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During angiogenesis, growing neovessels must effectively navigate through the tissue space as they elongate and subsequently integrate into a microvascular network. While time series microscopy has provided insight into the cell activities within single growing neovessel sprouts, less is known concerning neovascular dynamics within a large angiogenic tissue bed. Here, we developed a time-lapse imaging technique that allowed visualization and quantification of sprouting neovessels as they form and grow away from adult parent microvessels in three dimensions over cubic millimeters of matrix volume during the course of up to 5 days on the microscope. Using a new image acquisition procedure and novel morphometric analysis tools, we quantified the elongation dynamics of growing neovessels and found an episodic growth pattern accompanied by fluctuations in neovessel diameter. Average elongation rate was 5 μm/h for individual vessels, but we also observed considerable dynamic variability in growth character including retraction and complete regression of entire neovessels. We observed neovessel-to-neovessel directed growth over tens to hundreds of microns preceding tip-to-tip inosculation. As we have previously described via static 3D imaging at discrete time points, we identified different collagen fibril structures associated with the growing neovessel tip and stalk, and observed the coordinated alignment of growing neovessels in a deforming matrix. Overall analysis of the entire image volumes demonstrated that although individual neovessels exhibited episodic growth and regression, there was a monotonic increase in parameters associated with the entire vascular bed such as total network length and number of branch points. This new time-lapse imaging approach corroborated morphometric changes in individual neovessels described by us and others, as well as captured dynamic neovessel behaviors unique to days-long angiogenesis within the forming neovascular network.

Unlabelled: Adipose-derived regenerative and stem cells, defined collectively as the stromal vascular fraction (SVF), support the formation of neovascular networks at the site of implantation. The effect of advancing age on SVF cell population effectiveness towards stimulated neovascularization was evaluated., Methods: SVF was enzymatically isolated from adipose of young (ySVF, 4 months) or old (oSVF, 24 months) Fisher-344 rats, combined with type I collagen and polymerized. Encapsulated SVF was implanted subcutaneously into young Rag1 mice for two or four weeks. Angiogenic function of age-dependent SVF was also extensively evaluated in vitro using standard assays., Results: In vitro studies indicated no difference in angiogenic function between ySVF and oSVF (viability, proliferation, migration, and tube-formation). At two weeks post-implantation, there was no age-related difference in percent apoptosis in explanted constructs. By four weeks post-implantation, oSVF implants displayed 36% less total vessels/mm(2), 43% less perfused vessels/mm(2), and exhibited greater percent apoptosis compared to ySVF (n ≥ 12). Blocking thrombospondin-1 (Thbs-1), a protein found to be highly expressed in oSVF but not ySVF, increased the percent of perfused vascular volume and vessel diameters in oSVF constructs after two weeks compared to oSVF implants treated with control antibody., Conclusions: Advancing donor age reduces the potential of adipose-derived SVF to derive a mature microcirculation, but does not hinder initial angiogenesis. However, modulation of Thbs-1 may improve this outcome. This data suggests that greater pruning, dysfunctional structural adaptation and/or poor maturation with initiation of blood flow may occur in oSVF., (Copyright © 2015. Published by Elsevier Inc.)

The details of the mechanical factors that modulate angiogenesis remain poorly understood. Previous in vitro studies of angiogenesis using microvessel fragments cultured within collagen constructs demonstrated that neovessel alignment can be induced via mechanical constraint of the boundaries (i.e., boundary conditions). The objective of this study was to investigate the role of mechanical boundary conditions in the regulation of angiogenic alignment and growth in an in vitro model of angiogenesis. Angiogenic microvessels within three-dimensional constructs were subjected to different boundary conditions, thus producing different stress and strain fields during growth. Neovessel outgrowth and orientation were quantified from confocal image data after 6 days. Vascularity and branching decreased as the amount of constraint imposed on the culture increased. In long-axis constrained hexahedral constructs, microvessels aligned parallel to the constrained axis. In contrast, constructs that were constrained along the short axis had random microvessel orientation. Finite element models were used to simulate the contraction of gels under the various boundary conditions and to predict the local strain field experienced by microvessels. Results from the experiments and simulations demonstrated that microvessels aligned perpendicular to directions of compressive strain. Alignment was due to anisotropic deformation of the matrix from cell-generated traction forces interacting with the mechanical boundary conditions. These findings demonstrate that boundary conditions and thus the effective stiffness of the matrix regulate angiogenesis. This study offers a potential explanation for the oriented vascular beds that occur in native tissues and provides the basis for improved control of tissue vascularization in both native tissues and tissue-engineered constructs., (Copyright © 2014 the American Physiological Society.)

