EphrinB2/EphB4 signaling regulates non‐sprouting angiogenesis by VEGF

Article11 April 2018Open Access Transparent process EphrinB2/EphB4 signaling regulates non-sprouting angiogenesis by VEGF Elena Groppa Elena Groppa Department of Biomedicine, University Hospital, University of Basel, Basel, Switzerland Department of Surgery, University Hospital, Basel, Switzerland Search for more papers by this author Sime Brkic Sime Brkic Department of Biomedicine, University Hospital, University of Basel, Basel, Switzerland Department of Surgery, University Hospital, Basel, Switzerland Search for more papers by this author Andrea Uccelli Andrea Uccelli Department of Biomedicine, University Hospital, University of Basel, Basel, Switzerland Department of Surgery, University Hospital, Basel, Switzerland Search for more papers by this author Galina Wirth Galina Wirth A. I. Virtanen Institute, University of Eastern Finland, Kuopio, Finland Search for more papers by this author Petra Korpisalo-Pirinen Petra Korpisalo-Pirinen A. I. Virtanen Institute, University of Eastern Finland, Kuopio, Finland Search for more papers by this author Maria Filippova Maria Filippova Department of Biomedicine, University Hospital, University of Basel, Basel, Switzerland Department of Surgery, University Hospital, Basel, Switzerland Search for more papers by this author Boris Dasen Boris Dasen Department of Biomedicine, University Hospital, University of Basel, Basel, Switzerland Department of Surgery, University Hospital, Basel, Switzerland Search for more papers by this author Veronica Sacchi Veronica Sacchi Department of Biomedicine, University Hospital, University of Basel, Basel, Switzerland Department of Surgery, University Hospital, Basel, Switzerland Search for more papers by this author Manuele Giuseppe Muraro Manuele Giuseppe Muraro Department of Biomedicine, University Hospital, University of Basel, Basel, Switzerland Department of Surgery, University Hospital, Basel, Switzerland Search for more papers by this author Marianna Trani Marianna Trani Department of Biomedicine, University Hospital, University of Basel, Basel, Switzerland Department of Surgery, University Hospital, Basel, Switzerland Search for more papers by this author Silvia Reginato Silvia Reginato Department of Biomedicine, University Hospital, University of Basel, Basel, Switzerland Department of Surgery, University Hospital, Basel, Switzerland Search for more papers by this author Roberto Gianni-Barrera Roberto Gianni-Barrera Department of Biomedicine, University Hospital, University of Basel, Basel, Switzerland Department of Surgery, University Hospital, Basel, Switzerland Search for more papers by this author Seppo Ylä-Herttuala Seppo Ylä-Herttuala A. I. Virtanen Institute, University of Eastern Finland, Kuopio, Finland Heart Center, Kuopio University Hospital, Kuopio, Finland Search for more papers by this author Andrea Banfi Corresponding Author Andrea Banfi [email protected] orcid.org/0000-0001-5737-8811 Department of Biomedicine, University Hospital, University of Basel, Basel, Switzerland Department of Surgery, University Hospital, Basel, Switzerland Search for more papers by this author Elena Groppa Elena Groppa Department of Biomedicine, University Hospital, University of Basel, Basel, Switzerland Department of Surgery, University Hospital, Basel, Switzerland Search for more papers by this author Sime Brkic Sime Brkic Department of Biomedicine, University Hospital, University of Basel, Basel, Switzerland Department of Surgery, University Hospital, Basel, Switzerland Search for more papers by this author Andrea Uccelli Andrea Uccelli Department of Biomedicine, University Hospital, University of Basel, Basel, Switzerland Department of Surgery, University Hospital, Basel, Switzerland Search for more papers by this author Galina Wirth Galina Wirth A. I. Virtanen Institute, University of Eastern Finland, Kuopio, Finland Search for more papers by this author Petra Korpisalo-Pirinen Petra Korpisalo-Pirinen A. I. Virtanen Institute, University of Eastern Finland, Kuopio, Finland Search for more papers by this author Maria Filippova Maria Filippova Department of Biomedicine, University Hospital, University