Heme Oxygenase-1 Gene Expression Attenuates Angiotensin II-Mediated DNA Damage in Endothelial Cells

Abstract

Heme oxygenase (HO) catalyzes the conversion of heme to biliverdin with the release of iron and carbon monoxide. HO-1 is inducible by inflammatory conditions, which cause oxidative stress in endothelial cells. Overexpression of human HO-1 in endothelial cells may have the potential to provide protection against a variety of agents that cause oxidative stress. We investigated the physiological significance of human HO-1 overexpression using a retroviral vector on attenuation of angiotensin II (Ang II)-mediated oxidative stress. Comet and glutathione (GSH) levels were used as indicators of the levels of oxidative stress. Comet assay was performed to evaluate damage on DNA, whereas GSH levels were measured to determine the unbalance of redox potential. Pretreatments with inducers, such as heme 10 μM, SnCl₂ 10 μM, and inhibitors, such as tin-mesoporphyrin 10 μM was followed by treatment with Ang II 200 ng/ml. Pretreatment with heme or SnCl₂ provoked significant reductions (P < 0.01) of tail moment in the comet assay. Opposite effects were evident by pretreatment for 16 hr with tin-mesoporphyrin. A decrease in tail moment levels was found in human endothelial cells transduced with the human HO-1 gene. The addition of Ang II (200 ng/ml) to human dermal microvessel endothelial cel1-1 for 16 hr resulted in a significant (P < 0.05) reduction of GSH contents control endothelial cells but not in endothelial cells transduced with HO-1 gene. The results presented indicated that stimulation or overexpression of HO-1 attenuated DNA damages caused by exposures of Ang II.

Oxidative stress has been increasingly implicated in the pathogenesis of many disease states and important biological processes, including aging, atherosclerosis, carcinogenesis, ischemia-reperfusion tissue injury, and acute and chronic inflammatory disorders. Toxic reactive oxygen species (ROS), including the superoxide and hydroxyl radicals, and hydrogen peroxide (H₂O₂), generated from normal cellular respiration and aerobic metabolism or exogenous oxidants, can cause cellular damage by oxidizing nucleic acids, proteins and membrane lipids (1). In cultured cells, oxidative stress can elicit the induction of antioxidant enzymes, such as superoxide dismutase, catalase, glutathione peroxidase, and genes that include stress-response proteins, such as heat shock proteins, metallothionein, and heme oxygenase-1 (HO-1)(2, 3). The primary function of these enzymes and proteins is to scavenge ROS and to help attenuate the pro-oxidant state of the cell to maintain normal cellular homeostasis (3–7).

HO-1 is considered one of the most important indicators of cellular stress and it is very sensitive to various chemicals and stimuli, including the glutathione (GSH) depletion (2, 8). Furthermore, it has been demonstrated already that GSH depletion, either by direct chemical agent or by use of a specific inhibitor of GSH synthesis, causes an increase in transcript level of HO-1.

Angiotensin (Ang) II is an important factor in cardiovascular homeostasis and exerts many actions on vascular tone, hormone secretion, neuronal effects, and the central nervous system (9). Ang II is produced by the action of angiotensin-converting enzyme on angiotensinogen. The effects of Ang II are mediated by two receptors: AT₁ and AT₂. They both share a seven-transmembrane domain topology; however, they have differential pharmacological and biochemical properties and appear to exert opposite effects in terms of cardiovascular hemodynamics and cell growth (10). Ang II may induce oxidant stress via activation of the NADPH/NADH oxidase system and generation of ROS as superoxide anion (O₂ ^•−), and hydroxyl radical (HO). Additionally, Ang II increased lipid peroxidation (11) and may stimulate the production of a number of cytokines, many of which are pro oxidant (12–14). Ang II also induces cellular adhesion molecules, chemotactic, and proinflammatory cytokines, all of which participate in the induction of an inflammatory response in the vessel wall (15–17); and in the generation of ROS in smooth muscle cells via the activation of NADH/NADPH oxidases (18, 19).

