Choroidal neovascularization (CNV) is a leading threat to severe vision loss, particularly in patients with age-related macular degeneration (AMD). In CNV, newly formed blood vessels sprout from the choroid to the sub-retinal space, where leakage and bleeding of the abnormal vessels lead to photoreceptor death and subsequent vision loss. It is believed that CNV is mediated by growth factors (e.g. vascular endothelial growth factor {VEGF}) produced by the retinal pigment epithelium (RPE) under pathological states (e.g. hypoxia). Current treatments for CNV aiming at countering VEGF only help decrease leakage and inhibit formation of CNV, but none of them is curative and the recurrence rate remains high. In order to find other more powerful potential therapeutic targets, the regulations of VEGF signaling in the pathophysiology of CNV is the focus of numerous translational investigations. Previously, Hypoxia-inducible factor-1 (HIF-1), a crucial transcriptional factor in response to hypoxia, is identified as the master transcriptional factor controlling VEGF expression in the RPE promoting CNV. Alpha-enolase (ENO1), a key glycolytic enzyme, is known to be over expressed in several types of carcinomas also under the regulation of HIF-1. ENO1 has been reported to be closely associated with cancer progression, angiogenesis, and venous invasion. The molecular events of ENO1 in the pathogenesis of promoting angiogenesis are of interest but still barely understood. Recently, the association of ENO1 antibodies with retina has been seen in patients with AMD. We hypothesize that ENO1 expression in the RPE may play a role in the development of CNV, participating in the regulation of VEGF. Hypoxia is an important pathological condition in the formation of CNV. Here, we first determined ENO1 expression and cell death in a human RPE cell line, ARPE-19, under cobalt (II) chloride (CoCl2)-induced hypoxia or anoxia (95% N2, 5% CO2). To further investigate the regulation of ENO1 in CNV, HIF-1α-diminished RPE cells were generated using small interfering RNA (siRNA) and the change of ENO1 expression in response to hypoxic injury was determined. Upon 24 hr of treatment with CoCl2-induced hypoxia or anoxia, the expression of ENO1 and VEGF increased significantly along with HIF-1α in ARPE-19 cells, both of which could in turn be significantly down-regulated by HIF-1α siRNA. Interestingly, cell death remained low in ARPE-19 cells, even after 24 hr of CoCl2-induced hypoxia or anoxia. To further study the role of ENO1 in CNV, we started by investigating the relationship between ENO1 and VEGF. SiRNA was used to knock down the expression of ENO1 in ARPE-19 cells. Upon transfection with the siRNA, ENO1 expression was successfully down-regulated when treated with CoCl2-induced hypoxia. However, VEGF secretions from the ENO1-diminished ARPE-19 cells under CoCl2-induced hypoxia remained unchanged. Double knockdown of ENO1 together with HIF-1α by siRNA also did not help to further suppress VEGF secretion in the hypoxic ARPE-19 cells. Hence, ENO1 was demonstrated to be activated and up-regulated by HIF-1 in RPE cells responding to hypoxia, suggesting a potential role of ENO1 in favoring the formation of CNV, but not through influencing VEGF secretion. / published_or_final_version / Ophthalmology / Master / Master of Philosophy

Both white and brown adipose tissues exhibit extensive vascularity. Increased angiogenesis in brown adipose tissue (BAT) is crucial for brown fat activation and thermogenesis in animals during cold acclimation. BAT can be similarly activated by food intake to generate heat through cellular respiration, in a process known as diet induced thermogenesis. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that regulates both pathological and physiological angiogenesis and can stimulate cell proliferation, migration, survival and vessel permeability. However, VEGF has also been shown to affect an increasing number of non-vascular cells such as skeletal muscle and kidney podocytes. The expression and function of VEGF in white and brown adipocytes are not fully understood. We have previously shown that the expression of VEGF is concomitantly regulated with skeletal muscle differentiation. Here we show that VEGF is expressed in BAT and all major white adipose depots in mice. VEGF expression was increased during white and brown adipocyte differentiation and was regulated in cultured brown adipocytes by the \(PPAR\gamma\) agonist troglitazone and by \(PGC1\alpha\) in BAT in vivo. Systemic VEGF neutralization led to brown adipocyte apoptosis in vivo, loss of mitochondrial cristae and increased mitophagy and was associated with increased inflammation and fibrosis. VEGFR2 was expressed in both brown preadipocytes and adipocytes. Blockade of VEGF signaling using anti-VEGFR2 antibody DC101 increased brown adipocyte apoptosis in vitro. VEGF also functioned as a mitogen and survival factor for brown preadipocytes. VEGF 164 and VEGF 188, isoforms that can bind heparan sulfate proteoglycans, comprise >98% of total VEGF in BAT, subcutaneous and perigonadal fat depots. Embryos that lacked VEGF 164 and 188 displayed abnormal BAT development with fewer brown adipocytes, lower levels of mitochondrial uncoupling protein 1 and Cox IV. These results indicate a direct role for VEGF signaling in brown adipocytes and preadipocytes and suggest the importance of heparan sulfate binding VEGF isoforms in BAT development. Elucidation of the role of VEGF signaling in adipocytes is vital to understanding adipose tissue expansion and activation and may reveal novel therapeutic targets for the activation of brown fat in humans.

