[0001] This application claims the benefit of U.S. Provisional Application No.

62/437,263, filed on December 21, 2016, the entire contents of which are incorporated herein by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Grant Numbers

HL067330, HL089934, and HL130826 awarded by the National Institutes of Health. The government has certain rights in the invention. FIELD OF THE DISCLOSURE

[0003] This disclosure generally relates to tissue generation and the treatment of fibrotic tissue. More specifically, this disclosure relates to methods for generating vascular tissue, ameliorating fibrosis and treating hepatic disorders by administration of certain therapeutic agents to a subject. As such, certain therapeutic methods for the treatment of hepatic disorders are disclosed. BACKGROUND

[0004] Hepatic fibrosis is an excessive accumulation of extracellular protein matrix in the presence of cellular stress or organ damage. The progression of hepatic fibrosis is slow and the parameters for evaluating hepatic function are not universally accepted. There is also no standard treatment for hepatic fibrosis. As such, liver diseases culminate in cirrhosis and loss of liver function, posing a major health problem worldwide. See Friedman SL, et al. Sci Transl Med.2013; 5(167); and Friedman SL, Nat. Rev.

Gastroenterol.Hepatol.2010; 7 pp.425-436. Therefore, effective strategies to stimulate liver regeneration are sought after. It is known resection of 70% of liver mass in mammals by partial hepatectomy (PH) induces rapid regrowth of functional liver tissue. Therefore, liver tissue has the capacity to regenerate after damage caused by cellular stress or hepatic disease. See, e.g., Huang W, et al. Science.2006; 312(5771) pp.233- 236. However, hepatic tissue regeneration requires complex interactions between replicating hepatocytes and expanding non-parenchymal cells such as hepatic stellate cells (HSC) (see Seki E, et al., Nature medicine.2007; 13(11) pp.1324-1332), vascular endothelial cells (ECs) (see, e.g., Matsumoto K, et al. Science.2001; 294(5542) pp.559- 563), and hematopoietic cells (see, e.g., Boulter L, et al. Nature medicine.2012; 18(4) pp. 572-579). The complex nature of hepatic tissue regeneration has hindered the

development of effective therapeutics. For example, certain studies showing that disruption of hepatocyte-endothelium signaling in injured liver frequently results in impaired regeneration and maladaptive healing (see Ding BS, et al., Nature.

2014;505(7481) pp.97-102; and Zeisberg EM, et al., Nature medicine.2007; 13(8) pp. 952-61), which leads to the formation of scar tissue (fibrosis), and eventually cirrhosis of the liver.

[0005] Regenerating liver tissue relies on regrowth of the functional sinusoidal vascular network that distributes blood flow between systemic and portal circulation. A dysfunctional hepatic vascular system not only suppresses the metabolic activity of the liver but also induces thrombotic and fibrotic responses. As such, functional remodeling of replicating liver sinusoidal endothelial cells (LSEC) to connect with the existing vascular system is crucial for liver regeneration. However, how hepatic sinusoidal vascular expansion and remodeling are regulated during liver regeneration and fibrogenesis is not currently understood.

[0006] Sphingosine 1-phosphate (S1P) is a membrane-derived lysophospholipid that acts primarily as an extracellular signaling molecule. Signals initiated by S1P are transduced by five G protein-coupled receptors (GPCR), named S1P _1-5 . The lipid mediator, S1P regulates diverse endothelial functions such as barrier function, vascular maturation and flow signaling. See, for example, Blaho VA, et al. Nature.2015; 523(7560) pp.342-346. Cellular and temporal expression of the sphingosine-1-phosphate receptors (S1PR) determine these receptors specific roles in various organ systems, but they are

particularly critical for regulation of the cardiovascular, immune, and nervous systems, with the most well-known contributions of S1PR signaling being modulation of vascular barrier function, vascular tone, and regulation of lymphocyte trafficking. For example, plasma born S1P can act as a chaperone that signals S1P receptors, such as the S1P receptor 1 (S1P ₁ ), which is highly expressed in cardiac ECs. See Galvani S, et al. Science signaling.2015; 8(389). However, knowledge of S1P receptor biology in the liver is incomplete due in-part to the complexity of S1PR mediated physiological networks. Furthermore, viable therapeutic uses for modulators of S1P ₁ have been hindered by findings that repeated administration results in increased vascular leak, bradycardia and atrioventricular block. See Shea BS, et al. Am J. Respir. Cell Mol. Biol.2010; 43 pp. 662-673; Kappos, L., et al. N. Engl. J. Med.2010; 362 pp. 387-401; and Cohen, JA., et al., N. Engl. J. Med.2010; 362 pp.402-415.

[0007] Accordingly, identification of key cellular and molecular mechanisms involved in hepatic regeneration is an important goal in the development of novel therapeutic strategies to control liver-related diseases. SUMMARY OF THE DISCLOSURE

[0008] Regeneration of hepatic sinusoidal vasculature is essential for non-fibrotic liver tissue growth and restoration of metabolic capacity in the liver of subjects having a hepatic disorder. Without being bound by any one particular theory, the present disclosure reveals that activation of endothelial sphingosine-1-phosphate receptor-1 (S1P ₁ ) by its natural ligand (S1P), and S1P bound to apolipoprotein M (ApoM)- containing HDL (HDL-S1P) induces liver tissue growth and regeneration, as well as inhibits hepatic fibrosis. As shown herein, the absence of HDL-S1P or S1P ₁ mediated signaling in the liver endothelium results in aberrant liver tissue regeneration and vascular remodeling, thrombosis and perisinusoidal fibrosis. In contrast, it is also shown, for the first time, that enhancing the amount of HDL-S1P in a subject or administration of an S1P ₁ -specific agonist, e.g., 5-[4-Phenyl-5-(trifluoromethyl)thiophen-2-yl]-3-[3- (trifluoromethyl)phenyl]1,2,4-oxadiazole (SEW2871), results in the regeneration of metabolically functional liver vasculature and alleviates hepatic fibrosis in vivo. [0009] As such, the present disclosure provides novel methods for treating a subject with a hepatic disorder by administering an S1P ₁ -selective agent to the subject to stimulate liver tissue development, vascularization and/or inhibit hepatic fibrosis, such that the S1P ₁ -selective agent is capable of activating S1P ₁ -mediated cell signaling when bound to S1P ₁ on LESCs.

[0010] In certain embodiments of the present disclosure, methods for treating a hepatic disorder or inhibiting the onset of thrombosis or hepatic fibrosis are provided. Hepatic disorders that can be treated by the present methods include any disorder marked by decreased liver function. In some embodiments, the hepatic disorder is a disorder that results in a partial hepatoectomy (PH) or requires regeneration of hepatic tissue. For example hepatic disorders suitable for treatment in the present methods include, but are not limited to, cancer, hepatitis, cirrhosis, fatty liver disease, cholestatic liver injury (cholestasis) and genetic disorders. In certain embodiments, the hepatic disorder is cholestasis or cirrhosis of the liver, i.e., hepatic cirrhosis.

[0011] In certain embodiments, the methods for treating a hepatic disorder include administering an effective amount of a therapeutic agent to a subject that selectively binds to S1P ₁ and activates S1P ₁ -mediated signaling in the endothelium of the liver, i.e., an S1P ₁ -selective agent. The therapeutic agent can be, for example, a synthetic or natural ligand to S1P ₁ (such as S1P or HDL-S1P), a peptide, a nucleic acid, an antibody, a small molecule, or a chemical compound that binds S1P ₁ . In specific embodiments, the therapeutic agent is a small molecule selected from the group consisting of S1P, 5-[4- Phenyl-5-(trifluoromethyl)thiophen-2-yl]-3-[3-(trifluorometh yl)phenyl]1,2,4-oxadiazole (SEW2871), 2-Amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol (Fingolimod), 5-(3- {(1S)-1-[(2-Hydroxyethyl)amino]-2,3-dihydro-1H-inden-4-yl}-1 ,2,4-oxadiazol-5-yl)-2- isopropoxybenzonitrile (Ozanimod), and isomers or analogs thereof . In other embodiments, the therapeutic agent is a peptide that includes ApoM or a high-density lipoprotein (HDL) alone or bound to S1P. In certain instances, the S1P ₁ -selective agent includes a combination of peptides and small molecule. For example, the S1P ₁ -selective agent can include ApoM-bound HDL and S1P. [0012] In some instances, the therapeutic agent can be linked to a carrier or targeting moiety capable of, for example, increasing the serum half-life of the agent and/or selectively delivering the agent to liver endothelial cells.