Ang II-induced apoptosis of human venous endothelial cells, myocyte, and vascular smooth muscular cells by caspase cascade activation and the blockade of Ang II type AT₁ and AT₂ receptor prevents Ang II-induced apoptosis, whereas selective agonistic stimulation of the AT₂ receptor alone induces apoptosis (20). In endothelial cells, Ang II induces apoptosis via ERK1/2 dephosphorylation (21). Furthermore, Ang II increases HO-1 expression in the kidney of the hypertensive rat (22), suggesting a possible protective role of HO-1 in response to the Ang II-induced renal injury. A similar response was shown in the aortas of rats made hypertensive by Ang II infusion (23).

ROS are directly or indirectly responsible for various clinical disorders, such as atherosclerosis (15), reperfusion injury, neurodegenerative diseases (25) and cancer (25). ROS induce cell injury to act on different cellular targets, such as DNA, proteins, and membrane lipids. ROS may cause DNA-protein cross-links, alterations to the deoxyribose-phosphate backbone by chemical modification of purine and pyrimidine bases (26, 27), protein oxidation products and carbonyl derivatives from oxidative modifications of amino acid side chains, reactive oxygen-mediated peptide cleavage, and lipid peroxidation.

The objective of this study was to examine the cytoprotective role of HO-1 against Ang II-mediated oxidative stress. GHS levels and DNA degradation (COMET) were measured to determine the injurious effect of Ang II. We examined the effect of HO-1 inducers and inhibitors as well as delivery of human HO-1 gene in sense orientation on endothelial cell DNA integrity and cellular redox levels as measured by GSH. Because antioxidants have the potential to limit ROS effects, we have measured the inner cellular reduced GSH levels to assess the recruitment of HO-1 by a scavenger mechanism GSH independent pathway. Our results show that induction of HO-1 prevented Ang II-mediated DNA degradation and may act as antioxidants.

The results demonstrated that the inducers of HO-1, such as SnCL2 and HO-1 gene transfer, prevent DNA degradation by Ang II. In contrast, inhibitors of HO activity decreased GSH and enhanced COMET levels.

Materials and Methods

Cell Culture Conditions.

Human dermal microvessel endothelial cells (HMECs) were a kind gift from Dr. Michael Dillon (National Center for Infectious Diseases, Atlanta, GA) and grown in MCDB131 medium (GIBCO-BRL, Grand Island, NY) supplemented with 10% fetal bovine serum, 10 ng/ml EGF (Sigma, St. Louis, MO), and 1 μg/ml hydrocortisone (Sigma). The cells were incubated at 37°C in a 5% CO₂-humidified atmosphere and maintained at subconfluency by passaging with Trypsin-EDTA (GIBCO-BRL).

Development of Recombinant Retroviral Vectors and Viral Propagation.

The LSN-HOP-human HO-1 was constructed as previously described (28). Briefly, the retroviral vector pGEM-HOP was constructed by cloning a 1519 bp (+19 to −1500) human HO-1 transcriptional-regulated sequence (HOP) from the plasmid A-CAT at the XhoI and HindIII sites of the plasmid pGEM-7zf (+) (Promega, Madison, WI). The retroviral vector LSN-HOP was constructed by cloning the XhoI-EcoRI HOP sequence of the pGEM-HOP at the EcoRI and XhoI sites of the retroviral vector LXSN. The 987 bp (−63 to +924 bp) HindIII human HO-1 cDNA fragment from the pRc-CMV-human HO-1 (29) was end-blunted and inserted at the end-blunted BamHI site of the LSN-HOP. After clone selection, the transcription-oriented construct was designated as LSN-HOP-human HO-1.

The amphotropic retroviral packaging cell line PT67 (Clontech Laboratories, Inc., Palo Alto, CA) was used for generation of replication-deficient recombinant retroviruses. The PT67 cells were grown in Dulbecco’s modified Eagle’s medium (GIBCO-BRL) supplemented with 10% heat-inactivated fetal bovine serum and transfected with the retroviral vector (LSN-HOP-human HO-1) using Lipofectamine reagent (Life Technologies, Inc., Grand Island, NY). HMECs were infected by supernatants of the retroviral packaging cells (PT67/LSN-HOP-human HO-1) and HMEC-HO-1 (expressing human HO-1) were obtained after clone selection with G418.

