Endotoxin (lipopolysaccharide, LPS), is a constituent of the outer membrane of Gram-negative bacteria, activates macrophages to release cytokines that can cause local or systemic inflammatory responses. Plasma leakage and polymorphonuclear leukocyte infiltration are characteristic features of inflammation. This study examined the effect of LPS to induce subcutaneous inflammatory lesions, including time course of changes in plasma extravasation and level of leukocyte influx into the tissue interstitium. To investigate LPS-induced plasma leakage in the skin, LPS (500 £gg/site) was administered by subcutaneous injection in the hindpaw skin. India ink (1 ml/kg) was used as tracer dye to measure the area density of ink-labeled leaky blood vessels. Our results showed that the postcapillary venules were leaking immediately at five minutes after LPS. The area density of India ink-labeled leaky vessels was 33.9 %¡Ó5.6 % (n=5) after the administration of LPS. The magnitude of plasma leakage was 2 times as the value of saline control (16.6 %¡Ó1.8 %, n=5). Plasma leakage peaked at 30 min (42.5 %¡Ó2.5 %, n=11) after LPS. Staining of the microvasculature by silver nitrate showed endothelial gap formation in venules and indicated the positive relevance to plasma leakage. Leukocytes (neutrophils and eosinophils) in hindpaw skin whole mounts were stained by a histochemical reaction for myeloperoxidase and the numbers of leukocytes quantified. LPS caused a severe response in leukocyte adhesion and accumulation. The number of leukocytes after LPS was 5 times as the number after saline. It is concluded that local injection of LPS in the skin caused formation of endothelial gaps and leukocyte infiltration that resulted in an increase in local vascular permeability.

This research assessed the role of Hatha yoga in the modulation of vascular health. In study one, Hatha yoga practitioners were compared to sedentary controls to whom they were matched for age and body mass index. Practitioners of Hatha yoga were no different from sedentary individuals in terms of arterial stiffness or vascular endothelial function. Yoga practitioners possessed lower HbA1c (P < 0.05) levels and lower pulse pressure (P < 0.05) than their sedentary counterparts. Practitioners of Hatha yoga had lower body fat percentages, but this observed trend did not reach statistical significance (P = 0.052). In study two, a 12-week Hatha yoga intervention resulted in reductions in HbA1c levels (P < 0.05) and total cholesterol (P < 0.05) in previously sedentary adults. No changes were observed in carotid artery compliance or brachial artery flow-mediated dilation as a result of the intervention. In study three, obese and lean, apparently healthy adults completed an 8-week Bikram yoga intervention. Reductions in total- and LDL-cholesterol were observed in the lean subjects (P < 0.05), with no changes in lipid profiles in the obese group. The homeostasis model assessment of insulin resistance (HOMA-IR) decreased in the lean subjects, but this trend did not attain statistical significance (P = 0.06). Although an observed trend was shown at 60 minutes during the oral glucose tolerance test (P = 0.07), glucose tolerance remained unchanged in the obese subjects. Brachial artery flow-mediated improved by approximately 2% in the obese subjects, but this observed change did reach statistical significance (P = 0.10). Flexibility increased in both groups as a result of the Bikram yoga intervention. Therefore Hatha yoga improved lipid profiles and glycemic control in sedentary adults, but no effects on vascular health were demonstrated. / text

The homeobox transcription factor PROX1 is the mammalian ortholog of the Drosophila gene Prospero. Expression of PROX1 in a subset of venous endothelial cells changes their fate to lymphatic endothelial cells (LEC). PROX1 is required for lymphatic development as Prox1 null mice lack all lymphatic vasculature. PROX1 has been shown to have cell-type dependent roles in regulating the cell cycle. We hypothesize that PROX1 functions as a key cell cycle regulator in LECs and promotes their cell cycle progression. In this study, immunocytochemistry, western blotting and luciferase assays were used to characterize PROX1 mediated activation of the mouse Ccne1 promoter. Following deletion of the Prospero 1 domain (PD1∆), the resulting PROX1 protein is localized to both the nucleus and the cytoplasm. We have determined that PROX1 requires both E2F binding sites located in the Ccne1 promoter to activate transcription of the gene. We observed that siRNA knockdown of Prox1 reduced CYCLIN E1 protein levels as well as decreased cellular proliferation in LECs. In contrast, overexpression of a version of PROX1 in which the homeodomain and Prospero domain 2 (HDPD2Δ) were deleted increased CYCLIN E1 protein levels in human umbilical vein endothelial cells (HUVEC), but resulted in the arrest of cells in the G1 phase. We have also established that PROX1 is phosphorylated in primary human LECs. We have shown a role for the PD1 domain in mediating PROX1 subcellular localization and we have observed that the expression of the HDPD2Δ version of PROX1 blocks proliferation in HUVECs. We are the first to demonstrate a role for PROX1 as a transcriptional co-activator and to establish that PROX1 is phosphorylated in LECs.

