Luca Domenico D'Andrea

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.

In vivo properties of the proangiogenic peptide QK

The main regulator of neovascularization is Vascular Endothelial Growth Factor (VEGF). We recently demonstrated that QK, a de novo engineered VEGF mimicking peptide, shares in vitro the same biological properties of VEGF, inducing capillary formation and organization. On these grounds, the aim of this study is to evaluate in vivo the effects of this small peptide. Therefore, on Wistar Kyoto rats, we evaluated vasomotor responses to VEGF and QK in common carotid rings. Also, we assessed the effects of QK in three different models of angiogenesis: ischemic hindlimb, wound healing and Matrigel plugs. QK and VEGF present similar endothelium-dependent vasodilatation. Moreover, the ability of QK to induce neovascularization was confirmed us by digital angiographies, dyed beads dilution and histological analysis in the ischemic hindlimb as well as by histology in wounds and Matrigel plugs. Our findings show the proangiogenic properties of QK, suggesting that also in vivo this peptide resembles the full VEGF protein. These data open to new fields of investigation on the mechanisms of activation of VEGF receptors, offering clinical implications for treatment of pathophysiological conditions such as chronic ischemia.

Assignment of the Binding Site for Haptoglobin on Apolipoprotein A-I

Haptoglobin (Hpt) was previously found to bind the high density lipoprotein (HDL) apolipoprotein A-I (ApoA-I) and able to inhibit the ApoA-I-dependent activity of the enzyme lecithin:cholesterol acyltransferase (LCAT), which plays a major role in the reverse cholesterol transport. The ApoA-I structure was analyzed to detect the site bound by Hpt. ApoA-I was treated by cyanogen bromide or hydroxylamine; the resulting fragments, separated by electrophoresis or gel filtration, were tested by Western blotting or enzyme-linked immunosorbent assay for their ability to bind Hpt. The ApoA-I sequence from Glu113 to Asn184 harbored the binding site for Hpt. Biotinylated peptides were synthesized overlapping such a sequence, and their Hpt binding activity was determined by avidin-linked peroxidase. The highest activity was exhibited by the peptide P2a, containing the ApoA-I sequence from Leu141 to Ala164. Such a sequence contains an ApoA-I domain required for binding cells, promoting cholesterol efflux, and stimulating LCAT. The peptide P2a effectively prevented both binding of Hpt to HDL-coated plastic wells and Hpt-dependent inhibition of LCAT, measured by anti-Hpt antibodies and cholesterol esterification activity, respectively. The enzyme activity was not influenced, in the absence of Hpt, by P2a. Differently from ApoA-I or HDL, the peptide did not compete with hemoglobin for Hpt binding in enzyme-linked immunosorbent assay experiments. The results suggest that Hpt might mask the ApoA-I domain required for LCAT stimulation, thus impairing the HDL function. Synthetic peptides, able to displace Hpt from ApoA-I without altering its property of binding hemoglobin, might be used for treatment of diseases associated with defective LCAT function.

Structural Determinants of the Unusual Helix Stability of a De Novo Engineered Vascular Endothelial Growth Factor (VEGF) Mimicking Peptide

