USE OF CDON INHIBITORS FOR THE TREATMENT OF ENDOTHELIAL DYSFUNCTION

Abstract

Endothelial dysfunction is a hallmark of peripheral arterial disease which is defined as vascular occlusion below the level of the inguinal ligament, and which is one of the most severe complications of diabetes and inflammatory conditions such as sepsis. Evidences accumulated within the past decades, identified Hedgehog (Hh) signaling as a new regulator of micro-vessel integrity. The purpose of the inventors was to investigate whether Hh co-receptors Gas1 and Cdon may be used as therapeutic targets to modulate Dhh signaling in ECs. The inventors demonstrated that both Gas1 and Cdon are expressed in adult ECs and relied on either siRNAs or EC specific conditional KO mice to investigate their role. They found that Gas1 deficiency mainly photocopies Dhh deficiency especially by inducing VCAM-1 and ICAM-1 overexpression while Cdon deficiency has opposite effects by promoting endothelial junction integrity. At a molecular level, Cdon prevents Dhh binding to Ptch1 and thus acts a decoy receptor for Dhh, while Gas1 promotes Dhh binding to Smo and as a result potentiates Dhh effects. Since Cdon is overexpressed in ECs treated by inflammatory cytokines including TNFα and Il1β, the inventors then tested whether Cdon inhibition would promote endothelium integrity in acute inflammatory conditions and found that both fibrinogen and IgG extravasation were decreased in association with an increased Cdh5 expression in the brain cortex of EC specific Cdon KO mice administered locally with Il1β. Altogether these results demonstrate that Cdon is a negative regulator and justify that Cdon blocking molecules may be used to promote endothelium integrity at least in inflammatory conditions.

Claims

1. A method of treating endothelial dysfunction in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a Cdon inhibitor.

2. The method of claim 1 wherein the patient suffers from a systemic inflammatory response syndrome or sepsis.

3. The method of claim 1 wherein the patient suffers diabetes mellitus.

4. The method of claim 1 wherein the patient suffers from diabetic micro- and/or macroangiopathy.

5. The method of claim 1 wherein the patient suffers from diabetic nephropathy, diabetic dermopathy, diabetic retinopathy and diabetic neuropathy.

6. The method of claim 1 wherein the patient suffers from peripheral arterial disease.

7. The method of claim 6 wherein the peripheral arterial disease is selected from the group consisting of acute and chronic critical limb ischemia, Buerger's disease and critical limb ischemia in diabetes.

8. The method of claim 1 wherein the Cdon inhibitor is an antibody having binding affinity for Cdon.

9. The method of claim 1 wherein the Cdon inhibitor is an antibody directed against the extracellular domain of Cdon.

10. The method of claim 1 wherein the Cdon inhibitor is an antibody having binding affinity for the region of Cdon which binds to Dhh.

11. The method of claim 10 wherein the Cdon inhibitor is an antibody that binds to Fibronectin type-III 3 domain of Cdon.

12. The method of claim 1 wherein the Cdon inhibitor is an antibody having binding affinity for the amino acid sequence ranging from the amino acid residue at position 826 to the amino acid residue at position 926 in SEQ ID NO: 1.

13. The method of claim 1 wherein the Cdon inhibitor is an inhibitor of expression that directly blocks the translation of Cdon mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation.

14. The method of claim 13 wherein the inhibitor of expression is a siRNA, an antisense oligonucleotide or a ribozyme.

15. The method of claim 13 wherein the inhibitor of expression is an endonuclease.

Description

FIGURES

[0036] FIG. 1: Cdon deficiency in ECs prevents Il1β-induced BBB disruption. Both Cdh5-Cre.sup.ERT2; Cdon.sup.Flox/Flox (Cdon.sup.ECKO) and Cdon.sup.Flox/Flox (control) mice were administered in the cerebral cortex with adenoviruses encoding Il1β (n=7 and 5 mice respectively). Mice were sacrificed 7 days later. (A) Cdh5 expression was quantified as the Cdh5+ surface area. (B) Fibrinogen extravasation was quantified as the fibrinogen+ surface area. (C) IgG extravasation was quantified as the fibrinogen+ surface area. *: p≤0.05; **: p≤0.01; ***: p≤0.001. Mann Withney test.

