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Title: Could Dietary Peroxidized Lipids provoke an Intestinal Inflammatory Response?.
Creator: Doomra, Mitsushita, Parthasarathy, Sampath, Jewett, Mollie, Singla, Dinender, University of Central Florida
Abstract / Description: Inflammatory Bowel Disease and Crohn's disease represent chronic intestinal inflammatory diseases. It is suspected that bacterial infection is one of the causes of gut inflammation. Studies from others as well as from our laboratory have indicated that peroxidized lipids and their decomposition products are pro-inflammatory. As we consume considerable amounts of dietary oxidized lipids (arising from deep frying of vegetable oils), we hypothesize that dietary peroxidized lipids may also lead...
Show moreInflammatory Bowel Disease and Crohn's disease represent chronic intestinal inflammatory diseases. It is suspected that bacterial infection is one of the causes of gut inflammation. Studies from others as well as from our laboratory have indicated that peroxidized lipids and their decomposition products are pro-inflammatory. As we consume considerable amounts of dietary oxidized lipids (arising from deep frying of vegetable oils), we hypothesize that dietary peroxidized lipids may also lead to intestinal inflammation. To test this hypothesis, intestine from C57BL/6J mice were collected and used in this study. The intestinal epithelial tissue as well as intestinal lymphoid tissues [Peyer's Patches (PP)] were identified and harvested. Both the tissue samples were incubated with 13-Hydroperoxyoctadecadienoic acid (HPODE, a simple form of peroxidized fatty acid) or oxidized phosphatidyl choline (Ox-PL) or minimally modified LDL (mmLDL) or bacterial lipopolysaccharide (LPS) at 37(&)deg;C. After 6 hours of incubation, RNA was extracted and RT-PCR analysis was performed to determine inflammatory markers using mouse primers for the gene expression of cytokines. We noted an increased basal gene expressions of inflammatory cytokines in PP tissues as opposed to the epithelial tissue. An increase in inflammatory cytokines gene expression was observed in LPS/POL treated intestinal tissues as compared to untreated tissues. Overall, our findings might suggest additional potential sources of gut inflammation as well as an active participation of epithelial cells in the inflammatory process. These might also offer novel targets for the control of inflammation of the gut in patients suffering from gut inflammatory diseases.
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Date Issued: 2016
Identifier: CFE0006683, ucf:51901
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0006683

Title: Malondialdehyde (MDA) and Glutathione Peroxidase (GPx) are elevated in Crohns disease-associated with Mycobacterium avium subspecies paratuberculosis (MAP).
Creator: Qasem, Ahmad, Naser, Saleh, Masternak, Michal, Parthasarathy, Sampath, Andl, Claudia, University of Central Florida
Abstract / Description: Inflamed tissue in Crohn's disease (CD) are continuously producing toxic oxygen metabolites leading to cellular injury and apoptosis. Here, we are evaluating the role of Mycobacterium avium subspecies paratuberculosis (MAP) in oxidative stress in CD by evaluation of lipid peroxidation and antioxidant defense activity. Specifically, we measured malondialdehyde (MDA) level and selenium-dependent glutathione peroxidase (GPx) activity in the plasma from patients and cattle infected with MAP. The...
Show moreInflamed tissue in Crohn's disease (CD) are continuously producing toxic oxygen metabolites leading to cellular injury and apoptosis. Here, we are evaluating the role of Mycobacterium avium subspecies paratuberculosis (MAP) in oxidative stress in CD by evaluation of lipid peroxidation and antioxidant defense activity. Specifically, we measured malondialdehyde (MDA) level and selenium-dependent glutathione peroxidase (GPx) activity in the plasma from patients and cattle infected with MAP. The level of MAP antibodies in bovine sera was determined by IDEXX kit whereas detection of MAP DNA was performed by IS900-based nPCR. A total of 42 cattle (21 infected with MAP and 21 healthy controls), 27 CD subjects, 27 of CD-healthy relatives, 66 subjects with various diseases and 34 non-related healthy subjects were investigated. Overall, GPx activity was significantly higher in MAP infected humans (0.80941(&)#177;0.521) versus MAP (-ve) samples (0.42367(&)#177;0.229 units/ml), P(<)0.01. Similarly, the average of GPx activity in cattle infected with MAP was 1.59(&)#177;0.65 units/ml compared to 0.46907(&)#177;0.28 units/ml in healthy cattle (P(<)0.01). Although it was not statistically significant, MDA average level was higher in MAP infected human samples versus MAP (-ve) controls (1.11(&)#177;0.185 nmol/ml versus 0.805(&)#177;0.151 nmol/ml, respectively). Similarly, MDA average level in CD samples that are MAP+ (1.703(&)#177;0.231 nmol/ml) was higher than CD samples that are MAP (-ve) (1.429(&)#177;0.187 nmol/ml). In cattle, MDA average level in MAP infected samples was significantly higher at 3.818(&)#177;0.45 nmol/ml compared to 0.538(&)#177;0.18 nmol/ml in healthy cattle (P(<)0.01). Clearly, the data demonstrated that MAP infection is associated with oxidative stress and resulting in the pathophysiology of worsening of the condition of CD patients.
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Date Issued: 2016
Identifier: CFE0006699, ucf:51906
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0006699

