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OR13,74 Longevity and age-related pathology of mice deficient in pregnancy-associated plasma protein-A
Oral Presentations / Growth Hormone & IGF Research 20 (2010) S1–S38
in vivo. In our experiment mAb 263 showed 13.3% effect of GH maximum at concentration of 24.1 nM and its agonist effect could be significantly improved by the second antibodies. In contrast to mAb GHRA1, mAb 263 showed no antagonist effect. This study indicated that only GHR mAbs with epitopes on subdomain 1 are able to act as agonists in Baf/B03 cell proliferation assay. The fact that both mAb GHRA1 and mAb 263 could activate GHR implied that GH signaling could be induced through other ligands binding to a broader region of GHR than the GH binding site. However, a suitable conformation change of GHR similar as induced by GH might be needed.
Thursday, October 7, 2010 Oral session 13. IGFs and IGFBPs in aging and disease OR13,73 A novel mechanism to inhibit IGF-I and prevent the development of diabetic retinopathy L.A. Maile1 , K. Gollahon1 , L. Allen1 , C. Wai1 , P. Dunbar1 , D. Clemmons1 . 1 Medicine, University of North Carolina, Chapel Hill, NC, USA IGF-I is implicated in the initiation and progression of diabetic retinopathy. We have determined that under hyperglycemic conditions IGF-I signaling is regulated by the association between two transmembrane proteins, IAP and SHPS-1. The aims of this study were to 1)characterize the cross-talk between IGF and VEGF in regulating endothelial cell permeability and tube formation two critical events in the development of diabetic retinopathy and 2) to determine whether inhibiting IAP association with SHPS-1 is an effective strategy for preventing the development of diabetic retinopathy by inhibiting IGF-I signaling. Methods: In vitro assays to measure endothelial cell permeability and tube formation were performed in hyperglycemic conditions in the presence or absence of IGF-I, VEGF and inhibitors of IGF-I and VEGF signaling and an antibody which disrupts the association between IAP and SHPS-1. To measure vascular permeability in vivo, diabetic rats were treated every 72 hours with the anti-IAP antibody for 3 weeks, then anethestized and infused with Evans Blue dye for 1 hour prior to enucleation and preparation of the retina. Evans blue was extracted from each retina and the amount of Evans Blue in each retina calculated and corrected for the total Evans blue in the circulation as a measure of vascular permeabilty. Results: We determined that IGF-I stimulates a significant increase in VEGF production and activation of the VEGF receptor in endothelial cells maintained in hyperglycemic conditions. Both VEGF and IGF-I stimulated an increase in vascular permeability. However, the ability of IGF-I to stimulate an increase in vascular permeability was dependent upon VEGF as it was inhibited in the presence of a VEGF inhibitor. Inhibiting IGF-I signaling by disrupting IAP association with SHPS-1 blocked both IGF and VEGF stimulated increase in vascular permeability. Both VEGF and IGF-I stimulated an increase in endothelial cell tube formation. However, IGF-I stimulated tube formation even in the presence of VEGF inhibitors. Both IGF-I and VEGF stimulated tube formation was blocked when the association between IAP and SHPS-1 was disrupted. In the rat model of diabetes there was a 1.9±0.4 fold increase in Evans Blue leakage in the hyperglycemic animals compared with control and this was normalized to control levels in rats treated with the anti-IAP antibody. Conclusion: Taken together our results suggest that the hyperglycemia induced increase in IAP plays a significant role in IGF-I stimulated vascular permeability and endothelial cell tube
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formation in hyperglycemic conditions. Our data also demonstrate that while IGF-I is required for VEGF signaling IGF-I can function in both a VEGF dependent and independent manner. Importantly, blocking IGF-I signaling through disruption of IAP association with SHPS-1 also inhibited the effects of VEGF. Therefore inhibiting the association of IAP with SHPS-1 is a potential strategy for the treatment of diabetic retinopathy. OR13,74 Longevity and age-related pathology of mice deficient in pregnancy-associated plasma protein-A C.A. Conover1 , L.K. Bale2 , J.A. Grell2 , J.R. Mader2 , E.J. Mason2 , M.A. Mason2 , K.P. Keenan3 , R.J. Marler4 , K. Gollahon5 , L. Allen5 . 