Measurement of HO Activity.

Microsomal HO activity was assayed as previously described (30). In brief, bilirubin, which is the product of HO degradation, was extracted with chloroform and its concentration was determined spectrophotometrically using the difference in absorbance from λ 460 to λ 530 nm with an absorption coefficient of 40 mM⁻¹ and cm⁻¹.

DNA Analysis by Comet Assay.

The presence of DNA fragmentation was examined by single-cell electrophoresis (Comet assay). Briefly, 0.5–0.8 × 10⁵ cells were mixed with 75 μl of 0.5% low-melting agarose and spotted on slides. The “minigels” were maintained in lysis solution (N-laurosil–sarcosine 1%, NaCl 2.5 M, Na₂EDTA 100 mM, DMSO 10%, pH 10) for 1 hr at 4°C, then denatured in a high pH buffer (NaOH 300 mM, Na₂EDTA 1 mM) for 20 min and finally electrophoresed in the same buffer at 25 V for 50–60 min. At the end of the run the minigels were neutralized in Tris-HCl 0.4 M, pH 7.5, stained with 100 μl of ethidium bromide (2 μg/ml) for 10 min, and scored using a Nikon fluorescence microscope (Nikon Labophot) interfaced with a computer. Software Scion Image with a Comet 1.3 version macro (free download from the National Institute of Health website) allowed us to analyze and quantify DNA damage by measuring: (a) tail length (TL), (b) tail DNA percentage (TDNA). These parameters are employed by the software to determine the level of DNA damage as: tail moment (TMOM) expressed as the product of TL and TDNA.

GSH Levels.

The contents of GSH were quantitatively measured by spectrophotometric method. The GSH contents were expressed in nmol of GSH per mg of proteins. Briefly, 300 μl of lysate was mixed with 10 μl of trichloroacetic acid (30% w/v) in an Eppendorf tube for 15 min in ice. The mixture is centrifuged (3000g for 10 min at 4°C), and 100 μl of supernatant was mixed with 0.880 ml of Tris (0.25 M)–EDTA (20 mM) (TE) buffer (pH 8.2) and 20 μl of 10 mM 2,2-dithiobisnitrobenzoic acid (DTNB). The color is developed for 15–20 min at room temperature. The absorbance of the supernatant was measured at 412 nm and subtracted from a DTNB-TE blank.

Statistical Analysis.

The data are presented as mean ± SE for the number of experiments. Statistical significance (P < 0.05) between the experimental groups was determined by the Fisher methods for multiple comparisons. For comparison between treatment groups, the null hypothesis was tested by single factor analysis of variance (ANOVA) for multiple groups or unpaired t test for two groups.

Results

Effects of Ang II on COMET Levels.

We compared the effect of Ang II (200 ng/ml for 16 hr) on COMET levels and the effect of HO inhibitors such as SnMP 10 μM for 16 hr. As seen in Figure 1A, Ang II increased tail moment (TMOM) levels from TMOM of 58.96 ± 6.3 to 168.96 ± 16.4 in exposed cells. Similarly, addition of H₂O₂ increased TMOM to higher levels than in the control. SnMP, Inhibitors of HO, exacerbated the effect of Ang II on COMET activity. In contrast, preincubation of endothelial cells with inducers of HO-1, SnCl₂ 10 μM attenuated Ang II-mediated increase in TMOM levels (Fig. 1B ).

Addition of sodium nitroprusside, SNP, an NO donor (SNP 500 μM for 16 h), or H₂O₂ as positive controls resulted in significant increased TMOM levels. TMOM levels in H₂O₂ pretreated cells were 230 ± 23.4 and in cells treated with SNP the TMOM were 235.99 ± 31.8 (P < 0.01). Pretreatment with HO-1 inducers, SnCl₂, led to a significant decrease of DNA damage, which was caused by the additional of SNP. The addition of SnCl₂ followed by treatment with H₂O₂ lowered the TMOM levels to 199.22 ± 20.8 compared to cells treated with H₂O₂ at 230 ± 23.4 (P > 0.05; Fig. 2A ). Inhibition of HO activity with SnMP elicited higher TMOM levels in cells treated with H₂O₂ and SNP alone (Fig. 2B ).

