Differing calcification processes in cultured vascular smooth muscle cells and osteoblasts

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.

Apoptosis in the Extraosseous Calcification Process

“…Despite the similarities between vascular and bone mineralization, it has to be pointed out that, besides the pathologic vs. physiologic consequences, VC is characterized by localized mineralized areas of the elastic component, whereas collagen is rarely involved. Moreover, calcifying VSMCs exhibit reduced viability with increased apoptosis and higher expression of calcification markers (e.g., TNAP or Runx2) although at lower levels compared to bone cells [ 228 ].…”

Mechanisms of the Osteogenic Switch of Smooth Muscle Cells in Vascular Calcification: WNT Signaling, BMPs, Mechanotransduction, and EndMT

“…Osteogenic genes were upregulated in the calcified VSMCs, but the amount of mRNA was much lower than that in the osteoblasts. They concluded that even when calcified, the VSMCs are in the early stages of osteoblast-like differentiation [28]. This transition typically occurs in environments with high serum phosphate and calcium levels.…”

“…A study done by Patel et al showed that calcifying VSMCs have higher rates of apoptosis and lower cellular viability than control VSMCs and osteoblasts [ 28 ]. The study showed how calcification damages VSMCs and reduces compliance and structural integrity, leading to heart attack or stroke.…”

Correlativity between osteocalcin and coronary artery calcification in middle-aged and elderly population

“…As people get older, the calcium lost from bones is deposited in the cardiovascular system 6 . Vascular calcification (VC) is a recognized risk factor for cardiovascular morbidity and mortality 7 and is currently considered an active process [8][9][10][11] .VC has some similarities to bone mineralization and is related to the transformation of vascular smooth muscle cells (VSMCs) into osteoblast-like phenotypes 12 .…”