The inhibitory effect of tyrosine kinase inhibitors on angiogenesis in zebrafish and interrelationship with the activation of the PI3K/AKT pathway

Abstract

Background

To evaluate the effects of tyrosine kinase inhibitors (TKIs) Dovitinib, Lapatinib, and Telatinib on angiogenesis in zebrafish, and to explore the regulatory mechanism of PI3K/AKT pathway activator SC79 on these inhibitory effects.

Methods

Transgenic Fli-1:EGFP zebrafish embryos were used to observe the development and morphological changes of intersegmental vessels (ISVs) by fluorescence microscopy. Real-time fluorescence quantitative PCR was employed to detect the expression of angiogenesis-related genes including kdrl, flt1, vegfaa, akt2, and pik3r1. After determining the maximum tolerated concentration (MTC) of each compound, the effects of single-drug treatment and SC79 co-treatment on vascular development and gene expression were observed.

Results

Dovitinib (0.488 µM) and Telatinib (0.031 µM) significantly reduced the number (by 16.9% and 33.8%, respectively, P < 0.001) and length (by 14.9% and 23.3%, respectively, P < 0.001) of complete ISVs, while Lapatinib (1.95 µM) showed no significant inhibitory effect. Gene expression analysis revealed that Dovitinib mainly downregulated flt1 expression (P < 0.01), whereas Telatinib more broadly downregulated kdrl (P < 0.05), flt1 (P < 0.001), and akt2 (P < 0.01) expression. SC79 (2.44 nM) promoted angiogenesis, increasing ISV number (P < 0.01) and length (P < 0.05), while upregulating kdrl (2.03-fold, P < 0.001), flt1 (1.31-fold, P < 0.05), vegfaa (1.45-fold, P < 0.01), akt2 (1.64-fold, P < 0.01), and pik3r1 (1.48-fold, P < 0.05) expression. Co-treatment experiments showed that all three TKIs could counteract SC79’s pro-angiogenic effects to varying degrees, with Telatinib showing the strongest effect. Under SC79 co-treatment conditions, all TKIs significantly downregulated kdrl expression (P < 0.001), but had differential effects on other genes: Dovitinib mainly downregulated flt1 (P < 0.05), Lapatinib specifically downregulated vegfaa (P < 0.01), akt2 (P < 0.05), and pik3r1 (P < 0.05), while Telatinib simultaneously downregulated flt1 (P < 0.01), akt2 (P < 0.05), and pik3r1 (P < 0.01).

Conclusions

Dovitinib and Telatinib exhibit significant anti-angiogenic effects, while Lapatinib is less effective. Different TKIs inhibit angiogenesis by regulating different signaling molecules: Dovitinib primarily targets VEGF receptors, Telatinib targets both receptors and the downstream PI3K/AKT pathway, and Lapatinib shows inhibitory effects on VEGF ligand expression in the SC79 background. SC79 promotes angiogenesis by activating the PI3K/AKT pathway and partially reverses the inhibitory effects of TKIs. These findings provide new insights for targeted therapy of abnormal angiogenesis in ophthalmic diseases, suggesting that precise regulation of angiogenesis may be achieved by modulating the balance between TKIs and the PI3K/AKT signaling pathway.

Background

Angiogenesis is a crucial physiological process in the body, involving the proliferation, migration, and maturation of endothelial cells, and plays a role in embryonic development, tissue repair, and disease progression [1]. In various diseases, such as neovascular age-related macular degeneration and diabetic retinopathy, abnormal angiogenesis is a significant cause of vision loss [2]. For these diseases, therapeutic strategies targeting the inhibition of angiogenesis have become a major research focus.

The vascular endothelial growth factor (VEGF) pathway is a key regulatory pathway in angiogenesis [3]. VEGF primarily binds to vascular endothelial growth factor receptors (VEGFRs), including VEGFR1 (Flt1) and VEGFR2 (KDR/Flk1, known as kdrl in zebrafish), activating downstream signaling pathways such as PI3K/AKT and MAPK/ERK, thereby promoting endothelial cell proliferation, migration, and survival [3, 4]. Among these receptors, VEGFR2 is the most critical receptor mediating the biological effects of VEGF [2]. Its activation enhances downstream signaling pathways, including PI3K/AKT and MEK/ERK, which in turn promote endothelial cell migration, proliferation, and vascular formation [1, 3]. Recent clinical evidence suggests that PI3K/AKT pathway activation contributes to TKI resistance in approximately 5% of cases, highlighting the need for combination therapeutic strategies.

Tyrosine kinase inhibitors (TKIs) are a class of small-molecule inhibitors targeting tyrosine kinase receptors, which have been widely used in the treatment of various diseases [5, 6]. Among them, Dovitinib, Lapatinib, and Telatinib are multi-target TKIs capable of inhibiting multiple receptor tyrosine kinases, including VEGFR, FGFR, and PDGFR [7–9]. Dovitinib not only inhibits various tyrosine kinases but also exhibits inhibitory effects on topoisomerase I and II [7]. It has demonstrated potent anti-angiogenic activity in corneal neovascularization models [10]. Lapatinib, as a dual-target inhibitor of EGFR/HER2, has shown anti-angiogenic effects alongside its ability to inhibit breast cancer cell growth [11, 12]. Although these TKIs exhibit broad potential in anti-tumor therapy and angiogenesis inhibition, common adverse effects such as diarrhea, skin rash, and cardiotoxicity in clinical applications remain a concern [6, 11, 13].

