CIGB-300, a proapoptotic peptide, inhibits angiogenesis in vitro and in vivo

We have previously demonstrated that a proapoptotic cyclic peptide CIGB-300, formerly known as P15-Tat delivered into the cells by the cell-penetrating peptide Tat, was able to abrogate the CK2-mediated phosphorylation and induce tumor regression when injected directly into solid tumors in mice or by systemic administration. In this work, we studied the role of CIGB-300 on the main events that take place in angiogenesis. At non-cytotoxic doses, CIGB-300 was able to inhibit adhesion, migration, and tubular network formation induced by human umbilical vein endothelial cells (HUVEC) growing upon Matrigel in vitro. Likewise, we evaluated the cellular penetration and localization into the HUVEC cells of CIGB-300. Our results confirmed a quick cellular penetration and a cytoplasmic accumulation in the early minutes of incubation and a translocation into the nuclei beginning at 12 h of treatment, with a strong presence in the perinuclear area. A microarray analysis was used to determine the genes affected by the treatment. We observed that CIGB-300 significantly decreased four genes strongly associated with tubulogenesis, growth, and differentiation of endothelial cells. The CIGB-300 was tested in vivo on chicken embryo chorioallantoic membranes (CAM), and a large number of newly formed blood vessels were significantly regressed. The results suggested that CIGB-300 has a potential as an antiangiogenic treatment. The mechanism of action may be associated with partial inhibition of VEGF and Notch pathways.     

Synthesis and activities of cyclic thrombin-receptor-derived peptide analogues of the Ser42-Phe-Leu-Leu-Arg46 motif sequence containing d-Phe and/or d-Arg

Summary Cyclic analogues of the active thrombin receptor peptide SFLLR (TRP_42–46) containingd-Phe and/ord-Arg have been prepared by the solid-phase method, purified by reversed-phase HPLC and bioassayed in a rat smooth muscle contractile assay. Cyclization was achieved by forming an amide linkage between the-NH₂ and-COOH groups of the two leucine residues located at the N- and C-terminal positions of the linear protected precursor H₂N-Leu-Arg(Pmc)-Y-Phe-Leu-OH (Y=Gly,Acp) using 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluoroborate borate (HBTU) or 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) as coupling reagents andN,N-diisopropylethylamine (DIPEA) in high dilution. Their structure was confirmed by fast by fast atom bombardment mass spectrometry and NMR methods. The cyclic peptides c-fLLrG, c-fLLRG, c-FLLrG and c-fLLrAcp, c-FLLrAcp so synthesized were assessed for their contractile activity in a rat gastric longitudinal muscle bioassay system which has been used previously to evaluate the biological activities of linear thrombin-receptor-derived polypeptides such as SFLLR (P5) and SFLLR-NH₂ (P5-NH₂).     

Inhibition of the angiogenesis by the MCP-1 (monocyte chemoattractant protein-1) binding peptide

The CC chemokine, monocyte chemoattractant protein-1 (MCP-1), plays a crucial role in the initiation of atherosclerosis and has direct effects that promote angiogenesis. To develop a specific inhibitor for MCP-1-induced angiogenesis, we performed in vitro selection employing phage display random peptide libraries. Most of the selected peptides were found to be homologous to the second extracellular loops of CCR2 and CCR3. We synthesized the peptide encoding the homologous sequences of the receptors and tested its effect on the MCP-1 induced angiogenesis. Surface plasmon resonance measurements demonstrated specific binding of the peptide to MCP-1 but not to the other homologous protein, MCP-3. Flow cytometry revealed that the peptide inhibited the MCP-1 binding to THP-1 monocytes. Moreover, CAM and rat aortic ring assays showed that the peptide inhibited MCP-1 induced angiogenesis. Our observations indicate that the MCP-1-binding peptide exerts its anti-angiogenic effect by interfering with the interaction between MCP-1 and its receptor.     

