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Angiogenesis and intravasation of PC-3 dissemination variants
Abstracts
8 Induction of physiologic and pathologic angiogenesis by TIMP-free proMMP-9 derived uniquely from inflammatory neutrophils James P. Quigley Department of Cell Biology, The Scripps Research Institute, La Jolla, CA, United States Angiogenesis, the process by which normal or tumor-infiltrated tissues become newly vascularized, is generally believed to be induced by a number of well-established growth factors such as VEGF. Molecules that might inductively operate directly upstream of the angiogenic growth factors are not so wellestablished. Our laboratory, using both chick embryo and mouse models, has demonstrated that proMMP-9 is a potent angiogenesis inducer at subnanomolar levels. In order to function as an inducer of angiogenesis, the proMMP-9 must be completely free of its normally tightly-associated TIMP-1 molecule. A purified preparation of the proMMP-9-TIMP-1 complex, which is the natural form of MMP-9 produced by most cell types, is not angiogenic even when added at 20 fold molar excess over the TIMP-1-free proMMP-9. Inflammatory neutrophils appear to be one of the only cell types that produce proMMP-9 in the TIMP-1-free form. These results are consistent with a large amount of data from a number of laboratories demonstrating a strong link between rapidly infiltrating inflammatory neutrophils and subsequent angiogenesis in those infiltrated tissues. We recently explored mechanistically the biochemical features and reasons for the angiogenic potency of neutrophil TIMPfree proMMP-9. Employing domain variants and mutants of human MMP-9, it appears that an intact catalytic site and the presence of the hemopexin domain are required for MMP-9's angiogenic ability. The proMMP-9 zymogen must be activated and manifest its catalytic activity within the first 24 h of a 70–80 hour in vivo angiogenesis assay. The proMMP-9 in the natural stoichiometric complex with TIMP-1 fails to undergo either proteolytic or chemical activation, and this we suggest is the mechanistic reason for TIMP-1's angiogenic dampening abililty. Interestingly, the catalytic activity of activated TIMP-1-free MMP-9 induces a significant release of basic fibroblast growth factor (FGF-2) over that of VEGF. Furthermore, MMP-9's pronounced angiogenic capabilities are blocked by specific antibodies directed to FGF-2 and the FGF receptor, FGFR2. Finally, neutrophil TIMP-1-free proMMP-9 stimulates angiogenesis into a non-aggressive, non-angiogenic tumor developing on the chick embryo and the induced angiogenesis is blocked by anti-FGF-2 antibodies. In summary, inflammatory neutrophils rapidly deliver a potent angiogenic inducer to tissues that are to undergo neovascularization. The inducer, TIMP-1-free proMMP-9, is rapidly activated and proteolytically enhances the influx and generation of a number of protein factors, one of which, FGF-2, appears to be, within our in vivo models, the major downstream inducer of new blood vessel formation. doi:10.1016/j.matbio.2008.09.223
9 Fibulin-5 inhibits integrin-induced ROS production Marie K. Schlutermana, Shelby L. Chapmanb, Grzegorz Korpantya, Hiromi Yanagisawab, Rolf A. Brekkena a Hamon Center for Therapeutic Oncology Research and Department of Surgery, UT-Southwestern, Dallas, TX 75390, United States b Department of Molecular Biology, UT-Southwestern, Dallas, TX 75390, United States Fibulin-5 (Fbln5), an integrin-binding matricellular protein, is expressed by vascular cells and is critical for the appropriate deposition and function of the vascular extracellular matrix. Here, we demonstrate that Fbln5 inhibits the production of reactive oxygen species (ROS) in an integrin-dependent manner and through this pathway exerts control over the angiogenic process. We found that cells lacking endogenous Fbln5 produced higher levels of ROS in a β1 integrin and fibronectin-dependent manner in vitro. Additionally, we identified that elevated ROS in Fbln5-deficient (Fbln5−/−) animals is critical for vascular invasion of matrigel implants but detrimental to angiogenesis and growth of syngeneic tumors. Importantly, both effects were reverted with anti-oxidant treatment. We conclude that Fbln5 is a central and critical control factor in the regulation of the level of ROS in the vascular microenvironment, alteration of