[0013] In certain embodiments, the methods of the present disclosure include

administering a therapeutic agent selective to S1P ₁ along with at least one additional therapeutic agent. The at least one additional therapeutic agent can be administered as part of the same composition or administered separately. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIGS.1A-1G. Regeneration of liver mass and vascular structure in the absence of S1P ₁ -ligand component, ApoM in partial hepatectomy murine model. Recovery of liver weight (A), body weight (B), mouse survival rate (C), restoration of hepatic function (D), and extent of liver parenchymal injury (E, F) in Apom ^-/- and wild-type mice at indicated time points after hepatic disorder (PH) induction. Levels of plasma bilirubin and serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured to examine hepatic function and liver damage. N = 6-8 mice per group. Each dot in the dot plot indicates individual animal throughout all figures. Statistical relevance was determined by one-way ANOVA. (G) Cell proliferation after PH in the liver was determined by staining of incorporated BrdU. Scale bar = 50 µm.

[0015] FIG.2. Sinusoidal vascular regeneration is impaired in Apom ^-/- mice, compared to control groups. Ultrastructure of LSEC in wild-type (WT) and Apom ^-/- mice after induction of hepatic disease (PH) was analyzed by transmission electron microscopy. After PH induction, Apom ^-/- mice exhibited distorted morphology of LSECs, increased LSEC-hepatocyte distance (dashed line), and a higher level of perivascular matrix protein (inset). Black arrow and red arrowhead indicate the borders of LSEC and hepatocyte, respectively. Scale bar = 5 µm.

[0016] FIGS.3A-3D. Fibrosis in Apom knockout (Apom ^-/- ) and transgenic mice that over-express ApoM protein (Apom TG) after bile duct ligation (BDL)-induced cholestasis. Hepatic disease progression was assessed in all mouse models and sirius red staining (A) and serum levels of AST and ALT (B) were used examine collagen deposition and liver injury of indicated mouse groups, respectively. Scale bar = 50 µm. N = 6-8 mice per group. Statistical difference was determined by one-way ANOVA. Fibrosis and vascular development (ultrastructure) were analyzed by transmission electron microscopy (TEM) to show aberrant vascular development and fibrotic tissue in the epithelium of diseased mice without ApoM (C, D) quantification of platelet-deposited region in mouse liver after bile hepatic disease induction. P < 0.05 between WT and Apom TG groups; N = 7-8 mice per group.

[0017] FIG.4. S1P ₁ signaling in hepatic endothelium modulates vascular regeneration after hepatic disease induction (PH). After partial hepatoectomy (PH), S1P ₁ -mediated signaling (GFP) was localized in VEGFR3 ⁺ LSECs, revealing the role of LSEC S1P ₁ - mediated signaling in proper liver tissue regeneration and vascularization. Scale bar = 50 µm.

[0018] FIGS.5A-5F. Liver regeneration is suppressed in mice with inducible endothelial cell-specific deletion of S1P ₁ (S1pr1 ^iΔEC/iΔEC ). Recovery of liver mass (A), body weight (B) and survival rate (C) of hepatectomized control and hepatectomized mice lacking the S1P1 receptor (S1pr1 ^iΔEC/iΔEC ). N= 7-8 animals per group. Statistical difference was determined by one-way ANOVA. Levels of plasma bilirubin (D), serum AST (E) and serum ALT (F) hepatectomized mice lacking the S1P1 receptor and control mice. N= 7-9 animals per group.

[0019] FIGS.6A-6C. Development of fibrosis in hepatectomized mice lacking S1P1. (A) Transmission electronic microscopy was used to examine the morphology of liver sinusoids after PH (A). Scale bar = 5 µm. Accumulation of fibrin clots and fibronectin protein in the liver of indicated mouse groups after disease induction. Immunoblot of fibrin and fibronectin (B) were used to measure thrombosis and matrix deposition in the liver. Protein levels were quantified (C) and statistical relevance was determined by one- way ANOVA. Scale bar = 50 µm. N = 7-8 mice per group.

[0020] FIGS.7A-7F. Effect of S1P ₁ -selective compounds (SEW2871) on liver regeneration and fibrosis in the absence of ApoM. (A-C) Mouse survival rate (A), recovery of liver weight (B), and restoration of hepatic function (C) were tested in Apom- ^/- mice after oral administration of an exemplary S1P ₁ agonist, SEW2871 or control (vehicle) compositions after induction of hepatic disease (PH). Plasma bilirubin level was measured to examine the functional recovery of liver mass. N = 6-8 mice per group. 10 mg/kg/day SEW2871 was administered via oral gavage for seven days immediately after BDL and for another seven days between day 14 and day 21. P < 0.05 between vehicle and SEW2871 treated groups in (B) and (C). Statistical difference was determined by one-way ANOVA. (D-F) Sinusoidal vascular structure and platelet cell deposition in hepatectomized ApoM ^-/- mice treated with SEW2871. Vascular perfusion and platelet distribution in the liver were determined by staining of LSEC marker VEGFR3, intravenously injected B4-isolectin (D, E), and platelet marker CD41 (F). Immunostaining images are shown in (D), while perfused LSEC number was measured by quantification of VEGFR3 ⁺ Isolectin ⁺ area percentage in (E). SEW2871 improved the perfusion of VEGFR3 ⁺ LSEC in Apom ^-/- mice.

[0021] FIGS.8A-8B. SEW2871 reduced aberrant vascular remodeling and associated fibrosis independent of ApoM in diseased mice. Vascular morphology alteration in treated Apom ^-/- mice was assessed by electron microscopy (A), and collagen deposition was measured by sirius red staining (B). Cholestasis induction disrupted the sinusoidal vasculature structure in Apom ^/- mice, and SEW2871 attenuated this maladaptive vascular remodeling, resulting in a proper sinusoidal structure displaying unperturbed cell junction (arrows) and endothelial morphology. Scale bar = 5 µm (A) and 50 µm (B).

[0022] FIGS.9A-9D. S1P ₁ -selective therapeutic agents promote liver repair in wild-type mice after cholestasis induction (BDL injury). (A) Activation of S1P ₁ signaling by SEW2871 in murine LSEC after cholestasis induction. Here, S1P ₁ -GFP mice were treated with SEW2871 and subjected to bile duct ligation (BDL) to induce cholestasis; GFP was co-stained with LSEC marker VEGFR3. Scale bar = 50 µm. (B)

Administration of SEW2871 prevented liver damage, decreased thrombosis and fibrosis in mice following BDL. Thrombosis was assessed by immunostaining of CD41 (C), and liver fibrosis was determined by Sirius red staining (D). Scale bar = 50 µm. N = 11 mice per group. Statistical difference was determined by one-way ANOVA.

