PPARβ/δ priming enhances mesenchymal stromal cell anti-apoptotic properties on chondrocytes via ANGPTL4 and improves their therapeutic effects in osteoarthritis

Figure 1.. Activation of PPARβ/δ enhances therapeutic efficacy of murine mesenchymal stromal cell (MSC) in a collagenase-induced osteoarthritis (CIOA) murine model in vivo. (A) On Day 0 and 2, mice received intra-articular injection (IA) of 1 unit of type VII collagenase. On Day 7, 2.5 × 105 MSC control (MSCControl) or pretreated with 1 µm PPAR agonist (MSCAgo) were IA injected. After 42 days, mice were euthanized and knee joints were harvested and fixed for histological analysis. (B) Representative 3D reconstructed images of medial plateau of articular cartilage post confocal laser scanning microscopy analysis. (C) Quantitative histomorphometric evaluation of 3D reconstructed images displaying volume, surface degradation and thickness of articular cartilage. (D) Representative histological sections of tibial articular cartilage stained with Safranin-O-Fast green. (E) Osteoarthritis (OA) average and maximal score measured in both medial and lateral tibia plateaus. Data are presented as mean ± SD; n = 10-13 mice per group; *P < .05, **P < .01, ***P < .001, ****P < .0001. Created in BioRender. Djouad, F. (2026) https://BioRender.com/b2xcplu.

Figure 2.. Treatment with peroxisome proliferator-activated receptor (PPAR) agonist does not affect the immunomodulatory capacity of mesenchymal stromal cells (MSCs). (A) Concanavalin A-activated splenocytes proliferation and representative histogram. (B) Gating strategy for macrophages analysis. Cells were first selected based on their size and granularity, then double events were excluded, as well as dead cells, finally cells were gated on CD45+CD11b+F4/80+ population, considered as differentiated macrophages. (C) The median fluorescence intensity (MFI) of pro-inflammatory-associated markers (CD80 and MHC-II) and anti-inflammatory-associated marker (CD206) was analyzed. Data are presented as the mean fluorescence intensity ± SEM; n = 3. (D) Supernatants from macrophages cultures were analyzed by ELISA to determine the presence of prof-inflammatory (tumor necrosis factor alpha [TNFα]) and anti-inflammatory (interleukin-10 [IL-10]) mediators. (E) Relative mRNA expression of inflammatory/chemotactic genes (CCL2, CCL5, IL1B, IL8, and TNFA) on SW982 synovial cells was quantified by RT-qPCR and normalized to ACTB. Data are shown as fold change relative to the Naive condition and represent mean…

Figure 3.. PPARβ/δ priming protects mesenchymal stromal cells from H2O2-induced apoptosis. (A) MSCControl or MSCAgo were exposed to H2O2 for 16 hours, then apoptosis was measured by DNA fragmentation assay. (B) Bar plots representing specific DNA fragmentation after H2O2 exposure results are expressed as fold change (enrichment factor) relative to untreated cells (MSCalive), indicated by the dotted line. Data are presented as the mean ± SEM; n = 5. ****P < .0001. Created in BioRender. Djouad, F. (2026) https://BioRender.com/b2xcplu.

Figure 4.. MSC PPARAgo exhibit an enhanced anti-apoptotic effect on chondrocytes. (A) TC28a2 human chondrocytes were exposed to 100 µm H2O2 for 2 hours to induce apoptosis, then MSCControl or MSCAgo were co-cultured in transwell inserts at 1:2 ratio for an additional 14 hours (16 hours in total). Apoptosis in chondrocytes was quantified using a cell death detection ELISA. (B) Bar plots show specific DNA fragmentation after H2O2 exposure. Results are expressed as fold change (enrichment factor) relative to untreated cells (TC28Alive), indicated by the dotted line. Data are presented as mean ± SEM (n = 5). ****P < .0001. Created in BioRender. Djouad, F. (2026) https://BioRender.com/b2xcplu.

Figure 5.. Mechanisms involved in the enhanced properties of PPARβ/δ-primed MSCs. (A) Volcano plot of the 13 753 variables identified by RNA seq analysis on murine MSC. Volcano plot shows the significance (non-adjusted P-value) converted to –Log10(P-value) versus the fold change converted to Log2(fold change). For the analysis, a gene was considered significantly differentially expressed when P-value ≤ 0.05 and absolute fold change ≥ 1.5. (B) The expression of Angptl4 in murine MSC was validated by qRT-PCR. (C) Volcano plots of human MSC derived from bone marrow (hBM), adipose tissue (hASC), and umbilical cord (hUC). (D) The expression of ANGPTL4 was validated by qRT-PCR in human MSC derived from hBM, hASC, and hUC. Data are presented as the mean ± SD; n = 4. ***P < .001; ****P < .0001.

Figure 6.. ANGPTL4 mediates the antiapoptotic effect induced by PPARβ/δ agonist priming. Mesenchymal stromal cell (MSC) were silenced with specific siRNA against Angptl4 and primed or not with peroxisome proliferator-activated receptor (PPAR) agonist. (A) MSCs pre-treated or not were exposed to H2O2 for 16 hours, and then apoptosis was quantified using a Cell Death ELISA kit. (B) Chondrocytes were exposed to H2O2 for 2 hours to induce apoptosis, then MSC were co-cultured in transwell inserts at 1:2 ratio for an additional 14 hours (16 hours in total). Bar plots show specific DNA fragmentation after H2O2 exposure. Results are expressed as fold change (enrichment factor) relative to untreated cells (dotted line). Data are presented as the mean ± SEM; n = 5. *P < .05; ****P < .0001.