Master of Science / Department of Animal Sciences and Industry / Timothy G. Rozell / Since vascularization of the theca layer increases as follicles progress in size through preantral and antral stages, the principal angiogenic factor, vascular endothelial growth factor A (VEGFA), may influence follicle growth via regulation of angiogenesis. However, VEGFA may also influence follicular development through nonangiogenic mechanisms since its expression has been localized to nonvascular follicles and cells. Alternative mRNA splicing of 8 exons from the VEGFA gene results in the formation of different VEGFA isoforms. Each isoform has unique properties and is identified by the number of amino acids within the mature protein. Proangiogenic isoforms are encoded by exon 8a while a sister set of isoforms with antiangiogenic properties are encoded by exon 8b. The antiangiogenic isoforms comprise the majority of VEGFA expressed in most tissues while expression of the proangiogenic VEGFA isoforms is upregulated in tissues undergoing active angiogenesis. The Vegfa angiogenic isoforms (Vegfa_120, Vegfa_164, and Vegfa_188) were detected in developing rat ovaries, and quantitative RT-PCR determined that Vegfa_120 and Vegfa_164 mRNA was more abundant after birth, while Vegfa_188 mRNA was highest at embryonic day 16. The antiangiogenic isoforms, Vegfa_165b and Vegfa_189b, were amplified and sequenced from rat ovaries and quantitative RT-PCR determined that Vegfa_165b mRNA was more abundant around embryonic day 18, but Vegfa_189b lacked a distinct pattern of abundance. VEGFA and its receptors were localized to pregranulosa and granulosa cells of all follicle stages and to theca cells of advanced-stage follicles. Antiangiogenic VEGFA isoforms were localized to pregranulosa and granulosa cells of all follicle stages and to theca cells of advanced-stage follicles. To determine the role of VEGFA in developing ovaries, postnatal day 3/4 rat ovaries were cultured with VEGFR-TKI, a tyrosine kinase inhibitor that blocks signaling through the VEGFA receptors, FLT1 and KDR. Ovaries treated with VEGFR-TKI had vascular development reduced by 94%. In addition, treated ovaries had more primordial follicles, fewer early primary, transitional, and secondary follicles, and greater total follicle numbers compared with control ovaries. This suggests that VEGFA promotes follicle recruitment and early follicular development. These effects may be dependent upon increased ovarian vascularization or they may be mediated by nonvascular mechanisms.

Atherosclerosis is a leading cause of morbidity and mortality worldwide and type 2 diabetes and obesity-associated metabolic syndrome, both characterized by insulin resistance, are potent risk factors. These conditions also increase the risk for restenosis after revascularization procedures used for treatment of atherosclerosis. Studies have shown that insulin and resveratrol (RSV), a red wine polyphenol, decrease neointimal growth after vessel injury in models of restenosis, demonstrating a protective effect on the vasculature. However, oral glucose and sucrose were used in insulin studies to maintain normoglycemia, and their effect on neointimal formation was not assessed. Several studies have shown that nitric oxide (NO) production is stimulated by insulin and RSV, and since NO can decrease neointimal growth, the objective of this thesis was to address the mechanism of action of insulin or RSV to protect against restenosis, and determine whether NO production mediates these effects. To examine this, we treated rats with insulin or RSV and performed arterial balloon injury. In Study 1, insulin reduced neointimal area after injury in rats receiving oral glucose but not oral sucrose. Oral glucose alone had no effect on neointimal formation or insulin sensitivity whereas oral sucrose increased neointimal growth and induced insulin resistance. In Study 2, insulin decreased neointimal area and cell migration, and increased re-endothelialization. These effects were abolished by nitric oxide synthase (NOS) inhibition. In addition, insulin increased eNOS protein expression in the vessel. In Study 3, RSV reduced neointimal growth, cell proliferation, and migration after injury, without affecting re-endothelialization. Most of these effects were abolished by NOS inhibition, except for the decrease in cell migration. Insulin sensitivity and systolic blood pressure were not affected by RSV. Together, the results demonstrate that insulin, independent of glycemic effects, and RSV have a protective effect on the vessel against restenosis, which is mediated by NO. Since both insulin and RSV decrease neointimal formation without negatively impacting re-endothelialization, insulin or RSV treatment could provide some advantage over anti-mitogenic agents currently used in drug-eluting stents, which delay re-endothelialization. These studies suggest that insulin or RSV may have clinical potential in the prevention of restenosis after angioplasty.

