PONE-D-22-13587Low Grade Intravascular Hemolysis Associates with Peripheral Nerve Injury in Type 2 DiabetesPLOS ONE

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: The manuscript is well written and authors have focused on important topic of peripheral nerve injury in type 2 diabetes patients. The authors have concluded T2D was associated with low-grade IVH. Plasma absorbance may constitute a novel biomarker of peripheral neuropathy in T2D, while flow cytometry focusing on large EV may be maladapted to characterize RBC EV in T2D. Moreover, therapeutics limiting IVH or neutralizing RBC breakdown products might bolster

vasculoprotection in T2D. Spectrophotometric markers of IVH might eventually help stratify the risk of peripheral nerve damage during T2D, and identify patients who could benefit from earlier prophylaxis and IVH-targeted personalized therapy. The findings made by the authors are novel and scientifically sound.

Reviewer #2: In this manuscript Sylvain Le Jeune et al. investigated whether Type2 diabetes (T2D) promotes intravascular hemolysis (IVH). Spectrophotometric analysis of T2D plasma revealed that low grade IVH occurs in T2D, especially in T2D obese patients. They found that red blood cells (RBCs) from T2D patients have a greater potential to release heme-loaded extracellular vesicles (EVs). Circulating EVs in T2D plasma are smaller than EVs from control subjects. Because of abnormal small size of EVs, classical FACS is less appropriate to measure them in T2D plasma. They found that heme-related absorbance of T2D plasma is independently associated with peripheral sensory neuropathy, a specific microvascular complication of T2D. The authors showed that T2D RBC-derived EV triggers production of reactive oxygen species (ROS) in endothelial cells and thrombin activation in a phosphatidylserine- and heme-dependent manner.

T2D is a global health burden with huge impact. It is long known that T2D modifies hemoglobin (Hb), red blood cell (RBC) deformability and impairs hemorheology. RBC lysis is an etiopathogenic factor in many hemolytic diseases, mainly due to the pro-oxidant and pro-inflammatory actions of hemoglobin oxidation and break-down products. But the involvement of RBC lysis and Hb-derived species in T2D remained mainly unexplored.

Therefore this study is highly relevant, well-designed and provide new information.

I have only one concern: in the methods the authors write the following: „We did not attempt to specify the different forms of ferrous, ferryl and deoxy-Hb which are all expected to contribute to Abs575.” Pathological consequences of IVH are maily due to the production and actions of oxidized Hb forms. I am missing the description of Hb oxidation, and the specific pro-oxidant and pro-inflammatory actions of the different products (metHb, ferrylHb, covalently crosslinked Hb forms, free heme). I understand that measuring the concentration of these products are quite challenging and it was not the goal of the current work, but this should be discussed properly to encourage further work in this field.

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Reviewer #1: No

Reviewer #2: Yes: Viktória Jeney

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Low Grade Intravascular Hemolysis Associates with Peripheral Nerve Injury in Type 2 Diabetes

PONE-D-22-13587R1

Dear Dr. BLANC-BRUDE,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Alok Raghav, PhD

Academic Editor

PLOS ONE

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