{"gene":"ACTA2","run_date":"2026-06-09T22:02:39","timeline":{"discoveries":[{"year":2015,"finding":"The R258C mutation in smooth muscle α-actin (SM α-actin, ACTA2) disrupts actin filament stability: R258C filaments are less stable than WT, more susceptible to severing by cofilin, and smooth muscle tropomyosin provides little protection from cofilin cleavage of mutant filaments. Profilin binds tighter to the R258C monomer, increasing the pool of G-actin. In an in vitro motility assay, smooth muscle myosin moves R258C filaments more slowly than WT, and under loaded conditions small ensembles of myosin are unable to produce force on R258C actin-tropomyosin filaments, suggesting tropomyosin occupies an inhibitory position on mutant actin. These defects are allosteric—many cannot be explained by direct interaction with the mutated residue.","method":"TIRF microscopy (single-filament growth assay), in vitro motility assay, baculovirus-expressed recombinant protein, cofilin severing assay, profilin-binding assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted in vitro with purified mutant protein, multiple orthogonal functional assays (TIRF, motility, severing, binding), rigorous mechanistic dissection in a single focused study","pmids":["26153420"],"is_preprint":false},{"year":2021,"finding":"The common ACTA2 variant p.Arg149Cys (R149C) causes increased retention of mutant SM α-actin in the chaperonin-containing TCP1 (CCT) folding complex, reducing the amount of mutant protein that reaches functional levels in smooth muscle cells. This explains reduced penetrance: enhanced CCT binding lowers mutant monomer levels, minimizing its effect on SMC function. In vitro motility assays confirmed decreased interaction of R149C mutant filaments with SM myosin. TIRF microscopy showed enhanced nucleation of R149C SM α-actin by formin, correlating with disorganized and reduced actin filaments in Acta2R149C/+ SMCs.","method":"CRISPR/Cas9 knock-in mouse model, in vitro motility assay, TIRF microscopy, chaperonin-binding retention assay, aortic contraction assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (TIRF, motility, CCT-binding retention, in vivo mouse model) in one rigorous study establishing a novel molecular mechanism","pmids":["34600884"],"is_preprint":false},{"year":2025,"finding":"The ACTA2 R179H pathogenic variant causes a dramatic phenotypic switch in human iPSC-derived smooth muscle cells from a contractile to a synthetic state, associated with increased proliferation and migration and reduced contractility. CRISPR adenine base editing (ABE8e-SpCas9-VRQR) correcting R179H prevented this phenotypic switch and restored normal SMC function in vitro. In humanized R179H mice, in vivo AAV9-delivered base editing rescued aortic dilation/dissection, bladder enlargement, gut dilation, and hydronephrosis.","method":"iPSC-derived SMC differentiation, CRISPR adenine base editing, AAV9 in vivo delivery, functional contractility/migration/proliferation assays, humanized knock-in mouse model","journal":"Circulation","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — reconstitution with iPSC-derived SMCs plus in vivo rescue in humanized mice, multiple orthogonal functional endpoints, mechanistic causality established","pmids":["40378078"],"is_preprint":false},{"year":2025,"finding":"SMCs carrying the Acta2 R179C mutation fail to fully differentiate and maintain stem cell-like features including increased migration and elevated glycolytic flux compared to WT SMCs. Boosting mitochondrial oxidative respiration with nicotinamide riboside (NR) drives differentiation and decreases migration of mutant SMCs. In an Acta2SMC-R179C/+ mouse carotid injury model, mutant mice develop intraluminal SMC accumulation causing moyamoya-like occlusive lesions, neurological symptoms, and neuron loss; NR treatment prevents all of these phenotypes.","method":"SMC-specific knock-in mouse model, carotid artery injury, nicotinamide riboside treatment, migration/differentiation/metabolic flux assays, histology, neurological scoring","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — SMC-specific in vivo knock-in model with carotid injury, pharmacological rescue with mechanistic metabolic endpoint, multiple orthogonal phenotypic readouts","pmids":["40603847"],"is_preprint":false},{"year":2013,"finding":"Purine-rich element binding protein B (Purβ) represses ACTA2 transcription by cooperatively binding the sense (purine-rich) strand of the ACTA2 5′ promoter-enhancer MCAT cis-element as a homodimer with three separate ssDNA-binding modules formed by inter- and intramolecular subdomain interactions. Purβ knockdown in mouse embryo fibroblasts promoted myofibroblast-like morphology, increased ACTA2 expression (confirmed by promoter-reporter assay), and increased cell migration. Discrete Purβ subdomains mediating ssDNA binding, protein-protein interaction with corepressor YBX1, and ACTA2 enhancer inhibition were mapped.","method":"shRNA stable knockdown, promoter-reporter assay, recombinant truncation mutants, biochemical/biophysical ssDNA binding assays, computational structural modeling","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — multiple orthogonal biochemical and cell-based methods (knockdown + reporter + biophysical binding), structural subdomain mapping, single lab","pmids":["23724822"],"is_preprint":false},{"year":2016,"finding":"Purβ represses ACTA2 transcription through electrostatic and hydrophobic interactions with the purine-rich ssDNA of the MCAT element in the Acta2 promoter. Site-directed mutagenesis of basic residues R267 (intermolecular subdomain) and K82/R159 (intramolecular subdomains) reduced both ssDNA binding affinity and Acta2 repressor activity in fibroblast promoter-reporter assays. R267A mutation additionally impaired binding to the Acta2 corepressor YBX1.","method":"Site-directed mutagenesis, quantitative ssDNA binding assays (salt/pH/detergent titration), promoter-reporter assay in fibroblasts, purified recombinant Purβ variants","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstitution with purified mutant proteins, mutagenesis + functional validation in cells, mechanistic residue-level dissection; single lab","pmids":["27064749"],"is_preprint":false},{"year":2013,"finding":"ACTA2 is required for myofibroblast cell motility and contraction in hepatic stellate cells. Inhibition of Acta2 by multiple knockdown techniques reduced cellular motility and contraction without affecting other cytoplasmic actin isoforms. Acta2 knockdown was also associated with a significant reduction in ERK1/2 phosphorylation, indicating ACTA2 regulates signaling (MAPK pathway) in addition to its structural role.","method":"In vitro wound healing and contraction assays, siRNA/shRNA knockdown, ERK1/2 phosphorylation (Western blot), in vivo liver injury model","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — multiple knockdown approaches and two orthogonal functional readouts (motility + contraction + signaling), single lab","pmids":["24204762"],"is_preprint":false},{"year":2022,"finding":"Cardiac fibroblast-specific deletion of Acta2 does not prevent myofibroblast differentiation or impair post-MI cardiac repair. Acta2-null cardiac myofibroblasts show normal proliferation, migration, and contractility and a normal total filamentous actin level because deletion triggers compensatory transcriptional upregulation of non-Acta2 actin isoforms (particularly Actg2 and Acta1). MRTF-A is critical for myofibroblast differentiation but is not required for this compensatory response.","method":"Tamoxifen-inducible cardiac fibroblast-specific Acta2 knockout mouse, post-MI survival/histology/function, proliferation/migration/contractility assays, RT-qPCR for actin isoforms","journal":"Journal of molecular and cellular cardiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional cell-type-specific KO with multiple functional readouts and mechanistic follow-up (isoform compensation), well-controlled in vivo study","pmids":["36007455"],"is_preprint":false},{"year":2015,"finding":"TGF-β2 treatment of lens epithelial cells increases histone H4 acetylation specifically at the ACTA2 promoter region (assessed by ChIP), correlating with increased ACTA2 mRNA and protein expression and EMT. The HDAC inhibitor trichostatin-A (TSA) suppresses TGF-β2-induced ACTA2 upregulation and EMT while globally elevating acetylated H4 (but reducing H4 acetylation at the ACTA2 promoter under TGF-β2 stimulation).","method":"Chromatin immunoprecipitation (ChIP) for H4 acetylation at ACTA2 promoter, RT-qPCR/Western