Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2010. / Härtill 4 uppsatser.

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Αγγειογένεση καλείται ο σχηματισμός νέων αγγείων από προϋπάρχοντα δίκτυα αιμοφόρων αγγείων σε προηγουμένως ανάγγειες δομές. Η αγγειογένεση συμμετέχει σε μια πλειάδα νοσημάτων (π.χ. καρκίνος, ρευματοειδής αρθρίτιδα) συμβάλλοντας καθοριστικά στην παθογένεια αυτών των καταστάσεων και στην ανάπτυξη επιπλοκών. Στον οφθαλμό, η παθολογική αγγειογένεση στις διάφορες στιβάδες του οργάνου αποτελεί την κύρια αιτία μη αναστρέψιμης απώλειας της όρασης στον Δυτικό κόσμο, καθιστώντας επιτακτική την ανάγκη ανάπτυξης νέων αντι-αγγειογενετικών παραγόντων, ικανών να κατανεμηθούν ενδοφθαλμίως και να αναστείλουν αποτελεσματικά τόσο την εν εξελίξει όσο και την εγκατεστημένη αγγειογένεση. Η Παρστατίνη αποτελεί το ελεύθερο πεπτίδιο 41 αμινοξέων που προκύπτει από την πρωτεολυτική διάσπαση του αμινοτελικού άκρου του ανθρώπινου υποδοχέα PAR1 κατά την ενεργοποίησή του από τη θρομβίνη. Προηγούμενες πειραματικές μελέτες έχουν καταδείξει ότι η Παρστατίνη καταστέλλει την αγγειογένεση στον οφθαλμό και ασκεί καρδιοπροστατευτική δράση σε βλάβες εξ ισχαιμίας/επαναιμάτωσης. Επίσης, έχει δειχθεί ότι τα πεπτιδικά υπομόρια της Παρστατίνης, Π1-26 και Π24-41, παρουσιάζουν εξειδικευμένη δράση και επιδρούν εκλεκτικά στην προστασία του μυοκαρδίου και στην αναστολή της οφθαλμικής νεοαγγειογένεσης, αντιστοίχως. Σύμφωνα με αυτά τα πειράματα, οι δράσεις των Π1-26 και Π24-41 είναι σημαντικά ισχυρότερες από αυτές του μητρικού μορίου, Παρστατίνη. Σκοπός της τρέχουσας μελέτης είναι η διερεύνηση της ιστικής κατανομής in vivo της Παρστατίνης και των αναλόγων-πεπτιδίων (Π1-26, Π24-41) αυτής στον οφθαλμό και στον νεφρό επίμυος. Η Παρστατίνη και τα ανάλογα-πεπτίδιά της (Π1-26, Π24-41) σημασμένα με FITC χορηγήθηκαν υπό τον βολβικό επιπεφυκότα ή ενδοφλεβίως, σε υγιείς αρσενικούς επίμυες. Ακολούθως τα πειραματόζωα θανατώθηκαν σε διαφορετικά χρονικά διαστήματα και οι ιστοί (οφθαλμοί, νεφροί, μυοκάρδιο) απομονώθηκαν χειρουργικά. Κατόπιν κατάλληλης επεξεργασίας, ελήφθησαν τομές ιστών νωπού παρασκευάσματος, πάχους 10 μm, οι οποίες παρατηρήθηκαν σε μικροσκόπιο φθορισμού. Κατά την παρατήρηση των οφθαλμικών τομών με FITC-Παρστατίνη και αυτών με FITC-Π1-26, ισχυρό σήμα φθορισμού ανιχνεύτηκε στις θέσεις χορήγησης και στους περιοφθαλμικούς ιστούς. Δεν παρατηρήθηκε σήμα φθορισμού στις εσώτερες στιβάδες του οφθαλμού. Ειδικότερα για την FITC-Παρστατίνη, η κατανομή του σήματος φθορισμού δεν μεταβλήθηκε σε διαφορετικά χρονικά διαστήματα που κυμάνθηκαν από 30 λεπτά έως 19 ώρες. Συγκριτικά με τα παραπάνω αποτελέσματα, οι αντίστοιχες οφθαλμικές τομές με FITC-Π24-41 ανέδειξαν ισχνό σήμα φθορισμού στις θέσεις χορήγησης και στους περιοφθαλμικούς ιστούς, χωρίς να συνοδεύεται από σήμα φθορισμού στις έσω ανατομικές δομές του οργάνου. Στις τομές νεφρού και μυοκαρδίου, ο ισχυρός αυτοφθορισμός δεν επέτρεψε τη μελέτη της ιστικής κατανομής των πεπτιδίων. Συμπερασματικά, καταδεικνύεται ότι η χορήγηση υπό το βολβικό επιπεφυκότα της Παρστατίνης και των πεπτιδικών αναλόγων της, του Π1-26 και του Π24-41 δεν αποτελεί την ενδεδειγμένη οδό χορήγησης για την ενδοφθάλμια κατανομή των χορηγούμενων ουσιών. / Angiogenesis refers to the formation of new blood vessels from pre-existing vasculature in previously avascular structures. Angiogenesis is an important component of numerous diseases (such as cancer, rheumatoid arthritis etc), contributing in the pathogenesis of these conditions. In the western world, pathological ocular angiogenesis constitutes the major cause of irreversible vision loss. Consequently, this fact gives rise to the need for the development of new anti-angiogenic agents, characterized by successful intraocular delivery and effective inhibition of developing angiogenesis and regression of already established vessels. Parstatin is a 41-amino acid peptide, released through proteolytic cleavage of the human PAR1 receptor N-terminal region upon activation by thrombin. Experimental studies have demonstrated that Parstatin suppresses ocular neovascularization and has a potential therapeutic role in the protection of myocardium by ischaemia and reperfusion injury. In addition, it has been shown that the Parstatin fragments, P1-26 and P24-41, have