A novel peptide inhibitor of Dll4-Notch1 signalling and its pro-angiogenic functions

Abstract

Background and Purpose:

The Dll4-Notch1 signalling pathway plays an important role in sprouting angiogenesis, vascular remodelling and arterial or venous specificity. Genetic or pharmacological inhibition of Dll4-Notch1 signalling leads to excessive sprouting angiogenesis. However, transcriptional inhibitors of Dll4-Notch1 signalling have not been described.

Experimental Approach:

We designed a new peptide targeting Notch signalling, referred to as TAT-ANK, and assessed its effects on angiogenesis. In vitro, tube formation and fibrin gel bead assay were carried out, using human umbilical vein endothelial cells (HUVECs). In vivo, Matrigel plug angiogenesis assay, a developmental retinal model and tumour models in mice were used. The mechanisms underlying TAT-ANK activity were investigated by immunochemistry, western blotting, immunoprecipitation, RT-qPCR and luciferase reporter assays.

Key Results:

The amino acid residues 179–191 in the G-protein-coupled receptor-kinase-interacting protein-1 (GIT1-ankyrin domain) are crucial for GIT1 binding to the Notch transcription repressor, RBP-J. We designed the peptide TAT-ANK, based on residues 179–191 in GIT1. TAT-ANK significantly inhibited Dll4 expression and Notch 1 activation in HUVECs by competing with activated Notch1 to bind to RBP-J. The analyses of biological functions showed that TAT-ANK promoted angiogenesis in vitro and in vivo by inhibiting Dll4-Notch1 signalling.

Conclusions and Implications:

We synthesized and investigated the biological actions of TAT-ANK peptide, a new inhibitor of Notch signalling. This peptide will be of significant interest to research on Dll4-Notch1 signalling and to clinicians carrying out clinical trials using Notch signalling inhibitors. Furthermore, our findings will have important conceptual and therapeutic implications for angiogenesis-related diseases.

1 |. INTRODUCTION

The Notch signalling pathway is evolutionarily conserved in both invertebrates and vertebrates. In mammals, there are five DSL ligands, Delta-like 1 (Dll1), Delta-like 3 (Dll3), Delta-like 4 (Dll4), Jagged1 (Jag1) and Jagged2 (Jag2), and four transmembrane receptors, NOTCH1-NOTCH4. Once DSL ligands bind to the receptors, Notch receptors undergo two proteolytic cleavages by the proteases of the ADAM/TACE/kuzbanian family and γ-secretase, followed by release of the Notch intracellular domains (NICDs). NICDs translocate into the nucleus, replace co-repressors and interact with the transcription factor RBP-J/CSL and the coactivator Mastermind (Mam) (Adams & Alitalo, 2007; Phng & Gerhardt, 2009; Roca & Adams, 2007) to regulate Notch target gene expression. Among the DSL ligands, Dll4 has received increasing attention due to the following reasons: (a) restricted expression: Dll4 is confined to endothelial cells (ECs), especially in arteries but not in veins (Benedito et al., 2009; Duarte et al., 2004; Krebs et al., 2000); (b) haplo-insufficient lethality due to defects of sprouting angiogenesis and arteriovenous formation in Dll4 heterozygous mice. To date, Dll4 is the second gene with haplo-insufficient lethality, the first described gene being that for vascular endothelial growth factor (VEGF), indicating the indispensable roles of Dll4 and VEGF in angiogenesis (Benedito et al., 2009, 2012; Duarte et al., 2004; Krebs et al., 2000). (c) Anti-tumour effects: Dll4-Notch1 signalling is widely investigated in the developmental and tumour angiogenesis. Inhibition of the Dll4-Notch1 pathway promotes excessive sprouts and endothelial proliferation in the murine neonatal retina (Benedito et al., 2009, 2012; Hellström et al., 2007; Suchting et al., 2007). Blocking Dll4-Notch1 signalling using a Dll4 neutralizing antibody reduces tumour growth, as neovascularization by tumour vessels is non-functional (Hoey et al., 2009; Ridgway et al., 2006). (d) Self-propagation: unlike the other ligands, Dll4 is cell-contact dependent and could be propagated by activation of Notch signalling in a positive feedback mechanism (Benedito et al., 2009; Caolo et al., 2010; Majumder et al., 2016).

Recently, our group demonstrated that the protein, G-protein-coupled receptor-kinase-interacting protein-1 (GIT1), is a strong endogenous inhibitor of the Dll4-Notch1 signalling pathway. Resembling the effect of Dll4-Notch1 signalling in sprouting angiogenesis, GIT1 knockout mice showed impaired pulmonary and retinal vascular development (Majumder et al., 2016; Pang et al., 2009). Tumour angiogenesis in GIT1 KO mice is also reduced (Majumder et al., 2014). Mechanistically, the ankyrin (ANK) domain of GIT1 is responsible for repressing Notch signalling through competition with the NICD to bind the RBP-J transcription factor (Majumder et al., 2016).

Small peptides targeting protein-protein interactions (PPIs) have made a very significant expansion in drug discovery by increasing the number of new therapeutic targets, previously regarded as ‘undruggable’ (Bakail & Ochsenbein, 2016). Given the critical role of Dll4-Notch signalling in angiogenesis, we designed a peptide based on the sequence of the ANK domain in GIT1, to inhibit Dll4-Notch signalling through binding to RBP-J by competition with NICD. To facilitate the delivery of this peptide, we fused ANK peptides with a cell-penetrating peptide, TAT-PTD (trans-acting activator of transcription [TAT] protein transduction domain [PTD]) (Caron et al., 2001; Flinterman et al., 2009; Gratton et al., 2003) and named this fused peptide as TAT-ANK. In the current study, we have investigated the role of TAT-ANK in sprouting angiogenesis and related mechanisms. Our results showed that the TAT-ANK peptide significantly promoted angiogenesis in vitro and in vivo, by inhibiting Dll4 and Notch1 activation in ECs.

2 |. METHODS

2.1 |. Peptide design

The ANK peptide (Figure 1a) was designed by the website ‘Blocking’, developed by the Fred Hutchinson Cancer Research Center (http://130.88.97.239/bioactivity/newblocksrch.html), based on the peptide sequence of the ANK domain (amino acids 132–228, conserved across various species) in GIT. Unfortunately, the website is no longer maintained. The scramble peptide for ANK (Sc-ANK) was generated by Scramble-Peptide Library (MIMOTOPS, http://www.mimotopes.com/peptideLibraryScreening.asp?id=97). The peptides were dissolved in 50% DMSO and 50% sterile H₂O for our experiments.

