In-vitro analysis of normal and aneurismal human ascending aortic tissues using FT-IR microspectroscopy

FTIR microspectroscopy has shown to be a proven tool in the investigation of many tissue types. We have used this spectroscopic approach to analyse structural differences between normal and aneurismal aortic tissues and also aortas from patients with congenital anomalies like aortic bicuspid valves. Spectral analysis showed important variations in amide I and II regions, related to changes in alpha-helix and beta-sheet secondary structure of proteins that seem to be correlated to structural modifications of collagen and elastin. These proteins are the major constituents of the aortic wall associated to smooth muscular cells. The amide regions have thus been identified as a marker of structural modifications related to these proteins whose modifications can be associated to a given aortic pathological situation. Both univariate (total absorbance image and band ratio) and multivariate (principal components analysis) analyses of the spectral information contained in the infrared images have been performed. Differences between tissues have been identified by these two approaches and allowed to separate each group of aortic tissues. However, with univariate band ratio analysis, the pathological group was found to be composed of samples from aneurismal aortas associated or not with an aortic bicuspid valve. In contrast, PCA was able to separate these two types of aortic pathologies. For other groups, PCA and band ratio analysis can differentiate between normal, aneurismal, and none dilated aortas from patients with a bicuspid aortic valve.     

FTIR protein secondary structure analysis of human ascending aortic tissues

The advent of moderate dilatations in ascending aortas is often accompanied by structural modifications of the main components of the aortic tissue, elastin and collagen. In this study, we have undertaken an approach based on FTIR microscopy coupled to a curve‐fitting procedure to analyze secondary structure modifications in these proteins in human normal and pathological aortic tissues. We found that the outcome of the aortic pathology is strongly influenced by these proteins, which are abundant in the media of the aortic wall, and that the advent of an aortic dilatation is generally accompanied by a decrease of parallel β‐sheet structures. Elastin, essentially composed of β‐sheet structures, seems to be directly related to these changes and therefore indicative of the elastic alteration of the aortic wall. Conventional microscopy and confocal fluorescence microscopy were used to compare FTIR microscopy results with the organization of the elastic fibers present in the tissues. This in‐vitro study on 6 patients (three normal and three pathologic), suggests that such a spectroscopic marker, specific to aneurismal tissue characterization, could be important information for surgeons who face the dilemma of moderate aortic tissue dilatation of the ascending aortas. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Analysis of structural changes in normal and aneurismal human aortic tissues using FTIR microscopy

Aortic aneurisms are frequently asymptomatic but can induce dramatic complications. The diagnosis is only based on the aortic diameter and not on a structural and compositional basis. In this preliminary study, we propose infrared microspectroscopy to nondestructively probe normal and aneurismal human aortas. Spectra from 19 human ascending aortic biopsies (10 normal and 9 aneurismal) were acquired using infrared microspectroscopy. A 1500 x 150 microm(2) area of each 7-microm thick cryosection was investigated using a 30-microm spatial resolution with a total of about 200 spectra per sample. Spectral differences between normal and aneurismal tissues were mainly located in spectral regions related to proteins, such as elastin and collagen, and proteoglycans (1750-1000 cm(-1)). Tissue heterogeneity and sample classification have been evaluated using hierarchical cluster analysis of individual or mean spectra and their second derivative. Using spectral range related to proteins, 100% of good classification was obtained whereas the proteoglycan spectral range was less discriminant. This in vitro study demonstrates the potential of such technique to differentiate between normal and aneurismal aortas using selected spectral ranges. Future investigations will be focused on these specific spectral regions to determine the role of elastin and collagen in the discrimination of normal and pathological aortas.     

