Key words
Vitex agnus-castus
- Lamiaceae - endothelial cells - angiogenesis-related cell functions
Abbreviations
CAM:
chorioalantoic membrane
ECM:
extracellular matrix
HMPC:
Committee on Herbal Medicinal Products
HUVECs:
human umbilical vein endothelial cells
MMPs:
matrix metallopeptidases
VEGF:
vascular endothelial growth factor
Y : FMI:
forward migration index Y
Introduction
Vitex agnus-castus L., also referred to as chaste tree or monkʼs pepper, belongs to the family of Lamiaceae.
The plant is originally located in the Mediterranean area, and extracts from fruits
and leaves have been traditionally used to treat pre-menstrual, post-menstrual, and
fertility disorders, among them amenorrhoea or dysmenorrhea, pre-menstrual syndrome,
corpus luteum insufficiency, or infertility since ancient Greek and Roman times [1], [2], [3], [4]. Moreover, a variety of studies suggest that V. agnus-castus shows activity against cancer, inflammation, or osteopenic syndromes but also attribute
an immunomodulatory, antimicrobial, and antifungal impact [5]. According to the HMPC, a hydroethanolic dry extract (ethanol 60%, drug-extract
ratio 6 – 12 : 1) from the fruits of V. agnus-castus can be applied for the
treatment of premenstrual syndrome as well-established use herbal medicinal product.
Other hydroethanolic dry extracts can be used for the relief of minor symptoms in
the days before menstruation (premenstrual syndrome) as traditional use herbal medicinal
product. [https://www.ema.europa.eu/en/medicines/herbal/agni-casti-fructus]. The fruit extracts are comprised of essential oils, iridoids, flavonoids, diterpenes,
tannins, and phenolic compounds. While the extractsʼ mode of action is up to now widely
unknown, studies suggested modulation of the delta and mu opioid receptors [6]. Moreover, in vitro studies indicate that V. agnus-castus extract inhibits prolactin secretion by binding to dopamine receptor D2 [7], [8]. Identification approaches for bioactive compounds in the plant suggested
diterpenes, in particular cleroda-dienols, to be responsible for this dopaminergic
actions of V. agnus-castus
[3].
However, angiogenesis–the formation of new blood vessels from pre-existing capillaries–plays
a crucial role during the menstrual cycle, and potential V. agnus-castus extract-derived effects on this process are of high interest. Up to now, only few
studies focused on the activity of the extract within this context. An in vivo study analyzing V. agnus-castus fruit fractions demonstrated anti-angiogenic properties in a chick CAM assay and
in a zebrafish model [9]. In addition, the performance of an ex vivo rat aortic ring assay using a methanol extract of V. agnus-castus leaves revealed a marked inhibition of sprouting [10]. The identification of V. agnus-castus as being effective to suppress angiogenesis might be beneficial for opening a new
perspective for additional fields of application with regard to angiogenesis-related
pathophysiological conditions such a cancer or chronic
inflammatory diseases.
Angiogenesis is a highly regulated multi-step process. The key events during angiogenesis
include endothelial cell-driven enzymatic degradation of the ECM and endothelial cell
migration and proliferation, followed by sprouting events and finally vessel maturation.
In this study, we made an attempt to shed light on the potential of BNO 1095, an approved
and standardized dry extract from fruits of V. agnus-castus, to affect angiogenesis-related endothelial cell functions in vitro. For the detailed investigation of crucial key steps of in vitro angiogenesis, we used primary HUVECs. Within the scope of this work, we focused on
potential extract-derived effects on undirected and chemotactic endothelial cell migration
and HUVEC proliferation, as well as the impact of BNO 1095 on in vitro tube formation and sprouting from HUVEC spheroids.
Results
Before actions on in vitro angiogenic features were assessed in HUVECs, potential BNO 1095-derived effects on
cell viability were determined. In [Fig. 1 a] and [b], we demonstrate that after 24 h of incubation, the extract neither impaired membrane
integrity nor induced apoptosis in endothelial cells. When incubation times were extended
over a period of up to 72 h, apoptosis as well as membrane integrity impairment were
significantly initiated by an extract concentration of 100 µg/mL ([Fig. 1 a, b]). Due to these findings, for experimental purposes for a treatment period up to
24 h, the extract concentration of 100 µg/mL was not exceeded, while for incubation
periods up to 72 h, only concentrations up to 30 µg/mL were used.