blot for ACTA2, TSA treatment, cell migration assay","journal":"Eye (London, England)","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — ChIP directly at ACTA2 promoter is Tier 2, but single lab with limited follow-up","pmids":["25853442"],"is_preprint":false},{"year":2016,"finding":"EGFR/HER2 dimerization induces ACTA2 expression through a JAK2/STAT1 signaling pathway in breast cancer cells. HER2 overexpression increases both STAT1 and ACTA2 protein levels; STAT1 inhibition (fludarabine) or JAK2 inhibition (AG490) decreases basal ACTA2 expression, and STAT1 overexpression increases ACTA2. ACTA2 knockdown suppresses cell motility in vitro and reduces lung metastatic nodules in vivo.","method":"HER2 transient/stable overexpression, siRNA knockdown, JAK2/STAT1 inhibitors, ACTA2 shRNA, in vivo mouse lung metastasis model","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — pathway placement by pharmacological inhibition and genetic OE/KD, in vivo validation; single lab","pmids":["28881584"],"is_preprint":false},{"year":2013,"finding":"ACTA2 expression in lung adenocarcinoma cells is required for metastatic potential: ACTA2 knockdown impairs in vitro migration, invasion, clonogenicity, and transendothelial penetration without affecting proliferation, and reduces in vivo metastatic potential. ACTA2 downregulation reduces c-MET and FAK expression in lung adenocarcinoma cells and is accompanied by loss of mesenchymal characteristics.","method":"shRNA/siRNA knockdown of ACTA2, in vitro migration/invasion/transendothelial assays, in vivo metastasis model (PC14PE6 cells), Western blot for c-MET and FAK","journal":"Clinical cancer research","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — loss-of-function with multiple functional readouts and in vivo validation plus downstream pathway (c-MET/FAK) analysis; single lab","pmids":["23995859"],"is_preprint":false},{"year":2018,"finding":"Deletion of ACTA2 in mice promotes angiotensin II-induced aortic lumen dilation, with increased expression of osteopontin (OPN), elevated Bax/Bcl-2 ratio, increased VSMC apoptosis, and phenotypic modulation of VSMCs compared to WT mice receiving AngII. Baseline ACTA2 knockout mice had no severe vascular phenotype.","method":"ACTA2 knockout mouse model, AngII osmotic minipump infusion, ultrasound lumen measurement, RT-qPCR/Western blot, TUNEL apoptosis assay, immunohistochemistry","journal":"Journal of thoracic disease","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — in vivo KO model with defined phenotypic readouts but single lab and limited mechanistic depth","pmids":["30233845"],"is_preprint":false},{"year":2020,"finding":"ACTA2 downregulation in neural stem cells (NSCs) inhibits migration by impeding actin filament polymerization via increased RhoA expression and decreased Rac1 expression, placing ACTA2 upstream of RhoA/Rac1 GTPase balance in NSC cytoskeletal regulation.","method":"siRNA knockdown of ACTA2 in primary NSCs, migration assay, RT-PCR/immunostaining for ACTA2, RhoA/Rac1 expression analysis","journal":"Stem cells international","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single knockdown approach, signaling pathway inferred from expression changes rather than direct epistasis experiments","pmids":["32508931"],"is_preprint":false},{"year":2021,"finding":"ACTA2 pathogenic variants (ACTA2, MYH11) in transdifferentiated VSMC-like cells show impaired migration velocity and reduced contractility (ACTA2) and decreased SMAD2 phosphorylation in ACTA2 cells, providing functional evidence that ACTA2 mutations directly impair SMC contractile and migratory function.","method":"Fibroblast transdifferentiation to SMC-like cells, cytoskeletal integrity assessment, TGFβ signaling (SMAD2 phosphorylation) assay, migration velocity assay, contraction assay","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — patient-derived cells with multiple functional assays and pathway readout; single lab with orthogonal methods","pmids":["34244757"],"is_preprint":false},{"year":2024,"finding":"Novel ACTA2 missense variants associated with TAAD act through a dominant-negative mechanism on yeast actin, disrupting actin cytoskeletal organization and mitochondrial distribution. Wild-type yeast expressing heterozygous mutant ACTA2 alleles showed significant increases in cells with abnormal mitochondrial distribution and abnormal actin cytoskeleton organization, consistent with dominant-negative interference with WT actin function.","method":"S. cerevisiae heterozygous expression assay, spot growth test, fluorescence microscopy of actin cytoskeleton and mitochondrial morphology","journal":"European journal of human genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — yeast model with functional cytoskeletal and organellar readouts, establishes dominant-negative mechanism; single lab, heterologous system","pmids":["38486025"],"is_preprint":false},{"year":2015,"finding":"3D molecular modeling of the actin filament structure revealed that the R179 residue is positioned at the interface between the two strands of filamentous actin, and the R179H mutation destabilizes inter-strand bundling, providing a structural explanation for the severe vascular phenotype associated with this mutation.","method":"Actin three-dimensional molecular modeling (computational structural analysis), correlated with histopathological findings in patient tissue","journal":"Acta neuropathologica communications","confidence":"Low","confidence_rationale":"Tier 4 / Weak — computational modeling only, no experimental structural validation; correlation with pathology is descriptive","pmids":["26637293"],"is_preprint":false},{"year":1990,"finding":"The vascular smooth muscle actin gene (ACTSA/ACTA2) was assigned to human chromosome 10, specifically the long arm at q22-q24, by Southern blot analysis of rodent-human somatic cell hybrids and in situ hybridization.","method":"Southern blot of somatic cell hybrids, chromosomal in situ hybridization","journal":"Jinrui idengaku zasshi. The Japanese journal of human genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — two orthogonal cytogenetic methods (somatic cell hybrids + in situ hybridization), foundational chromosomal assignment","pmids":["2398629"],"is_preprint":false},{"year":2022,"finding":"Transient angiotensin II infusion causes sustained downregulation of ACTA2 (α-smooth muscle actin) in aortic tissue beyond AngII withdrawal, associated with increased H3K27me3 at aortic nuclei and decreased myocardin (MYOCD) expression, indicating epigenetic silencing of ACTA2 as a 'vascular memory' mechanism. This was reproduced in cultured human aortic VSMCs.","method":"Mouse AngII infusion model with post-infusion follow-up, RNAseq aortic profiling, RT-qPCR/immunohistochemistry validation, H3K27me3 immunostaining, human VSMC cell culture","journal":"Frontiers in cardiovascular medicine","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — in vivo and in vitro with epigenetic endpoint (H3K27me3), replicated in human cells; mechanistic depth limited to correlation","pmids":["35360022"],"is_preprint":false},{"year":2023,"finding":"In Hirschsprung disease (HSCR) aganglionic segments, ACTA2 expression is abnormally elevated specifically in circular smooth muscle beginning at embryonic day E15.5 in Ednrb−/− mice. siRNA knockdown of Acta2 in intestinal smooth muscle cells (iSMCs) weakens their contraction ability, demonstrating that elevated ACTA2 directly drives hyperactive contraction in aganglionic bowel.","method":"Immunohistochemistry in HSCR patients and Ednrb−/− mice, siRNA knockdown of Acta2 in iSMCs, contraction functional assay","journal":"Pediatric surgery international","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — loss-of-function with direct contractility readout in disease-relevant cells, corroborated by in vivo mouse model; single lab","pmids":["37278766"],"is_preprint":false},{"year":2014,"finding":"RHOA knockdown significantly downregulates ACTA2 gene expression in both osteoblast-like and osteoclast-like cells, placing RHOA upstream of ACTA2 in a bone cell regulatory pathway.","method":"siRNA knockdown of RHOA, microarray analysis, qRT-PCR validation in multiple human bone cell lines","journal":"PloS one","confidence":"Low","confidence_rationale":"Tier 3 / Weak — knockdown with expression readout only, no direct functional assay on ACTA2 protein; single lab","pmids":["24840563"],"is_preprint":false}],"current_model":"ACTA2 encodes smooth muscle α-actin (SM α-actin), the predominant actin isoform in vascular smooth muscle cells and myofibroblasts, where it forms stable filaments in the contractile apparatus; pathogenic variants (e.g., R258C, R149C, R179H) disrupt filament stability, reduce myosin-driven force generation, impair SMC differentiation/quiescence, and promote aberrant SMC proliferation and migration—mechanisms established by in vitro reconstitution assays, TIRF microscopy, knock-in mouse models, and iPSC-derived SMC studies—while ACTA2 transcription is repressed by Purβ homodimers binding the MCAT enhancer element through electrostatic and hydrophobic ssDNA interactions and is epigenetically regulated by histone acetylation at its promoter and H3K27 methylation under angiotensin II stimulation."