WO2025174646A1 - Inhibitors of beta-catenin c-terminal domain and methods of use - Google Patents

Abstract

Provided herein are compositions that inhibit activity of the C-terminal domain of beta-catenin. Also provided herein are methods of using such inhibitors of the C-terminal domain of beta-catenin for treating disease in a subject in need thereof, including, but not limited to, methods of preventing or treating cardiovascular disease.

Description

INHIBITORS OF ΒETA-CATENIN C-TERMINAL DOMAIN AND METHODS OF USE STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH [0001] This invention was made with government support under 1R01HL133861 awarded by the National Institutes of Health (NIH) and under 1R01HL163635 awarded by the NIH. The government has certain rights in the invention. FIELD [0002] The present disclosure relates generally to the field of molecular biology and medicine. More particularly, the methods and compositions herein are useful for treating disease, including cardiovascular disease. BACKGROUND [0003] Cardiovascular disease is the leading cause of death globally, and is responsible for significant morbidity and health-care costs worldwide. Vascular remodeling is a hallmark of cardiovascular diseases including atherosclerosis, stenosis after angioplasty and stent placement, vein graft disease, and transplant arteriosclerosis. As a result, vascular remodeling can lead to target organ damage and fatal cardiovascular events. [0004] Smooth muscle cells (SMCs) contribute substantially to vascular remodeling induced by injury or disease. In normal arteries, SMCs are typically confined to the medial layer and regulate vessel tone or diameter. After vascular injury or during atherosclerosis, SMCs undergo phenotypic switching from a quiescent, differentiated, contractile, and non- migratory phenotype to a dedifferentiated, migratory, proliferative, and secretory phenotype. These changes promote SMC proliferation and migration from the media into the intima, and lead to neointima formation that may occlude the arterial lumen. The molecular mechanisms underlying these processes are, however, not fully understood, which limits implementation of therapies to reduce vascular remodeling and disease. [0005] Accordingly, new compositions and methods that reduce SMC proliferation and neointima formation are urgently needed. SUMMARY [0006] Provided herein are compositions that inhibit activity of the C-terminal domain of beta-catenin and methods of using such compositions for the treatment of disease. 166682443.1 [0007] Provided is a method of preventing or reducing restenosis in a blood vessel in a subject in need thereof, the method comprising administering to the subject a compound of Formula I or a pharmaceutically acceptable salt thereof. [0008] Provided is a method of preventing, inhibiting, or reducing vascular remodeling in the blood vessel of a subject in need thereof, the method comprising administering to the subject a compound of Formula I or a pharmaceutically acceptable salt thereof. [0009] Provided is a method of preventing, inhibiting, or reducing thickening of the intima in a blood vessel in a subject in need thereof, the method comprising administering to the subject a compound of Formula I or a pharmaceutically acceptable salt thereof. [0010] Provided is a method of decreasing proliferation of smooth muscle cells in a blood vessel in a subject in need thereof, the method comprising administering to the subject a compound of Formula I or a pharmaceutically acceptable salt thereof. [0011] Provided is a method of preventing, inhibiting, or reducing the formation and/or expansion of atherosclerotic lesions in a blood vessel in a subject in need thereof, the method comprising administering to the subject a compound of Formula I or a pharmaceutically acceptable salt thereof. [0012] Provided is a method of maintaining patency of a blood vessel in a subject, the method comprising administering to the subject a compound of Formula I or a pharmaceutically acceptable salt thereof. [0013] In one embodiment, the subject has undergone angioplasty of the blood vessel. In one embodiment, the subject has undergone placement of a stent in the subject’s blood vessel. In one embodiment, the subject has undergone endarterectomy. [0014] In some embodiments, the blood vessel is a coronary blood vessel, a peripheral artery, a carotid artery, or a cerebral artery. In one embodiment, the blood vessel is in a arterio- venous fistula (AVF). In one embodiment, the patient has undergone a blood vessel graft. In one embodiment, the blood vessel graft is a vein graft. In one embodiment, the subject has undergone interposition vein grafting into the arterial circulation or bypass surgery. [0015] In one embodiment, the subject has a cardiovascular disease or is at risk of developing cardiovascular disease. In one embodiment, the subject has atherosclerosis or is at risk of developing atherosclerosis. [0016] Provided is a method of preventing or reducing transplant-associated vasculopathy in a subject in need thereof, the method comprising administering to the subject a compound of Formula I or a pharmaceutically acceptable salt thereof. 2 166682443.1 [0017] Provided is a method of preventing, inhibiting, or reducing progression of atherosclerosis in a subject in need thereof, the method comprising administering to the subject a compound of Formula I or a pharmaceutically acceptable salt thereof. [0018] Provided is a method of surgically inserting a stent of into the lumen of a blood vessel in a subject in need thereof, wherein the stent comprises (i) a scaffold coated with a polymer, which polymer is impregnated with, adsorbed with, or coated with a compound of Formula I, or (ii) a scaffold which is impregnated with, adsorbed with, or coated with, a compound of Formula I. [0019] In one embodiment, the subject is a mammal. In one embodiment, the subject is a human. BRIEF DESCRIPTION OF THE FIGURES [0020] Figs.1A, 1B, and 1C illustrate the analysis of smooth muscle ^-catenin C-terminus- deficient (SMβC^{^C}) mice. Fig. 1A. Western blotting was performed for ^-catenin in total arterial protein lysates isolated from aortas of wild-type (SMβC^WT/-), smooth muscle ^-catenin C-terminus-deficient (SMβC^{^C}), and smooth muscle ^-catenin knockout (SMβC^-/-) mice after tamoxifen injection. Different ^-catenin antibodies targeting the C- or N-terminal domains (C- term or N-term, respectively) of the protein were used. Arterial lysates from smooth muscle ^- catenin knockout (SMβC^-/-) mice were used as a negative control. Shown is the densitometric analysis of ^-catenin protein levels evaluated by using distinct antibodies targeting the C- or N-terminal domain of the protein, normalized to GAPDH (n=3). Statistical analysis: unpaired Student’s t-test. *P<0.05. Data shown as mean ± SEM of independent biological replicates. Fig.1B. Immunostaining for ^-catenin and red fluorescent protein (RFP) in carotid arteries 21 days after ligation. Different antibodies targeting the C- or N-terminal domains (C-term or N- term, respectively) of ^-catenin were used. Shown is the percentage of smooth muscle cells (SMCs) positive for ^-catenin according to the antibodies used. Statistical analysis: unpaired Student’s t-test. n=3. *P<0.05. ns, not significant. Data shown as mean ± SEM of independent biological replicates. Fig. 1C. Percentage of SMCs positive for Axin2. Statistical analysis: unpaired Student’s t-test. n=3. *P<0.05. Data shown as mean ± SEM of independent biological replicates. [0021] Figs. 2A, 2B, and 2C illustrate that smooth muscle ^-catenin C-terminus signaling promotes neointima formation after carotid artery ligation. Carotid artery ligation was performed one week after the first tamoxifen injection in male and female SMβC^{^C} and 3 166682443.1 littermate control (SMβC^wt/-) mice. Ligated and uninjured arteries were harvested at 21 days after surgery. Morphometric measurements of the medial area (Fig.2A), intimal area (Fig.2B), and a ratio of intimal to medial area (Fig.2C) are shown. Each dot represents a single mouse. Statistical analysis: unpaired Student’s t-test. Values are represented as mean ± SEM of independent biological replicates. n=11 for SMβC^WT/- males, n=7 for SMβC^WT/- females, and n=10 for SMβC^{^C} males and females. [0022] Figs.3A, 3B, 3C, and 3D illustrate that loss of SMC ^-catenin C-terminal signaling decreases SMC proliferation and dedifferentiation and increases apoptosis. Fig. 3A. Immunostaining for PCNA, a marker of proliferation, and RFP, SMC lineage tracer, in carotid arteries 21 days after ligation was performed. Percentage of SMCs positive for PCNA is shown. Statistical analysis: unpaired Student’s t-test. n=3. Data shown as mean ± SEM of independent biological replicates. Fig. 3B. Immunostaining for Ki67 and RFP, SMC lineage tracer, in carotid arteries 21 days after ligation was performed. Percentage of SMCs positive for Ki67 is shown. Statistical analysis: Mann-Whitney test. n=4 for SMβC^WT/- and n=5 for SMβC^{^C}. Data shown as mean ± SEM of independent biological replicates. Fig. 3C. Immunostaining for cleaved caspase 3 (Casp3), a marker of apoptosis, and RFP in carotid arteries 21 days after ligation was performed. Percentage of SMCs positive for cleaved caspase 3 is shown. Statistical analysis: unpaired Student’s t-test. n=6 for SMβC^WT/- and n=7 for SMβC^{^C}. Data shown as mean ± SEM of independent biological replicates. Fig.3D. Immunostaining for smooth muscle actin (SMA) and RFP in carotid arteries 21 days after ligation was performed. Percentage of SMCs positive for SMA in uninjured (control) and ligated carotid arteries is shown. Statistical analysis: unpaired Student’s t-test. n=3 for SMβC^WT/- and n=5 for SMβC^{^C}. Data shown as mean ± SEM of independent biological replicates. [0023] Figs. 4A, 4B, 4C, 4D, and 4E illustrate that loss of SMC ^-catenin C-terminal signaling reduces SMC growth and downregulates S1pr1 expression. Fig.4A. Cell population growth of βC^Control and βC^ΔC MASMCs. *P<0.05, 2-way ANOVA with Šídák’s multiple comparison test. n=6. Data shown as mean ± SEM of independent biological replicates. Fig. 4B. Principal component analysis from RNA-seq of βC^Control (dots on the right) and βC^ΔC (dots on the right) MASMCs. Each dot represents an independent sample. Fig. 4C. Volcano plot. Each data point represents a gene. The log2 fold change of each gene is represented on the x- axis and the log10 of its adjusted p-value (padj) is on the y-axis. Dots on the right indicate significantly upregulated genes (log2 fold change>1 and padj<0.05), while dots on the left represent significantly downregulated genes (log2 fold change<-1 and padj<0.05) in βC^ΔC 4 166682443.1 MASMCs compared to βC^Control. The black dots in the middle represent genes that do not satisfy the above conditions. Fig. 4D. Significantly enriched canonical signaling pathways identified by IPA and ranked by p-value. Positive and negative z-scores indicate up- and down-regulated signaling pathways, respectively, in βC^ΔC vs. βC^Control MASMCs. Fig. 4E. qPCR for Axin2, S1pr1, and S1pr3, which were significantly downregulated in βC^ΔC MASMCs in RNA-seq analysis. Rpl13 and β-actin were used as housekeeping controls. Statistical analysis: unpaired Student’s t-test. n=6. ns, not significant. Data shown as mean ± SEM of independent biological replicates. βC^ΔC (right bars), βC^Control (left bars). [0024] Figs. 5A, 5B, 5C, 5D, 5E, 5F, 5G and 5H illustrate that S1PR1 is a transcriptional target of β-catenin and rescues the defective growth of βC^ΔC MASMCs towards control levels. Fig. 5A. Densitometric analysis of S1PR1 in βC^Control and βC^ΔC MASMCs (assessed by Western blotting), normalized to GAPDH. *P<0.05, unpaired Student’s t-test. n=4. Fig. 5B. Densitometric analysis of S1PR1 and β-catenin in βC^Control and βC^ΔC MASMCs electroporated with empty vector or β-catenin^S33Y, a constitutively active form of β-catenin (assessed by Western blotting). *P<0.05, 2-way ANOVA with Šídák’s multiple comparison test. n=3. ns, not significant. βC^ΔC (right two bars in each panel), βC^Control (left two bars in each panel). Fig. 5C. Densitometric analysis of S1PR1 and β-catenin in βC^Control and βC^ΔC MASMCs (assessed by Western blotting) treated with CHIR99021 (CHIR), a small molecule that prevents β-catenin degradation. *P<0.05, 1-way ANOVA with Tukey’s multiple comparison test. n=3. βC^ΔC (right three bars in each panel), βC^Control (left three bars in each panel). Fig. 5D. βC^Control and βC^ΔC MASMCs were co-electroporated with a human S1pr1-driven luciferase reporter (pGL3- S1pr1-promoter) and empty vector or β-catenin^S33Y. Forty-eight hours post-transfection, cells were harvested for luciferase assay. Luminescence (Lum) was normalized to β-galactosidase activity (β-gal) to control for transfection efficiency and expressed relative to empty vector in βC^Control MASMCs. *P<0.05, 2-way ANOVA with Šídák’s multiple comparison test. n=6. βC^ΔC (right two bars), βC^Control (left two bars). Fig.5E. Cleavage under targets & release using nuclease (CUT&RUN) assay was conducted in MASMCs using anti-β-catenin, anti-TCF4 and anti-IgG antibodies and analyzed by q-PCR using primers that amplify different regions in the S1pr1 promoter containing (+) or not (-) a consensus TCF binding motif, 5′-TCAAAG. Indicated locations are relative to transcription start site. *P<0.05, unpaired Student’s t-test. n=4. ns, not significant. IGG (left bars), beta-catenin (middle bars), TCF4 (right bars). Fig.5F. Cleavage under targets & release using nuclease (CUT&RUN) assay was conducted in MASMCs using anti-β-catenin and anti-IgG antibodies and analyzed by quantitative polymerase chain reaction (qPCR) using primers that amplify a region in the S1pr1 promoter 5 166682443.1 containing a consensus TCF binding motif, 5′-TCAAAG (fragment -939/-790). Statistical analysis: unpaired Student’s t-test. n=3-6. ns, not significant. Data shown as mean ± SEM of independent biological replicates. IGG (left bars), beta-catenin (right bars). Fig. 5G. qPCR using controls for CUT&RUN assay. qPCR for IGG, ^-catenin and TCF4 was performed using a negative control primer set that amplifies an 82 base pair fragment in a gene desert on mouse chromosome 6. Anti-H3 antibody and RPL30 (ribosomal protein L30) primers were used as a positive control for the assay technique and reagent integrity. n=4. Values are represented as mean ± SEM of independent biological replicates. Fig.5H. Cell population growth of βC^Control, βC^ΔC (bottom trace), βC^Control S1PR1^{Gain-of-function (GOF)} and βC^ΔC S1PR1^GOF MASMCs. *P<0.05 compared to other 2- with 5I. Cell population (10 nM) and treated with CHIR (1 ^M) or vehicle. Statistical analysis: 2-way ANOVA with Šídák’s multiple comparison test. n=6. *P<0.05 compared to other groups. Data shown as mean ± SEM of independent biological replicates. siControl + CHIR (top trace). [0025] Figs. 6A. 6B, 6C, and 6D illustrate that reestablishing S1PR1 expression in SMCs rescues injury induced-neointima formation in ^-catenin C-terminus-deficient mice. Fig. 6A. Immunostaining for S1PR1 and RFP was performed in carotid arteries 21 days after ligation. Percentage of SMCs positive for S1PR1 in the media and neointima area of carotid arteries is shown. Statistical analysis: unpaired Student’s t-test. n=5. Data shown as mean ± SEM of independent biological replicates. SMβC^WT/- (left bar), SMβC^{^C} (right bar). Figs. 6B and 6C. Morphometric measurements of the intimal area (Fig.6B), and a ratio of intimal to medial area (Fig.6C). Each dot represents a single mouse. Statistical analysis: 2-way ANOVA with Šídák’s multiple comparison test. n=6 for SMβC^WT/- males and females, n=7 for SMβC^{^C} males and females, n=7 for SMβC^WT/- SM-S1PR1^GOF