Elucidating endothelin signalling in post-COVID skeletal sequelae

Abstract

The COVID-19 pandemic has posed a significant global challenge, with skeletal disorders emerging as common post-acute sequelae. Symptoms such as persistent joint aches and bone loss have been reported months after SARS-CoV-2 infection, but the exact pathomechanism remains unclear, and no effective cure has been found. Endothelial dysfunction and activation of endothelin signalling have been linked to the high prevalence of post-COVID-19 symptoms in patients. Notably, SARS-CoV-2 spike antigen has been observed in individuals with long COVID and detected in bronchial cartilage. However, the impact of endothelins and the spike protein on joint cartilage is still uncertain. This study hypothesizes that the synergistic effect of ET-1 and viral spike protein leakage from injured vessels could damage articular cartilage and growth plate in the sub-acute phase of SARS-CoV-2 infection. This research aimed to elucidate the role of endothelins in bone and joint after SARS-CoV-2 infection and explore the feasibility of using endothelin receptor blockers to treat post-COVID skeletal sequelae. We demonstrated vascular damage with upregulation of serum ET-1 after SARS-CoV­-2 infection in individuals and identified pathological damage in the vascular architecture of various organs with activation of endothelin signalling in the sub-acute phase of viral infection in hamsters. Additionally, we investigated bone and joint injuries over time in a hamster model after SARS-CoV-2 infection. In the joint, we observed cyst formation at the osteochondral junction and chondrocyte dropout with systemic activation of endothelin signalling as early as 4 days post-infection (dpi), persisting for at least 30 days. In the bone, we observed growth plate disturbance and cyst formation after viral infection, with viral spike persistence in the bone at 30 dpi, causing chondrocyte death and growth plate thinning with upregulation of ET-1 and its receptors. We also demonstrated that bone turnover was inhibited after SARS-CoV-2 infection, reducing the new bone formation. Moreover, our study deciphered that endothelin signalling increased with endothelial dysfunction, leading to the leakage of spike protein into the bone marrow cavity, resulting in osteoblast precursor and chondrocyte loss. Using a 3D micromass model, we identified that the combination of SARS-CoV-2 anti-spike protein S1, viral spike protein, and ET-1 contributed to caspase-driven chondrocyte death. We also demonstrated that viral spike RBD-induced deterioration of joint structure, chondrocyte and osteoblast precursor loss with upregulated ET-1 in a mouse model. Inhibition of endothelin signalling could mitigate these effects in vitro and in vivo. Furthermore, our data showed that in the acute phase of viral infection, inhibiting endothelin receptors using the FDA-approved medicine Macitentan ameliorated cystic lesions, mitigated growth plate disorganization, preserved chondrocyte numbers, and activated osteoblast precursors. Even delayed Macitentan treatment during the sub-acute infection phase, it still suppressed caspase-driven chondrocyte death, rescued the growth plate disturbance, and activated bone remodelling. Collectively, our findings provided a unique insight into understanding joint cartilage and growth plate defects under the synergistic interaction of viral spike and endothelin­-1 in the sub-acute phase of viral infection. Macitentan emerges as a repurposable medication candidate for treating bone and joint damage in post-acute COVID-19.