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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

SS induces cell death in endothelial cells exposed to 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 represents the intracellular content determination of phosphorylated histone 2AX in cell exposed to Bleo. Data are expressed as percent of control. * = p<0.05. b) The graph on the left depicts the result in which cell viability was determined by propidium-iodide staining and FACS analysis. Values are expressed as fold increase compared to static control. The right panel shows the result of free nucleosome evaluation performed by ELISA. Values are expressed in optical density (O.D.). Each experiments was performed three times in duplicate. * = p<0.05; ** = p<0.01. c) A representative western blotting analysis (WB) of caspase 3 cleavage is shown on the left. The inactive precursors (pro-casp-3) and the cleaved form (casp-3) are indicated. The experiments were performed in static culture (ST), in the presence or absence of Bleo (B) and or laminar SS (SS). The right panel shows the average densitometric analysis of three independent experiments. Data are expressed in arbitrary units. * = p<0.05.
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pone-0006603-g001: SS induces cell death in endothelial cells exposed to 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 represents the intracellular content determination of phosphorylated histone 2AX in cell exposed to Bleo. Data are expressed as percent of control. * = p<0.05. b) The graph on the left depicts the result in which cell viability was determined by propidium-iodide staining and FACS analysis. Values are expressed as fold increase compared to static control. The right panel shows the result of free nucleosome evaluation performed by ELISA. Values are expressed in optical density (O.D.). Each experiments was performed three times in duplicate. * = p<0.05; ** = p<0.01. c) A representative western blotting analysis (WB) of caspase 3 cleavage is shown on the left. The inactive precursors (pro-casp-3) and the cleaved form (casp-3) are indicated. The experiments were performed in static culture (ST), in the presence or absence of Bleo (B) and or laminar SS (SS). The right panel shows the average densitometric analysis of three independent experiments. Data are expressed in arbitrary units. * = p<0.05.

Mentions: HUVEC were exposed to a SS of 12 dyne/cm2 for 16 hours (h) in the presence or absence of a sub-lethal concentration of Bleo (10 µg/ml). In this experimental condition the endothelial cell treatment with Bleo alone promoted the chromatin incorporation of the phosphorylated isoform of histone H2AX (γH2AX) (Figure 1a). Figure 1b, left and right panels, shows the results of experiments in which cell death and apoptosis were evaluated by propidium-iodide staining/FACS analysis (left) and free nucleosomes release (right) respectively. Both were found significantly increased in endothelial cells exposed to SS and Bleo. The evidence of a proapoptotic program activation was further supported by the presence of caspase 3 cleavage (Figure 1c, left panel) which reached the highest level, two fold above static control and about seven fold above SS alone, (Figure 1c, right panel) in the presence of Bleo and SS.


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)

SS induces cell death in endothelial cells exposed to 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 represents the intracellular content determination of phosphorylated histone 2AX in cell exposed to Bleo. Data are expressed as percent of control. * = p<0.05. b) The graph on the left depicts the result in which cell viability was determined by propidium-iodide staining and FACS analysis. Values are expressed as fold increase compared to static control. The right panel shows the result of free nucleosome evaluation performed by ELISA. Values are expressed in optical density (O.D.). Each experiments was performed three times in duplicate. * = p<0.05; ** = p<0.01. c) A representative western blotting analysis (WB) of caspase 3 cleavage is shown on the left. The inactive precursors (pro-casp-3) and the cleaved form (casp-3) are indicated. The experiments were performed in static culture (ST), in the presence or absence of Bleo (B) and or laminar SS (SS). The right panel shows the average densitometric analysis of three independent experiments. Data are expressed in arbitrary units. * = p<0.05.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2719102&req=5

pone-0006603-g001: SS induces cell death in endothelial cells exposed to 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 represents the intracellular content determination of phosphorylated histone 2AX in cell exposed to Bleo. Data are expressed as percent of control. * = p<0.05. b) The graph on the left depicts the result in which cell viability was determined by propidium-iodide staining and FACS analysis. Values are expressed as fold increase compared to static control. The right panel shows the result of free nucleosome evaluation performed by ELISA. Values are expressed in optical density (O.D.). Each experiments was performed three times in duplicate. * = p<0.05; ** = p<0.01. c) A representative western blotting analysis (WB) of caspase 3 cleavage is shown on the left. The inactive precursors (pro-casp-3) and the cleaved form (casp-3) are indicated. The experiments were performed in static culture (ST), in the presence or absence of Bleo (B) and or laminar SS (SS). The right panel shows the average densitometric analysis of three independent experiments. Data are expressed in arbitrary units. * = p<0.05.
Mentions: HUVEC were exposed to a SS of 12 dyne/cm2 for 16 hours (h) in the presence or absence of a sub-lethal concentration of Bleo (10 µg/ml). In this experimental condition the endothelial cell treatment with Bleo alone promoted the chromatin incorporation of the phosphorylated isoform of histone H2AX (γH2AX) (Figure 1a). Figure 1b, left and right panels, shows the results of experiments in which cell death and apoptosis were evaluated by propidium-iodide staining/FACS analysis (left) and free nucleosomes release (right) respectively. Both were found significantly increased in endothelial cells exposed to SS and Bleo. The evidence of a proapoptotic program activation was further supported by the presence of caspase 3 cleavage (Figure 1c, left panel) which reached the highest level, two fold above static control and about seven fold above SS alone, (Figure 1c, right panel) in the presence of Bleo and SS.

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