Autotaxin May Have Lysophosphatidic Acid-Unrelated Effects on Three-Dimension (3D) Cultured Human Trabecular Meshwork (HTM) Cells

Round 1

Reviewer 1 Report

Minor Comments:

• In the abstract, the authors described results of TGFb2 and PGF2a. However, there are no figures in the manuscript showing these results. Please rectify.
• Figure legend 1 describes exposure of 3D HTM spheroids to different concentrations of LPA? Did the authors meant HTM 3D spheroids were treated with LPA at varying time points, not concentrations?
• The description in Figure legend 2 is quite confusing. “As the barrier function of LPA (200nM) untreated (NT) or treated (LPA)”. It may be better to say “2D cultured HTM monolayers were either untreated (NT) or treated with 200nM LPA. Same case in Figure legend 3 –> “non-treated control with autotaxin (NT)”. Please describe as “…spheroids were compared with non-treated control (NT).”
• Line 508, ATX is an enzyme not receptor; “confirmed the presence of ATX and LPA receptors in 3D HTM spheroids”.

Major comments

The authors made an interesting observation that ATX and LPA seems to differentially regulate the expression of several genes related to ECM, TIMPs, MMPs, LOX, HIF (1&2) and ER-stress related factors. They hypothesize that this could be attributed to ATX binding to cell surface integrins via the SMB domains. Have the authors looked at exogenous treatment with the catalytically inactive ATX enzyme (mutation at the catalytic site T210A) and compare the effects to wild type ATX enzyme? Also, LPA is broken down rapidly by lipid phosphate phosphatases. What are the expression levels LPP enzymes by HTM cells? Which LPA species is used in these experiments? Are there differences in the effects of LPA species (18:1, 18:0, 16:0 etc) on the gene expression studies?

Author Response

Dear Editor,

Thank you very much for the constructive comments concerning our manuscript; " Comparison of the drug induced effects of a selective EP2 ago-nist, Omidenepag isopropyl, with PGF2α, on TGF-β2-treated human trabecular meshwork (HTM) cells “. We carefully examined all of the comments from the Reviewer and have made a series of specific changes to our manuscript as follows;

 

During the course of preparing the 1^st draft of the manuscript and this correction, the author contribution was significantly changed. Therefore, we request that the order of authors be changed (Megumi Watanabe, Masato Furuhashi, Yuri Tsugeno,Yosuke Ida, Fumihito Hikage, Hiroshi Ohguro,) and the corresponding author is Hiroshi Ohguro. In addition, Masato Furuhashi made substantial contributions to this study, as did Megumi Watanabe. Therefore, both of these authors contributed equally.

 

Reviewer 1

Minor Comments:

1. In the abstract, the authors described results of TGFb2 and PGF2a. However, there are no figures in the manuscript showing these results. Please rectify.

Answer; Thank you for this comment. I am sorry for this. I confused these data with findings from another study using PGF2a, Therefore, the abstract was appropriately rewritten and the unnecessary material was deleted; “Abstract: Purpose: The objective of the current study was to evaluate the effects of the autotaxin (ATX)-lysophosphatidic acid (LPA) signaling axis on human trabecular meshwork (HTM), two-dimension (2D) and three-dimension (3D) cultures of HTM cells. Methods: The effects were characterized by transendothelial electrical resistance (TEER) and FITC-dextran permeability (2D), measurements of size and stiffness (3D), and the expression of several genes including extracellular matrix (ECM) molecules, their modulators, and endoplasmic reticulum (ER) stress related factors. Results: A one-day exposure to 200 nM LPA induced significant down-sizing effects of the 3D HTM spheroids, and these effects were enhanced slightly on longer exposure. The TEER and FITC-dextran permeability data indicate that LPA induced an increase in the barrier function of the 2D HTM monolayers. A one-day exposure to a 2 mg/L solution of ATX also resulted in a significant decrease in the sizes of the 3D HTM spheroids and an increase in stiffness was also observed. The gene expression of several ECMs, their regulators and ER-stress related factors by the 3D HTM spheroids were altered by both ATX or LPA but in different manners. Conclusions: The findings presented herein suggest that ATX may have additional roles toward human TM in addition to the ATX-LPA signaling axis.”.

 

2. Figure legend 1 describes exposure of 3D HTM spheroids to different concentrations of LPA? Did the authors meant HTM 3D spheroids were treated with LPA at varying time points, not concentrations?

