elastin and Calcinosis

Calcinosis cutis, defined as sub-epidermal deposition of calcium salts, is a major clinical problem in patients with SSc, affecting 20-40% of patients. A number of recognized factors associated with calcinosis have been identified, including disease duration, digital ischaemia and acro-osteolysis. Yet, to date, the pathogenesis of SSc-related calcinosis remains unknown, and currently there is no effective disease-modifying pharmacotherapy. Following onset of SSc, there are marked changes in the extracellular matrix (ECM) of the skin, notably a breakdown in the microfibrillar network and accumulation of type I collagen. Our hypothesis is that these pathological changes reflect a changing cellular phenotype and result in a primed microenvironment for soft tissue calcification, with SSc fibroblasts adopting a pro-osteogenic profile, and specific driving forces promoting tissue mineralization. Considering the role of the ECM in disease progression may help elucidate the mechanism(s) behind SSc-related calcinosis and inform the development of future therapeutic interventions.

Topics: Calcinosis; Cell Differentiation; Cell Hypoxia; Cellular Microenvironment; Collagen Type I; Disease Progression; Elastin; Extracellular Matrix; Fibrillin-1; Fibroblasts; Glucose Transporter Type 1; Humans; Mesenchymal Stem Cells; Myofibroblasts; Osteoblasts; Osteogenesis; Osteolysis; Phenotype; Phosphates; Scleroderma, Systemic; Skin; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Vascular calcification is an independent risk factor for the development of cardiovascular disease and is classified into two types based on the site of calcification : intimal atherosclerotic calcification and Mönckeberg's medial calcification. Matrix vesicles released from macrophages and vascular smooth muscle cells (VSMC) during apoptosis play a pivotal role in formation of fine granular calcification, while osteogenic differentiation of VSMC contributes to progression of advanced calcification. Recent noninvasive imaging studies of atherosclerotic calcification provide robust evidence that inflammation precedes active calcification, leading to establish the inflammation-dependent calcification paradigm. On the other hand, elastin degradation by increased elastolytic activities and disturbance of regulatory systems of extracellular pyrophosphate metabolism play an important role in development of Mönckeberg's medial calcification.

Topics: Animals; Apoptosis; Calcinosis; Cardiovascular Diseases; Cellular Senescence; Diphosphates; Elastin; Extracellular Matrix; Humans; Macrophages; Mice; Muscle, Smooth, Vascular; Osteogenesis; Risk Factors; Vascular Calcification

Calcific aortic stenosis is a progressive disease characterized by massive fibrosis andmineralization of the valve leaflets. The aim of this study was to determine whether the onset of native calcific aortic stenosis is associated primarily with matrix remodelling events, and particularly with elastin degradation.. The immunohistochemical expression profile of matrix degradating enzymes and tenascin-C was investigated in both healthy and native calcified aortic valves. Collagen and elastic tissue were studied by light microscopy and electron microscopy. Immunophenotypic analysis of inflammatory cells was carried out by using monoclonal antibodies to macrophages, T and B lymphocytes. Immunoreactivity for tenascin-C and matrix metalloproteinase-12 (MMP-12) was associated with areas of dense mineralization, which were characterized by fibrosis, fragmentation and calcification of elastic fibres a positive reaction was also found around small islands of calcification. MMP-11 was not detected in the diseased valves. Osteopontin and osteonectin were also found at sites of mineralization. All calcified valves examined showed inflammatory cell infiltration.. Our results demonstrate the direct involvement of MMP-12 in native aortic valve stenosis. MMP-mediated degradation of elastic fibres might contribute actively to valve mineralization by inducing calcium deposition onto fragmented elastin.

Topics: Aged; Aged, 80 and over; Aortic Valve; Aortic Valve Stenosis; Calcinosis; Elastin; Female; Humans; Immunohistochemistry; Inflammation; Male; Matrix Metalloproteinase 12; Microscopy, Electron, Transmission; Middle Aged; Osteonectin; Osteopontin; Tenascin

Disturbances in bone and mineral metabolism in patients with chronic kidney disease (CKD) affect not only the bone diseases but also other organ disorders in the whole body and deteriorate the survival of these patients. The term CKD-Mineral and Bone Disorder (CKD-MBD) has been established describing a broader clinical syndrome that develops as a systemic disorder of mineral and bone metabolism due to CKD. Vascular calcification and secondary hyperparathyroidism are major diseases in CKD-MBD.

Topics: Apoptosis; Bone Density; Bone Diseases, Metabolic; Calcinosis; Chronic Disease; Elastin; Humans; Hyperparathyroidism, Secondary; Kidney Diseases; Muscle, Smooth, Vascular; Receptors, Calcitriol; Receptors, Calcium-Sensing; Vascular Diseases

Vascular calcification is an active process similar to physiological mineralization of skeletal tissues. Not only apoptosis, cellular senescence, and osteochondrogenic transdifferentiation of vascular smooth muscle cells, but also degeneration and degradation of elastin may play an important role in its developmental process. Vascular calcification is induced by the interactions among various regulatory molecules for calcification in the progression of atherosclerosis or arteriosclerosis.

Topics: Alkaline Phosphatase; Animals; Apoptosis; Atherosclerosis; Calcinosis; Cytokines; Disease Progression; Elastin; Extracellular Matrix; Humans; Mice; Muscle, Smooth, Vascular; Osteopontin; Oxidative Stress; Phosphates; RANK Ligand; Vascular Diseases

Vascular calcification occurred as the last step of arteriosclerosis makes a lot of disturbances on vascular function and should influence on the worsening of the vascular diseases. Calcium is the main component of the vascular calcification like bone, and one of causes of vascular calcification should be the hypoparathyroidism due to the lowering of serum calcium and the following calcium paradox seen in osteoporosis. Bone calcium must shift to the arterial wall from the bone. Medial calcification could be formed under the molecular regulatory control like in bone by differentiated osteoblast or chondroblast from pericyte like cell origin smooth muscle cell. Many substances such as osteopontine, osteocalcine, bone morphogenetic protein 2, matrix Gla protein and alkaliphosphatase were found in calcified area. In intimal calcification, degenerated elastin and macrophage originated calcification were found. In the process of degeneration of elastin polypentapeptide structure in elastin can be easily conbined to Ca(2+), elastin-Ca(2+) complex is neutralized by PO4(2-) and calcium phosphate is accumulated in degenerated elastin.

Topics: Alkaline Phosphatase; Aortic Diseases; Arteriosclerosis; Bone Morphogenetic Protein 2; Calcinosis; Calcium; Calcium-Binding Proteins; Elastin; Extracellular Matrix Proteins; Humans; Hypoparathyroidism; Macrophages; Matrix Gla Protein; Osteocalcin; Osteopontin; Osteoporosis

Animal models of large artery wall stiffness fall into two categories: firstly those that slowly develop multifactorial vascular dysfunction spontaneously, such as the ageing rat. The second type of model consists of those in which a specific pathology is induced by surgical, chemical, or genetic means. Such models are based on a short-term, highly traumatic insult to the arterial wall of a young animal and its acute reaction to such insult. This is very different from the human situation in which changes in wall stiffness arise from the long-term accumulation of relatively minor episodes of vascular insult in the vulnerable elderly.

Topics: Animals; Arteries; Arteriosclerosis; Calcinosis; Collagen; Disease Models, Animal; Elastin; Humans; Hypertension; Neurotransmitter Agents; Pancreatic Elastase; Renin-Angiotensin System; Vascular Resistance

Conduit arteries become stiffer with age due to alterations in their morphology and the composition of the their major structural proteins, elastin and collagen. The elastic lamellae undergo fragmentation and thinning, leading to ectasia and a gradual transfer of mechanical load to collagen, which is 100-1000 times stiffer than elastin. Possible causes of this fragmentation are mechanical (fatigue failure) or enzymatic (driven by matrix metallo proteinases (MMP) activity), both of which may have genetic or environmental origins (fetal programming). Furthermore, the remaining elastin itself becomes stiffer, owing to calcification and the formation of cross-links due to advanced glycation end-products (AGEs), a process that affects collagen even more strongly. These changes are accelerated in the presence of disease such as hypertension, diabetes and uraemia and may be exacerbated locally by atherosclerosis. Raised MMP activity, calcification and impaired endothelial function are also associated with a high level of plasma homocysteine, which itself increases with age. Impaired endothelial function leads to increased resting vascular smooth muscle tone and further increases in vascular stiffness and mean and/or pulse pressure. The effect of increased stiffness, whatever its underlying causes, is to reduce the reservoir/buffering function of the conduit arteries near the heart and to increase pulse wave velocity, both of which increase systolic and pulse pressure. These determine the peak load on the heart and the vascular system as a whole, the breakdown of which, like that of any machine, depends more on the maximum loads they must bear than on their average. Reversing or stabilising the increased arterial stiffness associated with age and disease by targeting any or all of its causes provides a number of promising new approaches to the treatment of systolic hypertension and its sequelae, the main causes of mortality and morbidity in the developed world.

Topics: Aging; Arteries; Blood Pressure; Calcinosis; Elasticity; Elastin; Endothelium, Vascular; Fetal Development; Glycation End Products, Advanced; Homocysteine; Humans; Matrix Metalloproteinases; Stress, Mechanical

Topics: Aging; Arteriosclerosis; Blood Pressure; Calcinosis; Cholesterol; Collagen; Diastole; Elastin; Endothelium, Vascular; Humans; Myocardial Infarction

Vascular calcification occurs at two distinct sites within the vessel wall: the intima and the media. Intimal calcification occurs in the context of atherosclerosis, associated with lipid, macrophages and vascular smooth muscle cells, whereas medial calcification can exist independently of atherosclerosis and is associated with elastin and vascular smooth muscle cells.. In this review we compare intimal and medial calcification, particularly discussing the mechanisms which may be responsible for each type of calcification. Similar mechanisms probably initiate and regulate both forms of calcification including the generation of matrix vesicles/apoptotic bodies and local expression of mineralization-regulating proteins. However, since different modifying agents such as lipids in the intima and elastin in the media are present at the sites of calcification and are associated with particular diseases, this implies that the etiologies of these processes differ. For example, intimal calcification is associated with atherosclerosis while medial calcification occurs commonly in the diabetic neuropathic leg.. Since both types of calcification correlate with significant morbidity and mortality, we discuss the different types of calcification in terms of their clinical importance.

Topics: Adult; Age Factors; Aged; Animals; Apoptosis; Arteriosclerosis; Calcinosis; Cells, Cultured; Coronary Artery Disease; Diabetic Neuropathies; Elastin; Endothelium, Vascular; Gene Expression; Humans; Lipid Metabolism; Mice; Mice, Knockout; Muscle, Smooth, Vascular; Phenotype; Risk Factors; Tunica Intima

In studies concerning risk factors for cardiovascular diseases, a number of reports have emphasized the influence of lipids, but the role of dietary minerals other than sodium has been less studied. However, epidemiological studies have suggested that dietary intake of magnesium and potassium may be involved in such pathogenesis. Studies of the influence of magnesium deficiency on arteriosclerosis include its effect on the initial lesion, altered metabolism of elastin, proliferation of collagen, calcification, lipid metabolism, platelet aggregation and hypertension. Magnesium and potassium metabolism are closely related and magnesium is required for maintaining the level of cellular potassium. As a consequence, magnesium and potassium deficiency frequently occur together and potassium deficiency may be an aggravating factor in pathogenesis. The development of the initial lesion in the arterial wall may be facilitated by loss of cellular magnesium and potassium. Experimental magnesium deficiency induces arterial damage, a loss of magnesium and potassium and an increase in the calcium and sodium content of the cell. Experimental models that have been used to produce cardiovascular lesions induce similar changes and losses of major intracellular cations may affect the main metabolic processes of the cell. This report summarizes the experimental evidence that magnesium deficiency may affect several different stages involved in arteriosclerosis and that potassium deficiency may exacerbate this. Magnesium deficiency results in vascular calcification. Experiments indicate that elastin is the site of the initial calcification and the metabolism of elastin is altered. This vascular lesion then brings about an increase in the collagen content of the wall. Low magnesium status could probably affect this process by slowing collagen resorption and lead to an irreversible accumulation of connective tissue. Results showing a different distribution of the various types of lipoprotein during experimental magnesium deficiency strongly suggest that lipid exchange between the vessel walls and blood can be modified. Severe magnesium deficiency in weanling rats produces a marked hypertriglyceridemia, a decrease in the percentage of cholesterol transported by HDL lipoprotein and a reduction in LCAT activity. The decreased clearance of circulatory triglycerides appears to be the major mechanism contributing to hyperlipemia. Magnesium deficiency could therefore contribute to accu

Topics: Animals; Arteriosclerosis; Blood Platelets; Calcinosis; Collagen; Diabetes Mellitus; Diet; Elastin; Humans; Hypertension; Lipid Metabolism; Lipoproteins; Magnesium; Magnesium Deficiency; Muscle, Smooth, Vascular; Potassium; Potassium Deficiency; Rats; Thrombosis

Topics: Aging; Amino Acids; Animals; Aorta, Thoracic; Arteriosclerosis; Calcinosis; Cattle; Chemical Phenomena; Chemistry; Chickens; Dogs; Elastin; Extracellular Space; Hot Temperature; Humans; Macromolecular Substances

Topics: Arteries; Arteriosclerosis; Calcinosis; Calcium; Elastin; Glycoproteins; Humans; Lipid Metabolism; Macromolecular Substances; Myofibrils; Pancreatic Elastase

Topics: Aged; Aging; Amino Acids; Aorta; Arteriosclerosis; Calcinosis; Calcium; Chemical Phenomena; Chemistry; Child; Elastic Tissue; Elastin; Fluorescence; Glycosaminoglycans; Humans; Lipids; Pulmonary Artery

Topics: Alkaline Phosphatase; Biochemical Phenomena; Biochemistry; Calcification, Physiologic; Calcinosis; Calcium; Cartilage; Collagen; Elastin; Glycosaminoglycans; Humans; Lipoproteins; Mucoproteins; Phosphates; Physiology

Topics: Amino Acids; Aorta; Arteries; Arteriosclerosis; Autoradiography; Calcinosis; Chemical Phenomena; Chemistry; Cholesterol; Clinical Trials as Topic; Elastin; Humans; Hydrolysis; In Vitro Techniques; Lipid Metabolism; Lipoproteins; Proteins; Tritium

The causes of heart valve bioprosthetic calcification are still not clear. In this paper, we compared the calcification in the porcine aorta (Ao) and the bovine jugular vein (Ve) walls, as well as the bovine pericardium (Pe). Biomaterials were crosslinked with glutaraldehyde (GA) and diepoxide (DE), after which they were implanted subcutaneously in young rats for 10, 20, and 30 days. Collagen, elastin, and fibrillin were visualized in non-implanted samples. Atomic absorption spectroscopy, histological methods, scanning electron microscopy, and Fourier-transform infrared spectroscopy were used to study the dynamics of calcification. By the 30th day, calcium accumulated most intensively in the collagen fibers of the GA-Pe. In elastin-rich materials, calcium deposits were associated with elastin fibers and localized differences in the walls of Ao and Ve. The DE-Pe did not calcify at all for 30 days. Alkaline phosphatase does not affect calcification since it was not found in the implant tissue. Fibrillin surrounds elastin fibers in the Ao and Ve, but its involvement in calcification is questionable. In the subcutaneous space of young rats, which are used to model the implants' calcification, the content of phosphorus was five times higher than in aging animals. We hypothesize that the centers of calcium phosphate nucleation are the positively charged nitrogen of the pyridinium rings, which is the main one in fresh elastin and appears in collagen as a result of GA preservation. Nucleation can be significantly accelerated at high concentrations of phosphorus in biological fluids. The hypothesis needs further experimental confirmation.

