Poster D7 - Mechanical susceptibility of chondrocytes by Piezo1 modulators and inflammatory cytokines in situ

Friday, October 25, 2024

3:30 PM – 4:30 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Johann Kintzel (he/him/his)

Research Scientist/Lab Technician
University of Rochester
Rushville, New York, United States

Co-Author(s)

SL

Suk Hee Lee

Post Doctoral Associate
University of Rochester Medical Center, United States

Primary Investigator(s)

Whasil Lee, PhD

Assist Professor
University of Rochester, NY
Rochester, New York, United States

Introduction: Osteoarthritis (OA) is considered one of the most prevalent chronic diseases that is characterized by progressive degradation of the articular cartilage and joint inflammation (1,2). It has been established that Piezo1 plays a crucial role in mechanosensitivity of articular chodrocytes (3). It has also been established that Piezo1 modulator GsMTx4 decreases mechano-susceptibility in chondrocytes (4). To further understand this disease, it is also important to focus on the inflammation that is produced. This can be accomplished using pro-inflammatory and anti-inflammatory cytokines such as interleukin-1β (IL-1β) and interleukin-4 (IL-4), respectively (5). We and others used interleukin-1α (IL-1α) induced OA model which augments Piezo1 expression similar to OA (3,4). From this we can hypothesize that Piezo1 modulators GsMTx4 and Yoda1, Piezo1 specific agonist, will cause an increase and decrease in mechano-susceptibility of chondrocytes, respectively. Additionally, IL-1β and IL-4 will increase and decrease in mechano-susceptibility of chondrocytes, respectively.

Materials and

Methods: Porcine cartilage explants were harvest from the tibiofemoral joint from the regions including the femoral condyle and trochlea using a 4mm biopsy punch (Fig.1A). IL-4 and IL-1β Treatment Cartilage plugs (trochlea) were placed in a 12 well plate per 4 pig cohort, 5-6 plugs per well. In a serial dilution from 100ug/ml stock of IL-4 and IL-1β using feed media to dilute, a 1ng/mL treated feed media is created for both IL-4 and IL-1β. Cartilage plugs are treated in this new media for 120 hours (5 days), the media being renewed as needed. Mechano-death assay Calcein AM live dye/propidium iodide kits (Invitrogen) were used to image chondrocyte vulnerability post-1mm biopsy punch injury. Chondrocyte mechano-vulnerability was quantified in ImageJ by measuring the width of death rings (Fig1.B, C; Fig2.A). Piezo1 Modulator incubation Plugs are taken from feed media and follows mechano-death assay with additional 15-minute incubation of 40μM GsMTx4 or 20μM Yoda1. Post-biopsy drop samples sit in treatment for 3 hours before being imaged again (Fig1.D).

Results, Conclusions, and Discussions: First, articular cartilage expressed greater mechano-vulnerability in OA cartilage versus healthy cartilage (n = 8-11 explants, **p < 0.01) (Fig1.C). Second, Yoda1 expressed greater mechano-susceptibility over 3 hours, compared to control (n = 7-10 explants, **p < 0.01) (Fig1.D-E). Third, GsMTx4 expressed lower mechano-susceptibility over 3 hours, compared to control (n = 6 explants) (Fig1.D-F). Fourth, IL-4-treated plugs showed lower mechano-vulnerability in comparison to IL-1β and control (untreated) samples (n = 8 pigs, **p < 0.01) (Fig2.A-B). Finally, IL-4-treated samples displayed lower cell area compared to IL-1β treated samples (n = 8 pigs, **p < 0.01) (Fig2.A, C). Articular cartilage showed greater mechano-vulnerability in more osteoarthritic samples, based on chondrocyte morphology, suggesting chondrocytes are not operating at homeostasis thus more susceptible to mechanical impact. GsMTx4 displayed smaller increase in mechano-susceptibility over 3 hours, compared to untreated control, suggesting that there is rescue by GsMTx4 or inhibition of OA to some degree. Yoda1 displayed a greater increase in mechano-susceptibility over 3 hours, compared to untreated control, suggesting that Piezo1 activation increased cell death. IL-4 samples expressed significantly lower mechano-vulnerability suggesting inflammation is a key contributing factor to OA conditions and chondrocyte function in OA conditions. Although there were no significant findings made using IL-1β compared to control, there is still a clear, subtle trend that IL-1β is altering the chondrocytes in the direction of OA. To further understand Piezo1 and the tested cytokines, IHC can be utilized, in the future, to find the Piezo1 expression in both IL-1β and IL-4.

Acknowledgements (Optional): The authors thank Dr. Alexander Kotelsky for building cartilage impact device, and to Dr. Thomas Suchyna for the GsMTx4 peptides. This work was supported by NIH/NIAMS R01AR082349, NIH/NIGMS R35GM147054, and URMC/CMSR Discover award.