Title: Role of Lipid Peroxide Derived Dicarboxylic Acids in Atherosclerotic Calcification.
Creator: Riad, Aladdin, Parthasarathy, Sampath, Altomare, Deborah, Masternak, Michal, Naser, Saleh, University of Central Florida
Abstract / Description: Cardiovascular diseases, including atherosclerosis, are the leading cause of death in the United States. Atherosclerotic lesions are formed by deposition of lipids in the intima of arteries. Upon exposure to oxidative stresses, low-density lipoprotein (LDL) is converted to highly atherogenic oxidized LDL (ox-LDL) particles, contributing to disease development and progression. Advanced disease stages may result in calcification of lesions. This calcification process is important, as it has...
Show moreCardiovascular diseases, including atherosclerosis, are the leading cause of death in the United States. Atherosclerotic lesions are formed by deposition of lipids in the intima of arteries. Upon exposure to oxidative stresses, low-density lipoprotein (LDL) is converted to highly atherogenic oxidized LDL (ox-LDL) particles, contributing to disease development and progression. Advanced disease stages may result in calcification of lesions. This calcification process is important, as it has been shown to be associated with stable plaques that are less prone to rupture. Calcification is present in lipid rich domains of lesions, however neither the composition of the mineralized calcium deposits nor its relationship to lipid peroxidation or the lipid rich atherosclerotic core has previously been identified. This study provides evidence that the lipid peroxide derived dicarboxylic acid (DCA), azelaic acid (AzA) induces calcification in smooth muscle cells, thereby providing the link between calcification and overall plaque burden, and association of calcification with the lipophilic region of the lesion. The potential of lipid peroxide-derived lipophilic DCAs to promote calcification upon exposure to vascular smooth muscle cells was tested. 13-hydroperoxylinoleic acid (HPODE) treatment resulted in the cellular conversion to 9-oxononanoic acid (ONA) and AzA as determined by mass spectrometry analysis. Delivery of AzA via lysophosphatidylcholine (Lyso-PtdCho) micelles induced calcification of human aortic smooth muscle cells (HASMC). AzA was identified in calcified human and mouse atherosclerotic plaques. Calcification of HASMC due to AzA treatment resulted in a less inflammatory and oxidative environment as indicated by genetic expression. These results demonstrate that DCAs may contribute to atherosclerotic calcification thus accounting for the latter's relationship to plaque burden and association with lipids. This study also challenges the dogma that arterial calcification represents the deposition of calcium phosphate and has implications with the development of new therapeutic strategies in treating late stage atherosclerosis.
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Date Issued: 2018
Identifier: CFE0007413, ucf:52730
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0007413

Title: VO-OHpic Treatment Reduces Cardiac Remodeling in Doxorubicin-Induced Cardiomyopathy.
Creator: Johnson, Taylor, Singla, Dinender, Parthasarathy, Sampath, Naser, Saleh, University of Central Florida
Abstract / Description: Doxorubicin (Doxo) is one of multiple anthracycline drugs used to effectively treat various forms of cancer. Unfortunately, Doxo treatment, as a side effect, induces cardiomyopathy and subsequent heart failure. We have previously demonstrated that transplanted embryonic stem (ES) cells and their conditioned medium (CM) modulate the PTEN pathway and reduce apoptosis, fibrosis and hypertrophy in a Doxo induced cardiomyopathy (DIC) model. However, mechanisms of inhibited apoptosis mediated...
Show moreDoxorubicin (Doxo) is one of multiple anthracycline drugs used to effectively treat various forms of cancer. Unfortunately, Doxo treatment, as a side effect, induces cardiomyopathy and subsequent heart failure. We have previously demonstrated that transplanted embryonic stem (ES) cells and their conditioned medium (CM) modulate the PTEN pathway and reduce apoptosis, fibrosis and hypertrophy in a Doxo induced cardiomyopathy (DIC) model. However, mechanisms of inhibited apoptosis mediated through PTEN pathway are completely unknown. Therefore, we used VO-OHpic (VO), a potent PTEN inhibitor to understand the mechanism of apoptosis as well as its effect on cardiac remodeling in DIC. Animals were divided into three groups; Group 1: Control (Saline), Group 2: Doxo (12 mg/kg, cumulative dose) and Group 3: Doxo+VO (30ug/kg cumulative dose). Animals were studied at one week and eight weeks post-DIC. Mice were subjected to echocardiography to examine cardiac function, sacrificed and hearts were harvested for further analysis. Immunohistochemistry staining revealed a significant (p (<) 0.05) decrease in apoptotic cardiomyocytes in Doxo+VO treated hearts compared with Doxo group. Furthermore, Hematoxylin and Eosin (H(&)E) and Masson's Trichrome histological stains confirmed reduced hypertrophy and fibrosis in Doxo+VO treated subjects compared to Doxo group. Western Blotting confirmed the reduction of p-PTEN levels and the increase in p-AKT cell survival protein expression in Doxo+VO subjects. In addition, VO-OHpic administration was shown to reduce the number of pro-inflammatory macrophages and increase the number of anti-inflammatory M2 macrophages that may further be involved in reduced apoptosis and fibrosis. Finally, heart function was improved in mice treated with VO compared to Doxo group. Collectively, our data suggests that VO-OHpic treatment reduces apoptosis, cardiac fibrosis and the process is mediated through the PTEN/AKT and inflammatory mechanisms with improved heart function in the DIC heart.
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Date Issued: 2016
Identifier: CFE0006690, ucf:51924
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0006690

