Platelet Activation Is Triggered by Factors Secreted by Senescent Endothelial HMEC-1 Cells In Vitro

Round 1

Reviewer 1 Report

1. Figure 1C: The control panel is not visible. Please update with the high-resolution image.
2. Have you done cell apoptosis assay to further confirm the high concentration of Doxorubicin id harmful to the cells?
3. Why authors only checked 24 and 48 hours for IL-Beta levels in the conditioned media? The optimum hour was 72 that you used for earlier experiments? Please explain.
4. Have you measured the ROS?
5. It is very important to analyze the cell cycle? Have you done it?
6. The authors did not mention about the replicates for their in vitro study. 
7. There is no immunoblotting/immunostaining supporting the existing results. 

Author Response

Dear Reviewer,
Thanks for your comments and observations, these have allowed us to improve our work. We work hard to answer your questions and we hope the answers will solve your doubts.
Major text modifications are highlighted in yellow in the new version of the manuscript.
Best regards,

Response to the reviewers

Reviewer 1

• Figure 1C: The control panel is not visible. Please update with the high-resolution image.

Response: As requested, high resolution images were included in the new version of the manuscript.

• Have you done cell apoptosis assay to further confirm the high concentration of Doxorubicin id harmful to the cells?

Response: the reviewer’s suggestion is, of course, an important one. Before selecting low doses of Doxorubicin as our senescence inducer we assessed other alternatives, including oxygen peroxide and Palbociclib (an inhibitor of CDK4/6 that induces preferentially senescence in other cellular systems). For each one of these compounds, as well as for Doxorubicin, curves of dose-response and assays of trypan blue exclusion were carried out. From these early characterizations it was evident that non-viable cells rapidly accumulated upon exposure to increasing concentrations of oxygen peroxide and Palbociclib, as well as high doses (> 2 µM) of Doxorubicin. Based on these studies, we then became confident that 0.05 µM Doxorubicin induced senescence without compromising cell viability. Accordingly, we did not check the status of apoptosis or necrosis in our cellular system. If necessary, however, we would be willing to complement our data with experiments aimed to detect apoptosis (immunoblotting for the detection of cleaved caspase 3 or PARP). These experiments, however, will take some time as experimental work in our lab has been restricted in order to confront the current sanitary emergency imposed by the COVID-19 pandemic.

• Why authors only checked 24 and 48 hours for IL-Beta levels in the conditioned media? The optimum hour was 72 that you used for earlier experiments? Please explain.

Response: we apologize for the confusion. After showing that the mRNA expression of IL-1β in senescent cells was 28 times higher than in non-senescent cells, IL-1β protein levels in conditioned media were determined by ELISA. To this end, HMEC-1 cells were cultured for 72 hours in the presence of vehicle (0.01% DMSO) or Doxorubicin (0.05 µM). After this time, media were replaced with minimum volumes of serum- and Doxorubicin-free media and cells were cultured for an additional 24 or 48 hours. Therefore, the 24 or 28 hours corresponded to the time in which cells were cultured in the absence of serum and Doxorubicin “after” a full period of senescence induction (previous 72 hours). This was necessary because 1) the presence of serum factors would interfere with measurements of peptides in conditioned media, and 2) the presence of Doxorubicin in conditioned media could be a confounding factor in subsequent platelet functional assays. In order to make this clearer, we have incorporated a new sub-heading “Determination of IL-1β in conditioned media” in the Materials and Methods section.

• Have you measured the ROS?

Response: The reviewer’s suggestion is interesting. We know that, among several mechanisms of action of Doxorubicin, the generation of ROS is key. Indeed, Doxorubicin-associated cardiotoxicity observed in clinical settings is likely caused by local ROS production. Similarly, ROS production has been suggested as one of the main mechanisms through which Doxorubicin induces senescence in endothelial cells in vitro (De Falco et al., 2016; He at al., 2020; Kotamraju et al., 2000). However, measuring ROS (and assessing, for example, the effects of ROS scavengers on the senescent phenotype) was beyond the scope of this report. Nevertheless, we have now highlighted ROS production as a key event in the induction of senescence in endothelial cells (see the Discussion section and supporting references).

References

De Falco, Elena, et al. "Role of NOX2 in mediating doxorubicin-induced senescence in human endothelial progenitor cells." Mechanisms of Ageing and Development 159 (2016): 37-43.

