Anna Łabędź-Maslowska

Luisa Vergori

Sylwia Kędracka-Krok

Elżbieta Karnas

Sylwia Bobis-Wozowicz

Malgorzata Sekula-Stryjewska

Michal Sarna

Ramaroson Andriantsitohaina
email: ramaroson.andriantsitohaina@inserm.fr.

Ewa K Zuba-Surma
email: ewa.zuba-surma@uj.edu.pl

Anna Łabędź-Masłowska

Luisa Vergori #

Małgorzata Sekuła-Stryjewska

Michał Sarna

Ewa K Zuba

Mesenchymal stem cell-derived extracellular vesicles exert pro-angiogenic and pro-lymphangiogenic effects in ischemic

Keywords: EVs Extracellular vesicles, MSCs Mesenchymal stem/stromal cells, BM Bone marrow MSC-EVs Mesenchymal stem/stromal cell-derived extracellular vesicles, ECs Endothelial cells, MCECs Mouse cardiac endothelial cells, LECs Lymphatic endothelial cells, HLI Hind limb ischemia, WT Wild type, ECM Extracellular matrix, Itgα5 Integrin α5, NRP1 Neuropilin- Angiogenesis, Extracellular vesicles, Ischemic tissue regeneration, Lymphangiogenesis, Mesenchymal stem cells

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Introduction

Ischemic tissue injuries and diseases accompanied with progressing adverse impact on the patients functional condition, became a leading and growing cause of disabilities and deaths worldwide [START_REF] Virani | Heart disease and stroke statistics-2020 update[END_REF]. One of the key aspects in the treatment of such diseases include restoration of blood flow in injured tissues via inducing angiogenesis [START_REF] Bian | Therapeutic angiogenesis using stem cell-derived extracellular vesicles: an emerging approach for treatment of ischemic diseases[END_REF]. Since pharmacological and surgical treatments are insufficient to stimulate vascular remodeling, it has been believed that stem cells (SCs)-based therapies may provide a novel approach for ischemic tissue repair [START_REF] Bian | Therapeutic angiogenesis using stem cell-derived extracellular vesicles: an emerging approach for treatment of ischemic diseases[END_REF]. Mesenchymal stem/stromal cells (MSCs) exhibiting capacity to self-renewal and multilineage differentiation potential, are emerging as promising and safe therapeutic cells for ischemic diseases, which was confirmed in several studies [START_REF] Gnecchi | Mesenchymal stem cell therapy for heart disease[END_REF]. Nowadays, close to 1500 clinical trials has been registered in the ClinicalTrials.gov database as studies using MSCs of various origin in the treatment of several human tissue injuries. The pro-regenerative effects observed following the MSC transplantation may be a result of their: (i) differentiation into mature cells of certain tissue, including into endothelial cells, (ii) immunomodulatory properties, (iii) paracrine activity including the release of pro-angiogenic and cytoprotective agent and (iv) extracellular vesicles (EVs) production [START_REF] Nagata | Cardiac adiposederived stem cells exhibit high differentiation potential to cardiovascular cells in C57BL/6 mice[END_REF][START_REF] Paliwal | Regenerative abilities of mesenchymal stem cells through mitochondrial transfer[END_REF]. Since the retention of transplanted MSCs in injured tissues is relatively low [START_REF] Cortez-Toledo | Enhancing retention of human bone marrow mesenchymal stem cells with prosurvival factors promotes angiogenesis in a mouse model of limb ischemia[END_REF][START_REF] Toma | Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart[END_REF], the paracrine activity and EV release may predominantly drive the structural and functional recovery of injured tissues observed following their administration. These has recently increased interest in cell-free therapies employing EVs, which may carry bioactive compounds produced by their parental cells including SCs such as MSCs [START_REF] Harrell | Molecular mechanisms responsible for therapeutic potential of mesenchymal stem cell-derived secretome[END_REF]. EVs play an important role in cell-to-cell communication ensuring an exchange of bioactive compounds between different cells and affecting their functions [START_REF] Adamiak | Induced pluripotent stem cell (iPSC)-derived extracellular vesicles are safer and more effective for cardiac repair than iPSCs[END_REF]. Growing number of reports indicate that EVs secreted by various types of SCs may modulate functions of other cells including not only endogenous mature cells, but also progenitors and stem cells residing in tissues, thereby improving regenerative processes in injured organs [START_REF] Adamiak | Induced pluripotent stem cell (iPSC)-derived extracellular vesicles are safer and more effective for cardiac repair than iPSCs[END_REF][START_REF] Grange | Stem cellderived extracellular vesicles inhibit and revert fibrosis progression in a mouse model of diabetic nephropathy[END_REF]. Importantly, EVs are smaller than cells, which minimize a risk of blocking of small capillaries and vascular embolism after i.v. injection. It has been shown that bone marrow MSC derived EVs may promote tissue repair by stimulating angiogenesis in vitro and in vivo [START_REF] Wang | Mesenchymal stem cellsderived extracellular vesicles, via miR-210, improve infarcted cardiac function by promotion of angiogenesis[END_REF][START_REF] Gangadaran | Extracellular vesicles from mesenchymal stem cells activates VEGF receptors and accelerates recovery of hindlimb ischemia[END_REF], however the mechanisms of their action are still not fully explored. Importantly, it remains an open question if MSC-EVs may stimulate other proregenerative mechanisms in ischemic tissues such as lymphangiogenesis representing other arm of tissue vasculature development.

Although single studies showing the molecular characteristics of human MSC-EVs at the protein [START_REF] Anderson | Comprehensive proteomic analysis of mesenchymal stem cell exosomes reveals modulation of angiogneesis via NFkB signaling[END_REF] or microRNA level [START_REF] Collino | Exosome and microvesicle-enriched fractions isolated from mesenchymal stem cells by gradient separation showed different molecular signatures and functions on renal tubular epithelial cells[END_REF] have been reported, there are no results presenting an indepth multi-faceted analysis of MSC-EVs strictly correlated with EVs potential biological activity relevant for ischemic tissue repair. Thus, in this study, we meticulously analyzed the molecular composition of murine MSC-EVs in the context of their functional interaction with target cells critical for ischemic tissue recovery such as endothelial cells in vitro. Importantly, we for the first time demonstrate here a crucial role of VEGF-C-Itgα5 and VEGF-C-NRP1 axes in promoting processes of lympangiogenesis in vitro. Moreover, we evaluated the proregenerative potential of systematically/intravenously (i.v) administrated MSCEVs in murine model of ischemic tissue injury such as hindlimb ischemia (HLI) in vivo. We indicated the potential mechanisms of action of MSC-EVs based on their molecular composition and effects observed in tissues at histological and biochemical level after their administration in vivo. Besides angiogenic potential of MSC-EVs, we examined also an impact of MSC-EVs on lymphangiogenesis in vivo, which has never been described during regeneration following MSC-EVs treatment. Since there is a growing interest in developing novel EV-based approaches in the treatment of various tissue injuries (following e.g. stroke, myocardial infarction, burns), the results presented in this study may greatly contribute to this field indicating the future direction for the development of novel pro-angiogenic cell-free medicinal products.

Materials and methods

The extended materials and methods are available in Additional file 1.

Animals

Two strains of 4 to 8 week old mice were used in this study: [START_REF] Virani | Heart disease and stroke statistics-2020 update[END_REF] C57Bl/6 wild-type (WT) strain and (2) transgenic eGFP-expressing mice (C57Bl/6-Tg(GFP)J). All procedures, including the isolation of mouse tissues, were performed at the Jagiellonian University (JU) in Krakow in accordance with the approval of the Ethical Committee for Animal Experiments at the JU (approval number: 183/2012). Tissue regeneration and in vivo biodistribution experiments were performed at the Universite d'Angers in Angers, France, in accordance with the guidelines and authorization of the French Ministry of Agriculture, based on the European Community standards for the care and use of laboratory animals.

Cell culture

Isolation and culture of murine BM-derived MSCs

Murine bone marrow (BM) cells were harvested from tibiae and femurs by rinsing the bone cavities with Dulbecco's modified Eagle's medium/nutrient mixture F-12 ham (DMEM/F12; #D8437, Merck). BM-derived cells were centrifuged (350×g, 6 min, RT), resuspended in complete culture medium-DMEM/F12 supplemented with 10% fetal bovine serum (FBS; #F2442, Merck), 100 U/mL penicillin, and 100 μg/mL streptomycin (P/S; Gibco, ThermoFisher Scientific)-and seeded into tissue culture flasks (BD Falcon, Corning). The rinsed bones were fragmented and enzymatically digested with a mixture of collagenase type I and II (each at a concentration of 1 mg/mL; Merck) for 1.5 h at 37 °C. Collagenases were inactivated by adding DMEM/ F12 supplemented with 10% FBS, and cells were then centrifuged (350×g, 6 min, RT), resuspended in complete culture medium, and transferred to culture flasks containing previously rinsed BM-derived cells. Cells were cultured for 72 h and non-adherent cells were removed. Passaging was performed with 0.25% trypsin/EDTA (Gibco, ThermoFisher Scientific) when cell confluence reached approx. 90%. Cells with the highest viability (> 98%) were used for further experiments including EV harvest.

Culture of MCECs

Mouse cardiac endothelial cells (MCECs) were purchased from CELLutions Biosystems Inc. (#CLU510, Ontario, Canada) and cultured in Dulbecco's modified Eagle's medium (DMEM; #D5796, Merck) supplemented with 10 mmol/L HEPES (HyClone, GE Healthcare Life Sciences) and 5% FBS (#F2442, Merck) on 0.1% gelatin-coated cell culture dishes (BD Falcon, Corning). Cells were passaged with Tryple Select Enzyme 1× (ThermoFisher Scientific) after reaching a confluence of app. 80-90%.

Culture of LECs

Mouse primary dermal lymphatic endothelial cells (LECs) from the C57BL/6 mouse strain were purchased from Cell Biologics (#C57-6064L, Chicago, USA) and cultured in tissue culture flasks precoated with gelatinbased coating solution (#6950, Cell Biologics) in Culture Complete Mouse Endothelial Cell Medium (#M1168, Cell Biologics). Cells were passaged after reaching a confluence of approx. 80%.

Isolation of EVs

Murine MSC-derived extracellular vesicles (MSCEVs) were isolated from cell culture supernatants of BM-derived MSCs (at passages 4, 5, or 6) using a sequential centrifugation method including a double ultracentrifugation step. Briefly, MSCs reaching 80% confluence were washed with PBS (w/o Ca2+, Mg2+, HyClone) and cultured in ultracentrifuged medium DMEM/F12 containing 10% FBS (to remove EVs and other small particles of FBS origin) for 48 h. The collected cell-conditioned media were centrifuged at 350×g for 10 min at 4 °C to remove individual detached cells. The supernatants were transferred to new tubes (Sarstedt) and centrifuged at 500×g for 10 min at 4 °C to remove residual cells and cell debris. The supernatants were then immediately processed or frozen at -80 °C. In the next step, the fresh or frozen/thawed cell-conditioned media were centrifuged at 2000×g for 20 min at 4 °C to remove small cellular debris and apoptotic bodies. Finally, EVs (and large protein/RNA complexes) were collected by ultracentrifugation of the supernatants in PP Oak Ridge tubes (Thermo Fisher) at 100,000×g for 75 min at 4 °C (in a T-865 fixed angle rotor, Thermo Fisher; in an Optima XPB ultracentrifuge, Beckman Coulter), washed once in ultrapure PBS (#BE17-512F, Lonza) and pelleted (centrifuged at 100,000×g for 75 min at 4 °C using the rotor and centrifuge described above). Freshly isolated MSC-EVs were resuspended in PBS (#BE17-512F, Lonza) and transferred to Protein LoBind Tubes (Eppendorf). Protein content of MSC-EV samples was quantified by the Bradford assay.

