Antioxidant Glutathione Analogues UPF1 and UPF17 Modulate the Expression of Enzymes Involved in the Pathophysiology of Chronic Obstructive Pulmonary Disease

Round 1

Reviewer 1 Report

A well designed and investigational work on synthetic GSH analogue peptides as a potential COPD therapy against excessive oxidative stress, which on further experiments, over a large cohort, may be effectively applied for similar disease conditions. Authors, therefore, deserve deep sense of appreciation. However, following minor suggestions / modifications may be incorporated

While recruiting the COPD patients (AE-COPD & stable COPD) for this study, the authors must have followed exclusion criteria for cardiovascular and other metabolic diseases. The same may be reflected in material & methods under 2.1 Study Individuals.

Chronic Obstructive Pulmonary Disease has been abbreviated as COPD in the abstract itself, therefore such repetition may be avoided in the succeeding paragraphs/ text

Table -2 line 161 : It is UPF17 instead of UPF 7, authors are advised to incorporate correction.

Table-2 & 3  : Unit of measurement of Pro and Anti-inflammatory enzymes and GSH metabolism enzymes should  be given for better understanding of the readers.

Author Response

We thank the reviewer for the valuable time that has been taken in thorough analysis of the manuscript and making useful recommendations for improvement of our manuscript “Antioxidant Glutathione Analogues UPF1 and UPF17 Modulate the Expression of Enzymes Involved in the Pathophysiology of COPD”.

We have provided point-by point answers to the comments and revised our manuscript in the light of all suggestions and requirements. Modifications in the manuscript have been marked in red. English language revisions are marked in blue. Please see the attachment.

Reviewer #1

A well designed and investigational work on synthetic GSH analogue peptides as a potential COPD therapy against excessive oxidative stress, which on further experiments, over a large cohort, may be effectively applied for similar disease conditions. Authors, therefore, deserve deep sense of appreciation. However, following minor suggestions / modifications may be incorporated

1. While recruiting the COPD patients (AE-COPD & stable COPD) for this study, the authors must have followed exclusion criteria for cardiovascular and other metabolic diseases. The same may be reflected in material & methods under 2.1 Study Individuals.

Answer: Thank you! We definitively agree that highlighting exclusion criteria are important for a study like this. We have supplemented the main document in Methods 2.1 Study Individuals, where exclusion criteria have now been mentioned: “Exclusion criteria for patients with both stable COPD and AE-COPD included mechanical ventilation, hospitalization into an intensive care unit, unstable coronary artery disease and presence of active cancer.” Please find this supplementation at page 3, lines 83-85

We also added a description in the Methods 2.1 Study Individuals section “All study individuals were required to be without upper and lower respiratory tract infections (including acute bronchitis, bronchiolitis and pneumonia) during the study and for at least 4 weeks before the study with the exception of patients with AE-COPD in whom acute respiratory infections other than pneumonia by were allowed provided these infections had a causative relation to the current COPD exacerbation as judged by the treating pulmonary physician.” Please look at page 3, lines 89-94.

2. Chronic Obstructive Pulmonary Disease has been abbreviated as COPD in the abstract itself, therefore such repetition may be avoided in the succeeding paragraphs/ text

Answer: Thank you for reading our manuscript with great care. However, despite the abbreviation “COPD” has been explained after first use in abstract, we need to follow the guidelines laid down by the Journal (CIMB). Therefore, we need to repeat the spelling wherever it appears first again: in the main text and in headers and footers of tables and figures etc.

(From CIMB Instructions for Authors: “Acronyms/Abbreviations/Initialisms should be defined the first time they appear in each of three sections: the abstract; the main text; the first figure or table. When defined for the first time, the acronym/abbreviation/initialism should be added in parentheses after the written-out form.”)

As a result, we have removed the repeating explanation of COPD, GOLD and GSH from Table 2, Table 3 and Figure 2. Look at page 5, lines 162, 164, 165; page 6, lines 184, 186; page 9, line 224.

3. Table -2 line 161 : It is UPF17 instead of UPF 7, authors are advised to incorporate correction.

Answer: We are extremely sorry for the typo. UPF7 has been replaced with UPF17 in Table 2. Table 3, Figure 1, Figure 2 and Supplementary Figure 1 headers. Look at page 5, line 162; page 6, line 184; page 8, line 201; page 9, line 224; page 13, line 378.  

4. Table-2 & 3  : Unit of measurement of Pro and Anti-inflammatory enzymes and GSH metabolism enzymes should  be given for better understanding of the readers.

Answer: This is a very valuable point for the readers indeed. However, the mRNA expression levels of inflammatory and GSH metabolism enzymes do not have a unit of measurement of its own and is referred to as an amount of PCR product. mRNA expression level is based on real-time PCR efficiencies (E) and the crossing point (CP) difference (Δ) of the enzyme under study versus a control enzyme (DCP_{control – sample}).

