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Versican is an extracellular matrix (ECM) molecule that interacts with other ECM components to influence ECM organization, stability, composition, and cell behavior. Versican is known to increase in a number of cancers, but little is known about how versican influences the amount and organization of the ECM components in the tumor microenvironment. In the present study, we modulated versican expression using siRNAs in the human leiomyosarcoma (LMS) smooth muscle cell line SK-LMS-1, and observed the formation of elastin and elastic fibers in vitro and also in vivo in a nude mouse tumor model. Constitutive siRNA-directed knockdown of versican in LMS cells resulted in increased levels of elastin, as shown by immunohistochemical staining of the cells in vitro, and by mRNA and protein analyses. Moreover, versican siRNA LMS cells, when injected into nude mice, generated smaller tumors that had significantly greater immunohistochemical and histochemical staining for elastin when compared to control tumors. Additionally, microarray analyses were used to determine the influence of versican isoform modulation on gene expression profiles, and to identify genes that influence and relate to the process of elastogenesis. cDNA microarray analysis and TaqMan low density array validation identified previously unreported genes associated with downregulation of versican and increased elastogenesis. These results highlight an important role for the proteoglycan versican in regulating the expression and assembly of elastin and the phenotype of LMS cells.

Elastin and collagen levels in tissues are frequently difficult to measure because of each protein's limited solubility. This chapter provides detailed methodology for the determination of elastin, collagen, and total protein levels in a single tissue sample. All three assays start with an acid hydrolysate of the tissue, which breaks the tissue-associated proteins down to their component amino acids. Marker amino acids unique to each protein (desmosine for elastin and hydroxyproline for collagen) are then quantified. Total protein content, useful as a denominator for data normalization, can also be measured from a portion of the hydrolysate using an assay for free amino groups. These measurements are performed using convenient 96-well assay plates and require only a plate reader to determine absorbance.

Elastin plays a pivotal role in lung development. We therefore queried if elastin haploinsufficient newborn mice ( Eln+/−) would exhibit abnormal lung structure and function related to modified extracellular matrix (ECM) composition. Because mechanical ventilation (MV) has been linked to dysregulated elastic fiber formation in the newborn lung, we also asked if elastin haploinsufficiency would accentuate lung growth arrest seen after prolonged MV of neonatal mice. We studied 5-day-old wild-type ( Eln+/+) and Eln+/− littermates at baseline and after MV with air for 8–24 h. Lungs of unventilated Eln+/− mice contained ∼50% less elastin and ∼100% more collagen-1 and lysyl oxidase compared with Eln+/+ pups. Eln+/− lungs contained fewer capillaries than Eln+/+ lungs, without discernible differences in alveolar structure. In response to MV, lung tropoelastin and elastase activity increased in Eln+/+ neonates, whereas tropoelastin decreased and elastase activity was unchanged in Eln+/− mice. Fibrillin-1 protein increased in lungs of both groups during MV, more in Eln+/− than in Eln+/+ pups. In both groups, MV caused capillary loss, with larger and fewer alveoli compared with unventilated controls. Respiratory system elastance, which was less in unventilated Eln+/− compared with Eln+/+ mice, was similar in both groups after MV. These results suggest that elastin haploinsufficiency adversely impacts pulmonary angiogenesis and that MV dysregulates elastic fiber integrity, with further loss of lung capillaries, lung growth arrest, and impaired respiratory function in both Eln+/+ and Eln+/− mice. Paucity of lung capillaries in Eln+/− newborns might help explain subsequent development of pulmonary hypertension previously reported in adult Eln+/− mice.

Rationale: At physiologic pressures, elastic fibers constrain artery diameter. Local treatment of atherosclerotic arteries with PRT-201, a recombinant type I elastase, could result in fragmentation and removal of elastin fibers and increased vessel diameter. Objective: To investigate the use of PRT-201 as a treatment for human atherosclerotic arteries. Methods and Results: Arteries were harvested from donor legs amputated due to severe peripheral artery disease or from recently deceased persons who donated their bodies to science. Three- to four-centimeter artery segments were studied on a perfusion myograph to obtain baseline diameter data. After treatment with PRT-201 3.6 mg/mL or saline for 30 minutes myography was repeated. PRT-201 treatment resulted in an increase in vessel diameter across a range of transmural pressures. Average anterior tibial artery diameter increased by 0.78 ± 0.21 mm (27% ± 12%), whereas average posterior tibial artery diameter increased by 0.58 ± 0.30 mm (21% ± 11%), both P < 0.001. Elastin content as measured by desmosine radioimmunoassay was reduced by approximately 50%, P < 0.001. Conclusions: The results suggest that PRT-201 treatment of atherosclerotic peripheral arteries in patients could increase artery diameter, and thus luminal area, possibly alleviating some of the symptoms of peripheral artery disease.

International audience; Arterial wall elastic fibers, made of 90% elastin, are arranged into elastic lamellae which are responsible for the resilience and elastic properties of the large arteries (aorta and its proximal branches). Elastin is synthesized only in early life and adolescence mainly by the vascular smooth muscles cells (VSMC) through the cross-linking of its soluble precursor, tropoelastin. In normal aging, the elastic fibers become fragmented and the mechanical load is transferred to collagen fibers, which are 100-1000 times stiffer than elastic fibers. Minoxidil, an ATP-dependent K+ channel opener, has been shown to stimulate elastin expression in vitro, and in vivo in the aorta of male aged mice and young adult hypertensive rats. Here, we have studied the effect of a 3-month chronic oral treatment with minoxidil (120 mg/L in drinking water) on the abdominal aorta structure and function in adult (6-month-old) and aged (24-month-old) male and female mice. Our results show that minoxidil treatment preserves elastic lamellae integrity at both ages, which is accompanied by the formation of newly synthesized elastic fibers in aged mice. This leads to a generally decreased pulse pressure and a significant improvement of the arterial biomechanical properties in female mice, which present an increased distensibility and a decreased rigidity of the aorta. Our studies show that minoxidil treatment reversed some of the major adverse effects of arterial aging in mice and could be an interesting anti-arterial aging agent, also potentially usable for female-targeted therapies.

Desmosine, a crosslinking amino acid unique to elastin, was investigated as a possible biomarker for cancer. Twenty-eight normal controls, median age 67 years, had a median value for urine desmosine of 43.5 picomoles desmosine/mg creatinine. The median for 19 untreated cancer subjects of similar age was significantly higher (175 picomoles desmosine/mg creatinine, p

Background: Vessel injury at the time of Arteriovenous Fistula (AVF) creation may lead to neointimal hyperplasia that impairs AVF maturation. Vonapanitase, a recombinant human chymotrypsin-like elastase family member 1, is an investigational drug under development to improve AVF maturation and patency. The current studies were designed to document vonapanitase effects in human cephalic veins that are used in AVF creation. Methods: Human cephalic veins were mounted on a perfusion myograph. Vonapanitase 1.2, 4, 13.2, and 40 μg/ml or saline was applied drop wise on the vein followed by saline rinse. Vein segments were cut into rings for elastin content determination by desmosine radioimmunoassay and histology. Fluorescently-labelled vonapanitase was applied to veins and adventitial imaging was performed using laser scanning confocal microscopy. In vivo time course experiments were performed by treating rabbit jugular veins and harvesting 1 h and 4 h after vonapanitase treatment. Results / Conclusion: Vonapanitase reduced desmosine content in a dose-related manner. Histology also confirmed a dose-related reduction in elastic fiber staining. Fluorescently-labelled vonapanitase persistently localized to elastic fibers in the vein adventitia. In vivo experiments showed a reduction in desmosine content in jugular veins from 1 h to 4 h following treatment. These data suggest that vonapanitase targets elastin in elastic fibers in a dose related manner and that elastase remains in the vessel wall and has catalytic activity for at least 1 h.

