Aberrant methylation and expression of TNXB promotes chondrocyte apoptosis and extracullar matrix degradation in hemophilic arthropathy via AKT signaling

Reviewer #1 (Public Review):

Summary:
Chen and colleagues first compared the cartilage tissues collected from OA and HA patients using histology and immunostaining. Then, a genome-wide DNA methylation analysis was performed, which informed the changes of a novel gene, TNXB. IHC confirmed that TNXB has a lower expression level in HA cartilage than OA. Next, the authors demonstrated that TNXB levels were reduced in the HA animal model, and intraarticular injection of AAV carrying TNXB siRNA induced cartilage degradation and promoted chondrocyte apoptosis. Based on KEGG enrichment, histopathological analysis, and western blot, the authors also showed the relationship between TNXB and AKT phosphorylation. Lastly, AKT agonist, specifically SC79 in this study, was shown to partially rescue the changes of in vitro-cultured chondrocytes induced by Tnxb knock-down. Overall, this is an interesting study and provided sufficient data to support their conclusion.

Strengths:
(1) Both human and mouse samples were examined.
(2) The HA model was used.
(3) Genome-wide DNA methylation analysis was performed.

Weaknesses:
(1) In some experiments, the selection of the control groups was not ideal.
(2) More details on analyzing methods and information on replicates need to be included.
(3) Discussion can be improved by comparing findings to other relevant studies.
(4) The use of transgenic mice with conditional Tnxb depletion can further define the physiological roles of Tnxb.

Reviewer #2 (Public Review):

Summary:
This manuscript mainly studied the biological effect of tenascin XB (TNXB) on hemophilic arthropathy (HA) progression. Using bioinformatic and histopathological approaches, the authors identified the novel candidate gene TNXB for HA. Next, the authors showed that TNXB knockdown leads to chondrocyte apoptosis, matrix degeneration, and subchondral bone loss in vivo/vitro. Furthermore, AKT agonists promoted extracellular matrix synthesis and prevented apoptosis in TNXB knockdown chondrocytes.

Strengths:
In general, this study significantly advances our understanding of HA pathogenesis. The authors utilize comprehensive experimental strategies to demonstrate the role of TNXB in cartilage degeneration associated with HA. The results are clearly presented, and the conclusions appear appropriate.

Weaknesses:
Additional clarification is required regarding the gender of the F8-/- mouse in the study. Is the mouse male or female?

Reviewer #3 (Public Review):

Summary:
The manuscript by Dr. Chen et al. investigates the genes that are differentially methylated and associated with cartilage degeneration in hemophilia patients. The study demonstrates the functional mechanisms of the TNXB gene in chondrocytes and F8-/- mice. The authors first showed significant DNA methylation differences between hemophilic arthritis (HA) and osteoarthritis through genome-wide DNA methylation analysis. Subsequently, they showed a decreased expression of the differentially methylated TNXB gene in cartilage from HA patients and mice. By knocking down TNXB in vivo and in vitro, the results indicated that TNXB regulates extracellular matrix homeostasis and apoptosis by modulating p-AKT. The findings are novel and interesting, and the study presents valuable information in blood-induced arthritis research.

Strengths:
The authors adopted a comprehensive approach by combining genome-wide DNA methylation analysis, in vivo and in vitro experiments using human and mouse samples to illustrate the molecular mechanisms involved in HA progression, which is crucial for developing targeted therapeutic strategies. The study identifies Tenascin XB (TNXB) as a central mediator in cartilage matrix degradation. It provides mechanistic insights into how TNXB influences cartilage matrix degradation by regulating the activation of AKT. It opens avenues for future research and potential therapeutic interventions using AKT agonists for cartilage protection in hemophilic arthropathy. The conclusions drawn from the study are clear and directly tied to the findings.

Weaknesses:
(1) The study utilizes a small sample size (N=5 for both osteoarthritis and hemophilic arthropathy). A larger sample size would enhance the generalizability and statistical power of the findings.
(2) The use of an animal model (F8-/- mouse) to investigate the role of TNXB may not fully capture the complexity of human hemophilic arthropathy. Differences in the biology between species may affect the translatability of the findings to human patients.
(3) The study primarily focuses on TNXB as a central mediator, but it might overlook other potentially relevant factors contributing to cartilage degradation in hemophilic arthropathy. A more holistic exploration of genetic and molecular factors could provide a broader understanding of the condition.