The PI3K/AKT pathway is a crucial downstream signaling pathway of multiple receptor tyrosine kinases, playing a pivotal role in regulating cell survival, proliferation, and metabolism [14, 15]. During angiogenesis, the activation of the PI3K/AKT pathway is essential for endothelial cell proliferation, migration, and survival [4, 14]. SC79, a specific AKT activator, promotes AKT phosphorylation and activation by binding to its PH domain [16–18]. It has demonstrated protective effects in various disease models, including hepatoprotection [16], neuroprotection [17, 18], and promotion of liver regeneration [19].

Zebrafish have become an ideal model for studying angiogenesis due to their transparent embryos, ease of observing vascular development, and high genomic homology with humans [20, 21]. Particularly, the transgenic Fli-1:EGFP zebrafish strain, in which vascular endothelial cells specifically express green fluorescent protein, allows direct observation of vascular development in living organisms [21, 22]. Intersegmental vessels (ISVs), which grow dorsally from the dorsal aorta (DA) in the trunk region of zebrafish, serve as a hallmark structure for studying vascular development and screening inhibitors [21].

In this study, we utilized a zebrafish embryo model to evaluate the effects of Dovitinib, Lapatinib, and Telatinib on angiogenesis and to investigate the regulation of these TKIs’ inhibitory effects by the PI3K/AKT pathway activator SC79. The aim is to gain deeper insights into the mechanisms underlying TKI-mediated angiogenesis inhibition and its relationship with the PI3K/AKT signaling pathway, thereby providing a new theoretical foundation for targeted therapies against abnormal angiogenesis in ocular diseases.

Materials and methods

Materials

Compounds

Dovitinib, Lapatinib, Telatinib, and SC79 were dissolved in dimethyl sulfoxide (DMSO, Lot No. I2229063, Shanghai Aladdin Biochemical Technology Co., Ltd., China) to prepare stock solutions. In all treatment groups, the final concentration of DMSO in the zebrafish water was maintained at 0.1% v/v. The control group was treated with a solution containing 0.1% DMSO as the solvent control.

Experimental animals

Transgenic Fli-1:EGFP zebrafish expressing enhanced green fluorescent protein in vascular endothelial cells and wild-type AB strain zebrafish were maintained in standard fish water at 28℃ (conductivity: 450–550 µS/cm; pH: 6.5–8.5; hardness: 50–100 mg/L CaCO₃). The fish were bred and provided by the Hangzhou Hunter Biotech limited liability company. Prior to the experiment, zebrafish larvae were euthanized by gradual immersion in an ice-water mixture to slow their heartbeats to a stop. The use of experimental animals was permitted under license number: SYXK (Zhe) 2022-0004. Husbandry management met the requirements of the international AAALAC accreditation (Certification No.: 001458). Ethical review by the Institutional Animal Care and Use Committee (IACUC) was approved under protocol number: IACUC-2023-6895-01.

Equipment and reagents

Imaging equipment included a stereomicroscope (SZX7, OLYMPUS, Japan), a CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China), and a motorized focus zoom fluorescence microscope (AZ100, Nikon, Japan). Molecular biology equipment included a high-speed refrigerated centrifuge (Heraeus Fresco17, ThermoFisher, Germany), a spectrophotometer (Nanodrop2000, Thermo, USA), an automated nucleic acid extraction system (Auto-Pure32A, Hangzhou Aosheng Instruments Co., Ltd., China), a PCR thermal cycler (T100, BIO-RAD, Singapore), and a real-time PCR system (CFX Connect, BIO-RAD, Singapore).

Key reagents included ChamQ Universal SYBR qPCR Master Mix (Lot No. 7E760L3, Vazyme, China), Hifair III 1st Strand cDNA Synthesis SuperMix for qPCR (Lot No. H9305270, Yeasen Biotechnology (Shanghai) Co., Ltd., China), and a pre-packaged magnetic bead-based universal RNA extraction kit (Lot No. TL2312001643C, Foshan Aowei Biotechnology Co., Ltd., China).

Methods

Determination of maximum tolerated concentration (MTC)

Randomly selected 2 h post-fertilization (hpf) zebrafish embryos, placed in 6-well plates, with 30 embryos per well and treated with different concentrations of compounds dissolved in system water containing 0.1% DMSO. The control group was treated with a solution containing only 0.1% DMSO. Embryos were maintained at 28 °C for 48 h, after which mortality and developmental abnormalities were assessed to determine the MTC of each compound.