Analysis of Extracellular Superoxide Dismutase and Akt in Ascending Aortic Aneurysm With Tricuspid or Bicuspid Aortic Valve

Ascending aortic aneurysm (AsAA) is a consequence of medial degeneration (MD), deriving from apoptotic loss of smooth muscle cells (SMC) and fragmentation of elastin and collagen fibers. Alterations of extracellular matrix structure and protein composition, typical of medial degeneration, can modulate intracellular pathways. In this study we examined the relevance of extracellular superoxide dismutase (SOD3) and Akt in AsAA pathogenesis, evaluating their tissue distribution and protein levels in ascending aortic tissues from controls (n=6), patients affected by AsAA associated to tricuspid aortic valve (TAV, n=9) or bicuspid aortic valve (BAV, n=9). The results showed a significant reduction of SOD3, phospho-Akt and Akt protein levels in AsAA tissues from patients with BAV, compared to controls, whereas the differences observed between controls and patients with TAV were not significant. The decreased levels of SOD3 and Akt in BAV aortic tissues are associated with decreased Erk1/Erk2 phosphorylation and MMP-9 levels increase. The authors suggest a role of decreased SOD3 protein levels in the progression of AsAA with BAV and a link between ECM modifications of aortic media layer and impaired Erk1/Erk2 and Akt signaling in the late stages of the aortopathy associated with BAV.Key words: Ascending aortic aneurysm, bicuspid aortic valve, medial degeneration, smooth muscle cells, extracellular superoxide dismutase, Akt, tri-cuspid aortic valve     

Systolic fluid–structure interaction model of the congenitally bicuspid aortic valve: assessment of modelling requirements

A transient fluid–structure interaction (FSI) model of a congenitally bicuspid aortic valve has been developed which allows simultaneous calculation of fluid flow and structural deformation. The valve is modelled during the systolic phase (the stage when blood pressure is elevated within the heart to pump blood to the body). The geometry was simplified to represent the bicuspid aortic valve in two dimensions. A congenital bicuspid valve is compared within the aortic root only and within the aortic arch. Symmetric and asymmetric cusps were simulated, along with differences in mechanical properties. A moving arbitrary Lagrange–Euler mesh was used to allow FSI. The FSI model requires blood flow to induce valve opening and induced strains in the region of 10%. It was determined that bicuspid aortic valve simulations required the inclusion of the ascending aorta and aortic arch. The flow patterns developed were sensitive to cusp asymmetry and differences in mechanical properties. Stiffening of the valve amplified peak velocities, and recirculation which developed in the ascending aorta. Model predictions demonstrate the need to take into account the category, including any existing cusp asymmetry, of a congenital bicuspid aortic valve when simulating its fluid flow and mechanics.     

Unraveling divergent gene expression profiles in bicuspid and tricuspid aortic valve patients with thoracic aortic dilatation: the ASAP study

Thoracic aortic aneurysm (TAA) is a common complication in patients with a bicuspid aortic valve (BAV), the most frequent congenital heart disorder. For unknown reasons TAA occurs at a younger age, with a higher frequency in BAV patients than in patients with a tricuspid aortic valve (TAV), resulting in an increased risk for aortic dissection and rupture. To investigate the increased TAA incidence in BAV patients, we obtained tissue biopsy samples from nondilated and dilated aortas of 131 BAV and TAV patients. Global gene expression profiles were analyzed from controls and from aortic intima-media and adventitia of patients (in total 345 samples). Of the genes found to be differentially expressed with dilation, only a few (<4%) were differentially expressed in both BAV and TAV patients. With the use of gene set enrichment analysis, the cell adhesion and extracellular region gene ontology sets were identified as common features of TAA in both BAV and TAV patients. Immune response genes were observed to be particularly overexpressed in the aortic media of dilated TAV samples. The divergent gene expression profiles indicate that there are fundamental differences in TAA etiology in BAV and TAV patients. Immune response activation solely in the aortic media of TAV patients suggests that inflammation is involved in TAA formation in TAV but not in BAV patients. Conversely, genes were identified that were only differentially expressed with dilation in BAV patients. The result has bearing on future clinical studies in which separate analysis of BAV and TAV patients is recommended.     