Effect of Human HO-1 Gene Transfer on COMET Activity.

Because the effect of inducers of HO-1, such as SnCl₂, may not be very specific, we used retroviral vector to over express human HO-1. The addition of Ang II (200 ng/ml) to cell overexpressing HO-1 (Fig. 3A ) elicits a lower TMOM levels compared with control cells (Fig. 1A ). The TMOM levels in cell overexpressing HO-1 was 91.41 ± 9.7 compared with control endothelial cells, 168.96 ± 16.4, described in Figure 1A (P < 0.01). Inhibition of HO activity by SnMP increased TMOM levels to 257.27 ± 21.8 compared to cells treated with Ang II alone.

Morphological Measurements of COMET in Cells Treated with Ang II.

We measured the morphological changes in cells treated with Ang II and assessed the effect of inducers on COMET changes. In control nontreated cells, no DNA damage was detected. In fact the DNA remained intact as shown in Figure 4A . After treatment with Ang II 200 ng/ml for 16 hr, which induced a DNA single-strand breaks, distinct COMETs were observed (Fig. 4B ). The extent of DNA damage was quantitated by image analysis to produce a TMOM, defined as the product of the percentage DNA in the comet tail and the distance between the means of the head and tail distributions, based on the definition (31). As seen in Figure 3A and B, the COMET appearance is clearly seen at a higher levels compared with control cells. Cells pretreated with SnCl₂ showed a similar profile to the nontreated cells (Fig. 4C ). Pretreatment with SnCl₂ attenuated COMET levels (Fig. 4C ). Inhibition of HO activity by SnMP reversed the effect of SnCl₂, suggesting the HO-1 expression was a key player in attenuating Ang II-mediated increases in COMET. Addition of bilirubin to cell treated with Ang II decreased COMET levels (data not shown).

Effect of Ang II on GSH Content.

Determination of reduced GSH content aims to evaluate imbalance in redox potential. We measured GSH content in control endothelial cells and cells overexpressing HO-1 or cell pretreated with inducer of HO-1, SnCl₂ in the presence or absence of Ang II 200 ng/ml for 16 hr (Fig. 5 ). Ang II caused a significant decrease of GSH levels than control (P < 0.05). Inhibition of HO-1 by SnMP resulted in a significant decreased in GSH content compared with control. Similarly, Overexpression of HO-1 resulted in increased of GSH in control cells. GSH levels were 1.68 and 1.52 in cells overexpressing GO-1 and cells treated with SnMP, respectively, (P < 0.05). Cells treated with biliverdin increased GSH levels compared to the control < 0.01).

Effect of Ang II on HO Activity in Endothelial Cells.

To investigate the significance of modulation of HO-1 in the oxidative stress–DNA damage, we examined the effect of the role of Ang II, SnCl₂, and SnMP on HO activity. The basal level of HO activity in endothelial cells treated with SnCl₂ or Ang II was increased respectively by 2.5- and 1.7-fold (P < 0.05). The increase in HO activity was inhibited by addition of SnMP (P < 0.05) (Fig. 6 ). Pretreatments with both Ang II and SnCl₂ resulted in additional increase HO activity compared with either Ang II or SnCl2 treatment alone.

Discussion

In the present study, we described the physiological effect of the functional expression of upregulation of HO-1 gene by inducers, such as SnCl₂, or by the human HO-1 gene transfer on DNA damage and GSH levels. To detect DNA damage at the individual cell level. We used the COMET assay, a rapid and sensitive fluorescence microscopic method. The damaged DNA migrates during electrophoresis from the nucleus towards the anode, forming a shape of a “comet” with a head (cell nucleus with intact DNA) and a tail (relaxed and broken DNA) (32). Cells overexpressing HO-1 by SnCl₂ or cells transduced with a retroviral-mediated human HO-1 gene displayed enhanced bilirubin and CO formation in cell cultures. Overexpression of the HO-1 gene in endothelial cells also provided protection against Ang II-mediated DNA degradation as measure by COMET assay distribution and cell cycle progression.