Effects on vascular development

Fli-1:EGFP transgenic zebrafish embryos (2 hpf) were treated in 6-well plates with different concentrations of test compounds or 0.1% DMSO (control) (30 embryos per well, 3 mL medium per well). After incubation at 28 °C for 48 h, 10 larvae were randomly selected from each treatment group, anesthetized in embryo medium with 0.016% buffered tricaine for 1–2 min, positioned laterally, and imaged using a fluorescence microscope.

To ensure standardized measurements, we analyzed the trunk region spanning five complete somites starting from the yolk extension end. Within this region, we quantified the number of complete ISVs fully extending from the DA to the dorsal longitudinal anastomotic vessel (DLAV), the average length of ISVs (measured in pixels from the DA to the DLAV) and the overall size of the embryo (measured as the distance from the anterior end to the tail tip).

For co-treatment experiments, embryos were simultaneously exposed to SC79 (2.44 nM) and the corresponding MTC of each test compound. All imaging and measurements were performed on 48 hpf embryos.

Gene expression analysis

Wild-type AB zebrafish embryos (2 hpf) were treated as described above. At 48 hpf, total RNA was extracted from pooled embryos (30 embryos per treatment group) using a magnetic bead-based RNA extraction kit according to the manufacturer’s protocol. RNA concentration and purity were determined by spectrophotometry (acceptable A260/A280 ratio: 1.8–2.2).

For each sample, 2.0 µg of total RNA was reverse-transcribed into cDNA using Hifair III 1st Strand cDNA Synthesis SuperMix. Quantitative PCR was performed using ChamQ Universal SYBR qPCR Master Mix with the following cycling conditions: initial denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s.

Gene-specific primers used were as follows:

β-Actin: Forward 5’-TCGAGCAGGAGATGGGAACC-3’, Reverse 5’-CTCGTGGATACCGCAAGATTC-3’.

kdrl: Forward 5’-TCATGTCCCGAGCGATGAAGA-3’, Reverse 5’-CCAGCGCAGAGCATGTCAGA-3’.

flt1: Forward 5’-GAGCGATGCTCCCGTTATCA-3’, Reverse 5’-CACAACTCCACTCTCCCTGG-3’.

vegfaa: Forward 5’-TCCCGACAGAGACACGAAAC-3’, Reverse 5’-CATCTTGGCTTTTCACATCTTTCT-3’.

akt2: Forward 5’-CCGCACCAAAGTGACCATGA-3’, Reverse 5’-AGGAAAGGATGCCGTGTGTT-3’.

pik3r1: Forward 5’-TGGAGGGAATGGAGGAGAGA-3’, Reverse 5’-ACAGAGTGCTCAAGCTTTGCT-3’.

Relative gene expression was calculated using the 2^(-ΔΔCt) method, with β-actin as the internal control. For comparisons between the control and treatment groups, the control group was set as the reference (expression value = 1). For comparisons between the SC79 and co-treatment groups, the SC79 group was set as the reference (expression value = 1).

Statistical analysis

Data are presented as mean ± standard error (SE). Each experiment was performed independently three times (n = 3), with 30 embryos per treatment group for morphological analysis and 10 randomly selected embryos for imaging quantification in each independent experiment. For gene expression analysis, RNA was extracted from pooled embryos (30 embryos per treatment group) with three biological replicates per condition. Statistical analyses were performed using Graphpad Prism 9.5.1 software. Differences between groups were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. A P-value < 0.05 was considered statistically significant, while a P-value < 0.01 was considered highly significant.

Results

MTC of test compounds

To determine appropriate doses for subsequent experiments, we first identified the MTC of each test compound in zebrafish embryos. Transgenic Fli-1 zebrafish embryos at 2 hpf were exposed to varying concentrations of Dovitinib, Lapatinib, and Telatinib. After 48 h of treatment, survival rates and morphological features were assessed. As shown in Table 1, the MTCs for Dovitinib, Lapatinib, and Telatinib were determined to be 0.488 µM, 1.95 µM, and 0.031 µM, respectively. At these concentrations, no mortality was observed, and embryonic development was normal, comparable to the control group. Concentrations exceeding these thresholds resulted in increased mortality and impaired developmental status.

Table 1.

Determination of MTC of tyrosine kinase inhibitors in zebrafish embryos (n = 30)

Group	Concentration (µM)	Mortality (%)	Phenotype
Control	-	0	No obvious abnormalities observed
Dovitinib	0.122	0	Similar to the control group
	0.244	0	Similar to the control group
	0.488	0	Similar to the control group
	0.977	7	Developmental Impairment
	1.95	7	Developmental Impairment
Lapatinib	0.122	0	Similar to the control group
	0.244	0	Similar to the control group
	0.488	0	Similar to the control group
	0.977	0	Similar to the control group
	1.95	0	Similar to the control group
Telatinib	0.004	0	Similar to the control group
	0.008	0	Similar to the control group
	0.015	0	Similar to the control group
	0.031	0	Similar to the control group
	0.061	10	Developmental Impairment

Similarly, the MTC of the PI3K/AKT pathway activator SC79 was determined to be 2.44 nM. At this concentration, zebrafish embryos exhibited normal development with no observable mortality (Table 2).

