RETRACTED ARTICLE: ß2-adrenoreceptor Inverse Agonist Down-regulates Muscarine Cholinergic Subtype-3 Receptor and Its Downstream Signal Pathways in Airway Smooth Muscle Cells in vitro
Authors: Zhu Luo
Wei Luo
Jian Luo
Yuan-Hua Liu
Chun-Tao Liu
Journal: Scientific Reports (2017)
Discipline: Multidisciplinary
Keywords: β2-adrenoreceptor; inverse agonist; bronchoprotectiveness; ICI118,551; formoterol
Affiliations: Department of Respiratory Diseases, West China School of Medicine and West China Hospital, Sichuan University, Chengdu, 610041, China., Department of Respiratory Diseases, The First Affiliated Hospital, Zhengzhou University, Zhengzhou, 450052, China., Department of Respiratory Diseases, The First Affiliated Hospital, Chongqing Medical University, Chongqing, 400016, China.

Abstract:  Mechanisms underlying β2-adrenoreceptor (β2AR) inverse agonist mediated bronchoprotectiveness remain unknown. We incubated ICI118,551, formoterol, budesonide, and formoterol plus budesonide, as well as ICI118,551 or pindolol plus formoterol, ICI118,551 plus forskolin, SQ22,536 or H89 plus formoterol in ASMCs to detect expressions of M3R, PLCβ1 and IP3. The level of M3R in the presence of 10-5 mmol/L ICI118,551 were significantly decreased at 12 h, 24 h and 48 h (P &lt; 0.05), and at 24 h were significantly reduced in ICI118,551 with concentration of 10-5 mmol/L, 10-6 mmol/L, 10-7 mmol/L, and 10-8 mmol/L (P &lt; 0.05). The level of IP3 in 10-5 mmol/L ICI118,551 was significantly diminished at 24 h (P &lt; 0.01), except for that at 1 h, neither was in the level of PLCβ1. A concentration of 10-5 mmol/L ICI118,551 at 24 h showed a significant reduction of M3R level compared to formoterol (P &lt; 0.01), budesonide (P &lt; 0.01), and formoterol + budesonide (P &lt; 0.05), but significant reduction of PLCβ1 and IP3 was only found between 10-5 mmol/L ICI118,551 and formoterol at 24 h, but not in the comparison of budesonide or formoterol + budesonide. Pindolol and H89 could not inhibit the formoterol-induced expression of M3R (P &gt; 0.05), but SQ22,536 significantly antagonized the formoterol-induced M3R expression (P &lt; 0.05). In conclusions, β2AR inverse agonist, ICI118,551, exerts similar bronchoprotective effects to corticosteroids via decreasing the expression of M3R and inhibiting the production of IP3. 

Retracted: Yes

PubPeer comments:
 Figures 4 and 5 statistics Given the data distribution and n=5 or less, what statistical method could find the significant differences indicated by asterisks? No statistical method is given in the paper. Furthermore, could the authors explain the lack of a dose effect? Increasing the dose by a factor of 2 was less effective. ![](http: ) ![file] ![file] ![](http: ) , 
 This article refers to and uses the data presented in a previous publication doi:10.1093 gerona glx056 The Methods section in paper indicates that this was a non-blinded non-randomized trial which obviously did not have a placebo prong , just 3 doses of cells. It does not refer to statistical methods. Interestingly, one of the authors affiliated to the University of Miami Miller School of Medicine declares being a share holder in Longeveron, a company which currently offers this unproven treatment to patients traveling to the Bahamas . , 
 The figures in this review article are reproduced from 2 original articles in the Journal of Gerontology: https: pubmed.ncbi.nlm.nih.gov 28977399 https: pubmed.ncbi.nlm.nih.gov 28444181 They are the peer reviewed publications corresponding to a phase 1 trial that did not have a placebo group, and a phase 2 that did have a placebo randomization. All data were monitored by a third party which also conducted the statistics. Methods for the statistics are included in the data supplement: https: academic.oup.com biomedgerontology article 72 11 1513 3977809#supplementary-data With regard to dosing, this is an interesting issue. A new trial is underway under IND, with third party monitoring to rigorously examine dosing using a placebo controlled design. The new trial results will be announced shortly and will give new insights into dosing. , 
 The response by the author is interesting in several ways. 1. It reveals that the figures are not original but are reproduced from two previous publications, while the figure legends of figures 4 and 5 in Florea et al. 2019 do not state that fact. Only in the text is there a vague reference to the source of the data. 2. In the 2017 papers referenced by Dr. Harte, it is stated that the statistical analysis had been outsourced to the Emmes Corporation, a company providing full service support for clinical studies including not only statistical analysis but also protocol development and even u201cmedical writing.u201d 3. The 2017 papers describe (insufficiently) the statistical analysis in the supplemental material section by naming a few statistical tests, including the Wilcoxon signed rank test. 4. However, if one uses, for example, the data in Figure 4B in Florea et al. 2019, identical to Figure 2A in Golpanian et al. 2017, and performs the Wilcoxon test, no significance at the 0.05 level can be detected, which is contrary to the claim in the referenced papers. Also, the apparent N=4 in these studies is not conducive to a rank order test. 5. The N=4 is curious since all other data are represented with an N of 5, as stated. 6. The odd phenomenon that increasing the dose of stem cells by a factor of 2 abolishes the u201ceffectu201d is not properly addressed in the reply. Instead of giving an explanation, the author cryptically refers to a new study. Does that mean the old data were interpreted erroneously?. 7. The supplemental material to which the author refers for statistical information describes proprietary (i.e. hidden) methods used by the Emmes Corp., so it is not possible to determine how the analysis was done. The supplement does say that for missing data,  imputation methods may be used. This means that if data are missing, new data from somewhere will be added, presumably for the purposes of analysis. We are not told if this was done, just that it might have been done. , 
 The difficulties with the hidden statistics and questionable statistical significance of the published studies reported by the first commenter in two posts are particularly serious given the importance for the authors ongoing clinical trials. The biggest problem as I see it is the senior authoru2019s conflict of interest in these studies, not mentioned in the review, since he is a major shareholder in the company that produces the cells used in the clinical trials with nearly 8 million shares worth tens of millions of dollars. Moreover, the company is already using the cells to treat paying patients at a private clinic in the Bahamas (Why Are Scientists and Patients Visiting This Island Paradise? - leaps.org) under a special arrangement with the company, Longeveron, as the Bahamas Registry Trial (Clinical Trials (longeveron.com)). Thus the senior author is set to profit from the outcomes of these studies. These relationships should be examined more closely. , 
 The senior authoru2019s response to the lack of statistical significance of the published studies reported by the first commenter is somewhat surprising. Typically, reviews are more read and cited than original papers these days. Thus, proper reference to data sources and potential conflicts of interest should be even more important for this type of publication. Furthermore, this raises several questions since the senior author is a major shareholder in the company that produces the cells used in the clinical trials. The company is already using the cells to treat paying patients at a private clinic in the Bahamas (Why Are Scientists and Patients Visiting This Island Paradise? - leaps.org) under a special arrangement as the Bahamas Registry Trial (Clinical Trials (longeveron.com)). Why does the review not cite the corresponding author s association with the company that produces the cells? ,