males and females and n=8 for SMβC^{^C} SM- S1PR1^GOF males and females. Values are represented as mean ± SEM of independent biological replicates. SMβC^WT/- (left bars), SMβC^{^C} (right bars). Fig.6D. Effect of restoring sphingosine- 1 phosphate receptor-1 (S1PR1) expression on medial area of carotid arteries after vascular injury. Measurements of the medial area of carotid arteries of wild-type (SMβC^WT/-), smooth muscle ^-catenin C-terminus-deficient (SMβC^{^C}), smooth muscle-specific S1PR1 gain-of- function (GOF) (SMβC^WT/- SM-S1PR1^GOF), and smooth muscle-specific ^-catenin C- terminus-deficient, S1PR1 GOF (SMβC^{^C} SM-S1PR1^GOF) mice 21 days after ligation. Each dot represents a single mouse. Statistical analysis: 2-way ANOVA with Šídák’s multiple comparison test. n=6 for SMβC^WT/- males and females, n=7 for SMβC^{^C} males and females, 6 166682443.1 n=7 for SMβC^WT/- SM-S1PR1^GOF males and females and n=8 for SMβC^{^C} SM-S1PR1^GOF males and females. Values are represented as mean ± SEM of independent biological replicates. [0026] Figs. 7A, 7B, 7C, 7D, 7E, 7F, 7G, 7H, and 7I illustrate that pharmacologic inhibition of ^-catenin C-terminal signaling attenuates neointimal formation and reduces S1PR1 expression in vivo. Fig. 7A. The ^-catenin C-terminus inhibitor E7386 at 100 nM inhibits cell population growth of βC^Control MASMCs but has no effect on βC^ΔC MASMCs. Statistical analysis: 2-way ANOVA with Šídák’s multiple comparison test. n=6. *P<0.05 compared to other groups. Data shown as mean ± SEM of independent biological replicates. βC^Control Vehicle (top trace). Figs. 7B and 7C. The ^-catenin C-terminus inhibitor E7386 reduces cell growth and the sphingosine-1 phosphate receptor-1 (S1PR1) expression in mouse aortic smooth muscle cells (MASMCs). E7386 at 50 nM (Fig.7B) and 10 nM (Fig.7C) inhibits cell population growth of βC^Control MASMCs but has no effect on βC^ΔC MASMCs. Statistical analysis: 2-way ANOVA with Šídák’s multiple comparison test. n=6. *P<0.05 for βC^Control vehicle compared to other groups. #P<0.05 for βC^Control E7386 compared to other groups. Data shown as mean ± SEM of independent biological replicates. Fig. 7D. Densitometric analysis of Axin2, a typical target of ^-catenin signaling (as assessed by Western blotting), and S1PR1 in MASMCs treated with E7386 (100 nM) or vehicle for 48h, normalized to GAPDH. Statistical analysis: unpaired Student’s t-test. n=3. Data shown as mean ± SEM of independent biological replicates. Fig. 7E. E7386 reduces in vivo expression of Axin2, a typical target ^- catenin signaling. Percentage of SMA⁺ cells positive for Axin2 is shown. n=5 for each condition. Figs. 7F, 7G, and 7H. Morphometric measurements of the medial area (Fig. 7F), intimal area (Fig. 7G), and a ratio of intimal to medial area (Fig. 7H). Each dot represents a single mouse; n=7 for each group except n=6 for E7386-treated females. Statistical analysis: unpaired Student’s t-test. Values are represented as mean ± SEM of independent biological replicates. Fig. 7I. E7386 reduces in vivo expression of S1PR1. Percentage of SMA⁺ cells positive for S1PR1 is shown. n=4 for each condition. Scale bar: 50 μm. Statistical analysis: unpaired Student’s t-test. Values are represented as mean ± SEM of independent biological replicates. [0027] Figs.8A, 8B, 8C, 8D, 8E, 8F, 8G, 8H, 8I, and 8J illustrate that the pharmacologic inhibition of ^-catenin C-terminal signaling limits atherosclerosis and decreases S1PR1 expression in SMCs from atherosclerotic lesions. Fig.8A. Axin2 expression in carotid arteries of ApoE^-/- mice fed with Western diet and treated with vehicle or E7386. Immunostaining was performed for Axin2 and smooth muscle actin (SMA) in carotid arteries of ApoE^-/- mice fed 7 166682443.1 with Western diet and treated with vehicle or E738625 mg/kg for 21 days. Percentage of SMA⁺ cells positive for Axin2 is shown. Each dot represents a single mouse. Statistical analysis: unpaired Student’s t-test. n=6 (vehicle) and 5 (E7386). Data shown as mean ± SEM of independent biological replicates. Fig. 8B. Body weight of ApoE^-/- mice before and at the end of the atherosclerosis protocol. Statistical analysis: unpaired Student’s t-test. n=12 (vehicle) and 14 (E7386). Data shown as mean ± SEM of independent biological replicates. Fig. 8C. Total serum cholesterol levels of ApoE^-/- mice fed with Western diet and treated with vehicle or E738625 mg/kg for 21 days. Statistical analysis: unpaired Student’s t-test. n=4 (vehicle) and 6 (E7386). Data shown as mean ± SEM of independent biological replicates. Figs.8D, 8E, 8F, 8G, 8H, and 8I. Morphometric measurements of the medial area (Fig. 8D), lesion area (Fig. 8E), ratio of lesion to medial area (Fig. 8F), lumen area (Fig. 8G), necrotic core area (Fig. 8H), and fibrous cap thickness (Fig. 8I). Each dot represents a single mouse; n=11 (vehicle) and n=14 (E7386). Statistical analysis: unpaired Student’s t-test. Values are represented as mean ± SEM of independent biological replicates. Fig.8J. Percentage of SMA+ cells positive for S1PR1 in atherosclerotic lesions is shown. Each dot represents a single mouse; n=5 for each condition. Statistical analysis: unpaired Student’s t-test. Values are represented as mean ± SEM of independent biological replicates. DETAILED DESCRIPTION [0028] Provided herein are compositions that inhibit activity of the C-terminal domain of beta-catenin and methods of using such compositions for the treatment of disease. [0029] Beta-catenin [0030] The Wnt/beta-catenin signaling pathway belongs to a small group of highly conserved signaling networks that support normal development and adult tissue homeostasis. The terms beta-catenin and β-catenin are used herein interchangeably. [0031] Beta-catenin (Armadillo in Drosophila) is a multifunctional, 90 kD protein that serves as a central downstream effector of the Wnt/beta-catenin signaling pathway. Stimulation of beta-catenin by Wnt ligands prevents cytoplasmic beta-catenin degradation, which in turn allows its nuclear translocation and interaction with the transcription factor T-cell factor 4 (TCF4, also known as Tcf7l2) to induce transcription of β-catenin target genes. Additionally, beta-catenin plays a structural role in cell and tissue integrity as part of the cadherin-mediated cell-cell adhesion complex. Imbalance in the structural and signaling properties of β-catenin 8 166682443.1 (also referred to as CTNNB) often results in disease and deregulated growth connected to cancer and metastasis. [0032] Beta-catenin (781 aa residues in humans) is a member of the Armadillo (ARM) repeat protein superfamily. Each ARM repeat of its central region comprises ∼42 residues, forming three helices arranged in triangular shape. As a result, all ARM repeats form a superhelix that features a long, positively charged groove. Beta-catenin consists of a central region (residues 141–664) comprising twelve imperfect Armadillo repeats (R1–12). This central region is flanked by distinct N- and C-terminal domains, NTD and CTD, respectively. A specific conserved helix (Helix-C) is located between the last Armadillo repeat and the flexible part of the CTD. Both the NTD and the CTD may be structurally flexible, whereas the central region forms a relatively rigid scaffold. The central scaffold serves as an interaction platform for many β-catenin binding partners, including at the membrane, in cytosol, and in the nucleus. The C-terminus of β-catenin binds a variety of complexes promoting β-catenin- mediated transcription. For the adhesive functions of β-catenin the central Armadillo repeats are essential, while N- and C-terminal domain are dispensable. β-catenin signaling activity, on the other hand, requires the Armadillo repeats as well as the N- and/or C-terminal domains. [0033] Provided herein are compositions that inhibit the beta-catenin by interacting with beta-catenin’s CTD as well as methods of using such compositions to treat disease. [0034] Inhibitors of beta-catenin [0035] In one aspect, provided is a method of using an inhibitor of beta-catenin’s CTD for treating a disorder or a disorder in a subject in need thereof. As used herein, an “inhibitor of beta-catenin’s CTD” is a natural or synthetic compound that has a biological effect to reduce, eliminate, or otherwise interfere with the activity of the CTD of beta-catenin and/or to reduce, eliminate, or otherwise interfere with binding of the CTD of beta-catenin with one or more interaction partners. The inhibitor may reduce the activity of the CTD of beta-catenin and/or binding of the CTD of beta-catenin with one or more interaction partners by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% as compared to the corresponding activity and/or binding prior to treatment with the inhibitor. The inhibitor may perform this biological function by interaction with beta-catenin’s CTD directly or indirectly. The inhibitor may be a small molecule, e.g., a molecule of molecular weight less than about 1.5 kilodaltons (kDa). The inhibitor may be a polypeptide, including, but not limited to, an antibody or antigen-binding fragment thereof. The inhibitor may be a polypeptide analog. 9 166682443.1 Methods for determining whether a specific compound is an inhibitor of beta-catenin’s CTD are known in the art and described herein. [0036] In one embodiment, the inhibitor comprises a compound of Formula I or a pharmaceutically acceptable salt thereof: (Formula I) I is also referred to as E7386, a β-catenin C-terminus inhibitor that can be orally administered and has shown beneficial effects for different tumors. See Yamada et al, E7386, a Selective Inhibitor of the Interaction between β-Catenin and CBP, Exerts Antitumor Activity in Tumor Models with Activated Canonical Wnt Signaling. Cancer Res. 2021 Feb 15;81(4):1052-1062; Kanda et al, NF-κB suppression synergizes with E7386, an inhibitor of CBP/β-catenin interaction, to block proliferation of patient-derived colon cancer spheroids. Biochem Biophys Res Commun. 2022 Jan 1;586:93-99, both of which are incorporated herein in their entireties. However, E7386 has not been previously tested in the context of vascular biology or disease. [0038] In one embodiment, the inhibitor comprises a compound of Formula II or a pharmaceutically acceptable salt thereof: (Formula II) of Formula II is also referred to as ICG-001. See Janssens et al, The Wnt-dependent signaling pathways as target in oncology drug discovery. Invest New Drugs. 2006 Jul;24(4):263-80, which is incorporated herein in its entirety. [0040] In one embodiment, the inhibitor comprises a compound of Formula III or a pharmaceutically acceptable salt thereof: 10 166682443.1 (Formula III) [00 mula III is also referred to as OP-724 or PRI-724. See Ouchi et al., Anti-inflammatory and antifibrotic effects of CBP/β-catenin inhibitor for hepatocytes: small molecular inhibitor, OP-724 possibly improves liver function. Med Mol Morphol.2023 Jun;56(2):94-105; Hirakawa et al., β-catenin signaling inhibitors ICG-001 and C-82 improve fibrosis in preclinical models of endometriosis. Sci Rep. 2019 Dec 27;9(1):20056, which are incorporated herein in their entireties. [0042] Also contemplated or pharmaceutically acceptable salts of an inhibitor of beta- catenin’s CTD. [0043] In some embodiments, the inhibitor of beta-catenin’s CTD contains a basic functional group, such as amino or alkylamino, and is, thus, capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable acids. The term “pharmaceutically acceptable salts” refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds disclosed herein. These salts can be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting a purified compound disclosed herein in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed during subsequent purification. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like (see, e.g., Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci.66:1-19). The pharmaceutically acceptable salts of the subject compounds include the conventional nontoxic salts or quaternary ammonium salts of the compounds, e.g., from non-toxic organic or inorganic acids. For example, such conventional nontoxic salts include those derived from inorganic acids such as hydrochloride, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, 11 166682443.1 hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like. [0044] In other cases, the inhibitor of beta-catenin’s CTD contains one or more acidic functional groups and, thus, is capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term “pharmaceutically acceptable salts” in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of the compounds disclosed herein. These salts can likewise be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a pharmaceutically-acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like (see, e.g., Berge et al., supra). [0045] Pharmaceutical compositions [0046] Provided herein are pharmaceutically acceptable compositions which comprise a therapeutically effective amount of an inhibitor of beta-catenin’s CTD, formulated together with one or more pharmaceutically acceptable excipients. As described below, the pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained- release formulation; (3) topical application, for example, as a cream, ointment, or a controlled- release patch or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8) nasally. [0047] The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals with toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit/risk ratio. The phrase “pharmaceutically-acceptable carrier” as used 12 166682443.1 herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier should be be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; and (22) other non-toxic compatible substances employed in pharmaceutical formulations. [0048] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions. [0049] Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. [0050] Formulations contemplated herein include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient 13 166682443.1 which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 0.1 per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent. [0051] In certain embodiments, a formulation comprises an excipient selected from the group consisting of cyclodextrins, celluloses, liposomes, micelle forming agents, e.g., bile acids, and polymeric carriers, e.g., polyesters and polyanhydrides; and an inhibitor of beta- catenin’s CTD. In certain embodiments, an aforementioned formulation renders orally bioavailable the inhibitor of beta-catenin’s CTD. [0052] Methods of preparing these formulations or compositions include the step of bringing into association the inhibitor of beta-catenin’s CTD with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the inhibitor of beta-catenin’s CTD with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product. [0053] Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges, powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of an inhibitor of beta-catenin’s CTD as an active ingredient. The inhibitor of beta-catenin’s CTD may also be administered as a bolus, electuary or paste. [0054] In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, trouches and the like), the active ingredient is mixed with one or more pharmaceutically-acceptable excipients including a pharmaceutically-acceptable carrier, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds and surfactants, such as poloxamer and sodium lauryl sulfate; (7) wetting agents, such as, for example, cetyl alcohol, glycerol monostearate, and non-ionic surfactants; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, 14 166682443.1 calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, zinc stearate, sodium stearate, stearic acid, and mixtures thereof; (10) coloring agents; and (11) controlled release agents such as crospovidone or ethyl cellulose. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-shelled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like. [0055] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface- active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. [0056] The tablets, and other solid dosage forms of the pharmaceutical compositions, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical- formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be formulated for rapid release, e.g., freeze-dried. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in micro- encapsulated form, if appropriate, with one or more of the above-described excipients. [0057] Liquid dosage forms for oral administration of the compounds disclosed herein include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in 15 166682443.1 particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. [0058] Besides diluents, the oral compositions can also include additional excipients such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. [0059] Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof. [0060] Formulations of the pharmaceutical compositions disclosed herein for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing the inhibitor of beta-catenin’s CTD with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound. [0061] Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate. [0062] Dosage forms for the topical or transdermal administration of an inhibitor of beta- catenin’s CTD include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required. [0063] The ointments, pastes, creams and gels may contain, in addition to the inhibitor of beta-catenin’s CTD, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof. [0064] Powders and sprays can contain, in addition to the inhibitor of beta-catenin’s CTD, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane. 16 166682443.1 [0065] Transdermal patches have the added advantage of providing controlled delivery of the inhibitor of beta-catenin’s CTD to the body. Such dosage forms can be made by dissolving or dispersing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel. [0066] Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this disclosure. [0067] Pharmaceutical compositions suitable for parenteral administration comprise the inhibitor of beta-catenin’s CTD in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. [0068] Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the inhibitor of beta-catenin’s CTD include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. [0069] These compositions may also contain additional excipients such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms upon the subject compounds may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin. [0070] In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of 17 166682443.1 dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle. [0071] Injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue. [0072] When the compounds disclosed herein are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99% (more preferably, 10 to 30%) of active ingredient in combination with a pharmaceutically acceptable carrier. [0073] Routes of administration and dose [0074] The inhibitor of beta-catenin’s CTD or pharmaceutical composition comprising the inhibitor of beta-catenin’s CTD may be given orally, parenterally, topically, or rectally. The inhibitor is given in a form suitable for the chosen administration route. For example, the inhibitor is administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. Oral administrations are preferred. [0075] The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. [0076] The phrases “systemic administration,” “administered systemically,” “peripheral administration” and “administered peripherally” as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration. [0077] The inhibitors disclosed herein may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, 18 166682443.1 a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually. [0078] Regardless of the route of administration selected, the inhibitors disclosed herein, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions disclosed herein, may be formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art. [0079] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the inhibitor of beta-catenin’s CTD may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. [0080] The selected dosage level will depend upon a variety of factors including the activity of the inhibitor of beta-catenin’s CTD employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the rate and extent of absorption, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. [0081] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the inhibitor of beta-catenin’s CTD employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. [0082] In general, a suitable daily dose of the inhibitor of beta-catenin’s CTD will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Generally, oral, intravenous, intracerebroventricular and subcutaneous doses of the inhibitor of beta-catenin’s CTD for a patient, when used for the indicated analgesic effects, will range from about 0.0001 to about 100 mg per kilogram of body weight per day. [0083] In certain embodiments, a dose of a compound or a composition is administered to a subject every day, every other day, every couple of days, every third day, once a week, twice a week, three times a week, or once every two weeks. If desired, the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage 19 166682443.1 forms. In some embodiments, a dose(s) of a compound or a composition is administered for 2 days, 3 days, 5 days, 7 days, 14 days, or 21 days. In certain embodiments, a dose of a compound or a composition is administered for 1 month, 1.5 months, 2 months, 2.5 months, 3 months, 4 months, 5 months, 6 months or more. [0084] The above-described administration schedules are provided for illustrative purposes only and should not be considered limiting. A person of ordinary skill in the art will readily understand that all doses are within the scope of the disclosure. [0085] The compounds for use in the methods disclosed herein can be administered as such or in admixtures with pharmaceutically acceptable carriers and can also be administered in conjunction with antimicrobial agents such as penicillins, cephalosporins, aminoglycosides and glycopeptides. Conjunctive therapy thus includes sequential, simultaneous and separate administration of the active compound in a way that the therapeutical effects of the first administered one is not entirely disappeared when the subsequent is administered. [0086] The addition of the active compound of the inhibitor of beta-catenin’s CTD to animal feed is preferably accomplished by preparing an appropriate feed premix containing the active compound in an effective amount and incorporating the premix into the complete ration. [0087] Alternatively, an intermediate concentrate or feed supplement containing the active ingredient can be blended into the feed. The way in which such feed premixes and complete rations can be prepared and administered are described in reference books (such as "Applied Animal Nutrition", W.H. Freedman and CO., San Francisco, U.S.A., 1969 or "Livestock Feeds and Feeding" O and B books, Corvallis, Ore., U.S.A., 1977). [0088] Microemulsification technology may be employed to improve bioavailability of lipophilic (water insoluble) pharmaceutical agents. Examples include Trimetrine (Dordunoo, S. K., et al., Drug Development and Industrial Pharmacy, 17(12), 1685-1713, 1991) and REV 5901 (Sheen, P. C., et al., J Pharm Sci 80(7), 712-714, 1991). Among other things, microemulsification provides enhanced bioavailability by preferentially directing absorption to the lymphatic system instead of the circulatory system, which thereby bypasses the liver, and prevents destruction of the compounds in the hepatobiliary circulation. [0089] Controlled release [0090] The release characteristics of an inhibitor disclosed herein depend on the encapsulating material, the concentration of encapsulated drug, and the presence of release modifiers. Release can be manipulated to be pH dependent, for example, using a pH sensitive coating that releases only at a low pH, as in the stomach, or a higher pH, as in the intestine. An 20 166682443.1 enteric coating can be used to prevent release from occurring until after passage through the stomach. Multiple coatings or mixtures of cyanamide encapsulated in different materials can be used to obtain an initial release in the stomach, followed by later release in the intestine. Release can also be manipulated by inclusion of salts or pore forming agents, which can increase water uptake or release of drug by diffusion from the capsule. Excipients that modify the solubility of the drug can also be used to control the release rate. Agents which enhance degradation of the matrix or release from the matrix can also be incorporated. They can be added to the drug, added as a separate phase (i.e., as particulates), or can be co-dissolved in the polymer phase depending on the compound. Types of degradation enhancers include inorganic salts such as ammonium sulfate and ammonium chloride, organic acids such as citric acid, benzoic acid, and ascorbic acid, inorganic bases such as sodium carbonate, potassium carbonate, calcium carbonate, zinc carbonate, and zinc hydroxide, and organic bases such as protamine sulfate, spermine, choline, ethanolamine, diethanolamine, and triethanolamine and surfactants such as Tween® and Pluronic®. Pore forming agents which add microstructure to the matrices (i.e., water soluble compounds such as inorganic salts and sugars) are added as particulates. The range should be between one and thirty percent (w/w polymer). [0091] Uptake can also be manipulated by altering residence time of the particles in the gut. This can be achieved, for example, by coating the particle with, or selecting as the encapsulating material, a mucosal adhesive polymer. Examples include most polymers with free carboxyl groups, such as chitosan, celluloses, and especially polyacrylates (as used herein, polyacrylates refers to polymers including acrylate groups and modified acrylate groups such as cyanoacrylates and methacrylates). [0092] Methods [0093] Provided herein are method of using an inhibitor of beta-catenin’s CTD for the treatment or prevention of a disease or disorder in a subject in need thereof. The inhibitor of beta-catenin’s CTD may be an inhibitor disclosed herein. In some embodiments, the inhibitor comprises a compound of Formula I, II, or III (or a pharmaceutically acceptable salt thereof). In a preferred embodiment, the inhibitor comprises a compound of Formula I or a pharmaceutically acceptable salt thereof. [0094] As used herein, the term subject and patient may be used interchangeably. In some embodiments, the subject is a mammal. The term “mammal” as used herein is intended to include, but is not limited to, humans, laboratory animals (including, but not limited to mice, rats, dogs, pigs, and sheep), domestic pets, and farm animals. Mammals, include, but are not 21 166682443.1 limited to, a human or non-human mammal, such as a bovine, porcine, equine, canine, ovine, or feline, etc. In one embodiment, the subject is a human. [0095] In embodiments, the methods disclosed herein are useful for treating or preventing disease in a subject. The terms “treat,” “treated,” “treating,” or “treatment” as used herein refer to therapeutic treatment, wherein the object is to slow down (lessen) an undesired physiological condition, disorder or disease, or to obtain beneficial or desired clinical results. For the purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent of the condition, disorder or disease; stabilization (i.e., not worsening) of the state of the condition, disorder or disease; delay in onset or slowing of the progression of the condition, disorder or disease; amelioration of one or more symptoms of the condition, disorder or disease state; and remission (whether partial or total), or enhancement or improvement of the condition, disorder or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment. The terms “prevent”, “prevention”, and the like refer to acting prior to overt disease or disorder onset, to prevent the disease or disorder from developing or to minimize the extent of the disease or disorder or slow its course of development. In some embodiments, treatment refers to increased survival (e.g., survival time). For example, treatment can result in an increased life expectancy of a patient. In some embodiments, treatment results in an increased life expectancy of a patient by more than about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 105%, about 110%, about 115%, about 120%, about 125%, about 130%, about 135%, about 140%, about 145%, about 150%, about 155%, about 160%, about 165%, about 170%, about 175%, about 180%, about 185%, about 190%, about 195%, about 200% or more, as compared to the average life expectancy of one or more control individuals with a disease or disorder without treatment. In some embodiments, treatment results in an increased life expectancy of a patient by more than about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years or more, as compared to the average life expectancy of one or more control individuals with a disease or disorder without treatment. In some embodiments, treatment results in long term survival of a patient. As used herein, the term “long term survival” refers to a survival 22 166682443.1 time or life expectancy longer than about 40 years, 45 years, 50 years, 55 years, 60 years, or longer. [0096] Provided herein is a method of preventing or reducing restenosis in a blood vessel in a subject in need thereof, the method comprising administering to the subject an inhibitor of beta-catenin’s CTD or a pharmaceutical composition comprising an inhibitor of beta-catenin’s CTD. Restenosis may refer to the reduction in the diameter of the vessel lumen, e.g., after a patient has percutaneous coronary intervention. Complete narrowing of a blood vessel is also referred to as blood vessel occlusion. Provided herein is a method of preventing occlusion in a blood vessel in a subject in need thereof, the method comprising administering to the subject an inhibitor of beta-catenin’s CTD or a pharmaceutical composition comprising an inhibitor of beta-catenin’s CTD. Methods of measuring restenosis are known in the art and include, for example, duplex ultrasound, magnetic resonance angiography (MRA), computed tomography angiography (CTA), conventional catheter-based angiography, catheter-based digital subtraction angiography, direct measurement of an excised vessel or at surgery or post-mortem, histopathology. Methods of measuring occlusion are known in the art and include, for example, measurement of optical reflection and temperature changes, the use of Doppler ultrasound or pulse wave measurements, duplex ultrasound, magnetic resonance angiography (MRA), computed tomography angiography (CTA), conventional catheter-based angiography, catheter-based digital subtraction angiography, direct measurement of an excised vessel or at surgery or post-mortem, histopathology. Provided herein is a method of preventing, inhibiting, or reducing restenosis in a blood vessel in a subject in need thereof, the method comprising administering to the subject a compound of Formula I or a pharmaceutically acceptable salt thereof. Provided herein is a method of preventing occlusion in a blood vessel in a subject in need thereof, the method comprising administering to the subject a compound of Formula I or a pharmaceutically acceptable salt