Pharmacological activation of p53 induces dose-dependent changes in endothelial cell fate during angiogenic sprouting

Abstract

The cell cycle is a key regulator of endothelial cell specification into tip and stalk cell phenotypes, which are essential for angiogenesis in both normal development and pathological conditions. While the tumor suppressor p53 is known to regulate the cell cycle and influence cell fate, its role in modulating the cell fate of these phenotypes remains unclear. Using non-genotoxic small molecule and stapled peptide compounds to pharmacologically activate p53 via MDM2 inhibition, we demonstrate that graded levels of p53 induce distinct cellular fates in normal endothelial cells. Low levels of p53 induce reversible cell cycle arrest by reducing DNA replication, while high levels induce senescence and cell death. Surprisingly, all tested levels of p53 activation reduced the growth of venous blood vessels in vitro and in zebrafish embryo models. This reduction in sprouting may stem from distinct cellular responses in tip-like and non-tip-like cells to pharmacological p53 activation: low p53 levels primarily reduced proliferation in non-tip-like cells, whereas high levels decreased the frequency of tip-like cells and the expression of genes associated with tip and stalk cell identities. Our findings show for the first time that pharmacological p53 activation modulates endothelial cell fate in a dose-dependent manner during sprouting angiogenesis. They also highlight the potential of using graded p53 modulation as a therapeutic strategy to target abnormal tip or stalk cell development in pathological angiogenesis, such as in cancer.

Fig. 1. Pharmacological activation of p53 induces cell cycle arrest, cell death, and senescence in endothelial cells in a concentration-dependent manner.

A Concentration-dependent growth inhibition of human umbilical vein endothelial cells (HUVEC) by three p53-activators (MDM2 inhibitors navtemadlin and nutlin-3a; MDM2/MDMX inhibitor sulanemadlin), but not by non-specific control peptide. Cell growth was measured by live-cell imaging as the percent confluence normalized to untreated wells following 72 h treatment. Data points show averaged value from one experiment (n = 3 experiments) and are fitted with a best fit model for concentration-growth response. B Growth inhibition of human dermal microvascular endothelial cells (HDMEC) and normal human dermal fibroblasts (NHDF) by navtemadlin, as measured by live-cell imaging. Data points show averaged value from one experiment (n = 3 experiments) and are fitted with a best fit model for concentration-growth response. C Growth inhibition of HUVEC recovers within 48 h following an initial 24 h treatment using ≤ 0.1 μM navtemadlin. Cell growth was measured by live-cell imaging and quantified over 72 h as cell counts per well and normalized to counts at time 0. Data points show mean  ± SD (n = 3 independent experiments). *P_adj < 0.05, **P_adj < 0.01 using 1-way, repeated measures ANOVA with adjustment using Dunnett’s correction. D Morphological abnormalities are visible in phase-contrast images of venous ECs (HUVEC) and capillary ECs (HDMEC), but not in those of fibroblasts (NHDF) after 72 h of navtemadlin treatment, but not after 24 h. Scale bar = 200 μm. E Increased expression of proteins involved in p53 signaling (MDM2, clone IF2, 0.5 μg/mL; p53, clone DO-1, 0.4 μg/mL), cell cycle arrest (p21, clone 12D1, 0.24 μg/mL), and apoptosis (PUMA, clone D30C10, 0.96 μg/mL) in HUVEC following 24 h navtemadlin treatment, as determined by western blot analysis. Total protein controls correspond to distinct membranes (L1 or L2). Images are cropped from full-length blots of one biological experiment (see ‘Full Length Western Blots’) and are representative of at least two experiments. Further details regarding antibodies used are shown in SI Table 1. F Increased expression of p53 (clone DO-1, 12 μg/mL), cell cycle arrest (p21, clone 12D1, 1.22 μg/mL), apoptosis (PUMA, clone D30C10, 4.8 μg/mL), dead cells (Sytox green, 100 nM), and senescence (β-galactosidase), and reduced expression of active cell cycle (Ki67, clone SP6, 0.12 μg/mL), following 24 h treatment of HUVEC using navtemadlin as visualized by immunofluorescence, live-cell fluorescence imaging, and colorimetric staining. Scale bar = 50 μm. Further details regarding antibodies and concentrations used are shown in SI Table 1. G–K Quantification of fluorescence and colorimetric levels of protein markers, showing increased expression of p53, cell cycle arrest (p21), apoptosis (PUMA), cell death (Sytox green), and senescence, and reduced activity in cell cycle (Ki67). Data points indicate value from one experiment (n = 3 experiments). **P_adj < 0.01, ***P_adj < 0.001 using one-way ANOVA with adjustment using Dunnett’s correction. Horizontal black line indicates the mean value.

