Introduction

Oxidative damage is an important factor in the accumulation of DNA damage. It can lead to mutagenesis and carcinogenesis among other biological effects [9]. Increased levels of reactive oxygen species (ROS) are found in atherosclerosis in all layers of the affected artery wall [24, 30, 38]. Oxidative damage was also proposed to have an impact on telomeric DNA ends as the accumulation of ROS can cause an accelerated telomere shortening [7, 36]. Telomeres are specialized G-rich repeat sequences, playing an important role in the maintenance of genomic stability by preventing the natural ends from being recognized as damaged DNA [1, 3, 4, 13, 19]. Telomeric repeats are generated by a specialized reverse transcriptase, called telomerase, which is composed of an RNA component, serving as a template for the addition of telomeric repeats, and a protein component, which is the telomerase reverse-transcriptase catalytic subunit (Tert) [28]. The sensitivity of telomeric DNA ends to oxidative stress was attributed to their high guanine content [23, 24, 36]. As the single strand breaks at telomeric DNA cannot undergo a repair process, 8-oxo dG as a result of reactive oxygen species causes telomere shortening through replication [36]. Ataxia telangiectasia (A-T), an autosomal recessive disease, is an example of the relation between oxidative stress and telomere shortening [31].

ROS-related damage is known to be a reversible process induced by a number of antioxidant agents. We previously reported that balloon angioplasty in iliac arteries resulted in intimal hyperplasia due to proliferation of the smooth muscle cells, and small-size telomeric restrictional fragments were evident in injured arteries [22]. In the present study, we investigated the effects of oxidative stress on telomeric length regulation of proliferating vascular smooth muscle cells after balloon injury. We employed buthionine sulfoximine (BSO) to generate oxidative stress and taurine as the reversing agent by either single or combined treatment of the experimental animals with these two agents. Buthionine sulfoximine is a selective inhibitor of γ-glutamyl cysteine synthetase, which is responsible for the synthesis of glutathione (GSH) [34, 37]. Taurine, on the other hand, is involved in a variety of biological processes such as bile salt formation, osmoregulation, immunomodulation, diabetes, atherosclerosis, and oxidative stress inhibition [5, 21]. The effects of these agents were monitored by determining GSH concentrations, the ratio of [GSH] to [GSSG], glutathione peroxidase (GPx) activity, and telomeric restriction fragment (TRF) length, as well as morphometric and histochemical analyses.

Materials and methods

General

Telomeric length analyses were performed in a total of 12 New Zealand white rabbits (2.5–3.0 kg) of both sexes. Briefly, experimental animals were anesthetized with pentobarbital (25 mg/kg, iv), and balloon angioplasty was performed in the iliac artery of rabbits with a Fogarty 2.5-mm balloon catheter four times with 6-, 8-, 4-, and 10-atm pressure for 1 min [22, 29]. Whole injured and uninjured contralateral arteries (sham) were isolated for morphometric, immunohistochemical, and telomeric length analyses on day 14. Experimental animals were treated with BSO (75 mg/kg/day) (group I), taurine (10% w/v of total water intake) (group II), both BSO and taurine (group III). Group IV animals did not receive further treatment following balloon angioplasty. Blood samples were drawn on days 0 and 14 to determine selected oxidative stress markers, namely [GSH], [GSH]/[GSSG], and GPx activity. The animals were anesthetized with pentobarbital (25 mg/kg, iv) on the 14th day of treatment to excise iliac arteries (~100 mg) for morphometric, immunohistochemical, and telomeric length analyses. The study was approved by the Ethics Committee of Ege University, Izmir, TR. All the animals received care according to the criteria outlined in the “Guide for Care and Use of Laboratory Animals” prepared by the National Academy of Science, also adopted and implemented by Ege University, Izmir, TR.

Histological and immunohistochemical analyses

Histological and immunohistochemical analyses were carried out as described [21]. Four-micron-thick samples were analyzed through immunohistochemical staining using an antibody against proliferating cell nuclear antigen (PCNA) (PC10 clone, M0879, Dako, Glostrup, Denmark) [6, 27].

Biochemical analyses

Total GSH concentration, the ratio of reduced and oxidized glutathione (GSH/GSSG), and the activity of GPx were determined using Bioxytech kits, namely GSH-420, GSH/GSSG-412, and GPx-340 (Oxis, USA), respectively, according to the manufacturer’s specifications.

