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The molecular signaling cascades that regulate angiogenesis and microvascular remodeling are fundamental to normal development, healthy physiology, and pathologies such as inflammation and cancer. Yet quantifying such complex, fractally branching vascular patterns remains difficult. We review application of NASA’s globally available, freely downloadable VESsel GENeration (VESGEN) Analysis software to numerous examples of 2D vascular trees, networks, and tree-network composites. Upon input of a binary vascular image, automated output includes informative vascular maps and quantification of parameters such as tortuosity, fractal dimension, vessel diameter, area, length, number, and branch point. Previous research has demonstrated that cytokines and therapeutics such as vascular endothelial growth factor, basic fibroblast growth factor (fibroblast growth factor-2), transforming growth factor-beta-1, and steroid triamcinolone acetonide specify unique “fingerprint” or “biomarker” vascular patterns that integrate dominant signaling with physiological response. In vivo experimental examples described here include vascular response to keratinocyte growth factor, a novel vessel tortuosity factor; angiogenic inhibition in humanized tumor xenografts by the anti-angiogenesis drug leronlimab; intestinal vascular inflammation with probiotic protection by Saccharomyces boulardii, and a workflow programming of vascular architecture for 3D bioprinting of regenerative tissues from 2D images. Microvascular remodeling in the human retina is described for astronaut risks in microgravity, vessel tortuosity in diabetic retinopathy, and venous occlusive disease.

The Spaceflight Associated Neuro-ocular Syndrome (SANS), associated with the headward fluid shifts incurred in microgravity during long-duration missions, remains a high-priority health and performance risk for human space exploration. To help characterize the pathophysiology of SANS, NASA’s VESsel GENeration Analysis (VESGEN) software was used to map and quantify vascular adaptations in the retina before and after 70 days of bed rest at 6-degree Head-Down Tilt (HDT), a well-studied microgravity analog. Results were compared to the retinal vascular response of astronauts following 6-month missions to the International Space Station (ISS). By mixed effects modeling, the trends of vascular response were opposite. Vascular density decreased significantly in the 16 retinas of eight astronauts and in contrast, increased slightly in the ten retinas of five subjects after HDT (although with limited significance). The one astronaut retina diagnosed with SANS displayed the greatest vascular loss. Results suggest that microgravity is a major variable in the retinal mediation of fluid shifts that is not reproduced in this HDT bed rest model.

Purpose Ocular structural and functional changes, collectively termed spaceflight-associated neuro-ocular syndrome (SANS), have been described in astronauts undergoing long-duration missions in the microgravity environment of the International Space Station. We tested the hypothesis that retinal vascular remodeling, particularly by smaller vessels, mediates the chronic headward fluid shifts associated with SANS. Methods As a retrospective study, arterial and venous patterns extracted from 30° infrared Heidelberg Spectralis retinal images of eight crew members acquired before and after six-month missions were analyzed with NASA's recently released VESsel GENeration Analysis (VESGEN) software. Output parameters included the fractal dimension and overall vessel length density that was further classified into large and small vascular branching generations. Vascular results were compared with SANS-associated clinical ocular measures. Results Significant postflight decreases in Df, Lv, and in smaller but not larger vessels were quantified in 11 of 16 retinas for arteries and veins (P value for Df, Lv, and smaller vessels in all 16 retinas were ≤0.033). The greatest vascular decreases occurred in the only retina displaying clinical evidence of SANS by choroidal folds and optic disc edema. In the remaining 15 retinas, decreases in vascular density from Df and Lv ranged from minimal to high by a custom Subclinical Vascular Pathology Index. Conclusions Together with VESGEN, the Subclinical Vascular Pathology Index may represent a new, useful SANS biomarker for advancing the understanding of SANS etiology and developing successful countermeasures for long duration space exploration in microgravity, although further research is required to better characterize retinal microvascular adaptations.

