A preprint study has found that the stiffness of the extracellular matrix (ECM) itself encourages cells to undergo senescence.
An unexplored relationship
ECM stiffness and cellular senescence are both well-known aspects of aging. The cross-linked collagens, such as glucosepane, that lead to a less-flexible ECM accumulate with time and have long been the subject of aging research efforts. Senescent cells, which have lost their capacity to divide and do not always perform their proper functions, are among the most studied subjects in aging.
However, despite ECM stiffness being known to affect cellular function [1], particularly in the vasculature [2], the idea that ECM stiffness may be a direct contributor to senescence has gone almost entirely unexplored. This is not an easy relationship to study, as specialized modeling systems are required to properly isolate stiffness from confounding stimuli [3].
To that end, these researchers placed vascular endothelial cells (ECs) into a hydrogel scaffold that can have its stiffness changed on the fly without impacting ECs in unrelated ways. This allowed them to mimic different ECM conditions while keeping biochemical cues constant, thus ensuring that stiffness is the only variable being tested. The hydrogel stiffnesses used in this study were similar to the ECM stiffnesses of naturally aging mice.
When vascular cells stop forming vasculature
In their first experiment, the researchers cultivated colony-forming ECs for 48 hours in their hydrogel substrate, then altered the stiffnesses of different groups for another 48 hours. In a hydrogel where the ECM remained soft, the ECs continued to proliferate and form blood vessel networks; however, once the stiffness was increased to moderate or severe levels, vessel formation dropped dramatically, becoming minimal in the moderate group and nearly nonexistent in the severe group.
This change in vessel formation was matched by increases in gene expression levels of CDKN1A, which produces p21, and CDKN2A, which produces p16. Their proteins were similarly increased along with the well-known senescence marker SA-β-gal. However, they did not secrete the same phenotype as many other senescent cells do (the SASP); the SASP cytokines IL-6, IL-8, and CXCL1 were notably downregulated, while IL-33, IL-1α, and IFN-γ were upregulated instead. The researchers hypothesized that this suggests a “divergent SASP trajectory under mechanical stress” that uses different pathways.
Further experimentation confirmed this hypothesis. One of these alternative pathways involves Notch signaling, which was found to be increased with increasing ECM stiffness. Notch is associated with several other senesence-related pathways, including JNK; however, a different senescence pathway, c-JUN, was unaffected, which explains the lack of IL-6 and IL-8. Inhibiting Notch by administering nirogacestat mitigated stiffness-related senescence.
Similar effects in humans
Synthetic breast implants often cause localized areas of fibrosis, a stiff tissue that contains substantial quantities of senescent cells. RNA sequencing of this tissue found that Notch and senescence-related signaling were upregulated compared to the surrounding tissue. Perhaps most concerning was the finding that fibrosis-related genes were upregulated in the local ECs; while these cells are not the primary cause of fibrosis, this suggests that ECM stiffness may lead such cells to encourage its progression.
The researchers hold that ECM stiffness plays an upstream role in the progression of cellular senescence, at least in this circumstance. This means that addressing molecular cross-links through glucosepane breakers and other treatments may be more vital than previously anticipated; if these findings are validated, not only are such cross-links leading to the physical problems associated with tissue stiffness, they are also contributing to even more long-term damage in the process.
Literature
[1] Selman, M., & Pardo, A. (2021). Fibroageing: An ageing pathological feature driven by dysregulated extracellular matrix-cell mechanobiology. Ageing Research Reviews, 70, 101393.
[2] Schnellmann, R., Ntekoumes, D., Choudhury, M. I., Sun, S., Wei, Z., & Gerecht, S. (2022). Stiffening matrix induces age‐mediated microvascular phenotype through increased cell contractility and destabilization of adherens junctions. Advanced Science, 9(22), 2201483.
[3] Wang, E. Y., Zhao, Y., Okhovatian, S., Smith, J. B., & Radisic, M. (2022). Intersection of stem cell biology and engineering towards next generation in vitro models of human fibrosis. Frontiers in Bioengineering and Biotechnology, 10, 1005051.
View the article at lifespan.io
