The majority of cancer related deaths occur due to the invasive growth of metastatic lesions. In the early stages of metastasis, circulating cell interact with the endothelial cells to establish at a distant site. In inflammation endothelial activation results in induction of adhesion molecules on the endothelium that participate in the homing of leukocytes. Because of the interactions of metastatic cells with the endothelium, the question was whether some of the characteristic molecules of endothelial activation were induced during metastasis. In vivo pulmonary metastatic models were used to characterize the expression profile of endothelial activation. Immunohistochemistry identified VCAM-1 to be induced on the pulmonary endothelium following tumour cell arrest. VCAM-1 upregulation was not observed prior to tumour cells arrest or within the first hours. In contrast, tumour cell arrest appeared to be required for endothelial activation, arguing against a mechanism analogous to leukocyte homing. The upregulation of VCAM-1 upon tumour cell arrest corresponded with the initiation of platelet clot formation around the tumour cell and recruitment of leukocytes to the site, both previously shown to be essential for metastasis. Disruption of both phenomena, either through genetic or pharmacological manipulation, demonstrated that in contrast to the recruited leukocytes, platelets were involved in inducing endothelial activation. Another protein investigated was VAP-1. In contrast to VCAM-1, central to VAP-1 adhesive function is its enzymatic activity. Blocking the functions of either molecule highlighted their role in facilitating the recruitment of the leukocyte population to the tumour cell. Disruption of which led to a significant attenuation of metastasis. While VCAM-1 and VAP-1 function appears critical in the early steps of metastasis, their inhibition had no effect at later stages of pulmonary colonization.

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.

Tumors are composed not only of malignant cells, but also of various types of normal cells, including vascular cells and infiltrating immune cells, which drive tumor development and progression. The tumor vasculature is abnormal and dysfunctional due to sustained tumor angiogenesis driven by high levels of pro-angiogenic factors. Proteins differentially expressed in tumor vessels affect vascular function and the tumor microenvironment and may serve as targets for therapy. The tumor is also a site of sustained chronic inflammation. The recruitment and activation of inflammatory cells significantly influence tumor progression and regression. Targeting molecules regulating tumor angiogenesis and inflammation in the tumor microenvironment is therefore a promising strategy for the treatment of cancer. This thesis is aiming to understand and investigate the molecular regulation of these two processes in tumors. αB-crystallin is a heat shock protein previously proposed as a target for cancer therapy due to its role in increasing survival of tumor cells and enhancing tumor angiogenesis. In this thesis, we demonstrate a novel role of αB-crystallin in limiting expansion of CD11b+Gr1+ immature myeloid cells in pathological conditions, including tumor development. In addition, we show that αB-crystallin regulates leukocyte recruitment by promoting expression of adhesion molecules ICAM-1, VCAM-1 and E-selectin during TNF-α-induced endothelial activation. Therefore, targeting of αB-crystallin may influence tumor inflammation by regulating immature myeloid cell expansion and leukocyte recruitment. Abnormal, dysfunctional vessels are characteristic of glioblastomas, which are aggressive malignant brain tumors. We have identified the orphan G-protein coupled receptor ELTD1 as highly expressed in glioblastoma vessel and investigated its role in tumor angiogenesis. Interestingly, deficiency of ELTD1 was associated with increased growth of orthotopic GL261 glioma and T241 fibrosarcoma, but did not affect vessel density in any model. Further investigation is warranted to evaluate whether ELTD1 serves a suitable vascular target for glioblastoma treatment. Anti-angiogenic drugs targeting VEGF signaling is widely used in the clinic for various types of cancer. However, the influences of anti-angiogenic treatment on tumor inflammation have not been thoroughly investigated. We demonstrate that VEGF inhibits TNF-α-induced endothelial activation by repressing NF-κB activation and expression of chemokines involved in T-cell recruitment. Sunitinib, a small molecule kinase inhibitor targeting VEGF/VEGFR2 signaling increased expression of chemokines CXCL10, CXCL11, and enhanced T-lymphocyte infiltration into tumors. Our study suggests that anti-angiogenic therapy may improve immunotherapy by enhancing endothelial activation and facilitating immune cell infiltration into tumors.

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.

This study investigated the effects of daily consumption of Thompson seedless raisins on markers of inflammation, oxidative stress and endothelial activation in response to an acute high-fat meal in obese individuals. Seventeen overweight men and women consumed raisins or placebo (264 kcal/d) for 14 d in a randomized cross-over design while following a low-flavonoid, weight-maintenance diet. Four high-fat (53% fat) meals were consumed with the respective treatment pre and post interventions. Measures at fasting, and 2, 3 and 4 hours postprandial included markers of oxidative stress (urinary 8-isoPGF2α; serum Oxygen Radical Absorbance Capacity, ORAC), inflammation (serum C-reactive protein, CRP; interleukin-6, IL-6), endothelial function (serum soluble intercellular adhesion molecule-1, sICAM-1; soluble vascular adhesion molecule-1, sVCAM-1), and metabolic measures (free fatty acids (FFA), triacylglycerol (TAC), glucose, insulin). Urinary 8-isoPGF2α decreased 22% and ORAC increased 3% pre to post interventions combined. Postprandial metabolic responses differed by gender, males surpassed females for several measures: FFA, triacylglycerol, glucose, and sVCAM-1. Neither the meals nor treatment with raisins had any noteworthy influence on fasted measures of inflammation or endothelial dysfunction. Acute high fat meal consumption did not result in evidence of inflammation or oxidative stress in these relatively healthy, overweight individuals. Providing all food in regular pattern reduced measures of oxidative stress. Gender influenced metabolic responses to meals; males had a greater postprandial response in metabolic measures than females. / Master of Science

