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Published as doi: 10.1096/fj.07-098921.
 (The FASEB Journal. 2008;22:1849-1860.)
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Protective effects of phosphodiesterase-4 (PDE-4) inhibition in the early phase of pulmonary arterial hypertension in transgenic sickle cell mice

Lucia De Franceschi*, Orah S. Platt{ddagger}, Giorgio Malpeli{dagger}, Anne Janin§, Aldo Scarpa{dagger}, Christophe Leboeuf§, Yves Beuzard||, Emmanuel Payen|| and Carlo Brugnara{ddagger}

* Department of Clinical and Experimental Medicine, Section of Internal Medicine, and

{dagger} Department of Pathology, Section of Anatomic Pathology, University of Verona, Verona, Italy;

{ddagger} Labs of Medicine and Pathology, Children’s Hospital, Harvard Medical School, Boston, Massachusetts, USA; and

§ Laboratorie de Pathologie, INSERM 02 20, and

|| Laboratory of Gene Therapy and Hematological Diseases, INSERM U-733, Hospital Saint Louis, Paris, France

1Correspondence: Department of Clinical and Experimental Medicine, Section of Internal Medicine, University of Verona, Policlinico GB Rossi; P. le L. Scuro, 10; 37134 Verona, Italy. E-mail: lucia.defranceschi{at}univr.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pulmonary arterial hypertension (PAH) is one of the leading causes of morbidity and mortality in adult patients with sickle cell disease (SCD). Here, we developed a model to study the early stage of PAH in SCD. We exposed wild-type and transgenic sickle cell SAD (Hbbs/Hbbs) mice to hypoxia (8% O2) for 7 days. Prolonged hypoxia in SAD mice only induced 1) increased neutrophil count in both bronchoalveoal lavage (BAL) and peripheral circulation; 2) increased BAL IL1β, IL10, IL6, and TNF-{alpha}; and 3) up-regulation of the genes endothelin-1, cyclo-oxygenase-2, angiotensin-converting-enzyme, and IL-1β, suggesting that amplified inflammatory response and activation of the endothelin-1 system may contribute to the early phase of PAH in SCD. Since phosphodiesterases (PDEs) are involved in pulmonary vascular tone regulation, we evaluated gene expression of phosphodiesterase-4 (PDE-4) isoforms and of PDE-1, -2, -3, -7, -8, which are the main cyclic-adenosine-monophosphate hydrolyzing enzymes. In SAD mouse lungs, prolonged hypoxia significantly increased PDE-4 and -1 gene expressions. The PDE-4 inhibitor, rolipram, prevented the hypoxia-induced PDE-4 and -1 gene up-regulation and interfered with the development of PAH, most likely through modulation of both vascular tone and inflammatory factors. This finding supports a possible therapeutic use of PDEs inhibitors in the earlier phases of PAH in SCD.—De Franceschi, L., Platt, O. S., Malpeli, G., Janin, A., Scarpa, A., Leboeuf, C., Beuzard, Y., Payen, E., Brugnara, C. Protective effects of PDE-4 inhibition in the early phase of pulmonary arterial hypertension in transgenic sickle cell mice.


Key Words: inflammatory response • lung injury • vascular remodeling • hypoxia • endothelin-1


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SICKLE CELL DISEASE (SCD) IS CAUSED by a single point mutation in codon 6 of the human β-globin gene, which results in valine substituting the glutamic acid in position 6 of the beta globin chain. In homozygotes, the abnormal hemoglobin (Hb) [HbS ({alpha}2βS2)] forms polymers within red blood cells on deoxygenation, which thereby triggers erythrocyte sickling and dehydration (1 2 3 4 5) . Repeated cycles of HbS polymerization, red cell sickling, dehydration, and red cell membrane oxidative damage play a major role in the pathogenesis of both the chronic hemolytic anemia and the vaso-occlusive-ischemic events, which promote recurrent, painful crises and, ultimately, irreversible organ damage (5 6 7 8) . Vaso-occlusive events might result from the interactions between different factors: dense red cells, reticulocytes (stress reticulocytes), endothelial cells, platelets, and leukocytes, in particular neutrophils (3 , 5 , 9 , 10) . In addition, in SCD vascular endothelial cells appear to be abnormally activated as supported by the increase in vascular soluble adhesion molecules (5 , 8 9 10 11 12 13 14) but also by the transient increase in plasma levels of endothelin-1 (ET-1) during acute severe sickle cell vaso-occlusive crises (15 , 16) .