Vascular tissue contains abundant elastic fibers that contribute to vessel elasticity. Vonapanitase (formerly PRT‐201) is a recombinant human chymotrypsin‐like elastase family member 1 (CELA1) shown to cleave the elastin component of elastic fibers, resulting in increased vessel diameter. The purpose of these current studies was to determine vein diameter, wall thickness, elastin content, and vonapanitase potency in veins used in a model of arteriovenous fistula (AVF) and in patients undergoing AVF creation for hemodialysis access to guide dose selection for human trials. Rabbit linguofacial, maxillary, and external jugular veins, and human basilic and upper and lower arm cephalic veins were dissected postmortem and sectioned into 2 mm length rings. Rings were incubated in vonapanitase at 37°C at varying concentrations and times. Elastin content was estimated histologically and by quantifying desmosine, a protein cross‐link unique to elastin. Rabbit veins were substantially thinner and contained less elastin than human veins. In human veins, elastin content was greatest in basilic and least in lower arm cephalic. Vonapanitase removed elastin in a time‐ and concentration‐dependent manner in all vein types. A lower concentration of vonapanitase was required to remove elastin from rabbit relative to human veins. In summary, vonapanitase reduced the elastin content of rabbit and human veins but did so at a lower concentration in the rabbit veins. Rabbit models may overestimate the potency of vonapanitase in humans. These results indicate that human dose selection should be guided by human vein ring experiments.

Rationale Cystic fibrosis (CF) lung disease is characterized by structural changes and remodeling in airway architecture and lung parenchyma. Neutrophilic inflammation and infection lead to injury and breakdown of airway matrix constituents, including elastin. The non-invasive measurement of urinary desmosine (UDes), a breakdown product of elastin, may be reflective of ongoing lung injury and may serve as a biomarker of active short-term damage during pulmonary exacerbation. Our objectives were to measure desmosine in the urine of CF patients hospitalized for treatment of a pulmonary exacerbation and to explore the correlation between desmosine concentration and other markers of clinical improvement, including lung function and inflammatory mediators. Methods Urine and blood samples plus lung function measurements were collected at up to three points during hospitalization for treatment of a CF pulmonary exacerbation. We used a repeated measures model, adjusted for age and time between measurements, to compare log transformed urine desmosine concentrations across multiple time points and to correlate those concentrations with related clinical variables. Change in UDes concentration was investigated using a statistical model that incorporated normalization factors to account for variations in urinary concentration. Results Desmosine was measured by radioimmunoassay (RIA) in 155 spot urine samples from 53 CF patients hospitalized for 63 pulmonary exacerbations (range of results: 0–235 pmol Des/ml). Specific gravity (SG) adjusted UDes concentration decreased significantly during admission for CF pulmonary exacerbation, P

Objective— The goal of this study was to determine whether antagonizing microRNA (miR)-29 enhances elastin (ELN) levels in cells and tissues lacking ELN. Methods and Results— miR-29 mimics reduced ELN levels in fibroblasts and smooth muscle cells, whereas miR-29 inhibition increased ELN levels. Antagonism of miR-29 also increased ELN levels in cells from patients haploinsufficient for ELN and in bioengineered human vessels. Conclusion— miR-29 antagonism may promote increased ELN levels during conditions of ELN deficiencies.

Background: Age-related arterial alterations affecting cells, matrix and biomolecules are the main culprit for initiation and progression of cardiovascular disease. The objective of this study is to gain further insights into the complex mechanism of elastic tissue ageing in human aortic blood vessels. Methods: One hundred and nineteen human aortic tissue samples were collected from adult patients (101 males, 18 females; age 40–86 years) undergoing coronary artery bypass grafting. Overall extracellular matrix architecture was examined by multiphoton laser scanning microscopy and histology. Matrix metalloproteinases 2 and 9, corresponding tissue inhibitors 1 and 2 as well as desmosine were determined. mRNA levels of tropoelastin were assessed by quantitative RT-PCR. Results: Age-related destruction of the vascular elastic laminas as well as a loss of interlamina cross-links were observed by laser scanning microscopy. These results were confirmed by histology indicating increasing interlamina gaps. There were no significant differences in matrix turnover or desmosine content. A steady decrease in tropoelastin mRNA by about 50% per 10 years of age increase was observed. Conclusions: Our findings indicate that ageing is accompanied by a destruction of the elastic vascular structure. However, tropoelastin expression analysis suggests that elastogenesis occurs throughout life with constantly decreasing levels.

Heterozygous elastin gene mutations cause autosomal dominant cutis laxa associated with emphysema and aortic aneurysms. To investigate the molecular mechanisms leading to cutis laxa in vivo, we generated transgenic mice by pronuclear injection of minigenes encoding normal human tropoelastin (WT) or tropoelastin with a cutis laxa mutation (CL). Three independent founder lines of CL mice showed emphysematous pulmonary airspace enlargement. No consistent dermatological or cardiovascular pathologies were observed. One CL and one WT line were selected for detailed studies. Both mutant and control transgenic animals showed elastin deposition into pulmonary elastic fibers, indicated by increased desmosine levels in the lung and by colocalization of transgenic and endogenous elastin by immunostaining. CL mice showed increased static lung compliance and decreased stiffness of lung tissue. In addition, markers of transforming growth factor-β (TGFβ) signaling and the unfolded protein response (UPR) were elevated together with increased apoptosis in the lungs of CL animals. We conclude that the synthesis of mutant elastin in CL activates multiple downstream disease pathways by triggering a UPR, altered mechanical signaling, increased release of TGFβ and apoptosis. We propose that the combined effects of these processes lead to the development of an emphysematous pulmonary phenotype in CL.

A promising method to fabricate tissue-engineered blood vessels is to have cells synthesize the supportive extracellular matrix scaffold of the tissue-engineered blood vessel; however, a shortcoming of this method has been limited elastogenesis. Previously, we found that arterial smooth muscle cells (ASMCs) produced significant quantities of elastin when transduced with splice variant 3 of the proteoglycan versican (V3). In this study, we assessed whether elastogenesis and the structural properties of entirely cell-derived engineered vascular constructs could be improved by the incorporation of V3-transduced rat ASMCs. After 18 weeks of culture, V3 constructs had more tropoelastin, more elastin crosslinks, higher burst strengths, greater elasticity, and thicker collagen fiber bundles compared with empty-vector controls. The expression of elastin and elastin-associated proteins was increased in V3 and control ASMC monolayer cultures when ascorbic acid, which promotes collagen synthesis and inhibits elastogenesis, was removed from the medium. Engineered vascular constructs with ascorbate withdrawn for 14 weeks, after an initial 4-week exposure to ascorbate, exhibited increased elastin, desmosine content, elasticity, and burst strength compared with constructs exposed continuously to ascorbate. Our results show that V3 coupled with limited exposure to ascorbate promotes elastogenesis and improves the structural and functional properties of engineered vascular constructs.

Objective— Even though elastin and fibrillin-1 are the major structural components of elastic fibers, mutations in elastin and fibrillin-1 lead to narrowing of large arteries in supravascular aortic stenosis and dilation of the ascending aorta in Marfan syndrome, respectively. A genetic approach was therefore used here to distinguish the differential contributions of elastin and fibrillin-1 to arterial development and compliance. Methods and Results— Key parameters of cardiovascular function were compared among adult mice haploinsufficient for elastin ( Eln +/− ), fibrillin-1 ( Fbn1 +/− ), or both proteins ( dHet ). Physiological and morphological comparisons correlate elastin haploinsufficiency with increased blood pressure and vessel length and tortuosity in dHet mice, and fibrillin-1 haploinsufficiency with increased aortic diameter in the same mutant animals. Mechanical tests confirm that elastin and fibrillin-1 impart elastic recoil and tensile strength to the aortic wall, respectively. Additional ex vivo analyses demonstrate additive and overlapping contributions of elastin and fibrillin-1 to the material properties of vascular tissues. Lastly, light and electron microscopy evidence implicates fibrillin-1 in the hypertension-promoted remodeling of the elastin-deficient aorta. Conclusions— These results demonstrate that elastin and fibrillin-1 have both differential and complementary roles in arterial wall formation and function, and advance our knowledge of the structural determinants of vascular physiology and disease.

