35 results on '"Annes JP"'
Search Results
2. Sol-moiety: Discovery of a water-soluble prodrug technology for enhanced oral bioavailability of insoluble therapeutics.
- Author
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Karbasi AB, Barfuss JD, Morgan TC, Collins D, Costenbader DA, Dennis DG, Hinman A, Ko K, Messina C, Nguyen KC, Schugar RC, Stein KA, Williams BB, Xu H, Annes JP, and Smith M
- Subjects
- Animals, Administration, Oral, Mice, Humans, Paclitaxel pharmacokinetics, Paclitaxel administration & dosage, Hydrolysis, Pancreatic Neoplasms drug therapy, Pancreatic Neoplasms pathology, Female, Prodrugs pharmacokinetics, Prodrugs administration & dosage, Prodrugs chemistry, Solubility, Biological Availability, Water chemistry
- Abstract
Though conceptually attractive, the use of water-soluble prodrug technology to enhance oral bioavailability of highly insoluble small molecule therapeutics has not been widely adopted. In large part, this is due to the rapid enzymatic or chemical hydrolysis of prodrugs within the gastrointestinal tract, resulting in drug precipitation and no overall improvement in oral bioavailability relative to standard formulation strategies. We reasoned that an optimal water-soluble prodrug could be attained if the rate of prodrug hydrolysis were reduced to favor drug absorption rather than drug precipitation. In doing so, the rate of hydrolysis provides a pharmacokinetic control point for drug delivery. Herein, we report the discovery of a water-soluble promoiety (Sol-moiety) technology to optimize the oral bioavailability of highly insoluble small molecule therapeutics, possessing various functional groups, without the need for sophisticated, often toxic, lipid or organic solvent-based formulations. The power of the technology is demonstrated with marked pharmacokinetic improvement of the commercial drugs enzalutamide, vemurafenib, and paclitaxel. This led to a successful efficacy study of a water-soluble orally administered prodrug of paclitaxel in a mouse pancreatic tumor model., (© 2024. The Author(s).)
- Published
- 2024
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3. Enhancing Therapeutic Insulin Transport from Macroencapsulated Islets Using Sub-Minute Pressure at Physiological Levels.
- Author
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Thomson EA, Lee S, Xu H, Moeller H, Sands J, Lal RA, Annes JP, and Poon ASY
- Abstract
Cadaveric islet and stem cell-derived transplantations hold promise as treatments for type 1 diabetes. To tackle the issue of immunocompatibility, numerous cellular macroencapsulation techniques have been developed that utilize diffusion to transport insulin across an immunoisolating barrier. However, despite several devices progressing to human clinical trials, none have successfully managed to attain physiologic glucose control or insulin independence. Based on empirical evidence, macroencapsulation methods with multilayered, high islet surface density are incompatible with homeostatic, on-demand insulin delivery and physiologic glucose regulation, when reliant solely on diffusion. An additional driving force is essential to overcome the distance limit of diffusion. In this study, we present both theoretical proof and experimental validation that applying pressure at levels comparable to physiological diastolic blood pressure significantly enhances insulin flux across immunoisolation membranes-increasing it by nearly three orders of magnitude. This significant enhancement in transport rate allows for precise, sub-minute regulation of both bolus and basal insulin delivery. By incorporating this technique with a pump-based extravascular system, we demonstrate the ability to rapidly reduce glucose levels in diabetic rodent models, effectively replicating the timescale and therapeutic effect of subcutaneous insulin injection or infusion. This advance provides a potential path towards achieving insulin independence with islet macroencapsulation., One Sentence Summary: Towards improved glucose control, applying sub-minute pressure at physiological levels enhances therapeutic insulin transport from macroencapsulated islets.
- Published
- 2024
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4. Vascular Ensheathment Reflects Characteristic Migratory Behavior of Paragangliomas.
- Author
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Needleman L, Holsinger FC, and Annes JP
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- 2024
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5. Caution on the Use of 68 Ga-DOTATATE for the Diagnosis of Pheochromocytoma: A Report of 2 Cases.
- Author
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Needleman L, Enamandram S, and Annes JP
- Abstract
Pheochromocytomas are intra-adrenal sympathetic neuroendocrine tumors that arise from chromaffin cells. Paragangliomas similarly arise from chromaffin cells, although at extra-adrenal sites such as sympathetic paraganglia in the abdomen/thorax, or parasympathetic paraganglia in the head/neck. Collectively, pheochromocytomas and paragangliomas are important to diagnose and resect because they may secrete harmful levels of catecholamines, have mass effects, hemorrhage, and/or metastasize. Anatomic imaging of pheochromocytomas is usually completed with computed tomography or magnetic resonance imaging; however, functional imaging may be used to provide additional localization, staging, and/or biologic information. Accordingly, selection of the proper functional imaging modality can be critical to developing the optimal therapeutic strategy.
68 Gallium- and64 Copper-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-octreotate positron emission tomography computed tomography (68 Ga- and64 Cu-DOTATATE) are widely used in evaluating pheochromocytomas and paragangliomas, although data regarding the sensitivity for diagnosing pheochromocytoma are limited. We report 2 cases of pheochromocytoma that showed nondiagnostic68 Ga-DOTATATE uptake but were subsequently visualized using alternative functional imaging modalities. Additionally, we provide a review of the literature to highlight the underappreciated limitations of functional adrenal imaging with somatostatin-based compounds., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society.)- Published
- 2023
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6. Rare Presentation of Paroxysmal High B-Pee.
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Bradshaw C, Abounasr A, Brunsing RL, Kao CS, Reejhsinghani R, Annes JP, Chung BI, Mihm F, and Bhalla V
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- 2023
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7. On-demand electrochemically controlled compound release from an ultrasonically powered implant.
- Author
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Wang ML, Chamberlayne CF, Xu H, Mofidfar M, Baltsavias S, Annes JP, Zare RN, and Arbabian A
- Abstract
On-demand drug delivery systems are promising for a wide range of therapeutic applications. When combined with wireless implants for controlled drug delivery, they can reduce overall dosage and side effects. Here, we demonstrate release of fluorescein from a novel on-demand release system for negatively charged compounds. The release system is based on a modified electroresponsive polypyrrole nanoparticulate film designed to minimize ion exchange with the stored compound - a major passive leakage mechanism. We further designed an ultrasonically powered mm-sized implant to electronically control the on-demand drug delivery system in vivo . Release kinetics are characterized both in vitro and in vivo in mice using fluorescein as a model drug, demonstrating the feasibility of wireless, controllable drug release using an ultrasonically powered implant., Competing Interests: There are no conflicts of interest to declare., (This journal is © The Royal Society of Chemistry.)
- Published
- 2022
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8. β-Cell Succinate Dehydrogenase Deficiency Triggers Metabolic Dysfunction and Insulinopenic Diabetes.