FIGURE 1.

The design of TAT-ANK polypeptide. (a) Amino acids sequence alignment (designed by blocking website) of human GIT1 amino acids 179–191, with homologues in various species. (b) Immunoprecipitation (IP) was performed after 24 h post transfection with GFP-ANK or GFP-ANK (Δ179–191) deletion mutant in HEK293 cells to determine the binding of GFP-ANKs and RBP-J. (c) Sequence of GIT1-ANK amino acids 179–191, TAT-ANK peptide (TAT-ANK), TAT-scrambled peptide (TAT-Sc) and the TAT domain. (d) Human umbilical vein endothelial cells (HUVECs) were treated with biotin-conjugated peptides (30 μM, 6 h). Streptavidin-conjugated beads were added into cell lysates for 2 h, followed by immunoblot with anti RPB-J antibody. b-TAT-Sc, biotin-TAT-Sc; b-TAT-ANK, biotin-TAT-ANK. (e) After 6-h incubation with TAT-Sc or TAT-ANK, HUVECs were harvested for immunoprecipitation of NICD with RBP-J. WCL, whole cell lysis; IB, immunoblotting antibody. Similar results were observed in five independent experiments (n = 5)

2.2 |. Animal experiments

All animal care and experimental procedures complied with the NIH guidelines (Guide for the Care and Use of Laboratory Animals) and were approved by the Animal Care and Use Committees of University of Rochester. Animal studies are reported in compliance with the ARRIVE guidelines (Percie du Sert et al., 2020) and with the recommendations made by the British Journal of Pharmacology (Lilley et al., 2020).

Six- to eight-week-old C57BL/6 male mice (Jackson Labs, Bar Harbor, ME) were used for Matrigel plug assay. Littermates of C57BL/6 at post-natal day 5 (P5) were randomly divided into two groups for intravitreal injections of 0.5 μl of 30-μM Sc-ANK or ANK peptide and retinas were harvested at P6 for further experiments. Eight-week-old SCID female mice (Charles River, Wilmington, MA) were used for studying tumour angiogenesis. All mice used in the study were housed in a specific pathogen-free (SPF) facility at the University of Rochester or Tongji University. Mice (no more than four per cage) were accommodated in ventilated cages with food and water ad libitum under a 12-h light/dark cycle (lights on from 6 AM to 6 PM). The temperature and relative humidity of the animal room ranged from 64°F to 79°F and 30% to 70%, respectively. For murine anaesthesia, 2% isoflurane was used and mice were killed by overdose of sodium pentobarbital (100 mg·kg⁻¹, i.p.).

2.3 |. Intravitreal injection

Neonatal (P5) C57BL/6 mice from the same litters were anaesthetised with 2% isoflurane. TAT-Sc (0.5 μl 30 μM) or TAT-ANK (0.5 μl, 30 μM) was injected intravitreally (IVT) into the eyes using a 10-μl Hamilton syringe and a 36-gauge needle, under a dissecting microscope. Retinas were collected at P6 for whole-mount staining or RT-PCR analysis.

2.4 |. In vivo proliferation assay

To analyse the in vivo effect of TAT-ANK on the proliferation of ECs, we assayed the incorporation of 5-ethynyl-2′-deoxyuridine (EdU). EdU in PBS (30 μl of 2 mg·ml⁻¹) was injected i.p. in P6 murine pups and the pups killed 2 h later. Eyes were fixed and dissected retinas were blocked, as described above. Click-iT EdU Imaging Kits (Invitrogen, C10339) were used for labelling EdU nucleotides in retinal vessels together with IB4 staining.

2.5 |. Matrigel plug angiogenesis assay

Matrigel (500 μl Cat # 356234; BD Bioscience, Franklin Lakes, NJ) containing 50-ng·ml⁻¹ VEGF (Cat # 293-VE-050; R&D Systems, Minneapolis, MN) with SC-ANK (30 μM) was injected subcutaneously on the left side of the back of 6- to 8-week-old C57BL/6 mice, whereas Matrigel with ANK (30 μM) was injected on the right side of the back. Seven days after injections, Matrigel plugs were collected and divided into two parts. Half of the Matrigel plugs were fixed with 4% PFA, paraffin embedded and sectioned at 5 μm, then processed for haematoxylin and eosin (H&E) and immunofluorescent staining. The other halves were weighed and snap frozen in liquid nitrogen and stored in −80°C for further RT-qPCR analysis. For immunostaining of the Matrigel plug, paraffin sections were deparaffinized and rehydrated, followed by antigen retrieval in sodium citrate buffer (pH = 6) and stained with the endothelial nuclear marker ERG (Cat #ab92513 Abcam,Waltham, MA). Alexa Fluor 488-coupled secondary antibody (1:500; Jackson Immuno Research, West Grove, PA) was used, followed by mounting using aqueous mounting solution (Cat #F4680, Sigma, St Louis, MO) and analysis using an Olympus IX83 microscope. For analysing murine mRNA of the Matrigel plug, the previously reported protocol was performed with minor modification (Coltrini et al., 2013). Briefly, the weighed frozen Matrigel plug was homogenized in liquid nitrogen by a cylindrical pestle made of glass, followed by addition of the appropriate TRIzol Reagent (Invitrogen) containing 1 × 10⁴ human pulmonary fibroblasts (HPFs) per milligram of Matrigel plug. The combined murine and human mRNA was extracted according to the manufacture’s instruction (Invitrogen). The murine transcript CD31 or CDH5 was normalized to the human-specific housekeeping gene hypoxanthine guanine phosphoribosyltransferase (HPRT).

2.6 |. Tumour models

Eight-week-old SCID female mice were purchased from Charles River. One million of human colon carcinoma cells (Colo205) or murine lymphoma cells (EL4) mixed with 50% Matrigel (100 μl, BD bioscience, 356234) were subcutaneously injected into the right posterior flank of mice. Once tumour volume reached about 200 mm³, mice were randomly grouped for subcutaneous injection of TAT-Sc or TAT-ANK (10 mg·kg⁻¹). The peptides were injected every other day for EL4 tumour bearing mice. For Colo205 tumour model, we gave peptide injections once every 3 days. Tumours were harvested and processed for further experiments at the times indicated. Tumour volumes (V) were analysed by measuring the length (L) and width (W), V = L × W²/2. For investigating vessel patency in tumours, Lycopersicon esculentum (tomato) Lectin (LEL, TL)-DyLight® 488 (150 μg in 150-μl PBS; Vector, DL-1174-1) was injected via the tail vein and allowed to circulate 10 min before mice were killed. Tumours were fixed in 4% PFA and processed for frozen sections. After staining with CD31 (EC marker), tumour vessels were visualized by confocal microscopy for analysing vessel perfusion.