Fig. 2. Knockdown of TP53 reduces activation of p53 pathway induced by navtemadlin in HUVEC.

A–C Knockdown of TP53 reduces the activation of TP53 and its target genes CDKN1A and MDM2 induced by 6 h treatment with 1 μM navtemadlin in HUVEC, as measured by RT-qPCR. Cells were transfected for 24 h using Dicer-substrate short interfering RNA (DsiRNA) targeting a negative control sequence (“Ctrl”) or a pool of three DsiRNAs targeting TP53 before navtemadlin treatment in fresh medium. Data points represent values from one experiment (n = 3 independent experiments) and are plotted as log₁₀ fold change (FC). Bar height indicates mean value. Statistical analysis was performed on the delta Ct values, after normalization to housekeeping gene B2M. *P_adj < 0.05, **P_adj < 0.01, ***P_adj < 0.001 using two-way ANOVA with adjustment using Šidák’s correction. D Phase-contrast images of HUVEC following knockdown of TP53 show phenotypic rescue of morphological changes observed after 24 h treatment using 1 μM navtemadlin. Scale bar = 300 μm. E In HUVEC treated with TP53 DsiRNA, protein levels of p53, p21, and PUMA are reduced following 24 h navtemadlin, as determined by western blot analysis. Images are cropped from full-length blots of one biological experiment (see ‘Full Length Western Blots’) and are representative of at least three experiments. F In HUVEC treated with TP53 DsiRNA, protein levels of p53 and p21 are reduced following 24 h navtemadlin treatment, as visualized by immunofluorescence staining. Scale bar = 50 μm. G, H Quantification of fluorescence levels of p53 (G) and p21 (H) shows reduced expression in HUVEC treated with TP53 DsiRNA compared to those treated with control DsiRNA. Data points indicate averaged value from one experiment (n = 3 experiments). *P_adj < 0.05, **P_adj < 0.01, ***P_adj < 0.001 using two-way ANOVA with adjustment using Dunnett’s correction. Bar height indicates mean value. Statistical analysis was performed on log-transformed data, but plotted on linear scale to show differences more clearly.

Fig. 3. Low levels of p53 activation alter protein networks in DNA replication while high levels alter those involved in stress responses and ribosome assembly.

A Schematic of proteomics workflow in which HUVEC were treated with navtemadlin for 24 h, digested, and quantified using TMT-based mass spectrometry. B Protein abundances (mean-normalized) separate by treatment (DMSO vs navtemadlin), as assessed by principal component analysis. Percentage of variance is shown in parentheses. C Volcano plots showing differentially expressed proteins with decreased and increased abundance between DMSO-treated and navtemadlin-treated samples. Downregulated proteins include CDK1 (involved in cell cycle), THBS1 and PECAM (associated with angiogenesis), and KPNA2 (important for nuclear export), while upregulated proteins include those TIGAR and TP53I3 (involved in p53 signaling and apoptosis). FDR on P_adj < 0.03. D Heat map depicting the top 20 enriched terms within ontological biological processes following p53 activation by navtemadlin. Enriched terms are involved in p53 signaling, DNA replication, and ribosome assembly. Proteins were mapped to their corresponding gene symbols in Metascape for functional enrichment analysis using various ontologies (GO biological processes, GO molecular functions, KEGG pathways, Reactome, and canonical pathways). The color gradient represents P-values (as -log₁₀). E Circos plot depict partial overlap in ontology terms between low and high levels of p53 activation induced by navtemadlin. Dark orange segments indicate shared ontology terms between treatment groups, while light orange segments indicate ontology terms unique to each treatment. Proteins were mapped to their corresponding gene symbols for enrichment analysis using Metascape. F Different biological functions are affected at low and high levels of p53 activation induced by navtemadlin. After protein hits were converted to their respective gene symbols (using Metascape), functional analysis was performed to identify enrichment in ontology terms. The top 20 clusters of enriched ontology terms were then selected using a P-value < 0.01, a minimum count of 3 genes, and an enrichment factor > 1.5 (i.e., pathway is represented 1.5 more frequently in list than would be expected by chance). Each node (circle) in the network represents an ontology term comprising a group of genes that share a common biological function. The size of the node indicates the number of genes comprising that group, and the color of the pie charts within each node represents treatment groups. Nodes are grouped into clusters using the Kappa similarity score, so that clustered nodes are functionally related. Each cluster is labeled with a representative term summarizing its main biological function.