of Basel, Basel, Switzerland Department of Surgery, University Hospital, Basel, Switzerland Search for more papers by this author Boris Dasen Boris Dasen Department of Biomedicine, University Hospital, University of Basel, Basel, Switzerland Department of Surgery, University Hospital, Basel, Switzerland Search for more papers by this author Veronica Sacchi Veronica Sacchi Department of Biomedicine, University Hospital, University of Basel, Basel, Switzerland Department of Surgery, University Hospital, Basel, Switzerland Search for more papers by this author Manuele Giuseppe Muraro Manuele Giuseppe Muraro Department of Biomedicine, University Hospital, University of Basel, Basel, Switzerland Department of Surgery, University Hospital, Basel, Switzerland Search for more papers by this author Marianna Trani Marianna Trani Department of Biomedicine, University Hospital, University of Basel, Basel, Switzerland Department of Surgery, University Hospital, Basel, Switzerland Search for more papers by this author Silvia Reginato Silvia Reginato Department of Biomedicine, University Hospital, University of Basel, Basel, Switzerland Department of Surgery, University Hospital, Basel, Switzerland Search for more papers by this author Roberto Gianni-Barrera Roberto Gianni-Barrera Department of Biomedicine, University Hospital, University of Basel, Basel, Switzerland Department of Surgery, University Hospital, Basel, Switzerland Search for more papers by this author Seppo Ylä-Herttuala Seppo Ylä-Herttuala A. I. Virtanen Institute, University of Eastern Finland, Kuopio, Finland Heart Center, Kuopio University Hospital, Kuopio, Finland Search for more papers by this author Andrea Banfi Corresponding Author Andrea Banfi [email protected] orcid.org/0000-0001-5737-8811 Department of Biomedicine, University Hospital, University of Basel, Basel, Switzerland Department of Surgery, University Hospital, Basel, Switzerland Search for more papers by this author Author Information Elena Groppa1,2,5,‡, Sime Brkic1,2,‡, Andrea Uccelli1,2, Galina Wirth3, Petra Korpisalo-Pirinen3, Maria Filippova1,2, Boris Dasen1,2, Veronica Sacchi1,2,6, Manuele Giuseppe Muraro1,2, Marianna Trani1,2, Silvia Reginato1,2, Roberto Gianni-Barrera1,2, Seppo Ylä-Herttuala3,4 and Andrea Banfi *,1,2 1Department of Biomedicine, University Hospital, University of Basel, Basel, Switzerland 2Department of Surgery, University Hospital, Basel, Switzerland 3A. I. Virtanen Institute, University of Eastern Finland, Kuopio, Finland 4Heart Center, Kuopio University Hospital, Kuopio, Finland 5Present address: The Biomedical Research Centre, The University of British Columbia, Vancouver, BC, Canada 6Present address: Genomics Institute of the Novartis Research Foundation, San Diego, CA, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +41 61 265 3507; Fax: +41 61 265 3990; E-mail: [email protected] EMBO Reports (2018)19:e45054https://doi.org/10.15252/embr.201745054 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Vascular endothelial growth factor (VEGF) is the master regulator of angiogenesis, whose best-understood mechanism is sprouting. However, therapeutic VEGF delivery to ischemic muscle induces angiogenesis by the alternative process of intussusception, or vascular splitting, whose molecular regulation is essentially unknown. Here, we identify ephrinB2/EphB4 signaling as a key regulator of intussusceptive angiogenesis and its outcome under therapeutically relevant conditions. EphB4 signaling fine-tunes the degree of endothelial proliferation induced by specific VEGF doses during the initial stage of circumferential enlargement of vessels, thereby limiting their size and subsequently enabling successful splitting into normal capillary networks. Mechanistically, EphB4 neither inhibits VEGF-R2 activation by VEGF nor its internalization, but it modulates VEGF-R2 downstream signaling through phospho-ERK1/2. In vivo inhibitor experiments show that ERK1/2 activity is required for EphB4 regulation of VEGF-induced intussusceptive angiogenesis. Lastly, after clinically relevant VEGF gene delivery with adenoviral vectors, pharmacological stimulation of EphB4 normalizes dysfunctional vascular growth in both