Due to the high metabolic demand and low capacity for energy storage of the brain, neurons are vitally reliant on a constant oxygen supply. Under chronic mild hypoxic conditions (10% oxygen), angiogenesis is induced in the brain in an attempt to restore tissue oxygen tension to normal levels. In brain hypoxia, vascular endothelial growth factor (VEGF) plays a critical role in angiogenesis; however, the role of its homolog placental growth factor (PlGF) is unknown. Using PlGF knockout (PlGF-/-) mice exposed to whole body hypoxia (10% oxygen) for 7, 14 and 21-days, we show that PlGF-/- animals exhibit a delay in the angiogenic response of the brain to hypoxia. PlGF-/- microvessels had a significant increase in fibrinogen accumulation and extravasation, which correlated with disruption of the tight-junction protein claudin-5. These vessels displayed large lumens, were surrounded by reactive astrocytes, lacked mural cell coverage and endothelial VEGF expression, and regressed after 21 days of hypoxia. The lack of PlGF, in combination with reduced VEGF expression levels observed in the brain of PlGF-/- animals during the first 5 days of hypoxia, is likely the cause of the delayed angiogenic response and the prothrombotic phenotype of these mice. In vitro studies conducted to analyze mechanisms involved in the impaired angiogenic phenotype and enhanced astrocytic reactivity to hypoxia of PlGF-/- animals indicated that: i) PlGF-/- mouse brain endothelial cells exhibit alterations in intracellular signaling pathways associated with sprouting (ERK1/2) and vessel branching morphogenesis (GSK-3β) and ii) PlGF-/- astrocytes overexpress VEGF receptor-2 (VEGFR-2) which through activation of the ERK1/2 signaling pathway leads to a more proliferative astrocytic phenotype. These astrocytes were more resistant to oxygen and glucose deprivation (OGD) than PlGF+/+ astrocytes, a characteristic that was shown to be independent of the classical antiapoptotic VEGFR-2-dependent PI3K/Akt pathway. The findings presented in this thesis demonstrated a critical role of PlGF in vascular remodeling in the hypoxic brain.

Due to the high metabolic demand and low capacity for energy storage of the brain, neurons are vitally reliant on a constant oxygen supply. Under chronic mild hypoxic conditions (10% oxygen), angiogenesis is induced in the brain in an attempt to restore tissue oxygen tension to normal levels. In brain hypoxia, vascular endothelial growth factor (VEGF) plays a critical role in angiogenesis; however, the role of its homolog placental growth factor (PlGF) is unknown. Using PlGF knockout (PlGF-/-) mice exposed to whole body hypoxia (10% oxygen) for 7, 14 and 21-days, we show that PlGF-/- animals exhibit a delay in the angiogenic response of the brain to hypoxia. PlGF-/- microvessels had a significant increase in fibrinogen accumulation and extravasation, which correlated with disruption of the tight-junction protein claudin-5. These vessels displayed large lumens, were surrounded by reactive astrocytes, lacked mural cell coverage and endothelial VEGF expression, and regressed after 21 days of hypoxia. The lack of PlGF, in combination with reduced VEGF expression levels observed in the brain of PlGF-/- animals during the first 5 days of hypoxia, is likely the cause of the delayed angiogenic response and the prothrombotic phenotype of these mice. In vitro studies conducted to analyze mechanisms involved in the impaired angiogenic phenotype and enhanced astrocytic reactivity to hypoxia of PlGF-/- animals indicated that: i) PlGF-/- mouse brain endothelial cells exhibit alterations in intracellular signaling pathways associated with sprouting (ERK1/2) and vessel branching morphogenesis (GSK-3β) and ii) PlGF-/- astrocytes overexpress VEGF receptor-2 (VEGFR-2) which through activation of the ERK1/2 signaling pathway leads to a more proliferative astrocytic phenotype. These astrocytes were more resistant to oxygen and glucose deprivation (OGD) than PlGF+/+ astrocytes, a characteristic that was shown to be independent of the classical antiapoptotic VEGFR-2-dependent PI3K/Akt pathway. The findings presented in this thesis demonstrated a critical role of PlGF in vascular remodeling in the hypoxic brain.

