Marta Wawrzyniak

Christine Pich

Barbara Gross

Frédéric Schütz

Sébastien Fleury

Sandrine Quemener

Marie Sgandurra

Emmanuel Bouchaert

Catherine Moret

Lionel Mury

Wawrzyniak Marta

BSc Quemener Sandrine

MSc Sgandurra Marie

Moret Tech

Catherine

Mury Tech

Lionel

Rommens Tech

Corinne

Mottaz Tech

Hélène

Dombrowicz Tech

David

PhD Michalik Liliane
email: liliane.michalik@unil.ch

Dr Liliane Michalik

Dr David Dombrowicz
email: david.dombrowicz@pasteur-lille.fr

Endothelial but not smooth muscle PPAR/ regulates vascular permeability and anaphylaxis

Keywords: PPAR/, anaphylaxis, vascular permeability, endothelium, smooth muscle cells NON-STANDARD Peroxisome Proliferator-Activated Receptor, AVH: Acute Vascular Hyperpermeability, BVP: Basal Vascular Permeability, EC: Endothelial cell, VSMC Vascular smooth muscle cell, HUVEC: Human Umbilical Vein Endothelial Cell, SMA: Smooth Muscle Actin, AJ: Adherens junctions

est destinée au dépôt

INTRODUCTION

The structure of the vessel wall includes a layer of contractile cells (vascular smooth muscle cells -VSMCin large vessels, pericytes in arterioles and venules), and the internal endothelium. The endothelium performs critical functions in the control of vessel wall permeability, by controlling exchanges via the transcellular and the paracellular routes [START_REF] Komarova | Regulation of endothelial permeability via paracellular and transcellular transport pathways[END_REF][START_REF] Dejana | Vascular endothelial-cadherin and vascular stability[END_REF][START_REF] Goddard | Cellular and molecular regulation of vascular permeability[END_REF][START_REF] Giannotta | Ve-cadherin and endothelial adherens junctions: Active guardians of vascular integrity[END_REF] . The paracellular route (based on destabilization of cell-to-cell endothelial junctions) is believed to be of primary importance for vessel hyperpermeability in pathophysiological situations 2, 5 . The endothelium also regulates contractile cell functions, by releasing vasorelaxing and vasoconstricting signals [START_REF] Mackenzie | Endothelium-derived vasoactive agents, at1 receptors and inflammation[END_REF] , thereby regulating vessel tone and diameter, as well as blood pressure [START_REF] Fisher | Vascular smooth muscle phenotypic diversity and function[END_REF][START_REF] Armulik | Pericytes: Developmental, physiological, and pathological perspectives, problems, and promises[END_REF] .

Vasodilatation, vascular hyperpermeability and plasma extravasation are hallmarks of inflammatory disorders, like allergic reactions (type I immediate hypersensitivity in the older nomenclature). In sensitized allergic individuals, the prevalent mechanism through which allergens trigger an inflammatory reaction is the activation of mast cells and basophils upon binding of multivalent antigen to Immunoglobulin (Ig)E-loaded high affinity IgE receptors, FcRI 9-12 . Activated mast cells release preformed mediators such as histamine (also serotonin in rodents), proteases and neosynthetized arachidonic acid-derived prostaglandins and leukotrienes as well as cytokines, growth factors (like VEGF-A) and chemokines [START_REF] Brown | Anaphylaxis: Clinical patterns, mediator release, and severity[END_REF][START_REF] Voehringer | Protective and pathological roles of mast cells and basophils[END_REF][START_REF] Moon | Advances in mast cell biology: New understanding of heterogeneity and function[END_REF][START_REF] Theoharides | Mast cells and inflammation[END_REF] . Mediators such as histamine and VEGF-A decrease vessel tone and destabilize the endothelial barrier [START_REF] Kumar | Molecular mechanisms of endothelial hyperpermeability: Implications in inflammation[END_REF][START_REF] Triggiani | Allergy and the cardiovascular system[END_REF] , causing systemic plasma extravasation and vasodilatation, which results in edema, decreased blood volume and hypotension [START_REF] Triggiani | Allergy and the cardiovascular system[END_REF][START_REF] Peavy | Understanding the mechanisms of anaphylaxis[END_REF] . In its most severe form, systemic anaphylaxis, general vascular failure results is a life threatening altered tissue perfusion, which remains a major cause of lethality in systemic shocks [START_REF] Levy | Vascular hyporesponsiveness to vasopressors in septic shock: From bench to bedside[END_REF][START_REF] Panesar | The epidemiology of anaphylaxis in europe: A systematic review[END_REF] . Surprisingly, while the importance of the vasculature in inflammation manifestations and allergies is widely acknowledged, therapeutic strategies targeting the vasculature in order to improve existing treatments have received little attention.

The three peroxisome proliferator-activated receptor (PPAR) isotypes PPAR, / and  are ligand-inducible transcription factors that form a subfamily of the nuclear hormone receptors [START_REF] Varga | Ppars are a unique set of fatty acid regulated transcription factors controlling both lipid metabolism and inflammation[END_REF] . The canonical pathway by which PPARs regulate gene transcription involves activation by an agonist, heterodimerization with the Retinoid X Receptor (RXR), binding to a PPAR response element (PPRE) in the promoter region of their target genes, and recruitment of coactivators, which results in the activation of the target genes. PPARs are activated by natural fatty acids and fatty acid derivatives [START_REF] Varga | Ppars are a unique set of fatty acid regulated transcription factors controlling both lipid metabolism and inflammation[END_REF][START_REF] Poulsen | Ppars: Fatty acid sensors controlling metabolism[END_REF][START_REF] Wahli | Ppars at the crossroads of lipid signaling and inflammation[END_REF] .

