Patricia Parsons-Wingerter

The Cardioprotective Protein Apolipoprotein A1 Promotes Potent Anti-tumorigenic Effects

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. 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.

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 VEGF165, basic fibroblast growth factor, transforming growth factor β‐1, angiostatin and the clinical steroid triamcinolone acetonide induce ‘fingerprint’ or ‘signature’ changes in vascular patterning that provide unique readouts of dominant molecular signaling. Anat Rec, 292:320–332, 2009.

Selective Inhibition of Angiogenesis in Small Blood Vessels and Decrease in Vessel Diameter throughout the Vascular Tree by Triamcinolone Acetonide

PURPOSEnTo 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.nnnMETHODSnIncreasing 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.nnnRESULTSnVessel 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)).nnnCONCLUSIONSnBy VESGEN analysis, TA selectively inhibited the angiogenesis of smaller blood vessels, but decreased the vessel diameter of all vessels within the vascular tree.

Expression of Lymphatic Markers During Avian and Mouse Cardiogenesis

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 before 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. Anat Rec, 2010.

Probiotic yeast inhibits VEGFR signaling and angiogenesis in intestinal inflammation.

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<0.01) and histological damage (p<0.01) in DSS colitis. VESGEN analysis of angiogenesis showed significantly increased blood vessel density and volume in DSS-treated mice compared to control. Sb treatment significantly reduced the neo-vascularization associated with acute DSS colitis and accelerated mucosal recovery restoration of the lamina propria capillary network to a normal morphology. 2) Sb inhibited VEGF-induced angiogenesis in vivo in the mouse ear model. 3) Sb also significantly inhibited angiogenesis in vitro in the capillary tube assay in a dose-dependent manner (p<0.01). 4) In HUVEC, Sb reduced basal VEGFR-2 phosphorylation, VEGFR-2 phosphorylation in response to VEGF as well as activation of the downstream kinases PLCγ and Erk1/2. Conclusions Our findings indicate that the probiotic yeast S boulardii can modulate angiogenesis to limit intestinal inflammation and promote mucosal tissue repair by regulating VEGFR signaling.

Oscillation of Angiogenesis with Vascular Dropout in Diabetic Retinopathy by VESsel GENeration Analysis (VESGEN)

PURPOSEnVascular dropout and angiogenesis are hallmarks of the progression of diabetic retinopathy (DR). However, current evaluation of DR relies on grading of secondary vascular effects, such as microaneurysms and hemorrhages, by clinical examination instead of by evaluation of actual vascular changes. The purpose of this study was to map and quantify vascular changes during progression of DR by VESsel GENeration Analysis (VESGEN).nnnMETHODSnIn this prospective cross-sectional study, 15 eyes with DR were evaluated with fluorescein angiography (FA) and color fundus photography, and were graded using modified Early Treatment Diabetic Retinopathy Study criteria. FA images were separated by semiautomatic image processing into arterial and venous trees. Vessel length density (L(v)), number density (N(v)), and diameter (D(v)) were analyzed in a masked fashion with VESGEN software. Each vascular tree was automatically segmented into branching generations (G(1)...G(8) or G(9)) by vessel diameter and branching. Vascular remodeling status (VRS) for N(v) and L(v) was graded 1 to 4 for increasing severity of vascular change.nnnRESULTSnBy N(v) and L(v), VRS correlated significantly with the independent clinical diagnosis of mild to proliferative DR (13/15 eyes). N(v) and L(v) of smaller vessels (G(> or =6)) increased from VRS1 to VRS2 by 2.4 x and 1.6 x, decreased from VRS2 to VRS3 by 0.4 x and 0.6 x, and increased from VRS3 to VRS4 by 1.7 x and 1.5 x (P < 0.01). Throughout DR progression, the density of larger vessels (G(1-5)) remained essentially unchanged, and D(v1-5) increased slightly.nnnCONCLUSIONSnVessel density oscillated with the progression of DR. Alternating phases of angiogenesis/neovascularization and vascular dropout were dominated first by remodeling of arteries and subsequently by veins.