WO2024091485A1 - Compositions and methods for treating or preventing pulmonary fibrosis - Google Patents

Abstract

Disclosed herein are compounds and their use for treatment of pulmonary fibrosis and related diseases. Methods for identifying compound(s) using animal models of pulmonary fibrosis are described.

Description

FH Docket No.:UCH-34425 UCLA Ref. No.: [UCLA 2023-041-2] WO COMPOSITIONS AND METHODS FOR TREATING OR PREVENTING PULMONARY FIBROSIS CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application serial number 63/419,217, filed on October 25, 2022, the contents of which are hereby incorporated by reference in its entirety. GOVERNMENT SUPPORT This invention was made with government support under HL139675 and HL081397, awarded by the National Institutes of Health. The government has certain rights in the invention. This work was supported by the U.S. Department of Veterans Affairs, and the Federal government has certain rights in the invention. BACKGROUND Pulmonary fibrosis is a severe fibrotic lung disease, affecting over three million people causing high morbidity and mortality worldwide. In the progression of pulmonary fibrosis, extensive fibrogenesis occurs in the interstitium of lung tissue where it replaces normal structural components, damages alveolar units and disrupts gas exchanges. The survival time of patients with pulmonary fibrosis is quite poor due to the dramatic reduction of pulmonary function. Therefore, a need remains for developing treatments for treating pulmonary fibrosis. SUMMARY Disclosed herein are methods and compounds for treating a subject having one or more fibrotic diseases (e.g., pulmonary fibrosis), comprising administering to the subject a compound that reduces activity and/or expression of ALK5, and/or a compound that increases activity and/or expression of FoxA2. In some embodiments, the compound suppresses expression of ALK5. In some embodiments, the compound reduces the expression of ALK5. In certain embodiments, the compound induces transcription factor FoxA2. In certain embodiments, the compound is berbamine, or a pharmaceutically acceptable salt thereof. In certain embodiments, the invention described herein provides methods for promoting differentiation of EC-like myofibroblasts toward myofibroblasts by FH Docket No.:UCH-34425 UCLA Ref. No.: [UCLA 2023-041-2] WO administering a compound that reduces activity and/or expression of ALK5, and/or a compound that increases activity and/or expression of FoxA2, e.g., berbamine. The invention further provides use of a compound, e.g., berbamine, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for treating or preventing pulmonary fibrosis. As described herein, berbamine can redirect the differentiation of the EC-like myofibroblasts and reduce pulmonary fibrosis. The compositions and methods described herein can similarly be used to reduce the expression of enzymatic targets (e.g., ALK5), induce the expression of transcription factors (e.g., FoxA2), and/or to promote the differentiation of EC-like myofibroblasts toward myofibroblasts, thereby leading to the treatment or prevention of pulmonary fibrosis. The details of one or more embodiments of the invention are set forth in the accompanying drawings and description below. Other features, objects and advantages of the invention will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF THE FIGURES FIG. 1A shows Masson’s trichrome (MT) staining of pulmonary tissues (n=10). f/f, flox/flox. Bleomycin, wild type mice were injected with bleomycin. VE-cad, VE-cadherin. Scale bar, 100 µm. FIG. 1B shows quantification of MT staining of pulmonary tissues (n=9). The data were analyzed for statistical significance by ANOVA with post hoc Tukey’s test. The bounds of the boxes are upper and lower quartiles with data points. The line in the box is the median. Error bars are maximal and minimal values. FIG.1C shows mouse pulmonary tests (n=6). CO2, hypercapnia phase with 7% CO2, 21% O₂, and balanced N₂. RA, room air. The data were analyzed for statistical significance by ANOVA with post hoc Tukey’s test. The bounds of the boxes are upper and lower quartiles with data points. The line in the box is the median. Error bars are mean ± standard deviation (SD). ***, P<0.0001. FIG.2A shows immunostaining and FACS of pulmonary tissues of VE- cadherin^creMgp^flox/flox and Sm22α^creMgp^flox/flox mice (n=8). Mgp^flox/flox was used as control. Scale bar, 100 µm. The data were analyzed for statistical significance by unpaired 2-tailed Student’s t test. The bounds of the boxes are upper and lower quartiles with data points. The line in the box is the median. Error bars are maximal and minimal values. ***, P<0.0001. FH Docket No.:UCH-34425 UCLA Ref. No.: [UCLA 2023-041-2] WO FIG. 2B shows MT staining and immunostaining of the tissues of human pulmonary fibrosis (left), and quantification of CD34+ cells in human pulmonary fibrosis (right) (n=6). Control, healthy lungs. Scale bar, 100 µm. The data were analyzed for statistical significance by unpaired 2-tailed Student’s t test. The bounds of the boxes are upper and lower quartiles with data points. The line in the box is the median. Error bars are maximal and minimal values. ***, P<0.0001. FIG.3A shows UMAP for the cell populations subclustered from the whole population of CD34+CD45- pulmonary cells and violin plots of CD34 expression in the cell clusters. FIG.3B shows violin plots of the gene expression of the lineage markers in the cell clusters. Mgp: matrix Gla protein; PDGFR: platelet-derived growth factor receptor; Fn1, fibronectin 1; SMMHC: smooth muscle myosin heavy. FIG.3C shows pseudotemporal trajectories of the cell clusters. FIG.3D shows immunostaining of the lungs of wild-type mice and healthy humans (n=3). Scale bar=100 µm. PDGFR: platelet-derived growth factor receptor; ; DAPI: 4',6- diamidino-2-phenylindole. FIG.3E shows fluorescence-activated cell sorting (FACS) of whole lung cells isolated from bleomycin-injected mice, VE-cadherin^creMgp^flox/flox mice and Sm22α^creMgp^flox/flox mice (n=6). Mgp: matrix Gla protein; SMMHC: smooth muscle myosin heavy chain. FIG. 4A shows UMAP for cell populations subclustered from the whole population of CD34+CD45- pulmonary cells of healthy human lungs and human pulmonary fibrosis. FIG.4B shows violin plots of the gene expression of the lineage markers. Mgp: matrix Gla protein; vWF: von Willebrand factor; SMA: smooth muscle actin; Fn1, fibronectin 1. FIG.4C shows pseudotemporal trajectories of the cell clusters. FIG. 4D shows alterations in cell compositions of different populations in healthy human lungs and human pulmonary fibrosis. FH Docket No.:UCH-34425 UCLA Ref. No.: [UCLA 2023-041-2] WO FIG. 5A shows immunostaining of pulmonary tissues of Sm22α^creRosa^tdTomato mice (n=9). VE-cad, VE-cadherin. Scale, 50 µm. FIG. 5B shows expression of MGP, Col3a1, Fibronectin 1 (Fn1), VE-cadherin and vWF in tdTomato+VE-cadherin+ cells isolated from the lungs of SM22α^creRosa^tdTomato mice. The cells were transfected with MGP siRNA or scrambled siRNA (SCR) (n=8). The data were analyzed for statistical significance by ANOVA with post hoc Tukey’s test. The bounds of the boxes are upper and lower quartiles with data points. Error bars are maximal and minimal values. FIG.5C shows binding of BMP-1 to MGP shown by immunoprecipitation. BMP-1 (200 ng) and conditioned medium containing N-FLAG-MGP (100 μl; approximately 200 ng) were combined as indicated in the top panel, and the presence of the respective protein was confirmed by immunoblotting (IB) (top 2 blots). Interactions between the proteins were analyzed by immunoprecipitation (IP) followed by immunoblotting with antibodies as indicated. FIG. 5D shows binding of BMP-1 to MGP showed by chemical crosslinking. BMP- 1 (200 ng) and conditioned medium containing N-FLAG-MGP (100 μl; approximately 200 ng) were combined as indicated in lane 2 and 3. Interactions were analyzed using chemical crosslinking followed by immunoblotting with antibodies as indicated. FIG.5E shows Expression of Col3a1 and Fibronectin 1 in tdTomato+VE-cadherin+ cells isolated from the lungs of SM22α^creRosa^tdTomato mice and treated with BMP-1 (200ng/ml) with transfection of MGP siRNA or SCR (n=6). The data were analyzed for statistical significance by ANOVA with post hoc Tukey’s test. The bounds of the boxes are upper and lower quartiles with data points. Error bars are maximal and minimal values. FIG. 5F shows time-course immunoblotting of LTBP-1 in tdTomatoVE-cadherin+ cell lysates after treatment with