Antonio Di Grazia

The Frog Skin-Derived Antimicrobial Peptide Esculentin-1a(1-21)NH2 Promotes the Migration of Human HaCaT Keratinocytes in an EGF Receptor-Dependent Manner: A Novel Promoter of Human Skin Wound Healing?

One of the many functions of skin is to protect the organism against a wide range of pathogens. Antimicrobial peptides (AMPs) produced by the skin epithelium provide an effective chemical shield against microbial pathogens. However, whereas antibacterial/antifungal activities of AMPs have been extensively characterized, much less is known regarding their wound healing-modulatory properties. By using an in vitro re-epithelialisation assay employing special cell-culture inserts, we detected that a derivative of the frog-skin AMP esculentin-1a, named esculentin-1a(1-21)NH2, significantly stimulates migration of immortalized human keratinocytes (HaCaT cells) over a wide range of peptide concentrations (0.025–4 μM), and this notably more efficiently than human cathelicidin (LL-37). This activity is preserved in primary human epidermal keratinocytes. By using appropriate inhibitors and an enzyme-linked immunosorbent assay we found that the peptide-induced cell migration involves activation of the epidermal growth factor receptor and STAT3 protein. These results suggest that esculentin-1a(1-21)NH2 now deserves to be tested in standard wound healing assays as a novel candidate promoter of skin re-epithelialisation. The established ability of esculentin-1a(1-21)NH2 to kill microbes without harming mammalian cells, namely its high anti-Pseudomonal activity, makes this AMP a particularly attractive candidate wound healing promoter, especially in the management of chronic, often Pseudomonas-infected, skin ulcers.

Phospholipase D Regulates Myogenic Differentiation through the Activation of Both mTORC1 and mTORC2 Complexes

How phospholipase D (PLD) is involved in myogenesis remains unclear. At the onset of myogenic differentiation of L6 cells induced by the PLD agonist vasopressin in the absence of serum, mTORC1 complex was rapidly activated, as reflected by phosphorylation of S6 kinase1 (S6K1). Both the long (p85) and short (p70) S6K1 isoforms were phosphorylated in a PLD1-dependent way. Short rapamycin treatment specifically inhibiting mTORC1 suppressed p70 but not p85 phosphorylation, suggesting that p85 might be directly activated by phosphatidic acid. Vasopressin stimulation also induced phosphorylation of Akt on Ser-473 through PLD1-dependent activation of mTORC2 complex. In this model of myogenesis, mTORC2 had a positive role mostly unrelated to Akt activation, whereas mTORC1 had a negative role, associated with S6K1-induced Rictor phosphorylation. The PLD requirement for differentiation can thus be attributed to its ability to trigger via mTORC2 activation the phosphorylation of an effector that could be PKCα. Moreover, PLD is involved in a counter-regulation loop expected to limit the response. This study thus brings new insights in the intricate way PLD and mTOR cooperate to control myogenesis.

d-Amino acids incorporation in the frog skin-derived peptide esculentin-1a(1-21)NH2 is beneficial for its multiple functions

Naturally occurring antimicrobial peptides (AMPs) represent promising future antibiotics. We have previously isolated esculentin-1a(1-21)NH2, a short peptide derived from the frog skin AMP esculentin-1a, with a potent anti-Pseudomonal activity. Here, we investigated additional functions of the peptide and properties responsible for these activities. For that purpose, we synthesized the peptide, as well as its structurally altered analog containing two d-amino acids. The peptides were then biophysically and biologically investigated for their cytotoxicity and immunomodulating activities. The data revealed that compared to the wild-type, the diastereomer: (1) is significantly less toxic towards mammalian cells, in agreement with its lower α-helical structure, as determined by circular dichroism spectroscopy; (2) is more effective against the biofilm form of Pseudomonas aeruginosa (responsible for lung infections in cystic fibrosis sufferers), while maintaining a high activity against the free-living form of this important pathogen; (3) is more stable in serum; (4) has a higher activity in promoting migration of lung epithelial cells, and presumably in healing damaged lung tissue, and (5) disaggregates and detoxifies the bacterial lipopolysaccharide (LPS), albeit less than the wild-type. Light scattering studies revealed a correlation between anti-LPS activity and the ability to disaggregate the LPS. Besides shedding light on the multifunction properties of esculentin-1a(1-21)NH2, the d-amino acid containing isomer may serve as an attractive template for the development of new anti-Pseudomonal compounds with additional beneficial properties. Furthermore, together with other studies, incorporation of d-amino acids may serve as a general approach to optimize the future design of new AMPs.

