What determines P2RY12 receptor expression/activity specifically in VSMCs during atherosclerosis progression?
---
Mechanism: Krüppel-like factor 4 (KLF4) drives VSMC phenotypic modulation from contractile to synthetic phenotype, and may repress P2RY12 transcription during disease progression, linking VSMC dedifferentiation to foam cell susceptibility.
Target Gene/Protein/Pathway: KLF4 → P2RY12 promoter binding
Supporting Evidence:
- KLF4 is a master regulator of VSMC phenotypic switching (PMID: 29908848)
- KLF4 cooperates with myocardin/SRF to regulate VSMC-specific genes (PMID: 31302669)
- P2RY12 expression correlates with VSMC phenotypic state (PMID: 32160082)
Predicted Experiment: ChIP-qPCR/ATAC-seq in VSMCs from early vs. advanced atherosclerotic plaques to map KLF4 occupancy at the P2RY12 promoter. Rescue P2RY12 expression with KLF4 knockdown in ApoE⁻/⁻ mice.
Confidence: 0.65
---
Mechanism: Pro-inflammatory cytokines in advanced plaques (particularly TNF-α) activate NF-κB signaling, which binds to κB sites in the P2RY12 promoter, increasing transcription and amplifying foam cell formation in a feed-forward inflammatory loop.
Target Gene/Protein/Pathway: TNF-α → IKK/NF-κB → P2RY12 transcription
Supporting Evidence:
- TNF-α upregulates P2RY12 in platelets via NF-κB (PMID: 17244679, 24692168)
- NF-κB activation drives atherosclerotic inflammation (PMID: 25994186)
- Advanced plaques show elevated TNF-α and P2RY12 (PMID: 32160082)
Predicted Experiment: Treat VSMCs with TNF-α (10 ng/mL) ± IKK inhibitor (BAY 11-7082). Measure P2RY12 mRNA (RT-qPCR) and protein (Western blot). Perform luciferase assay with P2RY12 promoter constructs containing mutated κB sites.
Confidence: 0.70
---
Mechanism: Oxidized LDL accumulates in atherosclerotic lesions and engages lectin-like oxLDL receptor-1 (LOX-1) on VSMCs, generating reactive oxygen species that stabilize P2RY12 mRNA or activate transcription factors (AP-1, Nrf2) to upregulate P2RY12.
Target Gene/Protein/Pathway: oxLDL → LOX-1 → ROS/Nrf2 → P2RY12
Supporting Evidence:
- oxLDL induces foam cell formation via LOX-1 (PMID: 24816296)
- ROS modulates P2Y receptor signaling (PMID: 25047031)
- P2RY12 promotes oxLDL uptake in VSMCs (PMID: 32160082)
Predicted Experiment: VSMC treatment with oxLDL (50 μg/mL) ± LOX-1 blocking antibody or N-acetylcysteine (NAC, antioxidant). Assess P2RY12 expression and foam cell formation (Oil Red O staining).
Confidence: 0.65
---
Mechanism: The miR-143/145 cluster maintains VSMC contractile phenotype; loss of these miRNAs during phenotypic switching derepresses unknown target genes that transcriptionally activate P2RY12, or alternatively, a specific miRNA (e.g., miR-150) directly targets P2RY12 3'UTR to silence expression in healthy vessels.
Target Gene/Protein/Pathway: miR-143/145 → transcription factors (KLF4, Myocardin) → P2RY12 (indirect); or direct miRNA → P2RY12 3'UTR
Supporting Evidence:
- miR-143/145 regulate VSMC differentiation (PMID: 25446983)
- miRNA dysregulation occurs in atherosclerosis (PMID: 26888767)
- P2RY12 3'UTR contains predicted miRNA binding sites
Predicted Experiment: Bioinformatic prediction + dual-luciferase assay for miRNA-P2RY12 3'UTR interaction. Transfect VSMCs with miR-143/145 mimics or antagomirs; assay P2RY12 expression and autophagy markers (LC3-II, p62).
Confidence: 0.60
---
Mechanism: Activated platelets adhering to damaged endothelium release PDGF-BB, which activates VSMC PDGF receptors, triggering MAPK/ERK signaling that enhances P2RY12 promoter activity and primes VSMCs for ADP-induced foam cell formation.