BMP-1 (200 ng/ml) with or without conditioned medium containing N-FLAG-MGP (n=6). Fig. 5G shows levels of TGFβ1 in culture media of tdTomato+VE-cadherin+ cell after treatment with BMP-1 with or without conditioned medium containing N-FLAG-MGP (n=3). The data were analyzed for statistical significance by ANOVA with post hoc Tukey’s test. The bounds of the boxes are upper and lower quartiles with data points. Error bars are mean ± standard deviation (SD). ***, P<0.0001. FIG.5H shows a schematic diagram. FH Docket No.:UCH-34425 UCLA Ref. No.: [UCLA 2023-041-2] WO FIG.5I shows immunoblotting with densitometry of lung tissues of VE- cadherin^creMgp^flox/flox and Sm22α^creMgp^flox/flox and control mice (n=3). FIG.5J shows Masson's trichrome (MT) staining with quantification of the lungs of VE-cadherin^creMgp^flox/flox and Sm22α^creMgp^flox/flox mice and control mice after treatment with UK388367 (n=8). Scale bars=100 µm. Data were analyzed for statistical significance by ANOVA with post hoc Tukey's test. The bounds of the boxes are upper and lower quartiles with data points. Error bars are maximal and minimal values. FIG.6A shows UMAP and differential gene expression between EC-like myofibroblasts and ECs. FIG. 6B shows gene expression in tdTomatoVE+cadherin+ cells isolated from the lungs of SM22α^creRosa^tdTomato mice and treated with berbamine (20 µM) with transfection of MGP siRNA or SCR (n=6). Fn1, fibronectin 1. The data were analyzed for statistical significance by ANOVA with post hoc Tukey’s test. The bounds of the boxes are upper and lower quartiles with data points. The line in the box is median. Error bars are maximal and minimal values. ***, P<0.0001. FIG.6C shows MT staining of pulmonary tissues from VE-cadherin^creMgp^flox/flox and Sm22α^creMgp^flox/flox mice after berbamine treatment (100 ng/g, daily) (n=12). FIG. 6D shows quantification of MT staining and expression of Col3a1 and Fibronectin 1 in pulmonary tissues from VE-cadherin^creMgp^flox/flox and Sm22α^creMgp^flox/flox mice after berbamine treatment (n=9 for quantification of MT and n=6 for gene expression). The data were analyzed for statistical significance by ANOVA with post hoc Tukey’s test. The bounds of the boxes are upper and lower quartiles with data points. The line in the box is median. Error bars are maximal and minimal values. ***, P<0.0001. FIG. 7A shows expression of ALK5 in tdTomatoVE+cadherin+ cells isolated from the lungs of SM22α^creRosa^tdTomato mice and treated with berbamine (20 µM). The data were analyzed statistical significance by unpaired 2-tailed Student’s t test. The bounds of the boxes are upper and lower quartiles with data points. The line in the box is median. Error bars are maximal and minimal values. ***, P<0.0001. FIG. 7B shows gene expression in tdTomatoVE+cadherin+ cells transfected with MGP siRNA in combination with berbamine treatment with or without overexpression of ALK5 (n=5). CMV, cytomegalovirus promoter. Fn1, fibronectin 1. SCR. Scrambled siRNA. The data were analyzed for statistical significance by ANOVA with post hoc Tukey’s test. FH Docket No.:UCH-34425 UCLA Ref. No.: [UCLA 2023-041-2] WO The bounds of the boxes are upper and lower quartiles with data points. The line in the box is median. Error bars are maximal and minimal values. ***, P<0.0001. FIG.7C shows expression of FoxA2 in tdTomato+VE+cadherin+ cells isolated from the lungs of SM22α^creRosa^tdTomato mice and treated with berbamine (20 µM). The data were analyzed statistical significance by unpaired 2-tailed Student’s t test. The bounds of the boxes are upper and lower quartiles with data points. The line in the box is median. Error bars are maximal and minimal values. ***, P<0.0001. FIG.7D shows immunoblotting using tdTomatoVE-cadherin+ cell lysates treated with berbamine (Bbm) with or without overexpression of FoxA2 (CMV-FoxA2) (n=3). FIG.7E shows gene expression of endothelial markers in tdTomatoVE+cadherin+ cells treated with berbamine in combination with FoxA2 overexpression or FoxA2 knockdown (FoxA2 siRNA) (n=6). Data were analyzed for statistical significance by ANOVA with post hoc Tukey’s test. The bounds of the boxes are upper and lower quartiles with data points. The line in the box is median. Error bars are maximal and minimal values. ***, P<0.0001. FIG.7F shows immunoblotting with densitometry of the lungs of VE- cadherin^creMgp^flox/flox mice, Sm22α^creMgp^flox/flox mice and control mice after treatment of berbamine (Bbm) (n=6). FIG. 7G shows quantification of VE-cadherin-positive and platelet-derived growth factor receptor (PDGFR)α-positive (VE-cadherin+PDGFRα+) cells and VE-cadherin negative and PDGFRα positive (VE-cadherin−PDGFRα+) cells after immunostaining of the lungs of bleomycin-injected wild-type mice, where gene expression was examined by real- time PCR (n=8). Data were analysed for statistical significance by ANOVA with post hoc Tukey's test. The bounds of the boxes are upper and lower quartiles with data points; the line in the box is median; error bars are maximal and minimal values. FIG. 7H shows immunostaining of ALK5 and FoxA2 of human pulmonary fibrosis (n=3). FIG.8A shows a strategy of Mgp gene targeting for Generation of Mgpflox/flox mice. FIG. 8B shows genotyping of Mgpflox/flox (Mgpf/f) mice by PCR. +/+, wild type. fl/+, heterozygous. fl/fl, homozygous. FH Docket No.:UCH-34425 UCLA Ref. No.: [UCLA 2023-041-2] WO FIG.9A shows Masson's trichrome (MT) staining and immunostaining with cell quantification of the lungs of tamoxifen-injected VE-cadherin^cre/ERT2Rosa^tdTomato mice after administration of bleomycin (n=10). The data were analysed for statistical significance by unpaired two-tailed t-test. The bounds of the boxes are upper and lower quartiles with data points; the line in the box is the median; error bars are maximal and minimal values. PDGFR: platelet-derived growth factor receptor. ***: p<0.0001. FIG.9B shows MT staining and immunostaining with cell quantification of the lungs of Sm22α^creRosa^tdTomato mice after administration of bleomycin (n=10). The data were analysed for statistical significance by unpaired two-tailed t-test. The bounds of the boxes are upper and lower quartiles with data points; the line in the box is the median; error bars are maximal and minimal values. ***: p<0.0001. FIG.9C shows immunostaining with cell quantification of the lungs of Sm22α^creRosa^tdTomato and Sm22α^creRosa^tdTomatoMgp^Flox/Flox mice. The expression of Col3a1 and fibronectin (Fn)1 in ^tdTomato and VE-cadherin double-positive cells isolated from lungs of Sm22α^creRosa^tdTomato and Sm22α^creRosa^tdTomatoMgp^Flox/Flox mice (n=10). Scale bars=100 µm. The data were analysed for statistical significance by unpaired two- tailed t-test. The bounds of the boxes are upper and lower quartiles with data points; the line in the box is the median; error bars are maximal and minimal values. ***: p<0.0001. DETAILED DESCRIPTION Definitions Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art of the present disclosure. The following references provide one of skill with a general definition of many of the terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise. The term "subject" to which administration is contemplated includes, but is not limited to, humans (i.e., a male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult or senior FH Docket No.:UCH-34425 UCLA Ref. No.: [UCLA 2023-041-2] WO adult)) and/or other primates (e.g., cynomolgus monkeys, rhesus monkeys); mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and/or dogs; and/or birds, including commercially relevant birds such as chickens, ducks, geese, quail, and/or turkeys. Preferred subjects are humans. As used herein, an agent that “prevents” a disorder or condition refers to a compound that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample. The term “treating” includes prophylactic and/or therapeutic treatments. The term “prophylactic or therapeutic” treatment is art-recognized and includes administration to the subject of one or more of the disclosed compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the subject) then the treatment is prophylactic (i.e., it protects the subject against developing the unwanted condition), whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic, (i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof). The term “prodrug” is intended to encompass compounds which, under physiologic conditions, are converted into the agents of the present invention. A common method for making a prodrug is to include one or more selected