Browsing by Author "Santangelo, Kelly, committee member"
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Item Embargo Culture-expanded articular chondrocytes: a potential cellular therapeutic for osteoarthritis with MSC-like properties(Colorado State University. Libraries, 2022) Liebig, Bethany Ellen, author; Goodrich, Laurie, advisor; Kisiday, John, advisor; Regan, Daniel, committee member; Santangelo, Kelly, committee member; McGilvray, Kirk, committee member; Bahney, Chelsea, committee memberOsteoarthritis (OA) is a highly prevalent and debilitating joint disease in horses, dogs, and humans. OA affects more than 303 million people globally with an annual economic loss to Americans approaching $200 billion. It has a considerable impact on the patient, resulting in pain and disability and more than 1 million people undergo knee arthroscopy or joint replacement surgery each year due to end-stage OA in the United States. Therefore, OA therapies that produce lasting symptom- and disease-modifying effects are a medical priority. Mesenchymal stromal cells (MSCs) are considered 'medicinal signaling cells' that have been postulated to treat OA by reducing inflammation and restoring joint function. However, IA injection of MSCs into diseased human or companion animal joints has demonstrated only a modest benefit to date, as symptom-modifying effects are often temporary, and evidence of disease-modification has been minimal. It has been reported that culture-expanded chondrocytes (CECs) can assume many of the hallmark properties of MSCs, such as immunomodulation and immunophenotype. However, unlike MSCs, chondrocytes are known to thrive in suspension, which is important as IA injections release cells into synovial fluid. The goal of this research aims to characterize the growth, immunomodulatory properties, and gene expression of equine CECs as a function of expansion in vitro as well as CEC persistence in the joint after intra-articular injection using a validated model of OA in rats. Additional goals of this research are to 1) determine how CECs may (persistence) or may not (immunomodulation and molecular fingerprint) differ from bone marrow derived MSCs, and 2) compare cellular properties of CECs across age to determine an ideal donor for generating allogeneic therapies. The results shown in chapters 2 and 3 indicate that chondrocytes retain a strong propensity for immunomodulation, that increases with expansion and dedifferentiation does not coincide with other temporal changes in gene expression. Further, these data do not indicate a benefit of neonatal donors. Future in vitro studies should further characterize the immunomodulatory, redifferentiation (chondrogenic) and angiogenic potential of CECs. The preliminary results described in chapter 4 indicate that CECs may have greater persistence than MSCs in the first 3 days post IA injection. Future in vivo studies should focus on determining the symptom- and disease-modifying effects following IA injection of CECs in relevant preclinical models, such as the rodent, horse, and dog.Item Embargo From the ovine to human rotator cuff; tenocyte as to MSC derived exosomes for tendon healing(Colorado State University. Libraries, 2024) von Stade, Devin P., author; Regan, Daniel, advisor; McGilvray, Kirk, advisor; Santangelo, Kelly, committee member; Hollinshead, Fiona, committee memberTendinopathies comprise one of the most widespread and economically significant diseases in developed nations. The societal value of rotator cuff tear surgical intervention alone has been estimated at greater than 3.4 billion US dollars despite frequent repair failures (30-79%). This drives great interest in adjunct therapies; however, research is complicated by a limited understanding of the underlying pathogenesis. Recent data suggests that the primary driver is cell-to-cell communication during the acute and chronic stages of rotator cuff tears. Most notably, the paracrine signaling of macrophages, which are preferentially recruited earlier and persist longer than other immune cells, may direct the structural function of injured tendons. Extracellular vesicles (EVs) are the primary contributors to the paracrine signaling responsible for many successful cell therapy studies. Investigations into mesenchymal stromal cell (MSC) derived EVs have served as a launching point toward this end, however, cell origin can dramatically change the effect of EVs on target cells. To explore the effects of exosomes as a function of cell source on tendon healing, we have developed in vitro models in human and ovine cell lines to test the effects of tissue native, tenocyte derived EVs as they compare to MSC derived EVs on key effectors of rotator cuff tears, tenocytes and macrophages. The goal of this work is to (a) describe the direct effect of EV education, as a function of cell source, MSC vs tenocyte, on macrophage gene regulation and cytokine production and tenocyte bioactivity; (b) to then assess the indirect effects of such EV educated macrophages on tenocyte bioactivity. (c) Additionally, the underlying pathogenesis of tendinopathy and the animal models of rotator cuff tears we use will be explored and further defined