Histotopographic evidence that amyloid deposits in sclerocalcific heart valves and other chronic lesions of the cardiovascular system are related to old thrombotic material

Summary Deposition of amyloid in human sclero-calcific heart valves has been reported recently as a localized age-independant and dystrophic form of amyloidosis. Histochemical studies have shown that the deposits are permanganate resistant, contain tryptophan and P component and are immunologically unrelated to any known type of amyloid fibril protein. In this study histological observations from a series of four selected sclerotic heart valves show amyloid deposition in old thrombotic material covering fusing commissures or appositional collagen on the body of the leaflets. Similar cases from extravalvular sites have been added to the series: a partly hyalinized thrombus of the left atrium, a thrombotic aneurysm of the left ventricle, 2 thrombotic atherosclerotic aneurysms of the aorta and popliteal artery respectively, and an encapsulated haematoma of the scalp. The deposits are Congo red positive with typical green dichroism in polarized light, permanganate resistant and contain tryptophan. Electron microscopy of 3 cases displays small fibrils which are typical of amyloid.No patient showed evidence of systemic amyloidosis. The natural history of sclero-calcific valvulopathies and present observations favour the following pathogenesis: first, recurrent thrombotic deposition on thickened and fibrotic endocardium; second, degradation of a coagulation-related protein with potential during the aging of the clot with transformation into amyloid fibrils; finally, inclusion of the amyloid in sclerotic replacement tissue.     

Maturation of the extracellular material of the semilunar heart valves in the mouse

Summary The developmental changes of collagen and mucopolysaccharides in the semilunar valves of the mouse were studied during the embryonic, fetal and postnatal period. The valvular collagen was investigated using Van Gieson and Sirius red-polarization microscopy methods. These procedures showed that the establishment of the fibrosa layer of the cusps does not occur until the second week of the postnatal period.The nature and distribution of the valvular mucopolysaccharides were investigated by staining with Alcian blue at specific pH values and at various critical electrolyte concentrations, with the appropiate enzymatic controls using Streptomyces and testicular hyaluronidase. The results show that hyaluronate and chondroitin 4- and 6-sulphate are the major components during the embryonic and fetal period. In the older fetal stages and during the postnatal period the relative amount of hyaluronate decreases, while chondroitin sulphate increases.It is concluded from this study that the maturation of the valves occurs over a long period of the postnatal life.     

Intrinsic innervation of porcine semilunar heart valves

Summary In order to provide additional information on the morphology and the functional performance of semilunar valves, the presence of nerve fibers was investigated in the aortic and pulmonary leaflets by AChE techniques, formaldehyde-induced fluorescence methods, en bloc silver nitrate and gold chloride impregnation and electron microscopy.The results show that AChE positive and adrenergic nerve fibers are constantly present in every leaflet of all the animals examined. The pattern of innervation is similar in aortic and pulmonary valves and no differences exist between coronary and non coronary aortic leaflets. Nerve networks extend over the inferior two-thirds of the leaflets: they are composed of myelinated and non-myelinated axons and are mainly located in the ventricular side of the leaflets. Structures resembling free sensory endings can be shown both by light and electron microscopy.The presence in the leaflets of blood vessels and of an intrinsic smooth muscle system suggests that these two components are the most probable targets of the nerve fibers.Thanks are due to the management of the Vismara meat supply company for providing the pigs' hearts     

Histochemical and ultrastructural study on the innervation of human and porcine atrio-ventricular valves

Summary The presence of nerve fibers was investigated in porcine and human atrio-ventricular valves by AChE technique, formaldehyde-induced fluorescence, en bloc silver and gold chloride impregnation and electron microscopy.Elaborate nerve plexuses were observed in every leaflet and in some chordae tendineae of all the samples examined, without significant species differences in the pattern of innervation.The presence of a sensory innervation was inferred from the demonstration, in whole mount samples processed for acetylcholinesterase, of thick myelinated nerve fibers and of endings with dot-like or brush-like appearance. Moreover the results of the combined histochemical and ultrastructural methods showed the existence of both cholinergic and adrenergic efferent nerve fibers. Nerve varicosities with clear or dense-cored vesicles were frequently observed in proximity to blood vessels and to cardiac and smooth muscle bundles, which therefore can be considered as the targets of the efferent nerve supply.The complex pattern of the innervation herein demonstrated suggests the existence of a nervous control of valvular function through the regulation of contractile elements.     