Acute Respiratory Distress Syndrome (ARDS) is the most extreme form of acute lung injury and continues to have a significant morbidity and mortality. Unfortunately, the mechanisms involved in the recovery and repair of the lung following ARDS remain poorly understood. An understanding of these is pivotal to improving outcome from acute lung injury. Several observational studies have suggested a potential relationship between Vascular Endothelial Growth Factor (VEGF) in the lung and the development/outcome of ARDS. In this thesis, three potential mechanisms underlying these observations have been explored: 1. What is the anatomical distribution of VEGF receptor and isoform expression in normal and ARDS lung? How does this change at early and later time points following acute lung injury? 2. Are human type 2 alveolar epithelial (ATII) cells a source of and target for VEGF? How does exposure to a pro-inflammatory milieu modify their expression of VEGF isoforms and receptors? 3. Is there a relationship between a functional VEGF polymorphism and susceptibility to developing and severity of ARDS? I have demonstrated VEGF receptor expression on both sides of the alveolarcapillary membrane with upregulation in later ARDS. All three principal isoforms (VEGF121, VEGF165 and VEGF189) are expressed in normal human lung with uniform downregulation of all three in early ARDS, which normalises with increasing time following injury. I have not found any evidence of VEGF isoform switching. I have also demonstrated human ATII cells are both a significant cellular source of and a target for VEGF (via VEGF receptor expression) confirming autocrine VEGF activity in the lung. VEGF is an ATII cell survival factor. ATII cells differentially respond to pro-inflammatory stimuli by increasing VEGF isoform but not receptor expression, which may serve as a regulatory control mechanism. Finally, I have demonstrated the VEGF 936 T allele increases susceptibility to and the severity of lung injury. The T allele is associated with an increase in plasma VEGF level in ARDS patients but intra-alveolar levels are unaffected.