Table 2.

Determination of MTC of SC79 in zebrafish embryos (n = 30)

Group	Concentration (nM)	Mortality (%)	Phenotype
Control	-	0	No obvious abnormalities observed
SC79	2.44	0	Similar to the control group
	4.88	0	Developmental Impairment
	9.77	0	Developmental Impairment
	19.5	0	Developmental Impairment
	39.1	0	Developmental Impairment

Effects of tyrosine kinase inhibitors on vascular development

To evaluate the anti-angiogenic properties of the three tyrosine kinase inhibitors, we examined their effects on ISVs development in transgenic Fli-1 zebrafish embryos. After 48 h of treatment, we quantified two key parameters: the number of complete ISVs and the length of ISVs.

As shown in Fig. 1, Dovitinib exhibited dose-dependent inhibition of angiogenesis. At the highest tested concentration (0.488 µM), Dovitinib significantly reduced the number of complete ISVs (22.6 ± 1.07 vs. control 27.2 ± 0.200, p < 0.001) and the length of ISVs (245 ± 8.68 pixels vs. control 288 ± 6.11 pixels, p < 0.001). At the intermediate concentration (0.244 µM), moderate inhibition of ISV development was observed, with a statistically significant reduction in the number of complete ISVs (p < 0.05).

Fig. 1.

Effects of various concentrations of Dovitinib, Lapatinib, and Telatinib on angiogenesis in zebrafish. (A) Number of ISVs in zebrafish treated with different concentrations of Dovitinib, Lapatinib, and Telatinib; (B) Length of ISVs in zebrafish treated with different concentrations of Dovitinib, Lapatinib, and Telatinib; (C) Typical images of ISVs in zebrafish treated with different concentrations of Dovitinib, Lapatinib, and Telatinib; yellow arrows indicating complete vessels and red arrows indicating missing vessels. Control group exhibited a complete ISV, while both Dovitinib and Telatinib treatments induced dose-dependent reductions in vascular formation. Lapatinib demonstrated minimal effects on vascular development; (D) Typical images showing the length of ISVs in zebrafish treated with different concentrations of Dovitinib, Lapatinib, and Telatinib. Dovitinib and Telatinib treatments significantly reduced ISV length compared to controls, whereas Lapatinib showed negligible effects. Compared to the control group, *p < 0.05, **p < 0.01, ***p < 0.001. Each experiment was performed independently three times (n = 3)

Among the three compounds, Telatinib demonstrated the strongest anti-angiogenic activity. At a concentration of 0.031 µM, Telatinib significantly reduced the number of complete ISVs (18.0 ± 1.14 vs. control 27.2 ± 0.200, p < 0.001) and the length of ISVs (221 ± 7.34 pixels vs. control 288 ± 6.11 pixels, p < 0.001). Even at a lower concentration (0.008 µM), Telatinib significantly reduced ISV length (p < 0.05).

In contrast, Lapatinib had minimal effects on vascular development at all tested concentrations. Even at the highest concentration (1.95 µM), there were no significant differences in the number of complete ISVs or ISV length compared to the control group (p > 0.05).

Gene expression analysis of angiogenesis-related factors

To investigate the molecular mechanisms of the observed anti-angiogenic effects, we analyzed the expression of key genes involved in angiogenesis and the PI3K/AKT signaling pathway: kdrl (VEGFR2), flt1 (VEGFR1), vegfaa (VEGF-A), akt2, and pik3r1.

As shown in Fig. 2, Dovitinib treatment induced significant downregulation of flt1 expression at the lowest tested concentration (0.738 ± 0.009 vs. control 1.00 ± 0.045, p < 0.01), while its effects on other genes were not statistically significant. Notably, Dovitinib exhibited a non-linear dose-response pattern for flt1 expression. At the lowest tested concentration (0.244 µM), significant downregulation was observed (0.738 ± 0.009 vs. control 1.00 ± 0.045, p < 0.01), while higher concentrations (0.488 µM) showed less pronounced effects. This phenomenon may be attributed to receptor saturation effects or compensatory pathway activation at higher drug concentrations, consistent with Dovitinib’s multi-target inhibitory profile affecting topoisomerase I and II in addition to receptor tyrosine kinases. These results suggest that Dovitinib’s anti-angiogenic activity may primarily involve inhibition of VEGFR1-mediated signaling.

Fig. 2.

