Progressive leukoencephalopathy impairs neurobehavioral development in sialin-deficient mice.

Slc17a5-/- mice represent an animal model for the infantile form of sialic acid storage disease (SASD). We analyzed genetic and histological time-course expression of myelin and oligodendrocyte (OL) lineage markers in different parts of the CNS, and related this to postnatal neurobehavioral development in these mice. Sialin-deficient mice display a distinct spatiotemporal pattern of sialic acid storage, CNS hypomyelination and leukoencephalopathy. Whereas few genes are differentially expressed in the perinatal stage (p0), microarray analysis revealed increased differential gene expression in later postnatal stages (p10-p18). This included progressive upregulation of neuroinflammatory genes, as well as continuous down-regulation of genes that encode myelin constituents and typical OL lineage markers. Age-related histopathological analysis indicates that initial myelination occurs normally in hindbrain regions, but progression to more frontal areas is affected in Slc17a5-/- mice. This course of progressive leukoencephalopathy and CNS hypomyelination delays neurobehavioral development in sialin-deficient mice. Slc17a5-/- mice successfully achieve early neurobehavioral milestones, but exhibit progressive delay of later-stage sensory and motor milestones. The present findings may contribute to further understanding of the processes of CNS myelination as well as help to develop therapeutic strategies for SASD and other myelination disorders.

Automated PGP9.5 immunofluorescence staining: a valuable tool in the assessment of small fiber neuropathy?

BACKGROUND: In this study we explored the possibility of automating the PGP9.5 immunofluorescence staining assay for the diagnosis of small fiber neuropathy using skin punch biopsies. The laboratory developed test (LDT) was subjected to a validation strategy as required by good laboratory practice guidelines and compared to the well-established gold standard method approved by the European Federation of Neurological Societies (EFNS). To facilitate automation, the use of thinner sections. (16 µm) was evaluated. Biopsies from previously published studies were used. The aim was to evaluate the diagnostic performance of the LDT compared to the gold standard. We focused on technical aspects to reach high-quality standardization of the PGP9.5 assay and finally evaluate its potential for use in large scale batch testing. RESULTS: We first studied linear nerve fiber densities in skin of healthy volunteers to establish reference ranges, and compared our LDT using the modifications to the EFNS counting rule to the gold standard in visualizing and quantifying the epidermal nerve fiber network. As the LDT requires the use of 16 µm tissue sections, a higher incidence of intra-epidermal nerve fiber fragments and a lower incidence of secondary branches were detected. Nevertheless, the LDT showed excellent concordance with the gold standard method. Next, the diagnostic performance and yield of the LDT were explored and challenged to the gold standard using skin punch biopsies of capsaicin treated subjects, and patients with diabetic polyneuropathy. The LDT reached good agreement with the gold standard in identifying small fiber neuropathy. The reduction of section thickness from 50 to 16 µm resulted in a significantly lower visualization of the three-dimensional epidermal nerve fiber network, as expected. However, the diagnostic performance of the LDT was adequate as characterized by a sensitivity and specificity of 80 and 64 %, respectively. CONCLUSIONS: This study, designed as a proof of principle, indicated that the LDT is an accurate, robust and automated assay, which adequately and reliably identifies patients presenting with small fiber neuropathy, and therefore has potential for use in large scale clinical studies.

Elastin fragmentation in atherosclerotic mice leads to intraplaque neovascularization, plaque rupture, myocardial infarction, stroke, and sudden death.

