MD, MSc Alexandre Le Joncour

MD Anne-Claire Desbois

Elie S Aur

PhD Leroyer

PhD Edwige Tellier

PhD Paul R Egnier

PhD Anna Maciejewski-Duval

MD Clo E Comarmond

MD St Ephane Barete

MD Michel Arock

MD Patrick Bruneval

MD Jean-Marie Launay

MD Pierre Fouret

MD Ulrich Blank

MD Michelle Rosenzwajg

MD David Klatzmann

MD Mohamed Jarraya

MD Laurent Chiche

MD Fabien Koskas

MD, MSc Patrice Cacoub

MD Gilles Kaplanski

MD, PhD David Saadoun
email: david.saadoun@aphp.fr.

Mast cells drive pathologic vascular lesions in Takayasu arteritis

Keywords: nnn-nnn.) Large vessel vasculitis, mast cell, vascular remodeling, Takayasu

Background: Takayasu arteritis (TAK) is a large vessel vasculitis resulting in artery wall remodeling with segmental stenosis and/or aneurysm formation. Mast cells (MCs) are instrumental in bridging cell injury and inflammatory response. Objectives: This study sought to investigate the contribution of MCs on vessel permeability, angiogenesis, and fibrosis in patients with TAK. Methods: MC activation and their tissue expression were assessed in sera and in aorta from patients with TAK and from healthy donors (HDs). In vivo permeability was assessed using a modified Miles assay. Subconfluent cultured human umbilic vein endothelial cells and fibroblasts were used in vitro to investigate the effects of MC mediators on angiogenesis and fibrogenesis. Results: This study found increased levels of MC activation markers (histamine and indoleamine 2,3-dioxygenase) in sera of patients with TAK compared with in sera of HDs. Marked expression of MCs was shown in aortic lesions of patients with TAK compared with in those of noninflammatory aorta controls. Using Miles assay, this study showed that sera of patients with TAK significantly increased vascular permeability in vivo as compared with that of HDs. Vessel permeability was abrogated in MC-deficient mice. MCs stimulated by sera of patients with TAK supported neoangiogenesis (increased human umbilic vein endothelial cell proliferation and branches) and fibrosis by inducing increased production of fibronectin, type 1 collagen, and a-smooth muscle actin by fibroblasts as compared to MCs stimulated by sera of HD. Conclusions: MCs are a key regulator of vascular lesions in patients with TAK and may represent a new therapeutic target in large vessel vasculitis. (

Large vessel vasculitis (LVV) includes mainly Takayasu arteritis (TAK) and giant cell arteritis. LVV may lead to segmental stenosis, occlusion, dilatation, and/or aneurysm formation in the aorta and/or its main branches and is found in 50% to 70% of patients with TAK. [START_REF] Numano | Takayasu's arteritis[END_REF][START_REF] Chung | Patterns of aortic involvement in Takayasu arteritis and its clinical implications: evaluation with spiral computed tomography angiography[END_REF] In LVV, histological lesions are characterized by an inflammatory infiltrate located mainly in the inner part of the aortic wall (media) and the adventitia. Previous studies have provided evidence that LVV inflammation is driven by T H 1 and T H 17 cells in the peripheral blood and inflamed tissues. [START_REF] Saadoun | Th1 and Th17 cytokines drive inflammation in Takayasu arteritis[END_REF][START_REF] Deng | Th17 and Th1 T-cell responses in giant cell arteritis[END_REF][START_REF] Joncour | Targeting JAK/STAT pathway in Takayasu's arteritis[END_REF] This inflammatory reaction is associated with hyperplasia of vasa vasorum and subsequently with a medio-adventitia sclerosis and intimal proliferation. [START_REF] Kaiser | Formation of new vasa vasorum in vasculitis: production of angiogenic cytokines by multinucleated giant cells[END_REF][START_REF] Kurata | Difference in immunohistochemical characteristics between Takayasu arteritis and giant cell arteritis: it may be better to distinguish them in the same age[END_REF][START_REF] Inder | Accumulation of lymphocytes, dendritic cells, and granulocytes in the aortic wall affected by Takayasu's disease[END_REF] The inflow of inflammatory cells in arterial inflamed walls is mainly due to their interaction with specific adhesion molecules expressed on the inflamed endothelium. Neovessels and adventitial vasa vasorum are the main sites where adhesion molecules for leukocytes are expressed, and therefore the main sites through which circulating leucocytes are recruited into the vascular wall. The mechanisms resulting in the vascular remodeling in LVV are still unclear. Innate immune response is instrumental in bringing immune cells to the artery and in the tissue-damaging inflammation. Mast cells (MCs) are unique, multifunctional innate immune cells of our defense system, with characteristics such as widespread tissue distribution, expression of receptors capable of recognizing both endogenous and exogenous agents, and the capability to respond rapidly to triggering factors by selective mediator release. [START_REF] Beaven | Our perception of the mast cell from Paul Ehrlich to now[END_REF] Abbreviations used a-SMA: a-Smooth muscle actin HD: Healthy donor HUVEC: Human umbilic vein endothelial cell IDO: Indoleamine 2,3-dioxygenase LVV: Large vessel vasculitis MC: Mast cell PDGF: Platelet-derived growth factor qRT-PCR: Quantitative reverse transcriptase PCR TAK: Takayasu arteritis Of note, MCs are instrumental in remodeling vascular structures as highlighted in abdominal aortic aneurysm, [START_REF] Swedenborg | Mast cells: important players in the orchestrated pathogenesis of abdominal aortic aneurysms[END_REF] myocardial infarction, [START_REF] Levick | Cardiac mast cells: the centrepiece in adverse myocardial remodelling[END_REF] and atherosclerosis. [START_REF] Bot | Mast cells in atherosclerosis[END_REF] MCs and their mediators promote leucocyte infiltration by increasing vascular permeability, inducing expression of specialized adhesion molecules on endothelial cells and by secretion of proinflammatory molecules. MCs can also affect the behavior of structural cells within inflammatory sites such as fibroblasts. [START_REF] Bachelet | Mast cells: not only in allergy[END_REF] Moreover, MCs are capable of producing proangiogenic factors. [START_REF] De Souza Da | Mast cells interact with endothelial cells to accelerate in vitro angiogenesis[END_REF] In LVV, M€ ayr€ anp€ a€ a et al [START_REF] Trosien | Mast cells associate with neovessels in the media and adventitia of abdominal aortic aneurysms[END_REF][START_REF] Trosien | Mast cells associate with Tcells and neointimal microvessels in giant cell arteritis[END_REF] have shown a spatial association of activated MCs, T cells, and neovessels, suggesting that MCs may contribute to the pathogenesis of such diseases.

Because MCs represent an important link between cell injury and an inflammatory response that can lead to vicious circle driving vascular remodeling, we aimed to investigate the implication of MCs in vascular lesions of patients with TAK.

METHODS Patients

The study population consisted of 30 patients with TAK (median age at diagnosis: 32.4 [interquartile range, 27.2; 53.2] years), fulfilling the international criteria for TAK. [START_REF] Hunder | The American College of Rheumatology 1990 criteria for the classification of giant cell arteritis[END_REF] Clinical characteristics of patients with LVVare presented in Table I. Patients treated with biologics and/or steroids >10 mg/day were excluded. Blood samples from 20 healthy donors (HDs) >18 years of age and sex-matched, obtained from the Etablissement Franc ¸ais du Sang (Hôpital Piti e-Salpêtri ere), were used as controls (15 women (75%), median age, 35.2 [interquartile range, 29.0; 35.1] years). Aorta biopsies were obtained from patients with TAK undergoing surgical repair for aortic aneurysm or dissection in our hospital center. Six noninflammatory aorta biopsies were provided by Saint Louis Hospital's human tissue bank and obtained from vessels taken for transplant in multiorgan and tissue donors. The study was approved by the Paris VI ethics review board and was performed according to the Helsinki Declaration. All patients gave informed consent.

Patient involvement

We provided instructional support to foster knowledge of research concepts and terminology. If the patients asked, we provided them information about the course of the study.

Culture of human MCs

Human ROSA KITWT MCs [START_REF] Arock | Preclinical human models and emerging therapeutics for advanced systemic mastocytosis[END_REF][START_REF] Saleh | A new human mast cell line expressing a functional IgE receptor converts to tumorigenic growth by KIT D816V transfection[END_REF] were cultured exactly as previously described: they were cultured in Iscove's Modified Dulbecco's Medium (ref 21980-032, Gibco, Thermo Fisher Scientific, Waltham, Mass) including the following supplements (penicillin 1%, streptomycin 1%, sodium pyruvate 1%; Minimum Essential Medium Vitamin solution 1%; Minimum Essential Medium Non Essential Amino Acids Solution 2%; and L-Glutamine 1%, Insulin-Transferrin-Selenium 1%, Gibco; Bovine Serum Albumin 0.3% CHoKL 10%, Sigma-Aldrich, St Louis, Mo), in the presence or the absence of recombinant IL-33 (10 ng/mL) or of neutralizing anti-IL33 (10 mg/mL), IgE (2 mg/mL; ref 401152, Merck Millipore, Burlington, Mass). MCs were also cultured with quercetin (Q4951, Sigma-Aldrich), cromolyn (C0399, Sigma-Aldrich), and neutralizing IL-6 antibody (MAB2061, R&D Systems, Minneapolis, Minn). Levels of histamine (ELISA Kit, IBL International GmbH, Tecan Group, M€ annedorf, Switzerland) and total tryptase (a and b) (ImmunoCAP Tryptase, Thermo Fisher Scientific) were measured in culture supernatants 1 hour after stimulation. Levels of platelet-derived growth factor (PDGF) and of TGF-b (Milliplex, Merck Millipore) were measured 5 days after stimulation.

Immunohistochemistry

Detection of IL-33 1 , ST2 1 , and tryptase 1 cells was performed on fixed, paraffin-embedded samples (aorta) from patients with TAK and noninflammatory aorta controls. After dewaxing in baths of xylene and ethanol, slides were submitted to antigen retrieval by heating (1108C) in citrate buffer at pH 6.0. Before incubation with primary antibodies, Fc receptors were blocked with normal goat serum (3.3%). Slides were then incubated overnight with mouse monoclonal anti-IL-33 (dilution 1:1000; Enzo Life Sciences, Farmingdale, NY), rabbit polyclonal anti-ST2 (dilution 1:100; Sigma-Aldrich), monoclonal mouse anti-tryptase (1/200; Dako, Agilent, Santa Clara, Calif), or with isotype control: polyclonal rabbit IgG or monoclonal mouse IgG (Abcam, Cambridge, UK). Slides were then incubated for 2 hours at room temperature with Cy3conjugated goat anti-mouse immunoglobulin (working dilution 1:1000; Jackson ImmunoResearch, West Grove, Pa) and Alexa 488 donkey anti-rabbit Ig (working dilution 1:1000; Life Technologies, Thermo Fisher Scientific), mounted in Mowiol and evaluated under fluorescence microscopy (Leica Microsystems, Wetzlar, Germany). MCs were also quantified by counting the number of tryptase 1 cells per field in 100 fields per sample.

Gene expression quantification at the mRNA level in aortic tissues and fibroblasts

Quantification of mRNA expression was performed on paraffin-embedded samples from patients and controls with noninflammatory aorta aneurysms. Quantification of mRNA expression was also performed on fibroblast cultured with serum or with MC supernatant. TGF-b and PDGF gene expression was analyzed. The theoretical and practical aspects of real-time quantitative reverse transcriptase-PCR (qRT-PCR) using the ABI Prism 7900 Sequence Detection System (PerkinElmer Applied Biosystems, Foster City, Calif) have been described in detail elsewhere. Briefly, total RNA was extracted using the High Pure FFPET RNA Isolation Kit (Roche Diagnostics, Rotkreuz, Switzerland) and was reverse-transcribed before real-time PCR amplification. Quantitative values were obtained from the threshold cycle number at which the increase in the signal associated with exponential growth of PCR products began to be detected using analysis software, according to the recommendations of the manufacturer (PerkinElmer Applied Biosystems).