Title: Role of Kruppel-like Factor 8 (KLF8) in Cancer and Cardiomyopathy.
Creator: Lahiri, Satadru, Zhao, Jihe, Parthasarathy, Sampath, Masternak, Michal, Siddiqi, Shadab, University of Central Florida
Abstract / Description: Cancer and cardiovascular diseases are two most fatal diseases causing innumerable death each year. Understanding the mechanisms underlying these diseases is critical for developing proper therapeutic approach. Kr(&)#252;ppel-like factor 8 (KLF8) is a member of Kr(&)#252;ppel-like family transcription factors that is overexpressed in many types of cancers. There is no report on role of KLF8 in cardiovascular diseases to date. KLF8 transcriptionally activates or represses a host of target...
Show moreCancer and cardiovascular diseases are two most fatal diseases causing innumerable death each year. Understanding the mechanisms underlying these diseases is critical for developing proper therapeutic approach. Kr(&)#252;ppel-like factor 8 (KLF8) is a member of Kr(&)#252;ppel-like family transcription factors that is overexpressed in many types of cancers. There is no report on role of KLF8 in cardiovascular diseases to date. KLF8 transcriptionally activates or represses a host of target genes to promote cancer cell proliferation, migration, invasion and epithelial to mesenchymal transition during tumor progression. Studies proposed in this thesis identified a novel posttranslational modification of KLF8 essential for its role in promoting cancer cell migration and discovered a novel function of KLF8 in cardiomyopathy. In our first study, we identified serine 48 (S48) as a novel phosphorylation site on KLF8. Pharmacological and genetic manipulations of various potential kinases further revealed ERK2 as the kinase responsible for this novel phosphorylation. Functional studies indicated that this phosphorylation is crucial for protecting KLF8 protein from degradation in the nucleus and promoting cancer cell migration. Preclinical xenograft models have indicated an important role of KLF8 for tumor progression. To investigate role of KLF8 in spontaneous tumorigenesis better recapitulating pathology in patients, we established the first Cre-regulated conditional KLF8 transgenic mouse model. Upon induction of global expression of the KLF8 transgene, spontaneous mammary and testicular tumors were formed in a small population of the mice by their mid-age, as expected considering the long latency required for tumor progression. Surprisingly, however, nearly 100% of KLF8 the mice died with a significantly enlarged heart, which did not occur to any littermate control mouse. Further characterization of the mice revealed that the global expression of the transgene caused striking systolic dysfunction leading to fatal dilated cardiomyopathy. Importantly, these similar phenotypes were reproduced in heart-specific KLF8 transgenic mice. Cardiovascular disease PCR array identified a number of genes potentially mediating KLF8-induced cardiac pathology. These results identified a previously unimagined function of KLF8 in the heart, shed new light on the mechanisms of cardiac diseases and provide novel preclinical mouse models for future translational research.
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Date Issued: 2016
Identifier: CFE0006692, ucf:51914
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0006692

Title: Role of Mycobacterium avium paratuberculosis (MAP) and TNFSF15 SNPs on TL1A in CD.
Creator: Hassouneh, Sayf Al-Deen, Naser, Saleh, Yooseph, Shibu, Parthasarathy, Sampath, University of Central Florida
Abstract / Description: Tumor Necrosis Factor-Like Ligand 1a (TL1A) is a cytokine encoded by Tumor Necrosis Factor Super Family 15 gene (TNFSF15) gene mostly in endothelial cells which binds to T-cells and foments the production of pro-inflammatory cytokines including TNF-?, IL-6, IL-1b, IFN- ? and IL-13. TL1A level is elevated in inflammatory diseases including Crohn's Disease (CD). Although Single Nucleotide Polymorphisms (SNPs) in TNFSF15 have been reported in CD, no studies have investigated the effect of these...
Show moreTumor Necrosis Factor-Like Ligand 1a (TL1A) is a cytokine encoded by Tumor Necrosis Factor Super Family 15 gene (TNFSF15) gene mostly in endothelial cells which binds to T-cells and foments the production of pro-inflammatory cytokines including TNF-?, IL-6, IL-1b, IFN- ? and IL-13. TL1A level is elevated in inflammatory diseases including Crohn's Disease (CD). Although Single Nucleotide Polymorphisms (SNPs) in TNFSF15 have been reported in CD, no studies have investigated the effect of these SNPs on TL1A, inflammation, and susceptibility to Mycobacterium avium subspecies paratuberculosis (MAP) infection. MAP is a strong candidate in CD pathogenesis. This study is designed to elucidate the combined effect of MAP and SNPs in TNFSF15 (rs4263839, rs7848647, rs6478108, or rs6478109) on TL1A secretion and downstream effect on pro-inflammatory cytokines. Peripheral blood from CD and healthy subjects was analyzed for MAP DNA, TNFSF15 genotyping, circulating TL1A level, and IFN- ? and TNF-? gene expression. Our data is first to report that rs4263839, rs7848647, rs6478108, and rs6478109 in TNFSF15 resulted in increase in circulating TL1A level in healthy and CD samples. Specifically, in CD samples with rs7848647, the average TL1A level was 146.9 pg/mL (&)#177; 124.5 compared 62.4 pg/mL (&)#177; 82.8 in normal samples. Similarly, TL1A level in CD samples with rs6478109 was 141.9 pg/mL (&)#177; 127.7 compared to 71.5 pg/mL (&)#177; 88.4 in normal samples (p(<)0.05). All 4 SNPs resulted in significant elevation in TL1A level in healthy samples (p(<)0.05). Moreover, IFN-? expression was significantly higher, by approximately 1.6-fold in CD patients with SNPs relative to CD patients with no SNPs (p(<)0.05). Interestingly, SNPs in TNFS15 had no significant effect on TNF-? expression. MAP was detected in the blood of 63% of CD compared to 6% healthy subjects (p(<).001). The data did not support a correlation between MAP presence and circulating TL1A levels, and no correlation between SNPs in TNSF15 and MAP susceptibility. This study strongly suggests, that SNPs in TNFSF15 increase TL1A levels and may be a contributory factor to the inflammation experienced by CD patients. Over all, the study emphasizes the need for a pharmacogenomic approach in treatment delivery for patients with CD by using TNFSF15 SNPs to identify patients that would benefit from biologics targeting TL1A rather than TNF-? for more efficacious treatment regiments for CD patients.
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Date Issued: 2018
Identifier: CFE0007189, ucf:52263
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0007189

