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Am J Physiol Lung Cell Mol Physiol 291: L457-L465, 2006. First published April 7, 2006; doi:10.1152/ajplung.00462.2005
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Protective effects of S-nitrosoalbumin on lung injury induced by hypoxia-reoxygenation in mouse model of sickle cell disease

Lucia de Franceschi,1,* Giorgio Malpeli,2,* Aldo Scarpa,2 Anne Janin,3 Eva Maria Muchitsch,4 Paola Roncada,5 Christhope Leboeuf,3 Roberto Corrocher,1 Yves Beuzard,6 and Carlo Brugnara7

1Section of Internal Medicine, Department of Clinical and Experimental Medicine, and 2Section of Anatomic Pathology, Department of Pathology, University of Verona, Verona, Italy; 3Laboratorie de Pathologie, Institut National de la Santé et de la Recherche Médicale INSERM 02 20, Hopital Saint Louis, Paris, France; 4Department of Experimental Pharmacology, Baxter Bioscience, Vienna, Austria; 5Istituto Italiano Spallanzani, Milan, Italy; 6Laboratory of Hematopoietic Gene Therapy, INSERM U733, Centre Hayem, Hopital Saint Louis, Paris, France; and 7Department of Laboratory Medicine and Department of Pathology, Children's Hospital, Harvard Medical School, Boston, Massachusetts

Submitted 3 November 2005 ; accepted in final form 23 March 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Nitric oxide (NO) is a potential new therapeutic agent for sickle cell disease (SCD). We investigated the effects of NO donor on hypoxia-induced acute lung injury that occurs when transgenic sickle cell SAD mice are exposed to chronic hypoxia, a model for lung vasoocclusive sickle cell events. In wild-type and SAD mice, intraperitoneal injection of S-nitrosoalbumin (NO-Alb) produced no significant hematologic changes under room air conditions, whereas it induced mild temporary hypotension and inhibition of platelet aggregation. NO-Alb administration (300 mg/kg ip twice a day, equivalent to 7.5 µM NO) in wild-type and SAD mice exposed to 46 h of hypoxia (8% oxygen) followed by 2 h of normoxia resulted in 1) reduction of the hypoxia-induced increase in blood neutrophil count, 2) prevention of hypoxia-induced increased IL-6 and IL-1beta levels in bronchoalveolar lavage, 3) reduction of the lung injury induced by hypoxia-reoxygenation, 4) prevention of thrombus formation, and 5) prevention of hypoxia-induced increase of lung matrix metalloproteinase-9 gene expression. These effects provide new insights into the possible use of NO-Alb in the treatment of acute lung injury in SCD.

transgenic sickle cell SAD mice; hypoxic lung injury; nitric oxide; nitrite

NITRIC OXIDE (NO) is a potent vasodilator, an inhibitor of vascular remodeling, and a modulator of the cascade of events involved in leukocyte, platelet, and endothelial activation (8,9,21). Sickle cell disease patients show reduced NO metabolites during either vasoocclusive crisis associated with severe pain or acute chest syndrome as well as a decreased exhaled NO, suggesting a possible role of a relative NO lung deficiency in the pathogenesis of sickle cell-related pulmonary complications (25). Although the mechanisms of action of inhaled NO are still under investigation, its therapeutic role in sickle cell pain crisis has been suggested in a placebo-controlled trial (42). Sickle cell vasoocclusive events involve complex interactions between different cell types, including dense, dehydrated sickle cells, reticulocytes, abnormally activated endothelial cells, leukocytes, platelets, and plasma factors (11,22,23,30). Molecular and cellular evidence of the therapeutic effects of inhaled NO in treatment of acute sickle cell-related lung injury has been described in transgenic sickle cell SAD mice exposed to hypoxia-reoxygenation (4).

NO physiologically circulates also as S-mononitrosylated albumin (NO-Alb) nitrite, S-nitrosoamines, iron-nitrosyls, or nitrated lipids (37). However, the contribution to NO homeostasis of each one of these NO-derived species is still under investigation (6,37,41). Recent data showing the protective role of nitrite in heart and liver ischemic tissue damage support the hypothesis that even NO derivates may play beneficial effects in tissues where NO is deficient (6,37,41).

