{"gene":"GUCY1A1","run_date":"2026-06-10T01:55:21","timeline":{"discoveries":[{"year":2017,"finding":"The transcription factor ZEB1 binds preferentially to the non-risk allele at rs7692387 (an intronic DNase I hypersensitivity site in GUCY1A3), and ZEB1 knockdown reduces GUCY1A3 promoter activity and endogenous GUCY1A3 expression, establishing ZEB1 as a positive transcriptional regulator of GUCY1A3 through allele-specific chromatin binding.","method":"Allele-specific chromatin immunoprecipitation, siRNA knockdown, reporter gene assay","journal":"Circulation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal allele-specific ChIP plus reporter assay plus siRNA, single lab, orthogonal methods","pmids":["28487391"],"is_preprint":false},{"year":2017,"finding":"Pharmacological stimulation of soluble guanylyl cyclase (sGC, whose α1 subunit is encoded by GUCY1A3) inhibits vascular smooth muscle cell migration specifically in cells homozygous for the non-risk allele of rs7692387, demonstrating that GUCY1A3-dependent sGC activity suppresses VSMC migration.","method":"Vascular smooth muscle cell migration assay with sGC stimulation, genotype-stratified","journal":"Circulation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional cellular assay with genotype stratification, single lab","pmids":["28487391"],"is_preprint":false},{"year":2017,"finding":"Ex vivo platelets from GUCY1A3 non-risk allele homozygotes show enhanced inhibition of ADP-induced platelet aggregation by the NO donor sodium nitroprusside and the PDE5 inhibitor sildenafil, demonstrating that GUCY1A3 genotype modulates sGC-dependent platelet inhibition.","method":"Ex vivo platelet aggregation assay, genotype-stratified","journal":"Circulation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct functional assay on human ex vivo platelets stratified by genotype, single lab","pmids":["28487391"],"is_preprint":false},{"year":2016,"finding":"The GUCY1A3 missense variant Cys517Tyr (α1 subunit of soluble guanylate cyclase) produces a mutant protein with a significantly blunted cGMP signaling response to nitric oxide, establishing this as a loss-of-function allele that disrupts NO/cGMP signaling.","method":"Biochemical assay of cGMP production in response to NO stimulation using mutant protein","journal":"Clinical genetics","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — direct in vitro enzymatic assay with mutant protein, single lab, single method described in abstract","pmids":["26777256"],"is_preprint":false},{"year":2016,"finding":"Eight rare coding GUCY1A3 variants found in MI patients all dimerize with the β1 subunit of sGC, but five variants show significantly decreased cGMP production upon NO stimulation; one variant additionally reduces protein levels by 72%. Addition of the sGC stimulator BAY 41-2272 rescues cGMP production in all five activity-reduced variants to near wild-type levels.","method":"Co-immunoprecipitation (dimerization assay), immunoblotting (protein levels), cGMP radioimmunoassay after NO stimulation in HEK293 cells expressing GUCY1A3 variants","journal":"Basic research in cardiology","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic assay plus co-IP plus immunoblot, multiple variants tested, single lab","pmids":["27342234"],"is_preprint":false},{"year":2014,"finding":"The α1-A680T variant of GUCY1A3 (sGC α1 subunit), found in Kyrgyz highlanders without high-altitude pulmonary hypertension, confers enhanced sensitivity to nitric oxide and higher cGMP production compared to wild-type enzyme when expressed in reporter cells and in purified form in vitro.","method":"cGMP production assay in reporter cells and in vitro assay with purified recombinant protein","journal":"Circulation. Cardiovascular genetics","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic assay with both cell-based and purified protein systems, single lab","pmids":["25373139"],"is_preprint":false},{"year":2004,"finding":"Antisense knockdown of GUCY1A3 (or GUCY1B3) in glioma cell lines (CCF-STTG1, U-87MG) markedly reduced intracellular cGMP levels, decreased VEGF expression, inhibited HUVEC growth in vitro, and suppressed subcutaneous tumor formation and vascular index in nude mice, placing GUCY1A3-dependent cGMP production upstream of VEGF-mediated angiogenesis in glioma.","method":"Antisense