J.-M Launay

P Hervé

K Peoc'

C Tournois

J Callebert

C G Nebigil

N Etienne

L Drouet

M Humbert

G Simonneau

L Maroteaux

Cr C Bernard

Function of the serotonin 5-hydroxytryptamine 2B receptor in pulmonary hypertension

Primary pulmonary hypertension is a progressive and often fatal human disorder that results from an increase in pulmonary blood pressure associated with abnormal vascular proliferation. The use of dexfenfluramine increases the risk of pulmonary hypertension in humans and its active metabolite is a selective serotonin 5-HT 2B -receptor agonist. We have therefore investigated a possible contribution of 5-HT 2B -receptor to pulmonary hypertension pathogenesis. Using the chronic hypoxic mice model of pulmonary hypertension, we found that the hypoxia-dependent increase in pulmonary blood pressure and lung remodeling are associated with a rise in vascular proliferation, elastase activity and TGF-ß level and these parameters are potentiated by dexfenfluramine treatment. By contrast, hypoxic mice with genetically or pharmacologically inactive 5-HT 2B receptors manifested no change in any of these parameters. In both humans and mice, pulmonary hypertension is associated with a striking increase in 5-HT 2B receptor expression in pulmonary arteries. These data show that activation of 5-HT 2B receptors is a limiting step in the development of pulmonary hypertension.

Primary pulmonary hypertension (P-PH) is a rare condition characterized by an elevation in pulmonary arterial pressure that is associated with irreversible pulmonary remodeling [START_REF] Rabinovitch | Pulmonary hypertension: updating a mysterious disease[END_REF][START_REF] Rubin | Cellular and molecular mechanisms responsible for the pathogenesis of primary pulmonary hypertension[END_REF] . The hypoxia-induced changes in pulmonary artery pressure that result from vascular cell proliferation recapitulate, at least in part, the changes observed in P-PH indicating that it is a relevant model for this pathology [START_REF] Loscalzo | Genetic clues to the cause of primary pulmonary hypertension[END_REF][START_REF] Tucker | Role of vascular smooth muscle in the development of high altitude pulmonary hypertension: an interspecies evaluation[END_REF] . Several studies have suggested a role for serotonin (5-hydroxytryptamine, 5-HT) in the aetiology of PH [START_REF] Maclean | 5-hydroxytryptamine and the pulmonary circulation: receptors, transporters and relevance to pulmonary arterial hypertension[END_REF][START_REF] Michelakis | Anorectic drugs and pulmonary hypertension from the bedside to the bench[END_REF][START_REF] Egermayer | Role of serotonin in the pathogenesis of acute and chronic pulmonary hypertension[END_REF] . In response to hypoxia, 5-HT is released from pulmonary neuroendocrine cells and neuroepithelial bodies distributed throughout the airways. A sustained increase of 5-HT is observed in the plasma of PH patients [START_REF] Hervé | Increased plasma serotonin in primary pulmonary hypertension[END_REF][START_REF] Kéreveur | High plasma serotonin levels in primary pulmonary hypertension : effect of long-term epoprostenol (Prostacyclin) therapy[END_REF] . Furthermore, isolated smooth muscle and endothelial cells from pulmonary arteries express mRNAs for 5-HT 1B , 5-HT 2A , 5-HT 7 and 5-HT 2B receptors [START_REF] Ullmer | Expression of serotonin receptor mRNA in blood vessels[END_REF] . As a potent pulmonary vasoconstrictor [START_REF] Maclean | 5-hydroxytryptamine and the pulmonary circulation: receptors, transporters and relevance to pulmonary arterial hypertension[END_REF] , 5-HT might have a dual effect on pulmonary circulation, contributing to both hypoxia-induced acute vasoconstriction and chronic vascular remodeling by activating its cognate receptors.

When used for more than 3 months, the appetite-suppressants (anorexigens) derived from amphetamines, fenfluramine and dexfenfluramine (DF), increase the risk of developing PH by a factor of between 3.7 to 23-fold depending on the study consulted [START_REF] Rich | Anorexigens and pulmonary hypertension in the United States: results from the surveillance of North American pulmonary hypertension[END_REF][START_REF] Abenhaim | Appetite-suppressant drugs and the risk of primary pulmonary hypertension. International Primary Pulmonary Hypertension Study Group[END_REF] . DF binds weakly to the 5-HT 2A , 5-HT 2B , and 5-HT 2C receptors. However, the main metabolite of DF, norDF, behaves as a high affinity ligand for 5-HT 2B receptors (5-HT 2B R) and more moderately for 5-HT 2A and 5-HT 2C receptors [START_REF] Fitzgerald | Possible role of valvular serotonin 5-HT2B receptors in the cardiopathy associated with fenfluramine[END_REF] . Furthermore, in cells expressing recombinant 5-HT 2B R, norDF acts as a potent agonist, which stimulates phosphoinositide hydrolysis, intracellular Ca 2+ levels and the mitogenactivated protein (MAP) kinase cascade [START_REF] Fitzgerald | Possible role of valvular serotonin 5-HT2B receptors in the cardiopathy associated with fenfluramine[END_REF] . MAPkinase activation mediates the mitogenic actions of 5-HT, which is accomplished via the 5-HT 2B R by stimulating p60 Src and p21 ras signaling [START_REF] Launay | Ras involvement in signal transduction by the serotonin 5-HT2B receptor[END_REF][START_REF] Nebigil | 5-Hydroxytryptamine 2B receptor regulates cell-cycle progression: Cross talk with tyrosine kinase pathways[END_REF] . Functionally and immunohistologically, 5-HT 2B Rs are detected in lung endothelial and smooth muscle cells of various species including humans [START_REF] Ullmer | 5-HT2B receptormediated calcium release from ryanodine-sensitive intracellular stores in human pulmonary artery endothelial cells[END_REF][START_REF] Glusa | Further evidence that 5-HT-induced relaxation of pig pulmonary artery is mediated by endothelial 5-HT2B receptors[END_REF] . We have therefore tested the hypothesis of a direct implication of 5-HT 2B Rs in the development of PH. This work has revealed that 5-HT 2B R expression is significantly increased in PH and that the blockade of 5-HT 2B Rs using two independent approaches prevents completely the development of hypoxia-induced PH in mice.

The hypoxia-induced rise in pulmonary artery pressure is 5-HT 2B R-dependent. We first assessed the effect of a chronic exposure of mice to hypoxia (10% O 2 , 5 weeks) by monitoring their hemodynamic parameters. When compared with mice maintained under normoxic conditions, control mice (129PAS wild-type, +/+ ) exposed to hypoxia exhibited gradual increases in right ventricular systolic pressure (RVSP) such that, after 5 weeks, values were higher than those recorded after 2 weeks (not shown). After 5 weeks under hypoxia, the mean RVSP was significantly higher (165% of the control value) than in normoxic mice (Fig. 1a).

We recently generated mutant mice by homologous recombination of the 5-HT 2B R gene (5-HT 2B -/-). These mutant mice display cardiac defects leading to partial embryonic lethality [START_REF] Nebigil | Serotonin 2B receptor is required for heart development[END_REF] . Living adult 5-HT 2B -/-mice have normal systolic blood pressure despite their dilated cardiomyopathy [START_REF] Nebigil | Ablation of serotonin 5-HT2B receptors in mice leads to abnormal cardiac structure and function[END_REF] . In a first attempt to evaluate a potential role for the 5-HT 2B R in PH, we treated 5-HT 2B -/-adult mice with the same hypoxic protocol as described above. Surprisingly, none of the 5-HT 2B -/-mice exposed to hypoxia exhibited an increase in RVSP (Fig. 1a). The right-ventricle weight over left-ventricle-plusseptum weight ratio (RV/LV+S) was significantly increased only in +/+ mice exposed to hypoxia, reasserting the hypoxia-induced modifications of RVSP in +/+ mice and the absence of hypoxia-induced responses of 5-HT 2B -/-mice (Table I); the basal value of 5-HT 2B -/-mice, higher than in control animals, reflects directly their left ventricle dysfunctions. Our results indicate that the 5-HT 2B R is necessary for the chronic hypoxic response leading to increased RVSP.

Since DF increases the risk of developing PH in humans [START_REF] Abenhaim | Appetite-suppressant drugs and the risk of primary pulmonary hypertension. International Primary Pulmonary Hypertension Study Group[END_REF] , we evaluated the putative effect of "therapeutic" concentrations of DF in hypoxia-induced increases in pulmonary pressure in mice. The chronic exposure of +/+ mice to hypoxia and DF further increased significantly the RVSP (229% and 138% of normoxic and hypoxic values respectively) (Fig. 1a). The RV/LV+S was also significantly increased in +/+ mice exposed to hypoxia in the presence of DF when compared with both normoxic and hypoxic +/+ mice (Table I). Importantly, hypoxia in the presence of DF generated high lethality: out of 20 mice treated, four were found dead from obvious signs of PH. Furthermore, chronic exposure of 5-HT 2B -/-mice to DF in the presence of hypoxia or normoxia had no significant effect on the RVSP (Fig. 1a) or on the RV/LV+S (Table I).

To validate these observations, we used an independent pharmacological approach. We challenged +/+ mice with the recently developed compound, RS-127445 (2-amino-4-(4-fluoronaphth-1-yl)-6-isopropylpyrimidine, RS). This compound has a sub-nanomolar affinity for the 5-HT 2B R (pK i = 9.5 ± 0.1) and potently antagonizes the 5-HT 2B R with a 1,000 fold selectivity over numerous other molecules including all known 5-HT receptors and transporter [START_REF] Bonhaus | RS-127445: a selective, high affinity, orally bioavailable 5-HT2B receptor antagonist[END_REF] . In complete agreement with our first observations on 5-HT 2B -/-mice, simultaneous exposure of +/+ mice to hypoxia in the presence of RS prevented an increase in RVSP (Fig. 1a). Furthermore, chronic exposure to RS in the presence of normoxia in +/+ mice and of hypoxia or normoxia in 5-HT 2B -/mice had no significant effects on the RVSP (Fig. 1a). Combined, these data support the initial findings that the 5-HT 2B R is required for the chronic hypoxia-induced increase in RVSP.

