Anti‐angiogenic effect of quercetin and its 8‐methyl pentamethyl ether derivative in human microvascular endothelial cells

Abstract Angiogenesis is involved in many pathological states such as progression of tumours, retinopathy of prematurity and diabetic retinopathy. The latter is a more complex diabetic complication in which neurodegeneration plays a significant role and a leading cause of blindness. The vascular endothelial growth factor (VEGF) is a powerful pro‐angiogenic factor that acts through three tyrosine kinase receptors (VEGFR‐1, VEGFR‐2 and VEGFR‐3). In this work we studied the anti‐angiogenic effect of quercetin (Q) and some of its derivates in human microvascular endothelial cells, as a blood retinal barrier model, after stimulation with VEGF‐A. We found that a permethylated form of Q, namely 8MQPM, more than the simple Q, is a potent inhibitor of angiogenesis both in vitro and ex vivo. Our results showed that these compounds inhibited cell viability and migration and disrupted the formation of microvessels in rabbit aortic ring. The addition of Q and more significantly 8MQPM caused recoveries or completely re‐establish the transendothelial electrical resistance (TEER) to the control values and suppressed the activation of VEGFR2 downstream signalling molecules such as AKT, extracellular signal‐regulated kinase, and c‐Jun N‐terminal kinase. Taken together, these data suggest that 8MQPM might have an important role in the contrast of angiogenesis‐related diseases.


| INTRODUC TI ON
In the human retina, homeostasis is maintained by the blood retinal barrier (BRB). The retinal continuous endothelium forms the main structure of the BRB and leans on a basal lamina which is covered by the processes of astrocytes, Müller cells and pericytes. These cellular types all contribute to the maintenance of the BRB, 1 whose integrity is essential for proper vision. Physiologically, the intercellular spaces among the BRB endothelial cells are sealed by complex tight junctions and the cells themselves lack fenestrations and have few pinocytotic vesicles. These features result in the typical restricted paracellular permeability and high transendothelial electrical resistance (TEER). The BRB breakdown greatly contributes to the pathology and vision loss in retinal disorders such as uveitis, diabetic retinopathy, age-related macular degeneration and tumour. 2 Growth factors, such as vascular endothelial growth factor (VEGF) and pro-inflammatory cytokines have been implicated in the pathophysiology of these diseases and contribute to the clinically observed retinal inflammation, angiogenesis and vascular hyperpermeability. 3,4 The treatment with anti-VEGF agents significantly reduces the abnormal growth of microvessels. 5 Clinical trials have demonstrated the efficacy of these treatments in the advanced stages of neovascular eye diseases hence confirming the modulatory role of VEGF in the pathogenesis and progression of retinal aberrant neovascularization, which occurs in pathological conditions, 6 diabetic retinopathy, 7 and tumour. 8 The permeability-and proangiogenic-inducing effects of VEGF on endothelial cells are mainly mediated by VEGF receptor-2 (VEGFR-2), whereas VEGFR-1, in dependence of the tissue context, is both a negative and positive regulator of VEGFR-2 signalling. Activation of VEGFR-2 contributes to phosphorylation of multiple downstream signals such as ERK (extracellular signal-regulated), AKT (also termed protein kinase B), JNK (c-Jun N-terminal kinase), which cause proliferation, migration and tube formation of endothelial cells. 9,10 Quercetin (Q) is one of the most widely diffused flavonoids in fruits and vegetables, 11 usually present in the form of 3-O-glycoside with the d-glucose, galactose or rhamnose. It is an antioxidant and free radical scavenger, 12 and it has been shown to have antiinflammatory 13 and neuroprotective 14 effects. The inhibition of certain enzymes involved in proliferation and signal transduction pathway including tyrosine kinase, protein kinase C, 15 PI-3 kinase, 16 proline-directed protein kinase fatty acid in human prostate carcinoma cells 17 and JNK 18 and the induction of apoptosis and growth inhibition in lung cancer cells 19 by Q have already been highlighted.
Moreover, there exists a considerable number of data reporting on the capability of Q to induce cell cycle arrest and apoptosis and to being an inhibitor of carcinogenesis and angiogenesis. 20,21 Several studies have highlighted that the anti-cancerous property of Q is due to the down-regulation of the VEGF and Hypoxia-inducible factor-1 (HIF-1) expression. 22 It has been shown that Q has negative 23 as well as positive effects 24 on HIF-1 and VEGF expressions in different types of cell and, to date, several uncertainties have been raised regarding its absorption and availability in tumour. It has been demonstrated that Q inhibits angiogenesis by targeting VEGFR-2 regulated AKT/mTOR/P70S6K signalling pathway. 25 In previous experiments, we have shown that the tumour angiogenic stimulus is driven through the phosphorylation/activation of Protein kinase C α, ERK1/2 and cytosolic calcium-dependent and -independent phospholipases A 2 , all events required for cell proliferation and motility. [26][27][28] As mentioned above, several studies have confirmed that Q, likewise other common dietary flavonoids, inhibits the angiogenic process both in vitro and in vivo. 25 On the other hand, the derivatives of Q often manifest different effects on the activation of signalling pathways and in their consequent biological effects, for example angiogenesis. Unlike quercetin-3′-sulphate, for instance, quercetin-3-O-glucuronide (Q3GA), suppresses the in vivo VEGF-induced angiogenesis through ERK inhibition. 29 Quercetin has long received great interest in these studies but its poor bioavailability and low stability in aqueous media definitely limit the use of this flavonoid in clinical applications. For these reasons, in this study three derivatives of Q, namely, 6,8-dibromoquercetin (6,8-diBrQ), 8-methylquercetin pentamethyl ether (8MQPM) and quercetin dimer (QD; see Scheme 1) were prepared and tested along with Q as therapeutic agents. Quercetin was permethylated in order to improve its ability to cross the cell membranes. On the other hand, the bromo derivative (6,8-diBrQ ) is able to release small quantities of bromine in solution 30 which may produce interesting and useful effects on cells. In this work, human primary endothelial cells, isolated from retinal microcapillaries (HREC) as a BRB modelsystem, were used for the experiments. Human retinal endothelial cells were treated with conditioned medium (CM) from Y-79 human retinoblastoma cell line or were VEGFA-stimulated in order to reproduce the angiogenic environment of the above-mentioned retinal diseases and cancer. Our findings reveal that Q and even more so 8MQPM offer protection against the powerful pro-angiogenic stimulus present in our in vitro human BRB model.

