Nutrition & Food Science
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Item Role of Transient Receptor Potential Vanilloid 4 (TRPV4) Calcium-permeable Channels in Fibro-inflammatory Diseases(2021) Goswami, Rishov; Rahaman, Shaik O.; Nutrition; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Tissue fibrosis and foreign body response (FBR) have emerged as two major public health problems globally over the last few decades. While fibrosis is an outcome of a dysregulated wound healing process, FBR, a chronic inflammatory disease, develops when the body responds and reacts to the implantation of biological materials. Interestingly, recent studies have associated these non-specific inflammatory diseases with altering stiffness although the exact underlying mechanisms by which mechanical cues can regulate the diseases remain poorly understood. The objective of this thesis work is to determine how the changing of tissue stiffness and implant rigidity mediates disease progression of fibrosis and FBR respectively. Here we identify a novel role of a polymodal mechanosensitive calcium channel, Transient Receptor Potential Vanilloid 4 (TRPV4), as a potential cell membrane receptor/channel in the pathophysiology of FBR and skin fibrosis associated with Scleroderma, a multisystem idiopathic fibro-inflammatory connective tissue disorder. Our results showed that TRPV4 is over expressed in fibrotic skin tissue and colocalize with alpha-smooth muscle actin (a-SMA), a common myofibroblast marker. Using mouse model, we demonstrated that TRPV4 knockout mice are protected from bleomycin-induced skin fibrosis development. Additionally, in a separate mouse model, we showed that genetic ablation of the TRPV4 channel protects mice from implantation-induced macrophage foreign body giant cell (FBGC) formation, macrophage accumulation, and FBR development to biomaterials. The results of our studies also determined an essential role of TRPV4 for macrophage fusion and the mechanism by which TRPV4 and matrix stiffness leads to cytoskeletal remodeling through a feed-forward functional interaction generating cellular force to modulate FBGC formation. We also identified a mechanosensing domain of TRPV4 which is crucial for FBGC generation. Altogether, the results presented in this thesis suggest TRPV4 as a potential regulator of stiffness-dependent fibrosis and inflammation development, and multinucleated FBGC formation. The results of this thesis work proposes that interaction between TRPV4 and substrate stiffness leads to cytoskeletal remodeling and cellular force generation to modulate FBGC formation under FBR. Overall, the work presented in this thesis provides a better understanding about the role of mechanosensitive calcium channel TRPV4 in the regulation of fibro-inflammatory diseases and highlights the possibilities of therapeutically targeting of this channel for disease management.Item Identification and functional analysis of a biflavone as a novel inhibitor of TRPV4-dependent atherogenic process in macrophages(2021) Alharbi, Mazen Obaid; Rahaman, Shaik O.; Nutrition; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Cardiovascular disease is the major cause of death throughout the world. Atherosclerosis, a chronic inflammatory disease of large arteries, is the major contributor to the growing burden of cardiovascular disease-related mortality and morbidity throughout the world. During early atherogenesis, as a result of inflammation and endothelial dysfunction, monocytes transmigrate into the aortic intimal areas, and differentiate into lipid-laden macrophage foam cells, a critical process in atherosclerosis. Numerous natural compounds such as flavonoids and polyphenols are known to have anti-inflammatory and anti-atherogenic properties. Transient receptor potential vanilloid 4 (TRPV4), a non-selective Ca2+-permeant ion channel and a mechanosensor, is widely expressed in diverse cell types including macrophages. Accumulating reports from our laboratory and others on TRPV4 recognized this plasma membrane receptor/channel as an essential modulator of various physiological functions in cardiac, pulmonary, urinary, skeletal, digestive system, and central and peripheral nervous systems. Thus, it is expected that aberrant regulation of TRPV4 activity may lead to multiple pathological conditions such as cardiovascular disease, pulmonary disease, inflammation, neurological disorders, inflammatory bowel disease and wound healing. Previous studies by our group and others have reported that TRPV4 can be activated by numerous mechanical and biochemical stimuli including shear stress, osmolarity, temperature, and