Browsing by Author "Bidwell, Joseph"
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Item Generation and Exploration of a Novel Low Oxygen Landscape for Hematopoietic Stem and Progenitor Cells(2022-10) Dausinas, Paige Burke; Elmendorf, Jeffrey; O'Leary, Heather; Bidwell, Joseph; Wan, Jun; Zhang, JiHematopoietic stem (HSC) and progenitor (HSPC) cells reside in low oxygen (~1- 4%, low O2) bone marrow niches which provide critical signals for maintenance, selfrenewal, and differentiation. Exposure of HSC/HSPCs to air (~21%) for less than 10 minutes irreversibly diminishes numbers of phenotypic and functional stem cells, a phenomenon termed extra physiologic oxygen stress/shock. Yet, most studies harvest and analyze HSC/HSPCs in air and often in fixed cells, leaving endogenous signaling mechanisms unidentified. To better understand the endogenous mechanisms regulating HSCs and HSPCs, we generated the first low O2 landscape of phenotypic/functional/signaling alterations in live, low O2 harvested/sorted HSC/HSPCs utilizing novel technology. HSC (LSKCD150+) and HSC/HSPC (LSK) expression, frequency, and stem cell maintenance retention were enhanced in low O2 relative to historic data and our air data. Transcriptomics uncovered low O2 differential pathway regulation of HSC/HSPCs and HSCs with analysis identifying low O2 enrichment of genes/pathways including Ca2+ ion binding, altered sodium hydrogen (Na+/H+) activity, viral entry, and transmembrane receptor activity in both HSCs and HSPCs. In exploring the low O2 landscape, we investigated differential low O2 regulation of Ca2+ and SARS-CoV-2 related pathways/mechanisms in HSCs and HSPCs. Differential Ca2+ regulation was observed in our transcriptional/proteomic analysis corroborated by phenotypic/functional data demonstrating increases in low O2 of cytosolic and mitochondrial Ca2+ flux, ABC Transporter (ABCG2) and Na+/H+ (NHE1) expression, discovery of a novel low O2 Ca2+ high HSPC population that enhances HSC maintenance compared to Ca2+ low populations and blunting of this population and subsequent enhanced stem cell maintenance upon NHE1 inhibition (Cariporide). Multi-omics analyses also identified enhancements in COVID19-related pathways in low O2 that corresponded with enhanced expression of SARS-CoV-2 receptors/co-receptors, SARS-CoV-2 spike protein (SP) binding, and expansion of SP-bound HSC/HSPCs in low O2 compared to air, as well as enhanced stem cell maintenance of SP-bound, versus unbound, cells in low O2. Together, these data presented show low O2 harvest/retention of HSC/HSPCs enhances stem cell maintenance, which could be utilized to improve HSC expansion, and leads to differential pathway/signaling regulation of various biological pathways in HSC/HSPCs including Ca2+ and SARS-CoV-2/viral infection that results in phenotypic and functional consequences.Item Identification of Extracellular Wnt Inhibitors for Novel Synergistic Anabolic Action on the Skeleton(2023-07) Choi, Roy Byung-Jun; Robling, Alexander; Bidwell, Joseph; Thompson, William; Pavalko, Fredrick; White, KennethThe Wnt pathway has been an obvious target for designing skeletal therapies, mainly based on the high-bone-mass phenotypes in patients with activating mutations in the Wnt co-receptors Lrp5 and Lrp6, or with inactivating mutations in the Lrp5/6 inhibitor Sost. An attractive property of the Wnt pathway is that it stimulates anabolic action in bone cells. The powerful anabolic potential of manipulating Wnt signaling in bone has been demonstrated by the recent FDA approval of sclerostin antibody (Scl-mAb) EvenityTM (Romosozumab) for the treatment of osteoporosis. However, an increased risk of cardiovascular events was reported, triggering the FDA to issue a ‘black box warning’ requirement for romosozumab. One potential solution to lower the risk of adverse events is to reduce the medication dose. Reducing the dose of Scl-mAb, while maintaining the anabolic potential of the drug, will likely provide a safer and more cost-effective strategy to harness Wnt for fracture prevention. Here, we found that dual inhibition of sclerostin and Dkk1 produced extremely potent synergistic bone anabolic effects in the