Abstract 544: Diabetes Mellitus Impacts on Vascular Calcification in Peripheral Arterial Disease Patients but Does Not Further Activates Osteogenic Markers in Beta-Glycerophosphate Stimulated Vascular Smooth Muscle Cells

Abstract

Introduction: Diabetes mellitus (DM) accelerates vascular calcification (VC) in peripheral arterial disease (PAD), increasing limb ischemia and amputation risk. Although DM and VC implicates in PAD, the mechanisms underlying vascular smooth muscle cells (SMC) osteochondrogenic differentiation in this setting are scarce. Objectives: To assess VC and osteogenic mRNA/protein expression (Msx2, Runx2 and ALPL) in arteries and in primary SMC isolated from PAD patients with DM (n=3), without DM (n=2) and individuals without PAD (control [CTL], n=2) that underwent amputation. Methods: Ethical committee approved study and patients signed informed consent. 5-7 th passages SMC were incubated without (untreated) or with β-glycerophosphate 10mM (β-GP) for 48h (mRNA expression), 72h (protein expression) and 14 days (calcification). In addition, we performed tissue immunofluorescence and Alizarin Red S. Results: Untreated PAD+DM SMC increased calcification (4.6±1.2) in comparison to PAD without DM patients (2.5±0.2) and CTL individuals (1.0±0.07). β-GP further increased calcification in respective groups (51.3±4.7; 13.5±1.1 and 9.8±1.2). Msx2 mRNA expression decreased in CTL SMC after β-GP (48h) and did not change in PAD without DM and in PAD+DM SMC. Msx2 protein expression decreased in all groups after calcifying medium (72h). Moreover, Runx2 mRNA expression increased in β-GP-treated PAD+DM SMC and ALPL mRNA expression augmented in β-GP-treated SMC from PAD without DM patients. However, Runx2 and ALPL protein expression was not modulated by calcifying medium (72h) in all groups. Interestingly, immunofluorescence of arterial samples demonstrated increased osteochondrogenic protein expression (Msx2, Runx2 and ALPL) around calcifying foci in PAD+DM group (+++) versus PAD without DM (++) and versus CTL (+) patients. Coincidently, PAD+DM group showed augmented vascular calcification (19x10 4 ±10x10 4 ; 7x10 4 ±2x10 4 ; 3x10 3 ±10 -2 μm 2 ). Conclusion: We demonstrated increased calcification in arteries from amputated PAD+DM patients and in respective SMC without and with calcifying medium. Although we found augmented osteochondrogenic protein expression in the vessel wall from PAD+DM individuals this was not robustly shown in isolated SMC in vitro .