[0023] FIGS.10A-D. SEW2871 decreases liver fibrosis in chronic liver injury hepatic disease model. Chronic liver injury was induced in mice by injection of CCl ₄ every three days for 10 injections. Mice were sacrificed at day 40 after first injection. SEW2871 was given to mice after the third CCl ₄ injection. Administration of SEW2871 restored vascular perfusion and prevented liver fibrosis after repeated CCl ₄ injection. Vascular perfusion was tested by visualization of intravenously injected B4-isolectin (A, B) and fibrosis was determined by measuring levels of SMA (B), Collagen (C), and

hydroxyproline (D) in the injured liver. Veh = vehicle controls. Scale bar = 50 µm. N = 11 mice per group. Statistical relevance was calculated by one-way ANOVA. DETAILED DESCRIPTION OF THE DISCLOSURE

[0024] The present disclosure demonstrates that activation of the endothelial S1P ₁ receptor by either its natural ligand or by an agonist thereof promotes hepatic

regeneration and attenuates fibrosis. More specifically, as shown herein, S1P-bound ApoM-containing HDL (HDL-S1P) controls sinusoidal vessel assembly by activating S1P ₁ mediated signaling in LSECs to form metabolically functional vasculature during liver regeneration. In addition, this disclosure shows that administration of other agents that selectively bind to endothelial S1P ₁ and activates S1P ₁ -mediated signaling in the hepatic endothelium also drive the formation and remodeling of functional LSECs, leading to proper sinusoidal vessel assembly in liver tissue regeneration.

[0025] Without being bound by any one particular theory, it is shown here that a lack of ApoM, which corresponds to an absence of functional HDL-S1P ligand, causes severe vascular dysfunction and occlusion in a damaged liver due to impaired endothelial S1P ₁ signaling. FIGS.1A-3D. This vascular dysfunction exacerbates the symptoms of hepatic disorders, such as cholestatic liver injury and hepatic tissue loss (PH) by triggering more vascular damage, including fibrin clot formation, vascular occlusion, and deposition of perivascular matrix protein. Subsequently, increased vascular stress reinforces the liver injury and increases fibrosis progression. As a result, enhanced fibrosis in the liver markedly disrupts vascular perfusion.

[0026] As such, the present disclosure provides novel methods for treating a subject with a hepatic disorder by administering to the subject an S1P ₁ -selective agent in order to stimulate liver tissue development and vascularization, as well as to inhibit hepatic fibrosis.

[0027] An agent for use in the present methods can be, for example, a synthetic or natural ligand to S1P ₁ , such as an S1P compound, HDL-bound S1P, or a polypeptide comprising S1P-bound ApoM-containing HDL, a nucleic acid, an antibody or fragment thereof, or a chemical compound that binds S1P ₁ .

[0028] The term“S1P ₁ ” or“sphingosine-1-phosphate receptor-1” are used

interchangeably herein to refer to the G protein-coupled receptor that is highly expressed in endothelial cells and binds the ligand sphingosine-1-phosphate (S1P), which is also known as sphingosine-1-phosphate receptor subtype 1, endothelial differentiation gene 1 (Edg1), CD363; ECGF1; EDG-1; CHEDG1, S1PR1 and D1S3362. As used herein the term, S1P ₁ includes the peptide represented by RefSeq ID. No: NP_001307659.1, as well as homologs and orthologs thereof.

[0029] The term“agent” or“therapeutic agent” are used interchangeably herein to refer to any kind of compound or molecule and any combination thereof capable of binding to S1P ₁ and activating the biological function thereof. In certain embodiments the therapeutic agent selectively binds to S1P ₁ but does not bind to other sphingosine-1- phosphate receptors. In one embodiment of the disclosure, the agent is a small molecule. In another embodiment of the disclosure, the agent is a biological molecule, including, but not limited to, a protein peptide or a nucleic acid.

[0030] The term“binding”,“to bind”,“binds”,“bound” or any derivation thereof refers to any chemical bond between two or more molecules, including, but not limited to, covalent bonding, ionic bonding, and hydrogen bonding. Thus, this term also

encompasses hybridization between two nucleic acid molecules among other types of chemical bonding between two or more molecules.

[0031] The term“selectively binds” or“selective” as used herein shall mean an agent that will bind one molecule but not another. For example, an S1P ₁ -selective agent will bind to one sphingosine-1-phosphate receptor, such as S1P ₁ , but not another such as S1P _2-5 . [0032] As shown in FIGS.2 and 9A-10D, S1P ₁ agonists, such as HDL-S1P and

SEW2871 reduce liver damage and fibrosis in both cholestasis and chronic hepatotoxin- mediated injury models. It has also been shown that administration of the S1P ₁ -selective agent, SEW2871, also restores the defective liver regeneration after partial hepatectomy. The beneficial effect of SEW2871 in all tested liver repair models shows that

administration of an S1P ₁ -selective agent that activates S1P ₁ -mediated signaling in the liver of a subject effectively treats liver disorders by reducing liver damage, inhibiting fibrosis and/or promoting hepatic vascular development in a subject regardless of disease etiology.

[0033] As such an S1P ₁ -selective agent of the present disclosure is any small molecule, or compound capable of binding endogenous S1P ₁ .

[0034] The term“compound” as used herein refers to a chemical entity or biological product, or combination of chemical entities or biological products, administered to a subject to treat a hepatic disorder. The chemical entity or biological product is preferably, but not necessarily a low molecular weight compound, but may also be a larger compound, for example, an oligomer of nucleic acids (oligonucleotides), amino acids, chemical compositions or carbohydrates including without limitation proteins, glycoproteins, lipoproteins, aptamers and modifications and combinations thereof. A compound for use in the present methods can be capable of binding to an endogenous molecule, such as S1P ₁ . A compound of the present disclosure can be modified to enhance efficacy, stability, pharmaceutical compatibility, and the like.

[0035] In the context of this disclosure, the term“small molecule” refers to small organic compounds, including but not limited to, heterocycles, peptides, saccharides, steroids, antibodies and the like. The small molecules can have a molecular weight of less than about 1500 Daltons, 1200 Daltons, 1000 Daltons, or 800 Daltons. In some embodiments, a small molecule modulator is less than 500 Daltons. The small molecules can be modified to enhance efficacy, stability, pharmaceutical compatibility, and the like.

[0036] Candidate selective therapeutic agents for use in the present methods can be identified from libraries of synthetic or natural compounds using methods known to those of ordinary skill in the art. For example, synthetic compound libraries are commercially available from a number of companies. Combinatorial libraries are also readily available or can be prepared according to known synthetic techniques. Additionally, natural and synthetically produced libraries and agents may be further modified through conventional chemical and biochemical techniques.

[0037] In certain embodiments, the small molecule includes S1P or 5-[4-Phenyl-5- (trifluoromethyl)thiophen-2-yl]-3-[3-(trifluoromethyl)phenyl ]1,2,4-oxadiazole

(SEW2871) or an analog thereof. In specific embodiments the small molecule including S1P, is S1P bound to HDL, e.g., HDL-S1P.

[0038]“S1P” or“sphingosine-1-phosphate” are used interchangeably herein to refer to the lysophospholipid signaling sphingolipid small molecule having about a 380 Dalton molecular weight with the chemical formula, C ₁₈ H ₃₈ NO ₅ P, and structure,

. S1P is a known blood borne lipid mediator, which binds to lipoproteins such as high-density lipoprotein (HDL), a ligand of the G protein-coupled receptor, S1P ₁ .

[0039] In certain embodiments, the therapeutic agent includes a small molecule, such as 5-[4-Phenyl-5-(trifluoromethyl)thiophen-2-yl]-3-[3-(trifluor omethyl)phenyl]1,2,4- oxadiazole (SEW2871), 2-Amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol

(Fingolimod), 5-(3-{(1S)-1-[(2-Hydroxyethyl)amino]-2,3-dihydro-1H-inden-4- yl}-1,2,4- oxadiazol-5-yl)-2-isopropoxybenzonitrile (Ozanimod) and analogs thereof.

[0040] In other embodiments, S1P ₁ -selective agents for use in the present methods include small molecules identified in U.S. Patent No.8,039,674, the entire contents of which is incorporated herein by reference.

