Development Of Atheromatous Lesions Research Articles

Ultrasonic biomicroscopy and micro-Raman spectroscopy for the mechanochemical characterization of atheromatous lesions

Atherosclerosis is defined as a focal, inflammatory, and fibro-proliferative response to endothelial injury. The development of atheromatous lesions in the coronary tree is predominantly a quiescent asymptomatic process without any clinical manifestations. The unpredictable and acute nature of cardiovascular complications such as vulnerable plaque rupture makes diagnosis and treatment of this disease an outstanding medical challenge. We investigate non-invasive techniques that facilitate mechanical measurements at the microscopic level, which can then be directly correlated to biomarker localization within lesion sites. To characterize these sites in vitro, we used time-resolved scanning acoustic microscopy (TRSAM). This technique allows for non-invasive interrogation of tissue samples with optical resolution at the micrometer scale. Furthermore, we combined TRSAM with micro-Raman (micro-RS) spectroscopy to investigate plaque morphology with regard to specific biomarkers. We characterized mechanoelastic and biochemical regions containing high cholesterol, phosphate, and carbonate apatite that are characteristic of atherosclerotic lesions. The mechanoelastic evaluation of these regions was determined using TRSAM. Calcified lesions, for example, exhibit ultrasonic velocities of 1810 m/s ± 25 m/s and are more rigid and stiffer than normal blood vessel tissues.

Monitoring the oxidative modification of lipoprotein(a) by capillary zone electrophoresis

Lipoprotein(a) [Lp(a)] is a recognized risk factor for atherosclerotic cardiovascular disease. It is made of a lipoprotein particle containing apoB100 linked by a single disulfide bridge to apolipoprotein(a), a glycosylated protein with a variable mass. Some authors suggest that oxidative modification could explain the contribution of Lp(a) in the development of atheromatous lesions in a comparable way to low-density lipoproteins (LDL). Recently, the use of capillary electrophoresis to measure the variations in the relative electrophoretic mobility (REM) of LDL subjected to copper oxidation has been proposed. The aim of this work is to employ this method also to monitor the copper-induced oxidative modification of Lp(a). Migration of Lp(a) was monitored by absorption at 200 nm in a 50 mmol/L tricine, 100 mmol/L methylglucamine, pH 9.7 run buffer. Contrary to the conventional slab gel methods, our procedure provides a rapid and reproducible means to measure the electrophoretic mobility of Lp(a) (migration time <10 min with a CV% <0.5).

L -citrulline and l -arginine supplementation retards the progression of high-cholesterol-diet-induced atherosclerosis in rabbits

The objective of this study was to evaluate the influence of ingested l-arginine, l-citrulline, and antioxidants (vitamins C and E) on the progression of atherosclerosis in rabbits fed a high-cholesterol diet. The fatty diet caused a marked impairment of endothelium-dependent vasorelaxation in isolated thoracic aorta and blood flow in rabbit ear artery in vivo, the development of atheromatous lesions and increased superoxide anion production in thoracic aorta, and increased oxidation-sensitive gene expression [Elk-1 and phosphorylated cAMP response element-binding protein]. Rabbits were treated orally for 12 weeks with l-arginine, l-citrulline, and/or antioxidants. l-arginine plus l-citrulline, either alone or in combination with antioxidants, caused a marked improvement in endothelium-dependent vasorelaxation and blood flow, dramatic regression in atheromatous lesions, and decrease in superoxide production and oxidation-sensitive gene expression. These therapeutic effects were associated with concomitant increases in aortic endothelial NO synthase expression and plasma NO(2)(-)+NO(3)(-) and cGMP levels. These observations indicate that ingestion of certain NO-boosting substances, including l-arginine, l-citrulline, and antioxidants, can abrogate the state of oxidative stress and reverse the progression of atherosclerosis. This approach may have clinical utility in the treatment of atherosclerosis in humans.

Diffusion of gamma globulin into the arterial wall identifies localized entry of lipid and cells in atherosclerosis.

