Thesis (Ph. D.)--University of Wisconsin--Madison, 1970. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Angiogenesis is a highly regulated process orchestrated by the vascular endothelial growth factor-A (VEGF-A) system of ligands and receptors. Heparin/heparan sulfate (HS) proteoglycans and neuropilin-1 (NRP-1) have been identified as co-receptors for VEGF-A, yet the mechanisms of action have not been fully defined. In the present study, we characterized molecular interactions between receptors and co-receptors and the two major VEGF-A isoforms, using surface plasmon resonance (SPR) and in vitro binding assays. We found that VEGF dissociated 25-times faster from its major signaling receptor, VEGF receptor-2 (VEGFR-2) than from its “decoy” receptor, VEGF receptor-1 (VEGFR-1). We identified a potential mechanism for co-receptors to decrease the dissociation rate and prolong the signaling complex lifetime. Using a systematic approach, we obtained kinetic parameters for each individual interaction in an intercomparable way to measure the effect NRP-1 and HS have on complex stability. Additionally, we demonstrated that these binding events influence VEGF activity within endothelial cells. These parameters can be used in mathematical models to predict therapy outcomes in defined cellular contexts. Furthermore, we optimized a competition-based technique using SPR and structurally defined HS oligosaccharides and demonstrated that it can be used to rapidly measure affinities to HS-binding proteins. We used this method to define interactions and structural and length requirements for heparin/HS interactions with VEGFR-1, NRP-1, and VEGF165, the most relevant VEGF-A isoform, in complex with VEGFR-2 and NRP-1. We show that the structural requirements were distinct for each interaction. We further found that VEGF165, VEGFR-2 and monomeric NRP-1 bound weakly to heparin alone, yet binding to heparin increased synergistically when presented together. This enhanced binding correlated with alterations in VEGF signaling in endothelial cells. We found that soluble NRP-1 increased VEGF binding and activated phosphorylation of VEGFR-2 and Erk1/2 in endothelial cells, and that these effects required sulfated HS. These data suggest that the presence of HS/heparin and NRP-1 may dictate the specific receptor type activated by VEGF and ultimately determine the biological output. The ability of co-receptors to fine-tune VEGF responsiveness suggests the possibility that VEGF-mediated angiogenesis can be selectively stimulated or inhibited by targeting HS/heparin and NRP-1.

nÃo hà / Com a cirurgia de catarata moderna realizada em cÃrneas normais, independente da tÃcnica cirÃrgica utilizada, hà uma perda celular endotelial mÃdia de 10 a 20%. SubstÃncias viscoelÃsticas foram desenvolvidas com a funÃÃo de manter os espaÃos naturais do olho e conferir proteÃÃo mecÃnica Ãs estruturas intraoculares durante a cirurgia. O objetivo deste estudo foi realizar uma anÃlise comparativa do efeito de duas formulaÃÃes viscoelÃsticas, metilcelulose a 2% e 4%, na proteÃÃo do endotÃlio corneano contra o dano secundÃrio à facoemulsificaÃÃo. Foi realizado um estudo prospectivo, randomizado e duplo cego com pacientes portadores de catarata senil, submetidos à cirurgia de facoemulsificaÃÃo com Implante de Lente Intraocular (LIO). Todos os pacientes foram submetidos a uma avaliaÃÃo oftalmolÃgica completa, incluindo microscopia especular. ApÃs o exame inicial, os pacientes foram randomizados em dois grupos: no grupo A (n=63) foi utilizada a metilcelulose 2% e no grupo B (n=63) a metilcelulose 4%. A mÃdia da Densidade de CÃlulas Endoteliais (DCE) mostrou-se significativamente reduzida (p<0,05), tanto na anÃlise