thereof. [0097] Provided herein is a method of preventing, reducing, or inhibiting vascular remodeling in the blood vessel of a subject in need thereof, the method comprising administering to the subject an inhibitor of beta-catenin’s CTD or a pharmaceutical composition comprising an inhibitor of beta-catenin’s CTD. During vascular remodeling (e.g., after vascular injury or during atherosclerosis), smooth muscle cells (SMCs) undergo phenotypic switching from a quiescent, differentiated, contractile, and non-migratory phenotype to a dedifferentiated, migratory, proliferative, and secretory phenotype. These changes promote SMC proliferation and migration from the media into the intima, and lead to neointima formation (intimal expansion) that may occlude the arterial lumen. As such, vascular 23 166682443.1 remodeling can result in vascular wall thickening and the gradual loss of luminal patency. Provided herein is a method of preventing, inhibiting, or reducing thickening of the intima in a blood vessel in a subject in need thereof, the method comprising administering to the subject an inhibitor of beta-catenin’s CTD or a pharmaceutical composition comprising an inhibitor of beta-catenin’s CTD. Also provided herein is a method of decreasing proliferation of SMCs in a blood vessel in a subject in need thereof, the method comprising administering to the subject an inhibitor of beta-catenin’s CTD or a pharmaceutical composition comprising an inhibitor of beta-catenin’s CTD. Methods of measuring neointimal hyperplasia, vascular remodeling, and proliferation of SMCs are known in the art and include histopathological staining, intra- vascular ultrasound, optical coherence tomography staining, and other imaging modalities. Provided herein is a method of preventing, reducing or inhibiting vascular remodeling in the blood vessel of a subject in need thereof, the method comprising administering to the subject a compound of Formula I or a pharmaceutically acceptable salt thereof. Provided herein is a method of preventing, inhibiting, or reducing thickening of the intima in a blood vessel in a subject in need thereof, the method comprising administering to the subject a compound of Formula I or a pharmaceutically acceptable salt thereof. Provided herein is a method of decreasing proliferation of smooth muscle cells in a blood vessel in a subject in need thereof, the method comprising administering to the subject a compound of Formula I or a pharmaceutically acceptable salt thereof. [0098] Provided herein is a method of preventing, inhibiting, or reducing the formation and/or expansion of atherosclerotic lesions in a blood vessel in a subject in need thereof, the method comprising administering to the subject an inhibitor of beta-catenin’s CTD or a pharmaceutical composition comprising an inhibitor of beta-catenin’s CTD. Atherosclerotic lesions may refer to deposits observed during atherosclerosis. For example, early lesions of atherosclerosis may consist of subendothelial accumulations of cholesterol-engorged macrophages, called “foam cells.” Such lesions may also be referred to as “fatty streak” lesions. More advanced lesions are characterized by the accumulation of lipid-rich necrotic debris and SMCs. These ”fibrous lesions” usually comprise a “fibrous cap” consisting of SMCs and extracellular matrix that encloses a lipid-rich “necrotic core.” Even more advanced lesions may be referred to as plaques, which have a more complex composition and can comprises fatty substances, cholesterol, cellular waste products, calcium, and/or fibrin. Methods of assessing the presence of atherosclerotic lesions are known in the art. In embodiments, the compositions disclosed herein reduce the size of atherosclerotic plaques and/or necrotic cores. In embodiments, the compositions disclosed herein preserve fibrous cap thickness. In 24 166682443.1 embodiments, the compositions disclosed herein do not cause a change in serum lipid levels. Provided herein is a method of preventing, inhibiting, or reducing the formation and/or expansion of atherosclerotic lesions in a blood vessel in a subject in need thereof, the method comprising administering to the subject a compound of Formula I or a pharmaceutically acceptable salt thereof. [0099] Provided herein is a method of maintaining patency of a blood vessel in a subject, the method comprising administering to the subject an inhibitor of beta-catenin’s CTD or a pharmaceutical composition comprising an inhibitor of beta-catenin’s CTD. Blood vessel patency relates to the degree to which a blood vessel is not blocked or obstructed. Greater patency indicates a lesser degree of blockage or obstruction. Methods of measuring patency are known in the art. Provided herein is a method of maintaining patency of a blood vessel in a subject, the method comprising administering to the subject a compound of Formula I or a pharmaceutically acceptable salt thereof. [0100] The blood vessel may be a coronary blood vessel. The blood vessel may be a peripheral artery. The blood vessel may be a carotid artery. The blood vessel may be a cerebral artery. The blood vessel may be part of an arterio-venous fistula (AVF) (i.e., a connection between an artery and a vein). The methods provided herein may also apply to a blood vessel graft. A blood vessel graft or vascular graft is a blood vessel that is implanted into the patient, for example, to bridge an obstruction in the patient’s blood vessel(s) or as part of an AVF. In some embodiments, the blood vessel graft is an autologous graft from the patient, which can, for example, be taken from the saphenous vein from the leg or the internal thoracic artery in the chest wall. A blood vessel graft may also be obtained from another human (i.e., an allogeneic donor) or a non-human donor animal (if compatible). A blood vessel graft can be bio-engineered using a method known to a person skilled in the art, including, but not limited to grafts made of out polyester fibers such as Dacron, or grafts generated by 3D-printing. In one embodiment, the blood vessel graft is a vein graft (i.e., a portion of a vein is grafted). In one embodiment, the blood vessel graft is an artery graft. In one embodiment, the blood vessel graft is the graft of a saphenous vein. In one embodiment, the blood vessel graft is an internal mammary artery graft. In some embodiments, the blood vessel graft is a graft of a radial artery, an internal thoracic artery, or a gastroepiploic artery. The final surgical graft created may be achieved using a variety of approaches including, but not limited to, interpositional grafting, grafting where the proximal end of the graft remains naturally attached to the arterial system but the distal end is anastomosed onto the arterial tree beyond a blockage (classically an internal 25 166682443.1 mammary artery graft), the direct connection of a vein to an artery such as with an AVF, or various other approaches. [0101] In one embodiment, the subject has a cardiovascular disease or is at risk of developing cardiovascular disease. In some embodiments, the patient has one or more of the following or is at risk of developing one or more of the following: coronary artery diseases (including, e.g., angina, myocardial infarction (MI), heart attack, heart failure), hypertensive heart disease, rheumatic heart disease, cardiomyopathy, arrhythmia, congenital heart disease, valvular heart disease, carditis, aortic aneurysms, peripheral artery disease, thromboembolic disease, venous thrombosis, pulmonary embolism, aorta disease, Marfan syndrome, vascular disease (including obstructive vascular disease), cerebrovascular disease, atherosclerosis, carotid artery disease, peripheral artery disease, or vein graft disease. In one embodiment, the subject has arteriosclerosis after receiving a transplant. In one embodiment, the subject has transplant-associated vasculopathy or is at risk of developing transplant-associated vasculopathy. In one embodiment, the subject has suffered an injury to a blood vessel. [0102] In one embodiment, the subject has undergone angioplasty. In one embodiment, the subject has undergone placement of a stent in the subject’s blood vessel. [0103] In some embodiments, the subject has undergone endarterectomy. The endarterectomy may have been performed to remove plaque buildup from narrowed or blocked arteries. Endarterectomy may be a treatment for carotid artery disease or for peripheral artery disease. [0104] In one embodiment, the patient has undergone AVF surgery. In one embodiment, the patient is receiving dialysis. [0105] In some embodiments, the patient has undergone interposition vein grafting into the arterial circulation or bypass surgery. [0106] Provided herein is a method of preventing or reducing transplant-associated vasculopathy in a subject in need thereof, the method comprising administering to the subject an inhibitor of beta-catenin’s CTD or a pharmaceutical composition comprising an inhibitor of beta-catenin’s CTD. Vasculopathy may refer to a disease affecting blood vessels. Provided is a method of reducing or preventing transplant-associated vasculopathy in a subject in need thereof, the method comprising administering to the subject a compound of Formula I or a pharmaceutically acceptable salt thereof. [0107] Provided herein is a method of preventing, inhibiting, or reducing progression of atherosclerosis in a subject in need thereof, the method comprising administering to the subject an inhibitor of beta-catenin’s CTD or a pharmaceutical composition comprising an inhibitor of 26 166682443.1 beta-catenin’s CTD. Atherosclerosis may refer to an occlusive disease of the vasculature that is associated with the deposition of lipid-laden plaques. The plaques may comprise fatty substances, cholesterol, cellular waste products, calcium, and/or fibrin. Further, the plaques may cause arteries to narrow, blocking blood flow, and resulting in blood clots. Atherosclerosis can result in coronary artery disease, stroke, peripheral artery disease, or kidney disorders. Provided herein is a method of preventing, inhibiting, or reducing progression of atherosclerosis in a subject in need thereof, the method comprising administering to the subject a compound of Formula I or a pharmaceutically acceptable salt thereof. [0108] Also provided herein is a method of surgically inserting a stent of into the lumen of a blood vessel in a subject in need thereof, wherein the stent comprises (i) a scaffold coated with a polymer, which polymer is impregnated with, adsorbed with, or coated with an inhibitor of beta-catenin’s CTD, or (ii) a scaffold which is impregnated with, adsorbed with, or coated with, an inhibitor of beta-catenin’s CTD. Provided is a method for performing an angioplasty in a subject in need thereof, the method comprising surgically inserting a stent into the blood vessel and expanding the stent therein so as perform the angioplasty, wherein stent comprises (i) a scaffold coated with a polymer, which polymer is impregnated with, adsorbed with, or coated with an inhibitor of beta-catenin’s CTD, or (ii) a scaffold which is impregnated with, adsorbed with, or coated with, an inhibitor of beta-catenin’s CTD. Provided is a method of preventing or reducing restenosis in blood vessel in a subject in need thereof, the method comprising surgically inserting a stent into the lumen of the blood vessel at the site deemed at risk of restenosis, so as to reduce the risk of restenosis, wherein stent comprises (i) a scaffold coated with a polymer, which polymer is impregnated with, adsorbed with, or coated with an inhibitor of beta-catenin’s CTD, or (ii) a scaffold which is impregnated with, adsorbed with, or coated with, an inhibitor of beta-catenin’s CTD. In one embodiment, the stent is a shaped to be suitable as an endovascular stent. In one embodiment, at least 50% the inhibitor of beta- catenin’s CTD elutes from the stent. In one embodiment, at least 90% of the inhibitor of beta- catenin’s CTD elutes from the stent over a time period of 1 to 150 days. In one embodiment, the inhibitor of beta-catenin’s CTD elutes from the stent in a moist environment in contact with the stent. In one embodiment, the moist environment comprises a blood vessel. In one embodiment, the stent comprises a scaffold impregnated with, adsorbed with, or coated with, an inhibitor of beta-catenin’s CTD. In one embodiment, the stent comprises a scaffold coated with a polymer impregnated with, adsorbed with, or coated with an inhibitor of beta-catenin’s CTD. In one embodiment, the inhibitor of beta-catenin’s CTD is adsorbed within the polymer and/or adsorbed to a surface of the polymer. In one embodiment, the inhibitor of beta-catenin’s 27 166682443.1 CTD is coated on the polymer. In one embodiment, the polymer is not prothrombotic. In one embodiment, the polymer is a synthetic polymer. In one embodiment, it is a hydrocarbon-based polymer. In one embodiment, the polymer comprises one or more of poly(n-butyl methacrylate), poly(ethylene-co-vinyl acetate), poly(styrene-b-isobutylene-b-styrene) or poly(vinylidene fluoride-co-hexafluoropropylene). In one embodiment, the polymer comprises phosphorylcholine. In one embodiment, the polymer is durable. In one embodiment, the polymer is biodegradable. In one embodiment, the stent is bioresorbable. In one embodiment, the stent is balloon-expandable or self-expandable. Preferably, the inhibitor of beta-catenin’s CTD comprises a compound of Formula I or a pharmaceutically acceptable salt thereof. [0109] It is to be understood that this invention is not limited to the particular molecules, compositions, methodologies, or protocols described, as these may vary. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention. It is further to be understood that the disclosure of the invention in this specification includes all possible combinations of such particular features. For example, where a particular feature is disclosed in the context of a particular aspect or embodiment of the invention, or a particular claim, that feature can also be used, to the extent possible, in combination with and/or in the context of other particular aspects and embodiments of the invention, and in the invention generally. [0110] Where reference is made herein to a method comprising two or more defined steps, the defined steps can be carried out in any order or simultaneously (except where the context excludes that possibility), and the method can include one or more other steps which are carried out before any of the defined steps, between two of the defined steps, or after all the defined steps (except where the context excludes those possibilities). [0111] All other literature references, patents and applications are incorporated herein by reference in their entireties. Furthermore, where a definition or use of a term in a reference, which is incorporated by reference herein is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply. [0112] To facilitate a better understanding of the present invention, the following Examples of specific embodiments are given. The following examples should not be read to limit or define the entire scope of the invention. 