Particle size analysis

Tunable resistive pulse sensing (TRPS, qNano, Izon) was used to analyze the size distribution of nanoparticles in MSC-EV samples using NP200 nanopore (analytical range 85-500 nm; Izon Science) at 20 or 10 mbar pressure. Samples were analyzed for 5 min or until 1000 particles were counted. Data processing and analysis were performed using Izon Control Suite software v2.2 (Izon Science).

Analysis of EV morphology

To analyze the morphology of MSC-EVs according to the recommendations published by the International Society for Extracellular Vesicles (ISEV) [START_REF] Théry | Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines[END_REF], Atomic Force Microscopy (AFM) technique was used. Images of the topography of the mika surface containing attached MSC-EVs were captured using the PeakForce Tapping (PFT) mode.

Flow cytometric analysis of EV antigenic profile

Surface markers on MSC-EVs were analyzed using Apogee A50-Micro flow cytometer (Apogee Flow Systems, UK). The following fluorochrome-conjugated antibodies against murine antigens were used according to the manufacturer's protocols: CD29-APC (clone: HMβ1-1, Biolegend), CD44-APC (clone: IM7, Biolegend), CD81-APC (clone: Eat2, BD Bioscence), CD90-APC (clone: 30-H12, Biolegend), CD309-APC (clone: Avas12, Biolegend) and Sca-1-APC (clone: E13-161.7, Biolegend) as well as the following isotype controls: Armenian hamster IgG-APC (clone: HTK888, Biolegend), Rat IgG2a, κ-APC (clone: RTK2758, Biolegend) and rat IgG2b, κ-APC (clone: RTK4530, Biolegend). MSC-EVs were co-stained with SYTO RNA Select dye (ThermoFisher Scientific), which binds RNA molecules. Staining was conducted for 30 min in the dark at 4 °C. The obtained results were analyzed using Apogee Histogram software (Apogee Flow Systems). In order to confirm the presence of the indicated antigens on MSCEVs, an ImageStream X Mark II imaging cytometer (Merck) was additionally used.

Western blotting

Western blotting was used to analyze the presence of EV-specific markers in the isolated MSC-EV samples. Briefly, proteins were separated by SDS-PAGE and transferred to PVDF membranes (Merck). The blocked PVDF membranes were immunolabeled with the following antibodies: rabbit anti-mouse CD9 (1:1000, AntibodyGenie), goat anti-mouse calnexin (1:500, ThermoFisher Scientific), rabbit anti-mouse syntenin (1:1000, ThermoFisher Scientific), or mouse anti-mouse β-actin (1:2000, ThermoFisher Scientific) overnight at 4 °C. Horseradish peroxidase-conjugated goat anti-rabbit (1:4000, Dako), rabbit anti-goat (1:4000, ThermoFisher Scientific), or goat anti-mouse (1:4000, ThermoFisher Scientific) antibodies were then incubated with PVDF membranes for 1 h. Product detection was performed using Supersignal West Dura chemiluminescent substrate (Pierce). Imaging of specific bands was performed using a ChemiDoc MP system (Bio-Rad) and Image Lab software (version 5.1, Bio-Rad).

Global proteomic analysis of EV content

Lysed MSC-EV samples were prepared for global proteomic analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using the filter assisted sample preparation (FASP) method described by Wisniewski et al. [START_REF] Wisniewski | Universal sample preparation method for proteome analysis[END_REF]. The obtained peptides were then fractionated according to the protocol also described by Wisniewski et al. [START_REF] Wiśniewski | Proteomic workflow for analysis of archival formalin-fixed and paraffin-embedded clinical samples to a depth of 10000 proteins[END_REF]. The MSC-EV-derived peptides were analyzed by mass spectrometry (MS) using high-resolution Q-Exactive mass spectrometer (Thermo Scientific) coupled to an UltiMate 3000RS LC nanoSystem (Thermo Scientific). In the experiment performed, 3 samples (biological replicates) of MSC-EVs were profiled. The data obtained from LC-MS/MS data were analyzed using Proteome Discoverer 1.4 (Thermo Scientific). Briefly, an in-house MASCOT server (v. 2.5.1, Matrix Science, Boston, MA, USA) was used for searching against the Swissprot_201509 database, which was restricted to the Mus musculus taxonomy. Validation of the search results was performed in Proteome Discoverer 1.4 using the Percolator algorithm according to Kall et al. [START_REF] Käll | Semi-supervised learning for peptide identification from shotgun proteomics datasets[END_REF]. The false discovery rate (FDR) was set below 1%.

The total number of proteins identified was more than 2200. Only proteins identified in at least 2 of the 3 MSC-EV samples, based on 2 or more unique peptides for each protein, were considered for further analysis. For the proteins identified in MSC-EVs, GO enrichment analysis was performed in DAVID Functional Annotation Bioinformatics Microarray Analysis (https:// david. ncifcrf. gov/ home. jsp). A p-value threshold of 0.05 was used in the analysis. In addition, proteins identified in MSCEVs were also analyzed for their association with specific pathways based on GO: Panther Pathways annotation, using FDR below 5% or 1%. In the next step, interaction analysis was performed on the proteins identified in MSC-EVs using the commercially available STRING Ver. 10.5 application (https:// string-db. org/). In addition, the ExoCarta database [START_REF] Keerthikumar | ExoCarta: a web-based compendium of exosomal cargo[END_REF] was used to confirm or exclude the presence of the top 100 proteins frequently identified in small EVs (previously called exosomes).

Analysis of microRNA content in EVs Real-time qRT-PCR profiling was used to determine the microRNA content of MSC-EVs. Briefly, total RNA (including small RNAs) was purified from MSC-EV samples using miRCURY RNA Isolation Kit-Cell & Plant (Exiqon) according to the manufacturer's protocol. Samples were treated with Turbo DNAse (according to the manufacturer's instructions; Ambion, ThermoFisher Scientific) to ensure complete removal of genomic DNA contamination. The purity and RNA concentration were determined using a Nano Photometer (Implen). Only RNA preparations with a concentration greater than 5 ng/μL and a purity of approximately 2.00, as measured by the absorbance ratio A260/280, were used for further analysis. Next, cDNA synthesis was performed using the Universal manufacturer's instructions in a C1000 Touch Thermal Cycler (Bio-Rad). The cDNA products were then diluted and transferred to the Ready-to-use Mouse and Rat microRNA (miRNA) PCR Panel I + II, V3.M (Exiqon) and quantified using real-time qPCR based on Power SYBR Green Master Mix (Applied Biosystems, ThermoFisher Scientific) and locked nucleic acid (LNA)-enhanced miRNA-specific primers. The qPCRs were run on the QS6 Real-Time PCR System (ThermoFisher Scientific) using the reaction parameters recommended by Exiqon. The data were analyzed using GeneEx software (bioMCC, Freising, Germany) and further using mirPath v.3 (DIANA-Lab, University of Thessaly, Greece) dedicated to the assessment of miRNA regulatory roles and the identification of controlled pathways as described by Vlachos et al. [START_REF] Vlachos | DIANA-miRPath v3.0: deciphering microRNA function with experimental support[END_REF]. Three (3) independent samples (biological replicates) of MSC-EVs were analyzed in this study. For the most enriched miRNAs in MSC-EVs (fold change in expression ≥ 2.0 when compared to parental MSCs), the heat-map showing their involvement in functional pathways was generated with DIANA mirPath web tool.

Internalization of EVs by endothelial cells in vitro

Fluorescent labeling of MSC-EVs

To examine MSC-EV uptake by target cells such as vascular endothelial cells in vitro, MSC-EV samples were stained with lipophilic Vibrant DiO Cell-Labeling Solution (ThermoFisher Scientific) and SYTO RNA Select green fluorescent cell stain (ThermoFisher Scientific) according to the manufacturer's protocols to label membrane lipids and RNA within the vesicles, respectively. MSC-EVs were stained for 20 min in the dark at RT and freshly used for experiments.

Analysis of cellular uptake of MSC-EVs

MCEC cells were seeded on glass bottom dishes (O 35 mm, WillCo Wells B.V.) at a density of 5 × 104 cells/dish in complete DMEM medium (#D5796, Merck) supplemented with 5% FBS (#F2442, Merck). After 24 h post seeding, cells were washed with PBS (w/o Ca2+, Mg2+, HyClone) and divided into two groups:

1) Control-further culture of MCECs in complete DMEM medium with 5% FBS for the following 24 h; 2) Microenvironment mimicking inflammation and hypoxia-further culture of MCECs in complete DMEM medium with 1% FBS, 10 ng/mL IL-1β (#211-11B, Peprotech) and 10 ng/mL TNF-α (#315-01, Peprotech) for the following 24 h. Fluorescently-labelled MSC-EVs (50 μg) were then added into each dish and MCECs were further incubated at 37 °C in humidified atmosphere containing 5% CO2 and 21% O2 (Control) or 5% CO2 and 2% O2 (microenvironment of inflammation and hypoxia). Internalization of MSC-EVs by MCEC cells was examined after 2, 4 and 24 h of incubation with EVs by using Leica DMI6000B inverted fluorescence microscope (ver. AF7000, Leica Microsystems) equipped with Leica Application Suite X software (Leica Microsystems). The mean fluorescence intensity of MCECs was measured using ImageJ software (Maryland, USA).

Capillary-like tube formation assay in vitro

Assessment of pro-angiogenic potential of MCECs

To investigate an effect of MSC-EVs on the proangiogenic potential of vascular endothelial cells-MCECs, capillary formation was assessed in vitro. Briefly, 24-well plates (BD Falcon, Corning) were coated with Matrigel Matrix Grow Factor Reduced (100 μL/well, Corning) and incubated at 37 °C for 30 min. MCECs were seeded at a density of 5 × 104 cells/well in the EBM-2 basal medium supplemented with EGM-2MV SingleQuots Kit (both from Clonetics, Lonza). The seeded MCECs were divided into the following four subgroups: 1) Control-further incubation of MCECs in EBM-2 supplemented with EGM-2MV SingleQuots Kit;

2) Microenvironment of inflammation and hypoxia-further incubation of MCECs in EBM-2 supplemented with EGM-2MV SingleQuots Kit, 10 ng/mL IL-1β (#211-11B, Peprotech) and 10 ng/mL TNF-α (#315-01, Peprotech);

3) Control + MSC-EVs-further incubation of MCECs in EBM-2 supplemented with EGM-2MV SingleQuots Kit and 10 μg/mL MSC-EVs; 4) Microenvironment of inflammation and hypoxia + MSC-EVs-further incubation of MCECs in EBM-2 supplemented with EGM-2MV SingleQuots Kit, 10 ng/mL IL-1β (#211-11B, Peprotech), 10 ng/mL TNF-α (#315-01, Peprotech) and 10 μg/mL MSC-EVs.

Capillary-like structure formation by MCECs was examined and recorded at 37 °C in a 5% CO2 chamber (PeCon GmbH, Erbach, Germany) mounted on a Leica DMI6000B inverted microscope equipped with a dry 10×, NA 0.25 objective, integrated modulation contrast optics and a DFC360FX digital CCD camera (Leica Microsystems). The capillary-like tube formation of MCECs was time-lapse recorded for 8 h using Leica Application Suite X software (Leica Microsystems). The total tube length was quantified using the Image J software.

