Metformin impairs rho gtpase signaling and inhibits the survival and migration of neuroblastoma cells

EFFECTS OF DIRECT STIMULATION OF THE CGMP/PKG SIGNALING PATHWAY VERSUS ANTIHYPERTENSIVE TREATMENT ON THE UTERINE VASCULATURE IN PREGNANT, NO-INHIBITED RATS Richard D. Adams, MD, Nicole Maille, BS, and George Osol, PhD Department of Obstetrics, Gynecology & Reproductive Sciences University of Vermont, Burlington, VT Background: The physiological stress of pregnancy on the maternal systemic and uterine vascular systems demands significant vascular remodeling to adapt to the increased blood volume and uteroplacental blood flow. Nitric Oxide (NO) is a key signaling molecule necessary for normal vascular remodeling, while inhibition of NO attenuates the remodeling process. Evidence exists for NO to mediate effects by mechanisms other than the canonical sGC-cGMPPKG pathway. The primary goal was to determine whether treatment with a NO-independent stimulator of soluble Guanylate Cyclase (sGC) could re-institute the cGMP-PKG pathway, reverse hypertension, and restore vascular remodeling in NO-inhibited rats. The secondary goal was to determine whether vascular remodeling could be restored by treatment with an antihypertensive alone. The tertiary goal was to evaluate vessel reactivity when exposed to a potent NO donor. Methods: Pregnant Sprague-Dawley rats were treated with L-NAME (NOS inhibitor) or cotreated with either BAY41-2272 (sGC stimulator) or Diltiazem (antihypertensive) during the second half of gestation. Measurements taken included mean arterial pressure (MAP) at midgestation as well as main uterine artery (MUA) and arcuate artery diameters, and pup & placenta weights upon sacrifice of the animal on Day 20 for the following groups: Control (C:n=11), LNAME (L:n=14), L-NAME+BAY (LB:n=9), and L-NAME+DILT (LD:n=9) treated rats. Arcuate arteries were dissected, mounted on an arteriograph in a physiologic solution, and treated with DETA/NO (NO donor) in a dose-response fashion to evaluate vessel reactivity. Results: Co-treatment with BAY41-2272 prevented the development of hypertension (C: 96 ± 5.4 mmHg; L: 129±2.8 mmHg; L+B: 94±2.4 mmHg; L+D: 96±2.9 mmHg p<0.05) and fully restored MUA remodeling (C: 228±10 μm; L: 172±12 μm; L+B: 222 ± 7 μm) while BP control with Diltiazem normalized BP but not remodeling (L+D:170±12 μm; p<0.05). A similar pattern was seen with arcuate artery remodeling, and no between-group differences in pup/placental weights. The L-NAME and L+D vessels showed increased sensitivity to NO compared to controls (EC50 values: L: 11.6±5.2 μM; L+D: 6.3 ± 2.1 μM; C: 34.9±3.6 μM), while the L+B showed an intermediate response (EC50 values: L+B: 16.4±7.2 μM). Conclusion: In summary, NO signaling via the sGC/PKG pathway is essential for normal uterine vascular adaptation in pregnancy. Contact Information Category: Basic Science Richard D. Adams, MD Division of Maternal-Fetal Medicine USC Department of Obstetrics and Gynecology Columbia, SC 29203 803 545-5700 Richard.Adams@uscmed.sc.edu CpG METHYLATION OF HPV16 L2 IN A COHORT OF COLLEGE-AGE WOMEN Erin L. Anderson, Carolyn E. Banister, Susanna Kassler, Amy Messersmith , Lucia PirisiCreek, Michael D. Wyatt, Kim E. Creek Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Department of Pharmaceutical Sciences, College of Pharmacy, Presbyterian College, Department of Pathology, Microbiology and Immunology, School of Medicine, University of South Carolina Abstract Background and Significance: It is unknown why HPV infections clear in most women yet persist in others. Women with persistent high-risk HPV (HR-HPV) infection are at the greatest risk of cervical dysplasia and cervical cancer. There is a clinical need to identify biomarkers that are predictive of the small number of HR-HPV infections that will persist. In the Carolina Women’s Care Study (CWCS) at the University of South Carolina we followed a cohort of 467 freshman women over four years of college with the goal of identifying biomarkers of HPV persistence. Methods: DNA from exfoliated cervical cells was collected in biannual visits, which allowed for the identification of participants who rapidly cleared HPV infections along with others who had persistent infections. We hypothesized that alterations in the methylation status of HPV DNA may ultimately determine viral persistence or clearance. Therefore we determined the methylation status of 5 CpG sites in the L2 genes from 222 unique HPV16-positive visits from women who were categorized as “clearers” or “persisters”. DNA samples were bisulfite converted and amplified with the EpiTect® Whole Bisulfitome kit. PCR was performed for individual biotinylated amplicons containing CpG sites of interest. Pyrosequencing of samples to determine the extent of methylation was carried out by EpigenDx and analyzed with PyroMark® Software. Discussion: There were interesting variations in methylation status at the individual CpG’s that were not statistically significant with any