The GEF Trio controls endothelial cell size and arterial remodeling downstream of Vegf signaling in both zebrafish and cell models

Abstract

Arterial networks enlarge in response to increase in tissue metabolism to facilitate flow and nutrient delivery. Typically, the transition of a growing artery with a small diameter into a large caliber artery with a sizeable diameter occurs upon the blood flow driven change in number and shape of endothelial cells lining the arterial lumen. Here, using zebrafish embryos and endothelial cell models, we describe an alternative, flow independent model, involving enlargement of arterial endothelial cells, which results in the formation of large diameter arteries. Endothelial enlargement requires the GEF1 domain of the guanine nucleotide exchange factor Trio and activation of Rho-GTPases Rac1 and RhoG in the cell periphery, inducing F-actin cytoskeleton remodeling, myosin based tension at junction regions and focal adhesions. Activation of Trio in developing arteries in vivo involves precise titration of the Vegf signaling strength in the arterial wall, which is controlled by the soluble Vegf receptor Flt1.

Fig. 1. Arterial Flt1 determines vessel lumen dimensions.

a, b In vivo confocal imaging of trunk vascular architecture in WT (a) and vegfaa^musc transgenic embryos (b). Note disrupted vascular development in vegfaa^musc transgenics. c, d Whole mount immune staining with anti-HA antibody in TgTm(flt1_E3_HAHA)^ka611 embryos at 32 hpf (c) and 48 hpf (d) to show Flt1 protein distribution. Arrows indicate aISVs and box shows zoom. e–g Immunestaining with anti-HA antibody showing sFlt1 protein distribution in Tg(flt1^enh:sflt1_Δ7-HAHA)^ka612; Tg(kdrl:hsa.HRAS-mcherry)^s916 double transgenic embryos. aISV express sFlt1 protein (green; e); kdrl is shown in red (f) and merge shows colocalization of sFlt1 and kdrl in aISV (g, blue arrows). The white squared inset in e indicates control staining. h–k Confocal imaging of aISV in WT (h), flt1^−/− mutant, flt1^ka601 (i), plgf GOF transgenic plgf^musc (j), and vegfba GOF transgenic vegfb^musc (k). Upper panels show overview; lower panels show detail of red boxed area. l Quantification of aISV diameter for indicated genotype; mean ± s.e.m, unpaired two-sided students t-test, n = 18, 16, 16, 10 aISVs per genotype. ***p < 0.001. m Confocal imaging of aISV in WT injected with flt1 targeting morpholino. Area indicated by the red dotted box in the upper panel is displayed at higher magnification in the lower panel. n Relative aISV diameter change in embryos injected with flt1 targeting morpholino (WT = 100%). mean ± s.e.m, unpaired two-sided students t-test, n = 20, 25 aISVs for indicated scenario, ***p < 0.001. o, p Imaging of plasma extravasation in WT and plgf^musc (n = 14, 16 embryos) injected with 70kD Dextran Texas-Red from 3 biologically independent experiments. q Quantification of images in o,p. mean ± s.e.m; unpaired two-sided students t-test, n = 6, 8 embryos per indicated group. ns, not significantly different. Scale bar, 50 μm in a–g, o, p; 25 µm in h–k, m. aISV intersegmental artery, DA dorsal aorta, PCV posterior cardinal vein. hpf hours post fertilization, MO morpholino, GOF gain of function.

Fig. 2. mFlt1 is not required for plgf-induced arterial diameter growth.

a–c In vivo confocal imaging of aISV in WT (a); mft1^−/− mutant, mflt^ka605, (b); mft1^−/− combined with plgf gain of function plgf^musc (c); n = 58,64 and 57 aISVs per genotype derived from three autonomous experiments. The areas indicated by the red dotted boxes are displayed at higher magnification in the lower panels. d Quantification of images in a–c. Mean ± s.e.m, unpaired two-sided students t-test, n = 7,13, and 24 aISVs for indicated scenario. **p = 0.0051, ***p < 0.001. Note: significantly increased aISV diameter in mflt1^−/−+plgf^musc scenario. e–g In vivo confocal imaging of aISV in plgf^musc (e), plgf^musc concomitant with morpholino knock-down of flt1 (f; plgf^musc + flt1-MO), flt1^−/− mutant, flt1^ka601 (g). The areas indicated by the red dotted boxes are displayed at higher magnification in the lower panels. h Quantification of images in e–g. mean ± s.e.m, unpaired two-sided students t-test, n = 40,19, and 13 aISVs for indicated scenario. ns not significant, **p < 0.01. Scale bar; 25 µm in all panels. hpf hours post fertilization, aISV intersegmental artery, MO morpholino.

