Z24 - Modeling Macrophage-Mediated Inflammation after Hypoxic Injury in a Heart-Attack-on-a-Chip Model

Friday, October 10, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

BK

Brennan Kandalaft, MS

PhD Student
University of Southern California
Los Angeles, California, United States

Primary Investigator(s)

Megan L. McCain, PhD

Professor of Biomedical Engineering and Stem Cell Biology & Regenerative Medicine
University of Southern California
Los Angeles, California, United States

Co-Author(s)

JY

Justin Yuen

Masters Student
University of Southern California, United States
TN

Tiana Nejatpoor

Undergraduate
University of Southern California, United States

Introduction: : Acute myocardial infarction is caused by a blockage in the coronary vessels of the heart, resulting in localized ischemic shock to the myocardium and a spatially heterogeneous infarct border zone, characterized by an oxygen gradient. After myocardial infarction, the infarcted tissue undergoes a cellular remodelling process with three temporal phases: the inflammatory, proliferative, and maturation phases. Each phase in this process is characterized by precise interplay between infarcted cardiomyocytes (CMs), immune cells, and cardiac fibroblasts (FBs). The spatial and temporal components of post-infarct remodeling likely directly influence cardiac recovery post-infarction, influencing the size and expansion of the infarcted region and the likelihood of heart failure. Because cardiac function is dependent on how myocardial cells translate and respond to these heterogeneous microenvironmental cues, manipulating cell-cell interactions post-infarction could have therapeutic value in mediating wound repair and mitigating the progression to heart failure.
Our lab previously established a Heart-Attack-on-a-Chip model that mimics hypoxia-induced cardiac injury using a microfluidic device to induce an oxygen gradient. Previously, this model was used to co-culture hypoxic/normoxic CMs and FBs, which led to the discovery that expression of pro-inflammatory genes by these cells is upregulated in an oxygen gradient. Our long-term goal is to advance this model by introducing macrophages (MPs) to the engineered infarct model. We hypothesize that MPs will polarize into a pro-inflammatory phenotype when exposed to hypoxia and will initiate polarization in normoxic MPs via paracrine crosstalk. Here, our goal is to characterize the polarization of macrophages in response to uniform hypoxia.

Materials and

Methods: : THP-1 Human Acute Monocytic Leukemia (AML) cells were cultured in suspension until differentiation with Phorbol 12-myristate 13-acetate (PMA). PMA (8.1nM) was added for 24 hours to differentiate these cells into adherent macrophages, followed by 72 hours of rest in control media. Macrophages were polarized to M1 (pro-inflammatory) or M2 (anti-inflammatory) phenotype using LPS (250ng/mL) and IFN-𝛄 (20 ng/mL) or IL-4 (20 ng/mL) and IL-13 (20 ng/mL), respectively, for 48 hours as positive controls. Polarization was measured by immunostaining MPs for M1 [CD86] or M2 markers [CD206] and also by RT-qPCR (Qiagen RNeasy Mini Kit). Next, MPs were exposed to hypoxia for 4 hours or 48 hours in a StemCell Technologies Hypoxia Chamber. MPs were immediately fixed and stained for HIF-1ɑ (Figure 1) in addition to the polarization markers described above (CD206, CD86). Images were collected on a Nikon fluorescent microscope and mean fluorescence intensity of CD206, CD86, and HIF-1a was quantified using custom-built workflows in CellProfiler and Ilastik. Data was analyzed using Graphpad Prism and statistics were generated using one-way ANOVA.

Results, Conclusions, and Discussions:: THP-1 cells were differentiated into adherent MPs. Image-based fluorescence quantification of macrophage surface markers showed that MPs treated with LPS (without IFN-𝛄) had a decreased CD206/CD86 ratio while MPs treated with IL-4 (without IL-13) had an increased CD206/CD86 ratio. This suggests that THP-1 macrophages were effectively polarized with extrinsic stimulatory molecules and that macrophage polarization can be measured using image-based fluorescence quantification.
Preliminary hypoxia studies show nuclear localization of HIF-1α in hypoxic macrophages (increased nuclear/cytoplasmic HIF-1α) at both 4 hours and 48 hours timepoints. Furthermore, when assayed for polarization markers, macrophages displayed a lower CD206/CD86 ratio, indicating pro-inflammatory polarization after exposure to hypoxia for 48 hours.
Ongoing studies are focused on assaying MP polarization after hypoxic culture using RT-qPCR to further validate and benchmark our results.
Future studies will focus on culturing MPs in our hypoxia device to quantify HIF-1α localization and cell polarization as a function of a controlled oxygen gradient. Additionally, we will integrate MPs onto our hypoxia device to evaluate hypoxic-normoxic crosstalk. We will also evaluate both homotypic (MP-MP) and heterotypic (MP-CM, MP-FB, MP-CM-FB) crosstalk within our device. Our model system will systematically uncover mechanisms governing post-infarction multicellular tissue remodeling and help inform future development of therapeutics.

Acknowledgements and/or References (Optional): :