Effects of down-regulated carbonic anhydrase 8 on cell survival and glucose metabolism in human colorectal cancer cell lines.

Carbonic anhydrase 8 (CA8) is a member of the α-carbonic anhydrase family but does not catalyze the reversible hydration of carbon dioxide. In the present study, we examined the effects of CA8 on two human colon cancer cell lines, SW480 and SW620, by suppressing CA8 expression through shRNA knockdown. Our results showed that knockdown of CA8 decreased cell growth and cell mobility in SW620 cells, but not in SW480 cells. In addition, downregulated CA8 resulted in a significant decrease of glucose uptake in both SW480 and SW620 cells. Interestingly, stable downregulation of CA8 decreased phosphofructokinase-1 expression but increased glucose transporter 3 (GLUT3) levels in SW620 cells. However, transient downregulation of CA8 fails to up-regulate GLUT3 expression, indicating that the increased GLUT3 observed in SW620-shCA8 cells is a compensatory effect. In addition, the interaction between CA8 and GLUT3 was evidenced by pull-down and IP assays. On the other hand, we showed that metformin, a first-line drug for type II diabetes patients, significantly inhibited cell migration of SW620 cells, depending on the expressions of CA8 and focal adhesion kinase. Taken together, our data demonstrate that when compared to primary colon cancer SW480 cells, metastatic colon cancer SW620 cells respond differently to downregulated CA8, indicating that CA8 in more aggressive cancer cells may play a more important role in controlling cell survival and metformin response. CA8 may affect glucose metabolism- and cell invasion-related molecules in colon cancer, suggesting that CA8 may be a potential target in future cancer therapy.

IL-4-induced decrease in both the number and CTLA-4 expression of Treg impairs suppression of Th2 type inflammation in severe atopic dermatitis.

BACKGROUND: Treg plays a pivotal role in the suppression of Th2 cell and the maintenance of immune homeostasis. The precise molecular mechanism underlying the disruption of Treg suppression of Th2 cell and the promotion of Th2 type inflammation in allergic diseases remains elusive. OBJECTIVE: This study aims to investigate the molecular mechanism underlying quantitative and functional changes of Treg in AD. METHODS: The molecular mechanism was investigated using flow cytometry, mRNA sequencing, co-culture experiments, co-immunoprecipitation, chromatin immunoprecipitation, and bisulfite sequencing in vitro or in AD mice model and patients with AD. RESULTS: Increased proportion of Treg was detected in mild and moderate AD. Conversely, characteristic decrease in both the number and CTLA-4 expression of Treg was relevant to serum IL-4 level in severe AD patients, which was verified under a high concentration of IL-4 treatment in vitro. The underlying mechanism is that IL-4/pSTAT6 pathway recruits DNMT1 and HDAC2 to inhibit transcriptional regulation of Foxp3 and CTLA-4 loci. High level of IL-4 impaired the suppression of Treg against Th2 cell differentiation mediated by CTLA-4, and blockade of IL-4Rα signaling in Treg restored Treg number and suppression of Th2 cell in AD model mice and patients with AD. CONCLUSION: The number of Treg is relevant to stratification of severity and serum IL-4 level in patients with AD. Abnormal high level of IL-4 epigenetically triggers a decrease in both the number and CTLA-4 expression of Treg. The reduced expression of CTLA-4 on Treg induced by IL-4 impairs suppression of Th2 cell differentiation.

Dynamic pathological analysis reveals a protective role against skin fibrosis for TREM2-dependent macrophages.

Rationale: Systemic sclerosis (SSc) is a chronic and incurable autoimmune disease with high mortality rates, and skin fibrosis is one of distinguishing hallmarks in the pathogenesis. However, macrophage heterogeneity regulating skin fibrosis remain largely unknown. Methods: We established mouse disease model and performed single-cell RNA-sequencing (scRNA-seq) to resolve the dynamic and heterogenous characteristics of macrophages in skin fibrosis, and the role of TREM2-dependent macrophages in the pathological process was investigated using knockout mice and intraperitoneal transferring TREM2+ macrophages combining with functional assays. Results: We show that TREM2-expressing macrophages (TREM2+ MФs) accumulate in injured skin of mice treated by bleomycin (BLM) and human SSc, and their gene signatures and functional pathways are identified in the course of disease. Genetic ablation of Trem2 in mice globally accelerates and aggravates skin fibrosis, whereas transferring TREM2hi macrophages improves and alleviates skin fibrosis. Amazingly, we found that disease-associated TREM2+ MФs in skin fibrosis exhibit overlapping signatures with fetal skin counterparts in mice and human to maintain skin homeostasis, but each has merits in skin remodeling and development respectively. Conclusion: This study identifies that TREM2 acts as a functional molecule and a major signaling by which macrophage subpopulations play a protective role against fibrosis, and disease-associated TREM2+ MФs in skin fibrosis might undergo a fetal-like reprogramming similar to fetal skin counterparts.