},"narrative":{"mechanistic_narrative":"ACTA2 encodes smooth muscle α-actin, the dominant actin isoform of vascular smooth muscle cells and myofibroblasts, where it provides the structural filament that supports myosin-driven contraction and cell motility [PMID:26153420, PMID:24204762]. Pathogenic missense variants act largely through allosteric and dominant-negative effects on filament behavior: R258C destabilizes filaments, sensitizes them to cofilin severing, expands the G-actin pool by tighter profilin binding, and slows myosin-driven motility [PMID:26153420]; R149C is additionally retained by the CCT chaperonin folding complex, lowering mutant monomer levels and reducing penetrance while enhancing aberrant formin-driven nucleation [PMID:34600884]; and modeling of R179 places it at the inter-strand filament interface, consistent with its severe phenotype [PMID:26637293]. Functionally, these variants drive a contractile-to-synthetic phenotypic switch in SMCs, increasing proliferation and migration while reducing contractility — a switch that is reversible by base-editing correction of R179H in iPSC-SMCs and humanized mice, and by metabolic rescue (boosting oxidative respiration with nicotinamide riboside) in R179C SMCs [PMID:40378078, PMID:40603847, PMID:34244757]. Beyond its structural role, ACTA2 loss perturbs cytoskeletal signaling, reducing ERK1/2 phosphorylation in myofibroblasts and altering RhoA/Rac1 balance [PMID:24204762]. ACTA2 transcription is repressed by the purine-rich element binding protein Purβ, which binds the purine-rich strand of the promoter MCAT cis-element as a homodimer through electrostatic and hydrophobic ssDNA interactions and recruits the corepressor YBX1 [PMID:23724822, PMID:27064749], and is epigenetically tuned by histone H4 acetylation at its promoter and by H3K27 trimethylation that mediates sustained angiotensin II-induced silencing [PMID:25853442, PMID:35360022]. In some cell types Acta2 is functionally dispensable, as cardiac fibroblast-specific deletion is compensated by upregulation of other actin isoforms [PMID:36007455].","teleology":[{"year":1990,"claim":"Established the genomic location of the vascular smooth muscle actin gene, providing the foundation for linking ACTA2 to human disease loci.","evidence":"Southern blot of somatic cell hybrids and chromosomal in situ hybridization","pmids":["2398629"],"confidence":"High","gaps":["No functional or mechanistic characterization","Does not address protein-level activity"]},{"year":2013,"claim":"Resolved how ACTA2 transcription is held in check, showing Purβ homodimers cooperatively bind the purine-rich strand of the MCAT promoter element and recruit YBX1 to repress expression.","evidence":"shRNA knockdown, promoter-reporter assays, recombinant truncation mutants, and biophysical ssDNA binding in fibroblasts","pmids":["23724822"],"confidence":"High","gaps":["Repression characterized in fibroblasts, not vascular SMCs","Physiological signals controlling Purβ occupancy not defined"]},{"year":2013,"claim":"Demonstrated that ACTA2 is functionally required for myofibroblast motility and contraction independent of other actin isoforms, and unexpectedly modulates MAPK signaling.","evidence":"Multiple knockdown approaches with motility/contraction assays and ERK1/2 Western blot in hepatic stellate cells plus liver injury model","pmids":["24204762"],"confidence":"Medium","gaps":["Mechanism linking ACTA2 to ERK1/2 phosphorylation unresolved","Single lab"]},{"year":2013,"claim":"Extended ACTA2's role beyond contraction to cancer cell metastasis, showing it is required for migration, invasion, and transendothelial penetration in lung adenocarcinoma.","evidence":"shRNA/siRNA knockdown with in vitro invasion assays and in vivo metastasis model, c-MET/FAK Western blot","pmids":["23995859"],"confidence":"Medium","gaps":["Whether c-MET/FAK changes are direct or downstream of motility loss unclear","Single lab"]},{"year":2015,"claim":"Defined the molecular pathophysiology of a TAAD-causing variant, showing R258C destabilizes filaments and impairs myosin force generation through allosteric effects rather than direct contacts at the mutated residue.","evidence":"TIRF single-filament assays, in vitro motility, cofilin severing and profilin binding with baculovirus-expressed recombinant protein","pmids":["26153420"],"confidence":"High","gaps":["Allosteric structural basis not resolved atomically","Single variant studied in reconstitution"]},{"year":2015,"claim":"Connected histone acetylation to ACTA2 induction, showing TGF-β2 increases H4 acetylation at the promoter and HDAC inhibition blocks ACTA2 upregulation and EMT.","evidence":"ChIP for H4 acetylation at the ACTA2 promoter with TSA treatment and migration assays in lens epithelial cells","pmids":["25853442"],"confidence":"Medium","gaps":["Specific HAT/HDAC enzymes not identified","Limited follow-up, single lab"]},{"year":2016,"claim":"Resolved the chemical basis of Purβ repression to specific basic residues mediating ssDNA contacts and YBX1 corepressor binding.","evidence":"Site-directed mutagenesis of R267/K82/R159 with quantitative ssDNA binding titrations and promoter-reporter assays","pmids":["27064749"],"confidence":"High","gaps":["In vivo relevance of individual residues not tested","Single lab"]},{"year":2016,"claim":"Placed ACTA2 downstream of a receptor signaling cascade, showing EGFR/HER2 dimerization induces ACTA2 via JAK2/STAT1 to drive breast cancer cell motility and metastasis.","evidence":"HER2 overexpression, JAK2/STAT1 pharmacological inhibition, STAT1 overexpression, ACTA2 shRNA and in vivo metastasis model","pmids":["28881584"],"confidence":"Medium","gaps":["Direct STAT1 binding at ACTA2 promoter not demonstrated","Single lab"]},{"year":2018,"claim":"Tested the in vivo consequence of ACTA2 loss under hemodynamic stress, showing knockout mice develop AngII-induced aortic dilation with VSMC apoptosis and phenotypic modulation.","evidence":"ACTA2 knockout mice with AngII infusion, ultrasound, TUNEL apoptosis and OPN/Bax-Bcl2 analysis","pmids":["30233845"],"confidence":"Medium","gaps":["No baseline phenotype, mechanism of stress sensitivity unclear","Limited mechanistic depth"]},{"year":2020,"claim":"Linked ACTA2 to RhoA/Rac1 GTPase balance in neural stem cell migration.","evidence":"siRNA knockdown in primary NSCs with migration assay and RhoA/Rac1 expression analysis","pmids":["32508931"],"confidence":"Low","gaps":["Signaling inferred from expression changes rather than epistasis","Single knockdown approach"]},{"year":2021,"claim":"Explained reduced penetrance of a common variant by showing R149C is retained in the CCT chaperonin, lowering functional mutant levels while still perturbing filament organization.","evidence":"CRISPR knock-in mouse, CCT-binding retention assay, in vitro motility, TIRF nucleation assay and aortic contraction","pmids":["34600884"],"confidence":"High","gaps":["How CCT retention varies across variants not generalized","Quantitative threshold for pathogenicity undefined"]},{"year":2021,"claim":"Provided patient-derived functional evidence that ACTA2 mutations directly impair SMC contractility and migration and reduce SMAD2 phosphorylation.","evidence":"Fibroblast transdifferentiation to SMC-like cells with cytoskeletal, TGFβ/SMAD2 and migration/contraction assays","pmids":["34244757"],"confidence":"Medium","gaps":["Link between actin defect and SMAD2 signaling unresolved","Single lab"]},{"year":2022,"claim":"Revealed cell-type-specific dispensability, showing cardiac fibroblast-specific Acta2 deletion is tolerated through compensatory upregulation of other actin isoforms.","evidence":"Tamoxifen-inducible cardiac fibroblast-specific knockout with post-MI functional readouts and RT-qPCR for actin isoforms","pmids":["36007455"],"confidence":"High","gaps":["Whether vascular SMCs mount similar compensation untested","Trigger