growth factors, as well as by alterations in matrix stiffness in vitro and in vivo. Recently, we reported that oxidized low-density lipoprotein-mediated and matrix stiffness-induced macrophage foam cell formation, a critical pathological process in atherosclerosis, is regulated in a TRPV4-dependent manner. Given that TRPV4 is a mechanosensitive channels and mechanical factors like hypertension, disrupted laminar flow of blood, and matrix stiffening are recognized pro-atherogenic factors, makes TRPV4 an important target for therapeutic intervention of atherosclerosis. The objectives of this proposal is to: i) identify natural inhibitor (s) of TRPV4 utilizing a fluorometric imaging plate reader-supported Ca2+ influx assay, ii) functionally characterize the identified compound, and iii) determine the mechanisms by which the identified compound blocks pro-atherogenic/inflammatory TRPV4 activity in macrophages. We expect that the results of this study may strengthen the rationale for the use of natural compounds for developing therapeutic and/or chemopreventive molecules.Item THE ROLE OF ESE-1 IN NON-SMALL CELL LUNG CANCER (NSCLC) CELLS(2020) Lou, Zhiyuan; Lee, Seong-Ho; Nutrition; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Lung cancer is the most life-threatening cancer in the world. The identification of the effective molecular target is essential for lung cancer prevention and therapy. Epithelial Specific ETS-1 (ESE-1) is a transcription factor associated with several types of cancer. However, the significance of ESE-1 in human non-small cell lung cancer (NSCLC) remains unclear. The objective of this dissertation was to investigate if ESE-1 expression influences the tumorigenic and metastatic activity of human non-small cell lung cancer (NSCLC) and to explore the mechanisms associated with tumorigenesis and epithelial mesenchymal transition (EMT). Overexpression of ESE-1 repressed the anchorage-independent growth of human NSCLC cells (H1299 and H1703) and led to an increase of G1 arrest and apoptosis, additionally, to repress invasion and migration. Xenograft study indicated that ESE-1 expression inhibited the formation and development of the tumor. In terms of mechanistic studies, overexpression of ESE-1 downregulates NF-κB transcriptional activity in both H1299 and H1703 cells. The downregulation might be associated with inhibition of NF-κB-p65 phosphorylation. ESE-1 is a downstream target of TGF-β-stimulated EMT. Downregulation of ESE-1 by TGF-β is dependent on Smad2/3, but not on Smad4 and other alternative pathways, including ERK, p38 MAPK, JNK, RAS, GSK3, PI3K, NF-ĸB, CDC42, PKC, and Rock signaling. We identified two putative Smad responsive elements (SRE) in the ESE-1 promoter. After cloning internal deletion and point mutated clones lacking distal and proximal SRE, which were localized at the distal and proximal regions of the ESE-1 promoter between -1500 to -713, the double mutation responsible for ESE-1 transcriptional downregulation with TGF-β induction. Moreover, EMT downstream target Snail reciprocally interacts with ESE-1. Our findings indicate that ESE-1 serves as a tumor repressor in ESE-1-null NSCLC cells, and we propose a potential use of ESE-1 as a target of lung cancer chemoprevention.Item TRPV4, A CALCIUM-PERMEABLE CHANNEL, PLAYS A ROLE IN MATRIX STIFFNESS INDUCED MACROPHAGE POLARIZATION(2020) Dutta, Bidisha; Rahaman, Shaik Ohidar; Nutrition; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Phenotypic polarization of macrophages is deemed essential in innate immunity and various pathophysiological processes, but little is known about how mechanical forces like matrix stiffness regulate the polarization and the associated signaling mechanisms. Here we report that calcium ion channel Transient Receptor Potential Vanilloid 4 (TRPV4), a mechanosensitive receptor/channel, mediates the substrate stiffness-induced macrophage polarization. Using atomic force microscopy, we show that fibrosis-induced tissue stiffness is dependent on TRPV4. M1 macrophages were found to be the predominant macrophage subtype in stiffer tissues and loss of TRPV4 significantly decreased the level of M1 macrophages. These findings were further validated by our in vitro assays indicating that increase in substrate stiffness leads to an increased secretion of M1 proinflammatory mediators, which is further enhanced by the addition of soluble factors. Taken together, these findings provide new insights about the role of TRPV4 in matrix stiffness-induced macrophage polarization that can be explored in tissue engineering and in the development of targeted