INTRODUCTION: Osteoarthritis (OA) is a debilitating disease of the joints, decreasing the quality of life among all populations. Although many of the factors that cause cartilage breakdown during OA are understood, the root cause for the secretion of these molecules is yet to be determined. Recently, our lab has studied endothelin-1 (ET1) in search of a possible regulator for OA. ET1 has been associated with pathogenesis in multiple tissues, however, its specific role in OA remains unclear. This study will investigate a link between ET1 and OA by analyzing mRNA transcripts, protein expression, and extracellular matrix (ECM) deposition in human mesenchymal stem cells (hMSCs), chondrocytes, and osteoblasts after ET1 exposure. Catabolic factors that break down cartilage and bone during OA include metalloproteinases (MMPs) and aggrecanases (ADAMTS). Specifically, MMP-13, MMP-2, and ADAMTS-4 are of interest for their role in disrupting cartilage components and altering mechanical properties. 


METHODS: Four donor hMSC lines were utilized for the in vitro component of this study. Each biological replicate was studied with three technical replicates. These cells were obtained from tissue that was donated via informed consent after total hip replacement surgery. After isolating and expanding the multipotent stem cells from the donor tissue, they were differentiated into both bone and cartilage for 21 days. The control group was not exposed to ET1 during expansion or differentiation, while the experimental group was pretreated with ET1 for two passages during expansion and treated throughout differentiation. Expansion and differentiation medium were changed every 72 hours, along with fresh administration of ET1 in the experimental group. Gene expression of ECM components, MMPs and ADAMTS were analyzed via real-time PCR. Quantification of ECM deposition and histology staining were performed to corroborate mRNA expression. Glycosaminoglycan content was analyzed via DMMB assay in chondrogenic pellets and alcian blue staining was used to view proteoglycan localization. Calcium mineralization in osteogenic tissue culture plates was extracted with HCL and quantified; alizarin red staining was used to image calcium deposition. Immunofluorescent staining of chondrogenic pellets was used to image MMP13 localization.   



RESULTS SECTION: All experiments were performed with three technical replicates and four biological replicates. Statistical significance was determined by Tukey’s post hoc t-test, with alpha as 0.05. Culture of hMSCs with ET1 did not produce any mitogenic effects during expansion culture. In vitro differentiation of hMSCs to chondrocytes in the presence of ET1 yielded significant reduction in MMP2, MMP13, and ADAMTS4 in comparison to the control. This effect was shown to be proportional to ET1 concentration in culture medium; a dose-dependent reduction in MMP13 expression after chondrogenesis, with p < 0.01 for the 0.01 µM group, and p < 0.001 for the 0.1 µM group when compared to the control. In vitro differentiation of hMSCs to osteoblasts in the presence of ET1 yielded a significant reduction in MMP13 (p<0.01). GAG and calcium quantification showed significant increase in ECM deposition for treated groups (p<0.05). This was corroborated by denser alcian blue and alizarin red staining. MMP13 localization via anti-MMP13 displayed reduced MMP13 presence at the protein level for chondrogenic pellets treated with ET1. 


DISCUSSION: While it is known that MMPs and ADAMTS are upregulated during OA, the root causes for these changes in expression are unknown. In our previous work, treatment with ET1 yielded significant reductions in MMP2, MMP13 and ADAMTS4 expression in hMSCs that were differentiated to chondrocytes. This result was confirmed during osteoblast differentiation using the same cell lines. Because MMPs and ADAMTS contribute to bone and cartilage degradation, it is reasonable to suggest that ET1 combats these events as they occur in patients with OA. It is well established that MMP13 concentrations are increased in aged or damaged joints, which results in cartilage loss and joint instability. Healthy bone and cartilage are constantly being synthesized and degraded to remodel and adapt to mechanical stressors; proper resorption of bone and cartilage is required for this process to be effective, but overactivity causes joint instability. Studies have also shown that both ET1 and MMP13 are increased in the joint space of a patient with OA. This association is one of the motivators behind this abstract. Whether ET1 attenuates the damaging metalloproteinase-directed degradation or disrupts the delicate remodeling of bone and cartilage is unknown. Thus far, we have shown ET1 plays a clear role in ADAMTS and MMP expression in bone and cartilage. In vivo studies will provide additional context to this question and provide a biological model for the true role of ET1 in joint disease.


SIGNIFICANCE/CLINICAL RELEVANCE: Osteoarthritis is the leading cause of physical disability in the United States, but the mechanisms behind the disease are yet to be discovered. Given the large combination of contributing factors and sheer complexity of joint disease, we are far from understanding this debilitating problem. If ET1 truly alters the catabolic processes in cartilage and bone via mesenchymal stem cells, it is worth pursuing for potential therapeutic benefits.

0389 - Investigating Endothelin-1 as a Possible Biomarker and Regulator of Osteoarthritis