Understanding how an amino acid sequence folds into a well organized three-dimensional structure remains a challenge. The interest in protein folding comes from the possibility to predict the protein structure from genome-derived sequence, design proteins with new fold and understand protein misfolding. Peptide helix is a simple model system in which various contributions to helix formation can be dissected and understood qualitatively. Many strategies have been pursued to design peptide helices and notable results have been achieved even with very short sequences, but mainly these methods rely on the use of nonnatural amino acids or introducing constraints. In this paper, we report on the stability characterization, using CD, NMR and MD studies, of a designed, a-helical, 15-mer peptide (named QK), composed only of natural amino acids (sequence AcKLTWQELYQLKYKGI-NH2), which activates the VEGFdependent angiogenic response. The QK peptide shows an unusual thermal stability, whose structural determinants have been determined. These results could have implication in the field of protein folding and in the design of helical structured scaffolds for the realization of peptides for applications in chemical biology. As recently described, the NMR structure of QK in pure water presents a central helical sequence (residues 4–12), which corresponds to the VEGF N-terminal helix (residues 17–25), flanked by Nand C-capping regions. The helical conformation of QK represents an important prerequisite for its biological activity, since the isolated peptide, corresponding to the helix region of VEGF, does not assume a helical conformation and does not have significant biological activity. Interestingly, QK represents one of the very few examples of bioactive helical designed peptides, composed of only natural amino acids. To gain an insight into the molecular determinants of QK helical propensity, we examined the effect of the temperature on the QK structure through NMR and CD analyses. Primarily, the aggregation state of the peptide under conditions identical to those used in the NMR structure determination was confirmed by NMR DOSY experiments (see Supporting Information). The DOSY-derived diffusion coefficient value of 1.98@10 10 ms 1 is consistent with a QK monomer state. QK structure variations upon temperature increase were followed by TOCSY experiments. In the 298– 343 K range only small changes of the backbone chemical shifts were observed (Table 1 Supporting Information). The temperature dependences of Ha chemical shift deviations from the random coil values (DdHa) are reported in Figure 1a. Unusually, the chemical shift index (CSI) analysis indicates that at 343 K the peptide retains at least the 80% of the helix conformation at 298 K and the slight reduction occurs uniformly in 4–12 region (Figure 1a). The thermal behavior was also analyzed by CD spectroscopy which allowed [a] D. Diana, Prof. Dr. R. Fattorusso Dipartimento di Scienze Ambientali, Seconda UniversitC di Napoli via Vivaldi 43, 81100 Caserta (Italy) Fax: (+39)0823-274605 E-mail : roberto.fattorusso@unina2.it [b] B. Ziaco, Prof. Dr. C. Pedone, Dr. L. D. DIAndrea Istituto di Biostrutture e Bioimmagini, CNR, via Mezzocannone, 16 80134 Napoli (Italy) Fax: (+39)081-2534574 E-mail : ldandrea@unina.it [c] Dr. G. Colombo, Dr. G. Scarabelli Istituto di Chimica del Riconoscimento Molecolare, CNR via Bianco, 9, 20131 Milano (Italy) [d] Dr. A. Romanelli Dipartimento delle Scienze Biologiche UniversitC di Napoli “Federico II” via Mezzocannone 16, 80134 Napoli (Italy) Supporting information for this article is available on the WWW under http://www.chemistry.org or from the author: Peptide synthesis, circular dichroism, nmr spectroscopy and molecular dynamic simulations.

A vascular endothelial growth factor mimetic accelerates gastric ulcer healing in an iNOS-dependent manner

Angiogenesis is crucial to all types of wound healing, including gastric ulcer healing. The most potent promoter of angiogenesis is vascular endothelial growth factor (VEGF). We hypothesized that a 15-amino acid peptide designed to mimic the angiogenic action of VEGF would accelerate gastric ulcer healing. Gastric ulcers were induced in mice by serosal application of acetic acid. Treatment with the VEGF mimetic accelerated gastric ulcer healing when administered orally or intraperitoneally, at a dose of 50 ng/kg or greater. Such healing was not observed when the reverse sequence pentadecapeptide or the full-length VEGF protein was administered. Contrary to our hypothesis, the VEGF mimetic did not significantly increase angiogenesis in the ulcerated stomach. The enhancement of ulcer healing by the VEGF mimetic occurred independently of cyclooxygenase-2 (COX-2) activity but was blocked by inhibitors of inducible nitric oxide synthase (iNOS). These results demonstrate that a VEGF mimetic is a potent stimulus for gastric ulcer healing, even when given orally. The effects of the mimetic were independent of stimulatory effects on angiogenesis and COX-2 activity but were dependent on iNOS-derived NO production.

β-Hairpin Peptide That Targets Vascular Endothelial Growth Factor (VEGF) Receptors DESIGN, NMR CHARACTERIZATION, AND BIOLOGICAL ACTIVITY

VEGF receptors have been the target of intense research aimed to develop molecules able to inhibit or stimulate angiogenesis. Based on the x-ray structure of the complex placental growth factor-VEGF receptor 1D2, we designed a VEGF receptor-binding peptide reproducing the placental growth factor β-hairpin region Gln87–Val100 that is involved in receptor recognition. A conformational analysis showed that the designed peptide adopts the expected fold in pure water. Moreover, a combination of NMR interaction analysis and cell binding studies were used to demonstrate that the peptide targets VEGF receptors. The VEGF receptor 1D2-interacting residues were characterized at the molecular level, and they correspond to the residues recognizing the placental growth factor sequence Gln87–Val100. Finally, the peptide biological activity was characterized in vitro and in vivo, and it showed a VEGF-like behavior. Indeed, the peptide activated VEGF-dependent intracellular pathways, induced endothelial cell proliferation and rescue from apoptosis, and promoted angiogenesis in vivo. This compound is one of the few peptides known with proangiogenic activity, which makes it a candidate for the development of a novel peptide-based drug for medical applications in therapeutic angiogenesis.

