Hemophilia is an X-linked genetic disorder that prevents blood from clotting normally due to a deficiency in either coagulation factor VIII or factor IX. Uncontrolled internal bleeding can occur during surgery and from blunt force trauma-induced breakage of blood vessels in the synovial membrane. Recurrent episodes of hemarthrosis lead to complete destruction of the articular cartilage, known as hemophilic arthropathy. While the exact mechanisms of blood-induced cartilage damage remain unclear, apoptosis is implicated as the primary form of regulated cell death (RCD) in chondrocytes due to elevated concentrations of pro-inflammatory cytokines. However, other forms of RCD may play roles in inducing damage to cartilage as well as the surrounding synovium. Ferroptosis is a recently discovered, iron-dependent, nonapoptotic form of RCD associated with excessive intracellular accumulation of lipid hydroperoxides formed from free hydroxyl radicals and polyunsaturated fatty acids. This proposal explores ferroptosis as a potential mechanism of joint tissue damage caused by excess intra-articular iron released from blood. In addition, ferroptosis inhibitors such as Ferrostatin-1, which prevent the formation of lipid hydroperoxides, are explored as potential therapeutics against blood-induced cell death. This new F31 proposal will fill a large gap in the current understanding of hemophilic arthropathy. Better understanding of the dose-response of blood exposure and duration may yield potential therapeutic windows of intervention that abrogate the sequela of joint bleeding. The role of physiologic joint loading and the synovium on blood-induced cartilage damage will also be addressed using a synovial joint model system coupled with modern bioengineering and molecular biology techniques (e.g., metabolomics). Isolating the effects of blood on cartilage, synovium, and their co-culture will inform new targets aimed at chondroprotection from joint bleeding. Hypothesis 1: Blood-induced cartilage damage is due in part to ferroptosis of articular chondrocytes.
Specific Aim 1 : A) Perform dose-response to blood on mechanical and biochemical properties of articular cartilage. Assess relative contribution of necrosis, apoptosis and ferroptosis in blood-induced cartilage damage. B) Study respective contribution of blood constituents to joint tissue damage and compare with ferroptosis inducers. C) Subject blood to fluid-induced shear and monitor erythrocyte viability and blood-cell related products in the synovial fluid. D) Assess ability of Ferrostatin-1 to mitigate blood-induced changes to cartilage. Hypothesis 2: Blood related cartilage damage is exacerbated by blood-induced changes to synovium.
Specific Aim 2 : Repeat Specific Aim 1 on synovium and cartilage-synovium co-culture. A) Perform reciprocating shear of synovium-on-cartilage and cartilage-on-cartilage. B) Perform conditioned media experiments that transfer media from reciprocal shear loading of cartilage-on-glass to synovium culture or synovium-on-glass to cartilage culture. Perform no-loading controls.
Although hemophilic arthropathy is the biggest cause of morbidity in patients suffering from hemophilia, the exact mechanisms behind blood-induced cartilage damage remain unclear. Elucidating specific causes of chondrocyte cell death as well as the detrimental doses and timing of blood exposure will inform therapeutic windows for treatment and novel targets for chondroprotection.
