Protective effects of PDGF-AB/BB against cellular senescence in human intervertebral disc

Reviewer #1 (Public review):

The authors, Zhang et al., demonstrate the beneficial effects of treating degenerate human primary intervertebral disc (IVD) cells with recombinant human PDGF-AB/BB on the senescence transcriptomic signatures. Utilizing a combination of degenerate cells from elderly humans and experimentally induced senescence in young, healthy IVD cells, the authors show the therapeutic effects on mRNA transcription as well as cellular processes through informatics approaches.

One notable strength of this study is the use of human primary cells and recombinant forms of human PDGF-AB/BB proteins, which increases the translational potential of these in vitro studies. The manuscript is well-written, and the informatics analyses are thorough and clearly presented.

However, in its current form, the study does not provide sufficient experimental details, and clarifications are needed. These are as follows:

(1) The source of PDGF-AB/BB proteins is not detailed.
(2) The irradiation parameters are not adequately reported - the authors should consider (PMCID: PMC5495460) for the parameters that should be reported.
(3) The criteria for young and old patient donors are not explicitly described - though from the table, one presumes the cut-off for young is 27 years old.
(4) What is the rationale for using different concentrations of PDGF-AB/BB in the degenerate cell and irradiation experiments?

There are also a number of other issues the authors could consider. First, in the title and throughout the manuscript, the effects of PDGF-AB/BB are described as protective, yet in all the experiments, PDGF-AB/BB appears to be administered following either in vivo degeneration or in vitro irradiation, where protective effects (e.g., administration prior to insult) were not tested. Therefore, the effects of PDGF-AB/BB may be more accurately described as mitigating or therapeutic rather than protective.

The authors state that the focus on NP (nucleus pulposus) cell studies is due to NP being the first site impacted during degeneration. However, this reviewer believes that this is because changes in the NP are more clinically evident (by imaging methods), despite degeneration often initiating from the AF (annulus fibrosus), e,g. through tears/microtears.

A prior study has examined the effects of X-ray irradiation on NF-kB signaling in young and aged IVDs (PMCID: PMC5495460), and the authors may wish to consider this work.

Reviewer #2 (Public review):

Summary:

This work highlights a novel role for platelet-derived growth factor (PDGF) in mitigating cellular senescence associated with age-related and painful intervertebral disc degeneration. Prior literature has demonstrated the importance of the accumulation of senescent cells in mediating many of the pathological effects associated with the degenerate disc joint such as inflammation and tissue breakdown. In this study, the authors treat clinically relevant human nucleus pulposus and annulus fibrosus cells from patients undergoing discectomy with recombinant PDGF-AB/BB for 5 days and then deep phenotyped the outcomes using bulk RNA sequencing. In addition, they irradiated healthy human disc cells which they subsequently treated with PDGF-AB/BB examining the expression of SASP-related markers and also PDGFRA receptor gene expression. Overall PDGF was able to down-regulate many senescent-associated pathways and the degenerate phenotype in IVD cells. Altered pathways were associated with neurogenesis, mechanical stimuli, metabolism, cell cycle, reactive oxygen species, and mitochondrial dysfunction. Overall the authors achieved their aims and the results by and large support their conclusions although improvements could be made to enhance the rigor of the study and findings.

Strengths:

A major strength of this study is the use of human cells from patients undergoing discectomy for disc herniation as well as access to healthy human cells. Investigating the role of PDGF regarding cellular senescence in the degenerate disc joint is a novel and underexplored area of research which is a significant contribution to the field of spine. This study highlights a potential target for addressing cellular senescence where most of the prior focus has been on senolytic drugs. Such studies have broad implications for other age-related diseases where senescence plays a major role. The use of transcriptomics and therefore an unbiased approach to investigating the role of PDGF is also considered a strength as are the follow-up studies involving irradiating healthy human disc cells and treating these cells with PDGF. The combined assessment of both nucleus pulposus and annulus fibrosus cells in the context of these studies adds to the impact.

Weaknesses:

A weakness of these studies lies in the lack of experimental details provided in the methodology including the rationale for such methods/conditions. Many details such as the specific culture models utilized, substrates, cell density, and media components are missing which impacts rigor. Such details would strengthen the manuscript and the ability to replicate and build on such work/findings. An additional weakness relates to qualitative data presented such as the B-galactosidase assay and immunofluorescence of senescence-associated proteins such as P21 and P16. Quantification of such data sets would greatly strengthen the studies and lend further support to the hypotheses. The study in its current form could be strengthened by the inclusion of mechanistic studies probing the downstream PDGF receptor-associated pathways for example specifically targeting or modulating the activity of the PDGF receptor PDGFRA including validation of the gene data for PDGFRA with protein level data to determine if the transcripts are being translated to protein. The claim that in annulus fibrosus cells, PDGF do not mediate their effects via the PDGFRA does not appear to be supported by the current data as only gene expression for the receptor was assessed with no functional or mechanistic studies being performed. Further discussion, interpretation, and direct comparison of the nucleus pulposus and annulus fibrosus data sets would be helpful for the readers. The magnitude of changes related to the effects of PDGF-BB on the S-phase in irradiated NP and AF cells between control and treated groups seem small making interpretation of these findings challenging.