Title

 

Authors

V. Vellecco¹, M. Bucci¹, L Cigliano², A. Rossi¹, L. Sautebin¹, F. Roviezzo¹, P. Abrescia², C. Pedone³ and G. Cirino^1
 
1Dept. of Experimental Pharmacology, Faculty of Pharmacy, University of Naples “Federico II”, via D. Montesano 49, 80131 Naples, Italy. ²Dept. of Biological Sciences  University of Naples “Federico II”, via Mezzocannone 8, 80134 Naples, Italy. ³Biostructures and Bioimaging Institute, Consiglio Nazionale delle Ricerche, via Mezzocannone 16, 80134 Naples, Italy. 

 

Abstract

Recently, evidence have accumulated towards a protective role for high density lipoproteins (HDL) in atherosclerosis based on their ability to promote the reverse cholesterol transport (Rader; 2006). Moreover it has been demonstrated that the acute phase protein haptoglobin (Hpt) binds the HDL protein components apolipoprotein A-I (ApoA-I) and apolipoprotein E (ApoE), impairing their key functions in reverse cholesterol transport (Salvatore et al., 2009). In particular ApoA-I, the major protein component of HDL, stimulates the efflux of cellular cholesterol, and activates the enzyme, Lecithin-Cholesterol Acyl-Transferase (LCAT), which in turn converts the free cholesterol into cholesteryl esters (CE) addressing them to HDL for transport into the circulation (Rader et al., 2008). Therefore the binding of Hpt to ApoA-I inhibits LCAT activity and reduces ApoA-I-mediated delivery of cholesterol to hepatocytes (Cigliano et al., 2009). We have previously shown that an ApoA-I mimetic peptide,P2a, with amino acid sequence overlapping the stimulatory site for LCAT, displaced Hpt from ApoA-I restoring the enzyme activity in vitro. Aim of this study was to evaluate whether P2a displaces Hpt from ApoA-I in vivo, and if this event is linked to an anti-inflammatory activity. To pursue this goal we have used a mouse model of inflammation like mouse paw oedema. Mice received subplantar injection of carrageenan. Paw volume was measured before the injection and 2, 4, 6, 24, 48, 72 and 96h thereafter. Each group of animals received intraperitoneally administration of P2a peptide (0.3, 0.6, 1 mg/kg), scramble peptide (1mg/kg), or vehicle. All peptides  were administrated immediately before the injection of carrageenan and 24h thereafter. In the same time points, concentration of HDL-cholesterol (C) and cholesterol esters (CE) was measured by HPLC, while Hpt and ApoA-I levels were evaluated by ELISA. Western blotting analysis on paw homogenates for NOS and COX isofoms were also performed. In the early phase of carrageenan-induced inflammation (0-6 h), no differences were found in the CE/C ratio compared to the vehicle. On the contrary, in the late phase of inflammation (24-48 h), the values of the CE/C ratio significantly decreased in carrageenan-treated mice (21.5 ±1 at time 0 versus 12 ±0.6 at 24 hs, P< 0. 01; 21.5±1 at time 0 versus 12 ±0.6 at 48hs, P< 0. 01) whereas no change was detected in controls. The Hpt/ApoA-I ratio was found negatively correlated with the CE/C ratio in fact Hpt levels reached a peak after 48 h (66.6 ±4 in inflamed mice versus 13.8 ±2.8 µM in controls; P< 0.01) and declined at 144 h (14.2 ±3 in inflamed mice versus 13.4 ±2.1 µM in controls). P2a administration significantly restored the CE/C ratio.Contemporary, P2a displayed an anti-inflammatory effect, it significantly and dose-dependently inhibited the late phase of paw oedema (24-96h). The anti-inflammatory effect of P2a involved COX-2 enzyme, particularly after 48h from carrageenan injection, P2a (1mg/kg) significantly reduced COX-2 expression as well as PGE₂ levels, implying that, in presence of P2a, CE/C ratio rescue and oedema inhibition are strictly related. These data suggest that the P2a effect is secondary to its binding to Hpt with consequent displacement of ApoA-I that exerts the anti-inflammatory action. Therefore it is feasible to design drugs that could enhance the physiological endogenous protective role of ApoA-I.

Rader (2006). J Clin Invest. 116: 3090-3100.
Salvatore et al., (2009). Journal of Neurochemistry 110: 255-263
Rader et al., (2008). J Lipid Res. 50: S189-194
Cigliano et al.,  (2009). FEBS Journal 276: 6158-6171.