Assessment of pro-lymphangiogenic potential of LECs

The formation of three-dimensional lymphatic capillary-like structures by endothelial cells of lymphatic vessels-LECs treated with MSC-EVs was examined by performing a Matrigelbased tube formation assay. Each well of a μ-slide (ibidi GmbH) was filled with 10 μL of ECMgel (#E1270, Sigma ECMgel, Merck), which was allowed to polymerize for 30 min at 37 °C. The LECs were seeded at a density of 1 × 104 cells/well and cultured in endothelial medium in the presence or absence of 100 ng/mL VEGF C (#SRP4634, Merck). The LECs were treated with MSCEVs and/or recombinant murine IL-1β (10 ng/mL) (#211-11B, Peprotech), recombinant murine TNF-α (10 ng/mL) (#315-01, Peprotech) to induce inflammatory conditions.

In another set of experiments, mouse/rat antineuropilin-1 antibody (#AF566, R&D System) and antiintegrin alpha 5 (CD49e) antibody (#103908, BioLegend) were preincubated with MSC-EVs for 1 h at 4 °C, and then centrifuged to remove excess of blocking antibody, before being added to the cells. A positive control was performed with 20 ng/mL PMA (#P8139, Merck). After 18 h of incubation, the tube formation images were captured at fourfold magnification using the Olympus CK40 (Olympus CK40, Olympus Corporation). The total tube length and number of tubes were quantified using the Image J software.

Expression of EC activation markers in vitro

To investigate the level of expression of markers of activated vascular endothelial cells under different conditions, immunofluorescence staining and flow cytometry analysis of cells were performed.

Briefly, MCECs were seeded at a density of 10 5 cells/dish (O 60 mm, Falcon, Corning) in complete DMEM medium (#D5796, Merck) supplemented with 5% FBS (#F2442, Merck). Cells were washed with PBS (w/o Ca2+, Mg2+, HyClone) after 24 h and divided into two groups:

1) Control-further culture of MCECs in complete DMEM with 5% FBS for the following 24 h; 2) Microenvironment mimicking inflammation and hypoxia-further culture of MCECs in DMEM with 1% FBS, 10 ng/mL IL-1β (#211-11B, Peprotech) and 10 ng/mL TNF-α (#315-01, Peprotech) for the following 24 h.

The following factors were then added to MCECs incubated in the inflammatory and hypoxic microenvironment: 10 ng/mL IL-1β, 10 ng/mL TNF-α or 10 ng/mL IL-1β, 10 ng/mL TNF-α and 10 μg/mL MSC-EVs. Cell culture medium was removed after 4 or 24 h of incubation and MCECs were washed with PBS (w/o Ca2+, Mg2+, HyClone), and passaged. Cells were further centrifuged (300×g, 5 min, RT), resuspended in staining medium (DMEM supplemented with 1% FBS) and stained with the following fluorochrome-conjugated antimouse antibodies: CD54-APC (clone: YN1/1.7.4, Biolegend), CD62E-PE (clone: 10E9.6, BD Bioscence), CD62P-Alx647 (clone: RB40.34, BD Bioscence), CD106-APC (clone: 429, Biolegend) for 30 min in the dark at 4 °C. Samples were then washed with PBS (w/o Ca2+, Mg2+, HyClone), centrifuged (300×g, 6 min, RT) and resuspended in staining medium. DAPI (3 μM, ThermoFisher Scientific) was added 10 min prior to analysis to exclude dead cells from further analysis. Cells were analyzed using LSR Fortessa flow cytometer (Becton Dickinson).

Murine model of hind limb ischemia (HLI) in vivo

To induce unilateral hind limb ischemia (HLI), the ligation of the left femoral artery (FA) was performed according to the procedure described by Niiyama et al. established at the Stanford University, CA, USA [START_REF] Niiyama | Murine model of hindlimb ischemia[END_REF]. In the male C57BL/6 mice (Charles River Laboratories, L'Arbresle, France) at the aged of 6 to 8 weeks, the distal and proximal ends of the FA were occluded with 6-0 silk suture (Ethicon). The segment of FA between the distal and proximal knots were carefully transected. The wound was then closed and dressed. Mice were randomly divided into two subgroups (N = 8-9/group): (1) vehicle (PBS, control group) and (2) MSC-EVs (EV-treated group). Respectively, PBS or MSC-EVs (10 μg/mL of blood), respectively, were administered intravenously through the tail vein every 3 days for 21 days of the experiment.

The total blood volume for each mouse was calculated based on the consideration that the total blood volume in the mouse is in the range of 6-8 mL/100 g of body weight [START_REF] Hoff | Methods of blood collection in the mouse[END_REF] as shown in the Additional file 1: Table S1. At 7, 14 and 21 days after FA occlusion, blood flow was measured in both limbs as described below. At 21 days after HLI, animals were sacrificed and muscle tissues were harvested for further biochemical and histological analysis. The in vivo experimental design is presented in Additional file 1: Fig. S1.

Analysis of blood flow in tissues in vivo

To provide functional evidence of ischemia and further recovery of blood perfusion, laser Doppler perfusion imaging technique based on dynamic light scattering in tissue was used [START_REF] Couffinhal | Mouse model of angiogenesis[END_REF]. Briefly, mice were anesthetized with Aerrane Isoflurane (Baxter) and placed on a heating plate to maintain a stable cutaneous temperature in order to minimize temperature variation throughout the experiments. Limb perfusion was measured using a laser Doppler flow probe (PF 408, Perimed). A minimum of 3 flow measurements were performed per mouse. Limb blood flow was expressed as the ratio of left (ischemic) to right (non-ischemic) limb perfusion, as described by Limbourg et al. [START_REF] Limbourg | Evaluation of postnatal arteriogenesis and angiogenesis in a mouse model of hind-limb ischemia[END_REF].

Analysis of EV biodistribution in vivo

To investigate the retention of MSC-EVs in different organs after i.v. administration, animal tissues were analyzed using an in vivo fluorescence imaging system. Briefly, male C57BL/6 mice were injected with 100 μg GFP-positive MSC-EVs or PBS via the tail vein 24 h after HLI. Mice were sacrificed 4 or 24 h after injection of MSC-EVs or PBS. Blood, liver, spleen, lung, kidney, and limb were then isolated. Multispectral imaging system MAESTRO In-Vivo Fluorescence Imaging System (Cambridge Research & Instrumentation; Woburn, MA, USA) was used to analyze the biodistribution of GFP-positive MSC-EVs. For plasma isolation, blood was centrifuged at 350×g, 7 min, RT and transferred to a new 1.5 mL tube. The respective fluorescence of isolated organs, plasma and limbs was analyzed using the MAESTRO In-Vivo Fluorescence Imaging System software (Cambridge Research & Instrumentation; Woburn, MA USA).

Immunofluorescence staining and confocal microscopy

To prepare frozen sections for semiquantitative analysis of neovascularization in MSC-EVtreated tissues, both ischemic and non-ischemic gastrocnemius muscles were dissected and embedded in Tissue-Tek O.C.T compound (Sakura Finetek), frozen in liquid N2 and stored at -80 °C until further processing. Frozen tissues were sectioned using Leica CM3050S cryostat (Leica Biosystems). Subsequently, cryosections (7 μm) were fixed in 100% methanol (Merck) for 5 min at -20 °C, and non-specific binding sites were saturated with blocking buffer containing 5% BSA (Euromedx) in PBS for 1.5 h at RT. Tissue sections were then incubated with rat anti-mouse CD31 antibody (marker of blood vessel endothelial cells of s; 1:100, BD Biosciences) and rabbit anti-mouse Lyve1 antibody (marker of lymphatic vessel endothelial cells; 1:200, Acris Antibodies) overnight at 4 °C. The sections were then washed tree times with PBS and incubated (for 1 h at RT) with goat anti-rat FITC-conjugated IgG (1:100, Southern Biotech) to identify blood vessels or goat anti-rabbit Alexa546 (1:500, Interchim) secondary antibody to detect Lyve1 expression. To visualize nuclei, tissue sections were stained with DAPI (300 nM, Santa Cruz Biotechology) for 3 min at RT. After final washes, the coverslips were mounted. LSM 700 confocal (Zeiss) was used for the optical sectioning of the tissue sections. Digital imaging was performed using the Laser Sharp software. Vessels were quantified using ImageJ software and counted in at least five randomly selected fields for each muscle section, and the mean value for each section was calculated (magnification 40×).

Analysis of NO and O2 -determination production in tissues

Detection of nitric oxide (NO) and superoxide anion ( O2-) production in mouse tissues was performed using electronic paramagnetic resonance (EPR) with Fe2+ diethyldithiocarbamate (DETC) as a spin trap. At 21 days after FA ligation, animals were euthanized, and aortas and both ischemic and nonischemic muscles were dissected and incubated in Krebs-HEPES buffer containing Fe(DETC)2 solution for analysis of NO production or deferoxamine, DETC and CMH solution for analysis of O2production for 45 min at 37 °C.

The tissues for NO and O2production analysis were then immediately frozen in liquid N2. Measurement of NO and O2production was performed using a benchtop x-band spectrometer Miniscope (Magnettech, MS200, Berlin, Germany). Recordings were made at 77 °K using a Dewar flask. The instrument setting was as follow: 10 mW of microwave power, 1 mT of amplitude modulation frequency, 60 s of sweep time and 5 scans.

Analysis of angiogenesis-related proteins in tissues

In each experiment, the aorta and the skeletal muscle from both ischemic and nonischemic limbs were removed and frozen in liquid N2 to further evaluate the expression of angiogenesis-related proteins after MSC-EV treatment. Briefly, homogenized tissue samples were separated by SDS-PAGE and transferred onto PVDF membranes (Merck). The blocked PVDF membranes were incubated overnight at 4 °C with one of the following primary monoclonal antibodies: anti-endothelial NOS (eNOS; 1:2500, BD Biosciences), anticaveolin-1 (1000, BD Biosciences), anti-phospho-eNOS (Ser 1177, 1:1000, Cell Signaling Technology), anti-Akt (1:1000, Cell Signaling Technology), anti-phospho-Akt (Ser 473, 1:2000, Cell Signaling Technology), and anti-VEGF (1:1000, R&D Systems). A rabbit polyclonal anti-actin antibody (1:5000, Merck) was used to visualize protein loading on the gel. The membranes were then washed at least three times in Tris buffer containing 0.05% Tween-20 and incubated for 1 h at RT with the appropriate horseradish peroxidase (HRP)conjugated secondary antibody (Amersham, Piscataway, NJ). The protein-antibody complexes were detected by ECL-Plus Chemiluminescence kit (Santa Cruz Biotechnology, Santa Cruz, CA) according to manufacturer's protocol using Image Reader Las-3000 (Fujifilm).

Statistical analysis

Data are presented as mean ± SD or SEM as indicated. Statistical analyses were performed by Student's t-test or Mann-Whitney U-test (nonparametric) using Prism software package 8.00 or 5.00 (GraphPAD Software, San Diego, CA). P < 0.05 was considered to be statistically significant.

