The lymphatic system complements the blood circulatory system in absorption and transport of nutrients, and in the maintenance of homeostasis. Historically, the angiogenesis field has advanced faster and farther than the field of lymphangiogenesis. The discovery of lymphatic markers and the emerging evidence implicating the lymphatic system as a central player in a variety of pathological conditions has attracted research interest and driven the field forward. Research efforts have produced the observation that regulators of the blood endothelium are frequently members of the same protein families of regulators of the lymphatic endothelium. More importantly, these regulators do not act discretely, restricting their regulatory activities to one endothelial cell (EC) type. Two examples of regulators that behave in this manner are the VEGF and the Angiopoietin families of proteins, which have cell-type-dependent effects on EC processes such as migration, proliferation and survival. The study of these regulators therefore requires an in vitro EC system capable of accommodating the simultaneous characterization of the signaling pathways downstream of these shared molecular regulators in venous, arterial and lymphatic endotheliums. To build such an in vitro system, I isolated and validated lymphatic, venous, and arterial ECs derived from vessels of bovine mesentery. The proteomes of the three cell types were comparatively studied using two-dimensional polyacrylamide gel electrophoresis followed by mass spectrometric identification. The three cell types were used in a subtractive immunization scheme for the production of a monoclonal antibody selectively reactive to a potentially novel surface protein marker of lymphatic ECs. The studies recorded herein all share the common goal of identifying and characterizing unique molecular signatures that distinguish lymphatic ECs from blood ECs, and that may underline the cellular biology of the lymphatic endothelium as distinct from the blood endothelium.

Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 5 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2005. / Härtill 4 uppsatser.

Thesis (M.S.)--University of Missouri-Columbia, 2008. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Thesis advisor: Dr. William Durante. "December 2008" Includes bibliographical references.

Diet-derived polyphenols are believed to have health benefits on account of their antioxidant properties. Cardiovascular health is considered to be a suitable target for antioxidant therapy because oxidative stress is implicit in atherogenesis – the disease process that underpins heart attacks, ischaemic strokes and peripheral vascular disease. Numerous in vitro, in vivo and clinical studies indicate that polyphenols are protective in cardiovascular disease, but their mechanism of action still remains ambiguous. This thesis describes a wide range of studies to characterise the activity and stability of a key polyphenol, delphinidin, found in widely consumed berries, and ultimately to test the hypothesis that delphinidin, at physiologically relevant concentrations (~1 µM), protects cultured human umbilical vein endothelial cells (HUVECs) against oxidative damage via a mechanism that is independent of direct antioxidant activity. Delphinidin aglycone was found to be unstable in tissue culture medium, in which it decomposed rapidly to simple phenolic compounds, including gallic acid. Electron paramagnetic resonance spectroscopy indicated that, if anything, both delphinidin and gallic acid were pro-oxidant rather than antioxidant. Moreover, high concentrations of both delphinidin and gallic acid induced rapid morphological changes in HUVECs, most notably in the formation of vacuoles or vesicles. Treatment of HUVECs with a range of concentrations (1 nM - 100 µM) of delphinidin and gallic acid showed that high (100 µM) concentrations of both were cytotoxic. However, both agents were found to have a protective effect in cells exposed to oxidative stress when present at concentrations of ~1 µM – too low to be due to direct antioxidant activity. Deeper examination of cells treated with delphinidin and gallic acid indicated that the protective effect was perhaps partially mediated by changes in expression of the intracellular antioxidant, glutathione. Taken together, the results in this thesis suggest that metabolic products of delphinidin might be responsible for the antioxidant effects seen on account of initiating cellular defence responses.

