Ataxia Telangiectasia Mutated (ATM)-mediated DNA Damage Response in Oxidative Stress-induced Vascular Endothelial Cell Senescence*

Oxidative stress regulates dysfunction and senescence of vascular endothelial cells. The DNA damage response and its main signaling pathway involving ataxia telangiectasia mutated (ATM) have been implicated in playing a central role in mediating the actions of oxidative stress; however, the role of the ATM signaling pathway in vascular pathogenesis has largely remained unclear. Here, we identify ATM to regulate oxidative stress-induced endothelial cell dysfunction and premature senescence. Oxidative stress induced senescence in endothelial cells through activation/phosphorylation of ATM by way of an Akt/p53/p21-mediated pathway. These actions were abrogated in cells in which ATM was knocked down by RNA interference or inhibited by specific inhibitory compounds. Furthermore, the in vivo significance of this regulatory pathway was confirmed using ATM knock-out mice in which induction of senescent endothelial cells in the aorta in a diabetic mouse model of endothelial dysfunction and senescence was attenuated in contrast to pathological changes seen in wild-type mice. Collectively, our results show that ATM through an ATM/Akt/p53/p21-dependent signaling pathway mediates an instructive role in oxidative stress-induced endothelial dysfunction and premature senescence.

The DNA damage response is activated in response to stimuli ranging from oxidative stress, oncogenic stress to ionizing radiation to determine which cells remain viable after cytopathogenic insult. Recent reports suggest that the DNA damage response in addition to its classical role in regulating the cell cycle checkpoint in cancer to also play a major regulatory role in nononcogenic fields such as in aging/senescence (1)(2)(3). One field in which the DNA damage response has remained poorly addressed is the cardiovasculature.
Aging is known to be a major cardiovascular risk factor (4). Premature senescence in endothelial cells is induced by proatherogenic and proinflammatory factors such as hydrogen peroxide (H 2 O 2 ), oxidized LDL or TNF-␣ by telomeric inactivation through an Akt-dependent mechanism (5,6). Because regulation of aging/senescence of the vasculature, notably through the endothelial cell, contributes to mechanisms of arteriosclerosis and other age-related cardiovascular diseases (7), we questioned whether the DNA damage response might play a role in regulation of endothelial regulation of aging/ senescence. For this, we focused on the role of ataxia telangiectasia mutated (ATM), 4 which is the central effector molecule in the DNA damage response pathway.
ATM belongs to the phosphoinositide 3-kinase (PI3-kinase)related protein kinase (PIKK) family which has been identified as the product mutated or inactivated in ataxia telangiectasia (A-T) patients. The DNA damage response and its main signaling pathway involving ATM have been implicated in playing a central role in mediating the actions of oxidative stress (8 -10). However, the role of the ATM signaling pathway in vascular pathogenesis has remained unclear. Further, pathogenic mechanisms of the vascular pathologies associated with mutated ATM (telangiectasia, premature coronary artery disease) remain obscure.
In the present study, we examined the effects of ATM-mediated oxidative stress-induced senescence in vascular pathologies through actions on endothelial cells. Our results show that ATM through an ATM/Akt/p53/p21-dependent signaling pathway mediates an instructive role in oxidative stress-induced endothelial dysfunction and premature senescence.

EXPERIMENTAL PROCEDURES
Cell Culture-Human umbilical vein endothelial cells (HUVECs) were purchased from Sanko Junyaku (Tokyo, Japan) and maintained with endothelial cell basal medium-2 containing EGM-2 supplement as purchased from Cambrex Bio Science (Rockland, MD) in humidified air with 5% CO 2 at 37°C. All experiments were performed between passages 4 and 6.
