Background-Inflammatory responses contribute to vascular remodeling during tissue repair or ischemia. We hypothesized that inflammatory cell recruitment and endothelial cell activation during vasculogenesis and ischemia-mediated arteriogenesis could be temporally assessed by noninvasive molecular imaging. Methods and Results-Contrast ultrasound perfusion imaging and molecular imaging with microbubbles targeted to activated neutrophils, ␣ 5 -integrins, or vascular cell adhesion molecule (VCAM-1) were performed in murine models of vasculogenesis (subcutaneous matrigel) or hind-limb ischemia produced by arterial occlusion in wild-type or monocyte chemotactic protein-1-deficient mice. In subcutaneous matrigel plugs, perfusion advanced centripetally between days 3 and 10. On targeted imaging, signal enhancement from ␣ 5 -integrins and VCAM-1 coincided with the earliest appearance of regional blood flow. Targeted imaging correlated temporally with histological evidence of channel formation by ␣ 5 -integrinpositive monocytes, followed by the appearance of spindle-shaped cells lining the channels that expressed VCAM-1. In ischemic hind-limb tissue, skeletal muscle blood flow and arteriolar density increased progressively between days 2 and 21 after arterial ligation. Targeted imaging demonstrated early signal enhancement for neutrophils, monocyte ␣ 5 -integrin, and VCAM-1 at day 2 when blood flow was very low (Ͻ20% control). The neutrophil signal declined precipitously between days 2 and 4, whereas VCAM-1 and monocyte signal persisted to day 7. In mice deficient for monocyte chemotactic protein-1, monocyte-targeted signal was severely reduced compared with wild-type mice (

Clerk LH, Vincent MA, Barrett EJ, Lankford MF, Lindner JR. Skeletal muscle capillary responses to insulin are abnormal in latestage diabetes and are restored by angiogensin-converting enzyme inhibition. Am J Physiol Endocrinol Metab 293: E1804-E1809, 2007. First published October 2, 2007; doi:10.1152/ajpendo.00498.2007.-Acute physiological hyperinsulinemia increases skeletal muscle capillary blood volume (CBV), presumably to augment glucose and insulin delivery. We hypothesized that insulin-mediated changes in CBV are impaired in type 2 diabetes mellitus (DM) and are improved by angiotensin-converting enzyme inhibition (ACE-I). Zucker obese diabetic rats (ZDF, n ϭ 18) and control rats (n ϭ 9) were studied at 20 wk of age. One-half of the ZDF rats were treated with quinapril (ZDF-Q) for 15 wk prior to study. CBV and capillary flow in hindlimb skeletal muscle were measured by contrast-enhanced ultrasound (CEU) at baseline and at 30 and 120 min after initiation of a euglycemic hyperinsulinemic clamp (3 mU ⅐ min Ϫ1 ⅐ kg Ϫ1 ). At baseline, ZDF and ZDF-Q rats were hyperglycemic and hyperinsulinemic vs. controls. Glucose utilization in ZDF rats was 60 -70% lower (P Ͻ 0.05) than in controls after 30 and 120 min of hyperinsulinemia. In ZDF-Q rats, glucose utilization was impaired at 30 min but similar to controls at 120 min. Basal CBV was lower in ZDF and ZDF-Q rats compared with controls (13 Ϯ 4, 7 Ϯ 3, and 9 Ϯ 2 U, respectively). With hyperinsulinemia, CBV increased by about twofold in control animals at 30 and 120 min, did not change in ZDF animals, and increased in ZDF-Q animals only at 120 min to a level similar to controls. Anatomic capillary density on immunohistology was not different between groups. We conclude that insulin-mediated capillary recruitment in skeletal muscle, which participates in glucose utilization, is impaired in animals with DM and can be partially reversed by chronic ACE-I therapy. contrast ultrasound; microbubbles; blood flow; blood volume THERE IS INCREASING EVIDENCE that abnormal vascular responses to insulin play a role in the pathogenesis of type 2 diabetes mellitus (DM). In normal subjects, insulin increases skeletal muscle blood flow in a dose-dependent fashion (1,3,8,26,29). In the distal microcirculation, insulin increases skeletal muscle blood flow in part by increasing the number of capillaries actively perfused (8,10,26). Capillary recruitment occurs relatively rapidly after the development of physiological hyperinsulinemia or after a carbohydrate-rich meal and is thought to increase surface area for glucose and insulin entry into muscle tissue (26,32,34). Impairment of these capillary responses may contribute to the development of insulin resistance, hyperglycemia, and diabetic complications. Therefore, improvement in microvascular responses may be a novel therapeutic target for patients with insulin resistance, although routine methods for evaluating capillary reactivity are lacking.We (8, 10, 32-34) have recently used skeletal muscle contrast-enhanced ultrasound (CEU) to charac...

