CARMA: A platform for analyzing microarray datasets that incorporate replicate measures

BACKGROUND: The incorporation of statistical models that account for experimental variability provides a necessary framework for the interpretation of microarray data. A robust experimental design coupled with an analysis of variance (ANOVA) incorporating a model that accounts for known sources of experimental variability can significantly improve the determination of differences in gene expression and estimations of their significance. RESULTS: To realize the full benefits of performing analysis of variance on microarray data we have developed CARMA, a microarray analysis platform that reads data files generated by most microarray image processing software packages, performs ANOVA using a user-defined linear model, and produces easily interpretable graphical and numeric results. No pre-processing of the data is required and user-specified parameters control most aspects of the analysis including statistical significance criterion. The software also performs location and intensity dependent lowess normalization, automatic outlier detection and removal, and accommodates missing data. CONCLUSION: CARMA provides a clear quantitative and statistical characterization of each measured gene that can be used to assess marginally acceptable measures and improve confidence in the interpretation of microarray results. Overall, applying CARMA to microarray datasets incorporating repeated measures effectively reduces the number of gene incorrectly identified as differentially expressed and results in a more robust and reliable analysis

Vessel Arterial-Venous Plasticity in Adult Neovascularization

OBJECTIVE: Proper arterial and venous specification is a hallmark of functional vascular networks. While arterial-venous identity is genetically pre-determined during embryo development, it is unknown whether an analogous pre-specification occurs in adult neovascularization. Our goal is to determine whether vessel arterial-venous specification in adult neovascularization is pre-determined by the identity of the originating vessels. METHODS AND RESULTS: We assessed identity specification during neovascularization by implanting isolated microvessels of arterial identity from both mice and rats and assessing the identity outcomes of the resulting, newly formed vasculature. These microvessels of arterial identity spontaneously formed a stereotypical, perfused microcirculation comprised of the full complement of microvessel types intrinsic to a mature microvasculature. Changes in microvessel identity occurred during sprouting angiogenesis, with neovessels displaying an ambiguous arterial-venous phenotype associated with reduced EphrinB2 phosphorylation. CONCLUSIONS: Our findings indicate that microvessel arterial-venous identity in adult neovascularization is not necessarily pre-determined and that adult microvessels display a considerable level of phenotypic plasticity during neovascularization. In addition, we show that vessels of arterial identity also hold the potential to undergo sprouting angiogenesis

Chronic hindlimb ischemia impairs functional vasodilation and vascular reactivity in mouse feed arteries

Vasodilation of lower leg arterioles is impaired in animal models of chronic peripheral ischemia. In addition to arterioles, feed arteries are a critical component of the vascular resistance network, accounting for as much as 50% of the pressure drop across the arterial circulation. Despite the critical importance of feed arteries in blood flow control, the impact of ischemia on feed artery vascular reactivity is unknown. At 14 days following unilateral resection of the femoral–saphenous artery–vein pair, functional vasodilation of the profunda femoris artery was severely impaired, 11 ± 9 versus 152 ± 22%. Although endothelial and smooth muscle-dependent vasodilation were both impaired in ischemic arteries compared to control arteries (Ach: 40 ± 14 versus 81 ± 11%, SNP: 43 ± 12 versus and 85 ± 11%), the responses to acetylcholine and sodium nitroprusside were similar, implicating impaired smooth muscle-dependent vasodilation. Conversely, vasoconstriction responses to norepinephrine were not different between ischemic and control arteries, −68 ± 3 versus −66 ± 3%, indicating that smooth muscle cells were functional following the ischemic insult. Finally, maximal dilation responses to acetylcholine, ex vivo, were significantly impaired in the ischemic artery compared to control, 71 ± 9 versus 97 ± 2%, despite a similar generation of myogenic tone to the same intravascular pressure (80 mmHg). These data indicate that ischemia impairs feed artery vasodilation by impairing the responsiveness of the vascular wall to vasodilating stimuli. Future studies to examine the mechanistic basis for the impact of ischemia on vascular reactivity or treatment strategies to improve vascular reactivity following ischemia could provide the foundation for an alternative therapeutic paradigm for peripheral arterial occlusive disease

A Mechanostimulation System for Revealing Intercellular Calcium Communication in HUVEC Networks

Abstract -This paper reports a mechanostimulation system for studying mechanically induced intercellular calcium signaling in networks of human umbilical vein endothelial cells (HUVECs). By incorporating a capacitive (comb drive) force probe and plasma lithography cell patterning, the roles of biophysical factors, including force, duration, and network architecture, in calcium intercellular communication can be investigated systematically. Particularly, we observed cancellation of calcium waves in linear networks and bi-directional splitting in cross junctions. The effects of key biophysical factors on intercellular calcium wave propagation were studied. These results demonstrate the applicability of the mechanostimulation system in studying intercellular calcium signaling and reveal the robustness of calcium signaling in HUVEC networks, which mimics the vasculature

Inaugural Charles River World Congress on Animal Models in Drug Discovery and Development

https://deepblue.lib.umich.edu/bitstream/2027.42/138112/1/12967_2017_Article_1274.pd

Cell-matrix interactions of microvessel endothelial cells in response to basic fibroblast growth factor.

Vertebrate tissues consist of parenchyma and vascular elements all of which are necessary for the specific form and function of these tissues. In a unique process termed angiogenesis, vessels invade forming tissues to provide for proper tissue perfusion. Much is known about the molecular and cellular elements of angiogenesis, however, it is not clear how these elements are coordinated to produce specific microvascular beds. In an effort to answer this question, the effects of basic fibroblast growth factor (bFGF) on human microvessel endothelial cell (HMVEC) interactions with collagen I were examined. HMVEC migration on collagen I was chosen as the model angiogenic response. Utilizing two distinct migration assays, bFGF either induced migration or had no effect. Examination of HMVEC adhesion with two separate assays revealed that HMVEC adhesion to collagen I was altered by bFGF treatment and depended on the density of HMVEC at the time of treatment. Adhesion of HMVEC with or without bFGF treatment was mediated entirely by β1 integrins as demonstrated with a blocking antibody studies. Experiments were performed to determine the mechanism by which bFGF can alter HMVEC adhesion and focused on low density HMVEC. The reduction in adhesion of low density HMVEC following bFGF treatment correlated with no change in β1 integrin surface expression, delayed cell spreading, altered organization of β1 integrin into substrate contacts, and serine/threonine phosphorylation of the β1 subunit. To evaluate the coordinated effects of bFGF on angiogenesis, an in vitro model simulating a microvascular environment was developed utilizing isolated microvessel fragments from rat adipose tissue cultured in three dimensional collagen I gels. The addition of crude basic fibroblast growth factor to the cultures resulted in the growth of significantly longer microvessels and the expression of an endothelial cell protein, von Willebrand factor. Based on this work, it is apparent that cellular responses to physiological signals during angiogenesis are multifactorial and are sensitive to many coincidental environmental factors such as cell density. The influence of these environmental factors is such as to substantially alter the effects of a signalling factor acting alone

Vascularized Tissue Organoids

Tissue organoids hold enormous potential as tools for a variety of applications, including disease modeling and drug screening. To effectively mimic the native tissue environment, it is critical to integrate a microvasculature with the parenchyma and stroma. In addition to providing a means to physiologically perfuse the organoids, the microvasculature also contributes to the cellular dynamics of the tissue model via the cells of the perivascular niche, thereby further modulating tissue function. In this review, we discuss current and developing strategies for vascularizing organoids, consider tissue-specific vascularization approaches, discuss the importance of perfusion, and provide perspectives on the state of the field