OBJECTIVE: While it has long been known that vascular endothelial growth factor A (VEGF-A) plays a role in vascular homeostasis, only recently have its effects been explored in adipose tissue. As perivascular adipose tissue (PVAT) is in close proximity with the aorta and coronary arteries and is known to contribute to vasodilation, it may influence vascular function via secretion of VEGF-A. The objective of this study is to analyze the effects of brown-adipocyte deletion of VEGF-A on circulating VEGF-A levels and distribution of VEGF-A isoforms. We hypothesize that ablation of VEGF-A in brown adipocytes will affect perivascular adipocyte and vascular function. MATERIALS/ METHODS: Mice harboring a brown adipose-specific VEGF deficiency, UCP1cre.VEGFflox/flox mice, were maintained on a chow diet. Primary adipocytes were isolated from brown adipose tissue (BAT) and thoracic PVAT by collagenase digestion and culturing. Gene expression was measured by RT-PCR from RNA extracted from tissues of UCP1cre.VEGFflox/flox mice. Circulating and tissue VEGF-A levels were quantified by ELISA. RESULTS: While VEGF-A ablation using the UCP1 promoter decreases VEGF- protein A levels in BAT and PVAT, it does not affect VEGF-A levels in the circulation. CONCLUSION: This study confirms the functional utility of the UCP1cre.VEGFflox/flox mouse model, as it selectively reduces VEGF-A levels in BAT and PVAT without affecting other tissues or circulating levels. As previous studies using VEGF ablation in all adipose tissues demonstrate an impaired thermogenic response and brown-adipocyte dysfunction, further study of the brown adipose-specific mouse model is warranted. Because PVAT provides protection against vascular stiffness, modulation of VEGF-A in PVAT may be a viable treatment for obesity-associated vascular complications.

Background: Hypertension is one of the most common diseases in the United Kingdom and it remains an important risk factor for cardiovascular morbidity and mortality. Dietary sodium is an important trigger for hypertension and humans show a heterogeneous blood pressure (BP) response to salt intake. The mechanisms for this have not been fully explained, with renal sodium handling thought to play a central role. Animal studies have shown that dietary salt loading results in Na+ accumulation and lymphangiogenesis in skin mediated by vascular endothelial growth factor-C (VEGF-C), both attenuating the rise in BP. This represents an additional system for maintaining BP and volume homeostasis in response to salt load. The focus of this thesis is to determine whether these dermal mechanisms exist in humans. Methods: The technique of measuring skin Na+ and K+ using inductively coupled plasma optical emission spectrometry was developed in a pilot study of healthy adults. In a further study in healthy adults, the effects of dietary salt modulation on skin Na+, the effect of sex and the relationship between skin Na+ and haemodynamic parameters and plasma VEGF-C were studied. Skin Na+ concentrations were expressed as the ratio Na+:K+ to correct for variability in sample hydration. The effect of dietary salt intake on skin gene expression of factors that potentially influence BP such as VEGF-C and the hypoxia inducible factor (HIF) transcription system was assessed, exploring possible mechanisms linking skin Na+ to haemodynamic variables. Results: Skin Na+:K+ increased with dietary salt loading and this effect appeared to be greater in men while only women showed a rise in ambulatory mean BP. Skin Na+:K+ correlated with blood pressure, stroke volume and peripheral vascular resistance in men, but not in women. No change was noted in plasma vascular endothelial growth factor-C. Conclusions: These findings suggest that the skin may buffer dietary Na+, reducing the hemodynamic consequences of increased salt and this may be influenced by sex. Skin Na+ may influence blood pressure, stroke volume and PVR.

