F‐domain valency determines outcome of signaling through the angiopoietin pathway
2021; Springer Nature; Volume: 22; Issue: 12 Linguagem: Inglês
10.15252/embr.202153471
ISSN1469-3178
AutoresYan Ting Zhao, Jorge A. Fallas, Shally Saini, George Ueda, Logeshwaran Somasundaram, Ziben Zhou, Infencia Xavier Raj, Chunfu Xu, Lauren Carter, Samuel Wrenn, Julie Mathieu, Drew L. Sellers, David Baker, Hannele Ruohola‐Baker,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoArticle26 October 2021free access Source DataTransparent process F-domain valency determines outcome of signaling through the angiopoietin pathway Yan Ting Zhao Yan Ting Zhao orcid.org/0000-0001-9230-7066 Department of Biochemistry, University of Washington, Seattle, WA, USA Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA Oral Health Sciences, School of Dentistry, University of Washington, Seattle, WA, USA Search for more papers by this author Jorge A Fallas Jorge A Fallas orcid.org/0000-0002-1431-820X Department of Biochemistry, University of Washington, Seattle, WA, USA Institute for Protein Design, University of Washington, Seattle, WA, USA Search for more papers by this author Shally Saini Shally Saini orcid.org/0000-0002-0430-8788 Department of Biochemistry, University of Washington, Seattle, WA, USA Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA Search for more papers by this author George Ueda George Ueda orcid.org/0000-0002-9792-7149 Department of Biochemistry, University of Washington, Seattle, WA, USA Institute for Protein Design, University of Washington, Seattle, WA, USA Search for more papers by this author Logeshwaran Somasundaram Logeshwaran Somasundaram orcid.org/0000-0003-1924-8156 Department of Biochemistry, University of Washington, Seattle, WA, USA Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA Search for more papers by this author Ziben Zhou Ziben Zhou orcid.org/0000-0001-8829-5330 Department of Biochemistry, University of Washington, Seattle, WA, USA Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA Search for more papers by this author Infencia Xavier Raj Infencia Xavier Raj orcid.org/0000-0002-4889-4643 Department of Biochemistry, University of Washington, Seattle, WA, USA Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA Search for more papers by this author Chunfu Xu Chunfu Xu orcid.org/0000-0002-8668-0566 Department of Biochemistry, University of Washington, Seattle, WA, USA Search for more papers by this author Lauren Carter Lauren Carter orcid.org/0000-0002-9837-9068 Department of Biochemistry, University of Washington, Seattle, WA, USA Institute for Protein Design, University of Washington, Seattle, WA, USA Search for more papers by this author Samuel Wrenn Samuel Wrenn Department of Biochemistry, University of Washington, Seattle, WA, USA Institute for Protein Design, University of Washington, Seattle, WA, USA Search for more papers by this author Julie Mathieu Julie Mathieu orcid.org/0000-0002-3826-3549 Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA Department of Comparative Medicine, University of Washington, Seattle, WA, USA Search for more papers by this author Drew L Sellers Corresponding Author Drew L Sellers [email protected] orcid.org/0000-0003-1799-5908 Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA Department of Bioengineering, University of Washington, Seattle, WA, USA Search for more papers by this author David Baker Corresponding Author David Baker [email protected] orcid.org/0000-0001-7896-6217 Department of Biochemistry, University of Washington, Seattle, WA, USA Institute for Protein Design, University of Washington, Seattle, WA, USA Department of Bioengineering, University of Washington, Seattle, WA, USA Search for more papers by this author Hannele Ruohola-Baker Corresponding Author Hannele Ruohola-Baker [email protected] orcid.org/0000-0002-5588-4531 Department of Biochemistry, University of Washington, Seattle, WA, USA Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA Oral Health Sciences, School of Dentistry, University of Washington, Seattle, WA, USA Department of Bioengineering, University of Washington, Seattle, WA, USA Search for more papers by this author Yan