Translational Science Theses and Dissertations

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Anti-Vasculogenic Effect of Mycophenolic Acid
(2018-10) Go, Ellen Lao; O'Neil, Kathleen M.; Yoder, Mervin C.; Paczesny, Sophie
Hydrogen sulfide therapy improves intestinal recovery through endothelial nitric oxide dependent mechanisms
(2017) Jensen, Amanda; Markel, Troy
H2S is a gaseous mediator that acts as an anti-inflammatory agent contributing to gastrointestinal mucosal defense. It promotes vascular dilation, mucosal repair, and resolution of inflammation following intestinal ischemia and may be exploited as a novel therapeutic agent. It is unclear if H2S works through nitric oxide-dependent pathways in the intestine. We appreciated that H2S was able to improve post-ischemic recovery of mesenteric perfusion, mucosal integrity, and inflammation. The beneficial effects of H2S appear to be mediated through endothelial nitric oxide-dependent pathways.
Microrna 21 targets B Cell Lymphoma 2 (Bcl2) Mrna to increase beta cell apoptosis and exosomal Microrna 21 could serve as a biomarker of developing Type 1 Diabetes Mellitus
(2018) Sims, Emily K.
The role of beta cell miR-21 in Type 1 Diabetes (T1D) pathophysiology has been controversial. Here, we sought to define the context of beta cell miR-21 upregulation in T1D and the phenotype of beta cell miR-21 overexpression through target identification. Furthermore, we sought to identify whether circulating extracellular vesicle (EV) beta cell-derived miR-21 may reflect inflammatory stress within the islet during T1D development.. Results suggest that beta cell miR-21 is increased in in-vivo models of T1D and cytokine-treated cells/islets. miR-21 overexpression decreased cell count and viability, and increased cleaved caspase-3 levels, suggesting increased cell death. In silico prediction tools identified the anti-apoptotic mRNA B Cell Lymphoma 2 (BCL2) as a conserved miR-21 target. Consistent with this, miR-21 overexpression decreased BCL2 transcript and protein expression, while miR-21 inhibition increased BCL2 protein levels and reduced cleaved caspase-3 levels following cytokine-treatment. miR-21-mediated cell death was abrogated in 828/33 cells, which constitutively overexpress BCL-2. Luciferase assays suggested a direct interaction between miR-21 and the BCL2 3’untranslated region. With miR-21 overexpression, PRP revealed a shift of BCL-2 message toward monosome-associated fractions, indicating inhibition of BCL2 translation. Finally, overexpression in dispersed human islets confirmed a reduction in BCL2 transcripts and increased cleaved caspase 3 production. Analysis of EVs from human beta cells and islets exposed to cytokines revealed a 3-5-fold increase in miR-21. Nanoparticle tracking analysis showed no changes in EV quantity in response to cytokines, implicating specific changes within EV cargo as responsible for the miR-21 increase. Circulating EVs from diabetic non-obese diabetic (NOD) mice displayed progressive increases in miR-21 that preceded diabetes onset. To validate relevance to human T1D, we assayed serum samples collected from 19 pediatric T1D subjects at the time of diagnosis and 16 healthy controls. Consistent with our NOD data, EV miR-21 was increased 5-fold in T1D samples. In conclusion, in contrast to the pro-survival role reported in other systems, our results demonstrate that miR-21 increases beta cell death via BCL2 transcript degradation and inhibition of BCL2 translation. Furthermore, we propose that EV miR-21 may be a promising marker of developing T1D.
Distinct cachexia phenotypes and the importance of adipose tissue loss on survival of patients with advanced pancreatic cancer on FOLFIRINOX chemotherapy
(2017-12) Kays, Joshua; Zimmers, Teresa A.; Koniaris, Leonidas G.