There is an enduring need for vascularization of bioprinted constructs with vascular networks optimized for distribution of nutrient-containing fluids, both for in vitro applications and in vivo implantation. However, most of the efforts in this field were directed so far towards generation of simple linear channels, often lined with endothelial cells only, and thus lacking the anatomical details of real vascular networks. To start addressing this need, here we explored the possibility of using actual vascular patterns derived from human ocular fundus for instructing the 3D printing activity. In order to assign to these patterns the organ-specific topology, and eventually vessel branch-defined cellular composition, we describe the use of the branching analysis program VESGEN 2D for planning a workflow that links the primary vascular images with their 3D printing with bioinks. To this end, we show how to process flat vascular images and, for an even more realistic representation, how to retro-engineer concave retinal patterns from flat images and to print them in a supporting hydrogel. This work opens the possibility of bioprinting more anatomically realistic vascular networks, and thus to eventually improve the vascularization of living tissue-engineered constructs.

Vascular patterning is a key, genetically responsive phylogenetic classifier of tissues in major organisms flown in space, such as the wings of Drosophila melanogaster (the fruit fly), mouse retina, and leaves of Arabidopsis thaliana. Phenotypes of increasingly abnormal ectopic wing venation in the highly stereotyped Drosophila wing generated by overexpressing the H-C2 construct of Notch antagonist Hairless (Johannes and Preiss, 2002) were mapped and quantified by NASA’s VESsel GENeration Analysis (VESGEN) software. By several confirming vascular parameters, the eight stereotyped wing veins remained quite constant in wild type compared to Class 5 H-C2, the most perturbed category of the H-C2 overexpression phenotypes. However, ectopic veins increased in number from 1 in the wild type, to 18 in Class 5 H-C2. We therefore demonstrate the feasibility of using VESGEN to quantify microscopic images of altered wing venation in Drosophila melanogaster. We further determined that several of the signal transduction pathways affecting wing vein patterning were altered by spaceflight, according to gene expression differences observed in our transcriptomic data from a previous shuttle flight experiment. Future studies will help characterize the extent to which these gene expression changes can cause even subtle developmental changes using model organisms, such as Drosophila. Therefore, we propose that the sensitive analyses provided by VESGEN software will not only serve as a useful tool to map the genetics of wing vein patterning for terrestrial applications, but also for future phenotypic studies with Drosophila for spaceflight missions.

Angiotensin-converting enzyme 2 (ACE2) is the primary enzyme of the vasoprotective axis of the renin angiotensin system (RAS). We tested the hypothesis that loss of ACE2 would exacerbate diabetic retinopathy by promoting bone marrow dysfunction. ACE2–/y were crossed with Akita mice, a model of type 1 diabetes. When comparing the bone marrow of the ACE2–/y-Akita mice to that of Akita mice, we observed a reduction of both short-term and long-term repopulating hematopoietic stem cells, a shift of hematopoiesis toward myelopoiesis, and an impairment of lineage–c-kit+ hematopoietic stem/progenitor cell (HS/PC) migration and proliferation. Migratory and proliferative dysfunction of these cells was corrected by exposure to angiotensin-1-7 (Ang-1-7), the protective peptide generated by ACE2. Over the duration of diabetes examined, ACE2 deficiency led to progressive reduction in electrical responses assessed by electroretinography and to increases in neural infarcts observed by fundus photography. Compared with Akita mice, ACE2–/y-Akita at 9-months of diabetes showed an increased number of acellular capillaries indicative of more severe diabetic retinopathy. In diabetic and control human subjects, CD34+ cells, a key bone marrow HS/PC population, were assessed for changes in mRNA levels for MAS, the receptor for Ang-1-7. Levels were highest in CD34+ cells from diabetics without retinopathy. Higher serum Ang-1-7 levels predicted protection from development of retinopathy in diabetics. Treatment with Ang-1-7 or alamandine restored the impaired migration function of CD34+ cells from subjects with retinopathy. These data support that activation of the protective RAS within HS/PCs may represents a therapeutic strategy for prevention of diabetic retinopathy.

