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Title: TRANSPLANTATION OF IPS CELLS REDUCES APOPTOSIS AND FIBROSIS AND IMPROVES CARDIAC FUNCTION IN STREPTOZOTOCIN-INDUCED DIABETIC RATS.
Creator: Neel, Sarah, Singla, Dinender, University of Central Florida
Abstract / Description: Background: Streptozotocin (STZ) induced diabetes leads to various complications including cardiomyopathy. Recent data suggests transplanted bone marrow stem cells improve cardiac function in diabetic cardiomyopathy. However, whether modified ES, iPS cells, or factors released from these cells can inhibit apoptosis and fibrosis remains completely unknown. The present study was designed to determine the effects of transplanted ES cells overexpressing pancreatic transcription factor 1 a (Ptf1a)...
Show moreBackground: Streptozotocin (STZ) induced diabetes leads to various complications including cardiomyopathy. Recent data suggests transplanted bone marrow stem cells improve cardiac function in diabetic cardiomyopathy. However, whether modified ES, iPS cells, or factors released from these cells can inhibit apoptosis and fibrosis remains completely unknown. The present study was designed to determine the effects of transplanted ES cells overexpressing pancreatic transcription factor 1 a (Ptf1a), a pro-pancreatic endodermal transcription factor, iPS cells, or their respective conditioned media (CM) on diabetic cardiomyopathy. Methods: Experimental diabetes was induced in male Sprague Dawley rats (8-10 weeks old) by intraperitoneal STZ injections (65 mg/kg body weight for 2 consecutive days). Animals were divided into six experimental groups including control, treated with sodium citrate buffer IP, STZ, STZ + ES-Ptf1a cells, STZ + iPS cells, STZ + ES-Ptf1a CM and STZ + iPS CM. Following STZ injections, appropriate cells (1 X 106/mL/injection/day) or CM (2 mL injection/day) were given intravenously for 3 consecutive days. Animals were sacrificed and hearts were harvested at day 28. Histology, TUNEL staining, and Caspase-3 activity were used to assess apoptosis and fibrosis. ERK1/2 phosphorylation was quantified using ELISAs. M-mode echocardiography fractional shortening was used to assess cardiac function. Results: Animals transplanted with ES cells, iPS cells, or both CMs showed a significant (p<0.05) reduction in interstitial fibrosis, and apoptosis compared with STZ group. ERK expression was not significantly different compared with STZ. Echocardiography showed a significant (p<0.05) improvement in fractional shortening in cell and media transplanted groups compared with STZ. Conclusions: Our data suggest that ES cells, iPS cells, and/or CMs inhibit apoptosis, reduce fibrosis, and improve cardiac function in STZ-treated diabetic rats.
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Date Issued: 2010
Identifier: CFE0003512, ucf:48964
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0003512

Title: GENETICALLY MODIFIED ES CELLS ENHANCE CARDIAC REPAIR AND REGENERATION IN THE INFARCTED HEART.
Creator: Glass, Carley, Singla, Dinender, University of Central Florida
Abstract / Description: Transplanted embryonic stem (ES) cells following myocardial infarction (MI) contribute to limited cardiac repair and regeneration with improved function. Therefore novel strategies are still needed to enhance the efficacy by which ES cells differentiate into cardiac cell types and inhibit adverse remodeling in the infarcted myocardium. Our studies evaluate whether genetic manipulation of transplanted ES cells employing miR-1, a pro-cardiac microRNA, and TIMP-1, an anti-apoptotic and anti...
Show moreTransplanted embryonic stem (ES) cells following myocardial infarction (MI) contribute to limited cardiac repair and regeneration with improved function. Therefore novel strategies are still needed to enhance the efficacy by which ES cells differentiate into cardiac cell types and inhibit adverse remodeling in the infarcted myocardium. Our studies evaluate whether genetic manipulation of transplanted ES cells employing miR-1, a pro-cardiac microRNA, and TIMP-1, an anti-apoptotic and anti-fibrotic protein, will enhance cardiac myocyte differentiation, inhibit native cardiac apoptosis, and reduce fibrosis in the infarcted myocardium. Furthermore, we assess levels of associated pro-(caspase-3, PTEN) and anti-(Akt) apoptotic proteins as well as a pro-fibrotic protein (MMP-9) in the post-MI and cell transplanted heart. microRNAs (miRs) have emerged as critical regulators of various physiological processes including development, differentiation, metabolism, and death. Indeed, miR-1 plays an integral role in early cardiac development in Drosophila and mice as well as mediates differentiation of cardiac myocytes in vitro. To that end, we generated ES cells overexpressing miR-1 (miR-1-ES cells), transplanted them into the infarcted myocardium, and evaluated their impact on cardiac myocyte differentiation, myocardial repair, and left ventricular dysfunction post-MI. We provide evidence demonstrating enhanced cardiac myocyte commitment of transplanted miR-1-ES cells in the mouse infarcted heart as compared to ES cell and culture media transplanted hearts. Assessment of apoptosis revealed overexpression of miR-1 in transplanted ES cells protected host myocardium from MI-induced apoptosis through activation of p-Akt and inhibition of caspase-3, PTEN, and superoxide anion production. A significant reduction in interstitial and vascular fibrosis was quantified in miR-1-ES and ES cell transplanted groups compared with control MI. However, no statistical significance between miR-1-ES cell and ES cell groups was observed. Finally mice receiving miR-1-ES cell transplantation post-MI had significantly improved heart function compared with respective controls. Our data suggests miR-1 drives cardiac myocyte differentiation from transplanted ES cells and inhibits apoptosis post-MI ultimately giving rise to enhanced cardiac repair, regeneration, and function. Next, we assessed the role of miR-1-ES cells in a chronic model of MI as research has shown that apoptosis occurs not only hours but months following ischemia. 4 weeks following transplantation into the infarcted myocardium, we provide evidence demonstrating reduced cardiac apoptosis in miR-1-ES cell transplanted hearts compared to respective controls. Moreover, we show significant elevation of p-Akt levels and diminished PTEN levels in hearts transplanted with miR-1-ES cells as determined by enzyme-linked immunoassays. Finally, using echocardiography, we reveal mice receiving miR-1-ES cell transplantation post-MI had significantly improved cardiac function compared with animals transplanted with ES cell and culture media. Our data suggests that miR-1, when overexpressed in transplanted ES cells, has the capacity to inhibit apoptosis long term while attenuating contractility loss. In addition to enhancing cardiac-specific donor cell differentiation, improving the efficacy by which stem cells promote cell survival and repair in the host myocardium is imperative in the pursuit of refining and optimizing stem cell therapy. To that end, we overexpressed TIMP-1, an endogenous inhibitor of apoptosis and fibrosis, in ES cells (TIMP-1-ES cells), transplanted them into infarcted myocardium, and evaluated their impact on adverse cardiac remodeling. Immunofluorescence, TUNEL staining, caspase-3 activity, ELISAs, histology, and echocardiography were used to assess apoptosis, fibrosis, and heart function. Hearts transplanted with TIMP-1-ES cells demonstrated a reduction in apoptosis as well as an increase in p-Akt activity compared with ES cells or culture media controls. Interstitial and vascular fibrosis was significantly decreased in the TIMP-1-ES cell group compared to controls. Furthermore, MMP-9, a key pro-fibrotic protein, was significantly reduced following TIMP-1-ES cell transplantation. Echocardiography data showed fractional shortening and ejection fraction were significantly improved in the TIMP-1-ES cell group compared with respective controls. Our data suggest that transplanted ES cells overexpressing TIMP-1 attenuate adverse myocardial remodeling and improve cardiac function compared with ES cells. Overall, our data suggest that genetic manipulation of ES cells following transplantation in the infarcted heart enhances cardiac myocyte differentiation, inhibits apoptosis and fibrosis as well as improves cardiac function.
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Date Issued: 2011
Identifier: CFE0003936, ucf:48705
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0003936