Results

MSC-EVs carry proteins and miRNAs that regulate pro-regenerative processes in injured tissues

Following ISEV recommendations for EV characterization, we found that our isolates of murine bone marrow MSC-EVs represent heterogeneous populations of particles with size ranged between 70 and 430 nm and the mean diameter of 172.3 ± 14.4 nm (Fig. 1A,B). Analysis of basic protein content in our MSC-EV samples showed that they contained subpopulations of vesicles carrying parental cell specific antigens (e.g. CD29, CD44, CD90 and Sca-1) and tetraspanins (e.g. CD9, CD63 and CD81) (Fig. 1C-E, Additional file 1: Table S2) representing 1 st category of proteins occurring in all EVs [START_REF] Théry | Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines[END_REF]. MSC-EVs contained also cytosolic proteins such as syntenin, annexins, actin, tubulin (Fig. 1C, Additional file 1: Table S2) representing 2nd category of proteins occurring in all EVs [START_REF] Théry | Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines[END_REF]. Interestingly, calnexin being major component of non-EV co-isolated structures [START_REF] Théry | Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines[END_REF] was not detected in EV specimens (Fig. 1C), whereas our MSC-EV population were enriched in small EVs (resembling mostly exosomes) as shown by the presence of 82 out of the top 100 most identified small EV marker proteins published in ExoCarta database (Additional file 1: Table S2) as well as 14 out of the 15 hallmark proteins identified by different investigators in MSC-EVs with smaller size (≤ 200 nm) (Additional file 1: Table S3). Subpopulation of MSC-EVs also carried VEGF receptor 2 (CD309) on their surfaces (Fig. 1D,E).

Extending the studies on molecular composition of MSC-EVs, we conducted further global proteomic analysis of MSC-EV content by mass spectrometry followed by bioinformatics analysis of the identified proteins. We identified more than 2200 proteins to be carried by MSC-EVs and only proteins identified in at least 2 out of the 3 MSC-EV samples based on 2 or more unique peptides were considered for further analyses.

With regard to biological pathways, the analysis conducted by Gene Ontology (GO) indicated the presence in MSC-EVs of multitude proteins involved in several signaling pathways including the ones known to play pro-regenerative role such as e.g. involved in regulation of inflammation, cell response after EGF, FGF, VEGF or PDGF binding, as well as angiogenesis (Fig. 2A). The analysis of a role of the identified proteins in biological processes (conducted by DAVID Functional Annotation Bioinformatics Microarray Analysis tool) revealed enrichment of MSC-EVs in proteins involved in or regulating e.g., metabolic processes such as glycolysis, protein catabolic processes, receptor-mediated endocytosis, cell to-matrix and cell-to-cell adhesion, cell migration as well as pro-regenerative processes such as wound healing, angiogenesis and their positive regulation (Fig. 2B).

With reference to molecular function, MSC-EVs carried a wide range of proteins essential for interactions and binding several other proteins, ions, DNA, RNA, ECM or cell adhesion molecules (Fig. 3A). The proteins identified in MSC-EV samples and indicated by DAVID Functional Annotation Bioinformatics Microarray Analysis tool as involved in several cellular processes as well as those involved in cell adhesion have also been grouped in the STRING database (Search Tool for the Retrieval of Interacting Genes/Proteins). These analyses revealed a clear network of connections and interaction between these proteins (Additional file 1: Fig. S2) indicating their functional importance for MSC-EV activity. Moreover, extensive interaction network was also detected between proteins involved in cell adhesion, angiogenesis and wound healing (Additional file 1: Fig. S3). Among these proteins, we identified integrin α5 (Itgα5) involved in inflammatory lymphangiogenesis [START_REF] Dietrich | Inhibition of inflammatory lymphangiogenesis by integrin α5 blockade[END_REF] and neuropilin-1 (NRP1) which acts as a key modulator of vascular, lymphatic, and inflammatory cell responses [START_REF] Saban | Neuropilin-VEGF signaling pathway acts as a key modulator of vascular, lymphatic, and inflammatory cell responses of the bladder to intravesical BCG treatment[END_REF]. Taken together, the in-depth proteomic analysis revealed that MSC-EVs carry proteins that play a significant role in the regulation of multiple cellular processes including those associated with pro-regenerative events in injured tissues.

We further characterized protein contents of MSC-EVs with reference to the interactions which affect functions of recipient cells by mechanisms involving focal adhesion as well as extracellular matrix (ECM) interactions. Indeed, we found MSC-EVs carried 79 cell adhesion proteins (e.g., integrins, CD44 antigen) as well as miRNAs involved in ECM-receptor interaction (Additional file 1: Table S4, Fig. 2; respectively) suggesting their possible interaction with target cells including inflamed endothelial cells. Moreover, MSC-EVs contained proteins of signaling pathways linked to focal adhesion that participate in modulation of cell motility, proliferation and survival (Additional file 1: Fig. S4, Table S5). Finally, we also identified MSC-EV proteins involved in ECMreceptor interactions (Additional file 1: Fig. S5).

Global miRNA profiling by qRT-PCR showed several miRNAs to be present in MSC-EVs corresponding to the parental MSC content, as reported previously [START_REF] Guduric-Fuchs | Selective extracellular vesicle-mediated export of an overlapping set of microRNAs from multiple cell types[END_REF]. Interestingly, ten [START_REF] Grange | Stem cellderived extracellular vesicles inhibit and revert fibrosis progression in a mouse model of diabetic nephropathy[END_REF] miRNAs were detected in MSC-EVs only, but they were not detected in MSCs (Additional file 1: Fig. S6) that may suggest that they are under detection limit in parental cells. The analysis of miRNA types enriched in MSC-EVs (with fold change ≥ 2 compared to MSCs; conducted by miRPath tool) revealed their possible involvement in TGF-β signaling (e.g. miR-276a-3p, 761, 369-3p, 344d-3p), signaling regulating pluripotency of stem cells (miR-344d-3p, 187-3p, 376a-3p) or ECM-receptor interactions (miR-325-3p, 202-3p) (Fig. 3B). Moreover, miRNA-382-5p, 132-3p and 126-3p that contributed to pro-regenerative processes [START_REF] Seok | MicroRNA-382 induced by HIF-1α is an angiogenic miR targeting the tumor suppressor phosphatase and tensin homolog[END_REF][START_REF] Liu | The role of miRNA-132 against apoptosis and oxidative stress in heart failure[END_REF][START_REF] Chen | Mesenchymal stem cells overexpressing MiR-126 enhance ischemic angiogenesis via the AKT/ERK-related pathway[END_REF] were also detected in MSC-EVs (Additional file 1: Fig. S6).

Together, these data show that MSC-EVs are enriched in MSC-derived bioactive molecules including proteins and miRNA that may regulate phenotypic change and function of other target cells including endothelial and immune cells under normal or inflamed state.

MSC-EVs increase angiogenic potential and viability of ECs under normal and ischemic tissue conditions

In our in vitro studies, we found that under conditions mimicking ischemic tissue (where proinflammatory factors and hypoxic conditions were present), the internalization of MSC-EVs by mouse vascular endothelial cells-MCECs was significantly higher when compared those obtained under control conditions, which was especially observed shortly after incubation (after 4 h) (Fig. 4A). Interestingly, in the same time, no significant change in the expression of selected adhesion molecules such as e.g. CD62E or CD62P, was observed on the surface of endothelial cells in normal and tissue injury mimicking conditions, but the density of CD54 and especially CD106 molecule (interacting with β1 integrin containing ligand) were significantly increased on the MSC-EVs (Fig. 4B) and induced a time-dependent increase of capillary formation by endothelial cells in both control and ischemic tissue injury-mimicking conditions (Fig. 4C). Interestingly, no impact of pro-inflammatory factors on angiogenic potential of MCEC was observed when compared to control (Fig. 4C). However, we found that tissue injured conditions mimicked with inflammatory cytokines, IL-1β and TNF-α significantly reduced endothelial cell viability by increasing the late phase of apoptosis, which was reversed when MSC-EVs were added to the cells (Fig. 4D). Thus, we found that under adverse conditions of injured and inflamed tissue, the internalization of MSCEVs by endothelial cells was significantly enhanced allowing the molecular transfer of the EV cargo and MSC-EVs increased angiogenic activity of these cells and their survival by inhibiting apoptosis (Fig. 4A-D). The enhanced EV internalization in such conditions may be dependent on the interaction of CD106 (VCAM-1) molecule with β1 integrin (CD29) representing common MSC marker, also present on fraction of MSC-EVs (Fig. 1D).

MSC-EVs increase capillary-forming capacity of lymphatic ECs via VEGF3 signaling

As reported above, global proteomic analysis revealed that MSC-EVs carry proteins such as Itgα5 involved in inflammatory lymphangiogenesis and neuropilin-1 (NRP1), which act as a key modulator of vascular, lymphatic, and inflammatory cell responses [START_REF] Dietrich | Inhibition of inflammatory lymphangiogenesis by integrin α5 blockade[END_REF][START_REF] Saban | Neuropilin-VEGF signaling pathway acts as a key modulator of vascular, lymphatic, and inflammatory cell responses of the bladder to intravesical BCG treatment[END_REF].

We found that under normal conditions, MSC-EVs increased capillary length of lymphatic endothelial cells (LEC) and this in absence or in presence of VEGFC (Fig. 5). Interestingly, neutralizing the interaction with blocking specific antibody of either Itgα5 (MSCEVsItgα5-) or NRP1 (MSC-EVsNRP1-) alone or in combination (MSC-EVsItgα5-/NRP1-) prevented the ability of MSC-EVs to increase the capillary length structure both in absence and in presence of VEGF-C (Fig. 5).

In the absence of VEGF-C and in inflammatory conditions, none of the MSC-EVs had an effect on capillary length of LEC. However, in the presence of VEGF-C, inflammatory conditions significantly increased total tube length whereas MSC-EVs were not able to induce a further increase in capillary length (Fig. 5B,D). Blockade either of Itgα5 or NRP1 alone, using MSC-EVsItgα5-or MSC-EVsNRP1-significantly reduced the ability of inflammatory conditions to increase LEC tube formation in the presence of VEGF-C (Fig. 5B,D). Interestingly, concomitant blockade of Itgα5 or NRP1 with MSC-EVsItgα5-/ NRP1completely abrogated the effect of inflammatory conditions in presence of VEGF-C (Fig. 5B,D).

Collectively, we demonstrate for the first time a mandatory role of VEGF3 signaling and Itgα5 and NRP1 carried by MSC-EVs in regulating lymphangiogenesis depending on lymphatic ECs.

MSC-EVs exhibit retention into ischemic tissues in vivo

Next, the in vivo biodistribution study of MSC-EVs were conducted following i.v. injection, in order to verify whether the EVs exhibit tropism into the site of ischemic injury. For such purpose, C57Bl/6J mice underwent hindlimb ischemia injury and were i.v. injected with GFP positive MSC-EVs (100 μg) to investigate biodistribution of MSC-EVs in the plasma and selected organs at 4 or 24 h post administration. At 4 h, MSC-EVs were mostly distributed to the kidneys with lower level in the liver, spleen and lungs (Additional file 1: Fig. S7A,B). Twenty four hours' post-injection, the lower fluorescence signal was detected in liver, kidneys and lungs when compared to the level observed at 4 h post-injection. Importantly, we found increased localization of MSC-EVs in ischemic limbs at 24 h post-injection, when compared to control untreated limb, indicating enhanced retention of the EVs in injured inflamed and hypoxic tissues (Fig. 6A).