28 166682443.1 EXAMPLES [0113] Example 1: Materials and Methods for Examples 2-8 [0114] Mice [0115] Ctnnb1^{^C/flox}, SMA-CreER^T2, and Rosa26^LSL-S1pr1 mice were generated and validated using methods known in the art. Ctnnb1^flox/flox (B6.129-Ctnnb1tm2Kem/KnwJ) and Rosa26^{LSL- TdTomato/LSL-TdTomato} (Gt(ROSA)26Sor^{tm9(CAG-tdTomato)Hze}; also referred to as RFP) mice were obtained from the Jackson Laboratory. Ctnnb1^WT/WT mice were crossed with Ctnnb1^flox/flox to generate Ctnnb1^WT/flox mice. Next, Ctnnb1^WT/flox mice were crossed with SMA-CreER^T2 and Rosa26^{LSL-TdTomato/LSL-TdTomato} mice to generate Ctnnb1^WT/flox;Rosa26^{LSL-TdTomato/WT};SMA- CreER^T2 mice. Finally, Ctnnb1^{^C/flox} mice were crossed with Ctnnb1^WT/flox;Rosa26^{LSL- TdTomato/WT};SMA-CreER^T2 mice to generate Ctnnb1^{^C/flox}; Rosa26^{LSL-TdTomato/WT};SMA-CreER^T2 mice and their littermate controls Ctnnb1^WT/flox;Rosa26^{LSL-TdTomato/WT};SMA-CreER^T2. These mice were treated with tamoxifen to remove the β-catenin flox allele, resulting in mice bearing the ^C allele (referred to as SMβC^{^C} mice) or the WT allele (referred to as SMβC^wt/- mice) as the only sources of β-catenin in SMCs. In addition, tamoxifen administration also activates RFP for SMC lineage tracing. For tamoxifen inducible, SMC-specific gain-of-function of S1pr1 gene, Ctnnb1^WT/flox;Rosa26^{LSL-TdTomato/WT};SMA-CreER^T2 mice were initially crossed with Rosa26^LSL-S1pr1 mice to generate Ctnnb1^WT/flox;Rosa26^{LSL-TdTomato/LSL-S1pr1};SMA-CreER^T2 mice. Next, Ctnnb1^{^C/flox}; Rosa26^{LSL-TdTomato/WT} mice were crossed with Ctnnb1^WT/flox;Rosa26^{LSL-TdTomato/LSL-S1pr1};SMA-CreER^T2 mice that yielded Ctnnb1^{^C/flox};Rosa26^{LSL-TdTomato/LSL-S1pr1};SMA-CreER^T2 mice (referred to as SMβC^{^C} SM- S1PR1^GOF mice after tamoxifen administration) and their littermate controls Ctnnb1^WT/flox;Rosa26^{LSL-TdTomato/LSL-S1pr1};SMA-CreER^T2 (referred to as SMβC^wt/- SM- S1PR1^GOF mice after tamoxifen administration). Male and female 8-16-week-old mice were used as breeders for timed mating. Methods for mouse tail genotyping by PCR for Ctnnb1^{^C/flox}, Rosa26^LSL-S1pr1, Ctnnb1^flox/flox and SMA-CreER^T2 mice are known in the art, and genotyping for Rosa26^LSL-TdTomato mice was performed according to a Jackson Laboratory protocol. Wild- type C57BL/6J (Strain #000664) and ApoE^−/− (Strain #002052) mice used in the pharmacologic studies were purchased from the Jackson Laboratory. All animals were housed in pathogen-free conditions. 29 166682443.1 [0116] Tamoxifen administration [0117] Under pathogen-free conditions, 100 mg of tamoxifen (STEMCELL Technologies No. 72662) was dissolved in 0.5 ml of ethanol (Decon Laboratories, Inc. 22032601), and 9.5 ml of corn oil (Sigma C8267) was added to achieve a final concentration of 10 mg/ml. Eight- to 9-week-old male and female mice were given 100 μl (1 mg) of tamoxifen solution via intraperitoneal injection daily for 5 consecutive days. Carotid artery ligation was performed 1 week after the first injection. [0118] Carotid artery ligation [0119] Briefly, 9–10-week-old male and female mice were anesthetized with ketamine/xylazine (90 mg/kg and 10 mg/kg, respectively) via intraperitoneal injection. A 5 mm skin incision on the base of the neck and blunt dissection were performed until the left common carotid artery was exposed. The artery was separated from surrounding tissues and ligated with 6-0 silk (Ethicon K889H) just proximal to the bifurcation. The right common carotid artery served as control. The incision was closed and mice were allowed to recover. Meloxicam 5 mg/kg was administered subcutaneously after carotid ligation for pain relief. Carotid arteries were harvested 21 days after ligation. Mice were euthanized by ketamine/xylazine injection plus thoracotomy, and the systemic circulation was flushed with phosphate buffer solution (PBS) and then perfused with 10% formalin phosphate buffer (Fisher Scientific SF100-4) for 7 minutes. Left and right carotid arteries were removed and post-fixed in 10% formalin phosphate buffer overnight, followed by 70% ethanol. Tissues required for DNA, RNA, or protein isolation were harvested, snap frozen, and kept at -80 ºC until processing. [0120] E7386 treatment after carotid artery ligation [0121] For in vivo administration, E7386 (Chemietek CT-E7386) was dissolved in 0.01 mol/L HCl. After carotid artery ligation, 9–10-week-old male and female WT C57BL/6J mice were given E738650 mg/kg or vehicle (0.01 mol/L HCl) by oral gavage twice daily for 14 days. Carotid arteries were harvested 14 days after ligation, following the same procedures described above. [0122] Processing and morphometric analysis of injured carotid arteries [0123] Fixed carotid arteries were trimmed under a Stemi 2000-C stereomicroscope (Zeiss) such that 3 mm of vessel adjacent to the ligation point was left available for analysis. Trimmed arteries were processed and paraffin embedded with the ligature on top. Paraffin blocks were trimmed until a full cross-section of the ligated artery was visualized; thereafter, in sequence, the next 400 μm of tissue was discarded, 180 μm was collected for analysis (5 μm-thick sections, 3 sections per slide, 12 slides), 400 μm discarded, 180 μm collected for analysis, 400 30 166682443.1 μm discarded, and 180 μm collected for analysis. This protocol allowed the evaluation of vascular remodeling at three consistent distances from the ligation site in each artery. Arterial cross-sections were stained with hematoxylin and eosin (H&E), and photographed with a Leica DMi8 inverted microscope. Morphometric analysis of carotid arteries was performed using ImageJ software as follows: the area of the lumen, the area inside the internal elastic lamina (IEL), and the area inside the external elastic lamina (EEL) were measured in pixels. The area of the intima was calculated by subtracting the area of the lumen from the area inside the IEL. The area of the media was calculated by subtracting the area inside the IEL from the area inside the EEL. Finally, the intima/media ratio was calculated. [0124] Atherosclerosis studies [0125] Disturbed flow-induced atherosclerosis in the common carotid artery was induced in 8–10-week-old male ApoE^-/- mice by partial carotid artery ligation and Western-type diet feeding. Briefly, mice were anesthetized with ketamine/xylazine (90 mg/kg and 10 mg/kg, respectively) via intraperitoneal injection. A 5 mm skin incision on the base of the neck and blunt dissection were performed until the left common carotid artery was exposed. Three of four caudal branches of the left common carotid artery (left external carotid, internal carotid, and occipital artery) were ligated with 6–0 silk (Ethicon K889H), while the superior thyroid artery was left intact. The incision was closed and mice were allowed to recover. Meloxicam 5 mg/kg was administered subcutaneously after carotid ligation for pain relief. After surgery, mice were placed 21 days on Western diet (Envigo TD.88137; 42% kcal from fat, 0.2% total cholesterol, saturated fat >60% total fat, and high sucrose) and orally administered with E7386, 25 mg/kg or vehicle (0.01 mol/L HCl) by oral gavage twice daily for 21 days. Blood and carotid arteries were harvested 21 days after ligation. Mice were euthanized by ketamine/xylazine injection plus thoracotomy, and the systemic circulation was flushed with phosphate buffer solution (PBS) and then perfused with 10% formalin phosphate buffer (Fisher Scientific SF100-4) for 7 minutes. Left and right carotid arteries were removed and post-fixed in 10% formalin phosphate buffer overnight, followed by 70% ethanol. Blood samples were obtained by cardiac puncture before PBS perfusion, and serum was obtained by centrifugation at 6,000g for 10 min at 4 °C, snap-frozen and stored at – 80 ˚C until further use. Serum levels of total cholesterol were measured using the Cholesterol Quantification Assay kit (Sigma CS0005- 1KT). [0126] Processing and morphometric analysis of atherosclerotic lesions [0127] Fixed carotid arteries were processed and paraffin embedded with the ligature on top. Paraffin blocks were trimmed until a full cross-section of the ligated artery was visualized; 31 166682443.1 thereafter, in sequence, the next 240 μm of tissue was collected for analysis (5 μm-thick sections, 4 sections per slide, 12 slides), 500 μm discarded, 240 μm collected for analysis, 500 μm discarded, and 240 μm collected for analysis, 500 μm discarded, 240 μm collected for analysis, 500 μm discarded, and 240 μm collected for analysis. This protocol allowed the evaluation of atherosclerotic lesions at five consistent distances from the ligation site in each artery. Arterial cross-sections were stained with H&E, and photographed with a Leica DMi8 inverted microscope. Morphometric analysis of carotid arteries was performed using ImageJ software as follows: the area of the lumen, the area inside the IEL, and the area inside the EEL were measured in pixels. The lesion area was calculated by subtracting the area of the lumen from the area inside the IEL. The area of the media was calculated by subtracting the area inside the IEL from the area inside the EEL, and the lesion/media ratio was calculated. Necrotic cores were quantified by defining the H&E-negative acellular areas in the intima. The fibrous cap thickness was quantified by choosing the largest necrotic core in a section and measuring the thinnest part of the cap, from its outer edge to the necrotic core boundary. [0128] Vascular homeostasis studies [0129] Baseline blood pressure and body weight of 8–9-week-old male and female mice were recorded just prior the first tamoxifen administration and followed for 12 weeks after last tamoxifen injection, at 2-week intervals. Blood pressure was measured as described in the next section. Thoracic aortas and carotid arteries were harvested 12 weeks after last tamoxifen injection. Mice were euthanized by ketamine/xylazine injection plus thoracotomy, and the vascular system was flushed with phosphate buffer solution (PBS) and then perfused with 10% formalin phosphate buffer (Fisher Scientific SF100-4) for 7 minutes. Aortas and carotid arteries were removed and post-fixed in 10% formalin phosphate buffer overnight, followed by 70% ethanol and then embedded in paraffin. The serial cuts at 5 μm-thick sections were done using the paraffin blocks. The cuts were stained with H&E and photographed with a Leica DMi8 inverted microscope. Morphometric analysis of medial area of aortas and carotid arteries was performed using ImageJ software. The area inside the IEL, and the area inside the EEL were measured in pixels. The area of the media was calculated by subtracting the area inside the IEL from the area inside the EEL. [0130] Blood pressure measurements [0131] Systolic blood pressure was measured in conscious male and female mice using the tail-cuff method (Kent Scientific, Inc.). Briefly, animals were placed in a mouse restrainer (RTBP007, Kent Scientific, Inc.), and a mouse occlusion cuff (RTBP050, Kent Scientific, Inc.) and mouse plethysmographic cuff (XBP051, Kent Scientific, Inc.) were applied to the tail of 32 166682443.1 the mouse. Mice were trained for one week to become accustomed to the new handling and environment. Systolic blood pressure measurements were taken using the XBP1000 apparatus (Kent Scientific, Inc.) connected to a data acquisition system. Five consecutive measurements were performed per mouse, per time-point, and the values were averaged. Measurements were done at the same time of the day at the indicated time points. [0132] Immunofluorescence of arteries [0133] Tissue sections were deparaffinized and rehydrated, and antigen retrieval performed by boiling in sodium citrate solution (Vector Labs H-3300). Tissues were blocked in 0.3% Triton X-100, 2% BSA and 5% normal horse serum in PBS for 1 hour at room temperature. Tissues were incubated overnight at 4 °C in blocking solution with the following primary antibodies: anti-β-catenin targeting the C-terminus (Santa Cruz sc-7963, 1:50 dilution); anti-β- catenin targeting the N-terminus (Cell Signaling 9562S, 1:100 dilution); anti-RFP anti-rabbit (Rockland 600-401-379, 1:200 dilution), anti-RFP anti-mouse (Invitrogen MA5-15257, 1:100 dilution), anti-Axin2 (Invitrogen PA5-21093, 1:100 dilution), anti-CD31 (Abcam 28364, 1:50 dilution), anti-SMA (Santa Cruz sc-32251, 1:200 dilution); anti-Ki67 (Abcam 15580, 1:50 dilution); anti-PCNA (Invitrogen 13-3900, 1:100 dilution), anti-cleaved caspase 3 (Cell Signaling 9661, 1:50 dilution); anti-S1PR1 (Invitrogen PA1-1040; 1:100 dilution). After washing, sections were incubated for 1 hour at room temperature with fluorochrome- conjugated secondary antibodies Alexa 488 goat anti-mouse (Invitrogen A32723, 1:200 dilution) and Alexa 546 donkey anti-rabbit (Invitrogen A10040, 1:400 dilution) in blocking solution. After washing, sections were stained with DAPI during mounting (FLUORO-GEL II, with DAPI, Electron Microscopy Sciences 17985-50). Fluorescence emission was visualized with a Leica DMi8 inverted microscope. Subsequent image processing and quantification of fluorescent signal were achieved by using the NIH Fiji program. Specificity of staining was confirmed by omission of the primary antibody. [0134] Mouse aortic smooth muscle cells [0135] Primary mouse aortic smooth muscle cells (MASMCs) were isolated from 5–6 week old male and female Ctnnb1^{^C/flox} and Ctnnb1^{^C/flox}; Rosa26^{LSL-S1pr1/LSL-TdTomato} mice using collagenase-elastase digestion, maintained in Dulbecco’s modified eagle medium (DMEM) plus 20% fetal bovine serum (FBS), 100 U/ml of penicillin, 100 μg/ml of streptomycin, and 2 mM L-glutamine, and subcultured weekly. Passage 2 of cells isolated from Ctnnb1^{^C/flox} mice was transduced with adenovirus expressing GFP (Ad5.CMV-GFP) or Cre (Ad5.CMV-Cre) to generate control (βC^Control) or β-catenin C-terminus-deficient (βC^ΔC) cells, respectively. 33 166682443.1 Passage 2 of cells isolated from Ctnnb1^{^C/flox}; Rosa26^{LSL-S1pr1/LSL-TdTomato} mice was transduced with adenovirus expressing GFP (Ad5.CMV-GFP) or Cre (Ad5.CMV-Cre) to generate control (βC^Control S1PR1^Control) or βC^ΔC cells with S1PR1 gain-of-function (βC^ΔC S1PR1^GOF), respectively. Passages 3 to 6 were used for experiments. To evaluate how the inhibition of β- catenin degradation affects S1PR1 expression, βC^Control and βC^ΔC MASMCs with CHIR99021 (Cell Signaling 54290S) were treated for 48 h. CHIR99021 was dissolved in DMSO to obtain a stock solution at 10 mM. Dilutions from the stock solution in culture medium were performed to test final concentrations from 0.1 to 1 μM. Same dilution protocols were used with only DMSO to generate vehicle controls specific for each concentration. To evaluate the effects of the pharmacologic inhibition of β-catenin C-terminus, βC^Control and βC^ΔC MASMCs were treated with E7386 (Chemietek CT-E7386), which was dissolved in DMSO to obtain a stock solution at 10 mM. Dilutions from the stock solution in culture medium were performed to test final concentrations from 10 to 100 nM. Same dilution protocols were used with only DMSO to generate vehicle controls specific for each concentration. [0136] Cell population growth [0137] To evaluate the effect of genetic interventions on growth of MASMCs, 4 x 10³ cells per well were plated in 96-well plates with a minimum of four independent wells per group. Evaluation of cell growth in culture was performed with the AlamarBlue assay (BUF012A, BIO-RAD), and absorbances at 570 nm and 600 nm were measured on a Varioskan LUX multimode microplate reader (Thermo Scientific). AlamarBlue reduction correlates with the number of viable cells, and was calculated according to the manufacturer’s instructions and expressed as fold-change with respect to baseline. [0138] Cell transfection were transfected with pcDNA3-β-catenin^S33Y vector using the electroporation method. The electroporation was performed using the Neon transfection system (Invitrogen). Electroporated cells were plated in antibiotic-free medium in a 24-well plate for 24 h and then switched to regular medium. Triplicates for every group and condition were used every time. Cell lysates were collected 72 h after electroporation for Western blotting experiments. [0140] Small interfering RNA (siRNA) [0141] The siRNA targeting mouse S1PR1 was synthesized by Thermo Fisher (Silencer® Select 4390771, assay ID s65292). Transient transfection of