The leaf venation of angiosperms is key to their terrestirial dominance. These higher land plants include maple, corn, and ISS model organism Arabidopsis thaliana. The venation-dependent photosynthetic capacity of angiosperm leaves is largely responsible for terrestrial production of glucose and atmospheric oxygen, and may be fundamentally important to long-term space colonization. Leaf studies in orbit, where human-tended experiments are limited, can be enhanced by quantifying complex venation patterning. VESsel GENeration Analysis (VESGEN), a beta-level NASA software that analyzes vertebrate and human vascular branching for biomedical applications, is therefore being modified to map the branching venation patterns of dicot angiosperm leaves. By physiological branching rules, VESGEN decomposes a continuously connected vascular tree into its structural (dendritic) branching and reticulate (networked) capillary components. For an arabidopsis juvenile leaf flown on NASA Space Shuttle Mission (STS)-130, the venation patterning of larger structural vessel orders 1°-2° remained relatively constant compared to normal gravity (by vessel number density Nv , 1.24E-5/micron2, and 1.29E-5/micron2, respectively). However, as a measure of increased venation maturity, Nv of smaller reticulate orders ≥ 3° increased considerably from 7.7E-6/micron2 in ground control to 1.74E-5/micron2 in the STS-130 leaf. Vascular geometric complexity is another feature of plant development that is governed in part by changes in gene expression patterns responding to environmental influence. We therefore propose that the mapping of leaf venation patterns by VESGEN can provide additional insight into plant responses to the spaceflight environment.

Here, we show that apolipoprotein A1 (apoA1), the major protein component of high density lipoprotein (HDL), through both innate and adaptive immune processes, potently suppresses tumor growth and metastasis in multiple animal tumor models, including the aggressive B16F10L murine malignant melanoma model. Mice expressing the human apoA1 transgene (A1Tg) exhibited increased infiltration of CD11b+ F4/80+ macrophages with M1, anti-tumor phenotype, reduced tumor burden and metastasis, and enhanced survival. In contrast, apoA1-deficient (A1KO) mice showed markedly heightened tumor growth and reduced survival. Injection of human apoA1 into A1KO mice inoculated with tumor cells remarkably reduced both tumor growth and metastasis, enhanced survival, and promoted regression of both tumor and metastasis burden when administered following palpable tumor formation and metastasis development. Studies with apolipoprotein A2 revealed the anti-cancer therapeutic effect was specific to apoA1. In vitro studies ruled out substantial direct suppressive effects by apoA1 or HDL on tumor cells. Animal models defective in different aspects of immunity revealed both innate and adaptive arms of immunity contribute to complete apoA1 anti-tumor activity. This study reveals a potent immunomodulatory role for apoA1 in the tumor microenvironment, altering tumor-associated macrophages from a pro-tumor M2 to an anti-tumor M1 phenotype. Use of apoA1 to redirect in vivo elicited tumor-infiltrating macrophages toward tumor rejection may hold benefit as a potential cancer therapeutic. Background: ApoA1, a component of HDL, promotes anti-inflammatory, immunomodulatory, and cardioprotective functions. Results: ApoA1 suppresses tumor growth and metastasis, primarily via modulation of innate and adaptive immune responses. Conclusion: ApoA1 impacts tumor biology at multiple levels, which appear to be linked to immunomodulatory function. Significance: ApoA1 redirects elicited immune cells toward tumor suppression and rejection and may hold benefit as a cancer therapeutic.

The adult heart has been reported to have an extensive lymphatic system, yet the development of this important system during cardiogenesis is still largely unexplored. The nuclear-localized transcription factor Prox-1 identified a sheet of Prox-1-positive cells on the developing aorta and pulmonary trunk in avian and murine embryos just prior to septation of the four heart chambers. The cells coalesced into a branching lymphatic network that spread within the epicardium to cover the heart. These vessels eventually expressed the lymphatic markers LYVE-1, VEGFR-3, and podoplanin. Before the Prox-1-positive cells were detected in the mouse epicardium, LYVE-1, a homologue of the CD44 glycoprotein, was primarily expressed in individual epicardial cells. Similar staining patterns were observed for CD44 in avian embryos. The proximity of these LYVE-1/CD44-positive mesenchymal cells to Prox-1-positive vessels suggests that they may become incorporated into the lymphatics. Unexpectedly, we detected LYVE-1/PECAM/VEGFR-3-positive vessels within the embryonic and adult myocardium which remained Prox-1/podoplanin-negative. Lymphatic markers were surprisingly found in adult rat and embryonic mouse epicardial cell lines, with Prox-1 also exhibiting nuclear-localized expression in primary cultures of embryonic avian epicardial cells. Our data identified three types of cells in the embryonic heart expressing lymphatic markers: (1) Prox-1-positive cells from an extracardiac source that migrate within the serosa of the outflow tract into the epicardium of the developing heart, (2) individual LYVE-1-positive cells in the epicardium that may be incorporated into the Prox-1-positive lymphatic vasculature, and (3) LYVE-1-positive cells/vessels in the myocardium that do not become Prox-1-positive even in the adult heart.