Ang II causes a rapid deterioration in the integrity of DNA and caused DNA damage as result of activation of NAD(P)H oxidase complex, leading to the generation of O₂ ^• and H₂O₂ (33). These changes in DNA degradation and decreased GSH levels after Ang II exposure were more dramatically increased in cells with low HO-1 activity, as seen in cells pretreated with SnMP, but on the contrary are diminished by overexpression of HO-1. Superoxide generated by Ang II can be dismutated spontaneously by superoxide dismutase to yield H₂O₂ or can be metabolized by catalase to form oxygen and water, or a fenton reaction may occur in the presence of iron, resulting in formation of hydroxyl radical. Thus, it is relevant that an increase in HO-1 activity and generation of bilirubin, antioxidant and hydroxyl radical scavenger, suggest a possible protective role of HO-1 against DNA degradation and decreased in GSH levels.

Although, the mechanism by which increased in HO-1 gene expression is still not clearly defined. In these pathways, HO activity and its product, bilirubin and CO, may play an important role. The reduction of CO formation by the inhibitor of HO activity, SnMP (34), reversed the protective effect of human HO-1 gene transfer against tumor necrosis factor-α (35, 36). Roles for ferritin and bilirubin synthesis, which are associated with upregulation of HO activity in the phenomenon observed, are not excluded. The Fe release resulting from HO activity is believed to be the cause of the increased expression of ferritin synthesis, which serves to sequester the iron, thus rendering this potential cellular oxidant inactive (37). Bilirubin and biliverdin both act as antioxidants in vitro and in vivo (38) and their increased local concentrations, after HO induction, may be beneficial in protecting endothelial cells from injury. The upregulation of HO-1 has been shown in our experiments with endothelial cells to be cytoprotective (28). The gene products of HO-1 protect the cells from tumor necrosis factor-mediated apoptosis and preserve DNA integrity and prevent abnormal cell cycle progression (35, 36). However, delivery of human HO-1 gene promotes somatic growth in whole rats spontaneous hypertensive rats (39) and in human normal microvessel endothelial cells promotes cell proliferation (28) and protects against oxidant and inflammatory cytokine stimulated apoptosis (40). It is also possible that amelioration of induced oxidative stress is, in part, results from the increased levels of bilirubin (41, 42).

More recently, we reported that elevation of CO in endothelial cells enhances cell proliferation (27) and (43) signifies the important role of this gas in cell growth via protection of DNA synthesis. Others have shown that when an inhibitor of HO blocks HO activity or the action of carbon monoxide is inhibited by hemoglobin, heme oxygenase activity no longer prevents endothelial cell apoptosis (44). Our study defines a novel function of human HO-1 in endothelial cell proliferation and protection against Ang II-mediated DNA degradation and supports the notion that induction of HO-1 and formation of bilirubin, ferritin, and CO are central features of this antioxidative mechanism. We have reported that H₂O₂ or heme elicits cell death and that this effect can be reversed by elevation of HO-derived bilirubin levels (45). In contrast, HO inhibitors enhance cell death, an effect that can be prevented by pre-elevation of endogenous bilirubin (45). We hypothesize that the antigrowth arrest effects of HO-1-, not HO-2-, derived bilirubin and CO attenuates DNA degradation. Figure 7 presents a schematic representation of the hypothesis that overexpression of HO attenuates the cytostatic effects of Ang II on generation of oxidants and superoxide associated with an increased in DNA degradation (COMET) and a decrease in GSH content. The capacity of vascular endothelial cells to generate bilirubin and CO via the heme-HO system is of potential clinical importance. Collectively, our findings suggest that the HO system and products generated through its catalytic activity, via formation of bilirubin and/or CO, attenuate Ang II-mediated oxidative injury and DNA degradation. Up regulation of HO-1 is a major protective mechanism in attenuating ANG II induced oxidant mediated cell injury.