MSC-EVs enhance recovery of blood flow and vasculature in ischemic tissues in vivo

To evaluate pro-regenerative potential of MSC-EVs in murine model of HLI, mice underwent hindlimb ischemia injury by ligating-excising the left FA and were further i.v. injected either with MSC-EVs or vehicle (PBS) as a control every 3 days up to 21 days (Additional file 1: Fig. S1). Tissue blood perfusion was measured before FA occlusion (baseline) as well as at day 7, 14 or 21 post HLI, by laser Doppler perfusion imaging technique (Fig. 6B). Importantly, we found significant improvement in blood tissue perfusion in ischemic limb following MSC-EV administration at day 7, 14 and 21 after HLI injury, when compared to the vehicle-treated mice (Fig. 6B). We found also greater number of capillaries in ischemic skeletal muscle tissue harvested form animals treated with MSCEVs when compared to the vehicle-treated mice, as indicated by immunohistochemical staining against the endothelial cell marker CD31 (Fig. 6C).

Taken together, these results demonstrate pro-angiogenic potential of MSC-EVs in ischemic tissue in vivo, which was associated with increased capillary densities leading to improve blood tissue perfusion in vivo.

MSC-EVs enhance NO production and endothelial NO synthase (eNOS) activation in ischemic limbs

The production of reactive oxygen species (O2-) and nitric oxide (NO) in skeletal muscle following the HLI and MSC-EV administration were analyzed by EPR technique. We found significantly enhanced production of NO, without modification of O2-, in the ischemic skeletal muscles in MSC-EV-treated mice at 21 days post HLI, when compared to vehicletreated mice (Fig. 7A,B).

Western blotting analysis revealed that MSC-EV treatment enhanced phosphorylation of NO producing enzyme: eNOS on its activator site (Ser 1177) and decreased caveolin-1 expression showing that increased NO production was due to the activation of eNOS pathway. In contrast, neither expression of VEGF and Akt nor activation of Akt was influenced by MSC-EV treatment. In comparison, in non-ischemic tissues such as aorta we did not observe any increase in NO production or eNOS and VEGF expressions. The level of phosphorylated form of Akt was lower after MSC-EV treatment when compared to vehicle-treated group (Additional file 1: Fig. S8).

Taken together, MSC-EV treatment stimulates NO production in ischemic tissues, which may be involved in NO-mediated angiogenesis in injured tissues.

MSC-EVs promote lymphangiogenesis in ischemic tissues in vivo

Immunohistochemical staining of skeletal muscle tissue sections for the endothelial cells (CD31) and lymphatic endothelial (Lyve) markers were analyzed by confocal microscopy. Importantly, we observed increased number of not only CD31, but also Lyve1 expressing cells in ischemic muscle sections following MSC-EV treatment, when compared to vehicletreated animals (Fig. 8). As shown in Fig. 8A, many lymphatic and blood vessels coexisted in intimate contact, indeed no significant overlap was noted between Lyve1 and CD31 staining. These data demonstrate that MSC-EVs not only induce angiogenesis, but also lymphangiogenesis in injured tissues in vivo.

Discussion

In the present study, we have described the proangiogenic and pro-lymphangiogenic roles of MSCEVs on ECs under ischemic conditions, which may play their role via the transfer of bioactive cargo consisting of various microRNAs and proteins including Itgα5 and NRP1. Although MSCs secrete various soluble active factors [START_REF] Alvites | Mesenchymal stem/stromal cells and their paracrine activity-immunomodulation mechanisms and how to influence the therapeutic potential[END_REF], we focused exclusively on MSC-EVs that were isolated using a sequential centrifugation method including double ultracentrifugation step.

Importantly for the functional effects of MSC-EVs in tissue repair, our proteomic data revealed the presence in MSC-EVs of proteins involved in many biological pathways including those involved in tissue repair and angiogenesis. We found that MSC-EVs were enriched in proteins involved the regulation of several metabolic processes including glycolysis, cellular protein catabolism, receptor-mediated endocytosis, cell-matrix and cellcell adhesion, cell migration as well as pro-regenerative processes such as wound healing, angiogenesis and their positive regulation. Among such proteins, we identified Itgα5, which is known to be involved in inflammatory lymphangiogenesis [START_REF] Dietrich | Inhibition of inflammatory lymphangiogenesis by integrin α5 blockade[END_REF] and NRP1, which acts as a key modulator of vascular, lymphatic, and inflammatory cell responses [START_REF] Saban | Neuropilin-VEGF signaling pathway acts as a key modulator of vascular, lymphatic, and inflammatory cell responses of the bladder to intravesical BCG treatment[END_REF]. Furthermore, microRNA profiling showed that MSC-EVs are enriched in miRNAs involved in TGF-β signaling, regulation of stem cells pluripotency, focal adhesion signaling and ECM-receptor interactions.

Thus, we found that MSC-EVs are enriched in MSC derived bioactive molecules such as proteins and miRNA that can regulate phenotype, function and homing of target cells including endothelial and immune cells under normal or inflamed state. Indeed, we found that MSC-EVs enhanced angiogenic properties and viability of capillary ECs under normal and inflammatory/ischemic conditions via the inhibition of the late phase of apoptosis. Importantly, we report here for the first time that MSC-EVs enhanced capillaryformation capacity of lymphatic ECs via regulation of the VEGFR3 signaling pathway with a mandatory role of Itgα5 and NRP1 under normal and "tissue injured" conditions. Thus, in our multiple in vitro assays, we demonstrated that MSC-EVs induced both angiogenesis and lymphangiogenesis, which are essential for the improvement of organ function after injury. Importantly, in mouse model of ischemic hind limb injury, we found greater recovery of blood flow in MSC-EVs-treated mice when compared to controls, which was associated with an increase in NO production in ischemic muscles. Indeed, EVs increased eNOS phosphorylation and decreased caveolin-1 expression in ischemic muscles.

Bio-distribution analysis of EVs revealed increased staining of MSC-EVs-GFP in the ischemic hind limb compared to non-ischemic muscles. Interestingly, dual CD31 and Lyve1 immunostaining in muscle cryosections, indicating angiogenesis and lymphangiogenesis, respectively, showed that MSC-EVs enhanced both processes in ischemic tissues in vivo. Thus, the present study provides repertoire of MSCEV proteins and miRNA bioactive factors to meet therapeutic needs to rescue organ ischemic injury via the enhancement of both angiogenesis and lymphangiogenesis processes. These findings also highlight Itgα5 and NRP1 carried by MSC-EVs as potential targets to promote lymphangiogenesis.

The functional effects of EVs depend on several factors including their origin and harvesting. The method used in this study to isolate MSC-EVs by differential centrifugation was performed in accordance with the recommendations of the MISEV position and allowed us to isolate mixed population of small and medium/large EVs from MSCs [START_REF] Théry | Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines[END_REF]. Deun et al. confirmed relatively high efficiency of EV isolation by differential centrifugation, which correlated with relatively low contamination with other proteins [START_REF] Van Deun | The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling[END_REF]. Furthermore, the isolation was performed using MSCs-derived conditioned media free of exogenous serumderived EVs in accordance with the approach recommended by many investigators [START_REF] Urzì | The dark side of foetal bovine serum in extracellular vesicle studies[END_REF][START_REF] Shelke | Importance of exosome depletion protocols to eliminate functional and RNA-containing extracellular vesicles from fetal bovine serum[END_REF]. Thus, MSC-EVs obtained under these experimental conditions allow in depth analysis the content and biological potential a whole population of BM-derived MSC-EVs with a good assessment of vesicle purity [START_REF] Urzì | The dark side of foetal bovine serum in extracellular vesicle studies[END_REF][START_REF] Shelke | Importance of exosome depletion protocols to eliminate functional and RNA-containing extracellular vesicles from fetal bovine serum[END_REF]. The size distribution of murine MSC-EVs ranged from 70 to 430 nm and their antigenic profile are similar to that of human umbilical cord-derived MSC-EVs [START_REF] Bobis-Wozowicz | Diverse impact of xeno-free conditions on biological and regenerative properties of hUC-MSCs and their extracellular vesicles[END_REF]. Using the Apogee A50 system followed by image flow cytometry, we confirmed expression of parental cell-specific antigens (CD29, CD44, CD90 and Sca-1) and tetraspanin (CD81), representing 1st category of proteins present in all EVs [START_REF] Théry | Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines[END_REF] as well as receptor 2 for VEGF (CD309) on their surface. Western blotting revealed expression of syntenin (representing 2nd category of proteins present in all EVs [START_REF] Théry | Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines[END_REF]) and lack of calnexin. Subsquently, high through put global proteomic analysis revealed presence of 82 proteins from the 100 most identified small EV markers published in ExoCarta database [START_REF] Mathivanan | ExoCarta 2012: database of exosomal proteins, RNA and lipids[END_REF], confirming the enrichment in small EVs as well as the presence of other 1st and 2nd category proteins present in all EVs [START_REF] Théry | Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines[END_REF]. Balkom et al. compared proteomic profile of MSC-EVs (≤ 200 nm) published by 10 research groups and defined the following MSC-EVs hallmark proteins: COL1A1, COL1A2, LRP1, ACTN1, ALDOC, ANPEP, COL6A1, COL6A2, COL6A3, FLNB, FLNC, HBB, Thy1, VAT1, VIM [START_REF] Van Balkom | Proteomic signature of mesenchymal stromal cell-derived small extracellular vesicles[END_REF]. Interestingly, 14 of the 15 proteins mentioned above were detected in MSC-EV samples obtained during current study.

Importantly, bioinformatic analysis of LC-MS/MS data and microarray analysis underscored that MSCEVs contain a repertoire of proteins involved in wide range of signaling pathways involved in pro-regenerative processes such as angiogenesis. In addition, MSC-EVs were enriched with proteins involved in several biological processes, including glycolysis, receptor-mediated endocytosis, cell-matrix and cell-cell adhesion, cell migration and wound healing. Interestingly, a robust association of network proteins involved in both focal adhesion and ECM interactions was established. Taken together, in-depth proteomic analysis revealed that MSCEVs carry proteins with significant roles in the regulation of multiple cellular processes including those associated with pro-regenerative events. Global miRNA profiling highlighted the enrichment of MSC-EVs in 10 miRNAs involved in TGF-β signaling, Hippo signaling, signaling regulating stem cell pluripotency or ECM-receptor interaction. For example, miRNA-382-5p upregulation is associated with increased proliferation, migration and tube formation of vascular endothelial cells [START_REF] Seok | MicroRNA-382 induced by HIF-1α is an angiogenic miR targeting the tumor suppressor phosphatase and tensin homolog[END_REF], miRNA-132-3p overexpression is associated with increased antioxidant stress and anti-apoptotic ability of cardiomyoblast cells [START_REF] Liu | The role of miRNA-132 against apoptosis and oxidative stress in heart failure[END_REF], whereas transplantation of miRNA-126-overexpressing MSCs improves angiogenesis in the infarcted area and cardiac function of mice [START_REF] Chen | Mesenchymal stem cells overexpressing MiR-126 enhance ischemic angiogenesis via the AKT/ERK-related pathway[END_REF]. Together, these data show that MSC-EVs are enriched with bioactive molecules (proteins, miRNA) that regulate phenotype, function and homing of target cells including endothelial cells.

The formation of new blood capillaries is an important component of pathological tissue repair in response to ischemia. The development of an effective collateralization in the ischemic zone involves angiogenesis, which is the sprouting of new capillaries, and arteriogenesis, the development of arterial structures from small preexisting collateral vessels. In addition, the lymphatic vasculature is essential for maintaining tissue fluid homeostasis, immune cell trafficking and nutritional lipid uptake. Therapeutic stimulation of cardiac lymphangiogenesis after myocardial infarction results in accelerated resolution of myocardial edema and inflammation, thereby promoting cardiac recovery [START_REF] Brakenhielm | Cardiac lymphatics in health and disease[END_REF]. We found that MSC-EVs increased capillary-like tube formation and protected the viability of MCECs under normal and inflammatory conditions via the inhibition of the late phase of apoptosis.