intra como na intergrupo no 1Â, 15 e 30 dias de pÃs-operatÃrio, representando uma perda de cÃlulas endoteliais de 15,26% e 6,79% nos grupos A e B, respectivamente. A Espessura Central da CÃrnea (ECC) mostrou-se aumentada no 1 dia pÃs operatÃrio (DPO) no grupo A, sendo este dado estatisticamente significante (p<0,05). Houve aumento no Coeficiente de VariaÃÃo da tamanho celular (CV) e diminuiÃÃo no Percentual de CÃlulas Hexagonais (6 A), no entanto, sem significÃncia estatÃstica para esses dois parÃmetros. ConcluÃmos que, à despeito das vantagens da metilcelulose a 4% em relaÃÃo à diminuiÃÃo de perdas de cÃlulas endoteliais apÃs facoemulsificaÃÃo, ambos os viscoelÃsticos sÃo semelhantes em relaÃÃo a parÃmetros clÃnicos como ECC, CV e 6A, os quais refletem a manutenÃÃo de condiÃÃes fisiologicamente adequadas para o bom funcionamento do endotÃlio. No entanto, devido à pequena diferenÃa de custo entre os dois produtos e os benefÃcios da metilcelulose 4%, esta pode ser considerada um produto de escolha em cirurgias de catarata de rotina, uma vez que as indicaÃÃes de facoemulsificaÃÃo estÃo sendo cada vez mais precoces, devido Ãs exigÃncias visuais dos pacientes, promovendo, desta forma, cÃrneas mais saudÃveis por longo prazo. / Modern cataract surgery in normal corneas, independent of the surgical technique used, causes a mean 10 to 20% loss of endothelial cells. Viscoelastic substances were developed with the function of maintaining the natural spaces of the eye and providing mechanical protection to the intraocular structures during surgery. The objective of this study was to perform a comparative analysis of the effect of two viscoelastic compositions, methylcellulose 2% and 4%, in corneal endothelial protection against secondary damage to the phacoemulsification procedure. We performed a prospective, randomized, double-blind study with senile cataract patients, submitted to phacoemulsification surgery with intraocular lens (IOL) implant. All patients were submitted to a complete ophthalmological assessment, including specular microscopy. After initial assessment, the patients were randomized into two groups: in Group A (n=63) we used methylcellulose 2%, and in Group B (n=63) methylcellulose 4%. There was no statistically significant difference for the parameters analyzed between groups before surgery. However, at 30 days after surgery, mean density of endothelial cells (DCE) was 2128.78Â277.55 cells/mm in Group A and 2395.79Â262.16 cells/mm in Group B, representing a 15.15 and 6.76% loss of endothelial cells in Groups A and B, respectively. Mean DCE was significantly reduced (p<0.05), in both the inter- and inter-group analysis on the 1st, 15th, and 30th day after surgery. ECC increased on the 1st day after surgery in group A, this datum was statistically significant (p<0.05). There was no statistical difference for the other parameters assessed. In this study, we conclude that, despite the advantages of methylcellulose 4% over 2% in regards to the reduction of endothelial cell loss after phacoemulsification, both viscoelastic substances are similar in relation to clinical parameters such as central corneal thickness, coefficient of variation in cell size and percentage of hexagonal cells, which reflect corneal preservation and maintain adequate physiological conditions for proper endothelial function. However, due to the slight difference in cost between the two products and the benefits of methylcellulose 4% on endothelial cell preservation, this may be considered a choice product in routine cataract surgery, as indications for phacoemulsification are increasingly early, due to patientsâ visual demands, promoting, in this manner, healthier corneas in the long term.

