Competitive ELISA-Based Screening of Plant Derivatives for the Inhibition of VEGF Family Members Interaction with Vascular Endothelial Growth Factor Receptor 1

• Abstract
• Introduction
• Materials and Methods
• Plant material
• Extraction and isolation
• ELISA-based assays
• Results and Discussion
• Acknowledgements
• References

Abstract

The activation of vascular endothelial growth factor receptor-1 (VEGFR-1, also known as Flt-1) is crucial in many physiological and pathological conditions, like angiogenesis, cancer, inflammation, hematopoiesis, bone marrow precursors/stem cells recruitment in tumor angiogenesis, and metastasis formation. Many recent reports indicate that molecules able to antagonize Flt-1 activity have gained a strong interest in the view of therapeutic approaches. In order to identify new compounds able to interfere in the Flt-1 recognition by VEGFs family members, we have developed a highly sensitive competitive ELISA-based screening to study plant extracts and derivatives. Several fractions of the n-butanol extract of Pteleopsis suberosa leaves and of the chloroform extract of Parinari campestris leaves demonstrated by a bioassay-guided fractionation an evident inhibition of VEGF-A or placental growth factor (PlGF) interaction with Flt-1, with an inhibition over 50 % in particular for the VEGF-A/Flt-1 interaction at a concentration of 100 μg/mL. This activity seems be due to the presence of a combination of compounds acting synergistically.

Introduction

A complicated tuning of several growth factors families and related receptors regulates the formation of new vessels [1]. Among these players, the activation of two vascular endothelial growth factor (VEGF) receptors, VEGF receptor 1 (Flt-1) and VEGF receptor 2 (Flk-1 in mouse, KDR in human) represents a crucial event in both physiological and pathological angiogenesis [2]. Three members of the VEGF family involved in angiogenesis bind and activate VEGF receptors: VEGF-A is able to bind to both Flt-1 and KDR receptors, while two other VEGF family members, VEGF-B and placental growth factor (PlGF), recognize specifically the Flt-1 receptor. The signals activated by the VEGF-A/KDR interaction are crucial both in physiological and pathological angiogenesis while the activity of PlGF [3] and VEGF-B [4] through the interaction with Flt-1 appears to be restricted to pathological conditions [5].

The biological activity following the interaction between VEGF and KDR receptor has been until now the main target for the development of anti-angiogenic therapeutic approaches. Moreover, phenotype analysis of knockout mice and in vivo biochemical interactions strongly suggest that the inhibition of Flt-1 activity constitutes an alternative target for therapeutic modulation of angiogenesis as well as inflammatory disorders and metastatic process [6], [7]. In particular, the block of Flt-1 activation performed with neutralizing anti-Flt-1 monoclonal antibody strongly inhibits the neovascularization in tumors as well in models of ischemic retina and age-related macular degeneration. In the same manner, it has been recently reported that neutralizing monoclonal antibody anti-PlGF inhibits xenograft tumor growth in mice with an extent comparable to that observed for blocking the VEGF/Flk-1 interaction [8].

The activation of Flt-1, in particular by PlGF, is not only crucial for ECs stimulation during the neo-angiogenesis process, but also plays a fundamental role in stabilization of neo-vessels through the recruitment of SMC and in the recruitment and differentiation of monocyte-macrophage cells [3]. Flt-1 activation is crucial also in the recruitment of bone marrow-derived endothelial and hematopoietic precursors in tumor angiogenesis [9].

Taken together these data strongly indicate the requirement of molecules able to modulate the activation of Flt-1 for therapeutic approaches. On the basis of these findings, we developed a competitive ELISA-based screening of medicinal plant extracts to discover small molecules able to inhibit VEGF-A and/or PlGF interaction with Flt-1 receptor.

Materials and Methods

Plant material

S. dominica L. leaves, S. menthaefolia Ten. leaves, S. sclarea L. roots, (Lamiaceae), P. suberosa Engl. Et Diels leaves (Combretaceae), P. graeca L. small branches (Asclepiadaceae), and P. campestris Aublet Pl. leaves (Chrysobalanaceae) were supplied as described in our previous papers [10], [11], [12], [13].