Fig. 3. Vegfaa—Vegf receptor-2/Kdrl signaling drives outward arterial lumen remodeling.

a–d aISV in WT (upper panels) and plgf^musc embryos (lower panels) treated with DMSO vehicle - control (a), low dose Vegf-R2 inhibitor (b), high dose Vegf-R2 inhibitor (c), or vegfaa ATG targeting morpholino (d). Red dotted box is displayed at higher magnification in panel below. e Quantification of aISV diameter at 50 hpf upon R2 inhibition. Mean ± s.e.m, one-way ANOVA, and post-hoc bonferroni, n = 18, 20, 20, 15, 20, 20 aISVs for indicated scenarios. **p = 0.0026, ***p < 0.001. f Quantification of aISV diameter at 48 hpf upon morpholino-mediated knockdown of vegfaa. Mean ± s.e.m, one-way ANOVA, and post-hoc bonferroni, n = 15, 22, 14, 28 aISVs for indicated scenarios. ***p < 0.001. g–j In vivo confocal imaging of WT (g), flt4^−/− mutant, flt4^mu407(h), flt4^−/− mutant injected with flt1 ATG targeting morpholino (i), flt4^−/− mutant injected with plgf^musc plasmid (j). k Quantification of images in g–j. Left panel, absolute aISV diameter (n = 21, 16, 42, 34 aISVs per indicated genotype). Right panel, aISV diameter change relative to WT. Mean ± s.e.m, unpaired two-sided students t-test. ns not significant, ***p ≤ 0.001. l, m In vivo confocal imaging of developing aISVs prior to the onset of aISV perfusion in WT (l) and plgf^musc embryos (m). Note: larger aISV prior to onset of flow in plgf^musc. n Quantification of images in l, m. Mean ± s.e.m, unpaired two-sided students t-test, n = 16 aISVs/group. ***p < 0.001. o–q Imaging of WT (o), WT treated with L-type calcium channel blocker nifedipine (p), and plgf^musc treated with nifedipine (q). r Quantification of images in o–q. Mean ± s.e.m, unpaired two-sided students t-test, n = 11, 20, and 15 aISVs per treatment group. ***p < 0.001. s–u Confocal cross-section to show lumen dimensions of aISV in WT (s), WT injected with tnnt2 targeting morpholino (t), and plgf^musc embryo injected with tnnt2 targeting morpholino (u). v Quantification of images in s–u. Mean ± s.e.m, unpaired two-sided students t-test, n = 20 aISVs per group. ***p < 0.001. Scale bar: 25 μm in all images. aISV intersegmental artery, MO morpholino, hpf hours post fertilization, RI remodeling index, tnnt2 cardiac muscle troponin T2.

Fig. 4. Plgf-induced arterial diameter growth requires the Rho GEF Trio.

a, b endothelial cell (EC) nuclei distribution in green (eGFP) during aISV formation in WT (a) and in plgf^musc embryos (b). ECs derived from migration events numbered in yellow, from proliferation events in purple. c Quantification of migration (yellow) and proliferation (purple) events during aISV formation; mean ± s.e.m, unpaired two-sided students t-test, n = 10,11 aISVs for indicated genotype. *p = 0.0190, ***p < 0.001. d Time-lapse imaging in Tg(fli1a:lifeactEGFP)^mu240 showing endothelial actin in aISV of WT (upper panels) and plgf^musc embryos (lower panels). Red box in the left panel at higher magnification in the right panels. Arrowheads at 41hpf delineate an individual EC. e EC surface area changes in WT (blue circles) and plgf^musc embryos (red squares); mean ± s.e.m, unpaired two-sided students t-test, n = 6 ECs for each genotype. f Left panel, arterial ISV diameter in plgf^musc embryos (magenta bar) treated with the actin polymerization inhibitor Latrunculin B (LatB, pink bar), Rac1 inhibitor CAS 1177865-17-6 (green bar), Trio inhibitor ITX3 (yellow bar). Right panel, RI upon indicated inhibitor treatment. Mean ± s.e.m, one-way ANOVA, post-hoc bonferroni, n = 25, 38, 30, 60, 59 aISVs for indicated condition. ***p < 0.001. g–i aISV in WT (g), WT injected with 1.7 ng Trio targeting morpholino (h), and WT injected with 5 ng Trio targeting morpholino (i). j–l aISV in plgf^musc (j), plgf^musc injected with 1.7 ng Trio targeting morpholino (k), and plgf^musc injected with 5 ng Trio targeting morpholino (l). m Quantification of images in g–l. Mean ± s.e.m, ANOVA & post-hoc bonferroni, n = 17, 21, 18, 15, 17, and 16 aISVs/group. ***p < 0.001. n–p aISV in WT (n), WT injected with 1.7 ng Trio targeting morpholino (o), and WT injected with 5 ng Trio targeting morpholino (p). q–s aISV in plgf^musc (q), plgf^musc injected with 1.7 ng Trio targeting morpholino (r), and plgf^musc injected with 5 ng Trio targeting morpholino (s). t Quantification of images in n–s. Mean ± s.e.m, ANOVA & post-hoc bonferroni, n = 18, 14, 14, 16, 18, and 18 aISVs per group. ns not significant, **p < 0.01, ***p < 0.001. Scale bar, 25 µm in all panels. aISV intersegmental artery, MO morpholino.

