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The present invention refers to the field of molecular biology and more in particular to the use of

HMGBl protein inhibitors and antagonists for the preparation of therapeutic agents able to inhibit atherosclerosis pathogenesis and development .

In the international application No . PCT/IT02/00153 , in the name of the same applicants , the role of HMGBl as chemoattractant signal for smooth muscle cells and fibroblasts has been underlined; therefore , inhibitors and antagonist , to be usefully used for the preparation of pharmaceutical agents able to positively and negatively regulate the regeneration of connective tissues and for the treatment of vascular diseases that occur after coronary and/or' carotid angioplasty surgery with or without stent positioning and angiography, have been identify.

Subsequent further studies have shown that HMGBl induces endothelial cell proliferation. Italian patent application No . RM2004A000058 , corresponding to PCT/IT2005/000039 , disclosed the use of molecules able to modulate the interaction between HMGBl and its receptors for the preparation of therapeutic agents for the treatment of vascular diseases , linked to endothelial and smooth muscle cells proliferation, due to atherosclerosis and restenosis occurring after blood vessels damages or that occur after coronary and/or carotid angioplasty with or without stent positioning,

^' angiography and every surgical operation using catheters on or through blood vessels .

In view of the prior art and considering what previously underlined by the same authors , the attention has been drawn up to the analysis of atherosclerosis pathophysiology and the role of HMGBl in the atherosclerotic plaque .

In particular, conducted studies intended ' to attest if HMGBl role was restricted to atherosclerosis due to vascular damages .

Obj ect of the present invention is the identification of active molecules to be used for the preparation of pharmaceutical agents for the prevention and the cure of atherosclerosis of any origin . Further features of the invention will be readily apparent from the following detailed description with reference to the accompanying drawings where :

Figure 1 shows western blot analysis of protein- free medium from incubation of atherosclerotic plaque from endarterectomy.

Figure 2 shows the results of immunohistochemistry on sections of human carotid artery atherosclerotic plaque tissue (A-F) and on section of human artery (G-

I) (control) stained with anti-HMGBl antibody and counterstained with hematoxylin .

Figure 3 shows the results of immunofluorescence on human carotid artery atherosclerotic plaque sections .

Figure 4 shows the results of immunofluorescence on human SMCs from atherosclerotic carotid and normal

aorta arteries (control) after incubation with or without cholesterol and the results of SDS-PAGE on SMCs lysate and supernatant .

Figure 5 shows the results of immunofluorescence on human carotid atherosclerotic plaque sections .

Figure 6 shows the results of migration essays on human aorta SMCs after stimulation with HMGBl , PDGFBB and FCS . Data represent the mean +_ SD of four experiments in duplicate , Atherosclerotic plaque secrete HMGBl

Samples of human atherosclerotic plaque and human artery (control) were put into culture plates inserts and incubated in protein-free culture medium (RPMI , 1% HEPES , 1% penicillin, streptomycin, amphotericin) for 3 days at 37 ⁰ C . The culture medium, was collected every 24 hours and analyzed by immunoblot to detect HMGBl and lactate dehydrogenase (LHD) , the cellular lysate was analysed similarly as control .

The results , shown in figure 1 , underline that the large amount of HMGBl in the supernatant are released by atherosclerotic plaque and not by the normal vascular tissue . Moreover, the absence of cellular necrosis marker (LDH) in the supernatant demonstrates that HMGBl is actively secreted and not released by dead cells .

HMGBl is expressed in the cytosol of cells within Atherosclerotic plaque

Histologic sections of atherosclerotic plaques and human arteries (control) were processed for immunohistochemistry and immunofluorescence .

The results shown in figure 2 confirm that HMGBl is contained in both nuclei and cytosol of endothelial cells , of foam cells derived by macrophages in the neointima and surprisingly in the cytosol of SMCs , (mostly localized in the neointima) (A-F) . In the control (G-I ) , HMGBl is , as expected, abundant only in the nuclei of endothelial cells and present at low level only in the nuclei of SMCs .

The presence of cytosolic HMGBl in SMCs of the atherosclerotic plaque supposes a secretory activity of these cells .

