Laurence Bessueille

Lynn Kawtharany

Thibaut Quillard

Claudia Goettsch

Anne Briolay

Nirina Taraconat

Stéphane Balayssac

Véronique Gilard

Saîda Mebarek

Olivier Peyruchaud

PhD David Magne

Saida Mebarek

François Duboeuf

Caroline Bouillot

Anthony Pinkerton

Laura Mechtouff

René Buchet

Eva Hamade

Kazem Zibara

Caroline Fonta

Emmanuelle Canet-Soulas

Jose Luis Millan

Monzur Murshed
email: monzur.murshed@mcgill.ca

Olga Martinez-Augustin

Kawtharany

Quillard

Hamade E 10

Zibara K 10

Fonta C 11

Millan Jl

Metabolism

Inhibition of alkaline phosphatase impairs dyslipidemia and protects mice from atherosclerosis

Keywords: Atherosclerosis, calcification, inflammation, TNAP, liver, metabolic syndrome positron emission tomography, PPi: inorganic pyrophosphate, SUVR: standardized uptake value ratio, TNAP: tissue-nonspecific alkaline phosphatase, VSMC: vascular smooth muscle cell Atherosclerosis, calcification, inflammation, TNAP, liver, metabolic syndrome

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

We recently published a review article in Cardiovascular Research explaining that alkaline phosphatase, the necessary enzyme in bone mineralization, is increasingly suspected to participate in cardiovascular calcification (Goettsch et al, Cardiovasc Res 2022). In the present experimental article, we report that alkaline phosphatase is locally associated with calcification in human and mouse atherosclerotic plaques, and that its pharmacological inhibition with the novel inhibitor SBI-425, prevents calcification in human vascular smooth muscle cell cultures in vitro, and in vivo in aortic plaques of mice with atherosclerosis. More unexpectedly and interestingly, we found that hepatic alkaline phosphatase is activated during the progression of liver steatosis, and that its inhibition reduces the release of triglycerides and cholesterol in the blood, and consequently impairs the whole development of atherosclerotic plaques. We identified phosphocholine as a new substrate of liver alkaline phosphatase, whose dephosphorylation normally participates in the release of lipids from the liver to the blood. Collectively, these data indicate that SBI-425 protects from atherosclerosis through the inhibition of both liver and arterial alkaline phosphatase. They moreover strengthen the notion that alkaline phosphatase inhibition may be a promising therapeutic option for atherosclerosis, especially since we observed that SBI-425 administration reduces inflammation and preserves the bone architecture. This article is not under consideration for publication elsewhere. All co-authors have read and approved the manuscript.

Yours faithfully, cover letter

Brief Commentary

Background:

Our study provides the first demonstration that alkaline phosphatase inhibition prevents atherosclerotic plaque calcification and ameliorates atherosclerosis. Moreover, it identifies an unsuspected function of liver alkaline phosphatase, which is to participate in VLDL release through phosphocholine dephosphorylation.

Translational Significance:

Alkaline phosphatase inhibition has recently emerged as a promising option to block arterial calcification in patients with chronic kidney disease (CKD). Our study suggests that it will also ameliorates atherosclerosis, in patients with, and without CKD. Finally, it provides an unexpected function of liver alkaline phosphatase, whose release in the blood is clinically routinely measured in clinics, but whose function is obscure.

Introduction

Cardiovascular diseases are responsible for 17.9 million deaths each year, which represents 31% of all deaths worldwide (https://www.who.int/health-topics/cardiovascular-diseases). Rupture of an atherosclerotic plaque is considered the primary reason for cardiovascular death, accounting for most myocardial infarction cases and about 20% of ischemic strokes [START_REF] Ornello | Distribution and Temporal Trends From 1993 to 2015 of Ischemic Stroke Subtypes: A Systematic Review and Meta-Analysis[END_REF] . Acknowledged features of vulnerability include a large lipid and necrotic core, a thin fibrous cap and intraplaque hemorrhage [START_REF] Silvestre-Roig | Atherosclerotic plaque destabilization: mechanisms, models, and therapeutic strategies[END_REF] .

In addition, recent data suggested that microcalcification, characterized by a size less than 10 µm, but not macrocalcification, negatively impact plaque stability [START_REF] Karlöf | Correlation of computed tomography with carotid plaque transcriptomes associates calcification with lesion-stabilization[END_REF][START_REF] Shioi | Plaque Calcification During Atherosclerosis Progression and Regression[END_REF][START_REF] Bos | Atherosclerotic Carotid Plaque Composition and Incident Stroke and Coronary Events[END_REF] . Clinically, microcalcification can be discriminated from macrocalcification with [START_REF] Harmey | Concerted regulation of inorganic pyrophosphate and osteopontin by akp2, enpp1, and ank: an integrated model of the pathogenesis of mineralization disorders[END_REF] F-NaF positron emission tomography (PET) [START_REF] Irkle | Identifying active vascular microcalcification by (18)F-sodium fluoride positron emission tomography[END_REF] , because fluoride ions can replace hydroxyl ions in newly formed apatite crystals, but not in more mature, inert macrocalcification. To date, several ex vivo and in vivo studies have shown that [START_REF] Harmey | Concerted regulation of inorganic pyrophosphate and osteopontin by akp2, enpp1, and ank: an integrated model of the pathogenesis of mineralization disorders[END_REF] F-NaF accumulates in plaques with hallmarks of vulnerability, predicting future events [START_REF] Joshi | F-fluoride positron emission tomography for identification of ruptured and high-risk coronary atherosclerotic plaques: a prospective clinical trial[END_REF][START_REF] Vesey | F-Fluoride and[END_REF][START_REF] Mechtouff | Simultaneous assessment of microcalcifications and morphological criteria of vulnerability in carotid artery plaque using hybrid[END_REF] . Microcalcification may destabilize plaques through pro-inflammatory and mechanical mechanisms. In human macrophage cultures, crystals measuring from 1 µm to 15 µm induce the release of tumor necrosis factor (TNF)-α at levels inversely associated with crystal size [START_REF] Nadra | Proinflammatory activation of macrophages by basic calcium phosphate crystals via protein kinase C and MAP kinase pathways: a vicious cycle of inflammation and arterial calcification?[END_REF][START_REF] Nadra | Effect of particle size on hydroxyapatite crystal-induced tumor necrosis factor alpha secretion by macrophages[END_REF] . Microcalcification can also exert a harmful mechanical stress and favor plaque rupture, especially when it develops within the fibrous cap [START_REF] Kelly-Arnold | Revised microcalcification hypothesis for fibrous cap rupture in human coronary arteries[END_REF][START_REF] Maldonado | A mechanistic analysis of the role of microcalcifications in atherosclerotic plaque stability: potential implications for plaque rupture[END_REF][START_REF] Vengrenyuk | A hypothesis for vulnerable plaque rupture due to stress-induced debonding around cellular microcalcifications in thin fibrous caps[END_REF] . Nonetheless, in the absence of calcification inhibitors that could be used in vivo, the real impact of calcification on plaque progression still remains debatable. In this regard, the recent development of an effective specific and in vivo inhibitor of tissue-nonspecific alkaline phosphatase (TNAP) should help fill this gap in knowledge 15 . TNAP, the enzyme necessary for physiologic bone and tooth mineralization [START_REF] Millán | Alkaline Phosphatase and Hypophosphatasia[END_REF] , has emerged as a likely suspect responsible for plaque calcification [reviewed in [START_REF] Goettsch | TNAP as a therapeutic target for cardiovascular calcification -a discussion of its pleiotropic functions in the body[END_REF]. TNAP is normally expressed under the control of the transcription factor RUNX2 in hypertrophic chondrocytes and osteoblasts, where it hydrolyzes the constitutive mineralization inhibitor inorganic pyrophosphate (PPi), to allow progression of mineralization onto the extracellular matrix [START_REF] Harmey | Concerted regulation of inorganic pyrophosphate and osteopontin by akp2, enpp1, and ank: an integrated model of the pathogenesis of mineralization disorders[END_REF][START_REF] Hessle | Tissue-nonspecific alkaline phosphatase and plasma cell membrane glycoprotein-1 are central antagonistic regulators of bone mineralization[END_REF] . Interestingly, in atherosclerotic Apolipoprotein (Apo)E-deficient mice, vascular smooth muscle cells (VSMCs) transdifferentiate into chondrocytes and express TNAP before calcification can be detected [START_REF] Rattazzi | Calcification of advanced atherosclerotic lesions in the innominate arteries of ApoEdeficient mice: potential role of chondrocyte-like cells[END_REF][START_REF] Rosenfeld | Advanced atherosclerotic lesions in the innominate artery of the ApoE knockout mouse[END_REF][START_REF] Aikawa | Osteogenesis associates with inflammation in early-stage atherosclerosis evaluated by molecular imaging in vivo[END_REF] . Moreover, VSMC-specific deletion of RUNX2 in these mice [START_REF] Sun | Smooth muscle cell-specific runx2 deficiency inhibits vascular calcification[END_REF] , or in atherosclerotic mice deficient for low density lipoprotein receptor (LDLr -/-) [START_REF] Lin | Runx2 deletion in smooth muscle cells inhibits vascular osteochondrogenesis and calcification but not atherosclerotic lesion formation[END_REF] , strongly reduces calcification, further suggesting that TNAP is a key player. In humans, where plaque calcification does not seem to involve cartilage formation, and bone formation within only a minority of plaques [START_REF] Espitia | Implication of molecular vascular smooth muscle cell heterogeneity among arterial beds in arterial calcification[END_REF] , TNAP from the circulation is suspected to stimulate plaque formation [START_REF] Tonelli | Relation between alkaline phosphatase, serum phosphate, and all-cause or cardiovascular mortality[END_REF][START_REF] Panh | Association between serum alkaline phosphatase and coronary artery calcification in a sample of primary cardiovascular prevention patients[END_REF] . As its name indicates, TNAP is indeed relatively ubiquitous. In humans, while predominantly expressed in the skeleton and teeth during physiologic mineralization, it is also expressed in the liver and released into the blood in increasing amounts with cholestasis and with the development of metabolic syndrome (MetS) [START_REF] Tonelli | Relation between alkaline phosphatase, serum phosphate, and all-cause or cardiovascular mortality[END_REF][START_REF] Lammers | Levels of alkaline phosphatase and bilirubin are surrogate end points of outcomes of patients with primary biliary cirrhosis: an international follow-up study[END_REF][START_REF] Abramowitz | Serum alkaline phosphatase and phosphate and risk of mortality and hospitalization[END_REF][START_REF] Krishnamurthy | Associations of serum alkaline phosphatase with metabolic syndrome and mortality[END_REF][START_REF] Wannamethee | Alkaline phosphatase, serum phosphate, and incident cardiovascular disease and total mortality in older men[END_REF][START_REF] Filipowicz | Associations of serum skeletal alkaline phosphatase with elevated C-reactive protein and mortality[END_REF] . Since genetic deficiency in PPi generation leads to vascular calcification [START_REF] Rutsch | Mutations in ENPP1 are associated with 'idiopathic' infantile arterial calcification[END_REF][START_REF] Nitschke | Generalized arterial calcification of infancy and pseudoxanthoma elasticum can be caused by mutations in either ENPP1 or ABCC6[END_REF] , it is suspected that increased circulating TNAP in MetS exacerbates plaque calcification.