The Role of Rubella in the Etiology of Supravalvular Aortic Stenosis

The possibility of an etiological relationship between rubella embryopathy and sporadic forms of supravalvular aortic stenosis is considered. A case is presented of a patient with rubella embryopathy and supravalvular aortic stenosis associated with pulmonary valvular and peripheral pulmonary artery stenosis, bicuspid aortic valve, aortic valve stenosis and subendothelial myocardial fibrosis. A review of the literature revealed many clinical and pathological features common to both syndromes. The hypothesis that rubella virus produced germ-cell mutation and subsequent persistence of rubella in the zygote produced further fetal damage is presented to explain these observations.     

Coronary artery anomalies and aortic valve morphology in the Syrian hamster

In the Syrian hamster, anomalies in the origin of the left coronary artery are significantly associated with the bicuspid condition of the aortic valve. In this species, bicuspid aortic valves are expressions of a trait, the variation of which takes the form of a phenotypic continuum, ranging from a tricuspid aortic valve with no commissural fusion to a bicuspid aortic valve with the aortic sinuses located in ventrodorsal orientation and devoid of any raphe. The intermediate stages of the continuum are represented by tricuspid aortic valves with a more or less extensive fusion of the ventral commissure and bicuspid aortic valves with a more or less developed raphe located in the ventral aortic sinus. The present study was designed to decide whether there is a gap between tricuspid and bicuspid aortic valves regarding the incidence of coronary artery anomalies, or whether this incidence varies according to the different tricuspid and bicuspid morphotypes of the continuum. The study was carried out in Syrian hamsters belonging to a single inbred family with a high incidence of tricuspid aortic valves with fusion of the ventral commissure, bicuspid aortic valves, and anomalies in the origin of the left coronary artery, i.e. single right coronary artery ostium in aorta, anomalous origin of the left coronary artery from the pulmonary artery, and anomalous origin of the left coronary artery from the dorsal aortic sinus. The specimens were examined by means of a stereomicroscope and, in several cases, scanning electron microscopy was also used. The relationships between anomalous coronary artery patterns and aortic valve morphologies were tested using a logistic regression model. The results obtained indicate that there is no discontinuity between tricuspid and bicuspid aortic valves regarding the incidence of coronary artery anomalies. The probability of occurrence of anomalous coronary artery patterns increases continuously according to the deviation degree of the aortic valve from its normal (tricuspid) design. The present findings suggest that in the Syrian hamster, the morphogenetic mechanisms involved in the formation of congenital anomalous aortic valves and anomalies in the origin of the left coronary artery, respectively, are strongly related from an aetiological viewpoint.     

Bicuspid aortic valve aortopathies: An hemodynamics characterization in dilated aortas

Bicuspid aortic valve (BAV) aortopathy remains of difficult clinical management due to its heterogeneity and further assessment of related aortic hemodynamics is necessary. The aim of this study was to assess systolic hemodynamic indexes and wall stresses in patients with diverse BAV phenotypes and dilated ascending aortas. The aortic geometry was reconstructed from patient-specific images while the aortic valve was generated based on patient-specific measurements. Physiologic material properties and boundary conditions were applied and fully coupled fluid-structure interaction (FSI) analysis were conducted. Our dilated aortic models were characterized by the presence of abnormal hemodynamics with elevated degrees of flow skewness and eccentricity, regardless of BAV morphotype. Retrograde flow was also present. Both features, predicted by flow angle and flow reversal ratios, were consistently higher than those reported for non-dilated aortas. Right-handed helical flow was present, as well as elevated wall shear stress (WSS) on the outer ascending aortic wall. Our results suggest that the abnormal flow associated with BAV may play a role in aortic enlargement and progress it further on already dilated aortas.     