of isoform compensation unknown"]},{"year":2022,"claim":"Identified an epigenetic 'vascular memory' mechanism in which transient AngII causes sustained ACTA2 silencing via H3K27me3 and reduced myocardin.","evidence":"Mouse AngII infusion with post-infusion follow-up, RNAseq, H3K27me3 immunostaining, replicated in human aortic VSMCs","pmids":["35360022"],"confidence":"Medium","gaps":["Methyltransferase responsible not identified","Causal role of H3K27me3 correlative"]},{"year":2023,"claim":"Showed that abnormally elevated ACTA2, not only its loss, is pathogenic, driving hyperactive contraction in aganglionic Hirschsprung bowel.","evidence":"Immunohistochemistry in HSCR patients and Ednrb−/− mice with siRNA knockdown and contraction assay in intestinal SMCs","pmids":["37278766"],"confidence":"Medium","gaps":["Upstream cause of ACTA2 elevation undefined","Single lab"]},{"year":2024,"claim":"Established a dominant-negative mechanism for TAAD-associated missense variants using a heterologous system, showing heterozygous mutant ACTA2 disrupts actin organization and mitochondrial distribution.","evidence":"S. cerevisiae heterozygous expression with spot growth and fluorescence microscopy of actin and mitochondria","pmids":["38486025"],"confidence":"Medium","gaps":["Yeast system may not capture mammalian SMC context","Mechanism of mitochondrial mispositioning unclear"]},{"year":2025,"claim":"Demonstrated reversibility of the disease phenotype, showing base-editing correction of R179H prevents the contractile-to-synthetic switch in iPSC-SMCs and rescues multi-organ pathology in humanized mice.","evidence":"iPSC-derived SMC differentiation, CRISPR adenine base editing, AAV9 in vivo delivery and humanized knock-in mouse rescue","pmids":["40378078"],"confidence":"High","gaps":["Durability and off-target profile of in vivo editing not fully defined","Single variant corrected"]},{"year":2025,"claim":"Linked the ACTA2-mutant phenotype to metabolic state, showing R179C SMCs fail to differentiate and rely on glycolysis, with oxidative-respiration boosting (nicotinamide riboside) restoring differentiation and preventing occlusive lesions.","evidence":"SMC-specific knock-in mouse with carotid injury, metabolic flux assays and nicotinamide riboside rescue","pmids":["40603847"],"confidence":"High","gaps":["Mechanism coupling actin mutation to glycolytic shift unresolved","Single variant studied"]},{"year":null,"claim":"How specific ACTA2 variants are mechanistically partitioned between loss-of-function, dominant-negative filament interference, and chaperonin-mediated reduction — and how these map onto the divergent vascular, cerebrovascular, gut, and bladder phenotypes — remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No unifying genotype-mechanism-phenotype framework across variants","Structural basis of allosteric defects not resolved atomically","Coupling between cytoskeletal defects and metabolic/signaling reprogramming undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,6,14]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0,1]}],"localization":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[0,1,14]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0]}],"pathway":[{"term_id":"R-HSA-397014","term_label":"Muscle contraction","supporting_discovery_ids":[0,1,13]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[4,5,8,17]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[2,3]}],"complexes":["CCT/TCP1 chaperonin (transient client)"],"partners":["MYH11","CCT","TPM (SMOOTH MUSCLE TROPOMYOSIN)","CFL (COFILIN)","PFN (PROFILIN)"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P62736","full_name":"Actin, aortic smooth muscle","aliases":["Alpha-actin-2","Cell growth-inhibiting gene 46 protein"],"length_aa":377,"mass_kda":42.0,"function":"Actins are highly conserved proteins that are involved in various types of cell motility and are ubiquitously expressed in all eukaryotic cells","subcellular_location":"Cytoplasm, cytoskeleton","url":"https://www.uniprot.org/uniprotkb/P62736/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ACTA2","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/ACTA2","total_profiled":1310},"omim":[{"mim_id":"620475","title":"THROMBOCYTOPENIA 8, WITH DYSMORPHIC FEATURES AND DEVELOPMENTAL DELAY; THC8","url":"https://www.omim.org/entry/620475"},{"mim_id":"620093","title":"ACTIN MATURATION PROTEASE; ACTMAP","url":"https://www.omim.org/entry/620093"},{"mim_id":"619656","title":"LOEYS-DIETZ SYNDROME 6; LDS6","url":"https://www.omim.org/entry/619656"},{"mim_id":"619365","title":"MEGACYSTIS-MICROCOLON-INTESTINAL HYPOPERISTALSIS SYNDROME 4; MMIHS4","url":"https://www.omim.org/entry/619365"},{"mim_id":"614042","title":"MOYAMOYA DISEASE 5; MYMY5","url":"https://www.omim.org/entry/614042"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Actin filaments","reliability":"Approved"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"blood vessel","ntpm":10509.9},{"tissue":"smooth 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Le journal canadien des sciences neurologiques","url":"https://pubmed.ncbi.nlm.nih.gov/30975232","citation_count":5,"is_preprint":false},{"pmid":"40378078","id":"PMC_40378078","title":"Genomic Editing of a Pathogenic Sequence Variant in ACTA2 Rescues Multisystemic Smooth Muscle Dysfunction Syndrome in Mice.","date":"2025","source":"Circulation","url":"https://pubmed.ncbi.nlm.nih.gov/40378078","citation_count":4,"is_preprint":false},{"pmid":"40785968","id":"PMC_40785968","title":"Human Amniotic Epithelial Stem Cell Exosomes Regulate Chondrocyte Ferroptosis through ACTA2-AS1-Targeted Binding to ACSL4 for Osteoarthritis Intervention.","date":"2025","source":"Research (Washington, D.C.)","url":"https://pubmed.ncbi.nlm.nih.gov/40785968","citation_count":4,"is_preprint":false},{"pmid":"38044429","id":"PMC_38044429","title":"A highly penetrant ACTA2 mutation of thoracic aortic disease.","date":"2023","source":"Journal of cardiothoracic surgery","url":"https://pubmed.ncbi.nlm.nih.gov/38044429","citation_count":4,"is_preprint":false},{"pmid":"36053285","id":"PMC_36053285","title":"Expanding the genetic and phenotypic spectrum of ACTA2-related vasculopathies in a Dutch cohort.","date":"2022","source":"Genetics in medicine : official journal of the American College of Medical Genetics","url":"https://pubmed.ncbi.nlm.nih.gov/36053285","citation_count":4,"is_preprint":false},{"pmid":"33342581","id":"PMC_33342581","title":"Refractory cerebral infarction in a child with an ACTA2 mutation.","date":"2020","source":"Brain & development","url":"https://pubmed.ncbi.nlm.nih.gov/33342581","citation_count":4,"is_preprint":false},{"pmid":"32595813","id":"PMC_32595813","title":"ACTA2 leukovasculopathy: A rare pediatric white matter disorder.","date":"2020","source":"Radiology case reports","url":"https://pubmed.ncbi.nlm.nih.gov/32595813","citation_count":4,"is_preprint":false},{"pmid":"34437965","id":"PMC_34437965","title":"The natural history of a family with aortic dissection associated with a novel ACTA2 variant.","date":"2021","source":"Annals of vascular surgery","url":"https://pubmed.ncbi.nlm.nih.gov/34437965","citation_count":4,"is_preprint":false},{"pmid":"36607831","id":"PMC_36607831","title":"Neonatal diagnosis of ACTA2-related disease: A case report and review of literature.","date":"2023","source":"American journal of medical genetics. Part A","url":"https://pubmed.ncbi.nlm.nih.gov/36607831","citation_count":3,"is_preprint":false},{"pmid":"32452246","id":"PMC_32452246","title":"Aneurysmal Dilatation of Ductus Arteriosus and Pulmonary Artery in Association With ACTA2 Mutation.","date":"2020","source":"World journal for pediatric & congenital heart surgery","url":"https://pubmed.ncbi.nlm.nih.gov/32452246","citation_count":3,"is_preprint":false},{"pmid":"40603847","id":"PMC_40603847","title":"Immature Acta2R179C/+ smooth muscle cells cause moyamoya-like cerebrovascular lesions in mice prevented by boosting OXPHOS.","date":"2025","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/40603847","citation_count":2,"is_preprint":false},{"pmid":"19586843","id":"PMC_19586843","title":"[Cloning and expression analysis of porcine ACTA2 gene and its association with production traits].","date":"2009","source":"Yi chuan = Hereditas","url":"https://pubmed.ncbi.nlm.nih.gov/19586843","citation_count":2,"is_preprint":false},{"pmid":"37886459","id":"PMC_37886459","title":"Augmenting Mitochondrial Respiration in Immature Smooth Muscle Cells with an ACTA2 Pathogenic Variant Mitigates Moyamoya-like Cerebrovascular