Rheumatoid arthritis (RA) is a chronic disabling disease that is associated with a shortened life span. Cardiovascular disease (CVD) contributes to this increased mortality, and also to a great extent to the co-morbidity observed in patients with RA. This thesis aimed to investigate these issues further. The incidence of, and prognosis after an acute myocardial infarction (AMI) /or stroke in a cohort of RA patients was compared with that in the general population within the northern Sweden MONICA register. The standard incidence ratio (SIR) for AMI was 2.9 and for stroke 2.7 in RA patients compared with the general population (p&lt;0.05 for both). During the first 10 years following an event, RA patients had a higher overall case fatality (CF) compared with controls (HR for AMI=1.67, 95%CI [1.02, 2.71], HR for stroke=1.65, 95%CI [1.03, 2.66]). An elevated level of homocysteine is regarded to be a risk marker for CVD. The effects of treatment with B vitamins on the homocysteine level in patients with RA were studied in a consecutive cohort of patients with RA. Sixty-two patients with RA having a homocysteine level of 12 mol were randomized to receive either a placebo or a combination of the vitamins B6, B12 and folic acid. The patients were treated and evaluated in a double-blind manner over 12 months. The homocysteine level was found to be significantly decreased in the B-vitamin treated patients compared with the placebo group (p&lt;0.0001). To evaluate the progression of sub-clinical atherosclerosis in patients with very early RA compared with controls, all patients from the three most northern counties of Sweden newly diagnosed with RA and aged ≤60 years were consecutively recruited. Age and sex matched controls from the general population were also included. Intima media thickness (IMT) of the common carotid artery and endothelium dependent flow mediated dilation (ED-FMD) of the brachial artery were measured using ultrasonography. After 18 months the same measurements were undertaken in a sub-group of the patients with early RA and the relevant controls. There were no differences between patients with early RA and controls in terms of IMT or ED-FMD at inclusion into the study. However, after 18 months there was a significant increase in the IMT among the patients with early RA (p&lt;0.05); no such increase occurred in the control group. Biomarkers of endothelial activation that may reflect the early atherosclerosis that occurs in RA were also evaluated. At inclusion, both IMT and ED-FMD among the patients with early RA related significantly to several of the biomarkers of endothelial activation. Furthermore, markers of inflammation (e.g., DAS28) were significantly related to biomarkers of endothelial activation. In conclusion, RA patients had a higher incidence of CVD and a higher CF after a CV event. The increased homocysteine level among patients with RA was as easy to decrease as in the general population. At the time of diagnosis of RA there were no differences in atherosclerosis between patients and controls, however the patients with RA had a more rapid progression of atherosclerosis than the control subjects. Moreover, there were implications of endothelial activation already in patients with very early RA. Taken together, these results emphasize the necessity of optimizing the preventive, diagnostic and caring strategies for CVD in patients with RA.

A study of hyperglycemia and its effects on endothelial activation, macrophage recruitment, and atherosclerotic plaque development in mice. Hyperglycemic mice demonstrated greater VCAM but not ICAM expression along the endothelium, increased macrophage presence within the subendothelial space of these regions, and a greater volume of plaque in adulthood. / Cardiovascular disease is the leading cause of death in the world today. Atherosclerosis is the formation of plaque in the arteries and a major underlying cause of these fatalities. Type I and II diabetes are each strong independent risk factors for atherosclerosis. This study examines the effects of hyperglycemia on early atherosclerosis. Hyperglycemia did not promote atherosclerosis in the absence of hypercholesterolemia. Hyperglycemic mice demonstrated greater VCAM, but not ICAM, expression in regions of the endothelium susceptible to atherogenesis, prior to initiation of plaque development. Regions correlating to upregulation of VCAM exhibited a greater quantity of macrophages infiltrating the intima. This study suggests a unique and important role for VCAM in early atherosclerotic development and may explain the accelerated atherosclerotic plaque progression seen in hyperglycemic mice. This study also identifies VCAM as a potential target for the development of therapies to block or slow atherosclerotic plaque development in people with diabetes. / Thesis / Master of Science (MSc) / Cardiovascular disease is the leading cause of death in the world today. A major underlying cause of cardiovascular disease is atherosclerosis – a condition involving the thickening of the artery wall. Type I and II diabetes are each strong independent risk factors for atherosclerosis. The purpose of this study is to examine the effects of high blood glucose (hyperglycemia) on early events leading to atherosclerosis. This study found that hyperglycemia was not sufficient to promote atherosclerosis unless plasma cholesterol levels were also elevated. Hyperglycemia appeared to induce atherosclerosis by increasing the expression of factors responsible for recruiting white blood cells to the artery wall. This is consistent with the observation that hyperglycemic mice also had significantly more macrophages in the sites of plaque development. This study implicates one macrophage-recruitment factor in particular, vascular cell adhesion molecule (VCAM), as playing an important and unique role in the initiation of atherosclerosis by hyperglycemia. Therefore, VCAM is a possible target for the development of therapies to block or slow the development of atherosclerosis in individuals with diabetes.