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: Role of Single Nucleotide Polymorphisms (SNPs) in PTPN2/22 and Mycobacterium avium subspecies paratuberculosis (MAP) in Rheumatoid Arthritis and Crohn's Disease.
Creator: Sharp, Robert, Naser, Saleh, Parks, Griffith, Roy, Herve, Singla, Dinender, University of Central Florida
Abstract / Description: Both genetic pre-disposition and potential environmental triggers are shared between Rheumatoid arthritis (RA) and Crohn's disease (CD). We hypothesized that single nucleotide polymorphisms (SNPs) in the negative T-cell regulators Protein Tyrosine Phosphatase Non-receptor type 2 and 22 (PTPN2/22) lead to a dysregulated immune response as seen in RA and CD. To test the hypothesis, peripheral leukocytes samples from 204 consented subjects were TaqMan genotyped for 9 SNPs in PTPN2/22. The SNPs...
Show moreBoth genetic pre-disposition and potential environmental triggers are shared between Rheumatoid arthritis (RA) and Crohn's disease (CD). We hypothesized that single nucleotide polymorphisms (SNPs) in the negative T-cell regulators Protein Tyrosine Phosphatase Non-receptor type 2 and 22 (PTPN2/22) lead to a dysregulated immune response as seen in RA and CD. To test the hypothesis, peripheral leukocytes samples from 204 consented subjects were TaqMan genotyped for 9 SNPs in PTPN2/22. The SNPs effect on PTPN2/22 and IFN-y expression was determined using RT-PCR. Blood samples were analyzed for the Mycobacterium avium subspecies paratuberculosis (MAP) IS900 gene by nPCR. T-cell proliferation and response to phytohematoagglutonin (PHA) mitogen and MAP cell lysate were determined by BrdU proliferation assay. Out of 9 SNPs, SNP alleles of PTPN2:rs478582 occurred in 79% RA compared to 60% control (p-values ? 0.05). SNP alleles of PTPN22:rs2476601 occurred in 29% RA compared to 6% control (p-values ? 0.05). For the haplotype combination of PTPN2:rs478582/PTPN22rs2476601, 21.4% RA had both SNPs (C-A) compared to 2.4% control (p-values ? 0.05). PTPN2/22 expression in RA was decreased by an average of 1.2 fold. PTPN2:rs478582 upregulated IFN-y in RA by an average of 1.5 fold. Combined PTPN2:rs478582/PTPN22:rs2476601 increased T-cell proliferation by an average of 2.7 fold when treated with PHA. MAP DNA was detected in 34% RA compared to 8% controls (p-values ? 0.05), where samples with PTPN2:rs478582 and/or PTPN22:rs2476601 were more MAP positive. PTPN2:rs478582/PTPN22:rs2476601 together with MAP infection significantly increased T-cell response and IFN-y expression in RA samples. The same experimental approach was followed on blood samples from CD patients. Both PTPN2:rs478582/PTPN22:rs2476601 affected PTPN2/22 and IFN-y expression along with T-cell proliferation significantly more than in RA. MAP DNA was detected in 64% of CD. This is the first study to report the correlation between SNPs in PTPN2/22, IFN-y expression and MAP in autoimmune disease.
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Date Issued: 2018
Identifier: CFE0007371, ucf:52094
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0007371

Title: Investigating changes in quiescence in oral and esophageal epithelium in response to injury.
Creator: Rothaus, Alexandra, Andl, Claudia, Chakrabarti, Ratna, Singla, Dinender, University of Central Florida
Abstract / Description: More than 570,000 new cases of esophageal cancer are estimated to be diagnosed annually worldwide. Risk factors include gender, age, tobacco use and dietary habits leading to tissue injury and ultimately cancer. While prognoses for other cancers have improved, the 5-year survival for patients with esophageal cancer is only 20%. During the repair process, cell proliferation is increased and is associated with inflammation. Slow-cycling lifetime residential stem cells, called quiescent cells,...
Show moreMore than 570,000 new cases of esophageal cancer are estimated to be diagnosed annually worldwide. Risk factors include gender, age, tobacco use and dietary habits leading to tissue injury and ultimately cancer. While prognoses for other cancers have improved, the 5-year survival for patients with esophageal cancer is only 20%. During the repair process, cell proliferation is increased and is associated with inflammation. Slow-cycling lifetime residential stem cells, called quiescent cells, facilitate repair but are thought to accumulate mutations during DNA replication eventually giving rise to cancer. We hypothesize that esophageal stem cells become activated upon injury and are regulated by Transforming Growth Factor beta 1 (TGF?1), a known regulator of cell proliferation and differentiation. We established an in vitro model of quiescence using normal esophageal epithelial (STR) and oral (OKF6) cells treated with recombinant human TGF?1. Flow cytometry showed increases in cells arrested in G1/G0 phase of the cell cycle in TGF?1 treated cells for both cell lines (STR p(<)0.01, OKF6 p(<)0.05). EdU (5-ethynyl-2'-deoxyuridine) positive recovery cells indicated quiescence in both cell lines (p(<)0.01). Analysis of TGF?1 regulation of putative stem cell markers via western blot and qRT-PCR showed increases in ITGB1, PDPN and K15 as well as XPC, and MeCP2 in treated cells. To apply our in vitro findings, we performed immunohistochemistry staining on tissue microarrays. Proliferation marker Ki67 increased in disease progression from normal to inflammation to hyperplasia (p(<)0.001) while TGF?1 target markers decrease. Our data indicate that the onset of cancer-associated inflammation correlates with the loss of TGF?1 mediated stemness markers and increased basal proliferation suggesting cancer is a stem cell disease.
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Date Issued: 2019
Identifier: CFE0007903, ucf:52754
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0007903