Western Blot Analysis and Antibodies-HUVECs (3 ϫ 10 5 cells/well) were treated with 100 M H 2 O 2 in the absence or presence of N-acetyl-L-cysteine (NAC) (Sigma-Aldrich), caffeine (Wako, Osaka, Japan), or KU-55933 (Calbiochem). Cells were washed with cold phosphate-buffered saline (PBS) and lysed with 2ϫ NuPAGE LDS sample buffer (Invitrogen) supplemented with 1 mM sodium orthovanadate (Sigma-Aldrich) and 1 mM sodium fluoride (Wako). Lysates were then boiled for 10 min and centrifuged for 2 min at 4°C. Equal amounts of protein were separated by NuPAGE 3-8% Tris acetate mini gel electrophoresis or NuPAGE 10% BisTris gel (Invitrogen) and then transferred onto a polyvinylidine difluoride (PVDF) membrane (Invitrogen). Membranes were blocked with 5% skim milk in PBS at room temperature for 1 h and then subsequently probed with primary antibodies at a predetermined optimal concentration for 2-4 h at room temperature or overnight at 4°C. After rinsing with TBS containing 0.1% Triton X-100 (TBS-T), membranes were incubated with appropriate horseradish peroxidase-conjugated anti-rabbit (Cell Signaling Technology) or anti-mouse IgG (GE Healthcare) for 1 h at room temperature. After three washes with TBS-T, immunoblots were detected using the ECL Plus Western blotting Detection System (GE Healthcare) and exposed to x-ray film (Fuji medical x-ray film, Tokyo, Japan). Anti-phospho-ATM (Ser 1981 ) antibody was purchased from Millipore; anti-ATM (2C1) and anti-p53 (DO-1) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA); and anti-phospho-p53 (Ser 15 ), anti-phospho-Akt (Ser 473 ), and anti-Akt antibodies were from Cell Signaling Technology. Anti-GAPDH antibody (Life Technologies) was used as a loading control. Purified mouse anti-p21 antibody was obtained from BD Pharmingen. See also supplemental Experimental Procedures.
Immunofluorescence Microscopy-Cells were grown on coverslips at a density of 1 ϫ 10 5 per slide. After treatment with 100 M H 2 O 2 for 1 h, cells were fixed in 3% paraformaldehyde in PBS for 10 min. Chamber slides were washed three times with PBS and then permeabilized with 0.25% Triton X-100 in PBS for 10 min. After washing twice with PBS and blocking for 5 min with 0.1% gelatin, slides were incubated with primary antibody (1:100) in 0.1% gelatin in PBS for 1 h in a humidified chamber at 37°C. Cells were blocked three times with 0.1% gelatin, and then samples were incubated with secondary antibody using Alexa Fluor 488 green or Alexa Fluor 635 red (Life Technologies) in 0.1% gelatin in PBS for 1 h in a humidified chamber at 37°C. Antibodies used for immunofluorescence were ATM-Ser 1981 , ATM, p53-Ser 15 , p53, p21, and 53BP1 (Cell Signaling Technology). Nuclei were stained with Hoechst 33258 and mounted with DakoCytomation Fluorescent Mounting Medium (Dako Japan, Kyoto, Japan) and then visualized with a Carl Zeiss LSM510 confocal microscope (Carl Zeiss, Jena, Germany).
RNA Interference-Small interference RNA (siRNA) constructs were obtained as siGENOME SMARTpool reagent from Dharmacon. See also supplemental Experimental Procedures. A siRNA pool specific for ATM (siGENOME SMARTpool Human ATM, M-003201020005) and control siRNA (siGENOME nontargeting siRNA pool, D-0012061320) were used. HUVECs (3 ϫ 10 5 cells/well) were grown to 80% confluence in 6-well culture dishes. Transient transfections of 10, 50, and 100 pmol of ATM siRNA or nontarget siRNA were performed by a liposome-mediated method using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen). The indicated amounts of siRNA and 5 l of Lipofectamine 2000 were, respectively, diluted in 0.25 ml of Opti-MEM without serum. After incubation for 5 min, diluted siRNA and Lipofectamine 2000 were combined, and the mixture was incubated for 20 min at room temperature to allow the DNA⅐Lipofectamine 2000 complexes to form. 0.5 ml of complex was added to each well. After 5 h of transfection, the medium with complexes was removed, and endothelial cell basal medium-2 supplemented with EGM-2 was added. At 72 h following transient transfection, total RNA was extracted and submitted to reverse transcription-PCR (RT-PCR) experiments using oligonucleotide primers specific to ATM and 18 S rRNA. In ATM knock-down cells, the levels of total ATM, phospho-p53 (Ser 15 ), phospho-Akt (Ser 473 ), total Akt, or GAPDH proteins were analyzed by Western blotting, or SA-␤-gal activity was measured.