Target Gene/Protein/Pathway: PDGF-BB → PDGFRβ → MAPK/ERK → P2RY12
Supporting Evidence:
- PDGF-BB drives VSMC migration and proliferation in atherosclerosis (PMID: 27477582)
- Platelet-VSMC crosstalk promotes atherosclerotic progression (PMID: 29615459)
- P2RY12 mediates ADP-driven foam cell formation (PMID: 32160082)
Predicted Experiment: VSMC treatment with recombinant PDGF-BB (20 ng/mL) ± PDGFR inhibitor (CP-673451). RNA-seq to identify PDGF-regulated P2RY12 and related genes. Co-culture VSMCs with thrombin-activated platelets.
Confidence: 0.55
---
Mechanism: LRP1 (low-density lipoprotein receptor-related protein 1) normally suppresses pro-atherogenic signaling in VSMCs; its downregulation during atherosclerosis removes this inhibition, permitting upregulation of P2RY12 and consequent autophagy inhibition and foam cell accumulation.
Target Gene/Protein/Pathway: LRP1 →下游信号 → P2RY12 transcription/autophagy regulation
Supporting Evidence:
- LRP1 deficiency in VSMCs accelerates atherosclerosis (PMID: 24218264)
- LRP1 regulates autophagy in vascular cells (PMID: 26582122)
- P2RY12 inhibits autophagy (PMID: 32160082)
Predicted Experiment: VSMC-specific LRP1 knockout (LRP1^fl/fl;SM22α-Cre) in ApoE⁻/⁻ mice. Measure P2RY12 expression, autophagy flux, and plaque phenotype. Co-immunoprecipitation to test LRP1-P2RY12 protein interaction.
Confidence: 0.55
---
Mechanism: During atherosclerosis, global DNA hypomethylation in VSMCs leads to demethylation of CpG sites in the P2RY12 promoter, increasing chromatin accessibility and transcription factor binding, thereby elevating P2RY12 expression in advanced lesions.
Target Gene/Protein/Pathway: DNMTs/TETs → P2RY12 promoter methylation → transcriptional silencing/derepression
Supporting Evidence:
- DNA methylation patterns are altered in atherosclerotic vessels (PMID: 28871001)
- TET2 mutations promote cardiovascular disease (PMID: 30361495)
- Epigenetic regulation of P2Y receptors has precedent (PMID: 29042452)
Predicted Experiment: Bisulfite sequencing of P2RY12 promoter in laser-captured VSMCs from early vs. advanced human atherosclerotic plaques. Treat VSMCs with 5-azacytidine (DNMT inhibitor) and assess P2RY12 expression and foam cell formation.
Confidence: 0.50
---
| # | Hypothesis | Confidence | Key Therapeutic Target |
|---|------------|------------|------------------------|
| 1 | KLF4-mediated repression | 0.65 | KLF4, myocardin |
| 2 | TNF-α/NF-κB axis | 0.70 | IKKβ, p65, TNF-α |
| 3 | oxLDL/LOX-1/ROS | 0.65 | LOX-1, Nrf2 |
| 4 | miR-143/145 dysregulation | 0.60 | miR-143/145, miR-150 |
| 5 | Platelet PDGF-BB | 0.55 | PDGFRβ, MAPK pathway |
| 6 | LRP1 deficiency | 0.55 | LRP1 |
| 7 | DNA hypomethylation | 0.50 | DNMTs, TETs |
Highest Priority for Investigation: Hypothesis 2 (TNF-α/NF-κB) has the strongest mechanistic support and most direct translational potential, as NF-κB inhibitors are clinically available (e.g., salsalate) and could be repurposed to target VSMC P2RY12 in atherosclerosis.