Title: Embryonic Stem Cell-Derived Exosomes Inhibit Doxorubicin-Induced Pyroptosis in Cell Culture Models.
Creator: Tavakoli Dargani, Zahra, Singla, Dinender, Masternak, Michal, Siddiqi, Shadab, Steward, Robert, University of Central Florida
Abstract / Description: Doxorubicin (Dox) is a potent chemotherapeutic drug used for the treatment of various cancers. Unfortunately, its use is limited as Dox induces adverse cardiotoxicity (DIC) and muscle toxicity (DIMT), which are mediated through oxidative stress, ER stress, and inflammation. However, it remains unknown whether Dox induces an inflammation mediated cell death, called (")pyroptosis("). The current study is designed to determine whether Dox induces pyroptosis in cardiac and muscle cell culture...
Show moreDoxorubicin (Dox) is a potent chemotherapeutic drug used for the treatment of various cancers. Unfortunately, its use is limited as Dox induces adverse cardiotoxicity (DIC) and muscle toxicity (DIMT), which are mediated through oxidative stress, ER stress, and inflammation. However, it remains unknown whether Dox induces an inflammation mediated cell death, called (")pyroptosis("). The current study is designed to determine whether Dox induces pyroptosis in cardiac and muscle cell culture models. Moreover, the protective effects of embryonic stem cell-derived exosomes (ES-Exos) in inhibiting pyroptosis will also be determined. For this purpose, we designed two different cell culture models using H9c2 cadiomyoblasts and Sol 8 cells. For the DIC model, H9c2 were exposed to Dox to induce pyroptosis and then treated with exosomes. Cells were divided into 4 groups: Control, Dox, Dox+ES-Exos, and Dox+MEF-Exos (negative control). Furthermore, to generate the DIMT model, Sol 8 cells were incubated with Dox+THP-1 conditioned medium (TCM) to induce toxicity and inflammation, which was followed by exosomes treatment. We assigned cells into 5 groups: Control, Dox+TCM, Dox+TCM+ES-Exos, Dox+TCM+MEF-Exos (negative control), and Dox+TCM+ES-Exos+GW4869 compound (exosomes inhibitor, negative control). Our data shows that Dox treatment significantly increased pyroptotic marker expression including TLR-4, NLRP3, caspase-1, IL1-?, Caspase-11, and gasdermin-D as well as increased pro-inflammatory TNF-? and IL-6 expression in H9c2 cells. There was also a significant increase in caspase-1, IL1-?, and IL-18 expression in Dox+TCM treated Sol 8 cells. Conversely, increased pyroptosis and inflammation post-Dox treatment were inhibited by ES-Exos in both culture models. No significant changes observed upon MEF-Exos and GW4869 compound treatments. In conclusion, our data shows Dox induces pyroptosis and inflammation within cardiac and skeletal muscle cells, which can be inhibited following treatment with ES-exosomes. This is a novel study with new mechanistic observations on the pathophysiological role of pyroptosis in Dox-induced cardio and muscle toxicities.
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Date Issued: 2018
Identifier: CFE0007416, ucf:52700
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0007416

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: Bone Morphogenetic Protein 7 Inhibits Pyroptotic Cell Death in Vascular Smooth Muscle Cells of Atherosclerotic Apolipoprotein E -/- Mice.
Creator: Garner, Kaley, Singla, Dinender, Zhao, Jihe, Pourmoghadam, Kamal, University of Central Florida
Abstract / Description: Atherosclerosis (ATH) is an inflammation-mediated disease in which cell death underlies the formation of lesions along the intima layer of vascular walls resulting in vessel narrowing, decreased blood flow, and increased risk of lesion rupture leading to myocardial infarction and stroke. The current study was undertaken to investigate whether inflammation in ATH can induce pyroptosis in vascular smooth muscle cells (SMC's). We therefore hypothesized that pyroptosis occurs and is inhibited by...
Show moreAtherosclerosis (ATH) is an inflammation-mediated disease in which cell death underlies the formation of lesions along the intima layer of vascular walls resulting in vessel narrowing, decreased blood flow, and increased risk of lesion rupture leading to myocardial infarction and stroke. The current study was undertaken to investigate whether inflammation in ATH can induce pyroptosis in vascular smooth muscle cells (SMC's). We therefore hypothesized that pyroptosis occurs and is inhibited by bone morphogenetic protein 7 (BMP7). We examined SMC pyroptosis at acute (D5) and midstage (D28) following disturbed flow-induced hemodynamic injury to the vascular wall using our partial left carotid artery ligation (PLCA) model. ApoE -/- mice (11(&)#177;1 week old) were divided into three groups: Sham, PLCA, PLCA+BMP7 (200?g/kg; i.v) and arterial tissue was collected for immunohistochemical staining (IHC) and western blot (WB) analysis. At D5 and D28, IHC data demonstrated that PLCA significantly upregulated Toll-like receptor 4 (TLR4) and NLRP3 inflammasome components (NLRP3 and Caspase-1), indicating the initiation and activation of pyroptosis in SMC's (p(<)0.05). Further, maturation of pro-IL-1? and pro-IL-18 released through cell membrane pores mediated by Caspase-11 were investigated. Our data shows a significant increase at D5 and D28 in IL-1?, IL-18, and Caspase-11 expression following PLCA, which was significantly improved upon treatment with BMP7 (p(<)0.05). Western blot analysis supported these findings demonstrating initiation of pyroptosis via TLR4, upregulation of inflammasome components (Caspase-1 and NLRP3), and release of proinflammatory cytokines, IL-1? and IL-18 at D28, but not at D5. Overall, this study demonstrates that pyroptosis occurs in vascular smooth muscle cells in our PLCA model and that BMP7 administration attenuates pyroptosis significantly.
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Date Issued: 2019
Identifier: CFE0007635, ucf:52472
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0007635

