The tyrosine kinase receptor Tie-2 is expressed on the surface of endothelial cells and is necessary for angiogenesis and vascular stability. To date, the best characterized ligands for Tie-2 are Angiopoietin-1 (Ang-1) and Angiopoietin-2 (Ang-2). Ang-1 has been identified as the main activating ligand for Tie-2 while the role of Ang-2 has been controversial since its discovery; some studies reported Ang-2 as a Tie-2 antagonist while others described Ang-2 as a Tie-2 agonist. The purpose of this thesis was to understand: (1) how the receptor Tie-2 is regulated by Ang-1 and Ang-2 in endothelial cells, (2) to compare the effects of Ang-1 and Ang-2, and (3) to determine the arrangement and distribution of Tie-2 in endothelial cells. The research presented in this thesis indicates that Tie-2 is arranged in variably sized clusters on the endothelial cell surface. Clusters of Tie-2 were expressed on all surfaces of cells: on the apical plasma membrane, on the tips of microvilli, and on the basolateral plasma membrane. When endothelial cells were stimulated with Ang-1, Tie-2 was rapidly internalized and degraded. Upon Ang-1 stimulation, Tie-2 localized to clathrin-coated pits on all surfaces of endothelial cells indicating that one pathway mediating Tie-2 internalization is through clathrin-coated pits. After activation of Tie-2, Ang-1 dissociates from the endothelial cell surface and accumulates in the surrounding medium. When experiments were repeated with Ang-2, it was discovered that Ang-2 induced all of the same effects on Tie-2 as Ang-1 but at a much reduced level and rate, indicating that Ang-2 likely functions as a partial agonist for Tie-2 in endothelial cells. / PhD

The development of a functional vascular system is impinged upon the restructuring of a primitive vasculature into a more complex and mature vessel network via a process known as angiogenesis. Of particular importance to this vascular remodeling process is the function of the Tek/Tie-2 receptor tyrosine kinase. Mouse gene-targeting studies have shown that Tie-2 deficient embryos succumb to embryonic death at embryonic day 9.5 due to insufficient sprouting and remodeling of the primary capillary plexus. Over the years, the functions and the signaling pathways downstream of Tie-2 receptor have been elucidated; however, the repertoire of genes controlled by Tie-2 signaling leading to angiogenesis had not been studied. To identify the underlying genetic mechanisms, transcriptomes from Tie-2 wild-type (WT) and knockout (KO) embryonic day 8.5 yolk sac tissues were quantitatively analyzed using a gene expression profiling technique called Serial Analysis of Gene Expression (SAGE). Tie-2 WT and KO SAGE libraries were constructed, sequenced and compared to identify genes that were differentially expressed. A list of candidate genes was selected for further validation using semi-quantitative PCR that included 4933402E13Rik, a novel transcript encoding a protein product containing the melanoma-associated antigen (MAGE) domain. Initial characterization of 4933402E13Rik suggested a murine-specific expression profile restricted to the yolk sac, embryo, placenta, testis, endothelial and embryonic stem cells. The expression of 4933402E13Rik in mouse endothelial cells was found to be regulated by Tie-2 signaling since down-regulation of Tie-2 level via siRNA knockdown resulted in decreased 4933402E13Rik mRNA expression. In contrast, stimulation of Tie-2 in mouse endothelial cells using its ligand, Angiopoietin-1, increased 4933402E13Rik mRNA levels. Additionally, 4933402E13Rik expression was found to be modulated through epigenetics especially by histone deacetylation. Mouse endothelial cells treated with Trichostatin A, a potent inhibitor of histone deacetylase, led to an increase in the expression of 4933402E13Rik. Taken together, the results of this study shed new insight on the repertoire of genes implicated in Tie-2 signaling. The identification of 4933402E13Rik as a novel gene modulated by Tie-2 provides a new avenue of research on Tie-2 signaling that may contribute further to our understanding of vascular development.

