Reduced-gravity environment hardware demonstrations of a prototype miniaturized flow cytometer and companion microfluidic mixing technology.

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If humans are to travel beyond low Earth orbit, a transition towards space-ready, point-of-care (POC) testing is required. Such testing needs to be comprehensive, easy to perform in a reduced-gravity environment, and unaffected by the stresses of launch and spaceflight. Countless POC devices have been developed to mimic laboratory scale counterparts, but most have narrow applications and few have demonstrable use in an in-flight, reduced-gravity environment. In fact, demonstrations of biomedical diagnostics in reduced gravity are limited altogether, making component choice and certain logistical challenges difficult to approach when seeking to test new technology. To help fill the void, we are presenting a modular method for the construction and operation of a prototype blood diagnostic device and its associated parabolic flight test rig that meet the standards for flight-testing onboard a parabolic flight, reduced-gravity aircraft. The method first focuses on rig assembly for in-flight, reduced-gravity testing of a flow cytometer and a companion microfluidic mixing chip. Components are adaptable to other designs and some custom components, such as a microvolume sample loader and the micromixer may be of particular interest. The method then shifts focus to flight preparation, by offering guidelines and suggestions to prepare for a successful flight test with regard to user training, development of a standard operating procedure (SOP), and other issues. Finally, in-flight experimental procedures specific to our demonstrations are described.

VESGEN 2D: automated, user-interactive software for quantification and mapping of angiogenic and lymphangiogenic trees and networks.

Quantification of microvascular remodeling as a meaningful discovery tool requires mapping and measurement of site-specific changes within vascular trees and networks. Vessel density and other critical vascular parameters are often modulated by molecular regulators as determined by local vascular architecture. For example, enlargement of vessel diameter by vascular endothelial growth factor (VEGF) is restricted to specific generations of vessel branching (Parsons-Wingerter et al., Microvascular Research72: 91, 2006). The averaging of vessel diameter over many successively smaller generations is therefore not particularly useful. The newly automated, user-interactive software VESsel GENeration Analysis (VESGEN) quantifies major vessel parameters within two-dimensional (2D) vascular trees, networks, and tree-network composites. This report reviews application of VESGEN 2D to angiogenic and lymphangiogenic tissues that includes the human and murine retina, embryonic coronary vessels, and avian chorioallantoic membrane. Software output includes colorized image maps with quantification of local vessel diameter, fractal dimension, tortuosity, and avascular spacing. The density of parameters such as vessel area, length, number, and branch point are quantified according to site-specific generational branching within vascular trees. The sole user input requirement is a binary (black/white) vascular image. Future applications of VESGEN will include analysis of 3D vascular architecture and bioinformatic dimensions such as blood flow and receptor localization. Branching analysis by VESGEN has demonstrated that numerous regulators including VEGF(165), basic fibroblast growth factor, transforming growth factor beta-1, angiostatin and the clinical steroid triamcinolone acetonide induce 'fingerprint' or 'signature' changes in vascular patterning that provide unique readouts of dominant molecular signaling.

Selective inhibition of angiogenesis in small blood vessels and decrease in vessel diameter throughout the vascular tree by triamcinolone acetonide.

PURPOSE: To quantify the effects of the steroid triamcinolone acetonide (TA) on branching morphology within the angiogenic microvascular tree of the chorioallantoic membrane (CAM) of quail embryos. METHODS: Increasing concentrations of TA (0-16 ng/mL) were applied topically on embryonic day (E) 7 to the chorioallantoic membrane (CAM) of quail embryos cultured in petri dishes and incubated for an additional 24 or 48 hours until fixation. Binary (black/white) microscopic images of arterial end points were quantified by generational analysis of vessel branching (VESGEN) software to obtain major vascular parameters that include vessel diameter (D(v)), fractal dimension (D(f)), tortuosity (T(v)), and densities of vessel area, length, number, and branch point (A(v), L(v), N(v), and Br(v)). For assessment of specific changes in vascular morphology induced by TA, the VESGEN software automatically segmented the vascular tree into branching generations (G(1)... G(10)) according to changes in vessel diameter and branching. RESULTS: Vessel density decreased significantly up to 34% as the function of increasing concentration of TA according to A(v), L(v), Br(v), N(v), and D(f). TA selectively inhibited the growth of new, small vessels because L(v) decreased from 13.14 +/- 0.61 cm/cm(2) for controls to 8.012 +/- 0.82 cm/cm(2) at 16 ng TA/mL in smaller branching generations (G(7)-G(10)) and for N(v) from 473.83 +/- 29.85 cm(-2) to 302.32 +/- 33.09 cm(-2). In contrast, vessel diameter (D(v)) decreased throughout the vascular tree (G(1)-G(10)). CONCLUSIONS: By VESGEN analysis, TA selectively inhibited the angiogenesis of smaller blood vessels, but decreased the vessel diameter of all vessels within the vascular tree.

