Differing calcification processes in cultured vascular smooth muscle cells and osteoblasts

Jessal J Patel,Lucie E Bourne,Bethan K Davies,Timothy R Arnett,Vicky E Macrae,Caroline Pd Wheeler-Jones,Isabel R Orriss

doi:10.1016/j.yexcr.2019.04.020

Abstract

Arterial medial calcification (AMC) is the deposition of calcium phosphate mineral, often as hydroxyapatite, in the medial layer of the arteries. AMC shares some similarities to skeletal mineralisation and has been associated with the transdifferentiation of vascular smooth muscle cells (VSMCs) towards an osteoblast-like phenotype. This study used primary mouse VSMCs and calvarial osteoblasts to directly compare the established and widely used in vitro models of AMC and bone formation. Significant differences were identified between osteoblasts and calcifying VSMCs. First, osteoblasts formed large mineralised bone nodules that were associated with widespread deposition of an extracellular collagenous matrix. In contrast, VSMCs formed small discrete regions of calcification that were not associated with collagen deposition and did not resemble bone. Second, calcifying VSMCs displayed a progressive reduction in cell viability over time (≤7-fold), with a 50% increase in apoptosis, whereas osteoblast and control VSMCs viability remained unchanged. Third, osteoblasts expressed high levels of alkaline phosphatase (TNAP) activity and TNAP inhibition reduced bone formation by to 90%. TNAP activity in calcifying VSMCs was ∼100-fold lower than that of bone-forming osteoblasts and cultures treated with β-glycerophosphate, a TNAP substrate, did not calcify. Furthermore, TNAP inhibition had no effect on VSMC calcification. Although, VSMC calcification was associated with increased mRNA expression of osteoblast-related genes (e.g. Runx2, osterix, osteocalcin, osteopontin), the relative expression of these genes was up to 40-fold lower in calcifying VSMCs versus bone-forming osteoblasts. In summary, calcifying VSMCs in vitro display some limited osteoblast-like characteristics but also differ in several key respects: 1) their inability to form collagen-containing bone; 2) their lack of reliance on TNAP to promote mineral deposition; and, 3) the deleterious effect of calcification on their viability.

Highlights

Vascular calcification is a common consequence of ageing, atherosclerosis, diabetes and chronic kidney disease
vascular smooth muscle cell (VSMC) cultured with 2 mM sodium orthophosphate formed discrete regions of calcification that were much smaller than osteoblast bone nodules and did not appear to be associated with collagenous matrix (Fig. 1F)
The development of Arterial medial calcification (AMC) is widely accepted to be associated with a phenotypic transdifferentiation of VSMCs which results in them taking on characteristics usually associated with bone-forming osteoblasts [2,8,10,12]

Summary

Introduction

Vascular calcification is a common consequence of ageing, atherosclerosis, diabetes and chronic kidney disease. It is the pathological deposition of calcium phosphate mineral, usually as hydroxyapatite, in the medial and/or intimal layer of the arteries and heart valves. Arterial medial calcification (AMC) refers to the calcification that occurs within the tunica media of blood vessels and is characterised by increased vessel stiffness and reduced blood flow [1]. AMC was thought to be a passive process caused by high serum levels of phosphate and calcium. It is accepted that vascular calcification is a complex cell-mediated process that is thought to share some similarities with physiological bone formation [2]. Histological analysis of calcified arteries from patients with end stage renal disease has shown the expression of several bone-associated proteins including osteopontin (OPN), tissue non-specific alkaline phosphatase (TNAP) and Runx2 [3,4]

Methods

Results

Conclusion