In vivo permeability assays

All animal experiments were performed in compliance with the guidelines for the care and use of laboratory animals and were approved by the Local Ethics Committee (approval 13-14122011). In vivo permeability was assessed using a modified Miles assay. [START_REF] Scheppke | Retinal vascular permeability suppression by topical application of a novel VEGFR2/Src kinase inhibitor in mice and rabbits[END_REF] Briefly, Evans blue dye (Sigma-Aldrich) was retro-orbitally injected into C57BL/6 mice (used as wild-type controls) and into MC-deficient C57BL/6 KitW-sh/W-sh mice (carrying a natural mutation in the MC growth factor cKIT) 21 at 8 weeks of age (100 mL of 1% Evans blue in 0.9% NaCl). After 10 minutes, 40 mL of serum was injected intradermally. Twenty minutes later, the shaved dorsal skin at the injection area was excised using a calibrator ring, dye was extracted with formamide overnight at 558C, and the dye absorbance was further measured at 630 nm. For IL-33 inhibition experiments, 5 mg/mL neutralizing anti-human IL-33 antibody (AF3625, R&D Systems) or of its corresponding isotype antibody control (AB-108C, R&D Systems) were incubated with serum for 1 hour before the injection.

In vitro angiogenesis assay

Proliferation. Subconfluent human umbilic vein endothelial cells (HUVECs) were starved for 12 hours and were then incubated for 24 hours with serum (1:10) or MC culture supernatant (1:10). Proliferation was assayed by 5-bromo-2-deoxyuridine incorporation using the Cell Proliferation Assay Kit (Roche Diagnostic).

Tube formation. Subconfluent HUVECs were starved for 12 hours.

Ninety-six-well plates were coated with 50 mL of growth factor-reduced Matrigel (BD Biosciences, San Jose, Calif) for 30 minutes at 378C. HUVECs (20,000 cells/well) were seeded on Matrigel for 12 hours in presence of serum (1:10) or MC supernatant (1:10). Capillary-like structures and branch points were counted using ImageJ version 1.44 software (National Center for Biotechnology Information, Bethesda, Md). Each experiment was run in triplicate.

In vitro culture of fibroblasts

Arterial fibroblasts were isolated from the adventitia of noninflammatory arteries. Briefly, the adventitial layer was separated from the media layer and then digested with collagenase solution for 3 hours at 378C. Filtered cells were washed and then cultured. Fibroblasts (20,000 per well) were thereafter incubated for 5 days with TAK or HD serum (10%). Experiments were performed with P2 or P3 fibroblasts. Arterial fibroblasts were cultured in an endothelial cell growth medium with supplements (C-22211, Endothelial Cell Growth Medium 2 Kit, PromoCell, Heidelberg, Germany).

To assess the impact of MCs on the differentiation of fibroblasts, arterial fibroblasts were also incubated for 5 days with supernatants (10%) of MCs previously cultured with TAK or HD serum for 24 hours. Production of collagen (COL1A), fibronectin, a-smooth muscle actin (a-SMA), TGF-b, and PDGF-a was assessed by qRT-PCR. Briefly, total RNA was extracted by TRIzol reagent (Invitrogen, Thermo Fisher Scientific) and reverse-transcribed using SuperScript VILO cDNA Synthesis Kit (Invitrogen) both according to the manufacturer's instruction. Gene expression was determined by real-time PCR. Each cDNA sample was amplified in triplicate using SYBR Green (Applied Biosystems, Thermo Fisher Scientific) on 7500 FAST Real-time PCR System (Applied Biosystems). The thermal cycling conditions comprised an initial denaturation step at 958C for 10 minutes, followed by 40 cycles at 958C for 15 seconds and 658C for 1 minute. Quantitative values were obtained from the threshold cycle number at which the increase in the signal associated with exponential growth of PCR products began to be detected. GADPH gene was used as an endogenous control.

Statistics

Continuous variables were presented with the median and extreme values or with the mean 6 SEM. Categorical variables were presented with counts and proportions. Statistical comparisons were performed by using the Mann-Whitney test for quantitative unpaired data, and the Wilcoxon matched pairs test for quantitative paired data. All statistical tests were 2-tailed with a significance level of .05. Statistical significance was evaluated using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, Calif).

RESULTS

MCs are activated and abundant in inflammatory lesions of TAK.

In vivo, we observed an increased level of MC activation markers in sera of patients with TAK compared with in sera of HDs. Histamine level (11.23 6 1.3 vs 7.15 6 0.5 nmol/L; P 5 .008), indoleamine 2,3-dioxygenase (IDO) activity (6.4% 6 2.7% vs 3.6% 6 0.6%; P 5 .009), and TNF-a (795 6 633 vs 239 6 135 m/mL; P 5 .001) were increased in patients with TAK compared with in HDs, respectively (Fig 1,A). Level of tryptase was not statistically different in patients with TAK and HDs (P 5 .4). We then confirmed these results in vitro. Human MC (ROSA KIT WT cell line) stimulated with 10% sera from patients with TAK produced an increased level of histamine and IDO activity as compared to that of HDs (11.19 6 7.5 vs 3.2 6 0.2 mmol/L; P < .001; and 5.9% 6 2.2% vs 2.9% 6 0.3%; P 5 .003, respectively) (Fig 1 ,B). Level of TNF-a was not different. As observed in sera, tryptase level was not statistically different in patients with TAK and in HDs (P 5 .08). In the aorta, we confirmed that MCs were markedly increased in patients with TAK as compared to noninflammatory aorta controls. Indeed, number of tryptase1 cells per field in 100 fields was 51. 6 Quantification of tryptase mRNA from aorta by qRT-PCR indicated that tryptase mRNA expression aorta was increased in aorta from patients with LVV as compared to those from noninflammatory aorta controls, respectively (8.5 6 10.2 vs 1.3 6 0.2; P 5.01) (Fig 1,E).

Increased vessel permeability in TAK is dependent on MCs

We previously showed that MCs are activated and abundant in arterial inflammatory lesions of patients with TAK. Because MCs can be an important player in vascular permeability, [START_REF] Oschatz | Mast cells increase vascular permeability by heparin-initiated bradykinin formation in vivo[END_REF] we next examined the effects of TAK sera and MCs on vascular permeability by performing a Miles assay to study Evans blue extravasation. [START_REF] Scheppke | Retinal vascular permeability suppression by topical application of a novel VEGFR2/Src kinase inhibitor in mice and rabbits[END_REF] We showed that injection of sera from patients with TAK induce increased vessel permeability as compared to sera of HDs (0.42 6 0.03 vs 0.2 6 0.1 OD; P 5 .0001) (Fig 2, A andB). To demonstrate that vessel permeability was dependent on MCs, we then performed the same Miles assay on MC-deficient mice. Evans blue extravasation after injection of TAK sera was dramatically decreased in MC-deficient mice as compared to that observed in wild-type mice (0.2 6 0.02 vs 0.42 6 0.05 OD; P < .0001) (Fig 2 ,C).

We further explored the vascular permeability mechanisms induced by TAK sera and MC dependence. Because IL-33 is a well-known cytokine involved in vessel permeability and MC activation, [START_REF] Allakhverdi | Cutting edge: the ST2 ligand IL-33 potently activates and drives maturation of human mast cells[END_REF] we assessed IL-33 levels in TAK sera. MC activation markers (ie, histamine and tryptase levels) were also investigated in supernatant from MC previously stimulated with and without recombinant IL-33. IL-33 sera level was increased in patients with TAK compared with in HDs ( 155

MCs support neo-angiogenesis in TAK

We next investigated whether MCs could have a functional effect on angiogenesis in TAK. We first assessed HUVEC proliferation and showed that supernatant of MCs incubated with TAK sera significantly increased HUVEC proliferation (1.77 6 0.03 OD) as compared to supernatant of MC incubated with HD sera (1.6 6 0.03 OD; P 5 .03) and to TAK and HD sera alone (0.70 6 0.02 OD; P 5 .002; and 0.67 6 0.01 OD; P < .001, respectively) (Fig 4,A). Incubation of the MC supernatants with an IL-33 neutralizing antibody decreased in HUVEC proliferation induced by MC supernatant preincubated with TAK sera (1.8 6 0.03 vs 1.7 6 0.04 OD; P 5 .003), whereas such treatment had no effect on MC supernatant incubated with HD sera (1.6 6 0.02 vs 1.6 6 0.02 OD; P 5 .43) (Fig 4,A). Thus, HUVEC proliferation is increased by MC supernatant stimulated by TAK sera and is partially dependent on IL-33. Similarly, in Matrigel assays, we showed that MC supernatant incubated with TAK sera significantly increased HUVEC branches (58 6 7.8) as compared to MC supernatant incubated with HD sera (30 6 8.4; P 5 .04) and to LVV sera alone (32.7 6 5.9; P 5.02) (Fig 4 ,B). In addition, treatment of MC supernatant preincubated with TAK sera with IL-33 neutralizing antibody decreased HUVEC branches formation as compared to MC supernatant preincubated with TAK sera (46.7 6 8.5 vs 26.7 6 4.7; P 5 .01), whereas it had no effect on MC supernatant incubated with HD sera (34.3 6 8.0 vs 16.6 6 3.8; P 5.1) (Fig 4 ,B). We then showed that MC supernatant incubated with TAK sera tend to increase HUVEC pseudotube formation (65 6 6.5) as compared to MC supernatant incubated with HD sera (46 6 8.2; P 5 .09) and to TAK sera alone (44 6 6.9; P 5.05) (Fig 4 ,C). Finally, IL-33 neutralizing antibody decreased HUVEC pseudotube formation stimulated by MC supernatant preincubated with TAK sera (56.1 6 8.3 vs 37.1 6 5.3; P 5 .03), whereas it had no effect on MC supernatant incubated with HD sera (45.7 6 8.2 vs 35.2 6 7.1; P 5 .9) (Fig 4, C andD). Interestingly, supernatant of IgE-stimulated MCs enhanced HUVEC branches and pseudotubes formation as compared to other conditions but had little effect on HUVEC proliferation (Fig 4, A-C). Altogether, these data show that increased neoangiogenesis induced by TAK sera is dependent on MC activation.