in the context of contemporary histologic and biomechanical methods.Item Open Access Glial inflammation as a key regulator and therapeutic target for prion disease(Colorado State University. Libraries, 2023) Hay, Arielle, author; Zabel, Mark, advisor; Moreno, Julie, advisor; Tjalkens, Ronald, committee member; Chanda, Soham, committee member; Santangelo, Kelly, committee memberPrion diseases are lethal neurodegenerative diseases characterized by the misfolding of the cellular prion protein, PrPC, into the infectious PrPSc. PrPSc accumulation in the brain contributes to the activation of microglia and the subsequent increase in reactive astrocytes, which together contribute to neuroinflammation. PrPSc aggregation triggers and leads to the dysregulation of a variety of cellular stress pathways, including the oxidative stress response, unfolded protein response, ubiquitin-proteosome system, autophagy and lysosomal degradation. Most critically, PrPSc contributes to neuronal toxicity and death, but the mechanism behind this is poorly understood. Prion diseases affect humans and a variety of mammalian species, with no available treatments. The majority of therapeutics developed to combat these diseases have targeted the prion protein itself. As these have been unsuccessful, it is time to turn our attention to treatments that target the cellular pathways and neuroinflammation caused by PrPSc accumulation in the brain. The overarching goal of this work is to identify glial-induced inflammation as a candidate for therapeutic intervention of prion diseases. We assessed the use of mesenchymal stromal cells (MSCs), which are potent regulators of inflammatory signaling and glial polarization, in cell culture and animal models of prion disease. Additionally, we investigate the role of a key inflammatory signaling pathway, Nuclear Factor-Kappa B (NF-κB) in microglial response to prion infection. Our findings both characterize contributions of specific glial cells to prion-induced inflammation, as well as uncovering novel targets for the treatment of prion diseases. First, we assessed the therapeutic potential of adipose-derived mesenchymal stromal cells (AdMSCs) in a cell culture model of glial prion infection. MSCs are known for their ability to migrate to sites of inflammation and produce immunomodulators. We evaluated the ability of cultured AdMSCs to respond to molecular factors present in brain homogenates from prion-infected animals. We found that these cells upregulate anti-inflammatory genes in response to both specific inflammatory cytokines and crude prion brain homogenates. Moreover, AdMSCs migrate towards prion brain homogenates in an in vitro model. Co-culturing AdMSCs with prion-treated BV2 cells or infected primary mixed glial cultures resulted in a significant decrease in markers of inflammation and disease-associated microglia and reactive astrocyte markers. These findings were independent of PrPSc, as AdMSCs had no effect on prion accumulation in mixed glial cultures. Collectively, these findings highlight AdMSCs as an intriguing candidate for modulating glial-induced inflammation in prion disease. Next, we evaluated AdMSCs in a mouse model of prion disease. Prior to delivery into prion-infected mice, AdMSCs were stimulated with TNFα, which we show increases their upregulation of anti-inflammatory molecules and growth factors. Stimulated AdMSCs were delivered intranasally to prion-infected mice every two weeks beginning from early in infection (10 weeks post-infection (wpi)) and ending late in infection (20 wpi). A cohort of mice was euthanized at various stages in infection, at 14 wpi, 16 wpi and 18 wpi. We show that AdMSCs are able to migrate throughout the brain when delivered intranasally, with the most cells being found in the hippocampus and thalamus. Although AdMSCs were not successful in improving behavior or increasing survival in prion-infected mice, they did induce changes in prion pathology at early time points in disease. A decrease was seen in inflammatory cytokines and markers of glial activation. No changes were seen in PrPSc accumulation or neuronal loss compared to untreated controls. However, at both 16- and 18 wpi, we identified significant changes in both glial numbers as well as morphology, indicating that AdMSCs attenuate reactivity in microglia and astrocytes. Together, these findings highlight AdMSCs as potent regulators of prion-induced glial inflammation, and warrants further investigation to optimize these cells as a treatment for prion disease. In addition to assessing therapeutics that decrease inflammation and reprogram glial cells to a homeostatic phenotype, we wanted to better characterize specific inflammatory pathways and understand how these were being regulated in glial cells in response to prion infection. NF-κB-related genes have long been identified in the brains of animal models with prion disease, but studies that have investigated its role in prion pathogenesis have focused on neurons and astrocytes. Microglia are critical innate immune regulators in the brain, and interact closely with both neurons and astrocytes to regulate inflammation and cell survival. Therefore, we saw an immediate