1 Endocrine Research, Mayo Clinic Foundation, Rochester, MN, USA; 2 Endocrinology, Mayo Clinic Foundation, Rochester, MN, USA; 3 Pathology Associates, Charles River Laboratories, Frederick, MD, USA; 4 Comparative Medicine, Mayo Clinic Foundation, Scottsdale, AZ, USA; 5 Medicine, University of North Carolina, Chapel Hill, NC, USA Pregnancy-associated plasma protein-A (PAPP-A) is a zinc metalloproteinase that enhances insulin-like growth factor (IGF)-I bioactivity through degradation of inhibitory IGF binding proteins in the pericellular microenvironment in vitro and in vivo. Conversely, inhibition or loss of PAPP-A suppresses IGF-I receptor-mediated action without altering IGF or IGF-I receptor expression. Thus, the PAPP-A knock-out (KO) mouse is a model of reduced local IGF-I activity with normal circulating IGF-I levels and was used in this study to shed light on the role of autocrine/paracrine distinct from endocrine regulation of aging by IGF-I. PAPP-A KO mice had significantly increased mean (27%), median (27%), and maximum (35%) lifespan compared to wild-type (WT) littermates (P < 0.0001). End-of-life pathology was performed to assess probable cause-of-death. Incidence of hematopoietic tumors, hepatocellular carcinoma, and bronchio-alveolar carcinoma was not significantly different in the two groups of mice. However, presumably fatal neoplastic disease occurred in older aged PAPP-A KO compared to WT mice. Furthermore, PAPP-A KO mice were less likely to show degenerative changes of age, such as atrial thrombosis and nephropathy, even though the average age at death was higher. Approximately 30% of PAPP-A KO mice and 6% of WT mice died without histological evidence of lethal pathologic changes. Analyses of the pathology of PAPP-A KO and WT mice at 78, 104, and 130 weeks-of-age indicated that WT mice, in general, had more degenerative changes and tumors earlier than PAPP-A KO mice. This was particularly true for abnormalities in heart, testes, brain, kidney, spleen and thymus. In summary, the major contributors to the extended lifespan of PAPP-A KO mice are delayed occurrence of fatal neoplasias and decreased incidence of agerelated degenerative changes. Thus, survival curves and pathology suggest that PAPP-A KO mice have an extended healthy lifespan. OR13,75 Role of IGF-I and IGFBPs in the fasting-induced protection of mice versus malignant cells against chemotherapy C. Lee1 , F. Safdie2 , M. Wei2 , F. Madia2 , E. Parrella2 , D. Hwang1 , P. Cohen3 , V. Longo2 . 1 Pediatric Endocrinology, UCLA, Los Angeles, CA, USA; 2 Gerontology, University of Southern California, Los Angeles, CA, USA; 3 Pediatric Endocrinolgy, UCLA, Los Angeles, CA, USA Most chemotherapy agents cause considerable damage to normal cells, leading to toxicity which is dose limiting and causes both short- and long-term side effects in patients. Although drug development has reduced these side effects with a succession of selective anti-tumor agents such as antibodies that target specific antigens on cancer or agents with a wider therapeutic index, toxicity continues to limit cancer treatment. Thus, interventions that reduce the undesired toxic side-effects could increase the efficacy of many chemotherapy drugs. Chemoprotectants such
in vivo. In our experiment mAb 263 showed 13.3% effect of GH maximum at concentration of 24.1 nM and its agonist effect could be significantly improved by the second antibodies. In contrast to mAb GHRA1, mAb 263 showed no antagonist effect. This study indicated that only GHR mAbs with epitopes on subdomain 1 are able to act as agonists in Baf/B03 cell proliferation assay. The fact that both mAb GHRA1 and mAb 263 could activate GHR implied that GH signaling could be induced through other ligands binding to a broader region of GHR than the GH binding site. However, a suitable conformation change of GHR similar as induced by GH might be needed.
Thursday, October 7, 2010 Oral session 13. IGFs and IGFBPs in aging and disease OR13,73 A novel mechanism to inhibit IGF-I and prevent the development of diabetic retinopathy L.A. Maile1 , K. Gollahon1 , L. Allen1 , C. Wai1 , P. Dunbar1 , D. Clemmons1 . 1 Medicine, University of North Carolina, Chapel Hill, NC, USA IGF-I is implicated in the initiation and progression of diabetic retinopathy. We have determined that under hyperglycemic conditions IGF-I signaling is regulated by the association between two transmembrane proteins, IAP and SHPS-1. The aims of this study were to 1)characterize the cross-talk between IGF and VEGF in regulating endothelial cell permeability and tube formation two critical events in the development of diabetic retinopathy and 2) to determine whether inhibiting IAP association with SHPS-1 is an effective strategy for preventing the development of diabetic retinopathy by inhibiting IGF-I signaling. Methods: In vitro assays to measure endothelial cell permeability and tube formation were performed in hyperglycemic conditions in the presence or absence of IGF-I, VEGF and inhibitors of IGF-I and VEGF signaling and an antibody which disrupts the association between IAP and SHPS-1. To measure vascular permeability in vivo, diabetic rats were treated every 72 hours with the anti-IAP antibody for 3 weeks, then anethestized and infused with Evans Blue dye for 1 hour prior to enucleation and preparation of the retina. Evans blue was extracted from each retina and the amount of Evans Blue in each retina calculated and corrected for the total Evans blue in the circulation as a measure of vascular permeabilty. Results: We determined that IGF-I stimulates a significant increase in VEGF production and activation of the VEGF receptor in endothelial