normoxic and ischemic muscle. These results identify EphB4 as a druggable target to modulate the outcome of VEGF gene delivery and support further investigation of its therapeutic potential. Synopsis EphrinB2/EphB4 signalling between pericytes and endothelium regulates the switch from normal to aberrant angiogenesis caused by increasing Vascular endothelial growth factor (VEGF) doses. Pharmacologic stimulation of EphB4 ensures exclusively physiological vessel growth under therapeutically relevant conditions of VEGF gene delivery. The endothelial tyrosine kinase receptor EphB4 finely tunes the degree of endothelial proliferation by specific VEGF doses in vivo. EphB4 does not affect VEGF-R2 activation or internalization, but regulates its downstream signaling through p-ERK1/2. EphB4 stimulation limits the size of circumferential vessel enlargement induced by VEGF, thereby enabling splitting into normal capillaries and preventing aberrant growth into angiomas. Introduction Angiogenesis plays a key role in the pathophysiology of a widespread variety of human diseases, both degenerative and neoplastic, as well as in physiological tissue regeneration 1. Vascular endothelial growth factor-A (VEGF) is the master regulator of vascular growth in development and postnatal life, and it is therefore the key molecular target to promote the growth of new blood vessels in ischemic diseases, such as myocardial infarction, stroke, or peripheral vascular disease 2, 3. However, simple VEGF gene delivery for therapeutic angiogenesis has failed to prove clinical efficacy to date, despite the clear biological activity of the factor 2, 4, highlighting the need to better understand the mechanisms of physiological vascular growth by VEGF, especially under therapeutically relevant conditions of factor delivery. The best-understood mode of angiogenesis is sprouting, which is mostly studied during development, when specialized endothelial tip cells migrate from pre-existing vessels, followed by proliferating stalk cells, to invade surrounding avascular tissue 5. However, blood vessels can also grow by the alternative mechanism of intussusception, or splitting angiogenesis, whereby rows of intraluminal endothelial pillars split pre-existing vessels longitudinally into new ones 6. Intussusception is increasingly recognized as a therapeutically important mode of angiogenesis, both in tumor resistance to anti-angiogenic treatments and in reparative vascular growth 7-9, but very little is known about its molecular regulation due to a paucity of appropriate models. Taking advantage of a cell-based platform that we developed for the controlled expression of specific and homogeneous doses of angiogenic factors in vivo, we previously found that (i) VEGF can induce either normal and functional capillary networks or aberrant angioma-like vascular structures depending on its concentration in the microenvironment around each producing cell in vivo 10 and (ii) VEGF doses required for therapeutic efficacy 11, induce robust vascular growth in skeletal muscle essentially through intussusception 9. Interestingly, both normal and aberrant vascular structures form through a first stage of circumferential enlargement within the first 4 days, followed by intussusceptive remodeling by 7 days 9, whereas the transition from normal to aberrant angiogenesis is determined by the retention or loss of pericytes during the initial stage of vascular enlargement 12. Here, we took advantage of this unique and well-characterized model of VEGF dose-dependent intussusceptive angiogenesis to investigate its molecular regulation. We dissected the role of specific pericyte-mediated signaling pathways, and we identified a critical function for ephrinB2/EphB4 signaling, but not TGF-β or angiopoietin signaling. Specifically, we show that the endothelial receptor EphB4 controls the outcome of intussusceptive angiogenesis by fine-tuning the degree of endothelial proliferation caused by specific VEGF doses and therefore the size of initial vascular enlargement, without directly affecting VEGF-R2 activation, but rather modulating its downstream signaling through MAPK/ERK. Together, these results identify the ephrinB2/EphB4 