- Author
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Lee S, Xu H, Van Vleck A, Mawla AM, Li AM, Ye J, Huising MO, and Annes JP
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- Animals, Electron Transport Complex II deficiency, Glucose metabolism, Insulin-Secreting Cells, Metabolism, Inborn Errors, Mice, Mitochondrial Diseases, TOR Serine-Threonine Kinases metabolism, Diabetes Mellitus, Type 2 metabolism, Succinate Dehydrogenase deficiency, Succinate Dehydrogenase genetics
- Abstract
Mitochondrial dysfunction plays a central role in type 2 diabetes (T2D); however, the pathogenic mechanisms in pancreatic β-cells are incompletely elucidated. Succinate dehydrogenase (SDH) is a key mitochondrial enzyme with dual functions in the tricarboxylic acid cycle and electron transport chain. Using samples from human with diabetes and a mouse model of β-cell-specific SDH ablation (SDHBβKO), we define SDH deficiency as a driver of mitochondrial dysfunction in β-cell failure and insulinopenic diabetes. β-Cell SDH deficiency impairs glucose-induced respiratory oxidative phosphorylation and mitochondrial membrane potential collapse, thereby compromising glucose-stimulated ATP production, insulin secretion, and β-cell growth. Mechanistically, metabolomic and transcriptomic studies reveal that the loss of SDH causes excess succinate accumulation, which inappropriately activates mammalian target of rapamycin (mTOR) complex 1-regulated metabolic anabolism, including increased SREBP-regulated lipid synthesis. These alterations, which mirror diabetes-associated human β-cell dysfunction, are partially reversed by acute mTOR inhibition with rapamycin. We propose SDH deficiency as a contributing mechanism to the progressive β-cell failure of diabetes and identify mTOR complex 1 inhibition as a potential mitigation strategy., (© 2022 by the American Diabetes Association.)
- Published
- 2022
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9. SDHB knockout and succinate accumulation are insufficient for tumorigenesis but dual SDHB/NF1 loss yields SDHx-like pheochromocytomas.
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Armstrong N, Storey CM, Noll SE, Margulis K, Soe MH, Xu H, Yeh B, Fishbein L, Kebebew E, Howitt BE, Zare RN, Sage J, and Annes JP
- Subjects
- Animals, Carcinogenesis genetics, Cell Transformation, Neoplastic, Mice, Succinate Dehydrogenase genetics, Succinate Dehydrogenase metabolism, Succinates, Succinic Acid metabolism, Adrenal Gland Neoplasms genetics, Adrenal Gland Neoplasms metabolism, Adrenal Gland Neoplasms pathology, Dioxygenases metabolism, Pheochromocytoma genetics, Pheochromocytoma metabolism, Pheochromocytoma pathology
- Abstract
Inherited pathogenic succinate dehydrogenase (SDHx) gene mutations cause the hereditary pheochromocytoma and paraganglioma tumor syndrome. Syndromic tumors exhibit elevated succinate, an oncometabolite that is proposed to drive tumorigenesis via DNA and histone hypermethylation, mitochondrial expansion, and pseudohypoxia-related gene expression. To interrogate this prevailing model, we disrupt mouse adrenal medulla SDHB expression, which recapitulates several key molecular features of human SDHx tumors, including succinate accumulation but not 5hmC loss, HIF accumulation, or tumorigenesis. By contrast, concomitant SDHB and the neurofibromin 1 tumor suppressor disruption yields SDHx-like pheochromocytomas. Unexpectedly, in vivo depletion of the 2-oxoglutarate (2-OG) dioxygenase cofactor ascorbate reduces SDHB-deficient cell survival, indicating that SDHx loss may be better tolerated by tissues with high antioxidant capacity. Contrary to the prevailing oncometabolite model, succinate accumulation and 2-OG-dependent dioxygenase inhibition are insufficient for mouse pheochromocytoma tumorigenesis, which requires additional growth-regulatory pathway activation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2022
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10. Novel Pathogenic De Novo INS p.T97P Variant Presenting With Severe Neonatal DKA.
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Lal RA, Moeller HP, Thomson EA, Horton TM, Lee S, Freeman R, Prahalad P, Poon ASY, and Annes JP
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- Diabetes Mellitus, Humans, Infant, Insulin metabolism, Male, Models, Molecular, Proinsulin chemistry, Proinsulin genetics, Proinsulin metabolism, Protein Folding, Diabetic Ketoacidosis genetics, Insulin genetics, Mutation, Missense
- Abstract
Pathogenic INS gene mutations are causative for mutant INS-gene-induced diabetes of youth (MIDY). We characterize a novel de novo heterozygous INS gene mutation (c.289A>C, p.T97P) that presented in an autoantibody-negative 5-month-old male infant with severe diabetic ketoacidosis. In silico pathogenicity prediction tools provided contradictory interpretations, while structural modeling indicated a deleterious effect on proinsulin folding. Transfection of wildtype and INS p.T97P expression and luciferase reporter constructs demonstrated elevated intracellular mutant proinsulin levels and dramatically impaired proinsulin/insulin and luciferase secretion. Notably, proteasome inhibition partially and selectively rescued INS p.T97P-derived luciferase secretion. Additionally, expression of INS p.T97P caused increased intracellular proinsulin aggregate formation and XBP-1s protein levels, consistent with induction of endoplasmic reticulum stress. We conclude that INS p.T97P is a newly identified pathogenic A-chain variant that is causative for MIDY via disruption of proinsulin folding and processing with induction of the endoplasmic reticulum stress response., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
- Published
- 2022
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11. Intracardiac paragangliomas: surgical approach and perioperative management.
- Author
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Guenthart BA, Trope W, Keeyapaj W, Weiel JJ, Edmonson A, MacArthur JW, Annes JP, Woo YJ, and Lui NS
- Subjects
- Female, Humans, Middle Aged, Paraganglioma, Vena Cava, Superior
- Abstract
Intracardiac paragangliomas most commonly arise from the left atrium and are often infiltrative and densely adherent to surrounding structures. Given their rarity, only scattered reports exist in the literature and standardized perioperative and surgical management is not well established. We describe a case of a 60-year-old woman with a mildly functioning intracardiac paraganglioma in which division of the superior vena cava improved exposure and enabled a complex limited resection. Further, we provide an overview of the diagnostic workup, perioperative medical management, surgical approach, and surveillance strategy in patients with these challenging tumors.
- Published
- 2021
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12. Probability of positive genetic testing in patients diagnosed with pheochromocytoma and paraganglioma: Criteria beyond a family history.
