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Thrombospondin-1 regulates blood pressure and cardiac response
S32
Abstracts
cell morphology. A direct matrix adhesion is mediated through the binding of uPAR to vitronectin and this event is followed by downstream effects including changes in the cytoskeletal organization. However, it remains unclear if the adhesion through uPAR-vitronectin is the only event capable of initiating these morphological rearrangements, or if lateral interactions between uPAR and other membrane proteins can induce the same response. Here we show that both of these triggering mechanisms can be operative and that uPAR dependent modulation of cell morphology can indeed occur independently of vitronectin binding. Expression of wildtype uPAR on HEK-293 cells led to pronounced vitronectin adhesion and cytoskeletal rearrangements whereas a mutant uPAR, uPARW32A with defective vitronectin binding, failed to induce both phenomena. However, upon saturation of uPARW32A with the protease ligand, pro-uPA or its receptor-binding domain, the ability to induce cytoskeletal rearrangements was restored even though this did not rescue the vitronectin binding and adhesion capability. On the other hand, using other uPAR variants, we could show that uPAR-vitronectin adhesion is indeed capable and sufficient to induce the same morphological rearrangements.
techniques allow the extraction of peptides from FFPE samples for proteomic analysis. We used these methods to test the hypothesis that tumors arising from different tissues can be distinguished by the ECM proteins they make. We chose to study osteosarcoma and chondrosarcoma, both neoplasms of mesenchymal origin, but believed to arise from osteoblasts and chondrocytes, respectively. Importantly, it is often difficult to differentiate chondrosarcoma from osteosarcoma with standard histopathology. Making an accurate diagnosis between the two malignancies is clinically critical because these sarcomas respond differently to chemotherapy. Laser capture microscopy was used to isolate only ECM from precise regions for each tumor type (N = 3). This strategy results in samples of lower complexity, and increases the capacity to detect differences. Samples were treated with proprietary solutions (Expression Pathology, Inc.), trypsinized, 2D liquid chromatographic separation linked to tandem MS/MS. Distinct ECM protein signatures were identified for the two tumor types. This demonstrates that proteomic analysis of FFPE tumor ECM is possible, and has potential for basic and translational research, and for improving clinical diagnostic accuracy.
doi:10.1016/j.matbio.2008.09.307 doi:10.1016/j.matbio.2008.09.309
93 Novel role of Cdk4 in leukocyte adhesion and trafficking Lynn M. Schnappa, Li Liua, Barbara Schwartza, Yu-Hua Chowa, Yoshiaki Tsubotab, Elaine W. Rainesb, John M. Harlana a Department of Medicine, United States b Department of Pathology, University of Washington, Seattle, WA, United States Leukocyte emigration is a tightly regulated process essential for an appropriate inflammatory response. The current paradigm describes discrete steps in the process: rolling, activation, adhesion, and migration through subendothelial extracellular matrix (ECM). We report a new adhesion pathway that may be important in post-trafficking, i.e. mediating leukocyte interactions to and migration through subendothelial ECM. We found several novel features of this pathway that distinguish it from “conventional” stimulated adhesion: it is dependent on Cdk4 activity; it requires microtubules; it does not require exogenous leukocyte activation; and it does not require Rap-1 activity. Because this novel pathway allows leukocyte adhesion to physiological relevant substrates such as exposed endothelial matrix in the absence of exogenous stimulation, we have termed it “Ligand-induced Adhesion”. Monocytes and lymphocytes, but not neutrophils, are capable of LIA using several ligands, including purified FN and endothelial cell-derived ECM. The known substrates of Cdk4, Rb and Smad, are not required for ligandinduced adhesion suggesting the involvement of a novel Cdk4 substrate. Furthermore, we show that mice lacking Cdk4 have impaired recruitment of lymphocytes in bronchoalveolarlavage fluid following bleomycin induced lung injury. Thus, Cdk4 may be a novel therapeutic target for regulating lymphocyte recruitment during lung injury and inflammation. Further characterization of this novel pathway and its role during injury will be critical to our understanding of leukocyte trafficking. doi:10.1016/j.matbio.2008.09.308
94 Tumor typing by proteomic analysis of formalin fixed sample ECM Timothy M. Rittya, Elizabeth Frauenhofferb, Edward J. Foxa, Bruce Stanleyc Orthopaedics, PSU Cancer Inst., Penn St. Coll. of Medicine, Hershey, PA, United States b Path., Penn St. Coll. of Medicine, Hershey, PA, United States c Proteomics Facility, Penn St. Coll. of Medicine, Hershey, PA, United States
