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Background: Abdominal aortic aneurysm (AAA) is a prevalent disease affecting around 5% of the population aged more than 65 years. The exact etiology and physiopathology of AAA still raises questions, and elective surgery is currently the only treatment option for this often progressive disease. In this study, we hypothesized and tested a pathophysiological model that depicts AAA as an inflammation-triggered autoimmune disease with remnant vessel wall peptide fragments as the antigen., Methods: A pilot study with male AAA patients (n = 14) and male controls (n = 8) was conducted. In both study groups, peripheral blood monocytes and plasma were separated from whole blood by centrifugation. An ELISpot test was performed on cultured white blood cells for the presence of elastin-specific T-lymphocytes. An Enzyme-linked immuno sorbent assay (ELISA) was performed on plasma for the presence of elastin-specific IgG molecules., Results: ELISpot interferon-gamma secretion in AAA (7.7 ± 9.5%) and control (4.6 ± 3.5%) and ELISA anti-elastin IgG titer in AAA (77.5 ± 17.8%) and control (78.2 ± 31.5%) were not significantly different (P = 0.94 and P = 0.55, respectively). Both results are expressed as a percentage relative to the respective positive and negative control., Conclusions: The results of our pilot study did not indicate a clear and invariable autoimmune process directed against remnant elastin peptide fragments. Further research into the model mechanics and a possible antigen is still necessary. In the mean time, the model as presented here already offers a pathophysiological framework to further research into the possible remnant epitope-driven AAA etiology., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background and Objective: A correlation between the levels of antibodies to alpha-elastin (alpha-AEAb) and tropoelastin (tropo-AEAb) and the corresponding peptide concentration is found in human serum in health and disease. Serum elastin peptide and anti-elastin antibodies (AEAb) levels are age-related and vary with the stages of atherosclerotic vascular damage. This study aims to determine if elastin metabolism (assessed by the ratio of tropo-AEAb to alpha-AEAb) differs in patients with symptomatic carotid stenosis versus subjects with asymptomatic stenosis., Patients and Methods: Alpha-AEAb and tropo-AEAb were measured by ELISA in blood sera of 65 patients with ultrasound verified high-grade symptomatic carotid stenosis (resulting in stroke 1-7 days before measurement) compared to 51 patients with asymptomatic stenosis., Results: Serum anti-alpha-elastin IgG levels are extremely increased in symptomatic versus asymptomatic carotid stenosis. The ratio of tropo-AEAb (reflecting elastin synthesis) to alpha-AEAb (a function of elastin degradation) was 3.7 in symptomatic stenosis versus 14.2 in asymptomatic stenosis (p<0.001)., Conclusions: There is a significant difference in elastin metabolism in patients with symptomatic carotid stenosis versus asymptomatic stenosis. The ratio of tropo-AEAb to alpha-AEAb as an index of elastin synthesis/degradation proves useful in investigation of atherosclerotic lesions and may represent a new immunologic marker for carotid plaque destabilization., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: After deep excision of burn eschar down to the muscle fascia patients have a non-reversible loss of the skin and underlying subcutaneous tissue. These patients would benefit from the development of a sufficient epidermal, dermal, and hypodermal tissue-engineered replacement provided by new technologies of tissue engineering. The aim of the present study was to determine whether keratinocytes and preadipocytes grow simultaneously on a bovine-derived collagen-elastin matrix under in vitro conditions in order to obtain a multi-layer skin substitute., Methods: Human keratinocytes as well as human preadipocytes were seeded onto a collagen-elastin matrix (Matriderm®). Human preadipocytes were isolated from human subcutaneous adipose tissue and seeded onto the scaffold directly after isolation. Keratinocytes were isolated from fresh human split-thickness skin harvests and seeded onto the surface of the scaffold after 4 days of proliferation. Twenty one days after seeding all scaffolds were histologically evaluated, using hematoxylin eosin, immunohistochemical staining with collagen IV as well as immunofluorescence labeling with anti-Ki67 antibody and DAPI (4',6-diamidino-2-phenylindole)., Results: Simultaneous growth of keratinocytes and preadipocytes could be observed on the collagen-elastin matrix. Keratinocytes adhered well to the surface of the matrix and formed a confluent epidermis-like layer. Preadipocytes adhered well and also penetrated into the deeper layers of the matrix., Conclusion: In this study, a collagen-elastin matrix served as a suitable scaffold for simultaneous culturing of preadipocytes and keratinocytes. Preadipocytes showed good penetration into deeper layers of the scaffold, whereas keratinocytes attached only to the uppermost surface of the matrix. This approach towards a multi-layered skin substitute might be a useful asset for future reconstructive surgery., (Copyright © 2010 Elsevier Ltd and ISBI. All rights reserved.)