Extraction and isolation

The powdered dried materials of Salvia species (300 g) were extracted as reported before [10] (see also Supporting Information). The leaves of P. suberosa were extracted and the n-butanol extract was chromatographed to give fourteen pooled fractions (A - N) as reported in a previous paper [11] (see also Supporting Information). Compound 2 was obtained from fraction H, compounds 3 and 4 from fraction J, 6 from fraction I, and 5 from fraction L, respectively. Fraction A (279 mg) was submitted to RP-HPLC with a C₁₈ μ-Bondapak column (30 cm × 7.8 mm, flow rate 2 mL/min) using MeOH-H₂O (25 : 75) to obtain compound 1 (2.0 mg, t _R = 10 min). Fraction C (200 mg) was purified by RP-HPLC using MeOH-H₂O (33 : 67) to give compounds 8 (9.9 mg, t _R = 9 min), 7 (10.8 mg, t _R = 20 min), and 9 (2.5 mg, t _R = 26 min). The dried, powdered small branches of P. graeca were extracted as reported previously and the n-BuOH extract was chromatographed over a Sephadex LH-20 column to obtain six major fractions (AA - FF) [12] (see also Supporting Information). The leaves of P. campestris (470 g) were extracted with solvents of increasing polarity and the CHCl₃ extract was submitted to silica gel flash column chromatography and grouped into fifteen fractions (AAA - PPP) as reported in our previous paper [13] (see also Supporting Information). Fraction DDD yielded compound 18, LLL compound 13, and PPP compounds 19 and 20, respectively. Fraction GGG (260 mg) was purified by RP-HPLC on a C₁₈ μ-Bondapak column (30 cm × 7.8 mm, flow rate 2.0 mL/min) with MeOH-H₂O (3 : 2) to give compounds 11 (3.0 mg, t _R = 20 min), 10 (5.5 mg, t _R = 23 min), and 12 (4.2 mg, t _R = 40 min). Fraction NNN (226 mg) was subjected to RP-18 silica gel column chromatography (2 g, 40 - 63 μm) with mixtures of MeOH-H₂O to yield compound 15 eluting with MeOH-H₂O (3 : 7) (elution volume 25 - 35 mL), compound 14 eluting with MeOH-H₂O (6 : 4) (elution volume 200 - 205 mL), and a fraction eluted with MeOH-H₂O (4 : 1) (elution volume 315 - 325 mL) that was further purified by RP-HPLC on a C₁₈ μ-Bondapak column (30 cm × 7.8 mm, flow rate 2.0 mL/min) using MeOH-H₂O (9 : 1) to give compounds 16 (2.0 mg, t _R = 7 min) and 17 (2.3 mg, t _R = 12 min).