Fig. 5. Trio-GEF1 domain targets Rac1 and RhoG increase endothelial cell size.

a–f Confocal images showing mCherry (a, c, e) and F-actin distribution (b, d, f) in ECs transfected with mCherry, mCherry-TrioN (TrioN), or mCherry-TrioGEF1 (TrioGEF1). Scale bar, 25 µm. g Endothelial cell surface area in ECs expressing mCherry, mCherry-TrioN, or mCherry-TrioGEF1. Mean ± s.e.m, unpaired two-sided students t-test, n = 59, 69, 63 cells per indicated group. ***p < 0.001. h–k Confocal images of ECs transfected with mCherry, photo-activatable Rac1 (mCherry-Rac1-PA-WT), mutated photo-activatable Rac1 (mCherry-Rac1-PA-C450A), or constitutively active Rac1 construct (mCherry-Rac1-Q61L). Red arrowheads indicate cell–cell junctions. Scale bar, 25 µm. l Quantification of images in h–k. Mean ± s.e.m, two-sided Mann–Whitney U test, n = 67, 56, 65, 74 cells per group. *p = 0.0477, ***p < 0.001. m Surface area of ECs transfected with control plasmid or constitutive active RhoG Q61L. Mean ± s.e.m, unpaired two-sided students t-test, n = 89, 83 cells per group, from three independent experiments. ***p < 0.001. n Western blots for Rac1 and RhoG; control of knockdown efficiency for shRNA shRac1 and shRhoG as used in O, P and actin for protein loading control, as indicated. o Change in EC size upon TrioN overexpression after silencing of Rac1. Mean ± s.e.m, unpaired two-sided students t-test, n = 73, 97 cells from three independent experiments. ***p < 0.001. p Change in EC size upon TrioN overexpression after silencing of RhoG. Mean ± s.e.m, unpaired two-sided students t-test, n = 101, 146 cells per group from three independent experiments. ***p < 0.001. q–s Endothelial cells transfected with control plasmid (ctrl) and stained for Tiam1 (q), F-actin with phalloidin (r) and VE-Cadherin as junction marker (s). Tiam1 localizes cytosolic and to a lesser extent to junction regions. Scale bar, 20 μm. t–v Endothelial cells transfected with TrioN and stained for Tiam1 (t), F-actin with phalloidin (u) and VE-Cadherin as junction marker (v). Tiam1 localizes at junction regions (arrowheads). Scale bar, 20 μm. w Endothelial cell size of ECs transfected with Tiam1, TrioN, or transfected with both Tiam1 and TrioN. Cell size was measured of n = 78, 82, 103 and 80 cells derived from three separate experiments. Mean ± s.e.m, unpaired two-sided students t-test. ns not significant, ***p < 0.001.

Fig. 6. Trio activates Rac1 and induces tension in endothelial junctional regions.

