Integrative transcriptomics and human tibial artery validation identify ADAM12, PDK4, and RUNX1 as peripheral artery disease-associated genes enriched in calcified tibial arteries

Abstract

Background

Medial arterial calcification of tibial vessels is a major obstacle to limb salvage in peripheral artery disease (PAD), yet the molecular drivers remain poorly defined. We used publicly available transcriptomic data to nominate candidate genes relevant to vascular calcification and then tested those candidates in human tibial arteries and in primary PAD-derived vascular cells.

Methods

Differential gene expression was analyzed across publicly available PAD/chronic limb-threatening ischemia (CLTI) transcriptomic datasets, comparing PAD/CLTI tissue to non-PAD controls (limma and DESeq² adjusted P < .05, |log₂ fold-change| > 1.5). Candidates were prioritized based on predicted roles in calcification processes in endothelial cells and vascular smooth muscle cells. Expression was validated by quantitative polymerase chain reaction in isolated human tibial arteries harvested from patients with PAD/CLTI undergoing below-the-knee amputations and non-PAD control vessels. Following the preparation of primary PAD-derived endothelial cells and vascular smooth muscle cells, the cells were cultured under high-glucose or osmotic control conditions for 7 days to assess the effects of a hyperglycemic challenge.

Results

Analysis of 14 datasets identified 89 differentially expressed genes enriched in inflammatory, osteogenic, and metabolic pathways. Fourteen candidates were selected for validation. Among them, ADAM12, PDK4, and RUNX1 were confirmed in PAD tibial arteries as the most relevant candidates from the prespecified panel, consistent with their prioritization from the transcriptomic and literature-guided candidate-selection strategy.

Conclusions

A candidate-first translational strategy integrating public transcriptomic prioritization with validation in human tibial arteries confirmed PDK4 and RUNX1 as significantly upregulated medial arterial calcification-associated genes in PAD tibial arteries, and identified ADAM12 as a biologically prioritized candidate showing a concordant but statistically nonsignificant trend in the same direction. Their expression in diseased tissue, together with the absence of acute glucose responsiveness in primary PAD-derived vascular cells, supports a model in which these genes are linked to chronic vascular remodeling rather than transient hyperglycemic signaling. These findings refine the molecular landscape of PAD-associated calcification and establish a focused framework for potential mechanistic studies.

Clinical relevance

Tibial medial arterial calcification is a major barrier to limb salvage in patients with peripheral artery disease (PAD)/chronic limb-threatening ischemia because it makes revascularization more difficult and less durable. By validating candidate genes in human tibial arteries and primary PAD-derived vascular cells, this study links diseased tissue biology to pathways that may help explain why some patients develop severe calcification and experience poor clinical outcomes. These findings provide a disease-specific framework for identifying potential biomarkers and therapeutic targets to improve infrapopliteal revascularization outcomes and reduce limb loss.