MCs support fibrosis in TAK

As we have shown that MC could promote endothelial cells neo-angiogenesis, we next hypothesized that MC could also modulate the phenotype of fibroblasts. We first highlighted that fibroblasts stimulated with MC supernatant incubated with TAK sera expressed more fibronectin, collagen 1, and a-SMA than did fibroblasts simulated with TAK sera alone (fibronectin, 21 6 2.9 vs 10.2 6 1.3; P 5 .005; collagen 1, 6.1 6 0.8 vs 1.7 6 0.2; P < .001; and a-SMA, 0.016 6 0.003 vs 0.006 6 0.001; P 5 .001) (Fig 5,A). These profibrotic molecules (ie, fibronectin, collagen 1, and a-SMA) were also expressed markedly higher by fibroblasts stimulated with MC supernatant stimulated with TAK sera as compared to MC supernatant incubated with HD sera (fibronectin, 21 6 2.9 vs 3.4 6 2.5; P 5 .002; collagen 1, 6.1 6 0.8 vs 1.5 6 0.8; P 5 .001; and a-SMA, 0.016 6 0.003 vs 0.005 6 0.003; P 5 .03) (Fig 5,A). Interestingly, supernatant of IgE-stimulated MC enhanced fibronectin and a-SMA production by fibroblast but seems to have little effect on collagen 1 production (Fig 5,A We then aimed to determine the mechanism that could modulate fibroblast phenotype. We found by qRT-PCR that the relative expressions of 2 major profibrotic cytokines, PDGF and TGF-b, were higher in aorta specimens obtained from patients with TAK compared with those from noninflammatory aorta (10.7 6 1.9 vs 2.5 6 0.8 relative expression; P 5 .0047; and 209 6 23.8 vs 84 6 20.7; P 5 .01, respectively) (Fig 5 ,B). We then determined the levels of PDGF and TGF-b in supernatant of MCs incubated with TAK sera as compared to those incubated with HD sera. Production of PDGF and TGF-b in supernatant of MCs cultured with TAK sera was higher than those of MCs cultured with HD sera (590 6 91 vs 241.6 6 46.6 pg/mL; P 5 .003; and 8352 6 367 vs 6240 6 490 pg/mL; P 5 .003, respectively) (Fig 5 ,C). IL-33 blockade did not modify production of PDGF and TGF-b in supernatant of MCs cultured with TAK sera (Fig E2 ,C).

To provide more insight into the nature of the primary MC activating factor found in the serum of patients with TAK, we performed the same experiment using inhibitors of MC degranulation (quercetin and cromolyn) and IL-6 blockade (a major inflammatory cytokine involved in TAK). We did not see a consistent effect of either blocking MC degranulation or anti-IL-6 (Fig E3).

Altogether, these data show that MC activated by TAK sera support fibrosis and suggest that these effects could be dependent of TGF-b and PDGF production.

DISCUSSION

Inflammation and angiogenesis are closely associated in a number of pathological processes. With regard to LVV, immunemediated damage to aorta results in vascular remodeling such as segmental stenosis, occlusion, dilatation, and/or aneurysm formation. [START_REF] Kaiser | Formation of new vasa vasorum in vasculitis: production of angiogenic cytokines by multinucleated giant cells[END_REF][START_REF] Cid | Cell adhesion molecules in the development of inflammatory infiltrates in giant cell arteritis: inflammation-induced angiogenesis as the preferential site of leukocyte-endothelial cell interactions[END_REF] In the present report, we identified MCs as a novel player in driving vascular lesions in TAK. Although MCs are not circulating cells, we first highlighted an increased level of MC activation markers (ie, histamine and IDO) in TAK sera. MCs are an important source of TNF-a and may act on fibroblast and endothelial cells. [START_REF] Izadi | Identification of TNFR2 and IL-33 as therapeutic targets in localized fibrosis[END_REF] We found high levels of TNF-a in sera of patients with TAK but these levels were similar in MC supernatant stimulated with TAK sera or HD sera. As histamine may be produced by basophils and IDO by dendritic cells, we confirmed these results in MC supernatant. We then identified a marked increase in MC numbers in TAK arterial lesions. MCs localized mostly in the adventitia layer, indicating that these cells are optimally positioned for sensing danger signals in the arterial wall, via the vasa vasorum, which are restricted to the adventitia layer. The high density of MCs in aorta is unexpected because MCs are mainly found at sites close to the external environment, such as the skin and mucosal surfaces of the lung and gut. Along this line, the presence of MCs has been pointed out in temporal arteries affected by giant cell arteritis in patients. [START_REF] Trosien | Mast cells associate with Tcells and neointimal microvessels in giant cell arteritis[END_REF] MCs have also been implicated in abdominal aortic aneurysm, 10 myocardial infarction, [START_REF] Levick | Cardiac mast cells: the centrepiece in adverse myocardial remodelling[END_REF] and atherosclerosis [START_REF] Bot | Mast cells in atherosclerosis[END_REF] lesions, by promoting leucocyte infiltration through increasing vascular permeability.

In addition, in the present report, we provide strong evidence, using a Miles assay (Evans blue extravasation), that treatment of MCs with sera from patients with TAK is able to induce vascular leakage and that significantly more than treatment with sera from HDs can do. In addition, vascular permeability induced by injection of sera from patients with TAK was dramatically decreased in MC-deficient mice as compared to wild-type mice, underlining the critical role of MCs in such phenomenon.

We then showed that MCs stimulated by sera of patients with TAK have angiogenic properties by promoting HUVEC proliferation, pseudotubes, and branch formation. Several factors can enhance MC-mediated angiogenesis. Indeed, MCs produce a large spectrum of proangiogenic factors such as vascular endothelial growth factor, TGF-b, fibroblast growth factor 2, vascular permeability factor. [START_REF] Ribatti | Mast cells, angiogenesis, and tumour growth[END_REF] Moreover, it has been shown that tryptase secreted by MCs greatly enhanced angiogenesis [START_REF] Blair | Human mast cells stimulate vascular tube formation: tryptase is a novel, potent angiogenic factor[END_REF] and that histamine stimulates new vessel formation by acting through both H1 and H2 receptors. [START_REF] S€ Orbo | Mast-cell histamine is angiogenic through receptors for histamine1 and histamine2[END_REF] Lastly, MCs also contain preformed matrix metalloproteinases, such as MMP-2 and MMP-9, which enable MCs to directly modulate extracellular matrix degradation. This, in turn, allows for tissue release of extracellular matrix-bound angiogenic factors. [START_REF] Ribatti | Mast cells, angiogenesis, and tumour growth[END_REF] Because IL-33 is a potent regulator of MC functions [START_REF] Allakhverdi | Cutting edge: the ST2 ligand IL-33 potently activates and drives maturation of human mast cells[END_REF][START_REF] Nian | Epithelial cells expressed IL-33 to promote degranulation of mast cells through inhibition on ST2/PI3K/mTOR-mediated autophagy in allergic rhinitis[END_REF] and is involved in both vessel permeability and angiogenesis, we examined its role in vascular homeostasis in TAK. We pointed out the ability of IL-33 to activate MCs. Because we observed that MCs and ST2 (the IL-33 receptor) colocalized in inflamed aorta of patients with TAK, we sought to investigate the effect of blocking in IL-33 effects on MCs in such context. Interestingly, we observed that blocking IL-33 inhibited the MC-dependent vascular permeability and angiogenesis induced by TAK sera. Altogether, these data showed that IL-33 and MCs might constitute an important link between cell injury and an inflammatory response that can lead to restoration of tissue function and homeostasis, but might, under other circumstances, contribute to a vicious circle driving tissue remodeling. In an inflammatory context, MCs can promote vascular permeability and thus can amplify the recruitment of inflammatory cells. [START_REF] Kunder | Mast cell modulation of the vascular and lymphatic endothelium[END_REF] In TAK, inflammatory response is followed by hyperplasia of vasa vasorum and subsequently by medio-adventitia fibrosis and intimal proliferation. However, the inflammatory and stenotic phases of the disease coexist frequently. MCs can promote fibrosis through various mechanism such as fibroblast proliferation and activation, fibroblast recruitment, and induction of secretion of matrix protein by fibroblasts. [START_REF] Bradding | The controversial role of mast cells in fibrosis[END_REF] Indeed, MCs have been associated with numerous fibrotic conditions, such as idiopathic lung fibrosis, cardiac fibrosis, renal fibrosis, scleroderma, chronic graft-versus-host disease, and liver cirrhosis. [START_REF] Legere | Mast cells in cardiac fibrosis: new insights suggest opportunities for intervention[END_REF][START_REF] Brown | The immunopathogenesis of fibrosis in systemic sclerosis[END_REF][START_REF] Weiskirchen | Mast cells in liver fibrogenesis[END_REF][START_REF] Conti | Critical role of inflammatory mast cell in fibrosis: potential therapeutic effect of IL-37[END_REF] MC degranulation and proliferation have been documented in fibrotic processes in vivo, and several MC mediators such as PDGF and TGF-b have been found to exert fibrogenic effects by causing fibroblast proliferation or by increasing collagen synthesis. [START_REF] Levi-Schaffer | Mast cell/fibroblast interactions in health and disease[END_REF][START_REF] Garbuzenko | Human mast cells stimulate fibroblast proliferation, collagen synthesis and lattice contraction: a direct role for mast cells in skin fibrosis[END_REF] Interestingly, we demonstrated here that MCs stimulated by sera of patients with TAK led to increased production of collagen, fibronectin, and a-SMA by fibroblasts. In addition, MCs stimulated with TAK sera secreted higher levels of TGF-b and PDGF than those stimulated with HD sera did, suggesting a role for MCs in the profibrotic phenotype of fibroblasts. Furthermore, the mRNA expression of these 2 mediators was markedly increased in inflamed aorta of patients with TAK. The mechanisms involved in vascular remodeling in TAK are unknown. Unlike in vessel permeability and angiogenesis, we did not find any effect of IL-33 on fibrosis. To provide more insight into the nature of the primary MC activating factor, we used inhibitors of MC degranulation (quercetin/cromolyn) to assess the effect of MC degranulation and IL-6 blockade (a major inflammatory cytokine involved in TAK) but did not find any effect on MC-mediated fibrosis in TAK. Moreover, the effects of LVV sera in endothelial cells and fibroblasts did not fully phenocopy the IgE effect. This supports the concept of a novel MC activation pathway present in LVV. Further studies are needed to find the missing activating factor(s) present in TAK sera and involved in MC-mediated fibrosis. In the late phase of TAK, scarring can be seen in the tunica media with disruption and disorganization of the remaining elastic fibers. Dense adventitial fibrosis is also often present. However, the precise mechanisms involved in vascular remodeling in TAK were largely unknown.

In conclusion, we provide here strong evidence that MCs play a critical role in regulating vessel permeability, angiogenesis, endothelial activation, and fibrogenesis in TAK. IL-33 and MCs may constitute and important link between cell injury and an inflammatory response in such diseases.

  6 53.5 versus 3.3 6 3.5 (P 5 .005) in TAK versus control samples, respectively (Fig 1, C and D). Chromogenic immunohistochemistry of TAK aorta sections showed that MCs localize mostly in the adventitia layer and to a lesser extent in the intima (see Fig E1 in this article's Online Repository at www.jacionline.org).

  6 70 vs 21 6 19.6 pg/ mL; P 5 .001) (Fig 3, A). Recombinant IL-33 significantly increased the secretion of histamine (10% vs 1.6%; P 5 .001) and of tryptase (8.2% vs 0.5%; P 5 .002) by MCs stimulated with TAK sera with compared those stimulated with HD sera (Fig 3, B). Moreover, IL-33 blockade in sera with an IL-33 neutralizing antibody significantly reduced levels of histamine and tryptase in MC supernatant (see Fig E2, A in this article's Online Repository at www.jacionline.org). In addition, MCs and ST2 (the IL-33 receptor) colocalized in aortic lesions of patients with LVV (Fig 3, C). Lastly, Evans blue extravasation was significantly decreased by the addition of neutralizing anti-IL-33 antibody (anti-IL33) (0.42 6 0.03 vs 0.32 6 0.03 OD; P 5 .001) (Fig 3, D and E). Altogether, these data showed that increased vessel permeability in TAK is dependent on MCs and at least partially on mediators such as IL-33.

FIG 1 .

