Methods and compositions for treating respiratory disorders

Abstract

The present disclosure generally relates methods and compositions comprising VEGF-D, which methods and compositions can be used in the treatment of respiratory disorders. In some instances, the methods and compositions can be used for treating acute lung injury (ALI) or acute respiratory distress syndrome (ARDS).

Description

[0001] METHODS AND COMPOSITIONS FOR TREATING RESPIRATORY DISORDERS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority under 35 U.S. C. § 119(e) to U.S. Provisional Application No. 63 / 518,349, filed August 9, 2023; U.S. Provisional Application No. 63 / 508,428, filed June 15, 2023; and U.S. Provisional Application No. 63 / 508,140, filed June 14, 2023; the entire contents of each of which are hereby incorporated by reference.

[0004] REFERENCE TO SEQUENCE LISTING

[0005] The Sequence Listing concurrently submitted herewith as an XML-formatted text file named “047162-7469WOl_sequence_listing.xml” created on June 7, 2024, and having a size of 2,206 bytes is herein incorporated by reference in its entirety pursuant to 37 C.F.R. 1.52(e)(5).

[0006] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0007] This invention was made with government support under 1K99HL 159261 awarded by National Institutes of Health. The government has certain rights in the invention.

[0008] BACKGROUND

[0009] Acute lung injury (ALI), or its more severe form, acute respiratory distress syndrome (ARDS), is a major cause of morbidity and mortality in Western countries. The incidence of ALLARDS in the United States alone is over 200,000 cases per year, with a death rate approaching 40%. The primary cause of ALI / ARDS involves systemic sepsis, traumatic injury, or severe bacterial or viral pneumonia, including the novel coronavirus disease-19 (COVID-19), in which the vascular barrier in the lung is disrupted, accompanied by an increased pulmonary edema, and impaired gas exchange. However, currently, there is no therapy available for such diseases.

[0010] BRIEF SUMMARY

[0011] In one aspect, the present disclosure relates to a method of treating, ameliorating, and / or preventing a respiratory disorder in a subject in need thereof. In some aspects, the method comprises administering an effective amount of vascular endothelial growth factor D (VEGF-D) to the subject in need thereof. Tn some aspects, the respiratory disorder is respiratory distress syndrome. In some aspects, the respiratory disorder is acute lung injury. In some aspects, the respiratory disorder is acute respiratory distress syndrome. In some aspects, the respiratory disorder is cystic fibrosis. In some aspects, the respiratory disorder is pulmonary tuberculosis. In some aspects, the respiratory disorder is cough. In some aspects, the respiratory disorder is bronchial asthma. In some aspects, the respiratory disorder is cough based on increased airway hyperreactivity. In some aspects, the respiratory disorder is flu syndrome. In some aspects, the respiratory disorder is cough hypersensitivity. In some aspects, the respiratory disorder is airway hyperreactivity. In some aspects, the respiratory disorder is tuberculosis disease. In some aspects, the respiratory disorder is asthma. In some aspects, the respiratory disorder is chronic obstructive pulmonary disease. In some aspects, the respiratory disorder is emphysema. In some aspects, the respiratory disorder is pulmonary fibrosis. In some aspects, the respiratory disorder is idiopathic pulmonary fibrosis. In some aspects, the respiratory disorder is cough. In some aspects, the respiratory disorder is reversible airway obstruction. In some aspects, the respiratory disorder is adult respiratory disease syndrome. In some aspects, the respiratory disorder is pigeon fancier's disease. In some aspects, the respiratory disorder is farmer's lung. In some aspects, the respiratory disorder is bronchopulmonary dysplasia. In some aspects, the respiratory disorder is airway disorder. In some aspects, the respiratory disorder is emphysema. In some aspects, the respiratory disorder is allergic bronchopulmonary aspergillosis. In some aspects, the respiratory disorder is allergic bronchitis bronchiectasis. In some aspects, the respiratory disorder is COVID- 19. In some aspects, the respiratory disorder is occupational asthma. In some aspects, the respiratory disorder is reactive airway disease syndrome. In some aspects, the respiratory disorder is interstitial lung disease. In some aspects, the respiratory disorder is parasitic lung disease. In some aspects, the respiratory disorder is acute lung injury (ALT) or acute respiratory distress syndrome (ARDS). In some aspects, the ALT or ARDS is caused by systemic sepsis, traumatic injury, or bacterial or viral infection. In some aspects, the VEGF-D administered to the subject is encoded by a polynucleotide. In some aspects, the polynucleotide comprises recombinant DNA. In some aspects, the polynucleotide comprises recombinant RNA. In some aspects, the VEGF-D administered to the subject comprises a recombinant polypeptide comprising VEGF-D. In some aspects, the VEGF-D is delivered to the subject on an encoded vector, wherein the vector encodes the VEGF-D. In some aspects, the vector is an expression vector. In some aspects, the VEGF-D comprises human VEGF-D, or a variant thereof. In some aspects, the VEGF-D comprises mouse VEGF-D, or a variant thereof. In some aspects, the VEGF-D comprises rat VEGF-D, or a variant thereof. In some aspects, the VEGF-D comprises chicken VEGF-D, or a variant thereof. In some aspects, the VEGF-D comprises Rhesus monkey VEGF-D, or a variant thereof. In some aspects, the VEGF-D comprises chimpanzee VEGF-D, or a variant thereof. In some aspects, the VEGF-D comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 1. In some aspects, the VEGF-D comprises the amino acid sequence of SEQ ID NO: 1. In some aspects, the treatment improves the endothelial integrity of the subject as compared to a subject in need of treatment but not treated with the effective amount of VEGF-D. In some aspects, the subject is a mammal. In some aspects, the mammal is a human. In some aspects, the method further comprises contacting a cell with the VEGF-D. In some aspects, the cell is an endothelial cell. In some aspects, the endothelial cell is a lung endothelial cell. In some aspects, the lung endothelial cell is a human lung microvascular endothelial cell. In some aspects, treatment with VEGF-D reduces barrier resistance of the cells compared to untreated cells as measured by ECIS assay. In some aspects, treatment with VEGF-D reduces barrier resistance of the cells compared to cells treated with slit2, BMP5, or secretin as measured by ECIS assay. In some aspects, the treatment with VEGF-D reduces endothelial permeability compared to untreated cells as measured by transwell assay. In some aspects, the treatment with VEGF-D reduces endothelial permeability compared to cells treated with slit2, BMP5, or secretin as measured by transwell assay. In some aspects, treatment with VEGF-D reduces vascular permeability as compared to PBS control cells in an LPS-induced lung injury model. In some aspects, the VEGF-D is administered acutely or chronically to the subject. In some aspects, the VEGF-D is administered locally, regionally, parenterally, or systemically to the subject. In some aspects, the VEGF-D is administered to the subject by at least one route selected from the group consisting of subcutaneous, oral, aerosol, inhalational, rectal, vaginal, transdermal, subcutaneous, intranasal, buccal, sublingual, parenteral, intrathecal, intragastrical, ophthalmic, pulmonary, and topical. In some aspects, the VEGF-D is administered to the subject as a pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier. In one aspect, the present disclosure relates to a method of promoting the protection of endothelial integrity. In some aspects, the method comprises contacting an endothelial cell with an effective amount of vascular endothelial growth factor D (VEGF-D). In some aspects, contacting the endothelial cell comprises contacting the endothelial cell of a subject in need of treatment. In some aspects, the subject has a respiratory disorder. In some aspects, the respiratory disorder is respiratory distress syndrome. In some aspects, the respiratory disorder is acute lung injury. In some aspects, the respiratory disorder is acute respiratory distress syndrome. In some aspects, the respiratory disorder is cystic fibrosis. In some aspects, the respiratory disorder is pulmonary tuberculosis. In some aspects, the respiratory disorder is cough. In some aspects, the respiratory disorder is bronchial asthma. In some aspects, the respiratory disorder is cough based on increased airway hyperreactivity. In some aspects, the respiratory disorder is flu syndrome. In some aspects, the respiratory disorder is cough hypersensitivity. In some aspects, the respiratory disorder is airway hyperreactivity. In some aspects, the respiratory disorder is tuberculosis disease. In some aspects, the respiratory disorder is asthma. In some aspects, the respiratory disorder is chronic obstructive pulmonary disease. In some aspects, the respiratory disorder is emphysema. In some aspects, the respiratory disorder is pulmonary fibrosis. In some aspects, the respiratory disorder is idiopathic pulmonary fibrosis. In some aspects, the respiratory disorder is cough. In some aspects, the respiratory disorder is reversible airway obstruction. In some aspects, the respiratory disorder is adult respiratory disease syndrome. In some aspects, the respiratory disorder is pigeon fancier's disease. In some aspects, the respiratory disorder is farmer's lung. In some aspects, the respiratory disorder is bronchopulmonary dysplasia. In some aspects, the respiratory disorder is airway disorder. In some aspects, the respiratory disorder is emphysema. In some aspects, the respiratory disorder is allergic bronchopulmonary aspergillosis. In some aspects, the respiratory disorder is allergic bronchitis bronchiectasis. In some aspects, the respiratory disorder is COVID-19. In some aspects, the respiratory disorder is occupational asthma. In some aspects, the respiratory disorder is reactive airway disease syndrome. In some aspects, the respiratory disorder is interstitial lung disease. In some aspects, the respiratory disorder is parasitic lung disease. In some aspects, the respiratory disorder is acute lung injury (ALI) or acute respiratory distress syndrome (ARDS). In some aspects, the ALI or ARDS is caused by systemic sepsis, traumatic injury, or bacterial or viral infection. In some aspects, the VEGF-D is encoded by a polynucleotide. In some aspects, the polynucleotide comprises recombinant DNA. In some aspects, the polynucleotide comprises recombinant RNA. In some aspects, the VEGF-D comprises a recombinant polypeptide comprising VEGF-D. In some aspects, the VEGF-D is encoded by a vector. In some aspects, the vector is an expression vector. In some aspects, the VEGF-D comprises human VEGF-D, or a variant thereof. In some aspects, the VEGF-D comprises mouse VEGF-D, or a variant thereof. In some aspects, the VEGF-D comprises rat VEGF-D, or a variant thereof. In some aspects, the VEGF-D comprises chicken VEGF-D, or a variant thereof. In some aspects, the VEGF-D comprises Rhesus monkey VEGF- D, or a variant thereof. In some aspects, the VEGF-D comprises chimpanzee VEGF-D, or a variant thereof. In some aspects, the VEGF-D comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 1. In some aspects, the VEGF-D comprises the amino acid sequence of SEQ ID NO: 1. In some aspects, the endothelial cell is a lung endothelial cell. In some aspects, the lung endothelial cell is a human lung microvascular endothelial cell. In some aspects, contacting the endothelial cell with VEGF-D reduces barrier resistance of the cells compared to untreated cells as measured by ECIS assay. In some aspects, contacting the endothelial cell with VEGF-D reduces barrier resistance of the cells compared to cells treated with slit2, BMP5, or secretin as measured by ECIS assay. In some aspects, contacting the endothelial cell with VEGF-D reduces endothelial permeability compared to untreated cells as measured by transwell assay. In some aspects, contacting the endothelial cell with VEGF-D reduces endothelial permeability compared to cells treated with slit2, BMP5, or secretin as measured by transwell assay. In some aspects, contacting the endothelial cell with VEGF-D reduces vascular permeability as compared to PBS control cells in an LPS-induced lung injury model.