Importantly, PPAR and  are the therapeutic targets of fibrates, (lipid-lowering drugs), and thiazolidinediones (insulin-sensitizing compounds), respectively, although the use of thiazolidinediones has recently been restricted due to major side effects [START_REF] Lamers | Therapeutic modulators of peroxisome proliferator-activated receptors (ppar): A patent review (2008-present)[END_REF][START_REF] Ahmadian | Ppargamma signaling and metabolism: The good, the bad and the future[END_REF] . PPAR/ is involved in the control of energy homeostasis, lipid metabolism, inflammation and skeletal and heart muscle functions [START_REF] Madrazo | The ppar trio: Regulators of myocardial energy metabolism in health and disease[END_REF][START_REF] Bojic | Peroxisome proliferator-activated receptor delta: A multifaceted metabolic player[END_REF] , as well as in the development of the white adipose tissue, skin, gut and placenta [START_REF] Barak | Ppar signaling in placental development and function[END_REF][START_REF] Abbott | Review of the expression of peroxisome proliferator-activated receptors alpha (ppar alpha), beta (ppar beta), and gamma (ppar gamma) in rodent and human development[END_REF][START_REF] Nadra | Differentiation of trophoblast giant cells and their metabolic functions are dependent on peroxisome proliferator-activated receptor beta/delta[END_REF] . We have identified PPAR/ as a key actor in skin repair [START_REF] Montagner | New insights into the role of ppars. Prostaglandins, leukotrienes, and essential fatty acids[END_REF][START_REF] Michalik | Roles of the peroxisome proliferators-activated receptor (ppar) alpha and beta/delta in skin wound healing[END_REF] and cancer [START_REF] Montagner | Src is activated by the nuclear receptor peroxisome proliferator-activated receptor beta/delta in ultraviolet radiation-induced skin cancer[END_REF] , and in the regulation of keratinocyte functions [START_REF] Michalik | Roles of the peroxisome proliferators-activated receptor (ppar) alpha and beta/delta in skin wound healing[END_REF][START_REF] Tan | The nuclear hormone receptor peroxisome proliferator-activated receptor beta/delta potentiates cell chemotactism, polarization, and migration[END_REF][START_REF] Chong | Regulation of epithelial-mesenchymal il-1 signaling by pparbeta/delta is essential for skin homeostasis and wound healing[END_REF] . Recent results show that PPAR/ also regulates vasculature functions. In endothelial and/or vascular smooth muscle cells, PPAR/ performs pro-angiogenic, anti-inflammatory, anti-atherosclerotic and antihypertensive functions [START_REF] Duan | Ppars: The vasculature, inflammation and hypertension[END_REF][START_REF] Bishop-Bailey | Ppars and angiogenesis[END_REF][START_REF] Plutzky | The ppar-rxr transcriptional complex in the vasculature: Energy in the balance[END_REF][START_REF] Cheang | Pleiotropic effects of peroxisome proliferator-activated receptor gamma and delta in vascular diseases[END_REF] .

In this paper, we have shown that, whereas PPAR/ is dispensable for the development of mature microvessels, its activity is a key factor for acute vascular hyperpermeability (AVH) and vasodilatation.

METHODS

Animals:

Animal procedures were performed upon approval by the cantonal veterinary service of the Canton of Vaud or by the Ethical committee of the Nord-Pas-de Calais Region. PPARβ/ germline-deficient animals were described previously [START_REF] Nadra | Differentiation of trophoblast giant cells and their metabolic functions are dependent on peroxisome proliferator-activated receptor beta/delta[END_REF] . PPAR/ -/- and control PPAR/ +/+ animals were littermates obtained from heterozygous mating.

An endothelial cell-specific PPAR/-deficient mouse line was obtained by crossing Tie1-Cre recombinase hemizygous animals (Pr R. Fässler; Max Planck Institute, Martinsried, Germany Miles assay: Acute vascular permeability was assessed using the Miles assay. Evans blue dye (EBD; 100 µl, 5% solution in 0.9% NaCl) was injected in the tail vein of 6months old females. After 10 minutes, 50 ul of VEGF-A (PeproTech PEPR100-20D-10UG, 100 ng/ml), thrombin (Sigma-Aldrich AG T6884-100UN, 10 U/ml), histamine (Sigma-Aldrich AG H7125-1G, 100 µmole/l) or the control (PBS) were injected intradermally into the flank skin (PBS in the left side flank skin; vasoactive agents in the right side flank skin). After 30 minutes, the animals were euthanized, and full-thickness flank skin samples were collected (standardized 1 x 1cm surfaces, which included the entire injection site), minced, and incubated in a fixed volume of 1 ml formamide for 48 hours at 60°C and extravasated EBD was quantified (OD 620 nm).

Anaphylaxis: Females (8-12 weeks old) were injected with 100µg/ml anti-DNP IgE (Sigma D 8406) or PBS (200µl per animal) intravenously (tail vein). 24 hours later, the anaphylaxis reaction was initiated by injection (i.v. tail vein) of 5mg/ml DNP-KLH (Calbiochem 324121-100MG) / 2% EBD (200µl). Hypothermia was monitored using an electronic thermometer with a rectal probe. At the end of the experiment (based on temperature recovery: 50 min for the PPARβ/ germline mouse strain, 100min for Tie1-PPARβ/ and SM22-PPARβ/ mouse strains), animals were euthanized and full thickness ear tissue samples (standardized surfaces with an 8 mm punch biopsy device) were collected, minced, and incubated in a fixed volume of 1 ml formamide, for 48 hours at 60°C) and extravasated EBD was quantified (OD 620 nm) [START_REF] Dombrowicz | Abolition of anaphylaxis by targeted disruption of the high affinity immunoglobulin e receptor alpha chain gene[END_REF][START_REF] Mazuc | A novel druglike spleen tyrosine kinase binder prevents anaphylactic shock when administered orally[END_REF] .

Cell cultures and transfection: Human umbilical vein endothelial cells (HUVECs;

PromoCell C2519A; passages four to seven) were cultured in complete Endothelial Cell Growth Medium (PromoCell 211 0101). Cells were seeded in TPP plastic 6-well plates (0.1% gelatin coating), except for immunofluorescent stainings, for which cells were seeded on glass-bottom culture dishes (10ug/ml fibronectin coating). For siRNA transfection, 30-50% confluent HUVECs were treated with RNAimax (Life Tehnology 13778150) and 25nM control (Thermo Scientific, scrambled sequence) or PPARβ/ siRNA (PPAR/ loss-of-function; Thermo Scientific, GACCUGGCCCUAUUCAUUG). Technology 2118) were detected as follows: 5 ug of proteins were separated on SDS-PAGE and immunolabelled using western blotting. Primary antibodies were incubated for 2h at room temperature. Secondary antibody (Anti-rabbit IgG-horseradish peroxidase ; 1/30000, Promega W4011) was incubated for 1h at room temperature. All antibodies were diluted in Tris-buffer/ 1% Tween-20/ 1% bovine serum albumin.