He H, Wang L, Qiao Y, Zhou Q, Li H, Chen S, Yin D, Huang Q, He M. Doxorubicin Induces Endotheliotoxicity and Mitochondrial Dysfunction via ROS/eNOS/NO Pathway. Front Pharmacol. 2020 Jan 10;10:1531. doi: 10.3389/fphar.2019.01531. eCollection 2019.

Kotamraju, S., Konorev, E. A., Joseph, J., and Kalyanaraman, B. (2000). Doxorubicin induced apoptosis in endothelial cells and cardiomyocytes is ameliorated by nitrone spin traps and ebselen. Role of reactive oxygen and nitrogen species. J. Biol. Chem. 275, 33585–33592. doi: 10.1074/jbc.M003890200.

• It is very important to analyze the cell cycle? Have you done it?

Response: In order to assess induction of senescence, both proliferation and SA-beta-Gal activity were tested in Doxorubicin-treated HMEC-1 cells. In addition, the expression of p21, a cell cycle inhibitor that suppresses the G1-S cell cycle transition, was determined. Combined, these approaches strongly support cell cycle exit in our experimental system. While cell cycle profiles can be determined, it would require time and an important logistic effort (experimental work in our lab is currently restricted due to the sanitary emergency imposed by the COVID-19 pandemic).

• The authors did not mention about the replicates for their in vitro study.

Response: We apologize for the misunderstanding. In the new version of the manuscript, we have added the number of replicates in the legends of Figures 1 and 2.

• There is no immunoblotting/immunostaining supporting the existing results.

Response: In its present form, we believe our results provide enough information to support our conclusions. We could certainly complement the data with immunoblotting or immunostaining, but we are currently heavily limited by time and lab access due to sanitary restrictions.

Reviewer 2 Report

It is a well-designed in vitro study examining the effect of low concentrations of chemotherapy (Doxorubicin) on cell senescence using immortalized endothelial cells (HMEC-1). The results showed that low concentrations doxorubicin induce senescence in HMEC-1, senescent HMEC-1 cells resulted in increased production and secretion in the media of selected components of the SASP capable of inducing platelet activation and aggregation.

Major Concern:

• What is the relevance to in vivo situations to support the relevance to the role of endothelial cells in increased risk of thrombosis (platelet activation) versus impact of chemotherapy on other circulating cells that are prone to produce cytokines and chemokines such as Monocyte, platelet itself, and other circulating cells?
• What is the effect of Doxorubicin on native endothelial cells versus immortalized HMEC-1?
• What is the effect of Doxorubicin at those low concentrations on platelet functions?

Minor Comments:

• No references should be cited in the results section, it is not a discussion section
• English and grammars need to be checked.

Author Response

Dear Reviewer,
Thanks for your comments and observations, these have allowed us to improve our work. We work hard to answer your questions and we hope the answers will solve your doubts.
Major text modifications are highlighted in yellow in the new version of the manuscript.
Best regards,

Response to the reviewers

Reviewer 2

• What is the relevance to in vivo situations to support the relevance to the role of endothelial cells in increased risk of thrombosis (platelet activation) versus impact of chemotherapy on other circulating cells that are prone to produce cytokines and chemokines such as Monocyte, platelet itself, and other circulating cells?

Response: The reviewer alludes to a fundamental but largely unsolved question, namely the contribution of drug-induced senescent endothelial cells to pathological processes in which other cell types may also be involved. More generally, this is the question of how our in vitro findings inform what is occurring in vivo. We do not have an answer to this question. Our study only “suggests” a rather simple and unidirectional mechanism (from senescent endothelial cells to platelets) that might contribute to platelet activation/aggregation in the context of endothelial cells that have become senescent due to the acute effects of a drug. We certainly cannot rule out other, more complex, scenarios that may arise in vivo.

• What is the effect of Doxorubicin on native endothelial cells versus immortalized HMEC-1?