By the traditional definition of unintended weight loss, cachexia develops in ~80% of patients with pancreatic ductal adenocarcinoma (PDAC). Here we measure the longitudinal body composition changes in patients with advanced PDAC undergoing FOLFIRINOX therapy. We performed a retrospective review of 53 patients with advanced PDAC on FOLFIRINOX as first line therapy at Indiana University Hospital from July 2010 to August 2015. Demographic, clinical, and survival data were collected. Body composition measurement, trend, univariate and multivariate analysis were performed. Three cachexia phenotypes were identified. The majority of patients, 64%, had Muscle-and-Fat Wasting (MFW), while 17% had Fat-Only Wasting (FW) and 19% had No Wasting (NW). NW had significantly improved overall median survival (OMS) of 22.6 months vs. 13.0 months for FW and 12.2 months for MFW (p=0.02). FW (HR=5.2; 95%CI=1.5-17.3) and MFW (HR=1.8; 95%CI=1.1-2.9) were associated with an increased risk of mortality compared to NW. OMS and risk of mortality did not differ between FW and MFW. Progression of disease, sarcopenic obesity at diagnosis, and primary tail tumors were also associated with decreased OMS. On multivariate analysis cachexia phenotype and chemotherapy response were independently associated with survival. Three phenotypes of cachexia were observed. Moreover, three phenotypes suggests molecular or genetic heterogeneity of host or tumor. Identifying these differences will be vital to defining optimal treatment for cachexia. Survival among FW was as poor as MFW suggesting adipose tissue plays a crucial role in cachexia. Blunting or possibly preventing cachexia may confer a significant survival advantage in patients with advanced PDAC.
GDF11 mediates cardiac and skeletal muscle dysfunction and cachexia
(2016-08) Liang, Tiffany; Koniaris, Leonidas George; Murphy, Michael P.; Zimmers, Teresa Audrey
Growth differentiation factor 11 (GDF11) is important in regulating early fetal development of the axial skeleton and various visceral organs. Its actions on the adult body are less clear, and recent studies have led to conflicting accounts of GDF11’s ability to affect cardiac hypertrophy and skeletal muscle regeneration. If boosting GDF11 levels in adults had the ability to rejuvenate tissues and reverse the effects of aging, then the therapeutic possibilities are potentially vast. We attempted to provide clarification of this controversial topic by studying the effects of supraphysiologic levels of GDF11 in a mouse model using injected Chinese hamster ovary cells producing GDF11. We found that increasing endogenous levels of GDF11 in this in vivo mouse model resulted in overall bodily wasting, specifically with evidence of cardiac and skeletal muscle atrophy. In light of these results, caution must be exercised if GDF11 is ever considered as a potential therapeutic agent.
Characterization of antibody binding to swine leukocyte antigen class II
(2016-05-26) Ladowski, Joseph Matthew; Tector, A. Joseph; Tector, Matthew; Blum, Janice S.
Though the elimination of carbohydrate xenoantigens has reduced the antibody barrier to clinical xenotransplantation, identification of additional targets of rejection could further increase the immunologic compatibility of pig tissues with humans. Many patients in need of organ transplantation have antibodies to proteins encoded by the human major histocompatibility complex (MHC) which have high similarity to their swine homologs. The goal of this thesis was to determine if the class II genes of the swine MHC can bind human antibodies. To characterize antibody binding effect to class II swine leukocyte antigens (SLA), a constitutively positive SLA class II cell was created through transfection with the human class II transactivator (CIITA). Cells expressing only SLA-DR or SLA-DQ were also created using the CRISPR/Cas9 gene knockout tools. These various lines were incubated with human sera and tested for binding to IgM and IgG in a flow cytometry crossmatch (FCXM). The results demonstrate reliable antibody binding to each of the SLA class II –DR and –DQ derivatives. A two-way paired t-test revealed statistical difference in total sera binding between to the DR(+)DQ(+) and DR(-)DQ(-) clones for IgG (p = 0.0059) but not IgM (p = 0.2460). Looking at the subset of individuals with and without anti-HLA class II sensitization, statistical difference was noted for IgG (p = 0.0229) but not IgM (p = 0.3045). Examining further the role of DR(+) vs DQ(+), statistical analysis revealed difference in the DR(+)DQ(-) vs. the DR(-)DQ(+) FCXM (p = 0.0099), the DR(+)DQ(-) vs. the DR(+)DQ(+) FCXM (p = 0.0192), and the DR(-)DQ(-) parent vs. DR(+)DQ(+) FCXM (p = 0.0329). No difference was found in the DR(-)DQ(+) vs. DR(+)DQ(+) FCXM (p = 0.1601). The results of this project suggest that SLA class II, specifically SLA-DQ, could be a target of antibody binding and cross-reactive anti-HLA class II antibodies may be capable of binding SLA class II.