Wallis MG, Lankford MF, Keller SR. Vasopressin is a physiological substrate for the insulin-regulated aminopeptidase IRAP. Am J Physiol Endocrinol Metab 293: E1092-E1102, 2007. First published August 7, 2007; doi:10.1152/ajpendo.00440.2007.-Insulin-regulated aminopeptidase (IRAP) is a membrane aminopeptidase and is homologous to the placental leucine aminopeptidase, P-LAP. IRAP has a wide distribution but has been best characterized in adipocytes and myocytes. In these cells, IRAP colocalizes with the glucose transporter GLUT4 to intracellular vesicles and, like GLUT4, translocates from these vesicles to the cell surface in response to insulin. Earlier studies demonstrated that purified IRAP cleaves several peptide hormones and that, concomitant with the appearance of IRAP at the surface of insulin-stimulated adipocytes, aminopeptidase activity toward extracellular substrates increases. In the present study, to identify in vivo substrates for IRAP, we tested potential substrates for cleavage by IRAP-deficient (IRAP Ϫ/Ϫ ) and control mice. We found that vasopressin and oxytocin were not processed from the NH2 terminus by isolated IRAP Ϫ/Ϫ adipocytes and skeletal muscles. Vasopressin was not cleaved from the NH2 terminus after injection into IRAP Ϫ/Ϫ mice and exhibited a threefold increased half-life in the circulation of IRAP Ϫ/Ϫ mice. Consistent with this finding, endogenous plasma vasopressin levels were elevated twofold in IRAP Ϫ/Ϫ mice, and vasopressin levels in IRAP Ϫ/Ϫ brains, where plasma vasopressin originates, showed a compensatory decrease. We further established that insulin increased the clearance of vasopressin from control but not from IRAP Ϫ/Ϫ mice. In conclusion, we have identified vasopressin as the first physiological substrate for IRAP. Changes in plasma and brain vasopressin levels in IRAP Ϫ/Ϫ mice suggest a significant role for IRAP in regulating vasopressin. We have also uncovered a novel IRAP-dependent insulin effect: to acutely modify vasopressin.peptide hormone cleavage; insulin-regulated aminopeptidase-deficient mice; insulin action THE INSULIN-REGULATED AMINOPEPTIDASE (IRAP) is a member of the family of zinc-dependent membrane aminopeptidases and is the homologue of the human placental leucine aminopeptidase (P-LAP) (16). IRAP has a wide tissue distribution (18, 34) but in each of the tissues is expressed only in specific cell types (28). In all cells so far examined, IRAP is found in an intracellular location and is recruited to the cell surface in response to different stimuli (reviewed in Ref. 16). The subcellular localization of IRAP and regulation thereof have been best characterized in adipocytes and muscle cells (reviewed in Ref. 16). In these cells, under basal conditions, IRAP is sequestered within intracellular vesicles that also harbor the insulin-responsive glucose transporter GLUT4. In response to insulin, IRAP, like GLUT4, translocates from the intracellular vesicles to the cell surface. In adipocytes, this results in a 6-to 8-and 10-to 20-fold increase of IRAP and GLUT4, ...

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