Temporins A and B Stimulate Migration of HaCaT Keratinocytes and Kill Intracellular Staphylococcus aureus

ABSTRACT The growing number of microbial pathogens resistant to available antibiotics is a serious threat to human life. Among them is the bacterium Staphylococcus aureus, which colonizes keratinocytes, the most abundant cell type in the epidermis. Its intracellular accumulation complicates treatments against resulting infections, mainly due to the limited diffusion of conventional drugs into the cells. Temporins A (Ta) and B (Tb) are short frog skin antimicrobial peptides (AMPs). Despite extensive studies regarding their antimicrobial activity, very little is known about their activity on infected cells or involvement in various immunomodulatory functions. Here we show that Tb kills both ATCC-derived and multidrug-resistant clinical isolates of S. aureus within infected HaCaT keratinocytes (80% and 40% bacterial mortality, respectively) at a nontoxic concentration, i.e., 16 μM, whereas a weaker effect is displayed by Ta. Furthermore, the peptides prevent killing of keratinocytes by the invading bacteria. Further studies revealed that both temporins promote wound healing in a monolayer of HaCaT cells, with front speed migrations of 19 μm/h and 12 μm/h for Ta and Tb, respectively. Migration is inhibited by mitomycin C and involves the epidermal growth factor receptor (EGFR) signaling pathway. Finally, confocal fluorescence microscopy indicated that the peptides diffuse into the cells. By combining antibacterial and wound-healing activities, Ta and Tb may act as multifunctional mediators of innate immunity in humans. Particularly, their nonendogenous origin may reduce microbial resistance to them as well as the risk of autoimmune diseases in mammals.

Video evaluation of the kinematics and dynamics of the beating cardiac syncytium: An alternative to the Langendorff method

Many important observations and discoveries in heart physiology have been made possible using the isolated heart method of Langendorff. Nevertheless, the Langendorff method has some limitations and disadvantages such as the vulnerability of the excised heart to contusions and injuries, the probability of preconditioning during instrumentation, the possibility of inducing tissue edema, and high oxidative stress, leading to the deterioration of the contractile function. To avoid these drawbacks associated with the use of a whole heart, we alternatively used beating mouse cardiac syncytia cultured in vitro in order to assess possible ergotropic, chronotropic, and inotropic effects of drugs. To achieve this aim, we developed a method based on image processing analysis to evaluate the kinematics and the dynamics of the drug-stimulated beating syncytia starting from the video recording of their contraction movement. In this manner, in comparison with the physiological no-drug condition, we observed progressive positive ergotropic, positive chronotropic, and positive inotropic effects of 10 μM isoproterenol (β-adrenergic agonist) and early positive ergotropic, negative chronotropic, and positive inotropic effects of 10 μM phenylephrine (α-adrenergic agonist), followed by a late phase with negative ergotropic, positive chronotropic, and negative inotropic trends. Our method permitted a systematic study of in vitro beating syncytia, producing results consistent with previous works. Consequently, it could be used in in vitro studies of beating cardiac patches, as an alternative to Langendorffs heart in biochemical and pharmacological studies, and especially when the Langendorff technique is inapplicable (e.g., in studies about human cardiac syncytium in physiological and pathological conditions, patient-tailored therapeutics, and syncytium models derived from induced pluripotent/embryonic stem cells with genetic mutations). Furthermore, the method could be helpful in heart tissue engineering and bioartificial heart research to “engineer the heart piece by piece.” In particular, the proposed method could be useful in the identification of a suitable cell source, in the development and testing of “smart” biomaterials, and in the design and use of novel bioreactors and microperfusion systems.

Novel α-MSH Peptide Analogues with Broad Spectrum Antimicrobial Activity

Previous investigations indicate that α-melanocyte-stimulating hormone (α-MSH) and certain synthetic analogues of it exert antimicrobial effects against bacteria and yeasts. However, these molecules have weak activity in standard microbiology conditions and this hampers a realistic clinical use. The aim in the present study was to identify novel peptides with broad-spectrum antimicrobial activity in growth medium. To this purpose, the Gly10 residue in the [DNal(2′)-7, Phe-12]-MSH(6–13) sequence was replaced with conventional and unconventional amino acids with different degrees of conformational rigidity. Two derivatives in which Gly10 was replaced by the residues Aic and Cha, respectively, had substantial activity against Candida strains, including C. albicans, C. glabrata, and C. krusei and against gram-positive and gram-negative bacteria. Conformational analysis indicated that the helical structure along residues 8–13 is a key factor in antimicrobial activity. Synthetic analogues of α-MSH can be valuable agents to treat infections in humans. The structural preferences associated with antimicrobial activity identified in this research can help further development of synthetic melanocortins with enhanced biological activity.

Naturally occurring peptides from Rana temporaria: Antimicrobial properties and more

The extensive search for alternative therapeutics against microbial pathogens has led to the discovery of cationic peptides as new anti-infectives with a novel mode of action. Particular interest has been devoted to small linear peptides that can be efficiently made by chemical synthesis at competitive costs. The most promising originate from a large family of short, naturally occurring peptides found in the skin of amphibia of Rana genus, i.e. the temporins. This review is mainly focused on the recent structure-function studies of the earliest known temporin isoforms (TA, TB and TL) and their potential clinical role as novel antimicrobial agents. The development of novel antibiotics is an urgent public health concern due to the increased resistance of microorganisms to conventional antibiotics, particularly in the hospital setting.

