Abstract.A new cookie test was developed for the simultaneous evaluation of multiple risk factors such as glucose intolerance, hyperinsulinemia, insulin resistance and postprandial dyslipidemia. The cookie consisting of 75 g carbohydrate and 25 g fat is ingested and the blood samples are obtained at 0, 1 and 2 hours later. When the two carbohydrate sources, liquid glucose and test cookie, were compared as a glucose load within 3 months, the 2 hr plasma glucose levels were not statistically different, proposing the use of the same criteria at 2 hour glucose level for the diagnosis of diabetes and impaired glucose tolerance (IGT) in subjects without exocrine pancreatic dysfunction. In addition, hyperinsulinemia, insulin resistance (AUC insulin, and/or AUC insulin X AUC glucose), and postprandial hyperlipidemia (DTG, Triglyceride; DRLP, remnant like particles) have been simultaneously uncovered. Reactive hypoglycemia with adverse epigastric discomfort was observed in 26.3% of the control subjects with liquid glucose, while it was observed in only 1 case (5.3%) without any symptom with cookie tests. In fact, one reactive hypoglycemia out of 5 with liquid glucose turned out to be IGT with cookie test. In 64 subjects with lifestyle-related diseases, cookie test revealed hyperinsulinemia and insulin resistance in 56% respectively, postprandial hyperlipidemia in 39%, diabetes and IGT in 22-23% of each of the subjects and all showed at least one abnormal value. In contrast, in university students with exercise habit, all showed normal results with cookie test. In addition, improved insulin sensitivity over non-exercise group was obverved. In summary, the cookie test provided more informations compared with OGTT using liquid glucose and with fewer side effects. Simultaneous evaluation of glucose intolerance, hyperinsulinemia, insulin resistance, and postprandial hyperlipidemia was also possible. SIMULTANEOUS determination of glucose intolerance including diabetes mellitus (DM), hyperinsulinemia, insulin resistance and postprandial dyslipidemia is beneficial for the early detection of metabolic factors involved in the lifestyle-related diseases [1]. Oral glucose tolerance test (OGTT) can reveal glucose intolerance, hyperinsulinemia, and apparent insulin resistance [2], but false reactive hypoglycemia has often been observed with adverse epigastric symptoms and does not reflect the daily blood glucose excursion and insulin responses. The cookie was ingested and the blood glucose, insulin and TG or RLP responses were measured in the newly developed cookie test. With the exception of exocrine pancreatic dysfunction, starch and 15% maltose are thought to be well digested and no significant difference has been reported for the postprandial 2 hr blood glucose levels [3]. Thus after ingestion of the cookie, postprandial hyperglycemia using the same criteria as OGTT, as well as postprandial dyslipidemia can be simultaneously evaluated. Measurement of endogenous insulin gives additional information regarding hyperinsulinemia and in...

Transforming growth factor-␤ (TGF-␤) is involved in vascular formation through activin receptor-like kinase (ALK)1 and ALK5. ALK5, which is expressed ubiquitously, phosphorylates Smad2 and Smad3, whereas endothelial cell (EC)-specific ALK1 activates Smad1 and Smad5. Because ALK5 kinase activity is required for ALK1 to transduce TGF-␤ signaling via Smad1/5 in ECs, ALK5 knockout (KO) mice were not able to give us the precise mechanisms by which TGF-␤/ALK5/Smad2/3 signaling is implicated in angiogenesis. To delineate the role of Smad2/3 signaling in endothelium, the Smad2 gene in Smad3 KO mice was selectively deleted in ECs using Tie2-Cre transgenic mice, termed EC-specific Smad2/3 double KO (EC-Smad2/3KO) mice. EC-Smad2/3KO embryos revealed hemorrhage leading to embryonic lethality around E12.5. EC-Smad2/3KO embryos exhibited no abnormality of vasculogenesis and angiogenesis in both the yolk sac and the whole embryo, whereas vascular maturation was incomplete because of inadequate assembly of mural cells in the vasculature. Wide gaps between ECs and mural cells could be observed in the vasculature of EC-Smad2/3KO mice because of reduced expression of Ncadherin and sphingosine-1-phosphate receptor-1 (S1PR1) in ECs from those mice. These results indicated that Smad2/3 signaling in ECs is indispensable for maintenance of vascular integrity via the fine-tuning of N-cadherin, VEcadherin, and S1PR1 expressions in the vasculature.(Blood. 2012;119(22): 5320-5328) IntroductionAberrant vascularization leads to a number of diseases including atherosclerosis, tumorigenicity, and retinopathy, 1,2 whereas angiogenesis is essential during embryonic development as well as in adulthood. Angiogenesis is mediated by sprouting of new vessels from preexisting ones or by intussusceptive microvascular growth. In general, vascular formation is quiet in adulthood, although angiogenesis involved in wound healing, inflammation, ischemia, and the female reproductive cycle can be observed. Angiogenesis is divided into 2 phases: the activation phase and the resolution phase. The balance between physiologic stimulators (eg, vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF-2), angiopoietins, and hypoxia) and inhibitors (eg, angiostatin, endostatin, and interferon-␣) is strategic to tuning of the angiogenic switch. Proliferation of endothelial cells (ECs), increase in vascular permeability, and degradation of extracellular matrix components can be observed during the activation phase. Consequently, ECs make new capillary sprouts. In the resolution phase, the proliferation and migration of ECs ceases and is followed by reconstitution of the basement membrane and maturation of the vessels. 3 Transforming growth factor-␤ (TGF-␤) is a pivotal cytokine that contributes to the behaviors and activities of most cells from the embryonic to the adult stage. The TGF-␤ signal is initiated when the ligand binds to its own TGF-␤ type II receptor (T␤RII); thereafter, the TGF-␤ type I receptor (T␤RI or activin receptor-like kinase [ALK]...