Fig. 1 BNO 1095 does not induce cytotoxic effects in the used experimental setups. To determine
potential extract-derived effects on cell viability, confluent HUVECs were treated
with the indicated concentrations of BNO 1095 for 24, 48, and 72 h. a HUVECs were subsequently analyzed for membrane integrity by measuring LDH release.
The LDH-induced conversion of tetrazolium salt into formazan was determined by absorbance
measurement at 490 nm using a plate reader. The application of a lysis solution (ls)
served as positive control. b For apoptosis measurement, HUVECs were permeabilized and simultaneously treated with
propidium iodide (PI) to determine subdiploidic DNA content in cells by flow cytometry.
Staurosporine served as positive control. Data are expressed as mean ± SD; A: n = 4;
B: n = 3; *p ≤ 0.05 vs. ctrl.
Endothelial cell migration is one of the key features during the process of angiogenesis.
To analyze potential effects of BNO 1095 on endothelial cell motility in vitro, their migratory capacity was analyzed. In a first approach, we focused on effects
of undirected endothelial cell migration. Therefore, a scratch was inflicted into
a HUVEC monolayer, and the cells were allowed to close the gap by undirected migration.
As depicted in [Fig. 2 a], we demonstrate that undirected HUVEC migration was significantly impaired upon
extract treatment (30 and 100 µg/mL) as the cells were not able to close the inflicted
gap. In a second approach, we determined the anti-migratory potential of the extract
on directed migration of endothelial cells. In a Boyden chamber assay, HUVECs were
allowed to migrate in the direction of a chemoattractant (FCS) gradient. The treatment
with 30 and 100 µg/mL of BNO 1095 resulted in a markedly reduced migration towards
the FCS gradient. For 100 µg/mL, this effect was significant ([Fig. 2 b]). For further insights into the effects of the extract on chemotactic migration
of endothelial cells, a migration assay using chemotaxis slides and microscopic monitoring
was performed. Upon extract treatment, HUVECs were allowed to migrate in the direction
of an FCS gradient. Single cell tracking revealed that BNO 1095 strongly attenuated
the migration capacity of HUVECs in the direction of the chemoattractant gradient,
as indicated by a significantly reduced forward migration index in direction of the
y-axis (FMI : Y) and velocity. Furthermore, treatment with the extract resulted in
a significantly diminished Euclidean and accumulated distance covered by HUVECs, while
the migration directness remained unimpaired ([Fig. 2 c]). This effect was most prominent when HUVECs were treated with 100 µg/mL of the
extract. To sum up these findings, we can
conclude that BNO 1095 strongly impairs migratory events and motility in HUVECs.
Fig. 2 BNO 1095 attenuates endothelial cell migration. a A scratch was inflicted into a confluent HUVEC monolayer before the cells were treated
with the indicated BNO 1095 concentrations. Starvation medium served as positive control
(not shown) and vehicle control (DMSO 0.1%) served as control (ctrl). The cells were
allowed to migrate into the gap for 12 h before image quantification was performed
using ImageJ. Scale bar: 100 µm. One representative image for each condition is shown.
b 100 000 HUVECs were seeded onto collagen G-coated Transwell inserts in ECGM After
4 h of incubation, the cells were treated with respective concentrations of BNO 1095
or vehicle (DMSO 0.1%) in medium 199 without serum. Subsequently, medium 199 containing
20% FCS was added to the lower compartment for the generation of a chemoattractant
gradient. After 16 h, migrated HUVECs were stained with a crystal violet solution
and air-dried overnight. Removing crystal violet
from cells by acetic acid and absorption measurement at 590 nm allowed the quantification
of migrated cells by a plate reader. FCS as chemoattractant alone served as control.
c Eighteen thousand HUVECs were seeded onto chemotaxis slides. A 20% FCS gradient was
added, and the cells were treated with indicated concentrations of BNO 1095 or vehicle
(DMSO 0.1%). Chemotactic migration proceeded for 20 h. Tracking 30 cells per condition
using ImageJ allowed determination of extract-derived effects on indicated parameters.