trend analysis attempted. Analysis by ethnicity in this subset presented differences in methylation status trends. While not statistically significant, the data was not analyzed under the context of global methylation which has been shown by others to be lower in African American women. Finally, we analyzed the SNP MTHFR rs1801131, a key regulator in folate metabolism. In the entire CWCS cohort, participants with the AA homozygous allele were found to be 0.4x less likely to be a persister than the CC or AC allele. Our subset showed a decreased percentage of African Americans with the AA allele, however the statistical correlation was not to persistence but ethnicity. Discussion: Although there are tantalizing suggestions of trends, limited sample numbers prevent the achievement of statistical significance. It is not possible to rule in or out the potential predictive benefit of measuring HPV methylation long before the appearance of abnormal cytology. ______________________________________________________________________ Contact Information Category: Basic Science Erin L. Anderson, Research Associate Principal Investigators: Michael Wyatt, PhD and Kim Creek, PhD Department of Drug Discovery and Biomedical Sciences South Carolina College of Pharmacy, University of South Carolina Phone: 919-522-5157 Email: andere@sccp.sc.eduBackground and Significance: It is unknown why HPV infections clear in most women yet persist in others. Women with persistent high-risk HPV (HR-HPV) infection are at the greatest risk of cervical dysplasia and cervical cancer. There is a clinical need to identify biomarkers that are predictive of the small number of HR-HPV infections that will persist. In the Carolina Women’s Care Study (CWCS) at the University of South Carolina we followed a cohort of 467 freshman women over four years of college with the goal of identifying biomarkers of HPV persistence. Methods: DNA from exfoliated cervical cells was collected in biannual visits, which allowed for the identification of participants who rapidly cleared HPV infections along with others who had persistent infections. We hypothesized that alterations in the methylation status of HPV DNA may ultimately determine viral persistence or clearance. Therefore we determined the methylation status of 5 CpG sites in the L2 genes from 222 unique HPV16-positive visits from women who were categorized as “clearers” or “persisters”. DNA samples were bisulfite converted and amplified with the EpiTect® Whole Bisulfitome kit. PCR was performed for individual biotinylated amplicons containing CpG sites of interest. Pyrosequencing of samples to determine the extent of methylation was carried out by EpigenDx and analyzed with PyroMark® Software. Discussion: There were interesting variations in methylation status at the individual CpG’s that were not statistically significant with any trend analysis attempted. Analysis by ethnicity in this subset presented differences in methylation status trends. While not statistically significant, the data was not analyzed under the context of global methylation which has been shown by others to be lower in African American women. Finally, we analyzed the SNP MTHFR rs1801131, a key regulator in folate metabolism. In the entire CWCS cohort, participants with the AA homozygous allele were found to be 0.4x less likely to be a persister than the CC or AC allele. Our subset showed a decreased percentage of African Americans with the AA allele, however the statistical correlation was not to persistence but ethnicity. Discussion: Although there are tantalizing suggestions of trends, limited sample numbers prevent the achievement of statistical significance. It is not possible to rule in or out the potential predictive benefit of measuring HPV methylation long before the appearance of abnormal cytology. ______________________________________________________________________ Contact Information Category: Basic Science Erin L. Anderson, Research Associate Principal Investigators: Michael Wyatt, PhD and Kim Creek, PhD Department of Drug Discovery and Biomedical Sciences South Carolina College of Pharmacy, University of South Carolina Phone: 919-522-5157 Email: andere@sccp.sc.edu Poster presented at 30th International Papillomavirus Conference September 2015 NIH grants R21 CA169998 and P20 MD001770 ATTITUDES AND PRACTICE PATTERNS REGARDING TRIAL OF LABOR AFTER CESAREAN DELIVERY Fran Bailey,MD, Bo Cai, PhD and Courtney Brooks, MD 1Department of Obstetrics and Gynecology, University of South Carolina School of Medicine, Columbia, SC 2 Department of Epidemiology and Biostatistics, University of South Carolina, Columbia, SC Abstract Background and significance: In the United States the rate of cesarean deliveries has increased drastically over the last 30 years. Repeat cesarean plays a large role in increasing the overall cesarean rate. Trial of labor after previous cesarean delivery (TOLAC) is a feasible option for many women who have had a prior cesarean.