Accumulation of misfolded protein within the endoplasmic reticulum (ER) is a central event leading to cell death that contributes to disease pathogenesis. Although proteins exhibit oxidative damage in diverse disease states, the relationship between protein misfolding and oxidative stress has not been explored. To elucidate the relationship between protein misfolding and oxidative stress, we have analyzed expression of the clotting factor VIII (FVIII), the protein deficient in hemophilia A and prone to misfolding. Although hemophilia A patients are treated by frequent infusions of plasma-derived or recombinant-derived FVIII, significant limitations remain. It is hoped that FVIII gene therapy will solve these problems. Unfortunately, limited clinical studies have not demonstrated expression of FVIII at therapeutic levels. At the cellular level FVIII expression is limited due to protein misfolding and retention in the ER. As a consequence, FVIII expression induces transcription of ER stress response genes, through an intracellular signaling pathway called the unfolded protein response (UPR). Our studies have shown that the chronic unresolved accumulation of unfolded FVIII in the ER leads to apoptosis in a manner that requires the proapoptotic transcription factor C/EBP homologous protein CHOP. Recently, we have demonstrated that FVIII misfolding causes oxidative stress and induces an inflammatory response. In addition, oxidative stress causes FVIII misfolding, thereby creating a vicious cycle between FVIII misfolding and oxidative stress. Profoundly, anti-oxidant treatment to reduce oxidative stress improves FVIII secretion and reduces apoptosis. These findings provide the basis of the proposed research to elucidate how FVIII induces apoptotic, oxidative, and inflammatory response pathways. The studies will test the hypothesis that FVIII expression is limited due to induction of these stress responses. Studies will test whether intervention to prevent these toxic responses may improve therapeutic efficacy in FVIII gene delivery for hemophilia A. The findings from the proposed studies will provide fundamental new insights toward elucidating how protein misfolding in the ER signals a cell death response and should have impact on a number of disease states associated with ER stress.