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Homeodomain interacting protein kinase 2 activation compromises endothelial cell response to laminar flow: protective role of p21(waf1,cip1,sdi1).

Mattiussi S, Lazzari C, Truffa S, Antonini A, Soddu S, Capogrossi MC, Gaetano C - PLoS ONE (2009)

Bottom Line: Experiments with the inducible nitric oxide synthase (iNOS) inhibitor GW274150 significantly reduced the SS-dependent apoptosis indicating that the production of NO was relevant for this effect.At molecular level, the ataxia-telangectasia-mutated (ATM) kinase, the homeodomain-interacting protein kinase-2 (HIPK2) and p53 were found activated along a pro-apoptotic signalling pathway while p21(waf1,cip1,sdi1) was prevented from its protective action.RNA interference experiments revealed that HIPK2 and p53 were both important for this process, however, only the forced expression p21(waf1,cip1,sdi1) fully restored the SS-dependent pro-survival function.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio di Patologia Vascolare, Istituto Dermopatico dell' Immacolata, Rome, Italy.

ABSTRACT

Background: In the cardiovascular system, laminar shear stress (SS) is one of the most important source of endothelial protecting signals. Physical and chemical agents, however, including ionising radiations and anticancer drugs, may injure endothelial cells determining an increase in oxidative stress and genotoxic damage. Whether the SS protective function remains intact in the presence of strong oxidants or DNA damage is currently unclear.

Methods and results: To investigate this aspect a series of experiments were performed in which HUVEC were exposed to sub-lethal doses of the radio-mimetic compound Bleomycin (Bleo; 10 microg/ml) which generated free radicals (ROS) without significantly compromising cell survival. Remarkably, the application of a SS of 12 dyne/cm(2) did not protect endothelial cells but markedly accelerated apoptosis compared to controls kept in static culture and in the presence of Bleo. Experiments with the inducible nitric oxide synthase (iNOS) inhibitor GW274150 significantly reduced the SS-dependent apoptosis indicating that the production of NO was relevant for this effect. At molecular level, the ataxia-telangectasia-mutated (ATM) kinase, the homeodomain-interacting protein kinase-2 (HIPK2) and p53 were found activated along a pro-apoptotic signalling pathway while p21(waf1,cip1,sdi1) was prevented from its protective action. RNA interference experiments revealed that HIPK2 and p53 were both important for this process, however, only the forced expression p21(waf1,cip1,sdi1) fully restored the SS-dependent pro-survival function.

Conclusions: This study provides the first evidence that, in the presence of genotoxic damage, laminar flow contributes to endothelial toxicity and death and identifies molecular targets potentially relevant in endothelial dysfunction and cardiovascular disease pathogenesis.

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Related in: MedlinePlus

p21waf1,cip1,sdi1 is regulated by iNOS and protects cells from SS-dependent cell death in the presence of Bleo.HUVEC were treated for 16 h with Bleo (10 ug/ml) in static condition ST or exposed to SS. Control cells (open bars); Bleo treated cells (striped bars). a) The picture shows a representative western blotting analysis of p21waf1,cip1,sdi1 levels in cells cultured in static conditions (ST), in the presence of laminar flow (SS), with or without Bleo (B) and/or the iNOS inhibitor GW274150. The relative p21waf1,cip1,sdi1 band is indicated on the left. Red Ponceau is shown as loading control (Ponceau). The graph on the right shows the average value of p21waf1,cip1,sdi1 levels in the different experimental conditions. Data were generated from three independent experiments. * = p<0.05. b) The graphs shows the effect of the adenovirus (Ad.p21) mediated p21waf1,cip1,sdi1 overexpression (open bars) in cells culture in static condition (ST), in the presence of laminar flow (SS) with or without Bleo (B). A GFP adenovirus was used as control (black bars). The inset on the left shows a western blotting analysis of p21waf1,cip1,sdi1 in control (GFP) and Ad.P21 infected cells (p21). * = <p0.05. ** = p<0.01.
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pone-0006603-g005: p21waf1,cip1,sdi1 is regulated by iNOS and protects cells from SS-dependent cell death in the presence of Bleo.HUVEC were treated for 16 h with Bleo (10 ug/ml) in static condition ST or exposed to SS. Control cells (open bars); Bleo treated cells (striped bars). a) The picture shows a representative western blotting analysis of p21waf1,cip1,sdi1 levels in cells cultured in static conditions (ST), in the presence of laminar flow (SS), with or without Bleo (B) and/or the iNOS inhibitor GW274150. The relative p21waf1,cip1,sdi1 band is indicated on the left. Red Ponceau is shown as loading control (Ponceau). The graph on the right shows the average value of p21waf1,cip1,sdi1 levels in the different experimental conditions. Data were generated from three independent experiments. * = p<0.05. b) The graphs shows the effect of the adenovirus (Ad.p21) mediated p21waf1,cip1,sdi1 overexpression (open bars) in cells culture in static condition (ST), in the presence of laminar flow (SS) with or without Bleo (B). A GFP adenovirus was used as control (black bars). The inset on the left shows a western blotting analysis of p21waf1,cip1,sdi1 in control (GFP) and Ad.P21 infected cells (p21). * = <p0.05. ** = p<0.01.