Title: Embryonic Stem Cell Derived Exosomes Enhance Cardiac Stem Cell Differentiation into Heart Cells.
Creator: Hammond, Jamillah, Singla, Dinender, Masternak, Michal, Davidson, Victor, University of Central Florida
Abstract / Description: Transplantation of embryonic stem (ES) cells into the ischemic and infarcted heart has proven to repopulate cardiac cell populations, attenuate structural cardiac remodeling, and rescue cardiac function. Unfortunately, the pluripotency of ES cells increases risk of teratoma formation in vivo. Exosomes, smaller in comparison to ES cells, are cell free carriers of miRNA, proteins, and lipids, and do not suggest risk of teratoma formation. Exosomes have been proposed to mediate and attenuate...
Show moreTransplantation of embryonic stem (ES) cells into the ischemic and infarcted heart has proven to repopulate cardiac cell populations, attenuate structural cardiac remodeling, and rescue cardiac function. Unfortunately, the pluripotency of ES cells increases risk of teratoma formation in vivo. Exosomes, smaller in comparison to ES cells, are cell free carriers of miRNA, proteins, and lipids, and do not suggest risk of teratoma formation. Exosomes have been proposed to mediate and attenuate regeneration following myocardial infarction (MI), however, the role of exosomes derived from ES cells (ES-Exo) in activating resident cardiac stem cells (CSCs) to undergo cardiac differentiation is not established. In the present study, Stem cell antigen 1 positive (Sca-1+ve) CSCs were isolated, incubated with exosomes, and evaluated for differentiation into the major heart cell types in vitro. Observations of in vitro cardiac differentiation were further established in an in vivo model of MI. Ligation of the coronary artery, or a sham surgery was performed in C57BL/6 mice 8-12 weeks of age. Mice were split among four study groups: sham, MI, MI + H9c2-Exo (a cell line control), (&) MI + ES-Exo. ES-Exo were transplanted via intramyocardial (IM) injection immediately following coronary artery ligation. At day 14 (D14), echocardiography was used to evaluate cardiac function. Differentiation into the major heart cells was determined by sarcomeric ?-actin (cardiomyocytes) and smooth muscle ?-actin (vascular smooth muscle cells) immunostaining. Hematoxylin and Eosin and Masson's Trichrome staining assessed cardiomyocyte hypertrophy and fibrosis, respectively. Immunostaining for major heart cellular markers revealed significant activation of resident Sca-1+ve CSCs to undergo cardiac differentiation after ES-Exo treatment. Cardiomyocyte hypertrophy and myocardial fibrosis were significantly increased following coronary artery ligation. Results from histological staining revealed significantly decreased levels of hypertrophy and fibrosis in hearts transplanted with ES-Exo following coronary ligation. In summary, our findings advocate ES-Exo as a viable treatment option to repopulate the myocardium with viable heart cells, attenuate cardiac remodeling, and rescue cardiac function.
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Date Issued: 2018
Identifier: CFE0007188, ucf:52254
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0007188

Title: TRANSPLANTATION OF PLURIPOTENT STEM CELLS CONFERS CARDIAC PROTECTION IN DOX-INDUCED HEART FAILURE THROUGH NOTCH-1 PATHWAY.
Creator: Merino-Chavez, Hilda, Singla, Dinender, Zervos, Antonis, Naser, Saleh, University of Central Florida
Abstract / Description: Doxorubicin (DOX) is the antineoplastic drug of preference used to treat a wide variety of malignancies, with high survival rates among treated patients. However, the benefits of this drug have become less appealing due to the side effects that occur such as DOX-induced cardiomyopathy (DIC) and an increased risk of myocardial infarction (MI). Therefore, there is an urgent need to explore the therapeutic options to treat DIC. In this context, adult stem cells have been used as a source to...
Show moreDoxorubicin (DOX) is the antineoplastic drug of preference used to treat a wide variety of malignancies, with high survival rates among treated patients. However, the benefits of this drug have become less appealing due to the side effects that occur such as DOX-induced cardiomyopathy (DIC) and an increased risk of myocardial infarction (MI). Therefore, there is an urgent need to explore the therapeutic options to treat DIC. In this context, adult stem cells have been used as a source to reduce cardiomyocyte apoptosis in DIC; however, the effects of transplanted embryonic stem (ES) cells and induced pluripotent stem (iPS) cells in DIC post MI are unknown. As a result, we wanted to understand how transplanted ES and iPS cells and the factors released by them inhibit apoptosis and improve cardiac function in DIC post MI. C57BL/6 mice were divided into five groups: Sham, DOX-MI, DOX-MI+cell culture (CC) media, DOX-MI+ES cells, and DOX-MI+iPS cells. Mice were treated with DOX (12 mg/kg, cumulative dose) followed by left coronary artery ligation to induce MI. ES or iPS cells (5 x 104) were delivered into the peri-infarct region. At day 14 post-MI, echocardiography was performed, mice sacrificed, and hearts harvested for further analyses. To investigate if protective effects are provided by factors released from ES and iPS cells in DIC, we performed in vitro studies using condition media (CM) obtained from ES or iPS cells to treat DOX-induced cardiotoxicity in H9c2 cells. Our data reveal that apoptosis was significantly inhibited in the ES and iPS cell transplanted hearts as well as ESCM and iPSCM treated cells compared with the untreated controls. Furthermore, a significant increase in levels of Notch-1, Hes1, and pAkt survival protein were observed. Decreased levels of PTEN, a negative regulator of Akt pathway, along with improved heart function were also observed in the stem cell transplanted groups. In conclusion, our data show that transplantation of ES and iPS cells blunt DOX-induced apoptosis in vivo, which is associated with improved cardiac function. Moreover, decreased apoptosis in both in vitro and in vivo models is mediated by the Notch pathway.
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Date Issued: 2012
Identifier: CFE0004577, ucf:49213
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0004577