a
A vast and mostly unused resource of tumors and normal stroma is harvested daily by surgical resection, and is formalin fixed/paraffin embedded (FFPE) for pathology. Molecular analysis of FFPE tumors is difficult due to covalent cross-linking induced by formalin. Consequently, these samples are an untapped fund of molecular information. New
95 Thrombospondin-1 regulates blood pressure and cardiac response Jeff S. Isenberga, Yan Qinb, Daryl Despresc, Russell W. Bandlea, Jurgen Schnermannb, William A. Frazierd, David D. Robertsa a Laboratory of Pathology, National Cancer Institute, NIH, Bethesda, MD 20892, United States b Kidney Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD 20892, United States c Mouse Imaging Facility, National Institutes of Health, Bethsda, MD, United States d Department of Biochemistry and Molecular Biophysics, Washington Uni. School of Med., St. Louis, MO, United States Aims Nitric oxide (NO) regulates regional vascular resistance and systemic blood pressure by modulating blood vessel tone. The ability of thrombospondin-1 via its receptor CD47 to locally limit NO-mediated vasodilation and alter regional blood flow suggested that it may also regulate systemic cardiovascular physiology. Methods Wild type, thrombospondin-1 and CD47-null mice underwent analysis of several cardiovascular endpoints including cardiac output, ejection fraction and telemetric blood pressure before and after vasoactive challenge. Analysis of skin blood flow and core temperature change following vasoactive challenge was also performed. Results Mice lacking thrombospondin-1 exhibit an activity-associated increase in diastolic blood pressure and have decreased pulse pressure. CD47deficient mice have elevated resting blood pressure. Both null mice show exaggerated decreases in MAP and pulse pressure and increased cardiac output and ejection fraction in response to NO. They are also resistance to the hypertensive effects of epinephrine. Conversely, autonomic blockade induce exaggerated hypotensive responses in thrombospondin-1 null mice. In combination, these agents cause premature cardiovascular collapse and death of thrombospondin-1 null mice. Conclusions Thrombospondin-1 signaling via CD47 is an acute physiological regulator of blood pressure and maintains blood pressure through resistance of nitric oxide stimulated vasodilation. doi:10.1016/j.matbio.2008.09.310
Abstracts
cell morphology. A direct matrix adhesion is mediated through the binding of uPAR to vitronectin and this event is followed by downstream effects including changes in the cytoskeletal organization. However, it remains unclear if the adhesion through uPAR-vitronectin is the only event capable of initiating these morphological rearrangements, or if lateral interactions between uPAR and other membrane proteins can induce the same response. Here we show that both of these triggering mechanisms can be operative and that uPAR dependent modulation of cell morphology can indeed occur independently of vitronectin binding. Expression of wildtype uPAR on HEK-293 cells led to pronounced vitronectin adhesion and cytoskeletal rearrangements whereas a mutant uPAR, uPARW32A with defective vitronectin binding, failed to induce both phenomena. However, upon saturation of uPARW32A with the protease ligand, pro-uPA or its receptor-binding domain, the ability to induce cytoskeletal rearrangements was restored even though this did not rescue the vitronectin binding and adhesion capability. On the other hand, using other uPAR variants, we could show that uPAR-vitronectin adhesion is indeed capable and sufficient to induce the same morphological rearrangements.
techniques allow the extraction of peptides from FFPE samples for proteomic analysis. We used these methods to test the hypothesis that tumors arising from different tissues can be distinguished by the ECM proteins they make. We chose to study osteosarcoma and chondrosarcoma, both neoplasms of mesenchymal origin, but believed to arise from osteoblasts and chondrocytes, respectively. Importantly, it is often difficult to differentiate chondrosarcoma from osteosarcoma with standard histopathology. Making an accurate diagnosis between the two malignancies is clinically critical because these sarcomas respond differently to chemotherapy. Laser capture microscopy was used to isolate only ECM from precise regions for each tumor type (N = 3). This strategy results in samples of lower complexity, and increases the capacity to detect differences. Samples were treated with proprietary solutions (Expression Pathology, Inc.), trypsinized, 2D liquid chromatographic separation linked to tandem MS/MS. Distinct ECM protein signatures were identified for the two tumor types. This demonstrates that proteomic analysis of FFPE tumor ECM is possible, and has potential for basic and translational research, and for improving clinical diagnostic accuracy.