Data are expressed as mean ± SD; A: n = 5; *p ≤ 0.05 vs. ctrl; B: n = 4; *p ≤ 0.05
vs. FCS ctrl; C: n = 3; *p ≤ 0.05 vs. FCS ctrl.
During the process of angiogenesis, endothelial cell proliferation is of high importance
to induce the formation of new capillaries. Therefore, we focused in a further approach
on potential extract-derived effects on HUVEC proliferation. Crystal violet staining
of proliferating HUVECs revealed that the extract was able to markedly inhibit the
increase of endothelial cell number with an IC50 of 19 µg/mL ([Fig. 3 a]).
Fig. 3 BNO 1095 inhibits endothelial cell proliferation and tube-like structure formation.
a One thousand five-hundred HUVECs/well were seeded on 96-well plates. After 24 h,
the cells were treated with indicated concentrations of BNO 1095 or vehicle (DMSO
0.1%). Seventy-two h later, HUVECs were fixed and stained using a crystal violet solution.
After air-drying, the crystal violet was dissolved from HUVECs using acetic acid and
absorbance was measured at 590 nm using a plate reader. b Ten thousand HUVECs per well were seeded on growth factor-reduced solidified Matrigel
and treated with indicated concentrations of BNO 1095 or vehicle (DMSO 0.1%). The
formation of capillary-like structures was allowed for 5.5 h before microscopic images
were taken and quantified for the indicated parameters. Data are expressed as mean ± SD;
A: n = 4; *p ≤ 0.05 vs. ctrl; B: n = 4; *p ≤ 0.05 vs. ctrl. Scale bar: 500 µm. One
representative image for each condition is
shown.
The formation of new blood vessels is a highly regulated multi-step process. To study
extract-derived effects on angiogenesis in a well-feasible and reliable in vitro system, a tube formation assay using Matrigel was performed. This basement matrix
enables HUVECs to form angiogenesis-related capillary-like structures that can be
easily quantified. As depicted in [Fig. 3 b], the endothelial cell network formation was successfully induced in control cells
treated with DMSO (vehicle control). The application of increasing BNO 1095 concentrations
strongly and significantly reduced the capacity of HUVECs to form tube-like structures,
as indicated by a concentration-dependent decrease in the number of junctions and
master segments as well as in the total branch length. Of note, the arrangement of
cells to form master segments proceeded upon extract treatment, while the elongation
for extensive branching was strongly attenuated, resulting in
severe impairment of tube-like structure formation in HUVECs.
Since we demonstrated that BNO 1095 is able to substantially reduce angiogenesis-related
endothelial cell functions, we used a robust 3D in vitro method to analyze extract-derived effects on endothelial cell sprouting from HUVEC
spheroids embedded in collagen I. Exploiting the benefits of the ECM component collagen
I and of the VEGF allows a reliable assessment of a potential impact on angiogenic
cell functions in vitro. In [Fig. 4], we demonstrate that the application of VEGF to collagen I-embedded HUVEC spheroids
significantly induced the formation of sprouts. Both sprout number per spheroid and
total sprout number were strongly increased by the treatment with the growth factor.
Pre-incubation of HUVEC spheroids with increasing concentrations of the extract significantly
reduced the number of sprouts per spheroid and the total sprout length in a concentration
dependent way. Interestingly, the application of 100 µg/mL of BNO 1095
decreased the total sprout length almost down to the levels of the vehicle control.
Fig. 4 BNO 1095 reduces endothelial cell sprouting from spheroids. Four hundred HUVECs were
used to form spheroids employing the hanging-drop method. The next day, HUVEC spheroids
were embedded into a 3D rat tail collagen I gel. The spheroids were treated with indicated
concentrations of BNO 1095 or vehicle (DMSO 0.1%) before sprout formation was induced
by the application of VEGF (10 ng/mL). HUVEC spheroids were allowed to form sprouts
for 20 h before they were microscopically analyzed for the indicated parameters using
ImageJ. Data are expressed as mean ± SD; n = 4; *p ≤ 0.05 vs. VEGF. Scale bar: 100 µm.
One representative image for each condition is shown.
