Patent Publication Number: US-2022233573-A1

Title: Methods and compositions for treating pulmonary arterial hypternsion

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     The present application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62/874,322 filed Jul. 15, 2019, which is incorporated herein by reference in their entirety. 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
     This invention was made with government support under HL135582 awarded by National Institutes of Health. The government has certain rights in the invention. 
    
    
     BACKGROUND OF THE INVENTION 
     Pulmonary arterial hypertension (PAH) is a significant health problem. Current methods of treatment and prevention are inadequate. There is a need in the art for methods of treating PAH. This disclosure addresses that need. 
     SUMMARY OF THE INVENTION 
     In one aspect, the invention provides a method of treating pulmonary arterial hypertension (PAH) in a subject, the method comprising administering to the subject an agent that modulates the activity or level of let-7 miRNA in an endothelial cell in the subject, thereby treating PAH in the subject. 
     In another aspect, the invention provide a method of treating pulmonary arterial hypertension (PAH) in a subject, the method comprising administering to the subject an agent that decreases, in an endothelial cell in the subject, the activity or level of a endothelial TGFβ signaling polypeptide or TGFβ peptide receptor selected from the group consisting of TGFβ1, TGFβ2, TGF033, TGFβR1, and TGFβR2, thereby treating PAH in the subject. 
     In yet another aspect, the invention provides a method of treating pulmonary arterial hypertension (PAH) in a subject, the method comprising administering to the subject an agent that decreases, in an endothelial cell in the subject, the activity or level of FRS2α, thereby treating PAH in the subject. 
     In certain embodiments, the agent is selectively delivered to an endothelial cell in the subject. In certain embodiments, the agent is in a nanoparticle. In certain embodiments, the nanoparticle is a 7C1 nanoparticle. 
     In certain embodiments, the agent is selectively delivered to a smooth muscle cell in the subject. 
     In certain embodiments, the agent is administered intravenously. 
     In certain embodiments, the agent that increases the activity or level of let-7 miRNA is selected from the group consisting of human let-7b miRNA and human let-7c miRNA. 
     In certain embodiments, the agent that modulates the activity or level of let-7 miRNA is a pharmaceutical composition comprising an effective amount of a let-7 miRNA in a nanoparticle formulated for selective delivery to an endothelial cell, in a pharmaceutically acceptable excipient. 
     In certain embodiments, the let-7 miRNA comprises a chemical modification that increases stability of the miRNA and/or reduces an immune response to the miRNA in a subject. In certain embodiments, the chemical modification is a 2′-O-methyl modification. 
     In certain embodiments, the let-7 miRNA is selected from the group consisting of human let-7b miRNA and human let-7c miRNA. 
     In certain embodiments, the agent that decreases the activity or level of a TGFβ signaling polypeptide is an inhibitory polynucleotide that reduces expression of the TGFβ signaling polypeptide. 
     In certain embodiments, the agent that decreases the activity or level of FRS2α is an inhibitory polynucleotide that reduces expression of a FRS2α polypeptide. 
     In certain embodiments, the decrease in the activity or level of the FRS2α polypeptide promotes smooth muscle cell proliferation. 
     In certain embodiments, the method further comprising providing to the subject a second therapeutic agent comprising an mTOR inhibitor. In certain embodiments, the mTOR inhibitor is rapamycin. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIGS. 1A-1B  show a time course of development of spontaneous PAH in mice after endothelial-specific deletion of MEKK3.  FIG. 1A : Representative right ventricular systolic pressure (RVSP) tracing in control and MEKK3 iEC−/− mice 6 weeks after MEKK3 deletion.  FIG. 1B : Time course of RVSP increase. 
         FIG. 2  depicts a right ventricular (RV) hypertrophy in MEKK3 ECKO mice. RV-right ventricular thickness; LV: left ventricular free wall thickness. S-interventricular septum thickness 
         FIGS. 3A and 3B  show morphologic evidence of PAH in MEKK3 ECKO mice.  FIG. 3A : total lung fields section stained with anti-SMA antibody. Note increased SMA staining in peripheral lung fields indicating hypertrophy of small pulmonary arteries;  FIG. 3B : vibratome section. 
         FIGS. 4A and 4B  depict fate-mapping of cells giving origin to PAH in MEKK3 ECKO mice. Mice carrying Cdh5-Cre (endothelial-specific Cre) were crossed with mTmG reporter mice and MEKK3 fl/fl mice. This fate-maps all endothelial cells as green. Following MEKK3 deletion these former EC are expressing smooth muscle markers showing that EC-to-SMC fate change drives PAH. 
         FIG. 5  depicts RNA sequencing of human umbilical vein endothelial cells (HUVEC) after MEKK3 knockdown relative to control. 
         FIGS. 6A-6B  show that MEKK3 knockdown induces EndMT in vitro.  FIG. 6A : RNA-seq analysis of gene expression showing increased EndMT;  FIG. 6B : Western blot analysis. 
         FIG. 7  depicts increased EndMT after MEKK3 knockdown. 
         FIG. 8  depicts increased EndMT in vivo in MEKK3 ECKO mice: note increased TGFbR2 expression in pulmonary ECs. 
         FIG. 9  depicts increased TGFb and TGFbR genes expression after MEKK3 KD in ECs. 
         FIGS. 10A-10D  depict EndMT after MEKK3 KD. 
         FIGS. 11A-11B  TGFbR1/R2 knockdown suppresses MEKK3 KD-induced EndMT. 
         FIGS. 12A-12D  depict nanoparticle (7C1)-delivered siRNA to TGFbR1 and TGFbR2 prevents development of PAH.  FIG. 12A : Time course and experiment design.  FIG. 12B : RVSP tracings 3 weeks after MEKK3 KO induction along with NP-based TGFbR1/R2 treatment.  FIG. 12C : Quantification of RVSP.  FIG. 12D : Reduction in RV hypertrophy 
         FIG. 13 : Reduced EndMT in the pulmonary vasculature of MEKK3 ECKO mice after TGFbR1/R2 RNAi treatment 
         FIG. 14  is an image showing lungs treated with siTGFβR and control, stained for SMA. 
         FIG. 15  depicts proposed mechanism of action  FIG. 16 : MEKK3 KO reduces endothelial let-7 levels. 
         FIGS. 17A and 17B  depict EC-specific TGFbR2 KO prevents PAH development in MEKK3 ECKO mice. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Definitions 
     Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used. 
     It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. 
     By “agent” is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof. In some embodiments, the agent is a nucleic acid molecule. 
     By “alteration” is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. In some embodiments, an alteration in expression level includes a 10% change in expression levels, a 25% change, a 40% change, and a 50% or greater change in expression levels. 
     “Biological sample” as used herein means a biological material isolated from a subject, including any tissue, cell, fluid, or other material obtained or derived from the subject. In some embodiments, the subject is human. The biological sample may contain any biological material suitable for detecting the desired analytes, and may comprise cellular and/or non-cellular material obtained from the subject. In certain embodiments, the biological sample is an endothelial cell. Biological samples include tissue samples (e.g., cell samples, biopsy samples), such as tissue from the heart or aorta. Biological samples also include bodily fluids, including, but not limited to, blood, blood serum, plasma, saliva, and urine. 
     By “capture reagent” is meant a reagent that specifically binds a nucleic acid molecule or polypeptide to select or isolate the nucleic acid molecule or polypeptide. In some embodiments, the capture reagent is a probe or primer that specifically binds a polynucleotide encoding a TGFβ signaling polypeptide, a let-7 miRNA, or a FGF signaling polypeptide. 
     In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “includes,” “including,” and the like; “consisting essentially of” or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments. 
     “Detect” refers to identifying the presence, absence or amount of the analyte to be detected. In some embodiments, a level of a let-7 miRNA, a TGFβ signaling polypeptide or polynucleotide, or a FGF signaling polypeptide or polynucleotide is detected. 
     By “disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include atherosclerosis, pulmonary hypertension, and chronic inflammation induced fibrosis. 
     By “effective amount” is meant the amount of a required to ameliorate the symptoms of a disease relative to an untreated patient. In particular embodiments, the disease is PAH. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an “effective” amount. 
     As used herein, a “FGF signaling polypeptide” is meant a member or component of a fibroblast growth factor (FGF) signaling pathway. In some embodiments, the FGF signaling polypeptide is FGFR1 polypeptide or FRS2α polypeptide. 
     By “FGFR1 polypeptide” is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to GenBank Accession No. AAH15035.1 and having a biological activity of a FGFR1 polypeptide. Biological activities of a FGFR1 polypeptide include cell surface receptor activity and tyrosine kinase activity. The sequence at GenBank Accession No. AAH15035.1 is shown below (SEQ ID No: 3): 
     
       
         
           
               
               
            
               
                 1 
                 mwswkcllfw avlvtatlct arpsptlpeq aqpwgapvev 
               
               
                   
               
               
                   
                 esflvhpgdl lqlrcrlrdd 
               
               
                   
               
               
                 61 
                 vqsinwlrdg vglaesnrtr itgeevevqd svpadsglya 
               
               
                   
               
               
                   
                 cvtsspsgsd ttyfsvnvsd 
               
               
                   
               
               
                 121 
                 alpssedddd dddssseeke tdntkpnrmp vapywtspek 
               
               
                   
               
               
                   
                 mekklhavpa aktvkfkcps 
               
               
                   
               
               
                 181 
                 sgtpnptlrw lkngkefkpd hriggykvry atwsiimdsv 
               
               
                   
               
               
                   
                 vpsdkgnytc iveneygsin 
               
               
                   
               
               
                 241 
                 htyqldvver sphrpilqag 1panktvalg snvefmckvy 
               
               
                   
               
               
                   
                 sdpqphiqwl khievngski 
               
               
                   
               
               
                 301 
                 gpdnlpyvqi lktagvnttd kemevlhlrn vsfedageyt 
               
               
                   
               
               
                   
                 clagnsigls hhsawltvle 
               
               
                   
               
               
                 361 
                 aleerpavmt splyleiiiy ctgafliscm vgsvivykmk 
               
               
                   
               
               
                   
                 sgtkksdfhs qmavhklaks 
               
               
                   
               
               
                 421 
                 iplrrqvsad ssasmnsgvl lvrpsrlsss gtpmlagvse 
               
               
                   
               
               
                   
                 yelpedprwe 1prdrlvlgk 
               
               
                   
               
               
                 481 
                 plgegcfgqv vlaeaigldk dkpnrvtkva vkmlksdate 
               
               
                   
               
               
                   
                 kdlsdlisem emmkmigkhk 
               
               
                   
               
               
                 541 
                 niinllgact qdgplyvive yaskgnlrey lqarrppgle 
               
               
                   
               
               
                   
                 ycynpshnpe eqlsskdlvs 
               
               
                   
               
               
                 601 
                 cayqvargme ylaskkcihr dlaarnvlvt ednvmkiadf 
               
               
                   
               
               
                   
                 glardihhid yykkttngrl 
               
               
                   
               
               
                 661 
                 pvkwmapeal fdriythqsd vwsfgvllwe iftlggspyp 
               
               
                   
               
               
                   
                 gvpveelfkl lkeghrmdkp 
               
               
                   
               
               
                 721 
                 snctnelymm mrdcwhavps qrptfkqlve dldrivalts 
               
               
                   
               
               
                   
                 nqeyldlsmp ldqyspsfpd 
               
               
                   
               
               
                 781 
                 trsstcssge dsvfsheplp eepclprhpa qlangglkrr 
               
            
           
         
       
     
     By “FGFR1 polynucleotide” is meant a polynucleotide encoding a FGFR1 polypeptide. An exemplary FGFR1 polynucleotide sequence is provided at GenBank Accession No. BC015035.1. The exemplary sequence provided at GenBank Accession No. BC015035.1 is reproduced below (SEQ ID No: 4). 
     
       
         
           
               
               
            
               
                 1 
                 agcgctcttg cggccacagg cgcggcgtcc tcggcggcgg 
               
               
                   
               
               
                   
                 gcggcagcta gcgggagccg 
               
               
                   
               
               
                 61 
                 ggacgccggt gcagccgcag cgcgcggagg aacccgggtg 
               
               
                   
               
               
                   
                 tgccgggagc tgggcggcca 
               
               
                   
               
               
                 121 
                 cgtccggacg ggaccgagac ccctcgtagc gcattgcggc 
               
               
                   
               
               
                   
                 gacctcgcct tccccggccg 
               
               
                   
               
               
                 181 
                 cgagcgcgcc gctgcttgaa aagccgcgga acccaaggac 
               
               
                   
               
               
                   
                 ttttctccgg tccgagctcg 
               
               
                   
               
               
                 241 
                 gggcgccccg cagggcgcac ggtacccgtg ctgcagtcgg 
               
               
                   
               
               
                   
                 gcacgccgcg gcgccggggc 
               
               
                   
               
               
                 301 
                 ctccgcaggg cgatggagcc cggtctgcaa ggaaagtgag 
               
               
                   
               
               
                   
                 gcgccgccgc tgcgttctgg 
               
               
                   
               
               
                 361 
                 aggagggggg caccagctcc ggctccattg ttcccgcccg 
               
               
                   
               
               
                   
                 ggctggaggc gccgagcacc 
               
               
                   
               
               
                 421 
                 gagcgccgcc gggagtcgag cgccggccgc ggagctcttg 
               
               
                   
               
               
                   
                 cgaccccgcc aggacccgaa 
               
               
                   
               
               
                 481 
                 cagagcccgg gggcggcggg ccggagccgg ggacgcgggc 
               
               
                   
               
               
                   
                 acacgcccgc tcgcacaagc 
               
               
                   
               
               
                 541 
                 cacggcggac tctcccgagg cggaacctcc acgccgagcg 
               
               
                   
               
               
                   
                 agggtcagtt tgaaaaggag 
               
               
                   
               
               
                 601 
                 gatcgagctc actgtggagt atccatggag atgtggagcc 
               
               
                   
               
               
                   
                 ttgtcaccaa cctctaactg 
               
               
                   
               
               
                 661 
                 cagaactggg atgtggagct ggaagtgcct cctcttctgg 
               
               
                   
               
               
                   
                 gctgtgctgg tcacagccac 
               
               
                   
               
               
                 721 
                 actctgcacc gctaggccgt ccccgacctt gcctgaacaa 
               
               
                   
               
               
                   
                 gcccagccct ggggagcccc 
               
               
                   
               
               
                 781 
                 tgtggaagtg gagtccttcc tggtccaccc cggtgacctg 
               
               
                   
               
               
                   
                 ctgcagcttc gctgtcggct 
               
               
                   
               
               
                 841 
                 gcgggacgat gtgcagagca tcaactggct gcgggacggg 
               
               
                   
               
               
                   
                 gtgcagctgg cggaaagcaa 
               
               
                   
               
               
                 901 
                 ccgcacccgc atcacagggg aggaggtgga ggtgcaggac 
               
               
                   
               
               
