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Laboratory of Cellular Pharmacology of Atherosclerosis  

Lab. Corsini

 

Head
Prof. Alberto CORSINI                  alberto.corsini@unimi.it

Team
Stefano Bellosta, Assistant Professor stefano.bellosta@unimi.it
Ferri Nicola, Research Associate  nicola.ferri@unimi.it
Lorenzo Arnaboldi, Research Associate lorenzo.arnaboldi@unimi.it
Sergio K Bernini, Ph.D. Student sergiokevin@gmail.com
Ilaria Giunzioni, Ph.D. Student Ilaria.giunzioni@unimi.it
Agnese Granata, Laboratory Technician  agnese.granata@unimi.it

 

The research group has focused its interest on the physiopathology, biochemistry and pharmacological regulation of atherosclerosis and cardiovascular diseases. Experience is in molecular and pharmacological studies, including expertise in vascular cell biology, molecular biology, inflammatory processes, biochemistry of lipids, and lipoproteins.

Lines of research

Investigating the role of pro-protein convertase subtilisin/kexin type 9 (PCSK9) released from smooth muscle cells in atherogenesis
Proprotein convertase subtilisin kexin type 9 (PCSK9) is an important regulator of hepatic low-density lipoprotein (LDL) receptors and plasma cholesterol levels. Although PCSK9 is mainly of hepatic origin, we have recently documented that it is also secreted by the smooth muscle cells (SMC) of the arterial wall and it is detectable in human atherosclerotic plaques. By using in vitro cultured cells we demonstrated that PCSK9, secreted by SMCs, reduces macrophages LDLR levels. Additionally, preliminary data indicates that PDGF strongly induces PCSK9, and that PCSK9 is required for proper SMC migration. From these evidences it is tempting to speculate that PCSK9, expressed locally in the atherosclerotic plaque, may play a role on atherogenesis. In the present project we will define, by using both in vitro and in vivo experimental approaches, the role of PCSK9 in atherogenesis and coronary heart disease.

Design and synthesis of novel inhibitors of protein Rac1 and their pharmacological evaluation within the cardiovascular disease therapy
Rho GTPases of the Ras superfamily are proteins involved in the regulation of multiple cell functions and have been implicated in the pathogenesis of cardiovascular diseases and cancer. Several evidences have indicated that Rho proteins play a pivotal role in the pathogenesis of atherosclerosis, thus supporting Rho proteins as potential pharmacological targets for cardiovascular diseases. Recently, starting from the structure-function information on Rac1 we identified five new Rac1 inhibitors through a virtual screening strategy. In the present project, starting from thosefive hits, it will be identified new derivatives to be biologically tested, in order to dispose an adequate set for a Structure ActivityRelationship (SAR) analysis and consequent hit-to-lead optimization. The identified compounds will be subjected to several in vitro biological assays in order to obtain a completeinformation about the pharmacological and toxicological profiles of the new compounds. Finally, the best drug candidates selected by in vitro testing will besubjected to an in vivo study by evaluating the potential activity on cardiac hypertrophy in a specific animal model, thus obtaining a rather complete description of thepotentiality of the new compounds to be used for the treatment of atherosclerosis.

Design and synthesis of novel inhibitors of protein STAT3 and their pharmacological evaluation
The research program deals with the synthesis, structural analysis, biological evaluation and a thorough investigation of the mechanism of action of novel compounds as potential antitumor agents able to inhibit STAT3 (Signal Trasducer and Activator of Transciption 3), a factor being significantly involved in tumor progression and metastasis. Recently we identified an oxadiazole derivative (MD77) as a potential lead, able to inhibit STAT3 in a dose dependent manner (Dual-luciferase assay in HCT-116 cells) and to bind STAT3 SH2 domain (AlphaScreen-based assay). The compound displayed a significant growth inhibitory activity on a panel of 58 human cellular lines derived from 9 different types of tumor cells. Aim of the project is to obtain SAR data to optimize the pharmacophore what would allow the synthesis of antitumor agents provided with high selectivity and low toxicity. In parallel, on the bases of the encouraging results displayed both in vitro and in vivo by platinum complexes as direct and selective STAT3 inhibitors, the introduction of a methylamino group at position 4 of MD77 will be considered in order to coordinate platinum. The biological evaluation and the investigation of the mechanism of action of all the synthesized compounds will be performed in our Laboratory