[0041] In contrast to other S1P receptor-binding agents, SEW2871 exhibits limited functionality as an antagonist of immune cell trafficking by antagonism of lymphocyte S1P ₁ receptor function. As such, in specific embodiments, the S1P ₁ -selective agent for use in the present methods is SEW2871, an isomer thereof, and analogs thereof.

[0042] SEW2871 is a small molecule having about a 440 Dalton molecular weight with the chemical formula of C ₂₀ H ₁₀ F ₆ N ₂ OS and following structure: SEW2871 also can include, an analog thereof, a prodrug thereof, thereof, a pharmaceutically acceptable salt thereof (e.g., a non-toxic salt), and a hydrate thereof. Included within the definition of SEW2871, are all isomers and analogs of SEW2871. For example, alkyl, alkenyl, alkynyl, alkyloxy, alkoxy, alkenyloxy, alkynyloxy, alkylthio, alkylsulfonyl, alkylene, alkenylene, alky nylene, acyl, and acyloxy include straight chain and branched ones. Moreover, all of isomers and analogs due to double bond, ring, and fused ring (e.g., cis- and trans-forms), isomers due to presence of asymmetric carbon(s) or the like (R-, S-form, alpha and beta

configurations, enantiomers, and diastereomers), optically active materials having optical rotation (D-, L-, d- and l-forms), polar compounds by chromatographic separation, equilibrium compounds, rotamers, a mixture thereof in any proportion, and a racemic mixture are included. All tautomers are also included in the present definition.

[0043] In certain embodiments, the S1P ₁ -selective agent is a compound such as a peptide or antibody, or a fragment thereof that binds to S1P ₁ in the hepatic endothelium.

[0044] The term“peptide” as used herein refers to a compound comprising a linear series of amino acid residues linked to one another by peptide bonds between the alpha-amino and carboxy groups of adjacent amino acid residues. The term peptide includes polypeptides, such as multimeric proteins or antibodies. In some embodiments, the peptide is an antibody or fragment thereof. The term“synthetic peptide” is intended to refer to a chemically derived chain of amino acid residues linked together by peptide bonds. The term synthetic peptide is also intended to refer to recombinantly produced peptides.

[0045] In some embodiments, the S1P ₁ -selective agent includes a peptide or an antibody. An S1P ₁ -selective protein can be produced, for example using an in vitro cell-free translation system, available commercially e.g., through Life Technology or any other appropriate source. Both prokaryotic and eukaryotic cell-free translation systems can be used to develop S1P ₁ -selective proteins. Typically, a cell-free translation system utilizes extracts prepared from cells engaged in a high rate of protein synthesis, such as rabbit reticulocytes, wheat germ and E. coli, which contain the macromolecular components (70S or 80S ribosomes, tRNAs, aminoacyl-tRNA synthetases, initiation, elongation and termination factors, for example) required for translation. The extract is generally supplemented with amino acids, energy sources (ATP, GTP), energy regenerating systems (creatine phosphate and creatine phosphokinase for eukaryotic systems, and phosphoenol pyruvate and pyruvate kinase for the E. coli lysate), and other co-factors (Mg2+, K+, etc.). Some exemplary protein translation systems, such as reticulocyte lysates and wheat germ extracts, use RNA as a template, whereas other exemplary systems start with DNA templates, which are transcribed into RNA then translated to form the desired S1P ₁ -selective protein.

[0046] In certain embodiments, the S1P ₁ -selective agent includes the ApoM protein. “Apolipoprotein M” or“ApoM” as used herein refers to a 22-kDa HDL-associated apolipoprotein and a member of the lipocalin family of proteins which mainly resides in the plasma HDL fraction of blood. It is known that ApoM acts as a lipid anchor attaching ApoM to the phospholipid layer of lipoproteins, thereby keeping it in circulation and preventing filtration of ApoM.

[0047] In specific embodiments, the agent includes ApoM protein and a high-density lipoprotein (HDL). In other embodiments, the S1P ₁ -selective agent that includes ApoM is bound to a compound, such as S1P. In specific embodiments, the S1P ₁ - selective agent including ApoM comprises ApoM-bound HDL, which is bound to S1P.

[0048] The formation of ApoM-bound S1P ₁ -selective agent can be achieved, for example, by producing ApoM protein in an in vitro translation system in the presence of another protein, such as HDL and incubating the ApoM protein and HDL to permit binding between the two molecules. Alternatively, ApoM- containing HDLs can be purified from human plasma as described, e.g., by Christoffesen, et al. J Lipid Res. (2006) 47 pp.1833-1843.

[0049] In some embodiments, ApoM is allowed to form a complex with S1P prior to therapeutic use. Soluble isolated ApoM proteins, or ApoM-bound HDL peptides can be incubated with SIP to allow the formation of the ApoM-bound HDL-S1P agents. See, e.g., LEE et al., Science, 279: 1552-1555 (1998).

[0050] In other embodiments, an S1P ₁ -selective agent is a pharmaceutical composition suitable for administration for therapeutic use is composed of substantially purified native HDLs containing ApoM proteins.

[0051] In other embodiments, an ApoM containing S1P ₁ -selective agent does not include S1P, and instead and recruits endogenous plasma borne S1P in the subject after administration. For example, S1P is stored in relatively high concentrations in human platelets, which lack the enzymes responsible for its catabolism, and is released into the blood stream upon activation of physiological stimuli, such as growth factors, cytokines, and receptor agonists and antigens commonly released as a result of hepatic injury.

[0052] In certain embodiments, the methods of the present disclosure include

administering a therapeutic agent selective to S1P ₁ is administered alone. In other embodiments, the S1P ₁ -selective agent is administered to a subject with at least one additional therapeutic agent. The at least one additional therapeutic agent can be administered as part of the same composition or administered separately. For example, a second S1P ₁ -selective agent that is different from the first S1P ₁ -selective agent administered or being administered. Other suitable additional agents include, but are not limited to, cytotoxic agents, chemotherapeutic agents, hormones, steroidal anti- inflammatory drugs (e.g., prednisone and corticosteroids), non-steroidal anti- inflammatory drugs (e.g., NSAIDs, aspirin, and acetaminophen) or combinations thereof.

[0053] In some instances, the therapeutic agent can be linked to a carrier or targeting moiety capable of, for example, increasing the serum half-life of the agent and/or selectively delivering the agent to liver endothelial cells. Accordingly, an agent that selectively binds S1P ₁ can be combined with a pharmaceutically acceptable carrier prior to administration to a subject. As used herein, a pharmaceutically acceptable carrier includes any and all solvents, dispersion media, isotonic agents and the like. Except insofar as any conventional media, agent, diluent or carrier is detrimental to the recipient or to the therapeutic effectiveness of the active ingredient(s), i.e., the therapeutic agent, contained therein, its use in practicing the methods of the present disclosure is appropriate. The carrier can be liquid, semi-solid, e.g. pastes, or solid carriers. Examples of carriers include oils, water, saline solutions, alcohol, sugar, gel, lipids, liposomes, resins, porous matrices, binders, fillers, coatings, preservatives and the like, or combinations thereof.

[0054] An active ingredient, i.e., a therapeutic agent, such as an S1P ₁ -selective agent that binds to S1P ₁ in the liver endothelium or that specifically targets and binds the

S1P ₁ receptor, can be combined with the carrier in any convenient and practical manner, e.g., by admixture, solution, suspension, emulsification, encapsulation, absorption and the like, and can be made in formulations such as tablets, capsules, powder, syrup, suspensions, liquid drops, that are suitable for injections, implantations, inhalations, ingestions or any other appropriate application.

[0055] Administration of the S1P ₁ -selective agents described herein may be

accomplished by any acceptable method which allows such S1P ₁ -selective agents to reach its target on the surface of hepatic endothelial cells. Any acceptable method known to one of ordinary skill in the art may be used to administer an agent to the subject. The administration may be localized (i.e., to a particular region, physiological system, tissue, organ, or cell type) or systemic, depending on the hepatic disorder being treated. In certain embodiments, the targeted tissue is an endothelial cell. In specific embodiments, the targeted tissue is a liver sinusoidal endothelial cells (LSEC).

