Annarita Del Gatto

Peptide-based Molecules in Angiogenesis

Angiogenesis refers to the process of remodeling the vascular tissue characterized by the branching out of a new blood vessel from a pre‐existing vessel. Angiogenesis is particularly active during embryogenesis, while during adult life it is quiescent and limited to particular physiologic phenomena. Recently, the study of molecular mechanisms of angiogenesis has stirred renewed interest due to the recognition of the role played by angiogenesis in several pathologies of significant medical impact, such as cancer and cardiovascular disease, and due to the pharmacologic interest rising from the possibility of modulating these phenomena. Antibodies, peptides and small molecules targeting active endothelial cells represent an innovative tool in therapeutic and diagnostic fields. In this study, we reviewed the literature of peptide and peptidomimetics in angiogenesis and their potential applications. Two specific protein systems, namely the vascular endothelial growth factor and its receptor and integrins, will be discussed in detail.

AKAP121 downregulation impairs protective cAMP signals, promotes mitochondrial dysfunction, and increases oxidative stress

AIMS The aim of the present study was to determine the function and the role of the scaffold protein AKAP121, tethering cAMP dependent protein kinase A to the outer wall of mitochondria, in neonatal ventricular myocytes and the heart. METHODS AND RESULTS Competitive peptides displacing AKAP121 from mitochondria in the tissue and in the cells were used to investigate the role of AKAP121 in mitochondrial function, reactive oxygen species (ROS) generation, and cell survival. Displacement of AKAP121 from mitochondria by synthetic peptides triggers the death program in cardiomyocytes. Under pathological conditions in vivo, in a rat model of cardiac hypertrophy induced by ascending aorta banding, the levels of AKAP121 are significantly down-regulated. Disappearance of AKAP121 is associated with mitochondrial dysfunction, high oxidative stress, and apoptosis. In vivo delocalization of AKAP121 by competitive peptides replicates some of the molecular signatures induced by pressure overload: mitochondrial dysfunction, increased mitochondrial ROS, and apoptosis. CONCLUSION These data suggest that AKAP121 regulates the response to stress in cardiomyocytes, and therefore AKAP121 downregulation might represent an important event contributing to the development of cardiac dysfunction.

Evaluation of the anti-angiogenic properties of the new selective αVβ3 integrin antagonist RGDechiHCit.

BackgroundIntegrins are heterodimeric receptors that play a critical role in cell-cell and cell-matrix adhesion processes. Among them, αVβ3 integrin, that recognizes the aminoacidic RGD triad, is reported to be involved in angiogenesis, tissue repair and tumor growth. We have recently synthesized a new and selective ligand of αVβ3 receptor, referred to as RGDechiHCit, that contains a cyclic RGD motif and two echistatin moieties.MethodsThe aim of this study is to evaluate in vitro and in vivo the effects of RGDechiHCit. Therefore, we assessed its properties in cellular (endothelial cells [EC], and vascular smooth muscle cells [VSMC]) and animal models (Wistar Kyoto rats and c57Bl/6 mice) of angiogenesis.ResultsIn EC, but not VSMC, RGDechiHCit inhibits intracellular mitogenic signaling and cell proliferation. Furthermore, RGDechiHCit blocks the ability of EC to form tubes on Matrigel. In vivo, wound healing is delayed in presence of RGDechiHCit. Similarly, Matrigel plugs demonstrate an antiangiogenic effect of RGDechiHCit.ConclusionsOur data indicate the importance of RGDechiHCit in the selective inhibition of endothelial αVβ3 integrin in vitro and in vivo. Such inhibition opens new fields of investigation on the mechanisms of angiogenesis, offering clinical implications for treatment of pathophysiological conditions such as cancer, proliferative retinopathy and inflammatory disease.

Peptides for tumour therapy and diagnosis: current status and future directions.

The use of peptides as targeting tools has been validated in different applications. In particular radiolabelled peptides with adequate stability, receptor-binding properties and biokinetic behaviour have been investigated as an important class of radiopharmaceuticals for cancer pathology imaging and therapy. This review focuses on recent progress in design and synthetic modifications of small biologically active peptides used in diagnosis and therapy. In particular, we report the current development and optimization of suitable peptides for targeting three relevant biological receptors (CCK, somatostatin, and integrin receptors) involved in specific tumour diseases.