Integrin alpha5beta1 is among the proteins overexpressed on tumor vessels and is a potential target for diagnostics and therapeutics. Here, we mapped the distribution of alpha5beta1 integrin in three murine tumor models and identified sites of expression that are rapidly accessible to intravascular antibodies. When examined by conventional immunohistochemistry, alpha5beta1 integrin expression was strong on most blood vessels in RIP-Tag2 transgenic mouse tumors, adenomatous polyposis coli (apc) mouse adenomas, and implanted MCa-IV mammary carcinomas. Expression increased during malignant progression in RIP-Tag2 mice. However, immunoreactivity was also strong in normal pancreatic ducts, intestinal smooth muscle, and several other sites. To determine which sites of expression were rapidly accessible from the bloodstream, we intravenously injected anti-alpha5beta1 integrin antibody and 10 minutes to 24 hours later examined the amount and distribution of labeling. The injected antibody strongly labeled tumor vessels at all time points but did not label most normal blood vessels or gain access to pancreatic ducts or intestinal smooth muscle. Intense vascular labeling by anti-alpha5beta1 integrin antibody co-localized with the uniform CD31 immunoreactivity of tumor vessels and contrasted sharply with the patchy accumulation of nonspecific IgG at sites of leakage. This strategy of injecting antibodies revealed the uniform overexpression and rapid accessibility of alpha5beta1 integrin on tumor vessels and may prove useful in assessing other potential therapeutic targets in cancer.

Evaluation of normal and abnormal vascular pattern in the human retina using a novel method: quantitative region-based fractal analysis.Binary (black/white) vascular patterns of the human retina originating at the optic disc were obtained by semi-automatic computer processing of digital images from 60-degree fundus fluorescein angiography of 5 normal eyes and 5 eyes with non-proliferative diabetic retinopathy (NPDR). As determined by image resolution, vascular patterns included vessels with diametersor=50 microm and excluded small vessels and capillaries. The density of linearized (i.e., skeletonized) vascular patterns in the macular region versus paramacular region (termed "region-based" linearized vascular pattern) was quantified with the fractal dimension (D(f)) and confirmed by grid intersection (rho(v)).By region-based quantification, D(f) and rho( v) were significantly higher in the normal macular region than in the NPDR macular region (p = 0.008 and p = 0.019, respectively). However, differences in D(f) and rho(v) between the normal and NPDR paramacular regions were not strongly significant (p = 0.168 and p = 0.337, respectively).Results from the retrospective analytical study demonstrate the feasibility of using quantitative region-based fractal analysis of early-stage vascular disease in the human retina. The results are encouraging for a broader study of diverse patient populations.

Challenges to long-duration space exploration and colonization in microgravity and cosmic radiation environments by humans include poorly understood risks for gastrointestinal function and cancer. Nonetheless, constant remodeling of the intestinal microvasculature is critical for tissue viability, healthy wound healing, and successful prevention or recovery from vascular-mediated inflammatory or ischemic diseases such as cancer. Currently no automated image analysis programs provide quantitative assessments of the complex structure of the mucosal vascular system that are necessary for tracking disease development and tissue recovery. Increasing abnormalities to the microvascular network geometry were therefore mapped with VESsel GENeration Analysis (VESGEN) software from 3D tissue reconstructions of developing intestinal inflammation in a dextran sulfate sodium (DSS) mouse model. By several VESGEN parameters and a novel vascular network linking analysis, inflammation strongly disrupted the regular, lattice-like geometry that defines the normal microvascular network, correlating positively with the increased recruitment of dendritic cells during mucosal defense responses.