Impact of different concentrations of Dovitinib, Lapatinib, and Telatinib on angiogenesis-related gene expression in zebrafish. (A) Relative expression of the flt1 gene after treatment with different concentrations of Dovitinib, Lapatinib, and Telatinib; (B) Relative expression of the kdrl gene after treatment with different concentrations of Dovitinib, Lapatinib, and Telatinib; (C) Relative expression of the vegfaa gene after treatment with different concentrations of Dovitinib, Lapatinib, and Telatinib; (D) Relative expression of the akt2 gene after treatment with different concentrations of Dovitinib, Lapatinib, and Telatinib; (E) Relative expression of the pik3r1 gene after treatment with different concentrations of Dovitinib, Lapatinib, and Telatinib. Compared to the control group, *p < 0.05, **p < 0.01, ***p < 0.001. Each experiment was performed independently three times (n = 3)

Telatinib exhibited broader effects on gene expression, significantly downregulating kdrl (0.708 ± 0.015 vs. control 1.00 ± 0.049, p < 0.05), flt1 (0.419 ± 0.014 vs. control 1.00 ± 0.045, p < 0.001), and akt2 (0.745 ± 0.033 vs. control 1.00 ± 0.040, p < 0.01) at various concentrations. The potent suppression of both VEGF receptors (VEGFR1 and VEGFR2) likely contributes to Telatinib’s robust anti-angiogenic effects.

Consistent with morphological observations, Lapatinib did not significantly alter the expression of any angiogenesis-related genes examined, further confirming its limited anti-angiogenic activity in this model.

Effects of PI3K/AKT pathway activator alone and in combination with TKIs on vascular development

To investigate the role of the PI3K/AKT pathway in angiogenesis, we treated zebrafish embryos with the specific AKT activator SC79. As shown in Fig. 3, At a concentration of 2.44 nM, SC79 significantly enhanced angiogenesis compared to the control group. SC79-treated embryos demonstrated a significant increase in both the number (24.6 ± 0.306 vs. 22.9 ± 0.407 in controls, p < 0.01) and the length (632 ± 11.8 pixels vs. 575 ± 17.6 pixels in controls, p < 0.05) of complete ISVs. These findings clearly indicate the pro-angiogenic properties of SC79.

Fig. 3.

Effects of SC79 combined with TKIs on ISV development in zebrafish. (A) Number of ISVs in zebrafish after treatment with SC79 alone and in combined with TKIs; (B) Length of ISVs in zebrafish after treatment with SC79 alone and in combined with TKIs; (C) Typical images of ISVs in zebrafish after treatment with SC79 alone and in combined with TKIs, yellow arrows indicating complete vessels and red arrows indicating missing vessels. SC79 treatment enhanced vascular formation compared to control, while co-treatment with tyrosine kinase inhibitors counteracted this effect to varying degrees; (D) Typical images showing the length of ISVs in zebrafish after treatment with SC79 alone and in combined with TKIs. SC79 treatment increased vascular length compared to controls, while co-treatment with tyrosine kinase inhibitors (particularly Telatinib) reduced this effect. Each experiment was performed independently three times (n = 3)

To investigate whether TKIs could counteract the pro-angiogenic effects of PI3K/AKT pathway activation, zebrafish embryos were co-treated with SC79 (2.44 nM) and each of the three TKIs. Comparative analysis revealed in Fig. 3 that Dovitinib (0.488 µM) significantly suppressed SC79-induced angiogenesis on the number(22.5 ± 0.671 vs. 24.6 ± 0.306 in SC79 alone, p < 0.05) and the length(545 ± 7.89 pixels vs. 632 ± 11.8 pixels in SC79 alone, p < 0.001) of complete ISVs. Telatinib (0.015 µM) exhibited the most potent inhibition on the number(18.1 ± 0.657 vs. 24.6 ± 0.306 in SC79 alone, p < 0.001) and the length(456 ± 14.2 pixels vs. 632 ± 11.8 pixels in SC79 alone, p < 0.001) of complete ISVs. Lapatinib (1.95 µM) showed selective effects, significant reduction in the length of ISVs(572 ± 16.4 pixels vs. 632 ± 11.8 pixels in SC79 alone, p < 0.01), but no significant change in the number of complete ISVs (p > 0.05).

Gene expression analysis of SC79 monotherapy versus combination therapy with TKIs

Consistent with morphological findings, SC79 treatment significantly upregulated the expression of genes involved in angiogenesis and the PI3K/AKT pathway. As shown in Fig. 4, Compared to controls, embryos treated with SC79 at its maximum tolerated concentration (2.44 nM) showed increased expression of akt2 (1.64 ± 0.172 vs. control 1.00 ± 0.101, p < 0.01) and pik3r1 (1.48 ± 0.152 vs. control 1.00 ± 0.021, p < 0.05). Further analysis revealed that SC79 also upregulated the expression of kdrl, flt1, and vegfaa, indicating that PI3K/AKT pathway activation promotes angiogenesis by enhancing VEGF and its receptor expression.

Fig. 4.

Impact of SC79 alone and in combined with TKIs on angiogenesis-related gene expression in zebrafish. (A) Relative expression of the kdrl gene after treatment with SC79 combined with TKIs; (B) Relative expression of the flt1 gene after treatment with SC79 combined with TKIs; (C) Relative expression of the vegfaa gene after treatment with SC79 combined with TKIs; (D) Relative expression of the akt2 gene after treatment with SC79 combined with TKIs; (E) Relative expression of the pik3r1 gene after treatment with SC79 combined with TKIs. Compared to the control group, *p < 0.05, **p < 0.01, ***p < 0.001. Each experiment was performed independently three times (n = 3)

Compared to SC79 monotherapy, all three TKIs in combination treatment significantly downregulated kdrl expression (Dovitinib: 0.371 ± 0.020, p < 0.001; Lapatinib: 0.451 ± 0.046, p < 0.001; Telatinib: 0.332 ± 0.019, p < 0.001), indicating that inhibition of VEGFR2-mediated signaling represents a common mechanism.