pathway as a key regulator of intussusceptive angiogenesis and a druggable target to modulate the outcome of VEGF delivery. Results Generation and validation of blockers of pericyte-endothelium paracrine signaling To determine whether and which pericyte-derived signals may control normal vascular morphogenesis induced by moderate VEGF doses, we blocked the three main signaling pathways responsible for the cross-talk between pericytes (P) and endothelial cells (EC), that is, the TGF-β1, angiopoietin (Ang)/Tie2, and ephrinB2/EphB4 axes. A clonal myoblast population that homogeneously expresses moderate VEGF levels (V-low = 61 ± 2.9 ng/106 cells/day) was selected to induce normal angiogenesis 9, 13, or myoblasts that do not express VEGF as control (Ctrl). Both populations were transduced with retroviral vectors co-expressing soluble blockers of the TGF-β1 (latency-associated peptide, LAP), Ang/Tie2 (sTie2Fc), and ephrinB2/EphB4 (sEphB4) signaling, together with a truncated version of CD4 (trCD4) in a bicistronic cassette (Fig EV1A) as a FACS-quantifiable surface marker 13 (Fig EV1B). ELISA measurements confirmed that all blocker-expressing V-low populations maintained a similar VEGF production as the original V-low clone (V-low = 64 ± 3, V-low LAP = 64 ± 6, V-low sTie2Fc = 79 ± 4, V-low sEphB4 = 62 ± 5 ng/106 cells/day). Specific expression of each blocker was confirmed by RT–PCR on the in vitro cultured myoblast populations (Fig EV1C), while the functional activity of the secreted proteins was verified by appropriate in vitro assays on myoblast conditioned media (Fig EV1D–F). Click here to expand this figure. Figure EV1. Development and validation of soluble blockers Retroviral construct carrying a bicistronic cassette coding for one of three signaling blockers (LAP, sEphB4, or sTie2Fc) linked to a truncated version of rabbit CD4 (tr.rbCD4), as a convenient cell surface FACS-sortable marker, through an internal ribosomal entry site sequence (IRES). LTR = retroviral long terminal repeats. Blocker-expressing myoblast populations, generated from control cells (Ctrl) or a clone expressing low VEGF (V-low), were FACS-sorted, and their purity was determined by analysis of CD4 expression (black curves) vs. isotype control (gray curves). Expression specificity was determined by RT–PCR on RNA isolated from each population, using primers specific for LAP (L), sEphB4 (E), and sTie2Fc (T), amplifying products of 781, 963, and 1,519 bp, respectively. Functional activity of the LAP blocker. HEK293N cells were transfected with a TGF-β reporter construct, expressing luciferase under a SMAD-dependent promoter. Conditioned medium from LAP-expressing myoblasts (LAP) inhibited luciferase activity induced by stimulation with 0.1 and 1 ng/ml of TGF-β1 compared to control conditioned medium from CD4 myoblasts (Ctrl). R.L.U. = relative light units. Mean ± SEM; n = 3/condition; ***P < 0.001 (one-way ANOVA with Bonferroni multiple comparisons test, after data normalization by logarithmic-transformation). Functional activity of the sTie2Fc blocker. Treatment of RAW264.7 macrophages with LPS causes upregulation of TNFα, which is inhibited by COMP-Ang1. Real-time qRT–PCR analysis of Tnfa gene expression shows that conditioned medium from sTie2Fc myoblasts (LPS + sTie2Fc) prevented this inhibition by 50 ng/ml COMP-Ang1 compared to control conditioned medium from CD4 myoblasts (LPS + Ctrl). Mean ± SEM; n = 6/condition; **P < 0.01 (one-way ANOVA with Bonferroni multiple comparisons test, after data normalization by logarithmic-transformation). Functional activity of the sEphB4 blocker. Human umbilical vein endothelial cells were treated with ephrinB2-Fc or control Fc, and phosphorylation of the EphB4 receptor was measured by ELISA. Conditioned medium from sEphB4 myoblasts (sEphB4) inhibited EphB4 phosphorylation compared to control conditioned medium from CD4 myoblasts (Ctrl). O.D. = optical density units. Mean ± SEM; n = 3/condition; *P < 0.05 (one-way ANOVA with Bonferroni multiple comparisons test, after data normalization by logarithmic-transformation). Download figure Download PowerPoint Blockade of ephrinB2/EphB4 signaling, but not of TGF-β1/TGF-βR or angiopoietin/Tie2, switches