Due to the high metabolic demand and low capacity for energy storage of the brain, neurons are vitally reliant on a constant oxygen supply. Under chronic mild hypoxic conditions (10% oxygen), angiogenesis is induced in the brain in an attempt to restore tissue oxygen tension to normal levels. In brain hypoxia, vascular endothelial growth factor (VEGF) plays a critical role in angiogenesis; however, the role of its homolog placental growth factor (PlGF) is unknown. Using PlGF knockout (PlGF-/-) mice exposed to whole body hypoxia (10% oxygen) for 7, 14 and 21-days, we show that PlGF-/- animals exhibit a delay in the angiogenic response of the brain to hypoxia. PlGF-/- microvessels had a significant increase in fibrinogen accumulation and extravasation, which correlated with disruption of the tight-junction protein claudin-5. These vessels displayed large lumens, were surrounded by reactive astrocytes, lacked mural cell coverage and endothelial VEGF expression, and regressed after 21 days of hypoxia. The lack of PlGF, in combination with reduced VEGF expression levels observed in the brain of PlGF-/- animals during the first 5 days of hypoxia, is likely the cause of the delayed angiogenic response and the prothrombotic phenotype of these mice. In vitro studies conducted to analyze mechanisms involved in the impaired angiogenic phenotype and enhanced astrocytic reactivity to hypoxia of PlGF-/- animals indicated that: i) PlGF-/- mouse brain endothelial cells exhibit alterations in intracellular signaling pathways associated with sprouting (ERK1/2) and vessel branching morphogenesis (GSK-3β) and ii) PlGF-/- astrocytes overexpress VEGF receptor-2 (VEGFR-2) which through activation of the ERK1/2 signaling pathway leads to a more proliferative astrocytic phenotype. These astrocytes were more resistant to oxygen and glucose deprivation (OGD) than PlGF+/+ astrocytes, a characteristic that was shown to be independent of the classical antiapoptotic VEGFR-2-dependent PI3K/Akt pathway. The findings presented in this thesis demonstrated a critical role of PlGF in vascular remodeling in the hypoxic brain.

Due to the high metabolic demand and low capacity for energy storage of the brain, neurons are vitally reliant on a constant oxygen supply. Under chronic mild hypoxic conditions (10% oxygen), angiogenesis is induced in the brain in an attempt to restore tissue oxygen tension to normal levels. In brain hypoxia, vascular endothelial growth factor (VEGF) plays a critical role in angiogenesis; however, the role of its homolog placental growth factor (PlGF) is unknown. Using PlGF knockout (PlGF-/-) mice exposed to whole body hypoxia (10% oxygen) for 7, 14 and 21-days, we show that PlGF-/- animals exhibit a delay in the angiogenic response of the brain to hypoxia. PlGF-/- microvessels had a significant increase in fibrinogen accumulation and extravasation, which correlated with disruption of the tight-junction protein claudin-5. These vessels displayed large lumens, were surrounded by reactive astrocytes, lacked mural cell coverage and endothelial VEGF expression, and regressed after 21 days of hypoxia. The lack of PlGF, in combination with reduced VEGF expression levels observed in the brain of PlGF-/- animals during the first 5 days of hypoxia, is likely the cause of the delayed angiogenic response and the prothrombotic phenotype of these mice. In vitro studies conducted to analyze mechanisms involved in the impaired angiogenic phenotype and enhanced astrocytic reactivity to hypoxia of PlGF-/- animals indicated that: i) PlGF-/- mouse brain endothelial cells exhibit alterations in intracellular signaling pathways associated with sprouting (ERK1/2) and vessel branching morphogenesis (GSK-3β) and ii) PlGF-/- astrocytes overexpress VEGF receptor-2 (VEGFR-2) which through activation of the ERK1/2 signaling pathway leads to a more proliferative astrocytic phenotype. These astrocytes were more resistant to oxygen and glucose deprivation (OGD) than PlGF+/+ astrocytes, a characteristic that was shown to be independent of the classical antiapoptotic VEGFR-2-dependent PI3K/Akt pathway. The findings presented in this thesis demonstrated a critical role of PlGF in vascular remodeling in the hypoxic brain.

