Browsing by Subject "Proliferation"

Now showing 1 - 8 of 8
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    Biphasic alterations in coronary smooth muscle Ca2+ regulation in a repeat cross-sectional study of coronary artery disease severity in metabolic syndrome
    (Elsevier, 2016-06) McKenney-Drake, Mikaela L.; Rodenbeck, Stacey D.; Owen, Meredith K.; Schultz, Kyle A.; Alloosh, Mouhamad; Tune, Johnathan D.; Sturek, Michael; Department of Cellular and Integrative Physiology, School of Medicine
    BACKGROUND AND AIMS: Coronary artery disease (CAD) is progressive, classified by stages of severity. Alterations in Ca(2+) regulation within coronary smooth muscle (CSM) cells in metabolic syndrome (MetS) have been observed, but there is a lack of data in relatively early (mild) and late (severe) stages of CAD. The current study examined alterations in CSM Ca(2+) regulation at several time points during CAD progression. METHODS: MetS was induced by feeding an excess calorie atherogenic diet for 6, 9, or 12 months and compared to age-matched lean controls. CAD was measured with intravascular ultrasound (IVUS). Intracellular Ca(2+) was assessed with fura-2. RESULTS: IVUS revealed that the extent of atherosclerotic CAD correlated with the duration on atherogenic diet. Fura-2 imaging of intracellular Ca(2+) in CSM cells revealed heightened Ca(2+) signaling at 9 months on diet, compared to 6 and 12 months, and to age-matched lean controls. Isolated coronary artery rings from swine fed for 9 months followed the same pattern, developing greater tension to depolarization, compared to 6 and 12 months (6 months = 1.8 ± 0.6 g, 9 months = 5.0 ± 1.0 g, 12 months = 0.7 ± 0.1 g). CSM in severe atherosclerotic plaques showed dampened Ca(2+) regulation and decreased proliferation compared to CSM from the wall. CONCLUSIONS: These CSM Ca(2+) regulation data from several time points in CAD progression and severity help to resolve the controversy regarding up-vs. down-regulation of CSM Ca(2+) regulation in previous reports. These data are consistent with the hypothesis that alterations in sarcoplasmic reticulum Ca(2+) contribute to progression of atherosclerotic CAD in MetS.
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    Coronary artery disease in metabolic syndrome: a role for the sarcoplasmic reticulum Ca2+ ATPase
    (2016-05-10) Rodenbeck, Stacey Dineen; Sturek, Michael S.; Day, Richard N.; Evans-Molina, Carmella; Mather, Kieren; Tune, Johnathan D.
    Coronary artery disease (CAD) is a leading cause of death among Americans and is fueled by underlying metabolic syndrome (MetS). The prevalence and lethality of CAD necessitates rigorous investigations into its underlying mechanisms and to facilitate the development of effective treatment options. Coronary smooth muscle (CSM) phenotypic modulation from quiescent to synthetic, proliferative, and osteogenic phenotypes is a key area of investigation, with underlying mechanisms that remain poorly understood. Using a well-established pre-clinical model of CAD and MetS, the Ossabaw miniature swine, we established for the first time the time course of Ca2+ dysregulation during MetS-induced CAD progression. In particular, we used the fluorescent Ca2+ dye, fura-2, to examine alterations in CSM intracellular Ca2+ regulation during CAD progression, as perturbations in intracellular Ca2+ regulation are implicated in several cellular processes associated with CAD pathology, including CSM contractile responses and proliferative pathways. These studies revealed that the function of several CSM Ca2+ handling proteins is elevated in early CAD, followed by loss of function in severe atherosclerotic plaques. Decreased intracellular Ca2+ regulation occurred concurrently with reductions in CSM proliferation, measured with Ki-67 staining. In particular, alterations in sarcoplasmic reticulum (SR) Ca2+ store together with altered function of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) were associated with induction of proliferation. Organ culture of coronary arterial segments revealed that culture-induced medial thickening was prevented by SERCA inhibition with cyclopiazonic acid (CPA). Activation of SERCA with the small molecule activator, CDN1163, increased CSM proliferation, which was attenuated by treatment with CPA, thus establishing upregulated SERCA function as a proximal inducer of CSM proliferation. Further, we demonstrated that in vitro treatment of CSM from lean Ossabaw swine with the glucagon-like peptide-1 (GLP-1) receptor agonist, exenatide, increased SERCA function. However, in vivo treatment of Ossabaw swine with MetS with the GLP-1 receptor agonist, AC3174, had no effect on CAD progression and in vitro examination revealed resistance of SERCA to GLP-1 receptor agonism in MetS. These findings further implicate SERCA in CAD progression. Collectively, these are the first data directly linking SERCA dysfunction to CSM proliferation and CAD progression, providing a key mechanistic step in CAD progression.