VEGF-induced angiogenesis from normal to aberrant Simultaneous blockade of all three pathways of the P-EC cross-talk was achieved by co-implanting the individual blocker-expressing populations into hindlimb muscles of adult mice (Fig 1A). After 2 weeks, myoblasts expressing only the blockers in the absence of VEGF (Ctrl 3b) did not perturb the pre-existing vasculature compared to controls (Ctrl CD4). Low levels of VEGF induced the growth of normal mature capillaries, tightly associated with NG2+/α-SMA− pericytes, but co-expression of the three soluble inhibitors converted these into aberrant vascular structures, characterized by enlarged and irregular diameters, and covered by a patchy layer of SMA+ smooth muscle cells instead of pericytes (V-low 3b), similar to the angioma-like structures induced by another monoclonal myoblast population expressing high VEGF levels alone 10 (V-high = 137.7 ± 1.6 ng/106 cells/day). Figure 1. Blockade of ephrinB2/EphB4 signaling switches VEGF-induced angiogenesis from normal to aberrant A, B. Immunofluorescence staining of endothelium (CD31, red), pericytes (NG2, green), smooth muscle cells (α-SMA, cyan), and nuclei (DAPI, blue) on frozen sections of limb muscles injected with myoblast clones expressing different VEGF levels (V-low and V-high, respectively) or co-expressing low VEGF with blockers of the TGF-β1, angiopoietin/Tie2, and ephrinB2/EphB4 pathways together (V-low 3b) or each individually (LAP, sTie2Fc, or sEphB4). Cells expressing only CD4 surface marker (Ctrl CD4) or blockers (Ctrl 3b) served as controls. Normal angiogenesis induced by V-low was switched to aberrant, enlarged, and smooth muscle-covered vessels, similar to those induced by high VEGF alone (V-high), in the presence of all three blockers or selectively by inhibition of ephrinB2/EphB4 signaling alone. Scale bar = 25 μm. C, D. Quantification of vessel diameters, displayed as distribution (C) or mean ± SEM (D). A population of aberrantly enlarged vessels > 10 μm is induced by ephrinB2/EphB4 blockade. n = 3 mice/group (Ctrl sEphB4 and V-low), n = 5 mice (V-low sEphB4); *P < 0.05 (Mann–Whitney test). E. Immunofluorescence staining for mural cell markers (NG2 or α-SMA, both green) and basal lamina (laminin, purple) shows that aberrant vessels induced by ephrinB2/EphB4 blockade are associated with smooth muscle (α-SMA+ outside the basal lamina) rather than pericytes (NG2+ embedded inside the basal lamina). White arrows indicate an NG2+ pericyte (in V-low left panels) and an α-SMA+ smooth muscle cell (in the V-low sEphB4 right panels); *lumen of aberrant structure. Scale bar = 25 μm. F. Quantification of mural cell coverage of vessels induced by V-low or V-low sEphB4, shown as the ratio of NG2+/CD31+ and α-SMA+/CD31+ areas, or the ratio between the two markers (NG2/SMA). n = 3 mice (V-low), n = 6 mice (V-low sEphB4); *P < 0.05 (Mann–Whitney test). Download figure Download PowerPoint To determine whether any of the three signaling pathways was individually responsible for the switch, each blocker-secreting V-low population was injected separately (Fig 1B). By 2 weeks, ephrinB2/EphB4 blockade caused the appearance of irregularly enlarged aberrant vascular structures, similar to those induced by high VEGF alone, whereas neither TGF-β1/TGF-βR nor Ang/Tie2 blockade affected the normal angiogenesis induced by V-low. Quantification of vessel diameter distributions showed that V-low induced angiogenesis characterized by homogeneous capillary-size vessels with a median of 4.0 μm and 90th percentile of 6.1 μm. However, inhibition of ephrinB2/EphB4 signaling gave rise to a fraction of significantly enlarged structures, with 13% of vessels having diameter > 10 μm, compared to 2 and 1% that could be observed in muscles implanted with control cells expressing only sEphB4 and no VEGF, or with V-low cells alone, respectively (Fig 1C). The average size of vessels induced by V-low was also significantly increased by EphB4 blockade (V-low = 4.4 ± 0.2 μm vs. V-low sEphB4 = 6.6 ± 0.5 μm, P < 0.05; Fig 1D). The nature of mural cells associated with vessels induced by V-low alone or with sEphB4 was further investigated by co-staining for the vascular basal