The extracellular matrix (ECM), as the native cellular substrate, provides necessary mechanical and biological signals to cells. Cells exert forces in the nanonewton range, which when applied over time can strain extracellular matrix fibers until breakage. Cells and tissues inherently interact mechanically with their surrounding matrix, so tissue engineering materials would benefit from the ability to fully exploit mechanical-biochemical interactions to enhance integration with the human body. In this work, I developed an increased understanding of ECM fiber mechanical and mechano-biochemical properties. First, I generated novel composite ECM fibers that can be used to study combinations of ECM proteins in a controlled way. I determined how protein composition impacts mechanical properties of novel single ECM fibers in a hydrated state and showed how mechanical properties can be tuned through composition. Next, I assayed for strain and heparin-sensitive allosteric binding of ligands to fibronectin and fibrin fibers, and determined that the binding of two key growth factors is impacted by strain and heparin. Finally, I investigated the impact of fiber strain, heparin-pretreatment, and growth factor interactions on endothelial cell migration. The novel contributions of this project are the generation of new composite extracellular matrix fiber types with tunable mechanical properties, as well as the identification of extracellular matrix protein mechanosensitive and heparin-sensitive interactions with growth factors and their impact on endothelial cell migration, which could be used to aid in the design of protein-based biomaterials for cardiovascular applications. / 2024-05-23T00:00:00Z

This body of work encompasses two studies: the collection of canine tears via a novel polyester<br />rod and the comparison of VEGF-A concentrations in tears from dogs with normal and<br />vascularized corneas. The first study used polyester rods for tear collection in dogs. Fluid volume and VEGF recovery characteristics, as well as potential binding of VEGF to the rod, were determined. Tears were harvested from normal dogs using rods and glass capillary tubes. Tears were assayed for tear film VEGF using a commercial canine VEGF sandwich ELISA kit. Dilutions of VEGF standard were wicked into the rods or drawn into capillary tubes, eluted, and assayed. Percent volume recovery is adequate for polyester rods as is percent VEGF recovery. VEGF is detectable in normal canine tears.The second study harvested tear samples from eyes of dogs with vascularizing corneal disease, as well as the contralateral unaffected eye of unilaterally diseased dogs, and normal dogs. Vascularization scores were assigned to diseased eyes and tear film VEGF concentration was assayed as above. Mean tear film VEGF concentration of diseased eyes did not differ from control eyes, and was not correlated with disease process, extent of vascularization, or other parameters. Tear film VEGF in unaffected eyes was significantly higher than control and vascularized eyes. Canine tear film VEGF exceeds biologically active concentrations, but does not correlate with state of corneal vascularization. VEGF-related control of corneal vascularization may be mediated by other proangiogenic factors. / Master of Science

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The vascular endothelial growth factor (VEGF) family is prominently involved in angiogenesis, the formation of new vasculature. Overexpression has been linked to pathological conditions including solid tumor growth. Responses to VEGF are mediated by Flt-1 and KDR, two cell-surface receptors selectively expressed by vascular endothelial cells. A secreted form of Flt-1 (sFlt1) retains high affinity for VEGF and can compete for VEGF binding, thereby inhibiting angiogenic responses. In this study, a computational model of this ligand-receptor binding system is used to quantitatively describe the activation of KDR signaling by VEGF and its inhibition by sFlt-1. The model consists of a system of 12 ordinary differential equations, based on mass action kinetics, which describe, based on mass-action kinetics, the formation and disappearance of receptor-ligand complexes competent to transduce a VEGF signal. Cell-based assays were used to test predictions of the in silico model by measuring dynamics of 1) KDR tyrosine hosphorylation and 2) generation of intracellular second messengers in response to VEGF stimulation in the presence and absence of sFlt-1. Results indicate that the model predicts well the maximal activation in the presence of VEGF-A or the KDR-selective agonist VEGF-E. Subsequent characteristics of binding, such as time-to-peak activations, were less realistically modeled, suggesting that the mathematical descriptions of certain receptor fates (receptor internalization, sequestration or externalization) may need refinement. Also presented are the results of KDR activation experiments in the simultaneous presence of VEGF-A, sFlt1, and the Flt1-selective agonist placental growth factor (PlGF). Here, the model predicts accurately the percentage of inhibition of maximal activation when sFlt-1 is present in the system. Finally, a revised model that includes receptor heterodimerization is presented that suggests sFlt-1 can more effectively inhibit VEGF binding and activation when acting in a dominant negative fashion. Overall, the findings support the idea that the early events in VEGF signaling can be modeled successfully. A natural extension of the model, refined based on experimental testing, will be its application to normal physiological systems. / Ph. D.