moieties which are hydrolyzed under physiologic conditions to reveal the desired molecule. In other embodiments, the prodrug is converted by an enzymatic activity of the subject. For example, esters or carbonates (e.g., esters or carbonates of alcohols or carboxylic acids) are preferred prodrugs of the present invention. In certain embodiments, some or all of the disclosed agents in a formulation represented above can be replaced with the corresponding suitable prodrug, e.g., wherein a hydroxyl in the parent compound is presented as an ester or a carbonate or carboxylic acid. An “effective amount”, as used herein, refers to an amount that is sufficient to achieve a desired biological effect. A “therapeutically effective amount”, as used herein, refers to an amount that is sufficient to achieve a desired therapeutic effect. For example, a therapeutically effective amount can refer to an amount that is sufficient to improve at least one sign or symptom of a fibrotic disease or disorder. FH Docket No.:UCH-34425 UCLA Ref. No.: [UCLA 2023-041-2] WO Methods of Use Recent studies have shown that several types of cells contribute to the stimulus of extracellular matrix deposition, such as alveolar type 2 epithelial cells and alveolar macrophages. The studies reveal that these cells secrete a number of growth factors and cytokines that activate myofibroblasts, which produce aberrant compositions of extracellular fibrotic matrix leading to pulmonary fibrosis. Myofibroblasts were initially discovered in the granulation tissue during tissue repair. Advanced studies have observed that myofibroblasts contain the features of both fibroblasts and smooth muscle cells, and play a critical role in quickly responding and producing the collagens needed to repair a wound site. Beyond normal tissue repair, overloaded myofibroblasts are found in almost all fibrotic organs, including the liver, kidneys, heart, and lungs, where myofibroblasts persistently produce and accumulate unwanted fibrotic components to form scar tissue. In pulmonary fibrosis, excessively activated myofibroblasts have been shown to generate deleterious extracellular matrix that are dominated by fibrillar collagens, fibronectin and other fibrotic proteomes. Although studies suggest that several cell lineages may be involved in the differentiation of myofibroblasts, the origin of myofibroblasts remains unclear. Matrix Gla Protein (MGP) is a bone morphogenic protein (BMP) antagonist and highly expressed in pulmonary cells. MGP is essential for endothelial-epithelial interactions to direct pulmonary specification. Conventional deletion of Mgp gene in mice causes an imbalance between the pulmonary vasculature and the airways, leading to vascular malformations and underdeveloped lungs. Mutations in the human Mgp gene cause Keutel syndrome, which is characterized by peripheral pulmonary stenoses and other developmental defects. With warfarin treatment that renders MGP non-functional by interfering with g-carboxylation, pulmonary fibrosis deteriorates sharply. MGP has also been found to support normal endothelial differentiation in progenitor cells and prevent ECs from unwanted differentiation. In this study, it was revealed that cell-specific deletion of Mgp gene causes severe pulmonary fibrosis. Previously unknown endogenous EC-like myofibroblasts, which can differentiate into myofibroblasts in normal lungs were identified. The studies showed that EC-like myofibroblasts significantly contribute myofibroblasts to pulmonary fibrosis. MGP FH Docket No.:UCH-34425 UCLA Ref. No.: [UCLA 2023-041-2] WO was found to bind to a unique member of the BMP family, BMP-1, to inhibit its activity for in the differentiation of EC-like myofibroblasts. A small molecule that redirects the differentiation of EC-like myofibroblasts and reduces pulmonary fibrosis was also identified. For example, the compound and methods disclosed herein may treat or prevent fibrosis resulting from conditions including but not limited to pulmonary interstitial fibrosis, drug-induced sarcoidosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, asthma, chronic obstructive pulmonary disease, diffuse alveolar damage disease, pulmonary hypertension, neonatal bronchopulmonary dysplasia, chronic asthma, and emphysema. In some embodiments, the compound and methods disclosed herein may treat or prevent fibrosis resulting from pulmonary artery stenosis. In some embodiments, the compound and methods disclosed herein may treat or prevent pulmonary arteriovenous malformations (AVMs). In some embodiments, the compound and methods disclosed herein may treat or prevent pulmonary fibrosis that accompanies a respiratory infection, such as a coronavirus, rhinovirus, or influenza infection. Pharmaceutical Compositions In certain embodiments, the present invention provides a pharmaceutical preparation suitable for use in a human patient, comprising any of the agents shown above, and one or more pharmaceutically acceptable excipients. In certain embodiments, the pharmaceutical preparations may be for use in treating or preventing a condition or disease as described herein. Any of the disclosed agents may be used in the manufacture of medicaments for the treatment of any diseases or conditions disclosed herein. The compositions and methods of the present invention may be utilized to treat a subject in need thereof. In certain embodiments, the subject is a mammal such as a human, or a non-human mammal. When administered to subject, such as a human, the composition or the agent is preferably administered as a pharmaceutical composition comprising, for example, an agent of the invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In a FH Docket No.:UCH-34425 UCLA Ref. No.: [UCLA 2023-041-2] WO preferred embodiment, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration (i.e., routes, such as injection or implantation, that circumvent transport or diffusion through an epithelial barrier), the aqueous solution is pyrogen-free, or substantially pyrogen-free. The excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection or the like. The composition can also be present in a transdermal delivery system, e.g., a skin patch. The composition can also be present in a solution suitable for topical administration, such as an eye drop. A pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of an agent such as agent of the invention. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can have incorporated therein, for example, an agent of the invention. Liposomes, for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer. The phrase "pharmaceutically acceptable" is employed herein to refer to those agents, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be FH Docket No.:UCH-34425 UCLA Ref. No.: [UCLA 2023-041-2] WO "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. 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) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations. A pharmaceutical composition (preparation) can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); anally, rectally or vaginally (for example, as a pessary, cream or foam); parenterally (including intramuscularly, intravenously, subcutaneously or intrathecally as, for example, a sterile solution or suspension); nasally; intraperitoneally; subcutaneously; transdermally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin, or as an eye drop). The agent may also be formulated for inhalation. In certain embodiments, an agent may be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for same can be found in, for example, U.S. Pat. Nos.6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896, as well as in patents cited therein. 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 subject being treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a FH Docket No.:UCH-34425 UCLA Ref. No.: [UCLA 2023-041-2] WO single dosage form will generally be that amount of the agent which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent. Methods of preparing these formulations or compositions include the step of bringing into association an active agent, such as an agent of the invention, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association an agent of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product. Formulations of the invention suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), lyophile, 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 agent of the present invention as an active ingredient. Compositions or agents may also be administered as a bolus, electuary or paste. To prepare solid dosage forms for oral administration (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, 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; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents, such as, modified and FH Docket No.:UCH-34425 UCLA Ref. No.: [UCLA 2023-041-2] WO unmodified cyclodextrins; and (11) coloring agents. In the case of capsules (including sprinkle capsules and gelatin 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-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like. 