AIMS: There is a need for animal models of plaque rupture. We previously reported that elastin fragmentation, due to a mutation (C1039G(+/-)) in the fibrillin-1 (Fbn1) gene, promotes atherogenesis and a highly unstable plaque phenotype in apolipoprotein E deficient (ApoE(-/-)) mice on a Western-type diet (WD). Here, we investigated whether plaque rupture occurred in ApoE(-/-)Fbn1(C1039G+/-) mice and was associated with myocardial infarction, stroke, and sudden death. METHODS AND RESULTS: Female ApoE(-/-)Fbn1(C1039G+/-) and ApoE(-/-) mice were fed a WD for up to 35 weeks. Compared to ApoE(-/-) mice, plaques of ApoE(-/-)Fbn1(C1039G+/-) mice showed a threefold increase in necrotic core size, augmented T-cell infiltration, a decreased collagen I content (70 ± 10%), extensive neovascularization, intraplaque haemorrhage, and a significant increase in matrix metalloproteinase-2, -9, -12, and -13 expression or activity. Plaque rupture was observed in 70% of ascending aortas and in 50% of brachiocephalic arteries of ApoE(-/-)Fbn1(C1039G+/-) mice. In ApoE(-/-) mice, plaque rupture was not seen in ascending aortas and only in 10% of brachiocephalic arteries. Seventy percent of ApoE(-/-)Fbn1(C1039G+/-) mice died suddenly, whereas all ApoE(-/-) mice survived. ApoE(-/-)Fbn1(C1039G+/-) mice showed coronary plaques and myocardial infarction (75% of mice). Furthermore, they displayed head tilt, disorientation, and motor disturbances (66% of cases), disturbed cerebral blood flow (73% of cases; MR angiograms) and brain hypoxia (64% of cases), indicative of stroke. CONCLUSIONS: Elastin fragmentation plays a key role in plaque destabilization and rupture. ApoE(-/-)Fbn1(C1039G+/-) mice represent a unique model of acute plaque rupture with human-like complications.

Asymptomatic small fiber neuropathy in diabetes mellitus: investigations with intraepidermal nerve fiber density, quantitative sensory testing and laser-evoked potentials.

This study aimed at evaluating the performance of a battery of morphological and functional tests for the assessment of small nerve fiber loss in asymptomatic diabetic neuropathy (DNP). Patients diagnosed for ≥10 years with type 1 (n = 10) or type 2 (n = 13) diabetes mellitus (DM) without conventional symptoms or signs of DNP were recruited and compared with healthy controls (n = 18) and patients with overt DNP (n = 5). Intraepidermal nerve fiber density (IENFd) was measured with PGP9.5 immunostaining on punch skin biopsies performed at the distal leg. Functional tests consisted of quantitative sensory testing (QST) for light-touch, cool, warm and heat pain detection thresholds and brain-evoked potentials with electrical (SEPs) and CO(2) laser stimulation [laser-evoked potentials (LEPs)] of hand dorsum and distal leg using small (0.8 mm(2)) and large (20 mm(2)) beam sizes. Results confirmed a state of asymptomatic DNP in DM, but only at the distal leg. Defining a critical small fiber loss as a reduction of IENFd ≤-2 z scores of healthy controls, this state prevailed in type 2 (30%) over type 1 DM (10%) patients despite similar disease duration and current glycemic control. LEPs with the small laser beam performed best in terms of sensitivity (91%), specificity (83%) and area-under-the ROC curve (0.924). Although this performance was not statically different from that of warm and cold detection threshold, LEPs offer an advantage over QST given that they bypass the subjective report and are therefore unbiased by perceptual factors.

The time course of CO2 laser-evoked responses and of skin nerve fibre markers after topical capsaicin in human volunteers.

OBJECTIVE: To assess the temporal relationship between skin nerve denervation and regeneration (dermal and intra-epidermal fibres, IENF) and functional changes (CO(2) laser-evoked potentials, LEPs, and quantitative sensory tests, QST) after topical cutaneous application of capsaicin. METHODS: Capsaicin (0.075%) was applied to the lateral calf for three consecutive days. QST, LEPs and skin biopsies were performed at baseline and time intervals up to 54days post-capsaicin treatment. Biopsies were immunostained with antibodies for PGP9.5, TRPV1, and GAP-43 (marker of regenerating nerve fibres), and analyzed for IENFs and dermal innervation (for GAP-43). RESULTS: At 1day post-capsaicin, cutaneous thermal sensitivity was reduced, as were LEPs. PGP9.5, TRPV1, and GAP-43 immunoreactive-nerve fibres were almost completely absent. By Day 12, LEPs had fully recovered, but PGP9.5 and TRPV1 IENF continued to be significantly decreased 54days post-capsaicin. In contrast, dermal GAP-43 immunoreactivity closely matched recovery of LEPs. CONCLUSIONS: A good correlation was observed between LEPs and GAP-43 staining, in contrast to PGP9.5 and TRPV1. Laser stimulation is a non-invasive and sensitive method for assessing the initial IENF loss, and regenerating nerve fibres. SIGNIFICANCE: Assessing skin biopsies by PGP9.5 immunostaining alone may miss significant diagnostic and prognostic information regarding regenerating nerve fibres, if other approaches are neglected, e.g. LEPs or GAP-43 immunostaining.