Ting Zhao Yan Ting Zhao orcid.org/0000-0001-9230-7066 Department of Biochemistry, University of Washington, Seattle, WA, USA Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA Oral Health Sciences, School of Dentistry, University of Washington, Seattle, WA, USA Search for more papers by this author Jorge A Fallas Jorge A Fallas orcid.org/0000-0002-1431-820X Department of Biochemistry, University of Washington, Seattle, WA, USA Institute for Protein Design, University of Washington, Seattle, WA, USA Search for more papers by this author Shally Saini Shally Saini orcid.org/0000-0002-0430-8788 Department of Biochemistry, University of Washington, Seattle, WA, USA Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA Search for more papers by this author George Ueda George Ueda orcid.org/0000-0002-9792-7149 Department of Biochemistry, University of Washington, Seattle, WA, USA Institute for Protein Design, University of Washington, Seattle, WA, USA Search for more papers by this author Logeshwaran Somasundaram Logeshwaran Somasundaram orcid.org/0000-0003-1924-8156 Department of Biochemistry, University of Washington, Seattle, WA, USA Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA Search for more papers by this author Ziben Zhou Ziben Zhou orcid.org/0000-0001-8829-5330 Department of Biochemistry, University of Washington, Seattle, WA, USA Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA Search for more papers by this author Infencia Xavier Raj Infencia Xavier Raj orcid.org/0000-0002-4889-4643 Department of Biochemistry, University of Washington, Seattle, WA, USA Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA Search for more papers by this author Chunfu Xu Chunfu Xu orcid.org/0000-0002-8668-0566 Department of Biochemistry, University of Washington, Seattle, WA, USA Search for more papers by this author Lauren Carter Lauren Carter orcid.org/0000-0002-9837-9068 Department of Biochemistry, University of Washington, Seattle, WA, USA Institute for Protein Design, University of Washington, Seattle, WA, USA Search for more papers by this author Samuel Wrenn Samuel Wrenn Department of Biochemistry, University of Washington, Seattle, WA, USA Institute for Protein Design, University of Washington, Seattle, WA, USA Search for more papers by this author Julie Mathieu Julie Mathieu orcid.org/0000-0002-3826-3549 Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA Department of Comparative Medicine, University of Washington, Seattle, WA, USA Search for more papers by this author Drew L Sellers Corresponding Author Drew L Sellers [email protected] orcid.org/0000-0003-1799-5908 Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA Department of Bioengineering, University of Washington, Seattle, WA, USA Search for more papers by this author David Baker Corresponding Author David Baker [email protected] orcid.org/0000-0001-7896-6217 Department of Biochemistry, University of Washington, Seattle, WA, USA Institute for Protein Design, University of Washington, Seattle, WA, USA Department of Bioengineering, University of Washington, Seattle, WA, USA Search for more papers by this author Hannele Ruohola-Baker Corresponding Author Hannele Ruohola-Baker [email protected] orcid.org/0000-0002-5588-4531 Department of Biochemistry, University of Washington, Seattle, WA, USA Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA Oral Health Sciences, School of Dentistry, University of Washington, Seattle, WA, USA Department of Bioengineering, University of Washington, Seattle, WA, USA Search for more papers by this author Author Information Yan Ting Zhao1,2,3,†, Jorge A Fallas1,4,†, Shally Saini1,2, George Ueda1,4, Logeshwaran Somasundaram1,2, Ziben Zhou1,2, Infencia Xavier Raj1,2, Chunfu Xu1, Lauren Carter1,4, Samuel Wrenn1,4, Julie Mathieu2,5, Drew L Sellers *,2,6, David Baker *,1,4,6 and Hannele Ruohola-Baker *,1,2,3,6 1Department of Biochemistry, University of Washington, Seattle, WA, USA 2Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA 3Oral Health Sciences, School of Dentistry, University of Washington, Seattle, WA, USA 4Institute for Protein Design, University of Washington, Seattle, WA, USA 5Department of Comparative