2.7 |. Whole-mount immunostaining of retinal vasculature

Retinal whole-mount staining was carried out as described earlier (Majumder et al., 2016; Zhu et al., 2018). Briefly, eyes were fixed for 2 h at 4°C immersed in 4% PFA in PBS. Dissected retinas were blocked with 1% bovine serum albumin (BSA), 0.3% Triton X-100 dissolved in PBS for 1 h at room temperature. Retinas were incubated overnight at 4°C in the blocking buffer with biotinylated isolectin B4 (IB4) (1:100; Vector Labs, B-1205) and following primary antibodies: rabbit anti-ERG (1:100; ab92513, Abcam), rabbit anti-collagen IV (1:100; Abcam, ab6585), rabbit anti-desmin (1:100; Abcam, ab15200), mouse anti-aSMA-Cy3 (1:500; Sigma, C6198), goat anti-Dll4 (1:100; R&D Systems, AF1389) and goat anti-Jag1 (1:100; Sigma, J4127). After wash in PBS with three times, appropriate Alexa Fluor streptavidin-conjugated (1:200; Invitrogen) and Alexa Fluor-coupled secondary antibodies (1:500; Jackson Immuno Research) were incubated in the blocking buffer at room temperature for 2 h. Retinas were further washed with PBS for three times, followed by mounting and imaging by confocal microscopy (Zeiss LSM 710). The immunofluorescence intensity was measured with the ImageJ software.

2.8 |. Active NICD staining in retinal vessels

Highly sensitive immunostaining for the Notch1 active form NICD (CST; Val 1744, #2421) has been described earlier (Del Monte et al., 2007). Briefly, after 2-h fixation with 4% PFA in PBS, retinas were dissected and refixed overnight in 100% Methanol at −20°C. Retinas were briefly washed with PBS, followed by antigen retrieving in 95°C water bath for 15 min in sodium citrate (10 mM, pH 6.0). After cooling down to room temperature, retinas were twice washed in distilled water and PBS, 5 min per wash. H₂O₂ (1% in 100% methanol) was used to quench endogenous peroxidase activity by incubating with retinas for 40 min at room temperature. Retinas were twice washed in PBS and Triton X-100/PBS for 5 min each. After blocking for 1 h in Histoblock solution (1% BSA, 20-mM Mgcl2, 0.3% Triton X-100, 5% goat serum in PBS), retinas were incubated with NICD (1:500; CST, #2421) and FITC-conjugated IB4 (20 μg·ml⁻¹; Sigma, L-2140) for overnight at 4°C in Histoblock buffer. Next, retinas were washed in PBS and 0.3% Triton X-100/PBS for 5 min twice. Retinas were incubated with biotinylated goat anti-rabbit antibody (1:100; Vector, BA-1000-1.5) for 1 h in 1% BSA/PBS at room temperature. After two washes in PBS and 0.3% Triton X-100/PBS for 5 min each, retinas were first amplified by avidin/biotin-HRP (ABC kit; Vector, PK-6100) for 1 h. Retinas were washed twice in PBS and 0.3% Triton X-100/PBS, 5 min per wash. TSA-Cy3 (1:50; Perkin Elmer, NEL744001KT) was incubated with retinas for magnifying the NICD immunological signal. After brief wash in PBS, retinas were mounted and analysed as described above.

2.9 |. Cell culture

Human umbilical vein endothelial cells (HUVECs) and HPFs were purchased from Sciencell (Carlsbad, CA). HUVECs were grown in ECM (Sciencell, # 1001), and the HPFs were cultured in FM (Sciencell, #2301). For spheroid assays, HUVECs were cultured in the EGM-2 complete medium supplemented with the basal medium (Cat # CC-3156, Lonza, Rockland, ME) and the supplements (Lonza, CC-4147). HEK293 cells (CRL. 1573™; ATCC®, Manassas, VA), Colo205 cells (ATCC® CCL-222™) and EL4 cells (ATCC® TIB-39) were grown in DMEM (Gibco) containing 10% fetal bovine serum (FBS), 1% penicillin and streptomycin. To activate Notch signalling, anti-Fc (1 îg·mr⁻¹) or Dll4 ligand (R&D Systems, 1389-D4; 1 îg-mr⁻¹) was precoated in a six-well plate for 4 h, followed by plating 5 × 10⁵ HUVECs per well for 12 h. Next, HUVECs were treated with 30-μM TAT-Sc or TAT-ANK for 12 h and then harvested for further analysis. To inhibit Notch signalling, DMSO or 10-μM DAPT was incubated with HUVECs for 24 h, followed by 6-h treatment of 30-μM TAT-Sc or TAT-ANK for subjecting to RT-qPCR.

2.10 |. Cell viability and cytotoxicity assay

HUVECs at passage 3 or Colo205 tumour cells were seeded in a 96-well plate (10,000 cells per well) overnight, followed by treatment of indicated concentration of TAT-Sc or TAT-ANK for 12 h. Next, LDH cytotoxicity Assay (Sigma, 96992) and CellTiter-Glo® Luminescent Cell Viability Assay (Cat # G7570,Promega, Madison, WI) were performed according to the manufacturer’s protocol.

2.11 |. Tube formation assay

Details of the tube formation assay have been described previously (Arnaoutova & Kleinman, 2010). Briefly, HUVECs at Passage 3 were resuspended at a concentration of 1.5 × 10⁵·ml⁻¹. Then, 100-μl cell suspensions contained 30-μM TAT-Sc or TAT-ANK were added to the Matrigel (Corning, 354230) coated corresponding well with VEGF (50 ng·ml⁻¹) or without VEGF in the 96-well plate. After 18 h, networks were imaged using an inverted microscope, and ImageJ software was used to quantify tube lengths.