- Author
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Alobuia WM, Ammar S, Tyagi M, Ghosh C, Gunda V, Annes JP, and Kebebew E
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- Adolescent, Adrenal Gland Neoplasms genetics, Adrenal Gland Neoplasms pathology, Adrenal Gland Neoplasms surgery, Adrenal Glands pathology, Adrenal Glands surgery, Adrenalectomy, Adult, Age Factors, DNA Mutational Analysis statistics & numerical data, Female, Genetic Predisposition to Disease, Germ-Line Mutation, Humans, Logistic Models, Male, Medical History Taking statistics & numerical data, Middle Aged, Odds Ratio, Paraganglioma genetics, Paraganglioma pathology, Paraganglioma surgery, Pheochromocytoma genetics, Pheochromocytoma pathology, Pheochromocytoma surgery, Preoperative Period, ROC Curve, Retrospective Studies, Tumor Burden, Young Adult, Adrenal Gland Neoplasms diagnosis, Biomarkers, Tumor genetics, Genetic Testing statistics & numerical data, Paraganglioma diagnosis, Pheochromocytoma diagnosis
- Abstract
Background: Genetic testing for germline pheochromocytoma and paraganglioma susceptibility genes is associated with improved patient management. However, data are currently sparse on the probability of a positive testing result based on an individual's clinical presentation. This study evaluates clinical characteristics for association with testing positive for known pheochromocytoma and paraganglioma susceptibility genes., Methods: This retrospective analysis examined 111 patients with a diagnosis of pheochromocytoma and paraganglioma who underwent genetic testing. Logistic regression and receiver operating characteristic analyses were performed to identify factors associated with a positive genetic testing result. Probabilities were then calculated for combinations of significant factors to determine the likelihood of a positive test result in each group., Results: Of 32 patients with a family history of pheochromocytoma and paraganglioma, 31 (97%) had a germline mutation detected. Of 79 patients without a family history, 24 (30%) had a pathogenic germline mutation detected. In multivariate analysis, a positive family history, aged ≤47 years, and tumor size ≤2.9 cm were independent factors associated with a positive genetic testing result. Patients meeting all 3 criteria had a 100% probability compared with 13% in those without any of the criteria. In addition to a positive family history, having either aged ≤47 years or tumor size ≤2.9 cm resulted in a 90% and 100% probability of a positive result, respectively. In the absence of a family history, the probability in patients who were aged ≤47 years and had a tumor size ≤2.9 cm was 60%., Conclusion: In addition to a family history of pheochromocytoma and paraganglioma, aged ≤47 years, and tumor size ≤2.9 cm are associated with a higher probability of testing positive for a pheochromocytoma and paraganglioma susceptibility gene mutation. Patients meeting all 3 criteria have a 100% probability of a positive genetic testing result., (Copyright © 2020 Elsevier Inc. All rights reserved.)
- Published
- 2021
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13. Protocol for determining zinc-dependent β cell-selective small-molecule delivery in mouse pancreas.
- Author
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Horton TM, Kraemer BR, and Annes JP
- Subjects
- Animals, Insulin-Secreting Cells cytology, Mice, Chelating Agents chemistry, Chelating Agents pharmacology, Drug Delivery Systems, Insulin-Secreting Cells metabolism, Zinc chemistry, Zinc pharmacology
- Abstract
Targeted drug delivery to pancreatic islet β cells is an unmet clinical need. β cells possess a uniquely high Zn
2+ concentration, and integrating Zn2+ -binding activity into a small molecule can bias drug accumulation and activity toward β cells. This protocol can be used to evaluate a molecule's capacity to chelate islet Zn2+ , accumulate in islets, and stimulate β cell-selective replication in mouse pancreas. One obstacle is establishing an LC-MS/MS-based method for compound measurement. Limitations include target compound ionizability and the time-sensitive nature of some experimental assay steps. For complete details on the use and execution of this protocol, please refer to Horton et al. (2019)., Competing Interests: The authors declare no competing interests., (© 2020 The Author(s).)- Published
- 2021
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14. PAM staining intensity of primary neuroendocrine neoplasms is a potential prognostic biomarker.
- Author
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Horton TM, Sundaram V, Lee CH, Hornbacker K, Van Vleck A, Benjamin KN, Zemek A, Longacre TA, Kunz PL, and Annes JP
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- Aged, Aged, 80 and over, Female, Follow-Up Studies, Humans, Male, Middle Aged, Neuroendocrine Tumors metabolism, Neuroendocrine Tumors surgery, Prognosis, Survival Rate, Amidine-Lyases metabolism, Biomarkers, Tumor metabolism, Immunohistochemistry methods, Mixed Function Oxygenases metabolism, Neuroendocrine Tumors pathology
- Abstract
Neuroendocrine neoplasms (NENs) are rare epithelial tumors with heterogeneous and frequently unpredictable clinical behavior. Available biomarkers are insufficient to guide individual patient prognosis or therapy selection. Peptidylglycine α-amidating monooxygenase (PAM) is an enzyme expressed by neuroendocrine cells that participates in hormone maturation. The objective of this study was to assess the distribution, clinical associations and survival implications of PAM immunoreactivity in primary NENs. Of 109 primary NENs, 7% were PAM-negative, 25% were PAM-low and 68% were PAM-high. Staining intensity was high in small bowel (p = 0.04) and low in stomach (p = 0.004) NENs. PAM staining was lower in higher grade tumors (p < 0.001) and patients who died (p < 0.001) but did not vary by tumor size or stage at surgery. In patients who died, time to death was shorter in patients with reduced PAM immunoreactivity: median times to death were 11.3 (PAM-negative), 29.4 (PAM-low) and 61.7 (PAM-high) months. Lower PAM staining was associated with increased risk of death after adjusting for disease stage [PAM negative, HR = 13.8 (CI: 4.2-45.5)]. PAM immunoreactivity in primary NENs is readily assessable and a potentially useful stage-independent predictor of survival.
- Published
- 2020
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15. Generation of highly potent DYRK1A-dependent inducers of human β-Cell replication via Multi-Dimensional compound optimization.
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Allegretti PA, Horton TM, Abdolazimi Y, Moeller HP, Yeh B, Caffet M, Michel G, Smith M, and Annes JP
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- Binding Sites, Cell Proliferation drug effects, Humans, Insulin metabolism, Insulin-Secreting Cells cytology, Insulin-Secreting Cells drug effects, Insulin-Secreting Cells metabolism, Kinetics, Molecular Docking Simulation, Naphthyridines chemical synthesis, Naphthyridines chemistry, Naphthyridines pharmacology, Protein Kinase Inhibitors metabolism, Protein Kinase Inhibitors pharmacology, Protein Serine-Threonine Kinases antagonists & inhibitors, Protein Structure, Tertiary, Protein-Tyrosine Kinases antagonists & inhibitors, Small Molecule Libraries metabolism, Small Molecule Libraries pharmacology, Structure-Activity Relationship, Dyrk Kinases, Protein Kinase Inhibitors chemistry, Protein Serine-Threonine Kinases metabolism, Protein-Tyrosine Kinases metabolism, Small Molecule Libraries chemistry
- Abstract
Small molecule stimulation of β-cell regeneration has emerged as a promising therapeutic strategy for diabetes. Although chemical inhibition of dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) is sufficient to enhance β-cell replication, current lead compounds have inadequate cellular potency for in vivo application. Herein, we report the clinical stage anti-cancer kinase inhibitor OTS167 as a structurally novel, remarkably potent DYRK1A inhibitor and inducer of human β-cell replication. Unfortunately, OTS167's target promiscuity and cytotoxicity curtails utility. To tailor kinase selectivity towards DYRK1A and reduce cytotoxicity we designed a library of fifty-one OTS167 derivatives based upon a modeled structure of the DYRK1A-OTS167 complex. Indeed, derivative characterization yielded several leads with exceptional DYRK1A inhibition and human β-cell replication promoting potencies but substantially reduced cytotoxicity. These compounds are the most potent human β-cell replication-promoting compounds yet described and exemplify the potential to purposefully leverage off-target activities of advanced stage compounds for a desired application., (Copyright © 2019 Elsevier Ltd. All rights reserved.)