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 agent moistened with an inert liquid diluent. The tablets, and other solid dosage forms of the pharmaceutical compositions, such as dragees, capsules (including sprinkle capsules and gelatin 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 sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that 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 that 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. Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, 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, cyclodextrins and derivatives thereof, solubilizing agents and emulsifiers, such as ethyl FH Docket No.:UCH-34425 UCLA Ref. No.: [UCLA 2023-041-2] WO alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. Suspensions, in addition to the active agents, 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. Formulations of the pharmaceutical compositions for rectal, vaginal, or urethral administration may be presented as a suppository, which may be prepared by mixing one or more active agents 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 agent. Formulations of the pharmaceutical compositions for administration to the mouth may be presented as a mouthwash, or an oral spray, or an oral ointment. Alternatively or additionally, compositions can be formulated for delivery via a catheter, stent, wire, or other intraluminal device. Delivery via such devices may be especially useful for delivery to the bladder, urethra, ureter, rectum, or intestine. 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. Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active agentmay be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required. The ointments, pastes, creams and gels may contain, in addition to an active agent, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, FH Docket No.:UCH-34425 UCLA Ref. No.: [UCLA 2023-041-2] WO cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof. Powders and sprays can contain, in addition to an active agent, 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. 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, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. Pharmaceutical compositions suitable for parenteral administration comprise one or more active agents 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 antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention 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. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms 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 FH Docket No.:UCH-34425 UCLA Ref. No.: [UCLA 2023-041-2] WO compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin. 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 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. Injectable depot forms are made by forming microencapsulated matrices of the subject agents 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 that are compatible with body tissue. For use in the methods of this invention, active agents can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier. Methods of introduction may also be provided by rechargeable or biodegradable devices. Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinacious biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of an agent at a particular target site. Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of factors including the activity of the particular agent, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular agent being employed, the duration FH Docket No.:UCH-34425 UCLA Ref. No.: [UCLA 2023-041-2] WO of the treatment, other drugs, compounds and/or materials used in combination with the particular agent employed, the age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well known in the medical arts. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the pharmaceutical composition or agent 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. By “therapeutically effective amount” is meant the concentration of an agent that is sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the agent will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount may include, but are not limited to, the severity of the subject's condition, the disorder being treated, the stability of the agent, and, if desired, another type of therapeutic agent being administered with the agent of the invention. A larger total dose can be delivered by multiple administrations of the agent. Methods to determine efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison’s Principles of Internal Medicine 13 ed., 1814-1882, herein incorporated by reference). In general, a suitable daily dose of an active agent used in the compositions and methods of the invention will be that amount of the agent that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. If desired, the effective daily dose of the active agent may be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In certain embodiments of the present invention, the active agent may be administered two or three times daily. In preferred embodiments, the active agent will be administered once daily. In certain embodiments, agents of the invention may be used alone or conjointly administered with another type of therapeutic agent. As used herein, the phrase “conjoint administration” refers to any form of administration of two or more different therapeutic compounds such that the second compound is administered while the previously administered therapeutic compound is still effective in the body (e.g., the two compounds FH Docket No.:UCH-34425 UCLA Ref. No.: [UCLA 2023-041-2] WO are simultaneously effective in the subject, which may include synergistic effects of the two compounds). For example, the different therapeutic compounds can be administered either in the same formulation or in a separate formulation, either concomitantly or sequentially. In certain embodiments, the different therapeutic compounds can be administered within one hour, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, or a week of one another. Thus, a subject who receives such treatment can benefit from a combined effect of different therapeutic compounds. In certain embodiments, conjoint administration of agents of the invention with one or more additional therapeutic agent(s) (e.g., one or more additional therapeutic agent(s)) provides improved efficacy relative to each individual administration of the agent of the invention or the one or more additional therapeutic agent(s). In certain such embodiments, the conjoint administration provides an additive effect, wherein an additive effect refers to the sum of each of the effects of individual administration of the agent of the invention and the one or more additional therapeutic agent(s). This disclosure includes the use of pharmaceutically acceptable salts of compounds of the disclosure in the compositions and methods of the present disclosure. The term “pharmaceutically-acceptable salts” in this respect, refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or by separately reacting a purified compound of the invention in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed. 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, for example, 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 FH Docket No.:UCH-34425 UCLA Ref. No.: [UCLA 2023-041-2] WO acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2- acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like. In other cases, the compounds of the present invention may contain one or more acidic functional groups and, thus, are 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 compounds of the present invention. These salts can likewise be prepared in situ during the final isolation and purification of the compounds, 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, for example, Berge et al., supra). The pharmaceutically acceptable acid addition salts can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates can also be prepared. The source of such solvate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent. 