Fig. 4. Pharmacological activation of p53 reduces growth of venous vessels in vitro and in vivo at all tested concentrations.

A Schematic of the endothelial spheroid sprouting assay. HUVEC were embedded in a fibrin gel matrix and treated for 24 h using solvent (DMSO) or increasing concentrations of navtemadlin (navt). Vascular endothelial growth factor (VEGF, 20 ng/mL) was used to stimulate angiogenic sprouting. B Fluorescence microscopy images show reduced sprouting in HUVEC spheroids treated with increasing navtemadlin concentrations; the images are brightness and contrast adjusted for clarity. Insets show cell filopodia in sprouts. Scale bars = 100 µm. C–E All tested concentrations of navtemadlin reduce (C) total sprout length, (D) fraction of sprouts associated with spheroid body (a measure of connectivity), and (E) total sprout number of HUVEC spheroids. Each data point in violin plot indicates one spheroid (n = 64 spheroids for baseline; 61 spheroids for VEGF; 66 spheroids for 0.1 and 1 μM navt; 84 spheroids for 10 μM navt; pooled from three experiments). *P_adj < 0.05, ** P_adj < 0.01, ***P_adj < 0.001, **** P_adj < 0.0001 using Kruskal-Wallis with Dunn’s correction. Note y-axis is shown on log scale. F Schematic of endothelial sprouting assay following 24 h transfection with control or TP53-targeting DsiRNAs (pool). After embedding, spheroids were treated for 24 h using DMSO or navtemadlin in the presence of VEGF. G Fluorescence microscopy images show that navtemadlin does not further reduce sprouting of HUVEC spheroids after TP53-knockdown; the images are brightness and contrast adjusted for clarity. Scale bars = 100 µm. H Quantification of total sprout length in drug-treated HUVEC spheroids following transfection with control or TP53-targeting DsiRNAs (pooled). Each data point in violin plot indicates one spheroid (n = 66 spheroids for DMSO and 82 spheroids for 1 μM navt in Ctrl DsiRNA; 90 spheroids for DMSO and 65 spheroids for 1 μM navt in TP53 DsiRNA spheroids/group pooled from three experiments). *P_adj < 0.05, ** P_adj < 0.01, using Kruskal-Wallis with Dunn’s correction. Note y-axis is shown on log scale. I A zebrafish embryo (Danio rerio) model expressing fluorescent vasculature (Tg(fli1:eGFP)) was used to measure effects of p53 activation in developing subintestinal vessels (SIV) in vivo. Embryos were treated with vehicle (DMSO), sunitinib (positive control), sulanemadlin (stapled peptide activator of p53), or control peptide for 48 h, starting at 20 h post-fertilization (hpf). Cropped maximum intensity projections of the subintestinal vessels and corresponding vessel masks (binary images below) are shown; the images are brightness and contrast adjusted for clarity. Arrows (orange) on the binary masks indicate ectopic sprouts. Scale bar of cropped images = 150 µm. J–L Treatment with p53 activator sulanemadlin leads to (J) reduction in subintestinal vessel area, (K) an increase in number of endothelial cell tip extensions, and (J) no measurable change in fish size. Each data point in violin plot represents one embryo (n = 26 embryos for DMSO; 30 embryos for sunitinib; 32 embryos for sulanemadlin; 31 embryos for control peptide; pooled from two independent experiments). ***P_adj < 0.001, ****P_adj < 0.0001 for vessel areas using Brown-Forsythe ANOVA with Dunnett’s T3 correction and *P_adj < 0.05, **P_adj < 0.01 for sprout number using Kruskal-Wallis non-parametric test with Dunn’s correction for multiple testing.