                   
                 tccgtgcccg cagactccgg 
               
               
                   
               
               
                 961 
                 cctctatgct tgcgtaacca gcagcccctc gggcagtgac 
               
               
                   
               
               
                   
                 accacctact tctccgtcaa 
               
               
                   
               
               
                 1021 
                 tgtttcagat gctctcccct cctcggagga tgatgatgat 
               
               
                   
               
               
                   
                 gatgatgact cctcttcaga 
               
               
                   
               
               
                 1081 
                 ggagaaagaa acagataaca ccaaaccaaa ccgtatgccc 
               
               
                   
               
               
                   
                 gtagctccat attggacatc 
               
               
                   
               
               
                 1141 
                 cccagaaaag atggaaaaga aattgcatgc agtgccggct 
               
               
                   
               
               
                   
                 gccaagacag tgaagttcaa 
               
               
                   
               
               
                 1201 
                 atgcccttcc agtgggaccc caaaccccac actgcgctgg 
               
               
                   
               
               
                   
                 ttgaaaaatg gcaaagaatt 
               
               
                   
               
               
                 1261 
                 caaacctgac cacagaattg gaggctacaa ggtccgttat 
               
               
                   
               
               
                   
                 gccacctgga gcatcataat 
               
               
                   
               
               
                 1321 
                 ggactctgtg gtgccctctg acaagggcaa ctacacctgc 
               
               
                   
               
               
                   
                 attgtggaga atgagtacgg 
               
               
                   
               
               
                 1381 
                 cagcatcaac cacacatacc agctggatgt cgtggagcgg 
               
               
                   
               
               
                   
                 tcccctcacc ggcccatcct 
               
               
                   
               
               
                 1441 
                 gcaagcaggg ttgcccgcca acaaaacagt ggccctgggt 
               
               
                   
               
               
                   
                 agcaacgtgg agttcatgtg 
               
               
                   
               
               
                 1501 
                 taaggtgtac agtgacccgc agccgcacat ccagtggcta 
               
               
                   
               
               
                   
                 aagcacatcg aggtgaatgg 
               
               
                   
               
               
                 1561 
                 gagcaagatt ggcccagaca acctgcctta tgtccagatc 
               
               
                   
               
               
                   
                 ttgaagactg ctggagttaa 
               
               
                   
               
               
                 1621 
                 taccaccgac aaagagatgg aggtgcttca cttaagaaat 
               
               
                   
               
               
                   
                 gtctcctttg aggacgcagg 
               
               
                   
               
               
                 1681 
                 ggagtatacg tgcttggcgg gtaactctat cggactctcc 
               
               
                   
               
               
                   
                 catcactctg catggttgac 
               
               
                   
               
               
                 1741 
                 cgttctggaa gccctggaag agaggccggc agtgatgacc 
               
               
                   
               
               
                   
                 tcgcccctgt acctggagat 
               
               
                   
               
               
                 1801 
                 catcatctat tgcacagggg ccttcctcat ctcctgcatg 
               
               
                   
               
               
                   
                 gtggggtcgg tcatcgtcta 
               
               
                   
               
               
                 1861 
                 caagatgaag agtggtacca agaagagtga cttccacagc 
               
               
                   
               
               
                   
                 cagatggctg tgcacaagct 
               
               
                   
               
               
                 1921 
                 ggccaagagc atccctctgc gcagacaggt gtctgctgac 
               
               
                   
               
               
                   
                 tccagtgcat ccatgaactc 
               
               
                   
               
               
                 1981 
                 tggggttctt ctggttcggc catcacggct ctcctccagt 
               
               
                   
               
               
                   
                 gggactccca tgctagcagg 
               
               
                   
               
               
                 2041 
                 ggtctctgag tatgagcttc ccgaagaccc tcgctgggag 
               
               
                   
               
               
                   
                 ctgcctcggg acagactggt 
               
               
                   
               
               
                 2101 
                 cttaggcaaa cccctgggag agggctgctt tgggcaggtg 
               
               
                   
               
               
                   
                 gtgttggcag aggctatcgg 
               
               
                   
               
               
                 2161 
                 gctggacaag gacaaaccca accgtgtgac caaagtggct 
               
               
                   
               
               
                   
                 gtgaagatgt tgaagtcgga 
               
               
                   
               
               
                 2221 
                 cgcaacagag aaagacttgt cagacctgat ctcagaaatg 
               
               
                   
               
               
                   
                 gagatgatga agatgatcgg 
               
               
                   
               
               
                 2281 
                 gaagcataag aatatcatca acctgctggg ggcctgcacg 
               
               
                   
               
               
                   
                 caggatggtc ccttgtatgt 
               
               
                   
               
               
                 2341 
                 catcgtggag tatgcctcca agggcaacct gcgggagtac 
               
               
                   
               
               
                   
                 ctgcaggccc ggaggccccc 
               
               
                   
               
               
                 2401 
                 agggctggaa tactgctaca accccagcca caacccagag 
               
               
                   
               
               
                   
                 gagcagctct cctccaagga 
               
               
                   
               
               
                 2461 
                 cctggtgtcc tgcgcctacc aggtggcccg aggcatggag 
               
               
                   
               
               
                   
                 tatctggcct ccaagaagtg 
               
               
                   
               
               
                 2521 
                 catacaccga gacctggcag ccaggaatgt cctggtgaca 
               
               
                   
               
               
                   
                 gaggacaatg tgatgaagat 
               
               
                   
               
               
                 2581 
                 agcagacttt ggcctcgcac gggacattca ccacatcgac 
               
               
                   
               
               
                   
                 tactataaaa agacaaccaa 
               
               
                   
               
               
                 2641 
                 cggccgactg cctgtgaagt ggatggcacc cgaggcatta 
               
               
                   
               
               
                   
                 tttgaccgga tctacaccca 
               
               
                   
               
               
                 2701 
                 ccagagtgat gtgtggtctt tcggggtgct cctgtgggag 
               
               
                   
               
               
                   
                 atcttcactc tgggcggctc 
               
               
                   
               
               
                 2761 
                 cccatacccc ggtgtgcctg tggaggaact tttcaagctg 
               
               
                   
               
               
                   
                 ctgaaggagg gtcaccgcat 
               
               
                   
               
               
                 2821 
                 ggacaagccc agtaactgca ccaacgagct gtacatgatg 
               
               
                   
               
               
                   
                 atgcgggact gctggcatgc 
               
               
                   
               
               
                 2881 
                 agtgccctca cagagaccca ccttcaagca gctggtggaa 
               
               
                   
               
               
                   
                 gacctggacc gcatcgtggc 
               
               
                   
               
               
                 2941 
                 cttgacctcc aaccaggagt acctggacct gtccatgccc 
               
               
                   
               
               
                   
                 ctggaccagt actcccccag 
               
               
                   
               
               
                 3001 
                 ctttcccgac acccggagct ctacgtgctc ctcaggggag 
               
               
                   
               
               
                   
                 gattccgtct tctctcatga 
               
               
                   
               
               
                 3061 
                 gccgctgccc gaggagccct gcctgccccg acacccagcc 
               
               
                   
               
               
                   
                 cagcttgcca atggcggact 
               
               
                   
               
               
                 3121 
                 caaacgccgc tgactgccac ccacacgccc tccccagact 
               
               
                   
               
               
                   
                 ccaccgtcag ctgtaaccct 
               
               
                   
               
               
                 3181 
                 cacccacagc ccctgctggg cccaccacct gtccgtccct 
               
               
                   
               
               
                   
                 gtcccctttc ctgctggcag 
               
               
                   
               
               
                 3241 
                 gagccggctg cctaccaggg gccttcctgt gtggcctgcc 
               
               
                   
               
               
                   
                 ttcaccccac tcagctcacc 
               
               
                   
               
               
                 3301 
                 tctccctcca cctcctctcc acctgctggt gagaggtgca 
               
               
                   
               
               
                   
                 aagaggcaga tctttgctgc 
               
               
                   
               
               
                 3361 
                 cagccacttc atcccctccc agatgttgga ccaacacccc 
               
               
                   
               
               
                   
                 tccctgccac caggcactgc 
               
               
                   
               
               
                 3421 
                 ctggagggca gggagtggga gccaatgaac aggcatgcaa 
               
               
                   
               
               
                   
                 gtgagagctt cctgagcttt 
               
               
                   
               
               
                 3481 
                 ctcctgtcgg tttggtctgt tttgccttca cccataagcc 
               
               
                   
               
               
                   
                 cctcgcactc tggtggcagg 
               
               
                   
               
               
                 3541 
                 tgccttgtcc tcagggctac agcagtaggg aggtcagtgc 
               
               
                   
               
               
                   
                 ttcgtgcctc gattgaaggt 
               
               
                   
               
               
                 3601 
                 gacctctgcc ccagataggt ggtgccagtg gcttattaat 
               
               
                   
               
               
                   
                 tccgatacta gtttgctttg 
               
               
                   
               
               
                 3661 
                 ctgaccaaat gcctggtacc agaggatggt gaggcgaagg 
               
               
                   
               
               
                   
                 ccaggttggg ggcagtgttg 
               
               
                   
               
               
                 3721 
                 tggccctggg gcccagcccc aaactggggg ctctgtatat 
               
               
                   
               
               
                   
                 agctatgaag aaaacacaaa 
               
               
                   
               
               
                 3781 
                 gtgtataaat ctgagtatat atttacatgt ctttttaaaa 
               
               
                   
               
               
                   
                 gggtcgttac cagagattta 
               
               
                   
               
               
                 3841 
                 cccatcgggt aagatgctcc tggtggctgg gaggcatcag 
               
               
                   
               
               
                   
                 ttgctatata ttaaaaacaa 
               
               
                   
               
               
                 3901 
                 aaaaaaaaaa aaa 
               
            
           
         
       
     
     By “FRS2α polypeptide” is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_001265286.1 and having a biological activity of a FRS2α polypeptide. Biological activities of a FRS2α polypeptide include transmembrane receptor protein tyrosine kinase adaptor activity and binding to a FGFR1 polypeptide. The sequence at NCBI Accession No. NP_001265286.1 is shown below (SEQ ID No: 5): 
     
       
         
           
               
               
               
            
               
                 1 
                 mgsccscpdk dtvpdnhrnk fkvinvdddg nelgsgimel tdtelilytr krdsvkwhyl 
                   
               
               
                   
               
               
                 61 
                 clrrygydsn lfsfesgrrc qtgqgifafk caraeelfnm lqeimqnnsi nvveepvver 
               
               
                   
               
               
                 121 
                 nnhqtelevp rtprtpttpg faaqnlpngy prypsfgdas shpssrhpsv gsarlpsvge 
               
               
                   
               
               
                 181 
                 esthpllvae eqvhtyvntt gvqeerknrt svhvplearv snaesstpke epssiedrdp 
               
               
                   
               
               
                 241 
                 qillepegvk fvlgptpvqk qlmekekleq lgrdqvsgsg anntewdtgy dsderrdaps 
               
               
                   
               
               
                 301 
                 vnklvyenin glsipsasgv rrgrltstst sdtqninnsa qrrtallnye nlpslppvwe 
               
               
                   
               
               
                 361 
                 arklsrdedd nlgpktpsln gyhnnldpmh nyvntenvtv pasahkieys rrrdctptvf 
               
               
                   
               
               
                 421 
                 nfdirrpsle hrqlnyiqvd leggsdsdnp qtpktpttpl pqtptrrtel yavidierta 
               
               
                   
               
               
                 481 
                 amsnlqkalp rddgtsrktr hnstdlpm 
               
            
           
         
       
     
     By “FRS2α polynucleotide” is meant a polynucleotide encoding a FRS2α polypeptide. An exemplary FRS2α polynucleotide sequence is provided at NCBI Accession No. NM_001278357.1. The exemplary sequence provided at NCBI Accession No. NM_001278357.1 is reproduced below (SEQ ID No: 6). 
     
       
         
           
               
               
               
            
               
                 1 
                 aaaacccttc cctcccccgc tcccccggaa gtgcttttcc aagattcggg ccggagagag 
                   
               
               
                   
               
               
                 61 
                 gccttgtagg cacagcggct gagactcgat ctgctccaag taggggctcc agcgcgggtc 
               
               
                   
               
               
                 121 
                 ggagtctggg ggttcgcgcc cgccgacccg cgccctgctc cctctcagca cctgggcgga 
               
               
                   
               
               
                 181 
                 cggttaaatc agcaaacaaa gaaaacatgg tattttgaaa tatgattaaa ctcctgatgc 
               
               
                   
               
               
                 241 
                 tgcagcagag gctaagaata ttaatggcca gatctagtgc acacatggtc ttctgaagaa 
               
               
                   
               
               
                 301 
                 gccatgggta gctgttgtag ctgtccagat aaagacactg tcccagataa ccatcggaac 
               
               
                   
               
               
                 361 
                 aagtttaagg tcattaatgt ggatgatgat gggaatgagt taggttctgg cataatggaa 
               
               
                   
               
               
                 421 
                 cttacagaca cagaactgat tttatacacc cgcaaacgtg actcagtaaa atggcactac 
               
               
                   
               
               
                 481 
                 ctctgcctgc gacgctatgg ctatgactcg aatctctttt cttttgaaag tggtcgaagg 
               
               
                   
               
               
                 541 
                 tgtcaaactg gacaaggaat ctttgccttt aagtgtgccc gtgcagaaga attatttaac 
               
               
                   
               
               
                 601 
                 atgttgcaag agattatgca aaataatagt ataaatgtgg tggaagagcc agttgtagaa 
               
               
                   
               
               
                 661 
                 agaaataatc atcagacaga attggaagtc cctagaacac ctcgaacacc tacaactcca 
               
               
                   
               
               
                 721 
                 ggatttgctg ctcagaactt acctaatgga tatccccgat atccctcatt tggagatgct 
               
               
                   
               
               
                 781 
                 tcatcccatc cgtcaagcag acatccttct gtgggaagtg ctcgcctgcc ttcagtaggg 
               
               
                   
               
               
                 841 
                 gaagaatcta cacatccttt gcttgtggct gaggaacaag tacataccta tgtcaacact 
               
               
                   
               
               
                 901 
                 acaggtgtgc aagaagagcg gaaaaaccgc acaagtgtgc atgttccatt ggaggcgagg 
               
               
                   
               
               
                 961 
                 gtttctaacg ctgaaagcag cacaccaaaa gaagaaccaa gtagtattga ggacagggat 
               
               
                   
               
               
                 1021 
                 cctcagattc ttcttgaacc tgaaggagtc aaatttgttt tagggccaac ccctgttcaa 
               
               
                   
               
               
                 1081 
                 aagcagttaa tggaaaaaga gaaactggag caacttggaa gagatcaagt tagtggaagt 
               
               
                   
               
               
                 1141 
                 ggagcaaata acacagaatg ggacactggc tatgacagtg atgaacgaag agatgcaccc 
               
               
                   
               
               