The histological remodeling triggered by hypoxia requires 5-HT 2B R.

Exposure to hypoxia dramatically increases structural remodeling of the pulmonary arteries resulting from vascular cell proliferation that generates most of the RVSP increase [START_REF] Belknap | Hypoxia increases bromodeoxyuridine labeling indices in bovine neonatal pulmonary arteries[END_REF] . Chronic hypoxic exposure of +/+ mice induced obvious modifications of lung morphology revealed by the degree of pulmonary artery muscularization (Fig. 1b-2ghi); the statistical analysis of lung morphology revealed that increased RVSP is associated with a significant increase in pulmonary artery muscularization; DF further and significantly enhanced this remodeling (Fig. 1b-2jklnp). Alternatively, no significant modification of the pulmonary artery muscularization could be revealed in hypoxic 5-HT 2B -/-mice with, or without, RS or DF, or following RS treatment of hypoxic +/+ mice (Fig. 1b-2def). In addition, staining of lung sections revealed that the hyperplasic cell population consisted mainly of smooth muscle cells as they expressed smooth musclespecific actin (Fig. 2n) and also demonstrated the presence of 5-HT 2B Rs expression in hypoxic +/+ mice (Fig. 2p).

To get further insights into the molecular pathways involved in hypoxic responses, we developed isolated lung vasculature explant culture of hypoxia-(or normoxia-) treated mice. Compared with normoxic animals, basal thymidine incorporation was significantly increased (1.95 times basal) in the lung vessels from hypoxic +/+ mice (Fig. 3a). This increase was completely prevented in the hypoxic lungs of +/+ mice treated by RS, while DF treatment significantly potentiated DNA synthesis (2.5 times vs. basal and 1.3 times vs. hypoxia). By contrast, lung vessels of 5-HT 2B -/-mice did not exhibit any hypoxia-induced nor DF-potentiated increase in DNA synthesis. These data support the notion that 5-HT 2B R is a limiting factor in chronic hypoxia-induced pulmonary vascular proliferation.

Progression of PH is associated with increased serine elastase activity [START_REF] Rabinovitch | EVE and beyond, retro and prospective insights[END_REF] , which can activate storage sites for growth factors such as basic fibroblast growth factor and transforming growth factor-beta (TGF-ß) [START_REF] Vu | Matrix metalloproteinases: effectors of development and normal physiology[END_REF] . Supernatants of lung vessels cultures from hypoxic +/+ mice revealed a significant increase in elastase activity (11.6 times basal) and in TGF-ß levels (3.9 times basal), both of which were further potentiated after DF treatment (16.9 and 4.9 times basal, respectively). Conversely, there was no detectable change in hypoxia-induced elastase activity and TGF-ß levels in supernatants from vessels of hypoxic +/+ mice in the presence of RS or of 5-HT 2B -/-mice after either treatment (Fig. 3b-c). These results indicate that both hypoxia-increased remodeling factors, pulmonary elastase activity and TGF-ß levels, are dependent on 5-HT 2B R activity.

The 5-HT 2B R is not involved in acute hypoxia-dependent vasoconstriction Acute hypoxic responses are important triggers in the establishment of chronic PH [START_REF] Maclean | Pulmonary hypertension, anorexigens and 5-HT: pharmacological synergism in action?[END_REF] and thus, the limiting functions of 5-HT 2B R in chronic response to hypoxia could rely on direct acute effects of hypoxia on 5-HT 2B R. We therefore investigated whether 5-HT 2B R participates in acute responses to hypoxia. Following repeated acute hypoxic stimuli [START_REF] Eddahibi | Effect of dexfenfluramine treatment in rats exposed to acute and chronic hypoxia[END_REF] , we observed a moderate but significant increase in RSVP in both +/+ and 5-HT 2B -/-mice, indicating that 5-HT 2B Rs are not required for short-term increases in RVSP (Fig. 3d). The result of the same experimental setup in the presence of DF was also independent of 5-HT 2B Rs. Plasma DF and norDF measurements (Supplementary Data) clearly demonstrated that short term exposure is not sufficient enough to produce significant amounts of norDF and confirmed that the effect of DF is mediated by its metabolite norDF at 5-HT 2B R. When testing for elastase activity following the same acute hypoxic stimuli, we observed a significant increase in elastase activity in +/+ but not in 5-HT 2B -/-mice lungs (Fig. 3e), indicating that effectors of the 5-HT 2B R (after chronic hypoxia) are regulated by acute hypoxic stimulus. These results clearly suggest that 5-HT 2B R influence is not at the level of acute vasoconstrictive responses but rather that of the signaling mechanisms that lead to the expression of factors important in vascular remodeling.

Pulmonary artery 5-HT 2B R expression is increased during PH.

The absence of 5-HT transporter (5-HTT) in pulmonary vessels reduces chronic hypoxia-induced PH [START_REF] Eddahibi | Attenuated hypoxic pulmonary hypertension in mice lacking the 5hydroxytryptamine transporter gene[END_REF] . We tested therefore the 5-HT uptake activity in cultures of lung vasculature. In +/+ hypoxic vessels, we observed a trend toward a decrease in 5-HT uptake while, in 5-HT 2B -/-mice, an increase in basal 5-HTT activity was detected. The 5-HT uptake activity was almost entirely blocked by DF in both +/+ and 5-HT 2B -/hypoxic vessels (Fig. 4a). From this latter observation, it can be concluded that defective 5-HT uptake is not sufficient to induce pulmonary hypertensive response in 5-HT 2B -/-mice.

To elucidate how 5-HT receptors control hypoxic responses, we investigated their expression in PH lung vasculature. While 5-HT 2A R expression was not modified by exposure of +/+ mice to hypoxia and DF, 5-HT 1B R expression increased 1.75 ± 0.1 times (P <0.01, n = 6), though this could be prevented by RS. More strikingly, hypoxia itself and in the presence of DF or of RS increased significantly the mean 5-HT 2B R-specific binding (2.6 ± 0.2 times, 2.9 ± 0.2 times, and 2.2 ± 0.1 times, respectively) in +/+ mice (Fig. 4b). Furthermore, we observed significant (P < 0.0001) correlations between the entire individual 5-HT 2B R binding and RVSP values (Fig. 4c) and between the basal thymidine incorporation indexes in lungs and both 5-HT 2B R binding and RVSP values but only in mice submitted to hypoxia and in the presence of active 5-HT 2B R (Fig. 4de). These correlations further substantiate the evidence that 5-HT 2B R is required for a hypoxic increase in RVSP and thus for the PH pathogenesis. Accordingly, a preliminary screening of 5-HT receptors expression in biopsies of human pulmonary artery indicates that the numbers of binding sites are markedly higher in PH patients than in non-PH patients for 5-HT 1B R and 5-HT 2B R (1.98±0.05 times and 4.32±0.76 times, respectively, P <0.01, n = 5), but not for 5-HT 2A R (0.99±0.10 times, n = 5, NS).

Discussion

The identification of specific pathways involved in the development of PH is dependent upon pharmacological, transgenic or targeted gene disruption approaches on animal models. The only commonly utilized mouse model of PH is the chronic hypoxic case. Mice with genetic ablation of known vasodilators, endothelial NO synthase [START_REF] Steudel | Sustained pulmonary hypertension and right ventricular hypertrophy after chronic hypoxia in mice with congenital deficiency of nitric oxide synthase 3[END_REF] or prostacyclin receptor [START_REF] Hoshikawa | Prostacyclin receptor-dependent modulation of pulmonary vascular remodeling[END_REF] , appear more sensitive to hypoxia. By contrast, targeted disruption of genes involved in pulmonary vasoconstriction appears to reduce hypoxiainduced PH in mice. Mice deficient for 5-HT 1B R (5-HT 1B -/-) developed less severe PH and vascular remodeling than +/+ mice [START_REF] Keegan | Contribution of the 5-HT1B receptor to hypoxia-induced pulmonary hypertension. Converging evidence using 5-HT1B-receptor knockout mice and the 5-HT1B/1D-receptor antagonist GR127935[END_REF] . The 5-HT 1B Rs play a role in the development of PH via enhanced 5-HT 1B R-mediated contraction of the pulmonary arterial circulation [START_REF] Maclean | Pulmonary hypertension, anorexigens and 5-HT: pharmacological synergism in action?[END_REF] , but this activity is not limiting in the development of PH since 5-HT 1B -/-mice are still responding to hypoxia [START_REF] Keegan | Contribution of the 5-HT1B receptor to hypoxia-induced pulmonary hypertension. Converging evidence using 5-HT1B-receptor knockout mice and the 5-HT1B/1D-receptor antagonist GR127935[END_REF] . However, using the chronic hypoxic mice model, we are able to demonstrate the complete absence of PH and vascular remodeling in hypoxic 5-HT 2B R-inactive mice indicating that 5-HT 2B Rs are limiting for chronic hypoxic responses, though independently of acute hypoxic vasoconstrictive responses.