The development of a functional vascular system is impinged upon the restructuring of a primitive vasculature into a more complex and mature vessel network via a process known as angiogenesis. Of particular importance to this vascular remodeling process is the function of the Tek/Tie-2 receptor tyrosine kinase. Mouse gene-targeting studies have shown that Tie-2 deficient embryos succumb to embryonic death at embryonic day 9.5 due to insufficient sprouting and remodeling of the primary capillary plexus. Over the years, the functions and the signaling pathways downstream of Tie-2 receptor have been elucidated; however, the repertoire of genes controlled by Tie-2 signaling leading to angiogenesis had not been studied. To identify the underlying genetic mechanisms, transcriptomes from Tie-2 wild-type (WT) and knockout (KO) embryonic day 8.5 yolk sac tissues were quantitatively analyzed using a gene expression profiling technique called Serial Analysis of Gene Expression (SAGE). Tie-2 WT and KO SAGE libraries were constructed, sequenced and compared to identify genes that were differentially expressed. A list of candidate genes was selected for further validation using semi-quantitative PCR that included 4933402E13Rik, a novel transcript encoding a protein product containing the melanoma-associated antigen (MAGE) domain. Initial characterization of 4933402E13Rik suggested a murine-specific expression profile restricted to the yolk sac, embryo, placenta, testis, endothelial and embryonic stem cells. The expression of 4933402E13Rik in mouse endothelial cells was found to be regulated by Tie-2 signaling since down-regulation of Tie-2 level via siRNA knockdown resulted in decreased 4933402E13Rik mRNA expression. In contrast, stimulation of Tie-2 in mouse endothelial cells using its ligand, Angiopoietin-1, increased 4933402E13Rik mRNA levels. Additionally, 4933402E13Rik expression was found to be modulated through epigenetics especially by histone deacetylation. Mouse endothelial cells treated with Trichostatin A, a potent inhibitor of histone deacetylase, led to an increase in the expression of 4933402E13Rik. Taken together, the results of this study shed new insight on the repertoire of genes implicated in Tie-2 signaling. The identification of 4933402E13Rik as a novel gene modulated by Tie-2 provides a new avenue of research on Tie-2 signaling that may contribute further to our understanding of vascular development.

Tie-2 expressing monocytes (TEMs) were identified to be a subset of monocytes that was potent inducer of tumor angiogenesis in various types of cancer. On the other hand, monocytes and macrophages are crucially involved in the innate immune response, but the function of TEMs in this process is not well understood. Therefore, we studied the role of Tie-2 in regulating the inflammatory response initiated by lipopolysaccharide in murine macrophages. First, we observed that a small percentage of bone marrow-derived macrophages express Tie-2. Furthermore, we observed that Tie-2 ligands were able to induce phosphorylation of signalling proteins in macrophages. However, we found that Tie-2 ligands were not able to regulate pro-inflammatory cytokine secretion, nor were the ligands able to prevent apoptosis induced by lipopolysaccharide in macrophages. These results suggest that contrary to current evidence in the literature, Tie-2 ligands may not play a role in modulating the pro-inflammatory response in TEMs.

Tie-2 expressing monocytes (TEMs) were identified to be a subset of monocytes that was potent inducer of tumor angiogenesis in various types of cancer. On the other hand, monocytes and macrophages are crucially involved in the innate immune response, but the function of TEMs in this process is not well understood. Therefore, we studied the role of Tie-2 in regulating the inflammatory response initiated by lipopolysaccharide in murine macrophages. First, we observed that a small percentage of bone marrow-derived macrophages express Tie-2. Furthermore, we observed that Tie-2 ligands were able to induce phosphorylation of signalling proteins in macrophages. However, we found that Tie-2 ligands were not able to regulate pro-inflammatory cytokine secretion, nor were the ligands able to prevent apoptosis induced by lipopolysaccharide in macrophages. These results suggest that contrary to current evidence in the literature, Tie-2 ligands may not play a role in modulating the pro-inflammatory response in TEMs.

