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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

Laminar SS increases iNOS expression and NO production in the presence of Bleomycin.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 graph shows the average NO production, in cells cultured in control conditions or exposed to Bleo, SS or both in combination, determined by DAF incorporation and FACS analysis. Data are represented in arbitrary units and are the results of three independent experiments performed in duplicate. * = p<0.05; ** = p<0.01. b) The picture on the left shows a representative western blotting analysis (WB) depicting iNOS protein level in cells kept in static culture (ST) or exposed to laminar SS (SS) in the presence of Bleo (B). Densitometric analysis of three independent experiments expressed in arbitrary units is shown on the right. * = p<0.05. c) The graph on the left shows the effect of the NO donor DETA/NO (500 µM), administered to endothelial cells cultured in static conditions in the presence or absence of Bleo, on cell survival determined by propidium-iodide incorporation and FACS analysis. Data, expressed in arbitrary units, are the results of three independent experiments. Ns = not significant; *** = p<0.001. d) The graph shows the effect of the iNOS inhibitor GW274150 on endothelial cell survival determined by propidium iodide incorporation and FACS analysis. Data are represented in arbitrary units and are the average of three independent experiments performed in static culture (ST) or in the presence of laminar flow (SS). * = p<0.05. ** = p<0.01. # = p<0.005. e) The graph shows the effect of the iNOS inhibitor GW274150 on NO production determined by DAF incorporation and FACS analysis in endothelial cells culture in the presence of Bleo (B). Data are represented in arbitrary units and are the average of three independent experiments performed in static culture (ST) or in the presence of laminar flow (SS). *** = p<0.001. f) The picture shows a representative western blotting analysis (WB) performed by using an anti-nitrotyrosine antibody on total lysates obtained from cells cultured in static condition (ST) or exposed to laminar flow (SS) and/or Bleo (B). The Red Ponceau staining is shown below as loading control (Ponceau). The right panels indicates the average densitometric values expressed as arbitrary units. Data were generated by three independent experiments. * = p<0.05.
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pone-0006603-g002: Laminar SS increases iNOS expression and NO production in the presence of Bleomycin.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 graph shows the average NO production, in cells cultured in control conditions or exposed to Bleo, SS or both in combination, determined by DAF incorporation and FACS analysis. Data are represented in arbitrary units and are the results of three independent experiments performed in duplicate. * = p<0.05; ** = p<0.01. b) The picture on the left shows a representative western blotting analysis (WB) depicting iNOS protein level in cells kept in static culture (ST) or exposed to laminar SS (SS) in the presence of Bleo (B). Densitometric analysis of three independent experiments expressed in arbitrary units is shown on the right. * = p<0.05. c) The graph on the left shows the effect of the NO donor DETA/NO (500 µM), administered to endothelial cells cultured in static conditions in the presence or absence of Bleo, on cell survival determined by propidium-iodide incorporation and FACS analysis. Data, expressed in arbitrary units, are the results of three independent experiments. Ns = not significant; *** = p<0.001. d) The graph shows the effect of the iNOS inhibitor GW274150 on endothelial cell survival determined by propidium iodide incorporation and FACS analysis. Data are represented in arbitrary units and are the average of three independent experiments performed in static culture (ST) or in the presence of laminar flow (SS). * = p<0.05. ** = p<0.01. # = p<0.005. e) The graph shows the effect of the iNOS inhibitor GW274150 on NO production determined by DAF incorporation and FACS analysis in endothelial cells culture in the presence of Bleo (B). Data are represented in arbitrary units and are the average of three independent experiments performed in static culture (ST) or in the presence of laminar flow (SS). *** = p<0.001. f) The picture shows a representative western blotting analysis (WB) performed by using an anti-nitrotyrosine antibody on total lysates obtained from cells cultured in static condition (ST) or exposed to laminar flow (SS) and/or Bleo (B). The Red Ponceau staining is shown below as loading control (Ponceau). The right panels indicates the average densitometric values expressed as arbitrary units. Data were generated by three independent experiments. * = p<0.05.

Mentions: As part of its physiological mechanism of action, laminar flow increases the intracellular content of NO by eNOS activation[22]. In our experiments SS increased about 2.5 fold, above basal level, the production of NO in cells kept in normal condition, however, this effect was further enhanced up to 4.5 fold by SS administered in the presence of Bleo (Fig. 2a). In patho-physiological contexts, oxidative stress or hypoxia increase the expression of the inducible NOS isoform which synthesizes high levels of NO[23] independently of phosphorylation signals. Figure 2b, left and right panels, shows that iNOS expression was induced about 2.5. fold above control by Bleo and even further by the coincident treatment of SS and Bleo. We reasoned that these results could be explained by the Bleo-dependent production of ROS which could be intensified in the presence of SS. Supplemental Figure S1, panels a and b, shows that Bleo and SS in combination determined a significant increase in the intracellular content of oxygen free radicals evaluated by dichlorofluorescein (DCF) and dihydroethidium (DHE) staining. Notably, DCF, but not DHE, is also able to detect NO[24] which may explain the significant increase in fluorescent signals occurring in the presence of SS and detectable in absence of Bleo as depicted in the supplemental Figure S1, panel a.


