ACKR1-high endothelial cells enhance CXCL8 responsiveness and reveal a therapeutic entry point in calcific aortic valve disease

Background

Valvular endothelial dysfunction is increasingly recognised as an initiating event in calcific aortic valve disease (CAVD). However, the endothelial programmes that enhance inflammatory responsiveness and create therapeutic vulnerabilities remain poorly defined, hindering the development of disease-modifying therapies.

Purpose

To characterise a novel ACKR1 hi valvular endothelial cell (VEC) subset and elucidate its role in amplifying CXCL8 responsiveness, metabolic reprogramming, and endothelial-to-mesenchymal transition (EndoMT) during CAVD progression.

Methods

We integrated single-cell RNA sequencing (scRNA-seq) datasets from 24 patients (171,628 cells) to resolve endothelial heterogeneity and intercellular communication. Plasma CXCL8 was quantified in a prospective cohort of patients with haemodynamically severe CAVD (n=80) versus matched controls (n=40). Mechanistic studies employed gain-of-function assays in primary human VECs and endothelial-specific Ackr1 silencing in a rat CAVD model. Finally, leveraging the ACKR1 cryo-EM structure, we utilised deep learning–based DeepDocking and biolayer interferometry (BLI) to identify and validate high-affinity pharmacological inhibitors.

Results

scRNA-seq integration identified a previously unrecognised ACKR1 hi VEC subset significantly enriched in fibro-calcific valves. Convergent ligand–receptor analyses (CellChat and CellPhoneDB) identified the ACKR1–CXCL8 axis as the dominant chemokine signalling pathway, with VECs serving as the principal responding compartment. Clinically, elevated plasma CXCL8 correlated with haemodynamic severity (Vmax and mean gradient).

Functionally, while wild-type VECs remained largely quiescent under low-level CXCL8 (15 pg/mL), ACKR1 hi VECs exhibited markedly enhanced responsiveness, triggering robust EndoMT and metabolic reprogramming. Multi-omics profiling demonstrated reduced fatty acid oxidation (FAO), consistent with the metabolic–EndoMT phenotype observed in human ACKR1 hi VECs and associated with increased local CXCL8 availability.

In vivo, endothelial-specific Ackr1 silencing significantly blunted transvalvular peak velocity progression and reduced valvular fibrosis. Guided by the cryo-EM structure, DeepDocking identified a lead compound, E957-0282, which exhibited nanomolar binding affinity (via BLI) and effectively rescued the pathological VEC phenotype, leading to improved haemodynamic outcomes.

Conclusions

The emergence of an ACKR1 hi VEC subset acts as a tissue-level signal amplifier that sensitises the endothelium to systemic inflammatory cues. By integrating single-cell discovery with structure-based drug design, we identify ACKR1 as a tractable therapeutic target for disease modification in CAVD.