Biomarkers measured in the blood can provide information about disease pathophysiology, diagnosis and prognosis. Pronounced proinflammatory responses are characteristic of severe malaria, and excessive activation of the immune system is central to the pathophysiology of both cerebral malaria and placental malaria. Severe malaria is characterized by cytoadherence of parasitized erythrocytes to the microvasculature; impaired tissue perfusion; dysregulated inflammatory responses; and activation of the complement system, mononuclear cells, and endothelium. Despite the availability of effective antimalarial drugs, the mortality rate in severe malaria remains unacceptably high. To glean further insight into malaria pathophysiology, we investigated host biomarkers of immune activation in the blood of subjects with different manifestations of severe disease. C5 has been identified as being necessary and sufficient for the development of experimental cerebral malaria. We hypothesized that C5a (a terminal component of the complement cascade with potent inflammatory properties) may mediate its action by inducing and exacerbating inflammatory processes in severe malaria, leading to endothelial activation and dysregulated angiogenesis. I tested this hypothesis in vitro, and found that C5a interacted with malaria toxin PfGPI to drive deleterious inflammatory and anti-angiogenic responses. As C5a and anti-angiogenic factor sFlt-1 have been implicated in models of pathologic pregnancies, we asked whether increased levels of C5a in subjects with placental malaria were associated with altered angiogenesis and poor birth outcomes. Our results suggest that C5a impairs angiogenic remodelling in placental malaria leading to vascular insufficiency and fetal growth restriction. Further, altered profiles of inflammatory and angiogenic biomarkers in the periphery may identify occult placental malaria infections. We extended these observations to cerebral malaria where similar pathogenic pathways contribute to disease pathophysiology. In adults and children with cerebral malaria, altered profiles of angiogenic proteins were associated with disease severity and mortality and represent putative diagnostic and prognostic biomarkers in severe malaria.

Discovering novel compounds that stimulate or abrogate angiogenesis can lead to development of new therapeutic agents that may effectively treat diseases with pathological angiogenesis. The zebrafish model allows for a whole-organism approach to drug discovery. Advantages over other animal models include small embryo size, fecundity, rapid embryonic development, optical clarity and easy accessibility of the embryos. My goal is to validate the therapeutic efficacy and identify the molecular mechanisms of action of three compounds identified from our previous chemical genetic screens. Fenretinide promoted angiogenesis in zebrafish embryos but inhibited the angiogenesis-dependent process of fin regeneration. The pro-angiogenic effects of fenretinide appear secondary to the stimulation of somitogenesis. I3M potently inhibited angiogenesis and fin regeneration, and may act partially through the notch pathway. Lastly, I validated the anti-angiogenic effect of a novel compound DHM. Comprehensively, my studies support the utility of zebrafish as a versatile tool for anti-angiogenic drug discovery.

Skeletal muscle is one of the largest tissues in humans and provides many pivotal functions to support life. Abnormality in skeletal muscle functions can lead to disease. For example, insulin resistance in skeletal muscle leads to type II diabetes. The underlying mechanisms that control energy balance in skeletal muscle remain largely elusive, especially at the genetic level. Here in the second chapter, I showed that MyoD mediated the transcriptional regulation of ACSL5, a mitochondrial protein, in C2C12 myoblasts via two E-box elements. A SNP rs2419621 (T) created a de novo E-box that together with the two pre-existing proximal E-boxes strongly enhances ACSL5 expression in both CV1 and C2C12 cells. In the third chapter, I identified a novel VGLL4-interacting protein IRF2BP2 and verified the interaction with co-immunoprecipitation and mammalian two-hybrid assays. Functionally, overexpression of IRF2BP2 and transcription factor TEAD1 activates mouse VEGF-A promoter in CV1 cells and enhances the biosynthesis of VEGF-A in C2C12 myoblasts. In vivo studies showed that ischemia induced the expression of IRF2BP2 by more than three fold, suggesting that IRF2BP2 could play a pivotal role during tissue ischemia. IRF2BP2 is a nuclear protein in both mouse cardiac myocytes and C2C12 myoblasts as demonstrated by immunohistochemistry and immunocytochemistry, respectively. Therefore, I sought to delineate the mechanism for the nuclear shuttling of IRF2BP2 in the fourth chapter. With various DNA alternations, I mapped the NLS to an evolutionarily conserved sequence 354ARKRKPSP361 in IRF2BP2. Deletion of the positively charged amino acids resulted in the abolishment of the NLS signal. Next, I showed that phosphorylation of serine 360 (S360) mediates the nuclear import of the protein. Whereas an alanine substitution (S360A) at the site resulted in perinuclear accumulation of the protein, an aspartic acid substitution (S360D) forced the nuclear accumulation. Nevertheless, the forced accumulation of the S360D mutant did not enhance the activation of VEGF-A promoter in CV1 cells as did the wild-type protein. My studies revealed two novel mechanisms by which skeletal muscle could harvest energy, thus providing new insight into the energy metabolism in skeletal muscle

