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.

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. AngiogenesisAngiogenesis is a physiologic process which refers to the remodeling of the vascular tissue characterized by the branching out of a new blood vessel from a pre-existing vessel. It is intimately associated with endothelial cell (EC) migration and proliferation. ECs are particularly active during embryonic development while during adult life EC turnover is very low and limited to particular physiologic phenomena (1). In a healthy individual angiogenesis is finely tuned by proangiogenic and anti-angiogenic factors (Table 1), the shift from this equilibrium (angiogenic switch), under specific stimuli such as hypoxia, is related to several human diseases (pathologic angiogenesis) (2). The prevalence of proangiogenic factors (excessive angiogenesis), is associated with cancer, proliferating retinopathy, rheumatoid arthritis, and psoriasis. Whereas, insufficient angiogenesis is at the basis of coronary diseases, ischemia and a reduced capacity for tissue regeneration (3).The mechanism of angiogenesis is very complex and its description is beyond the aim of this review. Some excellent studies (1,4) have been published on this topic and the reader can refer to them for a detailed description.In a simplified overview, angiogenesis starts with the release of angiogenic factors which bind to their cognate receptors present on the membrane of ECs, allowing the transmission of the signal which leads to the transcription of several genes. Following angiogenic stimulus, the ECs express integrins, in particular a v b 3 , thus facilitating the ECs adhesion to the extracellular matrix (ECM) components and their migration. Moreover, ECs secrete and activate many proteolytic factors, such as matrix metalloproteases (MMP), which degrade the ECM facilitating the migration of ECs. As a functional vascular network is formed, it is necessary that the newly formed vessel be remodeled to form a mature vessel. The release of platelet-derived growth factor (PDGF) is fundamental for this maturation, as it...

The critical role of integrins in tumor progression and metastasis has stimulated intense efforts to identify pharmacological agents that can modulate integrin function. In recent years, αv β3 and αv β5 integrin antagonists were demonstrated to be effective in blocking tumor progression. RGDechi-hCit, a chimeric peptide containing a cyclic RGD motif linked to an echistatin C-terminal fragment, is able to recognize selectively αv β3 integrin both in vitro and in vivo. High-resolution molecular details of the selective αv β3 recognition of the peptide are certainly required, nonetheless RGDechi-hCit internalization limited the use of classical in cell NMR experiments. To overcome such limitations, we used WM266 isolated cellular membranes to accomplish a detailed NMR interaction study that, combined with a computational analysis, provides significant structural insights into αv β3 molecular recognition by RGDechi-hCit. Remarkably, on the basis of the identified molecular determinants, we design a RGDechi-hCit mutant that is selective for αv β5 integrin.

Purpose: To test whether a novel bifunctional chimeric peptide comprising a cyclic ArgGly-Asp pentapeptide covalently bound to an echistatin domain can discriminate α v β 3 from α v β 5 integrin, thus allowing the in vivo selective visualization of α v β 3 expression by single-photon and positron emission tomography (PET) imaging. Experimental Design: The chimeric peptide was preliminarily tested for inhibition of α v β 3 -dependent cell adhesion and competition of 125

In the funneled landscape, proteins fold to their native states through a stochastic process in which the free energy decreases spontaneously and unfolded, transition, native, and possible intermediate states correspond to local minima or saddle points. Atomic description of the folding pathway appears therefore to be essential for a deep comprehension of the folding mechanism. In metallo-proteins, characterization of the folding pathways becomes even more complex, and therefore, despite their fundamental role in critical biological processes, little is known about their folding and assembly. The study of the mechanisms through which a cofactor influences the protein folding/unfolding reaction has been the rationale of the present study aimed at contributing to the search for cofactors' general roles in protein folding reactions. In particular, we have investigated the folding pathway of two homologous proteins, Ros87, which contains a prokaryotic zinc finger domain, and Ml452-151, lacking the zinc ion. Using a combination of CD, DSC and NMR techniques, we determined the thermodynamics and the structural features, at an atomic level, of the thermal unfolding of Ros87 and compared them to the behavior of Ml452-151. Our results, also corroborated by NMR (1)H/(2)H exchange measurements, show that the presence of the structural Zn(II) in Ros87 implies a switch from the Ml452-151 fully cooperative to a two-step unfolding process in which the intermediate converts to the native state through a downhill barrierless transition. This observation, which has never been reported for any metal ion so far, may have a significant role in the understanding of the protein misfolding associated with the presence of metal ions, as observed in neurodegenerative diseases.

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.

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.

Among RGD-dependent integrins, the alpha(v)beta3 receptor has recently received increasing attention as a therapeutic target because of its critical role in tumor-induced angiogenesis and metastasis formation. Here, we describe a new peptide antagonist of alpha(v)beta3 receptor, designed on the basis of the crystal structure of integrin alpha(v)beta3 in complex with c(RGDf[NMe]V) and the NMR structure of echistatin. Cell adhesion assays have demonstrated that the peptide is a potent and selective antagonist of the alpha(v)beta3 receptor.