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Item Age and sex affect TGFβ2-induced ocular hypertension in C57BL/6J mice(Elsevier, 2022) Sugali, Chenna Kesavulu; Rayana, Naga Pradeep; Dai, Jiannong; Peng, Michael; Mao, Weiming; Ophthalmology, School of MedicineGlaucoma is a leading cause of blindness worldwide. The loss of vision in glaucoma patients is due to optic nerve damage. The most important risk factor of glaucoma is elevated intraocular pressure (IOP) which is due to glaucomatous changes in the trabecular meshwork. Animal models, especially mouse models for ocular hypertension (OHT), are important for studying glaucoma. Published studies showed that 2.5X107 PFU adenoviral vectors expressing the biologically active form of human TGFβ2 elevate IOP in female C57BL/6J mice when they are intravitreally delivered. In this study, we found that 2.5X107 PFU adenoviral TGFβ2 vector did not elevate IOP in 3- or 5-month old male C57BL/6J mice. In contrast, 5X107 PFU of the same viral vectors elevated IOP in both 3- and 5-month old male C57BL/6J mice. Also, 5-month old mice showed earlier OHT and higher IOP compared to 3-month old mice. In summary, our data showed that age and sex play roles in adenoviral vector-mediated TGFβ2-induced OHT in C57BL/6J mice.Item Angiogenic gene signature in human pancreatic cancer correlates with TGF-beta and inflammatory transcriptomes(2016-04-11) Craven, Kelly E.; Korc, Murray; Liu, Yunlong; Mosley, Amber L.; Quilliam, Lawrence A.Pancreatic ductal adenocarcinoma (PDAC), which comprises 85% of pancreatic cancers, is the 4th leading cause of cancer death in the United States with a 5-year survival rate of 8%. While human PDACs (hPDACs) are hypovascular, they also overexpress a number of angiogenic growth factors and receptors. Additionally, the use of anti-angiogenic agents in murine models of PDAC leads to reduced tumor volume, tumor spread, and microvessel density (MVD), and improved survival. Nonetheless, clinical trials using anti-angiogenic therapy have been overwhelmingly unsuccessful in hPDAC. On the other hand, pancreatic neuroendocrine tumors (PNETs) account for only 2% of pancreatic tumors, yet they are very vascular and classically angiogenic, respond to anti-angiogenic therapy, and confer a better prognosis than PDAC even in the metastatic setting. In an effort to compare and contrast the angiogenic transcriptomes of these two tumor types, we analyzed RNA-Sequencing (RNA-Seq) data from The Cancer Genome Atlas (TCGA) and found that a pro-angiogenic gene signature is present in 35% of PDACs and that it is mostly distinct from the angiogenic signature present in PNETs. The pro-angiogenic PDAC subgroup also exhibits a transcriptome that reflects active TGF-β signaling, less frequent SMAD4 inactivation than PDACs without the signature, and up-regulation of several pro-inflammatory genes, including members of JAK signaling pathways. Consequently, targeting the TGF-β receptor type-1 kinase with SB505124 and JAK1/2 with ruxolitinib blocks proliferative crosstalk between human pancreatic cancer cells (PCCs) and human endothelial cells (ECs). Additionally, treatment of the KRC (oncogenic Kras, homozygous deletion of Rb1) and KPC (oncogenic Kras, mutated Trp53) genetically engineered PDAC mouse models with ruxolitinib suppresses murine PDAC (mPDAC) progression only in the KRC model, which shows superior enrichment and differential expression of the human pro-angiogenic gene signature as compared to KPC tumors. These findings suggest that targeting both TGF-β and JAK signaling in the 35% of PDAC patients whose cancers exhibit an pro-angiogenic gene signature should be explored in a clinical trial.Item Differential effects of Epigallocatechin-3-gallate containing supplements on correcting skeletal defects in a Down syndrome mouse model(Wiley, 2016-04) Abeysekera, Irushi; Thomas, Jared; Georgiadis, Taxiarchis M.; Berman, Alycia G.; Hammond, Max