In 1899, the German surgeon Max Wilms hypothesised that different cell types in a variety of childhood kidney cancers were all derived from the mesodermal layer during embryonic development. Nearly a century later, the WT1 gene was identified on the short arm of chromosome 11, and was thought to be inactive in ~20% of nephroblastomas (Wilms’ tumours). The expression of WT1 after birth appears to be restricted to a finite number of tissues, namely, the glomerular podocytes, mesothelium and ~1% of bone marrow cells. Emerging evidence suggests WT1 is required not only for development, but also for tissue homeostasis, regeneration, repair and angiogenesis. Interestingly, WT1 has been implicated in the response to myocardial infarction and tumour angiogenesis, yet its precise role remains unclear. This thesis aims to address the hypothesis that activation of the WT1 gene in the vascular endothelium is essential for physiological and pathophysiological angiogenesis in the adult. In order to assess whether Wt1 was expressed in quiescent endothelial cells (ECs) immunofluorescence was used to analyse a variety of tissues in the adult mouse. Whilst Wt1 was detected in renal podocytes, no endothelial Wt1 expression was discovered in the lung, heart, kidney, spleen and gastrocnemius muscle. In contrast, tissues known to undergo physiological angiogenesis (endometrium and breast) did exhibit Wt1 expression in the vascular endothelium. Moreover, tubular EC outgrowths generated by aortic rings embedded in collagen ex vivo were positive for Wt1. The role of Wt1 in ischaemic angiogenesis was assessed using models of hind-limb and coronary ischaemia in the mouse. Wt1 was detected in ECs and non-vascular cells following ischaemic injury by a combination of immunofluorescence and qualitative real-time polymerase chain reaction (qRT-PCR). Using a time course analysis of these experimental models the chronology of this relationship was demonstrated, alongside the association with key angiogenic factors, such as Vegf. Given the findings in ischaemic tissue the C3(1)/Tag transgenic mammary cancer model was used to test the hypothesis that Wt1 would be upregulated in the tumour vasculature. Endothelial Wt1 was up regulated in these tumours compared to healthy control tissue. This finding was mirrored in a sub-set of aggressive breast cancers, confirming that the results obtained in mice can be translated to humans. Quantitative PCR revealed no association between histopathological grade of the tumours, oestrogen receptor status, and WT1 expression. In order to delineate the cell types involved in vessel formation, Wt1+ cells were sorted using fluorescent activated cell sorting (FACS) from transgenic mice with a green fluorescent protein knocked into the Wt1 locus following sponge implantation. Distinct sub-populations of Wt1+ cells were identified, some of which expressed EC and pericyte markers. Moreover, these Wt1+ sub-populations changed in composition and number over time. These findings were confirmed by genetic fate mapping of Wt1+ cells in this model. Finally, a conditional knockout mouse was generated to allow the selective deletion of Wt1 from vascular ECs in the sponge model of angiogenesis. The results demonstrated that deletion of Wt1 from this cellular compartment led to a dramatic reduction in vessel formation supporting a potential role in regulating angiogenesis. These results support the hypothesis that expression of WT1 in the vascular endothelium contributes to the regulation of angiogenesis in tumours and ischaemic tissue, and provides evidence that selective deletion of the gene inhibits new vessel formation. This suggests that targeting WT1 may have a therapeutic benefit in cancer and could aid regeneration of ischaemic tissues following injury in conditions such as myocardial infarction and critical limb ischaemia.

No description available.

No description available.

Pulmonary arterial hypertension (PAH) is a lethal disease characterized by excessive proliferation of pulmonary vascular cells, such as endothelial cells (ECs). Hereditary (H) PAH is mainly caused by ―loss-of-function‖ mutations in the gene coding for the bone morphogenetic protein type II receptor (BMPR2). However, the mechanisms by which these mutations cause PAH remain unclear. The hypothesis of this thesis was that BMPR2 mutations produce an imbalance in EC protein expression and/or activity that is integrally related to the development of abnormalities in lung vascular function and structure in HPAH. Patient-specific blood-outgrowth endothelial cells (BOECs) expanded ex vivo from peripheral blood mononuclear cells from patients with HPAH and healthy subjects were used to examine the consequences of BMPR2 mutations on the BOEC protein expression profile as well as on their functionality. Functional analyses of the BOECs revealed that HPAH-derived BOECs are more susceptible to apoptosis and more proliferative compared with healthy controls. Protein isolates of BOECs from patients with HPAH and from healthy subjects were subjected to 2-D gel electrophoresis and stained for total proteins and phosphoproteins, and to a quantitative computerassisted analysis. Differentially regulated proteins were identified by mass spectrometry (LC-MS/MS). Of the 416 total proteins detected under basal conditions, 11 were significantly downregulated in HPAH-derived BOECs and 11, including the translationally controlled tumor protein (TCTP), were upregulated. TCTP has previously been shown to be involved in systemic arteriolar remodeling, inflammation and growth. Therefore, the potential role of TCTP in PAH was studied in vivo in the SU5416 rat model of severe angioproliferative PAH. Immunofluorescence staining revealed high expression of TCTP in arteriolar ECs of PAH lungs tightly localized to proliferating cells within occlusive intimal lesions; whereas, only minimal TCTP expression was seen in vascular ECs of normal lungs. Similarly, abundant TCTP immunostaining was also seen in human PAH lung sections, again associated with complex vascular lesions. In BOECs, TCTP was found to participate in cell growth and survival. These data suggest that TCTP could play an important role in PAH by mediating pro-survival and growth signaling in vascular cells, contributing to occlusive pulmonary vascular remodeling triggered by EC apoptosis.

