Thank you very much for giving me the opportunity to present our research with you today. My name is Julie Oakes and I am an OMS3 at NYIT-COM. This project was funded in part by NYIT-COM Academic Medicine Scholars Program. Recent publications illustrated a correlation between warfarin, a leading prescribed anticoagulant, and increased vascular calcification. Based on literature review, DBA2J mice are susceptible to vascular calcification and can reproduce vascular side effects of warfarin use. Using this animal model, we aim to investigate the changes in cardiac structure and function in response to warfarin-induced calcification 10 weeks after discontinuation of warfarin treatment and determine if this process is influenced by sex. Identifying long-term consequences is critical to managing cardiovascular disease in long-term warfarin users. This study aims to examine if the discontinuation of warfarin will lead to the persistence of calcification and changes in cardiac function. We also aim to compare warfarin-induced calcification cardiovascular response in male and female mice. DBA2J mice heterozygous for the LDLR genotype obtained from the Jackson Laboratory were intercrossed and genotyped. 46 mice at 28 weeks old were randomly assigned to 4 groups, 12 animals per group, 50% males. After 20 weeks of treatment on 3 mg per gram of warfarin and 1.5% vitamin K supplemental diet, mice were placed on a strictly Western diet. Echocardiogram imaging has been utilized to assess cardiac and vascular parameters. Echocardiogram imaging consisted of a long-axis B-mode, short-axis B-mode, M-mode, and Doppler using the Vivo 3100 ultrasound system. Mice were anesthetized with 3% isoflurane for induction, ranging from 1 to 2% for maintenance, while heart rate and respiration were monitored on the heated platform at 40 to 42 degrees Celsius. LD mass was obtained by echocardiogram of the left ventricle by isolating the long axis, then moving the probe 90 degrees to the short axis. Ascending aortic peak velocity was used as a measure of aortic valve vascular stiffness. Micro-CT methodology was used to quantify calcification. Aortic arches were dissected, scanned, and reconstructed using Enricon and segmented using Dragonfly software. We obtained the volume of calcification based on segmentation of the images above the set intensity threshold corresponding to calcification. All procedures and practices were approved and performed according to IACUC regulatory standards. Pearson correlation matrix was used to identify significant associations. Single and multilinear regression models were performed and based on significant parameters identified, including sex, genotype, treatment, body weight, heart rate, ejection fraction, cardiac output adjusted for body weight, LV mass adjusted for body weight, aortic valve velocity, and calcification volume in the univariate correlation matrix. Two groups were compared with Student T-Test or Mann-Whitney U-Test. Figure 1, panel A depicts micro-CT imaging of the aortic arch representative of female treated mice compared to controls, while panel B shows calcification volume by treatment and panel C illustrating calcification volume by sex. Figure 2 shows the Pearson correlation matrix. Significant p-values are recorded. The blue coloring illustrates a positive correlation while red illustrates a negative correlation. This figure demonstrates that aortic calcification was associated with warfarin treatment, a p-value of less than 0.001, and ascending aortic peak velocity was associated with sex with a p-value of 0.048. LV mass adjusted for body weight was not associated with ascending aortic peak velocity. And in figure 3, the linear regression analysis of the effects of ascending aortic calcification on left ventricular mass normalized to body weight, ejection fraction, cardiac output normalized to body weight, and peak aortic valve velocity. As illustrated, ascending aortic calcification was significantly correlated with LV mass when adjusted for body weight with a p-value of 0.022. In the multivariate regression analysis, the ascending aortic calcification remained associated with the LV mass to body weight ratio independent of sex, genotype, and treatment group assignment. In conclusion, withdrawal of warfarin treatment for 10 weeks resulted in continued ascending aortic calcification. Calcification was associated with warfarin treatment. The extent of the warfarin-induced calcification was not affected by sex. Withdrawal of warfarin did not result in the resolution of ascending aortic calcification. There was a positive relationship between warfarin treatment and normalized LV mass. However, under the experimental conditions, cardiac ejection fraction and cardiac output were not affected by the ascending aortic calcification. In conjunction with the lack of correlation in the ascending aortic peak velocity and normalized LV mass, these results suggest that additional physiologic compensatory mechanisms are involved. Further research can investigate potential physiologic responses such as aortic root dilatation. I would like to thank you very much for your time today. I would also like to give a special thank you to the NYIT COM Academic Medicine Scholars Program, the NYIT COM Animal Facility, the NYIT Micro-CT Facility, and of course our mentor Dr. Savanova for making this work possible. Thank you.

warfarin

vascular calcification

DBA2J mice

cardiovascular impacts

NYIT-COM