<p>Angiogenesis, de novo synthesis of blood vessels from the pre-existing vasculature, is required both during embryonic development and in pathophysiological conditions. In particular, tumor growth needs new capillary vessels in order to both deliver oxygen and nutrients and to remove toxin and metabolites. Growth of most solid tumors would be restricted to a microscopic size in the absence of neovascularization. Angiogenesis ensues as a result of a shift in the balance between pro- and anti-angiogenic molecules.</p><p>Histidine-rich glycoprotein (HRGP) is a heparin-binding plasma protein. We showed that HRGP inhibits endothelial cell migration and adhesion to vitronectin. As a consequence, HRGP attenuates growth and vascularization of mouse model tumors. The anti-angiogenic effect of HRGP is mediated by the central histidine/proline (His/Pro)-rich domain, which must be released from the parent molecule to exert its effect. A 35-amino acid residue peptide denoted HRGP330, derived from the His/Pro-rich domain, was identified as a minimal active anti-angiogenic domain of HRGP. HRGP330 induces disruption of molecular interactions required for cell motility, such as the integrin-linked kinase/paxillin complex. Moreover, HRGP330 inhibits VEGF-induced tyrosine phosphorylation of α-actinin, a focal adhesion kinase (FAK) substrate. Consequently, the motility of endothelial cells is arrested. By use of a signal transduction antibody array, we identified FAK, paxillin and growth factor receptor-bound 2 (Grb2) as tyrosine phosphorylated in HRGP330-treated cells. We confirmed that HRGP targets focal adhesions in endothelial cells, thereby disrupting the cytoskeletal organization and the ability of endothelial cells to assemble into vessel structures. A critical role of FAK in HRGP-inhibition of angiogenesis was validated using a FAK inhibitor, geldanamycin, which allowed rescue of endothelial cell actin rearrangement.</p><p>We identified another potential mechanism in the HRGP/HRGP330 anti-angiogenic effects, exerted through regulation of tumor-associated macrophages (TAMs). HRGP/HRGP330 treatment led to reduced TAM infiltration, which in turn caused a marked decrease in VEGF and MMP-9 levels in the tumor. </p><p>Taken together, our present studies show that HRGP/HRGP330 target endothelial cell adhesion, migration, focal adhesions, and furthermore, that HRGP is involved in regulation of macrophage infiltration.</p>

Angiogenesis, de novo synthesis of blood vessels from the pre-existing vasculature, is required both during embryonic development and in pathophysiological conditions. In particular, tumor growth needs new capillary vessels in order to both deliver oxygen and nutrients and to remove toxin and metabolites. Growth of most solid tumors would be restricted to a microscopic size in the absence of neovascularization. Angiogenesis ensues as a result of a shift in the balance between pro- and anti-angiogenic molecules. Histidine-rich glycoprotein (HRGP) is a heparin-binding plasma protein. We showed that HRGP inhibits endothelial cell migration and adhesion to vitronectin. As a consequence, HRGP attenuates growth and vascularization of mouse model tumors. The anti-angiogenic effect of HRGP is mediated by the central histidine/proline (His/Pro)-rich domain, which must be released from the parent molecule to exert its effect. A 35-amino acid residue peptide denoted HRGP330, derived from the His/Pro-rich domain, was identified as a minimal active anti-angiogenic domain of HRGP. HRGP330 induces disruption of molecular interactions required for cell motility, such as the integrin-linked kinase/paxillin complex. Moreover, HRGP330 inhibits VEGF-induced tyrosine phosphorylation of α-actinin, a focal adhesion kinase (FAK) substrate. Consequently, the motility of endothelial cells is arrested. By use of a signal transduction antibody array, we identified FAK, paxillin and growth factor receptor-bound 2 (Grb2) as tyrosine phosphorylated in HRGP330-treated cells. We confirmed that HRGP targets focal adhesions in endothelial cells, thereby disrupting the cytoskeletal organization and the ability of endothelial cells to assemble into vessel structures. A critical role of FAK in HRGP-inhibition of angiogenesis was validated using a FAK inhibitor, geldanamycin, which allowed rescue of endothelial cell actin rearrangement. We identified another potential mechanism in the HRGP/HRGP330 anti-angiogenic effects, exerted through regulation of tumor-associated macrophages (TAMs). HRGP/HRGP330 treatment led to reduced TAM infiltration, which in turn caused a marked decrease in VEGF and MMP-9 levels in the tumor. Taken together, our present studies show that HRGP/HRGP330 target endothelial cell adhesion, migration, focal adhesions, and furthermore, that HRGP is involved in regulation of macrophage infiltration.