[0012] BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings.

[0014] FIG. 1A-FIG. ID present schematics and results related to single-cell RNA-seq analysis of cell populations within human pulmonary microvascular niche. FIG. 1A presents a schematic of an overview of the experimental design, wherein connectome in the healthy human lung microvascular niche was generated, followed by validation through functional testing. FIG. IB presents a UMAPPlot of the cell populations (221,171 cells) in microvascular niche from 68 control lungs; where each dot represents a single cell, and cells are labeled as 1 of 24 discrete cell types. FIG. 1C presents a VlnPlot of representative markers of each cluster of FIG. IB for microvascular endothelium and mesenchyme. FIG. ID presents a VlnPlot of representative markers of each cluster of FIG. IB for alveolar epithelium, myeloid, and lymphoid.

[0015] FIG. 2A-FIG. 2D present data and results related to EC intercellular communication with other cells in the lung microvascular niche. FIG. 2A presents a visual representation of a small subset of the connectomic analysis. Circos plot of top 50 edges by edge weight with capillary endothelial cells (EC) as signal sender is presented. FIG. 2B presents a visual representation of a small subset of the connectomic analysis. Circos plot of top 50 edges by edge weight with capillary EC as a signal receiver is presented. FIG. 2C presents images related to immunostaining of SLIT2-ROBO4, ADM-RAMP2, BMP5-BMPR2, and VEGFD-KDR pairs in a human lung section. FIG. 2D presents graphical representations of the secretion levels of SLIT2, ADM, BMP5, and VEGFD as determined by corresponding ELISAs.

[0016] FIG. 3A-FIG. 3D present data and results related to analysis of ADM, ANG1, and VEGFD in barrier resistance of human lung microvascular endothelial cells (HLMVECs). FIG. 3A presents the results of electric cell-substrate impedance sensing (ECIS) assays analyzing the effects of ADM, Angiopoietin 1, and VEGFD on barrier function. FIG. 3B presents the results of transwell assays analyzing the effects of ADM, Angiopoietin 1, and VEGFD on barrier function. FIG. 3C presents a graphical representation of results analyzing the effects of ILip, TNFa, or thrombin challenge on VEGF-D treated cells. FIG. 3D presents images relating to the results of ILip or TNFa on VEGF-D treated cells.

[0017] FIG. 4A-FIG. 4B present results related to assays evaluating human lung endothelial cell barrier function. FIG. 4A presents a graphical representation of an siRNA assay measuring mRNA expression of VEGFR2 and FLT4 when using the denoted siRNA constructs. FIG. 4B presents a graphical representation of the normalized resistance of cells in an ECIS assays using various different siRNA constructs administered in conjunction with VEGF-D.

[0018] FIG. 5A-FIG. 5D present schematics and results related to the impact of VEGF-D in lipopolysaccharide (LPS) injured lungs. FIG. 5A presents a schematic of the experimental design used to test the impact of VEGF-D in LPS injured lungs. FIG. 5B presents a graphical representation of results related to the treatment with VEGFD in LPS-injured lungs. FIG. 5C presents a graphical representation of results related to the treatment with VEGFD in LPS- injured lungs. FIG. 5D presents a graphical representation of results related to the treatment with VEGFD in LPS-injured lungs.

[0019] FIG. 6A-FIG. 6D present results related to the impact of VEGF-D in LPS injured lungs. FIG. 6A presents a graphical representation of results related to the treatment with VEGFD in LPS-injured lungs on permeability. FIG. 6B presents a graphical representation of results related to the treatment with VEGFD in LPS-injured lungs on permeability white blood cell count. FIG. 6C presents a graphical representation of results related to the treatment with VEGFD in LPS- injured lungs on permeability red blood cell count. FIG. 6D presents a graphical representation of results related to the treatment with VEGFD in LPS-injured lungs on permeability platelet count.

[0020] DETAILED DESCRIPTION

[0021] Acute lung injury (ALI), or its more severe form, acute respiratory distress syndrome (ARDS), is a major cause of morbidity and mortality in Western countries. In particular, ALI and ARDS are known to negatively affect epithelial cells, such as lung endothelial cells. Oftentimes, advanced ALI and / or ARDS can result in a subject needing to use a mechanical ventilator as a result of the damage sustained by the lungs and / or pulmonary system of the subject. Currently there is no therapy or treatment for effectively treating such diseases as ALI and ARDS.

[0022] VEGF-D has generally been considered an agonist in angiogenesis and lymphangiogenesis, and further has been shown to impair lung vascular integrity. However, as discussed in the present application, surprisingly, the data presented herein demonstrate that VEGF-D significantly improved and / or promoted the protection of endothelial integrity, in particular lung endothelial integrity. For instance, such improvement and / or promotion of protection in endothelial integrity was demonstrated by the surprising and unexpected effects of treatment with VEGF-D significantly increasing the barrier function of human lung endothelial cells, as described herein. Additionally, such improvement and / or promotion of protection was demonstrated by the surprising and unexpected effects of VEGF-D exerting barrier-protective and anti-inflammatory properties against tumor necrosis factor-a, interleukin 1-b, and lipopolysaccharide challenges, as described herein. Also, such improvement and / or promotion of protection was demonstrated by the surprising effects of administration of VEGF-D significantly reducing permeability and immune cell infiltration in a lipopolysaccharide-induced lung injury animal model, as described herein. The studies presented herein therefore demonstrate the therapeutic potential of VEGF-D in treating ALVARDS in subjects in need thereof as well as the promotion of protection of endothelial integrity, in particular lung endothelial integrity, when using VEGF-D as a treatment for ALI / ARDS.

[0023] As such, in one aspect, the present disclosure generally relates to a method of treating a respiratory disorder in a subject in need thereof, wherein the treatment comprises administering an effective amount of vascular endothelial growth factor D (VEGF-D) to the subject in need thereof. Furthermore, in one aspect, the present disclosure generally relates to a method of promoting the protection of endothelial integrity, wherein the method comprises contacting an endothelial cell with an effective amount of vascular endothelial growth factor D (VEGF-D). In some aspects, the respiratory disorder is acute lung injury (ALI) or acute respiratory distress syndrome (ARDS). In some aspects, the VEGF-D comprises a recombinant DNA encoding the VEGF-D, a recombinant RNA encoding the VEGF-D, and / or a recombinant polypeptide comprising VEGF-D. In some aspects, the VEGF-D comprises human VEGF-D.

[0024] DEFINITIONS

[0025] Unless otherwise defined, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of “or” means “and / or” unless stated otherwise. The use of the term “including,” as well as other forms, such as “includes” and “included,” is not limiting.

[0026] Generally, nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry, and nucleic acid hybridization described herein is well-known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well- known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0027] Furthermore, the experiments described herein, unless otherwise indicated, use conventional molecular and cellular biological and immunological techniques within the skill of the art. Such techniques are well known to the skilled worker, and are explained fully in the literature. See, e.g., Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2008), including all supplements, Molecular Cloning: A Laboratory Manual (Fourth Edition) by MR Green and J. Sambrook and Harlow et al., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013, 2nd edition).

[0028] That the disclosure may be more readily understood, select terms are defined below.

[0029] As used herein, the terms "a," "an," or "the" are used to include one or more than one unless the context clearly dictates otherwise. By way of example, “an element” means one element or more than one element. The term "or" is used to refer to a nonexclusive "or" unless otherwise indicated. The statement "at least one of A and B" or "at least one of A or B" has the same meaning as "A, B, or A and B."

[0030] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0031] The term “therapeutic” as used herein means a treatment and / or prophylaxis. A therapeutic effect is obtained by any degree of suppression, remission, or eradication of a disease state.

[0032] To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0033] As used herein, to “alleviate” a disease means reducing the severity of one or more symptoms of the disease. Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 and so forth, as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0034] In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. patent law and can mean “includes,” “including,” and the like; “consisting essentially of’ or “consists essentially” likewise has the meaning ascribed in U.S. patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

[0035] The term “subject” is intended to include living organisms that may be subjected to treatment for a given disease, e.g., mammals. A “subject” or “patient,” as used herein, can be a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals, as well as simian and non- human primate mammals. Preferably, the subject is human.

[0036] As used herein, the terms “respiratory disorder” and “respiratory disease” generally refer to a disease or condition that is physically manifested in the respiratory tract including, but not limited to, cystic fibrosis, respiratory distress syndrome, acute lung injury, acute respiratory distress syndrome, pulmonary tuberculosis, cough, bronchial asthma, cough based on increased airway hyperreactivity (bronchitis, flu syndrome, asthma, obstructive pulmonary disease, and the like), flu syndrome, cough hypersensitivity, airway hyperreactivity, tuberculosis disease, asthma (airway inflammatory cell infiltration, increased airway hyperresponsiveness, bronchoconstriction, mucus hypersecretion, and the like), chronic obstructive pulmonary disease, emphysema, pulmonary fibrosis, idiopathic pulmonary fibrosis, cough, reversible airway obstruction, adult respiratory disease syndrome, pigeon fancier's disease, farmer's lung, bronchopulmonary dysplasia, airway disorder, emphysema, allergic bronchopulmonary aspergillosis, allergic bronchitis bronchiectasis, occupational asthma, reactive airway disease syndrome, interstitial lung disease, parasitic lung disease, and the like. In some aspects, the respiratory disorder is acute lung injury or acute respiratory distress syndrome. In some aspects, the ALI or ARDS is caused by systemic sepsis, traumatic injury, or bacterial or viral infection, such as bacterial or viral pneumonia, including the novel coronavirus disease-19 (COVID-19).