2.12 |. Spheroid sprouting assay

The HUVEC fibrin gel bead assay was performed as described (Nakatsu & Hughes, 2008). Briefly, EGM-2 complete medium was used to culture HUVECs 1 day before coating onto Cytodex 3 beads (Cat # C3275, Amersham Pharmacia Biotech, Erie, PA). About 400 HUVECs per bead were added to the FACS tube and were inverted to mix every 20 min for 4 h, followed by incubating overnight in T25 flasks. The next day, 500 cell-coated beads per well were mixed with fibrin gel composed of 2-μg·ml⁻¹ fibrinogen (Sigma, F3879), 22-μ·ml⁻¹ aprotinin (Sigma, A4529) and 0.625-U·ml⁻¹ thrombin (Sigma, T6884). After clotting, 4000 HPFs in 1-ml EGM-2 complete medium containing the TAT-Sc or TAT-ANK were added to each fibrin gel. Medium containing SC-ANK or ANK was changed every day. The inverted microscope and ImageJ software were used to photograph and quantify sprouts. For isolating the mRNA of HUVECs in the fibrin gel, 3- and 4-mg·ml⁻¹ trypsin (Sigma, T1426) were used to remove the pulmonary fibroblasts and fibrin gel, respectively. After complete digestion, the cell pellets from HUVECs from 12-well plate were resuspended in the TRIzol for RNA extraction.

2.13 |. Plasmids and mutation generation

The sequence of GIT1-ANK domain and its mutant were synthesized by MAP Biotech (Shanghai, China), followed by cloning to pEGFP-C2 (Cat #6083-1, Clontech, Mountain View, CA). HEK293 cells were used for plasmids transfection by FuGENE®HD (Promega). Immunoprecipitation (IP) and immunoblotting analysis were performed 24 h after transfection.

2.14 |. Luciferase reporter assay

HEK293 cells in a 24-well plate were cotransfected with 100 ng per well Hey1 luciferase reporter gene, 300 ng per well pCMV-NICD (generous gift from Dr. Eileen M. Redmond, Department of Surgery, University of Rochester School of Medicine and Dentistry) and 100 ng per well CMV-β-galactosidase (β-gal) using FuGENE® HD (Promega). After 12-h transfection, cells were treated with 30-μM TAT-Sc or TAT-ANK for another 12 h. A luciferase assay system with reporter lysis buffer (Promega, E4030) was used to detect the luciferase activity. β-Gal activity was measured by β-gal enzyme assay system with reporter lysis buffer (Promega, E2000).

2.15 |. Western blotting, immunoprecipitation and RT-qPCR

HUVECs were lysed in cell lysis buffer (Cat # 9803,Cell Signaling Technology, Danvers,MA) supplemented with protease inhibitors (Sigma, P2714). Protein concentration was quantified by the BCA assay prior to immunoblotting. The following antibodies were used for immunoblotting: Hes1 (CST, 11988), Dll4 (CST, 2589), NICD (CST, 4147S), Jag1 (CST, 28H8), RBP-J (Santa Cruz, sc-27118), RBP-J (CST, 5313S), GFP (CST, 2955S), VEGFR-2 (CST, 9698S), NRP-1(Abcam, ab81321), P44/42 MAPK (CST, 4695S), phospho-P44/42 MAPK (CST, 4370S), phospho-VEGFR2(CST, 2478S), VEGFR2(CST, 2479S), β-actin (CST, 58169S) and tubulin (Sigma, T5168).

For immunoprecipitation (IP), cells were lysed in IP buffer (50-mM HEPES, 150-mM KCl, 1% NP-40, 0.5-mM DTT, 2-mM EDTA, 1-mM NaF) with protease inhibitors (Roche, 04693159001). The lysates were centrifuged at 12,000 g for 10 min at 4°C to remove cell debris. The primary antibody GFP (GeneTex, GTX113617, 1:100) or RBP-J (CST, 5313S) was added to the cell lysate for 2 h on the rotator at 4°C, followed by incubating an additional 2 h with the prewashed goat anti-rabbit IgG magnetic beads (NEB, S1432S). For detecting protein bound to biotin-conjugated peptides, streptavidin magnetic beads (BIOTNT, L-1012) were used. The immunocomplex-conjugated beads were washed by IP buffer four times, heated at 95°C for 5 min with 1× Laemmli buffer, then analysed by SDS-PAGE.

Total RNA of HUVECs, Matrigel plugs, retinas or tumour tissues was isolated by TRIzol Reagent (Invitrogen) and 1 μg per reaction was transcribed into cDNA by a PrimeScript RT Reagent Kit (Takara, Mountain View, CA). Quantitative PCR was conducted with the SYBR Premix Ex Taq Kit (Takara). The primer sequences can be found in Table S1. Relative gene expression was normalized to the endogenous housekeeping gene HPRT.

2.16 |. Data and statistical analysis

The data and statistical analysis in this study comply with the recommendations on experimental design and analysis in the British Journal of Pharmacology (Curtis et al., 2018). All animal experiments were designed with equal group sizes, randomization and blinding. Group data were generated by five or more independent experiments and analysed by SPSS version 20.0 (SPSS Inc). Values are presented as mean ± SEM, unless stated otherwise. Unpaired Student’s t test was used for statistical analysis between two groups. One-way ANOVA was applied for analysing statistical significance by appropriate post hoc tests if more than two groups were compared. P < 0.05 was considered significant.

2.17 |. Materials

The Sc-ANK and ANK peptides or biotin-conjugated Sc-ANK and ANK fused with TAT (Figure 1c) were synthesized and purified by reverse-phase chromatography (HPLC) by Biomatik Corporation (Kitchener, Canada). DAPT (D5042) was supplied by Sigma.

2.18 |. Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org and are permanently archived in the Concise Guide to PHARMACOLOGY 2021/22 (Alexander, Fabbro et al., 2021a,b; Alexander, Kelly et al., 2021).