An increase in blood flow associated shear stress results in an endothelial dependent increase in vessel diameter (flow mediated vasodilation (FMD)). Assessment of FMD can provide an index of endothelial function. The stimulus profiles that have been used to investigate FMD in human conduit arteries fall into two categories: reactive hyperemia and sustained stimuli. Stimulus-response specificity proposes that the stimulus characteristics (e.g. magnitude, pattern) are essential determinants of the response characteristics (e.g. magnitude, mechanisms). Purpose: To investigate four specific aspects of FMD stimulus-response specificity: 1) The relative importance of the peak vs. the duration of reactive hyperemia in determining FMD response magnitude. 2) The nitric oxide (NO) dependence of FMD following different durations of reactive hyperemia. 3) The impact of sustained shear stress stimulus magnitude on FMD response dynamics and magnitude. 4) FMD dynamics and magnitude in response to steady vs. oscillatory shear stress evoked passively or via exercise. Methods: Doppler ultrasound was applied to the brachial or radial artery to measure blood flow velocity. Vessel diameter was measured with automated edge detection software. Shear rate, an estimate of shear stress was calculated as the blood flow velocity/vessel diameter. Results: 1) The duration of reactive hyperemia is an important determinant of peak FMD magnitude while the independent contribution of the peak shear to FMD is minimal. 2) NO is not obligatory to FMD following either a five or a ten minute duration occlusion. 3) FMD in response to a sustained stimulus is characterized by a generally bi-phasic response with a fast first phase followed by a slower final phase. 4) The endothelium transduces the mean shear stress when it is exposed passive or handgrip exercise induced oscillations in shear stress. Conclusions: The results indicate that future reactive hyperemia studies must account for the stimulus duration when interpreting FMD results. Further, they demonstrate that the role of NO in FMD is unclear and caution against oversimplified conceptual models of FMD mechanisms. FMD in response to sustained stimuli provides information distinct from reactive hyperemia investigations and exercise may provide a valuable stimulus creation technique. / Thesis (Ph.D, Kinesiology & Health Studies) -- Queen's University, 2007-08-16 18:11:18.941

Flow-mediated dilation (FMD) can be used to assess the risk of atherosclerosis; however, an inverse relationship between vessel size and FMD has been identified using reactive hyperemia (RH) to create a shear stress (SS) stimulus in human conduit arteries. RH creates a transient and uncontrolled SS stimulus that is inversely related to baseline arterial diameter. It is therefore unclear whether differences in FMD between groups with non-uniform artery sizes are indicative of differences in vascular health or due to the creation of a greater SS stimulus in smaller vessels. Unlike RH, exercise can effectively create sustained and controlled increases in conduit artery SS. The purpose of this study was to compare the FMD responses of two differently sized upper limb arteries (brachial (BA) and radial artery (RA)) to matched graded levels of SS. Using exercise, three distinct sustained shear rate stimuli were created ((SR)=blood flow velocity/vessel diameter; estimate of SS) in the RA and BA. Artery diameter and mean blood flow velocity were assessed with echo and Doppler ultrasound respectively in 15 healthy male subjects (19-25yrs). Data are means ±SE. Subjects performed 6-min each of adductor pollicis and handgrip exercise to increase SR in the RA and BA respectively. Exercise intensity was modulated to achieve uniformity of SR between the RA and BA at three SR targets (40s-1, 60s-1, 80s-1). Three distinct SR levels were successfully created (steady state exercise: 39.8±0.6s-1, 57.3±0.7s-1, 72.4±1.2s-1; p<0.001 between SR levels). The %FMD at the end of exercise was greater in the RA vs. BA (SR40 RA: 5.4±0.8%, BA: 1.0±0.2%; SR60 RA: 9.8±1.0%, BA: 2.5±0.5%; SR80 RA: 15.7±1.5%, BA: 5.4±0.7%; p<0.001). The mean slope of the within-subject FMD-SR dose-response regression lines was significantly greater in the RA (RA: 0.33±0.04, BA: 0.13±0.02; p<0.001) and a strong within-subjects relationship between FMD and SR was observed in both arteries (RA r2: 0.92±0.02; BA r2: 0.90±0.03). These findings suggest that the response to SS is not uniform across differently sized vessels, which is in agreement with previous RH studies. Future research is required to investigate the potential mechanisms that mediate the functional differences observed between differently sized vessels. / Thesis (Master, Kinesiology & Health Studies) -- Queen's University, 2010-09-22 11:01:26.028

