Poly(methacrylic acid -co- methyl methacrylate) beads were previously shown to have a therapeutic effect on wound closure through the promotion of angiogenesis. However, it was unclear how this polymer elicited its beneficial properties. The goal of this thesis was to characterize the host response to MAA beads by identifying molecules of interest involved in MAA-mediated angiogenesis (in comparison to poly(methyl methacrylate) beads, PMMA). Using a model of diabetic wound healing and a macrophage-like cell line (dTHP-1), eight molecules of interest were identified in the host response to MAA beads. Gene and/or protein expression analysis showed that MAA beads increased the expression of Shh, IL-1β, IL-6, TNF-α and Spry2, but decreased the expression of CXCL10 and CXCL12, compared to PMMA and no beads. MAA beads also appeared to modulate the expression of OPN. In vivo, the global gene expression of OPN was increased in wounds treated with MAA beads, compared to PMMA and no beads. In contrast, dTHP-1 decreased OPN gene expression compared to PMMA and no beads, but expressed the same amount of secreted OPN, suggesting that the cells decreased the expression of the intracellular isoform of OPN. Interestingly, MAA beads had no effect on the expression of pro-angiogenic growth factors VEGF, bFGF and PDGF-B in vivo or in vitro, suggesting that MAA beads do not induce angiogenesis by simply increasing the expression of pro-angiogenic factors, but use more subtle mechanisms. It was hypothesized that these mechanisms may involve modulation of toll-like receptor signaling in macrophages interacting with the protein layer adsorbed on to MAA beads, in a manner distinct from PMMA beads and no beads. Taken together, the results suggest that MAA beads promote angiogenesis through increased expression of Shh, decreased expression of CXCL10 and modulation of the expression of OPN, but not through increased expression of typical pro-angiogenic growth factors. The resulting vessel-rich “alternative foreign body reaction” has exciting clinical implications as the polymer itself was found to exert a therapeutic effect in the absence of bioactive components or transplanted cells. Understanding the mechanism could lead to new applications for this material and others designed on similar principles.

Multiple sclerosis (MS) is an inflammatory autoimmune disease of the central nervous system. We hypothesize that angiogenesis results in new vessels which serve as a conduit for immune cell recruitment in MS, and contribute to inflammation through the pro-inflammatory properties of angiogenic regulators. This study is one of the first to explore regulation of angiogenesis in a murine model of MS (experimental autoimmune encephalomyelitis, EAE), and its potential as a therapeutic target. Angiogenesis was apparent 21 days following disease induction and correlated with clinical and pathologic scores. We documented alterations in the VEGF and Ang/Tie signaling pathways. Expression of VEGF increased at day 14 then reduced by day 21. At this time point, Ang-2 levels were elevated due to expression by infiltrating macrophages. Ang-1 was significantly reduced at day 14 and increased at day 21 due to expression by CD3+ T-cells. The same expression pattern was validated in inflammatory cells within human MS tissue. Vascular permeability increased at day 14 and returned to control levels at day 21. The volume of permeable tissue weakly correlated with angiogenesis. VEGF blockage with bevacizumab suppressed angiogenesis and reduced clinical scores and vascular permeability. Retention of CD4+ T-cells in peripheral lymph nodes and reduced T-cell proliferation was noted following treatment. Bevacizumab reduced mononuclear cell infiltration into spinal cord. Isolated CD4+ T-cells showed reduced expression of IL-17 and IFN-??. B20-4.1.1 (a monoclonal antibody against murine VEGF) reduced clinical scores and suppressed angiogenesis as did treatment with angiostatin (an inhibitor of endothelial cell proliferation). B20-4.1.1 reduced vascular permeability, induced retention of CD4+ T-cells in peripheral lymph nodes, and inhibited T-cell proliferation, while angiostatin had no effect. Isolated lymphoid cells from mice treated with both agents showed reduced secretion of IL-17, but B20-4.1.1 had no effect on Tcell proliferation or IL-17 secretion when combined with angiostatin. ?? We conclude that these angiogenesis inhibitors are effective in EAE and act by suppressing angiogenesis with a secondary effect on peripheral T-cell activation. To the extent that EAE replicates changes occurring in MS, we have demonstrated that modulation of angiogenesis may represent a promising strategy in the management of disease progression.

