Master of Science / Department of Kinesiology / Carl Ade / Adjuvant systemic chemotherapy for the treatment of certain cancers, particularly breast and lymphoma, adversely impacts cardiovascular health. However, the extent to which it impairs endothelial function is not well understood. Therefore, the purpose of this study was to determine if microvascular and macrovascular endothelial-dependent vasoreactivity is attenuated in breast cancer and lymphoma patients currently being treated with chemotherapy compared to healthy counterparts. With laser Doppler imaging, cutaneous microvascular function was evaluated via changes in cutaneous vascular conductance (CVC) in response to iontophoresis of acetylcholine (ACh). Endothelium-dependent flow-mediated dilation (FMD) was evaluated in the brachial artery via ultrasonography. CVC responses to iontophoresis of ACh in the cutaneous microcirculation was significantly lower in cancer patients than in control subjects (cancer (n=7): 959.9 ± 187.3%; control (n=7): 1556.8 ± 222.2%; P = 0.03). Furthermore, FMD was significantly lower in cancer patients than in control subjects (cancer: 2.2 ± 0.6%; control: 6.6 ± 1.4%; P = 0.006). These data provide evidence of microvascular and macrovascular dysfunction in breast cancer and lymphoma patients currently undergoing adjuvant chemotherapy, which may contribute to the increased long-term risk of cardiovascular disease morbidity and mortality in those treated for cancer.

Background: Vascular endothelial dysfunction plays a major role in the pathogenesis of atherosclerosis. As such, the study of endothelial cells has sought to identify causal pathways and novel therapeutic approaches to promote vascular repair. Induced pluripotent stem (iPS) cell technology may be a particularly useful tool, and could be used to derive endothelial cells and their progenitors from individuals with endothelial dysfunction to explore these pathways and develop novel strategies for vascular regeneration. Whilst iPS cells are conventionally obtained from the reprogramming of dermal fibroblasts, it was hypothesised that endothelial cells could also be reprogrammed, and that these pluripotent cells would have enhanced capacity for endothelial differentiation and vascular regeneration. Objectives: To generate iPS cells from human fibroblasts and endothelial cells and to assess their potential for endothelial differentiation and vascular regeneration. Methods and Results: A) Reprogramming: Dermal fibroblasts and endothelial outgrowth cells from blood were obtained from healthy donors (n=5) and transfected with episomal vectors containing six reprogramming factors: Sox2, Klf4, Oct3/4, L-Myc, Lin28 and Shp53. Successfully reprogrammed fibroblast-derived iPS (fiPS) and endothelial cell-derived iPS (eiPS) arose as colonies, and were isolated and expanded. Reprogrammed cells expressed pluripotency markers SSEA3, SSEA4, TRA 1 60, Oct3/4 and NANOG, and developed into all three germ layers following embryoid body formation. B) Endothelial differentiation: iPS and ES cell lines were aggregated into embryoid bodies in stem cell growth media containing mesoderminducing cytokines. Embryoid bodies were then disaggregated and cultured in endothelial medium supplemented with VEGF. After seven days, a population of CD31+ cells was isolated and further cultured. Mature endothelial cell antigen expression was confirmed by flow cytometry. CD31+ cells were similar to mature endothelial cells in functional assays of proliferation, migration, nitric oxide production and angiogenesis. C) Comparison of fiPS versus eiPS: eiPS differentiated into endothelial cells with greater efficiency than fiPS (21±3% versus 3±2%, P < 0.05). fiPS-derived endothelial cells and eiPS-derived endothelial cells expressed similar levels of endothelial markers CD146, CD31, VEFGR2 and CD34 compared to control endothelial cells. When grown on Matrigel, they formed tubule-like structures with a similar number of vessel connections. In vivo, endothelial cells derived from fiPS and eiPS increased neovasculogenesis in a nude mouse model: vessel density was increased after implantation of endothelial cells from fiPS and eiPS by 3.50 vessel counts (P≤0.001) and 3.47 vessel counts (P≤0.001) respectively, when compared to controls. By comparison control endothelial cells did not increase vessel density compared to control (P > 0.05). Conclusions: Endothelial cells can be isolated from blood and reprogrammed to form pluripotent stem cells with enhanced capacity to differentiate into endothelial cells than those derived from dermal fibroblasts. Endothelial cells derived from both sources promote angiogenesis in vivo, and have major potential for therapeutic applications in vascular regeneration.