Elastic fibres are essential extracellular matrix components of the skin, contributing to its resilience and elasticity. In the course of skin ageing, elastin synthesis is reduced, and elastase activity is accelerated, resulting in skin sagging and reduced skin elasticity. Our studies show that non-denatured Glycine max (soybean) extracts induced elastin promoter activity, inhibited elastase activity and protected elastic fibres from degradation by exogenous elastases in vitro. Mouse and swine skins topically treated with soybean extracts showed enhanced elastic fibre network and increased desmosine content. Elastin expression was also augmented in human skin transplanted onto SCID mice in response to soy treatment. These data suggest that non-denatured soybean extracts may be used as skin care agents to reduce the signs of skin ageing.

It is known that ATP-sensitive potassium (KATP) channels regulate the membrane potential of smooth muscle cells and vascular tone. Because their activity is altered during ageing, many pharmacological treatments aimed at improving KATP channel and cardiovascular functions have been evaluated. Nicorandil, a KATP channel opener, nitric oxide (NO) donor and anti-oxidant, induces vasodilation, decreases blood pressure and exhibits cardioprotection in ageing, as well as after ischaemia–reperfusion. In the present study, using tension myography and biochemical and histological techniques, we investigated the effects of chronic (2 months) low-dose nicorandil (0.1 mg/kg per day) treatment on the function of rat aorta during ageing (in 4-, 12- and 24-month old rats). The results showed that chronic nicorandil treatment significantly improves mechanical relaxation and noradrenaline-induced vasoconstriction in aged rats. At all ages, the nicorandil-induced vasodilation was primarily mediated by its NO donor group. Nicorandil treatment resulted in an additional 0.5–1 elastic lamella in the aorta and decreased total protein, collagen and elastin content in the aortic wall at all ages. However, in 4-month-old rats, nicorandil significantly increased the elastin : total protein ratio by 19%. In contrast with results of previous studies that used high doses of nicorandil (i.e. 60 mg/kg per day), low-dose nicorandil treatment in the present study did not lead to a progressive desensitization to nicorandil and may be beneficial in improving arterial function in ageing or cardiovascular diseases. Keywords: ageing, arterial mechanics, arteries, ATP-dependent potassium channels, cardiovascular system, collagen, elastic fibres, elastin, nicorandil, vasoactivity INTRODUCTION Ageing large arteries remodel, classically exhibiting accelerated endothelial cell turnover, smooth muscle cell hypertrophy, increased diameter and wall thickness, as well as elastic fibre fragmentation and collagen accumulation. These events progressively lead to wall stiffening and increases in blood pressure.1 Large arteries of aged animals are also more contracted, although less responsive to vasoactive agents/pathways,2 such as noradrenaline/α-adrenoceptors,3–6 acetylcholine (ACh)/nitric oxide (NO)7,8 or membrane K+ channel opening. The latter mechanism blocks voltage-dependent membrane calcium channels and induces vasodilation9 and partly mediates ACh-induced vasodilation.10,11 The ATP-dependent K+ (KATP) channels have the additional role of cell metabolism sensors.12 The function of KATP channels from different cell types is progressively altered because of an age-dependent oxidation of thiol groups from the intracellular portion of the channel.13 This oxidation inhibits both the spontaneous and agonist-triggered activities of the channels,13 leading to age-dependent decreases in arterial reactivity to different vasoregulatory molecules, including catecholamines and ACh.14,15 Because K+ channel dysfunction contributes substantially to the age-dependent arterial dysfunctions described above,14,15 many studies have investigated the relevance of treatments whose purpose was to restore the responsiveness of K+ channels, in particular KATP channels, in ageing or cardiovascular diseases.16–19 Nicorandil (N-(2-hydroxyethyl)-nicotinamide nitrate) is a drug of choice in these treatments because its combined molecular structure and function targets several deleterious mechanisms involved in arterial ageing, namely KATP channel oxidation and dysfunction and NO pathway impairment. Nicorandil is a KATP channel opener molecule combining an organic nitrate and a nicotinamide group, which confer the additional properties of an NO donor and anti-oxidant, respectively.18,20 Acute low doses of nicorandil (range 10 μg/kg to 1 mg/kg) have been shown to lower blood pressure, reduce oxidized KATP channels and restore their activity,21 induce dilation of different types of arteries through NO delivery and KATP channel opening22,23 and improve the global condition of patients with angina pectoris.24 Acute higher doses of nicorandil (25–100 μg/kg) in a cardiac infarction model in rats have been shown to result in decreased formation of microvascular obstruction and a reduction in infarct size.25 In humans with cardiac infarction, acute nicorandil has been shown recently to suppress the infarction-induced increase in plasma levels of matrix metalloproteinase activity and to prevent left ventricular remodelling.26 However, chronic treatment with high doses of nicorandil (60 mg/kg per day) produces opposite effects in the long term (i.e. 2–4 weeks), namely desensitization of KATP channels to agonists and inactivation of the cGMP pathway/vascular smooth muscle cell relaxation.17 Previously, we showed that chronic treatment of rats with a low dose of nicorandil (0.1 mg/kg per day for 2 months) improved cardiac function in ageing, under both physiological and experimentally induced pathological conditions. Heart function (developed pressure, action potential duration, survival rate) was protected during ageing and ischaemia–reperfusion experiments because of a higher activity of KATP channels.27 The aim of the present study was to investigate the effects of chronic treatment with a low dose of nicorandil on arterial structure and function during ageing in rats. The results show that low-dose nicorandil treatment induces aortic remodelling and improves function in aged animals.

Contains fulltext : 80686.pdf (Publisher’s version ) (Closed access) We recently demonstrated aberrant staining of fibrillin-1 in lung tissue specimens with emphysematous lesions. In this study, we have extended this observation by an elaborate analysis of the elastic fibre. Using domain-specific antibodies to fibrillin-1, and to other elastin fibre-associated molecules, lung tissue derived from patients without obvious clinical emphysema, but harbouring various degrees of microscopical emphysematous lesions, was analysed. In addition, the fibrillin-regulated growth factor TGF-beta was studied. Electron microscopy and biochemical analysis of desmosine (a marker for elastin) were also performed. Results were compared with lung tissue derived from patients with clinical emphysema. Domain-specific antibodies recognizing the C-terminal, N-terminal, and middle part of fibrillin-1 showed aberrant staining patterns associated with increasing degrees of microscopical emphysema. Staining for elastin, emilin-1, and fibulin-2 was, however, not aberrant. TGF-beta staining was markedly increased. On the electron microscopic, but not light microscopical, level, initial elastic fibre degradation was noticed in specimens with microscopical emphysema. Lung specimens from patients with clinical emphysema also displayed fragmented fibrillin-1 staining and, in addition, displayed extensive degradation of the elastic fibre. The results suggest that fibrillin-1 anomalies and TGF-beta overexpression are associated with initial events occurring during the emphysematous process. Based on these and other data, a mechanism for emphysematogenesis is proposed.