Medicine, University of Washington, Seattle, WA, USA 6Department of Bioengineering, University of Washington, Seattle, WA, USA † These authors contributed equally to this work *Corresponding author. Tel: +1 206 616 8023; E-mail: [email protected] *Corresponding author. Tel: +1 206 616 7542; E-mail: [email protected] *Corresponding author. Tel: +1 206 543 8468; E-mail: [email protected] EMBO Reports (2021)22:e53471https://doi.org/10.15252/embr.202153471 PDFDownload PDF of article text and main figures.AM PDF 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 Angiopoietins 1 and 2 (Ang1 and Ang2) regulate angiogenesis through their similar F-domains by activating Tie2 receptors on endothelial cells. Despite the similarity in the underlying receptor-binding interaction, the two angiopoietins have opposite effects: Ang1 induces phosphorylation of AKT, strengthens cell–cell junctions, and enhances endothelial cell survival while Ang2 can antagonize these effects, depending on cellular context. To investigate the molecular basis for the opposing effects, we examined the phenotypes of a series of computationally designed protein scaffolds presenting the Ang1 F-domain in a wide range of valencies and geometries. We find two broad phenotypic classes distinguished by the number of presented F-domains: Scaffolds presenting 3 or 4 F-domains have Ang2-like activity, upregulating pFAK and pERK but not pAKT, while scaffolds presenting 6, 8, 12, 30, or 60 F-domains have Ang1-like activity, upregulating pAKT and inducing migration and vascular stability. The scaffolds with 6 or more F-domains display super-agonist activity, producing stronger phenotypes at lower concentrations than Ang1. Tie2 super-agonist nanoparticles reduced blood extravasation and improved blood–brain barrier integrity four days after a controlled cortical impact injury. Synopsis An array of synthetic ligands designed computationally allows to precisely delineate the molecular basis of the Angiopoietin-Tie2 pathway and the role of receptor oligomerization and transmembrane signaling. F-domain valency determines the activation of pAKT, pERK, and pFAK downstream of Tie2. High valency F-domain superagonists promote integrin colocalization and actin rearrangement. F-domain scaffold superagonists promote vascular stabilization in vitro and in vivo. Introduction Ang1 and Ang2 both contain a Tie2-binding carboxy-terminal fibrinogen-like domain (F), a coiled-coil domain, and an amino-terminal super clustering domain for multimerization (Davis et al, 2003). The binding modes of the Ang1 and Ang2 F-domains to Tie2 are very similar (carbon alpha r.m.s.d. of 0.8 Å; pdb id 4k0v (Yu et al, 2013) and 2gy7 (Barton et al, 2006)), and they have nearly identical tertiary structure (Yu et al, 2013) and similar Tie2-binding affinity (Maisonpierre et al, 1997), and thus, it has been speculated that differences in Ang/Tie2 signaling outputs produced by Ang1 and Ang2 are due to differences in their oligomerization state. Crystal structures of the Tie2 extracellular region (ECR) coupled with solution X-ray scattering experiments have shown that the ECR forms a dimer in solution and suggest two different receptor association modes involving the membrane-proximal FNIII domains (Souma et al, 2016; Leppänen et al, 2017; Moore et al, 2017), and it has been proposed that binding of multivalent Ang1-like ligands leads to the generation of a regular lattice of Tie2 receptors from constitutively formed dimers (Leppänen et al, 2017; Moore et al, 2017). EM characterization of the Ang1 and Ang2 oligomerization states suggests greater tendency of the former to self-associate (Davis et al, 2003; Cho et al, 2004; Kim et al, 2005), which has been proposed to stem from a cysteine residue in Ang1 that is not present in Ang2 (Kim et al, 2005). Constructs displaying multiple F-domains, such as Comp-Ang (Cho et al, 2004), have been generated that have Ang1-like activity, but correlating receptor-binding valency to signaling output is not straightforward since both Ang1 or Ang2 (Kim et al, 2005), an Ang1 surrogate (Davis et al, 2003), and Comp-Ang (Cho et al, 2004) can occupy a range of oligomeric states. Similarly, an