lamina. As can be seen in Fig 1E, normal capillaries induced by low VEGF were associated with NG2+ pericytes that were completely embedded in the laminin-positive basal lamina, whereas the mural cells associated with the aberrant vascular structures induced in the presence of sEphB4 were both α-SMA+ and positioned externally to the basement membrane and were therefore identified as smooth muscle cells rather than pericytes. Quantification of NG2+ and α-SMA+ mural cell coverage (Fig 1F) showed that the transition from pericytes to smooth muscle cells by EphB4 blockade did not result in a loss of NG2, as both mural cell types retain expression of this marker. However, the α-SMA/CD31 ratio was significantly increased with sEphB4, as pericytes do not express α-SMA (V-low = 0.1 ± 0.0 vs. V-low sEphB4 = 0.7 ± 0.1, P < 0.05), as well as the α-SMA/NG2 ratio (V-low = 0.2 ± 0.0 vs. V-low sEphB4 = 1.4 ± 0.1, P < 0.05). Intravascular staining by FITC-labeled tomato lectin, which binds to the luminal surface of endothelial structures only if they are connected to the systemic circulation, co-localized with endothelium staining (CD31), indicating that the aberrant structures caused by V-low sEphB4 cells were not simply endothelial clusters, but were functionally perfused (Fig EV2). This is in agreement with previous findings for angioma-like structures induced by high VEGF alone 11. Further, staining for the apical- and basal-specific markers podocalyxin 14 and laminin confirmed that endothelium in both normal and aberrant vascular structures induced by V-low, V-low 3b, and V-low sEphB4 was functionally polarized into luminal and basal compartments (Appendix Fig S1). Lastly, to determine the evolution of the morphological changes caused by ephrinB2/EphB4 blockade, tissues were analyzed after 12 weeks, showing that the aberrant structures observed by 2 weeks continued growing in size (Appendix Fig S2). Click here to expand this figure. Figure EV2. Blockade of ephrinB2/EphB4 signaling does not affect perfusion of VEGF-induced vesselsMice received intravenous injections of FITC-lectin 2 weeks after implantation of myoblast clones expressing low VEGF levels alone (V-low) or co-expressing the sEphB4 blocker (V-low sEphB4). Frozen sections of limb muscles were immunostained for CD31 (endothelium, red), and perfused structures were visualized by FITC-lectin co-localization (green). Vascular perfusion was similar in both conditions. Scale bar = 25 μm. Download figure Download PowerPoint Altogether, these results suggest that the ephrinB2/EphB4 pathway, but not TGF-β1/TGF-βR and Ang/Tie2, has a function in the development of normal angiogenesis by low VEGF doses and its blockade causes the switch to an aberrant phenotype resembling the angioma-like vascular structures induced by high VEGF alone. Activation of EphB4 signaling prevents aberrant angiogenesis induced by high VEGF doses To complement the ephrinB2/EphB4 inhibition data above, we asked whether the pharmacological activation of EphB4 might prevent aberrant angiogenesis by high VEGF levels. A recombinant ephrinB2-Fc chimeric protein, whereby fusion with the immunoglobulin Fc portion enables the formation of dimers of ephrinB2 extracellular domains, was used to activate the EphB4 receptor 15. V-high clonal myoblasts were injected in leg muscles of adult mice that were treated systemically with ephrinB2-Fc or Fc control protein by intraperitoneal injection 16. Two weeks later, high VEGF induced heterogeneous enlarged vascular structures associated with smooth muscles cells (Fig 2A). As normal muscle capillaries have homogeneous sizes smaller than 10 μm, vessel diameter distribution was quantified and showed that 26% of induced structures were larger than 10 μm (Fig 2B). On the other hand, treatment with ephrinB2-Fc yielded networks of pericyte-covered normal capillaries (Fig 2A), similar to those induced by V-low alone (Fig 1B) and with a homogeneous diameter distribution (Fig 2B; median = 5.1 μm and 6% of vessels larger than 10 μm). The average vessel size was also significantly reduced by ephrinB2-Fc treatment (V-high + Fc = 9.5 ± 0.3 μm vs. V-high + ephrinB2-Fc = 5.8 ± 0.2 μm, P < 0.05; Fig 2C). Again, podocalyxin