Bioprosthetic heart valve (BHV) cusps have a complex architecture consisting of an anisotropic arrangement of collagen, glycosaminoglycans (GAGs) and elastin. Glutaraldehyde (GLUT) is used as a fixative for all clinical BHV implants; however, it only stabilizes the collagen component of the tissue, and other components such as GAGs and elastin are lost from the tissue during processing, storage or after implantation. We have shown previously that the effectiveness of the chemical crosslinking can be increased by incorporating neomycin trisulfate, a hyaluronidase inhibitor, to prevent the enzyme-mediated GAG degradation. In the present study, we optimized carbodiimide-based GAG-targeted chemistry to incorporate neomycin into BHV cusps prior to conventional GLUT crosslinking. This crosslinking leads to enhanced preservation of GAGs during in vitro cyclic fatigue and storage. The neomycin group showed greater GAG retention after both 10 and 50 million accelerated-fatigue cycles and after 1 year of storage in GLUT solution. Thus, additional binding of neomycin to the cusps prior to standard GLUT crosslinking could enhance tissue stability and thus heart valve durability.

Fibulin-5 is a 66 kDa modular, extracellular matrix protein that localizes to elastic fibers. Although in vitro protein-protein binding studies have shown that fibulin-5 binds many proteins involved in elastic fiber formation, the specific role of fibulin-5 in elastogenesis remains unclear. To provide a more detailed analysis of elastic fiber assembly in the absence of fibulin-5, the dermis of wild-type and fibulin-5 gene knockout (Fbln5(-/-)) mice was examined with electron microscopy (EM). Although light microscopy showed apparently normal elastic fibers near the hair follicles and the absence of elastic fibers in the intervening dermis of the Fbln5(-/-) mouse, EM revealed the presence of aberrantly assembled elastic fibers in both locales. Instead of the elastin being incorporated into the microfibrillar scaffold, the elastin appeared as globules juxtaposed to the microfibrils. Desmosine analysis showed significantly lower levels of mature cross-linked elastin in the Fbln5(-/-) dermis, however, gene expression levels for tropoelastin and fibrillin-1, the major elastic fiber components, were unaffected. Based on these results, the nature of tropoelastin cross-linking was investigated using domain specific antibodies to lysyl oxidase like-1 (LOXL-1). Immunolocalization with an antibody to the N-terminal pro-peptide, which is cleaved to generate the active enzyme, revealed abundant staining in the Fbln5(-/-) dermis and no staining in the wild-type dermis. Overall, these results suggest two previously unrecognized functions for fibulin-5 in elastogenesis; first, to limit the extent of aggregation of tropoelastin monomers and/or coacervates and aid in the incorporation of elastin into the microfibril bundles, and second, to potentially assist in the activation of LOXL-1.

Objective To investigate a function of fibulin-5 in the elastic fiber formation, we studied the molecular interactions among elastin, fibrillin-1, and fibulin-5 in the extracellular space and the maturation of tropoelastin using retinal pigment epithelial cells (ARPE-19). Design and methods Bacterial recombinant tropoelastin (rTE) was added to ARPE-19 cells overexpressing V5-tagged fibulin-5 (ARPE-Fibulin-5). These elastic fibers were evaluated by immunofluorescence staining, the quantitative analysis of cross-linked amino acids, and semi-quantitative analysis of matrix-associated tropoelastin. Results Immunoprecipitation assays revealed that fibulin-5 is able to separately interact with tropoelastin or fibrillin-1 in the culture medium. Moreover, immunofluorescent staining showed that elastin, fibrillin-1, and fibulin-5 co-localize in the extracellular matrix. Desmosine levels were significantly increased in ARPE-Fibulin-5 relative to untransfected cells in spite of equal deposition of tropoelastin by enzyme-linked immunosorbent assay. The addition of a tropoelastin isoform, which lacked the peptide encoded by exon 26A (Δ26A) and could bind to fibulin-5 strongly, led to a larger increase in cross-linking amino acids compared to tropoelastin containing the exon 26A peptide sequence. Conclusion These data provide new insights into the initial steps of elastic fiber assembly and identify fibulin-5 and tropoelastin isoforms as potential targets for the regeneration of elastic fibers in vivo.

Galactosamine-containing glycosaminoglycans (GAGs), such as the chondroitin sulfate chains of the proteoglycan versican, have been shown to inhibit elastogenesis. Another proteoglycan that may influence elastogenesis is biglycan, which possesses two GAG chains. To assess the importance of these chains on elastogenesis in blood vessels, rat aortic smooth muscle cells were transduced with a GAG-deficient biglycan cDNA-containing retroviral vector (LmBSN). Control cells were transduced with either biglycan or empty vector. Transduced cells were characterized in vitro and then seeded into balloon-injured rat carotid arteries to determine the effects on neointimal structure. Cultured cells overexpressing LmBSN showed marked up-regulation of tropoelastin and fibulin-5 mRNAs, increased amounts of desmosine and insoluble elastin, and increased deposition of elastic fibers as compared with empty vector- and biglycan-transduced cells. Conversely, collagen alpha(1) synthesis and the deposition of collagen fibers were both markedly decreased in LmBSN cultures. In vivo, neointimae formed from cells that overexpressed LmBSN and showed increased deposits of elastin that aggregated into parallel nascent fibers, generally arranged circumferentially. Neointimae that formed from cells with biglycan or empty vector contained fewer and less aggregated deposits of elastin. These findings suggest that the GAG chains of biglycan serve as inhibitors of elastin synthesis and assembly, and that biglycan can act as an important modulator of the composition of the extracellular matrix of blood vessels.

The assembly of elastic fibers in tissues that undergo repeated cycles of extension and recoil, such as the lungs and blood vessels, is dependent on the proper interaction and alignment of tropoelastin with a microfibrillar scaffold. Here, we describe in vivo histopathological effects of neuraminidase-1 (Neu1) deficiency on elastin assembly in the lungs and aorta of mice. These mice exhibited a tight-skin phenotype very similar to the Tsk mouse. Normal septation of Neu1-null mice did not occur in neonatal mice, resulting in enlarged alveoli that were maintained in adults. The abnormal development of elastic fibers was remarkable under electron microscopy and confirmed by the overlapping distribution of elastin, fibrillin-1, fibrillin-2, and fibulin-5 (Fib-5) by the light microscopy immunostainings. Fib-5 fibers appeared diffuse and unorganized around the alveolar walls and the apex of developing secondary septal crests. Fibrillin-2 deposition was also abnormal in neonatal and adult lungs. Dispersion of myofibroblasts appeared abnormal in developing lungs of Neu1-null mice, with a random distribution of myofibroblast around the alveolar walls, rather than concentrating at sites of elastin synthesis. The elastic lamellae in the aorta of the Neu1-null mice were thinner and separated by hypertrophic smooth muscle cells that were surrounded by an excess of the sialic acid-containing moieties. The concentration of elastin, as measure by desmosine levels, was significantly reduced in the aorta of Neu1-null mice. Message levels for tropoelastin and Fib-5 were normal, suggesting the elastic fiber defects in Neu1-null mice result from impaired extracellular assembly.

Elastin, the main component of elastic fibers, is synthesized only in early life and provides the blood vessels with their elastic properties. With aging, elastin is progressively degraded, leading to arterial enlargement, stiffening, and dysfunction. Also, elastin is a key regulator of vascular smooth muscle cell proliferation and migration during development since heterozygous mutations in its gene (Eln) are responsible for a severe obstructive vascular disease, supravalvular aortic stenosis, isolated or associated to Williams syndrome. Here, we have studied whether early elastin synthesis could also influence the aging processes, by comparing the structure and function of ascending aorta from 6- and 24-month-old Eln+/- and Eln+/+ mice. Eln+/- animals have high blood pressure and arteries with smaller diameters and more rigid walls containing additional although thinner elastic lamellas. Nevertheless, longevity of these animals is unaffected. In young adult Eln+/- mice, some features resemble vascular aging of wild-type animals: cardiac hypertrophy, loss of elasticity of the arterial wall through enhanced fragmentation of the elastic fibers, and extracellular matrix accumulation in the aortic wall, in particular in the intima. In Eln+/- animals, we also observed an age-dependent alteration of endothelial vasorelaxant function. On the contrary, Eln+/- mice were protected from several classical consequences of aging visible in aged Eln+/+ mice, such as arterial wall thickening and alteration of alpha(1)-adrenoceptor-mediated vasoconstriction. Our results suggest that early elastin expression and organization modify arterial aging through their impact on both vascular cell physiology and structure and mechanics of blood vessels.