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| Hypothesis | Original | Revised | Key Issue |
|------------|----------|---------|-----------|
| 1: KLF4 repression | 0.65 | 0.35 | Logical inversion—KLF4 activity should suppress P2RY12 trajectory |
| 2: TNF-α/NF-κB | 0.70 | 0.60 | Strongest hypothesis but assumes VSMC promoter structure |
| 3: oxLDL/LOX-1 | 0.65 | 0.50 | Directionality ambiguous; Nrf2 paradox unexplained |
| 4: miRNA dysregulation | 0.60 | 0.40 | Dual-mechanism hedge undermines testability |
| 5: PDGF-BB | 0.55 | 0.40 | Functional mismatch (proliferation vs. lipid accumulation) |
| 6: LRP1 deficiency | 0.55 | 0.45 | Mechanism gap; epistasis not established |
| 7: DNA hypomethylation | 0.50 | 0.35 | Specificity problem; correlation vs. causation |
1. Mechanism-of-action gap: All hypotheses focus on transcriptional regulation, but P2RY12 activity is also controlled post-translationally (receptor trafficking, desensitization, ADP availability). Non-transcriptional mechanisms (e.g., reduced endocytosis increasing surface expression) should be considered as alternatives.
2. Cell-type specificity: P2RY12 upregulation in VSMCs specifically—as opposed to platelets or macrophages—requires demonstrating that proposed mechanisms operate in VSMCs, not merely in other cell types where P2RY12 is well-characterized.
3. Causal vs. correlative evidence: None of the hypotheses provide direct causal evidence linking upstream regulators to P2RY12 in VSMCs during atherosclerosis progression. The strongest test would be genetic epistasis: Does perturbation of candidate upstream regulator in VSMC-specific knockout mice alter P2RY12 expression and modify foam cell formation or plaque phenotype?
After critical evaluation, three hypotheses survive with sufficient testability. I assess each across druggability, biomarkers/model systems, clinical development constraints, safety, and realistic timeline/cost parameters.
---
| Target Level | Agent Class | Status | VSMC Specificity Challenge |
|--------------|-------------|--------|---------------------------|
| TNF-α | Infliximab, adalimumab, etanercept | Approved (autoimmune) | Systemically immunosuppressive; cannot isolate vascular effect |
| IKKβ | BAY 11-7082, ML120B | Preclinical/Phase I | Requires nanoparticle delivery to VSMCs; systemic NF-κB inhibition causes immunosuppression |
| p65 (RELA) | Selective peptidic inhibitors | Research stage | Could theoretically achieve better cell-type selectivity |
Key constraint: Systemic NF-κB inhibition is clinically untenable for cardiovascular prevention. The only viable path is local vascular delivery—catheter-based nanoparticle formulations (e.g., dextran nanoparticles conjugated to NF-κB inhibitor) that concentrate in atherosclerotic lesions. This technology exists in preclinical models but has never been scaled for chronic human use.
| System | Readout | Validation Status |
|--------|---------|-------------------|
| In vitro | P2RY12 promoter κB sites → luciferase activity | Definitive mechanistic test |
| p65 ChIP-seq | Direct P2RY12 promoter binding in TNF-α-treated VSMCs | Gold standard |
| ApoE⁻/⁻ + IKKβ inhibitor | P2RY12 expression, foam cell burden | Demonstrates causality |
| Human plaques | Correlation between p65 activity and P2RY12 | Observational only |
Critical biomarker gap: No validated biomarker measures local vascular NF-κB activity in living patients. Peripheral blood mononuclear cell (PBMC) p65 phosphorylation does not reflect vessel wall signaling. This is a major clinical development obstacle.
1. Mechanism validation in humans requires vascular sampling (carotid endarterectomy or coronary atherectomy)—invasive, not generalizable to early disease.
2. Phase II endpoint challenge: VSMC P2RY12 expression is not measurable in vivo; surrogate imaging (PET with NF-κB-targeted tracers) is experimental.
3. Regulatory path: Reformulating NF-κB inhibitors for local vascular delivery is essentially a new drug entity requiring full safety package.
| Risk | Severity | Mitigation |
|------|----------|------------|
| Systemic immunosuppression (NF-κB inhibition) | High | Local delivery eliminates this risk |
| Heart failure exacerbation (TNF-α inhibitors) | Moderate | Avoid anti-TNF antibodies; use IKKβ inhibitors instead |
| Off-target bleeding | Low | P2RY12 inhibition on platelets is separate mechanism |
Conclusion: Local vascular delivery could mitigate systemic toxicity, but this approach has never been approved for cardiovascular indications and carries substantial development risk.
```
Preclinical validation: 18 months, $4M
Toxicology/pharmacology (local delivery): 24 months, $12M
Phase I safety (first-in-human, local vascular): 18 months, $15M
Phase II efficacy (imaging endpoints): 30 months, $40M
─────────────────────────────────────────────────────────
Total estimated: 7-8 years, $70-100M
```
High-risk investment with uncertain regulatory precedent.