In SCD, the lungs are particularly vulnerable to vaso-occlusive events because of their anatomic features. In fact, in pulmonary microcirculation, dehydrated and sickled red cells are trapped or adhere to the abnormally activated vascular endothelium before reoxygenation and unsickling can occur, which promotes frequent and diffuse microinfarction that results in severe acute and chronic lung disease, ultimately leading to the development of pulmonary artery hypertension (PAH) (17 , 18) . Hsu et al. (12) have recently shown the presence of spontaneous pulmonary arterial hypertension without thrombosis or vascular remodeling in a mouse model for SCD, which seems to correlate with chronic hemolysis, abnormalities in nitric oxide (NO), homeostasis, and endothelial dysfunction. In another sickle cell mouse model, we have shown that the lungs develop a relative NO-deficient state under hypoxic conditions (19) .

Pulmonary vascular tone is determined by the balance between vasodilators, such as NO or prostacyclin, and vasocontrictors, such as endothelin-1, angiotensin-II or thromboxane (20 21 22 23 24) . An abnormally prolonged state of pulmonary vasoconstriction due to an imbalance between vasodilators and vasoconstrictors might contribute to vascular remodeling and subsequently to PAH (23 24 25) . In SCD, the lungs are characterized by reduced NO bioavaibility, which results in chronic perturbation of vascular homeostasis in the lungs, and by temporarily increased ET-1 plasma levels during acute vaso-occlusive crisis (15) . Thus, acute and chronic pathological events related to SCD may profoundly alter this balance and result in acute and chronic lung damage. Pulmonary arterial hypertension has emerged as one of the leading causes of morbidity and mortality in adult sickle cell patients (18) . In the past few years, two possible pathogenetic mechanisms have been identified: the recurrence of vaso-occlusive episodes, with progressive loss of the vascular bed, and chronic hemolysis, with chronic release of free hemoglobin scavenging nitric oxide and catalyzing the formation of oxygen-free radicals (26) .

In SCD, pharmacological treatment of pulmonary arterial hypertension is actually limited to the synthetic prostacyclin (26 , 27) and more recently to the phoshodiesterase-5 (PDE-5) inhibitor, sildenafil (18 , 28 29 30 31) . Several members of the PDEs superfamily (PDE-1, -2, -3, -4, -5) have been identified in pulmonary vasculature regulating the vascular metabolism of cyclic-guanine-5-monophosphate (cGMP) and cyclic adenine-5-monophosphate (cAMP) (32) . PDE-5 inhibitors block cGMP degradation, while phosphodiesterase-4 (PDE-4) inhibitors block the degradation of cAMP; both cAMP and cGMP participate in integrating signaling pathways in different cell types (25 , 33 34 35 36 37) . The increase in cAMP and cGMP seems to be involved in both vascular tone and vascular remodeling (38 39 40 41 42) .

Although progress on the pathogenesis of PAH in SCD has been made, little is known about the early phase of PAH, in particular how the crosstalk between vasoactive agents and proinflammatory mediators leads to the generation of pulmonary arterial hypertension. We present here a model to study the early stages of pulmonary arterial hypertension in SCD. We exposed transgenic sickle cell SAD mice to hypoxia (8% oxygen) for 7 days, which induced vascular remodeling supported by migration of smooth muscle cells, increased vascular congestion, and increased wall thickness of pulmonary small vessels, compatible with pulmonary arterial hypertension. In addition, we observed increased mRNA levels of PDE-4 isoforms and PDE-1 during prolonged hypoxia. Then, we investigated the effects of a PDE-4 inhibitor, rolipram, on this model, testing whether the inhibition of PDE-4 may display beneficial effects on sickle cell lung injury. Lastly, we studied its mechanisms of action.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Transgenic Hbbs/Hbbs SAD mice, which include the three human mutations βS, βAntilles, and βD-Punjab (βSAD), and C57B6/6J control (wild-type) mice aged between 4 and 6 months were used for these studies (female and male, 20–25 g body weight) (19) .