Genital prolapse is a debilitating manifestation of pelvic floor dysfunction. The cause of this condition has not been elucidated. The purpose of this study was to determine elastin content and RNA expression of related enzymes of elastin synthesis in uterosacral ligament biopsies from women with severe prolapse, and controls with normal pelvic support.Biopsies were taken from the uterosacral ligament tissue of 31 women with Grade III or greater prolapse and 29 women with normal pelvic support. Elastin content was assessed by measuring desmosine using radioimmunoassay, and quantitative real time PCR was performed to quantify mRNA levels of lysyl oxidase (LOX), lysyl oxidase like-1 (LOXL1), LOXL2 and fibulin-5 (FIB-5).The mean desmosine concentration found in uterosacral ligaments of women with prolapse (n =26) was 103.3+/-59.3 pmolD/mgP compared to controls (n =29) 120.5+/-47.4 pmolD/mgP (p =0.1943). In the subgroup of subjects with complete procidentia (n =8), mean desmosine concentration was 50.6+/-25.8 and 127.1+/-42.2 pmolD/mgP in age-matched controls (n =12) (p0.05). In tissue from subjects with more than 2 vaginal deliveries (n =18), the mean desmosine concentration was 99.9+/-60.7 and 133.0+/-44.0 pmolD/mgP in controls (n =17) (p0.05). Expression of LOX, LOXL1 and LOXL2 decreased 8.2-fold+/-3.4, 5.0-fold+/-1.7 and 15.2-fold+/-5.2, respectively (mean+/-SD) in cases versus controls (p0.05). Expression of FIB-5 was increased 3.1-fold+/-0.7 compared to controls (p0.05).Significantly decreased desmosine content was measured in the uterosacral ligament tissue from women with prolapse versus controls in women with parity2 and in women with complete procidentia. Suppression of mRNA for LOX and two LOX isoenzymes was correspondingly present. These results suggest that altered elastin metabolism is present in women with uterine prolapse.

Elastic fibers play a key role in the structure and function of numerous organs that require elasticity. Elastogenesis is a complex process in which cells first produce a microfibrillar scaffold, composed of numerous structural proteins, upon which tropoelastin assembles to be cross-linked into polymeric elastin. Recently, it was demonstrated that low concentrations of free iron upregulate elastin gene expression in cultured fibroblasts. The present studies were conducted to assess whether low-iron diets would affect the deposition of elastic fibers in an in vivo model. One-day-old chicks were fed semipurified diets containing 1.3 (low), 12 (moderate), and 24 (control) mg/kg of iron. After 3 wk, chicks in the low-iron group were underweight and anemic. Their aortas were smaller with significantly thinner walls than control chicks, yet elastin or collagen content did not decrease relative to total protein. They also demonstrated a significantly lower stress-strain resistance than the controls. Electron microscopy demonstrated that aortic and lung smooth muscle cells were vacuolated and surrounded by loose extracellular matrix and disorganized elastic lamellae with diffuse and fragmented networks of elastic fibers and microfibrils. Immunohistology demonstrated that fibrillin-3 (FBN3) was disorganized and markedly reduced in amount in aortas of the low-iron chicks. Elastin messenger RNA levels were not downregulated in the tissues from the low-iron-fed chicks; however, there was a significant reduction in expression of the FBN1 and FBN3 genes compared with control chicks. The studies indicate that iron deficiency had a pronounced negative effect on elastic fiber development and suggests that fibrillin may have an important role in this pathology.

Background—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.Methods and Results—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.Conclusions—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.

Pelvic organ prolapse is strongly associated with a history of vaginal delivery. The mechanisms by which pregnancy and parturition lead to failure of pelvic organ support, however, are not known. Recently, it was reported that mice with null mutations in lysyl oxidase-like 1 (LOXL1) develop pelvic organ prolapse. Elastin is a substrate for lysyl oxidase (LOX) and LOXL1, and LOXL1 interacts with fibulin-5 (FBLN5). Therefore, to clarify the potential role of elastic fiber assembly in the pathogenesis of pelvic organ prolapse, pelvic organ support was characterized in Fbln5-/- mice, and changes in elastic fiber homeostasis in the mouse vagina during pregnancy and parturition were determined. Pelvic organ prolapse in Fbln5-/- mice was remarkably similar to that in primates. The temporal relationship between LOX mRNA and protein, processing of LOXL1 protein, FBLN5 and tropoelastin protein, and desmosine content in the vagina suggest that a burst of elastic fiber assembly and cross linking occurs in the vaginal wall postpartum. Together with the phenotype of Fbln5-/- mice, the results suggest that synthesis and assembly of elastic fibers are crucial for recovery of pelvic organ support after vaginal delivery and that disordered elastic fiber homeostasis is a primary event in the pathogenesis of pelvic organ prolapse in mice.

Naturally occurring mutations are useful in identifying domains that are important for protein function. We studied a mutation in the elastin gene, 800−3G>C, a common disease allele for SVAS (supravalvular aortic stenosis). We showed in primary skin fibroblasts from two different SVAS families that this mutation causes skipping of exons 16–17 and results in a stable mRNA. Tropoelastin lacking domains 16–17 (Δ16–17) was synthesized efficiently and secreted by transfected retinal pigment epithelium cells, but showed the deficient deposition into the extracellular matrix compared with normal as demonstrated by immunofluorescent staining and desmosine assays. Solid-phase binding assays indicated normal molecular interaction of Δ16–17 with fibrillin-1 and fibulin-5. However, self-association of Δ16–17 was diminished as shown by an elevated coacervation temperature. Moreover, negative staining electron microscopy confirmed that Δ16–17 was deficient in forming fibrillar polymers. Domain 16 has high homology with domain 30, which can form a β-sheet structure facilitating fibre formation. Taken together, we conclude that domains 16–17 are important for self-association of tropoelastin and elastic fibre formation. This study is the first to discover that domains of elastin play an essential role in elastic fibre formation by facilitating homotypic interactions.

Desmosine is a stable breakdown product of elastin that can be reliably measured in urine samples. We tested the hypothesis that higher baseline urine desmosine would be associated with higher mortality in 579 of 861 patients included in the recent Acute Respiratory Distress Syndrome Network trial of lower tidal volume ventilation ( 1 ). We also correlated urine desmosine levels with indexes of disease severity. Finally, we assessed whether urine desmosine was lower in patients who received lower tidal volumes. Desmosine was measured by radioimmunoassay in urine samples from days 0, 1, and 3 of the study. The data were expressed as a ratio of urine desmosine to urine creatinine to control for renal dilution. The results show that higher baseline ( day 0) urine desmosine-to-creatinine concentration was associated with a higher risk of death on adjusted analysis (odds ratio 1.36, 95% confidence interval 1.02–1.82, P = 0.03). Urine desmosine increased in both ventilator groups from day 0 to day 3, but the average rise was higher in the 12-ml/kg predicted body weight group compared with the 6-ml/kg predicted body weight group ( P = 0.053, repeated-measures model). In conclusion, patients with acute lung injury ventilated with lower tidal volumes have lower urine desmosine levels, a finding that may reflect reduced extracellular matrix breakdown. These results illustrate the value of evaluating urinary biological markers that may have prognostic and pathogenetic significance in acute lung injury.