RNA transfection, cGMP measurement, VEGF expression analysis, in vitro angiogenesis assay (HUVEC growth), in vivo xenograft model","journal":"Oncology reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (in vitro + in vivo), single lab","pmids":["15201957"],"is_preprint":false},{"year":2025,"finding":"In cardiac ischemia-reperfusion injury, the sGC-cGMP-PKG pathway (with GUCY1A1 as the α1 subunit of sGC) suppresses endothelial ferroptosis: PKG phosphorylates LDHA at threonine 95, activating LDHA's moonlighting kinase function to phosphorylate GPX4 at serine 131, thereby reducing chaperone-mediated autophagy-dependent degradation of GPX4. EC-specific GUCY1A1 knockout increased the no-reflow area and infarct size, while GUCY1A1 overexpression or the activator vericiguat alleviated microvascular dysfunction.","method":"Endothelial cell-specific conditional knockout and AAV-mediated overexpression mice; mass spectrometry identification of phosphorylation sites; CRISPR-Cas9 mutagenesis of phosphorylation sites; co-immunoprecipitation; cardiac ischemia-reperfusion model","journal":"Circulation research","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstitution of phosphorylation cascade with MS, mutagenesis, co-IP, and in vivo conditional KO/OE model, multiple orthogonal methods","pmids":["40856046"],"is_preprint":false},{"year":2023,"finding":"Loss of Gucy1a3 in mice following permanent middle cerebral artery occlusion increased infarct volume, aggravated neurological deficits, reduced microvessel density, and decreased VEGFA and HIF-1α protein expression, placing GUCY1A3 upstream of the HIF-1α/VEGFA angiogenic signaling pathway in post-stroke recovery.","method":"Gucy1a3 knockout mice, pMCAO model, TTC staining, CD31 immunohistochemistry, western blotting","journal":"Journal of stroke and cerebrovascular diseases","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO with defined phenotypic readout and pathway marker analysis, single lab","pmids":["38064974"],"is_preprint":false},{"year":2026,"finding":"In Gucy1a3 knockout mice, loss of the α1 subunit of sGC did not produce large-artery stenosis typical of moyamoya disease under basal conditions, but caused significant rarefaction of leptomeningeal vascular networks (reduced branching and density) and reduced cortical microvessel density and diameter, indicating that GUCY1A3 is required for small-vessel but not large-vessel integrity in early adult mice.","method":"7.0T MR angiography, cerebral vascular casting, H&E/EVG staining, α-SMA immunohistochemistry, CD31 immunohistochemistry, vascular skeletonization/topology analysis in Gucy1a3-/- mice","journal":"Frontiers in neurology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO with multiple imaging and histological readouts, single lab","pmids":["42051768"],"is_preprint":false},{"year":2026,"finding":"Gucy1a3 siRNA knockdown in chondrocytes phenocopied the anti-inflammatory effect of DAPT (Notch inhibitor) by suppressing iNOS and MMP13, and DAPT suppressed Gucy1a3 expression, identifying a reciprocal positive feedback loop between NO-Gucy1a3 signaling and Notch activation in chondrocytes.","method":"siRNA knockdown of Gucy1a3 in chondrocytes, Notch inhibitor DAPT treatment, gene expression analysis, rat medial meniscus resection model","journal":"Drug design, development and therapy","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, siRNA knockdown with gene expression readout, mechanistic loop inferred without direct biochemical reconstitution","pmids":["42157792"],"is_preprint":false},{"year":2023,"finding":"A homozygous GUCY1A3 missense variant (c.1778G>A) located in the catalytic domain of sGC is predicted to disrupt the 3D structure of the domain and cause loss of enzymatic function, associated with moyamoya angiopathy and early-onset arterial hypertension in a consanguineous proband.","method":"Exome sequencing, western blot (protein expression in endothelial progenitor cells), 3D protein structure analysis","journal":"Human genomics","confidence":"Low","confidence_rationale":"Tier 3 / Weak — western blot and computational structural prediction, functional enzymatic activity not directly measured for this variant","pmids":["36941667"],"is_preprint":false}],"current_model":"GUCY1A1 (GUCY1A3) encodes the α1 subunit of soluble guanylate cyclase (sGC), which heterodimerizes with the β1 subunit to generate cGMP upon nitric oxide stimulation; transcription factor ZEB1 positively regulates its expression through allele-specific promoter binding; downstream, the sGC–cGMP–PKG axis suppresses endothelial ferroptosis by directing PKG to phosphorylate LDHA (T95), which then phosphorylates GPX4 (S131) to prevent its autophagic degradation, and also suppresses VSMC migration and platelet aggregation, inhibits VEGF-driven angiogenesis via cGMP, and supports post-stroke and post-ischemia vascular integrity partly through HIF-1α/VEGFA signaling."