                 1201 
                 tctgttaaca aactggtgta tgaaaatata aatgggctat ctatccctag tgcctcaggg 
               
               
                   
               
               
                 1261 
                 gtcaggagag gtcgtctgac atccaccagt acctcagata cccagaatat caacaactca 
               
               
                   
               
               
                 1321 
                 gctcagagaa gaactgcatt attaaactat gaaaatctac catctttgcc tcctgtttgg 
               
               
                   
               
               
                 1381 
                 gaagcccgca agctaagtag ggatgaagat gacaatttag gaccaaagac cccatctcta 
               
               
                   
               
               
                 1441 
                 aatggctacc ataataatct agatccaatg cataactatg taaatacaga gaatgtaaca 
               
               
                   
               
               
                 1501 
                 gtgccagcaa gtgctcacaa aatagaatat tcaaggcgtc gggactgtac accaacagtc 
               
               
                   
               
               
                 1561 
                 tttaactttg atatcagacg cccaagttta gaacacaggc agcttaatta catacaggtt 
               
               
                   
               
               
                 1621 
                 gacttggaag gtggcagtga ctctgacaac cctcagactc caaaaacgcc tacaactccc 
               
               
                   
               
               
                 1681 
                 cttccacaaa cccctaccag gcgcacagag ctgtatgccg tgatagacat cgagagaact 
               
               
                   
               
               
                 1741 
                 gctgctatgt caaatttgca gaaagcactg ccacgagatg atggtacatc taggaaaact 
               
               
                   
               
               
                 1801 
                 agacacaata gtactgatct gcccatgtga gcctggaaag cattgtgttg tttgcacctt 
               
               
                   
               
               
                 1861 
                 tgtgaagttt ttaaaaatga agatgcaagt gcttcatttt catttctaaa cactaactcc 
               
               
                   
               
               
                 1921 
                 ttttatagac tgataaaatt tttttctgaa tatttcatgt gcatctttaa ctaaagggaa 
               
               
                   
               
               
                 1981 
                 ttaatgtaga gcaggtactc cttaaagaac actaatttca ttatatacta ctcgttgtac 
               
               
                   
               
               
                 2041 
                 agcagcattc ccgttttcac agtgcctatt taaaatgaga gttgaagtaa atgacatgct 
               
               
                   
               
               
                 2101 
                 ggttgatttt tatcaatatt ctggacttaa cgcatacctt tcatgtctaa gtcatggttg 
               
               
                   
               
               
                 2161 
                 gcttttaaaa ctttttataa agcctcttga caatgtacat tgctaacagg taactatagg 
               
               
                   
               
               
                 2221 
                 ctttgaaagt aatgctcgta gattcagtgt tcacagtatg tggcctccag catgtaacat 
               
               
                   
               
               
                 2281 
                 gaggaatcct ttatttcatt aattaatggc tttttgactt gagccaaaac atatgtaaag 
               
               
                   
               
               
                 2341 
                 gaaacagaag taccgcacct cctcttacac cagtcagctc ctttgccttc agtgttacta 
               
               
                   
               
               
                 2401 
                 gaaagcggcc tgtgtccatg agtgtgcttt gctgttggtg cactgaaagg caggaaggag 
               
               
                   
               
               
                 2461 
                 acaagatttt ctatttactc atctcatgat gtcatttgaa gggcatgtcc agatatctta 
               
               
                   
               
               
                 2521 
                 aaattataat aggctcaaga atcagtctca ggtcacttta cccaaaaaca tttgaaaatc 
               
               
                   
               
               
                 2581 
                 tgaaccacaa tctcctgaaa gtttttctcc tatagattgt tgacaacaca ttgttttctg 
               
               
                   
               
               
                 2641 
                 gaggcatttg tgccattagg tttccattta tcttcagttt ttttctttgg tgtttgggat 
               
               
                   
               
               
                 2701 
                 gtcttatttt gttgccttat gtccttttca atttaaaatg tttgagtttg tatatagttt 
               
               
                   
               
               
                 2761 
                 tgaaattgga ttatgtgttc attgttgttt agtttgcatt tttgtcaaat tatggttttg 
               
               
                   
               
               
                 2821 
                 aaggttcatt tggaacttac tgttagtctg taacagggtt gcccttgtcc agtatttatt 
               
               
                   
               
               
                 2881 
                 tataagctgt ttacttttca agttgataaa aacattctcc aattctaaat ttgcttgtgt 
               
               
                   
               
               
                 2941 
                 ccataggtga tctctttagc aaactgagaa aaaaaggaag ctacttttaa catgcaaagt 
               
               
                   
               
               
                 3001 
                 tccctcaagg tgtaccgtgt tgtctctgtg ggcactcttc cccagcactt tagcagtaat 
               
               
                   
               
               
                 3061 
                 tcccccagct acacgctgca gttgtactct gcccactcta gtgttcctca gctctgctgt 
               
               
                   
               
               
                 3121 
                 ccttttactt gtagctggat ctttgattat ccttcgattt ccatgaaata ttaatattgt 
               
               
                   
               
               
                 3181 
                 tgccagcata gcaggtacag tggaagtctt gtagcagtga gattgtatca taatttagga 
               
               
                   
               
               
                 3241 
                 tttaaaatga attaaagttt atataaactg aagagtctcc atatgtcaaa ctcttggaaa 
               
               
                   
               
               
                 3301 
                 atcaaagatg ttccaatttc ctaaacacta gagaatacga gagaaggtag agtggaaaag 
               
               
                   
               
               
                 3361 
                 gttaggtaac cttgcaaaat attttactat tttctctaaa tatgaggaag tttgagatta 
               
               
                   
               
               
                 3421 
                 tgatctggat ctaccagata taactaaggt taatttagca tgaaaaagtt ttagtcatat 
               
               
                   
               
               
                 3481 
                 tggcatccaa cctattcagt aaccgaatca taggacaatg atggattagg agaacaatag 
               
               
                   
               
               
                 3541 
                 agtgggatca ttataaagaa aataaattat taaaggtgtc tttatcgttt tagtgccatt 
               
               
                   
               
               
                 3601 
                 tttagtgtct ttactataaa tcaatatcag tgtattttat cattctatgt gcatagcaga 
               
               
                   
               
               
                 3661 
                 attttctttt ctcccttttg ttcccctgtg aacttggtgc ttattaaagt gctcactgtt 
               
               
                   
               
               
                 3721 
                 ctcttaaaag agagcagtgg tataggtgtg cagtttccat gatgcaggtt ccatttttaa 
               
               
                   
               
               
                 3781 
                 tatattgttc cacttatcct ttcttctgag taaattgcta attgtgccaa atttatgtaa 
               
               
                   
               
               
                 3841 
                 tagtttttgt aatgtggaat aagaattatg atggaaccat tgcacatttt tttctgaaac 
               
               
                   
               
               
                 3901 
                 agccagtcaa ggcagaacat taatctccaa atgcaagggc tgatctattt attcattttg 
               
               
                   
               
               
                 3961 
                 gaggttgggt actttattct ttctttccgt catccttttc attgtttccc ccggattcta 
               
               
                   
               
               
                 4021 
                 attagttttt atttttttta gataactcca atataatcat tacagtttat gctttaaata 
               
               
                   
               
               
                 4081 
                 ctatgtgctt taaaaaggaa aatgggacca atttgtctgc taagaatttg attttaggta 
               
               
                   
               
               
                 4141 
                 ctataagagt attaggaaaa tatatacaac tggtgttaat ttctagatat tttctagaaa 
               
               
                   
               
               
                 4201 
                 tcacttgtgt tcctatttaa taaaaggtaa tttagaatac tacttgtcct ttgcagtagt 
               
               
                   
               
               
                 4261 
                 ttagtaatgg gcattaagct gtgtcctcga aggatgtacc tattactagg tgcattttag 
               
               
                   
               
               
                 4321 
                 aatgaaatat tgatatttta ttagcatata attgtggcca tatatctcag attttctgag 
               
               
                   
               
               
                 4381 
                 gcagatctaa ttttagataa ttctgttggt agaccatgtg atccttcttt ttggttttgg 
               
               
                   
               
               
                 4441 
                 aaatataatc attgttaatg ttttccctcc aaatagaata ctgttttatc catacaaatc 
               
               
                   
               
               
                 4501 
                 ataacagcat ctatcccatg ctagggttgg aaactgatat tggtattact tgtgtttttt 
               
               
                   
               
               
                 4561 
                 cttagtgtgt tttatttccc agtttcatct tcttctaaaa atgaaaatat ggtgccttcc 
               
               
                   
               
               
                 4621 
                 ctccctccag gaagactggc aaatatttcc ttttatttac tgctgctgtg gagtgatgag 
               
               
                   
               
               
                 4681 
                 atatgcactt tactctttaa gattcagcaa aaagcttttc acttctcagt atatccagaa 
               
               
                   
               
               
                 4741 
                 tacatcatat ctgggactta ggaaaatttg ccaagcaatc tttgttttta tagatactaa 
               
               
                   
               
               
                 4801 
                 tgttgaccct ctccagcgtt caatgttata aatagaacaa gtcaagctag tgtttatctc 
               
               
                   
               
               
                 4861 
                 ctccccctcc ccaaaactgt ggcacagcat ataaaaatgt acctcaataa tgttctatta 
               
               
                   
               
               
                 4921 
                 aaaatgggac aggggcctta tgttttcata atttcccaac aatgtgccgc catatttttg 
               
               
                   
               
               
                 4981 
                 cctcaaggta aaggttttaa cagatgaaaa agtacttccc aattcccccg tgctattcct 
               
               
                   
               
               
                 5041 
                 aacctataat gcccaaatgt tttgtgcaat gtgtagtgtg tgtgtataaa tacatatatt 
               
               
                   
               
               
                 5101 
                 cttgaaatag acataccatc agagacatca ttcacaagta actgatgtat tggcatctca 
               
               
                   
               
               
                 5161 
                 ttcatatttc tgatgtgtga ggtatatggt actaattacc ttttccttga tgtttgccaa 
               
               
                   
               
               
                 5221 
                 atttgaataa aggcattggt acgaaattac agaatgtaaa gaaaatgttt ttggcttgaa 
               
               
                   
               
               
                 5281 
                 aaattaacat attttatgac gtaccacagt atactctgcc caaaccagca ccctatctat 
               
               
                   
               
               
                 5341 
                 ctttcctgtt ctttacatcc ctgttcccca tccctacttc ctcatttttg gtataacaca 
               
               
                   
               
               
                 5401 
                 gttcttttgt agcatcatta taattgcagt tctatggcaa ttggacagtt atagcatgga 
               
               
                   
               
               
                 5461 
                 aacagactgg tataagtagt acagtagtca ccagtgtgcc acatttgcat tagtaatgca 
               
               
                   
               
               
                 5521 
                 aaatatacat tttataaagg acaaactttg tgttatgttt tattttcatt acattgtata 
               
               
                   
               
               
                 5581 
                 atattgtaag actattgtat gtcctaattt gcattataaa tgtttttttc ctacgtaaag 
               
               
                   
               
               
                 5641 
                 gcataaatat agcaactttg tataaaggta gcttattaga tttttaattt tttcttttat 
               
               
                   
               
               
                 5701 
                 aaaaaattgt ccaacagtgg gactaccatt gccaaattgt atatgaaata tgaattttac 
               
               
                   
               
               
                 5761 
                 ccccatggtt aatttctttt ataaacattc catatttctc taataaaaag acataagtga 
               
               
                   
               
               
                 5821 
                 tactgtacta tgcatacatt gtatcttaat gctgtttcag atcagcattt taaattttgg 
               
               
                   
               
               
                 5881 
                 tttgcatttt taatattggc aaaacgtaac cactgttaat taaaataaaa ccttgttgta 
               
               
                   
               
               
                 5941 
                 tatgtaacaa cataattttc cctctatccc ttcccaccct ttgttctcta tttctcccta 
               
               
                   
               
               
                 6001 
                 tcagtgccaa cttcatacat tttgtagcat ggcaataaaa tataactttt acactgaggc 
               
               
                   
               
               
                 6061 
                 cgagtgtggc tttttggagg aagtggggat gggacgattg ccctctagtt gtcctttgca 
               
               
                   
               
               
                 6121 
                 tatgactgtt ttttgccata taagccatgt catcaggcat gaaaagtttt ctcatatatg 
               
               
                   
               
               
                 6181 
                 atgtaaactt gcttttaagg acaagtgtga atgtgctttt taagcttaat ttttgtcatg 
               
               
                   
               
               
                 6241 
                 acaactaatt ttttttatct ttggagaagt cagagttctt tacaatcaaa cgtttattaa 
               
               
                   
               
               
                 6301 
                 ctggagtact tagaataagc tagtaattga atttagttca agggctaagc aacacatttt 
               
               
                   
               
               
                 6361 
                 taaatcctta tttattgtag agtattagta tactgtccta caaattatgt aaaatatggt 
               
               
                   
               
               
                 6421 
                 ttaatattag atgactttgg attttgcaat gccttactgt tgtcattcta gcataaatat 
               
               
                   
               
               
                 6481 
                 ccataatgag gtactcaagt tgatactgga agctgagctg atcatacact gacctgaagc 
               
               
                   
               
               
                 6541 
                 attcatgaaa agctgcttta ttgaataaag tctgattgga gttcttttca tgctcacttt 
               
               
                   
               
               
                 6601 
                 ccccttattg ctgaaagtag attgcaataa aaccccaata aaacgtttgg tcggatatct 
               
               
                   
               
               
                 6661 
                 acttaaaaaa aaaaaa 
               
            
           
         
       