Thesis (Ph.D.)--Virginia Commonwealth University, 2010. / Prepared for: Dept. of Biochemistry. Title from title-page of electronic thesis. Bibliography: leaves 106-117

A atresia biliar (AB) é uma doença da infância caracterizada por uma colangiopatia esclerosante progressiva que leva à obstrução biliar. O tratamento de escolha é a portoenterostomia, cujo prognóstico é relacionado à idade do paciente na época da cirurgia e a variáveis histológicas como a extensão da fibrose e da reação ductular. O espessamento da túnica média (TM) sugere uma arteriopatia na patogenia da AB. Nós avaliamos a expressão do sistema angiopoietinas (ANGPT)/receptor tirosinaquinase com domínios imunoglobulina e EGF(endotelial growth fator) like (Tie2) no fígado de pacientes com AB e com colestase intra-hepática (CIH), correlacionando com espessamento da TM, com variáveis associadas a gravidade da doença e com o prognóstico pós operatório. Métodos - A expressão da ANGPT1, ANGPT2 e Tie2 foi feita com o método de PCR quantitativo em amostras de fígado obtidas de pacientes com AB (n23) na ocasião da portoenterostomia e de crianças de idade semelhante com CIH (n7). As variáveis histológicas foram analisadas por método morfométrico. Resultados - A ANGPT1 e a ANGPT2 apresentaram expressão aumentada no grupo com AB em comparação com o grupo com CIH (P=0,024 e P=0,029, respectivamente). No grupo com AB, a expressão das ANPTs correlacionouse positivamente com a espessura da TM (ANGPT1: rs=0,59, P=0,013; ANGPT2: rs=0,52, P=0,032) e não teve correlação com variáveis associadas a gravidade da doença. O Tie2 e as ANGPTs correlacionaram-se negativamente (ANGPT1: rs=-0,73, P<0,001; ANGPT2: rs=-0,54, P=0,007). Conclusão - Na AB há uma expressão aumentada da ANGPT1 e da ANGPT2 e uma correlação positiva desta expressão com a espessura da TM, mas, não com a idade na ocasião da portoenterostomia ou com variáveis histológicas associadas com a gravidade da doença na época do procedimento. / Background- Biliary atresia (BA) is an infantile disorder characterized by progressive sclerosing cholangiopathy leading to biliary obstruction. First line treatment of BA is hepatoporto-enterostomy, whose prognosis is related to age at surgery and to histologic variables such as extent of fibrosis and ductular reaction. Hepatic arterial medial thickening (MT) suggests an arteriopathy in BA pathogenesis. We evaluated the expression of angiopoietin (ANGPT)/tyrosine kinase with immunoglobulin-like and EGF-like domains 2 (Tie2) system in livers from patients with BA, correlating with MT, variables associated with disease severity and postoperative prognosis. Methods- ANGPT1, ANGPT2 and Tie2 expressions were assessed by qPCR in liver samples obtained from BA patients (n=23) at portoenterostomy and age-matched infants with intrahepatic cholestasis (IHC, n=7). Histologic variables were morphometrically assessed. Results- ANGPT1 and ANGPT2 were overexpressed in BA in comparison with IHC (respectively, P=0.024 and P=0.029). In BA, ANGPTs′ expression was positively correlated with MT (ANGPT1:rs=0.59, P=0.013; ANGPT2:rs=0.52, P=0.032), not with variables associated with disease severity. Tie2 and ANGPTs′ expressions were negatively correlated (ANGPT1: rs=-0.73, P<0.001; ANGPT2: rs=- 0.54, P=0.007). Conclusion- In BA there is overexpression of both ANGPT1 and ANGPT2 correlated with MT but not with age at portoenterostomy or with the histological variables associated with disease severity at the procedure.