Inhibition of inflammatory angiogenesis by distant subcutaneous tumor in mice

We investigated angiogenesis, inflammatory cells accumulation and endogenous production of cytokines in sponge implants of tumor-bearing mice. Seven days after inoculation of Ehrlich tumor cells (2.5 x 10(6)), sponge discs were implanted subcutaneously in the dorsa of mice to induce the formation of fibrovascular tissue. The implants of tumor-bearing and non tumor-bearing animals were assessed for neovascularization and leukocyte accumulation, together with levels of relevant cytokines, vascular endothelial growth factor VEGF), tumor necrosis factor alpha (TNF-alpha), CXCL1-3/KC and CCL2/JE. In the implants of tumor-bearing animals angiogenesis (assessed by hemoglobin content and VEGF levels in the implants) and leukocyte accumulation (assessed by myeloperoxidase -MPO- and N- acetylglucosaminidase-NAG-enzyme activities) were all significantly less than those in the implants of non tumor-bearing animals. Although the chemokine CXCL1-3/KC was lower in the implants of tumor-bearing animals, the chemokine CCL2/JE was increased in this group. The production of TNF-alpha in the implants was not modified by the presence of the subcutaneous tumor. The combination of the methodologies used in this study has provided a novel approach to investigate the interaction between two distinct proliferating tissues that share common features (angiogenesis, cell recruitment, inflammation) and has shown that the predominant inhibitory effect of a tumor mass over repair process is associated with altered cytokine production.     

Dimethyl phenyl piperazine iodide (DMPP) induces glioma regression by inhibiting angiogenesis

1,1-Dimethyl-4-phenyl piperazine iodide (DMPP) is a synthetic nicotinic acetylcholine receptor (nAChR) agonist that could reduce airway inflammation. In this study, we demonstrated that DMPP could dramatically inhibit glioma size maintained on the chick embryonic chorioallantoic membrane (CAM). We first performed MTT and BrdU incorporation experiments on U87 glioma cells in vitro to understand the mechanism involved. We established that DMPP did not significantly affect U87 cell proliferation and survival. We speculated that DMPP directly caused the tumor to regress by affecting the vasculature in and around the implanted tumor on our chick CAM model. Hence, we conducted detailed analysis of DMPP's inhibitory effects on angiogenesis. Three vasculogenesis and angiogenesis in vivo models were used in the study which included (1) early chick blood islands formation, (2) chick yolk-sac membrane (YSW) and (3) CAM models. The results revealed that DMPP directly suppressed all developmental stages involved in vasculogenesis and angiogenesis – possibly by acting through Ang-1 and HIF-2α signaling. In sum, our results show that DMPP could induce glioma regression grown on CAM by inhibiting vasculogenesis and angiogenesis.     

NC-100717: a versatile RGD peptide scaffold for angiogenesis imaging

Targeting the molecular pathways associated with angiogenesis offers great potential in detecting disease pathology using in vivo imaging technologies. Initiation of angiogenesis requires activation and migration of endothelial cells in order for neovascularization to proceed. Endothelial cells associate with the extracellular matrix through specific interactions with a variety of cell adhesion receptors known as integrins. Peptides containing the tripeptide sequence RGD are known to bind with high affinity to the vβ3 and vβ5 integrins associated with angiogenesis. We present herein the synthesis and in vitro binding affinity of the RGD-containing peptide NC-100717 and a range of molecular probes derived from this intermediate.     

Properties of a novel chemotactic esapeptide,an analogue of the prototypical N-formylmethionyl peptide

The new disulphur-bridged peptide, for-Met-Leu-Cys(OMe)-Cys(OMe)-Leu-Met-for, has been synthesized and its biological properties resulting from its binding to the formyl-peptide receptor of human neutrophils characterized. Three activities resulting from this interaction were measured: directed cell migration (i.e., chemotaxis); superoxide anion production; and lysozyme enzyme release. The properties were compared with those observed for the prototypical peptide, for-Met-Leu-Phe-OMe. Chemotaxis is strongly triggered while both superoxide anion production and lysosomal enzyme release are elicited only at high concentrations and never reach the response peak observed for the prototype peptide at physiologically relevant concentrations. The derivative appears to bind with a good affinity to the formyl-peptide receptors. These results provide new information regarding the structure-activity relationship of the formyl-peptide receptor.     