Hematological parameters
Hematological parameters were measured as described previously (19) . Whole blood was collected by retro-orbital venipuncture in mice anesthetized by methoxyfluorane. Hematocrit (Hct), hemoglobin (Hb), reticulocyte count, white cell count, and neutrophils were measured at baseline and after 7 days hypoxia (19) .

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 7 days hypoxia (8% oxygen). One group from each strain was left untreated during hypoxia, while another hypoxic SAD mouse group was treated with the PDE-4 inhibitor rolipram, at the dosage of 30 mg/kg once a day by gavage (43 44 45 46) . Treatment was started 48 h before hypoxia and maintained during hypoxia. Mice did not show major side effects related to rolipram treatment. In the hypoxic SAD mouse group, 5 animals were alive at 7 days, while all wild-type and rolipram-treated mice were alive and well after 7 days hypoxia. As we previously reported, the gas mixture for hypoxia was blended using separately regulated and calibrated mass flow-meters (RDM 280, Airliquid, Paris-La-Defense, France), maintaining a gas flow rate constant of ~1.5 L/min through a 5 L exposure cage (19) . The FiO2 was kept constant at 0.21 in baseline experiments and at 0.08 in hypoxia experiments, using a polarographic electrode (Oxygen Analyzer, Ohmeda 5120, Englewood, CO, USA). Mice were given free access to water and food.

Bronchoalvear (BAL) fluid, cells, and cytokines content
BAL fluids were collected by instilling and withdrawing a total volume of 2 ml of sterile PBS in 500 µl aliquots, 4x, via intratracheal cannula. Cells were recovered by centrifugation and counted by microcytometry. The percentage of neutrophils was determined by cytospin centrifugation, fixation, and staining. Remaining BAL samples were centrifuged at 1500 g for 10 min at 4°C. The supernatants were used to measure the following cytokines: TNF-{alpha}, IL-1β, IL-6, and IL-10 by commercial ELISA (R&D Systems Europe, Abingdon, UK; Amersham, Oxford, UK), according to the manufacturer’s instructions (19) .

Histopathology and molecular studies
Lung tissue histology
One lung was immediately frozen in liquid nitrogen, while the other was fixed in formalin by perfusion followed by submersion and embedding in paraffin. Multiple (≥5) 3 µm whole-mount sections were obtained for each paraffin-embedded lung and stained with hematoxylin eosin, Masson’s trichome, and May-Grünwald-Giemsa. Morphological analysis was performed blindly and independently by two pathologists and consisted of the evaluation of the tissue architecture and changes induced by hypoxia and/or treatment regimens. The interobserver difference measure was <5%. Vessels were evaluated for the presence of congestion and thrombi in small pulmonary vessels, whereas the bronchi were evaluated for the presence of mucus and inflammatory cell infiltrate. Quantitative data were obtained with PALM robot software version 1.2.1 (PALM.microlaser technologies AG, Bernried, Germany), on an Olympus Provis AX70 microscope with wide-field eyepiece number 26.5 (Olympus, Tokyo, Japan), providing a field size of 0.344 mm2 at x400. Vascular congestion was evaluated by measuring the percentage of hematoxylin-eosine stained lung sections containing red cells (47 , 48) . The wall thickness of lung arteries was measured on hematoxylin-eosine stained lung sections at x400. Ten different lung arteries were evaluated for each mouse with SIS software (analysisSIS, v.3.2; SiS Corp., Hsin-Chu, Taiwan), allowing the calculation of the mean thickness of each artery from 30 separate determinations. Results were expressed in square micrometers. Mucus filling the mouse bronchi was quantified by measuring the percentage of bronchus section in hematoxylin-eosin stained lungs that contained mucus. Quantification of inflammatory cell infiltrate and neutrophils was expressed as the mean number of cells per field at x400, which resulted from the analysis of at least four different fields on each hematoxylin-eosin-stained whole lung section.

{alpha}-Smooth muscle actin immunohistochemistry (IHC) on lungs
Samples were fixed for 2 h in alcohol, formol, and acetic acid (AFA; Labonord, Templemars, France) and further processed for paraffin embedding. Sequential 5-µm-thick sections were realized on a microtome with water flow (HM 350 S Niagara; Microm, Francheville, France). After removal of paraffin with xylene and rehydration of the tissue sections in alcohol, an indirect immunoperoxydase procedure was performed with mouse anti {alpha}-smooth muscle actin antibody (clone 1A4; Sigma Chemical Co., Saint Louis, MO, USA) at the dilution of 1:2000 and a secondary biotinylated antibody (Ventana Nexes; Ventana Medical Systems, Tucson, AZ, USA). Two pathologists independently performed the procedure with an Olympus AX70 microscope at x200 and x400 (49 , 50) .

