Poster H9 - Development of an In Vitro System to Model Pulmonary Capillary Stress Failure due to Pulmonary Hypertension

Thursday, October 24, 2024

2:45 PM - 3:45 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

An Tran (she/her/hers)

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

Co-Author(s)

Tasneem Mustafa, BS (she/her/hers)

Graduate Student
University of California, Irvine, United States
JN

Jimmy Nguyen

Undergraduate Researcher
University of California, Irvine, United States

Primary Investigator(s)

Naomi Chesler, PhD (she/her/hers)

UCI Chesler Lab Principal Investigator
University of California, Irvine
Irvine, California, United States

Introduction: Left heart failure (LHF) impacts over 6 million people in the U.S., with a 5-year mortality rate of 50% in older adults [1, 2]. LHF progresses to pulmonary hypertension (PH-LHF) for 36-83% of the population [3]. PH-LHF begins as isolated post-capillary PH (Ipc-PH) when blood pressure in the pulmonary veins elevates and progresses to combined pre-/post-capillary PH (Cpc-PH), increasing both morbidity and mortality. Because they are critical for gas exchange, the pulmonary capillary walls are thin. Subject to PH, the capillary walls can rupture, resulting in pulmonary capillary stress failure [4]. The mechanisms of pulmonary capillary stress failure have been underexplored in the context of PH-LHF. In particular, the role of the endothelial glycocalyx (eGCX) in pulmonary capillary stress failure has not been studied. The eGCX is made of glycoproteins, proteoglycans, and glycosaminoglycans, and sits on top of the endothelial cell monolayer [5]. The eGCX plays an important role in mechanotransduction and its degradation has been linked to inflammation [6]. In disease conditions, the pulmonary capillary basement membrane is known to become fibrotic and stiff [7]. An in vitro system can be created by simulating pathological and physiological conditions of the basement membrane using substrate stiffnesses and induced cyclic stretch. Developing this in vitro system to study the role of the eGCX in pulmonary capillary stress failure will advance understanding of disease progression from Ipc-PH to Cpc-PH.

Materials and

Methods: Two types of substrates were fabricated to mimic healthy (soft) and diseased (stiff) capillary basement membrane. The soft and stiff substrates were made from 15g of PDMS (Sylgard 184, Dow Chemical Company) at 50:1 w/w polymeric base to curing agent for soft substrates and 25:1 w/w for stiff, respectively. Substrates were cured in an oven at 80°C for 3 hours (soft) or 5 hours (stiff). The substrate stiffnesses were tested on a biaxial tensile tester (BioTester 5000, CellScale) under 10%, 20%, and 30% stretch conditions. The data was analyzed through a MATLAB code to calculate Young’s Modulus.
HUVECs obtained from Lonza were cultured, and passage 5 cells were used for this experiment. Before seeding on the PDMS, HUVECs were plated into a T75 flask previously coated with gelatin and placed in a 37 °C humidified incubator to allow the cells to adhere. The PDMS substrates were cut into circles with a 20mm diameter, placed into a 12-well culture plate, and sterilized with a UV/ozone treatment. The substrates were also coated with gelatin to promote cell attachment. The HUVECs, once confluent in the T75 flask, were seeded in the 12-well plate on three conditions: soft substrate, stiff substrate, and hard, gelatin-coated cell culture plastic. Once confluent in the well plate, supernatant and cell lysate were collected and frozen down.

Results, Conclusions, and Discussions: Through tensile testing, the average Young’s Moduli of three stiff and soft substrates were determined to be 165 kPa and 49 kPa, respectively. Initially, PDMS fabrication yielded uncured and uneven substrates that were not suitable for cell adherence. Several optimizations were made for this process to enhance substrate reproducibility, such as increasing the curing time and placing PDMS on a low-speed shaker to even the surface. These changes yielded more viable and even substrates. A static experiment was conducted using HUVECs on cell culture plastic, stiff (25:1), and soft (50:1) PDMS substrates. Healthy cell growth was observed, and cells grew to 80-90% confluency. Supernatant was allowed to sit for 48 hours and collected for analysis along with cell lysate. Future directions include placing PDMS substrates under 10% cyclic stretch conditions, performing ELISA for tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β) inflammatory cytokines, and immunofluorescent staining for image analysis.
With this system, we plan to investigate the impact of two pressure-induced mechanical forces on eGCX degradation -- cyclic stretch and substrate stiffness. We hypothesize that both increased cyclic stretch and increased substrate stiffnesses will increase the concentration of inflammatory cytokines, including TNF-α and IL-1β. By studying mechanisms of pulmonary capillary stress failure, we hope to advance disease understanding, prevention, and intervention practices.

Acknowledgements (Optional): Special thanks to the UCI Grosberg Lab for their project guidance and resources. Images made in BioRender.

References:
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[4] N. Villalba, S. Baby, and S.Y. Yuan, “The Endothelial Glycocalyx as a Double-Edged Sword in Microvascular Homeostasis and Pathogenesis,” Frontiers in Cell and Developmental Biology, vol. 9, Jul. 2021. https://doi.org/10.3389/fcell.2021.711003
[5] Y. Yao, A. Rabodzey, and C.F. Dewey Jr., “Glycocalyx modulates the motility and proliferative response of vascular endothelium to fluid shear stress,” American Journal of Physiology-Heart and Circulatory Physiology, vol. 293, no. 2, pp. H1023-H1030, Aug. 2007. https://doi.org/10.1152/ajpheart.00162.2007
[6] J.B. West, “Invited Review: Pulmonary capillary stress failure,” Journal of Applied Physiology, vol. 89, no. 6, pp. 2483-2489, Dec. 2000. https://doi.org/10.1152/jappl.2000.89.6.2483
[7] M. Loscertales, et al, “Type IV collagen drives alveolar epithelial–endothelial association and the morphogenetic movements of septation,” BMC Biology, vol. 14, no. 73, Sep. 2016.
https://doi.org/10.1186/s12915-016-0297-7