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Blockade of endothelin receptors mitigates SARS-CoV-2-induced osteoarthritis

Nature Microbiology volume 9, pages 2538–2552 (2024)Cite this article

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A Publisher Correction to this article was published on 16 September 2024

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Abstract

Joint pain and osteoarthritis can occur as coronavirus disease 2019 (COVID-19) sequelae after infection. However, little is known about the damage to articular cartilage. Here we illustrate knee joint damage after wild-type, Delta and Omicron variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in vivo. Rapid joint injury with cystic lesions at the osteochondral junction was observed in two patients with post-COVID osteoarthritis and recapitulated in a golden Syrian hamster model. SARS-CoV-2-activated endothelin-1 signalling increased vascular permeability and caused viral spike proteins leakage into the subchondral bone. Osteoclast activation, chondrocyte dropout and cyst formation were confirmed histologically. The US Food and Drug Administration-approved endothelin receptor antagonist, macitentan, mitigated cystic lesions and preserved chondrocyte number in the acute phase of viral infection in hamsters. Delayed macitentan treatment at post-acute infection phase alleviated chondrocyte senescence and restored subchondral bone loss. It is worth noting that it could also attenuate viral spike-induced joint pain. Our work suggests endothelin receptor blockade as a novel therapeutic strategy for post-COVID arthritis.

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Fig. 1: Rapid joint destruction in patients with COVID—case report.
Fig. 2: SARS-CoV-2 infection induces osteochondral damage in hamsters.
Fig. 3: SARS-CoV-2 spike causes endothelial dysfunction and chondrocyte senescence.
Fig. 4: Viral spike contributes to joint pain and chondrocyte senescence.
Fig. 5: Early macitentan treatment partially rescues SARS-Cov-2-associated osteochondral damage.
Fig. 6: Delayed macitentan treatment restores subchondral bone remodelling at sub-acute phase of SARS-CoV-2 infection.

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Data availability

The authors declare that the data supporting the findings of this study are available within the Article and Supplementary Information tables. RNA-seq data are available under GEO Accession Viewer with GSE267009. All the raw data are also available on Sequence Read Archive with BioProject number PRJNA1104007. The graphs derived from the datasets that are used in the manuscript were also uploaded to GitHub at https://github.com/MZ1808/DataDeposit/tree/main. The public scRNA sequencing datasets used in this study can be downloaded from NCBI Gene Expression Omnibus (GEO) (https://www.ncbi.nlm.nih.gov/geo/) at GSE122960, GSE149878 and GSE171668. Source data are provided with this paper.

Code availability

Custom code for bulk RNA and single-cell sequencing analysis performed in this study are publicly available via Zenodo at https://zenodo.org/doi/10.5281/zenodo.12561715 (ref. 56) and https://zenodo.org/doi/10.5281/zenodo.12565023 (ref. 57).

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Acknowledgements

This work was supported by Health and Medical Research Fund (16172691# to C.W. and 07210107# to S.Y.), the Food and Health Bureau, The Government of the Hong Kong Special Administrative Region; General Research Fund (PolyU 151061/20M, PolyU15100821M, 15100324 to C.W.), Collaborative Research Fund (C7060-21G and C7002-23Y to S.Y.) and Theme-Based Research Scheme (T11-709/21-N to S.Y.), the Research Grants Council of the Hong Kong Special Administrative Region; The National Natural Science Foundation of China (NSFC) and the Research Grants Council (RGC) (NFSC/RGC) schemes (N_PolyU 520/20 to C.W. and N_HKU767/22 to S.Y.); The Innovation and Technology Fund Mainland-Hong Kong Joint Funding Scheme (ITF MHKJFS) (MHP/011/20 to C.W.); the Hong Kong Polytechnic University (HKPU) Project of Strategic Importance (ZE2C to C.W.); Health@InnoHK, Innovation and Technology Commission, the Government of the Hong Kong Special Administrative Region; National Key Research and Development Program of China (2021YFC0866100 and 2023YFC3041600 to S.Y.); National Natural Science Foundation of China (32322087 and 32300134 to S.Y.); and PolyU SZRI Seed Fund (I2022A006 to C.W.). We thank D. Chan from the HKU, L. Rong from Sun Yat-Sen University and M. Yang from the HKPU for the cell lines used in this study. We also thank E. Lau and M. Yuen from the University Research Facility in Life Sciences of the HKPU for providing technical support of micro-MRI scanning and high-resolution microscopy. We likewise thank J. Liu and Y. Zhang from the Department of Biomedical Engineering of HKPU for providing imaging assistance for micro-MRI and µCT scanning.

