(C-89) Thymidine-Induced Senescence is not Regulated by ROCK Signaling

Introduction:: In the last century, the average lifespan has been extended due to improvements in healthcare and treatment options. In the United States it is projected that by 2030 the number of people over the age of 65 will grow to be 71 million. Yet, loss of tissue and organ function associated with aging-associated disease (AAD) continues to be a critical medical challenge. Changes in tissue stiffness and cellular mechanics are hallmarks of physiological and pathological aging, but the role of mechanics in aging remains underexplored. In aged tissues, homeostasis is dysregulated through the progressive accumulation of senescent cells that have permanently exited the cell cycle. Senescent cells play an important role in tumor suppression and wound healing. However, the senescence-associated secretory phenotype (SASP) modifies the cellular microenvironment through the upregulation of growth factors, chemokines, and other molecules. SASP has been linked to a wide array of tissue dysfunction, and specifically in the propagation of senescence to neighboring, healthy cells (termed bystander senescence). Further, there is a growing appreciation that senescent cells are biomechanically altered with more pronounced cytoskeletons, however, it remains unknown if this contributes to the pathology in aging tissues. We hypothesized the downregulation of cytoskeletal tension via ROCK signaling inhibition would mitigate the onset of senescence.

Materials and Methods:: HMEC1 cells (endothelial, human) were routinely cultured in complete MCDB131. They were seeded in a 24 well plate on glass coverslips at 75k cells/well and allowed to attach overnight. Senescence was induced by incubating cells in 7 mM thymidine for 72 hr. To assess the effects of ROCK inhibition, treatment groups were dosed with 7 mM thymidine and 5 or 10 μM Y-27632 for a 72 hr period, followed by a 72 hr recovery period. The cells were then fixed in 4% paraformaldehyde and 0.25% Triton-X 100 and counterstained for f-actin and nuclei. To evaluate the onset of senescence we stained against p16^ink4a (p16). Additionally, we assessed the effect of ROCK inhibition on the downstream target YAP via immunofluorescence. Confocal microscopy was used to image a 4 mm x 4 mm x 0.5 mm region. Custom MATLAB algorithms were implemented to quantify nuclear and cytosolic intensity p16 and YAP.


Results, Conclusions, and Discussions:: Results and Discussion: Normalized to vehicle control, treatment with 7 mM thymidine resulted in a mean fold increase of 2.25±0.459 in nuclear/cytosolic p16 intensity (p < 0.05), indicative of senescence. Cells treated with thymidine and 10 μM Y-27632 demonstrated a mean 2.07±0.253-fold increase in nuclear/cytosolic intensity (p < 0.05). There was no significant difference in p16 intensities between cells treated with thymidine or Y-2632/thymidine (Figure 1A), demonstrating that ROCK inhibition does not attenuate cellular entry into senescence. Similarly, when normalized to vehicle control, thymidine treated cells have a 1.77±0.130 fold increase in nuclear/cytosolic YAP intensity (p < 0.01). Cells treated with thymidine and 10 μM Y-27632 have a 1.53±0.159 fold increase in nuclear/cytosolic YAP intensity (p < 0.05) (Figure 1B). Interestingly, YAP activation observed in senescent cells appears to be occurring independent of ROCK signaling. There was no significant difference in YAP activation between cells treated with thymidine or Y-27632/thymidine. Similar results were observed when 5 µM Y-27632 was used (data not shown).



Conclusion: Together these data indicate that ROCK inhibition does not attenuate the onset of senescence. Additionally, senescence-associated YAP activation appears to occur independently of ROCK signaling in this context. Future experiments will focus on elucidating the mechanisms driving the onset and paracrine spread of senescence in vascular endothelial cells. Understanding what drives bystander senescence and loss of tissue homeostasis with age may aid in the development of novel therapeutic targets for aging-associated disease.


Acknowledgements (Optional): : IAB is supported in part by a TRANSCEND fellowship from the California Institute of Regenerative Medicine (Award # EDUC4-12752).

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