Background: Antenatal pulmonary inflammation is associated with reduced risk for respiratory distress syndrome but with an increased risk for bronchopulmonary dysplasia (BPD) with impaired alveogenesis., Objective: We hypothesized that fetal systemic inflammation induced by intravenous (IV) lipopolysaccharide (LPS) would affect lung development in utero., Study Design: Twenty-one fetal sheep were instrumented (107 days gestational age). Control fetuses received saline (N = 12) and 9 in the study group received 100 ng of LPS IV 3 days after surgery. Animals were assessed for lung maturation and structure after 3 (N = 5) and 7 (N = 4) days., Results: Interleukin-6 concentration increased in the bronchoalveolar lavage more than 40-fold 3 days after LPS IV. Processing of pro-surfactant protein (SP)-B to mature SP-B and increased SP-B concentrations were shown 7 days after LPS IV. Deposition of elastin fibers at sites of septation was disturbed within 3 days after LPS IV., Conclusion: Lung maturation and disturbed lung structure occurred after short-term exposure to fetal inflammation and suggests new targeted therapies for BPD.

Elastin breakdown products are found in the serum of all human subjects. The presence of these elastin-derived peptides (EDP) and the corresponding antibodies in circulation leads to formation of circulating immune complexes (CIC). The aim of this study was to determine if serum level of free-EDP (unbound in CIC) correlate with the development of microvascular complications in children with Type 1 (insulin-dependent) diabetes mellitus. To this end we used a method for detecting immune complexes (CIF-ELISA) in combination with an ELISA for detection of EDP. The levels of free EDP were studied in sera of 81 diabetic children (mean age 13.46+/-3.51 years, diabetes duration 5.17+/-4.21 years). Forty-two of the children had vascular complications (group 1) and 39 were without vascular complications (group 2). Twenty-one healthy children (mean age 12.6+/-2.47 years) were used as controls. Diabetics showed significantly higher levels of free EDP (68.1+/-25 ng/ml versus 51+/-12.5 ng/ml; p=0.003) compared to the control group. In group 1, free EDP showed significantly higher levels than controls (78.9+/-25.6 ng/ml versus 51+/-12.5 ng/ml; p=0.0001). About 38 of 81 (47%) patients were positive for free EDP (30/42--71% in group 1 and 8/39--21% in group 2). Free EDP levels in all diabetics showed a correlation with insulin dose (r=0.23; p=0.041), and microalbuminuria (r=0.57; p=0.0001). Patients who had vascular pathology showed a correlation of free EDP with microalbuminuria (r=0.41; p=0.0081), retinopathy (r=0.32; p=0.041), insulin dose (r=0.37; p=0.02), HbA1c (r=0.35; p=0.03), systolic blood pressure (r=0.30; p=0.045) and total cholesterol (r=0.36; p=0.02). These findings suggest that elevated levels of free EDP are associated with the development of diabetic vascular complications in children.

Background: Chronic inflammation is a characteristic feature of abdominal aortic aneurysms (AAAs), but the molecular signals responsible for recruiting monocytes into the outer aortic wall are unresolved. The purpose of this study was to examine whether AAA tissues elaborate chemotactic activity for mononuclear phagocytes and to determine whether this activity is attributable to interactions between elastin degradation peptides (EDPs) and their cell surface receptor, the 67-kD elastin binding protein (EBP)., Material and Methods: Soluble proteins were extracted from human AAA tissues, and chemotactic activity for differentiated U937 mononuclear phagocytes was measured by use of a modified Boyden chamber. Chemotactic activity induced by N -formyl-Met-Leu-Phe was used as a positive control and checkerboard analysis was used to distinguish chemotaxis from chemokinesis. Inhibition of chemotaxis was tested by peptide competition, blocking antibodies and galactosugar-mediated dissociation of the 67-kD EBP., Results: AAA extracts stimulated a concentration-dependent increase in monocyte migration that reached up to 24% of the maximal effect induced by N -formyl-Met-Leu-Phe. Checkerboard analysis demonstrated that AAA extracts stimulated chemotaxis without a chemokinetic effect. AAA-derived chemotactic activity was eliminated by competition with Val-Gly-Val-Arg-Pro-Gly (VGVAPG), a repetitive peptide found in human elastin that binds to cellular elastin receptors, and decreased nearly 40% in the presence of BA-4, an antielastin monoclonal antibody that can block EDP-mediated chemotactic activity. Monocyte chemotaxis in response to both VGVAPG and AAA extracts was abolished in the presence of lactose, a galactosugar that specifically dissociates the 67-kD EBP, but it was unaffected by either glucose, fructose, or mannose., Conclusions: These findings indicate that soluble EDPs released within human AAA tissue can subsequently attract mononuclear phagocytes through ligand-receptor interactions with the 67-kD EBP, thereby providing a plausible molecular mechanism to explain the inflammatory response that accompanies aneurysmal degeneration. Better understanding of factors regulating inflammatory cell recruitment may lead to novel forms of therapy for early stages of aneurysmal degeneration.

