José e Fernandes

Changes Related to Age and Cerebrovascular Symptoms in the Extracellular Matrix of Human Carotid Plaques

Background and Purpose— Many processes involved in the pathogenesis of atherosclerosis result in modifications of the extracellular matrix. These changes not only determine the mechanical stability of atherosclerotic lesions but can directly or indirectly influence further development of the lesions. The purpose of the present study was to compare the matrix composition of human carotid plaques from symptomatic patients with those obtained from patients without symptoms. Furthermore, matrix changes related to age were studied. Methods— Thirty atherosclerotic carotid plaques were removed by endarterectomy from 27 patients and divided into 2 groups on the basis of the presence of ipsilateral symptoms. The plaques were homogenized, and the total levels of the major components of the extracellular matrix were determined. Results— Plaques associated with symptoms were characterized by increased levels of elastin (1.58±0.46 versus 1.24±0.40 mg/g wet wt;P =0.03) and decreased levels of hydroxyapatite (45.1±46.3 versus 131.4±111.7 mg/g wet wt;P =0.02) compared with asymptomatic plaques. The increase in elastin in plaques from symptomatic patients was due to elevated levels of an intermediate-size fraction, as determined by liquid chromatography. Collagen and sulfated glycosaminoglycans were present in equal amounts in both groups. Elastin content in carotid plaques decreased with age. Conclusions— Carotid plaques from symptomatic patients have lower levels of hydroxyapatite than those from asymptomatic patients. The present study also raises the possibility that non–cross-linked forms of elastin, increased in plaques associated with symptoms, could be a marker of plaque vulnerability and/or directly induce harmful cellular activities or increase lipoprotein retention in the vascular wall.

Elastin and Calcium Rather Than Collagen or Lipid Content Are Associated With Echogenicity of Human Carotid Plaques

Background and Purpose— Echolucent carotid plaques have been associated with increased risk for stroke. Histological studies suggested that echolucent plaques are hemorrhage- and lipid-rich, whereas echogenic plaques are characterized by fibrosis and calcification. This is the first study to relate echogenicity to plaque composition analyzed biochemically. Methods— Echogenicity of human carotid plaques was analyzed by standardized high-definition ultrasound and classified into echolucent, with gray-scale median (GSM) <32 and echogenic with GSM ≥32. The biochemical composition of the plaques was assessed by fast-performance liquid chromotography and high-performance thin-layer chromotography. Results— As assessed biochemically (milligrams per gram [mg/g]), echolucent plaques contained less hydroxyapatite (43.8 [SD 41.2] mg/g versus 121.6 [SD 106.2] mg/g; P=0.018), more total elastin (1.7 [SD 0.4] mg/g versus 1.2 [SD 0.4] mg/g; P=0.008), and more intermediate-size elastin forms (1.2 [SD 0.3] mg/g versus 0.8 [SD 0.4] mg/g; P=0.018). There was no difference in collagen amount between echogenic and echolucent plaques, neither biochemically (15.3 [SD 3.7] mg/g versus 14.4 [SD 3.4] mg/g) nor histologically (13.4 [SD 4.9] % versus 13.0 [SD 5.6] %). Cholesterol esters, unesterified cholesterol, and triglycerides were increased in plaques associated with symptoms (22.5 [SD 23.3] mg/g versus 13.3 [SD 3.2]; P=0.04), but no differences were detected between echolucent and echogenic plaques (13.5 [SD 4.0] versus 20.2 [SD 21.5] mg/g). Similar results were obtained by Oil Red O staining (symptomatic 7.6 [SD 4.7] % versus asymptomatic 4.2 [SD 3.6] %; P=0.03; echolucent 5.9 [SD 4.1] % versus echogenic 5.0 [SD 4.0] % of area). Conclusions— Echogenicity of carotid plaques is mainly determined by their elastin and calcium but not collagen or lipid content. In addition, echolucency is associated to higher elastin content.