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. 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 FH Docket No.:UCH-34425 UCLA Ref. No.: [UCLA 2023-041-2] WO acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. EXEMPLIFICATION The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention. Materials and Methods Animals VE-cadherin^cre (B6.FVB-Tg (Cdh5-cre)7Mlia/J), SM22α^cre (B6.129S6-Taglntm2 (cre)Yec/J) and Rosa^tdTomato (B6;129S6-Gt(ROSA)26Sortm9 (CAG-tdTomato)Hze/J) mice on C57BL/6J background were purchased from the Jackson Laboratory. Genotypes were confirmed by PCR, and experiments were performed with generation F4-F6. Littermates were used as wild type controls. All mice were fed a standard chow diet (Diet 8604, Harlan Teklad Laboratory). Tissue culture The tdTomatoVE-cadherin+ cells were isolated by FACS and cultured as previously described. Berbamine (Sigma-Aldrich, 547190), TGFβ1 (R&D system, 7666-MB) and BMP- 1 (R&D system, RDC2450) treatments were performed as described in the main text. Lentiviral vectors containing CMV-ALK5, CMV-FoxA2, MGP or FoxA2 siRNA were all purchased from GeneCopeia^TM and applied to the cells as per the manufacturer’s protocols. Vector construction The Flag-hMGP vector was constructed as previously described. Briefly, to construct the N-terminally FLAG-tagged human MGP (hMGP) vector, a fragment containing the coding region for hMGP was amplified by PCR. The FLAG tag was placed in the N terminus of the secreted, mature protein by subcloning a synthesized FLAG-coding DNA fragment between the coding regions for the signal peptide and the mature protein. The FLAG- FH Docket No.:UCH-34425 UCLA Ref. No.: [UCLA 2023-041-2] WO containing hMGP DNA fragment was amplified by PCR and subcloned into the NheI and XhoI sites of pcDNA3.1(+) (Invitrogen) using restriction sites in the primers. Pulmonary function test Mouse pulmonary function tests were performed as previously described. Mice were weighed and placed into a single chamber with a volume of 0.8 L where they were allowed to move freely and acclimate for at least 15 minutes. To provide a baseline reading, a room air-reading was taken as follows: compressed air was supplied to the chamber at a flow rate of 1 L/min for 45 minutes. At this point, the chamber was completely sealed, with air flow momentarily stopped. The changes in pressure caused by breathing was recorded and amplified by the software. Subsequently, the mice were allowed to rest for at least 5 minutes or until the breathing returned to baseline before conducting the hypercapnia phase. In the hypercapnia phase, a gas mixture containing 7% CO₂, 21% O₂ and balanced N₂ was supplied to the chamber at a flow of 1 L/min. After 5 minutes, the chamber was sealed and ventilatory patterns recorded. During the breathing room-air and hypercapnia phase, the average tidal volume (TV) and respiratory rate (RR) were measured for a period of at least 10 consecutive breaths. To avoid an excessive build-up in CO₂ concentration within the chamber due to re- breathing, a Bias Flow Regulator (Buxco Electronics, Inc.) was used. The Bias Flow Regulator provided a quiet, constant, and smooth flow through the animal chamber that prevented CO2 build-up. The following parameters were recorded: respiratory rate (RT), tidal volume (TV), peak inspiration flow (PIF), peak expiratory flow (PEF), and minute ventilation. The results were calculated and corrected for body weight. Average values were calculated one per minute for each serial 10 minutes. The machine was sanitized with alcohol between uses. RNA analysis Real-time PCR analysis was performed as previously described. Glyceraldehyde 3- phosphate dehydrogenase (GAPDH) was used as a control gene. Primers and probes for mouse MGP, Col3a1, Fibronectin 1, VE-cadherin, Flk1, ALK5, FoxA2 and von Willebrand factor (vWF) were obtained from Applied Biosystems as part of Taqman^® Gene Expression Assays. FH Docket No.:UCH-34425 UCLA Ref. No.: [UCLA 2023-041-2] WO Single cell RNA-sequencing (scRNA-seq) 10x Library Preparation, Sequencing, and Alignment: scRNA-seq libraries were generated with the Chromium Single Cell 3′ v3 assay (10x Genomics). The libraries were sequenced using Illumina NextSeq 550 platform with a depth of 378 million reads. Raw reads were aligned to the mouse genome (mm10). The cellranger (v3.0.0) mkfastq function was used to generate FASTQ files and cells were called using the cellranger count function. Cell Clustering and Cell Type Annotation: The R package Seurat version 4.0.1 was used for cell clustering and differential gene expression analysis. Cells were first filtered to have more than 500 detected genes and less than 10% of mitochondrial genes. Natural-log transformation was then applied to the gene counts and data were scaled to regress out total number of RNA counts and the percentage of mitochondrial reads. The FindVariableFeatures function was used to select variable genes with default parameters and principal component analysis (PCA) was performed using the RunPCA function on the variable genes. The first 25 PCs were used for cell clustering with a resolution of 0.5. Uniform manifold approximation and projection (UMAP) dimensional reduction was applied using the RunUMAP function. The FindAllMarkers function was used to identify marker genes for each cluster. Cell identities were defined by known marker genes. The FindMarkers function was performed for differential gene expression analysis between two cell types using Wilcoxon Rank Sum test with at least log-fold difference of 0.25 between the two groups of cells. Pseudotime Trajectory Construction: R package Monocle (v2.18.0) was used for pseudotime trajectory construction. Genes with average expression greater than 0.5 were retained for trajectory analysis in a subset of data. Variable genes were adopted from variable features identified by the Seurat FindVariableFeatures function, then were used as ordering genes. The trajectory was constructed by the reduceDimension function with default parameters. Differential expression in pseudotime was carried out by the differentialGeneTest function using likelihood ratio tests. CMAP and compound identification From each cell type in scRNA-seq analysis, top 100 marker genes were selected. Connectivity Map (CMAP) from the Broad Institute is a database with a collection of gene expression profiles that were obtained from nine human cell lines treated with various small FH Docket No.:UCH-34425 UCLA Ref. No.: [UCLA 2023-041-2] WO compounds. To identify a small molecule from 2429 compounds in the LINCS database (L1000), the next-generation CMap (CLUE) platform was used to directly explore the connectivity of gene signatures among perturbagens with Touchstone tool. The drug similarity was ranked according to the CMap connectivity score (from −100 to 100). Connectivity scores above 95 or below −95 were considered as strong scores to predict small candidate molecule corresponding to the query of selected gene expression signatures between different cell types. Fluorescence-activated cell sorting (FACS) FACS analysis was performed as previously described. The cells were stained with fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, or Alexa Fluor 488 (AF-488)- conjugated antibodies against CD34 and VE-cadherin (all from BD biosciences, 553731 and 562243). Nonspecific fluorochrome- and isotype-matched IgGs (BD Pharmingen) served as controls. Immunoblotting and immunofluorescence Immunoblotting was performed as previously described. Equal amounts of tissue lysates were used for immunoblotting. Blots were incubated with specific antibodies to BMP- 1 and LTBP-1 (Abcam, ab205394 and ab78294), Flag (Sigma-Aldrich, F3165), ALK5 (R&D system, MAB5871), Col3a1 and Fibronectin 1 (Novus Biologicals, NB600 and NBP1- 91258). β-Actin (Sigma-Aldrich, A2228) was used as a loading control. Immunofluorescence was performed as previously described in detail. Specific antibodies to CD34 (BD Bioscience, 553731), Nkx2.1 (Abcam, ab76013) and VE-cadherin (BD biosciences, 562243) were used. The nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, Sigma- Aldrich, D9564). Masson’s trichrome (MT) staining Sections were deparaffinized, rehydrated and stained with Trichrome Stain Kit (Abcam, ab150686) as per the manufacturer’s protocol. After being washed in distilled water, the sections were dehydrated and cleared