A.; Dria, Karl J.; Wallace, Joseph M.; Roper, Randall J.; Biology, School of ScienceSCOPE: Down syndrome (DS), caused by trisomy of human chromosome 21 (Hsa21), is characterized by a spectrum of phenotypes including skeletal abnormalities. The Ts65Dn DS mouse model exhibits similar skeletal phenotypes as humans with DS. DYRK1A, a kinase encoded on Hsa21, has been linked to deficiencies in bone homeostasis in DS mice and individuals with DS. Treatment with Epigallocatechin-3-gallate (EGCG), a known inhibitor of Dyrk1a, improves some skeletal abnormalities associated with DS in mice. EGCG supplements are widely available but the effectiveness of different EGCG-containing supplements has not been well studied. METHODS AND RESULTS: Six commercially available supplements containing EGCG were analyzed, and two of these supplements were compared with pure EGCG for their impact on skeletal deficits in a DS mouse model. The results demonstrate differential effects of commercial supplements on correcting skeletal abnormalities in Ts65Dn mice. Different EGCG-containing supplements display differences in degradation, polyphenol content, and effects on trisomic bone. CONCLUSION: This work suggests that the dose of EGCG and composition of EGCG-containing supplements may be important in correcting skeletal deficits associated with DS. Careful analyses of these parameters may lead to a better understanding of how to improve skeletal and other deficits that impair individuals with DS.Item Dissecting the cellular and molecular mechanisms mediating neurofibromatosis type 1 related bone defects(2013-06) Rhodes, Steven David; Yang, Feng-Chun; Clapp, D. Wade; Robling, Alexander G.; Bidwell, Joseph P.Skeletal manifestations including short stature, osteoporosis, kyphoscoliosis, and tibial dysplasia cumulatively affect approximately 70% of patients with neurofibromatosis type 1 (NF1). Tibial pseudarthrosis, the chronic non-union of a spontaneous fracture, is a debilitating skeletal malady affecting young children with NF1. These non-healing fractures respond poorly to treatment and often require amputation of the affected limb due to limited understanding of the causative mechanisms. To better understand the cellular and molecular pathogenesis of these osseous defects, we have established a new mouse model which recapitulates a spectrum of skeletal pathologies frequently observed in patients with NF1. Nf1flox/-;Col2.3Cre mice, harboring Nf1 nullizygous osteoblasts on a Nf1+/- background, exhibit multiple osseous defects which are closely reminiscent of those found in NF1 patients, including runting (short stature), bone mass deficits, spinal deformities, and tibial fracture non-union. Through adoptive bone marrow transfer studies, we have demonstrated that the Nf1 haploinsufficient hematopoietic system pivotally mediates the pathogenesis of bone loss and fracture non-union in Nf1flox/-;Col2.3Cre mice. By genetic ablation of a single Nf1 allele in early myeloid development, under the control of LysMCre, we have further delineated that Nf1 haploinsufficient myeloid progenitors and osteoclasts are the culprit lineages mediating accelerated bone loss. Interestingly, conditional Nf1 haploinsufficiency in mature osteoclasts, induced by CtskCre, was insufficient to trigger enhanced lytic activity. These data provide direct genetic evidence for Nf1’s temporal significance as a gatekeeper of the osteoclast progenitor pool in primitive myelopoiesis. On the molecular level, we found that transforming growth factor-beta1 (TGF-β1), a primary mediator in the spatiotemporal coupling of bone remodeling, is pathologically overexpressed by five- to six- fold in both NF1 patients and in mice. Nf1 deficient osteoblasts, the principal source of TGF-β1 in the bone matrix, overexpress TGF-β1 in a gene dosage dependent fashion. Moreover, p21Ras dependent hyperactivation of the Smad pathway accentuates responses