Elastic fibers form a complex network that contributes to the elasticity of connective tissues. Alterations in the elastic fiber network are involved in several disease affecting organs in which compliance of the connective tissue is essential: skin, main vasculature, lung, joints, muscle, and ligament. The aim of our work was to study the deposition, maturation, and organization of elastic fiber components in a dermal equivalent model consisting of collagen-GAG-chitosan seeded with fibroblasts. The influence of keratinocytes was studied in parallel, thus constituting a skin equivalent model. These models were examined by transmission electron microscopy (TEM) and by immunohistochemistry to determine the staining patterns of fibrillin-1 and elastin proteins representative of the microfibrillar framework and of the elastic fibers, respectively. After 2 mo of fibroblast culture in the dermal equivalent, elastin was undetectable, whereas fibrillin-1 staining was weak and microfibrils were infrequently observed by TEM. In the skin equivalent, fibrillin-1 and elastin were detected by immunostaining 15 d after epidermization and TEM revealed the typical structure and organization of the elastic network in the dermis, with elastin deposition on the microfibrillar scaffold. This in vitro skin equivalent model is to our knowledge the first in which elastic fibers have been detected, thus demonstrating the influence of keratinocytes on the maturation and organization of the elastic network.

A search for antibodies reactive against a total human aorta extract and its main protein components such as elastin, fibronectin and collagen was attempted by electroimmunetransference and ELISA. Thirty five sera from clinically and angiographically proven diagnosis of Takayasu Arteritis patients were compared with 32 sera from people without abnormalities. Non specific binding was found on electroimmune transference and no difference was shown in optical density readings in ELISA, therefore, we did not demonstrate the presence of antiaorta specific antibodies in this vasculitic condition. Our findings are in agreement with several authors, the contribution of humoral immunity to pathogenesis of Takayasu Arteritis has not been proved yet.

Discrepancies exist between the reported values for the mean elastin peptide (EP) concentration in human sera. In order to understand these discrepancies, several EP preparations were obtained in vitro and monoclonal and polyclonal antibodies were produced against them. These different EP preparations and antibodies were used in an enzyme-linked immunosorbent assay (ELISA) to study cross-reactivity between EP preparations and to quantitate EP concentration in human sera. The method of purification of elastin, the method of hydrolysis of elastin and the molecular weight of EP influence their reactivity with antibodies and the results of EP measurements in human sera. However, there is a good correlation between EP measurements carried out in several human sera with the different EP preparations and different antibodies. Although absolute values of the EP concentrations varied with the EP preparation and antibodies used for the ELISA, the variations of this EP concentration measured from one human serum to another are significant.

Markers of elastin metabolism were estimated in sera of children from families with a high risk of atherosclerosis (ATH). There was no statistically significant difference in the serum elastase-like activity between the groups studied. The concentration of elastin-derived peptides was statistically significantly elevated in the ATH group. Anti-elastin antibodies were found to be present in 73% of ATH children, while they circulated in 5% of control subjects only. Antibodies observed in the youngest ATH children were of the IgM type, suggesting the initial stage of the autoimmunization to elastin. The data obtained in this study may indicate an enhanced metabolism of elastin in ATH children.

Association of group A streptococci with acute rheumatic fever and valvular heart disease is well established; however the basis of valve injury remains unclear. In this study, anti-streptococcal monoclonal antibodies (MAbs) cross-reactive with myocardium were reacted with sections from 22 rheumatic valves, nine normal, five endocarditic, one 'floppy,' and one Marfan valve. In immunohistochemical studies, MAb reactivity was observed with cardiac myocytes, smooth muscle cells, cell surface and cytoplasm of endothelial cells lining valves, and valvular interstitial cells. Endothelial basement membrane and elastin fibrils reacted with the MAbs, whereas collagen was unreactive. Similar reactivity was seen with sera from acute rheumatic fever patients. The anti-streptococcal MAbs reacted with intravalvular myosin and vimentin in Western blots, and purified elastin competitively inhibited the binding of the anti-streptococcal MAbs to whole group A streptococci. The data show that human heart valves have numerous sites of immunoreactivity with anti-streptococcal MAbs and acute rheumatic fever sera of potential importance in the pathogenesis of rheumatic valvular injury.