Importantly, we also found that MSC-EVs enhanced the capillary formation capacity of lymphatic ECs in normal or "injured tissue environment". Interestingly, neutralizing the interaction of Itgα5 and NRP1 using blocking antibodies prevented the ability of MSC-EVs to increase capillary-like structures. Thus, the interaction of Itgα5 and NRP1 carried by MSC-EVs with a cell-surface receptor and/or their internalization by LECs is essential for lymphangiogenesis. Both Itgα5 and Nrp1 participate in VEGFR-3-mediated cell signaling independent of VEGFC stimulation. Indeed, the VEGFR3/VEGFR2/ NRP1 complex is known to be involved in VEGFR3 signaling to the lymphatic endothelium by controlling AKT activation [START_REF] Deng | Molecular controls of lymphatic VEGFR3 signaling[END_REF]. Likewise, VEGFR-3 selectively associates with integrin Itgα5 to promote the growth of LECs through the activation of phosphatidylinositol 3 kinase/ Akt signaling pathway [START_REF] Zhang | Extracellular matrix regulates endothelial functions through interaction of VEGFR-3 and integrin α5β1[END_REF]. Thus, we report an obligatory role of Itgα5 and NRP1 carried by MCS-EVs in stimulating lymphangiogenesis under normal and injured tissue-mimicking conditions as a potential mechanism for tissue repair. Future studies using knockout of the indicated factors would be interesting to further evaluate this phenomenon.

Finally, we provide in vivo evidence that MSC-EV treatment induces an increased homing of these EVs to ischemic tissues and stimulates blood flow recovery in ischemic limbs after femoral artery ligation. Intravenously administered MSC-EVs also showed retention in liver, kidneys, lung and spleen which is consistent with observations made by other investigators [START_REF] Kang | Biodistribution of extracellular vesicles following administration into animals: a systematic review[END_REF]. EVs remain in the circulation for a short time [START_REF] Kang | Biodistribution of extracellular vesicles following administration into animals: a systematic review[END_REF] and their blood concentration is determined by a balance between abundant secretion and rapid clearance [START_REF] Matsumoto | Blood concentrations of small extracellular vesicles are determined by a balance between abundant secretion and rapid clearance[END_REF]. The observed potential of MSC-EVs for retention in injured tissues may create new applications for cellfree drugs and, importantly this phenomenon may contribute to an efficient therapeutic outcome [START_REF] Shi | Preclinical efficacy and clinical safety of clinical-grade nebulized allogenic adipose mesenchymal stromal cells-derived extracellular vesicles[END_REF]. Interestingly, other stem cell-derived EVs have been shown to accumulate at sites of injury and confer substantial protection [START_REF] Han | Highly efficient magnetic labelling allows MRI tracking of the homing of stem cell-derived extracellular vesicles following systemic delivery[END_REF], similarly to the observations in the current study.

We found that MSC-EV treatment promoted the production of NO in the ischemic area, which is an important proangiogenic factor. This was associated with increased phosphorylation of eNOS on the activator site and decreased expression of caveolin-1, which acts as negative regulator of eNOS activity and NO production. On the other hand, the molecular content of MSCEVs may on the other hand suggest that MSC-EVs may also promote angiogenesis by transferring their protein repertoire to target cells such as EGF, FGF, PDGF and VEGF as well as miRNA molecules including miRNA-382-5p, miRNA-132-3p and miRNA-126. Regarding lymphangiogenesis, to the best of our knowledge, we describe for the first time, to our knowledge, that MSC-EV treatment increased Lyve1 expression in ischemic skeletal muscle. As shown in our in vitro study, we found an obligatory role of Itgα5 and NRP1 carried by MSC-EVs in the process of capillary formation by lymphatic ECs under normal and injured tissue mimicking conditions. We hypothesize that MSC-EVs may improve ischemic limb recovery by limiting both edema and inflammation through regulation of NRP1/or Itgα5/VEGFR-3 signaling, which requires further studies.

Conclusions

The present study provides evidence for pro-angiogenic and pro-lymphangiogenic properties of MSC-EVs, which may be exploited for ischemic tissue repair. The results highlight a role of protein and miRNA content of MSCEVs, including Itgα5 and NRP1, in the regulation of such processes and indicate them as potential targets to promote lymphangiogenesis in injuries and pathologies associated with peripheral ischemia.

The beneficial effect of the application of MSC-EVs in a murine model of hindlimb ischemia provides a potentially interesting therapeutic perspective for patients suffering from ischemic diseases to induce therapeutic angiogenesis and lymphangiogenesis. Based on promising preclinical data regarding the pro-regenerative efficacy and clinical safety of MSC-EVs [START_REF] Shi | Preclinical efficacy and clinical safety of clinical-grade nebulized allogenic adipose mesenchymal stromal cells-derived extracellular vesicles[END_REF], they may be an interesting future therapeutic product for cell-free therapy of ischemic tissues in human patients. However, further investigations would be required for a deeper understanding of the differential in effects of MSC-EVs on the processes of angiogenesis and lymphangiogenesis.

.

Acknowledgements

This work was supported by the National Science Centre of Poland under grants: MAESTRO no. 2019/34/A/NZ3/00134 to E.Z-S. and ETIUDA o.2016/20/T/ NZ3/00516 to A.L.-M., and INSERM Institution. Graphical abstract was created using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license (https:// creativeco mmons.org/licen ses/ by/3. 0/). The open-access publication has been supported by the Faculty of Biochemistry, Biophysics and Biotechnology under the Strategic Programme Excellence Initiative at Jagiellonian University in Krakow, Poland.

Funding

The open-access publication has been supported by the Faculty of Biochemistry, Biophysics and Biotechnology under the Strategic Programme Excellence Initiative at Jagiellonian University in Krakow, Poland.

Data availability

The underlying data for this article can be found in the article and its Additional file.

Author contributions

AL-M and LV contributed equally to this work as first authors. RA and EZ-S contributed equally to conceptualization of the study as senior corresponding authors. The authors would like to thank Prof. Stefan Chłopicki from The Jagiellonian Centre for Experimental Therapeutics, Jagiellonian University, Krakow, Poland for access to IZON qNano System. Conceived and designed the experiments: AL-M, LV, EZ-S, RA. Performed the experiments: AL-M, LV, SK-K, EK, MS-S, MS. Analyzed the data: AL-M, LV, SK-K, SB-W, EZ-S, RA. Wrote the paper: AL-M, LV, EZ-S, RA. Revised the paper for scientific content: EZ-S, RA. All authors agree and approve the publication of the article.

Declarations Ethics approval and consent to participate

The affiliated INSERM U1063, Oxidative Stress and Metabolic Pathologies, Angers University in France and Faculty of Biochemistry Biophysics and Biotechnology, Department of Cell Biology, Jagiellonian University in Krakow, Poland possessed appropriate approvals of the Animal Committee for the experiments involving mice.

Consent for publication

Not applicable.

Competing interests

There is no competing interest between the authors.


Rémi Diesler
email: remi.diesler@chu-lyon.fr

Marie Legendre

Salim Si-Mohamed

Pierre-Yves Brillet

Lidwine Wemeau

Effrosyni D Manali

Frédéric Gagnadoux

Sandrine Hirschi

Gwenaël Lorillon

Martine Reynaud-Gaubert

Vanessa Bironneau

Elodie Blanchard

Arnaud Bourdin

Stéphane Dominique

Aurélien Justet

Julie Macey

Sylvain Marchand-Adam

Hélène Morisse-Pradier

Hilario Nunes

Spyros A Papiris

Julie Traclet

Ibrahim Traore

Bruno Crestani

Serge Amselem

Nadia Nathan

Raphaël Borie

Vincent Cottin

; Orphalung

Rola Abou Taam

Julien Bermudez

Philippe Bonniaud

Alexandre Brudon

Pierrick Cros

Céline Delestrain

Ralph Epaud

Helen Fouquet

Anne Gondouin

Alice Hadchouel Duverge

Stéphane Jouneau

Karine Juvin

Jérôme Le Pavec

Jean-Claude Meurice

Mouhamad Nasser

Jean Pastre

Clément Picard

Mathilde Provost

Adrien Tissot

Yurdagul Uzunhan

Dominique Valeyre

Rémi Diesleremail

Similarities and differences of interstitial lung disease associated with pathogenic variants in SFTPC and ABCA3 in adults

Keywords: ABCA3, SFTPC, adults, fibrosing interstitial lung disease, interstitial lung disease, surfactant

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INTRODUCTION

Variants in surfactant-related genes have been linked to interstitial lung disease (ILD) in children1and adults.2,3ILDcaused by variants in SFTPC, encoding the surfactant pro-tein (SP) C, are rare and may be underestimated in adults due to the lack of specific presentation and/or absence of family history.4-12The inheritance pattern is autosomaldominant with incomplete penetrance. Variants inABCA3,encoding the lipid transporter ATP-binding cassette subfamily A member 3, can cause neonatal fatal respiratory distress syndrome13and ILD in children and adults.14Inher-itance pattern is autosomal recessive and heterozygosity forABCA3variants may play a disease modifier role in other surfactant-related ILDs.7,10Despite belonging to surfactant-related genes associated with onset of ILD in children,ABCA3andSFTPCdiffer regarding the supposed pathophysiology of ILD.ABCA3pathogenic variants are loss of functionvariants,15whereas p.(Ile73Thr), the most frequent SFTPC variant, is responsible for protein dysfunction and cell toxicity.16We hypothesized thatSFTPC-andABCA3-associated ILD in adults may share an identifiable phenotype but with distinct characteristics. Here, we report the phenotypical description of ILD in a cohort of adult patients carrying pathogenic variants in eitherSFTPCorABCA3.

METHODS

Subjects

A multicentre retrospective study was conducted within the French rare pulmonary diseases network OrphaLung. Data from participating centres were matched with those from the molecular reference genetic centre for surfactant-related diseases (Armand Trousseau Hospital, Paris, France) to include all consecutive patients diagnosed with ILD andSFTPCorABCA3variants from1 January 2002 until 1 June 2022. The inclusion criteria were patients >18 years of age, the presence of ILD on chest computed tomography (CT) and pathogenic/likelypathogenicSFTPCorABCA3variant(s). Exclusion criteria were missing radiologic or pulmonary function data, absence of ILD on CT and the presence of only monoalle-licABCA3variant.