 1 FIG 1. MCs are activated and abundant in inflammatory lesions of TAK. Levels of histamine, tryptase, IDO, and TNF-a were quantified (A) in the serum of HDs (n 5 9) and of patients with TAK (n 5 17) and (B) in the supernatant of human MCs cultured for 5 days with 10% of serum from patients with LVV (n 5 11) or of HDs (n 5 5). (C) Representative immunofluorescence staining of aorta obtained from 3 patients with LVV and 3 HDs. Tryptase 1 MCs are stained in red (white arrows) and 49-6-diamidino-2-phenylindole, dihydrochloride (DNA) in blue. Original magnification 6403. (D) Number of MCs (tryptase 1 cells) counted per 100 fields of aorta section of 12 patients with LVV and of 6 noninflammatory controls. (E) Quantification of MCs (tryptase RNA) by qPCR from aorta obtained from patients with LVV (n 5 11) and noninflammatory controls (n 5 4). Data are shown as mean 6 SEM. For statistical analyses, Mann-Whitney test was used. *P < .05; **P < .01; ***P < .001. NS, Not significant.

  ). Unlike for permeability and angiogenesis, serum IL-33 blockade did not modified fibronectin, collagen 1, and a-SMA production by fibroblast cultured with TAK serum (Fig E2, B) nor with MC supernatant incubated with TAK serum plus anti IL-33 (see Fig E3 in this article's Online Repository at www.jacionline.org).

FIG 2 .

 2 FIG 2. Increased vessel permeability in TAK is dependent on MCs. Vessel permeability induced by sera was evaluated by performing a Miles assay. Extravasation of Evans blue dye, as a measure of vascular leakiness, was analyzed using spectrophotometry. (A) Miles assay on wild-type (WT) mice after injection of sera obtained from patients with TAK (n 5 14) and HDs (n 5 6) (PBS and histamine were injected as a negative and positive controls, respectively). (B) Representative illustration of Miles assay performed on WT mice using injection in duplicate of sera from a patient with TAK (left) or from a HD (right). (C) Miles assay on WT and MC-deficient mice. Vascular permeability induced by injection of sera from patients with TAK was significantly decreased in MC-deficient mice compared with in WT mice (PBS and histamine were injected as a negative and positive control, respectively) (n 5 20 in each group). Data are shown as mean 6 SEM. For statistical analyses, Mann-Whitney test was used;.*P < .05; ***P < .001; ****P < .0001. KO, Knockout.

FIG 3 .

 3 FIG 3. MC-induced vessel permeability in TAK is partially mediated by IL-33. (A) Level of IL-33 in sera of HDs and patients with TAK. (B) Levels of histamine and of tryptase in the supernatant of MCs cultured for 5 days with 10% TAK (n 5 11) or HD (n 5 6) serum previously incubated 1 hour with or without human recombinant IL-33. (C) Immunofluorescence staining of TAK inflammatory lesions showed expression of ST2 (in green) on MCs (tryptase, in red) (white arrows). Original magnification 6403. (D) Miles assay were performed on WT mice as described in Fig 2. Vascular permeability induced by injection of sera from patients with LVV was decreased when sera from patients were previously incubated 1 hour with neutralizing anti-IL-33 antibody. (E) Representative illustration of Miles assay (Evans blue extravasation) performed on WT mice using injection in duplicate of sera from a patient with TAK with isotypic control antibody (left) or with anti-IL-33 antibody (right). Data are shown as mean 6 SEM. For statistical analyses, Mann-Whitney test and Wilcoxon matched pairs test for paired groups were used. ***P < .001.

FIG 4 .

 4 FIG 4. MCs support neoangiogenesis in TAK through IL-33. (A) Percentage of proliferation rate as measured by 5-bromo-2-deoxyuridine incorporation assay in HUVECs treated for 24 hours with TAK (n 5 17) or HD (n 5 6) sera and with supernatant of MCs cultured for 5 days with 10% TAK (n 5 12) or HD (n 5 6) sera with or without anti IL-33. (B and C) In vitro angiogenesis assay on growth factor reduced Matrigel. HUVECs treated for 12 hours with TAK or HD (n 5 5) sera and MC supernatant cultured for 5 days with 10% TAK (n 5 9) and HD (n 5 6) sera previously incubated 1 hour with or without anti-IL-33. (D) Representative experiment of capillary tube formation by HUVECs treated with TAK sera preincubated with neutralizing anti-IL-33 or the same TAK sera preincubated with isotype antibodies. Data are shown as mean 6 SEM. For statistical analyses, Mann-Whitney test and Wilcoxon matched pairs test for paired groups were used.

FIG 5 .

 5 FIG 5. MCs support fibrosis in TAK. (A) Production of fibronectin, type 1 collagen, and a-SMA by fibroblasts cultured for 5 days with TAK (n 5 14) or HD (n 5 7) sera and MC supernatant cultured for 5 days with 10% TAK (n 5 9) and HD (n 5 6) sera. (B) PDGF and TGF-b mRNA quantification in aorta obtained from patients with TAK (n 5 37) and HDs (n 5 4) by qRT-PCR. (C) PDGF and TGF-b were measured in the supernatant of MCs cultured for 5 days with 10% of HD (n 5 5) or TAK (n 5 13) sera. Data are shown as mean 6 SEM. For statistical analyses, Mann-Whitney test and Wilcoxon matched pairs test for paired groups were used *P < .05; **P < .01; ****P < .0001.

FIG E1 .

 E1 FIG E1. MCs localize mostly in the adventitia layer. Chromogenic immunohistochemistry of 3 TAK aorta sections showing that MCs localize mostly in the adventitia layer and to a lesser extent in the intima.

TABLE I .

 I Main demographical and clinical characteristics of the cohort of patients with TAK studied

		TAK N 5 30
	Demographic features
	Age, median (IQR)	32.4 (27.2; 53.2)
	Female	24 (79.6)
	Geographic origin
	Caucasian	11 (33)
	African	8 (27)
	North African	8 (27)
	Other	3 (10)
	Clinical features
	Numano classification
	I	4 (13)
	II	5 (17)
	III	3 (10)
	IV	1 (3)
	V	17 (57)
	Ischemic cardiac disease	3 (10)
	Stroke	6 (20)
	Aortic aneurysms	11 (37)
	Treatment
	Steroids <10 mg/d	20 (66)
	DMARDS	15 (50)
	Biologics	0 (0)
	DMARDs, Disease modifying antirheumatic drugs; IQR, interquartile range.
	Values are n (%) unless otherwise indicated.

*These authors contributed equally to this work. A.C. Desbois was supported by grant from the Fondation pour la Recherche M edicale. A.

Le Joncour was supported by grant from INSERM.


S Alaoui Haroni

" M Alifriqui

Vincent Simonneaux

S Alaoui
email: alaoui_safia@yahoo.fr

Haroni Ae

M Alifriqui

Ae V Simonneaux

Á M Alifriqui

Recent dynamics of the wet pastures at Oukaimeden plateau (High Atlas mountains, Morroco)

Keywords: Wet grasslands, Cartography, Dynamic, GIS, Biodiversity, High Atlas mountains, Morocco

Wetlands are a very important universal heritage; in the Moroccan High Atlas mountains the geomorphology and the slopes evolution determine the existence of the numerous projecting ledges of pozzines on plateaus and oozing. This is particularly the case in Oukaimeden site located at 75 km south of Marrakesh. These wet grasslands have been developed on small surface areas supported by soils water accumulation; and close to the sources and on some banks constitute wetlands of high floristic richness, which makes them very coveted pastures by local populations. These wet pastures are highly sensitive to climatic factors. Recent climatic changes, the anthropic and pastoral pressures and the global environmental changes, affect these zones at the level of their structure (areas, soil characteristics…) and their ecological functions. In the Oukaimeden plateau, the use of aerial photography made it possible to carry out a detailed follow up of the hydraulic adjustments (dam construction in the 1970s) as well as the setting up of new adjustments (tracks, paths, drain channel) on the structure and functioning of these grasslands.

Introduction

All over the world, wetlands are of particular scientific interest, for their great ecological value as ecosystems containing a very characteristic flora and fauna. Particularly in Morocco, this is practically the richest country in wetlands among all the North African countries. Moroccan wetlands are characterized by a highly degree of biodiversity [START_REF] Galland | Dynamique de la ve ´ge ´tation et des formations superficielles dans les montagnes du bassin occidental de la me ´diterrane ´e: e ´tude des formations a gene ´vrier thurife `re et xe ´rophytes e ´pineuses en coussinet des atlas marocains; doctorat d'e ´tat; universite ´Paul Sabatier[END_REF]).

In the High Atlas mountains, the wetlands subject of this study are present at the altitudes ranging between 2,500 and 3,000 m, and occupy depressions and projecting ledges where water, fine materials (sand, clay) and humic matter accumulate [START_REF] Que ´zel | Peuplement ve ´ge ´tal des hautes montagnes de l'Afrique du nord. Encyclope ´die bioge ´ographique et e ´cologique[END_REF]Gauquelin 1988). They are mountainous vegetal formations named pozzines; this term was introduced into the scientific literature by the botanist Briquet in 1910 to indicate wetland vegetal formations of the High Corsican mountains.

Those pozzines occupy the projecting ledges which accumulate fine materials that allow a good retention of water precipitations and snow melt; these elements come from the framing slopes colonized by xeric thornyshrubs. They are the only summit vegetal groupings where recognition of sometimes complex soil is possible, but often without discernible strata (Gauquelin 1988). Therefore, these pozzines were described and recognized as being a specialized grouping related to the local climatic and edaphic conditions [START_REF] Galland | Dynamique de la ve ´ge ´tation et des formations superficielles dans les montagnes du bassin occidental de la me ´diterrane ´e: e ´tude des formations a gene ´vrier thurife `re et xe ´rophytes e ´pineuses en coussinet des atlas marocains; doctorat d'e ´tat; universite ´Paul Sabatier[END_REF].

The vegetation of the wet grasslands consists of an endemic flora, particularly rich in alpine and boreal plants. In this location, these wetlands which have been dominated by centuries-old pastoral exploitation, are still very coveted for the pasture and undergo a particular traditional management [START_REF] Berque | Structures sociales du Haut Atlas[END_REF]. Considering their vulnerability, permanent or intermittent wetlands are very affected by climatic changes [START_REF] Harris | Ecological restoration and global climate change[END_REF], by the anthropic and pastoral pressure [START_REF] He | Zoning grassland protection area using remote sensing and cellular automata modeling. A case study in Xilingol steppe grassland in northern China[END_REF]. Indeed, because of their strict water determinism and soil, the altitudinal wet grasslands are highly affected by the recent climatic changes [START_REF] Hulme | Adapting to climate change: is there scope for ecological management in the face of a global threat?[END_REF][START_REF] Harris | Ecological restoration and global climate change[END_REF] and for this reason constitute very good witnesses of the environmental changes that the site undergoes.

Materials and methods

Geographical situation

The Oukaimeden plateau (W 7°52 0 , N 31°12 0 ) is located at 75 km south of Marrakesh (Fig. 1); it rises at an altitude of 2,650 m.

Geology

The site of Oukaimeden belongs to the High siliceous Atlas mountains with an acid eruptive Precambrian substrate comprising Rhyolites and Andesites, is limited to the North by red sandy substrates of Jbel Tizrag, from permotriasic age [START_REF] Dresch | Recherches sur l'e ´volution du relief dans le massif central du grand Atlas, le Haouz et le Sous[END_REF][START_REF] Que ´zel | Peuplement ve ´ge ´tal des hautes montagnes de l'Afrique du nord. Encyclope ´die bioge ´ographique et e ´cologique[END_REF].

Climate

The Oukaimeden site is dominated by the semiarid Mediterranean bioclimate with cold winter variant. Rainfall varies between 400 and 500 mm/year (Fig. 2). Snowfall is significant between December and March; freezing days are estimated between 82 and 139 days/ year according to years. The temperatures are rather low, the minima average in the coldest month is evaluated at -2.69°C and the average maxima of the hottest month reached 23.5°C.