anti-Ang antibody, ABTAA, potentiates Tie2 activation, but the oligomerization state of the active signaling complexes is unclear (Han et al, 2016). Further complicating the picture, work with chimeric mouse angiopoietins generated by swapping the F-domains of Ang1 and Ang2 suggested the F-domain is the determinant for Tie2 activation, not the oligomeric state (Procopio et al, 1999). Here we generate and characterize computationally designed protein scaffolds presenting the Ang1 F-domain in a wide range of valencies and geometries and show that scaffolds presenting 3 or 4 F-domains have Ang2-like activity, while scaffolds presenting 6, 8, 12, 30, or 60 F-domains have Ang1-like activity. The Tie2 agonist nanoparticles improved blood–brain barrier integrity four days after a controlled cortical impact injury. Results We set out to systematically investigate the molecular basis of the Ang1 and Ang2 signaling differences by generating a series of computationally designed ligands which display F-domains in a wide range of valencies (3, 4, 6, 8, 12, 30, and 60 copies of F-domain) and symmetries (cyclic, tetrahedral, and icosahedral) (Fig 1A). We employed previously designed trimeric (H3, Tet1-A, Tet1-A.2, and Icos1-A), tetrameric (C4 and AkC4), hexameric (H6), octameric (H8) cyclic oligomers with 3, 4, 6, and 8 subunits, and tetrahedral (Tet1 and Tet2) and icosahedral (Icos1 and Icos2) with 12 and 60 copies of F-domains, respectively (Bale et al, 2016; Fallas et al, 2017; Xu et al, 2020). To ensure that the F-domain was identical across all the different presentation formats, we covalently conjugated SpyTagged F-domain to a SpyCatcher domain fused to the N- or C-terminus of the designed scaffold subunits (Fig 1A and Appendix Fig S1A) (Zakeri et al, 2012). To examine the effect of F-domain spacing independent of oligomerization state, we produced four different trimeric configurations in which the distance between the F-domain attachment points ranged from 2.2 nm to 8.0 nm. Protein sequences and details on protein production, purification, conjugation, and characterization are described in the methods and supporting information sections (Appendix Fig S4A–D and Appendix Table S1). The SpyTag–SpyCatcher conjugation efficiency was ˜80% for the tetrameric scaffolds and 90% or greater for all the remaining scaffolds (Fig 1B). The homogeneity of the designed scaffolds is illustrated for F-domain-conjugated Icos1 in Fig 1C—the negative stain electron micrographs show spherical particles of the expected size (˜30nm) and shape with small spikes corresponding to the SpyCatcher domain displayed on the particle surface. Figure 1. Computationally designed scaffolds present Ang1 F-domain in wide range of geometries and valencies A. Designed F-domain scaffold structures. Red and purple dots indicate N- and C-terminal sites of F-domain conjugation, respectively. N is the maximum number of conjugated F-domains, and the dashed lines are the distance between F-domain conjugation sites. Top two rows: Cyclic homo-oligomers. The trimeric nanocage subunits Icos1-A, Tet1-A, and Tet1-A.2 allow precise testing of the effect of valency on signaling independent of geometry as they can be tested as trimers alone or as nanocages upon addition of the other component of these two-component nanoparticles. C4 and AkC4 are tetrameric scaffolds, and H3, H6, and H8 are helical bundle scaffolds with nearly identical geometry but different valency. Third row: Tetrahedral nanoparticle scaffolds Tet1 and Tet2. Fourth row: Icosahedral nanoparticles. Icos1 and Icos2 present sixty copies of the F-domain on the trimer and pentamer subunits, respectively. B. Average conjugation efficience maximum number of F-domains for each scaffold. C. Negative stain electron micrograph of Icos1 scaffold with 60 F-domain conjugation sites. Scale bar is 1,000 nm. D. Serum-starved HUVECs were stimulated with 18 nM of angiopoietins or PBS (vehicle) for 15 min, and phosphorylation levels were analyzed by immunoblotting for pAKT(S473), pERK1/2 (T202/Y204), pFAK(Y397), and β-Actin. Download figure Download PowerPoint Akt phosphorylation correlates with F-domain valency We evaluated the