Distortion to hemodynamic, ischemic and metabolic stimuli during low load resistance exercise with blood flow restriction (BFR) may influence regional vascular adaptation. This thesis investigated the acute response and chronic adaptations of the peripheral vasculature to low load resistance exercise with BFR. The methodology utilised Doppler ultrasound, strain gauge plethysmography and muscle biopsy for insightful measures of the vasculature at different regions of the arterial tree. Short term (4-6 weeks) localised low load (30-40% 1RM) resistance exercise with BFR increased brachial (3.1%) and popliteal (3.3%) artery maximal diameter (in response to ischemic exercise), forearm (29%) and calf (24%) post-occlusive blood flow, and calf filtration capacity (14%). These findings indicate potential vascular remodelling at the conduit (chapters 3, 4) resistance (chapter 4) and capillary (chapter 4) level of the vascular tree. Regional, rather than systemic, factors are responsible for these adaptations as evidenced by an absent response in the contralateral control limb. Transient improvements in popliteal artery FMD% occurred at week 2 before increased maximal diameter at week 6, suggesting functional changes precede structural remodelling (chapter 4). Maximal brachial artery diameter and forearm post-occlusive blood flow returned to baseline values after a 2 week detraining period, signifying rapid structural normalisation after stimulus removal (chapter 3). Enhanced capillarity, despite low training loads, could be explained by augmentation of VEGF (~7 fold), PGC-1α (~6 fold) and eNOS (~5 fold) mRNA, and upregulation VEGFR-2 (~5 fold) and HIF-1α (~2.5 fold) mRNA with BFR (chapter 5). This indicates a targeted angiogenic response potentially mediated through enhanced metabolic, ischemic and shear stress stimuli. Large between subject variability in the level of BFR was observed during upper and lower limb cuff inflation protocols. Adipose tissue thickness and mean arterial pressure were the largest independent determinants of upper and lower limb BFR, respectively (Chapter 6). In conclusion, this thesis demonstrates that low load resistance exercise with BFR induces adaptation in the conduit, resistance and capillary vessels. The mediators of this response are likely to be the hemodynamic and chemical signals elicited by repeated bouts of BFR resistance exercise, although confirmation of these mechanisms is required. The functional significance of these adaptations is unknown and warrants further investigation.

Obesity affects about 520 million people world-wide and more recently studies have shown that fat cells produce proteins called adipokines which have various influences on the human metabolism and has helped to change the perspectives of researchers on the concept of the adipose tissue being just a store of energy. As a result of this, adipokines have been reported to represent a connection between obesity and cardiovascular diseases (CVD) and diabetes mellitus. The concentrations and the bases of the effects of the adipokines in beta cell failure of diabetes mellitus and endothelial cell dysfunction of cardiovascular diseases are still not fully understood. The effect of leptin and adiponectin, which are two adipokines with opposing effects, has been explored in this study. In the present study, therefore, the concentrations of leptin and adiponectin with significant effect on beta cell and endothelial cell function and the basis of these functions were explored. Also, attempts were made in the present study to correlate the concentrations of leptin and adiponectin with possible clinical pointers to complications. In order to achieve this, beta cells (BTC) were grown, made into pseudo-islets (which are said to produce more insulin) and treated with various concentrations of leptin and adiponectin and cells assayed for insulin and amylin (to investigate the role of amylin in insulin secretion). Also the cells were collected and mRNA extracted from these cells, reverse transcription PCR carried out to find out the role of protein phosphatase 1 (PP-1) in the effect of leptin on insulin secretion. PP-1 is a substrate that increases insulin secretion by allowing calcium influx into the cell and is said to be blocked by leptin). Leptin at 500ng/ml was found to significantly (p<0.05) inhibit the secretion of insulin and the expression of PP1 gene, thus supporting this as a basis for the effect of leptin on insulin secretion. Adiponectin however increased insulin secretion significantly but was not as consistent in its effect as leptin was in inhibiting insulin secretion. In order to explore the role of adipokines in cardiovascular diseases, EAHY human endothelial cells were cultured and treated with various concentrations of adiponectin and leptin both individually and in combinations and cells collected and mRNA extracted in order to carry out a reverse transcription PCR for the expression of angiogenic (TIMP2, TIMP3 and MMP2) genes and atherosclerotic (LPA and LPL) genes. Leptin (1nM) was shown to increase the expression of atherosclerotic and angiogenic genes while adiponectin (100nM) inhibited the expression of the atherosclerotic and angiogenic genes. A combination of leptin and adiponectin caused a reduction in the stimulatory effect of leptin on the expression of atherosclerotic and angiogenic genes. This shows that leptin may predispose to CVD while adiponectin reduces the risk of CVD. The clinical part of this study involved recruiting 150 patients with diabetes after the ethical approval for the clinical study was granted. The data collected from the patients included their age, sex, race, and physical parameters like the body mass index (BMI). Also blood samples were collected to measure the clinical indicators for CVD and renal function such as cholesterol, HDL levels, eGFR, albumin levels and their retinopathy status checked as these are the common complications seen in diabetic patients. The blood samples were also assayed in the laboratory for leptin and adiponectin levels and the leptin, adiponectin and the leptin/adiponectin ratio (LAR) were then correlated with the laboratory determinants of CVD, renal and retinopathy risks. It was found that the LAR and the leptin levels correlates significantly with the BMI, while the leptin levels were significantly correlated with the risk of nephropathy in diabetic patients while adiponectin levels correlated significantly with a reduced risk for developing CVD. The role of the enzymes in the leptin and adiponectin signaling pathway was also explored and it was discovered that ERK, P38 and AMPK all had roles in the effect of leptin and adiponectin on the expression of atherosclerotic and angiogenic genes. These data indicate that leptin and adiponectin play significant roles in the beta cell and endothelial cell function and are links between obesity and CVD and diabetes mellitus.