cancellous compartment, using both genetic and pharmacological models. Further, much lower total doses of dual antibody treatment, given at optimized proportions, generated equivalent trabecular bone anabolic effects as Scl-mAb alone. On the contrary, we looked for other candidates that might potentiate the cortical effects of sclerostin inhibition. We found that either Sostdc1 or Notum deletion results in high bone mass, specifically in the cortical compartment, with little to no effect in the cancellous compartment. Inhibition/deletion of Sostdc1 or Notum alone had no detectable effects (Sostdc1) or mild (Notum) cortical effects, but suppression of either target while co-supressing/deleting Sost improved bone mass disproportionately. In summary, these findings highlight the potential therapeutic role that combinational inhibition of different Wnt inhibitors generates, resulting in synergistic bone anabolic action in different/select skeletal compartments.Item Nmp4 Suppresses Osteoanabolic Potency(2023-07) Heim, Crystal Noelle; Bidwell, Joseph; Wek, Ronald; White, Kenneth; Robling, Alexander; Plotkin, LilianTreating severe osteoporosis is limited to two strategies: 1. Stimulation of the parathyroid hormone receptor with analogs for parathyroid hormone (PTH) or parathyroid hormone related peptide, and 2. Stimulation of Wnt signaling via neutralization of sclerostin, a natural inhibitor of this pathway, with a monoclonal antibody (romosozumab-aqqg, Scl-mAb). Despite mobilizing distinct molecular and cellular pathways to stimulate bone gain, all their efficacies rapidly diminish. Identifying the barrier to enhancing potency is a clinical priority. We recently reported that mice harboring the conditional loss of the transcription factor Nmp4 (Nuclear Matrix Protein 4) in mesenchymal stem/progenitor cells (MSPCs) exhibited no measurable baseline effect on the skeleton but showed a significantly enhanced increase in bone formation during PTH therapy. Remarkably, (and unexpectedly) skeletal response to PTH therapy was not improved when Nmp4 was conditionally disabled at the osteoblast or osteocyte stages. For the present study, we hypothesized that the potency of any osteoanabolic drug is pre-programmed (and can be re-programmed) in osteoprogenitors. To test this hypothesis, we treated our global Nmp4-/- mice, various conditional knockout mice, and their controls with Scl-mAb. We observed a similar pattern of improved bone response in our mouse models, which we previously observed with the PTH therapy. That is, removal of Nmp4 early in osteoblast differentiation (MSPC) was required for an exaggerated bone-formation response to Scl-mAb therapy. Disabling Nmp4 later in osteogenic differentiation did not increase the potency of Scl-mAb. These data suggest that Nmp4 is part of a common barrier to improving the efficacy of any osteoanabolic. Potential pathways and actors that comprise the re-programming of Nmp4-/- MSPCs to support the exaggerated osteoanabolic effect on the skeleton are discussed.Item Nmp4, a Regulator of Induced Osteoanabolism, Also Influences Insulin Secretion and Sensitivity(Springer, 2022) Bidwell, Joseph; Tersey, Sarah A.; Adaway, Michele; Bone, Robert N.; Creecy, Amy; Klunk, Angela; Atkinson, Emily G.; Wek, Ronald C.; Robling, Alexander G.; Wallace, Joseph M.; Evans-Molina, Carmella; Anatomy, Cell Biology and Physiology, School of MedicineA bidirectional and complex relationship exists between bone and glycemia. Persons with type 2 diabetes (T2D) are at risk for bone loss and fracture, however, heightened osteoanabolism may ameliorate T2D-induced deficits in glycemia as bone-forming osteoblasts contribute to energy metabolism via increased glucose uptake and cellular glycolysis. Mice globally lacking Nuclear Matrix Protein 4 (Nmp4), a transcription factor expressed in all tissues and conserved between humans and rodents, are healthy and exhibit enhanced bone formation in response to anabolic osteoporosis therapies. To test whether loss of Nmp4 similarly impacted bone deficits caused by diet induced obesity, male wild type (WT) and Nmp4−/− mice (8wks) were fed either low-fat diet (LFD) or high-fat diet (HFD) for 