3 |. RESULTS

3.1 |. The characterization of ANK peptide derived from the GIT1-ankyrin domain

Our earlier experiments demonstrated that the protein GIT1 competed with NICD to bind to RBP-J, through its ANK domain (in human GIT1, that comprises amino acids 132–228) and thus inhibited Dll4-Notch1 signalling (Majumder et al., 2016). Given the critical role of Dll4-Notch signalling in vascular development and diseases and based on the sequence of the GIT1 ANK domain, we designed a blocking peptide to inhibit Notch signalling. The peptide was designed based on the sequence of GIT1 ANK domain to bind to RBP-J and prevent association of NICD and RBP-J. The ANK peptide (human GIT1 amino acids 179–191) and its scrambled control were designed by the ‘Blocking’ website. The sequence of ANK peptide is conserved across a number of species (Figure 1a). To determine the role of the polypeptide aa179–191 in binding of GIT1-ANK with RBP-J, we transfected HEK293 cells with GFP tagged-GIT1-ANK (aa132–228) or GIT1-ΔANK (the deletion mutation of ANK, Δ179–191). As we expected, ANK showed robust interaction with RBP-J, whereas the association was markedly reduced by deletion of amino acids 179–191 (Figure 1b), indicating that this sequence of 13 amino acids (179–191; ANK peptide) is essential for the ANK domain to bind to RBP-J. As the ANK peptide comprises 13 amino acids, it cannot enter living cells. We therefore fused the known cell-penetrating polypeptide, TAT, with both scramble and ANK peptide. We synthesized the penetrating ANK peptide (TAT-ANK) which consisted of TAT-PTD attached to the 13 amino acids (179–191) of the GIT1-ANK domain and the penetrating scrambled peptide (TAT-Sc) with the same 13 amino acids randomly arranged and fused to TAT-PTD (Figure 1c). To determine the interaction of TAT-ANK and RPB-J, biotin-conjugated TAT-ANK and TAT-sc were synthesized and were incubated with HUVECs for 6 h. Then immunoprecipitation was performed using streptavidin beads. As we expected, we observed RPB-J bound to biotin-conjugated TAT-ANK (Figure 1d). Meanwhile, we treated HUVECs with TAT-ANK for 6 h and detected the association of RPB-J with N1-ICD and found that TAT-ANK reduced the binding of RPB-J with N1-ICD (Figure 1e). Our results indicated that TAT-ANK could be a potent inhibitor of Notch signalling by competing with N1-ICD to bind to RBP-J.

3.2 |. TAT-ANK inhibited Dll4-Notch1 signalling in ECs

To determine the effect of TAT-ANK on Notch signalling, we chose to use HUVECs, aa they are frequently used primary endothelial cells. Both Dll4 and Notch1 are enriched and restricted in ECs (Benedito & Hellstrom, 2013; Krebs et al., 2000). To determine the safety range of TAT-ANK, we first performed cytotoxicity and cell viability assay after exposure to TAT-ANK in different concentrations (0, 10, 30, 50, 70 and 90 μM, the maximum concentration of TAT-ANK is 90 μM due to the solubility). Incubation with TAT-ANK, even at the highest concentration (90 μM), had no effects on cell viability (Figure S1A,B), suggesting that the safety range of TAT-ANK was, at least, up to 90 μM.

To optimize the doses of peptides on Notch signalling, we treated HUVECs with a lower concentration of TAT-Sc or TAT-ANK peptide (0, 30 and 50 μM) for 6 h. TAT-Sc showed no effects on Dll4 or Hey1 mRNA expression, compared with untreated HUVECs, whereas the TAT-ANK peptide induced a dose-dependent decrease of Dll4 and Hey1 transcripts, with maximal inhibition achieved at 30 μM (Figure 2a,b). Next, we treated HUVECs with 30-μM peptides for different times and observed marked inhibition of Dll4 and Hey1 transcripts (3.2 ± 0.67- and 1.6 ± 0.1-fold decreased), starting at 6 h, with this effect sustained for longer treatments (12, 24 and 48 h) (Figure 2c,d). Consistent with these findings, TAT-Sc had no effects on Dll4 or NICD expression in HUVECs, whereas incubation with TAT-ANK for 12, 24 and 48 h inhibited Dll4 protein expression by 31% ± 5.2%, 79% ± 5.3% and 38% ± 1.7%, respectively, and Notch1 activation (detected by specific antibody for N1-ICD) by 41% ± 8.3%, 83% ± 3.7% and 33% ± 4.9%, compared with the treatment of TAT-Sc (Figure 2e–j). Moreover, TAT-ANK also inhibited Dll4 ligand-induced Notch1 activation in HUVECs (Figure S2A,B).

FIGURE 2.

TAT-ANK inhibited Dll4-Notch1 signalling. (a,b) Human umbilical vein endothelial cells (HUVECs) were treated using different concentrations of peptide, followed by analysis with RT-qPCR for Dll4 and Hey1 transcripts. Untreated HUVECs were marked as control (Ctrl) (n = 6). (c,d) Dll4 and Hey1 transcripts of HUVECs were analysed by RT-qPCR at indicated time points after addition of TAT-Sc or TAT-ANK (n = 6). (e–j) HUVECs were treated by Ctrl (no treatment), 30-μM TAT-Sc or TAT-ANK for 12, 24, and 48 h. Dll4 and NICD were detected by Western blots (n = 6). (k) HEK293 cells were cotransfected with NICD, β-galactosidase (β-gal) and Hey1 luciferase reporter gene for 12 h, followed by addition of TAT-Sc or TAT-ANK for another 12 h. Quantification of the luciferase and β-gal enzyme activities was performed (mean ± SEM; n = 6). All data presented are means ± SEM. *P < 0.05, significantly different as indicated

To define the role of these peptides on transcriptional activity of Notch target gene Hey1, we cotransfected HEK293 cells with Hey1-luciferase (Hey1-luc) reporter construct, pCMV-NICD and CMV-β-gal. Overexpression of NICD increased Heyl-luc activity by 22.2 ± 1.7-fold; TAT-Sc had no effect on NICD-induced Hey1 activation. In contrast, TAT-ANK significantly reduced the luciferase activity of Hey1 (Figure 2k). We also detected expression of another important Notch1 ligand, Jag1, after incubation with 30-μM TAT-ANK for 24 h, and found no significant changes in HUVECs (Figure S3A,B). Taken together, our findings demonstrated that TAT-ANK could robustly inhibit Dll4-Notch1 signalling in HUVECs.

3.3 |. TAT-ANK promoted angiogenesis in vitro

It is well established that inhibition of Dll4-Notch1 signalling promotes sprouting angiogenesis (Benedito et al., 2012; Hellström et al., 2007; Lobov et al., 2007). Therefore, we performed the tube formation assay to determine the role of TAT-ANK in angiogenesis. Interestingly, TAT-ANK significantly increased the capillary-like tubes in Matrigel containing VEGF (50 ng·ml⁻¹) compared with TAT-Sc, measured by the relative total tube length (Figure 3a,b). Similar results were observed even without VEGF stimulation (Figure S4A,B). HUVECs in fibrin gels co-cultured with HPFs were used to generate endothelial sprouts for in vitro sprouting angiogenesis (Arnaoutova & Kleinman, 2010; Newman et al., 2011). Addition of the TAT-ANK to the culture medium massively enhanced sprouts, as quantified by cumulative sprout length compared with TAT-Sc peptide-treated cells (Figure 3c,d). To confirm the role of TAT-ANK on Notch signalling, the transcripts of Dll4 and Hey1 of HUVECs in the fibrin gels were assayed and showed significant decreases in the presence of TAT-ANK (Figure 3e). These results support the pro-angiogenic effect of TAT-ANK in vitro.

FIGURE 3.