[0056] In some embodiments, S1P ₁ -selective agents can be administered to a subject by injection. Injections can be, for example, intravenous, intradermal, subcutaneous, intramuscular, or intraperitoneal. In certain embodiments, the S1P ₁ -selective agents are administered via intravenous injection.

[0057] In other embodiments, the S1P ₁ -selective agents can be delivered by implantation. Implantation can include inserting implantable drug delivery systems, e.g., microspheres, hydrogels, polymeric reservoirs, cholesterol matrixes, polymeric systems, e.g., matrix erosion and/or diffusion systems and non-polymeric systems, e.g., compressed, fused, or partially-fused pellets. [0058] In further embodiments, the S1P ₁ -selective agents can be delivered by inhalation. Inhalation can include administering the S1P ₁ -selective agents with an aerosol in an inhaler, either alone or attached to a carrier that can be absorbed by the subject.

[0059] Other suitable delivery systems for the administration of S1P ₁ -selective agents include, but are not limited to, time-release, delayed release, sustained release, or controlled release delivery systems (e.g., tablets, capsules, etc.). Such systems may avoid repeated administrations in many cases, increasing convenience to the subject. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include, for example, polymer-based systems such as polylactic and/or polyglycolic acids, polyanhydrides, polycaprolactones, copolyoxalates, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and/or combinations of these. Microcapsules of the foregoing polymers containing therapeutic agents are described in, for example, U.S. Pat. No.5,075,109. Other examples include nonpolymer systems that are lipid- based including sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-, di- and triglycerides; hydrogel release systems; liposome-based systems; phospholipid based-systems; silastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; or partially fused implants. Specific examples include, but are not limited to, erosional systems in which a synthetic compound is contained in a formulation within a matrix (for example, as described in U.S. Pat. Nos.4,452,775, 4,675,189, 5,736,152, 4,667,013, 4,748,034 and 5,239,660), or diffusional systems in which an active component controls the release rate (for example, as described in U.S. Pat. Nos.3,832,253, 3,854,480, 5,133,974 and 5,407,686).

[0060] In certain embodiments, the delivery system administering the S1P ₁ -selective agents may allow sustained or controlled release of the agent to occur, for example, through control of the diffusion or erosion/degradation rate of the formulation containing the S1P ₁ -selective agent.

[0061] The phrase“subject” as used herein refers to any mammalian subject in need of a treatment. Particularly, a subject of the present disclosure has been diagnosed with, or is at risk of developing, a hepatic disorder. The methods disclosed herein can be practiced on any mammalian subject that has a hepatic disorder or is at risk of developing hepatic fibrosis. Particularly, the methods described herein are most useful when practiced on humans.

[0062] The term“treating” or“treating a hepatic disorder”, as used herein, means that one or more of the clinical symptoms of a relevant disorder (i.e., hepatic disorder) is ameliorated or reduced or prevented, the duration that the symptom presents a subject is shortened, or the frequency of the occurrence of the symptom(s) is reduced. For example, treatment of a hepatic disorder can include restoring vascular perfusion in the liver, preventing fibrosis or thrombosis and/or generating healthy hepatic tissue.

[0063] In some embodiments, an S1P ₁ -selective agent can be administered to a subject for preventing or reducing the likelihood of developing a condition associated with a hepatic disorder, especially a patient at risk of developing such a condition. In other embodiments, an S1P ₁ -selective agent is administered to a subject suffering from a condition associated with a hepatic disorder.

[0064] Hepatic disorders that can be treated by the present methods include any disorder marked by decreased liver function. Hepatic disorders to be treated by the present methods will be readily identified by one of ordinary skill in the art. For example, hepatic disorders that can be treated using the present methods include, but are not limited to, cancer, hepatitis, cirrhosis, fatty liver disease, cholestatic liver injury

(cholestasis) and genetic disorders.

[0065] In certain embodiments, the hepatic disorder being treated by the present methods is hepatocellular carcinoma (HCC). In some embodiments, a subject having HCC or at risk of having HCC can be treated using the present methods before, after or during additional treatments for HCC. For example, the subject may be treated according to the present methods after partial hepatoectomy or before. In other embodiments, a subject having HCC may be administered a chemotherapeutic agent, or radiation therapy prior to, concurrently with or after being administered an S1P ₁ -selective agent according to the present methods.

[0066] In some embodiments, the hepatic disorder to be treated by the present methods is hepatitis, such as viral hepatitis (e.g., hepatitis A, hepatitis B, and hepatitis C), autoimmune hepatitis and hepatitis schistosomiasis. In some embodiments, a subject having hepatitis or whom is at risk of having hepatitis can be treated using the present methods before, after or during undergoing additional treatments for hepatitis. For example, the subject may be treated according to the present methods can be administered a therapeutic agent known by one of ordinary skill in the art to treat hepatitis concurrently with or after being administered an S1P ₁ -selective agent.

[0067] In other embodiments, the subject being treated by the present methods can have or be at risk of having fatty liver disease, such as non-alcoholic fatty liver disease (NAFLD), i.e., non-alcoholic steatohepatitis (NASH), or alcoholic liver disease (alcoholic steatohepatitis). By way of example, fatty liver disease can exhibit inflammation and liver cell damage, as well as fat in the liver and lead to cirrhosis or hepatic fibrosis.

[0068] In yet other embodiments, the subject being treated by the present methods can have or be at risk of having cholestasis, drug induced liver injury (DILI), Sclerosing cholangitis, or a genetic liver disorder, e.g., Wilson disease, Alagille syndrome, cystic fibrosis and hemochromatosis.

[0069] In certain embodiments, the hepatic disorder being treated is cholestasis.

Cholestasis is defined by a decrease in bile flow from the liver due to impaired secretion by hepatocytes or obstruction of bile flow through intra-or extrahepatic bile ducts. Therefore, cholestasis is any condition in which substances normally excreted into bile are retained, such as biliary atresia or primary sclerosing cholangitis.

[0070] In specific embodiments, the hepatic disorder being treated is cirrhosis of the liver, i.e., hepatic cirrhosis. Cirrhosis is a condition in which the liver deteriorates and is unable to function normally due to chronic, or long lasting, injury. Subjects with cirrhosis develop scar tissue replacing healthy liver tissue, which (at least partially) blocks the flow of blood through the liver.

[0071] In some embodiments, the hepatic disorder being treated is fibrosis. Fibrosis is marked by scarring of the liver, which can lead to cirrhosis or chornic liver failure. The buildup of scar tissue that causes fibrosis is usually a slow and gradual process. In the early stages of fibrosis, the liver continues to function. However, as fibrosis progresses scar tissue replaces healthy hepatic tissue and the liver begins to fail. As such, in certain instances the present methods can be employed to inhibit the progression of hepatic fibrosis.

[0072] In accordance with the present methods, hepatic disorders are treated by administering to a subject an agent that selectively binds to S1P ₁ causing activation of S1P ₁ -mediated signaling in the endothelium of the liver, or an agent that targets S1P ₁ on LESCs. The term“activate S1P ₁ -mediated signaling” can refer to amount of S1P bound to S1P ₁ , as well as the S1P ₁ ’s ability to interact with its effectors and modulate endothelial cell processes such as, restoring vascular perfusion, preventing fibrosis or thrombosis, generating healthy hepatic tissue.

[0073] In certain instances, activation of S1P ₁ -mediated signaling can be determined, as set forth in Example 8, by detecting one or more physiological markers or processes in the subject and comparing the same to a control level of the same (e.g., a level of the physiological marker or process prior to administration of the S1P ₁ -selective agent to the subject). In certain embodiments, physiological markers that can be detected to determine treatment efficacy using the present methods can include, plasma bilirubin, fibrin, fibronectin, vascular endothelial growth factor-3 (VEGFR3), phosphorylated myosin light chain (MCL), CD41, serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), collagen, smooth muscle actin (SMA) and HDL-S1P.