and laminin staining confirmed proper apico-basal polarization of the endothelial structures (Appendix Fig S1). Figure 2. Activation of EphB4 by ephrinB2-Fc prevents aberrant angiogenesis A–F. A high VEGF dose was delivered to limb muscles of mice either by genetically modified myoblasts (V-high, A–C) or as fibrin-bound recombinant protein (fibrin-High V, D–F), and animals were treated intraperitoneally with ephrinB2-Fc or control Fc recombinant protein. Immunostaining (A, D) of frozen sections for endothelium (CD31, red), pericytes (NG2, green), smooth muscle cells (α-SMA, cyan), and nuclei (DAPI, blue) showed that, with both delivery platforms, ephrinB2-Fc treatment prevented the induction of aberrant vascular structure by high VEGF and yielded only normal capillary networks. *lumens of aberrant structures in (D); scale bar = 25 μm. Quantification (B, C, E, and F) of vessel diameters showed a consistent and significant decrease in vessel sizes after treatment with ephrinB2-Fc. Results are shown as diameter distributions (B, E) and mean ± SEM (C, F). Red arrows and numbers indicate the fraction of vessel diameters > 10 μm. n = 4 mice; *P < 0.05 (Mann–Whitney test). Download figure Download PowerPoint These results were confirmed independently of cell-based VEGF delivery, using an optimized fibrin-based platform that we recently developed for controlled release of VEGF recombinant protein at specific doses and with duration up to 4 weeks in skeletal muscle 17. An engineered version of murine VEGF164 was fused to the transglutaminase substrate octapeptide NQEQVSPL (α2-PI1–8-VEGF), to allow its covalent cross-linking into fibrin hydrogels by the coagulation factor XIIIa and release only by enzymatic cleavage 18, 19. Fibrin hydrogels containing a high dose of α2-PI1–8-VEGF (50 μg/ml), which we previously found to induce aberrant angiogenesis 17, were injected in gastrocnemius muscles and the animals were treated systemically with ephrinB2-Fc. In agreement with the myoblast-based experiments, ephrinB2-Fc treatment prevented the appearance of heterogeneous, enlarged, and smooth muscle-covered vascular structures induced by the high VEGF dose, yielding instead networks of pericyte-covered capillaries by 7 days (Fig 2D), with a more homogeneous size distribution (Fig 2E) and significantly smaller diameters (fibrin-High V + Fc = 10.0 ± 0.6 μm vs. fibrin-High V+ephrinB2-Fc = 6.9 ± 0.5 μm, P < 0.05; Fig 2F). The observed prevention of aberrant vascular structures could be due to either their switch to a normal phenotype or to their regression. Since regressing vessels leave behind their basal lamina, a staining for laminin was performed to detect so-called empty sleeves of vascular basement membrane, which provide a sort of historical record of pre-existing vessels 20. As shown in Fig EV3, by 7 days after injection of V-high myoblasts we could not identify laminin sleeves in the tissues treated with ephrinB2-Fc compared with the controls treated with Fc only. On the other hand, many empty sleeves were clearly visible in positive control tissues treated with the potent VEGF blocker aflibercept, which caused the regression of vascular structures induced by high VEGF, suggesting that EphB4 stimulation could prevent the formation of aberrant structures by regulating VEGF-induced vascular morphogenesis. Click here to expand this figure. Figure EV3. EphB4 stimulation does not cause vessel regressionMouse limb muscles were implanted with the V-high myoblast clone, while treating animals systemically with ephrinB2-Fc or control Fc protein. As a positive control for vessel regression, mice implanted with the V-high clone were treated with the potent VEGF blocker aflibercept (V-high + aflibercept). Immunofluorescence staining for endothelium (CD31, green), basal lamina (laminin; lam, red), and nuclei (DAPI, blue) showed that ephrinB2-Fc treatment did not cause any vessel regression, as all basal laminas were associated with endothelial tubes. By contrast, after aflibercept treatment widespread vessel regression was evident through the detection of empty sleeves of basal lamina (white arrows) devoid of endothelium, remnants of disappeared vessels. Scale bar: 25 μm.