Abstract Body: Objectives: Vascular Ehlers-Danlos syndrome (VEDS), caused by pathogenic COL3A1 variants, compromises vascular tissue integrity. We aimed to characterize genotype-specific alterations in dermal collagen fibril architecture and type III collagen content, using the extracellular matrix (ECM) as a window into arterial wall biology, to elucidate mechanisms of substrate vulnerability in VEDS.

Methods: Dermal biopsies were obtained from 11 individuals with VEDS (2 in-frame deletions, 3 alanine, 4 serine, and 2 valine substitutions of glycine) and 11 controls. Transmission electron microscopy (TEM) measured collagen fibril diameters across dermal layers using a customized, semi-automated Fiji/ImageJ macro for standardized, high-throughput measurement. Fibril diameters were analyzed by COL3A1 variant type. In parallel, type III collagen content from 8 VEDS samples was quantified using liquid chromatography–mass spectrometry (LC-MS), including allele-specific peptide analysis.

Results: Multimodal analysis revealed marked, genotype-dependent heterogeneity in ECM organization. Quantitative LC-MS (n = 8 VEDS) showed reduced type III collagen relative to total collagen (type I + III) in VEDS versus controls (17.8% vs 27.1%, p = 0.01), providing the first direct quantification of type III collagen dermal deficiency. Allele-specific peptide analysis showed near-equimolar mutant/wild-type expression in alanine substitutions (~55% variant) but reduced variant peptide expression in valine substitutions (16.7%), indicating variant-specific effects on ECM incorporation. TEM revealed smaller mean fibril diameters in large-side-chain variants and deletions (0.081 ± 0.013 μm) compared to those with small-side-chain substitutions (0.092 ± 0.014 μm), particularly in the reticular dermis. Although group differences were not statistically significant, large effect sizes (Hedges’ g = 0.80) support a biologically meaningful remodeling signature.

Conclusion: VEDS is a genotype-specific disorder of ECM organization, not merely a type III collagen deficiency. Distinct COL3A1 variants yield divergent fibrillar architecture not predicted by type III collagen levels alone, implicating mutant protein incorporation as a key driver of ECM remodeling. These findings hold significance for understanding fibrillogenesis, refining risk stratification, and guiding surgical decision-making.