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.

Characterization of a designed vascular endothelial growth factor receptor antagonist helical peptide with antiangiogenic activity in vivo.

Angiogenesis is a fundamental process underlining physiological and pathological conditions. It is mainly regulated by the vascular endothelial growth factor (VEGF) and its receptors, which are the main targets of molecules able to modulate the angiogenic response. Pharmaceutical therapies based on antiangiogenic drugs represent a promising approach for the treatment of several socially important diseases. We report the biological and structural characterization of a VEGF receptor binder peptide designed on the N-terminal helix of VEGF. The reported experimental evidence shows that the peptide assumes in water a well-defined helical conformation and indicates that this peptide is a VEGF receptor antagonist and possesses antiangiogenic biological activity. In particular, it inhibits VEGF stimulated endothelial cell proliferation, activation, and survival, as well as angiogenesis and tumor progression in vivo. This peptide is a candidate for the development of novel peptide-based drugs for the treatment of diseases associated with excessive VEGF-dependent angiogenesis.

Structural Analysis of a Helical Peptide Unfolding Pathway

The analysis of the folding mechanism in peptides adopting well-defined secondary structure is fundamental to understand protein folding. Herein, we describe the thermal unfolding of a 15-mer vascular endothelial growth factor mimicking alpha-helical peptide (QK(L10A)) through the combination of spectroscopic and computational analyses. In particular, on the basis of the temperature dependencies of QK(L10A) H(alpha) chemical shifts we show that the first phase of the thermal helix unfolding, ending at around 320 K, involves mainly the terminal regions. A second phase of the transition, ending at around 333 K, comprises the central helical region of the peptide. The determination of high-resolution QK(L10A) conformational preferences in water at 313 K allowed us to identify, at atomic resolution, one intermediate of the folding-unfolding pathway. Molecular dynamics simulations corroborate experimental observations detecting a stable central helical turn, which represents the most probable site for the helix nucleation in the folding direction. The data presented herein allows us to draw a folding-unfolding picture for the small peptide QK(L10A) compatible with the nucleation-propagation model. This study, besides contributing to the basic field of peptide helix folding, is useful to gain an insight into the design of stable helical peptides, which could find applications as molecular scaffolds to target protein-protein interactions.

In vivo and in vitro characterization of CCK8 bearing a histidine-based chelator labeled with 99mTc-tricarbonyl

The development of receptor targeting radiolabeled ligands has gained much interest in recent years for diagnostic and therapeutic applications in nuclear medicine. Cholecystokinin (CCK) receptors have been shown to be overexpressed in a subset of neuroendocrine and other tumors. We are evaluating binding and biodistribution properties of a CCK8 peptide derivative labeled with 99mTc(I)‐tricarbonyl. The CCK8 peptide was modified at its N‐terminus by adding to its N‐terminus two lysine–histidine modules (KH), where histidine is coupled to the side chain of the lysine ((KH)2‐CCK8). 99mTc(I)‐tricarbonyl was generated with the IsoLink™ kit. A431 cells stably transfected with a cDNA encoding for the human CCK2 receptor were utilized to determine binding affinity, internalization, and retention of the labeled peptide, in comparison with wild‐type A431 cells. A nude mouse tumor model was obtained by generating A431‐CCK2R and A431‐control tumors in opposite flanks of the animals. High specific activity labeling with 99mTc was achieved. In A431‐CCK2R cells, specific saturable binding was observed as well as evident internalization of the radiolabeled peptide after binding. Biodistribution experiments showed rapid, specific localization of (KH)2‐CCK8 on A431‐CCK2R xenografts compared with control tumors, although absolute uptake values were not markedly higher compared with background activity. Clearance of unbound radioactivity was both urinary and hepatobiliary. In imaging experiments, while targeting to CCK2R positive tumors could be appreciated, there was poor contrast between target and nontarget areas. (KH)2‐CCK8 shows adequate in vitro and in vivo properties for CCK2R targeting although improvement of biodistribution warrant further development.