Vegetation

The Oukaimeden grasslands flora is remarkable mountainous vegetation, rich in endemic species and supplemented with plants of alpine and boreal origin. It is consist of vegetal Oukaimeden plateau's whole flora (highly wet grasslands ? pozzines, wet grasslands, fairly wet grasslands and dry grasslands) (Table 1) is characterised by a specific richness estimated at 198 species divided into 122 genus and 38 families.

The wet entity which includes highly wet grasslands ? pozzines, wet grasslands, fairly wet grasslands represent a great floristic biodiversity estimated at 134 species, 97 genus and 34 families (Table 2). This represents more than 60% of the total specific richness of the Oukaimeden grasslands with a rate of endemism exceeding 25% (29 species are endemics of Morocco only, 5 are endemics of Morocco and Iberian peninsula, 1 specie endemic of Morocco and Algeria and 1 specie endemic of Morocco, Iberian peninsula and Algeria); and degree of species rarity exceeding 20% (15 are rare species, 8 are suspect rare species, 14 are highly rare species and 1 species is suspect highly rare) (Fig. 3). This flora extremely supplemented with plants of Euro-Siberian-Boreo-American origin (23%) divided on 19 species of Circum-boreal origin and 13 species of Eurasiatic origin (Fig. 4), which confers to these milieus a significant floristic and landscape originality (Alaoui [START_REF] Haroni | Altitudinal wet pastures: threats and conservation means; the case of Oukaimeden plateau (High Atlas Mountains, morocco)[END_REF].

More specifically, these spaces are very coveted for their pastoral richness. Indeed, this study site corresponds to a collective rangeland called ''Agdal:'' ''A traditional Institution shape of collective pastures management of the stockbreeders in the High Atlas'' [START_REF] Mahdi | Scale-dependent relationships between plant diversity and aboveground biomass in temperate grasslands, south-eastern Mongolia[END_REF]. This particular traditional management of area and resources consists of limiting rights of access and (or) uses of the customary law [START_REF] Ilahiane | The berber Agdal institution: indigenous range management in the Atlas mountains[END_REF][START_REF] Auclair | Les ressources sylvopastorales au Maghreb. Structure segmentaire et appropriation communautaire[END_REF]), aiming to allow the milieu reconstitution in order to ensure the perenniality of the resource.

Methodology

In recent years, geographic information systems (GIS) have increasingly been used in a wide array of application contexts for development cooperation particularly in mountain areas [START_REF] Heinimann | The challenge of applying geographic information system to sustainable mountain development[END_REF]. In this study, cartography techniques such as GIS capabilities aims to carry out dynamic map of Oukaimeden wet grasslands. This cartography required the compilation of a whole data base which included:

• Aerial photos of the 1964, 1986 and 2002 missions (Table 3).

• The satellite imagery (spot-vegetation in 2000) of the study site.

• Topographic maps digitalized and georeferenced by the software ''Envi.'' • The digital terrain model. The approach rests on two principal stages

• Pre treatment and data preparation

The various missions of the aerial photographs underwent a pre treatment by the software ''Envi'' which consisted of the orthorectification of these photos in order to project them On the aerial photos mission of 1964, 1986 and 2002 the visual photo-interpretation by stereoscope consists of delimiting homogeneous zones from the grasslands humidity point of view, which based on the distinction of the levels of gray on the photo (more the grassland is humid more it appears gray dark on the photo), the limits of the grasslands can be traced by GIS-Arcview 3.2 [START_REF] Kraak | GIS-cartography: visual decision support for spatio-temporal data handling[END_REF][START_REF] Wang | Spatial accuracy of orthorectified IKONOS imagery and historical aerial photographs across five sites in China[END_REF]. Thus, the updating of 2002 mission and the verification were done on the terrain via GPS points made it possible to obtain actual Oukaimeden grasslands map ( 2004).

The maps of 1964, 1986 and 2004 so ready will be superimposed via the GIS. The crossing between 1986 and 2004 gives rise to the dynamic map of the Oukaimeden wet grasslands and the crossing between 1964 and 1986 on only the plateau zone in the dam neighbourhoods made it possible to evaluate his contribution in the extension of the wet grasslands surfaces, we cannot carried out a whole wet grasslands evolution between 1964 and 1986 because 1964 aerial photo did not cover all the wet grasslands in the study site but only in the plateau.

Results

The monochronic cartography (Fig. 5) of Oukaimeden grasslands shows that the geomorphology and the gradient of humidity allow the individualization of the various facies (Fig. 6). This can be found from the highly wet pozzines occupants of small surfaces, to the very wide dry grasslands which mark the transition towards xeric element.

The diachronic cartography based on crossing monochromic maps 1964, 1986 and 2004 (Fig. 7, Table 4) enabled us to evaluate the spatiotemporal evolution of wet grasslands.

Between 1964 and 1986 the evolution of wet grasslands on only the plateau zone in the dam neighbourhoods made it possible to evaluate the contribution of dam construction in the extension of the wet grasslands area. So, for do it we consider various groupings of wet grasslands in 1986 as being only one wet entity.

During this period, wet grasslands knew a very significant progression. It is evaluated at 20.784 ha, the plateau grasslands areas passed from 3.24 ha at 1964 to 24.024 ha at 1986. This progression is due to the installation of the Oukaimeden plateau dam in 1970s. This was very beneficial for the wet grasslands; it is allowed the water refill of the neighbouring soils and consequently the extension of the wet grasslands area, the dam construction allowed not only the extension of wet grasslands on the Oukaimeden plateau but also appearance of new wetlands around dam margin, indeed, wetlands in the plateau due to the dam installation are of two kinds:

• A wet grassland and pozzines that are the same as the one of origin with the same floristic content (Narcissus bulbocodium L. ssp. obesus (Salisb.) Maire, Nardus stricta L., Dactylorhiza elata (Poiret) Soo …) • And new wetland (Fig. 8) that never had place on the site before the dam construction; it is about flooding zone of the dam margin containing a very characteristic flora of this environment type (Crassula vaillantii (Willd.) Roth, Eleocharis palustris (L.)…), and also vegetal species that are common with the pozzines and wet grasslands (Lepidium hirtum L. ssp. atlanticum (Ball), Poa trivialis L.) (Table 5). 1964, 1986 and 2004 Pozzines's species did not colonize the new wetland yet, may be because of their mode of dissemination or because of the milieu conditions (soil nature, water retention by soil…) which do not allow the installation of these species.

Between 1986 and 2004, the dynamic map of Oukaimeden wet grasslands (Fig. 9, Table 6) translates the whole Oukaimeden wet grasslands evolution.

According to the dynamic map 8% (6.598 ha) of Oukaimeden wet grasslands is in regression. This regression is localised in the plateau near roads and in the valleys near sheepfolder and human activities.

The regression is of two types:

Regression type 1: represents 35% and corresponds to the regressions of grasslands grouping to another grouping while remaining in the wet entity: such as the regression of the highly wet groupings to a lesser wet groupings. Regression type 2: estimates at 65% and corresponds to the elimination of the wet entity in favour of the dry grasslands or to the total disappearance of the wet grasslands.

On the dynamic map also appears the zones which are in progression, they are estimated at 0.94 ha (1%) and are located in the plateau near dam and along stream pouring out in the dam.

Alopecurus pratensis

L.

Characteristic species

Botrychium lunaria

L.

Crassula vaillantii

(Willd.) Roth
Apium nodiflorum (L.) Koch.

Ranunculus lateriflorus

DC.

Eryngium variifolium

Coss.

Eleocharis palustris

Sedum surculosum

Coss.

Gentiana atlantica

Litard. & Maire

Trifolium humile L.

Persicaria bistrota L. ssp. bistrota

Rumex atlanticus

Batt.

Alchemilla atlantica

Lindb.

Table 5 continued Pozzines and highly wet grasslands

Dam margin

Parnassia palustris

L.

Euphrasia willkomii

Freyn

Parentucellia viscosa

(L). Caruel

Veronica anagallis aquatica L.

Veronica repens

DC.

Viola palustris

L.

Narcissus bulbocodium

L. ssp. obesus (Salisb.)

Maire

Carex echinata

Mur.

Carex intricata

Tineo

Carex leporina

L.

Juncus fontanesii Gay ssp. brachyanthus

Trab.

Luzula multiflora (Retz) ssp. congesta (Thuill.)

Luzula spicata (L.) DC.

Dactylorrhiza elata (Poiret) Soo

Orchis coriophora L.

Orchis mascula

Link.

Agrostis atlantica

Maire & Trab.

Anthoxanthum odoratum

L.

Avenula bromoides

Discussion

The High mountains wet grasslands are wetlands with great ecological, economic and socio-cultural interest. These vegetal formations characterised by their significant floristic richness contain a very vulnerable flora, rich in endemic species and plants of alpine and boreal origin. The characteristic of these zones as a climatic and botanic intersection land, situated by a Mediterranean zone from the north and a strong southern influence, represents a great phytogeographical originality. The dam installation in the 1970s was beneficial for the Oukaimeden plateau wet grasslands; it is allowed the extension of wet grasslands and the creation of new habitats for wetlands characteristic flora and fauna. Although, the spatiotemporal evolution shows some stability between 1986 and 2004 and regression representing only 8%, the global environmental changes that the site knows all the same threaten one of the mountainous biodiversity hottest spot of Morocco and North Africa (Que ´zel 1957;[START_REF] Galland | Dynamique de la ve ´ge ´tation et des formations superficielles dans les montagnes du bassin occidental de la me ´diterrane ´e: e ´tude des formations a gene ´vrier thurife `re et xe ´rophytes e ´pineuses en coussinet des atlas marocains; doctorat d'e ´tat; universite ´Paul Sabatier[END_REF]. Because of their strict water determinism and soil, these vegetal units are very affected by the recent climatic changes which threatens our planet [START_REF] Hulme | Adapting to climate change: is there scope for ecological management in the face of a global threat?[END_REF][START_REF] Sebastia | Plant guilds drive biomass response to global warming and water availability in subalpine grassland[END_REF].

These climatic changes, increasingly amplified by more human activity, jeopardise the rangeland productivity (particularly biomass production) and consequently ecosystem functions [START_REF] Mahdi | Scale-dependent relationships between plant diversity and aboveground biomass in temperate grasslands, south-eastern Mongolia[END_REF]. Indeed, At Oukaimeden plateau the most recent climatic changes are manifested by a considerable drop of snowfall last decades especially in spring (Fig. 10) also the Oukaimeden site knew a drop of rainfall; it was described by Que ´zel in 1957 in the ombrothermic diagram like a station with subhumid climate with cold winter that is the annual rainfall average between 700 and 800 mm but nowadays Oukaimeden is a station with semiarid climate (annual rainfall average between 400 and 500 mm). These climatic changes make wet grasslands more sensitive to anthropogenic pressure.

