published_or_final_version / Biological Sciences / Master / Master of Philosophy

No description available.

No description available.

No description available.

No description available.

No description available.

The lymphatic system complements the blood circulatory system in absorption and transport of nutrients, and in the maintenance of homeostasis. Historically, the angiogenesis field has advanced faster and farther than the field of lymphangiogenesis. The discovery of lymphatic markers and the emerging evidence implicating the lymphatic system as a central player in a variety of pathological conditions has attracted research interest and driven the field forward. Research efforts have produced the observation that regulators of the blood endothelium are frequently members of the same protein families of regulators of the lymphatic endothelium. More importantly, these regulators do not act discretely, restricting their regulatory activities to one endothelial cell (EC) type. Two examples of regulators that behave in this manner are the VEGF and the Angiopoietin families of proteins, which have cell-type-dependent effects on EC processes such as migration, proliferation and survival. The study of these regulators therefore requires an in vitro EC system capable of accommodating the simultaneous characterization of the signaling pathways downstream of these shared molecular regulators in venous, arterial and lymphatic endotheliums. To build such an in vitro system, I isolated and validated lymphatic, venous, and arterial ECs derived from vessels of bovine mesentery. The proteomes of the three cell types were comparatively studied using two-dimensional polyacrylamide gel electrophoresis followed by mass spectrometric identification. The three cell types were used in a subtractive immunization scheme for the production of a monoclonal antibody selectively reactive to a potentially novel surface protein marker of lymphatic ECs. The studies recorded herein all share the common goal of identifying and characterizing unique molecular signatures that distinguish lymphatic ECs from blood ECs, and that may underline the cellular biology of the lymphatic endothelium as distinct from the blood endothelium.

Objective:  Obesity has become the leading cause of mortality worldwide; however, the fundamental pathophysiology underlying this association remains unclear. The discovery of adipokines, i.e., cytokines produced by adipose cells (adipocytes), revealed that adipose tissue is a highly endocrine organ, thus opening new lines of investigation. The prototypical adipokine leptin increases in obesity, and leptin receptors are found in vascular cells. However, results are contradictory regarding the role of leptin in vascular and endothelial functions. Leptin has been shown to elicit vasodilatation, but has also been linked with atherosclerotic and thrombotic disease. The main aim of the present thesis was to study the association of circulating levels of leptin with markers of endothelial function, and to analyze the effects of leptin infusion in vivo  on vasomotor function and endogenous fibrinolysis. Material:  Four associative studies and two interventional studies were conducted. The former included DISARM (studies 1 and 2), the PIVUS study (study 3), and the Scottish post-infarction study (study 4). The DISARM studies and study 4, respectively, recruited 20 men and 83 men and women with stable ischemic heart disease. Study 3 included a random sample of 1016 subjects (54% women, 70 years old) living in the community of Uppsala, Sweden. For the interventional studies (studies 5 and 6), 10 healthy men were recruited for each study. Methods:  In all studies, endothelial function was estimated based on forearm blood flow (FBF) as measured by strain-gauge venous occlusion plethysmography, at rest or during infusion of vasodilators. In study 3, additional measurement techniques were used, such as brachial ultrasound flow-mediated dilation (FMD) and the aortic augmentation index (AoAIx) by tonometry in the radial artery. Fibrinolytic status was estimated based on basal and stimulated levels of tissue plasminogen activator antigen (t-PA), and by assessment of the endothelial release of t-PA (net t-PA release). Plasma leptin levels were measured by radioimmunoassay. In the associative studies, endothelial function and fibrinolytic status were related to circulating plasma leptin levels. In the experimental studies, exogenous leptin was administered in the brachial artery and endothelial function was assessed by strain-gauge plethysmography Results:  In elderly men and women, leptin was independently associated with decreased endothelial-dependent and -independent vasodilatation, reflecting disturbed endothelial function in resistance vessels. This association was attenuated after adjustment for BMI, and when analyzed among subjects with high plasma leptin levels. FMD (a measure of endothelial function in conduit vessels) was not associated with leptin. Exogenous leptin infusion did not alter vasomotor tone, but the endothelium-dependent and -independent vasodilatation was impaired during concomitant infusion of leptin and vasodilators. Infused leptin in the forearm did not affect blood pressure or pulse rate. Chronic hyperleptinemia, but not acutely induced hyperleptinemia, was associated with release of endothelial tissue plasminogen activator (net t-PA). Conclusions:  In humans, leptin was associated with impaired vasodilatation. However, this relationship was blunted after adjustment for BMI, suggesting that leptin could be the mediator between obesity and impaired vascular function. Furthermore, the observed lack of association in hyperleptinemic subjects may reflect a state of leptin resistance. The experimental result showing attenuated vascular reactivity following leptin infusion is in accordance with the results of the associative studies. The augmented net t-PA release in patients with chronic hyperleptinemia may indicate a state of “vascular activation,” which was not observed in healthy endothelium during a short period of leptin infusion. This thesis addresses several controversial issues regarding the action of leptin on vascular tissue in humans. The final results indicate that the in vivo action of leptin on vascularity is complex and mediated by several mechanisms. Our findings suggest that leptin is an important mediator between obesity and endothelial dysfunction, and should stimulate further investigation of this matter. / Manuel Cruz Gonzalez

