Bruton’s tyrosine kinase (BTK) is a Tec family tyrosine kinase that plays an essential part in the B cell receptor (BCR) signaling pathway, which is responsible for proliferation, survival, and maturation (Satterthwaite and Witte, 2000; Mohamed et al., 2009; Buggy and Elias, 2012). Ibrutinib is an orally bioavailable, irreversible selective BTK inhibitor, which has demonstrated impressive therapeutic effects in patients with B cell malignancies, such as mantle cell lymphoma and chronic lymphocytic leukemia (Byrd et al., 2013; Wang et al., 2013; Herman et al., 2014; de Rooij et al., 2015). Although ibrutinib is generally well tolerated, toxicities and adverse events have been reported (Paydas, 2019; Lasica and Tam, 2020), including atrial fibrillation (Thompson et al., 2016; Shanafelt et al., 2017), bleeding (Kunk et al., 2016; Caron et al., 2017), hypertension (Dickerson et al., 2019), and infections (Sun et al., 2015). The undesirable side effects of ibrutinib can be partly attributed to the off-target effects caused by non-specific interactions with other kinases. Besides BTK, ibrutinib also binds to proteins that are involved in other important signaling pathways, such as bone marrow expressed kinase (BMX), epidermal growth factor receptor (EGFR), interleukin-2-inducible T cell kinase (ITK), and Janus kinase 3 (JAK3) (Liang et al., 2018; Kim, 2019). Thus, more studies on the toxicological effect of ibrutinib in living organisms are needed to provide clinical guidelines for ibrutinib application.

The formation of the cardiovascular system plays a pivotal role in the development of the vertebrate embryo. During embryogenesis, hemangioblasts give rise to two cell populations, primitive hematopoietic cells, and vascular endothelial precursor cells, which are also called angioblasts (Eilken et al., 2009; Lancrin et al., 2009). The development of the vascular system involves two processes, vasculogenesis and angiogenesis. Vasculogenesis defines the process by which angioblasts form de novo blood vessels, while angiogenesis is the formation of new blood vessels sprouting from pre-existing ones (Risau et al., 1988). Several key factors are known to be crucial for vascular formation. Previous studies demonstrate that the vascular endothelial growth factor (VEGF) family and their receptors (VEGFR) are required for both vasculogenesis and angiogenesis (Carmeliet et al., 1996; Ferrara et al., 2003). Vascular development, especially angiogenesis, is closely related to the pathogenesis of several chronic diseases, such as cancer, arthritis, and diabetic retinopathy (Carmeliet, 2003; Carmeliet, 2005). Angiogenesis is an effective target in the therapies of these diseases. On the other hand, the clinical uses of anti-angiogenic agents are associated with the increased risk of vascular toxicities, including bleeding, hypertension, and thrombosis (Kabbinavar et al., 2003; Motzer et al., 2006; Horsley et al., 2012). A recent study suggests that ibrutinib-treated macrophages reduce the tube formation of endothelial cells (ECs) in vitro (Ping et al., 2017), and structure-based drug repositioning predicts that ibrutinib may be a VEGFR2 inhibitor (Adasme et al., 2020). However, there is no further evidence or in vivo study to confirm whether ibrutinib can influence vascular development.

Zebrafish has been widely used as a model organism in studying embryonic development and the toxicological effects of drugs (Sukardi et al., 2011; MacRae and Peterson, 2015). Furthermore, zebrafish are known to be a successful model for investigating vascular development because the basic framework of vascular anatomy and the molecular mechanisms of vascular formation are conserved between zebrafish and humans (Ellertsdottir et al., 2010; Gore et al., 2012). In the present study, we demonstrate the adverse effects of ibrutinib on vascular development in zebrafish embryos. We reveal that ibrutinib strongly suppresses the sprouting and formation of new vessels and reduces vascular lumen size. In addition, we verify that ibrutinib exposure leads to abated proliferation and excessive apoptosis of vascular endothelial cells (VECs). Moreover, we show that the expression of crucial genes associated with vascular development is altered by ibrutinib. Overall, this study demonstrates that ibrutinib exposure exhibits vascular toxicity during embryogenesis, providing an important basis for the safety guidelines of ibrutinib in clinical application.