Gold nanoparticles capped by a GC-containing peptide functionalized with an RGD motif for integrin targeting.

Gold nanoparticles were obtained by reduction of a tetrachloroaurate aqueous solution in the presence of a RGD-(GC)(2) peptide as stabilizer. As comparison, the behavior of the (GC)(2) peptide has been studied. The (GC)(2) and RGD-(GC)(2) peptides were prepared ad hoc by Fmoc synthesis. The colloidal systems have been characterized by UV-visible, TGA, ATR-FTIR, mono and bidimensional NMR techniques, confocal and transmission (TEM) microscopy, ζ-potential, and light scattering measurements. The efficient cellular uptake of Au-RGD-(GC)(2) and Au-(GC)(2) stabilized gold nanoparticles into U87 cells (human glioblastoma cells) were investigated by confocal microscopy and compared with the behavior of (GC)(2) capped gold nanoparticles. A quantitative determination of the nanoparticles taken up has been carried out by measuring the pixel brightness of the images, a measure that highlighted the importance of the RGD termination of the peptide. Insight in the cellular uptake mechanism was investigated by TEM microscopy. Various important evidences indicated the selective uptake of RGD-(GC)(2) gold nanoparticles into the nucleus.

Imaging of αvβ3 Expression by a Bifunctional Chimeric RGD Peptide not Cross-Reacting with αvβ5

Purpose: To test whether a novel bifunctional chimeric peptide comprising a cyclic Arg-Gly-Asp pentapeptide covalently bound to an echistatin domain can discriminate αvβ3 from αvβ5 integrin, thus allowing the in vivo selective visualization of αvβ3 expression by single-photon and positron emission tomography (PET) imaging. Experimental Design: The chimeric peptide was preliminarily tested for inhibition of αvβ3-dependent cell adhesion and competition of 125I-echistatin binding to membrane of stably transfected K562 cells expressing αvβ3 (Kαvβ3) or αvβ5 (Kαvβ5) integrin. The chimeric peptide was then conjugated with diethylenetriaminepentaacetic acid and labeled with 111In for single-photon imaging, whereas a one-step procedure was used for labeling the full-length peptide and a truncated derivative, lacking the last five C-terminal amino acids, with 18F for PET imaging. Nude mice bearing tumors from Kαvβ3, Kαvβ5, U87MG human glioblastoma, and A431 human epidermoid cells were subjected to single-photon and PET imaging. Results: Adhesion and competitive binding assays showed that the novel chimeric peptide selectively binds to αvβ3 integrin and does not cross-react with αvβ5. In agreement with in vitro findings, single-photon and PET imaging studies showed that the radiolabeled chimeric peptide selectively localizes in tumor xenografts expressing αvβ3 and fails to accumulate in those expressing αvβ5 integrin. When 18F-labeled truncated derivative was used for PET imaging, αvβ3- and αvβ5-expressing tumors were visualized, indicating that the five C-terminal amino acids are required to differentially bind the two integrins. Conclusion: Our findings indicate that the novel chimeric Arg-Gly-Asp peptide, having no cross-reaction with αvβ5 integrin, allows highly selective αvβ3 expression imaging and monitoring. (Clin Cancer Res 2009;15(16):5224–33)

Peptides Targeting Angiogenesis Related Growth Factor Receptors

Growth factors (GFs) are extracellular signaling polypeptides regulating cell proliferation, differentiation and survival. They exert a wide spectrum of biological activities selectively binding to and activating specific membrane receptors which then transfer the message to cell interior inducing specific biochemical pathways. GFs are especially involved in the regulation of angiogenesis, a physiological process underlining several pathologies. Molecules able to modulate angiogenesis, interfering with the molecular recognition between a GF and its receptor, have a big pharmacologic interest. Either GF and the receptor are potential drug target. Peptides are useful molecules to develop new lead compounds disrupting protein-protein interface for pharmacological applications. In this review we describe peptides targeting the receptors of the pro-angiogenic growth factors FGF, PDGF and VEGF. The biological function and the structure of each growth factor/receptor system are discussed, as well as the molecular interaction between peptides and the receptors. Finally, we highlight the pharmacological and diagnostic applications of these peptides in angiogenesis related diseases.

