Fibroblast Activation Protein Mediates Vascular Restenosis After Surgery for Peripheral Arterial Disease

Technical Summary: Fibroblast Activation Protein Mediates Vascular Restenosis After Surgery for Peripheral Arterial Disease

Problem Statement
Postoperative vascular restenosis remains a critical limitation to the long-term efficacy of surgical interventions for peripheral arterial disease (PAD), particularly following percutaneous transluminal angioplasty (PTA). The primary pathological driver of this restenosis is neointimal hyperplasia, caused by the abnormal migration and proliferation of vascular smooth muscle cells (VSMCs) shifting from a quiescent contractile phenotype to a synthetic phenotype. Despite the use of drug-coated balloons and drug-eluting stents, restenosis rates remain high (25%–63% in femoropopliteal lesions), and current therapies lack specific molecular targets to inhibit neointimal formation effectively. The molecular regulators governing this VSMC phenotypic switching and migration in the context of PAD restenosis are not fully defined.

Methodology
The study employed a multi-modal approach combining bioinformatics, in vitro cellular assays, in vivo animal models, and transcriptomic sequencing:

1. Bioinformatic Screening: Transcriptomic datasets related to neointimal hyperplasia were retrieved from the Gene Expression Omnibus (GEO) (specifically GSE164050 and GSE220512). Differentially expressed genes (DEGs) were identified, and overlapping upregulated genes were subjected to Gene Ontology (GO) enrichment analysis to identify candidate regulators.
2. In Vitro Characterization: Primary human vascular cells (VSMCs, endothelial cells, fibroblasts, macrophages) were isolated to assess baseline FAP distribution. Rat VSMCs were stimulated with 10% fetal bovine serum (FBS) or platelet-derived growth factor-BB (PDGF-BB) to induce phenotypic switching. FAP expression was quantified via qRT-PCR and Western blot.
3. Functional Validation (In Vitro): FAP expression was suppressed in VSMCs using small interfering RNA (siRNA). Cell migration was assessed via scratch wound-healing assays, and phenotypic markers (α-SMA, SM22α, CNN1) were analyzed via Western blot.
4. In Vivo Validation: Two vascular injury models were established in mice: the carotid artery wire injury model (CAWIM) and the partial carotid ligation (PCL) model. Adeno-associated virus (AAV9) mediated RNA interference was used to knock down FAP in vivo. Neointimal hyperplasia was evaluated 4 weeks post-injury using Hematoxylin and Eosin (HE) staining to measure the intima-to-media (I/M) ratio and luminal stenosis.
5. Transcriptomic Analysis: RNA sequencing (RNA-seq) was performed on FAP-knockdown and control VSMCs under both serum-stimulated and non-stimulated conditions to identify downstream signaling pathways.

Key Results

• Identification and Expression: Bioinformatic analysis identified Fibroblast Activation Protein (FAP) as a candidate hub gene upregulated in neointimal hyperplasia. FAP expression was found to be low in normal arteries and quiescent VSMCs but significantly elevated in injured vessels and synthetic VSMCs.
• Correlation with Migration: FAP expression correlated positively with VSMC migration. Stimulation with FBS or PDGF-BB increased FAP protein levels and VSMC migratory capacity while inducing a phenotypic switch (decreased SM22α and CNN1).
• Loss-of-Function Effects:
  • In Vitro: siRNA-mediated knockdown of FAP significantly reduced VSMC migration at 24 hours and preserved the contractile phenotype (increased SM22α and CNN1) without altering α-SMA levels.
  • In Vivo: AAV-mediated FAP knockdown in both CAWIM and PCL models resulted in a significant reduction in the intima-to-media area ratio and luminal stenosis compared to controls.
• Mechanistic Insight: RNA-seq analysis of FAP-deficient VSMCs under serum stimulation revealed significant downregulation of the Net1–RhoA–ROCK signaling axis. Specifically, FAP knockdown led to decreased expression of Net1, RhoA, Arhgef2, and ROCK1, while increasing Arhgap6 (a negative regulator of RhoA). This suggests FAP promotes migration by activating this specific signaling cascade.

Key Contributions

• This study is the first to identify FAP as a critical mediator of vascular restenosis following PAD surgery, expanding its known role beyond tumor stroma and fibrosis.
• It establishes a direct causal link between FAP upregulation, VSMC migration, and neointimal hyperplasia through both in vitro and in vivo loss-of-function experiments.
• The research elucidates a potential molecular mechanism wherein FAP facilitates VSMC migration via the Net1–RhoA–ROCK signaling axis.

Significance and Claims
The authors claim that FAP is upregulated in injured arterial tissues and actively promotes VSMC migration to facilitate neointimal hyperplasia and subsequent restenosis. Consequently, the study positions FAP as a potential biomarker and a novel therapeutic target for preventing restenosis after peripheral arterial revascularization. The findings suggest that targeting FAP could offer new strategies for managing lower extremity in-stent restenosis (ISR), particularly in complex cases where current therapies show limited efficacy. The authors note that while the Net1–RhoA–ROCK axis appears central, further investigation is required to determine if FAP directly interacts with or regulates the stability of these signaling molecules.