distinct effects in protecting myocardium and inhibiting ocular neovascularization, respectively. In fact, these effects are more potent than the native Parstatin. The aim of the present study was the in vivo evaluation of tissue-distribution of Parstatin and its peptide fragments (P1-26, P24-41) in rat eyes and kidneys. To address this question, Parstatin and its peptide analogues (P1-26, P24-41) were conjugated with FITC. Pathogen-free male rats received subconjunctival or intravenous injections of the FITC-labeled peptides. Following euthanasia of rats at different time points after the injections were performed, the tissues (eyes, kidneys, hearts) were surgically excised from the respective animals. After treatment, the fresh-frozen tissues were sectioned sagittally into 10 μm thick slices and observed using fluorescence microscope. When sections of eyes treated with FITC-Parstatin and those ones treated with FITC-P1-26 were examined, intensive FITC signal was detected at the sites of administration and at periocular tissues. FITC signal was not observed in internal ocular layers. Regarding FITC-Parstatin, the distribution of specific signal showed no variability during different time intervals which ranged from 30 minutes to 19 hours. As compared to the above results, the respective ocular sections with FITC-P24-41 revealed weak FITC signal at the injection sites and at periocular tissues, accompanied by no detection of intraocular localization. On the other hand, high autofluorescence present in kidney sections and heart sections did not allow the investigation of tissue-distribution of the peptide molecules. In the present study, it appeared that subconjunctival administration of Parstatin and of its peptide fragments, P1-26 and P24-41, is not the appropriate route for the drug delivery into the internal ocular tissues.

Cigarette smoking (CS) represents the main causative agent underlying development and progress of COPD. Recently, involvement of CS in the pathogenesis of COPDassociated muscle abnormalities is becoming increasingly evident. Nevertheless, involved triggers and underlying mechanisms remain largely unknown. This study was conceived in order to examine effects of cigarette smoke exposure on skeletal muscle morphology, vascular supply and function. For this purpose, we have specifically designed murine COPD/emphysema model and gastrocnemius muscle was examined, while in vitro experiments were conducted using murine C2C12 skeletal muscle myocytes. In addition to the mild emphysematous changes present in the lungs of CS-exposed mice, our results demonstrated evident signs of muscle atrophy reflected by decreased fiber cross-sectional area, profound fiber size variation and reduced body mass. Furthermore, we have observed impairment in terminal myogenesis and lower number of myonuclei in skeletal muscles of CS-exposed animals despite evident activation of muscle repair process. Additionally, our results demonstrate capillary rarefaction in skeletal muscles of CS-exposed animals which was associated with deregulation of hypoxia-angiogenesis signaling, reduced levels of angiogenic factors such as HIF1-α and VEGF and enhanced expression of VHL and its partner proteins PHD2 and Ube2D1. The results of our in-vitro experiments demonstrated that VHL and its ubiquitination machinery can be synergistically regulated by TNF and hypoxia consequentially impairing angiogenic potential of skeletal muscle myocytes. Finally, we have shown that CS elicits chronic ER stress in murine skeletal muscles which is associated with activation of ERAD and apoptotic pathways as mirrored by elevated expression of Usp19, caspase 12 and caspase 3 in skeletal muscles of CSexposed animals. Moreover, molecular and morphological alterations in CS-exposed mice resulted in impairment of muscle function as reflected by their impaired exercise capacity. Taken together, from our results it is evident that cigarette smoke exposure elicits set of morphological, vascular and functional changes highly resembling those observed in COPD. Additionally, CS induces wide range of molecular alterations and signaling pathway deregulations suggesting profound effects of cigarette smoke exposure on skeletal muscle cell homeostasis.

