Retrovirus-mediated HO gene transfer into endothelial cells protects against oxidant-induced injury

Liming Yang1, Shuo Quan1, and Nader G. Abraham1,2

2 Laboratory of Pharmacology, The Rockefeller University, New York 10021; and 1 Department of Pharmacology, New York Medical College, Valhalla, New York 10595


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Heme oxygenase (HO)-1 is a stress protein that has been implicated in defense mechanisms against agents that may induce oxidative injury, such as endotoxins, heme, and cytokines. Overexpression of HO-1 in cells might, therefore, protect against oxidative stress produced by certain agents, specifically heme, by catalyzing its degradation to bilirubin, which by itself has antioxidant properties. We report for the first time the successful transduction of human HO-1 gene into rat lung microvessel endothelium using replication-defective retroviral vector. Cells transduced with human HO-1 gene exhibited a 2.1-fold increase in HO-1 protein level, which was associated with a 2.3-fold elevation in enzyme activity compared with that in nontransduced cells. The cGMP content in transduced endothelial cells was increased by 2.9-fold relative to that in nontransduced cells. Moreover, human HO-1 gene-transduced endothelial cells acquired substantial resistance to toxicity produced by exposure to heme and H2O2 compared with that in nontransduced cells. The protective effect of enhancement of HO-1 activity against heme and H2O2 was reversed by pretreatment with stannic mesoporphyrin, a competitive inhibitor of HO. These data demonstrate that the induction of HO-1 in response to injurious stimuli represents an important mechanism for moderating the severity of cell damage. Regulation of HO activity in this manner may have clinical applications.

retroviral vector; stress protein; guanosine 3',5'-cyclic monophosphate


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE CELLULAR RESPONSE to a variety of oxidants such as ultraviolet (UV) irradiation, heavy metals, heme, and H2O2 involves the production of a number of cellular mediators, including acute-phase proteins, eicosanoids, and various cytokines (2, 4, 17). Accumulating evidence suggests that heme oxygenase (HO), the initial rate-limiting enzyme in heme catabolism, plays a vital role in diverse biological processes including cell respiration, energy generation, oxidative biotransformation, and cell growth and differentiation (7, 36). It is well known that HO-1 can be induced by various oxidant stresses, especially its substrate heme. Enhanced HO-1 activity has been associated with cellular protection against UVA radiation (7), hemoglobin (3), hypoxia (18), and hyperoxia (10). Conversely, deficient HO expression in mammalian cells contributed to a reduced stress defense (25, 26). More recently, Hancock et al. (14) have shown that HO-1 induction prevents oxidant-stressed endothelial upregulation of adhesion molecules and development of transplant arteriosclerosis in normal mice (14). We have previously demonstrated that overexpression of HO-1 in coronary endothelial cells resulted in resistance against hemoglobin-induced injury (3), suggesting that moderate increases in HO activity may be beneficial in resistance to oxidants.

HO catalyzes the degradation of heme to biliverdin, which is subsequently reduced to bilirubin by biliverdin reductase (32, 33). In this process, iron and carbon monoxide (CO) are released. To date, three HO isoforms (HO-1, HO-2, and HO-3) that catalyze this reaction have been identified (19, 20, 31). HO-1 is a 32-kDa heat shock protein (30) that is inducible by numerous noxious stimuli (11, 12). HO-2 is a constitutively synthesized 36-kDa protein that is abundant in brain and testis (20, 28, 35). HO-3 is related to HO-2 but is the product of a different gene, and its ability to catalyze heme degradation is lower than that of HO-1 (21). Increasing data reveal that HO and its metabolized product, CO, play an important role in numerous biological processes, especially in cellular antioxidative reactions (15, 24, 36-38), whereas the evidence that the suppression of HO-1 can lead to cell protection is also accumulating (16, 40). The mechanism for this paradox is not clear, but the evidence showed that HO can mediate both cell protection and cell injury depending on experimental conditions (9, 10).