Megastigman-7-ene-3,5,6,9-tetrol 9-O-β-D-glucopyranoside (1), [α]_D ²⁵: -55 (c 0.5, MeOH) [14]; gallocatechin (2), [α]_D ²⁵: + 150 (c 1, MeOH); myricetin 3-O-β-D-xylopyranoside (3), [α]_D ²⁵: -30 (c 0.4, MeOH); myricetin 3-O-α-L-arabinopyranoside (4), [α]_D ²⁵: -55 (c 0.1, MeOH); myricetin 3-O-(6′′-galloyl)-β-D-glucopyranoside (5), [α]_D ²⁵: -50 (c 0.8, MeOH); myricetin 3-O-α-L-arabinofuranoside (6), [α]_D ²⁵: -160 (c 1, EtOH); myricetin 3-O-neohesperidoside (7), [α]_D ²⁵: -81 (c 0.7, MeOH) [11]; 3,4-dihydroxybenzoic acid (8), [15]; quercetin 3-O-β-D-glucopyranoside (9), [α]_D ²⁵: -69 (c 0.1, MeOH) [16]; 10α,13α,16α,17-tetrahydroxy-9α-methyl-15-oxo-20-nor-kauran-19-oic acid γ-lactone (10), [α]_D ²⁵: + 24 (c 0.1, MeOH) [17]; 15-oxozoapatlin-13α-yl-10′α,16′α-dihydroxy-9′α-methyl-20′-nor-19′-oic acid γ-lactone-17′-oate (11), [α]_D ²⁵: -44 (c 0.1, MeOH) [13]; 2α-hydroxyursolic acid (12), [α]_D ²⁵: + 50 (c 0.3, CHCl₃) [18]; 1β,16α,17-trihydroxy-ent-kaurane (13), [α]_D ²⁵: + 6 (c 0.1, MeOH) [17]; arjunolic acid (14), [α]_D ²⁵: + 35 (c 0.1, MeOH) [19]; megastigman-7-ene-3,9-diol (15), [α]_D ²⁵: -11 (c 0.3, MeOH) [20]; platanic acid (16), [α]_D ²⁵: -45 (c 1, CHCl₃) [21]; stigmasta-5,24(28)-dien-3β-ol (17), [α]_D ²⁵: -42 (c 1, CHCl₃) [22]; 13-hydroxy-15-oxozoapatlin (18), [α]_D ²⁵: -26 (c 1, CHCl₃); 3α,10α,13α,16α,17-pentahydroxy-9α-methyl-15-oxo-20-nor-kauran-19-oic acid γ-lactone (19), [α]_D ²⁵: + 5 (c 0.1, MeOH); 2α,10α,13α,16α,17-pentahydroxy-9α-methyl-15-oxo-20-nor-kauran-19-oic acid (19,10)-lactone (20), [α]_D ²⁵: + 18 (c 0.1, MeOH) [19] were identified by comparison of their spectral data (¹H- and ¹³C-NMR and MS data) with literature values. Purity of all compounds based on ¹H-NMR spectra and HPLC analysis was between 95 and 98 %.

ELISA-based assays

The ELISA-based assay was performed by coating a 96-well plate with a recombinant form of Flt-1(VEGFR-1-Fc chimera; R&D Systems) at 0.5 μg/mL, 100 μL/well, 16 h at room temperature. Wells were washed five times with PBS containing 0.004 % Tween-20 (PBT) and the plate was then blocked for three h at room temperature with 1 % bovine serum albumin (BSA) in PBS, 200 μL/well. Wells were washed as above, and a recombinant form of human PlGF (R&D Systems) at 5 ng/mL or human VEGF (R&D Systems) at 10 ng/mL in PBS containing 0.1 % BSA, 5 mM EDTA, 0.004 % Tween 20 (PBET), 100 μL/well, were added and incubated for 1 h at 37 °C followed by 1 h at room temperature. Plant extracts, fractions and compounds 1 - 20 dissolved in DMSO (Sigma) were properly diluted and added to the wells pre-mixed with the ligand. Biotinylated anti-human PlGF or anti-human VEGF polyclonal antibodies (R&D Systems), diluted in PBET at 300 ng/mL, 100 μL/well, were added to the wells and incubated for 1 h at 37 °C followed by 1 h at room temperature. A solution containing a preformed avidin and biotinylated HRP macromolecular complex (Vectastain elite ABC kit) was added to each well and incubated for 1 h at rom temperature. After the last wash, 100 μL of HRP substrate composed of 1 mg/mL of ortho-phenylenediamine in 50 mM citrate phosphate buffer pH 5, 0.006 % of H₂O₂, were added and incubated for 40 min in the dark at room temperature. The reaction was blocked by adding 30 μL/well of 4 N H₂SO₄ and the absorbance measured at 490 nm on a microplate reader (Biorad BenchMark; BioRad).

Each point was done in triplicate and each experiment was repeated two times. As positive control of inhibition, we used the synthetic peptide (CP) with sequence GNQWFI [23] at 20 μM. The peptide was synthesized at PRIMM (Milano, Italy) and had a purity of 95 %.

