Caratterizzazione istomorfometrica ed immunoistochimica delle alterazioni coronariche in un modello sperimentale di aterogenesi accelerata: ruolo dell'infiammazione e della proliferazione cellulare

L’ aterosclerosi è una patologia infiammatoria/degenerativa delle arterie di medio e grosso calibro, frequentemente associata ad eventi cardiovascolari avversi quali ischemia ed infarto miocardico, ictus cerebrale , etc. Nelle fasi iniziali del processo aterosclerotico la frazione LDL del colesterolo si accumula nello strato sottoendoteliale delle arterie per aumentata permeabilità e/o disfunzione dell’endotelio, cellule del sistema immunitario sono reclutate dal circolo per fagocitare l’LDL modificato e i monociti aderiscono alla parete vascolare e da questa passano nello spazio sub-endoteliale dove avviene la loro maturazione in cellule dendritiche e macrofagiche. I macrofagi, e più precisamente i fattori di crescita e le citochine da questi rilasciate, innescano il processo di crescita della placca. Il conseguente stato infiammatorio provoca, inoltre, l’attivazione delle cellule muscolari lisce della tunica media vasale (SMC), che a loro volta acquisiscono la capacità di proliferare e di migrare accumulandosi all’interno della regione sub-endoteliale. A differenza della patologia aterosclerotica avanzata (placche coronariche e carotidee umane complicate), lo studio dei quadri iniziali di nascita e formazione della lesione si basa generalmente sull’impiego di modelli sperimentali animali di aterogenesi accelerata mediante somministrazione di diete ad alto contenuto di grassi saturi e colesterolo. All’interno di questi modelli, il maiale costituisce, per la somiglianza istologica delle lesioni e per alcune omologie metaboliche e genetiche, l’animale più appropriato per la traslazione clinica dei risultati. Lo scopo di questo studio è valutare, attraverso la caratterizzazione morfometrica ed immunoistochimica, i fattori morfologici e molecolari di infiammazione/proliferazione associati ai diversi stadi evolutivi delle lesioni aterosclerotiche coronariche e periferiche in un modello sperimentale (maiale) di aterogenesi coronarica accelerata (dieta iperlipidica ed ipercolesterolemica di 2 e 4 mesi. Attraverso la combinazione di caratterizzazione istomorfometrica, composizione cellulare prevalente ed espressione tissutale di markers di infiammazione e proliferazione cellulare (mediante immunoistochimica) nelle diverse fasi del processo di aterogenesi, ci si propone di chiarire la sequenza temporale ed il ruolo prevalente della migrazione/ differenziazione monocitaria rispetto alla dedifferenziaione/proliferazione delle cellule muscolari lisce nella progressione ed evoluzione della placca aterosclerotica coronarica e periferica

Proteome dissection of the atherosclerotic plaque: a deep immersion into the extracellular matrix ocean.

Background- Carotid plaque rupture, leading to atherothrombosis, is the third most common cause of death and it is responsible for 20% of all strokes. Plaque core-specific molecular factors of the extracellular matrix (ECM) can be identified by proteomics analysis and may be exploited as clinically relevant specific biomarkers of vulnerability. Aim of this study was to map ECM proteins and fragments from carotid plaque specimens. Methods-The ECM profile of atherosclerotic internal carotid artery (ICA) derived from endoarterectomy was obtained by using HPLC coupled with mass spectrometry (LC-MSMS). ICA samples were treated by different extraction methods and identified proteins in all fractions were evaluated and compared. All fractions were analysed by LC-MSMS prior and after fractionation by gel electrophoresis. Histology and immunohistochemistry were performed to characterize plaque core. Results-The LC-MSMS approach enabled the identification of more than 150 ECM and ECM associated proteins (according to Gene Ontology database). Among these the most represented families were proteoglycans and collagens. Gel electrophoresis fractionation evidenced peptides of Collagen alpha 1 and 2 (193 and 129 kDa respectively) at low MW regions (< 30 kDa) suggesting an intense intraplaque enzyme activity. Conclusions-An optimized workflow, allowing a detailed matrix protein profile of carotid plaque, may assist and improve biomarker discovery of molecular factors specifically related to disease complication. The proportion between intact protein and its fragments into the ECM plaque core could represent a quantitative indirect measure of intralesional protease activity and therefore provide a clinically novel and potentially more reliable index of “the chemical rupture risk” for vulnerable plaques