Tracking organelle activities through efficient and stable root genetic transformation system in woody plants.

Due to the protracted transgenic timeline and low efficiency in stable genetic transformation of woody plants, there has been limited exploration of real-time organelle imaging within stable transgenic woody plant cells. Here, we established an efficient in vivo genetic transformation system for woody plants using an Agrobacterium rhizogenes-mediated approach. This system was successfully validated in multiple perennial woody species. Using citrus as a model, we introduced organelle-targeted fluorescent reporters via genetic transformation and investigated their subcellular localization and dynamics using advanced imaging techniques, such as confocal microscopy and live-cell imaging. Moreover, we subjected transgenic MT-GFP-labeled mitochondria in root cells to stress conditions simulating agricultural adversities faced by fruit crops. The stress-induced experiments revealed notable alterations in mitochondrial morphology. Our study contributes novel insights into membrane trafficking processes, protein localization dynamics, and cellular physiology in woody plants, while also providing stable and efficient genetic transformation methods for perennial woody species.

Systematic review and meta-analysis of reproductive outcomes after high-intensity focused ultrasound (HIFU) treatment of adenomyosis.

High-intensity focused ultrasound (HIFU) has emerged as a promising uterus-sparing and possibly fertility-sparing treatment modality for women with adenomyosis, especially those who desire to conceive. We conducted this systematic review and performed a meta-analysis on clinical studies aimed to improve reproduction in women with adenomyosis. After extensive search of PubMed and CNKI, we identified 10 studies published in English and Chinese involving a total of 557 patients with adenomyosis who desired to conceive after HIFU treatment. We found a pooled estimate of pregnancy rate of 53.4% and of the live birth rate of 35.2%, and there was a substantial heterogeneity among these studies. While there is a potential for HIFU treatment to improve fertility for patients with adenomyosis who desired to conceive, such evidence is very weak as of now. Comparative studies with much higher methodological rigor, preferably randomized clinical trials, are badly needed to further illuminate this issue.

Can the Blended Application of Controlled-Release and Common Urea Effectively Replace the Common Urea in a Wheat-Maize Rotation System? A Case Study Based on a Long-Term Experiment.

The one-time application of blended urea (BU), combining controlled-release urea (CRU) and uncoated urea, has proven to be a promising nitrogen (N) management strategy. However, the long-term sustainability of blending urea remains largely unexplored. To assess whether a single application of blended urea could effectively replace split uncoated urea applications, a long-term field experiment was conducted in the North China Plain (NCP). The results indicated that, when compared to common urea (CU) at the optimal N rate (180 kg N ha-1), BU achieved comparable grain yields, N uptake and NUE (61% vs. 62). BU exhibited a 12% higher 0-20 cm soil organic nitrogen stock and a 9% higher soil organic carbon (C) stock. Additionally, BU reduced life-cycle reactive N (Nr) losses and the N footprint by 10%, and lowered greenhouse gas (GHG) emissions and the C footprint by 7%. From an economic analysis perspective, BU demonstrated comparable private profitability and a 3% greater ecosystem economic benefit. Therefore, BU under the optimal N rate has the potential to substitute split applications of common urea in the long-term and can be regarded as a sustainable N management strategy for wheat and maize production in the NCP.

Hyperplastic Human Macromass Cartilage for Joint Regeneration.