Topics: Animals; Bioprosthesis; Calcinosis; Calcium; Cattle; Collagen; Elastin; Glutaral; Heart Valve Diseases; Heart Valve Prosthesis; Pericardium; Phosphorus; Rats; Swine

Patients with thoracic aortopathy are at increased risk of catastrophic aortic dissection, carrying with it substantial mortality and morbidity. Although granular medial calcinosis (medial microcalcification) has been associated with thoracic aortopathy, its relationship to disease severity has yet to be established.. One hundred one thoracic aortic specimens were collected from 57 patients with thoracic aortopathy and 18 control subjects. Standardized histopathologic scores, immunohistochemistry, and nanoindentation (tissue elastic modulus) were compared with the extent of microcalcification on von Kossa histology and 18F-sodium fluoride autoradiography.. Microcalcification content was higher in thoracic aortopathy samples with mild (n=28; 6.17 [2.71-10.39];. Medial microcalcification is a marker of aortopathy, although progression to severe aortopathy is associated with loss of both elastin fibers and microcalcification.

Topics: Aorta; Calcinosis; Elastin; Humans; Severity of Illness Index; Sodium Fluoride

Calcinosis cutis is defined as abnormal deposition of calcium salts in the skin and subcutaneous tissues. Dystrophic calcification, the most common form of calcinosis cutis, is associated with autoimmune connective tissue diseases. This condition is associated with severe pain and can affect the patient's quality of life and lead to long-term disability. Treatment is often challenging, and there is a very limited evidence base for potential treatments of calcinosis cutis associated with systemic sclerosis and dermatomyositis. Inkless tattoo is very similar to microneedling, a minimally invasive procedure stimulating the wound-healing cascade contributing to elastin and collagen formation as well as neovascularization. This technique has not been reported as a potential therapeutic option for calcinosis cutis. Here, we present a patient with calcinosis cutis in the setting of dermatomyositis that responded dramatically to inkless tattoo application. Our results support the need for future studies of microneedling in patients with this disorder.

Topics: Calcinosis; Calcium; Collagen; Dermatomyositis; Elastin; Humans; Quality of Life; Salts; Skin Diseases; Tattooing

Medial calcification has been associated with diabetes, chronic kidney disease, and genetic disorders like pseudoxanthoma elasticum. Recently, we showed that genetic reduction of arterial elastin content reduces the severity of medial calcification in matrix Gla protein (MGP)-deficient and Eln haploinsufficient Mgp-/-;Eln+/- mice. This study suggests that there might be a direct effect of elastin amount on medial calcification. We studied this using novel in vitro systems, which are based on elastin or elastin-like polypeptides. We first examined the mineral deposition properties of a transfected pigmented epithelial cell line that expresses elastin and other elastic lamina proteins. When grown in inorganic phosphate-supplemented medium, these cells deposited calcium phosphate minerals, which could be prevented by an N'-terminal peptide of MGP (m3pS) carrying phosphorylated serine residues. We next confirmed these findings using a cell-free elastin-like polypeptide (ELP

Topics: Calcinosis; Calcium-Binding Proteins; Elastin; Extracellular Matrix Proteins; Humans; Matrix Gla Protein; Minerals; Peptides

Despite its long half-life and physiological role, elastin undergoes irreversible changes (i.e elastolysis and/or calcification) impairing resilience of soft connective tissues. At present, it is still undefined: 1) to which extent elastin fibers have to be fragmented in order to increase their susceptibility to calcify; 2) which is the contribution of ionic environment on elastin mineralization; 3) why, in the same tissue area, mineralized coexist with non-mineralized fibers. The in vitro mineralization process was investigated on insoluble elastin, hydrolyzed or not-hydrolyzed, and incubated in different cell-free ionic environments. Mineral deposition is favored on hydrolyzed fibrillar structures due to exposure of multiple charged sites increasing the adsorption of Ca

Topics: Animals; Biodegradation, Environmental; Calcification, Physiologic; Calcinosis; Calcium; Caseins; Cattle; Durapatite; Elastin; Humans; Hydrolysis; Ions; Microscopy, Electron, Scanning; Spectrometry, X-Ray Emission; X-Ray Diffraction

This study evaluated the ability of bisphosphonates (BPAs) of different molecular structures to mitigate the calcification of porcine aortic wall (PAW) and bovine jugular vein wall (BJVW). Tissues cross-linked with glutaraldehyde (GA) or diepoxide (DE) were modified with pamidronic acid (PAM), alendronic acid (ALE), neridronic acid (NER) (type 1 BPAs); 2-(2'-carboxyethylamino)ethylidene-1,1-bisphosphonic acid (CEABA), 2-(5-carboxypentylamino)ethylidene-1,1-bisphosphonic acid (CPABA) (type 2); and zoledronic acid (ZOL) (type 3). After implanting the tissue samples subcutaneously in 100 rats, calcification was examined using atomic absorption spectrophotometry (60-day explants) and light microscopy after von Kossa staining (10- and 30-day explants). The calcium contents in GA-BJVW and GA- and DE-PAW increased up to 100-120 mg/g after 60 days, while being 3 times lower in DE-BJVW. In modified and nonmodified PAW samples, calcium phosphates appeared by day 10 and were associated with elastic fibers and devitalized cellular elements. In all groups of BJVW samples, mineralization began in elastic fibers near the subendothelial layer. In addition, calcified collagen was found in the GA-BJVW samples. Minimal calcification was found in GA-PAW treated with type 1 BPAs and CEABA. For DE-PAW and GA-BJVW, the calcium level significantly decreased with PAM and CEABA. Meanwhile, ALE and NER were effective for DE-BJVW.

Topics: Animals; Biocompatible Materials; Bioprosthesis; Calcinosis; Cattle; Diphosphonates; Elastin; Swine

Pore size is generally small in nanofibrous scaffolds prepared by electrospinning polymeric solutions. Increase of scaffold thickness leads to decrease in pore size, causing impediment to cell infiltration into the scaffolds during tissue engineering. In contrast, comparatively larger pore size can be realized in microfibrous scaffolds prepared from polymeric solutions at higher concentrations. Further, microfibrous scaffolds are conducive to infiltration of reparative M2 phenotype macrophages during in vivo/in situ tissue engineering. However, rise of mechanical properties of a fibrous scaffold with the increase of polymer concentration may limit the functionality of a scaffold-based, tissue-engineered heart valve. In this study, we developed microfibrous scaffolds from 14%, 16% and 18% (wt/v) polycaprolactone (PCL) polymer solutions prepared with chloroform solvent. Porcine valvular interstitial cells were cultured in the scaffolds for 14 d to investigate the effect of microfibers prepared with different PCL concentrations on the seeded cells. Further, fresh microfibrous scaffolds were implanted subcutaneously in a rat model for two months to investigate the effect of microfibers on infiltrated cells. Cell proliferation, and its morphologies, gene expression and deposition of different extracellular matrix proteins in the in vitro study were characterized. During the in vivo study, we characterized cell infiltration, and myofibroblast and M1/M2 phenotypes expression of the infiltrated cells. Among different PCL concentrations, microfibrous scaffolds from 14% solution were suitable for heart valve tissue engineering for their sufficient pore size and low but adequate tensile properties, which promoted cell adhesion to and proliferation in the scaffolds, and effective gene expression and extracellular matrix deposition by the cells in vitro. They also encouraged the cells in vivo for their infiltration and effective gene expression, including M2 phenotype expression.

Topics: Animals; Aortic Valve; Aortic Valve Stenosis; Calcinosis; Cell Adhesion; Cell Proliferation; Cells, Cultured; Collagen; Elastin; Extracellular Matrix; Glycosaminoglycans; Mesenchymal Stem Cells; Nanofibers; Polyesters; Polymers; Rats; Rats, Sprague-Dawley; Stress, Mechanical; Swine; Tensile Strength; Tissue Engineering; Tissue Scaffolds; Vimentin

Glutaraldehyde fixed xenogeneic heart valve prosthesis are hindered by calcification and lack of growth potential. The aim of tissue decellularization is to remove tissue antigenicity, avoiding the use of glutaraldehyde and improve valve integration with low inflammation and host cell recolonization. In this preliminary study, we investigated the efficacy of a NaOH-based process for decellularization and biocompatibility improvement of porcine pulmonary heart valves in comparison to a detergent-based process (SDS-SDC0, 5%).. Native cryopreserved porcine pulmonary heart valves were treated with detergent and NaOH-based processes. Decellularization was assessed by Hematoxylin and eosin/DAPI/alpha-gal/SLA-I staining and DNA quantification of native and processed leaflets, walls and muscles. Elongation stress test investigated mechanical integrity of leaflets and walls (n = 3 tests/valve component) of valves in the native and treated groups (n = 4/group). Biochemical integrity (collagen/elastin/glycosaminoglycans content) of leaflet-wall and muscle of the valves (n = 4/group) was assessed and compared between groups with trichrome staining (Sirius Red/Miller/Alcian blue). Secondly, a preliminary in vivo study assessed biocompatibility (CD3 and CD68 immunostaining) and remodeling (Hematoxylin and eosin/CD31 and ASMA immunofluorescent staining) of NaOH processed valves implanted in orthotopic position in young Landrace pigs, at 1 (n = 1) and 3 months (n = 2).. Decellularization was better achieved with the NaOH-based process (92% vs 69% DNA reduction in the wall). Both treatments did not significantly alter mechanical properties. The detergent-based process induced a significant loss of glycosaminoglycans (p < 0,05). In vivo, explanted valves exhibited normal morphology without any sign of graft dilatation, degeneration or rejection. Low inflammation was noticed at one and three months follow-up (1,8 +/- 3,03 and 0,9836 +/- 1,3605 CD3 cells/0,12 mm. NaOH-based process offers better porcine pulmonary valve decellularization than the detergent process. In vivo, the NaOH processed valves showed low inflammatory response at 3 months and partial recellularization. Regarding additional property of securing, this treatment should be considered for the new generation of heart valves prosthesis. Graphical abstract of the study.

Topics: Animals; Biomechanical Phenomena; Bioprosthesis; Calcinosis; Collagen; Cryopreservation; Detergents; Elastin; Glycosaminoglycans; Heart Valve Prosthesis; Heart Valve Prosthesis Implantation; Heterografts; Pulmonary Valve; Sodium Hydroxide; Swine; Tissue Engineering

The lifetime of bioprosthetic heart valves (BHVs) is limited by the mechanical damage and calcification. The major components of BHVs are collagen and elastin. Collagen could be well protected by glutaraldehyde (GLUT) crosslinking, while elastin is not stabilized and has a high risk of degradation, which could lead to the calcification of BHVs. We aimed to develop methods for stabilizing elastin and decreasing calcification. We investigated the combined tannic acid (TA) or epigallocatechin gallate (EGCG) with ferric chloride to stabilize elastin and prevent calcification. We found that the amount of TA/EGCG bound to elastin was in a time-dependent pattern and this reaction showed better efficiency in acidic condition and ethanol-water mixed solvents. Moreover, Fe

Topics: Animals; Bioprosthesis; Calcinosis; Catechin; Chlorides; Collagen; Cross-Linking Reagents; Elastin; Ferric Compounds; Glutaral; Heart Valve Prosthesis; Male; Pericardium; Polyphenols; Protein Stability; Rats, Sprague-Dawley; Swine; Tannins; Tensile Strength

Valvular heart diseases lead to over 300,000 heart valve replacements worldwide each year. Commercially available bioprosthetic heart valves (BHVs) are mostly made from porcine or bovine pericardiums which were crosslinked by glutaraldehyde (GLUT). However, valve failures can occur within 10 years due to progressive degradation and calcification. GLUT could crosslink collagen but it fails to stabilize elastin. In this current study, we developed a new BHVs preparation strategy named as "GLUT/TE/LOXL/EGCG" that utilizes exogenous tropoelastin (TE)/lysyl oxidase (LOXL) and epigallocatechin gallate (EGCG) to increase the elastin content as well as the stabilization of elastin. The feeding ratios of tropoelastin and lysyl oxidase were optimized. The contents of desmosine and insoluble elastin, biomechanics, cytotoxicity, hemocompatibility, in vivo componential stability and anti-calcification potential were characterized. Pericardiums with increased elastin content had improved the mechanical properties. GLUT/TE/LOXL/EGCG-treated pericardiums had similar cytotoxicity and coagulation properties compared to GLUT and GLUT/EGCG control. We demonstrated that GLUT/TE/LOXL/EGCG-treated pericardiums had high amount of insoluble elastin in 90 days' rat subdermal implantation model, and better resistance for calcification. This new tropoelastin and lysyl oxidase treatments strategy would be a promising method to make BHVs which have better structural stability and anti-calcification properties.

Topics: Animals; Biomechanical Phenomena; Bioprosthesis; Calcinosis; Catechin; Cross-Linking Reagents; Elastin; Glutaral; Heart Valve Prosthesis; Male; Pericardium; Protein Stability; Protein-Lysine 6-Oxidase; Rats, Sprague-Dawley; Swine; Tropoelastin

Topics: Animals; Aorta; Aortic Aneurysm; Calcinosis; Elastin; Extracellular Signal-Regulated MAP Kinases; Humans; Marfan Syndrome; Mice; Mitogen-Activated Protein Kinase Kinases; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle

Elastin degradation is a key feature of emphysema and may have a role in the pathogenesis of atherosclerosis associated with chronic obstructive pulmonary disease (COPD). Circulating desmosine is a specific biomarker of elastin degradation. We investigated the association between plasma desmosine (pDES) and emphysema severity/progression, coronary artery calcium score (CACS) and mortality.pDES was measured in 1177 COPD patients and 110 healthy control subjects from two independent cohorts. Emphysema was assessed on chest computed tomography scans. Aortic arterial stiffness was measured as the aortic-femoral pulse wave velocity.pDES was elevated in patients with cardiovascular disease (p<0.005) and correlated with age (rho=0.39, p<0.0005), CACS (rho=0.19, p<0.0005) modified Medical Research Council dyspnoea score (rho=0.15, p<0.0005), 6-min walking distance (rho=-0.17, p<0.0005) and body mass index, airflow obstruction, dyspnoea, exercise capacity index (rho=0.10, p<0.01), but not with emphysema, emphysema progression or forced expiratory volume in 1 s decline. pDES predicted all-cause mortality independently of several confounding factors (p<0.005). In an independent cohort of 186 patients with COPD and 110 control subjects, pDES levels were higher in COPD patients with cardiovascular disease and correlated with arterial stiffness (p<0.05).In COPD, excess elastin degradation relates to cardiovascular comorbidities, atherosclerosis, arterial stiffness, systemic inflammation and mortality, but not to emphysema or emphysema progression. pDES is a good biomarker of cardiovascular risk and mortality in COPD.