Site-Specific Secretome Map Evidences VSMC-Related Markers of Coronary Atherosclerosis Grade and Extent in the Hypercholesterolemic Swine

A major drawback in coronary atherosclerosis (ATS) research is the difficulty of investigating early phase of plaque growth and related features in the clinical context. In this study, secreted proteins from atherosclerotic coronary arteries in a hypercholesterolemic swine model were characterized by a proteomics approach and their expression was correlated to site-specific ATS stage and extent. A wide coronary artery map of secreted proteins has been obtained in high fat (HF) diet induced ATS swine model and a significantly different expression of many proteins related to vascular smooth muscle cell (VSMC) activation/migration has been identified. Significant associations with ATS stage of HF coronary lesions were found for several VSMC-derived proteins and validated for chitinase 3 like protein 1 (CHI3L1) by tissue immunoexpression. A direct correlation R2=0.85 was evidenced with intima to media thickness ratio values and ELISA confirmed the higher blood concentrations of CHI3L1 in HF cases. These findings confirmed the pivotal role of VSMCs in coronary plaque development and demonstrated a strong site-specific relation between VSMC-secreted CHI3L1 and lesion grade, suggesting that this protein could be proposed as a useful biomarker for diagnosing and staging of atherosclerotic lesions in coronary artery disease

Quantitative micro-CT based coronary artery profiling using interactive local thresholding and cylindrical coordinates

Micro-CT is an established imaging technique for high-resolution non-destructive assessment of vascular samples, which is gaining growing interest for investigations of atherosclerotic arteries both in humans and in animal models. However, there is still a lack in the definition of micro-CT image metrics suitable for comprehensive evaluation and quantification of features of interest in the field of experimental atherosclerosis (ATS). OBJECTIVE: A novel approach to micro-CT image processing for profiling of coronary ATS is described, providing comprehensive visualization and quantification of contrast agent-free 3D high-resolution reconstruction of full-length artery walls. METHODS: Accelerated coronary ATS has been induced by high fat cholesterol-enriched diet in swine and left coronary artery (LCA) harvested en bloc for micro-CT scanning and histologic processing. A cylindrical coordinate system has been defined on the image space after curved multiplanar reformation of the coronary vessel for the comprehensive visualization of the main vessel features such as wall thickening and calcium content. A novel semi-automatic segmentation procedure based on 2D histograms has been implemented and the quantitative results validated by histology. RESULTS: The potentiality of attenuation-based micro-CT at low kV to reliably separate arterial wall layers from adjacent tissue as well as identify wall and plaque contours and major tissue components has been validated by histology. Morphometric indexes from histological data corresponding to several micro-CT slices have been derived (double observer evaluation at different coronary ATS stages) and highly significant correlations (R2 &lt; 0.90) evidenced. Semi-automatic morphometry has been validated by double observer manual morphometry of micro-CT slices and highly significant correlations were found (R2 &lt; 0.92). CONCLUSION: The micro-CT methodology described represents a handy and reliable tool for quantitative high resolution and contrast agent free full length coronary wall profiling, able to assist atherosclerotic vessels morphometry in a preclinical experimental model of coronary ATS and providing a link between in vivo imaging and histology

Nédemax mese (Leucoselect, Lymphaselect, Bromelain) in the treatment of chronic venous disease: a multicenter , obbservational study

BACKGROUND: Chronic venous disease (CVD)is major health concern; however,there remains a need to improve treatment approaches.Nédemax Mese , a nutritional supplementation consisting of Leucoselect 300 mg,Lymphaselect 100 mg and Bromelain 100 mg, is a patented formulation thah may have a role in the treatment of CVD. In this prospective , multicenter study conducted at 54 Italian centers, we investigated the effectiveness of Nédemax Mese in a large sample of CVD patients

Nédemax® Mese (Leucoselect®, Lymphaselect®, Bromelain) in the treatment of chronic venous disease: a multicenter, observational study

BACKGROUND: Chronic venous disease (CVD)is major health concern; however,there remains a need to improve treatment approaches.Nédemax Mese , a nutritional supplementation consisting of Leucoselect 300 mg,Lymphaselect 100 mg and Bromelain 100 mg, is a patented formulation thah may have a role in the treatment of CVD. In this prospective , multicenter study conducted at 54 Italian centers, we investigated the effectiveness of Nédemax Mese in a large sample of CVD patients