---
| Target | Agent | Status | Clinical Trial History |
|--------|-------|--------|----------------------|
| LOX-1 | Blocking antibodies | Preclinical | Previously failed in atherosclerosis trials |
| Nrf2 | Bardoxolone methyl, dimethyl fumarate | Approved (diabetic nephropathy, MS) | Bardoxolone: increased cardiovascular mortality in BEACON trial |
| General ROS | NAC, edaravone | Approved/generic | Failed consistently in CV prevention trials |
The Nrf2 Paradox (Critical Obstacle): Nrf2 activators failed catastrophically in clinical trials for conditions overlapping with atherosclerosis (diabetes, CKD). Bardoxolone's cardiovascular mortality signal suggests Nrf2 activation in patients with metabolic disease may be harmful or reflect confounded population risk.
Viable path: Vascular-selective antioxidants targeting NADPH oxidase-4 (Nox4) specifically in VSMCs. Nox4 is relatively atheroprotective; Nox1/2 are pro-atherogenic. Selective Nox1 inhibitors are in development but lack VSMC specificity data.
| System | Utility |
|--------|---------|
| OxLDL ELISA | Available but reflects whole-body oxidative modification; does not distinguish vascular source |
| 8-OHdG (urine) | Systemic oxidative stress marker; poor correlation with vascular pathology |
| Nrf2 target genes (HO-1, NQO1) in PBMCs | Indicates systemic Nrf2 activation—not VSMC-specific |
| LOX-1 KO × ApoE⁻/⁻ mice | Definitive genetic test; failed to show dramatic benefit in prior studies |
Critical gap: No biomarker distinguishes LOX-1-mediated signaling from other oxLDL uptake pathways (CD36, SR-A) in vivo.
1. Prior LOX-1 antibody failure suggests this pathway may not be dominant in human disease—foam cell formation proceeds despite LOX-1 blockade.
2. Nrf2 activator history creates regulatory headwind; demonstrating safety in cardiovascular population will require extensive Phase II data.
3. Antioxidant class has been thoroughly discredited for cardiovascular prevention (Vitamin E, beta-carotene, NAC trials)—novel mechanism must clearly distinguish from failed approaches.
| Agent | Safety Concern | Mitigation |
|-------|----------------|------------|
| Bardoxolone | CV mortality, hepatotoxicity | Abandon in favor of VSMC-selective approach |
| Dimethyl fumarate | GI intolerance, lymphopenia | Poorly suited for chronic CV prevention |
| NAC | Low toxicity but low efficacy | Acceptable safety but unlikely to demonstrate benefit |
Conclusion: Safety profile of existing agents is acceptable for short-term use but unacceptable for chronic cardiovascular prevention given prior trial failures.
```
Repurposing Nrf2 activators: 4-5 years, $30-50M (existing safety data)
Novel Nox1 inhibitor development: 6-8 years, $80-120M
─────────────────────────────────────────────────────
Total: $30-120M depending on strategy
```
Lower confidence due to prior clinical failure of mechanistically related approaches.
---
| Approach | Feasibility | Problem |
|----------|-------------|---------|
| Restore LRP1 expression | Low | No identified pathway to pharmacologically increase LRP1 transcription in VSMCs |
| Inhibit LRP1 cleavage | Low | γ-secretase inhibitors lack specificity; LRP1 shedding is downstream of many proteolytic events |
| Gene therapy | Very low | AAV9 does not efficiently transduce medial VSMCs in vivo; no validated VSMC-targeting serotype |
| Target downstream pathway | Moderate | Identify transcriptional repressors that silence LRP1 in atherosclerosis |
Most tractable angle: Identify the transcriptional repressor(s) that suppress LRP1 during VSMC phenotypic switching. If a druggable transcription factor (e.g., YAP/TAZ, inflammatory STATs) mediates LRP1 suppression, inhibitor development becomes feasible.
| System | Utility |
|--------|---------|
| Soluble LRP1 (sLRP1) | Cleaved receptor fragment detectable in plasma; elevated sLRP1 correlates with cardiovascular events (independent validation needed) |
| LRP1^fl/fl;SM22α-Cre; ApoE⁻/⁻ mice | Definitive genetic model; demonstrate foam cell phenotype |
| iPSC-derived VSMCs from LRP1 variant carriers | Human genetic validation; could identify LRP1 expression modulators |
| Co-IP (LRP1-P2RY12) | Mechanistically misframed—tests protein interaction rather than transcriptional regulation |
Critical limitation: sLRP1 is a proxy biomarker for LRP1 processing, not expression or transcriptional regulation. Does not prove that P2RY12 is downstream of LRP1 loss.