Ibrutinib is widely used in the treatment of patients with B cell malignancies due to its marked efficacy, high safety, and good tolerance in clinical testing (Brown, 2018). Therefore, studies of ibrutinib mainly focus on patients or animal disease models (Dubovsky et al., 2014; Woyach et al., 2014; Singh et al., 2018). However, because adverse effects have also been demonstrated (Paydas, 2019; Lasica and Tam, 2020), studies on the toxicity of ibrutinib in normal development are needed. In the present study, we investigated the toxicological effect of ibrutinib on embryonic development by using zebrafish as a model organism. The results indicate that ibrutinib has potent developmental toxicity on zebrafish embryos. Embryos treated with ibrutinib show obvious malformation (Figures 1A,B) and could not survive (Figure 1C). Since ibrutinib could bind to proteins in many important signaling pathways, including BTK, BMX, EGFR, ITK, and JAK3 (Liang et al., 2018; Kim, 2019), we speculate that ibrutinib might induce developmental deficiency by affecting some crucial signaling pathways during embryogenesis.

The vascular development of vertebrates is commonly subdivided into two different processes, vasculogenesis, whereby aortas and veins are formed, and angiogenesis, whereby new blood vessels sprout from pre-existing ones (Risau et al., 1988). In zebrafish, vasculogenesis commences at approximately 12 hpf, when angioblasts start migrating from the lateral plate mesoderm to the midline. Subsequently, angioblasts differentiate into arterial precursor cells and venous precursor cells and then form the DA and PCV, respectively, at the 22-somite stage (about 20 hpf). After major axial vessels are established, sprouting angiogenesis occurs from the DA at approximately 22 hpf. These sprouting vessels, which are the future ISVs, grow dorsally and ultimately reach the level of the dorsal neural tube to form the dorsal longitudinal anastomotic vessel (DLAV) by fusing with neighboring vessels at 28–30 hpf (Childs et al., 2002; Hogan and Schulte-Merker, 2017). In this study, embryos treated with ibrutinib at 12 hpf, just before the vasculogenesis initiation, could still form the DA and PCV (Figure 2A), implying that ibrutinib might not affect vasculogenesis. In contrast, the cerebral vasculature and the ISVs in the trunk were strongly impeded (Figures 2A,B), suggesting that angiogenesis was perturbed by ibrutinib. To further explore the time window of ibrutinib inhibiting angiogenesis, embryos were exposed at different stages. Angiogenesis was severely suppressed in embryos exposed with ibrutinib at 12 or 24 hpf as expected (Figures 2C,D), considering that ISV sprouting occurs at around 22 hpf. Interestingly, embryos exposed at 36 hpf, when the primary ISV network is established, also exhibited remarkable angiogenesis deficiency (Figures 2C,D), implying that ibrutinib not only inhibits the sprouting of ISVs but also impaired existing ISVs. Moreover, embryos exposed at 12 hpf showed intact ISV networks when ibrutinib was washed out at 24 hpf, or weakly perturbed angiogenesis when ibrutinib was washed out at 36 hpf (Figures 2C,D). According to these results, we reasoned that ibrutinib might influence single or multiple proteins which were constantly needed for the whole angiogenesis process. Once ibrutinib was removed, the angiogenesis process would recover even if it had been suppressed before. The target protein by which ibrutinib inhibits angiogenesis is not thought to be BTK, the primary target of ibrutinib, because a direct link between BTK and angiogenesis is rarely reported. Besides, we verified this supposition by treatment with spebrutinib, another well-known BTK inhibitor, as well as btk gene knockdown by MO in zebrafish embryos. The effect on angiogenesis was inappreciable in both experiments (Supplementary Figures S1A,B).