Our goal in this study was to augment HO activity by transducing rat lung microvessel (RLMV) endothelial cells via retrovirus-mediated human (h) HO-1 gene transfer to distinguish expression of the transduced HO-1 from that of rat HO-1 and to determine whether the transduced hHO-1 gene could function to protect the endothelial cells against the effects of free H2O2 and heme. We have used retrovirus and adenovirus to transduce target genes into a variety of cell lines (1, 6, and S. Quan, E. Feldman, L. Yang, F. A. D. T. G. Wagener, T. J. Farley, T. Ahmed, and N. G. Abraham, unpublished observations); other investigators also utilized similar vectors with success in vitro and in vivo (23, 37). Our data show that it is possible to transduce rat endothelial cells with hHO-1 gene and to achieve selective hHO-1 expression to levels significantly higher than those of endogenous rat HO-1 expression. The overexpression of hHO-1 in rat endothelial cells was associated with an elevation in cGMP. Our data also reveal that augmentation of hHO-1 activity in transduced cells conferred substantial protection against cellular toxicity produced by H2O2 and heme. These data directly demonstrate the protective effect of hHO-1 gene against the cytotoxic effect of heme and H2O2, which are known to participate in inflammatory reactions at sites of thrombosis, trauma, and hypoxia-ischemia.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture condition. The amphotropic retroviral packaging cell line PA317 (ATCC) was used for the generation of helper-free recombinant retroviruses. PA317 and RLMV endothelial cells were grown in DMEM (GIBCO BRL, Grand Island, NY) supplemented with 10% heat-inactivated FBS (GIBCO BRL), 100 U/ml penicillin, and 100 µg/ml streptomycin (P/S; GIBCO BRL). NIH/3T3 fibroblasts (ATCC) were cultured in DMEM with 10% calf serum (GIBCO BRL) and P/S. All cells were incubated at 37°C in a 5% CO2 humidified atmosphere and were maintained at subconfluency by passaging with trypsin-EDTA (GIBCO BRL).

Reagents. Stannic mesoporphyrin (SnMP) was obtained from Porphyrin Products (Logan, UT). All other reagents were purchased from Sigma Chemical (St. Louis, MO) unless otherwise specified.

Construction of the retroviral vector LSN-hHO. The hHO1-expressing replication-deficient retrovirus vector LSN-hHO was constructed as follows: 987-bp Hind III-Hind III hHO-1 cDNA fragment was released from plasmid pRC-CMV-hHO (2) and was inserted at the Hind III site of pGEM-7zf(+) vector (Promega). After digestion with Apa I, the transcription-orientational clones were selected and named as pGEM7-hHO. LSN-hHO was constructed by cloning the hHO cDNA fragment from pGEM7-hHO at the EcoR I-BamH I sites of retroviral vector LXSN (Fig. 1).


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Fig. 1.   Schematic diagram of retroviral vectors LSN-hHO and LXSN. hHO, human (h) heme oxygenase (HO)-1 cDNA; neor, neomycin-resistant gene; SV40P, Simian virus 40 promoter; LTR, long terminal repeat.

Transduction and virus production. PA317 retroviral packaging cells (3 × 105/ml) were seeded in 60-mm tissue culture dishes and were incubated for 24 h. The attached cells were washed two times with serum-reduced Opti-MEM (GIBCO BRL). The cells transfected with 5 µg of retroviral vectors and 20 µl of lipofectamine (GIBCO BRL) were incubated for 5 h at 37°C, and then 3 ml of DMEM containing 20% FBS were added to the culture dishes and incubated for 18 h. The culture medium was replaced with fresh complete medium and was incubated for another 24 h. The cells were selected for neomycin resistance (neor) in medium containing G418 (600 µg/ml; GIBCO BRL). After 14 days of culture at 37°C in 5% CO2, individual G418-resistant clones were selected.

For each isolated clone, the viral titer was determined by infection of NIH/3T3 fibroblasts (19). A clone of packaging cell line PA317/LSN-hHO (PA317/hHO) producing the highest viral titer of 1.4 × 106 colony-forming units (CFU)/ml was employed in the experiments described below. Simultaneously, we received NIH/3T3/LXSN and NIH/3T3/LSN-hHO (NIH/3T3/hHO) cell lines. Using supernatants of the PA317/LXSN and PA317/LSN-hHO cells, we infected RLMV cells. After selection with G418, transduced endothelial cells RLMV/LXSN and RLMV/LSN-hHO (RLMV/hHO) were obtained.