The need for simple and efficacious therapeutic tools in treatment of acute ischemic events involving the lungs such as acute respiratory distress syndrome or major thorax surgery has prompted the evaluation of NO-Alb and to a lesser extent human serum albumin (sh-Alb) as radical scavengers (15,19,24,28,40). NO-Alb and polynitrosylated albumin (PNA) have been evaluated in in vitro and in vivo animal models of ischemic tissue injury (2,7,10,15,18,26,28,31,35). Because lungs of transgenic sickle cell mice exposed to hypoxia-reoxygenation are characterized by relative NO reduction and microvascular dysfunction, requiring an increase in both NO delivery and colloid osmotic pressure and effective NO delivery for this correction, we hypothesized that NO-Alb may provide two distinct beneficial effects, representing a possible new therapeutic tool for acute pulmonary sickle cell events.

In this study, we evaluated NO-Alb, nitrosylated at position 34, under normoxic conditions in wild-type and transgenic sickle cell SAD mice. We also investigated the effects of NO-Alb during acute vasoocclusive sickle cell events occurring when SAD mice are exposed to chronic hypoxia, based on our previous studies (4) showing this to be a pertinent model for lung vasoocclusive sickle cell events.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Transgenic Hbbs/Hbbs SAD mice and C57B6/6J control (wild type) mice were used for these studies (female and male, 20–25 g in body wt) (4,5,33,34). Mice were handled according to the Institutional and National guidelines for the care and use of laboratory animals. The study protocol was reviewed and approved by University of Verona Animal Care and Use Committee and by the Ministero della Salute (Italy).

Generation of NO-Alb

The starting material for generating NO-Alb was Cohn fractionation V. After dissolution and filtration caprylate was added for stabilization. The liberation of the SH- group in cysteine 34 of albumin was achieved by the addition of a 20-fold molar excess of L-cysteine at pH 6.1. Afterwards, NaNO2 was added at a concentration of 21 mol reagent/mol protein under acidic conditions. This high concentration is necessary as residual L-cysteine in the solution will also react with NaNO2. After nitrosation, the solution was neutralized with 1 M NaOH, diafiltered against water to remove NO-cysteine and residual NaNO2, and concentrated to 10%. NO-Alb was then pasteurized in the presence of stabilizers 0.08 mM caprylate and 0.08 mM acetyltryptophan and lyophilized. The measured S-nitrosation in the final container was ~85%.

Normoxia Studies

Single-dose hematologic studies. Wild-type (n = 4) and SAD (n = 4) mice were treated with NO-Alb or with sh-Alb as placebo solution, with one single dose of 100, 300, or 1,000 mg/kg injected intraperitoneally. Hematologic parameters were measured as previously described (5,33,34). Methemoglobin (MetHb) level was calculated from the optical spectrum recorded at the end of the oxygen equilibrium measurements (4). Hematocrit (Hct), hemoglobin (Hb), MetHb, and reticulocyte count were measured at baseline and 30, 60, and 120 min after drug administration.

Multiple-dose hematologic studies. The same parameters described above were evaluated at baseline and after 4-day administration of either sh-Alb or NO-Alb at a dose of 300 mg/kg (equal to 7 µM NO/kg) twice a day intraperitoneally.

Measurements of platelet aggregation in mice treated with NO-Alb. NO-Alb or sh-Alb solutions were tested in wild-type mice (n = 101 divided as follows: 44 in the NO-Alb group and 57 in the sh-Alb group) with one single dose of 300 mg/kg intravenously (2,7,18). Mice were anesthetized by intraperitoneal injection of 80 mg/kg pentobarbital sodium; blood was collected via the inferior vena cava before and 6, 10, 15, 30, and 60 min after NO-Alb or sh-Alb administration. Aggregation in platelet-rich plasma (PRP) was measured with a Chrono-Log whole blood aggregometer (model 570-VS, Coulter Electronics) by the turbidimetric method. PRP was prepared by centrifugation of citrated blood (collected into 3.8% trisodium citrate at a ratio of 1:9) at 120 g for 6 min. PRP from three or four mice was pooled to obtain the volume necessary for aggregation assay. A 2.5-µl mixture of 1.01 µM ADP and 2.56 µg/ml collagen was added to 245 µl of PRP. At each time point one to four PRP samples were analyzed. Measurements were carried out either in duplicate or in triplicate. Aggregation was expressed as the initial aggregation rate (%/min) comparing the two treatment groups (17).