The aortic valve (AV) functions in arguably the most demanding mechanical environment in the body. The AV experiences fluid shear stress, cyclic pressure and mechanical strain in vivo. Recent evidence has shown the progression of degenerative aortic valve disease (AVD) to be an active cellular mediated process, altering the conception of the AV as a passive tissue. AVD has shown a strong correlation with altered hemodynamics and tissue mechanics. Aortic valve endothelial cells (AVECs) line the fibrosa (aortic facing) and ventricularis (left ventricle facing) surfaces of the valve. AVECs sense and respond to circulating stimuli in the blood stream while maintaining a non-thrombogenic layer. AVEC activation has been implicated in the initiation and progression of AVD, but the role of cyclic strain has yet to be elucidated. The hypothesis of this dissertation is that altered mechanical forces have a causal relationship with aortic valvular endothelial cell activation. To test this hypothesis 1) the role of in vitro cyclic strain in regulating expression of pro-inflammatory adhesion molecule was elucidated 2) cyclic strain-dependent activation of side-specific aortic valve endothelial cells was investigated 3) a novel stretch bioreactor was developed to dramatically increase the ability to correlate valvular endothelium response to physiologically relevant applied planar biaxial loads. The results from this study further the field of heart valve mechanobiology by correlating AVEC physiological and pathophysiological function to cellular and tissue level strain. Elucidating the AVEC response to an altered mechanical environment may result in novel clinical diagnostic and therapeutic approaches to the initiation and progression of degenerative AVD. Furthermore, a cardiovascular health outreach program, Bulldogs for Heart Health, has been designed and implemented to combat the startling rise in childhood obesity in the state of Mississippi. It is the hope that these results, novel methods, and outreach initiatives developed will significantly impact the study of the mechanobiology of the aortic valve endothelial cell and potential treatment and prevention of cardiovascular disease.

Vascular hyperpermeability is a common characteristic among many tumour types, especially those that grow in ascites form. With these, the exudate that flows out of the circulation collects as ascites fluid in the cavities within which these tumours are growing. In the past, this hyperpermeability has been attributed to the production of vascular permeability factor (VPF) by tumours. VPF has been found to bind to endothelial cells and lead to an increased vascular permeability. In the present study, the role of polymorphonuclear leukocytes (neutrophils) in tumour vascular hyperpermeability was investigated. Hey-3 tumour cells were grown into masses on the chick embryo chorioallantoic membrane (CAM). Interstitial neutrophilia was found to be a common feature at the tumour-host interface. Horseradish peroxidase was injected into the circulation and allowed to perfuse for five minutes. The density of labelled vesicles within the endothelial cell cytoplasm was calculated to be 0.99 +/-0.28 vesicles/μm². This vesicular density was comparable to that of N-formyl-methionine-leucine-phenylalanine (a chemotactic peptide for neutrophils)-treated CAM (1.04 +/-0.09 vesicles/μm²), but very different from control CAM (0.51 +/- 0.09 vesicles/μm²). In order to rule out any immune response to foreign cells, immune hepatocyte masses were grown on the CAM and vesicular density was calculated to be 0.54 +/-0.03 vesicles/μm². Through chemotaxis assays with the Boyden chamber, it was observed that Hey-3 tumour cells in culture were producing a chemotactic factor that is an attractant for human neutrophils. Once in the area, neutrophils do possess the potential to increase vascular permeability. Thus, neutrophils play a role in vascular endothelial hyperpermeability in tumours. / Thesis / Master of Science (MS)

