Steppogenin exhibits antiangiogenic activity through inhibition of DLL4 and Notch1 in endothelial cells

doi:10.21203/rs.3.rs-4174241/v1

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Steppogenin exhibits antiangiogenic activity through inhibition of DLL4 and Notch1 in endothelial cells

https://doi.org/10.21203/rs.3.rs-4174241/v1

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In vascular sprouting, tip endothelial cells (ECs) express delta-like 4 (DLL4), and stalk ECs express neurogenic locus notch homolog protein 1 (NOTCH1). The DLL4/NOTCH1 signaling regulates EC migration and proliferation in angiogenesis. Steppogenin (2), a flavanone isolated from Morus alba L., has a significant inhibitory property against DLL4 in ECs. This study aimed to identify natural compounds that can inhibit the DLL4/NOTCH1 signaling pathway in the presence of VEGF in ECs. Ten natural compounds including flavanone derivatives were screened. 2 inhibited DLL4 and NOTCH1 activities. However, sanggenon F (4) only suppressed DLL4 activity, and dehydrovomifoliol (9) inhibited NOTCH1 activity alone. The inhibitory effects of sanggenon F (4) and steppogenin (2) against EC migration was better than those of dehydrovomifoliol (9). However, EC proliferation was suppressed by steppogenin (2), sanggenon F (4), and dehydrovomifoliol (9). Therefore, DLL4 had a better regulatory effect on EC migration than NOTCH1. Dehydrovomifoliol inhibited EC sprouting by 60% compared with VEGF alone. Compared to 9, 2 and 4 showed more inhibitory activity on 3D sprouting ability, thereby indicating that DLL4 activity strongly mediates EC sprouting in angiogenesis. DLL4 and NOTCH1 inhibition by steppogenin significantly enhanced antiangiogenic activity. Further, DLL4 and NOTCH1 inhibition might be more efficient than DLL4 or NOTCH1 inhibition alone for treating angiogenic diseases, such as cancer.

steppogenin

VEGF

DLL4

NOTCH1

sprouting angiogenesis

Angiogenesis is a complex biological process in which new blood vessels are formed from pre-existing blood vessels via the vascular sprouting of endothelial cells (ECs) and the splitting ECs, called intussuceptive angiogenesis [1, 2]. It plays a fundamental role in both normal physiology and disease [3, 4]. In specific pathological conditions, such as cancer, age-related macular degeneration, and chronic inflammatory diseases, angiogenesis can be dysregulated and excessive [5]. Newly formed blood vessels supply oxygen and nutrients to the tissues; thereby, facilitating its expansion and spread by releasing signaling molecules, referred to as angiogenic factors [3].

In ECs, the tip cell and stalk cell are two distinct cellular phenotypes involved in angiogenesis [6]. Tip cells are specialized ECs that are responsible for the sprouting of new blood vessels. They are found at the leading edge or the tip of growing blood vessel sprouts. These cells are highly migratory and have a more invasive phenotype than stalk cells. Further, they are guided by various signaling molecules and cues, such as vascular endothelial growth factor (VEGF) gradients. In contrast, stalk cells respond to guidance cues from the tip cells and help establish and maintain the integrity of the vessel structure during angiogenesis. These cells have a more proliferative phenotype than tip cells, and they cleave to generate new ECs, thereby contributing to vessel growth. The interaction and coordination between tip cells and stalk cells are important for the proper formation and remodeling of blood vessels during angiogenesis [6, 7]. Delta-like 4 (DLL4) is expressed on the surface of tip cells, and it acts as a ligand and binds to the neurogenic locus notch homolog protein 1 (NOTCH1) receptor in neighboring stalk cells. This interaction between DLL4 and NOTCH1 initiates a signaling cascade, which also regulates the expression of other downstream genes involved in angiogenesis [8, 9]. However, some reports on the angiogenic role of DLL4 and/or NOTCH1 have contrasting results. The DLL4/NOTCH1 signaling pathway prevents excessive angiogenesis by inhibiting branching and triggering vessel maturation [10–13]. Meanwhile, blocking this signaling pathway in vivo significantly delays tumor growth by suppressing branching and triggering vessel maturation [14]. Thus, this mechanism can be used in a novel class of anticancer agents.