Analyses of telomeric restriction fragments

Total DNA was isolated using “Qiagen DNeasy® Tissue Kit” according to the manufacturer’s specifications (QIAGEN, Haan, Germany). Following spectrophotometric (UV-1208, Shimadzu, Kyoto, Japan) quantification of isolated DNA, comparable amounts of DNA (~5 μg) dissolved in Tris–ethylenediaminetetraacetic acid (EDTA) (10 mM Tris–Cl, 1 mM EDTA, pH 8.0) were fragmented using 10 U of each of HinfI and RsaI enzymes at 37°C for 2 h in a total volume of 20 μL. Reaction products were analyzed on 1% agarose gels in TBE buffer (5 V/cm) and photographed under UV light after staining with ethidium bromide (EtBr) (0.5 μg/mL). Southern transfer and hybridizations were carried out as described [18, 25]. Transferred DNA was cross-linked to the membrane (HYBOND, Escondido, CA, USA) in a UV cross-linker (Vilber Lourmat, Cedex, France) for hybridization with the telomeric probe labeled with digoxigenin (digoxigenin 3′-end-labeled 5′-CCTAAA-3′). Membranes were visualized using an X-Ray developer (Kodak X-Omat, Plano, TX, USA). The signal strength of the DNA bands were quantified from gel photographs using Bio-Rad Multianalyst (ver. 1.1), and mean telomeric fragment lengths of digested samples were calculated according to the following equation:

$$ \sum {\left( {\hbox{ODi}} \right)/\sum {\left( {{\hbox{ODi}}/{\hbox{Li}}} \right.} )} $$

where ODi and Li stand for the signal intensity and the length of the given fragment, respectively [12, 32]. All reactions were carried out in DNase-free 1.5-mL microcentrifuge tubes. To check for reproducibility, all reactions were repeated at least once. The values of the injured arteries were given as percentage of the uninjured arteries [15].

Statistical analysis

Values are represented as mean ± standard error. One-way analysis of variance was carried out using GraphPad Prism Software (Ver 3.0), and the significance of differences between the groups was assessed using Bonferroni test. Significance was defined at 95% confidence interval.

Results and discussion

We used the balloon angioplasty model to study oxidative stress in relation to telomeric length regulation of proliferated vascular smooth muscle cells. Figure 1 shows representative vascular smooth muscle segments obtained from balloon-injured iliac arteries (Fig. 1a) treated either with BSO (Fig. 1b) or taurine (Fig. 1c). The result of treatment when the animals received both agents is given in Fig. 1d. Balloon angioplasty resulted in a remarkable thickness of intimal area, which was further increased by BSO treatment. We then measured intimal area of individual samples to quantify the detected intimal thickness of the samples (Fig. 2). As seen in Fig. 2, taurine treatment reversed the intimal thickening together with BSO. Our results were attributed to balloon angioplasticity, as there was no difference between the treatments among uninjured arteries. On the other hand, our analyses on luminal area to a considerable degree supported the results we obtained with intimal area (Fig. 3). As was to be expected while balloon angioplasticity resulted in remarkable thickness of intimal area (Fig. 2), it also caused a significant decrease in luminal area (Fig. 3). However, as seen in Figs. 2 and 3, taurine treatment significantly inhibited BSO-induced intimal thickening whereas no differences were observed in luminal area. Taking these findings into consideration, our results suggests that the effect of BSO together with balloon injury seems to be more pronounced in intimal thickening than in luminal area.

Fig. 1
figure 1

Hematoxylin–eosin-stained iliac artery tissue sections: a only balloon-injured, b balloon-injured + BSO-treated, c balloon-injured + taurine-treated, d balloon-injured + BSO- and taurine-treated, e uninjured and untreated contralateral artery (×10 magnification)

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Fig. 2
figure 2

Intimal area (mm2) after balloon injury (Control uninjured and untreated contralateral artery, B balloon-injured, BSO + B balloon-injured + BSO-treated, B + T balloon-injured + taurine-treated, BSO + B + T balloon-injured + BSO- and taurine-treated)

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Fig. 3
figure 3

Luminal area (mm2) after balloon injury (Control uninjured and untreated contralateral artery, B balloon-injured, BSO + B balloon-injured + BSO-treated, B + T balloon-injured + taurine-treated, BSO +  B +  T balloon-injured + BSO- and taurine-treated)

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Figure 4 gives the results of immunohistochemical analyses to detect the proliferation in the smooth muscle cells as a consequence of treatments in the same order as Fig. 1. We employed antibody against PCNA as proliferation indicator and detected positive staining in the nuclear area following balloon injury, independent of either BSO or taurine treatment. However, there was no staining in uninjured arteries (Fig. 4). We also detected smooth muscle-specific α-actin expression in our samples (data not shown).

