- Email: [email protected]
Endothelial cell to cell junctions and the control of vascular permeability
Abstracts
models. Furthermore, a combination of antibodies that inhibit ligand binding and receptor dimerization by an allosteric effect was found to provide improved anti-angiogenic efficacy. Our studies indicate that VEGF-C and VEGFR-3 provide new targets to complement current antiangiogenic therapies. Because of their ability to attenuate angiogenic sprouting and inhibit lymphatic metastasis, VEGFR-3 blocking antibodies are now being tested in phase I clinical (safety) trials. Furthermore, our studies indicate that antibody combinations may be used for increased efficacy of inhibition of angiogenic signal transduction pathways. 1. Tammela T. and Alitalo, K. Lymphangiogenesis: molecular mechanisms and future promise. Cell, 140: 460–76, 2010. 2. Leppänen et al., Structural determinants of growth factor binding and specificity by VEGF receptor 2. Proc. Natl. Acad. Sci. USA, 107: 2425–30, 2010. 3. Tvorogov D. Et al., Effective suppression of vascular network formation by combination of antibodies blocking VEGFR ligand binding and receptor dimerization. Cancer Cell, 18: 630–40, 2010. 4. Leppänen VM, Jeltsch M, Anisimov A, Tvorogov D, Aho K, Kalkkinen N, Toivanen P, Ylä-Herttuala S, Ballmer-Hofer K, Alitalo K: Structural determinants of vascular endothelial growth factor-D — receptor binding and specificity. Blood, 117: 1507–15, 2011. 5. Lähteenvuo M, Honkonen K, Tervala T, Tammela T, Suominen E, Lähteenvuo J, Kholová I, Alitalo K, Ylä-Herttuala S, Saaristo A. Growth factor therapy and autologous lymph node transfer in lymphedema. Circulation, 123: 613–20, 2011. 6. Tammela, T. Saaristo, A. Holopainen, T. Ylä-Herttuala, S. Andersson, L. Virolainen, S. Immonen, I., Alitalo, K. Photodynamic ablation of lymphatic vessels and intralymphatic cancer cells prevents metastasis. Science Translational Medicine, 3: 69ra11, 2011. doi:10.1016/j.vph.2011.08.006
PL.1.3 Endothelial cell to cell junctions and the control of vascular permeability Elisabetta Dejana IFOM, FIRC Institute of Molecular Oncology and University of Milan, Milan, Italy E-mail address: [email protected] The control of vascular permeability has a pivotal importance in several vascular pathologies which include inflammatory reactions, interstitial edema and leukocyte extravasation. Different agents such as histamine or bradykinin act on endothelial cells to induce a quick and reversible increase in permeability. Plasma fluids can cross the endothelial barrier through specialized cell to cell junctions which include tight and adherens junctions (AJ). At AJ, adhesion is mediated by members of the cadherin family and, more specifically, in the endothelium by a cell specific cadherin, called vascular endothelial cadherin (VEC). VEC promotes homophilic adhesion and forms zipper like structures along endothelial cell to cell contacts. The cytoplasmic domain of VEC interacts with several intracellular partners including beta-catenin, plakoglobin and p120 which transfer intracellular signals and modulate the interaction of AJs with actin cytoskeleton. Blocking antibodies to VEC induce a dramatic increase in lung and heart permeability accompanied by edema and hemorrhages. More subtle changes in AJ organization and strength are associated with tyrosine phosphorylation of VEC cytoplasmic domain. Most of our knowledge is based on experiments performed on cultured endothelial cells while vessels respond quite differently to permeability increasing factors (PIFs) in vivo. Venules are the typical targets of PIFs in inflammatory conditions while small caliber arteries are poorly affected. We know little about the molecular basis of the different A/V responses which may be due to
307
intrinsic properties of the endothelium or to the hemodynamic conditions to which the vessels are exposed. Using specific anti phospho-VEC antibodies we found that VEC is constantly tyrosine phosphorylated in vivo but only in veins and not in arteries. VEC phosphorylation is mediated by a mechanosensor which activates junctional src under venous but not arterial shear stress. Phosphorylation makes VEC particularly dynamic and sensitive to BK-induced endocytosis which, in turn, mediates a rapid increase in permeability. Angiopoietin 1, a known inhibitor of vascular permeability does not inhibit VEC phosphorylation but blocks its endocytosis. We conclude that phosphorylation of VEC in veins is a prerequisite for a quick endocytosis and recycling of the protein at cell to cell junctions in vivo. These data reconcile a large set of previous observations within a coherent sequence of events and set the basis to understand, at a molecular level, the different response of arteries and veins to permeability. doi:10.1016/j.vph.2011.08.007