Northern analysis. Total RNA was isolated either by the guanidinium thiocyanate-phenol extraction method or with the use of TRIzol reagent (GIBCO BRL) following the instructions provided by the manufacturer. Total RNA (20 µg/lane) was denatured, electrophoresed on 1.2% agarose formaldehyde gels, transferred to a positively charged nylon membrane (Hybond N+; Amersham), and UV cross-linked (Stratalinker; Stratagene). Membranes were prehybridized for 1-2 h at 60°C and were subsequently hybridized overnight at 60°C with random-primer [32P]dCTP-labeled hHO cDNA or glyceraldehyde-3-phosphate dehydrogenase cDNA (Clontech). The blots were washed three times with a solution containing 0.5% BSA, 5% SDS, and 1 mM EDTA in 0.2× saline-sodium citrate at 56-60°C and then were exposed to X-ray film at -80°C.

RT-PCR detection. RT was carried out using either the First-Strand cDNA Synthesis Kit (Pharmacia Biotech) or the Advantage RT-for-PCR Kit (Clontech). Oligo(dT) were used as reverse transcription primers. Specific primers for hHO cDNA fragment were as follows: primer 1, 5'-CAGGCAGAGAATGCTGAGTTC-3'; primer 2, 5'-GATGTTGAGCAGGAACGCAGT-3'. PCR was performed using the AmpliTaq PCR kit (Perkin-Elmer). For each RT-PCR, a sample without reverse transcriptase was processed in parallel and served as a negative control.

Cycling parameters for amplifying RT products were as follows: 95°C for 1 min; 60°C for 1 min; 72°C for 2 min, for 30 cycles, and then extension at 72°C for another 5 min. In some PCR assays, [32P]dCTP was added to the PCR reaction tube. After amplification, PCR products were electrophoresed on 1.2% agarose gel, stained with ethidium bromide, and visualized under UV light. Alternatively, [32P]dCTP-incorporated PCR products were separated on 8% nondenaturing polyacrylamide gel, dried on a gel dryer (Bio-Rad), and exposed to X-ray film.

Cell viability. RLMV cells were seeded in 12-well culture plates (6 × 104 cells/well) and were grown for 24 h. The next day, subconfluent cells were treated with different reagents (heme, H2O2, or SnMP) for another 24 h. Next, the cells were trypsinized with 0.2 ml of 0.1% trypsin-EDTA and neutralized with 0.8 ml of culture medium. Cell suspensions (50 µl) were mixed with an equal volume of 0.5% trypan blue (GIBCO BRL) and were allowed to stand for 4 min at room temperature. Cells were counted by using a hemocytometer. Cell viability was calculated as the percentage of viable (unstained) cells of total (stained and unstained) cells.

Western blot. For detection of HO-1-immunoreactive protein, 20-µg aliquots of cell lysates were electrophoresed on a 12% polyacrylamide gel. Proteins were transferred to enhanced chemiluminescence (ECL) membrane for 1 h with a Bio-Rad transblot apparatus. Equal loading of protein samples was assessed by Coomassie brilliant blue in gels and by Ponceau (Sigma) solution in transblotted membrane. Blots were blocked overnight at 4°C in 5% nonfat milk, washed briefly with Tris-buffered saline containing 0.1% Tween 20 (TBS-T), incubated with a 1:2,000 dilution in 3% nonfat milk TBS-T of mouse anti-HO-1 monoclonal antibody (Stressgen) for 1 h, washed with 20 ml of TBS-T three times, and incubated with a 1:4,000 dilution of peroxidase-labeled rabbit anti-mouse antibody for 1 h. The membrane was rinsed three times, detected with ECL reagent, and exposed to an X-ray film for 0.5-15 min.