Quantitative analysis of vascular generational branching demonstrated that transforming growth factor-beta1 (TGF-beta1), a multifunctional cytokine and angiogenic regulator, strongly inhibited angiogenesis in the arterial tree of the developing quail chorioallantoic membrane (CAM) by inhibition of the normal increase in the number of new, small vessels. The cytokine was applied uniformly in solution at embryonic day 7 (E7) to the CAMs of quail embryos cultured in petri dishes. After 24 h the rate of arterial growth was inhibited by as much as 105% as a function of increasing TGF-beta1 concentration. Inhibition of the rate of angiogenesis in the arterial tree by TGF-beta1 relative to controls was measured in digital images by three well-correlated, computerized methods. The first computerized method, direct measurement by the computer code VESGEN of vascular morphological parameters according to branching generations G(sub 1) through G(sub greater than or equal to 5), revealed that TGF-beta1 selectively inhibited the increase in the number density of small vessels, N(sub v greater than or equal to 5), (382 plus or minus 85 per square centimeter) for specimens treated with 1 microgram TGF-beta1/CAM for 24 h, compared to 583 plus or minus 99 per square centimeter for controls), but did not significantly affect other parameters such as average vessel length or vessel diameter. The second and third methods, the fractal dimension (D(sub f)) and grid intersection (rho (sub v)), are statistical descriptors of spatial pattern and density. According to D(sub f) and rho(sub v), arterial density increased in control specimens from 1.382 plus or minus 0.007 and 662 plus or minus 52 per square centimeters at E7 (0 h) to 1.439 plus or minus 0.013 and 884 plus or minus 55 per square centimeters at E8 (24 h), compared to 1.379 plus or minus 0.039 and 650 plus or minus 111 per square centimeter for specimens treated with 1 microgram TGF-beta1/CAM for 24 h. TGF-beta1 therefore regulates vascular pattern and the rate of angiogenesis in a unique fingerprint manner, as do other major angiogenic regulators that include VEGF, FGF-2 (bFGF), and angiostatin. TGF-beta1 did not stimulate angiogenesis significantly at low cytokine concentrations, which suggests that this quail CAM model of angiogenesis is not associated with an inflammatory response.

We hypothesized that oxygen gradients and hypoxia-responsive signaling may play a role in the patterning of neural or vascular cells recruited to the developing heart. Endothelial progenitor and neural cells are recruited to and form branched structures adjacent to the relatively hypoxic outflow tract (OFT) myocardium from stages 27-32 (ED6.5-7.5) of chick development. As determined by whole mount confocal microscopy, the neural and vascular structures were not anatomically associated. Adenoviral delivery of a VEGF trap dramatically affected the remodeling of the vascular plexus into a coronary tree while neuronal branching was normal. Both neuronal and vascular branching was diminished in the hearts of embryos incubated under hyperoxic conditions. Quantitative analysis of the vascular defects using our recently developed VESGEN program demonstrated reduced small vessel branching and increased vessel diameters. We propose that vascular and neural patterning in the developing heart share dependence on tissue oxygen gradients but are not interdependent.