- Published
- 2020
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16. Zinc-Chelating Small Molecules Preferentially Accumulate and Function within Pancreatic β Cells.
- Author
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Horton TM, Allegretti PA, Lee S, Moeller HP, Smith M, and Annes JP
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- Aminoquinolines analysis, Aminoquinolines chemistry, Aminoquinolines metabolism, Animals, Chelating Agents metabolism, Chromatography, High Pressure Liquid, Dithizone chemistry, Dithizone metabolism, Ethylenediamines analysis, Ethylenediamines chemistry, Ethylenediamines metabolism, Humans, Insulin-Secreting Cells cytology, Male, Mice, Mice, Inbred C57BL, Protein Kinase Inhibitors chemical synthesis, Protein Kinase Inhibitors chemistry, Protein Kinase Inhibitors metabolism, Protein Serine-Threonine Kinases antagonists & inhibitors, Protein Serine-Threonine Kinases metabolism, Protein-Tyrosine Kinases antagonists & inhibitors, Protein-Tyrosine Kinases metabolism, Tandem Mass Spectrometry, Tosyl Compounds analysis, Tosyl Compounds chemistry, Tosyl Compounds metabolism, Dyrk Kinases, Chelating Agents chemistry, Insulin-Secreting Cells metabolism, Zinc chemistry
- Abstract
Diabetes is a hyperglycemic condition characterized by pancreatic β-cell dysfunction and depletion. Whereas methods for monitoring β-cell function in vivo exist, methods to deliver therapeutics to β cells are lacking. We leveraged the rare ability of β cells to concentrate zinc to preferentially trap zinc-binding molecules within β cells, resulting in β-cell-targeted compound delivery. We determined that zinc-rich β cells and islets preferentially accumulated TSQ (6-methoxy-8-p-toluenesulfonamido-quinoline) in a zinc-dependent manner compared with exocrine pancreas. Next, we asked whether appending a zinc-chelating moiety onto a β-cell replication-inducing compound was sufficient to confer preferential β-cell accumulation and activity. Indeed, the hybrid compound preferentially accumulated within rodent and human islets in a zinc-dependent manner and increased the selectivity of replication-promoting activity toward β cells. These data resolve the fundamental question of whether intracellular accumulation of zinc-chelating compounds is influenced by zinc content. Furthermore, application of this principle yielded a proof-of-concept method for β-cell-targeted drug delivery and bioactivity., (Copyright © 2018 Elsevier Ltd. All rights reserved.)
- Published
- 2019
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17. CC-401 Promotes β-Cell Replication via Pleiotropic Consequences of DYRK1A/B Inhibition.
- Author
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Abdolazimi Y, Zhao Z, Lee S, Xu H, Allegretti P, Horton TM, Yeh B, Moeller HP, Nichols RJ, McCutcheon D, Shalizi A, Smith M, Armstrong NA, and Annes JP
- Subjects
- Adult, Animals, Cell Cycle Proteins drug effects, Cell Cycle Proteins metabolism, Cyclin-Dependent Kinase Inhibitor p27 drug effects, Cyclin-Dependent Kinase Inhibitor p27 metabolism, Female, Forkhead Box Protein M1 drug effects, Forkhead Box Protein M1 metabolism, Humans, In Vitro Techniques, Kv Channel-Interacting Proteins drug effects, Kv Channel-Interacting Proteins metabolism, Male, Middle Aged, NFATC Transcription Factors drug effects, NFATC Transcription Factors metabolism, Rats, Repressor Proteins drug effects, Repressor Proteins metabolism, Trans-Activators drug effects, Trans-Activators metabolism, Transcription Factors drug effects, Transcription Factors metabolism, Dyrk Kinases, Cell Proliferation drug effects, Glycogen Synthase Kinase 3 beta antagonists & inhibitors, Insulin-Secreting Cells drug effects, JNK Mitogen-Activated Protein Kinases antagonists & inhibitors, Protein Serine-Threonine Kinases antagonists & inhibitors, Protein-Tyrosine Kinases antagonists & inhibitors, Pyrazolones pharmacology, Receptor, Transforming Growth Factor-beta Type I antagonists & inhibitors
- Abstract
Pharmacologic expansion of endogenous β cells is a promising therapeutic strategy for diabetes. To elucidate the molecular pathways that control β-cell growth we screened ∼2400 bioactive compounds for rat β-cell replication-modulating activity. Numerous hit compounds impaired or promoted rat β-cell replication, including CC-401, an advanced clinical candidate previously characterized as a c-Jun N-terminal kinase inhibitor. Surprisingly, CC-401 induced rodent (in vitro and in vivo) and human (in vitro) β-cell replication via dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) 1A and 1B inhibition. In contrast to rat β cells, which were broadly growth responsive to compound treatment, human β-cell replication was only consistently induced by DYRK1A/B inhibitors. This effect was enhanced by simultaneous glycogen synthase kinase-3β (GSK-3β) or activin A receptor type II-like kinase/transforming growth factor-β (ALK5/TGF-β) inhibition. Prior work emphasized DYRK1A/B inhibition-dependent activation of nuclear factor of activated T cells (NFAT) as the primary mechanism of human β-cell-replication induction. However, inhibition of NFAT activity had limited effect on CC-401-induced β-cell replication. Consequently, we investigated additional effects of CC-401-dependent DYRK1A/B inhibition. Indeed, CC-401 inhibited DYRK1A-dependent phosphorylation/stabilization of the β-cell-replication inhibitor p27Kip1. Additionally, CC-401 increased expression of numerous replication-promoting genes normally suppressed by the dimerization partner, RB-like, E2F and multivulval class B (DREAM) complex, which depends upon DYRK1A/B activity for integrity, including MYBL2 and FOXM1. In summary, we present a compendium of compounds as a valuable resource for manipulating the signaling pathways that control β-cell replication and leverage a DYRK1A/B inhibitor (CC-401) to expand our understanding of the molecular pathways that control β-cell growth.
- Published
- 2018
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18. Hyaluronan content governs tissue stiffness in pancreatic islet inflammation.