| Chemicals
All reagents and solvents for the synthesis of 8MQPM were purchased from Sigma-Aldrich and were used without further purification. The 13 C and 1 H Nuclear magnetic resonance spectroscopy (NMR) spectra were recorded at 400.13 MHz ( 1 H) and 100.62 MHz ( 13 C) in CDCl3 solutions at 298 K on a Bruker AvanceTM 400 spectrometer. Electrospray ionization mass spectrometers (ESI-MS) spectra were recorded with a GC-MS QP5050A Shimadzu spectrometer.

| Synthesis of quercetin derivatives
Quercetin dihydrate (HPLC) ≥98%, 6, 8-diBrQ and QD were available from previous studies. The syntheses of 6, 8-diBrQ and QD along with the NMR spectra are reported in references 31 and 32,33 respectively. The synthesis of 8MQPM and its carbon and proton NMR spectra are instead reported below.
One gram of quercetin dihydrate (3.0 mmol) was solubilized in a mixture of 60 mL of Tetrahydrofuran and 5 mL of Dimethylformamide. Then, 6.5 g (20 mmol) of Cs 2 CO 3 and 3 mL (48 mmol) of CH 3

| CM collection
Human Y-79 retinoblastoma cells (0.45-5 × 10 6 cells/mL) were cultured in serum free medium at 37°C for 24 hours to obtain CM. 34 Conditioned medium was collected, centrifuged at 1000× g for 5 minutes, filtered with 0.2 μm filter and stored at −80°C until use. Conditioned medium was used without any dilution. 26

| Wound healing assay
HREC were seeded in 24 well tissue culture plates to a final density

| TEER of cell layer
Transendothelial electrical resistance was measured with the Millicell-ERS system (MERS 000 01; Millipore AG, Volketswil, Switzerland) as previously described. 27 Values were expressed as ω × cm 2 and calculated by the formula: (the average resistance of experimental wells − the average resistance of blank wells] × 0.33 (the area of the transwell membrane).