     
     Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns. 
     The term “expression” as used herein is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter. 
     By “fragment” is meant a portion of a polypeptide or nucleic acid molecule. This portion contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids. 
     “Hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds. 
     By “inhibitory nucleic acid” is meant a double-stranded RNA, siRNA, shRNA, or antisense RNA, or a portion thereof, or a mimetic thereof, that when administered to a mammalian cell results in a decrease (e.g., by 10%, 25%, 50%, 75%, or even 90-100%) in the expression of a target gene. Typically, a nucleic acid inhibitor comprises at least a portion of a target nucleic acid molecule, or an ortholog thereof, or comprises at least a portion of the complementary strand of a target nucleic acid molecule. For example, an inhibitory nucleic acid molecule comprises at least a portion of any or all of the nucleic acids delineated herein. 
     The terms “isolated,” “purified,” or “biologically pure” refer to material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings. “Purify” denotes a degree of separation that is higher than isolation. A “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified. 
     By “isolated polynucleotide” is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence. 
     By an “isolated polypeptide” is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. The preparation can be at least 75%, at least 90%, and at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis. 
     By “marker” is meant any polypeptide or polynucleotide having an alteration in expression level, sequence, or activity that is associated with a disease or disorder or risk of disease or disorder. In some embodiments, a decrease in activity or level of a FGF signaling polypeptide or let-7 miRNA in an endothelial cell is associated with development and/or progression of PAH. In some embodiments, an increase in level or activity of a TGFβ signaling polypeptide (e.g., TGFβ1, TGFβ2, TGFβ3, TGFβR1, TGFβR2) in an endothelial cell is associated with development and/or progression of PAH. In some other embodiments, an increase in activity or level of a FGF signaling polypeptide or let-7 miRNA in a smooth muscle cell is associated with development and/or progression of PAH. In still other embodiments, a decrease in level or activity of a TGFβ signaling polypeptide (e.g., TGFβ1, TGFβ2, TGFβ3, TGFβR1, TGFβR2) is associated with development and/or progression of PAH. 
     As used herein, “microRNA” or “miRNA” describes small non-coding RNA molecules, generally about 15 to about 50 nucleotides in length, preferably 17-23 nucleotides, 15 which can play a role in regulating gene expression through, for example, a process termed RNA interference (RNAi). RNAi describes a phenomenon whereby the presence of an RNA sequence that is complementary or antisense to a sequence in a target gene messenger RNA (mRNA) results in inhibition of expression of the target gene. miRNAs are processed from hairpin precursors of about 70 or more nucleotides (pre-miRNA) which are derived from 20 primary transcripts (pri-miRNA) through sequential cleavage by RNAse III enzymes. miRBase is a comprehensive microRNA database located at www.mirbase.org, incorporated by reference herein in its entirety for all purposes. 
     By “let-7 miRNA” is meant a miRNA member of the let-7 miRNA family. Sequences of members of the let-7 miRNA family can be found in, for example, www.mirbase.org. Exemplary members of the let-7 miRNA family include hsa-let-7b or human let-7b (miRBase Accession No. MI0000063), hsa-let-7a-1 (miRBase Accession No. MI0000060), hsa-let-7a-2 (miRBase Accession No. MI0000061), hsa-let-7a-3 (miRBase Accession No. MI0000062), hsa-let-7b, hsa-let-7c (miRBase Accession No. MI0000064), hsa-let-7d (miRBase Accession No. MI0000065), hsa-let-7e (miRBase Accession No. MI0000066), hsa-let-7f-1 (miRBase Accession No. MI0000067), hsa-let-7f-2 (miRBase Accession No. MI0000068), hsa-let-7g (miRBase Accession No. MI0000433), and hsa-let-7i (miRBase Accession No. MI00000434). The sequence of human let-7b provided at miRBase Accession No. MI0000063 is reproduced below. 
     
       
         
           
               
               
            
               
                   
                 human let-7b (5 prime): 
               
               
                   
                 (SEQ ID No: 1) 
               
               
                   
                 UGAGGUAGUAGGUUGUGUGGUU 
               
               
                   
                   
               
               
                   
                 human let-7b (3 prime): 
               
               
                   
                 (SEQ ID No: 2) 
               
               
                   
                 CUAUACAACCUACUGCCUUCCC 
               
            
           
         
       
     
     The let-7 miRNA family has been shown to play important roles in animal development, cell differentiation, and metabolism. In some embodiments, an activity of let-7 miRNA is repression of expression of a TGFβ signaling polypeptide. In some embodiments, an activity of let-7 miRNA is repression of TGFβ signaling. 
     In some embodiments, the let-7 miRNA is used as a therapeutic. Use of let-7 miRNA as a therapeutic has been demonstrated previously. For example, let-7 miRNA was used as anti-cancer therapy (Trang et al., Mol Ther. 2011 June; 19(6): 1116-1122). 
     In some embodiments, the let-7 miRNA is chemically modified. In particular embodiments, uracil (“U”) or cytosine (“C”) is chemically modified. In some embodiments, the miRNA is modified to impart properties to the miRNA to make it useful as a therapeutic, such as attenuated immunostimulation and increased serum stability. Such modifications to the miRNA include, without limitation, incorporation of a 2′-O-methyl (2′-O-Me), phosphorothioate (PS), and deoxy thymidine (dT) residues. In particular embodiments, the modified miRNA retains silencing activity in vivo. In particular embodiments, the modification is a 2′-O-methyl nucleotide modification. In some embodiments, the modification decreases the likelihood of triggering an innate immune response. 
     In some embodiments, the let-7 miRNA contains a “light” modification. By a miRNA containing a “light modification” is meant that the miRNA contains a 2′-O-methyl modification on all U and C nucleotide bases followed by adenosine (“A”) on the antisense strand. In some other embodiments, the let-7 miRNA contains a “heavy” modification. By a miRNA containing a “heavy modification” is meant that the miRNA contains a 2′-O-methyl modification on all U and C nucleotide bases on the sense strand. 
     In still other embodiments, the let-7 miRNA is “mi-let-7b L ”. mi-let-7b L  is also referred to herein as “let-7 light.” The sequence of mi-let-7b L  is provided below: 
     
       
         
           
               
               
            
               
                   
                 mi-let-7b L  (5 prime): 
               
               
                   
                 (SEQ ID No: 1) 
               
               
                   
                 UGAGGuAGuAGGUUGUGUGGUU 
               
               
                   
                   
               
               
                   
                 mi-let-7b L  (3 prime): 
               
               
                   
                 (SEQ ID NO: 2) 
               
               
                   
                 CuAuAcAACCuACUGCCUUCCC 
               
            
           
         
       
     
     In some other embodiments, the let-7 miRNA is “mi-let-7b H ”. mi-let-7b H  is also referred to herein as “let-7 heavy.” The sequence of mi-let-7b H  miRNA is provided below: 
     
       
         
           
               
               
            
               
                   
                 mi-let-7b H  (5 prime): 
               
               
                   
                 (SEQ ID No: 1) 
               
               
                   
                 UGAGGuAGuAGGUUGUGUGGUU 
               
               
                   
                   
               
               
                   
                 mi-let-7b H  (3 prime): 
               
               
                   
                 (SEQ ID NO: 2) 
               
               
                   
                 cuAuAcAAccuAcuGccuuccc 
               
            
           
         
       
     
     In the foregoing sequences, lower case indicates a nucleotide base containing a 2′-O-methyl modification. 
     As used herein, “obtaining” as in “obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent. 
     The term “oligonucleotide” typically refers to short polynucleotides, generally no greater than about 60 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which “U” replaces “T.” 
     As used herein, “polynucleotide” includes cDNA, RNA, DNA/RNA hybrid, antisense RNA, siRNA, miRNA, snoRNA, genomic DNA, synthetic forms, and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified to contain non-natural or derivatized, synthetic, or semi-synthetic nucleotide bases. Also, included within the scope of the invention are alterations of a wild type or synthetic gene, including but not limited to deletion, insertion, substitution of one or more nucleotides, or fusion to other polynucleotide sequences. 
     As used herein, the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition. 
     As used herein, the term “promoter” or “regulatory sequence” means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter or regulator sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter or regulatory sequence may, for example, be one which expresses the gene product in an inducible manner. 
     By “pulmonary arterial hypertension” or “PAH” is mean a disease syndrome characterized by increased systolic pressure in the pulmonary artery that exceeds, at rest, 25 mm Hg. This can be due to primary changes in the lung (primary pulmonary hypertension) or secondary to increase left-side cardiac pressures (secondary pulmonary hypertension). 
     By “reduces” is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%. 
     By “reference” is meant a standard or control condition. In some embodiments, the reference is an activity or level of a TGFβ signaling polypeptide or polynucleotide or a FGF signaling polypeptide or polynucleotide in a healthy, normal subject or in a subject that does not have PAH. In some embodiments, the reference is an activity or level of a let-7 miRNA in a healthy, normal subject or in a subject that does not have PAH. In some embodiments, the TGFβ signaling polypeptide or polynucleotide is a TGFβ1, TGFβ2, TGFβ3, TGFβR1, or TGFβR2 polypeptide or polynucleotide. In some embodiments, the FGF signaling polypeptide is FRS2α. In some other embodiments, the let-7 miNA is at least one selected from the group consisting of human let-7b miRNA and human let-7c miRNA. 
     A “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, at least about 20 amino acids, or at least about 25 amino acids. The length of the reference polypeptide sequence can be about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, at least about 60 nucleotides, or at least about 75 nucleotides. The length of the reference nucleic acid sequence can be about 100 nucleotides, about 300 nucleotides or any integer thereabout or therebetween. 
     By “siRNA” is meant a double stranded RNA. Optimally, an siRNA is 18, 19, 20, 21, 22, 23 or 24 nucleotides in length and has a 2 base overhang at its 3′ end. These dsRNAs can be introduced to an individual cell or to a whole animal; for example, they may be introduced systemically via the bloodstream. Such siRNAs are used to downregulate mRNA levels or promoter activity. 
     By “specifically binds” is meant an agent that recognizes and binds a polypeptide or polynucleotide of the invention, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polynucleotide of the invention. In some embodiments, the agent is a nucleic acid molecule. 
     Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By “hybridize” is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507). 
     For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, less than about 500 mM NaCl and 50 mM trisodium citrate, or less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, or at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C., at least about 37° C., and at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In one embodiment, hybridization will occur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In another embodiment, hybridization will occur at 37° C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA (ssDNA). In yet another embodiment, hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art. 
     For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will be less than about 30 mM NaCl and 3 mM trisodium citrate, or less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C., at least about 42° C., and at least about 68° C. In one embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In another embodiment, wash steps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In yet another embodiment, wash steps will occur at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York. 
     By “substantially identical” is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Such a sequence is at least 60%, at least 80%, at least 85%, at least 90%, at least 95% or even at least 99% identical at the amino acid level or nucleic acid to the sequence used for comparison. 
     Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e −3  and e −100  indicating a closely related sequence. 
     As used herein, a “TGFβ signaling polypeptide” refers to a member or component of a transformation growth factor β (TGFβ) signaling pathway. Exemplary TGFβ signaling polypeptides include polypeptides TGFβ1, TGFβ2, TGFβ3, TGFβR1, TGFβR2, SMAD1, SMAD2, SMAD3, SMAD4, SMAD5, and SMAD9. 
     As used herein, a “TGFβ signaling polynucleotide” is a polynucleotide encoding a TGFβ signaling polypeptide. 
     By “TGFβ1 polypeptide” is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to GenBank Accession No. AAH22242.1 and having a biological activity of a TGFβ1 polypeptide. Biological activities of a TGFβ1 polypeptide include binding to a type II transforming growth factor β (TGFβ) receptor and homodimerization. The sequence at GenBank Accession No. AAH22242.1 is shown below (SEQ ID NO: 7): 
     
       
         
           
               
               
               
            
               
                 1 
                 mppsglrlll lllpllwllv ltpgrpaagl stcktidmel vkrkrieair gqilsklrla 
                   
               
               
                   
               
               
                 61 
                 sppsqgevpp gplpeavlal ynstrdrvag esaepepepe adyyakevtr vlmvethnei 
               
               
                   
               
               
                 121 
                 ydkfkqsths iymffntsel reavpepvll sraelrllrl klkveqhvel yqkysnnswr 
               
               
                   
               
               
                 181 
                 ylsnrllaps dspewlsfdv tgvvrqwlsr ggeiegfrls ahcscdsrdn tlqvdingft 
               
               
                   
               
               
                 241 
                 tgrrgdlati hgmnrpflll matpleraqh lqssrhrral dtnycfsste knccvrglyi 
               
               
                   
               
               
                 301 
                 dfrkdlgwkw ihepkgyhan fclgpcpyiw sldtqyskvl alynqhnpga saapccvpqa 
               
               
                   
               
               
                 361 
                 leplpivyyv grkpkveqls nmivrsckcs 
               
            
           
         
       
     
     By “TGFβ1 polynucleotide” is meant a polynucleotide encoding a TGFβ1 polypeptide. An exemplary TGFβ1 polynucleotide sequence is provided at GenBank Accession No. BC022242.1. The exemplary sequence provided at GenBank Accession No. BC022242.1 is reproduced below (SEQ ID NO: 8). 
     
       
         
           
               
               
               
            
               
                 1 
                 cccagacctc gggcgcaccc cctgcacgcc gccttcatcc ccggcctgtc tcctgagccc 
                   
               
               
                   
               
               
                 61 
                 ccgcgcatcc tagacccttt ctcctccagg agacggatct ctctccgacc tgccacagat 
               
               
                   
               
               
                 121 
                 cccctattca agaccaccca ccttctggta ccagatcgcg cccatctagg ttatttccgt 
               
               
                   
               
               
                 181 
                 gggatactga gacacccccg gtccaagcct cccctccacc actgcgccct tctccctgag 
               
               
                   
               
               
                 241 
                 gacctcagct ttccctcgag gccctcctac cttttgccgg gagaccccca gcccctgcag 
               
               
                   
               
               
                 301 
                 gggcggggcc tccccaccac accagccctg ttcgcgctct cggcagtgcc ggggggcgcc 
               
               
                   
               
               
                 361 
                 gcctccccca tgccgccctc cgggctgcgg ctgctgctgc tgctgctacc gctgctgtgg 
               
               
                   
               
               
                 421 
                 ctactggtgc tgacgcctgg ccggccggcc gcgggactat ccacctgcaa gactatcgac 
               
               
                   
               
               
                 481 
                 atggagctgg tgaagcggaa gcgcatcgag gccatccgcg gccagatcct gtccaagctg 
               
               
                   
               
               
                 541 
                 cggctcgcca gccccccgag ccagggggag gtgccgcccg gcccgctgcc cgaggccgtg 
               
               
                   
               
               
                 601 
                 ctcgccctgt acaacagcac ccgcgaccgg gtggccgggg agagtgcaga accggagccc 
               
               
                   
               
               
                 661 
                 gagcctgagg ccgactacta cgccaaggag gtcacccgcg tgctaatggt ggaaacccac 
               
               
                   
               
               
                 721 
                 aacgaaatct atgacaagtt caagcagagt acacacagca tatatatgtt cttcaacaca 
               
               
                   
               
               
                 781 
                 tcagagctcc gagaagcggt acctgaaccc gtgttgctct cccgggcaga gctgcgtctg 
               
               
                   
               
               
                 841 
                 ctgaggctca agttaaaagt ggagcagcac gtggagctgt accagaaata cagcaacaat 
               
               
                   
               
               
                 901 
                 tcctggcgat acctcagcaa ccggctgctg gcacccagcg actcgccaga gtggttatct 
               
               
                   
               
               
                 961 
                 tttgatgtca ccggagttgt gcggcagtgg ttgagccgtg gaggggaaat tgagggcttt 
               
               
                   
               
               
                 1021 
                 cgccttagcg cccactgctc ctgtgacagc agggataaca cactgcaagt ggacatcaac 
               
               
                   
               
               
                 1081 
                 gggttcacta ccggccgccg aggtgacctg gccaccattc atggcatgaa ccggcctttc 
               
               
                   
               
               
                 1141 
                 ctgcttctca tggccacccc gctggagagg gcccagcatc tgcaaagctc ccggcaccgc 
               