Lumen enhancement influences absolute noncalcific plaque density on multislice computed tomography coronary angiography: ex-vivo validation and in-vivo demonstration.

AIM: The purpose of this study was to define the in-vitro and in-vivo effects of intracoronary enhancement on the absolute density values of coronary plaques during multislice computed tomography. METHODS: We studied seven ex-vivo left coronary artery specimens surrounded by olive oil and filled with isotonic saline and four solutions with decreasing dilutions of contrast material: control (isotonic saline), 1/200, 1/80, 1/50, and 1/20. The multislice computed tomography protocol was: slice/collimation 32 x 2 x 0.6 mm and rotation time 330 ms. The attenuation (Hounsfield units) value of atherosclerotic plaques was measured for each dilution in lumen, plaque (noncalcified coronary wall thickening), calcium, and surrounding oil. In-vivo assessment was performed in 12 patients (nine men; mean age 58.7 +/- 9.9 years) who underwent two subsequent multislice computed tomography scans (arterial and delayed) after intravenous administration of a single bolus of contrast material. The attenuation values of lumen and plaques during arterial and delayed computed tomography were compared. The results were compared with one-way analysis of variance and correlated with Pearson's test. RESULTS: Mean lumen (45 +/- 38-669 +/- 151 HU) and plaque (11 +/- 35-101 +/- 72 HU) attenuation differed significantly (P < 0.001) among the different dilutions. The attenuation of lumen and plaque of coronary plaques showed moderate correlation (r = 0.54, P < 0.001). The mean attenuation value in vivo for the arterial and delayed phase scans differed significantly (P < 0.001) for lumen (325 +/- 70 and 174 +/- 46 HU, respectively) and plaque (138 +/- 71 and 100 +/- 52 HU, respectively). CONCLUSION: Coronary plaque attenuation values are significantly modified by differences in lumen contrast densities both ex vivo and in vivo. This should be taken into account when considering the distinction between lipid and fibrous plaques.

Stent thrombosis is not always stent thrombosis: de novo atherosclerosis in a stented coronary segment.

We discuss a case of late thrombosis, 9 years after implantation of overlapping bare metal stents in a circumflex artery. The patient presented with an acute ST segment elevation infero-lateral myocardial infarction. The coronary angiogram revealed a sub-occlusive thrombus within the boundaries of the stents. Aspiration of the material was performed and pathological analysis showed, together with fibrin thrombus and platelet aggregates, fragments of an atherosclerotic plaque (parts of necrotic core with cholesterol clefts and inflammatory cells such as macrophages) including iron deposition, suggestive for plaque rupture. We conclude that this event occurred because of de novo atherosclerotic formation of a vulnerable, rupture-prone plaque within the boundaries of the stents.

Quantitative ex vivo and in vivo comparison of lumen dimensions measured by optical coherence tomography and intravascular ultrasound in human coronary arteries.

INTRODUCTION AND OBJECTIVES: The relationship between the lumen dimensions obtained in human coronary arteries using intravascular ultrasound (IVUS) and those obtained using optical coherence tomography (OCT) is not well understood. The objectives were to compare the lumen measurements obtained ex vivo in human coronary arteries using IVUS, OCT and histomorphometry, and in vivo in patients using IVUS and OCT with and without balloon occlusion. METHODS: Ex vivo study: the lumen areas of matched anatomical sections of human coronary arteries were measured using IVUS, OCT and histology. In vivo study: the lumen areas in matched sections were measured using IVUS and OCT with and without occlusion. RESULTS: Ex vivo: in the eight specimens studied, the lumen area obtained using OCT and IVUS was larger than that obtained using histomorphometry: mean difference 0.8+/-1 mm(2) (28%) for OCT and 1.3+/-1.1 mm(2) (40%) for IVUS. In vivo: in the five vessels analyzed, the lumen area obtained using IVUS was larger than that obtained using OCT: mean difference 1.67+/-0.54 mm(2) (33.7%) for IVUS relative to OCT with occlusion and 1.11+/-0.53 mm(2) (21.5%) relative to OCT without occlusion. The lumen area obtained using OCT without occlusion was larger than that obtained using OCT with occlusion: mean difference 0.61+/-0.23 mm(2) (13%). CONCLUSIONS: In fixed human coronary arteries, both IVUS and OCT overestimated the lumen area compared with histomorphometry. In vivo the lumen dimensions obtained using IVUS were larger than those obtained using OCT, with or without occlusion. Moreover, the OCT image acquisition technique (i.e. with or without occlusion) also had an impact on lumen measurement.

