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Aquaporin (AQP) 1 null mice have a defect in the renal concentrating gradient because of their inability to generate a hyperosmotic medullary interstitium. To determine the effect of vasopressin on renal medullary gene expression, in the absence of high local osmolarity, we infused 1-deamino-8-D-arginine vasopressin (dDAVP), a [V.sub.2] receptor ([V.sub.2]R)-specific agonist, in AQP1 null mice for 7 days. cDNA microarray analysis was performed on the renal medullary tissue, and 5,140 genes of the possible 12,000 genes on the array were included in the analysis. In the renal medulla of AQPI null mice, 245 transcripts were identified as increased by dDAVP infusion and 200 transcripts as decreased (1.5-fold or more). Quantitative real-time PCR measurements confirmed the increases seen for cyclin D1, early growth response gene 1, and activating transcription factor 3, genes associated with changes in cell cycle/growth. Changes in mRNA expression were correlated with changes in protein expression by semiquantitative immunoblotting; cyclin D1 and ATF3 were increased significantly in abundance following dDAVP infusion in the renal medulla of AQP1 null mice (161 and 461%, respectively). A significant increase in proliferation of medullary collecting ducts cells, following [V.sub.2]R activation, was identified by proliferating cell nuclear antigen immunohistochemistry; colocalization studies with AQP2 indicated that the increase in proliferation was primarily observed in principal cells of the inner medullary collecting duct (IMCD). [V.sub.2]R activation, via dDAVP, increased AQP2 and AQP3 protein abundance in the cortical collecting ducts of AQP1 null mice. However, [V.sub.2]R activation did not increase AQP2 protein abundance in the IMCD of AQP1 null mice. cyclin D1; microarray; activating transcription factor 3

The extracellular matrix (ECM) plays a critical role in angiogenesis by providing biochemical and positional cues, as well as by mechanically influencing microvessel cell behavior. Considerable information is known concerning the biochemical cues relevant to angiogenesis, but less is known about the mechanical dynamics during active angiogenesis. The objective of this study was to characterize changes in the material properties of a simple angiogenic tissue before and during angiogenesis, During sprouting, there was an overall decrease in tissue stiffness followed by an increase during neovessel elongation. The fall in matrix stiffness coincided with peak matrix metalloproteinase mRNA expression and elevated proteolytic activity. An elevated expression of genes for ECM components and cell-ECM interaction molecules and a subsequent drop in proteolytic activity (although enzyme levels remained elevated) coincided with the subsequent stiffening. The results of this study show that the mechanical properties of a scaffold tissue may be actively modified during angiogenesis by the growing microvasculature, biomechanics; matrix metalloprotease

To better understand interstitial matrix remodeling during angiogenesis, we probed endogenous optical signatures of collagen fibrils and cells with multiphoton microscopy to noninvasively visualize, in real-time, changes to fibril organization around angiogenic sprouts and growing neovessels. From analyses of the second-harmonic generation signal from fibrillar collagen and two-photon excited fluorescence, as well as coherent transmitted light from vascular cells, we found that microvessel fragments interacting with the collagen matrix exhibited two key features: a strong association of fibrillar collagen around the parent vessel fragment during vessel construct reconstitution and a substantial collagen fibril reorganization by sprout and neovessel tips. Results indicate that angiogenic sprouts and growing neovessels actively and differentially remodel existing collagen fibrils. This imaging approach to assess local changes in matrix organization may have a broader impact on tissue biology and mechanics during angiogenesis and allow for new insights in cardiovascular, diabetes, and cancer research. extracellular matrix; multiphoton microscopy; second harmonic generation doi:10.1152/ajpheart.01234.2006

To identify novel gene targets of vasopressin regulation in the renal medulla, we performed a cDNA microarray study on the inner medullary tissue of mice following a 48-h water restriction protocol. In this study, 4,625 genes of the possible ~12,000 genes on the array were included in the analysis, and of these 157 transcripts were increased and 63 transcripts were decreased by 1.5-fold or more. Quantitative, real-time PCR measurements confirmed the increases seen for 12 selected transcripts, and the decreases were confirmed for 7 transcripts. In addition, we measured transcript abundance for many renal collecting duct proteins that were not represented on the array; aquaporin-2 (AQP2), AQP3, Pax-8, and [alpha]- and [beta]-Na-K-ATPase subunits were all significantly increased in abundance; the [beta]- and [gamma]-subunits of ENaC and the vasopressin type 1A receptor were significantly decreased. To correlate changes in mRNA expression with changes in protein expression, we carried out quantitative immunoblotting. For most of the genes examined, changes in mRNA abundances were not associated with concomitant protein abundance changes; however, AQP2 transcript abundance and protein abundance did correlate. Surprisingly, aldolase B transcript abundance was increased but protein abundance was decreased following 48 h of water restriction. Several transcripts identified by microarray were novel with respect to their expression in mouse renal medullary tissues. The steroid hormone enzyme 3[beta]-hydroxysteroid dehydrogenase 4 (3[beta]HSD4) was identified as a novel target of vasopressin regulation, and via dual labeling immunofluorescence we colocalized the expression of this protein to AQP2-expressing collecting ducts of the kidney. These studies have identified several transcripts whose abundances are regulated in mouse inner medulla in response to an increase in endogenous vasopressin levels and could play roles in the regulation of salt and water excretion. vasopressin; microarray