to pathological TGF-β1 signals in Nf1 deficient bone cells. As a proof of concept, we demonstrate that pharmacologic TβRI kinase inhibition can rescue bone mass defects and prevent tibial fracture non-union in Nf1flox/-;Col2.3Cre mice, suggesting that targeting TGF-β1 signaling in myeloid lineages may provide therapeutic benefit for treating NF1 skeletal defects.Item Epigallocatechin-3-gallate (EGCG) consumption in the Ts65Dn model of Down syndrome fails to improve behavioral deficits and is detrimental to skeletal phenotypes(Elsevier, 2017-08-01) Stringer, Megan; Abeysekera, Irushi; Thomas, Jared; LaCombe, Jonathan; Stancombe, Kailey; Stewart, Robert J.; Dria, Karl J.; Wallace, Joseph M.; Goodlett, Charles R.; Roper, Randall J.; Psychology, School of ScienceDown syndrome (DS) is caused by three copies of human chromosome 21 (Hsa21) and results in phenotypes including intellectual disability and skeletal deficits. Ts65Dn mice have three copies of ~50% of the genes homologous to Hsa21 and display phenotypes associated with DS, including cognitive deficits and skeletal abnormalities. DYRK1A is found in three copies in humans with Trisomy 21 and in Ts65Dn mice, and is involved in a number of critical pathways including neurological development and osteoclastogenesis. Epigallocatechin-3-gallate (EGCG), the main polyphenol in green tea, inhibits Dyrk1a activity. We have previously shown that EGCG treatment (~10mg/kg/day) improves skeletal abnormalities in Ts65Dn mice, yet the same dose, as well as ~20mg/kg/day did not rescue deficits in the Morris water maze spatial learning task (MWM), novel object recognition (NOR) or balance beam task (BB). In contrast, a recent study reported that an EGCG-containing supplement with a dose of 2-3mg per day (~40-60mg/kg/day) improved hippocampal-dependent task deficits in Ts65Dn mice. The current study investigated if an EGCG dosage similar to that study would yield similar improvements in either cognitive or skeletal deficits. Ts65Dn mice and euploid littermates were given EGCG [0.4mg/mL] or a water control, with treatments yielding average daily intakes of ~50mg/kg/day EGCG, and tested on the multivariate concentric square field (MCSF)-which assesses activity, exploratory behavior, risk assessment, risk taking, and shelter seeking-and NOR, BB, and MWM. EGCG treatment failed to improve cognitive deficits; EGCG also produced several detrimental effects on skeleton in both genotypes. In a refined HPLC-based assay, its first application in Ts65Dn mice, EGCG treatment significantly reduced kinase activity in femora but not in the cerebral cortex, cerebellum, or hippocampus. Counter to expectation, 9-week-old Ts65Dn mice exhibited a decrease in Dyrk1a protein levels in Western blot analysis in the cerebellum. The lack of beneficial therapeutic behavioral effects and potentially detrimental skeletal effects of EGCG found in Ts65Dn mice emphasize the importance of identifying dosages of EGCG that reliably improve DS phenotypes and linking those effects to actions of EGCG (or EGCG-containing supplements) in specific targets in brain and bone.Item The Exploration of an Effective Medical Countermeasure Enhancing Survival and Hematopoietic Recovery and Preventing Immune Insufficiency in Lethally-Irradiated Mice(2020-08) Wu, Tong; Orschell, Christie M.; Basile, David P.; Unthank, Joseph L.; Haneline, Laura S.; Pelus, Louis M.; MacVittie, Thomas J.There is an urgent demand for effective medical countermeasures (MCM) in the event of high-dose radiation exposure ranging from nuclear plant disasters to potential nuclear warfare. Victims of lethal-dose radiation exposure face multi-organ injuries including the hematopoietic acute radiation syndrome (H-ARS) and the delayed effects of acute radiation exposure (DEARE) years after irradiation. Defective lymphocyte reconstitution and its subsequent immune insufficiency are some of the most serious consequences of