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    Effect of Adenomatous Polyposis Coli Loss on Tumorigenic Potential in Pancreatic Ductal Adenocarcinoma
    (MDPI, 2019-09-14) Cole, Jennifer M.; Simmons, Kaitlyn; Prosperi, Jenifer R.; Biochemistry and Molecular Biology, School of Medicine
    Loss of the Adenomatous Polyposis Coli (APC) tumor suppressor in colorectal cancer elicits rapid signaling through the Wnt/β-catenin signaling pathway. In contrast to this well-established role of APC, recent studies from our laboratory demonstrated that APC functions through Wnt-independent pathways to mediate in vitro and in vivo models of breast tumorigenesis. Pancreatic ductal adenocarcinoma (PDAC) has an overall median survival of less than one year with a 5-year survival rate of 7.2%. APC is lost in a subset of pancreatic cancers, but the impact on Wnt signaling or tumor development is unclear. Given the lack of effective treatment strategies for pancreatic cancer, it is important to understand the functional implications of APC loss in pancreatic cancer cell lines. Therefore, the goal of this project is to study how APC loss affects Wnt pathway activation and in vitro tumor phenotypes. Using lentiviral shRNA, we successfully knocked down APC expression in six pancreatic cancer cell lines (AsPC-1, BxPC3, L3.6pl, HPAF-II, Hs 766T, MIA PaCa-2). No changes were observed in localization of β-catenin or reporter assays to assess β-catenin/TCF interaction. Despite this lack of Wnt/β-catenin pathway activation, the majority of APC knockdown cell lines exhibit an increase in cell proliferation. Cell migration assays showed that the BxPC-3 and L3.6pl cells were impacted by APC knockdown, showing faster wound healing in scratch wound assays. Interestingly, APC knockdown had no effect on gemcitabine treatment, which is the standard care for pancreatic cancer. It is important to understand the functional implications of APC loss in pancreatic cancer cells lines, which could be used as a target for therapeutics.
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    The Effects of a Pyk2 Kinase Inhibitor on the Proliferation and Differentiation of Human Dental Pulp Stem Cells
    (2021) McIntyre, Patrick; Bruzzaniti, Angela; Ehrlich, Ygal; Bringas, Josef; Spolnik, Kenneth
    Introduction: Regenerative endodontic procedures are an effective treatment option for immature teeth with infected necrotic pulps to allow for healing and potential continued root development, yet challenges to ideal treatment outcomes remain. Consistent development of root length and width of dentin remains a challenge, as does development of the pulp-dentin complex. Previous in vitro studies have assessed the role of different growth factors and bioactive molecules in combination with scaffolds to potentially facilitate continued development of the pulp-dentin complex using dental pulp stem cells (DPSCs). The proline-rich tyrosine kinase 2 (Pyk2) is linked with osteoblast activity and the regulation of bone mass. Further, the Pyk2 inhibitor PF-4618433 (PF-46) has been shown in previous studies to enhance osteoblast activity and mineral deposition in vitro. However, whether Pyk2 targeting promotes the osteogenic differentiation of DPSCs remains unknown. Objective: The purpose of this study was to investigate the effect of a Pyk2 inhibitor, PF-46, on the proliferation, differentiation, and mineralization of human DPSCs. Materials and Methods: Human DPSCs were cultured in 24-well plates with α-MEM with 10% FBS, and containing 0 μM (vehicle control) or 0.1 μM, 0.3 μM, or 0.6 μM PF-46. Fresh media and treatments were replaced every 2-3 days. After 1 day incubation, cytotoxic effects were evaluated by using an MTS proliferation assay. After 4 days of treatment, direct cell counting was performed. To induce osteogenic differentiation, ascorbic acid and β-glycerol phosphate were added to the culture media and the DPSCs were cultured with PF-46 for 14 days. Then, an alkaline phosphatase (ALP) assay and mineral deposition assay were performed. Differences between treatment groups were analyzed by a one-way ANOVA followed by pair-wise tests conducted using Tukey’s multiple comparisons procedure with a 5% significance level. Results: The 0.6 μM PF-46 group had a significantly higher cell count, ALP activity and mineral deposition when compared to 0 μM PF-46. The 0.1 and 0.3 