Metformin protects against vascular calcification through the selective degradation of Runx2 by the p62 autophagy receptor.

Vascular calcification is associated with aging, type 2 diabetes, and atherosclerosis, and increases the risk of cardiovascular morbidity and mortality. It is an active, highly regulated process that resembles physiological bone formation. It has previously been established that pharmacological doses of metformin alleviate arterial calcification through adenosine monophosphate-activated protein kinase (AMPK)-activated autophagy, however the specific pathway remains elusive. In the present study we hypothesized that metformin protects against arterial calcification through the direct autophagic degradation of runt-related transcription factor 2 (Runx2). Calcification was blunted in vascular smooth muscle cells (VSMCs) by metformin in a dose-dependent manner (0.5-1.5 mM) compared to control cells (p < 0.01). VSMCs cultured under high-phosphate (Pi) conditions in the presence of metformin (1 mM) showed a significant increase in LC3 puncta following bafilomycin-A1 (Baf-A; 5 nM) treatment compared to control cells (p < 0.001). Furthermore, reduced expression of Runx2 was observed in the nuclei of metformin-treated calcifying VSMCs (p < 0.0001). Evaluation of the functional role of autophagy through Atg3 knockdown in VSMCs showed aggravated Pi-induced calcification (p < 0.0001), failure to induce autophagy (punctate LC3) (p < 0.001) and increased nuclear Runx2 expression (p < 0.0001) in VSMCs cultured under high Pi conditions in the presence of metformin (1 mM). Mechanistic studies employing three-way coimmunoprecipitation with Runx2, p62, and LC3 revealed that p62 binds to both LC3 and Runx2 upon metformin treatment in VSMCs. Furthermore, immunoblotting with LC3 revealed that Runx2 specifically binds with p62 and LC3-II in metformin-treated calcified VSMCs. Lastly, we investigated the importance of the autophagy pathway in vascular calcification in a clinical setting. Ex vivo clinical analyses of calcified diabetic lower limb artery tissues highlighted a negative association between Runx2 and LC3 in the vascular calcification process. These studies suggest that exploitation of metformin and its analogues may represent a novel therapeutic strategy for clinical intervention through the induction of AMPK/Autophagy Related 3 (Atg3)-dependent autophagy and the subsequent p62-mediated autophagic degradation of Runx2.

hox gene expression predicts tetrapod-like axial regionalization in the skate, Leucoraja erinacea.

The axial skeleton of tetrapods is organized into distinct anteroposterior regions of the vertebral column (cervical, trunk, sacral, and caudal), and transitions between these regions are determined by colinear anterior expression boundaries of Hox5/6, -9, -10, and -11 paralogy group genes within embryonic paraxial mesoderm. Fishes, conversely, exhibit little in the way of discrete axial regionalization, and this has led to scenarios of an origin of Hox-mediated axial skeletal complexity with the evolutionary transition to land in tetrapods. Here, combining geometric morphometric analysis of vertebral column morphology with cell lineage tracing of hox gene expression boundaries in developing embryos, we recover evidence of at least five distinct regions in the vertebral skeleton of a cartilaginous fish, the little skate (Leucoraja erinacea). We find that skate embryos exhibit tetrapod-like anteroposterior nesting of hox gene expression in their paraxial mesoderm, and we show that anterior expression boundaries of hox5/6, hox9, hox10, and hox11 paralogy group genes predict regional transitions in the differentiated skate axial skeleton. Our findings suggest that hox-based axial skeletal regionalization did not originate with tetrapods but rather has a much deeper evolutionary history than was previously appreciated.