Developing a Senomorphic Treatment Strategy in Osteoarthritis

Osteoarthritis (OA) is the most common form of arthritis in the population worldwide, resulting in significant disabilities. Currently, no treatments can prevent or reverse the development of OA. Cellular senescence has been identified as a major contributing factor to OA. Therefore, a therapy targeting senescence could be an effective treatment for OA. Several strategies have been proposed to target senescence in OA, including neutralizing agents for the senescence-associated phenotypes (SASPs), senolytics for eliminating senescent cells, and senomorphics for modifying the senescence phenotype. In fact, a senolytic, UBX0101, showed a protective effect for post-traumatic OA (PTOA) development in mouse models by reducing both OA histological grading and OA related pain. However, it failed to meet the primary endpoint of relieving symptoms in a clinical phase 2 trial. The failure of the human trial may be related to a high placebo response rate from the control group or, more likely, the heterogeneous phenotypes involved in human OA disease differ from the PTOA in the mouse model. The lack of available senescent-specific biomarkers, which could be used to refine the phenotype of the subject enrollment or to monitor the occurrence of senescence presents a challenge to evaluate a trial successfully. Alternatively, senolytics may be detrimental in tissue with a higher proportion of senescent cells. After treatment, the remaining cells may not be able to maintain the integrity of the cartilage. Therefore, in the present study, we investigated the association of cellular senescence with OA disease severity, identified a biomarker dipeptidyl peptidase-4 (DPP4) for chondrocyte senescence and OA progression, and proposed a senomorphic treatment using chromobox 4 (CBX4) for modulating cell function of the replicative senescent model WI-38 cells and human osteoarthritic chondrocytes.First, we investigated the association of OA disease severity in human knee joints with the percentage of cells expressing senescence-associated β-galactosidase activity (SA-β-gal) and p16. We assessed three regions within the tibial plateau of the knee corresponding to a gradient of disease severities in tissue procured from human medial-dominant OA. We found that SA-β-gal and p16 were positively correlated with OA severity. Our result suggested cellular senescence could be involved in OA progression, and targeting senescence could be a promising treatment for OA. Subsequently, we identified Dipeptidyl-peptidase 4 (DPP4, also known as CD26) as a potential biomarker for OA senescence. We isolated chondrocytes from knee OA cartilage and determined the association of DPP4 expression with senescence markers, SA-β-gal and p16, by flow-cytometry. We also compared the expression of anabolic and catabolic genes, senescence-related genes, and senescence-associated secretory phenotypes (SASPs) in DPP4+ and DPP4- cells, isolated by two different methods: fluorescence-activated cell sorting and magnetic-activated cell sorting. Additionally, we quantified soluble DPP4 in synovial fluid (SF) and assessed its association with radiographic knee OA. DPP4 expression was associated with higher SA-β-gal, p16 expression, senescence-related gene and catabolic gene expression, SASPs secretion, and lower anabolic gene expression in chondrocytes. In addition, SF DPP4 was significantly associated with radiographic knee OA progression (β=4.657 p=0.015). Next, we identified the senomorphic effect of CBX4 in WI-38 cells. We first observed the decrease of CBX4 protein expression and increased senescence markers and gene expression during WI-38 serial culture. We next evaluated the presence of senescence markers and expression of senescence-related genes in the CBX4 activation and CBX4 knockdown compared to controls in pre-senescent WI-38. Compared to the control group, knockdown of CBX4 increased cellular senescence, whereas activation of CBX4 decreased senescence in the pre-senescent WI-38 cells. Based on these results, we identified that CBX4 regulates replicative senescence in WI-38 cells and functions as a senomorphic and potential anti-senescence target. Additionally, we explored the mechanisms of senescence regulation from CBX4 domains by using CBX4 mutated lentiviral particles and compared them with CBX4 wildtype in WI-38 cells. CDM, SIMs, and C-box domains are all involved in the regulation of senescence by CBX4; where CDM is mainly involved in cell cycle regulation, SIMs are involved in the cell proliferation, DNA damage repair, and SASPs secretion, and C-box is related to cell proliferation and SASPs secretion. Taken together, CBX4 is a multi-functional protein, and these mutants elucidated the different non-overlapping functions in senescence regulation. Finally, we identified the CBX4 senomorphic effect in osteoarthritic chondrocytes by comparing CBX4 wildtype and control transduced cells. Compared to control, CBX4 overexpressed chondrocytes demonstrated lower DPP4 expression and higher proliferation marker EdU. Overall, our study demonstrated that cellular senescence is positively correlated with OA disease severity, identified DPP4 as a potential biomarker for cellular senescence in OA, and explored CBX4 as a potential senomorphic treatment in human WI-38 fibroblasts and OA chondrocytes.