Reconstructive flaps are a mainstay form of treatment for anatomical defects in plastic surgery, and despite extensive progress in the areas of flap anatomy and design, the mechanism of flap healing and the factors that regulate this process are poorly understood. This thesis investigates the regulation of flap healing, and tests the hypothesis that the introduction of genes for angiogenic growth factors can be used to augment the vascularisation and wound healing of ischaemic flaps. Using a modified McFarlane ischaemic skin flap model in Sprague Dawley rats, endogenous angiogenic regulatory factors that included the VEGF and angiopoietin families and their receptors were investigated. Twelve specific quantitative real-time PCR assays documented a general up-regulation of angiogenic genes and their receptors with time following flap elevation. There was not a readily identifiable “master regulator”. Angiogenic protein levels were more variable with a decrease VEGF-A and TNF-α levels along the flap. Debridement studies of the necrotic distal flaps demonstrated for the first time that VEGF-A164 and TNF-α protein levels stabilised, while angiogenic genes of VEGF-A164, VEGF-A120, angiopoietins and their receptors were down-regulated and VEGF-B186 and HIF-1α mRNA increased, compared to non-debrided flaps. Leucocyte proteolysis in devitalised tissue is discussed as a possible mechanism for reduced angiogenic proteins levels in ischaemic flaps. The impact of angiogenic gene therapy using adenoviral vectors in the flap model revealed for the first time that recombinant adenoviruses containing the VEGF-B186 transgene could significantly augment neovascularisation and improve flap survival. This neovascularisation correlated with up-regulation of the expression of multiple endogenous angiogenic genes that included VEGF-A164, the angiopoietins and their receptors. Erythematous plaques were documented as a side effect of Ad VEGF-A165 and Ad VEGF-B186 treatment of rat skin, although in the latter treatment they were very mild. Weals induced by the presence of VEGF-A165 transgene were associated with a marked acute inflammatory cell infiltrate and oedema consistent with the increased vascular permeability effects of VEGF-A165. Ad VEGF-A165 plus Ad ANG-1* induced weals were less prominent with reduced oedema highlighting the stabilising effect of Ad ANG-1* on vascular permeability.

Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 4 uppsatser.

Diss. Uppsala : Univ., 2003.