Answer; Thank you for this comment. It is true that treated HTM 3D spheroids with fixed concentrations of LPA (200 nM) at varying time points. Therefore, the figure legend was rewritten to read; “During the 6-day culture of the 3D HTM spheroids, 200 nM LPA was exposed during Day 1 through 6 (5D LPA), Day 3 through 6 (3D LPA) or Day 5 through 6 (1D LPA). The mean sizes (panel A) and stiffness (panel B) of the HTM 3D spheroids under three different exposure conditions of fixed concentration of LPA (200 nM) were compared with non-treated controls with LPA (NT). Data are presented as the arithmetic mean ± standard error of the mean (SEM). *P<0.05, **P<0.01, ***P<0.005 (ANOVA followed by a Tukey’s multiple comparison test).”.

3. The description in Figure legend 2 is quite confusing. “As the barrier function of LPA (200nM) untreated (NT) or treated (LPA)”. It may be better to say “2D cultured HTM monolayers were either untreated (NT) or treated with 200nM LPA. Same case in Figure legend 3 –> “non-treated control with autotaxin (NT)”. Please describe as “…spheroids were compared with non-treated control (NT).”

Answer; Thank you so much for this comment. As suggested, the legend for Fig. 2 was rewritten and now reads as follows; “Among conditions in which the 2D cultured HTM monolayers were either untreated (NT) or treated with 200nM LPA, TEER (Ωcm2) values were measured at different time points (0, 0.5, 1, 3, 6 and 24 hours) and plotted (panel A). Alternatively, 2D cultured HTM monolayers were either untreated (NT) or treated with 200 nM LPA for 24 hours, and was FITC-dextran permeability was measured and the data plotted in panel B. All experiments were performed in triplicate using fresh preparations (n=4). Data are presented as the arithmetic mean ± standard error of the mean (SEM). **P<0.01, ***P<0.005 (ANOVA followed by a Tukey’s multiple comparison test).”, and Fig. 3 legend was rewritten; “During the 6-day culture of the 3D HTM spheroids, they were exposed to a 2 mg/L solution of ATX during Day 5 through 6, and the mean sizes (µm2, panel A) and stiffness (µN/µm, panel B) of the HTM 3D spheroids were compared with non-treated control (NT). Data are presented as the arithmetic mean ± standard error of the mean (SEM). ***P<0.005 (ANOVA followed by a Tukey’s multiple comparison test).”.

4. Line 508, ATX is an enzyme not receptor; “confirmed the presence of ATX and LPA receptors in 3D HTM spheroids”.

Answer; Thank you for this comment. As suggested, corresponding sentence and figure legend of supplemental figure 1 were changed appropriately.

Major comments

5. The authors made an interesting observation that ATX and LPA seems to differentially regulate the expression of several genes related to ECM, TIMPs, MMPs, LOX, HIF (1&2) and ER-stress related factors. They hypothesize that this could be attributed to ATX binding to cell surface integrins via the SMB domains. Have the authors looked at exogenous treatment with the catalytically inactive ATX enzyme (mutation at the catalytic site T210A) and compare the effects to wild type ATX enzyme? Also, LPA is broken down rapidly by lipid phosphate phosphatases. What are the expression levels LPP enzymes by HTM cells? Which LPA species is used in these experiments? Are there differences in the effects of LPA species (18:1, 18:0, 16:0 etc) on the gene expression studies?

Answer; Thank you so much for very constructive comments regarding several ideas for further investigating our current hypothesis that this could be attributed to ATX binding to cell surface integrins via the SMB domains. Nevertheless, as of this writing, such experiments have not done yet, but we plan to do these proposed investigations as our next project. Therefore, this information is included in the last paragraph of discussion “In conclusion, based upon our current study, we hypothesize that unrelated effects of ATX with LPA could be attributed to ATX binding to cell surface integrins via the SMB domains. However, as of writing this, to prove this hypothesis, we will be required for further investigations, such as 1) effects of exogenous treatment with the catalytically inactive ATX enzyme (mutation at the catalytic site T210A), 2) effects of phosphatase to break down LPA, 3) roles of several species of LPAs (18:1, 18:0, 16:0 etc) within HTM cells, and others. Therefore, those are our next future projects.”.

 

Author Response File: Author Response.docx

Reviewer 2 Report

In this paper the authors reported the in vitro effects of   the autotaxin (ATX)-lysophosphatidic acid (LPA) signaling axis   on 2D and 3 D human trabecular meshwork (HTM). 

For this aim, the authors selected several relevant  end points , but  some were analysed  on 2D culture and other on 3D spheroids. So it is  difficult to consider the real predictivity of the reported results. As known, the results obtained in a 2D model can be very different from those from  a 3D culture that we know to be more realistic than the condition in vivo.