Appreciable amounts of glycosaminoglycans have been found by immunocytochemistry within mature elastin fibers of human dermis. On thin sections, elastin fibers showed antigenic sites for monoclonal antibodies recognizing the unsaturated units remaining after digestion of hyaluronic acid with Streptomyces hyaluronidase and after digestion of dermatan and chondroitin sulfates with chondroitinase ABC. Moreover, sectioned elastin fibers were positive towards antibodies raised against synthetic peptides corresponding to amino acid sequences near the N-terminus of the protein core of small matrix proteoglycans PGI and PGII, respectively (Fisher et al., J. Biol. Chem. 262, 9702-9708 (1987)). This is the first demonstration that highly hydrophylic molecules are strictly associated with normally cross-linked elastin. The presence of highly hydrated molecules within the elastin polymer could greatly influence its physiological properties and behavior in pathology.

We describe the generation of a monoclonal antibody library to ocular zonule components and the characterization of three monoclonal antibodies: 1) one specific for microfibrillar associated glycoprotein (MAGP), a component of both ocular zonules and microfibrils of elastin fibers, 2) an antibody to an as yet unidentified 70,000 dalton antigen that is present in abundance in the extracellular matrix (ECM) of elastin-producing cells, and 3) an antibody reacting with the 67000 dalton subunit of the elastin receptor. The presence of antigenic determinants common to the ocular zonule and elastic fiber microfibrils suggests that zonules, which can be obtained in relatively pure form, can provide a valuable resource for characterizing proteins common to both microfibrillar structures.

The development of immunity to homologous connective tissue antigens was studied with respect to aging in the tight-skin (Tsk) mouse mutant. A delayed-type hypersensitivity (DTH) response to elastase-solubilized lung peptides in Tsk/+ mice, which became evident at 10 weeks of age and increased in intensity until 22 weeks, was observed. Tsk mice did not demonstrate significant DTH responses when challenged with type I or IV collagen, and normal (+/+) littermates of all ages did not respond to any of the antigens under study. DTH responses could be adoptively transferred to normal +/+ and C57BL/6 mice with spleen cells from 30-week-old Tsk/+ mice; treatment with anti-Thy 1.2 antibodies plus complement significantly reduced the ability of these Tsk/+ cells to transfer DTH reactivity. No antibody activity to the antigens under study could be detected in the sera of Tsk/+ or +/+ mice at any age. These results are discussed with regard to the pathological manifestations observed in the Tsk/+ mutant mouse.

Research on the pathogenesis of experimental emphysema has involved studies of the distribution of and destruction of elastin in the alveolar interstitium. The ill-defined organization of elastin in the alveolar interstitium makes it difficult to identify the elastin specifically by staining procedures ordinarily used for electron microscopy. This problem becomes more significant when the elastic tissue is fragmented during emphysema development and localization of the elastin fragments is essential. Therefore, a specific technique using high-titer antibodies against purified canine lung elastin was developed. The primary antibody was used on preembedded or etched postembedded sections. Localization of the antielastin IgG was accomplished with ferritin-labeled rabbit antisheep IgG as the secondary antibody. Treatment with the preimmune serum gave negligible ferritin background staining. The antielastin antibody did not react with lung connective tissue proteins such as the microfibrillar component of elastin or collagen or proteoglycan. The antielastin antibody appeared to be species specific. The method may be useful for studies of experimental emphysema.

Rat aortae were fixed, reduced into fragments, fixed again according to the fracture-label method from Pinto da Silva et al. (1981) and labelled with anti-elastin antibodies. We thus demonstrate that this method, extensively used with lectins, is also usable with antibodies to investigate components of the extracellular matrix.