1. Gene therapy: No validated delivery system targets VSMCs in humans. Stent-based AAV delivery has been attempted but yields inefficient, transient transduction.
2. No lead compound identified: Unlike NF-κB or Nrf2 pathways, there is no identified small molecule that restores LRP1 expression.
3. Epistasis not established: Even if LRP1 deficiency upregulates P2RY12, whether P2RY12
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This hypothesis contains a critical logical inversion: since P2RY12 increases in advanced plaques alongside KLF4 activity, repression by KLF4 contradicts observed trajectories. Revised model may suggest KLF4 indirectly primes VSMCs for P2RY12 upregulation through chromatin remodeling rather than direct transcriptional repression.","target_gene":"KLF4","dimension_scores":{"evidence_strength":0.38,"novelty":0.68,"feasibility":0.55,"therapeutic_potential":0.48,"mechanistic_plausibility":0.32,"druggability":0.52,"safety_profile":0.58,"competitive_landscape":0.62,"data_availability":0.55,"reproducibility":0.42},"composite_score":0.44,"evidence_for":[{"claim":"KLF4 is a master regulator of VSMC phenotypic switching","pmid":"29908848"},{"claim":"KLF4 cooperates with myocardin/SRF to regulate VSMC-specific genes","pmid":"31302669"},{"claim":"P2RY12 expression correlates with VSMC phenotypic state","pmid":"32160082"}],"evidence_against":[{"claim":"KLF4 activity increases in advanced plaques but so does P2RY12 - trajectories should be inversely correlated if KLF4 represses","pmid":"29908848"},{"claim":"KLF4 is generally pro-atherogenic - co-upregulation is more parsimonious","pmid":"N/A"},{"claim":"No direct evidence linking KLF4 to P2RY12 promoter binding","pmid":"N/A"}]},{"title":"Platelet-Derived PDGF-BB Primes VSMCs for P2RY12 Upregulation","description":"Activated platelets adhering to damaged endothelium release PDGF-BB, activating VSMC PDGFRβ and triggering MAPK/ERK signaling that enhances P2RY12 promoter activity. 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However, the specificity problem is severe—global hypomethylation affects thousands of genes, and methylation changes in plaques may reflect proliferative history rather than active regulatory mechanisms driving P2RY12 expression.","target_gene":"DNMT1, TET2","dimension_scores":{"evidence_strength":0.38,"novelty":0.72,"feasibility":0.45,"therapeutic_potential":0.38,"mechanistic_plausibility":0.35,"druggability":0.35,"safety_profile":0.68,"competitive_landscape":0.65,"data_availability":0.40,"reproducibility":0.38},"composite_score":0.44,"evidence_for":[{"claim":"DNA methylation patterns are altered in atherosclerotic vessels","pmid":"28871001"},{"claim":"TET2 mutations promote cardiovascular disease","pmid":"30361495"},{"claim":"Epigenetic regulation of P2Y receptors has precedent","pmid":"29042452"}],"evidence_against":[{"claim":"Global hypomethylation affects thousands of genes - specificity not established","pmid":"N/A"},{"claim":"Methylation changes may be passive marker of proliferation, not active regulatory mechanism","pmid":"N/A"},{"claim":"Cellular heterogeneity in laser-captured VSMCs may confound results","pmid":"N/A"}]}],"knowledge_edges":[{"source_id":"H2","source_type":"hypothesis","target_id":"RELA","target_type":"gene","relation":"direct_transcriptional_activation"},{"source_id":"H2","source_type":"hypothesis","target_id":"IKBKB","target_type":"gene","relation":"upstream_kinase"},{"source_id":"H2","source_type":"hypothesis","target_id":"TNF","target_type":"gene","relation":"extracellular_ligand"},{"source_id":"H3","source_type":"hypothesis","target_id":"OLR1","target_type":"gene","relation":"receptor_mediating_uptake"},{"source_id":"H3","source_type":"hypothesis","target_id":"NFE2L2","target_type":"gene","relation":"transcription_factor_downstream"},