Disease.","date":"2023","source":"Research square","url":"https://pubmed.ncbi.nlm.nih.gov/37886459","citation_count":2,"is_preprint":false},{"pmid":"35248443","id":"PMC_35248443","title":"Triple bypass for multisystem smooth muscle dysfunction syndrome due to Arg179His ACTA2 mutation.","date":"2022","source":"Journal of stroke and cerebrovascular diseases : the official journal of National Stroke Association","url":"https://pubmed.ncbi.nlm.nih.gov/35248443","citation_count":2,"is_preprint":false},{"pmid":"35420309","id":"PMC_35420309","title":"Clinical and neuroimaging features of a familial pathogenic ACTA2 variant as a model of a vascular neurocristopathy.","date":"2022","source":"Neuroradiology","url":"https://pubmed.ncbi.nlm.nih.gov/35420309","citation_count":2,"is_preprint":false},{"pmid":"32464348","id":"PMC_32464348","title":"Expanding the cerebrovascular phenotype of the p.R258H variant in ACTA2 related hereditary thoracic aortic disease (HTAD).","date":"2020","source":"Journal of the neurological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/32464348","citation_count":2,"is_preprint":false},{"pmid":"28343608","id":"PMC_28343608","title":"Management of Bilateral Axillary Aneurysms, Threatened Limb, and Diffuse Vasculopathy in a Patient With ACTA2 Mutation.","date":"2017","source":"The Canadian journal of cardiology","url":"https://pubmed.ncbi.nlm.nih.gov/28343608","citation_count":2,"is_preprint":false},{"pmid":"33299681","id":"PMC_33299681","title":"ACTA2 Mutation: Microsurgeon Beware!","date":"2020","source":"Plastic and reconstructive surgery. Global open","url":"https://pubmed.ncbi.nlm.nih.gov/33299681","citation_count":1,"is_preprint":false},{"pmid":"34546411","id":"PMC_34546411","title":"Nonsurgical treatment of cerebral ischemia associated with ACTA2 cerebral arteriopathy: a case report and literature review.","date":"2021","source":"Child's nervous system : ChNS : official journal of the International Society for Pediatric Neurosurgery","url":"https://pubmed.ncbi.nlm.nih.gov/34546411","citation_count":1,"is_preprint":false},{"pmid":"37672683","id":"PMC_37672683","title":"Aortic Dissection and a Previously Unreported ACTA2 Missense Variant Mutation in a Young Patient: A Case Report.","date":"2023","source":"Pediatric and developmental pathology : the official journal of the Society for Pediatric Pathology and the Paediatric Pathology Society","url":"https://pubmed.ncbi.nlm.nih.gov/37672683","citation_count":1,"is_preprint":false},{"pmid":"38486025","id":"PMC_38486025","title":"A yeast based assay establishes the pathogenicity of novel missense ACTA2 variants associated with aortic aneurysms.","date":"2024","source":"European journal of human genetics : EJHG","url":"https://pubmed.ncbi.nlm.nih.gov/38486025","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":50439,"output_tokens":5771,"usd":0.118941,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":14418,"output_tokens":5168,"usd":0.100645,"stage2_stop_reason":"end_turn"},"total_usd":0.219586,"stage1_batch_id":"msgbatch_0179Z2pxRYBsXi2xADneafRw","stage2_batch_id":"msgbatch_01LaY9ZaVdgjWArcZo9eqQE5","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2015,\n      \"finding\": \"The R258C mutation in smooth muscle α-actin (SM α-actin, ACTA2) disrupts actin filament stability: R258C filaments are less stable than WT, more susceptible to severing by cofilin, and smooth muscle tropomyosin provides little protection from cofilin cleavage of mutant filaments. Profilin binds tighter to the R258C monomer, increasing the pool of G-actin. In an in vitro motility assay, smooth muscle myosin moves R258C filaments more slowly than WT, and under loaded conditions small ensembles of myosin are unable to produce force on R258C actin-tropomyosin filaments, suggesting tropomyosin occupies an inhibitory position on mutant actin. These defects are allosteric—many cannot be explained by direct interaction with the mutated residue.\",\n      \"method\": \"TIRF microscopy (single-filament growth assay), in vitro motility assay, baculovirus-expressed recombinant protein, cofilin severing assay, profilin-binding assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted in vitro with purified mutant protein, multiple orthogonal functional assays (TIRF, motility, severing, binding), rigorous mechanistic dissection in a single focused study\",\n      \"pmids\": [\"26153420\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The common ACTA2 variant p.Arg149Cys (R149C) causes increased retention of mutant SM α-actin in the chaperonin-containing TCP1 (CCT) folding complex, reducing the amount of mutant protein that reaches functional levels in smooth muscle cells. This explains reduced penetrance: enhanced CCT binding lowers mutant monomer levels, minimizing its effect on SMC function. In vitro motility assays confirmed decreased interaction of R149C mutant filaments with SM myosin. TIRF microscopy showed enhanced nucleation of R149C SM α-actin by formin, correlating with disorganized and reduced actin filaments in Acta2R149C/+ SMCs.\",\n      \"method\": \"CRISPR/Cas9 knock-in mouse model, in vitro motility assay, TIRF microscopy, chaperonin-binding retention assay, aortic contraction assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (TIRF, motility, CCT-binding retention, in vivo mouse model) in one rigorous study establishing a novel molecular mechanism\",\n      \"pmids\": [\"34600884\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The ACTA2 R179H pathogenic variant causes a dramatic phenotypic switch in human iPSC-derived smooth muscle cells from a contractile to a synthetic state, associated with increased proliferation and migration and reduced contractility. CRISPR adenine base editing (ABE8e-SpCas9-VRQR) correcting R179H prevented this phenotypic switch and restored normal SMC function in vitro. In humanized R179H mice, in vivo AAV9-delivered base editing rescued aortic dilation/dissection, bladder enlargement, gut dilation, and hydronephrosis.\",\n      \"method\": \"iPSC-derived SMC differentiation, CRISPR adenine base editing, AAV9 in vivo delivery, functional contractility/migration/proliferation assays, humanized knock-in mouse model\",\n      \"journal\": \"Circulation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — reconstitution with iPSC-derived SMCs plus in vivo rescue in humanized mice, multiple orthogonal functional endpoints, mechanistic causality established\",\n      \"pmids\": [\"40378078\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SMCs carrying the Acta2 R179C mutation fail to fully differentiate and maintain stem cell-like features including increased migration and elevated glycolytic flux compared to WT SMCs. Boosting mitochondrial oxidative respiration with nicotinamide riboside (NR) drives differentiation and decreases migration of mutant SMCs. In an Acta2SMC-R179C/+ mouse carotid injury model, mutant mice develop intraluminal SMC accumulation causing moyamoya-like occlusive lesions, neurological symptoms, and neuron loss; NR treatment prevents all of these phenotypes.\",\n      \"method\": \"SMC-specific knock-in mouse model, carotid artery injury, nicotinamide riboside treatment, migration/differentiation/metabolic flux assays, histology, neurological scoring\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — SMC-specific in vivo knock-in model with carotid injury, pharmacological rescue with mechanistic metabolic endpoint, multiple orthogonal phenotypic readouts\",\n      \"pmids\": [\"40603847\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Purine-rich element binding protein B (Purβ) represses ACTA2 transcription by cooperatively binding the sense (purine-rich) strand of the ACTA2 5′ promoter-enhancer MCAT cis-element as a homodimer with three separate ssDNA-binding modules formed by inter- and intramolecular subdomain interactions. Purβ knockdown in mouse embryo fibroblasts promoted myofibroblast-like morphology, increased ACTA2 expression (confirmed by promoter-reporter assay), and increased cell migration. Discrete Purβ subdomains mediating ssDNA binding, protein-protein interaction with corepressor YBX1, and ACTA2 enhancer inhibition were mapped.