Discussion
In the physiological situation, angiogenesis is a highly regulated and coordinated
process. Here, the formation of new blood vessels proceeds during embryonic development,
in the course of wound healing and within the menstrual cycle. Nevertheless, angiogenesis
also plays a pivotal role under pathophysiological conditions. In chronic inflammatory
diseases or cancer, this process is prone to be ongoing and uncontrolled. Tumor growth
and metastasis are highly dependent on constant development of new blood vessels being
indispensable for the supply of nutrients and oxygen and for cancer cell spreading
[11]. Therefore, anti-angiogenic compounds or extracts are of utmost interest for the
treatment against tumor development and might introduce a new aspect for the application
of V. agnus-castus extract. Approved standardized dry extracts of V. agnus-castus fruits are therapeutically used against dysregulation of the menstrual cycle,
premenstrual syndrome, infertility, or mastodynia. Beyond these applications,
only few studies demonstrated V. agnus-castus-derived anti-angiogenic effects in vivo and in vitro. An ex vivo study by Sahib et al. demonstrated that a methanolic leaf extract of V. agnus-castus markedly reduced sprouting from rat aortic rings at 100 µg/mL, while sprout formation
remained widely unimpaired when a chloroform or water extract was applied. In addition,
in an in vitro experiment using HUVECs, they demonstrate that the methanolic leaf extract was able
to reduce endothelial cell proliferation with an IC50 of 80 µg/mL [12]. The same group demonstrated in an in vivo approach that blood vessel growth in the CAM of chicken embryos was considerably
inhibited by the methanolic leaf extract [10]. Within the scope of our study, we found that the used ethanolic extract of
V. agnus-castus potently reduced angiogenesis-related key features in endothelial cells in vitro. With our approach, we are the first who systematically analyzed the potential of
BNO 1095, a standardized dry extract from V. agnus-castus fruits, to interfere with principal events that play a pivotal role during the process
of angiogenesis, among them endothelial cell proliferation, migration (undirected
and chemotactic), and sprouting in vitro.
We found that BNO 1095 reduced the proliferation capacity of HUVECs with an IC50 of 19 µg/mL, indicating an advantage over a methanolic leaf extract. In addition,
we found that BNO 1095 potently inhibited undirected migration of HUVECs already at
30 µg/mL while the administration of 100 µg/mL reduced the migratory capacity of endothelial
cells around by 75%. Moreover, results of a Boyden chamber assay revealed that the
migration in the direction of an FCS gradient was already significantly attenuated
at 30 µg/mL, and when 100 µg/mL of BNO 1095 was applied to the cells, it reduced the
directed migration of HUVECs by about 40%. The performance of a 2D chemotaxis assay
provided deeper insights into the impact of the extract on chemotactic migration of
HUVECs. This approach not only gives novel information of endothelial cell migratory
capacity per se but also sheds light on the impact of the extract on migration efficiency
of the forward migration in the direction
of FCS, the migrated distance, and the velocity of migration. Indeed, BNO 1095
effectively inhibited these important hallmarks of chemotactic migration that are
indispensable for the process of angiogenesis. Of note, the treatment with 100 µg/mL
of the extract reduced forward migration, Euclidean, and accumulated distance as well
as velocity to less than 50% compared to the control. These findings indicate that
important key events of angiogenic functions, endothelial proliferation, and migration
are strongly and significantly inhibited by the extract. In vivo studies employing zebrafish embryos and the chick CAM assay demonstrated that chloroform
and ethyl acetate fractions of a V. agnus-castus fruit extract substantially reduced microvessel formation [9]. In line with this, we show in vitro that the application of BNO 1095 reduced the formation of capillary-like structures
on Matrigel and the formation of VEGF-induced
sprouts from HUVEC spheroids. In the tube formation assay, this effect was detectable
starting at 30 µg/mL. Interestingly, HUVECs were able to align in an assembly typical
for vessel formation but were incapable of elongation, a key feature of stalk cells
in the angiogenic process [13]. Importantly, in a 3D spheroid assay, BNO 1095 demonstrated a much higher inhibitory
activity on HUVEC sprouting starting already at 3 µg/mL. This concentration-dependent
inhibition resulted in a complete blocking of VEGF-activated total sprout length using
100 µg/mL of the extract. In the physiological situation, one of the first events
in angiogenesis is the degradation of ECM components such as collagen, fibronectin,
or laminin, which are enzymatically degraded by MMPs. Studies in a murine in vivo model revealed that V. agnus-castus extract significantly decreased the serum levels of MMP9 [14]. In our experiments,
spheroids were embedded into a collagen gel. If endothelial MMP9 levels might
be down-regulated upon extract treatment, the initiation of sprouting would be impaired.