Although congenital cardiac defects increase the risk of developing PH [START_REF] Rabinovitch | Problems of pulmonary hypertension in children with congenital cardiac defects[END_REF] , LV dysfunction in 5-HT 2B -/-mice [START_REF] Nebigil | Ablation of serotonin 5-HT2B receptors in mice leads to abnormal cardiac structure and function[END_REF] could impact on the cardiovascular remodeling responses, but this can be ruled out in 5-HT 2B -/-mice since: (i) The cardiac phenotype of 5-HT 2B -/-mice is characterized by LV dysfunctions but no apparent RV alterations [START_REF] Nebigil | Ablation of serotonin 5-HT2B receptors in mice leads to abnormal cardiac structure and function[END_REF] . (ii) Basal pulmonary pressure in 5-HT 2B -/-mice is indistinguishable from +/+ mice. (iii) No PH developed in +/+ mice exposed to chronic hypoxia in the presence of 5-HT 2B R antagonist (RS), clearly demonstrating that the activation of 5-HT 2B Rs is required for hypoxia-induced PH independently of any prior cardiac phenotype. Furthermore, some mutant mice strains developing cardiomyopathy such as mice lacking natriuretic peptide receptor A have an increased sensitivity to hypoxia of their lung vasculature [START_REF] Zhao | NPR-A-Deficient mice show increased susceptibility to hypoxia-induced pulmonary hypertension[END_REF] . Combined, these observations strongly support the conclusion that the absence of hypoxic response of 5-HT 2B -/-mice cannot be attributed to their cardiomyopathy. Some 5-HTT blockers and substrates such as DF are implicated in PH pathogenesis, while others, e.g. m-CPP are not [START_REF] Buczko | Effect of fenfluramine on 5hydroxytryptamine uptake and release by rat blood platelets[END_REF][START_REF] Rothman | Evidence for possible involvement of 5-HT2B receptors in the cardiac valvulopathy associated with fenfluramine and other serotonergic medications[END_REF] . The DF effect on PH was initially suggested to result from 5-HTT inhibition since a change in platelet 5-HT uptake could explain the raised 5-HT plasma level observed in PH patients [START_REF] Egermayer | Role of serotonin in the pathogenesis of acute and chronic pulmonary hypertension[END_REF][START_REF] Hervé | Increased plasma serotonin in primary pulmonary hypertension[END_REF][START_REF] Kéreveur | High plasma serotonin levels in primary pulmonary hypertension : effect of long-term epoprostenol (Prostacyclin) therapy[END_REF][START_REF] Humbert | Pulmonary arterial hypertension and type-I glycogen-storage disease: the serotonin hypothesis[END_REF] . Other 5-HTT blockers, however, such as 5-HT-specific-reuptake-inhibitors or tricyclic antidepressants, have not been associated with pulmonary pathology. Upon 5-HT 2B Rs, the EC 50 of DF is over 1 µM, but the major metabolite of DF, norDF, which acts as a potent agonist with nanomolar affinity (pEC 50 = 8.06 ± 0.16) [START_REF] Fitzgerald | Possible role of valvular serotonin 5-HT2B receptors in the cardiopathy associated with fenfluramine[END_REF] , requires chronic treatments to be detected (Supplementary Data). Other high affinity agonists at 5-HT 2B Rs do not appear to be associated with PH in humans [START_REF] Rothman | Evidence for possible involvement of 5-HT2B receptors in the cardiac valvulopathy associated with fenfluramine and other serotonergic medications[END_REF] and thus a combinatorial effect at different receptors with transporters may be required for PH development. However, the significant DF potentiation of a hypoxia-dependent increase in RVSP and remodeling is completely eliminated in 5-HT 2B -/-mice. Since the 5-HT uptake activity is largely reduced in lungs of 5-HT 2B -/-mice exposed to hypoxia and DF and which do not develop PH, blocking 5-HT uptake activity is not sufficient to trigger hypoxiadependent PH. Consistent with this observation is the fact that mice deficient for 5-HTT (5-HTT -/-) are still responsive to hypoxia though they develop less severe PH and vascular remodeling when compared with the +/+ mice [START_REF] Eddahibi | Attenuated hypoxic pulmonary hypertension in mice lacking the 5hydroxytryptamine transporter gene[END_REF] .

In animal models, DF does not induce PH per se, but it can potentiate hypoxiainduced PH: dogs chronically treated with DF revealed increased pulmonary vascular resistance upon exposure to hypoxia [START_REF] Naeije | Effects of chronic dexfenfluramine treatment on pulmonary hemodynamics in dogs[END_REF] . Rats exposed to a 4-week DF treatment in normoxia followed by 2-week hypoxia developed stronger PH than the controls, suggesting also that DF pre-treatment promotes the development of PH in rats [START_REF] Eddahibi | Dexfenfluramine-associated changes in 5-hydroxytryptamine transporter expression and development of hypoxic pulmonary hypertension in rats[END_REF] . The recently proposed mechanism by which DF-like drugs may promote pulmonary vascular remodeling by stimulating 5-HTT expression [START_REF] Eddahibi | Serotonin transporter overexpression is responsible for pulmonary artery smooth muscle hyperplasia in primary pulmonary hypertension[END_REF] cannot fully explain why patients developing PH after DF treatment reveal the same proportion of 5-HTT promoter polymorphism as do the PH patients in general. The lack of a higher incidence of the 5-HTT polymorphism in the group that ingested DF and developed PH suggests also that other factors are required for DF-induced PH to develop [START_REF] Rabinovitch | Linking a serotonin transporter polymorphism to vascular smooth muscle proliferation in patients with primary pulmonary hypertension[END_REF] . In our hypoxic mouse model, the need of active 5-HT 2B Rs to reveal the DF potentiating action demonstrates that, although a contribution of 5-HTT to DF effects is likely, the DF potentiation of hypoxia-induced PH requires 5-HT 2B R activation.

A lung vascular proliferative response occurs in response to 5-HT, but it remains debatable whether the 5-HT mitogenic effect is initiated through a 5-HT 2 R or the 5-HTT, or both [START_REF] Fanburg | A new role for an old molecule: serotonin as a mitogen[END_REF][START_REF] Lee | Serotonin stimulates mitogenactivated protein kinase activity through the formation of superoxide anion[END_REF] . The observation that lung tissue from PH patients without the long 5-HTT promoter polymorphism revealed proliferative response to 5-HT higher than normal [START_REF] Eddahibi | Serotonin transporter overexpression is responsible for pulmonary artery smooth muscle hyperplasia in primary pulmonary hypertension[END_REF] , indicates also that other components of the 5-HT pathway, such as receptors or downstream effectors, are needed to promote PH [START_REF] Rabinovitch | Linking a serotonin transporter polymorphism to vascular smooth muscle proliferation in patients with primary pulmonary hypertension[END_REF] . In lung vascular cells, 5-HT induced DNA synthesis seemed dependent on the 5-HTT, since it was prevented by some 5-HTT inhibitors, but unaltered by ketanserin, a 5-HT 2A R antagonist [START_REF] Fanburg | A new role for an old molecule: serotonin as a mitogen[END_REF] . However, in lung smooth muscle cells, the calcium transients due to 5-HT persisted in Na + -free conditions (no 5-HT uptake) and 5-HTT inhibitors were still inhibiting 5-HT-induced mitogenesis [START_REF] Pitt | Serotonin increases DNA synthesis in rat proximal and distal pulmonary vascular smooth muscle cells in culture[END_REF] . Thus, the 5-HT 2 R rather than the 5-HTT initiated the 5-HT effect, indicating that 5-HTT inhibitors have 5-HT 2 R antagonist properties as recently confirmed [START_REF] Sanchez | Comparison of the effects of antidepressants and their metabolites on reuptake of biogenic amines and on receptor binding[END_REF] . In contrast to what is observed in hypoxic +/+ mice lung vasculature, the total lack of increased basal thymidine incorporation in hypoxic 5-HT 2B -/-mice tissue indicates an absolute requirement of 5-HT 2B R for chronic hypoxia-induced mitogenesis. This finding is validated by the complete prevention of proliferation and vascular remodeling upon treatment of hypoxic +/+ mice with the 5-HT 2B R antagonist RS. Although a direct contribution of 5-HT 2B R to the chronic hypoxia-induced mitogenesis remains to be firmly established, it is likely since (i) we have previously reported that activation of 5-HT 2B R can initiate cell cycle progression [START_REF] Nebigil | 5-Hydroxytryptamine 2B receptor regulates cell-cycle progression: Cross talk with tyrosine kinase pathways[END_REF] and (ii) the basal thymidine incorporation indexes in lungs are significantly correlated with both 5-HT 2B R binding and RVSP values, only when mice are submitted to hypoxia and in the presence of active 5-HT 2B R. Other serotonergic molecules [START_REF] Maclean | 5-hydroxytryptamine and the pulmonary circulation: receptors, transporters and relevance to pulmonary arterial hypertension[END_REF][START_REF] Eddahibi | Attenuated hypoxic pulmonary hypertension in mice lacking the 5hydroxytryptamine transporter gene[END_REF] may indeed participate in the hypoxic mitogenic process of PH, but our animal model provides the first direct evidence that 5-HT 2B R activation is a limiting, non-redundant step, required for the hypoxia-induced vascular proliferation in mice.