H-ARS and DEARE. In order to investigate potential MCMs to protect or mitigate these radiation injuries, the prolonged tissue-specific immunosuppression at all levels of lymphocyte development in established murine H-ARS and DEARE models was defined, along with unique sex-related and age-related changes present in some tissues but not others. The “double hits” of irradiation and age-related stress on lymphopoiesis led to significant myeloid skew and long-term immune involution. Different kinds and different combinations of hematopoietic growth factors, some in combination with angiotensin converting enzyme inhibitor, were administered to lethally irradiated mice. These radiomitigators were found to significantly increase survival and enhance hematopoiesis in H-ARS, but they did little to alleviate the severity of DEARE including immune insufficiency. 16,16 dimethyl-prostaglandin E2 (dmPGE2), a long-acting formulation of PGE2 with similar biological effects as PGE2, was found to enhance survival and hematopoiesis in lethal-irradiated mice when used as radiomitigator or radioprotectant. The optimum time window for administration of radioprotectant and radiomitigator dmPGE2 was defined, which is -3hr to -15min prior to irradiation and +6hr to +30hr post irradiation. Significant survival efficacy of radioprotectant dmPGE2 was also demonstrated in pediatric and geriatric mice. Using specific PGE2 receptor (EP) agonists, the EP4 receptor was defined as the PGE2 receptor potentially responsible for dmPGE2 radioprotection. Radioprotectant dmPGE2 was also found to prevent radiation-induced thymic involution and to ameliorate the long-term immune suppression in radiation survivors in the DEARE phase via promoting hematopoietic stem cell differentiation towards to the lymphoid lineage. This is the first report of an effective MCM for H-ARS which also targets long-term thymic involution and lymphoid lineage reconstitution.Item G-CSF in stem cell mobilization: new insights, new questions(AME Publishing Company, 2017-07) Patterson, Andrea M.; Pelus, Louis M.; Microbiology and Immunology, School of MedicineItem Gene-metabolite networks associated with impediment of bone fracture repair in spaceflight(Elsevier, 2021-06-08) Chakraborty, Nabarun; Zamarioli, Ariane; Gautam, Aarti; Campbell, Ross; Mendenhall, Stephen K.; Childress, Paul J.; Dimitrov, George; Sowe, Bintu; Tucker, Aamir; Zhao, Liming; Hammamieh, Rasha; Kacena, Melissa A.; Orthopaedic Surgery, School of MedicineAdverse effects of spaceflight on musculoskeletal health increase the risk of bone injury and impairment of fracture healing. Its yet elusive molecular comprehension warrants immediate attention, since space travel is becoming more frequent. Here we examined the effects of spaceflight on bone fracture healing using a 2 mm femoral segmental bone defect (SBD) model. Forty, 9-week-old, male C57BL/6J mice were randomized into 4 groups: 1) Sham surgery on Ground (G-Sham); 2) Sham surgery housed in Spaceflight (FLT-Sham); 3) SBD surgery on Ground (G-Surgery); and 4) SBD surgery housed in Spaceflight (FLT-Surgery). Surgery procedures occurred 4 days prior to launch; post-launch, the spaceflight mice were house in the rodent habitats on the International Space Station (ISS) for approximately 4 weeks before euthanasia. Mice remaining on the Earth were subjected to identical housing and experimental conditions. The right femur from half of the spaceflight and ground groups was investigated by micro-computed tomography (µCT). In the remaining mice, the callus regions from surgery groups and corresponding femoral segments in sham mice were probed by global transcriptomic and metabolomic assays. µCT confirmed escalated bone loss in FLT-Sham compared to G-Sham mice. Comparing to their respective on-ground counterparts, the morbidity gene-network signal was inhibited in sham spaceflight mice but activated in the spaceflight callus. µCT