Resistance artery narrowing and stiffening are key elements in the pathogenesis of essential hypertension, but their origin is not completely understood. In mesenteric resistance arteries (MRA) from spontaneously hypertensive rats (SHR), we have shown that inward remodeling is associated with abnormal elastic fiber organization, leading to smaller fenestrae in the internal elastic lamina. Our current aim is to determine whether this alteration is an early event that precedes vessel narrowing, or if elastic fiber reorganization in SHR arteries occurs because of the remodeling process itself. Using MRA from 10-day-old, 30-day-old, and 6-mo-old SHR and normotensive Wistar Kyoto rats, we investigated the time course of the development of structural and mechanical alterations (pressure myography), elastic fiber organization (confocal microscopy), and amount of elastin (radioimmunoassay for desmosine) and collagen (picrosirius red). SHR MRA had an impairment of fenestrae enlargement during the first month of life. In 30-day-old SHR, smaller fenestrae and more packed elastic fibers in the internal elastic lamina were paralleled by increased wall stiffness. Collagen and elastin levels were unaltered at this age. MRA from 6-mo-old SHR also had smaller fenestrae and a denser network of adventitial elastic fibers, accompanied by increased collagen content and vessel narrowing. At this age, elastase digestion was less effective in SHR MRA, suggesting a lower susceptibility of elastic fibers to enzymatic degradation. These data suggest that abnormal elastic fiber deposition in SHR increases resistance artery stiffness at an early age, which might participate in vessel narrowing later in life.

Background Transplantation of cryopreserved allografts represents a well-established valve replacement option. Despite their clinical use for more than 40 years, the integrity of the extracellular matrix (ECM) of these valves after thawing has not been determined. The purpose of this study was to investigate and compare ECM structures of fresh and cryopreserved porcine heart valve leaflets with special emphasis on the condition of collagenous and elastic fibers. Methods Pulmonary valves were excised from unprocessed porcine hearts under sterile conditions. After treatment with antibiotics, the valves were incubated in a cryoprotective solution, cryopreserved stepwise, and stored at –196°C for 1 week. Two groups of heart valves (fresh untreated and thawed cryopreserved [each, n=8]) were analyzed using biochemical (collagen, elastin, desmosine), histologic (hematoxylin-eosin, Movat-pentachrome, resorcin-fuchsin), and immunohistochemical (antibodies against collagen I, III, IV, and elastin) methods. Near-infrared femtosecond multiphoton laser scanning microscopy and second harmonic generation were used for high-resolution three-dimensional imaging of ECM structures. Results Biochemical testing demonstrated similar amounts of collagen and desmosine, but a minor loss of elastin in the cryopreserved specimens. Conventional histology revealed almost comparable cell and ECM formations in fresh and cryopreserved valve leaflets. In contrast, laser-induced autofluorescence imaging showed substantial ultrastructural deterioration and disintegration of most collagenous structures. Second harmonic generation was not inducible. Conclusions Conventional cryopreservation of heart valves is accompanied by serious alterations and destruction of leaflet ECM structures, specifically demonstrated by multiphoton imaging. Further in-depth studies to clarify the impact of alternative cryopreservation techniques proposed for clinical use, such as vitrification, are crucial.

Elastic fibers provide tissues with elasticity which is critical to the function of arteries, lungs, skin, and other dynamic organs. Loss of elasticity is a major contributing factor in aging and diseases. However, the mechanism of elastic fiber development and assembly is poorly understood. Here, we show that lack of fibulin-4, an extracellular matrix molecule, abolishes elastogenesis. fibulin-4−/− mice generated by gene targeting exhibited severe lung and vascular defects including emphysema, artery tortuosity, irregularity, aneurysm, rupture, and resulting hemorrhages. All the homozygous mice died perinatally. The earliest abnormality noted was a uniformly narrowing of the descending aorta in fibulin-4−/− embryos at embryonic day 12.5 (E12.5). Aorta tortuosity and irregularity became noticeable at E15.5. Histological analysis demonstrated that fibulin-4−/− mice do not develop intact elastic fibers but contain irregular elastin aggregates. Electron microscopy revealed that the elastin aggregates are highly unusual in that they contain evenly distributed rod-like filaments, in contrast to the amorphous appearance of normal elastic fibers. Desmosine analysis indicated that elastin cross-links in fibulin-4−/− tissues were largely diminished. However, expression of tropoelastin or lysyl oxidase mRNA was unaffected in fibulin-4−/− mice. In addition, fibulin-4 strongly interacts with tropoelastin and colocalizes with elastic fibers in culture. These results demonstrate that fibulin-4 plays an irreplaceable role in elastogenesis.

Objective: Liver growth factor (LGF), a mitogen for liver cells, reduces fibrosis in a rat model of cirrhosis. The present study assesses the possible vascular antifibrotic and antihypertensive effects of LGF treatment on spontaneously hypertensive rats (SHR). Methods: Six-month-old male SHR and normotensive Wistar Kyoto rats (WKY) were treated with LGF (4.5 μg LGF/rat i.p. twice a week for 2 weeks). Haemodynamic parameters were measured in anaesthetized rats. Vascular structure and function were studied in carotid arteries using optical and confocal microscopy, radioimmunoassay for desmosine, and isometric tension recording. Results: LGF reduced systolic and diastolic blood pressure only in SHR. When compared to those of untreated SHR, carotid arteries from LGF-treated SHR showed: 1) a 50% reduction in collagen area and an increase in vascular smooth muscle cell number in the media, 2) no difference in total elastin content, but an increase in size of fenestrae in the internal elastic lamina, and 3) enhanced relaxation to acetylcholine, sodium nitroprusside, and forskolin. These effects were specific for SHR, since no changes were observed in LGF-treated WKY. Conclusion: Short-term treatment with a low dose of LGF induced a large improvement in vascular structure and function and significantly reduced blood pressure in a rat model of essential hypertension. The present results could open future research to explore the vascular effects of this endogenous factor in order to determine its potential as an antifibrotic and antihypertensive agent in humans.

Elastic fibers play an important role in characteristic elastic properties of tissues such as skin, lungs, ligaments, and blood vessels. Many of the disorders related to elastic fibers are due to errors in assembly, yet the basic mechanisms of this process remain unclear. Cutis laxa, which is caused by an elastin gene mutation, includes a rare and heterogeneous group of the disorders characterized by lax, inelastic skin. To investigate whether elastic fiber assembly insufficiency is a possible factor in cutis laxa, we compared normal tropoelastin (nTE) and frameshift-mutated tropoelastin (fmTE) from a patient with autosomal dominant cutis laxa using an in vitro elastic fiber assembly model, in which purified recombinant tropoelastin was added to the culture medium. Assembly was evaluated by immunofluorescence staining, the quantitative analysis of cross-linked amino acids, and semi-quantitative analysis of matrix-associated tropoelastin. Immunofluorescence microscopy indicated an approximate 50% decrease in the deposition of fmTE in the extracelluar matrix, in contrast to that of nTE. The amount of cross-linked amino acids unique to mature insoluble elastin in the fmTE assembly was also decreased by approximately 20%. In addition, we clarified that the molecular interaction between fmTE and the amino-terminal domain of fibrillin-1 or full-length fibulin-5 was significantly decreased. Our results suggest that the defect of fmTE fiber assembly is due, at least in part, to decreased molecular interactions between tropoelastin and the microfibrillar components fibulin-5 and fibrillin.