Measurements of cardiovascular parameters after intraperitoneal or intravenous injection of NO-Alb in mice. The hemodynamic effects of NO-Alb were evaluated by measuring mean arterial blood pressure (MAP) and heart rate (HR). Mice were anesthetized by intraperitoneal injection of pentobarbital sodium (80 mg/kg). The trachea was intubated, and a catheter was firmly placed into the carotid artery. After baseline pressure and HR values were obtained, NO-Alb or sh-Alb was injected. In the single-dose experiment, pulsatile arterial pressure and HR were recorded before and continuously after injection up to 15 min (intravenous route) or 30 min after injection (intraperitoneal route). Systolic and diastolic blood pressures were recorded on a polygraph with an electromechanical pressure transducer and used to calculate MAP. HR was measured from the pulse waves. Changes in blood pressure and HR were calculated as a percentage of the individual baseline values ({Delta}%): NO-Alb or sh-Alb was tested in wild-type mice (n = 4–7/group) with one single intraperitoneal dose of either 300 or 1,000 mg/kg. In a second set of experiments, repeated doses of 300 mg/kg were administered intraperitoneally twice a day for 4 days. Effects on MAP and HR after intravenous injection of 300 or 1,000 mg/kg NO-Alb were assessed in single mice only for direct comparison of the intravenous and intraperitoneal routes of administration. In the repeated-dose experiments, cardiovascular measurements took place after repeated administration of NO-Alb or the albumin placebo formulation buffer on day 4, immediately before injection of the last dose and continuously for 1 h afterwards.

Hypoxia Studies

Wild-type and SAD mice were divided into groups of six mice each. One group from each strain was used to determine baseline parameters in the normoxic state; the other groups were exposed to 46 h of hypoxia (8% oxygen) followed by 2 h of normoxia (4). One group from each strain was left untreated during hypoxia; of the remaining two hypoxic SAD mouse groups one group was treated with sh-Alb at a dosage of 300 mg/kg twice a day intraperitoneally and the other with NO-Alb at a dosage of 300 mg (equal to 7.5 µM NO)/kg twice a day intraperitoneally. Treatment was administrated 24 h before hypoxia and during hypoxia.

As we previously reported (4,5), the gas mixture for hypoxia was blended with separately regulated and calibrated mass flowmeters (RDM 280, Airliquid, Paris-La-Defense, France), maintaining a gas flow rate constant of ~1.5 l/min through a 5-l exposure cage. Inspired O2 fraction was kept constant at 0.21 in baseline experiments and 0.08 in hypoxia experiments with a polarographic electrode (oxygen analyzer, Ohmeda 5120, Englewood, CO). Mice were given free access to water and food (4,5).

Bronchoalveolar Lavage Fluid and Cytokine Content

Bronchoalveolar lavage (BAL) fluids were collected by instilling and withdrawing 500 µl of sterile PBS four times via the intratracheal cannula. Cells were recovered by centrifugation and counted by microcytometry. The percentage of neutrophils was determined by cytospin centrifugation, fixation, and staining (38,44). The remaining BAL samples were centrifuged at 1,500 g for 10 min at 4°C. The supernatants were used to measure the cytokines TNF-{alpha}, IL-1beta, IL-6, and IL-10 by commercial ELISA (R&D Systems Europe, Abingdon, UK; Amersham, Oxford, UK), according to manufacturer's instructions (4,38,44).

