# Targeting mechanosensitive EphA2 phase separation to alleviate arterial stiffening

## TL;DR

A new mechanism involving EphA2 protein phase separation is found to cause arterial stiffening, and a peptide treatment shows promise in reducing this condition in mice.

## Contribution

The study identifies EphA2 phase separation as a novel mechanosensitive mechanism in arterial stiffening and proposes a peptide-based therapeutic strategy.

## Key findings

- EphA2 forms mechanosensitive condensates that activate ERK1/2-CREB-NR4A3 signaling in stiffened arteries.
- A retro-reversed peptide targeting EphA2 disrupts phase separation and reduces VSMC dysfunction.
- Nanoparticle delivery of the peptide alleviates arterial calcification and stiffening in mice.

## Abstract

Arterial stiffening, a major cardiovascular risk factor, is driven by aberrant mechanotransduction in vascular smooth muscle cells (VSMCs), yet the critical mechanoreceptors and underlying mechanisms remain elusive. Here, we identified Ephrin receptor A2 (EphA2) as a significantly upregulated mechanosensitive receptor in stiffened arteries from a 5/6 nephrectomy mouse model. Genetic deletion of Epha2 in VSMCs markedly attenuated arterial stiffening. Utilizing polyacrylamide gels of varying stiffness and in situ stiffening bioclick hydrogels, we demonstrated that matrix stiffening directly induces EphA2 phase separation, forming a biomolecular condensate that serves as a signaling hub to recruit and activate ERK1/2. This leads to phosphorylation of the transcription factor CREB and subsequent upregulation of the pro-remodeling nuclear receptor NR4A3. To translate this discovery, we designed a retro-reversed peptide targeting the intrinsically disordered regions (IDRs) of EphA2, which effectively disrupted phase separation and mitigated VSMCs dysfunction in vitro. Crucially, in vivo delivery of this peptide via VAPG-modified nanoparticles significantly alleviated arterial calcification and stiffening in mice. Our study establishes EphA2 phase separation as a pivotal mechanism in vascular mechanotransduction and unveils a novel EphA2-ERK1/2-NR4A3 signaling axis, thereby presenting a promising therapeutic strategy for combating arterial stiffening by targeting pathological biomolecular condensates.

Image 1

•EphA2 forms mechanosensitive biomolecular condensates that drive arterial stiffening via ERK1/2-CREB-NR4A3 signaling.•A retro‑reversed peptide targeting EphA2’s disordered region inhibits its phase separation and downstream signaling.•Nanoparticle-delivered peptide disrupts EphA2 phase separation in vivo, alleviating vascular stiffening and calcification.

EphA2 forms mechanosensitive biomolecular condensates that drive arterial stiffening via ERK1/2-CREB-NR4A3 signaling.

A retro‑reversed peptide targeting EphA2’s disordered region inhibits its phase separation and downstream signaling.

Nanoparticle-delivered peptide disrupts EphA2 phase separation in vivo, alleviating vascular stiffening and calcification.