S11
which can promote or suppress angiogenesis. These results suggest that Fbln5 may provide a unique target for therapy of angiogenesis-dependent disease. doi:10.1016/j.matbio.2008.09.224
10 Angiogenesis and intravasation of PC-3 dissemination variants Erin M. Conna,b, Elena I. Deryuginaa, James P. Quigleya a Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, United States b University of California–San Diego, Molecular Pathology Graduate Program, La Jolla, CA 92037, United States Intravasation, i.e. the entry of aggressive tumor cells into the vasculature, is one early and potentially rate-limiting step in the metastatic cascade. To analyze this process, our lab has selected in vivo high and low disseminating cell variants derived from the human PC-3 prostate carcinoma cell line, i.e., PC-hi/diss and PClo/diss, using the human tumor-chick embryo spontaneous metastasis model. In this model, human tumor cells are grafted onto the chorioallantoic membrane (CAM) where they rapidly form tumors and intravasate into the vasculature. Cells disseminated to distant sites can be quantified by Alu-qPCR analysis. Using both in vitro and in vivo assays to recapitulate processes involved in intravasation, we have dissected out individual rate-limiting steps that are more successfully completed by PC-hi/diss. Notably, coinciding with enhanced intravasation potential, only PChi/diss induces a high level of tumor angiogenesis in chick and murine models, mediated by VEGF and IL-8 pathways. The new angiogenic vessels recruited by PChi/diss may serve as conduits for tumor cell intravasation and dissemination. The contributions of neoangiogenesis to PC-hi/diss dissemination can be determined by analyzing the functional relationships between these processes. In addition, PChi/diss is more migratory and invasive in vitro, suggesting enhanced ability to escape from the primary tumor and invade local stroma in vivo. Analyses of levels and activation of specific matrix proteases, performed to examine the mechanism of this differential, have revealed a functionally important increase in uPA expression in PC-hi/diss. Overall, the in vivo selection and comparative analysis of PC-3 intravasation variants is useful for identification of individual rate-limiting steps and key molecular changes ultimately leading to tumor cell intravasation. doi:10.1016/j.matbio.2008.09.225
11 Endorepellin evokes SHP1 activity in endothelial cells Alexander Nystroma, Zabeena P. Shaika, Ambra Pozzib, Thomas Kriegc, Beate Eckesc, Renato V. Iozzoa a Thomas Jefferson University, Philadelphia, PA, United States b Vanderbilt University, Nashville, TN, United States c University of Cologne, Cologne, Germany Endorepellin, the C-terminal domain of perlecan, is a powerful inhibitor of angiogenesis. Here we aimed to further dissect the mechanisms of endorepellin-mediated endothelial silencing. Antibody arrays against tyrosine kinase receptors showed that endothelial cells exposed to endorepellin exhibited widespread reduction in overall receptor phosphorylation, especially affecting key receptors involved in regulating angiogenesis. Functionblocking antibodies against the integrin α2β1 abrogated the global dephosphorylation evoked by endorepellin suggesting that endorepellin activated tyrosine phosphatases via α2β1 integrin outside–in signaling. Tyrosine phosphatase assays showed an increase in general phosphatase activity after endorepellin treatment. Strong genetic evidence was provided by the lack of response in microvascular endothelial cells derived from α2β1 integrin null mice. SHP1, a tyrosine phosphatase, co-precipitated with the integrin α2 subunit and was phosphorylated in a dynamic fashion after endorepellin stimulation. Immunoblot of organs from integrin α2β1-deficient mice revealed a significant decrease in SHP1 content as compared to wild-type organs. Conversely, siRNAmediated knockdown of the integrin α2 subunit caused a dose-dependent reduction of SHP1. Hence, both biochemical and genetic evidence strongly