Selection of Reference Genes for Quantitative Real Time PCR (qPCR) Assays in Tissue from Human Ascending Aorta

Dilatation of the ascending aorta (AAD) is a prevalent aortopathy that occurs frequently associated with bicuspid aortic valve (BAV), the most common human congenital cardiac malformation. The molecular mechanisms leading to AAD associated with BAV are still poorly understood. The search for differentially expressed genes in diseased tissue by quantitative real-time PCR (qPCR) is an invaluable tool to fill this gap. However, studies dedicated to identify reference genes necessary for normalization of mRNA expression in aortic tissue are scarce. In this report, we evaluate the qPCR expression of six candidate reference genes in tissue from the ascending aorta of 52 patients with a variety of clinical and demographic characteristics, normal and dilated aortas, and different morphologies of the aortic valve (normal aorta and normal valve n = 30; dilated aorta and normal valve n = 10; normal aorta and BAV n = 4; dilated aorta and BAV n = 8). The expression stability of the candidate reference genes was determined with three statistical algorithms, GeNorm, NormFinder and Bestkeeper. The expression analyses showed that the most stable genes for the three algorithms employed were CDKN1β, POLR2A and CASC3, independently of the structure of the aorta and the valve morphology. In conclusion, we propose the use of these three genes as reference genes for mRNA expression analysis in human ascending aorta. However, we suggest searching for specific reference genes when conducting qPCR experiments with new cohort of samples.     

Techniques of aortic valve repair

Similar to mitral repair, newer methods of aortic valve reconstruction are achieving excellent outcomes with an 85% to 90% freedom from valve-related complications at 10 years. The goal of this review is to illustrate these newer and more stable techniques of aortic valve repair. Most patients with aortic insufficiency from either trileaflet or bicuspid aortic valves are candidates for repair, in addition to selected patients with mixed aortic stenosis/insufficiency and aortic root aneurysms. Initially, aggressive commissural annuloplasty is performed to reduce measured valve diameter to 19 to 21 mm. Leaflet prolapse is corrected with plication stitches placed in the free edge of each leaflet adjacent to the Nodulus Arantius. In this regard, the leaflet free edge functions as the chorda tendinea of the aortic valve, and shortening with plication stitches raises the leaflet to a proper "effective height." Leaflet defects are augmented with gluteraldehyde-fixed autologous pericardium, and mild-to-moderate strategically placed spicules of calcium are removed with the cavitron ultrasonic surgical aspirator. Using these methods, most insufficient aortic valves, and many with mixed lesions, can be satisfactorily repaired. Six cases are illustrated in this review, spanning the spectrum of pathologies from annular dilatation without leaflet defects, to standard congenital bicuspid valve with prolapse, to trileaflet prolapse, to unusual bicuspid pathology with calcification, to a moderately calcified trileaflet valve with mixed lesions, and to aortic root aneurysms with severe aortic insufficiency. All valves were repaired using the techniques described above with trivial residual leak and minimal gradients. All repairs have been followed with yearly echocardiography, and valve reconstruction with these methods is now quite stable with excellent late outcomes. Most insufficient aortic valves now can undergo stable repair with minimal late valve-related complications. Greater application of aortic valve repair seems indicated.     

Compositional and structural alterations of chondroitin and dermatan sulfates during the progression of atherosclerosis and aneurysmal dilatation of the human abdominal aorta

Glycosaminoglycans participate in several biological functions in the arterial wall through their specific structures. They undergo specific compositional and structural modifications during the development of vascular diseases. The present study was performed to determine the variations in the concentration and the structural characteristics of galactosaminoglycans--chondroitin sulfate (CS) and dermatan sulfate (DS)--during the progression of atherosclerosis and aneurysmal dilatation of the human abdominal aorta. The concentration of CS was increased 24% (p < or = 0.05) in atherosclerotic type II aortas, but it was significantly decreased (29%, p < or = 0.05) in atherosclerotic type V aortas and aneurysmal aortas (65%, p < or = 0.01). In contrast, the concentration of DS was almost constant in all stages of arterial disease examined. Significant structural alterations were detected in the disaccharide composition of galactosaminoglycans. The ratio of 6-sulfated to 4-sulfated disaccharides was increased in atherosclerotic type II aortas (4.0 instead of 3.1 in normal aortas) due to the marked increase of CS in this tissue. This ratio was significantly decreased in atherosclerotic type V and aneurysmal aortas (2.1 and 1.6, respectively) due to the significant reduction of CS in the respective tissues. In addition, significant decrease of the oversulfated disaccharides, which are mainly located in DS chains, was recorded in atherosclerotic and aneurysmal aortas. Particularly, deltadi-di(2,6)S were decreased 32% (p < or = 0.01) and 86% (p < or = 0.01) in atherosclerotic type II and V aortas and 88% (p < or = 0.01) in aneurysm. Deltadi-di(2,4)S were increased in atherosclerotic type II aortas (21%, p < or = 0.01), but significantly decreased in atherosclerotic type V (33%, p < or = 0.01) and aneurysmal aortas (56%, p < or = 0.01). The amounts of deltadi-di(4,6)S were not markedly affected in the diseased tissues. These results suggest that the concentration of galactosaminoglycans is differentially affected during the progression of atherosclerosis. Furthermore, the development of vascular disease is associated with specific structural modifications, especially with the significant reduction of particular types of oversulfated disaccharides, which may play essential biological roles in the arterial wall.     