To confirm these observations , histologic sections of atherosclerotic plaques were treated with specific markers of HMGBl , endothelial cells , foam cells and SMCs , and analysed by immunofluorescence . As shown in figure 3 , HMGBl was found in the nuclei and cytosol of endothelial cells and foam cells , respectively CD31- and CD68-positive cells (Figure 3A, 3B) . Moreover, double labelling for HMGBl and alfa-actin confirmed that HMGBl is expressed in SMCs of neointima . The presence of HMGBl in the cytoplasm of some SMCs (figure 3C) is remarkable and suggest that HMGBl can be secreted not only by endothelial cells and foam cells but by SMCs as well . Findings on SMCs are 'completely unexpected, in fact as normal SMCs contain a very low level of HMGBl and most of the cell types contain high level .

SMCs secrete HMGBl after treatment with cholesterol

In order to directly demonstrate that SMCs can secrete HMGBl , said cells were exposed to cholesterol , a atherogenic stimuli .

SMCs isolated from normal arteries and atherosclerotic plaques were stimulated with 10 μg/ml cholesterol complexed to methyl-beta-cyclodextrin for

72 hours; the results are shown in figure 4. All SMCs accumulate cholesterol droplets in the cytosol (Oil Red

0-staining) (figure 4A) , express the CD68 macrophagic marker (figure 4B) and release HMGBl in the cytosol

(figure 4C) .

Those findings demonstrate that cholesterol , a typical atherogenic stimuli , promotes HMGBl secretion by SMCs in vitro . The results in figure 5 confirm in vitro findings : in the atherosclerotic plaque SMCs that contain cytoplasmatic HMGBl also express CD68.

HMGBl is a chemoattractant and a growth factor for SMCs . The mitogenic effect exerted by HMGBl was tested on human SMCs , as shown in figure 6.

SMCs were incubated for 16 hours at 37 ⁰ C and HMGBl was added to the serum-free medium. As well shown in

Figure 6B shows there is a significant increase in the number of cells after stimulation with HMGBl for 2 days .

After, whether in response to extracellular HMGBl human SMCs can secrete HMGBl themselves was tested .

Human SMCs were starved for 16 hours at 37 ⁰ C in absence of serum, and then increasing concentration of HMGBl box B were added to the medium.

As shown in figure 6C, after 48 hours a significantly increase of SMCs HMGBl expression and secretion in response to HMGBl-boxB in a dose-dependent manner was found . The absence of LDH in the supernatan demonstrated that HMGBl was not released by lysis of necrotic cells .

The analysis of the obtained experimental results , as previously described, lead to the experimental assumption that HMGBl is secreted in much larger quantities in atherosclerotic plaques as compared to normal arterial walls . In particular, extracellular HMGBl has not only a necrotic origin but it is actively secreted .

All three cells types in the atherosclerotic plaques : endothelial cells , foam cells and, surprisingly SMCs contain and actively secrete cytoplasmatic HMGBl .

In case of SMCs of atherosclerotic plaque , experimental findings were completely different from expected data coming from normal SMC that , as normal , show very low levels of cytosolic HMGBl .

SMCs of the atherosclerotic plaque actively secrete HMGBl when loaded with atherogenic stimuli like cholesterol . Extracellular HMGBl itself induces an additional secretion of endogen HMGBl by SMCs . Furthermore , HMGBl induces SMCs migration and proliferation. Thus , the ability of SMCs to secrete HMGBl (endogen) and contemporarily to respond to extracellular HMGBl creates the conditions for an auto-stimulation system (the so called autocrine loop) .

In view of the obtained experimental results , previously described, it is evident that all the molecules that avoid or modulate the interaction between HMGBl and its receptor (or receptors if they are more than one) can be advantageously used for the preparation of pharmaceutical agents that inhibit atherosclerosis pathogenesis and development .

What previously mentioned has recently found a further confirmation in Mitola S . et al , 2006 , J . Immunol . , 176 : 12 -15 that discloses experimental results proving the angiogenic effect of HMGBl in vitro and in vivo .

In particular, all the molecules, belonging to the class of HMGBl inhibitors (antibodies or fragments thereof and four-way DNA) , that bind to HMG-box, also bind to hexogen and endogen extracellular HMGBl , and by block the interaction with its receptors, avoid the positive stimulation of SMCs .

At the same time, molecules having sequence homology with HMG box, that belong to the class of antagonists (HMGBl fragments or fragments of molecules having similar sequence) , bind to HMGBl receptors and render said receptors unavailable for their activation by HMGBl . Obj ect of the present invention is the use of inhibitors and antagonists for the preparation of pharmaceutical agents able to block the autocrine loop system that is the base of atherosclerosis pathogenesis and development .

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