Comparación cuantitativa ex vivo e in vivo de las dimensiones del lumen medidas por tomografía de coherencia óptica y ecografía intravascular en arterias coronarias humanas / Quantitative Ex Vivo and in Vivo Comparison of lumen Dimensions Measured by optical Coherence tomography and intravascular ultrasound in Human Coronary arteries

Introducción y objetivos. La relación entre las dimensiones del lumen medidas por ecografía intravascular (IVUS) y tomografía de coherencia óptica (OCT) en arterias coronarias humanas no es bien conocida. Los objetivos son comparar las dimensiones del lumen en IVUS, OCT e histología en arterias coronarias humanas ex vivo, y comparar in vivo las dimensiones del lumen obtenidas en pacientes con IVUS, OCT con oclusión y OCT sin oclusión. Métodos. Estudio ex vivo: el área luminal se midió en secciones anatómicas correspondientes en IVUS, OCT e histología en arterias coronarias humanas. Estudio in vivo: el área luminal se midió en regiones correspondientes en IVUS y OCT con y sin oclusión. Resultados. Ex vivo: en las 8 muestras estudiadas, el área del lumen fue más grande en IVUS y OCT que en histología ¿diferencia media, 0,8 ± 1 mm2 (28%) para OCT y 1,3 ± 1,1 mm2 (40%) para IVUS¿. In vivo: en los cinco vasos analizados las dimensiones del lumen fueron más grandes en IVUS que en OCT ¿diferencia área media del lumen, 1,67 ± 0.54 mm2 (33,7%) para IVUS y OCT con oclusión y 1,11 ± 0.53 mm2 (21,5%) para IVUS y OCT sin oclusión¿. Las dimensiones del lumen fueron más grandes en OCT sin oclusión que en OCT con oclusión ¿diferencia media, 0,61 ± 0,23 mm2 (13%). Conclusiones. En arterias coronarias humanas fijadas, IVUS y OCT sobrestimaron el área del lumen en comparación con la histología. In vivo, las dimensiones del lumen fueron más grandes en IVUS que en OCT con o sin oclusión. La técnica de adquisición de OCT (con o sin oclusión) influye en las dimensiones del lumen (AU)

Introduction and objectives. The relationship between the lumen dimensions obtained in human coronary arteries using intravascular ultrasound (IVUS) and those obtained using optical coherence tomography (OCT) is not well understood. The objectives were to compare the lumen measurements obtained ex vivo in human coronary arteries using IVUS, OCT and histomorphometry, and in vivo in patients using IVUS and OCT with and without balloon occlusion.Methods. Ex vivo study: the lumen areas of matched anatomical sections of human coronary arteries were measured using IVUS, OCT and histology. In vivo study: the lumen areas in matched sections were measured using IVUS and OCT with and without occlusion.results. Ex vivo: in the eight specimens studied, the lumen area obtained using OCT and IVUS was larger than that obtained using histomorphometry: mean difference 0.8±1 mm2 (28%) for OCT and 1.3±1.1 mm2 (40%) for IVUS. In vivo: in the five vessels analyzed, the lumen area obtained using IVUS was larger than that obtained using OCT: mean difference 1.67±0.54 mm2 (33.7%) for IVUS relative to OCT with occlusion and 1.11±0.53 mm2 (21.5%) relative to OCT without occlusion. The lumen area obtained using OCT without occlusion was larger than that obtained using OCT with occlusion: mean difference 0.61±0.23 mm2 (13%).Conclusions. In fixed human coronary arteries, both IVUS and OCT overestimated the lumen area compared with histomorphometry. In vivo the lumen dimensions obtained using IVUS were larger than those obtained using OCT, with or without occlusion. Moreover, the OCT image acquisition technique (i.e. with or without occlusion) also had an impact on lumen measurement (AU)

Evaluation of cell death markers in severe calcified aortic valves.