Topics: Adult; Aged; Biomarkers; Body Composition; Bronchodilator Agents; Calcinosis; Cardiovascular Diseases; Case-Control Studies; Coronary Vessels; Desmosine; Disease Progression; Elastin; Emphysema; Female; Forced Expiratory Volume; Humans; Inflammation; Male; Middle Aged; Pulmonary Disease, Chronic Obstructive; Pulmonary Emphysema; Pulse Wave Analysis; Respiratory Function Tests; Risk Factors; Smoking; Vascular Stiffness

Topics: Aged; Calcinosis; Elastic Tissue; Elastin; Female; Humans; Image-Guided Biopsy; Muscle, Smooth, Vascular; Retinal Artery Occlusion; Temporal Arteries

Glutaraldehyde cross-linked bioprosthetic heart valves fail within 12-15 years of implantation due to limited durability. Glutaraldehyde does not adequately stabilize extracellular matrix components such as glycosaminoglycans and elastin, and loss of these components could be a major cause of degeneration of valve after implantation. We have shown earlier that neomycin-based cross-linking stabilizes glycosaminoglycans in the tissue but fails to stabilize elastin component. Here, we report a new treatment where neomycin and pentagalloyl glucose (PGG) were incorporated into glutaraldehyde cross-linking neomycin-PGG-Glutaraldehyde (NPG) to stabilize both glycosaminoglycans and elastin in porcine aortic valves. In vitro studies demonstrated a marked increase in extracellular matrix stability against enzymatic degradation after cross-linking and 10 month storage in NPG group when compared to glutaraldehyde controls. Tensile properties showed increased lower elastic modulus in both radial and circumferential directions in NPG group as compared to glutaraldehyde, probably due to increased elastin stabilization with no changes in upper elastic modulus and extensibility. The enhanced extracellular matrix stability was further maintained in NPG-treated tissues after rat subdermal implantation for three weeks. NPG group also showed reduced calcification when compared to glutaraldehyde controls. We conclude that NPG cross-linking would be an excellent alternative to glutaraldehyde cross-linking of bioprosthetic heart valves to improve its durability.

Topics: Bioprosthesis; Calcinosis; Calorimetry, Differential Scanning; Elastin; Glycosaminoglycans; Heart Valve Prosthesis; Hydrolyzable Tannins; Neomycin; Protein Denaturation

Tissue engineering techniques have emerged that allow bioresorbable grafts to be implanted that restore function and transform into biologically active arteries. However, these implants are susceptible to calcification during the remodeling process. The objective of this study was to evaluate the role of pore size of bioabsorbable grafts in the development of calcification.. Two types of grafts were prepared: a large-pore graft constructed of poly(L-lactic acid) (PLA) fibers coated with poly(L-lactide-co-ε-caprolactone) (PLCL) (PLA-PLCL), and a small-pore graft made of electrospun PLA nanofibers (PLA-nano). Twenty-eight PLA-PLCL grafts and twenty-five PLA-nano grafts were implanted as infra-renal aortic interposition conduits in 8-week-old female SCID/Bg mice, and followed for 12 months after implantation.. Large-pore PLA-PLCL grafts induced a well-organized neointima after 12 months, and Alizarin Red S staining showed neointimal calcification only in the thin neointima of small-pore PLA-nano grafts. At 12 months, macrophage infiltration, evaluated by F4/80 staining, was observed in the thin neointima of the PLA-nano graft, and there were few vascular smooth muscle cells (VSMCs) in this layer. On the other hand, the neointima of the PLA-PLCL graft was composed of abundant VSMCs, and a lower density of macrophages (F4/80 positive cells, PLA-PLCL; 68.1 ± 41.4/mm(2) vs PLA-nano; 188.3 ± 41.9/mm(2), p = 0.007). The VSMCs of PLA-PLCL graft expressed transcription factors of both osteoblasts and osteoclasts.. These findings demonstrate that in mouse arterial circulation, large-pore PLA-PLCL grafts created a well-organized neointima and prevented calcific deposition compared to small-pore, electrospun PLA-nano grafts.

Topics: Animals; Arteries; Calcinosis; Collagen; Elastin; Female; Inflammation; Lactic Acid; Macrophages; Mice; Mice, SCID; Microscopy, Electron, Scanning; Models, Animal; Neointima; Osteoblasts; Osteoclasts; Osteogenesis; Polyesters; Polymers; Porosity; Tissue Engineering; Transcription Factors; Vascular Grafting

A number of beta-thalassemia (β-thal) patients in the course of the disease exhibit ectopic calcification affecting skin, eyes and the cardiovascular system. Clinical and histopathological features have been described similar to those in pseudoxanthoma elasticum (PXE), although different genes are affected in the two diseases. Cultured dermal fibroblasts from β-thal patients with and without PXE-like clinical manifestations have been compared for parameters of redox balance and for the expression of proteins, which have been already associated with the pathologic mineralisation of soft connective tissues. Even though oxidative stress is a well-known condition of β-thal patients, our results indicate that the occurrence of mineralized elastin is associated with a more pronounced redox disequilibrium, as demonstrated by the intracellular increase of anion superoxide and of oxidized proteins and lipids. Moreover, fibroblasts from β-thal PXE-like patients are characterized by decreased availability of carboxylated matrix Gla protein (MGP), as well as by altered expression of proteins involved in the vitamin K-dependent carboxylation process. Results demonstrate that elastic fibre calcification is promoted when redox balance threshold levels are exceeded and the vitamin K-dependent carboxylation process is affected decreasing the activity of MGP, a well-known inhibitor of ectopic calcification. Furthermore, independently from the primary gene defect, these pathways are similarly involved in fibroblasts from PXE and from β-thal PXE-like patients as well as in other diseases leading to ectopic calcification, thus suggesting that can be used as markers of pathologic mineralisation.

Topics: Adult; Advanced Oxidation Protein Products; beta-Thalassemia; Blotting, Western; Calcinosis; Calcium-Binding Proteins; Carboxylic Acids; Dermis; DNA Methylation; Elastic Tissue; Elastin; Extracellular Matrix Proteins; Female; Fibroblasts; Flow Cytometry; Glutathione Peroxidase; Glutathione Transferase; Humans; Lipid Peroxides; Male; Malondialdehyde; Matrix Gla Protein; Oxidative Stress; Pseudoxanthoma Elasticum; Superoxide Dismutase; Vitamin K

Glutaraldehyde cross-linked porcine aortic valves, referred to as bioprosthetic heart valves (BHVs), are often used in heart valve replacements. Glutaraldehyde does not stabilize glycosaminoglycans (GAGs) and they are lost during preparation, in vivo implantation, cyclic fatigue, and storage. We report that binding of neomycin, a hyaluronidase inhibitor, to the tissues with carbodiimide cross-linking improves GAG retention without reducing collagen and elastin stability. It also led to improved biomechanical properties. Neomycin carbodiimide cross-linking did not significantly reduce calcification in a rat subdermal implantation model when they were stored in formaldehyde after cross-linking. Removal of formaldehyde storage significantly reduced calcification.

Topics: Animals; Aortic Valve; Biocompatible Materials; Biomechanical Phenomena; Bioprosthesis; Calcinosis; Carbodiimides; Collagen; Cross-Linking Reagents; Elastin; Glutaral; Glycosaminoglycans; Heart Valve Prosthesis; Male; Materials Testing; Neomycin; Protein Stability; Rats; Rats, Sprague-Dawley; Sus scrofa; Tissue Fixation

Calcinosis cutis is a cutaneous disorder characterised by abnormal deposition of calcium in the dermis. Treatment of this condition has variable success rates and includes medical, topical and surgical management. Here we describe a case of a woman with a painful calcinosis lesion on the buttocks, treated with excision and application of a bovine collagen-elastin dermal regeneration template, a thin, porous membrane consisting of a native bovine type I, II and V collagen-fibre template coated with elastin hydrolysate. The patient's wound healed without complication and without the use of a skin graft. She remains recurrence free at 10-month follow up, with satisfactory outcome.

Topics: Acellular Dermis; Buttocks; Calcinosis; Collagen; Elastin; Female; Humans; Middle Aged; Scleroderma, Systemic

This study aimed to investigate the effect of decellularization plus photooxidative crosslinking and ethanol pretreatment on bioprosthetic tissue calcification. Photooxidatively crosslinked acellular (PCA) bovine jugular vein conduits (BJVCs) and their photooxidized controls (n = 5 each) were sterilized in a graded concentration of ethanol solutions for 4 h, and used to reconstruct dog right ventricular outflow tracts. At 1-year implantation, echocardiography showed similar hemodynamic performance, but obvious calcification for the photooxidized BJVC walls. Further histological examination showed intense calcium deposition colocalized with slightly degraded elastic fibers in the photooxidized BJVC walls, with sparsely distributed punctate calcification in the valves and other areas of walls. But PCA BJVCs had apparent degradation of elastic fibers in the walls, with only sparsely distributed punctate calcification in the walls and valves. Content assay demonstrated comparable calcium content for the two groups at preimplantation, whereas less calcium for the PCA group in the walls and similar calcium in the valvular leaflets compared with the photooxidized group at 1-year retrieval. Elastin content assay presented the conduit walls of PCA group had less elastin content at preimplantation, but similar content at 1-year retrieval compared with the photooxidized group. Phospholipid analysis showed phospholipid extraction by ethanol for the PCA group was more efficacious than the photooxidized group. These results indicate that PCA BJVCs resist calcification in right-side heart implantation owing to decellularization, further photooxidative crosslinking, and subsequent phospholipid extraction by ethanol at preimplantation.

Topics: Animals; Calcinosis; Calcium; Cardiomyopathies; Cattle; Cross-Linking Reagents; Dogs; Elastin; Heart Valve Prosthesis; Heart Valves; Heart Ventricles; Immunohistochemistry; Jugular Veins; Light; Oxidation-Reduction; Phospholipids; Prosthesis Implantation; Staining and Labeling; Ultrasonography

Age-associated central arterial wall stiffness is linked to extracellular matrix remodeling, including fibrosis and vascular calcification. Angiotensin II induces both matrix metalloproteinase 2 (MMP2) and calpain-1 expression and activity in the arterial wall. However, the role of calpain-1 in MMP2 activation and extracellular matrix remodeling remains unknown. Dual histo-immunolabeling demonstrates colocalization of calpain-1 and MMP2 within old rat vascular smooth muscle cells. Overexpression of calpain-1 induces MMP2 transcripts, protein levels, and activity, in part, by increasing the ratio of membrane type 1 MMPs to tissue inhibitor of metalloproteinases 2. These effects of calpain-1 overexpression-induced MMP2 activation are linked to increased collagen I and III production and vascular calcification. In addition, overexpression of calpain-1 also induces transforming growth factor-β1/Smad signaling, elastin degradation, alkaline phosphatase activation, and total calcium content but reduces the expression of calcification inhibitors, osteopontin, and osteonectin, in cultured vascular smooth muscle cells in vitro and in carotid artery rings ex vivo. Furthermore, both calpain-1 and collagen II increase with aging within human aortic intima. Interestingly, in aged human aortic wall, both calpain-1 and collagen II are highly expressed in artherosclerotic plaque areas compared with grossly normal areas. Cross-talk of 2 proteases, calpain-1 and MMP2, leads to secretion of active MMP2, which modulates extracellular matrix remodeling via enhancing collagen production and facilitating vascular calcification. These results establish calpain-1 as a novel molecular candidate to retard age-associated extracellular matrix remodeling and its attendant risk for hypertension and atherosclerosis.

Topics: Adolescent; Aged; Aging; Animals; Aorta; Blotting, Western; Calcinosis; Calpain; Cells, Cultured; Collagen; Elastin; Enzyme Activation; Fibrosis; Humans; Male; Matrix Metalloproteinase 2; Middle Aged; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Rats; Rats, Inbred BN; Rats, Inbred F344; Reverse Transcriptase Polymerase Chain Reaction; Tissue Inhibitor of Metalloproteinase-2; Young Adult

Pseudoxanthoma elasticum (PXE) is a heritable disorder characterized by mineralization of connective tissue, which leads to pathology in eye, skin and blood vessels. The disease is caused by mutations in ABCC6. To learn more about PXE eye pathology, we analyzed Bruch's membrane (BM) of the eye of an Abcc6 knockout mouse. With age, BM differences between Abcc6-/- and wild type mice became apparent. At two years of age, von Kossa staining indicated clear calcification of BM in Abcc6-/- mice, and not in healthy controls. Electron microscopy revealed BM changes as early as at 10 months of age: Fibrous structures with abnormal high electron-density were present in the central layers of BM of Abcc6-/- mice. EDX (Energy Dispersive X-ray) analysis demonstrated that these structures contained elevated levels of Ca, P and O. Since some of these electron-dense structures showed a banding pattern with periodicity of about 50 nm, they most likely represent calcified collagen fibers. Immunoelectron microscopy showed that the calcified structures were positive for collagen III. Remarkably, the elastic layer of BM appeared to have a normal ultrastructure, even in 2.5 year old Abcc6-/- mice. Our results suggest that Abcc6 deficiency in the mouse causes calcification of BM. While PXE is considered to affect primarily the elastic fibers, we found predominantly mineralization of collagen fibers.

Topics: Animals; ATP-Binding Cassette Transporters; Bruch Membrane; Calcinosis; Collagen Type III; Disease Models, Animal; Elastin; Female; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Immunoelectron; Multidrug Resistance-Associated Proteins; Pseudoxanthoma Elasticum; Spectrometry, X-Ray Emission

Arterial medial calcification (AMC), a hallmark of vascular disease in uremic patients, is highly correlated with serum phosphate levels and cardiovascular mortality. To determine the mechanisms of AMC, mice were made uremic by partial right-side renal ablation (week 0), followed by left-side nephrectomy at week 2. At 3 weeks, mice were switched to a high-phosphate diet, and various parameters of disease progression were examined over time. Serum phosphate, calcium, and fibroblast growth factor 23 (FGF-23) were up-regulated as early as week 4. Whereas serum phosphate and calcium levels declined to normal by 10 weeks, FGF-23 levels remained elevated through 16 weeks, consistent with an increased phosphate load. Elastin turnover and vascular smooth muscle cell (VSMC) phenotype change were early events, detected by week 4 and before AMC. Both AMC and VSMC loss were significantly elevated by week 8. Matrix metalloprotease 2 (MMP-2) and cathepsin S were present at baseline and were significantly elevated at weeks 8 and 12. In contrast, MMP-9 was not up-regulated until week 12. These findings over time suggest that VSMC phenotype change and VSMC loss (early phosphate-dependent events) may be necessary and sufficient to promote AMC in uremic mice fed a high-phosphate diet, whereas elastin degradation might be necessary but is not sufficient to induce AMC (because elastin degradation occurred also in uremic mice on a normal-phosphate diet, but they did not develop AMC).