Author information

Author notes

  1. These authors contributed equally: Man Ting Au, Junguo Ni, Kaiming Tang.

Authors and Affiliations

  1. Department of Biomedical Engineering, The Hong Kong Polytechnic University, Hong Kong Special Administrative Region, China

    Man Ting Au, Junguo Ni, Wei Wang, Lanlan Zhang, Hantang Wang, Fangyi Zhao, Zhan Li, Charlie Yuli Zhang, Tianshu Jiang, Marianne Lauwers & Chunyi Wen

  2. State Key Laboratory of Emerging Infectious Diseases, Department of Microbiology, School of Clinical Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong Special Administrative Region, China

    Kaiming Tang, Peng Luo, Cuiting Luo, Jasper Fuk-Woo Chan & Shuofeng Yuan

  3. Department of Orthopaedics and Traumatology, School of Clinical Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong Special Administrative Region, China

    Lawrence Chun-Man Lau & Ping-Keung Chan

  4. Department of Biology, School of Medicine, Southern University of Science and Technology, Shenzhen, China

    Bo Zhou

  5. State Key Laboratory of Environmental and Biological Analysis, Hong Kong Baptist University, Hong Kong Special Administrative Region, China

    Lin Zhu

  6. Department of Infectious Disease and Microbiology, The University of Hong Kong-Shenzhen Hospital, Shenzhen, China

    Jasper Fuk-Woo Chan & Shuofeng Yuan

  7. Centre for Virology, Vaccinology and Therapeutics, Hong Kong Science and Technology Park, Hong Kong Special Administrative Region, China

    Jasper Fuk-Woo Chan & Shuofeng Yuan

  8. Research Institute for Smart Ageing, The Hong Kong Polytechnic University, Hong Kong Special Administrative Region, China

    Chunyi Wen

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Contributions

C.W., S.Y., M.T.A. and J.N. conceived and designed the experiments. M.T.A., J.N., K.T., H.W., F.Z., Z.L., C.L., L. Zhu, C.Y.Z., T.J. and M.L. performed experiments and analysed data. M.T.A., C.W., L.C.-M.L., S.Y. and K.T. wrote the manuscript. L.C.-M.L., W.W. and P.-K.C. did the retrospective clinical study. B.Z., L. Zhang and P.L. performed sequencing analysis. C.W., S.Y. and J.F.-W.C. oversaw the conception, supervised the study and provided writing advice. C.W. and S.Y. provided the grant support.

Corresponding authors

Correspondence to Shuofeng Yuan or Chunyi Wen.

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The authors declare no competing interests.

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Nature Microbiology thanks Suresh Mahalingam and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Clinical CT images showing cyst formation at the osteochondral junction of COVID patients.

X-ray images of knee joints from 5 individuals. Cysts formation (yellow rectangle) was observed in medial compartment of knee in COVID patients. Collapse of tibial plateau on medial side in the post-COVID OA patient was shown. Scale bar, 1 cm.

Source data

Extended Data Fig. 2 Structural change of knee joint and chondrocyte senescence upon infection of different SARS-CoV-2 variants.