We report that the 5-HT 2B R effect is not at the level of acute vasoconstrictive responses but rather the signaling mechanisms that control factors important in vascular remodeling such as elastase. PH vascular remodeling is associated with increased serine elastase activity working either directly or via activation of matrix metalloproteinases, and elastase inhibitors can reverse advanced pulmonary vascular disease [START_REF] Cowan | Complete reversal of fatal pulmonary hypertension in rats by a serine elastase inhibitor[END_REF] . The matrix metalloproteinases are a family of proteases implicated in extracellular matrix turnover and hence contribute to vascular smooth muscle and endothelial cell migration and proliferation [START_REF] Rabinovitch | EVE and beyond, retro and prospective insights[END_REF] . Furthermore, elastase can increase TGF-ß availability, optimizing the mitogenic response in cultured vascular cells [START_REF] Vu | Matrix metalloproteinases: effectors of development and normal physiology[END_REF] . Our data indicate that 5-HT 2B Rs are crucial for hypoxia-induced remodeling factors by their regulation of elastase activity and TGF expression in mice lung vasculature. In humans, heterozygous mutations within the bone morphogenetic protein type II receptor (BMPR-II) gene of the TGF-ß cell-signaling superfamily have recently been identified in 25% of sporadic and 55% of familial cases of PH [START_REF] Loscalzo | Genetic clues to the cause of primary pulmonary hypertension[END_REF] . The low penetrancy of these mutations (10-20%) suggests a need for other factors (genetic, environmental, pathologic) in PH pathogenesis, see for review [START_REF] Loscalzo | Genetic clues to the cause of primary pulmonary hypertension[END_REF] . Recent evidence has shown that both TGF-ß and BMPs proliferative functions are altered in smooth muscles from PH patients [START_REF] Morrell | Altered growth responses of pulmonary artery smooth muscle cells from patients with primary pulmonary hypertension to transforming growth factorbeta(1) and bone morphogenetic proteins[END_REF] . In familial forms of PH, the primary event appears to be vascular-cell proliferation 3 . However, hypoxia is a preponderant environmental factor that generates PH in humans [START_REF] Tucker | Role of vascular smooth muscle in the development of high altitude pulmonary hypertension: an interspecies evaluation[END_REF] . Vascular proliferative changes, which may represent adaptive (maladaptive) responses to this exogenous stimulus are associated with increased 5-HT 2B R expression and require activation of 5-HT 2B Rs. In complete agreement with the mice model is the increase in 5-HT 2B R expression found in lungs of PH patients. Furthermore, this finding could explain the proliferative response to 5-HT, higher than normal, observed in PH patients [START_REF] Eddahibi | Serotonin transporter overexpression is responsible for pulmonary artery smooth muscle hyperplasia in primary pulmonary hypertension[END_REF] and independent of their 5-HTT genotype [START_REF] Rabinovitch | Linking a serotonin transporter polymorphism to vascular smooth muscle proliferation in patients with primary pulmonary hypertension[END_REF] .

In conclusion, our work supports the scheme presented (Fig. 5) in which 5-HT 2B Rs hypoxia-dependent increased expression has a central role in pulmonary hypertensive responses in mice. Subsequently, active 5-HT 2B Rs are an absolute necessity for lung vascular proliferation, elastase-and TGF-ß-dependent remodeling. In addition, DF action at 5-HTT acts as an amplifier of norDF agonist action at 5-HT 2B Rs. All these activities contribute toward increases in RVSP and PH pathogenesis in mice. Similar increased 5-HT receptor expression in lungs of PH patients suggests that comparable mechanisms exist in humans and indicate that 5-HT 2B R expression may be used as a diagnostic criterion for susceptibility to PH. The 5-HT 2B Rs should now be considered as a potential target for developing new treatments of PH in humans.

'Methods'. Hypoxia.

The 5-HT 2B R mutant mice (5-HT 2B -/-) were generated in the 129PAS pure genetic background that was used as controls ( +/+ ). Mice 6 weeks of age (20-25 g) were randomly divided into two groups of 5 males and 5 females, one maintained in room air (21% O 2 ) and the other exposed to hypoxia, for 2 or 5 weeks. Normobaric hypoxic chambers (21-23°C) maintained by 5.5 l/min flow of hypoxic air (10% O 2 , 90% N 2 ) were opened twice a day for 15 min for cage cleaning, food and water supplies. The oxygen concentrations were monitored continuously using blood gas analyzers and soda lime was used to reduce the concentration of carbon dioxide. Normoxic mice were kept in the same room with the same 12/12 light-dark cycle. The vehicle, RS at 1 mg/kg/day or DF at the "therapeutic" dose of 2.5 mg/kg/day was delivered by miniosmotic pump (Alzet) at the beginning of the hypoxic (or normoxic) treatment. Acute hypoxic experiments consisted of the exposure of conscious mice to five successive hypoxic (10% O 2 ) challenges [START_REF] Eddahibi | Effect of dexfenfluramine treatment in rats exposed to acute and chronic hypoxia[END_REF] , hemodynamic variables were recorded first while the animal was breathing air, then after 10 min of exposure to the hypoxic gas mixture and finally 20 min after returning to normoxia. Five similar successive hypoxic challenges were repeated for each mouse. All animal care and procedures were in accordance with institutional guidelines and European regulations.

Cardiovascular evaluations.

Mice were anesthetized with intraperitoneal ketamine hydrochloride (60 mg/kg) and xylazine (8 mg/kg). Right ventricular systolic pressure (RVSP) was measured by insertion into the right ventricle of a 26-gauge needle connected to a pressure transducer. After a blood sample was collected by cardiac puncture for DF and norDF measurements (Supplementary Data), the pulmonary artery was cannulated through an incision in the right ventricle and perfused with Earle's balanced salt solution (37°C, 20 cm H 2 O pressure). The heart and lungs were removed en bloc, and the airways were distended with 10% formaldehyde solution and fixed for 3 days. For the assessment of right ventricular hypertrophy, weights of the right ventricular free wall (RV) and the left ventricle together with the septum (LV + S) were measured separately. For morphometric analysis, one sagittal section (5 µm in thickness) was obtained from each left lung and after paraffin embedding, stained with hematoxylin-phloxin-saffron and orcein-picroindigo-carmine. In each section, a total of 55 to 65 vessels was examined. Pulmonary arteries (25-100 µm) associated with an airway distal to the respiratory bronchiole were counted. The arteries were considered as muscularized if they possessed a distinct double-elastic lamina visible for the complete diameter in the vessel cross-section [START_REF] Keegan | Contribution of the 5-HT1B receptor to hypoxia-induced pulmonary hypertension. Converging evidence using 5-HT1B-receptor knockout mice and the 5-HT1B/1D-receptor antagonist GR127935[END_REF] . If they were seen in smaller part of the arterial circumference, they were recorded as partially muscular arteries and if absent as non-muscular arteries. These observations were done in a blinded setup vs. genotypes and treatments.

Explants.

Lung vascular bed around pulmonary arteries was collected and prepared for organ culture [START_REF] Cowan | Regression of hypertrophied rat pulmonary arteries in organ culture is associated with suppression of proteolytic activity, inhibition of tenascin-C, and smooth muscle cell apoptosis[END_REF] . Each explant (10-15 mg) was maintained in DMEM supplemented with 5-HT-depleted serum (10%) for a maximal period of 3 days. Fragments (1-2 mg each) seeded onto tissue culture vessels for 24-h were washed with HBSS and grown for 24-h in serum-free medium {DMEM/F-12 (1:1) with 5 µg/ml insulin, 5 µg/ml transferrin, 30 nM selenium, 20 nM progesterone, and 100 µM putrescine} before thymidine incorporation was performed within next 12-h 14 . Conditioned media from all cultures were collected and stored. For elastolytic activities, conditioned media were incubated at 37°C with [ 3 H]-elastin [START_REF] Cowan | Complete reversal of fatal pulmonary hypertension in rats by a serine elastase inhibitor[END_REF] . TGF-ß was also measured in conditioned media with an ELISA using a pan-specific anti-TGF-ß polyclonal antibody (R&D Systems catalog # AB-100-NA). To measure 5-HT uptake, after incubation for 1 min with 10 -7 M [ 3 H]-5-HT binoxalate, each lung fragment was lysed by addition of 0.1 N NaOH and radioactivity was counted. Specific 5-HT uptake assessed as the difference between total uptake and uptake in the presence of 1 µM paroxetine, was expressed in picomoles of 5-HT taken up per milligram of protein per minute. Measurements of the 5-HT receptor expression was performed using a 5-HT 2B R-specific tritiated radioligand (LY266070), a 5-HT 1B R-specific iodinated radioligand ( 125 I-GTI), or for 5-HT 2A R the fraction of 5-HT 2 R-specific iodinated radioligand ( 125 I-DOI) insensitive to LY266070 as previously described [START_REF] Nebigil | Serotonin 2B receptor is required for heart development[END_REF] .

Statistics.

