Use of the recombinant fibrinogen-like domain of angiopoietin-like 4 for treating sepsis capillary leak syndrome

Abstract

Microvascular leak plays a critical role in the outcome of sepsis. Counteracting vascular leakage has recently raised a huge interest in the field but therapeutic targets and translation studies are crucially lacking. The inventors hypothesized that ANGPTL4 might counteract lipopolysaccharide-induced vascular hyperpermeability. Mechanistically, the inventors show that the C-terminal fragment of ANGPTL4 recapitulates full-length ANGPTL4 inhibition of vascular permeability by stabilizing endothelial cell adherent junctions whereas the N-terminus has no such effect. The inventors further demonstrate that giving human recombinant c- ANGPTL4 to mice prevents microvascular leak and mortality induced by LPS. In humans, the inventors describe the association between ANGPTL4 plasma level at time of inclusion and 90- day mortality in sepsis or septic shock patients French and European Outcome Registry in Intensive Care Units (FROG-ICU). In conclusion, the inventors demonstrate that suppressing vascular permeability by c-ANGPTL4 could be a novel therapeutic for the treatment of sepsis capillary leak syndrome. Either alone or in combination with existing drugs, c-ANGPTL4 could contribute to the reduction of mortality in patients with shock states.

Description

[0001] USE OF THE RECOMBINANT FIBRINOGEN-LIKE DOMAIN OF ANGIOPOIETIN-LIKE 4 FOR TREATING SEPSIS CAPILLARY LEAK SYNDROME FIELD OF THE INVENTION: The present invention is in the field of medicine, in particular critical care medicine. BACKGROUND OF THE INVENTION: The endothelium plays a crucial role via regulation of metabolic and gas exchanges, cell trafficking, blood coagulation, vascular tone, blood flow and fluid extravasation. Uponpathogen recognition during sepsis, the activation of the endothelium1 leads to phenotypicplasticity and transition towards endothelial cell (EC) activation and distinct specializedphenotypes2which contribute to thromboinflammation3and vascular permeability, leading to sepsis-associated multiple organ dysfunction syndrome (MODS) characterized by tissue hypoxia, tissue injury and multiple organ failure. Fluid balance (difference between fluid input and output) is regulated by an impaired capillary permeability which allows fluid to leave the circulation and redistribute into the extravascular space, thereby affecting microvascularperfusion and tissue oxygenation, independently correlates with mortality during septic shock4.Though, despite intensive research, current therapeutic approaches are focused on antibiotics, fluid resuscitation, vasopressors and oxygen / ventilatory support5. The molecular mechanisms that control blood endothelium maintenance and phenotypic plasticity / transition in sepsis and how these mechanisms are involved in controlling blood capillary leakage might open newavenues for new drug development. Counteracting vascular permeability in sepsis has thusraised a huge interest and previous efforts have focused on Vascular endothelial (VE)-cadherin,the major component of endothelial adherens junctions. It has been shown that Slit prevents dissociation of VE-cadherin and p120 catenin and its enhancement of vascular integrity and reduction of mortality was achieved independently of a widespread inflammation6. Robo-4-mediated suppression of vascular permeability was also shown as an effective strategy toimprove the mortality of severe infectious diseases7. Bb15-42 (FX-06), a 28 amino acid cleavage product of fibrin that interacts with VE-Cadherin and stabilizes endothelial barriers8was shown to be an attractive adjuvant in the treatment of septic shock9. Clinical translation ofthese findings is still pending as no treatments that specifically target the endothelium andmicrovascular leak have reached the clinic. As an example, FX06 did not lower SARS-CoV-2- induced pulmonary vascular leakage multicenter in a double-blinded, randomized unique- dosing-regimen trial included adults with COVID-19-associated acute respiratory distress syndrome10. Angiopoietins are also crucial regulators of the endothelial barrier. Various compounds that activate Tie2 and mimick Angiopoietin-1 were shown to improve mortality in mouse models of sepsis11–13. Angiopoietin-like proteins (ANGPTLs) share common motifs to angiopoietins but do not bind the Tie2 receptor. ANGPTL3, ANGPTL4 and ANGPTL8 caninhibit LPL and thus raise plasma triglycerides levels. These proteins are composed of a N-terminal coiled-coil domain (CCD or n-ANGPTL) that contains the lipoprotein lipase (LPL)-binding region, a linker region, and a C-terminal fibrinogen-like domain (FLD or c-ANGPTL)14. Full-length ANGPTL4 (fl-ANGPTL4) is cleaved by proprotein convertases attheir recognition motif in the linker region14. The N-terminal domain (n-ANGPTL4) isresponsible for the inhibitory effects on LPL, converting the active form of LPL into an inactiveform15. In contrast, the C-terminal fibrinogen-like domain (c-ANGPTL4) has been involved innon-metabolic processes such as context-dependent regulation of metastasis and vascularleakiness 16. Altogether, results on fl-ANGPTL4 suggest a context-, cell type- and tissue-specific activity and thus underscore the need to specifically address the function of ANGPTL4 in ECs17. Indeed, a recent demonstrates fl-ANGPTL4 is required for maintaining EC metabolic function vital for vascular permeability and angiogenesis (Chaube, B., Citrin, K.M., Sahraei, M. et al. Suppression of angiopoietin-like 4 reprograms endothelial cell metabolism andinhibits angiogenesis. Nat Commun 14, 8251 (2023)) in line with our previous findings showingthat ANGPTL4 is induced by hypoxia in ECs18. In humans, Angptl4 mRNA is highly expressed in ischemic tissues from patients with peripheral artery disease19and is also a specific marker of renal cell carcinoma20. In vitro, ECM-bound ANGPTL4 inhibits EC adhesion, migration,and sprouting21, and soluble ANGPTL4 modulates the growth of tubule-like structures22,23. Wehave shown that ANGPTL4 binds directly to integrin αvβ3, leading to Src recruitment and its sequestration away from VEGF receptor 2 (VEGFR2), resulting in diminished Src signalling downstream of VEGFR2, and thereby inducing stabilization of both VEGFR2-VE-cadherin andVEGFR2-αvβ3 complexes16. Also, in vivo injection of recombinant human ANGPTL4protected VEGF-driven dissociation of the VEGFR2 / VE-cadherin complex, reduced myocardial infarct size, and the extent of no-reflow24. In ischemic stroke models, ANGPTL4 also counteracts the loss of vascular integrity, thus reducing oedema, infarct size, neuronal loss, and improving mice behaviour. These results suggest that ANGPTL4 constitutes a relevanttarget for vasculoprotection in ischemic diseases but its interest for the treatment of sepsiscapillary leak syndrome has never been investigated. SUMMARY OF THE INVENTION: The present invention is defined by the claims. In particular, the present invention relates to theuse of the recombinant fibrinogen-like domain of angiopoietin-like 4 for treating sepsiscapillary leak syndrome. DETAILED DESCRIPTION OF THE INVENTION: Microvascular leak plays a critical role in the outcome of sepsis. Counteracting vascular leakage has recently raised a huge interest in the field but therapeutic targets and translation studies arecrucially lacking. The inventors hypothesized that ANGPTL4 might counteractlipopolysaccharide-induced vascular hyperpermeability. Mechanistically, the inventors showthat the C-terminal fragment of ANGPTL4 recapitulates full-length ANGPTL4 inhibition ofvascular permeability by stabilizing endothelial