Hypothesis

Core claim: The substrate hierarchy of autophagy in vascular smooth muscle cells (VSMCs) determines tropoelastin expression by regulating mitochondrial ROS‑dependent YAP/TAZ activity. When the hierarchy is perturbed—specifically when mitophagy is delayed relative to ER‑phagy or protein aggregate clearance—mitochondrial ROS accumulates, drives nuclear translocation of YAP/TAZ, and YAP/TAZ‑TEAD complexes recruit HDAC2 to the ELN promoter, suppressing tropoelastin transcription and impairing elastic fiber assembly. Restoring the proper order (i.e., prioritizing mitophagy) rescues elastin synthesis even if overall autophagic flux is reduced.

Mechanistic rationale

1. Hierarchy matters more than bulk flux – Autophagy adapters (SQSTM1/p62, BNIP3, FUNDC1) compete for limited LC3 pools; substrate stiffness and mTORC1 signaling bias the order toward damaged mitochondria first (12). If this order is inverted, damaged mitochondria persist.
2. ROS‑YAP axis – Mitochondrial ROS oxidizes cysteine residues on LATS1/2, inhibiting the Hippo kinase cascade and allowing YAP/TAZ nuclear entry (3). Nuclear YAP/TAZ binds TEAD transcription factors and brings HDAC2 to the ELN promoter, deacetylating histones and repressing ELN transcription.
3. Elastin consequence – Reduced tropoelastin limits elastin fiber cross‑linking, promoting VSMC osteogenic shift and arterial calcification, as observed when autophagy is defective (45).
4. Feedback – Elastin fragmentation further increases VSMC mechanosensing, activating mTORC1 and worsening autophagy misprioritization, creating a vicious loop.

Testable predictions

• Prediction 1: VSMCs with CRISPR‑mediated knockout of the mitophagy receptor BNIP3 (but intact p62‑mediated autophagy) will show ↓ ELN mRNA and protein, ↑ mitochondrial ROS, and ↑ nuclear YAP/TAZ, despite normal LC3‑II turnover.
• Prediction 2: Pharmacological enhancement of mitophagy (e.g., urolithin A) in VSMCs treated with rapamycin (to suppress overall autophagy) will restore ELN expression and elastin deposition to control levels.
• Prediction 3: In vivo, VSMC‑specific BNIP3 knockout mice will develop increased aortic pulse wave velocity and elastin fragmentation, whereas VSMC‑specific overexpression of BNIP3 will attenuate age‑related stiffening even when global autophagy is inhibited by heterozygous Atg7 loss.

Falsifiability

If manipulating mitophagy alone fails to alter ELN expression or elastic fiber integrity while global autophagy flux is changed, the hypothesis is falsified. Conversely, if rescuing mitophagy does not normalize YAP/TAZ localization or ELN transcription under conditions of mitochondrial ROS overload, the proposed ROS‑YAP‑ELN link is invalid.

Experimental outline (brief)

1. Culture primary human aortic VSMCs; transfect with siRNA against BNIP3 or p62; measure LC3‑II, mito‑ROS (MitoSOX), nuclear YAP/TAZ (IF/WB), ELN qPCR, and tropoelastin secretion (ELISA).
2. Treat with urolithin A or MitoTEMPO to dissect ROS dependence.
3. Perform ChIP for YAP/TAZ and HDAC2 at the ELN promoter.
4. In vivo: generate SM22α‑Cre;BNIP3^fl/fl and SM22α‑Cre;BNIP3^OE mice; assess PWV, elastin staining (Verhoeff‑Van Gieson), and autophagy flux (LC3‑II/p62) at 3, 12, 24 months.
5. Statistical analysis: ANOVA with post‑hoc tests; n≥6 per group.

Implications

Redefining autophagy defects as a loss of substrate‑specific triage rather than a global degradation shortfall shifts therapeutic focus toward selective autophagy modulators (mitophagy inducers, ER‑phagy inhibitors) to preserve arterial elastin and mitigate age‑related vascular stiffening.