here, I observed an up-regulation of syndecan-4 on endothelial cells in angiogenic settings both in vitro and in vivo. Furthermore, through analysis of in vivo models of pathological angiogenesis, I observed that syndecan-4 is critical for VEGF-A-mediated angiogenesis. This effect was partially explained by impaired activation of VEGFR2 in syndecan-4-null endothelial cells. This data suggests that syndecan- 4 is the main heparan sulphate proteoglycan to act as VEGFR2 co-receptor during pathological angiogenesis. Collectively, the findings of this research have identified previously unknown roles for syndecans in the regulation of angiogenesis

A Study On The Roles And Effects Of Syndecans On Endothelial Cell Biology During Angiogenesis

PhD (md res)Angiogenesis, the formation of new blood vessels from pre-existing ones, is a key process during development, wound healing and the female reproductive cycle. However, it is also a pathological feature of many diseases such as cancer, age-related macular degeneration and chronic inflammatory pathologies. Given the complexity underlying the fine regulation of this process, despite the great number of studies, our understanding of it is not yet exhaustive. Syndecans are heparan sulphate proteoglycans with roles in cell adhesion, migration and growth factor interactions. The aim of this body of work was to investigate the role of these molecules in the regulation of angiogenesis. For this purpose, in the first part of my project, I investigated the effects of the extracellular core proteins of all four syndecans on endothelial cell functions. These studies identified that all four proteins are able to inhibit angiogenesis and strongly suppress endothelial cell migration, invasion and tube formation. On the basis of these discoveries, I focused on the syndecan-2 extracellular core protein and showed that it is shed during inflammation and can interact with the endothelial cell surface tyrosine phosphatase receptor CD148. Upon binding to CD148, the syndecan-2 extracellular core protein reduces the expression of active β1 integrins on endothelial cell surface and inhibits endothelial cell migration. This provides a mechanism through which shed syndecan-2 can regulate angiogenesis. Critically, this is an entirely novel pathway for the reduction of angiogenesis which is independent to the main angiogenic factor VEGF. Lastly, since previous works suggested that the syndecan-4-null mouse may exhibit angiogenic defects, I set out to determine the role of syndecan-4 in angiogenesis. Here, I observed an up-regulation of syndecan-4 on endothelial cells in angiogenic settings both in vitro and in vivo. Furthermore, through analysis of in vivo models of pathological angiogenesis, I observed that syndecan-4 is critical for VEGF-A-mediated angiogenesis. This effect was partially explained by impaired activation of VEGFR2 in syndecan-4-null endothelial cells. This data suggests that syndecan- 4 is the main heparan sulphate proteoglycan to act as VEGFR2 co-receptor during pathological angiogenesis. Collectively, the findings of this research have identified previously unknown roles for syndecans in the regulation of angiogenesis

Dermoscopy and methyl aminolevulinate: A study for detection and evaluation of field cancerization

Actinic keratosis (AK) is a keratinocyte intraepidermal neoplasia UV light – induced that frequently appears in sun-exposed areas of the skin. Although historically AK was de fi ned as “ precancerous ” , actually it is considered as the earliest stage of squamous cell carcinoma (SCC) in situ. Since AKs can progress into invasive SCC, their treatment isrecommended. AKsrarely developasa singlelesion;usually multiplelesions commonly affect anen- tire area of chronically actinic damaged skin. This has led to the concept of “ fi eld cancerization ” , an area chroni- cally sun-exposed that surrounds peripherally visible lesions, in which are individualized subclinical alterations. One of the main principles endpoint in the management of AKs is the evaluation and the treatment of fi eld cancerization. In this view, in order to detect and quantify fi eld cancerization, we employed a method based on the topical application of methyl aminolevulinate (MAL) and the detection of the fl uorescence emitted by its metabolite Protoporphyrin IX (PpIX); then, considering the extension and the intensity of measured fl uores- cence, we create a score of fi eld cancerization. The results show that patients underwent to daylight PDT had a reduction of total score, from T0 to T2. Whereas in the group untreated we observed a stability of total score or a slightly worse. So, the method and the score used allows to evaluate with a good approximation the dimension of fi eld cancerization and show the modi fi cation of it after treatment

Dynamic heterogeneity in random and gradient copolymers: A computational investigation

