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SILAC Analysis Reveals Increased Secretion of Hemostasis-Related Factors by Senescent Cells

cellular senescence aging homeostasis coagulation clotting thrombosis chemotherapy sasp

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#1 Engadin

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Posted 01 October 2019 - 12:29 PM


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F U L L   T E X T   S O U R C E :   Cell

 

 

 

 

 

Highlights
 
 
•  An unbiased SILAC screen identifies >300 proteins secreted by senescent human cells
 
•  Forty-four of these proteins have reported roles in hemostasis
 
•  Conditioned media from senescent cells promotes platelet activation
 
•  Eliminating senescent cells ameliorates pro-coagulation side effects of doxorubicin
 
 
Summary
 
Cellular senescence irreversibly arrests cell proliferation, accompanied by a multi-component senescence-associated secretory phenotype (SASP) that participates in several age-related diseases. Using stable isotope labeling with amino acids (SILACs) and cultured cells, we identify 343 SASP proteins that senescent human fibroblasts secrete at 2-fold or higher levels compared with quiescent cell counterparts. Bioinformatic analysis reveals that 44 of these proteins participate in hemostasis, a process not previously linked with cellular senescence. We validated the expression of some of these SASP factors in cultured cells and in vivo. Mice treated with the chemotherapeutic agent doxorubicin, which induces widespread cellular senescence in vivo, show increased blood clotting. Conversely, selective removal of senescent cells using transgenic p16-3MR mice showed that clearing senescent cells attenuates the increased clotting caused by doxorubicin. Our study provides an in-depth, unbiased analysis of the SASP and unveils a function for cellular senescence in hemostasis.
 
 
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Introduction
 
Cellular senescence permanently arrests cell proliferation in response to a variety of stresses, including DNA damage, mitochondrial dysfunction, and oncogene activation (Campisi, 2013, Wiley et al., 2016). Senescent cells participate in a wide range of biological processes (Childs et al., 2015, Muñoz-Espín and Serrano, 2014), playing beneficial or deleterious roles, depending on the triggers of senescence, as well as the tissue and cell types. Senescent cells accumulate with age and drive a variety of age-related pathologies, at least in mice (Baar et al., 2017, Baker et al., 2016, Chang et al., 2016). The senescence response can impair tissue regeneration (Chang et al., 2016, Jeon et al., 2017) and promote cancer cell proliferation and invasion (Krtolica et al., 2001, Laberge et al., 2015). In contrast, senescence suppresses tumorigenesis (Prieur and Peeper, 2008), accelerates wound healing (Demaria et al., 2014), and fine-tunes embryogenesis (Muñoz-Espín et al., 2013, Storer et al., 2013). Although the mechanisms are incompletely understood, senescent cells exert some of these effects by secreting numerous growth factors, cytokines, chemokines, and proteases, known as the senescence-associated secretory phenotype (SASP) (Acosta et al., 2013, Coppé et al., 2008, Kuilman et al., 2008).
 
To better understand the biological consequences of cellular senescence, we embarked on a comprehensive, unbiased method to profile the SASP using stable isotope labeling with amino acids (SILACs), a mass spectrometry-based technique that uses non-radioactive, stable isotope labeling of cells. Surprisingly, this screen revealed several SASP factors, expressed by cells induced to senesce by DNA damage, that were predicted to participate in hemostasis.
 
Hemostasis is a tightly regulated, complex process that prevents excessive bleeding after injury. Hemostasis has primary and secondary major components. Primary hemostasis forms a platelet plug at the injured site. Upon vascular damage, platelets adhere to the injured vessel, become activated, and aggregate to arrest bleeding (Tanaka et al., 2009, Versteeg et al., 2013). However, because the primary plug is not stable, secondary hemostasis then stabilizes the clot by depositing insoluble fibrin, which is generated by the coagulation cascade. These two processes happen simultaneously and interact in a dynamic fashion. Multiple anti-clotting mechanisms, such as circulating anticoagulants and fibrinolysis, which removes the plug after the vessel is repaired, also play critical roles in hemostasis (Tanaka et al., 2009, Versteeg et al., 2013). An impaired balance between pro- and anti-elements of coagulation can lead to pathological bleeding or, conversely, thrombosis.
 
An increased risk for thrombosis is a common age-associated complication and major risk factor for mortality (Sepúlveda et al., 2015). Thrombosis is also a common side effect of radio- and genotoxic chemotherapies, which significantly increase the risk for post-therapy mortality and decrease quality of life (Bosch et al., 2014, Vergati et al., 2013). Though it is known that DNA-damaging cancer therapies induce cellular senescence (Demaria et al., 2017, Ewald et al., 2010), an association between cellular senescence and thrombosis has not yet been made.
 
Our finding that the secretion of a subset of hemostasis-related factors increases significantly when human fibroblasts undergo senescence suggests that senescent cells may contribute to the etiology of age- and genotoxic therapy-induced thrombosis. We show that senescent cells promote blood clotting in mice treated with the genotoxic anti-cancer chemotherapeutic doxorubicin, whereas removing senescent cells from such mice attenuates clotting. Furthermore, the pro-coagulation effect of senescent cells is associated with both increased platelet number and reactivity. Our findings identify an additional function of senescent cells and provide potentially important insights into genotoxic therapy-induced thrombosis.
 
