IF a combinatorial intervention consisting of: (i) two cycles of oral Dasatinib (5 mg/kg) + Quercetin (50 mg/kg) administered on days 1–3 and 15–17 ("D+Q pulse"), followed by (ii) a single tail-vein injection of AAV9-CMV-TFEB (1×10¹¹ vg/mouse) at day 21,

is administered to aged (24-month-old) male C57BL/6J mice,

THEN the aortic homocitrulline-to-hydroxyproline molar ratio—quantified by stable-isotope-dilution LC-MS/MS after complete acid hydrolysis of aortic tissue—will be reduced by ≥30% relative to aged vehicle controls at 12 weeks post-AAV delivery, with a concomitant reduction in pulse wave velocity (≥15%) and restoration of autophagic flux (LC3-II/LC3-I ratio) in aortic smooth muscle cells toward young-reference values,

BECAUSE the following causal chain is operative:

1. Senescent VSMC burden in aged aorta drives both SASP-mediated ECM dysregulation and local cyanate production via upregulated arginase/urea cycling, the immediate precursor of protein carbamylation. Senolytic D+Q clears p16^Ink4a^-positive senescent cells in aged tissue, reducing the cellular source of reactive isocyanic acid and SASP factors (including TGF-β and MMPs) that collectively impair ECM quality. (Senolytics improve physical function and increase lifespan)[https://doi.org/10.1038/s41591-018-0092-9]

2. Senescent cells also actively suppress autophagic flux in neighboring VSMCs via paracrine IL-6 and IL-1β signaling, creating a proteostatic "bystander collapse." D+Q pulse therapy, by eliminating the senescent cell population, removes this paracrine suppression and thus partially restores the cellular capacity for matrix protein internalization and lysosomal processing—but is insufficient alone to clear accumulated, long-lived carbamylated collagen deposits already embedded in the arterial wall. [SPECULATIVE — paracrine autophagy suppression by SASP in VSMCs is mechanistically plausible but not directly quantified in aortic tissue]

3. TFEB is the master transcriptional activator of the CLEAR (Coordinated Lysosomal Expression and Regulation) network; AAV9-mediated overexpression in vascular cells drives coordinated upregulation of lysosomal biogenesis genes (LAMP1, CTSD, ATP6V1), autophagy initiation (BECN1, ULK1), and receptor-mediated endocytosis pathways. Enhancement of this system in macrophages and smooth muscle cells of the atherosclerotic vascular wall has been demonstrated to reduce plaque burden and restore proteostasis. (TFEB and trehalose drive the macrophage autophagy-lysosome system to protect against atherosclerosis)[https://doi.org/10.1038/ncomms15750]

4. The expanded lysosomal compartment, activated downstream of TFEB overexpression, processes internalized ECM fragments—including carbamylated collagen peptides taken up via scavenger receptors (SR-A1, CD36) and macropinocytosis—through cathepsin-mediated hydrolysis. Cathepsin K, B, and L are TFEB transcriptional targets and the principal intracellular collagenolytic enzymes; the...

SENS category: GlycoSENS

Key references: • doi.org/10.1038/s41591-018-0092-9]. • doi.org/10.1038/s41593-019-0372-9]. • doi.org/10.1038/s41591-018-0092-9] • doi.org/10.1038/ncomms15750] • doi.org/10.1038/s41593-019-0372-9]