Topics: Animals; Calcinosis; Cell Death; Diet; Disease Models, Animal; Disease Progression; Elastin; Enzyme Activation; Fibroblast Growth Factor-23; Immunohistochemistry; Kidney Failure, Chronic; Matrix Metalloproteinases; Mice; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Phenotype; Phosphates; Time Factors; Tunica Media; Uremia

Vascular calcification is the most important cause of cardiovascular disease in patients with chronic kidney disease (CKD). Medial layer vascular calcification, which is recognized to be an active process (i.e. the transformation of vascular smooth muscle cells into osteoblast-like cells), is common in CKD patients. We have recently reported the possibility of an interaction between elastin degradation and medial layer vascular calcification. Matrix metalloproteinase-2 (MMP-2), which induces the degradation of elastin, has been implicated in the elastic calcification in arteries of dialysis patients; however, the precise mechanisms by which elastin degradation interacts with the development of vascular calcification remain to be studied. To clarify the mechanisms by which elastin degradation is involved in the development of medial layer vascular calcification in the uremic milieu, we induced aortic medial layer calcification in 5/6 nephrectomized uremic rats (male Sprague-Dawley rats) fed a diet containing high phosphate (1.2%) and lactate (20%). After 10 weeks, the rats were euthanized for the measurement of serum chemistry profiles and histological analyses. The uremic rats showed significant increases in blood pressure, serum creatinine, phosphate, and parathyroid hormone levels compared with normal rats. Von Kossa staining showed medial layer aortic calcification in the uremic rats. In calcified lesions, thin elastic lamellae were observed by elastin staining, indicating that elastin degradation could occur in the area. Furthermore, MMP-2 expression determined by immunohistochemistry was also observed in the same area. Elastin degradation accompanied by MMP-2 expression might be involved in the development of medial layer vascular calcification in uremic rats.

Topics: Animals; Aorta; Blood Pressure; Calcinosis; Cardiovascular Diseases; Chronic Disease; Creatinine; Elastin; Kidney Diseases; Male; Matrix Metalloproteinase 2; Parathyroid Hormone; Phosphates; Rats; Rats, Sprague-Dawley; Uremia

Calcification and fibrosis reduce vascular compliance in arteriosclerosis. To better understand the role of osteopontin (OPN), a multifunctional protein upregulated in diabetic arteries, we evaluated contributions of OPN in male low-density lipoprotein receptor (LDLR)-/- mice fed a high-fat diet.. OPN had no impact on high-fat diet-induced hyperglycemia, dyslipidemia, or body composition. However, OPN-/-;LDLR-/- mice exhibited an altered time-course of aortic calcium accrual-reduced during initiation but increased with progression-versus OPN+/+;LDLR-/- controls. Collagen accumulation, chondroid metaplasia, and mural thickness were increased in aortas of OPN-/-;LDLR-/- mice. Aortic compliance was concomitantly reduced. Vascular reexpression of OPN (SM22-OPN transgene) reduced aortic Col2A1 and medial chondroid metaplasia but did not affect atherosclerotic calcification, Col1A1 expression, collagen accumulation, or arterial stiffness. Dosing with the proinflammatory OPN fragment SVVYGLR upregulated aortic Wnt and osteogenic gene expression, increased aortic β-catenin, and restored early-phase aortic calcification in OPN-/-;LDLR-/- mice.. OPN exerts stage-specific roles in arteriosclerosis in LDLR-/- mice. Actions phenocopied by the OPN metabolite SVVYGLR promote osteogenic calcification processes with disease initiation. OPN limits vascular chondroid metaplasia, endochondral mineralization, and collagen accumulation with progression. Complete deficiency yields a net increase in arteriosclerotic disease, reducing aortic compliance and conduit vessel function in LDLR-/- mice.

Topics: Amino Acid Sequence; Animals; Aorta; Arteriosclerosis; beta Catenin; Calcinosis; Calcium; Collagen; Diabetic Angiopathies; Elastin; Fibrosis; Male; Matrix Metalloproteinases; Mice; Mice, Knockout; Microfilament Proteins; Muscle Proteins; Osteopontin; Peptide Fragments; Promoter Regions, Genetic; Receptors, LDL; Signal Transduction; Vascular Resistance

Vascular calcification is a major contributor to cardiovascular disease, a leading cause of death in patients with chronic kidney disease. Mechanistic studies highlight the importance of dysregulated mineral metabolism, vascular osteochondrogenic processes, apoptosis, and deficiencies in calcification inhibitors as potential mediators of calcification in renal disease. However, the contribution of the extracellular matrix in vascular calcification associated with chronic kidney disease is less understood. Here we examine evidence that suggests important roles for elastin and elastin-degrading enzymes as potential key regulators of calcification. Additional studies aimed at further understanding their role are critical for the design of therapeutic interventions.

Topics: Animals; Calcinosis; Elastin; Humans; Matrix Metalloproteinases; Renal Insufficiency, Chronic; Vascular Diseases

Plaque with dense inflammatory cells, including macrophages, thin fibrous cap and superficial necrotic/lipid core is thought to be prone-to-rupture. We report a time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) technique for detection of such markers of plaque vulnerability in human plaques.. The autofluorescence of carotid plaques (65 endarterectomy patients) induced by a pulsed laser (337 nm, 0.7 ns) was measured from 831 distinct areas. The emission was resolved spectrally (360-550 nm range) and temporally (0.3 ns resolution) using a prototype fiber-optic TR-LIFS apparatus. Lesions were evaluated microscopically and quantified as to the % of different components (fibrous cap, necrotic core, inflammatory cells, foam cells, mature and degraded collagen, elastic fibers, calcification, and smooth muscle cell of the vessel wall).. We determined that the spectral intensities and time-dependent parameters at discrete emission wavelengths (1) allow for discrimination (sensitivity >81%, specificity >94%) of various compositional and pathological features associated with plaque vulnerability including infiltration of macrophages into intima and necrotic/lipid core under a thin fibrous cap, and (2) show a linear correlation with plaque biochemical content: elastin (P<0.008), collagen (P<0.02), inflammatory cells (P<0.003), necrosis (P<0.004).. Our results demonstrate the feasibility of TR-LIFS as a method for the identification of markers of plaque vulnerability. Current findings enable future development of TR-LIFS-based clinical devices for rapid investigation of atherosclerotic plaques and detection of those at high-risk.

Topics: Calcinosis; Carotid Artery Diseases; Carotid Artery, Common; Collagen; Elastin; Endarterectomy, Carotid; Feasibility Studies; Fiber Optic Technology; Fibrosis; Foam Cells; Humans; Lasers; Lipids; Necrosis; Predictive Value of Tests; Reproducibility of Results; Rupture; Sensitivity and Specificity; Spectrometry, Fluorescence; Time Factors

Clinical studies have demonstrated that 50% of individuals with chronic renal disease (CRD) die of cardiovascular causes, including advanced calcific arterial and valvular disease; however, the mechanisms of accelerated calcification in CRD remain obscure, and no therapies can prevent disease progression. We recently demonstrated in vivo that inflammation triggers cardiovascular calcification. In vitro evidence also indicates that elastin degradation products may promote osteogenesis. Here, we used genetically modified mice and molecular imaging to test the hypothesis in vivo that cathepsin S (catS), a potent elastolytic proteinase, accelerates calcification in atherosclerotic mice with CRD induced by 5/6 nephrectomy.. Apolipoprotein-deficient (apoE(-/-))/catS(+/+) (n=24) and apoE(-/-)/catS(-/-) (n=24) mice were assigned to CRD and control groups. CRD mice had significantly higher serum phosphate, creatinine, and cystatin C levels than those without CRD. To visualize catS activity and osteogenesis in vivo, we coadministered catS-activatable and calcification-targeted molecular imaging agents 10 weeks after nephrectomy. Imaging coregistered increased catS and osteogenic activities in the CRD apoE(-/-)/catS(+/+) cohort, whereas CRD apoE(-/-)/catS(-/-) mice exhibited less calcification. Quantitative histology demonstrated greater catS-associated elastin fragmentation and calcification in CRD apoE(-/-)/catS(+/+) than CRD apoE(-/-)/catS(-/-) aortas and aortic valves. Notably, catS deletion did not cause compensatory increases in RNA levels of other elastolytic cathepsins or matrix metalloproteinases. Elastin peptide and recombinant catS significantly increased calcification in smooth muscle cells in vitro, a process further amplified in phosphate-enriched culture medium.. The present study provides direct in vivo evidence that catS-induced elastolysis accelerates arterial and aortic valve calcification in CRD, providing new insight into the pathophysiology of cardiovascular calcification.

Topics: Animals; Aorta; Aortic Valve; Aortic Valve Stenosis; Apolipoproteins E; Calcinosis; Carotid Artery Diseases; Cathepsins; Cells, Cultured; Creatinine; Cystatin C; Elastin; Humans; Kidney Failure, Chronic; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Muscle, Smooth, Vascular; Nephrectomy; Osteogenesis; Phosphates; Tunica Intima; Tunica Media

The heterotrimeric G protein alpha-subunit G(s)alpha links receptors to stimulation of cAMP/protein kinase A signaling, which inhibits skin fibroblast proliferation and collagen synthesis. We now describe the development of fibrous tumors in mice with heterozygous disruption of the Gnas gene, which encodes G(s)alpha and other gene products.. Disruption of Gnas exon 2 on either the maternal or paternal allele (Gnas(E2-/+)) results in fibromas or angiofibromas on the ears, paws and tail beginning at 4 months of age. The tumors were composed of fibroblastic cell proliferation with collagen and elastin deposition and calcification, and seemed to be associated with mechanical skin damage. The presence of calcification was associated with greater amounts of matrix metalloproteinase-2, suggesting an association between calcium deposition and extracellular matrix degradation. Osteoblast-specific markers were absent, consistent with the calcification not being secondary to ossification. Molecular studies showed that the tumors were not associated with deletion of the wild-type allele, making it unlikely that these tumors resulted from homozygous loss of G(s)alpha.. These findings provide in vivo evidence that G(s)alpha pathways inhibit fibroblast and endothelial proliferation and matrix deposition.

Topics: Angiofibroma; Animals; Calcinosis; Chromogranins; Collagen; Elastin; Fibroma; GTP-Binding Protein alpha Subunits, Gs; Mice; Skin Neoplasms

Medial layer vascular calcification is common in patients with end-stage kidney disease. Inorganic phosphate has been shown to accelerate the transformation of vascular smooth muscle cells (VSMCs) into osteoblast-like cells, which is thought to be a major process of medial layer calcification. Although elastin degradation is associated with medial layer calcification, the linkage between elastin degradation and the transformation of VSMCs remains to be clarified. We investigated the involvement of elastin degradation in the transformation of VSMCs. Rat VSMCs were isolated and cultured with a normal- (NP, 1.0 mM) or high- (HP, 2.5 mM) phosphate medium. An elastin-derived peptide, alpha-elastin (500 microg/ml), was also added to the normal- (NP + E) or high- (HP + E) phosphate medium. After a culture period of 2 weeks, von Kossa staining revealed mineralization in the HP group, which was accelerated by alpha-elastin, whereas alpha-elastin did not affect the mineralization at a normal phosphate concentration. The gene expression of osteoblastic differentiation factors, i.e., Runx2 or osteocalcin (OC), in VSMCs was significantly increased in the HP (Runx2 P < 0.05, OC P < 0.05) and HP + E (OC P < 0.05) groups compared with the NP and NP + E groups. Both gene and protein expressions of tissue-nonspecific alkaline phosphatase (TNAP) were significantly increased in the HP group compared with the NP and NP + E groups (P < 0.01, respectively). This increment was augmented in the HP + E group (P < 0.01). These results suggest that elastin degradation would accelerate or stabilize the process of VSMC transformation, which is induced by high phosphate through the upregulation of TNAP.

Topics: Animals; Calcinosis; Cells, Cultured; Elastin; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Osteoblasts; Phosphates; Rats; Rats, Sprague-Dawley; Up-Regulation

This study was undertaken to establish a Peyronie's disease model with penile curvature by using recombinant transforming growth factor-beta1 (TGF-beta1) protein or adenovirus (ad-TGF-beta1). Four-month-old male Sprague-Dawley rats were divided into seven groups (n = 18 per group): G1 received a single injection of saline into the tunica albuginea (0.1 mL); G2, repeated injections of ad-LacZ (days 0, 3, and 6; 1 x 10(10) particles/0.1 mL respectively); G3, a single injection of recombinant TGF-beta1 protein (700 ng/0.1 mL); G4, repeated injections of recombinant TGF-beta1 protein (days 0, 3 and 6; 700 ng/0.1 mL respectively); G5, a single injection of low-dose ad-TGF-beta1 (1 x 10(10) particles/0.1 mL); G 6, a single injection of high-dose ad-TGF-beta1 (3 x 10(10) particles/0.1 mL); and G7, repeated injections of low-dose ad-TGF-beta1 (days 0, 3, and 6; 1 x 10(10) particles/0.1 mL respectively). Penile curvature was evaluated 30, 45 and 60 days after treatment, and the penis was then harvested for histological examination. Repeated injection of low-dose ad-TGF-beta1 not only induced fibrous scar in the tunica, which lasted up to 60 days after injection, but also resulted in significant penile curvature by artificial erection test 45 days after treatment. A peculiar histological finding in this group was trapping of inflammatory cells in the tunica, subsequent fibrosis, and formation of cartilage and calcification as well as loss of elastin fibres. This model involving repeated injection of ad-TGF-beta1 may contribute to further investigation of the pathogenesis of Peyronie's disease and the development of new therapeutics targeting this pathway.