Data collection

Data were collected using a standardized and anonymous form. For patients diagnosed before the age of 18-years, paediatric descriptive data (functional, radiological, histopathological and treatment-related) were censored, and the radiological analysis was carried out from the first chest CT available after the age of 18-years. The retrospective analysis of histopathology was based on the biopsy reports and the multidisciplinary discussion reports provided by the reporting centre for each patient. Lung biopsies had been reviewed by expert pathologists in all cases. Pulmonary hypertensionat right heart catheterization (RHC) was defined as perguidelines.17

Genetic analysis

Genetic data were reviewed retrospectively by molecular geneticists (ML, SA). Variants were classified as class 4 (likely pathogenic) or class 5 (pathogenic) variants according to the international classification.18

Radiological analysis

Chest CT scans were reviewed by two independent thoracic radiologists (SSM, PYB) blinded to the clinical and genetic data, using a pre-determined list of features. Whenever possible, patterns were categorized according to the 2018 international guidelines on idiopathic pulmonary fibrosis (IPF),19 the 2013 classification of idiopathic interstitial pneumonias20,21or other patterns such as pleuroparenchymal fibroelastosis (PPFE).22 Radiologic patterns of fibrosing ILD that did not fit into existing categories were termed' fibrotic ILD, unclassifiable pat-tern '23,24and classified between three subgroups based on the presence of (1) predominant ground glass opacities(GGO) (unclassifiable pattern-GGO), (2) prominent cystic changes (unclassifiable pattern-cysts) and (3) combination of GGO and cystic changes (unclassifiable pattern-mixed). The extent of ILD was determined by visual assessment between four categories (0%-25%,26%-50%, 51%-75% and 75%-100% of the lung parenchyma). Discrepancies between the independent radiological reviews were resolved by consensus.

Statistical analysis

Comparisons between groups were made with Wilcoxon test for quantitative variables and Fisher's exact test for qualitative variables. A random effect linear model was used to evaluate forced vital capacity (FVC) and diffusing capacity for carbon monoxide (DLCO) changes over time. Survival was analysed with the Kaplan-Meier method, using the date of ILD diagnosis as baseline (including for cases diagnosed in childhood). All statistical analyses were per-formed using the RStudio software (RStudio, version2022.02.0 Build 443).

RESULTS

Study population and characteristics

Fifty-four patients were initially reported by clinicians (FigureS1in the Supporting Information). Eighteen patients were excluded: 11 with a variant in SFTPC and 7 with variants inABCA3. Reasons for exclusion in the SFTPC group were death before the age of 18 years (n=1), lack of data available (n=3), lack of data available prior to lung transplantation (n=2), SFTPC variant of uncertain significance (class 3, n=3) and absence of ILD in asymptomatic carriers (n=2). Reasons for exclusion in the ABCA3 group were the presence of only monoallelic variant ofABCA3 (n=6) and ABCA3 variants of uncertain significance (class 3, n=1).Thirty-six patients from 29 families were included (22 in the SFTPC group and 14 in the ABCA3 group), 12 of whom belonging to 5 kindreds (all in the SFTPC group). Eight patients diagnosed before the age of 18-years had reached the adult age and were included (five in the SFTPC group and three in theABCA3 group). Only one patient (in the SFTPC group) presented with an asymptomatic ILD, discovered during familial screening. Six patients 25-30 were previously reported.

Genetic findings

Variants in SFTPCare reported in TableS1in the Supporting Information. The most frequent mutation was the p.(Ile73Thr) (12 patients in 10 families, 54.5%). Variants inABCA3are reported in TableS2in the Supporting Information. Three patients (21%) had homozygous pathogenic variants: p.[(Gly974Asp;Ala1027Pro)], p.(Thr1173Arg) and p.(Asp253His), respectively. The other patients were com-pound heterozygous. The most common variant was p.(Glu292Val), found in five patients (35.7%).

Clinical findings

The main clinical characteristics are reported in Table1.Median age of ILD diagnosis was 38.9 years (IQR 23.2-47.6 years) in the SFTPC group and 31.5 years (IQR19.1-40.6 years) in the ABCA3 group (p=0.24). Both groups were similar except for sex (77% of males in the SFTPC group vs. 21.4% in theABCA3group,p=0.002)and the familial history of ILD (81.8% in the SFTPC groups vs. 28.6% in the ABCA3 group, p=0.004), compatible with the autosomal dominant pattern of inheritance of SFTPC variants. Dyspnoea at presentation and inspiratory crackles were common on examination, found each in86.1% of the patients. Cough and clubbing were present in more than half of the cases. Seven patients (19.4%) had pectus excavatum. Overall, 27.8% of patients were smokers or former smokers.

Pulmonary function tests

Baseline PFTs are reported in Table2. Out of the 35 patients with available FVC at baseline, 29 (82.9%) had a FVC <80% predicted (15 out of 21 in the SFTPC group and all 14 in the ABCA3 group). Two patients presented an obstructive pat-tern. Twenty-eight patients had available baseline DLco, which was <80%predicted for 27 (96.4%) of them. Forced expiratory volume in 1 second (FEV1, %predicted) and DLco were lower in theABCA3group as compared to SFTPC (Table2).

Radiological findings

The chest CT characteristics of 34 patients are provided inTable3, and representative examples are showed in Figures1and2.

In 85.3% of patients, the ILD pattern was categorized as unclassifiable, including 44.1% percent of patients with a nun-classifiable pattern with predominance of GGO, 8.8%with an unclassifiable pattern with predominance of cysts, and 32.4% with an unclassifiable pattern with both GGO and cysts. Other patterns were found in five patients (14.7%): two patients with a pattern suggesting PPFE, one with a radiological pattern of fibrosing nonspecific interstitial pneumonia (NSIP) (Figure2E), one with cystic changes with minimal fibrosis in the SFTPC group and one with a pattern of both PPFE and GGO in theABCA3group.There was no significant difference of predominant pat-terns between groups. Pulmonary cysts, present in all CT scans except two, were oval or irregularly shaped (Figure1). In half of the patients, cysts showed an upper lobe predominance, with no difference between groups. GGO were present in 31 patients (91.2%), were patchy in21 patients (67.7%) and colocalized with the cysts in most of the cases (Figure1), with no difference between groups. Thirteen patients (38.2%) had emphysematous lesions on CT scan with random distribution. The presence of emphysema was not associated with tobacco use (p=0.17) or age at diagnosis of ILD (p=0.85). Features of lung fibrosis, including bronchial distortion and reticulations (Figure1), were present in 91.2% of patients. Honey combing was absent from all CT scans. Fibrotic lesions were reported as the main feature on CT scan in only five patients (14.7%), all in the SFTPC group. Upper lobe sub-pleural consolidations evoking PPFE lesions were seen in30 patients (88.2%; Figure 2C,D,F).

Histopathological findings

Seventeen patients (47.2%) underwent video-assisted thoracoscopic surgical lung biopsy, nine in the SFTPC group and eight in theABCA3group. Data from an additional patient in the SFTPC group were available on the explanted lung after transplantation. Fifteen histopathological results (9 in the SFTPC group and 7 in theABCA3group) were included in the analysis because one biopsy in each group were per-formed in patients <18-year-old at the time and therefore censored. The most frequent histopathological pattern observed was NSIP (one patient in the SFTPC group and five in theABCA3 group). Usual interstitial pneumonia (UIP) was reported in three patients, and end-stage fibrotic lung disease in two patients (all in the SFTPC group). Additional reported findings included inflammatory fibrosis, emphysema, cysts and fibroelastosis foci (Tables S1, S2 and S3 in the Supporting Information). Cysts were described as subpleural with either thin walls or walls thickened by fibrosis. Two patients had accessory histological features of alveolar proteinosis. No patient had available data regarding bronchoalveolar lavage fluid staining with periodic acid-Schiff.

Outcomes

The median follow-up duration was 9 years (IQR 3-14.5 years)in the overall population, 7.7 years (IQR 2.4-11.3 years) in the SFTPC group and 10.6 years (IQR 4.6-15.9 years) in theABCA3group. Drugs prescribed for ILD are reported inTableS4in the Supporting Information. Nine patients (25% underwent lung transplantation (eight in the SFTPC group and one in the ABCA3 group). Median time from diagnosis to transplantation was 3.2 years (IQR 2.4-9 years). Five patients (four in the SFTPC group and one in theABCA3group) died without undergoing lung transplantation. Death was attributed to acute exacerbation of ILD (n=3), acute respiratory insufficiency due to bilateral pneumothorax (n=1) and evolution of the ILD (n=1). Median time from diagnosis to death was 5.7 years (IQR 2.1-9.5 years) in the overall population. Figure 3 shows the survival estimates of the two groups from diagnosis to a composite event of death or transplantation. Median time to the composite event was 10 years in the SFTPC group and was not reached at the end of follow-up in theABCA3group. Survival to death or lung transplantation tended to be worse in the SFTPC group but the difference did not reach statistical significance (p=0.07) Follow-up data on FVC were available for 21 patients in the SFTPC group and 14 patients in theABCA3group while follow-up data on DLCO were available for 20 patients in the SFTPC group and 13 patients in theABCA3group.FigureS4in the Supporting Information shows the individual decline of lung function in both groups. Using random effect linear model, FVC declined annually by 1.87% of predicted (CI95% [2.58; 1.16], p< 0.001) in the SFTPC group and by 0.72% of predicted (CI95% [1.43;0.004], p=0.048) in theABCA3group (absolute p=0.014for comparison between groups). DLCO declined annually by 2.43% of predicted (CI95% [3.24; 1.62],p< 0.001) in the SFTPC group and by 0.95% of predicted (CI95% [1.83;0.07],p=0.03) in theABCA3group (absolute change, p=0.004 for comparison between groups).

Fifteen patients (41.6%) presented at least one exacerbation of ILD (45.5% in the SFTPC group vs. 35.7% in the ABCA3 group, p=0.73). In eight patients, pulmonary hypertension was diagnosed by RHC (seven in the SFTPC group, 31.8%; one in theABCA3group, 7.1%,p=0.12).

DISCUSSION

The present study describes the characteristics of adult patients with ILD associated with pathogenic variants inSFTPCorABCA3. The main findings of the study were that SFTPC-andABCA3-associated ILD shares unusual CT features distinct from UIP, with three predominant radiological patterns combining fibrotic features with GGO and cysts in a young adult, yet with differences between SFTPC and ABCA3.

Despite good descriptions in paediatric series, few studies to date have reported ILD associated with SFTPC or ABCA3 pathogenic variants in adults. Therefore, one purpose of our study was to provide information to pulmonologists to better recognize the possibility of ILD caused by such variants. SP-C is a small, extremely hydrophobic, valine-rich protein of 35 amino-acid residues.31, 32 Its hydrophobic proper-ties help to stabilize surfactant at low lung volumes, thus preventing alveolar endexpiratory collapse. The SFTPC gene, located on chromosome 8, encodes a 197-amino-acidprecursor protein selectively expressed in the alveolar type II epithelial cells of the lung alveoli.33The COOH flanking domain of the precursor protein can be separated into a35 amino acids linker domain and a 104 amino acids BRI-CHOS domain. Mutations in the BRICHOS domain of other proteins have been linked to various diseases including dementia, chondrosarcoma and stomach cancer. [START_REF] Sanchez-Pulido | BRICHOS: a conserved domain in proteins associated with dementia, respiratory distress and cancer[END_REF] The ABCA3 gene encodes a 1704-amino acids protein consisting of two homologous sequences comprising 12 transmembrane helices, the signature ATP-binding cassette motif, two nucleotide binding domains, two regulatory domains and two extracellular domains.35, 36 ABCA3 is involved in the formation of lamellar bodies and is located at their outer mem-brane where it drives the transportation of phospholipids. 37

Variants in SFTPC induce different cellular effects depending on the localization of the mutation. Proposed mechanisms of cellular injury include blockade of macroautophagy, 38 impairment of endosomal machinery39and induction of endo-plasmic reticulum stress. Mutations in the BRICHOS domain alter the correct SP-C folding and result in aggregation in endosomal compartments, also inducing endoplasmic reticulum stress.40In line with a dominant dysfunction resulting in cell toxicity, all the pathogenic variants identified in SFTPC in our study were missense ones, except one three amino-acid deletion and one late frameshift mutation (p.(Ser 153 Leu fs*27)) that may escape nonsense-mediated mRNA decay and result in the production of a moderately truncated protein. VariantsinABCA3can affect the cellular localization, the function of the protein or both.41Some variants lead to retention of the mutant protein in the endoplasmic reticulum, inducing endo-plasmic reticulum stress and cellular toxicity, 41, 42 where as others impair lipid transport43or ATPase activity.44