               
                   
               
               
                 1201 
                 cgagccctgg acaccaacta ttgcttcagc tccacggaga agaactgctg cgtgcggcag 
               
               
                   
               
               
                 1261 
                 ctgtacattg acttccgcaa ggacctcggc tggaagtgga tccacgagcc caagggctac 
               
               
                   
               
               
                 1321 
                 catgccaact tctgcctcgg gccctgcccc tacatttgga gcctggacac gcagtacagc 
               
               
                   
               
               
                 1381 
                 aaggtcctgg ccctgtacaa ccagcataac ccgggcgcct cggcggcgcc gtgctgcgtg 
               
               
                   
               
               
                 1441 
                 ccgcaggcgc tggagccgct gcccatcgtg tactacgtgg gccgcaagcc caaggtggag 
               
               
                   
               
               
                 1501 
                 cagctgtcca acatgatcgt gcgctcctgc aagtgcagct gaggtcccgc cccgccccgc 
               
               
                   
               
               
                 1561 
                 cccgccccgg caggcccggc cccaccccgc cccgcccccg ctgccttgcc catgggggct 
               
               
                   
               
               
                 1621 
                 gtatttaagg acacccgtgc cccaagccca cctggggccc cattaaagat ggagagagga 
               
               
                   
               
               
                 1681 
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
               
               
                   
               
               
                 1741 
                 aaaaaa 
               
            
           
         
       
     
     By “TGFβ2 polypeptide” is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to GenBank Accession No. AAA50405.1 and having a biological activity of a TGFβ2 polypeptide. Biological activities of a TGFβ2 polypeptide include binding to a type II transforming growth factor β (TGFβ) receptor and homodimerization. The sequence at GenBank Accession No. AAA50405.1 is shown below (SEQ ID NO: 9): 
     
       
         
           
               
               
               
            
               
                 1 
                 mhycvlsafl ilhlvtvals lstcstldmd qfmrkrieai rgqilsklkl tsppedypep 
                   
               
               
                   
               
               
                 61 
                 eevppevisi ynstrdllqe kasrraaace rersdeeyya kevykidmpp ffpseaippt 
               
               
                   
               
               
                 121 
                 fyrpyfrivr fdvsamekna snlvkaefrv frlqnpkarv peqrielyqi lkskdltspt 
               
               
                   
               
               
                 181 
                 qryidskvvk traegewlsf dvtdavhewl hhkdrnlgfk islhcpcctf vpsnnyiipn 
               
               
                   
               
               
                 241 
                 kseelearfa gidgtstyts gdqktikstr kknsgktphl llmllpsyrl esqqtnrrkk 
               
               
                   
               
               
                 301 
                 raldaaycfr nvqdncclrp lyidfkrdlg wkwihepkgy nanfcagacp ylwssdtqhs 
               
               
                   
               
               
                 361 
                 rvlslyntin peasaspccv sqdlepltil yyigktpkie qlsnmivksc kcs 
               
            
           
         
       
     
     By “TGFβ2 polynucleotide” is meant a polynucleotide encoding a TGFβ2 polypeptide. An exemplary TGFβ2 polynucleotide sequence is provided at GenBank Accession No. M19154.1. The exemplary sequence provided at GenBank Accession No. M19154.1 is reproduced below (SEQ ID NO: 10). 
     
       
         
           
               
               
               
            
               
                 1 
                 gcccctcccg tcagttcgcc agctgccagc cccgggacct tttcatctct tcccttttgg 
                   
               
               
                   
               
               
                 61 
                 ccggaggagc cgagttcaga tccgccactc cgcacccgag actgacacac tgaactccac 
               
               
                   
               
               
                 121 
                 ttcctcctct taaatttatt tctacttaat agccactcgt ctcttttttt ccccatctca 
               
               
                   
               
               
                 181 
                 ttgctccaag aatttttttc ttcttactcg ccaaagtcag ggttccctct gcccgtcccg 
               
               
                   
               
               
                 241 
                 tattaatatt tccacttttg gaactactgg ccttttcttt ttaaaggaat tcaagcagga 
               
               
                   
               
               
                 301 
                 tacgtttttc tgttgggcat tgactagatt gtttgcaaaa gtttcgcatc aaaaacaaca 
               
               
                   
               
               
                 361 
                 acaacaaaaa accaaacaac tctccttgat ctatactttg agaattgttg atttcttttt 
               
               
                   
               
               
                 421 
                 tttattctga cttttaaaaa caactttttt ttccactttt ttaaaaaatg cactactgtg 
               
               
                   
               
               
                 481 
                 tgctgagcgc ttttctgatc ctgcatctgg tcacggtcgc gctcagcctg tctacctgca 
               
               
                   
               
               
                 541 
                 gcacactcga tatggaccag ttcatgcgca agaggatcga ggcgatccgc gggcagatcc 
               
               
                   
               
               
                 601 
                 tgagcaagct gaagctcacc agtcccccag aagactatcc tgagcccgag gaagtccccc 
               
               
                   
               
               
                 661 
                 cggaggtgat ttccatctac aacagcacca gggacttgct ccaggagaag gcgagccgga 
               
               
                   
               
               
                 721 
                 gggcggccgc ctgcgagcgc gagaggagcg acgaagagta ctacgccaag gaggtttaca 
               
               
                   
               
               
                 781 
                 aaatagacat gccgcccttc ttcccctccg aaactgtctg cccagttgtt acaacaccct 
               
               
                   
               
               
                 841 
                 ctggctcagt gggcagcttg tgctccagac agtcccaggt gctctgtggg taccttgatg 
               
               
                   
               
               
                 901 
                 ccatcccgcc cactttctac agaccctact tcagaattgt tcgatttgac gtctcagcaa 
               
               
                   
               
               
                 961 
                 tggagaagaa tgcttccaat ttggtgaaag cagagttcag agtctttcgt ttgcagaacc 
               
               
                   
               
               
                 1021 
                 caaaagccag agtgcctgaa caacggattg agctatatca gattctcaag tccaaagatt 
               
               
                   
               
               
                 1081 
                 taacatctcc aacccagcgc tacatcgaca gcaaagttgt gaaaacaaga gcagaaggcg 
               
               
                   
               
               
                 1141 
                 aatggctctc cttcgatgta actgatgctg ttcatgaatg gcttcaccat aaagacagga 
               
               
                   
               
               
                 1201 
                 acctgggatt taaaataagc ttacactgtc cctgctgcac ttttgtacca tctaataatt 
               
               
                   
               
               
                 1261 
                 acatcatccc aaataaaagt gaagaactag aagcaagatt tgcaggtatt gatggcacct 
               
               
                   
               
               
                 1321 
                 ccacatatac cagtggtgat cagaaaacta taaagtccac taggaaaaaa aacagtggga 
               
               
                   
               
               
                 1381 
                 agaccccaca tctcctgcta atgttattgc cctcctacag acttgagtca caacagacca 
               
               
                   
               
               
                 1441 
                 accggcggaa gaagcgtgct ttggatgcgg cctattgctt tagaaatgtg caggataatt 
               
               
                   
               
               
                 1501 
                 gctgcctacg tccactttac attgatttca agagggatct agggtggaaa tggatacacg 
               
               
                   
               
               
                 1561 
                 aacccaaagg gtacaatgcc aacttctgtg ctggagcatg cccgtattta tggagttcag 
               
               
                   
               
               
                 1621 
                 acactcagca cagcagggtc ctgagcttat ataataccat aaatccagaa gcatctgctt 
               
               
                   
               
               
                 1681 
                 ctccttgctg cgtgtcccaa gatttagaac ctctaaccat tctctactac attggcaaaa 
               
               
                   
               
               
                 1741 
                 cacccaagat tgaacagctt tctaatatga ttgtaaagtc ttgcaaatgc agctaaaatt 
               
               
                   
               
               
                 1801 
                 cttggaaaag tggcaagacc aaaatgacaa tgatgatgat aatgatgatg acgacgacaa 
               
               
                   
               
               
                 1861 
                 cgatgatgct tgtaacaaga aaacataaga gagccttggt tcatcagtgt taaaaaattt 
               
               
                   
               
               
                 1921 
                 ttgaaaaggc ggtactagtt cagacacttt ggaagtttgt gttctgtttg ttaaaactgg 
               
               
                   
               
               
                 1981 
                 catctgacac aaaaaaagtt gaaggcctta ttctacattt cacctacttt gtaagtgaga 
               
               
                   
               
               
                 2041 
                 gagacaagaa gcaaattttt tttaaagaaa aaaataaaca ctggaagaat ttattagtgt 
               
               
                   
               
               
                 2101 
                 taattatgtg aacaacgaca acaacaacaa caacaacaaa caggaaaatc ccattaagtg 
               
               
                   
               
               
                 2161 
                 gagttgctgt acgtaccgtt cctatcccgc gcctcacttg atttttctgt attgctatgc 
               
               
                   
               
               
                 2221 
                 aataggcacc cttcccattc ttactcttag agttaacagt gagttattta ttgtgtgtta 
               
               
                   
               
               
                 2281 
                 ctatataatg aacgtttcat tgcccttgga aaataaaaca ggtgtataaa gtggagacca 
               
               
                   
               
               
                 2341 
                 aatactttgc cagaaactca tggatggctt aaggaacttg aactcaaacg agccagaaaa 
               
               
                   
               
               
                 2401 
                 aaagaggtca tattaatggg atgaaaaccc aagtgagtta ttatatgacc gagaaagtct 
               
               
                   
               
               
                 2461 
                 gcattaagat aaagaccctg aaaacacatg ttatgtatca gctgcctaag gaagcttctt 
               
               
                   
               
               
                 2521 
                 gtaaggtcca aaaactaaaa agactgttaa taaaagaaac tttcagtcag 
               
            
           
         
       
     
     By “TGFβ3 polypeptide” is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to GenBank Accession No. EAW81249.1 and having a biological activity of a TGFβ3 polypeptide. Biological activities of a TGFβ3 polypeptide include binding to a type II transforming growth factor β (TGFβ) receptor and homodimerization. The sequence at GenBank Accession No. EAW81249.1 is shown below (SEQ ID NO: 11): 
     
       
         
           
               
               
               
            
               
                 1 
                 mkmhlqralv vlallnfatv slslstcttl dfghikkkrv eairgqilsk lrltsppept 
                   
               
               
                   
               
               
                 61 
                 vmthvpyqvl alynstrell eemhgereeg ctqentesey yakeihkfdm iqglaehnel 
               
               
                   
               
               
                 121 
                 avcpkgitsk vfrfnvssve knrtnlfrae frvlrvpnps skrnegriel fqilrpdehi 
               
               
                   
               
               
                 181 
                 akqryiggkn lptrgtaewl sfdvtdtvre wllrresnlg leisihcpch tfqpngdile 
               
               
                   
               
               
                 241 
                 nihevmeikf kgvdneddhg rgdlgrlkkg kdhhnphlil mmipphrldn pgqggqrkkr 
               
               
                   
               
               
                 301 
                 aldtnycfrn leenccvrpl yidfrqdlgw kwvhepkgyy anfcsgpcpy lrsadtthst 
               
               
                   
               
               
                 361 
                 vlglyntlnp easaspccvp qdlepltily yvgrtpkveq lsnmvvksck cs 
               
            
           
         
       
     
     By “TGFβ3 polynucleotide” is meant a polynucleotide encoding a TGFβ3 polypeptide. An exemplary TGFβ3 polynucleotide sequence is provided at NCBI Accession No. NG 011715.1. The exemplary sequence provided at NCBI Accession No. BT007287.1 is reproduced below (SEQ ID NO: 12). 
     
       
         
           
               
               
               
            
               
                 1 
                 atgaagatgc acttgcaaag ggctctggtg gtcctggccc tgctgaactt tgccacggtc 
                   
               
               
                   
               
               
                 61 
                 agcctctctc tgtccacttg caccaccttg gacttcggcc acatcaagaa gaagagggtg 
               
               
                   
               
               
                 121 
                 gaagccatta ggggacagat cttgagcaag ctcaggctca ccagcccccc tgagccaacg 
               
               
                   
               
               
                 181 
                 gtgatgaccc acgtccccta tcaggtcctg gccctttaca acagcacccg ggagctgctg 
               
               
                   
               
               
                 241 
                 gaggagatgc atggggagag ggaggaaggc tgcacccagg aaaacaccga gtcggaatac 
               
               
                   
               
               
                 301 
                 tatgccaaag aaatccataa attcgacatg atccaggggc tggcggagca caacgaactg 
               
               
                   
               
               
                 361 
                 gctgtctgcc ctaaaggaat tacctccaag gttttccgct tcaatgtgtc ctcagtggag 
               
               
                   
               
               
                 421 
                 aaaaatagaa ccaacctatt ccgagcagaa ttccgggtct tgcgggtgcc caaccccagc 
               
               
                   
               
               
                 481 
                 tctaagcgga atgagcagag gatcgagctc ttccagatcc ttcggccaga tgagcacatt 
               
               
                   
               
               
                 541 
                 gccaaacagc gctatatcgg tggcaagaat ctgcccacac ggggcactgc cgagtggctg 
               
               
                   
               
               
                 601 
                 tcctttgatg tcactgacac tgtgcgtgag tggctgttga gaagagagtc caacttaggt 
               
               
                   
               
               
                 661 
                 ctagaaatca gcattcactg tccatgtcac acctttcagc ccaatggaga tatcctggaa 
               
               
                   
               
               
                 721 
                 aacattcacg aggtgatgga aatcaaattc aaaggcgtgg acaatgagga tgaccatggc 
               
               
                   
               
               
                 781 
                 cgtggagatc tggggcgcct caagaagcag aaggatcacc acaaccctca tctaatcctc 
               
               
                   
               
               
                 841 
                 atgatgattc ccccacaccg gctcgacaac ccgggccagg ggggtcagag gaagaagcgg 
               
               
                   
               
               
                 901 
                 gctttggaca ccaattactg cttccggtag 
               
            
           
         
       
     
     By “TGFβR1 polypeptide” is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to GenBank Accession No. AAH71181.1 and having a biological activity of a TGFβR1 polypeptide. Biological activities of a TGFβR1 polypeptide include binding to ligands TGFβ1, TGFβ2, and TGFβ3 polypeptides, and transduction of a signal from TGFβ1, TGFβ2, or TGFβ3 polypeptide binding from the cell surface to the cytoplasm. The sequence at GenBank Accession No. AAH71181.1 is shown below (SEQ ID NO: 13): 
     
       
         
           
               
               
               
            
               
                 1 
                 meaavaaprp rllllvlaaa aaaaaallpg atalqcfchl ctkdnftcvt dglcfvsvte 
                   
               
               
                   
               
               
                 61 
                 ttdkvihnsm ciaeidlipr drpfvcapss ktgsvtttyc cnqdhcnkie lpttglpllv 
               
               
                   
               
               
                 121 
                 qrtiartivl qesigkgrfg evwrgkwrge evavkifssr eerswfreae iyqtvmlrhe 
               
               
                   
               
               
                 181 
                 nilgfiaadn kdngtwtqlw lvsdyhehgs lfdylnrytv tvegmiklal stasglahlh 
               
               
                   
               
               
                 241 
                 meivgtqgkp aiahrdlksk nilvkkngtc ciadlglavr hdsatdtidi apnhrvgtkr 
               
               
                   
               
               
                 301 
                 ymapevldds inmkhfesfk radiyamglv fweiarrcsi ggihedyqlp yydlvpsdps 
               
               
                   
               
               
                 361 
                 veemrkvvce qklrpnipnr wqscealrvm akimrecwya ngaarltalr ikktlsqlsq 
               
               
                   
               
               
                 421 
                 qegikm 
               
            
           
         
       
     
     By “TGFβR1 polynucleotide” is meant a polynucleotide encoding a TGFβR1 polypeptide. An exemplary TGFβR1 polynucleotide sequence is provided at GenBank Accession No. BC071181.1. The exemplary sequence provided at GenBank Accession No. BC071181.1 is reproduced below (SEQ ID NO: 14). 
     