Lung Tissue Histology

One lung was immediately frozen in liquid nitrogen, and the other was fixed in formalin and embedded in paraffin. Multiple (at least 5) 3-µm whole mount sections were obtained for each paraffin-embedded lung and stained with hematoxylin and eosin, Masson's trichrome, and May-Grünwald-Giemsa. Morphological analysis was performed in a blinded manner and independently by two pathologists and consisted of the evaluation of the tissue architecture and changes induced by hypoxia and/or treatment regimens. Interobserver difference was <5%. Pathological criteria for morphological assessment of pulmonary vessels and bronchi are detailed inTable 1. Vessels were evaluated for the presence and entity of congestion and thrombi, whereas bronchi were evaluated for the presence and entity of mucus and inflammatory cell infiltrate. The latter quantification was expressed as the mean number of cells per field at magnification 250x, resulting from the analysis of at least four different fields on each hematoxylin and eosin-stained whole lung section on an Olympus Provis AX 70 microscope equipped with a wide-field eyepiece no. 26.5 (4).


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  		Table 1. Effects of NO-Alb and sh-Alb on hematologic parameters in wild-type and transgenic sickle cell SAD mice exposed to hypoxia-reoxygenation

 
Gene Expression Analysis of Lung Tissue

Total RNA was isolated from frozen lung tissues by the cesium chloride gradient method. One microgram of total RNA was converted to cDNA with random primers with a first-strand cDNA synthesis kit (AMV; Roche Diagnostic, Mannheim, Germany). Real-time quantitative RT-PCR analysis was performed on an ABI Prism 7000 SDS (Applied Biosystems, Foster City, CA) with the SYBR Green I dye contained in the SYBR Green Master Mix (Applied Biosystems) as a probe. All PCR reactions contained 1x Master Mix, each specific primer at 200 nM, and 5 ng of cDNA (total RNA equivalent) in a 25-µl total volume. Thermal cycling included an initial incubation at 95°C for 10 min and then 45 cycles of 15 s at 95°C for denaturation and 1 min at 60°C for extension. After each run, PCR amplification products were checked by melting curves and electrophoresis. Sequences of oligonucleotide primers of Actb, Ace, Nfe2l2, Hspcb, Hspca, and Cxcl2 genes were previously published (4). Primer sequences for Mmp9 were the following: forward 5'-CAGGGATGGGCGCCTC-3' and reverse 5'-GAACAGGCTGTACCCTTGGTC-3'. All samples were amplified in triplicate, and the mean of the threshold cycle was used for further calculations. The probe signal was normalized to an internal reference, and the expression level of genes was calculated by relative quantification using Actb (actin, beta) transcript level as endogenous reference. Expression data were analyzed following the comparative method as indicated in Applied Biosystems User Bulletin no. 2.

Statistical Analysis for Soluble and Hematologic Parameters

The two-way ANOVA algorithm for repeated measures between treatment schedules was used for data analysis. Differences with P < 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of NO-Alb on Hematologic Parameters, Platelet Aggregation, and Hemodynamic Response Under Ambient Air Conditions in Wild-Type and SAD Mice

We first verified the effects of NO-Alb on hematologic parameters with a dose-response curve under ambient air conditions. Sh-Alb and NO-Alb at doses of 100, 300, and 1,000 mg/kg were administered intraperitoneally in a single dose to wild-type and transgenic sickle cell SAD mice. No significant changes in Hct, Hb, or MetHb could be detected at 30, 60, and 120 min after NO-Alb or sh-Alb administration (data not shown).

On the basis of the inhibitory effects of NO on platelet aggregation described both in vitro and in vivo, we assessed platelet aggregation after a single intravenous injection of 300 mg/kg of either NO-Alb or sh-Alb in wild-type control mice. A marked inhibition of platelet aggregation was detected between 6 and 30 min after the administration of NO-Alb, with no significant change with sh-Alb (Fig. 1).


Figure 1
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  		Fig. 1. Effect of S-nitrosoalbumin (NO-Alb) on platelet aggregation measured in mouse platelet-rich plasma (PRP). A 2.5-µl mixture of 1.01 µM ADP and 2.56 µg/ml collagen was added to 245 µl of PRP. At each time point 1–4 PRP samples were analyzed. Measurements were carried out either in duplicate or in triplicate. Platelet aggregation is presented as maximal aggregation rate in %/min over time. Wild-type mice were injected intravenously with 300 mg/kg of either NO-Alb or human serum albumin (sh-Alb).