\",\n      \"method\": \"shRNA stable knockdown, promoter-reporter assay, recombinant truncation mutants, biochemical/biophysical ssDNA binding assays, computational structural modeling\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — multiple orthogonal biochemical and cell-based methods (knockdown + reporter + biophysical binding), structural subdomain mapping, single lab\",\n      \"pmids\": [\"23724822\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Purβ represses ACTA2 transcription through electrostatic and hydrophobic interactions with the purine-rich ssDNA of the MCAT element in the Acta2 promoter. Site-directed mutagenesis of basic residues R267 (intermolecular subdomain) and K82/R159 (intramolecular subdomains) reduced both ssDNA binding affinity and Acta2 repressor activity in fibroblast promoter-reporter assays. R267A mutation additionally impaired binding to the Acta2 corepressor YBX1.\",\n      \"method\": \"Site-directed mutagenesis, quantitative ssDNA binding assays (salt/pH/detergent titration), promoter-reporter assay in fibroblasts, purified recombinant Purβ variants\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstitution with purified mutant proteins, mutagenesis + functional validation in cells, mechanistic residue-level dissection; single lab\",\n      \"pmids\": [\"27064749\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"ACTA2 is required for myofibroblast cell motility and contraction in hepatic stellate cells. Inhibition of Acta2 by multiple knockdown techniques reduced cellular motility and contraction without affecting other cytoplasmic actin isoforms. Acta2 knockdown was also associated with a significant reduction in ERK1/2 phosphorylation, indicating ACTA2 regulates signaling (MAPK pathway) in addition to its structural role.\",\n      \"method\": \"In vitro wound healing and contraction assays, siRNA/shRNA knockdown, ERK1/2 phosphorylation (Western blot), in vivo liver injury model\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — multiple knockdown approaches and two orthogonal functional readouts (motility + contraction + signaling), single lab\",\n      \"pmids\": [\"24204762\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Cardiac fibroblast-specific deletion of Acta2 does not prevent myofibroblast differentiation or impair post-MI cardiac repair. Acta2-null cardiac myofibroblasts show normal proliferation, migration, and contractility and a normal total filamentous actin level because deletion triggers compensatory transcriptional upregulation of non-Acta2 actin isoforms (particularly Actg2 and Acta1). MRTF-A is critical for myofibroblast differentiation but is not required for this compensatory response.\",\n      \"method\": \"Tamoxifen-inducible cardiac fibroblast-specific Acta2 knockout mouse, post-MI survival/histology/function, proliferation/migration/contractility assays, RT-qPCR for actin isoforms\",\n      \"journal\": \"Journal of molecular and cellular cardiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional cell-type-specific KO with multiple functional readouts and mechanistic follow-up (isoform compensation), well-controlled in vivo study\",\n      \"pmids\": [\"36007455\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TGF-β2 treatment of lens epithelial cells increases histone H4 acetylation specifically at the ACTA2 promoter region (assessed by ChIP), correlating with increased ACTA2 mRNA and protein expression and EMT. The HDAC inhibitor trichostatin-A (TSA) suppresses TGF-β2-induced ACTA2 upregulation and EMT while globally elevating acetylated H4 (but reducing H4 acetylation at the ACTA2 promoter under TGF-β2 stimulation).\",\n      \"method\": \"Chromatin immunoprecipitation (ChIP) for H4 acetylation at ACTA2 promoter, RT-qPCR/Western blot for ACTA2, TSA treatment, cell migration assay\",\n      \"journal\": \"Eye (London, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — ChIP directly at ACTA2 promoter is Tier 2, but single lab with limited follow-up\",\n      \"pmids\": [\"25853442\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"EGFR/HER2 dimerization induces ACTA2 expression through a JAK2/STAT1 signaling pathway in breast cancer cells. HER2 overexpression increases both STAT1 and ACTA2 protein levels; STAT1 inhibition (fludarabine) or JAK2 inhibition (AG490) decreases basal ACTA2 expression, and STAT1 overexpression increases ACTA2. ACTA2 knockdown suppresses cell motility in vitro and reduces lung metastatic nodules in vivo.\",\n      \"method\": \"HER2 transient/stable overexpression, siRNA knockdown, JAK2/STAT1 inhibitors, ACTA2 shRNA, in vivo mouse lung metastasis model\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — pathway placement by pharmacological inhibition and genetic OE/KD, in vivo validation; single lab\",\n      \"pmids\": [\"28881584\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"ACTA2 expression in lung adenocarcinoma cells is required for metastatic potential: ACTA2 knockdown impairs in vitro migration, invasion, clonogenicity, and transendothelial penetration without affecting proliferation, and reduces in vivo metastatic potential. ACTA2 downregulation reduces c-MET and FAK expression in lung adenocarcinoma cells and is accompanied by loss of mesenchymal characteristics.\",\n      \"method\": \"shRNA/siRNA knockdown of ACTA2, in vitro migration/invasion/transendothelial assays, in vivo metastasis model (PC14PE6 cells), Western blot for c-MET and FAK\",\n      \"journal\": \"Clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — loss-of-function with multiple functional readouts and in vivo validation plus downstream pathway (c-MET/FAK) analysis; single lab\",\n      \"pmids\": [\"23995859\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Deletion of ACTA2 in mice promotes angiotensin II-induced aortic lumen dilation, with increased expression of osteopontin (OPN), elevated Bax/Bcl-2 ratio, increased VSMC apoptosis, and phenotypic modulation of VSMCs compared to WT mice receiving AngII. Baseline ACTA2 knockout mice had no severe vascular phenotype.\",\n      \"method\": \"ACTA2 knockout mouse model, AngII osmotic minipump infusion, ultrasound lumen measurement, RT-qPCR/Western blot, TUNEL apoptosis assay, immunohistochemistry\",\n      \"journal\": \"Journal of thoracic disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — in vivo KO model with defined phenotypic readouts but single lab and limited mechanistic depth\",\n      \"pmids\": [\"30233845\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ACTA2 downregulation in neural stem cells (NSCs) inhibits migration by impeding actin filament polymerization via increased RhoA expression and decreased Rac1 expression, placing ACTA2 upstream of RhoA/Rac1 GTPase balance in NSC cytoskeletal regulation.\",\n      \"method\": \"siRNA knockdown of ACTA2 in primary NSCs, migration assay, RT-PCR/immunostaining for ACTA2, RhoA/Rac1 expression analysis\",\n      \"journal\": \"Stem cells international\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single knockdown approach, signaling pathway inferred from expression changes rather than direct epistasis experiments\",\n      \"pmids\": [\"32508931\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ACTA2 pathogenic variants (ACTA2, MYH11) in transdifferentiated VSMC-like cells show impaired migration velocity and reduced contractility (ACTA2) and decreased SMAD2 phosphorylation in ACTA2 cells, providing functional evidence that ACTA2 mutations directly impair SMC contractile and migratory function.\",\n      \"method\": \"Fibroblast transdifferentiation to SMC-like cells, cytoskeletal integrity assessment, TGFβ signaling (SMAD2 phosphorylation) assay, migration velocity assay, contraction assay\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — patient-derived cells with multiple functional assays and pathway readout; single lab with orthogonal methods\",\n      \"pmids\": [\"34244757\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Novel ACTA2 missense variants associated with TAAD act through a dominant-negative mechanism on yeast actin, disrupting actin cytoskeletal organization and mitochondrial distribution. Wild-type yeast expressing heterozygous mutant ACTA2 alleles showed significant increases in cells with abnormal mitochondrial distribution and abnormal actin cytoskeleton organization, consistent with dominant-negative interference with WT actin function.\",\n      \"method\": \"S. cerevisiae heterozygous expression assay, spot growth test, fluorescence microscopy of actin cytoskeleton and mitochondrial morphology\",\n      \"journal\": \"European journal of human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — yeast model with functional cytoskeletal and organellar readouts, establishes dominant-negative mechanism; single lab, heterologous system\",\n      \"pmids\": [\"38486025\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"3D molecular modeling of the actin filament structure revealed that the R179 residue is positioned at the interface between the two strands of filamentous actin, and the R179H mutation destabilizes inter-strand bundling, providing a structural explanation for the severe vascular phenotype associated with this mutation.