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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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Overall DAPK expression for pre- and post-sheared experimental groups. A: DAPK protein expression for the treatment and pre-sheared experimental groups. B: DAPK protein expression for the treatment 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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Fig2: Overall DAPK expression for pre- and post-sheared experimental groups. A: DAPK protein expression for the treatment and pre-sheared experimental groups. B: DAPK protein expression for the treatment 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: First, we explored the effect of pre-conditioning cells with shear stress exposure (12 dynes/cm2) in samples with and without each of the stimuli (TNFα, H2O2, and serum depletion). Cell lysates from each group were collected and analyzed for overall DAPK expression in Western blots (Figure 1B). Samples were duplicated in three independent experiments, and bands were quantified and analyzed for statistical comparison (Figure 2A). Western analysis showed DAPK activity significantly decreased in cells exposed to pre-shearing followed by stimuli incubation compared to stimulants alone (static + stimulus) (Figure 1B). DAPK expression was quantified using the relative intensity of DAPK bands normalized to a loading control, actin (Figure 2A). After quantitative analysis, we consistently observed a statistically significant increase in DAPK expression after exposure to TNFα, H2O2, or serum depletion, as compared to control BAEC (P < 0.05). Also, we saw a significant decrease in DAPK expression in cells pre-sheared prior to stimuli incubation, compared with the static cells exposed to each stimuli (P < 0.05). The decrease due to shear stress is observed independent of the apoptosis trigger, which all activated DAPK expression. Based on our quantitative analysis of Western bands, the addition of shear stress before apoptosis trigger decreased DAPK by 27% for TNFα, 24.33% for H2O2, or 18.58% for serum depletion. Similarly, for the post-sheared experiments, BAEC cells were incubated with each stimulus for 18 hrs prior to 6 hr shear stress. Western blots were carried out to evaluate DAPK expression (Figure 1C), and quantified using the relative band intensity compared to actin (Figure 2B). Again, DAPK expression significantly increased for apoptosis-induced static cells. With the addition of shear stress following apoptosis induction, we observed significant decrease in DAPK expression, compared to cells incubated with stimulants alone (P < 0.05). Cells sheared for 6 hrs after exposure to TNFα, H2O2, or serum depletion still resulted in an increase of total DAPK expression, but less significantly than static cells under the same stimulus conditions (Figure 2B). However, the decrease in DAPK expression by post-induction shear stress was less than that of pre-shearing before apoptotic induction. Based on our quantitative Western analysis, the addition of shear stress after apoptosis trigger decreased DAPK by 14.05% for TNFα, 22% for H2O2, or 17.22% for serum depletion. Overall, our data suggest that exposure to shear stress mitigates the increase in endothelial DAPK expression brought on by the addition of cytokine TNFα, H2O2, and serum depletion on compared to the static induction cases.Figure 2


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)

Overall DAPK expression for pre- and post-sheared experimental groups. A: DAPK protein expression for the treatment and pre-sheared experimental groups. B: DAPK protein expression for the treatment 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

Fig2: Overall DAPK expression for pre- and post-sheared experimental groups. A: DAPK protein expression for the treatment and pre-sheared experimental groups. B: DAPK protein expression for the treatment 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: First, we explored the effect of pre-conditioning cells with shear stress exposure (12 dynes/cm2) in samples with and without each of the stimuli (TNFα, H2O2, and serum depletion). Cell lysates from each group were collected and analyzed for overall DAPK expression in Western blots (Figure 1B). Samples were duplicated in three independent experiments, and bands were quantified and analyzed for statistical comparison (Figure 2A). Western analysis showed DAPK activity significantly decreased in cells exposed to pre-shearing followed by stimuli incubation compared to stimulants alone (static + stimulus) (Figure 1B). DAPK expression was quantified using the relative intensity of DAPK bands normalized to a loading control, actin (Figure 2A). After quantitative analysis, we consistently observed a statistically significant increase in DAPK expression after exposure to TNFα, H2O2, or serum depletion, as compared to control BAEC (P < 0.05). Also, we saw a significant decrease in DAPK expression in cells pre-sheared prior to stimuli incubation, compared with the static cells exposed to each stimuli (P < 0.05). The decrease due to shear stress is observed independent of the apoptosis trigger, which all activated DAPK expression. Based on our quantitative analysis of Western bands, the addition of shear stress before apoptosis trigger decreased DAPK by 27% for TNFα, 24.33% for H2O2, or 18.58% for serum depletion. Similarly, for the post-sheared experiments, BAEC cells were incubated with each stimulus for 18 hrs prior to 6 hr shear stress. Western blots were carried out to evaluate DAPK expression (Figure 1C), and quantified using the relative band intensity compared to actin (Figure 2B). Again, DAPK expression significantly increased for apoptosis-induced static cells. With the addition of shear stress following apoptosis induction, we observed significant decrease in DAPK expression, compared to cells incubated with stimulants alone (P < 0.05). Cells sheared for 6 hrs after exposure to TNFα, H2O2, or serum depletion still resulted in an increase of total DAPK expression, but less significantly than static cells under the same stimulus conditions (Figure 2B). However, the decrease in DAPK expression by post-induction shear stress was less than that of pre-shearing before apoptotic induction. Based on our quantitative Western analysis, the addition of shear stress after apoptosis trigger decreased DAPK by 14.05% for TNFα, 22% for H2O2, or 17.22% for serum depletion. Overall, our data suggest that exposure to shear stress mitigates the increase in endothelial DAPK expression brought on by the addition of cytokine TNFα, H2O2, and serum depletion on compared to the static induction cases.Figure 2

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