Shear stress is the frictional force exerted on the vascular wall by blood flowing through an artery. It is a major regulator of endothelial cell function, which is essential for vasoprotection and local regulation of vascular tone. Using handgrip exercise (HGEX) to increase shear stress is an increasingly popular method for assessing brachial artery (BA) endothelial cell function via flow-mediated dilation (FMD, dilation which increases with improved endothelial function). However, different exercise duty-cycles (ratio of handgrip relaxation to contraction in seconds) produce different patterns of BA shear stress with different anterograde and retrograde flow magnitudes. PURPOSE: To determine the impact of HGEX duty-cycle on BA %FMD while maintaining a constant mean shear stress. METHODS: N=16 healthy males. BA diameter (BAD) and blood velocity (BV) were assessed via echo and Doppler ultrasound. Shear stress was estimated as shear rate (SR=BV/BAD) and reported as mean SR during the last minute of baseline (target 10 s 1) and each minute of HGEX (75 s-1). Subjects performed 3 six minute HGEX trials on each of 2 separate days (like trials averaged). Each trial was one of 3 randomly ordered HGEX duty-cycles (1:1, 3:1, 5:1). %FMD was calculated as the increase in BAD from baseline to the end of HGEX and at each minute (subset N=10) during HGEX. RESULTS: Data are means ± SD. As intended, mean SR was similar between duty-cycles (main effect, p=0.835), despite significant differences in anterograde and retrograde SR (P<0.001). There was no impact of duty cycle on blood pressure (p=0.188) or heart rate (p=0.131) responses. End exercise %FMD (4.0 ± 1.3%, 4.1 ± 2.2%, 4.2 ± 1.4%, p=0.860) and minute by minute %FMD (main effect p=0.939; interaction, p=0.545) were also not different between duty-cycles. CONCLUSION: Distinct HGEX duty-cycles create markedly different shear stress patterns in the BA. However, duty cycle had no impact on %FMD magnitude suggesting that mean shear stress is the most important stimulus for FMD in the BA. Using a 5:1 duty cycle may yield the best vessel image and diameter measurement quality due to the long period of arm stability between contractions. / Thesis (Master, Kinesiology & Health Studies) -- Queen's University, 2012-09-06 14:31:50.467

CD146 is a cell adhesion molecule that has been shown to regulate cell adhesion, migration and proliferation of different cell types. It is highly expressed in blood endothelial cells (BECs), but is only lowly expressed in lymphatic endothelial cells (LECs). The PROX1 homeobox gene is a master regulator of lymphangiogenesis and its expression is necessary and sufficient to drive venous endothelial cells into a LEC phenotype. The highly permeable nature of the lymphatic vessels may partially derive from PROX1 mediated repression of CD146 transcription. We hypothesize that PROX1 promotes lymphatic differentiation by repressing CD146 transcription. In gain of function studies, Human Umbilical Vein Endothelial Cells (HUVECs) were infected with adenoviruses encoding EGFP, wild type PROX1 (AdProx1) or a Homeo-Prospero domain deleted version of PROX1 (AdHDPD), which cannot bind DNA. In order to knockdown PROX1, LECs were transfected with PROX1 specific siRNA. When compared to EGFP infected HUVECs, AdProx1 infected HUVECs had decreased CD146 expression both at protein and mRNA levels. In contrast, AdHDPD infected HUVECs had increased levels of CD146 expression. In support of a role for PROX1 in repressing CD146, PROX1 siRNA transfected LECs express higher levels of CD146 as compared to mock transfected LECs or LECs transfected with control siRNA. Based on these results, we predict that CD146 expression is kept at basal levels by an unknown repressor bound to the CD146 promoter. By interacting with this unknown repressor, PROX1 further represses CD146 expression. On the other hand, the DNA binding-deficient ΔHDPD version of PROX1 binds the unknown repressor and sequesters it from the CD146 promoter, thereby relieving the repression of CD146 expression in ECs. Different levels of CD146 expression between BECs and LECs might reflect the structural and functional differences between blood and lymphatic vessels. Since CD146 plays a critical role in EC adhesion, regulation of CD146 expression in ECs might be one of the key factors regulating vessel permeability.