Ovarian cancer (OC) is the second most common gynecologic cancer; however it is responsible for the most gynecologic cancer-related deaths. Apoptosis evasion is an important mechanism in OC tumorigenesis, and the prototypic anti-apoptotic protein, B-cell lymphoma 2 (Bcl-2), is often overexpressed in OC tumors. Gaining a better understanding of the mechanism(s) behind Bcl-2 overexpression and potential extra-anti-apoptotic functions of Bcl-2 could elucidate the importance of elevated Bcl-2 in OC. In the current study, I show through immunohistochemical analysis of normal, benign, and OC tissue sections, that both epithelial and stromal Bcl-2 expression decreases with OC progression. However, the number of Bcl-2-positive lymphocyte nests and the size of these lymphocyte nests increase dramatically with OC progression. Additionally, this study shows that lysophosphatidic acid (LPA), a glycerophospholipid frequently elevated in serum and ascites fluid of OC patients, upregulates Bcl-2 in OC cells. Bcl-2 enzyme-linked immunosorbant assay (ELISA), western blot analysis, reverse transcriptase polymerase chain reaction (RT-PCR), and luciferase reporter assays reveal that LPA increases Bcl-2 promoter, messenger RNA (mRNA), and protein levels in OC cells, but not in normal immortalized ovarian surface epithelial (IOSE) cells. LPA also increases secreted levels of Bcl-2. In vitro human umbilical vein endothelial cell (HUVEC) tube formation assays show that OC-derived Bcl-2 or recombinant human (rh) Bcl-2 promotes aberrant formation of tube-like structures. Though extracellular Bcl-2 does not affect HUVEC cell viability, it may cause aberrant tube formation by inhibiting HUVEC migration. Finally, Bcl-2 ELISA reveals that urinary Bcl-2 levels in OC patients are higher than those in normal individuals and patients with benign gynecologic disease. Urinary Bcl-2 also complements serum CA125 when the two are compared in parallel samples. Furthermore, urinary Bcl-2 decreases following cytoreductive surgery. Altogether, the results suggest that Bcl-2 is important in OC tumorigenesis and angiogenesis. Additionally, urinary Bcl-2 may be a valuable non-invasive biomarker for OC diagnosis and/or screening. Consequently, further elucidation of mechanisms of Bcl-2 overexpression and its extra-apoptotic functions could lead to improved treatment and diagnostic strategies for OC patients.

Pyruvate kinase M2 (PKM2) is a glycolytic enzyme expressed in highly proliferating cells. Studies of PKM2 have been focused on its function of promoting cell proliferation in cancer cells. Our laboratory previously discovered that extracellular PKM2 released from cancer cells promoted angiogenesis by activating endothelial cell proliferation and migration. PKM2 activated endothelial cells through integrin αvβ3. Angiogenesis and myofibroblast differentiation are key processes during wound healing. In this dissertation, I demonstrate that extracellular PKM2 released from activated neutrophils promotes angiogenesis and myofibroblast differentiation during wound healing. PKM2 activates dermal fibroblasts through integrin αvβ3 and PI3K signaling pathway. I also claim that extracellular PKM2 plays a role during liver fibrosis. PKM2 protects hepatic stellate cells from apoptosis by activating the survival signaling pathway.