activity of F-domain scaffolds by determining their signaling profiles in Human Umbilical Vein Endothelial Cells (HUVECs). To set a baseline for these studies, serum-starved HUVECs were treated with Ang1 or Ang2 (18nM) for 15 min, and the activation of downstream signaling pathways was analyzed by western blot. Consistent with previous studies, Ang1 treatment increased phosphorylation of AKT (S473), FAK (Y397), and ERK1/2 (T202, Y204), whereas Ang2 only activated FAK and ERK (Fig 1D). The multivalent F-domain displaying designed scaffolds were incubated with HUVEC cells, and protein lysates were analyzed for AKT (S473), ERK1/2 (T202/Y204), FAK (Y397), and Tie2 (Y992) phosphorylations using immunoblotting. We observed a significant increase in Tie2 phosphorylation following F-domain scaffold administration, suggesting that the synthetic ligands act through similar pathways as natural ligands (Appendix Fig S5A and B). The differences in AKT activation between the different valencies were notable. The four trimeric scaffolds failed to activate AKT, as did the tetrameric scaffolds (Fig 2A, D, E, H, I, L, and Appendix Fig S1C and D). In contrast, all the scaffolds displaying six or more SpyCatcher domains strongly activated AKT. To confirm that the F-domain scaffolds signal through Tie2 receptors, we knocked down Tie2 expression in HUVECs using siRNA. This significantly reduced H8-dependent pAKT levels compared with cells stimulated with H8 but without siRNA transfection (Fig 2Q and R), showing that the multivalent F-domain scaffolds act through the Tie2 receptor. The distance between F-domains in the scaffolds had relatively little impact on signaling—the range of distances among the trimeric and tetrameric scaffolds was similar to those of the higher valency scaffolds. Several direct comparisons highlight the importance of F-domain valency over that of geometry. First, the series of scaffolds H3, H6, and H8 have nearly identical geometry (cyclic helical bundles), but the trimeric H3 failed to induce AKT phosphorylation while the hexameric H6 and octameric H8 both strongly induced phosphorylation (Fig 2A and D). The nanoparticles are comprised of two self-assembling components, one of which is conjugated to the F-domain using SpyTag and SpyCatcher as described previously. The F-domain-conjugated nanoparticle components Tet1-A and Icos1-A failed to activate AKT on their own (Fig 2E and I). However, when combined with the partner nanoparticle component which results in a tetrahedral (Tet1 and Tet2) or icosahedral (Icos1 and Icos2) assembly, respectively, both nanoparticles strongly activate AKT. While there was a strong effect of valency on AKT activation, almost all scaffolds upregulated FAK (Fig 2C, G and K) and ERK phosphorylation (Fig 2B, F and J). FAK phosphorylation appears to be somewhat sensitive to geometry as Tet1-A activated FAK phosphorylation more than the other trimeric and tetrameric constructs (Fig 2G). These results suggest that induction of clusters of 3–4 F-domains is sufficient to induce phosphorylation of FAK and ERK, but phosphorylation of AKT requires clustering of six or more F-domains. Figure 2. F-domain valency determines level of activation of AKT phosphorylation A–L. Serum-starved HUVECs were stimulated with designed scaffolds normalized to 18 nM of F-domains for 15 min and analyzed by immunoblotting for pAKT (S473), pERK1/2 (T202/Y204), and pFAK (Y397) phosphorylations. (A, E, I) pAKT, (B, F, J) pERK1/2, and (C, G, K) pFAK phosphorylation levels were quantified and normalized to Icos-cage signaling levels. Appendix Fig S1B shows the detection of two bands expected for pERK1/2. Representative immunoblot gels of cells stimulated with or without scaffolds at 18 nM of F-domain: (D) helical bundles, (H) trimeric and tetrameric homo-oligomers and tetrahedral nanocages, (L) Icosahedral nanocages and trimeric subunit. M–P. Dose–response curves, EC50, and EMAX for pAKT and pERK1/2 activation. The curves, EC50 and EMAX values are calculated using Prism, GraphPad. (N, P) Darker red color indicates lower EC50 and higher EMAX values. Q, R. Quantification (Q) and representative gel (R) for Tie2 knockdown using 40 