Cigarette smoke and atherogenesis: role of monocyte/macrophages
The research deals with the effect(s) of cigarette smoke in atherogenesis. Cigarette smoke is a risk factor for atherosclerosis, but it is not clear how it can affect monocyte behavior in atherosclerotic lesions. Circulating monocytes are a key component of the atherogenic process: they adhere to the endothelium and migrate into the underlying intima in response to chemokines. Once recruited into the plaque, monocytes differentiate to macrophages and contribute to its growth by secreting pro-inflammatory cytokines and accumulating intracellular lipids. The aim of our studies is to evaluate the effects of cigarette smoke on monocyte/macrophage functions in atherogenesis. We utilize human monocytes and endothelial cells to characterize the effects of cigarette smoke on monocyte adhesion and transmigration through an endothelial cell layer, on proinflammatory cytokines and metalloproteinases expression and on intracellular accumulation of cholesterol.

Cholesterol-induced transdifferentiation of smooth muscle cells to macrophage-like cells
In the initial steps of atherogenesis, cells of the artery wall, such as macrophages, may accumulate free cholesterol (FC) and cholesteryl esters becoming foam cells. Although the majority of foam cells in the atherosclerotic lesion are thought to be derived from macrophages, some of them may also originate from smooth muscle cells (SMCs). It was shown that cholesterol-loaded SMCs assume a foam cell-like appearance and lose the expression of SMC markers. The aim of our study is to further characterize the transdifferentiation of SMCs to macrophages. Aortic SMCs, isolated from wild type and loaded with FC complexed to methyl-beta-cyclodextrin (Chol:MβCD), rapidly assume a foam cell-like appearance; they lose the expression of the SMC marker alpha-actin while the expression of the macrophage marker Mac-2 and of ABCA1, ABCG1 and SRBI is increased. We are currently evaluating the role of ABCA1, ABCG1 and HDL3 in modulating these processes in both WT SMC and in SMC isolated from ABCA1 knock-out mice.

Effect of everolimus immunosuppressive regimens on plasma lipids, lipoprotein phenotype and inflammatory markers in de novo heart transplant patients
Hyperlipidemia, one of the preminent complications after organ transplantation, strongly contributes to atherosclerosis development. Unfortunately, this phenomenon is exacerbated by immunosuppressants like mTOR inhibitors and, despite relevant evidence, the mechanism of this dyslipidemia is not well-described. Since the mTOR inhibitor everolimus is widely used in combination with low-dose cyclosporine in preventing acute rejections, in this project we propose to study the dyslipidemia in plasma samples of de-novo heart transplanted patients treated with cyclosporine, steroids, plus everolimus (immediately or after 4-6 weeks of mofetyl mycophenolate) and fluvastatin as lipid-lowering drug. On samples drawn at transplantation and after 1, 3 and 6 months, beyond following the time-course of the transplant-induced dyslipidemia, we will assess the pharmacological effect of everolimus and of fluvastatin by measuring: a) plasma lipids (total-, HDL-, LDL, non-HDL cholesterol, triglycerides), lipoprotein profile (VLDL, LDL, HDL) and their lipid content, b) apolipoprotein concentrations (apoB, apoA-I, apoC-II), c) CETP activity and d) inflammation markers (CD40L, matrix metalloproteases), by chromatographic and colorimetric techniques. These results may help to understand the mechanism(s) of the dyslidipidemia and to assess the effectiveness of everolimus-fluvastatin treatment in heart transplanted patients.

NO donors and antioxidant hybrids as a new approach in atheroprevention. In vitro effects on smooth muscle cell proliferation
Atherosclerosis is a multifactorial disease, in which LDL oxidation, nitric oxide unbalance and smooth muscle cell (SMC) proliferation play essential roles. In order to find a novel multiple approach to control atherogenesis, we synthesized NO-donor molecules (furoxans) which demonstrated to be able in relaxing rat aorta strips in a NO-dependent fashion. We then joined these compounds with different antioxidant moieties for the generation of NO donor-antioxidant hybrids and we evaluated their effect in inhibiting cell proliferation. These compounds, albeit with different potency, inhibited SMC proliferation, by a selective effect on G1/S phase transition. To understand whether the antiproliferative mechanism of these hybrids was due to the furoxan moiety, we split them in their native pharmacophores and we found that furoxans, but not the antioxidant moieties, decreased SMC proliferation. We demonstrated that both the inhibitory potency on SMC proliferation and on vasodilation paralleled with the electron-attractor capacity of the group in position 3 of the heteroring and also that the opening of the furoxan ring is essential for growth-inhibition. The molecular basis of this effect is neither cGMP- nor polyamine-dependent, the two main NO-mediated pathways involved in SMC proliferation, suggesting a by-stander role for NO in this process. We are actually performing experiments aimed at understanding the mechanism of this effect by different proteomic approaches; once clarified, furoxans, thanks to their adjustable pharmacological properties (e.g. NO donor ability, inhibition of SMC proliferation) may be proper candidates to be coupled with other pharmacophores in generating hybrids aimed at preventing or healing different pathologies.