- Author
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Nagy N, de la Zerda A, Kaber G, Johnson PY, Hu KH, Kratochvil MJ, Yadava K, Zhao W, Cui Y, Navarro G, Annes JP, Wight TN, Heilshorn SC, Bollyky PL, and Butte MJ
- Subjects
- Animals, Autoimmune Diseases metabolism, Diabetes Mellitus, Type 1 metabolism, Extracellular Matrix drug effects, Extracellular Matrix metabolism, Humans, Hydrogels, Hymecromone pharmacology, Mice, Microscopy, Atomic Force, Hyaluronic Acid metabolism, Inflammation metabolism, Islets of Langerhans drug effects, Islets of Langerhans metabolism
- Abstract
We have identified a novel role for hyaluronan (HA), an extracellular matrix polymer, in governing the mechanical properties of inflamed tissues. We recently reported that insulitis in type 1 diabetes of mice and humans is preceded by intraislet accumulation of HA, a highly hygroscopic polymer. Using the double transgenic DO11.10 × RIPmOVA (DORmO) mouse model of type 1 diabetes, we asked whether autoimmune insulitis was associated with changes in the stiffness of islets. To measure islet stiffness, we used atomic force microscopy (AFM) and developed a novel "bed of nails"-like approach that uses quartz glass nanopillars to anchor islets, solving a long-standing problem of keeping tissue-scale objects immobilized while performing AFM. We measured stiffness via AFM nanoindentation with a spherical indenter and found that insulitis made islets mechanically soft compared with controls. Conversely, treatment with 4-methylumbelliferone, a small-molecule inhibitor of HA synthesis, reduced HA accumulation, diminished swelling, and restored basal tissue stiffness. These results indicate that HA content governs the mechanical properties of islets. In hydrogels with variable HA content, we confirmed that increased HA leads to mechanically softer hydrogels, consistent with our model. In light of recent reports that the insulin production of islets is mechanosensitive, these findings open up an exciting new avenue of research into the fundamental mechanisms by which inflammation impacts local cellular responses., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2018
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19. Erratum. Genetic Disruption of Adenosine Kinase in Mouse Pancreatic β-Cells Protects Against High-Fat Diet-Induced Glucose Intolerance. Diabetes 2017;66:1928-1938.
- Author
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Navarro G, Abdolazami Y, Zhao Z, Xu H, Lee S, Armstrong NA, and Annes JP
- Published
- 2017
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20. Genetic Disruption of Adenosine Kinase in Mouse Pancreatic β-Cells Protects Against High-Fat Diet-Induced Glucose Intolerance.
- Author
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Navarro G, Abdolazimi Y, Zhao Z, Xu H, Lee S, Armstrong NA, and Annes JP
- Subjects
- Animals, Blotting, Western, Fluorescent Antibody Technique, Glucose Intolerance metabolism, In Vitro Techniques, Insulin Resistance, Insulin-Secreting Cells pathology, Islets of Langerhans metabolism, Islets of Langerhans pathology, Mice, Mice, Knockout, Organ Size, Weight Gain, Adenosine Kinase genetics, Blood Glucose metabolism, Diet, High-Fat, Glucose Intolerance genetics, Insulin-Secreting Cells metabolism
- Abstract
Islet β-cells adapt to insulin resistance through increased insulin secretion and expansion. Type 2 diabetes typically occurs when prolonged insulin resistance exceeds the adaptive capacity of β-cells. Our prior screening efforts led to the discovery that adenosine kinase (ADK) inhibitors stimulate β-cell replication. Here, we evaluated whether ADK disruption in mouse β-cells affects β-cell mass and/or protects against high-fat diet (HFD)-induced glucose dysregulation. Mice targeted at the Adk locus were bred to Rip-Cre and Ins1-Cre/ERT
1Lphi mice to enable constitutive (βADKO) and conditional (iβADKO) disruption of ADK expression in β-cells, respectively. Weight gain, glucose tolerance, insulin sensitivity, and glucose-stimulated insulin secretion (GSIS) were longitudinally monitored in normal chow (NC)-fed and HFD-fed mice. In addition, β-cell mass and replication were measured by immunofluorescence-based islet morphometry. NC-fed adult βADKO and iβADKO mice displayed glucose tolerance, insulin tolerance and β-cell mass comparable to control animals. By contrast, HFD-fed βADKO and iβADKO animals had improved glucose tolerance and increased in vivo GSIS. Improved glucose handling was associated with increased β-cell replication and mass. We conclude that ADK expression negatively regulates the adaptive β-cell response to HFD challenge. Therefore, modulation of ADK activity is a potential strategy for enhancing the adaptive β-cell response., (© 2017 by the American Diabetes Association.)- Published
- 2017
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21. Electrically controlled release of insulin using polypyrrole nanoparticles.
- Author
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Hosseini-Nassab N, Samanta D, Abdolazimi Y, Annes JP, and Zare RN
- Abstract
Conducting polymers present an opportunity for developing programmable, adjustable, spatially, and temporally controllable drug delivery systems. While several small molecule drugs have been released from thin conductive polymeric films successfully, delivering large molecule therapeutics, such as polypeptides and nucleic acids, has remained a significant challenge. Poor drug loading (∼ng cm
-2 ) of thin films coupled with film instability has, in many cases, made conducting polymer films refractory to clinical development. To address these limitations, we have utilized conductive polymer nanoparticulate backbones to controllably release insulin, a high molecular weight, clinically relevant polypeptide. We find that the interaction between insulin and the polymer scaffold can be described by a simple Langmuir-type adsorption model. By modifying the ratio of the amount of nanoparticles to the amount of insulin, we have obtained drug loading percentages estimated to be as high as 51 wt% percent. In vivo experiments in mice confirmed retained bioactivity of the released insulin after electrical stimulation.- Published
- 2017
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22. A High-content In Vitro Pancreatic Islet β-cell Replication Discovery Platform.
- Author
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Zhao Z, Abdolazimi Y, Armstrong NA, and Annes JP
- Subjects
- Cell Cycle, Cell Line, Humans, Cell Culture Techniques, Insulin-Secreting Cells cytology, Islets of Langerhans cytology
- Abstract
Loss of insulin-producing β-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet β-cells remains an attractive strategy. β-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain β-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify β-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a β-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on β-cells and the molecular mechanisms that regulate β-cell growth.
- Published
- 2016
- Full Text
- View/download PDF
23. Repurposing cAMP-modulating medications to promote β-cell replication.
- Author
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Zhao Z, Low YS, Armstrong NA, Ryu JH, Sun SA, Arvanites AC, Hollister-Lock J, Shah NH, Weir GC, and Annes JP
- Subjects
- Animals, Cell Division physiology, Insulin-Secreting Cells physiology, Male, Norepinephrine pharmacology, Pancreas physiology, Rats, Rats, Sprague-Dawley, Cell Division drug effects, Insulin-Secreting Cells drug effects, Pancreas drug effects, Phosphodiesterase Inhibitors pharmacology, Regeneration drug effects
- Abstract
Loss of β-cell mass is a cardinal feature of diabetes. Consequently, developing medications to promote β-cell regeneration is a priority. cAMP is an intracellular second messenger that modulates β-cell replication. We investigated whether medications that increase cAMP stability or synthesis selectively stimulate β-cell growth. To identify cAMP-stabilizing medications that promote β-cell replication, we performed high-content screening of a phosphodiesterase (PDE) inhibitor library. PDE3, -4, and -10 inhibitors, including dipyridamole, were found to promote β-cell replication in an adenosine receptor-dependent manner. Dipyridamole's action is specific for β-cells and not α-cells. Next we demonstrated that norepinephrine (NE), a physiologic suppressor of cAMP synthesis in β-cells, impairs β-cell replication via activation of α(2)-adrenergic receptors. Accordingly, mirtazapine, an α(2)-adrenergic receptor antagonist and antidepressant, prevents NE-dependent suppression of β-cell replication. Interestingly, NE's growth-suppressive effect is modulated by endogenously expressed catecholamine-inactivating enzymes (catechol-O-methyltransferase and l-monoamine oxidase) and is dominant over the growth-promoting effects of PDE inhibitors. Treatment with dipyridamole and/or mirtazapine promote β-cell replication in mice, and treatment with dipyridamole is associated with reduced glucose levels in humans. This work provides new mechanistic insights into cAMP-dependent growth regulation of β-cells and highlights the potential of commonly prescribed medications to influence β-cell growth.