Degenerative aortic valve disease is the most frequent acquired valve disease. Especially in the elderly, its prevalence is increasing. Once the disease becomes symptomatic, it is rapidly fatal. The disease cannot be considered a result of aging alone. The condition is an active process, which occurs with rapid progression, especially when calcification can be documented. This calcification can be the end result of cellular mechanisms involving cell death pathways (such as autophagy) and cellular matrix remodeling. These processes are beginning to be unraveled in the initiation and propagation of the disease. Autophagy could be the common step through which these mechanisms lead to this pathway of cell death in this disease. Autophagy can be detected by procedures described hereafter.

Compositional volumetry of non-calcified coronary plaques by multislice computed tomography: an ex vivo feasibility study.

AIMS: Non-invasive quantitative compositional analysis of coronary plaque would be a major advantage to study coronary artery disease. This study explores the application to use the Hounsfield units (HU) distribution of coronary plaques imaged by multislice computed tomography-coronary angiography (MSCT-CA). METHODS AND RESULTS: A dedicated computer-assisted method was developed to measure the HU distribution within a coronary plaque by MSCT-CA. To test the feasibility of the method, an ex vivo left anterior descending (LAD) coronary specimen, excised during autopsy, was imaged both by non-enhanced and enhanced MSCT-CA. Quantitative histology was used as a reference. To test the feasibility of the new volumetric analytic method, the MSCT-CA data were compared with volumetric histopathology. The coronary specimen, with a heterogeneously distributed plaque composition without large areas of calcification, was histologically sampled at five different locations, 5 mm apart, where at each location 15 sections were taken at 100 microm intervals, resulting in 75 individual histology sections. Tri-chrome Masson staining was used for histology quantification of three plaque/tissue components: smooth muscle cells (SMC), collagen and calcium. MSCT plaque composition was defined as "lower-HU" or "higher-HU" plaque and "calcium" based on the HU distribution. Comparison of the MSCT defined tissue components against histology showed a good relationship without significant differences. CONCLUSIONS: This ex vivo study shows the feasibility of using the Hounsfield unit distribution to perform compositional coronary plaque volumetry by MSCT-CA. The results are encouraging.

Three-dimensional and quantitative analysis of atherosclerotic plaque composition by automated differential echogenicity.

OBJECTIVE: To validate automated and quantitative three-dimensional analysis of coronary plaque composition using intracoronary ultrasound (ICUS). BACKGROUND: ICUS displays different tissue components based on their acoustic properties in 256 grey-levels. We hypothesised that computer-assisted image analysis (differential echogenicity) would permit automated quantification of several tissue components in atherosclerotic plaques. METHODS AND RESULTS: Ten 40-mm-long left anterior descending specimens were excised during autopsy of which eight could be successfully imaged by ICUS. Histological sections were taken at 5 mm intervals and analyzed. Since most of the plaques were calcified and having a homogeneous appearance, one specimen with a more heterogeneous composition was further examined: at each interval of 5 mm, 15 additional sections (every 100 microm) were evaluated. Plaques were scored for echogenicity against the adventitia: brighter (hyperechogenic) or less bright (hypoechogenic). Areas of hypoechogenicity correlated with the presence of smooth muscle cells. Areas of hyperechogenicity correlated with presence of collagen, and areas of hyperechogenicity with acoustic shadowing correlated with calcium. None of these comparisons showed statistical significant differences. CONCLUSION: This ex vivo feasibility study shows that automated three-dimensional differential echogenicity analysis of ICUS images allows identification of different tissue types within atherosclerotic plaques. This technology may play a role as an additional tool in longitudinal studies to trace possible changes in plaque composition.

Autophagy in cardiovascular disease.