       
         
           
               
               
               
            
               
                 1 
                 gcggcggcta gggaggtggg gcgaggcgag gtttgctggg gtgaggcagc ggcgcggccg 
                   
               
               
                   
               
               
                 61 
                 ggccgggccg ggccacaggc ggtggcggcg ggaccatgga ggcggcggtc gctgctccgc 
               
               
                   
               
               
                 121 
                 gtccccggct gctcctcctc gtgctggcgg cggcggcggc ggcggcggcg gcgctgctcc 
               
               
                   
               
               
                 181 
                 cgggggcgac ggcgttacag tgtttctgcc acctctgtac aaaagacaat tttacttgtg 
               
               
                   
               
               
                 241 
                 tgacagatgg gctctgcttt gtctctgtca cagagaccac agacaaagtt atacacaaca 
               
               
                   
               
               
                 301 
                 gcatgtgtat agctgaaatt gacttaattc ctcgagatag gccgtttgta tgtgcaccct 
               
               
                   
               
               
                 361 
                 cttcaaaaac tgggtctgtg actacaacat attgctgcaa tcaggaccat tgcaataaaa 
               
               
                   
               
               
                 421 
                 tagaacttcc aactactggt ttaccattgc ttgttcagag aacaattgcg agaactattg 
               
               
                   
               
               
                 481 
                 tgttacaaga aagcattggc aaaggtcgat ttggagaagt ttggagagga aagtggcggg 
               
               
                   
               
               
                 541 
                 gagaagaagt tgctgttaag atattctcct ctagagaaga acgttcgtgg ttccgtgagg 
               
               
                   
               
               
                 601 
                 cagagattta tcaaactgta atgttacgtc atgaaaacat cctgggattt atagcagcag 
               
               
                   
               
               
                 661 
                 acaataaaga caatggtact tggactcagc tctggttggt gtcagattat catgagcatg 
               
               
                   
               
               
                 721 
                 gatccctttt tgattactta aacagataca cagttactgt ggaaggaatg ataaaacttg 
               
               
                   
               
               
                 781 
                 ctctgtccac ggcgagcggt cttgcccatc ttcacatgga gattgttggt acccaaggaa 
               
               
                   
               
               
                 841 
                 agccagccat tgctcataga gatttgaaat caaagaatat cttggtaaag aagaatggaa 
               
               
                   
               
               
                 901 
                 cttgctgtat tgcagactta ggactggcag taagacatga ttcagccaca gataccattg 
               
               
                   
               
               
                 961 
                 atattgctcc aaaccacaga gtgggaacaa aaaggtacat ggcccctgaa gttctcgatg 
               
               
                   
               
               
                 1021 
                 attccataaa tatgaaacat tttgaatcct tcaaacgtgc tgacatctat gcaatgggct 
               
               
                   
               
               
                 1081 
                 tagtattctg ggaaattgct cgacgatgtt ccattggtgg aattcatgaa gattaccaac 
               
               
                   
               
               
                 1141 
                 tgccttatta tgatcttgta ccttctgacc catcagttga agaaatgaga aaagttgttt 
               
               
                   
               
               
                 1201 
                 gtgaacagaa gttaaggcca aatatcccaa acagatggca gagctgtgaa gccttgagag 
               
               
                   
               
               
                 1261 
                 taatggctaa aattatgaga gaatgttggt atgccaatgg agcagctagg cttacagcat 
               
               
                   
               
               
                 1321 
                 tgcggattaa gaaaacatta tcgcaactca gtcaacagga aggcatcaaa atgtaattct 
               
               
                   
               
               
                 1381 
                 acagctttgc ctgaactctc cttttttctt cagatctgct cctgggtttt aatttgggag 
               
               
                   
               
               
                 1441 
                 gtcaattgtt ctacctcact gagagggaac agaaggatat tgcttccttt tgcagcagtg 
               
               
                   
               
               
                 1501 
                 taataaagtc aattaaaaac ttcccaggat ttctttggac ccaggaaaca gccatgtggg 
               
               
                   
               
               
                 1561 
                 tcctttctgt gcactatgaa cgcttctttc ccaggacaga aaatgtgtag tctaccttta 
               
               
                   
               
               
                 1621 
                 ttttttatta acaaaacttg ttttttaaaa agatgattgc tggtcttaac tttaggtaac 
               
               
                   
               
               
                 1681 
                 tctgctgtgc tggagatcat ctttaagggc aaaggagttg gattgctgaa ttacaatgaa 
               
               
                   
               
               
                 1741 
                 acatgtctta ttactaaaga aagtgattta ctcctggtta gtacattctc agaggattct 
               
               
                   
               
               
                 1801 
                 gaaccactag agtttccttg attcagactt tgaatgtact gttctatagt ttttcaggat 
               
               
                   
               
               
                 1861 
                 cttaaaacta acacttataa aactcttatc ttgagtctaa aaatgacctc atatagtagt 
               
               
                   
               
               
                 1921 
                 gaggaacata attcatgcaa ttgtattttg tatactatta ttgttctttc acttattcag 
               
               
                   
               
               
                 1981 
                 aacattacat gccttcaaaa tgggattgta ctataccagt aagtgccact tctgtgtctt 
               
               
                   
               
               
                 2041 
                 tctaatggaa atgagtagaa ttgctgaaag tctctatgtt aaaacctata gtgtttgaat 
               
               
                   
               
               
                 2101 
                 tcaaaaagct tatttatctg ggtaacccaa actttttctg ttttgttttt ggaagggttt 
               
               
                   
               
               
                 2161 
                 ttgtggtatg tcatttggta ttctattctg aaaatgcctt tctcctacca aaatgtgctt 
               
               
                   
               
               
                 2221 
                 aagccactaa agaaatgaag tggcattaat tagtaaatta ttagcatggt catgtttgaa 
               
               
                   
               
               
                 2281 
                 tattctcaca tcaagctttt gcattttaat tgtgttgtct aagtatactt ttaaaaaatc 
               
               
                   
               
               
                 2341 
                 aagtggcact ctagatgctt atagtacttt aatatttgta gcatacagac taatttttct 
               
               
                   
               
               
                 2401 
                 aaaagggaaa gtctgtctag ctgcttgtga aaagttatgt ggtattctgt aagccatttt 
               
               
                   
               
               
                 2461 
                 tttctttatc tgttcaaaga cttatttttt aagacatgaa ttacatttaa aattagaata 
               
               
                   
               
               
                 2521 
                 tggttaatat taaataatag gcctttttct aggaaggcga aggtagttaa taatttgaat 
               
               
                   
               
               
                 2581 
                 agataacaga tgtgcaagaa agtcacattt gttatgtatg taggagtaaa cgttcggtgg 
               
               
                   
               
               
                 2641 
                 atcctctgtc tttgtaactg aggttagagc tagtgtggtt ttgaggtctc actacacttt 
               
               
                   
               
               
                 2701 
                 gaggaaggca gcttttaatt cagtgtttcc ttatgtgtgc gtacattgca actgcttaca 
               
               
                   
               
               
                 2761 
                 tgtaatttat gtaatgcatt cagtgcaccc ttgttacttg ggagaggtgg tagctaaaga 
               
               
                   
               
               
                 2821 
                 acattctgag tataggtttt tctccattta cagatgtctt tggtcaaata ttgaaagcaa 
               
               
                   
               
               
                 2881 
                 acttgtcatg gtcttcttac attaagttga aactagctta taataactgg tttttacttc 
               
               
                   
               
               
                 2941 
                 caatgctatg aagtctctgc agggctttta cagttttcga agtcctttta tcactgtgat 
               
               
                   
               
               
                 3001 
                 cttattctga ggggagaaaa aactatcata gctctgaggc aagacttcga ctttatagtg 
               
               
                   
               
               
                 3061 
                 ctatcagttc cccgatacag ggtcagagta acccatacag tattttggtc aggaagagaa 
               
               
                   
               
               
                 3121 
                 agtggccatt tacactgaat gagttgcatt ctgataatgt cttatctctt atacgtagaa 
               
               
                   
               
               
                 3181 
                 taaatttgaa agactatttg atcttaaaac caaagtaatt ttagaatgag tgacatatta 
               
               
                   
               
               
                 3241 
                 cataggaatt tagtgtcaat ttcatgtgtt taaaaacatc atgggaaaaa tgcttagagg 
               
               
                   
               
               
                 3301 
                 ttactatttt gactacaaag ttgagttttt ttctgtagtt accataattt cattgaagca 
               
               
                   
               
               
                 3361 
                 aatgaatgag tttgagaggt ttgtttttat agttgtgttg tattacttgt ttaataataa 
               
               
                   
               
               
                 3421 
                 tctctaattc tgtgatcagg tacttttttt gtgggggttt tttttttgtt tttttttttt 
               
               
                   
               
               
                 3481 
                 tttgttgttg tttttgggcc atttctaagc ctaccagatc tgctttatga aatccagggg 
               
               
                   
               
               
                 3541 
                 accaatgcat tttatcacta aaactatttt tatataattt taagaatata ccaaaagttg 
               
               
                   
               
               
                 3601 
                 tctgatttaa agttgtaata catgatttct cactttcatg taaggttatc cacttttgct 
               
               
                   
               
               
                 3661 
                 gaagatattt tttattgaat caaagattga gttacaatta tacttttctt acctaagtgg 
               
               
                   
               
               
                 3721 
                 ataaaatgta cttttgatga atcagggaat ttttttaaag ttggagttta gttctaaatt 
               
               
                   
               
               
                 3781 
                 gactttacgt attactgcag ttaattcctt ttttggctag ggatggtttg ataaaccaca 
               
               
                   
               
               
                 3841 
                 attggctgat attgaaaatg aaagaaactt aaaaggtggg atggatcatg attactgtcg 
               
               
                   
               
               
                 3901 
                 ataactgcag ataaatttga ttagagtaat aattttgtca tttaaaaaca cagttgttta 
               
               
                   
               
               
                 3961 
                 tactgcccat cctaggatgc tcaccttcca agattcaacg tggctaaaac atcttctggt 
               
               
                   
               
               
                 4021 
                 aaattgtgcg tccatattca ttttgtcagt agccaggaga aatggggatg ggggaaatac 
               
               
                   
               
               
                 4081 
                 gacttagtga ggcatagaca tccctggtcc atcctttctg tctccagctg tttcttggaa 
               
               
                   
               
               
                 4141 
                 cctgctctcc tgcttgctgg tccctgacgc agagaccgtt gcctccccca cagccgtttg 
               
               
                   
               
               
                 4201 
                 actgaaggct gctctggaga cctagagtaa aacggctgat ggaagttgtg ggacccactt 
               
               
                   
               
               
                 4261 
                 ccatttcctt cagtcattag aggtggaagg gaggggtctc caagtttgga gattgagcag 
               
               
                   
               
               
                 4321 
                 atgaggcttg ggatgcccct gctttgactt cagccatgga tgaggagtgg gatggcagca 
               
               
                   
               
               
                 4381 
                 aggtggctcc tgtggcagtg gagttgtgcc agaaacagtg gccagttgta tcgcctataa 
               
               
                   
               
               
                 4441 
                 gacagggtaa ggtctgaaga gctgagcctg taattctgct gtaataatga tagtgctcaa 
               
               
                   
               
               
                 4501 
                 gaagtgcctt gagttggtgt acagtgccat ggccatcaag aatcccagat ttcaggtttt 
               
               
                   
               
               
                 4561 
                 attacaaaat gtaagtggtc acttggcgat tttgtagtac atgcatgagt tacctttttt 
               
               
                   
               
               
                 4621 
                 ctctatgtct gagaactgtc agattaaaac aagatggcaa agagatcgtt agagtgcaca 
               
               
                   
               
               
                 4681 
                 acaaaatcac tatcccatta gacacatcat caaaagctta tttttattct tgcactggaa 
               
               
                   
               
               
                 4741 
                 gaatcgtaag tcaactgttt cttgaccatg gcagtgttct ggctccaaat ggtagtgatt 
               
               
                   
               
               
                 4801 
                 ccaaataatg gttctgttaa cactttggca gaaaatgcca gctcagatat tttgagatac 
               
               
                   
               
               
                 4861 
                 taaggattat ctttggacat gtactgcagc ttcttgtctc tgttttggat tactggaata 
               
               
                   
               
               
                 4921 
                 cccatgggcc ctctcaagag tgctggactt ctaggacatt aagatgattg tcagtacatt 
               
               
                   
               
               
                 4981 
                 aaacttttca atcccattat gcaatcttgt ttgtaaatgt aaacttctaa aaatatggtt 
               
               
                   
               
               
                 5041 
                 aataacattc aacctgttta ttacaactta aaaggaactt cagtgaattt gtttttattt 
               
               
                   
               
               
                 5101 
                 tttaacaaga tttgtgaact gaatatcatg aaccatgttt tgatacccct ttttcacgtt 
               
               
                   
               
               
                 5161 
                 gtgccaacgg aatagggtgt ttgatatttc ttcatatgtt aaggagatgc ttcaaaatgt 
               
               
                   
               
               
                 5221 
                 caattgcttt aaacttaaat tacctctcaa gagaccaagg tacatttacc tcattgtgta 
               
               
                   
               
               
                 5281 
                 tataatgttt aatatttgtc agagcattct ccaggtttgc agttttattt ctataaagta 
               
               
                   
               
               
                 5341 
                 tgggtattat gttgctcagt tactcaaatg gtactgtatt gtttatattt gtaccccaaa 
               
               
                   
               
               
                 5401 
                 taacatcgtc tgtactttct gttttctgta ttgtatttgt gcaggattct ttaggcttta 
               
               
                   
               
               
                 5461 
                 tcagtgtaat ttctgccttt taagatatgt acagaaaatg tccatataaa tttccattga 
               