In the present study, we explored the involvement of TNAP in plaque development and calcification in different experimental designs: using ApoE-deficient mice, a biocollection of human carotid plaques as well as cultured human VSMCs. To take into account the ubiquitous nature of TNAP, we also analyzed the effects of its inhibition on the skeleton and liver. We report that systemic TNAP inhibition decreased blood triglyceride and cholesterol levels, and hindered atherosclerosis development, as demonstrated by reduced plaque growth, inflammation and calcification. Importantly, SBI-425 administration exerted these beneficial effects without producing any negative effect on mouse bone architecture.

Materials and methods

Animal experiments

The present project received authorization from the ethics committee of the French Ministry of Education, Research and Innovation (APAFIS#15387-2018060708052279). Two cohorts of mice were used to characterize plaque calcification and TNAP activation (Suppl. Fig. 1A). Six-week-old ApoEdeficient male mice (Charles River, Lyon, France) were housed at the animal facility of INSA (Lyon, France; Cetil n°C2EA-102), and placed in conventional cages with 5 mice per cage. Mice were maintained on a 12-hrs light-dark cycle (7:00 am-7:00 pm) and were supplied with food and water ad libitum. From 10 weeks of age, they were fed ad libitum with a high fat diet (HFD, D12108C, Research Diets, New Brunswick, USA) containing 40% fat (kcal) and 1.25% cholesterol (weight). In cohort 1, 8 mice were sacrificed every 2 weeks between weeks 17 to 31, after overnight fasting, for histological and molecular analyses. Total body weight was measured weekly after starting the HFD. The sacrifice was done by CO2 inhalation after anesthesia with isoflurane and intracardiac blood puncture. The abdominal aorta was used for quantitative polymerase chain reaction (PCR) analyses. We used mice from cohort 2 (n=5 per group) to analyze aortic calcification with [START_REF] Harmey | Concerted regulation of inorganic pyrophosphate and osteopontin by akp2, enpp1, and ank: an integrated model of the pathogenesis of mineralization disorders[END_REF] F-NaF µPET and µCT and to compare the in vivo data with the ex vivo results obtained in cohort 1. According to the results obtained in cohorts 1 and 2, in the next cohorts (cohorts 3 and 4), we administered the TNAP inhibitor SBI-425 (30 mg/kg/day, formulated as a food admixture in the HFD) from 10 weeks of age [START_REF] Romanelli | Overexpression of tissue-nonspecific alkaline phosphatase (TNAP) in endothelial cells accelerates coronary artery disease in a mouse model of familial hypercholesterolemia[END_REF] . Treatment was stopped at 25 weeks of age, when microcalcification is detected in all mice analyzed in cohorts 1 and 2. Mice in cohort 3 (2 groups of 13 mice) were used for ex vivo analysis, and mice in cohort 4 (2 groups of 12 mice) for in vivo imaging.

In vivo imaging

Animals were transferred from the animal facility to the Cermep imaging platform (Lyon/Bron, France).

Mice were placed into a chamber connected to an isoflurane anesthesia unit. Anesthesia was induced using an airflow rate of 0.8 L/min and 4% isoflurane, airflow rate was then maintained to 0.6 L/min with 2% isoflurane during the scans. After induction, mice were injected with 0.36 MBq/g of 18 F-NaF in 0.1mL saline via tail vein (19 scans) or in retro-orbital venous sinus (16 scans) when the tail veins were not available. After injection, animals were placed in the prone position on the scanning bed. PET-CT images were obtained using a micro PET-CT scanner (Inveon PET-CT, Siemens, Germany). Mice were positioned into the CT system and imaged using the low magnification acquisition settings (acquisition parameters: attenuation mode; projection: 120 ; rotation: 200° ; estimated scan time: 492s ; binning 4x4; effective voxel size: 0.111 x 0.111 x 0.111 mm 3 ; voltage: 80 kV; current: 500 µA; filter thickness: 0.5 mm; exposure: 200 ms). After CT acquisition, mice were moved into the center of the PET scanner (axial field of view: 127mm). PET acquisition started exactly one hour after 18 F-NaF injection, for a total scan time of twenty minutes. Then, mice were allowed to recover into a lead shielded cage. Static images of the 20-min PET acquisitions were reconstructed with attenuation and scatter correction by 3D ordinary Poisson ordered subsets expectation maximization (OP-OSEM3D) with 4 iterations and a zoom factor of 2. The reconstructed volume was constituted of 159 slices of 128 × 128 matrix voxels, with voxel size 0.388 × 0.388 × 0.796 mm 3 . CT data were reconstructed using a Feldkamp algorithm with a down sample of 1 (Inveon Acquisition Workplace, Siemens). After reconstruction, the PET and CT data were coregistered. Images were analyzed using Inveon Research workplace (Siemens). The presence of atherosclerotic plaques was evaluated both in CT and PET images. Due to the higher resolution of the CT scan, regions-of-interest (ROI) of the aortic arch were drawn on CT images. Only voxels above the threshold of 390 Hounsfield Unit (calcified structure) were considered. The ROIs were exported onto the coregistered PET images in order to measure [START_REF] Harmey | Concerted regulation of inorganic pyrophosphate and osteopontin by akp2, enpp1, and ank: an integrated model of the pathogenesis of mineralization disorders[END_REF] F-NaF uptake. Standardized uptake value ratio (SUVR) were calculated using the whole body SUV as reference.

Tissue harvest and processing

After euthanasia, the heart and vasculature were perfused with sterile 0.9% NaCl. The aortic root and the aortic arch, with the major arteries that stem from them (brachiocephalic, left common carotid and left subclavian), were dissected under a microscope and frozen in optimal cutting temperature medium (OCT, Thermo Scientific) and stored at -80 °C before serial cryo-sectioning. Abdominal aortas were dissected, rinsed once with sterile phosphate-buffered saline (PBS) pH 7.4, and frozen until RNA extraction. Cryostat sections (5-μm thick) were cut at -21 °C using a Leica CM3050 S cryostat (Leica), mounted on glass slides (SuperFrost Plus Gold glass slides, Thermo scientific) and stored at -80°C until use. Four sections of 5-µm thickness were harvested per slide, yielding 25 to 45 slides per mouse. Prior to analysis, frozen tissue sections were air-dried for 10 min at room temperature and eight consecutive sections were taken from each mouse at 15-µm intervals. Calcification was imaged by near-infrared fluorescence (NIRF) with the calcium tracer osteosense (OS) (PerkinElmer, ex/em 650/680 nm) as previously described [START_REF] Goettsch | Sortilin mediates vascular calcification via its recruitment into extracellular vesicles[END_REF] . Briefly, frozen sections were treated overnight with OS 680 (1:100) before NIRF imaging. Quantification was performed by computer image analysis using Image J software (NIH, Bethesda, MD) by averaging of measurements from 2 sections of 15 µm apart. Alkaline phosphatase activity was stained in serial sections using nitro blue tetrazolium/5-Bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) solution [START_REF] Lencel | Cell-specific effects of TNF-α and IL-1β on alkaline phosphatase: implication for syndesmophyte formation and vascular calcification[END_REF] .

To perform CD68 immunohistochemical staining to detect macrophages, serial frozen sections of aortic arch were fixed in acetone (-20 °C) for 10 min and were air-dried afterwards for 10 min. Endogenous peroxidase activity was neutralized with 0.3% H2O2 for 10 min followed by 3 washing steps of 5 min each using Tris buffered saline (TBS) pH 7.4 containing 0.025% Triton-X100. Sections were incubated with TBS containing 5% normal goat serum (Abcam) and 1% bovine serum albumin (BSA) for 1 h to reduce non-specific background staining. Sections were subsequently incubated overnight at 4 °C with anti-CD68 antibody (Abcam ab125212) diluted in TBS containing 1% BSA. Horseradish peroxidaseconjugated polyclonal anti-rabbit secondary antibody (Abcam) was applied for 1 h and then revealed with a DAB staining kit (Abcam). Preparations were counterstained with hematoxylin-eosin staining (Mayer's hematoxylin solution, Sigma). Negative control sections were analyzed using a similar procedure excluding the primary antibody. Images of the sections were captured with an Eclipse TI-E microscope fitted with a DSiQ2 digital camera combined with Nikon's NIS Elements imaging software (Nikon Instruments Inc.). At least 2 sections per mouse were examined for CD68 immunostaining.

For oil Red O staining of neutral lipids, sections were rehydrated in PBS for 5 min, fixed with 10% neutral buffered formalin for 5 min, extensively washed with water, and then treated with propylene glycol for 2 min. The sections were then stained with oil red O solution (0.5% in propylene glycol) for 15 min at 60°C and extensively washed with water. After the oil red O staining procedure, the sections were counterstained with hematoxylin-eosin staining (Mayer's hematoxylin solution, Sigma). Images were captured with an Eclipse TI-E microscope and digital camera (Nikon), combined with Nikon's NIS Elements imaging software. To visualize cartilage glycosaminoglycans, sections were stained with Alcian blue 8GX (Sigma) at pH 2.5 (1% in 3% acetic acid).

Blood biochemistry

Blood was collected from mice by cardiac puncture under anesthesia with isoflurane. After blood coagulation, serum was obtained through centrifugation of the blood for 15 min at 1,500 g at 4°C, frozen in liquid nitrogen and stored at -80°C before analysis. Concentration of total cholesterol and triglycerides were measured using a colorimetric (MAK043, Sigma) and a fluorimetric assay (MAK266, Sigma), respectively. Total bile acids were assayed with a commercial enzyme-based colorimetric kit (Abcam Ab239702). Serum IL-6 and haptoglobin levels were determined using the commercially available ELISA kits (Fischer Scientific 10110623 and Abcam 157714, respectively). The alkaline phosphatase activity assay was adapted from a previously published protocol and performed in a 96well plate [START_REF] Sergienko | A method for direct assessment of tissue-nonspecific alkaline phosphatase (TNAP) inhibitors in blood samples[END_REF] . Briefly, sera were mixed with a concentrated buffer at a ratio of 2.85:1 (vol/vol) in order to get final concentrations of 0.56 M 2-amino-2-methyl-1-propanol buffer pH 10.5, 1 mM MgCl2 and 10 mM p-nitrophenylphosphate. Absorbance was acquired for 5 min at 405 nm (Tecan plate reader).