Normal and abnormal development of the aortic wall and valve: correlation with clinical entities

Dilation of the wall of the thoracic aorta can be found in patients with a tricuspid (TAV) as well as a bicuspid aortic valve (BAV) with and without a syndromic component. BAV is the most common congenital cardiovascular malformation, with a population prevalence of 0.5–2 %. The clinical course is often characterised by aneurysm formation and in some cases dissection. The non-dilated aortic wall is less well differentiated in all BAV as compared with TAV, thereby conferring inherent developmental susceptibility. Furthermore, a turbulent flow, caused by the inappropriate opening of the bicuspid valve, could accelerate the degenerative process in the aortic wall. However, not all patients with bicuspidy develop clinical complications during their life. We postulate that the increased vulnerability for aortic complications in a subset of patients with BAV is caused by a defect in the early development of the aorta and aortic valve. This review discusses histological and molecular genetic aspects of the normal and abnormal development of the aortic wall and semilunar valves. Aortopathy associated with BAV could be the result of a shared developmental defect during embryogenesis.     

Experimental characterization of rupture in human aortic aneurysms using a full-field measurement technique

The present study aims at investigating biomechanical failure behaviour of human aneurismal aortic tissues so as to diagnose the rupture risk of aneurysms more accurately. An inflation test is performed on aneurismal aortic tissues up to failure and full-field measurements are achieved using stereo digital image correlation. Then, an appropriate constitutive model derived from histological structure of arteries is adopted to retrieve the Cauchy stress. The virtual fields method is used as an inverse procedure to identify material parameters. Next, the Cauchy stress components are calculated from the identified parameters and the measured Lagrange strain fields. Finally, an important stress parameter which can quantify the strength of aneurismal tissues is derived from the failure stress of aneurismal tissues.     

Origin of the left anterior descending coronary artery from the pulmonary artery: An unusual cause of angina in a middle-aged woman

A 48-year-old woman was admitted to our institution with angina pectoris and a systolic murmur. At cardiac catheterization, she was found to have an anomalous origin of the left anterior descending coronary artery from the pulmonary trunk. There was also an associated atrial septal defect and a bicuspid aortic valve.     

Fluid Dynamics of Coarctation of the Aorta and Effect of Bicuspid Aortic Valve

Up to 80% of patients with coarctation of the aorta (COA) have a bicuspid aortic valve (BAV). Patients with COA and BAV have elevated risks of aortic complications despite successful surgical repair. The development of such complications involves the interplay between the mechanical forces applied on the artery and the biological processes occurring at the cellular level. The focus of this study is on hemodynamic modifications induced in the aorta in the presence of a COA and a BAV. For this purpose, numerical investigations and magnetic resonance imaging measurements were conducted with different configurations: (1) normal: normal aorta and normal aortic valve; (2) isolated COA: aorta with COA (75% reduction by area) and normal aortic valve; (3) complex COA: aorta with the same severity of COA (75% reduction by area) and BAV. The results show that the coexistence of COA and BAV significantly alters blood flow in the aorta with a significant increase in the maximal velocity, secondary flow, pressure loss, time-averaged wall shear stress and oscillatory shear index downstream of the COA. These findings can contribute to a better understanding of why patients with complex COA have adverse outcome even following a successful surgery.     