[0037] FIG. 2: Cdon blocking antibodies can be used to promote endothelium integrity. HUVECs were treated or not with 10 ng/mL TNFα in the presence of 1.5 μg/mL Cdon blocking antibodies or 1.5 μg/mL unspecific IgGs. Cdh5 localization was quantified as the mean junction thickness using Image J software. The experiment was repeated at least 4 times. **: p≤0.01; ***: p≤0.001. NS: not significant. One way ANOVA followed by Bonferroni's multiple comparisons test.

EXAMPLE

[0038] Material & Methods

[0039] Mice

[0040] Cdon Floxed (Cdon.sup.Flox) mice were generated at the “Institut Clinique de la Souris” through the International Mouse Phenotyping Consortium (IMPC) from a vector generated by the European conditional mice mutagenesis program, EUCOMM. Gas1.sup.tm3,1Fan (Gas1.sup.Flox) mice (Jin et al. 2015, 1) were kindly given by C. M. Fan and Tg(Cdh5-cre/ERT2)1Rha (Cdh5-CreERT2) mice (Azzoni et al. 2014) were a gift from RH. Adams.

[0041] Animal experiments were performed in accordance with the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes and approved by the local Animal Care and Use Committee of Bordeaux University.

[0042] The Cre recombinase in Cdh5-Cre.sup.ERT2 mice was activated by intraperitoneal injection of 1 mg tamoxifen for 5 consecutive days at 8 weeks of age. Mice were phenotyped 2 weeks later. Successful and specific activation of the Cre recombinase has been verified before (Caradu et al. 2018). Both males and females were used in equal proportions. At the end of experiments animal were sacrificed via cervical dislocation.

[0043] Mouse Corneal Angiogenesis Assay

[0044] Pellets were prepared as previously described (Kenyon et al. 1996). Briefly, 5 μg of VEGFA (Shenandoah biotechnology diluted in 10 μL sterile phosphate-buffered saline (PBS) was mixed with 2.5 mg sucrose octasulfate-aluminum complex (Sigma-Aldrich Co., St. Louis, Mo., USA), and 10 μL of 12% hydron in ethanol was added. The suspension was deposited on a 400-μm nylon mesh (Sefar America Inc., Depew, N.Y., USA), then both sides of the mesh were covered with a thin layer of hydron and allowed to dry.

[0045] Female mice were anesthetized with an intraperitoneal (IP) injection of ketamine 100 mg/kg and xylazine 10 mg/kg. The eyes of the mice eyes were topically anesthetized with 0.5% Proparacaine™ or similar ophthalmic anesthetic. The globe of the eye was proptosed with jeweler's forceps taking care to not damage the limbus vessel surrounding the base of the globe. Sterile saline was also be applied directly to each eye as needed during the procedure to prevent excessive drying of the cornea and to facilitate insertion of the pellet into the lamellar pocket of the eyes. Using an operating microscope, a central, intrasomal linear keratotomy was performed with a surgical blade parallel to the insertion of the lateral rectus muscle. Using a modified von greafe knife, a lamellar micro pocket was made toward the temporal limbus by ‘rocking’ the von greafe knife back and forth.

[0046] Hh containing or control pellet was placed on the cornea surface with jeweler's forceps at the opening of the lamellar pocket. A drop of saline was applied directly to the pellet, and using the modified von greafe knife, the pellet was gently advanced to the temporal end of the pocket. Buprenorphine was given at a dose of 0.05 mg/kg subcutaneously on the day of surgery.

[0047] Nine days after pellet implantation, mice were sacrificed, and then eyes were harvested and fixed with 2% paraformaldehyde. Capillaries were stained with rat anti-mouse CD31 antibodies (BMA Biomedicals, Cat #T-2001), primary antibodies were visualized with Alexa 568-conjugated anti-rat antibodies (Invitrogen). Pictures were taken under 50× magnification. Angiogenesis was quantified as the CD31+surface area.

