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PODOPLANIN GENE DEFICIENCY CAUSES LYMPHATIC MALFORMATION AND LYMPHOCYTE DEVELOPMENTAL DEFECTS
Cardiovascular Pathology 13 (2004) S3 – S16
Oral Abstracts Presentations Workshop 1
Workshop 2
O01
O02
PODOPLANIN GENE DEFICIENCY CAUSES LYMPHATIC MALFORMATION AND LYMPHOCYTE DEVELOPMENTAL DEFECTS. Pavel Uhrin, Jan Zaujec, Miroslav Bauer, Mario Hilpert, Heinrich Kowalski, Elke Furtbauer, Martin Bilban, Hannes Stockinger, Dontscho Kerjaschki, Bernd R. Binder. Department of Vascular Biology and Thrombosis Research, Department of Pathology, Institute of Immunology, Institute of Medical and Chemical Laboratory Diagnostics, University of Vienna, Vienna, Austria.
RHOA-DEPENDENT NUCLEAR TRANSLOCATION OF MYOCARDIN RELATED TRANSCRIPTION FACTOR A IS IMPORTANT FOR SMC-SPECIFIC TRANSCRIPTION. Jeremiah S. Hinson, Dionne Morris, Kashelle Lockman, Matthew D. Medlin, Joan M. Taylor, Christopher P. Mack. University of North Carolina, Chapel Hill, NC.
Lymphatic vessels are essential tissue for homeostasis, fat absorption and for immune surveillance. Podoplanin, a mucin-like membrane-bound glycoprotein, has been defined as a novel phenotypic marker of lymphatic endothelial cells, but is also expressed in other organs, such as the thymus. In order to analyze podoplanin function in more detail, we generated podoplanin / mice in 129S/v : Swiss background.The lack of podoplanin resulted in loss of about 30 % of the embryos. Within the first week, approximately 55% of born podoplanin / mice died, but 20% of born mice survived till adulthood. Both male and female podoplanin / adult mice were fertile. With respect to the lymphatic vasculature, young podoplanin / mice showed enlarged and tortuous lymphatic vessels, while the lymphatic network was impaired. Additionally, throughout the small intestine a network of dilated, tortuous and leaky lymphatic vessels resulting in the chylous ascitis and hyperemic Peyer´s patches were found. The lymphnodes of podoplanin / mice were also hyperemic. Lymphatic fluid transport, as revealed by intradermal injection of Chicago sky blue dye, was impaired in the knockouts. These data indicate that formation of the lymphatic vessel system is erroneous in lymphoid and non-lymphoid tissues in podoplanin / mice. Young podoplanin / mice suffered from dramatic weight loss, in spite of normal glucose levels and increased triglycerides levels observed within postnatal day 3 – 4, thus indicating a wasting syndrome. Additionally, in the thymus and spleen, disruption of podoplanin was accompanied by an increase of a secondary lymphoid tissue chemokine CCL21, previously shown to be co-localized with podoplanin. This wasting syndrome occurred despite not increased TNF-alpha levels, but in the presence of a population of cytotoxic T-cells not found in the wild-type controls, expressing markers of activation and indicating a possible role of this T-cell population to the podoplanin / phenotype. To prove this, we thymectomized 2 – 3 days old mice and in fact could rescue podoplanin / mice from wasting syndrome completely so that they survived the first week and reached the fertility age. Thymectomy had, however, no effect on the lymphatic vascular phenotype that remained changed also in adult podoplanin / mice. In summary, our results indicate that podoplanin is not only necessary for normal lymphatic vessel development, but is also critical for normal lymphocyte development.