[0037] As used herein, the terms “effective amount” and “therapeutically effective amount” are used interchangeably and refer to the amount required to reduce or improve at least one symptom or change in a clinical marker of a disease, e.g., a respiratory disorder, relative to an untreated patient, and / or the amount required to promote the protection of endothelial cell integrity, e.g., lung endothelial cell integrity. The effective amount of the treatment, e.g., VEGF-D, used for therapeutic treatment of the disease, e.g., respiratory disorder, varies depending upon the manner of the specific disorder, condition or disease, extent of the disorder, condition or disease, and administration of the cells, as well as the age, body weight, and general health of the subject. The effective amount is capable of achieving a particular desired biological result and / or provides a therapeutic or prophylactic benefit.

[0038] The term “endothelial cells” as used herein refers to the thin layer of squamous cells that form the endothelium. The term “endothelium” as used herein refers to a type of epithelium that lines the interior surface of blood vessels and lymphatic vessels, forming an interface between circulating blood or lymph in the lumen and the rest of the vessel wall. Endothelial cells in direct contact with blood are called vascular endothelial cells, whereas those in direct contact with lymph are known as lymphatic endothelial cells. Vascular endothelial cells line the entire circulatory system, from the heart to the smallest capillaries. These cells have unique functions in vascular biology. These functions include fluid filtration, such as in the glomerulus of the kidney, blood vessel tone, hemostasis, neutrophil recruitment, and hormone trafficking. Endothelium is mesodermal in origin. Endothelial cells are involved in many aspects of vascular biology, including barrier function, blood clotting (thrombosis & fibrinolysis), inflammation, formation of new blood vessels (angiogenesis), vasoconstriction and vasodilation (and hence the control of blood pressure). Endothelial dysfunction, or the loss of proper endothelial function, is a hallmark for vascular diseases, and is often regarded as a key early event in the development of atherosclerosis. Impaired endothelial function, causing hypertension and thrombosis, is often seen in patients with coronary artery disease, diabetes mellitus, hypertension, hypercholesterolemia, as well as in smokers. Endothelial dysfunction has also been shown to be predictive of future adverse cardiovascular events, and is also present in inflammatory disease such as rheumatoid arthritis and systemic lupus erythematosus. In some aspects, the endothelial cells are lung endothelial cells.

[0039] As used herein, the term “promoting the protection of endothelial integrity” and the like generally refer to any one or more of the following effects on an endothelial cell: increasing barrier function, such as lung vascular barrier function, exerting barrier-protective and antiinflammatory properties during inflammation; reducing permeability and immune cell infiltration during lung inflammation, such as in a lipopolysaccharide-induced lung injury animal model; reducing barrier damage during endotoxin-induced lung inflammation; and / or reducing junctional protein disruption such as VE-Cadherin. Such effects can be observed and / or measured using any of the assays and guidance provided in the present disclosure, in particular as described in the working examples infra.

[0040] As used herein, the term “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as 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 (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene 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.

[0041] As used herein, the term “expression” as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0042] As used herein, the term “expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., Sendai viruses, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0043] As used herein, the term “identity” refers to the subunit sequence identity between two polymeric molecules particularly between two amino acid molecules, such as, between two polypeptide molecules. When two amino acid sequences have the same residues at the same positions; e.g., if a position in each of two polypeptide molecules is occupied by an arginine, then they are identical at that position. The identity or extent to which two amino acid sequences have the same residues at the same positions in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions; e.g., if half (e.g., five positions in a polymer ten amino acids in length) of the positions in two sequences are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 of 10), are matched or identical, the two amino acids sequences are 90% identical.

[0044] As used herein, the terms "conservative variation" or "conservative substitution" generally refers to the replacement of an amino acid residue by another, biologically similar residue. Conservative variations or substitutions are not likely to change the shape of the peptide chain. Examples of conservative variations, or substitutions, include the replacement of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine.

[0045] As used herein, the term “isolated” means altered or removed from the natural state. For example, a nucleic acid or a polypeptide naturally present in a living animal is not “isolated,” but the same nucleic acid or polypeptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0046] An "isolated nucleic acid" refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, i.e., a DNA fragment which has been removed from the sequences that are normally adjacent to the fragment, i.e., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to nucleic acids that have been substantially purified from other components which naturally accompany the nucleic acid, i.e., RNA or DNA or proteins, which naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule i.e., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

[0047] The term "recombinant polypeptide" as used herein is defined as a polypeptide produced by using recombinant DNA methods.

[0048] The term "recombinant DNA" as used herein is defined as DNA produced by joining pieces of DNA from different sources.

[0049] "Variant" as the term is used herein, is a nucleic acid sequence or a peptide sequence that differs in sequence from a reference nucleic acid sequence or peptide sequence respectively, but retains essential properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions and truncations. Changes in the sequence of peptide variants are typically limited or conservative, so that the sequences of the reference peptide and the variant are closely similar overall and, in many regions, identical. A variant and reference peptide may differ in amino acid sequence by one or more substitutions, additions, or deletions in any combination. A variant of a nucleic acid or peptide may be a naturally occurring such as an allelic variant, or may be a variant that is not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides may be made by mutagenesis techniques or by direct synthesis.

[0050] By the term “modified” as used herein, is meant a changed state or structure of a molecule or cell of the invention. Molecules may be modified in many ways, including chemically, structurally, and functionally. Cells may be modified through the introduction of nucleic acids.

[0051] As used herein, the term “oligonucleotide” typically refers to short polynucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, C, G), this also includes an RNA sequence (i.e., A, U, C, G) in which “U” replaces “T.” 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 an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

[0052] “Parenteral” administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, or infusion techniques.

[0053] The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, “nucleic acid” and “polynucleotide” as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides” and which comprise one or more “nucleotide sequence(s)”. The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences (z.c., “nucleotide sequences”) which are obtained by any means available in the art, including, without limitation, recombinant means, z.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.

[0054] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0055] A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, Sendai viral vectors, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

[0056] VEGF-D

[0057] VEGF-D is a member of the VEGF / PDGF family of structurally-related proteins, and is regarded as a potent angiogenic cytokine. VEGF-D promotes endothelial cell growth, promotes lymphangiogenesis, and can also affect vascular permeability. VEGF-D is highly expressed in the lung, heart, small intestine and fetal lung, and at lower levels in the skeletal muscle, colon, and pancreas. VEGF-D can form cell surface-associated, non-covalent, disulfide-linked homodimers, and can bind and activate both VEGFR-2 (flkl) and VEGFR-3 (flt4) receptors. VEGF-D can also exist as a non-covalent homodimer as well as a monomer. During embryogenesis, VEGF-D may play a role in the formation of the venous and lymphatic vascular systems. VEGF-D has been found to be over-expressed in certain cancers, and the resulting elevated levels of VEGF-D tends to correlate with increased lymphatic metastasis. See, for instance, Karnezis, T., et al., VEGF-D Promotes Tumor Metastasis by Regulating Prostaglandins Produced by the Collecting Lymphatic Endothelium, Cancer Cell, 21 :2, 181-195 (2012), which describes the role of VEGF-D in promoting tumor metastasis, in particular into lymph nodes.

[0058] As such, and as discussed above, it was surprising and unexpected that treatment with VEGF-D could significantly increase the barrier function of human lung endothelial cells, as described herein. Additionally, it was surprising and unexpected that VEGF-D exerted barrier- protective and anti-inflammatory properties against tumor necrosis factor-a, interleukin 1-b, and lipopolysaccharide challenges, as described herein. It was also surprising that administration of VEGF-D significantly reduced permeability and immune cell infiltration in a lipopolysaccharide- induced lung injury animal model, as described herein.

[0059] In some aspects, the VEGF-D comprises human VEGF-D, mouse VEGF-D, rat VEGF-D, chicken VEGF-D, Rhesus monkey VEGF-D, chimpanzee VEGF-D, or a variant thereof. In some aspects, the VEGF-D comprises human VEGF-D, or a variant thereof. In some aspects, the VEGF-D comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 1.

[0060] Polynucleotides and Vectors

[0061] In some aspects, the VEGF-D, such as the VEGF-D administered to a subject and / or used to contact a cell, is encoded by a polynucleotide. In some aspects, the polynucleotide comprises recombinant or isolated or synthetic DNA. In some aspects, the polynucleotide comprises recombinant or isolated or synthetic RNA. In some aspects, the polynucleotide encodes a recombinant or synthetic polypeptide comprising VEGF-D. In some aspects, a vector encodes the VEGF-D. In some aspects, the vector is an expression vector.

[0062] In some aspects, the polynucleotide encoding VEGF-D is operably linked to a transcriptional control element, e.g., a promoter, and enhancer, etc. Suitable promoter and enhancer elements are known to those of skill in the art. In some aspects, the polynucleotide encoding VEGF-D is operably linked to a promoter. For expression in a bacterial cell, suitable promoters include, but are not limited to, lacl, lacZ, T3, T7, gpt, lambda P and trc. For expression in a eukaryotic cell, suitable promoters include, but are not limited to, light and / or heavy chain immunoglobulin gene promoter and enhancer elements; cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; promoter present in long terminal repeats from a retrovirus; mouse metallothionein-I promoter; and various art-known tissue specific promoters. Suitable reversible promoters, including reversible inducible promoters are known in the art. Such reversible promoters may be isolated and derived from many organisms, e.g., eukaryotes and prokaryotes. Modification of reversible promoters derived from a first organism for use in a second organism, e.g., a first prokaryote and a second a eukaryote, a first eukaryote and a second a prokaryote, etc., is well known in the art. Such reversible promoters, and systems based on such reversible promoters but also comprising additional control proteins, include, but are not limited to, alcohol regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator proteins (AlcR), etc.), tetracycline regulated promoters, (e.g., promoter systems including TetActivators, TetON, TetOFF, etc.), steroid regulated promoters (e.g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid promoter systems, thyroid promoter systems, ecdysone promoter systems, mifepristone promoter systems, etc.), metal regulated promoters (e.g., metallothionein promoter systems, etc.), pathogenesis- related regulated promoters (e.g., salicylic acid regulated promoters, ethylene regulated promoters, benzothiadiazole regulated promoters, etc.), temperature regulated promoters (e.g., heat shock inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), light regulated promoters, synthetic inducible promoters, and the like.