Title: Bone Morphogenetic Protein-7 (BMP-7) Polarizes Monocytes into M2 Macrophages.
Creator: Rocher, Crystal, Singla, Dinender, Siddiqi, Shadab, Sugaya, Kiminobu, University of Central Florida
Abstract / Description: Atherosclerosis is an inflammatory disease in which an accumulation of fatty acids and cholesterol occurs to form a plaque in small and large arteries. Monocyte polarization to classic M1 macrophages or alternative M2 macrophages is an important area of research that can determine the severity of disease progression. BMP-7 is a key growth factor responsible for directing differentiation of mesenchymal stem cells into brown fat cells, suggesting a role of BMP-7 in cellular plasticity; however,...
Show moreAtherosclerosis is an inflammatory disease in which an accumulation of fatty acids and cholesterol occurs to form a plaque in small and large arteries. Monocyte polarization to classic M1 macrophages or alternative M2 macrophages is an important area of research that can determine the severity of disease progression. BMP-7 is a key growth factor responsible for directing differentiation of mesenchymal stem cells into brown fat cells, suggesting a role of BMP-7 in cellular plasticity; however, its role in monocyte polarization is yet to be revealed. In the current study, we hypothesize that monocyte treatment with BMP-7 will significantly result in increased polarization of monocytes into M2 macrophages and increased expression of anti-inflammatory cytokines. To that effect, we have established a stress induced cell culture system with monocytes (THP-1 cells) and apoptotic conditioned medium (ACM), simulating injury, to understand the effects of BMP-7 on M2 macrophage polarization from monocytes. Our data demonstrates that the BMP type 2 receptor (BMPR2) is found on monocytes and its activation is significantly (p(<)0.05) increased in both monocytes and M2 macrophages following treatment with BMP-7. Furthermore, a significant (p(<)0.05) increase of M2 macrophages in the BMP-7 treated group was shown following immunostaining with CD206 and arginase-1, two M2 macrophage markers, whereas a significant (p(<)0.05) decrease of iNOS expression, an M1 macrophage marker, was shown. Moreover, treatment with BMP-7 resulted in significantly (p(<)0.05) increased expression of IL-10 and IL-1ra, two anti-inflammatory cytokines, but significantly (p(<)0.05) decreased levels of the pro-inflammatory cytokines, MCP-1, IL-6 and TNF-?. We also hypothesize that polarization of monocytes to M2 macrophages occurs through activation of SMAD1/5/8 and PI3K-Akt-mTOR pathways. Upon BMP-7 binding to its receptor, BMPR2, activation of SMAD1/5/8 occurs which then activates the p85 subunit of PI3K resulting in downstream activation of Akt and mTOR. Our data shows that following treatment with BMP-7, expression of p-SMAD1/5/8, p-PI3K, p-Akt and p-mTOR is significantly (p(<)0.05) increased compared to controls whereas p-PTEN, an inhibitor of the PI3K pathway, is significantly (p(<)0.05) decreased in the BMP-7 treated group compared to controls. In conclusion, our data reveals that BMP-7 polarizes monocytes into M2 macrophages and it achieves this through activation of the PI3K-Akt-mTOR pathway, which will have significant applications for atherosclerosis treatment.
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Date Issued: 2013
Identifier: CFE0004922, ucf:49617
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0004922

Title: BMP-7 Inhibits p38 and JNK Pathways and Increases M2 Macrophage Differentiation to Reduce Atherosclerosis in Apolipoprotein E-/- Mice.
Creator: Shoulders, Heidi, Singla, Dinender, Cheng, Zixi, Naser, Saleh, University of Central Florida
Abstract / Description: We have previously shown that treating atherosclerosis with bone morphogenetic protein-7 (BMP-7) affects the presence of macrophage subtypes in vitro, however it remains unknown whether BMP-7 treatment affects development and progression of atherosclerosis in vivo at an early and mid-stage of the disease. We therefore performed a Day 5 (D5) and Day 28 (D28) study to examine BMP-7's potential to affect monocyte differentiation. Atherosclerotic plaque formation was developed using our standard...
Show moreWe have previously shown that treating atherosclerosis with bone morphogenetic protein-7 (BMP-7) affects the presence of macrophage subtypes in vitro, however it remains unknown whether BMP-7 treatment affects development and progression of atherosclerosis in vivo at an early and mid-stage of the disease. We therefore performed a Day 5 (D5) and Day 28 (D28) study to examine BMP-7's potential to affect monocyte differentiation. Atherosclerotic plaque formation was developed using our standard method and ApoE-/- mice were sacrificed at D5 and D28 post-surgery. Treatment animals received intravenous injections of BMP-7 at 200(&)#181;g/kg of bodyweight. Hematoxylin and Eosin morphological stain shows that BMP-7 is capable of significantly reducing plaque accumulation at D28 post-surgery vs. PLCA group, p(<)0.05. At D5, plaque formation was reduced but not significant. Immunohistochemistry staining was performed to determine BMP-7's effect on monocytes (CD14), inflammatory M1 (iNOS) and anti-inflammatory M2 (CD206, Arginase-1) macrophages. Immunohistochemistry results show BMP-7 administration reduced pro-inflammatory monocytes and M1 macrophages at D5 and D28 compared to PLCA animals; however, monocytes were not statistically lower at D28. The anti-inflammatory M2 macrophage population was significantly less in PLCA animals compared to SHAM animals at D5 and D28. There was no significant difference in M2 macrophages between PLCA and PLCA + BMP7 animals at D5, however, by D28, PLCA + BMP7 animals showed a significant increase in M2 macrophages compared to PLCA animals. Western blot analysis confirms a significant increase in pro-survival kinase ERK and a significant reduction in pro-inflammatory kinases p38 and JNK in BMP-7 treated mice (D5 and D28, p(<)0.05). ELISA showed a significant reduction in pro-inflammatory cytokines IL-6, MCP-1, and TNF-? (D5 and D28, p(<)0.05) and a significant increase in anti-inflammatory cytokine IL-10 in BMP-7 treated mice (D5 and D28, p(<)0.05). In summary, our data indicate BMP-7 treatment induces monocyte to M2 macrophage differentiation, increases anti-inflammatory cytokine levels (IL-1ra and IL-10), and improves blow flow velocity (D5 and D28, p(<)0.05) compared to untreated animals. The mechanisms of monocyte to M2 macrophage differentiation appear to be mediated by the p38, JNK, and ERK pathways. This study suggests BMP-7 is capable of reducing inflammation and slowing progression of atherosclerosis at both an early and mid-stage of the disease.
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Date Issued: 2016
Identifier: CFE0006504, ucf:51388
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0006504