doi:10.1016/j.matbio.2008.09.307 doi:10.1016/j.matbio.2008.09.309
93 Novel role of Cdk4 in leukocyte adhesion and trafficking Lynn M. Schnappa, Li Liua, Barbara Schwartza, Yu-Hua Chowa, Yoshiaki Tsubotab, Elaine W. Rainesb, John M. Harlana a Department of Medicine, United States b Department of Pathology, University of Washington, Seattle, WA, United States Leukocyte emigration is a tightly regulated process essential for an appropriate inflammatory response. The current paradigm describes discrete steps in the process: rolling, activation, adhesion, and migration through subendothelial extracellular matrix (ECM). We report a new adhesion pathway that may be important in post-trafficking, i.e. mediating leukocyte interactions to and migration through subendothelial ECM. We found several novel features of this pathway that distinguish it from “conventional” stimulated adhesion: it is dependent on Cdk4 activity; it requires microtubules; it does not require exogenous leukocyte activation; and it does not require Rap-1 activity. Because this novel pathway allows leukocyte adhesion to physiological relevant substrates such as exposed endothelial matrix in the absence of exogenous stimulation, we have termed it “Ligand-induced Adhesion”. Monocytes and lymphocytes, but not neutrophils, are capable of LIA using several ligands, including purified FN and endothelial cell-derived ECM. The known substrates of Cdk4, Rb and Smad, are not required for ligandinduced adhesion suggesting the involvement of a novel Cdk4 substrate. Furthermore, we show that mice lacking Cdk4 have impaired recruitment of lymphocytes in bronchoalveolarlavage fluid following bleomycin induced lung injury. Thus, Cdk4 may be a novel therapeutic target for regulating lymphocyte recruitment during lung injury and inflammation. Further characterization of this novel pathway and its role during injury will be critical to our understanding of leukocyte trafficking. doi:10.1016/j.matbio.2008.09.308
94 Tumor typing by proteomic analysis of formalin fixed sample ECM Timothy M. Rittya, Elizabeth Frauenhofferb, Edward J. Foxa, Bruce Stanleyc Orthopaedics, PSU Cancer Inst., Penn St. Coll. of Medicine, Hershey, PA, United States b Path., Penn St. Coll. of Medicine, Hershey, PA, United States c Proteomics Facility, Penn St. Coll. of Medicine, Hershey, PA, United States
a
A vast and mostly unused resource of tumors and normal stroma is harvested daily by surgical resection, and is formalin fixed/paraffin embedded (FFPE) for pathology. Molecular analysis of FFPE tumors is difficult due to covalent cross-linking induced by formalin. Consequently, these samples are an untapped fund of molecular information. New
95 Thrombospondin-1 regulates blood pressure and cardiac response Jeff S. Isenberga, Yan Qinb, Daryl Despresc, Russell W. Bandlea, Jurgen Schnermannb, William A. Frazierd, David D. Robertsa a Laboratory of Pathology, National Cancer Institute, NIH, Bethesda, MD 20892, United States b Kidney Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD 20892, United States c Mouse Imaging Facility, National Institutes of Health, Bethsda, MD, United States d Department of Biochemistry and Molecular Biophysics, Washington Uni. School of Med., St. Louis, MO, United States Aims Nitric oxide (NO) regulates regional vascular resistance and systemic blood pressure by modulating blood vessel tone. The ability of thrombospondin-1 via its receptor CD47 to locally limit NO-mediated vasodilation and alter regional blood flow suggested that it may also regulate systemic cardiovascular physiology. Methods Wild type, thrombospondin-1 and CD47-null mice underwent analysis of several cardiovascular endpoints including cardiac output, ejection fraction and telemetric blood pressure before and after vasoactive challenge. Analysis of skin blood flow and core temperature change following vasoactive challenge was also performed. Results Mice lacking thrombospondin-1 exhibit an activity-associated increase in diastolic blood pressure and have decreased pulse pressure. CD47deficient mice have elevated resting blood pressure. Both null mice show exaggerated decreases in MAP and pulse pressure and increased cardiac output and ejection fraction in response to NO. They are also resistance to the hypertensive effects of epinephrine. Conversely, autonomic blockade induce exaggerated hypotensive responses in thrombospondin-1 null mice. In combination, these agents cause premature cardiovascular collapse and death of thrombospondin-1 null mice. Conclusions Thrombospondin-1 signaling via CD47 is an acute physiological regulator of blood pressure and maintains blood pressure through resistance of nitric oxide stimulated vasodilation. doi:10.1016/j.matbio.2008.09.310