We have previously shown that RhoA signaling regulates serum response factor (SRF)-dependent transcription of the smooth muscle cell (SMC)specific genes. The recent observation that nuclear localization of the Myocardin-Related Transcription Factor A (MRTFA) is regulated by RhoA signaling suggests that MRTFA may be an important factor in agonist-mediated regulation of SMC-specific transcription. MRTFA is expressed in SMC, 10T1/2, and NIH3T3 cells as measured by quantitative RT PCR and Western Blot. Overexpression of MRTFA strongly activated the SM22, SM a-actin, and SM MHC promoter in 10T1/2 cells as well as in NIH3T3 fibroblasts. MRTFA also weakly trans-activated the CArGcontaining c-fos promoter in both of these cell-types. Transactivation of SM a-actin by MRTFA required the presence of the two higher affinity SRF binding elements, CArG B and the intronic CArG, but not the low affinity CArG A element. Sphingosine-1-phosphate (S1P), a known activator of RhoA signaling, significantly increased SM22 promoter activity in serum starved 10T1/2 cells and this effect was inhibited by a dominant negative form of MRTFA. To monitor MRTFA localization we used immunofluorescence techniques in cells expressing myc-tagged MRTFA or an MRTFA-GFP fusion protein. Results demonstrated significant increases in MRTFA nuclear localization upon 10% serum and S1P treatment. However, there were also significant cell-type-specific differences in MRTFA localization under serum free conditions and in MRTFA translocation following stimulation. The Rho-kinase inhibitor, Y-27632, decreased MRTFA nuclear localization and partially decreased the effects of overexpressed MRTFA on promoter activity. Taken together these results indicate that MRTFA may play a role in the regulation of SMCspecific gene expression and that MRTFA may serve as an important link between the extrinsic cues that regulate SMC function and the transcriptional machinery that ultimately determines SMC phenotype. NIH 1RO1HL70953-01
O03 OSTEOPROTEGERIN AN AVB3-INDUCED ENDOTHELIAL CELL SURVIVAL MOLECULE. Marta Scatena, Laura Pritzker, Cecilia M. Giachelli. Department of Bioengineering, University of Washington, Seattle, WA. Osteoprotegerin (OPG), a soluble member of the TNF-receptor superfamily, is known to regulate osteoclast differentiation, activation, and survival. We found that OPG expression was induced by avb3 ligation in a NF-kBdependent manner in growth factor-deprived rat and human endothelial cells. NF-kB activation is required for avb3-dependent endothelial cell survival following serum-deprivation induced apoptosis. OPG prevented endothelial cells growth factor withdrawal-induced apoptosis even when
1054-8807/04/$ – see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.carpath.2004.03.002
Oral Abstracts Presentations Workshop 1
Workshop 2
O01
O02
PODOPLANIN GENE DEFICIENCY CAUSES LYMPHATIC MALFORMATION AND LYMPHOCYTE DEVELOPMENTAL DEFECTS. Pavel Uhrin, Jan Zaujec, Miroslav Bauer, Mario Hilpert, Heinrich Kowalski, Elke Furtbauer, Martin Bilban, Hannes Stockinger, Dontscho Kerjaschki, Bernd R. Binder. Department of Vascular Biology and Thrombosis Research, Department of Pathology, Institute of Immunology, Institute of Medical and Chemical Laboratory Diagnostics, University of Vienna, Vienna, Austria.
RHOA-DEPENDENT NUCLEAR TRANSLOCATION OF MYOCARDIN RELATED TRANSCRIPTION FACTOR A IS IMPORTANT FOR SMC-SPECIFIC TRANSCRIPTION. Jeremiah S. Hinson, Dionne Morris, Kashelle Lockman, Matthew D. Medlin, Joan M. Taylor, Christopher P. Mack. University of North Carolina, Chapel Hill, NC.