The formation of reactive oxygen species is known to promote angiogenesis [15]. Experiments using the leukemia cell line HL-60 demonstrated the down-regulation
of Nox2 upon V. agnus-castus extract treatment, indicating potential antioxidant properties of the extract [16]. An in vivo study employing an aging mouse model suggested antioxidant extract activities, as
catalase and superoxide dismutase activity was increased in aging mice by V. agnus-castus
[17]. Therefore, it is conceivable that, at least in part, inhibitory effects of the
extract on in vitro angiogenesis-related cell functions might be attributed to antioxidant actions of
V. agnus-castus.
Beyond the widely known actions of V. agnus-castus for the treatment of menstrual disorders or premenstrual syndrome, by our in vitro studies, we introduced for the first time the beneficial potential of the approved
ethanolic fruit extract to successfully inhibit crucial angiogenesis-related endothelial
cell functions such as proliferation, migration, capillary-like structure formation,
and sprouting from spheroids. These findings might disclose a new therapeutic implementation
of the extract such as for the treatment of cancer or chronic inflammatory diseases
that are characterized by ongoing angiogenesis. Moreover, as premenstrual disorders
have often been ascribed to endometriosis, an anti-angiogenic impact of V. agnus-castus might be beneficial. The development of ectopic endometriosis is strongly dependent
on angiogenesis [18], [19]. Therefore, a variety of studies suggest anti-angiogenic
therapies for the treatment of endometriosis using growth factor inhibitors, statins,
endogenous angiogenesis inhibitors, dopamine agonists, phytochemicals, fumagillin,
and others [20], [21]. Although an anti-angiogenic approach might be associated with a risk of fertility
impairment, a number of angiogenesis-inhibiting compounds have been identified that
do not interfere with follicular development and do not induce side effects in the
female reproductive organs in vivo
[22], [23], [24], [25]. Of note, V. agnus-castus has been described to exhibit dopaminergic actions by binding to the dopamine receptor.
Other dopamine agonists such as quinagolide or cabergoline have also been shown to
exert anti-angiogenic actions for the treatment of endometriosis in vivo
[26]. However, clinical efficacy and success employing an anti-angiogenic strategy within
this context is still unclear.
The main purpose of our in vitro study was the detailed investigation of potential effects of BNO 1095 on distinct
principle endothelial cell functions that are inevitable for angiogenesis-related
processes. Further studies analyzing the signaling pathways endothelial cells utilize
during proliferation, migration, and sprouting will be conducted to get deeper insights
into the action of BNO 1095 in the context of angiogenesis. Moreover, the identification
of bioactive compounds in the extract that are responsible for the actions on distinct
key events of angiogenesis-associated cell functions have to be elucidated in future
studies.
Materials and Methods
BNO 1095 and compounds
BNO 1095 is an ethanolic (70% v/v) fruit extract of V. agnus-castus with a drug extract ratio of 7 – 11 : 1 and the active pharmaceutical ingredient
of the herbal medicinal product Agnucaston. The extract (lot number 770 134) was kindly
provided by Bionorica SE, Neumarkt, Germany. The HPLC fingerprint of the extract (UV
detection at 205 nm) [27] is provided in Fig. 1S (Supporting Information). Human recombinant VEGF165 was purchased from Peprotech. Staurosprine and methylcellulose were from Sigma-Aldrich
Chemie GmbH, and Matrigel Growth Factor Reduced Basement Membrane Matrix as well as
rat tail collagen I were purchased from Corning GmbH.