},"narrative":{"mechanistic_narrative":"GUCY1A1 (GUCY1A3) encodes the α1 subunit of soluble guanylate cyclase (sGC), which heterodimerizes with the β1 subunit to convert nitric oxide stimulation into cGMP production [PMID:27342234]; rare coding variants from myocardial infarction patients retain β1 dimerization yet show blunted NO-stimulated cGMP output, and a Cys517Tyr loss-of-function allele as well as a catalytic-domain variant confer reduced signaling, while sGC stimulators such as BAY 41-2272 restore cGMP production [PMID:26777256, PMID:27342234, PMID:36941667]. Expression of GUCY1A3 is positively controlled by the transcription factor ZEB1, which binds allele-specifically at an intronic regulatory site (rs7692387) to drive promoter activity [PMID:28487391]. Functionally, sGC-derived cGMP suppresses vascular smooth muscle cell migration and potentiates NO-dependent inhibition of platelet aggregation in an allele-dependent manner [PMID:28487391]. The downstream sGC–cGMP–PKG axis protects the endothelium from ferroptosis: PKG phosphorylates LDHA at Thr95, activating a moonlighting kinase function that phosphorylates GPX4 at Ser131 to block its chaperone-mediated autophagic degradation, and endothelial-specific GUCY1A1 loss enlarges no-reflow and infarct area in cardiac ischemia-reperfusion [PMID:40856046]. GUCY1A3-dependent cGMP also acts upstream of VEGF/HIF-1α angiogenic signaling, supporting microvascular integrity in glioma vascularization, post-stroke recovery, and small-vessel networks [PMID:15201957, PMID:38064974, PMID:42051768].","teleology":[{"year":2004,"claim":"Established that GUCY1A3-dependent cGMP production sits upstream of VEGF-driven angiogenesis rather than acting only as a vasorelaxant effector.","evidence":"Antisense knockdown in glioma cell lines with cGMP/VEGF readouts, HUVEC growth assay, and nude-mouse xenograft vascular index","pmids":["15201957"],"confidence":"Medium","gaps":["Did not define the molecular link between cGMP and VEGF transcription","Antisense effects not confirmed with genetic knockout"]},{"year":2014,"claim":"Showed that natural GUCY1A3 coding variation tunes enzyme sensitivity, with an α1-A680T variant conferring enhanced NO responsiveness and higher cGMP output.","evidence":"cGMP production assays in reporter cells and with purified recombinant protein","pmids":["25373139"],"confidence":"Medium","gaps":["Physiological consequence in highlanders inferred from association, not demonstrated mechanistically","Structural basis of enhanced sensitivity not resolved"]},{"year":2016,"claim":"Defined GUCY1A3 loss-of-function alleles in MI patients, showing variants dimerize normally with β1 but produce less cGMP, and that pharmacological sGC stimulation can rescue activity.","evidence":"Co-IP dimerization, immunoblot protein-level quantification, and cGMP radioimmunoassay of variant proteins in HEK293; plus a separate Cys517Tyr biochemical assay","pmids":["27342234","26777256"],"confidence":"Medium","gaps":["In vitro HEK293 activity may not reflect native vascular cell context","Causal link from reduced cGMP to MI risk not demonstrated in vivo"]},{"year":2017,"claim":"Connected a coronary-disease risk locus to GUCY1A3 regulation and function by showing ZEB1 drives allele-specific expression and that genotype modulates sGC-dependent VSMC migration and platelet inhibition.","evidence":"Allele-specific ChIP, siRNA knockdown, reporter assay, genotype-stratified VSMC migration and ex vivo platelet aggregation assays","pmids":["28487391"],"confidence":"Medium","gaps":["Single lab","Whether ZEB1 is the principal in vivo regulator across vascular tissues unknown","Mechanism linking VSMC/platelet phenotype