Gérard Y Perret

Identification of a peptide blocking vascular endothelial growth factor (VEGF)‐mediated angiogenesis

Vascular endothelial growth factor (VEGF) binding to the kinase domain receptor (KDR/FLK1 or VEGFR‐2) mediates vascularization and tumor‐induced angiogenesis. Since there is evidence that KDR plays an important role in tumor angiogenesis, we sought to identify peptides able to block the VEGF–KDR interaction. A phage epitope library was screened by affinity for membrane‐expressed KDR or for an anti‐VEGF neutralizing monoclonal antibody. Both strategies led to the isolation of peptides binding KDR specifically, but those isolated by KDR binding tended to display lower reactivities. Of the synthetic peptides corresponding to selected clones tested to determine their inhibitory activity, ATWLPPR completely abolished VEGF binding to cell‐displayed KDR. In vitro, this effect led to the inhibition of the VEGF‐mediated proliferation of human vascular endothelial cells, in a dose‐dependent and endothelial cell type‐specific manner. Moreover, in vivo, ATWLPPR totally abolished VEGF‐induced angiogenesis in a rabbit corneal model. Taken together, these data demonstrate that ATWLPPR is an effective antagonist of VEGF binding, and suggest that this peptide may be a potent inhibitor of tumor angiogenesis and metastasis.

HTLV-1 uses HSPG and neuropilin-1 for entry by molecular mimicry of VEGF165

Human T-cell lymphotropic virus type 1 (HTLV-1) entry involves the interaction between the surface (SU) subunit of the Env proteins and cellular receptor(s). Previously, our laboratories demonstrated that heparan sulfate proteoglycans (HSPGs) and neuropilin-1 (NRP-1), a receptor of VEGF(165), are essential for HTLV-1 entry. Here we investigated whether, as when binding VEGF(165), HSPGs and NRP-1 work in concert during HTLV-1 entry. VEGF(165) binds to the b domain of NRP-1 through both HSPG-dependent and -independent interactions, the latter involving its exon 8. We show that VEGF(165) is a selective competitor of HTLV-1 entry and that HTLV-1 mimics VEGF(165) to recruit HSPGs and NRP-1: (1) the NRP-1 b domain is required for HTLV-1 binding; (2) SU binding to target cells is blocked by the HSPG-binding domain of VEGF(165); (3) the formation of Env/NRP-1 complexes is enhanced by HSPGs; and (4) the HTLV SU contains a motif homologous to VEGF(165) exon 8. This motif directly binds to NRP-1 and is essential for HTLV-1 binding to, internalization into, and infection of CD4(+) T cells and dendritic cells. These findings demonstrate that HSPGs and NRP-1 function as HTLV-1 receptors in a cooperative manner and reveal an unexpected mimicry mechanism that may have major implications in vivo.

Low Molecular Weight Fucoidan Increases VEGF165-induced Endothelial Cell Migration by Enhancing VEGF165 Binding to VEGFR-2 and NRP1

Therapeutic induction of angiogenesis is a potential treatment for chronic ischemia. Heparan sulfate proteoglycans are known to play an important role by their interactions with proangiogenic growth factors such as vascular endothelial growth factor (VEGF). Low molecular weight fucoidan (LMWF), a sulfated polysaccharide from brown seaweeds that mimic some biological activities of heparin, has been shown recently to promote revascularization in rat critical hindlimb ischemia. In this report, we first used cultured human endothelial cells (ECs) to investigate the possible ability of LMWF to enhance the actions of VEGF165. Data showed that LMWF greatly enhances EC tube formation in growth factor reduced matrigel. LMWF is a strong enhancer of VEGF165-induced EC chemotaxis, but not proliferation. In addition, LMWF has no effect on VEGF121-induced EC migration, a VEGF isoform that does not bind to heparan sulfate proteoglycans. Then, with binding studies using 125I-VEGF165, we observed that LMWF enhances the binding of VEGF165 to recombinant VEGFR-2 and Neuropilin-1 (NRP1), but not to VEGFR-1. Surface plasmon resonance analysis showed that LMWF binds with high affinity to VEGF165 (1.2 nm) and its receptors (5-20 nm), but not to VEGF121. Pre-injection of LMWF on immobilized receptors shows that VEGF165 has the highest affinity for VEGFR-2 and NRP1, as compared with VEGFR-1. Overall, the effects of LMWF were much more pronounced than those of LMW heparin. These findings suggested an efficient mechanism of action of LMWF by promoting VEGF165 binding to VEGFR-2 and NRP1 on ECs that could help in stimulating therapeutic revascularization.