12wks. Endpoint parameters included bone architecture, structural and estimated tissue level mechanical properties, body weight/composition, glucose-stimulated insulin secretion, glucose tolerance, insulin tolerance and metabolic cage analysis. HFD diminished bone architecture and ultimate force and stiffness equally in both genotypes. Unexpectedly, the Nmp4−/− mice exhibited deficits in pancreatic β-cell function and were modestly glucose intolerant under normal diet conditions. Despite the β-cell deficits, the Nmp4−/− mice were less sensitive to HFD-induced weight gain, increases in % fat mass, and decreases in glucose tolerance and insulin sensitivity. We conclude that Nmp4 supports pancreatic β-cell function but suppresses peripheral glucose utilization, perhaps contributing to its suppression of induced skeletal anabolism. Selective disruption of Nmp4 in peripheral tissues may provide a strategy for improving both induced osteoanabolism and energy metabolism in comorbid patients.Item Novel insights into the mechanistic gene regulation of STAT3 in bone cells(2015-06-25) Corry, Kylie A.; Li, Jiliang; Bidwell, Joseph; Na, SungsooMany cells are involved in the orchestra that is bone homeostasis--particularly osteoclasts and osteoblasts, which mediate remodeling of bones. This creates a balance that must be kept in check, otherwise pathologies arise. The JAK-STAT signaling pathway is crucial to maintaining this balance. It has long been known that the transcription factor STAT3 has more profound effects on bone homeostasis than other members of the STAT family of proteins. Recently, a genetic condition called Job’s Syndrome has been specifically linked to point mutations in the Stat3 gene. These patients present with severe bone abnormalities, including prominent foreheads, broad nasal bridges, and abnormal eye spacing. For this reason, our lab has extensively studied conditional knockouts of Stat3 in all three types of bones cells in mice and observed severe deficiencies in numerous parameters of normal bone phenotypes. STAT3 seems to play a principal role in the signaling that takes place upon mechanical loading of bone tissues and calling cells into action where they are needed. Furthermore, STAT3 has been found to be up-regulated in the early-response gene cluster following mechanical loading. Our current approach to studying STAT3’s effects on bone includes both in vivo and in vitro comparisons of WT and KO STAT3 models. The conditional knock-out of STAT3 in 8-week old mice revealed significant phenotypic variations as compared to the WT controls, while no significant differences were observed in cKO newborn pups. We also looked at immortalized WT and STAT3 KO cell lines. The STAT3 KO cells had diminished proliferation rates and decreased differentiation capabilities. Furthermore, STAT3 KO cells showed significantly reduced mRNA levels of both Wnt3a and Wnt5a when exposed to fluid shear stress. By employing available ChIP-seq data, we were able to elucidate the genome-wide binding patterns of STAT3. From the peak distribution, we can begin to uncover novel downstream effectors of STAT3 signaling that are responsible for the observed phenotypes in our conditional knockout mouse model. A preliminary look at the ChIP-seq data reveals Wnt and Nrf2 signaling to be under the putative control of STAT3. In our further research, we endeavor to experimentally confirm the ChIP-seq data for STAT3 with RNA-seq experiments in the hopes of finding potential therapeutic targets for bone pathologies.Item Orthodontic Mechanotransduction and the Role of the P2X7 Receptor(2009) Viecilli, Rodrigo F.; Katona, Thomas R.; Chen, Jie; Roberts, W. Eugene, Jr.; Hartsfield, James K., Jr.; Bidwell, JosephThe first part of the study describes the development of a microCT based engineering model to study orthodontic responses. The second part investigated the relationship between orthodontic stimulus, root resorption and bone modeling. It was hypothesized that stress magnitudes are insufficient to portray the mechanical environment and explain the clinical response; directions also play a role. An idealized tooth model was constructed for finite element analysis. The