[0063] For expression in a yeast cell, a suitable promoter is a constitutive promoter such as an ADH1 promoter, a PGK1 promoter, an ENO promoter, a PYK1 promoter and the like; or a regulatable promoter such as a GALI promoter, a GAL 10 promoter, an ADH2 promoter, a PHOS promoter, a CUP1 promoter, a GALT promoter, a MET25 promoter, a MET3 promoter, a CYC1 promoter, a HIS3 promoter, an ADH1 promoter, a PGK promoter, a GAPDH promoter, an ADC1 promoter, a TRP1 promoter, a URA3 promoter, a LEU2 promoter, an ENO promoter, a TP1 promoter, and AOX1 (e.g., for use in Pichia). Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art. Suitable promoters for use in prokaryotic host cells include, but are not limited to, a bacteriophage T7 RNA polymerase promoter; a trp promoter; a lac operon promoter; a hybrid promoter, e.g., a lac / tac hybrid promoter, a tac / trc hybrid promoter, a trp / lac promoter, a T7 / lac promoter; a trc promoter; a tac promoter, and the like; an araBAD promoter; in vivo regulated promoters, such as an ssaG promoter or a related promoter (see, e.g., U.S. Patent Publication No. 20040131637), a pagC promoter (Pulkkinen and Miller, J. Bacteriol. (1991) 173(1): 86-93; Alpuche- Aranda et al., Proc. Natl. Acad. Sci. USA (1992) 89(21): 10079-83), a nirB promoter (Harborne et al. Mol. Micro. (1992) 6:2805-2813), and the like (see, e.g., Dunstan et al., Infect. Immun. (1999) 67:5133-5141; McKelvie et al., Vaccine (2004) 22:3243-3255; and Chatfield et al., Biotechnol. (1992) 10:888- 892); a sigma70 promoter, e.g., a consensus sigma70 promoter (see, e.g., GenBank Accession Nos. AX798980, AX798961, and AX798183); a stationary phase promoter, e.g., a dps promoter, an spv promoter, and the like; a promoter derived from the pathogenicity island SPL2 (see, e.g., WO96 / 17951); an actA promoter (see, e.g., Shetron-Rama et al., Infect. Immun. (2002) 70:1087- 1096); an rpsM promoter (see, e.g., Valdivia and Falkow Mol. Microbiol. (1996). 22:367); a tet promoter (see, e.g., Hillen, W. and Wissmann, A. (1989) In Saenger, W. and Heinemann, U. (eds), Topics in Molecular and Structural Biology, Protein— Nucleic Acid Interaction. Macmillan, London, UK, Vol. 10, pp. 143-162); an SP6 promoter (see, e.g., Melton et al., Nucl. Acids Res. (1984) 12:7035); and the like. Suitable strong promoters for use in prokaryotes such as Escherichia coli include, but are not limited to Trc, Tac, T5, T7, and PLambda. Non-limiting examples of operators for use in bacterial host cells include a lactose promoter operator (LacI repressor protein changes conformation when contacted with lactose, thereby preventing the Lad repressor protein from binding to the operator), a tryptophan promoter operator (when complexed with tryptophan, TrpR repressor protein has a conformation that binds the operator; in the absence of tryptophan, the TrpR repressor protein has a conformation that does not bind to the operator), and a tac promoter operator (see, c. ., deBoer et al., Proc. Natl. Acad. Sci. U.S.A. (1983) 80:21-25).

[0064] Other examples of suitable promoters include the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. Other constitutive promoter sequences may also be used, including, but not limited to a simian virus 40 (SV40) early promoter, a mouse mammary tumor virus (MMTV) or human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, a MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, the EF-1 alpha promoter, as well as human gene promoters such as, but not limited to, an actin promoter, a myosin promoter, a hemoglobin promoter, and a creatine kinase promoter. Further, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0065] In some aspects, the polynucleotide or vector containing the suitable promoter is irreversibly switched through the induction of an inducible system. Suitable systems for induction of an irreversible switch are well known in the art, e.g., induction of an irreversible switch may make use of a Cre-lox-mediated recombination (see, e.g., Fuhrmann-Benzakein, et al., Proc. Natl. Acad. Sci. USA (2000) 28:e99, the disclosure of which is incorporated herein by reference). Any suitable combination of recombinase, endonuclease, ligase, recombination sites, etc. known to the art may be used in generating an irreversibly switchable promoter. Methods, mechanisms, and requirements for performing site-specific recombination, described elsewhere herein, find use in generating irreversibly switched promoters and are well known in the art, see, e.g., Grindley et al. Annual Review of Biochemistry (2006) 567-605; and Tropp, Molecular Biology (2012) (Jones & Bartlett Publishers, Sudbury, Mass ), the disclosures of which are incorporated herein by reference.

[0066] A polynucleotide encoding VEGF-D of the present disclosure can be present within an expression vector and / or a cloning vector. An expression vector can include a selectable marker, an origin of replication, and other features that provide for replication and / or maintenance of the vector. Suitable expression vectors include, e.g., plasmids, viral vectors, and the like. Large numbers of suitable vectors and promoters are known to those of skill in the art; many are commercially available for generating a subject recombinant construct. The following vectors are provided by way of example and should not be construed in anyway as limiting: Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pST-Ki, pST-KiT, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).

[0067] Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker operative in the expression host may be present. Suitable expression vectors include, but are not limited to, viral vectors (e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest. Opthalmol. Vis. Sci. (1994) 35: 2543-2549; Borras et al., Gene Ther. (1999) 6: 515-524; Li and Davidson, Proc. Natl. Acad. Sci. USA (1995) 92: 7700-7704; Sakamoto et al., H. Gene Ther. (1999) 5: 1088-1097; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655); adeno-associated virus (see, e.g., Ali et al., Hum. Gene Ther. (1998) 9: 81-86, Flannery et al., Proc. Natl. Acad. Sci. USA (1997) 94: 6916-6921; Bennett et al., Invest. Opthalmol. Vis. Sci. (1997) 38: 2857- 2863; Jomary et al., Gene Ther. (1997) 4:683 690, Rolling et al., Hum. Gene Ther. (1999) 10: 641-648; Ali et al., Hum. Mol. Genet. (1996) 5: 591-594; Srivastava in WO 93 / 09239, Samulski et al., J. Vir. (1989) 63: 3822-3828; Mendelson et al., Virol. (1988) 166: 154-165; and Flotte et al., Proc. Natl. Acad. Sci. USA (1993) 90: 10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., Proc. Natl. Acad. Sci. USA (1997) 94: 10319- 23; Takahashi et al., J. Virol. (1999) 73: 7812-7816); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like.

[0068] Additional expression vectors suitable for use are, e.g., without limitation, a lentivirus vector, a gamma retrovirus vector, a foamy virus vector, an adeno-associated virus vector, an adenovirus vector, a pox virus vector, a herpes virus vector, an engineered hybrid virus vector, a transposon mediated vector, and the like. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, and lentiviruses.

[0069] In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No. 6,326,193).

[0070] In some aspects, an expression vector is used to introduce the VEGF-D into a cell. Accordingly, an expression vector (e.g., a lentiviral vector) of the present invention may comprise a nucleic acid encoding for VEGF-D. In some aspects, the expression vector comprises additional elements that will aid in the functional expression of the VEGF-D encoded therein. In some aspects, an expression vector comprising a nucleic acid encoding for VEGF-D further comprises a mammalian promoter. In one aspect, the vector further comprises an elongationfactor-1 -alpha promoter (EF-la promoter). Use of an EF-la promoter may increase the efficiency in expression of downstream transgenes. Physiologic promoters (e.g., an EF-la promoter) may be less likely to induce integration mediated genotoxicity, and may abrogate the ability of the retroviral vector to transform stem cells. Other physiological promoters suitable for use in a vector are known to those of skill in the art and may be incorporated into a vector of the present invention. In some aspects, the vector further comprises a non-requisite cis acting sequence that may improve titers and gene expression. One non-limiting example of a nonrequisite cis acting sequence is the central polypurine tract and central termination sequence (cPPT / CTS) which is important for efficient reverse transcription and nuclear import. Other non- requisite cis acting sequences are known to those of skill in the art and may be incorporated into a vector (e.g., lentiviral vector) of the present invention. In some aspects, the vector further comprises a posttranscriptional regulatory element. Posttranscriptional regulatory elements may improve RNA translation, improve transgene expression and stabilize RNA transcripts. One example of a posttranscriptional regulatory element is the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Accordingly, in some aspects a vector for the present invention further comprises a WPRE sequence. Various posttranscriptional regulator elements are known to those of skill in the art and may be incorporated into a vector of the present invention. A vector of the present invention may further comprise additional elements such as a rev response element (RRE) for RNA transport, packaging sequences, and 5’ and 3’ long terminal repeats (LTRs). The term “long terminal repeat” or “LTR” refers to domains of base pairs located at the ends of retroviral DNAs which comprise U3, R and U5 regions. LTRs generally provide functions required for the expression of retroviral genes e ., promotion, initiation and polyadenylation of gene transcripts) and to viral replication. In one aspect, a vector of the present invention includes a 3’ U3 deleted LTR. Accordingly, a vector of the present invention may comprise any combination of the elements described herein to enhance the efficiency of functional expression of transgenes.

[0071] In some aspects, a polynucleotide encoding VEGF-D is RNA, e.g., in vitro synthesized RNA. Methods for in vitro synthesis of RNA are known to those of skill in the art; any known method can be used to synthesize RNA comprising a sequence encoding VEGF-D of the present disclosure. Methods for introducing RNA into a host cell are known in the art. See, e.g., Zhao et al. Cancer Res. (2010) 15: 9053. Introducing RNA comprising a nucleotide sequence encoding VEGF-D of the present disclosure into a host cell can be carried out in vitro, ex vivo or in vivo. For example, a host cell can be electroporated in vitro or ex vivo with RNA comprising a nucleotide sequence encoding a VEGF-D of the present disclosure.