Background: Pulmonary arterial hypertension (PAH) is a multifaceted disease characterized by elevated pulmonary arterial pressure, right ventricular hypertrophy, and a poor prognosis. Pathological hallmarks of PAH include pulmonary vascular remodelling, pre-capillary arterial obliteration, and plexiform lesions. Over the past 15 years, pulmonary endothelial cell (EC) apoptosis has been repeatedly implicated as a key trigger of occlusive arterial remodelling in PAH. While it has been hypothesized that pulmonary EC apoptosis gives rise to the emergence of growth-dysregulated, apoptosis- resistant ECs involved in arterial remodelling, the molecular mechanisms linking these two events has not yet been fully elucidated. Recently, our lab identified translationally controlled tumour protein (TCTP) as one of several significantly dysregulated proteins in culture-derived blood-outgrowth endothelial cells (BOECs) isolated from hereditable PAH (HPAH) patients harbouring mutations in the gene encoding for bone morphogenetic protein receptor type 2. Immunohistological analyses indicated that TCTP expression was associated with intra-luminal pulmonary ECs and inflammatory cells in the remodelled vessels of both human PAH patients and SU5416 rats. Furthermore, TCTP silencing abrogated excessive HPAH BOEC proliferation and promoted apoptosis in vitro. Hypothesis: We hypothesized that TCTP represents a central molecular mechanism linking pulmonary arterial EC damage and apoptosis to the emergence of growth- dysregulated lung vascular cells and complex arterial remodelling in PAH.Purpose: The purpose of the present thesis was to examine the effects TCTP inhibition on EC survival and TCTP abundance in vitro as well as on pulmonary hemodynamic changes and arterial remodelling in vivo using a well-validated rat model of severe PAH. Methods: Inhibition of TCTP was accomplished using two TCTP small molecule inhibitors, sertraline and thioridazine. In vitro, rat lung microvascular ECs (RLMVECs) were exposed to thioridazine and assayed for TCTP abundance, survival, and markers of apoptosis. In vivo, PAH was induced in male Sprague Dawley rats using SU5416 combined with 3 weeks of chronic hypoxia (SU/CH). After 4 weeks, right ventricle systolic pressure (RVSP) was measured by direct catheterization and osmotic pumps containing either thioridazine or sertraline were implanted subcutaneously. Following 3 weeks of small molecule delivery, RVSP was re-evaluated, cardiac function/structure was determined using transthoracic echocardiography, and histological analyses of vascular remodelling and inflammation were performed. Results: Our in vitro experiments demonstrated that thioridazine was able to significantly down-regulate TCTP levels and induce an apoptotic phenotype in RLMVECs. In the SU/CH rat model of severe PAH, both thioridazine and sertraline failed to have any effect on pulmonary hemodynamics, right ventricle structure/function, or vascular remodelling. Moreover, neither small molecule was able to detectably down-regulate TCTP levels in the lungs of SU/CH rats. Immunofluorescence staining revealed that TCTP expression occasionally corresponded with the expression of macrophage/monocyte marker CD68 in the lungs of SU/CH rats, consistent with its expression by inflammatory cells; however, no significant differences were found in adventitial cell clearance in the presence or absence of the inhibitors. Conclusions: Our findings support previous reports that thioridazine is able to significantly down-regulate TCTP and induce apoptosis in vitro. In contrast, both small molecule inhibitors failed to down-regulate lung TCTP levels or have any beneficial effects on the progression of PAH in SU/CH rats.