Adherens junctions immunolabelling:

Positive signals were detected using WesternBright Quantum-HRP (Advansta K-12042-D20) and Fusion FX (Vilber Lourmat), and quantified with the Bio1D software (Vilber Lourmat). The number of ear dermal blood and lymph vessels (vessel density) was counted on three standardized, non adjacent, microscopic fields of 0.7 mm 2 . For the quantification of vessel dilatation, the surface occupied by the blood or lymph vessels (which reflects the vessel size) was quantified with computer-assisted morphometry (ImageJ software), standardized to the number of vessels and presented as a % of the total dermis surface subject to analysis. Three non-adjacent sections per animal, 6 animals per group, were analyzed.

Histology and tissue immunostaining:

Statistical analysis:

Results were presented as mean values  standard deviation (SD) or standard error of mean (SEM) as indicated. Unless mentioned otherwise, the statistical comparison between groups was performed by using two-way ANOVA followed by Bonferroni`s multiple comparison test. Probability was considered to be significant at p < 0.05 (*** p<0.001, ** p<0.01, * p<0.05). For Figures 4A, 5A and 6A: 2way ANOVA was used to assess the effect of the genotype over time. Time points were sampled at regular intervals in order to reduce the auto-correlation effect. Both models with and without interactions were tested, and yielded similar results (with no interaction effect). Pools of independent biological replicates are shown as indicated in the legends.

AVAILABLE AS SUPPLEMENTARY METHODS

Histamine induced hypothermia

Epidermal and dermal cell isolation

Mast cell degranulation

Reverse transcription and real-time PCR

Primer sequences and reference 10

RESULTS

PPAR/ loss-of-function severely compromises acute vascular hyperpermeability (AVH) and microvessel dilatation in PPAR/ -/-mice.

In order to evaluate the impact of PPAR/ on the development and maturation of the skin microvasculature, we compared dermal microvessels in PPAR/ -/-and PPAR/ +/+ control animals. PPAR/ -/-and PPAR/ +/+ animals had a similar dermal microvessel density (Figure 1A). Next, we examined the dermal microvessel wall architecture, which is expected to comprise a quiescent endothelium lined with a continuous basement membrane, covered by quiescent mural cells. Labeling with the Ki67 proliferation marker revealed no proliferating cells in the vessel wall of PPAR/ -/- or PPAR/ +/+ skins, therefore confirming the absence of activated microvessels in the unchallenged tissues (Figure 1B andE1A). Labeling with the collagen IV basement membrane marker showed that this layer was tightly surrounding the endothelium in a continuous manner in both PPAR/ -/-and PPAR/ +/+ dermises, while detection of smooth muscle actin (SMA), a marker for vascular mural cells, revealed similar microvessel wrapping in PPAR/ -/-and PPAR/ +/+ unchallenged dermises (Figure 1B). These observations were confirmed in whole mount staining of dermal vessels harvested from PPAR/ -/-and PPAR/ +/+ mouse skin (data not shown).

In order to explore the role of PPAR/ in the acute increase of permeability and vasodilatation of the microvasculature, we induced an acute response by injecting VEGF-A in the dermis of PPAR/ -/-and PPAR/ +/+ mice. We then quantified AVH as well as basal vessel permeability (BVP) with the Miles assay. In the unchallenged dermal microvessels, we observed no significant difference at the baseline between the PPAR/ -/-and PPAR/ +/+ mice (Figure 1C). In response to the intradermal injection of VEGF-A, however, PPAR/ +/+ animals exhibited a 2.5 and 5 fold increase in AVH in the ear (data not shown) and the flank dermis (Figure 1C), respectively, compared to PBS injected controls. In contrast, VEGF-A injections failed to significantly stimulate microvessel AVH in the dermis of PPAR/ -/-mice. This suggests that VEGF-A-induced dermal AVH is PPAR/-dependent (Figure 1C).

We further determined whether the lack of response of PPAR/ -/-dermal vessels was specifically due to impaired VEGF-A signaling in the PPAR/ -/-vessel wall by comparing the AVH response of PPAR/ -/-and PPAR/ +/+ microvessels to the intradermal injection of two other well-characterized activators of vascular permeability, thrombin and histamine. Although thrombin is not involved in anaphylactic shocks, it was used as an independent vasoactive inflammatory mediator known to induce 11 hyperpermeability

5

. Whereas PPAR/ +/+ dermal microvessels showed a significant increased in AVH in response to the dermal injection of VEGF-A (3 fold), thrombin (2.5 fold) and histamine (3.5 fold), PPAR/ -/-dermal microvessels showed no significant AVH in response to any of the three vasoactive mediators (Figure 1D). Although lower expression levels of VEGFR-1 and 2 in the skin of PPAR/ -/-may account for a lack of VEGF-A-induced AVH (Figure E1C), the absence of histamine and thrombin-induced AVH shows that the compromised response of the PPAR/ microvessels to vasoactive mediators is not limited to VEGF-A and involves more general mechanisms.

VEGF-A is also known to stimulate vasodilatation. We therefore investigated dermal microvessel enlargement in PPAR/ -/-skin. Microvessel vasodilatation was quantified by computer-assisted morphometry on ear sections of PPAR/ -/-and PPAR/ +/+ mice, following the intradermal injection of VEGF-A or vehicle (PBS), and labeling of the microvessels with the endothelial (CD31) and lymph endothelial (LYVE-1) markers (Figure 1E). In the PBS-injected dermis, no significant difference was observed in the number and size of the PPAR/ -/-and PPAR/ +/+ blood vessels (Figure 1E, left and middle panels) or lymph vessels (Figure 1E, left and right panels). Upon stimulation with VEGF-A, PPAR/ +/+ mice displayed a significant 2.2 fold increase in blood dermal microvessel size, whereas PPAR/ -/-blood dermal microvessels did not enlarge (Figure 1E, left and middle panels). As expected in response to VEGF-A, lymph vessels, whose diameter is generally much more variable compared to blood vessels, remained unaffected in both PPAR/ -/-and PPAR/ +/+ animals (Figure 1E, left and right panels).