need to characterize NF-κB signaling in microglia, and its contribution to prion-induced neuroinflammation. IKβ kinase (IKK) is a complex that responds to cell stressors and is critical for NF-κB signaling to occur. We utilized a primary mixed glial model containing wild-type (WT) astrocytes and IKK KO microglia. Upon infecting these mixed glial cultures with prions, we saw a drastic decrease in NF-κB-related genes compared to cultures containing WT astrocytes and WT microglia. Despite this, cultures containing IKK KO microglia still contribute neurotoxic signals that induce neuronal cell death. Moreover, we found that cultures with IKK KO microglia showed significantly more PrPSc accumulation, suggesting that these cells may have impaired autophagy. This work implicates microglial NF-κB-signaling and IKK as a potent inducer of inflammation and regulator of autophagy in prion disease.Item Open Access Iron, hepcidin, and microcytosis in canine hepatocellular carcinoma(Colorado State University. Libraries, 2021) Polak, Klaudia Zofia, author; Olver, Christine, advisor; Avery, Anne, committee member; Santangelo, Kelly, committee member; Shropshire, Sarah, committee memberHepatocellular carcinoma (HCC) is the most common primary liver tumor found in dogs. There is evidence that iron dysregulation is associated with HCC pathogenesis in both humans and dogs. Anemia and thrombocytosis were common hematologic abnormalities detected in about half of dogs with massive HCC, and microcytosis was present in approximately 31% of dogs in one study. Additionally, humans with hereditary hemochromatosis have an increased risk of HCC. The liver is the major organ site for iron storage and metabolism containing numerous iron regulatory proteins which may play an important role in canine HCC. Since microcytosis is associated with iron restricted erythropoiesis, our first objective was to determine whether neoplastic hepatocytes exhibit differential expression of iron regulatory genes as well as hepatic iron stores in normocytic versus microcytic HCC cases in an initial pilot study. Next, we aimed to quantify and compare expressions of a larger set of iron regulatory and human HCC-related genes among canine HCC tumor tissue, adjacent peritumoral liver tissue, non-specific reactive hepatitis liver tissue from non-HCC dogs, and normal liver tissue, as well as to quantify and compare estimated hepatic iron stores. We hypothesized that canine HCC tumor tissue exhibits iron overloading and higher expression of hepcidin and its upstream regulators (IL-6 and BMP6), which would promote intracellular iron availability for neoplastic hepatocyte proliferation. We also hypothesized that microcytic HCC cases would exhibit higher expression levels of hepcidin in tumor tissue compared to tumors from normocytic dogs. Additionally, we explored associations between clinical parameters and RNA levels of iron regulatory genes as well as estimated hepatocellular iron stores in both HCC tumor and the adjacent, peritumoral tissues. We expected to find gene expression patterns in canine HCC tumor tissue related to abnormal regulation of iron metabolism and other pathways similar to what has been described in human malignancies. Cases were selected from a database search for canine HCC and included if complete pre-operative blood work was available and there was adequate formalin-fixed paraffin-embedded (FFPE) tissue for RNA isolation for all cases. Hematologic and clinical parameters were recorded and used for correlation studies. All liver sections were reviewed by a board-certified veterinary anatomic pathologist. RNA was isolated from FFPE blocks and NanoString nCounter platform was used to quantify RNA counts for selected genes. Sections were stained with Perls Prussian Blue stain and hepatocytic iron stores were estimated using NIS-Elements software. Contrary to our hypotheses, all canine HCC tumors had markedly decreased expression of hepcidin (HAMP) and depletion of hepatocellular iron stores. Other iron-related genes down-regulated in canine HCC tumor tissue included TfR2 (an upstream regulator of hepcidin), STEAP2, LTF, HMOX1, CYBRD1and SFXN5. Tumor tissue overexpressed TfR1, STEAP3, and LCN2. No significant differences in RNA levels or iron stores were found between tumors of microcytic and normocytic HCC cases, but the adjacent peritumoral tissue was markedly iron loaded and exhibited negative correlation between hepcidin RNA levels and mean cell volume (MCV) as well as serum iron. Microcytic HCC cases were associated with noteworthy clinical findings such as increased ALT, lower HCT and serum iron, and histologically more poorly differentiated tumors. Differential expression of genes involved in Wnt signaling and ferroptosis was observed in canine HCC tumor versus the adjacent peritumoral liver tissue.Item Open Access Testing the metabolic sink postulate: subcutaneous adipose tissue the protective depot(Colorado State University. Libraries, 2017) Booth-Kalajian, Andrea Deborah, author; Foster, Michelle, advisor; Wier, Tiffany, committee member; Melby, Chris, committee member; Santangelo, Kelly, committee memberAdipose tissue distribution and not body mass index is the