No description available.

The homeobox transcription factor PROX1 is the mammalian ortholog of the Drosophila gene Prospero. Expression of PROX1 in a subset of venous endothelial cells changes their fate to lymphatic endothelial cells (LEC). PROX1 is required for lymphatic development as Prox1 null mice lack all lymphatic vasculature. PROX1 has been shown to have cell-type dependent roles in regulating the cell cycle. We hypothesize that PROX1 functions as a key cell cycle regulator in LECs and promotes their cell cycle progression. In this study, immunocytochemistry, western blotting and luciferase assays were used to characterize PROX1 mediated activation of the mouse Ccne1 promoter. Following deletion of the Prospero 1 domain (PD1∆), the resulting PROX1 protein is localized to both the nucleus and the cytoplasm. We have determined that PROX1 requires both E2F binding sites located in the Ccne1 promoter to activate transcription of the gene. We observed that siRNA knockdown of Prox1 reduced CYCLIN E1 protein levels as well as decreased cellular proliferation in LECs. In contrast, overexpression of a version of PROX1 in which the homeodomain and Prospero domain 2 (HDPD2Δ) were deleted increased CYCLIN E1 protein levels in human umbilical vein endothelial cells (HUVEC), but resulted in the arrest of cells in the G1 phase. We have also established that PROX1 is phosphorylated in primary human LECs. We have shown a role for the PD1 domain in mediating PROX1 subcellular localization and we have observed that the expression of the HDPD2Δ version of PROX1 blocks proliferation in HUVECs. We are the first to demonstrate a role for PROX1 as a transcriptional co-activator and to establish that PROX1 is phosphorylated in LECs.

CD146 is a cell adhesion molecule that has been shown to regulate cell adhesion, migration and proliferation of different cell types. It is highly expressed in blood endothelial cells (BECs), but is only lowly expressed in lymphatic endothelial cells (LECs). The PROX1 homeobox gene is a master regulator of lymphangiogenesis and its expression is necessary and sufficient to drive venous endothelial cells into a LEC phenotype. The highly permeable nature of the lymphatic vessels may partially derive from PROX1 mediated repression of CD146 transcription. We hypothesize that PROX1 promotes lymphatic differentiation by repressing CD146 transcription. In gain of function studies, Human Umbilical Vein Endothelial Cells (HUVECs) were infected with adenoviruses encoding EGFP, wild type PROX1 (AdProx1) or a Homeo-Prospero domain deleted version of PROX1 (AdHDPD), which cannot bind DNA. In order to knockdown PROX1, LECs were transfected with PROX1 specific siRNA. When compared to EGFP infected HUVECs, AdProx1 infected HUVECs had decreased CD146 expression both at protein and mRNA levels. In contrast, AdHDPD infected HUVECs had increased levels of CD146 expression. In support of a role for PROX1 in repressing CD146, PROX1 siRNA transfected LECs express higher levels of CD146 as compared to mock transfected LECs or LECs transfected with control siRNA. Based on these results, we predict that CD146 expression is kept at basal levels by an unknown repressor bound to the CD146 promoter. By interacting with this unknown repressor, PROX1 further represses CD146 expression. On the other hand, the DNA binding-deficient ΔHDPD version of PROX1 binds the unknown repressor and sequesters it from the CD146 promoter, thereby relieving the repression of CD146 expression in ECs. Different levels of CD146 expression between BECs and LECs might reflect the structural and functional differences between blood and lymphatic vessels. Since CD146 plays a critical role in EC adhesion, regulation of CD146 expression in ECs might be one of the key factors regulating vessel permeability.