to atherosclerotic risk not established"]},{"year":2023,"claim":"Demonstrated in vivo that Gucy1a3 loss impairs post-stroke vascular recovery via reduced HIF-1α/VEGFA signaling, and that GUCY1A3 is required for small-vessel but not large-artery integrity.","evidence":"Gucy1a3 knockout mice in pMCAO model with infarct, microvessel-density and pathway-marker analysis; separate KO with MR angiography, vascular casting and topology analysis","pmids":["38064974","42051768"],"confidence":"Medium","gaps":["Cell-type origin of the angiogenic defect not isolated","Absence of large-artery stenosis leaves the moyamoya link unexplained","Direct mechanism coupling sGC to HIF-1α not defined"]},{"year":2025,"claim":"Resolved a downstream effector cascade by which endothelial sGC-cGMP-PKG suppresses ferroptosis through a PKG→LDHA(T95)→GPX4(S131) phosphorylation relay that stabilizes GPX4 against autophagic degradation.","evidence":"Endothelial-specific conditional KO and AAV overexpression mice, mass spectrometry phosphosite mapping, CRISPR phosphosite mutagenesis, co-IP, and cardiac ischemia-reperfusion model with vericiguat","pmids":["40856046"],"confidence":"High","gaps":["Generality of the LDHA moonlighting kinase function beyond endothelium unknown","Quantitative contribution of this axis versus canonical vasorelaxation not parsed"]},{"year":null,"claim":"How GUCY1A3 loss-of-function alleles and the sGC-cGMP axis mechanistically cause moyamoya angiopathy and arterial hypertension in patients remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No in vivo model reproduces large-artery moyamoya stenosis","Catalytic-domain disease variant assessed only by structural prediction and western blot, not direct enzymatic assay"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0009975","term_label":"cyclase activity","supporting_discovery_ids":[3,4,5]},{"term_id":"GO:0016829","term_label":"lyase activity","supporting_discovery_ids":[4,5]}],"localization":[],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[4,7]}],"complexes":["soluble guanylate cyclase (α1/β1 heterodimer)"],"partners":["GUCY1B1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q02108","full_name":"Guanylate cyclase soluble subunit alpha-1","aliases":["Guanylate cyclase soluble subunit alpha-3","GCS-alpha-3","Soluble guanylate cyclase large subunit"],"length_aa":690,"mass_kda":77.5,"function":"","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q02108/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/GUCY1A1","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/GUCY1A1","total_profiled":1310},"omim":[{"mim_id":"615750","title":"MOYAMOYA DISEASE 6 WITH OR WITHOUT ACHALASIA; MYMY6","url":"https://www.omim.org/entry/615750"},{"mim_id":"139397","title":"GUANYLATE CYCLASE, SOLUBLE, BETA-1; GUCY1B1","url":"https://www.omim.org/entry/139397"},{"mim_id":"139396","title":"GUANYLATE CYCLASE, SOLUBLE, ALPHA-1; GUCY1A1","url":"https://www.omim.org/entry/139396"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/GUCY1A1"},"hgnc":{"alias_symbol":["GC-SA3"],"prev_symbol":["GUC1A3","GUCY1A3"]},"alphafold":{"accession":"Q02108","domains":[{"cath_id":"3.90.1520","chopping":"72-179_186-198_207-244","consensus_level":"high","plddt":81.0018,"start":72,"end":244},{"cath_id":"3.30.450.260","chopping":"280-355_363-404","consensus_level":"high","plddt":85.7872,"start":280,"end":404},{"cath_id":"3.30.70.1230","chopping":"470-661","consensus_level":"high","plddt":92.7219,"start":470,"end":661},{"cath_id":"1.20.5","chopping":"406-458","consensus_level":"medium","plddt":91.3215,"start":406,"end":458}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q02108","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q02108-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q02108-F1-predicted_aligned_error_v6.png","plddt_mean":77.44},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=GUCY1A1","jax_strain_url":"https://www.jax.org/strain/search?query=GUCY1A1"},"sequence":{"accession":"Q02108","fasta_url":"https://rest.uniprot.org/uniprotkb/Q02108.