cells maintained in hyperglycemic conditions. Both VEGF and IGF-I stimulated an increase in vascular permeability. However, the ability of IGF-I to stimulate an increase in vascular permeability was dependent upon VEGF as it was inhibited in the presence of a VEGF inhibitor. Inhibiting IGF-I signaling by disrupting IAP association with SHPS-1 blocked both IGF and VEGF stimulated increase in vascular permeability. Both VEGF and IGF-I stimulated an increase in endothelial cell tube formation. However, IGF-I stimulated tube formation even in the presence of VEGF inhibitors. Both IGF-I and VEGF stimulated tube formation was blocked when the association between IAP and SHPS-1 was disrupted. In the rat model of diabetes there was a 1.9±0.4 fold increase in Evans Blue leakage in the hyperglycemic animals compared with control and this was normalized to control levels in rats treated with the anti-IAP antibody. Conclusion: Taken together our results suggest that the hyperglycemia induced increase in IAP plays a significant role in IGF-I stimulated vascular permeability and endothelial cell tube
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formation in hyperglycemic conditions. Our data also demonstrate that while IGF-I is required for VEGF signaling IGF-I can function in both a VEGF dependent and independent manner. Importantly, blocking IGF-I signaling through disruption of IAP association with SHPS-1 also inhibited the effects of VEGF. Therefore inhibiting the association of IAP with SHPS-1 is a potential strategy for the treatment of diabetic retinopathy. OR13,74 Longevity and age-related pathology of mice deficient in pregnancy-associated plasma protein-A C.A. Conover1 , L.K. Bale2 , J.A. Grell2 , J.R. Mader2 , E.J. Mason2 , M.A. Mason2 , K.P. Keenan3 , R.J. Marler4 , K. Gollahon5 , L. Allen5 . 1 Endocrine Research, Mayo Clinic Foundation, Rochester, MN, USA; 2 Endocrinology, Mayo Clinic Foundation, Rochester, MN, USA; 3 Pathology Associates, Charles River Laboratories, Frederick, MD, USA; 4 Comparative Medicine, Mayo Clinic Foundation, Scottsdale, AZ, USA; 5 Medicine, University of North Carolina, Chapel Hill, NC, USA Pregnancy-associated plasma protein-A (PAPP-A) is a zinc metalloproteinase that enhances insulin-like growth factor (IGF)-I bioactivity through degradation of inhibitory IGF binding proteins in the pericellular microenvironment in vitro and in vivo. Conversely, inhibition or loss of PAPP-A suppresses IGF-I receptor-mediated action without altering IGF or IGF-I receptor expression. Thus, the PAPP-A knock-out (KO) mouse is a model of reduced local IGF-I activity with normal circulating IGF-I levels and was used in this study to shed light on the role of autocrine/paracrine distinct from endocrine regulation of aging by IGF-I. PAPP-A KO mice had significantly increased mean (27%), median (27%), and maximum (35%) lifespan compared to wild-type (WT) littermates (P < 0.0001). End-of-life pathology was performed to assess probable cause-of-death. Incidence of hematopoietic tumors, hepatocellular carcinoma, and bronchio-alveolar carcinoma was not significantly different in the two groups of mice. However, presumably fatal neoplastic disease occurred in older aged PAPP-A KO compared to WT mice. Furthermore, PAPP-A KO mice were less likely to show degenerative changes of age, such as atrial thrombosis and nephropathy, even though the average age at death was higher. Approximately 30% of PAPP-A KO mice and 6% of WT mice died without histological evidence of lethal pathologic changes. Analyses of the pathology of PAPP-A KO and WT mice at 78, 104, and 130 weeks-of-age indicated that WT mice, in general, had more degenerative changes and tumors earlier than PAPP-A KO mice. This was particularly true for abnormalities in heart, testes, brain, kidney, spleen and thymus. In summary, the major contributors to the extended lifespan of PAPP-A KO mice are delayed occurrence of fatal neoplasias and decreased incidence of agerelated degenerative changes. Thus, survival curves and pathology suggest that PAPP-A KO mice have an extended healthy lifespan. OR13,75 Role of IGF-I and IGFBPs in the fasting-induced protection of mice versus malignant cells against chemotherapy C. Lee1 , F. Safdie2 , M. Wei2 , F. Madia2 , E. Parrella2 , D. Hwang1 , P. Cohen3 , V. Longo2 . 1 Pediatric Endocrinology, UCLA, Los Angeles, CA, USA; 2 Gerontology, University of Southern California, Los Angeles, CA, USA; 3 Pediatric Endocrinolgy, UCLA, Los Angeles, CA, USA Most chemotherapy agents cause considerable damage to normal cells, leading to toxicity which is dose limiting and causes both short- and long-term side effects in patients. Although drug development has reduced these side effects with a succession of selective anti-tumor agents such as antibodies that target specific antigens on cancer or agents with a wider therapeutic index, toxicity continues to limit cancer treatment. Thus, interventions that reduce the undesired toxic side-effects could increase the efficacy of many chemotherapy drugs. Chemoprotectants such