8 Induction of physiologic and pathologic angiogenesis by TIMP-free proMMP-9 derived uniquely from inflammatory neutrophils James P. Quigley Department of Cell Biology, The Scripps Research Institute, La Jolla, CA, United States Angiogenesis, the process by which normal or tumor-infiltrated tissues become newly vascularized, is generally believed to be induced by a number of well-established growth factors such as VEGF. Molecules that might inductively operate directly upstream of the angiogenic growth factors are not so wellestablished. Our laboratory, using both chick embryo and mouse models, has demonstrated that proMMP-9 is a potent angiogenesis inducer at subnanomolar levels. In order to function as an inducer of angiogenesis, the proMMP-9 must be completely free of its normally tightly-associated TIMP-1 molecule. A purified preparation of the proMMP-9-TIMP-1 complex, which is the natural form of MMP-9 produced by most cell types, is not angiogenic even when added at 20 fold molar excess over the TIMP-1-free proMMP-9. Inflammatory neutrophils appear to be one of the only cell types that produce proMMP-9 in the TIMP-1-free form. These results are consistent with a large amount of data from a number of laboratories demonstrating a strong link between rapidly infiltrating inflammatory neutrophils and subsequent angiogenesis in those infiltrated tissues. We recently explored mechanistically the biochemical features and reasons for the angiogenic potency of neutrophil TIMPfree proMMP-9. Employing domain variants and mutants of human MMP-9, it appears that an intact catalytic site and the presence of the hemopexin domain are required for MMP-9's angiogenic ability. The proMMP-9 zymogen must be activated and manifest its catalytic activity within the first 24 h of a 70–80 hour in vivo angiogenesis assay. The proMMP-9 in the natural stoichiometric complex with TIMP-1 fails to undergo either proteolytic or chemical activation, and this we suggest is the mechanistic reason for TIMP-1's angiogenic dampening abililty. Interestingly, the catalytic activity of activated TIMP-1-free MMP-9 induces a significant release of basic fibroblast growth factor (FGF-2) over that of VEGF. Furthermore, MMP-9's pronounced angiogenic capabilities are blocked by specific antibodies directed to FGF-2 and the FGF receptor, FGFR2. Finally, neutrophil TIMP-1-free proMMP-9 stimulates angiogenesis into a non-aggressive, non-angiogenic tumor developing on the chick embryo and the induced angiogenesis is blocked by anti-FGF-2 antibodies. In summary, inflammatory neutrophils rapidly deliver a potent angiogenic inducer to tissues that are to undergo neovascularization. The inducer, TIMP-1-free proMMP-9, is rapidly activated and proteolytically enhances the influx and generation of a number of protein factors, one of which, FGF-2, appears to be, within our in vivo models, the major downstream inducer of new blood vessel formation. doi:10.1016/j.matbio.2008.09.223
9 Fibulin-5 inhibits integrin-induced ROS production Marie K. Schlutermana, Shelby L. Chapmanb, Grzegorz Korpantya, Hiromi Yanagisawab, Rolf A. Brekkena a Hamon Center for Therapeutic Oncology Research and Department of Surgery, UT-Southwestern, Dallas, TX 75390, United States b Department of Molecular Biology, UT-Southwestern, Dallas, TX 75390, United States Fibulin-5 (Fbln5), an integrin-binding matricellular protein, is expressed by vascular cells and is critical for the appropriate deposition and function of the vascular extracellular matrix. Here, we demonstrate that Fbln5 inhibits the production of reactive oxygen species (ROS) in an integrin-dependent manner and through this pathway exerts control over the angiogenic process. We found that cells lacking endogenous Fbln5 produced higher levels of ROS in a β1 integrin and fibronectin-dependent manner in vitro. Additionally, we identified that elevated ROS in Fbln5-deficient (Fbln5−/−) animals is critical for vascular invasion of matrigel implants but detrimental to angiogenesis and growth of syngeneic tumors. Importantly, both effects were reverted with anti-oxidant treatment. We conclude that Fbln5 is a central and critical control factor in the regulation of the level of ROS in the vascular microenvironment, alteration of