Cartilage damage affects millions of people worldwide. Tissue engineering strategies hold the promise to provide off-the-shelf cartilage analogs for tissue transplantation in cartilage repair. However, current strategies hardly generate sufficient grafts, as tissues cannot maintain size growth and cartilaginous phenotypes simultaneously. Herein, a step-wise strategy is developed for fabricating expandable human macromass cartilage (macro-cartilage) in a 3D condition by employing human polydactyly chondrocytes and a screen-defined serum-free customized culture (CC). CC-induced chondrocytes demonstrate improved cell plasticity, expressing chondrogenic biomarkers after a 14.59-times expansion. Crucially, CC-chondrocytes form large-size cartilage tissues with average diameters of 3.25 ± 0.05 mm, exhibiting abundant homogenous matrix and intact structure without a necrotic core. Compared with typical culture, the cell yield in CC increases 2.57 times, and the expression of cartilage marker collagen type II increases 4.70 times. Transcriptomics reveal that this step-wise culture drives a proliferation-to-differentiation process through an intermediate plastic stage, and CC-chondrocytes undergo a chondral lineage-specific differentiation with an activated metabolism. Animal studies show that CC macro-cartilage maintains a hyaline-like cartilage phenotype in vivo and significantly promotes the healing of large cartilage defects. Overall, an efficient expansion of human macro-cartilage with superior regenerative plasticity is achieved, providing a promising strategy for joint regeneration.

Cardiac tropoini T (TNNT2) plays a potential oncogenic role in colorectal carcinogenesis.

PURPOSE: Colorectal cancer (CRC) is the third most common cancer in the world. The purpose of this study was to investigate the role of TNNT2 in the proliferation, migration and invasion of CRC cells and its expression in CRC tissues to better understand the regulatory role of TNNT2 in CRC. METHODS: Western blotting (WB) and qPCR were used to detect the expression of TNNT2 in colorectal cancer tissues and paracancerous tissues. CCK-8, colony formation, Transwell and other experiments were used to clarify the role of TNNT2 in the proliferation, migration and invasion of colorectal cancer cells. Changes in TNNT2, EGFR and HER2 mRNA transcription levels were detected by SYBR Real-Time PCR assay, and the effects of TNNT2 overexpression or knockdown on the expression of EGFR, HER2 and EMT-related proteins in CRC cells were determined by WB. TNNT2 and EGFR intreaction was carried out in HCT116 cells by coimmunoprecipitation experiments. RESULTS: The protein and mRNA expression level of TNNT2 in CRC tissues were higher than those in paracancerous tissues. The CCK-8 results suggested that overexpression of TNNT2 significantly promoted the proliferation of HCT116 and RKO cells, and TNNT2 konckdown gets the opposite result; and the colony formation results were the same as tthose of CCK-8 assay. Transwell invasion and migration experiments showed that overexpression of TNNT2 promoted the migration and invasion of HCT116 and PKO cells, and TNNT2 konckdown suppressed the migration and invasion of the these cells. The SYBR Green I real-time PCR method revealed that them RNA levels of TNNT2, EGFR and HER2 in the TNNT2 overexpression group were higher than those in RKO cells. WB showed that overexpressing TNNT2 increased the expression of EGFR and HER2 in HCT16 and RKO cells,decreased the expression of EMT marker E-cadherin, and increased the expression of Vimentin and N-cadherin. Konckdown of TNNT2 decreased the expression of EGFR and HER2, increased the expression of E-cadherin, and decreased the expression of Vimentin and N-cadherin in HCT16 and RKO cells. The immunocoprecipitation experiment showed that there was an interaction between EGFR and TNNT2. CONCLUSION: TNNT2 can promote the proliferation, invasion and metastasis of colorectal cancer cells. There is an interaction between TNNT2 and EGFR protein. TNNT2 can upregulate EGFR and HER2-related proteins in colorectal cancer cells and promote the occurrence of EMT. Therefore, TNNT2 can promote the invasion and metastasis of CRC cells through the EGFR/HER2/EMT signal axis, suggesting that TNNT2 is a potential target of CRC treatment.

Sodium thiosulfate inhibits epithelial-mesenchymal transition in melanoma via regulating the Wnt/ß-catenin signaling pathway.