[0048] In Vivo Permeability Assessment (Miles Assay)

[0049] The back of female mice was shaved. 72 hours, later mice were administered with 100 μL 1% Evans blue via retro orbital injection. Subsequently they were administered with 50 μL NaCl 0.9% containing or not 20 ng VEGFA (Shenandoah biotechnology) subcutaneously at 6 spots on their back.

[0050] 30 minutes later mice were sacrificed, skin biopsy around each injection point were then harvested to quantify Evans blue extravasation. Evans blue dye was extracted from the skin by incubation at 65° C. with formamide. The concentration of Evans blue dye extracted was determined spectrophotometrically at 620 nm with a reference at 740 nm. Buprenorphine was given at a dose of 0.05 mg/kg subcutaneously on the day of surgery.

[0051] Ad-Il1β Stereotaxic Injections

[0052] Mice were anaesthetized using isoflurane and placed into a stereotactic frame (Stoelting). To prevent eye dryness, an ophthalmic ointment was applied at the ocular surface to maintain eye hydration during the time of surgery. The skull was shaved and the skin incised on 1 cm to expose the skull cap. Then, a hole was drilled into the cerebral cortex and 3 μL of an AdIL-1 (Horng et al. 2017) or AdDL70 control (AdCtrl), (10.sup.7 pfu) solution was microinjected at y=1 mm caudal to Bregma, x=2 mm, z=1.5 mm using a Hamilton syringe, into the cerebral cortex and infused for 3 minutes before removing the needle from the skull hole (Argaw et al. 2009). Mice received a subcutaneous injection of buprenorphine (0.05 mg/kg) 30 minutes before surgery and again 8 hours post-surgery to assure a constant analgesia during the procedure and postoperatively. Mice were sacrificed 7 days post-surgery. For histological assessment, brains were harvested and fixed in formalin for 3 hours before being incubated in 30% sucrose overnight and OCT embedded. Then, for each brain, the lesion area identified by the puncture site was cut into 7 μm thick sections.

[0053] Immunostaining

[0054] Prior to staining, heart, brain, and lung tissues were fixed in methanol; paraffin embedded and cut into 7 μm thick sections. Whole mount corneas were fixed with 2.5% Formaline for 10 minutes and cultured cells were fixed with 10% formaline for 10 minutes.

[0055] Capillaries were identified using rat anti-mouse CD31 antibodies (BMA Biomedicals, Cat #T-2001). Neutrophils were stained with a rat anti-Ly6G (GR1) antibody (BD Pharmingen Inc, Cat #551459). Human Cdh5 was stained using mouse anti-human Cdh5 antibodies (Santa Cruz Biotechnology, Inc, Cat #sc-9989). Mouse Cdh5 was stained using goat anti-mouse Cdh5 antibodies (R&D systems, Cat #AF1002). Albumin and fibrinogen were stained using sheep anti-albumin antibodies (Abcam, Cat #ab8940) and rabbit anti-fibrinogen antibodies (Dako, Cat #A0080) respectively. Mouse IgGs were stained with Alexa Fluor 568 conjugated donkey anti-mouse IgG (Invitrogen, Cat #A-10037). Pan-leucocytes were identified using rat anti-mouse CD45 antibodies (BD Pharmingen Inc, Cat #550539). CD11b+ microglia and macrophages were identified using rat anti-CD11b antibodies (ThermoFisher, cat #14-0112-82). GFAP was stained using rabbit anti-GFAP antibodies (ThermoFisher, Cat #OPA1-06100). Neurons were identified using anti-NeuN antibodies (Millipore, Cat #ABN78). Cdon was stained using goat anti-mouse Cdon antibodies (R&D systems, Cat #AF2429). Gas1 was stained using goat anti-human Gas1 antibodies (R&D systems, Cat #AF2636). Dhh was stained using mouse anti-Dhh antibodies (Santa Cruz Biotechnology, Inc, Cat #sc-271168). Ptch1 was stained using rabbit anti-Ptch1 antibodies (Abcam, Cat #ab53715). For immunofluorescence analyzes, primary antibodies were resolved with Alexa Fluor®-conjugated secondary polyclonal antibodies (Invitrogen, Cat #A-21206, A-21208, A-11077, A-11057, A-31573, A-10037) and nuclei were counterstained with DAPI (1/5000). For both immunohistochemical and immunofluorescence analyses, negative controls using secondary antibodies only were done to check for antibody specificity.