Immunization of rabbits with soluble elastin peptides (kappa 1-elastin) in complete Freund's adjuvant resulted in morphological and biochemical modifications in aorta and in lung arterioles. The elastic fibers of both tissues appeared fragmented at the light microscopical and ultrastructural levels. The presence of IgG at the site of lysed elastic fibers could be evidenced by immunological techniques. In agreement with these findings, a significantly increased elastase-type protease activity could be demonstrated in aorta extracts from the immunized animals as compared to those obtained from control animals. The biosynthetic activities of aorta explants of rabbits immunized with kappa 1-elastin maintained in organ culture conditions were considerably reduced, as shown by the decrease of incorporation of [14C]lysine and [14C]glucosamine in aorta macromolecules. These results show that anti-elastin antibodies may well be involved in the pathological modifications of the arterial wall and especially in the triggering of the degradation of elastic lamellae.

The nature of elastotic material in sun-damaged human skin was investigated by indirect immunofluorescence. Antibodies were used against the following components of the dermis: type I and type VI collagens, aminopropeptide of type I and type III procollagens, fibronectin, elastin, microfibrillar proteins, and basement membrane represented by the 7S domain of type IV collagen, laminin, and nidogen. The elastotic material exhibited marked fluorescence for elastin and microfibrillar proteins which codistributed with fibronectin. The presence of type I and VI collagens and procollagen type III were demonstrated to a lesser extent within the elastotic material. These results suggest that solar elastosis is primarily derived from elastic fibers and not from preexisting or newly synthesized collagens.

A modified version of the enzyme-linked immunosorbent assay (ELISA), utilising human insoluble aortic elastin, was used for determination of anti-elastin antibodies in serum from normal and atherosclerotic subjects. The age-related changes in their level among healthy persons were investigated. Anti-elastin antibodies were found in all the tested human sera, showing the highest level at the age of 18-20 and the lowest at the age over 60 and especially among atherosclerotic patients. The possible role of the immune system in the turnover of elastin is discussed.

Rabbits immunized with kappa elastin produced arteriosclerosis and antibodies that bound to target-structures (elastic fiber sheaths, endothelial and smooth muscle cells). These antibodies were cytotoxic for cultured rabbit or rat arterial smooth muscle cells. Absorption of the antielastin antiserum with pig aorta or human serum glycoproteins inhibited its binding to target-structures and suppressed its in vitro cytotoxicity. These data are discussed.

Immunization of rabbits with elastin peptides prepared from purified bovine ligamentum nuchae elastin produces calcified arteriosclerotic lesions and fragmentation of elastic lamellae. Simultaneous administration of porcine calcitonin largely prevents the development of lesions. Experiments were carried out to clarify the mechanisms involved in the development of lesions as well as those involved in the preventive effect of calcitonin. Control experiments were carried out using bovine serum albumin (BSA) as antigen. Circulating antibodies and soluble immune complexes increased steadily in the sera of animals immunized with elastin peptides or BSA. The cellular immune reaction was weak as assessed by [3H]thymidine incorporation into lymphocytes in the presence of antigen or phytohemagglutinin. Arterial lesions appeared only in the animals immunized with elastin peptides, not in those immunized with BSA. Ion flux measurements were also carried out on strips of aorta obtained from immunized and control animals. Immunization with elastin peptides significantly increased the ouabain-insensitive 22Na+ efflux, the 86Rb efflux (indicator of K+ efflux), and the 45Ca2+ influx. Simultaneous calcitonin administration prevented the increase in Ca2+ influx but did enhance passive permeability to Na+ and K+ as well as the sodium pump. When calcitonin was administered without immunization, it decreased arterial smooth muscle permeability to Na+ and K+ and also decreased the basal Ca2+ influx. It is concluded that the pathological modifications of the arterial wall triggered by immunization with elastin peptides is at least partly mediated by the effect of antielastin antibodies and immune complexes on the ion permeability of arterial smooth muscle. Prevention of the increased Ca2+ influx by calcitonin is probably a key effect in the prevention of the development of lesions. The fact that calcitonin alone can modify the ion permeability of arterial smooth muscle suggests that this hormone may play a role in the regulation of vascular homeostasis.

The effect of elastase on the extracellular matrix of neonatal rat aortic smooth muscle cell cultures was monitored both chemically and ultrastructurally. Porcine pancreatic elastase was shown to decrease the elastin content in these cultures. Although chemically no distinction could be made between the elastin remaining in the culture matrix after elastase when compared to that in the nontreated cultures, the elastin was dramatically altered morphologically. The elastin assumed a "mottled" appearance after elastase treatment similar to that seen in vivo in emphysema models. A highly sensitive immunogold staining technique was used to detect elastin at the earliest stages of accumulation. Pulse experiments demonstrated an increase in protein synthesis by the cells 20 hr after elastase exposure. The culture system described here provides a model for probing in vivo elastase effects on elastin-containing tissues.