Relative mRNA expressions were analyzed by RT-qPCR and ΔΔCt calculations were used. To normalize the results, endogenous hypoxanthine phosphoribosyltransferase-1 (HPRT-1) was used as a housekeeping gene for mRNA. HPRT-1 is adequately expressed and shows minimal variability in expression between samples and under different experimental conditions in PBMC.

Methods part has been updated to explain it better: “The comparative Ct method (DCt value) was used for quantification of mRNA, where the amount of target transcript was normalized to the level of endogenous housekeeper gene HPRT-1“.  Page 4, lines 126-128.

Reviewer 2 Report

The study addresses a significant gap in understanding the role of antioxidants in modulating systemic consequences of COPD, shedding light on potential therapeutic avenues.

The study is comprehensive, detailed and well-structured. The discussion effectively interprets authors' findings in the context of existing literature, discussing potential implications for COPD pathogenesis and therapeutic strategies.

However, several major concerns need to be addressed before ensuring publication of the manuscript:

1. -The study acknowledges limitations regarding small cohort sizes in certain subgroups, potentially limiting the generalizability of the findings. Addressing this concern may involve increasing the sample size or collaborating with multiple centers to validate the results across diverse populations. Please address this point in discussion.

2.  

3. -The study acknowledges that the treatment part was performed ex vivo, which may not fully recapitulate the in vivo physiological conditions. Future studies should aim to validate these findings in vivo to better understand the clinical relevance and potential therapeutic efficacy of UPF1 and UPF17. Please address this point in discussion.

4.  

5. -While mRNA expression analysis provides valuable insights, confirming the findings through functional proteomics or enzymatic analysis would strengthen the reliability and significance of the results. Please address this limitation.

6.  

7. - While the study highlights the potential of UPF1 and UPF17 as lead compounds for COPD therapy, the discussion should provide more nuanced insights into the clinical relevance and translational potential of these findings.

- I found several english errors throughout the manuscript. Please have a deep language revision.

Moderate revision required. 

Author Response

We thank the reviewer for the valuable time that has been taken in thorough analysis of the manuscript and making useful recommendations for improvement of our manuscript “Antioxidant Glutathione Analogues UPF1 and UPF17 Modulate the Expression of Enzymes Involved in the Pathophysiology of COPD”.

We have provided point-by point answers to the comments and revised our manuscript in the light of all suggestions and requirements. Modifications in the manuscript are marked in red. English language revisions are marked in blue. Please see the attachment.

Reviewer #2

The study addresses a significant gap in understanding the role of antioxidants in modulating systemic consequences of COPD, shedding light on potential therapeutic avenues.

The study is comprehensive, detailed and well-structured. The discussion effectively interprets authors' findings in the context of existing literature, discussing potential implications for COPD pathogenesis and therapeutic strategies.

However, several major concerns need to be addressed before ensuring publication of the manuscript:

1. The study acknowledges limitations regarding small cohort sizes in certain subgroups, potentially limiting the generalizability of the findings. Addressing this concern may involve increasing the sample size or collaborating with multiple centers to validate the results across diverse populations. Please address this point in discussion.

Answer: Thank you for pointing out this issue. Small cohort sizes in certain subgroups and the study being a single-centre study could indeed reduce the power of such a study. We completely agree that collaborating with multiple centers can provide larger sample sizes among other benefits including giving us diverse population coverage and increased generalizability. On the other hand, it could also compromise the validity of the study due to methodological and statistical challenges (Das MK. Multicenter Studies: Relevance, Design and Implementation. Res Methodol. 2022 Jan; 59:571–579). As this was the first step to see if glutathione analogues UPF1 and UPF17 have any effect on antioxidant, pro- and anti-inflammatory enzymes in PBMC from patients with COPD, single-center study was deemed sufficient to show that further studies with larger cohort sizes from different hospitals are justified to provide us with better understanding of the effects of UPF1 and UPF17.

A sentence was also added to discuss the issue of this study being a single-center study: “Collaborating with multiple centers would have provided us with more diverse population coverage and increased generalizability.” (Please refer to page 12, lines 336-338).