cell adherent junctions whereas the N-terminushas no such effect. The inventors further demonstrate that giving human recombinant c-ANGPTL4 to mice prevents microvascular leak and mortality induced by LPS. In humans, theinventors describe the association between ANGPTL4 plasma level at time of inclusion and 90-day mortality in sepsis or septic shock patients French and European Outcome Registry inIntensive Care Units (FROG-ICU). In conclusion, the inventors demonstrate that suppressingvascular permeability by c-ANGPTL4 could be a novel therapeutic for the treatment of sepsis capillary leak syndrome. Either alone or in combination with existing drugs, c-ANGPTL4 couldcontribute to the reduction of mortality in patients with shock states.Main definitions: As used herein, the term “polypeptide” has its general meaning in the art and refers to a polymer of amino acids of any length. The polymer can comprise modified amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art. As used herein, the term “polynucleotide” as used herein refers to polymers of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. This term refers to the primary structure of the molecule. Thus, the term includes triple-,double- and single-stranded deoxyribonucleic acid (“DNA”), as well as triple-, double- andsingle-stranded ribonucleic acid (“RNA”). It also includes modified, for example by alkylation, and / or by capping, and unmodified forms of the polynucleotide. More particularly, the term “polynucleotide” includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, siRNA and mRNA,whether spliced or unspliced, any other type of polynucleotide which is an N- or C-glycosideof a purine or pyrimidine base, and other polymers containing normucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids “PNAs”) and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA. In some embodiments, the polynucleotide comprises an mRNA. In other aspect, the mRNA is a synthetic mRNA. In some embodiments, the synthetic mRNA comprises at least one unnatural nucleobase. In some embodiments, all nucleobases of a certain class have been replaced with unnatural nucleobases (e.g., all uridines in a polynucleotide disclosed herein can be replaced with an unnatural nucleobase, e.g., 5-methoxyuridine). In some embodiments, the polynucleotide (e.g., a synthetic RNA or a synthetic DNA) comprises only natural nucleobases, i.e., A, C, T and G in the case of a synthetic DNA, or A, C, T, and U in the case of a synthetic RNA. As used herein, the term "encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as, for example, a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase “nucleotide sequence that encodes a protein or a RNA” may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s). As used herein, the “percent identity” between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below. The percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (Needleman, Saul B. & Wunsch, Christian D. (1970). "A general method applicable to the search for similarities in the amino acid sequence of two proteins". Journal of Molecular Biology.48 (3): 443–53). The percent identity between two nucleotide or amino acid sequences may also be determined using for example algorithms such as EMBOSS Needle (pair wise alignment; available at www.ebi.ac.uk). For example, EMBOSS Needle may be used with a BLOSUM62 matrix, a “gap open penalty” of 10, a “gap extend penalty” of 0.5, a false “end gap penalty”, an “end gap open penalty” of 10 and an “end gap extend penalty” of 0.5. In general, the “percent identity” is a function of the number of matching positions divided by the number of positions compared and multiplied by 100. For instance, if 6 out of 10 sequence positions are identical between the two compared sequences after alignment, then the identity is 60%. The % identity is typically determined over the whole length of the query sequence on which the analysis is performed. Two molecules having the same primary amino acid sequence or nucleic acid sequence are identical irrespective of any chemical and / or biological modification. According to the invention a first amino acid sequence having at least 90% of identity with a second amino acid sequence means that the first sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second amino acid sequence. As used herein, the term “ANGPTL4” has its general meaning in the art and refers to theAngiopoietin-like protein 4 encoded by the ANGPTL4 gene. An exemplary human aminosequence for ANGPTL4 is SEQ ID NO:1. Human ANGPTL4 consists of 406 amino acids. Its protein structure is common to the angiopoietins, with a signal peptide directing secretion, an amino-terminal coiled-coil domain (CCD), a linker, and a carboxyterminal fibrinogen-like domain (FLD). ANGPTL4 undergoes processing, releasing CCD and FLD-containing fragments. The soluble CCD binds to lipoprotein lipase and converts the catalytically active dimeric form of the enzyme into inactive monomers, whereas the soluble FLD regulatesvascular biology. The CCD fragments consists of the amino acid sequence which ranges fromthe amino acid residue at position 22 to the amino acid residue at position 170. The FLDfragment consists of the amino acid sequence which ranges from the amino acid residue atposition 186 to the amino acid residue at position 406. SEQ ID NO:1 >sp|Q9BY76|ANGL4_HUMAN Angiopoietin-related protein 4 OS=Homo sapiens OX=9606 GN=ANGPTL4 PE=1 SV=2 MSGAPTAGAALMLCAATAVLLSAQGGPVQSKSPRFASWDEMNVLAHGLLQLGQGLREHAE RTRSQLSALERRLSACGSACQGTEGSTDLPLAPESRVDPEVLHSLQTQLKAQNSRIQQLF HKVAQQQRHLEKQHLRIQHLQSQFGLLDHKHLDHEVAKPARRKRLPEMAQPVDPAHNVSR LHRLPRDCQELFQVGERQSGLFEIQPQGSPPFLVNCKMTSDGGWTVIQRRHDGSVDFNRP WEAYKAGFGDPHGEFWLGLEKVHSITGDRNSRLAVQLRDWDGNAELLQFSVHLGGEDTAY SLQLTAPVAGQLGATTVPPSGLSVPFSTWDQDHDLRRDKNCAKSLSGGWWFGTCSHSNLN GQYFRSIPQQRQKLKKGIFWKTWRGRYYPLQATTMLIQPMAAEAASAs used herein, the term “subject”, “individual” or “patient" is used interchangeably andrefers to any subject for whom diagnosis, treatment, or therapy is desired, particularly humans. Other subjects may include cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and the like. In some preferred embodiments, the subject is a human. As used herein, the term “sepsis capillary leak syndrome” refers to a condition that is associated with sepsis and characterized by abnormal leakage of fluid from the blood vessels into the interstitial spaces, resulting in hypotension, edema, and organ dysfunction. Sepsis capillary leak syndrome is a life-threatening complication that requires prompt diagnosis and treatment. As used herein, the term “sepsis” has its general meaning in the art and represents a serious medical condition that is characterized by a whole-body inflammatory state. In addition to symptoms related to the provoking infection, sepsis is characterized by presence of acute inflammation present throughout the entire body, and is, therefore, frequently associated with fever and elevated white blood cell count (leukocytosis) or low white blood cell count andlower-than- average temperature, and vomiting. In particular, sepsis is defined as a deregulatedimmune response to infection, translating into life-threatening organs dysfunction, defined by a Sequential Organ Failure Assessment score of 2 more. Infection can be suspected or proven, or a clinical syndrome pathognomonic for infection. Septic shock is defined by infection and the need for vasopressors to maintain mean blood pressure ≥65mmHg and arterial lactate levels >2mmol / l. As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment. As used herein, the term "reduce" or other forms of the word, such as "reducing" or "reduction," is meant lowering of an event or characteristic (e.g., vascular leakage). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. As used herein, the terms "inhibit" and "inhibition" also refer to a reduction or preventionof vascular leakage. Vascular leakage is considered to be "reduced" when vascular leakage isreduced by at least 10% in a given permeability assay and preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, up to and including complete inhibition, or 100%.Methods for assessing vascular leakage are well known in the art (see e.g. Wollborn, Jakob, etal. "Diagnosing capillary leak in critically ill patients: development of an innovative scoring instrument for non-invasive detection." Annals of Intensive Care 11.1 (2021): 1-13.). As used herein, the term "prevent" or other forms of the word, such as "preventing" or "prevention," is meant to stop a particular event or characteristic (e.g., vascular leakage), to stabilize or delay the development or progression of a particular event or characteristic (e.g., vascular leakage), or to minimize the chances that a particular event or characteristic (e.g., vascular leakage) will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something couldbe prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed.As used herein, the term "therapeutically effective amount" is meant a sufficient amount ofthe active ingredient for treating or reducing the symptoms at reasonable benefit / risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination with the active ingredients; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. Methods of therapy: The first object of the present invention relates to a method of treating sepsis capillary leaksyndrome comprising administering to the patient a therapeutically effective amount of i) apolypeptide comprising an amino acid sequence having at least 90% of identity with the aminoacid sequence ranging from the amino acid residue at position 186 to the amino acid residue atposition 406 in SEQ ID NO:1 or ii) a polynucleotide encoding for a polypeptide comprising anamino acid sequence having at least 90% of identity with the amino acid sequence ranging fromthe amino acid residue at position 186 to the amino acid residue at position 406 in SEQ IDNO:1. The method of the present invention is particularly suitable for reducing and / or preventingvascular leakage in a patient suffering from sepsis. More particularly, the method of the presentinvention is suitable for reducing and / or preventing lipopolysaccharide-induced vascular hyperpermeability.As used herein, the term “lipopolysaccharide-induced vascular hyperpermeability” is acondition in which the blood vessels become more permeable or leaky due to the exposure to lipopolysaccharides (LPS), which are components of the outer membrane of gram-negative bacteria. LPS can trigger an inflammatory response in the body, leading to the activation of various signaling pathways and the release of cytokines and chemokines that affect the endothelial cells lining the blood vessels. As a result, the endothelial cells become more prone to contraction, retraction, and apoptosis, creating gaps between them that allow fluid and molecules to escape from the blood into the surrounding tissues. This can cause edema, hypotension, organ dysfunction, and shock, which are common symptoms of sepsis. The method of the present invention is particularly suitable for preventing acute respiratory distress syndrome. As used herein, the term "acute respiratory distress syndrome" (abbreviated ARDS) relates to a severe, life-threatening medical condition characterized by presence of a risk factor (e.g. pneumonia, pancreatitis, etc.), bilateral pulmonary infiltrates, and oxygen impairment not fully explained by cardiac failure. More specifically, the term ARDS as used herein relates to acute respiratory distress syndrome as convened in 2011 in the Berlin definition (ARDS Definition Task Force et al.2012 JAMA 307(23): 2526-2533). More particularly, the present invention is particularly suitable for preventing death in a patient suffering from sepsis capillary leak syndrome.In some embodiments, the polypeptide of the present invention further comprises the sequenceof a signal peptide. As used herein, the term "signal peptide" has its general meaning in the art and refers to a pre-peptide which is present as an N-terminal peptide on a precursor form of a protein. The function of the signal peptide is to facilitate translocation of the expressed polypeptide to which it is attached into the endoplasmic reticulum. The signal peptide is normally cleaved off in the course of this process. The signal peptide may be heterologous orhomologous to the organism used to produce the polypeptide. In some embodiments, thepolypeptide of the present invention comprises the signal peptide that ranger from the amino acid residue at position 1 to the amino acid residue at position 25 in SEQ ID NO:1. In some embodiments, the polypeptide of the present invention comprises the amino acid sequences as set forth in SEQ ID NO:1 having or more conservative mutation(s), preferably one or more conservative substitutions. As used herein, the term "conservative mutations" refers to amino acid modifications that do not significantly affect or alter the biologic function of the protein containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into a protein by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A “conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. Amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine. Amino acid substitutions may further be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and / or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid;positively charged amino acids include lysine and arginine; and amino acids with unchargedpolar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. Other families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).In some embodiments, it is contemplated that the polypeptides of the invention are modified inorder to improve their therapeutic efficacy. Such modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution. For example, the toxicity of potentially important therapeutic compounds can be decreased significantly bycombination with a variety of drug carrier vehicles that modify biodistribution. A strategy forimproving drug viability is the utilization of water-soluble polymers. Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake,change the permeability through physiological barriers; and modify the rate of clearance fromthe body. To achieve either a targeting or sustained-release effect, water-soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or aspendent groups on the polymer chain. Polyethylene glycol (PEG) has been widely used as adrug carrier, given its high degree of biocompatibility and ease of modification. Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity. PEG can be coupled to active agents through the hydroxyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule. In a different approach, copolymers of PEG and amino acids were explored as novel biomaterials which would retain the biocompatibility properties of PEG, but which would have the added advantage of numerous attachment points per molecule (providing greater drug loading), and which could be synthetically designed to suit a variety of applications. In some embodiments, the polypeptide of the invention is fused a Fc domain of an immunoglobulin. Suitable immunoglobins are IgG, IgM, IgA, IgD, and IgE. IgG and IgA are preferred IgGs are most preferred, e.g. an IgG1. Said Fc domain may be a complete Fc domain or a function-conservative variant thereof. The polypeptide of the invention may be linked to the Fc domain by a linker. The linker may consist of about 1 to 100, preferably 1 to 10 amino acid residues. According to the invention, the polypeptide of the invention may be produced by conventional automated peptide synthesis methods or by recombinant expression. General principles fordesigning and making proteins are well known to those of skill in the art. The polypeptides ofthe invention may be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available. The polypeptides of the invention may also be synthesized by solid-phase technology employing an exemplary peptide synthesizer such as a Model 433A from Applied Biosystems Inc. The purity of any given protein; generated through automated peptide synthesis or through recombinant methods may be determined using reverse phase HPLC analysis. Chemical authenticity of eachpeptide may be established by any method well known to those of skill in the art. As an alternative to automated peptide synthesis, recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a protein of choice is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression as described herein below. A variety of expression vector / host systems may be utilized to contain and express the peptide or protein coding sequence. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus, see Ghosh et al., 2002); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmid); or animal cell systems. Mammalian cells that are useful in recombinant protein productions include but are not limited to VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells (such as COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and 293 cells. Mammalian host systems for the expression of recombinant proteins also are well known to those of skill in the art. Host cell strains may be chosen for a particular ability to process the expressed protein or produce certain post-translation modifications that will be useful in providing protein activity. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing which cleaves a "prepro" form of the protein may also be important for correct insertion, folding and / or function. Different host cells such as CHO, HeLa, MDCK, 293, WI38, and the like have specific cellular machinery and characteristic mechanisms for such post-translational activities and maybe chosen to ensure the correct modification and processing of the introduced, foreign protein.In some embodiments, the polynucleotide of the present invention is a messenger RNA (mRNA).In some embodiments, the polynucleotide comprises a sequence that has been codon optimizedfor expression in a mammalian cell. Codon optimization refers to the discovery that the frequency of occurrence of synonymous codons (i.e., codons that code for the same amino acid) in coding DNA is biased in different species. Such codon degeneracy allows an identical polypeptide to be encoded by a variety of nucleotide sequences. A variety of codon optimization methods is known in the art, and include, e.g., methods disclosed in at least U.S. Pat. Nos. 5,786,464 and 6,114,148. In order to confirm the presence of the polynucleotide in the donor cell, a variety of assays may be performed. Such assays include, for example, "molecular biological" assays well known such as Southern and Northern blotting, RT-PCR and quantitative PCR; or "biochemical" assays, such as detecting the presence or absence of a particular peptide. In some embodiments, the polynucleotide of the present invention is operatively linked to oneor more control sequences . As used herein, the term "control sequence'" refers collectively topromoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translatedin an appropriate host cell. In particular, the control sequence is a "promoter" sequence, whichis used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3'-direction) coding sequence. Transcription promoters can include "inducible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), "repressible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and "constitutive promoters”. In some embodiments, the polypeptide or the polynucleotide of the present invention is incorporated into a particle. As used herein, the term “particle” refers to a small object that behaves as a whole unit with respect to its transport and properties i.e. a discrete unit of matter, where the atoms or molecules from which it is formed essentially embody the particle. As used herein, the term “incorporate” refers to the introduction or insertion of a substance or objectinto or onto a particle of the present invention. As used herein, the term “incorporating” refersto introducing or inserting a substance or object into or onto the particle of the present invention.Any particles which have been described in the art for cargo delivery into cells may be used.Such nanoparticles include for example liposomes, micelles, extracellular vesicles,nanospheres, and nanoparticles. Examples of these include viral particles (which normally have a size of 20 nm to 300 nm), virus-like particles (e.g. particles that are composed of only the shell of a viral particle), HDL and LDL nanoparticles (which normally have a size of 5-30 nm), self-assembled nanoparticles, bacterial particles, and extracellular vesicles. In some embodiments, the nanoparticles comprises at least a core with one or more polymers, or their copolymer, such as, e.g., one or more of dextran, carboxymethyl dextran, chitosan, trimetylchitosan, polyvinylalcohol (PVA), polyanhydrides, polyacylates, polymethacrylates, polyacylamides, cellulose, hydromellose, starch, dendrimers, polyamino acids, polyethyleneglycols, polyethyleneglycol-co-propyleneglycol, aliphatic polyesters, including poly(lactic acid (PLA), poly(glycolic acid), and their copolymers including poly(lactic-co- glycolylic)acid (PLGA), or poly(^-caprolactone). Other suitable polymers may comprise polyamino acid selected from the group consisting of poly(g-glutamic acid), poly(a-aspartic acid), poly(e-lysine), poly(a-glutamic acid), poly(a-lysine), poly-asparagine, or derivatives thereof, and mixtures thereof. In general the surface of the nanoparticles may also be functionalised or coated to produce a desirable physical characteristic such as solubility, biocompatibility, and for facilitating chemical linkages with other biomolecules, such as the syncytin-1 fusion protein of the present invention. In some embodiments, the surface of the nanoparticles can be functionalized by incorporating one or more chemical linkers such as, without limitation: carboxyl groups, amine groups, carboxyl / amine, hydroxyl groups, polymers such as silane, dextran or PEG or their derivatives.In some embodiments, the particle is a virus particle or a virus-like particle. As it is readilyunderstood by the one skilled in the art, a virus particle that is used according to the invention may be selected in a group comprising Moloney murine leukemia virus-derived vector particles, Bovine immunodeficiency virus-derived particles, Simian immunodeficiency virus-derived vector particles, Feline immunodeficiency virus-derived vector particles, Human immunodeficiency virus-derived vector particles, Equine infection anemia virus-derived vector particles, Caprine arthritis encephalitis virus-derived vector particle, Baboon endogenous virus- derived vector particles, Rabies virus-derived vector particles, Influenza virus-derived vector particles, Norovirus-derived vector particles, Respiratory syncytial virus-derived vector particles, Hepatitis A virus-derived vector particles, Hepatitis B virus-derived vector particles, Hepatitis E virus-derived