Furthermore, both Dovitinib (0.769 ± 0.027, p < 0.05) and Telatinib (0.458 ± 0.076, p < 0.01) significantly reduced flt1 (VEGFR1) expression, while Lapatinib showed no significant effect on flt1 levels. This differential regulation of VEGFR1 may partially explain the observed variations in compound efficacy.

Regarding downstream PI3K/AKT pathway, both Lapatinib and Telatinib significantly downregulated akt2 (Lapatinib: 0.843 ± 0.018, p < 0.05; Telatinib: 0.785 ± 0.033, p < 0.05) and pik3r1 (Lapatinib: 0.929 ± 0.018, p < 0.05; Telatinib: 0.814 ± 0.027, p < 0.01) expression compared to SC79 monotherapy. In contrast, Dovitinib showed no significant effects on these genes, suggesting its primary mechanism involves upstream VEGF receptor inhibition rather than direct PI3K/AKT pathway modulation.

Notably, Lapatinib uniquely suppressed vegfaa expression (0.700 ± 0.060 vs. SC79 alone 1.00 ± 0.019, p < 0.01), while Dovitinib and Telatinib exhibited no such effect. This distinct inhibition of VEGF-A may contribute to Lapatinib’s ability to counteract SC79-induced angiogenesis, despite its limited effects on vascular development.

Discussion

This study evaluated the anti-angiogenic effects of three TKIs (Dovitinib, Lapatinib, and Telatinib) using a zebrafish model and investigated the regulatory role of the PI3K/AKT pathway activator SC79 in this process. The results demonstrated that Dovitinib and Telatinib significantly inhibited angiogenesis in zebrafish, whereas Lapatinib exhibited relatively weaker inhibitory effects. Furthermore, SC79, as a PI3K/AKT pathway activator, promoted angiogenesis by upregulating vascular-related gene expression and partially rescued the inhibitory effects of these TKIs.

Dovitinib and Telatinib significantly inhibited both the length and number of ISVs in zebrafish, consistent with their multi-target inhibitory effects on angiogenesis-related receptors including VEGFR and FGFR [7–9]. Our data revealed that Dovitinib at 0.488 µM reduced the number of complete ISVs from 27.2 ± 0.20 (control) to 22.6 ± 1.07 (16.9% reduction) and the length from 288 ± 6.11 pixels to 245 ± 8.68 pixels (14.9% reduction), Telatinib at 0.031 µM demonstrated superior efficacy, reducing the number of complete ISVs to 18.0 ± 1.14 (33.8% reduction), and the length to 221 ± 7.34 pixels (23.3% reduction). This dose-dependent inhibition pattern confirms these TKIs act through specific targeting rather than non-specific toxicity. These findings align with previous reports of Dovitinib’s anti-angiogenic activity in hepatocellular carcinoma and triple-negative breast cancer models [8, 9], as well as Telatinib’s established potency as a VEGFR inhibitor [5]. Notably, morphological analysis showed that while TKIs-treated embryos exhibited reduceing ISVs’ parameters, DLAVs remained intact and embryonic body size showed no significant difference versus controls. This confirms the observed vascular inhibition represents specific anti-angiogenic effects rather than generalized developmental delay.

The novelty of this study lies in the systematic comparison of three distinct TKIs under identical experimental conditions and the use of SC79 to simulate clinical resistance scenarios. According to clinical evidence, approximately 5% of TKI resistance cases are associated with PI3K/AKT pathway activation, making our experimental model particularly relevant for understanding resistance mechanisms and developing combination therapeutic strategies.

Gene expression analysis further elucidated the distinct molecular mechanisms through which these TKIs exert their anti-angiogenic effects. Our results revealed differential targeting strategies among the three TKIs. Dovitinib primarily downregulated flt1 expression (0.738 ± 0.009 vs. control 1.00 ± 0.045, p < 0.01). Notably, Dovitinib exhibited stronger flt1 inhibition at lower concentrations, which may reflect compensatory mechanisms triggered by its multi-target profile affecting topoisomerase I and II in addition to receptor tyrosine kinases [7]. Telatinib exhibited broader suppression, significantly reducing the expression of kdrl (0.708 ± 0.015 vs. control 1.00 ± 0.049, p < 0.05), flt1 (0.419 ± 0.014 vs. control 1.00 ± 0.045, p < 0.001) and akt2 (0.745 ± 0.033 vs. control 1.00 ± 0.040, p < 0.01). Telatinib’s simultaneous targeting of multiple components, including VEGF receptors and PI3K/AKT pathway elements, explains its superior anti-angiogenic efficacy and suggests potential advantages in preventing resistance development. These differential gene expression patterns suggest compound-specific mechanisms of action. VEGFR2 (kdrl), as the primary VEGF receptor, mediates endothelial cell proliferation, migration and survival [1, 3]. VEGFR1 (flt1) primarily regulates angiogenic sprouting and endothelial maturation [23]. Telatinib’s potent dual inhibition of both receptors (particularly 58% flt1 suppression) likely underlies its superior anti-angiogenic efficacy. Its additional suppression of akt2 indicates direct PI3K/AKT pathway modulation, consistent with this pathway’s established role in angiogenesis [3, 14, 15].