Results and Discussion

The ELISA assay allowed strong detection of the interaction of 0.5 - 1.0 ng of soluble growth factors with 50 ng of immobilized recombinant Flt-1 receptor, and permits us to use high concentration of DMSO, up to 25 %, without loss of binding activity.

In a first screening we tested several extracts from Salvia dominica, Salvia menthaefolia, Salvia sclarea, Pteleopsis suberosa, Periploca graeca, and Parinari campestris. The results showed a moderate activity for the P. suberosa and P. graeca n-butanol extracts and for the P. campestris chloroform residue (data not shown). Therefore, these extracts were submitted to a bioassay-guided fractionation. The inhibitory activity of the fractions obtained from the bioactive extracts, assayed at 1 mg/mL on PlGF/Flt-1 and VEGF/Flt-1 interactions, is reported in [Fig. 1]. In the ELISA-based assay for PlGF/Flt-1 interaction, four fractions from P. suberosa (A, I, J, and L) and four from P. campestris (DDD, GGG, LLL, and NNN) were able to inhibit the interaction by more than 60 % ([Fig. 1] A). The same fractions and in addition C and PPP were able to inhibit the VEGF/Flt-1 interaction by more than 70 % ([Fig. 1] B). None of the fractions obtained from P. graeca (AA, CC, EE) was able to inhibit both PlGF and VEGF interaction with Flt-1 receptor. The most active fractions were then assayed in dose-dependent experiments at concentrations ranging from 500 to 20 μg/mL. Among those from P. suberosa, only A was able to inhibit the PlGF/Flt-1 interaction in a dose-dependent manner ([Fig. 2] A), whereas different fractions of the same species (A, C, I, J, and H) inhibited VEGF/Flt-1 interaction ([Fig. 2] B). Moreover, four fractions from the P. campestris chloroform extract (DDD, GGG, LLL, and NNN) were able to inhibit in a dose-dependent manner both PlGF and VEGF interaction with Flt-1 receptor; DDD, GGG, and LLL showed more than 50 % of inhibition of PlGF/Flt-1 interaction at 100 μg/mL, while all fractions of P. campestris were able to inhibit VEGF/Flt-1 interaction with more than 50 % of inhibition again at 100 μg/mL. The active fractions were examined for identification of the compounds to which this inhibitory activity could be attributed and to determine the dose-effect curves.

Chromatographic separation of P. suberosa fractions led to the isolation of compounds 1 - 9, identified as six flavonoid derivatives (2 - 7 and 9), one phenolic compound (8), and one megastigmane derivative (1) ([Fig. 3]). From P. campestris fractions, six diterpenes (10, 11, 13 and 18 - 20), one megastigmane (15), one sterol (17), and three triterpenes (12, 14 and 16) were separated and characterized ([Fig. 3]). All isolated compounds were thus submitted to the ELISA based assays at 100 μg/mL. At this concentration, the single compounds did not show any inhibitory properties (not shown). To verify the loss of inhibitory activity, the compounds were also assayed a 500 μg/mL, and as showed in [Fig. 4], only two compounds (2 and 12) gave inhibition of more than 50 % on VEGF/Flt-1 interaction. Whereas compound 2 isolated from fraction H of n-butanol extracts of P. suberosa, was able able to inhibit only VEGF/Flt-1 interaction ([Fig. 2]), compound 12 was purified from fraction GGG of P. campestris, was able to inhibit both interactions investigated. The differences in inhibitory properties are probably due to the existing diversity in the interaction of VEGF and PlGF to Flt-1 that occurs in a similar but not identical manner [24].

On the basis of our results, we could hypothesize that the strong inhibitory activity of PlGF and VEGF interaction with the Flt-1 receptor exerted by some extracts and fractions as reported in [Figs. 1] and [2] may be due to the presence of a combination of compounds acting synergistically or as vehicles enhancing the biological activity. However, we cannot rule out the possibility that the activity of the extracts and fractions could be due to a very minor compound not isolated.

Acknowledgements

The authors thank Vincenzo Mercadante for technical assistance. S.D.F is supported by AIRC (Italian Association for Cancer Research), grant number 4840.