BACKGROUND: Melanoma is the most common form of skin cancer. Given its high metastasis and high recurrence, its therapies are constantly updated. OBJECTIVE: The study aims to prove the efficacy of sodium thiosulfate (STS), an antidote to cyanide or nitroprusside poisoning, in melanoma treatment. METHODS: We tested the effect of STS by culturing melanoma cells (B16 and A375) in vitro and establishing melanoma mouse models in vivo. The proliferation and viability of melanoma cells were measured by the CCK-8 test, cell cycle assay, apoptosis analysis, wound healing assay, and transwell migration assay. The expression of apoptosis-related molecules, epithelial-mesenchymal transition (EMT)-associated molecules, and the Wnt/ß-catenin signaling pathway-related molecules were determined by Western blotting and immunofluorescence. RESULTS: The high metastasis of melanoma is considered to be linked to the EMT process. The scratch assay using B16 and A375 cells also showed that STS could inhibit the EMT process of melanoma. We demonstrated that STS inhibited the proliferation, viability, and EMT process of melanoma by releasing H2S. STS-mediated weakening of cell migration was related to the inhibition of the Wnt/ß-catenin signaling pathway. Mechanistically, we defined that STS inhibited the EMT process via the Wnt/ß-catenin signaling pathway. CONCLUSIONS: These results suggest that the negative effect of STS on melanoma development is mediated by the reduction of EMT via the regulation of the Wnt/ß-catenin signaling pathway, which provides a new clue to treating melanoma.

Tetrel-Bond Interactions Involving Metallylenes TH2 (T = Si, Ge, Sn, Pb): Dual Binding Behavior.

The dual binding behavior of the metallylenes TH2 (T = Si, Ge, Sn, Pb) with some selected Lewis acids (T'H3F, T' = Si, Ge, Sn, Pb) and bases (N2, HCN, CO, and C6H6) has been investigated by using the high-level quantum chemical method. Two types (type-A and type-B) of tetrel-bonded complexes can be formed for TH2 due to their ambiphilic character. TH2 act as Lewis bases in type-A complexes, and they act as Lewis acids in type-B ones. CO exhibits two binding modes in the type-B complexes, one of which is TH2···CO and the other is TH2···OC. The TH2···OC complexes possess a weaker binding strength than the other type-B complexes. The TH2···OC complexes are referred to as the type-B2 complexes, and the other type-B complexes are referred to as the type-B1 complexes. The type-A complexes exhibit a relatively weak binding strength with Eint (interaction energy) values ranging from -7.11 to -15.55 kJ/mol, and the type-B complexes have a broad range of Eint values ranging from -9.45 to -98.44 kJ/mol. The Eint values of the type-A and type-B1 complexes go in the order SiH2 > GeH2 > SnH2 > PbH2. The AIM (atoms in molecules) analysis suggests that the tetrel bonds in type-A complexes are purely closed-shell interactions, and those in most type-B1 complexes have a partially covalent character. The EDA (Energy decomposition analysis) results indicate that the contribution values of the three energy terms go in the order electrostatic > dispersion > induction for the type-A and type-B2 complexes, and this order is electrostatic > induction > dispersion for the type-B1 complexes.

Intermolecular interactions between the heavy-atom analogues of acetylene T2H2 (T = Si, Ge, Sn, Pb) and HCN.

METHODS: The intermolecular interactions between the heavy-atom analogues of acetylene T2H2 (T = Si, Ge, Sn, Pb) and HCN have been investigated by theoretical calculations at the CCSD(T)/aug-cc-pVTZ//MP2/aug-cc-pVDZ level. RESULTS: The global energy minimum of T2H2 is the butterfly structure A, and another energy minimum is the planar structure B. Both structures A and B exhibit the dual behavior when binding with HCN. The various hydrogen bond (HB), dihydrogen bond (DB) and tetrel bond (TB) complexes can be found according to the MEP maps of T2H2. One TB and three HB complexes formed between structure A and HCN can be located for Si2H2 and Ge2H2. One TB, two HB and one DB complexes formed between structure A and HCN can be located for Sn2H2 and Pb2H2. Four TB and one HB complexes formed between structure B and HCN can be located for all the T2H2. The geometries and binding strengths of the complexes are compared and analyzed. CONCLUSIONS: The interactions in these complexes are generally weak, and the interaction energies of these complexes range from -0.53 to -8.23 kcal/mol. The interaction energies of the TB complexes are larger than those of the corresponding HB and DB complexes for structure A···HCN systems. The relative binding strength of the four TB complexes exhibits different order for different structure B···HCN systems, which is consistent with the MEP maps of the isolated monomers.

Seamless and early gap healing of osteochondral defects by autologous mosaicplasty combined with bioactive supramolecular nanofiber-enabled gelatin methacryloyl (BSN-GelMA) hydrogel.