Hemorheological parameters are related to subclinical atherosclerosis in systemic lupus erythematosus and rheumatoid arthritis patients

OBJECTIVES Rheological characteristics of blood are strongly linked to atherothrombosis in the general population, but its contribution to atherosclerosis in systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) is currently unclear. This work examines the relationship between blood rheology, traditional cardiovascular (CV) risk factors, inflammation and subclinical atherosclerosis in SLE and RA. METHODS Whole blood viscosity (WBV), plasma viscosity (PV), erythrocyte deformability (ED), aggregation (EA) and erythrocyte NO production were measured in 197 patients (96 SLE and 101 RA) and compared to 97 controls, all females without previous CV events. Clinical information was obtained and fasting lipids and acute phase reactants were measured. The relationship between hemorheological parameters, CV risk factors and inflammation was assessed in patients and the impact of these variables on carotid intima-media thickness (cIMT) was evaluated in univariate followed by multivariate regression analyses. RESULTS WBV and ED are significantly lower in patients, while EA is elevated as compared with controls. Hemorheological disturbances correlate with CV risk factors and markers of inflammation and are more profound in patients with metabolic syndrome. Multivariable analysis showed that menopause (OR 34.72, 95%CI 4.44-271.77), obesity (OR 4.09, 95%CI 1.00-16.68) and WBV (OR 3.98; 95%CI 1.23-12.83) are positively associated whereas current corticosteroid dose (OR 0.87; 95%CI 0.78-0.98), and erythrocyte NO production (OR 0.16; 95%CI 0.05-0.52) are negatively associated with cIMT. CONCLUSION Disturbed hemorheological parameters in SLE and RA women are related to the presence of CV risk factors and inflammation. WBV and erythrocyte NO are independently associated with the early stages of atherosclerosis.

Activation of calpain-1 in human carotid artery atherosclerotic lesions

BackgroundIn a previous study, we observed that oxidized low-density lipoprotein-induced death of endothelial cells was calpain-1-dependent. The purpose of the present paper was to study the possible activation of calpain in human carotid plaques, and to compare calpain activity in the plaques from symptomatic patients with those obtained from patients without symptoms.MethodsHuman atherosclerotic carotid plaques (n = 29, 12 associated with symptoms) were removed by endarterectomy. Calpain activity and apoptosis were detected by performing immunohistochemical analysis and TUNEL assay on human carotid plaque sections. An antibody specific for calpain-proteolyzed α-fodrin was used on western blots.ResultsWe found that calpain was activated in all the plaques and calpain activity colocalized with apoptotic cell death. Our observation of autoproteolytic cleavage of the 80 kDa subunit of calpain-1 provided further evidence for enzyme activity in the plaque samples. When calpain activity was quantified, we found that plaques from symptomatic patients displayed significantly lower calpain activity compared with asymptomatic plaques.ConclusionThese novel results suggest that calpain-1 is commonly active in carotid artery atherosclerotic plaques, and that calpain activity is colocalized with cell death and inversely associated with symptoms.

Asymptomatic Carotid Disease—A New Tool for Assessing Neurological Risk

Active carotid plaques are associated with atheroembolism and neurological events; its identification is crucial for stroke prevention. High‐definition ultrasound (HDU) can be used to recognize plaque structure in carotid bifurcation stenosis associated with plaque vulnerability and occurrence of brain ischemic events. A new computer‐assisted HDU method to study the echomorphology of the carotid plaque and to determine a risk score for developing appropriate symptoms is proposed in this study. Plaque echomorphology characteristics such as presence of ulceration at the plaque surface, juxta‐luminal location of echolucent areas, echoheterogeneity were obtained from B‐mode ultrasound scans using several image processing algorithms and were combined with measurement of severity of stenosis to obtain a clinical score—enhanced activity index (EAI)—which was correlated with the presence or absence of ipsilateral appropriate ischemic symptoms. An optimal cutoff value of EAI was determined to obtain the best separation between symptomatic (active) from asymptomatic (inactive) plaques and its diagnostic yield was compared to other 2 reference methods by means of receiver‐operating characteristic (ROC) analysis. Classification performance was evaluated by leave‐one‐patient‐out cross‐validation applied to a cohort of 146 carotid plaques from 99 patients. The proposed method was benchmarked against (a) degree of stenosis criteria and (b) earlier proposed activity index (AI) and demonstrated that EAI yielded the highest accuracy up to an accuracy of 77% to predict asymptomatic plaques that developed symptoms in a prospective cross‐sectional study. Enhanced activity index is a noninvasive, easy to obtain parameter, which provided accurate estimation of neurological risk of carotid plaques.

Ultrasonographic characterization and identification of symptomatic carotid plaques

Carotid plaques are the main cause of neurological symptoms due to distal embolization or flow reduction. An objective classification of such lesions into symptomatic or asymptomatic is crucial for optimal treatment planning.

