Myxomatous mitral valve degeneration (MMVD) is the single most common cardiac disease of the dog, and is analogous to Mitral Valve Prolapse in humans. Very little is known about the aetiopathogenesis of this disease or the changes in valvular interstitial cell populations in diseased valves. The aim of this study was to identify morphological, cellular and molecular changes associated with MMVD. Mitral valve leaflets from both normal and varying grades (Whitney’s 1-4) of diseased dogs were subject to image analysis, immunophenotyping, proteomics and RT-PCR. Image analysis - leaflet thickening due to accumulation of glycosaminoglycan was significant in this disease. MMVD is associated with loss of connective tissue, reduction in cell numbers but no change in cell shape in the overtly myxomatous area. Near the surface, increase in valvular interstitial cells (VIC) towards the damaged endothelium in concert with destruction of collagen and building up of ground substance was manifested during the disease process. Immunophenotyping - activated myofibroblasts were increased and fibroblast-like VICs were reduced without any change in desmin and myosin expression in MMVD compared to clinical normal dogs. In addition, other cell types like macrophage, adipocyte, chondrocyte, mast cell, and stem cell were identified and their possible role in MMVD is discussed. Proteomics - a protein expression profile was established, with 64 proteins being positively identified from dog’s mitral valve using 1-D SDS PAGE LC/MS. Amongst them 44 proteins were differentially expressed comparing normal and severely diseased. Two actin binding proteins, tropomyosin alpha and myosin light chain-2 were found to be differentially expressed in the normal but down regulated in the diseased. RT-PCR was used to assess the expression of 8 genes of interest. Their expression was compared with 3 different housekeeping genes.

<p>Approximately 50% of all patients with cancer receive radiation therapy at some point during the course of their illness. Despite advances in radiation delivery and treatment planning, normal tissue toxicity often limits the ability of radiation to eradicate tumors. The tumor microenvironment consists of tumor cells and stromal cells such as endothelial cells that contribute to tumor initiation, progression and response to therapy. Although endothelial cells can contribute to normal tissue injury following radiation, the contribution of stromal cells to tumor response to radiation therapy remains controversial. To investigate the contribution of endothelial cells to the radiation response of primary tumors, we have developed the technology to contemporaneously mutate different genes in the tumor cells and stromal cells of a genetically engineered mouse model of soft tissue sarcoma. Using this dual recombinase technology, we deleted the DNA damage response gene <italic>Atm</italic> in sarcoma and heart endothelial cells. Although deletion of <italic>Atm</italic> increased cell death of proliferating tumor endothelial cells, <italic>Atm</italic> deletion in quiescent endothelial cells of the heart did not sensitize mice to radiation-induced myocardial necrosis. In addition, the ATM inhibitor NVP-BEZ235 selectively radiosensitized primary sarcomas, demonstrating a therapeutic window for inhibiting ATM during radiation therapy. Sensitizing tumor endothelial cells to radiation by deleting <italic>Atm</italic> prolonged tumor growth delay following a non-curative dose of radiation, but failed to increase local control. In contrast, deletion of <italic>Atm</italic> in tumor parenchymal cells increased the probability of tumor eradication. These results demonstrate that tumor parenchymal cells rather than endothelial cells are the critical targets that regulate tumor eradicaiton by radiation therapy.</p> / Dissertation