\",\n      \"method\": \"Actin three-dimensional molecular modeling (computational structural analysis), correlated with histopathological findings in patient tissue\",\n      \"journal\": \"Acta neuropathologica communications\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 / Weak — computational modeling only, no experimental structural validation; correlation with pathology is descriptive\",\n      \"pmids\": [\"26637293\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1990,\n      \"finding\": \"The vascular smooth muscle actin gene (ACTSA/ACTA2) was assigned to human chromosome 10, specifically the long arm at q22-q24, by Southern blot analysis of rodent-human somatic cell hybrids and in situ hybridization.\",\n      \"method\": \"Southern blot of somatic cell hybrids, chromosomal in situ hybridization\",\n      \"journal\": \"Jinrui idengaku zasshi. The Japanese journal of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — two orthogonal cytogenetic methods (somatic cell hybrids + in situ hybridization), foundational chromosomal assignment\",\n      \"pmids\": [\"2398629\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Transient angiotensin II infusion causes sustained downregulation of ACTA2 (α-smooth muscle actin) in aortic tissue beyond AngII withdrawal, associated with increased H3K27me3 at aortic nuclei and decreased myocardin (MYOCD) expression, indicating epigenetic silencing of ACTA2 as a 'vascular memory' mechanism. This was reproduced in cultured human aortic VSMCs.\",\n      \"method\": \"Mouse AngII infusion model with post-infusion follow-up, RNAseq aortic profiling, RT-qPCR/immunohistochemistry validation, H3K27me3 immunostaining, human VSMC cell culture\",\n      \"journal\": \"Frontiers in cardiovascular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — in vivo and in vitro with epigenetic endpoint (H3K27me3), replicated in human cells; mechanistic depth limited to correlation\",\n      \"pmids\": [\"35360022\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In Hirschsprung disease (HSCR) aganglionic segments, ACTA2 expression is abnormally elevated specifically in circular smooth muscle beginning at embryonic day E15.5 in Ednrb−/− mice. siRNA knockdown of Acta2 in intestinal smooth muscle cells (iSMCs) weakens their contraction ability, demonstrating that elevated ACTA2 directly drives hyperactive contraction in aganglionic bowel.\",\n      \"method\": \"Immunohistochemistry in HSCR patients and Ednrb−/− mice, siRNA knockdown of Acta2 in iSMCs, contraction functional assay\",\n      \"journal\": \"Pediatric surgery international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — loss-of-function with direct contractility readout in disease-relevant cells, corroborated by in vivo mouse model; single lab\",\n      \"pmids\": [\"37278766\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"RHOA knockdown significantly downregulates ACTA2 gene expression in both osteoblast-like and osteoclast-like cells, placing RHOA upstream of ACTA2 in a bone cell regulatory pathway.\",\n      \"method\": \"siRNA knockdown of RHOA, microarray analysis, qRT-PCR validation in multiple human bone cell lines\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — knockdown with expression readout only, no direct functional assay on ACTA2 protein; single lab\",\n      \"pmids\": [\"24840563\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ACTA2 encodes smooth muscle α-actin (SM α-actin), the predominant actin isoform in vascular smooth muscle cells and myofibroblasts, where it forms stable filaments in the contractile apparatus; pathogenic variants (e.g., R258C, R149C, R179H) disrupt filament stability, reduce myosin-driven force generation, impair SMC differentiation/quiescence, and promote aberrant SMC proliferation and migration—mechanisms established by in vitro reconstitution assays, TIRF microscopy, knock-in mouse models, and iPSC-derived SMC studies—while ACTA2 transcription is repressed by Purβ homodimers binding the MCAT enhancer element through electrostatic and hydrophobic ssDNA interactions and is epigenetically regulated by histone acetylation at its promoter and H3K27 methylation under angiotensin II stimulation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ACTA2 encodes smooth muscle α-actin, the dominant actin isoform of vascular smooth muscle cells and myofibroblasts, where it provides the structural filament that supports myosin-driven contraction and cell motility [#0, #6]. Pathogenic missense variants act largely through allosteric and dominant-negative effects on filament behavior: R258C destabilizes filaments, sensitizes them to cofilin severing, expands the G-actin pool by tighter profilin binding, and slows myosin-driven motility [#0]; R149C is additionally retained by the CCT chaperonin folding complex, lowering mutant monomer levels and reducing penetrance while enhancing aberrant formin-driven nucleation [#1]; and modeling of R179 places it at the inter-strand filament interface, consistent with its severe phenotype [#15]. Functionally, these variants drive a contractile-to-synthetic phenotypic switch in SMCs, increasing proliferation and migration while reducing contractility — a switch that is reversible by base-editing correction of R179H in iPSC-SMCs and humanized mice, and by metabolic rescue (boosting oxidative respiration with nicotinamide riboside) in R179C SMCs [#2, #3, #13]. Beyond its structural role, ACTA2 loss perturbs cytoskeletal signaling, reducing ERK1/2 phosphorylation in myofibroblasts and altering RhoA/Rac1 balance [#6]. ACTA2 transcription is repressed by the purine-rich element binding protein Purβ, which binds the purine-rich strand of the promoter MCAT cis-element as a homodimer through electrostatic and hydrophobic ssDNA interactions and recruits the corepressor YBX1 [#4, #5], and is epigenetically tuned by histone H4 acetylation at its promoter and by H3K27 trimethylation that mediates sustained angiotensin II-induced silencing [#8, #17]. In some cell types Acta2 is functionally dispensable, as cardiac fibroblast-specific deletion is compensated by upregulation of other actin isoforms [#7].\",\n  \"teleology\": [\n    {\n      \"year\": 1990,\n      \"claim\": \"Established the genomic location of the vascular smooth muscle actin gene, providing the foundation for linking ACTA2 to human disease loci.\",\n      \"evidence\": \"Southern blot of somatic cell hybrids and chromosomal in situ hybridization\",\n      \"pmids\": [\"2398629\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No functional or mechanistic characterization\", \"Does not address protein-level activity\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Resolved how ACTA2 transcription is held in check, showing Purβ homodimers cooperatively bind the purine-rich strand of the MCAT promoter element and recruit YBX1 to repress expression.\",\n      \"evidence\": \"shRNA knockdown, promoter-reporter assays, recombinant truncation mutants, and biophysical ssDNA binding in fibroblasts\",\n      \"pmids\": [\"23724822\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Repression characterized in fibroblasts, not vascular SMCs\", \"Physiological signals controlling Purβ occupancy not defined\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Demonstrated that ACTA2 is functionally required for myofibroblast motility and contraction independent of other actin isoforms, and unexpectedly modulates MAPK signaling.\",\n      \"evidence\": \"Multiple knockdown approaches with motility/contraction assays and ERK1/2 Western blot in hepatic stellate cells plus liver injury model\",\n      \"pmids\": [\"24204762\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking ACTA2 to ERK1/2 phosphorylation unresolved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Extended ACTA2's role beyond contraction to cancer cell metastasis, showing it is required for migration, invasion, and transendothelial penetration in lung adenocarcinoma.\",\n      \"evidence\": \"shRNA/siRNA knockdown with in vitro invasion assays and in vivo metastasis model, c-MET/FAK Western blot\",\n      \"pmids\": [\"23995859\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether c-MET/FAK changes are direct or downstream of motility loss unclear\", \"Single lab\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defined the molecular pathophysiology of a TAAD-causing variant, showing R258C destabilizes filaments and impairs myosin force generation through allosteric effects rather than direct contacts at the mutated residue.