Zinc to cadmium replacement in the A. thaliana SUPERMAN Cys2His2 zinc finger induces structural rearrangements of typical DNA base determinant positions

Among heavy metals, whose toxicity cause a steadily increasing of environmental pollution, cadmium is of special concern due to its relatively high mobility in soils and potential toxicity at low concentrations. Given their ubiquitous role, zinc fingers domains have been proposed as mediators for the toxic and carcinogenic effects exerted by xenobiotic metals. To verify the structural effects of zinc replacement by cadmium in zinc fingers, we have determined the high resolution structure of the single Cys₂ His₂ zinc finger of the Arabidopsis thaliana SUPERMAN protein (SUP37) complexed to the cadmium ion by means of UV-vis and NMR techniques. SUP37 is able to bind Cd(II), though with a dissociation constant higher than that measured for Zn(II). Cd-SUP37 retains the ββα fold but experiences a global structural rearrangement affecting both the relative orientation of the secondary structure elements and the position of side chains involved in DNA recognition: among them Ser17 side chain, which we show to be essential for DNA binding, experiences the largest displacement.

Integrin-targeting with peptide-bioconjugated semiconductor-magnetic nanocrystalline heterostructures

Binary asymmetric nanocrystals (BNCs), composed of a photoactive TiO2 nanorod joined with a superparamagnetic γ-Fe2O3 spherical domain, were embedded in polyethylene glycol modified phospholipid micelle and successfully bioconjugated to a suitably designed peptide containing an RGD motif. BNCs represent a relevant multifunctional nanomaterial, owing to the coexistence of two distinct domains in one particle, characterized by high photoactivity and magnetic properties, that is particularly suited for use as a phototherapy and hyperthermia agent as well as a magnetic probe in biological imaging. We selected the RGD motif in order to target integrin expressed on activated endothelial cells and several types of cancer cells. The prepared RGD-peptide/BNC conjugates, comprehensively monitored by using complementary optical and structural techniques, demonstrated a high stability and uniform dispersibility in biological media. The cytotoxicity of the RGD-peptide/BNC conjugates was studied in vitro. The cellular uptake of RGD-peptide conjugates in the cells, assessed by means of two distinct approaches, namely confocal microscopy analysis and emission spectroscopy determination in cell lysates, displayed selectivity of the RGD-peptide-BNC conjugate for the αvβ3 integrin. These RGD-peptide-BNC conjugates have a high potential for theranostic treatment of cancer.

RGDechi-hCit: αvβ3 Selective Pro-Apoptotic Peptide as Potential Carrier for Drug Delivery into Melanoma Metastatic Cells

αvβ3 integrin is an important tumor marker widely expressed on the surface of cancer cells. Recently, we reported some biological features of RGDechi-hCit, an αvβ3 selective peptide antagonist. In the present work, we mainly investigated the pro-apoptotic activity of the molecule and its ability to penetrate the membrane of WM266 cells, human malignant melanoma cells expressing high levels of αvβ3 integrin. For the first time we demonstrated the pro-apoptotic effect and the ability of RGDechi-hCit to enter into cell overexpressing αvβ3 integrin mainly by clathrin- and caveolin-mediated endocytosis. Furthermore, we deepened and confirmed the selectivity, anti-adhesion, and anti-proliferative features of the peptide. Altogether these experiments give insight into the biological behavior of RGDechi-hCit and have important implications for the employment of the peptide as a new selective carrier to deliver drugs into the cell and as a therapeutic and diagnostic tool for metastatic melanoma. Moreover, since the peptide shows a pro-apoptotic effect, a great perspective could be the development of a new class of selective systems containing RGDechi-hCit and pro-apoptotic molecules or other therapeutic agents to attain a synergic action.