therapeutics.Item Identification of ESE-1 as a novel molecular target of chemopreventive agents for colon cancer prevention(2019) Lee, Jihye; Lee, Seong-Ho; Nutrition; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Epithelial specific ETS-1 (ESE-1) is one of the E26 transformation-specific transcription factor superfamily and could be considerable interest as a possible target for regulating diverse types of human cancer. Despite its clinical importance, the reported biological role of ESE-1 on cancer development and progression are still controversial and its underlying mechanisms of tumorigenesis remains unclear. The objectives of this dissertation are to elucidate the role of ESE-1 in tumorigenesis. With the evidence in cancer phenotypes, the underlying mechanisms of ESE-1 in colon cancer was also investigated. ESE-1 knockout mice increased azoxymethane (AOM) -induced and dextran sulfate sodium (DSS)-promoted formation of aberrant crypt foci (ACF) compared to wild type mice. Overexpression of ESE-1 suppressed anchorage-independent growth and migration/invasion in human colon cancer cells and while knockdown of ESE-1 reversed anti-cancer activity. Full length ESE-1 was abundantly found in the nucleus, and internal deletion of nuclear localization sequence 2 (NLS2) decreased the amount of nuclear ESE-1. Three lysine residues (318KKK320) in the NLS2 were critical for nuclear localization of ESE-1 and mediates tumor suppressive activity of ESE-1 through reduced beta-catenin transcriptional activity. We identified two anti-cancer natural compounds, epigallocatechin-3-gallate (EGCG) and patchouli alcohol as ESE-1 inducers. Both EGCG and patchouli alcohol increased expression of ESE-1 protein and mRNA in human colon cancer cells. Patchouli alcohol showed reduced the number of tumors and tumor load in Apcmin/+ colon cancer animal model although protein expression level of ESE-1 did not show significant difference. These findings suggest a potential use of ESE-1 as a novel and potential molecular target of natural anti-cancer phytochemicals for colon cancer prevention.Item TRPV4, a calcium-permeable channel, regulates oxidized LDL-induced macrophage foam cell formation(2017) Goswami, Rishov; Rahaman, Shaik O.; Nutrition; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Accumulation of lipid-laden “macrophage foam cell” in the arterial wall is the hallmark of atherosclerosis that leads to the highest number of cardiovascular disease-related deaths in United States. Membrane scavenger receptors such as SR-A, and CD36 play important role in controlling oxidized low-density lipoprotein binding and uptake, and, thereby, in macrophage foam cell formation. Recent studies also put emphasis on the role of mechanical factors, such as matrix stiffness, in the regulation of macrophage function and atherogenesis. However, the identity of a plasma membrane mechanosensor and the underlying mechanisms that may promote atherogenesis is yet to be identified. We have found that a calcium-permeable plasma membrane protein TRPV4, a mechanosensor, may play an essential role in regulating macrophage foam cell formation, a critical process in atherosclerosis. We have also found that TRPV4 is essential for oxLDL uptake, but not for its binding. Altogether, herein, we demonstrate that TRPV4 plays a critical role in macrophage-foam-cell formation by regulating oxLDL uptake in cells.Item ANTICANCER MECHANISM OF TOLFENAMIC ACID IN COLORECTAL CANCER(2016) Lou, Zhiyuan; Lee, Seong-Ho; Nutrition; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Colorectal cancer (CRC) is the third leading cause of cancer-related death in the United States. Chemopreventive therapies could be effective way to treat CRC. Tolfenamic acid, one of the NSAIDs, shows anti-cancer activities in several types of cancer. Aberrant Wnt/β-catenin regulation pathway is a major mechanism of colon tumorigenesis. Here, we sought to better define the mechanism by which tolfenamic acid suppresses colorectal tumorigenesis focusing on regulation of β-catenin pathway. Treatment of tolfenamic acid led to a down-regulation of β-catenin expression in dose dependent manner in human colon cancer cell lines without changing mRNA. MG132 inhibited tolfenamic acid-induced downregulation of β-catenin and exogenously overexpression β-catenin was stabilized in the presence of tolfenamic acid. Tolfenamic acid induced an ubiquitin-mediated proteasomal degradation of β-catenin. In addition, tolfenamic acid treatment decreased transcriptional activity of β-catenin and expression of Smad2 and Smad3 while overexpression of Smad 2 inhibited tolfenamic acid-stimulated