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Item Differential HDAC6 Activity Modulates Ciliogenesis and Subsequent Mechanosensing of Endothelial Cells Derived from Pluripotent Stem Cells(Elsevier, 2018-07-24) Smith, Quinton; Macklin, Bria; Chan, Xin Yi; Jones, Hannah; Trempel, Michelle; Yoder, Mervin C.; Gerecht, Sharon; Pediatrics, School of MedicineSummary The role of primary cilia in mechanosensation is essential in endothelial cell (EC) shear responsiveness. Here, we find that venous, capillary, and progenitor ECs respond to shear stress in vitro in a cilia-dependent manner. We then demonstrate that primary cilia assembly in human induced pluripotent stem cell (hiPSC)-derived ECs varies between different cell lines with marginal influence of differentiation protocol. hiPSC-derived ECs lacking cilia do not align to shear stress, lack stress fiber assembly, have uncoordinated migration during wound closure in vitro, and have aberrant calcium influx upon shear exposure. Transcriptional analysis reveals variation in regulatory genes involved in ciliogenesis among different hiPSC-derived ECs. Moreover, inhibition of histone deacetylase 6 (HDAC6) activity in hiPSC-ECs lacking cilia rescues cilia formation and restores mechanical sensing. Taken together, these results show the importance of primary cilia in hiPSC-EC mechano-responsiveness and its modulation through HDAC6 activity varies among hiPSC-ECs.Item Expression of Nuclear Lamin Proteins in Endothelial Cells is Sensitive to Cell Passage and Fluid Shear Stress(Springer, 2018-02) Jiang, Yizhi; Ji, Julie Y.; Engineering Technology, School of Engineering and TechnologyIntroduction Vascular cells are regulated by continuous hemodynamic forces in vivo, and mechanical forces such as shear stress are proposed to involve in the progression of cardiovascular diseases such as atherosclerosis. Lamin A/C makes up the nuclear lamina, which structurally supports the nucleus while also functionally participates in chromatin organization and gene transcription. Diseases caused by lamin or other nuclear proteins are called laminopathies. One example, Hutchinson Gilford Progeria Syndrome (HGPS) where young patients show signs of accelerated aging, is caused by de novo mutations on the lamin A/C gene. Vasculature of HGPS patients shares many similarities with people of advanced age, suggesting a role for lamin in vascular aging. Methods In this study, we examined how arterial shear stress affects lamin A/C expression in bovine aortic endothelial cells at different population doubling levels (PDL). We also used fluorescence image analysis to examine nuclear shape changes with shear stress and PDL. Results Our results suggest that laminar shear stress downregulated lamin A/C expression in low PDL cells, but the effect was reversed in high PDL cells. Nuclear shape changes were more prominent after shear stress in low PDL cells. Moreover, lamin A/C accumulated more at the nuclear periphery after exposure to shear stress. Conclusions Overall, our results indicate that both shear stress and cell passage can have an impact on lamin expressions at transcriptional and translational levels, as we continue to understand the effect of shear stress on endothelial lamina as part of the vascular aging process.Item Mechano-sensitivity of nuclear lamin proteins in endothelial cells(2016-07-22) Jiang, Yizhi; Ji, Julie Ying Hui; Na, Sungsoo; Wallace, JosephAtherosclerosis is a chronic disease that happens mostly in aged people, and recently studies have showed many similarities between Hutchinson Gilford Progeria Syndrome (HGPS) cells and aging cells, implicating dysfunctions of lamin A/C in aging process and atherosclerosis, as HGPS is caused by a mutated form of lamin A/C. Blood flow in arteries is generating shear stress that is mostly applied on endothelial cells that align along inner blood vessel wall. At the same time, endothelial cells are also under stretch by periodic arterial pulses. Considering the fact that atherosclerosis is prone to developing at arterial branches with disturbed shear and increased stretch, it is highly possible that laminar flow and proper stretch force are regulating endothelium to function appropriately. In this thesis, the investigation of what effects laminar flow or cyclic stretch can bring to endothelial cells was conducted, and examination of lamin A/C expression under mechanical forces were elaborated and incorporated with cell senescence. Results showed that laminar shear stress and stretch force can regulate lamin A/C expression in different patterns, which were impaired in senescent cells.Item Shear stress attenuates apoptosis due to TNFα, oxidative stress, and serum depletion via deathassociated protein kinase (DAPK) expression(BioMed Central, 2015-03) Rennier, Keith; Ji, Julie Y.; Department of Biomedical Engineering, School of Engineering and TechnologyBackground Misdirected apoptosis in endothelial cells participates in the development of pathological conditions such as atherosclerosis. Tight regulation of apoptosis is necessary to ensure normal cell function. The rate of cell turnover is increased at sites prone to lesion development. Laminar shear stress is protective against atherosclerosis, and helps suppress apoptosis induced by cytokines, oxidative stress, and serum depletion. Current Studies have shown that the pro-apoptotic DAPK expression and function to be regulated in part by shear stress, and that shearing cells already treated with cytokine tumor necrosis factor (TNF) α significantly reduced apoptosis. We investigate further the suppression of endothelial apoptosis by shear stress with other apoptotic triggers, and the involvement of DAPK and caspase 3/7. Results We have shown that exposure to shear stress (12 dynes/cm2 for 6 hrs) suppressed endothelial apoptosis triggered by cytokine (TNFα), oxidative stress (H2O2), and serum depletion, either before or after a long term (18 hr) induction. This is correlated with a parallel decrease of DAPK expression and caspase activity compared to non-sheared cells. We found similar modulation of DAPK and apoptosis by shear stress with other pro-apoptotic signals. Changes in DAPK and caspase 3/7 are directly correlated to changes in apoptosis. Interestingly, shear stress applied to cells prior to induction with apoptosis agents resulted in a higher suppression of apoptosis and DAPK and caspase activity, compared to applying shear stress post induction. This is correlated with a higher expression and activation of DAPK in cells sheared at the end of 24-hr experiment. Also, shear stress alone also induced higher apoptosis and DAPK expression, and the effect is sustained even after 18 hrs incubation in static condition, compared to non-sheared cells. Conclusions Overall, we show that laminar shear stress inhibits various apoptosis pathways by modulating DAPK activity, as well as caspase activation, in a time-dependent manner. Shear stress could target DAPK as a converging point to exert its effects of suppressing endothelial apoptosis. The temporal shear stress stimulation of DAPK and its role in different apoptosis pathways may help identify key mechanisms of the endothelial mechanotransduction pathway.