Determination of SU5416, a novel angiogenesis inhibitor, in human plasma by liquid chromatography

A high-performance liquid chromatographic (HPLC) assay with UV detection has been developed for the quantitative determination of the antiangiogenic agent SU5416 in human plasma. Sample pretreatment involved a single protein-precipitation step with acetonitrile containing the internal standard, chrysin. Separation of the compounds of interest was achieved on a column packed with HP Zorbax C(8) material (5microm particle size; length: 150mm; i.d.: 4.6mm) using a dual solvent system of 0.01M aqueous ammonium acetate and acetonitrile delivered as a nonlinear gradient at a flow-rate of 1.00ml/min. Simultaneous UV detection was performed at 440nm (SU5416) and 268nm (chrysin). The calibration graph was fit to log-transformed response-concentration data over a range of 10-5000ng/ml. Values for accuracy and precision, obtained from six quality controls analyzed on different days in replicates of 3 or 6, ranged 92.9-109 and 0.8-6.2%, respectively. The developed method was successfully applied to study the pharmacokinetics of SU5416 in a cancer patient receiving the drug as a 1h infusion.     

Angiogenic activity of syndecan-binding laminin peptide AG73 (RKRLQVQLSIRT)

The AG73 peptide (RKRLQVQLSIRT, mouse laminin alpha 1 chain 2719-2730) promotes cell adhesion and tumor metastasis, and interacts with transmembrane syndecan proteoglycans. Here, we demonstrate AG73 peptide angiogenic activity using in vitro, ex vivo, and in vivo models. AG73 induced murine endothelial cell (SVEC4-10) tube formation on Cultrex Basement Membrane Extract (Cultrex BME) and stimulated sprouting of aortic rings. None of the homologous sequences from the laminin alpha2, alpha3, alpha4, or alpha5 chains was as active as AG73 in promoting sprouting formation. AG73 also mediated angiogenesis in the chick chorioallantonic membrane (CAM) assay. Using subcutaneously injected Cultrex BME supplemented with AG73, we observed a large angiogenic response. Furthermore, AG73-conjugated to a chitosan membrane promoted a strong angiogenic response in the CAM assay. These results indicate that the AG73 peptide is a potent syndecan-binding angiogenesis stimulator and may be useful for therapeutic application to treat ischemic injuries.     

Inhibition of new vessel growth in mouse model of laser-induced choroidal neovascularization by adiponectin peptide II

We have investigated the effect of adiponectin (APN) peptide II on new vessel growth in mouse model of choroidal neovascularization (CNV) or wet type age-related macular degeneration (AMD). Mice were injected intraperitoneally with APN peptide II, control peptide, or PBS on day 1-7 or day 5-14. APN, AdipoR1, PCNA, and VEGF localization was investigated using confocal microscopy, immunohistochemistry, and RT-PCR. APN peptide II decreased the relative area of FITC-dextran perfused vessels by 4-fold, PCNA expression by 3-fold, and the number of PCNA stained HUVEC and MAVEC cells by 38 and 46%, respectively. We concluded that APN peptide II inhibits CNV size on days 7 and 14 by inhibiting the proliferation of endothelial cells in vivo and in vitro. APN peptide II may have therapeutic potential to inhibit CNV or wet AMD.     

Inhibition of angiogenesis by a mouse sprouty protein

Sprouty negatively modulates branching morphogenesis in the Drosophila tracheal system. To address the role of mammalian Sprouty homologues in angiogenesis, another form of branching morphogenesis, a recombinant adenovirus engineered to express murine Sprouty-4 selectively in endothelial cells, was injected into the sinus venosus of embryonic day 9.0 cultured mouse embryos. Sprouty-4 expression inhibited branching and sprouting of small vessels, resulting in abnormal embryonic development. In vitro, Sprouty-4 inhibited fibroblast growth factor and vascular endothelial cell growth factor-mediated cell proliferation and migration and prevented basic fibroblast growth factor and vascular endothelial cell growth factor-induced MAPK phosphorylation in endothelial cells, indicating inhibition of tyrosine kinase-mediated signaling pathways. The ability of constitutively activated mutant Ras(L61) to rescue Sprouty-4 inhibition of MAPK phosphorylation suggests that Sprouty inhibits receptor tyrosine kinase signaling upstream of Ras. Thus, Sprouty may regulate angiogenesis in normal and disease processes by modulating signaling by endothelial tyrosine kinases.     

