The intersection of protein and lipid biology is of growing importance for understanding how cells address structural challenges during adhesion and migration. While protein complexes engaged with the cytoskeleton play a vital role, support from the phospholipid membrane is crucial for directing localization and assembly of key protein complexes. During angiogenesis, dramatic cellular remodeling is necessary for endothelial cells to shift from a stable monolayer to invasive structures. However, the molecular dynamics between lipids and proteins during endothelial invasion are not defined. Here, we utilized cell culture, immunofluorescence, and lipidomic analyses to identify a novel role for the membrane binding protein Annexin A2 (ANXA2) in modulating the composition of specific membrane lipids necessary for cortical F‐actin organization and adherens junction stabilization. In the absence of ANXA2, there is disorganized cortical F‐actin, reduced junctional Arp2, excess sprout initiation, and ultimately failed sprout maturation. Furthermore, we observed reduced filipin III labeling of membrane cholesterol in cells with reduced ANXA2, suggesting there is an alteration in phospholipid membrane dynamics. Lipidomic analyses revealed that 42 lipid species were altered with loss of ANXA2, including an accumulation of phosphatidylcholine (16:0_16:0). We found that supplementation of phosphatidylcholine (16:0_16:0) in wild‐type endothelial cells mimicked the ANXA2 knock‐down phenotype, indicating that ANXA2 regulated the phospholipid membrane upstream of Arp2 recruitment and organization of cortical F‐actin. Altogether, these data indicate a novel role for ANXA2 in coordinating events at endothelial junctions needed to initiate sprouting and show that proper lipid modulation is a critical component of these events.

Rodent husbandry requires careful consideration of environmental factors that may impact colony performance and subsequent physiological studies. Of note, recent reports have suggested corncob bedding may affect a broad range of organ systems. As corncob bedding may contain digestible hemicelluloses, trace sugars, and fiber, we hypothesized that corncob bedding impacts overnight fasting blood glucose and murine vascular function. Here, we compared mice housed on corncob bedding, which were then fasted overnight on either corncob or ALPHA-dri® bedding, a virgin paper pulp cellulose alternative. Male and female mice were used from two non-induced, endothelial-specific conditional knock-out strains (Cadherin 5-cre/ERT2, floxed hemoglobin alpha 1 [Hba1 fl/fl] or Cadherin 5-cre/ERT2, floxed cytochrome B5 reductase 3 [CyB5R3 fl/fl]) on a C57BL/6J genetic background. After fasting overnight, initial fasting blood glucose was measured, and mice were anesthetized with isoflurane for measurement of blood perfusion via laser speckle contrast analysis using a PeriMed PeriCam PSI NR system. After a 15-minute equilibration, the mice were injected intraperitoneally with the alpha-1 adrenergic receptor agonist, phenylephrine (5 mg/kg) or saline and monitored for changes in blood perfusion. After a 15-minute response period, blood glucose was remeasured post-procedure. In both strains, mice fasted on corncob bedding had higher blood glucose than the pulp cellulose group. In the CyB5R3 fl/fl strain, mice housed on corncob bedding displayed a significant reduction in phenylephrine-mediated change in perfusion. In the Hba1 fl/fl strain, phenylephrine-induced change in perfusion was not different in the corncob group.

The intersection of protein and lipid biology is of growing importance for understanding how cells address structural challenges during adhesion and migration. While protein complexes engaged with the cytoskeleton play a vital role, support from the phospholipid membrane is crucial for directing localization and assembly of key protein complexes. During angiogenesis, it is well observed that dramatic cellular remodeling is necessary for endothelial cells to shift from a stable monolayer to invasive structures. However, the molecular dynamics between lipids and proteins during endothelial invasion are not defined. Here, we utilized cell culture, immunofluorescence, and lipidomic analyses to identify a novel role for the membrane binding protein Annexin A2 (ANXA2) in modulating the composition of specific membrane lipids necessary for cortical F-actin organization and adherens junction stabilization. In the absence of ANXA2, there is disorganized cortical F-actin, reduced junctional Arp2, excess sprout initiation, and ultimately failed sprout maturation. Further, we observed reduced filipin III labeling of membrane cholesterol in cells with reduced ANXA2, suggesting there is an alteration in phospholipid membrane dynamics. Lipidomic analyses reveal that 42 lipid species are altered with loss of ANXA2, including an accumulation of phosphatidylcholine (16:0_16:0). We find that supplementation of phosphatidylcholine (16:0_16:0) in wild-type endothelial cells mimics the ANXA2 knock-down phenotype, indicating that ANXA2 regulates the phospholipid membrane upstream of Arp2 recruitment and organization of cortical F-actin. Altogether these data indicate a novel role for ANXA2, and show that proper lipid modulation is a critical component of endothelial sprouting.