vector particles, Newcastle disease virus-derived vector particles, Norwalk virus-derived vector particles, Parvovirus-derived vector particles, Papillomavirus- derived vector particles, Yeast retrotransposon-derived vector particles, Measles virus-derivedvector particles, and bacteriophage-derived vector particles. In some embodiments, the virusparticle of the present invention is a retrovirus-derived particle. In some embodiments, the virus particle of the present invention is a lentivirus-derived particle. In some embodiments, the polypeptide or polynucleotide of the present invention is formulated using one or more lipid-based structures that include but are not limited to liposomes, lipoplexes, or lipid nanoparticles (Paunovska, Kalina, David Loughrey, and James E. Dahlman. "Drug delivery systems for RNA therapeutics." Nature Reviews Genetics (2022): 1-16). In some embodiments, the particle is a lipid nanoparticle. As used herein, the term "lipid nanoparticle" or "LNP" has its general meaning in the art and refers to a nanoscale particle composed of lipids that can encapsulate various payloads, such as nucleic acids, proteins, peptides, or small molecules. Lipid nanoparticles are widely used for drug delivery and gene therapy applications, as they can protect the payload from degradation, enhance its stability and solubility, and facilitate its cellular uptake and release. Lipid nanoparticles typically have a core-shell structure, with a hydrophobic core surrounded by a hydrophilic shell that containsone or more lipid bilayers. The composition and properties of the lipid nanoparticle can betailored to the specific payload and target tissue. Lipid nanoparticle formulations typicallycomprise a lipid, in particular, an ionizable cationic lipid, and further comprise a neutral lipid, a sterol and a molecule capable of reducing particle aggregation, for example a PEG or PEG- modified lipid. The lipid can be selected from, but is not limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, PEGylated lipids and amino alcohol lipids. In some embodiments, the lipid is a cationic lipid such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA and amino alcohol lipids. The amino alcohol cationic lipid can be the lipids described in and / or made by the methods described in US Patent Publication No. US20130150625. As a non-limiting example, the cationic lipid can be 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1- yloxy]-2-{[(9Z,2Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (Compound 1 in US20130150625); 2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-1- yloxy]methyl}propan-1-ol (Compound 2 in US20130150625); 2-amino-3-[(9Z,12Z)-octadeca- 9,12-dien-1-yloxy]-2-[(octyloxy)methyl]propan-1-ol (Compound 3 in US20130150625); and 2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,12Z)-octadeca-9,12- dien-1-yloxy]methyl}propan-1-ol (Compound 4 in US20130150625); or any pharmaceutically acceptable salt or stereoisomer thereof. Nanoparticle formulations of the present disclosure can be coated with a surfactant or polymer in order to improve the delivery of the particle. In some embodiments, the nanoparticle is coated with a hydrophilic coating such as, but not limited to, PEG coatings and / or coatings that have a neutral surface charge. The hydrophilic coatings can help to deliver nanoparticles with larger payloads such as, but not limited to, polynucleotides within the central nervous system. As a non-limiting example nanoparticles comprising a hydrophilic coating and methods of making such nanoparticles are described in US Patent Publication No. US20130183244.Typically the active ingredient of the present invention (i.e. the polypeptide, polynucleotide orparticle of the present invention) is combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. As used herein, the term “pharmaceutical composition” refers to a composition described herein, or pharmaceutically acceptable salts thereof, with other agents such as carriers and / or excipients. The pharmaceutical compositions as provided herewith typically include a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical-Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. FIGURES:Figure 1 shows the structure of the different testes constructs and their production.Figure 2 shows that c-ANGPTL4 fragment is required to counteract thrombin-induced permeability in HUVEC. Figure 3 shows that c-ANGPTL4 fragment is required to counteract thrombin-induced permeability in HPMEC.Figure 4 shows that c-ANGPTL4 fragment reduces capillary leakage in LPS shock model.Figure 5 shows that c-ANGPTL4 fragment improves survival rate in LPS shock model.Figure 6 shows the effect of fl-ANPGTL4 and c-ANPGTL4 on triglycerides level in theLPS model.Figure 7 shows that ANGPTL4 plasma levels are associated with higher survival rate andcorrelated with pro-inflammatory biomarkers in septic patients. EXAMPLE: Material & Methods:1. Cell culture and reagentsHuman umbilical vein endothelial cells (HUVEC) were prepared and grown as previously reported16in Endothelial Cell Growth Medium 2 (ECGM2, Promocell, Heidelberg, Germany). Human Pulmonary Microvascular Endothelial Cells (HPMEC) were from Promocell(Heidelberg, Germany) and cultured in Endothelial Cell Growth Medium MV (MV, PromocellHeidelberg, Germany). Experiments were performed between passages 2 and 6 For coatingexperiments, collagen I (Corning™, Waltham, MA, USA) wase diluted in 20 mM acetic acidbefore coating culture dishes for 30 min at 37°C. Human full-length Angiopoietin-like 4 (fl-ANGPTL4), n-ANGPTL4 (extending from residues 22 to 70) and c-ANGPTL4 (extendingfrom residues 171 to 406) were produced and purified as previously described14.2. Measurement of cell permeabilityLabel-free real-time measurement of transendothelial resistance was measured using an xCELLigence RTCA (Real Time Cell Analysis) from Agilent (Santa Clara, CA, USA). HUVEC and HPMEC were cultured for for 15-20h on gold-electrode‐coated plates E-Plate 16 until they formed a confluent monolayer in a 37 °C incubator in the presence of 5% CO2 upon constant impedance monitoring. Once a stable cell monolayer formed, the E-Plate 16 was then removed from the RTCA station and supplemented media ECGM2 (HUVEC) or MV (HPMEC)was replaced by basal medium ECBM2 (100µL) for 2 hours, before treatment with 2U / mLthrombin (Sigma Aldrich, Saint Quentin Fallavier, France) + / - human full-length ANGPTL4(fl-ANGPTL4), Coiled-coiled domain (n-ANGPTL4) and Fibrinogen-like domain (c-ANGPTL4) at 90 µM directly in the incubator. Cells permeability was monitored every 10 minfor 3-4h.3. Immunostaining of adherens junctions and images analysesCells were seeded at confluency in 8 wells-ibiTreat µ-slides (Ibidi, Gräfelfing, Germany) andcultured for 72h before fixation using 4% paraformaldehyde (PFA) and permeabilization with0.5% Triton-X-100. Non-specific binding was saturated using PBS containing 10% normal goat serum (ThermoFisher Scientific™, Waltham, MA, USA). Alexa Fluor™488 monoclonal VE- cadherin antibody (53-1449-42, Invitrogen, Carlsbad, CA, USA) was incubated in the presence of 1% normal goat serum and 0.01% Triton-X-100. Images were acquired with Z1 Axio Observer epifluorescence microscope equipped with an Apotome2 using 63x objective equipped with Zen 2 Pro software (Zeiss, Oberkochen, Germany).4. Quantification of adherens junctionsThe analysis of adherens junctions morphology was performed in confluent monolayers ofHUVECs stained for VE- cadherin. Between 5 and 10 images of 0.045 mm2 per condition perexperiment were acquired. Four morphological categories were defined: straight, reticular, fingers and thick / reticular using custom macros25written for the Fiji software26(v2.14.0 / 1.54f, Open-source software, USA), and ilastik27(v1.4.0, Open-source software, Germany). First, Find Focused Slices plugin28was used to select images with optimal focus quality within the stack (i.e. with a variance larger than 80% of the maximum variance). A maximum intensity Z-projection of these slices was performed. The image was then segmented using the PixelClassification workflow provided by ilastik. The first step consisted in selecting (between 5 and 10) training images and annotating them manually by associating pixels to a class. 