Mentions: Our prior work revealed that the ability of laminar flow to regulate p21waf1,cip1,sdi1 expression is important protecting endothelial cells from growth factor deprivation and hypoxia-dependent cell death [3]. Although little information is available about the role of p21waf1,cip1,sdi1 in the presence of oxidative stress and genotoxic damage, the experiments depicted in figure 5a, left and right panels, show that, the iNOS inhibitor GW274150 fully restored the SS-dependent regulation of p21waf1,cip1,sdi1 suggesting that iNOS, as well as HIPK2, is important to prevent p21waf1,cip1,sdi1 induction. Figure 5b demonstrates that adenovirus-mediated p21waf1,cip1,sdi1 over-expression prevents endothelial cell death in all the conditions tested indicating an appropriate ratio between basal and SS-inducible levels of p21waf1,cip1,sdi1 may play an important role in the SS-dependent protective function of endothelial cells.


Homeodomain interacting protein kinase 2 activation compromises endothelial cell response to laminar flow: protective role of p21(waf1,cip1,sdi1).

Mattiussi S, Lazzari C, Truffa S, Antonini A, Soddu S, Capogrossi MC, Gaetano C - PLoS ONE (2009)

p21waf1,cip1,sdi1 is regulated by iNOS and protects cells from SS-dependent cell death in the presence of Bleo.HUVEC were treated for 16 h with Bleo (10 ug/ml) in static condition ST or exposed to SS. Control cells (open bars); Bleo treated cells (striped bars). a) The picture shows a representative western blotting analysis of p21waf1,cip1,sdi1 levels in cells cultured in static conditions (ST), in the presence of laminar flow (SS), with or without Bleo (B) and/or the iNOS inhibitor GW274150. The relative p21waf1,cip1,sdi1 band is indicated on the left. Red Ponceau is shown as loading control (Ponceau). The graph on the right shows the average value of p21waf1,cip1,sdi1 levels in the different experimental conditions. Data were generated from three independent experiments. * = p<0.05. b) The graphs shows the effect of the adenovirus (Ad.p21) mediated p21waf1,cip1,sdi1 overexpression (open bars) in cells culture in static condition (ST), in the presence of laminar flow (SS) with or without Bleo (B). A GFP adenovirus was used as control (black bars). The inset on the left shows a western blotting analysis of p21waf1,cip1,sdi1 in control (GFP) and Ad.P21 infected cells (p21). * = <p0.05. ** = p<0.01.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2719102&req=5