As a dual EGFR/HER2-targeting TKI, Lapatinib showed no significant inhibitory effects on zebrafish angiogenesis in this study. Even at its maximum tolerated concentration (1.95 µM), neither the number and length of ISVs nor gene expression exhibited statistically significant differences compared to controls. This observation aligns with Lapatinib’s primary targeting of EGFR rather than VEGFRs [11, 12]. Notably, while previous studies reported Lapatinib’s efficacy in suppressing VEGF expression and neovascularization in corneal angiogenesis models [12], such effects likely occur through indirect EGFR-VEGF crosstalk mechanisms that may require higher drug concentrations and longer treatment durations.However, under SC79-activated PI3K/AKT conditions, Lapatinib demonstrated the ability to suppress VEGF ligand expression, suggesting it may be more effective in diseases with specific signaling activation profiles, which is consistent with its clinical use in HER2-positive cancers where PI3K/AKT activation is common.

A key finding of this study is that the PI3K/AKT pathway activator SC79 not only promotes zebrafish angiogenesis but also partially rescues TKI-mediated inhibition. Treatment with SC79 (2.44 nM) significantly increased the number of ISVs (24.6 ± 0.306 vs. control 22.9 ± 0.407, p < 0.01) and the length (632 ± 11.8 pixels vs. control 575 ± 17.6 pixels, p < 0.05). Gene expression profiling revealed SC79 markedly upregulated multiple angiogenesis-related genes, such as kdrl (2.03 fold, p < 0.001), flt1 (1.31 fold, p < 0.05), vegfaa (1.45 fold, p < 0.01), akt2 (1.41 fold, p < 0.05), pik3r1 (1.41 fold, p < 0.01). These results demonstrate that SC79 enhances angiogenic signaling through PI3K/AKT pathway activation [3, 16]. As a specific AKT activator, SC79 has shown protective effects in various disease models [16–18]. Our findings further confirm its pro-angiogenic role, aligning with Wang et al.‘s report that Catalpol promotes angiogenesis via the VEGF-PI3K/AKT pathway [4].

Co-treatment experiments with SC79 and TKIs revealed that while SC79 could partially rescue the anti-angiogenic effects of TKIs, the degree of reversal varied significantly among compounds. Compared to SC79 monotherapy, SC79 combined with Dovitinib reduced the number of ISVs by 8.5% (22.5 ± 0.671 vs. 24.6 ± 0.306, p < 0.05) and a 13.8% decrease in length (545 ± 7.89 vs. 632 ± 11.8 pixels, p < 0.001). SC79 combined with Telatinib reduced 26.4% in the number of ISVs (18.1 ± 0.657 vs. 24.6 ± 0.306, p < 0.001) and 27.8% decrease in length (456 ± 14.2 vs. 632 ± 11.8 pixels, p < 0.001). SC79 combined with Lapatinib showed no significant effect on the number of ISVs, but reduced 9.5% in the length of ISV (572 ± 16.4 vs. 632 ± 11.8 pixels, p < 0.01). These results demonstrate that PI3K/AKT pathway activation by SC79 can partially, but not completely, overcome TKIs-mediated angiogenesis inhibition, particularly for Telatinib, which maintained the strongest suppression even in the presence of SC79.

Gene expression analysis uncovered finer mechanistic insights into the differential effects of TKIs. All TKIs cotreated with SC79 significantly downregulated kdrl expression versus SC79 treated alone, this confirms VEGFR2 as a common target for TKIs-mediated angiogenesis inhibition. In addition, SC79 cotreated with Dovitinib downregulated flt1, SC79 cotreated with Telatinib suppressed flt1, akt2 and pik3r1, SC79 cotreated with Lapatinib uniquely inhibited vegfaa, alongside akt2 and pik3r1. These differential patterns reveal distinct mechanisms, Dovitinib primarily targets VEGF receptors, Telatinib additionally modulates PI3K/AKT pathway components and Lapatinib, while minimally effective alone, suppresses VEGF ligand and downstream effectors in SC79-activated conditions. The results demonstrate that TKIs inhibit angiogenesis by suppressing receptor tyrosine kinases (e.g., VEGFRs) and downstream PI3K/AKT signaling [3, 15, 24]. SC79, as an AKT activator, bypasses receptor-level inhibition by directly promoting AKT phosphorylation, thereby partially rescuing TKIs effects [16, 17]. The observed variations in SC79-mediated reversal efficacy among TKIs likely stem from their differential target specificity and inhibitory potency, such as Telatinib maintains strong anti-angiogenic activity even with SC79 co-treatment due to its superior VEGFR inhibitory potency [5]. While Lapatinib primarily targets EGFR/HER2 with weak direct VEGFR inhibition [11, 12], it demonstrates a novel capacity to suppress vegfaa expression under SC79-induced PI3K/AKT activation. This phenomenon deserves further exploration.