a EC transfected with mCherry and Rac1 biosensor (Cer3, cerulean3 channel) and stained for VE-Cadherin. FRET signals depicted as warm colors. b EC transfected with mCherry-TrioN and Rac1 biosensor (Cer3) and stained for VE-Cadherin. FRET signals depicted as warm colors. Arrowheads indicate FRET signal in junctional regions. c, d Rac1 activation for indicated scenario. Mean ± s.e.m, two-sided Mann–Whitney U test, n = 69, 59, 65, 69 cells per indicated group (c). Ratio junctional to cytoplasmic Rac1 activation (d): mean ± s.e.m, two-sided Mann–Whitney-test, n = 69, 59, 65, 69 cells per group, respectively. ***p < 0.001. e Kymograph illustrating the actin cytoskeleton dynamics along indicated line in control and TrioN-transfected cells. f Quantification of protrusion lifetime (left panel). Mean ± s.e.m, unpaired two-sided students t-test, n = 50, 110 cells. (right panel) Quantification of protrusion dynamics expressed as protrusion number per hour from data in left panel. ***p < 0.001. g EC transfected with mCherry and GFP-Myosin II, stained for F-actin and VE-cadherin. Myosin-II localizes at actin bundles (arrowheads). h EC transfected with mCherry-TrioN and GFP-Myosin II, stained for F-actin and VE-cadherin. Myosin-II localizes at junction actin bundles (arrowheads). i EC transfected with GFP stained for pMLC-S19, F-actin, and VE-cadherin. j EC transfected with GFP-TrioN, stained for pMLC-S19, F-actin and VE-cadherin. Arrowheads show pMLC-19 at junctional actin bundles. k EC transfected with GFP, stained for pMLC-T18S19 and F-actin. l EC transfected with GFP-TrioN, stained for pMLC-T18S19 and F-actin. Arrowheads show pMLC-T18S19 at junctional regions. m, n distribution of F-actin (m) and VE-Cadherin (n) in TrioN overexpressing EC. o Intracellular distribution of central, cortical and junctional F-actin bundles in ECs. p F-actin recoil response upon laser ablation. Shown are pre-ablation (left panel) and post-ablation states (right panel); yellow arrowheads indicate the extent of recoil. q–s Recoil distance in central (q), cortical (r) and junctional (s) F-actin bundles, in GFP and GFP-TrioN-transfected ECs; mean ± s.e.m, unpaired two-sided students t-test, n = 11, 7, 17, 13, 11 and 25 cells per indicated group. ns not significant ***p < 0.001. Scale bar, 25 µm in a, b, m, n; 20 µm in e, g–l, 10 µm in o, 5 µm in p.

Fig. 7. Trio acts cell-autonomous to increase endothelial cell size.

a Electrical Cell-Substrate impedance (ECIS) array in control and TrioN overexpressing EC. Experiment in triplicate, repeated three independent times. Mean ± s.e.m, unpaired two-sided students t-test, ***p < 0.001. b ECIS array in control plasmid or TrioN-transfected EC, and co-transfected with control shRNA or VE-Cadherin silencing shRNA. Experiment in duplicate, repeated three independent times. Mean ± s.e.m, unpaired two-sided students t-test, **p = 0.0063, ***p < 0.001. c Relative impedance change for indicated scenario based on data in b. Experiment carried out in duplicate, three independent times. Mean ± s.e.m, unpaired two-sided students t-test. ns not significant. d–f VE-Cadherin (d), F-actin (e), and mCherry (f) distribution in EC transfected with mCherry plasmid and control shRNA (shCTRL). g–i VE-Cadherin (g), F-actin (h), and mCherry-TrioN (i) distribution in ECs transfected with shCTRL, and co-transfected with TrioN. Arrowheads show colocalization of VE-Cadherin, F-actin and TrioN at cell–cell junctions. j–l VE-Cadherin (j), F-actin (k), and mCherry-TrioN (l) distribution in ECs transfected with shVE-Cadherin, and co-transfected with TrioN. Arrowheads show intact F-actin bundles and TrioN expression at junctions. m EC size for indicated scenario. mean ± s.e.m, two-sided Mann–Whitney U test, n = 69, 26, and 22 cells per group derived from four independent experiments. ***p < 0.001. n EC size of TrioN expressing cell, when in contact with TrioN expressing neighbouring cell (homogenous expression) or when in contact with control-transfected neighbouring cells (mosaic expression). Cell-sizes from 52 cells per condition, and two independent experiments. Mean ± s.e.m, unpaired two-sided students t-test. ns not significant, ***p < 0.001. o, p Distribution of focal adhesions, labeled with phospho-Paxillin (o); and F-actin (p) in control-transfected ECs. q, r Distribution of focal adhesions, labeled with phospho-Paxillin (q); and F-actin (r) in TrioN-transfected ECs. s Focal adhesion density in control and TrioN-transfected ECs. Mean ± s.e.m, unpaired two-sided students t-test, n = 7, 9 cells per indicated group. ***p < 0.001. t Integrin-alpha-5 and F-actin expression in GFP control-transfected EC. u Integrin-alpha-5 and F-actin expression in GFP-TrioN-transfected EC. Arrowheads indicate junctional region. v Integrin-beta-1 and F-actin expression in GFP control-transfected EC. w Integrin-beta-1 and F-actin expression in GFP-TrioN-transfected EC. Arrowheads indicate junctional region. Scale bar, 25 μm in all panels.