RNA Extraction and Quantitative RT-PCR-Total RNA from HUVECs (3 ϫ 10 5 cells/well) was extracted by the RNeasy Mini kit (Qiagen) according to the manufacturer's instructions. RNA concentration and purity were determined on a spectrophotometer (Ultrospec 3000; GE Healthcare) by calculating the ratio of optical density at 260 nm to 280 nm. One microgram of total RNA sample was used to generate first-strand complementary DNA by using powerscript reverse transcriptase (Clontech) according to the manufacturer's recommended procedures. PCR was then performed for ATM from the same complementary DNA samples using HotStarTaq (Qiagen), 10ϫ PCR buffer, and 2.5 mM dNTP mix. The forward primer 5Ј-GATGTTGTTGTCCCTAC-TATGG-3Ј and the reverse primer 5Ј-GCTACACT-GCGCGTATAAGCC-3Ј corresponded to human ATM. 18 S rRNA (Life Technologies) was used as an internal control. Amplification was initiated by 15 min of denaturation at 95°C for 1 cycle followed by 30 cycles at 95°C for 30 s, 57°C for 30 s, and 72°C for 40 s. After the last cycle of amplification, samples were incubated at 72°C for 10 min in a GeneAmp TM PCR system (Applied Biosystems). PCR products, a 589-bp ATM fragment and a 350-bp 18 S fragment, were then visualized by UV illumination after electrophoresis by 2% agarose gels containing 0.5 g/ml ethidium bromide.
Animal Experiments-ATM knock-out mice (129S6/ SvEvTac-Atm tm1Awb /J) were obtained from Jackson Laboratories. All generations were from matings of heterozygous parents. The following sequence, as recommended by Jackson Laboratories, was used for genotyping: oIMR640, 5Ј-GCTGC-CATACTTGATCAATG-3Ј; oIMR641, 5Ј-TCCGAATTTG-CAGGAGTG-3Ј; oIMR0013, 5Ј-CTTGGGTGGAGAGGC-TATTC-3; and oIMR0014, 5Ј-AGGTGAGATGACAGGAG-ATC-3Ј. Age-matched 10-week-old SPF male ATM wild-type ( ϩ/ϩ ), heterozygote ( ϩ/Ϫ ), and homozygote ( Ϫ/Ϫ ) mice (n ϭ 6, respectively, weighing ϳ15-25 g) were used. Hyperglycemia was induced by a single intraperitoneal injection of streptozotocin (STZ) (180 mg/kg; Sigma-Aldrich). Tail blood glucose was assayed 3 days after injection using glucose test strips (Roche Applied Science). All diabetic animals had blood glucose values Ͼ300 mg/dl. Mice were housed under constant temperature (23 Ϯ 1°C) with a 12-h light and 12-h dark cycle for 10 days with free access to water and chow and were killed by cervical dislocation. The aorta was removed after systemic perfusion with PBS for histological examination. The aorta was fixed for 30 min at room temperature in 2% formaldehyde/ 0.2% glutaraldehyde, washed, incubated at 37°C for 24 h (without CO 2 ) with fresh SA-␤-gal stain solution, and then embedded in OTC compound before freezing in liquid nitrogen. The samples were stored at Ϫ80°C until sample slides were prepared. The proportion of SA-␤gal-positive cells were analyzed by Scion Image software. Serial crosssections (10 m) were obtained from each sample and stained with kernechtrot staining solution (Muto, Tokyo, Japan) or prepared for immunohistochemistry. All experimental protocols complied with the guidelines for animal experiments of the University of Tokyo.