Our previous report has found that steppogenin, a flavanone compound, inhibits DLL4 expression in ECs [15]. Thus, this study aimed to identify natural compounds having inhibitory activity for DLL4/NOTCH1. And further, we investigated whether DLL4 and/or NOTCH1 inhibition is specific for suppressing certain angiogenic processes with these natural compounds.

Materials

The natural compounds used in this study (isolated from various natural medicinal herbs) were provided by Prof. Nam-In Baek from the nature compound library of Kyung Hee University (College of Life Science, Kyung Hee University, Korea). Selected compounds are, 1: Liquiritigenin, 2: Steppogenin, 3: 3'-O-methyltaxifolin, 4: Sanggenone F, 5: Sanggenol O, 6: Moracin E, 7: Lyoniresinol, 8: Sesamolin, 9: Dehydrovomifoliol, and 10: Panaxytriol. The tested compounds (1–10) were isolated from medicinal plants and were considered as potential compounds. 1 was isolated from the roots of Glycyrrhiza glabra [16], 2–9 from the barks of M. alba [17], and 10 from the roots of Panax ginseng [18]. Each compound was dissolved in dimethyl sulfoxide (DMSO) to produce a 10-mM stock concentration.

Animals

The study is reported in accordance with ARRIVE guidelines. Animal handling and experimental procedures were conducted and approved according to the Guidelines for the Care and Use of Laboratory Animals issued by the Institutional Ethical Animal Care Committee of Kyungpook National University (protocol number: KNU 2022 − 0415). Six-week-old C57BL/6J mice were purchased from Japan SLC, Inc. (Japan).

Cell culture

A permanent human endothelial cell line, EA.hy926 cells [19–22] (ATCC, Manassas, VA, the USA) were cultured in Dulbecco’s Modified Eagle’s Medium (Hyclon, Logan, UT, the USA) containing 10% fetal bovine serum (FBS) (Hyclon) and 1% antibiotics (100 units/mL of penicillin and 100 mg/mL of streptomycin; Invitrogen, Carlsbad, CA, the USA). All cells were cultured under normoxic conditions (5% CO₂ and 95% air).

Cell viability assay

EA.hy926 cells were seeded in a 96-well plate (5 × 10³ cells/well) at 37°C with 95% air and 5% CO₂ saturated humidity conditions. The 0.5% FBS starvation medium containing 1% DMSO was used in the control group. Cells were incubated for an additional 20 and 44 h, and 20 µL of the MTT reagent (0.5 mg/mL) was added to each well for 4 h at 37°C. Next, the culture medium containing the MTT reagent was removed, and 1 mL of DMSO was added to each well to dissolve the formazan.

Dual-luciferase reporter assay

EA.hy926 cells were seeded at a density of 5 × 10³ cells/well on a 96-well plate and cultured at 37°C for 24 h. The cells were co-transfected with pCS2-4xCSL luciferase (a gift from Prof. Hee-Sae Park from Chonnam National University) [23] or pGL3-DLL4 luciferase vector with pRL-SV40 Renilla vector by Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, the USA), as described in a previous study [15]. After being cultured for 6 h at 37°C, the cells were incubated with fresh medium for an additional 18 h at 37°C. Before stimulation with VEGF, the cells were incubated with 10 µM concentrations of natural compounds for 12 h. Then, the cells were incubated for 24 h in the presence or absence of VEGF (10 ng/mL). Two luminescence of each CSL- and DLL4-promoter activity was detected using a microplate reader with the Dual-Luciferase Reporter Assay kit (Promega, Madison, WI, the USA), according to the manufacturer’s instructions. The luciferase activity of natural compounds was normalized to the Renilla luciferase activity [24].