References

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• 21 Mayer R. Three lupane derivates from Leptospermum scoparium. .  Arch Pharm. 1996;  329 447-50
  CrossrefPubMedSearch in Google Scholar
• 22 McInnes A G, Walter J A, Wright J LC. Carbon-13 NMR spectra of Δ 24(28)-phytosterols.  Org Magn Reson. 1980;  13 302-3
  CrossrefPubMedSearch in Google Scholar
• 23 Bae D G, Kim T D, Li G, Yoon W H, Chae C B. Anti-flt1 peptide, a vascular endothelial growth factor receptor 1-specific hexapeptide, inhibits tumor growth and metastasis.  Clin Cancer Res. 2005;  11 651-61.
  PubMedSearch in Google Scholar
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  PubMedSearch in Google Scholar

Dr. Sandro De Falco

Angiogenesis Lab and Stem Cell Fate Lab

Institute of Genetics and Biophysics ‘Adriano Buzzati-Traverso’

CNR,

Via P. Castellino 111

80131 Naples

Italy

Phone: +39-081-613-2354

Fax: +39-081-613-2595

Email: defalco@igb.cnr.it

References

• 1 Yancopoulos G D, Davis S, Gale N W, Rudge J S, Wiegand S J, Holash J. Vascular-specific growth factors and blood vessel formation.  Nature. 2000;  407 242-8
  CrossrefPubMedSearch in Google Scholar
• 2 Shibuya M. Differential roles of vascular endothelial growth factor receptor-1 and receptor-2 in angiogenesis.  J Biochem Mol Biol. 2006;  39 469-8
  CrossrefPubMedSearch in Google Scholar
• 3 Carmeliet P, Moons L, Luttun A, Vincenti V, Compernolle V, De Mol M. et al . Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions.  Nat Med. 2001;  7 575-3
  CrossrefPubMedSearch in Google Scholar
• 4 Bellomo D, Headrick J P, Silins G U, Paterson C A, Thomas P S, Gartside M. et al . Mice lacking the vascular endothelial growth factor-B gene (Vegfb) have smaller hearts, dysfunctional coronary vasculature, and impaired recovery from cardiac ischemia.  Circ Res. 2000;  86 E29-35
  PubMedSearch in Google Scholar
• 5 Carmeliet P. Angiogenesis in health and disease.  Nat Med. 2003;  9 653-60
  CrossrefPubMedSearch in Google Scholar
• 6 Luttun A, Tjwa M, Carmeliet P. Placental growth factor (PlGF) and its receptor Flt-1 (VEGFR-1): novel therapeutic targets for angiogenic disorders.  Ann N Y Acad Sci. 2002;  979 80-93
  PubMedSearch in Google Scholar
• 7 Kaplan R N, Riba R D, Zacharoulis S, Bramley A H, Vincent L, Costa C. et al . VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche.  Nature. 2005;  438 820-7
  CrossrefPubMedSearch in Google Scholar
• 8 Fischer C, Jonckx B, Mazzone M, Zacchigna S, Loges S, Pattarini L. et al . Anti-PlGF inhibits growth of VEGF(R)-inhibitor-resistant tumors without affecting healthy vessels.  Cell. 2007;  131 63-75
  PubMedSearch in Google Scholar
• 9 Lyden D, Hattori K, Dias S, Costa C, Blaikie P, Butros L. et al . Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth.  Nat Med. 2001;  7 1194-201
  CrossrefPubMedSearch in Google Scholar
• 10 Fiore G, Nencini C, Cavallo F, Capasso A, Bader A, Giorgi G. et al . In vitro antiproliferative effect of six Salvia species on human tumor cell lines.  Phytotherapy Res. 2006;  20 01-3
  CrossrefPubMedSearch in Google Scholar