The effects of TAT-ANK on angiogenesis in vitro. (a) Human umbilical vein endothelial cells (HUVECs) were embedded in Matrigel and treated with 50-ng·nl⁻¹ VEGF together with 30-μM TAT-Sc or TAT-ANK in serum-depleted medium. After 18 h, representative images of tube formation were shown. (b) Quantification data of relative total tube length (data from five independent experiments with three replicates). (c) Cytodex beads with absorbed HUVECs were embedded into the fibrin gels, followed by coculture with human pulmonary fibroblasts on top gels. Sprouting was present after 5 days when cultured with 30-μM TAT-Sc or TAT-ANK. (d) Relative sprout length was quantified (data from five independent experiments with total 30 beads). (e) Dll4 and Hey1 transcripts of HUVECs in the fibrin gels were analysed by RT-qPCR (n = 6). All data presented are means ± SEM. *P < 0.05, significantly different as indicated

3.4 |. TAT-ANK enhanced angiogenesis in Matrigel plugs

To determine the role of TAT-ANK on angiogenesis in vivo, Matrigel plug assays were performed. HE staining showed that administration of TAT-ANK in gel plugs led to more capillaries compared with that in gel plugs containing TAT-Sc (Figure 4a). Moreover, immunostaining demonstrated that TAT-ANK increased capillary density by 81.5% ± 8.6% in the plug, compared with that induced by TAT-Sc, indicated by enhanced numbers of cells positively stainined for ERG, the EC nuclear marker (Figure 4b,c). To further confirm the findings from immunostaining, we analysed the transcripts of endothelial markers including CD31 and CDH5 genes in plugs, by an improved RT-qPCR method reported by Presta’s group (Coltrini et al., 2013). Indeed, compared with TAT-Sc plugs, we observed seven- to eight-fold increases of CD31 and CDH5 transcripts in the TAT-ANK plugs normalized by human-specific HPRT mRNA levels (Figure 4d). Furthermore, murine Dll4 and Hey1 transcripts in the TAT-ANK plugs normalized with murine endothelial marker CDH5 were also significantly decreased (Figure 4e). These findings suggested TAT-ANK promotes angiogenesis in vivo by inhibiting the Dll4-Notch1 signalling pathway.

FIGURE 4.

TAT-ANK promotes angiogenesis in Matrigel plugs. (a) Matrigel plugs contained 50-ng·ml⁻¹ VEGF together with 30-μM TAT-Sc or TAT-ANK were subcutaneously injected into back of mice. After 7 days, Matrigel plugs were harvested for paraffin sectioning, followed by staining with haematoxylin and eosin (H&E). (b,c) Double immunostaining of endothelial cell (EC) nuclear marker ERG (green) and DAPI (blue) was performed. Vessel density was measured by counting vessel numbers per field after immunohistochemistry with ERG antibody. Quantification was performed on three different fields of the largest Matrigel cross-sections (n = 6). D. Matrigel plug was weighed and homogenized in TRIzol together with 1 × 10⁴ human pulmonary fibroblasts per milligram of plug. Then, murine transcripts CD31 and CDH5 were measured and normalized to human HPRT (n = 8). E. Murine Dll4 and Hey1 transcripts in the Matrigel plugs were normalized to murine CDH5 by RT-qPCR analysis (n = 6). All data presented are means ± SEM. *P < 0.05, significantly different from TAT-Sc

3.5 |. TAT-ANK increased sprouting angiogenesis in mouse retinal vasculature

Mouse retinal vasculature is a well-recognized and commonly used animal model for sprouting angiogenesis (Nowak-Sliwinska et al., 2018; Pitulescu et al., 2010). To further confirm the proangiogenic effect of TAT-ANK in vivo, we intravitreally injected 0.5 μl (30 μM) TAT-Sc or TAT-ANK at P5 and harvested the eyes at P6. The retinal vasculature was visualized by IB4 (a dye for rodent ECs) staining. The TAT-ANK-treated mice showed increases by 44.8% ± 6.7% in EC coverage, 38.9% ± 4.8% in branch points and 89.0% ± 9.6% in filopodia number (Figure 5a–d), respectively, compared with TAT-Sc-treated mice. ERG (EC nuclei maker) and IB4 staining demonstrated that TAT-ANK enriched ECs in the angiogenic front (Figure 5e,g; increased by 38.6% ± 5.0%). The incorporation of EdU in vivo showed that the proliferating ECs (EdU+/IB4+) in the angiogenic front were enhanced by 42.6% ± 8.2% after treatment of TAT-ANK (Figure 5f,h). The proliferating non-ECs (EdU+/IB4−) were not affected (Figure 5f,i). During angiogenesis, newly formed vasculature undergoes regression to adapt to the microenvironment (Franco et al., 2015). Thus, we performed double staining of IB4 (EC marker) and collagen IV (matrix secreted by EC), and TAT-ANK injection had no significant effects on vessel regression indicated by the number of empty sleeves (IB4+ collagen IV−) compared with TAT-Sc group (Figure S5A–C). Because it is well known that mural cells (vSMCs and pericytes) are responsible for vessel stabilization and maturation, we also determined the role of TAT-ANK in vSMC coverage by α-smooth actin (α-SMA) and IB4 staining and found that TAT-ANK reduced vSMC coverage in central plexus (Figure S6A,C). We also observed that desmin-positive pericytes were decreased in angiogenic fronts but not in middle plexus (Figure S6B,D,E). These findings are consistent with previous studies showing that inhibition of Notch signalling impaired pericyte coverage (Nadeem et al., 2020; Ridgway et al., 2006).

FIGURE 5.