Topics: Adenoviridae; Animals; Calcinosis; Cartilage; Disease Models, Animal; Disease Progression; Elastin; Fibrosis; Gene Transfer Techniques; Genetic Vectors; Injections; Male; Penile Erection; Penile Induration; Penis; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Swine; Time Factors; Transforming Growth Factor beta1

Isolated systolic hypertension is associated with increased elastase activity, vascular calcification, and vascular stiffness. We sought to determine the importance of elastase activity and matrix degradation in the development of elastocalcinosis.. Elastocalcinosis was induced in vivo and ex vivo using warfarin. Hemodynamic parameters, calcium deposition, elastin degradation, transforming growth factor (TGF)-beta signaling, and elastase activity were evaluated at different time points in the in vivo model. Metalloproteinases, serine proteases, and cysteine proteases were blocked to measure their relative implication in elastin degradation. Gradual elastocalcinosis was obtained, and paralleled the elastin degradation pattern. Matrix metalloproteinase (MMP)-9 activity was increased at 5 days of warfarin treatment, whereas TGF-beta signaling was increased at 7 days. Calcification was significantly elevated after 21 days. Blocking metalloproteinases activation with doxycycline and TGF-beta signaling with SB-431542 were able to prevent calcification.. Early MMP-9 activation precedes the increase of TGF-beta signaling, and overt vascular elastocalcinosis and stiffness. Modulation of matrix degradation could represent a novel therapeutic avenue to prevent the gradual age-related stiffening of large arteries, leading to isolated systolic hypertension.

Topics: Animals; Aorta, Abdominal; Calcinosis; Collagen; Disease Models, Animal; Durapatite; Elastin; Femoral Artery; Male; Matrix Metalloproteinase 9; Osteopontin; Rats; Rats, Wistar; Signal Transduction; Transforming Growth Factor beta; Vascular Diseases; Warfarin

Maintaining the integrity of arterial elastin is vital for the prevention of abdominal aortic aneurysm (AAA) development. We hypothesized that in vivo stabilization of aortic elastin with pentagalloyl glucose (PGG), an elastin-binding polyphenol, would interfere with AAA development.. Safety and efficacy of PGG treatment were first tested in vitro using cytotoxicity, elastin stability, and PGG-elastin interaction assays. For in vivo studies, the efficacy of PGG was evaluated within a well-established AAA model in rats on the basis of CaCl2-mediated aortic injury. With this model, PGG was delivered periadventitially at 2 separate time points during the course of AAA development; aortic diameter, elastin integrity, and other pathological aspects were monitored and evaluated in PGG-treated aortas compared with saline-treated control aortas. Our results show that a one-time periadventitial delivery of noncytotoxic levels of PGG inhibits elastin degeneration, attenuates aneurysmal expansion, and hinders AAA development in rats without interfering with the pathogenic mechanisms typical of this model, namely inflammation, calcification, and high metalloproteinase activities. PGG binds specifically to arterial elastin and, in doing so, preserves the integrity of elastic lamellae despite the presence of high levels of proteinases derived from inflammatory cells.. Periadventitial administration of PGG hinders the development of AAA in a clinically relevant animal model. Stabilization of aortic elastin in aneurysm-prone arterial segments offers great potential toward the development of safe and effective therapies for AAAs.

Topics: Administration, Topical; Animals; Aorta, Abdominal; Aortic Aneurysm, Abdominal; Calcinosis; Calcium Chloride; Cells, Cultured; Disease Models, Animal; Disease Progression; Drug Evaluation, Preclinical; Elastin; Fibroblasts; Hydrolyzable Tannins; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Protein Denaturation; Rats; Rats, Sprague-Dawley

Calcification of vascular elastin occurs in patients with arteriosclerosis, renal failure, diabetes, and vascular graft implants. We hypothesized that pathological elastin calcification is related to degenerative and osteogenic mechanisms. To test this hypothesis, the temporal expression of genes and proteins associated with elastin degradation and osteogenesis was examined in the rat subdermal calcification model by quantitative real-time reverse transcription-polymerase chain reaction and specific protein assays. Purified elastin implanted subdermally in juvenile rats exhibited progressive calcification in a time-dependent manner along with fibroblast and macrophage infiltration. Reverse transcription-polymerase chain reaction analysis showed that relative gene expression levels of matrix metalloproteinases (MMP-2 and MMP-9) and transforming growth factor-beta1 were increased in parallel with calcification. Gelatin zymography showed strong MMP activities at early time points, which were associated with high levels of soluble elastin peptides. Gene expression of core binding factor alpha-1, an osteoblast-specific transcription factor, increased in parallel with elastin calcification and attained approximately 9.5-fold higher expression at 21 days compared to 3 days after implantation. Similarly, mRNA levels of the bone markers osteopontin and alkaline phosphatase also increased progressively, but osteocalcin levels remained unchanged. We conclude that degenerative and osteogenic processes may be involved in elastin calcification.

Topics: Alkaline Phosphatase; Animals; Calcinosis; Core Binding Factor Alpha 1 Subunit; Disease Models, Animal; Elastin; Fibroblasts; Gene Expression Regulation; Heart; Macrophages; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Osteogenesis; Osteopontin; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sialoglycoproteins; Swine; Transforming Growth Factor beta; Transforming Growth Factor beta1; Up-Regulation

Collagen-elastin scaffolds may be valuable biomaterials for tissue engineering because they combine tensile strength with elasticity. In this study, the tissue response to and the calcification of these scaffolds were evaluated. In particular, the hypothesis was tested that calcification, a common phenomenon in biomaterials, may be due to microfibrils within the elastic fibre, and that these microfibrils might generate a tissue response. Four scaffolds were subcutaneously implanted, viz. collagen, collagen + pure elastin, collagen+microfibril-containing, and collagen + pulverised elastic ligament (the source for elastin). Explants were evaluated at day 3, 7 and 21. In young Sprague Dawley rats, collagen + ligament calcified substantially, whereas collagen + elastin (with and without microfibrils) calcified less, and collagen did not. Calcification started at elastic fibres. In both Sprague Dawley and Wistar adult rats, however, none of the scaffolds calcified. Mononuclear cell infiltration was prominent in young and adult Sprague Dawley rats. In adult Wistar rats, this infiltration was associated with the presence of microfibrils. Degradation of scaffolds and new matrix formation were related with cellular influx and degree of vascularisation. In conclusion, absence of microfibrils from the elastic fibre does not prevent calcification in young Sprague Dawley rats, but does reduce the tissue response in adult Wistar rats. Cellular response and calcification differs with age and strain and therefore the choice of animal model is of key importance in biomaterial evaluation.

Topics: Aging; Animals; Biocompatible Materials; Calcinosis; Collagen; Elastin; Foreign-Body Reaction; Implants, Experimental; Male; Materials Testing; Rats; Rats, Sprague-Dawley; Rats, Wistar; Species Specificity; Tissue Engineering

Progressive degeneration and calcification of glutaraldehyde (Glut) fixed tissues used in cardiovascular surgery restrict their long-term clinical performance. This limited biological stability may be attributable to the inability of Glut to adequately protect certain tissue components such as elastin from enzymatic attack. The aim of our studies was to develop novel tissue-processing techniques targeted specifically at elastin stabilization by using tannic acid (TA), a plant polyphenol capable of protecting elastin from digestion by specific enzymes. In present studies we demonstrated that Glut does not adequately protect porcine aorta from elastase-mediated degradation in vitro. The addition of TA to the Glut fixation process increased the stability of Glut-fixed aorta to elastase digestion by 15-fold and also decreased calcification in the rat subdermal model by 66%. TA was found to be chemically compatible with Glut fixation and did not hinder collagen crosslinking as shown by minor changes in thermal denaturation temperatures, resistance to collagenase and mechanical properties. In vitro and in vivo studies also revealed that TA binding to aortic wall was stable over an extended period of time. TA-mediated elastin stabilization in Glut-fixed cardiovascular implants may significantly extend the clinical durability of these tissue replacements.

Topics: Animals; Aorta; Bioprosthesis; Calcinosis; Cattle; Drug Combinations; Elasticity; Elastin; Glutaral; Heart Valve Prosthesis; In Vitro Techniques; Male; Organ Preservation Solutions; Pancreatic Elastase; Rats; Rats, Sprague-Dawley; Tannins; Tensile Strength; Tissue Preservation

The present study was undertaken to systematically investigate whether calcification of elastic fibers occurs in human atherosclerotic plaques. Fourteen carotid artery segments obtained by endarterectomy were examined by a combination of electron microscopy and cytochemistry. The analysis demonstrated that calcification of elastic fibers occurred in all 14 specimens. Two distinct types of calcification of elastic fibers were identified. In type I calcification, elastin itself was observed to undergo calcification and no visible structural alterations preceded the calcification. In type II of calcification, structural alteration of elastin preceded calcification of elastic fibers and included vacuolization of elastin accompanied by the accumulation of neutral lipids and unesterified cholesterol within altered elastic fibers. In type II calcification, calcified deposits were found to form in an association with unesterified cholesterol. Type II calcification was widespread throughout the plaque matrix while type I calcification occurred only in the deep portions of plaques.

Topics: Aged; Arteriosclerosis; Calcinosis; Carotid Arteries; Elastin; Female; Humans; Immunohistochemistry; Male; Microscopy, Electron; Middle Aged

A method is described that could be of potential use for the rapid ultrastructural identification of abnormal and fragmented elastic fibers in very small wet samples of dermal biopsies from patients affected by Pseudoxanthoma elasticum (PXE). Moreover, the method, which consists of the use of sealed capsules transparent to electrons, allows the rapid and accurate localization and detection of mineralized areas in PXE patients and of their ion composition by X-ray microanalysis. This methodology could be of great help in any tissue disorder, especially in connective tissue disorders, characterized by structural alterations associated with ion precipitation.

Topics: Biopsy; Calcinosis; Connective Tissue Diseases; Elastic Tissue; Elastin; Electron Probe Microanalysis; Humans; Ions; Pseudoxanthoma Elasticum; Skin

We have investigated the long-term effect of aluminum chloride (AlCl(3)) treatment on the calcification and inflammatory reaction of a porcine elastin-derived biomaterial (PEB) in a novel subdermal adult mouse model. Untreated PEB disks and PEB treated with AlCl(3) were implanted subdermally in BALB/c mice for 30, 60, and 180 days. The calcification of the elastin disks was examined with histological analysis and atomic absorption analysis of calcium content. The inflammatory reaction was evaluated both with histological analysis of explants and by an enzyme-linked immunosorbent assay of the serum in each mouse to determine the production of antielastin antibodies. Robust calcification was evident in all untreated PEBs with calcium levels of 107.1 +/- 11.8, 151.4 +/- 14.4, and 227.2 +/- 23.8 microg/mg for 30, 60, and 180 days, respectively. AlCl(3) treatment only temporarily prevented the calcification of the elastin disks for 30 days. By 60 and 180 days, the AlCl(3)-treated materials had significant calcification with 88.7 +/- 17.4 and 105.3 +/- 27.0 microg/mg calcium, respectively. The inflammatory reaction was moderate for both types of implants. The AlCl(3)-treated implants displayed significantly more macrophage and lymphocyte infiltration at 180 days after implantation, and a trend to higher humoral responses at 30 and 60 days when compared with untreated PEBs. We conclude that PEBs extensively calcify in the adult mice model. AlCl(3) treatment of elastin enhances the long-term immunological response to xenogenic elastin implants and merely delays the onset of calcification.

Topics: Aluminum Chloride; Aluminum Compounds; Amino Acids; Animals; Aorta; Biocompatible Materials; Calcinosis; Chlorides; Disease Models, Animal; Elastin; Female; Implants, Experimental; Mice; Mice, Inbred BALB C; Random Allocation; Skin; Swine

In cases of vascular calcification, the expression of tropoelastin is down-regulated, which most likely decreases elastic fiber formation. However, the function of tropoelastin in vascular calcification remains unknown. We investigated whether tropoelastin affects the induction of vascular calcification. Calcification was induced using inorganic phosphate in cultured bovine aortic smooth muscle cells. The increase in tropoelastin due to the addition of recombinant bovine tropoelastin (ReBTE; 1 or 10 microg/ml) or beta-aminopropionitrile (25 microg/ml) significantly inhibited calcification at day 6, as assessed by the o-cresolphthalein complexone method. The addition of an elastin-derived peptide, VGVAPG peptide (0.1-1,000 nM), inhibited calcification at day 6 in a dose-dependent manner. In addition, these responses of beta-aminopropionitrile, ReBTE, and VGVAPG peptide were confirmed using von Kossa staining. To examine whether ReBTE inhibited calcium deposition via the elastin binding protein, lactose and elastin-specific antibody were used. The combination of lactose (20 mM) or this antibody (50 microg/ml) with ReBTE (10 microg/ml) attenuated the inhibition of calcification. These results suggest that increased tropoelastin inhibits vascular calcification in this model via the interaction between tropoelastin and elastin binding protein.

Topics: Animals; Aorta; Calcinosis; Calcium; Cattle; Cells, Cultured; Down-Regulation; Elastin; Models, Animal; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Receptors, Cell Surface; Tropoelastin

Elastin calcification is a widespread feature of vascular pathology, and circumstantial evidence exists for a correlation between elastin degradation and calcification. We hypothesized that matrix metalloproteinase (MMP)-mediated vascular remodeling plays a significant role in elastin calcification.. In the present studies, we determined that short-term periadventitial treatment of the rat abdominal aorta with low concentrations of calcium chloride (CaCl2) induced chronic degeneration and calcification of vascular elastic fibers in the absence of aneurysm formation and inflammatory reactions. Furthermore, the rate of progression of calcification depended on the application method and concentration of CaCl2 applied periarterially. Initial calcium deposits, associated mainly with elastic fibers, were persistently accompanied by elastin degradation, disorganization of aortic extracellular matrix, and moderate levels of vascular cell apoptosis. Application of aluminum ions (known inhibitors of elastin degradation) before the CaCl2-mediated injury significantly reduced elastin calcification and abolished both extracellular matrix degradation and apoptosis. We also found that MMP-knockout mice were resistant to CaCl2-mediated aortic injury and did not develop elastin degeneration and calcification.. Collectively, these data strongly indicate a correlation between MMP-mediated elastin degradation and vascular calcification.

Topics: Animals; Aorta, Abdominal; Aortic Diseases; Calcinosis; Calcium; Calcium Chloride; Capillary Permeability; Desmosine; Elastic Tissue; Elastin; Endothelium, Vascular; Extracellular Matrix; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Mice, Knockout; Rats; Rats, Sprague-Dawley; Tunica Media

Williams-Beuren syndrome is a rare and usually sporadic genetic anomaly with an estimated frequency of 1:25,000, that also has cardiac defects due to the effect on the elastin locus of a deletion on the 7th chromosome. Identical twin boys presented with exercise-induced syncope. Echocardiographic examination revealed severe calcification at the aortic valves, mitral anterior leaflets, and mitral annuli in both cases. A basal interventricular septum was also involved in one case. Doppler evaluation demonstrated severe aortic stenosis with a peak gradient of 112 and 118 mmHg in both cases. Moderate mitral stenosis was also detected in one twin. We performed aortic mechanical valve replacement and dilated the aortic annulus with Nick's procedure and evaluated the diagnosis and therapy methods in light of the literature. As a result, we determined that these boys are the first monozygotic twins who were diagnosed during childhood.