Background: Strategies to maintain BBB integrity in diseases with a hypoxia/reoxygenation (H/R) component involve preventing glutathione (GSH) loss from endothelial cells. GSH efflux transporters include multidrug resistance proteins (Mrps). Therefore, characterization of Mrp regulation at the BBB during H/R is required to advance these transporters as therapeutic targets. Our goal was to investigate, in vivo, regulation of Abcc1, Abcc2, and Abcc4 mRNA expression (i.e., genes encoding Mrp isoforms that transport GSH) by nuclear factor E2-related factor (Nrf2) using a well-established H/R model. Methods: Female Sprague-Dawley rats (200-250 g) were subjected to normoxia (Nx, 21% O-2, 60 min), hypoxia (Hx, 6% O-2, 60 min) or H/R (6% O-2, 60 min followed by 21% O-2, 10 min, 30 min, or 1 h) or were treated with the Nrf2 activator sulforaphane (25 mg/kg, i.p.) for 3 h. Abcc mRNA expression in brain microvessels was determined using quantitative real-time PCR. Nrf2 signaling activation was examined using an electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) respectively. Data were expressed as mean +/- SD and analyzed via ANOVA followed by the post hoc Bonferroni t test. Results: We observed increased microvascular expression of Abcc1, Abcc2, and Abcc4 mRNA following H/R treatment with reoxygenation times of 10 min, 30 min, and 1 h and in animals treated with sulforaphane. Using a biotinylated Nrf2 probe, we observed an upward band shift in brain microvessels isolated from H/R animals or animals administered sulforaphane. ChIP studies showed increased Nrf2 binding to antioxidant response elements on Abcc1, Abcc2, and Abcc4 promoters following H/R or sulforaphane treatment, suggesting a role for Nrf2 signaling in Abcc gene regulation. Conclusions: Our data show increased Abcc1, Abcc2, and Abcc4 mRNA expression at the BBB in response to H/R stress and that Abcc gene expression is regulated by Nrf2 signaling. Since these Mrp isoforms transport GSH, these results may point to endogenous transporters that can be targeted for BBB protection during H/R stress. Experiments are ongoing to examine functional implications of Nrf2-mediated increases in Abcc transcript expression. Such studies will determine utility of targeting Mrp isoforms for BBB protection in diseases with an H/R component.

La croissance tumorale et le processus de métastatisation dépendent de la néoformation de vaisseaux sanguins ou néoangiogenèse. Parmi les molécules intervenant dans ce processus, l'adrenomédul1ine (AM) est un peptide, dont l'expression est corrélée à l'agressivité de certaines tumeurs, et qui représente un maillon «clé» dans les interactions entre les cellules tumorales et les cellules du microenvironnement. Les résultats spectaculaires qu'offre le traitement des xénogreffes de cellules issues de glioblastomes (GBM) humains par les anticorps dirigés contre l'AM ou son récepteur sont très encourageants, puisque la tumeur traitée régresse en quelques semaines, la vascularisation tumorale s'en trouve touchée de manière spécifique. C'est dans ce contexte, que nous avons choisi de poursuivre notre travail sur les mécanismes d'action de l'AM dans la néoangiogenèse. Grâce à des études in vitro et in vivo, nous avons pu montrer que l'AM est impliquée dans plusieurs étapes de la néoangiogenèse tumorale : migration des cellules endothéliales, stabilisation des contacts endothéliaux et endothélio-péricytaires, recrutement des cellules mésenchymateuses. Nos résultats démontrent que nous sommes en présence d'une molécule d'AM qui agit sur diverses cibles moléculaires et cellulaires, régulant la stabilité du complexe d”adhésion intercellulaire VE-cadhérine/-caténine, nécessaire à la protection des interactions homotypiques et hétérotypiques de l°endothélium nouvellement formé. Ainsi, l'étude des mécanismes d'action de l'AM réalisée pennettra d'établir ue stratégie thérapeutique autour de l'AM. / Tumoral growth and process of metastatization depend on the formation of new blood vessels or angiogenesis. Among the molecules implicated in this process, adrenomedullin (AM) is a peptide, which expression is correlated with the aggressiveness of tumors, and which represents a "key" link in the interactions between tumoral cells and the microenvironment cells. The spectacular results offered by the treatment of human glioblastoma (GBM) xenograft by antibodies directed against the AM or its receptor are very encouraging, as the treated tumor declines in some weeks, and the tumoral vascularization is also touched in a specific way. In this context, we chose to pursue our work on the mechanisms of action of AM in angiogenesis. In vitro and in vivo studies showed that AM is involved in several stages of tumoral angiogenesis : migration of endothelial cells, stabilization of endothelial contacts, stabilization of the pericyte coverage, recruitment of multipotent cells. Our results demonstrate that we are in presence of a molecule of AM which acts on diverse molecular and cellular targets, regulating the stability of the VE-cadherin/β-catenin complex, required for the protection of the homotypics and heterotypics interactions of the newly formed endothelium. The study of the mechanisms of action of AM realized will allow us to establish a therapeutic strategy around AM.