Title: Plasticity of Central and Peripheral Nervous System: Effects of Oxygen-Glucose Deprivation (OGD), Chronic Intermittent Hypoxia (CIH) and hSOD1 Overexpression.
Creator: Chen, Jin, Cheng, Zixi, Naser, Saleh, Singla, Dinender, University of Central Florida
Abstract / Description: Transient receptor potential canonical 6 (TRPC6) channels are permeable to Na+ and Ca2+ and are widely expressed in the brain. In this study, we investigated the role of TRPC6 following ischemia/reperfusion (I/R) and oxygen-glucose deprivation (OGD). We found that TRPC6 expression was increased in wild type (WT) mice cortical neurons following I/R and in primary neurons with OGD, and that deletion of TRPC6 reduced the I/R-induced brain infarct in mice and the OGD- /neurotoxin-induced neuronal...
Show moreTransient receptor potential canonical 6 (TRPC6) channels are permeable to Na+ and Ca2+ and are widely expressed in the brain. In this study, we investigated the role of TRPC6 following ischemia/reperfusion (I/R) and oxygen-glucose deprivation (OGD). We found that TRPC6 expression was increased in wild type (WT) mice cortical neurons following I/R and in primary neurons with OGD, and that deletion of TRPC6 reduced the I/R-induced brain infarct in mice and the OGD- /neurotoxin-induced neuronal death. Using live-cell imaging to examine intracellular Ca2+ levels ([Ca2+]i), we found that OGD induced a significant higher increase in glutamate-evoked Ca2+ influx compared to untreated control and such an increase was reduced by TRPC6 deletion. Enhancement of TRPC6 expression using AdCMV-TRPC6-GFP infection in WT neurons increased [Ca2+]i in response to glutamate application compared to AdCMV-GFP control. Inhibition of N-methyl-d-aspartic acid receptor (NMDAR) with MK801 decreased TRPC6-dependent increase of [Ca2+]i, indicating that such a Ca2+ influx was NMDAR dependent. Furthermore, TRPC6-dependent Ca2+ influx was blunted by blockade of Na+ entry. Finally, OGD-enhanced Ca2+ influx was reduced, but not completely blocked, in the presence of voltage dependent Na+ channel blocker tetrodotoxin (TTX) and dl???amino?3?hydroxy?5?methyl?4?isoxazole propionic acid (AMPA) blocker CNQX. Altogether, we concluded that I/R-induced brain damage was, in part, due to upregulation of TRPC6 in cortical neurons. We postulate that overexpression of TRPC6 following I/R may induce neuronal death partially through TRPC6-dependent Na+ entry which activated NMDAR, thus leading to a damaging Ca2+ overload. These findings may provide a potential target for future intervention in stroke-induced brain damage. Obstructive sleep apnea (OSA) is a highly prevalent sleep disorder that is associated with many cardiovascular complications, such as autonomic dysfunctions, stroke and heart failure. Chronic intermittent hypoxia (CIH) is a prominent feature of OSA. In CIH exposed rodents (a model for OSA), CIH induces the similar cardiovascular complications as seen in OSA patients. In particular, OSA impairs baroreflex control of the heart rate (HR), which is used as an independent indicator for heart failure. Since the baroreflex control arc includes the aortic depressor nerve (ADN), vagal efferent and central components, we hypothesize that CIH induces dysfunctions of all three components. Since mice can be genetically manipulated, an understanding of the effects of CIH on multiple neural components in the baroreflex arc in wild type mice may lead to a future study of treatments. In this study, we have examined the effects of CIH on baroreceptor afferent, central and vagal efferent components of the baroreflex circuitry in normal wild type C57BL/6J mice. Mice (4-5 months) were exposed to room air (RA) or CIH for 35-50 days and were then anesthetized with isoflurane, ventilated and catheterized for measurement of mean arterial blood pressure (MAP) and HR. Baroreceptor function was characterized by measuring percent changes of integrated ADN activity (Int ADNA) relative to the baseline value in response to the vasodilator sodium nitroprusside and the vasoconstrictor phenylephrine-induced changes in MAP. Data were fitted to a sigmoid logistic function curve. HR responses to electrical stimulation of the left ADN and the right vagus nerve were assessed under anesthesia. Compared with RA controls, CIH significantly increased maximum baroreceptor gain or maximum slope, maximum Int ADNA, and Int ADNA range (maximum-minimum Int ADNA). In addition, CIH maintained the maximum amplitude of the bradycardic response to vagal efferent stimulation. In contrast, CIH significantly reduced the maximum amplitude of bradycardic response to left ADN stimulation. Thus, CIH decreased central mediation of the baroreflex, but augmented the baroreceptor afferent function and maintained vagal efferent control of HR in mice. Excessive reactive oxygen species (ROS) (such as the superoxide radical) is commonly associated with cardiac autonomic dysfunctions. Though superoxide dismutase 1 (SOD1) overexpression may protect against ROS damage to the autonomic nervous system, superoxide radical reduction may change normal physiological functions. Previously, we demonstrated that human SOD1 (hSOD1) overexpression did not change baroreflex bradycardia and tachycardia, but increased aortic depressor nerve activity (ADNA) in responses to arterial pressure changes in C57B6SJL-Tg (SOD1)2 Gur/J mice. Since the barorelfex arc includes afferent, central and efferent components, the objective of this study was to determine whether hSOD1 overexpression alters the central and vagal efferent mediation of the heart rate (HR) responses. Our data indicate that SOD1 overexpression decreased HR responses to vagal efferent nerve stimulations but did not change HR responses to aortic nerve stimulation. Along with the previous study, we suggest that SOD1 overexpression preserves the normal baroreflex function but may alter the functions of aortic depressor nerve, vagal efferent and central components differently. While SOD1 overexpression likely enhanced aortic depressor nerve function and central mediation of bradycardia, it decreased vagal efferent control of HR. Currently, we are using the hSOD1 overexpressing mouse model to determine whether hSOD1 overexpression can preserve normal afferent, efferent, and central components of the baroreflex arc in the CIH model of sleep apnea.
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Date Issued: 2017
Identifier: CFE0006576, ucf:51334
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0006576