Fig. 5. p53 activation differentially modulates cell fate in tip-like and non-tip-like cells.

A Epifluorescence microscopy images show altered sprout morphology of HUVEC spheroids treated using sunitinib or increasing concentrations of the p53 activator navtemadlin (navt; 0.05 µM, 0.1 µM, 1 µM) in the presence of VEGF (20 ng/mL). DMSO was used as solvent control. Spheroids were stained for nuclei (green pseudo-color, Hoechst) and sprouts (magenta pseudo-color, phalloidin). Scale bar: 100 µm. B, C Reduction in the total number of (B) tip cells and (C) stalk cells quantified per spheroid upon treatment with sunitinib or high concentrations of navtemadlin (1 µM) in the presence of VEGF (20 ng/mL). Each symbol in violin plot represents one spheroid (n = 30 spheroids for baseline; 31 spheroids for VEGF; 25 spheroids for sunitinib; 38 spheroids for 0.05 μM navt; 25 spheroids for 0.1 μM navt; 30 spheroids for 1 μM navt; pooled from two independent experiments). ***P_adj < 0.001, ****P_adj < 0.0001 using Kruskal-Wallis with Dunn’s correction. D Treatment with high concentrations of navtemadlin (1 μM) reduces the VEGF-induced increase in the percentage of CD34+ (tip-like) cells, as measured by flow cytometry analysis of treated HUVEC monolayers. Each data point represents value from one experiment (n = 4 experiments). Bar height indicates mean value ± SD. ***P_adj < 0.001, ****P_adj < 0.0001 using one-way ANOVA with Dunnett’s correction. E, F Activation of p53 by high concentrations of navtemadlin (1 μM) induces significant p21 activity in CD34− (non-tip-like) cells. The change in the median fluorescence intensity of (E) p53 and (F) p21 were measured by flow cytometry in CD34+ (tip-like) and CD34− cells following 24 h treatment of HUVEC monolayers in the absence and presence of navtemadlin and VEGF (20 ng/mL). Each data point represents value from one experiment (n = 3 experiments). Bar height indicates mean value ± SD. ***P_adj < 0.001, ****P_adj < 0.0001 using two-way ANOVA with Dunnett’s correction. G, H Navtemadlin treatment significantly alters cell cycle distribution of CD34− cells at low concentrations (0.05 μM), and of both CD34+ and CD34− cells at high concentrations (1 μM). The proportion of cells in the different phases of the cell cycle (SubG₁, G₁, S, G₂/M), as well as the total number of cells analyzed in each treatment, were measured using flow cytometry. Bar graphs represent summed values from all experiments (n = 4 experiments). ***P_adj < 0.001, ****P_adj < 0.0001 using two-way ANOVA with Dunnett’s T3 correction. I, J In HUVEC monolayers, treatment with navtemadlin (1 µM) primarily alters the expression of genes associated with tip cells. Heatmaps depict fold changes (FC) in mRNA expression of tip cell genes (ANGPT2, CD34, CXCR4, DLL4) and stalk cell genes (HES1, JAG1, FLT1) following 24 h treatment with sunitinib or navtemadlin, in the absence or presence of VEGF (20 ng/mL). Expression is normalized to DMSO controls and analyzed using NORMA-Gene [50]. Each square represents the averaged value from one experiment (n = 3 experiments). ** FDR < 0.01.