A key challenge in tissue engineering is overcoming cell death in the scaffold interior due to the limited diffusion of oxygen and nutrients therein. We hypothesized here that immobilizing a gradient of vascular endothelial growth factor (VEGF-165) would guide endothelial cells into the interior of the scaffold thereby enhancing angiogenesis. The protein was immobilized onto a collagen scaffold through carbodiimide chemistry by one of the three methods experimented: placing 5 µl of the solution at the center of the scaffold to create a ~2 ng/ml/mm gradient in a radial direction. D4T endothelial cells were observed to be guided by this VEGF-165 gradient deep into the center of the scaffold compared to both uniformly immobilized VEGF-165 and VEGF-free controls. We concluded that the VEGF-165 gradient scaffolds promoted the migration, and not proliferation, of cells deep into the scaffold. These gradient scaffolds provide the foundation for future in vivo tissue engineering studies.

A key challenge in tissue engineering is overcoming cell death in the scaffold interior due to the limited diffusion of oxygen and nutrients therein. We hypothesized here that immobilizing a gradient of vascular endothelial growth factor (VEGF-165) would guide endothelial cells into the interior of the scaffold thereby enhancing angiogenesis. The protein was immobilized onto a collagen scaffold through carbodiimide chemistry by one of the three methods experimented: placing 5 µl of the solution at the center of the scaffold to create a ~2 ng/ml/mm gradient in a radial direction. D4T endothelial cells were observed to be guided by this VEGF-165 gradient deep into the center of the scaffold compared to both uniformly immobilized VEGF-165 and VEGF-free controls. We concluded that the VEGF-165 gradient scaffolds promoted the migration, and not proliferation, of cells deep into the scaffold. These gradient scaffolds provide the foundation for future in vivo tissue engineering studies.

La DMAE es la principal causa de ceguera en pacientes por encima de los 65 años. La investigación de esta enfermedad está adquiriendo gran importancia desde hace años por su alta prevalencia a nivel mundial. Desde la introducción de los fármacos anti-VEGF ha mejorado de manera sustancial el pronóstico de los pacientes afectos por la forma húmeda de esta enfermedad. El propósito de esta tesis es evaluar nuestra experiencia en un centro terciario de referencia con el uso de los anti- VEFG en la práctica clínica diaria para el tratamiento de esta enfermedad. En concreto nuestros objetivos son estudiar los resultados anatómico funcionales con nuestra pauta de tratamiento basada en 2+ PRN, comparar los principales fármacos utilizados ( Bevacizumab y Ranibizumab) y evaluar algunos factores de riesgo generales, locales y maculares para predecir si los mismos pueden influir en el resultado funcional final. Inicialmente se realiza una revisión de la literatura. Se profundiza en la etiopatogenia, epidemiología, factores de riesgo y clasificación de la enfermedad, para terminar revisando de manera amplia la evolución de los diferentes tratamientos utilizados para el tratamiento de la DMAE neovascular. El estudio diseñado es prospectivo, intervencional, comparativo y no randomizado y comprende una muestra de 94 ojos, homogénea a nivel de características demográficas y oculares basales, con valores medios en cuanto a las características poblacionales coincidentes con los de la típica población utilizada en la mayoría de los grandes estudios randomizados, todos los casos cuentan con un seguimiento mínimo de 6 meses. Se utiliza en todos los pacientes una pauta de 2+ PRN y como tratamiento se administra Bevacizumab, Ranibizumab o bien tratamiento combinado. Como conclusión los resultados obtenidos nos muestran que nuestra pauta utilizada de 2 inyecciones en la fase de carga presenta mayor número de recidivas y peores resultados visuales finales que las pautas de 3 inyecciones +mantenimiento usadas en la mayoría de series publicadas y que no hay diferencias entre los dos fármacos principales a estudio Ranibizumab y Bevacizumab a nivel de resultados anatómicos y funcionales finales, pero parece que el Ranibizumab es más efectivo a corto plazo. En cuanto al estudio de los factores de riesgo, se observa que la hipertensión arterial, el colesterol total y la edad son los factores que influyen de manera directa en el pronóstico funcional final. / AMD is the leading cause of blindness in patients over 65 years. The investigation of this disease has gained considerable importance over the years by its high prevalence worldwide. Since the introduction of anti-VEGF drugs have substantially improved the prognosis of patients affected by the wet form of the disease. The purpose of this thesis is to evaluate our experience in a tertiary referral center with the use of anti-VEGF in clinical practice for the treatment of this disease. Specifically our objectives are to study the functional anatomical results with our treatment regimen based on 2 + PRN, comparing the main drugs used (Bevacizumab and Ranibizumab) and evaluate some general risk factors, local and macular to predict whether they can influence the final functional outcome. Initially we review the literature. It delves into the pathogenesis, epidemiology, risk factors and disease classification, to finish reviewing comprehensively the evolution of the different treatments used to treat neovascular AMD. The study design is prospective, interventional, nonrandomized comparative and includes a sample of 94 eyes, homogeneous level of baseline demographic and ocular, with mean values, in terms of population characteristics, that match those of the typical population used in most of large randomized studies, all cases have a follow up 6 months or more. It is used in all patients a pattern of 2 + PRN and as treatment is administered Bevacizumab, Ranibizumab or combination therapy. In conclusion the results show that our regimen of 2 injections used in the loading phase shows more relapses and worse final visual results than patterns of 3 injections + manteinance used in most published series. They also indicate that there is no difference between the two main drugs Lucentis and Avastin in late anatomical and functional results, but it seems that Ranibizumab is more effective the short-term. As for the study of risk factors, we observe that high blood pressure, total cholesterol and age are factors that directly influence the functional prognosis.