Fig. 4
figure 4

PCNA staining after balloon injury: a balloon injury, b balloon injury + BSO, c balloon injury + taurine, d balloon injury + BSO + taurine, e uninjured, untreated contralateral artery (×10 magnification)

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Measurement of oxidative stress parameters in blood samples of days 0 and 14 is summarized in Table 1. As seen in Table 1, BSO diminished the GSH concentration, while this agent, when applied together with taurine, significantly reversed this effect (p < 0.001). Likewise, the ratio of [GSH] to [GSSG] was decreased upon BSO treatment with a considerable reversal by taurine (p < 0.01) (Table 1). Finally, BSO-related [GSH] depletion decreased the activity of GPx (Table 1) while BSO treatment together with taurine normalized the depleted [GSH], [GSH]/[GSSG], and decreased GPx activity (Table 1).

Table 1 Levels of oxidative stress parameters
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Our previous study showed no detectable telomerase activity following balloon angioplasty [22]. Absence of telomerase activity is a common property of the vast majority of adult somatic cells [8, 14, 26]. We, therefore, analyzed telomeric restrictional fragment length distribution using Southern blot to determine mean telomeric length. Southern blot is a highly sensitive methodology in estimation of desired fragments transferred from agarose gels, provided that hybridization of the membrane is carried out using specific probes in stringent conditions [25]. We used digoxigenin-labeled telomere-specific oligonucleotide of 15 mer as telomere-specific probe. The isolated DNAs were fragmented using the restriction enzymes, HinfI and RsaI, and applied on 1% agarose gel. A representative gel, accommodating fragmented telomeric DNAs, is given in Fig. 5. High molecular weight DNA samples were detectable at the upper left and upper right panels of the gel due to uneven sample loading (Fig. 5). However, the small-sized DNA molecules representing telomeric DNA repeats were visualized throughout the groups (Fig. 5). Although the DNA content of the gels were transferred by Southern blot, we used the negatives of the EtBr-stained gel photos to calculate the average band intensities to eliminate any variation due to underexposure or overexposure of the X-rays. Our results showed a significant decrease in the length of mean telomeric fragments upon BSO treatment (72.3% vs 56.7%, for control and BSO groups, respectively) (p < 0.01) (Table 2). Taurine effectively reversed the decrease in telomere shortening (56.7% vs 66.7%, for BSO and BSO + taurine groups, respectively) (p < 0.05) (Table 2). The MTF obtained in taurine-treated group was no different from that of the control samples (72.3% and 77.0% for control and taurine groups, respectively) (Table 2).

Fig. 5
figure 5

Southern blot of HinfI and RsaI fragments hybridized with telomeric oligonucleotide probe (mw molecular weight marker, B balloon-injured artery, N balloon-uninjured contralateral artery)

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Table 2 Relative mean telomere fragments of control, BSO, BSO + taurine, and taurine groups following balloon injury on day 14
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In conclusion, our study showed that exposure to oxidative stress increased balloon injury, whereas taurine treatment significantly diminished intimal hyperplasia. This result was consistent with our previous study [10]. We also report that there was a remarkable shortening of telomere length after balloon injury. In vivo oxidative stress accelerated this shortening of the telomere length, and antioxidant taurine partially reversed the shortening. Several studies have shown that senescence can be triggered either by telomeric DNA instability resulting from telomere erosion (replicative senescence) or following exposure to multiple types of stress (stress-induced senescence), such as oxidative stress [33], DNA damage, and mutagenic stress [2, 35]. In vitro, chronic oxidative stress results in increased cellular turnover that promotes telomere erosion and senescence of endothelial [16] and vascular smooth muscle cells [17, 20]. Previously, it was shown that glutathione depletion by buthionine sulfoximine induces oxidative damage to DNA in organs of rabbits [11]. Our findings suggest that, in vivo, telomeres in vascular smooth muscle cells shorten after balloon injury, which may result in a replicative senescence and shortening of the telomeres. This process is accelerated by oxidative stress, which may result from an oxidative damage to telomeric DNA, and prevention of cellular senescence with antioxidant agents like taurine may be a novel therapeutic target in atherosclerosis. The mechanisms of cellular senescence caused by telomeric erosion and possible oxidative damage to telomeric DNA will be included within the scope of our future research.