A VEGF165-induced phenotypic switch from increased vessel density to increased vessel diameter and increased endothelial NOS activity
Introduction
Vascular endothelial growth factor-165 (VEGF165), generally the most angiogenic isoform of VEGF-A, is a regulator of key endothelial activities that include cell proliferation, migration, maturation and permeability (for reviews, see Neufeld et al., 1999, Ferrara et al., 2003, Nagy et al., 2003). Effects of the five VEGF family members and their associated isoforms on microvascular morphology are not highly quantified, because of the complex, three-dimensional (3D) spatial patterns of most branching vascular trees. Although increased expression of the VEGFs during angiogenesis and lymphangiogenesis is well established, their pleiomorphic activities in vivo are complex and sometimes contradictory (see reviews cited above). Nitric oxide, produced by the nitric oxide synthases and localized to endothelial caveolae with VEGF receptor-2 (VEGFR-2), is a powerful vasodilator and permeability agent that is upregulated in response to VEGF during angiogenesis (Ziche et al., 1997, Spyridopoulos et al., 2002, Sonveaux et al., 2004). Nitric oxide, however, is reported to be both a downstream effector and an upstream signal of VEGF (Brennan et al., 2002, Xu et al., 2002). VEGF and nitric oxide synthase (NOS) activities are coordinated during the normal angiogenesis that occurs in embryonic development (Nurkiewicz and Boegehold, 2004), pregnancy (Vonnahme et al., 2005), muscular adaptation (Milkiewicz et al., 2005) and wound-healing of ischemic injury (Wang et al., 2005, Yu et al., 2005) and burns (Altavilla et al., 2005), and during pathological angiogenesis critical to disease progression in heart failure (Haider et al., 2005), cancer (Karihtala et al., 2004, Brouet et al., 2005, Shang and Li, 2005), and diabetes (Musicki et al., 2005). However, the dose-dependent relationships between VEGF and NOS activities during angiogenesis have not yet been well characterized and quantified.
We previously described a model of angiogenesis in vivo in which the 2D blood vessels of the transparent, rapidly developing quail chorioallantoic membrane (CAM) are easily and uniformly exposed to angiogenic cytokines and regulators in solution (Parsons-Wingerter et al., 1998, Parsons-Wingerter et al., 2000a, Parsons-Wingerter et al., 2000b). Cytokine regulators of angiogenesis tested previously in this CAM model, including the stimulator basic fibroblast growth factor (bFGF or FGF-2) and inhibitor transforming growth factor-β1 (TGF-β1), elicited simple, robust, unimodal vascular patterns that were characterized by strong statistical confidence (i.e., by low sample variation). Both bFGF and TGF-β1 regulated angiogenesis by selectively altering the number density of new, small vessels on the vascular tree. Complex spatial patterns of the branching vascular tree and associated capillary network are readily visualized by light and fluorescence microscopy and thereafter quantified by fractal-based generational branching analysis. By fractal-based analysis of skeletonized (linearized) vessels in both the chicken and quail CAM (Kirchner et al., 1996, Parsons-Wingerter et al., 1998), angiogenesis during CAM development is highly linear, supporting the straightforward quantification of CAM angiogenesis as a linear rate process (Parsons-Wingerter et al., 1998).
Fractal analysis, a new, non-Euclidean mathematical tool (Mandelbrot, 1983), is useful for the quantification of complex spatial patterns such as branching vascular trees. Fractal geometry is common in nature and includes objects such as vascular and botanical trees, coastlines, snowflakes and even complex spatiotemporal phenomena of vascular-based physiological scaling (West et al., 1997, Bassingthwaighte et al., 1994). A fractal dimension (Df) is a non-Euclidean dimension (i.e., a fractional, nonintegral number), that increases directly according to the increasing space-filling density of an object. Generally a fractal object achieves most of its space-filling capacity by utilizing the geometric property of self-similarity, which is the repetition of a spatial pattern, such as vascular bifurcational branching, at increasingly smaller length scales. In 2D binary (black/white) images, Df is limited by the Euclidean dimensions of 1 and 2, and is sensitive to small, early-stage changes in the vascular tree (Parsons-Wingerter et al., 1998, Parsons-Wingerter et al., 2000a, Parsons-Wingerter et al., 2000b, Avakian et al., 2002).
We now report that vascular response to VEGF165 in the quail CAM was complex and multimodal. Increased vessel density and increased vessel diameter reached maximal frequencies at lower and higher VEGF concentrations, respectively. By fractal and generational branching analysis with the computer code VESGEN, increased regulation of angiogenesis by VEGF, as for bFGF and TGF-β1, also resulted from selective targeting of the growth of new, small vessels within the vascular tree. By assay of NOS activities, endothelial NOS (eNOS) activity increased maximally in response to high concentrations of VEGF and hence was associated with increased vessel diameter, but response of inducible NOS (iNOS) activity to VEGF application was insignificant. The switch from a VEGF-induced phenotype of increased vessel density displaying normal vascular morphology, to a phenotype of increased vessel diameter displaying abnormal vascular morphology that correlated positively with increased eNOS activity, was controlled by increasing concentration of VEGF165. Regulation by VEGF165 therefore resulted in complex, multimodal vascular change, compared to strong stimulation or inhibition of vascular density as a sole, unimodal response of CAM vessels to bFGF or TGF-β1, respectively (Parsons-Wingerter et al., 2000a, Parsons-Wingerter et al., 2000b).
Section snippets
Materials and methods
Embryonic culture, assay, mounting, imaging, and fractal/VESGEN branching analysis have been described previously (Parsons-Wingerter et al., 1998, Parsons-Wingerter et al., 2000a, Parsons-Wingerter et al., 2000b).
Vascular response to VEGF by Fractal/VESGEN analysis
Qualitative observations of experimental images revealed that VEGF165 strongly stimulated complex, multimodal change during ongoing angiogenesis in the CAM when applied at E7 for 24 h, as illustrated by binary (black/white) and skeletonized patterns of arterial end points in the vascular tree (Fig. 1). All results reported here are for E8 specimens treated at E7 for 24 h with VEGF or with control vehicle. Increased vessel density and vessel diameter were measured by several confirming
Discussion
By fractal-based complexity analysis of generational vascular branching pattern, we report that increasing concentration of VEGF165 triggered a phenotypic switch from predominant stimulation of increased vessel density at lower VEGF concentration, to predominant stimulation of increased vessel diameter at higher concentration. In general, we report that VEGF165 stimulated complex, multimodal change in vascular morphology during the middle stage of embryonic development (E7–E8) in the quail CAM,
Acknowledgments
The authors gratefully acknowledge undergraduate student Van Le Thuy for excellent image processing and data analysis. We thank Dr. Frank Peale, Genentech, for kind support in obtaining the VEGF reagent; the reviewers for helpful suggestions, and Glenn L. Williams, NASA Glenn Research Center; Steven Zyzanski, Case Western Reserve University, Elaine Raines, Department of Pathology and Biostatistical Consultation, Department of Biostatistics, University of Washington, and Alanna Ruddell, Fred
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Present Address: Department of Internal Medicine, Health Sciences Center, School of Medicine, University of New Mexico, Albuquerque, NM 87131, USA.