Runx2 and Vascular Calcification

Thesis (Ph.D.)--University of Washington, 2016-06

Complications arising from vascular calcification, the inappropriate deposition of calcium phosphate crystals in the vasculature, have become more evident in recent years as human longevity has increased. Arterial calcification is frequently categorized as two types, depending on where it occurs: arterial medial calcification (AMC), commonly associated with aging, type 2 diabetes mellitus, and CKD, and arterial intimal calcification (AIC), characterized by plaque accumulation and occlusive lesions in the vasculature. Runx2 (runt-related transcription factor 2) is a critical regulator of bone development, early chondrocyte proliferation, and hypertrophic chondrocyte formation. Surprisingly, Runx2 has been found to be upregulated in arteries of patients with atherosclerosis (intimal calcification) and CKD (medial calcification). Because SMCs can undergo lineage reprogramming toward osteoblastic and chondrocytic fates during vascular calcification, we propose to determine whether SMC-specific Runx2 expression is required for development of AMC and AIC. We developed a mouse with smooth muscle-specific removal of Runx2 (Runx2ΔSM). We observed that in AMC, Runx2 was required for pathological AMC development and prevented osteogenic phenotype change. Similar to our in vivo experiments, SMCs isolated from Runx2ΔSM mice were much less susceptible to elevated phosphate-induced calcification. In AIC, on the other hand, Runx2 inhibited not only osteoblastic differentiation of vascular SMCs but also SMC-derived chondrocyte maturation, leading to a 50% reduction in calcification. Our studies showed that while SMC expression of Runx2 was not required for initial chondrogenesis, it was necessary for maturation of chondrocytes to a pro-mineralizing, hypertrophic state. Lastly, we proposed to investigate the effect of Runx2 on development of atherosclerosis and polarization of lesional macrophages. Removal of Runx2 did not affect monocyte/macrophage recruitment and atherosclerotic lesion size. Within lesions, we observed a strong shift in macrophage polarization toward an M2 polarization in atherosclerotic aortas. Using a co-culture model of vascular SMCs and RAW264.7 cells to study the effect of Runx2-mediated smooth muscle signaling of macrophages, we found significant increases in expression of two M2 markers: MRC1 and IR-1R2. To our knowledge, these results are the first to show that SMCs can directly alter macrophage polarization, and have promising implications for treatments looking to regulate macrophages in atherosclerotic lesions.