Browsing by Author "Wu, Danielle"
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Item BIGH3 mediates apoptosis and gap junction failure in osteocytes during renal cell carcinoma bone metastasis progression(Elsevier, 2024) Pan, Tianhong; Liu, Fengshuo; Hao, Xiaoxin; Wang, Shubo; Wasi, Murtaza; Song, Jian H.; Lewis, Valerae O.; Lin, Patrick P.; Moon, Bryan; Bird, Justin E.; Panaretakis, Theocharis; Lin, Sue-Hwa; Wu, Danielle; Farach-Carson, Mary C.; Wang, Liyun; Zhang, Ningyan; An, Zhiqiang; Zhang, Xiang H. -F.; Satcher, Robert L.Renal cell carcinoma (RCC) bone metastatis progression is driven by crosstalk between tumor cells and the bone microenvironment, which includes osteoblasts, osteoclasts, and osteocytes. RCC bone metastases (RCCBM) are predominantly osteolytic and resistant to antiresorptive therapy. The molecular mechanisms underlying pathologic osteolysis and disruption of bone homeostasis remain incompletely understood. We previously reported that BIGH3/TGFBI (transforming growth factor-beta-induced protein ig-h3, shortened to BIGH3 henceforth) secreted by colonizing RCC cells drives osteolysis by inhibiting osteoblast differentiation, impairing healing of osteolytic lesions, which is reversible with osteoanabolic agents. Here, we report that BIGH3 induces osteocyte apoptosis in both human RCCBM tissue specimens and in a preclinical mouse model. We also demonstrate that BIGH3 reduces Cx43 expression, blocking gap junction (GJ) function and osteocyte network communication. BIGH3-mediated GJ inhibition is blocked by the lysosomal inhibitor hydroxychloroquine (HCQ), but not osteoanabolic agents. Our results broaden the understanding of pathologic osteolysis in RCCBM and indicate that targeting the BIGH3 mechanism could be a combinational strategy for the treatment of RCCBM-induced bone disease that overcomes the limited efficacy of antiresorptives that target osteoclasts.Item Dynamic Assembly of Human Salivary Stem/Progenitor Microstructures Requires Coordinated α1β1 Integrin-Mediated Motility(Frontiers, 2019) Wu, Danielle; Witt, Robert L.; Harrington, Daniel Anton; Farach-Carson, Mary C.A tissue engineering approach can provide replacement salivary gland structures to patients with hyposalivation disorders and xerostomia. Salivary human stem/progenitor cells (hS/PCs) were isolated from healthy regions of parotid glands of head and neck surgery patients, expanded, then encapsulated in biocompatible hyaluronate (HA)-based hydrogels. These bioactive hydrogels provide a surrogate territorial matrix suitable for the dynamic assembly, growth and reorganization of salivary gland components. This study examined the dynamics of salivary microstructure formation, growth, and reorganization using time-lapse imaging over 15 h. Immunofluorescence detection monitored production of individual basement membrane components forming around developing microstructures, and Ki67 assessed proliferation. Dynamic movements in hydrogels were quantified by measuring angular velocity (ω) of rotating salivary microstructures and changes in basement membrane architecture during microstructure growth. Integrin involvement in the dynamic reassembly was assessed using knockdown and inhibitor approaches. Single hS/PCs expanded over 5 days into spherical microstructures typically containing 3–10 cells. In larger macrostructures, proliferation occurred near the peripheral basement membrane that underwent growth-associated cycles of thinning and collapse. De novo secretion of laminin/collagen IV from reorganizing hS/PCs preceded that of perlecan/HSPG2. Microstructures routinely expressed β1 integrin-containing complexes at basement membrane-associated regions and exhibited spontaneous and coordinated rotation during basement membrane maturation. β1 integrin siRNA knockdown at the single-cell state prevented hS/PC microstructure growth. After microstructure formation, β1 integrin knockdown reduced rotation and mean ω by 84%. Blockade of the α1 integrin subunit (CD49a) that associates with β1 reduced mean ω by 66%. Studies presented here show that initial hS/PC structure growth and basement membrane maturation depends on α1β1-integrin mediated signaling. Coordinated cellular motility during neotissue reorganization reminiscent of salivary gland acini was critically dependent both on hS/PC-secretion of laminin,collagen type-IV, and perlecan/HSPG2 and the force-driven interactions of α1β1-integrin activation. We conclude that α1β1-integrin plays a critical role in establishing human salivary gland coordinated structure and