Measurement of HO enzyme activity. Enzyme activity was assessed using cell sonicates of NIH/3T3 fibroblasts or RLMV cells nontransfected or transfected with retroviral vector LSN-hHO or LXSN. After cells were harvested, the cell sonicates were incubated with the following reagents: heme (50 µM), rat liver cytosol (1 mg/ml), MgCl2 (1 mM), glucose-6-phosphate dehydrogenase (3 units), glucose 6-phosphate (1 mM), and NADP+ (2 mM) in 0.5 ml of 0.1 M potassium phosphate buffer (pH 7.4) at 37°C for 30 min. The reaction was stopped by placing the tubes on ice. The amount of bilirubin generated was estimated with a scanning spectrophotometer and was defined as the difference between 463 and 520 nm. The HO activity is expressed as nanomoles of bilirubin per milligram of protein per hour.

Measurements of cGMP concentrations. Cellular cGMP content was determined with a commercial ELISA kit (BIOMOL) following the instructions provided by the manufacturer. Briefly, the cells were washed with ice-cold PBS one time and then incubated with 0.1 M HCl for 10 min. The cells were collected using a scraper and were centrifuged at 8,000 rpm for 5 min at 4°C. Supernatants were stored at -80°C until used. cGMP concentration was normalized to protein content as determined by a dye-binding assay (Bio-Rad).

Statistical analysis. Data are presented as means ± SD for the number of experiments. Statistical significance (P < 0.05) between the experimental groups was determined by means of ANOVA and Student's t-test.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

hHO-1 gene expression in transduced PA317, NIH/3T3, and RLMV endothelial cells. PA317 retroviral packaging cells were transfected with retroviral vector LSN-hHO and control retroviral vector LXSN. G418-resistant clones (PA317/hHO and PA317/LXSN) were isolated and tested for their potential to produce retrovirus in culture medium. The results showed that the highest retroviral titers could reach 1.4 × 106 and 1.2 × 106 CFU/ml in PA317/hHO and PA317/LXSN cell clones, respectively. The neor gene was expressed in both PA317/hHO and PA317/LXSN cells, whereas only hHO-1 gene expression was detected in PA317/hHO cells as confirmed by RT-PCR (Fig. 2A, lane 6). We used the supernatants of PA317/hHO-1 and PA317/LXSN cells to infect the NIH/3T3 cells and RLMV endothelial cells. After selection with G418, G418-resistant cell clones were tested for their ability to express hHO-1 mRNA. Total RNA isolated from NIH/3T3 cells and from endothelial cells was assessed for the presence of hHO-1 mRNA by RT-PCR and Northern blot (data not shown). As shown in Fig. 2A, the presence of hHO-1 mRNA in transduced NIH/3T3 cells is indicated in lanes 2 and 4. Similarly, Fig. 2B showed that retroviral construct LSN-hHO was able to transduce RLMV endothelial cells and express hHO-1 mRNA. In contrast, endothelial cells nontransduced or transduced with control retrovirus LXSN did not show a detectable band for hHO-1 (Fig. 2B, lanes 1 and 2). To determine the expression of viral vector sequence (neor) in retrovirus-infected cells, we performed RT-PCR analysis using neor sequence-specific primers. The results demonstrated the expression of neor mRNA in all infected NIH/3T3 (Fig. 2A, lanes 2-4) and endothelial cells (Fig. 2B, lanes 1 and 3). The above analyses showed that hHO-1 mRNA was efficiently expressed in NIH/3T3 and endothelial cells after infection with the LSN-hHO retroviral supernatant. Similar results were noted in several human cell lines such as human umbilical vein endothelial cells and human microvessel endothelial-1 cells (data not shown).