[0056] Cell Culture

[0057] In vitro experiments were performed using human umbilical vein endothelial cells (HUVECs) (Lonza), human dermal microvascular endothelial cells (HMVECs-D) (Lonza) or human brain microvascular Endothelial Cells (HBMECs) (Alphabioregen). HUVECs and HBMECs were cultured in endothelial basal medium-2 (EBM-2) supplemented with EGM™-2 BulletKits™ (Lonza). HMVECs-D were cultured in endothelial basal medium-2 (EBM-2) supplemented with EGM™-2 MV BulletKits™ (Lonza). Cell from passage 3 to passage 6 were used. Before any treatment cells were serum starved in 0.5% fetal bovine serum medium for 24 hours. HeLa ATCC®CCL-2™ cells were cultured in Roswell Park Memorial Institute medium (RPMI) supplemented with 10% fetal bovine serum.

[0058] siRNA/Transfection

[0059] HUVECs were transfected with human Gas1 siRNA, human Cdon siRNA, human Dhh siRN, human Ptch1 siRNA or universal scrambled negative control siRNA duplex (Origen) using JetPRIME™ transfection reagent (Polyplus Transfection), according to the manufacturer's instructions.

[0060] Plasmids/Transfection

[0061] The human Gas1 encoding vector, pcDNA3-Gas1 was kindly given by C. M. Fan (Lee, Buttitta, et Fan 2001a), the GFP tagged-mouse Cdon encoding vector, pCA-mCdonEGFP, was a gift from A. Okada (Okada et al. 2006), the myc-tagged human Ptch1, Ptch1-1B-myc was kindly given by R. Toftgard (Kogerman et al. 2002) and the human full length Dhh was previously described (Caradu et al. 2018). A myc tag was added by PCR at the N-terminal of human full length Dhh to generate the myc-tagged Dhh encoding vector.

[0062] HeLa cells were transfected using JetPRIME™ transfection reagent (Polyplus Transfection), according to the manufacturer's instructions.

[0063] Quantitative Reverse-Transcription Polymerase Chain Reaction (RT-PCR)

[0064] Following manufacturer's instructions, RNAs were isolated and homogenized, from 3×10.sup.5 cells or from tissues previously snap-frozen in liquid nitrogen, using Tri Reagent® (Molecular Research Center Inc). For quantitative RT-PCR analyzes, total RNA was reverse transcribed with M-MLV reverse transcriptase (Promega) and amplification was performed on a DNA Engine Opticon®2 (MJ Research Inc) using B-R SYBER® Green SuperMix (Quanta Biosciences). The relative expression of each mRNA was calculated by the comparative threshold cycle method and normalized to β-actin mRNA expression.

[0065] Immunoprecipitation/Western Blot Analysis

[0066] Prior to western blot analysis, Dhh, Ptch1 ou Smo were immunoprecipitated with mouse anti-Dhh antibodies (Santa Cruz Biotechnology, Cat #sc-271168), anti myc-tag antibodies (Millipore, Cat #05-724) or mouse anti-Smo antibodies (Santa Cruz Biotechnology, Cat #sc-166685).

[0067] Expression of Cdon, Gas1, Dhh, Ptch1 and Smo were evaluated by SDS PAGE using goat anti-mouse Cdon antibodies (R&D systems, Cat #AF2429), goat anti-human Gas1 antibodies (R&D systems, Cat #AF2636), mouse anti-Dhh antibodies (Santa Cruz Biotechnology, Inc, Cat #sc-271168), rabbit anti-Ptch1 antibodies (Abcam, Cat #ab53715) and mouse anti-Smo antibodies (Santa Cruz Biotechnology, Cat #sc-166685) respectively.