Title: Neurological profile of older ApoE-PON1 double knockout mice.
Creator: Mitra, Connie, Parthasarathy, Sampath, Kim, Yoon-Seong, Zhao, Jihe, University of Central Florida
Abstract / Description: Atherosclerosis is a cardiovascular disease where plaques made up of lipids in the form of cholesterol ester build up in the carotid and innominate arteries that supply blood to the brain. Accumulation of the plaques limit the flow of blood and nutrients to the brain, leading to diminished oxygen supply, increased oxidative stress and cell death. All these have been implicated in Alzheimer's disease (AD). Alzheimer's disease, a chronic, progressive, age related neurodegenerative disorder is...
Show moreAtherosclerosis is a cardiovascular disease where plaques made up of lipids in the form of cholesterol ester build up in the carotid and innominate arteries that supply blood to the brain. Accumulation of the plaques limit the flow of blood and nutrients to the brain, leading to diminished oxygen supply, increased oxidative stress and cell death. All these have been implicated in Alzheimer's disease (AD). Alzheimer's disease, a chronic, progressive, age related neurodegenerative disorder is the most common form of dementia in the elderly accounting for 60-80% of the cases. Clinically, Alzheimer's disease is characterized by loss of memory, damage of brain tissues, and neuronal and synaptic loss. Pathologically, it is delineated by accumulation of amyloid beta and tau proteins forming senile plaques and neurofibrillary tangles respectively. Apolipoprotein E (ApoE) polymorphism, increased oxidative stress and products of lipid peroxidation are associated with atherosclerosis and Alzheimer's disease. ApoE is a glycosylated protein that mediates plasma lipoprotein metabolism. ApoE isoforms have differential effect on amyloid beta aggregation and clearance, thus playing an important role in Alzheimer's pathology. Serum paraoxonase 1 (PON1) is a lipoprotein associated antioxidant enzyme that prevents lipid peroxidation. S100B protein is a plasma biomarker, altered expression of which has been implied in AD. We propose the hypothesis that combined deficiencies in apolipoprotein E and antioxidant defense (established by the lack of PON1), together with dyslipidemia and development of carotid atherosclerosis in aging mice would reflect Alzheimer's pathology. The brains of young and old ApoE-PON1 double knockout (DKO) mice and control C57BL/6J mice were harvested. Atherosclerotic lesions were quantified by Image J. RNA was isolated, cDNA was synthesized and quantitative RT-PCR was performed to detect mRNA levels of S100B. Blood levels of S100B protein was measured by ELISA. Brain tissues were stained with Hematoxylin and Eosin stain and 4G8 immunostain to detect histopathological changes. The blood brain barrier (BBB) is altered in AD resulting in increased permeability and vascular dysfunction. The vascular permeability of BBB was analyzed by Evans Blue Dye (EBD) assay. The results showed that the older DKO mice had severe carotid atherosclerosis, increased levels of serum S100B protein and elevated mRNA levels of S100B. Histological examination showed the presence of characteristic hallmarks of AD. The leakage of EBD into brain parenchyma indicated disruption of BBB. The results suggest that diminished blood flow and nutrient supply to the brain due to atherosclerosis and increased oxidative stress might contribute to Alzheimer's pathology. We suggest that older ApoE-PON1 DKO mice may serve as a model of Alzheimer's disease and prevention of atherosclerosis might promote regression of Alzheimer's disease.
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Date Issued: 2016
Identifier: CFE0006483, ucf:51407
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0006483

Title: Molecular Mechanisms involved in inflammatory angiogenesis induced by monocyte chemotactic protein induced protein-1 (MCPIP1).
Creator: Roy, Arpita, Kolattukudy, Pappachan, Ebert, Steven, Parthasarathy, Sampath, Self, William, University of Central Florida
Abstract / Description: Major diseases such as cardiovascular diseases, diabetes, obesity and tumor growth are known to involve inflammatory angiogenesis. MCP-induced protein 1 (MCPIP1) encoded by ZC3H12A gene, was reported to promote angiogenesis and is addressed in my dissertation as MCPIP. The mechanism/s involved in the angiogenic differentiation induced by MCPIP was however unknown. The aim of this study was to bridge this gap in our knowledge and delineate the molecular mechanisms and sequential processes...
Show moreMajor diseases such as cardiovascular diseases, diabetes, obesity and tumor growth are known to involve inflammatory angiogenesis. MCP-induced protein 1 (MCPIP1) encoded by ZC3H12A gene, was reported to promote angiogenesis and is addressed in my dissertation as MCPIP. The mechanism/s involved in the angiogenic differentiation induced by MCPIP was however unknown. The aim of this study was to bridge this gap in our knowledge and delineate the molecular mechanisms and sequential processes involved in angiogenesis mediated via MCPIP. To determine if angiogenesis induced by inflammatory cytokines, TNF-?, IL-1? and IL-8 is mediated via induction of MCPIP, knockdown of MCPIP by its specific siRNA, in human umbilical vein endothelial cells was performed. Oxidative stress, ER stress and autophagy are known to be involved in mediating inflammation. We hypothesized that MCPIP-induced angiogenic differentiation is mediated via induction of oxidative stress, ER stress and autophagy. Chemical inhibitors and specific gene knockdown approach were used to inhibit each process postulated. Oxidative stress was inhibited by apocynin or cerium oxide nanoparticles or knockdown of NADPH oxidase subunit, phox47. Endoplasmic reticulum (ER) stress was blocked by tauroursodeoxycholate or knockdown of ER stress signaling protein IRE-1 and autophagy was inhibited by the use of 3?methyl adenine, or LY 294002 or by specific knockdown of beclin1. Matrigel assay was used as an in vitro tool to assay angiogenic differentiation. Inhibition of each step inhibited the subsequent steps postulated. The results reveal that angiogenesis induced by inflammatory agents is mediated via sequential induction of MCPIP that causes oxidative and nitrosative stress resulting in ER stress leading to autophagy required for angiogenesis. MCPIP has deubiquitinase and anti-dicer RNase activities. If and how the dual enzymatic activities of MCPIP mediate angiogenesis was unknown. Our results showed that hypoxia-induced angiogenesis is mediated via MCPIP. MCPIP deubiquitinated ubiquitinated hypoxia-inducible factor (HIF-1?) and the stabilized HIF-1? entered the nucleus to promote the transcription of its target genes, cyclooxygenase-2 and vascular endothelial growth factor causing the activation of p38 MAP kinase involved in angiogenesis. MCPIP expression promoted angiogenesis by inhibition of thrombospondin-1 synthesis via induction of silent information regulator (SIRT)-1 and/or via suppression of VEG-inhibitor levels caused by inhibition of NF-?B activation. MCPIP inhibited the production of the anti-angiogenic microRNAs (miR)-20b and miR-34a that repress the translation of HIF-1? and SIRT-1, respectively. Cells expressing the RNase-dead mutant of MCPIP, D141N, that had lost the ability to induce angiogenesis had deubiquitinase activity but did not inhibit the production of miR-20b and miR-34a. Mimetics of miR-20b and miR-34a inhibited MCPIP-induced angiogenesis. These results show for the first time that both deubiquitinase and anti-dicer RNase activities of MCPIP are involved in inflammatory angiogenesis. Results from our study delineate key processes that could be potential targets for therapeutic intervention against inflammatory angiogenesis.
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Date Issued: 2012
Identifier: CFE0004793, ucf:49760
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0004793