Made available in DSpace on 2014-06-11T19:27:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-04-30Bitstream added on 2014-06-13T18:31:06Z : No. of bitstreams: 1 agostini_dls_me_bauru.pdf: 3608842 bytes, checksum: 9f88482e6ed9f6d122b753891fe26c0a (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / As membranas de borracha natural são usadas frequentemente como material biológico na indução de angiogênese e neoformação. Neste trabalho foram realizados estudos com objetivo de identificar os componentes do látex que são responsáveis pelo processo de angiogênese e neoformação. O látex, as fases centrifugadas do látex e as membranas da borracha natural, tratados termicamente em 60, 85 e 120ºC, foram liofilizados para a redução de água nos mesmos. A caracterização de tais materiais foi realizada por espectroscopia infravermelha (FTIR), micro-Raman, ressonância magnética nuclear (NMR - 1H e 13C), difração de raio x (DRX), microscopia eletrônica de varredura (MEV) e microscopia de força atômica (AFM), análise do termogravimétrica (TG) acoplado com infravermelho (TG/FT-IR), calorimetria exploratória diferencial (DSC), análise dinâmico mecânica (DMA) e o método de Bradford. Nos resultados de espectroscopia no infravermelho, micro de Raman, NMR (1H e 13C), de raios x e TG/FT-IR; os componentes químicos do látex, das fases centrifugadas do látex e das membranas da borracha natural foram identificados. A técnica de TG foi utilizada para avaliar a estabilidade térmica e os resultados mostraram que as membranas obtidas a 60ºC possuem maior estabilidade. Nos resultados de DSC notou-se que a transição vítrea acontece em ~ -68ºC, para todos os materiais que contêm isopreno e a degradação estrutural ocorrem em torno de 376ºC. As transições de vítreas foram confirmadas através dos resultados de DMA. O látex centrifugado apresenta três fases: partículas de borracha (F1), lutóides (F2) e Frey-Wyssling (F3). Nas frações F2 e F3 o isopreno é ausente, ou apresenta pequena proporção, mas contêm diversos componentes químicos: proteínas, ácidos aminados e grupos funcionais que podem induzir o angiogênese e a neoformação em tecidos biológicos... / Natural latex and natural rubber membranes are frequently used as biomaterial in the angiogenesis induction and neoformation of biological tissues. Our studies aimed to study and identify the latex components that are responsible for angiogenesis and neoformation processes. The natural latex, the centrifuged latex phases and the natural rubber membranes were thermally treated at 60, 85 and 120ºC and afterward all samples were lyophilized. The characterization of such materials were carried out using infrared (FT-IR), micro Raman spectroscopy, nuclear magnetic resonance (NMR - 1H e 13C), X-ray diffraction, scanning electronic microscopy (SEM) and atomic force microscopy (AFM), thermogravimetry analysis coupled with infrared (TG/FT-IR), differential scanning calorimeter (DSC), dynamic mechanical analysis (DMA) and the method of Bradford. From the results of infrared and micro Raman spectroscopy, NMR (1H e 13C), X-rays and TG/FT-IR the chemical components present in the latex, in the centrifuged latex phases and in natural rubber membranes were identified. TG technique was used to evaluate the thermal stability and results showed that membranes obtained at 60ºC present greater stability, up to 350ºC. From DSC results it was found that the glass transition happens at –68ºC, for all materials containing isoprene, and the structural degradation occurs at 376ºC. Glass transitions were confirmed also by DMA results. Centrifuged latex presents three phases: rubber particles (F1), lutoids (F2) and Frey-Wyssling (F3). In the fractions F2 and F3 the isoprene is absent but they contain several chemical components: proteins, amino acids and functional groups that can induce angiogenesis and neoformation on biological tissues. The treated membrane 60ºC of natural rubber showed the highest angiogenesis and neoformation activities. Furthermore, membranes containing pores favor the cicatrization process and the vascularization processes.