The slope was calculated to determine TNAP activity levels in each sample and normalized to protein amounts measured with the BCA protein assay (Pierce).

Bone parameters

3D microarchitecture of the distal metaphyseal femur and cortical midshaft were carried out using a Skyscan 1176 micro-CT scanner (Skyscan Inc.). The X-ray excitation voltage was set to 50 kV with a current of 500 mA. A 0.5 mm aluminum filter was used to reduce beam-hardening artifacts. Samples were scanned in 70% ethanol with a fixed voxel size of 9.08 μm. Section images were reconstructed with NRecon software (version 1.6.1.8, Skyscan). The region of interest (ROI) to delineate trabecular bone was drawn manually, away from the endocortical surface, starting at 0.5 mm underneath the growth plate and ending at 1.5 mm. For cortical analysis, 0.5 mm on either side of the femur midshaft were reconstructed. The global threshold was set at 0.394 g HA/cm 3 . Three-dimensional modeling and analysis of bone volume to tissue volume ratio (BV/TV), trabecular number, trabecular separation, cortical thickness, total porosity, were obtained with the CTAn (version 1.9) and CTVol (version 2.0) software.

Liver biochemistry

For the determination of cholesterol, triglycerides, and total bile acids in the liver, 40-mg pieces of liver were homogenized in 1 mL of 5% NP40 solution in a Dounce homogenizer. The hydrophobic components were extracted twice at 80°C for 5 minutes. Samples were centrifuged and the supernatants were assayed for cholesterol, triglycerides and bile acids with the same kits used for blood. In order to quantify hydroxyproline, 100-mg pieces of liver were homogenized in 1 mL of pure water in a dounce homogenizer and a perchlorate-free kit was used (Sigma, MAK357).

Metabolomics analysis

Liver extraction and NMR measurement: The frozen tissues were weighed and then extracted according to Beckonert's procedure [START_REF] Beckonert | Metabolic profiling, metabolomic and metabonomic procedures for NMR spectroscopy of urine, plasma, serum and tissue extracts[END_REF] using the methanol-chloroform-water system [2:2:1.425 (v/v/v)], and including absorbance measurement using the Bradford reagent with BSA as standard. The upper methanol/water phase was collected, dried, lyophilized and stored at -80°C. Deuterated phosphate buffer at pH = 7.4 (200 mM) was added to the dried aqueous extract with TSP (sodium 2,2,3,3-tetradeutero-3-trimethylsilylpropionate) (Sigma-Aldrich) as internal chemical shift and quantification reference. [START_REF] Ornello | Distribution and Temporal Trends From 1993 to 2015 of Ischemic Stroke Subtypes: A Systematic Review and Meta-Analysis[END_REF] Metabolite concentrations (ng/mg liver tissue) were given by mx = AX/ATSP. 9/NX. 112.Mx/mL with AX and ATSP being the areas of the signals of metabolite X and TSP, 9 and NX the number of protons of TSP and metabolite X evoking the signals, 112 the number of nmol of TSP in the solution analyzed, Mx the molecular weight of metabolite X and m the weight (mg) of tissue analyzed.

Human samples

Analysis in human samples has been performed on healthy and diseased carotid arteries. Carotid plaques [n=101 calcified, and 14 non-calcified from the ECLAGEN biocollection [START_REF] Espitia | Implication of molecular vascular smooth muscle cell heterogeneity among arterial beds in arterial calcification[END_REF] ] were removed as a single specimen by endarterectomy at the bifurcation from within the lumen. The ECLAGEN biocollection was collected in accordance to the principles outlined in the Declaration of Helsinki. All samples were 1-2 cm long. For histology, we analyzed sections of the lesion core present in each arterial sample. Sample collection and handling were performed in accordance with our institutional medical ethics committee (research protocol#PFS09-014, n°DC-2008-402), and all patients participating in the study provided written informed consent. In the case of organ donors (healthy arteries), the absence of patient opposition to organ donation and an informed and signed consent was required from the patient's family. The arterial samples were fixed in 10% formalin for 48 h, decalcified in 4.13% EDTA-0.2% paraformaldehyde pH 7.4 over 4 days in KOS sw10 (Milestone), and embedded in paraffin. Following heat-induced epitope retrieval in EDTA pH 9, serial sections (4-µm thick) were stained with TNAP (Abcam), and Runx2 (Abcam) antibodies. Imaging of the sections was obtained with the NanoZoomer device (Hamamatsu Photonics).

Human VSMC cultures

Human coronary artery VSMCs (PromoCell) were grown in SMC growth medium 2 (SMC-GM2, PromoCell) supplemented with epidermal growth factor (0.5 ng/mL), insulin (5 μg/mL), basic fibroblast growth factor-β (2 ng/mL), and fetal bovine serum (5%) at 37 °C in humidified 5% CO2. Cells were used between passages 3 and 9. Cells from 4-5 independent donors were used. VSMCs were cultured for up to 21 days in the presence of either control medium (DMEM, 10% FBS, 1% penicillin/streptomycin) or osteogenic medium (consisting of control medium supplemented with 10 nM dexamethasone, 10 mM β-glycerophosphate, and 100 µM L-ascorbate phosphate). Media were changed 3 times per week.

Mycoplasma contamination test was routinely performed (once a month). TNAP activity was inhibited by the TNAP inhibitor MLS-0038949 (Merck, CAS 496014-13-2), the parent compound of SBI-425 [START_REF] Pinkerton | Discovery of 5-((5-chloro-2-methoxyphenyl)sulfonamido)nicotinamide (SBI-425), a potent and orally bioavailable tissue-nonspecific alkaline phosphatase (TNAP) inhibitor[END_REF] .

TNAP activity was determined by the Alkaline Phosphatase Activity Colorimetric Assay Kit (BioVision, San Francisco, USA) according to the manufacturer's instructions. TNAP activity was normalized to the protein amount. To visualize calcification, cells were washed and fixed with 4% paraformaldehyde for 10-15 min at room temperature. The mineralized matrix was visualized by 2% (w/v) Alizarin Red staining (Sigma Aldrich).

Quantitative polymerase chain reaction (PCR) analysis

Mouse tissues: total RNAs were extracted from grinded abdominal aorta or liver with the NucleoSpin RNA II kit (Macherey-Nagel) following the manufacturer's protocol. The quantity and quality of RNA were determined at 260 and 280 nm absorbance with a NanoDrop. RNA (1 µg) was retro-transcribed into cDNA with Superscript II reverse transcriptase (Life Technologies). PCR analysis was conducted using SYBR green Supermix (Biorad) on the iCycler Real-Time Detection system (Biorad). Fold change in mRNA expression was calculated using the 2 -ΔCt method by the CFX Manager software (Biorad). Data were normalized by 3 housekeeping genes (HPRT, Actin and Rpl13a) levels. Primers and PCR conditions are given in Table I. The obtained products were checked by sequencing.

Human VSMCs: total RNA was isolated using TriZol (Life Technologies) and mRNA levels were determined by TaqMan-based real-time PCR (Life Technologies). The following TaqMan probes were used: HS01047978_m1 (human Runx2), HS99999902_m1 (human Rplp0). The expression levels were normalized to Rplp0. Results were calculated using the ΔΔCt method, and presented as fold increase relative to control.

In vitro dephosphorylation of phosphocholine and phosphoethanolamine by TNAP

Results

Atherosclerotic plaque calcification is associated with TNAP activity in mouse and human lesions

Our first objective was to determine whether atherosclerotic plaque calcification is associated with TNAP activity in the serum, in the plaques, or in both compartments. To this purpose, we thoroughly characterized where and when calcification initiates in aortas from ApoE -/-mice. Aorta calcification was first imaged in vivo at 19, 25, 31 and 34 weeks using µCT to quantify calcium volume, and [START_REF] Harmey | Concerted regulation of inorganic pyrophosphate and osteopontin by akp2, enpp1, and ank: an integrated model of the pathogenesis of mineralization disorders[END_REF] F-NaF µPET to determine fluoride incorporation in nascent crystals, and therefore calcification activity (Suppl. Fig. 1A and Fig. 1A). µCT data showed that calcium deposition in the aorta progressed in an almost exponential manner from 19 weeks to 34 weeks of age (Fig. 1B and Suppl. Fig. 1B). Calcification activity also increased from 25 to 31 weeks of age, then it slowed-down between 31 and 34 weeks (Fig. 1C and Suppl. Fig. 1C). Histologically, using the calcium fluorescent dye osteosense, microcalcification of about 10-20 µm in size was detected in the aortic arch, in mice aged 19-21 weeks (Fig. 1D). These first calcified regions grew and fused in the arch until 31 weeks (Fig. 1D,E), to sometimes fill the whole aortic arch (Suppl. Fig. 1D). In parallel, new calcifications appeared in other arteries, such as the left common carotid (Fig. 1D). The total surface of calcifications determined ex vivo increased exponentially (Fig. 1F), and similarly to the total volume of calcium measured in vivo with µCT (Fig. 1B). We then asked whether TNAP activation in the plaques and/or in the blood was associated with plaque calcification. Strikingly, calcification was consistently associated with TNAP activity in atherosclerotic plaques (Fig. 1G). Moreover, TNAP activity was commonly detected in aortic plaques from 17-19 week-old mice (Fig. 1H), suggesting that TNAP activation preceded microcalcification, and was responsible for its formation. On the other hand, serum TNAP activity remained unchanged from 17 to 31 weeks (Suppl. Fig. 1E). These results strongly suggested that local TNAP, but not liver or circulating TNAP, participated in plaque calcification in ApoE -/-mice.

We next wondered which cells expressing TNAP might be involved in atherosclerotic plaque calcification. In ApoE -/-mice, calcification was shown to develop into cartilaginous metaplasia [START_REF] Rattazzi | Calcification of advanced atherosclerotic lesions in the innominate arteries of ApoEdeficient mice: potential role of chondrocyte-like cells[END_REF] .