- Published
- 2014
- Full Text
- View/download PDF
24. Adult tissue sources for new β cells.
- Author
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Nichols RJ, New C, and Annes JP
- Subjects
- Adult, Animals, Cell Transplantation trends, Diabetes Mellitus therapy, Humans, Cell Transplantation methods, Insulin-Secreting Cells physiology
- Abstract
The diabetes pandemic incurs extraordinary public health and financial costs that are projected to expand for the foreseeable future. Consequently, the development of definitive therapies for diabetes is a priority. Currently, a wide spectrum of therapeutic strategies-from implantable insulin delivery devices to transplantation-based cell replacement therapy, to β-cell regeneration-focus on replacing the lost insulin-producing capacity of individuals with diabetes. Among these, β-cell regeneration remains promising but heretofore unproved. Indeed, recent experimental work has uncovered surprising biology that underscores the potential therapeutic benefit of β-cell regeneration. These studies have elucidated a variety of sources for the endogenous production of new β cells from existing cells. First, β cells, long thought to be postmitotic, have demonstrated the potential for regenerative capacity. Second, the presence of pancreatic facultative endocrine progenitor cells has been established. Third, the malleability of cellular identity has availed the possibility of generating β cells from other differentiated cell types. Here, we review the exciting developments surrounding endogenous sources of β-cell production and consider the potential of realizing a regenerative therapy for diabetes from adult tissues., (Copyright © 2014 Mosby, Inc. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
25. The influence of sodium- and calcium-regulatory hormone interventions on adipocytokines in obesity and diabetes.
- Author
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Vaidya A, Underwood PC, Annes JP, Sun B, Williams GH, Forman JP, and Williams JS
- Subjects
- Adult, Aged, Angiotensin II pharmacology, Arterial Pressure drug effects, Biomarkers, Cholecalciferol metabolism, Female, Glycated Hemoglobin metabolism, Humans, Inflammation metabolism, Infusions, Intravenous, Male, Middle Aged, Renin-Angiotensin System physiology, Resistin metabolism, Young Adult, Adipokines metabolism, Calcium metabolism, Diabetes Mellitus metabolism, Hormones metabolism, Obesity metabolism, Sodium metabolism
- Abstract
Objective: The renin-angiotensin-aldosterone system (RAAS), vitamin D, and parathyroid hormone have all been implicated as regulators of adipocytokines and inflammation. We evaluated human interventional study protocols to investigate whether controlled modulations of these calcium- and sodium-regulatory hormones could influence adipocytokines and inflammation in obesity and diabetes., Methods: Post-hoc analyses of two separate human protocols (Protocol 1, n=14; Protocol 2, n=24) conducted in a clinical research setting after rigorous control of diet, posture, medications, and diurnal rhythm, were performed. Protocol 1 evaluated obese hypertensives with vitamin D deficiency who received an infusion of angiotensin II (AngII) before and after 1month of vitamin D3 therapy. Protocol 2 evaluated obese subjects with type 2 diabetes who also received AngII. Adipocytokines and inflammatory markers were measured before and after vitamin D3 therapy, and also before and after infusions of AngII., Results: Vitamin D3 therapy significantly raised 25(OH)D and 1,25(OH)2D concentrations, and lowered parathyroid hormone, but had no effect on concentrations of adiponectin, resistin, leptin, IL-6, PAI-1, urinary TGFβ1, or HOMA-IR. AngII infusions, despite significant elevations in blood pressure and serum aldosterone, did not influence adipocytokine concentrations in either protocol., Conclusion: In contrast to prior studies conducted in healthy populations, or those that could not control major regulators of the RAAS or adipocytokines, we observed that robust modulations in calcium- and sodium-regulatory hormones did not influence adipocytokines or inflammation in obesity or diabetes. Adipose-tissue physiology in these conditions may alter the hormonal regulation of inflammatory parameters., (Copyright © 2013 Elsevier Inc. All rights reserved.)
- Published
- 2013
- Full Text
- View/download PDF
26. Adenosine kinase inhibition selectively promotes rodent and porcine islet β-cell replication.
- Author
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Annes JP, Ryu JH, Lam K, Carolan PJ, Utz K, Hollister-Lock J, Arvanites AC, Rubin LL, Weir G, and Melton DA
- Subjects
- Animals, Female, Fibroblasts metabolism, Glucagon-Like Peptide-1 Receptor, Glucose metabolism, Hepatocytes cytology, Insulin metabolism, Islets of Langerhans cytology, Mice, Mice, Inbred C57BL, Rats, Rats, Sprague-Dawley, Receptors, Glucagon metabolism, Swine, TOR Serine-Threonine Kinases metabolism, Adenosine Kinase pharmacology, Gene Expression Regulation, Insulin-Secreting Cells cytology
- Abstract
Diabetes is a pathological condition characterized by relative insulin deficiency, persistent hyperglycemia, and, consequently, diffuse micro- and macrovascular disease. One therapeutic strategy is to amplify insulin-secretion capacity by increasing the number of the insulin-producing β cells without triggering a generalized proliferative response. Here, we present the development of a small-molecule screening platform for the identification of molecules that increase β-cell replication. Using this platform, we identify a class of compounds [adenosine kinase inhibitors (ADK-Is)] that promote replication of primary β cells in three species (mouse, rat, and pig). Furthermore, the replication effect of ADK-Is is cell type-selective: treatment of islet cell cultures with ADK-Is increases replication of β cells but not that of α cells, PP cells, or fibroblasts. Short-term in vivo treatment with an ADK-I also increases β-cell replication but not exocrine cell or hepatocyte replication. Therefore, we propose ADK inhibition as a strategy for the treatment of diabetes.
- Published
- 2012
- Full Text
- View/download PDF
27. In vivo screening for secreted proteins that modulate glucose handling identifies interleukin-6 family members as potent hypoglycemic agents.