No description available.

Formation of functional collateral circulation, to repair blocked or damaged arterial blood flow, is an important process in amending adverse outcomes after acute coronary occlusion events. Inadequate capillary growth during pressure overloads impairs myocardial perfusion, often contributing to the progression of coronary heart disease and ischaemia. Considered to be the critical rate-limiting step in physiological angiogenesis, the binding of VEGF (vascular endothelial growth factor) to VEGFR (vascular endothelial growth factor receptors) is essential for the growth and repair of arteries. Conversely, VEGF mediated angiogenesis has also been shown to promote atherosclerosis through arterial wall thickening. However, an alternatively spliced soluble form of VEGFR-1 (sFlt-1) has been shown to inhibit VEGF activity. sFlt-1 binds and sequesters free extracellular VEGF and/or heterodimerizes with VEGFR preventing the angiogenic pathway occurring. As a result, the primary pathway of angiogenesis does not occur. In recent years this has led to debate over the nature of sFlt-1 in the VEGF system. However, the level of sFlt-1 found in cardiovascular disease (CVD) patients, as well as its stability in plasma, has allowed for current research into its involvement with ischemic disorders to take place. Enhanced T-cell activity that results in increased production of interferon-γ has been shown to have involvement in the pathogenesis of CVD. 7,8-dihydroneopterin (7,8 NP) production by monocytes and macrophages is primarily in response to stimulation by interferon-γ (IFN-γ) released by activated T-lymphocytes. When combined with neopterin, the oxidised product of 7,8 NP, the total neopterin is accounted for which is a measure of the total macrophage activation by interferon-γ. Therefore, the levels of total neopterin observed may reflect the level of cell-mediated immunity within individuals which could contribute to mortality post CVD event. Progression of coronary heart disease is often clinically silent, without signs or symptoms. For this reason, the ability of markers to monitor progression is a powerful tool for predicting cardiovascular risk and the level of preventative treatment required. This study shows, that in 514 stable post-ACS (MI or unstable angina) patients, above median baseline sFlt-1, total neopterin and 7,8 NP levels, were strong predictors of mortality over a median 5 year period. Furthermore, above median sFlt-1 levels were specifically predictive of CVD death (p=0.001). This suggests that sFlt-1, total neopterin and 7,8 NP may be useful markers for risk prediction in CVD patients, post-acute event, with potential to aid prognosis in previously diagnosed patients. In support of these findings, levels of sFlt-1 measured in plasma taken from patients, immediately prior to undergoing carotid endarterectomy procedures (n=27), were significantly raised in comparison to age and gender matched healthy controls (p<0.001). Furthermore, levels of sFlt-1 in patient and control groups were shown to be independent of both age and gender. Another aspect of the study, analysis of excised live plaque tissue from carotid endarterectomy patients, showed the presence of live inflammatory cell populations. Macrophages, in the plaque sections, could be stimulated in the presence of IFN-γ to produce significantly elevated (p<0.01) levels of the antioxidant 7,8 NP. Since bivariate analysis of 7,8 NP and sFlt-1, in plasma from the endarterectomy patients, yields a positive correlation (r=0.323, p<0.01), further analysis of live plaque may give insight into the association between inflammation and hypoxic up-regulation of sFlt-1. It is now generally accepted, in diseases with complex pathogenesis, that particular biomarkers are predominantly indicative of only a single variable in a wide range of contributing factors. The data generated in this study highlights the potential for sFlt-1, neopterin and 7,8 NP to be used as contributing biomarkers in the prognosis of patients suffering from CVD, which if confirmed, may have important clinical implications in the medical community.