μM PF-46 groups also had significantly higher ALP activity compared to the 0 μM PF-46 group after 14 days of incubation. There was a general trend of increased differentiation and mineral deposition as the concentration of PF-46 increased from 0.1 μM to 0.6 μM. Conclusion: There was a general concentration-dependent increase in cell count, differentiation, and mineral deposition by human DPSCs as the concentration of PF-46 increased from 0 μM up to 0.6 μM, with the highest activity observed with 0.6 μM PF-46. Although further research is needed, these results suggest that strategies that target Pyk2 may potentially be used to improve the osteogenic differentiation of DPSCs to aid endodontic regeneration.
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    Epidermal growth factor receptor restoration rescues the fatty liver regeneration in mice
    (American Physiological Society, 2017-10-01) Zimmers, Teresa A.; Jin, Xiaoling; Zhang, Zongxiu; Jiang, Yanlin; Koniaris, Leonidas G.; Surgery, School of Medicine
    Hepatic steatosis is a common histological finding in obese patients. Even mild steatosis is associated with delayed hepatic regeneration and poor outcomes following liver resection or transplantation. We sought to identify and target molecular pathways that mediate this dysfunction. Lean mice and mice made obese through feeding of a high-fat, hypercaloric diet underwent 70 or 80% hepatectomy. After 70% resection, obese mice demonstrated 100% survival but experienced increased liver injury, reduced energy stores, reduced mitoses, increased necroapoptosis, and delayed recovery of liver mass. Increasing liver resection to 80% was associated with mortality of 40% in lean and 80% in obese mice (P < 0.05). Gene expression profiling showed decreased epidermal growth factor receptor (EGFR) in fatty liver. Meta-analysis of expression studies in mice, rats, and patients also demonstrated reduction of EGFR in fatty liver. In mice, both EGFR and phosphorylated EGFR decreased with increasing percent body fat. Hydrodynamic transfection of EGFR plasmids in mice corrected fatty liver regeneration, reducing liver injury, increasing proliferation, and improving survival after 80% resection. Loss of EGFR expression is rate limiting for liver regeneration in obesity. Therapies directed at increasing EGFR in steatosis might promote liver regeneration and survival following hepatic resection or transplantation.
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    Kinetic analyses of vasculogenesis inform mechanistic studies
    (American Physiological Society, 2017-04-01) Varberg, Kaela M.; Winfree, Seth; Chu, Chenghao; Tu, Wanzhu; Blue, Emily K.; Gohn, Cassandra R.; Dunn, Kenneth W.; Haneline, Laura S.; Cellular and Integrative Physiology, School of Medicine
    Vasculogenesis is a complex process by which endothelial stem and progenitor cells undergo de novo vessel formation. Quantitative assessment of vasculogenesis is a central readout of endothelial progenitor cell functionality. However, current assays lack kinetic measurements. To address this issue, new approaches were developed to quantitatively assess in vitro endothelial colony-forming cell (ECFC) network formation in real time. Eight parameters of network structure were quantified using novel Kinetic Analysis of Vasculogenesis (KAV) software. KAV assessment of structure complexity identified two phases of network formation. This observation guided the development of additional vasculogenic readouts. A tissue cytometry approach was established to quantify the frequency and localization of dividing ECFCs. Additionally, Fiji TrackMate was used to quantify ECFC displacement and speed at the single-cell level during network formation. These novel approaches were then implemented to identify how intrauterine exposure to maternal diabetes mellitus (DM) impairs fetal ECFC vasculogenesis. Fetal ECFCs exposed to maternal DM form fewer initial network structures, which are not stable over time. Correlation analyses demonstrated that ECFC samples with greater division in branches form fewer closed network structures. Additionally, reductions in average ECFC movement over time decrease structural connectivity. Identification of these novel phenotypes utilizing the newly established methodologies provides evidence for the cellular mechanisms contributing to aberrant ECFC vasculogenesis.