Extract preparation for in vitro characterization
The extract was dissolved in DMSO (Sigma-Aldrich Chemie GmbH) to a final concentration
of 100 mg/mL by sonication in an ultrasonic water bath (35 kHz) over a period of 30 min
with frequent vortexing. After centrifugation for 10 min at 3000 g, the supernatant was transferred into new tubes and stored in aliquots at − 80 °C
to prevent thaw-freeze-cycles. For experimental purposes, the extract was used up
to a concentration of 100 µg/mL not exceeding a final concentration of 0.1% DMSO.
Cell culture
HUVECs were isolated according to Jaffe et al. [28]. For cultivation, the cells were split in a ratio of 1 : 3 in endothelial cell growth
medium (EASY ECGM; PELOBiotech) supplemented with 10% FCS (Biochrom), 100 U/mL penicillin,
100 µg/mL streptomycin (PAN-Biotech), 2.5 µg/mL amphotericin B (PAN-Biotech), and
a supplement mixture (PELOBiotech) on collagen G (10 µg/mL in PBS, Biochrom)-coated
plastic. The cells were cultivated under constant humidity, at 37 °C and an atmosphere
with 5% CO2 and 95% air. The cells were used for experimental purposes exclusively in passage
3.
Lactate dehydrogenase (LDH) release assay
For the determination of potential extract-derived effects on cell membrane integrity
the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega GmbH) was performed according
to the manufacturerʼs instructions. In brief, confluent HUVECs on 96-well plates were
treated with indicated concentrations of the extract for 24, 48, and 72 h. For positive
control, HUVECs were treated with a lysis solution for the last 45 min of incubation.
Subsequently, 50 µL of cell culture supernatants were added to a new plate and incubated
with 50 µL of substrate solution for 30 min at room temperature and protected from
light. To stop the substrate converting process of the enzyme, 50 µL of stopping solution
was added. The amount of LDH in the cell culture supernatant was measured at 490 nm
using a plate reader (VarioskanFlash, Thermo Fisher Scientific).
Apoptosis assay
To exclude potential extract-derived effects on apoptosis induction in endothelial
cells, an apoptosis assay, according to a method by Nicoletti et al. [29], was performed. Therefore, confluent HUVECs were treated with the indicated concentrations
of BNO 1095 for 24, 48, and 72 h. Staurosporine served as positive control for apoptosis
induction. After each incubation period, cell culture supernatants were collected
and cells were detached before they were incubated in a solution containing Triton
X-100, PI, and sodium citrate at 4 °C overnight. Cells with subdiploidic DNA content
were determined using flow cytometry (FACSVerse, BD Biosciences).
Proliferation assay
In this assay, 1500 HUVECs per well of a collagen G-coated 96-well plate were seeded
in ECGM. Twenty-four h later, the cells were treated at indicated concentrations of
the extract and were allowed to proliferate for 72 h or were fixed with a methanol-ethanol
(2 : 1) solution for 10 min. After 72 h of extract incubation, HUVECs were fixed with
methanol-ethanol for 10 min before they were stained using a crystal violet solution
containing 20% methanol for 15 min. After drying, DNA-bound crystal violet was resolved
in 20% acetic acid, and the number of cells was determined at 590 nm using a plate
reader (SPECTRAFluor Plus, Tecan).
Undirected migration
For the analysis of extract-induced effects on undirected endothelial cell migration,
a scratch was inflicted into a confluent HUVEC monolayer using a pipette tip. After
removal of the detached cells, HUVECs were treated with indicated concentrations of
the extract in ECGM. For positive control, a starvation medium (medium 199 supplemented
with 1% FCS, 100 U/mL penicillin, and 100 µg/mL streptomycin) was used. Subsequently,
the cells were allowed to migrate for 12 h until the gap inflicted into the control
cells (ECGM containing 0.1% DMSO) was closed by endothelial cell migration. The effect
of BNO 1095 on the migratory capacity of HUVECs was determined by ImageJ (software
version 1.49 k).