5-pixel classes are defined: background and straight, reticular, fingers and thick / reticular. The Random Forest classifier was then trained, including all available 2D pixel features (e.g. intensity, edge, and texture) across all given scales. Once the model trained, it was applied to all images. Given the large variations in intensity that can occur between two image acquisition batches, the model was re-trained for each new batch of images. A 2D median filter (radius = 5) was applied onthe resulting image in order to smoothen pixel classification. The area covered by each adherensjunctions type was extracted from the segmentation result. More precisely, the image was divided into squares of 64x64 pixels. Squares were assigned to one of the four morphologies. If at least 50% of the square area is covered by one unique junction type, it was thus set to belong to the corresponding morphology. Finally, the percentage of squares belonging to each junction morphology was computed (data not shown).5. Animal procedures5.1. Animal careExperiments were performed in accordance with the ethical guidelines for animal care of theEuropean Community Council (directive 2010 / 63 / UE) and were approved by the local ethicalcommittee (institutional Animal Care and Use Committee of Collège de France) and theMinistère de l’Éducation Nationale, de l’Enseignement Supérieur et de la Recherche(APAFIS#37706-2022061517327918). All experiments were performed using 8-week-oldmale wild-type (WT) C57BL / 6J mice purchased from Janvier Labs (Saint Berthevin, France).Mice were housed in a controlled environment (light from 7am to 7pm, constant humidity andtemperature). Buprenorphine (Buprécare® 0.3mg / ml Solution, Axiences, Pantin, France) wasadministrated 30 mn prior the experiments.5.2. Experimental endotoxemic shock modelAbdominal endotoxemia was modeled by intraperitoneal (IP) LPS injection in mice. Every setof experiments included a control which received IP injection of LPS-free phosphate bufferedsaline (PBS, vehicle). Briefly, purified LPS of Escherichia coli serotype 055: B5 (SigmaAldrich, Saint Quentin Fallavier, France) was suspended in endotoxin-free PBS and vigorouslymixed before use. C57BL / 6 mice received a single injection (25mg / kg of body weight) of LPSintraperitoneally followed or not by three injections of c-ANGPTL4 (75 µg / kg of body weight)intraperitoneally every 2 hours after LPS injection; i.e. t= 2h; 4h; 6h. At time point 8h, micewere sacrificed by cervical dislocation and organs, blood, lungs and bronchoalveolar lavagefluid (BALF) were collected.For survival studies, Mice were monitored every 2 hours for 3 days. Mouse body temperaturewere monitored every 2 hours for 3 days using an Infrared camera (Teledyne FLIR E8,Wilsonville, Oregon, USA). Blood was collected by intracardiac puncture in EDTA-coated tubes. Plasmas were separatedby centrifugation at 8,000 x g for 10 min at 4°C and were stored at -20°C until use. Organs(lungs and hearts) were weighed and then dried in a ventilated oven at 56 °C for 72 hours beforeweighing again. Wet / Dry ratios were calculated as a measure of tissue edema.BALF collection was performed as follows: the thorax was opened and a cannula was introduced to the trachea. The fluid collection was performed by using a total of 300 µL PBSdivided into two washes. Then, plasmas and BALF were gently mixed with CryoStor® CS10(Stem Cell Technologies, Vancouver, Canada) solution (ratio 1:18) and stored at -80°C until use for flow cytometry analyses.Lungs were fixed by 4% PFA overnight, dehydrated, paraffin-embedded and sectioned at 5 μmand immunostained using CD45 monoclonal antibody (14-0451-82, Invitrogen, Carlsbad, CA,USA). Whole sections were captured using a slide scanner Axioscan 7 equipped with Zen 3.0Slidescan software (Zeiss, Oberkochen, Germany). Image analyses and quantifications were performed using QuPath software (v0.4.4, Open-source software, UK). Briefly, CD45 channelwas segmented using the pixel classification tool provided by QuPath. Three classes weredefined: CD45-positive cells, tissue and background. A random trees pixel classifier was trained on annotations done on several images, to improve robustness of the model to variations in intensity between images. Gaussian, Laplacian of Gaussian, and Gradient magnitude pixelfeatures are included, across scale 1.5.3. Plasma biochemical analysesCytokines concentrations were determined by ELISA according to manufacturer protocols(MCP-1 / CCL2; RAB0055 Sigma Aldrich, Saint Quentin Fallavier, France and TNF- α;BMS607-3 ThermoFisher Scientific™, Waltham, MA, USA).5.4. Flow cytometryWhole blood and bronchioalveolar lavage fluid (BALF) samples were cryopreserved at -80°C in CryoStor® CS10 following the manufacturer’s instructions (Stemcell Technologies, Saint- Égrève, France). Samples were thawed at 37°C for 1 minute before preparation for flow cytometry staining. Samples were stained using 2 panels (data not shown) in 50 µL of staining buffer for 30 minutes at 4°C, protected from light. Panel 1 was used to analyze differentleukocyte populations. 100 µL of sample material (whole blood or BAL) was used, andsubjected to red blood cell lysis, staining, and washing as previously described29. FoxP3Intracellular staining was carried out using the eBioscience™ Foxp3 / Transcription FactorStaining Buffer Set (ThermoFisher Scientific™, Waltham, MA, USA).Panel 2 was used toquantify red blood cells and platelets.20 µL of sample material (whole blood or BAL) was used and subjected to staining and washing as previously described. Fully stained samples wereresuspended in 200 µL FACS buffer. Panel 1 was acquired on an ID7000™ Spectral CellAnalyzer (Sony Inc., Tokyo, Japon). Unmixing was done using the Sony ID7000™ software(v1.1.10). Panel 2 was acquired on a MACSQuant10® Analyzer (Miltenyi Biotec, BergischGladbach, Germany). FCS files were exported and analyzed in FlowJo v10.7.1 (v.10.7.3, BDBiosciences, Heidelberg, Germany).6. Clinical study (FROG-ICU Cohort)6.1. Study design and patientsWe analyzed data from the French and European Outcome Registry in Intensive Care Units (FROG-ICU). The FROG-ICU study (www.clinicaltrials.gov / show / NCT01367093) was a prospective, observational, multicenter cohort study, designed to assess all-cause one-year mortality after ICU discharge and to identify the mortality risk factors during the year following discharge from the ICU30. The study protocol has been previously published31. Briefly, the study was conducted in France and in Belgium following and was approved by the ethical committee according to the French law32. The study involved ICUs of 21 centers. The study cohort included 2087 consecutive patients, who were admitted to the ICU in any of the participating centers from August 2011 to June 2013 when the following inclusion criteria were met: invasive mechanical ventilation support for at least 24h and / or treatment with a positive inotropic agent for more than 24h. Non-inclusion keys criteria were: <18 years old, severe brain injury or brain death or a persistent