{"source_id":"H1","source_type":"hypothesis","target_id":"KLF4","target_type":"gene","relation":"transcriptional_repressor_candidate"},{"source_id":"H4","source_type":"hypothesis","target_id":"MIR143","target_type":"gene","relation":"epigenetic_regulator"},{"source_id":"H4","source_type":"hypothesis","target_id":"MIR145","target_type":"gene","relation":"epigenetic_regulator"},{"source_id":"H5","source_type":"hypothesis","target_id":"PDGFB","target_type":"gene","relation":"paracrine_ligand_source"},{"source_id":"H5","source_type":"hypothesis","target_id":"PDGFRB","target_type":"gene","relation":"receptor_tyrosine_kinase"},{"source_id":"H6","source_type":"hypothesis","target_id":"LRP1","target_type":"gene","relation":"transmembrane_receptor_downregulation"},{"source_id":"H7","source_type":"hypothesis","target_id":"DNMT1","target_type":"gene","relation":"epigenetic_modifier"},{"source_id":"H2","source_type":"hypothesis","target_id":"P2RY12","target_type":"gene","relation":"target_of_regulation"},{"source_id":"H3","source_type":"hypothesis","target_id":"P2RY12","target_type":"gene","relation":"target_of_regulation"},{"source_id":"H1","source_type":"hypothesis","target_id":"P2RY12","target_type":"gene","relation":"target_of_regulation"},{"source_id":"H4","source_type":"hypothesis","target_id":"P2RY12","target_type":"gene","relation":"target_of_regulation"},{"source_id":"H5","source_type":"hypothesis","target_id":"P2RY12","target_type":"gene","relation":"target_of_regulation"},{"source_id":"H6","source_type":"hypothesis","target_id":"P2RY12","target_type":"gene","relation":"target_of_regulation"},{"source_id":"H7","source_type":"hypothesis","target_id":"P2RY12","target_type":"gene","relation":"target_of_regulation"}],"synthesis_summary":"The four-persona debate converges on two surviving mechanistically plausible hypotheses with translational potential. First, the TNF-α/NF-κB axis (composite score 0.65) emerges as the strongest candidate given direct precedent from platelet studies demonstrating P2RY12 regulation via NF-κB, co-elevation of TNF-α and P2RY12 in advanced plaques, and the availability of local vascular delivery strategies (nanoparticle IKKβ inhibitors) that could mitigate systemic immunosuppression concerns. However, this hypothesis requires critical validation: direct demonstration of κB sites in the VSMC P2RY12 promoter, p65 ChIP-seq confirming binding, and demonstration that local (not systemic) NF-κB inhibition reduces VSMC P2RY12 expression and foam cell burden in vivo. Second, the oxLDL/LOX-1/ROS pathway (composite score 0.55) provides biological plausibility through a reinforcing feed-forward loop but faces substantial translational obstacles: prior LOX-1 antibody failures, the Nrf2 paradox (atheroprotective Nrf2 driving pro-atherogenic P2RY12), and the catastrophic cardiovascular mortality signal from Nrf2 activator bardoxolone in the BEACON trial. The remaining five hypotheses are substantially weakened by logical inconsistencies (KLF4), dual-mechanism hedges reducing testability (miR-143/145), poor druggability (LRP1), temporal discordance (PDGF-BB), and specificity problems (DNA hypomethylation). Across all hypotheses, a critical gap emerges: none address post-translational regulation of P2RY12 activity (receptor trafficking, desensitization, ADP availability), and all require VSMC-specific validation rather than extrapolation from other cell types where P2RY12 is well-characterized. The highest-priority investigation combines these: verify NF-κB binding to the VSMC P2RY12 promoter (mechanistic), then test whether oxLDL-induced P2RY12 upregulation depends on NF-κB activation (mechanistic integration), and finally test whether local nanoparticle delivery of IKKβ inhibitors reduces P2RY12+ foam cells in advanced plaques (translational)."}