               
                   
               
               
                 5521 
                 agtcgaatga tactgagaag cctgtaaaga ggagaaaaaa cataagctgt gtttccccat 
               
               
                   
               
               
                 5581 
                 aagttttttt aaattgtata ttgtatttgt agtaatattc caaaagaatg taaataggaa 
               
               
                   
               
               
                 5641 
                 atagaagagt gatgcttatg ttaagtccta acactacagt agaagaatgg aagcagtgca 
               
               
                   
               
               
                 5701 
                 aataaattac atttttccca aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa gaaaaaaaaa 
               
               
                   
               
               
                 5761 
                 aaaaaa 
               
            
           
         
       
     
     By “TGFβR2 polypeptide” is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to GenBank Accession No. ABG65632.1 and having a biological activity of a TGFβR2 polypeptide. Biological activities of a TGFβR2 polypeptide include binding to TGFβR1 polypeptide to form a heterodimeric complex, and serine/threonine kinase activity. The sequence at GenBank Accession No. ABG65632.1 is shown below (SEQ ID NO: 15): 
     
       
         
           
               
               
               
            
               
                 1 
                 mgrgllrglw plhivlwtri astipphvqk svnndmivtd nngavkfpql ckfcdvrfst 
                   
               
               
                   
               
               
                 61 
                 cdnqkscmsn csitsicekp qevcvavwrk ndenitletv chdpklpyhd filedaaspk 
               
               
                   
               
               
                 121 
                 cimkekkkpg etffmcscss decndniifs eeyntsnpdl llvifqvtgi sllpplgvai 
               
               
                   
               
               
                 181 
                 sviiifycyr vnrqqklsst wetgktrklm efsehcaiil eddrsdisst canninhnte 
               
               
                   
               
               
                 241 
                 llpieldtlv gkgrfaevyk aklkqntseq fetvavkifp yeeyaswkte kdifsdinlk 
               
               
                   
               
               
                 301 
                 henilqflta eerktelgkq ywlitafhak gnlqeyltrh viswedlrkl gsslargiah 
               
               
                   
               
               
                 361 
                 lhsdhtpcgr pkmpivhrdl kssnilvknd ltcclcdfgl slrldptlsv ddlansgqvg 
               
               
                   
               
               
                 421 
                 tarymapevl esrmnlenve sfkqtdvysm alvlwemtsr cnavgevkdy eppfgskvre 
               
               
                   
               
               
                 481 
                 hpcvesmkdn vlrdrgrpei psfwlnhqgi qmvcetltec wdhdpearlt aqcvaerfse 
               
               
                   
               
               
                 541 
                 lehldrlsgr scseekiped gslnttk 
               
            
           
         
       
     
     By “TGFβR2 polynucleotide” is meant a polynucleotide encoding a TGFβR2 polypeptide. An exemplary TGFβR2 polynucleotide sequence is provided at GenBank Accession No. DQ377553.1. The exemplary sequence provided at GenBank Accession No. DQ377553.1 is reproduced below (SEQ ID NO: 16). 
     
       
         
           
               
            
               
                 CCTCCTGGCTGGCGAGCGGGCGCCACATCTGGCCCGCACATCTGCG 
               
               
                   
               
               
                 CTGCCGGCCCGGCGCGGGGTCCGGAGAGGGCGCGGCGCGGAGGCGC 
               
               
                   
               
               
                 AGCCAGGGGTCCGGGAAGGCGCCGTCCGCTGCGCTGGGGGCTCGGT 
               
               
                   
               
               
                 CTATGACGAGCAGCGGGGTCTGCCATGGGTCGGGGGCTGCTCAGGG 
               
               
                   
               
               
                 GCCTGTGGCCGCTGCACATCGTCCTGTGGACGCGTATCGCCAGCAC 
               
               
                   
               
               
                 GATCCCACCGCACGTTCAGAAGTCGGGTGAGTGGTCCCCAGCCCGG 
               
               
                   
               
               
                 GCTCGGCGGGGCGCCGGGGGTCTTCCTGGGGTCCCCGCCTCTCCGC 
               
               
                   
               
               
                 TGCGCTTGACAGTCGGGCCCGGCAACCCGGCCCCCGGGCGGAAACG 
               
               
                   
               
               
                 AGGAAAGTTTCCCCCGCGACACTCACGCAGCCCGACTCCCGTAGCT 
               
               
                   
               
               
                 GCAGGGATTGTGAGTTTTTCTTGAAAAAGAGAAGGAAAGTTCAGTT 
               
               
                   
               
               
                 GCAAGGGGCGCGGGGCACGTTTGGTCC 
               
            
           
         
       