| Tube formation assay
The ability of cells to migrate and organize into capillary-like structures was evaluated by using the Matrigel assay (

| Immunoblots
After treatments, the cells were detached by scraping, collected by centrifugation and lysed as previously described. 38 The membranes were incubated with primary antibodies (4°C, o/n) and then with secondary antibodies (1 hour, room temperature). The membranes, after anti p-VEGFR2, VEGFR2, p-AKT, AKT, p-ERK and ERK antibodies, were successfully stripped and re-probed for β-actin, to test the equal protein loading. The same procedure was followed for p-JNK and JNK. The immunocomplexes were detected by enhanced chemiluminescence reagent (Amersham).

| Statistical analysis
Statistical significance between two groups was analysed by Student's t test. One-way and two-way ANOVA, followed by Tukey's post hoc test, were used for multiple comparisons. P values <0.05 were considered statistically significant.

| Quercetin and its derivatives affect HREC viability
In order to assess the toxicity of Q and of its derivatives dose-(25-, 50-and 100 μmol/L concentrations in serum-starved medium) and time-dependent HREC viability were assayed by the MTT rapid colorimetric test, after 24 and 48 hours incubation (Figure 1). Quercetin and its derivatives caused a significant decrease in cell viability at the doses 50 and 100 μmol/L, both at 24 and 48 hours. At the con-

| Quercetins and HREC proliferation
BrdU assay experiments were performed to investigate the cellular proliferative response after 24 hours incubation with Q and its

| Quercetins and HREC migration
Endothelial cell migration is a critical process for wound healing and angiogenesis. The migration was evaluated by the wound-healing assay ( Figure 2). In panel A, contrast phase representative photo- at 100 (panel B). As expected from our previous data, 28 both the growth factor and the retinoblastoma CM stimulated HREC motility so quantitatively similar by 1.8-fold. Q significantly decreased such migration by 53% and even more so, 8MQPM by almost 55%, in the presence of CM. The 6,8-diBrQ and QD reduced the migration by 17% and 15%, respectively, proving that their effect is not comparable to those of Q and 8MQPM. The individual compounds had no effect in the absence of VEGF-A and CM (data not shown).

| Effects of Q and its derivatives on bloodretinal barrier: The TEER
The TEER represents the electrical resistance across the endothe-

| Quercetin and 8MQPM inhibit angiogenesis by Matrigel assay
Since an imbalance in the new blood vessel formation from those pre-existing contributes to numerous infectious, inflammatory, immune, and, above all, malignant disorders, 39

| Quercetin and 8MQPM inhibit angiogenesis in a rabbit aortic ring ex vivo model
In the ex vivo rabbit aortic ring assay, 40   Interestingly, 8MQPM was more effective in reducing the phosphorylation levels (by 70%, 47% and 72%, for p-Akt, p-ERK and p-JNK, respectively). These data shed light on the issue that Q and even more 8MQPM inhibit the primary HREC proliferation, migration, spatial organization and maturation by affecting Ras downstream cascade of MEK/ERK, MEK/JNK and PI3-K/AKT pathways.