Title: Bone Morphogenetic Protein-7 Attenuates Inflammation and Apoptosis and Improves Cardiac Function in Diabetes.
Creator: Urbina, Princess, Singla, Dinender, Naser, Saleh, Zhao, Jihe, University of Central Florida
Abstract / Description: Bone Morphogenetic Protein-7 (BMP-7) belongs to the transforming growth factor-? (TGF?) family of cytokines has is known to have potent anti-inflammatory properties. It has been used in patients to treat osteoporosis clinically and has been reported to treat diabetic nephropathy in murine models. Moreover, studies show that inflammation is up-regulated in patients with pre-diabetes (PD). We, therefore, hypothesize that the administration of BMP-7 will attenuate inflammation in the heart of...
Show moreBone Morphogenetic Protein-7 (BMP-7) belongs to the transforming growth factor-? (TGF?) family of cytokines has is known to have potent anti-inflammatory properties. It has been used in patients to treat osteoporosis clinically and has been reported to treat diabetic nephropathy in murine models. Moreover, studies show that inflammation is up-regulated in patients with pre-diabetes (PD). We, therefore, hypothesize that the administration of BMP-7 will attenuate inflammation in the heart of Streptozotocin (STZ)-induced PD mice. In this study, we divided C57Bl/6 mice into three groups: CONTROL, PD, and PD+BMP-7. CONTROL mice received intraperitoneal (i.p.) injections of Sodium Citrate Buffer while PD and PD+BMP-7 groups received i.p. injections of Streptozotocin (STZ) for two days. In addition, PD+BMP-7 mice received intravenous injections (i.v.) of BMP-7 (200(&)#181;g/kg) on the last day of STZ injection and for the following two days. Animals were sacrificed 21 days post last injection and examined for levels of oxidative stress, inflammatory immune response, apoptosis, fibrosis and cardiac function. Our results indicate significant glucose intolerance in PD mice (p(<)0.05), which was attenuated in the PD+BMP-7 group (p(<)0.05). We also observed increased oxidative stress (p(<)0.001) and secretion of pro-inflammatory cytokines (p(<)0.05), interleukin-6 (IL-6) and tumor necrosis factor-? (TNF-?), in PD mice as compared with the controls. PD+BMP-7 mice revealed significant up-regulation of M2 macrophages (p(<)0.05) and secretion of anti-inflammatory cytokines (p(<)0.05), interleukin-10 (IL-10) and interleukin-1RA (IL-1RA), as compared to PD mice. This was observed with a concomitant down-regulation of pro-inflammatory cytokines, IL-6 and TNF-?, as compared to the PD group. Moreover, we observed significantly increased cardiac apoptosis and fibrosis in PD mice (p(<)0.001) as compared to the control group. These observations, however, were down-regulated upon treatment with BMP-7. Lastly, analysis of echocardiograms revealed significantly depressed cardiac function in PD mice as compared with controls, while the PD+BMP-7 group presented improved cardiac function compared to PD mice. In conclusion, our data suggest that treatment with BMP-7 is effective in alleviating cardiac inflammation, inhibiting apoptosis, blunting cardiac remodeling and improving cardiac function in the hearts of STZ-induced PD mice. This reveals the potential of BMP-7 as a therapy in PD patients who present an increased inflammatory immune response.
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Date Issued: 2013
Identifier: CFE0004765, ucf:49799
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0004765

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: Identification of proteins regulating VLDL sorting into the VLDL Transport Vesicle (VTV) and involved in the biogenesis of the VTV.
Creator: Tiwari, Samata, Siddiqi, Shadab, Zervos, Antonis, Singla, Dinender, Naser, Saleh, University of Central Florida
Abstract / Description: Increased secretion of very low-density lipoprotein (VLDL), a triglyceride-rich lipoprotein, by the liver causes hypertriglyceridemia, which is a major risk factor for the development of atherosclerosis. The rate of VLDL-secretion from the liver is determined by its controlled transport from the endoplasmic reticulum (ER) to the Golgi. The ER-to-Golgi transport of newly synthesized VLDL is a complex multi-step process and is mediated by the VLDL transport vesicle (VTV). Once a nascent VLDL...
Show moreIncreased secretion of very low-density lipoprotein (VLDL), a triglyceride-rich lipoprotein, by the liver causes hypertriglyceridemia, which is a major risk factor for the development of atherosclerosis. The rate of VLDL-secretion from the liver is determined by its controlled transport from the endoplasmic reticulum (ER) to the Golgi. The ER-to-Golgi transport of newly synthesized VLDL is a complex multi-step process and is mediated by the VLDL transport vesicle (VTV). Once a nascent VLDL particle is synthesized in the lumen of the ER, it triggers the process of VTV-biogenesis and this process requires coat complex II (COPII) proteins that mediate the formation of classical protein transport vesicles (PTV). Even though, both VTV and PTV bud off the same ER at the same time and require the same COPII proteins, their cargos and sizes are different. The VTV specifically exports VLDL to the Golgi and excludes hepatic secretory proteins such as albumin and the size of the VTV is larger (~ 100 -120 nm) than PTV to accommodate VLDL-sized particles. These observations indicate (i) the existence of a sorting mechanism at the level of the ER; and (ii) the involvement of proteins in addition to COPII components. This doctoral thesis is focused on identification of proteins regulating VLDL sorting into the VTV and involved in the biogenesis of the VTV. In order to identify proteins present exclusively in VTV, we have characterized the proteome of VTV, which suggest CideB (cell death-inducing DFF45-like effector b) and SVIP (small VCP/P97 interacting protein) as candidates, present in VTV but excluded from PTV. We further confirmed the finding by performing co-immunoprecipitation studies and confocal microscopy studies. CideB, a 26-kDa protein was found to interact with apolipoprotein B100 (apoB 100), the structural protein of VLDL. Moreover, CideB interacts with two of the COPII components, Sar1 and Sec24. VTV generation was examined after blocking CideB by specific antibodies and by silencing CideB in rat primary hepatocytes. Knockdown of CideB in primary hepatocytes showed significant reduction in VTV generation, however, CideB was concentrated in VTV as compared with the ER suggesting its functional role in the sorting of VLDL into the VTV. SVIP, a small (~ 9-kDa) protein was found to interact with Sar1, a COPII component that initiates the budding of vesicles from ER membrane. SVIP has sites for myristoylation and we found increased recruitment of SVIP on ER membrane upon myristic acid (MA) treatment. Sar1 that lacks sites for myristoylation also is recruited more on ER upon myristoylation indicating that SVIP promotes Sar1 recruitment on ER. Additionally, our data suggest that Sar1 interacts with SVIP and forms a multimer that facilitates the biogenesis of VTV. Interestingly, silencing of SVIP reduced the VTV generation significantly. Conversely, incubation with MA increased the VTV budding, suggesting recruitment of SVIP on ER surface facilitates the VTV budding. We conclude that SVIP recruits Sar1 on ER membrane and makes an intricate COPII coat leading to the formation of a large vesicle, the VTV. Overall, the data presented in this thesis, determines the role of CideB and SVIP in regulating VLDL sorting and VTV biogenesis.
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Date Issued: 2013
Identifier: CFE0005270, ucf:50553
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0005270