pone-0006603-g005: p21waf1,cip1,sdi1 is regulated by iNOS and protects cells from SS-dependent cell death in the presence of Bleo.HUVEC were treated for 16 h with Bleo (10 ug/ml) in static condition ST or exposed to SS. Control cells (open bars); Bleo treated cells (striped bars). a) The picture shows a representative western blotting analysis of p21waf1,cip1,sdi1 levels in cells cultured in static conditions (ST), in the presence of laminar flow (SS), with or without Bleo (B) and/or the iNOS inhibitor GW274150. The relative p21waf1,cip1,sdi1 band is indicated on the left. Red Ponceau is shown as loading control (Ponceau). The graph on the right shows the average value of p21waf1,cip1,sdi1 levels in the different experimental conditions. Data were generated from three independent experiments. * = p<0.05. b) The graphs shows the effect of the adenovirus (Ad.p21) mediated p21waf1,cip1,sdi1 overexpression (open bars) in cells culture in static condition (ST), in the presence of laminar flow (SS) with or without Bleo (B). A GFP adenovirus was used as control (black bars). The inset on the left shows a western blotting analysis of p21waf1,cip1,sdi1 in control (GFP) and Ad.P21 infected cells (p21). * = <p0.05. ** = p<0.01.
Mentions: Our prior work revealed that the ability of laminar flow to regulate p21waf1,cip1,sdi1 expression is important protecting endothelial cells from growth factor deprivation and hypoxia-dependent cell death [3]. Although little information is available about the role of p21waf1,cip1,sdi1 in the presence of oxidative stress and genotoxic damage, the experiments depicted in figure 5a, left and right panels, show that, the iNOS inhibitor GW274150 fully restored the SS-dependent regulation of p21waf1,cip1,sdi1 suggesting that iNOS, as well as HIPK2, is important to prevent p21waf1,cip1,sdi1 induction. Figure 5b demonstrates that adenovirus-mediated p21waf1,cip1,sdi1 over-expression prevents endothelial cell death in all the conditions tested indicating an appropriate ratio between basal and SS-inducible levels of p21waf1,cip1,sdi1 may play an important role in the SS-dependent protective function of endothelial cells.

Bottom Line: Experiments with the inducible nitric oxide synthase (iNOS) inhibitor GW274150 significantly reduced the SS-dependent apoptosis indicating that the production of NO was relevant for this effect.At molecular level, the ataxia-telangectasia-mutated (ATM) kinase, the homeodomain-interacting protein kinase-2 (HIPK2) and p53 were found activated along a pro-apoptotic signalling pathway while p21(waf1,cip1,sdi1) was prevented from its protective action.RNA interference experiments revealed that HIPK2 and p53 were both important for this process, however, only the forced expression p21(waf1,cip1,sdi1) fully restored the SS-dependent pro-survival function.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio di Patologia Vascolare, Istituto Dermopatico dell' Immacolata, Rome, Italy.

ABSTRACT

Background: In the cardiovascular system, laminar shear stress (SS) is one of the most important source of endothelial protecting signals. Physical and chemical agents, however, including ionising radiations and anticancer drugs, may injure endothelial cells determining an increase in oxidative stress and genotoxic damage. Whether the SS protective function remains intact in the presence of strong oxidants or DNA damage is currently unclear.

Methods and results: To investigate this aspect a series of experiments were performed in which HUVEC were exposed to sub-lethal doses of the radio-mimetic compound Bleomycin (Bleo; 10 microg/ml) which generated free radicals (ROS) without significantly compromising cell survival. Remarkably, the application of a SS of 12 dyne/cm(2) did not protect endothelial cells but markedly accelerated apoptosis compared to controls kept in static culture and in the presence of Bleo. Experiments with the inducible nitric oxide synthase (iNOS) inhibitor GW274150 significantly reduced the SS-dependent apoptosis indicating that the production of NO was relevant for this effect. At molecular level, the ataxia-telangectasia-mutated (ATM) kinase, the homeodomain-interacting protein kinase-2 (HIPK2) and p53 were found activated along a pro-apoptotic signalling pathway while p21(waf1,cip1,sdi1) was prevented from its protective action. RNA interference experiments revealed that HIPK2 and p53 were both important for this process, however, only the forced expression p21(waf1,cip1,sdi1) fully restored the SS-dependent pro-survival function.

Conclusions: This study provides the first evidence that, in the presence of genotoxic damage, laminar flow contributes to endothelial toxicity and death and identifies molecular targets potentially relevant in endothelial dysfunction and cardiovascular disease pathogenesis.

Show MeSH
Related in: MedlinePlus