[0072] In order to assess the expression of a polypeptide or portions thereof, the expression vector to be introduced into a cell may also contain either a selectable marker gene or a reporter gene, or both, to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In some aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, without limitation, antibiotic-resistance genes.

[0073] Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assessed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include, without limitation, genes encoding luciferase, betagalactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82).

[0074] METHODS OF TREATMENT, AMELIORATION, AND / OR PREVENTION

[0075] In one aspect, the present disclosure generally relates to a method of treating, ameliorating, and / or preventing a respiratory disorder in a subject in need thereof. I some aspects, the method comprises administering an effective amount of vascular endothelial growth factor D (VEGF-D) to the subject in need thereof. In some aspects, the respiratory disorder is respiratory distress syndrome, acute lung injury, acute respiratory distress syndrome, cystic fibrosis, pulmonary tuberculosis, cough, bronchial asthma, cough based on increased airway hyperreactivity, flu syndrome, cough hypersensitivity, airway hyperreactivity, tuberculosis disease, asthma, chronic obstructive pulmonary disease, emphysema, pulmonary fibrosis, idiopathic pulmonary fibrosis, cough, reversible airway obstruction, adult respiratory disease syndrome, pigeon fancier's disease, farmer's lung, bronchopulmonary dysplasia, airway disorder, emphysema, allergic bronchopulmonary aspergillosis, allergic bronchitis bronchiectasis, COVID- 19, occupational asthma, reactive airway disease syndrome, interstitial lung disease, or parasitic lung disease. In some aspects, the respiratory disorder is acute lung injury (ALI) or acute respiratory distress syndrome (ARDS). In some aspects, the ALI or ARDS is caused by systemic sepsis, traumatic injury, or bacterial or viral infection.

[0076] In some aspects, the VEGF-D administered to the subject comprises a recombinant polypeptide comprising VEGF-D. In some aspects, the VEGF-D is delivered on an encoded vector to the subject, wherein the vector encodes the VEGF-D. In some aspects, the vector is an expression vector. In some aspects, the treatment improves the endothelial integrity of the subject as compared to a subject in need of treatment but not treated with the effective amount of VEGF-D. In some aspects, the subject is a mammal. In some aspects, the mammal is a human.

[0077] In some aspects, the method further comprises contacting a cell with the VEGF-D. In some aspects, the cell is an endothelial cell. In some aspects, the endothelial cell is a lung endothelial cell. In some aspects, the lung endothelial cell is a human lung microvascular endothelial cell. In some aspects, treatment with VEGF-D reduces barrier resistance of the cells compared to untreated cells as measured by ECIS assay. In some aspects, treatment with VEGF- D reduces barrier resistance of the cells compared to cells treated with slit2, BMP5, or secretin as measured by ECIS assay. In some aspects, the treatment with VEGF-D reduces endothelial permeability compared to untreated cells as measured by transwell assay. In some aspects, the treatment with VEGF-D reduces endothelial permeability compared to cells treated with slit2, BMP5, or secretin as measured by transwell assay. In some aspects, treatment with VEGF-D reduces vascular permeability as compared to PBS control cells in an LPS-induced lung injury model.

[0078] In some aspects, the VEGFD is administered acutely or chronically to the subject. In some aspects, the VEGFD is administered locally, regionally, parenterally, or systemically to the subject. In some aspects, the VEGFD is administered to the subject by at least one route selected from the group consisting of subcutaneous, oral, aerosol, inhalational, rectal, vaginal, transdermal, subcutaneous, intranasal, buccal, sublingual, parenteral, intrathecal, intragastrical, ophthalmic, pulmonary, and topical. In some aspects, the VEGFD is administered to the subject as a pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier.

[0079] In some aspects, treatment with VEGF-D is used to regulate pulmonary microvascular cell functions. In some aspects, treatment with VEGF-D is used to protect the vascular barrier. In some aspects, treatment with VEGF-D is used to reduce vascular barrier damage during endotoxin-induced lung inflammation.

[0080] Moreover, in one aspect, the present disclosure generally relates to a method of promoting the protection of endothelial integrity, wherein the method comprises contacting an endothelial cell with an effective amount of vascular endothelial growth factor D (VEGF-D). In some aspects, contacting the endothelial cell comprises contacting the endothelial cell of a subject in need of treatment. In some aspects, the subject has a respiratory disorder or disease. In some aspects, the respiratory disorder or disease is respiratory distress syndrome, acute lung injury, acute respiratory distress syndrome, cystic fibrosis, pulmonary tuberculosis, cough, bronchial asthma, cough based on increased airway hyperreactivity, flu syndrome, cough hypersensitivity, airway hyperreactivity, tuberculosis disease, asthma, chronic obstructive pulmonary disease, emphysema, pulmonary fibrosis, idiopathic pulmonary fibrosis, cough, reversible airway obstruction, adult respiratory disease syndrome, pigeon fancier's disease, farmer's lung, bronchopulmonary dysplasia, airway disorder, emphysema, allergic bronchopulmonary aspergillosis, allergic bronchitis bronchiectasis, COVID-19, occupational asthma, reactive airway disease syndrome, interstitial lung disease, or parasitic lung disease. In some aspects, the respiratory disorder is acute lung injury (ALI) or acute respiratory distress syndrome (ARDS). In some aspects, the ALI or ARDS is caused by systemic sepsis, traumatic injury, or bacterial or viral infection.

[0081] In some aspects, the endothelial cell is a lung endothelial cell. In some aspects, the lung endothelial cell is a human lung microvascular endothelial cell. In some aspects, contacting the endothelial cell with VEGF-D reduces barrier resistance of the cells compared to untreated cells as measured by ECIS assay. In some aspects, contacting the endothelial cell with VEGF-D reduces barrier resistance of the cells compared to cells treated with slit2, BMP5, or secretin as measured by ECIS assay. In some aspects, contacting the endothelial cell with VEGF-D reduces endothelial permeability compared to untreated cells as measured by transwell assay. In some aspects, contacting the endothelial cell with VEGF-D reduces endothelial permeability compared to cells treated with slit2, BMP5, or secretin as measured by transwell assay. In some aspects, contacting the endothelial cell with VEGF-D reduces vascular permeability as compared to PBS control cells in an LPS-induced lung injury model.

[0082] In some aspects, the VEGF-D used to contact the cell is any VEGF-D as described herein, such as, for instance, human VEGF-D, or a variant thereof.

[0083] PHARMACEUTICAL COMPOSITIONS

[0084] Also provided herein are compositions comprising VEGF-D, such as compositions comprising a polynucleotide encoding VEGF-D and / or compositions comprising an isolated or recombinant VEGF-D polypeptide. Among the compositions are pharmaceutical compositions and formulations for administration, such as for treating a respiratory disease or disorder. Also provided are therapeutic methods for administering the VEGF-D and compositions to subjects, e.g., patients. In one aspect, the present disclosure generally relates to a pharmaceutical composition comprising VEGF-D and at least one pharmaceutically acceptable carrier.

[0085] Also provided are compositions for administration, including pharmaceutical compositions and formulations, such as unit dose form compositions including the amount of VEGF-D for administration in a given dose or fraction thereof. The pharmaceutical compositions and formulations generally include one or more optional pharmaceutically acceptable carrier or excipient. In some aspects, the composition includes at least one additional therapeutic agent.

[0086] The term “pharmaceutical formulation” or “pharmaceutical composition” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative. In some aspects, the choice of carrier is determined in part by the particular cell and / or by the method of administration. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0087] Buffering agents in some aspects are included in the compositions. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

[0088] The formulations can include aqueous solutions. The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated, where the respective activities do not adversely affect one another. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. Thus, in some aspects, the pharmaceutical composition further includes other pharmaceutically active agents or drugs. The pharmaceutical composition in some aspects contains the VEGF-D in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some aspects is monitored by periodic assessment of treated subjects. The desired dosage can be delivered by a single administration of the VEGF-D, by multiple administrations of the VEGF-D, or by continuous administration of the VEGF-D.

[0089] Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some aspects, the VEGF-D is administered parenterally. The term “parenteral,” as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some aspects, the cells are administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection. Compositions in some aspects are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.

[0090] Sterile injectable solutions can be prepared by incorporating the cells in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, and / or colors, depending upon the route of administration and the preparation desired. Standard texts may in some aspects be consulted to prepare suitable preparations.

[0091] Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, and sorbic acid. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0092] The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e. ., by fdtration through sterile filtration membranes.

[0093] TABLE 1 - SEQUENCE LISTING EXAMPLES

[0094] The instant specification further describes in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless so specified. Thus, the instant specification should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0095] Materials and Methods

[0096] More detailed Materials and Methods for the experiments described herein are now provided.

[0097] Cell culture

[0098] In the examples using human lung microvascular endothelial cells (HLMVECs), the HLMVECs were prepared as follows. HLMVECs were seeded onto fibronectin-coated tissue culture flasks at a density of 10,000 cells / cm2and cultured in endothelial culture medium (VascuLife® VEGF Endothelial Medium Complete Kit, Frederick, MD) supplemented with 1.5% antibiotics / antimycotics and 10% FBS. Cells at passage 5 - 7 were used in the current study.

[0099] Immuiiostainin

[0100] In the examples using immunostaining, the immunostaining proceeded as follows. Immunostaining was performed as previously described (Yuan et al., Epac agonist improves barrier function in iPSC-derived endothelial colony forming cells for whole organ tissue engineering. Biomaterials. 2019;200:25-34; Schupp et al., Integrated Single-Cell Atlas of Endothelial Cells of the Human Lung. Circulation. 2021;144(4):286-302). Formalin fixed paraffin embedded (FFPE) blocks were cut at 5 pm, de-paraffinized by soaking in antigen retrieval solution (0.05% Tween-20, ImM EDTA, and 10 mM Tris (pH=9)) at 75 °C for 20 minutes, followed by blocking with 10% FBS and 0.2% Triton X-100 for 20 minutes at RT. Human lung samples were then stained with antibodies overnight at 4 °C followed by staining with secondary antibodies (RT, 1 hour) and DAPI (RT, 5 min). All samples were then washed, mounted, and assessed with confocal microscopy (Leica SP5). To determine the cytoskeletal and junction proteins, 5 X 104HLMVEC / well were seeded on fibronectin-coated coverslips in 12 well-plates. After 3 days of culture, adherent cells were fixed with 2% paraformaldehyde (containing Ca2 / Mg2) for 15 minutes, permeabilized with 0.1% Triton-X in PBS for 3 minutes, and blocked with 5% FBS PBS for 1 hour at RT. The fixed cells were then probed for VE-Cadherin overnight at 4 °C, followed by a 1.5 hour incubation with a secondary antibody conjugated with Alexa Fluor 488, and by rhodamine phalloidin and counterstained with DAPI for 5 minutes.