Transgenic mice overexpressing the calcium binding protein, S100A4/Mts1, occasionally develop severe pulmonary vascular obstructive disease. To understand what underlies this propensity, we compared the pulmonary vascular hemodynamic and structural features of S100A4/Mts1 with control C57Bl/6 mice at baseline, following a 2-week exposure to chronic hypoxia, and after 1 and 3 months “recovery” in room air. S100A4/Mts1 mice had greater right ventricular systolic pressure and right ventricular hypertrophy at baseline, which increased further with chronic hypoxia and was sustained after 3 months “recovery” in room air. These findings correlated with a heightened response to acute hypoxia and failure to vasodilate with nitric oxide or oxygen. S100A4/Mts1 mice, when compared with C57Bl/6 mice, also had impaired cardiac function judged by reduced ventricular elastance and decreased cardiac output. Despite higher right ventricular systolic pressures with chronic hypoxia, S100A4/Mts1 mice did not develop more severe PVD, but in contrast to C57Bl/6 mice, these features did not regress on return to room air. Microarray analysis of lung tissue identified a number of genes differentially upregulated in S100A4/Mts1 versus control mice. One of these, fibulin-5, is a matrix component necessary for normal elastin fiber assembly. Fibulin-5 was localized to pulmonary arteries and associated with thickened elastic laminae. This feature could underlie attenuation of pulmonary vascular changes in response to elevated pressure, as well as impaired reversibility.

We have previously developed a method for estimating elastin content and organization in resistance arteries, where it is a minor component. The aim of the present study was to validate the method against a quantitative assay and to determine the relative importance of elastin content and organization for intrinsic elasticity of small arteries. Mesenteric third order branches (from 10-day-old, 1- and 6-month-old rats) and middle cerebral arteries (from 6-month-old rats) were pressurized. beta-Values were calculated from stress-strain relationships and used as indicators of intrinsic stiffness. The same pressure-fixed arteries were used to estimate elastin content and organization in the internal elastic lamina with confocal microscopy. Collagen and elastin contents were determined by Picrosirius Red staining and radioimmunoassay for desmosine, respectively. Confocal and desmosine assays gave similar results: no difference in elastin content of mesenteric vessels from 1- and 6-month-old rats, and a significant reduction in cerebral compared to mesenteric arteries. For all parameters (elastin and collagen content, fenestrae area and internal elastic lamina thickness) the best correlation was found between beta-values and fenestrae size. These data suggest that in small arteries: (1) confocal microscopy can be used as a method for the simultaneous study of changes in elastin content and organization; and (2) elastin organization might be a key determinant of intrinsic elastic properties.

Dermal elastic fibers are believed to have a primary role in providing elastic stretch and recoil to the skin. Here we compare the structural arrangement of dermal elastic fibers of chick skin and different animal species. Most elastic fibers in chick skin are derived from cells that line the feather follicle and/or smooth muscle that connects the pterial and apterial muscle bundles to feather follicles. Elastic fibers in the dermis of animals with single, primary hair follicles are derived from cells lining the hair follicle or from the ends of the pili muscle, which anchors the muscle to the matrix or to the hair follicle. Each follicle is interconnected with elastic fibers. Follicles of animals with primary and secondary (wool) hair follicles are also interconnected by elastic fibers, yet only the elastic fibers derived from the primary follicle are connected to each primary follicle. Only the primary hair follicles are connected to the pili muscle. Human skin, but not the skin of other primates, is significantly different from other animals with respect to elastic fiber organization and probably cell of origin. The data suggest that the primary role for elastic fibers in animals, with the possible exception of humans, is movement and/or placement of feathers or hair.

Homocysteinemia in animals is associated with disruption of the elastic fiber component of the extracellular matrix, resulting in vascular complications. The authors have utilized both avian and murine models to investigate the effects of homocysteinemia on lung development and repair following injury. Days old chicks were fed a diet containing 2% methionine for 3 weeks. Pregnant mice were given 2% methionine in the diet and feeding continued for up to 6 weeks after birth. The lungs were removed and examined for defects in elastin fiber formation. Methionine levels were elevated 20-fold in the serum from chicks receiving the methionine and 10-fold in pregnant mice. The elastic fibers in the parabronchi and air capillaries of chicks receiving methionine were thin and clearly disrupted. In the 2% methionine neonatal pups, normal lung development was prevented and the alveoli were significantly enlarged. However, after the pups reached 10 days of age the 2% methionine lungs did not differ histologically from the normal controls. Fetal mice reflected the same serum methionine levels as the dams fed the 2% methionine diet, yet after birth the serum levels of the neonates returned to control levels within 3 days. The authors found that the high serum methionine levels of the dams were not transferred to the milk, allowing the pups to reverse the histopathology observed early and then develop normally. The ability of the lung to replace elastin following elastase injury was not different in mice raised on the 2% methionine diet compared to controls. The studies show that continuous exposure of the developing lung to high circulating levels of methionine/homocysteine can result in major disruptions of elastic fibers and lung architecture. However, young mammals such as the mouse are protected from extended lung pathology because toxic levels of methionine are not transferred through the mothers milk.

Background— 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. Methods and Results— In the present studies, we determined that short-term periadventitial treatment of the rat abdominal aorta with low concentrations of calcium chloride (CaCl 2 ) 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 CaCl 2 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 CaCl 2 -mediated injury significantly reduced elastin calcification and abolished both extracellular matrix degradation and apoptosis. We also found that MMP-knockout mice were resistant to CaCl 2 -mediated aortic injury and did not develop elastin degeneration and calcification. Conclusion— Collectively, these data strongly indicate a correlation between MMP-mediated elastin degradation and vascular calcification.

Objective: To study autologous tissue engineered blood vessels (TEBV) in the descending aorta of juvenile sheep. Methods: Autologous vascular smooth muscle cells (vSMC) and endothelial cells were obtained from ovine carotid arteries. vSMC were seeded on bioresorbable scaffolds and dynamically cultured for 14 days. Following endothelialization an additional external ovine small intestinal submucosa wrapping was applied. Constructs were implanted in the descending aorta of juvenile sheep and removed after 1, 3, 6, 12 and 24 weeks for evaluation with histological, microscopical and biochemical techniques. Results: Up to 3 months after implantation, grafts were fully patent, without any signs of dilatation, occlusion or intimal thickening. Scanning electron microscopy revealed a confluent luminal endothelial cell layer. In contrast, the 6 months graft displayed significant dilatation and partial thrombus formation. Histology displayed layered tissue formation resembling native aorta. Extracellular matrix (ECM) stains, immunostaining and Transmission Electron Microscopy (TEM) revealed alternating layers of vSMC and extracellular matrix consisting of collagen, elastin and glycosaminogycans. Compared to native aorta, the elastin content of the TE grafts was significantly reduced. Conclusion: In this study, we report for the first time, the implantation of a TEBV in the descending aorta in a large animal model. TEBV were fully functional for up to 3 months. At 6 months the graft remained functional but significantly dilated, most likely caused by an insufficient elastic fiber synthesis. Hence, future studies need to focus on the stimulation of elastin synthesis in TEBV.

Elastic fibers are components of the extracellular matrix and confer resilience1. Once laid down, they are thought to remain stable2, except in the uterine tract where cycles of active remodeling occur3. Loss of elastic fibers underlies connective tissue aging and important diseases including emphysema4,5,6,7. Failure to maintain elastic fibers is explained by a theory of antielastase-elastase imbalance8, but little is known about the role of renewal. Here we show that mice lacking the protein lysyl oxidase–like 1 (LOXL1) do not deposit normal elastic fibers in the uterine tract post partum and develop pelvic organ prolapse, enlarged airspaces of the lung, loose skin and vascular abnormalities with concomitant tropoelastin accumulation. Distinct from the prototypic lysyl oxidase (LOX), LOXL1 localizes specifically to sites of elastogenesis and interacts with fibulin-5. Thus elastin polymer deposition is a crucial aspect of elastic fiber maintenance and is dependent on LOXL1, which serves both as a cross-linking enzyme and an element of the scaffold to ensure spatially defined deposition of elastin.

