Role of Stromal Cell-Derived Factor-1 in Neoangiogenesis in Endometriosis Lesions

Endometriosis affects 5-10% of women and is characterized by the growth of endometrial tissue outside of the uterus. Treatment for endometriosis primarily focuses on symptom relief, is short term with severe side effects and often leads to recurrence of the condition. Establishing new blood supply is a fundamental requirement for endometriosis lesions growth. This has led to the idea that antiangiogenic therapy may be a successful approach for inhibiting endometriosis. Recent evidence indicates that endothelial progenitor cells (EPCs) contribute to neoangiogenesis of endometriotic lesions. These EPCs are recruited to the lesion site by stromal cell-derived factor-1 (SDF-1). We hypothesize that SDF-1 is central to the neoangiogenesis and survival of endometriotic lesions and that administration of SDF-1 blocking antibody will inhibit lesion growth by inhibiting angiogenesis in a murine model of endometriosis. Immunohistochemistry for SDF-1 and CD34 was performed on human endometriosis and normal endometrial samples. Quantification of SDF-1 and EPCs was performed in the blood of endometriosis patients and controls using ELISA and flow cytometry, respectively. A new mouse model of endometriosis was developed using BALB/c-Rag2-/-/IL2rg-/- mice to investigate role of SDF-1 in neoangiogenesis. Either SDF-1 blocking antibody or an isotype control was administered on a weekly basis for four weeks. Weekly samples of peripheral blood from mice were analyzed for SDF-1, other cytokines of interest and EPCs. Mice were euthanized at seven weeks to observe lesion growth and blood vessel development. Our results indicate overabundance of SDF-1 and CD34+ progenitor cells in human endometriotic lesions compared to eutopic endometrium. In the mouse model, SDF-1 and circulating EPC levels decreased from pre-treatment levels after one week, and remained constant over the course of the treatment in both SDF-1 blocking antibody and isotype control groups. In the SDF-1 blocking group, reduced vascularity of lesions, identified by immunofluorescence staining for CD31, was revealed compared to isotype controls. These findings suggest that SDF-1 may be responsible for CD34+ progenitor cell recruitment to the neoangiogenic sites in endometriosis. Blocking of SDF-1 reduces neovascularization of human endometriotic lesions in a mouse model. Further studies on blocking SDF-1 in combination with other antiangiogenic agents are needed.

The Comparative Effectiveness of EEG Biomarkers in Antidepressant Response and Illness Prediction in Major Depressive Disorder

Background: There is growing evidence that individual EEG differences may aid in classifying patients with major depressive disorder (MDD) and also help predict clinical response to antidepressant treatment. This study aims to compare the effectiveness of EEG frequency band power, alpha asymmetry and prefrontal theta cordance towards escitalopram response prediction and MDD diagnosis, in a multi-site initiative. Methods: Resting EEG (eyes open and closed) was recorded from 64 electrodes in 44 depressed patients and 20 healthy controls at baseline, 2 weeks post-treatment and 8 weeks post-treatment. Clinical response was measured as change from baseline MADRS of 50% or more. EEG measures were analyzed (1) at baseline (2) at 2 weeks post-treatment and (3) as an ‘‘early change” variable defined as change in EEG from baseline to 2 weeks post-treatment. Results: At baseline, responders exhibited greater absolute alpha power in the left hemisphere versus the right while non-responders showed the opposite. Responders further exhibited a cortical asymmetry of greater right relative to left activity in parietal areas. Groups also differed in baseline relative delta power with responders showing greater power in the right hemisphere versus the left while non-responders showed the opposite. At 2 weeks post-treatment, responders exhibited greater absolute beta power in the left hemisphere relative to right and the opposite was noted for non-responders. The opposite pattern was noted for absolute and relative delta power at 2 weeks post-treatment. Responders exhibited early reduction in relative alpha power and early increments in relative theta power. Non-responders showed a significant early increase in prefrontal theta cordance. Absolute delta power helped distinguish MDD patients from healthy controls. Conclusions: Hemispheric asymmetries in the alpha and delta bands at pre-treatment baseline and at 2 weeks post-treatment have moderate to moderately strong predictive utility towards antidepressant treatment response. These findings have significant potential for improving clinical practice in psychiatry by eventually guiding clinical choice of treatments. This would greatly benefit patients awaiting relief from depressive symptoms as treatment optimization would help overcome problems associated with delayed recovery. Our results also indicate that resting EEG activity may have clinical utility in predicting MDD diagnosis.