in xylene, then mounted with resinous mounting medium. FH Docket No.:UCH-34425 UCLA Ref. No.: [UCLA 2023-041-2] WO Statistical analysis The analyses were performed using GraphPad Instat®, version 3.0 (GraphPad Software). Data was analyzed by either unpaired 2-tailed Student’s t test or one-way ANOVA with Tukey’s multiple-comparisons test for statistical significance. Results Specific MGP deletion causes pulmonary fibrosis Conventional gene deletion of Mgp has been shown to disrupt pulmonary cell differentiation. To perform the cell-specific deletion of Mgp, Mgp^flox/flox mice were generated, in which the genomic region of exon 2 to 4 of Mgp gene was floxed by two loxP sites (Figure 8a-b). Mgp^flox/flox mice with VE-cadherin^cre or Smooth muscle 22α^cre (SM22α^cre) mice were bred. At 10 weeks of age, Masson's trichrome staining showed striking pulmonary fibrosis in both VE-cadherin^creMgp^flox/flox mice and SM22α^creMgp^flox/flox mice (Figure 1a-b). At one year of age, both mice had severe pulmonary fibrosis (Figure 1a-b). The pulmonary function of the mice at one year of age using unrestricted whole body barometric plethysmography was examined. The results showed that the respiratory rates of VE-cadherin^creMgp^flox/flox mice and SM22α^creMgp^flox/flox mice were significantly higher than that of Mgp^flox/flox control mice during room air breathing and hypercapnia phase (Figure 1c). VE-cadherin^creMgp^flox/flox and SM22α^creMgp^flox/flox mice also displayed a significant decrease in tidal volume and lower peaks of expiratory and inspiratory flow (Figure 1c). Bleomycin-injected wild type mice and Mgp^flox/flox mice were used as controls. The results suggested that progressive pulmonary fibrosis occurred in both VE-cadherin^creMgp^flox/flox mice and SM22α^creMgp^flox/flox mice. Endogenous EC-like myofibroblasts in normal lungs Interestingly, when the expression of endothelial markers were examined, immunostaining and fluorescence-activated cell sorting (FACS) both showed a robustly increased CD34+ cell population in the lungs of VE-cadherin^creMgp^flox/flox mice and SM22α^creMgp^flox/flox mice (Figure 2a). Immunostaining also showed an increase of the CD34+ cell population in human pulmonary fibrosis (Figure 2b). FH Docket No.:UCH-34425 UCLA Ref. No.: [UCLA 2023-041-2] WO The lungs of wild type mice were isolated and FACS was used to select pulmonary CD34+ population, in which immune cells were excluded. The transcriptional profile of CD34+CD45- cells were examined at single-cell resolution and single-cell RNA sequencing (scRNA-seq) uncovered specific cell clusters in the CD34+CD45- population (Figure 3a). Differential gene expression divided these clusters into three major types of cells: 1) ECs that only expressed endothelial markers; 2) EC-like myofibroblasts that highly expressed endothelial and myofibroblast markers, MGP, and SM22α, Snai1, Snai2, Zeb1, Zeb2; 3) myofibroblasts that only expressed myofibroblast markers and Twist1 and 2 (Figure 3a-b). Cell differentiation trajectory projected a clear differential path from ECs to EC-like myofibroblasts ending with myofibroblasts (Figure 3c). Interestingly, none of the clusters expressed the typical smooth muscle cell marker smooth muscle myosin heavy chain (SMMHC), ruling out the involvement of smooth muscle cells (figure 3a). Wild-type mouse lungs and healthy human lungs were examined, and immunostaining showed the co- localisation of VE-cadherin and platelet-derived growth factor receptor (PDGFR)α around the vessels and small airways in the mouse lungs (Figure 3d). The co-localisation was also identified in the structures of alveolar units in healthy human lungs (Figure 3d). The results suggested that the endogenous EC-like myofibroblasts in normal lungs contribute to myofibroblasts. CD34+CD45− cells in the lungs of VE-cadherin^cre Mgp^flox/flox, SM22α^creMgp^flox/flox and bleomycin-injected wild-type mice were examined. FACS showed an increased number of CD34+CD45− cells in all three models (Figure 3e), suggesting a similar induction of CD34+CD45− cells in mouse models of pulmonary fibrosis. EC-like myofibroblasts contribute to human pulmonary fibrosis To determine the role of EC-like myofibroblasts in pulmonary fibrosis, publicly available scRNA-seq data of 10 healthy human lungs and 20 patients with pulmonary fibrosis were re-analyzed. The analysis identified the cluster of EC-like myofibroblasts that co-expressed endothelial and myofibroblast markers, MGP, and SM22α, in healthy lungs and pulmonary fibrosis (Figure 4a-b). Cell differential trajectory showed that EC-like myofibroblasts were potentially derived from ECs and differentiated into myofibroblasts (Figure 4a-c). Strikingly, differential expression revealed lower expression of endothelial markers, VE-cadherin and von Willebrand factor (vWF) and higher expression of FH Docket No.:UCH-34425 UCLA Ref. No.: [UCLA 2023-041-2] WO myofibroblast markers, a-smooth muscle actin, Col3a1 and fibronectin 1 (Fn1) in EC-like myofibroblasts of pulmonary fibrosis samples compared to those of healthy control samples (Figure 4b). A decrease of EC differentiation together with an increase of EC-like myofibroblasts and myofibroblasts was also detected in pulmonary fibrosis (Figure 4d). The results suggested that EC-like myofibroblasts contributed myofibroblasts to human pulmonary fibrosis. Severe pulmonary fibrosis after COVID-19 infection has become a critical issue for late-stage and long COVID-19 patients. In the analysis of the publicly available scRNA-seq data, it was observed that patients with severe pulmonary fibrosis after COVID-19 infection showed a robust elevation of EC-like myofibroblasts in these cases, and identified COVID-19 infection as a force that drives ECs to EC-like myofibroblasts and myofibroblasts. To determine if other viral infection affected pulmonary endothelial lineage cells, another set of publicly available scRNA-seq data obtained from mouse lungs with influenza A viral infection were analyzed, and similar differentiation trajectories of ECs and EC-like myofibroblasts were found in mouse lungs after influenza A virus infection. The results suggest that the contribution of ECs and EC-like myofibroblasts to fibrosis after a variety of viral infections may have similar mechanisms (Data not shown). MGP inhibits BMP-1 to regulate TGFβ1 maturation for controlling EC-like myofibroblasts toward myofibroblast differentiation To explore the differentiation of EC-like myofibroblasts in lungs, SM22α^creRosa^tdTomato mice, in which SM22α promoter-driven Cre activated tdTomato expression were generated. Using these mice, the tdTomato-labeled cells that reflected the SM22α expression were traced. At 10 weeks of age, the lungs of SM22α^creRosa^tdTomato mice were examined where immunostaining showed that tdTomato colocalized with VE- cadherin in the lung tissue (Figure 5a). FACS also revealed a pulmonary cell population that co-expressed tdTomato and the endothelial marker VE-cadherin (Figure 5b), confirming that endogenous EC-like myofibroblasts were present in normal lung tissues. tdTomato and VE-cadherin double positive cells (tdTomato+VE-cadherin+) from the lungs of SM22α^creRosa^tdTomato mice and depleted MGP in these cells using specific siRNA were isolated (Figure 5b). The cells were then treated with TGFβ1 and the myofibroblast markers Col3a1 and Fibronectin, and endothelial markers VE-cadherin and vWF were examined. Real-time PCR showed the induction of myofibroblast markers with the FH Docket No.:UCH-34425 UCLA Ref. No.: [UCLA 2023-041-2] WO reduction of endothelial markers in MGP-depleted or TGFβ1-treated tdTomato+VE- cadherin+ cells. The combination of MGP-depletion and excess TGFβ1 significantly magnified the alterations in the expression of these markers (Figure 5b). The results suggested that lack of MGP or excess TGFβ1 promoted the differentiation of EC-like myofibroblasts toward myofibroblasts. tdTomato+VE-cadherin+ cells were isolated from the lungs of SM22α^creRosa^tdTomato mice and the cells were treated with BMP-1 in company with the over-expression of Flag- tagged MGP. Co-immunoprecipitation followed by immunoblotting uncovered a direct binding between BMP-1 and MGP (Figure 5c). Chemical crosslinking of BMP-1 and Flag- tagged MGP resulted in the formation of a complex detected by immunoblotting with both anti-Flag and anti-BMP-1 antibodies (Figure 5d), strongly supporting a direct interaction between BMP-1 and MGP. BMP-1 was used to treat tdTomato+VE-cadherin+ cells with or without MGP deletion. The