The key role of the PI3K/AKT pathway in angiogenesis has been extensively studied [14, 15, 25]. This pathway participates in various stages of angiogenesis by regulating endothelial cell proliferation, migration, and survival [3, 14]. Our study found that SC79 upregulated the expression of PI3K/AKT pathway related genes such as akt2 and pik3r1, further confirming the promoting role of AKT activation in angiogenesis. In addition, we also observed that SC79 can upregulate the expression of vegfaa, indicating that AKT activation may enhance VEGF signaling through a positive feedback mechanism [3], which is consistent with the results reported in studies that the PI3K/AKT pathway can regulate VEGF expression [1, 3].

It is worth noting that our research mainly focuses on physiological angiogenesis during development, while there may be differences in the regulation of signaling pathways in pathological angiogenesis related to diseases [1, 3]. For example, in the tumor microenvironment, excessive activation of VEGF signaling can lead to abnormal angiogenesis, forming a structurally disordered and dysfunctional vascular network [3]. The role of the PI3K/AKT pathway in tumor angiogenesis is more complex, affecting not only endothelial cells but also tumor cells and immune cells, jointly shaping the tumor microenvironment [15, 25]. Therefore, when extending the results of this study to pathological angiogenesis, these differences need to be considered.

Several limitations of this study warrant discussion and future investigation. Firstly, our conclusions regarding pathway modulation are primarily based on mRNA expression analysis. While gene expression changes provide valuable mechanistic insights, protein-level validation through Western blotting for phosphorylated AKT, VEGFR2, and ERK would strengthen these conclusions. Future studies should incorporate immunofluorescence analysis to visualize spatial distribution of pathway activation in vascular endothelial cells. Secondly, the zebrafish model, while excellent for developmental angiogenesis studies, may not fully recapitulate the complex tumor microenvironment where these TKIs are clinically applied. The temporal dynamics of drug action may also differ between embryonic development (48 h) and chronic disease states. Extended treatment protocols (72–96 h) may be necessary to observe the full spectrum of EGFR-VEGF crosstalk effects, particularly for Lapatinib. Thirdly, we focus on the PI3K/AKT pathway, while mechanistically important, representing only one component of the complex angiogenic network. As highlighted by Claesson-Welsh and Welsh [3], angiogenesis involves intricate interactions between VEGF, MAPK/ERK, Notch, and other signaling pathways. Future studies should examine these additional pathways to provide a more comprehensive mechanistic understanding. Fourthly, while our SC79 model provides insights into PI3K/AKT-mediated resistance, it does not capture other resistance mechanisms such as MET amplification or ErbB2 overexpression reported in clinical settings. Establishing zebrafish models with these specific resistance mutations would provide more clinically relevant insights.

Conclusions

Overall, this study revealed significant inhibitory effects of Dovitinib and Telatinib on zebrafish angiogenesis, and confirmed the promoting role of PI3K/AKT pathway activator SC79 in regulating angiogenesis. Our findings provide a new approach for targeted therapy of diseases related to abnormal angiogenesis, which may achieve precise regulation of angiogenesis by regulating the balance between TKIs and the PI3K/AKT signaling pathway [26–28]. Future research directions include: (1) validating the anti angiogenic effects of these TKIs in disease-specific models; (2) Explore the combined application strategy of TKIs with other signaling pathway modulators; (3) Develop angiogenesis regulatory drugs specific to different diseases [2, 22].

Abbreviations

TKIs

Tyrosine kinase inhibitors

ISVs

Intersegmental vessels

MTC

Maximum tolerated concentration

VEGF

Vascular endothelial growth factor

VEGFRs

Vascular endothelial growth factor receptors

DA

Dorsal aorta

hpf

Hours post-fertilization

DLAV

Dorsal longitudinal anastomotic vessel

Author contributions

YL: Conceptualization, Data curation and Analysis, Funding acquisition, Methodology, Project administration, Writing-original draft, Writing-review and editing. LX: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing-review and editing.

Funding

This study was supported by funding from the Medical Health Science and Technology Project of Zhejiang Province (grant no. 2024KY364).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Transgenic Fli-1:EGFP and wild-type AB zebrafish were bred and provided by the Hangzhou Hunter Biotech limited liability company. The use of experimental animals was permitted under license number: SYXK (Zhe) 2022-0004. Husbandry management met the requirements of the international AAALAC accreditation (Certification No.: 001458). Ethical review by the Institutional Animal Care and Use Committee (IACUC) was approved under protocol number: IACUC-2023-6895-01.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.