[0074] Physiological markers, their amount and concentration can be measured by any method commonly known in the art. This includes, for example, methods involving mass spectrometry, high pressure liquid chromatography (HPLC), combined gas

chromatography-mass spectrometry, and liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (see, for example, De Marchi et al., Lipids Health Dis.2:5, 2003), immunoblotting (e.g., Western blotting), or microscopy (e.g., immunostaining, fluorescent microscopy). The activation of S1P ₁ -mediated signaling by an S1P ₁ -selective agent can be determined, for example by measuring an increase or decrease of the concentration of one of the foregoing physiological markers in a subject, by detecting the marker in samples taken at different times, such as before and after administration of an S1P ₁ -selective agent to the subject. Measurements can be conducted in samples taken from, for example, hepatic tissue samples or biopsy.

[0075] In certain embodiments, physiological processes that can be detected to determine treatment efficacy using the present methods. Physiological processes that can be used to determine the efficacy of the therapeutic methods of the present disclosure can include, for example, vascularization in the liver endothelium, hepatic vascular perfusion, liver function, platelet distribution in the hepatic endothelium, fibrosis, thrombosis and Rho signaling activation.

[0076] In certain embodiments, vascularization of the hepatic endothelium includes an increase in the amount of VEGF3 positive endothelial cells in the liver of a subject, when compared to amounts prior to administration of an S1P ₁ -selective agent.

[0077] In one embodiment, hepatic vascular perfusion can be determined by detection of a reduced amount of plasma bilirubin, serum AST and/or Serum ALT in the subject, when compared to amounts of the same prior to administration of an S1P ₁ -selective agent.

[0078] In some embodiments, liver function can be determined by detecting a higher amount Rho signaling (i.e., increased p-MLC in LESC) and/or detection of a lower number of LESC in the subject, when compared to amounts of the same prior to administration of an S1P ₁ -selective agent.

[0079] In other embodiments, fibrosis can be determined by detecting an increased amount of one or more of the following: fibrin (e.g., fibrin beta-sheets) and fibronectin in the endothelial cells of the liver, when compared to amounts of the same prior to administration of an S1P ₁ -selective agent.

[0080] In some emobodiments, thrombosis can be determined by detecting an increased amount of platelets (i.e., CD41 positive cells) in a subject, when compared to amounts of the same prior to administration of an S1P ₁ -selective agent.

[0081] Accordingly, the term“administering a therapeutically effective amount” means the administration of an S1P ₁ -selective agent in an amount that is sufficient to treat a hepatic disorder in the subject. [0082] Administration can be accomplished via single or divided doses over time. As such, the methods of the present disclosure contemplate single as well as multiple administrations, given either simultaneously or over an extended period of time.

[0083] Dosages for a particular subject can be determined by one of ordinary skill in the art using conventional considerations, (e.g. by means of an appropriate, conventional pharmacological protocol). So long as, the dose administered to a subject is sufficient to effect a beneficial therapeutic response in the subject over time. Precise dosages depend on the type of formulations, the route of administration, the timing and frequency of the administration, and the condition of the recipient, for example. For example when the subject is an animal, or a human a veterinarian or physician, respectively may prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. The dose can be determined by the efficacy of the particular formulation, and the activity, stability or serum half-life of the S1P ₁ -selective agent employed and the condition of the subject, as well as the body weight or surface area of the subject to be treated. The size of the dose can also be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular composition in a particular subject.

[0084] Therapeutic compositions comprising one or more S1P ₁ -selective agent can optionally be tested in one or more appropriate in vitro and/or in vivo animal models of disease, to confirm efficacy, tissue metabolism, and to determine dosages, according to methods well known in the art. In particular, dosages can be initially determined by activity, stability or other suitable measures of treatment vs. non-treatment (e.g., comparison of treated vs. untreated cells or animal models), in a relevant assay.

Formulations are administered at a rate determined by the LD50 of the relevant formulation, and/or observation of any side-effects of the S1P ₁ -selective agent at various concentrations, e.g., as applied to the mass and overall health of the patient.

[0085] Dosages may include about 2 mg/kg of bodyweight/day, about 5 mg/kg of bodyweight/day, about 10 mg/kg of bodyweight/day, about 15 mg/kg of bodyweight/day, about 20 mg/kg of bodyweight/day, about 25 mg/kg of bodyweight/day, about 30 mg/kg of bodyweight/day, about 40 mg/kg of bodyweight/day, about 50 mg/kg of bodyweight/day, about 60 mg/kg of bodyweight/day, about 70 mg/kg of bodyweight/day, about 80 mg/kg of bodyweight/day, about 90 mg/kg of bodyweight/day, about 100 mg/kg of bodyweight/day, about 125 mg/kg of bodyweight/day, about 150 mg/kg of

bodyweight/day, about 175 mg/kg of bodyweight/day, about 200 mg/kg of

bodyweight/day, about 250 mg/kg of bodyweight/day, about 300 mg/kg of

bodyweight/day, about 350 mg/kg of bodyweight/day, about 400 mg/kg of

bodyweight/day, about 500 mg/kg of bodyweight/day, about 600 mg/kg of

bodyweight/day, about 700 mg/kg of bodyweight/day, about 800 mg/kg of

bodyweight/day, and about 900 mg/kg of bodyweight/day.

[0086] The methods described herein can supplement treatment conditions by any known conventional method for treating a liver disorder, including, but not limited to, antibody administration, vaccine administration, administration of cytotoxic agents, natural amino acid polypeptides, nucleic acids, nucleotide analogues, and biologic response modifiers. EXAMPLES

Example 1. Materials and Methods.

[0087] Endothelial cell-specific gene deletion. C57BL/6J mice were obtained from Jackson Laboratories. S1Pr1 ^f/f mice were previously described (Jung B, et al.

Developmental cell.2012; 23(3) pp.600-610) and crossed with Cdh5-(PAC)-Cre ^ERT2 mice (Wang Y, et al. Nature.2010; 465(7297) pp.483-486) to establish the Cre ⁺ S1Pr1 ^f/f mice and control S1Pr1 ^f/f mice.

[0088] To induce endothelial cell-specific knockdown of S1P ₁ , Cre ⁺ S1PR1 ^f/f mice and control S1Pr1 ^f/f mice were treated with tamoxifen.6-week-old male and female mice were treated with tamoxifen intraperitoneally at a dose of 200 mg kg ⁻¹ in sunflower oil for 6 days, and interrupted for 3 days after the third dose. After 3 days of rest, the fourth dose was injected for an additional 3 days. After three weeks of tamoxifen treatment, deletion of target genes in ECs was corroborated by quantitative PCR, and mice were used for partial hepatectomy (PH), bile duct ligation (BDL), and CCl ₄ treatment.

[0089] Mouse liver regeneration model. Mouse PH model was used to induce liver regeneration Ding BS, et al. Nature.2010; 468(7321) pp.310-315. Mice were anaesthetized by 100 mg/kg intraperitoneal ketamine and 10 mg/kg xylazine. Midline laparotomy was performed in the anaesthetized mice, and three most anterior lobes (right medial, left medial and left lateral lobes) containing 70% of the liver weight were surgically removed. Briefly, after opening the upper abdomen and the exposure of the liver, the left lobe to be removed was lifted. A 5-0 silk suture tie (Roboz) was placed under the lobe and positioned to the origin of the lobe. After three knots were tied, the tied lobe distal to the suture was resected by a microdissecting scissor. This surgical procedure was then repeated for the other median lobes to complete PH procedure.

Following surgical removal of 70% of liver mass, the peritoneum was re-approximated, and the skin was closed. Sham-operated mice underwent laparotomy without lobe resection.