Structure–function analysis of the antiangiogenic ATWLPPR peptide inhibiting VEGF165 binding to neuropilin-1 and molecular dynamics simulations of the ATWLPPR/neuropilin-1 complex

Heptapeptide ATWLPPR (A7R), identified in our laboratory by screening a mutated phage library, was shown to bind specifically to neuropilin-1 (NRP-1) and then to selectively inhibit VEGF(165) binding to this receptor. In vivo, treatment with A7R resulted in decreasing breast cancer angiogenesis and growth. The present work is focused on structural characterization of A7R. Analogs of the peptide, obtained by substitution of each amino acid with alanine (alanine-scanning) or by amino acid deletion, have been systematically assayed to determine the relative importance of the side chains of each residue with respect to the inhibitory effect of A7R on VEGF(165) binding to NRP-1. We show here the importance of the C-terminal sequence LPPR and particularly the key role of C-terminal arginine. In solution, A7R displays significant secondary structure of the backbone adopting an extended conformation. However, the functional groups of arginine are very flexible in the absence of NRP-1 pointing to an induced fit upon binding to the receptor. A MD trajectory of the A7R/NRP-1 complex in explicit water, based on the recent tuftsin/NRP-1 crystal structure, has revealed the hydrogen-bonding network that contributes to A7Rs binding activity.

β-Adrenoceptor subtype expression and function in rat white adipocytes

The pharmacological features of rat white adipocyte β‐adrenoceptor subtypes were investigated by saturation and β‐agonist competition studies with [3H]‐CGP 12177 and by lipolysis induced by β‐agonists as well as their inhibition by CGP 20712A (selective β1‐antagonist) and ICI 118551 (selective β2‐antagonist) in an attempt to establish a relationship between the functionality and binding capacity of β‐adrenoceptor subtypes. Two populations of binding sites were identified on adipocyte membranes, one with high affinity (0.22±0.07 nm) and the other with low affinity (23±7 nm). The low affinity binding sites constituted 90% of the total binding sites. The competition curves, with 15 nm [3H]‐CGP 12177, for the β‐agonists, isoprenaline (Iso), noradrenaline (NA) and adrenaline (Ad), and the selective β3‐agonist, BRL 37344 (BRL), were clearly biphasic (P<0.001). The rank orders of agonist potency (pKi) in competing for [3H]‐CGP 12177 high affinity and low affinity binding sites, respectively, were Iso (9.28±0.24)>NA (8.90±0.12)>Ad (8.65±0.12)>>BRL (4.53±0.17) and BRL (7.38±0.19)>>Iso (2.96±0.26)NA (2.80±0.17)>Ad (2.10±0.11) indicating the expression of β1‐ and β3‐adrenoceptor subtypes on rat white adipocytes, respectively. Inversely, competition studies with the selective β1‐agonist, xamoterol (Xam), provided evidence for a single homogeneous population of binding sites with low density (81±9 fmol mg−1) and high pKi value (7.23±0.26) confirming the presence of β1‐adrenoceptors. To assess a possible contribution of the β2‐subtype, procaterol (Proc), a selective β2‐agonist, was used to compete with 2 nm [3H]‐CGP 12177. A single low affinity (4.61±0.07) population of binding sites was identified. The density of these sites (71±12 fmol mg−1) was similar to the one obtained with Xam, suggesting that Proc displaced [3H]‐CGP 12177 from the β1‐subtype. The functional potency (pD2) order with BRL (9.07±0.20) and catecholamines (Iso: 7.26±0.06, NA: 6.89±0.02 and Ad: 6.32±0.07) was the same as that found for the low affinity binding sites in competition studies. Xam induced lipolysis with greater potency than dobutamine (Dob), 6.31±0.06 and 5.66±0.10, respectively. Proc stimulated lipolysis with a low potency (5.59±0.21). The lipolytic response to 0.001 μm BRL was inhibited by both, selective β1‐ and β2‐antagonist, in a monophasic manner with low potencies (CGP 20712A pKi: <4.5 and ICI 118551 pKi: 5.57±0.13). Similar monophasic profiles were obtained for inhibition of Xam‐ and Dob‐induced lipolysis. In this case, CGP 20712A was more potent (>10 times) than ICI 118551. The monophasic inhibition was also observed with ICI 118551 in the presence of 0.05 μm Iso or 0.13 μm NA. In contrast, two populations of sites were identified with CGP 20712A in the presence of Iso as well as NA. The pKi values for the first sites were 8.41±0.09 and 8.58±0.17, respectively, and for the second population of sites 4.73±0.22 and 4.27±0.27, respectively. The proportion of the first sites was low: 19±4 and 22±5%, respectively. Biphasic curves were obtained with both antagonists using 2.5 μm Proc (CGP 20712A: pKi1: 8.17±0.08, site1: 23±6%, pKi2: 4.77±0.14; ICI 118551: pKi1: 7.78±0.03, site1: 37±2%, pKi2: 5.35±0.25). Our results show that the radioligand [3H]‐CGP 12177 allows the characterization of β1‐ and β3‐adrenoceptor subtypes on rat white adipocytes. Lipolysis is highly dependent on β1‐ and β3‐adrenoceptors. Finally, binding and functional studies confirm that lipolysis is mainly driven by the β3‐subtype.