Overall, these data suggest that PPAR/ is dispensable for normal dermal vascularization, but is required for the acute response of dermal microvessels to vasoactive signals such as VEGF-A, histamine and thrombin. The appropriate invalidation of the PPAR/ gene was evidenced in the dermis of Tie1-PPAR/ -/-mice but not in the epidermis of these mice, neither in the Tie1-PPAR/ fl/fl dermis or epidermis, as expected for an endothelial-targeted inactivation (Figure E2B).

Selective deletion of PPAR/ in endothelial cells is sufficient to compromise AVH and microvessel dilatation in

Furthermore, PPAR/ expression was decreased as expected in Tie1-PPAR/ -/- thoracic aortas and total skin, the remaining PPAR/ expression levels being attributed to the non-endothelial cells of these tissues (Figure E2C). The density of dermal microvessels in the unchallenged skin of Tie1-PPAR/ -/-animals was similar to that of the Tie1-PPAR/ fl/fl controls (Figure 2A). In addition, the vessel wall was similar in animals of both genotypes, exhibiting quiescent endothelial and mural cell layers (Ki67), continuous lining of the endothelium with basement membrane (collagen IV)

and similar wrapping with mural cells (SMA) (Figure 2B).

Then, we compared the acute response of skin dermal microvessels following VEGF-A dermal injection. In vehicle (PBS)-injected flank skin, average BVP was similar in Tie1-PPAR/ fl/fl and Tie1-PPAR/ -/-animals (Figure 2C). In response to an intradermal injection of VEGF-A, the dermal microvessels of Tie1-PPAR/ fl/fl flank skin showed a significant 2 fold increase in AVH, which in contrast, was not observed in the vast majority of Tie1-PPAR/ -/-flank skin (Figure 2C). Furthermore, we observed a 1.8 fold increase in the VEGF-A-stimulated enlargement of the dermal blood microvessels in the Tie1-PPAR/ fl/fl dermis, but no response in the skin from Tie1-PPAR/ -/-mice (Figure 2D, left and middle panels). Lymph vessels did not show any significant dilatation in either animal (Figure 2D, left and right panels).

These data show that the absence of PPAR/ in the vascular endothelium is sufficient to compromise the acute response of microvessels to VEGF-A exposure in Tie1-PPAR/ -/-animals.

PPAR/ loss-of-function compromises the dynamics of the endothelial cell-tocell junction and the activity of kinases signaling pathways.

Since our data show that endothelial PPAR/ expression is a key factor for the stimulation of AVH, and since VEGF-A, histamine and thrombin are known to destabilize endothelial cell-to-cell junctions, thereby stimulating the paracellular permeability route, we examined whether PPAR/ might activate AVH by regulating endothelial paracellular permeability. We performed a siRNA-mediated PPAR/ knockdown in confluent human endothelial cells (HUVECs). PPAR/ siRNA reduced the PPAR/ mRNA level by 75% compared to scrambled control siRNA (Ctrl siRNA) (Figure E3A). The fact that no increase was observed in the expression of ANGTL-4 (a well-known PPAR/ target gene) in PPAR/ siRNA-transfected HUVECs, after treatment with a PPAR/ agonist, demonstrated that the transcriptional activity of PPAR/ was inhibited concomitantly with its decreased expression level (Figure E3A).

Neither the PPAR/ agonist treatment, nor the siRNA-mediated PPAR/ knockdown affected the expression levels of adherens junctions (AJ) proteins, i.e. VE-cadherin and associated -catenin, -catenin and p120-catenin (Figure E3B). However, the expression of one of the VEGF-A receptors was slightly decreased (Figure E3C). Taken together, these data demonstrate that PPAR/ loss-of-function compromises VEGF-A-induced dismantling of AJ in confluent HUVEC monolayers, through a mechanism which may involve changes in signaling through ERK1/2 and Akt, also observed following histamine stimulation.

Germline and endothelial-specific invalidation of PPAR/ attenuates IgEmediated systemic anaphylaxis in vivo.

Next, we investigated whether PPAR/ was also able to regulate systemic AVH associated with IgE-dependent passive systemic anaphylaxis, in which several mediators released by mast cells play (histamine, serotonin in rodent, PAF in humans...)

or might play (VEGF) a role.

The allergic reaction was induced in PPAR/-proficient and deficient animals and Evans blue dye extravasation as well as hypothermia were evaluated. In sensitized and challenged PPAR/ +/+ proficient animals, hypothermia was more pronounced with a maximal temperature decrease of about 4°C, which was achieved after 15 minutes while PPAR/ -/-animals exhibited an average decrease of 2.2°C at that time point and recovered faster (Figure 4A). Similarly, dye extravasation at the time of sacrifice was also 50 % lower in the absence of PPAR/ (Figure 4B). These results thus suggest that PPAR/ expression regulates the magnitude of a prototypic allergic reaction. As 14 PPAR/ is ubiquitously expressed and as the IgE-mediated anaphylactic reaction is primarily dependent on mast cell degranulation, we assessed the effect of PPAR/ deficiency in vitro using bone marrow derived mast cells (BMMC). BMMC differentiated normally from PPAR/-deficient haematopoietic progenitors and expressed comparable levels of FcRI (data not shown). Furthermore, IgE-induced degranulation was comparable in PPAR/ +/+ and PPAR/ -/-BMMC (Figure E1D, left panel), and the specific PPAR/ agonist (GW0742) or antagonist (GSK0660) had no impact on IgEmediated BMMC degranulation (Figure E1D, right panel). This suggests that PPAR/ regulates the magnitude of systemic anaphylaxis through the control of vascular permeability. We further confirmed the key role of endothelial PPAR/ expression in this process by inducing IgE-mediated anaphylaxis in Tie1-PPAR/ fl/fl and Tie1-PPAR/ -/-animals. The specific absence of PPAR/ in endothelial cells slightly affected maximal hypothermia (about 3.5°C in PPAR/-deficient, 4°C in PPAR/proficient mice), led to a faster recovery (Figure 5A), and to a significantly 50% lower peripheral plasma extravasation (Figure 5B; 1.7-fold increase in PPAR/-deficient, 3fold increase in PPAR/-proficient mice). In line, the Tie1-PPAR/ -/-animals also recovered faster from histamine-induced hypothermia (Figure E4). In contrast, smooth muscle cell (SMC) PPAR/ did not appear to play a regulatory role in this pathophysiological context, as SMC-specific PPAR/ deletion in SM22-PPAR/ -/- animals led to a similar IgE-mediated anaphylaxis as WT mice, measured by the induced hypothermia and plasma extravasation reactions (Figure 6A and B, Figure E2D-F). This suggests that PPAR/ regulated IgE-mediated anaphylaxis through its expression on endothelial cells at least in part through control of their sensitivity/response to histamine.