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Τα ρετινοειδή αποτελούν μια μεγάλη οικογένεια οργανικών μορίων που μοιάζουν δομικά με τη βιταμίνη Α. Το all-trans ρετινοϊκό οξύ (atRA) είναι γνωστό πως ρυθμίζει ένα μεγάλο εύρος κυτταρικών βιολογικών διεργασιών μέσω της πρόσδεσής του και της ενεργοποίησης των υποδοχέων του, τους υποδοχείς ρετινοϊκού οξέος (RAR) και τους υποδοχείς ρειτνοειδών Χ (RXR). Κάθε κατηγορία υποδοχέων περιλαμβάνει τρία μέλη, α, β και γ. Τα ρετινοειδή χρησιμοποιούνται για τη θεραπεία πολλών ασθενειών, από την κοινή ακμή ως την οξεία προμυελωτική λευχαιμία. Η χρήση τους, παρ’ όλα αυτά, περιορίζεται εξαιτίας των σοβαρών ανεπιθύμητων ενεργειών τους, και είναι επιτακτική ανάγκη να συντεθούν ανάλογα με λιγότερες ανεπιθύμητες δράσεις. Προς αυτήν την κατεύθυνση έχουν συντεθεί πολλά ανάλογα, ανάμεσα τους και το N12-bis(all-trans-retinoyl)spermine (RA2SPM), ένα σύζευγμα δύο μορίων atRA με σπερμίνη, το οποίο συντέθηκε από την ερευνητική ομάδα του καθηγητή Δ. Παπαϊωάννου, στο Τμήμα Χημείας του Πανεπιστημίου Πατρών. Στην παρούσα εργασία μελετήθηκε η δράση του atRA και του RA2SPM στην αγγειογένεση και την ανάπτυξη καρκινικών κυττάρων προστάτη. Το atRA ανέστειλε με δοσο-εξαρτώμενο τρόπο την αγγειογένεση στο μοντέλο της χοριοαλλαντοϊκής μεμβράνης (CAM) εμβρύου όρνιθας, παρόλο που η δράση του ήταν χαμηλότερη από αυτή που είχε δείξει παλιότερα το RA2SPM στο ίδιο σύστημα. Παρ’ όλο που το atRA προκάλεσε επαγωγή στην έκφραση του αγγειογενετικού αυξητικού παράγοντα πλειοτροπίνη (PTN) στην CAM, ανέστειλε την αλληλεπίδρασή της με τον υποδοχέα της RPTPβ/ζ, κάτι που συνάδει με μειωμένη επαγωγική δράση της PTN στην αγγειογένεση της CAM εμβρύου όρνιθας. Τα δύο ρετινοειδή μείωσαν των αριθμό των ανθρώπινων ενδοθηλιακών κυττάρων φλέβας ομφαλίου λώρου (HUVEC) και των προστατικών καρκινικών κυττάρων LNCaP και PC3, με δοσο-εξαρτώμενο τρόπο. Σε όλες τις περιπτώσεις, το RA2SPM ήταν πιο αποτελεσματικό σε σχέση με το atRA. Η δράση και των δύο ρετινοειδών στα καρκινικά κύτταρα προστάτη ήταν μεγαλύτερη από τη δράση τους στα ενδοθηλιακά κύτταρα. Αυτές οι δράσεις φαίνεται να σχετίζονται με αύξηση των επιπέδων του mRNA του ογκοκαταστατλικού γονιδίου RARβ και από τις δύο ουσίες που μελετήθηκαν. Ενδιαφέρον είναι ότι η δράση του atRA και του RA2SPM βρέθηκε να διαμεσολαβείται από τον RARα και ήταν μεγαλύτερη στα PC3 κύτταρα, τα οποία δεν εκφράζουν τον RARβ και θεωρούνται πιο επιθετικά. Στα κύτταρα που εκφράζουν τον RARβ, δηλαδή στα HUVEC και στα LNCaP, οι δύο ουσίες ήταν λιγότερο αποτελεσματικές. Τέλος, τόσο το atRA όσο και το RA2SPM μείωσαν τα επίπεδα mRNA και πρωτεΐνης της PTN, η οποία είναι γνωστό πως παίζει σημαντικό ρόλο στην ανάπτυξη των καρκινικών κυττάρων προστάτη PC3. Συμπερασματικά, τα αποτελέσματα της παρούσας εργασίας υποδεικνύουν ότι το RA2SPM φαίνεται να είναι πιο αποτελεσματικό από το atRA στην αναστολή της αγγειογένεσης και της ανάπτυξης των καρκινικών κυττάρων προστάτη. Επίσης, καταδεικνύουν τον RARα ως τον υποδοχέα μέσω του οποίου τα δύο υπό μελέτη ρετινοειδή προκαλούν αύξηση της έκφρασης του RARβ και αναστολή της ανάπτυξης των καρκινικών κυττάρων προστάτη. / Retinoids constitute a large family of organic compounds structurally related to the naturally occurring vitamin A. All-trans-retinoic acid (atRA) is known to modulate a wide range of cellular biological processes through binding to and activation of its specific receptors, retinoic acid receptors (RAR) and retinoid X receptors α, β and γ. Retinoids are being used for the treatment of various diseases, ranging from acne vulgaris to acute promyelotic leukemia. Their use however is limited due to serious adverse effects and there is a great need for analogues with minimized adverse effects. Towards this direction, many analogues are being synthesized, among which N1,N12-bis(all-trans-retinoyl)spermine (RA2SPM), a conjugate of all-trans retinoic acid with spermine, which has been synthesized by the research group of Prof. D. Papaioannou at the Dept. of Chemistry of the University of Patras. In the present work, the effect of atRA and RA2SPM on angiogenesis and prostate cancer cell growth has been studied. ATRA dose-dependently inhibited angiogenesis in the chicken embryo chorioallantoic membrane (CAM) model, although its effect was lower that the previously shown effect of RA2SPM on the same system. Although atRA increased the expression of the angiogenic growth factor pleiotrophin (PTN) in the CAM, it decreased its interaction with its receptor RPTPβ/ζ, in line with a possible decreased effect of PTN in stimulating CAM angiogenesis. Both retinoids decreased the number of human umbilical vein endothelial (HUVEC) and prostate cancer LNCaP and PC3 cells in a concentration-dependent manner. In all cases, RA2SPM was more effective and potent compared with atRA and the effect of both retinoids on prostate cancer cells was higher than their corresponding effect on endothelial cells. These effects seem to be related to an increase of the mRNA of the RARβ tumour repressing gene by both substances tested. Interestingly, the effect of atRA and RA2SPM was mediated by RARα and was higher in PC3 cells that do not express RARβ and are considered more aggressive; in cells that express RARβ, i.e. HUVEC and LNCaP, both substances were less effective. Finally, both atRA and RA2SPM decreased mRNA and protein levels of PTN, which is known to be significant for prostate cancer PC3 cell growth. These data suggest that RA2SPM seems to be more effective than atRA in decreasing angiogenesis and prostate cancer cell growth and identify RARα as the receptor through which it causes RARβ up-regulation and decrease of prostate cancer cell growth.