Limits...
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.

Show MeSH

Related in: MedlinePlus

Experimental design setup and representative DAPK Western blots. A: Subset design to analyze the effects of simultaneous shear stress and stimuli. Cells are treated to shear stress either before or after treatment with apoptotic stimuli, which includes TNFα, H2O2, and serum depletion. B: Western blot of DAPK in pre-sheared cells versus cells treated with stimulus (TNFα, H2O2, or serum depletion). C: Western blot of DAPK in cells sheared post treatment with stimulus (TNFα, H2O2, or serum depletion).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Experimental design setup and representative DAPK Western blots. A: Subset design to analyze the effects of simultaneous shear stress and stimuli. Cells are treated to shear stress either before or after treatment with apoptotic stimuli, which includes TNFα, H2O2, and serum depletion. B: Western blot of DAPK in pre-sheared cells versus cells treated with stimulus (TNFα, H2O2, or serum depletion). C: Western blot of DAPK in cells sheared post treatment with stimulus (TNFα, H2O2, or serum depletion).

Mentions: We examined the time-dependent effect of shear stress on apoptosis and DAPK expression following stress stimulus. Since our preliminary data showed increased DAPK and apoptosis level after 6 hr shearing alone, we decided to incorporate 6 hr shearing either before or subsequent to apoptotic stimulus, to fully evaluate its effect on DAPK and apoptosis. Cells were categorized as either exposed to shear stress before or after stimulation with an apoptotic trigger. Experiments were carried out based on the following 6 groups: Control BAEC; static incubation with stimulus for 24 hrs (Static + stimulus); 6 hr shear stress then incubation in regular media for 18 hrs (6 hr Pre-shear); 6 hr exposure to shear stress first followed by application of the stimulus for 18 hrs (6 hr Pre-shear + stimulus); incubation with the stimulus for 18 hrs followed by 6 hr shear stress (stimulus + 6 hr Post-shear); and static condition followed by 6 hr post-shear (6 hr Post-shear). They are also represented in the diagram in Figure 1A. For biochemical apoptotic triggers, we chose either cytokine (TNFα treatment at 25 ng/ml), oxidative stress (0.5 mM H2O2), or serum depletion (low level 0.5% FBS), as the “stimulus” for apoptosis. We will apply the same 24 hr time frame to all groups to capture apoptotic and DAPK results (Figure 1A).Figure 1


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)

Experimental design setup and representative DAPK Western blots. A: Subset design to analyze the effects of simultaneous shear stress and stimuli. Cells are treated to shear stress either before or after treatment with apoptotic stimuli, which includes TNFα, H2O2, and serum depletion. B: Western blot of DAPK in pre-sheared cells versus cells treated with stimulus (TNFα, H2O2, or serum depletion). C: Western blot of DAPK in cells sheared post treatment with stimulus (TNFα, H2O2, or serum depletion).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Experimental design setup and representative DAPK Western blots. A: Subset design to analyze the effects of simultaneous shear stress and stimuli. Cells are treated to shear stress either before or after treatment with apoptotic stimuli, which includes TNFα, H2O2, and serum depletion. B: Western blot of DAPK in pre-sheared cells versus cells treated with stimulus (TNFα, H2O2, or serum depletion). C: Western blot of DAPK in cells sheared post treatment with stimulus (TNFα, H2O2, or serum depletion).
Mentions: We examined the time-dependent effect of shear stress on apoptosis and DAPK expression following stress stimulus. Since our preliminary data showed increased DAPK and apoptosis level after 6 hr shearing alone, we decided to incorporate 6 hr shearing either before or subsequent to apoptotic stimulus, to fully evaluate its effect on DAPK and apoptosis. Cells were categorized as either exposed to shear stress before or after stimulation with an apoptotic trigger. Experiments were carried out based on the following 6 groups: Control BAEC; static incubation with stimulus for 24 hrs (Static + stimulus); 6 hr shear stress then incubation in regular media for 18 hrs (6 hr Pre-shear); 6 hr exposure to shear stress first followed by application of the stimulus for 18 hrs (6 hr Pre-shear + stimulus); incubation with the stimulus for 18 hrs followed by 6 hr shear stress (stimulus + 6 hr Post-shear); and static condition followed by 6 hr post-shear (6 hr Post-shear). They are also represented in the diagram in Figure 1A. For biochemical apoptotic triggers, we chose either cytokine (TNFα treatment at 25 ng/ml), oxidative stress (0.5 mM H2O2), or serum depletion (low level 0.5% FBS), as the “stimulus” for apoptosis. We will apply the same 24 hr time frame to all groups to capture apoptotic and DAPK results (Figure 1A).Figure 1

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