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Fig. 2.   A: detection of hHO-1, neor, and beta -actin transcripts by RTPCR in RNA extracted from PA317 and NIH/3T3 cells nontransduced or transduced with retroviral vectors LSN-hHO or LXSN. RT-PCR products were amplified in the RNA extracted from NIH/3T3 fibroblasts (lane 1) and NIH/3T3/hHO (lanes 2 and 4), NIH/3T3/LXSN (lane 3), PA317/LXSN (lane 5), PA317/hHO (lane 6), and PA317 (lane 7) cells. [alpha -32P]dCTP-incorporated RT-PCR was performed to amplify hHO, neor, and beta -actin transcripts. After electrophoresis in 8% nondenaturing polyacrylamide gel, the dried gel was exposed to X-ray film. B: detection of hHO-1 transcripts by RT-PCR analysis in RNA isolated from rat lung microvessel (RLMV) endothelial cells nontransduced or transduced with retroviral vectors (LSNhHO or LXSN). Lane 1, RLMV/LXSN; lane 2, RLMV; and lane 3, RLMV/hHO.

Enhancement of HO activity in endothelial cells transduced with hHO-1 gene. We next examined the expression of total hHO-1 protein and HO activity in endothelial cells nontransduced or transduced with retroviral vector LSN-hHO or control vector LXSN, respectively (Fig. 3). The basal levels of HO activity in nontransduced or LXSN-transduced endothelial cells were not significantly different (P > 0.05). HO activity in hHO-1 gene-transduced endothelial cells was increased by 2.3-fold compared with that in nontransduced cells (P < 0.05; Fig. 3B). To ascertain the characteristics of the expressed protein, we tested the effect of SnMP, a potent inhibitor of HO activity (5), on cultured endothelial cells. Addition of 10 µM SnMP to cell cultures inhibited the enzyme activity (data not shown). To assess that the increase in hHO-1 mRNA and HO activity was associated with the elevation in HO-1 protein, Western blot analysis was performed on cell lysates obtained from nontransduced and transduced cells. Using HO antibodies that recognize both human and rat HO-1 protein, we found that hHO-1 gene-transduced endothelial cells exhibited a strong signal for HO-1 protein. The quantitative evaluation of HO-1 protein was measured by scanning densitometry. The results showed that hHO-1 protein was increased 2.1-fold (Fig. 3A). Comparison of the levels of HO-1 protein with HO activity showed that HO activity was increased at a comparable level to HO-1 protein. These findings indicated that LSN-hHO-transduced endothelial cells generate increased levels of HO-1 protein and activity compared with those in nontransduced cells.


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Fig. 3.   A: relatively immunoreactive HO-1 protein in RLMV endothelial cells nontransduced or transduced with LSN-hHO or LXSN by Western blot-enhanced chemiluminescence analysis. Values are expressed as means ± SD of 3 experiments. B: HO activity in RLMV endothelial cells nontransduced or transduced with retroviral vectors LSN-hHO or LXSN. HO activity is expressed as means ± SD of 3 experiments. * P < 0.05 vs. control RLMV cells.

Enhanced cell survival to heme- and H2O2-mediated cell injury in hHO gene-transduced endothelial cells. Because the oxidative agents heme and H2O2 are able to damage vascular endothelial cells (2) and fibroblasts (10), we examined the effect of heme and H2O2 on cell viability in endothelial cells nontransduced or transduced with the hHO-1 gene. The impact of H2O2 (200-400 µM) and heme (100-200 µM) exposure on endothelial cell survival was assayed using trypan blue exclusion. Endothelial cells nontransduced or transduced with hHO-1 genes were exposed to sublethal or lethal doses of heme and H2O2 and were evaluated for cell survival after 24 h. Nontransduced or LXSN-transduced endothelial cells without exposure to heme (Fig. 4A) and H2O2 (Fig. 4B) were >98% viable, whereas treatment of both cells with heme or H2O2 decreased cell survival in a concentration-dependent fashion. A significant decrease in cell survival was observed after exposure to 200 µM H2O2 or 100 µM heme (P < 0.05) relative to nontreated cells. However, the endothelial cells transduced with hHO-1 gene were remarkably protected and resistant to both heme and H2O2. As shown in Fig. 4, cell survival of transduced endothelial cells with hHO-1 gene was more pronounced at high concentrations of heme (>100 µM) and H2O2 (>200 µM). In contrast, inclusion of the HO inhibitor SnMP decreased cell survival in all endothelial cells treated with heme (Fig. 4C) or with H2O2 (Fig. 4D). These results demonstrate that the overexpression of HO-1 in endothelial cells is associated with a substantial attenuation of cell toxicity elicited by exposure to heme and H2O2. Inhibition of HO activity resulted in reversal of cell resistance to oxidant-induced cell death.