Inhibition of type 5 phosphodiesterase counteracts β2-adrenergic signalling in beating cardiomyocytes

AIMS Compartmentalization of cAMP and PKA activity in cardiac muscle cells plays a key role in maintaining basal and enhanced contractility stimulated by sympathetic nerve activity. In cardiomyocytes, activation of adrenergic receptor increases cAMP production, which is countered by the hydrolytic activity of selective phosphodiesterases (PDEs). The intracellular regional dynamics of cAMP production and hydrolysis modulate downstream signals resulting in different biological responses. The interplay between beta receptors (βARs) signalling and phosphodiesterase 5 (PDE5) activity remains to be addressed. METHODS AND RESULTS Using combined strategies with pharmacological inhibitors and genetic deletion of PDEs and βAR isoforms, we revealed a specific pool of cAMP that is under dual regulation by PDE2 and, indirectly, PDE5 activity. Inhibition of PDE5 with sildenafil produces a cGMP-dependent activation of PDE2 that attenuates cAMP generation induced by βAR agonists, with concomitant modulation of stimulated contraction rate and calcium transients. PDE2 haploinsufficiency abolished the effects of sildenafil. The negative chronotropic effect of PDE5 inhibition through PDE2 activation was also observed in sinoatrial node tissue from adult mice. PDE5 inhibition selectively lowered contraction rate stimulated by β2AR, but not β1AR activation, supporting a compartmentalization of the cGMP-modulated pool of cAMP. CONCLUSION These data identify a new effect of PDE5 inhibitors on the modulation of cardiomyocyte response to adrenergic stimulation via PDE5-PDE2-mediated cross-talk.

Anti-Candida activity of 1–18 fragment of the frog skin peptide esculentin-1b: in vitro and in vivo studies in a Caenorhabditis elegans infection model

Candida albicans represents one of the most prevalent species causing life-threatening fungal infections. Current treatments to defeat Candida albicans have become quite difficult, due to their toxic side effects and the emergence of resistant strains. Antimicrobial peptides (AMPs) are fascinating molecules with a potential role as novel anti-infective agents. However, only a few studies have been performed on their efficacy towards the most virulent hyphal phenotype of this pathogen. The purpose of this work is to evaluate the anti-Candida activity of the N-terminal 1–18 fragment of the frog skin AMP esculentin-1b, Esc(1–18), under both in vitro and in vivo conditions using Caenorhabditis elegans as a simple host model for microbial infections. Our results demonstrate that Esc(1–18) caused a rapid reduction in the number of viable yeast cells and killing of the hyphal population. Esc(1–18) revealed a membrane perturbing effect which is likely the basis of its mode of action. To the best of our knowledge, this is the first report showing the ability of a frog skin AMP-derived peptide (1) to kill both growing stages of Candida; (2) to promote survival of Candida-infected living organisms and (3) to inhibit transition of these fungal cells from the roundish yeast shape to the more dangerous hyphal form at sub-inhibitory concentrations.

Identification of Murine Phosphodiesterase 5A Isoforms and their Functional Characterization in HL-1 Cardiac Cell Line

Phosphodiesterase 5A (PDE5A) specifically degrades the ubiquitous second messenger cGMP and experimental and clinical data highlight its important role in cardiac diseases. To address PDE5A role in cardiac physiology, three splice variants of the PDE5A were cloned for the first time from mouse cDNA library (mPde5a1, mPde5a2, and mPde5a3). The predicted amino acidic sequences of the three murine isoforms are different in the N‐terminal regulatory domain. mPDE5A isoforms were transfected in HEK293T cells and they showed high affinity for cGMP and similar sensitivity to sildenafil inhibition. RT‐PCR analysis showed that mPde5a1, mPde5a2, and mPde5a3 had differential tissue distribution. In the adult heart, mPde5a1 and mPde5a2 were expressed at different levels whereas mPde5a3 was undetectable. Overexpression of mPDE5As induced an increase of HL‐1 number cells which progress into cell cycle. mPDE5A1 and mPDE5A3 overexpression increased the number of polyploid and binucleated cells, mPDE5A3 widened HL‐1 areas, and modulated hypertrophic markers more efficiently respect to the other mPDE5A isoforms. Moreover, mPDE5A isoforms had differential subcellular localization: mPDE5A1 was mainly localized in the cytoplasm, mPDE5A2 and mPDE5A3 were also nuclear localized. These results demonstrate for the first time the existence of three PDE5A isoforms in mouse and highlight their potential role in the induction of hypertrophy.