Vascular endothelial growth factor A (VEGF) is a potent mediator of blood vessel formation throughout the body. Intraocular diseases characterized by inflammation, hypoxia or neoplasia induce new blood vessel formation within the eye. The end result of such blood vessel formation may be blinding sequellae such as glaucoma from outflow obstruction or hyphema from intraocular hemorrhage. Elevated VEGF concentrations in the aqueous humor and vitreous are documented in a number of human intraocular disease processes, including tumors, retinal detachment and uveitic glaucoma. Pharmacotherapy inhibiting VEGF expression demonstrates promise for control of some of these ophthalmic conditions. We quantified and compared VEGF concentrations in canine aqueous humor samples from 13 dogs with normal eyes and 226 eyes from 178 dogs with a variety of ophthalmic diseases by ELISA. Dogs with primary cataract, diabetic cataract, primary glaucoma, uveitic glaucoma, aphakic/pseudophakic glaucoma, retinal detachment, lens luxation and neoplasia were evaluated. Elevated VEGF concentrations were found in all disease conditions tested as compared to normal dogs excepting cataracts and diabetic cataracts. Elevated aqueous humor VEGF concentrations were found in dogs with pre-iridal fibrovascular membranes (PIFM) as compared to dogs without PIFM. These results are consistent with the hypothesis that VEGF has a role in the causation or progression of a variety of canine ocular disorders. / Master of Science

Les facteurs de croissance endothéliaux (VEGFs) sont produits par les tumeurs qui sont hypoxiques. Principaux responsables de la néo-vascularisation pathologique, ils régulent le stroma tumoral. Les nouvelles stratégies qui ciblent et inhibent le VEGF ouvrent vers la thérapie anti-cancéreuse moderne. Elles ont pour but de contrôler l’angiogenèse tumorale plutôt que la détruire. Le défi est donc de piéger sélectivement le VEGF produit en excès, dans le microenvironnement tumoral, sous l’effet de l’hypoxie. La thèse présentée dans ce manuscrit est consacrée à la réalisation d’une nouvelle stratégie ciblante par l’intermédiaire de cellules, aussi appelée « Cheval de Troie ». Elle combine dans la même entité, une unité de ciblage et un système de délivrance spécifique d’un gène/molécule thérapeutique. Dans le but d’adresser la thérapie aux cellules cancéreuses sans affecter les cellules saines, un modèle de cellules endothéliales de type précurseur (CEPs) a été utilisé comme cellules ciblantes capables d’atteindre spécifiquement le site tumoral. Les CEPs ont été « armées » pour exprimer un gène thérapeutique chargé d’inhiber le VEGF. La neutralisation a été obtenue par la production d’une forme soluble du récepteur-2 du VEGF (VEGFR2 soluble), agissant comme inhibiteur. Caractéristique des tumeurs solides se développant, l’hypoxie a été choisie pour déclencher/éteindre l’expression et la sécrétion du VEGFR2 soluble, en introduisant, en amont du gène thérapeutique, une séquence régulatrice : HRE. Adressé au site tumoral par les CEPs, le régulateur de l’angiogenèse qu’est la forme soluble du VEGFR2, est exprimé de manière conditionnée et réversible, à l’hypoxie. Ceci ouvre à de nouvelles stratégies de normalisation contrôlée et stable des vaisseaux tumoraux en vue de l’utilisation de thérapies combinées. / Vascular endothelial growth factors (VEGFs) are over-expressed upon hypoxia in solid tumors. Major actors directing pathologic neo-vascularisation, they regulate the stromal reaction. Novel strategies that target and inhibit VEGF bring promise to modern anti-cancer therapies. They aim to control rather than destroy tumor angiogenesis. Consequently, the challenge is to selectively trap VEGFs, over-produced upon hypoxia, in the tumor microenvironment. The thesis presented in this manuscript focuses on the design of a novel cell-based targeting strategy, so-called “Trojan Horse”, combining in the same engineered entity, a targeting unit and a specific drug/gene delivery system. Aiming to address the therapy to cancer cells without affecting healthy cells, a model of endothelial precursor cell (EPCs) was used as targeting cell able to reach specifically the tumor site. EPCs were “armed” to express a therapeutic gene to inhibit VEGF. Trapping was attempted based on the production of a soluble form of the VEGF receptor-2 (sVEGFR2) as a candidate inhibitor. Hypoxia, a hallmark of developing solid tumors, was chosen to turn on/off the sVEGFR2 expression and secretion by introducing, upstream of the therapeutic gene, a hypoxia response element (HRE) regulating sequence. Properly addressed by the EPCs to the tumor site, such angiogenesis regulator as the soluble form of VEGFR2 is, was chosen to be expressed in a hypoxia-conditioned and reversible manner. This opens new strategies for a stably controlled normalization of tumor vessels in view of adjuvant design for combined therapies.