The localization of atheromatous lesions in vulnerable arteries and their relatively rare occurrence in other arteries of the same subject cannot be explained by current theories of the aetiology of atherosclerosis. To determine whether abnormal diffusion of gamma globulin into the arterial wall from the lumen will identify defects of barrier function allowing localized entry of lipid and cells in atherosclerosis. Paraffin sections of left anterior descending coronary arteries and corresponding internal thoracic arteries from 80 human subjects aged 1-65 years were stained for gamma globulin by the immunoperoxidase technique. Duplicate sections were stained with orcein to demonstrate the elastin structure. The barrier function of the luminal surface of the thickened intima was associated with the presence of an elastin lamina beneath the endothelial cells. With advancing age, the coronary arteries exhibited breakdown of this barrier function in localized areas with entry into the arterial wall of gamma globulin, lipid and cells. This was rare in the internal thoracic artery. Lack of continuity or incomplete formation of this sub-endothelial lamina, which was seen particularly in the coronary artery, was associated with localized entry into the arterial wall of gamma globulin, lipid and cells from the circulating blood and with the development of atheromatous lesions.

Centrolobular liver fibrosis in the hypercholesterolemic rabbit.

During a study on the development of atheromatous lesions in rabbits fed a diet with a low or high cholesterol supplement, we found a moderate to pronounced centrolobular liver fibrosis. This fibrosis developed in three stages. Early after supplementation of cholesterol, we observed increased immunoreactivity of collagen types I, III, and IV, and fibronectin, around central veins and in adjacent sinusoids. In the second stage, we observed further increase of collagen and fibronectin immunoreactivity, together with the appearance of alpha-smooth muscle actin (alpha-SM actin)-positive cells and anti-rabbit macrophage monoclonal antibody (RAM 11)-positive cells. In the third stage, we observed large numbers of alpha-SM actin-positive cells, together with heavy deposition of connective tissue proteins in pericentral sinusoids, in addition to focal atrophy of parenchymal cells. By transmission electron microscopy (TEM), fat-storing cells in the pericentral regions were shown to be enlarged, to lose their lipid-droplets, and to acquire dilated rough endoplasmic reticulum corresponding to an activated phenotype. Parenchymal cells were either normal or contained numerous small lipid-droplets. They sometimes were smaller and distorted. Northern hybridization performed on total RNA of whole liver showed an increased level of collagen alpha1(I), alpha1(III), and alpha1(IV) messenger RNA (mRNA) after 24 weeks of low dietary cholesterol supplementation. These data show enhanced expression of extracellular matrix proteins. We conclude that cholesterol overload induces pericentral liver fibrosis in rabbits. The diet clearly activates fat-storing cells to become fibrogenic effector cells. At present, we have no explanation why hypercholesterolemia induces phenotypic transition of fat-storing cells.

Centrolobular liver fibrosis in the hypercholesterolemic rabbit

During a study on the development of atheromatous lesions in rabbits fed a diet with a low or high cholesterol supplement, we found a moderate to pronounced centrolobular liver fibrosis. This fibrosis developed in three stages. Early after supplementation of cholesterol, we observed increased immunoreactivity of collagen types I, III, and IV, and fibronectin, around central veins and in adjacent sinusoids. In the second stage, we observed further increase of collagen and fibronectin immunoreactivity, together with the appearance of alpha-smooth muscle actin (alpha-SM actin)-positive cells and anti-rabbit macrophage monoclonal antibody (RAM 11)-positive cells. In the third stage, we observed large numbers of alpha-SM actin-positive cells, together with heavy deposition of connective tissue proteins in pericentral sinusoids, in addition to focal atrophy of parenchymal cells. By transmission electron microscopy (TEM), fat-storing cells in the pericentral regions were shown to be enlarged, to lose their lipid-droplets, and to acquire dilated rough endoplasmic reticulum corresponding to an activated phenotype. Parenchymal cells were either normal or contained numerous small lipid-droplets. They sometimes were smaller and distorted. Northern hybridization performed on total RNA of whole liver showed an increased level of collagen alpha1(I), alpha1(III), and alpha1(IV) messenger RNA (mRNA) after 24 weeks of low dietary cholesterol supplementation. These data show enhanced expression of extracellular matrix proteins. We conclude that cholesterol overload induces pericentral liver fibrosis in rabbits. The diet clearly activates fat-storing cells to become fibrogenic effector cells. At present, we have no explanation why hypercholesterolemia induces phenotypic transition of fat-storing cells. (Hepatology 1996 Oct;24(4):939-46)

Modified lipoproteins, cytokines and macrovascular disease in non-insulin-dependent diabetes mellitus.