Cell proliferation assay

The cell proliferation assay was performed using the colorimetric BrdU proliferation kit, according to the manufacturer’s instructions (Roche, IN, the USA). Briefly, 5 x 10³ EA.hy926 cells/well were seeded on a 96-well plate. On the next day, the cells were pretreated with the vehicle (DMSO) or natural compounds in DMEM containing 0.5% FBS for 12 h. Cells were then stimulated with human recombinant VEGF-A (20 ng/mL, Miltenyi Biotec, Germany) for 6 h and labeled with 10 µM of BrdU for 6 h. The BrdU-labeled DNA conjugate was detected via the anti-BrdU-antibody reaction. Absorbance was measured at 370 nm using a microplate reader (Infinite M200 Pro; TECAN, Mannedorf, Switzerland). Optical density could be proportional to cell proliferation.

Wound healing migration assay

Confluent EA.hy926 cells were starved in 0.5% FBS-containing media for 12 h with or without the natural compounds and treated with mitomycin C (0.5 µg/ml) for 1 h. The cells were wounded by scraping with a 200-µl pipette tip, washed with PBS, and treated with or without of VEGF (10 ng/mL) for 12 h. The images of migrated cells were taken at 0 and 12 h using an inverted microscope (ECLIPSE Ts2, Nikon, Japan).

Spheroid sprouting assay

EA.hy926 cells (4 × 10³) were mixed with a 1.2% methylcellulose solution (Sigma-Aldrich) and the M199 medium containing 0.1% FBS. The cell suspension was distributed into a 96-well U-shaped culture plate (BD Biosciences) and cultured overnight. On the next day, the spheroids were harvested and mixed with diluted rat tail collagen (BD Biosciences). One milliliter of the spheroid-containing collagen solution was aliquoted into each well in a prewarmed 24-well culture plate. After 1 h of collagen polymerization at 37°C, VEGF was applied to the polymerized collagen gel. Endothelial sprouting was observed, and an image was taken under the microscope (ECLIPSE Ts2, Nikon, Japan).

Matrigel plug assay

Six-week-old C57BL/6J mice (SLC, Japan) were housed in a specific pathogen-free facility with individual ventilation systems at Kyungpook National University. Matrigel (300 µl) containing heparin (60 U/mL, Sigma, MO, the USA) and VEGF (100 ng/mL) with or without natural compounds (10 µM) were injected subcutaneously into the mice’s flank area using an ice-cold syringe. Seven days after Matrigel inoculation, the mice were euthanized via isoflurane inhalation, and Matrigel plugs were harvested. The extent of vascularization was quantified by measuring the hemoglobin content of the plugs. The hemoglobin content was measured using the Drabkin Reagent Kit 525 (Sigma, MO, the USA). The hemoglobin concentration was calculated from the amount of hemoglobin assayed in parallel.

Statistical analysis

Data were presented as means ± standard deviations (SDs) from at least three samples. All statistical analyses were performed using GraphPad Prism (version 8.0; GraphPad Software, La Jolla, CA). Student’s t-test or one-way ANOVA with Tukey's multiple comparisons was used to compare mean values.

Screening of potential DLL4 and NOTCH1 inhibitors among 10 natural compounds

Based on our previous study, steppogenin (2), a flavanone compound isolated from the root bark of Morus alba L., has an inhibitory effect against DLL4 in ECs [15]. Thus, we selected three flavanones (liquiritigenin [1], 3'-O-methyltaxifolin [3], sanggenone F [4]) or phenolic compounds (sanggenol O [5], moracin E [6], lyoniresinol [7], and sesamolin [8]) and evaluated their DLL4/NOTCH1 inhibitory activity in ECs during angiogenic sprouting. We also screened other natural compounds (dehydrovomifoliol [9], panaxytriol [10]) to compare flavanones and phenolics with different structural derivatives (Fig. 1). The tested compounds (1–10) were isolated from medicinal plants and were considered as potential compounds. 1 was isolated from the roots of Glycyrrhiza glabra [16], 2–9 from the barks of M. alba [17], and 10 from the roots of Panax ginseng [18].