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Fig. 4.   Survival of RLMV endothelial cells nontransduced or transduced with retroviral vectors (LSN-hHO or LXSN) after exposure to several reagents. Cell survival was assayed by trypan blue exclusion. Data are shown as means ± SD of 3-5 independent experiments. A: treatment with heme for 24 h; B: treatment with H2O2 for 24 h; C: pretreatment with stannic mesoporphyrin (SnMP; 10 µM) for 4 h followed by exposure to heme for 24 h; D: pretreatment with SnMP (10 µM) for 4 h followed by exposure to H2O2 for 24 h. * P < 0.05 vs. control RLMV cells.

Evaluation of cellular cGMP. To determine whether overexpression of the HO-1 gene affects the endogenous cellular cGMP level, we measured cGMP content in hHO-1 gene-transduced and nontransduced endothelial cells. Basal cGMP levels in endothelial cells transduced with hHO-1 gene (43.6 ± 11.4 pmol/mg protein) were significantly increased 2.9-fold compared with cGMP content (14.9 ± 8.8 pmol/mg protein) in nontransduced cells (P < 0.05). However, there was no significant difference in cGMP content between nontransduced and LXSN-transduced RLMV endothelial cells (16.2 ± 8.4 pmol/mg protein). To demonstrate that the elevated cGMP level is mediated by the overexpression of HO, we treated endothelial cells with SnMP. The results showed that the addition of SnMP could reduce the cellular cGMP level (Fig. 5), indicating that HO overexpression contributed to the elevated cGMP content.


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Fig. 5.   Overexpression of hHO-1 gene increases cGMP level in RLMV endothelial cells. cGMP concentrations are means ± SD of 4-6 experiments performed in duplicate. +SnMP, cells treated with SnMP (10 µM) for 24 h before cGMP was assayed. * P < 0.05 vs. control RLMV cells.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Because the adenoviral vector has the advantage of high infection efficiency and high expression (8), it has been used in numerous gene transfer and gene therapy projects (13, 29). The viral titer of adenovirus can reach 1010 to 1012 plaque-forming units/ml, which is much higher than that of retrovirus (105 to 107 CFU/ml). This feature renders adenoviral vector a good vehicle for in vivo gene transfer experiments. However, because the transgene delivered by adenovirus is located at an extrachromosomal site, its expression will rapidly decrease during cell replication (29). Therefore, adenoviral vectors are only appropriate for transient gene transfection and expression strategies. Retrovirus, on the other hand, can be used to stably express target genes in host cells due to its characteristic of incorporation of the gene into the host cell chromosomes. Although retroviral infection efficiency is generally not sufficient to meet the need of in vivo gene transfer, specific gene transfer methods, for example, catheter-directed gene transfer or ex vivo gene transfer, might be used to overcome retroviral shortcomings. Indeed, this laboratory has successfully developed several cell lines that specifically express certain target genes in host cells with a long-term expression (1, 3).

In the present study, we describe for the first time the construction of the retroviral vector LSN-hHO for gene transfer of HO-1, a key enzyme in the degradation of heme/hemoglobin and a potential candidate for vascular endothelial cell gene therapy. Our results showed that LSN-hHO retrovirus could efficiently infect rat microvessel endothelial cells and lead to the expression of functional HO-1 gene at high levels. Our studies on protein expression and enzyme activity document the effectiveness of retroviral vector LSN-hHO for transferring HO-1 gene into cultured endothelial cells. In our developed RLMV/hHO endothelial cell line, HO activity was increased 2.3-fold compared with that in transduced cells. The data from Western blot confirmed that immunoreactive HO-1 protein increased 2.1-fold in RLMV/hHO endothelial cells relative to nontransduced cells. Furthermore, the specific hHO-1 mRNA transcription in the above hHO-1 gene-transduced cells was confirmed by RT-PCR (Fig. 2B).