In the study by van Moorsel et al. the authors identified SFTPC variants in 5 adults in a cohort of 20 patients with familial pulmonary fibrosis, including the p.(Ile73Thr) variant in three patients.11 The chest CT scans of all variant carriers showed unclassifiable patterns of ILD, reported as cystic ILD with diffuse GGO and nodular septal thickening. The histological analysis available in four patients showed a UIP pattern in all.11

To our knowledge, only 12 adult patients with ILD associated with bi-allelic variants inABCA3were previouslyreported. [START_REF] Klay | ABCA3 mutations in adult pulmonary fibrosis patients: a case series and review of literature[END_REF][START_REF] Bullard | ABCA3 mutations associated with pediatric interstitial lung disease[END_REF][START_REF] Flamein | Molecular and cellular characteristics of ABCA3 mutations associated with diffuse parenchymal lung diseases in children[END_REF][START_REF] Manali | Bi-allelic missense ABCA3 mutations in a patient with childhood ILD who reached adulthood[END_REF][START_REF] Epaud | Combined pulmonary fibrosis and emphysema syndrome associated with ABCA3 mutations[END_REF][START_REF] Kroner | Lung disease caused by ABCA3 mutations[END_REF][START_REF] Campo | A large kindred of pulmonary fibrosis associated with a novel ABCA3 gene variant[END_REF] Consistent with our findings, chest CT scan often showed diffuse GGO with or without super-imposed reticulonodular opacities. Wambach et al. suggested that the clinical phenotype of lung disease associatedwithABCA3mutations relates to the type of mutation. Null/null ABCA3 genotypes seem incompatible with life, while null/other or other/other genotypes display phenotypicalheterogeneity. [START_REF] Wambach | Genotype-phenotype correlations for infants and children with ABCA3 deficiency[END_REF] In line with this assessment, no patient in our study had a null/null genotype. Of note, 5 out of 14 patients carried the pathogenic hypomorphic p. (Glu292-Val) mutation in the compound heterozygous state. This is in keeping with the relatively high frequency of this variant in the general population (e.g., about 0.86% of controls from European descent are heterozygous for this variant in the gnomAD database).

Although the imaging features did not belong to an existing category, we were able to subcategorize radiologic patterns of fibrosing ILD according to the predominance of GGO, cysts or both. Almost all patients presented GGO and pulmonary cysts. It has been previously hypothesized that ground-glass opacities on chest CT scan in children with SFTPC variants are the radiological expression of alveolar septal thickening, alveolar accumulation of macrophages and type II pneumocytes hyperplasia, whereas cystic lesions may be related to dilatation of the bronchioles and alveolarducts.48Moreover, sustained chronic lung inflammation and activation of the NFκB pathway could explain the fibrotic phenotype observed in most patients.16, 49 Emphysema was a common feature and may lead to combined pulmonary fibrosis with emphysema syndrome, as previously described in adults withSFTPCorABCA3variants.27, 29, 50 Emphysema was not associated with tobacco use or age at diagnosis, suggesting different pathogenic effects in patients sharing the same variant. Additionally, alveolar injury could also con-tribute to emphysema.51 Of note, two seemingly different subsets of patients can be distinguished based on imaging: some patients display retraction of the lung, with reduced lung volumes and chest wall deformity, and some patients display lung distension from cysts and emphysema (FigureS2in the Supporting Information). No patient presented a UIP pattern on chest CT. Although few histological results were available, only a small number of patients had histologic UIP. Therefore, ILD associated with mutations in SFTPC and ABCA3 can generally be distinguished from IPF. Interestingly, there seems to be a continuum between the centrilobular and perilobular in filtration (i.e., GGO and reticular pattern). We hypothesize that imaging abnormalities may follow the subsequent progression: GGO, then cystic changes often intertwined with reticulations, possibly leading to emphysema and/or established fibrotic changes (Figure S3 in the Supporting Information).

Based on our results, we propose that a diagnosis of ILD associated with variants inSFTPCorABCA3should be suspected in a patient presenting with:

•A relatively young age at diagnosis (before the seventh decade),

•A CT pattern of fibrosing ILD with pulmonary cysts and GGO,

•A familial history of ILD (for SFTPC variants, or for ABCA3 variants in inbred individuals).

In our series, ILD was severe with a significant number of patients experiencing disease progression to death or lung transplantation, and nearly half of them had an acute exacerbation of ILD. However, the median survival was 10 years after diagnosis in patients with SFTPC variant, while it is less than 5 years in IPF.52Patients with variants in SFTPC more often presented with a fibrosing phenotype than those withABCA3variants, and more frequently received antifibrotic medications (Table S4 in the Supporting Information). [START_REF] Thomas | Heterozygosity for a surfactant protein C gene mutation associated with usual interstitial pneumonitis and cellular nonspecific interstitial pneumonitis in one kindred[END_REF][START_REF] Abou Taam | Familial interstitial disease with I73T mutation: a mid-and long-term study[END_REF][START_REF] Van Moorsel | Surfactant protein C mutations are the basis of a significant portion of adult familial pulmonary fibrosis in a dutch cohort[END_REF][START_REF] Lawson | Genetic mutations in surfactant protein C are a rare cause of sporadic cases of IPF[END_REF] Patients with ABCA3 variants tended to be younger at ILD onset and more severe but with better prognosis. These results are consistent with the recent reported Dutch cohort of 12 SFTPC and 3ABCA3 adult variant carriers with ILD.53 Moreover, they indicate that further research is warranted to develop specific targeted therapies based on both the genotype and the pulmonary phenotype.

Although this study is the first to characterize a sizable number of adult patients with ILD associated with variants in both these genes, it is however limited by its retrospective design and heterogeneity. Whilst chest imaging was centrally reviewed, lung biopsies were not. Functional assessment of the variants was not performed. Respiratory functional decline analysis must be interpreted with caution, owing to the lack of data for a proportion of patients and the small observed difference between groups, which can be skewed by intraindividual measurement variability. The changes of clinical practices over the large period of data collection, the presence of patients diagnosed during childhood or adulthood and the heterogeneity of the treatment strategies used in this study population hinder its ability to describe the effect of treatments. Prospective studies are needed to define the best possible course of action in managing these patients.

In conclusion, variants in the surfactant-related genesSFTPCandABCA3are associated with a non-UIP phenotype of fibrotic ILD in adult patients, with severe prognosis. A radiologic pattern of fibrosing ILD with ground-glass opacities and/or cysts is frequently found in these rare conditions.

ACKNOWLEDGEMENTS

We thank Professor Anna Karakatsani, National and Kapodistrian University of Athens, for patient care; and Mrs Shanez HAOUARI for linguistic corrections. OrphaLung network Collaborators: Rola Abou Taam, Julien Bermudez, Philippe Bonniaud, Alexandre Brudon, Pierrick Cros, Céline Delestrain, Ralph Epaud, Helen Fouquet, Anne Gondouin, Alice Hadchouel Duverge, Stéphane Jouneau, Karine Juvin, Jérôme Le Pavec, Jean-Claude Meurice, Mouhamad Nasser, Jean Pastre, Clément Picard, Mathilde Provost, Adrien Tissot, Yurdagul Uzunhan and Dominique Valeyre.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.

FUNDING INFORMATION

No funding was received for conducting this study.

CONFLICT OF INTEREST STATEMENT

Rémi Diesler (0000-0003-0746-357X) reported support for attending meetings and/or travel by Asdia.

Pierre-Yves Brillet reported payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events, support for attending meetings and/or travel by Boehringer Ingelheim and Sanofi, and participation on a Data Safety Monitoring Board or Advisory Board for Boehringer Ingelheim.

Effrosyni D. Manali reported payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events, support for attending meetings and/or travel by Boehringer Ingelheim, Hoffman-La Roche and CSL Behring, and support for attending meetings and/or travel by Boehringer Ingelheim and CSL Behring.

Frédéric Gagnadoux reported payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events, support for attending meetings and/ortravel by Boehringer Ingelheim. Sandrine Hirschi reported payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events by Boehringer Ingelheim and support for attending meetings and/or travel by Boehringer Ingelheim, CSL Behring and ISIS Medical.

Arnaud Bourdin (0000-0002-4645-5209) reported grants or contracts from Astra Zeneca, GSK and Boehringer Ingelheim, consulting fees from Astra Zeneca, GSK, Chiesi, Sanofi, Regeneron, AB Science and Novartis, payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events by Astra Zeneca, GSK, Novartis, Chiesi, Sanofi, Regeneron and AB Science, support for attending meetings and/or travel by AstraZeneca, GSK, Novartis, Chiesi, Sanofi, Regeneron, participation on a Data Safety Monitoring Board or Advisory Board by AB Science.

Stéphane Dominique reported consulting fees from Astra Zeneca and Boehringer Ingelheim, payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events by Boehringer Ingelheim and support for attending meetings and/or travel by Boehringer Ingelheim and Chiesi. Spyros A. Papiris reported payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events, support for attending meetings and/or travel by Boehringer Ingelheim, Hoffman-La Roche and CSL Behring, and support for attending meetings and/or travel by Boehringer Ingelheim. Bruno Crestani reported grants or contracts from Boehringer Ingelheim, royalties or licences from XN one, consulting fees from Apellis, BMS, Boehringer Ingelheim and Sanofi, payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events by Apellis, Astra Zeneca, BMS, Boehringer Ingelheim, Novartis and Sanofi, payment for expert testimony by XN one, support for attending meetings and/or travel by AstraZeneca, BMS, Boehringer Ingelheim and Sanofi, patents planned, issued or pending by X None, participation on a Data Safety Monitoring Board or Advisory Board by Apellis, BMS, Boehringer Ingelheim and Sanofi, membership of the board of trustee of the Fondation du Souffle (a French charity).

Nadia Nathan (0000-0001-5149-7975) reported grants or contracts from Legs poix de la Chancellerie des Universités 2022 no. 2022000594. Raphaël Borie reported grants or contracts from Boehringer Ingelheim, payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events by Boehringer Ingelheim and Sanofi and support for attending meetings and/or travel by Boehringer Ingelheim.

Vincent Cottin (0000-0002-5591-0955) reported grants or contracts from Boehringer Ingelheim, consulting fees from Astra Zeneca, Boehringer Ingelheim, Celgene/BMS,CSL Behring, Ferrer/United Therapeutics, GSK, Pliant, Pure Tech, RedX, Roche, Sanofi and Shionogi, payment or honoraria for lectures, presentations, speakers bureaus, manuscript. writing or educational events by Boehringer Ingelheim, Ferrer/United Therapeutics and Roche, support for attending meetings and/or travel by Boehringer Ingelheim and Roche, participation on a Data Safety Monitoring Board or Advisory Board by Galapagos, Galecto and GSK, and leadership or fiduciary role in other board, society, committee or advocacy group, paid or unpaid by Fibrogen.

HUMAN ETHICS APPROVAL DECLARATION

The study was registered in the Commission Nationale Informatique et Liberté of the Hospices Civils de Lyon and approved by the Institutional Review Board (number21_636). All patients signed an informed consent for genetic analysis.