The role of TAT-ANK on retinal angiogenesis. (a–h) P6 retinas were harvested after TAT-Sc or TAT-ANK treatment for 24 h. (a) The upper panel shows overview retinas at low magnification, and the white boxed areas in the upper panel are magnified and indicated for retinal angiogenic fronts at high magnification (lower panel). (b,c) Quantification of endothelial cell (EC) area, branch points and numbers of angiogenic filopodia in the TAT-Sc- or ATAT-ANK-treated retinas. (e,f) Double immunostaining of IB4 (green) and ERG (red) in the TAT-ANK-injected retinas compared with the controls. (g,h) Quantification of ERG+/IB4, EDU+/IB4+ and EDU+/IB4− cells (n = 6). All data presented are means ± SEM. *P < 0.05, significantly different from TAT-Sc

To confirm the role of TAT-ANK on Dll4-Notch1 signalling, we measured Dll4 and cleaved Notch1 (NICD Val1744, activated Notch1) by immunofluorescent staining or signalling amplify system, as we have already published (Zhu et al., 2018). In the TAT-Sc-injected group, Dll4 was expressed in tip cells and arteries (Figure 6a), consistent with the others and our previous reports (Benedito et al., 2012; Majumder et al., 2014; Pitulescu et al., 2017; Zhu et al., 2018). NICD mainly localized in arteries and angiogenic plexus but not in veins (Figure 6b). TAT-ANK treatment substantially reduced Dll4 and NICD levels in both angiogenic and central plexus (Figure 6a–f). The transcripts of Notch1 target genes like Hey1, Hes1 and Dll4 were also significantly reduced by 21.6% ± 7.0%, 27.7% ± 6.5% and 22.7% ± 4.9%, respectively (Figure S7), 24h after TAT-ANK injection. However, Jagged1 (Jag1, an important Notch ligand in ECs, has opposing effects on Dll4-Notch1 pathway) protein expression in retinas was comparable between TAT-ANK or TAT-Sc peptide administration for 24 h (Figure S8A–C). Therefore, these results implied that TAT-ANK promoted angiogenesis by repressing Dll4-Notch1 signalling pathway.

FIGURE 6.

TAT-ANK reduced Dll4-Notch1 signalling in retinal vessels. (a–f) Double immunostaining of IB4 (green) and Dll4 (red) or NICD (red) in P6 retinas treated by TAT-Sc or TAT-ANK for 24 h. A. Confocal images of IB4 and Dll4 co-staining in the angiogenic front and central plexus of the scrambled or TAT-ANK-injected retinas. (b) Improved double staining of NICD and IB4 in the retinal vessels treated by the indicated peptide. (c–f) Quantification of Dll4 or NICD signal in IB4+ angiogenic front and central plexus in retinas after control or TAT-ANK treatment (n = 6). All data presented are means ± SEM. *P < 0.05, significantly different from TAT-Sc

3.6 |. TAT-ANK repressed tumour growth and vessel patency

Dll4-Notch1 signalling plays an important role on tumour angiogenesis (McKeage et al., 2018; Noguera-Troise et al., 2006; Ridgway et al., 2006). Repressing Dll4-Notch1 signalling shows marked inhibition of tumour growth in various tumour models (Noguera-Troise et al., 2006; Ridgway et al., 2006). To exclude the direct effects of TAT-ANK on the viability of tumour cells, we treated Colo205 with TAT-ANK for 12 h and found that TAT-ANK had effects comparable to those of TAT-Sc (Figure S9). To further explore the effect of TAT-ANK on tumour growth, we generated two tumour models by injecting comparable number of human colon carcinoma (Colo205) cells or EL4 murine lymphoma cells into flanks of eight-week-old male SCID mice, and treated the mice, with TAT-Sc or TAT-ANK, when tumour volume reached about 200 mm³ and harvested the tumours after 1–3 weeks. We found that treatment with TAT-ANK reduced the Colo205 tumour size by 28.9% ± 4.1% (Figure 7a,b) and murine lymphoma tumour size by 23.2% ± 2.4% (Figure S10A,B). Next, CD31 staining was performed for evaluating tumour vasculature density. Interestingly, TAT-ANK-treated Colo205 and lymphoma tumour had a marked increase of ECs (Figure 7c,d, 2.9 ± 0.2-fold in Colo205 tumour; Figure S5C,D, 1.8 ± 0.15-fold in lymphoma tumour). To evaluate the blood perfusion in the two types of tumour, the L. esculentum (tomato) Lectin-DyLight® 488, an EC dye, was injected systemically. TAT-ANK significantly reduced lectin-labelled ECs in colon tumours (Figure 7e) and in lymphoma tumours (Figure S10E), compared with TAT-Sc, which indicates decreased blood perfusion after TAT-ANK treatment. To define the Dll4-Notch signalling changes in tumours, we detected Notch target gene expression. The transcripts of Notch1 target genes, such as Hey1, Hes1 and Dll4, were strongly inhibited in TAT-ANK-treated Colo205 tumour tissue (Figure 7f; decreased by 27.2% ± 6.8%, 53.4% ± 7.4% and 54.7% ± 4.7%, respectively). Similar results were observed in lymphoma tumour (Figure S10F). All these data showed that TAT-ANK inhibited tumour growth by impaired tumour angiogenesis.

FIGURE 7.

The effect of TAT-ANK on tumour growth and blood perfusion. TAT-Sc- or TAT-ANK-treated tumours from the SCID mice were harvested at Day 24 after subcutaneously injection of Colo205 cells. (a) Tumour sizes were significantly reduced in the TAT-ANK treated mice. (b) Tumours volume was analysed by measuring the length and width (n = 6). C. Immunostaining of CD31 from Colo205 tumour sections was performed. (d) Quantification of CD31 staining in the TAT-Sc- or TAT-ANK-treated tumours. (e) Lycopersicon esculentum (tomato) Lectin-DyLight® 488 perfusion and CD31 (red) staining in the Colo205 frozen sections. (f) Dll4, Hey1 and Hes1 transcripts in the Colo205 tumour samples were quantified by RT-qPCR analysis (n = 6). All data presented are means ± SEM. *P < 0.05, significantly different from TAT-Sc

4 |. DISCUSSION

The major finding of the current study is that the polypeptide TAT-ANK, which was designed based on the ANK domain of GIT1, promoted angiogenesis in vitro and in vivo by repressing Dll4-Notch signalling by binding to RBP-J. TAT-ANK treatment significantly repressed the mRNA and protein expression of Notch target genes, including Hey1 and Dll4. Decrease of Dll4 led to reduced Notch1 activation, as detected by NICD. Mechanistically, TAT-ANK directly bound to RBP-J by competing with NICD. Biological functional studies demonstrated that TAT-ANK increased angiogenesis in vitro and in vivo. The inhibition of Notch signalling was further confirmed in vivo.