vegetative state, pregnancy or breastfeeding, transplantation in the past 12 months, not expected to survive or to leave the hospital and / or no social security coverage30.6.2. Data collection and biological samplesIn this FROG-ICU post hoc study, the following patient data were collected at the time of inclusion: demographics, past medical history, hemodynamic, and severity of disease classification scores. In addition, biological routine parameters were collected at inclusion. A biobank was created and stored at -80°C with blood samples collected within 24h after patient inclusion. The following biomarkers were centrally measured a posteriori: plasma levels of hs troponin I (Abbott, Abbott Park, IL, USA), N-Terminal pro-Brain Natriuretic Peptide (NT- proBNP, Roche Diagnostics GmbH, Mannheim, Germany), proenkephalin A 119–159 (penKid, Sphingotec GmbH, Hennigsdorf, Germany), galectin-3 (Abbott, Abbott Park, IL, USA), interleukin 6 (IL-6, Elecsys, Roche, Penzberg Germany), dipeptidyl peptidase 3 (DPP3- luminometric immunoassay, 4TEEN4 Pharmaceuticals GmbH ), bioactive-adrenomedullin (Adrenomed GmbH, Hennigsdorf, Germany), pro-septin (4TEEN4 Pharmaceuticals GmbH), Angiopoietin-like 4 DuoSet ELISA (DY3485, R&D Systems, Minneapolis, USA) according tothe manufacturer’s protocol.6.3. Inclusion dataPatients with a diagnostic of sepsis or septic shock according to Sepsis 2 definition and plasma available at inclusion were included in this post hoc analysis.6.4. ObjectivesThe primary objective of this post hoc analysis was to describe the association between ANGPTL4 plasma level at time of inclusion and 90-day mortality in sepsis or septic shock patients.6.5. Statistical analysisData were expressed as median (inter-quartile range, IQR) for continuous variables andfrequency (percentage) for categorical variables. Numerical data were compared using Kruskal-Wallis test, while categorical variables were compared using χ2 test or Fisher’s exact test, asappropriate. Ninety-day survival curves were drawn according to tercile of ANGPTL4 atinclusion. Association between ANGPTL4 at inclusion on 90-day mortality in a Cox unadjustedmodel using restricted cubic splines with 3 knots placed at 10th, 50th, 90th percentiles ofANGPTL4 was performed. The reference was ANGPTL4 at 0 pg / ml. A Principal ComponentAnalysis (PCA) was employed to reduce the dimensionality of the dataset and identify patternsin the variance of 10 selected biomarkers. This technique transformed the original variablesinto a new set of uncorrelated components, with the first two components capturing themaximum variance. Hazard ratio with 95% confidence interval were computed withoutadjustment. No imputation of missing data was performed. A two-tailed p-value of less than0.05 was considered significant. Statistical analyses were performed using R v4.3.1 (RFoundation for Statistical Computing, Vienna, Austria).7. Statistical analysesData were collected using Microsoft Excel. All data are shown as mean values ± SEM. Prism9.2.0 software (GraphPad, San Diego, CA, USA) was used to perform statistical analyses.Shapiro-Wilk test was used to test the normality of value distribution before performing parametric or non-parametric statistical analyses. Mann-Whitney U test or Student’s t test were used to assess the significance of differences between two groups. In the presence of more thantwo groups, a One-ANOVA or Kruskall-Wallis’ test was performed and a Dunn’s correctionwas applied when groups had different variances. A value of *p<0.05 was consideredstatistically significant (ns, not significant; *p<0.05, **p<0.01, ***p<0.001 and****p<0.0001). Results: Coiled-coil domain (CCD) and Fibrinogen-like domain (FLD) of ANGPTL4. Recombinant CCD (n-ANGPTL4) and FLD (c-ANGPTL4) mutants of full-length ANGPTL4 (fl-ANGPTL4) were generated and purified as previously described14(Fig. 1). Briefly, themutant proteins were produced in stably transfected Chinese hamster ovary (CHO) cell lineslacking the dihydrofolate reductase (CHODHFR) and purified as previously described14.Coomassie blue staining shows high degree of purity of fl-ANGPTL4 and n-ANGPTL422-170,c-ANGPTL4171-406 domains (Data not shown). c-ANGPTL4 fragment is required to counteract thrombin-induced permeability in HUVEC and HPMEC.To explore whether fl-ANGPTL4, n-ANGPTL4 and c-ANGPTL4 fragments have an effect onbarrier function of endothelial monolayers, we conducted real-time impedance measurementsusing the xCELLigence system. First, HUVEC were treated with 2U / ml thrombin, a well-known permeability enzyme, leading to a maximal decrease in impedance in 25-30 min and anoverall duration of approximately 3h, around which impedance has stabilized at a level slightlyunder baseline (Fig. 2A, C, E) The calculation of area under curve (AUC) showed a significantreduction in impedance with thrombin (-1.13 ± 0.07 u.a.; p<0.0001) compared to the control(Fig. 2B, D). The addition of fl-ANGTPL4 at 90 µM counteracted the thrombin-induced effecton cell permeability (-0.80 ± 0.09 u.a.; p<0.01) (Fig. 2A, B). When HUVEC were co-treatedwith thrombin and n-ANGPTL4 fragment at 90 µM, no significant effect was observed on cellpermeability as compared to thrombin only (Fig. 2C, D). In contrast, the c-ANGPTL4 at 90µM was able to counteract thrombin effect (-0.58 ± 0.05 and -1.15 ± 0.04 u.a., respectively;p<0.0001) (Fig 2.E, F). We then conducted the same experiments using HPMEC. Similarly to HUVEC, 2U / mlthrombin decreased the impedance at 25-30 min (Fig. 3A, C, E) which resulted in a significantreduction in AUC (-1.55 ± 0.16 u.a.; p<0.001) compared to the control (Fig. 3B, D, F). Co-treatment with 90 µM fl-ANGPTL4 showed an increase in impedance (-0.98 ± 0.20 u.a.; p<0.05) (Fig.3A, B). n-ANGPTL4 had no effect on thrombin effect in HPMEC (Fig.3C, D).Treatment with 90 µM c-ANGPTL4 counteracted thrombin effect (-0.76 ± 0.17 u.a.; p<0.01)(Fig.3E, F). These data confirm the prominent role of ANGPTL4 and particularly its c-terminaldomain in cell permeability regulation.c-ANGPTL4 restores thrombin-induced changes in adherent junctions morphology ofHUVEC and HPMEC. We then assess whether thrombin-induced effect on permeability could be linked to amodulation of junctions morphology in endothelial cells, we defined four junctional categories :straight, reticular, fingers and thick / reticular. The effect of 90 µM c-ANGPTL4 on junctionswas also explored in HUVEC and HPMEC by immunostaining of VE-cadherin (Fig. 4, 5,respectively). First, images of HUVEC adherent junctions were acquired (Data not shown) andthen quantified (Data not shown). Whereas control cells showed mostly reticular junctions,thrombin reduced reticular and straight and junctions (15.78 ± 1.39% vs. 46.04 ± 1.90% ;p<0.0001 and 3.50 ± 0.58% vs. 21.87 ± 3.18%; p<0.001, respectively) and increased finger andthick / reticular ones (68.53 ± 1.95% vs 27.58 ± 3.66% and 12.19 ± 1.44% vs 4.51 ± 0.98% respectively; p<0.0001 and p<0.001). Interestingly, the addition of 90 µM c-ANGPTL4 restored morphological changes towards control values by increasing straight and reticular junctions (14.36 ± 4.09% and 25.47 ± 5.84% respectively; p<0.05) and decreasing fingers and thick / reticular junctions ones (53.06 ± 7.61% and 7.11 ± 1.73% respectively; p<0.05) (Data not shown). To summarize, in basal conditions, straight junctions represented 21.87% of totaladherent junctions, reticular junctions 46.04%, fingers junctions 27.58% and thick / reticularjunctions 4.51% vs. 3.50%, 15.78%, 68.53% and 12.19%, respectively, in cells treated withthrombin only. Treatment with c-ANGPTL4 restored straight and reticular junctions (14.36% and 25.47%, respectively) but decreased