Impairment of an Endothelial NAD+-H2S Signaling Network Is a Reversible Cause of Vascular Aging
Authors: Lindsay E Wu
Abhirup Das
Eric O Williams
Catherine Li
Zolt Arany
Michael B Schultz
Leonard Guarente
Tzong-Tyng Hung
Jose Humberto Treviño-Villarreal
Lynn-Jee Kim
David A Sinclair
James R Mitchell
Sanket Joshi
Nigel Turner
Yuancheng Lu
Myung-Jin Kang
Alban Longchamp
Brendan Lee
Michael S Bonkowski
Brenna Osborne
George X Huang
Masaki Igarashi
Journal: Cell (2018)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: NAD(+); aging; angiogenesis; endurance; exercise
Affiliations: Paul F. Glenn Center for the Biological Mechanisms of Aging, Department of Genetics, Harvard Medical School, Boston, MA 02115, USA; Laboratory for Ageing Research, Department of Pharmacology, School of Medical Sciences, The University of New South Wales, Sydney, NSW 2052, Australia; Paul F. Glenn Center for Science of Aging Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA., Paul F. Glenn Center for the Biological Mechanisms of Aging, Department of Genetics, Harvard Medical School, Boston, MA 02115, USA; Cardiovascular Institute, Perelman School of Medicine, University of Pennsylvania, 3400 Civic Center Boulevard, Bldg. 421, Philadelphia, PA 19104, USA., Paul F. Glenn Center for the Biological Mechanisms of Aging, Department of Genetics, Harvard Medical School, Boston, MA 02115, USA., Department of Genetics and Complex Diseases, Harvard T.H. Chan School of Public Health, Boston, MA, USA., Laboratory for Ageing Research, Department of Pharmacology, School of Medical Sciences, The University of New South Wales, Sydney, NSW 2052, Australia., Mitochondrial Bioenergetics Laboratory, Department of Pharmacology, School of Medical Sciences, The University of New South Wales, Sydney, NSW 2052, Australia., Biological Resources Imaging Laboratory, Mark Wainwright Analytical Centre, The University of New South Wales, Sydney, NSW 2052, Australia., Paul F. Glenn Center for Science of Aging Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA., Cardiovascular Institute, Perelman School of Medicine, University of Pennsylvania, 3400 Civic Center Boulevard, Bldg. 421, Philadelphia, PA 19104, USA. Electronic address: ., Paul F. Glenn Center for Science of Aging Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Electronic address: ., Paul F. Glenn Center for the Biological Mechanisms of Aging, Department of Genetics, Harvard Medical School, Boston, MA 02115, USA; Laboratory for Ageing Research, Department of Pharmacology, School of Medical Sciences, The University of New South Wales, Sydney, NSW 2052, Australia. Electronic address: .

Abstract:  A decline in capillary density and blood flow with age is a major cause of mortality and morbidity. Understanding why this occurs is key to future gains in human health. NAD precursors reverse aspects of aging, in part, by activating sirtuin deacylases (SIRT1-SIRT7) that mediate the benefits of exercise and dietary restriction (DR). We show that SIRT1 in endothelial cells is a key mediator of pro-angiogenic signals secreted from myocytes. Treatment of mice with the NAD+ booster nicotinamide mononucleotide (NMN) improves blood flow and increases endurance in elderly mice by promoting SIRT1-dependent increases in capillary density, an effect augmented by exercise or increasing the levels of hydrogen sulfide (H2S), a DR mimetic and regulator of endothelial NAD+ levels. These findings have implications for improving blood flow to organs and tissues, increasing human performance, and reestablishing a virtuous cycle of mobility in the elderly. 