Στην χρόνια νεφρική ανεπάρκεια, η μειωμένη διήθηση του πλάσματος οδηγεί στην αυξημένη συγκέντρωση τοξινών στο αίμα. Αυτό προκαλεί δυσλειτουργία του ενδοθηλίου, η οποία περιλαμβάνει την φλεγμονή, την αθηροσκλήρωση και τελικά την υπέρταση. Στο πλαίσιο της μελέτης της δράσης των μη διηθούμενων τοξινών, χρησιμοποιήθηκε ορός ασθενών πριν και μετά την αιμοκάθαρση. Ως in vitro σύστημα μελέτης, χρησιμοποιήθηκε πρωτογενής καλλιέργεια ενδοθηλιακών κυττάρων από ομφάλια φλέβα ανθρώπου (HUVEC) και ελέγχθηκε η επίδραση ορού πριν και μετά την αιμοκάθαρση, στον πολλαπλασιασμό, την απόπτωση, την μετανάστευση και την «επούλωση πληγών» των κυττάρων αυτών. Επίσης, μελετήθηκε η έκφραση των μεταλλοπρωτεϊνασών MMP-2 και MMP-9 και των αναστολέων τους TIMP-1 και 2 σε επίπεδο mRNA και πρωτεΐνης καθώς και η έκφραση του κολλαγόνου IV και της ελαστίνης. Χρόνο- και δοσο-εξαρτώμενα πειράματα έδειξαν ότι ο μετά-ορός, σε σχέση με τον προ-ορό, επάγει τον πολλαπλασιασμό, την μετανάστευση και την διαδικασία της «επούλωσης πληγών» των ενδοθηλιακών κυττάρων με στατιστικά σημαντικό τρόπο, ενώ βρέθηκε να μειώνει την απόπτωση. Επίσης βρέθηκε ότι ο προ- ορός, σε σχέση με τον μετά- ορό, επάγει με στατιστικά σημαντικό τρόπο, την έκφραση και ενεργότητα των MMP-2 και MMP-9, ενώ καταστέλλει την έκφραση καθώς και τα επίπεδα των αναστολέων TIMP-1 και TIMP-2. Τέλος καταστέλλει την έκφραση των βασικών συστατικών της εξωκυττάριας ύλης, του κολλαγόνου IV και της ελαστίνης. Συμπερασματικά, ο ορός πριν την αιμοκάθαρση φαίνεται να συμμετέχει στην δυσλειτουργία του ενδοθηλίου, καταστέλλοντας τον πολλαπλασιασμό και την μετανάστευση και επάγοντας ταυτόχρονα την απόπτωση των ενδοθηλιακών κυττάρων. Παράλληλα ο προ-ορός καταστρέφει την εξωκυττάρια ύλη των ενδοθηλιακών κυττάρων επάγοντας τις MMP-2 και -9, καταστέλλοντας τους αναστολείς τους TIMP-1 και -2 και μειώνοντας ταυτόχρονα την έκφραση του κολλαγόνου IV και της ελαστίνης. / In chronic renal disease, reduced glomerular filtration results to increased toxin concentration in blood. This leads, among others, to endothelial dysfunction which concludes inflammation, atherosclerosis and finally hypertension. In this study, in order to investigate the effect of non-filtrated toxins, we used pre-haemodialysis and post-haemodialysis serum (control) from end-stage renal patients. As in vitro system of study we used the primary endothelial cell culture from human umbilical vein (HUVEC) and the effect of pre- and post- haemodialysis serum on cell proliferation, migration, apoptosis and wound healing was investigated. In addition, the expression of matrix metalloproteases MMP-2 and MMP-9 and their inhibitors TIMP-1 and TIMP-2 was examined. Time course and dose-response experiments revealed that pre-serum reduces proliferation, migration and wound healing, while induced apoptosis in a statistically significant manner. Pre-, compared to post-haemodialysis serum, induces the expression and activity of MMP-2 and MMP-9, while inhibits the expression of TIMP-1 and TIMP-2. Concluding, uremic toxins lead to endothelial dysfunction, inhibiting cell proliferation, cell migration and inducing apoptosis. In addition, uremic toxins contribute to the degradation of extracellular matrix, inducing MMP-2 and MMP-9 and inhibiting TIMP-1 and TIMP-2.

During embryonic vasculogenesis, endothelial cells form in the mesoderm , assemble into cord-like structures and then undergo tube formation. Previous studies have shown that signaling by members of the hedgehog family of secreted growth factors is essential for normal development of embryonic blood vessels. Embryos lacking hedgehog function show the presence of abundant endothelial cells but the cells fail to assemble into vascular cords and lumenized endothelial tubes do not form. At present it is not known whether active hedgehog signaling is required for both cord and tube formation or only for the initial step. To address this question, we have used small molecule inhibitors and agonists to the alter activity of the hedgehog signaling pathway in the chick embryo. If development is allowed to proceed until endothelial cells of the future dorsal aortae have assembled into cords, subsequent inhibition of hedgehog signaling, using cyclopamine, does not prevent aortal cells from forming endothelial tubes, however, it does lead to a reduction in cross sectional area of the aorta and to a loss of density of the adjacent vascular plexus. In contrast, activation of the hedgehog pathway with SAG leads to formation of enlarged aortae and increased density of the plexus. Very little, if any, of the observed effects are due to differences in number of endothelial cells in the treated embryos. Examination of endothelial cells during vascular plexus formation shows that inhibition of hedgehog signaling with cyclopamine inhibits formation of filopodia while treatment with SAG increases the number of filopodial extensions. These studies show that hedgehog signaling levels must be tightly regulated for normal vascular patterning to be achieved.