Autologous mosaicplasty is a common approach used to treat osteochondral defects in clinical practice. Gap integration between host and transplanted plugs requires bone tissue reservation and hyaline cartilage regeneration without uneven surface, graft necrosis and sclerosis. However, poor gap integration is a serious concern, which eventually leads to deterioration of joint function. To deal with such complications, this study has developed a strategy to effectively enhance integration of the gap region following mosaicplasty by applying injectable bioactive supramolecular nanofiber-enabled gelatin methacryloyl (GelMA) hydrogel (BSN-GelMA). A rabbit osteochondral defect model demonstrated that BSN-GelMA achieved seamless osteochondral healing in the gap region between plugs of osteochondral defects following mosaicplasty, as early as six weeks. Moreover, the International Cartilage Repair Society score, histology score, glycosaminoglycan content, subchondral bone volume, and collagen II expression were observed to be the highest in the gap region of BSN-GelMA treated group. This improved outcome was due to bio-interactive materials, which acted as tissue fillers to bridge the gap, prevent cartilage degeneration, and promote graft survival and migration of bone marrow mesenchymal stem cells by releasing bioactive supramolecular nanofibers from the GelMA hydrogel. This study provides a powerful and applicable approach to improve gap integration after autologous mosaicplasty. It is also a promising off-the-shelf bioactive material for cell-free in situ tissue regeneration.

Computational decision-support tools for urban design to improve resilience against COVID-19 and other infectious diseases: A systematic review.

The COVID-19 pandemic highlighted the need for decision-support tools to help cities become more resilient to infectious diseases. Through urban design and planning, non-pharmaceutical interventions can be enabled, impelling behaviour change and facilitating the construction of lower risk buildings and public spaces. Computational tools, including computer simulation, statistical models, and artificial intelligence, have been used to support responses to the current pandemic as well as to the spread of previous infectious diseases. Our multidisciplinary research group systematically reviewed state-of-the-art literature to propose a toolkit that employs computational modelling for various interventions and urban design processes. We selected 109 out of 8,737 studies retrieved from databases and analysed them based on the pathogen type, transmission mode and phase, design intervention and process, as well as modelling methodology (method, goal, motivation, focus, and indication to urban design). We also explored the relationship between infectious disease and urban design, as well as computational modelling support, including specific models and parameters. The proposed toolkit will help designers, planners, and computer modellers to select relevant approaches for evaluating design decisions depending on the target disease, geographic context, design stages, and spatial and temporal scales. The findings herein can be regarded as stand-alone tools, particularly for fighting against COVID-19, or be incorporated into broader frameworks to help cities become more resilient to future disasters.

Chlamydia repurposes the actin-binding protein EPS8 to disassemble epithelial tight junctions and promote infection.

Invasive microbial pathogens often disrupt epithelial barriers, yet the mechanisms used to dismantle tight junctions are poorly understood. Here, we show that the obligate pathogen Chlamydia trachomatis uses the effector protein TepP to transiently disassemble tight junctions early during infection. TepP alters the tyrosine phosphorylation status of host proteins involved in cytoskeletal regulation, including the filamentous actin-binding protein EPS8. We determined that TepP and EPS8 are necessary and sufficient to remodel tight junctions and that the ensuing disruption of epithelial barrier function promotes secondary invasion events. The genetic deletion of EPS8 renders epithelial cells and endometrial organoids resistant to TepP-mediated tight junction remodeling. Finally, TepP and EPS8 promote infection in murine models of infections, with TepP mutants displaying defects in ascension to the upper genital tract. These findings reveal a non-canonical function of EPS8 in the disassembly of epithelial junctions and an important role for Chlamydia pathogenesis.

Msx1+ stem cells recruited by bioactive tissue engineering graft for bone regeneration.

Critical-sized bone defects often lead to non-union and full-thickness defects of the calvarium specifically still present reconstructive challenges. In this study, we show that neurotrophic supplements induce robust in vitro expansion of mesenchymal stromal cells, and in situ transplantation of neurotrophic supplements-incorporated 3D-printed hydrogel grafts promote full-thickness regeneration of critical-sized bone defects. Single-cell RNA sequencing analysis reveals that a unique atlas of in situ stem/progenitor cells is generated during the calvarial bone healing in vivo. Notably, we find a local expansion of resident Msx1+ skeletal stem cells after transplantation of the in situ cell culture system. Moreover, the enhanced calvarial bone regeneration is accompanied by an increased endochondral ossification that closely correlates to the Msx1+ skeletal stem cells. Our findings illustrate the time-saving and regenerative efficacy of in situ cell culture systems targeting major cell subpopulations in vivo for rapid bone tissue regeneration.

