Modulation of cGMP by human HO-1 retrovirus gene transfer in pulmonary microvessel endothelial cells

Nader G. Abraham1, Shuo Quan1, Paul A. Mieyal1, Liming Yang1, Theresa Burke-Wolin1, Christopher J. Mingone2, Alvin I. Goodman3, Alberto Nasjletti1, and Michael S. Wolin2

Departments of 1 Pharmacology, 2 Physiology, and 3 Medicine, New York Medical College, Valhalla, New York 10595


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Carbon monoxide (CO) stimulates guanylate cyclase (GC) and increases guanosine 3',5'-cyclic monophosphate (cGMP) levels. We transfected rat-lung pulmonary endothelial cells with a retrovirus-mediated human heme oxygenase (hHO)-1 gene. Pulmonary cells that expressed hHO-1 exhibited a fourfold increase in HO activity associated with decreases in the steady-state levels of heme and cGMP without changes in soluble GC (sGC) and endothelial nitric oxide synthase (NOS) proteins or basal nitrite production. Heme elicited significant increases in CO production and intracellular cGMP levels in both pulmonary endothelial and pulmonary hHO-1-expressing cells. Nomega -nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, significantly decreased cGMP levels in heme-treated pulmonary endothelial cells but not heme-treated hHO-1-expressing cells. In the presence of exogenous heme, CO and cGMP levels in hHO-1-expressing cells exceeded the corresponding levels in pulmonary endothelial cells. Acute exposure of endothelial cells to SnCl2, which is an inducer of HO-1, increased cGMP levels, whereas chronic exposure decreased heme and cGMP levels. These results indicate that prolonged overexpression of HO-1 ultimately decreases sGC activity by limiting the availability of cellular heme. Heme activates sGC and enhances cGMP levels via a mechanism that is largely insensitive to NOS inhibition.

guanosine 3',5'-cyclic monophosphate; retroviral vector; soluble guanylate cyclase; heme oxygenase


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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CELLULAR LEVELS OF HEME are regulated by the rates of heme synthesis and degradation. Heme catabolism occurs by oxidative cleavage of the alpha -methene bridge of the tetrapyrrole, which eventually leads to the formation of equimolar amounts of biliverdin and carbon monoxide (CO) and the release of the contained iron atom. Biliverdin is then rapidly reduced to form bilirubin (3). The heme oxygenase (HO) system controls the rate-limiting step in heme catabolism (3). To date, three HO isoforms have been identified: HO-1, HO-2, and HO-3. HO-1 is a 32-kDa heat shock protein (29, 34) that is inducible by numerous noxious stimuli (8, 21, 23, 30); HO-2 is a constitutively synthesized 36-kDa protein that is abundant in the brain and testis (26, 35); and HO-3 exhibits 90% homology to HO-2; however, it lacks significant catalytic activity (27).

Soluble guanylate cyclase (sGC) is a heme-dependent enzyme that catalyzes the formation of guanosine 3',5'-cyclic monophosphate (cGMP) after the binding of nitric oxide (NO) to the iron of the heme group (11, 14, 15). Heme binds to sGC with a stoichiometry of approximately one molecule per sGC apoenzyme (14, 15). The heme prosthetic group of sGC is essential for the expression of catalytic activity and the synthesis of cGMP. The purified heme-containing form of sGC has also been reported to be activated by CO (36).

CO exhibits physiological properties similar to NO, and it is believed that these actions are mediated in part by the ability of CO to act as an activator of sGC (24). Like NO, CO binds to the heme moiety of sGC and results in its activation as well as an increase in cGMP levels (18). This mechanism also appears to account for the ability of CO to inhibit platelet aggregation (6). After induction of HO-1 in rat aorta, the tissue cGMP content is greatly enhanced (33). Similar findings have been reported for other tissues (8, 9). The ability of CO to activate sGC and increase cGMP is believed to be part of the mechanism that underlies CO vasodilatory activity (33, 39). Upregulation of heme-HO-derived CO has been shown to elicit vasodilatation in the ductus arteriosus vessels (10) and the gracilis muscle arterioles (19) and to attenuate vasoconstrictor responses in renal arterial vessels (17). The cardiovascular system has a high capacity for producing CO, because HO-2 is constitutively expressed in endothelial and smooth muscle cells (25, 41) and HO-1 can be greatly upregulated (12). Thus the substrate of HO, heme, is readily available for catalysis in both vascular and myocardial tissues (22). Accordingly, variations in the levels of heme and CO affect the amount of catalytically active sGC that is present in cells.

Owing to the relative ease of incorporation of the transgene into the host-cell chromosomes and stable transfection, retroviral vectors are suitable for transferring and expressing the HO-1 gene in the host. The objective of this study was to examine the feasibility of utilizing the retrovirus-mediated transfer of a human HO-1 (hHO-1) gene to modulate heme-HO-1 expression and function and thus permit the study of the effects of heme oxidation and CO synthesis on cGMP levels over the long term. Our data demonstrate that selective delivery of the hHO-1 gene into rat endothelial cells results in accelerated catabolism of cellular heme and that this effect ultimately modulates cGMP levels. This report also demonstrates that supplementation of endothelial cells that express the hHO-1 gene with heme resulted in a rapid increase in cGMP levels. Thus the heme-HO system may greatly contribute to CO-mediated upregulation of cGMP levels as long as cellular heme is not a limiting factor.