Fig. 8. Trio augments endothelial cell size and arterial diameter in vivo.

a, b Imaging of aISV in WT (a) and in flt1^enh:TrioN embryos (b). c, d EC nuclei distribution in WT (c) and flt1^enh:TrioN embryos (d). Note: six ECs in both scenarios and larger aISV diameter upon TrioN. e Quantification of aISV diameter for indicated scenario. Mean ± s.e.m, two-sided Mann–Whitney U test, n = 35 aISVs per genotype. ***p < 0.001. f Quantification of EC surface area in aISVs for indicated scenario. Mean ± s.e.m, two-sided Mann–Whitney U test, n = 34, 48 aISVs per indicated genotype. ***p < 0.001. g Quantification of EC nuclei number in aISVs for indicated scenario. mean ± s.e.m, two-sided Mann–Whitney U test, n = 51, 70 aISVs per genotype. ns not significant. h–k aISV in WT (h), in plgf^musc (i), plgf^musc combined with flt1^enh:TrioN (j), and plgf^musc combined with flt1^enh:TrioNmut (k). l–n Transverse optical section of aISV vessel lumen in WT (l), plgf^musc (m), and plgf^musc+flt1^enh:TrioN scenario (n). The lumen area is color indicated (lower panels). o Left panel: Quantification of aISV diameter in WT, plgf^musc, plgf^musc+flt1^enh:TrioN, and plgf^musc+flt1^enh:TrioNmut scenario. Mean ± s.e.m, unpaired two-sided students t-test, n = 18, 19, 13, 14 aISVs per group. ns, not significant; ***p < 0.001. Right panel: Quantification of EC size in WT, plgf^musc, plgf^musc+flt1^enh:TrioN, and plgf^musc+flt1^enh:TrioNmut scenario. Mean±s.e.m, unpaired two-sided students t-test, n = 21, 24, 15, 12 cells per group. ns not significant; ***p < 0.001. p–s aISV in WT embryo (p), flt1^enh:TrioN embryo (q), WT embryo injected with ve-cadherin (cdh5) targeting morpholino (r), flt1^enh:TrioN embryo injected with ve-cadherin (cdh5) targeting morpholino (s). t Quantification of images in p–s; mean ± s.e.m, unpaired two-sided students t-test, n = 15, 21, 16, and 13 cells per indicated genotype. ***p < 0.001. u Dynamic changes in EC surface area in plgf^musc + flt1^enh:TrioN embryo, plgf^musc embryo, WT embryo injected with flt1 targeting morpholino, and WT embryo. n = 12, 12, 10, 18 cells per indicated genotype, mean ± s.e.m. v, w Aorta diameter (v) and EC surface area (w) in kdrl:TrioN or fli1a:TrioN scenario. Mean ± s.e.m, unpaired two-sided students t-test, n = 8, 10, 10 (v) and n = 8, 16, 14 (w) biologically independent embryos/group. *p < 0.05, **p = 0.0061. Scale bars: a–d, h–k, p–s, 25 μm, l–n 10 μm.

Fig. 9. Trio and endoglin interact to determine 3dpf aortic EC size.

a Dorsal aorta diameter at 3dpf in endoglin morphants (magenta bar), endoglin morphants injected with 0.8 ng Trio targeting morpholino (blue bar), endoglin morphants treated with Trio inhibitor ITX3 (dark green bar) and WT treated with ITX3 (light green bar). Note that loss of Trio or inhibiting Trio reduced diameter growth in endoglin morphants. Mean ± s.e.m, unpaired two-sided students t-test, n = 8, 9, 8, 10 independent embryos per group. **p = 0.0011, ***p = 0.001. b Aorta EC surface area at 3dpf in endoglin morphants (magenta bar), endoglin morphants injected with 0.8 ng Trio targeting morpholino (blue bar), endoglin morphants treated with Trio inhibitor ITX3 (dark green bar) and WT treated with ITX3 (light green bar). Note that loss of Trio or inhibiting Trio reduced EC surface area in endoglin morphants. Mean ± s.e.m, unpaired two-sided students t-test, n = 18, 20, 22, 16 cells derived from six independent embryos per group; ***p < 0.001; **p = 0.0055; *p = 0.0100.

Fig. 10. Schematic representation of arterial lumen increase.

a Increase in arterial lumen dimensions normalized to WT (WT = 1.0). Artery specific TrioN gain of function fish show 1.5 fold, flt1 mutants show 1.6 fold, plgf^musc 1.9 fold, vegfba^musc 2.0 fold, and plgf^musc+artery specific TrioN 2.5-fold larger arterial lumen diameter dimensions compared to WT. b Promoting endothelial cell enlargement results in larger arterial diameter. Simultaneously promoting endothelial cell number and size, and creating arteries with multiple enlarged ECs results in even more pronounced arterial diameter increase.