Purpose: It is thought that local ischemia and oxygen radicals are responsible for fibroblast-to-myofibroblast cell transformation and proliferation. We hypothesized that hypoxia could differentially activate the contractility of fibroblasts from normal human palmar fascia and from fibroblasts-myofibroblasts of Dupuytren cords., Methods: Normal palmar fascia from 5 patients with carpal tunnel syndrome and Dupuytren cords from 5 patients were harvested. Cells were cultured from all tissue samples, and collagen lattices were prepared containing these cells. Oxygen treatment subgroups were created and incubated under hypoxic (1% O(2), 5% CO(2), and 94% N(2)), normoxic (21% O(2), 5% CO(2), and 74% N(2)), and hyperoxic (100% oxygen using 2.4 atm pressure twice a day for 7 d) conditions. After 7 days, each subgroup was photographed, and lattices were released from dishes. Postrelease photographs were taken immediately, 5 minutes after release, and after 1 hour. Areas of the lattices at each time point were calculated using MetaMorph software. Actin staining and live/dead cell analysis was performed. Linear repeated measures analysis of variance was used for data analysis given that contraction levels were measured over 3 distinct time points., Results: We found a statistically significant difference between normal samples and Dupuytren samples in mean contraction levels over time. There was no statistically significant difference between tissue groups over the 3 time periods based on the oxygen treatment received., Conclusions: Our results showed a greater degree of contractility in Dupuytren disease cells than normal fibroblasts. However, the contraction in either group was not affected by oxygen level. Future in vivo research is needed to better understand the nature of pathophysiology of Dupuytren disease., (Copyright © 2013 American Society for Surgery of the Hand. Published by Elsevier Inc. All rights reserved.)

Calcium signaling in the diverse vascular structures is regulated by a wide range of mechanical and biochemical factors to maintain essential physiological functions of the vasculature. To properly transmit information, the intercellular calcium communication mechanism must be robust against various conditions in the cellular microenvironment. Using plasma lithography geometric confinement, we investigate mechanically induced calcium wave propagation in networks of human umbilical vein endothelial cells organized. Endothelial cell networks with confined architectures were stimulated at the single cell level, including using capacitive force probes. Calcium wave propagation in the network was observed using fluorescence calcium imaging. We show that mechanically induced calcium signaling in the endothelial networks is dynamically regulated against a wide range of probing forces and repeated stimulations. The calcium wave is able to propagate consistently in various dimensions from monolayers to individual cell chains, and in different topologies from linear patterns to cell junctions. Our results reveal that calcium signaling provides a robust mechanism for cell-cell communication in networks of endothelial cells despite the diversity of the microenvironmental inputs and complexity of vascular structures., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A three-dimensional tissue construct was created using adipose-derived stromal vascular fraction (SVF) cells and evaluated as a microvascular protection treatment in a myocardial infarction (MI) model. This study evaluated coronary blood flow (BF) and global left ventricular function after MI with and without the SVF construct. Fischer-344 rats were separated into four groups: sham operation (sham), MI, MI Vicryl patch (no cells), and MI SVF construct (MI SVF). SVF cells were labeled with green fluorescent protein (GFP). Immediately postinfarct, constructs were implanted onto the epicardium at the site of ischemia. Four weeks postsurgery, the coronary BF reserve was significantly decreased by 67% in the MI group and 75% in the MI Vicryl group compared with the sham group. The coronary BF reserve of the sham and MI SVF groups in the area at risk was not significantly different (sham group: 83 ± 22% and MI SVF group: 57 ± 22%). Griffonia simplicifolia I and GFP-positive SVF immunostaining revealed engrafted SVF cells around microvessels in the infarct region 4 wk postimplant. Overall heart function, specifically ejection fraction, was significantly greater in MI SVF hearts compared with MI and MI Vicryl hearts (MI SVF: 66 ± 4%, MI: 37 ± 8%, and MI Vicryl: 29 ± 6%). In conclusion, adipose-derived SVF cells can be used to construct a novel therapeutic modality for treating microvascular instability and ischemia through implantation on the epicardial surface of the heart. The SVF construct implanted immediately after MI not only maintains heart function but also sustains microvascular perfusion and function in the infarct area by sustaining the coronary BF reserve.