Topics: Aortic Valve; Aortic Valve Stenosis; Calcinosis; Diseases in Twins; Elastin; Gene Deletion; Heart Valve Prosthesis Implantation; Humans; Male; Mitral Valve Stenosis; Twins, Monozygotic; Williams Syndrome

Isolated systolic hypertension is the predominant form of hypertension in the elderly population. Reduction of arterial compliance appears to contribute to the elevation of pulse pressure (PP) and among potential mechanisms, gradual vascular calcification, fragmentation of elastic lamellae, and augmentation of rigid component like collagen could contribute to increase aortic stiffening. Few experimental models of the disease are currently available.. To induce large artery calcification, rats were treated with warfarin and vitamin K(1) (WK) for 4 and 8 weeks, to inhibit the maturation of matrix Gla protein. The impact of chronic PP elevation was determined on large artery and cardiac remodeling and on aortic endothelial function.. The WK treatment led to aortic medial calcification and a proportional elevation of PP, attributable mainly to a selective elevation of systolic blood pressure. The chronic treatment also increased collagen, whereas elastin decreased in the aorta. Pulse wave velocity, an index of aortic stiffening, increased in rats treated with WK. However, indices of left ventricular and aortic hypertrophy and remodeling remained normal. In addition, the WK treatment did not modify the vasoconstriction to norepinephrine and endothelin-1, and the vasodilatory response to acetylcholine and sodium nitroprusside.. Chronic treatment with WK represents a new model of isolated systolic hypertension with several characteristics of the human disease. The relative ease to induce calcification in this model may help to foster more fundamental research, which is lacking in this type of hypertension.

Topics: Animals; Anticoagulants; Antifibrinolytic Agents; Aorta; Calcinosis; Calcium; Collagen; Disease Models, Animal; Drug Administration Schedule; Elastin; Hemodynamics; Hypertension; Male; Rats; Rats, Wistar; Renin; Systole; Vascular Diseases; Vasomotor System; Vitamin K 1; Warfarin

Calcification of bioprosthetic heart valves fabricated from glutaraldehyde (GA)-pretreated heterograft tissue is frequently responsible for the clinical failure of these devices. Stentless bioprostheses fabricated from GA-fixed porcine aortic valves pose an important challenge in this regard, as pathologic calcification can affect not only the bioprosthetic cusps, but also the aortic wall segment.. A synergistic approach was used to prevent bioprosthetic cusp and aortic wall calcification. Ethanol pretreatment of bioprosthetic heart valves was shown to inhibit cuspal calcification due to multiple mechanisms, including alterations of collagen structure and lipid extraction. AlCl3 pretreatment of bioprostheses to prevent calcification was also investigated; this alters elastin structure, inhibits alkaline phosphatase, and complexes with phosphoesters, thereby inhibiting aortic wall mineralization.. Experimental data from rat subdermal implants and sheep mitral replacements showed successful synergism with co-pretreatment of porcine aortic valve bioprostheses with ethanol and AlCl3. Significant inhibition of both cusp and aortic wall calcification was achieved by differential pretreatments that restrict AlCl3 to only the aortic wall, and not the cusp, accompanied by ethanol cuspal exposure. Sequential exposure of bioprostheses, first to AlCl3 and then to ethanol, led to unexpectedly severe cuspal calcification.. Differential pretreatment of stentless bioprostheses with ethanol and AlCl3 can effectively inhibit both cuspal and aortic wall calcification.

Topics: Aluminum; Animals; Aortic Valve; Bioprosthesis; Calcinosis; Coated Materials, Biocompatible; Collagen Type I; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Synergism; Drug Therapy, Combination; Elastin; Ethanol; Heart Valve Diseases; Heart Valve Prosthesis; Lipid Metabolism; Magnetic Resonance Spectroscopy; Male; Models, Cardiovascular; Rats; Rats, Sprague-Dawley; Sheep; Solvents

Stentless aortic heart valve substitutes, manufactured from biological tissues, are fixed with glutaraldehyde to cross-link collagen, reduce antigenicity, and sterilize the tissue. Despite improved cross linking, reduced antigenicity, and various anticalcification measures, the aortic wall tissue present in these prostheses tends to calcify. The aim of this study was to assess the morphology, collagen cross-link stability, and calcification potential of glutaraldehyde-preserved kangaroo aortic wall tissue as opposed to porcine aortic wall tissue. Porcine and kangaroo aortic wall tissues were fixed in 0.625% buffered glutaraldehyde. Histology and cross-link stability were examined. Calcification potential was determined in the subcutaneous rat model. Kangaroo aortic wall tissue was significantly (p < 0.01) less calcified than porcine aortic wall tissue (26.67 +/- 6.53 versus 41.959 +/- 2.75 microg/mg tissue) at 8 weeks. In conclusion, the histological differences between kangaroo and porcine aortic wall tissue correlate well with the reduced calcification potential of kangaroo aortic wall tissue. The reduced calcification potential could result in improved long-term durability of stentless kangaroo heart valves as bioprostheses.

Topics: Animals; Bioprosthesis; Calcinosis; Calcium; Collagen; Cross-Linking Reagents; Elastin; Fixatives; Glutaral; Heart Valve Prosthesis; Macropodidae; Male; Materials Testing; Rats; Rats, Wistar; Sus scrofa; Tissue Fixation

Bioprosthetic heart valves fabricated from glutaraldehyde crosslinked porcine aortic valves often fail because of calcific degeneration. Calcification occurs in both cusp and aortic wall portions of bioprosthetic heart valves. The purpose of this study was to discern the role of different aortic wall components in the calcification process. Thus, we selectively extracted cells and other extracellular matrix proteins from porcine aorta using trypsin/DNase/RNase, cyanogen bromide (CNBr), and sodium hydroxide (NaOH) treatments and subdermally implanted these pretreated aortas in young rats. Total DNA and phospholipid data showed complete removal of cells by CNBr and NaOH treatments, whereas trypsin/DNase/RNase treatment was effective in removing DNA but not phospholipids. As shown by amino acid data and Masson's trichrome staining, collagen was removed in CNBr and NaOH treatments. Control fresh porcine aorta calcified significantly after 21 days of implantation (Ca 26.4 +/- 2.4 microg/mg). Removal of cells and collagen from the aorta by CNBr treatment did not lead to a statistically significant reduction in aortic calcification (Ca 20.8 +/- 3.0 microg/mg). Moreover, partial degradation of elastin fibers caused by NaOH (during extraction) and trypsin treatment (after implantation) of the aorta significantly increased elastin-oriented calcification (Ca 94.4 +/- 9.3 and 58.4 +/- 4.6 microg/mg, respectively). Our results indicate that the elastin component of the aorta may undergo independent calcification irrespective of devitalized cell-mediated calcification observed in glutaraldehyde crosslinked aortas. Our results also demonstrate the importance of studying elastin-oriented calcification in decellularized elastin-rich aortic matrices currently used in tissue-engineering applications.

Topics: Amino Acids; Animals; Aorta; Aortic Diseases; Bioprosthesis; Calcinosis; Cross-Linking Reagents; Cyanogen Bromide; Dermis; DNA; Elastic Tissue; Elastin; Extracellular Matrix; Glutaral; Heart Valve Prosthesis; Male; Phospholipids; Postoperative Complications; Rats; Sodium Hydroxide; Swine; Transplantation, Heterologous; Transplantation, Heterotopic; Trypsin

Calcification of the media of arteries is common in patients with end-stage renal disease (ESRD) undergoing haemodialysis and is a major cause of arteriosclerosis. The aim of this study was to clarify the role of glycoxidative modification of elastin in the calcification of aortic media in this group of patients.. Samples of tunica media were obtained from non-atherosclerotic areas of the aortas of cadavers of seven non-diabetic patients with ESRD (age 65.5 +/- 10.6 years) and 10 age-matched controls (age 61.1 +/- 10.3 years). The localization of pentosidine, a major glycoxidation product, and calcium deposits in the media were examined using immunohistochemical and von Kossa staining, followed by orcein staining for elastin fibres. Tissue levels of pentosidine and calcium were measured in elastase-digested media using reversed high-performance liquid chromatography and atomic absorption spectrophotometry, respectively.. In aortic media, but not intima, immunostained pentosidine was observed along elastin fibres or in the extracellular spaces between them. Early calcification was manifest as small punctate calcified deposits along elastin fibres in the media. Advanced calcification was found as large, confluent calcified deposits in extracellular spaces between elastin fibres. Double staining showed co-localization of pentosidine and calcified deposits in the media. Both the staining density of pentosidine and calcification were more prominent in ESRD patients than in controls. The mean medial contents of both elastin-associated pentosidine and calcium were significantly higher in ESRD patients than in controls. In ESRD patients, the level of calcium in elastase-digested media correlated significantly with pentosidine levels, which increased in parallel with the duration of haemodialysis.. Our results indicate that glycoxidative modification of elastin in aortic media may be involved in the enhancement of medial calcification in ESRD patients on haemodialysis.

Topics: Aged; Aorta; Arginine; Calcinosis; Calcium; Elastin; Female; Humans; Immunohistochemistry; Kidney Failure, Chronic; Lysine; Male; Middle Aged; Tunica Media

Isolated systolic hypertension (ISH) is the predominant form of hypertension in the elderly population and drug treatment is unsatisfactory. We compared the efficacy of an endothelin-receptor antagonist (darusentan), an angiotensin-receptor blocker (irbesartan) and a thiazide diuretic (hydrochlorothiazide, HCTZ) to prevent and regress pulse pressure (PP) elevation and remodeling of large and small arteries, in a rat model of ISH obtained by the chronic administration of warfarin and vitamin K1 (WK).. Warfarin and vitamin K1 treatment for 4 or 8 weeks led to an elevation of PP, associated with increases in aortic calcium deposition and the ratio of collagen to elastin (C/E). Despite these changes in the composition of the aortic wall, the global structure of the aorta was unchanged. In contrast, an outward hypertrophic remodeling was observed in the middle cerebral artery. An early treatment with all drugs (darusentan, irbesartan, HCTZ) prevented PP elevation, changes of aortic media composition and the development of vascular remodeling. However, after 4 weeks of ISH, only darusentan and irbesartan reduced PP when administered from week 4 to 8. Darusentan was the most effective to regress existent aortic calcification, while only irbesartan reversed small artery hypertrophic remodeling.. During the development of ISH, drug treatment appears more beneficial when started early. Indeed, the three agents prevented PP elevation, aortic calcification and C/E increase in the aorta, and hypertrophy in small arteries. In contrast, once the disease is established, endothelin appears crucial in the maintenance of aortic calcification, while angiotensin II sustains small artery hypertrophy.

Topics: Animals; Antihypertensive Agents; Aorta, Thoracic; Arteries; Biphenyl Compounds; Calcinosis; Collagen; Disease Models, Animal; Elastin; Hydrochlorothiazide; Hypertension; Irbesartan; Male; Middle Cerebral Artery; Phenylpropionates; Pyrimidines; Rats; Rats, Wistar; Tetrazoles

The role of elastin in the aortic wall calcification and involved mechanisms were investigated. The major hypothesis of this work is that elastin is one of the major components to regulate calcification of bioprosthetic heart valve (BHV). The relationship between the elastin content and the calcification rate of the aortic wall was established using collagen-elastin matrices (CEM) made of varying ratios of collagen and elastin (90 and 10, 50 and 50, and 20 and 80). Biophysical characterization of CEM was performed by water content measurement and the tensile strength test. The conformational changes of the calcifiable matrix were evaluated as a function of elastin content using Fourier transform infrared (FTIR) spectroscopy. The calcium contents in CEM implanted in the rat subcutaneous model for 7 days were measured using atomic absorption (AA) spectroscopy. As the concentration of elastin in CEM increased from 10 to 80%, the total amount of calcium accumulated in CEM also increased. The calcium level in CEM containing the collagen and an elastin ratio of 20:80 was 20.16 +/- 0.70 microg/mg compared with 1.96 +/- 0.04 microg/mg in CEM containing the collagen and an elastin ratio of 90:10. The calcification rate of CEM pretreated with ethanol increased, as the elastin concentration in the CEM. However, the calcification rate of CEM pretreated with ethanol is significantly lower than that of the untreated control. The permeation rate of ethanol through CEM with the collagen and an elastin ratio of 20:80 (0.37 +/- 0.13 mmol/cm(2)/h) is significantly smaller than that through CEM with 90:10 (0.94 +/- 0.27 mmol/cm(2)/h). These results indicate that elastin has a significant role in tissue calcification and that elastin's resistance to ethanol penetration partially contributes less effectiveness of ethanol on aortic wall calcification.

Topics: Aorta; Calcinosis; Collagen; Elastin; Ethanol; Spectroscopy, Fourier Transform Infrared; Tensile Strength

Calcification of elastin occurs in many pathological cardiovascular diseases including atherosclerosis. We have previously shown that purified elastin when subdermally implanted in rats undergoes severe calcification and aluminum chloride (AlCl(3)) pretreatment of elastin inhibits calcification. In the present study we investigated whether matrix metalloproteinase (MMP) binding to elastin and elastin degradation is prevented by AlCl(3) pretreatment. Subdermal implantation of AlCl(3)-pretreated elastin showed significantly lower MMP-9 and MMP-2 activity surrounding the implant as compared to the control implants. AlCl(3) pretreatment also significantly inhibited elastin implant calcification at the seven-day implant period (AlCl(3)-pretreated 4.07 +/- 1.27, control 23.82 +/- 2.24 microg/mg; p<0.0001). Moreover, elastin gel zymography studies showed that gel pretreatment with AlCl(3) inhibited elastolysis by MMP-9. We also demonstrate significant suppression of MMP-2 activity in aortic wall segments of AlCl(3)-pretreated porcine bioprosthetic heart valve implants as compared to control valve implants in sheep mitral valve replacement studies. AlCl(3) pretreatment also significantly inhibited calcification of elastin in this model. Thus, we conclude that aluminum ion binding to elastin prevents MMP-mediated elastolysis and thus prevents elastin calcification.

Topics: Aluminum Chloride; Aluminum Compounds; Animals; Aorta; Calcinosis; Chlorides; Connective Tissue Diseases; Elastic Tissue; Elastin; Heart Valve Prosthesis; Immunohistochemistry; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Mitral Valve; Rats; Rats, Sprague-Dawley; Sheep; Swine

Elastin, a major extracellular matrix protein present in arterial walls provides elastic recoil and resilience to arteries. Elastin is prone to calcification in a number of cardiovascular diseases including atherosclerosis and bioprosthetic heart valve mineralization. We have recently shown that purified elastin when implanted subdermally in rats undergoes severe calcification. In the present study, we used this elastin implant model to investigate the molecular mechanisms underlying elastin calcification. Intense matrix metalloproteinase (MMP-2) and tenascin-C (TN-C) expression were seen in the proximity of the initial cal-cific deposits at 7 days. Gelatin zymography studies showed both MMP-2 (latent and active form) and MMP-9 expression within the implants. To investigate the role of MMPs in calcification, rats were administered a MMP inhibitor, (2S:-allyl-N:-hydroxy-3R:-isobutyl-N:-(1S:-methylcarbamoyl-2-ph enylet hyl)-succinamide (BB-1101) by daily injection, either systemically or at the implant site. The site-specific BB-1101 administration almost completely suppressed TN-C expression, as shown by immunohistochemical staining, within the implants. The systemic BB-1101 injections also significantly reduced TN-C expression within the elastin implants. Moreover, calcification of elastin implants was significantly reduced in the site-specific administration group (5.43 +/- 1.03 microg/mg Ca for BB-1101 group versus 21.71 +/- 1.19 for control group, P: < 0.001). Alizarin Red staining clearly showed that the elastin fibers were heavily calcified in the control group, whereas in BB-1101 group the calcification was scarce with few fibers showing initial calcification deposits. The systemic administration of BB-1101 also significantly reduced elastin calcification (28.07 +/- 5.81 control versus 16.92 +/- 2.56 in the BB-1101 group, P: < 0.05), although less than the site-specific administration. Thus, the present studies indicate that MMPs and TN-C play a role in elastin-oriented calcification.