analyses of spaceflight callus revealed increased trabecular spacing and decreased trabecular connectivity. Activated apoptotic signals in spaceflight callus were synchronized with inhibited cell migration signals that potentially hindered the wound site to recruit growth factors. A major pro-apoptotic and anti-migration gene network, namely the RANK-NFκB axis, emerged as the central node in spaceflight callus. Concluding, spaceflight suppressed a unique biomolecular mechanism in callus tissue to facilitate a failed regeneration, which merits a customized intervention strategy.Item Generation of the first Autosomal Dominant Osteopetrosis Type II (ADO2) disease models(Elsevier B.V., 2014-02) Alam, Imranul; Gray, Amie K.; Chu, Kang; Ichikawa, Shoji; Mohammad, Khalid S.; Capannolo, Marta; Capulli, Mattia; Maurizi, Antonio; Muraca, Maurizio; Teti, Anna; Econs, Michael J.; Del Fattore, Andrea; Department of Orthopaedic Surgery, IU School of MedicineAutosomal Dominant Osteopetrosis Type II (ADO2) is a heritable osteosclerotic disorder dependent on osteoclast impairment. In most patients it results from heterozygous missense mutations in the chloride channel 7 (CLCN7) gene, encoding for a 2Cl−/1H+ antiporter. By a knock-in strategy inserting a missense mutation in the Clcn7 gene, our two research groups independently generated mouse models of ADO2 on different genetic backgrounds carrying the homolog of the most frequent heterozygous mutation (p.G213R) in the Clcn7 gene found in humans. Our results demonstrate that the heterozygous model holds true presenting with higher bone mass, increased numbers of poorly resorbing osteoclasts and a lethal phenotype in the homozygous state. Considerable variability is observed in the heterozygous mice according with the mouse background, suggesting that modifier genes could influence the penetrance of the disease gene.Item In vivo tumor growth of high-grade serous ovarian cancer cell lines(Elsevier, 2015-08) Mitra, Anirban; Davis, David A.; Tomar, Sunil; Roy, Lynn; Gurler, Hilal; Xie, Jia; Lantvit, Daniel D.; Cardenas, Horacio; Fang, Fang; Liu, Yueying; Loughran, Elizabeth; Yang, Jing; Stack, M. Sharon; Emerson, Robert E.; Dahl, Karen D. Cowden; Barbolina, Maria; Nephew, Kenneth P.; Matei, Daniela; Burdette, Joanna E.; Department of Medicine, IU School of MedicineOBJECTIVE: Genomic studies of ovarian cancer (OC) cell lines frequently used in research revealed that these cells do not fully represent high-grade serous ovarian cancer (HGSOC), the most common OC histologic type. However, OC lines that appear to genomically resemble HGSOC have not been extensively used and their growth characteristics in murine xenografts are essentially unknown. METHODS: To better understand growth patterns and characteristics of HGSOC cell lines in vivo, CAOV3, COV362, KURAMOCHI, NIH-OVCAR3, OVCAR4, OVCAR5, OVCAR8, OVSAHO, OVKATE, SNU119 and UWB1.289 cells were assessed for tumor formation in nude mice. Cells were injected intraperitoneally (i.p.) or subcutaneously (s.c.) in female athymic nude mice and allowed to grow (maximum of 90 days) and tumor formation was analyzed. All tumors were sectioned and assessed using H&E staining and immunohistochemistry for p53, PAX8 and WT1 expression. RESULTS: Six lines (OVCAR3, OVCAR4, OVCAR5, OVCAR8, CAOV3, and OVSAHO) formed i.p xenografts with HGSOC histology. OVKATE and COV362 formed s.c. tumors only. Rapid tumor formation was observed for OVCAR3, OVCAR5 and OVCAR8, but only OVCAR8 reliably formed ascites. Tumors derived from OVCAR3, OVCAR4, and OVKATE displayed papillary features. Of the 11 lines examined, three (Kuramochi, SNU119 and UWB1.289) were non-tumorigenic. CONCLUSIONS: Our findings help further define which HGSOC cell models reliably generate tumors and/or ascites, critical information for preclinical drug development, validating in vitro findings, imaging and prevention studies by the OC research community.