This thesis is an investigation of the use of thrombospondin-1 mimetic peptides for the treatment of epithelial ovarian cancer. The current standard of care for women diagnosed with ovarian cancer is surgical de-bulking followed by chemotherapeutics. Initially, this treatment regimen results in a reduction in the primary tumor, unfortunately chemoresistance and disease recurrence are problematic. Recent data has suggested a potential role for anti-angiogenic therapy for the treatment of various cancers. Therefore, the purpose of this study was to investigate the use of mimetics consisting of the anti-angiogenic domain of thrombospondin-1 (TSP-1) for the treatment of epithelial ovarian cancer (EOC) using a mouse model of the disease. The peptides were applied at various stages of tumor progression and a significant reduction in tumor size following treatment was observed. We found that not only were the peptides capable of slowing down tumor progression but they also played a role in reducing the size of established tumors. Treatment with TSP-1 mimetics also resulted in a significant reduction in secondary lesions and ascites fluid in the peritoneal cavity of animals. A significant increase in disease-free survival was also identified following long-term treatment with the peptide. Various histological studies revealed that the anti-angiogenic peptide was in fact inducing apoptosis of the endothelial cells and also re-organizing the vasculature. To determine whether this resulted in increased blood vessel profusion we applied standard chemotherapeutics in combination with TSP-1 mimetics. Experiments with radiolabelled and fluorescent chemotherapeutics demonstrated that pre-treating with TSP-1 mimetics allowed the vasculature to become normalized and resulted in an increased uptake of chemotherapeutics. Lastly, we investigated the mechanism of action of anti-angiogenic peptides. Most of the anti-tumor effects appeared to be due to the apoptotic effects of TSP-1 mimetics on the vasculature. A direct apoptotic effect on epithelial cells also was observed; however, it is uncertain how much of a role this plays. In conclusion, this study was important for identifying TSP-1 mimetic peptides as a potential therapeutic treatment for women suffering from EOC.

Low Dose Metronomic (LDM) chemotherapy, combined with VEGF pathway inhibitors, is a highly effective strategy to coordinately inhibit angiogenesis and tumor growth. We have tested the efficacies of daily oral LDM topotecan alone and in combination with pazopanib, in three pediatric extracranial solid tumors mouse models. We also investigated the effect of prolonged combination therapy with the combination on tumor behavior in a neuroblastoma mouse xenograft model. In-vitro dose-response study of topotecan and pazopanib was conducted on several cell lines. In-vivo antitumor efficacies of drugs, as single agents and combination, were tested in immunodeficient mice models. For studying the mechanisms of resistance to our therapy, a time-response study (28, 56 and 80 days) was conducted in SK-N-BE(2) xenografts model, treated in same way as earlier. In vitro, topotecan caused a dose-dependent decrease in viabilities of all cell lines, while pazopanib did not. In vivo, the combination of topotecan and pazopanib demonstrated significant anti-tumor activity compared to the respective single agents in all models. Reductions in the levels of viable Circulating Endothelial Progenitors and/or Circulating Endothelial Cells and tumor microvessel density were correlated with tumor response and therefore confirmed the antiangiogenic activity of the regimens. However, the combination also caused significantly higher myelotoxicity than single agents. Pharmacokinetic study did not reveal any interaction between the two co-administered drugs. In the time-response study, we found that only combination treated animals survived till 80 days. However, tumors in these animals started growing gradually after 50 days. Unlike single agents, all three durations of combination treatment significantly lowered tumor microvessel densities, compared to the control. However, tumors treated with the combination for 56 and 80 days had higher pericyte coverage. The combination increased the hypoxia, angiogenic expression and proliferative index and caused metabolic reprogramming of tumor cells. We conclude that the combination of LDM topotecan and pazopanib has superior efficacy than either single agents, which is attributed to superior antiangiogenic activity. However, prolonged treatment with the combination can have additive myelotoxicity and may encounter adaptive resistance associated with metabolic reprogramming and increased proliferation of the tumor cells.

Prospective epidemiological studies indicate that regular exercise during the year prior to conception reduces preeclampsia risk, whereas exercise during affects pregnancy reduces preeclampsia risk only at specific dosages, or in specific subpopulations. The risk of severe preeclampsia is increased among women who exercise for more than 270 minutes/week in early pregnancy. Physiology studies are needed to identify mechanisms through which regular exercise may influence preeclampsia risk. This dissertation examined the effects of pregnancy (30-36 weeks gestation), and regular exercise participation, on two important pathophysiological features of preeclampsia; circulating anti-angiogenic markers, represented by soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng), and endothelial dysfunction. The results demonstrate that regularly exercising, pregnant non-smoking women have higher levels of serum placental growth factor (PlGF), lower levels of serum sFlt-1 and sFlt-1:PlGF, and are less likely to experience high serum sEng levels, than sedentary women. The effects of exercise on PlGF and sFlt-1:PlGF are more pronounced among women exercising less than 270 minutes/week in pregnancy. Anti-angiogenic changes that could contribute to preeclampsia were not observed immediately after short-duration, moderate-intensity exercise in the third trimester. Flow-mediated dilation (FMD) and the shear stimulus for FMD are not affected by pregnancy, however the time to peak FMD was increased in pregnancy. Regular exercise did not affect FMD or its shear stimulus in healthy pregnant or non-pregnant women. FMD and its shear stimulus were positively correlated in active, but not inactive, pregnant and non-pregnant women. Pregnancy and physical activity do not affect radial artery low flow-mediated constriction (L-FMC). L-FMC is artery dependent, occurring in the radial, but not the brachial, artery of healthy pregnant and non-pregnant women. The positive correlation between L-FMC and FMD suggests that L-FMC and FMD are not independent measurements. The results of this thesis suggest that physical activity and exercise may reduce preeclampsia risk by reducing concentrations of angiogenic markers. Although exercise participation did not affect conduit artery vascular function in healthy pregnant women, future studies should investigate the effects of exercise on vascular function in women with endothelial dysfunction, or risk factors for preeclampsia. / Thesis (Ph.D, Kinesiology & Health Studies) -- Queen's University, 2009-06-11 12:41:53.466