- Author
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Chen CA, Carolan PJ, and Annes JP
- Subjects
- Animals, Female, Gene Expression, Genes, Reporter, Glucose Tolerance Test, Hepatocytes cytology, Hepatocytes metabolism, Histocytochemistry, Hydrodynamics, Hypoglycemic Agents metabolism, Injections, Intravenous, Interleukin-6 genetics, Interleukin-6 metabolism, Liver cytology, Liver metabolism, Luciferases, Mice, Mice, Inbred ICR, Microscopy, Fluorescence, Plasmids, Tail, Glucose metabolism, Hepatocytes drug effects, Hypoglycemic Agents administration & dosage, Interleukin-6 administration & dosage, Liver drug effects, Transfection methods
- Abstract
Diabetes is a disease of abnormal glucose homeostasis characterized by chronic hyperglycemia and a broad array of consequent organ damage. Because normal glucose homeostasis is maintained by a complex interaction between behavior (feeding and physical activity) and metabolic activity that is modulated by inter-organ signaling through secreted factors, disease modeling in vitro is necessarily limited. In contrast, in vivo studies allow complex metabolic phenotypes to be studied but present a barrier to high throughput studies. Here we present the development of a novel in vivo screening platform that addresses this primary limitation of in vivo experimentation. Our platform leverages the large secretory capacity of the liver and the hepatocyte transfection technique of hydrodynamic tail vein injection to achieve supraphysiologic blood levels of secreted proteins. To date, the utility of hydrodynamic transfection has been limited by the deleterious impact of the variable transfection efficiency inherent to this technique. We overcome this constraint by co-transfection of a secreted luciferase cDNA whose product can be easily monitored in the blood of a living animal and used as a surrogate marker for transfection efficiency and gene expression levels. To demonstrate the utility of our strategy, we screened 248 secreted proteins for the ability to enhance glucose tolerance. Surprisingly, interleukin-6 and several of its family members but not other well-recognized insulin sensitizing agents were identified as potent hypoglycemic factors. We propose this experimental system as a powerful and flexible in vivo screening platform for identifying genes that modulate complex behavioral and metabolic phenotypes.
- Published
- 2012
- Full Text
- View/download PDF
28. Erdheim-Chester disease presenting with cutaneous involvement: a case report and literature review.
- Author
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Volpicelli ER, Doyle L, Annes JP, Murray MF, Jacobsen E, Murphy GF, and Saavedra AP
- Subjects
- Adult, Alcoholism complications, Erdheim-Chester Disease physiopathology, Humans, Male, Obesity complications, Panic Disorder complications, Skin Diseases physiopathology, Sleep Apnea Syndromes complications, Erdheim-Chester Disease complications, Erdheim-Chester Disease pathology, Skin Diseases etiology, Skin Diseases pathology
- Abstract
Erdheim-Chester disease (ECD) is a rare, systemic, non-familial histiocytic disorder, first described by Jakob Erdheim and William Chester in 1930. Most patients have multiple sites of involvement at presentation. The most common site of involvement is the long bones of the axial skeleton, which is seen almost universally, followed by the nervous system, heart, lungs, orbit and retroperitoneum, which are seen in up to 50% of cases. Cutaneous involvement is rarely a presenting symptom of ECD, with two reported cases in the English literature. The diagnosis of ECD is rarely made by skin biopsy because of the relative rarity of cutaneous involvement as a presenting feature, and also perhaps because of the difficulty in distinguishing the histopathological appearance from potential mimics. The importance of distinguishing ECD from other cutaneous disorders with similar pathology lies in the implications for both treatment and prognosis. ECD is an aggressive, often fatal disorder, with death from disease occurring in greater than 50% of patients., (Copyright © 2010 John Wiley & Sons A/S.)
- Published
- 2011
- Full Text
- View/download PDF
29. Risks of presymptomatic direct-to-consumer genetic testing.
- Author
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Annes JP, Giovanni MA, and Murray MF
- Subjects
- Aryl Hydrocarbon Hydroxylases genetics, Genetic Predisposition to Disease, Genetic Testing standards, Government Regulation, Humans, United States, United States Food and Drug Administration, Device Approval, Genetic Testing legislation & jurisprudence
- Published
- 2010
- Full Text
- View/download PDF
30. Integrin alphaVbeta6-mediated activation of latent TGF-beta requires the latent TGF-beta binding protein-1.
- Author
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Annes JP, Chen Y, Munger JS, and Rifkin DB
- Subjects
- Amino Acid Sequence physiology, Animals, CHO Cells, Cricetinae, Latent TGF-beta Binding Proteins, Molecular Sequence Data, Peptides metabolism, Protein Isoforms metabolism, Protein Structure, Tertiary physiology, Antigens, Neoplasm metabolism, Carrier Proteins metabolism, Cell Membrane metabolism, Extracellular Matrix metabolism, Integrins metabolism, Intracellular Signaling Peptides and Proteins, Transforming Growth Factor beta metabolism
- Abstract
Transforming growth factor-betas (TGF-beta) are secreted as inactive complexes containing the TGF-beta, the TGF-beta propeptide, also called the latency-associated protein (LAP), and the latent TGF-beta binding protein (LTBP). Extracellular activation of this complex is a critical but incompletely understood step in TGF-beta regulation. We have investigated the role of LTBP in modulating TGF-beta generation by the integrin alphaVbeta6. We show that even though alphavbeta6 recognizes an RGD on LAP, LTBP-1 is required for alphaVbeta6-mediated latent TGF-beta activation. The domains of LTBP-1 necessary for activation include the TGF-beta propeptide-binding domain and a basic amino acid sequence (hinge domain) with ECM targeting properties. Our results demonstrate an LTBP-1 isoform-specific function in alphaVbeta6-mediated latent TGF-beta activation; LTBP-3 is unable to substitute for LTBP-1 in this assay. The results reveal a functional role for LTBP-1 in latent TGF-beta activation and suggest that activation of specific latent complexes is regulated by distinct mechanisms that may be determined by the LTBP isoform and its potential interaction with the matrix., (Copyright the Rockefeller University Press)
- Published
- 2004
- Full Text
- View/download PDF
31. Annexin II-mediated plasmin generation activates TGF-beta3 during epithelial-mesenchymal transformation in the developing avian heart.
- Author
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Krishnan S, Deora AB, Annes JP, Osoria J, Rifkin DB, and Hajjar KA
- Subjects
- Animals, Biological Assay, Blotting, Northern, Blotting, Western, Chick Embryo, Epithelium embryology, Epithelium physiology, Immunohistochemistry, In Situ Hybridization, Transforming Growth Factor beta3, alpha-2-Antiplasmin metabolism, Annexin A2 metabolism, Fibrinolysin biosynthesis, Heart Valves embryology, Mesoderm physiology, Transforming Growth Factor beta metabolism
- Abstract
Epithelial-mesenchymal transformation (EMT), the process by which epithelial cells are converted into motile, invasive mesenchymal cells, is critical to valvulogenesis. Transforming growth factor-beta3 (TGF-beta3), an established mediator of avian atrioventricular (AV) canal EMT, is secreted as a latent complex. In vitro, plasmin-mediated proteolysis has been shown to release active TGF-betas from the latent complex. Annexin II, a co-receptor for tissue plasminogen activator (tPA) and plasminogen, promotes cell-surface generation of the serine protease plasmin. Here, we show that annexin II-mediated plasmin activity regulates release of active TGF-beta3 during chick AV canal EMT. Primary embryonic endocardial-derived cells express annexin II which promotes plasminogen activation in vitro. Incubation of heart explant cultures with either alpha(2)antiplasmin (alpha(2)AP), a major physiological plasmin inhibitor, or anti-annexin II IgG, blocked EMT by approximately 80%, and 50%, respectively. Anti-annexin II IgG-mediated inhibition of EMT was overcome by the addition of recombinant TGF-beta3. Upon treatment with anti-annexin II IgG or alpha(2)AP, conditioned medium from heart explant cultures showed absence of the active fragment of TGF-beta3 by Western blot analysis and a approximately 50% decrease in TGF-beta specific bioactivity. Our results suggest that annexin II-mediated plasmin activity regulates the release of active TGF-beta during cardiac valve development in the avian heart.