Evaluation of right ventricular hypertrophy
The hearts were fixed with 10% formaldehyde for 24 h. The right ventricular (RV) free wall was separated from the left ventricular with septum (LV+S) under a dissection microscope. RV and LV+S were separately weighed and used to calculate the ratios RV/(LV+S) and RV/body weight (49 50 51) .

Quantitative RT (reverse-transcription) -PCR analysis
Total RNA was isolated from frozen lung tissues with the guanidinum thiocyanate/cesium chloride method. Total RNA (1 µg) was converted to cDNA by random primers and AMV retrotranscriptase (Roche Diagnostic, Mannheim, Germany). Real-time quantitative RT-PCR (qPCR) analysis was performed on an ABI Prism 7000 SDS (PE Applied Biosystems, Foster City, CA, USA) using the SYBR Green I dye contained in the SYBR Green Master Mix (PE Applied Biosystems) as a probe. All PCR reactions contained 1x Master Mix, 200 nM each primer, and 5 ng cDNA (total RNA equivalent) in 25 µl final volume. Samples were analyzed in triplicate. Primers were designed to prevent genomic DNA amplification using Primer Express software (PE Applied Biosystems). The primers used are shown in Table 1 . 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 annealing and extension. At the end of each run, PCR products were checked by melting curves analysis and agarose gel electrophoresis. The probe signal was normalized to an internal reference, and a cycle threshold (Ct) was taken significantly above the background fluorescence. Calibration curves for each pairs of primers were obtained by running five serial cDNA dilutions in triplicates and by plotting the corresponding fractional Ct in the function of the logarithm of the input cDNA. PCR efficiencies (calculated as 10–(1/slope)) deducted from calibration curves were ~90%. The relative expression level of genes was calculated by comparative method using Gapd transcript level as an endogenous reference. Data were analyzed as indicated in User Bulletin 2 (PE Applied Biosystems).


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  		Table 1. Genes studied and primers used in quantitative RT-PCR experiments

Statistical analysis
The 2-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
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Transgenic sickle SAD cell mice exposed to prolonged hypoxia developed pulmonary arterial hypertension
Transgenic sickle cell SAD mice exposed to prolonged hypoxia showed histopathological damage in the lungs and severe inflammatory response, with an extensive ischemic component, characterized by severe vascular congestion, increased thickness in the walls of small pulmonary arteries, the presence of thrombi in small vessels, the presence of mucus filling the bronchus section area, and increased numbers of inflammatory cells associated with a significant amount of neutrophils (Table 2 , Fig. 1 ). In addition, we observed partial pulmonary vein muscularization only in SAD mice exposed to 7 days hypoxia, as supported by the presence of smooth muscle {alpha}-actin linear deposits in the walls of pulmonary veins, which extended also to the muscle layers of lung arteries (Fig. 1) .


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  		Table 2. Lung pathology of wild-type and SAD mice exposed to hypoxia and effects of PDE4 inhibitor (rolipram) treatment


Figure 1
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  		Figure 1. Lung histopathology of transgenic sickle cell SAD mice exposed to 7 days hypoxia and effects of PDE-4 inhibitor (rolipram). The left panels show representative examples of wild-type (A) and SAD (B) mouse pulmonary arteries under ambient air conditions. The thickness of arterial walls is increased on hypoxia in both wild-type (C) and SAD (D) mice. In hypoxic SAD mice treated with the PDE-4 inhibitor rolipram (G), the arterial wall thickness was reduced compared to untreated hypoxic SAD mice (D). This figure also shows representative examples of partial muscularinization of the pulmonary vein in SAD mice exposed to 7 days hypoxia. Smooth muscle {alpha}-actin linear deposits (arrowheads) were found in the walls of pulmonary veins (E) and in the muscle layer (arrowheads) of a lung artery (F); indirect immunoperoxydase with an antibody directly against smooth muscle {alpha}-actin was used.