[0090] Regeneration of liver mass and function was assessed by measuring the weight of residual liver lobes and mouse body weight and levels of plasma bilirubin, serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) at indicated time points after PH.

[0091] Liver injury and fibrosis models. Six to eight-week-old mice were subjected to bile duct ligation to induce mouse cirrhotic liver injury model. Ding BS, et al. Nature. 2014; 505(7481) pp.97-102. To perform bile duct ligation (BDL), mice were subjected to a mid-abdominal incision, under general anesthesia. The common bile duct was ligated in two adjacent positions approximately 1 cm from the porta hepatis. The duct was then severed by incision between the two sites of ligation. Repeated injections of CCl ₄ were used to induce chronic liver injury. Here, CCl ₄ was diluted in oil to yield a concentration of 40% (0.64 mg/ml) and injected every three days at a dose of 1.6 mg/kg. Mice were sacrificed 10 days after the 10th CCl ₄ injection, and the livers were harvested for analysis of morphology and fibrosis.

[0092] To selectively activate S1P ₁ -mediated signaling, 10 mg/kg/day of an exemplary S1P ₁ -selective agent, SEW2871, was administered orally. Here, stock solution of SEW2871 was dissolved in 10% DMSO/25% Tween 20 (v/v). This solvent was used as vehicle for comparison (negative control). 10 mg/kg/day SEW2871 was administered to both Apom knockout and wild-type mice via oral gavage for seven days immediately after BDL and for another seven days between day 14 and day 21. 10 mg/kg SEW2871 was also administered into wild-type mice after a third injection of CCl ₄ and every two days thereafter till last injection. Mice were killed and whole liver tissues were harvested for fibrosis analysis. Collagen I deposition was tested by Sirius red staining, and SMA (5 μg/ml, Abcam, CA), fibronectin (5 μg/ml, Abcam, CA) protein levels were measured by immunoblot. Fibrin β-chain was detected by monoclonal antibody Clone 350 from

American Diagnostica (5 μg/ml), which recognizes fibrin neotope on beta-chain.

[0093] Measurement of fibrosis. Liver fibrosis was assessed after CCl ₄ injection to emulate chronic liver injury disease and BDL to emulate cholestasis hepatic disorders. Collagen deposition was determined by Sirius red staining, and hepatic hydroxyproline level was measured. Liver lobes were weighed, homogenized, and baked in 12 N hydrochloric acid. Obtained samples were added to 1.4% chloramine T in 0.5 M sodium acetate/10% isopropanol (Sigma) and then incubated with Erlich’s solution at 65 ^ο C. Absorbance at 540 nm wavelength was measured and comparing to hydroxyproline standard curve. Content of hydroxyproline in tissue lysate was quantified based on the liver weight. Sirius red staining on liver section was carried out, according to standard protocols.

[0094] Immunostaining and histological analysis of liver cryosections. Liver tissues were harvested for histological analysis. Mouse tissues were fixed with 4% PFA and cryopreserved in OCT. For immunofluorescent (IF) microscopy, the liver sections (10 μm) were blocked (5% donkey serum/0.3% Triton X-100) and incubated in primary Abs: anti-CD41 (mAb, 5 μg/ml, BD Biosciences, CA), anti-VE-cadherin polyclonal Ab (10 μg/ml, R&D Systems, MN), anti-VEGFR3 (mAb, 5 μg/ml, Imclone, NY), anti-fibronectin (5 μg/ml, Abcam, CA), anti-SMA (5 μg/ml, Abcam, CA), and anti-pMLC (5 μg/ml, Cell Signaling). After incubation in fluorophore-conjugated secondary antibodies (2.5 μg/ml, Jackson ImmunoResearch, PA), sections were counterstained with DAPI (Invitrogen, CA).

[0095] Image acquisition and analysis. Histology analysis and Sirius red staining of liver slides were captured with Olympus BX51 microscope (Olympus America, NY), and fluorescent images were recorded on AxioVert LSM710 confocal microscope (Zeiss).

Co-staining of VE-cadherin with SMA and desmin was carried out. Digital images were analyzed using Image J (NIH, MD). Investigators that performed mouse liver regeneration and repair experiments and who determined the extent and pattern of cell proliferation and activation were randomly assigned with animal samples from different experimental groups and were blinded to the genotype of samples.

[0096] Statistics. All data were presented as the mean ± standard error of mean (S.E.M). Comparisons between different groups were made using one-way ANOVA. Statistical significance was set at P < 0.05.

Example 2. Deficiency of ApoM-bound HDL inhibited liver regeneration in mice that have undergone partial hepatectomy. [0097] An Apom ^-/- murine model for partial hepatectomy was studied to test the contribution of ApoM-bound HDL-S1P in liver regeneration. The mice, which lack HDL S1P-binding component, Apoliprotein M (ApoM), and control wild-type (WT) mice underwent partial hepatectomy as above. Hepatic tissue regeneration after PH was significantly reduced in Apom ^-/- mice compared to the control group (WT), as evidenced by decreased liver weight, increased animal lethality, and elevated levels of plasma bilirubin, serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) (FIGS.1A-1F). These data show that ApoM-bound HDL-S1P promotes functional recovery of liver mass after hepatic injury, such as PH.

[0098] Proper regeneration of hepatic tissue requires the assembly of nascent LSECs into perfused sinusoidal vessels to allow hepatic blood circulation, which requires complex interactions between existing vascular system and surrounding parenchymal and stromal cells. To measure whether the hepatic vasculature is perfused by blood after PH, 2 mg/kg of Griffonia simplicifolia lectin (B4-isolectin) was intravenously administered to hepatectomized mice. Isolectin signal was visualized in liver cryosections after co- staining with LSEC marker VEGFR3. The emergence of functional LSECs perfused by hepatic blood flow was determined by identifying VEGFR3 ⁺ LSECs bound by isolectin. After PH, the liver of Apom ^-/- mice exhibited morphologically distorted and functionally non-perfused VEGFR3 ⁺ sinusoidal vasculature, as evidenced in FIG.1G. Therefore, ApoM contributes to the proper regeneration of perfused liver vasculature and restoration of functional liver tissue. Example 3. Impaired new vascular formation in hepatectomized Apom ^-/- mice is associated with perivascular fibrosis and thrombosis.

[0099] The hepatic vascular ultrastructure after PH was analyzed by transmission electron microscopy (TEM) and compared to that of control liver samples. As shown in FIG.2, control livers from healthy mice (WT) exhibit properly positioned LSECs and hepatocytes, while LSEC morphology of in the absence of ApoM (Apom ^-/- ) are fragmented with enlarged sinusoidal lumen. In addition, increased LSEC-hepatocyte distance and perivascular deposition of extracellular matrix (ECM) were observed, indicating that lack of HDL-S1P leads to the development of hepatic fibrosis. These data confirm that ApoM-bound HDL-S1P ligand toS1P ₁ is an essential component in the regeneration of functional sinusoidal vasculature after liver injury because the absence of ApoM-bound HDL-S1P protein leads to aberrant vascular remodeling, perivascular fibrosis, thrombosis, and formation of non-functional dilated sinusoidal vasculature. Example 4. ApoM exhibits anti-fibrotic function in cholestatic mouse model.

[00100] The bile duct ligation (BDL), a clinically relevant liver cholestasis model, was utilized to elucidate how ApoM affects fibrosis in the diseased liver. Common bile duct was ligated and resected to cause biliary epithelial damage in all conditions tested. As shown in FIGS 3A-B, when compared to wild-type cholestatic mice and cholestatic mice over expressing ApoM (TG), cholestatic mice lacking ApoM (Apom-/-) exhibit significant collagen deposition, enhanced serum levels of AST and ALT after BDL, as measured by Sirius red staining (FIG.3A) and immunoblot analysis (FIG.3B). Taken together, these data show that circulating ApoM reduces fibrogenesis in subjects with cholestasis.