[0068] Expression of human ICAM-1 and VCAM-1 expression were evaluated by SDS PAGE using mouse anti-human I-CAM1 antibodies (Santa Cruz Biotechnology, Cat #sc-8439) and rabbit anti-VCAM-1 (Abcam, Cat #ab134047) respectively.

[0069] Protein loading quantity was controlled using a monoclonal anti-α-tubulin antibody (Sigma). Secondary antibodies were from Invitrogen, Cat #A-21039, A-21084, A-21036). The signal was then revealed by using an Odyssey Infrared imager (LI-COR).

[0070] In Vitro Permeability Assay

[0071] 100 000 cells were seeded in Transwell® inserts. The day after, 0.5 mg/mL 70 kDa FITC-Dextran (Sigma) was added to the upper chamber. FITC fluorescence in the lower chamber was measured 20 minutes later.

[0072] Migration Assay

[0073] Cell migration was evaluated with a chemotaxis chamber (Neuro Probe, Inc., Gaithersburg, Md., USA). Briefly, a polycarbonate filter (8-μm pore size) (GE Infrastructure, Fairfield, Conn., USA) was coated with a solution containing 0.2% gelatin (Sigma-Aldrich Co.) and inserted between the chambers, then 5×104 cells per well were seeded in the upper chamber, and the lower chamber was filled with EBM-2 medium containing 0.5% FBS. Cells were incubated for 8 hours at 37° C. then viewed under 20× magnification, and the number of cells that had migrated to the lower chamber were counted in 3 HPFs per well; migration was reported as the mean number of migrated cells per HPF. Each condition was assayed in triplicate and each experiment was performed at least three times.

[0074] Methyl Thiazolyl Tetrazolium (MTT) Cell Proliferation Assay

[0075] 5×10.sup.3 cells per well were seeded in a 96-well plate. At the indicated time points, 10 of 5 mg/mL MTT were added to each wells. Cells were incubated 3-4 hours at 37° C. then culture medium was replaced by 100 μL DMSO. OD was read at 590 nm with a reference at 620 nm. Each condition included eight wells in each experiment and each experiment was performed at least three times.

[0076] Statistics

[0077] Results are reported as mean±SEM. Comparisons between groups were analyzed for significance with the non-parametric Mann-Whitney test or a one way ANOVA test followed by Bonferroni's multiple comparison test (for than two groups) using GraphPad Prism v7.0 (GraphPad Inc, San Diego, Calif.). Differences between groups were considered significant when p<0.05 (*: p<0.05; **: p<0.01; ***: p<0.001).

[0078] Results

[0079] ECs Express Cdon, Gas1 and Hhip but not Boc.

[0080] First, we searched for Cdon, Boc, Gas1 and Hhip expression in human EC from different origin, including HUVECs, HMVECs-D and HBMECs via RT-PCR. We show that human ECs express Hhip, Cdon, and Gas1 while they barely express Boc (data not shown). Notably, Gas1 is not detected in HBMECs. Since the role of endothelial Hhip has already been reported in several papers (Agrawal, Kim, et Kwon 2017; Sekiguchi et al. 2012; Nie et al. 2016), we focused our investigations on Gas1 and Cdon and confirmed their endothelial expression via immunostaining (data not shown). Interestingly, while TNFα inhibits Gas1 mRNA expression in HUVECs (data not shown), it increases Cdon mRNA expression (data not shown). Moreover, TNFα-induced Cdon mRNA expression depends on NF-κB activity (data not shown).

[0081] Next, we performed co-immunoprecipitation assays to verify that Dhh is able to bind these receptors. While we found that Dhh binds Gas1 directly (data not shown), Cdon alone cannot bind Dhh (data not shown). However, Dhh can bind Cdon in the presence of Ptch1 (data not shown). Consistently, Cdon co-localizes with Dhh only in the presence of Ptch1 while Gas1 co-localizes with Dhh both in the presence and absence of Ptch1 (data not shown)

[0082] With the aim to further investigate the role of Gas1 and Cdon in ECs we performed a series of in vitro and in vivo assays using siRNAs and EC-specific conditional KO mice respectively.