The reported data represent the mean of individual values ± SEM (n = number of individuals at the end of treatments as indicated in the text). Comparisons were performed using the non-parametric Kruskal-Wallis test. All statistical difference vs. normoxic untreated control values is indicated by an asterisk and vs. chronic hypoxia values by a cross and significance was set at P < 0.05. This level of significance also applies to correlation between individual values, assessed by Kendall rank coefficients. For the groups of pulmonary artery muscularization, statistical comparisons were made by one way ANOVA. When statistical significance was attained (P < 0.05), difference between groups was established using the Newman-Keuls multiple comparison test. -/-mice exposed to hypoxia for 5 weeks (n = 7) exhibits any change in RVSP vs. vehicle-treated normoxic mutant mice (n = 9), neither in the presence of RS (n = 9) nor DF (n = 9). Chronic exposure of normoxia to these compounds alone is ineffective for both genotypes. Any statistical difference from normoxic untreated control values is indicated by an asterisk, and from chronic hypoxia values by a cross (P < 0.05). b A significantly different degree of muscularization (muscular, M, partially muscular, PM, or non-muscular, NM) is observed in hypoxic pulmonary arteries compared with normoxic +/+ mice (P < 0.05). The degree of muscularization in the DF plus hypoxia mice is also significantly more severe than the plain hypoxia or the vehicle-treated mice (P < 0.05). Alternately, the degree of pulmonary artery muscularization does not differ significantly from controls after RS treatment of hypoxic +/+ mice or in hypoxia-treated 5-HT 2B -/-mice with, or without, RS or DF. c, m, o, +/+ V), hypoxic +/+ mice (g, h, i, +/+ H5W), mice treated by hypoxia in the presence of DF, +/+ mice (j, k, l, n, p, +/+ H5W+DF) or 5-HT 2B -/-mice (d, e, f, -/- H5W+DF) are presented. Sections of lung tissue stained with haematoxilin at low magnification (a, d, g, j), and intermediate magnification (b, e, h, k), illustrate the altered lung morphology and increased cell proliferation. At higher magnification, when compared with normoxic +/+ mice (c, m, o, +/+ V), the thickness of pulmonary vessels (arrowheads) media is clearly increased in hypoxic +/+ mice (i, +/+ H5W), and is further increased in the presence of DF (l, n, p, +/+ H5W+DF) whereas no modification of lung morphology was detected in normoxic 5-HT 2B -/-mice, in 5-HT 2B -/-mice after hypoxia with DF (f, -/-H5W+DF) or without (not illustrated). The pulmonary vessels media is illustrated by van Giesen staining of elastin (c, f, i, l) or by staining with antibodies against smooth muscle alpha-actin (m, n, actin) or against 5-HT 2B R (o, p, 5-HT 2B ). This staining reveals that the proliferating cell population are 5-HT 2B R positive whereas only few 5-HT 2B R positive cells could be observed in normoxic +/+ mice. Bars = 500 µm, (a, d, g, j). Bars = 125 µm, (b, e, h, k). Bars = 25 µm,(c,f,i,l,m,n,o,p). -/-mice. b In +/+ lung vessel explant supernatants, total elastolytic activity is significantly increased after chronic hypoxic exposure (344 ± 90 cpm/mg of tissue proteins, n = 10 vs. 33 ± 4 for normoxia, n = 19). A further significant increase is observed after chronic hypoxia in the presence of DF (496 ± 55 cpm/mg prot, n = 15) while near normoxic values are obtained in the presence of RS (67 ± 4 cpm/mg prot, n = 10). By contrast, hypoxia does not modify the elastolytic activity in supernatants of 5-HT 2B -/-mice following either treatment. c In +/+ lung vessel explant supernatants, TGF-ß levels are significantly increased upon hypoxia (322 ± 72 pg/ml vs. 83 ± 10 for normoxia, n = 10). This increase is potentiated by DF (401 ± 56 pg/ml) but blocked by RS (114 ± 8 pg/ml). No significant TGF-ß level modification can be detected in supernatants of hypoxic lungs of 5-HT 2B -/-mice after either treatment. d Exposure to acute hypoxic challenges (AH) triggers a significant increase in RVSP in both 5-HT 2B R -/-(22.5 ± 1.0 mm Hg, n = 10) and +/+ (21.6 ± 1.0 mm Hg, n = 10) mice, when compared with vehicle treated (AV) control mice (18.9 ± 1.0 mm Hg, n = 10 and 18.4 ± 0.5 mm Hg, n = 29, respectively). DF administered 5 min before the hypoxic period (ADF) further increases these values independently of the mice genotype. e In +/+ lungs, total elastolytic activity is significantly increased following acute hypoxic challenges (43.4 ± 6.1 cpm/mg of tissue proteins vs. 33.0 ± 3.6 for normoxia) though it is not significantly modified in 5-HT 2B R -/-mice (26.7 ± 4.2 cpm/mg of tissue proteins vs. 21.9 ± 4.5 for normoxia). normoxic controls (V5W), corresponding to a 28.6 % decrease of the maximal initial rate of uptake and no change in affinity for 5-HT. For the 5-HT 2B -/-mice, basal 5-HTT activity is slightly higher (by 24%) than normoxic +/+ values but is unaffected by hypoxia or RS (RS5W). DF treatment (DF5W) almost completely blocked the 5-HT uptake in the hypoxic +/+ and 5-HT 2B -/-mice. b Hypoxia increases significantly the number of 5-HT 2B R specific binding sites (64.7 ± 4.7 fmoles/mg prot, n = 10 vs. 25.3 ± 1.5 for normoxic lungs, n = 19). This increase in 5-HT 2B R binding sites is not significantly affected by either DF (71.9 ± 5.8 fmoles/mg prot, n = 15) or RS (56.0 ± 2.0 fmoles/mg prot, n = 10). Indeed, 5-HT 2B R-specific sites were not detected in 5-HT 2B -/mice (nd). c,d,e Correlation between parameters involved in PH development. A highly significant correlation (P < 0.0001) is observed between all individual values of the number of 5-HT 2B R specific binding sites and the RVSP increase (c). Moreover, only when +/+ mice are exposed to normoxia (+/+V5W, diamonds), hypoxia alone (+/+H5W, circles) or with DF (+/+H5W+DF5W, squares) but not RS (+/+H5W+RS5W, triangles) (i.e. active 5-HT 2B R), the thymidine incorporation index correlates significantly with the number of 5-HT 2B R binding sites (r = 0.987 for +/+H5W+DF5W and 0.868 for +/+H5W, both P < 0.01) (d) or the RVSP (r = 0.952 for +/+H5W+DF5W, 0.939 for +/+H5W and 0.827 for +/+V5W, in each case P < 0.01) (e). Abbreviations: +/+ mice exposed to hypoxia for 5 weeks ( +/+ H5W), vehicle-treated normoxic +/+ mice ( +/+ V). +/+ mice exposed to hypoxia in the presence of DF ( +/+ H5WDF), 5-HT 2B -/-mice exposed to hypoxia for 5 weeks ( -/-H5W), vehicle-treated normoxic 5-HT 2B -/-mice ( -/-V), in the presence of DF ( -/-H5WDF), and mice exposed chronically to these compounds alone ( +/+ DF5W, -/-DF5W). Any statistical difference from normoxic untreated control values is indicated by an asterisk and from chronic hypoxia values by a cross (P < 0.05, Kruskal-Wallis test). Abbreviations: +/+ mice exposed to hypoxia for 5 weeks ( +/+ H5W), vehicle-treated normoxic +/+ mice ( +/+ V). +/+ mice exposed to hypoxia in the presence of DF ( +/+ H5WDF), 5-HT 2B -/-mice exposed to hypoxia for 5 weeks ( -/-H5W), vehicle-treated normoxic 5-HT 2B -/-mice ( -/-V), in the presence of DF ( -/-H5WDF), and mice exposed chronically to these compounds alone ( +/+ DF5W, -/-DF5W), mice exposed to five successive acute hypoxic challenges in 5-HT 2B R -/-( -/-AH) or +/+ ( +/+ AH) mice and mice exposed to five successive acute hypoxic challenges in the presence of DF administered 5 min before the hypoxic period ( +/+ AHDF, -/-AHDF). Reference: Midha, K.K. et al. The identification of two new urinary metabolites of fenfluramine in man. Xenobiotica 13, 31-38. (1983).

Figure 1

 1 Figure1Requirement of 5-HT 2B R for hypoxia-induced RVSP and vascular muscularization. a In +/+ mice exposed to hypoxia for 5 weeks (H5W), RVSP is significantly higher (30.6 ± 3.8 mm Hg, n = 10) than in vehicle-treated normoxic +/+ mice (V5W, 18.5 ± 0.4 mm Hg n = 28). Simultaneous exposure of +/+ mice to hypoxia in the presence of the specific 5-HT 2B R antagonist RS (RS5W) prevents the increase in RVSP (23.6 ± 1.5 mm Hg, n = 10) whereas the presence of DF (DF5W) further increases significantly the RVSP (42.3 ± 4.7 mm Hg, n = 15). By contrast, none of the 5-HT 2B -/-mice exposed to hypoxia for 5 weeks (n = 7) exhibits any change in RVSP vs. vehicle-treated normoxic mutant mice (n = 9), neither in the presence of RS (n = 9) nor DF (n = 9). Chronic exposure of normoxia to these compounds alone is ineffective for both genotypes. Any statistical difference from normoxic untreated control values is indicated by an asterisk, and from chronic hypoxia values by a cross (P < 0.05). b A significantly different degree of muscularization (muscular, M, partially muscular, PM, or non-muscular, NM) is observed in hypoxic pulmonary arteries compared with normoxic +/+ mice (P < 0.05). The degree of muscularization in the DF plus hypoxia mice is also significantly more severe than the plain hypoxia or the vehicle-treated mice (P < 0.05). Alternately, the degree of pulmonary artery muscularization does not differ significantly from controls after RS treatment of hypoxic +/+ mice or in hypoxia-treated 5-HT 2B -/-mice with, or without, RS or DF.

Figure 2

 2 Figure2Requirement of 5-HT 2B R for hypoxia-induced lung remodeling. Sections of lung tissue of different genotype following different treatments, normoxic +/+ mice (a, b, c, m, o, +/+ V), hypoxic +/+ mice (g, h, i, +/+ H5W), mice treated by hypoxia in the presence of DF, +/+ mice (j, k, l, n, p, +/+ H5W+DF) or 5-HT 2B -/-mice (d, e, f, -/- H5W+DF) are presented. Sections of lung tissue stained with haematoxilin at low magnification (a, d, g, j), and intermediate magnification (b, e, h, k), illustrate the altered lung morphology and increased cell proliferation. At higher magnification, when compared with normoxic +/+ mice (c, m, o, +/+ V), the thickness of pulmonary vessels (arrowheads) media is clearly increased in hypoxic +/+ mice (i, +/+ H5W), and is further increased in the presence of DF (l, n, p, +/+ H5W+DF) whereas no modification of lung morphology was detected in normoxic 5-HT 2B -/-mice, in 5-HT 2B -/-mice after hypoxia with DF (f, -/-H5W+DF) or without (not illustrated). The pulmonary vessels media is illustrated by van Giesen staining of elastin (c, f, i, l) or by staining with antibodies against smooth muscle alpha-actin (m, n, actin) or against 5-HT 2B R (o, p, 5-HT 2B ). This staining reveals that the proliferating cell population are 5-HT 2B R positive whereas only few 5-HT 2B R positive cells could be observed in normoxic +/+ mice. Bars = 500 µm, (a, d, g, j). Bars = 125 µm, (b, e, h, k). Bars = 25 µm, (c, f, i, l, m, n, o, p).