a, Experimental design to study the effect of different variants of SARS-CoV-2 on knee joints of golden Syrian hamsters at 4 dpi. b, c, Representative MRI images and quantitative measurement of synovium area in mock and infected hamsters. Segmentation of each sample was shown at the bottom right corner. n = 3 for each group, both hind limbs were scanned excluding one leg from Delta group due to distorted joint capsule. The arrow indicates the hyperintensive region in synovium after virus infection. Scale bar, 1 mm. Mann-Whitney test. d, MicroCT and 3D rendering of microCT images showing bulges on the subchondral bone plate surface. Each data point represents the number of bulges per sample. n = 3 for each group. Data are mean ± S.E.M. Kruskal-Wallis test with Dunn’s multiple comparisons test. Scale bar, 200 µm. e, MicroCT images of knee joints at comparable positions showing no meniscal extrusion after SARS-CoV-2 infection. Scale bar, 1 mm. f, Representative immunostaining images and quantification for p15INK4b and p21 in mock-infected group and different time points after infection. n = 5 for each group. Data are mean ± S.E.M. One-way ANOVA with Tukey’s post hoc test unless otherwise specified.

Extended Data Fig. 3 Expression pattern of inflammatory cytokines at local joint tissue.

Representative immunostaining images for IL-1β, IL-6 and TNF-α at (a) articular cartilage, (b) subchondral bone and (c) synovium underwent mock or wildtype SARS-CoV-2 infection with macitentan treatment. Mock, n = 5; 4 dpi PBS, n = 5; 4 dpi Mac, n = 4; 30 dpi PBS, n = 5; 30 dpi Mac; n = 5 for IL-6 and TNF-α. Only n = 1 for each group was performed for IL-1β.

Extended Data Fig. 4 Integrative analysis of public scSeq datasets of lungs from normal subjects and COVID patients.

a, UMAP visualization of endothelial sub-groups from healthy subjects and COVID-19 patients. b, Boxplot showing individual proportions of endothelial sub-groups in healthy (n = 8) and COVID-19 patients (n = 11). c, Boxplot showing differential gene expression between healthy (n = 8) and COVID-19 patients (n = 11) in IL7R+ population. The boxplot compactly displays the distribution of a continuous variable. It visualises five summary statistics (the median, two hinges and two whiskers). The upper and lower hinges correspond to the first and third quartiles. The upper and lower whiskers extend from the hinge to the highest or lowest value that is within 1.5 * IQR of the hinge, where IQR is the inter-quartile range. The data in panels b and c are mean ± S.E.M. Wilcoxon rank-sum test.

Extended Data Fig. 5 SARS-CoV-2 viral proteins damage endothelial cells and accumulate near blood vessels.

a, b, Representative images of (a) CellROX and (b) MitoTracker staining showing accumulation of oxidative stress and changes in mitochondrial dynamics in HUVEC after SP RBD (10 µg/ml) treatment for 1 hour. Hydrogen peroxide at 100µM was used as positive control. Blob form structures were indicated by arrows. Three independent experiments. c, Representative immunostaining for vWF and SARS-CoV-2 nucleocapsid protein (NP) in showing nucleocapsid proteins located near dysfunctional blood vessels after infection. Arrows indicate nucleocapsid proteins. d, e, Representative images of (d) SARS-CoV-2 nucleocapsid protein (NP) (brown) and (e) SARS-CoV-2 spike protein (SP) (brown) in subchondral bone marrow cavity at different time points after infection. n = 5 per time point. Semi-quantification of NP and SP was performed based on integrated optical density (IOD). The grading method is shown in Supplementary Table 1.

Extended Data Fig. 6 Distribution of viral copies and components in different parts of the joint after SARS-CoV-2 infection.

a, b, c, Relative mRNA expression of (a) RdRp, (b) envelop gene and (c) nucleocapsid gene detected in different parts of a hamster knee joint after 4 days of mock or SARS-CoV-2 infection. Each data point represents sample obtained from one animal, except 3 animals were pooled for one data point for articular cartilage to get sufficient RNA. Multiple unpaired t-tests. d, Representative immunostaining images for spike protein in different regions of hamster knee joints with and without macitentan treatment after SARS-CoV-2 infection. Arrowheads indicate positive signals. Magnified figures are shown in the bottom left corner of each figure. Mock, n = 5; 4 dpi PBS, n = 5; 4 dpi Mac, n = 4; 30 dpi PBS, n = 5; 30 dpi Mac; n = 5.