DISCUSSION

PPAR/ is an important regulator of endothelial and smooth muscle cell activities.

PPAR/ supports angiogenesis in tumor growth and ischemia recovery [START_REF] Han | Peroxisome proliferator-activated receptor-delta agonist enhances vasculogenesis by regulating endothelial progenitor cells through genomic and nongenomic activations of the phosphatidylinositol 3-kinase/akt pathway[END_REF][START_REF] Muller-Brusselbach | Deregulation of tumor angiogenesis and blockade of tumor growth in pparbeta-deficient mice[END_REF][START_REF] Abdollahi | Transcriptional network governing the angiogenic switch in human pancreatic cancer[END_REF][START_REF] Khazaei | Role of peroxisome proliferator-activated receptor beta agonist on angiogenesis in hindlimb ischemic diabetic rats[END_REF] , it displays anti-inflammatory and anti-atherogenic properties in EC and VSMC [START_REF] Duan | Ppars: The vasculature, inflammation and hypertension[END_REF][START_REF] Han | Peroxisome proliferator-activated receptor-delta agonist enhances vasculogenesis by regulating endothelial progenitor cells through genomic and nongenomic activations of the phosphatidylinositol 3-kinase/akt pathway[END_REF][START_REF] Takata | Ppar{delta}-mediated antiinflammatory mechanisms inhibit angiotensin ii-accelerated atherosclerosis[END_REF][START_REF] Meissner | Peroxisome proliferator-activated receptor {delta} activators induce il-8 expression in nonstimulated endothelial cells in a transcriptional and posttranscriptional manner[END_REF][START_REF] Lim | Ppar delta agonist l-165041 inhibits rat vascular smooth muscle cell proliferation and migration via inhibition of cell cycle[END_REF][START_REF] Kim | Ppardelta inhibits il-1beta-stimulated proliferation and migration of vascular smooth muscle cells via up-regulation of il-1ra[END_REF][START_REF] Piqueras | Activation of ppar{beta}/{delta} inhibits leukocyte recruitment, cell adhesion molecule expression, and chemokine release[END_REF][START_REF] Bojic | Peroxisome proliferator-activated receptor delta agonist gw1516 attenuates diet-induced aortic inflammation, insulin resistance, and atherosclerosis in low-density lipoprotein receptor knockout mice[END_REF] , and PPAR/ agonists were reported to be vasorelaxing compounds [START_REF] Harrington | The pparbeta/delta agonist gw0742 relaxes pulmonary vessels and limits right heart hypertrophy in rats with hypoxia-induced pulmonary hypertension[END_REF][START_REF] Jimenez | Endothelium-dependent vasodilator effects of peroxisome proliferator-activated receptor beta agonists via the phosphatidyl-inositol-3 kinase-akt pathway[END_REF] able to reduce blood pressure and heart rate in spontaneously hypertensive rats [START_REF] Zarzuelo | Antihypertensive effects of peroxisome proliferator-activated receptor-beta activation in spontaneously hypertensive rats[END_REF] .

Despite this body of evidence in several pathophysiological conditions, PPAR/ involvement in vascular responses to the acute release of vasoactive mediators occurring in inflammation and allergies has not attracted any attention to date. Our work provides evidence that endothelial PPAR/ expression promotes local acute vasodilatation and AVH in response to allergic and inflammatory mediators. When exposed to such vasoactive mediators, the endothelium responds by increasing microvessel permeability to fluid, which provokes edema, and by releasing vasorelaxing agents, which relaxes VSMC and pericytes [START_REF] Fisher | Vascular smooth muscle phenotypic diversity and function[END_REF][START_REF] Armulik | Pericytes: Developmental, physiological, and pathological perspectives, problems, and promises[END_REF] . In this paper, we have shown that endothelial-specific PPAR/ loss-of-function plays a protective role by reducing acute local vasodilatation and edema in response to VEGF-A and histamine.

This was not due to a lower expression of the VEGF or histamine receptors, which were expressed at similar, or even higher levels in the vena cava of PPAR/-deficient animals (Figure E1B). Although the exact mechanisms remain to be unveiled, the and Akt are affected by PPAR/ deficiency, thereby preventing these signaling pathways from activating VE-cadherin dynamics and, in turn, AVH. One likely candidate is the kinase cSrc, a direct PPAR/ target gene [START_REF] Montagner | Src is activated by the nuclear receptor peroxisome proliferator-activated receptor beta/delta in ultraviolet radiation-induced skin cancer[END_REF] , known to be a major actor involved in VE-cadherin phosphorylation and AVH [START_REF] Dejana | The role of adherens junctions and vecadherin in the control of vascular permeability[END_REF][START_REF] Paul | Src deficiency or blockade of src activity in mice provides cerebral protection following stroke[END_REF][START_REF] Weis | Src blockade stabilizes a flk/cadherin complex, reducing edema and tissue injury following myocardial infarction[END_REF][START_REF] Eliceiri | Src-mediated coupling of focal adhesion kinase to integrin alpha(v)beta5 in vascular endothelial growth factor signaling[END_REF] . Alternatively, actin cytoskeleton dynamics, which is involved in VEGF-induced permeability [START_REF] Bates | Vascular endothelial growth factors and vascular permeability[END_REF] , is known to be compromised in the absence of PPAR/ [START_REF] Tan | The nuclear hormone receptor peroxisome proliferator-activated receptor beta/delta potentiates cell chemotactism, polarization, and migration[END_REF][START_REF] Iglesias | Pparbeta/delta affects pancreatic beta cell mass and insulin secretion in mice[END_REF] . . It is to be noted that PPAR/ agonists were shown to indirectly restore NO bioavailability in vessels affected by chronic endothelial dysfunctions [START_REF] Uscio | Activation of ppardelta prevents endothelial dysfunction induced by overexpression of amyloid-beta precursor protein[END_REF][START_REF] Quintela | Activation of peroxisome proliferator-activated receptor-beta/-delta (pparbeta/delta) prevents endothelial dysfunction in type 1 diabetic rats[END_REF][START_REF] Tian | Ppardelta activation protects endothelial function in diabetic mice[END_REF][START_REF] Santhanam | Ppardelta agonist gw501516 prevents uncoupling of endothelial nitric oxide synthase in cerebral microvessels of hph-1 mice[END_REF] .