A atresia biliar (AB) é uma doença da infância caracterizada por uma colangiopatia esclerosante progressiva que leva à obstrução biliar. O tratamento de escolha é a portoenterostomia, cujo prognóstico é relacionado à idade do paciente na época da cirurgia e a variáveis histológicas como a extensão da fibrose e da reação ductular. O espessamento da túnica média (TM) sugere uma arteriopatia na patogenia da AB. Nós avaliamos a expressão do sistema angiopoietinas (ANGPT)/receptor tirosinaquinase com domínios imunoglobulina e EGF(endotelial growth fator) like (Tie2) no fígado de pacientes com AB e com colestase intra-hepática (CIH), correlacionando com espessamento da TM, com variáveis associadas a gravidade da doença e com o prognóstico pós operatório. Métodos - A expressão da ANGPT1, ANGPT2 e Tie2 foi feita com o método de PCR quantitativo em amostras de fígado obtidas de pacientes com AB (n23) na ocasião da portoenterostomia e de crianças de idade semelhante com CIH (n7). As variáveis histológicas foram analisadas por método morfométrico. Resultados - A ANGPT1 e a ANGPT2 apresentaram expressão aumentada no grupo com AB em comparação com o grupo com CIH (P=0,024 e P=0,029, respectivamente). No grupo com AB, a expressão das ANPTs correlacionouse positivamente com a espessura da TM (ANGPT1: rs=0,59, P=0,013; ANGPT2: rs=0,52, P=0,032) e não teve correlação com variáveis associadas a gravidade da doença. O Tie2 e as ANGPTs correlacionaram-se negativamente (ANGPT1: rs=-0,73, P<0,001; ANGPT2: rs=-0,54, P=0,007). Conclusão - Na AB há uma expressão aumentada da ANGPT1 e da ANGPT2 e uma correlação positiva desta expressão com a espessura da TM, mas, não com a idade na ocasião da portoenterostomia ou com variáveis histológicas associadas com a gravidade da doença na época do procedimento. / Background- Biliary atresia (BA) is an infantile disorder characterized by progressive sclerosing cholangiopathy leading to biliary obstruction. First line treatment of BA is hepatoporto-enterostomy, whose prognosis is related to age at surgery and to histologic variables such as extent of fibrosis and ductular reaction. Hepatic arterial medial thickening (MT) suggests an arteriopathy in BA pathogenesis. We evaluated the expression of angiopoietin (ANGPT)/tyrosine kinase with immunoglobulin-like and EGF-like domains 2 (Tie2) system in livers from patients with BA, correlating with MT, variables associated with disease severity and postoperative prognosis. Methods- ANGPT1, ANGPT2 and Tie2 expressions were assessed by qPCR in liver samples obtained from BA patients (n=23) at portoenterostomy and age-matched infants with intrahepatic cholestasis (IHC, n=7). Histologic variables were morphometrically assessed. Results- ANGPT1 and ANGPT2 were overexpressed in BA in comparison with IHC (respectively, P=0.024 and P=0.029). In BA, ANGPTs′ expression was positively correlated with MT (ANGPT1:rs=0.59, P=0.013; ANGPT2:rs=0.52, P=0.032), not with variables associated with disease severity. Tie2 and ANGPTs′ expressions were negatively correlated (ANGPT1: rs=-0.73, P<0.001; ANGPT2: rs=- 0.54, P=0.007). Conclusion- In BA there is overexpression of both ANGPT1 and ANGPT2 correlated with MT but not with age at portoenterostomy or with the histological variables associated with disease severity at the procedure.