However, there is still some uncertainty as to whether plaque calcification in these mice is induced by hypertrophic chondrocytes, or whether like in humans, in association with calcifying macrophages [START_REF] Kawakami | S100A9-RAGE Axis Accelerates Formation of Macrophage-Mediated Extracellular Vesicle Microcalcification in Diabetes Mellitus[END_REF] , or with macrophage debris [START_REF] Jinnouchi | Calcium deposition within coronary atherosclerotic lesion: Implications for plaque stability[END_REF] . Like in Rattazzi's characterization of ApoE -/-mice [START_REF] Rattazzi | Calcification of advanced atherosclerotic lesions in the innominate arteries of ApoEdeficient mice: potential role of chondrocyte-like cells[END_REF] , macrocalcification unambiguously developed within cartilaginous metaplasia, as firstly demonstrated by the colocalization of osteosense and alcian blue staining (Fig 2A). Moreover, the expression pattern of the chondrocyte differentiation markers Sox9, Acan, Runx2 and Alpl (Fig. 2B) was very similar to the calcification activity measured with [START_REF] Harmey | Concerted regulation of inorganic pyrophosphate and osteopontin by akp2, enpp1, and ank: an integrated model of the pathogenesis of mineralization disorders[END_REF] F-NaF µPET, with a rise from 25 weeks followed by a plateau (Fig. 1C). In these cartilaginous metaplasia, macrocalcification likely relied on TNAP expressed by hypertrophic chondrocytes. Indeed, the evolution of Alpl transcripts encoding TNAP was very similar to that of the chondrocyte marker Acan, and strongly associated with that of the transcription factor Runx2, known to stimulate chondrocyte hypertrophy and Alpl expression (Fig. 2B,C). Finally, these cartilaginous macrocalcifications were devoid of CD68-positive cells (Fig. 2A), suggesting that macrophages did not directly participate in their growth. On the other hand, microcalcification was often observed in locations enriched in macrophages, and not yet positive for alcian blue staining for cartilage glycosaminoglycans (Fig. 2A). Therefore, it is possible that, before growing within cartilaginous metaplasia, microcalcification formed in association with TNAP in macrophage-like cells 42 . In conclusion, these first data collectively suggested that TNAP, expressed in plaques by hypertrophic chondrocytes and/or macrophages, was responsible for plaque calcification in ApoE -/- mice.

We then sought to investigate whether TNAP is also locally associated with calcifications in human carotid plaques. This was more uncertain because cartilage metaplasia has never been reported in human plaques, and because bone metaplasia occurs less frequently, and predominantly in peripheral arteries [START_REF] Espitia | Implication of molecular vascular smooth muscle cell heterogeneity among arterial beds in arterial calcification[END_REF] . We analyzed a biocollection of human carotid plaques in which the calcification morphology has been characterized in details [START_REF] Espitia | Implication of molecular vascular smooth muscle cell heterogeneity among arterial beds in arterial calcification[END_REF] . In these lesions and independently of the calcification size and morphology, TNAP was consistently located at the proximal periphery of calcifications (Fig. 3).

Interestingly, TNAP was expressed in both RUNX2-positive and RUNX2-negative cells. Considered together, these results in humans and mice strongly suggested that TNAP is involved in plaque calcification. To validate this hypothesis in ApoE -/-mice, we chose to inhibit TNAP in a preventive manner, from 10 weeks of age, by administrating 30 mg/kg/day of SBI-425 admixed in the HFD [START_REF] Romanelli | Overexpression of tissue-nonspecific alkaline phosphatase (TNAP) in endothelial cells accelerates coronary artery disease in a mouse model of familial hypercholesterolemia[END_REF] . This treatment was stopped at 25 weeks to determine the involvement of TNAP in the initiation of plaque calcification.

TNAP inhibition in mice decreases cholesterolemia and reduces atherosclerotic plaque development

As recently shown in other disease models [START_REF] Sheen | Pathophysiological role of vascular smooth muscle alkaline phosphatase in medial artery calcification[END_REF][START_REF] Tani | Inhibition of tissue-nonspecific alkaline phosphatase protects against medial arterial calcification and improves survival probability in the CKD-MBD mouse model[END_REF] , SBI-425 administration was well-tolerated, with no effect on food intake, weight gain, or all-cause mortality (Suppl. Fig. 2A). Moreover, not only TNAP inhibition did not deteriorate trabecular or cortical bone architecture, but also it tended to preserve it, although not at a statistically significant level (Suppl. Fig. 2B). Even more unexpectedly, while TNAP inhibition had no significant effect on serum glucose and non-esterified fatty acids (NEFAs), it strongly decreased the levels of triglycerides (TGs) and cholesterol (Fig. 4). Since hypercholesterolemia is the driving force for atherosclerosis in ApoE -/-mice [START_REF] Meir | Atherosclerosis in the apolipoprotein-E-deficient mouse: a decade of progress[END_REF] , SBI-425 treatment also affected atherogenesis. If the plaques of mice treated with SBI-425 presented similar levels of lipid content (%) (Fig. 5A,B), they were significantly smaller (Figure 5A,C). Moreover, TNAP inhibition also decreased aortic inflammation, as demonstrated by the significant reduction in the relative levels of the inflammatory markers Cd68, Rankl, Opn, Il8, Il1b, Il6 (Fig. 5D), and tended to reduce serum IL-6 concentration (Fig. 5E). Calcium accumulation and calcification activity were also impaired, as determined in vivo with µCT and 18 F-NaF µPET (Fig. 5F and 5G respectively). Histological analyses with osteosense, which in our hands was the most sensitive method for this imaging, detected calcification in only 4 mice out of 12 treated with SBI-425 (33%), whereas all control mice presented with calcification (Fig. 5H). The reduction of plaque calcification in mice treated with SBI-425 probably resulted from the beneficial effect of SBI-425 on cholesterolemia and plaque development. Indeed, TNAP inhibition not only prevented calcification, but it also prevented cartilage formation, as illustrated by the absence of alcian blue-positive cartilaginous metaplasia in aortic plaques that were devoid of calcification (Fig. 5B).

Nevertheless, SBI-425 also likely prevented calcification by directly inhibiting TNAP activity in plaques.

We already reported that TNAP inhibition prevents calcification in cultured mouse VSMCs transdifferentiated into chondrocytes [START_REF] Bessueille | Glucose stimulates chondrocyte differentiation of vascular smooth muscle cells and calcification: A possible role for IL-1β[END_REF] . We report here that TNAP was activated in human VSMCs stimulated to trans-differentiate into osteochondrocyte-like cells, and that its inhibition also prevented calcification (Suppl. Fig. 3). Collectively, these data suggested that SBI-425 administration impaired atherosclerosis by reducing blood cholesterol levels, and locally inhibited plaque calcification. Since in ApoE -/-mice, blood cholesterol is predominantly transported by liver-derived very low density lipoproteins (VLDLs) [START_REF] Meir | Atherosclerosis in the apolipoprotein-E-deficient mouse: a decade of progress[END_REF] , we next explored the effects of SBI-425 on the liver.

Liver TNAP reduces hypercholesterolemia by dephosphorylating phosphocholine

Transcript levels of Alpl, encoding TNAP, showed a broad peak between 19 and 27 weeks in the liver of ApoE -/-mice (Fig. 6A), which was concomitant with the accumulation of cholesterol (Fig. 6B) and TGs (Fig. 6C) in the liver, and the release of TGs in the blood (Fig. 6D). Moreover, there was a positive correlation between Alpl transcript levels and the TG content in the liver in 25-week-old mice (Fig. 6D).

Inhibition of TNAP from 10 to 25 weeks did not change the liver content of cholesterol (Fig. 6F) and TGs (Fig. 6G). Since TNAP inhibition reduced the levels of TGs and cholesterol in the circulation but not in the liver, these results suggested that liver TNAP participated in their release. We therefore hypothesized that TNAP inhibition induced liver inflammation or fibrosis to exert these effects through VLDL release. However, SBI-425 administration had no effect on liver inflammation, as revealed by the levels of Ly6g, Tnfa, Il1b or Hb transcripts (Fig. 6H), and by the concentration of the liver acute phase protein haptoglobin in the blood (Fig. 6I). Moreover, it did not impact liver fibrosis, as demonstrated by the similar contents of hydroxyproline in treated and untreated mice (Fig. 6J).

Then, to further identify the mechanisms explaining how TNAP inhibition decreased serum lipid levels while unaffecting lipid content in the liver, we used a metabolomics approach to determine hepatic metabolites whose levels were significantly impacted by SBI-425 administration. Multivariate statistical analysis on metabolomics data (PLS-DA) for the 30 detected metabolites, did not show any major impact of TNAP inhibition on liver metabolism (Suppl. Fig. 2C). Nevertheless, TNAP inhibition seemed to decrease phosphocholine levels in the liver (Fig. 6K, p=0.07), a phosphorylated compound and thus a putative TNAP substrate. Extracellular phosphocholine dephosphorylation in the liver is necessary to allow choline uptake in hepatocytes [START_REF] Morita | Structure and biological function of ENPP6, a choline-specific glycerophosphodiester-phosphodiesterase[END_REF] , intracellular synthesis of phosphocholine, and generation of phosphatidylcholine, which is required for VLDL production and TG and cholesterol release in the blood [START_REF] Li | Phosphatidylcholine and choline homeostasis[END_REF] . This mechanism explains why choline-deficient diets or genetic deficiency in liver phosphatidylcholine production result in liver steatosis, reduced VLDL levels and impaired atherosclerosis in ApoE-deficient mice [START_REF] Corbin | Choline metabolism provides novel insights into nonalcoholic fatty liver disease and its progression[END_REF][START_REF] Cole | Impaired phosphatidylcholine biosynthesis reduces atherosclerosis and prevents lipotoxic cardiac dysfunction in ApoE-/-Mice[END_REF] . This possible decrease in liver phosphocholine was moreover intriguing considering the recent report that TNAP-deficient mice develop exacerbated liver steatosis when fed a HFD but not a choline-deficient diet [START_REF] Gámez-Belmonte | Deficiency in Tissue Non-Specific Alkaline Phosphatase Leads to Steatohepatitis in Mice Fed a High Fat Diet Similar to That Produced by a Methionine and Choline Deficient Diet[END_REF] . We therefore believe that TNAPdependent inhibition of phosphocholine dephosphorylation in the liver by SBI-425 administration, likely contributed to the beneficial effects of SBI-425 on serum lipids and atherosclerosis. Indeed, we observed a significant inverse relationship between the content of phosphocholine and the levels of cholesterol and TGs in the liver, indicating that when phosphocholine content was the most reduced by SBI-425 administration, the liver content of TGs and cholesterol was the highest (Fig. 6L). Moreover, we provide here evidence that phosphocholine is a TNAP substrate. The quantification of Pi release from phosphocholine in presence or absence of the parental inhibitor of SBI-425 (MLS-0038949), indicated that mouse and human recombinant TNAP were equally potent in dephosphorylating phosphocholine (Fig. 6M). Finally, the cholesterol that was not released in VLDLs in mice treated with SBI-425 was probably transformed into bile acids (BAs) since the liver content of BA was almost significantly increased by SBI-425 treatment (Fig. 6N). Unfortunately, we did not harvest the bile and could not explore whether bile excretion was increased by TNAP inhibition in ApoE -/-mice, as it is in rats [START_REF] Alvaro | The function of alkaline phosphatase in the liver: regulation of intrahepatic biliary epithelium secretory activities in the rat[END_REF] .