results showed that excess BMP-1 induced Col3a1 and Fibronectin. With MGP depletion, BMP-1 treatment further elevated the induction of Col3a1 and Fibronectin (Figure 5e), suggesting that MGP interfered with BMP-1 and limited its ability to induce the differentiation of EC-like myofibroblasts. BMP-1 has been shown to cleave latent TGFβ1 binding protein-1 (LTBP-1) for TGFβ1 maturation. In this study, BMP-1 was used to treat tdTomato+VE-cadherin+ cells with or without the over-expression of MGP and LTBP-1 in the cell matrix was examined. Immunoblotting showed that excess BMP-1 efficiently cleaved LTBP-1, and the over- expression of MGP inhibited the BMP-1 cleavage of LTBP-1 (Figure 5f). The levels of TGFβ1 in the media were further examined. Enzyme-linked immunosorbent assay (ELISA) showed that BMP-1 increased TGFβ1 level and the over-expression of MGP decreased the TGFβ1 level even with BMP-1 treatment (Figure 5g). The expression of BMP-1 and TGFβ1 in the lung tissues of VE- cadherin^creMgp^flox/flox and SM22α^creMgp^flox/flox mice was examined. Immunoblotting showed higher levels of TGFβ1 in VE-cadherin^creMgp^flox/flox and SM22α^creMgp^flox/flox mice than in Mgp^flox/flox control mice (Figure 5i). Increased phosphorylation of SMAD2/3 confirmed the induction of TGFβ1 signalling (Figure 5i). No change in BMP-1 expression was detected, supporting that the interaction between MGP and BMP-1 inhibited the BMP-1 activity. To determine whether inhibition of BMP-1 affected the pulmonary fibrosis in the new mouse FH Docket No.:UCH-34425 UCLA Ref. No.: [UCLA 2023-041-2] WO models, VE-cadherin^creMgp^flox/flox and SM22α^creMgp^flox/flox mice were treated with the BMP- 1 inhibitor UK383367 (5 µg·g^-1, daily) for 2 weeks. Masson's trichrome staining showed a reduction of pulmonary fibrosis in both VE-cadherin^creMgp^flox/flox and SM22α^creMgp^flox/flox mice after the treatment with UK383367 (Figure 5j). Together, the results suggested that MGP interacted with BMP-1, inhibited the production of mature TGFβ1 and in turn regulated the differentiation of EC-like myofibroblasts towards myofibroblasts (Figure 5h). Berbamine prevents the shift of EC-like myofibroblasts toward myofibroblasts and ameliorates pulmonary fibrosis in mouse models In these studies, the scRNA-seq revealed a clear trajectory of cell differentiation from ECs towards EC-like myofibroblasts and myofibroblasts. By analyzing the differential expression between myofibroblasts and ECs, 537 genes with increased expression and 668 genes with decreased expression using adjusted p value less than 0.01 cutoff were identified (Figure 6a). These alterations in gene expression detailed the genetic signature of the shift from ECs to myofibroblasts. To identify a compound that prevented this cell shift, the differential expression profile was loaded into the Connectivity Map (CMap) platform. CMap platform is generated to connect genetic variants with small molecule treatments. Using a connectivity score from designed measurements of expression profiles, a query of CMap can be made to search compounds that cause similar genetic perturbations. With this advantage, CMap platform provides a possible approach to identify compounds that modify the transcriptional landscape towards desired differentiation direction. To find a compound to reverse the genetic alteration from ECs to myofibroblasts, the top 100 differential expression profiles with an opposite direction of alteration was loaded to generate a novel query, which allowed the platform to find the compound with the capability to reverse the genetic alteration. CMap identified a small molecule, berbamine, as a potential candidate. To determine if berbamine affected the differentiation of EC-like myofibroblasts, tdTomato+VE-cadherin+ cells from the lungs of SM22α^creRosa^tdTomato mice were isolated. The cells were treated with 20 µM berbamine for 24 hours and a reduction of myofibroblast markers and an increase of endothelial markers were found (Figure 6b). MGP in the cells were also depleted and the cells were treated with berbamine. Real-time PCR showed that FH Docket No.:UCH-34425 UCLA Ref. No.: [UCLA 2023-041-2] WO berbamine prevented the induction of myofibroblast markers and restored the expression of endothelial markers in MGP-depleted tdTomato+VE-cadherin+ cells (Figure 6b). The results suggested that berbamine redirected EC-like myofibroblasts back to EC differentiation. VE-cadherin^creMgp^flox/flox mice and SM22α^creMgp^flox/flox mice at 10 weeks of age were treated with berbamine (100 ng/g, daily) for 4 weeks. Masson’s trichrome staining showed that berbamine significantly decreased the pulmonary fibrosis of both mice (Figure 6c-d). Real-time PCR of lung tissues also showed that the expression of Col3a1 and Fibronectin was much lower in berbamine-treated group than in saline-treated controls (Figure 6d). The results suggested that berbamine reduced pulmonary fibrosis. To determine if berbamine affects TGFβ signaling, tdTomato+VE-cadherin+ cells were isolated from the lungs of SM22α^creRosa^tdTomato mice and the cells were treated with berbamine (20 µM). Real-time PCR and immunoblotting both showed that berbamine reduced the expression of the TGFβ1 receptor activin receptor-like kinase 5 (ALK5) (Figure 7a). Lentiviral vectors containing cytomegalovirus (CMV) promoter-driven ALK5 were used to infect the cells and the cells were treated with berbamine. Real-time PCR showed that excess ALK5 restored the induction of Col3a1 and Fibronectin in MGP-depleted tdTomato+VE-cadherin+ cells (Figure 7b), suggesting that berbamine-reduced ALK5 prevented the differentiation of EC-like myofibroblasts to myofibroblasts. Interestingly, excess ALK5 did not affect berbamine-induced endothelial markers (Figure 7b), suggesting that berbamine targets an additional factor to drive EC-like myofibroblasts towards EC differentiation. Berbamine has previously been identified as a calmodulin 1 inhibitor, a calcium channel blocker. Calmodulin 1 in tdTomato+VE-cadherin+ cells was depleted and no change in the expression of endothelial or myofibroblast markers was found (Data not shown), suggesting that inhibition of calmodulin 1 was not involved in the redirection of myofibroblasts. However, berbamine was found to significantly induced FoxA2 expression (Figure 7c) and overexpression of FoxA2 alone reduced the expression of ALK5 and the myofibroblast markers (Figure 7d). The combination of berbamine and FoxA2 overexpression further reduced ALK5 and the myofibroblast markers (Figure 7d). FH Docket No.:UCH-34425 UCLA Ref. No.: [UCLA 2023-041-2] WO FoxA2 in tdTomato+VE-cadherin+ cells was overexpressed or depleted and the cells were treated with berbamine. The results showed that the overexpression of FoxA2 alone induced the endothelial markers (Figure 7e). FoxA2 overexpression with berbamine treatment enhanced the induction of endothelial markers whereas FoxA2 depletion abolished the induction of endothelial markers in tdTomatoVE-cadherin+ cells (Figure 7e). The expression of ALK5 and FoxA2 were examined in the lungs of VE-cadherin^creMgp^flox/flox mice and SM22α^creMgp^flox/flox mice after berbamine treatment. Immunoblotting showed that berbamine increased FoxA2 expression and reduced ALK5 expression in the lungs of these mice (Figure 7f). Wild-type mice were then treated with berbamine (100 ng·g⁻¹, daily) for 4 weeks after bleomycin administration. Berbamine significantly decreased the numbers of both PDGFRα+ cells and PDGFRα+VE-cadherin+ cells in the lungs of bleomycin-treated mice. Real-time PCR showed a reduction of PDGFRα, Col1a1, Col3a1 and Fn1 (Figure 7g ). In addition, the results showed that berbamine induced FoxA2, but had no effect on bleomycin-reduced MGP expression (Figure 7g). To determine whether ALK5 and FoxA2 were expressed in human pulmonary fibrosis, immunostaining that showed strong expression of ALK5 and less of FoxA2 was performed (Figure 7h). These results suggested that berbamine induced FoxA2 to suppress ALK5 expression, in turn redirecting EC-like myofibroblasts towards EC differentiation. Cell lineage tracings reveal EC-like myofibroblasts contributing to pulmonary fibrosis To further determine if endogenous ECs and EC-like myofibroblasts contributed to pulmonary fibrosis, cell lineage tracings in the mouse model of bleomycin-induced pulmonary fibrosis were performed. VE-cadherin^cre/ERT2Rosa^tdTomato mice were used, where founder cells expressing VE-cadherin could be traced by detecting ^tdTomato after tamoxifen injections. SM22α^creRosa^tdTomato