transcriptional activity of β-catenin. Moreover, tolfenamic acid decreased β-catenin target gene such as vascular endothelial growth factor (VEGF) and cyclin D1. In summary, tolfenamic acid is a promising therapeutic drug targeting Smad 2-mediated downregulation of β-catenin in CRC.Item Nutritional roles of selenium in cellular and mouse aging(2013) Wu, Tsung-Yu; Cheng, Wen-Hsing; Nutrition; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Oxidative stress and persistent DNA damage response can lead to cellular senescence and aging. The ATM kinase and p53 protein play critical roles in the DNA damage response to reactive oxygen species and other DNA-damaging agents. Although the majority of selenoproteins carry antioxidant activities, little is known about the nutritional role of selenium (Se) in aging. Previous studies indicated that selenoprotein H (SelH) is very sensitive to dietary Se deficiency. Moreover, SelH is a nuclear selenoprotein that is proposed to carry redox domains and to transactivate redox genes including one for glutathione biosynthesis. To determine the role of SelH in genome maintenance, SelH and scrambled shRNA knockdown were stably established in MRC-5 human diploid fibroblast or immortalized cancer cells. SelH shRNA MRC-5 cells showed more pronounced induction of β-galactosidase expression, autofluorescence, growth inhibition, and ATM pathway activation (γH2AX and phospho-ATM Ser-1981) as compared to scrambled shRNA cells. Interestingly, the slow proliferation in SelH shRNA MRC-5 cells was alleviated in the presence of ATM kinase inhibitors KU 55933 and KU 60019, by p53 shRNA knockdown, or by maintaining the cells in 3% O2 incubator (vs. ambient O2). Phospho-ATM Ser-1981 and γH2AX induction by H2O2 treatment (20 M) was temporally exacerbated in SelH shRNA but reversed in the scrambled shRNA MRC-5 cells 1-5 days after recovery. GFP-SelH did not relocalize to sites of oxidative DNA damage. Results from cologenic assays indicated that SelH shRNA HeLa cells were hypersensitive to paraquat and H2O2 but not to other clastogens including hydroxyurea, neocarzinostatin or camptothecin. The H2O2-induced cell death was attenuated in the presence of N-acetyl cysteine (NAC), a glutathione analogue, in SelH but not in scrambled shRNA HeLa cells. In conclusion, SelH protects against cellular senescence specifically to oxidative stress through a genome maintenance pathway involving ATM and p53. While recent research has demonstrated that mice unable to express selenoproteins in epidermal cells or in osteo-chondroprogenitor cells showed an apparently aging phenotype characterized by alopecia or bone abnormality, respectively. Thus, a role of selenium, particularly at nutritional levels of intake, in aging is largely unknown. What is lacking is an appropriate aging model of dietary Se deprivation displaying many features of normal aging. Telomere attrition provokes DNA damage response and, subsequently, replicative senescence. Because the chromosomes of mice carry longer telomeres than those of humans, the proposed hypothesis is that lengthy telomeres preclude mice deprived of Se to display aging phenotypes and age-related disorders. To test this hypothesis, weanling late generation Terc-/- mice were fed a Se-deficient diet or the diet supplemented with selenate (0.15 ppm) throughout their life. The objectives are to elucidate the role of Se in reducing age-related loss of function and begin to identify the key molecular mediators and selenoproteins during the aging process. As evidenced by changes in metabolic markers (body weight, glucose intolerance, insulin resistance and bone structure) and aging phenotypes (gray hair, alopecia, wound healing and telomere attrition), these data strongly indicate health span deterioration by dietary Se deficiency in the short telomere mice. MicroRNAs (miRNAs) are regulators of messenger RNA stability and translation and have been proposed as biomarkers for a variety of diseases and physiological conditions, including aging. A high-throughput platform, TaqMan low density array, was used to profile more than 800 miRNAs in plasma whose expression were validated by using individual quantitative PCR. The expression of a couple of miRNAs were induced both by dietary Se deprivation and aging. Altogether, a very interesting model of aging is established in this project by deprivation of Se that displays many hallmarks of human aging and can reveal the roles of Se at nutritional levels, in contrast with previous approaches, in which these essential roles in delaying health span deterioration may have been masked by lengthy telomeres.