Theiler's murine encephalomyelitis virus (TMEV) is classified as a Cardiovirus in the Picornaviridae family. An enteric virus, TMEV, spreads within the mouse population by the fecal-oral route. The neurovirulent GDVII strain of Theiler's virus causes a fatal encephalitis in all strains of mice following intra-cranial infection of the virus. Persistent BeAn strain of Theiler's virus causes a demyelinating disease in susceptible strains of mice, which is similar to the human disease - Multiple Sclerosis (MS). Although a well-recognized model for MS, the route of entry of the virus into the central nervous system (CNS) following natural infection has not been well understood. One of the proposed portals of entry includes the blood-brain barrier (BBB). This report indicates the ability of both the neurovirulent and the persistent strains of Theiler's virus to induce apoptosis in the functional units of the BBB - the cerebrovascular endothelial cells (CVE) both in vitro and in vivo. Induction of apoptosis in CVE was demonstrated by Annexin staining, electron microscopy, DNA fragmentation assay, Hoechst staining and by caspase-3 staining. Corresponding to results by other authors, GDVII is a stronger inducer of apoptosis in CVE compared to BeAn. Induction of apoptosis is dependent on the MOI of the virus. UV-inactivated virus is not capable of inducing apoptosis and induction of apoptosis appears to be an internal event not requiring activation of death receptors. Determining the pathway of induction of apoptosis by TMEV in CVE indicated the involvement of a Ca2+ dependent pathway for apoptosis - the calpain pathway. Involvement of calpain in apoptosis has been reported in MS. Induction of apoptosis in CVE in vivo was also demonstrated following the intra-peritoneal inoculation of Theiler's virus. Induction of apoptosis in CVE following Theiler' virus infection could lead to a breach of the BBB and entry of inflammatory cells as well as virus into the central nervous system. This finding could aid understanding the neuropathogenesis of Theiler's virus.

Transcapillary exchange is the movement of fluid and molecules through the porous capillary wall, and is important in maintaining homeostasis of bodily tissues. The classical view of this process is that of Starling's hypothesis, in which the forces driving filtration or absorption are the hydrostatic and osmotic pressure differences across the capillary wall. However, experimental evidence has emerged suggesting the importance of the capillary wall ultrastructure, and thus rather than the global differences between capillary and tissue, it is the local difference across a structure lining the capillary wall known as the endothelial glycocalyx that determines filtration. Hu and Weinbaum presented a detailed cellular level microstructural model of this phenomenon which was able to explain some experimental discrepancies. In this Thesis, rather than describing the microstructural details, the capillary wall is treated as a poroelastic material. The assumptions of poroelasticity theory are such that the detailed pore structure is smeared out and replaced by an idealized homogeneous system in which the fluid and solid phases coexist at each point. The advantage of this approach is that the mathematical problem is greatly simplified such that analytical solutions of the governing equations may be obtained. This approach also allows calculation of the stress and strain distribution in the tissue. We depart from classical poroelasticity, however, due to the fact that since there are concentration gradients within the capillary wall, the filtration is driven by both hydrostatic and osmotic pressure gradients. The model predictions for the filtration flux as a function of capillary pressure compares favourably with both experimental observations and the predictions of the microstructural models. An important factor implicated in transcapillary exchange is the endothelial glycocalyx, which was shown experimentally to protect against edema formation. Using our theory in combination with the experimental measurements of glycocalyx thickness and pericapillary space dimension (PSD), we make a quantitative comparison for the excess flow as a result of a deteriorated glycocalyx, which shows reasonably good agreement with the data. Since many of the parameters in the model are difficult to measure, a sensitivity analysis was performed on the most important of these. Finally, since there was variation in the measurements of glycocalyx thickness and PSD, we used probability distributions to represent the data, and performed further calculations to obtain ranges of likely values for the various parameters. This work could find applications in cardiovascular disease, where the glycocalyx is degraded or absent, and in cancer research, where the abnormal vasculature is an impediment to the efficient delivery of anti-cancer drugs.