Angiogenesis supports tumor growth and facilitates metastasis, the leading cause of patient mortality in breast and other types of solid tumors. Angiopoietin-2 (Ang-2) is an angiogenic factor whose overexpression is associated with increased tumor vascularity, metastasis, and decreased patient survival. We assessed the effects of Ang-2 on breast tumor vasculature by comparing vascular morphology and metastasis of orthotopically implanted metastatic breast carcinoma line MDA-MB-231 that either lacked or overexpressed Ang-2. Methods: Luciferase-tagged MDA-MB-231 breast carcinoma cells designated hAng2 were engineered to overexpress human Ang-2. The control line expressed hAng-2 in reverse orientation. Stable production of hAng-2 was confirmed by RT-PCR, qRT-PCR, Western blot, and ELISA. Functionality of recombinant hAng-2 was assessed by migration and proliferation assays. MDA-MB-231 tumors, hAng2 and control, were orthotopically implanted into female Nu/Nu and SCID mice. Metastasis to lymph node and lung were determined by measuring luciferase activity in tissue extracts. Tumor blood vessel morphology was analyzed by immunohistochemistry (IHC) using antibodies against MECA-32, smooth muscle actin (SMA-alpha), VEGFR-3 and Notch-1. Results: MDA-MB-231 hAng2 lines were stably generated to produce 0-370 ng/ml of hAng-2 while expression in control line was undetectable. Tumor-produced hAng-2 was functional as demonstrated by a dose-dependent induction of migration of lymphatic endothelial cells and mouse mesenchymal stem cells with a maximum increase of 3-fold. Overexpression of Ang-2 had no significant effect on proliferation of cultured MDA-MB-231 cells, growth rate of hAng2 tumors or blood vessel density. In contrast, we detected substantial differences in vascular morphology of Ang-2 overexpressing tumors including 1.7-fold increase in blood vascular area, 2.8-fold increase in number of vessels with open lumen, and 6-fold increase in lumens' cross-sectional area. Blood vascular pericyte coverage shown by SMA-α staining decreased (p>0.001) in hAng2 tumors, demonstrating vessel destabilization. Blood vascular invasion by tumor cells and pulmonary metastasis increased in Ang-2 overexpressing tumors by 500% and 1100%, as compared with control tumors. Blood vessels in hAng2 tumors, but not lymphatic vessels, displayed significantly upregulated Notch-1 and VEGFR-3 expression amplified by a 2.2-fold. Conclusions: These data suggest that Ang-2 increases metastasis because of suppression of pericytes' recruitment that leads to destabilization of the tumor vessels, which facilitates the entry of tumor cells into the vessels and increases metastatic spread. These effects are associated with up-regulation of Notch-1 and VEGFR-3 on tumor vasculature suggesting that signaling of these proteins underlie the morphologic changes in Ang-2 overexpressing tumors. This is consistent with prior data demonstrating up-regulation of VEGFR-3 on tumor blood vessels and association of both proteins with increased metastasis. This study demonstrates that Ang-2 plays a key role in hematogenous metastasis and suggests that Ang-2 might represent novel a target for inhibition of breast cancer metastasis.

One of the critical periods of time for the establishment of pregnancy is after the onset of implantation but before the establishment of the placenta. In fact, evidence strongly suggests that when abnormalities occur during this period of pregnancy in humans there may be dire consequences such as termination of pregnancy, pre-eclampsia later in pregnancy and small for gestational weight offspring that are less healthy in later life. Therefore, what occurs in the endometrium during the progression of implantation can be crucial for proper fetal development later in pregnancy as well as the health of the offspring. One of the evolving ideas coming from research on the biology of what occurs during early pregnancy is the existence of key bi-directional communications between the conceptus and uterus that may be required for successful pregnancy. Amazingly, some common aspects of this signaling might exist to some extent between mammalian species that have very different modes of implantation and where quite different placentas are formed. Although some conceptus-uterine communication during the progression of decidualization is starting to be worked out, we still have much to learn about the precise details of the entire repertoire of the molecular signals responsible. In the rodent we still have to clearly find and define the biology of important paracrine factors produced by the conceptus that target cells of the endometrium during this period of time when the endometrium is undergoing decidualization. This review focuses on some of the approaches that have been used in the past and what has been learned about the effect of the conceptus on the decidualization of the rodent uterus. Where possible, this is compared and contrasted to what is currently known in other species that exhibit quite different modes of implantation.

