Fibulin-1 Drives Vascular Aging Through a ZNF384–TGF-β Senescence Loop

Vascular stiffness—measurable as increased pulse wave velocity (PWV)—is a hallmark of arterial aging and a major risk factor for heart attack, stroke, and heart failure. Despite its clinical importance, the precise molecular mechanisms driving arterial stiffening remain incompletely understood, and no disease-modifying therapies exist. This study set out to identify key extracellular matrix proteins dysregulated in vascular stiffness and map their upstream regulators and downstream effectors.

The researchers began with plasma proteomic profiling of a human family pedigree showing hereditary accelerated arteriosclerosis (PWV ≥ 1400 cm/s). Fibulin-1 (Fbln1), an ECM glycoprotein abundant in arterial walls, emerged as significantly elevated in affected versus unaffected family members. Fbln1 was also upregulated in aged human vascular tissue and in two established murine vascular stiffness models: natural aging and chronic angiotensin II (AngII) infusion (1000 ng/kg/min for 28 days). This dual-model approach strengthened the biological relevance of Fbln1 across distinct pathological triggers.

To establish causality, the team generated inducible systemic Fbln1 knockout mice (Fbln1−/−) using a tamoxifen-activated CAG-Cre ERT2 system. In both aging and AngII models, Fbln1 deletion significantly reduced PWV, attenuated collagen deposition (Masson staining), preserved elastic fiber integrity (EVG staining), and reversed VSMC senescence markers including SA-β-galactosidase activity, p21, and p16 expression. Vascular tension assays confirmed improved vasodilatory function. These findings established Fbln1 as a functionally important contributor to—not merely a marker of—vascular stiffening.

Using DNA pull-down assays and dual-luciferase reporter assays, ZNF384 was identified as a direct transcriptional activator of Fbln1, binding its promoter region. ZNF384 overexpression in VSMCs increased Fbln1 levels and promoted senescence and collagen production, while ZNF384 knockdown had the opposite effects. RNA sequencing of Fbln1-overexpressing VSMCs revealed enrichment of TGF-β/Smad3 pathway genes. Mechanistic experiments confirmed that Fbln1 activates TGF-β receptor signaling, leading to Smad3 phosphorylation (p-Smad3), nuclear translocation, and transcriptional upregulation of senescence- and fibrosis-associated genes. Pharmacological inhibition of TGF-β/Smad3 signaling (using SB505124 or siSmad3) completely abolished Fbln1-driven senescence and ECM remodeling in VSMCs, confirming pathway dependency.

This work establishes a coherent molecular axis: ZNF384 transcriptionally activates Fbln1, which in turn drives VSMC senescence and collagen fibrosis via TGF-β/Smad3, culminating in arterial stiffening. Each node represents a potential therapeutic target. Limitations include the use of systemic rather than VSMC-specific Fbln1 knockout, relatively small human cohort size, and the absence of pharmacological Fbln1 inhibition studies in vivo. Nonetheless, the convergence of human proteomic data, two independent animal models, and mechanistic cell studies makes this a compelling framework for future drug development targeting age-related vascular pathology.