Retracted: No

PubPeer comments:
 The Tubulin in the lungs in Fig. 2C resembles the Tubulin in the Thymus in Fig. S2, if it is flipped horizontally and rotated a touch. ![file] , 
 Eagle eye Carlina. Looks like more than a mere resemblance. The notch in the second-from-left tubulin band looks identical in both blots, making it likely that thymus and lung tubulin blots are duplicates flipped horizontally. , 
 The authors identified the error immediately after publication and sent a correction to the journal on April 9th, 2018, where it, and the original blots, were vetted. Below is the corrected figure and statement we sent in. We are grateful for the opportunity to rectify the error and apologize for any confusion.  We wish to correct panels 2D and S2F below. This does not change the findings. To avoid any confusion, please note that some images of cell sprouting are intentionally shown twice for comparison because they represent controls within the same experiment split between a main figure and a supplemental figure (c.f. Fig. 3C and Fig. S5A; Fig. S3C and Fig. S5E).u201d ![file] , 
 Thank you for that clarification, Dr Sinclair. It is a shame that, having vetted the correction, Cell did not feel any urgency to alert their readers of the outcome of that vetting. In the meantime, maybe you could post the full-length original for that gel? Presumably it was made available to the Cell editors? In fact, maybe you could share the originals for all of the gels in the paper, including the triplicates? , 
 #5 Don t get greedy Prostanthera Hindii. Savor this opportunity. Chew on the new blot for a while, since Tubulin is now just a bar of overexposure instead of duplicated blots. Like a ratchet mechanism, you can work your way up to the triplicates in due time. , 
 According to the Daily Mail (AKA *teh Daily Fail* which is, it must be acknowledged and notwithstanding some very strong competition, Brexit Landu2019s least reliable organ when it comes to hyping advances in science and medicine), a 47 year old male of the Ozzie subspecies who has been ingesting prodigious quantities of NMN is now 15 years younger. Struth cobber! http: www.dailymail.co.uk health article-4370092 Is-pink-pill-elixir-youth.html However, despite this individualu2019s excitingly rejuvenated and youthful body, it might also be the case that NMN overdosing by ordinary Galahs does not lead to improved brain function, at least as regards figure preparation for scientific publication: ![image] , 
 ![file] The NAD+ increase from NMN in liver on figure S6A is extremely large. Is the baseline NAD+ in the liver correct? Is GA supposed to have so much more NAD+ than Liver? , 
 Here is the link to those muscularly corrected figure image duplications https: www.ncbi.nlm.nih.gov pubmed ?term=30735637 Hopefully the authors also identified this additional figure duplication immediately after publication and its correction is in progress, if a little slow. Otherwise *Phosphate-buffered saline* is the new *Vascular endothelial growth factor* ![image] , 
 #8 Condylocarpon, I don t claim to be certain, but I wonder if there is no real mix-up here but instead just a case where the labeling and explanation are not fully clear. Looking through Figs 3, S3, 4, 5, and S5, it seems that these assays were performed under a variety of conditions, including - + VEGF, - + NMN, and - + siRNA. The control for VEGF is BSA, whereas the control for NMN is PBS. Probably in panel S3C all samples lacked NMN treatment, whereas in panel S5E all samples were treated with VEGF. If so, there would be no discrepancy. (Certainly, more complete legends would be helpful.) , 
 #9 Leucanella, See #4 The duplication is acknowledged but claimed to be the same and not admitted to be differently labelled. I do not think that this is appropriate. It should have been included in the correction. Also mentioned in #4 is a second reuse for 3C and S5A. In that case, I do think they might be representing the same things, though they are differently described. I do not like it when the text labelling obfuscates the reason for the similarities. , 
 #10 RE: * The duplication is acknowledged but claimed to be the same and not admitted to be differently labelled. I do not think that this is appropriate. * I don t quite follow this; it seems they were  claimed to be the same because they are the same. Are you saying that you already knew that these were the same treatment combination, but portrayed them as different in your post #8 ?? If so, consider me confused. Regardless, as I said, I would agree the descriptions should be clear. , 
 #11 Leucanella, Perhaps my words so far lack clarity, which would not be for the first time, so I will try again. The Y-axis labels refer to what has been administered to the aortic rings from two different genetic backgrounds (the X-axis labels). So, regarding the reused image depicted in #8 **VEGF is the experiment, whereas BSA is the control** And in the other figure **PBS is the control, whereas NMN is the experiment.** Therefore, to be as clear as possible: **VEGF != PBS** Never, in a month of Sundays, could the relation **VEGF = PBS** hold true. Observe that, in both figures, the top two images are the controls and the lower two images are the experimental condition. #4 acknowledges the image reuse but asserts that this is not a problem: *u201cTo avoid any confusion, please note that some images of cell sprouting are intentionally shown twice for comparison because they represent controls **within the same experiment** split between a main figure and a supplemental figure (c.f. Fig. 3C and Fig. S5A; **Fig. S3C and Fig. S5E**).u201d* Regarding S3C and S5E, this statement is simply not true. And since the duplication had been noted by the authors, it should have been included in the correction, given that the authors knew about it at the time. In my opinion, either their text is an attempt at obfuscation, or they have not understood their own experiments. , 
 #12 Well, perhaps it was me who was unclear. In #9, I described it this way:  Probably in panel S3C all samples lacked NMN treatment, whereas **in panel S5E all samples were treated with VEGF**. To put it differently, in each cases there are 2 relevant treatments, not just 1. Specifically, the reused image you are highlighting would be the following: +VEGF +PBS Even more specifically, as I read it, the presumed 4 conditions in each panel are as follows: Fig S3C: * WT +BSA +PBS * KO +BSA +PBS * WT +VEGF +PBS * KO +VEGF +PBS Fig. S5E: * WT +VEGF +PBS * KO +VEGF +PBS * WT +VEGF +NMN * KO +VEGF +NMN Or, to put it in graphical form: ![file] (Note that the legends to Fig S5 E,G,H,K confirm that the experiments testing the effects of NMN are all performed in the presence of VEGF. I.e., **the comparisons of PBS vs. NMN include VEGF stimulation**.) So, I am still unsure whether we disagree about the experimental conditions, or if we are in agreement but you are criticizing the unclear labeling. But hopefully this post helps clarify my interpretation. , 
 #13 I am indeed criticising the experiment itself in addition to the dodgy labelling strategy. If the experiment was the same, **the matching WT controls must themselves be the same too**. But they aren t are they? , 
 #14 Again, the fact that the samples in Fig S5E were all treated with VEGF is stated directly in the legend. The same is true for S5C, S5D, and S5K -- in all cases the VEGF treatment is not indicated by labeling in the figure itself, but instead is described in the figure legend. I can t agree that this is  dodgy ; rather, it seems sufficiently clear, and simply requires reading the legend, which is not an unreasonable expectation. I had hoped that pointing this out would help clarify matters, yet it does not seem to be helping, so I will not continue any further. ,