2. The study acknowledges that the treatment part was performed ex vivo, which may not fully recapitulate the in vivo physiological conditions. Future studies should aim to validate these findings in vivo to better understand the clinical relevance and potential therapeutic efficacy of UPF1 and UPF17. Please address this point in discussion

Answer: Thank you for this valuable annotation. UPF enzymes have been previously studied in many different cell types, including in a human erythroleukemia cells K562 (Kairane C, et al. Diverse Effects of Glutathione and UPF Peptides on Antioxidant Defense System in Human Erythroleukemia Cells K562. Int J Pept. 2012; 2012: 124163), human brain mitochondrial fraction (Kairane C, The Effects of Different Antioxidants on the Activity of Cerebrocortical MnSOD and Na,K-ATPase from post mortem Alzheimer’s Disease and Age-matched Normal Brains, Current Alzheimer Research, 2014, 11, 79-85), isolated rat heart (Kals J, et al Antioxidant UPF1 attenuates myocardial stunning in isolated rat hearts. 2008, International Journal of Cardiology. 125(1):133-5), rat cerebellar granule cells (Kazarjan J, Investigation of the surfactant type and concentration effect on the retention factors of glutathione and its analogues by micellar electrokinetic chromatography. J Sep Sci. 2015 Oct;38(19):3461-8). This current study was the first to measure UPF1 and UPF17 effect in freshly isolated PBMC of COPD patients, non-obstructive smokers and non-smokers. PBMC cells were isolated and treated with UPF peptides no more than 2h after phlebotomy.

Ex vivo studies definitely set limitations for interpreting UPF1 and UPF17 clinical relevance and potential therapeutic efficacy. Further studies in vivo are necessary, but before we can move on to in vivo studies, the exact mechanism of UPF-s should be further studied. Due to us not knowing the exact mechanisms, we have added a sentence in our conclusion: “Therefore, these peptides cannot possibly be used directly as themselves, but could nevertheless serve as a lead for designing potential COPD therapies against excessive OS.”  Please refer to page 12, lines 348-350.

A sentence was added to the discussion part: “Future studies in vivo could help better understand the clinical relevance and potential therapeutic efficacy of UPF1 and UPF17.”  (Page 12, lines 339-341)

3. While mRNA expression analysis provides valuable insights, confirming the findings through functional proteomics or enzymatic analysis would strengthen the reliability and significance of the results. Please address this limitation

Answer: That is a great observation. Indeed, as we do not support our findings through functional proteomics or enzymatic analysis. We agree that the exact functions and abilities of UPF1 and UPF17 need to be further investigated, in addition cytokines involvement should be measured to support and increase the significance of our findings.

This has been mentioned in page 12, lines 341-343: “The gene expression data should be confirmed by functional proteomics or enzymatic analysis, to elucidate the levels of the cytokines involved in the respective cascades or clarify the ability to scavenge ROS.”

4. While the study highlights the potential of UPF1 and UPF17 as lead compounds for COPD therapy, the discussion should provide more nuanced insights into the clinical relevance and translational potential of these findings

Answer: We thank this reviewer for picking up this issue. In parallel with agreeing with this, we also want to emphasize the relevance of UPF peptides’ ability to increase the level of intracellular GSH and due to that, its roles and functions in the fight against OS. Although the exact mechanisms of the effect of UPF1 and UPF17 remains unclear, what is known by now is that UPF1 modulates the activity of the G-proteins in the brain tissue (Karelson E, Mahlapuu R, Zilmer M, Soomets U, Bogdanovic N, Langel U. Possible signaling by glutathione and its novel analogue through potent stimulation of fontocortical G proteins in normal aging and in Alzheimer's disease. Ann N Y Acad Sci 2002;973:537–40) and can act as a scavenger (Põder P, Zilmer M, Starkopf J, et al. An antioxidant tetrapeptide UPF1 in rats has a neuroprotective effect in transient global brain ischemia. Neurosci Lett 2004;370:45–50) or increase the level of intracellular glutathione (GSH) via Nrf2 (Hansen M, Porosk R, Mahlapuu R, et al. GSH Synthetic Analogue O-Methyl-L-Tyrosinylglutathione Regulates Nrf2-Mediated Expression of GCLc and GCLm. J Chem. 2019;2019:1-8). Collectively, the biggest potential of UPF peptides is to increase the fight against ROS and OS (Altraja S, Mahlapuu R, Soomets U, et al. Cigarette smoke-induced differential regulation of glutathione metabolism in bronchial epithelial cells is balanced by an antioxidant tetrapeptide UPF1. Exp Toxicol Pathol. 2013 Sep;65(6):711-7).

This has been added to the discussion: “Altraja et al. showed that UPF1 is able to entirely restore the intracellular GSH levels in human bronchial epithelial cells under oxidative stress caused by CS condensate [48].” (Page 11, lines 317-319)

The reference list has also been updated appropriately (ref nr 48 - Altraja S, Mahlapuu R, Soomets U, et al. Cigarette smoke-induced differential regulation of glutathione metabolism in bronchial epithelial cells is balanced by an antioxidant tetrapeptide UPF1. Exp Toxicol Pathol. 2013 Sep;65(6):711-7).

5. I found several english errors throughout the manuscript. Please have a deep language revision.

Answer: The manuscript has been revised for English language. All language corrections have been marked in blue.

Round 2

Reviewer 2 Report

Authors fully replied to my comments. Ok to accept for me now.