Obesity is a serious health problem worldwide. Consumption of fat rich food is a common cause of obesity. Some of the food components (i.e. saturated free fatty acids (SFAs)) have been identified as inflammatory inducers (Egger G at al., 2010). After a meal, absorbed free fatty acids (FFAs) will be stored in the liver and adipose tissue. On the luminal surfaces of endothelium in adipose tissue microcirculation, lipoprotein lipase hydrolyses absorbed triglycerides into FFAs. Then, in order to be available for adipocyte storage, FFAs have to cross the capillary endothelium barrier, which connected by tight junctions (Stremmel W et al., 2001). Increased leukocyte infiltration is a featured sign of adipose tissue inflammation found in obesity. Endothelial adhesion molecules up-regulation contributes to leukocyte infiltration during inflammation. Some clinical data suggested an increase of leukocyte-endothelium interaction in healthy volunteers after ingestion of high-fat meals (Shimabukuro M et al., 2007). Other lab results also showed that neutrophil infiltration occurred at a very early stage with high-fat feeding in mice (Talukdar S et al., 2012). However, the detailed mechanism of the above phenomena is still unknown. This thesis provides exciting preliminary data which will guide the further study in this area. First of all, we successfully established a stable protocol that CD31 antibody conjugated microbeads were used to isolate primary microvascular endothelial cells from fresh mice lung tissue. After second sorting, CD31+ cells reach 83.3% by FACS analysis. Previous literatures showed that FFAs activate recruitment of inflammatory cells through up-regulation of endothelial adhesion molecules via reduced eNOS derived eNO production (Rizzo NO et al., 2010; Davenpeck KL et al., 1994; Ahluwalia A et al., 2004). In this thesis, it was found that SFAs palmitate exposure dose dependently reduced endothelial AMPK thr172 and eNOS ser1177 phosphorylation by western blot. Moreover, our study demonstrated that endothelial calpain, a calcium dependent protease associated with endothelial dysfunction, was activated by palmitate, specifically its μ-calpain isoform. Altogether, these data suggested that a new role of calpain as a key mediator of palmitate induced endothelial dysfunction and indicated both AMPK and eNOS1177 phosphorylation contribute to this pathological process. Further investigations are still needed to explore connections among those molecules. This thesis may also lead to a novel way of clinical treatment for the obese related vascular diseases. / Physiology

Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen involved in angiogenesis, the formation of new blood vessels. sFlt-1, a secreted form of the signal-transducing VEGF receptor Flt-1, can inhibit cellular responses to VEGF both in vitro and in vivo. sFlt-1 is generated by alternative pre-mRNA processing; removal of Flt-1 intron 13 by splicing produces the mRNA for transmembrane Flt-1, whereas cleavage/polyadenylation within this intron, preserving the exon 13/intron 13 junction, yields sFlt-1 mRNA. Despite the likely importance of sFlt-1 in VEGF signaling, little is known about the regulation of its expression. Previous studies using an Flt-1 minigene (pFIN13) revealed that intronic cleavage/polyadenylation signals can affect Flt-1 expression, and, conversely, that 3' intronic splice signals can affect sFlt-1 expression. The goal of present work was to test the hypothesis that splicing and cleavage/polyadenylation factors compete functionally on Flt-1 transcripts, by 1) assessing the influence of exon 13/14 splicing determinants on expression of Flt-1 RNA processing variants in a transfected cell model system; 2) determining the effects of altering the relative abundance of proteins principally involved in splicing or cleavage/polyadenylation; and 3) characterizing a previously-unknown splice variant, predicted to encode a novel sFlt-1 protein isoform, in cells overexpressing the spliceosomal RNA binding protein U2AF65. When the upstream exon in pFIN13 was decreased from 2135 to 309 bp, the sFlt-1:Flt-1 mRNA ratio decreased 8.9-fold and an aberrant 5'UTR/exon 14 splice decreased 60-fold, indicating that "exon definition" is a key parameter of successful Flt-1 RNA processing. Mutation of 5' or 3' intronic splice signals had little effect on Long sFlt-1:Total sFlt-1 mRNA ratio, suggesting that splicing and cleavage/polyadenylation factors may not compete physically for Flt-1 transcripts. Although co-transfection with RNA processing factor cDNAs did not generally produce the predicted pattern of effects on sFlt-1:Flt-1 mRNA ratio, a cryptic exon within intron 13 was revealed in cells overexpressing U2AF65. sFlt-1 protein apparently can be encoded by mRNAs either cleaved/polyadenylated within intron 13 or, surprisingly, by splicing of the cryptic exon "13b." Thus, the cellular decision to produce sFlt-1 or Flt-1 from a nascent RNA can no longer be viewed as a simple choice between cleavage/polyadenylation and splicing. / Ph. D.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 107-130).