We evaluated the presence of RV hypertrophy in mice exposed to hypoxia. No changes were observed in wild-type mice exposed to hypoxia, whereas in SAD mice exposed to hypoxia, we observed a slight but not significant increase in the RV/(LV+S) ratio (4 , 26) (normoxia: 0.22±0.02, n=6 vs. hypoxia: 0.28±0.08, n=6; P>0.05). These findings were consistent with the early stage of pulmonary arterial hypertension related to SCD. We observed only modest vascular congestion, no thrombi and no significant changes in the wall thickness of the small pulmonary artery in wild-type mice (Fig. 1 , Table 2 ), and no evidence of muscularization in the pulmonary veins (data not shown). These data agree with reports in the literature showing that wild-type mice need a longer exposure to hypoxia to develop pulmonary arterial hypertension (52) .

Prolonged hypoxia induced a significant increase in Hct and Hb levels, and reticulocyte counts, which was compatible with the effect of hypoxia on erythropoiesis in both wild-type and SAD mice. However, the increase in Hct and Hb was significantly blunted in SAD mice compared to wild-type mice, despite similar increases in reticulocyte count (Table 3 ). In addition, exposure of SAD mice to hypoxia resulted in an increase in spleen size, ranging from 0.16–0.24 g under normoxia to 0.34–0.39 g under prolonged hypoxia (n=5; P<0.05); and a significant increase in plasma hemoglobin (normoxia: 54.3±12.9 mg/dl vs. hypoxia: 164±23 mg/dl; n=5; P<0.05), suggesting a worsening of hemolysis.


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  		Table 3. Hematological parameters in wild-type and SAD mice exposed to hypoxia and effects of PDE-4 inhibitor (rolipram) treatment

Early phase pulmonary arterial hypertension was associated with the up-regulation of PDE-4 isoforms and the modulation of PDE-1, -2, -8 in the lungs of transgenic sickle cell SAD mice
Pulmonary vascular tone depends on the balance between intracellular levels of cyclic nucleotides such as cyclic adenosine monophosphate (cAMP) or cyclic guanosine monophosphate (cGMP), acting as second messengers in response to extracellular stimuli (25 , 33 34 35 36 37) . PDE enzymes hydrolyze cyclic nucleotides and control their intracellular concentrations maintaining a balance between production and degradation. PDE families 4, 7, 8 have been described to hydrolize cAMP, while PDE families 1, 2, 3 hydrolyze both cAMP and cGMP as substrates but with different affinities (34 , 53) . Thus, we first evaluated mRNA levels of PDE-4 isoforms in the lungs of both wild-type and transgenic sickle cell SAD mice under normoxia and exposed to prolonged hypoxia (Fig. 2 ). In transgenic sickle cell SAD mice, three PDE-4b isoforms and one PDE-4d isoform RNA level were increased, respectively, by 3- to 4-fold and 1.8-fold after prolonged hypoxia (Fig. 2B ). In addition, one of the PDE-4a isoform and one of the PDE-4b isoform RNA levels were down-regulated after prolonged hypoxia (Fig. 2B ). No significant changes were observed in wild-type mice (Fig. 2A ). Then, we evaluated mRNA levels of the phosphodiesterase isoenzymes PDE-1, PDE-2, PDE-3, PDE-7, and PDE-8 in the lungs of both wild-type and transgenic sickle cell SAD mice under normoxia and exposed to prolonged hypoxia (Fig. 3 ). Prolonged hypoxia significantly increased PDE-1 and PDE-8 mRNA levels in transgenic sickle cell SAD mice and down-regulated one of the PDE-2 isoforms (Fig. 3B ). No significant changes were observed in wild-type mice (Fig. 3A ).


Figure 2
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  		Figure 2. Effects of prolonged hypoxia on PDE-4 isoforms in wild-type (WT; A) and transgenic sickle cell SAD mice (SAD; B) under ambient air conditions (normoxia) and after prolonged hypoxia (7 days hypoxia), with and without treatment with the PDE-4 inhibitor rolipram. Data are reported as means ± SD, n = 6/group; *P < 0.05 vs. normoxic mice; °P < 0.05 vs. hypoxic untreated SAD mice.