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q02108/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q02108"}},"corpus_meta":[{"pmid":"28487391","id":"PMC_28487391","title":"Functional Characterization of the GUCY1A3 Coronary Artery Disease Risk Locus.","date":"2017","source":"Circulation","url":"https://pubmed.ncbi.nlm.nih.gov/28487391","citation_count":82,"is_preprint":false},{"pmid":"34381413","id":"PMC_34381413","title":"RNF213 and GUCY1A3 in Moyamoya Disease: Key Regulators of Metabolism, Inflammation, and Vascular Stability.","date":"2021","source":"Frontiers in neurology","url":"https://pubmed.ncbi.nlm.nih.gov/34381413","citation_count":70,"is_preprint":false},{"pmid":"26777256","id":"PMC_26777256","title":"Disrupted nitric oxide signaling due to GUCY1A3 mutations increases risk for moyamoya disease, achalasia and hypertension.","date":"2016","source":"Clinical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/26777256","citation_count":65,"is_preprint":false},{"pmid":"15201957","id":"PMC_15201957","title":"Inhibition of angiogenesis in human glioma cell lines by antisense RNA from the soluble guanylate cyclase genes, GUCY1A3 and GUCY1B3.","date":"2004","source":"Oncology reports","url":"https://pubmed.ncbi.nlm.nih.gov/15201957","citation_count":32,"is_preprint":false},{"pmid":"31228190","id":"PMC_31228190","title":"Genetic variation at the coronary artery disease risk locus GUCY1A3 modifies cardiovascular disease prevention effects of aspirin.","date":"2019","source":"European heart journal","url":"https://pubmed.ncbi.nlm.nih.gov/31228190","citation_count":31,"is_preprint":false},{"pmid":"25373139","id":"PMC_25373139","title":"α1-A680T variant in GUCY1A3 as a candidate conferring protection from pulmonary hypertension among Kyrgyz highlanders.","date":"2014","source":"Circulation. 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allele-specific chromatin binding.\",\n      \"method\": \"Allele-specific chromatin immunoprecipitation, siRNA knockdown, reporter gene assay\",\n      \"journal\": \"Circulation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal allele-specific ChIP plus reporter assay plus siRNA, single lab, orthogonal methods\",\n      \"pmids\": [\"28487391\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Pharmacological stimulation of soluble guanylyl cyclase (sGC, whose α1 subunit is encoded by GUCY1A3) inhibits vascular smooth muscle cell migration specifically in cells homozygous for the non-risk allele of rs7692387, demonstrating that GUCY1A3-dependent sGC activity suppresses VSMC migration.\",\n      \"method\": \"Vascular smooth muscle cell migration assay with sGC stimulation, genotype-stratified\",\n      \"journal\": \"Circulation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional cellular assay with genotype stratification, single lab\",\n      \"pmids\": [\"28487391\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Ex vivo platelets from GUCY1A3 non-risk allele homozygotes show enhanced inhibition of ADP-induced platelet aggregation by the NO donor sodium nitroprusside and the PDE5 inhibitor sildenafil, demonstrating that GUCY1A3 genotype modulates sGC-dependent platelet inhibition.\",\n      \"method\": \"Ex vivo platelet aggregation assay, genotype-stratified\",\n      \"journal\": \"Circulation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct functional assay on human ex vivo platelets stratified by genotype, single lab\",\n      \"pmids\": [\"28487391\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The GUCY1A3 missense variant Cys517Tyr (α1 subunit of soluble guanylate cyclase) produces a mutant protein with a significantly blunted cGMP signaling response to nitric oxide, establishing this as a loss-of-function allele that disrupts NO/cGMP signaling.\",\n      \"method\": \"Biochemical assay of cGMP production in response to NO stimulation using mutant protein\",\n      \"journal\": \"Clinical genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — direct in vitro enzymatic assay with mutant protein, single lab, single method described in abstract\",\n      \"pmids\": [\"26777256\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Eight rare coding GUCY1A3 variants found in MI patients all dimerize with the β1 subunit of sGC, but five variants show significantly decreased cGMP production upon NO stimulation; one variant additionally reduces protein levels by 72%. Addition of the sGC stimulator BAY 41-2272 rescues cGMP production in all five activity-reduced variants to near wild-type levels.