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

Microvessel segments of arterial identity form a competent, hierarchical microcirculation when implanted.

<p>A–D, Confocal microscopy images (Z-projections) of microvascular networks in MVCs derived from total isolates (tMVC) or arterial-enriched isolates (aMVC) and implanted for 6 weeks. In all cases, microvessel segments were isolated from brain tissue of GFP-expressing transgenic rats. The scale bar in (A) = 100 mm and applies to all panels. (A, B). Host mice were injected intravenously with dextran-TRITC (red) to identify implant microvessels (green) that were perfusion-competent or (C, D) MVC implants were immunostained for alpha smooth muscle actin (red) to identify perivascular cell morphology. Arrows indicate arterioles, arrowheads indicate venules and capillaries. (E) Analysis of perfusion-competence of microvessels in total- (tMVC) and arterial-derived (aMVC) implanted MVCs 6 weeks post-implantation. Vessel perfusion was measured as the % fraction of green vessels exhibiting red dye in their lumens. (F) The distribution of vessel diameters, as an index of network architecture, in 4 week total (tMVC) and arterial (aMVC) MVC implants plotted as the frequency of diameters placed into bins of 5 mm increments. Vessel diameter distribution from tMVC is Reprinted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027332#pone.0027332-Nunes1" target="_blank">[16]</a> with permission from Elsevier.</p

Isolation of arteriole segments with magnetic beads yields a functional, highly enriched arterial fraction.

<p>A and B, Isolated brain microvessel segments preloaded with magnetic beads prior to (A) and following (B) extraction with a magnet. Scale bar = 200 mm. C, Repeated use of a magnet to pull out bead-loaded microvessels highly enriches the isolate for segments expressing the Efnb2-GFP reporter. (n = 3), *p<0.05 by Student's t-test. Phase (D) and fluorescence (E) images of cultured, bead-loaded microvessel segments with sprouting neovessels (arrows). Scale bar = 100 µm.</p

Angiogenic neovessels express arterial and venous genetic markers.

<p>A–C, Confocal images (Z-projections) of angiogenic neovessels in tMVC implants during the sprouting angiogenic phase (week 1 post-implantation) obtained from (A) <i>Efnb2</i>- or (B) <i>Ephb4</i>-reporter mice. Microvessels were identified by labeling endothelial cells with TRITC-conjugated GS-I lectin (red). <i>Ephb4</i>-driven LacZ expression shown in panel B was detected with an anti-β-galactosidase-FITC antibody. C, Determination of the % fraction of angiogenic neovessels positive for either the <i>Efnb2</i>- or <i>Ephb4</i>-reporter in tMVCs prepared as in A and B. D, Co-expression of the <i>Efnb2</i>-reporter gene and EphB4 in individual angiogenic neovessels in 1 week tMVC implants prepared from <i>Efnb2</i>-reporter mice (green) and immunostained with an anti-EphB4 antibody (red). Scale bar in all panels = 100 mm. E, Real-time PCR measurement of mRNA expression of EphrinB2, EphB4, COUP-TFII, Delta-like4 (Dll4), Jag1 and Notch4 genes in freshly isolated, specified microvessels (mature) and angiogenic neovessels in tMVCs (angiogenic). * p<0.05, n = 3 by Student's t-test.</p

Primer sequences for real-time PCR.

<p>Primer sequences for real-time PCR.</p

Angiogenic neovessels exhibit reduced arterial and venous marker protein expression.

<p>A, Protein expression of EphrinB2 and COUP-TFII in mature microvessels and angiogenic neovessels in tMVCs as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027332#pone-0027332-g003" target="_blank">Fig. 3E</a>. B, EphrinB2 phosphorylation from similar preparations as in A. * p<0.05, n = 3 by Student's t-test.</p

Mature microvessels derived from arterial segments express arterial and venous markers.

<p>A–C, Representative confocal images (Z-projections) of vessel networks in MVCs derived from arterial-isolates obtained from <i>Efnb2</i>-reporter mice implanted for 6 weeks expressing the arterial reporter (<i>Efnb2</i> in green) and filled with a blood tracer (perfusion in red). Scale bar = 100 mm. D, The % of perfused microvessels (red) positive for the Efnb2-reporter (green). * p<0.05 by Student's t-test. E and F, Histology sections of 6 week-aMVC implants derived from an <i>Ephb4</i>-reporter transgenic mouse. Explants were sectioned and incubated with the β-galactosidase substrate X-gal to produce a blue precipitate as a marker of <i>Ephb4</i> expression (arrows in E, scale bar = 50 mm.) or no precipitate indicating an <i>Ephb4</i>-reporter negative vessel (arrows in F, scale bar = 25 mm).</p