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• Response to ‘Correspondence on ‘Digoxin targets low density lipoprotein receptor-related protein 4 and protects against osteoarthritis’ by Kai-di Wang’ by Cheng et al
  Kai-di Wang, Xiang Ding and Chuan-ju Liu
  Published on: 10 March 2022
• Digoxin targets low density lipoprotein receptor-related protein 4 and protects against osteoarthritis
  Yunzhong Cheng, Lijin Zhou and Yong Hai
  Published on: 10 March 2022

• Published on: 10 March 2022

  Response to ‘Correspondence on ‘Digoxin targets low density lipoprotein receptor-related protein 4 and protects against osteoarthritis’ by Kai-di Wang’ by Cheng et al

  • Kai-di Wang, Post doctoral fellow Department of Orthopedic Surgery, NYU Grossman School of Medicine, New York, New York, USA
  • Other Contributors:
    • Xiang Ding, PhD student
    • Chuan-ju Liu, Professor

  We thank Cheng et al1 for their interest in our paper ‘Digoxin targets low density lipoprotein receptor-related protein 4 and protects against osteoarthritis’.2 Below we provide point-by-point replies to the comments.
  First, we would like to clarify the roles of digoxin and ouabain in this study. We performed three rounds of drug screening and found that only ouabain could robustly induce the expressions of anabolic marker genes, including COL2A1, aggrecan (ACAN) and cartilage oligomeric matrix protein (COMP).3,4 The fact that ouabain belongs to a family of cardenolides prompted us to determine whether other cardenolides also possess the similar anabolic effects in chondrocytes, which led to the isolation of digoxin as another cardenolide that could also induce the expressions of anabolic marker genes in chondrocytes. We appreciate the author’s statement that ouabain demonstrated better protection against OA in some assays mentioned in their correspondence. However, digoxin also showed significant protective effects against OA in multiple analyses performed, quite similar to ouabain. For instance, there is no difference in the osteophyte number between digoxin and ouabain treatment groups (Figure 2F and 2G). More importantly, digoxin is known to be the only safe inotropic drug for oral use that improves hemodynamics.5 This led us to hypothesize that records related to digoxin might be accessible in general practitioner based medical records databases. Indeed, we foun...

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  We thank Cheng et al1 for their interest in our paper ‘Digoxin targets low density lipoprotein receptor-related protein 4 and protects against osteoarthritis’.2 Below we provide point-by-point replies to the comments.
  First, we would like to clarify the roles of digoxin and ouabain in this study. We performed three rounds of drug screening and found that only ouabain could robustly induce the expressions of anabolic marker genes, including COL2A1, aggrecan (ACAN) and cartilage oligomeric matrix protein (COMP).3,4 The fact that ouabain belongs to a family of cardenolides prompted us to determine whether other cardenolides also possess the similar anabolic effects in chondrocytes, which led to the isolation of digoxin as another cardenolide that could also induce the expressions of anabolic marker genes in chondrocytes. We appreciate the author’s statement that ouabain demonstrated better protection against OA in some assays mentioned in their correspondence. However, digoxin also showed significant protective effects against OA in multiple analyses performed, quite similar to ouabain. For instance, there is no difference in the osteophyte number between digoxin and ouabain treatment groups (Figure 2F and 2G). More importantly, digoxin is known to be the only safe inotropic drug for oral use that improves hemodynamics.5 This led us to hypothesize that records related to digoxin might be accessible in general practitioner based medical records databases. Indeed, we found that digoxin was the only cardenolides drug included in the data from The Health Improvement Network (THIN). Results indicated that digoxin use was associated with reduced risk of knee or hip OA-associated joint replacement among patients with atrial fibrillation. Therefore, we chose both ouabain and digoxin as the representatives of cardenolides in this study and emphasized the potential importance of digoxin in treating patients with OA in clinics.
  Second, it would be ideal and probably more valuable to focus on digoxin use in OA patients without other diseases, including atrail fibrillation. Unfortunately, such information is unavailable in the available database, including The Health Improvement Network (THIN), this is because of the fact that digoxin is only used in patients with atrial fibrillation with or without OA.
  Both ouabain and digoxin are FDA-approved drugs and their side effects have been well established. In addition, both ouabain and digoxin did not show detectable toxicity in our in vitro and in vivo assays (online supplemental figure 11). Having said that, we do appreciate the comments that combined use of digoxin and ouabain with lower dosages of drugs may exert synergistic protective effects against OA and may reduce their side effects, which worthwhile further investigations in future.
  REFERENCES
  1 Cheng YZ, Zhou LJ and Hai Y. Correspondence on ‘Digoxin targets low density lipoprotein receptor-related protein 4 and protects against osteoarthritis’ by Kai-di Wang. Ann Rheum Dis 2022.
  2 Wang KD, Ding X, Jiang N, et al. Digoxin targets low density lipoprotein
  receptor-related protein 4 and protects against osteoarthritis. Ann Rheum Dis 2021
  De1: annrheumdis-2021-221380.
  3 Fu WY, Hettinghouse A, Chen Y., et al., 14-3-3 epsilon is an intracellular component of TNFR2 receptor complex and its activation protects against osteoarthritis. Ann Rheum Dis 2021;80(12): p. 1615-1627.
  4 Liu, RH, Chen YH, Fu WY, et al., Fexofenadine inhibits TNF signaling through targeting to cytosolic phospholipase A2 and is therapeutic against inflammatory arthritis. Ann Rheum Dis 2019;78(11): p. 1524-1535.
  5 Kjeldsen K, Nørgaard A, Gheorghiade M. Myocardial Na,K-ATPase: the molecular
  basis for the hemodynamic effect of digoxin therapy in congestive heart failure.
  Cardiovasc Res 2002;55:710–3.