Title: Establishment of Methods for Isolation of Pnmt+ Cardiac Progenitor Cells.
Creator: Varudkar, Namita, Ebert, Steven, Parthasarathy, Sampath, Muller, Mark, University of Central Florida
Abstract / Description: Cardiovascular disease is the leading cause of death in the United States. Millions of patients suffer each year from endothelial dysfunction and/or debilitating myocardial damage resulting in decreased quality of life and increased risk of death or disablement. Current pharmacological approaches are only partly effective at treating cardiovascular disease, and hence, better strategies are needed to provide significant improvements in treatment options. Cardiac stem/progenitorcells have the...
Show moreCardiovascular disease is the leading cause of death in the United States. Millions of patients suffer each year from endothelial dysfunction and/or debilitating myocardial damage resulting in decreased quality of life and increased risk of death or disablement. Current pharmacological approaches are only partly effective at treating cardiovascular disease, and hence, better strategies are needed to provide significant improvements in treatment options. Cardiac stem/progenitorcells have the potential to regenerate myocardial tissue and repair damaged heart muscle. There are many different types of cardiac progenitor cells, and each may have certain unique properties and characteristics that would likely be useful for particular clinical applications. A current challengein the field is to identify, isolate, and test specific cardiac stem/progenitor cell populations for their ability to repair/regenerate myocardial tissue. Our laboratory has discovered a new type of cardiac progenitor cell that expresses the enzyme, Phenylethanolamine-n-methyltransferase (Pnmt). My initial studies focused on identification of Pnmt+ cells based on knock-in of a nuclear-localized Enhanced Green Fluorescent Protein (nEGFP) reporter gene into exon 1 of the Pnmt gene in a stable recombinant Pnmt-nEGFP mouse embryonic stem cell (mESC) line. These cells were differentiated into cardiomyocytes, and I identified nEGFP+ cells using fluorescence, immunofluorescence, and phase-contrast microscopy techniques. Our results showed that only about 0.025% ( 1 per 4000) of the cardiac-differentiating stem cells expressed the nEGFP+ marker. Because of the relative rarity of these cells, optimization of isolation methods proved initially challenging. To overcome this technical barrier, I used a surrogate cell culture system to establish the methodsof isolation based on expression of either a fluorescent cell marker (EGFP), or a unique cell surface receptor represented by an inactivated (truncated) version of the human low-affinity nerve growth factor receptor (LNGFR). Plasmid DNA containing these reporter genes was transiently transfected into a permissive cell line (RS1), and reporter gene expression was used to identify and isolate transfected from non-transfected cells using either Fluorescence-Activated Cell Sorting(FACS) or Magnetic-Activated Cell Sorting (MACS) methods. The main objective of the study was to establish the isolation techniques based on the expression of reporter genes (EGFP and LNGFR) in RS1 cells. Following transfection, EGFP+ cells were successfully isolated via FACS as verified by flow cytometric and microscopic analyses, which showed that approximately 96% of the isolated cells were indeed EGFP+. Despite the relative purity of the isolated cell population, however, their viability in culture following FACS was substantially compromised ( 50% attrition). In contrast, MACS enabled efficient isolation of LNGFR+ cells, and the vast majority of these ( 90%) retained viability in culture following MACS. The LNGFR expression was verified using RT-PCR. Further, MACS methods enabled isolation of marked cells in about 5-7 mins, whereas it took 2-4 hours to using FACS to perform similar isolations from the same amount of starting material (10^6 cells). In addition, MACS is a more economical method in that it does not require the use of an expensive laser-based instrument to perform the sorting. These results suggest that MACS was a more efficient, gentle, and feasible technique than FACS for isolation of reporter-tagged mammalian cells. Consequently, future studies aimed at isolation of Pnmt+ cardiac progenitor cells will thus primarily focus on MACS methods.
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Date Issued: 2014
Identifier: CFE0005558, ucf:50287
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0005558