A atresia biliar (AB) é uma doença da infância caracterizada por uma colangiopatia esclerosante progressiva que leva à obstrução biliar. O tratamento de escolha é a portoenterostomia, cujo prognóstico é relacionado à idade do paciente na época da cirurgia e a variáveis histológicas como a extensão da fibrose e da reação ductular. O espessamento da túnica média (TM) sugere uma arteriopatia na patogenia da AB. Nós avaliamos a expressão do sistema angiopoietinas (ANGPT)/receptor tirosinaquinase com domínios imunoglobulina e EGF(endotelial growth fator) like (Tie2) no fígado de pacientes com AB e com colestase intra-hepática (CIH), correlacionando com espessamento da TM, com variáveis associadas a gravidade da doença e com o prognóstico pós operatório. Métodos - A expressão da ANGPT1, ANGPT2 e Tie2 foi feita com o método de PCR quantitativo em amostras de fígado obtidas de pacientes com AB (n23) na ocasião da portoenterostomia e de crianças de idade semelhante com CIH (n7). As variáveis histológicas foram analisadas por método morfométrico. Resultados - A ANGPT1 e a ANGPT2 apresentaram expressão aumentada no grupo com AB em comparação com o grupo com CIH (P=0,024 e P=0,029, respectivamente). No grupo com AB, a expressão das ANPTs correlacionouse positivamente com a espessura da TM (ANGPT1: rs=0,59, P=0,013; ANGPT2: rs=0,52, P=0,032) e não teve correlação com variáveis associadas a gravidade da doença. O Tie2 e as ANGPTs correlacionaram-se negativamente (ANGPT1: rs=-0,73, P<0,001; ANGPT2: rs=-0,54, P=0,007). Conclusão - Na AB há uma expressão aumentada da ANGPT1 e da ANGPT2 e uma correlação positiva desta expressão com a espessura da TM, mas, não com a idade na ocasião da portoenterostomia ou com variáveis histológicas associadas com a gravidade da doença na época do procedimento. / Background- Biliary atresia (BA) is an infantile disorder characterized by progressive sclerosing cholangiopathy leading to biliary obstruction. First line treatment of BA is hepatoporto-enterostomy, whose prognosis is related to age at surgery and to histologic variables such as extent of fibrosis and ductular reaction. Hepatic arterial medial thickening (MT) suggests an arteriopathy in BA pathogenesis. We evaluated the expression of angiopoietin (ANGPT)/tyrosine kinase with immunoglobulin-like and EGF-like domains 2 (Tie2) system in livers from patients with BA, correlating with MT, variables associated with disease severity and postoperative prognosis. Methods- ANGPT1, ANGPT2 and Tie2 expressions were assessed by qPCR in liver samples obtained from BA patients (n=23) at portoenterostomy and age-matched infants with intrahepatic cholestasis (IHC, n=7). Histologic variables were morphometrically assessed. Results- ANGPT1 and ANGPT2 were overexpressed in BA in comparison with IHC (respectively, P=0.024 and P=0.029). In BA, ANGPTs′ expression was positively correlated with MT (ANGPT1:rs=0.59, P=0.013; ANGPT2:rs=0.52, P=0.032), not with variables associated with disease severity. Tie2 and ANGPTs′ expressions were negatively correlated (ANGPT1: rs=-0.73, P<0.001; ANGPT2: rs=- 0.54, P=0.007). Conclusion- In BA there is overexpression of both ANGPT1 and ANGPT2 correlated with MT but not with age at portoenterostomy or with the histological variables associated with disease severity at the procedure.

Förster Resonance Energy Transfer (FRET)-based techniques are gaining an increasing importance in cell biology and cell-matrix adhesion studies because they allow both the detection of conformational changes of target proteins and their localisation in cells. Frequency Domain-Fluorescence Lifetime Microscopy (FD-FLIM) is currently considered one of the most reliable methods to measure FRET in live cells. However, due to its dependence on many technical prerequisites, its use is not yet widespread. The purpose of this work was to first establish FD-FLIM measurements of FRET on a new FD-FLIM microscope module. Then we aimed to apply FD-FLIM-FRET measurements to the study of conformational changes of the cell matrix-adhesion proteins vinculin and integrin and of the growth factor receptor Tie-2. In the first part of the work, published FRET probes including distance-sensors and two sets of vinculin-based probes were extensively tested with FD-FLIM, sensitised emission and ratiometric FRET. FD-FLIM was shown to be the most accurate method in approximating molecular distances between fluorophores. Moreover this study unveiled specific caveats associated with both existing vinculin FRET probes. FD-FLIM was then used to study conformational changes of the extracellular matrix receptor alphavβ3 integrin and of the angiopoietin receptor Tie-2 using specific FRET probes designed by us. While data showed that the alphav-integrin-FRET probe localised to adhesion sites, more experiments will be required to evaluate its full functionality. The Tie-2-FRET probe was fully functional and, upon ligand binding, allowed the detection of a bending movement of the extracellular domain towards the cell membrane. Finally, a combination of FRET, immunofluorescence and tension release experiments were used to show that intracellular tension is not required to maintain integrins in their activated conformation. However, intracellular tension is required to recruit other key proteins such as vinculin, talin and tensin to adhesions sites. Overall this work demonstrates the importance of FD-FLIM-FRET as a tool to investigate conformational changes of adhesion proteins and transmembrane receptors within the cell environment.