nM of siRNA before starvation and F-domain scaffold at 18 nM of F-domain or PBS stimulation. S, T. Quantification (S) and representative gel (T) for inhibition of Ang1-dependent pAKT signaling by Ang2, H3, Tet1-A, or Icos1-A; pAKT band intensities were normalized to Ang1-dependent pAKT level). Representative immunoblots for H3, Tet1-A, and Icos1-A competition experiments are in Appendix Fig S1M–O. Quantification of pERK1/2 and pFAK level for the competition assay is in Appendix Fig S1P and Q. Partial valency analysis of icosahedral nanocages is in Appendix Fig S1R–T. Data information: Each point on all graphs represents a biological replicate, n > 3, bars represent the mean ± SEM. One-way ANOVA with Bonferroni test was used for multiple comparisons to calculate P-values. (****) indicates P < 0.0001. P-values are also indicated in Appendix Table S2. Download figure Download PowerPoint To compare the potency of the synthetic ligands to Ang1 and assess the effect of multivalency on cell binding affinity/avidity, we investigated the concentration dependence of AKT activation for a subset of scaffolds alongside Ang1 and Ang2. Serum starved HUVECs were treated with H3, H6, H8, Icos1, or Icos2, and AKT activation was analyzed using immunoblotting (Fig 2M and N and Appendix Fig S1F–L). All scaffolds with six or more copies of F-domain had lower half-maximal (EC50) AKT activation than Ang1; the H8 scaffold exhibited the lowest EC50 (1.4 nM F-domain), 19-fold lower than Ang1 (EC50 = 27 nM F-domain). AKT activation was much less variable with the synthetic scaffolds than Ang1. The higher valency designed scaffolds, on average, produced a higher level of maximum pAKT (Avg. EMAX = 1.3) compared with Ang1 (EMAX = 0.8). No AKT phosphorylation was observed in H3 titrations, even at high concentrations. We similarly compared dose–response curves for activation of ERK1/2 phosphorylation by the designed scaffolds with those of Ang1 and Ang2 (Fig 2O and P). The designed high valency F-domain scaffolds activated pERK1/2 to a greater extent (higher EMAX) than Ang1 and Ang2. H8 was again the most active (EMAX = 1.2) compared with Ang1 (EMAX = 0.7) and Ang2 (EMAX = 0.6). The H8 scaffold also had the lowest EC50 (1.2 nM F-domain). The efficiency and maximum signaling level of H3 and Ang2 to activate pERK1/2 are very similar with EMAX = 0.5 and 0.6, respectively, and EC50 = 12 and 10 nM F-domain, respectively. Overall, the designed, high valency F-domain scaffolds behave as super-agonists that activate the Tie2 pathway more strongly than Ang1 (for pAKT and pERK activations) or Ang2 (for pERK activation); the synthetic ligands are also considerably more robust. Trimeric scaffolds are Tie2-dependent pAKT antagonists Ang2 antagonizes Ang1 activity (Maisonpierre et al, 1997; Yuan et al, 2009), and to explore the mechanism of this antagonism, we investigated the effect of the F-domain scaffolds that do not activate pAKT on Ang1-induced pAKT phosphorylation. Consistent with expectation, in the HUVEC system, there was considerable attenuation of Ang1-dependent pAKT signaling in the presence of higher concentrations of Ang2 (9 nM Ang1 and 45 nM Ang2; Fig 2S and T). We found that the trimeric scaffolds H3 and Tet1-A reduced Ang1-induced pAKT expression at 27 and 45 nM F-domain concentrations (Appendix Fig S1M–O). H3 was more effective than Ang2 in reducing Ang1 signaling at 45 nM F-domain. We conclude that the trimeric scaffolds cannot activate pAKT but can block Ang1-dependent pAKT activation. The simplest explanation for this observation is that the F-domain scaffolds bind to available Tie2 receptors, blocking Ang1-induced activation by direct competition for receptor-binding sites, but we cannot rule out the possibility that this is cell type dependent. Scaffolds with six or more F-domains increase endothelial cell migration in vitro Angiogenesis requires sprouting, proliferation, and migration of endothelial cells (Mazurek et al, 2017), and activation of Tie2 by Ang1 promotes cell migration (Fukuhara et al, 2008; Saharinen et al, 2008; Xue & Hemmings, 2013). To evaluate the capacity of the F-domain scaffolds to promote wound healing, we evaluated them