EXAMINATION OF VALPROIC ACID-INDUCED PERTURBATIONS TO DNA REPAIR, NF-ΚB SIGNALING AND CBP/P300 TRANSCRIPTIONAL CO-ACTIVATORS

Exposure to the antiepileptic drug valproic acid (VPA) is associated with an increased risk of congenital malformations including heart, skeletal and most frequently neural tube defects. Although the mechanisms contributing to its teratogenesis are not well understood, VPA was previously shown to increase homologous recombination (HR)-mediated DNA repair and decrease protein expression of the transcription factor NF-κB/p65. The studies in this thesis utilized in vivo and in vitro models to evaluate the expression of HR mediators, investigate the implications of decreased p65 including DNA binding and transcriptional activation, and the expression and histone acetyltransferase activity of Cbp/p300 with an aim to provide mechanistic insight into VPA-mediated alterations. The first study demonstrated that following maternal administration of VPA, mouse embryonic mRNA expression of HR mediators Rad51, Brca1 and Brca2 exhibited temporal and tissue-specific alterations. Protein expression of Rad51 was similarly altered and preceded increased cleavage of caspase-3 and PARP; indicative of apoptosis. The second study confirms previous findings of decreased total cellular p65 protein using P19 cells, but is the first to demonstrate that nuclear p65 protein is unchanged. NF-κB DNA binding was decreased following VPA exposure and maybe mediated by decreased p50 protein, which dimerizes with p65 prior to DNA binding. Transcriptional activity of NF-κB was also increased with VPA exposure which was not due to increased p65 phosphorylation at Ser276. Furthermore, the transcriptional activation capacity was unaffected by VPA exposure as combined exposure to VPA and TNFα additively increased NF-κB activity. The third study demonstrated that VPA exposure in P19 cells decreased Cbp/p300 total cellular and nuclear protein attributed primarily to ubiquitin proteasome-mediated degradation. Histone acetyltransferase (HAT) activity of p300 was decreased proportionately to nuclear protein following VPA exposure. Inhibition of Cbp/p300 HAT activity decreased p65 total cellular protein, increased caspase-3 cleavage and ROS similar to VPA exposures. Furthermore, pre-treatment with the antioxidant enzyme catalase attenuated the increase in caspase-3 cleavage, but not p65 protein. Overall, this thesis demonstrates that VPA exposure impacts the expression and activity of the transcription factor NF-κB and transcriptional co-activators/HATs Cbp/p300, which has implications for downstream VPA targets including Rad51, Brca1 and Brca2.

CARBON MONOXIDE AND PREGNANCY: A SEARCH FOR A POSSIBLE THERAPEUTIC IN THE TREATMENT OF PRE-ECLAMPSIA

Pre-eclampsia (PE) is a pregnancy disorder that affects roughly 5-7% of all pregnancies and is a leading cause of both maternal and fetal/neonatal morbidity and mortality. With no present cure for the disease, researchers are interested in the lower incidence of PE observed among the cigarette smoking pregnant population. However, women who use smokeless tobacco do not experience the same decreased incidence of PE, leading to hypothesis of protection against PE from the largest combustible product of cigarette smoke, carbon monoxide (CO). Studies evaluated levels of CO in PE women and found that they were statistically lower than those of healthy pregnancy. Researchers have found CO to possess many cytoprotective and regulatory properties and specifically within the placenta, it has been found to increase perfusion pressure, decrease oxidative stress, decreases ischemia/reperfusion induced apoptosis and maintain endothelial functioning. The idea for use of CO as a possible therapeutic for PE has thus become a real possibility. This study determined CO levels in pregnant women ± smoking as well as in PE women±smoking, as to discover a possible therapeutic range for future treatments. The best correlated automated CO measurement device with blood CO levels was determined, for use in future clinical studies. This thesis also sought a possible CO delivery concentration, in order to achieve the CO levels observed in the human correlation study. A threshold level of maternal CO exposure in a murine animal model was found, for which fetal and maternal negative toxicities were not observed. The results of this thesis lend a few more pieces to the complicated puzzle involving CO and PE and offer another step toward the possibility of a therapeutic treatment/prevention using this gaseous molecule.