[0083] Cdon Promotes EC Proliferation, Migration and Angiogenesis

[0084] The role of Gas1 and Cdon in angiogenesis was investigated using the mouse corneal angiogenesis assay. Mice deficient for Gas1 or Cdon expression in EC together with their respective control littermates were implanted with VEGFA containing pellets. While VEGFA-induced angiogenesis was not different in Gas1.sup.ECKO mice from their control littermates (data not shown), VEGFA-induced angiogenesis was significantly-inhibited in Cdon.sup.ECKO mice compared to their control littermates (data not shown). Consistently, in vitro experiments performed in HUVECs showed that both EC proliferation (data not shown) and VEGFA-induced EC migration (data not shown) were decreased after Cdon knock down (KD). Gas1 KD did not modify EC proliferation or VEGFA-induced migration. However, Gas1 KD did promote EC migration in the absence of VEGF (data not shown).

[0085] This set of data demonstrates that Cdon is pro-angiogenic. Notably this effect works in the opposite direction to Dhh anti-angiogenic effect (Hollier et al. 2020).

[0086] Gas1 Prevent EC Activation and LPS-Induced Neutrophil Recruitment.

[0087] To investigate the role of Gas1 and Cdon in regulating EC immune quiescence, HUVECs were transfected with Gas1, Cdon or control siRNAs. VCAM-1 and ICAM-1 expression was measured via both RT-qPCR and western blot analyses. While Gas1 KD significantly increased both VCAM-1 and ICAM-1 expression (data not shown), Cdon KD did not (data not shown). These results were confirmed in TNFα treated cells (data not shown). Finally, to assess the functional consequences of EC activation in vivo, we quantified neutrophils recruitment in the lungs of mice that were administered with LPS. As expected, Neutrophils density in the lung of Gas1.sup.ECKO mice was significantly increased (data not shown) compared to their control littermates while we found no difference between Cdon.sup.ECKO mice and control littermates (data not shown).

[0088] Altogether these data demonstrate that Gas1 prevents EC activation similarly to Dhh (Caradu et al. 2018). On the contrary, Cdon does not seem to participate in the regulation of EC activation.

[0089] Cdon Disrupts Adherens Junction Integrity.

[0090] The role of Gas1 and Cdon in controlling endothelial intercellular junction integrity was first investigated in vitro. HUVECs were transfected with Gas1, Cdon or control siRNAs. Adherens junction integrity was quantified after Cdh5 immunostaining (data not shown) and endothelium permeability using Transwells. We found that Gas1 KD disrupts Cdh5-dependent junction integrity (data not shown) while Cdon KD prevents EC permeability (data not shown). Gas1 KD did not show any effects in the permeability assay test suggesting a very mild effect of Gas1 on adherens junction integrity. Consistently, in the Miles assay in vivo, VEGFA-induced vascular permeability was not different between Gas1.sup.ECKO and control mice (data not shown) while it was significantly decreased in the absence of endothelial Cdon (data not shown).

[0091] This last set of data demonstrate that Cdon strongly increases vascular permeability unlike Dhh (Hollier et al. 2020; Caradu et al. 2018) and that Gas 1 may slightly modify Cdh5 junction organization without functional consequences.

[0092] To conclude on this first set of results, Gas1 may promote Hh signaling in ECs since Gas1 KD mostly recapitulates the effects of Dhh KD. On the contrary, Cdon most likely inhibits Hh signaling since Cdon KD induces opposite effect to those of Dhh KD. Notably, Cdon has been previously identified as a Hh decoy receptor in the zebrafish optic vesicle (Cardozo et al. 2014).

[0093] Therefore, in the second part of this study, we chose to perform a series of experiments aiming to investigate whether and how Gas1 and Cdon modulate Hh signaling in ECs.

