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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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Quantified cell apoptosis results based on caspase activity. A: Analysis of Caspase-3 and −7 activity in pre-sheared cells versus cells treated with stimulus alone (TNFα, H2O2, or serum depletion). B: Analysis of Caspase-3 and −7 activity in cells sheared post treatment with stimulus versus cells treated with stimulus alone (TNFα, H2O2, or serum depletion). For all caspase data: * P < 0.01 compared to Control BAEC, + P < 0.01 compared to Static + TNFα, # P < 0.01 compared to Static + H2O2, ∆ P < 0.01 compared to Static – Serum.
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Fig5: Quantified cell apoptosis results based on caspase activity. A: Analysis of Caspase-3 and −7 activity in pre-sheared cells versus cells treated with stimulus alone (TNFα, H2O2, or serum depletion). B: Analysis of Caspase-3 and −7 activity in cells sheared post treatment with stimulus versus cells treated with stimulus alone (TNFα, H2O2, or serum depletion). For all caspase data: * P < 0.01 compared to Control BAEC, + P < 0.01 compared to Static + TNFα, # P < 0.01 compared to Static + H2O2, ∆ P < 0.01 compared to Static – Serum.

Mentions: To confirm the TUNEL stain apoptosis results, we also examined caspase activity in each sample (Figure 5). Caspase activity is downstream from DAPK in the apoptosis pathway, and would further link DAPK activity to apoptosis under each treatment. We have shown previously that corresponding caspase 3/7 activity is related to shear regulated DAPK activation [31,32]. Using the same experimental groups, we found that pre-shearing cells similarly decreased caspase activity when compared to each of the stimulant alone treatments (Figure 5A). This data correlate well to our TUNEL results (P < 0.01), further confirming activation of the apoptotic pathway. Shearing cells after apoptosis induction also mitigated the overall increase in caspase activity when compared to the static cells exposed to apoptosis inductors (P < 0.01) (Figure 5B). Although, shearing cells alone for 6 hrs, without induction with apoptotic trigger, induced a considerable increase in apoptosis and caspase production compared to control cells (no shearing or stimulus) (Figures 4 and 5). These results correlate with our previous discovery that shear stress induced an increase in overall DAPK and decrease of phospho-DAPK, which promotes DAPK activity and subsequent apoptosis.Figure 5


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)

Quantified cell apoptosis results based on caspase activity. A: Analysis of Caspase-3 and −7 activity in pre-sheared cells versus cells treated with stimulus alone (TNFα, H2O2, or serum depletion). B: Analysis of Caspase-3 and −7 activity in cells sheared post treatment with stimulus versus cells treated with stimulus alone (TNFα, H2O2, or serum depletion). For all caspase data: * P < 0.01 compared to Control BAEC, + P < 0.01 compared to Static + TNFα, # P < 0.01 compared to Static + H2O2, ∆ P < 0.01 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

Fig5: Quantified cell apoptosis results based on caspase activity. A: Analysis of Caspase-3 and −7 activity in pre-sheared cells versus cells treated with stimulus alone (TNFα, H2O2, or serum depletion). B: Analysis of Caspase-3 and −7 activity in cells sheared post treatment with stimulus versus cells treated with stimulus alone (TNFα, H2O2, or serum depletion). For all caspase data: * P < 0.01 compared to Control BAEC, + P < 0.01 compared to Static + TNFα, # P < 0.01 compared to Static + H2O2, ∆ P < 0.01 compared to Static – Serum.
Mentions: To confirm the TUNEL stain apoptosis results, we also examined caspase activity in each sample (Figure 5). Caspase activity is downstream from DAPK in the apoptosis pathway, and would further link DAPK activity to apoptosis under each treatment. We have shown previously that corresponding caspase 3/7 activity is related to shear regulated DAPK activation [31,32]. Using the same experimental groups, we found that pre-shearing cells similarly decreased caspase activity when compared to each of the stimulant alone treatments (Figure 5A). This data correlate well to our TUNEL results (P < 0.01), further confirming activation of the apoptotic pathway. Shearing cells after apoptosis induction also mitigated the overall increase in caspase activity when compared to the static cells exposed to apoptosis inductors (P < 0.01) (Figure 5B). Although, shearing cells alone for 6 hrs, without induction with apoptotic trigger, induced a considerable increase in apoptosis and caspase production compared to control cells (no shearing or stimulus) (Figures 4 and 5). These results correlate with our previous discovery that shear stress induced an increase in overall DAPK and decrease of phospho-DAPK, which promotes DAPK activity and subsequent apoptosis.Figure 5

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