The pastoral pressure consisting of more than 5,000 heads of livestock per year constitutes a considerable threat and causes a pressure on the natural resources (Pazos et al. 1 9 9 0 1 9 9 1 1 9 9 2 1 9 9 3 1 9 9 4 1 9 9 5 1 9 9 6 1 9 9 7 1 9 9 8 1 9 9 9 2 0 0 0 2 0 0 1 2 0 0 2 2 0 0 3 2 0 0 4 2 0 0 5 2 0 0 6 2 0 0 7 Snowfall Moving average for 5 periods (snowfall) 0 100 200 300 400 500 600 700 800 900 1 9 8 9 1 9 9 0 1 9 9 1 1 9 9 2 1 9 9 3 1 9 9 4 1 9 9 5 1 9 9 6 1 9 9 7 1 9 9 8 1 9 9 9 2 0 0 0 2 0 0 1 2 0 0 2 2 0 0 3 2 0 0 4 2 0 0 5 2 0 0 6 2 0 0 7 Snowfall Moving average for 5 periods (snowfall) Fig. 11 The impact of livestock trampling on the wet grasslands closure of the latest species vital cycle. The correspondence between the opening date and the resource regeneration proves the efficiency of this kind of management that aims for the milieu reconstitution and the resource safeguard. However, the increasing promotion of the tourist activity and in particular, mountain tourism directly affects these wetlands. Indeed, Oukaimeden is a station of skiing with touristic vocation, this tourist activity requires the adjustment of various infrastructures: drain off canals of snow melting waters, rains and sewages, reception facilities: hotels, chalets, restaurants… These various adjustments of construction draw their raw materials (removal of soils and gravels) from extended neighbouring wet grasslands. The impact does not stop there, in addition to the building materials removal (Fig. 12); the wet grasslands also receive waste construction, turning the space into a real discharge (Fig. 13).

Furthermore, the opening of road and path in order to facilitate the moving between different ''douars'' (villages) in the neighbourhoods of the Oukaimeden plateau created the drainage pits (Fig. 14) that leads to the drainage of soil water reserve ending in drying up of wet grasslands. This had as consequence the extension of the dry grasslands to the detriment of the oozing wet grasslands. The original vegetal communities, characterised by a strong representation of a particular mountainous flora of Morocco, cannot support such disturbances. But, the regression that knew the Oukaimeden wet grasslands has affected these zones at the level of their superficies and areas, we did not note the disappearance of characteristic taxa even the rarest ones.

Conclusion and perspective conservation

Altitudinal wet grasslands present a very significant ecological and biological inheritance.

These wet grasslands flora is supplemented by endemic and boreo-alpine species and constitute one of the primordial natural resources that are still allowing to maintain populations in such a marginal and sensitive space. Even if these wet grasslands know some temporal stability they deserve all the same that we act as soon as for their preservation and threats protection. Thus, we propose the following means of conservation:

• The valorisation of the indigenous knowledge as well as the community management for the access to the pastorals resources (Agdal). This type of ancestral practice allows the milieu reconstitution and the conservation of these pastoral spaces. Especially, front of the failure of the nature conservation strategies in Morocco, common protected area like pastoral Agdal appear better conserved, managed by local population and having rights with a collective way guaranteeing the durability of the resource. Thus, the registration of the site as protected area according to the common laws with the establishment of green track rigorously respected can constitute a very judicious means of valorisation of this site with all these ecological components, biological and human. • The valorisation of these wet pastures by educative tourism, by the creation in situ of school camps allowing children to develop a sense of naturalism. • The recognition of Oukaimeden site as an important plant area (IPA) or as a site of biological and ecological interest (SIBE) (new type of protected areas in Morocco), forcing the local actors to take measures to safeguard and conserve this biological and ecological heritage. • The scientific valorisation of the site by creating an observation center of the biodiversity evolution of the High Atlas wetlands sites, and at the same time, be able to adopt a global and comparative approach between these various wet sites.

Fig. 1

 1 Fig. 1 Map of the Oukaimeden site situation

  ; HWG, Highly wet grasslands; WG, Wet grasslands; OOZ, Oozing; FWG, Fairly wet grasslands; DG, Dry grasslands; D. M., Dam margins

Fig. 3 Fig. 4

 34 Fig. 3 Status of Oukaimeden wet grasslands species. E, endemics of Morocco; I, endemics of Morocco an Iberian peninsula; A, endemic of Morocco and Algeria; IA, endemic of Morocco, Iberian Peninsula and Algeria; R, Rare species; R?, Suspect rare species; RR, Highly rare species; RR?, Suspect highly rare species; SWS, Species without specific status

Fig. 5

 5 Fig. 6 Importance of Oukaimeden grasslands facies

Fig. 9

 9 Fig. 9 Dynamic map of Oukaimeden wet grasslands

Fig. 10

 10 Fig. 10 Snowfall in Oukaimeden plateau between 1989 and 2007

Fig. 13

 13 Fig. 13 Discharge on the wet grasslands

Fig. 14

 14 Fig. 14 Drainage pits in the wet grasslands

Table 1

 1 Characteristics of the various facies of the Oukaimeden wet grasslands(Gauquelin 1988)

	Facies	Substrate nature	Humidity	Slope	Recovery
			(in the first 10 cm
			in the soil)
	Highly wet	Peaty soil	100%	\5%	100%
	grassland ? pozzine
	Wet grasslands	Marly clayey soil	80% to 100%	\5%	100%
	Fairly wet grasslands	Sandy clayey soil	60% to 80%	\5%	90% to 80%
	Dry grasslands	Sandy soil with some	Less than 40%	More than 5% 80%
		stones on the surface

Table 2

 2

		Biogeographic	origin	Circum-boreal	Mediterranean					Eurasiatic					Mediterranean		Mediterranean			Mediterranean				Mediterranean			Eurasiatic
		Endemism Species	rarity	RR			E		E	R E		E	R ? E					R? E	E					E	E		E
		Facies	POZ OOZ D.M HWG WG FWG DG	* * *	* *	* * *	* *	*	* * *	* * *	*	* *	* *	* *	* *	*	* *	* * *	* *	* *	* *	*	* *	* *	* *	* *	* * *
	Table 2 List of Oukaimeden wet grasslands species	Family Species		Ophioglossaceae Botrychium lunaria L.	Apiaceae Apium nodiflorum (L.) Koch.	Apiaceae Apium repens Jacq.	Apiaceae Bunium alpinum Waldst. & Kit. ssp. atlanticum (Boiss)	Apiaceae Conium maculatum L.	Apiaceae Eryngium variifolium Coss.	Apiaceae Heracleum sphondylium (L.) Litard. var. embergeri Maire	Asteraceae Anacyclus pyrethrum L.	Asteraceae Bellis caerulescens Coss.	Asteraceae Cirsium chrysacanthum (Ball) Jahand.	Asteraceae Crepis pulchra L.	Asteraceae Hieracium pseudopillosella Ten.	Asteraceae Hypochoeris arachnoidea Poir.	Asteraceae Jurinea humilis DC.	Asteraceae Lactuca virosa L. ssp. cornigera (Pau & Font Quer)	Asteraceae Leontodon atlanticus (Ball.)	Asteraceae Mantisalca salmantica (L.) Briq. Et Cavill.	Asteraceae Senecio giganteus Desf.	Asteraceae Sonchus maritimus L. ssp. aquatilis (Pourr.) Nyman	Asteraceae Tarxacum sp.	Asteraceae Tolpis barbata (L.) Gaertn. ssp. liouvillei	Boraginaceae Cynoglossum watieri (Batt. & Maire) Br-Bl. & Maire	Boraginaceae Echium plantagineum L.	Boraginaceae Myosotis atlantica Vestergr.

Table 3

 3 Characteristics of aerial photographs in used

	Mission	Scale	Coverage	Photographs	Organism
	1964	1/50,000	NH29 XXII XXIII	185-189	IGN
	1986	1/20,000	BD 154	123-127	IGN
	2002	1/20,000	BD 05	05-11	DREF
			BD 06	07-11

Table 4

 4 Soil occupation between 1986 and 2004, expressed by the area at hectare

	Facies of grasslands	1986	2004
	Highly wet grasslands and pozzines	18,452	14,569
	Wet grasslands	25,849	23,952
	Fairly wet grasslands	34,849	34,120

Table 6

 6 Results of diachronic cartography of wet grasslands(between 1986 and 2004)

	Evolution type	Area (ha)	Percentage (%)
	Progression	940	1
	Regression	6,598	8
	Stable	71,612	91

Acknowledgements This work comes from the framework of the research program «Les Agdal du Haut Atlas marocain: biodiversite ´et gestion communautaire de l'acce `s aux espaces sylvopastoraux». Many thanks for its financial support and scientific (Convention of IRD-IFB financing/INRA N 2886). We gratefully acknowledge the SudMed project carried out by the IRD and the University Cadi Ayyad of Marrakesh and the CAF (French Alpine Club) for the logistics and climatic data that they provided us.


Marina Deng

Chhandak Mukherjee

Nil Davy

Virginie Nodjiadjim

Muriel Riet

Colin Mismer

Jérémie Renaudier

Magali De Matos

Cristell Maneux

InP DHBT characterization up to 500 GHz and compact model validation towards THz circuit design

Keywords: Indium-Phosphide (InP), InP/InGaAs, Double heterojunction bipolar transistor (DHBT), on-wafer characterization, millimeter-wave, sub-millimeter-wave

We report on-wafer characterization results up to 500 GHz on a 0.4×5 µm² InP/InGaAs DHBT. The measurements were performed using an on-wafer Thru-Reflect-Line (TRL) calibration kit especially developed in this technology. The resulting measurements allowed to validate both the RF performance of the 0.4-µm InP/InGaAs DHBT fabricated by III-V Lab, featuring 390/600 GHz fT/fmax, and the HiCuM compact model towards future circuit design at submillimeter-wave frequencies thanks to 90% typical yield reached in this technology.

I. INTRODUCTION

Nowadays, the multiplication of communication services, such as remote work, e-learning, e-healthcare and intensification of social relations supported by massive data transmission, pushes to develop the whole value chain from component to circuit, including the compact modeling of components, in order to increase transmission network capacities with Tb/s/channel objective [START_REF] Konczykowska | Recent advances in highspeed and large-swing integrated circuits implemented in InGaAs and GaAsSb InP DHBT for terabit-class optical coomunications[END_REF].

The Indium Phosphide (InP) double heterojunction bipolar transistor (DHBT) has become a promising candidate for terahertz (THz) integrated circuit design, by combining both maximum oscillation frequencies (fmax) above 0.8 THz and common-emitter breakdown voltage above 4.2 V [2]- [START_REF] Nodjiadjim | 0.7-μm InP DHBT Technology With 400-GHz fT and fMAX and 4.5-V BVCE0 for High Speed and High Frequency Integrated Circuits[END_REF].

Nevertheless, to ensure such high RF potential, the accurate RF characterization of these THz transistors is crucial to confirm the high cut-off frequencies as well as to precisely extract and validate the device compact model, thus enabling millimeter and sub-millimeter-wave IC design. On-wafer measurements are particularly difficult beyond 110 GHz, as discussed in [START_REF] Yau | Device and IC Characterization Above 100 GHz[END_REF] and [START_REF] Krozer | On-wafer small-signal and largesignal measurements up to sub-THz frequencies[END_REF], since unwanted parasitic effects become predominant, which are not totally removed by calibration and de-embedding techniques, and may mask the intrinsic behavior of the nanoscale device. Above 110 GHz, two strategies may be adopted and combined in order to perform accurate on-wafer device measurements at submillimeter-wave frequencies: i) optimizing the test structures' design in order to minimize the unwanted parasitics, ii) developping on-wafer calibration standards on the same substrate as the device to characterize. In this paper, we demonstrate that these strategies proved their efficiency for the characterization and compact modelling of an InP DHBT up to 500 GHz.

II. SUB-MILLIMETER WAVE CHARACTERIZATION STRATEGY

A. Technology Overview

The transistor vertical structure was grown by IntelliEPI on a 3'' semi-insulated InP substrate using Solid Source Molecular Beam Epitaxy. It includes a 40-nm InP emitter and a 28-nm highly C-doped. The highly doped level of this layer (~ 8x10 19 cm -3 ) leads to a low sheet resistance of 750Ω/□. The 130-nm thick collector is composed of a non-intentionally doped spacer, 20-nm thick highly doped InP region and a low doped InP layer (< 2.10 16 cm -3 ) to obtain a high breakdown voltage. An In-rich and highly doped InGaAs cap emitter layer is used in order to minimize the emitter contact resistivity (< 3Ω.µm 2 ).