Percutaneous coronary intervention (PCI) acutely improves coronary blood flow and myocardial perfusion but at the expense of endovascular laceration and endothelial denudation. PCI associated vascular injury is associated with intense inflammation and a loss of vascular function that may lead to significant in-stent restenosis (ISR), and the potentially catastrophic, acute stent thrombosis. Reendothelialisation is essential to the restoration of normal homeostasis and facilitating vascular healing. Attention has recently focused on a novel mechanism of reendothelialisation mediated by bone marrow-derived precursor or stem cells: endothelial progenitor cells (EPC). EPC are thought to home to, and reendothelialise sites of endothelial denudation, and therefore offer the potential to provide exciting new developments in the management of cardiovascular disease. Understanding the role of EPC following vascular injury may help us to enhance vascular repair following PCI. The following studies were performed to clarify the relationships between putative EPC and vascular injury associated with PCI. In studies of patients undergoing elective PCI for stable anginal symptoms I found that concentrations of traditional circulating phenotypic EPC expressing CD34+VEGFR-2+ were unaffected, unlike CD34+CD45- cell concentrations, which were transiently increased six hours following PCI, subsequently returning to normal by 24 hours, notably without an increase in CD34+ adhesion molecule expression or VEGF-A production. However, the purported progeny of CD34+VEGFR-2+ cells, endothelial cell-colony forming units (EC-CFU), were mobilised at 24 hours, commensurate with a systemic inflammatory response. Interestingly the concentration of circulating CD34+VEGFR-2+ cells and EC-CFU were unrelated to each other, emphasising the distinction between these two cell populations. Although EC-CFU contained proliferating cells and exhibited some endothelial characteristics, EC-CFU predominantly expressed the leukocyte antigen CD45 in addition to the lymphocyte markers CD4 and CD8, and most intensely, the surface markers CD68 and CD105, epitopes commonly expressed on macrophages. Notably, EC-CFU were a potent stimulus for the migration of mononuclear cells. However, despite being mobilised in the context of an acute systemic inflammatory response and being composed of leukocytes, isolated systemic inflammation in healthy volunteers (induced by Salmonella Typhus vaccination) in the absence of vascular injury did not cause selective mobilisation of EC-CFU or indeed of putative phenotypic EPC. It is therefore likely that EC-CFU mobilisation is a relatively specific inflammatory response to cardiovascular injury. In a cohort of 201 patients undergoing coronary angiography, traditional circulating phenotypic EPC (CD34+VEGFR-2+ and CD34+VEGFR-2+CD133+) were very rare indeed and were not increased in response to an acute coronary syndrome (ACS). Furthermore traditional EPC concentrations bore no relation to atheroma burden or clinical outcome. In contrast, concentrations of CD34+CD45- cells were increased in patients with coronary artery disease compared to those with normal coronary arteries and were increased in association with more severe coronary disease. Increased concentrations of circulating CD34+CD45- cells were also associated with a shorter cumulative event-free survival. Both EC-CFU and angiogenic monocytes expressing Tie-2 and VEGFR-2 were increased following acute myocardial infarction but did not relate to coronary atheroma or clinical outcome. These studies examine the behavior of putative EPC in response to both discrete vascular injury and myocardial infarction, and isolated inflammation in the absence of vascular injury. I have identified novel characteristics of the EC-CFU assay and determined that specific factors associated with cardiovascular injury likely trigger EC-CFU mobilisation. The clinical relevance of the traditional phenotypic EPC population is uncertain, but a novel CD34+CD45- population is mobilised acutely following discrete vascular injury and is significantly associated with coronary atheroma and clinical events. It is probable that the circulating CD34+CD45- concentration reflects vascular injury and atheroma burden, and I suggest that CD34+CD45- cells are released directly from the vessel wall following PCI, and do not reflect a reparatory response. In order to determine the impact of EPC populations on vascular healing, prospective studies examining the impact of periprocedural EPC concentrations on vascular healing following PCI are required.