Initially, we evaluated the cytotoxicity of the 10 compounds in ECs at 10 µM. There were no cytotoxic compounds in ECs (Fig. 2A). Next, to identify the inhibitors of DLL4 and NOTCH1 in ECs, the dual-luciferase reporter assay was performed with 10 natural compounds using the promoter activity of DLL4 as a tip cell marker and the CSL promoter activity of NOTCH1 as a stalk cell marker in the presence of VEGF. Among the phenolic compounds (1–8), 2, 4, and 6 presented with 88%, 70%, and 47% inhibitory activity, respectively, against DLL4 (Fig. 2B). Only 2 had a 55% inhibitory activity against NOTCH1, and only 9 presented with 39% inhibitory activity (Fig. 2C). Results showed that 2 and 4 did not present with the 2-hydroxyl group on their flavanone structure. Hence, this moiety is important in the inhibitory activity against DLL4 and NOTCH1.

DLL4 versus NOTCH1 inhibition against EC migration

4 was selected as an effective DLL4 inhibitor, 9 as a NOTCH1 inhibitor, and 2 as a dual inhibitor of DLL4 and NOTCH1. Next, to investigate cells with a greater contribution to vascular sprouting, the wound healing migration, proliferation, and three-dimensional sprouting assays were performed after treatment with these natural compounds. In the wounding migration assay, 4 inhibited at 50% compared to VEGF-treated group, whereas 9 inhibited at 22% on EC migration. Similar to 4, 2 inhibited VEGF-induced migration (Fig. 3A). Therefore, we can suggest that the NOTCH1 inhibitory effect of 2 might not regulate EC migration. In contrast, EC proliferation was inhibited at a similar degree by all these molecules (2, 4, 9) at an inhibition rate of approximately 50% (Fig. 3B). Therefore, DLL4 initially mediates EC migration. Then, NOTCH1 regulates stalk cell proliferation. Moreover, the inhibition of both DLL4 and NOTCH1 may not synergistically potentiate EC migration and proliferation, respectively.

Inhibition of in vitro and in vivo angiogenesis by suppressing DLL4 and/or NOTCH1 activity

Sprouting is a coordinated behavior of degrading matrix, migration, proliferation, and venule formations in ECs [25]. Therefore, we tested the sprouting activity of ECs by these natural compound inhibitors in vitro and in mice. EC spheroids were treated with VEGF and the natural compounds in the presence of VEGF. Further, VEGF significantly increased the number of spouts from spheroids. However, 2, 4, and 9 significantly inhibited VEGF-induced sprouting almost to the basal control level. 2 had the strongest inhibitory activity among the three compounds (Fig. 4). For the in vivo confirmation of these results, the Matrigel plug assay was performed on mice. VEGF significantly increased the formation of neovessels inside the Matrigel plug from the neighboring blood vessels. Nevertheless, 2, 4, and 9 significantly inhibited VEGF-induced angiogenesis in vivo (Fig. 5). 2 had the strongest effect on in vitro sprouting and in vivo angiogenesis. Hence, the inhibition of DLL4 and/or NOTCH1 significantly suppressed VEGF-induced in vivo and in vitro sprouting angiogenesis.