Diabetic retinopathy is the leading cause of visual loss among working-aged adults in the United States.1,2 The diagnosis and management of diabetic retinopathy are based on grading of features obtained from clinical examination, as was performed in the Early Treatment Diabetic Retinopathy Study (ETDRS).3–5 Although progression of diabetic retinopathy results from adverse vascular remodeling that includes vascular dropout, ischemia, and finally neovascularization, current diagnosis relies on the grading of secondary vascular effects, such as microaneurysms, leakage, and exudates. It is challenging to directly evaluate changes in retinal blood vessels because of the morphologic complexity of the overlapping, highly branching arterial and venous trees within the human retina. The goal of this study was to investigate changes in the branching patterns of the arterial and venous trees during the progression of diabetic retinopathy. To study vascular remodeling directly, we used VESsel GENeration Analysis (VESGEN) software6–11 to map and quantify arterial and venous trees extracted from clinical images obtained by fluorescein angiography (FA). The VESGEN computer program analyzes major vascular branching parameters in a binary (black/white) image of a vascular tree, vascular network, or tree-network composite. Mapping and quantification by VESGEN analysis automatically segments vessels within a tree into branching generations (G1, G2, … Gx) according to coordinate change in vessel diameter and branching. A recent review11 of VESGEN applications mapped and quantified vascular trees and networks in the human retina, transgenic mouse retina, and chorioallantoic membrane (CAM), an avian model of angiogenesis and lymphangiogenesis. Coronary vessel development was analyzed as an immature vasculogenic network, a transitional network-tree composite, and a mature tapering vascular tree. To develop VESGEN mapping capabilities, proangiogenesis and antiangiogenesis factors were first studied in the CAM. By VESGEN analysis, basic fibroblast growth factor (bFGF) stimulated specifically the robust growth of many small vessels.6 Vascular endothelial growth factor (VEGF)-A, however, had a more complex effect.9 At low concentrations, VEGF stimulated the growth of new small vessels in a manner resembling stimulation by bFGF. At high concentrations, regulation by VEGF resulted in a more pathologic regulatory phenotype, in which the diameter of larger vessels was significantly dilated (accompanied by vascular leakage). Inhibition of angiogenesis by transforming growth factor (TGF) β-1 retained a normal vascular morphology,7 whereas inhibition by angiostatin rendered the vascular tree highly abnormal and irregular.12 The steroid drug triamcinolone acetonide (TA) also inhibited the growth of small vessels and, furthermore, thinned the diameters of all vessels throughout the vascular tree except those of the smallest vessels.10 We have previously quantified, with the use of fractal analysis, vascular morphology in the retinas of patients with normal eyes and those with mild nonproliferative diabetic retinopathy (NPDR).13 That study showed that compared with the normal retina, the combined density of arteries and veins in the NPDR retina decreased in the macula but was unchanged in peripheral regions. In our present study of progressive vascular remodeling during diabetic retinopathy, VESGEN software mapped and quantified branching characteristics of separated arterial and venous trees to reveal generation-specific changes of oscillating vessel density.

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.

The effects of microvascular branching on blood flow were investigated in vivo by microscopic particle imaging velocimetry (micro-PIV). We use micro-PIV to measure blood flow by tracking red blood cells (RBC) as the moving particles. Velocity flow fields, including flow pulsatility, were analyzed for the first four branching orders of capillaries, postcapillary venules and small veins of the microvascular network within the developing avian yolksac at embryonic day 5 (E5). Increasing volumetric flowrates were obtained from parabolic laminar flow profiles as a function of increasing vessel diameter and branching order. Maximum flow velocities increased approximately twenty-fold as the function of increasing vessel diameter and branching order compared to flow velocities of 100 - 150 micron/sec in the capillaries. Results from our study will be useful for the increased understanding of blood flow within anastomotic, heterogeneous microvascular networks.

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 (αSMA) 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, αSMA (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. Published 2006 Wiley-Liss, Inc.

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 μg/CAM) and correlated positively with increased eNOS activity. Relative to control specimens, eNOS activity increased maximally to 60% following VEGF treatment at 5 μg/CAM, compared to 10% at 1.25 μg/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.

In a novel assay of angiogenesis in the quail chorioallantoic membrane (CAM), we measured vascular pattern and angiogenic rate after homogeneous exposure of the entire vascular tree to recognized modulators of vessel growth. In comparison to phosphate-buffered saline (PBS)-treated controls, the vascular stimulator, basic fibroblast growth factor (bFGF or FGF-2), increased the rate of angiogenesis by a maximum of 72%, whereas a recently discovered angiogenic inhibitor, angiostatin, decreased the rate of vascular growth by a maximum of 68%. The perturbants were applied in PBS to the CAM of 7-day-old embryos (E7) cultured in petri dishes, and the embryos were cultured further until fixation at E8 or E9. For morphometry of the quasi-two-dimensional CAM vasculature, digital images of arterial endpoints from the middle region of the CAM were acquired in grayscale at a magnification of 10×, binarized to black/white, and skeletonized. The pattern of vessel branching was assessed by measurement of the fractal dimension ( D f ), and vessel density (ρ v ), with the method of grid intersection. Correlations between these two statistical techniques were linear ( r 2 ranged from 0.967 to 0.985). For skeletonized images at E9, D f and ρ v of bFGF-treated samples were 1.55 ± 0.01 and 782 ± 26/cm 2 , respectively (relative to 1.49 ± 0.02 and 583 ± 60/cm 2 for controls), and of angiostatin-treated samples, 1.43 ± 0.02 and 424 ± 74/cm 2 (relative to 1.50 ± 0.02 and 616 ± 59/cm 2 for controls). To establish normalization values for rates of angiogenesis, we analyzed untreated CAMs of E6 to E12. From E7 to E10 in skeletonized images, D f increased linearly from 1.37 ± 0.01 to 1.54 ± 0.01 and ρ v from 311 ± 67 to 746 ± 124/cm 2 (in both cases, r 2 = 1.000). Thus, the rates of normal angiogenic growth as measured by D f and ρ v were 0.06/day and 138/cm 2 -day, respectively. From E10 to E12, D f and ρ v declined slightly. Differences between the vasculature of untreated and PBS-treated CAMs were statistically insignificant. In conclusion, vascular branching pattern and density in the quail CAM were stimulated by bFGF and inhibited by angiostatin. We quantified these changes with statistical significance by D f and ρ v , which are expressed relative to the rates of normal developmental angiogenesis measured for the two parameters in untreated quail embryos.