Discussion

The main finding of this study is that the systemic pharmacological inhibition of TNAP prevented dyslipidemia and protected ApoE -/-mice from atherosclerosis and plaque calcification. These beneficial effects most likely relied on the inhibition of liver TNAP. Like in humans, whom liver TNAP production and release in the blood are associated with the development of MetS [START_REF] Tonelli | Relation between alkaline phosphatase, serum phosphate, and all-cause or cardiovascular mortality[END_REF][START_REF] Panh | Association between serum alkaline phosphatase and coronary artery calcification in a sample of primary cardiovascular prevention patients[END_REF][START_REF] Lammers | Levels of alkaline phosphatase and bilirubin are surrogate end points of outcomes of patients with primary biliary cirrhosis: an international follow-up study[END_REF][START_REF] Abramowitz | Serum alkaline phosphatase and phosphate and risk of mortality and hospitalization[END_REF][START_REF] Krishnamurthy | Associations of serum alkaline phosphatase with metabolic syndrome and mortality[END_REF][START_REF] Wannamethee | Alkaline phosphatase, serum phosphate, and incident cardiovascular disease and total mortality in older men[END_REF][START_REF] Filipowicz | Associations of serum skeletal alkaline phosphatase with elevated C-reactive protein and mortality[END_REF] , hepatic TNAP expression in ApoE -/-mice showed a similar trend as TG levels in liver and serum. We provide here evidence that a function of this hepatic TNAP is to participate in the release of lipids from hepatocytes into circulating VLDLs. Indeed, when TNAP was inhibited, the levels of lipids in the blood were significantly reduced.

TNAP may be the yet unknown phosphatase that dephosphorylates phosphocholine in the liver, to allow the uptake of choline in hepatocytes [START_REF] Morita | Structure and biological function of ENPP6, a choline-specific glycerophosphodiester-phosphodiesterase[END_REF] . In the mouse liver, TNAP is mainly expressed in endothelial cells [START_REF] Linder | Isozyme profile and tissueorigin of alkaline phosphatases in mouse serum[END_REF] , where probably reside ENPP6, the enzyme that generates phosphocholine from glycerophosphocholine, and where is suspected to be the phosphatase hydrolyzing phosphocholine into choline [START_REF] Morita | Structure and biological function of ENPP6, a choline-specific glycerophosphodiester-phosphodiesterase[END_REF] . Following its uptake in hepatocytes, intracellular choline is then rephosphorylated into phosphocholine to generate phosphatidylcholine, which is the main phospholipid involved in VLDL production [START_REF] Li | Phosphatidylcholine and choline homeostasis[END_REF] . This hypothesis is appealing because choline deficiency or genetic deficiency in liver phosphatidylcholine production reduces blood cholesterol levels and impairs atherosclerosis in ApoEdeficient mice [START_REF] Li | Phosphatidylcholine and choline homeostasis[END_REF][START_REF] Corbin | Choline metabolism provides novel insights into nonalcoholic fatty liver disease and its progression[END_REF][START_REF] Cole | Impaired phosphatidylcholine biosynthesis reduces atherosclerosis and prevents lipotoxic cardiac dysfunction in ApoE-/-Mice[END_REF] . Phosphocholine levels in the liver showed a strong tendency to decrease in response to SBI-425 administration in vivo. In addition, mouse and human TNAP were equally potent in dephosphorylating phosphocholine in vitro. However, why TNAP inhibition did not exacerbate liver steatosis in ApoE -/-mice remains elusive, since choline-deficient diets induce liver steatosis [START_REF] Corbin | Choline metabolism provides novel insights into nonalcoholic fatty liver disease and its progression[END_REF] . We suspect that steatosis was prevented because of the previously described liver TNAP function in bile excretion. In addition to being active in liver endothelial cells, hepatic TNAP is also localized in the membrane of hepatocytes and cholangiocytes facing bile canaliculi and ducts [START_REF] Linder | Isozyme profile and tissueorigin of alkaline phosphatases in mouse serum[END_REF][START_REF] Hoshi | Immunolocalization of tissue non-specific alkaline phosphatase in mice[END_REF][START_REF] Araki | Redistribution and fate of colchicine-induced alkaline phosphatase in rat hepatocytes: possible formation of autophagosomes whose membrane is derived from excess plasma membrane[END_REF][START_REF] Chida | Localization of alkaline phosphatase and proteins related to intercellular junctions in primary cultures of fetal rat hepatocytes[END_REF] . At this location, TNAP is thought to reduce the secretory processes of the intrahepatic biliary epithelium, as demonstrated by the significant increase in bicarbonate secretion and basal bile flow induced by TNAP inhibition in rats [START_REF] Alvaro | The function of alkaline phosphatase in the liver: regulation of intrahepatic biliary epithelium secretory activities in the rat[END_REF] . Since in our experiments, TNAP inhibition tended to increase the content of bile acids in the liver, we suspect that cholesterol catabolism into bile acids and bile acid excretion were also enhanced, thereby preventing steatosis.

To summarize, TNAP inhibition limited atherosclerosis development, likely through impaired phosphocholine dephosphorylation, decreased choline uptake and phosphatidylcholine production in hepatocytes, and reduced TG and cholesterol release in VLDLs. This reduction in atherosclerotic development was reflected in mice treated with SBI-425 by smaller plaques in aortas, with less inflammation, and less calcification. While the decrease in plaque size and inflammation likely resulted from the inhibition of liver TNAP, the strong reduction in the number of aortic calcifications in mice treated with SBI-425 probably also relied on the inhibition of local TNAP blockade. Indeed, we observed that in mouse and human plaques, TNAP expression preceded calcification, and colocalized with calcium deposits once they had formed. Moreover, TNAP inhibition in human VSMCs stimulated to calcify totally prevented calcium deposition. This result is consistent with previous report by Tani et al, showing that SBI-425 treatment efficiently blocked vascular calcification in a mouse model of chronic kidney disease (CKD) [START_REF] Tani | Inhibition of tissue-nonspecific alkaline phosphatase protects against medial arterial calcification and improves survival probability in the CKD-MBD mouse model[END_REF] .

In conclusion, TNAP inhibition might represent a promising therapeutic option, not only to prevent vascular calcification in patients with CKD, but also to reduce hypercholesterolemia and atherosclerosis development in individuals with MetS, in whom TNAP levels are associated with mortality risk [START_REF] Tonelli | Relation between alkaline phosphatase, serum phosphate, and all-cause or cardiovascular mortality[END_REF][START_REF] Panh | Association between serum alkaline phosphatase and coronary artery calcification in a sample of primary cardiovascular prevention patients[END_REF][START_REF] Lammers | Levels of alkaline phosphatase and bilirubin are surrogate end points of outcomes of patients with primary biliary cirrhosis: an international follow-up study[END_REF][START_REF] Abramowitz | Serum alkaline phosphatase and phosphate and risk of mortality and hospitalization[END_REF][START_REF] Krishnamurthy | Associations of serum alkaline phosphatase with metabolic syndrome and mortality[END_REF][START_REF] Wannamethee | Alkaline phosphatase, serum phosphate, and incident cardiovascular disease and total mortality in older men[END_REF][START_REF] Filipowicz | Associations of serum skeletal alkaline phosphatase with elevated C-reactive protein and mortality[END_REF] . In our experiments in ApoE -/-mice, SBI-425 administration for 15 weeks did not induce negative effects in plaques, blood, liver or bones. While TNAP activation during liver disease in mice fed a HFD was proposed to participate in the dephosphorylation and detoxification of lipopolysaccharide (LPS) [START_REF] Schippers | Phosphate Groups in the Lipid A Moiety Determine the Effects of LPS on Hepatic Stellate Cells: A Role for LPS-Dephosphorylating Activity in Liver Fibrosis[END_REF] , we observed no increase in serum LPS levels in animals treated with SBI-425 as compared to controls (data not shown). Moreover, administration of SBI-425 tended to decrease, and not increase, circulating IL-6 levels, and those of the acute phase protein haptoglobin, indicating that systemic and liver inflammation were probably reduced, and not increased by SBI-425.

Finally, SBI-425 administration not only did not lead to bone architecture degradation, but instead also tended to increase the bone volume as compared to untreated animals. Mice treated with SBI-425 seemed globally healthier than untreated mice; they were less dyslipidemic and less inflammatory, explaining why their bones were preserved despite the important function of TNAP in bone mineralization. Supplemental Figure 3

  and Molecular Mechanisms ICBMS, CNRS UMR 5246 Bâtiment Raulin ; 43 Bd du 11 novembre 1918 69622 Villeurbanne Cedex, France Phone: 0033-4-27-46-57-20 Email: david.magne@univ-lyon1.fr 2022-03-16 Dear Professor Jeffrey, On behalf of all co-authors, I propose for publication in Translational Research an article entitled "Inhibition of alkaline phosphatase impairs dyslipidemia and protects mice from atherosclerosis".