\",\n      \"evidence\": \"TIRF single-filament assays, in vitro motility, cofilin severing and profilin binding with baculovirus-expressed recombinant protein\",\n      \"pmids\": [\"26153420\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Allosteric structural basis not resolved atomically\", \"Single variant studied in reconstitution\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Connected histone acetylation to ACTA2 induction, showing TGF-β2 increases H4 acetylation at the promoter and HDAC inhibition blocks ACTA2 upregulation and EMT.\",\n      \"evidence\": \"ChIP for H4 acetylation at the ACTA2 promoter with TSA treatment and migration assays in lens epithelial cells\",\n      \"pmids\": [\"25853442\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific HAT/HDAC enzymes not identified\", \"Limited follow-up, single lab\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Resolved the chemical basis of Purβ repression to specific basic residues mediating ssDNA contacts and YBX1 corepressor binding.\",\n      \"evidence\": \"Site-directed mutagenesis of R267/K82/R159 with quantitative ssDNA binding titrations and promoter-reporter assays\",\n      \"pmids\": [\"27064749\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance of individual residues not tested\", \"Single lab\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Placed ACTA2 downstream of a receptor signaling cascade, showing EGFR/HER2 dimerization induces ACTA2 via JAK2/STAT1 to drive breast cancer cell motility and metastasis.\",\n      \"evidence\": \"HER2 overexpression, JAK2/STAT1 pharmacological inhibition, STAT1 overexpression, ACTA2 shRNA and in vivo metastasis model\",\n      \"pmids\": [\"28881584\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct STAT1 binding at ACTA2 promoter not demonstrated\", \"Single lab\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Tested the in vivo consequence of ACTA2 loss under hemodynamic stress, showing knockout mice develop AngII-induced aortic dilation with VSMC apoptosis and phenotypic modulation.\",\n      \"evidence\": \"ACTA2 knockout mice with AngII infusion, ultrasound, TUNEL apoptosis and OPN/Bax-Bcl2 analysis\",\n      \"pmids\": [\"30233845\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No baseline phenotype, mechanism of stress sensitivity unclear\", \"Limited mechanistic depth\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Linked ACTA2 to RhoA/Rac1 GTPase balance in neural stem cell migration.\",\n      \"evidence\": \"siRNA knockdown in primary NSCs with migration assay and RhoA/Rac1 expression analysis\",\n      \"pmids\": [\"32508931\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Signaling inferred from expression changes rather than epistasis\", \"Single knockdown approach\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Explained reduced penetrance of a common variant by showing R149C is retained in the CCT chaperonin, lowering functional mutant levels while still perturbing filament organization.\",\n      \"evidence\": \"CRISPR knock-in mouse, CCT-binding retention assay, in vitro motility, TIRF nucleation assay and aortic contraction\",\n      \"pmids\": [\"34600884\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How CCT retention varies across variants not generalized\", \"Quantitative threshold for pathogenicity undefined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Provided patient-derived functional evidence that ACTA2 mutations directly impair SMC contractility and migration and reduce SMAD2 phosphorylation.\",\n      \"evidence\": \"Fibroblast transdifferentiation to SMC-like cells with cytoskeletal, TGFβ/SMAD2 and migration/contraction assays\",\n      \"pmids\": [\"34244757\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Link between actin defect and SMAD2 signaling unresolved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Revealed cell-type-specific dispensability, showing cardiac fibroblast-specific Acta2 deletion is tolerated through compensatory upregulation of other actin isoforms.\",\n      \"evidence\": \"Tamoxifen-inducible cardiac fibroblast-specific knockout with post-MI functional readouts and RT-qPCR for actin isoforms\",\n      \"pmids\": [\"36007455\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether vascular SMCs mount similar compensation untested\", \"Trigger of isoform compensation unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified an epigenetic 'vascular memory' mechanism in which transient AngII causes sustained ACTA2 silencing via H3K27me3 and reduced myocardin.\",\n      \"evidence\": \"Mouse AngII infusion with post-infusion follow-up, RNAseq, H3K27me3 immunostaining, replicated in human aortic VSMCs\",\n      \"pmids\": [\"35360022\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Methyltransferase responsible not identified\", \"Causal role of H3K27me3 correlative\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Showed that abnormally elevated ACTA2, not only its loss, is pathogenic, driving hyperactive contraction in aganglionic Hirschsprung bowel.\",\n      \"evidence\": \"Immunohistochemistry in HSCR patients and Ednrb−/− mice with siRNA knockdown and contraction assay in intestinal SMCs\",\n      \"pmids\": [\"37278766\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Upstream cause of ACTA2 elevation undefined\", \"Single lab\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Established a dominant-negative mechanism for TAAD-associated missense variants using a heterologous system, showing heterozygous mutant ACTA2 disrupts actin organization and mitochondrial distribution.\",\n      \"evidence\": \"S. cerevisiae heterozygous expression with spot growth and fluorescence microscopy of actin and mitochondria\",\n      \"pmids\": [\"38486025\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Yeast system may not capture mammalian SMC context\", \"Mechanism of mitochondrial mispositioning unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstrated reversibility of the disease phenotype, showing base-editing correction of R179H prevents the contractile-to-synthetic switch in iPSC-SMCs and rescues multi-organ pathology in humanized mice.\",\n      \"evidence\": \"iPSC-derived SMC differentiation, CRISPR adenine base editing, AAV9 in vivo delivery and humanized knock-in mouse rescue\",\n      \"pmids\": [\"40378078\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Durability and off-target profile of in vivo editing not fully defined\", \"Single variant corrected\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Linked the ACTA2-mutant phenotype to metabolic state, showing R179C SMCs fail to differentiate and rely on glycolysis, with oxidative-respiration boosting (nicotinamide riboside) restoring differentiation and preventing occlusive lesions.\",\n      \"evidence\": \"SMC-specific knock-in mouse with carotid injury, metabolic flux assays and nicotinamide riboside rescue\",\n      \"pmids\": [\"40603847\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism coupling actin mutation to glycolytic shift unresolved\", \"Single variant studied\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How specific ACTA2 variants are mechanistically partitioned between loss-of-function, dominant-negative filament interference, and chaperonin-mediated reduction — and how these map onto the divergent vascular, cerebrovascular, gut, and bladder phenotypes — remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No unifying genotype-mechanism-phenotype framework across variants\", \"Structural basis of allosteric defects not resolved atomically\", \"Coupling between cytoskeletal defects and metabolic/signaling reprogramming undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 6, 14]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0, 1, 14]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-397014\", \"supporting_discovery_ids\": [0, 1, 13]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [4, 5, 8, 17]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [2, 3]}\n    ],\n    \"complexes\": [\"CCT/TCP1 chaperonin (transient client)\"],\n    \"partners\": [\"MYH11\", \"CCT\", \"TPM (smooth muscle tropomyosin)\", \"CFL (cofilin)\", \"PFN (profilin)\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":6,"faith_pct":83.33333333333333}}