In mammalian cells, a variety of stress inducers, such as heme and heavy metals, oxidized lipoproteins, hyperthermia, UV and visible light, inflammatory cytokines, and hypoxia, can cause robust upregulation of HO-1 activity. The functional significance of HO-1 induction by oxidative stress is not well understood. However, numerous reports are supportive of the hypothesis that HO-1 induction plays an important role in cellular protection against both heme- and non-heme-mediated oxidant injury. Our previous study showed that, in rabbit coronary microvessel endothelial cells transduced with hHO-1 cDNA, an increase of HO-1 expression and HO activity was associated with cellular resistance to cytotoxic agents (3). In addition, Vile and colleagues (34) demonstrated that preirradiation of cultured skin fibroblasts with a sublethal dose of UVA, a treatment that induces HO-1 expression and HO enzymatic activity, provided cellular protection against a subsequent lethal dose. Suppression of HO activity by SnMP or inhibition of HO-1 expression by antisense oligonucleotides abolished the protective effect of preirradiation, further implicating HO-1 in the mechanism of cellular protection (34). In this study, we report that the cells transduced with hHO-1 gene acquired substantial tolerance to subsequent heme or H2O2 stimulation. The results from cell viability showed that hHO-1 gene-transduced RLMV endothelial cells could resist higher doses of heme and H2O2 compared with transduced cells. These data corroborate previous evidence that overexpression of HO-1 plays a crucial role in the cellular protective system.

The precise mechanisms by which HO-1 confers protection against cellular stress are still unclear. Several mechanisms whereby HO-1 activity could provide this protection have been proposed, including 1) depletion of the oxidant heme, 2) production of the antioxidants biliverdin and bilirubin, 3) elevation of intracellular free iron levels to facilitate ferritin upregulation (33), and 4) regulation of vascular tension through CO generation (21, 38). Poss and Tonegawa (26) analyzed the responses of cells from mice lacking functional HO-1 gene expression to oxidative challenges. Their results showed that cultured HO-1-deficient embryonic fibroblasts could produce higher oxygen free radicals when exposed to heme, H2O2, paraquat, or cadmium chloride and that they were hypersensitive to cytotoxicity caused by heme and H2O2. Our results further demonstrated that the overexpression of HO plays a crucial role in cellular protective systems against subsequent oxidative stress.

The results of this study also showed that retrovirus-mediated hHO-1 gene transfer into endothelial cells was associated with modulation of cGMP levels. The basal levels of cGMP were substantially increased in HO-1 gene-transduced endothelial cells compared with nontransduced cells, and the addition of SnMP could decrease cellular cGMP content. Moreover, we also observed a severalfold elevation in the cGMP level of endothelial cells nontransduced or transduced with the HO-1 gene after exposure to 10 µM heme for 24 h, and this elevated cGMP content could be partially reversed by the addition of SnMP. These results suggest that overexpression of HO-1, at least in part, contributes to the elevated cellular cGMP level. Further investigations are needed to clarify the exact mechanisms by which HO-1 overexpression modulates cGMP levels in HO-1 gene-transduced endothelial cells.

In summary, we have developed a retroviral packaging cell line, PA317/hHO, that can secrete high titers of the retrovirus LSN-hHO into culture medium. We demonstrated that infection with the LSN-hHO retrovirus resulted in the elevation of HO-1 protein and activity in RLMV endothelial cells and that the expressed HO was functional in response to heme- and H2O2-induced cytotoxicity. Retrovirus-mediated gene transfer techniques provide a novel tool for inducing high levels of HO gene expression in cultured vascular endothelial cells. The biological significance of elevated cellular cGMP levels after HO-1 gene transduction remains to be elucidated further.


    ACKNOWLEDGEMENTS

We thank Melody Steinberg for editing and preparing this manuscript.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant RO1 HL-S4318.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and correspondence: N. G. Abraham, Dept. of Pharmacology, New York Medical College, Valhalla, NY 10595 (E-mail: nader_abraham{at}nymc.edu).

Received 24 December 1998; accepted in final form 5 March 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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