SESSION 1 Mechanisms of vasculogenesis and angiogenesis L.1.1 Regulation of angiogenesis and vascular homeostasis through the Angiopoietin/Tie system Hellmut G. Augustin Vascular Biology, Medical Faculty Mannheim, Heidelberg University (CBTM), and German Cancer Research Center Heidelberg (DKFZ-ZMBH Alliance), Germany E-mail address: [email protected] It is well established that anti-VEGF/VEGFR therapies do not starve tumors to death by driving the tumor-associated vasculature into massive regression. Instead, anti-VEGF/VEGFR therapies prune the immature vascular tree within tumors to yield a thinned, more normally appearing and functioning vasculature. As such, the relative degree of vessel maturation has major impact on the efficacy and the overall therapeutic window of anti-VEGF/VEGFR therapies. Consequently, the manipulation of molecules affecting vessel maturation holds great promise to combine with established anti-VEGF/VEGFR therapies for an optimized rationally-designed combination therapy aimed at maximally exploiting the benefits of anti-angiogenic tumor intervention. The Angiopoietin/Tie system is a prototypic vessel maturation controlling molecular system. Angiopoietin-1 (Ang-1) acts as the agonistic ligand of the vascular RTK Tie2 controlling vessel remodeling, maturation and quiescence. In turn, Angiopoietin-2 (Ang-2) acts as antagonistic, vessel destabilizing ligand facilitating the response of exogenous cytokines. As such, it has been predicted that Ang-2 in the presence of angiogenic activity will facilitate the angiogenic response, whereas Ang-2 in the absence of angiogenic activity will drive vessels into regression. Such model is supported by genetic experiments which have shown that loss of Ang-2 blocks physiological vessel regression processes, e.g. leading to persistent hyaloid vessels or perturbed vessel regression in the cyclic ovary. In turn, Ang-2 has also been proposed to contextually promote angiogenesis. A number of studies have suggested that Ang-2 blocking reagents may significantly inhibit tumor angiogenesis and tumor growth. Recently, the combination of anti-VEGF/VEGFR and anti-Ang-2 has been proposed to exert excellent synergistic anti-angiogenic and anti-tumor effects which appears counterintuitive in light of the genetic data suggesting a vessel maintaining function of anti-Ang-2. This presentation will discuss Ang-2 functions in relation to endothelial cell phenotypes during angiogenesis and vascular homeostasis to discuss differential effects of Ang-2 on angiogenic tip cells vs. remodeling stalk cells vs. quiescent phalanx cells. A model of differential pro-angiogenic vs. vessel
models. Furthermore, a combination of antibodies that inhibit ligand binding and receptor dimerization by an allosteric effect was found to provide improved anti-angiogenic efficacy. Our studies indicate that VEGF-C and VEGFR-3 provide new targets to complement current antiangiogenic therapies. Because of their ability to attenuate angiogenic sprouting and inhibit lymphatic metastasis, VEGFR-3 blocking antibodies are now being tested in phase I clinical (safety) trials. Furthermore, our studies indicate that antibody combinations may be used for increased efficacy of inhibition of angiogenic signal transduction pathways. 1. Tammela T. and Alitalo, K. Lymphangiogenesis: molecular mechanisms and future promise. Cell, 140: 460–76, 2010. 2. Leppänen et al., Structural determinants of growth factor binding and specificity by VEGF receptor 2. Proc. Natl. Acad. Sci. USA, 107: 2425–30, 2010. 3. Tvorogov D. Et al., Effective suppression of vascular network formation by combination of antibodies blocking VEGFR ligand binding and receptor dimerization. Cancer Cell, 18: 630–40, 2010. 4. Leppänen VM, Jeltsch M, Anisimov A, Tvorogov D, Aho K, Kalkkinen N, Toivanen P, Ylä-Herttuala S, Ballmer-Hofer K, Alitalo K: Structural determinants of vascular endothelial growth factor-D — receptor binding and specificity. Blood, 117: 1507–15, 2011. 5. Lähteenvuo M, Honkonen K, Tervala T, Tammela T, Suominen E, Lähteenvuo J, Kholová I, Alitalo K, Ylä-Herttuala S, Saaristo A. Growth factor therapy and autologous lymph node transfer in lymphedema. Circulation, 123: 613–20, 2011. 6. Tammela, T. Saaristo, A. Holopainen, T. Ylä-Herttuala, S. Andersson, L. Virolainen, S. Immonen, I., Alitalo, K. Photodynamic ablation of lymphatic vessels and intralymphatic cancer cells prevents metastasis. Science Translational Medicine, 3: 69ra11, 2011. doi:10.1016/j.vph.2011.08.006