Hypoxia-induced pulmonary hypertension (HPH) is a progressive disease that is mainly caused by chronic exposure to high altitude, chronic obstructive lung disease, and obstructive sleep apnea. The increased pulmonary vascular resistance and increased pulmonary arterial pressure result in increased right ventricular afterload, leading to right heart failure and increased morbidity. There are several clinical reports suggesting a link between PH and diabetes, insulin resistance, or obesity; however, it is unclear whether HPH is associated with diabetes as a progressive complication in diabetes. The major goal of this study is to examine the effect of diabetic ''preconditioning'' or priming effect on the progression of HPH and define the molecular mechanisms that explain the link between diabetes and HPH. Our data show that HPH is significantly enhanced in diabetic mice, while endothelium-dependent relaxation in pulmonary arteries is significantly attenuated in chronically hypoxic diabetic mice (DH). In addition, we demonstrate that mouse pulmonary endothelial cells (MPECs) isolated from DH mice exhibit a significant increase in mitochondrial reactive oxygen species (ROS) concentration and decreased SOD2 protein expression. Finally, scavenging mitochondrial ROS by mitoTempol restores endothelium-dependent relaxation in pulmonary arteries that is attenuated in DH mice. These data suggest that excessive mitochondrial ROS production in diabetic MPECs leads to the development of severe HPH in diabetic mice exposed to hypoxia.

Atherosclerosis is complex inflammatory disease of the arterial wall with progressive accumulation of lipids and narrowing of the vessel. Increasing evidence suggest that inflammation plays an important role in plaque stability and often accelerate cardiovascular events such as myocardial infarction (MI). Among the vast number of inflammatory cytokines, IL-1β is known to be a key modulator in vessel wall inflammation and acceleration of the atherosclerotic process. The biologically active IL-1β is regulated by a multiprotein complex known as the NLRP3 inflammasome complex. In this thesis, we have focused on polymorphisms in the NLRP3 and CARD8 genes and their possible association to atherosclerosis and/or MI. We have also investigated the expression of inflammasome components NLRP3 and CARD8 in atherosclerosis and the role of genetic variants for the expression of these genes. The expression of NLRP3, CARD8, ASC, caspase-1, IL-1β, and IL-18 were found significantly upregulated in atherosclerotic lesions compared to normal arteries. Human carotid plaques not only express the NLRP3 inflammasome, but also release IL-1β upon exposure to lipopolysaccharide (LPS), adenosine triphosphate (ATP) and cholesterol crystals, which suggest NLRP3 inflammasome activation in human atherosclerotic lesions. Also, CARD8 was found to be important in the regulation of several inflammatory markers in endothelial cells, like RANTES, IP10 and ICAM-1. We further assessed the potential association of a CARD8 polymorphism and polymorphisms located downstream of the NLRP3 gene to the risk of MI in two independent Swedish cohorts. The CARD8 variant exhibited no association to risk of MI in either of the two cohorts. Some of the minor alleles of NLRP3 variants were associated with increased IL-1β levels and to NLRP3 mRNA levels in peripheral blood monocytic cells (PBMC). Taken together, the present thesis shows that NLRP3 inflammasome activation and increased expression of CARD8 in the atherosclerotic plaque might be possible contributors to the enhanced inflammatory response and leukocyte infiltration in the pathophysiology of atherosclerosis.

No description available.