Is Sentinel Lymph Node Mapping in Colorectal Cancer a Future Prognostic Factor? A Meta-analysis

The diagnostic value of sentinel lymph node mapping (SLNM) in patients with colorectal cancer (CRC) is controversial. Prognostic factors for CRC must be detected to improve its treatment. A PubMed query (key words: colorectal cancer, sentinel node) provided 182 studies on the sentinel lymph node (SLN) for CRC, the abstracts of which were reviewed. Altogether, 48 studies dealing with the diagnostic value of SLNM were selected from PubMed, and 6 other studies were retrieved from reviews. We compared the diagnostic value of SLNM with that of conventional histopathologic examination. We used the diagnostic accuracy odds ratio (DAOR) method. Because of significant heterogeneity, we chose the random effect model (Der Simonian and Laird). Statistics were performed on 33 studies, including 1794 patients (1201 colon and 332 rectum cancers). The mean SLNM failure rate was 10%. The global sensitivity and specificity of the SLNM were, respectively, 70% and 81%. The pooled DAOR was 10.7 (95% confidence interval 7.0–16.5). That means that a patient whose SLN is invaded has 10.7 times more risk to be node-positive than an SLN-negative patient. Lymphatic mapping appears to be readily applicable to CRC. One of the main reasons for the heterogeneity is the performance of the SLNM by Saha et al., whose data had better sensitivity (90%) than those in other studies. The SLNM technique should be better standardized in future studies. Understanding the cause of false-negative SLNs (9%) is a major issue to resolve before routinely using this technique in CRC management. The prognostic implication of micrometastases found in SLNs requires further evaluation.

Glycosaminoglycans and their synthetic mimetics inhibit RANTES-induced migration and invasion of human hepatoma cells

The CC-chemokine regulated on activation, normal T-cell expressed, and presumably secreted (RANTES)/CCL5 mediates its biological activities through activation of G protein–coupled receptors, CCR1, CCR3, or CCR5, and binds to glycosaminoglycans. This study was undertaken to investigate whether this chemokine is involved in hepatoma cell migration or invasion and to modulate these effects in vitro by the use of glycosaminoglycan mimetics. We show that the human hepatoma Huh7 and Hep3B cells express RANTES/CCL5 G protein–coupled receptor CCR1 but not CCR3 nor CCR5. RANTES/CCL5 binding to these cells depends on CCR1 and glycosaminoglycans. Moreover, RANTES/CCL5 strongly stimulates the migration and the invasion of Huh7 cells and to a lesser extent that of Hep3B cells. RANTES/CCL5 also stimulates the tyrosine phosphorylation of focal adhesion kinase and activates matrix metalloproteinase-9 in Huh7 hepatoma cells, resulting in increased invasion of these cells. The fact that RANTES/CCL5-induced migration and invasion of Huh7 cells are both strongly inhibited by anti-CCR1 antibodies and heparin, as well as by β-d-xyloside treatment of the cells, suggests that CCR1 and glycosaminoglycans are involved in these events. We then show by surface plasmon resonance that synthetic glycosaminoglycan mimetics, OTR4120 or OTR4131, directly bind to RANTES/CCL5. The preincubation of the chemokine with each of these mimetics strongly inhibited RANTES-induced migration and invasion of Huh7 cells. Therefore, targeting the RANTES-glycosaminoglycan interaction could be a new therapeutic approach for human hepatocellular carcinoma. [Mol Cancer Ther 2007;6(11):2948–58]

Overexpression of VEGF189 in breast cancer cells induces apoptosis via NRP1 under stress conditions