To give greater emphasis to the purpose of the paper and to better analyze the ATX-LPA signaling axis, and  in order to provide scientifically useful information, the authors have to analyze the same end points on both 2D and 3D models.
 

Author Response

Dear Editor,

Thank you very much for the constructive comments concerning our manuscript; " Comparison of the drug induced effects of a selective EP2 ago-nist, Omidenepag isopropyl, with PGF2α, on TGF-β2-treated human trabecular meshwork (HTM) cells “. We carefully examined all of the comments from the Reviewer and have made a series of specific changes to our manuscript as follows;

 

During the course of preparing the 1^st draft of the manuscript and this correction, the author contribution was significantly changed. Therefore, we request that the order of authors be changed (Megumi Watanabe, Masato Furuhashi, Yuri Tsugeno,Yosuke Ida, Fumihito Hikage, Hiroshi Ohguro,) and the corresponding author is Hiroshi Ohguro. In addition, Masato Furuhashi made substantial contributions to this study, as did Megumi Watanabe. Therefore, both of these authors contributed equally.

Reviewer 2

1. In this paper the authors reported the in vitro effects of the autotaxin (ATX)-lysophosphatidic acid (LPA) signaling axis on 2D and 3 D human trabecular meshwork (HTM). For this aim, the authors selected several relevant end points, but some were analysed on 2D culture and other on 3D spheroids. So it is difficult to consider the real predictivity of the reported results. As known, the results obtained in a 2D model can be very different from those from a 3D culture that we know to be more realistic than the condition in vivo. To give greater emphasis to the purpose of the paper and to better analyze the ATX-LPA signaling axis, and in rder to provide scientifically useful information, the authors have to analyze the same end points on both 2D and 3D models.

Answer; Thank you for this question and the proposal to analyze the same end points of both 2D and 3D models. I agree with this proposal. However, the effects of ATX and LPA on 2D HTM cells have already characterized by Honjyo and co-workers (J Biomed Sci. 2021 Jun 17;28(1):47. doi: 10.1186/s12929-021-00745-3., Mol Vis. 2021 Jan 20;27:61-77. eCollection 2021., Sci Rep. 2021 Jan 12;11(1):747. doi: 10.1038/s41598-020-80736-w., Sci Rep. 2020 Apr 14;10(1):6265. doi: 10.1038/s41598-020-63284-1., Invest Ophthalmol Vis Sci. 2018 Jan 1;59(1):21-30. doi: 10.1167/iovs.17-22807.) and Ho et al. (Invest Ophthalmol Vis Sci. 2018 Apr 1;59(5):1969-1984. doi: 10.1167/iovs.17-23702.). However, between these two groups, several different observations such as optimum ATX and LPA concentrations within AH specimens obtained from patients with glaucoma. In our current study, our goal was to elucidate new insights into the ATX-LPA signaling axis within HTM cells. Therefore, rather than repeating experiments using 2D cultured models, we mainly studied this issue using 3D spheroid HTM cells because this type of study has not yet been done. As the result, we obtained some quite new information showing that ATX may have additional roles toward human TM in addition to the ATX-LPA signaling axis, and based upon these, we hypothesized that this could be attributed to ATX binding to cell surface integrins via the SMB domains. To support this, we carried out several investigations using 2D and 3D cultured models from several cell sources, such as human orbital fibroblast, human conjunctival fibroblast, mouse preadipocyte and others, and the findings indicate that 2D and 3D culture models reveal quite different cell biological aspects, including the mRNA expression of ECM molecules, ECM regulatory genes, ER-stress related genes. Therefore, even if we analyzed the same endpoints at 2D as 3D, those results may be different from data collected in our previous studies in which different sources of cells were used, and the results showed that the effects were clearly different between 2D and 3D. However, at minimum, we also repeat similar experiments of TEER and FITC-dextran permeability of the 2D cultured HTM monolayer with Honjyo’s group to study LPA effects, and obtained almost identical results which serve to confirm the rapid effects by LPA. This information is important in terms of the design of an experimental protocol for the exposure of LPA and ATX to the 3D HTM spheroids. Therefore, this information was included last two sentences within the 2^nd paragraph of the Results section of the paper; “In fact, rapid effects by LPA on these analyses were also reported by Nakamura et al.[26]. Therefore, these results and the above information related to 3D spheroid sizes provide a scientific rationale for the design of subsequent experimental protocols for LPA or ATX periods of exposure on the stiffness measurements and mRNA expression analyses of the 3D HTM spheroids.”.     

 

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The authors have reviewed the article taking into account the comments of the referees.