    MATERIALS AND METHODS
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Cell culture conditions. The amphotropic retroviral packaging cell PA317 (American Type Culture Collection, Manassas, VA) was used for the generation of replication-deficient recombinant retroviruses. PA317 cells were grown in DMEM (GIBCO-BRL, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS). NIH/3T3 fibroblasts were cultured in DMEM with 10% calf serum. Rat pulmonary endothelial cells were cultured in MCDB 131 medium with 10% FBS, 10 ng/ml EGF, 1 µl/ml hydrocortisone, 0.1 mg/ml Endogro growth medium (VEC Technologies, Rensselaer, NY), and 90 µg/ml heparin (Sigma). All cells were incubated at 37°C in a 5% CO2 humidified atmosphere and maintained at subconfluency by passaging with trypsin-EDTA (GIBCO-BRL).

Production of retroviral constructs. PA317 retroviral packaging cells were transfected with the retroviral vectors LSN-hHO-1 or LXSN using Lipofectamine reagent (Life Technologies, Grand Island, NY). Individual G418-resistant clones were selected as described previously (40). For each isolated clone, the viral titer was determined by infection of NIH/3T3 fibroblasts. The clones of the packaging cell lines (PA317/LSN-hHO-1 and PA317/LXSN) with viral titers of (0.14-1.5) × 107 colony-forming units (cfu)/ml were employed in the experiments. Pulmonary endothelial cells were infected using the supernatants of the PA317 retroviral packaging cells. Pulmonary endothelial cells that expressed hHO-1 or an empty vector were obtained after selection with G418. This protocol yielded a pulmonary hHO-1 cell line that expresses the hHO-1 mRNA and exhibits elevated HO activity from passage to passage. The current study was performed on cells from passages 11-15.

Measurement of HO activity and CO levels. Microsomal HO activity was assayed by the method of Abraham et al. (5) in which bilirubin (the product of HO degradation) was extracted with chloroform and its concentration was determined spectrophotometrically using the differences in absorbances at wavelengths (lambda ) from 460 to 530 nm with an absorption coefficient of 40 mM-1cm-1.

To assess CO production by endothelial cells, cell cultures were incubated for 3 h in 2-ml vials that contained 1 ml of culture media. The concentration of CO in the headspace gas was then measured. CO analyses were performed using an HP-5989A mass spectrometer interfaced to an HP-5890 gas chromatograph. The separation of CO from other gases was carried out on a GS-Molesieve capillary column (30 m, 0.53 mm ID; J and W Scientific, Folsom, CA) kept at 40°C. Helium, which has a linear velocity of 0.3 m/s, was used as the carrier gas. CO was eluted at 3.6 min and fully separated from N2, O2, H2O, and CO2. The mass spectrometer parameters were as follows: ion source temperature, 120°C; electron energy, 31 eV; and transfer-line temperature, 120°C. Using a gas-tight syringe, 100-µl aliquots of headspace gas of either standard solutions or experimental samples were injected into a spitless injector that had a temperature of 120°C. Abundances of ions at mass-to-charge ratios (m/z) of 28, 29, and 31, which correspond to 12C16O, 13C16O, and 13C18O, respectively, were acquired via selected ion monitoring. The amount of CO in each cell-culture sample was calculated using standard curves that were constructed from ion abundances with m/z values of 28 and 29 or 31 as previously described (42).

Microsomal heme determination. Microsomal heme was determined as the pyridine hemochromogen by using the reduced minus the oxidized difference in absorbance at lambda  values of 400 and 600 nm with an absorption coefficient of 32.4 mM-1cm-1 (13).

Measurement of human and rat HO-1 and HO-2 mRNA. RT-PCR was performed as previously described (38, 40) to amplify hHO-1 and rat HO-1/HO-2 mRNA. Total RNA was extracted from cells using TRIzol reagent (GIBCO-BRL), and 10 µg of RNA were electrophoresced on gels containing 1% agarose and 1 M formaldehyde. RNA samples were then transferred to nylon membranes and hybridized with 32P-labeled probes. The probe used for hHO-1 was the 987-bp HindIII fragment prepared in the plasmid pGEM-hHO-1. The probe for glyceraldehyde-3-phosphate dehydrogenase (G-3-PDH) was purchased from Clontech. Autoradiography was performed for varying lengths of time at -80°C using XAR-5 film (Eastman Kodak, Rochester, NY) with Lighting-Plus intensifying screens (DuPont, Wilmington, DE). The relative amounts of HO-1 and G-3-PDH mRNA were determined by scanning the blots using NIH Image software. The density of G-3-PDH mRNA was used for normalization of the data.

Western Blot analysis. Cells were harvested using cell lysis buffer as described previously (40). The lysate was collected for Western blot analysis or HO activity measurement. Protein levels were visualized by immunoblotting with antibodies against hHO-1, rat HO-1, and rat HO-2 (Stressgen Biotechnologies, Victoria, BC, Canada). Briefly, 30 µg of lysate supernatant collected in HEPES buffer (that contained 50 mM HEPES, pH 7.4, 2 µM leupeptin, 2 µM pepstatin, 0.5 mM phenylmethylsulfonyl fluoride, and 1 mM sodium orthovanadate) were separated by 8% SDS-PAGE and transferred to a nitrocellulose membrane (Amersham, Piscataway, NJ) using a semidry transfer apparatus (Bio-Rad, Hercules, CA). The membranes were incubated with 5% milk in buffer that contained 10 mM Tris · HCl (pH 7.4), 150 mM NaCl, and 0.05% Tween 20 (TBST) at 4°C overnight. Membranes were then washed with TBST and incubated with a 1:2,000 dilution of anti-HO-1 or anti-HO-2 antibodies for 1 h at room temperature with constant shaking. The filters were washed and subsequently probed with horseradish peroxidase-conjugated donkey anti-rabbit IgG (Amersham) at a dilution of 1:2,000. Chemiluminescence detection was performed with the Amersham ECL detection kit according to the manufacturer's instructions.

The presence of sGC and endothelial NO synthase (eNOS) in cell lysates was detected using the anti-sGC and anti-eNOS antibodies followed by a donkey anti-rabbit secondary antibody conjugated to horseradish peroxidase (Transduction Laboratories, San Diego, CA). Peroxidase activity was determined using the Amersham ECL detection kit. The relative amount of sGC and eNOS was quantitated by laser densitometry and analyzed using an HP Scanjet II CX scanner. Image analysis was performed with SigmaScan image software (Jandel Scientific).

Measurement of cGMP concentrations. For measurement of cGMP levels in pulmonary endothelial and hHO-1-expressing endothelial cells, the cells were plated in 60-mm culture dishes and grown to ~80% confluence. Cellular cGMP content was determined using a commercial ELISA kit according to the instructions provided by the manufacturer. Briefly, the cells were washed once with ice-cold PBS and 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 use. The protein contents of samples were determined by the method of Bradford (Bio-Rad protein assay). The cGMP concentration was normalized and expressed as picomoles of cGMP per 106 cells. In experiments using Nomega -nitro-L-arginine methyl ester (L-NAME), it was added simultaneously with heme or vehicle.

Measurement of nitrite. Nitrite, the stable metabolite of NO, was quantitated colorimetrically via the Griess reaction. Sample aliquots (60 µl) were incubated for 10 min with 80 µl of 1% sulfanilamide in 4 N HCl at room temperature. After addition of 60 µl of 1% napthylethylenediamine in methanol, the incubation was continued for an additional 10 min. Nitrite concentration, which is proportional to absorbance at a lambda  of 540 nm, was determined with a standard curve using a microplate reader.

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


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Expression of hHO-1 mRNA in pulmonary cells. We used the supernatant of PA317 retroviral packaging cells to infect pulmonary endothelial cells. The retrovirus vector contains a neomycin resistance gene for selecting an endothelial cell clone that is resistant to the cytotoxic drug G418 and simultaneously expresses the hHO-1 gene. Cells were assessed by parallel determination of endogenous rat HO-1 mRNA and expressed hHO-1 mRNA. To measure the expression of the hHO-1 gene in pulmonary endothelial cells, we used an RT-PCR method followed by Southern blot to detect specific genes of interest in a small amount of tissue (37). As shown in Fig. 1A, RNA obtained from control endothelial cells (lane 1) and cells transduced with the empty vector (lane 2) did not yield RT-PCR products when amplified using hHO-1-specific primers. In contrast, RNA obtained from cells transduced with retrovirus-mediated hHO-1 yielded RT-PCR products hybridizable with the hHO-1 cDNA probe (lanes 3 and 4). Importantly, RT-PCR performed on total RNA samples using primers for the rat HO-1 gene demonstrated that both transduced and nontransduced cells yielded RT-PCR amplification products for the rat HO-1 (Fig. 1A). Western blot analysis confirmed that the hHO-1 gene transfer resulted in the expression of hHO-1 protein (lane 3, Fig. 1B). The hHO-1 gene expression did not affect the endogenous levels of rat HO-1 or HO-2 proteins (Fig. 1B). Control cells or cells transduced with the empty viral vector did not yield a significant change in the levels of rat HO-1 or HO-2 and were devoid of any detectable band of hHO-1 (Fig. 1B).


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Fig. 1.   A: detection of human and rat heme oxygenase (HO)-1 transcripts by RT-PCR analysis in RNA isolated from rat-lung microvessel (RLMV) endothelial cells transfected or nontransfected with retroviral vectors (LSN-hHO or LXSN). Lane 1, control endothelial cells; lane 2, cells transfected with empty vector; lane 3, cells transfected with human (h) HO-1 (11 passages); lane 4, cells transfected with hHO-1 (15 passages); and lane 5, positive controls. RT-PCR to amplify hHO-1 and rat HO-1 transcripts was performed as described in METHODS. B: representative immunoblots of HO isoforms. Homogenates (30 µg of protein) from control pulmonary endothelial cells (lane 1) and pulmonary endothelial cells transfected with either LXSN (lane 2) or LSN-hHO-1 (lane 3) were subjected to Western blot analysis using anti-rat HO-2 and HO-1 and anti-hHO-1 antibodies.

Effects of retrovirus-mediated hHO-1 gene transfer on HO activity in pulmonary endothelial cells. We examined HO enzymatic activity and heme content in pulmonary cells that were either nontransduced, transduced with control retrovirus vector, or transfected with retrovirus vector that contained the hHO-1 gene. The basal level of HO activity in pulmonary cells was not affected by the retroviral infection and selection processes because HO activity (0.77 ± 0.123 nmol · mg-1 · 30 min-1; n = 3) and heme content (263 ± 23 pmol/mg; n = 3) in cells infected with the empty vector were not different from those for control cells (Table 1). However, in cells that expressed hHO-1 via infection with the LSN-hHO-1 retrovirus, HO activity increased by 3.46-fold (Table 1). To further ascertain the characteristics of the expressed HO protein, we tested the effects of stannic mesoporphyrin (SnMP), which is a known inhibitor of HO activity, on cell homogenates from transfected cells. The addition of SnMP inhibited HO enzymatic activity in pulmonary cells from 0.71 ± 0.09 to 0.18 ± 0.05 nmol bilirubin · mg-1 · 30 min-1 and in hHO-1-expressing pulmonary cells from 2.46 ± 0.43 to 0.51 ± 0.07 nmol bilirubin · mg-1 · 30 min-1. The stable expression of hHO-1 in pulmonary cells reduced the basal steady-state levels of heme by ~50% compared with those of control pulmonary cells or cells transduced with the empty vector LXSN (Table 1).

                              
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Table 1.   Effect of retroviral-mediated human HO-1 transfection on heme content, HO activity, CO production, and cGMP levels

Effects of HO-1 transduction on CO production. The ability of the cells to produce CO was measured in the absence or presence of exogenously added heme. As shown in Table 1 and Fig. 2A, the basal level of CO in pulmonary cells that express hHO-1 was not significantly different from that in pulmonary endothelial cells. However, the addition of heme (30 µM) increased CO production in hHO-1-expressing cells to levels that were twofold of the amount produced by pulmonary endothelial cells (Fig. 2A), which indicates higher levels of HO enzymatic activity in pulmonary cells that express hHO-1. Heme-HO-mediated CO was inhibited by production with SnMP incubation in pulmonary endothelial cells from 0.45 ± 0.14 to 0.14 ± 0.04 nmol · 106 cells-1 · h-1 and in hHO-1-expressing cells from 0.88 ± 0.08 to 0.19 ± 0.04 nmol · 106 cells-1 · h-1.


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Fig. 2.   Carbon monoxide (CO) production (A) and guanosine 3',5'-cyclic monophosphate (cGMP) levels (B) in pulmonary endothelial (RLMV) and hHO-1-expressing pulmonary endothelial (RLMV hHO-1) cells. CO and cGMP measurements in cells treated with and without heme (30 µM) were performed as described in MATERIALS AND METHODS. Results are means ± SD, n = 3; *P < 0.05 vs. control; #P < 0.05 vs. pulmonary endothelial cells; Dagger P < 0.05 vs. HO-1-infected pulmonary endothelial cells.

Effects of HO-1 transfection on cellular cGMP levels. We measured the effects of HO-1 transduction on the basal level of cGMP in pulmonary cells and under conditions of elevated heme levels. Production of cGMP in cells transduced with HO-1 and grown in media that contained 0.5% FBS was significantly decreased compared with nontransduced cells, viz., 1.2 ± 0.4 and 15.3 ± 2.7 pmol/106 cells in hHO-1-expressing and pulmonary endothelial cells, respectively. The levels of cGMP greatly increased when cells were cultured in media that contained 10% FBS (8.4 ± 1.7 and 31.1 ± 5.6 pmol/106 cells) in hHO-1-expressing and control pulmonary endothelial cells, respectively; still, the amount of cGMP generated in HO-1-overexpressing cells was lower than in nontransduced cells, which suggests that depletion of cellular heme is the underlying reason for the decreased levels of cGMP in the HO-1-expressing cells. This is due to the levels of heme: 0.07 ± 0.03 µM in 0.5% FCS and 0.9 ± 0.07 µM in 10% FCS. Thus the rate of cGMP formation is dependent on the level of heme; the rate increased when extracellular levels of heme increased. Similar results were obtained when heme was added to the cultured media that contained 10% FBS. As seen in Fig. 2B, the levels of cGMP were markedly increased after addition of 30 µM heme to both transduced and nontransduced cells. However, in hHO-1-expressing cells, the levels of cGMP in the presence of heme were greater than the levels seen in control pulmonary endothelial cells treated with heme. Evidence that HO activity contributes in part to heme-induced cGMP levels in these cells was further documented by addition of the HO inhibitor SnMP to the incubation. SnMP inhibited heme-induced cGMP levels by 35% in both cell types. That SnMP inhibited heme-induced CO production by 60-70% whereas only 35% of heme-induced cGMP levels were inhibited by SnMP suggests that both heme itself and HO-heme-derived CO contribute to changes in sGC activity.

Under normal culture conditions (10% FBS), heme content of pulmonary endothelial cells was about twofold higher than that of hHO-1-expressing cells (260 ± 36 vs. 142 ± 53 pmol/mg protein, respectively; n = 3; P < 0.05). This difference in heme content corresponded to the differences in the levels of cGMP in these cells (Fig. 2). In the absence of heme addition, incubation with L-NAME did not significantly alter the levels of cGMP in either control pulmonary endothelial or hHO-1-expressing cells. However, in pulmonary endothelial cells treated with heme, the addition of L-NAME (50 µM) significantly decreased cGMP (Fig. 3), whereas the effect of L-NAME on cGMP levels elicited by heme in hHO-1-transfected cells did not reach statistical significance (Fig. 3). Hence, it is possible that a rapid degradation of exogenous heme by cells that overexpress HO-1 may generate amounts of CO sufficient for sGC activation and synthesis of cGMP insensitive to L-NAME.


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Fig. 3.   Effects of Nomega -nitro-L-arginine methyl ester (L-NAME) on cGMP levels in pulmonary endothelial and hHO-1-expressing pulmonary endothelial cells. Concentration of cGMP was determined in cells grown in media that contained 10% fetal bovine serum (FBS) and treated with heme (30 µM) both with and without L-NAME (50 µM) for 3 h. Results are means ± SD, n = 6; *P < 0.05 vs. control; #P < 0.05 vs. pulmonary endothelial cells.

Effects of HO-1 gene transfection on expression of sGC and NOS proteins. The finding that L-NAME did not decrease cGMP levels in heme-treated hHO-1-expressing cells suggested the possibility that HO-1 overexpression interferes with NOS expression or activity. Accordingly, we determined the levels of eNOS protein in cells transfected with the hHO-1 gene. As shown in Fig. 4, Western blot analysis demonstrated that the levels of immunoreactive eNOS were not different between pulmonary endothelial and hHO-1-expressing cells. Additional evidence that NOS activity is not impaired in cells that express hHO-1 is indicated by the fact that the nitrite levels were not significantly different from those in control pulmonary endothelial cells, i.e., 1,792 ± 378 and 2,100 ± 60 nmol nitrite · mg protein-1 · 24 h-1 in pulmonary endothelial and hHO-1-expressing cells, respectively. We also performed a Western blot analysis for sGC. As shown in Fig. 4, the levels of immunoreactive sGC protein in cell homogenates from pulmonary endothelial and hHO-1-expressing cells were similar, which further suggests that increased HO-1 expression does not interfere with the synthesis of sGC protein but rather influences its activity by controlling cellular heme and CO production.


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Fig. 4.   Western blot analyses of soluble guanylate cyclase (sGC) and endothelial nitric oxide synthase (eNOS) in endothelial cell homogenates. Cell homogenate protein samples of 30 µg were used. Western blot analysis and the relative amounts of sGC and eNOS were normalized to beta -actin. Immunoblots are representative of 3 immunoblots from 3 separate experiments.

Additional experiments were conducted to evaluate whether viral transfection induces inducible NOS (iNOS) in these cells. Western blot analysis using iNOS antibodies failed to detect iNOS immunoreactivity in either cells transduced or nontransduced with the retroviral hHO-1 vector or the empty vector (data not shown). This suggests that retroviral vectors do not induce an iNOS-generated NO pool.

Effects of acute and chronic HO-1 expression on cGMP, CO, and cellular heme. To investigate the differential effects of acute and chronic HO-1 expression on cGMP, we measured the effects of SnCl2, a potent inducer of HO-1 expression, on cGMP for 24, 48, and 72 h in pulmonary endothelial cells. As shown in Fig. 5A, cGMP levels were increased by sixfold in pulmonary endothelial cells exposed to SnCl2 (10 µM) for 24 h. Higher than basal levels of cGMP were maintained at 48 h postadministration of SnCl2; however, at 72 h, the levels of cGMP decreased to 60% of the basal levels. Pulmonary endothelial cells were found to contain ~240 ± 61 pmol heme/mg protein after 24 h of culture in 10% FCS. The SnCl2-induced increase in HO-1 resulted in a gradual decrease in cellular heme (Fig. 5B). The total content of heme per milligram of cellular protein decreased by 22, 38, and 65% after 24, 48, and 72 h of exposure to SnCl2, respectively.


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Fig. 5.   Effects of SnCl2 on cGMP levels (A) and heme content (B) in pulmonary endothelial cells. Cells were treated with SnCl2 (10 µM) for 24, 48, and 72 h. Results are means ± SD, n = 3; *P < 0.05 vs. control.

Incubation of pulmonary endothelial cells with SnCl2 for 24 h caused a 10-fold increase in CO production, and this increase was inhibited by 90% after coadministration with SnMP (Fig. 6). In one experiment, prolonged incubation with SnCl2 (48 and 72 h) markedly decreased CO production and reached basal levels at 72 h without affecting cell viability (data not shown).


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Fig. 6.   Effects of SnCl2 on CO production in pulmonary endothelial cells. CO production was determined in cells treated with and without SnCl2 (10 µM) and SnMP (10 µM) for 24 h. Results are means ± SD, n = 3; *P < 0.005 vs. control; #P < 0.01 vs. SnCl2 alone.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study we report the effects of long-term HO-1 gene expression on cellular cGMP in the presence or absence of heme supplementation using a retroviral delivery system. Cells transduced with the retroviral hHO-1 construct displayed enhanced heme oxidation activity, which resulted in a significant decrease in the amount of unmetabolized residual heme in cell cultures. By employing a microassay procedure for quantitating CO, it was determined that production of heme-driven CO was significantly enhanced, which is consistent with an increased rate of heme catabolism in the hHO-1-transduced cells. This effect was demonstrated in 11 sequential cell passages and can be presumed to be "permanent." Taken as a whole, the results indicate that it is possible to functionally express hHO-1 in cells for long periods of time with a retroviral hHO-1 gene construct.

This study clearly demonstrates that the heme-HO system affects cGMP production in endothelial cells presumably by influencing the availability of both heme and CO for the activation of sGC. Several observations support an important role for the heme-HO system in the regulation of cellular cGMP in endothelial cells. Induction of HO-1 by SnCl2 caused a rapid and striking increase in CO production as well as marked elevation in cellular cGMP levels. However, incubation of cells with SnCl2 for 72 h resulted in decreases of cellular heme, CO, and cGMP, which indicates that prolonged depletion of cellular heme that is brought about by sustained induction of HO-1 affects production of both CO and cGMP. In fact, cGMP levels remained elevated as long as the decrease in cellular heme was moderate. However, a further decrease of heme to levels that were 65% lower than the basal levels at 72 h resulted in a precipitous decrease in cGMP levels, which supports the notion that heme is a limiting factor not only for the production of CO but also for the activation of sGC and the generation of cGMP. This is further supported by results obtained in cultured endothelial cells in which the elevated functional expression of HO-1 was maintained by stable transfection of the hHO-1 gene. Although exhibiting a higher capacity to metabolize heme as indicated by increased HO enzymatic activity, these cells showed no increase in CO production under normal culture conditions and decreased cGMP levels. Whereas the level of HO-1 expression in hHO-1-expressing cells is relatively high, cellular heme levels were less than optimal to sustain CO and cGMP synthesis. Indeed, when fortified with heme, the hHO-1-transduced cells showed greater ability to produce CO and increase cGMP, which substantiates the notion that heme is a limiting factor in the HO-dependent regulation of cGMP. The attenuation of the heme-stimulated increase in cGMP by SnMP in hHO-1-expressing cells indicates that the modulation of sGC activity is partially HO dependent.

Another key finding is that long-term HO-1 gene expression does not modulate the level or activity of eNOS nor does it affect the level of sGC protein, which suggests that the effect of HO-1 overexpression on cGMP levels is due primarily to the direct effect of heme/CO on sGC activity. That heme-stimulated cGMP levels were not significantly affected by L-NAME in cells that overexpressed HO-1 suggests that the higher levels of heme/CO in these cells superseded NO in activating sGC.

The heme-stimulated increase in cGMP levels may be mediated by several factors or a combination of factors. Heme may be incorporated into sGC to result in increased sGC activity. Because heme readily binds to GC and restores activation by NO and CO, the restoration of heme is likely to directly affect the function of this enzyme (11). The ability of heme to restore the function of heme-depleted NOS is less well understood. Additionally, because heme is a substrate for HO-dependent CO production, sGC activity may also be stimulated by enhanced CO production. The heme-stimulated elevation of cGMP levels was partially blocked by L-NAME in control pulmonary endothelial cells. However, the enhanced cGMP production in endothelial cells transduced with hHO-1 was insensitive to L-NAME. Although the origin of a heme-induced stimulation of cGMP by NOS in pulmonary endothelial cells is not known, it may be a consequence of activation of a signaling mechanism that increases NO production in these cells. In contrast, it is likely that restoration of heme levels along with increased generation of CO via HO are the primary causes of the heme-stimulated increase in cGMP in endothelial hHO-expressing cells.

The availability of an hHO-1 gene-delivery system that carries sense nucleotide sequences offers many possibilities for experimental examination of the relationship of HO activity to various physiological processes. For example, the administration of HO-1 inducers is known to lower blood pressure (20, 32) in spontaneously hypertensive rats, whereas the administration of HO-1 inhibitors increases blood pressure and produces systemic vasoconstriction (16). These studies suggest a role for HO in the control of vascular tone and blood pressure. However, the use of metal, heme, and metalloporphyrins to manipulate HO activity is a far less desirable approach to specifically targeting HO. These agents are known for affecting processes unrelated to HO-1 expression and activity such as sGC activity and NO production, which by themselves can contribute to the effects seen in response to these agents. To this end, we (28) and others have embarked on developing the means to specifically target the expression and activity of HO in isoform- and site-specific manners. We have constructed an adenoviral vector carrying the hHO-1 gene and demonstrated its efficiency in increasing HO-1 gene expression and HO activity in vitro (2) and in vivo (1, 4). Although the adenoviral vector is efficient, it is short lasting (2-4 wk), whereas retroviral gene transfer offers long-lasting expression (7, 31). The availability of heme to cells that express the hHO-1 gene may be a determining factor for the generation of CO and cGMP synthesis. Additional studies are needed to evaluate the influence of HO-1 gene transfer on other aspects of the function of the hemoproteins sGC and NOS.

In summary, this study documents the regulatory action of the heme-HO system on cGMP production in endothelial cells. Acute upregulation of HO-1, which leads to elevation of CO, brings about an increase of cGMP. Chronic upregulation of HO-1 leads to a decrease in both heme and CO. Thus the impact of increased HO-1 overexpression on cGMP levels is dependent on the extent to which cellular heme levels are reduced. That supplementation of heme caused the activation of sGC and cGMP synthesis that was greater in cells overexpressing the HO-1 gene suggests that sustained overexpression of the HO-1 gene enables the cell to markedly increase cellular cGMP.


    ACKNOWLEDGEMENTS

The authors thank Drs. Michael Balazy and Houli Jiang for outstanding expertise in measurement of carbon monoxide, Sylvia Shenouda for technical assistance, and Jennifer Brown for excellent secretarial assistance.


    FOOTNOTES

This work was supported by American Heart Association Grant 50948T and National Heart, Lung, and Blood Institute Grants HL-55601, HL-31069, and HL-34300.

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

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. Section 1734 solely to indicate this fact.

July 3, 2002;10.1152/ajplung.00365.2001

Received 13 September 2001; accepted in final form 3 June 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abraham, NG, da Silva JL, Dunn MW, Kigasawa K, and Shibahara S. Retinal pigment epithelial cell-based gene therapy against hemoglobin toxicity. Int J Mol Med 1: 657-663, 1998[ISI][Medline].

2.   Abraham, NG, da Silva JL, Lavrovsky Y, Stoltz RA, Kappas A, Dunn MW, and Schwartzman ML. Adenovirus-mediated heme oxygenase-1 gene transfer into rabbit ocular tissues. Invest Ophthalmol Vis Sci 36: 2202-2210, 1995[Abstract].

3.   Abraham, NG, Drummond GS, Lutton JD, and Kappas A. The biological significance and physiological role of heme oxygenase. Cell Physiol Biochem 6: 129-168, 1996[ISI].

4.   Abraham, NG, Jiang S, Yang L, Zand BA, Marji J, Drummond GS, and Kappas A. Adenoviral vector-mediated transfer of human heme oxygenase in rats decreases renal heme-dependent arachidonic acid epoxygenase activity. J Pharmacol Exp Ther 293: 494-500, 2000[Abstract/Free Full Text].

5.   Abraham, NG, Lin JH, Dunn MW, and Schwartzman ML. Presence of heme oxygenase and NADPH cytochrome P-450c reductase in human corneal epithelium. Invest Ophthalmol Vis Sci 28: 1464-1472, 1987[Abstract].

6.   Brune, B, and Ullrich V. Inhibition of platelet aggregation by carbon monoxide is mediated by activation of guanylate cyclase. Mol Pharmacol 32: 497-504, 1987[Abstract].

7.   Chertkov, JL, Jiang S, Lutton JD, Harrison J, Levere RD, Tiefenthaler M, and Abraham NG. The hematopoietic stromal microenvironment promotes retrovirus-mediated gene transfer into hematopoietic stem cells. Stem Cells 11: 218-227, 1993[Abstract].

8.   Choi, AMK, and Alam J. Heme oxygenase-1: function, regulation, and implication of a novel stress-inducible protein in oxidant-induced lung injury. Am J Respir Cell Mol Biol 15: 9-19, 1996[Abstract].

9.   Christodoulides, N, Durante W, Kroll MH, and Schafer AI. Vascular smooth muscle cell heme oxygenases generate guanylyl cyclase-stimulatory carbon monoxide. Circulation 91: 2306-2309, 1995[Abstract/Free Full Text].

10.   Coceani, F, Kelsey L, Seidlitz E, Marks GS, McLaughlin BE, Vreman HJ, Stevenson DK, Rabinovitch M, and Ackerley C. Carbon monoxide formation in the ductus arteriosus in the lamb: implications for the regulation of muscle tone. Br J Pharmacol 120: 599-608, 1997[Abstract].

11.   Craven, PA, and DeRubertis FR. Restoration of the responsiveness of purified guanylate cyclase to nitrosoguanidine, nitric oxide, and related activators by heme and hemeproteins. Evidence for involvement of the paramagnetic nitrosyl-heme complex in enzyme activation. J Biol Chem 253: 8433-8443, 1978[Abstract].

12.   Durante, W, and Schafer AI. Carbon monoxide and vascular cell function. Int J Mol Med 2: 255-262, 1998[ISI][Medline].

13.   Fuhrop, JH, and Smith KM. Hemes: determination as pyridine hemochromes In: Porphyrins and Metalloporphyrins, , edited by Smith KM.. New York: Elsevier Scientific, 1975, p. 804-807.

14.   Gerzer, R, Bohme E, Hofmann F, and Schultz G. Soluble guanylate cyclase purified from bovine lung contains heme and copper. FEBS Lett 132: 71-74, 1981[ISI][Medline].

15.   Ignarro, LJ, Wood KS, and Wolin MS. Regulation of purified soluble guanylate cyclase by porphyrins and metalloporphyrins: a unifying concept. Adv Cyclic Nucleotide Protein Phosphorylation Res 17: 267-274, 1984[ISI][Medline].

16.   Johnson, RA, Lavesa M, Askari B, Abraham NG, and Nasjletti A. A heme oxygenase product, presumably carbon monoxide, mediates a vasodepressor function in rats. Hypertension 25: 166-169, 1995[Abstract/Free Full Text].

17.   Kaide, JI, Zhang F, Wei Y, Jiang H, Yu C, Wang WH, Balazy M, Abraham NG, and Nasjletti A. Carbon monoxide of vascular origin attenuates the sensitivity of renal arterial vessels to vasoconstrictors. J Clin Invest 107: 1163-1171, 2001[Abstract/Free Full Text].

18.   Kharitonov, VG, Sharma VS, Pilz RB, Magde D, and Koesling D. Basis of guanylate cyclase activation by carbon monoxide. Proc Natl Acad Sci USA 92: 2568-2671, 1995[Abstract].

19.   Kozma, F, Johnson RA, and Nasjletti A. Role of CO in heme-induced vasodilation. Eur J Pharmacol 323: R1-R2, 1997[ISI][Medline].

20.   Levere, RD, Martasek P, Escalante B, Schwartzman ML, and Abraham NG. Effect of heme arginate administration on blood pressure in spontaneously hypertensive rats. J Clin Invest 86: 213-219, 1990[ISI][Medline].

21.   Lutton, JD, da Silva JL, Moqattash S, Brown AC, Levere RD, and Abraham NG. Differential induction of heme oxygenase in the hepatocarcinoma cell line (Hep3b) by environmental agents. J Cell Biochem 49: 259-265, 1992[ISI][Medline].

22.   Maines, MD. The heme oxygenase system: a regulator of second messenger gases. Annu Rev Pharmacol Toxicol 37: 517-554, 1997[ISI][Medline].

23.   Maines, MD, and Kappas A. Cobalt induction of hepatic heme oxygenase with evidence that cytochrome P-450 is not essential for this enzyme activity. Proc Natl Acad Sci USA 71: 4293-4297, 1974[Abstract].

24.   Maines, MD, Mark JA, and Ewing JF. Heme oxygenase, a likely regulator of cGMP production in the brain: inductin of in vivo HO-1 compensates for depression in NO synthase activity. Mol Cen Neurosci 4: 398-405, 1993.

25.   Marks, GS, McLaughlin BE, Vreman HJ, Stevenson DK, Nakatsu K, Brien JF, and Pang SC. Heme oxygenase activity and immunohistochemical localization in bovine pulmonary artery and vein. J Cardiovasc Pharmacol 30: 1-6, 1997[ISI][Medline].

26.   McCoubrey, WK, Jr, Ewing JF, and Maines MD. Human heme oxygenase-2: characterization and expression of a full-length cDNA and evidence suggesting that the two HO-2 transcripts may differ by choice of polyadenylation signal. Arch Biochem Biophys 295: 13-20, 1992[ISI][Medline].

27.   McCoubrey, WK, Jr, Huang TJ, and Maines MD. Isolation and characterization of a cDNA from the rat brain that encodes hemoprotein heme oxygenase-3. Eur J Biochem 247: 725-732, 1997[Abstract].

28.   Minamino, T, Christou H, Hsieh CM, Liu Y, Dhawan V, Abraham NG, Perrella MA, Mitsialis SA, and Kourembanas S. Targeted expression of heme oxygenase-1 prevents the pulmonary inflammatory and vascular responses to hypoxia. Proc Natl Acad Sci USA 98: 8798-8803, 2001[Abstract/Free Full Text].

29.   Mitani, K, Fujita H, Sassa S, and Kappas A. Heat shock induction of heme oxygenase mRNA in human Hep3B hepatoma cells. Biochem Biophys Res Commun 165: 437-441, 1989[ISI][Medline].

30.   Neil, TK, Stoltz RA, Jiang S, Dunn MW, Levere RD, Kappas A, and Abraham NG. Modulation of corneal heme oxygenase expression by oxidative stress agents. J Ocul Pharmacol Ther 11: 455-468, 1995[ISI][Medline].

31.   Sabaawy, HE, Zhang F, Nguyen X, Elhosseiny A, Nasjletti A, Schwartzman M, Dennery P, Kappas A, and Abraham NG. Human heme oxygenase-1 gene transfer lowers blood pressure and promotes growth in spontaneously hypertensive rats. Hypertension 38: 210-215, 2001[Abstract/Free Full Text].

32.   Sacerdoti, D, Escalante B, Abraham NG, McGiff JC, Levere RD, and Schwartzman ML. Treatment with tin prevents the development of hypertension in spontaneously hypertensive rats. Science 243: 388-390, 1989[ISI][Medline].

33.   Sammut, IA, Foresti R, Clarck JE, Exon DJ, Vesely MJJ, Sarathchandra P, Green CJ, and Motterlini R. Carbon monoxide is a major contributor to the regulation of vascular tone in aortas expressing high levels of haeme oxygenase-1. Br J Pharmacol 125: 1437-1444, 1998[Abstract].

34.   Shibahara, S, Muller M, and Taguchi H. Transcriptional control of rat heme oxygenase by heat shock. J Biol Chem 262: 12889-12892, 1987[Abstract/Free Full Text].

35.   Shibahara, S, Yoshizawa M, Suzuki H, Takeda K, Meguro K, and Endo K. Functional analysis of cDNAs for two types of human heme oxygenase and evidence for their separate regulation. J Biochem (Tokyo) 113: 214-218, 1993[Abstract].

36.   Stone, JR, and Marletta MA. Soluble guanylate cyclase from bovine lung: activation with nitric oxide and carbon monoxide and spectral characterization of the ferrous and ferric states. Biochemistry 33: 5636-5640, 1994[ISI][Medline].

37.   Wagener, FADTG, Feldman E, de Witte T, and Abraham NG. Heme induces the expression of adhesion molecules ICAM-1, VCAM-1, and E selectin in vascular endothelial cells. Proc Soc Exp Biol Med 216: 456-463, 1997[Abstract].

38.   Wagner, CT, Durante W, Christodoulides N, Hellums JD, and Schafer AI. Hemodynaic forces induce the expression of heme oxygenase in cultured vascular smooth muscle cells. J Clin Invest 100: 589-596, 1997[Abstract/Free Full Text].

39.   Wang, R, Wang Z, and Wu L. Carbon monoxide-induced vasorelaxation and the underlying mechanisms. Br J Pharmacol 121: 927-934, 1997[Abstract].

40.   Yang, L, Quan S, and Abraham NG. Retrovirus-mediated HO gene transfer into endothelial cells protects against oxidant-induced injury. Am J Physiol Lung Cell Mol Physiol 277: L127-L133, 1999[Abstract/Free Full Text].

41.   Zakhary, R, Gaine SP, Dinerman JL, Ruat M, Flavahan NA, and Snyder SH. Heme oxygenase 2: endothelial and neuronal localization and role in endothelium-dependent relaxation. Proc Natl Acad Sci USA 93: 795-798, 1996[Abstract/Free Full Text].

42.   Zhang, F, Kaide JI, Wei Y, Jiang H, Yu C, Balazy M, Abraham NG, Wang W, and Nasjletti A. Carbon monoxide produced by isolated arterioles attenuates pressure-induced vasoconstriction. Am J Physiol Heart Circ Physiol 281: H350-H358, 2001[Abstract/Free Full Text].


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