\",\n      \"method\": \"Co-immunoprecipitation (dimerization assay), immunoblotting (protein levels), cGMP radioimmunoassay after NO stimulation in HEK293 cells expressing GUCY1A3 variants\",\n      \"journal\": \"Basic research in cardiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic assay plus co-IP plus immunoblot, multiple variants tested, single lab\",\n      \"pmids\": [\"27342234\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The α1-A680T variant of GUCY1A3 (sGC α1 subunit), found in Kyrgyz highlanders without high-altitude pulmonary hypertension, confers enhanced sensitivity to nitric oxide and higher cGMP production compared to wild-type enzyme when expressed in reporter cells and in purified form in vitro.\",\n      \"method\": \"cGMP production assay in reporter cells and in vitro assay with purified recombinant protein\",\n      \"journal\": \"Circulation. Cardiovascular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic assay with both cell-based and purified protein systems, single lab\",\n      \"pmids\": [\"25373139\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Antisense knockdown of GUCY1A3 (or GUCY1B3) in glioma cell lines (CCF-STTG1, U-87MG) markedly reduced intracellular cGMP levels, decreased VEGF expression, inhibited HUVEC growth in vitro, and suppressed subcutaneous tumor formation and vascular index in nude mice, placing GUCY1A3-dependent cGMP production upstream of VEGF-mediated angiogenesis in glioma.\",\n      \"method\": \"Antisense RNA transfection, cGMP measurement, VEGF expression analysis, in vitro angiogenesis assay (HUVEC growth), in vivo xenograft model\",\n      \"journal\": \"Oncology reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (in vitro + in vivo), single lab\",\n      \"pmids\": [\"15201957\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In cardiac ischemia-reperfusion injury, the sGC-cGMP-PKG pathway (with GUCY1A1 as the α1 subunit of sGC) suppresses endothelial ferroptosis: PKG phosphorylates LDHA at threonine 95, activating LDHA's moonlighting kinase function to phosphorylate GPX4 at serine 131, thereby reducing chaperone-mediated autophagy-dependent degradation of GPX4. EC-specific GUCY1A1 knockout increased the no-reflow area and infarct size, while GUCY1A1 overexpression or the activator vericiguat alleviated microvascular dysfunction.\",\n      \"method\": \"Endothelial cell-specific conditional knockout and AAV-mediated overexpression mice; mass spectrometry identification of phosphorylation sites; CRISPR-Cas9 mutagenesis of phosphorylation sites; co-immunoprecipitation; cardiac ischemia-reperfusion model\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstitution of phosphorylation cascade with MS, mutagenesis, co-IP, and in vivo conditional KO/OE model, multiple orthogonal methods\",\n      \"pmids\": [\"40856046\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Loss of Gucy1a3 in mice following permanent middle cerebral artery occlusion increased infarct volume, aggravated neurological deficits, reduced microvessel density, and decreased VEGFA and HIF-1α protein expression, placing GUCY1A3 upstream of the HIF-1α/VEGFA angiogenic signaling pathway in post-stroke recovery.\",\n      \"method\": \"Gucy1a3 knockout mice, pMCAO model, TTC staining, CD31 immunohistochemistry, western blotting\",\n      \"journal\": \"Journal of stroke and cerebrovascular diseases\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with defined phenotypic readout and pathway marker analysis, single lab\",\n      \"pmids\": [\"38064974\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"In Gucy1a3 knockout mice, loss of the α1 subunit of sGC did not produce large-artery stenosis typical of moyamoya disease under basal conditions, but caused significant rarefaction of leptomeningeal vascular networks (reduced branching and density) and reduced cortical microvessel density and diameter, indicating that GUCY1A3 is required for small-vessel but not large-vessel integrity in early adult mice.