Successful survival of large volume engineered tissues depends on the development of a vasculature to support the metabolic demands of donor tissue in vivo. Pancreatic islet transplantation is a cell therapy procedure to treat Type 1 diabetes that can potentially benefit from such a vascularization strategy. The treatment is limited as the majority of transplanted islets (60%) fail to engraft due to insufficient revascularization in the host(1, 2). Modular tissue engineering is a means of designing large volume functional tissues using micron sized tissues with an intrinsic vascularization. In this thesis, we explored the potential of endothelialized modules to drive vascularization in vivo and promote islet engraftment. Human endothelial cells (EC) covered modules were transplanted in the omental pouch of athymic rats and human EC formed vessels near implanted modules until 7 days when host macrophages were depleted. Rat endothelial cells covered modules were similarly transplanted in the omental pouch of allogeneic rats with and without immunosuppressants. When the drugs were administered, endothelialized modules significantly increased the vessel density. Moreover, donor GFP labelled EC formed vessels that integrated with the host vasculature and were perfusable until 60 days; this key result demonstrate for the first time that unmodified primary endothelial cells form stable vessels in an allograft model. Transplantation of islets in such endothelialized modules significantly improved the vessel density around transplanted islets. Donor endothelial cells formed vessels near transplanted islets in allogeneic immunesuppressed recipients. Meanwhile, there was an increase in islet viability with transplantation of endothelialized modules in syngeneic recipients but this difference was not significant. In summary, endothelialized modules were effective in promoting stable vascularization and improving transplanted islet vascularisation. Future work should promote faster maturity of donor vessels and modulate the host immune and inflammatory responses to significantly improve transplanted islet engraftment.

Successful survival of large volume engineered tissues depends on the development of a vasculature to support the metabolic demands of donor tissue in vivo. Pancreatic islet transplantation is a cell therapy procedure to treat Type 1 diabetes that can potentially benefit from such a vascularization strategy. The treatment is limited as the majority of transplanted islets (60%) fail to engraft due to insufficient revascularization in the host(1, 2). Modular tissue engineering is a means of designing large volume functional tissues using micron sized tissues with an intrinsic vascularization. In this thesis, we explored the potential of endothelialized modules to drive vascularization in vivo and promote islet engraftment. Human endothelial cells (EC) covered modules were transplanted in the omental pouch of athymic rats and human EC formed vessels near implanted modules until 7 days when host macrophages were depleted. Rat endothelial cells covered modules were similarly transplanted in the omental pouch of allogeneic rats with and without immunosuppressants. When the drugs were administered, endothelialized modules significantly increased the vessel density. Moreover, donor GFP labelled EC formed vessels that integrated with the host vasculature and were perfusable until 60 days; this key result demonstrate for the first time that unmodified primary endothelial cells form stable vessels in an allograft model. Transplantation of islets in such endothelialized modules significantly improved the vessel density around transplanted islets. Donor endothelial cells formed vessels near transplanted islets in allogeneic immunesuppressed recipients. Meanwhile, there was an increase in islet viability with transplantation of endothelialized modules in syngeneic recipients but this difference was not significant. In summary, endothelialized modules were effective in promoting stable vascularization and improving transplanted islet vascularisation. Future work should promote faster maturity of donor vessels and modulate the host immune and inflammatory responses to significantly improve transplanted islet engraftment.

Piperine, a pungent alkaloid found in the fruits of long and black pepper plants, has diverse physiological effects, including anti-inflammatory and anti-cancer activities. The effect of piperine on the function of T cells and endothelial cells, two important elements of inflammation, have not been examined previously and were the focus of this study. Piperine inhibited the proliferation of human endothelial cells, murine T cells, and IL-2-dependent CTLL-2 T cells, without affecting cell viability. Progression into the S phase of the cell cycle was inhibited in all three cell types. In T cells, piperine inhibited expression of the early activation marker CD25, production of IFN-?, IL-2, IL-4, and IL-17A, and the generation of cytotoxic effector cells. In endothelial cells, piperine inhibited migration and tubule formation in vitro and ex vivo, as well as breast cancer cell-induced angiogenesis in chick embryos. Piperine inhibited Akt phosphorylation in signaling pathways associated with growth factor receptors on endothelial cells, T cell receptor and CD28 on T cells, and IL-2 receptor on CTLL-2 cells. Additionally, piperine inhibited ERK1/2 and I?B phosphorylation in activated T cells, as well as STAT3, STAT5, and ERK1/2 phosphorylation in IL-2-stimulated CTLL-2 cells. However, piperine is not a broad-spectrum inhibitor of phosphorylation as it did not inhibit ZAP-70 phosphorylation in activated T cells or phosphorylation of JAK1 and JAK3 in IL-2-stimulated CTLL-2 cells. Piperine-mediated inhibition of T cell activation and IL-2 receptor signaling suppresses T cell proliferation and effector cell differentiation, suggesting possible utility in treating T cell-mediated autoimmune and chronic inflammatory conditions. Additionally, the potent anti-angiogenic activity of piperine warrants further study for the prevention of inflammation- and cancer-promoting angiogenesis.