Development of mouse semilunar valves

Summary The development of the semilunar valves of the great arteries of the mouse is studied by light and scanning electron microscopy. The earliest anlage of the valves is observed at day 11.5 of gestation, as three pairs of tubercles protruding towards the lumen of the truncus arteriosus. These early cusps consist of a core of mesenchymal tissue covered by the endocardium. From day 12.5 to 14 a process of excavation takes place in the arterial face of the cusps resulting in the achievement of their final shape. Our observations suggest that the excavation process is produced by a selective growth of the free edges of the cusps. Numerous pieces of evidence are reported suggesting that the selective growth of the cusps is due to an inductive interaction between the endocardium of the arterial face of the cusps and the underlying mesenchyme.The histogenesis of the cusps takes place very late in development and possibly continues into the postnatal period.The results are compared with studies made in other vertebrates.     

Amyloid substance within stenotic aortic valves promotes mineralization

Audet A, Côté N, Couture C, Bossé Y, Després J‐P, Pibarot P & Mathieu P
(2012) Histopathology  61, 610–619 Amyloid substance within stenotic aortic valves promotes mineralization Aims: Accumulation of apolipoproteins may play an important role in the pathobiology of calcific aortic valve disease (CAVD). We aimed to explore the hypothesis that apolipoprotein‐derived amyloid could play a role in the development of CAVD. Methods and results: In 70 explanted CAVD valves and 15 control non‐calcified aortic valves, we assessed the presence of amyloid by using Congo red staining. Immunohistochemistry was performed to document the presence of apolipoprotein AI (Apo‐AI). Apoptosis was documented by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) studies performed in control and CAVD valves. Control valves were free of amyloid. Deposition of amyloid was detected in all CAVD valves, and the amount was positively correlated with plasma high‐density lipoprotein and Apo‐AI levels. Apo‐AI within CAVD valves co‐localized with intense staining of fibrillar amyloid. In turn, deposition of amyloid co‐localized with apoptosis near mineralized areas. Isolation of amyloid fibrils confirmed that Apo‐AI is a major component of amyloid deposits in CAVD. In vitro, CAVD‐derived amyloid extracts increased apoptosis and mineralization of isolated aortic valvular interstitial cells. Conclusions: Apo‐AI is a major component of amyloid substance present within CAVD valves. Furthermore, amyloid deposits participate in mineralization in CAVD by promoting apoptosis of valvular interstitial cells.     

Classification of amyloid deposits in diagnostic cardiac specimens by immunofluorescence

BackgroundAt least 12 distinct forms of amyloidosis are known to involve the heart or great vessels. Patient treatment regimens require proper subtyping of amyloid deposits in small diagnostic cardiac specimens. A growing lack of confidence in immunohistochemical staining for subtyping amyloid has arisen primarily as a result of studies utilizing immunoperoxidase staining of formalin-fixed paraffin-embedded tissue. Immunofluorescence staining on fresh frozen tissue is generally considered superior to immunoperoxidase staining for subtyping amyloid; however, this technique has not previously been reported in a series of cardiac specimens.MethodsAmyloid deposits were subtyped in 17 cardiac specimens and 23 renal specimens using an immunofluorescence panel.ResultsAmyloid deposits were successfully subtyped as AL, AH, or AA amyloid by immunofluorescence in 82% of cardiac specimens and 87% of renal specimens. In all cases, the amyloid classification was in good agreement with available clinical and laboratory assessments. A cross-study analysis of 163 cases of AL amyloidosis reveals probable systemic misdiagnosis of cardiac AL amyloidosis by the immunoperoxidase technique, but not by the immunofluorescence technique.ConclusionsAmyloid deposits can be reliably subtyped in small diagnostic cardiac specimens using immunofluorescence. The practical aspects of implementing an immunofluorescence approach are compared with those of other approaches for subtyping amyloid in the clinical setting.     