Figure 3

 3 Figure3Requirement of 5-HT 2B Rs for lung remodeling factors but not for acute vasoconstriction. a After exposure to hypoxia, +/+ mice lung vasculature exhibits a significantly increased (H5W, 1.95 ± 0.06 times basal control, n = 10) tritiated thymidine incorporation (per mg of tissue proteins), when compared normoxic with lung vessels (V5W, 0.99 ± 0.05). The basal normoxic incorporation is 2112 ± 355 cpm/mg of tissue proteins. This increase is significantly potentiated by DF (DF5W, 2.51 ± 0.27 times basal, n = 10) and completely prevented by either RS treatment (RS5W) or by the elimination of 5-HT 2B R in 5-HT 2B -/-mice. b In +/+ lung vessel explant supernatants, total elastolytic activity is significantly increased after chronic hypoxic exposure (344 ± 90 cpm/mg of tissue proteins, n = 10 vs. 33 ± 4 for normoxia, n = 19). A further significant increase is observed after chronic hypoxia in the presence of DF (496 ± 55 cpm/mg prot, n = 15) while near normoxic values are obtained in the presence of RS (67 ± 4 cpm/mg prot, n = 10). By contrast, hypoxia does not modify the elastolytic activity in supernatants of 5-HT 2B -/-mice following either treatment. c In +/+ lung vessel explant supernatants, TGF-ß levels are significantly increased upon hypoxia (322 ± 72 pg/ml vs. 83 ± 10 for normoxia, n = 10). This increase is potentiated by DF (401 ± 56 pg/ml) but blocked by RS (114 ± 8 pg/ml). No significant TGF-ß level modification can be detected in supernatants of hypoxic lungs of 5-HT 2B -/-mice after either treatment. d Exposure to acute hypoxic challenges (AH) triggers a significant increase in RVSP in both 5-HT 2B R -/-(22.5 ± 1.0 mm Hg, n = 10) and +/+ (21.6 ± 1.0 mm Hg, n = 10) mice, when compared with vehicle treated (AV) control mice (18.9 ± 1.0 mm Hg, n = 10 and 18.4 ± 0.5 mm Hg, n = 29, respectively). DF administered 5 min before the hypoxic period (ADF) further increases these values independently of the mice genotype. e In +/+ lungs, total elastolytic activity is significantly increased following acute hypoxic challenges (43.4 ± 6.1 cpm/mg of tissue proteins vs. 33.0 ± 3.6 for normoxia) though it is not significantly modified in 5-HT 2B R -/-mice (26.7 ± 4.2 cpm/mg of tissue proteins vs. 21.9 ± 4.5 for normoxia).

Figure 4

 4 Figure 4 Hypoxia increases 5-HT 2B R expression in lungs. a The uptake activity of lung vasculature from +/+ hypoxic mice (H5W) is non-significantly reduced by 23.4 % vs.normoxic controls (V5W), corresponding to a 28.6 % decrease of the maximal initial rate of uptake and no change in affinity for 5-HT. For the 5-HT 2B -/-mice, basal 5-HTT activity is slightly higher (by 24%) than normoxic +/+ values but is unaffected by hypoxia or RS (RS5W). DF treatment (DF5W) almost completely blocked the 5-HT uptake in the hypoxic +/+ and 5-HT 2B -/-mice. b Hypoxia increases significantly the number of 5-HT 2B R specific binding sites (64.7 ± 4.7 fmoles/mg prot, n = 10 vs. 25.3 ± 1.5 for normoxic lungs, n = 19). This increase in 5-HT 2B R binding sites is not significantly affected by either DF (71.9 ± 5.8 fmoles/mg prot, n = 15) or RS (56.0 ± 2.0 fmoles/mg prot, n = 10). Indeed, 5-HT 2B R-specific sites were not detected in 5-HT 2B

Figure 5

 5 Figure5 Model of 5-HT 2B R-dependent hypoxia-induced RVSP in mice. Chronic hypoxia triggers a striking increase in 5-HT 2B R expression. Chronic DF (Dexfen) potentiates hypoxia by affecting RVSP through activation of 5-HT 2B R by norDF (norDexfen), with an amplification from 5-HTT blockage. Lung proliferation and remodeling also appear to be dependent on the 5-HT 2B R signaling activity and involve extracellular elastase activation and increase of TGF-ß levels.

Table I -Evaluation of RV/LV+S

 I

		+/+V +/+DF5W	+/+H5W	+/+H5WDF	-/-V -/-DF5W	-/-H5W -/-H5WDF
	RV/LV+S	0.21	0.19	0.29	0.39	0.46	0.45	0.47	0.46
	(%±sem)	±0.01 ±0.03	±0.03*	±0.04* †	±0.01 ±0.04	±0.02 ±0.02
	n	10	10	14	15	9	10	7	8

Table Supplementary Data -DF and norDF plasma levels (in nM ± SEM).

 Supplementary

		+/+V +/+DF5W	+/+H5WDF +/+AHDF	-/-V -/-5WDF	-/-H5WDF	-/-AHDF
	DF	0	6,4±0,8	7,2±0,5	425±65	0	13,5±16	8,9±1,1	391±43
	norDF	0	526±74	570±56	2,7±0,2	0	481±26	565±76	4,2±0,7
	n	10	10	6	9	9	10	8	10

DF and norDF plasma levels were measured by HPLC (Midha, K.

K. et al., 1983)

.

Acknowledgments

This work has been supported by funds from the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, the Hôpital Universitaire de Strasbourg, the Université Louis Pasteur, and by grants from the Fondation de France and from the Association pour la Recherche contre le Cancer. We thank P. Hickel for excellent technical assistance, J. Odillard for animal care, Dr. V. Mutel, Pr. J.-L. Laplanche, Pr. M. Mark, for critical reading of the manuscript and for helpful discussions, Drs. M. Pando and S. Brooks for english corrections, Drs. V. Mutel and D. Mc Namara for the gift of RS-127445 and Drs. F. Mentré and F. Godmard for statistical expertise.

Correspondence should be addressed to L.M. (e-mail: lucm@igbmc.u-strasbg.fr).


Francieli Zanon Boito

Rodrigo Kassick
email: rvkassick@inf.ufrgs.br

Philippe O A Navaux
email: navaux@inf.ufrgs.br

Yves Denneulin
email: yves.denneulin@imag.fr

Towards Fast Profiling of Storage Devices Regarding Access Sequentiality

Keywords: D, 4, 8 [Operating Systems]: Performance Storage Device

This work presents a tool for storage device profiling named SeRRa. Our tool obtains the sequential to random throughput ratio for reads and writes of different sizes. Several optimizations aim at adapting applications' access patterns in order to generate contiguous accesses for improved performance when accessing storage devices like hard disks. However, when considering other storage options like RAID arrays and SSDs, the access time ratio between contiguous and non-contiguous accesses may not compensate for these optimizations' cost. In this scenario, the information provided by our tool could be used to dynamically decide if optimizations are beneficial for performance, an important task to provide I/O throughput. In order to provide this information efficiently, SeRRa employs benchmarks to obtain the values for a subset of the parameter space and estimates the remaining values through a linear model. Extensive test campaigns with our tool on nine storage devices have shown median errors of approximately 5% (up to 55%) while taking only 1/168 of the originally needed time.

INTRODUCTION

For years, magnetic disks (Hard Disks Drives -HDDs) have been the main non-volatile storage device on personal computers and supercomputers for High Performance Computing (HPC). Because of that, most systems were adapted in order to obtain good performance when accessing these devices. For instance, disk schedulers employ elevator algo-SAC '2015, April 13 -17 2015, Salamanca, Spain http://dx.doi.org/10.1145/2695664.2695701.

rithms in order to minimize head movements [START_REF] Zhang | Self-learning disk scheduling[END_REF], improving data access performance. Several optimizations focus on generating sequential accesses instead of random ones, since this access pattern benefits HDDs.

Solid State Drives (SSDs) are a recent alternative to hard disks. These devices employ mainly flash memory to store data, with no mechanical parts. This characteristic makes them more resistant to falls and vibrations. Moreover, their advantages over HDDs include: size, noise generation, heat dissipation and energy consumption. However, despite the growing adoption of SSDs, their larger cost per byte still hampers their use on large-scale systems for HPC. Therefore, several parallel file system deployments on clusters still store data on hard disks. Nevertheless, hybrid solutions where both devices are used have been gaining popularity.

Another popular solution for storage on HPC systems is the use of RAID arrays that combine multiple hard disks onto a virtual unit for performance and reliability purposes. Data is striped among the disks and can be retrieved in parallel, which improves performance. Nonetheless, since both SSDs and RAID solutions are inherently different from HDDs, they should not be treated simply as "faster disks". Several assumptions about performance from HDDs do not hold when using SSDs and RAID arrays, and different requirements arise [START_REF] Rajimwale | Block management in solid-state devices[END_REF].

