Expression of osteogenic regulators in healthy and diseased vascular smooth muscle cells

Vascular calcification, a hallmark of chronic kidney disease (CKD), arises in part from vascular smooth muscle cell transdifferentiation, yet the mechanisms driving this osteogenic shift remain elusive. Using primary vascular smooth muscle cells (VSMC) from healthy Lewis and Nek8-mutant LPK rats, we examined the hypothesis that the LPK cells would have an increased propensity for osteogenic transdifferentiation due to their CKD inducing genetic mutation. VSMCs were serum-starved for 4-days, then cultured for 21-days in base media [α-MEM + 2% FBS] or osteogenic media (base media + 10mM β-glycerophosphate, 50μM ascorbic acid, and 100nM dexamethasone). Calcification was assessed via OsteoSense staining of hydroxyapatite and calcium quantification using an o-cresolphthalein assay. Osteogenic
transdifferentiation was assessed by qPCR for bone-morphogenetic protein two (BMP2) and runt-related transcription factor two (Runx2) mRNA expression, and BCIP/NBT staining for alkaline phosphatase (ALP) activity. Lewis and LPK VSMCs howed increased hydroxyapatite and calcium deposition following osteogenic treatment. BMP2 mRNA expression was upregulated in both Lewis and LPK osteogenic treated VSMCs, with no change in Runx2. ALP activity was significantly greater in LPK vs Lewis osteogenic treated VSMCs. VSMCs from the LPK rats exhibited elevated osteogenic potential, linked to increased ALP activity, suggesting this genetic CKD model provides a platform to study vascular calcification mechanisms and identify transdifferentiation-mitigating therapies.