 
We next evaluated the possible cardiovascular/hemodynamic effects of NO-Alb administered either intraperitoneally or intravenously. With intraperitoneal NO-Alb administration (300–1,000 mg/kg), a slight, not statistically significant decrease in MAP was observed (MAP –11.6 and –10.7 {Delta}%, respectively;Fig. 2A). A similarly minor, not statistically significant increase in MAP up to +10.8 {Delta}% was observed with single-dose intraperitoneal administration of sh-Alb (300–1,000 mg/kg). There was a slight, not statistically significant drop in HR 1 min after injection in all mouse groups, with a maximum decrease of –10.2 and –4.6 {Delta}% in mice treated with 300 and 1,000 mg/kg NO-Alb, respectively, and of –5.0 and –5.5 {Delta}% in animals treated with sh-Alb (Fig. 2B). With intravenous NO-Alb infusion as a single dose of 300 or 1,000 mg/kg, an immediate transient drop in MAP (–49.7 and –33.5 {Delta}% within 1 min after injection) was observed, followed by a gradual return toward baseline within the 15-min observation period (–10.9 and –18.3{Delta}%). Administration of 1,000 mg/kg of sh-Alb produced no changes in blood pressure (data not shown). Intravenous injection of either NO-Alb or sh-Alb produced no measurable changes in HR (data not shown).


Figure 2
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  		Fig. 2. A and B: effects of single intraperitoneal injection of either NO-Alb or sh-Alb in wild-type mice (Data are presented as means ± SD = 4–7 mice/group): mice were injected intraperitoneally with either 300 ({lozenge}) or 1,000 ({blacklozenge}) mg/kg NO-Alb or 300 ({circ}) or 1,000 (bullet) mg/kg sh-Alb placebo solution. Mean arterial blood pressure (MAP; A) and heart rate (HR; B) with % changes from baseline ({Delta}%) plotted vs. time are shown. C and D: effects of repeated intraperitoneal administration of NO-Alb or sh-Alb in wild-type mice (n = 10–11/group): mice were injected intraperitoneally with 300 mg/kg NO-Alb ({lozenge}) or sh-Alb ({circ}). MAP (C) and HR (D) with % changes from baseline ({Delta}%) plotted vs. time are shown.

 
To evaluate the effects of long-term administration of NO-Alb on hematologic or hemodynamic parameters, wild-type and SAD mice were treated with NO-Alb at a dose of 300 mg/kg twice a day intraperitoneally (equal to 7.5 µM NO/kg) for 4 days. In both wild-type and SAD mice, NO-Alb did not significantly modify Hct, Hb, or reticulocyte count (data not shown). MetHb did not increase significantly in either mouse strain.

MAP and HR slightly, but not significantly, increased with both NO-Alb and sh-Alb long-term treatment, when determined after the last dose on day 4. Maximal increase in MAP was +14.4 and +9.4 {Delta}% and +14.5 and 13.7 {Delta}% in HR for NO-Alb and sh-Alb, respectively (Fig. 2, C and D).

NO-Alb Reduces Hypoxia Proinflammatory Effects in SAD Mice Exposed to Hypoxia-Reoxygenation

We then asked whether, similar to inhaled NO, NO-Alb could also exert beneficial effects on lung injuries in transgenic sickle cell mice exposed to hypoxia-reoxygenation. We previously showed (4) that exposure of transgenic sickle cell SAD mice to 46 h of hypoxia (8% oxygen) followed by 2 h of reoxygenation is a valid model for acute vasoocclusive sickle cell lung injury.

Hematologic parameters of wild-type and SAD mice exposed to hypoxia-reoxygenation and treated with either sh-Alb or NO-Alb are shown inTable 1. No changes in Hct, Hb, MetHb, or reticulocyte count were observed in SAD mice treated with either sh-Alb or NO-Alb. NO-Alb prevented the hypoxia-induced increase in blood neutrophil count (Table 1). A marked reduction in the hypoxia-induced increase of both total leukocyte and neutrophil count in BAL was also observed in SAD mice treated with NO-Alb, whereas SAD mice treated with sh-Alb showed a slight but not significant decrease in BAL neutrophil count (Fig. 3B), suggesting the ability of NO-Alb to modulate the inflammatory response to hypoxia (Table 1).