Fibrin is an attractive material for regenerative medicine applications. It not only forms a polymer but also contains cryptic matrikines that are released upon its activation/degradation and enhance the regenerative process. Despite this advantageous biology associated with fibrin, commercially available systems (e.g. TISSEEL) display limited regenerative capacity. This limitation is in part due to formulations that are optimized for tissue sealant applications and result in dense fibrous networks that limit cell infiltration. Recent evidence suggests that polymerization knob 'B' engagement of polymerization hole 'b' activates an alternative polymerization mechanism in fibrin, which may result in altered single fiber mechanical properties. We hypothesized that augmenting fibrin polymerization through the addition of PEGylated knob peptides with specificity to hole 'b' (AHRPYAAC-PEG) would result in distinct fibrin polymer architectures with grossly different physical properties. Polymerization dynamics, polymer architecture, diffusivity, viscoelasticity, and degradation dynamics were analyzed. Results indicate that specific engagement of hole 'b' with PEGylated knob 'B' conjugates during polymerization significantly enhances the porosity of and subsequent diffusivity through fibrin polymers. Paradoxically, these polymers also display increased viscoelastic properties and decreased susceptibility to degradation. As a result, fibrin polymer strength was significantly augmented without any adverse effects on angiogenesis within the modified polymers., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Aim: Mechanical forces are important regulators of cell and tissue phenotype. We hypothesized that mechanical loading and boundary conditions would influence neovessel activity during angiogenesis., Methods and Results: Using an in vitro model of angiogenesis sprouting and a mechanical loading system, we evaluated the effects of boundary conditions and applied loading. The model consisted of rat microvessel fragments cultured in a 3D collagen gel, previously shown to recapitulate angiogenic sprouting observed in vivo. We examined changes in neovascular growth in response to four different mechanical conditions. Neovessel density, diameter, length and orientation were measured from volumetric confocal images of cultures exposed to no external load (free-floating shape control), intrinsic loads (fixed ends, no stretch), static external load (static stretch), or cyclic external load (cyclic stretch). Neovessels sprouted and grew by the third day of culture and continued to do so during the next 3 days of loading. The numbers of neovessels and branch points were significantly increased in the static stretch group when compared with the free-floating shape control group. In all mechanically loaded cultures, neovessel diameter and length distributions were heterogeneous, whereas they were homogeneous in shape control cultures. Neovessels were significantly more oriented along the direction of mechanical loading than those in the shape controls. Interestingly, collagen fibrils were organized parallel and adjacent to growing neovessels., Conclusion: Externally applied boundary conditions regulate neovessel sprouting and elongation during angiogenesis, affecting both neovessel growth characteristics and network morphometry. Furthermore, neovessels align parallel to the direction of stress/strain or internally generated traction, and this may be because of collagen fibril alignment induced by the growing neovessels themselves.

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.

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(2) receptor (V(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 AQP1 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(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(2)R activation, via dDAVP, increased AQP2 and AQP3 protein abundance in the cortical collecting ducts of AQP1 null mice. However, V(2)R activation did not increase AQP2 protein abundance in the IMCD of AQP1 null mice.

Epidemiological studies link arsenic exposure to increased risks of cancers of the skin, kidney, lung, bladder and liver. Additionally, a variety of non-cancerous conditions such as diabetes mellitus, hypertension, and cardiovascular disease have been associated with chronic ingestion of low levels of arsenic. However, the biological and molecular mechanisms by which arsenic exerts its effects remain elusive. Here we report increased renal hexokinase II (HKII) expression in response to arsenic exposure both in vivo and in vitro. In our model, HKII was up-regulated in the renal glomeruli of mice exposed to low levels of arsenic (10 ppb or 50 ppb) via their drinking water for up to 21 days. Additionally, a similar effect was observed in cultured renal mesangial cells exposed to arsenic. This correlation between our in vivo and in vitro data provides further evidence for a direct link between altered renal HKII expression and arsenic exposure. Thus, our data suggest that alterations in renal HKII expression may be involved in arsenic-induced pathological conditions involving the kidney. More importantly, these results were obtained using environmentally relevant arsenic concentrations.