Arteriolar arcades provide alternate pathways for blood flow after obstruction of arteries or arterioles such as occurs in stroke and coronary and peripheral vascular disease. When obstruction is prolonged, remaining vessels adjust their diameters chronically in response to altered hemodynamic and metabolic conditions. Here, the effectiveness of arcades in maintaining perfusion both immediately following obstruction and after structural adaptation was examined. Morphometric data from a vascular casting of the pig triceps brachii muscle and published data were used to develop a computational model for the hemodynamics and structural adaptation of the arcade network between two feed artery branches, FA1 and FA2. The predicted total flow to capillaries ([Q.sub.TA]) in the region initially supplied by FA2 decreased to 26% of the normal value immediately after FA2 obstruction but was restored to 78% of the normal value after adaptation. After obstruction of 1-10 randomly selected arcade segments, [Q.sub.TA] was on average 18% higher in the arcade network than in a corresponding two-tree network without arcades. Structural adaptation increased [Q.sub.TA] by an additional 16% in the arcade network but had almost no effect in the two-tree network. These results indicate that arcades can partially maintain blood flow after vascular blockage and that this effect is substantially enhanced by structural adaptation. collateralization; computational model; blood flow; shear stress

The goals of this study were to determine the time course and spatial dependence of structural diameter changes in the mouse gracilis artery after a redistribution of blood flow and to compare the observations with predictions of computational models for structural adaptation. Diameters were measured 1,2, 7, 14, 21, 28, and 56 days after resection of one of the two blood supplies to the artery. Overall average diameter, normalized with respect to diameters in untreated vessels, increased slightly during the first 7 days, then increased more rapidly, reaching a peak around day 21, and then decreased. This transient increase in diameter was spatially nonuniform, being largest toward the point of resection. A previously developed theoretical model, in which diameter varies in response to stimuli derived from local metabolic and hemodynamic conditions, was extended to include effects of time-delayed remodeling stimuli in regions of reduced perfusion. Predictions of this model were consistent with observed diameter changes, including the transient increase in diameters near the point of resection, when a remodeling stimulus with a time delay of ~7 days was included. The results suggest that delayed stimuli significantly influence the dynamic characteristics of vascular remodeling resulting from reduced blood supply. metabolic response; shear stress; vessel wall conduction; adaptation; computational model

Mice that lack the aquaporin-1 gene (AQP1) lack a functional countercurrent multiplier mechanism, fail to concentrate the inner medullary (IM) interstitium, and present with a urinary concentrating defect. In this study, we use DNA microarrays to identify the gene expression profile of the IM of AQP1 null mice and corresponding changes in gene expression resulting from a loss of a hypertonic medullary interstitium. An ANOVA analysis model, CARMA, was used to isolate the knockout effect while taking into account experimental variability associated with microarray studies. In this study 5,701 genes of the possible ~12,000 genes on the array were included in the ANOVA; 531 genes were identified as demonstrating a > 1.5-fold up- or downregulation between the wild-type and knockout groups. We randomly selected 35 genes for confirmation by real-time PCR, and 29 of the 35 genes were confirmed using this method. The overall pattern of gene expression in the AQP1 null mice was one of downregulation compared with gene expression in the renal medullas of the wild-type mice. Heat shock proteins 105 and 94, aldose reductase, adenylate kinase 2, aldolase B, aldehyde reductase 6, and p8 were decreased in the AQP1 null mice. Carboxylesterase 3, matrilin 2, lipocalin 2, and transforming growth factor-[alpha] were increased in IM of AQP1 null mice. In addition, we observed a loss of vasopressin type 2 receptor mRNA expression in renal medullas of the AQP1 null mice. Thus the loss of the hyperosmotic renal interstitium, due to a loss of the concentrating mechanism, drastically altered not only the phenotype of these animals but also their renal medullary gene expression profile. osmolarity; aquaporin; microarray; real-time PCR; vasopressin