[0101] Electric cell-substrate impedance sensing assay

[0102] For the examples using electric cell-substrate impedance sensing (ECIS) assays, the ECIS assays were performed as follows. ECIS was used to assess the barrier function of HLMVECs, as previously described ((Yuan et al., Epac agonist improves barrier function in iPSC-derived endothelial colony forming cells for whole organ tissue engineering. Biomaterials. 2019;200:25- 34). Briefly, electrodes in ECIS chamber were functionalized with 1 mM L-cysteine, followed by coating with fibronectin (2 pg / cm2) at 37 °C for 1 hour. HLMVECs were seeded into the ECIS chambers at 5 X 104cells / cm2, and the Chamber slides were inserted into the ECIS device (96W20idfPET, Applied Biophysics, Troy, NY) that was situated within a cell culture incubator. The transendothelial electrical resistance (TEER) was measured by ECIS system at real time. Transwell assay

[0103] For the examples using a transwell assay, the transwell assay was performed as follows. To determine EC permeability, a transwell assay was used as per previously described (Yuan et al., Epac agonist improves barrier function in iPSC-derived endothelial colony forming cells for whole organ tissue engineering. Biomaterials. 2019;200:25-34). In brief, 5 X 104cells / cm2of HLMVECs were cultured in the upper compartment of fibronectin-coated transwell inserts (polyester, 6.5 mm in diameter, pore size 0.4 pm) for 3 days. Individual molecules were applied for 30 minutes followed by adding 10 pL of FITC-Dextran (2.5 mg / mL, Sigma-Aldrich, 20 kDa) into the inserts. To assess the impact of VEGF-D during vascular injury, HLMVECs were treated with TNF-ot for 3 hours, Interleukin- 10 for 3 hours, or thrombin for 30 minutes to induce vascular leakage prior to adding VEGF-D. After 20 minutes of VEGF-D treatment, FITC- Dextran was added into the inserts to assess permeability.

[0104] ELISA For the examples using an ELISA assay, the ELISA assay was performed as follows. To measure secreted SLIT2, BMP5, ADM, and VEGFD protein, adult rat lungs were cut into small piece (<lmm3per piece) and cultured into serum-reduced culture medium for 20 hours. The supernatant of the ex vivo lung culture was collected at 1 hour, 6 hours, and 20 hours. SLIT2, BMP5, ADM, and VEGFD ELISA were performed using the corresponding Human ELISA kit as per manufacturer’s instructions.

[0105] Li opolysaccharide-induced acute lung injury

[0106] For the examples using a lipopolysaccharide-induced acute lung injury assay, the assay was performed as follows. Mice were anesthetized with ketamine / xyl azine (100 and 10 mg / kg). Lipopolysaccharide (LPS, 50 pl, 1 mg / ml, Escherichia coli Oi l :B4) was injected intrathecally while mice postures were maintained upright (Yuan et al., Pazopanib ameliorates acute lung injuries via inhibition of MAP3K2 and MAP3K3. Sci Transl Med. 2021; 13(591)). Eighteen hours after the induction of injury, 100 pl of fluorescein isothiocyanate-labeled albumin (10 mg / ml) was injected via retro-orbital vein, and 20 hours after the induction of injury, mice were euthanized for sample collection. To obtain BAL fluid, 0.75 ml of PBS was instilled into lungs and retrieved via a 1 ml syringe.

[0107] Statistical analysis

[0108] Comparisons between groups were performed with Student’s / -test or, for multi -group comparisons, by analysis of variance (ANOVA) followed by a post-hoc test. All analysis was performed with GraphPad Prism 7.0; values shown graphically are mean ± standard error. Results

[0109] More detailed results of the experiments described herein are now provided.

[0110] Example 1: scRNAseq of Cell Populations in Human Lung Microvascular Niche

[0111] In the present example, Connectome was used to analyze the ligand-receptor interactions within the cell populations in human lung microvascular niche and to identify locally acting soluble signals that had potential to improve vascular functions in the lung (FIG. 1A). The candidate targets identified could be used as treatments or biomarkers for lung diseases. The previously published single-cell dataset (Schupp et al. Integrated Single-Cell Atlas of Endothelial Cells of the Human Lung. Circulation. 2021 ; 144(4):286-302) was reanalyzed and reannotated in the present example. The analysis focused on 68 healthy adult individuals, including 29 females and 49 males, with a median age of 50 (range 32 - 61 years). To determine cell populations within the pulmonary microvascular niche, the cell populations were removed in large vessels and large and small airways from the dataset (FIG. IB). The final dataset includes 221,171 cells with 24 cell populations within the pulmonary microvascular niche expressed distinct canonical markers published previously (FIG. 1C and FIG. ID).

[0112] Referring now to FIG. IB, FIG. IB presents UMAPPlot of the cell populations (221,171 cells) in microvascular niche from 68 control lungs; each dot represents a single cell, and cells are labeled as 1 of 24 discrete cell types. Based on the clustering, within the distal lung vascular niche, there were two blood endothelial cell populations, two epithelial cell populations, three mesenchymal populations, and approximately 17 immune cell populations, consistent with previous literature (Schupp et al. Integrated Single-Cell Atlas of Endothelial Cells of the Human Lung. Circulation. 2021;144(4):286-302; Travaglini et al. A molecular cell atlas of the human lung from single-cell RNA sequencing. Nature. 2020 Nov;587(7835):619-625).

[0113] Referring now to FIG. 1C and FIG. ID, FIG. 1C and FIG. ID presents VlnPlot of representative markers of each cluster. The aeocyte capillary and general capillary endothelial cells expressed canonical markers HPGD and GPIHBP1, respectively. The mesenchymal cell populations expressed markers of their specific sub-types, including ITGA8 and DKK3 (fibroblast cluster), MSLN (mesothelium cluster), and COX4I2 (pericyte cluster). The type I and type II epithelium expressed canonical markers AGER and SFTPC, respectively. The myeloid and lymphoid cell populations were clustered by canonical markers, RBP4 and S100A8 for myeloid cells, and XCL1 and FGFBP2 for lymphoid cells.

[0114] Example 2: Ligand-Receptor Interactions within Human Pulmonary Microvascular Niche

[0115] In the present example, ligand-receptor interactions within the human pulmonary microvascular niche were analyzed as follows. To study the cell-cell communication signals in the microvascular niche, the previously published computational pipeline Connectome was applied to map the averaged gene expression per cell type to known ligand-receptor interactions cataloged in the NicheNet database (Raredon et al., Single-cell connectomic analysis of adult mammalian lungs. Sci Adv. 2019;5(12):eaaw3851; Browaeys et al., NicheNet: modeling intercellular communication by linking ligands to target genes. Nat Methods. 2020; 17(2): 159- 62). The analysis was focused on the interaction between pulmonary microvascular endothelial cell (EC) and non-EC cell types (FIGs. 2A and 2B).

[0116] Leveraging ligand-receptor connectome analysis, top signal interactions were identified between microvascular endothelium and surrounding cell populations, including SLIT2-ROBO4, ANGPT1-TEK, ADM-RAMP2, VEGFD-KDR, and BMP5-BMPR2 (FIG. 2B). Furthermore, their spatial localization and secretion levels were confirmed in native human distal lung samples. Canonical markers, such as ITGA8 and PRX, were used to label alveolar fibroblasts, and aerocyte capillary ECs, respectively. The immunostaining data revealed that all tested ligands and receptors were either co-localized or adjacent, suggesting a strong possibility of crosstalk (FIG. 2C). To further determine the secretion level of the identified ligands, the protein level in the supernatant in the ex vivo rat lung culture was measured as a surrogate. The level of SLIT2, ADM, BMP5, and VEGFD in the supernatant reached a biologically meaningful level as early as one hour (FIG. 2D). Thus, these methods validated the localization and secretion levels of the soluble ligand-receptor pairs, indicating their utility in regulating pulmonary microvascular cell functions.

[0117] Example 3: Analysis of ADM, ANG1, and VEGFD in Barrier Function in HLMVECs

[0118] In the present example, assays analyzing the effects of ADM, ANG1, and VEGFD in endothelial functions, such as barrier function, were performed. To evaluate the impact of these ligands on endothelial functions, electrical cell impendence sensing (ECIS) and transwell assays were applied to measure permeability. The results demonstrates that treatment with adrenomedullin (gene name: ADM), angiopoietin 1 (gene name: ANGPT1), and VEGF-D markedly improved barrier function in human lung microvascular endothelial cells in both assays, whereas treatment with other identified ligands, including recombinant proteins slit2, BMP5, and secretin (gene name: SCT), had modest or no effect on endothelial barrier function (FIGs. 3 A and 3B), which was consistent with previous findings (Weng et al., Slit2 / Robo4 signaling pathway modulates endothelial hyper-permeability in a two-event in vitro model of transfusion-related acute lung injury. Blood Cells Mol Dis. 2019;76:7-12; et al., Angiopoietin- 1 and vascular endothelial growth factor regulation of leukocyte adhesion to endothelial cells: role of nuclear receptor-77. Arterioscler Thromb Vase Biol. 2012;32(7): 1707-16; Geven and Pickkers, Adrenomedullin and Adrenomedullin-Targeted Therapy As Treatment Strategies Relevant for Sepsis. Front Immunol. 2018;9:292). In addition, the results demonstrated that VEGFD significantly reduced the permeability in the transwell assay after TNF-a, IL-lb, and thrombin insults (FIG. 3C). The results further demonstrated that the expression of the tight junction protein VE-Cadherin at the cell periphery changed from punctate to linear pattern (FIG. 3D), which indicated the protective effect of VEGF-D on junction protein during pro- inflammatory challenges

[0119] Example 4: Analysis of Human Lung Microvascular Endothelial Cell Barrier Function

[0120] The present example relates to the analysis of human lung microvascular endothelial cell barrier function as observed by siRNA assays. siRNA assays were performed to further analyze the ability of VEGF-D to improve barrier function. For instance, FIG. 4A presents a graphical representation of an siRNA assay measuring mRNA expression of VEGFR2 and FLT4 when using the denoted siRNA constructs, and FIG. 4B presents a graphical representation of the normalized resistance of cells in an ECIS assays using various different siRNA constructs administered in conjunction with VEGF-D. Taken together, the results of FIG. 4A-FIG. 4B demonstrated that VEGF-D improved human lung microvascular endothelial cell barrier function.