major predictor of risk for obesity-related chronic disease. Specifically, central adiposity, intra-abdominal/visceral adipose tissue accumulation, is associated with adverse metabolic outcomes such as, but not limited to, insulin resistance syndrome, cardiovascular disease, and hypertension [1, 2]. Conversely, peripheral adiposity, subcutaneous/gluteofemoral adipose tissue accumulation, is considered protective against metabolic disease [3, 4]. It is proposed that the subcutaneous adipose depot functions as a "metabolic sink" to sequester and store lipid from circulation, preventing ectopic deposition. Therefore, an individual with high overall fat mass primarily located in the lower body subcutaneous adipose depots could be metabolically healthy while obese. While subcutaneous adipose tissue (SAT) has been associated with improved insulin sensitivity and lower risk of adverse metabolic outcomes, it has not been fully examined for exact mechanisms or causality. The broad goal of this proposal was to identify and understand how adipose tissue contributes to the development, progression, and possibly resistance to metabolic disease. The specific goal of this dissertation was to examine how SAT protects against metabolic dysregulation. One of the protective properties of LBSAT is its ability to expand and proliferate with new/healthy, lipid-filling adipocytes. We examined adipose tissue compensation following intra-abdominal fat removal and glucose homeostasis. Peroxisome proliferator-activated receptor-γ (PPARγ; an activator of adipogenesis) knockout mice and control mice received either Sham surgery or intra-abdominal lipectomy. The inability of cell proliferation following lipectomy in PPARγ knockout mice induces glucose intolerance. Control mice with intra-abdominal lipectomy had increases in peripheral adipose mass, cell size redistribution, and improved glucose tolerance. The Foster lab previously demonstrated that removal of LBSAT caused skeletal muscle, but not liver, lipid accumulation in standard CHOW and high fat diet (HFD) mice. Additionally, LBSAT removal resulted in deterioration of systemic glucose tolerance and muscle insulin sensitivity in HFD animals only. Hence, we proposed that muscle triglyceride deposition per se was not sufficient to explain systemic glucose intolerance. One purpose of this dissertation was to further examine the protective properties of SAT and to investigate the fundamental mechanisms that contribute to impairment of glucose tolerance. We sought to extend our previous research with a systematic approach. We hypothesized that SAT has a dose-dependent association with systemic glucose regulation and maintenance of insulin sensitivity in nearby muscle. Our focus here was to examine the relation between peripheral adipose tissue and glucose homeostasis. This was accomplished with progressive removal of adipose tissue: ~20%, 40%, or 80% of the total SAT. Mice fed HFD for 13 weeks exhibit a dose-dependent decline in systemic glucose tolerance. This was accompanied by a decline in femoral muscle insulin response in the basal state but not the insulin-stimulated state. Muscle triglycerides were significantly high in all surgery groups. Other contributing factors were eliminated, including circulating factors, adipocyte distribution and compensation, or liver triglycerides. Therefore, we have demonstrated a dose-response effect of progressive SAT removal on glucose intolerance and basal muscle insulin insensitivity. In addition to metabolic outcomes, we seek to identify a lipid signature that is linked to diet-induced impairments in glucose tolerance. Liquid chromatography and mass spectrometry (LCMS) were used to identify differential diet patterns of lipid species between CHOW and HFD. Mice that did not have fat removed and were fed a healthy chow diet have intramuscular triglycerides that are consistent with longer chain fatty acids (more carbons) and a higher degree of unsaturation (less hydrogens). They also have high abundance of phosphatidylserine and phosphatidylinositol. Diet-induced obesity is associated with femoral muscle lipids that include diacylglycerides and sphingomyelin. Overall, when examining the total lipid profile in muscle, healthy fats were more influential than unhealthy ones. In summary, inhibition of adipocyte proliferation results in glucose intolerance following intra-abdominal fat removal. Progressive subcutaneous fat removal results in a dose-dependent deterioration of systemic and muscle glucose homeostasis. Thus, peripheral fat does indeed serve as a "metabolic sink" that sequesters excess energy and preserves metabolic regulation. Muscle lipid accumulation per se is not detrimental to health, but the types of lipids that are stored should be considered.Item Open Access Use of immune activated cellular therapy and risks with antibiotic administration in treatment of septic arthritis(Colorado State University. Libraries, 2021) Pezzanite, Lynn M., author; Dow, Steven, advisor; Goodrich, Laurie, advisor; Hendrickson, Dean, committee member; Santangelo, Kelly, committee member; Stoneback, Jason, committee memberTo view the abstract, please see the full text of the document.