Autophagy activation mediates resistance to FLT3 inhibitors in acute myeloid leukemia with FLT3-ITD mutation.

BACKGROUND: Autophagy plays a critical role in drug resistance in acute myeloid leukemia (AML), including the subtype with FLT3-ITD mutation. Yet how autophagy is activated and mediates resistance to FLT3 inhibitors in FLT3-ITD-positive AML remains unsure. METHODS: We detected the expression of autophagy markers in FLT3-ITD-positive leukemic cells after vs. before acquired resistance to FLT3 inhibitors; tested the stimulative effect of acquired D835Y mutation and bone marrow micro-environment (BME) on autophagy; explored the mechanism of autophagy mediating FLT3 inhibitor resistance. RESULTS: Sorafenib-resistant cells markedly overpresented autophagy markers in comparison with sorafenib-sensitive cells or the cells before sorafenib treatment. Both acquired D835Y mutation and BME activated cytoprotective autophagy to mediate FLT3 inhibitor resistance. Autophagy activation decreased the suppression efficacy of FLT3 inhibitors on FLT3 downstream signaling and then weakened their anti-leukemia effect. Inhibition of autophagy with CQ significantly enhanced the suppressive effect of FLT3 inhibitor on FLT3 downstream signaling, in the end overcame resistance to FLT3 inhibitors. CONCLUSIONS: Autophagy might be stimulated by acquired mutation or BME, and bypass activate FLT3 downstream signaling to mediate FLT3 inhibitor resistance in FLT3-ITD-positive AML. Targeting autophagy could be a promising strategy to overcome resistance.

A high-resolution route map reveals distinct stages of chondrocyte dedifferentiation for cartilage regeneration.

Articular cartilage damage is a universal health problem. Despite recent progress, chondrocyte dedifferentiation has severely compromised the clinical outcomes of cell-based cartilage regeneration. Loss-of-function changes are frequently observed in chondrocyte expansion and other pathological conditions, but the characteristics and intermediate molecular mechanisms remain unclear. In this study, we demonstrate a time-lapse atlas of chondrocyte dedifferentiation to provide molecular details and informative biomarkers associated with clinical chondrocyte evaluation. We performed various assays, such as single-cell RNA sequencing (scRNA-seq), live-cell metabolic assays, and assays for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), to develop a biphasic dedifferentiation model consisting of early and late dedifferentiation stages. Early-stage chondrocytes exhibited a glycolytic phenotype with increased expression of genes involved in metabolism and antioxidation, whereas late-stage chondrocytes exhibited ultrastructural changes involving mitochondrial damage and stress-associated chromatin remodeling. Using the chemical inhibitor BTB06584, we revealed that early and late dedifferentiated chondrocytes possessed distinct recovery potentials from functional phenotype loss. Notably, this two-stage transition was also validated in human chondrocytes. An image-based approach was established for clinical use to efficiently predict chondrocyte plasticity using stage-specific biomarkers. Overall, this study lays a foundation to improve the quality of chondrocytes in clinical use and provides deep insights into chondrocyte dedifferentiation.

Single cell analysis reveals inhibition of angiogenesis attenuates the progression of heterotopic ossification in Mkx-/- mice.

Tendon heterotopic ossification (HO) is characterized by bone formation inside tendon tissue, which severely debilitates people in their daily life. Current therapies fail to promote functional tissue repair largely due to our limited understanding of HO pathogenesis. Here, we investigate the pathological mechanism and propose a potential treatment method for HO. Immunofluorescence assays showed that the Mohawk (MKX) expression level was decreased in human tendon HO tissue, coinciding with spontaneous HO and the upregulated expression of osteochondrogenic and angiogenic genes in the tendons of Mkx-/- mice. Single-cell RNA sequencing analyses of wild-type and Mkx-/- tendons identified three cell types and revealed the excessive activation of osteochondrogenic genes during the tenogenesis of Mkx-/- tendon cells. Single-cell analysis revealed that the gene expression program of angiogenesis, which is strongly associated with bone formation, was activated in all cell types during HO. Moreover, inhibition of angiogenesis by the small-molecule inhibitor BIBF1120 attenuated bone formation and angiogenesis in the Achilles tendons of both Mkx mutant mice and a rat traumatic model of HO. These findings provide new insights into the cellular mechanisms of tendon HO and highlight the inhibition of angiogenesis with BIBF1120 as a potential treatment strategy for HO.