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)

Laminar SS increases iNOS expression and NO production in the presence of Bleomycin.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 graph shows the average NO production, in cells cultured in control conditions or exposed to Bleo, SS or both in combination, determined by DAF incorporation and FACS analysis. Data are represented in arbitrary units and are the results of three independent experiments performed in duplicate. * = p<0.05; ** = p<0.01. b) The picture on the left shows a representative western blotting analysis (WB) depicting iNOS protein level in cells kept in static culture (ST) or exposed to laminar SS (SS) in the presence of Bleo (B). Densitometric analysis of three independent experiments expressed in arbitrary units is shown on the right. * = p<0.05. c) The graph on the left shows the effect of the NO donor DETA/NO (500 µM), administered to endothelial cells cultured in static conditions in the presence or absence of Bleo, on cell survival determined by propidium-iodide incorporation and FACS analysis. Data, expressed in arbitrary units, are the results of three independent experiments. Ns = not significant; *** = p<0.001. d) The graph shows the effect of the iNOS inhibitor GW274150 on endothelial cell survival determined by propidium iodide incorporation and FACS analysis. Data are represented in arbitrary units and are the average of three independent experiments performed in static culture (ST) or in the presence of laminar flow (SS). * = p<0.05. ** = p<0.01. # = p<0.005. e) The graph shows the effect of the iNOS inhibitor GW274150 on NO production determined by DAF incorporation and FACS analysis in endothelial cells culture in the presence of Bleo (B). Data are represented in arbitrary units and are the average of three independent experiments performed in static culture (ST) or in the presence of laminar flow (SS). *** = p<0.001. f) The picture shows a representative western blotting analysis (WB) performed by using an anti-nitrotyrosine antibody on total lysates obtained from cells cultured in static condition (ST) or exposed to laminar flow (SS) and/or Bleo (B). The Red Ponceau staining is shown below as loading control (Ponceau). The right panels indicates the average densitometric values expressed as arbitrary units. Data were generated by three independent experiments. * = p<0.05.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2719102&req=5

pone-0006603-g002: Laminar SS increases iNOS expression and NO production in the presence of Bleomycin.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 graph shows the average NO production, in cells cultured in control conditions or exposed to Bleo, SS or both in combination, determined by DAF incorporation and FACS analysis. Data are represented in arbitrary units and are the results of three independent experiments performed in duplicate. * = p<0.05; ** = p<0.01. b) The picture on the left shows a representative western blotting analysis (WB) depicting iNOS protein level in cells kept in static culture (ST) or exposed to laminar SS (SS) in the presence of Bleo (B). Densitometric analysis of three independent experiments expressed in arbitrary units is shown on the right. * = p<0.05. c) The graph on the left shows the effect of the NO donor DETA/NO (500 µM), administered to endothelial cells cultured in static conditions in the presence or absence of Bleo, on cell survival determined by propidium-iodide incorporation and FACS analysis. Data, expressed in arbitrary units, are the results of three independent experiments. Ns = not significant; *** = p<0.001. d) The graph shows the effect of the iNOS inhibitor GW274150 on endothelial cell survival determined by propidium iodide incorporation and FACS analysis. Data are represented in arbitrary units and are the average of three independent experiments performed in static culture (ST) or in the presence of laminar flow (SS). * = p<0.05. ** = p<0.01. # = p<0.005. e) The graph shows the effect of the iNOS inhibitor GW274150 on NO production determined by DAF incorporation and FACS analysis in endothelial cells culture in the presence of Bleo (B). Data are represented in arbitrary units and are the average of three independent experiments performed in static culture (ST) or in the presence of laminar flow (SS). *** = p<0.001. f) The picture shows a representative western blotting analysis (WB) performed by using an anti-nitrotyrosine antibody on total lysates obtained from cells cultured in static condition (ST) or exposed to laminar flow (SS) and/or Bleo (B). The Red Ponceau staining is shown below as loading control (Ponceau). The right panels indicates the average densitometric values expressed as arbitrary units. Data were generated by three independent experiments. * = p<0.05.
Mentions: As part of its physiological mechanism of action, laminar flow increases the intracellular content of NO by eNOS activation[22]. In our experiments SS increased about 2.5 fold, above basal level, the production of NO in cells kept in normal condition, however, this effect was further enhanced up to 4.5 fold by SS administered in the presence of Bleo (Fig. 2a). In patho-physiological contexts, oxidative stress or hypoxia increase the expression of the inducible NOS isoform which synthesizes high levels of NO[23] independently of phosphorylation signals. Figure 2b, left and right panels, shows that iNOS expression was induced about 2.5. fold above control by Bleo and even further by the coincident treatment of SS and Bleo. We reasoned that these results could be explained by the Bleo-dependent production of ROS which could be intensified in the presence of SS. Supplemental Figure S1, panels a and b, shows that Bleo and SS in combination determined a significant increase in the intracellular content of oxygen free radicals evaluated by dichlorofluorescein (DCF) and dihydroethidium (DHE) staining. Notably, DCF, but not DHE, is also able to detect NO[24] which may explain the significant increase in fluorescent signals occurring in the presence of SS and detectable in absence of Bleo as depicted in the supplemental Figure S1, panel a.

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