Le vascular endothelial growth factor (VEGF) un facteur de croissance essentiel du système vasculaire exerce des fonctions multiples sur les cellules nerveuses en favorisant la neurogenèse, la plasticité synaptique ou encore l'apprentissage et la mémoire. Cependant, les mécanismes impliqués dans son action régulatrice de la transmission et la plasticité synaptiques restent à élucider. Nous avons récemment mis en évidence une nouvelle interaction entre VEGFR2, le récepteur principal du VEGF, et les récepteurs NMDA (NMDAR) au cours de la migration des neurones pendant le développement du cervelet. Comme les NMDAR sont des acteurs clés de la transmission et de la plasticité synaptique, nous avons exploré le rôle du VEGF dans la régulation de l'expression et de la fonction des NMDAR synaptiques dans l'hippocampe. Nos résultats révèlent que le VEGF et son récepteur sont exprimés dans les régions CA1 et CA3 de l'hippocampe et le domaine extracellulaire de VEGFR2 peut se lier à la sous-unité GluN2B des NMDAR. Le VEGF est capable d'augmenter la transmission synaptique dépendant des NMDAR en régulant l'adressage synaptique des récepteurs exprimant la sous-unité GluN2B. Il se produit également une augmentation du nombre de synapses en présence du VEGF. Ces effets du VEGF requièrent la co-activation des récepteurs VEGFR2 et NMDAR et conduisent à un enrichissement synaptique en récepteurs glutamatergiques de type AMPA qui dépend de l'activation de la CaMKII. Nos travaux démontrent pour la première fois un rôle direct de la signalisation VEGF/VEGFR2 dans la fonction de la synapse excitatrice glutamatergique / The vascular endothelial growth factor (VEGF) plays a critical role during vascular development but recent evidence indicates that it also regulates various neuronal processes in the nervous system, such as neurogenesis, hippocampal synaptic plasticity, learning and memory. Recently, we showed a novel interaction between the glutamate receptor NMDA (NMDAR) and the VEGF receptor VEGFR2 during neuronal migration in the developing cerebellum. As NMDAR have been widely implicated in synaptic transmission and plasticity, we hypothesized that VEGF might regulate NMDAR function in hippocampal synaptic transmission and plasticity, as well as in learning and memory. Our results revealed that VEGF and its receptor VEGFR2 are expressed in the CA1 and CA3 regions of the hippocampus. Biochemical exploration highlighted an interaction between the extracellular domain of VEGFR2 and the GluN2B subunit of NMDAR. In addition, whole-cell patch clamp experiments in acute hippocampal slices showed that VEGF potentiates post-synaptic GluN2B-expressing NMDAR responses. Furthermore NMDAR and VEGFR2 co-activation in hippocampal neurons increased the pool of synaptic GluN2B-NMDAR and affects synapse number. These processes are associated with an increase in AMPAR synaptic expression and an involvment of CaMKII signaling pathway. Altogether, our results demonstrated for the first time a direct effect of VEGF on the function of excitatory glutamatergic synapses

Objetivos: A avaliação da angiogênese no câncerde mama é um importante fator prognóstico e preditivo. A expressão do fator de crescimento vascular endotelial (VEGF) tem sido relatada como metodologia adequada posto sua relação com a densidade microvascular tumoral (MVD), a dosagem de VEGF plasmático assim como com a expressão de outros genes relacionados a angiogênese. Pacientes e Métodos: este estudo avaliou 30 pacientes com diagnóstico de carcinoma de mama localmente avançado tratadas inicialmente pela quimioterapia neoadjuvante e foi correlacionada a expressão da proteína VEGF e outros caracteres clínico-patológicos destas pacientes. Resultados: A expressão da proteína VEGF foi significamente correlacionada com a resposta patológica completa (p= 0,04). Não houve correlação entre expressão de VEGF e tamanho tumoral (p= 0,76), envolvimento axilar (p= 0,70), RP (p= 0,92), RE (p= 0,98), superexpressão de HER-2 (p= 0,79), grau tumoral (p= 0,68) ou menopausa (p= 0,07). Conclusões: A expressão de VEGF foi consistentemente associada a resposta patológica completa e pode ser utilizada como fator preditivo para selecionar pacientes com CMLA para tratamento primário pela quimioterapia. / Purpose: The assessment of angiogenesis in breast canceris a import predictive and prognostic factor. Vascular endothelial growth factor (VEGF) expression has been reported as reliable methodology to evaluate the relationship between microvascular density (MVD). VEGF plasma level and the expression of other angiogenesis related genes. Patients and Methods: This study examined 30 locally advanced breast cancer patients submitted to neoadjuvant chemotherapy. We analyzed the correlation between VEGF protein expression and clinicopathologicalcharacteristics. Results: VEGF expression was significantly correlated to complete pathological response (p= 0,04). There were no correlation between a VEGF expression and tumor size (p= 0,76), axillary involvement (p= 0,70), PR (p= 0,92), ER (p=0,96), HER-2 overexpression (p= 0,79), tumor grade (p=0,68) or menopausal status (p= 0,07). Conclusion: VEGF expression was consistently associated to complete pathological response and may be used as predictive factor to select patients to primary chemotherapy in LABC patients.