The generation of new lymphatic vessels through lymphangiogenesis has been implicated in many disease states. This process has some overlap with the better studied angiogenesis pathway, but is under distinct molecular control. Specifically, it has been shown that VEGFR-3 and neuropilin-2 are important mediators of lymphangiogenesis. A greater understanding of this process could lead to new therapies for cancer and lymphedemas. We investigated lymphatic vessel growth in a mouse model with a focus on the effects of neuropilin-2 knockout. First, we induced an immunogenic response via delayed-type hypersensitivity to examine lymphangiogenesis in the physiologic state. Our neuropilin-2 knockout mouse model displayed a decreased ability to resolve inflammation on exposure to an allergen. Next, we subcutaneously injected a highly invasive melanoma to examine lymphangiogenesis in the pathologic state. We noted significantly reduced tumor growth in our neuropilin-2 knockout. In addition, the neuropilin-2 knockout mice displayed reduced vessel area in comparison to their wild-type littermates, suggesting that inhibition of neuropilin-2 may prove a potent antitumor therapeutic strategy. These results highlight neuropilin-2's important role as a mediator of physiological and pathological angiogenesis and lymphangiogenesis.

In 1899, the German surgeon Max Wilms hypothesised that different cell types in a variety of childhood kidney cancers were all derived from the mesodermal layer during embryonic development. Nearly a century later, the WT1 gene was identified on the short arm of chromosome 11, and was thought to be inactive in ~20% of nephroblastomas (Wilms’ tumours). The expression of WT1 after birth appears to be restricted to a finite number of tissues, namely, the glomerular podocytes, mesothelium and ~1% of bone marrow cells. Emerging evidence suggests WT1 is required not only for development, but also for tissue homeostasis, regeneration, repair and angiogenesis. Interestingly, WT1 has been implicated in the response to myocardial infarction and tumour angiogenesis, yet its precise role remains unclear. This thesis aims to address the hypothesis that activation of the WT1 gene in the vascular endothelium is essential for physiological and pathophysiological angiogenesis in the adult. In order to assess whether Wt1 was expressed in quiescent endothelial cells (ECs) immunofluorescence was used to analyse a variety of tissues in the adult mouse. Whilst Wt1 was detected in renal podocytes, no endothelial Wt1 expression was discovered in the lung, heart, kidney, spleen and gastrocnemius muscle. In contrast, tissues known to undergo physiological angiogenesis (endometrium and breast) did exhibit Wt1 expression in the vascular endothelium. Moreover, tubular EC outgrowths generated by aortic rings embedded in collagen ex vivo were positive for Wt1. The role of Wt1 in ischaemic angiogenesis was assessed using models of hind-limb and coronary ischaemia in the mouse. Wt1 was detected in ECs and non-vascular cells following ischaemic injury by a combination of immunofluorescence and qualitative real-time polymerase chain reaction (qRT-PCR). Using a time course analysis of these experimental models the chronology of this relationship was demonstrated, alongside the association with key angiogenic factors, such as Vegf. Given the findings in ischaemic tissue the C3(1)/Tag transgenic mammary cancer model was used to test the hypothesis that Wt1 would be upregulated in the tumour vasculature. Endothelial Wt1 was up regulated in these tumours compared to healthy control tissue. This finding was mirrored in a sub-set of aggressive breast cancers, confirming that the results obtained in mice can be translated to humans. Quantitative PCR revealed no association between histopathological grade of the tumours, oestrogen receptor status, and WT1 expression. In order to delineate the cell types involved in vessel formation, Wt1+ cells were sorted using fluorescent activated cell sorting (FACS) from transgenic mice with a green fluorescent protein knocked into the Wt1 locus following sponge implantation. Distinct sub-populations of Wt1+ cells were identified, some of which expressed EC and pericyte markers. Moreover, these Wt1+ sub-populations changed in composition and number over time. These findings were confirmed by genetic fate mapping of Wt1+ cells in this model. Finally, a conditional knockout mouse was generated to allow the selective deletion of Wt1 from vascular ECs in the sponge model of angiogenesis. The results demonstrated that deletion of Wt1 from this cellular compartment led to a dramatic reduction in vessel formation supporting a potential role in regulating angiogenesis. These results support the hypothesis that expression of WT1 in the vascular endothelium contributes to the regulation of angiogenesis in tumours and ischaemic tissue, and provides evidence that selective deletion of the gene inhibits new vessel formation. This suggests that targeting WT1 may have a therapeutic benefit in cancer and could aid regeneration of ischaemic tissues following injury in conditions such as myocardial infarction and critical limb ischaemia.