Tissue Protein Extraction-Thoracic aorta of ATM knock-out mice (ATM ϩ/ϩ , ATM ϩ/Ϫ , ATM Ϫ/Ϫ mice) were dissected out of their thoracic aortas and snap frozen in liquid nitrogen. After thawing on ice, the thoracic aortas were homogenized mechanically at 25 Hz for 25 s five times on ice in 150 l of RIPA buffer (0.1% SDS, 0.5% Nonidet P-40, 0.1% sodium deoxycholate, 150 mM NaCl, 50 mM Tris-HCl, pH 7.9, and 1ϫ EDTA-free complete proteinase inhibitor (Roche Applied Science)). Samples were lysed gently on ice for 30 -60 min, and cellular debris was removed by centrifugation. Protein was then quantified using the BCA protein assay kit (Thermo). A 30-g aliquot of total protein was analyzed for ATM protein by Western blot analysis. Anti-GAPDH antibody was used as a loading control.
Immunohistochemistry-Immunostaining was performed using the Envision kit (Dako Japan). Frozen sections, 10 m thick, were fixed in acetone at 4°C, washed in TBS, and then blocked by 0.03% hydrogen peroxidase in methanol. After blocking nonspecific antibody-binding sites, the sections were incubated for 1 h with antibodies against von Willebrand factor (1:1,000; Abcam), p21 and p16 (Santa Cruz Biotechnology) as the primary antibody and then for 1 h with the peroxidase- Representative blots are shown in the upper panels whereas corresponding quantitation is shown in the lower panels. D-F, immunofluorescence for 53BP1, phosphorylated ATM-S1981, and total ATM is shown. Cells were exposed to 10 M etoposide or 100 M H 2 O 2 for 1 h and then immunostained for 53BP1 (D), ATM-S1981 (E) and total ATM (F). Cells incubated without etoposide or H 2 O 2 were used as controls. G, cells were exposed to 100 M H 2 O 2 for 3 h and then immunostained for p21. Hoechst 33258 was used as nuclear stain (blue). Original magnification, ϫ200 and ϫ630. Scale bars, 200 m and 10 m, respectively. Treatment with etoposide and immunofluorescence for 53BP1 (labeled by green fluorescence) was used for a positive control. Expression of phosphorylated ATM-S1981) and p21 labeled by red fluorescence in HUVECs and foci formation was significantly increased after H 2 O 2 treatment. Additional views of the photographs are shown in supplemental Fig. 1. labeled polymer. Finally, sections were incubated with diaminobenzidine tetrahydrochloride (Dako Japan), and the nuclei were counterstained with hematoxylin.
Statistical Analysis-All values are expressed as mean Ϯ S.E. Differences between two groups were analyzed using the twotailed Student's t test. Threshold of significance was taken as p Ͻ 0.05.

ATM Is Activated by Oxidative Stress in Vascular Endothelial
Cells-To investigate the involvement of ATM in mediating oxidative stress in the vasculature, we first examined the acti-vation and expression of ATM in cultured HUVECs treated with H 2 O 2 as an inducing agent of oxidative stress. HUVECs exposed to H 2 O 2 showed increased ATM phosphorylation at Ser 1981 (ATM-S1981) (Fig. 1, A and B), which releases it from an inhibitory homodimer structure leading to its activation and recruitment to sites of DNA double-strand breaks (DSBs) (12)(13)(14). We next investigated factors involved in the ATM signaling pathway. Because ATM mediates activation of Akt in response to insulin or ionizing radiation which then in turn results in radiosensitivity or resistance to insulin in cell lines derived from A-T patients and ATM knock-out mice (15), we first investigated the activation of Akt in response to oxidative stress. Akt phosphorylation at Ser 473 (Akt-S473) was increased in H 2 O 2treated HUVECs (Fig. 1, A and B). We then investigated involvement of p53, a downstream signaling molecule of ATM (10) and Akt (6), which showed phosphorylation at Ser 15 (p53-S15) after exposure to H 2 O 2 (Fig. 1, A and B). Furthermore, p21, a downstream target of p53, was increased in H 2 O 2 -treated HUVECs (Fig. 1C). Our results indicate that oxidative stress phosphorylates ATM and its involved genes, Akt and p53, with subsequent upregulation of p21 expression. Thus, the phosphorylation of ATM, Akt, and p53 and up-regulation of p21 expression mediate actions of oxidative stress in endothelial cells.