• 11 De Leo M, Braca A, Sanogo R, Cardile V, De Tommasi N, Russo A. Antiproliferative activity of Pteleopsis suberosa leaf extract and its flavonoid components in human prostate carcinoma cells.  Planta Med. 2006;  72 604-10
  Thieme ConnectPubMedSearch in Google Scholar
• 12 Siciliano T, Bader A, De Tommasi N, Morelli I, Braca A. Sulfated pregnane glycosides from Periploca graeca. .  J Nat Prod. 2005;  68 1164-8
  CrossrefPubMedSearch in Google Scholar
• 13 Braca A, Abdel-Razik A F, Mendez J, Morelli I. A new kaurane diterpene dimer from Parinari campestris. .  Fitoterapia. 2005;  76 614-9
  CrossrefPubMedSearch in Google Scholar
• 14 Otsuka H, Hirata E, Shinzato T, Takeda Y. Stereochemistry of megastigmane glucosides from Glochidion zeylanicum and Alangium premnifolium. .  Phytochemistry. 2003;  62 763-8
  CrossrefPubMedSearch in Google Scholar
• 15 Xu H X, Kadota S, Wang H, Kurokawa M, Shiraki K, Matsumoto T. et al . A new hydrolyzable tannin from Geum japonicum and its antiviral activity.  Heterocycles. 1994;  38 167-75
  PubMedSearch in Google Scholar
• 16 Agrawal P K. Carbon-13 NMR of flavonoids. Amsterdam; Elsevier 1989
  Search in Google Scholar
• 17 Braca A, Armenise A, Morelli I, Mendez J, Mi Q, Chai H -B. et al . Structure of kaurane-type diterpenes from Parinari sprucei and their potential anticancer activitiy.  Planta Med. 2004;  70 540-50
  Thieme ConnectPubMedSearch in Google Scholar
• 18 Ballesta-Acosta M C, Pascual-Villalobos M J, Rodriguez B. A new 24-nor-oleanane triterpenoid from Salvia carduacea. .  J Nat Prod. 2002;  65 1513-5
  CrossrefPubMedSearch in Google Scholar
• 19 De Felice A, Bader A, Leone A, Sosa S, Della Loggia R, Tubaro A. et al . New polyhydroxylated triterpenes and anti-inflammatory activity of Salvia hierosolymitana. .  Planta Med. 2006;  72 643-9
  Thieme ConnectPubMedSearch in Google Scholar
• 20 Morikawa T, Zhang Y, Nakamura S, Matsuda H, Muraoka O, Yoshikawa M. Bioactive contituents from Chinese natural medicines. XXII. Absolute structures of new megastigmane glycosides, sedumosides E₁, E₂, E₃, F₁, F₂, and G, from Sedum sarmentosum (Crassulaceae).  Chem Pharm Bull. 2007;  55 435-41
  CrossrefPubMedSearch in Google Scholar
• 21 Mayer R. Three lupane derivates from Leptospermum scoparium. .  Arch Pharm. 1996;  329 447-50
  CrossrefPubMedSearch in Google Scholar
• 22 McInnes A G, Walter J A, Wright J LC. Carbon-13 NMR spectra of Δ 24(28)-phytosterols.  Org Magn Reson. 1980;  13 302-3
  CrossrefPubMedSearch in Google Scholar
• 23 Bae D G, Kim T D, Li G, Yoon W H, Chae C B. Anti-flt1 peptide, a vascular endothelial growth factor receptor 1-specific hexapeptide, inhibits tumor growth and metastasis.  Clin Cancer Res. 2005;  11 651-61.
  PubMedSearch in Google Scholar
• 24 Errico M, Riccioni T, Iyer S, Pisano C, Acharya K R, Persico M G. et al . Identification of placenta growth factor determinants for binding and activation of Flt-1 receptor.  J Biol Chem. 2004;  279 43 929-39
  PubMedSearch in Google Scholar

Dr. Sandro De Falco

Angiogenesis Lab and Stem Cell Fate Lab

Institute of Genetics and Biophysics ‘Adriano Buzzati-Traverso’

CNR,

Via P. Castellino 111

80131 Naples

Italy

Phone: +39-081-613-2354

Fax: +39-081-613-2595

Email: defalco@igb.cnr.it

• Supplementary Material