Figure 3
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  		Figure 3. Effects of prolonged hypoxia on PDE-1, -2, -3, -7, -8 isoforms in wild-type (WT; A) and transgenic sickle cell SAD mice (SAD; B) under ambient air conditions (normoxia) and after prolonged hypoxia (7 days hypoxia), with and without treatment with the PDE-4 inhibitor rolipram. Data are reported as means ± SD, n = 6/group; *P < 0.05 vs. normoxic mice; °P < 0.05 vs. hypoxic untreated SAD mice.

Early phase pulmonary arterial hypertension was associated with increased inflammatory cells and cytokines in BAL
In both wild-type and SAD mice exposed to prolonged hypoxia, total leukocyte and neutrophil count in BAL significantly increased, with a larger change in SAD mice compared to wild-type mice, suggesting an amplification of the inflammatory response in transgenic sickle cell SAD mice (data not shown). Since cytokines play an immunomodulatory role in inflammatory response during lung injury, and because cytokines have been described as participating in the pathogenesis ofpulmonary arterial hypertension, we evaluated IL-6, IL-1β, IL-10, and TNF-{alpha} levels in BAL (54 , 55) . In both wild-type and SAD mice, BAL levels of IL-6, IL-1β, IL-10, and TNF-{alpha} significantly increased with hypoxia (Fig. 4 ). IL-1β and IL-10 BAL levels were higher in SAD mice exposed to hypoxia than in wild-type mice in the same experimental condition, supporting an amplified inflammatory response, which most likely contributed to the development of PAH in SAD mice (Fig. 4) (33 , 56 57 58 59 60 61) .


Figure 4
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  		Figure 4. BAL cytokines in prolonged hypoxia and effects of the PDE-4 inhibitor rolipram. BAL IL-6, IL1β, IL-10, and TNF-{alpha} levels were determined in wild type (WT) and transgenic sickle cell SAD mice (SAD) under ambient air conditions (normoxia) and after prolonged hypoxia (7 d hypoxia). Data are reported as means ± SD, n = 6/ group; *P < 0.05 vs. normoxic mice; °P < 0.05 vs. hypoxic untreated SAD mice.

The PDE-4 inhibitor rolipram reduced lung injury and modulated inflammatory response
Based on the pathological evidence that pulmonary arterial hypertension was present only in hypoxic sickle cell SAD mice and that prolonged hypoxia increased PDE-4 isoform RNA levels, we decided to study the effects of PDE-4 selective inhibitor, rolipram (62 63 64 65) , in SAD mice by comparison with the untreated SAD mouse population exposed to prolonged hypoxia.

In SAD mice exposed to prolonged hypoxia, the inhibition of PDE-4 by rolipram markedly reduced the vascular congestion and thicker wall of pulmonary small arteries, prevented thrombi formation in small pulmonary arteries, and reduced the inflammatory response, as supported by the absence of mucus in the bronchus section areas and the reduction of inflammatory cell infiltration (Table 2 , Fig. 1 ). In addition, PDE-4 inhibition reduced BAL leukocyte and neutrophil counts, suggesting a possible modulation of neutrophil chemotaxis in the lungs by rolipram (Fig. 1 , Table 2 ) (66) .

Rolipram treatment down-regulated the hypoxia-induced increased expression of PDE-4 isoforms, of one PDE-1a isoform, and of one PDE-2a isoform. No effects on PDE-8 were found, as expected, since PDE-8 is insensitive to rolipram treatment (Figs. 2B and 3B) (67) .

In SAD mice, PDE-4 inhibition also determined a marked reduction in BAL IL-6, IL-1β, and IL-10 levels compared to untreated hypoxic SAD mice. In particular, BAL IL-6 and IL-10 values became similar to those observed in untreated SAD mice under ambient air conditions (Fig. 4) , whereas the hypoxia-induced increase in BAL TNF-{alpha} levels was not modified by the rolipram treatment (Fig. 4) .

Prolonged hypoxia induced a significant increase in circulating neutrophils in both wild-type and SAD mice (Table 3) . The increase in neutrophil count was higher in SAD mice than in wild-type ones (Table 3) . The neutrophil count was significantly lowered by rolipram treatment in hypoxic SAD mice, indicating a possible systemic antiinflammatory effect of rolipram (Table 3) .

No changes were observed in the other hematological parameters evaluated in SAD mice under PDE-4 treatment compared to hypoxic untreated SAD mice (Table 3) .