Topics: Alkaline Phosphatase; Animals; Benzyl Compounds; Calcinosis; Dexamethasone; Disease Models, Animal; Drug Combinations; Elastin; Enzyme Inhibitors; Immunoenzyme Techniques; Implants, Experimental; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Pentoxifylline; Protease Inhibitors; Rats; Rats, Sprague-Dawley; Succinates; Tenascin

Elastin, an abundant structural protein present in the arterial wall, is prone to calcification in a number of disease processes including porcine bioprosthetic heart valve calcification and atherosclerosis. The mechanisms of elastin calcification are not completely elucidated. In the present work, we demonstrated calcification of purified elastin in rat subdermal implants (Ca(2+) = 89.73 +/- 9.84 microgram/mg after 21 days versus control, unimplanted Ca(2+) = 0.16 +/- 0.04 microgram/mg). X-ray diffraction analysis along with resolution enhanced FTIR spectroscopy demonstrated the mineral phase to be a poorly crystalline hydroxyapatite. We investigated the time course of calcification, the effect of glutaraldehyde crosslinking on calcification, and mechanisms of inhibition of elastin calcification by pretreatment with aluminum chloride (AlCl(3)). Glutaraldehyde pretreatment did not affect calcification (Ca(2+) = 89.06 +/- 17.93 microgram/mg for glutaraldehyde crosslinked elastin versus Ca(2+) = 89.73 +/- 9.84 microgram/mg for uncrosslinked elastin). This may be explained by radioactive ((3)H) glutaraldehyde studies showing very low reactivity between glutaraldehyde and elastin. Our results further demonstrated that AlCl(3) pretreatment of elastin led to complete inhibition of elastin calcification using 21-day rat subdermal implants, irrespective of glutaraldehyde crosslinking (Ca(2+) = 0.73-2.15 microgram/mg for AlCl(3) pretreated elastin versus 89.73 +/- 9.84 for untreated elastin). The AlCl(3) pretreatment caused irreversible binding of aluminum ions to elastin, as assessed by atomic emission spectroscopy. Moreover, aluminum ion binding altered the spatial configuration of elastin as shown by circular dichroism (CD), Fourier transform infrared (FTIR), and (13)C nuclear magnetic resonance (NMR) spectroscopy studies, suggesting a net structural change including a reduction in the extent of beta sheet structures and an increase in coil-turn conformations. Thus, it is concluded that purified elastin calcifies in rat subdermal implants, and that the AlCl(3)-pretreated elastin completely resists calcification due to irreversible aluminum ion binding and subsequent structural alterations caused by AlCl(3).

Topics: Aluminum Chloride; Aluminum Compounds; Amino Acids; Animals; Calcinosis; Cattle; Chlorides; Circular Dichroism; Disease Models, Animal; Durapatite; Elastin; Glutaral; Implants, Experimental; Magnetic Resonance Spectroscopy; Rats; Skin; Spectroscopy, Fourier Transform Infrared; X-Ray Diffraction

To examine the qualitative changes of elastin and the aorta related to calcification of human arteries, biochemical properties were measured, including calcium (Ca), phosphorus (P) and magnesium (Mg) contents in the aorta or in the elastin fraction in calcification, cholesterol content in atherosclerosis, desmosine content of cross-link, free thiol contents (free SH/total SH) and hydrophobic properties in the elastin fraction from the calcified portion, adjacent sites and another normal artery. The results from different sites of the calcified abdominal artery are as follows: The contents of Ca, P and Mg in aorta and the elastin fraction from the calcification site were higher than those at other sites. Moreover, Ca in the aorta and elastin fraction correlated positively with P and Mg. The content of cholesterol in the calcification site was the same as at other sites and did not correlate with Ca, P or Mg. The content of desmosine in the calcification site was significantly lower than that in different sites. In addition, its content was negatively associated with Ca and P in the elastin fraction and with the aortic Mg. The content of free thiol in the calcification site was similar to the other sites and correlated negatively with Ca and P in the aorta. The hydrophobicity in the calcification was similar to that at other sites, and was negatively associated with Ca and Mg in the elastin fraction.

Topics: Aged; Aged, 80 and over; Aorta; Aorta, Abdominal; Aortic Diseases; Calcinosis; Calcium; Cholesterol; Desmosine; Elastin; Humans; Magnesium; Middle Aged; Phosphorus; Sulfhydryl Compounds

Calcification has been examined in 250 samples of atherosclerotic lesions (types II to VI) of human carotid arteries using von Kossa and haematoxylin staining. Early calcification described as 'stippling' was first noted in stage III specimens, with intermediate and solid calcifications becoming increasingly prominent within advanced plaques, especially stages Vb and VI. Although the relative frequencies of stippling, intermediate and large calcified deposits varied between plaques of the same stage, the prevalent sites of calcification were recognized as the deeper regions of the intima and the atheroma. Immunolocalization and histochemical techniques were used to identify the associations of mast cells (MCs), macrophages, smooth muscle cells (SMCs), and elastin with the different stages of calcification. Early, dispersed stippling was commonly associated with local accumulations of macrophages (HAM56 and CD68-positive), MCs and extracellular MC tryptase, the presence of immunoreactive elastin, but the relative absence of SMCs. Intermediate stages of calcification described as 'morula' deposits were also associated with local increases in the numbers of macrophages and MCs. Larger calcified deposits, even within the same plaque specimen, showed no regular pattern of cellular or elastin associations. However, in the vast majority of specimens, macrophages represented the predominant cell type associated with different phases of calcification. By contrast, the calcification less frequently observed in the media beneath advanced plaques was commonly associated with SMCs and elastin; only rarely were macrophages or MCs present. These studies are the first to demonstrate that macrophages, MCs, and extracellular tryptase frequently occupy micro-environmental loci showing the first stages of calcification within the atherosclerotic plaque; similar associations with more advanced mineral deposits are discussed in relation to plaque rupture.

Topics: Aged; Aged, 80 and over; Arteriosclerosis; Calcinosis; Carotid Artery Diseases; Elastin; Female; Humans; Macrophages; Male; Mast Cells; Middle Aged; Muscle, Smooth, Vascular

Factors influencing the in vivo calcification of porcine collagen membranes containing elastic fibres were investigated by light and confocal microscopy. Two glutaraldehyde (GA) cross-linking protocols were used: a new one involving microwaving (NEWGA), and a conventional method using GA treatment at room temperature (OLDGA). We observed that the physical and chemical properties of implanted membranes will influence the acute inflammatory response, which initially checks the calcification process. The NEWGA membranes are superior in cases where rapid calcification is desired. In this context, it is proposed that NEWGA membranes may serve as a useful delivery system for bone morphogenetic protein (BMP).

Topics: Animals; Biocompatible Materials; Bone and Bones; Calcinosis; Collagen; Elastin; Glutaral; Inflammation; Male; Microscopy, Confocal; Microwaves; Prostheses and Implants; Rats; Rats, Wistar; Regeneration; Swine; Time Factors

The formation of calcified deposits in intimal thickenings of human aorta was studied by electron microscopy. Microzones of calcification were detected in about 20% of fatty streaks and were located predominantly in the deep musculoelastic layer of the intima. Calcified deposits formed only on previously existing structures including extracellular vesicles and unesterified cholesterol. Calcified deposits in the musculoelastic layer of the intima localised inside altered elastin fibres, but initiating the calcification of of elastin required the prior accumulation of cholesterol esters inside elastin fibres. Co-localization of calcified deposits and elastin fibres was followed by destruction of elastin. The present study suggests that at an early stage of development is atherosclerotic lesions, calcified deposits are formed by a physicochemical process which is not strongly controlled by the intimal cells. The recognition of calcified deposits in intimal thickenings support the hypothesis that a subset of fatty streaks might progress to fibrous plaques in human atherosclerosis.

Topics: Adolescent; Adult; Aorta, Thoracic; Aortic Diseases; Arteriosclerosis; Calcinosis; Elastin; Humans; Male; Middle Aged; Tunica Intima

Since structure-function correlations have not been determined for cryopreserved allograft heart valves, we studied 20 explanted valves in place several hours to nine years, as either orthotopic aortic valves/root replacements or right ventricle to pulmonary artery conduits. They were explanted primarily due to growth-related conduit or valve stenosis, valve regurgitation, or infection. Controls included seven unused cryopreserved allograft valves and 16 aortic valves removed from transplanted allograft hearts obtained at either autopsy (n = 15) or retransplantation (n = 1), two days to four years postoperatively, following myocardial rejection (n = 4), graft coronary arteriosclerosis (n = 4), and other (n = 8). Analysis included gross inspection, radiography, light microscopy, electron microscopy, and immunohistochemical studies (to allow identification/localization of endothelial cells, mononuclear inflammatory cells, and T and B lymphocyte subsets). Cryopreserved allograft valves implanted more than one day had progressively severe loss of normal structure and were devoid of surface endothelium and stainable deep connective tissue cells. Inflammatory cellularity varied from none (most valves) to prominent (primarily T lymphocytes in one valve). Transmission electron microscopy of three long term valvular allografts revealed no viable cells, remarkable preservation of the collagenous valve matrix and focal cell-oriented calcification. In contrast, aortic valves from transplanted hearts showed remarkable structural preservation, including layered architecture, endothelium and deep connective tissue cells; inflammatory infiltrates were generally sparse, even in cases with fatal myocardial rejection. We conclude that cryopreserved allograft valves are morphologically non-viable valves, whose structural basis for function seems primarily related to the largely preserved collagen.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Aortic Valve; Aortic Valve Insufficiency; Aortic Valve Stenosis; B-Lymphocyte Subsets; Calcinosis; Cell Survival; Collagen; Connective Tissue; Coronary Artery Disease; Cryopreservation; Elastin; Endocarditis, Bacterial; Endothelium; Follow-Up Studies; Graft Rejection; Humans; Immunohistochemistry; Leukocytes, Mononuclear; Microscopy, Electron; Pulmonary Valve; Pulmonary Valve Insufficiency; Pulmonary Valve Stenosis; Radiography; T-Lymphocyte Subsets; Tissue Preservation; Transplantation, Homologous

The rate of calcification within the human thoracic aorta from completion of body growth to advanced old age was examined. Fifty-eight aortae, obtained at necropsy, were dissected into four layers: the complete intima and the separated media, which was subdivided into three tissue samples of equal thickness, defined as the media-inner, -middle, and -outer layers. The sampling sites selected for analysis were from regions of the aortic surface that were free of atherosclerotic plaques. The calcium content within each tissue layer of the aorta was determined. Arterial wall thickness and the cholesterol content of the four layers were also measured. Intimal calcification increased progressively during aging: from 1.6 micrograms Ca/mg tissue at 20 years of age to 5.2 micrograms Ca/mg tissue by 90 years of age. When intima calcium concentration was expressed by tissue volume (w/v), no significant change during aging was found. Medial calcification, as w/v and by w/w, increased throughout aging. Calcium accumulation was most marked in the middle, elastin-rich layer of the media, increasing from 1.4 micrograms Ca/mg tissue at 20 years of age to 49.50 micrograms Ca/mg tissue by 90 years of age. Calcium levels also increased in the other media layers, but at a slower rate than that found within the middle media.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Aging; Aorta, Thoracic; Aortic Diseases; Arteriosclerosis; Calcinosis; Calcium; Cholesterol; Cohort Studies; Elastin; Female; Humans; Male; Middle Aged

Calcification frequently causes failure of porcine aortic valve bioprostheses; changes in collagen configuration induced by high-back-pressure fixation have deleterious effects on porcine aortic valve mechanics. Although modified porcine aortic valve bioprostheses that include the use of lower-pressure fixation and antimineralization treatments are used clinically, the morphologic characteristics of these valves are not known. We evaluated, by light and scanning and transmission electron microscopy, the comparative structural details of clinically processed Hancock Standard (no antimineralization treatment, 80 mm Hg fixation), Hancock II (T-6 antimineralization treatment, fixed initially at 1.5 mm Hg, then 80 mm Hg) and Intact (toluidine blue antimineralization treatment, zero-pressure-fixed [0 mm Hg]) porcine aortic valve bioprostheses as well as true low-pressure (1.5 mm Hg) fixed valves, zero-pressure-fixed porcine aortic valves (with no further treatment), and freshly fixed porcine aortic valve cusps as controls. Commercially processed valves had near-complete loss of endothelium and amorphous extracellular matrix and autolytic changes in the cuspal connective tissue cells. Both 80 and 1.5/80 mm Hg fixed valves, but not zero-pressure-fixed cusps (Intact valves, zero-pressure-fixed porcine aortic valves or immediately fixed porcine aortic valve cusps), had overall flattening and compression with near-complete loss of transverse cuspal ridges and collagen crimp; valves fixed at 1.5 mm Hg had intermediate features. T-6 and toluidine blue treatments induced no definite incremental microscopic changes attributable to the antimineralization treatment. No degenerative changes in collagen were noted in any valves that underwent antimineralization treatment. These studies indicate that valves fixed at zero but not at 80 or 1.5/80 mm Hg pressure retain collagen architecture virtually identical to that of relaxed native porcine aortic valve cusps and that the antimineralization treatments studied do not adversely affect collagen morphology.