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    Potential for Stem Cells Therapy in Alzheimer's Disease: Do Neurotrophic Factors Play Critical Role?
    (Bentham Science Publishers, 2017) Bali, Parul; Lahiri, Debomoy K.; Banik, Avijit; Nehru, Bimla; Anand, Akshay; Psychiatry, School of Medicine
    Alzheimer's disease (AD) is one of the most common causes of dementia. Despite several decades of research in AD, there is no standard disease- modifying therapy available and currentlyapproved drugs provide only symptomatic relief. Stem cells hold immense potential to regenerate damaged tissues and are currently tested in some brain-related disorders, such as AD, amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD). We review stem cell transplantation studies using preclinical and clinical tools. We describe different sources of stem cells used in various animal models and explaining the putative molecular mechanisms that can rescue neurodegenerative disorders. The clinical studies suggest safety, efficacy and translational potential of stem cell therapy. The therapeutic outcome of stem cell transplantation has been promising in many studies, but no unifying hypothesis can convincingly explain the underlying mechanism. Some studies have reported paracrine effects exerted by these stem cells via the release of neurotrophic factors, while other studies describe the immunomodulatory effects exerted by the transplanted cells. There are also reports which indicate that stem cell transplantation might result in endogenous cell proliferation or replacement of diseased cells. In animal models of AD, stem cell transplantation is also believed to increase expression of synaptic proteins.
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    Role of DHS in translation control of islet β-cell replication during high fat induced obesity and glucose intolerance
    (2017-07-12) Levasseur, Esther Marie; Mirmira, Raghu
    Insulin resistance in liver, muscle, and adipose tissue almost invariably occurs during obesity. To compensate, the insulin-producing β-cell increases insulin production by expanding cellular mass. The inability of the β-cell to fully compensate leads to hyperglycemia and ultimately type 2 diabetes. The enzyme deoxyhypusine synthase (DHS) catalyzes the spermidine-dependent posttranslational modification of Lys50 of eukaryotic translation initiation factor 5A (eIF5A) to form hypusine (Hyp). Studies have demonstrated this modification of eIF5A to contribute to cellular proliferation in cancerous cells, but its role in the physiologic proliferation of islet β-cells is unknown. I hypothesized eIF5A-Hyp to be required for the proliferation of islet β cells during the early phase of insulin resistance, allowing the β-cell to respond to the increased demand for insulin to maintain glucose homeostasis. To test this hypothesis, deletion of DHS was induced post-developmentally in β-cells by crossing Dhs-fl/fl mice with MIP1-CreERT mice, and animals were fed for 1 or 4 weeks with a 60% kcal from fat diet (HFD) or normal chow diet (NCD, 16% kcal from fat diet). NCD-fed and HFD-fed animals had normal glucose homeostasis after one week feeding, regardless of genotype. However, after 4 weeks of HFD, KO mice had significantly worse glucose intolerance compared to control mice. eIF5A-Hyp levels increased in β-cells of control animals and as expected remained low in the KO mice. β-cell proliferation was significantly increased after 1 week of HFD as measured by PCNA staining, however KO mice showed no increase. Cyclin D2 protein, but not mRNA, was increased in control animals fed a HFD; this protein increase was not observed in KO animals. Furthermore, polyribosomal profile of isolated islets of 1 week HFD-fed mice showed the Ccnd2 mRNA bound to the monoribosome fractions in the KO animals compared to the controls, resulting in changes of global translation. Interestingly, Ccnd1 polyribosome to monoribosome ratio showed no changes in translation compared to Ccnd2. Taken together, these results suggest that DHS (and, consequently, eIF5A-Hyp) is necessary for the adaptive proliferative and functional response of β-cells during high fat diet induced obesity and glucose intolerance.