Therapeutic angiogenesis is a promising technique to treat ischemia by creating new blood vessels. The aim of this thesis was to develop and characterize biodegradable elastomers for localized delivery of growth factors and to investigate the ability of released growth factors to induce angiogenesis. An osmotic delivery mechanism using photo-cross-linked elastomers based on trimethylene carbonate (TMC) was used to deliver vascular endothelial growth factor (VEGF165) and hepatocyte growth factor (HGF) alone or in combination at two different doses. It was hypothesized that elastomers made of TMC can provide an effective osmotic release using trehalose as a main osmotigen and that the use of TMC would eliminate the microenvironmental pH drop implicated in denaturing acid sensitive growth factors. To obtain an insight into the degrading zone in which growth factors will be released, the in vivo degradation mechanism and tissue response were investigated. The in vivo degradation of D,L-lactide/ε-caprolactone (DLLACL) elastomers that degrade by hydrolysis was investigated for comparison. Cross-link-density played a significant role in the degradation pattern of DLLACL elastomers. TMC and TMCCL elastomers degraded by surface erosion and oxidation played a significant role in their in vivo degradation. To obtain an efficient release, the mechanical properties of TMC elastomers were tailored by copolymerizing TMC with CL and DLLA and/or by controlling the cross-link density. The delivery device was able to provide a sustained release of growth factors for longer than two weeks with no initial burst. Cell based bioactivity assays indicated that released growth factors were highly bioactive over the entire release period. Microenvironmental pH studies using FITC-BSA indicated no significant drop in pH in TMC elastomers that contained small amounts of DLLA. Using 125I-VEGF165, it was found that the osmotic delivery can provide a direct in vivo-in vitro release correlation. Released growth factors were able to induce angiogenesis in rats when tested by subcutaneous implantation. Angiogenesis was dose dependent for both VEGF165 and HGF. Combined release of VEGF and HGF achieved the best results. The formed blood vessels were stable during the active release period, and they were normal looking and connected to the surrounding vasculature. / Thesis (Ph.D, Chemical Engineering) -- Queen's University, 2009-09-03 14:54:28.709

Numerous studies have identified that elevated plasma concentrations of lipoprotein(a) [Lp(a)] are an emerging risk factor for a variety of atherothrombotic disorders. Apolipoprotein(a) [apo(a)], the unique glycoprotein component of Lp(a), consists of tandem repeats of a plasminogen kringle (K) IV-like domain, followed by sequences homologous to the plasminogen KV and protease domains. Apo(a)/Lp(a) has been consistently shown to regulate endothelial function and inhibit plasminogen activation. In the present study, we have demonstrated that apo(a), signaling via integrin alphaVbeta3, is the functional unit in Lp(a) to stimulate in vitro endothelial cell (EC) proliferation and migration, and activate focal adhesion kinase (FAK) and mitogen-activated protein kinases (MAPK) in cultured ECs. Both apo(a) and Lp(a) have also been shown to reduce the levels of active and total transforming growth factor (TGF)-beta in cultured EC medium in an integrin alphaVbeta3–dependent manner. Despite the stimulatory effects of apo(a) on EC proliferation and migration, we have further confirmed an inhibitory effect of apo(a) on EC in vitro angiogenesis using a fibrin gel tube formation assay. We have provided evidence proving apo(a) inhibits angiogenesis through inhibition of plasminogen activation, and this inhibitory effect is dependent on the presence of apo(a) KV domain. Lastly, apo(a) is shown to reduce the protein levels of annexin A2 and S100A10 in ECs, which implies another potential mechanism by which apo(a)/Lp(a) could impair plasminogen activation on cell surface. In summary, we have discovered the first complete outside-in signaling pathway elicited by apo(a)/Lp(a) in ECs and have built up a connection between the ability of apo(a) to inhibit plasminogen activation and its inhibition of angiogenesis. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2009-09-25 18:29:47.106