By means of molecular dynamics simulations, we investigate the structural relaxation in disordered random copolymers and lamellar phases of gradient copolymers, containing chemical species of very different mobilities. Two models have been investigated: a generic bead-spring system and a MARTINI coarse-grained model of a polyester resin. The lamellar phase of the gradient copolymer is formed by domains rich in one species and poor in the other one, which are separated by broad interfaces. Unlike in strongly segregated block copolymers, there is a finite probability of finding monomers of a given species at any position within the domains rich in the other species. A direct consequence of this feature is that monomers can probe very different chemical environments, and because of the strong dynamic asymmetry between the two components, their relaxation are characterized by an extreme dynamic heterogeneity. This is confirmed by an analysis of dynamic correlators as a function of the distance to the interface. In the case of random copolymers long-range ordering is not possible, and local microsegregation results in a much weaker dynamic heterogeneity. The former features are consistent with the experimental observation of narrow glass transitions in random copolymers but extremely broad ones in lamellar gradient copolymers. © 2013 American Chemical Society.Part of this work was carried out under the HPC-Europa2 project (project number: 228398) with the support of the European Commission Capacities Area-Research Infrastructures Initiative. We also acknowledge support from Projects MAT2007-63681 (Spain), MAT2012-31088 (Spain), and IT-436-07 (GV, Spain).Peer Reviewe

LRG1: an emerging player in disease pathogenesis

The secreted glycoprotein leucine-rich α-2 glycoprotein 1 (LRG1) was first described as a key player in pathogenic ocular neovascularization almost a decade ago. Since then, an increasing number of publications have reported the involvement of LRG1 in multiple human conditions including cancer, diabetes, cardiovascular disease, neurological disease, and inflammatory disorders. The purpose of this review is to provide, for the first time, a comprehensive overview of the LRG1 literature considering its role in health and disease. Although LRG1 is constitutively expressed by hepatocytes and neutrophils, Lrg1-/- mice show no overt phenotypic abnormality suggesting that LRG1 is essentially redundant in development and homeostasis. However, emerging data are challenging this view by suggesting a novel role for LRG1 in innate immunity and preservation of tissue integrity. While our understanding of beneficial LRG1 functions in physiology remains limited, a consistent body of evidence shows that, in response to various inflammatory stimuli, LRG1 expression is induced and directly contributes to disease pathogenesis. Its potential role as a biomarker for the diagnosis, prognosis and monitoring of multiple conditions is widely discussed while dissecting the mechanisms underlying LRG1 pathogenic functions. Emphasis is given to the role that LRG1 plays as a vasculopathic factor where it disrupts the cellular interactions normally required for the formation and maintenance of mature vessels, thereby indirectly contributing to the establishment of a highly hypoxic and immunosuppressive microenvironment. In addition, LRG1 has also been reported to affect other cell types (including epithelial, immune, mesenchymal and cancer cells) mostly by modulating the TGFβ signalling pathway in a context-dependent manner. Crucially, animal studies have shown that LRG1 inhibition, through gene deletion or a function-blocking antibody, is sufficient to attenuate disease progression. In view of this, and taking into consideration its role as an upstream modifier of TGFβ signalling, LRG1 is suggested as a potentially important therapeutic target. While further investigations are needed to fill gaps in our current understanding of LRG1 function, the studies reviewed here confirm LRG1 as a pleiotropic and pathogenic signalling molecule providing a strong rationale for its use in the clinic as a biomarker and therapeutic target

Genetic Diversity of Old Chicken Breeds Kept in Poland

The aim of this study was to compare the genetic variation of five local chicken breeds reared in Poland. Twenty-seven microsatellite markers were investigated in 138 birds belonging to five breeds: Miniature Cochin (MCO), Gold Italian (GI), Green Legged Partridge (GLP), Silver Italian (SI) and White Leghorn (WL). One hundred eighty five alleles were detected in the overall population, with a mean number of 6.85 ± 3.32 alleles per locus. For the local breeds, the observed and expected heterozygosity ranged from a minimum of 0.287 to a maximum of 0.458 and from 0.397 to 0.499 for the GI and SI breeds, respectively. The overall population heterozygote deficiency was 0.430, the average Wright’s inbreeding coefficient (FIS) was 0.061 and the heterozygote deficiency due to breed subdivision was 0.393. Wright’s fixation index was slightly positive for all breeds excluding MCO (FIS = -0.476) and the estimated molecular inbreeding (fij) within breed ranged from 0.296 (GLP and SI) to 0.361 (WL) evidencing limited coancestry. Mean allelic richness, obtained with rarefaction method based on sixteen observations, was 2.12 being the WL the less variable (1.79). Tomiuk and Loeschcke’s DTL genetic distance values were used to draw a neighbornet network which separated the cluster made of MCO and GLP from the cluster of GI, WL and SI. The results arising from our microsatellites analysis represent a starting point for the valorization of these local Polish chicken breeds for monitoring and preserving their genetic variability

