Induction of cellular autophagy impairs TGF-β1-mediated extracellular matrix deposition in primary human knee fibroblasts

Listen to Lisa and Brian discuss the paper 'Induction of cellular autophagy impairs TGF-β1-mediated extracellular matrix deposition in primary human knee fibroblasts' published in the April 2025 issue of Bone & Joint Research.

Click here to read the paper.

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Transcript

[00:00:00] Welcome back to another episode of AI Talks with Bone & Joint from the publishers of Bone & Joint Research. Today we're mulling over the paper 'Induction of cellular autophagy impairs TGF-β1-mediated extracellular matrix deposition in primary human knee fibroblasts', published in April 2025 by OB Dilger and colleagues. I'm Lisa, and here with me is my co-host Brian.

Hello Lisa, and greetings to all our listeners. Today we're delving into a captivating study that explores how inducing cellular autophagy can affect the extracellular matrix deposition in knee fibroblasts. An important process relevant to arthrofibrosis.

Indeed, the primary aim of the study was to understand the role of autophagy and knee fibroblasts undergoing myofibroblast differentiation. This is particularly important because arthrofibrosis, a [00:01:00] complication following total knee arthroplasty involves excessive scar tissue formation that restricts joint movement.

The researchers sought to determine whether modulating autophagy could alleviate these fibrotic processes. They focused on primary human knee fibroblasts that were stimulated with transforming growth factor-beta 1 or TGF-β1, to induce a fibrotic state.

To induce autophagy, they employ two methods: pharmacologically with rapamycin and through amino acid deprivation.

Rapamycin is known to inhibit mTOR complex 1, thus promoting autophagy. They also deprive the fibroblasts of amino acids using Earle’s balanced salt solution to achieve a similar effect.

Absolutely and they assess the impact on collagen deposition using picrosirius red staining, a technique to visualize and quantify collagen fibers. Additionally, they used RT-qPCR and western blotting to evaluate changes in gene [00:02:00] expression related to fibrosis.

The results were quite compelling. They found that inducing autophagy with rapamycin significantly reduced TGF-β1-induced collagen deposition. Curiously, genes associated with fibrotic markers like ACTA2 were upregulated.

This discrepancy between extracellular matrix levels and gene expression is quite intriguing. While rapamycin reduced collagen deposition, it simultaneously increased the expression of several fibrotic markers. This implies that autophagy plays a complex role in moderating these pathways. And when they induced autophagy through amino acid deprivation, they observed a consistent reduction in both extracellular matrix levels and fibrotic gene expression, including ACTA2.

This suggests that different methods of inducing autophagy might have varying effects. It's also worth noting that the study found no evidence of apoptosis in the fibroblasts treated with [00:03:00] rapamycin. As indicated by the absence of caspase-3 cleavage products in their western blot analyses. This is significant because it implies that the antifibrotic effects were not a result of cell death.

The mechanistic pathways provide further insights. The study showed that while rapamycin required TGF-β1 to induce fibrotic gene expression, it did not enhance SMAD2 phosphorylation beyond what TGF-β1 alone could achieve. This rules out one potential pathway of hyperactivation.

In contrast, amino acid deprivation reduced SMAD two phosphorylation suggesting a suppression mechanism through canonical TGF-β signaling pathways. It's fascinating how different methods of inducing autophagy can lead to different outcomes in the same cells.

Indeed. To sum up, the key takeaway is that inducing autophagy, whether pharmacologically with rapamycin or through [00:04:00] amino acid deprivation, can modulate fibrotic pathways and knee fibroblasts. However, the underlying mechanisms and outcomes can vary significantly depending on the method of autophagy induction.

Precisely. These findings open up potential avenues for developing targeted therapies to prevent or treat arthrofibrosis. Following surgeries like total knee arthroplasty. Future research will need to delve deeper into these mechanisms and establish optimal strategies for clinical applications.

Thanks for tuning into this episode of AI Talks with Bone & Joint. Until next time, stay curious and keep exploring. Goodbye everyone.