[0094] Gas1 Promotes Dhh Interaction with Smo while Cdon Prevents Dhh Interaction with Ptch1.

[0095] First we tested whether Gas1 or Cdon modulate Dhh interaction with Ptch1 and Smo. Notably, Smo has been recently shown to be a receptor for Hh ligands especially in the case of cell autonomous signaling (Casillas et Roelink 2018). Interestingly, we found that Gas1 prevents Dhh interaction with Ptch1 but promotes Dhh interaction with Smo. On the contrary, Cdon prevents Dhh interaction with Ptch1 but does not modify Dhh interaction with Smo (data not shown).

[0096] Since Gas1 KD phenocopies most features of Dhh deficiency, we tested whether Gas1 effects on endothelial adherens junction's integrity and migration depend on Hh signaling. To do so, we performed rescue experiments. HUVECs were either transfected with Gas1 or control siRNAs and then treated or not with the Smo agonist SAG. In particular, we show that Gas1 KD-induced Cdh5 junction thickening was prevented in the presence of SAG (data not shown). Similarly, Gas1 KD failed to induce EC migration in the presence of SAG (data not shown). However, SAG had no effect on Gas1 KD-induced VCAM-1 and ICAM-1 (data not shown). Notably, VCAM-1 and ICAM-1 seem to be downstream of Ptch1 rather than Smo since Ptch1 KD is sufficient to increase their expression (data not shown).

[0097] Because Cdon has opposite effects to Dhh ones, we hypothesized that Cdon is a decoy receptor for Dhh at the surface of EC and thus tested whether siCdon-induced effects are prevented in the absence of Dhh. HUVECs were transfected with Cdon siRNAs alone or in combination with Dhh siRNAs. While siCdon alone decreased adherent junction thickness and endothelium permeability, in the siCdon+siDhh condition (data not shown), effects were no longer significant confirming our hypothesis.

[0098] Cdon Deficiency at the Endothelium Prevents Blood-Brain Barrier Opening in the Setting of Acute Inflammation

[0099] Finally, since Cdon appears to act as a negative regulator of Dhh-induced signaling in ECs, we hypothesized that blocking Cdon may promote Dhh-induced signaling in ECs and subsequently promote maintenance of endothelium integrity in pathological conditions.

[0100] To test such hypothesis, we administered adenoviruses encoding Il1β locally in the cortex of both Cdon.sup.ECKO mice and their control littermate to induce acute brain inflammation and BBB breakdown.

[0101] Notably, Cdon expression is significantly increased upon Il1β treatment in both HUVECs and HBMECs (data not shown). In accordance with our hypothesis, endothelial adherens junctions were preserved in the absence of Cdon, as attested by an increased Cdh5 expression in the cortical lesion area of Cdon.sup.ECKO mice injected with Il1β, compared to control littermates (FIGS. 1A-B). Consistently, both fibrinogen and IgG extravasation were decreased (FIGS. 1A, C). BBB tightness in Cdon.sup.ECKO mice was associated with a decreased leucocyte infiltration, a decreased microglia and astrocyte activation and finally with an increased neuron survival (data not shown).

[0102] These last results demonstrate that blocking Cdon might indeed be a working therapeutic strategy to preserve endothelium integrity in pathological setting such as acute neuro-inflammation.

[0103] Cdon Blocking Antibodies May be Used as a Therapeutic Tool to Maintain Endothelial Junctions in the Setting of Inflammation

[0104] We then tested whether Cdon antibodies may be used as a therapeutic tool to block Dhh binding to Cdon and improve endothelial integrity. To do so, HUVECs were treated or not with TNFα, in the presence or not of Cdon blocking antibodies. As shown in FIG. 2, TNFα-induced Cdh5 junction thickening is prevented in the presence of Cdon blocking antibodies.