Regarding spatial locality, HDDs are known for having better performance when accesses are done sequentially. Although RAID arrays are composed by hard disks and their performance is usually better with sequential accesses, their organization complicates this behavior. The alignment of accesses to the stripe size has a great impact and must be considered. On the other hand, works that aim at characterizing SSDs' performance behavior achieve different conclusions. Despite most SSDs presenting better performance for sequential writes than for random writes, read operations' performance follows no general rule. On some SSDs, there is no difference between sequential and random accesses, but on others this difference achieves orders of magnitude [START_REF] Chen | Understanding intrinsic characteristics and system implications of flash memory based solid state drives[END_REF]. The sequential to random throughput ratio on some SSDs surpasses what is observed on some HDDs [START_REF] Rajimwale | Block management in solid-state devices[END_REF].

Therefore, we cannot simply classify optimizations -such as I/O schedulers [START_REF] Boito | Agios: Application-guided i/o scheduling for parallel file systems[END_REF] -by saying they are only suitable for HDDs or SSDs. Approaches that aim at generating contiguous accesses (originally designed for HDDs) can greatly improve performance when used on SSDs that are also sensitive to access sequentiality. Furthermore, on any device, the performance improvement caused by the use of a specific optimization may not compensate its overhead. Hence, these optimizations could be classified according to the sequential to random throughput ratio that devices must present in order to benefit from them.

In this scenario, this paper proposes a tool for storage devices profiling named SeRRa. Our tool reports, for a storage device, the sequential to random throughput ratio for read and write operations with different requests sizes. Since I/O profiling of storage devices is a time consuming task, SeRRa aims at providing accurate results as fast as possible, facilitating its use. For that, benchmarks are executed only over a subset of all profiled requests sizes, and the remaining values are estimated through linear regressions.

This paper is organized as follows: the next section discusses related work. Section 3 presents the design of SeRRa. The error induced by the estimation process is discussed on Section 4, as well as results obtained with our tool for nine real storage devices that include HDDs, SSDs and RAID arrays. Finally, Section 5 brings final remarks.

RELATED WORK

With the growing adoption of solid state drives, several works focused at characterizing these devices by evaluating their performance over several access patterns [START_REF] Chen | Understanding intrinsic characteristics and system implications of flash memory based solid state drives[END_REF][START_REF] Rajimwale | Block management in solid-state devices[END_REF]. These works point at SSDs' project options, their impact on performance, and illustrate common phenomena as write amplification and stripe alignment. Differently, our work aims at measuring storage performance in a generic way, and not only on modeling or explaining SSDs' performance.

El Maghraoui et al. [START_REF] Maghraoui | Modeling and simulating flash based solid-state disks for operating systems[END_REF] propose a detailed model of SSDs' performance. Nonetheless, the model needs low-level information that must be profiled through micro-benchmarks. Moreover, the proposed model is a low-level model, focusing on the device only and not including higher levels of the I/O subsystem. A similar approach is used by Desnoyers [START_REF] Desnoyers | Analytic modeling of ssd write performance[END_REF]. Such models are suitable for evaluating project options for SSDs, for instance, contrary to what our tool does. We aim at providing a high level profiling of the storage system in order to make decisions about optimizations.

For similar reasons, device simulators like DiskSim [START_REF] Bucy | The disksim simulation environment version 4.0 reference manual[END_REF] and others [START_REF] Agrawal | Design tradeoffs for ssd performance[END_REF][START_REF] Kim | Flashsim: A simulator for nand flash-based solid-state drives[END_REF][START_REF] Yoo | Vssim: Virtual machine based ssd simulator[END_REF] have no use on our context. They allow the evaluation of devices parameters, but not the profiling of existing systems.

Although several benchmarking tools report access time and throughput on the access to files over different access patterns, we could not find any tool that reports the sequential to random throughput ratio. Other tools also do not estimate results for a large set of parameters from a few measurements, as SeRRa does through linear regressions. These reasons motivated the development of the tool, described in the next section.

A STORAGE DEVICE PROFILING TOOL

This section describes SeRRa, a storage device profiling tool. Its development was motivated by the need for a fast way to obtain the sequential to random throughput ratio from devices as HDDs, SSDs and RAID arrays. The main goals of SeRRa's project are:

• Performance: the information must be provided as quickly as possible; • Accuracy: the provided information must fairly reflect the real behavior of the profiled storage device;

• Generality: the tool must be easy to use and do not require user-provided information about the device. Keeping both performance and accuracy goals at the same time is a challenging problem because profiling a storage device adequately can take several hours. Table 1 presents the time spent to profile the devices used in this study. It includes time required for one execution of the tests and for the complete profiling (that includes several executions in order to provide statistical guarantees). Details about these devices and the executed tests will be presented in Section 4. From the times reported in Table 1, it is clear that these tests are time consuming, as the fastest profile took over 1.5 days, while the slowest one took almost 14 days.

Ideally, such a complete profiling would be needed only once for each storage device and its stored results could be used for a long time. However, even considering this fact, to dedicate the environment for days can be prohibitive. Moreover, since aspects other than the storage device like local file system, disk scheduler and operating system also affect performance, the reported results are dependent on a system configuration. Changing the operating system version, for instance, could require a new profiling.

The slow profiling whose times are presented in Table 1 consists of executing an I/O benchmark several times varying file size, requests size and operation (sequential read, random read, sequential write, and random write). Figure 1 shows an example of access times graph -obtained for 1200MB files with different requests sizes on a SSD. Similarly to what can be observed in this example, most of the access times graphs present a linear function appearance. Because of this observation, we have decided to use linear regressions on the design of our profiling tool. Therefore, the following steps compose SeRRa's execution:

1. Monte Carlo: request sizes inside a given interval are randomly picked. This interval is provided as a parameter, as well as the gap between every two consecutive sizes. For instance: if asked to approximate the results for the interval [8KB, 40KB] using 8KB gaps, the tool would pick points from the set {8KB, 16KB, 24KB, 32KB, 40KB}. The number of points to be selected for each interval is also defined by the user. Multiple intervals, with different gaps, could be provided. The points at the extremes of the interval (in this example, 8 and 40KB) are always included on this step.

2. Benchmark: the test is executed for the requests sizes that were picked on the previous step. We decided to use IOzone [START_REF] Norcott | Iozone filesystem benchmark[END_REF] because it is a widely adopted I/O benchmark, easy to install and use, and because it fits our needs. Other benchmarks that allow sequential and random accesses, like IOR [START_REF] Shan | Using ior to analyze the i/o performance for hpc platforms[END_REF], could be used with few modifications on the tool. The user also provides the file size to the tests.

3. Linear Regression: from the access times measured on the previous step, the complete set of access time results for each given interval is estimated through linear regression. Each test (read or write, or random) for each interval is estimated separately.

Report:

The sequential to random throughput ratios for the read and write tests are reported. The tool provides such values for all requests sizes, as well as averages, maximum and minimum values. The estimated access times curves are also informed.

The tool, implemented on Python, is open source and available at http://www.inf.ufrgs.br/~fzboito/serra.html.

RESULTS

This section presents the SeRRa profiling tool's evaluation. First, we describe the test environments and methodology on Section 4.1. Then Section 4.2 discusses the error induced by the estimation of access times through linear regression. Lastly, Section 4.3 evaluates the tool's results and discusses its trade-off between performance and accuracy.

Tests Environments and Methodology

This section describes the nine systems used as our test environments, listed in Table 2. These systems were selected by their variety, aiming at representing most available devices. Aside from the system named "Bali", all environments are nodes from Grid'5000 clusters [START_REF] Bolze | Grid5000: A large scale and highly reconfigurable experimental grid testbed[END_REF].

The tests were executed on Linux operating system, using IOzone 3.397. All tested devices were accessed through the Ext4 local file system, and the default cfq disk scheduler was kept. Both virtual memory's page size and file system's block size are 4KB. Caching was explicitly disabled through the O DIRECT POSIX flag ("-I" parameter for IOzone).

For the tests described in this paper, five different file sizes were used -40MB, 200MB, 400MB, 800MB and 1200MB. On all tested devices, we observed that the access time curves usually stabilize before 1200MB, not showing significant differences as we increase the file size further.

The requests size was increased from 8KB to 4MB. This range was divided into two intervals: from 8KB to 64KB with gaps of 8KB; and from 64KB to 4MB with gaps of 32KB. Most parallel file systems employ stripe sizes of at least 32KB for their data servers, therefore the requests they receive (and are object to optimizations) are usually multiples of this value. This happens because each large request issued by an application becomes a set of requests, that are multiples of the stripe size, to servers. We defined a smaller gap on the first interval in order to represent small requests.

Estimation error by linear approximation

This section aims at quantifying the error induced by estimating the access time curves through linear regressions.

In order to do that, we executed the complete profilingwithout estimations, executing the benchmarks for all the specified request sizes -on all tests environments with the parameters discussed on Section 4.1. All tests were repeated until a 90% confidence could be achieved with a t-student distribution -with at least six executions. The maximum accepted error was of 10%. The necessary time to obtain this complete profile per machine, up to 14 days, was previously presented in Table 1.

A modified version of SeRRa was then executed on all devices varying the number of measuring points per interval (number of requests sizes for which the benchmark is executed) from 2 to 8. In the end, instead of reporting the sequential to random throughput ratio, the modified tool compared its estimated curves with the measured ones, obtained through benchmarking only. The graphs from Figure 4.2 present the absolute values of estimation error for the four tests. For each tested environment (represented by a different color), we present results for both intervals (interval 1 -from 8KB to 64KB -represented by circles, and interval 2 -from 64KB to 4MB -by triangles). For each interval on each number of measuring points, the graphs show five points: one per file size, increasing from left to right. Each result is the median from the errors for all tested requests size. We chose to use the median when summarizing error values because we believe it is a better central tendency estimator, since it is more resistant to outliers than the arithmetic mean.