Cardiac output (Q) is an important determinant of the cardiovascular system‟s ability to meet the oxygen needs of the body. This dissertation addresses the non-invasive measurement of Q, in healthy children and those with heart and lung disease. 1) The correction factors for collision broadening, downstream difference and end tidal CO2 (PetCO2), used in the CO2 rebreathe (equilibrium) method, were evaluated. In lung disease, one is unable to assume a normal dead space to estimate arterial CO2 (PaCO2), and the use of any of these correction factors alone should be used with caution as they each exert a profound effect on the Q measurement. 2) A new equation to predict PaCO2 from PetCO2 in patients with CF was derived via multiple regression analysis, taking into account disease severity. 3) The validity and reliability of Q measures via the inert gas rebreathing technique (InnocorTM device) were evaluated. The highest intraclass correlation coefficients were attained during exercise (0.7-0.98), indicating excellent reliability of the device. Comparisons of Q measures from the InnocorTM (QInn) to the AMIS mass spectrometer system (QAmis) were made to assess validity. The bias (QInn-QAmis) and limits of agreement (±2SD) were 0.45 ± 1.9 L.min-1 and 0.27 ± 2.1 for children with congenital heart disease and healthy controls respectively, with no systematic differences between the two methods. 4) Assessment of cardiac output in Fontan patients demonstrated that an individualized, atrioventricular (AV) delay optimization was required. Moreover, there was a small but significant improvement in heart function with AV synchronized pacing (DDI) versus ventricular pacing (VVI), suggesting that further study with a larger sample of patients is warranted. The limitations and strengths of the measurement of non-invasive cardiac output in children, primarily via respiratory gas analysis, were delineated and recommendations were made for their use.

The synaptic remodeling of neural circuits is thought to underly memory formation. Both long-term memory formation and remote memory formation are thought to involve a process restructuring of synapses in specific areas of the brain. The transcription factor myocyte enhancer factor 2 (MEF2) has been shown to restrict spine growth in both in vivo and in vitro. It has been suggested that MEF2 is a critical molecule involved in memory formation, however, MEF2‘s role in adult memory formation it is largely unexplored. Thus, we have sought to characterize MEF2’s involvement in the formation of long-term and remote memory formation. We first showed that acute overexpression of MEF2 in the hippocampus blocks long-term spatial memory formation and activity-dependent spine formation. We next found that acute overexpression of MEF2 in the lateral amygdala likewise blocks long-term fear memory formation, suggesting that MEF2 is critical protein involved in synaptic plasticity necessary for long-term memory formation. We next demonstrated the bi-directionality of MEF2 by decreasing MEF2 function in the hippocampus and amygdala and showing a facilitation in memory formation. Together, these results suggest that MEF2 is a critical protein, which regulates spine formation important for the formation of long-term memories. We next investigated whether similar synaptic plasticity mechanisms are involved in the systems consolidation. We acutely overexpressed MEF2 in the anterior cingulate cortex at different time points following contextual fear conditioning. We noted that there was a critical window, where MEF2 blocks spine density increases in the ACC and remote memory formation. Results from this study, suggest that cortical synapses undergo a process of strengthening and remodeling and that MEF2 is a critical regulator involved. Our results demonstrate that MEF2 is involved in the synaptic consolidation of long-term and remote memories.

Postprandial overproduction of apolipoprotein B48 (apoB48)-containing lipoproteins has been observed in states of insulin resistance and is important to the sequelae of cardiovascular disease, but little is understood about factors that regulate their secretion. The glucagon-like peptides (GLPs) are released from ileal enteroendocrine L-cells following lipid ingestion. I hypothesized that the GLPs could acutely affect the production of apoB48-containing triglyceride (TG)-rich lipoproteins (TRL) in the small intestine. Using the Syrian golden hamster, I first characterized the gross effects of the GLPs on TRL secretion in response to an oral fat load and then continued to dissect the mechanisms of these changes using primary intestinal cell cultures and a variety of knockout mouse models. An exogenous GLP-1 receptor (GLP-1R) agonist was found to acutely inhibit chylomicron secretion in both hamsters and mouse models, and extending the bioactivity of endogenously-secreted GLP-1 with a dipeptidyl peptidase-4 inhibitor had suppressive effects in insulin-resistant fructose-fed hamsters. The insulinotropic and delayed gastric emptying functions do not completely account for the hypolipidemic effect of GLP-1R agonism, and the effect of the GLP-1R agonist exendin-4 could be seen directly in the apoB48 secretion of primary enterocytes. In contrast, the sister peptide GLP-2 was a potent acute stimulator of chylomicron secretion in hamsters and mice. The hyperlipidemic effect of GLP-2 could be attributed to an increased rate of luminal FA uptake mediated by the posttranslational modification of the FA transporter CD36, and CD36-deficient mice were found to be refractory to the stimulatory effects of GLP-2. The activity of nitric oxide synthase was also found to be essential to the hyperlipidemic action of GLP-2. I identified a set of intercellular communications that could contribute in mediating the action of GLP-2, in which GLP-2 secreted from the enteroendocrine L-cell stimulates intestinal subepithelial myofibroblasts to release vascular endothelial growth factor, which directly activated the enterocyte to secrete apoB48. In summary, this thesis demonstrates that two co-secreted postprandial hormones have considerable but completely opposite influences on chylomicron production. Changing the balance of the GLPs’ actions in vivo could provide a therapeutic strategy to combat postprandial dyslipidemia.