[0121] Example 5: VEGFD improves endothelial integrity in LPS injured lungs

[0122] In the present example, the effects of VEGFD on endothelial integrity in pulmonary vascular diseases was analyzed. To study the therapeutic effect of VEGFD in pulmonary vascular diseases, a lung inflammation animal model using lipopolysaccharides (LPS) was used (FIG. 5A). It has been well documented that the intratracheal injection of LPS could induce inflammation, damage vascular permeability, increase neutrophil infiltration, and cause pulmonary edema (Gross et al., LPS-induced Acute Lung Injury Involves NF-kappaB-mediated Downregulation of SOX18. Am J Respir Cell Mol Biol. 2018;58(5):614-24). After injecting VEGFD in the LPS-induced lung injury model, there was a significant reduction of vascular permeability, measured by FITC-albumin, and mouse albumin in BAL as compared to the group with PBS control (FIGs. 5B and 5C). Additionally, there was a modest reduction of white blood cell count in BAL (FIG. 5D). Taken together, the results demonstrated the ability of VEGF-D to reduce vascular barrier damage during endotoxin-induced lung inflammation. Furthermore, the results of FIG. 6A-FIG. 6D demonstrated that treatment with VEGFD in LPS-injured lungs reduced the number of FITC-albumin, WBC, RBC, and platelet count in BAL.

[0123] Enumerated Embodiments

[0124] In some aspects, the present invention is directed to the following non-limiting embodiments:

[0125] Embodiment 1 : A method of treating a respiratory disorder in a subject in need thereof, wherein the method comprises administering an effective amount of vascular endothelial growth factor D (VEGF-D) to the subject in need thereof.

[0126] Embodiment 2: The method of embodiment 1, wherein the respiratory disorder is respiratory distress syndrome, acute lung injury, acute respiratory distress syndrome, cystic fibrosis, pulmonary tuberculosis, cough, bronchial asthma, cough based on increased airway hyperreactivity, flu syndrome, cough hypersensitivity, airway hyperreactivity, tuberculosis disease, asthma, chronic obstructive pulmonary disease, emphysema, pulmonary fibrosis, idiopathic pulmonary fibrosis, cough, reversible airway obstruction, adult respiratory disease syndrome, pigeon fancier's disease, farmer's lung, bronchopulmonary dysplasia, airway disorder, emphysema, allergic bronchopulmonary aspergillosis, allergic bronchitis bronchiectasis, COVID- 19, occupational asthma, reactive airway disease syndrome, interstitial lung disease, or parasitic lung disease.

[0127] Embodiment 3: The method of embodiment 2, wherein the respiratory disorder is acute lung injury (ALI) or acute respiratory distress syndrome (ARDS).

[0128] Embodiment 4: The method of embodiment 3, wherein the ALI or ARDS is caused by systemic sepsis, traumatic injury, or bacterial or viral infection.

[0129] Embodiment 5: The method of any one of the foregoing embodiments, wherein the VEGF-D administered to the subject is encoded by a polynucleotide.

[0130] Embodiment 6: The method of embodiment 5, wherein the polynucleotide comprises recombinant DNA.

[0131] Embodiment 7: The method of embodiment 5 or embodiment 6, wherein the polynucleotide comprises recombinant RNA.

[0132] Embodiment 8: The method of any one of the foregoing embodiments, wherein the VEGF-D administered to the subject comprises a recombinant polypeptide comprising VEGF-D. Embodiment 9: The method of any one of the foregoing embodiments, wherein the VEGF-D is delivered to the subject on an encoded vector, wherein the vector encodes the VEGF- D.

[0133] Embodiment 10: The method of embodiment 9, wherein the vector is an expression vector.

[0134] Embodiment 11 : The method of any one of the foregoing embodiments, wherein the VEGF-D comprises human VEGF-D, mouse VEGF-D, rat VEGF-D, chicken VEGF-D, Rhesus monkey VEGF-D, chimpanzee VEGF-D, or a variant thereof.

[0135] Embodiment 12: The method of any one of the foregoing embodiments, wherein the VEGF-D comprises human VEGF-D, or a variant thereof.

[0136] Embodiment 13: The method of any one of the foregoing embodiments, wherein the VEGF-D comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 1.

[0137] Embodiment 14: The method of embodiment 13, wherein the VEGF-D comprises the amino acid sequence of SEQ ID NO: 1.

[0138] Embodiment 15: The method of any one of the foregoing embodiments, wherein the treatment improves the endothelial integrity of the subject as compared to a subject in need of treatment but not treated with the effective amount of VEGF-D.

[0139] Embodiment 16: The method of any one of the foregoing embodiments, wherein the subject is a mammal.

[0140] Embodiment 17: The method of embodiment 16, wherein the mammal is a human.

[0141] Embodiment 18: The method of any one of the foregoing embodiments, wherein the method further comprises contacting a cell with the VEGF-D.

[0142] Embodiment 19: The method of embodiment 18, wherein the cell is an endothelial cell.

[0143] Embodiment 20: The method of embodiment 19, wherein the endothelial cell is a lung endothelial cell.

[0144] Embodiment 21 : The method of embodiment 20, wherein the lung endothelial cell is a human lung microvascular endothelial cell. Embodiment 22: The method of any one embodiments 18-21, wherein treatment with VEGF-D reduces barrier resistance of the cells compared to untreated cells as measured by ECIS assay.

[0145] Embodiment 23: The method of any one embodiments 18-21, wherein treatment with VEGF-D reduces barrier resistance of the cells compared to cells treated with slit2, BMP5, or secretin as measured by ECIS assay.

[0146] Embodiment 24: The method of any one of embodiments 18-21, wherein the treatment with VEGF-D reduces endothelial permeability compared to untreated cells as measured by transwell assay.

[0147] Embodiment 25: The method of any one of embodiments 18-21, wherein the treatment with VEGF-D reduces endothelial permeability compared to cells treated with slit2, BMP5, or secretin as measured by transwell assay.

[0148] Embodiment 26: The method of any one of embodiments 18-21, wherein treatment with VEGF-D reduces vascular permeability as compared to PBS control cells in an LPS-induced lung injury model.

[0149] Embodiment 27: The method of any one of the foregoing embodiments, wherein the VEGF-D is administered acutely or chronically to the subject.

[0150] Embodiment 28: The method of any one of the foregoing embodiments, wherein the VEGF-D is administered locally, regionally, parenterally, or systemically to the subject.

[0151] Embodiment 29: The method of any one of the foregoing embodiments, wherein the VEGF-D is administered to the subject by at least one route selected from the group consisting of subcutaneous, oral, aerosol, inhalational, rectal, vaginal, transdermal, subcutaneous, intranasal, buccal, sublingual, parenteral, intrathecal, intragastrical, ophthalmic, pulmonary, and topical.

[0152] Embodiment 30: The method of any one of the foregoing embodiments, wherein the VEGF-D is administered to the subject as a pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier.

[0153] Embodiment 31 : A method of promoting the protection of endothelial integrity, wherein the method comprises contacting an endothelial cell with an effective amount of vascular endothelial growth factor D (VEGF-D).

[0154] Embodiment 32: The method of embodiment 31, wherein contacting the endothelial cell comprises contacting the endothelial cell of a subject in need of treatment. Embodiment 33: The method of embodiment 32, wherein the subject has a respiratory disorder.

[0155] Embodiment 34: The method of embodiment 33, wherein the respiratory disorder is respiratory distress syndrome, acute lung injury, acute respiratory distress syndrome, cystic fibrosis, pulmonary tuberculosis, cough, bronchial asthma, cough based on increased airway hyperreactivity, flu syndrome, cough hypersensitivity, airway hyperreactivity, tuberculosis disease, asthma, chronic obstructive pulmonary disease, emphysema, pulmonary fibrosis, idiopathic pulmonary fibrosis, cough, reversible airway obstruction, adult respiratory disease syndrome, pigeon fancier's disease, farmer's lung, bronchopulmonary dysplasia, airway disorder, emphysema, allergic bronchopulmonary aspergillosis, allergic bronchitis bronchiectasis, COVID- 19, occupational asthma, reactive airway disease syndrome, interstitial lung disease, or parasitic lung disease.

[0156] Embodiment 35: The method of embodiment 34, wherein the respiratory disorder is acute lung injury (ALI) or acute respiratory distress syndrome (ARDS).

[0157] Embodiment 36: The method of embodiment 35, wherein the ALI or ARDS is caused by systemic sepsis, traumatic injury, or bacterial or viral infection.

[0158] Embodiment 37: The method of any one of embodiments 31-36, wherein the VEGF-D is encoded by a polynucleotide.

[0159] Embodiment 38: The method of embodiment 37, wherein the polynucleotide comprises recombinant DNA.

[0160] Embodiment 39: The method of embodiment 37 or embodiment 38, wherein the polynucleotide comprises recombinant RNA.

[0161] Embodiment 40: The method of any one of embodiments 31-39, wherein the VEGF-D comprises a recombinant polypeptide comprising VEGF-D.

[0162] Embodiment 41 : The method of any one of embodiments 31-40, wherein the VEGF-D is encoded by a vector.

[0163] Embodiment 42: The method of embodiment 41, wherein the vector is an expression vector.

[0164] Embodiment 43: The method of any one of embodiments 31-42, wherein the VEGF-D comprises human VEGF-D, mouse VEGF-D, rat VEGF-D, chicken VEGF-D, Rhesus monkey VEGF-D, chimpanzee VEGF-D, or a variant thereof. Embodiment 44: The method of embodiment 43, wherein the VEGF-D comprises human VEGF-D, or a variant thereof.