Although ATM is predominantly present in the nucleus, a variable amount (Ͻ10%) has been reported in the cytoplasm, especially in neuronal cells. The major known role of nuclear ATM is to participate in the response to DSBs for DNA repair and cell cycle checkpoint activation (16). In response to agents that induce DSBs, ATM has been found to relocalize to the sites of breaks and in doing so forms large nuclear foci (17). To determine whether oxidative stress-induced ATM forms foci, immunofluorescence analyses for phospho-ATM (Ser 1981 ), total ATM, and p21 were performed. Increased fluorescence and foci formation were seen in response to H 2 O 2 for phosphorylated ATM (Fig. 1E and supplemental Fig. 1B) and increased p21 ( Fig.  1G and supplemental Fig. 1D) under conditions in which 53BP1 formed nuclear foci in response to etoposide as reported ( Fig. 1D and supplemental Fig. 1A) (18). Total ATM, in contrast, remained diffusely localized throughout the nucleus, and foci formation was not observed in H 2 O 2 -treated HUVECs (Fig. 1F  and supplemental Fig. 1C). Activated (phosphorylated) ATM localized to foci, which suggests that it might be recruited to sites of DNA damage caused by oxidative stress (17,18). However, the authors note that the antibody used (ATM-S1981) is known to also recognize a nonspecific protein, which makes conclusions based on experiments using this antibody inconclusive. We further investigated expression of 53BP1 and ␥-H2AX as markers of DSB formation in response to H 2 O 2 by immunostaining. In the H 2 O 2treated cells, both 53BP1 and ␥-H2AX formed nuclear foci (supplemental Fig. 3), which further supports that ATM is activated in response to DSB formation induced by H 2 O 2 .
Activation of the ATM-dependent DNA Damage Signaling Pathway Contributes to Endothelial Cell Senescence-To better confirm actions of activation of ATM by oxidative stress in endothelial cells, effects of inhibitors of oxidative stress and of ATM were tested. HUVECs were pretreated with NAC, an antioxidant and glutathione precursor that alters the redox state of cells (19), to assess whether ATM phosphorylation was caused by H 2 O 2 -induced redox imbalance. Western blot analysis showed that pretreatment of endothelial cells with NAC blocked the stimulatory effects of H 2 O 2 on the expression of ATM-S1981, Akt-S473, p53-S15 (lanes 3-5 in Fig. 2A) and p21 (lanes 3-5 in Fig. 2D). These results suggest a direct role of H 2 O 2 in ATM phosphorylation and activation of involved genes. We further employed two inhibitors, caffeine and KU-55933, to inhibit ATM kinase activity to examine whether Akt and p53 activation as well as p21 up-regulation induced by H 2 O 2 is dependent on the ATM protein kinase. Caffeine is known to disrupt ATM-dependent responses likely through direct inhibition of ATM kinase activity (20), and KU-55933 is a specific and potent inhibitor of ATM kinase (21). Both inhibitors inhibited H 2 O 2 -induced ATM (Ser 1981 ), Akt (Ser 473 ), and p53 (Ser 15 ) phosphorylation (lanes 3-5 in Fig. 2, B and C), in addition to p21 induction (lanes 6 -8 and lanes 11-13 in Fig. 2D). Thus, ATM kinase played a major role in transducing H 2 O 2induced DNA damage signaling in HUVECs. Because an Akt/ p53/p21-dependent pathway has been reported in endothelial cells (6), ATM thus likely regulates the cellular response to oxidative stress via phosphorylation of Akt and p53 then induction of p21 in sequence. Furthermore, NAC or KU-55933 also greatly reduced phosphorylated ATM nuclear fluorescence and foci formation (Fig. 3A). Collectively, H 2 O 2 induced DNA damage and stimulated autophosphorylation of ATM in addition to formation of discrete nuclear foci together with subsequent induction of phosphorylation of Akt, p53, and up-regulation of p21 expression in an ATM-dependent manner.