TAT-ANK is a potent Dll4-Notch1 inhibitor. Given the key role of Dll4-Notch signalling in angiogenesis, small molecules and antibodies targeting this pathway have been developed, including Dll4 neutralizing antibodies (OMP-21M18, demeiczumab) or γ-secretase inhibitors (MK-0752, AL101) in Phase I or II clinical trials (Cook et al., 2018; McCaw et al., 2021; McKeage et al., 2018; Smith et al., 2014). Our data demonstrated that TAT-ANK has similar blocking effects on Dll4-Notch signalling as other inhibitors (Benedito et al., 2012; Benedito & Hellstrom, 2013; Hellström et al., 2007; Ridgway et al., 2006; Serra et al., 2015; Shah et al., 2017). These effects were also consistent with the repression by GIT1 of the Dll4-Notch1 positive feedback loop as we previously reported (Majumder et al., 2016). Recently, Hurtado et al. identified a new RPB-J inhibitor-1 (RIN1) by high throughput screening on small molecules that disrupted RPB-J interacting with the scaffold protein SHARP or NICD. Although RPB-J is a transcription repressor of Notch signalling, it has opposite roles in Notch signalling by forming different complexes. For example, RPB-J binds to SHARP to form the repressive complex to inhibit Notch signalling whereas RPB-J binds to NICD to form the activating complex to increase Notch signalling. Therefore, RIN1 showed opposing effects on different Notch target genes and its effects on the proliferation of tumour cell lines and myoblast differentiation depend on the prevailing complex in the different context (Hurtado et al., 2019), whereas TAT-ANK, as a small peptide, mainly affects the interaction of NICD with RBP-J and only inhibits Notch signalling. Thus, as a potential therapeutic drug, TAT-ANK will be an optimal choice.

Inhibition of Dll4-Notch1 signalling pathway promotes angiogenesis (Benedito et al., 2009, 2012; Majumder et al., 2016; Pitulescu et al., 2017). In vitro, TAT-ANK significantly increased sprouting angiogenesis measured by tube formation and spheroid angiogenesis. In vivo, TAT-ANK led to the excessive sprouting angiogenesis in Matrigel plugs, retinal vasculatures and human and mouse tumours. The hyperproliferative ECs formed non-functional vasculature, which resulted in poor blood flow patency and reduced tumour growth. TAT-ANK demonstrated pro-angiogenic effects and anti-tumour growth which is similar to the effects of known Dll4-Notch1 inhibitors (Noguera-Troise et al., 2006; Ridgway et al., 2006). It is well recognized that VEGF signalling is critical for angiogenesis. Dll4 could up-regulate VEGFR-2 and NRP-1 in HUVECs (Williams et al., 2006). However, TAT-ANK treatment had no significant effect on VEGFR-2 and NRP-1 expression (Figure S3C–E) as well as VEGF signalling in ECs (Figure S3F–H), indicating that the pro-angiogenic effects of TAT-ANK is mainly mediated by repressing Notch signalling.

Compared with the small molecules and antibodies that inhibit Dll4-Notch signalling, TAT-ANK has several advantages. (a) Due to its smaller size, TAT-ANK is easy to synthesize and modify and does not cause significant immune responses. Also, (b) TAT-ANK can be degraded and will not accumulate in off-target organs. Therefore, side effects are expected to be minimal. (c) TAT-ANK has similar or greater efficacy compared with current Notch inhibitors (Hellström et al., 2007; Hoey et al., 2009; Hurtado et al., 2019; Majumder et al., 2016; Moellering et al., 2009; Noguera-Troise et al., 2006; Ridgway et al., 2006; Wu et al., 2010). (d) Small peptide synthesis in bulk is routine and cost effective.

The specificities of various Dll4-Notch inhibitors are different. γ-Secretase is expressed in all cell types and its inhibitors such as DAPT, block γ-secretase mediated Notch signalling and inhibit the cleavage of many other substrates such as the amyloid precursor protein (APP), ErbB4, N-cadherin and EphrinB (Chavez-Gutierrez et al., 2012; Georgakopoulos et al., 2006; Litterst et al., 2007; Marambaud et al., 2003; Wong et al., 2004). As Dll4 is restricted to ECs, Dll4 neutralizing antibodies mainly inhibit Dll4 mediated Notch signalling in ECs (Couch et al., 2016; Hoey et al., 2009; Ridgway et al., 2006). In contrast, TAT-ANK inhibits the Notch signalling pathway by competing with NICD to bind to RPB-J. The transcription factor RPB-J exists in all cell types. Therefore, the specificity of TAT-ANK depends on the major Notch receptor in the target cells. To investigate whether TAT-ANK could suppress the Notch signalling in other primary cells, in addition to ECs, we treated HPFs (Notch2 and Notch3 are major Notch receptors according to the LungMAP data base) with TAT-ANK. As expected, TAT-ANK also reduced the Notch target gene Hes1 protein expression in HPFs (Figure S11A,B).

There are two clear limitations of the TAT-ANK peptide, as used in the current study. Firstly, TAT-ANK has modest aqueous solubility, which limits administration of higher systemic doses. To improve its potential as a pharmacological agent, the solubility and stability of TAT-ANK need to be enhanced in further development. Secondly, the TAT-ANK may have potential side effects. For instance, Notch/RPB-J signalling is essential for lymphocyte development (Tanigaki & Honjo, 2007) and RPB-J deficiency also impaired neuron cell maturation (Fujimoto et al., 2009). However, mice heterozygous for either RBP-J or Notch1 have normal blood cell count and normal neurogenesis (Costa et al., 2003; Givogri et al., 2002; Mack et al., 2017). The possible adverse effects of TAT-ANK could be reduced or avoided by decreasing the concentration of the peptide. In addition, EC specificity of TAT-ANK could be improved by incorporating EC targeting sequences (Vivès et al., 2008).

In conclusion, our results suggest that TAT-ANK may be a promising pharmacological agent to treat angiogenesis-related diseases by inhibiting Notch signalling. Localized administration of TAT-ANK leads to functional angiogenesis and would benefit ischaemic states such as myocardial infarction. The anti-tumour effects of TAT-ANK could be expanded to more solid tumours with high levels of Dll4-Notch signalling in ECs, to increase the amount of non-functional vascular plexuses.

What is already known

• The Dll4-Notch1 signalling pathway is essential for sprouting angiogenesis

• GIT1 is an endogenous inhibitor of the Dll4-Notch1 signalling pathway

What does this study add

• We developed TAT-ANK, a novel polypeptide derived from GIT1, as an inhibitor of Notch signalling.

• TAT-ANK promoted angiogenesis in vitro and in vivo by inhibiting Dll4-Notch1 signalling.

What is the clinical significance

• TAT-ANK will have important conceptual and therapeutic implications for angiogenesis-related diseases.

Abbreviations:

Dll4

Delta-like 4

EC

endothelial cell

EdU

5-ethynyl-2′-deoxyuridine

GIT1

G-protein-coupled receptor-kinase-interacting protein-1

HPRT

hypoxanthine guanine phosphoribosyltransferase

NICD

Notch intracellular domain

RBP-J

recombining binding protein suppressor of hairless

vSMC

vascular smooth muscle cell

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.