Peripheral artery disease (PAD) is characterized by impaired blood flow to the lower extremities, resulting in ischemic limb injuries. Individuals with diabetes and PAD typically have more severe ischemic limb injuries and limb amputations, but the mechanisms involved are poorly understood. Previously, we identified BAG3 as a gene within a mouse genetic locus termed limb salvage QTL1 on mouse chromosome 7 that determined the extent of limb necrosis following ischemic injury in C57Bl/6 mice. Whether BAG3 deficiency plays a role in the severe ischemic injury observed in diabetic PAD is not known. In vitro, we found simulated ischemia enhanced BAG3 expression in primary human skeletal muscle cells, whereas BAG3 knockdown increased necroptosis markers and decreased cell viability. In vivo, ischemic skeletal muscles from hind limbs of high-fat diet (HFD)-fed mice showed poor BAG3 expression compared to normal chow diet (NCD)-fed mice, and this was associated with increased limb amputations. BAG3 overexpression in ischemic skeletal muscles from hind limbs of HFD mice rescued limb amputation and improved autophagy, necroptosis, skeletal muscle function and regeneration. Therefore, BAG3 deficiency in ischemic skeletal muscles contributes to the severity of ischemic limb injury in diabetic PAD, likely through autophagy and necroptosis pathways.

Background: Peripheral arterial disease affects >200 million people worldwide and is characterized by impaired blood flow to the lower extremities. There are no effective medical treatments available. Using the mouse hind‐limb ischemia model and miRNA sequencing, we identified a novel miRNA, miR‐6236, whose expression significantly elevated in ischemic mouse limbs compared with nonischemic limbs. The role of miR‐6236 in general or in postischemic angiogenesis is not known. Here we describe its role using in vivo and in vitro models of peripheral arterial disease. Methods and Results: In primary mouse and human endothelial cells, we studied the effect of simulated ischemia on miR‐6236 expression and assessed its role in cell viability, apoptosis, migration, and tube formation during ischemia. Furthermore, we developed miR‐6236 null mice and tested its role in postischemic perfusion recovery using the hind‐limb ischemia model. Lastly, using bioinformatics and gene expression analysis, we identified putative angiogenic miR‐6236 targets. In vitro simulated ischemia‐enhanced miR‐6236 expression in mouse and human endothelial cells, whereas its inhibition improved viability, migration, tube formation, and reduced apoptosis. In vivo ischemic mouse skeletal muscle tissue showed higher miR‐6236 expression compared with nonischemic muscles. Loss of miR‐6236 improved impaired postischemic perfusion recovery and poor angiogenesis associated with streptozotocin‐induced diabetes in mice. Six of the 8 miR‐6236 predicted angiogenic target mRNAs showed expression consistent with regulation by miR‐6236 in ischemic skeletal muscle. Conclusions: Our results show for the first time that miR‐6236 plays a key role in regulating postischemic perfusion recovery and angiogenesis.

12/15-lipoxygenase (12/15-LOX) plays an essential role in oxidative conversion of polyunsaturated fatty acids into various bioactive lipid molecules. Although 12/15-LOX's role in the pathophysiology of various human diseases has been well studied, its role in weight gain is controversial and poorly clarified. Here, we demonstrated the role of 12/15-LOX in high-fat diet (HFD)-induced weight gain in a mouse model. We found that 12/15-LOX mediates HFD-induced de novo lipogenesis (DNL), triglyceride (TG) biosynthesis and the transport of TGs from the liver to adipose tissue leading to white adipose tissue (WAT) expansion and weight gain via xanthine oxidase (XO)-dependent production of H₂O₂. 12/15-LOX deficiency leads to cullin2-mediated ubiquitination and degradation of XO, thereby suppressing H₂O₂ production, DNL and TG biosynthesis resulting in reduced WAT expansion and weight gain. These findings infer that manipulation of 12/15-LOX metabolism may manifest a potential therapeutic target for weight gain and obesity.