Class II α-isoform of phosphatidylinositol 3-kinases (PI3K-C2α) is localized in endosomes, the trans-Golgi network and clathrin-coated vesicles, however, its functional role is little understood. Global or endothelial cell (EC)-specific targeted disruption of PI3K-C2α resulted in embryonic lethality due to defects in sprouting angiogenesis and vascular maturation. PI3K-C2α knockdown in ECs induced decreased phospatidylinositol 3-phosphate-enriched endosomes, impaired endosomal trafficking, and defective delivery of VE-cadherin to EC junctions and its assembly. PI3K-C2α knockdown also impeded cell signaling including vascular endothelial growth factor receptor internalization and endosomal RhoA activation. These together led to defective EC migration, proliferation, tube formation and barrier integrity. Endothelial PI3K-C2α deletion suppressed post-ischemic and tumor angiogenesis, and diminished vascular barrier function, with greatly augmented susceptibility to anaphylaxis and a higher incidence of dissecting aortic aneurysm formation in response to angiotensin II infusion. Thus, PI3K-C2α plays a crucial role in vascular formation and barrier integrity, and represents a new therapeutic target for vascular diseases. 3Formation of the vascular network by vasculogenesis and angiogenesis is essential for embryonic development, repair and remodeling of tissues in adults, as well as tumor growth. The angiogenic response to vascular endothelial growth factor (VEGF) and other factors begins with vascular leakage and dissolution of the subendothelial basement membrane, followed by proliferation and migration of vascular EC 1,2 . Then, formation of the intercellular junctions results in initial sprouts from existing vessels. The newly formed endothelial tubes are associated with mural cells, i.e. smooth muscle cells (SMC) and pericytes, thus becoming mature and stabilized 3 . Tightness of the intercellular junctions, particularly adherens junctions composed of VE-cadherin, controls vascular permeability 4,5 . Quiescent, stabilized vasculature with intact barrier integrity dominates in the healthy condition. In contrast, in pathological conditions, such as tumors, the vasculature is generally inmaturate and leaky. In the case of vascular insult such as excessive angiotensin II (Ang II) activity, increased vascular permeability is asssociated with leukocyte infiltration in the vascular wall and vascular disruption 6,7 . Therefore, stabilization of the vasculature and maintenance of vascular integrity is essential for vascular and tissue homeostasis 8,9 .PI3Ks are an enzyme family that phosphorylates membrane inositol lipids at the 3' position of the inositol ring. The lipid products of PI3Ks serve as important intracellular messengers by interacting with effector proteins, which include protein kinases, guanine nucleotide exchangers for G proteins, and actin cytoskeleton-regulating proteins. Through these actions, PI3Ks regulate a diverse array of cellular processes 10-12 .PI3Ks comprise three classes. Class I PI...

Purpose: Solute carrier, OCTN1 (SLC22A4) is an orphan transporter, the physiologically important substrate of which is still unidentified. The aim of the present study was to examine physiological roles of OCTN1. Methods:We first constructed octn1 gene knockout (octn1 -/-) mice. Metabolome analysis was then performed to identify substrates in vivo. The possible association of the substrate identified with diseased conditions was further examined. Results:The metabolome analysis of blood and several organs indicated complete deficiency of a naturally occurring potent antioxidant ergothioneine in octn1 -/-mice among 112 metabolites examined.Pharmacokinetic analyses after oral administration revealed the highest distribution to small intestines and extensive renal reabsorption of [ 3 H]ergothioneine, both of which were much reduced in octn1 -/-mice.The octn1 -/-mice exhibited greater susceptibility to intestinal inflammation under the ischemia and reperfusion model. The blood ergothioneine concentration was also much reduced in Japanese patients with Crohn's disease, compared with healthy volunteers and patients with another inflammatory bowel disease, ulcerative colitis. 4Conclusions: These results indicate that OCTN1 plays a pivotal role for maintenance of systemic and intestinal exposure of ergothioneine which could be important for protective effect against intestinal tissue injuries, providing a possible diagnostic tool to distinguish the inflammatory bowel diseases.

Asparaginyl endopeptidase (AEP)/legumain, an asparagine-specific cysteine proteinase in animals, is an ortholog of plant vacuolar processing enzyme (VPE), which processes the exposed asparagine residues of various vacuolar proteins. In search for its physiological role in mammals, here we generated and characterized AEP-deficient mice. Although their body weights were significantly reduced, they were normally born and fertile. In the wild-type kidney where the expression of AEP was exceedingly high among various organs, the localization of AEP was mainly found in the lamp-2-positive late endosomes in the apical region of the proximal tubule cells. In these cells of AEP-deficient mice, the lamp-2-positive membrane structures were found to be greatly enlarged. These aberrant lysosomes, merged with the late endosomes, accumulated electron-dense and membranous materials. Furthermore, the processing of the lysosomal proteases, cathepsins B, H, and L, from the single-chain forms into the two-chain forms was completely defected in the deficient mice. Thus, the AEP deficiency caused the accumulation of macromolecules in the lysosomes, highlighting a pivotal role of AEP in the endosomal/lysosomal degradation system.