The goal of this study was to develop a modified fluorescent microsphere-based approach for measuring resting and hyperemic blood flows in individual mouse skeletal muscles. Absolute resting blood flow in the left gracilis posterior was 1.04+/-0.12 ml x min(-1).g(-1), while functional hyperemia following muscle activity was 5.94+/-1.33 ml x min(-1) x g(-1). Measuring absolute blood flow requires sampling arterial blood that serves as a flow-rate and concentration reference to the fluorescent microsphere (FMS) content in the tissue-of-interest for calculating the flow value. Because sampling arterial blood can impair cardiovascular function in the mouse, we also modified our FMS approach to determine relative blood flows in the left gracilis posterior by using the contralateral muscle as our reference in blood flow calculations. Absolute and relative hyperemia measurements detect similar increases in blood flow - 521.93+/-216.76% and 555.24+/-213.82%, respectively. However, sampling arterial blood during absolute blood flow measurements significantly decreased mean arterial pressure from the beginning to the end of our experiments, from 102.7+/-2.18 to 75.5+/-9.71 mm Hg. This decrease was not seen when measuring relative blood flows. This approach provides critical advantages over contemporary blood flow measurement approaches by allowing blood flow measurements in small and non-superficial tissues.

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.

We have combined confocal microscopy, image processing, and optimization techniques to obtain automated, accurate volumetric measurements of microvasculature. Initially, we made tissue phantoms containing 15-microm FocalCheck microspheres suspended in type I collagen. Using these phantoms we obtained a stack of confocal images and examined the accuracy of various thresholding schemes. Thresholding algorithms from the literature that utilize a unimodal histogram, a bimodal histogram, or an intensity and edge-based algorithm all significantly overestimated the volume of foreground structures in the image stack. Instead, we developed a heuristic technique to automatically determine good-quality threshold values based on the depth, intensity, and (optionally) gradient of each voxel. This method analyzed intensity and gradient threshold methods for each individual image stack, taking into account the intensity attenuation that is seen in deeper images of the stack. Finally, we generated a microvascular construct comprised of rat fat microvessel fragments embedded in collagen I gels and obtained stacks of confocal images. Using our new thresholding scheme we were able to obtain automatic volume measurements of growing microvessel fragments.

In prostate cancer progression, the basal lamina switches from predominantly laminin-5 to laminin-10. DU-145 prostate cancer cells were treated with either soluble laminin-5 (20 ng/ml) or laminin-10 (1 microg/ml) for 6, 24, and 48 hr. Total RNA was harvested for a 7,500 human cDNA microarray. Hybridizations were carried out in accordance with a 10 sample analysis of variance (ANOVA) statistical model. One thousand one hundred sixteen genes had measurable expression 2 standard deviations above background and 50% of spots for any given sample for all hybridizations were positive. Expression values of significantly varying genes were clustered and a list of 408 genes (P < 0.05) with a 1.5 or greater fold change in at least one time point were chosen for further analysis. Seventy eight changed in a time-dependent manner with laminin-10 treatment, 85 changed with laminin-5, and 13 showed changes with both treatments. The 408 genes that passed a paired t-test in at least one time-dependent category were further analyzed using Pathway Miner. One of the largest gene association networks involved signal transduction in the growth factor-MAP kinase pathways. EGFR was validated by real-time PCR and laminin-10 mediated cell adhesion activated EGFR in DU-145 cells. Both laminins appear to be important signal transducers in prostate cancer.

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 approximately 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 3beta-hydroxysteroid dehydrogenase 4 (3betaHSD4) 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.

