Platelet-Rich Plasma Activates Proinflammatory Signaling Pathways and Induces Oxidative Stress in Tendon Fibroblasts

Abstract

Background: Tendon injuries are one of the most common musculoskeletal conditions in active patients. Platelet-rich plasma (PRP) has shown some promise in the treatment of tendon disorders, but little is known as to the mechanisms by which PRP can improve tendon regeneration. PRP contains numerous different growth factors and cytokines that activate various cellular signaling cascades, but it has been difficult to determine precisely which signaling pathways and cellular responses are activated after PRP treatment. Additionally, macrophages play an important role in modulating tendon regeneration, but the influence of PRP on determining whether macrophages assume a proinflammatory or anti-inflammatory phenotype remains unknown.

Purpose: To use genome-wide expression profiling, bioinformatics, and protein analysis to determine the cellular pathways activated in fibroblasts treated with PRP. The effect of PRP on macrophage polarization was also evaluated.

Study design: Controlled laboratory study.

Methods: Tendon fibroblasts or macrophages from rats were cultured and treated with either platelet-poor plasma (PPP) or PRP. RNA or protein was isolated from cells and analyzed using microarrays, quantitative polymerase chain reaction, immunoblotting, or bioinformatics techniques.

Results: Pathway analysis determined that the most highly induced signaling pathways in PRP-treated tendon fibroblasts were TNFα and NFκB pathways. PRP also downregulated the expression of extracellular matrix genes and induced the expression of autophagy-related genes and reactive oxygen species (ROS) genes and protein markers in tendon fibroblasts. PRP failed to have a major effect on markers of macrophage polarization.

Conclusion: PRP induces an inflammatory response in tendon fibroblasts, which leads to the formation of ROS and the activation of oxidative stress pathways. PRP does not appear to significantly modulate macrophage polarization.

Clinical relevance: PRP might act by inducing a transient inflammatory event, which could then trigger a tissue regeneration response.

Keywords: autophagy; inflammation; oxidative stress; platelet-rich plasma; tendinopathy; tendon.

Figure 1

Microarray and bioinformatics analysis of tendon fibroblasts treated with PRP or PPP. (A) Microarray analysis identified 3296 genes of 36685 that were significantly different (P<0.05) between PRP and PPP treated cells. Of the 3296 genes that were significantly different, 315 genes were greater than 1.5 fold upregulated and 460 genes were greater than 1.5 fold downregulated in PRP treated cells compared to PPP. (B) Ingenuity Pathway Analysis identified two pathways that were predicted to be highly activated in cells treated with PRP compared to PPP, the TNFα pathway (P=6.6×10⁻⁶) and the NFκB pathway (P=9.5×10⁻¹⁹). The merged pathways are presented.

Figure 2

Gene expression of growth factor and cytokine transcripts from PPP or PRP treated tendon fibroblasts. Target gene expression was normalized to β-actin. Values are mean±SD. N=6 replicates from each group. *, significantly different from PPP group (P<0.05).

Figure 3

Gene expression of ECM structural and remodeling transcripts from PPP or PRP treated tendon fibroblasts. Target gene expression was normalized to β-actin. Values are mean±SD. N=6 replicates from each group. *, significantly different from PPP group (P<0.05).

Figure 4

Gene expression of cell proliferation, differentiation, autophagy and inflammatory transcripts from PPP or PRP treated tendon fibroblasts. Target gene expression was normalized to β-actin. Values are mean±SD. N=6 replicates from each group. *, significantly different from PPP group (P<0.05).

Figure 5

Immunoblots for carbonylation and NFκB activation. (A) Immunoblots for total carbonylated proteins and phospho NFκB activation from tendon fibroblasts treated with PRP or PPP. A Coomassie stained gel of the same samples is shown for validation of equal protein loading. Band densitometry analysis for (B) total carbonylated proteins and (C) phospho NFκB blots. Values are mean±SD. N=4 replicates from each group. *, significantly different from PPP group (P<0.05).

Figure 6

Gene expression of markers of (A) proinflammatory and (B) antiinflammatory polarization from PPP or PRP treated macrophages. Target gene expression was normalized to β-actin. Values are mean±SD. N=6 replicates from each group. *, significantly different from PPP group (P<0.05).