principal stress magnitudes and directions were calculated in tipping and translation. It was concluded that within the same region of root, PDL and bone, there can be compression in one structure, tension in another. At a given point in a structure, compression and tension can coexist in different directions. Magnitudes of compression or tension are typically different in different directions. Previously published data presenting only stress magnitude plots can be confusing, perhaps impossible to understand and/or correlate with biological responses. To avoid ambiguities, a reference to a principal stress should include its predominant direction. Combined stress magnitude/direction results suggest that the PDL is the initiator of mechanotransduction. The third part of this project tested the role of the P2X7 receptor in the dentoalveolar morphology of C57B/6 mice. P2X7R KO (knockout) mice were compared to C57B/6 WT to identify differences in a maxillary molar and bone. Tooth dimensions were measured and 3D bone morphometry was conducted. No statistically significant differences were found between the two mouse types. P2X7R does not have a major effect on alveolar bone or tooth morphology. The final part examines the role of the P2X7 receptor in a controlled biomechanical model. Orthodontic mechanotransduction was compared in wild-type (WT) and P2X7R knock-out (KO) mice. Using Finite Element Analysis, mouse mechanics were scaled to produce typical human stress levels. Relationships between the biological responses and the calculated stresses were statistically tested and compared. There were direct relationships between certain stress magnitudes and root resorption and bone formation. Hyalinization and root and bone resorption were different in WT and KO. Orthodontic responses are related to the principal stress patterns in the PDL and the P2X7 receptor plays a significant role in their mechanotransduction.Item The Role of Wnt Signaling in Bone Mechanotransduction(2019-11) Bullock, Whitney Ann; Robling, Alexander; Bidwell, Joseph; Plotkin, Lilian; Sankar, Uma; White, KennethThe aging US population is experiencing a growing incidence of osteoporosis, characterized by increased fracture risk and low bone mass. In skeletal tissue, canonical Wnt signaling is a critical regulator of bone mass, and dysregulation of the Wnt pathway has been implicated in numerous skeletal displasias. Some components of the Wnt signaling pathway have a clear role in bone homeostasis, particularly in the response of bone to altered mechanical environment. Other pathway components are more poorly defined. One important intracellular signal transduction node in the Wnt cascade is β- catenin, which modulates gene expression and cell-cell junctions, among other functions. During periods of disuse, β-catenin is degraded, leading to inhibition of Wnt targets. Here, I characterize the role of β-catenin in bone during a disuse challenge, using a genetic mouse model expressing an inducible constitively-active mutant form of β- catenin in the osteocyte population. I hypothesize that prevention of β-catenin degradation during disuse will prevent the bone wasting effects of mechanodeprivation. As a second goal, I focus on upstream (membrane-bound) modulation of Wnt. Here, I investigate the low-density lipoprotein receptor-related receptor 4 (Lrp4), in the regulation of bone mass and mechanotransduction. I generated an Lrp4 knockin mouse model harboring a missense mutation found among human patients with abnormally high bone mass. I hypothesize that the mutation compromises sclerostin action on bone cells. Understanding how each of these components of the Wnt signaling pathway interact, may lead to novel therapeutic targets for treatment of bone diseases.Item Targeting Bone Quality in Murine Models of Osteogenesis Imperfecta, Diabetes, and Chronic Kidney Disease(2024-05) Kohler, Rachel; Wallace, Joseph; Allen, Matthew; Bidwell, Joseph; Surowiec, RachelSkeletal fragility can be caused by a wide array of diseases and disorders, but the most difficult etiologies to clinically circumvent are those in which the body loses not just bone mass but the ability to create