1 H

 1 H NMR spectra were recorded at 298 K on a Bruker Avance spectrometer 500 equipped with a 5-mm TCI cryoprobe. The 1D pulse sequence (relaxation delay-pulse-acquisition) with pre-saturation for water (HOD) signal suppression was used. Acquisition parameters were as follows: relaxation delay 5.64 s, 30° pulse (4.5 µs) and acquisition time of 4.36 s. The spectral width of 15 ppm was used and 256 scans were collected. With this repetition time of 10 s, the 1 H resonances were fully relaxed. Data were processed using Bruker TopSpin software 3.2 with one level zero-filling and apodization (exponential, lb = 0.3 Hz) before Fourier transform, then phase correction, simple baseline correction and calibration (TSP = 0 ppm) were applied. Chemometric analysis of NMR data: Data NMR matrices were obtained using facilities included in KnowItAll® (Wiley Science Solutions) and NMRProcFlow® (https://nmrprocflow.org/)[START_REF] Jacob | NMRProcFlow: a graphical and interactive tool dedicated to 1D spectra processing for NMR-based metabolomics[END_REF] software. First, the bucket list was obtained by the bin area method using the intelligent variable size bucketing tool included in the KnowItAll® package according to NMR signals assignment. Second, NMRProcFlow® pipeline was used for local baseline correction of spectra, alignment of NMR signals and integration of NMR area by that bucket list. Then integrated regions were normalized by dividing their areas by that of the internal standard TSP and by the weight of tissue. Statistical analysis, supervised Partial Least Squares-Discriminant Analysis (PLS-DA) and the nonparametric Wilcoxon test were carried out using the SIMCA-P+ 13.0.3 (Sartorius, Aubagne, France) and Matlab® (MathWorks, Natick, Massachusetts, USA) softwares. NMR absolute quantification of metabolites: Metabolites were assigned with an in-house metabolite database at pH = 7.4. A total of 34 metabolites were identified; 30 could be quantified.

  25 µM of phosphocholine or phosphoethanolamine were incubated at 37°C with recombinant human TNAP (Biolegend 794504) or mouse TNAP (Biolegend 787804) in a Tris-HCl saline buffer pH 7.4 containing 1 mM MgCl2. After different time points, Pi release was determined using Malachite Green reagent. Statistics Data are expressed as the mean ± SEM or SD, as indicated in the Figure legends. Statistical analyses were performed with Past 3.20 software. Data were tested on normality and on equal variances with the Shapiro-Wilk test and Levene test, respectively. Then, data were analyzed on appropriate parametric or nonparametric test, as indicated in the Figure legends. P<0.05 was considered significant.

Figure 1 :

 1 Figure 1: TNAP activity is locally associated with plaque calcification in mice. Representative in vivo imaging of aortic calcification (red arrow) with µCT (top) and 18 F-NaF µPET/µCT (bottom) in a 34-weekold mouse (A), and its quantification by µCT (B) and 18 F-NaF µPET (C). Histological analysis of

Figure 2 :

 2 Figure 2: Calcification-associated TNAP is mainly produced by hypertrophic chondrocytes in mouse

Figure 3 :

 3 Figure 3: TNAP, expressed in RUNX2-positive and -negative cells, is locally associated with

Figure 4 Serum

 4 Figure 4

Aorta Acan levels Aorta Runx2 levels Aorta Alpl levels 25 31 19 21 23 27 29 Age (weeks) 17 25 31 19 21 23 27 29 Age (weeks) 17 25 31 19 21 23 27 29 Age (weeks) 17 Aorta Runx2 levels Aorta Alpl levels

SBI-425 CT SBI-425 CT SBI-425 CT SBI-425 CT SBI-425 CT SBI-425 CT SBI-425 CT SBI-425 CT SBI-425 CT SBI-425 CT SBI-425 β-Hydroxybutyrate (ng/mg)

Funding

This study was funded by European Research Area Network on Cardiovascular Diseases (ERA-CVD, Microexploration project 2018-2021) and by the "Fondation de France" (project 00086497). Part of the metabolomic studies were supported by the Association Hypophosphatasie Europe (contract 194679).

Conflict of interest

JLM and AP are co-inventors on a patent covering SBI-425 (PCT WO 2013126608). Other authors declare no competing financial or non-financial interests. magnification (x 2.5) photographs, the bar indicates 1 mm; on the high-magnification images (x 40), the bar indicates 50 µm. IHC: immunohistochemistry.


Lionel Perrier
email: perrierl@lyon.fnclcc.fr

Magali Morelle
email: morelle@lyon.fnclcc.fr

Nathalie Havet
email: havet@gate.cnrs.fr

Anthony Montella
email: montella@lyon.fnclcc.fr

Bertrand Favier
email: favier@lyon.fnclcc.fr

David Pérol
email: perold@lyon.fnclcc.fr

Frédéric Gomez
email: gomez@lyon.fnclcc.fr

Marie-Odile Carrère
email: mo.carrere@wanadoo.fr

Paul Rebattu
email: rebattu@lyon.fnclcc.fr

The effect of health care expenditures on survival in locally advanced and metastatic Non Small Cell Lung Cancer

Keywords: Cost, NSCLC, Oncology, Survival JEL code: I12 -health Production, I18 -Government Policy, Regulation, Public Health

Context: The significant survival benefit of chemotherapy over best supportive care for locally advanced and metastatic NSCLC has been amply demonstrated in the literature.

However, there is no clear evidence of the impact of the type of chemotherapy or of a superiority of combination chemotherapy over single-agent chemotherapy.

Objective: The present study empirically examines, in real-life practise and using multiple proxies, the impact of health care expenditures on overall survival in locally advanced and metastatic NSCLC in order to guide medical decision-making.

Methods: Disease characteristics, the resources used, the costs of treatment and survival data were retrospectively collected from the records of 175 patients treated between 2000 and 2004 at Léon Bérard Regional Cancer Center (Lyon, France). Survival data were modelled using multivariate Cox models and controlled for endogeneity with the instrumental variable method.

Results:

The median survival for the whole cohort was 289 days. The average total cost of treatment reached €35,160. Survival was significantly shorter for patients with stage IV disease, poor performance status, and past or concomitant cardiovascular disease and/or diabetes, for current smokers, and for patients with adenocarcinoma compared to large cell carcinoma. Survival duration was not significantly associated with the total cost of treatment per day of hospitalisation, the number of chemotherapy drugs administered, nor inpatient length of stay.

Conclusion:

Higher care expenditures do not appear to improve survival for patients with locally advanced or metastatic NSCLC. Hence, maintaining patient quality of life and tailoring therapy to stage, histology and comorbidities appears to be the less bad choice.

INTRODUCTION

At the macro-level, increased health care expenditure implies increased benefits for the patients (Cremieux et al., [1999]). However, this has not yet proven true at the micro-level.

Since the demonstration by Cochrane et al. that indicators of health care inputs are generally not associated with outcomes [START_REF] Cochrane | Health service 'input' and mortality 'output' in developed countries[END_REF]), the failure to identify a relationship between health care expenditure and health outcomes has become a persistent theme in the literature. Socioeconomic factors are recognized to be highly associated with health outcomes [START_REF] Nolte | Does care save lives? London: The Nuffield Trust[END_REF]; [START_REF] Young | An explanation of the persistent doctor mortality association[END_REF]; Saint [START_REF] Leger | The anomaly that finally went away[END_REF]). Lichtenberg has shown that cancers for which the stock of drugs increases more rapidly tend to have greater increases in survival rates, and the development of new cancer drugs generally increases the life expectancy of people diagnosed with cancer [START_REF] Lichtenberg | The expanding pharmaceutical arsenal in the war on cancer[END_REF]). Martin et al. have shown that health care expenditure has a strong positive effect on outcomes (Martin et al., [2007]).

They have found that a one percent increase in expenditure per head induces, ceteris paribus, a 0.38% reduction in years of life lost from cancer.

The present study focuses on the "real life" situations (as opposed to clinical trials) of patients with advanced or metastatic Non Small Cell Lung Cancer (NSCLC). NSCLC accounts for approximately 85% of all lung cancer cases, mostly late stage diseases with poor survival prognosis (Cella et al., [2000]; [START_REF] Burdett | Chemotherapy in addition to supportive care improves survival in advanced non-small-cell lung cancer: a systematic review and meta-analysis of individual patient data from 16 randomized controlled trials[END_REF]). Indeed, patients with locally advanced NSCLC are not candidates for surgery, their median survival is less than one year [START_REF] Galetta | Gemcitabine and docetaxel every 2 weeks in advanced non-small cell lung cancer: a phase II study of the Grupo oncologica Italia meridionale[END_REF]), and their 5-year survival rate is less than 5%. The modest survival rates, high toxicities, and high costs of treatment due to multiple chemotherapy regimens have prompted investigators to examine the potential benefits of new drugs. Clinical trials generally show that chemotherapy can improve survival, as compared to best supportive care, in stage IIIB and IV NSCLC (Hensing et al., [1999]; [START_REF] Thongprasert | Relationship between quality of life and clinical outcomes in advanced non-small cell lung cancer: best supportive care (BSC) versus BSC plus chemotherapy[END_REF]; [START_REF] Cullen | Mitomycin, ifosfamide, and cisplatin in unresectable non-small-cell lung cancer: effects on survival and quality of life[END_REF]; [START_REF] Burdett | Chemotherapy in addition to supportive care improves survival in advanced non-small-cell lung cancer: a systematic review and meta-analysis of individual patient data from 16 randomized controlled trials[END_REF]; [START_REF] Berhoune | Therapeutic strategy for treatment of metastatic non-small cell lung cancer[END_REF]). Economic studies have also shown that chemotherapy drugs are generally cost effective, as compared to best supportive care [START_REF] Carlson | Pharmacoeconomic evaluations in the treatment of non-small cell lung cancer[END_REF]). However, increasing the number of chemotherapy prescriptions, prolonging cytotoxic therapy, and using innovative and expensive chemotherapy drugs do not seem to improve overall survival (Socinsky et al., [2003]; [START_REF] Pujol | Gemcitabine-docetaxel versus cisplatin-vinorelbine in advanced or metastatic non-small-cell lung cancer: a phase III study addressing the case for cisplatin[END_REF]; Rubio-Terrés et al., [2002]; [START_REF] Burdett | Chemotherapy in addition to supportive care improves survival in advanced non-small-cell lung cancer: a systematic review and meta-analysis of individual patient data from 16 randomized controlled trials[END_REF]; [START_REF] Gridelli | Factorial phase III randomised trial of rofecoxib and prolonged constant infusion of gemcitabine in advanced non-small-cell lung cancer: the GEmcitabine-COxib in NSCLC (GECO) study[END_REF]).

In this context, and with the objective of guiding medical decision-making, we conducted a real-life practice study, based on retrospective data, to analyze the effects of health care expenditures on survival for patients with advanced NSCLC.

MATERIAL AND METHODS

Inclusion and exclusion criteria

All patients with stage IIIB or IV NSCLC treated with chemotherapy at the regional cancer centre of Lyon, France, between January 1, 2000 andDecember 31, 2004 were included in the study. Patients starting chemotherapy in another hospital, those receiving radiosensitizing chemotherapy alone, and those with stage I, II, or IIIA NSCLC were excluded from the study.

