H Nording(1,2,3), P Raddatz(3), A Lübken(3), A Fähnrich(4), J von Esebeck(1,2,3), M Leuner(1,2,8), I Eitel(2,5), O J Müller(1,2), M Meusel(1,2), H Langer(6,7)

1. Department of Internal Medicine V (Cardiology and Angiology), University of Kiel, and German Centre for Cardiovascular Research, Partner Site Hamburg/Kiel/Lübeck, Kiel, Germany; 2. DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Lübeck/Kiel, Lübeck, Germany; 3. Cardioimmunology Group, Medical Clinic II, University Heart Center Lübeck, Lübeck, Germany; 4. Institute of Experimental Dermatology, University of Luebeck,23562 Lübeck, Germany; 5. University Heart Center Lübeck, Medical Clinic II (Cardiology/Angiology/Intensive Care Medicine), University Hospital Schleswig-Holstein, Lübeck, Germany; 6. Department of Cardiology, Angiology, Haemostaseology, and Medical Intensive Care, University Medical Centre Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany; 7. German Centre for Cardiovascular Research (DZHK), Partner Site Heidelberg/Mannheim, Germany; 8: Institute of Human Genetics, Universitätsklinikum Schleswig-Holstein, University of Lübeck and University of Kiel, 23562, Lübeck, Germany

Atherosclerotic diseases remain a leading cause of global morbidity and mortality, driven by inflammatory narrowing of arteries. While traditional CAD risk factors predict the onset of atherosclerosis, clinical outcomes hinge on ischemic tissue damage—often modulated by the presence of collateral arteries. However, the mechanisms governing collateral vessel formation remain poorly understood, limiting therapeutic advances.

In the LUERPAD-Immuno study, we identified PAD patients with high-grade leg or groin stenoses but without ischemic walking pain using large-scale ABI screening, and matched these asymptomatic individuals by age and gender to symptomatic controls. We hypothesized that the absence of symptoms reflects enhanced collateral artery growth. To explore the underlying immune signature, we performed multi-colour flow cytometric phenotyping (43-colour panel) on 40 patients and single-cell RNA sequencing (using the Rhapsody system) on a balanced subgroup of 16 patients (8 asymptomatic and 8 symptomatic). We further validated our findings using a murine hindlimb ischemia model.

Our results revealed marked differences in circulating CD11c-high monocytes between the two groups. AI-based clustering demonstrated a strong correlation between their frequency and PAD symptoms, and single-cell transcriptomics confirmed a distinct transcriptional profile. In the murine hindlimb ischemia model, these monocytes rapidly migrated into ischemic tissue—explaining their lower circulating levels in patients with effective collateral formation—and further in vitro and in vivo experiments indicated they may supply TIMP1 to support vascular adaptation.

This study shows that integrating advanced AI with translational research uncovers novel immune mechanisms in cardiovascular disease, paving the way for innovative immune-based therapies.