Therefore, a possibility is that endothelial PPAR/ regulates acute vasodilatation via the regulation of NO and/or the prostacyclin release by endothelial cells. Importantly, the prevention of acute edema by endothelial PPAR/ loss-of-function is extended to the systemic level, since anaphylaxis-associated peripheral edema is reduced in PPAR/ -/-and endothelial PPAR/ -/-mice. The absence of PPAR/ from VSMC had no consequence on anaphylaxis and associated edema, which supports a major role of endothelial PPAR/ in the vascular response to this acute systemic allergic reaction.

Our results thus suggest that the silencing of endothelial PPAR/ may have a therapeutic benefit by reducing the magnitude of edema and vasodilatation.

Figure E4.

Histamine-induced hypothermia in Tie1-PPAR/ -/-compared to Tie1-PPAR/ fl/fl mice.

Data are expressed as mean  SEM (n=4 for PBS injected animals; n= 5-7 for histamine injected animals).

40 )

 40 and PPARβ/ fl/fl mice (Prs W. Wahli and B. Desvergne, University of Lausanne, Switzerland 41, 42 ). Mice genotypes are labeled as follows: Tie1-PPAR/ fl/fl are Cre recombinase-negative -PPAR/ proficient mice, PPAR/ exon 4 (encoding the N-terminal zinc finger of the DNA binding domain) is flanked by LOX-P sites; Tie1-PPAR/ -/-are Cre recombinase-positive mice, PPAR/ exon 4 is deleted and thereby PPAR/ is invalidated. A SM22-PPAR/ fl/fl Smooth muscle cell (SMC)-specific PPAR/-deficient mouse line (and littermate proficient controls) was obtained by crossing SM22-Cre recombinase animals (Jackson Laboratory; Strain STOCK Tg(Tagln-cre)1Her/J) and PPARβ/ fl/fl mice. Mice genotypes are labeled as follows: SM22-PPAR/ fl/fl are Cre recombinase-negative -PPAR/ proficient mice, PPAR exon 4 is flanked by LOX-P sites; SM22-PPAR/ -/-are Cre recombinasepositive mice, PPAR/ exon 4 is deleted and thereby PPAR/ is invalidated. All mouse strains are maintained on a mixed C57BL6/SV29 background. Genotyping (Figure E2; presence of the Cre recombinase transgene (472bp DNA fragment) of the PPAR/ invalidated allele (490bp DNA fragment), and of the PPAR/ floxed allele (400bp DNA fragment)) was performed by PCR of genomic DNA extracted from the ear (PCR positive control: Glut2 gene, 230bp DNA fragment). All animals were kept in a standard colony and in a temperature-and light-controlled environment, and were fed with a standard laboratory diet ad libitum.

  Skin tissues were fixed (Zinc Fixative, BD Pharmingen 550523) /24 hours, paraffin embedded, and 4-µm thick sections were used for standard hematoxylin and eosin or double immunostainings. Primary antibodies: CD31 (pan-endothelial marker, BD Pharmingen 557355; 1:50); Ki67 (proliferation marker, Abcam ab15580); collagen IV (basement membrane marker, Chemicon AB756P; 1:1000); SMA (smooth muscle cell marker, Sigma A2547; 1:400); LYVE-1 (lymph endothelial cell marker, Reliatech, 103-PA50AG) overnight at 4°C; secondary antibodies (all from Molecular Probes) goat anti-rat Alexa 568 (1:100); goat anti-mouse Alexa 488 (1:400); goat anti-rabbit Alexa 488 (1:400) during 40 minutes at room temperature. Nuclei were counterstained with DAPI (1:5000). Characterization of dermal microvessel density and enlargement: Dermal micro vessel density and enlargement following vasoactive treatments were quantified as follows. Ear sections were stained for CD31 (pan-endothelial marker, BD Pharmingen 557355; 1:50) and LYVE-1 (lymph endothelial cell marker, Reliatech, 103-PA50AG).

  Tie1-PPAR/ -/-mice. Next, we investigated the involvement of endothelial PPAR/ in the mediation of the AVH and vasodilatation responses to VEGF-A. Targeted deletion of PPAR/ in the endothelium was achieved by breeding Tie1-Cre recombinase transgenic mice 40 with a PPAR/ fl/fl mouse line. Characterization of the Tie1-Cre recombinase transgenic mice showed that Tie1-driven expression of Cre recombinase results in efficient and selective gene excision in endothelial cells 40 .