S11
which can promote or suppress angiogenesis. These results suggest that Fbln5 may provide a unique target for therapy of angiogenesis-dependent disease. doi:10.1016/j.matbio.2008.09.224
10 Angiogenesis and intravasation of PC-3 dissemination variants Erin M. Conna,b, Elena I. Deryuginaa, James P. Quigleya a Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, United States b University of California–San Diego, Molecular Pathology Graduate Program, La Jolla, CA 92037, United States Intravasation, i.e. the entry of aggressive tumor cells into the vasculature, is one early and potentially rate-limiting step in the metastatic cascade. To analyze this process, our lab has selected in vivo high and low disseminating cell variants derived from the human PC-3 prostate carcinoma cell line, i.e., PC-hi/diss and PClo/diss, using the human tumor-chick embryo spontaneous metastasis model. In this model, human tumor cells are grafted onto the chorioallantoic membrane (CAM) where they rapidly form tumors and intravasate into the vasculature. Cells disseminated to distant sites can be quantified by Alu-qPCR analysis. Using both in vitro and in vivo assays to recapitulate processes involved in intravasation, we have dissected out individual rate-limiting steps that are more successfully completed by PC-hi/diss. Notably, coinciding with enhanced intravasation potential, only PChi/diss induces a high level of tumor angiogenesis in chick and murine models, mediated by VEGF and IL-8 pathways. The new angiogenic vessels recruited by PChi/diss may serve as conduits for tumor cell intravasation and dissemination. The contributions of neoangiogenesis to PC-hi/diss dissemination can be determined by analyzing the functional relationships between these processes. In addition, PChi/diss is more migratory and invasive in vitro, suggesting enhanced ability to escape from the primary tumor and invade local stroma in vivo. Analyses of levels and activation of specific matrix proteases, performed to examine the mechanism of this differential, have revealed a functionally important increase in uPA expression in PC-hi/diss. Overall, the in vivo selection and comparative analysis of PC-3 intravasation variants is useful for identification of individual rate-limiting steps and key molecular changes ultimately leading to tumor cell intravasation. doi:10.1016/j.matbio.2008.09.225
11 Endorepellin evokes SHP1 activity in endothelial cells Alexander Nystroma, Zabeena P. Shaika, Ambra Pozzib, Thomas Kriegc, Beate Eckesc, Renato V. Iozzoa a Thomas Jefferson University, Philadelphia, PA, United States b Vanderbilt University, Nashville, TN, United States c University of Cologne, Cologne, Germany Endorepellin, the C-terminal domain of perlecan, is a powerful inhibitor of angiogenesis. Here we aimed to further dissect the mechanisms of endorepellin-mediated endothelial silencing. Antibody arrays against tyrosine kinase receptors showed that endothelial cells exposed to endorepellin exhibited widespread reduction in overall receptor phosphorylation, especially affecting key receptors involved in regulating angiogenesis. Functionblocking antibodies against the integrin α2β1 abrogated the global dephosphorylation evoked by endorepellin suggesting that endorepellin activated tyrosine phosphatases via α2β1 integrin outside–in signaling. Tyrosine phosphatase assays showed an increase in general phosphatase activity after endorepellin treatment. Strong genetic evidence was provided by the lack of response in microvascular endothelial cells derived from α2β1 integrin null mice. SHP1, a tyrosine phosphatase, co-precipitated with the integrin α2 subunit and was phosphorylated in a dynamic fashion after endorepellin stimulation. Immunoblot of organs from integrin α2β1-deficient mice revealed a significant decrease in SHP1 content as compared to wild-type organs. Conversely, siRNAmediated knockdown of the integrin α2 subunit caused a dose-dependent reduction of SHP1. Hence, both biochemical and genetic evidence strongly