Figure 5. PLOD1 pathogenicity in aortopathy is linked to vascular smooth muscle cell changes.

Left panel: Human vascular smooth muscle cells (VSMCs) were cultured and PLOD1 was intentionally silenced (91% si-RNA, *p< 0.05, A). Collagen gel-based assays were used to perform VSMC contraction studies, which showed that si-PLOD1 knockdown cells demonstrated hypercontractility (1.59 mm²±0.48 vs 2.59±0.312, p<0.01) (B–D). The same si-PLOD1 VSMCs demonstrated upregulation of contractile elements (ACTA2 (1.69±0.06 vs 1.01±0.09, p<0.01) (E) and MYH11 (Fig. S4), with insignificant reduction in the proliferative marker CCND1 (1.17+0.33 vs. 0.30+0.04, p=0.56) (F) and no significant difference in secretory elements COL1A1 and COL3A1 (G, H). Right panel: VSMCs were cultured and intentionally transfected with wildtype (WT, overexpression of PLOD1) or the p. (Ser178Arg) variant; WT and variant VSMCs demonstrated ~ 10,000-fold increase in PLOD1 and the variant, respectively (I). We compared contraction studies and genetic markers in control, wildtype and variant VSMCs. There was no difference in VSMC contractility in any of the cell lines (J, K). In WT cells there was no evidence of change in the contractile marker ACTA2 (L), however the proliferative marker CCND1 was increased in the variant, but not WT line (M). Finally, there was evidence of induction of a secretory phenotype in WT cells (increased COL1A1 and COL3A1), yet in the VSMCs transfected with the variant, this response was attenuated, consistent with a loss-of-function of PLOD1 (N, O). * p < 0.05.