Title: Is breakdown of fatty acid peroxides involved in the induction of apolipoprotein A1?.
Creator: Gupta, Rajat, Parthasarathy, Sampath, Siddiqi, Shadab, Jewett, Mollie, University of Central Florida
Abstract / Description: Over the past few years the number of deaths caused due to cardiovascular diseases has been increasing and is of major concern. In the United States, 75% of cardiovascular-related deaths have been attributed to atherosclerosis. Western diets containing large quantities of peroxidized lipids are considered atherogenic. Heated oil in the form of fried food brings high levels of peroxidized fat and its decomposition products in the diet. Peroxidized lipids are known to increase the...
Show moreOver the past few years the number of deaths caused due to cardiovascular diseases has been increasing and is of major concern. In the United States, 75% of cardiovascular-related deaths have been attributed to atherosclerosis. Western diets containing large quantities of peroxidized lipids are considered atherogenic. Heated oil in the form of fried food brings high levels of peroxidized fat and its decomposition products in the diet. Peroxidized lipids are known to increase the susceptibility of serum lipoproteins to undergo oxidation, thereby contributing to the progression of atherosclerosis. The intestinal cells are responsible for the absorption of dietary fatty acid peroxides (FAOOH) which has been reported to enhance anti-atherosclerotic effects by inducing apolipoprotein A1 (apoA1) gene and protein levels. Therefore, there is a void in the knowledge of when to expect (")harmful(") or (")beneficial(") effects of dietary lipid peroxides. The formation of toxic products like aldehydes from the decomposition of FAOOH is well documented. On the other hand, carboxylic acids particularly azelaic acid, formed as an end product of FAOOH decomposition has been reported to have anti-atherosclerotic effects. Hence, we hypothesize that intestinal cells may decompose FAOOH to aldehydes, which might get converted to carboxylic acids that can be transported across the intestine. Linoleic acid is the most abundant polyunsaturated fatty acid (PUFA) present in the diet. So, we will use peroxidized linoleic acid (13-HPODE) and incubate with intestine derived cells or Caco -2 cells as an in-vitro model for determining its decomposition to aldehydes and carboxylic acids. We propose that the decomposition products of FAOOH in the presence of intestinal cells might be responsible for causing an increase in apoA1 levels, which might suggest that lipid peroxidation derived products might actually be beneficial for reducing the progression of atherosclerosis as compared to the absorption of intact FAOOH.
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Date Issued: 2013
Identifier: CFE0004856, ucf:49700
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0004856

Title: Validation of a novel hypothesis of generating foam cells by its use to study reverse cholesterol transport.
Creator: Sengupta, Bhaswati, Parthasarathy, Sampath, Singla, Dinender, Jewett, Mollie, Rohde, Kyle, University of Central Florida
Abstract / Description: Generation of foam cells, an essential step for reverse cholesterol transport (RCT) studies, uses the technique of receptor dependent macrophage loading with radiolabeled acetylated Low Density Lipoprotein (Ac-LDL). In this study, we used the ability of a biologically relevant detergent molecule, Lysophosphatidylcholine (Lyso PtdCho), to form mixed micelles with cholesterol or cholesteryl ester (CE) to generate macrophage foam cells. Fluorescent or radiolabelled cholesterol / Lyso PtdCho...
Show moreGeneration of foam cells, an essential step for reverse cholesterol transport (RCT) studies, uses the technique of receptor dependent macrophage loading with radiolabeled acetylated Low Density Lipoprotein (Ac-LDL). In this study, we used the ability of a biologically relevant detergent molecule, Lysophosphatidylcholine (Lyso PtdCho), to form mixed micelles with cholesterol or cholesteryl ester (CE) to generate macrophage foam cells. Fluorescent or radiolabelled cholesterol / Lyso PtdCho mixed micelles were prepared and incubated with RAW 264.7 or mouse peritoneal macrophages. Results showed that such micelles were quite stable at 4(&)deg;C and retained the solubilized cholesterol during one month storage. Macrophages incubated with cholesterol or CE (unlabeled, fluorescently labeled or radiolabeled) / Lyso PtdCho mixed micelles accumulated CE as documented by microscopy, lipid staining, labeled oleate incorporation, and by thin layer chromatography (TLC). Such foam cells unloaded cholesterol when incubated with high density lipoprotein (HDL) and not with oxidized HDL (Ox-HDL). We propose that stable cholesterol or CE / Lyso PtdCho micelles would offer advantages over existing methods.Oxidative stress is associated with heart failure (HF). Previously our research group observed that the patients with low left-ventricular ejection fraction showed accumulation of high level of oxidized LDL (Ox-LDL) when compared with the heart failure patients with normal range of ejection fraction (EF). HDL is known to be atheroprotective and one of its important antioxidative functions is to protect LDL from oxidative modifications. However, HDL itself undergoes oxidation and Ox-HDL becomes functionally poor. It is expected to have a diminished ability to promote reverse cholesterol transport. Therefore, it was hypothesized that the quality of HDL present in the patients with EF would more compromised than those present in the patients with normal EF. Functionality of HDL was evaluated by measuring its cholesterol efflux capacity from foam cells generated in vitro. Functionality of HDL, which is strongly related to the oxidative modifications of HDL was further estimated by measuring paraoxonase 1 (PON1) enzyme activity associated with HDL. Higher the PON1 activity and RCT ability, better is the functionality of HDL.
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Date Issued: 2014
Identifier: CFE0005250, ucf:50596
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0005250