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: Dubious role of Mycobacterium paratuberculosis in pathogenesis of Type I diabetes.
Creator: Thanigachalam, Saisathya, Naser, Saleh, Singla, Dinender, Siddiqi, Shadab, Jewett, Travis, University of Central Florida
Abstract / Description: INTRODUCTION: Type 1 Diabetes Mellitus (T1DM) is a chronic disorder with unknown etiology and associated with insulin deficiency. Mycobacterium avium subsp. paratuberculosis (MAP), the etiologic agent of paratuberculosis in cattle, has been implicated in many autoimmune diseases including Crohn's disease, TIDM and others. We hypothesize that the molecular mimicry including epitope homology between MAP-Hsp65 and pancreatic Glutamic Acid Decarboxylase65 (GAD65) may play a role in the auto...
Show moreINTRODUCTION: Type 1 Diabetes Mellitus (T1DM) is a chronic disorder with unknown etiology and associated with insulin deficiency. Mycobacterium avium subsp. paratuberculosis (MAP), the etiologic agent of paratuberculosis in cattle, has been implicated in many autoimmune diseases including Crohn's disease, TIDM and others. We hypothesize that the molecular mimicry including epitope homology between MAP-Hsp65 and pancreatic Glutamic Acid Decarboxylase65 (GAD65) may play a role in the auto destruction of pancreatic beta cells leading to insufficient insulin production and the development of TIDM, following exposure to MAP. METHODOLOGY: Peptide sequences of MAP-Hsp65 and GAD65 were analyzed using BLAST and PyMOL bioinformatics tools. Cross reactivity between the two proteins were evaluated using immunoblot and ELISA. Furthermore, coded EDTA blood samples were collected from 18 subjects (12 DM and 6 controls) and investigated for the presence or exposure to MAP. Peripheral leukocytes were investigated for harboring viable MAP using long-term culture followed by nested PCR. Clinical plasma samples were used for measurement of anti-MAP IgG titer as well as glucose and Insulin concentrations. Moreover, coded bovine sera from 100 cattle (50 MAP infected and 50 healthy) were investigated for possible correlation between MAP infection and plasma levels of glucose and insulin. RESULT: Peptide BLAST analysis revealed a 44% identity between MAP Hsp65 and GAD65 proteins with 75% positive identities in a 16 amino acid region. PyMOL 3-D structural analyses identified a shared epitope region within the 16 amino acid motif which is known to be an antigenic site on GAD65 antigen. MAP DNA and anti-MAP IgG were detected in the blood of TD8, a TIDM subject. Strong cross reactivity was observed between plasma from TD8 and MAP Hsp65 in proteins samples from M. tuberculosis, and E. coli recombinant clone expressing MAP Hsp65. A weak cross reactivity was also observed between rat pancreatic tissue homogenate and rabbit anti-MAP IgG. Long term culture of leukocytes from 18 blood samples resulted in the detection of MAP in 3/10 (30%) TIDM and 4/8 (50%) control subjects whereas anti-MAP IgG were detected in 5/10 (50%) TIDM samples compared to 3/8 (37.5 %) controls. In MAP infected cattle, insulin level ranged from below 0.1ng/ml to 2.456 ng/ml with an average of 0.36 +/- 0.57ng/ml compared to 0.1ng/ml to 13.47ng/ml with an average of 2.86 +/- 3.00ng/ml in healthy cattle (P(<)0.0001). CONCLUSION: We identified and confirmed a shared epitope region between MAP Hsp65 and human pancreatic GAD65. The shared epitope is a known antigenic binding site. Although MAP DNA was detected in both TIDM and control subjects, a strong correlation was found between anti-MAP IgG titer and MAP-positive culture in clinical samples, regardless of diagnosis. The correlation between MAP infection and insulin level in cattle is significant. Overall the result is intriguing and requires further investigation of MAP in well-characterized clinical samples.
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Date Issued: 2012
Identifier: CFE0004608, ucf:49924
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0004608

BMP-7 (3)	+ -
Doxorubicin (3)	+ -
apoptosis (3)	+ -
Atherosclerosis (2)	+ -
Macrophages (2)	+ -

PTEN (2)	+ -
Pyroptosis (2)	+ -
TIMP-1 (2)	+ -
fibrosis (2)	+ -
myocardial infarction (2)	+ -
?-catenin (1)	+ -
Akt (1)	+ -
BMP7 (1)	+ -
CD (1)	+ -
CD63 (1)	+ -
Cardiac Remodeling (1)	+ -
Cardiac Stem Cells (1)	+ -
Cardiotoxicity (1)	+ -
Cholesterol (1)	+ -
Cholesterol efflux (1)	+ -
Cholesteryl ester (1)	+ -
Chronic Intermittent Hypoxia (CIH) (1)	+ -
CideB (1)	+ -
Coat complex II (1)	+ -
Crohn's Disease (1)	+ -
Cytokines (1)	+ -
Diabetes (1)	+ -
Diabetic Cardiomyopathy (1)	+ -
Dietary Peroxidized Lipids (1)	+ -
Dysfunctional HDL (1)	+ -

Singla, Dinender (18)	+ -
University of Central Florida (18)	+ -
Naser, Saleh (8)	+ -
Parthasarathy, Sampath (4)	+ -
Siddiqi, Shadab (4)	+ -

Cheng, Zixi (3)	+ -
Jewett, Mollie (3)	+ -
Masternak, Michal (2)	+ -
Zervos, Antonis (2)	+ -
Zhao, Jihe (2)	+ -
Abdelli, Latifa (1)	+ -
Andl, Claudia (1)	+ -
Chakrabarti, Ratna (1)	+ -
Chen, Jin (1)	+ -
Davidson, Victor (1)	+ -
Doomra, Mitsushita (1)	+ -
Garner, Kaley (1)	+ -
Glass, Carley (1)	+ -
Hammond, Jamillah (1)	+ -
Jewett, Travis (1)	+ -
Johnson, Taylor (1)	+ -
Merino-Chavez, Hilda (1)	+ -
Neel, Sarah (1)	+ -
Parks, Griffith (1)	+ -
Pourmoghadam, Kamal (1)	+ -
Rocher, Crystal (1)	+ -
Rohde, Kyle (1)	+ -
Rothaus, Alexandra (1)	+ -
Roy, Herve (1)	+ -
Sengupta, Bhaswati (1)	+ -

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