Modulation of the Notch Signaling Pathway in 3D Stem-Cell Derived Culture of Inner Ear Organoids
(2016-05-10) Elghouche, Alhasan Najib; Hashino, Eri; Nelson, Rick F.; Koehler, Karl Russell
Hearing loss and vestibular dysfunction are inner ear disease states that arise from an array of diverse etiologies that interfere with mechanosensory hair cell function, including: congenital syndromes, noise-induced trauma, ototoxic drugs, and aging. The investigation of normal inner ear development and the pathological aberrations that cause inner ear disease has been previously advanced through formation of an easily generated, scalable, accurate in vitro model system that readily facilitates experimental applications. This model utilizes a 3D floating cell culture protocol which guides differentiation of stem cell aggregates into inner ear organoids, which are vesicles containing a sensory epithelium with functioning mechanosensory hair cells. Inner ear organoid formation enables studying the effects of modulating the signaling pathways that guide developing inner ear structure and function. The Notch signaling pathway heavily influences the formation of the inner ear through two major mechanisms: lateral induction of sensory progenitor cells and lateral inhibition to determine which of those progenitors differentiate into mechanosensory hair cells. The effects of inhibiting Notch signaling within the inner ear organoid system were explored through application of the ɣ-secretase inhibitor MDL28170 (MDL) at a concentration of 25μM on day 8 of organoid culture. Aggregates were harvested on day 32, fixed, sectioned, and stained according to a standard immunohistochemistry protocol. Sections were stained for the mechanosensory hair cell markers Myosin7a (Myo7a) and Sox2. MDL-treated aggregates demonstrated statistically significant reductions in the total number of vesicles and the number of vesicles containing hair cells compared to control aggregates. In contrast to control aggregates which demonstrated two distinct organoid variants (protruding and embedded), MDL-treated aggregates only formed the embedded variant. Differences in the expression pattern of Sox2, which is also a marker of stemness and neural progenitor cells were also noted between the two conditions. MDL-treated aggregates demonstrated regions of ‘ectopic’ Sox2 expression whereas Sox2 expression in control aggregates was consistently expressed within Myo7a+ regions.
Targeting the protein tyrosine phosphatase, SHP2, and PI3K in FLT3-ITD+ leukemia
(2016-07) Bowling, Joshua D.; Chan, Rebecca J.; Paczesny, Sophie; Ferguson, Michael James
Internal tandem duplications in the fms-like tyrosine kinase receptor (FLT3-ITDs) cause constitutive activation of the receptor and confer a poor prognosis in acute myeloid leukemia (AML). We hypothesized that Shp2 interacts with FLT3-ITD via protein complexes at tyrosine (Y) 768, 955, and/or 969 and that Shp2 and PI3K work cooperatively to promote FLT3-ITD-induced leukemogenesis. Consistently, mutation of N51-FLT3 tyrosine 768 to phenylalanine reduced proliferation and levels of phospho-Erk compared to N51-FLT3-expressing cells while having no effect on levels of phospho-STAT5. In transplants, C3H/HeJ mice injected with either WT-FLT3-, N51-FLT3-, or N51-Y768F-expressing cells showed that mutation of Y768 had no effect on overall survival. In addition, pharmacologic inhibition of Shp2 with II-B08 or PI3K with GDC-0941 in N51-FLT3-expressing cells and primary patient samples showed decreased proliferation. A possible mechanistic explanation for reduced proliferation and selective reduction of P-Erk levels in the N51-FLT3-Y768-expressing cells is through decreased recruitment of Grb2, which participates with son of sevenless, SOS, to activate the RAS-Erk signaling pathway. The lack of improvement in overall survival could be due to preserved STAT5 signaling, as observed during in vitro experiments. Collectively, these data suggest that the tyrosine 768 residue plays an important role in phospho-Erk signaling in N51-FLT3-expressing cells, and that pharmacologic therapy with Shp2 or PI3K inhibitors may provide a novel treatment approach for FLT3-ITD positive AML. For future directions, we plan to treat mice with the Shp2 inhibitor, II-B08, the PI3K inhibitor, GDC-0941, or a combination to determine the effect on overall survival.