Lymphatic vessels are essential tissue for homeostasis, fat absorption and for immune surveillance. Podoplanin, a mucin-like membrane-bound glycoprotein, has been defined as a novel phenotypic marker of lymphatic endothelial cells, but is also expressed in other organs, such as the thymus. In order to analyze podoplanin function in more detail, we generated podoplanin / mice in 129S/v : Swiss background.The lack of podoplanin resulted in loss of about 30 % of the embryos. Within the first week, approximately 55% of born podoplanin / mice died, but 20% of born mice survived till adulthood. Both male and female podoplanin / adult mice were fertile. With respect to the lymphatic vasculature, young podoplanin / mice showed enlarged and tortuous lymphatic vessels, while the lymphatic network was impaired. Additionally, throughout the small intestine a network of dilated, tortuous and leaky lymphatic vessels resulting in the chylous ascitis and hyperemic Peyer´s patches were found. The lymphnodes of podoplanin / mice were also hyperemic. Lymphatic fluid transport, as revealed by intradermal injection of Chicago sky blue dye, was impaired in the knockouts. These data indicate that formation of the lymphatic vessel system is erroneous in lymphoid and non-lymphoid tissues in podoplanin / mice. Young podoplanin / mice suffered from dramatic weight loss, in spite of normal glucose levels and increased triglycerides levels observed within postnatal day 3 – 4, thus indicating a wasting syndrome. Additionally, in the thymus and spleen, disruption of podoplanin was accompanied by an increase of a secondary lymphoid tissue chemokine CCL21, previously shown to be co-localized with podoplanin. This wasting syndrome occurred despite not increased TNF-alpha levels, but in the presence of a population of cytotoxic T-cells not found in the wild-type controls, expressing markers of activation and indicating a possible role of this T-cell population to the podoplanin / phenotype. To prove this, we thymectomized 2 – 3 days old mice and in fact could rescue podoplanin / mice from wasting syndrome completely so that they survived the first week and reached the fertility age. Thymectomy had, however, no effect on the lymphatic vascular phenotype that remained changed also in adult podoplanin / mice. In summary, our results indicate that podoplanin is not only necessary for normal lymphatic vessel development, but is also critical for normal lymphocyte development.
We have previously shown that RhoA signaling regulates serum response factor (SRF)-dependent transcription of the smooth muscle cell (SMC)specific genes. The recent observation that nuclear localization of the Myocardin-Related Transcription Factor A (MRTFA) is regulated by RhoA signaling suggests that MRTFA may be an important factor in agonist-mediated regulation of SMC-specific transcription. MRTFA is expressed in SMC, 10T1/2, and NIH3T3 cells as measured by quantitative RT PCR and Western Blot. Overexpression of MRTFA strongly activated the SM22, SM a-actin, and SM MHC promoter in 10T1/2 cells as well as in NIH3T3 fibroblasts. MRTFA also weakly trans-activated the CArGcontaining c-fos promoter in both of these cell-types. Transactivation of SM a-actin by MRTFA required the presence of the two higher affinity SRF binding elements, CArG B and the intronic CArG, but not the low affinity CArG A element. Sphingosine-1-phosphate (S1P), a known activator of RhoA signaling, significantly increased SM22 promoter activity in serum starved 10T1/2 cells and this effect was inhibited by a dominant negative form of MRTFA. To monitor MRTFA localization we used immunofluorescence techniques in cells expressing myc-tagged MRTFA or an MRTFA-GFP fusion protein. Results demonstrated significant increases in MRTFA nuclear localization upon 10% serum and S1P treatment. However, there were also significant cell-type-specific differences in MRTFA localization under serum free conditions and in MRTFA translocation following stimulation. The Rho-kinase inhibitor, Y-27632, decreased MRTFA nuclear localization and partially decreased the effects of overexpressed MRTFA on promoter activity. Taken together these results indicate that MRTFA may play a role in the regulation of SMCspecific gene expression and that MRTFA may serve as an important link between the extrinsic cues that regulate SMC function and the transcriptional machinery that ultimately determines SMC phenotype. NIH 1RO1HL70953-01
O03 OSTEOPROTEGERIN AN AVB3-INDUCED ENDOTHELIAL CELL SURVIVAL MOLECULE. Marta Scatena, Laura Pritzker, Cecilia M. Giachelli. Department of Bioengineering, University of Washington, Seattle, WA. Osteoprotegerin (OPG), a soluble member of the TNF-receptor superfamily, is known to regulate osteoclast differentiation, activation, and survival. We found that OPG expression was induced by avb3 ligation in a NF-kBdependent manner in growth factor-deprived rat and human endothelial cells. NF-kB activation is required for avb3-dependent endothelial cell survival following serum-deprivation induced apoptosis. OPG prevented endothelial cells growth factor withdrawal-induced apoptosis even when
1054-8807/04/$ – see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.carpath.2004.03.002