Extended Data Fig. 7 SARS-CoV-2 caused upregulation of ET-1 and oxidative stress in chondrocytes.

a, Representative immunostaining images and quantification for ET-1 at articular cartilage at different times of infection. Treatment effect with macitentan was also shown. Mock, n = 5; 4 dpi PBS, n = 5; 4 dpi Mac, n = 4; 30 dpi PBS, n = 5; 30 dpi Mac; n = 5. Data are mean ± S.E.M. Data were excluded when they fell out of 1.5 IQR. One-way ANOVA with Tukey’s multiple-comparison. b, c, Representative images of (b) CellROX and (c) MitoTracker staining showing accumulation of oxidative stress and changes in mitochondrial dynamics in C28/I2 after SP RBD (10 µg/ml) treatment for 1 hour. C28/I2 cells were pre-treated with macitentan for 30 minutes before stimulation by SP RBD for 1 h. Three independent biological repeats.

Extended Data Fig. 8 RNA sequencing of human mesenchymal stem cells-differentiated chondrocytes after SARS-CoV-2 spike protein stimulation.

a, b, Top upregulated KEGG pathways comparing (a) SAE or (b) SP-treated hMSCs-differentiated chondrocytes with control. n = 3 for each group. Cellular senescence pathway was upregulated only in SAE-treated cells. c, Heatmap showing selected differentially expressed genes (DEGs) related to cellular senescence with p < 0.05, highlighting p15 and p21. d, Heatmap showing upregulation of inflammatory cytokines (yellow arrows) and increased OPG/RANKL ratio (red arrows) under stimulation by the combination of spike protein, anti-spike protein antibody S1 and endothelin-1 (SAE). Three biological replicates for each group.

Extended Data Fig. 9 Endothelin receptor antagonism restored spike protein-induced alteration of RANKL/OPG balance and bone microstructure.

a, Schematic design for the cellular experiment to study the effect of endothelin receptor blockers on chondrocyte catabolism. b, Relative mRNA expression of MMP1 and p16INK4a in C28/I2 after RBD stimulation with endothelin receptor blockers pre-treatment. n = 3. Two-way ANOVA with uncorrected Fisher’s LSD. c, Relative mRNA expression of OPG, RANKL and OPG/RANKL ratio in HUVEC upon stimulation of ET-1 and 10 µg/ml SP for 24 h. One-way ANOVA with Tukey’s multiple comparison. d, Relative mRNA ratio of OPG, RANKL and RANKL/OPG in C28/I2 upon stimulation by SP and ET-1, with the treatment effect of endothelin receptor blockers. Two-tailed unpaired t-test. Three independent biological repeats for c and d. e, Quantification based on micro-CT reconstruction of the proximal epiphysis of tibias. Vehicle (n = 4); SP RBD (n = 4); Low Mac (n = 4); High Mac (n = 5). One-tailed non-parametric Mann-Whitney test. The data in panels b-e are mean ± S.E.M.

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Supplementary Table 1

Semi-quantification of SARS-CoV-2 viral components. a, Grading of the presence of SARS-CoV-2 nucleocapsid protein (NP) in bone marrow cavity in subchondral bone region in tibias of hamsters based on integrated optical density (IOD) in per mille (‰). IODs of all sections were divided into three categories from ‘−’, indicating the lowest abundance, to ‘++’, indicating the highest abundance. b, Table showing the number of samples in each grade based on IOD. Chi-square test. c, Grading of the presence of SARS-CoV-2 SP in bone marrow cavity in subchondral bone region in tibias of hamsters based on IOD in percentage (%). IODs of all sections were divided into four categories from ‘−’, indicating the lowest abundance, to ‘+++’, indicating the highest abundance. d, Table showing the number of samples in each grade based on IOD. Mock (n = 5); 4 d.p.i. PBS (n = 5); 4 d.p.i. Mac (n = 4); 30 d.p.i. PBS (n = 5) and 30 d.p.i. Mac (n = 5). Chi-square test.

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Au, M.T., Ni, J., Tang, K. et al. Blockade of endothelin receptors mitigates SARS-CoV-2-induced osteoarthritis. Nat Microbiol 9, 2538–2552 (2024). https://doi.org/10.1038/s41564-024-01802-x

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