 

Experimental techniques

The laboratory has a cell culture facility fully equipped for isolation and maintenance of primary and established cell lines. The laboratory is equipped also with a real-time thermal cycler (ABI Prism 7000, Applied Biosystem) for quantification of gene expression, a gas-chromatographer (DANI GC 1000, Milan, Italy), an HPLC (Jasco), a Coulter counter for cell counting, and The iCELLigence System for monitoring real-time cell behavior.

In vitro experimental techniques:

Cell proliferation assay by cell counting with Coulter counter and by The iCELLigence System.

Boyden chamber chemotaxis assay;

Transwell migration assay;

Western blot and zymography analysis;

ELISA, EIA and G-LISA assays;

Quantitative RT-PCR analysis;

Gene overexpression and silencing in primary vascular cells (smooth muscle cells, macrophages and endothelial cells) by using retroviral vectors and siRNA.

In vivo experimental techniques:

Vascular injury through a periadventitial carotid placement of a non-occlusive collar in rabbit and mice.

Cardiac hypertrophy by infusion of Ang-II through osmotic pumps

Other experimental techniques:

Lipidomics (TLC, GLC, HPLC) of simple and complex lipids from different matrices (cell, media, tissues, plasma, lipoproteins).

 

Selected publications

Ferri N, Tibolla G, Pirillo A, Cipollone F, Mezzetti A, Pacia S, Corsini A and Catapano AL. (2012) Proprotein convertase subtilisin kexin type 9 (PCSK9) secreted by cultured smooth muscle cells reduces macrophages LDLR levels.. Atherosclerosis. 220(2):381-6. 

Bellosta S, Corsini A. (2012) Statin drug interactions and related adverse reactions. Expert Opinion On Drug Safety 11(6):933-46. 

Arnaboldi L, Corsini A. (2010) Do structural differences in statins correlate with clinical efficacy? Curr Opin Lipidol. 21(4):298-304 

Ferri N, Corsini A, Bottino P, Clerici F, and Contini A.  (2009) Virtual screening approach for the identification of new classes of Rac1 inhibitors. J Med Chem. 52(14):4087-90.

Arnaboldi L, Guzzetta M, Pazzucconi F, Radaelli G, Paoletti R, Sirtori CR, Corsini A. (2007) Serum from hypercholesterolemic patients treated with atorvastatin or simvastatin inhibits cultured human smooth muscle cell proliferation. Pharmacol Res. 56(6):503-8. 

Rizki G, Arnaboldi L, Gabrielli B, Yan J, Lee GS, Ng RK, Turner SM, Badger TM, Pitas RE, Maher JJ. (2006) Mice fed a lipogenic methionine-choline-deficient diet develop hypermetabolism coincident with hepatic suppression of SCD-1. J Lipid Res. 47(10):2280-90. 

Gizard F, Amant C, Barbier O, Bellosta S, Robillard R, Percevault F, Sevestre H, Krimpenfort P, Corsini A, Rochette J, Glineur C, Fruchart J-C, Torpier G, Staels B. (2005) PPARalpha inhibits vascular smooth muscle cell proliferation underlying intimal hyperplasia by inducing the tumor suppressor gene p16INK4a. J Clin Invest 115:3228-3238.  

Ferri N, Carragher NO, Raines EW. (2004) Role of Discoidin Domain Receptor 1 and 2 in human smooth muscle cell-mediated collagen remodeling: potential implications in lesions of atherosclerosis and lymphangioleimyomatosis. Am. J Pathol 164(5):1575-1585.

Bellosta S, Paoletti R, Corsini A. (2004) Safety of statins: Focus on clinical pharmacokinetics and drug interaction. Circulation 109(23 Suppl 1):III50-57.              

Bellosta S, Via D, Canavesi M, Pfister P, Fumagalli R, Bernini F. (1998) HMG-CoA reductase inhibitors reduce MMP-9 secretion by macrophages. Arterioscl. Thromb. Vasc. Biol. 18:1671-1678. 

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