Figure 3
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  		Fig. 3. A: bronchoalveolar lavage (BAL) total leukocyte count in wild-type and SAD mice exposed to hypoxia-reoxygenation and treatment with either NO-Alb or sh-Alb. B: BAL total neutrophil count in wild-type and SAD mice exposed to hypoxia-reoxygenation and treatment with either NO-Alb or sh-Alb. Data are means ± SD (n = 6 for each group). *P < 0.05 vs. baseline; {circ}P < 0.05 vs. sh-Alb-treated SAD mice.

 
Cytokines have been implicated in pulmonary inflammation and recruitment of inflammatory cells into the alveolar compartment, and as we have previously shown (4), they are modulated in transgenic SAD mice by hypoxia-reoxygenation. We evaluated BAL levels of IL-6, TNF-{alpha}, IL-1beta, and IL-10 in wild-type mice and SAD mice exposed to 46-h hypoxia-reoxygenation with and without NO-Alb treatment (Fig. 4) (4).


Figure 4
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  		Fig. 4. BAL IL-6 (A), IL-1beta (B), IL-10 (C), and TNF-{alpha} (D) levels in wild-type and SAD mice exposed to hypoxia-reoxygenation and treatment with either NO-Alb or sh-Alb. Data are presented as means ± SD. *P < 0.05 vs. baseline; {circ}P < 0.05 vs. sh-Alb-treated SAD mice.

 
BAL IL-6 and IL-1beta levels increased markedly with hypoxia-reoxygenation in both wild-type and SAD mice; similar to observations in SAD mice breathing NO, NO-Alb prevented the hypoxia-induced IL-6 increase (Fig. 4, A and B). BAL IL-10 levels were not significantly modified by hypoxia-reoxygenation in either wild-type or SAD mice (Fig. 4C). As previously reported (4), TNF-{alpha} increases only in the early phase of hypoxia; in fact, no significant changes in BAL TNF-{alpha} levels were present in either wild-type or SAD mice exposed to 46-h hypoxia-reoxygenation (Fig. 4D). These data indicate that NO, slowly released by NO-Alb, effectively decreases proinflammatory cytokines induced by hypoxia in BAL of SAD mice (16,36).

NO-Albumin Treatment Ameliorates SAD Mouse Hypoxia-Related Lung Injury and Normalizes Expression of Hypoxia-Induced Matrix Metalloproteinase-9 in SAD Mice

We then evaluated the effects of NO-Alb and sh-Alb on lung histopathological damage in SAD mice exposed to hypoxia-reoxygenation (Table 2). Hypoxia-reoxygenation resulted in a severe inflammatory response in SAD mice, with an extensive ischemic component, characterized by 1) severe vascular congestion, 2) presence of thrombi in small vessels, 3) presence of mucus filling >50% of the bronchus section area, and 4) increased inflammatory cells, associated with a significant amount of neutrophils.


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  		Table 2. Effects of NO-Alb and sh-Alb on lung pathology of wild-type and transgenic sickle cell SAD mice exposed to hypoxia-reoxygenation

 
Pulmonary vessel congestion and thrombi formation were reduced only in the SAD mouse group treated with NO-Alb (Table 2). Although both sh-Alb and NO-Alb reduced the mucus filling of bronchi, the inflammatory cell infiltrate was significantly reduced only by NO-Alb (Table 2;Fig. 5) (10,26). These data suggest that NO-Alb and, to a lesser extent, albumin reduce lung edema formation, whereas NO-Alb, similarly to inhaled NO, effectively provides protection from thrombus formation and attenuates the cell inflammatory infiltrates induced by hypoxia-reoxygenation events.


Figure 5
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  		Fig. 5. Effects of NO-Alb on lung histopathology of transgenic sickle cell SAD mice exposed to hypoxia-reoxygenation. Panels (same magnification) show that both arterial wall thickness and inflammatory infiltrate are significantly reduced with NO-Alb. Bars, 20 µm.