Title: TIMP-1 ACTIVATES A UNIQUE CARDIAC STEM CELL POPULATION, CD63+ve/C-KIT+ve, THEREBY ENHANCING CARDIAC DIFFERENTIATION, AND PROTECTS THE HEART FROM ADVERSE CARDIAC REMODELING FOLLOWING MYOCARDIAL INFARCTION.
Creator: Abdelli, Latifa, Singla, Dinender, Cheng, Zixi, Parthasarathy, Sampath, Jewett, Mollie, University of Central Florida
Abstract / Description: We previously demonstrated that embryonic stem (ES) cells over-expressing tissue inhibitor of metalloproteinase-1 (TIMP-1) have increased potential to engraft and differentiate into cardiac myocytes following transplantation into the infarcted heart. However, the ability of TIMP-1 to activate endogenous stem cells and enhance their differentiation into cardiac regenerative cell types is still unknown. We postulate that TIMP-1 may additionally activate a stem cell population that enhances...
Show moreWe previously demonstrated that embryonic stem (ES) cells over-expressing tissue inhibitor of metalloproteinase-1 (TIMP-1) have increased potential to engraft and differentiate into cardiac myocytes following transplantation into the infarcted heart. However, the ability of TIMP-1 to activate endogenous stem cells and enhance their differentiation into cardiac regenerative cell types is still unknown. We postulate that TIMP-1 may additionally activate a stem cell population that enhances cardiac cell type differentiation in the infarcted myocardium. To prove this hypothesis, we isolated c-kit+ve cells from four weeks old C57BL/6 mice and cultured them in vitro in presence of ES conditioned media (ESCM), ES-TIMP-1-CM or TIMP-1. Our immunostaining data validate the existence of a novel CD63+ve/c-kit+ve cells. When treated with TIMP-1, these cells showed significantly (p(<)0.05) increased proliferation and differentiation into cardiac myocytes, vascular smooth muscle cells, and endothelial cells. Western blot analysis revealed significantly (p(<)0.05) increased expression of CD63, phosphorylated and total ?-catenin proteins. Furthermore, our RT-PCR data showed increased cardiac gene expression (GATA-4, Mef2C, and Nkx-2.5) when compared to ESCM and control cells. Based on the in vitro findings, we investigated the effect of intramyocardial delivery of TIMP-1 on endogenous CD63+ve/c-kit+ve cells following myocardial infarction (MI). C57BL/6 and TIMP-1 KO mice underwent coronary artery ligation followed by intramyocardial delivery of 20(&)#181;l of culture media (CC), ESCM, ES-TIMP-1-CM or TIMP-1. Subsequent immunohistochemistry analysis demonstrated the presence of a CD63+ve/c-kit+ve cell population within the peri-infarct area and confirmed intramyocardial delivery of ES-TIMP-1-CM or TIMP-1 significantly (p(<)0.05) enhanced their proliferation. Percentage of CD63+ve/c-kit+ve cells was significantly (p(<)0.05) lower in TIMP-1 KO mice compared to C57BL/6 animals. RT-PCR analysis revealed TIMP-1 KO animals expressed significantly less CD63 and TIMP-1 mRNAs compared to C57BL/6 mice. Activated CD63+ve/c-kit+ve cells were also able to differentiate into major cardiac cell types as previously shown in vitro. The differentiation potential of these cells was however higher in C57BL/6 mice compared to TIMP-1 KO mice. We also demonstrate that CD63+ve/c-kit+ve cells differentiation is regulated by CD63/?-catenin pathway in vivo. Additionally, we provide evidence that TIMP-1 protects the heart from adverse cardiac remodeling through inhibition of cardiac apoptosis and fibrosis leading to significantly (p(<)0.05) improved contractile function. Collectively, our data show TIMP-1 plays a dual protective role in the MI heart. It activates a unique stem cell population, CD63+ve/c-kit+ve, which proliferates and differentiates into functional myocytes, smooth muscle cells and endothelial cells mediated through CD63/?-catenin pathway. TIMP-1 also protects the heart from adverse cardiac remodeling. Increased cardiac regeneration and inhibition of adverse cardiac remodeling consequently lead to restored cardiac function. ?
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Date Issued: 2015
Identifier: CFE0005750, ucf:50108
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0005750

Title: Regulation of Extra-Pituitary Prolactin in Monocytes and Macrophages.
Creator: Barrett, Richard, Parthasarathy, Sampath, McKinstry, Karl, Masternak, Michal, Zhao, Jihe, University of Central Florida
Abstract / Description: Recently it has been shown that leukocytes are capable of producing prolactin (PRL). Evidence of extra-pituitary PRL (ePRL) production is so far been limited to primates and is not shared across other mammal species such as mice and rats. While ePRL is characterized as an identical protein to traditional pituitary PRL, it is controlled under an alternative promoter and is thus regulated differently from pituitary PRL. Little is known about what regulates ePRL or its direct role in human...
Show moreRecently it has been shown that leukocytes are capable of producing prolactin (PRL). Evidence of extra-pituitary PRL (ePRL) production is so far been limited to primates and is not shared across other mammal species such as mice and rats. While ePRL is characterized as an identical protein to traditional pituitary PRL, it is controlled under an alternative promoter and is thus regulated differently from pituitary PRL. Little is known about what regulates ePRL or its direct role in human physiology, but given that PRL has well over 300 described functions, it is likely that the autocrine and paracrine effects of this hormone could have far reaching implications in overall physiology. This work takes some of the first steps in understanding how leukocyte ePRL is regulated. Our results show that, adrenergic hormones are one key stimulus in ePRL expression in monocytes/macrophages. This is particularly intriguing considering the opposing role of these two signals in settings such as adipose tissue where adipose tissue macrophages are constantly exposed to pro-lipolytic adrenergic hormones that would in turn stimulate production of an anti-lipolytic hormone, PRL. Further, our work shows that the inflammatory phenotype of the leukocytes influences basal expression of PRL and overall ePRL expression increases significantly as monocytes differentiate into macrophages, as is a common occurrence in adipose tissue. The final portion of our work shows how monocytes/macrophages also respond to preadipocytes directly. These stem cell precursors to mature adipose cells release an unknown factor that stimulates ePRL production in monocytes/macrophages. Analysis of our gene array shows many of the genes stimulated by adipose stem cells alongside PRL are important genes in tissue regeneration and remodeling, a possible role that fits well with known effects of PRL. Understanding such primate specific interactions between the immune system and major metabolic tissues such as adipose fills vital gaps in knowledge that may explain why so many treatments fail when transitioning from mouse models to humans.
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Date Issued: 2018
Identifier: CFE0007309, ucf:52164
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0007309