The existence of multiple VEGF-A isoforms raised the possibility that they may have distinct functions in tumor growth. We have previously published that VEGF189 and VEGF165 contribute to breast cancer progression and angiogenesis, but VEGF165 induced the most rapid tumor uptake. Since VEGF165 has been described as a survival factor for breast tumor cells, we questioned here the effects of VEGF189 on the survival/apoptosis of MDA-MB-231 cells. We used clones which overexpress VEGF189 (V189) or VEGF165 (V165) isoforms and compared them to a control one (cV). Overexpression of VEGF189 resulted in increased cell apoptosis, as determined by Annexin-V apoptosis assay, under serum starvation and doxorubicin treatment, while VEGF 165 was confirmed to be a survival factor. Since MDA-MB-231 highly express NRP1 (a co-receptor for VEGF-A), we used short hairpin RNA (shRNA) to knockdown NRP1 expression. V189shNRP1 clones were characterized by reduced apoptosis and higher necrosis, as compared to V189shCtl, under stress conditions. Unexpectedly, NRP1 knock-down had no effect on the survival or apoptosis of V165 cells. VEGF189 showed greater affinity towards NRP1 than VEGF165 using a BIAcore binding assay. Finally, since endogenously produced urokinase-type plasminogen (uPA) has been found to prevent apoptosis in breast cancers, we analyzed the level of uPA activity in our clones. An inhibition of uPA activity was observed in V189shNRP1 clones. Altogether, these results suggest a major role of NRP1 in apoptosis induced by VEGF189 in stress conditions and confirm VEGF165 as a survival factor.

In vivo effects of amiodarone on cardiac β-adrenoceptor density and heart rate require thyroid hormones

To assess if the anti-β-adrenergic effect and the bradycardia induced by amiodarone were mediated by thyroid hormone, we investigated these effects of amiodarone in euthyroid and hypothyroid rats. We studied control rats, thyroidectomized rats, control rats treated with amiodarone (50 mg/kg for 8 days), and thyroidectomized rats treated with amiodarone. At the end of the treatment, free thyroid hormone levels (FT4 and FT3) were determined, and cardiac β-receptor density (Bmax) and affinity (Kd) were assayed by using (-)-[125I]iodo-cyanopindolol as radioligand. Resting heart rate (rHR) was also assessed every day in control and thyroidectomized rats, before and after amiodarone. In hypothyroid rats, in which free thyroxine (FT4) was not detectable and free 3,5,3′-triiodothyronine (FT3) was only 16% that of euthyroid rats, Bmax (14.1 ± 2.5 fmol/mg, n = 7) and rHR (259 ± 9.7 beats/min, n = 6) were significantly lowered compared with euthyroid rats (Bmax:18.4 ± 3.4 fmol/mg, n = 7; rHR:277 ± 4.1 beats/min, n = 5). Amiodarone treatment decreased Bmax (13.6 ± 2.9 fmol/mg, n = 8) and rHR (252 ± 5.5 beats/min, n = 5) only in euthyroid rats and did not produce significant cardiac effects in hypothyroid rats. (Values are given as mean ± SD.) We conclude that a minimum serum thyroid hormone concentration is a necessary condition for amiodarone to produce some of its cardiac effects. An antagonistic reaction to thyroid hormones at the cellular level can be postulated as a mechanism of the cardiac anti-β-adrenergic action of amiodarone.

Discovery of novel inhibitors of vascular endothelial growth factor-A–Neuropilin-1 interaction by structure-based virtual screening

Neuropilin-1 (NRP-1), one of the most important co-receptors of vascular endothelial growth factor-A (VEGF-A), increases its angiogenic action in several chronic diseases including cancer by increasing the activity of associated tyrosine kinase receptors, VEGFR1 and VEGFR2. Binding of VEGF-A to NRP-1 plays a critical role in pathological angiogenesis and tumor progression. Today, targeting this interaction is a validated approach to fight against angiogenesis-dependent diseases. Only anti-NRP-1 antibodies, peptide and peptidomimetic drug-candidates or hits have been developed thus far. In order to identify potent orally active small organic molecules various experimental and in silico approaches can be used. Here we report, novel promising small drug-like molecules disrupting the binding of VEGF-A₁₆₅ to NRP-1. We carried out structure-based virtual screening experiments using the ChemBridge compound collection on the VEGF-A₁₆₅ binding pocket of NRP-1. After docking and two rounds of similarity search computations, we identified 4 compounds that inhibit the biotinylated VEGF-A₁₆₅ binding to recombinant NRP-1 with Ki of about 10 μM. These compounds contain a common chlorobenzyloxy alkyloxy halogenobenzyl amine scaffold that can serve as a base for further development of new NRP-1 inhibitors.