healthy bone tissue. While in conditions such as osteoporosis (the most prevalent cause of age-related skeletal fragility in which elevated resorption without compensatory elevated formation leads to bone loss), interventions can target bone remodeling pathways to protect and increase bone mass, many other diseases are characterized by genetic and metabolic crippling of the remodeling process, rendering those same mass-based interventions less effective at reducing fracture risk. Osteogenesis imperfecta (OI) is a class of genetic disorders in which gene mutations affect the formation of collagen, a crucial building block of bone tissue that makes up 90% of its organic matrix, leading to lost bone mass and quality. As the main genetic causes of OI cannot currently be directly treated, therapeutic OI treatments are needed that improve tissue-level material properties. Similarly, metabolic conditions such as diabetes, a disorder in which the body cannot properly regulate blood sugar due to loss of insulin production and/or efficacy, can have multi-organ impacts including increased risk of developing chronic kidney disease and skeletal fragility. Type 2 diabetes is especially notorious for increasing fracture risk despite maintained or even increased apparent bone mass, which is strong evidence that intrinsic bone material properties are impaired by the disease state. A possible solution to the bone quality problem may be treatments that increase bone water content, as amplifying the water content of bone can improve multi-scale material properties such as collagen fibril elasticity and whole-bone toughness. Therefore, increasing bone hydration could be a way of improving tissue-level material properties, despite being unable to eradicate the genetic or metabolic disorders that alter how collagen is produced and incorporated into the bone matrix. To that end, this dissertation presents several studies that characterize models of osteogenesis imperfecta and diabetic kidney disease in mice and investigate methods of rescuing skeletal fragility in these animals through treatments that target both bone mass and bone quality with ties to tissue hydration.Item The Effect of Estrogen Repletion on Osteoblast Differentiation and DNA Synthesis in Ovariectomized Rats(1995) Miller, Ronald L.; Garetto, Lawrence P.; Bidwell, Joseph; Katona, Thomas R.; Roberts, W. Eugene; Shanks, James; Wohlford, Mark E.Previous studies have demonstrated that estrogen plays a significant role in bone mass conservation. To investigate the role that estrogen plays in osteoblast differentiation, the fractional distribution of periodontal ligament (PDL) osteoblast precursor cells was determined. Twenty six-month old female rats (Charles River Co.) were divided into two groups. Both groups were ovariectomized (OVX). Thirty-five days after ovariectomy one group (OVX+E) received supplemental daily injections of estrogen (0. 1 mg/kg Ethinyl Estradiol) for three days. After sacrifice, PDL sections through the mesial root of the maxillary first molar were prepared for microscopic analysis. Using a nuclear morphometric assay, the fibroblast-like cells of the POL were identified as early osteoprogenitor (A+A'), preosteoblast (C+D) or nonosteogenic (B) cells (i.e., A+A'=40-79 μm3; 8=80-119 μm3; C+D>120 μm3). Comparison of the OVX and OVX+E groups showed that treatment with estrogen increased early osteoprogenitor (A+A') cell and decreased preosteoblast (C+D) cell fractional distributions. No changes were seen in the non-osteogenic (B) cell group (expressed as% cell type, mean±SEM for n=4-8 rats/group; *p<0.05). Specimens were also stained with 5-Bromo- 2'-deoxyuridine (BrdU) to localize cells undergoing DNA synthesis. Both OVX and OVX+E groups showed minimal random BrdU staining throughout the PDL. Group= OVX A+A’= 8.8 ± 1.8 B= 30.2 ± 2.3 C+D= 60.8 ± 2.6 Group= OVX+E A+A’= 21.9 ± 2.6* B= 36.0 ± 2.2 C+D= 41.6 ± 3.8* The data suggest a block in proliferation of both less-differentiated precursor cells and preosteoblasts in estrogen-deficient animals. Furthermore, they suggest that estrogen may be required for normal preosteoblast differentiation leading to osteoblast production.