Title: Consequences of Altered Short-Chain Carbon Metabolism in Heart Failure.
Creator: Horton, Julie, Estevez, Alvaro, Kelly, Daniel, Parthasarathy, Sampath, Crawford, Peter, University of Central Florida
Abstract / Description: Cardiovascular disease is currently the foremost cause of death within the United States. Heart failure (HF) is a syndrome defined by the inability of the heart to adequately execute requisite pump function in order to deliver nutrients and oxygen to peripheral tissues, irrespective of etiology. One of the most common causes of HF is chronic pressure overload due to hypertension. Ischemic heart disease is also a common driver of HF, often in conjunction with hypertension. Pressure overload...
Show moreCardiovascular disease is currently the foremost cause of death within the United States. Heart failure (HF) is a syndrome defined by the inability of the heart to adequately execute requisite pump function in order to deliver nutrients and oxygen to peripheral tissues, irrespective of etiology. One of the most common causes of HF is chronic pressure overload due to hypertension. Ischemic heart disease is also a common driver of HF, often in conjunction with hypertension. Pressure overload initially causes compensatory metabolic changes. Structural changes follow shortly thereafter typically resulting in left ventricular hypertrophy. Eventually, the heart loses the ability to compensate for the aberrant hemodynamic load and begins failing. The failing heart is unable to supply adequate adenosine triphosphate (ATP) for contractile function as evidenced by falling phosphocreatine (PCr) levels. This energy deficit occurs concurrently with a metabolic re-programming that results in a fuel utilization pattern resembling the fetal heart. Notably, enzymes involved in catabolism of fatty acids, the chief fuel substrate for ATP generation in the normal adult heart, are downregulated in the failing heart. However, the extent to which alternative fuels compensate for decreased fatty acid oxidation (FAO) is not well-known. Furthermore, consequences of the fuel substrate switches that occur in heart failure are not well established. In this work, we discover a new paradigm for alternate fuel utilization in the failing heart and define consequences of altered fuel metabolism in HF. We discovered a post-translational modification resultant from an accumulation of acetyl groups (C2) present in a mouse model of early-stage HF and human HF. Mitochondrial proteins were found to be hyperacetylated in the failing heart, and at least some of these alterations result in diminished electron-transport chain (ETC) capacity as shown by mutagenesis studies on succinate dehydrogenase A (SDHA). We also found an accumulation of C4-OH carnitine, a by-product of ketone oxidation in HF. This metabolite aggregation occurred alongside an increase in b-hydroxybutyrate dehydrogenase 1 (BDH1) transcript and protein levels. This signature suggested that the failing heart shifted to ketone bodies as a fuel. Subsequent experiments confirmed increased capacity for myocardial ketone oxidation in compensated cardiac hypertrophy and in HF. The consequences of increased ketone oxidation were then assessed using a cardiac-specific BDH1 knockout (BDH1 KO) mouse. Despite not having any apparent defect at baseline, we found BDH1 KO mouse hearts are completely unable to oxidize 3-hydroxybutyrate. The deficit for ketone oxidation capacity became consequential upon subjugation to transverse aortic constriction with a small apical myocardial infarction (TAC/MI). The BDH1 KO mice exhibit altered pathological cardiac remodeling compared to wild-type controls. These latter data suggest the increased reliance on ketone oxidation in HF, mediated by BDH1, is an adaptive response. Together the results of these studies provide important information regarding the consequences of altered fuel metabolism in HF. Recent reports of reduced HF mortality and elevated circulating ketone levels in patients prescribed Empagliflozin make cardiac ketone metabolism research in this dissertation particularly apropos.
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Date Issued: 2017
Identifier: CFE0006948, ucf:51663
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0006948

atherosclerosis (2)	+ -
?-catenin (1)	+ -
Alzheimer's disease (1)	+ -
Amyloid beta plaques (1)	+ -
Angiogenesis (1)	+ -

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Azelaic acid (1)	+ -
Blood Brain barrier (1)	+ -
CD63 (1)	+ -
Cancer (1)	+ -
Cardiac Progenitor Cells (1)	+ -
Cardiac Remodeling (1)	+ -
Cardiac Stem Cells (1)	+ -
Cardiomyopathy (1)	+ -
Cholesterol (1)	+ -
Cholesterol efflux (1)	+ -
Cholesteryl ester (1)	+ -
Crohn's disease (1)	+ -
Deubiquitinase (1)	+ -
Dietary Peroxidized Lipids (1)	+ -
Doxorubicin (1)	+ -
Dysfunctional HDL (1)	+ -
ER stress (1)	+ -
Evans Blue Dye (1)	+ -
Foam cells (1)	+ -
Genetically engineered mouse models (1)	+ -
Glutathione Peroxidase (1)	+ -
Granular cells (1)	+ -
HDL (1)	+ -
HPODE (1)	+ -

Parthasarathy, Sampath (14)	+ -
University of Central Florida (14)	+ -
Jewett, Mollie (4)	+ -
Masternak, Michal (4)	+ -
Naser, Saleh (4)	+ -

Singla, Dinender (4)	+ -
Zhao, Jihe (3)	+ -
Ebert, Steven (2)	+ -
Siddiqi, Shadab (2)	+ -
Abdelli, Latifa (1)	+ -
Altomare, Deborah (1)	+ -
Andl, Claudia (1)	+ -
Barrett, Richard (1)	+ -
Cheng, Zixi (1)	+ -
Crawford, Peter (1)	+ -
Doomra, Mitsushita (1)	+ -
Estevez, Alvaro (1)	+ -
Gupta, Rajat (1)	+ -
Hassouneh, Sayf Al-Deen (1)	+ -
Horton, Julie (1)	+ -
Johnson, Taylor (1)	+ -
Kelly, Daniel (1)	+ -
Kim, Yoon-Seong (1)	+ -
Kolattukudy, Pappachan (1)	+ -
Lahiri, Satadru (1)	+ -
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Mitra, Connie (1)	+ -
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