The transistors were fabricated using a wet-etch triple mesa technology including a self-aligned base-emitter metallization process similar to the one described in [START_REF] Nodjiadjim | 0.7-μm InP DHBT Technology With 400-GHz fT and fMAX and 4.5-V BVCE0 for High Speed and High Frequency Integrated Circuits[END_REF]. The emitter contact has a rectangular shape and a width of 0.4-or 0.5-µm. In comparison with previous transistor design [START_REF] Mukherjee | Towards Monolithic Indium Phosphide (InP)-Based Electronic Photonic Technologies for beyond 5G Communication Systems[END_REF], the base contact (WB) is reduced from 0.3-to 0.2-µm for some devices in order to minimize the base-collector capacitance. The transistor size is much more compact thanks the reduction of the distance between the base mesa and the collector contact as well as the minimization of the collector contact width. This allows to reduce parasitic elements such as collector access resistance, base-collector capacitance and emitter-collector capacitance. Figure 1 shows a scanning electron microphotograph of a 0.4µm emitter width DHBT before interconnection level, presenting an innovative rectangular emitter shape compared to conventional previous one. The typical fabrication yield of the 0.4-µm InP DHBT is above 90%.

B. On-wafer Calibration Kit Design

The on-wafer calibration kit was designed so as to apply the Thru-Reflect-Line (TRL) method, well known to be efficient for accurate S-parameter measurements beyond 110 GHz on both silicon and InP heterojunction transistors characterization [START_REF] Williams | A Prescription for Sub-Millimeter-Wave Transistor Characterization[END_REF]- [START_REF] Deng | Design of On-Wafer TRL Calibration Kit for InP Technologies Characterization up to 500 GHz[END_REF]. Well-known for its robustness and traceability, the TRL method, based on only three standards, is also easy to implement, since two transmission line, one Thru and one Line, need to be designed, as well as a symmetrical Reflect, either open or short-type. The only known parameters are the delays of the Thru and Line standards. In the III-V Lab InP process, 50 ohm coplanar waveguide (CPW) transmission lines were used for Thru and Lines standards, and one Pad-Open standard as the Reflect. As depicted in Fig. 2, the TRL calibration standards were located in the periphery of the chip. A continuous ground plane was implemented as recommended in [START_REF] Cabbia | Silicon Test Structures Design for Sub-THz and THz Measurements[END_REF] and [START_REF] Deng | Design of On-Wafer TRL Calibration Kit for InP Technologies Characterization up to 500 GHz[END_REF] with the support of electromagnetic simulation so as to minimize the coupling between the RF probes and the substrate. Then, all the ground pads of each test structure are connected together through this continuous ground plane. This on-wafer TRL calibration kit was used to perform Sparameter measurements from 1 to 500 GHz using four benches: 1-110 GHz, 140-220 GHz (G-band), 220-330 GHz (J-band) and 325-500 GHz. The ground-signal-ground RF pads were designed with a 50-100 µm probing pitch in order to be compatible with the different sets of RF probes in each measurement band.

C. De-embedding Test Structures Design And Characterization results

The transistor under test presented an emitter width of 0.4 µm and an emitter length of 5 µm. This geometry was selected for its high cut-off frequencies, with a fT/fmax couple of 390/600 GHz extracted at low frequencies, while maintaining a commonemitter breakdown voltage superior to 4.5 V.

The test structures associated to the transistor, as well as the dedicated open and short, are shown in Fig. 3. The CPW pads and access lines that connect the transistor are similar to the calibration structures. Consequently, the reference plane after calibration could be set along the access line as close as possible to the transistor terminals, as depicted in Fig. 3-b. The remaining interconnection parasitic effects were then removed using the short-open de-embedding.

Furthermore, the validity of the short-open de-embedding method rely on a lumped equivalent electrical circuit of the open and the short structures (see Fig. 4). To verify the frequency independence of the Open capacitances and Short inductances, these de-embedding test structures were measured up to 500 GHz, using on-wafer TRL calibration. The resulting capacitances and inductances were extracted using respectively Y and Z parameters (see Fig. 5). The open capacitances showed a relatively constant value in the overall 1-500 GHz measurement band, with no resonance in the highest frequency range and a good band continuity. The short measurements were quite difficult to carry out, especially in J-band. This was due to a high sensitivity to the contact in addition to the fact that the TRL calibration was made with the Pad-Open as Reflect, reducing the calibration accuracy for short measurements [START_REF] Yadav | On the Variation in Short-Open De-embedded S-parameter Measurement of SiGe HBT upto 500 GHz[END_REF]. However, an average constant value among the 1 to 500 GHz measurement range can be observed for the computed inductances. The validity of the Short-Open de-embedding method to apply to the transistor's measurement was then verified up to 500 GHz. 3. S-parameter measurement and simulation up to 500 GHz The HiCuM compact model was simulated and compared to the InP DHBT measurements up to 500 GHz after on-wafer TRL calibration and Short-Open de-embedding. The transistor under test was biased at peak fT/fmax, that is at VCE=1.6 V and VBE=0.94 V. A pretty good agreement was obtained in the overall 1-500 GHz frequency range. The deviation from the model is particularly observed above 325 GHz up to 500 GHz for all Sparameters. In the last band, indeed the transistor shows less gain while the on-wafer measurement setup is subject to significant losses in the connectors despite the maximum applied RF power. On-wafer TRL meas HiCUM sim measurements up to 500 GHz. The transistor was biased at a fixed VCE=1.6 V and the base voltage was swept from 890 to 940 mV. The H21 current gain and unilateral Mason gain were computed for both measured and simulated data, as well as their gain-bandwidth product so as to extract the transit frequency, fT, and the maximum oscillation frequency, fmax.

The expected 390/600 GHz fT/fmax was then confirmed by the measurements up to 500 GHz for fT and up to 400 GHz for fmax. This validates this InP DHBT technology as a promising candidate for THz MMIC design. Also, the satisfying agreement between the model and the measurement demonstrates the capability of the HiCuM compact model to fairly reproduce the transistor's behavior up to submillimeter-wave frequency range. Nevertheless, improvements still need to be done regarding the measurements' accuracy beyond 300 GHz, eventually by using EM simulation for a better understanding of the measurement artefacts or/and for further test structures optimization.

IV. CONCLUSION AND PERSPECTIVES

In this paper, on-wafer characterization of InP DHBT was achieved up to 500 GHz, using on-wafer TRL calibration and optimized test structures layout to minimize any parasitic effects due to the interconnects to the device and coupling between RF probes and substrate which strongly impacts the measurement accuracy at millimeter and sub-millimeter-wave frequency ranges. The validity of the lumped Short-Open de-embedding method was verified up to 500 GHz. This permitted accurate and reliable transistor measurements for both validation of the InP DHBT RF performances, featuring 390/600 GHz fT/fmax, and validation of the HiCuM compact model of InP DHBT up to 500 GHz, required to envisage successful first-pass THz MMIC design.

Fig. 1 .

 1 Fig. 1. SEM of 0.4×4-µm² InP DHBT before interconnection level

Fig. 2 .

 2 Fig. 2. Photography of the chip with all the test structures

Fig. 3 .Fig. 4 .Fig. 5 .Fig. 6 .Fig. 7 .

 34567 Fig. 3. Photography of the transistor (center) and its dedicated de-embedding Open (right) and Short (letft) test structures

Fig. 8 .Fig. 9 .

 89 Fig. 8. InP DHBT S-parameter measurement and simulation in magnitude up to 500 GHz -Transistor geometry: WE=0.4µm, LE=5µm, WB=0.2µm

Fig. 10 .Fig. 11 .

 1011 Fig. 10. InP DHBT gain curves: a) H21 current gain, b) Mason gain

This work was supported partly by the Agence Nationale de la Recherche in the frame of the project ULTIMATE (ANR-16-CE93-0007) and partly by the Région Nouvelle-Aquitaine authorities through the FAST project.


Nil Davy
email: nil.davy@3-5lab.fr

Virginie Nodjiadjim

Muriel Riet

Colin Mismer

Marina Deng

Mukherjee Chhandak

Jeremie Renaudier

Cristell Maneux

Marina Deng²

Chhandak Mukherjee²

Jérémie Renaudier

Cristell Maneux²

0.4-µm InP/InGaAs DHBT with a 380-GHz f T , > 600-GHz f MAX and BV CE0 > 4.5 V

Keywords: Double heterojunction bipolar transistor (DHBT), maximum oscillation frequency (fmax), Characterization, InP/InGaAs, millimeter-wave

    0.4-µm InP/InGaAs DHBT with a 380-GHz fT > 600-GHz fMAX and BVCE0 > 4.5 V

I. INTRODUCTION

To meet the growing demand of THz-range systems, be it for wireless communication, optical transmission or for imaging, various ultra-high-speed transistor technologies have emerged. InP heterojunction bipolar transistor (HBT are strong contenders for this competition. This technology has the potential to reach THz frequencies while combining high linearity and high breakdown voltage. InP/InGaAs/InP type-I DHBT have already been studied widely. Aggressive scaling of the emitter width has led to fMAX > 1 THz as shown in [START_REF] Rode | Indium Phosphide Heterobipolar Transistor Technology Beyond 1-THz Bandwidth[END_REF] and [START_REF] Urteaga | 130nm InP DHBTs with ft >0.52THz and fmax >1.1THz[END_REF] with a breakdown voltage (BVCE0) of 3.5 V. These performances have been achieved through engineering of the type-I base-collector heterojunction to improve both high frequency performances and high breakdown voltages. However, improving HBT performances through scaling is challenging as it increases the thermal resistance, which, in turn, negatively impacts the transistor performances, and decreases the breakdown voltage following collector thickness reduction.

In this paper we report 0.4 × 5 µm² InP/InGaAs DHBTs exhibiting fMAX = 605 GHz and fT = 380 GHz. The device shows a peak current gain of 29 and a common emitter breakdown voltage of 4.7 V. The improvement of RF performances is due to the reduction of the base-collector capacitance achieved through emitter size scaling and base contact width shrinking. The rest of this paper is organized as follows: section II details the device architecture and the fabrication process; section III includes device characterization results and small signal modeling, followed by the conclusion.

II. DEVICE STRUCTURE AND FABRICATION PROCESS

A. Epitaxial structure

The DHBT structure was grown on a 3-inch semi-insulated InP substrate by IntelliEPI using solid source molecular beam epitaxy. The current scheme of epitaxy is an optimized version of the one introduced in [START_REF] Nodjiadjim | 0.7-μ m InP DHBT Technology With 400-GHz fT and fMAX and 4.5-V BVCE0 for High Speed and High Frequency Integrated Circuits[END_REF]. The emitter contact is formed with highly n-doped In0.85Ga0.15As in order to have a low emitter contact resistance. The structure consists of a 40-nm InP emitter Si-doped at 5 × 10 17 cm -3 . The 28-nm thick InxGa1-xAs base is compositionally graded from x = 0.47 at the emitter side to x = 0.53 at the collector side. This grading in composition induces a 40-meV conduction band slope across the base that reduces the transit time. In order to minimize the base sheet resistance, the base layer is heavily C-doped (8 × 10 19 cm -3 ) without doping grading. A base sheet resistance of 730 Ω/□ is extracted from transmission line measurements. A 130-nm thick composite collector is used. It is composed of an InGaAs unintentionally doped spacer, a 20-nm thick heavily doped InP region (4 × 10 17 cm -3 ) and a lightly doped InP layer (1.5 × 10 16 cm -3 ). This low doping concentration has been chosen to keep a high breakdown voltage. A 5-nm heavily ndoped In0.53Ga0.47As layer is used as sub-collector. It allows a low thermal resistance while keeping a satisfying collector contact resistance.