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  Conflict of Interest:
  None declared.

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• Published on: 10 March 2022

  Digoxin targets low density lipoprotein receptor-related protein 4 and protects against osteoarthritis

  • Yunzhong Cheng, Orthopedic Surgeon Department of Orthopedic Surgery, Beijing Chao-Yang Hospital, Capital Medical University
  • Other Contributors:
    • Lijin Zhou, Orthopedic Surgeon
    • Yong Hai, Professor and Chairman

  We read with great interest the recent study by Kai-di Wang and colleagues1, published in the Annals of Rheumatic Disease, which showed LRP4 was isolated as a novel target of digoxin, and deletion of LRP4 abolished digoxin’s regulations of chondrocytes. We appreciate this meaningful research very much, and we believe that this study has significant guiding for providing new insights into the understanding of digoxin’s chondroprotective action and underlying mechanisms, but also present new evidence for repurposing digoxin for osteoarthritis (OA). However, we have some questions to discuss with the authors except for the limitations the authors mentioned in the study.
  As all we know, the authors used two cardenolides to conduct the experiments, ouabain and digoxin. We can know that ouabain and digoxin can both enhance chondrogenesis and stimulate chondrocyte anabolism from the result of Figure 1. More importantly, we found that the relative staining level in ouabain treated groups was much higher compared with that in digoxin group (Figure 1B). Moreover, the mRNA expressions of transcriptional levels of chondrogenic marker genes, such as COL2A1, Comp, ACAN, SOX5, SOX6, and SOX92-4 were also much higher compared with that in digoxin group (Figure 1C). The similar situation was showed in Figure 1D-G, which may indicate ouabain better enhance chondrogenesis and stimulate chondrocyte anabolism than that of digoxin. In addition, we may also speculate ouabain bette...

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  We read with great interest the recent study by Kai-di Wang and colleagues1, published in the Annals of Rheumatic Disease, which showed LRP4 was isolated as a novel target of digoxin, and deletion of LRP4 abolished digoxin’s regulations of chondrocytes. We appreciate this meaningful research very much, and we believe that this study has significant guiding for providing new insights into the understanding of digoxin’s chondroprotective action and underlying mechanisms, but also present new evidence for repurposing digoxin for osteoarthritis (OA). However, we have some questions to discuss with the authors except for the limitations the authors mentioned in the study.
  As all we know, the authors used two cardenolides to conduct the experiments, ouabain and digoxin. We can know that ouabain and digoxin can both enhance chondrogenesis and stimulate chondrocyte anabolism from the result of Figure 1. More importantly, we found that the relative staining level in ouabain treated groups was much higher compared with that in digoxin group (Figure 1B). Moreover, the mRNA expressions of transcriptional levels of chondrogenic marker genes, such as COL2A1, Comp, ACAN, SOX5, SOX6, and SOX92-4 were also much higher compared with that in digoxin group (Figure 1C). The similar situation was showed in Figure 1D-G, which may indicate ouabain better enhance chondrogenesis and stimulate chondrocyte anabolism than that of digoxin. In addition, we may also speculate ouabain better protect against OA in a surgically induced model in vivo than that of digoxin, especially from Figure 2H and Figure 2I. However, the study emphasized the importance of digoxin instead of ouabain from the beginning to the end. Therefor we are wondering what the role of ouabain played in the whole experiment procedure compared to digoxin on earth.
  On the other hand, Figure 4 demonstrated that digoxin use was associated with reduced risk of knee or hip OA-associated joint replacement among patients with atrial fibrillation. This is easy to understand. However, we want to know how many OA patients with atrial fibrillation in the world. What’s the percentage this kind of patients in the whole OA patients?
  This is why we are more interest in digoxin used in OA patients without atrial fibrillation. Whether digoxin can have the same protection among OA patients without atrial fibrillation.What’s more, What’s the side effect when using digoxin to OA patients with normal heart function?
  We would like to point out that since ouabain and digoxin can both enhance chondrogenesis and stimulate chondrocyte anabolism to protect against OA. We suppose whether we can use digoxin combined with ouabain at the same time to treat with OA patients. The most reason why this hypothesis we have is that we can perhaps reduce the side effect by using lower dose of digoxin, but do not affect the function of protecting against OA patients. And digoxin plus ouabain with different doses group can be planned to further experiments. Maybe we can find a better treatment to OA patients.
  References
  1 Wang KD, Ding X, Jiang N, et al. Digoxin targets low density lipoprotein receptor-related protein 4 and protects against osteoarthritis. Ann Rheum Dis. 2021 De1: annrheumdis-2021-221380.
  2. Nenna R, Turchetti A, Mastrogiorgio G, et al.COL2A1 Gene Mutations: Mechanisms of Spondyloepiphyseal Dysplasia Congenita. Appl Clin Genet. 2019;12:235-238.
  3. Lin EA, Liu C-J. The role of ADAMTSs in arthritis. Protein Cell. 2010;1:33–47.
  4. Liu C-ju, Zhang Y, Xu K, et al. Transcriptional activation of cartilage oligomeric matrix
  protein by SOX9, SOX5, and SOX6 transcription factors and CBP/p300 coactivators.
  Front Biosci.2007;12:3899–910.

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  Conflict of Interest:
  None declared.

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