Pre-eclampsia is a pregnancy-related disorder characterised by high blood pressure, proteinuria and oedema. The aetiology of the disease is unclear but evidence suggests that endothelial dysfunction is central to the development of the maternal syndrome. Kinins are endogenous peptides released by the endothelium that contribute to the regulation of cardiovascular homeostasis by inducing vasodilation, fibrinolysis and angiogenesis. Given that pre-eclampsia is associated with reduced endotheliumdependent relaxation, coagulation abnormalities and an angiogenic imbalance, it was hypothesised that alterations of kinin receptor-mediated responses may be involved in the pathogenesis of the condition. To investigate whether changes in kinin receptor activity are involved in the impairment of endothelium-dependent relaxation observed in pre-eclampsia, the effects of specific B2 and B1 receptor agonists and antagonists on myometrial vascular tone were tested on arteries from healthy pregnancy and pre-eclampsia. The results demonstrated that in addition to classical bradykinin B2 receptor-mediated relaxation, a subset of healthy patients exhibited nitric oxide-dependent relaxation to the B1 receptor agonist Lys-des- Arg9-BK (LDABK) which could not be inhibited by either B1 or B2 receptor antagonists. Also, vessels that exhibited this novel response to LDABK were more sensitive to bradykinin. Furthermore, this study revealed that patients with pre-eclampsia had an attenuated response to both bradykinin and LDABK. Immunolocalisation and mRNA expression of the kinin receptors in the myometrium revealed no differences between healthy pregnancy and pre-eclampsia suggesting that disturbances of kinin receptor signalling rather than changes in receptor distribution or expression levels may be involved in the reduction of kinin-mediated responses in these patients. The role of kinins in mediating placental angiogenesis in healthy pregnancy and preeclampsia was determined using the endothelial tube formation assay in primary human umbilical vein endothelial cells (HUVECs) isolated from healthy women and women with pre-eclampsia. B2 and B1 receptor agonists induced endothelial tube formation via a VEGF-dependent, nitric oxide-independent mechanism in healthy HUVECs cultured in normoxic conditions. HUVECs isolated from women with pre-eclampsia cultured under normoxia and HUVECs from healthy pregnancies cultured under hypoxia exhibited greater levels of angiogenic branching compared with healthy normoxic cells, but were unresponsive to bradykinin and LDABK. Incubation of these cells with a VEGF receptor inhibitor reduced the elevated levels of tube formation indicating that this effect may be due to hypoxic upregulation of VEGF or an intrinsic difference in their angiogenic capacity. Further studies are required to determine the cause for the differences in angiogenic potential between healthy and pre-eclamptic cells and the impact this could have on placental vascular development and the pathogenesis of preeclampsia.

The phenomenon known as immunothrombosis has garnered increased attention over the last few years. Much work has been done to characterize the cross talk between hemostasis and the innate immune system. This thesis outlines the role of the vascular endothelial cells during immunothrombotic events as regulators of coagulation, platelet-, and leukocyte recruitment. A newly developed method for investigating the interaction between endothelial cells and the blood compartment illustrated the procoagulant and proinflammatory effects elicited by tumor necrosis factor α activated endothelial cells upon exposure to whole blood. The method was utilized in evaluating treatment of endothelial dysfunction and disruption with a heparin conjugate. Damaged or hypoxic endothelial cells, in addition to basement membrane collagen, that were pretreated with the heparin conjugate prior to contact with blood were found to have reduced activation of coagulation, platelet-, and leukocyte recruitment; in contrast to unfractionated heparin, which had no effect on the aforementioned parameters. The treatment was then investigated in the setting of ischemia reperfusion injury during kidney transplantation and the heparin conjugate was found to bind cultured endothelial cells with high avidity under cold storage conditions. Furthermore, it was found to bind to the renal vasculature during static cold storage and was subsequently found to be beneficial with regard to early graft function in an experimental mouse model of syngeneic kidney transplantation. Recipients of kidneys treated with the heparin conjugate had reduced serum creatinine compared to controls 24 hours after transplantation. Lastly, the anticoagulant properties of the heparin conjugate were investigated in comparison to unfractionated heparin. While the conjugate exerted reduced capacity with regard to thrombin inhibition, it rapidly inhibited the binding of platelets to exposed collagen. The conjugate was furthermore found to preferentially locate to sites of endothelial cell activation at early stage during endotoxic shock in mice. In conclusion, this thesis demonstrates that disrupted functioning of the vascular endothelial cells actively contributes to immunothrombosis, and that it is possible to model endothelial cell function using whole blood assays. Furthermore, this thesis presents a treatment that enhances the hemocompatibility of damaged endothelial cells and subsequently improves the early renal function after kidney transplantation.

published_or_final_version / abstract / Surgery / Doctoral / Doctor of Philosophy