PL.1.3 Endothelial cell to cell junctions and the control of vascular permeability Elisabetta Dejana IFOM, FIRC Institute of Molecular Oncology and University of Milan, Milan, Italy E-mail address: [email protected] The control of vascular permeability has a pivotal importance in several vascular pathologies which include inflammatory reactions, interstitial edema and leukocyte extravasation. Different agents such as histamine or bradykinin act on endothelial cells to induce a quick and reversible increase in permeability. Plasma fluids can cross the endothelial barrier through specialized cell to cell junctions which include tight and adherens junctions (AJ). At AJ, adhesion is mediated by members of the cadherin family and, more specifically, in the endothelium by a cell specific cadherin, called vascular endothelial cadherin (VEC). VEC promotes homophilic adhesion and forms zipper like structures along endothelial cell to cell contacts. The cytoplasmic domain of VEC interacts with several intracellular partners including beta-catenin, plakoglobin and p120 which transfer intracellular signals and modulate the interaction of AJs with actin cytoskeleton. Blocking antibodies to VEC induce a dramatic increase in lung and heart permeability accompanied by edema and hemorrhages. More subtle changes in AJ organization and strength are associated with tyrosine phosphorylation of VEC cytoplasmic domain. Most of our knowledge is based on experiments performed on cultured endothelial cells while vessels respond quite differently to permeability increasing factors (PIFs) in vivo. Venules are the typical targets of PIFs in inflammatory conditions while small caliber arteries are poorly affected. We know little about the molecular basis of the different A/V responses which may be due to
307
intrinsic properties of the endothelium or to the hemodynamic conditions to which the vessels are exposed. Using specific anti phospho-VEC antibodies we found that VEC is constantly tyrosine phosphorylated in vivo but only in veins and not in arteries. VEC phosphorylation is mediated by a mechanosensor which activates junctional src under venous but not arterial shear stress. Phosphorylation makes VEC particularly dynamic and sensitive to BK-induced endocytosis which, in turn, mediates a rapid increase in permeability. Angiopoietin 1, a known inhibitor of vascular permeability does not inhibit VEC phosphorylation but blocks its endocytosis. We conclude that phosphorylation of VEC in veins is a prerequisite for a quick endocytosis and recycling of the protein at cell to cell junctions in vivo. These data reconcile a large set of previous observations within a coherent sequence of events and set the basis to understand, at a molecular level, the different response of arteries and veins to permeability. doi:10.1016/j.vph.2011.08.007
SESSION 1 Mechanisms of vasculogenesis and angiogenesis L.1.1 Regulation of angiogenesis and vascular homeostasis through the Angiopoietin/Tie system Hellmut G. Augustin Vascular Biology, Medical Faculty Mannheim, Heidelberg University (CBTM), and German Cancer Research Center Heidelberg (DKFZ-ZMBH Alliance), Germany E-mail address: [email protected] It is well established that anti-VEGF/VEGFR therapies do not starve tumors to death by driving the tumor-associated vasculature into massive regression. Instead, anti-VEGF/VEGFR therapies prune the immature vascular tree within tumors to yield a thinned, more normally appearing and functioning vasculature. As such, the relative degree of vessel maturation has major impact on the efficacy and the overall therapeutic window of anti-VEGF/VEGFR therapies. Consequently, the manipulation of molecules affecting vessel maturation holds great promise to combine with established anti-VEGF/VEGFR therapies for an optimized rationally-designed combination therapy aimed at maximally exploiting the benefits of anti-angiogenic tumor intervention. The Angiopoietin/Tie system is a prototypic vessel maturation controlling molecular system. Angiopoietin-1 (Ang-1) acts as the agonistic ligand of the vascular RTK Tie2 controlling vessel remodeling, maturation and quiescence. In turn, Angiopoietin-2 (Ang-2) acts as antagonistic, vessel destabilizing ligand facilitating the response of exogenous cytokines. As such, it has been predicted that Ang-2 in the presence of angiogenic activity will facilitate the angiogenic response, whereas Ang-2 in the absence of angiogenic activity will drive vessels into regression. Such model is supported by genetic experiments which have shown that loss of Ang-2 blocks physiological vessel regression processes, e.g. leading to persistent hyaloid vessels or perturbed vessel regression in the cyclic ovary. In turn, Ang-2 has also been proposed to contextually promote angiogenesis. A number of studies have suggested that Ang-2 blocking reagents may significantly inhibit tumor angiogenesis and tumor growth. Recently, the combination of anti-VEGF/VEGFR and anti-Ang-2 has been proposed to exert excellent synergistic anti-angiogenic and anti-tumor effects which appears counterintuitive in light of the genetic data suggesting a vessel maintaining function of anti-Ang-2. This presentation will discuss Ang-2 functions in relation to endothelial cell phenotypes during angiogenesis and vascular homeostasis to discuss differential effects of Ang-2 on angiogenic tip cells vs. remodeling stalk cells vs. quiescent phalanx cells. A model of differential pro-angiogenic vs. vessel