Globally, breast cancer is the most abundant cancer form in women, in Sweden about 20 women are diagnosed with breast cancer every day .Interactions between genetic and external factors are the contributing factors while metastasis formation is the leading cause of death. Cancer is in need of vessels,to get the nutrients and oxygen it needs in order to survive. Therefore,the aim of the study is to analyze and compare the groups of high and low differential cancer vessels of the respective form, and to see if any type contained more vessels than the other. The study is based on 20 invasive ductal breast cancer samples, ten of them were high differentiated and the other ten were low differentiated. To assess the number of vessels, immunhistochemical staining with CD31 antibody was performedCD31 is an adhesion molecule present on endothelial cells. The group of low differentiated gradebreast cancer tissue had significantly more vessels compared with the high differentiated breast cancer tissues. To prove these test results, more cancers must be analyzed.

Pyruvate kinase M2 (PKM2) has been studied for decades on its role in cancer metabolism. Recently, PKM2 is highlighted again for its new function: promoting gene transcription by acting as a protein kinase. Moreover, the PKM2 levels in patient circulation have been used as a diagnostic marker for many types of cancers. However, it remains unclear whether PKM2 in blood circulation has any physiological or pathological function. In my dissertation, I demonstrate that PKM2 released from cancer cells facilitates tumor growth by promoting tumor angiogenesis. Our experiments show that PKM2 promotes endothelial cell proliferation, migration and survival. Only the dimeric PKM2, not the tetrameric PKM2 possesses the activity in angiogenesis promotion. Our results further indicate that PKM2 regulates angiogenesis by integrin αvβ3 activation and integrin redistribution. I also found that PKM2 enhances drug resistance of cancer cells expressing integrin αvβ3.

Argument: Hypertension is a main contributing risk factor to many cardiovascular diseases and may be the cause or the result of cardiovascular dysfunction. Black Africans, especially, suffer from hypertension because of lifestyle changes that occur during westernisation, which may lead to sympatho-adrenal hyperactivity. Vascular endothelial growth factor-A (VEGF-A) and angiopoietin-2 (Ang-2) are regulators of angiogenesis and are significantly up regulated during states of vascular dysfunction. Levels of angiogenic factors are unknown for African people and may not be the same as levels thus far reported for Caucasians. Aims: The aim of this study is firstly, to determine whether differences exist regarding the levels of VEGF-A and Ang-2 in urbanised compared to rural black Africans and secondly, to determine whether increased levels of VEGF-A and Ang-2 factors are related to hypertension in black Africans. Methodology: This is a sub study that is based upon the Prospective Urban and Rural Epidemiological (PURE) study. Apparently healthy, fasting African men and women (N=272, aged 35 to 50 years) from the North-West province of South Africa were selected by a medical doctor to participate in this study. Groups were stratified according to gender and urbanisation status based upon information derived from sociodemographic questionnaires. Cardiovascular parameters (Omron HEM-757), pulse wave velocity (PWV) (Compiler SP), plasma angiogenic factor levels (ELISA) and anthropometric measures were determined. An independent t-test and Pearson Chi-square test were used to compare urban and rural data, followed by an analysis of covariance (ANCOVA) while correcting for confounders (age, body mass index, physical activity and tobacco usage). ANCOVAs (corrected for confounders) were applied where hypertensive and normotensive groups were compared within the whole group and urbanised groups. Correlations, correcting for confounders, between cardiovascular variables and angiogenic factors were determined within the whole group and urbanised groups. Results and conclusion: Plasma VEGF-A values for all black Africans were very low while the ANG-2 levels were elevated compared to control values for Caucasians (normotensive and hypertensive) in literature. Urbanised men were more overweight and indicated a higher incidence of hypertension (42.47%) and elevated VEGF-A levels, but lower Ang-2 levels compared to rural men. Urbanised women were generally overweight, physically less active and smoked less, but indicated higher diastolic blood pressure (BP), VEGF-A levels and lower PWV compared with their rural counterparts. Ang-2 levels indicate a negative relationship to diastolic BP data in rural women. No relationships between hypertensive individuals and high angiogenic factor levels were uncovered. Conclusive evidence suggested that angiogenic factor levels were affected more by urbanisation than by the state of hypertension. If low levels of VEGF-2 occur, ANG-2 stimulation and properties may be altered, thereby switching ANG-2 from an anti-angiogenic to a pro-angiogenic molecule, inferring blood vessel destabilisation and vascular dysfunction, such as is observed in hypertensive urbanised men. Higher ANG-2 levels may result in Tie-2 receptor down regulation, hence causing VEGF-A levels to be lower. Further study is needed to ascertain this mechanism since Tie-2 receptor activity was not determined in this study. / Thesis (M.Sc. (Physiology))--North-West University, Potchefstroom Campus, 2009.