Confluent

  HUVEC monolayers were then stimulated with VEGF-A, followed by VEcadherin labeling of AJ. In vehicle treated cells, VE-cadherin was localized along the intercellular contacts in Ctrl and PPAR/ siRNA treated monolayers, showing that the downregulation of PPAR/ expression did not interfere with AJ maturation (Figure 3A, left panel). In VEGF-A treated cells, computer-assisted analysis of AJ revealed a reorganization of VE-Cadherin from a continuous localization along the cell membranes to a diffuse distribution in Ctrl siRNA treated cells (white arrows in Figure 3A left panel, quantified in Figure 3A right panel), but not in cells transfected with the PPAR/ siRNA (Figure 3A). Importantly, siRNA mediated inactivation of PPAR/ in HUVECs affected VEGF-A and histamine-induced phosphorylation of ERK1/2 and Akt (Figure 3B and C).

  molecular basis underlying PPAR/-dependent regulation of AVH likely involves the ERK1/2 and Akt signaling pathways and the destabilization of endothelial cell-to-cell AJ. The ERK1/2 and Akt signaling pathways are two of the multiple pathways involved in the regulation of the endothelial permeability and of VE-cadherin-dependent cell-tocell adhesion 3, 61, 62 , 63-65 . The remodeling of VE-cadherin is affected by most if not all permeabilizing factors 1, 3, 62 , and correlates with reduced endothelial barrier integrity. Interestingly, we show that PPAR/ siRNA-mediated loss-of-function compromised the VEGF-A-induced destabilization of AJ. Moreover, PPAR/ deficiency exacerbated VEGF-A-, while it prevented histamine-dependent activation of ERK1/2 and Akt in human endothelial cell monolayers. Although an increase in ERK1/2 and Akt phosphorylation usually correlates with an increase in vessel permeability and VEcadherin dynamics, exacerbated activation of these pathways after PPAR/ loss-offunction is associated with compromised AVH and VE-cadherin reorganization. It is likely that, following stimulation by VEGF-A stimulation, factors downstream ERK1/2

  Compromised cSrc expression and/or actin cytoskeleton dynamics in PPAR/ loss-of-function conditions may therefore prevent VEGF-A induced AVH, despite activation of ERK and Akt signaling cascades. The exact molecular mechanism of endothelial PPAR/-dependent vasodilatation remains to be explored. The best-characterized vasorelaxing signals produced by endothelial cells are nitric oxide (NO) and prostacyclin, a ligand for 16

Figure 1 .

 1 Figure 1. PPAR/ -/-animals exhibit compromised vascular responses A) Dermal vessel density. B) Dermal vessels. Green: αSMA, collagen IV or Ki67. Red: CD31. Arrows: αSMA or collagen IV positive vessels. C-D) Miles assay, Evans blue extravasation. C) Dots: individual animals; Black lines: individual responses to treatment; Red lines: mean per group. E) Left: Dermal vessels following intradermal injection. Green: LYVE-1 (white points); Red: CD31 (white arrows). Middle and right: blood and lymph vessel size. Data are expressed as means  SD (n=6 (A-E), 9 (C), 3 (D) per group).

Figure 2 .

 2 Figure 2. Tie1-PPAR/ -/-animals exhibit compromised vascular responses A-B) Dermal vessels as in Fig 1A and B. C) Miles assay, Evans blue extravasation. Dots: individual animals; Black lines: individual responses to treatment; Red lines: mean per group. D) Left: Dermal vessels following intradermal injection. Green: LYVE-1 (white points); Red: CD31 (white arrows). Middle and right: blood and lymph vessel size. Data are expressed as mean ± SD (n=4 (A), 9 (C), 4-6 (D) per group).

Figure 3 .

 3 Figure 3. PPAR/ regulates cell-cell junction dismantling and kinase pathway activation. A) Left: VE-cadherin and nuclei (DAPI) stainings of HUVECs. Right: Area occupied by the VE-cadherin adherens junctions. Data expressed as mean ± SD (n= 7-9). B) Western blot of total ERK1/2, phosphorylated pERK1/2, total Akt, phosphorylated pAkt (upper band) from HUVECs treated as indicated. Loading control: GAPDH. C) Quantification of three independent experiments as shown in B). Data are expressed as mean ± SEM.

Figure 4 .

 4 Figure 4. Passive systemic anaphylaxis-induced hypothermia and edema are less severe in PPAR/ -/-compared to PPAR/ +/+ mice. A) Hypothermia in PPAR/ +/+ or PPAR/ -/-mice. Pool of 3 experiments B) Edema in the ears of PPAR/ +/+ and PPAR/ -/-mice. Data are expressed as mean  SEM (n=7-10 (A), 3-4 (B) for PBS injected animals; n= 10-15 (A), 4-6 (B) for IgE injected animals).

Figure 5 .

 5 Figure 5. Passive systemic anaphylaxis-induced hypothermia and edema are less severe in Tie1-PPAR/ -/-compared to Tie1-PPAR/ fl/fl mice.

Figure 6 . 8 R2Figure No. 3

 683 Figure 6. Smooth muscle cell specific PPAR/ deficiency does not affect passive systemic anaphylaxis-induced hypothermia and edema. A) Hypothermia in SM22-PPAR/ fl/fl or SM22-PPAR/ -/-mice. Pool of three experiments. B) Edema in the ears of SM22-PPAR/ fl/fl and SM22-PPAR/ -/-. Data are expressed as mean  SEM (n=7 (A), 3 (B) for PBS injected animals; n= 10 (A), 3 (B) for IgE injected animals).

  SM22-PPARβ/δ -/-IgE SM22-PPARβ/δ -/-PBS SM22-PPARβ/δ fl/fl IgE SM22-PPARβ/δ fl/fl PBS Time

  Figure E1. A) Proliferation marker positive control. E: epidermis; D: dermis. Green: Ki67. B) Expression levels of VEGF (VEGFR-1, VEGFR-2 and neuropilin (Nrp-1)) and histamine (H2) receptors in vena cava and C) total skin. Data are expressed as mean  SD; n=3 (B), 6 (C). D) Bone marrow-derived mast cells IgE-induced degranulation. Right: TNP-OVA (20ng/ml); Values are expressed as a percentage of total release ± S.D.

Figure E2 .

 E2 Figure E2. Characterization of Tie1-PPARβ/ and SM22-PPAR/ mice. A and D) PCR detection of the Cre recombinase transgene in genomic DNA (gDNA). PCR positive control: Glut2 gene. B and E) PCR detection of PPAR/ invalidated and floxed/proficient alleles in genomic DNA (gDNA) extracted from epidermis or dermis C and F) PPAR/ mRNA expression levels in thoracic aortas and total skin (n=3).

  Figure E3. A) siRNA-mediated PPAR/ silencing in HUVECs. Expression levels of PPAR/ (left) and of its target gene ANGPL-4 (right) in HUVECs. GW501516: PPAR/ agonist. B) Expression levels of adherens junction proteins (-catenin, -catenin, p120-catenin and VE-cadherin) and VEGF receptors (VEGFR-1, VEGFR-2 and co-receptor neuropilin (Nrp-1)) in HUVECs. Data are expressed as mean  SD (n=3).