Data source

All data related to the characteristics of the patients and the resources used ( , , , , ) were extracted from patient records kept by the Cancer Centre. The costs of irradiation sessions ( ) were obtained from the "Classification Commune des Actes

Médicaux" (CCAM), the costs of hospitalisation ( ) were calculated by the accounting

i c LOS , i r LOS , i o LOS , i m q i s q , s c h c
department of the Cancer Centre, and the price of chemotherapy drugs ( ) was given by the pharmacy of the Cancer Centre.

m p

Patient characteristics

We collected data on patient age and sex, smoking habits, performance status (PS), significant weight loss (10% of total body weight or more), as well as disease stage (IIIB versus IV) and histology (adenocarcinoma versus epidermoid or malpighian carcinoma versus large cell carcinoma). Past or concomitant cardiovascular disease and/or diabetes were also noted.

Health outcomes

The indicator used to evaluate treatment efficacy was overall survival from the date of diagnosis.

Cost evaluation

Costs were calculated from the hospital's point of view.

h i c n m m m i c LOS p q Cc i i × + × = ∑ = , 1 ( ) h i r s s i c LOS c q Cr i × + × = , h i o i c LOS Co × = , with

+ + =

We also determined the total cost of chemotherapy per cure, such as:

c i Cc i c i c i q Cc Cc =
where is the number of chemotherapy cures for patient i . And the total cost of radiotherapy per session:

i s i s i q Cr Cr =

Proxies of health care expenditures

Seven proxies were retained in this study, i.e. inpatient length of stay; number of outpatient health care procedures; number of chemotherapy drugs administrated; number of expensive and innovative chemotherapy drugs administrated (i.e. reimbursed by the French National Health Insurance in excess of DRG tariffs), number of irradiation sessions; total cost of chemotherapy drugs; total cost of treatment per day of hospitalisation .

d i

CT

Statistical and econometric analyses

Descriptive statistics

Mean and standard deviation for continuous variables, and percentage for categorical variables were used to analyze patient characteristics, the resources used (e.g. number of chemotherapy drugs administered, inpatient length of stay), and the costs of treatment.

Survival time was calculated from the date of diagnosis until the date of death or the date of the last follow-up when patients were still alive (survival time was in this case therefore censored). Univariate survival analyses were performed by calculating Kaplan-Meier estimates. Differences between survival distributions were tested using log-rank statistics.

Multivariate analyses

Several expenditure indicators (logarithm of the total cost of treatment per day of hospitalisation, , number of chemotherapy drugs administered, inpatient length of stay), and survival data were modelled using multiple regression analyses. Since health care spendings and survival were jointly determined, expenditures were predictive of survival and conversely. Due to this bi-directional relationship, we could suspect a risk of endogeneity [START_REF] Gujarati | Basic Econometrics 4th edition[END_REF]; [START_REF] Greene | Econometric Analysis 5th edition[END_REF]). Without any control for endogeneity, estimates from a standard Cox proportional hazard model for survival on the one hand, and from Ordinary Least Squares (OLS) for health expenditures models on the other hand, could be biased and inconsistent. Therefore we controlled endogeneity using the instrumental variable approach.

d i

CT

The choice of the appropriate instrument(s) was based on data from the medical literature, practising oncologists' expertise and on our intermediate results. For all analyses, statistical significance was set at 5%. Calculations were performed using Stata 10.0 software.

RESULTS

Descriptive statistics

Characteristics of patients

Main patient characteristics are described in table I. The sample consisted of a cohort of 175 patients of whom 73% were male. Mean age at inclusion was 60 years. A vast majority of patients (79%) had stage IV tumours. Adenocarcinoma was the main histological group (47% of the patients), followed by epidermoid or malpighian carcinoma (23%), and large cell carcinoma (30%). In total, 124 patients were current or former smokers (71%), 21 (12%) had concomitant or past cardiovascular disease and/or diabetes, and 35 (20%) reported significant weight loss. The prevalence of poor performance status was high (63% of the patients), with 37% of the patients scoring 2 or 3.

Health outcomes

At the time of the analysis (at least four years after diagnosis), 168 patients (96%) had died.

The median survival for the whole cohort was 289 days (95% Confidence Interval; [230-339 days]).

Proxies of health expenditures

Inpatient length of stay varied from 0 to 139 days. The median length of stay reached 25 days, with 10% of the patients staying three days or less and 90% 62 days or less. The number of outpatient health care procedures varied from 0 to 87, with a median of seven. Ten percent of the patients never received outpatient care, whereas 90 % came for 24 visits or less. The number of chemotherapy drugs administered varied from 0 to 8. A majority of patients received between 2 and 4 drugs. Amongst the patients receiving chemotherapy, 2% did not receive expensive and innovative drugs, i.e. chemotherapy drugs reimbursed by the French National Health Insurance in excess of DRG tariffs, and a majority received one drug of the formulary. 76% received radiotherapy with a median number of sessions of 14 [Table II].

Mean cost of chemotherapy drugs reached 4,596€, ranging from 0 to 42,705€. Mean total cost of treatment per day of hospitalisation reached 934€, ranging from 732€ to 1,715€ [Table III].

Other costs evaluation

The average total cost of treatment for patients with advanced or metastatic NSCLC ( CT ) reached €35,160 (n=174), ranging from €5,602 to €131,227. The average total cost of chemotherapy ( c C ) reached €14,741, i.e. 42% of CT . The average total cost of radiotherapy ( r C ) was €1,741, i.e. 5% of CT . However, as shown in table II, the average cost of radiotherapy for the 76 patients concerned reached €4,008. Results are detailed in table III.

Univariate analysis of survival

The median survival time was 8.0 months for men (CI 95%: [7.4; 10.0]) and 11.6 months for women (CI 95%: [9.6; 15.0]). The survival rate at 1 year was 36% for men and 47% for women. However, the overall survival of patients was not statistically different between sexes (p=0.06). The overall survival of patients younger than 70 years did not differ significantly from that of patients aged 70 and over (p=0.10). The overall survival of patients with PS ≤1 was significantly higher than that of patients with PS >1 (p=0.0003). The median survival time was 11.7 months for patients with PS ≤1 (CI 95%: [8.2; 14.8]), versus 7.6 months for patients with PS > 1 (CI 95%: [5.7; 9.5]). The survival rate at 1 year was 47% for patients with PS 1 and ≤ 25% for patients with PS> 1. The overall survival of patients was significantly higher for patients treated in combination with radiotherapy (p=0.04). The median survival time was 7.7 months (CI 95%: [6.7; 10.0]) for patients not treated with radiotherapy (33% survival at 12 months) and 11.6 months (CI 95%: [9.5; 15.0]) for patients treated (46% survival at 12 months). The Kaplan Meier survival curve stratified by stage (IIIB and IV) (log-rank p=0.1298) is presented in figure 1.

Multivariate analysis of survival

Results obtained with final survival models using instrumental variable analysis are provided in table IV. As mentioned above, modelling the interdependence between survival and expenditures may cause endogeneity problems and biased estimates. These problems could be overcome by using instrumental variables. The accuracy of the method relies on the relevance and validity of the instrumental variables available. An instrumental variable does not itself belong in the explanatory equation (here, does not have a direct effect on survival) for identification reason and must satisfy two requirements: (i) it must be strongly (or at least not weakly) correlated with the included endogenous variable (here, expenditures) conditional on the other covariates and (ii) it cannot be correlated with the error term in the explanatory equation [START_REF] Greene | Econometric Analysis 5th edition[END_REF]; [START_REF] Gujarati | Basic Econometrics 4th edition[END_REF]). According to medical literature and to medical practice, age could be a valuable instrument in our context, and an age limit of 70 years would have clinically relevant distinctive value. Indeed, several surveys have shown that the intensity of cancer treatments is usually reduced for elderly patients [START_REF] Gridelli | Treatment of advanced non-small-cell lung cancer in the elderly: results of an international expert panel[END_REF]).

These patients are generally more vulnerable to the toxicity of combined chemo-and radiation therapies, so we expected to find a negative correlation between higher age and overall treatment expenditures. In contrast, age was not expected to be a predictor of survival. Even if there is currently no elderly-specific trial (and if elderly people are under-represented in clinical trials), some published studies focusing on this topic have shown that, when all patients receive the same treatment, progression and survival rates do not differ between elderly and younger patients [START_REF] Sequist | Aggressive treatment for the fit elderly with non-small-cell lung cancer? Yes![END_REF]). Preliminary regressions of health expenditures and survival as a function of age, among other clinical variables, were ran in order to respectively assess the strength of this instrument in our empirical situation (cf.

requirement 1) and whether or not the equations of interest would be identifiable.

Our results suggested that both conditions hold when using inpatient length of stay, the number of chemotherapy drugs administered, and the total cost of treatment per day of hospitalisation as proxies of health care expenditures. Indeed, age was significantly correlated to each of these variables but not with survival. Unfortunately, this was not confirmed when using outpatient health care procedures, innovative and expensive chemotherapy drugs, the number of radiotherapy sessions, and the cost of chemotherapy drugs. The instrumental technique was not applicable to these latter cases.

However, the assumption that the instrument (age) was not correlated with the error term in the equation of interest was not testable (requirement 2). In practice, in the first stage, each endogenous covariate (here, each expenditure indicator) was regressed (with a OLS model or with an ordered logistic model, Tobit type I model, or Poisson regression, according the nature of the variable) on all of the exogenous variables. In the second stage, the survival regression was estimated as usual with a Cox model, except that in this stage the endogenous covariate (the considered expenditure) was replaced by the predicted values from the first stage model while the instrument (age) was excluded from the regression. The robust option in Stata was used to give consistent estimates of standard errors, since this method is known to affect the precision of yield estimates.

-Regarding the survival model as a function of inpatient length of stay, survival was, all other things being equal, significantly worse for patients with stage IV compared to stage IIIB (p=0.001), for patients with adenocarcinoma compared to large cell carcinoma (p=0.007), and for current smokers compared to never smokers (p=0.045). Survival duration was not significantly associated with inpatient length of stay (Table IV).

-Regarding the survival model as a function of the number of chemotherapy drugs administered, survival was, all other things being equal, significantly worse for patients with stage IV compared to stage IIIB disease (p=0.002), for those with past or concomitant cardiovascular disease and/or diabetes (p=0.033), and for current smokers compared to never smokers (p=0.031). Survival duration was not significantly associated with the number of chemotherapy drugs administered (Table IV).