In addition to its canonical role in aminoacylation, threonyl-tRNA synthetase (TARS) possesses pro-angiogenic activity that is susceptible to the TARS-specific antibiotic borrelidin. However, the therapeutic benefit of borrelidin is offset by its strong toxicity to living cells. The removal of a single methylene group from the parent borrelidin generates BC194, a modified compound with significantly reduced toxicity but comparable anti-angiogenic potential. Biochemical analyses revealed that the difference in toxicities was due to borrelidin's stimulation of amino acid starvation at ten-fold lower concentrations than BC194. However, both compounds were found to inhibit in vitro and in vivo models of angiogenesis at sub-toxic concentrations, suggesting a similar mechanism that is distinct from the toxic responses. Crystal structures of TARS in complex with each compound indicated that the decreased contacts in the BC194 structure may render it more susceptible to competition with the canonical substrates and permit sufficient aminoacylation activity over a wider concentration of inhibitor. Conversely, both borrelidin and BC194 induce identical conformational changes in TARS, providing a rationale for their comparable effects on angiogenesis. The mechanisms of TARS and borrelidin-based compounds on angiogenesis were subsequently tested using zebrafish and cell-based models. These data revealed ectopic branching, non-functional vessels, and increased cell-cell contracts following BC194-treatment or knockdown of TARS expression, suggesting a role for the enzyme in the maturation and guidance of nascent vasculature. Using various TARS constructs this function was found to be dependent on two interactions or activities associated with the TARS enzyme that are distinct from its canonical aminoacylation activity. Furthermore, observations that TARS may influence VEGF expression and purinergic signaling suggest the possibility for a receptor-mediated response. Taken together, the results presented here demonstrate a clear role for TARS in angiogenesis, independent of its primary function in translation. Although the exact molecular mechanisms through which TARS and borrelidin regulate this activity remain to be determined, these data provide a foundation for future investigations of TARS's function in vascular biology and its use as a target for angiogenesis-based therapy.

Exercise training promotes a beneficial endothelial cell (EC) phenotype and results in conduit vessel adaptation. The specific underlying mechanisms have been proposed (shear stress, circumferential stress, hypoxia, metabolic) but are yet to be fully elucidated. This thesis investigated the predominant stimuli responsible for conduit vessel adaptation with training. Further, it developed a method of in situ EC extraction to allow for determination of the cellular and molecular mechanisms underpinning these adaptations. The methodology utilised two-dimensional (2D) Doppler ultrasound, strain gauge plethysmography, immunocytochemistry and RT-qPCR to provide insight in to vascular characteristics, predominantly of the brachial artery and peripheral EC. Long-term repeated isometric forearm muscle contractions as performed by well-trained rock climbers promoted greater resting, peak (in response to 5 min ischaemia) and maximal (in response to ischaemic exercise) brachial artery diameters compared with controls. This structural response is dependent upon confounders associated with exercise additional to shear stress as evidenced by the lack of brachial artery remodelling in response to 8 weeks of ischaemic preconditioning (IPC). A transient increase in flow-mediated dilation (FMD)% was observed following 6 weeks exposure to IPC, which became significant when controlled for baseline artery diameter, despite an absence of augmentation following long-term (≥ 8 weeks) exposure to a shear stimulus. This is in line with the suggested timeline of conduit vessel adaptation to exercise training of a transient increase in function at 2-4 weeks. Underpinning molecular mechanisms responsible were not determined but may be further investigated given that the endovascular biopsy technique was developed and improved in this thesis. The endovascular biopsy successfully yields approximately 2100 ± 1700 EC per sample, providing sufficient material for determination of expression of both mRNA (RT-qPCR) and protein (immunocytochemistry). Specifically, type 2 diabetics (T2DM) with symptomatic cardiac abnormalities exhibited augmented eNOS mRNA and protein in brachial artery EC as compared with non-diabetic controls with symptomatic cardiac abnormalities. In conclusion, this thesis demonstrates that although shear stress promotes a transient trend for enhancement in function of the peripheral conduit arteries, additional factors are required for long-term structural adaptations. Further, the endovascular biopsy technique offers a novel method of extracting and analysing EC for genes and proteins of interest to vascular health. The use of this technique to decipher the underlying cellular and molecular mechanisms involved in vascular adaptations with exercise requires further investigation.

Cellular adhesion molecules (CAMs) and selectins are cell-surface proteins involved in the binding of cells to the vascular endothelium. Elevated levels of sCAMs and soluble E-selectin (sE-selectin) have been reported in patients with periodontitis. The aim of this study was to determine if periodontal scaling and root planing would influence the serum concentration of sICAM-1, sVCAM-1, and sE-selectin. Twenty-one subjects with chronic periodontitis received scaling and root planing in conjunction with blood serum sample analysis using enzyme-linked immunosorbent assay (ELISA), to determine if periodontal instrumentation results in changes in serum concentrations of sICAM-1, sVCAM-1, and sE-selectin. No change was observed in serum concentration of sICAM-1 or sVCAM-1. However, in a subset of 17 patients a statistically significant change in serum sE-selectin was observed (P < 0.05). This suggests that there is a decrease in endothelial activation following periodontal treatment.