using a commonly used in vitro cell migration assay (Liang et al, 2007). Confluent HUVECs were scratched in the center of the dish to mimic a wound and treated with scaffolds at 18 nM of F-domain for 12 h (Fig 3A). The change in the model wound area was followed to assess the extent of cell migration (Fig 3B). H6, H8, Icos1, and Icos2 increased cell migration (2- to 3-fold) compared with the vehicle (P < 0.0001). H8 and Icos1 increased considerably more migration than Ang1 (P < 0.05). In contrast, H3, Tet1-A, C4, and Icos1-A, like Ang2 (Harfouche & Hussain, 2006), did not increase cell migration (Fig 3C and Appendix Fig S2A). Figure 3. Designed scaffolds fall into Ang1- or Ang2-like classes depending on valency A. Schematic representation of the in vitro scratch cell migration/ wound-healing assay. B. Icos1 at 18 nM F-domain stimulates cell migration relative to PBS/vehicle control. Scale bars are 100 µm. C. Comparison of cell migration induced by Ang1, Ang2, and designed F-domain scaffolds after 12 h of treatment. The change in wound area was normalized to vehicle control. All experiments have at least three biological replicates. (*) is the comparison between F-domain scaffolds vs. Ang1, and (#) is the comparison between F-domain scaffolds vs. Vehicle. (####) indicated P < 0.0001. D. Schematic of in vitro tube formation assay; designed F-domain scaffolds were added at 90 nM F-domain and removed after 24 h; vascular stability was analyzed at 48-h and 72-h timepoints. E. Representative increase in vascular stability by Icos1. The number of nodes (blue), meshes (red), and tubes (green) was quantified using angiogenesis analyzer plug-in in ImageJ. Scale bar is 100 µm. F. Effect of F-domain scaffolds on the vascular stability at 48- and 72-h timepoints was assessed by taking the average of the number of nodes, meshes, and tubes and normalized to vehicle as log2 fold change. All experiments have at least three biological replicates except for Ang2 at 48-h timepoint and Ang2, Tet1, Tet2, akC4, and Icos2 at 72-h timepoint in tube formation assay that have two biological replicates. G. Designed F-domain scaffolds fall into Ang1- and Ang2-like classes. Color indicates highest (1) to lowest (0) pAKT, pERK1/2, and pFAK levels which were column normalized. Cell migration level and vascular stability (48-h timepoint) were also column normalized from highest to lowest. Data information: In C and F, the bars represent the mean ± SEM. One-way ANOVA with Bonferroni test was used for multiple comparisons to calculate P-values. P-values are also indicated in Appendix Table S2. Download figure Download PowerPoint High valency F-domain scaffolds stabilize HUVEC cell tubules We investigated the effect of the F-domain scaffolds on HUVEC tube formation, a simple but well-established in vitro angiogenesis assay (DeCicco-Skinner et al, 2014). HUVECs were plated on 100% Matrigel and incubated with F-domain scaffolds at 90 nM of F-domain for 24 h; thereafter, the scaffolds were removed, and the vascular stability was measured at 48- or 72-h timepoints (Fig 3D). Tubule networks were imaged, and the number of nodes, meshes, and tubes were quantified and averaged to evaluate vascular stability (DeCicco-Skinner et al, 2014) (Fig 3E and Appendix Fig S2B). The higher valency scaffolds H6, H8, Tet2, Icos1, and Icos2 enhanced vascular stability considerably compared with Ang1 at 48 (P < 0.01). H6, Icos1 and Icos2 are significantly better than Ang1 at 72 h (P < 0.01) (Fig 3F); Icos1 stabilizes vascular tubules 3-fold and 4-fold better than vehicle and 2.4-fold and 3-fold better than Ang1 at 48- and 72-h timepoints, respectively (Fig 3F). F-domain scaffolds presenting four or fewer SpyCatcher domains—Icos1-A, H3, Tet1-A, Tet1-A.2, C4, and AkC4— stabilized the tubules at levels less than the higher valency scaffolds but in the range of Ang1 and Ang2. In addition to cell migration and tube formation assays, we also investigated the effect F-domain scaffolds have on cell viability. HUVECs were serum starved for 16 h (Harfouche & Hussain, 2006), with or without H3 or H8 (18 nM of F-domains), and analyzed for vi
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