Thioredoxin-1 (Trx-1) is a redox protein that is overexpressed in many tumors where it is associated with tumor growth, inhibited apoptosis and decreased patient survival. Through redox reactions, Trx-1 is able to reduce a number of proteins including transcription factors. Sp1 activation has been implicated in the regulation of many genes involved in cellular growth and survival and its overexpression in certain cancer correlates with decreased patient survival. We demonstrate that Trx-1 is able to activate Sp1 in a redox dependent manner. Trx-1 overexpression increases Sp1 transactivation and DNA binding whereas a redox inactive Trx-1 has no effect on Sp1 DNA binding.Sp1 has been implicated in vascular endothelial growth factor regulation and we have shown that Trx-1 expression results in increased hypoxic VEGF expression and increased tumor permeability in vivo. Trx-1 overexpression results in an increase in VEGF expression that is dependent upon Sp1, as inhibition of Sp1 expression with siRNA prevented the induction of VEGF expression by Trx-1. These results suggest that Trx-1 increases VEGF expression under normoxic conditions through a redox dependent increase in the DNA binding of the Sp1 transcription factor. VEGF regulation by Sp1 could increase angiogenesis in relatively perfused areas contributing to the stimulation of tumor growth by Trx-1.We hypothesized that Trx-1 regulation of Sp1 may be part of the mechanism of Trx-1 induction of cellular growth. Sp1 regulates many genes involved in cellular growth including epidermal growth factor receptor (EGFR) and insulin-like growth factor I receptor (IGF-IR). These two growth factor receptors are important for cellular growth and have been shown to be important therapeutic targets for cancer treatment. We report that treatment with the Trx-1 inhibitor PX-12 results in decreased Sp1 DNA binding as well as decreased Sp1 activation and transactivation of VEGF, EGFR, and IGF-IR. These results indicate that Trx-1 promotes cellular growth and survival, in part, through the redox regulation of Sp1 responsive growth genes EGFR and IGF-IR. Inhibition of Trx-1, via PX-12, results in a decrease in EGFR and IGF-IR expression and suggests a new mechanism by which Trx-1 inhibition is clinically effective for treating cancer.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Preeclampsia is a pregnancy related disorder, characterized by proteinuria and hypertension. The pathogenesis of preeclampsia is poorly understood; however, two proteins, called sFlt-1 and sEng, were found to be highly elevated in the maternal plasma weeks prior to the onset of clinical symptoms. sFlt-1 and sEng are thought to inhibit VEGF and TGF-β receptor signalling respectively. In order to elucidate how these proteins may contribute to preeclampsia, we looked at their ability to affect the secretion of endothelin-1 (ET-1), a powerful vasoconstrictor, shown to be dysregulated in preeclampsia. We found that both TGF-β1 and BMP9, a recently described ligand for sEng, induce ET-1 secretion through Smad1/5/8 and p38 pathways. Moreover, we report that sEng and VEGF can efficiently block ET-1 secretion, induced by BMP9. We propose that the balance between VEGF and BMP9 signalling is disturbed during preeclampsia, leading to excessive release of ET-1, which in turn may cause hypertension.