\",\n      \"method\": \"7.0T MR angiography, cerebral vascular casting, H&E/EVG staining, α-SMA immunohistochemistry, CD31 immunohistochemistry, vascular skeletonization/topology analysis in Gucy1a3-/- mice\",\n      \"journal\": \"Frontiers in neurology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with multiple imaging and histological readouts, single lab\",\n      \"pmids\": [\"42051768\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Gucy1a3 siRNA knockdown in chondrocytes phenocopied the anti-inflammatory effect of DAPT (Notch inhibitor) by suppressing iNOS and MMP13, and DAPT suppressed Gucy1a3 expression, identifying a reciprocal positive feedback loop between NO-Gucy1a3 signaling and Notch activation in chondrocytes.\",\n      \"method\": \"siRNA knockdown of Gucy1a3 in chondrocytes, Notch inhibitor DAPT treatment, gene expression analysis, rat medial meniscus resection model\",\n      \"journal\": \"Drug design, development and therapy\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, siRNA knockdown with gene expression readout, mechanistic loop inferred without direct biochemical reconstitution\",\n      \"pmids\": [\"42157792\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"A homozygous GUCY1A3 missense variant (c.1778G>A) located in the catalytic domain of sGC is predicted to disrupt the 3D structure of the domain and cause loss of enzymatic function, associated with moyamoya angiopathy and early-onset arterial hypertension in a consanguineous proband.\",\n      \"method\": \"Exome sequencing, western blot (protein expression in endothelial progenitor cells), 3D protein structure analysis\",\n      \"journal\": \"Human genomics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — western blot and computational structural prediction, functional enzymatic activity not directly measured for this variant\",\n      \"pmids\": [\"36941667\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"GUCY1A1 (GUCY1A3) encodes the α1 subunit of soluble guanylate cyclase (sGC), which heterodimerizes with the β1 subunit to generate cGMP upon nitric oxide stimulation; transcription factor ZEB1 positively regulates its expression through allele-specific promoter binding; downstream, the sGC–cGMP–PKG axis suppresses endothelial ferroptosis by directing PKG to phosphorylate LDHA (T95), which then phosphorylates GPX4 (S131) to prevent its autophagic degradation, and also suppresses VSMC migration and platelet aggregation, inhibits VEGF-driven angiogenesis via cGMP, and supports post-stroke and post-ischemia vascular integrity partly through HIF-1α/VEGFA signaling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"GUCY1A1 (GUCY1A3) encodes the \\u03b11 subunit of soluble guanylate cyclase (sGC), which heterodimerizes with the \\u03b21 subunit to convert nitric oxide stimulation into cGMP production [#4]; rare coding variants from myocardial infarction patients retain \\u03b21 dimerization yet show blunted NO-stimulated cGMP output, and a Cys517Tyr loss-of-function allele as well as a catalytic-domain variant confer reduced signaling, while sGC stimulators such as BAY 41-2272 restore cGMP production [#3, #4, #11]. Expression of GUCY1A3 is positively controlled by the transcription factor ZEB1, which binds allele-specifically at an intronic regulatory site (rs7692387) to drive promoter activity [#0]. Functionally, sGC-derived cGMP suppresses vascular smooth muscle cell migration and potentiates NO-dependent inhibition of platelet aggregation in an allele-dependent manner [#1, #2]. The downstream sGC\\u2013cGMP\\u2013PKG axis protects the endothelium from ferroptosis: PKG phosphorylates LDHA at Thr95, activating a moonlighting kinase function that phosphorylates GPX4 at Ser131 to block its chaperone-mediated autophagic degradation, and endothelial-specific GUCY1A1 loss enlarges no-reflow and infarct area in cardiac ischemia-reperfusion [#7]. GUCY1A3-dependent cGMP also acts upstream of VEGF/HIF-1\\u03b1 angiogenic signaling, supporting microvascular integrity in glioma vascularization, post-stroke recovery, and small-vessel networks [#6, #8, #9].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Established that GUCY1A3-dependent cGMP production sits upstream of VEGF-driven angiogenesis rather than acting only as a vasorelaxant effector.\",\n      \"evidence\": \"Antisense knockdown in glioma cell lines with cGMP/VEGF readouts, HUVEC growth assay, and nude-mouse xenograft vascular index\",\n      \"pmids\": [\"15201957\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not define the molecular link between cGMP and VEGF transcription\", \"Antisense effects not confirmed with genetic knockout\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Showed that natural GUCY1A3 coding variation tunes enzyme sensitivity, with an \\u03b11-A680T variant conferring enhanced NO responsiveness and higher cGMP output.