Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study titled “NAD⁺ Enhancer Nicotinamide Riboside Alters Extracellular Purine Metabolism in Human Endothelial Cells” shows the effects of nicotinamide riboside (NR) on endothelial cell purine metabolism and leads to increased NAD+. The authors link the changes in NAD+ to the expression of adenosine cleavage ectoenzymes CD39, CD73 and eADA. NR treatment increased C39 and CD73 but reduced eADA effectively leading to complete ATP hydrolysis and reduced immune cell adhesion to the endothelial cells. This effect is reported as a potential mechanism through which NR could reduce inflammatory cell extravasation and reduce inflammatory load.

Figure 1:

In the caption, 1c is listed as NR and 1d as NAM but this order is reversed in the figure. This should be changed to reflect the caption.

The comparisons shown on the graphs are not uniform. The authors should make them uniform across groups and represent non-significant values with an ‘ns’. These comparisons should then be reflected in the results section clearly indicating the effect of NR treatment on changes in intracellular and extracellular downstream metabolites.

 

Figure 2:

In Figure 2c, the change in adenylate energy charge under forodesine or forodesine + NBTI treatment showed a decrease. However, the net change between the untreated and inhibitor combination is rather small and within the limits of AEC. The authors should include a strong positive control like oligomycin to trigger rapid loss of AEC and compare the effects of NR and the listed inhibitors.

Similar to Figure 1, the statistical comparisons are inconsistent across graphs. The authors should make the comparisons consistent with the claimed results.

 

Figures 3 and 4:

These two figures can be merged into one since their respective body text is confined to one paragraph.

Since CD39 and CD73 are implicated in the hydrolysis of ATP and AMP respectively, the authors should also show the changes in ATP hydrolysis using a relevant CD39 inhibitor similar to CD73 inhibitor experiments performed in figure 4.

The representative images provided for Figure 3b and Figure 4b do not have enough resolution and it is difficult to see the differences in control and +NR groups especially since CD39 and CD73 are membrane proteins. The scale bars are too small; these should be replaced with a larger font for better visibility. In the listed antibodies, these proteins can be reliably shown via western blots. The authors should supplement the immunofluorescence images with respective western blots to confirm the increase in these receptors in response to NR.

Figure 5:

This figure has a similar problem as figures 3 and 4. The immunofluorescence images are of low resolution, and it is difficult to clearly visualize the changes brought about by NR treatment. These images should be replaced with higher resolution images. eADA antibody is also listed to work on western blots. Using a representative western blot to show these changes would add more weight to the claim that NR reduces eADA expression and activity.

Figures 6, 7, 8:

In these three figures, the effect of NR on the adhesion properties of Jurkat, THP-1 and human platelets to endothelial cells were measured. The immune cells were tracked using fluorescein labeling. The representative images pose an issue wherein we cannot see the underlying endothelial cells. To better represent the interactions between immune cells and endothelial cells, they should be counter stained with a red membrane stain like wheat germ agglutinin (WGA) to clearly differentiate between the two cell types.

Next, the authors only used AOPCP as a control which is an inhibitor of CD73. The authors should include both AOPCP and a CD39 inhibitor to show the effects of these individual receptors and their effect on adhesion. Figures 6 and 7 include AOPCP but Figure 8 does not. The authors should make their comparisons consistent across figures. Furthermore, negative (no immune cells) and positive (activated immune cells via LPS or TNFa) controls should also be included to validate binding.

Overall, the authors provide evidence to show that nicotinamide riboside (NR) treatment alters endothelial NAD+ levels and leads to an accumulation of nicotinamide derived metabolites. They show that NR treatment increases CD39 and CD73 levels which are membrane receptors responsible for the hydrolysis of ATP and AMP respectively. They also show that eADA levels are decreased with NR leading to changes in immune cell adhesion to endothelial cells. They show that T cells (Jurkat), monocyte/macrophages (THP-1) and isolated human platelets change their adhesion properties upon NR treatment. The lowered adhesion potential leads to a reduced inflammatory state owing to the increased activities of both CD39 and CD73 thereby hydrolyzing ATP and AMP.

The study informs the effects of nicotinamide riboside on endothelial cells and its potential in reducing inflammation. However, there are several issues that need to be addressed to support their overall claim.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript studies the effects of nicotinamide riboside (NR) on intracellular NAD⁺ levels, extracellular purine metabolism, and immune cell adhesion in human microvascular endothelial cells (HMEC-1). While the manuscript presents several novel points, it has several research limitations as follows:

 

Major concerns:

1. Superficial mechanism exploration without direct verification

The study only infers that NR activates SIRT1 by elevating intracellular NAD⁺, which in turn regulates HIF-1α/NF-κB to affect ectoenzyme expression. However, it lacks direct experimental verification of this pathway through SIRT1 inhibitors/overexpression or HIF-1α/NF-κB knockdown. The proposed mechanism remains only a correlative speculation without causal evidence.