PDE-4 inhibitor down-regulates ET-1 and cyclo-oxygenase-2 (COX2) gene expression and modulates prolonged hypoxia-induced genes in the lungs of transgenic sickle cell SAD mice:
We focused our attention on the ET-1 system, which is involved in pulmonary microcirculation remodeling (21 , 25 , 33 , 59) . We evaluated mRNA levels of ET-1 in lungs of wild-type and SAD mice exposed to hypoxia-inducing pulmonary arterial hypertension and in hypoxic SAD mice treated with the PDE-4 inhibitor rolipram. In the same samples, we also evaluated the gene expressions of TNF-{alpha} and IL-1β as elements of inflammatory response to prolonged hypoxia and mRNA levels of COX2, which have been described to be induced by hypoxia and/or cytokines (23 , 68) . Since we previously showed that hypoxia, mimicking acute sickle cell vaso-occlusive crises, can induce up-regulation of angiotensin-converting enzyme (Ace) gene expression in SAD mice (19) and because Ace might participate in lung injury through increased production of angiotensin II (69) , we also measured lung Ace gene expression in SAD mice with early pulmonary arterial hypertension with and without rolipram treatment.

In SAD mice with early stage pulmonary arterial hypertension, ET-1, COX2, Ace, and Il1b mRNA levels were significantly increased, while no significant changes were evident for Tnf gene expression (Fig. 5 B). In wild-type mice, prolonged hypoxia up-regulated ET-1 and COX2 genes, while no differences were evident in lung mRNA levels for Ace, Il1b, or Tnf genes (Fig. 5B ). A comparison of the effects of hypoxia in the two mice strains showed a stronger up-regulation of ET-1 and COX2 and Ace in SAD mice vs. wild-type mice, suggesting an abnormal vascular endothelium response to hypoxia in transgenic sickle SAD mice (Fig. 5) .


Figure 5
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  		Figure 5. Quantitative RT-PCR expression profiles of ET-1, COX2, Ace, Tnf, and Il1b. Bars indicate the fold change in mRNA expression of the indicated genes in wild-type (WT; A) and in transgenic sickle cell SAD mice (SAD; B) under ambient air conditions (black bars), prolonged hypoxia (gray bars), and prolonged hypoxia with PDE-4 inhibitor (rolipram) treatment (white bars). Data are presented as means ± SD, n = 6/group; *P < 0.05 vs. normoxic mice; °P < 0.05 vs. hypoxic untreated SAD mice. For each gene, the expression levels obtained from the different experimental conditions were normalized relative to normoxic levels.

Treatment with rolipram significantly down-regulated the hypoxia-induced ET-1, COX2, Ace, and Il1b up-regulation in SAD mice, suggesting that PDE-4 inhibition may modulate local and systemic inflammatory responses and alter the pulmonary vascular tone most likely affecting the ET-1 system and COX2 network (Fig. 5B ).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
PAH is one of the newly identified leading causes of morbidity and mortality in adult patients with SCD (18 , 70 , 71) . The recurrence of vaso-occlusive episodes, with progressive loss of the vascular bed, and chronic hemolysis, with the release of free hemoglobin scavenging nitric oxide and catalyzing the formation of oxygen free radicals, are believed to play a crucial role in the pathogenesis of PAH (11 , 12 , 18 , 70 71 72 73) . Reduced NO bioavailability and severe vascular endothelial dysfunction have been described in SCD, which may facilitate further acute vaso-occlusive events and promote an amplified inflammatory response, with further release of vasoactive molecules, such as endothelin-1 (16 , 74 75 76) .

While substantial progress has been made in the clinical models and treatment of PAH in SCD, much less is known about the events involved in the early phase of PAH. In the present study, we analyzed the early phase of pulmonary arterial hypertension in a transgenic sickle cell mouse model (SAD). SAD mice have a relatively mild form of SCD, and they are better suited than other models for the study of lung pathology the development, since they do not normally show severe lung damage (19 , 48) . In SAD mice, prolonged hypoxia (7 days, 8% oxygen) induced pathological lung changes compatible with the early stages of pulmonary arterial hypertension, indicating that the sickle cell SAD hematological phenotype can induce the development of PAH. In contrast, we did not observe any signs of PAH in wild-type mice.