\",\n      \"evidence\": \"cGMP production assays in reporter cells and with purified recombinant protein\",\n      \"pmids\": [\"25373139\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological consequence in highlanders inferred from association, not demonstrated mechanistically\", \"Structural basis of enhanced sensitivity not resolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Defined GUCY1A3 loss-of-function alleles in MI patients, showing variants dimerize normally with \\u03b21 but produce less cGMP, and that pharmacological sGC stimulation can rescue activity.\",\n      \"evidence\": \"Co-IP dimerization, immunoblot protein-level quantification, and cGMP radioimmunoassay of variant proteins in HEK293; plus a separate Cys517Tyr biochemical assay\",\n      \"pmids\": [\"27342234\", \"26777256\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vitro HEK293 activity may not reflect native vascular cell context\", \"Causal link from reduced cGMP to MI risk not demonstrated in vivo\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Connected a coronary-disease risk locus to GUCY1A3 regulation and function by showing ZEB1 drives allele-specific expression and that genotype modulates sGC-dependent VSMC migration and platelet inhibition.\",\n      \"evidence\": \"Allele-specific ChIP, siRNA knockdown, reporter assay, genotype-stratified VSMC migration and ex vivo platelet aggregation assays\",\n      \"pmids\": [\"28487391\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Whether ZEB1 is the principal in vivo regulator across vascular tissues unknown\", \"Mechanism linking VSMC/platelet phenotype to atherosclerotic risk not established\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Demonstrated in vivo that Gucy1a3 loss impairs post-stroke vascular recovery via reduced HIF-1\\u03b1/VEGFA signaling, and that GUCY1A3 is required for small-vessel but not large-artery integrity.\",\n      \"evidence\": \"Gucy1a3 knockout mice in pMCAO model with infarct, microvessel-density and pathway-marker analysis; separate KO with MR angiography, vascular casting and topology analysis\",\n      \"pmids\": [\"38064974\", \"42051768\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cell-type origin of the angiogenic defect not isolated\", \"Absence of large-artery stenosis leaves the moyamoya link unexplained\", \"Direct mechanism coupling sGC to HIF-1\\u03b1 not defined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Resolved a downstream effector cascade by which endothelial sGC-cGMP-PKG suppresses ferroptosis through a PKG\\u2192LDHA(T95)\\u2192GPX4(S131) phosphorylation relay that stabilizes GPX4 against autophagic degradation.\",\n      \"evidence\": \"Endothelial-specific conditional KO and AAV overexpression mice, mass spectrometry phosphosite mapping, CRISPR phosphosite mutagenesis, co-IP, and cardiac ischemia-reperfusion model with vericiguat\",\n      \"pmids\": [\"40856046\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Generality of the LDHA moonlighting kinase function beyond endothelium unknown\", \"Quantitative contribution of this axis versus canonical vasorelaxation not parsed\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How GUCY1A3 loss-of-function alleles and the sGC-cGMP axis mechanistically cause moyamoya angiopathy and arterial hypertension in patients remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No in vivo model reproduces large-artery moyamoya stenosis\", \"Catalytic-domain disease variant assessed only by structural prediction and western blot, not direct enzymatic assay\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0009975\", \"supporting_discovery_ids\": [3, 4, 5]},\n      {\"term_id\": \"GO:0016829\", \"supporting_discovery_ids\": [4, 5]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [4, 7]}\n    ],\n    \"complexes\": [\"soluble guanylate cyclase (\\u03b11/\\u03b21 heterodimer)\"],\n    \"partners\": [\"GUCY1B1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"faith_supported":5,"faith_total":5,"faith_pct":100.0}}