-Regarding the survival model as a function of total cost of treatment per day of hospitalisation, survival time was, all other things being equal, significantly shorter for patients with stage IV compared to stage IIIB (p=0.003), poor performance status (p=0.006), and past or concomitant cardiovascular and/or diabetes co-morbidities (p=0.021). Moreover, current smokers had a shorter survival than never smokers (p=0.03). Survival was also significantly shorter for patients with adenocarcinoma compared to large cell carcinoma (p=0.003), whereas this significance was only borderline for weight loss (p=0.055).

Our results also showed major differences in the significance and magnitude of parameters estimated with and without controlling for endogeneity, leading to opposite conclusions.

Survival time was not significantly associated with the number of chemotherapy drugs administrated (p=0.281) and total cost of treatment per day of hospitalisation (p=0.264) when using instrumental variables (this finding further confirms the validity of our instrument), as was the case when the original variables were used (p=0.004 and p<0.001, respectively). These results confirmed our initial suspicion that seriously biased results were obtained when dealing with health expenditures variables, due to reciprocal causation between cost and survival (Table IV).

DISCUSSION

Choice of the health care expenditure proxies

Hospitalisation is described in the literature as the most expensive cost item for advanced and metastatic NSCLC [START_REF] Bordeleau | Pharmacoeconomics of systemic therapies for lung cancer[END_REF]). In Dedes et al. study, hospitalisation costs reach 68% and 74% of the total costs of treatment in stage III and stage IV patients, respectively, versus only 14% and 26% for chemotherapy drugs [START_REF] Dedes | Management and costs of treating lung cancer patients in a university hospital[END_REF]). Health care expenditures related to hospitalisation are largely integrated in our analysis, e.g. inpatient length of stay, number of outpatient health care procedures, total cost of treatment per day of hospitalisation.

Health care expenditures related to chemotherapy drugs are also detailed, e.g. number of chemotherapy drugs administered, number of expensive and innovative chemotherapy drugs administered, total cost of chemotherapy drugs). As radiotherapy can palliate the symptoms of poor-prognosis patients, the number of irradiation sessions was also taken into account [START_REF] Van Den Hout | Cost-utility analysis of short-versus long-course palliative radiotherapy in patients with non-small-cell lung cancer[END_REF]).

Because this is a retrospective study, rigorously assessing quality of life was not possible. The literature shows low quality of life scores in patients with locally advanced and metastatic NSCLC. Bordeleau reports a score of 0.34 [START_REF] Bordeleau | Pharmacoeconomics of systemic therapies for lung cancer[END_REF]). Van den Hout et al. report a total of 10% of negative utility measurements, indicating a health state worse than death in palliative NCSLC. However, toxicities (e.g. febrile neutropenia, diarrhoea, nausea and vomiting) could be introduced in order to reinforce the present analysis.

A real-life practise study of health care expenditures and survival

The effect of health care expenditures on survival in locally advanced and metastatic NSCLC was assessed in a real life situation with no previous selection of the patients, contrary to clinical trials. Hence, non surprisingly, the survival results obtained are worse than those of six clinical trials identified by a literature search of Medline for stage IIIB and IV NSCLC since 2000 [START_REF] Babiak | Mitomycin C and Vinorelbine for second-line chemotherapy in NSCLC-a phase II trial[END_REF]; [START_REF] Chen | Paclitaxel plus carboplatin, compared with paclitaxel plus gemcitabine, shows similar efficacy while more cost-effective: a randomized phase II study of combination chemotherapy against inoperable non-small-cell lung cancer previously untreated[END_REF]; [START_REF] Galetta | Gemcitabine and docetaxel every 2 weeks in advanced non-small cell lung cancer: a phase II study of the Grupo oncologica Italia meridionale[END_REF]; [START_REF] Perry | A phase III study of surgical resection and paclitaxel/carboplatin chemotherapy with or without adjuvant radiation therapy for resected stage III non-small-cell lung cancer: Cancer and Leukemia Group B 9734[END_REF]; [START_REF] Pujol | Gemcitabine-docetaxel versus cisplatin-vinorelbine in advanced or metastatic non-small-cell lung cancer: a phase III study addressing the case for cisplatin[END_REF]; [START_REF] Zinner | Trastuzumab in combination with cisplatin and gemcitabine in patients with Her2overexpressing, untreated, advanced non-small cell lung cancer: report of a phase II trial and findings regarding optimal identification of patients with Her2-overexpressing disease[END_REF]). The periods of inclusion of those clinical trials ranged from 1998 to 2001, except for the study reported by A. Babiak et al. which ran from 2002[START_REF] Gujarati | Basic Econometrics 4th edition[END_REF][START_REF] Babiak | Mitomycin C and Vinorelbine for second-line chemotherapy in NSCLC-a phase II trial[END_REF]). Except for this study, the median survival reported in clinical trials was higher than in our study. It ranged from 10.5 months [START_REF] Galetta | Gemcitabine and docetaxel every 2 weeks in advanced non-small cell lung cancer: a phase II study of the Grupo oncologica Italia meridionale[END_REF]) to 17.4 months [START_REF] Zinner | Trastuzumab in combination with cisplatin and gemcitabine in patients with Her2overexpressing, untreated, advanced non-small cell lung cancer: report of a phase II trial and findings regarding optimal identification of patients with Her2-overexpressing disease[END_REF]), compared to 9.6 months in this study.

The necessity to analyse the risk of endogeneity

Gravelle and Backhouse have highlighted the methodological difficulties associated with empirical investigations in health care, especially the associated endogeneity problem, and the possible time lag between expenditure and outcomes [START_REF] Gravelle | International cross-section analysis of the determination of mortality[END_REF]).

When the estimated regression equation consists of a mixture of potentially endogenous and exogenous variables, the coefficient on the endogenous variables may be biased when studies do not allow for endogeneity [START_REF] Martin | Does health care spending improve health outcomes? Evidence from English programme budgeting data[END_REF]). The risk of endogeneity and its impact on the relationship between health care expenditures and outcome appear clearly in this study.

When endogeneity is not taken into account, the higher the total cost of treatment per day of hospitalisation, the higher the survival rate, and the higher the number of chemotherapy drugs administrated, the higher the survival rate. This would lead to the conclusion that health care expenditures have a strong positive effect on survival in patients with advanced or metastatic NSCLC, which would bias the decision-making process in favour of expensive therapies.

Results homogenous with the literature in term of benefit regarding health care expenditures Survival time was not significantly associated with either total daily cost or the number of chemotherapy drugs administered and inpatient length of stay. These results reinforce the literature which shows that increasing the number of chemotherapy prescriptions, prolonging cytotoxic therapy, and using innovative and expensive chemotherapy drugs do not significantly improve survival in locally advanced and metastatic NSCLC [START_REF] Rubio-Terrès | Cost-minimisation analysis of three regimens of chemotherapy (docetaxel-cisplatin, paclitaxel-carboplatin) for advanced nonsmall-cell lung cancer[END_REF]; Socinsky et al., [2003]). Our results are also in agreement with those of a metaanalysis of 16 trials, which has failed to provide clear evidence of a difference in the effect of chemotherapy between different chemotherapy types or between trials that used combination chemotherapy and those that used single agent chemotherapy [START_REF] Burdett | Chemotherapy in addition to supportive care improves survival in advanced non-small-cell lung cancer: a systematic review and meta-analysis of individual patient data from 16 randomized controlled trials[END_REF]).

Implications in term of medical decision-making

Due to the short survival times observed in advanced or metastatic NSCLC, especially in the "real life situation", to the lack of consequences of health care expenditures on survival as measured by seven proxies, but also, as shown in the literature, to the significant benefit of chemotherapy on survival, the elaboration of recommendations for medical decision-making appears particularly difficult. The American Society for Clinical Oncology recommends, for example, the administration of a combination of two molecules of chemotherapy in first-line, whereas brief cyto-toxic treatments also appear optimal. Further studies encourage to maintain quality of life and to tailor therapy to patient characteristics and disease specificities (age, stage, histology and comorbidities). Based on the present study, we recommend not to multiply chemotherapy drugs and to encourage the use of less expensive drugs. This is particularly important within the French health care system because: (i) Expensive and innovative drugs are reimbursed to the hospitals by the national health insurance in excess of DRG tariffs, while the costs of other chemotherapy drugs are supported by the hospital as part of the DRG. (ii) Clinical phase IV trials are generally not performed. (iii) Cost effectiveness acceptability curves and willingness to pay thresholds are not specified by the French National insurance system.

CONCLUSION

Although there has been progress in the treatment of NSCLC, survival is still very modest for advanced and metastatic stages. In term of medical-decision making, the present study based on a "real life situation", using several proxies of health care expenditures and controlling the risk for endogeneity, encourages to limit the number of chemotherapy drugs administered and to promote the use of inexpensive drugs. This study also shows the importance of tailoring cytotoxic therapy based, according to the results of survival models, on age, stage, histology and comorbidities. Then, the focus of research on medical decision-making should be on (i) the observation of physicians' behaviour, in particular the reasons why they prescribe non efficient drugs (ii) the preservation of quality of life, based on the results of evaluations of targeted therapies which are pending in lung cancer [START_REF] Bordeleau | Pharmacoeconomics of systemic therapies for lung cancer[END_REF]; [START_REF] Glover | A review of small-molecule epidermal growth factor receptor-specific tyrosine kinase inhibitors in development for non-small cell lung cancer[END_REF]).

The mean estimated annual cost of targeted drugs may however reach $21,963 for Gefitinib, $31,000 for Erlotinib. As targeted cancer drugs are combined with standard regimens, the costs of these drugs add to already costly therapeutic packages. Hence, high-quality evaluations are required to provide clear information on the value and the acceptability of the costs of those treatments. Hopefully, targeted therapy could become a favourable option, and a societally viable solution, in advanced and metastatic NSCLC.

Table I Distribution of sociodemographic factors, smoking status and clinicopathologic variables (n=175 patients)

 I

	Characteristics	Mean ±SD or number of
		patients (%)
	Age (years)	59.8± 10.8
	Male / Female	128 (73%) / 47(27%)
	Tumor stage
	IIIb	37 (21%)
	IV	138 (79%)
	Histology
	Adenocarcinoma	82 (47%)
	Epidermoid or malpighian carcinoma	41 (23%)
	Large cell carcinoma	52 (30%)
	Performance status
	0	5 (3%)
	1	106 (60%)
	2	52 (30%)
	3	12 (7%)
	Smoking status
	Never	51 (29%)
	Former	42 (24%)
	Current	82 (47%)

Acknowledgment

We thank the financial and medical information departments of the Léon Bérard Cancer Centre, as well as M-D Reynaud for technical support.

CNIL authorisations: N°1257249