Computational modeling of the mechanics of cells and multicellular constructs with standard numerical discretization techniques such as the finite element (FE) method is complicated by the complex geometry, material properties and boundary conditions that are associated with such systems. The objectives of this research were to apply the material point method (MPM), a meshless method, to the modeling of vascularized constructs by adapting the algorithm to accurately handle quasi-static, large deformation mechanics, and to apply the modified MPM algorithm to large-scale simulations using a discretization that was obtained directly from volumetric confocal image data. The standard implicit time integration algorithm for MPM was modified to allow the background computational grid to remain fixed with respect to the spatial distribution of material points during the analysis. This algorithm was used to simulate the 3D mechanics of a vascularized scaffold under tension, consisting of growing microvascular fragments embedded in a collagen gel, by discretizing the construct with over 13.6 million material points. Baseline 3D simulations demonstrated that the modified MPM algorithm was both more accurate and more robust than the standard MPM algorithm. Scaling studies demonstrated the ability of the parallel code to scale to 200 processors. Optimal discretization was established for the simulations of the mechanics of vascularized scaffolds by examining stress distributions and reaction forces. Sensitivity studies demonstrated that the reaction force during simulated extension was highly sensitive to the modulus of the microvessels, despite the fact that they comprised only 10.4% of the volume of the total sample. In contrast, the reaction force was relatively insensitive to the effective Poisson's ratio of the entire sample. These results suggest that the MPM simulations could form the basis for estimating the modulus of the embedded microvessels through a parameter estimation scheme. Because of the generality and robustness of the modified MPM algorithm, the relative ease of generating spatial discretizations from volumetric image data, and the ability of the parallel computational implementation to scale to large processor counts, it is anticipated that this modeling approach may be extended to many other applications, including the analysis of other multicellular constructs and investigations of cell mechanics.

Microtubules are protein filaments that are emerging as potential building blocks in manufacturing nanoscale structures and systems such as interconnecting nanowires. Future development in using microtubules necessitates a control of their nucleation and growth. We report the controlled nucleation and growth of microtubules from functionalized gold on a hydrophilic oxidized silicon wafer. The gold substrate is functionalized with gamma-tubulin, a natural nucleating agent for microtubule growth. We show that the attached gamma-tubulin retains its biological functionality and leads to nucleation and assembly of microtubules from the functionalized gold surface. We also analyze the interplay between the geometry of the nucleating substrates and the morphology of microtubules arrays and networks grown from them. We consider two geometrical arrangements of the substrates: (a) a square lattice of small gold pads on a hydrophilic oxidized silicon wafer and (b) a large flat surface. Fluorescence microscopy and scanning electron microscopy are employed to provide a detailed characterization of the length and morphology of the nucleated and grown microtubules. The observed microtubule morphologies are modeled, analyzed and discussed within the context of reaction-diffusion and nucleation controlled processes.

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(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(TA) was on average 18% higher in the arcade network than in a corresponding two-tree network without arcades. Structural adaptation increased Q(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.

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 approximately 7 days was included. The results suggest that delayed stimuli significantly influence the dynamic characteristics of vascular remodeling resulting from reduced blood supply.

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 approximately 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.

Angiogenesis is the formation of new capillary blood vessels from preexisting vessels. It is involved in many normal and diseased conditions, as well as in the application of tissue-engineered products. There has been extensive effort made to develop strategies for controlling pathological angiogenesis and for promoting vascularization in biomedical engineering applications. Central to advancing these strategies is a mechanistic understanding of the angiogenic process. Angiogenesis is tightly regulated by local tissue environmental factors, including soluble molecules, extracellular matrices, cell-cell interactions, and diverse mechanical forces. Great advances have been made in identifying the biochemical factors and intracellular signaling pathways that mediate the control of angiogenesis. This review focuses on work that explores the biophysical aspect of angiogenesis regulation. Specifically, we discuss the role of cell-generated forces, counterforces from the extracellular matrix, and mechanical forces associated with blood flow and extravascular tissue activity in the regulation of angiogenesis. Because angiogenesis occurs in a mechanically dynamic environment, future investigations should aim at understanding how cells integrate chemical and mechanical signals so that a rational approach to controlling angiogenesis will become possible. In this regard, computational models that incorporate multiple epigenetic factors to predict capillary patterning will be useful.