We can see that most tests presented errors of up to approximately 20% (84% of the cases for sequential write, 86% for random write, 88% for sequential read, and 98% for random read). In general we did not observe improvements on the quality of the estimation by increasing the number of measuring points. In some cases, it is better to approximate the access time curve with only two points (the extremes of the interval) than including up to all points (Interval 1 has only 8 requests sizes). This happens because the real access time curve is not exactly a linear function, so slight variations from this linear format can impact the estimation, deviating it from the best approximation.

Since the values from the complete profiling are the average of several measurements (in order to provide statistical guarantees), the single benchmark executions made by our tool will hardly match them. We believe this inherent error is mostly responsible for the observed estimation error. This also helps to explain why including more points does not help estimation accuracy: more points potentially include more error.

From the graphs, we can observe that SSD and RAID devices were responsible for most of the cases where the error exceeded 40%. On the complete profiling, these devices took more repetitions in order to obtain statistical guarantees than the HDDs (17 repetitions on average were necessary for each test with HDDs, 43 with RAID arrays, and 26 with SSDs). Therefore, their results present more variability, and are harder to approximate. The machines with more variability were also the ones with incidence of high estimation errors (over 40%): Chimint (62 repetitions), Taurus (47), Bali-SSD (39), and Pastel (25). Table 3 presents the medians of all values from the graphs of Figure 4.2 separated by type of storage device. Results show that RAID arrays' random access time curves are harder to approximate by linear functions than HDDs'. SSDs' write time curves are the hardest to estimate. On SSDs, maintenance operations may be executed by the controller during write operations, affecting performance. This phenomenon, known as "write amplification", causes higher variability between repetitions of tests in SSDs, making the approximation on these devices more difficult.

SeRRa's Results Evaluation

The previous section discussed the error induced by approximating access times curves with linear functions. The presented results show that increasing the number of measuring points (requests sizes to run the benchmarks with) does not necessarily increase the quality of the estimation. Because of that, all ratios obtained with our tool in this section used only 2 measuring points per interval. Instead of increasing execution time by running tests with more requests sizes, in this section we evaluate the number of repetitions of the benchmarks. The arithmetic average of multiple executions' results is taken for each measuring point (instead of the result of a single execution).

The graphs from Figure 4.3 present the absolute error obtained by using SeRRa to measure the sequential to random throughput ratio (compared to the ratios obtained from the complete profiling) with 2 points per interval and up to 4 repetitions of each measurement. For each number of repetitions, there are 5 results for each machine: one per file size increasing from left to right, similar to the graphs on Figure 4.2. We can see that most tests presented errors of up to ≈ 20% (91% of the tests for write and 88% for read). The devices responsible for the cases with the highest errors were the same that presented the highest estimation errors on the previous section. 4.3 separated by type of storage device. In general, increasing the number of repetitions did not increase the results' accuracy significantly. It happened only for read tests on RAID arrays, and for write tests on SSDs. Despite SSDs presenting the highest estimation errors for write tests on the previous section, these devices presented the lowest ratio measurement errors. The time needed by SeRRa to obtain all sequential to random throughput ratios is presented in Table 5, along with its comparison to the time needed to obtain these ratios through the complete profiling discussed previously. With SeRRa, the slowest profiling (Pastel) takes 1.6 (with 1 repetition) or 6.3 (4 repetitions) hours instead of 14 days in order to provide a value whose error's median is around 5%.

We believe that more executions of the benchmark did not led to better results because 4 is still a small number of repetitions compared to what was necessary to provide the statistical guarantees defined on the complete profiling (from 13 to 62). In addition, increasing the number of executions can sometimes include outliers, perturbing the results. Since the decrease in time comes from both less repetitions of the tests and less requests sizes to be tested, running SeRRa to obtain this complete profiling with 62 repetitions, for instance, would be expected to take around 1/3 of the original time and provide results that are more accurate. The choice depends on the needed accuracy. We believe that a median error of approximately 5% (and up to 55%) would be enough for comparison between devices and optimizations' evaluation. Table 6 illustrates SeRRa's results by showing the sequential to random throughput ratios obtained with it for 1200M B files and 4 repetitions. The values are compared with results from the complete profiling.

FINAL REMARKS

This paper presented a tool for storage devices profiling regarding access sequentiality named SeRRa. It quantifies the difference between accessing files sequentially and randomly for a given device. In order to obtain this information quickly, the benchmarks are executed on a small subset of the reported values and the remaining ones are estimated through a linear model.

Storage devices present different performance behaviors, and this behavior cannot be simply stated for whole classes of devices. On the other hand, optimizations that work to adapt applications' access patterns impose overheads that may not be compensated by its performance improvement. On this context, the information provided by SeRRa can be used to decide which I/O optimizations will be beneficial for performance for a given storage device. The tool is open source and available on the Internet.

We presented results for a large space of parameters on 9 different storage devices, comprising HDDs, SSDs and RAID arrays. Our evaluation shows that SeRRa provides ratios with median errors of approximately 5% while taking 1/168 of the originally needed time.

Figure 1 :

 1 Figure 1: Profiling data from a SSD -Access time per request for several request sizes.

Figure 2 :

 2 Figure 2: Absolute error induced by using linear regressions to estimate the access time curves.

Figure 3 :

 3 Figure 3: Absolute error induced by using SeRRa to obtain the sequential to random throughput ratio.

Table 1 :

 1 Original time in hours to profile the storage devices used in this study.

	One test execu-	Total	profiling
	tion		time
	Pastel (HDD)	15.22		329.49
	Graphene (HDD)	12.26		120.38
	Bali (HDD)	10.38		126.10
	Chimint (RAID-0)	2.33		69.13
	Suno (RAID-0)	4.21		61.32
	Taurus (RAID-0)	2.43		57.95
	Edel (SSD)	3.09		40.44
	Graphite (SSD)	3.45		60.90
	Bali (SSD)	2.67		82.49

Table 2 :

 2 Configuration of the evaluated storage devices

	Cluster	RAM	Type	Storage Device Model	Capacity	Bus
	Pastel	8GB	HDD	Hitachi HDS7225SC	250GB	SATA 1.5Gb/s
	Graphene 16GB	HDD	Hitachi HDS72103	320GB	SATA 3Gb/s
	Bali	64GB	HDD	Seagate ST91000640NS	1T B	SATA 6Gb/s
	Chimint	16GB	RAID-0	2×Seagate ST3300657SS-H	2 × 136GB	SAS 3Gb/s
	Suno	32GB	RAID 0	2×Seagate ST9300653SS	2 × 300GB	SAS 3Gb/s
	Taurus	32GB	RAID-0	2×Seagate ST3300657SS	2 × 300GB	SAS 6Gb/s
	Edel	24GB	SSD	Micron C400	64GB	SATA 1.5Gb/s
	Graphite 256GB	SSD	Intel DC S3500 Series	300GB	SATA 6Gb/s
	Bali	64GB	SSD	SAMSUNG 840 Series MZ-7TD500BW	500GB	SATA 6Gb/s

Table 3 :

 3 Median from estimation errors.

		Write Random Write Read Random Read
	HDD	10%	8%	10%	3%
	RAID	10%	16%	9%	7%
	SSD	17%	19%	5%	2%
	All	9%	8%	7%	4%

Table 4 :

 4 Median of the errors with SeRRa.

			Write			Read
		1 rep. 2 rep. 4 rep. 1 rep. 2 rep. 4 rep.
	HDD	4.0%	5.7%	5.8%	3.4%	3.6%	3.3%
	RAID 6.7%	8.2%	9.0% 10.3% 8.9%	8.4%
	SSD	5.2%	3.6%	1.6%	1.5%	1.4%	2.0%
	All	5.2%	5.9%	5.2%	4.9%	4.1%	3.6%

Table 5 :

 5 SeRRa's time to measure (minutes).

		1 repetition	4 repetitions
	Pastel (HDD)	93.21 (1/212)	376.99 (1/52)
	Graphene (HDD)	85.69 (1/84)	341.50 (1/21)
	Bali (HDD)	74.03 (1/102)	282.79 (1/26)
	Chimint (RAID-0) 22.83 (1/181)	72.73 (1/57)
	Suno (RAID-0)	28.12 (1/130)	109.42 (1/33)
	Taurus (RAID-0)	16.03 (1/216)	64.54 (1/53)
	Edel (SSD)	5.92 (1/409)	23.58 (1/102)
	Graphite (SSD)	6.49 (1/562)	24.86 (1/146)
	Bali (SSD)	5.79 (1/855)	21.52 (1/229)
	Sum	338.10 (1/168) 1317.96 (1/43)
	Table 4 presents the medians of all values presented on
	Figure

Table 6 :

 6 Sequential do random ratio with 1200MB files -measured vs. estimated with SeRRa (4 repetitions).

			Pastel	Graphene	Bali	Chimint	Suno	Taurus	Edel	Graphite	Bali
			(HDD)	(HDD)	(HDD)	(RAID-0)	(RAID-0)	(RAID-0)	(SSD)	(SSD)	(SSD)
	Write	Measured SeRRa	2.02 1.98	1.89 1.75	1.74 1.68	1.95 1.80	1.71 1.55	1.62 1.32	0.99 0.97	1.01 1.04	1.38 1.64
	Read	Measured SeRRa	2.24 2.19	2.67 2.57	2.61 2.84	3.48 3.25	2.76 2.53	3.36 2.78	1.08 1.07	1.04 1.06	1.10 1.09

ACKNOWLEDGMENTS

This research has been partially supported by CNPq and CAPES-BRAZIL under the grants 5847/11-7 and Stic-Amsud 6132-13-8. The experiments presented in this paper were carried out on the Grid'5000 experimental test bed, being developed under the INRIA ALADDIN development action with support from CNRS, RENATER and several Universities as well as other funding bodies (see https: //www.grid5000.fr). This research was accomplished in the context of the International Joint Laboratory LICIA and of the HPC-GA project.