 
We next examined the expression of matrix metalloproteinase-9 (MMP-9), which was recently suggested to play a crucial role in lung diseases characterized by sustained inflammatory response and extensive alterations of lung extracellular matrix (ECM) (1,12,32,39). Lung matrix Mmp9 gene expression was increased 2.5-fold in SAD mice exposed to 46 h of hypoxia plus 2 h of reoxygenation (Fig. 6). NO-Alb treatment prevented the hypoxia-induced lung Mmp9 gene expression; sh-Alb per se reduced the Mmp9 gene expression in SAD mice exposed to hypoxia (Fig. 6).


Figure 6
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  		Fig. 6. Effect of NO-Alb administration on SAD mice lung gene expression. Bars indicate the relative expression of the indicated genes in SAD mice exposed to normoxia (black bars), 46 h of hypoxia + 2 h of normoxia (dark gray bars), 46 h of hypoxia + 2 h of normoxia and treatment with sh-Alb (light gray bars), or 46 h of hypoxia + 2 h of normoxia and treatment with NO-Alb (open bars). Data are presented as means ± SD of n = 6 mice for each condition. For each gene, the expression levels obtained from the different experimental conditions were normalized relative to normoxic levels.

 
We also evaluated the expression of genes involved in stress response and microvascular remodeling, previously reported to be affected by hypoxia and modulated by inhaled NO. The expression of heat shock protein 1beta and heat shock protein 1{alpha} (Hspcb, Hspca), which are early hypoxia (10 h)-induced stress genes, was unmodified by long-term hypoxia as well as by either sh-Alb or NO-Alb treatment. We had already reported (4) that inhaled NO had no effects on the expression of these genes at 46 h of hypoxia (Fig. 6). Nuclear factor erythroid-derived 2-like 2 (Nfe2l2) messenger RNA levels were slightly increased in SAD mice exposed to hypoxia-reoxygenation and were unaffected by either NO-Alb or sh-Alb treatment. We had previously reported (4) that inhaled NO was able to normalize Nfe2l2 expression after 46 h of hypoxia. Expression of macrophage-inducible protein 2 (Cxcl2), which we have shown to increase in the early phase of hypoxic lung damage (4), was unaffected by NO-Alb and increased by sh-Alb (Fig. 6). In SAD mice, hypoxia-reoxygenation increased Ace gene expression, which was unaffected by NO-Alb treatment, similar to that observed in hypoxic SAD mice breathing NO (4).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Therapeutic strategies to modulate NO homeostasis in sickle cell disease span from inhaled NO to oral L-arginine, which participates in NO metabolism. The mechanisms of therapeutic actions of inhaled NO and NO-related compounds in sickle cell disease are still under investigation. Clinical use of inhaled NO is limited by its high cost and the needs of specialized clinical units for patient treatment. Thus the identification of an effective NO donor to be used in the early phase of acute sickle cell vasoocclusive events may represent an alternative therapeutic strategy to inhaled NO.

Studies in various animal models for acute ischemic organ damage have shown the protective effects of NO-Alb and PNA in treatment of ischemia-reperfusion tissue injury (28,31,40,43). In the present study, we examined the effects of NO-Alb on the acute lung injury induced by hypoxia-reoxygenation in transgenic sickle cell SAD mice. Preliminary data on a transgenic sickle cell mouse model have shown that PNA positively interferes with hypoxia-induced microvascular stasis in dorsal skin chambers and with the activation of endothelial cells in lungs (3,13). However, no data on systemic effects, on BAL cell and cytokine composition, or on lung histopathology have been reported for PNA in transgenic sickle cell mice (3,13,14).

In the present study, NO-Alb reduced the hypoxia-induced increase in blood neutrophil count and reduced inflammatory cell infiltration, in particular neutrophil transmigration, suggesting a possible systemic anti-inflammatory effect (Table 2;Fig. 4) (26).