The formation and maintenance of blood vessel lumens by VECs are crucial for vascular function. In zebrafish, the DA begins to lumenize shortly after being assembled and forms a tube to allow blood flow at 24–26 hpf, followed by the lumenization of the PCV (Herbert et al., 2009; Ellertsdottir et al., 2010). In our study, the lumen size of the DA and PCV were both reduced in embryos treated with ibrutinib (Figure 3), implying that ibrutinib impaired vascular lumen maintenance. Blood circulation was prohibited in the treated embryos, as no erythrocytes were found in the collapsed blood vessels (Figure 3B). The morphology of VECs seemed to be normal in treated embryos (Figure 3C), thus we speculated that ibrutinib might affect the arrangement and junction of VECs to perturb vascular lumen maintenance. The proliferation and apoptosis of VECs were also examined in ibrutinib-exposed embryos. After treatment, the number of proliferated VECs was normal in the DA, PCV, and CV, but decreased in ISVs (Figures 4A,B), which was in line with the result that ibrutinib inhibited angiogenesis but not vasculogenesis. In contrast, apoptotic VECs were significantly increased in the DA, PCV, and CV, as well as ISVs (Figures 4C,D), suggesting that ibrutinib induced apoptosis of VECs, which might contribute to the impairment of lumen maintenance.

The VEGF/VEGFR signaling pathway is essential for multiple processes of vascular development and conserved among vertebrates. In zebrafish, the initial sprouting of angiogenesis occurs from the DA in a process dependent on the Vegfr2 paralogs, Kdr and Kdrl, as well as Vegfa, indicating that the Vegfa/Vegfr2 signals are critical for ISV formation (Covassin et al., 2006; Bahary et al., 2007). Flt1 also plays an important role in angiogenesis, because the knockdown of the flt1 gene is reported to result in ISV hyperbranching (Krueger et al., 2011). Flt4 is highly expressed in the tip cells of sprouting blood vessels, and the Vegfc/Flt4 pathway is negatively regulated by Notch signaling to limit angiogenesis (Villefranc et al., 2013; Le Guen et al., 2014). A recent study reveals that zebrafish Vegfd genetically interacts with Kdr and Flt4 to modulate angiogenesis (Bower et al., 2017). As indicated by our WISH results, the expression of early vascular marker genes, fli1, flt1, and kdrl, was dramatically suppressed in the sprouting ISVs of ibrutinib-exposed embryos at 24 hpf (Figure 5A), confirming that the primary angiogenesis was inhibited. However, the WISH signals of these genes were increased in the developing DA (Figure 5A). Considering that the sprouting of VECs from the DA was impeded, we reasoned that these VECs were probably accumulated, which led to the increased number of VECs in the DA. The qRT-PCR results revealed that the expression of genes from the VEGFR family, flt1, flt4, kdr, and kdrl, was decreased in the ibrutinib-exposed embryos, but the fold changes were not as great as expected (about 0.6–0.9) (Figure 5B). Moreover, the expression of genes from the VEGF family, vegfaa, vegfab, vegfc, and vegfd, was unaffected by ibrutinib (Supplementary Figure S2).These results indicated that VEGF/VEGFR pathway might not be the only pathway influenced by ibrutinib to impair vascular development. The inhibitory activity of ibrutinib on vascular development was independent of hematopoiesis, as verified by the WISH examination of hematopoietic markers in the treated embryos (Supplementary Figure S3).

In conclusion, we have demonstrated that the BTK inhibitor, ibrutinib, has adverse effects on the vascular development of zebrafish embryos. Ibrutinib exhibited a potent inhibitory activity on angiogenesis, but not vasculogenesis, during embryonic development. We also showed that ibrutinib exposure led to the collapsing of the vascular lumen, as well as abated proliferation and excessive apoptosis of VECs. The expression alteration in vascular development-related genes suggests that ibrutinib might influence the VEGF/VEGFR signaling pathway. However, further study is still needed to identify the precise targets of ibrutinib for perturbing vascular development. To the best of our knowledge, this is the first demonstration that ibrutinib exposure exhibits vascular toxicity in an animal model, and could provide the theoretical basis for safety guidelines in clinical treatment using ibrutinib.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by Ethical Committee of the Southern University of Science and Technology.

KW and HZ conceived and designed the project. KW and QX performed the experiments. KW wrote the manuscript.

This work was supported by a grant from the National Natural Science Foundation of China (31601166 and 31771603) and the Shenzhen Science and Technology Program (JCYJ20170817104455478).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.