DISCUSSION

[0105] Hedgehog signaling has been described to be regulated by several co-receptors including Hhip, Boc, Cdon and Gas1 especially in the setting of embryogenesis (Allen et al. 2011). The purpose of the present study was to investigate the role of Gas1 and Cdon in ECs in adults. Importantly, Hh signaling in ECs is original by several aspects. First, it exclusively involves non canonical signaling (Renault et al. 2010; Chinchilla et al. 2010), second, it is activated by full length unprocessed Dhh (FL-Dhh) (Hollier et al. 2020) and third, it occurs cell autonomously (Caradu et al. 2018). It is important to have in mind that full length unprocessed Hh ligands, may not only bind Ptch1 but also Smo directly (Casillas et Roelink 2018). In this particular setting, the present study demonstrates that Cdon prevents FL-Dhh binding to Ptch1. Gas1 also prevents FL-Dhh binding to Ptch1 but promotes FL-Dhh binding to Smo. By doing so, Cdon mainly acts as a negative regulator of FL-Dhh and destabilizes endothelial cell junctions to promote angiogenesis while Gas 1 is a positive regulator of FL-Dhh which prevents EC activation.

[0106] Cdon, Gas1 and Boc are typically believed to be positive regulators of Hh signaling (Ramsbottom et Pownall 2016) in line with the fact that Gas1, Cdon and Boc were shown to be equally capable of promoting Shh signaling during neural patterning since overexpression of any individual component results in ectopic ventral cell fate specification (Allen et al. 2011). Additionally, while genetic removal of Gas1, Cdon or Boc individually has only modest effects on Shh signaling, removal of any two components results in significantly reduced Shh-dependent ventral neural patterning (Allen et al. 2011). However, conflicting results have been published: Gas1 was first shown to bind Shh in 2001. However, it was first suggested to reduce the availability of active Shh in the somite based on ectopic expression studies (Lee, Buttitta, et Fan 2001b). In 2007, experiments using Gas1 deficient mice revealed, on the contrary, that Gas1 is a positive regulator of Shh signaling and facilitates Shh low level effects (Martinelli et Fan 2007). Similarly, Cdon was shown to positively regulate Shh-induced signaling especially in the developing brain (Tenzen et al. 2006; Zhang et al. 2006) while it was more recently shown to act as a Hedgehog decoy receptor during proximal-distal patterning of the optic vesicle (Cardozo et al. 2014). Whether Gas1 and Cdon are positive or negative regulators of Hh signaling may then most likely depend on the type of ligand and cell type involved.

[0107] Hedgehog signaling in ECs is still far from being fully understood (Candice Chapouly et al. 2019b). We have previously shown that Dhh prevents EC activation by downregulating VCAM-1 and ICAM-1 and protects adherens junction integrity by promoting Cdh5 interaction with β-catenin (Caradu et al. 2018). The present study suggests that Dhh regulates EC activation and EC junction integrity via distinct pathways. Indeed, while Cdon mainly affects Dhh regulation of endothelial junctions, Gas1 mainly regulates Dhh regulation of EC immune quiescence. In both cases, a dialogue between Ptch1 and Smo seems to be involved, since Cdon modulates Dhh interaction with Ptch1 to regulate EC junctions, while we previously found that Cdh5 junction integrity depends on Smo (Hollier et al. 2020). Similarly, Gas1 promotes Dhh binding with Smo to prevent EC activation, while we found that Ptch1 KD is sufficient to increase VCAM-1 and ICAM-1 expression in ECs. We then hypothesized that both dialogues going from Ptch1 to Smo and Smo to Ptch1 exist based on the reciprocal regulation of Ptch1 and Smo by Smurf family of E3 ubiquitin ligases (Li et al. 2018).

[0108] Finally, the main goal of this study was to investigate whether Hh co-receptors may be used to modulate Hh signaling in ECs for therapeutical purposes. By identifying Cdon as a negative regulator of Dhh in ECs, and by demonstrating that Cdon KO prevents BBB opening in the setting of brain inflammation, the present study offers the possibility of using Cdon blocking molecules including blocking antibodies (FIG. 2) as therapeutic tools to preserve endothelial integrity at least in the setting of inflammation. Notably, inflammatory cytokines including TNFα and Il1β increase Cdon expression in ECs.

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