Poster H11 - Investigating the Role of Estrogen Levels in Hemodynamic Function in a Rat Model of Pulmonary Arterial Hypertension

Friday, October 25, 2024

3:30 PM - 4:30 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Bardia Khosravi

Undergraduate Researcher
University of California, San Diego
Calabasas, California, United States

Primary Investigator(s)

DV

Daniela Valdez-Jasso, PhD

Associate Professor
University of California, San Diego, California, United States

Co-Author(s)

Giuditta Monti (she/her/hers)

Graduate Student
University of California, San Diego
La Jolla, California, United States
Yufan Lin, BS

Graduate Student
University of California, San Diego, United States
KG

Kristen Garcia, MS

Graduate Student
University of California, San Diego, United States

Introduction: Pulmonary arterial hypertension (PAH) is a vasculopathy, marked by stiffened and constricted pulmonary arteries (PAs) and sustained elevated mean pulmonary arterial pressure (mPAP > 20 mmHg).[1] In these narrowed PAs there is an increase in resistance, overloading the right ventricle (RV).[2] The RV can adapt but if left untreated, PAH leads to right heart failure and death.[1] PAH is also four times more prevalent in women.[3] However, a limited number of studies include female animals and the role of the estrous remains unclear. To address the underrepresentation of female animal models in PAH research, our lab focuses on studying the estrous cycle in conjunction with PAH progression. While the estrous cycle includes 4 stages, we focus on the stages with high levels of estrogen, proestrus, and the lowest levels, diestrus, and relate them to hemodynamic markers of RV function in normotensive and hypertensive rats.[4]

Materials and

Methods: Sprague-Dawley rats undergo a single injection of sugen 5416, a vascular endothelial growth factor receptor inhibitor[5], and are placed in hypoxia with 10% oxygen for three weeks. The rats are then kept in normoxia (21% oxygen) for either 1, 5, 9, 12 or 15 weeks. Control groups are kept in normoxia for the same time.

Hemodynamic measurements are made with an admittance pressure-volume (PV) catheter in the right ventricle during an open-chest procedure by measuring and distinguishing blood and muscle electrical properties.[6] These measurements are used to calculate hemodynamic metrics of mPAP, end-systolic and end-diastolic elastance, stroke volume and ejection fraction, cardiac output, end-systolic and end-diastolic pressures, and changes in pressure over time.

Estrous cycle stages are measured via vaginal cytology.[7] Daily vaginal swabs are collected using cotton swabs and streaked on gelatin-coated slides for eight consecutive days prior to hemodynamic measurements. These slides are then stained with crystal violet solution and imaged at 3.45 µm pixel resolution.[8] Each stage can be separated based on the observed cell types and densities: Proestrus is predominated by small nucleated epithelial cells, estrus mainly consists of anucleated epithelial cells, metestrus is characterized by both neutrophils and anucleated epithelial cells, and diestrus is identified by a combination of neutrophils and nucleated epithelial cells and fewer anucleated epithelial cells.[7] This protocol allows us to identify the stage of the estrous cycle. Uterine weight is used to confirm the estrous stage by correlating estrogen levels with uterus lining growth.[9],[10]

Results, Conclusions, and Discussions: For the PAH-induced group, most of the hemodynamic measurements were taken when the rat was in the diestrus stage of the estrous cycle. Diestrus, the longest stage lasting 24 to 72 hours, is characterized by low estrogen levels.[4] Using Microsoft Excel to perform a two-sample t-test, the observed average of uterus weights normalized to body weights of female PAH-induced and control rats were 2.23 ± 1.12 mg and 3.71 ± 1.25 mg, respectively, with a p-value of 0.16. Mean PA pressures were 33.7 ± 6.8 mmHg, approximately two times higher than the control group (14.0 ± 1.9 mmHg), with a p-value of 0.0002.

The p-value for the t-test for normalized uterus weights does not represent statistical significance to validate the differences in mean weight between the female PAH-induced and control groups, whereas there is statistical significance in the t-test concerning mPAP values. Notably, there are limited measurements from female rats in other stages of the estrous cycle, and future efforts are committed to collating more data from female rats in the proestrus stage corresponding to higher levels of estrogen.[4]

Estrogen has been shown to induce mitogenic effects in the inner lining of the uterus, increasing uterus size and weight.[9] It is known that elevated estrogen levels and increased uterus weights correlate with better hemodynamic metrics such as lower mPAP values, potentially explaining the higher survival rates of PAH in women.[11] The effects of this study aim to improve our understanding of PAH progression in female rats, possibly contributing to the development of more impactful treatments.

Acknowledgements (Optional):

References:
[1] Xue, L., Yang, Y., Sun, B., Liu, B., Zeng, Q., & Xiong, C. (2021). Mildly elevated pulmonary arterial pressure is associated with a high risk of progression to pulmonary hypertension and increased mortality: A systematic review and meta‐analysis. Journal of the American Heart Association, 10(7). https://doi.org/10.1161/jaha.120.018374

[2] Vélez‐Rendón, D., Zhang, X., Gerringer, J., & Valdez‐Jasso, D. (2018). Compensated right ventricular function of the onset of pulmonary hypertension in a rat model depends on chamber remodeling and contractile augmentation. Pulmonary Circulation, 8(4), 1–13. https://doi.org/10.1177/2045894018800439

[3] Ruopp, N. F., & Cockrill, B. A. (2022). Diagnosis and treatment of pulmonary arterial hypertension. JAMA, 327(14), 1379. https://doi.org/10.1001/jama.2022.4402

[4] Santmyire, B. R., Venkat, V., Beinder, E., & Baylis, C. (2010). Impact of the estrus cycle and reduction in estrogen levels with aromatase inhibition, on renal function and nitric oxide activity in female rats. Steroids, 75(12), 1011–1015. https://doi.org/10.1016/j.steroids.2010.06.016

[5] Transonic. (n.d.). Understanding PV loops: Technology & Theory. https://insidescientific.com/wp-content/uploads/2017/02/Understanding-PV-Loops-Ebook-RPV-2-wb.pdf

[6] Queen’s University. (n.d.). SOP 10.25. Estrus Cycle Monitoring in Rats. https://www.queensu.ca/animals-in-science/policies-procedures/sop/rats/10-25#:~:text=The%20estrous%20cycle%20is%20generally,estrus%2C%20metestrus%2C%20and%20diestrus

[7] EVOS M7000 imaging system. Thermo Fisher Scientific - US. (n.d.). https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cellular-imaging/evos-cell-imaging-systems/models/evos-m7000.html

[8] Ducsay, C. A., & Rhee, J. W. (1996). Steroid modulation of myometrial structure and function during pregnancy. (E. E. Bittar & T. Zakar, Eds.).Pregnancy and Parturition, 1, 175–193. https://doi.org/10.1016/s1569-2590(08)60072-5

[9] Vasilenko, P., Adams, W. C., & Frieden, E. H. (1981). Uterine size and glycogen content in cycling and pregnant rats: Influence of Relaxin. Biology of Reproduction, 25(1), 162–169. https://doi.org/10.1095/biolreprod25.1.162

[10] Sun, Y., Sangam, S., Guo, Q., Wang, J., Tang, H., Black, S. M., & Desai, A. A. (2021). Sex differences, estrogen metabolism and signaling in the development of Pulmonary Arterial Hypertension. Frontiers in Cardiovascular Medicine, 8. https://doi.org/10.3389/fcvm.2021.719058