Runx2 deletion in smooth muscle cells inhibits vascular osteochondrogenesis and calcification but not atherosclerotic lesion formation

Abstract

<div class="section"> <a class="named-anchor" id="d1568912e213"> <!-- named anchor --> </a> <h5 class="section-title" id="d1568912e214">Aims</h5> <p id="d1568912e216">Vascular smooth muscle cells (SMCs) are major precursors contributing to osteochondrogenesis and calcification in atherosclerosis. Runt-related transcription factor-2 (Runx2) has been found essential for SMC differentiation to an osteochondrogenic phenotype and subsequent calcification <i>in vitro.</i> A recent study using a conditional targeting allele that produced a truncated Runx2 protein in SMCs of <i>ApoE</i> ^−/− mice showed reduced vascular calcification, likely occurring via reduction of receptor activator of nuclear factor-κB ligand (RANKL), macrophage infiltration, and atherosclerotic lesion formation. Using an improved conditional Runx2 knockout mouse model, we have elucidated new roles for SMC-specific Runx2 in arterial intimal calcification (AIC) without effects on atherosclerotic lesion size. </p> </div><div class="section"> <a class="named-anchor" id="d1568912e227"> <!-- named anchor --> </a> <h5 class="section-title" id="d1568912e228">Methods and results</h5> <p id="d1568912e230">We used an improved targeting construct to generate <i>LDLr</i> ^−/− mice with <i>floxed-Runx2</i> alleles ( <i>LDLr</i> ^−/− <i>:Runx2 ^f/f </i>) such that Cre-mediated recombination ( <i>LDLr</i> ^−/− <i>:Runx2 ^ΔSM </i>) does not produce functional truncated Runx2 protein, thereby avoiding off-target effects. We found that both <i>LDLr</i> ^−/− <i>:Runx2 ^f/f </i> and <i>LDLr</i> ^−/− <i>:Runx2 ^ΔSM </i> mice fed with a high fat diet developed atherosclerosis. SMC-specific Runx2 deletion did not significantly reduce atherosclerotic lesion size, macrophage number, or expression of RANKL, MCP-1, and CCR2. However, it significantly reduced AIC by 50%. Mechanistically, Sox9 and type II collagen were unaltered in vessels of <i>LDLr</i> ^−/− <i>:Runx2 ^ΔSM </i> mice compared to <i>LDLr</i> ^−/− <i>:Runx2 ^f/f </i> counterparts, while type X collagen, MMP13 and the osteoblastic marker osteocalcin were significantly reduced. </p> </div><div class="section"> <a class="named-anchor" id="d1568912e316"> <!-- named anchor --> </a> <h5 class="section-title" id="d1568912e317">Conclusions</h5> <p id="d1568912e319">SMC autonomous Runx2 is required for SMC differentiation towards osteoblast-like cells, SMC-derived chondrocyte maturation and AIC in atherosclerotic mice. These effects were independent of systemic lipid metabolism, RANKL expression, macrophage infiltration, and atheromatous lesion progression. </p> </div>