In SAD mice, prolonged hypoxia up-regulated gene expression of PDE-4 and PDE-1 isoforms. These changes were prevented by rolipram treatment, supporting a role of PDEs in structural remodeling processes in the early phase of PAH in transgenic sickle cell SAD mice. In addition, a local and systemic inflammatory response was seen in SAD mice exposed to prolonged hypoxia, as supported by increased levels of cytokines (IL-6, IL-1β, IL-10, and TNF-{alpha}), neutrophil lung infiltrates, and circulating neutrophils (Table 3) . Previous reports have shown that inflammatory cytokines might contribute to the pathogenesis of PAH, most likely through the modulation of cAMP content in smooth muscle cells by IL-1β and IL-6 (77 , 78) , which seems also to be involved in this model of early phase PAH in SAD mice. In SAD mice, prolonged hypoxia up-regulated the gene expression of ET-1, Ace, and COX2 (Fig. 5) , suggesting that these genes may be important in the early phase of PAH in SCD.

Studies in different animal models have shown that hypoxia can up-regulate both ET-1 and COX2 production in pulmonary tissue (23 , 79) . ET-1 is a potent bronchovasoconstrictor, which also stimulates mucus secretion and modulates inflammatory response (80 , 81) . The hypoxia-induced COX2 gene expression leads to increased production of prostanoids (i.e., prostaglandins-PGE2) (22 , 49) , which has been shown to positively affect the balance between the ET-1 synthesis and ET-1 clearance in pulmonary capillaries (82 , 83) . In addition, it has been suggested that the increase in pulmonary prostanoids via COX2 might be limiting ET-1 induced pulmonary vaso-constriction more than acting as direct vasodilators (22 , 49 , 83 , 84) . In addition, the imbalance between prostacyclin and thromboxane levels may also contribute in sustained pulmonary vasocontriction (85 86 87) .

Thus, we hypothesize that in SCD the perturbation of pulmonary vascular endothelial tone related to the relative NO deficiency is enabled by the increase of the ET-1 system, an action only partially counteracted by the induction of COX2 and the increased production of prostanoids associated with amplified inflammatory response. Thus, therapeutic strategies for interfering with the development of PAH in SCD should also consider the balance between the ET-1 system and prostanoids but also with inflammatory factors.

In transgenic sickle cell SAD mice, the potent PDE-4 inhibitor rolipram protected SAD mice from development of PAH, reduced local and systemic inflammatory response, modulated the BAL cytokine levels, and down-regulated the hypoxia-induced ET-1 and COX2 genes. Thus, in SAD mice, the effects of the PDE-4 inhibitor rolipram are multiple: reduction of hypoxia related vasocontriction; modulation of neutrophil chemotaxis; and reduction of proinflammatory cytokines, such as IL-1β. These beneficial effects are also associated with the down-regulation of the hypoxia-induced up-regulation of ET-1 and COX2 genes, supporting the hypothesis of a functional crosstalk between ET-1 and COX2, most likely through the action of the prostanoids on the pulmonary vascular tone in transgenic sickle cell SAD mice during the early stage of PAH. The marked down-regulation of Ace gene expression by the PDE-4 inhibitor might be related to either a direct effect on Ace gene expression or most likely through an indirect effect by down-regulation of the ET-1 system. In addition, PDE-4 inhibition, by blocking cAMP degradation, has been shown in other models to mimic the effects of cAMP antagonist on vasoconstriction induce by thromboxane (85 , 87) , suggesting that ET-1 may not be the only final target of the PDE-4 inhibitor rolipram.

In conclusion, in transgenic sickle cell SAD mice, the PDE-4 inhibitor rolipram might interfere with the development of PAH by modulating vascular tone directly, via inhibition of cAMP cellular degradation, or indirectly, by reducing the magnitude of vasoconstriction events, but also by modulating the inflammatory response (20 , 88) . These data suggest that the inhibition of PDEs could have beneficial effects on the hypoxia-induced vascular-remodeling and possibly also assist in the early phase of PAH in SCD.


   ACKNOWLEDGMENTS
 
Preliminary data were presented at the 46th annual meeting of the American Society of Hematology, San Diego, California, USA, 2004. This study was supported by Finanziamento Ricerca di Base grant RBNE01XHME, PRIN to L.D.F., and U.S. National Institutes of Health grant DK504221 to L.D.F. and C.B.

Received for publication September 18, 2007. Accepted for publication January 3, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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