fingers and thick / reticular junctions (53.06% and 7.11%, respectively) (Data not shown).Thrombin also reduced the proportion of straight and reticular junctions (10.47 ± 0.85% vs26.95 ± 2.17% and 29.73 ± 1.78% and 52.09 ± 2.73% respectively; p<0.01) and increased thepercentage of fingers and thick / reticular ones (51.66 ± 4.07% vs 16.97 ± 4.30%, respectively;p<0.001) compared to control untreated cells. The addition of 90 µM c-ANGPTL4 increasedstraight and reticular junctions (23.29 ± 2.25 % and 36.94 ± 4.16% respectively; p<0.05 andns) and decreased fingers and thick / reticular proportions (33.11 ± 4.03 % and 6.66 ± 0.56%respectively; p<0.05 and ns) (Data not shown). Altogether, straight junctions represented 26.95%, reticular junctions 52.09%, fingers junctions 16.97% and thick / reticular junctions3.99% of total adherent junctions respectively in the control situation vs. 10.47%, 29.73%,51.66% and 8.14%, respectively, in cells treated with thrombin. Junction morphologicalchanges were reestablished towards control values by c-ANGPTL4 treatment (23.29% and36.94%, straight and reticular respectively) and (33.11% and 6.66%, fingers and thick / reticular,respectively) (Data not shown). c-ANGPTL4 fragment reduces capillary leakage in endotoxemic shock model and improves survival rate in mice.We further undertook the investigation c-ANGPTL4 effect on vascular permeability in vivo.Permeability was induced using a single intraperitoneal injection of LPS followed or not bythree injections of c-ANGPTL4 every 2 hours after LPS injection, i.e. t= 2h; 4h; 6h (Fig.4). At time point 8h, mice were sacrificed and organs, blood, lungs and bronchoalveolar lavage fluid(BALF) were collected (Fig.4A). Wet / Dry ratio in mice treated with c-ANGPTL4 issignificantly decreased in left and right lungs compared to the control group injected with LPSonly (4.40 ± 0.06 vs 4.69 ± 0.06 u.a., respectively, in left lung and 4.44 ± 0.04 vs 4.68 ± 0.06u.a., respectively, in right lung; p<0.05). No significant differences were observed in the heart (Fig.4B) In fact, our results showed no effects of c-ANGPTL4 injections on plasmas pro-inflammatory cytokines MCP-1 and TNF-α (data not shown), on CD45-positive cellsrecruitment in lungs (data not shown). Furthermore, c-ANGPTL4 did not affect circulatingimmune cell frequencies, red blood cells (RBC) and platelets counts whole blood (data notshown) and bronchioalveolar lavage in mice (data not shown).c-ANGPTL4 was also used to study the survival rate of mice treated with LPS (Fig. 5A).Seventy-five percent of mice treated with LPS only (control group) died within 28 to 72 hoursafter LPS injection, in contrast to 12.5% of mice treated with c-ANGPTL4. These data indicatedthat treatment with c-ANGPTL4 after LPS injection increased the survival rate (p=0.009) ofmice with LPS-induced endotoxemia.We also measured body temperature changes in mice after LPS injection (Fig.5B). There wasan initial hypothermic response that lasted about 20h, during which body temperature droppedto 28.75 ± 2.21°C and 27.66 ± 1.88°C in control group and c-ANGPTL4 treated group,respectively (Fig.5B). After this hypothermic response, body temperature increased and wasmore elevated in mice treated with c-ANGPTL4 as compared to control mice (Fig.5B). Figure7C shows final body temperatures measured in each mouse, thus indicating that mice treated with c-ANGPTL4 have a higher body temperature than their control counterparts which died when temperature dropped under 31°C. As fl-ANPGTL4 is in inhibitor of LPL, we next sought to evaluate the effect of fl-ANPGTL4and c-ANPGTL4 on triglycerides level in the LPS model (Figure 6). As reported, LPS induceslipolysis that translates to increased triglycerides level (LPS vs. Control). As expected, fl- ANGPTL4 injection inhibited LPL and led to further increased triglycerides level (LPS vs. LPS+fl-ANPGTL4). In contrast, c-ANGPTL4 injection did not affect triglycerides level neither in the control mice (control vs. control + c-ANPGTL4) nor in LPS-treated mice (LPS vs. LPS+c-ANPGTL4). The results strongly suggest that c-ANPGTL4 retains vasculo-protective effects as compared to fl-ANPGTL4 but its mechanism of action is independent of any effect on triglycerides levels. ANGPTL4 plasma levels are associated with higher survival rate and correlated with pro- inflammatory biomarkers in septic patients. Of the 2,087 patients at inclusion, 436 were diagnosed with sepsis or septic shock. Among these, plasma samples at the time of inclusion were available for 384 patients, who wereultimately included in the study. Table 1 describes the main characteristics at admissionaccording to tercile of ANGPTL4 values. Briefly, patients in medium and high terciles of ANGPTL4 at inclusion were older (p=0.018) and heavier (p=0.023). In their medical history, patients in medium and high terciles of ANGPTL4 presented also more hypertension (p=0.005 and peripheral vascular disease (p=0.005). At admission SOFA score and SAPSII were higher for patients in medium and high terciles of ANGPTL4. However, hemodynamic parameters (HR, SBP and DBP) were roughly similar within groups. Conversely, creatinine was increasedfor patients in high and medium compared to low ANGPTL4 terciles (p<0.0001). Figure 7Adescribed the 90-day survival according to the tercile of ANGPTL4 at inclusion. Medium and high tercile are sur imposed with, compared to the low tercile, a HR for medium tercile of 1.9895%CI [1.31 – 2.99] and high tercile of 2.00 95%CI [1.32 – 3.02]. Association betweenANGPTL4 values and 90-day survival is depicted in Figure 7B. In Figure 7C on PCA, thepositions of biomarkers relative to principal component 1 (variance 30%) and principal component 2 (variance 39%) axes provide insights into how these variables contribute to the variation (69%) captured in the data. The proximity of biomarkers to each other suggestscorrelations, and their distribution along the axes indicates the degree of their contribution toeach principal component. Briefly, ANGPTL4 is highly correlated with IL-6 and CRPbiomarkers and contributes importantly to the principal component 2 of the data. Table 1:

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Claims

CLAIMS:

1. A method of treating sepsis capillary leak syndrome comprising administering to thepatient a therapeutically effective amount of i) a polypeptide comprising an amino acid sequence having at least 90% of identity with the amino acid sequence ranging from the amino acid residue at position 186 to the amino acid residue at position 406 in SEQ ID NO:1 or ii) a polynucleotide encoding for a polypeptide comprising an amino acid sequence having at least 90% of identity with the amino acid sequence ranging from the amino acid residue at position 186 to the amino acid residue at position 406 in SEQ ID NO:1.

2. The method according to claim 1 for reducing and / or preventing vascular leakage in apatient suffering from sepsis.

3. The method according to claim 1 for reducing and / or preventing lipopolysaccharide-induced vascular hyperpermeability.

4. The method according to claim 1 for preventing acute respiratory distress syndrome.

5. The method according to claim 1 for preventing death in a patient suffering from sepsiscapillary leak syndrome.

6. The method according to any one of claim 1 to 5 wherein the polypeptide comprises theamino acid sequences as set forth in SEQ ID NO:1 having or more conservative mutation(s), preferably one or more conservative substitutions.

7. The method according to any one of claim 1 to 6 wherein the polynucleotide of thepresent invention is a messenger RNA (mRNA).

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