B. Fabrication process

The 0.4 µm emitter width DHBTs are processed using a wetetch self-aligned triple mesa technology. A base contact width (WB) of 0.2 µm is chosen to extends on each side of the emitter. The tasks that require high alignment accuracy like the emitter contact, the base contact and the base plug are processed using e-beam lithography. The other steps are processed using stepper lithography. The contacts are formed with Ti/Pd/Au stacks using vacuum evaporation. The first step consists of emitter contact deposition and emitter mesa etching. Under etching of the emitter results in an effective emitter width WE = 0.36 µm which has subsequently been taken into account for the calculation of all current densities. Then, the base contact is deposited followed by SiN encapsulation (which improve yield and reliability) and base mesa wet etching. Afterwards, the collector metallization is performed along with the base and the collector plugs followed by the collector mesa etching. Fig. 1 shows an scanning electron microscopy (SEM) view of a transistor after this etching step. Finally, the device is fully encapsulated with SiN and planarized with polyimide. The final step consists of the polyimide etching to form the interconnections of the device. The transistor fabrication yield is above 98% which is sufficiently high to consider using these devices for future realization of medium-scaled integrated circuits.

III. DEVICE CHARACTERISTICS

A. Measurement procedure

The RF measurements were performed from 250 MHz to 110 GHz with an Anritsu VectorStar network analyzer and 100µm pitch Picroprobe RF probes. The pad parasitic elements were de-embedded using on-wafer open and short standards. Difficulties in extracting the Mason's unilateral power gain U led to further investigations on the calibration methods. Three calibration methods have been used: the Short-Open-Load-Thru (SOLT), the Advanced Line-Reflect-Match (ALRM) and the multiline Thru-Reflect-Line [START_REF] Marks | A multiline method of network analyzer calibration[END_REF] (mTRL). All of them have been performed with off-wafer impedance standards. ALRM (also called LRM+) is a modified version of the LRM calibration method where an arbitrary known load is used. SOLT and ALRM require the same standards while mTRL requires other standards plus an additional one to cover the frequency range. The gain bandwidth product for the Mason's gain is plotted in Fig. 2 for the three studied calibration methods showing almost identical results up to 40 GHz, and then the ALRM results deviate from the other and overestimates the Mason's gain. Interestingly SOLT and mTRL give very close results. Even though often criticized for its validity for high frequency measurement the SOLT method has been chosen for the rest of the RF characterization. This is particularly because of the fact that this method requires a less number of standards than mTRL, the distance between probes does not need to be changed during calibration and that the validity of this method has already been affirmed up to 200GHz [START_REF] Fregonese | Comparison of On-Wafer TRL Calibration to ISS SOLT Calibration With Open-Short De-Embedding up to 500 GHz[END_REF]. Another important observation from Fig. 2 is that the gain-bandwidth product starts to collapse after 60-70 GHz for the three methods. A four step de-embedding (pad open, pad short, open, short), that allows to remove the coupling between probes and adjacent structures has also been tested which shows no impact on the curve's shape. Considering these observations, the collapse of the gain-bandwidth product can be attributed to the measurement environment, especially to the coupling between probes and structures. This phenomenon has been demonstrated for 100-µm pitch Picropobe in [START_REF] Yadav | Importance and Requirement of Frequency Band Specific RF Probes EM Models in Sub-THz and THz Measurements up to 500 GHz[END_REF] through EM simulations and is attributed to the crosstalk between port-1 and port-2 probes induced by the probes' design.

B. DC characterization

Fig. 3 shows the Gummel plots at VBC = 0 V of a 0.4 × 5 µm² DHBT showing a maximum static current gain of 29 at a collector current density JC of 4.6 mA/µm². It is a reasonable value considering the base thickness (28 nm) and high doping level (8 × 10 19 cm -3 ). The base and collector ideality factor have been extracted to be nB = 1.9 and nC = 1.15. The deviation of the collector ideality factor from unity is due to the type-I basecollector heterojunction. The Fig. 4 shows the common-emitter IC-VCE characteristics for a base current ranging from 0 to 825 µA with 75 µA steps. The common-emitter breakdown voltage BVCE0 is 4.7 V at a collector current density JC = 0.01 mA/µm². This breakdown voltage value is obtained thanks to the composite collector optimization. The transistor exhibits a turn-on offset voltage VCE,off of 200 mV.

C. RF characterization

As detailed in part A of this section, describing the RF characterization setup, Fig. 5 shows the short-circuit current gain, h21, and Mason's unilateral power gain, U. The extraction of fT and fMAX is performed using a single pole transfer function by fitting it against the measurement data up to 70 GHz. As discussed previously, the coupling between probes indeed disturbs the measurements at higher frequencies. This extraction on a 0.4 × 5 µm² DHBT leads to an fMAX = 605 GHz and an f T = 380 GHz at VCE = 1.6 V and JC = 5.8 mA/µm². The VCE value of 1.6 V is chosen since it gives the maximum value of fT. This WE = 0.4 µm DHBT technology has been designed to reach the highest fMAX possible with our epitaxial structure. For further analysis, the relationship between fT and fMAX [START_REF] Vaidyanathan | Extrapolated fmax of heterojunction bipolar transistors[END_REF], given by equation ( 1), has been considered.

 𝑓 𝑀𝐴𝑋 = √ 𝑓 𝑇 8𝜋(𝑅 𝐵 𝐶 𝐵𝐶 ) 𝑒𝑓𝑓   With (𝑅 𝐵 𝐶 𝐵𝐶 ) 𝑒𝑓𝑓 = 𝑅 𝐵𝑥 (𝐶 𝐵𝐶𝑥 + 𝐶 𝐵𝐶𝑖 ) + 𝑅 𝐵𝑖 𝐶 𝐵𝐶𝑖
To reach a 600 GHz fMAX, efforts were focused on reducing the product (RBCBC)eff in the denominator of (1), especially for reducing CBCx and CBCi. To achieve this the emitter width has been shrinked down to 0.4 µm and the width of the base contact on each side of the emitter has been reduced to WB = 0.2 µm. By shrinking the base contact the value of RBx is slightly increased, but the total RC product is reduced (in comparison with larger WB). This effect has already been studied in [START_REF] Arabhavi | Scaling of InP/GaAsSb DHBTs: A Simultaneous fT/fMAX=463/829 GHz in a 10 µm Long Emitter[END_REF]. Once again, our results highlights the importance of finding the right compromise between RB and CBC.

The RF figures of merit (fT and fMAX) of the transistor have been extracted at different collector currents IC and different collector-emitter voltages, VCE. Fig. 6 shows the results of this extraction. As explained before the maximum of fT is reached at VCE = 1.6 V, whereas, the maximum of fMAX (610 GHz) is reached at a higher voltage VCE = 1.8 V. This figures particularly highlight the fact that higher values of fT and fMAX (>75% of their maximum) can be reached with a wide range of the VCE. This illustrates the interest of this technology for monolithic microwave and high-speed integrated circuits. Circuit designers hence have a wide range of biasing points to exploit the transistor's performances.

D. Small-signal modeling up to 110 GHz

The extraction of the small-signal hybrid-π model [START_REF] Johansen | Improved External Base Resistance Extraction for Submicrometer InP/InGaAs DHBT Models[END_REF] parameters has been performed on the measurements presented in the preceding section at the peak of fT i.e. at VCE = 1.6 V and JC = 5.8mA/µm². Fig. 7 shows the results of this extraction. A good agreement between measurement and simulation is found at lower frequencies. However, some discrepancies start to appear at higher frequencies. This behavior is conforming to the deviation observed on the Mason's gain beyond 60 GHz which is also present in the measured S-parameters.

Extraction of the small-signal model is often performed at low frequency (< 30 GHz) for different reasons, especially because it is the cleanest part of the measurement. To understand the high-frequency discrepancies extraction of different parameters over the whole frequency range (250 MHz-110 GHz) has been performed. As the discrepancies are mainly observed in the Mason's gain, two parameters are of particular interest: the extrinsic base resistance RBx and the total basecollector capacitance CBC. Fig. 8 shows the extracted parameters up to 110 GHz. Ideally, these two parameters remain constant over the whole frequency range. However, the extrinsic base resistance shows significant variations over the frequency range. As expected these variations can be directly linked to the behavior of the gain bandwidth products (increase of RBx results in a decrease of the gain bandwidth product, see part A). Interestingly, this parameter makes it easier to identify the range of validity of the measurements: the frequency at which the value of RBx is no longer constant is the limit beyond which the measurements are no longer valid. Our measurements are not therefore useful for frequencies higher than 70 GHz. The extracted value of CBC, on the other hand, is constant over the whole frequency range and does not indicate any sign of the coupling. The same parameter extraction process can be applied to extract other parameters such as emitter-base capacitance and transit time that are expected to remain constant over the measured frequency range. However, similar to CBC, not all of these parameters might show coupling effects that limit the usable frequency band.

IV. CONCLUSION AND PERSPECTIVES

We reported a 0.4 × 5 µm² InP/InGaAs DHBT technology with simultaneous cutoff frequencies fT/fMAX = 380/605 GHz. The device exhibits a peak current gain of 29 and a common emitter breakdown voltage BVCE0 = 4.7 V. Comparison between different VNA calibration methods allowed us to choose the most appropriate method. Small-signal model simulation was performed that shows good agreement with measurements. Extraction of small-signal model parameters up to 110 GHz has allowed identifying a simple method to verify the range of measurement validity. This 0.4-µm DHBT technology demonstrates very high fMAX for a Type-I device without any aggressive emitter scaling. In order to improve fT while maintaining fMAX>fT, ongoing work focuses not only on vertical structure optimization thanks to multiscale physical simulations [START_REF] Wen | A Multiscale TCAD Approach for the Simulation of InP DHBTs and the Extraction of Their Transit Times[END_REF] but also on novel tungstenbased emitter process.

Fig. 1

 1 Fig. 1 SEM image of a 0.4 × 5 µm² DHBT after collector mesa etching

  bandwidth product (GHz) Frequency (GHz) Emitter size: 0.4 x 5 µm² J C = 5.8 mA/µm²

Fig. 2

 2 Fig. 2 Mason's gain bandwidth product as function of the frequency for the three studied calibration methods

Fig. 3 Fig. 4

 34 Fig. 3 Gummel plot and DC gain at VBC = 0 V of a 0.4 × 5 µm² DHBT.

Fig. 5

 5 Fig. 5 Short-circuit current gain h21 and Mason's unilateral power gain U measured at VCE = 1.6 V and JC = 5.8 mA/µm²

Fig. 6

 6 Fig. 6 fT (dashed lines) and fMAX (solid lines) as function of the current IC for different VCE values

Fig. 7

 7 Fig. 7 Hybrid-pi small-signal model of a 0.4 × 5 µm² transistor at VCE = 1.6 V and JC = 5.8 mA/µm². Comparison between measurement (blue) and simulation (red) is also plotted on the smith-chart.

Fig. 8

 8 Fig. 8 Extrinsic base resistance RBx (top) and Base-collector capacitance CBC (bottom) as function of the frequency. Red dash lines indicate the extracted values used in the small-signal model simulation.

ACKNOWLEDGMENT

This work was supported by ANR-FNS through ULTIMATE project (ANR-16-CE93-0007) and the European Commission through ICT TWILIGHT project (N°871741).

This work was supported by ANR-FNS through ULTIMATE project (ANR-16-CE93-0007) and the European Commission through ICT TWILIGHT project (N°871741).

.