Response: The cytotoxic effects of Doxorubicin on cardiac tissue and the endothelium have already been described (Singal and Lliskovic., 1998; Kotamraju et al., 2000; Wojcik et al., 2015; Soultati et al., 2012). In these cases, the generation of ROS seems to play a key pathophysiologic role. For example, a significant increase in ROS, accompanied with mitochondrial dysfunction and apoptosis, has been reported in endothelial cells of mice treated with Doxorubicin (He et al., 2020). Moreover, the effect of 1 mM Doxorubicin on HUVECs (human umbilical vein endothelial cells) leads to a 50% decrease in cell viability due to increased apoptosis (He et al., 2020). So far, however, no reports describing the effect of low doses of Doxorubicin on HMEC-1 cells. It is worth mentioning that the concentrations of Doxorubicin used in the experiments described by He et al. (2020) are 20 times higher than those used in our work (thus explaining higher toxicities).

References

Singal, P. K., and LIiskovic, N. (1998). Doxorubicin-induced cardiomyopathy. N. Engl. J. Med. 339, 900–905. doi: 10.1056/NEJM199809243391307

Kotamraju, S., Konorev, E. A., Joseph, J., and Kalyanaraman, B. (2000). Doxorubicin induced apoptosis in endothelial cells and cardiomyocytes is ameliorated by nitrone spin traps and ebselen. Role of reactive oxygen and nitrogen species. J. Biol. Chem. 275, 33585–33592. doi: 10.1074/jbc.M003890200

Wojcik, T., Szczesny, E., and Chlopicki, S. (2015). Detrimental effects of chemotherapeutics and other drugs on the endothelium: a call for endothelial toxicity profiling. Pharmacol. Rep. 67, 811–817. doi: 10.1016/j.pharep.2015.03.022

Soultati, A., Mountzios, G., Avgerinou, C., Papaxoinis, G., Pectasides, D., Dimopoulos, M. A., et al. (2012). Endothelial vascular toxicity from chemotherapeutic agents: preclinical evidence and clinical implications. Cancer Treat Rev. 38, 473–483. doi: 10.1016/j.ctrv.2011.09.002

He H, Wang L, Qiao Y, Zhou Q, Li H, Chen S, Yin D, Huang Q, He M. Doxorubicin Induces Endotheliotoxicity and Mitochondrial Dysfunction via ROS/eNOS/NO Pathway. Front Pharmacol. 2020 Jan 10;10:1531. doi: 10.3389/fphar.2019.01531. eCollection 2019.

• What is the effect of Doxorubicin at those low concentrations on platelet functions?

Response: We agree with the reviewer that this information is relevant, particularly if we wish to extend our findings to in vivo or clinical settings (in which not only endothelial cells but other cells, including platelets, are exposed to Doxorubicin). Of note, all the assays aimed to assess platelet function were performed in the presence of “drug-free” conditioned medium. Briefly, after a 72 hour of senescence induction, media were replaced with minimum volumes of serum- and Doxorubicin-free media, and cells were cultured for an additional 24 or 48 hours. These serum- and Doxorubicin-free conditioned media were subsequently used in assays of platelet function. Importantly, we did test the effects of 0.05 µM Doxorubicin in assays of platelet aggregation that were aimed to standardized experimental conditions. The results indicated that this concentration of the drug had no effect on platelet aggregation (data not shown). Nevertheless, high concentrations of Doxorubicin (ranging from 30 to 250 mM) have been shown to induce ROS-mediated platelet toxicity or procoagulant activities (Kim et al., 2009; Kim et al., 2011; Wang et al., 2015).

References

Kim, E‐J., et al. "Doxorubicin‐induced platelet cytotoxicity: a new contributory factor for doxorubicin‐mediated thrombocytopenia." Journal of thrombosis and haemostasis 7.7 (2009): 1172-1183.

Kim, Se-Hwan, et al. "Doxorubicin-induced platelet procoagulant activities: an important clue for chemotherapy-associated thrombosis." Toxicological Sciences 124.1 (2011): 215-224

Wang, Zhicheng, et al. "Mitochondria-derived reactive oxygen species play an important role in Doxorubicin-induced platelet apoptosis." International journal of molecular sciences 16.5 (2015): 11087-11100

• No references should be cited in the results section, it is not a discussion section.

Respuesta: We thank the reviewer for the observation. We have removed citations in the results section.

• English and grammars need to be checked.

Respuesta: the manuscript was thoroughly revised for typos and grammar errors.

Round 2

Reviewer 1 Report

The authors responded to all the queries raised by the reviewer. But the manuscript needs more supporting experiments. 

Reviewer 2 Report

It is acceptable, please add the data for the effect of doxorubicin at the low concentration used instead of data not shown (why not show it) if you have it.