人类纤维化心脏瓣膜噬菌体表达文库的构建及质量分析

目的: 各种病因引起的心脏瓣膜纤维化是导致心脏瓣膜功能丧失的主要原因,样本来源有限和不可重获性是其分子机制研究的瓶颈。本研究旨在成功获取患者纤维化瓣膜的遗传信息物质,为探明瓣膜病变的分子机制提供直接证据。方法: 心脏换瓣手术中取纤维化瓣膜组织,提取高质量RNA,用SMART技术合成cDNA第1链,再用长距离PCR合成双链cDNA,经 Sfi I酶切后过柱分级收集长片段cDNA,经T4 DNA连接酶催化连接入λTriplEx2载体,最后经λ噬菌体包装后构建成原始文库,用PCR、X-gal等方法进行文库质量鉴定。 结果: 成功构建了纤维化心脏瓣膜噬菌体表达文库,原始文库滴度为8× 10⁹ pfu/L,重组率为99%,外源基因片段大小在500到3 000 bp之间,其中81.25%片段大于1 000 bp。 结论: 成功构建了高质量的人类纤维化心脏瓣膜噬菌体表达文库,该文库的库容量足够代表人类基因的复杂性,高重组率能保障功能筛选的高效性,长片段更有助于全长基因的获得和表达。     

Artificial neural networks in computer-assisted classification of heart sounds in patients with porcine bioprosthetic valves

The paper describes the design, training and testing of a three-layer feedforward back-propagation neural network for the classification of bioprosthetic valve closure sounds. Forty-seven patients with a porcine bioprosthetic valve inserted in the aortic position were involved in the study. Twenty-four of them had a normal bioprosthetic valve, and the other 23 had a degenerated valve. Five features extracted from the Fourier spectra and 12 linear predictive coding (LPC) coefficients of the sounds were used separately as the input of two neural-network classifiers. The performance of the classifiers was tested using the leave-one-out method. Results show that correct classifications were 85 per cent using the spectral features, and 89 per cent using the LPC coefficients. The study confirms the potential of artificial networks for the classification of bioprosthetic valve closure sounds. Clinical use of this method, however, still requires further investigation.     

Differences in spectral composition between monostrut Bjork-Shiley and Carbomedics valves implanted in the aortic postion

The paper investigates the differences, in spectral composition between monostrut Bjork-Shiley and Carbomedics valves implanted in the aortic position using a modified overdetermined forward-backward Prony’s method. Spectral composition of the closing sounds produced by these two valves are compared in terms of the e number of spectral components and their relative energy. Results show that, for both valves, the major spectral components are concentrated below 2 kHz. In the case of the Carbomedics valve, it is found that 97% of the total energy lies below 120 Hz where the sensitivity of the human auditory system is poor. This is one of the main reasons why the Carbomedics valve is acoustically quieter than a monostrut Bjork-Shiley valve when in operation. In addition, a clear difference monostrut and Carbomedics valves has also been found in the 400 Hz-2 kHz sub-band. A distinct difference between normal and malfunctioning cases of each prostheses has been found in the frequency components beyond, 400 Hz. In the malfunctioning cases the frequency components in that region move to the lower regions of the spectrum and a large part of the energy is concentrated between 250 and 400 Hz.     

Isolated amyloidosis of the atrioventricular valves

Summary Autopsy of a 55-year-old man, whose death was due to coronary failure, revealed: 1) localized amyloidosis limited to the leaflets of the atrioventricular valves and their chordae tendinae, with no apparent relationship to atherosclerosis, and 2) deposits of crystallized vegetable hydrocarbons of dietary origin, mainly in the lungs, lymph nodes and liver.Observations of isolated amyloidosis in healthy valves are exceptional. The association with multi-organ deposition of vegetable wax hydrocarbons raises the question of a possible link between these two entities.Aldomet MSD     

Amyloid in surgical pathology

Amyloid is defined as a proteinaceous tissue deposit that shows a typical green birefringence in polarized light after staining with Congo red, the presence of non-branching linear fibrils of indefinite length with a mean diameter of 10 nm, and a distinct X-ray diffraction pattern consistent with Pauling's model of a cross -fibril. Amyloid may deposit locally or may present as a systemic disease. The origin of amyloid is diverse: 25 different fibril proteins have been described so far. The precursor proteins differ from each other in their primary structures and functions. The only common denominator is the propensity to form anti-parallel cross -fibrils under certain circumstances. Early diagnosis of amyloid is still a major challenge in surgical pathology. Histological proof can be obtained using Congo-red staining and polarization microscopy. However, small deposits may be difficult to discern, and sensitivity can be improved using fluorescence microscopy. Classification of amyloid is mandatory, since amyloid is treatable and different treatment regimens are applied to different amyloid diseases. This review focuses on the epidemiology, clinical features, pathology and diagnosis of amyloid in surgical pathology.     