Currently, there are no treatments for Alport syndrome, which is the second most commonly inherited kidney disease. Here we report the development of an exon-skipping therapy using an antisense-oligonucleotide (ASO) for severe male X-linked Alport syndrome (XLAS). We targeted truncating variants in exon 21 of the COL4A5 gene and conducted a type IV collagen α3/α4/α5 chain triple helix formation assay, and in vitro and in vivo treatment efficacy evaluation. We show that exon skipping enabled trimer formation, leading to remarkable clinical and pathological improvements including expression of the α5 chain on glomerular and the tubular basement membrane. In addition, the survival period was clearly prolonged in the ASO treated mice group. This data suggests that exon skipping may represent a promising therapeutic approach for treating severe male XLAS cases.

Endothelial cells are indispensable components of the vascular system, and play pivotal roles during development and in health and disease. Their properties have been studied extensively by in vivo analysis of genetically modified mice. However, further analysis of the molecular and cellular phenotypes of endothelial cells and their heterogeneity at various developmental stages, in vascular beds and in various organs has often been hampered by difficulties in culturing mouse endothelial cells. In order to overcome these difficulties, we developed a new transgenic mouse line expressing the SV40 tsA58 large T antigen (tsA58T Ag) under the control of a binary expression system based on Cre/loxP recombination. tsA58T Ag‐positive endothelial cells in primary cultures of a variety of organs proliferate continuously at 33 °C without undergoing cell senescence. The resulting cell population consists of blood vascular and lymphatic endothelial cells, which could be separated by immunosorting. Even when cultured for two months, the cells maintained endothelial cell properties, as assessed by expression of endothelium‐specific markers and intracellular signaling through the vascular endothelial growth factor receptors VEGFR–2 and VEGFR‐3, as well as their physiological characteristics. In addition, lymphatic vessel endothelial hyaluronan receptor‐1 (Lyve‐1) expression in liver sinusoidal endothelial cells in vivo was retained in vitro, suggesting that an organ‐specific endothelial characteristic was maintained. These results show that our transgenic cell culture system is useful for culturing murine endothelial cells, and will provide an accessible method and applications for studying endothelial cell biology.

Transforming growth factor (TGF)-b regulates vascular development through two type I receptors: activin receptor-like kinase (ALK) 1 and ALK5, each of which activates a different downstream Smad pathway. The endothelial cell (EC)-specific ALK1 increases EC proliferation and migration, whereas the ubiquitously expressed ALK5 inhibits both of these processes. As ALK1 requires the kinase activity of ALK5 for optimal activation, the lack of ALK5 in ECs results in defective phosphorylation of both Smad pathways on TGF-b stimulation. To understand why TGF-b signaling through ALK1 and ALK5 has opposing effects on ECs and whether this takes place in vivo, we carefully compared the phenotype of ALK5 knock-in (ALK5 KI/KI ) mice, in which the aspartic acid residue 266 in the L45 loop of ALK5 was replaced by an alanine residue, with the phenotypes of ALK5 knock-out (ALK5 À/À ) and wild-type mice. The ALK5 KI/KI mice showed angiogenic defects with embryonic lethality at E10.5-11.5. Although the phenotype of the ALK5 KI/KI mice was quite similar to that of the ALK5 À/À mice, the hierarchical structure of blood vessels formed in the ALK5 KI/KI embryos was more developed than that in the ALK5 À/À mutants. Thus, the L45 loop mutation in ALK5 partially rescued the earliest vascular defects in the ALK5 À/À embryos. This study supports our earlier observation that vascular maturation in vivo requires both TGF-b/ALK1/ BMP-Smad and TGF-b/ALK5/activin-Smad pathways for normal vascular development. Formation of mature blood vessels requires endothelial cell (EC) proliferation, the construction of a permanent basement membrane, and the recruitment of mural cells such as pericytes and vascular smooth muscle cells (VSMCs). Angiogenesis, the formation of new blood vessels, is controlled in two phases: the activation and the resolution phases. In the activation phase, vascular permeability and basement membrane degradation are increased, which allows ECs to proliferate, invade, and migrate into the extracellular space and form a new capillary lumen. In the resolution phase, ECs can no longer proliferate and migrate but are tightly bound to the extracellular matrix. Mesenchymal cells are then recruited onto the endothelial tube where they differentiate into pericytes or VSMCs surrounding the newly formed vessels. Multiple cytokines, including vascular endothelial growth factor, fibroblast growth factor, plateletderived growth factor, and transforming growth factor (TGF)-b, interplay with one another to regulate EC differentiation, vascular network formation, and the establishment and maintenance of vessel wall integrity.1,2 TGF-b is a multifunctional cytokine that regulates many cellular responses, including proliferation, differentiation, migration, and apoptosis. Aberrant TGF-b signaling is associated with tumorigenicity, cardiovascular abnormality, and anomalies in development of the gastrointestinal tract.