Introduction: The mechanism of cell activation by LPS in the absence of surface Toll-like receptor 4 (TLR4) is unclear. We hypothesize that ICAM-1 binds LPS on the cell surface, mediating cell activation independent of TLR4. Methods: The interaction between murine ICAM-1 and LPS was measured in a binding assay. Alveolar macrophages (AMs) isolated from TLR4 deficient mice were stimulated with LPS. Cell activation was measured by flow cytometry and cytokine production. The role of ICAM-1 in cell activation was determined by siRNA transfection. Results: Murine ICAM-1 binds LPS. TLR4 deficient AMs respond to LPS stimulation by upregulation of LPS binding sites, ICAM-1 expression and cytokine release. Cell activation is attenuated by treatment with polymyxin B and ICAM-1 gene silencing. Conclusions: ICAM-1 binds LPS and is important in TLR4-independent cell activation. Strategies devised to target ICAM-1 may have the potential to block the excessive inflammatory response seen in gram-negative sepsis.

Chronic iron overload (CIO) in patients leads to a cardiomyopathy characterized by conduction defects, including bradyarrhythmias. Using a murine model of CIO, we explored the effects of iron loading on the electrophyisology of the heart. Telemetric heart rate was reduced in conscious CIO mice compared to controls. Similarly, heart rates were depressed in both isolated CIO hearts and CIO mice following autonomic blockade, suggesting an intrinsic impairment of the SA node (SAN). Indeed, spontaneous action potential frequency was reduced in CIO SAN myocytes. The depressed pacing rate in CIO SAN myocytes was linked to reduced L-type Ca2+ current (ICa,L) density and a rightward shift in ICa,L activation, suggesting a selective reduction in α1D-mediated ICa,L. Western blot analysis demonstrates that the α1D isoform was reduced by ~ 89% in CIO atrial tissue. Therefore, the conduction defects under conditions of CIO are due to reductions in Cav1.3 channel expression in atrial tissue.

ChREBP has emerged as one of the key controllers of hepatic lipogenesis. While the function of ChREBP has been extensively investigated, mechanisms underlying its transcriptional regulation remain largely unknown. We located a conserved POU-binding site within mammalian ChREBP promoters, and demonstrated that the POU homeodomain protein Oct-1 binds to this site in the human HepG2 cell line. Oct-1 transfection significantly repressed ChREBP promoter activity 50-75%. Conversely, knockdown of Oct-1 expression with shRNA significantly increased ChREBP expression levels. Furthermore, insulin treatment resulted in a two-fold activation of ChREBP promoter activity, and stimulated endogenous ChREBP expression. We found that the stimulatory effect of insulin on the ChREBP promoter is at least partially dependent on the presence of the POU-binding site, and that insulin treatment reduced Oct-1 expression. Our observations identify Oct-1 as a transcriptional repressor of ChREBP, and suggest that insulin stimulates ChREBP expression via attenuating the repressive effect of Oct-1.

Four and a half LIM domains protein 1 (FHL1), consisting of 4.5 protein interaction mediating LIM domains, is a predominantly skeletal muscle protein that has consistently been upregulated in a variety of cardiovascular diseases. Since proteins mediate their functions in conjunction with other proteins, it was considered that delineation of interactions would provide insight into FHL1’s regulation and regulatory functions. We performed tandem affinity purification (TAP) from human embryonic kidney 293 (HEK-293) cells to purify tagged FHL1 and interacting proteins. Samples were analyzed using gel-free liquid chromatography mass spectrometry (LC-MS). 61 high confidence potential interactors were identified from multiple experiments. Validation of interactions was then performed by co-immunoprecipitation (co-IP) or streptavidin bead pull down, and supported by immunofluorescent colocalization studies. FHL1 interactions could thus be supported for four novel candidates: non-muscle α-actinin 1 (ACTN1), PDZ and LIM domain protein 1 (PDLIM1), cytoplasmic gelsolin (GSN), and ryanodine receptor 1 (RYR1).

Von Hippel Lindau (VHL) is a tumour suppressor protein classically known to target the α subunit of hypoxia inducible factor (HIF) for proteasomal degradation. Emerging evidence has underscored a novel role for VHL in both cell cycle regulation and extracellular matrix assembly. Herein, we provide evidence of VHL multitasking in normal and pathological placentation. Using ex vivo, first trimester human placental tissue and in vitro, JEG-3 choriocarcinoma cell line model we demonstrate that VHL plays a role in regulating the expression of cell cycle modulator CCND1 via a mechanism involving its inhibitor, p15 and HIF-2α. In addition, using a similar experimental strategy we provide evidence supporting a role for VHL in regulating the expression of fibronectin and its receptor integrin α5. Moreover, altered VHL expression observed in preeclampsia is associated with altered expression of cell cycle regulators and contributes to altered FN protein levels which are characteristic of this pathology.