Malformations of the semilunar valves produced in chick embryos by mechanical interference with cardiogenesis

Summary The purpose of the present work was to analyze the role of hemodynamics in the morphogenesis and histogenesis of the semilunar valves. To achieve this goal we have studied the development of the chick semilunar valves in conditions of abnormal local flow. To obtain an abnormal pattern of local flow we have induced alterations of the cardiac septation process by mechanical interference of the development of the conus cordis. The malformations obtained by this procedure consisted of a spectrum of alterations in the process of incorporation of the aortic conus into the left ventricle. These malformations ranged from a simple widening of the outflow tract of the left ventricle to severe forms of double-outlet right ventricle and ventricular septal defects. Malformations of the semilunar valves consisting of extensive thickening of the leaflets and lack of maturation of the valve tissues were very often present in the malformed hearts. The malformation of the valve leaflets was more frequent and severe in the aortic valve at more advanced stages of development and in the hearts showing more severe alteration of the septation process. The absence of alterations in the semilunar valves of the control embryos and in the experimental embryos without alteration of the cardiac septation suggest a close relationship between the semilunar valves anomalies and the hemodynamic alterations present in the malformed hearts.     

Cell shape and cytoskeletal organization of the endothelial cells of the semilunar heart valves in the developing chick

Summary The composition and arrangement of the cytoskeletal elements of the endothelium of the semilunar valves have been studied in the embryonic chick heart during the stages 30 to 38. Microtubules, vimentin intermediate filaments and actin microfilaments were constant components of the valvular endothelial cells in the studied stages. Scanning electron microscopy after Triton-X-100 extraction revealed significant differences in the tridimensional arrangement of the cytoskeleton in the course of valve development. In the ventricular face of the cusps the cytoskeletal elements displayed a progressive longitudinal alignment, while in the arterial face of the cusps the cytoskeleton maintained the appearance of a network. Transmission electron microscopy revealed that these differences were especially prominent for vimentin intermediate filaments, although a similar tendency was also observed for microtubules. Microfilaments were scarce in the endothelial cells of both faces of the cusps, and the stress fibers typical of the endothelial cells of the adult valves were not observed in the embryonic material. The significance of these results in valve morphogenesis and histogensis and a possible linkage with differences in the local characteristics of the blood flow are discussed.     

Hyaluronan turnover and hypoxic brown adipocytic differentiation are co-localized with ossification in calcified human aortic valves

The calcification process in aortic stenosis has garnered considerable interest but only limited investigation into selected signaling pathways. This study investigated mechanisms related to hypoxia, hyaluronan homeostasis, brown adipocytic differentiation, and ossification within calcified valves. Surgically explanted calcified aortic valves (n = 14) were immunostained for markers relevant to these mechanisms and evaluated in the center (NodCtr) and edge (NodEdge) of the calcified nodule (NodCtr), tissue directly surrounding nodule (NodSurr); center and tissue surrounding small “prenodules” (PreNod, PreNodSurr); and normal fibrosa layer (CollFibr). Pearson correlations were determined between staining intensities of markers within regions. Ossification markers primarily localized to NodCtr and NodEdge, along with markers related to hyaluronan turnover and hypoxia. Markers of brown adipocytic differentiation were frequently co-localized with markers of hypoxia. In NodCtr and NodSurr, brown fat and ossification markers correlated with hyaluronidase-1, whereas these markers, as well as hypoxia, correlated with hyaluronan synthases in NodEdge. The protein product of tumor necrosis factor-α stimulated gene-6 strongly correlated with ossification markers and hyaluronidase in the regions surrounding the nodules (NodSurr, PreNodSurr). In conclusion, this study suggests roles for hyaluronan homeostasis and the promotion of hypoxia by cells demonstrating brown fat markers in calcific aortic valve disease.     