function, and that its activation in tissue engineered systems is essential to tissue assembly.Item Gabapentin Disrupts Binding of Perlecan to the α2δ1 Voltage Sensitive Calcium Channel Subunit and Impairs Skeletal Mechanosensation(MDPI, 2022) Reyes Fernandez, Perla C.; Wright, Christian S.; Masterson, Adrianna N.; Yi, Xin; Tellman, Tristen V.; Bonteanu, Andrei; Rust, Katie; Noonan, Megan L.; White, Kenneth E.; Lewis, Karl J.; Sankar, Uma; Hum, Julia M.; Bix, Gregory; Wu, Danielle; Robling, Alexander G.; Sardar, Rajesh; Farach-Carson, Mary C.; Thompson, William R.Our understanding of how osteocytes, the principal mechanosensors within bone, sense and perceive force remains unclear. Previous work identified “tethering elements” (TEs) spanning the pericellular space of osteocytes and transmitting mechanical information into biochemical signals. While we identified the heparan sulfate proteoglycan perlecan (PLN) as a component of these TEs, PLN must attach to the cell surface to induce biochemical responses. As voltage-sensitive calcium channels (VSCCs) are critical for bone mechanotransduction, we hypothesized that PLN binds the extracellular α2δ1 subunit of VSCCs to couple the bone matrix to the osteocyte membrane. Here, we showed co-localization of PLN and α2δ1 along osteocyte dendritic processes. Additionally, we quantified the molecular interactions between α2δ1 and PLN domains and demonstrated for the first time that α2δ1 strongly associates with PLN via its domain III. Furthermore, α2δ1 is the binding site for the commonly used pain drug, gabapentin (GBP), which is associated with adverse skeletal effects when used chronically. We found that GBP disrupts PLNItem Immunosuppressed Miniswine as a Model for Testing Cell Therapy Success: Experience With Implants of Human Salivary Stem/Progenitor Cell Constructs(Frontiers Media S.A., 2021) Wu, Danielle; Lombaert, Isabelle M. A.; DeLeon, Maximilien; Pradhan-Bhatt, Swati; Witt, Robert L; Harrington, Daniel Anton; Trombetta, Mark G; Passineau, Michael J; Farach-Carson, Mary C.An urgent need exists to develop large animal models for preclinical testing of new cell therapies designed to replace lost or damaged tissues. Patients receiving irradiation for treatment of head and neck cancers frequently develop xerostomia/dry mouth, a condition that could one day be treated by cell therapy to repopulate functional saliva-producing cells. Using immunosuppression protocols developed for patients receiving whole face transplants, we successfully used immunosuppressed miniswine as a suitable host animal to evaluate the long-term stability, biocompatibility, and fate of matrix-modified hyaluronate (HA) hydrogel/bioscaffold materials containing encapsulated salivary human stem/progenitor cells (hS/PCs). An initial biocompatibility test was conducted in parotids of untreated miniswine. Subsequent experiments using hS/PC-laden hydrogels were performed in animals, beginning an immunosuppression regimen on the day of surgery. Implant sites included the kidney capsule for viability testing and the parotid gland for biointegration time periods up to eight weeks. No transplant rejection was seen in any animal assessed by analysis of the tissues near the site of the implants. First-generation implants containing only cells in hydrogel proved difficult to handle in the surgical suite and were modified to adhere to a porcine small intestinal submucosa (SIS) membrane for improved handling and could be delivered through the da Vinci surgical system. Several different surgical techniques were assessed using the second-generation 3D-salivary tissue (3D-ST) for ease and stability both on the kidney capsule and in the capsule-less parotid gland. For the kidney, sliding the implant under the capsule membrane and quick stitching proved superior to other methods. For the parotid gland, creation of a tissue “pocket” for placement and immediate multilayer tissue closure were well tolerated with minimal tissue damage. Surgical clips were placed as fiduciary markers for tissue harvest. Some implant experiments were conducted with miniswine 90 days post-irradiation when salivation decreased significantly. Sufficient parotid tissue remained to allow implant placement, and animals tolerated immunosuppression. In all experiments, viability of implanted hS/PCs was high with clear signs of both vascular and nervous system integration in the parotid implants. We thus conclude that the immunosuppressed miniswine is a high-value emerging model for testing human implants prior to first-in-human trials.