The processes of glycation and oxidation play a significant role in the acceleration of atherosclerosis in diabetes mellitus. Glycation is thought not only to increase the susceptibility of low-density lipoprotein (LDL) to oxidation but also to enhance the propensity of vessel wall structural proteins to bind extravasated plasma proteins, including LDL, and thus to contribute to a more marked oxidative modification of LDL. Glycated and oxidized lipoproteins induce cholesteryl ester accumulation in human macrophages and may promote platelet and endothelial cell dysfunction. Furthermore, these modified lipoproteins have the ability to trigger an autoimmune response that leads to the formation of autoantibodies and subsequently to the formation of immune complexes containing LDL. Both the modified lipoproteins and the immune complexes formed with autoantibodies reactive with modified lipoproteins may be responsible for several alternative and not mutually exclusive pathways leading to foam cell formation, macrophage activation and endothelial cell damage and may thus be of potential significance in initiating and/or contributing to the acceleration of the development of atherosclerosis. In this review we discuss how modified LDL affects lipoprotein metabolism, how immune complexes containing LDL induce the transformation of macrophages into foam cells and promote macrophage activation leading to the release of cytokines and thus initiating a sequence of events leading to endothelial cell damage and to the recruitment and activation of leucocytes. We also summarize our work showing that macrophage activation by LDL containing immune complexes leads to a paradoxical increase in LDL-receptor expression thus further impairing cholesterol homeostasis and enhancing the development of atheromatous lesions.

Effect of Thrombin, the Thrombin Receptor Activation Peptide, and Other Mitogens on Vascular Smooth Muscle Cell Urokinase Receptor mRNA Levels

AbstractBovine vascular smooth muscle cells (SMC) express the urokinase-type plasminogen activator receptor (u-PAR) claimed to be important in cell invasion. Receptor numbers and affinity are regulated by thrombin and several other mitogens involved in SMC proliferation. We investigated the effects of these mitogens on u-PAR mRNA levels. On continuous thrombin stimulation the u-PAR message in SMC was 10 ± 2.3-fold elevated reaching a maximum between 6 and 9 hours and declining to control values within 48 hours. Thrombin present for 30 minutes on the cell surface produced similar effects. Stimulation with the thrombin receptor activation peptide S-F-L-L-R-N representing the NH2-terminus of the tethered ligand also increased u-PAR mRNA levels with an identical time course. D-Phe-Pro-Arg-chloromethyl ketone (PPACK) active site blocked thrombin and the catalytically inactive thrombin mutant S205A did not affect u-PAR mRNA levels. Thrombin stimulation also resulted in a 2 ± 0.2-fold transient increase in thrombin receptor mRNA preceding the rise in u-PAR message. Transforming growth factor β1 (TGFβ1) and platelet-derived growth factor (PDGF) showed similar time courses for the elevation of u-PAR mRNA levels with a maximal 5.5 ± 0.9 and 12 ± 2.5-fold increase, respectively. Basic fibroblast growth factor (bFGF) and phorbol myristate acetate (PMA) showed a more prolonged effect increasing u-PAR mRNA levels 8 ± 2.0-fold and 12.3 ± 2.5-fold, respectively, within 6 hours but remaining 5 to 10-fold elevated at 48 hours. In order to decide if the u-PAR mRNA increase was due to message stabilization or a consequence of transcriptional activation we used the RNA polymerase II Inhibitor 5,6-di-chloro-1-β-D-ribofuranosyl benzimidazole (DRB) during the stimulation experiments, u-PAR mRNA levels on TGFβ1 stimulation of SMC decayed after the addition of DRB indicating that enhancement of transcriptional activity was involved in the induction. In contrast, the time course of u-PAR mRNA elevation on thrombin, bFGF, and PMA stimulation was not significantly altered in the presence of DRB suggesting that in these latter cases u-PAR mRNA message accumulation was at least in part due to mRNA stabilization. Increased transcriptional activity, mRNA stabilization and expression of u-PAR protein on the SMC surface in response to growth factors may facilitate enhanced cell surface protease activity, cell migration, and development of atheromatous lesions.