Because the DNA damage response pathway triggers senescence (22) and because p53 and p21 were described to be major players in the induction of senescence (23,24), we next addressed the functional effects of activation of the ATM/Akt/ p53/p21 pathway on endothelial cells, namely in the form of induction of senescence which is a consequence of endothelial dysfunction. We induced premature endothelial senescence by addition of H 2 O 2 to 100 M as shown by SA-␤-gal assay, a recognized surrogate index of senescent cells (25). We found that percentage of HUVECs positive for SA-␤-gal was markedly increased in cells treated with H 2 O 2 , compared with cells incubated without H 2 O 2 treatment (panel 2 in Fig. 3B and lane 2 in Fig. 3C). Furthermore, pretreatment with NAC, caffeine, or KU-55933 resulted in significant reduction of senescent (SA-␤-galpositive) cells compared with those exposed to H 2 O 2 alone (Fig. 3, B and  C). Thus, activation of ATM by H 2 O 2 promotes endothelial cell senescence and contributes to vascular pathogenesis.
Abrogation of ATM Blocks Effects of Oxidative Stress on Endothelial Cells-We further examined the requirement of ATM expression in H 2 O 2 -induced premature senescence by abrogating ATM in cells using RNA interference. Knockdown of ATM in HUVECs by siRNA reduced ATM mRNA and protein levels (Fig. 4, A and B). Cells transfected with ATM siRNA showed reduced ATM expression with inhibition of Akt and p53 phosphorylation (lanes 4 -6 in Fig. 4B) in addition to down-regulation of p21 expression (lanes 4 -6 in Fig. 4C). As expected, ATM nuclear fluorescence was also markedly decreased in cells with ATM knocked down (Fig. 5A). Furthermore, knockdown of ATM by siRNA suppressed increase in SA-␤-gal-positive cells induced by H 2 O 2 (panels 5 and 6 in Fig. 5B and lanes 5 and 6 in Fig. 5C), which were comparable with the changes seen when HUVECs were treated with antioxidant or ATM inhibitor. Moreover, down-regulation of Akt, p53, or p21 by siRNA also suppressed an increase in SA-␤-gal-positive cells induced by H 2 O 2 (supplemental Figs. 4 and 5). Thus, RNA interference experiments further confirmed that ATM and its downstream molecules, Akt, p53, and p21, mediate actions of oxidative stress on endothelial cells to induce senescence.
Abrogation of Senescent Phenotype in Aorta of ATM Knockout Mice-To test whether ATM mediates vascular endothelial cell senescence in vivo, we administered STZ to ATM knockout mice and wild-type littermates to induce hyperglycemiainduced endothelial dysfunction because vascular endothelial cell senescence has been previously documented to be induced in STZ-diabetic mice (26). Western blot analysis for ATM protein levels in the thoracic aortas of heterozygous knock-out mice showed marked reductions to levels almost comparable with those of homozygote knock-out mice in contrast to robust levels as seen in wild-type mice ( Fig. 6B and supplemental Fig. 7A) although the mechanisms of reduced protein levels in heterozygous knock-out mice are unclear. STZ-treated mice showed an elevation in blood glucose levels compared with STZ-untreated mice (lanes 2, 4, and 6 in Fig. 6C). SA-␤- gal activity was observed in the thoracic aortas of STZ-diabetic wild-type mice but not in STZ-diabetic ATM knockout mice (Fig. 6, D and E). Cross-sections of thoracic aortas stained with SA-␤-gal showed that positive areas were mostly localized to the luminal surface (Fig. 6F) which also stained positive for von Willebrand factor, indicating localization to vascular endothelial cells and not the extracellular matrix compared with normal rabbit IgG antibody ( Fig. 6G  and supplemental Fig. 6A). p21 and p16 are cyclin-dependent kinase inhibitor genes that are used as senescence markers. SA-␤-gal-positive areas of cross-sections of thoracic aortas stained positive for p21 and p16 ( Fig. 6H and 6I) but not for normal mouse IgG antibody used as a negative control (supplemental Fig. 6B). These results indicate that ATM is important for the induction of endothelial cell senescence in aortas of STZ-diabetic mice, which is consistent with in vitro cellular experiments.