Scanning and light microscope studies of the development of the chick embryo semilunar heart valves

Summary The development of the semilunar valves of the great arteries was studied by light and scanning electron microscopy in the chick embryo. The results show that three distinct developmental periods can be distinguished. The formation of the anlage of the valves takes place in the first period (stages 26–29). These early anlage consist of three pyramidal shaped cusps formed by a core of loosely packed mesenchymal cells covered by a flattened endothelium. In the second period (stages 30–35) the cusps undergo excavation on their distal face. Morphological evidence is reported suggesting that this excavation process is produced by an initial solid ingrowth of the endothelium of the arterial face of the cusps which is immediately luminated by detachment of cells towards the bloodstream and by cell death. The histogenesis of the valves takes place in the third period (from stage 36 until hatching). It was observed that during this period some myocardial cells of the outflow tracts of the ventricles invade the valvular tissue and that in the upper part of the cusps a prominent fibrous layer is formed.     

聚乙二醇改性去细胞猪主动脉瓣膜构建复合支架及其体外细胞毒性试验

目的 对去细胞猪主动脉瓣膜进行聚乙二醇(PEG)化改性,并评价改性后复合支架的细胞毒性.方法 合成功能基团为丙烯酰基的枝化状聚乙二醇衍生物,在去细胞猪主动脉瓣膜引入巯基,通过迈克尔加成反应完成PEG对去细胞猪主动脉瓣膜的交联,并作PEG改性前后瓣膜的生物力学测定.制作去细胞瓣膜的浸提液,浸提液分为单纯去细胞瓣膜组、PEG改性去细胞瓣膜组和阴性对照组(培养液),采用CCK-8法检测复合支架的浸提液对人脐静脉内皮细胞增殖率的影响,评价其细胞毒性.结果 PEG改性去细胞瓣膜反应条件温和,改性效果确切.复合瓣膜支架的抗拉强度明显高于去细胞瓣膜[(7.53±0.29)MPa比(5.65±0.24)MPa,P＜0.05],但与天然瓣膜[(7.68±0.20)MPa]差异无统计学意义(P＞0.05).复合支架和去细胞瓣膜毒性评级均0级,与阴性对照组差异无统计学意义(P＞0.05).人脐静脉内皮细胞经各浸提液的培养液培养后形态良好,增殖旺盛.结论 PEG改性能明显改善去细胞猪主动脉瓣膜的力学性能,且改性后的复合支架无细胞毒性,为进一步研究提供依据.     

Developmental aspects of the sinus valves and the sinus venosus septum of the right atrium in human embryos

Summary In 32 human embryos from 5 to 27 mm of length, stages 13 to 23 (according to the Carnegie system of stages), the contributions of the sinus venosus septum and the right sinus valve of the right atrium to the formation of the Eustachian and Thebesian valve were examined by scanning electron microscopy. The sinus septum takes part in the subdivision of the right sinus valve into the Eustachian and the Thebesian valves. From its first origin the sinus septum forms a septal structure between the orifices of the right hepatic vein (hepatic portion of the inferior caval vein), the precursor of the inferior caval vein, and the left horn of the sinus venosus, the precursor of the coronary sinus. Before the incorporation of the sinus venosus into the right atrium, it has an intra-sinusal position, and extends between the bases of the left and the right sinus valve. During the incorporation of the sinus venosus into the right atrium the sinus septum receives an intra-atrial position, and its positional relationships to the sinus valves and the orifices of the corresponding veins remain unchanged in principle. Due to the connection between the sinus septum and the right sinus valve, after completion of the incorporation of the sinus, the superior portion of the right sinus valve branches y-like into a lateral limb, (i.e. its original inferior portion) and into a medial limb, (i.e. the sinus septum). Thus, the Eustachian valve is formed by the superior portion of the right sinus valve and the sinus septum; whereas the Thebesian valve is formed by the inferior portion of the right sinus valve. The normal variations in the configuration of the Eustachian and the Thebesian valves are supposed to be caused by differential growth of the constituents of the different portions of the valves. This work was supported by the Stiftung Volkswagenwerk (I/63 363). Gratefully dedicated to Professor Erich Blechschmidt on the occasion of his 85th birthday