The Inhibitory Effect of a Novel Antiatheromatous Agent, E5050, on the Intimal Thickening of Aorta in Cholesterol-Fed Rabbit In Vivo.

The development of atheromatous lesions in the aortic arch of 0.5% cholesterol-fed rabbits was biochemically and morphologically examined. The animals were killed at week twelve (Cont-12W) or sixteen (Cont-16W). Both the micrographic and biochemical studies showed that the main atheromatous lesions in the Cont-12W group were fatty streaks, whereas those in the Cont-16W group were fibrous plaques. In these models, oral ingestion of 0.2% and 0.4% E5050, which has an antiproliferative effect on smooth muscle cells, had no effect on the surface involvement or the lipid content of the aortic arch at the sixteenth week, but reduced the degree of intimal thickening and the DNA content in the aortic arch in a dose-dependent manner. These results strongly suggest that E5050 suppresses the intimal thickening through its inhibitory effect on the proliferation of smooth muscle cells.

The Role of Internal Elastic Laminae Damage in the Development of Canine Arteriosclerosis

This study was designed to test the hypothesis that the extent of damage to the internal elastic lamina (IEL) may be a factor in determining the development of atheromatous lesions (lesion area). A bilateral iliac artery arteriosclerotic (AS) model in seven dogs prepared by intimal denudation and fracture of the IEL plus 5% cholesterol diet was investigated. The animals were sacrificed at 10-52 weeks (10, n = 1; 16, n = 3; 20, n = 1; 32, n = 1; 52, n = 1). Histologic analysis of sections (n = 42) of 14 canine iliac AS arteries harvested bilaterally from identical locations along the vessels (2, 7, and 12 cm from the aortic bifurcation) were compared with control segments of caudal and femoral arteries (n = 12) that had intact lamina and no luminal trauma. AS segments at all sites along the length of the traumatized iliac vessels demonstrated significant lesions, while control vessels had no arteriosclerotic changes. AS lesion area was calculated using ocular micrometry and a multiple regression model to test the predictability of the lesion area from the following independent variables: (1) proportion of intact IEL to total IEL (IEL/IELt), (2) sacrifice intervalens (3) frequency of disruptions to the IEL, (4) side and level of the arterial lesion, and (5) respective animals. This analysis revealed that none of the coefficients for the independent variables used in the multiple regression model was significant, suggesting that the extent of AS lesions is not related to the size, frequency, or location of breaks in the IEL, although a break in IEL is required to initiate the lesions. This observation suggests that the migration of cells from the arterial media through the broken laminae or the release of medial biochemical mediators which are normally contained by the elastic lamina is required to develop the AS lesions in this canine model, and that any size break in the lamina initiates the process.

A numerical study of pulsatile flow through stenosed canine femoral arteries

A numerical study is made of pulsatile flow through stenosed canine femoral arteries for lumen constrictions in the range 0–61%. Comparison of results with in vivo measurements indicates that the pressure drop across the stenosis and the time averaged peak wall shear may be sufficiently large when the areal restriction is 61% to result in the development of atheromatous lesions and endothelial damage proximal to the stenosis, but that these effects are unlikely when the restriction is 44% or less. Local flow reversal is observed in the wake of the stenosis during systole and during diastolic flow reversal, but the rate of development of the local reverse flow is very sensitive to stenosis height. For the more severe stenoses this local reverse flow results in a finite wall shear throughout the wake region, except at the point where the boundary of local reverse flow attaches at the stenosis. The amplitude and phase of the pressure gradient over the stenosis, relative to those in a normal section of artery, were found to be sensitive indicators of stenosis development.