Carotid Plaque 3D Compound Imaging and Echo-Morphology Analysis: a Bayesian Approach

This paper describes a method for volume reconstruction of the carotid plaque and presents a novel local characterization of its echo-morphology. The data is composed by a series of nearly parallel ultrasound images (3D Compound Imaging) and the acquisition is performed using traditional noninvasive ultrasound equipment available in most medical facilities, without need of a spatial locator device. The reconstruction algorithm uses the observed pixels inside the plaque, which were obtained in a pre-segmentation stage performed under medical guidance [1]. The paper proposes a Bayesian algorithm which estimates the underlying volume inside the plaque, by filtering and interpolating the data in order to remove speckle noise and fill non- observed regions, respectively. This volume is further used in plaque echo-morphology analysis. The observation model is based on the Rayleigh distribution, commonly used to model speckle noise in ultrasound images. A prior model based on the edge preserving Total Variation Gibbs distribution is also used to fill the gaps on non-evenly spaced observations. An energy function is derived from these models and an iterative algorithm computes its minimizer. The estimated function, defined in a given volume of interest, is used in global and local plaque characterization, namely to estimate its average levels of stenosis, echo-morphology and to identify vulnerable foci inside the plaque. The goal is to make atherosclerosis diagnosis more accurate and complete than using traditional 2D ultrasound analysis.

Elastin- and Collagen-Rich Human Carotid Plaques Have Increased Levels of the Cysteine Protease Inhibitor Cystatin C

Background: Cystatin C is a major inhibitor of the elastin- and collagen-degrading cysteine proteases and may therefore have an important role in preserving atherosclerotic plaque stability. In this study we analyzed the associations between human carotid plaque cystatin C expression and the plaque content of collagen and elastin. Methods: Thirty-one plaques were removed by endarterectomy and homogenized. Cystatin C levels were analyzed by densitometry of Western blots and elastin and collagen levels were determined colorimetrically. Results: The plaque content of cystatin C correlated with total elastin (r = 0.58, p = 0.001) and collagen (r = 0.50, p = 0.004), as well as with cross-linked forms of elastin (r = 0.42, p = 0.022) and collagen (r = 0.52, p = 0.003). Immunohistochemical analysis demonstrated that cystatin C colocalized with elastin and collagen. No correlation was seen between cystatin C and the amount of degraded elastin or collagen in plaques. Conclusion: The positive correlation between cystatin C levels and collagen and elastin levels in plaques supports the notion that cystatin C plays an important role in maintaining atherosclerotic plaque stability.

Direct association between diet and the stability of human atherosclerotic plaque.

Mediterranean diet has been suggested to explain why coronary heart disease mortality is lower in southern than northern Europe. Dietary habits can be revealed by isotope ratio mass spectrometry (IRMS) measurement of carbon (δ13C) and nitrogen (δ15N) in biological tissues. To study if diet is associated with human plaque stability, atherosclerotic plaques from carotid endarterectomy on 56 patients (21 Portuguese and 35 Swedish) were analysed by IRMS and histology. Plaque components affecting rupture risk were measured. Swedish plaques had more apoptosis, lipids and larger cores, as well as fewer proliferating cells and SMC than the Portuguese, conferring the Swedish a more rupture-prone phenotype. Portuguese plaques contained higher δ13C and δ15N than the Swedish, indicating that Portuguese plaques were more often derived from marine food. Plaque δ13C correlated with SMC and proliferating cells, and inversely with lipids, core size, apoptosis. Plaque δ15N correlated with SMC and inversely with lipids, core size and apoptosis. This is the first observational study showing that diet is reflected in plaque components associated with its vulnerability. The Portuguese plaques composition is consistent with an increased marine food intake and those plaques are more stable than those from Swedish patients. Marine-derived food is associated with plaque stability.

Rectus femoris muscle flap based on proximal insertion mobilization to cover a groin infected vascular graft

The rectus femoris (RF) muscle flap, which is widely used to cover groin infected vascular grafts, is usually harvested through distal tendon division and an extensive muscle elevation and transposition into the groin wound defect. A case of a vascular prosthetic graft infection in the groin was successfully controlled after coverage with an RF flap that was harvested based on proximal portion mobilization instead of the conventional distal one. This case suggests that the RF muscle flap based on proximal insertion mobilization is a feasible, effective, technically simpler, and less invasive alternative to cover infected vascular grafts in the groin.