siRNA (10 nM) was performed by using Lipofectamine RNAiMAX (Thermo Fisher 13778150), as recommended by the 34 166682443.1 manufacturer. Control cells were transfected with 10 nM Silencer Select negative control siRNA (Thermo Fisher 4390843). After 24 h, cells were treated with CHIR99021 (Cell Signaling 54290S) or vehicle, and the cell population growth was evaluated 48h and 72h after plating. [0142] Western blotting [0143] Protein lysates were extracted from SMCs or blood vessels using RIPA buffer (50 mM Tris-HCl pH 7.4, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 150 mM NaCl) plus protease inhibitors (Complete mini Roche Life Science 04693159001). A BCA protein assay kit (Pierce 23335) was used to measure protein concentrations, and equal amounts of protein were loaded (20 – 60 μg) and separated by 10% polyacrylamide gel electrophoresis. Proteins were transferred (Trans-Blot SD cell, BIORAD 170-3940) to 0.45 μm pore size PVDF membranes (Immobilon-P, Millipore), blocked for 1 hour at room temperature with TBST (Tris pH8.0, NaCl 150 mM, 0.1% Tween 20) plus 5% (wt/vol) nonfat milk or 6% bovine serum, and incubated overnight at 4 ºC in blocking solution with the following primary antibodies: anti-β- catenin targeting the C-terminus (Santa Cruz sc-7963, 1:500 dilution); anti-β-catenin targeting the N-terminus (Cell Signaling 9562S, 1:250 dilution); anti-S1PR1 (Millipore MABC94, 1:1000 dilution); anti-S1PR3 (Alomone ASR-013, 1:1000 dilution), anti-Axin2 (Invitrogen PA5-21093, 1:1000 dilution); anti-GAPDH (Santa Cruz sc-25778, 1:5000 dilution). After washing, membranes were incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature. After washing, signals were detected by adding ECL substrate (ECL Western Blotting Substrate, Pierce 32106) and exposing films to membranes. Densitometric analysis was performed with Image J. [0144] Standard RNA-Seq library preparation with rRNA depletion and HiSeq sequencing [0145] Total RNA was isolated from βC^Control and βC^ΔC MASMCs using Trizol reagent (Invitrogen 15596018) according to manufacturer’s instructions. RNA concentrations were calculated using NanoDrop technology (ThermoFisher Scientific ND-1000), and RNA integrity was evaluated using RNA 6000 Nano LabChips on an Agilent 2100 Bioanalyzer (Agilent Technologies). Sample quality control (QC), library preparations, and sequencing reactions for RNA-seq were conducted at GENEWIZ, LLC. (South Plainfield, NJ, USA). Ribosomal RNA depletion was performed using Ribo-Zero Gold Kit (Human/Mouse/Rat probe) (Illumina, San Diego, CA, USA). RNA-seq library preparation used NEBNext Ultra II RNA Library Preparation Kit for Illumina by following the manufacturer’s recommendations (NEB, Ipswich, MA, USA). Briefly, enriched RNAs were fragmented for 15 minutes at 94 °C. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were 35 166682443.1 end repaired and adenylated at 3’ ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment with limited cycle PCR. Sequencing libraries were validated using the Agilent Tapestation 4200 (Agilent Technologies, Palo Alto, CA, USA), and quantified using Qubit 4.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (Applied Biosystems, Carlsbad, CA, USA). The sequencing libraries were multiplexed and clustered onto a flow cell and loaded on the Illumina HiSeq4000 or equivalent instrument according to manufacturer’s instructions. The libraries were sequenced using a 2 x 150 Paired End (PE) configuration. Raw sequence data (.bcl files) generated from Illumina instrument were converted into FASTQ files and de-multiplexed using Illumina's bcl2fastq 2.17 software. One mismatch was allowed for index sequence identification. [0146] Standard RNA-Seq data analysis [0147] After investigating the quality of the raw data, sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality using Trimmomatic v.0.36. The trimmed reads were mapped to the Mus musculus reference genome available on ENSEMBL using the STAR aligner v.2.5.2b. The STAR aligner uses a splice aligner that detects splice junctions and incorporates them to help align the entire read sequences. BAM files were generated as a result of this step. Unique gene hit counts were calculated by using featureCounts from the Subread package v.1.5.2. Only unique reads that fell within exon regions were counted. Since a strand-specific library preparation was performed, the reads were strand-specifically counted. After extraction of gene hit counts, the gene hit counts table was used for downstream differential expression analysis. Using DESeq2, a comparison of gene expression between the groups of samples was performed. The Wald test was used to generate p-values and Log2 fold changes. Genes with adjusted p-values < 0.05 and absolute log2 fold changes > 1 were called as differentially expressed genes for each comparison. A principal component analysis (PCA) analysis was performed using the “plotPCA” function within the DESeq2 R package. The plot shows the samples in a 2D plane spanned by their first two principal components. The top 500 genes, selected by highest row variance, were used to generate the plot. Pathway analysis was performed using the IPA system (version 76765844, Qiagen) including the differentially expressed genes, with threshold defined as -log (P-value) >1.3. [0148] Real-time quantitative PCR [0149] Total RNA isolated from βC^Control and βC^ΔC MASMCs was reverse-transcribed to cDNA using Superscript III first strand synthesis system (Invitrogen 18080-051). cDNA was quantified using an SYBR Green qPCR kit (Applied Biosystems, Cat No.4309155) and ViiA7 36 166682443.1 Real-time PCR system (Applied Biosystems). The data was analyzed with 2^−ΔCt method in which ΔCt was calculated between the gene of interest and housekeeping gene, Rpl13 and β- actin. Primers were used for the following genes: Axin2, β-actin, Rpl13, S1pr1, and S1pr3. [0150] Luciferase assay [0151] βC^Control and βC^ΔC MASMCs (5 x 10⁴ per well) were electroporated with pCMV-β- gal (transfection control) and with pGL3-S1pr1-promoter (kindly provided by Dr. J. Garcia, University of Arizona). When indicated, pcDNA3-β-catenin^S33Y or empty vector were also included in the electroporation protocol. Electroporation was performed as described above. Cell lysates were collected 72 h after electroporation and luciferase activity was determined using the Glo-lysis buffer system (Promega) and the Varioskan LUX multimode microplate reader (Thermo Scientific). Luciferase activities were normalized to β-galactosidase activity for each well to control for transfection efficiency. [0152] Cleavage under targets & release using nuclease (CUT&RUN) assay [0153] CUT&RUN was performed using a CUT&RUN Assay Kit (Cell Signaling Technology, 86652). Briefly, 50,000 cells were washed in Wash Buffer and bound to 10 μl of activated Concanavalin A beads. Bead-bound cells were incubated with primary antibodies at 4°C overnight in antibody binding buffer. β-catenin (Antibodies Online, ABIN2855042), TCF4 (Cell Signaling Technology, 2569S), and rabbit IgG (Cell Signaling Technology, DA1E) primary antibodies validated for CUT&RUN were used. Then, the cell-bead mixture was washed with Wash Buffer and incubated with Protein A-MNase for 1 h at 4 °C. After washing with Wash Buffer, 2 mM CaCl₂ was added to the samples to activate Protein A-MNase digestion for 30 min on ice. The reaction was stopped with the addition of 2 x Stop Buffer containing 20 mM EDTA, 0.05% digitonin, and 5 mg/ml RNase A. Released chromatin fragments were purified using phenol/chloroform extraction and ethanol precipitation. The eluted DNA was analyzed by qPCR to determine β-catenin and TCF4 enrichment on the S1pr1 promoter. A primer set that amplifies an 82 base pair fragment in a gene desert on mouse chromosome 6 was used as a negative control (Active Motif, 71011). Anti-H3 antibody and RPL30 (ribosomal protein L30) primers provided by the kit were used as a positive control for the assay technique and reagent integrity. [0154] Statistics [0155] Data were graphed and statistics performed using GraphPad Prism version 9.4.1. The data presentation and statistical analyses are described in the figure legends. To determine the number of mice necessary for adequate statistical power, power analysis was performed using preliminary data sets. None of the animals were excluded from the study. Animals were 37 166682443.1 randomly assigned to vehicle or E7386 treatments. Data collection and analyses were conducted blinded to the samples. Blinding was removed only for the final statistical analysis. At least three independent experiments with minimum of 3 biological replicates were done. Results are presented as mean ± SEM of at least three independent biological replicates in all figures. Before statistical testing, normality was assessed using the Shapiro-Wilk test. For data confirmed to be normally distributed, Student’s unpaired 2-tailed t-test was used for comparisons between two groups and 1-way ANOVA or 2-way ANOVA with Tukey’s or Šídák’s multiple comparison test for data comparing 3 or more groups. For data not normally distributed, equivalent nonparametric tests were instead performed, as indicated in the figure legends. Statistically significant differences were defined as P<0.05. [0156] Example 2: SMC ^-catenin C-terminus signaling is required for neointima formation after vascular injury [0157] To test if the β-catenin C-terminal domain is required in SMCs for vascular remodeling, mice bearing a validated mutant β-catenin allele encoding a truncated β-catenin protein (^C) were studied. This truncated β-catenin protein is incapable of interaction with multiple transcriptional coactivators. Expression of β-catenin in SMCs is necessary for embryonic development. To mediate temporal and conditional inactivation of β-catenin expression, the tamoxifen-inducible smooth muscle α-actin (SMA)-CreER^T2 driver line was used. For lineage tracing, a Cre-activated fluorescent reporter (red fluorescent protein; RFP, also referred as TdTomato) transgene in the Rosa26 locus was used. The breeding strategy applied here generated mice bearing one β-catenin flox allele and either one knockin mutant (Ctnnb1^{^C/flox}) or WT (Ctnnb1^WT/flox; control) β-catenin allele, both carrying the SMA-CreER^T2 and RFP transgenes. In this system, tamoxifen administration removes the β-catenin flox allele, resulting in mice expressing either the mutant allele (designated as SMβC^{^C} mice) or the WT allele (designated as SMβC^wt/- mice) as the only source of β-catenin protein in SMCs (Table 1). 38 166682443.1 Table 1. Summary of transgenic mouse groups. Genotype Abbreviation β-catenin protein Functions in SMCs after expressed in SMCs tamoxifen ft r t m xif n ); al ge of al on ng , . found that all RFP⁺ cells in non-injured carotid arteries from both SMβC^{^C} and SMβC^wt/- mice are also positive for SMA and are restricted to the medial layer of the vessel (data not shown), indicating that, at least in the vascular wall, the SMA-CreER^T2 is selectively activated in SMCs, but not other cell types. As an additional control, non-injured carotid arteries from both SMβC^{^C} and SMβC^wt/- mice that did not receive tamoxifen were stained for RFP and SMA. Clear signals for SMA, but no signals for RFP were found (data not shown), indicating that autofluorescence or Cre-independent RFP expression are not relevant factors. [0159] Western analysis of aortic lysates using an antibody against the β-catenin C- terminus showed marked reduction of the β-catenin band, whereas an antibody against the β- catenin N-terminus yielded an 80 kDa band (apparent molecular weight consistent with the loss of the C-terminus) (Fig. 1A). Immunofluorescent studies with the anti-β-catenin N-terminal antibody showed a positive signal in RFP⁺ cells in carotid arteries of both SMβC^wt/- and SMβC^{^C} mice, while the anti-C-terminal antibody signal was lost in the latter (Fig.1B). Neither anti-N-terminal nor anti-C-terminal antibodies showed signals for β-catenin in RFP⁺ cells in carotid arteries of smooth muscle ^-catenin knockout (SMβC^-/-) mice, included as negative control (data not shown). In addition, the expression of Axin2 was evaluated, a typical target of ^-catenin signaling. It was found that Axin2 expression was highly induced in RFP⁺ cells from carotid arteries of SMβC^wt/- mice after injury, but markedly lower in injured arteries from SMβC^{^C} mice (Fig.1C). 39 166682443.1 [0160] These findings validate effective, SMC-selective genetic inactivation of β-catenin C-terminal signaling. [0161] Next, it was assessed if complete or C-terminus-specific β-catenin inactivation in SMCs in adulthood affected overall health or vascular homeostasis of unchallenged mice. The blood pressure and body weight of male and female SMβC^wt/-, SMβC^{^C} and SMβC^-/- mice was followed from just prior to first tamoxifen administration until 12 weeks after last tamoxifen injection. Normotensive blood pressures, no lethality or morbidity, and no change in body weight gain, regardless of sex or time-point, was observed in any of the groups (data not shown). Similarly, the structure of uninjured aorta and carotid arteries harvested at the 12-week timepoint appeared unchanged (data not shown). [0162] These findings indicate that full length or C-terminal domain β-catenin functions are not required – at least for 3 months – for overall health maintenance and vascular homeostasis of unchallenged mice. [0163] Next, the importance of SMC β-catenin C-terminal signaling in the response to vascular injury was assessed. To induce such an injury, carotid artery ligation was performed one week after the first tamoxifen injection in male and female SMβC^{^C} and littermate control (SMβC^wt/-) mice. Ligated and uninjured arteries were harvested at 21 days after surgery. Both male and female SMβC^{^C} mice exhibited marked decreases in neointima formation after injury compared to SMβC^wt/- mice. Reduced intima area (Fig. 2A) and decreased intima/media ratio (Fig. 2B) in SMβC^{^C} mice of both sexes were observed, with no differences in medial area (Fig. 2C). It was also found that expression of CD31, an endothelial marker, is limited to the layer of cells outlining the lumen of the vessel, while other cells forming the neointima express RFP in both SMβC^wt/- and SMβC^{^C} mice, indicating that SMCs and their derivatives are the main contributors to neointimal expansion. [0164] These observations indicate that β-catenin C-terminal signaling is required in SMCs for neointima formation after injury in adulthood. [0165] Example 3: Loss of SMC ^-catenin C-terminus signaling in vivo reduces cell proliferation and dedifferentiation but increases apoptosis [0166] Reduced neointimal formation in SMβC^{^C} mice could be due to decreased SMC proliferation and/or increased apoptosis. As such, expression of the proliferating cell nuclear antigen (PCNA) or Ki67, markers of cell proliferation, and cleaved caspase 3, a marker of apoptosis, in injured carotid artery sections was evaluated. Reduced percentages of PCNA⁺ or 40 166682443.1 Ki67⁺ SMCs in the neointima and media area of arteries from SMβC^{^C} mice were observed compared to SMβC^wt/- mice (Figs.3A and 3B). On the other hand, an increased percentage of SMCs positive for cleaved caspase 3 in the neointima and media areas of arteries from SMβC^{^C} mice was observed compared to SMβC^wt/- mice (Fig. 3C). Decreased expression of SMC differentiation markers is another hallmark of vascular remodeling. Thus, it was determined if this process was affected by loss of SMC β-catenin C-terminal function. Indeed, arterial injury decreased levels of the differentiation marker smooth muscle ^ actin (SMA) in RFP⁺ cells of ligated carotid arteries from SMβC^wt/- mice, but not in those of SMβC^{^C} mice (Fig.3D). [0167] Taken together, these findings indicate that the pro-proliferative, pro-survival and dedifferentiation functions of β-catenin in SMCs during vascular remodeling are mediated through its C-terminus. [0168] Example 4: Inhibition of SMC ^-catenin C-terminal signaling downregulates sphingosine-1 phosphate receptor-1 (S1PR1) expression [0169] In order to gain mechanistic insights, mouse aortic SMCs (MASMCs) were isolated from Ctnnb1^ΔC/flox mice and transduced with green fluorescent protein-expressing or Cre- expressing adenovirus to generate control (βC^Control) or β-catenin C-terminus-deficient (βC^ΔC) cells, respectively. Western analysis using antibodies against the β-catenin C- and N-terminal domains confirmed that C-terminus-deficient β-catenin is the only form of this protein expressed in βC^ΔC MASMCs (data not shown). Then, the phenotype of βC^Control and βC^ΔC MASMCs was characterized by evaluating cell population growth. It was bearing βC^{^C} as the only form of β-catenin showed reduced growth compared to βC^Control cells (Fig.4A). [0170] To explore the potential downstream molecular mechanisms underlying the effects of β-catenin C-terminal signaling in SMCs, RNA-sequencing (RNA-seq) analysis was performed. Principal component analysis of transcriptomes from βC^Control and βC^ΔC MASMCs appeared distinct (Fig. 4B), with the latter showing upregulation of 1518 genes and downregulation of 1925 genes compared with control (Fig. 4C). Evaluation on the Ingenuity Pathway Analysis (IPA) platform identified significant downregulation in βC^ΔC cells of canonical signaling pathways associated with pro-proliferative, pro-survival and dedifferentiated phenotypes, such as “Cardiac Hypertrophy Signaling”, “Wound Healing Signaling”, “STAT3 Pathway”, “ERK/MAPK Signaling” and “PTEN Signaling” (Fig. 4D). Both the broad “GPCR signaling” category and the more specific subset of “S1P Signaling” 41 166682443.1 were also identified. Closer examination of S1P-related transcripts revealed decreased S1pr1 and S1pr3 levels, with little change for S1pr2, S1pr5, and Sphk1 and 2 (the last two encoding sphingosine kinase 1 and 2, respectively). By quantitative reverse transcriptase-polymerase chain reaction (qPCR), the decrease in S1pr1 mRNA levels in βC^ΔC MASMCs compared to βC^Control was confirmed (Fig. 4E). On the other hand, both qPCR for S1pr3 (Fig. 4E) and Western blotting for S1PR3 protein (data not shown) showed no difference in expression. [0171] Together, these findings indicate that loss of β-catenin C-terminal signaling in SMCs reduces cell growth and downregulates S1pr1 expression, the latter effect with some selectivity within the S1P pathway. [0172] Example 5: S1PR1 is a ^-catenin transcriptional target that mediates ^-catenin- induced cell population growth in MASMCs [0173] As noted above, decreased expression of S1pr1 in β-catenin C-terminal-deficient MASMCs was observed. To extend this observation, Western analysis was performed, which showed reduced S1PR1 protein expression in βC^ΔC MASMCs compared to βC^Control (Fig.5A). The decreased S1PR1 expression observed in βC^ΔC MASMCs was restored when these cells were transfected with β-catenin^S33Y, a full-length, constitutively active form of β-catenin (Fig. 5B). In addition, treatment with CHIR99021, a small molecule that prevents β-catenin degradation, increased S1PR1 expression in a dose-dependent manner in βC^Control MASMCs, but did not affect S1PR1 expression in βC^ΔC MASMCs (Fig.5C), indicating that the β-catenin C-terminal domain is essential for S1PR1 expression in SMCs. [0174] The upstream factors that control S1PR1 expression in SMCs are not fully understood. To test if the β-catenin C-terminal domain is required for S1pr1 promoter activation, βC^Control and βC^ΔC MASMCs were transfected with a validated human S1PR1 promoter-driven luciferase reporter. Indeed, loss of β-catenin C-terminal signaling markedly reduced S1PR1 promoter activity, which recovered with co-transfection of a plasmid encoding β-catenin^S33Y (Fig.5D). It was further investigated if S1pr1 is a direct β-catenin transcriptional target. β-catenin lacks a DNA binding domain, and it partners with TCF4 in order to interact with target genes. To examine whether β-catenin and/or TCF4 bind to the S1pr1 promoter in MASMCs, a CUT&RUN (Cleavage Under Targets & Release Using Nuclease) assay was performed using anti-β-catenin and anti-TCF4 antibodies, plus qPCR to amplify different regions of the S1pr1 promoter. This assay revealed recruitment of β-catenin and TCF4 to a region of the S1pr1 promoter containing a consensus TCF binding motif, 5′-TCAAAG 42 166682443.1 (fragment -939/-790; Fig.5E). Such recruitment was not observed in other regions of the S1pr1 promoter that lack a consensus TCF binding motif (fragments -1973/-1802 and -638/-512; Fig. 5E). In addition, it was found that in serum-deprived, quiescent MASMCs, β-catenin is not recruited to the S1pr1 promoter (Fig.5F and 5G), consistent with reduced β-catenin and S1PR1 expression in these conditions (data not shown). [0175] Together, these findings show that under stimulated but not quiescent conditions, β- catenin and TCF4 bind to the S1pr1 promoter in SMCs, activating expression of S1PR1 through a β-catenin C-terminus-dependent function. [0176] Next, it was characterized how decreased S1PR1 expression contributes to the βC^ΔC SMC phenotype. MASMCs were isolated from Ctnnb1^{^C/flox} mice also bearing a Cre- conditional S1PR1 gain-of-function (GOF) allele. The cells were transduced with GFP or Cre- expressing adenovirus to generate control (βC^Control S1PR1^Control) or βC^ΔC cells with restored S1PR1 (βC^ΔC S1PR1^GOF), respectively. Western blotting validated the technical strategy (data not shown). Expression of S1PR1 in β-catenin C-terminus-deficient MASMCs (βC^ΔC S1PR1^GOF) restored cell growth towards control levels (Fig. 5H), thereby suppressing the growth phenotype observed in C-terminal-deficient MASMCs. In complementary studies, β- catenin gain-of-function were induced in MASMCs with CHIR99021 and knocked down S1PR1 using a small interfering RNA (siRNA) (data not shown). Loss of S1PR1 prevented the increase in cell growth promoted by CHIR99021 (Fig.5I), showing that S1PR1 is required for the increased SMC growth induced by β-catenin gain-of-function. [0177] Taken together, these observations are consistent with the idea that S1pr1 is a critical target gene for β-catenin C-terminal signaling that drives proliferation of SMCs in culture. [0178] Example 6:^-catenin C-terminus signaling promotes neointima formation after vascular injury by inducing S1PR1 expression in SMCs [0179] To test the in vivo relevance of the above findings, S1PR1 expression was examined in control and ligated carotid arteries from SMβC^{^C} and SMβC^wt/- mice. A clear signal for S1PR1 was found in the endothelial layer. S1PR1 expression could not be detected in SMCs of control, uninjured arteries from either SMβC^{^C} or SMβC^wt/- mice (data not shown). On the other hand, a high percentage of S1PR1⁺ SMCs was found in injured arteries from SMβC^wt/- mice, which was decreased in injured arteries from SMβC^{^C} mice (Fig.6A). Since it was found that loss of β-catenin C-terminal domain in SMCs reduced neointima formation after vascular injury (Fig. 2C) and this effect was associated with reduced S1PR1 expression (Fig. 6A), it 43 166682443.1 was examined if restoring S1PR1 expression in SMβC^{^C} mice could reestablish injury-induced neointima formation. To that end, the Cre-conditional S1PR1 GOF mouse line (Rosa26^{LSL- S1pr1}) was crossed with Ctnnb1^{^C/flox} SMA-CreER^T2 or Ctnnb1^WT/flox SMA-CreER^T2 mice to generate mice that upon tamoxifen administration express in SMCs a gain-of-function S1PR1 allele and the C-terminus-deficient β-catenin allele (designated as SMβC^{^C} SM-S1PR1^GOF mice) or a gain-of-function S1PR1 allele and a WT β-catenin allele (designated as SMβC^wt/- SM-S1PR1^GOF mice) (Table 1). Susceptibility to neointima formation was studied at 21 days after ligation of carotid arteries in the following four different groups: SMβC^wt/- SM-S1PR1^GOF, SMβC^{^C} SM-S1PR1^GOF, SMβC^wt/- and SMβC^{^C} mice. Increased expression of S1PR1 in SMCs within ligated carotid arteries of SMβC^wt/- SM-S1PR1^GOF and SMβC^{^C} SM-S1PR1^GOF mice was confirmed by immunofluorescence (data not shown). Greater neointima formation in both male and female SMβC^{^C} SM-S1PR1^GOF mice was observed compared to SMβC^{^C} mice. In particular, increased intima area (Fig. 6B) and a higher intima/media ratio (Fig. 6C) in male and female SMβC^{^C} SM-S1PR1^GOF mice compared to SMβC^{^C} mice was observed, with no differences in medial area (Fig. 6D). No difference in neointima size was observed between SMβC^{^C} SM-S1PR1^GOF mice and SMβC^wt/- mice (Figs.6B and 6C). [0180] These findings show that restoring expression of S1PR1 in SMCs reestablishes neointima formation in mice lacking the β-catenin C-terminus domain in SMCs. Altogether, these data demonstrate that signaling mediated by the C-terminus domain of β-catenin in SMCs is essential for vascular remodeling after injury, in part by inducing S1PR1 expression and function. [0181] Example 7: Pharmacologic inhibition of ^-catenin C-terminus signaling decreases S1PR1 expression in SMCs, attenuates neointimal formation, and limits atherosclerosis [0182] Next, the effects of pharmacological inhibition of ^-catenin C-terminus signaling on vascular remodeling were examined. To that end, E7386 was used, a first in class orally active ^-catenin C-terminus inhibitor that has been reported to inhibit the binding of the ^-catenin C- terminus to Creb-binding protein (CBP), a transcriptional coactivator, and to reduce canonical Wnt signaling-dependent gene expression. See Yamada et al, E7386, a Selective Inhibitor of the Interaction between β-Catenin and CBP, Exerts Antitumor Activity in Tumor Models with Activated Canonical Wnt Signaling. Cancer Res. 2021 Feb 15;81(4):1052-1062, which is incorporated herein in its entirety. 44 166682443.1 [0183] To confirm the specificity of E7386 for the C-terminal domain of ^-catenin, the effect of E7386 on βC^Control and βC^ΔC MASMCs was compared. It was found that E7386 at 10– 100 nM decreased cell growth of βC^Control MASMCs (Figs. 7A, 7B, and 7C). The magnitude of growth inhibition in βC^Control MASMCs treated with 100 nM E7386 was similar to that observed in MASMCs lacking the ^-catenin C-terminus (βC^ΔC MASMCs vehicle group; Fig. 7A), while E7386 at 50 nM and 10 nM yielded less pronounced but still significant inhibition (Figs. 7B and 7C). Notably, E7386 at 10–100 nM did not affect the cell growth of βC^ΔC MASMCs (Figs. 7A, 7B, and 7C), indicating that the inhibitory effect of E7386 on SMC growth requires the ^-catenin C-terminus. In addition, MASMCs treated with E7386 exhibited reduced expression of Axin2 (Fig. 7D), indicating that E7386 inhibits ^-catenin C-terminal transcriptional activity. Consistent with this idea, E7386 also reduced S1PR1 expression in MASMCs (Fig. 7D), since S1PR1 expression in these cells requires the ^-catenin C-terminal transcriptional activity (Fig.5). [0184] Taken together, these findings indicate that E7386 inhibits SMC proliferation by blocking interactions mediated by the ^-catenin C-terminus. [0185] To test the in vivo relevance of the above findings, vascular injury was induced by carotid artery ligation in male and female wild-type (WT) C57BL/6J mice, followed by treatment with E738650 mg/kg twice/day by oral gavage for 14 days. Immunostaining for Axin2 showed reduced expression of this protein in SMA⁺ cells of carotid arteries from mice treated with E7386, which is consistent with inhibition of ^-catenin C-terminus signaling in SMCs in vivo (Fig.7E). It was found that E7386 decreased the intimal area in females, trended toward a similar effect in males, and did not affect the media area in either sex (Figs.7F, 7G, and 7H). Notably, in both females and males E7386 reduced the intima/media ratio, a parameter of neointima formation that considers variation in blood vessel size (Fig.7H). E7386 also reduced expression of S1PR1 in SMA⁺ cells in the injured carotid arteries (Fig.7I). [0186] Altogether, these observations demonstrate that pharmacological inhibition of the ^- catenin C-terminal signaling decreases S1PR1 expression in SMCs and attenuates neointima formation after arterial injury. [0187] Example 8: Pharmacological inhibition of ^-catenin C-terminus signaling decreases S1PR1 expression in SMCs and limits atherosclerosis development [0188] Next, the effects of E7386 were tested in a model of atherosclerosis, a highly prevalent and important disease in which SMCs play a critical role. To this end, a mouse model 45 166682443.1 of disturbed flow-, hyperlipidemia-induced atherosclerosis was used. E7386 25 mg/kg or vehicle was administered by oral gavage twice/day for 21 days after initiation of altered blood flow. E7386 reduced Axin2 expression in SMA⁺ cells in atherosclerotic arteries (Fig. 8A), which is again consistent with inhibition of ^-catenin C-terminal signaling in vivo. Treatment with E7386 did not affect mouse body weight or serum levels of total cholesterol (Fig.8B and 8C), but reduced atherosclerotic lesion expansion. In particular, E7386 reduced atherosclerotic lesion area (Fig. 8E), reduced lesion/media ratio (Fig. 8F), increased lumen area (Fig. 8G), and reduced necrotic core area (Fig. 8H). Importantly, it preserved the lumen cross-sectional area (Fig. 8E) without affecting medial area (Fig. 8D) or fibrous cap thickness (Fig. 8I). In addition, E7386 decreased S1PR1 expression in SMA⁺ cells in atherosclerotic arteries (Fig. 8J). [0189] Collectively, these findings indicate that pharmacological inhibition of ^-catenin C- terminus signaling decreases S1PR1 expression in SMCs and limits atherosclerosis development in this preclinical model. [0190] In sum, the experiments shown herein uncover coordination between Wnt/β-catenin and S1PR1 signaling that is essential for vascular remodeling, demonstrating that expression of the β-catenin C-terminus in SMCs is required for neointima formation after vascular injury in both female and male mice. Evidence has been provided that β-catenin interacts with the S1pr1 promoter and activates it through its C-terminal domain, which promotes S1PR1 expression and ultimately leads to enhanced SMC growth and robust neointima formation in response to vascular injury. [0191] It was found that inactivation of total β-catenin or its C-terminus in SMCs was well tolerated in unchallenged adult mice over 3 months without obvious repercussions for body weight gain, blood pressure, or arterial structure and thus these signals are not necessary for maintenance of the unstressed, mature state of the vessel. When the adult arterial wall is disturbed by injury, however, the C-terminal activity of β-catenin becomes essential for SMC proliferation, survival, and neointima formation. The truncated β-catenin encoded by the ΔC allele retains cell adhesion function and N-terminal activity , so the findings herein also indicate that other functions of β-catenin (N-terminal signaling and adhesion) in SMCs are not sufficient to drive this phenotype. [0192] It was also shown that pharmacologic inhibition of β-catenin C-terminal signaling decreases SMC proliferation, decreases S1PR1 expression in SMCs, reduces neointima 46 166682443.1 formation after arterial injury, and limits atherosclerosis development. As such, inhibitors of the β-catenin C-terminus, at least in part by hindering the β-catenin C-terminus/S1PR1 axis, have therapeutic uses for vascular pathologies associated with SMC proliferation and intimal expansion, such as restenosis, atherosclerosis, and obstructive vascular disease. 47 166682443.1