[0165] Embodiment 45: The method of any one of embodiments 31-44, wherein the VEGF-D comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 1.

[0166] Embodiment 46: The method of embodiment 45, wherein the VEGF-D comprises the amino acid sequence of SEQ ID NO: 1.

[0167] Embodiment 47: The method of any one of embodiments 31-46, wherein the endothelial cell is a lung endothelial cell.

[0168] Embodiment 48: The method of embodiment 47, wherein the lung endothelial cell is a human lung microvascular endothelial cell.

[0169] Embodiment 49: The method of any one embodiments 31-48, wherein contacting the endothelial cell with VEGF-D reduces barrier resistance of the cells compared to untreated cells as measured by ECIS assay.

[0170] Embodiment 50: The method of any one embodiments 31-49, wherein contacting the endothelial cell with VEGF-D reduces barrier resistance of the cells compared to cells treated with slit2, BMP5, or secretin as measured by ECIS assay.

[0171] Embodiment 51 : The method of any one of embodiments 31-50, wherein the contacting the endothelial cell with VEGF-D reduces endothelial permeability compared to untreated cells as measured by transwell assay.

[0172] Embodiment 52: The method of any one of embodiments 31-51, wherein the contacting the endothelial cell with VEGF-D reduces endothelial permeability compared to cells treated with slit2, BMP5, or secretin as measured by transwell assay.

[0173] Embodiment 53: The method of any one of embodiments 31-51, wherein contacting the endothelial cell with VEGF-D reduces vascular permeability as compared to PBS control cells in an LPS-induced lung injury model.

[0174] The foregoing outlines features of several embodiments so that those skilled in the art may better understand the aspects of the present disclosure. Those skilled in the art should appreciate that they may readily use the present disclosure as a basis for designing or modifying other processes and structures for carrying out the same purposes and / or achieving the same advantages of the embodiments introduced herein. Those skilled in the art should also realize that such equivalent constructions do not depart from the spirit and scope of the present disclosure, and that they may make various changes, substitutions, and alterations herein without departing from the spirit and scope of the present disclosure. The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

[0175] In sum, while this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMS1. A method of treating a respiratory disorder in a subject in need thereof, wherein the method comprises administering an effective amount of vascular endothelial growth factor D (VEGF-D) to the subject in need thereof.

2. The method of claim 1, wherein the respiratory disorder is respiratory distress syndrome, acute lung injury, acute respiratory distress syndrome, cystic fibrosis, pulmonary tuberculosis, cough, bronchial asthma, cough based on increased airway hyperreactivity, flu syndrome, cough hypersensitivity, airway hyperreactivity, tuberculosis disease, asthma, chronic obstructive pulmonary disease, emphysema, pulmonary fibrosis, idiopathic pulmonary fibrosis, cough, reversible airway obstruction, adult respiratory disease syndrome, pigeon fancier's disease, farmer's lung, bronchopulmonary dysplasia, airway disorder, emphysema, allergic bronchopulmonary aspergillosis, allergic bronchitis bronchiectasis, COVID-19, occupational asthma, reactive airway disease syndrome, interstitial lung disease, or parasitic lung disease.

3. The method of claim 2, wherein the respiratory disorder is acute lung injury (AL1) or acute respiratory distress syndrome (ARDS).

4. The method of claim 3, wherein the ALI or ARDS is caused by systemic sepsis, traumatic injury, or bacterial or viral infection.

5. The method of any one of the foregoing claims, wherein the VEGF-D administered to the subject is encoded by a polynucleotide.

6. The method of claim 5, wherein the polynucleotide comprises recombinant DNA.

7. The method of claim 5 or claim 6, wherein the polynucleotide comprises recombinant RNA.

8. The method of any one of the foregoing claims, wherein the VEGF-D administered to the subject comprises a recombinant polypeptide comprising VEGF-D.

9. The method of any one of the foregoing claims, wherein the VEGF-D is delivered to the subject on an encoded vector, wherein the vector encodes the VEGF-D.

10. The method of claim 9, wherein the vector is an expression vector.

11. The method of any one of the foregoing claims, wherein the VEGF-D comprises human VEGF-D, mouse VEGF-D, rat VEGF-D, chicken VEGF-D, Rhesus monkey VEGF-D, chimpanzee VEGF-D, or a variant thereof.

12. The method of any one of the foregoing claims, wherein the VEGF-D comprises human VEGF-D, or a variant thereof.

13. The method of any one of the foregoing claims, wherein the VEGF-D comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 1.

14. The method of claim 13, wherein the VEGF-D comprises the amino acid sequence of SEQ ID NO: 1.

15. The method of any one of the foregoing claims, wherein the treatment improves the endothelial integrity of the subject as compared to a subject in need of treatment but not treated with the effective amount of VEGF-D.

16. The method of any one of the foregoing claims, wherein the subject is a mammal.

17. The method of claim 16, wherein the mammal is a human.

18. The method of any one of the foregoing claims, wherein the method further comprises contacting a cell with the VEGF-D.

19. The method of claim 18, wherein the cell is an endothelial cell.

20. The method of claim 19, wherein the endothelial cell is a lung endothelial cell.

21. The method of claim 20, wherein the lung endothelial cell is a human lung microvascular endothelial cell.

22. The method of any one claims 18-21, wherein treatment with VEGF-D reduces barrier resistance of the cells compared to untreated cells as measured by ECIS assay.

23. The method of any one claims 18-21 , wherein treatment with VEGF-D reduces barrier resistance of the cells compared to cells treated with slit2, BMP5, or secretin as measured by ECIS assay.

24. The method of any one of claims 18-21, wherein the treatment with VEGF-D reduces endothelial permeability compared to untreated cells as measured by transwell assay.

25. The method of any one of claims 18-21, wherein the treatment with VEGF-D reduces endothelial permeability compared to cells treated with slit2, BMP5, or secretin as measured by transwell assay.

26. The method of any one of claims 18-21, wherein treatment with VEGF-D reduces vascular permeability as compared to PBS control cells in an LPS-induced lung injury model.

27. The method of any one of the foregoing claims, wherein the VEGF-D is administered acutely or chronically to the subject.

28. The method of any one of the foregoing claims, wherein the VEGF-D is administered locally, regionally, parenterally, or systemically to the subject.

29. The method of any one of the foregoing claims, wherein the VEGF-D is administered to the subject by at least one route selected from the group consisting of subcutaneous, oral, aerosol, inhalational, rectal, vaginal, transdermal, subcutaneous, intranasal, buccal, sublingual, parenteral, intrathecal, intragastrical, ophthalmic, pulmonary, and topical.

30. The method of any one of the foregoing claims, wherein the VEGF-D is administered to the subject as a pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier.

31. A method of promoting the protection of endothelial integrity, wherein the method comprises contacting an endothelial cell with an effective amount of vascular endothelial growth factor D (VEGF-D).

32. The method of claim 31, wherein contacting the endothelial cell comprises contacting the endothelial cell of a subject in need of treatment.

33. The method of claim 32, wherein the subject has a respiratory disorder.

34. The method of claim 33, wherein the respiratory disorder is respiratory distress syndrome, acute lung injury, acute respiratory distress syndrome, cystic fibrosis, pulmonary tuberculosis, cough, bronchial asthma, cough based on increased airway hyperreactivity, flu syndrome, cough hypersensitivity, airway hyperreactivity, tuberculosis disease, asthma, chronic obstructive pulmonary disease, emphysema, pulmonary fibrosis, idiopathic pulmonary fibrosis, cough, reversible airway obstruction, adult respiratory disease syndrome, pigeon fancier's disease, farmer's lung, bronchopulmonary dysplasia, airway disorder, emphysema, allergic bronchopulmonary aspergillosis, allergic bronchitis bronchiectasis, COVID-19, occupational asthma, reactive airway disease syndrome, interstitial lung disease, or parasitic lung disease.

35. The method of claim 34, wherein the respiratory disorder is acute lung injury (ALI) or acute respiratory distress syndrome (ARDS).

36. The method of claim 35, wherein the ALI or ARDS is caused by systemic sepsis, traumatic injury, or bacterial or viral infection.

37. The method of any one of claims 31-36, wherein the VEGF-D is encoded by a polynucleotide.

38. The method of claim 37, wherein the polynucleotide comprises recombinant DNA.

39. The method of claim 37 or claim 38, wherein the polynucleotide comprises recombinant RNA.

40. The method of any one of claims 31-39, wherein the VEGF-D comprises a recombinant polypeptide comprising VEGF-D.

41. The method of any one of claims 31-40, wherein the VEGF-D is encoded by a vector.

42. The method of claim 41, wherein the vector is an expression vector.

43. The method of any one of claims 31-42, wherein the VEGF-D comprises human VEGF-D, mouse VEGF-D, rat VEGF-D, chicken VEGF-D, Rhesus monkey VEGF-D, chimpanzee VEGF-D, or a variant thereof.

44. The method of claim 43, wherein the VEGF-D comprises human VEGF-D, or a variant thereof.

45. The method of any one of claims 31-44, wherein the VEGF-D comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 1.

46. The method of claim 45, wherein the VEGF-D comprises the amino acid sequence of SEQ ID NO: 1.

47. The method of any one of claims 31-46, wherein the endothelial cell is a lung endothelial cell.

48. The method of claim 47, wherein the lung endothelial cell is a human lung microvascular endothelial cell.

49. The method of any one claims 31-48, wherein contacting the endothelial cell with VEGF- D reduces barrier resistance of the cells compared to untreated cells as measured by ECIS assay.

50. The method of any one claims 31-49, wherein contacting the endothelial cell with VEGF- D reduces barrier resistance of the cells compared to cells treated with slit2, BMP5, or secretin as measured by ECIS assay.

51. The method of any one of claims 31-50, wherein the contacting the endothelial cell with VEGF-D reduces endothelial permeability compared to untreated cells as measured by transwell assay.

52. The method of any one of claims 31-51, wherein the contacting the endothelial cell with VEGF-D reduces endothelial permeability compared to cells treated with slit2, BMP5, or secretin as measured by transwell assay.

53. The method of any one of claims 31-52, wherein contacting the endothelial cell with VEGF-D reduces vascular permeability as compared to PBS control cells in an LPS- induced lung injury model.

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