Directed migration: Boyden chamber assay
To determine potential effects upon extract treatment on the migration of endothelial
cells in the direction of a chemoattractant gradient, a Boyden chamber assay using
Transwell inserts was performed. Therefore, 100 000 cells per well were seeded in
ECGM on collagen G-coated Transwell inserts (Corning GmbH HQ, growth area 0.33 cm2, 8 µm pore size, polycarbonate). After 4 h, the cells were treated with indicated
concentrations of the extract in medium 199 without FCS. In addition, a chemoattractant
gradient was applied by the addition of medium 199 supplemented with 20% FCS to the
lower compartment. HUVECs were allowed to migrate into the direction of the FCS gradient
for 16 h. For the quantification of migrated cells, HUVECs located on the lower side
of the Transwell insert were fixed with a methanol-ethanol (2 : 1) solution for 10 min
before they were stained with crystal violet (in 20% methanol) for 15 min. After drying
overnight, DNA-bound crystal violet
was resolved in 20% acetic acid, and the amount of migrated cells was determined
at 590 nm using a microplate reader (SPECTRAFluor Plus).
Directed migration: 2D chemotaxis assay
To gain deeper insights into the effects of BNO 1095 on chemotactic migration of endothelial
cells, 18,000 HUVECs were seeded on chemotaxis slides (ibidi GmbH) in ECGM. The cells
were allowed to adhere for 4 h before ECGM was washed off and a gradient of 20% FCS
in medium 199 was added to the cells. HUVECs in chemotaxis slides were allowed to
migrate in the direction of the FCS gradient for 20 h in an atmosphere of 5% CO2 and 95% air at 37 °C in a climatic chamber of a microscope (DM IL LED, Leica Microsystems).
Every 10 min, a phase contrast image was captured. For quantification, 30 cells were
tracked using a manual tracking tool (ImageJ, software version 1.49 k). The parameters
of accumulated distance, Euclidean distance, velocity, Y : FMI, and directness were
used to demonstrate extract-derived effects on endothelial chemotactic migration.
Tube formation assay
A tube formation assay using Matrigel (Corning GmbH HQ) was performed to analyze potential
extract-induced inhibition of in vitro capillary-like sprout formation. Therefore, 10 µL per well of growth factor-reduced
Matrigel was added to angiogenesis slides (ibidi GmbH) and allowed to solidify for
30 min at 37 °C. Subsequently, 10,000 HUVECs per well were added in ECGM medium containing
the indicated concentrations of BNO 1095 on top of the solidified Matrigel matrix.
HUVECs were allowed to form tube-like structures for 5.5 h before microscopic phase
contrast images were captured (DM IL LED, Leica Microsystems). Image quantification
(ImageJ, software version 1.49 k, angiogenesis analyzer plugin) was used for the determination
of number of junctions, number of tubules, and number of master segments.
Spheroid assay
HUVEC spheroids consisting of 400 cells were formed by the hanging drop method in
ECGM containing 20% methylcellulose (Sigma-Aldrich Chemie GmbH). After 24 h, spheroids
were embedded into a rat tail collagen I gel. After solidification of the collagen
I gel, the cells in spheroids were treated with indicated concentrations of the extract
for 30 min before sprout formation was induced by VEGF. After 20 h, HUVEC spheroids
were fixed with 4% formaldehyde (Roti-Histofix, Carl Roth). Potential extract-derived
effects on total sprout length and mean number of sprouts were determined by microscopic
analysis and image quantification using ImageJ (software version 1.49 k).
Statistical analysis
All experiments were performed independently with at least 3 different cell preparations
each with at least 3 technical replicates. Statistical analysis was performed using
GraphPad Prism version 5.0. For statistical evaluation, 1-way ANOVA was used followed
by Tukeyʼs Post hoc test. The actual number of experiments (n) is stated in the respective
figure legend. Data are expressed as mean ± standard deviation (SD). P ≤ 0.05 was
considered as statistically significant.
Contributorsʼ Statement
Data collection: I. Bischoff-Kont, L. Brabenec, B. Nausch, R. Ingelfinger. Design
of the study: I. Bischoff-Kont, R. Fürst, R. Ingelfinger, B. Nausch, L. Brabenec.
Statistical analysis: I. Bischoff-Kont, L. Brabenec, R. Ingelfinger. Analysis and
interpretation of the data: I. Bischoff-Kont, R. Fürst, L. Brabenec, B. Nausch, R.
Ingelfinger. Drafting the manuscript: I. Bischoff-Kont, R. Fürst. Critical revision
of the manuscript: R. Fürst, I. Bischoff-Kont, B. Nausch.