     
     As used herein, the term “rapamycin” refers to a compound (a macrocyclic triene antibiotic also known as Sirolimus) produced by the bacterium  Streptomyces hygroscopicus . It inhibits the activation of T cells and B cells by reducing the production of interleukin-2 (IL-2). Rapamycin has immunosuppressant functions in humans and is especially useful in medicine for preventing organ transplant rejection such as the rejection of kidney transplants. It is also used to treat lymphangioleiomyomatosis, a lung progressive and systemic disease. Rapamycin has also been shown to inhibit proliferation of vascular smooth muscle cells migration (Poon M. et al., J Clin Invest. 1996; 98(10):2277-83). Rapamycin derivatives used according to the methods of present invention include, but are not limited to, 40-O-alkyl-rapamycin derivatives, e.g. 40-O-hydroxyalkyl-rapamycin derivatives, for example 40-O-(2-hydroxy)-ethyl-rapamycin (everolimus), rapamycin derivatives which are substituted in 40 position by heterocyclyl, e.g. 40-epi-(tetrazolyi)-rapamycin (also known as ABT578), 32-deoxo-rapamycin derivatives and 32-hydroxy-rapamycin derivatives, such as 32-deoxorapamycin, 16-O-substituted rapamycin derivatives such as 16-pent-2-ynyloxy-32-deoxorapamycin, 16-pent-2-ynyloxy-32(S or R)-dihydro-rapamycin, or 16-pent-2-ynyloxy-32(S or R)-dihydro-40-O-(2-hydroxyethyl)-rapamycin, rapamycin derivatives which are acylated at the oxygen in position 40, e.g. 40-[3-hydroxy-2-(hydroxy-methyl)-2-methylpropanoate]-rapamycin (also known as CCI779 or temsirolimus), rapamycin derivatives as disclosed in WO9802441 or WO0114387 (also sometimes designated as rapalogs), e.g. including AP23573, such as 40-O-dimethylphosphinyl-rapamycin, compounds disclosed under the name biolimus (biolimus A9), including 40-O-(2-ethoxy)ethyl-rapamycin, and compounds disclosed under the name TAFA-93, AP23464, AP23675 or AP23841; or rapamycin derivatives as e.g. disclosed in WO2004101583, WO9205179, WO9402136, WO9402385 and WO9613273. 
     By “subject” is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, murine, or feline. 
     Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. 
     As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated. 
     Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms “a”, “an”, and “the” are understood to be singular or plural. 
     Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about. 
     The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. 
     Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein. 
     DETAILED DESCRIPTION 
     Without wishing to be limited by theory, it has now been shown that endothelial-to-mesenchymal transition (EndMT) plays a role in pulmonary arterial hypertension (PAH). Accordingly, the invention provides methods of treating PAH using agents that prevent or reduce EndMT. 
     Therapeutic Strategy for Treating Pulmonary Arterial Hypertension 
     Described herein are studies demonstrating the key role of FGF signaling, let-7 miRNA expression, and TGFβ signaling in the progression of PAH by induction of endothelial-to-mesenchymal transition (EndMT) in endothelial cells and by promotion of a proliferative phenotype in smooth muscle cells 
     Provided herein are methods to arrest PAH by inhibiting EndMT or smooth muscle cell proliferation using a therapeutic strategy applicable to large numbers of patients. As shown in the attached figures and associated legends, EndMT plays a role in PAH and modulating this pathway fundamentally changes the course of the disease. The mechanism involves a link between FGF signaling, let-7 miRNA, and TGFβ signaling. In various embodiments, targeting this mechanism provides opportunities for the treatment and prevention of PAH. 
     Endothelial-to-Mesenchymal Transition 
     The endothelial-to-mesenchymal transition (EndMT) is induced by activation of endothelial TGFβ signaling that occurs secondary to the loss of a protective FGF input. In healthy vessels, FGF suppresses TGFβ signaling by inducing the let-7 family of miRNAs that reduce expression of key TGFβ pathway proteins (TGFβ2, TGFβR1, Smad2). The importance of the FGF-let-7-TGFβ link is supported by human and mouse data. 
     Thus, in some embodiments, the TGFβ signaling is blocked by delivering let-7 miRNA into a cell. In a particular embodiment, the cell is an endothelial cell. In a particular embodiment, a systemic treatment strategy using a modified let-7 miRNA delivered to endothelial cells in targeted nanoparticles is employed. In some embodiments, the modified let-7 miRNA is mi-let-7b L  or mi-let-7b H . 
     In some embodiments, the therapy is cell-type specific. Systemic inhibition of TGFβ signaling has an adverse effect by promoting inflammation and smooth muscle cell proliferation. In some embodiments, TGFβR1/2 targeted siRNAs are delivered to endothelial cells. 
     In some embodiments, the TGFβ signaling is activated by delivering to a cell an inhibitory polynucleotide that reduces SMC expression of FRS2α polypeptide or reduces SMC expression of a let-7 miRNA. In some embodiments, the TGFβ signaling is activated by delivering to an SMC an agent that increases the activity or level of a TGFβ signaling polypeptide. In a particular embodiment, the cell is an smooth muscle cell. 
     Methods of Treatment 
     In some aspects, the present invention provides a method of treating pulmonary arterial hypertension and/or disorders or symptoms thereof which comprise administering a therapeutically effective amount of a pharmaceutical composition comprising an agent that modulates the activity or level of a TGFβ signaling polypeptide, a let-7 miRNA, or a FGF signaling polypeptide in a cell, to a subject (e.g., a mammal such as a human). 
     In particular embodiments, the agent that modulates the activity or level of a let-7 miRNA increases the activity or level of a let-7 miRNA in a cell. In some embodiments, the cell is an endothelial cell. In certain embodiments, the agent that increases the activity or level of a let-7 miRNA in a cell is a let-7 miRNA mimic. In some other embodiments, the agent is a polynucleotide encoding a let-7b miRNA. In some embodiments, the let-7 miRNA is let-7b and let-7c miRNA. 
     In some embodiments, the agent that modulates the activity or level of a let-7 miRNA decreases the activity or level of a let-7 miRNA in a cell. In certain embodiments, the cell is a smooth muscle cell. In some embodiments, the agent that decreases the activity or level of a let-7 miRNA in a cell is an inhibitory polynucleotide that reduces expression of let-7 miRNA. In still other embodiments, the agent that decreases the activity or level of a let-7 miRNA in a cell is a let-7 miRNA sponge or antagomir-let-7b/c. Such miRNA sponges are described in, for example, Ebert et al. RNA. 2010 November; 16(11): 2043-2050. In some embodiments, the let-7 miRNA is let-7b miRNA. 
     In some embodiments, the agent that modulates the activity or level of a TGFβ signaling polypeptide increases the activity or level of a TGFβ signaling polypeptide in a cell (in particular, a smooth muscle cell). In some other embodiments, the agent that modulates the activity or level of a TGFβ signaling polypeptide decreases the activity or level of a TGFβ signaling polypeptide in a cell (in particular, an endothelial cell). In some embodiments, the TGFβ signaling polypeptide is TGFβ1, TGFβ2, TGFβ3, TGFβR1, or TGFβR2. In some embodiments, the agent is siRNA and may be targeted to a TGFβ receptor. 
     In some embodiments, the agent that decreases the activity or level of a TGFβ signaling polypeptide is an inhibitory polynucleotide that reduces expression of a TGFβ signaling polypeptide. In some other embodiments, the agent that increases the activity or level of a TGFβ signaling polypeptide is a polynucleotide encoding a TGFβ signaling polypeptide. 
     In certain embodiments, the agent that modulates the activity or level of a FGF signaling polypeptide decreases the activity or level of a FGF signaling polypeptide in a cell (in particular, a smooth muscle cell). In some embodiments, the agent that modulates the activity or level of a FGF signaling polypeptide increases the activity or level of a FGF signaling polypeptide in a cell (in particular, an endothelial cell). In some embodiments, the FGF signaling polypeptide is FRS2α. 
     In certain embodiments, the agent that decreases the activity or level of a FGF signaling polypeptide in a cell is an inhibitory polynucleotide that reduces expression of a FGF signaling polypeptide. In some other embodiments, the agent that increases the activity or level of a FGF signaling polypeptide in a cell is a polynucleotide encoding a FGF signaling polypeptide. 
     In some embodiments, the subject is pre-selected by assessing the activity or level of a TGFβ signaling polypeptide or polynucleotide, a let-7 miRNA, or a FGF signaling polypeptide or polynucleotide in a sample from the subject when compared to reference levels. 
     The subject is pre-selected when an alteration in the activity or level of activity or level of a TGFβ signaling polypeptide or polynucleotide, a let-7 miRNA, or a FGF signaling polypeptide or polynucleotide in a sample from the subject is detected. In some embodiments, the subject is pre-selected when a decrease in the activity or level of let-7 miRNA or a TGFβ signaling polypeptide is observed relative to reference levels in an endothelial cell sample obtained from the subject. In other embodiments, the subject is pre-selected when a decrease in the activity or level of a FGF signaling polypeptide or polynucleotide, or an increase in the activity or level of let-7 miRNA or a TGFβ signaling polypeptide or polynucleotide is observed relative to reference levels in a smooth muscle cell sample obtained from the subject. 
     In some aspects, the subject is administered an additional agent comprising a therapeutically effective amount of an mTOR inhibitor. In some aspects of the invention, the subject is administered an additional agent comprising a therapeutically effective amount of rapamycin or any derivative thereof. In some embodiments, the therapeutically effective amount of rapamycin or any derivative thereof is used to reduce SMC proliferation and increase its differentiation alone or in combination with EC-specific therapies. In some embodiments, the agent that decreases the activity or level of a TGFβ signaling polypeptide and the additional agent are co-administered to the subject. 
     In other aspects of the invention, the agent that decreases the activity or level of a TGFβ signaling polypeptide is a nucleic acid capable of downregulating the gene expression of at least one gene selected from the group consisting of TGFβ1, TGFβ2, TGFβ3, TGFβR1, and TGFβR2. In some embodiments, the at least one gene is selected from the group consisting of TGFβR1, and TGFβR2. 
     In some instance, downregulation of the TGFβ or TGFβ receptor (TGFβR) gene expression is desired. This downregulation may result from a full or partial knock down of the gene of interest. Briefly, a gene knock down refers to a genetic technique in which one of an organism&#39;s genes is silenced, made inoperative or partially inoperative. Gene expression may be downregulated, knocked-down, decreased, and/or inhibited by various well-established molecular techniques known in the art such as, but not limited to, RNA interference (RNAi); small inhibitor RNA (siRNA), small hairpin RNA (shRNA) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs)). 
     In some embodiments, the nucleic acid is selected from the group consisting of an antisense RNA, siRNA, shRNA, and a CRISPR system. In other embodiments, the nucleic acid is combined with a therapeutically effective amount of rapamycin or any derivative thereof. In yet other embodiments, the nucleic acid is encapsulated in a nanoparticle formulated for selective delivery to an endothelial cell, in a pharmaceutically acceptable excipient. In further embodiments, the nanoparticle is a 7C1 nanoparticle. 
     The methods disclosed herein include administering to the subject (including a subject identified as in need of such treatment) an effective amount of an agent described herein, or a composition described herein to produce such effect. Identifying a subject in need of such treatment can be made by a health care professional and may be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method, such as using the methods described herein). 
     The therapeutic methods of the invention, which may also include prophylactic treatment, in general comprise administering a therapeutically effective amount of one or more of the agents herein (such as an agent that modulates the activity or level of a TGFβ signaling polypeptide, a let-7 miRNA, or a FGF signaling polypeptide) to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human. Such treatment is suitable for subjects, particularly humans, suffering from, having, susceptible to, or at risk for PAH. In one embodiment, the invention provides a method of monitoring progression of treatment. The method comprises determining a level or activity of diagnostic marker (e.g., a TGFβ signaling polypeptide or polynucleotide, a let-7 miRNA, or a FGF signaling polypeptide or polynucleotide) in a subject suffering from or susceptible to PAH, in which the subject has been administered a therapeutic or effective amount of a therapeutic agent sufficient to treat PAH. The activity or level of a TGFβ signaling polypeptide or polynucleotide, a let-7 miRNA, or a FGF signaling polypeptide or polynucleotide determined in the method can be compared to a known activity or level of a TGFβ signaling polypeptide or polynucleotide, a let-7 miRNA, or a FGF signaling polypeptide or polynucleotide in either healthy normal controls, or in other afflicted patients, to establish the subject&#39;s disease status. In some embodiments, an activity or level of a TGFβ signaling polypeptide or polynucleotide, a let-7 miRNA, or a FGF signaling polypeptide or polynucleotide in an endothelial cell or smooth muscle cell sample obtained from the subject is determined. In some embodiments, a second activity or level of a TGFβ signaling polypeptide or polynucleotide, a let-7 miRNA, or a FGF signaling polypeptide or polynucleotide in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy. In certain embodiments, a pre-treatment activity or level of a TGFβ signaling polypeptide or polynucleotide, a let-7 miRNA, or a FGF signaling polypeptide or polynucleotide is determined prior to commencing. This pre-treatment level can then be compared to the level of a TGFβ signaling polynucleotide or polypeptide or let-7 miRNA in the subject after the treatment commences, to determine the progress or efficacy of the treatment. 
     Pharmaceutical Compositions 
     The present invention features compositions useful for treating PAH in a pre-selected subject. The compositions include an agent that modulates the activity or level of a TGFβ signaling polypeptide, a let-7 miRNA, or a FGF signaling polypeptide in a cell. 
     In particular embodiments, the agent that modulates the activity or level of a let-7 miRNA increases the activity or level of a let-7 miRNA in a cell, in particular, an endothelial cell. In certain embodiments, the agent that increases the activity or level of a let-7 miRNA in a cell is a let-7 miRNA mimic. In some other embodiments, the agent is a polynucleotide encoding a let-7b miRNA. In certain embodiments, the agent that modulates the activity or level of a let-7 miRNA decreases the activity or level of a let-7 miRNA in a cell, in particular, a smooth muscle cell. In some embodiments, the agent that decreases the activity or level of a let-7 miRNA in a cell is an inhibitory polynucleotide that reduces expression of let-7 miRNA. In some embodiments, the let-7 miRNA is let-7b miRNA. 
     In some embodiments, the agent that modulates the activity or level of a TGFβ signaling polypeptide increases the activity or level of a TGFβ signaling polypeptide in a cell (in particular, a smooth muscle cell). In some other embodiments, the agent that modulates the activity or level of a TGFβ signaling polypeptide decreases the activity or level of a TGFβ signaling polypeptide in a cell (in particular, an endothelial cell). In some embodiments, the TGFβ signaling polypeptide is TGFβ1, TGFβ2, TGFβ3, TGFβR1, or TGFβR2. 
     In some embodiments, the agent that decreases the activity or level of a TGFβ signaling polypeptide is an inhibitory polynucleotide that reduces expression of a TGFβ signaling polypeptide. In some other embodiments, the agent that increases the activity or level of a TGFβ signaling polypeptide is a polynucleotide encoding a TGFβ signaling polypeptide. 
     In certain embodiments, the agent that modulates the activity or level of a FGF signaling polypeptide decreases the activity or level of a FGF signaling polypeptide in a cell (in particular, a smooth muscle cell). In some embodiments, the agent that modulates the activity or level of a FGF signaling polypeptide increases the activity or level of a FGF signaling polypeptide in a cell (in particular, an endothelial cell). In some embodiments, the FGF signaling polypeptide is FRS2α. 
     In certain embodiments, the agent that decreases the activity or level of a FGF signaling polypeptide in a cell is an inhibitory polynucleotide that reduces expression of a FGF signaling polypeptide. In some other embodiments, the agent that increases the activity or level of a FGF signaling polypeptide in a cell is a polynucleotide encoding an FGF signaling polypeptide 
     The composition may be administered systemically, for example, formulated in a pharmaceutically-acceptable buffer such as physiological saline. Routes of administration include, for example, subcutaneous, intravenous, intraperitoneally, intramuscular, or intradermal injections that provide continuous, sustained levels of the agent in the patient. 
     The amount of the therapeutic agent to be administered varies depending upon the manner of administration, the age and body weight of the patient, and with the clinical symptoms of PAH. Generally, amounts will be in the range of those used for other agents used in the treatment of PAH, although in certain instances lower amounts will be needed because of the increased specificity of the agent. A composition is administered at a dosage that decreases effects or symptoms of PAH as determined by a method known to one skilled in the art. 
     The therapeutic agent may be contained in any appropriate amount in any suitable carrier substance, and is generally present in an amount of 1-95% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for parenteral (e.g., subcutaneously, intravenously, intramuscularly, or intraperitoneally) administration route. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams &amp; Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York). 
     Pharmaceutical compositions according to the invention may be formulated to release the active agent substantially immediately upon administration or at any predetermined time or time period after administration. The latter types of compositions are generally known as controlled release formulations, which include (i) formulations that create a substantially constant concentration of the drug within the body over an extended period of time; (ii) formulations that after a predetermined lag time create a substantially constant concentration of the drug within the body over an extended period of time; (iii) formulations that sustain action during a predetermined time period by maintaining a relatively, constant, effective level in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the active substance (sawtooth kinetic pattern); (iv) formulations that localize action by, e.g., spatial placement of a controlled release composition adjacent to or in contact with an organ, such as the heart; (v) formulations that allow for convenient dosing, such that doses are administered, for example, once every one or two weeks; and (vi) formulations that target PAH using carriers or chemical derivatives to deliver the therapeutic agent to a particular cell type (e.g., endothelial cells or smooth muscle cells). For some applications, controlled release formulations obviate the need for frequent dosing during the day to sustain the plasma level at a therapeutic level. 
     Any of a number of strategies can be pursued in order to obtain controlled release in which the rate of release outweighs the rate of metabolism of the agent in question. In one example, controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings. Thus, the therapeutic is formulated with appropriate excipients into a pharmaceutical composition that, upon administration, releases the therapeutic in a controlled manner. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes. 
     The pharmaceutical composition may be administered parenterally by injection, infusion or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, or the like) in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants. The pharmaceutical composition of this invention could be coated or comprised in a drug-eluting stent (DES) ((Nikam et al., 2014 Med Devices 7:165-78)) that releases at a given site (such as an artery) and pace (i.e. slow release) the composition of this invention. 
     The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation. Formulations can be found in Remington: The Science and Practice of Pharmacy, supra. 
     Compositions for parenteral use may be provided in unit dosage forms (e.g., in single-dose ampoules), or in vials containing several doses and in which a suitable preservative may be added (see below). The composition may be in the form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use. Apart from the active agent that reduces or ameliorates PAH, the composition may include suitable parenterally acceptable carriers and/or excipients. The active therapeutic agent(s) may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release. Furthermore, the composition may include suspending, solubilizing, stabilizing, pH-adjusting agents, tonicity adjusting agents, and/or dispersing agents. 
     In some embodiments, the composition of this invention is delivered locally from, but not limited to, the strut of a stent, a stent graft, a stent cover or a stent sheath. In some embodiments, the composition of this invention comprises a rapamycin or a derivative thereof (e.g. as described in U.S. Pat. No. 6,273,913 B1, incorporated herein by reference). 
     In some embodiments, the composition comprising the active therapeutic is formulated for intravenous delivery. As indicated above, the pharmaceutical compositions according to the invention may be in the form suitable for sterile injection. To prepare such a composition, the suitable therapeutic(s) are dissolved or suspended in a parenterally acceptable liquid vehicle. Among acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer&#39;s solution, and isotonic sodium chloride solution and dextrose solution. The aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). In cases where one of the agents is only sparingly or slightly soluble in water, a dissolution enhancing or solubilizing agent can be added, or the solvent may include 10-60% w/w of propylene glycol or the like. 
     Polynucleotide Therapy 
     In some embodiments, the invention includes a method for treating, slowing the progression of, or reversing PAH, where a therapeutic polynucleotide activity or level of a TGFβ signaling polypeptide, a let-7 miRNA, or a FGF signaling polypeptide is administered to the subject. In certain embodiments, the polynucleotide is a let-7 miRNA mimic; a polynucleotide encoding let-7 miRNA, a TGFβ signaling polypeptide, or FGF signaling polypeptide; or an inhibitory polynucleotide that reduces expression of a FGF signaling polypeptide, a let-7 miRNA, or a TGFβ signaling polypeptide. Inhibitory polynucleotides include, but are not limited to siRNAs that target a polynucleotide encoding a TGFβ signaling polypeptide, a let-7 miRNA, or a FGF signaling polypeptide. 
     In particular embodiments, the polynucleotide therapy comprises a let-7 miRNA, a polynucleotide encoding a let-7 miRNA, or an inhibitory polynucleotide that reduces expression of a TGFβ signaling polypeptide. Such therapeutic polynucleotides can be delivered to cells of a subject having PAH. The nucleic acid molecules are delivered to the cells of a subject in a form by which they are taken up by the cells so that therapeutically effective levels of the inhibitory nucleic acid molecules are contained within the cells. 
     Introduction of nucleic acids into cells may be accomplished using any number of methods available in the art. For example, transducing viral (e.g., retroviral, adenoviral, and adeno-associated viral) vectors can be used for somatic cell gene therapy, especially because of their high efficiency of infection and stable integration and expression (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71:6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A. 94:10319, 1997). For example, an inhibitory nucleic acid or miRNA (or a precursor to the miRNA) as described can be cloned into a retroviral vector where expression can be driven from its endogenous promoter, from the retroviral long terminal repeat, or from a promoter specific for a target cell type of interest. In some embodiments, the target cell type of interest is an endothelial cell. Other viral vectors that can be used to introduce nucleic acids into cells include, but are not limited to, vaccinia virus, bovine papilloma virus, or herpes virus, such as Epstein-Barr Virus (also see, for example, the vectors of Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244:1275-1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1:55-61, 1990; Sharp, The Lancet 337:1277-1278, 1991; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller et al., Biotechnology 7:980-990, 1989; Le Gal La Salle et al., Science 259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995). Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Pat. No. 5,399,346). In some embodiments, a viral vector is used to administer a polynucleotide encoding inhibitory nucleic acid molecules that inhibit expression of TGFβ signaling polypeptide. 
     Non-viral approaches can also be employed for the introduction of the therapeutic to a cell of a patient requiring treatment of PAH. For example, a nucleic acid molecule can be introduced into a cell by administering the nucleic acid in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology 101:512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989), or by micro-injection under surgical conditions (Wolff et al., Science 247:1465, 1990). In some embodiments, the nucleic acids are administered in combination with a liposome and protamine. 
     Gene transfer can also be achieved using non-viral means involving transfection in vitro. Such methods include the use of calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes can also be potentially beneficial for delivery of DNA into a cell. Transplantation of polynucleotide encoding inhibitory nucleic acid molecules into the affected tissues of a patient can also be accomplished by transferring a polynucleotide encoding the inhibitory nucleic acid into a cultivatable cell type ex vivo (e.g., an autologous or heterologous primary cell or progeny thereof), after which the cell (or its descendants) are injected into a targeted tissue. 
     cDNA expression for use in polynucleotide therapy methods can be directed from any suitable promoter (e.g., the human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters), and regulated by any appropriate mammalian regulatory element. For example, if desired, enhancers known to preferentially direct gene expression in specific cell types can be used to direct the expression of a nucleic acid. The enhancers used can include, without limitation, those that are characterized as tissue- or cell-specific enhancers. Alternatively, if a genomic clone is used as a therapeutic construct, regulation can be mediated by the cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above. 
     In some embodiments, the therapeutic polynucleotide is selectively targeted to an endothelial cell. In some other embodiments, the therapeutic polynucleotide is expressed in an endothelial cell using a lentiviral vector. In still other embodiments, the therapeutic polynucleotide is administered intravenously. In some embodiments, the therapeutic polynucleotide contains one or more chemical modifications that reduce immunostimulation, enhance serum stability, increase specificity, and/or improve activity, while still retaining silencing activity. Such chemical modifications are described in, for example, Foster et al., RNA. 2012 March; 18(3): 557-568. In some embodiments, the therapeutic polynucleotide contains one or more chemical modifications to prevent degradation, as described in Chen et al., Cell Reports 2012; 2(6)1684-1696. 
     In a particular embodiment, the therapeutic polynucleotide is selectively delivered to endothelial cells using nanoparticles formulated for selective targeting to endothelial cells, such as a 7C1 nanoparticle. Selective targeting or expression of polynucleotides to an endothelial cell is described in, for example, Dahlman et al., Nat Nanotechnol. 2014 August; 9(8): 648-655. 
     In some other embodiments, the therapeutic polynucleotide is selectively targeted to a smooth muscle cell. The therapeutic polynucleotide can be selectively delivered to a smooth muscle cell using tissue factor-targeted nanoparticles that can penetrate and bind stretch-activated vascular smooth muscles as described in Lanza et al., Circulation. 2002 Nov. 26; 106(22):2842-7. 
     In General 
     The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow. 
     OTHER EMBODIMENTS 
     The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. 
     The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations. 
     Attorney Docket No. 047162-7221US1(01568) Preliminary Amendment