ACKNOWLEDGEMENTS

We thank Pr Reinhard Fässler (Max Planck Institute of Biochemistry, Martinsried, Germany) for sharing the Tie1-cre recombinase transgenic mouse line, and to Pr Walter Wahli and Pr Béatrice Desvergne (Center for Integrative Genomics, University of Lausanne, Switzerland) for sharing the PPAR/ fl/fl transgenic mouse line. We are grateful to Dr Tatiana Petrova for helpful discussions and to Dr Valentina Triacca for excellent support. We thank Dr J. Ding, M. Husson, the Genotyping and Animal facility (Center for integrative genomics), the Mouse Metabolic Facility (University of Lausanne) and the Cellular Imaging Facility (University of Lausanne) for their excellent technical assistance.

Sources of funding

This work was supported by grants from the Swiss National Science Foundation (individual grant to L.M.), the Etat de Vaud and from EGID, ANR-10-LABX-46 (to D.D.). VEGFR1 (Flt1 QT00096292); VEGFR2 (Qiagen QuantiTect KDR QT00097020); NRP-1 (Qiagen QuantiTect QT0015); H2 (Qiagen

DISCLOSURES

SUPPLEMENTARY METHODS

Histamine induced hypothermia: Tie1-PPAR/ fl/fl and Tie1-PPAR/ -/-females (8-12 weeks old) were injected with 25mg/ml histamine (Sigma D 8406) or PBS (200µl per animal) intravenously (tail vein). Hypothermia was monitored using an electronic thermometer with a rectal probe.

Epidermal and dermal cell isolation: Full thickness dorsal skin was removed and washed in 70% ethanol and in sterile PBS. Narrow strips (2 mm) were cut and incubated in 1 ml Amonium thiocyanate (3.8% in PBS) at room temperature for 20 min.

The dermis was scraped with a scalpel blade. Epidermis and dermal cells were further used for genomic DNA or total RNA extraction.

Mast cell degranulation: Bone Marrow-derived mast cells (BMMC) were prepared from PPAR/-proficient and deficient mice using 10ng/ml SCF (Peprotech 250-03) and 10ng/ml IL-3 (Peprotech 213-13). Purity exceeded 95 % after 6 weeks of culture.

IgE-mediated BMMC degranulation was assessed by measuring -hexosaminidase release, as described previously, upon activation with 1 µg/ml anti-TNP IgE (BD Pharmingen 557079) followed by 0.01-1000 ng/ml ranging concentrations of TNP-OVA (Biosearch Technologies, Novato, CA. T-5051-100) as indicated in the figures [START_REF] Malbec | Peritoneal cell-derived mast cells: An in vitro model of mature serosal-type mouse mast cells[END_REF][START_REF] Dombrowicz | Allergy-associated fcrbeta is a molecular amplifier of ige-and igg-mediated in vivo responses[END_REF] .

The PPAR/ agonist GW0742 (Cayman Europe 10006798, 100 nM) and/or antagonist GSK0660 (Sigma Aldrich AG G5797, 1µM) were added to the culture medium 24 h before the degranulation was initiated. proprietary software based on [START_REF] Hellemans | Qbase relative quantification framework and software for management and automated analysis of real-time quantitative pcr data[END_REF] ) software were used to analyze the data.

Reverse transcription and real

Repository -Unmarked Text

Human primers:

VE-cadherin, -Catenin, -Catenin, p120-Catenin, ANGPTL4 were all purchased from Qiagen,sequence non provided.

Mouse primers:

SUPPLEMENTARY METHODS

Histamine induced hypothermia: Tie1-PPAR/ fl/fl and Tie1-PPAR/ -/-females (8-12 weeks old) were injected with 25mg/ml histamine (Sigma D 8406) or PBS (200µl per animal) intravenously (tail vein). Hypothermia was monitored using an electronic thermometer with a rectal probe.

Epidermal and dermal cell isolation: Full thickness dorsal skin was removed and washed in 70% ethanol and in sterile PBS. Narrow strips (2 mm) were cut and incubated in 1 ml Amonium thiocyanate (3.8% in PBS) at room temperature for 20 min.

The dermis was scraped with a scalpel blade. Epidermis and dermal cells were further used for genomic DNA or total RNA extraction.

Mast cell degranulation: Bone Marrow-derived mast cells (BMMC) were prepared from PPAR/-proficient and deficient mice using 10ng/ml SCF (Peprotech 250-03) and 10ng/ml IL-3 (Peprotech 213-13). Purity exceeded 95 % after 6 weeks of culture.

IgE-mediated BMMC degranulation was assessed by measuring -hexosaminidase release, as described previously, upon activation with 1 µg/ml anti-TNP IgE (BD Pharmingen 557079) followed by 0.01-1000 ng/ml ranging concentrations of TNP-OVA (Biosearch Technologies, Novato, CA. T-5051-100) as indicated in the figures [START_REF] Malbec | Peritoneal cell-derived mast cells: An in vitro model of mature serosal-type mouse mast cells[END_REF][START_REF] Dombrowicz | Allergy-associated fcrbeta is a molecular amplifier of ige-and igg-mediated in vivo responses[END_REF] .

The PPAR/ agonist GW0742 (Cayman Europe 10006798, 100 nM) and/or antagonist GSK0660 (Sigma Aldrich AG G5797, 1µM) were added to the culture medium 24 h before the degranulation was initiated. proprietary software based on [START_REF] Hellemans | Qbase relative quantification framework and software for management and automated analysis of real-time quantitative pcr data[END_REF] ) software were used to analyze the data.

Reverse transcription and real

Repository -Marked Text

Human primers: Unless mentioned otherwise, primers were purchased from Eurofins.

SUPPLEMENTARY FIGURE LEGENDS

Figure E4.

Histamine-induced hypothermia in Tie1-PPAR/ -/-compared to Tie1-PPAR/ fl/fl mice.

Data are expressed as mean  SEM (n=4 for PBS injected animals; n= 5-7 for histamine injected animals).