Autophagy is a major cytoprotective pathway that eukaryotic cells use to degrade and recycle cytoplasmic contents. Recent evidence indicates that autophagy under baseline conditions represents an important homeostatic mechanism for the maintenance of normal cardiovascular function and morphology. By contrast, excessive induction of the autophagic process by environmental or intracellular stress has an important role in several types of cardiomyopathy by functioning as a death pathway. As a consequence, enhanced autophagy represents one of the mechanisms underlying the cardiomyocyte dropout responsible for the worsening of heart failure. Successful therapeutic approaches that regulate autophagy have been reported recently, suggesting that the autophagic machinery can be manipulated to treat heart failure or to prevent rupture of atherosclerotic plaques and sudden death.

Selective clearance of macrophages in atherosclerotic plaques by autophagy.

OBJECTIVES: The purpose of this study was to investigate whether stent-based delivery of an inhibitor of mammalian target of rapamycin (mTOR) can selectively clear macrophages in rabbit atherosclerotic plaques. BACKGROUND: Current pharmacologic approaches to stabilize atherosclerotic plaques have only partially reduced the incidence of acute coronary syndromes and sudden death. Macrophages play a pivotal role in plaque destabilization, whereas smooth muscle cells (SMC) promote plaque stability. METHODS: Stents eluting the mTOR inhibitor everolimus were implanted in atherosclerotic arteries of cholesterol-fed rabbits. In addition, in vitro experiments using explanted atherosclerotic segments and cultured macrophages as well as SMC were performed. RESULTS: Stents eluting everolimus led to a marked reduction in macrophage content without altering the amount of SMC compared with polymer control stents. In vitro studies showed that everolimus treatment induced inhibition of translation in both cultured macrophages and SMC. However, cell death occurred only in macrophages and was characterized by bulk degradation of long-lived proteins, processing of microtubule-associated protein light chain 3, and cytoplasmic vacuolization, which are all markers of autophagy. Everolimus-induced autophagy was mediated by mTOR inhibition, because cell viability was not affected using tacrolimus, an mTOR-independent everolimus analog. Moreover, mTOR gene silencing was associated with selective induction of macrophage cell death. Autophagic macrophage cell death was confirmed by transmission electron microscopy both in cultured cells and in atherosclerotic explants. CONCLUSIONS: Stent-based delivery of everolimus selectively cleared macrophages in rabbit atherosclerotic plaques by autophagy, an mTOR inhibition-dependent and novel mechanism to induce cell death in mammalian cells.

Influence of convolution filtering on coronary plaque attenuation values: observations in an ex vivo model of multislice computed tomography coronary angiography.

Attenuation variability (measured in Hounsfield Units, HU) of human coronary plaques using multislice computed tomography (MSCT) was evaluated in an ex vivo model with increasing convolution kernels. MSCT was performed in seven ex vivo left coronary arteries sunk into oil followingthe instillation of saline (1/infinity) and a 1/50 solution of contrast material (400 mgI/ml iomeprol). Scan parameters were: slices/collimation, 16/0.75 mm; rotation time, 375 ms. Four convolution kernels were used: b30f-smooth, b36f-medium smooth, b46f-medium and b60f-sharp. An experienced radiologist scored for the presence of plaques and measured the attenuation in lumen, calcified and noncalcified plaques and the surrounding oil. The results were compared by the ANOVA test and correlated with Pearson's test. The signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) were calculated. The mean attenuation values were significantly different between the four filters (p < 0.0001) in each structure with both solutions. After clustering for the filter, all of the noncalcified plaque values (20.8 +/- 39.1, 14.2 +/- 35.8, 14.0 +/- 32.0, 3.2 +/- 32.4 HU with saline; 74.7 +/- 66.6, 68.2 +/- 63.3, 66.3 +/- 66.5, 48.5 +/- 60.0 HU in contrast solution) were significantly different, with the exception of the pair b36f-b46f, for which a moderate-high correlation was generally found. Improved SNRs and CNRs were achieved by b30f and b46f. The use of different convolution filters significantly modifief the attenuation values, while sharper filtering increased the calcified plaque attenuation and reduced the noncalcified plaque attenuation.

Autophagy as mechanism for cell death in degenerative aortic valve disease.