Claims

CLAIMS We claim: 1. A method of preventing or reducing restenosis in a blood vessel in a subject in need thereof, the method comprising administering to the subject a compound of Formula I or a pharmaceutically acceptable salt thereof: (Formula I).

2. A method of preventing, inhibiting, or reducing vascular remodeling in the blood vessel of a subject in need thereof, the method comprising administering to the subject a compound of Formula I or a pharmaceutically acceptable salt thereof.

3. A method of preventing, inhibiting, or reducing thickening of the intima in a blood vessel in a subject in need thereof, the method comprising administering to the subject a compound of Formula I or a pharmaceutically acceptable salt thereof.

4. A method of decreasing proliferation of smooth muscle cells in a blood vessel in a subject in need thereof, the method comprising administering to the subject a compound of Formula I or a pharmaceutically acceptable salt thereof.

5. A method of preventing, inhibiting, or reducing the formation and/or expansion of atherosclerotic lesions in a blood vessel in a subject in need thereof, the method comprising administering to the subject a compound of Formula I or a pharmaceutically acceptable salt thereof. 48 166682443.1

6. A method of maintaining patency of a blood vessel in a subject, the method comprising administering to the subject a compound of Formula I or a pharmaceutically acceptable salt thereof.

7. The method of any one of claims 1-6, wherein the subject has undergone angioplasty of the blood vessel.

8. The method of any one of claims 1-6, wherein the subject has undergone placement of a stent in the blood vessel.

9. The method of any one of claims 1-6, wherein the subject has undergone endarterectomy.

10. The method of any one of claims 1-6, wherein the blood vessel is a coronary blood vessel, a peripheral artery, a carotid artery, or a cerebral artery.

11. The method of any one of claims 1-6, wherein the blood vessel is in a arterio-venous fistula (AVF).

12. The method of any one of claims 1-6, wherein the subject has undergone a blood vessel graft.

13. The method of claim 12, wherein the blood vessel graft is a vein graft.

14. The method of claim 13, wherein the subject has undergone interposition vein grafting into the arterial circulation or bypass surgery.

15. The method of any one of claims 1-10 and 12-14, wherein the subject has a cardiovascular disease or is at risk of developing cardiovascular disease.

16. The method of any one of claims 1-10 and 12-14, wherein the subject has atherosclerosis or is at risk of developing atherosclerosis. 49 166682443.1

17. A method of preventing or reducing transplant-associated vasculopathy in a subject in need thereof, the method comprising administering to the subject a compound of Formula I or a pharmaceutically acceptable salt thereof.

18. A method of preventing, inhibiting, or reducing progression of atherosclerosis in a subject in need thereof, the method comprising administering to the subject a compound of Formula I or a pharmaceutically acceptable salt thereof.

19. A method of surgically inserting a stent of into the lumen of a blood vessel in a subject in need thereof, wherein the stent comprises (i) a scaffold coated with a polymer, which polymer is impregnated with, adsorbed with, or coated with a compound of Formula I, or (ii) a scaffold which is impregnated with, adsorbed with, or coated with, a compound of Formula I.

20. The method of any one of claims 1-19, wherein the subject is a mammal.

21. The method of claim 20, wherein the subject is a human. 50 166682443.1