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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

Schematic representation of SS-dependent signalling pathways activated in the presence of genotoxic damage leading to endothelial cell death and preventative interventions.Endothelial cells exposed to laminar shear stress (SS) and various sources of genotoxic damage (Bleo, Doxo, UV) generate reactive oxygen species (ROS) and up-regulate the inducible nitric oxide synthase at protein levels (iNOS) which, elevating the intracellular content of nitric oxide (NO), contributes to the increase of the intracellular ROS content. Genotoxic damage generates single and double DNA strand breaks which determine Ataxia-telangectasia mutated kinase (ATM) activation and phosphorylated histone 2AX incorporation (γH2AX) in DNA repair foci. ATM is known to regulate the homeodomain-interacting protein kinase 2 (HIPK2) which participate to the DNA repair process regulating p53 function by Serine 46 phosphorylation and activation along a DNA repair or proapoptotic signalling pathways. The latter regulated by proapoptotic members of the BCL2 protein family (BAX). In this condition, the SS-and p53-dependent cell cycle inhibitor and anti-apoptotic molecule p21waf1,cip1,sdi1 (p21) is inhibited contributing to endothelial cell death. Interventions aimed at reducing the ROS level, the production of NO or the intracellular content of HIPK2 and p53 reversed the SS-dependent endothelial cell death. Remarkably, the adenovirus-mediated (Ad.p21) p21waf1,cip1,sdi1 over-expression revealed an important protective role of this molecule preventing apoptosis in the presence of SS and genotoxic agents.
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pone-0006603-g006: Schematic representation of SS-dependent signalling pathways activated in the presence of genotoxic damage leading to endothelial cell death and preventative interventions.Endothelial cells exposed to laminar shear stress (SS) and various sources of genotoxic damage (Bleo, Doxo, UV) generate reactive oxygen species (ROS) and up-regulate the inducible nitric oxide synthase at protein levels (iNOS) which, elevating the intracellular content of nitric oxide (NO), contributes to the increase of the intracellular ROS content. Genotoxic damage generates single and double DNA strand breaks which determine Ataxia-telangectasia mutated kinase (ATM) activation and phosphorylated histone 2AX incorporation (γH2AX) in DNA repair foci. ATM is known to regulate the homeodomain-interacting protein kinase 2 (HIPK2) which participate to the DNA repair process regulating p53 function by Serine 46 phosphorylation and activation along a DNA repair or proapoptotic signalling pathways. The latter regulated by proapoptotic members of the BCL2 protein family (BAX). In this condition, the SS-and p53-dependent cell cycle inhibitor and anti-apoptotic molecule p21waf1,cip1,sdi1 (p21) is inhibited contributing to endothelial cell death. Interventions aimed at reducing the ROS level, the production of NO or the intracellular content of HIPK2 and p53 reversed the SS-dependent endothelial cell death. Remarkably, the adenovirus-mediated (Ad.p21) p21waf1,cip1,sdi1 over-expression revealed an important protective role of this molecule preventing apoptosis in the presence of SS and genotoxic agents.

Mentions: Taken altogether these data represent the first experimental evidence that SS properties are strongly influenced by the intracellular environment which determines the endothelial cell response to mechanical stimuli. Consequently, the presence of local flow alterations worsened by co-factors such as metabolic alterations, inflammatory processes or oxidative stress, may hamper the SS-dependent positive regulation of p21waf1,cip1,sdi1, determining the reversal of flow pro-survival functions (see schema in Figure 6). Noteworthy, molecular interventions aimed at restoring the appropriate intracellular levels of p21waf1,cip1,sdi1 successfully rescued the anti-apoptotic properties of SS. In conclusion p21waf1,cip1,sdi1 emerges from this study as an essential keystone molecule at the crossroad between homeostasis and pathogenesis of endothelial cell dysfunction.


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)

Schematic representation of SS-dependent signalling pathways activated in the presence of genotoxic damage leading to endothelial cell death and preventative interventions.Endothelial cells exposed to laminar shear stress (SS) and various sources of genotoxic damage (Bleo, Doxo, UV) generate reactive oxygen species (ROS) and up-regulate the inducible nitric oxide synthase at protein levels (iNOS) which, elevating the intracellular content of nitric oxide (NO), contributes to the increase of the intracellular ROS content. Genotoxic damage generates single and double DNA strand breaks which determine Ataxia-telangectasia mutated kinase (ATM) activation and phosphorylated histone 2AX incorporation (γH2AX) in DNA repair foci. ATM is known to regulate the homeodomain-interacting protein kinase 2 (HIPK2) which participate to the DNA repair process regulating p53 function by Serine 46 phosphorylation and activation along a DNA repair or proapoptotic signalling pathways. The latter regulated by proapoptotic members of the BCL2 protein family (BAX). In this condition, the SS-and p53-dependent cell cycle inhibitor and anti-apoptotic molecule p21waf1,cip1,sdi1 (p21) is inhibited contributing to endothelial cell death. Interventions aimed at reducing the ROS level, the production of NO or the intracellular content of HIPK2 and p53 reversed the SS-dependent endothelial cell death. Remarkably, the adenovirus-mediated (Ad.p21) p21waf1,cip1,sdi1 over-expression revealed an important protective role of this molecule preventing apoptosis in the presence of SS and genotoxic agents.
© Copyright Policy
Related In: Results  -  Collection

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pone-0006603-g006: Schematic representation of SS-dependent signalling pathways activated in the presence of genotoxic damage leading to endothelial cell death and preventative interventions.Endothelial cells exposed to laminar shear stress (SS) and various sources of genotoxic damage (Bleo, Doxo, UV) generate reactive oxygen species (ROS) and up-regulate the inducible nitric oxide synthase at protein levels (iNOS) which, elevating the intracellular content of nitric oxide (NO), contributes to the increase of the intracellular ROS content. Genotoxic damage generates single and double DNA strand breaks which determine Ataxia-telangectasia mutated kinase (ATM) activation and phosphorylated histone 2AX incorporation (γH2AX) in DNA repair foci. ATM is known to regulate the homeodomain-interacting protein kinase 2 (HIPK2) which participate to the DNA repair process regulating p53 function by Serine 46 phosphorylation and activation along a DNA repair or proapoptotic signalling pathways. The latter regulated by proapoptotic members of the BCL2 protein family (BAX). In this condition, the SS-and p53-dependent cell cycle inhibitor and anti-apoptotic molecule p21waf1,cip1,sdi1 (p21) is inhibited contributing to endothelial cell death. Interventions aimed at reducing the ROS level, the production of NO or the intracellular content of HIPK2 and p53 reversed the SS-dependent endothelial cell death. Remarkably, the adenovirus-mediated (Ad.p21) p21waf1,cip1,sdi1 over-expression revealed an important protective role of this molecule preventing apoptosis in the presence of SS and genotoxic agents.
Mentions: Taken altogether these data represent the first experimental evidence that SS properties are strongly influenced by the intracellular environment which determines the endothelial cell response to mechanical stimuli. Consequently, the presence of local flow alterations worsened by co-factors such as metabolic alterations, inflammatory processes or oxidative stress, may hamper the SS-dependent positive regulation of p21waf1,cip1,sdi1, determining the reversal of flow pro-survival functions (see schema in Figure 6). Noteworthy, molecular interventions aimed at restoring the appropriate intracellular levels of p21waf1,cip1,sdi1 successfully rescued the anti-apoptotic properties of SS. In conclusion p21waf1,cip1,sdi1 emerges from this study as an essential keystone molecule at the crossroad between homeostasis and pathogenesis of endothelial cell dysfunction.

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