Once degenerative aortic valve disease becomes symptomatic, valve replacement is necessary for prognostic and symptomatic reasons. In elderly patients, symptoms of degenerative aortic valve can often be doubtful. Therefore, it is difficult but important to distinguish patients who need surgery from those who do not. Estimation of the rate of the progression of this disease can be helpful herein because one needs to bear in mind that aortic valve degeneration is an active process, which can influence the rate of progression. Recently, autophagy was discovered as a mechanism of cell death in different cardiovascular diseases such as atherosclerosis, aortic valve degeneration, heart failure and at regions around heart infarctions. Thus understanding autophagy in all its details can be helpful to contribute insights into the cell death machinery of cardiovascular diseases. This could open ways for inhibition of cell death in cardiovascular disease and possibly define targets for future drug design.

Histopathology of calcific aortic valve stenosis.

Calcific aortic valve stenosis is the most common and increasing heart valve disease in the western world. In the last 30 years, diagnosis and management were revolutionized by the development of cardiac catheterisation, echocardiography, cardiac surgery, and medication. Recently, new strategies were introduced for aortic valve replacement using more sophisticated bioprosthetic heart valves. Moreover, tissue-engineered heart valves are under development to improve management strategies. In this article we review the current morphological and histopathological findings in the progression of calcific aortic valve stenosis. This is, to our understanding, important to contribute to the knowledge of fundamental management strategies of this disease.

The histopathology of varicose vein disease.

Varicosity is a complex venous pathology affecting the lower extremities. The exact etiology and physiopathology of varicose vein disease remain, however, unclear. Several theories exist from incompetence of the valves to a disturbance of the smooth muscle cells (SMC) and extra-cellular matrix (ECM) organization providing a weakness of the venous wall. Multiple studies have been performed to explain the underlying mechanisms of varicosity inducing alterations in the expression patterns of the endothelium, SMC, and ECM. In that respect, most attention has been focused on the alteration of the endothelium due to blood stasis and hypoxia inducing migration/proliferation of the medial SMC into the intima. Also, studies in the deformation of the ECM induced by alterations of the expression patterns of the metalloproteinases (MMP) and their inhibitors (TIMPs) have been put forward to explain the etiology of varicosity. However, less attention has been paid to the hormonal changes that occur during pregnancy and menopause, crucial factors to be involved in the etiology of varicosity. Since alteration of the estrogen receptor-b (ERb) expression could enhance directly the cellular volume of SMC and thus the disorganization of the contractile-elastic units, hypertrophy of SMC must be accounted a pivotal role that could induce the weakness of the venous wall. Altogether, this review summarizes an overview of the latest findings of varicosity with respect to the histopathological changes of the different cellular components of the varicose vein wall related to functional and morphologic alterations.

Histological evaluation of autophagic cell death in calcified aortic valve stenosis.

BACKGROUND AND AIM OF THE STUDY: Calcification in aortic valves is the most common valvular lesion in western populations. This event is correlated with cellular degeneration in the valvular cusps, although there is no exact evidence how these cells die: this requires further exploration. METHODS: Twelve human severely calcified aortic valves obtained during cardiac surgery were studied by semi-quantitative analysis, and results compared with data from 12 human control aortic valves obtained during autopsy. Tissue analysis was by hematoxylin and eosin and Alcian blue staining. Detection of neurons was by immunohistochemical staining of PGP9.5 and neurofilament. In order to detect autophagy, an immunohistochemical staining for ubiquitin was used. The TUNEL technique was used to detect apoptosis. Co-localization of Alizarin red with ubiquitin labeling was performed on non-decalcified aortic valves. RESULTS: Hematoxylin and eosin staining showed moderate to severe mineralization in 10 of 12 patients in the surgical group, but in only one of 12 in the autopsy group. No significant observations were made with regard to PGP9.5 and neurofilament staining. Moderate to severe ubiquitin labeling was found initially in the majority of the surgical resection group (9/12) compared to a minority in the autopsy group (1/12). TUNEL-positive labeling was very rare and found mostly at the endothelial layer of the valvular cusps. CONCLUSION: Immunohistochemical methods showed the main cell death mechanism involved in the calcification of aortic leaflets to be autophagy rather than apoptosis. These findings suggest that autophagic cell death might play a role in the release of matrix vesicles in early degenerative aortic valves, thereby attracting inflammatory cells, and this could eventually lead to mineralization.