Understanding angiogenesis has led to the development of therapeutic approaches that target this process [7, 26]. In cancer development, antiangiogenic therapies aim to inhibit the formation of new blood vessels, thereby starving and preventing the growth of tumors. These treatments have been successfully used in various types of cancers and other diseases characterized by excessive angiogenesis [27]. During EC sprouting from the pre-existing blood vessel, neighboring tip and stalk cells communicate via DLL4–NOTCH1 interaction. In response to VEGF, DLL4 is dominantly expressed in tip cells, which stimulates adjacent cells to express NOTCH1 target genes involved in proliferation and angiogenic activity [6, 7, 28]. The current study initially screened natural compounds with an inhibitory activity against DLL4 and/or NOTCH expression. Moreover, it investigated whether inhibitory compounds against DLL4 and/or NOTCH1 have a distinct role in migration, proliferation, and sprouting angiogenesis in vitro and in vivo angiogenesis. Based on our results, several effective natural compounds inhibited DLL4 and/or NOTCH1 by > 50% at 10 µM. Compared with flavanone compounds, nonflavanone compounds had a weaker inhibitory activity against DLL4 and/or NOTCH1 (Fig. 2). Flavanone compounds are part of a flavonoid class, which has a wide range of biological activities, such as antiinflammatory [29], antibacterial [30], anticancer [31], and antioxidant [32] activities. Thus, the flavonoid structure has been considered a biologically effective moiety [33]. This is one of the reasons why flavanone compounds are more effective than nonflavanone compounds in angiogenesis based on our results. For example, soy isoflavone genistein can be a potent angiogenesis inhibitor [34]. From our house library, we found that 2 and 4 were significantly effective in inhibiting DLL4. However, among the other compounds, only 9 inhibited NOTCH1. Interestingly, 2, previously considered as an inhibitor of DLL4 in ECs and hypoxia-inducible factor (HIF)-1α in cancer cells [15], inhibited NOTCH1 in ECs. Further 2, which is also known as (+)-norartocarpanone, has been used for centuries in traditional Chinese medicine for treating different conditions, including fever, inflammation, and pain. Moreover, it has an anti-inflammatory activity [35, 36]. Moreover, other flavonoids have an antioxidant activity [33]. Therefore, they might have an antioxidant or other various biological properties that induce antiangiogenic function. Here, we performed the biological activities, such as migration, proliferation, in vitro sprouting and in vivo angiogenesis assay based on the inhibitory activity of the natural compounds against DLL4 and NOTCH1 activity. As natural compounds have multiple biological responses in a context dependent manners and can have multiple targets [37], the molecular targets of effective compounds that we used here should be further evaluated to identify their mode of action.

The current study aimed to investigate the effect of the specific inhibition of tip and/or stalk cells on the migration, proliferation, and sprouting of ECs. Using natural compounds with inhibitory properties against DLL4 and NOTCH1, we found that migration ability can be regulated more efficiently by tip cells, and proliferative capacity can be mediated by stalk cells. As DLL4 can be an upstream regulator in EC sprouting, inhibition of DLL4 can then suppress neighboring cell NOTCH1 activity. As observed in the effect of compound 4 in DLL4 inhibition might affect the cell proliferation rate, even it has no effect on the NOTCH1 activity. However, the inhibition of both tip and stalk cells is more effective in sprouting angiogenesis even it is not synergistic.

In conclusion, DLL4 and/or NOTCH1 activities are inhibited by some natural compounds. Therefore, they can be candidate small molecules for the treatment of angiogenic diseases by suppressing EC sprouting in angiogenesis.

VEGF, vascular endothelial growth factor; DLL-4, delta-like protein 4; NOTCH1, neurogenic locus notch homolog protein 1; NICD, notch-intracellular domain

Consent for publication- Not applicable

Availability of data and materials

The data produced and analyzed in this study are available from the corresponding author upon reasonable request.

Competing interests

All authors certify that they have no relevant financial or non-finantial interests to disclose.

Funding

This study was supported by NRF-2020R1A5A2017323 from the Korean Ministry of SIT.

Author’s contribution

S.H. Ha: Data acquisition and analysis; J. Yu: Data presentation and analysis; H.G. Kim: Natural product isolation from the medicinal plants; Se Ha Kim: Data acquisition by experimentation; N.I. Baek: Compound curation and supervision; J.H. Jung: Structure curation and review; J. A. Kim: Conceptualization, review and editing, and Y.M. Lee: Conceptualization, funding, supervision, writing-review and editing.

Acknowledgements

Not applicable.

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No competing interests reported.

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Steppogenin exhibits antiangiogenic activity through inhibition of DLL4 and Notch1 in endothelial cells

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Version 1

Abstract

Figures

Introduction

Materials and Methods

Materials

Animals

Cell culture

Cell viability assay

Dual-luciferase reporter assay

Cell proliferation assay

Wound healing migration assay

Spheroid sprouting assay

Matrigel plug assay

Statistical analysis

Results

Screening of potential DLL4 and NOTCH1 inhibitors among 10 natural compounds

DLL4 versus NOTCH1 inhibition against EC migration

Discussion

Abbreviations

Declarations

References

Additional Declarations

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