- Published
- 2004
- Full Text
- View/download PDF
32. Making sense of latent TGFbeta activation.
- Author
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Annes JP, Munger JS, and Rifkin DB
- Subjects
- Animals, Carrier Proteins metabolism, Humans, Latent TGF-beta Binding Proteins, Macromolecular Substances, Models, Biological, Proteins metabolism, TNF Receptor-Associated Factor 2, Eukaryotic Cells metabolism, Extracellular Matrix metabolism, Intracellular Signaling Peptides and Proteins, Signal Transduction physiology, Transforming Growth Factor beta metabolism
- Abstract
TGFbeta is secreted as part of a latent complex that is targeted to the extracellular matrix. A variety of molecules, 'TGFbeta activators,' release TGFbeta from its latent state. The unusual temporal discontinuity of TGFbeta synthesis and action and the panoply of TGFbeta effects contribute to the interest in TGF-beta. However, the logical connections between TGFbeta synthesis, storage and action are obscure. We consider the latent TGFbeta complex as an extracellular sensor in which the TGFbeta propeptide functions as the detector, latent-TGFbeta-binding protein (LTBP) functions as the localizer, and TGF-beta functions as the effector. Such a view provides a logical continuity for various aspects of TGFbeta biology and allows us to appreciate TGFbeta biology from a new perspective.
- Published
- 2003
- Full Text
- View/download PDF
33. Latent TGF-beta binding protein-3 (LTBP-3) requires binding to TGF-beta for secretion.
- Author
-
Chen Y, Dabovic B, Annes JP, and Rifkin DB
- Subjects
- Animals, Carrier Proteins genetics, Cells, Cultured, Humans, Latent TGF-beta Binding Proteins, Mice, Protein Binding, Recombinant Fusion Proteins metabolism, Transfection, Adaptor Proteins, Signal Transducing, Carrier Proteins metabolism, Cysteine metabolism, Disulfides metabolism, Intracellular Signaling Peptides and Proteins, Transforming Growth Factor beta metabolism
- Abstract
Latent transforming growth factor-beta (TGF-beta) binding protein (LTBP)-1, which is easily secreted, has been shown to enhance the secretion of TGF-beta. Here we show that another member of the LTBP family, LTBP-3, is not secreted by several cell types, but secretion occurs after coexpression with TGF-beta. The secretion of LTBP-3 requires complexing of LTBP-3 with Cys33 of the TGF-beta propeptide.
- Published
- 2002
- Full Text
- View/download PDF
34. The integrin alphaVbeta6 binds and activates latent TGFbeta3.
- Author
-
Annes JP, Rifkin DB, and Munger JS
- Subjects
- Animals, Cell Line, Gene Deletion, Ligands, Mice, Mink, Oligopeptides genetics, Oligopeptides metabolism, Protein Binding, Protein Isoforms chemistry, Protein Isoforms genetics, Protein Isoforms metabolism, Protein Isoforms pharmacology, Proteins metabolism, Substrate Specificity, TNF Receptor-Associated Factor 2, Transfection, Transforming Growth Factor beta chemistry, Transforming Growth Factor beta genetics, Transforming Growth Factor beta pharmacology, Transforming Growth Factor beta1, Transforming Growth Factor beta3, Antigens, Neoplasm, Integrins metabolism, Transforming Growth Factor beta metabolism
- Abstract
Transforming growth factors-beta (TGFbeta1, 2 and 3) are secreted in a complex with their propeptides (latency-associated peptide 1 (LAP1), 2 and 3). TGFbeta signaling requires the dissociation of LAP and TGFbeta, a process termed latent TGFbeta activation. This process is a critical but incompletely understood step in the regulation of TGFbeta function. In particular, the extent to which activation mechanisms differ among the three TGFbeta isoforms is relatively unexplored. We show here that alphaVbeta6 binds and activates latent TGFbeta3.
- Published
- 2002
- Full Text
- View/download PDF
35. The latent transforming growth factor-beta-binding protein-1 promotes in vitro differentiation of embryonic stem cells into endothelium.
- Author
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Gualandris A, Annes JP, Arese M, Noguera I, Jurukovski V, and Rifkin DB
- Subjects
- Animals, Antibodies immunology, Biomarkers, Carrier Proteins genetics, Carrier Proteins immunology, Cell Differentiation, Cells, Cultured, Endothelium cytology, Endothelium metabolism, Extracellular Matrix metabolism, Gene Expression, Latent TGF-beta Binding Proteins, Mice, Stem Cells metabolism, Time Factors, Transforming Growth Factor beta immunology, Transforming Growth Factor beta physiology, Carrier Proteins physiology, Endothelium embryology, Intracellular Signaling Peptides and Proteins, Stem Cells cytology
- Abstract
The latent transforming growth factor-beta-binding protein-1 (LTBP-1) belongs to a family of extracellular glycoproteins that includes three additional isoforms (LTBP-2, -3, and -4) and the matrix proteins fibrillin-1 and -2. Originally described as a TGF-beta-masking protein, LTBP-1 is involved both in the sequestration of latent TGF-beta in the extracellular matrix and the regulation of its activation in the extracellular environment. Whereas the expression of LTBP-1 has been analyzed in normal and malignant cells and rodent and human tissues, little is known about LTBP-1 in embryonic development. To address this question, we used murine embryonic stem (ES) cells to analyze the appearance and role of LTBP-1 during ES cell differentiation. In vitro, ES cells aggregate to form embryoid bodies (EBs), which differentiate into multiple cell lineages. We analyzed LTBP-1 gene expression and LTBP-1 fiber appearance with respect to the emergence and distribution of cell types in differentiating EBs. LTBP-1 expression increased during the first 12 d in culture, appeared to remain constant between d 12 and 24, and declined thereafter. By immunostaining, fibrillar LTBP-1 was observed in those regions of the culture containing endothelial, smooth muscle, and epithelial cells. We found that inclusion of a polyclonal antibody to LTBP-1 during EB differentiation suppressed the expression of the endothelial specific genes ICAM-2 and von Willebrand factor and delayed the organization of differentiated endothelial cells into cord-like structures within the growing EBs. The same effect was observed when cultures were treated with either antibodies to TGF-beta or the latency associated peptide, which neutralize TGF-beta. Conversely, the organization of endothelial cells was enhanced by incubation with TGF-beta 1. These results suggest that during differentiation of ES cells LTBP-1 facilitates endothelial cell organization via a TGF-beta-dependent mechanism.
- Published
- 2000
- Full Text
- View/download PDF
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