DISCUSSION
Oxidative stress caused by reactive oxygen species plays an important causal role in senescence and age-related vascular diseases, including atherosclerosis and diabetic vasculopathy (7,27,28). Despite the wealth of knowledge on the effects and actions of oxidative stress on endothelial cells, characterization of involved regulatory pathways allowing for targeted molecular intervention with therapeutic intent had remained elusive.
We showed that ATM is involved in oxidative stress-induced endothelial dysfunction and premature senescencethroughanAkt/p53/p21dependent pathway. Cellular experiments using HUVECs showed that the ATM/Akt/p53/p21 pathway was involved in oxidative stress-induced cellular senescence in vitro. Experiments using antioxidant and specific ATM inhibitory compounds or siRNA against ATM inhibited oxidative stress-induced cellular senescence thus confirming involvement of ATM and its dependent pathway. Furthermore, ATM induced endothelial cellular senescence in vivo in the aorta of diabetic wild-type mice but not in ATM knock-out mice. STZ-diabetic ATM ϩ/Ϫ mice exhibit reduction in SA-␤-gal-positive cells to levels almost comparable with those seen in STZ-diabetic ATM Ϫ/Ϫ mice. Reduction of ATM protein expression in heterozygote knock-out mice may account for the difference seen in endothelial senescence between wild-type and heterozygous knockout mice. Collectively, the findings presented here indicate the importance of ATM in the induction of endothelial cell senescence induced by oxidative stress and abrogation of ATM resulting in a dysregulated response to pathophysiological stress in cardiovascular endothelial cells.
We further showed that ATM lies upstream from the Akt/ p53/p21 pathway and that oxidative stress as sensed through ATM possibly in response to oxidative DNA damage is the initiating "trigger" and plays an instructive role in activation of this signaling pathway. Previous studies also demonstrated that ATM is a major upstream activator of Akt through control of vive to later years are also known to be prone to ischemic heart disease (31). Although the precise underlying mechanism for the latter is unknown, in general, progression of coronary artery disease involves accelerated atherosclerosis in which oxidative stress of the endothelium and related cells plays a major contributory role. Response to oxidative stress in the vasculature may be disrupted in A-T patients. Dysregulation of endothelial cell function might lead to pathophysiological states such as vascular dysfunction in developmental states and accelerated atherosclerosis in adults. Better understanding the cellular response to oxidative stress with a focus on ATM is expected to shed further light on the pathogenesis of A-T and to clarify alternative pathological pathways that are activated in response to oxidative stress when deficient for ATM as well as to provide new insight into the molecular mechanisms underlying agerelated cardiovascular pathologies such as atherosclerosis in which ATM might pose a new therapeutic target for vascular pathologies involving oxidative stress.
In conclusion, we show that oxidative stress can induce cellular senescence in HUVECs as shown by staining for SA-␤-gal, which was associated with an ATM-dependent Akt/p53/p21 signaling pathway. Our findings might in part explain underlying mechanisms of the pathogenesis of the disease ataxia telangiectasia in which vascular dysplasia and dysfunction are seen, as well as suggesting that ATM may be a new therapeutic target for cardiovascular pathologies.