Inhibition of angiotensin-converting enzyme by angiotensin I analogue peptide inhibitors. A kinetic study

Angiotensin I analogues with a phosphonic acid group replacing the C-terminal carboxyl group were shown to be competitive inhibitors of angiotensin-converting enzyme. This new class of inhibitors was used to study the binding requirements of the angiotensin I-like ligands to the enzyme's active site. These studies indicate that angiotensin-converting enzyme recognizes at least five amino acid residues at the C-terminus of the peptide. The effect of pH on the binding of the most potent inhibitor peptide was compared to Captopril. The two inhibitors showed similar Ki-pH profiles despite their structural differences. Chloride enhanced the binding of the peptide inhibitor at both pH 9.0 and pH 6.5. At pH 9.0 the inhibitor peptide and the anion bind randomly to the enzyme, while at pH 6.5 the mechanism is ordered. In the latter case, the anion binds first to the enzyme.     

Controlled antibody immobilization onto immunoanalytical platforms by synthetic peptide

Antibody immobilization on a solid surface is inevitable in the preparation of immunochips/sensors. Antibody-binding proteins such as proteins A and G have been extensively employed to capture antibodies on sensor surfaces with right orientations, maintaining their full functionality. Because of their synthetic versatility and stability, in general, small molecules have more advantages than proteins. Nevertheless, no small molecule has been used for oriented and specific antibody immobilization. Here is described a novel strategy to immobilize an antibody on various sensor surfaces by using a small antibody-binding peptide. The peptide binds specifically to the Fc domain of immunoglobulin G (IgG) and, therefore, affords a properly oriented antibody surface. Surface plasmon resonance analysis indicated that a peptide linked to a gold chip surface through a hydrophilic linker efficiently captured human and rabbit IgGs. Moreover, antibodies captured by the peptide exhibited higher antigen binding capacity compared with randomly immobilized antibodies. Peptide-mediated antibody immobilization was successfully applied on the surfaces of biosensor substrates such as magnetic particles and glass slides. The antibody-binding peptide conjugate introduced in this work is the first small molecule linker that offers a highly stable and specific surface platform for antibody immobilization in immunoassays.     

Inhibition of adenovirus-mediated human MAGE-D1 on angiogenesis in vitro and in vivo

MAGE-D1 is a member of the MAGE family of proteins, and functions as an adaptor that mediates multiple signaling pathways. The current study for the first time provides evidence for a role of MAGE-D1 in the negative regulation of angiogenic activity in vitro and in vivo models. Our findings showed that MAGE-D1 over-expression significantly suppressed the angiogenic key events such as endothelial cell migration and invasion, adhesion on collagen I substrate, and in vitro differentiation into tube-like structures under both normoxic and hypoxic conditions. MAGE-D1 over-expression also inhibited in vivo angiogenesis in Matrigel plugs that were implanted subcutaneously in mice. With further experiments, we revealed that MAGE-D1 over-expression disrupted actin cytoskeleton organization and lamellipodia formation, and down-regulated HIF-1-dependent gene expression in endothelial cells under hypoxic conditions. These findings demonstrate a new function of MAGE-D1 in the regulation of angiogenesis and provide new insight into the ability of MAGE-D1 to suppress the growth and angiogenic response of endothelial cells by interfering with HIF-1-dependent gene expression, and actin cytoskeleton reorganization, suggesting that MAGE-D1 might be a novel inhibitor of angiogenesis in vitro and in vivo.     

Inhibition of virus-induced cell fusion by apolipoprotein A-I and its amphipathic peptide analogs.