Vascular endothelial cell activation and apoptosis (programmed cell death) are critical in inflammation and angiogenesis (the formation of new blood vessels). Tumor necrosis factor (TNF) is a pro-inflammatory cytokine known for its ability to induce endothelial cell activation and apoptosis. However, the ability of two death ligands in the TNF superfamily: TRAIL (TNF-Related Apoptosis-Inducing Ligand) and Fas ligand (FasL), to activate vascular endothelium is less well defined, and forms the basis of this work. We find that in the human endothelial cell line EA.hy926, TRAIL induces endothelial cell activation (activation of the transcription factor NF-?B with increased expression of the adhesion protein ICAM-1 and adhesion of human neutrophils) when it concurrently induces apoptosis. In addition, angiogenesis is implicated in diseases of the central nervous system, and its modulation represents an attractive therapeutic strategy. We investigated the modulatory potential of the two endogenous molecules TRAIL and FasL as well as an exogenous molecule resveratrol, a phytochemical present in red wine, in angiogenesis. We modeled cerebral angiogenesis with the human brain endothelial cell line hCMEC/D3 and primary bovine brain endothelial cells. Resveratrol inhibited several parameters of angiogenesis (proliferation, migration and tube formation) in human umbilical vein endothelial cells, however, neither TRAIL nor FasL had an effect on this model. By contrast, in hCMEC/D3 cells both resveratrol and TRAIL inhibited all parameters while FasL had minimal effects. Resveratrol did not induce apoptosis in hCMEC/D3 but arrested cell cycle progression to G2/M phase and inhibited phosphorylation of Akt/PKB, a key cell survival protein kinase. This leads to a reduction in cell growth, endothelial migration and tube formation, hence, inhibition of in vitro angiogenesis. TRAIL induced anti-angiogenic effects in hCMEC/D3 due to apoptosis. The data suggests that TRAIL primarily influences angiogenesis through induction of vascular endothelial apoptosis while resveratrol induces cell cycle arrest, both of which may lead to vessel regression. These are the first studies to report the modulation of different aspects of endothelial cell activation by TRAIL and resveratrol in several endothelial cell culture models, with a particular focus on the central nervous system.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

Indiana University-Purdue University Indianapolis (IUPUI) / Introduction: Regenerative endodontics (RE) is a treatment alternative for the infected immature tooth to establish an environment in the canal that enables continued root development and the growth of pulp or pulp-like tissue within the canal. A scaffold created in the canal encourages the formation of vital tissue. The porcine sub-intestinal-submucosa (SIS) membrane, Dynamatrix®, has the potential to serve as an endodontic scaffold. Research at Indiana University School of Dentistry (IUSD) has shown that Dynamatrix® can support the growth of human dental pulp stem cells (HDPSC) and human pulp fibroblasts (HPF). Positive angiogenic cytokine profiles were seen after these cells were seeded on Dynamatrix®. Endothelial cells play an important role in the formation of blood vessels and are a source of angiogenic cytokines. Exposure of these cells to DynaMatrix® may result in a positive angiogenic profile for both cytokines and matrix metalloproteinases (MMPs). Objective: The aim of this in-vitro study was to investigate if the exposure of human endothelial cells to the DynaMatrix® membrane would result in differences in the expression of cytokines and MMPs that play roles in angiogenesis. Materials and Methods: Human endothelial cells (HUVECs) were obtained from American Type Culture Collection (ATTC, Manassas, VA) and used in this study. Groups were established as follows: (a) Group 1: HUVECs seeded in culture media only, (b) Group 2: DynaMatrix® membrane incubated alone in the serum-media without any cells, and (c) Group 3: HUVECs seeded on DynaMatrix® membranes. After 72 hours of incubation, the conditioned media were collected and analyzed for the expression of 20 angiogenic cytokines and MMPs utilizing cytokine and MMP protein arrays. The density of each cytokine and MMP expressed was measured, averaged, and statistically analyzed by ANOVA. Results: Exposure of human umbilical vein endothelial cells (HUVECs) to the DynaMatrix® membrane resulted in a positive angiogenic profile for both cytokines and MMPs. Conclusion: This work furthers the evidence for the potential of DynaMatrix® to serve as a more predictable scaffold in RE.

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