| D ISCUSS I ON
The imbalance among the molecules that coordinate the neoangiogenesis is often the cause of inflammatory, immune, infectious and malignant disorders. 38 The newly formed blood vessels are fenestrated, permeable, tortuous and heterogeneous both in their structure and efficiency of perfusion. 43    These data confirm the results of previous studies done in different areas and model systems. 56,57 The first step in angiogenesis takes place with the formation of new sprouts off from the existing vasculature, mediated by cell proliferation and tip cell migration. 58 In wound-healing assays, 8MQPM much more than Q (see Figure 2) decreased the HREC migration induced by retinoblastoma CM, whereas 6,8-diBrQ and QD did not show any significant effects. Furthermore, 8MQPM and to a less extent Q were also able to inhibit the retinoblastoma-stimulated tube formation (see Figure 4). The tube total length and the number of branch points from single HREC were both significantly decreased compared to cells treated with retinoblastoma CM or VEGFA. Interestingly, 8MQPM was able to bring back the branch points number to the control values (HREC not stimulated with CM or growth factor). In ex vivo rabbit aortic ring experiments, the incubation of the pieces with Q or 8MQPM in the presence of CM decreased significantly both the stimulated tube length and the branch points. Again, the effects observed with 8MQPM were overwhelming those observed with Q (see Figure 5).
The quantification of TEER through the endothelium monolayer isolated from anatomical barriers is a widely accepted technique to measure the integrity of tight junction systems. The measurement of TEER  41 In clinical trials, the successful of the anti-angiogenic therapy often requires the simultaneous blockade of signalling downstream molecules from multiple pro-angiogenic factor receptors. 59 In our study, we found that 8MQPM blocked, much better than Q, the multiple downstream signalling components of VEGFR-2, such JNK, ERK and Akt, suggesting that these molecules exerted their anti-angiogenic activity by regulating the activation of VEGFR-2-mediated downstream F I G U R E 6 Effects of quercetin (Q) and 8MQPM on signal transduction. A representative blot is shown for p-VEGFR2, p-Akt, p-ERK, p-JNK and total expression (A). Densitometry analysis of phosphorylated/total protein ratio of each band (adu) was performed using Imagej software: p-VEGFR2/VEGFR2 (B), p-Akt/Akt (C), p-ERK/ERK (D), p-JNK/JNK (E). Values are expressed as the mean ± SD of three independent experiments (n = 3). ∆ P < 0.01 versus conditioned medium (CM)-stimulated human retinal endothelial cells; * P < 0.01 versus Q. One-way ANOVA followed by Tukey's test. VEGF, vascular endothelial growth factor; VEGFR-2, VEGF receptor-2 signalling cascade in retinal endothelial cells subjected to a tumorigenic stimulus. These data confirm previous results obtained from other cell types and model systems. 60 Moreover, it has been demonstrated, by docking simulations, that Quercetin compounds directly bind to the VEGFR2's active site, by forming four hydrogen bonds, with binding energies of −9.1 Kcal/mol. 61 A proposed model of Q and 8MQPM inhibitory effect on retinoblastoma CM-stimulated on angiogenesis is reported in Figure 7 (graphical abstract).

| CON CLUS IONS
This study shows that some derivatives of Q exhibit divergent effects on tumour-stimulated primary human retinal endothelium and on angiogenesis. We found that a permethylated form of Q, namely 8MQPM, more than the simple Q, is a potent inhibitor of angiogenesis both in vitro and ex vivo. These effects were not observed with other Q derivatives synthesized by us (6,8-diBrQ and QD) which demonstrated to be toxic at the concentration used in the experiments and unable to inhibit the activation of VEGFR-2/angiogenesis. The novelty of these results is that the treatment with 8MQPM and to a less extent with Q was able to inhibit the activation of VEGFR-2, thereby suppressing the HREC proliferation, migration, spatial organization and Ras downstream cascade of MEK/ERK, MEK/JNK and PI3-K/ AKT pathways. Further investigations, by using ex vivo and in vivo models, are needed to confirm that 8MQPM might be a promising retinal medication.

CO N FLI C T S O F I NTE R E S T
The authors declare no conflict of interest.

O RCI D
Carmelina Daniela Anfuso https://orcid.org/0000-0001-7951-1812 F I G U R E 7 Inhibition of the angiogenic events, induced by VEGF-A, with quercetin (Q) or 8MQPM. In the proposed model, Q and 8MQPM inhibit the VEGFR-2 activation and its downstream cascade of events, eventually leading to the inhibition of ERK1/2, Akt and JNK activation/phosphorylation. There is therefore a counteraction on migration, invasion and morphogenesis of HREC. 8MQPM inhibits angiogenesis more effectively than the native Q. ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; VEGF, vascular endothelial growth factor; VEGFR-2, VEGF receptor-2