The pulmonary response to hypoxia is sustained by activated lung macrophages and airway epithelial cells, which modulate various bronchoalveolar cytokines. In SAD mice, we showedpreviously (4) that there is a temporal sequence for BAL cytokine response to hypoxia: TNF-{alpha} may be the early stimulus (4–6 h hypoxia), whereas IL-6 (12–46 h hypoxia) may be the late stimulus. Here we evaluated the BAL cytokines involved in the long-term inflammatory response (late stimulus) in SAD mice exposed to chronic hypoxia (46 h). The hypoxia-induced increase in BAL IL-6 and IL-1beta levels is completely prevented by NO-Alb, suggesting that NO supplementation may influence the release of proinflammatory BAL cytokines (Fig. 4).

NO-Alb reduced the sickle cell-specific lung injury induced by hypoxia-reoxygenation, showing protective effects similar to those observed with inhaled NO in the same mouse model (4). It is further interesting to note that sh-Alb partially attenuates the hypoxic-reoxygenation lung injury in SAD mice, as supported by the reduction in vascular congestion, arterial wall thickness, and mucus filling of bronchi in the albumin-treated SAD mice. It is interesting to note that in SAD mice NO-Alb prevented the formation of thrombi, which may be related to either the interfering effects of NO-Alb on platelet aggregation or just the amelioration of hypoxia-induced lung injury.

The use of sh-Alb has been proposed in acute lung injury and in pulmonary resuscitation, based on its ability to reduce both birth transendothelial hydraulic conductivity and oxidant damage (40). However, there are no conclusive data on the therapeutic role of sh-Alb in acute lung injury.

In transgenic sickle cell SAD mice, hypoxia-reoxygenation induces lung expression of genes involved in stress response as well as microvascular and tissue remodeling (4). Recent studies have proposed a key role for MMPs in both acute and chronic lung damage (1,12,20,32). The MMPs are pivotal enzymes responsible for degradation of ECM components and are involved in tissue remodeling in different lung diseases (1,12,20,32). A close association has been described between lung expression of Mmp9 and early alveolar remodeling in acute lung injury (1,12,20), and Mmp9-knockout mice show decreased acute lung injury. This evidence indicates that the role of MMP-9 is not limited to the ability to break down ECM but is also extended to the modulation of cytokines such as IL-1 and IL-6 as well as to leukocyte migration (27,29,39). In addition, therapies aimed at reducing MMP-9 have been correlated with improved survival of mice exposed to acute lung injury (29). Excessive expression of MMPs and the related abnormal ECM deposition may play a role in the pathogenesis of pulmonary hypertension, a well-recognized long-term clinical complication of sickle cell disease (23). In transgenic sickle cell SAD mice, we have shown here that the hypoxia-reoxygenation increase in the expression of lung Mmp9 gene (Fig. 6) is completely prevented by NO-Alb, indicating that NO may affect the early alveolar remodeling events that are critical in the development of hypoxic pulmonary hypertension.

The systemic effects of NO-Alb might be related to the release of both NO and nitrite, both of which may beneficially affect the ischemic-reperfusion lung injury (6,37).

In conclusion, NO-Alb exhibits several beneficial effects in transgenic sickle cell SAD mice exposed to hypoxia-reoxygenation. These effects are most likely related to the combination of two distinct mechanisms: 1) an albumin-related NO-independent effect, based on increased transendothelial hydraulic conductivity, and 2) a NO-dependent effect, based on the local and systemic release of NO and NO derivative species, providing protection against ischemic-reperfusion tissue damage. These observations provide new insights into the possible use of NO-Alb in the treatment of acute sickle cell disease lung injury.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by Ministero Università e Ricerca (ex 60%, L. de Franceschi) and Fondazione Cassa di Risparmio di Verona (A. Scarpa and R. Corrocher).


   ACKNOWLEDGMENTS
 
We thank Prof. Hans Peter Schwarz (Baxter Bioscience, Vienna, Austria) for helpful discussion.

This work was presented at the 46th Annual Meeting of the American Society of Hematology, San Diego, CA, December, 2004, and an abstract was published (Blood 104, Suppl 1: 3580, 2004).


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. de Franceschi, Dept. of Clinical and Experimental Medicine, Section of Internal Medicine, Univ. of Verona, Policlinico GB Rossi, Piazzale L. Scuro, 10, 37134 Verona, Italy (e-mail: lucia.defranceschi{at}univr.it)

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.

* L. de Franceschi and G. Malpeli contributed equally to this work. Back


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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