Figure 1.

(A) Ang II-induced DNA damage. Effect of Ang II (200 ng/ml), SnMP (10 μM), H₂O₂ (300 μM), and pretreatment with SnMP (10 μM) in endothelial cells. The histograms demonstrated the DNA damage, as measured by COMET assay, and expressed as mean of TMOM. Treatment is representative of an average mean of TMOM ± SE of three individual slide determination experiment. On each slide, 50 COMETs were scored blindly for TMOM. Significantly different (*P < 0.05 vs control) from control values are determined by ANOVA multiple range test. (B) Endothelial cells were stimulated for 16 hr human by Ang II 200 ng/ml alone or in cells pretreated with SnCl₂. Each column is representative of an average mean of TMOM ± SE of three individual slides per treatment. On each slide, 50 COMETs were scored blindly for TMOM. Significantly different (*P < 0.05 vs control; **P < 0.05 vs control + Ang II) from control values are determined by ANOVA multiple range test.

Figure 2.

Protective effects of SnCl₂ on Ang II-DNA damage. Cells were stimulated for 16 hr human by Ang II 200 ng/ml alone or in cells pretreated with the HO-1 inducer, SnCl₂. Each column is representative of an average mean of TMOM ± SE of three individual slides per treatment. On each slide, 50 COMETs were scored blindly for TMOM. Significantly different (*P < 0.05 vs control; **P < 0.05 vs control + Ang II).

Figure 3.

Effect of SnMP on HO activity and Ang II-mediated DNA damage. An Inhibitor of HO activity, SnMP, was added alone or in combination with Ang II to control endothelial cells or cells transduced with human HO-1gene. Cells were stimulated for 16 hr with either SnMP or with Ang II 200 ng/ml alone or combinations. Each column is representative of an average mean of TMOM ± SE of three individual slides per treatment. On each slide, 50 Comets were scored blindly for TMOM. Significantly different (*P < 0.05 vs control; **P < 0.05 vs control + Ang II) from control as determined by ANOVA multiple range test.

Figure 4.

Morphological determination of COMET in cells treated with Ang II. Representative microphotographs of agarose low melting minigel of cells treated with vehicle solution “control” (A) Ang II 200 ng/ml for 16 hr alone (B) or cells pretreated HO-1 inducer, SnCl₂ (C). Damaged DNA migrates during electrophoresis from the nucleus towards the anode, forming a shape of a “comet” with a head (cell nucleus with intact DNA) and a tail (relaxed and broken DNA). Images were determined by a fluorescence microscope (rodamine filter) plugged with a digital camera after staining with ethidium bromure. Original magnification ×20.

Figure 5.

Determination of reduced GSH contents. GSH levels were measured in endothelial cells after treatment with Ang II, SnCl₂, or SnMP. GSH level measured spectrophotometrically, and expressed as mean of nmol of GSH/mg of protein. Each column is representative of an average mean of three experiments. Significantly different (*P < 0.05 vs control; **P < 0.05 vs control + Ang II) from control values are determined by ANOVA multiple range test.

Figure 6.

Effect of Ang II and HO modulators on HO activity. Cell lysate assayed by determining bilirubin levels. Result is expressed as the means ± SD of three experiments, respectively. Significantly different (*P < 0.05 vs control; **P < 0.05 vs control + Ang II) from control values are determined by ANOVA multiple range test.

Figure 7.

Schematic representation of the hypothesis that overexpression of HO-1 attenuates the cytostatic effects of Ang II. We hypothesize that the anti-DNA degradation effects of HO- activity are mediated by the generation of bilirubin, antixoidant and CO, a cell cycle regulator via a in cyclin kinase inhibition of p21/p27 (35, 36).

Footnotes

This report was supported by the National Institutes of Health Grants HL55601, HL-31069, and HL34300.

1

To whom requests for reprints should be addressed at New York Medical College, Valhalla, NY 10595. E-mail:

Acknowledgements

We thank Ms. Jennifer Brown for her excellent secretarial assistance.

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