Efflux pump genes of the resistance-nodulation-division family in Burkholderia cenocepacia genome

BACKGROUND: Burkholderia cenocepacia is recognized as opportunistic pathogen that can cause lung infections in cystic fibrosis patients. A hallmark of B. cenocepacia infections is the inability to eradicate the organism because of multiple intrinsic antibiotic resistance. As Resistance-Nodulation-Division (RND) efflux systems are responsible for much of the intrinsic multidrug resistance in Gram-negative bacteria, this study aims to identify RND genes in the B. cenocepacia genome and start to investigate their involvement into antimicrobial resistance. RESULTS: Genome analysis and homology searches revealed 14 open reading frames encoding putative drug efflux pumps belonging to RND family in B. cenocepacia J2315 strain. By reverse transcription (RT)-PCR analysis, it was found that orf3, orf9, orf11, and orf13 were expressed at detectable levels, while orf10 appeared to be weakly expressed in B. cenocepacia. Futhermore, orf3 was strongly induced by chloramphenicol. The orf2 conferred resistance to fluoroquinolones, tetraphenylphosphonium, streptomycin, and ethidium bromide when cloned and expressed in Escherichia coli KAM3, a strain lacking the multidrug efflux pump AcrAB. The orf2-overexpressing E. coli also accumulate low concentrations of ethidium bromide, which was restored to wild type level in the presence of CCCP, an energy uncoupler altering the energy of the drug efflux pump. CONCLUSION: The 14 RND pumps gene we have identified in the genome of B. cenocepacia suggest that active efflux could be a major mechanism underlying antimicrobial resistance in this microorganism. We have characterized the ORF2 pump, one of these 14 potential RND efflux systems. Its overexpression in E. coli conferred resistance to several antibiotics and to ethidium bromide but it remains to be determined if this pump play a significant role in the antimicrobial intrinsic resistance of B. cenocepacia. The characterization of antibiotic efflux pumps in B. cenocepacia is an obligatory step prior to the design of specific, potent bacterial inhibitors for the improved control of infectious diseases. Consequently, the topic deserves to be further investigated and future studies will involve systematic investigation on the function and expression of each of the RND efflux pump homologs

Structural basis of human LRG1 recognition by Magacizumab, a humanized monoclonal antibody with therapeutic potential

The formation of new dysfunctional blood vessels is a crucial stage in the development of various conditions such as macular degeneration, diabetes, cardiovascular disease, neurological disease and inflammatory disorders, as well as during tumor growth, eventually contributing to metastasis. An important factor involved in pathogenic angiogenesis is leucine-rich α-2-glycoprotein 1 (LRG1), the antibody blockade of which has been shown to lead to a reduction in both choroidal neovascularization and tumor growth in mouse models. In this work, the structural interactions between the LRG1 epitope and the Fab fragment of Magacizumab, a humanized function-blocking IgG4 against LRG1, are analysed, determining its specific binding mode and the key residues involved in LRG1 recognition. Based on these structural findings, a series of mutations are suggested that could be introduced into Magacizumab to increase its affinity for LRG1, as well as a model of the entire Fab–LRG1 complex that could enlighten new strategies to enhance affinity, consequently leading towards an even more efficient therapeutic

Combined X-ray and optical analysis to probe the origin of the plateau emission in γ\gamma-ray bursts afterglows

A large fraction of gamma-ray bursts (GRBs) shows a plateau phase during the X-ray afterglow emission, whose physical origin is still debated. In this work we define a sample of 30 GRBs with simultaneous X-ray and optical data during and after the plateau phase. Through a time-resolved spectral analysis of the X-ray plateaus, we test the consistency of the unabsorbed optical fluxes with those obtained via X-ray-to-optical spectral extrapolation by assuming a synchrotron spectrum. Combining X-ray with optical data, we find that 63% (19/30) GRBs are compatible with a single synchrotron spectrum thus suggesting that both the optical and X-ray radiations are produced from a single emitting region. For these GRBs we derive the temporal evolution of the break frequency and we compare it with the expectations predicted by several models. For 11/30 GRBs the optical emission is above the predicted range of values extrapolated from the X-rays in at least one temporal bin of the light curve. These GRBs may not be explained with a single zone emission, indicating the necessity of invoking two cooperating processes in order to explain the broad band spectral behaviour during X-ray plateaus. We discuss our findings in the framework of different scenarios invoked to explain the plateau feature, including the energy injection from a spinning-down magnetar and the high latitude emission from a structured jet.Comment: submitted to A&