[00101] Hepatic vascularisation was analyzed by TEM in wild-type cholestatic mice (WT), cholestatic mice over expressing ApoM (TG), and cholestatic mice lacking ApoM (Apom-/-). As shown in FIG.3C, in wild-type cholestatic mice, hepatic sinusoidal structure was perturbed by BDL, with a disruption of the LSEC layer and compromised cellular structure. However, this morphological abnormality was alleviated in cholestatic mice over expressing ApoM. See FIG.3C. In addition, cholestatic mice over expressing ApoM also exhibited significantly reduced fibrin clot accumulation in the liver than that of wild-type cholestatic mice (FIG.3D). In fact, sinusoidal vascular perfusion was improved in cholestatic mice over expressing ApoM, with ameliorated hepatic damage compared to wild-type cholestatic mice. Therefore, administration of HDL-bound S1P which is carried by ApoM, effectively treats hepatic disorders marked by abberant vascularisation and regeneration by alleviating fibrosis in cholestatic subjects.

Example 5. Activation of ApoM-S1P ₁ pathway in LSEC is essential for endothelial regeneration without causing fibrosis.

[00102] As shown herein, increased ApoM promotes proper liver repair by modulating LSEC function. Therefore, activation of S1P ₁ signaling in LSEC after PH was studied using an S1P ₁ signaling reporter murine model of hepatic disease, which measures S1P ₁ - mediated signaling. FIG.4. Using this clinically relevant model of hepatic injury it was determined that hepatic resection (PH) stimulated S1P ₁ (GFP) signaling in LSECs (VEGFR3 ⁺ ) _ENREF_33indicating that hepatic endothelial S1P ₁ signaling is an important therapeutic target for treating hepatic disorders after liver resection.

Example 6. Mice with endothelial cell-specific S1P ₁ deficiency rescues the disease phenotype of Apom ^-/- mice after partial hepatoectomy.

[00103] Endothelial cell (EC)-specific knockout of S1pr1 mice were generated by breeding floxed S1pr1 (S1pr1 ^f/f ) mice with mice carrying tamoxifen-responsive Cre ^ERT2 recombinase driven by EC-specific VE-cadherin/Cdh5 promoter (Cdh5-(PAC)-Cre ^ERT2 ). To induce EC-specific genetic deletion of S1pr1, resultant S1pr1 ^f/f Cdh5-(PAC)-Cre ^ERT2 mice were intraperitoneally treated with 200 mg/kg tamoxifen. This procedure generated mice with inducible EC-specific deletion of S1pr1 (S1pr1 ^iΔEC/iΔEC ). Tamoxifen-treated S1pr1 ^f/f Cre ^ERT2 mice (sex/age/weight matched littermate mice) were utilized as controls.

[00104] Compared to control mice, liver mass regeneration in S1pr1 ^iΔEC/iΔEC mice was significantly attenuated, which was accompanied by higher levels of plasma bilirubin, serum AST and ALT, as well as lower survival rate after PH, as shown in FIGS.5A-F. Moreover, livers from S1pr1 ^iΔEC/iΔEC mice exhibited a lower number of functional LSEC than those from control mice after PH, indicating the essential role of endothelial S1P ₁ signaling in stimulating the regeneration of functional LSEC in hepatic disease models. [00105] Fibrotic and thrombotic aberrations were examined in S1pr1 ^iΔEC/iΔEC mice after PH by transmission electron microscopy (FIG.6A), immunostaining and immunoblot of fibronectin (FIGS.6B-6C). As shown in FIG.6A, aberrant remodeling of hepatic sinusoidal vasculature was observed in S1pr1 ^iΔEC/iΔEC mice immunoblotting further demonstrated the increased perisinusoidal fibrosis and thrombosis in hepatectomized S1pr1 ^iΔEC/iΔEC mice. See also FIGS.6B-6C. Therefore, hepatic sinusoidal vascular regeneration in S1pr1 ^iΔEC/iΔEC mice recapitulates aberrant vascular regeneration in clinically relevant models of hepatic disease after hepatectomy.

Example 7. S1P ₁ agonist administration enhanced functional hepatic mass regeneration and suppressed fibrosis.

[00106] The similar phenotypes observed in both Apom ^-/- and S1pr1 ^iΔEC/iΔEC mice after induction of a hepatic disorder identify a direct relationship between HDL-S1P with endothelial S1P ₁ signaling. Thus, the defective liver regeneration seen in Apom ^-/- mice could be attributable to aberrant endothelial S1P ₁ -mediated signaling caused by the absence of S1P ₁ ligand, i.e. HDL-S1P. This was confirmed by activation of S1P ₁ - mediated signaling with chemical compounds, such as pharmacological agonists of S1P ₁ , which were shown to rescue the phenotype of Apom ^-/- mice. Notably, FIGS.7A-7C administration of an exemplary S1P ₁ -selective agent, S1P1 agonist SEW2871, enhanced the restoration of liver weight, prolonged animal survival, and augmented hepatic function recovery in Apom ^-/- mice after the induction of a hepatic disorder (PH), when compared to control (vehicle) mice. Additionally, proper regeneration of hepatic sinusoidal vasculature was also augmented by the administration of SEW2871, which was associated with decreased fibrosis and thrombosis. See FIGS.7D-7F. These data show that activation of S1P ₁ -mediated signaling in hepatic endothelial cells effectively regenerates liver tissue and inhibits the development of fibrotic tissue in the liver or thrombosis.

[00107] The efficacy of an exemplary S1P ₁ -selective therapeutic agent (SEW2871) on hepatic disorder-induced fibrosis was examined in in Apom ^-/- mice subjected to cholestasis (BDL). As shown in FIG.8A-8B, oral administration of SEW2871 prevented disruption of the sinusoidal vasculature in Apom ^-/- mice after disease induction (FIG.8A), while also preserving proper sinusoidal vascular perfusion (FIG.8B). Taken together, these data show that activation of S1P ₁ -mediated signaling results in proper hepatic vascular re-growth independent of the presence of endogenous ApoM.

Example 8. Effect of S1P ₁ agonist SEW2871 in WT mice after in cholestasis and chronic liver injury hepatic disease models.

[00108] To further establish the therapeutic value of administering S1P ₁ -selective agents to subjects having hepatic disorders, the efficacy of SEW2871 was examined in normal, wild-type mice (WT) mice in a cholestasis (BDL) hepatic disease model. Treatment of WT mice with SEW2871 promoted activation of endothelial S1P ₁ -mediated signaling (GFP expression in S1P ₁ -GFP mice) (FIG.9A) prevented hepatic parenchymal damage after induction of cholestasis, as demonstrated by lower levels of serum ALT, AST and bilirubin than vehicle-treated control mice (FIG.9B). In addition, both thrombosis and fibrosis in the diseased liver were alleviated by administration of the exemplary S1P ₁ - selective agent, SEW2871, as shown in FIGS.9C and 9D.

[00109] The therapeutic effects of S1P ₁ -selective agents were further assessed by the administration of SEW2871 assessed in a chronic liver injury hepatic disease model. To induce hepatic injury, hepatotoxin carbon tetrachloride (CCl ₄ ) was intraperitoneally administered to wild-type mice ten times to induce chronic liver injury. Fibrosis and vascular perfusion were compared between diseased mice administered SEW2871 and diseased mice administered a control agent (vehicle). AS shown in FIGS.10A-10D, intravenous administration of the exemplary S1P ₁ -selective agent, SEW2871, improved hepatic vascular perfusion and reduced smooth muscle actin (SMA) protein levels in the diseased livers (FIGS.10A-10B). Furthermore, as shown in FIGS.10C-10D, administration of SEW2871 lead to improved vascular function and reduced liver fibrosis. In view of the foregoing, it has been shown that administration of S1P ₁ -selective agents, such as SEW2871 effectively treats hepatic disorders by activating S1P ₁ signaling in LSEC.