Factor VIII (FVIII) is an essential blood-clotting protein and mutations in the FVIII gene are the cause of hemophilia A, a severe inherited bleeding disorder. FVIII synthesis has been observed in discreet endothelial sub-populations including liver sinusoidal endothelial cells and in selected microvascular beds. The mechanistic basis for this differential expression is unknown. Differences in shear stress are believed to play an important role in determining endothelial heterogeneity. In this study, we have evaluated the effect of various shear stress conditions on FVIII expression in blood outgrowth endothelial progenitor cells (BOECs) with an in vitro flow system. Under static conditions, BOECs do not express FVIII. In contrast, after exposure to laminar shear stress for 48 hrs, a significant increase in FVIII expression was documented by qRT-PCR, regardless of the magnitude of shear stress studied (1, 5, 15 and 30 dynes/cm2). To determine the effect of prolonged shear stress, laminar flow was applied over 120 hrs and FVIII mRNA levels returned to static levels. Induction of gene expression by laminar shear stress followed by repression after longer durations is common to other pro-coagulant genes induced by non-laminar or oscillatory flow (eg. tissue factor). BOECs exposed to 15 dyne/cm2 of shear stress, oscillating every 0.5 sec for 120 hrs, had FVIII mRNA levels 4.7-fold that of cells in static conditions. This was significantly higher than FVIII expression in BOECs exposed to 15 dyne/cm2 of laminar shear stress for the same duration. Expression of KLF2, a transcription factor that suppresses endothelial pro-coagulant gene expression under laminar shear stress, was significantly reduced in BOECs exposed to oscillatory as opposed to laminar shear stress. Finally, in BOECs exposed to oscillatory shear stress, FVIII protein was synthesized and co-localizes with its carrier protein VWF in Weibel-Palade bodies. These studies show that shear stress is a significant regulator of FVIII expression in BOECs, that FVIII expression is inversely correlated with that of KLF2, and that FVIII protein co-localizes with VWF in these cells. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2013-03-04 17:00:27.994

Background: Endothelial dysfunction (ED) is one of the earliest subclinical indicators of impaired cardiovascular health and several non-invasive tools have been developed to evaluate vascular function, including strain gauge plethysmography (SGP), brachial artery flow-mediated dilation (FMD) via ultrasound, and peripheral artery tonometry (PAT). While these tools have extensively been studied during a resting condition, the responses following acute exercise are not as well characterized. Purpose: The purpose of this study was to compare the pre- and post-exercise vascular function values obtained with SGP, FMD, and PAT. Relationships among the primary outcome variables obtained with each assessment tool were also evaluated. Methods: Vascular function was assessed in 17 sedentary, apparently healthy male subjects (24±4 yrs; 24.5±3.2 kg/m2) at rest and following an acute submaximal exercise bout with SGP, FMD, and PAT. Results: During rest, post-occlusion reactive hyperemia resulted in significant (p<0.05) increases in forearm blood flow (FBF; 2.13±1.03 vs 6.35 ± 2.90 mL/min/100 mL tissue) and area under the curve (AUC; 226.77 ± 111.20 vs 588.22 ±283.33 mL/min/100 mL) as determined by SGP. Brachial artery diameter (BAD) as assessed with FMD was increased by 5.3% (p<0.05). Resting reactive hyperemia index (RHI) as assessed by PAT was observed to be 1.73±0.34. Significant exercise-induced increases (p<0.05) were observed in baseline and post-occlusion FBF and baseline AUC values utilizing SGP. Additionally, FMD baseline blood velocity was significantly increased (91.8±11.1 vs 108.0±17.1 cm/sec, p<0.05) and the PAT augmentation index (AI) was significantly more negative (-8.8 ±9.4 vs -18.9±8.4%, p<0.05) after exercise. There were no significant correlations observed among the primary outcome measures obtained from each assessment technique. There was, however, a moderate correlation between pre-exercise vascular reactivity as assessed by SGP and change in blood velocity as assessed by FMD (r= 0.566, p= 0.035). Conclusions: The addition of an exercise stress to vascular function assessment may offer greater insight into the health of the vasculature. This initial study was undertaken to further evaluate the pre- to post-exercise responses obtained using three commonly employed vascular function assessment techniques in healthy individuals. Additional research as to the value of the addition of an exercise stress to vascular function assessment in individuals with traditional cardiovascular disease risk factors or known cardiovascular disease is warranted.