 

2. Single cell line model and lack of in vivo validation

The entire study only uses the immortalized HMEC-1 cell line, without employing primary human microvascular endothelial cells or validating the vascular protective effect of NR in animal models (e.g., atherosclerotic mice). Thus, the clinical relevance of the results are limited.

 

3. Insufficient analysis of mitochondrial and glycolytic functions

Although the study mentions that endothelial cells rely on glycolysis for energy supply and existing research has shown that NR improves mitochondrial function, it only reflects the energy status through adenine nucleotide detection, without directly measuring indicators such as glycolytic rate, mitochondrial respiratory function, and ROS levels. The specific regulatory effect of NR on the energy metabolism of endothelial cells remains unclear.

 

4. Lack of correlative validation with clinical samples

No clinical samples (e.g., serum from healthy individuals and atherosclerotic patients) were included to verify the effects of NR on human ectoenzyme activity, adenosine levels, and immune cell adhesion.

 

Minor flaws:

1. There are annotation errors in some figure legends (e.g., Figure 4c is incorrectly labeled as CD39 fluorescence quantification but actually represents CD73). Only P-values are marked for some statistical results without specifying the exact test statistics (e.g., t-value/F-value).

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript “NAD⁺ Enhancer Nicotinamide Riboside Alters Extracellular Purine Metabolism in Human Endothelial Cells” investigates the effects of nicotinamide riboside (NR) on intracellular NAD⁺ levels, extracellular purine metabolism, and immune cell adhesion in HMEC-1 endothelial cells. The topic is timely and relevant, and the experimental approach is generally sound. However, several conceptual, methodological, and interpretative weaknesses substantially limit the impact and translational relevance of the study. Addressing the points below would significantly improve the quality and robustness of the manuscript.

 

Major comments

1. The Introduction lacks sufficient critical framing and biological depth.
   While the background on NAD⁺ metabolism is extensive, the introduction is largely descriptive and does not sufficiently define a clear mechanistic or hypothesis-driven gap.
   The authors should more clearly articulate why extracellular purine metabolism is expected to be modulated by NR, and why this question is biologically and clinically relevant beyond descriptive observations.
   In particular, the link between intracellular NAD⁺ boosting and regulation of ecto-enzymes (CD39/CD73/eADA) is introduced too late and should be anticipated earlier.
2. The study remains largely correlative and does not establish causality.
   Although NR treatment increases NAD⁺ and alters ectoenzyme expression/activity, no direct mechanistic experiments are provided.
   The authors should demonstrate whether the observed effects on CD39/CD73/eADA are causally dependent on NAD⁺ elevation (e.g., by NAD⁺ depletion, NRK inhibition, or SIRT1 modulation).
   Without such experiments, the proposed mechanistic model in Figure 9 remains speculative.
3. The reliance on a single immortalized endothelial cell line limits generalizability.
   All experiments were performed exclusively in HMEC-1 cells, which is unacceptable.
   Primary human endothelial cells (e.g., HUVECs or microvascular ECs) must be included to validate the findings. Additional experiments are requested.
   
   
4. The extracellular adenosine hypothesis is not directly tested.
   Reduced immune cell and platelet adhesion is attributed to increased adenosine formation. However, adenosine levels were not directly measured during adhesion assays.
   Direct quantification of extracellular adenosine under adhesion assay conditions is necessary.
   Alternatively, pharmacological blockade of A2A/A2B receptors should be used to confirm adenosine-dependent effects.
5. The discussion overinterprets metabolic effects without functional validation.
   The manuscript suggests that NR may alter glycolysis and mitochondrial function in endothelial cells. However, no functional metabolic assays were performed.
   Claims regarding metabolic shifts should be toned down or supported by Seahorse analysis, lactate production, or mitochondrial respiration assays.
6. The translational relevance is insufficiently developed.
   While clinical trials of NR are cited, the implications of altered extracellular purine metabolism for vascular disease, inflammation, or thrombosis are not rigorously discussed.
   The authors should more clearly define how their in vitro findings could translate to pathological settings such as atherosclerosis, ischemia, or chronic inflammation.
7. Several typographical and formatting issues should be corrected throughout the manuscript.
   Examples include inconsistent spacing, unit formatting (e.g., µM, µL), and occasional grammatical errors.
   A thorough language revision is strongly recommended.
8. Figure presentation could be improved.
   Some figures are overly dense and difficult to interpret.
   Axes labels, statistical annotations, and legends should be clarified for improved readability.
9. Statistical reporting lacks full transparency.
   Exact p-values should be provided where possible instead of only significance thresholds.
   The number of biological versus technical replicates should be explicitly stated for each experiment.
10. Ethical considerations for platelet isolation should be clarified.
    The manuscript should explicitly state whether informed consent was obtained and whether the protocol was approved by an institutional ethics committee.

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

None