Background:
Cardiovascular disease, including peripheral artery disease (PAD), remains the leading global cause of death. Although PAD reflects advanced atherosclerosis and features high mortality, curative therapies are lacking. Interestingly, the vascular system can respond to atherosclerotic occlusion by forming bypass circuits. Clinical studies demonstrate that the degree of collateralization varies greatly between individuals. In asymptomatic PAD patients (silent high-grade PAD, sHG-PAD), vascular adaptations are so advanced that, despite significant arterial occlusion, no ischemic pain or tissue damage occur. Yet, the immune mechanisms enabling such adaptation are unclear. We hypothesised that sHG-PAD patients display a proangiogenic immune profile that favours collateral growth.
Materials and methods:
In the LUERPAD (Luebeck Registry of Peripheral Artery Disease)-IMMUNO study, we recruited symptomatic and asymptomatic PAD patients to investigate mechanisms of collateralisation. We comprehensively characterised 18 asymptomatic and 17 symptomatic patients using laboratory parameters, arterial haemodynamic profiles (ankle-brachial-index (ABI), stenosis severity, duplex-derived lower-limb flow profiles), questionnaires, and clinical assessments. To gain immunological insights, peripheral blood mononuclear cells (PBMCs) were analysed. First, we performed single-cell RNA sequencing (scRNA-seq) to explore the cellular transcriptomes in 12 patients (6 asymptomatic, 6 symptomatic). For further investigation of the immunophenotype and to uncover differences even on protein level we used full-spectrum flow cytometry, subsequently validated through FACS-sorting. T cells, B cells, NK cells, and monocytes were isolated, followed by RNA extraction, cDNA synthesis, and digital PCR (dPCR). Additionally, we conducted serological investigations using protein arrays and ELISAs to explore potential mechanisms and biomarkers associated with sHG-PAD.
Results:
In this cohort, arterial haemodynamic measures (ABI, stenosis severity, and duplex flow characteristics) showed no significant differences between asymptomatic and symptomatic groups, confirming equal degree of lower extremity atherosclerosis. Using scRNA-seq, we identified a novel lymphocyte subtype, enriched in sHG-PAD patients. This subtype exhibited a highly upregulated expression of the GABBR2 gene, as well as surface proteins involved in endothelial adhesion and angiogenesis.
Through full-spectrum flow cytometry using a custom-designed 41-colour panel, the hypothesis of distinct “GABAB”-lymphocytes was strengthened. AI-assisted clustering and annotation analyses identified the subtype as an NK-cell population, being upregulated in sHG-PAD patients. FACS-purified cells analysed by dPCR confirmed higher GABBR2 transcripts in NK cells than in T cells, B cells, or monocytes, an effect not observed in symptomatic patients. In vitro, PBMC stimulation with the GABAB-receptor agonist baclofen increased the release of pro-angiogenic mediators.
Conclusions:
These findings demonstrate that sHG-PAD patients exhibit a distinct immune signature marked by GABBR2-expressing NK cells, alongside a comparable macrovascular disease burden. It implicates NK-cell GABAB-receptor signalling as a candidate mechanism promoting collateral vessel formation and suggest testable immunomodulatory strategies for PAD.