Real time PCR and fluorescent in situ hybridization in the detection of the physical tsate of human papillomavirus 16 and 18 in paraffin embedded cervical tissue
(2015-07) Davis, Aisha; Brown, Darron; Ermel, Aaron C.; Johnson, Raymond
Human papillomaviruses (HPV) are the etiologic agents of most cervical dysplasia and all cervical carcinoma. Integration of high risk HPV into the human genome is thought to be a critical event in the progression from cervical dysplasia to invasive cervical carcinoma. The ability to use molecular assays in the detection and evaluation of HPV integration is essential in informing clinical models for early intervention and therapies. We therefore sought to determine the feasibility of real time-PCR (RT-PCR) as a molecular tool in detecting the physical state, episomal versus integration of HPV 16 and 18 DNA in cervical cancers. Tyramide amplified fluorescent DNA in situ hybridization (FISH) was used to look for evidence of HPV 16/18 integration using formalin-fixed, paraffin-embedded sections of cervical carcinomas. RT-PCR used the ratio of the E2 and E6 genes as a surrogate for determining the physical state of HPV 16 and 18 in 35 infected tissues. Results of RT-PCR showed that 16 cervical specimens (45.7%) contained episomal HPV, 17 cervical samples (48.6%) harbored the integrated form of HPV DNA, and 2 samples (5.7 %) contained both integrated and episomal forms of HPV. Results of the two assays were compared in 25 cervical carcinomas. For 13 of the 25 cervical samples there was an agreement in determining the physical state of HPV. RT-PCR, using the E2/E6 ratio as an assay for HPV integration appears to be promising and may prove to be an essential clinical method in the future.
The effect of hypoxia on ER-β expression in the lung and cultured pulmonary artery endothelial cells
(2014-03-12) Selej, Mona M.A.; Lahm, Tim; Petrache, Irina; Schweitzer, Kelly S.
17-β estradiol (E2) exerts protective effects in hypoxia-induced pulmonary hypertension (HPH) via endothelial cell estrogen receptor (ER)-dependent mechanisms. However, the effects of hypoxia on ER expression in the pulmonary-right ventricle (RV) axis remain unknown. Based on previous data suggesting a role of ER-β in mediating E2 protection, we hypothesized that hypoxia selectively up-regulates ER-β in the lung and pulmonary endothelial cells. In our Male Sprague-Dawley rat model, chronic hypoxia exposure (10% FiO2) resulted in a robust HPH phenotype associated with significant increases in ER- β but not ER-α protein in the lung via western blotting. More importantly, this hypoxia-induced ER-β increase was not replicated in the RV, left ventricle (LV) or in the liver. Hence, hypoxia-induced ER-β up-regulation appears to be lung-specific. Ex vivo, hypoxia exposure time-dependently up-regulated ER-β but not ER-α in cultured primary rat pulmonary artery endothelial cells (RPAECs) exposed to hypoxia (1% O2) for 4, 24 or 72h. Furthermore, the hypoxia induced ER-β protein abundance, while not accompanied by increases in its own transcript, was associated with ER-β nuclear translocation, suggesting increase in activity as well as post-transcriptional up-regulation of ER-β. Indeed, the requirement for ER-β activation was indicated in hypoxic ER-βKO mice where administration of E2 failed to inhibit hypoxia-induced pro-proliferative ERK1/2 signaling. Interestingly, HIF-1α accumulation was noted in lung tissue of hypoxic ER-βKO mice; consistent with previously reported negative feedback of ER-β on HIF-1α protein and transcriptional activation. In RAPECs, HIF-1 stabilization and overexpression did not replicate the effects of ER- β up-regulation seen in gas hypoxia; suggestive that HIF-1α is not sufficient for ER-β up- regulation. Similarly, HIF-1 inhibition with chetomin did not result in ER-β down-regulation. HIF-1α knockdown in RPAECs in hypoxic conditions is currently being investigated. Hypoxia increases ER- β, but not ER-α in the lung and lung vascular cells. Interpreted in context of beneficial effects of E2 on hypoxic PA and RV remodeling, our data suggest a protective role for ER-β in HPH. The mechanisms by which hypoxia increases ER-β appears to be post-transcriptional and HIF-1α independent. Elucidating hypoxia-related ER-β signaling pathways in PAECs may reveal novel therapeutic targets in HPH.

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