 
Results
 
SILAC Analysis Identifies a Potential Role for the SASP in Hemostasis
 
To obtain an unbiased profile of the SASP, we used SILACs to analyze conditioned media (CM) from quiescent and senescent primary human foreskin (HCA2) fibroblasts. Figure 1A and Figure S1A show the timeline for 13C6 lysine and 13C6 arginine labeling of quiescent cells and the schedule for inducing senescence in unlabeled cells by ionizing radiation (IR) and CM collection. We analyzed three independent biological replicates for each condition and confirmed the senescent and quiescent states by SA-β-gal staining and BrdU labeling (Figures S1B–S1C). The proteins we identified, and the magnitude of changes in secreted levels between quiescent and senescent cells, are shown in Tables S1A–S1C. Overall, we detected 1,047 proteins, identified by at least two peptides per protein, with 99% confidence (Table S1A).
 
 
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(A) A schematic outlining the strategy for SILAC-based identification of SASP proteins. See Figure S1 for time lines of how cells were made quiescent or senescent and how quiescent and senescent conditioned media (CM) were produced.
(B) Reactome pathway analysis of proteins secreted at >2-fold levels by senescent compared with quiescent HCA2 cells. Pathways related to hemostasis are identified by boxes.
© Heatmap showing individual levels of proteins, identified by SILACs, that participate in hemostasis and are secreted by quiescent (QUI) and senescent (SEN [IR]) HCA2 normal human fibroblasts.
(D) CM were collected from quiescent (QUI) and senescent (SEN [IR]) cells, concentrated, and analyzed using western blotting for the indicated SASP factors. Cells remaining on the plate at the time of CM collection were counted for normalization. Mindin protein levels and Ponceau S staining were used as loading controls.
Fold changes are mean ± SEM. p < 0.05 and ∗∗p < 0.01.

 

Among the SASP factors we identified, 343 showed a significant (>2-fold) increase in abundance in CM from senescent, compared with quiescent, cells (Table S1B). Reactome bioinformatic analyses showed that, notably, 5 of the top 25 pathways (highlighted in Figure 1B) were associated with hemostasis, blood clot formation, or clot dissolution. The SASP factors predicted to be hemostasis related by Reactome, and the magnitude of their changes, are presented as two heatmaps in Figure 1C. The left heatmap shows factors that are the most highly elevated (most yellow), and the right shows the second most highly elevated. Additional bioinformatic analyses identified the molecular functions, cellular components, and biological processes in which they are known to participate (Figures S1D–S1F). Together, the results suggest the SASP might include hemostasis-related factors.
 
We also observed increases in extracellular matrix (ECM) proteins in our initial dataset, which might warrant additional study (Figure 1B; Figures S1D–S1F). However, the concurrent increased secretion of proteinases may have artificially increased the abundance of peptides derived from the ECM.
 
For this reason, we chose to focus on hemostasis.
 
 
Validation of SILACs Results in Cultured Cells
 
To validate the SILAC results, we collected CM from three independent cultures of quiescent or IR-induced senescent HCA2 cells and determined by western blotting the levels of 11 hemostasis-related factors identified by SILACs (Figure 1D). These factors included vinculin (VCL), tissue inhibitor of metalloproteinase 1 (TIMP1), tissue factor pathway inhibitor (TFPI), thrombospondin 1 (THBS1), calumenin (CALU), serpin E1 (SERPINE1, PAI1), serpin E2 (SERPINE2), serpin B2 (SERPINB2, PAI2), serpin B6 (SERPINB6), filamin A-alpha (FLNA), and EH domain containing 2 (EHD2). All were present at increased levels in CM from senescent, compared with quiescent, cells in all three replicates, consistent with the SILAC results. These factors play important roles in regulating hemostasis. For example, THBS1 is a major protein released from platelets during activation, where it stimulates platelet aggregation by blocking nitric oxide/cGMP signaling (Isenberg et al., 2008).
 
Because thrombosis is a common unwanted side effect of genotoxic cancer treatments (Ashrani et al., 2016, Huang et al., 2011, Zagar et al., 2016), we compared factors from quiescent cells and cells induced to senesce by doxorubicin (DOXO), a DNA-damaging chemotherapeutic. Elevated secretion of many SASP factors correlates with increased mRNA abundance of the respective genes (Coppé et al., 2008). Indeed, mRNAs encoding several hemostasis-related factors increased after DOXO treatment (Figure 2A). We confirmed the induction of hemostasis-related genes upon senescence in another primary human fibroblast strain, WI-38 (Figure S2A). Because blood is more likely to be exposed to senescent vascular cells, we also tested senescent human umbilical vein endothelial cells (HUVECs) for ability to produce these factors (Figure S2B). In agreement with previous work (Coppé et al., 2008), the patterns were not identical. However, the expression of several major hemostasis factors increased upon irradiation of HUVECs, similar to fibroblasts, though PLAU, PLAUR, and FLNA were each lower in senescent HUVECs. Moreover, western blots of CM from DOXO-induced senescent HCA2 cells showed increased hemostatic SASP factors relative to quiescent controls, similar to IR-induced senescence (Figure 2B). Finally, DOXO can also deplete mtDNA. We therefore tested the levels of these factors in mitochondrial dysfunction-associated senescence (MiDAS) induced by mtDNA depletion (Wiley et al., 2016). Many hemostasis-related factors were also elevated in MiDAS (Figure 2C), indicating that multiple drivers of senescence potentially affect hemostasis

 

 

 

 

 

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Also tagged with one or more of these keywords: cellular senescence, aging, homeostasis, coagulation, clotting, thrombosis, chemotherapy, sasp

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