Apolipoprotein A-I (apoA-I), the major protein component of serum high-density lipoproteins (HDL), was found to inhibit herpes simplex virus (HSV)-induced cell fusion at physiological (approximately 1 microM) concentrations, whereas HDL did not exert any inhibitory effect. Lipid-associating, synthetic amphipathic peptides corresponding to residues 1-33 (apoA-I[1-33]) or residues 66-120 (apoA-I[66-120]) of apoA-I, also inhibited HSV-induced cell fusion, whereas a peptide corresponding to residues 8-33 of apoA-I (apoA-I[8-33]), which fails to associate with lipids, did not exert any inhibitory effect. These results suggest that lipid binding may be a prerequisite for peptide-mediated fusion inhibition. Consistent with this idea, a series of lipid-binding 22-amino-acid-residue-long synthetic amphipathic peptides that correspond to the amphipathic helical domains of apoA-I (A-I consensus series), or 18-residue-long model amphipathic peptides (18A series), were found to exert variable levels of fusion-inhibitory activity. The extent of fusion-inhibitory activity did not correlate with hydrophobic moment, hydrophobicity of the nonpolar face, helix-forming ability, or lipid affinity of the different peptides. Peptides in which the nonpolar face was not interrupted by a charged residue displayed greater fusion-inhibitory activity. Also, the presence of positively charged residues at the polar-nonpolar interface was found to correlate with higher fusion-inhibitory activity.     

Inhibition of thrombin by synthetic hirudin peptides

To investigate the role of different regions of hirudin in the interaction with the proteinase thrombin, segments of hirudin containing 15-51 residues were synthesized. The C-terminal segment 40-65 inhibited the fibrinogen clotting activity of thrombin but not amidolysis of tosyl-Gly-Pro-Arg-p-nitroanilide. Central peptide 15–42 was insoluble at pH 7, but peptide 15-65 inhibited fibrinogen clotting and amidolysis to an equal extent. The N-terminal loop peptide 1-15 had no inhibitory activity and did not affect the potency of peptide 15-65. These data suggest that the central region inhibits catalysis.     

Regulation of angiogenesis by the aging suppressor gene klotho

Advanced age is a major risk factor of peripheral artery disease. We examined the effects of the aging-suppressor gene klotho on angiogenesis in response to ischemia by introducing ischemic hindlimb model in mice heterozygously deficient for the klotho gene and in wild type mice. Blood flow recovery as assessed by laser doppler perfusion imaging and angiogenesis as assessed by density of PECAM-1/CD31-positive positive capillaries were markedly impaired in mice heterozygously deficient for the klotho gene (both <0.05). Our findings show that the aging-suppressor gene klotho affects angiogenesis and the possibility that age-related impairment of angiogenesis might be regulated by the klotho gene. Our results present a new possibility of therapeutic angiogenesis for patients of advanced age.     

Potentiating effect of heparin in the activation of procollagenase by a low-Mr angiogenesis factor

A low-Mr freely dialysable endothelial cell-stimulating angiogenesis factor (ESAF) from conditioned medium of a mouse lymphoma cell line has previously been shown to activate latent skin fibroblast procollagenase. Activation comparable with the maximum that can be achieved with trypsin is obtained with chemically undetectable amounts of the factor. We now show that when even smaller amounts of ESAF are used heparin is able to potentiate its action in this system. The relationship between this activity and the mechanism of angiogenesis, which is itself potentiated by heparin, is discussed.     

Inhibition of ischemia-induced angiogenesis by benzo[a]pyrene in a manner dependent on the aryl hydrocarbon receptor

We have investigated the effect of benzo[a]pyrene (B[a]P), a carcinogen of tobacco smoke and an agonist for the aryl hydrocarbon receptor (AHR), on hypoxia-induced angiogenesis. Ischemia was induced by femoral artery ligation in wild-type and AHR-null mice, and the animals were subjected to oral administration of B[a]P (125 mg/kg) once a week. Exposure to B[a]P up-regulated the expression of metallothionein in the ischemic hindlimb and markedly inhibited ischemia-induced angiogenesis in wild-type mice. The amounts of interleukin-6 and of vascular endothelial growth factor (VEGF) mRNA in the ischemic hindlimb of wild-type mice were reduced by exposure to B[a]P. These various effects of B[a]P were markedly attenuated in AHR-null mice. Our observations suggest that the loss of the inhibitory effect of B[a]P on ischemia-induced angiogenesis apparent in AHR-null mice may be attributable to maintenance of interleukin-6 expression and consequent promotion of angiogenesis through up-regulation of VEGF expression.