A retinal proteomics-based study identifies αA-crystallin as a sex steroid-regulated protein

Sex steroids influence the structural and functional organization of ocular tissues, promote survival in several pathological conditions including retinal neurodegeneration and have a prominent role in age-related eye diseases as well as neurodegenerative diseases. However, their underlying mechanisms are still elusive. We explored proteomic profiling of rat retinas following intravitreal injection of the bioactive 17β-estradiol or androgen dihydrotestosterone. Using narrow range 2-DE gels and MALDI-TOF-MS analysis, we identified three sex steroid-regulated proteins: the galectin-related-inter-fiber (GRIFIN) which is a galectin family member protein of unknown function, the fatty acid-binding protein epidermal-5 (FABP5) protein responsible for the fatty acid uptake and transport and the small heat shock αA-crystallin (CRYAA) protein involved in preventing aggregation of denatured or unfolded proteins. Changes in the expression of these proteins revealed a predominant estrogenic effect and the multiple CRYAA protein species reflected posttranslational modifications. Sex steroid-mediated modifications of CRYAA were confirmed by Western blotting analysis. This study provides new target proteins for sex steroids with a potential link to age-related diseases associated with proteotoxic stress.     

Fluid dynamics of aortic root dilation in Marfan syndrome

Aortic root dilation and propensity to dissection are typical manifestations of the Marfan Syndrome (MS), a genetic defect leading to the degeneration of the elastic fibres. Dilation affects the structure of the flow and, in turn, altered flow may play a role in vessel dilation, generation of aneurysms, and dissection. The aim of the present work is the investigation in-vitro of the fluid dynamic modifications occurring as a consequence of the morphological changes typically induced in the aortic root by MS. A mock-loop reproducing the left ventricle outflow tract and the aortic root was used to measure time resolved velocity maps on a longitudinal symmetry plane of the aortic root. Two dilated model aortas, designed to resemble morphological characteristics typically observed in MS patients, have been compared to a reference, healthy geometry. The aortic model was designed to quantitatively reproduce the change of aortic distensibility caused by MS. Results demonstrate that vorticity released from the valve leaflets, and possibly accumulating in the root, plays a fundamental role in redirecting the systolic jet issued from the aortic valve. The altered systolic flow also determines a different residual flow during the diastole.     

Influence of structural geometry on the severity of bicuspid aortic stenosis

Doppler-derived gradients may overestimate total pressure loss in degenerative and prosthetic aortic valve stenosis (AS) due to unaccounted pressure recovery distal to the orifice. However, in congenitally bicuspid valves, jet eccentricity may result in a higher anatomic-to-effective orifice contraction ratio, resulting in an increased pressure loss at the valve and a reduced pressure recovery distal to the orifice leading to greater functional severity. The objective of our study was to determine the impact of local geometry on the total versus Doppler-derived pressure loss and therefore the assessed severity of the stenosis in bicuspid valves. On the basis of clinically obtained measurements, two- and three-dimensional computer simulations were created with various local geometries by altering the diameters of the left ventricular outflow tract (LVOT; 1.8-3.0 cm), orifice diameter (OD; 0.8-1.6 cm), and aortic root diameter (AR; 3.0-5.4 cm). Jet eccentricity was altered in the models from 0 to 25 degrees. Simulations were performed under steady-flow conditions. Axisymmetric simulations indicate that the overall differences in pressure recovery were minor for variations in LVOT diameter (<3%). However, both OD and AR had a significant impact on pressure recovery (6-20%), with greatest recovery being the larger OD and the smaller recovery being the AR. In addition, three-dimensional data illustrate a greater pressure loss for eccentric jets with the same orifice area, thus increasing functional severity. In conclusion, jet eccentricity results in greater pressure loss in bicuspid valve AS due to reduced effective orifice area. Functional severity may also be enhanced by larger aortic roots, commonly occurring in these patients, leading to reduced pressure recovery. Thus, for the same anatomic orifice area, functional severity is greater in bicuspid than in degenerative tricuspid AS.