A VEGF165-induced phenotypic switch from increased vessel density to increased vessel diameter and increased endothelial NOS activity.

Although vascular endothelial growth factor-165 (VEGF(165)) regulates numerous angiogenic cellular activities, its complex effects on vascular morphology are not highly quantified. By fractal-based, multiparametric branching analysis of 2D vascular pattern in the quail chorioallantoic membrane (CAM), we report that vessel density increased maximally at lower VEGF concentrations, but that vessel diameter and activity of endothelial nitric oxide synthase (eNOS) increased maximally at higher VEGF concentrations. Following exogenous application of human VEGF(165) to the CAM at embryonic day 7, vessel density and diameter were measured after 24 h at arterial end points by the fractal dimension (D(f)) and generational branching parameters for vessel area density (A(v)), vessel length density (L(v)) and vessel diameter (D(v)) using the computer code VESGEN. The VEGF-dependent phenotypic switch from normal vessels displaying increased vessel density to abnormal, dilated vessels typical of tumor vasculature and other pathologies resulted from an approximate threefold increase in VEGF concentration (1.25 to 5 microg/CAM) and correlated positively with increased eNOS activity. Relative to control specimens, eNOS activity increased maximally to 60% following VEGF treatment at 5 microg/CAM, compared to 10% at 1.25 microg/CAM, and was accompanied by no significant change in activity of inducible NOS. In summary, VEGF(165) induced a phenotypic switch from increased vessel density associated with low VEGF concentration, to increased vessel diameter and increased eNOS activity at high VEGF concentration.

Lymphangiogenesis by blind-ended vessel sprouting is concurrent with hemangiogenesis by vascular splitting.

Development of effective vascular therapies requires the understanding of all modes of vessel formation involved in angiogenesis (here termed "hemangiogenesis") and lymphangiogenesis. Two major modes of vessel morphogenesis include sprouting of a new vessel from a preexisting vessel and splitting of a preexisting parent vessel into two offspring vessels. In the quail chorioallantoic membrane (CAM) during mid-development (embryonic days E6-E9), lymphangiogenesis progressed primarily via blind-ended vessel sprouting. Isolated lymphatic endothelial progenitor cells were recruited to the tips of growing vessels. During concurrent hemangiogenesis, parent blood vessels expanded from the capillary network and split into offspring vessels, accompanied by transient capillary expression of alpha smooth muscle actin (alphaSMA) and recruitment of polarized mural progenitor cells. Lymphatics and blood vessels were identified by confocal/fluorescence microscopy of vascular endothelial growth factor (VEGF) receptor VEGFR-2, alphaSMA (specific to CAM blood vessels), homeobox transcription factor Prox1 (specific to lymphatics), and the quail hematopoetic marker, QH-1. VEGFR-2 was expressed intensely in isolated cells and lymphatics, and moderately in blood vessels. Prox1 was absent from isolated progenitor cells prior to lymphatic recruitment. Exogenous vascular endothelial growth factor-165 (VEGF165) increased blood vessel density and anastomotic frequency without changing endogenous modes of vascular/lymphatic vessel formation or marker expression. Although VEGF165 is a key cellular regulator of hemangiogenesis and vasculogenesis, the role of VEGF165 in lymphangiogenesis is less clear. Interestingly, VEGF165 increased lymphatic vessel diameter and density as measured by novel Euclidean distance mapping, and the antimaturational dissociation of lymphatics from blood vessels, accompanied by lymphatic reassociation into homogeneous networks.