Background and Aims Saccharomyces boulardii (Sb) can protect against intestinal injury and tumor formation, but how this probiotic yeast controls protective mucosal host responses is unclear. Angiogenesis is an integral process of inflammatory responses in inflammatory bowel diseases (IBD) and required for mucosal remodeling during restitution. The aim of this study was to determine whether Sb alters VEGFR (vascular endothelial growth factor receptor) signaling, a central regulator of angiogenesis. Methods HUVEC were used to examine the effects of Sb on signaling and on capillary tube formation (using the ECMatrix™ system). The effects of Sb on VEGF-mediated angiogenesis were examined in vivo using an adenovirus expressing VEGF-A(164) in the ears of adult nude mice (NuNu). The effects of Sb on blood vessel volume branching and density in DSS-induced colitis was quantified using VESsel GENeration (VESGEN) software. Results 1) Sb treatment attenuated weight-loss (p

Angiogenesis requires specific molecular regulation of complex vascular branching morphology. The cleaved form of high molecular weight kininogen (HKa) has been shown to cause apoptosis of proliferating endothelial cells, and inhibit angiogenesis in vivo. However, critical morphological effects of HKa on the complex branching morphology of angiogenic vascular trees have not been defined. We used the quail chorioallantoic membrane (CAM) angiogenesis assay, in which angiogenesis agonists or antagonists cause uniform perturbation of angiogenesis throughout the entire vascular tree, to assess the effects of HKa on branching morphology. HKa (1.56 to 50 micrograms) was applied uniformly to the CAM, and its effect on angiogenesis determined after 24 hours of incubation. VESGEN vascular analysis software, currently under development at NASA Glenn Research Center, was used to quantify major vessel parameters in low-magnification images of vascular trees automatically extracted by VESGEN. Results derived from qualitative observation and initial quantification of two experiments demonstrates that HKa significantly inhibits vascular growth. Vessel length density (Lv) and branch point density (Br) in CAM specimens treated with 25 micrograms of HKa decreased by 23% and 31% respectively (24 ± 1 cm/cm2 and 441 ± 43 /cm2, compared to 31 ± 0 cm/cm2 and 677 ± 51 /cm2 in vehicle-treated controls; mean +/− S.E. for n = 3). The fractal dimension (Df) of skeletonized vascular images is a highly sensitive indicator of vessel density that ranges from 1.35 for strong angiogenesis inhibition, to 1.40 and 1.48 for controls and strong angiogenesis stimulation, respectively. Df decreased to 1.35 ± 0.01 in the HKa-treated specimens, relative to 1.39 ± 0.00 in controls. However, thickening of large vessels in response to HKa resulted in a virtually equivalent vessel area density (Av) of 0.167 ± 0.012 cm2/cm2, relative to 0.174 ± 0.015 cm2/cm2 in controls. Further studies of the site-specific effects of HKa on branching morphology within the vascular tree are in progress. These results demonstrate that the antiangiogenic effects of HKa result in a unique antiangiogenic ’fingerprint’ vascular morphology in comparison to other angiogenesis inhibitors that include transforming growth factor-β1 and angiostatin.