Exploiting the role of endothelin-1 and transforming growth factor beta-1 for subchondral bone remodeling in osteoarthritis

Abstract

Osteoarthritis (OA) is the most common type of arthritis and it is the leading cause of disability of the ageing population. As life expectancy increases along with medical advance, the demand for medical care will continue to grow as population ages and cause a great socioeconomic burden of health systems around the world. Radiography is widely used to confirm the diagnosis or assess the severity of OA. The radiographic hallmarks of knee OA include subchondral sclerosis and asymmetric joint cartilage loss. It was once believed that OA was just a simple “wear-and-tear” process. However, mounting evidence showed that it is associated with systemic inflammatory and other metabolic syndromes.

Endothelin-1 (ET-1), a potent vasoconstrictive peptide causing systemic hypertension, has been found to control the cellular behavior of osteoblast. It can increase the osteoblastic proliferation and differentiation and alter the mineralization process. ET-1 is also involved in fibrogenesis. It stimulates the myofibroblasts to secret abundant type I collagen and form scar tissues. Fibrogensis and OA pathogenesis share the characteristic in which the type I collagen is overproduced. Therefore, we suggested that ET-1 induces the fibrotic phenotype in osteoblast and potentially affect the OA progression. To test our hypothesis, in vitro and in vivo experiments were set up.

To exclude the effect of ETs on bone marrow mesenchymal stem cells in differentiating into non-osteogenic lineage, active osteoblastic cells from subchondral bone of OA patients were selected for studying the role of ET-1/transforming growth factor beta-1 (TGFβ1) with regards to OA development. The cells were first characterized by genes expression pattern, ALP activity and flow cytometry. Then, TGFβ1, a known inflammatory cytokines that associated with the onset of OA was added to the cell culture in addition with ET-1. Fibrogenic markers such as type I collagen and alpha smooth muscle actin (αSMA) were selected as the targeted proteins. The transcriptional and translational levels of the two markers were detected. The mRNA expression and protein synthesis of type I collagen and αSMA were found to be elevated under TGFβ1 and ET-1 treatments. Protein kinase B (PI3K) and mothers against decapentaplegic homolog protein (Smad) inhibitors were added to check potential pathways. Results showed that the enhancement of type I collagen and αSMA were mainly through Smad pathway.

In animal experiments, transgenic mouse line (TET) overexpressing ET-1 in endothelial cells was used. 8-week-old male TET mice were underwent anterior cruciate ligament transection (ACLT) surgery to induce OA phenotype. Knees with surgery in TET mice had a higher bone volume/ total volume ratio (BV/TV) and bone mineral density (BMD) when compared with the non-transgenic mice. The immunofluorescence data showed that TET mice had relatively more fibrogenic phenotype. The in vivo data suggested that ET-1 favors the sclerotic bone formation.

Taken together, our data suggested that a cooperative role of ET-1 and TGFβ1 in promoting fibrogenesis in subchondral osteoblastic cells and resulted in highly mineralized bone structure. Thus, it may provide clinical insights for fine-tuning systemic ET-1 in patients suffered from ET-1 related hypertension in compromising the OA progression.