mice were also used, in which the ^tdTomato- labelled cells that reflected the SM22α expression could be traced. At 6 weeks of age, VE-cadherin^cre/ERT2Rosa^tdTomato mice received tamoxifen injections (75µg·g⁻¹ daily) for five consecutive days to induce ^tdTomato expression. At 8 weeks of age, tamoxifen-treated VE-cadherin^cre/ERT2Rosa^tdTomato mice andSM22α^creRosa^tdTomato mice received bleomycin injections to induce pulmonary fibrosis. Saline-treated mice were used as controls. At 11 weeks of age, the lung tissues were examined. FH Docket No.:UCH-34425 UCLA Ref. No.: [UCLA 2023-041-2] WO Masson's trichrome staining showed pulmonary fibrosis in both tamoxifen-treated VE-cadherin^cre/ERT2Rosa^tdTomato mice and SM22α^creRosa^tdTomato mice after bleomycin injections (Figure 9a, Figure 9b), confirming the induction of pulmonary fibrosis. In tamoxifen-treatedVE-cadherin^cre/ERT2Rosa^tdTomato mice, immunostaining showed more ^tdTomato-positive cells expressing higher levels of PDGFRα and Fn1 in the bleomycin treated group than the controls, suggesting that ECs and EC-like myofibroblasts that expressed VE- cadherin contributed to bleomycin-induced pulmonary fibrosis (Figure 9a). In SM22α^creRosa^tdTomato mice, more tomato-positive cells were identified to express VE-cadherin and a higher level of Fn1 in the bleomycin-treated group than the controls, suggesting again that EC-like myofibroblasts contributed to the bleomycin- induced pulmonary fibrosis (Figure 9b). SM22α^creRosa^tdTomatoMgp^flox/flox mice were bred and examined. At 11 weeks of age, more tomato-positive cells were found that expressed VE-cadherin and a higher level of Fn1 than that in SM22α^creRosa^tdTomato mice (Figure 9c). ^tdTomato and VE-cadherin double positive cells were isolated from the lungs. Real- time PCR showed higher expression of Col3a1 and Fn1 in the cells isolated from SM22α^creRosa^tdTomatoMgp^flox/flox mice than from SM22α^creRosa^tdTomato mice (Figure 9c). Together, multiple cell lineage tracings in the mouse models revealed that EC-like myofibroblasts contributed to pulmonary fibrosis. Discussion Pulmonary fibrosis is a severe fibrotic lung disease causing high morbidity and mortality worldwide. Although the precise mechanism has not been determined, pulmonary fibrosis is characterized by progressive interstitial fibrogenesis that causes distortion of alveolar architecture leading to the loss of pulmonary function. Recent studies have shown ill-fated myofibroblasts emerging in pulmonary fibrosis. These unwanted myofibroblasts are strongly activated during interstitial fibrogenesis, in which they produce and deposit excessive amounts of extracellular fibrotic matrix in the tissues. In this study, it was found that cell-specific deletions of Mgp cause pulmonary fibrosis. MGP was shown to regulate the differentiation of a previously unknown population of EC-like myofibroblasts that significantly contribute to the differentiation of myofibroblasts in pulmonary fibrosis. A FH Docket No.:UCH-34425 UCLA Ref. No.: [UCLA 2023-041-2] WO small molecule which can redirect the differentiation of the EC-like myofibroblasts and reduce pulmonary fibrosis was also identified. Pulmonary ECs are essential in lung tissue. Endothelial defects result in poor induction of pulmonary lineages from progenitor cells and impaired epithelial repair after lung injury. Pulmonary ECs both support pulmonary epithelial cells and guide their differentiation and maturation. Previous studies have reported co-expression of endothelial and mesenchymal markers in pulmonary disease, suggesting that ECs transition into other lineages under disease conditions. In this study, a novel differentiation trajectory in normal lung tissue, where pulmonary ECs may differentiate into EC-like myofibroblasts and ultimately myofibroblasts was uncovered. It was shown that the dysregulation of this differentiation trajectory dramatically increases the production of myofibroblasts that when activated, contribute to pulmonary fibrosis. MGP, a BMP inhibitor, is essential for regulating vascular BMP activity. Loss of MGP causes arteriovenous malformation in cerebrum, lungs, kidneys, and retina in mice, which resemble the mouse model for hereditary hemorrhagic telangiectasia type 2. Mutations in the human Mgp gene cause an extremely rare disease, Keutel syndrome, that involves severe peripheral cardiovascular defects and pulmonary stenoses. However, full evaluations for pulmonary fibrosis have not been reported for these rare patients. In addition, severe side effects of warfarin treatment in pulmonary fibrosis patients suggest interference by MGP in pulmonary fibrosis. Warfarin inhibits the vitamin K–dependent γ-carboxylation, which is essential for the function of MGP. Warfarin treatment prevents the modification of Glu to Gla residues in MGP, resulting in impaired BMP binding. Warfarin treatment was reported to rapidly worsen the progression of pulmonary fibrosis and a ban of warfarin on the pulmonary fibrosis patients has been suggested. In this study, it was found that the Mgp deletion in VE-cadherin or SM22α positive cells causes pulmonary fibrosis in mice, suggesting an important role of MGP in the fibrotic process. These mice also provide new animal models for the study of pulmonary fibrosis. BMP-1 was initially classified as a BMP based on bone induction. However, BMP-1 encodes a protein with few similarities to other BMPs, and BMP-1 is not included in TGFβ superfamily. Unlike other BMPs, BMP-1 is a metalloprotease and executes its activity by modifying the protein precursors to mature proteins. For example, BMP-1 cleaves the BMP FH Docket No.:UCH-34425 UCLA Ref. No.: [UCLA 2023-041-2] WO antagonist Chordin to regulate BMP activity. BMP-1 also processes extracellular matrix proteins such as collagens, biglycan, and osteoglycin. Interestingly, fibroblasts strongly increase the deposition of collagens onto the insoluble extracellular matrix when incubated with BMP-1. In addition, BMP-1 cleaves latent TGFβ1 binding protein-1 (LTBP-1) to facilitate the maturation of TGFβ1, a master regulator of myofibroblast differentiation in pulmonary fibrosis. In this study, it was found that MGP binds to BMP-1 and reduces the production of mature TGFβ1, thereby regulating the differentiation of EC-like myofibroblasts to myofibroblasts. An interaction between MGP and BMP-1 was found. MGP has been previously shown to bind to BMP-2, 4 and 7 through Proline-64 and surrounding Gla residues. MGP has also been shown to interact with several other protein, such as fibronection, vitronectin, through its c-terminals and elastin with its N-terminals. Further studies would be necessary to identify what region of MGP interacts with BMP-1. Berbamine is a small molecule extracted from the plant named Berberis. Berbamine was initially identified as a calcium channel blocker with anti-arrhythmic effects and ischemic protective activity through the inhibition of calmodulin 1. Recent studies also found that berbamine inhibits the nuclear factor-kappa B (NF-kB) signaling pathway for anti- myeloma and reduces the activity of signal transducer and activator of transcription 3 (STAT3) in hepatocellular carcinoma. 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No.: [UCLA 2023-041-2] WO apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

FH Docket No.:UCH-34425 UCLA Ref. No.: [UCLA 2023-041-2] WO CLAIMS We claim: 1. A method for treating or preventing a fibrotic disease in a subject in need thereof, comprising administering to the subject a compound that suppresses expression of ALK5, or a pharmaceutical acceptable salt thereof. 2. A method for treating or preventing pulmonary fibrosis in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising a compound that reduces the expression of ALK5, or a pharmaceutical acceptable salt thereof. 3. The method of claim 1 or claim 2, whereby transcription factor FoxA2 is induced. 4. The method of any one of the preceding claims, whereby EC-like myofibroblasts are induced to differentiate toward myofibroblasts. 5. The method of any one of claims 1-4, wherein the compound is berbamine, or a pharmaceutically acceptable salt thereof. 6. The method of any one of claims 1-5, wherein the differentiation further comprises a reduction in expression of myofibroblast markers. 7. The method of any one of claims 1-5, wherein the differentiation further comprises an increased in expression of endothelial markers. 8. The method of any one of the preceding claims, wherein the subject is human.