Topics: Aortic Valve; Bioprosthesis; Biopsy; Calcinosis; Collagen; Elastin; Evaluation Studies as Topic; Glutaral; Heart Valve Prosthesis; Microscopy, Electron, Scanning; Microscopy, Electron, Scanning Transmission; Pressure; Tissue Preservation; Tolonium Chloride

The effects of glutaraldehyde pretreatment and allograft rejection in arterial grafts were assessed, using iso- and allografts in rats. An in situ glutaraldehyde fixation procedure was used to obtain homogeneous cross-linked vascular biografts. Ten Lewis rats were isografted, ten were isografted with a glutaraldehyde-treated aortic segment, ten were allografted with aortic segments from brown Norway (BN) inbred rats, and ten were allografted with glutaraldehyde-treated BN aortas. The macroscopic and microscopic appearances of the grafts were analyzed 3 weeks after the initial surgery. Immunological injury to the media and the intimal response were quantified morphometrically after monochromatic staining of cell nuclei (hematoxylin after periodic acid), elastin (orcein), and calcification (Von Kossa). Untreated isografts were normal. Untreated allografts showed the classical signs of arterial wall rejection: adventitial inflammatory granuloma, reduced medial thickness and smooth muscle cell density, and greatly increased intimal thickness (P < 0.005). Glutaraldehyde treatment significantly decreased the medial thickness in both iso- and allografts (P < 0.001) and prevented the intimal proliferative response (P < 0.005), but did not change adventitial inflammation. It also induced massive calcification mainly in isografts (P < 0.001). Histomorphological modifications of glutaraldehyde-treated grafts are consistent with a partial protective effect of glutaraldehyde against the rejection process, but also with an induction of a nonspecific inflammatory reaction. Glutaraldehyde-induced cross-linking of the extracellular matrix was responsible for ectopic calcification of the arterial grafts which was independent of the rejection process.

Topics: Animals; Aorta; Calcinosis; Elastin; Glutaral; Graft Rejection; Rats; Rats, Inbred BN; Rats, Inbred Lew; Transplantation, Homologous; Transplantation, Isogeneic

We have previously studied the process of calcification in bioprosthetic porcine heart valves crosslinked with glutaraldehyde. Observations using light microscopy had indicated that calcification of elastic fibers occurs in implanted heart valves, in addition to calcification associated with collagen fibers. To determine the contribution of elastin to the process of calcification, small pieces of rabbit aorta were cross-linked with 0.2% glutaraldehyde, rinsed in buffer, and implanted subcutaneously in young adult male rats. Cross-linked jugular vein implants were included as controls. After an implantation period of 1 month or longer, we observed many areas of calcification in the aortic media associated with elastin and fewer such areas associated with collagen. The elastin-rich aortic tissues accumulated more calcium than venous tissues. Calcium deposits appeared similar in both allogenic and xenogeneic implants. Calcified areas viewed under the electron microscope included intercellular nonfibrous material. Calcified areas involved predominantly the outer layers of elastic fibers. Calcific deposits included needle-like crystals of hydroxyapatite but often consisted of an amorphous flocculant material surrounded by crystals. The close spatial relationship of hydroxyapatite crystals and elastic membranes seen in this study may be relevant to the initiation of dystrophic calcification in glutaraldehyde cross-linked aortic grafts.

Topics: Animals; Calcinosis; Calcium; Cross-Linking Reagents; Disease Models, Animal; Elastin; Glutaral; Male; Microscopy, Electron; Prostheses and Implants; Rabbits; Rats; Vascular Diseases

Topics: Blood Vessels; Bruch Membrane; Calcinosis; Elastin; Fibrillins; Genes, Recessive; Humans; Microfilament Proteins; Microscopy, Electron; Mutation; Pseudoxanthoma Elasticum; Skin

In this paper we demonstrate that near infrared Fourier transform Raman spectroscopy provides unprecedented biochemical information about the extent of atherosclerosis in human aorta. In particular, elastin, collagen, cholesterol, cholesterol esters, lipids, carotenoids, and calcium apatite deposits all can be discerned by using this technique, permitting study of each stage in the disease process. Additionally, these moieties can be detected over 1.5 mm below the irradiated surface of the tissue, possibly allowing extraction of three-dimensional information about the histology of atherosclerotic plaques. We propose that this technique may be utilized for in situ optical histochemical analysis of atherosclerosis in particular and human disease in general.

Topics: Aorta; Apatites; Arteriosclerosis; Calcinosis; Calcium; Collagen; Elastin; Fourier Analysis; Humans; Lipids; Muscle, Smooth, Vascular; Reference Values; Spectrum Analysis, Raman

Intravascular ultrasound imaging is a new method in which high resolution images of the arterial wall are obtained with use of a catheter placed within an artery. An in vitro Plexiglas well model was used to validate measurements of the luminal area, and an excellent correlation was obtained. One hundred thirty segments of fresh peripheral arteries underwent ultrasound imaging and the findings were compared with the corresponding histopathologic sections. Luminal areas determined with ultrasound imaging correlated well with those calculated from microscopic slides (r = 0.98). Three patterns were identified on the ultrasound images: 1) distinct interface between media and adventitia, 2) indistinct interface between media and adventitia but different echo density layers, and 3) diffuse homogeneous appearance. The types of patterns depended on the relative composition of the media and adventitia. Calcification of intimal plaque obscured underlying structures. Atherosclerotic plaque was readily visualized but could not always be differentiated from the underlying media.

Topics: Adult; Aged; Aged, 80 and over; Arteries; Arteriosclerosis; Calcinosis; Collagen; Elasticity; Elastin; Humans; Middle Aged; Models, Structural; Muscle, Smooth, Vascular; Necrosis; Reference Values; Ultrasonography

Ectopic calcification of diseased tissues or around prosthetic implants can lead to serious disability. Therefore, calcification of implants of glutaraldehyde-cross-linked collagenous tissues and reconstituted collagen was compared with mineralization induced by demineralized bone matrix (DBM). Whereas implants of DBM accumulated large amounts of calcium and a bone-specific gamma-carboxyglutamic acid protein (BGP or osteocalcin) following implantation in both young and older rats, implants of cross-linked pericardium calcified with only traces of BGP. Glutaraldehyde-cross-linked DBM failed to calcify after implantation in 8-month-old rats for 2-16 weeks. Implants of cross-linked type I collagen exhibited small calcific deposits 2 weeks postimplantation but calcium content eventually dropped to levels equal to those of soft tissues as the implants were resorbed. The calcium content of DBM implanted in 1- and 8-month-old rats reached comparable levels after 4 weeks, but the BGP content was approximately twice as high in the younger animals than in the older ones. Glutaraldehyde-cross-linked implants of DBM, tendon, and cartilage calcified significantly in young but not in old animals. This form of dystrophic calcification was associated with only trace amounts of BGP. Alkaline phosphatase activity was high in implants of DBM and undetectable in implants of cross-linked collagenous tissues. These results show that implants of glutaraldehyde-cross-linked collagenous tissues and reconstituted collagen calcify to different extents depending upon their origin and the age of the host, and that the mechanism of dystrophic calcification differs significantly from the process of mineralization associated with bone induction as reflected by alkaline phosphatase activity and BGP accumulation.

Topics: Alkaline Phosphatase; Animals; Bone and Bones; Bone Development; Bone Matrix; Calcinosis; Calcium; Calcium-Binding Proteins; Collagen; Elastin; Glutaral; Male; Minerals; Osteocalcin; Prostheses and Implants; Proteoglycans; Rats; Rats, Inbred Strains

Lung calcification was detected in four out of 29 long-term dialysed patients on whom postmortem examinations were performed between 1967 and 1980. On light microscopy, calcification showed either a finely granular and linear localization along the alveolar septa, or a coarse and widespread parenchymal distribution. Histochemical studies revealed evidence of calcium, magnesium and phosphate ions in the deposits. Ultrastructural examination of the less severely involved alveolar septa showed selective deposition of calcium salts within an increased amount of elastin. The deposits consisted of electron dense roundish granules with a concentric laminar structure. They appeared either single or conglomerated in polycyclic formations, supposedly representing the progressive steps of the mineralization process, at first localized within elastin and progressively spreading outside it. The high magnesium content of the deposits suggests that the serum concentration of this ion may play an important role in visceral calcification of long-term dialysed patients.

Topics: Adult; Calcinosis; Calcium; Elastin; Female; Humans; Kidney Failure, Chronic; Lung; Lung Diseases; Magnesium; Male; Middle Aged; Renal Dialysis

Topics: Animals; Calcinosis; Calcium; Chemical Phenomena; Chemistry; Elastic Tissue; Elastin; Rats

Topics: Animals; Aorta; Arteriosclerosis; Calcinosis; Cholesterol; Demecolcine; Diet, Atherogenic; Elastin; Etidronic Acid; Mitosis; Organ Size; Rabbits

Topics: Amino Acid Sequence; Arteries; Calcification, Physiologic; Calcinosis; Elastin; Hot Temperature; Hydrogen Bonding; Lipid Metabolism; Membranes, Artificial; Models, Molecular; Protein Conformation; Solubility; Tropoelastin

Skin biopsy material obtained from 2 patients with pseudoxanthoma elasticum associated with congenital hyperphosphatasia was compared with skin biopsy material obtained from 3 patients with pseudoxanthoma elasticum unassociated with any other disease. Clinically normal skin showed calcification of normal-appearing elastin as the initial change in pseudoxanthoma elasticum. Elastin fibers become more ravelled as the disease progresses. A previously unreported early transient calcification of collagen was demonstrated. Other abnormal collagen forms may also be present. The Von Kossa stain appears to be the stain of choice for identifying these earliest histologic changes.

Topics: Adolescent; Adult; Biopsy; Calcinosis; Collagen; Elastin; Female; Humans; Middle Aged; Phosphoric Monoester Hydrolases; Pseudoxanthoma Elasticum; Skin

Topics: Arteriosclerosis; Binding Sites; Calcinosis; Calciphylaxis; Calcium; Circular Dichroism; Elastic Tissue; Elastin; Humans; Isoelectric Focusing; Microscopy, Electron; Models, Structural; Molecular Conformation; Molecular Weight; Peptide Chain Initiation, Translational; Peptide Initiation Factors; Spectrophotometry, Infrared; Templates, Genetic

Topics: Arteries; Arteriosclerosis; Binding Sites; Calcinosis; Calcium; Circular Dichroism; Elastin; Humans; Lipid Metabolism; Microscopy, Electron

Topics: Adult; Aged; Aging; Amino Acids; Animals; Aorta; Apatites; Arteriosclerosis; Calcinosis; Calcium; Calcium Radioisotopes; Carbonates; Copper; Elastic Tissue; Elastin; Humans; Male; Microscopy, Electron; Middle Aged; Phosphates; Rabbits; Tritium; X-Ray Diffraction

Topics: Angioid Streaks; Biopsy; Calcinosis; Calcium; Child; Elastin; Female; Histiocytes; Histocytochemistry; Humans; Male; Microscopy, Electron; Middle Aged; Phosphorus; Pseudoxanthoma Elasticum; Skin; Syndrome

Topics: Analysis of Variance; Animals; Aorta; Blood Pressure; Calcinosis; Calcium; Collagen; Connective Tissue; Elastin; Hypertension, Renal; Phosphates; Probability; Proteins; Rabbits; Regression Analysis; Vitamin D

Neutral, uncharged binding sites for calcium ions are proposed for elastin and collagen. The sites utilize, particularly from a conformational viewpoint, the most striking feature of the amino acid composition, that is, the high glycine content. Glycines favor the formation of beta-turns and associated conformations that are known, from studies on ion-transporting antibiotics, to interact with cations. By analogy with certain antibiotics, which are uncharged polypeptides and depsipeptides that bind cations by coordination with neutral acyl oxygens, it is proposed that calcium-ion binding also utilizes uncharged coordinating groups, i.e., neutral sites, in the protein matrix. The protein matrix, which becomes positively charged by virtue of the bound calcium ions, attracts neutralizing phosphate and carbonate ions, which then allow further calcium ion binding. The driving force is, therefore, the affinity of calcium ions for the neutral nucleation sites. The charge neutralization theory of calcification suggests a fundamental role of organic anions, for example sulfated mucopolysaccharides, in regulating bone formation and in retardation of atherosclerosis. The proposed mechanism contains elements that tend to unify several theories on the pathogenesis of atherosclerosis.

Topics: Arteriosclerosis; Binding Sites; Calcification, Physiologic; Calcinosis; Calcium; Carbonates; Collagen; Elastin; Glycine; Glycosaminoglycans; Models, Structural; Phosphates

Topics: Acetates; Animals; Binding Sites; Calcinosis; Calcium Chloride; Cattle; Chemical Phenomena; Chemistry; Circular Dichroism; Elastin; Ethanol; Fluoroacetates; Hydrochloric Acid; Sodium; Sodium Chloride

Topics: Adolescent; Adult; Aorta, Thoracic; Arteriosclerosis; Calcinosis; Calcium; Cholesterol; Elasticity; Elastin; Fatty Acids; Humans; Lipid Metabolism; Phosphates; Pressure; Protein Binding

Topics: Apatites; Calcinosis; Calcium; Calcium Isotopes; Chlorine; Elastin; Hydrogen-Ion Concentration; In Vitro Techniques; Ligaments; Protein Binding; Radioisotopes; Sodium Isotopes

Topics: Animals; Antibody Formation; Antigen-Antibody Reactions; Arteriosclerosis; Blood Proteins; Calcinosis; Cholesterol; Diet, Atherogenic; Elastic Tissue; Elastin; Rabbits

Topics: Animals; Aorta, Thoracic; Aortic Diseases; Calcinosis; Calcium; Carbon Isotopes; Elastin; Glycine; Heart Diseases; Kidney Diseases; Magnesium Deficiency; Male; Rats

Topics: Adrenal Glands; Age Factors; Animals; Aortic Diseases; Arteriosclerosis; Blood Vessels; Calcinosis; Cholesterol; Elastin; Endarteritis; Epinephrine; Female; Freund's Adjuvant; Glycoproteins; Glycosaminoglycans; Glycoside Hydrolases; Humans; Injections, Intravenous; Lipase; Lipid Metabolism; Male; Necrosis; Pancreatic Elastase; Rabbits; Sex Factors; Stress, Physiological; Thyroxine; Time Factors; Transplantation, Homologous

Topics: Aged; Aging; Aorta; Apatites; Calcinosis; Connective Tissue; Elastic Tissue; Elastin; Female; Glycosaminoglycans; Humans; Male; Microscopy, Electron; Middle Aged; Muscle, Smooth

Topics: Aortic Diseases; Calcinosis; Chemical Phenomena; Chemistry; Copper; Crystallization; Elastin; Hydroxyapatites; Solutions

Topics: Achilles Tendon; Animals; Calcification, Physiologic; Calcinosis; Calcium; Chemistry Techniques, Analytical; Collagen; Edetic Acid; Elastin; Eye; Histology; Rabbits; Rats; Research; Tendinopathy; Tendons; Transplantation

Topics: Biochemical Phenomena; Biochemistry; Calcinosis; Coloring Agents; Dermatomyositis; Elastin; Endopeptidases; Histological Techniques; Pathology; Specimen Handling; Staining and Labeling; Trypsin

Topics: Anti-Bacterial Agents; Antimetabolites; Aortic Diseases; Benzoates; Calcinosis; Calcium; Cyanides; Dicumarol; Dinitrophenols; Elastic Tissue; Elastin; Oligomycins; Ouabain; Pharmacology; Phosphorus; Physiology; Research; Sulfhydryl Compounds