The role of death-associated protein kinase in endothelial apoptosis under fluid shear stress

Endothelial cells are the interface between hemodynamic fluid flow and vascular tissue contact. They actively translate physical and chemical stimuli into intracellular signaling cascades which in turn regulate cell function, and endothelial dysfunction leads to inflammation and diseased conditions. For example, atherosclerosis, a chronic vascular disease, favorably develops in regions of disturbed fluid flow and low shear stress. Apoptosis, or programmed cell death, must be properly regulated to maintain homeostasis in the vascular wall. The loss of apoptosis control, as seen in low shear stress regions, is implicated in various diseases such as atherosclerosis and cancer. Death-associated protein kinase, DAPK is a pro-apoptotic regulator for various cell types that is localized in the cell cytoskeleton and regulates changes in cytoplasm associated with apoptosis. Yet its role in endothelial cells remains unclear. DAPK is a positive regulator in tumor necrosis factor &agr; (TNF&agr;) induced apoptotic pathway, and DAPK expression is lost in cancer cells. In this project, we begin to assess the effect of shear stress on endothelial cell apoptosis and DAPK.^ The potential role of DAPK and its corresponding signaling pathway in endothelial mechanotransduction and the role of nuclear lamina is further evaluated.^ Using bovine aortic endothelial cells, we have shown that laminar shear stress modulates DAPK expression. Initially, our study examined the time-dependent effects of conditioning cells with shear stress on apoptosis triggered by TNF&agr;, oxidative stress, and serum depletion; and the corresponding role of endothelial DAPK. Pre-conditioning cells with shear stress for 6 hours prior to apoptosis induction, decreased downstream caspase 3/7 activity, an apoptosis signaling constituent. Similarly, we also observed a corresponding decrease in DAPK in pre-sheared cells exposed to TNF&agr;, H2O 2, or serum starvation. Post-conditioning cells with 6 hours of shear after exposure to stimuli confirmed the protective effect of laminar shear stress in the presence of each apoptotic inducer. Our data suggest that shear stress and apoptosis agents may have competing effects on DAPK expression, and shear stress suppresses apoptosis by regulating DAPK in a time-dependent manner.^ Additionally, we transitioned our study to endothelial cells on non-glass substrates, such as flexible silicone membrane normally used for cyclic strain studies. We have shown a link between shear stress and DAPK expression and apoptosis in cells on membrane. Along with biochemical and molecular signals, the hemodynamic forces that the cells experience are also important regulators of endothelial functions. We found that adding shear stress significantly suppressed TNF&agr; induced apoptosis in cells; while shearing cells alone also increased apoptosis on either substrate. These data suggest that shear stress induced apoptosis in endothelial cells via increased DAPK expression and activation as well as caspase-3/7 activity. Most in vitro shear stress studies utilize the conventional parallel plate flow chamber where cells are cultured on glass, which is much stiffer than what cells encounter in vivo. Other mechanotransduction studies have utilized the flexible silicone membrane as substrate, for example, in cyclic stretch studies. Thus, this study bridges the gap between shear stress studies on cells plated on glass to future studies on different stiffness of substrates or mechanical stimulation such as cyclic strain.^ The nuclear lamina plays an important role in nuclear envelope structural support and connect to the cytoskeletal network through various co-localized proteins. Similarly, lamin is an integral part of the apoptosis process due to its direct linkage to the cytoskeleton and nuclear membrane. Conversely, lamin deficient cells lack nuclear structural integrity and the role of lamin in stress response is not completely understood. Mutations in lamin lead to changes in nuclear membrane mechanics eventually altering downstream signal response to stress. Therefore, we also looked at the impact of lamin deficiency in both MEF and BAEC cells on DAPK activity and apoptosis. We investigated the normally positive effects of shear on lamin deficient cells. The loss of lamin expression made cells more susceptible to shear stress and increased overall cell turnover compared to sheared control cells. Also, our results suggest the loss of lamin A/C expression mitigates TNF&agr;–induced apoptosis and affects its associated signaling pathways.^ In summary, the work presented in this dissertation has highlighted the role of DAPK in both mechanotransduction and endothelial apoptosis. Ultimately, this study demonstrates a key role lamin plays in mechanotransduction and apoptosis in response to both chemical and mechanical stimulation. By understanding how cells regulate phenotypic and genotypic changes in response to stimulation, we can better address disease and develop effective therapeutics

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival