Hypoxia-induced acute lung injury in murine models of sickle cell disease

Kirkwood A. Pritchard, Jr.,1,2,3,4 Jingsong Ou,1,3 Zhijun Ou,1,3 Yang Shi,1,3 James P. Franciosi,1 Paul Signorino,1 Sushma Kaul,1 Cathleen Ackland-Berglund,1 Karin Witte,1 Sandra Holzhauer,5 Narla Mohandas,6 Karen S. Guice,1,3 Keith T. Oldham,1,3,* and Cheryl A. Hillery5,*

1Department of Surgery, Division of Pediatric Surgery, 2Department of Pharmacology and Toxicology, 3Cardiovascular Center, 4Free Radical Research Center, and 5Department of Pediatrics, Division of Hematology-Oncology, Medical College of Wisconsin, Children's Hospital of Wisconsin, The Blood Center of Southeastern Wisconsin, Milwaukee, Wisconsin 53226; and 6New York Blood Center, New York, New York 10021

Submitted 21 August 2002 ; accepted in final form 11 September 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Vaso-occlusive events are the major source of morbidity and mortality in sickle cell disease (SCD); however, the pathogenic mechanisms driving these events remain unclear. Using hypoxia to induce pulmonary injury, we investigated mechanisms by which sickle hemoglobin increases susceptibility to lung injury in a murine model of SCD, where mice either exclusively express the human {alpha}/sickle {beta}-globin (h{alpha}{beta}S) transgene (SCD mice) or are heterozygous for the normal murine {beta}-globin gene and express the h{alpha}{beta}S transgene (m{beta}+/-, h{alpha}{beta}S+/-; heterozygote SCD mice). Under normoxia, lungs from the SCD mice contained higher levels of xanthine oxidase (XO), nitrotyrosine, and cGMP than controls (C57BL/6 mice). Hypoxia increased XO and nitrotyrosine and decreased cGMP content in the lungs of all mice. After hypoxia, vascular congestion was increased in lungs with a greater content of XO and nitrotyrosine. Under normoxia, the association of heat shock protein 90 (HSP90) with endothelial nitric oxide synthase (eNOS) in lungs of SCD and heterozygote SCD mice was decreased compared with the levels of association in lungs of controls. Hypoxia further decreased association of HSP90 with eNOS in lungs of SCD and heterozygote SCD mice, but not in the control lungs. Pretreatment of rat pulmonary microvascular endothelial cells in vitro with xanthine/XO decreased A-23187-stimulated nitrite + nitrate production and HSP90 interactions with eNOS. These data support the hypotheses that hypoxia increases XO release from ischemic tissues and that the local increase in XO-induced oxidative stress can then inhibit HSP90 interactions with eNOS, decreasing ·NO generation and predisposing the lung to vaso-occlusion.

vaso-occlusion; heat shock protein 90; nitric oxide; sickle hemoglobin; xanthine oxidase; pulmonary microvascular endothelial cell


VASO-OCCLUSIVE EVENTS ARE a major source of morbidity and mortality in sickle cell patients (9). The mechanisms by which sickle cell disease (SCD) promotes vascular dysfunction are complex and involve multiple sequential pathological steps related to the many primary and secondary effects of sickle hemoglobin (Hb S) on the erythrocyte membrane, vascular endothelium, and the inflammatory and coagulation pathways (27). Oxidative damage to the blood vessel wall is likely a critical component of the pathogenesis of vaso-occlusion in SCD (30, 37). Proposed mechanisms include the release of xanthine oxidase (XO) from livers damaged by acute episodic sickling-induced ischemic injury (2), sickle red blood cells that generate reactive oxygen species (25) and mononuclear cell production of superoxide anion (), which inactivates nitric oxide (·NO) (11).

·NO plays important roles in maintaining blood flow by promoting vasodilation and inhibiting platelet aggregation, leukocyte adhesion, and endothelial adhesion molecule expression (34). Whereas ·NO appears to slow Hb S polymerization (19, 24), loss of ·NO activity appears to accelerate red blood cell sickling, impair blood flow, and promote ischemic injury in SCD (2, 3, 24). Interestingly, low levels of ·NO metabolites correlate with higher pain scores for SCD patients who presented to the emergency room in acute painful crises, suggesting that a loss in ·NO production is associated with more severe vaso-occlusion (23). Inhibition of ·NO synthesis in rats that were transfused with sickle red blood cells induced vaso-occlusion in the central nervous system (15), providing further evidence that loss of ·NO contributes to sickle cell-induced ischemic injury. In addition, ·NO activity is decreased by scavenging with , which increases the formation of peroxynitrite, a potent oxidant that induces lipid peroxidation, DNA damage, and tyrosine nitration (18, 28, 44). When from XO bound to vascular endothelial cells scavenges endothelium-derived ·NO, peroxynitrite is formed, most likely in the lipid domain of the plasma membrane (59, 60), where it can oxidize phospholipids to form proinflammatory lipids (18) or critical plasma membrane proteins (59, 60). Interestingly, peroxynitrite-dependent nitration of tyrosyl-containing peptides increases with increasing depth of the tyrosyl residue in the lipid bilayers, whereas peroxidase-hydrogen peroxide-nitrite-induced tyrosyl nitration decreases with increasing depth of tyrosyl residues in the membrane (59, 60). As polymorphonuclear cell (PMN) activation, margination, and diapedesis have been observed in SCD, this mechanism of tyrosine nitration may also contribute to lung injury (4). Together, these mechanisms, both of which increase oxidative stress via generating nitrating species, may play critical roles in inducing pulmonary endothelial dysfunction and possibly with increasing susceptibility to acute chest syndrome (23). These findings suggest that perturbations in the ·NO pathway can contribute to sickle cell-induced vaso-occlusion and underscore the potential importance of ·NO in maintaining pulmonary vascular function in SCD.

One of the primary sources of ·NO in vascular tissues is endothelial nitric oxide synthase (eNOS) whose activity is influenced by posttranslation modifications, i.e., myristoylation, palmitoylation, and phosphorylation as well as by protein interactions with calmodulin, caveolin, and heat shock protein 90 (HSP90) (20). Nitric oxide synthases (NOSs) are homodimer enzymes composed of an L-arginine oxygenase domain containing binding sites for heme, tetrahydrobiopterin, L-arginine, and zinc, and a NADPH reductase domain containing binding sites for FMN, FAD, and NADPH. Upon binding calmodulin at its binding domain, electron flow from the reductase domain to the arginine oxygenase domain increases. Calcium-dependent calmodulin activation initiates ·NO generation in endothelial and neuronal NOS isoforms by a two-step process where electrons from NADPH reduce the heme iron, which then activates molecular oxygen that, in turn, oxidizes the guanidino N-group of arginine to generate ·NO, L-citrulline, and H2O (16, 26, 55). Failure to link the transfer of electrons from NADPH to L-arginine metabolism results in uncoupled enzyme activity, yielding rather than ·NO (16). The importance of this unique change in eNOS function is realized by the fact that for one ·NO made during coupled activity, two are generated when eNOS is uncoupled.

Disruption of HSP90 interactions with eNOS is associated with attenuated ·NO production (8, 17, 21, 32, 47) and uncoupling eNOS activity (42, 43, 51) in a variety of other systems. Antineoplastic agents that selectively inhibit conformational changes in HSP90 increase eNOS-dependent generation in stimulated endothelial cell cultures or in the endothelium of isolated arterial segments (42). In like fashion, angiostatin, a naturally occurring antineoplastic agent uncouples eNOS activity via decreasing HSP90-eNOS interactions to impair vasodilation (33). Further evidence of the importance of HSP90 to NOS is provided by in vitro electron paramagnetic resonance (EPR) data showing that HSP90 facilitates neuronal NOS generation of ·NO and inhibits production (43). Together, these reports suggest that a decrease in HSP90 interactions with eNOS impairs ·NO production, which may, in turn, contribute to acute lung injury in SCD.

In the present study, we examined the effects of SCD and hypoxia on lung injury in transgenic mice expressing Hb S. Our findings suggest that this genetic defect increases susceptibility to vaso-occlusion, which appears to be correlated with decreases in HSP90 association with eNOS in the lungs of the SCD mice.


    METHODS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Transgenic sickle cell mice and hypoxic conditions. Mice that exclusively express human Hb S (m{alpha}-/-, m{beta}-/-, Tg[h{alpha}h{beta}S]) or express both murine {beta}-globin and human Hb S (m{alpha}-/-, m{beta}+/-, Tg[h{alpha}h{beta}S]) were from a colony established at the Medical College of Wisconsin (MCW) Animal Research Care facility (7, 15, 41). The SCD mice (m{beta}-/-, h{alpha}{beta}S) have a moderately severe hemolytic anemia, circulating sickled red blood cells, and multiorgan pathology that closely mimics the pathobiology of SCD in humans, including areas of fibrosis/infarction in the liver, kidney, and spleen and the loss of urine concentrating ability (13, 36, 41). There is an excess of {alpha}-globin chain synthesis ({alpha}/{beta}S = 1.26), indicating that SCD mice also possess a mild to moderate {beta}-thalassemic phenotype. In contrast, heterozygote SCD mice (m{beta}+/-, h{alpha}{beta}S) have normal hemoglobin levels and have minimal organ pathology, with only mild Hb S-induced pathological changes of the kidney, similar to humans with sickle trait (41) (data not shown). Age-matched C57BL/6 mice were purchased as controls from Jackson Laboratory (Bar Harbor, ME). Cystamine hemoglobin cellulose acetate gel electrophoresis (57) and PCR of the murine {beta}-globin locus and deletion site (41) were used to confirm the genotypes of the mice. The number of copies of the h{alpha}{beta}S transgene was determined in heterozygote SCD mice by the relative quantities of h{alpha}{beta}S and chimeric h{alpha}m{beta}-hemoglobin on resolved cellulose acetate gels. All of the heterozygote SCD mice used for the experiments in the present study contained a single copy of the h{alpha}{beta}S transgene.

All experimental procedures involving the mice used in the present study were reviewed and approved by the Institutional Animal Care and Use Committee of the Medical College of Wisconsin. Before each experiment, the animals were weighed and labeled. Sickle (SCD), heterozygote SCD, or control mice were either left at ambient, normoxic air or exposed to hypoxia. For normoxic conditions, the mice were placed into a Plexiglas chamber receiving room air (FIO2 21%) for 80 min. For hypoxic conditions, N2 inflow was mixed with room air to decrease the FIO2% to 16% oxygen for 10 min, 12% for 10 min, and then 8% for 1 h. Mice were observed for any changes in activity or signs of respiratory distress. All of the mice used for experiments survived the experimental protocol for inducing hypoxic lung injury. After the mice were subjected to normoxic or hypoxic conditions, 0.5 ml of 2.5% Avertin (T4,840.2; Aldrich, St. Louis, MO) was injected intraperitoneally. After anesthesia was achieved, the chest wall was removed, the gross lung appearance was noted, and a butterfly needle was inserted into the left ventricle of the heart. The systemic and pulmonary circulations were immediately and gently perfused with phosphate-buffered saline (PBS, 20 ml, 37°C), which resulted in blanching of lung parenchyma, indicating that blood elements were flushed free from the pulmonary microcirculation. The lungs were dissected, transected into four portions per animal, and weighed, and three portions were snap-frozen in liquid N2. The snap-frozen samples were stored at -80°C until further analysis. The fourth lung sample (upper left lung) was placed into 4% paraformaldehyde in PBS and saved for histology studies.

Tissue homogenization. Flash-frozen lung tissue was pulverized in a stainless steel mortar and pestle prechilled with liquid N2. The powder was quickly transferred to a glass Dounce homogenizer and homogenized in modified radioimmunoprecipitation (RIPA) buffer (25 strokes) and centrifuged at 1,000 g for 5 min to remove debris as described (42, 43).

eNOS immunoprecipitation. An aliquot (400–500 µg of protein) of the lung homogenates was removed and precleared by incubation with protein A-Sepharose (40 µl of 50% slurry in modified RIPA buffer) with end-over-end rotation for 2 h at 4°C. The protein A-Sepharose beads were removed and discarded, and the supernatants were incubated with anti-eNOS antibody (mouse monoclonal IgG, 1 µg/100 µg lung protein, H32; Bio-Mol) overnight. The next morning protein A-Sepharose (60 µl) was added and incubated with end-over-end rotation for 2 h at 4°C to bind the immune complexes. The immunoprecipitated proteins were isolated by centrifugation (150 g, 2 min), beads were washed three times with Tris-buffered saline (TBS, 4°C), and proteins in the immune complex were removed from the beads with 2x Laemmli buffer (95°C, 5 min) after centrifugation.

Western analysis. The proteins in the Laemmli buffer were separated by SDS-PAGE (7.5–15%) and transferred onto nitrocellulose. The membrane was blocked in 5% nonfat dry milk in fresh TBS-Tween 20 (TBS-T, 1 h at room temperature) and then cut at 100 kDa. The membrane containing proteins <100 kDa was incubated overnight with anti-HSP90 antibody (H38220 [GenBank] , 1:1,000; Transduction Lab-oratories). Bands were visualized using the appropriate secondary horseradish peroxidase (HRP) antibodies and enhanced chemiluminescence (ECL) reagents from Amersham. The membrane containing proteins >100 kDa was incubated with anti-eNOS antibody [either 9D10, 33-4600 (1:250 dilution) from Zymed; N30020 [GenBank] (1:2,500 dilution) from Transduction Laboratories; or NOS3 (C-20), sc-654 (1: 1,000 dilution) from Santa Cruz]. Bands were visualized with the appropriate secondary HRP antibodies and ECL reagents from Amersham. Autoradiograms were scanned with a laser densitometer or a UMax flatbed scanner, and band densities were analyzed with NIH Image 1.62.

cGMP assays. Extracted mouse lung tissue was removed from -80°C, homogenized in cold 6% trichloroacetic acid, and centrifuged, and the supernatant was washed with diethyl ether to remove the trichloroacetic acid. The aqueous extract was lyophilized and dissolved in enzyme immunoassay (EIA) buffer provided with the EIA kit (cat. no. RPN 226, Amersham Life Sciences). Manufacture protocols were followed using 100-µl quantities of assay buffer, sample, and diluted antibody into the appropriate wells. cGMP peroxidase conjugate was incubated with the samples, and absorbance measurements were read at 630 nm. Measurements of cGMP content were determined as outlined in the cGMP EIA kit manual from a logarithmic curve generated using known cGMP standards provided with the kit.

Histology. Lung samples from each mouse were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and then stained with hematoxylin-eosin (H&E). Fluorescent immunohistochemistry was carried out on unstained sections by the protocols for detection of XO and nitrotyrosine as well as the appropriate nonimmune controls, as previously published by Aslan et al. (2). Briefly, paraffin-embedded lung sections from the experimental mice were processed for immunofluorescence by dewaxing with xylene and rehydrating with decreasing concentrations of ethanol and refixed with methanol. After being blocked with 1% BSA and 0.2% Tween 20, primary antibodies (Abs) directed against XO (mouse monoclonal Ab 145.4, 1:50; Lab Vision) or nitrotyrosine (rabbit polyclonal Ab, 1:200; Upstate Biotechnology) were incubated with sections for 60 min at 25°C. Bound Ab was detected with Alexa 594-conjugated goat anti-mouse IgG (1:100, Molecular Probes) to detect XO or Alexa 488-conjugated goat and rabbit (1:300, Molecular Probes) to detect nitrotyrosine. Nuclei were counterstained with Hoechst 33258 (3 µg/ml, Sigma). Images were obtained under a Nikon Eclipse TE 200 microscope with FITC, Texas red, and 4',6'-diamidino-2-phenylindole filters. Digital photographs were taken with a Spot Advance camera and software following manufacturers' instructions. Metamorph software (version 6.1; Universal Imaging, Downingtown, PA) was used to perform image analysis of digital photographs to obtain quantitative information concerning the intensity of XO and nitrotyrosine staining. Vascular congestion, as a marker of vaso-occlusion, was quantified by measuring the percent area of H&E-stained lung sections that contained red bloods cells. Red blood cells in digital images of the H&E sections were highlighted and converted to grayscale using Adobe Photoshop (version 7.0, Adobe Systems) followed by measurement of the percent red cell area using Metamorph software.

Endothelial cell culture. Rat pulmonary microvascular endothelial cells (PMVEC) were purchased from VEC Technologies (Rensselaer, NY). The cells, which arrived at passage 1, were expanded in MCDB-131 complete medium up to passage 3, harvested, and then cryopreserved in MCDB-131 complete medium containing 10% DMSO. For experiments, frozen stocks were thawed, transferred to a T75 cm2 flask, maintained until confluent, and then passaged onto the appropriate culture dishes as required for the experimental protocols.

Effects of xanthine/XO stress on stimulated nitrite ± nitrate production and on HSP90 interactions with eNOS. PMVEC nitrite + nitrate production was determined by ozone chemiluminescence using VCl3 as described (38). Complete MCDB-131 medium was diluted 1:20 in MCDB-131 media without serum to make serum-free medium. The PMVEC cultures in 60-mm dishes were serum-starved for 4–6 h in the diluted MCDB-131 medium and then incubated with vehicle or with XO (5 mU) + xanthine (10 µM) for 30 min (37°C). Next, the cultures were washed (3x) with HBSS and then incubated with HBSS containing L-arginine (10 µM) and A-23187 (5 µM) (stimulated activity) for 10 min. The HBSS was removed and saved for nitrite+nitrate analysis, and the cultures were lysed in modified RIPA buffer; cell proteins were processed, and eNOS was immunoprecipitated as described in Western analysis (38).

Statistical analysis. Statistical analysis was by analysis of variance or the Student's t-test. A P value of <0.05 was taken as being significant.


    RESULTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Representative H&E sections reveal that hypoxia, as provided in these experiments, does not induce histological evidence of vaso-occlusion in the lungs of control mice (Fig. 1, C and F). However, acute periods of hypoxia markedly increased lung injury in the SCD and heterozygote SCD mice relative to controls (Fig. 1). Under normoxic conditions, lungs from SCD mice contained focal areas of increased vascular congestion that increased in both the total area of involvement and the size of vessels affected after hypoxia (Fig. 1, A vs. D). Heterozygote SCD mice appeared slightly more susceptible to hypoxia-induced lung injury than control mice based on the number of sites of vascular congestion (Fig. 1, E vs. F). Yet heterozygote SCD mice did not appear to be as susceptible as SCD mice (Fig. 1, E vs. D). The percent area of the lung that contained red bloods cells is shown in Table 1, demonstrating a fourfold increase in vascular congestion in the lungs of SCD mice following exposure to hypoxia.



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Fig. 1. Increased vascular congestion in lungs from sickle and heterozygote sickle cell disease (SCD) mice after exposure to hypoxia. Sickle mice (m{alpha}-/-, m{beta}-/-, Tg[h{alpha}{beta}S]; sickle: A and D), heterozygote SCD mice (m{alpha}-/-, m{beta}+/-, Tg[h{alpha}{beta}S]; heterozygote: B and E) and wild-type C57BL/6 mice (control: C and F) were subjected to normoxia (21% FIO2, A–C) or hypoxia (8% FIO2, D–F) for 60 min followed by immediate perfusion and dissection of the lungs as described in METHODS. One portion of the lungs was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin-eosin. The photomicrographs were taken with a x40 objective and are representative of the 6 experimental groups. Arrows, vascular congestion in venules; *microvascular/capillary vascular congestion.

 

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Table 1. Quantitation of RBC congestion (Fig. 1) and XO/XOR (Fig. 2) and nitrotyrosine (Fig. 3) immunofluorescence

 



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Fig. 2. Increased xanthine oxidase (XO) in lung tissue from sickle and heterozygote SCD mice after exposure to hypoxia. Sickle mice (A and D), heterozygote SCD mice (B and E), and wild-type C57BL/6 mice (C and F) were subjected to normoxia (A–C) or hypoxia (D–F), and lungs were isolated as described in Fig. 1. Unstained lung sections were incubated with mouse MAb 145.4 directed against XO, and bound antibody was detected with Alexa 594-conjugated goat anti-mouse IgG as described in METHODS. Nuclei were counterstained with Hoechst reagent. Images were obtained under a Nikon Eclipse TE 200 microscope with FITC, Texas red, and 4',6'-diamidino-2-phenylindole filters. Digital photographs were taken with a x40 object with Spot Advance camera and software. Under normoxic conditions, lung sections of SCD mice contained patchy areas of intense XO staining compared with other areas of the same lung (A inset vs. A).

 


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Fig. 3. Increased nitrotyrosine levels in lung tissue from all groups of mice following exposure to hypoxia. Sickle mice (A and D), heterozygote SCD mice (B and E), and wild-type C57BL/6 mice (C and F) were subjected to normoxia (A–C) or hypoxia (D–F), and lungs were isolated as described in Fig. 1. Unstained lung sections were incubated with rabbit polyclonal antibody directed against nitrotyrosine, and bound antibody was detected with Alexa 488-conjugated goat and-rabbit IgG as described in METHODS. Nuclei were counterstained with Hoechst reagent. Images were obtained as in Fig. 2.

 
Under normoxic conditions, lung sections of SCD mice contained patchy areas of intense XO staining that were not seen in the lung sections of heterozygote SCD or control mice (Fig. 2A inset vs. 2, A–C). These observations suggest that under usual physiological conditions, SCD mice likely experience vascular injury that increases XO release as proposed by Aslan et al. (2). After 60 min of hypoxia, substantial increases in XO staining were detected throughout the lungs of SCD mice (Fig. 2D), and focal areas of mildly increased XO staining were seen in the lung sections of heterozygote SCD mice (Fig. 2E). In contrast, low levels of XO were detected in the lung sections of control mice under normoxic or hypoxic challenges (Fig. 2, C and F). Image analysis of the sections reveals that hypoxia increased the intensity of XO staining by approximately twofold in SCD mice (Table 1).

Under normoxic conditions, high levels of nitrotyrosine were detected in sections from SCD mice (Fig. 3A) compared with the low levels in sections from heterozygote SCD or control mice (Fig. 3, B and C, respectively). Hypoxia markedly increased nitrotyrosine staining in the lung sections from SCD mice (Fig. 3, A vs. D), which is in contrast to the modest increases in sections from heterozygote SCD mice and control (Fig. 3, B and E, and C and F, respectively). Image analysis of the sections reveals that hypoxia increased the intensity of nitrotyrosine staining by ~50% over baseline levels in SCD mice that were already increased 2.5-fold over the levels in control mice (Table 1).

We next measured cGMP levels in the lung because this signaling molecule is often considered an index of ·NO biological activity. Under normoxic conditions, the content of cGMP in lungs of SCD mice was increased compared with the content in lungs of heterozygote SCD and control mice (Fig. 4). Hypoxia reduced the content of cGMP in the lungs of all groups to essentially the same level.



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Fig. 4. Effects of hypoxia on cGMP content in lung tissue from all groups of mice. Control (C57BL/6), SCD, and heterozygote (Het) SCD mice were subjected to normoxia (N) and hypoxia (H), and lungs were isolated as described in METHODS. Portions of lungs were homogenized extracted to isolated cGMP, which was quantified by ELISA. Results are expressed as fmol/mg wet tissue wt. *P < 0.05; **P < 0.025.

 

As previous studies showed that HSP90 modulates eNOS activity in other systems (42, 43, 52), we immunoprecipitated eNOS from lung extracts and determined relative levels of HSP90 association. Under normoxic conditions the amount of HSP90 associated with eNOS in the lungs of SCD and heterozygote SCD mice was decreased compared with the levels of association in controls (Fig. 5, lanes 3 and 5 vs. 1). After hypoxic injury, the relative levels of HSP90 association with eNOS in lungs of SCD and heterozygote mice decreased, while the levels in the lungs of control mice remained relatively unchanged (Fig. 5, lane 1 vs. 2).



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Fig. 5. Effects of hypoxia on the association of heat shock protein (HSP) 90 with endothelial nitric oxide synthase (eNOS) in lung tissue from all groups of mice. Control (C, C57BL/6), SCD, and heterozygote SCD mice were subjected to normoxia and hypoxia, and lungs were isolated as described in METHODS. Portions of the lung were homogenized with modified radioimmunoprecipitation buffer, eNOS was immunoprecipitated (IP) with H32 anti-eNOS antibody, and coprecipitated proteins were detected by immunoblotting (IB) as outlined in METHODS. *P < 0.05; **P < 0.025.

 

Although XO, when it binds to vascular endothelium, can be considered a new enzymatic source of that inactivates endothelium-derived ·NO, XO-derived also increases oxidative stress on the endothelium such that subsequent stimulated ·NO generation may be impaired. To determine whether or not xanthine/XO treatments impaired ·NO generation, we pretreated PMVEC cultures with xanthine and XO and then stimulated them with the receptor-independent agonist A-23187, a calcium ionophore. Xanthine/XO pretreatments decreased stimulated PMVEC nitrite+nitrate production (Fig. 6A) and decreased levels of HSP90 association with eNOS in stimulated PMVEC cultures (Fig. 6B). In unstimulated cultures, pretreatment with xanthine/XO increased HSP90 association with eNOS and decreased phospho-eNOS (S1179) levels (Fig. 6B, lane 2 vs. 1). A-23187 stimulation of cultures pretreated with xanthine/XO decreased HSP90 association with eNOS and phospho-eNOS (S1179), which was in contrast to the marked increase in HSP90 association with eNOS and phospho-eNOS (S1179) that were observed in control PMVEC cultures (Fig. 6B, lane 4 vs. 3, bottom and top panels, respectively). These data are similar to the effects of xanthine/XO pretreatment of bovine coronary endothelial cells, with the exception that HSP90 association in the xanthine/XO pretreated PMVEC is essentially ablated upon stimulation, whereas in xanthine/XO-pretreated bovine aortic endothelial cells, HSP90 association with eNOS is attenuated upon stimulation (data not shown). Together, these findings suggest that xanthine/XO pretreatments increase oxidative stress, which in turn, alters critical cellular mechanisms governing eNOS activation.



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Fig. 6. Effects of XO on nitric oxide (·NO) and eNOS activation in pulmonary microvascular endothelial cell (PMVEC) cultures. A: effects on ·NO production. This bar graph shows the effects of effects of pretreating confluent rat PMVECs with xanthine (X, 10 µM) and XO (5 mU/ml) on basal and stimulated (A-23187, 5 µM, 10 min) ·NO production. Under basal conditions X/XO pretreatment decreased nitrite+nitrate accumulation in the HBSS assay buffer. Stimulation with the receptor-independent agonist A-23187 increases decreased nitrite+nitrate accumulation in controls, whereas X/XO pretreatment decreased nitrite+nitrate accumulation compared with stimulated controls (**P < 0.025, n = 3). B: effects on phosphorylation of eNOS (S1179) and HSP90 association. These immunoblots show the effects of pretreating confluent rat PMVECs with X and XO on HSP90 interactions with eNOS and the phosphorylation state of eNOS (p-eNOS S1179) under basal and A-23187-stimulated conditions as in A. IBs for p-eNOS, HSP90, and eNOS on eNOS IPs show that under basal conditions X/XO pretreatment increases HSP90 interactions with eNOS but decrease p-eNOS levels. Stimulation with the receptor-independent agonist A-23187 decreases HSP90 interactions with eNOS and decreases p-eNOS levels compared with stimulated controls (lane 4 vs. 3). Autoradiograms are representative of at least 3 independent analyses.

 


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study shows that SCD in mice increases susceptibility to hypoxia-induced pulmonary injury as evidenced by marked increases in vascular congestion. Histology confirms that even under normoxic conditions, lungs from SCD mice contain small regions of red cell congestion and higher levels of XO and nitrotyrosine than heterozygote SCD mice or control mice. Hypoxia increases red cell congestion, XO, and nitrotyrosine in the lungs of SCD mice to a much greater extent than in the lungs of heterozygote SCD and control mice. As might be expected, SCD mice were more susceptible than heterozygote SCD mice, which were more susceptible than control mice, to hypoxic injury. Although cGMP content in the lungs of SCD mice was increased under normoxic conditions, hypoxia decreased cGMP in the lungs of all mice to essentially the same level. Under normoxic conditions low levels of HSP90 association with eNOS were observed in the lungs of SCD and heterozygote SCD mice compared with control mice. Hypoxia decreased the association of HSP90 with eNOS in lungs from SCD and heterozygote SCD but not control mice. Although XO generates , which consumes ·NO, findings here demonstrate that the oxidative stress induced by XO can, in turn, decrease HSP90 interactions with eNOS in a way that subsequently impairs stimulated ·NO production.

In the present study, we used red cell congestion to assess pulmonary injury, taking not only the number but also the size of the sites into consideration, with larger vessels more commonly involved following hypoxia. Morphometric analysis assessing the percentage of the lung section area containing red cells confirms that SCD mice had preexisting lung pathology under normoxic conditions and that hypoxia induced marked increases in red cell congestion compared with the lungs of control and heterozygote SCD mice. Because the lungs of the mice were well perfused in situ before extraction, these sites likely represent vaso-occlusion, a common complication of patients with SCD whose pathogenesis is poorly understood.

Another index of tissue injury is XO, where plasma levels directly correlate with ischemic injury and liver levels inversely correlate with plasma levels in SCD (2). The importance of an increase in plasma XO to vascular function is that the endothelium avidly binds XO thereby establishing local regions of robust generation in vascular beds that are often quite distal from the original site of ischemic injury (1, 56). Image analysis of fluorescent intensities as published by Aslan et al. (2) revealed that the content of XO in lungs from SCD mice was increased compared with controls and heterozygote SCD mice. These observations are consistent with a report showing that SCD increases XO on the endothelium of aortas (2) and a recent report from our laboratory showing that treating SCD mice with L-4F, an apo-A-1 mimetic, improves endothelium- and eNOS-dependent vasodilation (40). In this study we observed that L-4F appeared to increase XO levels in the liver while decreasing XO on pulmonary endothelium of small arterioles (40). Interestingly, XO content in lungs from heterozygote SCD and control mice was not increased appreciably, in contrast to the marked increases in lungs from SCD mice. On the basis of our previous report (40) and the report by Aslan et al. (2), it is probable that the mechanism by which hypoxia increases XO staining in the lungs of SCD mice includes release from ischemic tissues. Because increased conversion of xanthine dehydrogenase to XO during hypoxia has been described in both the liver and the kidney in other murine models of SCD (29, 37), it is possible that the source of increased XO in the pulmonary vessels may also be derived from other organ sources. Regardless of where XO is derived, our findings are consistent with the observations that lungs from SCD mice are more susceptible to ischemic injury than lungs from heterozygote SCD and control mice.

The mechanism by which tyrosine nitration occurs is a source of some controversy (12, 22, 31, 46, 54). Although XO is capable of generating , which can react with ·NO to form peroxynitrite (46), it is also important to appreciate the role of vascular PMN activation, margination, and diapedesis nitration of tyrosine in vascular tissues (4, 5). When XO generates , it also produces measurable quantities of hydrogen peroxide that can, in the presence of heme containing peroxidases and nitrite, generate a nitrating species (54). Together, this information suggests that tyrosine can be nitrated in SCD by peroxidase- as well as peroxynitrite-dependent mechanisms. As both mechanisms play critical roles in lung injury, it seems logical that treatments or therapies aimed at preventing or limiting oxidative injury should target both XO and peroxidase.

Further evidence that SCD increases susceptibility of lungs to ischemic injury comes from nitrotyrosine studies. Image analysis of fluorescent intensity reveals that that hypoxia increased nitrotyrosine more notably in lungs from SCD mice than in lungs from heterozygote SCD or control mice. These findings are consistent with the observations that the lungs of SCD mice contain more sites of vaso-occlusion under normoxic conditions and after hypoxic injury than heterozygote SCD or control mice. Thus nitrotyrosine levels appear to correlate directly with red blood cell congestion and susceptibility to hypoxic injury in SCD.

Just as nitrotyrosine is a biochemical marker of oxidative stress (6), cGMP is often considered a physiological index of ·NO activity (43, 49). Logically, nitrotyrosine should increase under conditions of ·NO-derived oxidative stress, whereas cGMP should decrease. Although from XO bound to vascular endothelium is reported to be one of the major mechanisms by which ·NO is consumed in SCD (2), under normoxic conditions we observed that the highest cGMP content was found in lungs from SCD mice rather than control or heterozygote SCD mice. Even though such findings run counter to the idea that SCD increases ·NO consumption, they are consistent with a report by Nath et al. (35) showing that aortas isolated from SCD mice contained higher levels of cGMP than controls. A possible explanation for this paradoxical increase in cGMP in SCD mice where ·NO consumption is increased (2, 45) is the fact that guanylyl cyclase can also be activated by compound I, which is formed when hydrogen peroxide reacts with catalase (10, 58). Regardless of how cGMP levels are increased in SCD mice, hypoxia reduced cGMP content in all groups to essentially the same level. These data demonstrate that hypoxia shifts the balance of ·NO and and that the greatest shifts occur in SCD mice. Such shifts in ·NO balance seem to correlate with severity of vaso-occlusion in the lungs of SCD mice after hypoxia.

Recent reports suggest that HSP90 association with eNOS is one of the major mechanisms governing the balance of ·NO and from eNOS in vascular endothelium (33, 43, 52). Upon stimulation, HSP90 associates with eNOS, thereby increasing the efficiency of eNOS phosphorylation at S1179, which, in turn, increases the flux of electrons through the reductase domain of eNOS (14, 17, 21). An increase in eNOS phosphorylation at S1179 by itself, however, should not be interpreted as an increase ·NO production, in that, without the full support of HSP90, phospho-eNOS (S1179) also generates (39, 42). Additional evidence that HSP90 plays a physical role in modulating NOS function comes from in vitro EPR studies showing that native HSP90 decreases neuronal NOS generation, whereas boiled chaperone does not (50). With this information as background we investigated the effects of SCD and hypoxia on HSP90 interactions with eNOS in lung homogenates. We found that the levels of HSP90 associated with eNOS in control mice were significantly higher than the levels of association in heterozygote SCD and SCD mice and that hypoxia decreased the association in heterozygote SCD and SCD mice but not control mice. These observations parallel the increases in vaso-occlusion and staining of XO and nitrotyrosine that were greater in SCD mice than the heterozygote SCD and control mice after hypoxic injury. Findings here are consistent with previous reports in isolated hearts showing that enhanced HSP90 association with eNOS was cardioprotective (48). Together, these findings suggest that maintaining a high level of HSP90 association with eNOS affords greater lung protection.

Unlike the heart, which is predominantly cardiomyocytes by mass, the lung contains a wide variety of cells, epithelial, endothelial, smooth muscle, and mast cells. By mass, no one cell type dominates. To determine the effects of xanthine/XO on protein-protein interactions in a relevant endothelial cell population, we pretreated rat PMVEC with xanthine/XO before stimulation and then measured changes in stimulated nitrite + nitrate production and the association of HSP90 with eNOS. Pretreatment of PMVEC cultures with xanthine/XO decreased stimulated nitrite + nitrate production concomitantly with a decrease in the association of HSP90 with eNOS and phosphorylation of eNOS at S1179. These in vitro findings are consistent with in vivo data that the association of HSP90 with eNOS was decreased in the lungs from SCD and heterozygote SCD mice but not control mice. The observed decrease in the association of HSP90 with eNOS and in phospho-eNOS (S1179) levels in the stimulated cells is consistent with the hypothesis that both events are required to increase nitrite + nitrate production (17, 21). Our findings that XO impairs eNOS-dependent ·NO generation complement and extend a previous report showing that acute periods of hypoxia decreased HSP90 association with eNOS and impaired ·NO generation in pulmonary endothelial cells by a calpain-dependent mechanism (53). Our data suggest that the oxidative stress associated with XO binding to PMVEC represents another mechanism for decreasing eNOS-dependent ·NO and thereby worsens vaso-occlusion in SCD mice. In this respect, it might be possible to decrease pulmonary injury in acute chest syndrome by administration of an inhibitor of XO activity while providing oxygen.

The Berkeley murine model of SCD used in this study also expresses mild to moderate {beta}-thalassemia (13, 41). Although thalassemia decreases intracellular Hb S sickling, Hb S-{beta}0-thalassemia remains a clinically severe form of SCD in humans, indicating that {beta}0-thalassemia does not ameliorate many of the pathologies associated with SCD in humans. Red blood cells from individuals with {beta}-thalassemias have evidence of elevated in vivo oxidation, and so the combination of SCD and mild to moderate {beta}-thalassemia phenotype could potentially exaggerate susceptibility to oxidative damage. The fact that our findings confirm those in a different murine model that may have improved {alpha}/{beta}-globin ratio (2), suggests that our findings in the SCD mice are in large part due to Hb S. Because the heterozygote control mice also have thalassemia, the contribution of thalassemia can also be examined by comparing the heterozygote mice to the sickle mice.

A comment concerning the heterozygous SCD mice as a model of human sickle cell trait disease is in order. These mice have been found to segregate into two subpopulations containing low and high proportions of Hb S that are due to the presence of one and two copies of the human h{alpha}{beta}S transgenes, respectively (36). For this study, we used mice with only one copy of the human transgene for the heterozygote control group. Mice with only one copy of the Hb S transgene appear to be more sensitive to hypoxia due to acute sequestration of erythrocytes in the spleen, liver, and heart (36). Interestingly, these noted pathologies did not appear to be due to Hb S polymerization but possibly to a low oxygen hemoglobin oxygen affinity, a rare event in human disease (36). In addition, within different groups of heterozygote SCD mice, the responses to a standardized hypoxic challenge varied, suggesting a genetic component with each strain may also contribute to the syndrome (36). In this context, it should be noted that the heterozygote SCD mice used in the present study (from a colony established at MCW) all survived the hypoxic challenge and demonstrated an increase in red blood cell congestion in the lung compared with control mice, but not compared with the SCD mice.

In conclusion, SCD increases susceptibility to hypoxic injury by increasing XO release from ischemic tissues. Observations here are consistent with the fact that XO binding to PMVEC decreases ·NO activity by scavenging with and, by increasing oxidative stress on the endothelium such that when the cells are subsequently stimulated, eNOS-dependent ·NO generation is impaired. The cellular mechanisms mediating this impaired ·NO generation appear to be a decrease in HSP90 association with eNOS and a decrease in eNOS phosphorylation at S1179. Future studies investigating how XO alters HSP90-dependent signaling and eNOS phosphorylation may lead to new treatment modalities for preserving ·NO generation and preventing lung injury in SCD.


    ACKNOWLEDGMENTS
 
The authors thank Anthony Hudetz for helpful discussions.

GRANTS

This work was supported in part by Marie Z. Uihlein Endowed Chair award (to K. T. Oldham) from Children's Hospital Foundation (Milwaukee, WI), National Heart, Lung, and Blood Institute Grants HL-61417, HL-71214, PPO HL-68769 (to K. A. Pritchard), HL-44612 (to C. A. Hillery), and HL-31579 (to N. Mohandas).


    FOOTNOTES
 

Address for reprint requests and other correspondence: K. A. Pritchard, Jr., Medical College of Wisconsin, Div. of Pediatric Surgery, Cardiovascular Center M4060, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (E-mail: kpritch{at}mcw.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.

* K. T. Oldham and C. A. Hillery contributed equally to direction of the investigation. Back


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