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Shear stress attenuates apoptosis due to TNFα, oxidative stress, and serum depletion via death-associated protein kinase (DAPK) expression.

Rennier K, Ji JY - BMC Res Notes (2015)

Bottom Line: This is correlated with a parallel decrease of DAPK expression and caspase activity compared to non-sheared cells.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Indiana University Purdue University Indianapolis, 723 West Michigan Street, SL-220 J, Indianapolis, IN, 46202, USA. krennier@purdue.edu.

ABSTRACT

Background: 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/cm(2) 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.

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Phosphorylated DAPK for pre- and post-sheared experimental groups. A: Ratio of DAPKp308/Total DAPK protein expression for each experimental group, cells treated with TNFα, H2O2, or serum depletion versus pre-sheared exposure. B: Ratio of DAPKp308/Total DAPK protein expression for the treated and post-sheared experimental groups. For all figures: * P < 0.05 compared to Control BAEC, + P < 0.05 compared to Static + TNFα, # P < 0.05 compared to Static + H2O2, ∆ P < 0.05 compared to Static – Serum.
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Fig3: Phosphorylated DAPK for pre- and post-sheared experimental groups. A: Ratio of DAPKp308/Total DAPK protein expression for each experimental group, cells treated with TNFα, H2O2, or serum depletion versus pre-sheared exposure. B: Ratio of DAPKp308/Total DAPK protein expression for the treated and post-sheared experimental groups. For all figures: * P < 0.05 compared to Control BAEC, + P < 0.05 compared to Static + TNFα, # P < 0.05 compared to Static + H2O2, ∆ P < 0.05 compared to Static – Serum.

Mentions: As an inhibitory checkpoint, DAPK is auto-phosphorylated at serine 308 in its inactive state. When DAPK is dephosphorylated at serine 308, in the presence of calmodulin binding, DAPK becomes fully activated for its kinase function. The experimental setup previously described was used to investigate the effect of time-dependent shear stress on phospho-serine 308 DAPK (DAPK P308) in order to assess the level of DAPK activation in addition to expression changes. Western blots are duplicated in independent experiments, and bands were quantified and analyzed for statistical comparison. The values for overall phosphorylated DAPK (DAPK P308) are presented as fractions of the total DAPK within each experimental group (Figure 3). We found that fraction of DAPK P308 decreased after exposure to shear stress, but the decrease was significantly more after treatment with TNFα, H2O2, or serum depletion in static condition (P < 0.01). The decrease in phospho-308 DAPK in cells indicated an increased DAPK activation, and shear stress alone was not as effective as other environmental stimuli in activating DAPK activity. Furthermore, adding apoptotic stimulus after pre-shearing significant increased phospho-308 DAPK in treated cells compared to control with no pre-shearing (P < 0.05), indicating a decrease in activated DAPK (Figure 3A). Fraction of phospho- to total DAPK increased by 2.84 fold for TNFα, 2.72 fold for H2O2, or 2.95 fold for serum depletion.Figure 3


Shear stress attenuates apoptosis due to TNFα, oxidative stress, and serum depletion via death-associated protein kinase (DAPK) expression.

Rennier K, Ji JY - BMC Res Notes (2015)

Phosphorylated DAPK for pre- and post-sheared experimental groups. A: Ratio of DAPKp308/Total DAPK protein expression for each experimental group, cells treated with TNFα, H2O2, or serum depletion versus pre-sheared exposure. B: Ratio of DAPKp308/Total DAPK protein expression for the treated and post-sheared experimental groups. For all figures: * P < 0.05 compared to Control BAEC, + P < 0.05 compared to Static + TNFα, # P < 0.05 compared to Static + H2O2, ∆ P < 0.05 compared to Static – Serum.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4374420&req=5

Fig3: Phosphorylated DAPK for pre- and post-sheared experimental groups. A: Ratio of DAPKp308/Total DAPK protein expression for each experimental group, cells treated with TNFα, H2O2, or serum depletion versus pre-sheared exposure. B: Ratio of DAPKp308/Total DAPK protein expression for the treated and post-sheared experimental groups. For all figures: * P < 0.05 compared to Control BAEC, + P < 0.05 compared to Static + TNFα, # P < 0.05 compared to Static + H2O2, ∆ P < 0.05 compared to Static – Serum.
Mentions: As an inhibitory checkpoint, DAPK is auto-phosphorylated at serine 308 in its inactive state. When DAPK is dephosphorylated at serine 308, in the presence of calmodulin binding, DAPK becomes fully activated for its kinase function. The experimental setup previously described was used to investigate the effect of time-dependent shear stress on phospho-serine 308 DAPK (DAPK P308) in order to assess the level of DAPK activation in addition to expression changes. Western blots are duplicated in independent experiments, and bands were quantified and analyzed for statistical comparison. The values for overall phosphorylated DAPK (DAPK P308) are presented as fractions of the total DAPK within each experimental group (Figure 3). We found that fraction of DAPK P308 decreased after exposure to shear stress, but the decrease was significantly more after treatment with TNFα, H2O2, or serum depletion in static condition (P < 0.01). The decrease in phospho-308 DAPK in cells indicated an increased DAPK activation, and shear stress alone was not as effective as other environmental stimuli in activating DAPK activity. Furthermore, adding apoptotic stimulus after pre-shearing significant increased phospho-308 DAPK in treated cells compared to control with no pre-shearing (P < 0.05), indicating a decrease in activated DAPK (Figure 3A). Fraction of phospho- to total DAPK increased by 2.84 fold for TNFα, 2.72 fold for H2O2, or 2.95 fold for serum depletion.Figure 3

Bottom Line: This is correlated with a parallel decrease of DAPK expression and caspase activity compared to non-sheared cells.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Indiana University Purdue University Indianapolis, 723 West Michigan Street, SL-220 J, Indianapolis, IN, 46202, USA. krennier@purdue.edu.

ABSTRACT

Background: 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/cm(2) 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.

Show MeSH
Related in: MedlinePlus