Matrix metalloproteinases (MMPs) are degradative enzymes, which act to remodel tissue. Their activity is regulated by the tissue inhibitors of metalloproteinases (TIMPs). An imbalance in the degradation/inhibition activities has been associated with many diseases, including sepsis. We have previously shown that TIMP-3 knockout animals develop spontaneous, progressive air space enlargement. The objectives of this study were to determine the effects of a septic lung stress induced by cecal ligation and perforation (CLP) on lung function, structure, pulmonary surfactant, and inflammation in TIMP-3 null mice. Knockout and wild-type animals were randomized to either sham or CLP surgery, allowed to recover for 6 h, and then euthanized. TIMP-3 null animals exposed to sham surgery had a significant increase in lung compliance when compared with sham wild-type mice. Additionally, the TIMP-3 knockout mice showed a significant increase in compliance following CLP. Rapid compliance changes were accompanied by significantly decreased collagen and fibronectin levels and increased gelatinase (MMP-2 and -9) abundance and activation. Additionally, in situ zymography showed increased airway-associated gelatinase activity in the knockout animals enhanced following CLP. In conclusion, exposing TIMP-3 null animals to sepsis rapidly enhances the phenotypic abnormalities of these mice, due to increased MMP activity induced by CLP.

Processes involving self-assembly of monomeric units into organized polymeric arrays are currently the subject of much attention, particularly in the areas of nanotechnology and biomaterials. One biological example of a protein polymer with potential for self-organization is elastin. Elastin is the extracellular matrix protein that imparts the properties of extensibility and elastic recoil to large arteries, lung parenchyma, and other tissues. Tropoelastin, the approximately 70 kDa soluble monomeric form of elastin, is highly nonpolar in character, consisting essentially of 34 alternating hydrophobic and crosslinking domains. Crosslinking domains contain the lysine residues destined to form the covalent intermolecular crosslinks that stabilize the polymer. We and others have suggested that the hydrophobic domains are sites of interactions that contribute to juxtaposition of lysine residues in preparation for crosslink formation. Here, using recombinant polypeptides based on sequences in human elastin, we demonstrate that as few as three hydrophobic domains flanking two crosslinking domains are sufficient to support a self-assembly process that aligns lysines for zero-length crosslinking, resulting in formation of the crosslinks of native elastin. This process allows fabrication of a polymeric matrix with solubility and mechanical properties similar to those of native elastin.

Supravalvular aortic stenosis is an autosomal-dominant disease of elastin (Eln) insufficiency caused by loss-of-function mutations or gene deletion. Recently, we have modeled this disease in mice (Eln +/‐ ) and found that Eln haploinsufficiency results in unexpected changes in cardiovascular hemodynamics and arterial wall structure. Eln +/‐ animals were found to be stably hypertensive from birth, with a mean arterial pressure 25‐30 mmHg higher than their wild-type counterparts. The animals have only moderate cardiac hypertrophy and live a normal life span with no overt signs of degenerative vascular disease. Examination of arterial mechanical properties showed that the inner diameters of Eln +/‐ arteries were generally smaller than wild-type arteries at any given intravascular pressure. Because the Eln+/‐ mouse is hypertensive, however, the effective arterial working diameter is comparable to that of the normotensive wild-type animal. Physiological studies indicate a role for the reninangiotensin system in maintaining the hypertensive state. The association of hypertension with elastin haploinsufficiency in humans and mice strongly suggests that elastin and other proteins of the elastic fiber should be considered as causal genes for essential hypertension.

A number of strategies have been investigated to enhance the mechanical stability of engineered tissues. In this study, we utilized lysyl oxidase (LO) to enzymatically crosslink extracellular matrix (ECM) proteins, particularly collagen and elastin, to enhance the mechanical integrity of the ECM and thereby impart mechanical strength to the engineered tissue. Vascular smooth muscle cells (VSMCs) were liposomally transfected with the LO gene. Both Northern and Western analyses confirmed increased LO expression. Increased LO activity was demonstrated using a fluorescent enzyme substrate assay and by observation of the presence of increased levels of desmosine, a product of LO crosslinking, in the ECM. The mechanical effects of altered crosslink densities within tissue-engineered constructs were demonstrated in a VSMC-populated collagen gel model. When smooth muscle cells transfected with lysyl oxidase were seeded in collagen gels, the tensile strength and elastic modulus in these constructs increased by approximately two-fold compared to constructs seeded with mock-transfected VSMCs. Also, desmosine levels in the LO-populated collagen gels were higher than they were in mock-seeded gels, as demonstrated via immunohistochemical staining. Compositional analysis of the ECM deposited by the transformed cells showed similar collagen and elastin levels, and cell proliferation rates were similar as well, thus attributing increased mechanical properties to ECM crosslinking.

Lysyl oxidase (LOX) is an enzyme responsible for the cross-linking of collagen and elastin both in vitro and in vivo. The unique functions of the individual members of this multigene family have been difficult to ascertain because of highly conserved catalytic domains and overlapping tissue expression patterns. To address this problem of functional and structural redundancy and to determine the role of LOX in the development of tissue integrity, Lox gene expression was deleted by targeted mutagenesis in mice. Lox-targeted mice (LOX(-/-)) died soon after parturition, exhibiting cardiovascular instability with ruptured arterial aneurysms and diaphragmatic rupture. Microscopic analysis of the aorta demonstrated fragmented elastic fiber architecture in homozygous mutant null mice. LOX activity, as assessed by desmosine (elastin cross-link) analysis, was reduced by approximately 60% in the aorta and lungs of homozygous mutant animals compared with wild type mice. Immature collagen cross-links were decreased but to a lesser degree than elastin cross-links in LOX(-/-) mice. Thus, lysyl oxidase appears critical during embryogenesis for structural stability of the aorta and diaphragm and connective tissue development.

Homocysteinemia in humans is associated with vascular complications that increase the risk for atherosclerosis and stroke. Animal studies have shown that the disease is multifactorial and includes lesions associated with the elastin component of the extracellular matrix. In the following experiments we have used the aortas from rapidly growing chicks to assess the cause of the elastin defects resulting from homocysteinemia. Day-old chicks were fed diets containing varying amounts of DL-methionine, DL-homocysteine, homocysteine thiolactone or DL-cysteine for periods up to 9 wk. Three weeks after feeding 2% DL-methionine the plasma methionine was elevated 20-fold, whereas plasma homocysteine was more than 3-fold normal plasma values. The aortas showed severe histopathology, evidenced by the pronounced separation of elastic lamellae with marked smooth muscle proliferation and, in some instances, aneurysms. There was no evidence of decreased desmosine content or a significant reduction in lysyl oxidase in the aortas from the treated groups compared to those from controls. Increasing other dietary factors such as the vitamins required for methionine metabolism had no effect on the development of the vascular lesions. Twenty to 30% of the chicks fed the high methionine diets exhibited severe neurological problems, expressed as tonic contractions or seizures. Electron microscopy revealed disordered aortic elastic fibrils, associated with either an absence of or disrupted assembly of microfibrils. Immunohistochemical studies demonstrated a loss of fibrillin-2 immunoreactivity in the aortas of chicks fed 2% methionine. The studies suggest that elevated plasma methionine or its metabolites disrupt normal microfibril configuration, leading to the assembly of aberrant elastic fibers. J. Nutr. 132: 2143-2150, 2002.

Photodamage is characterized by degradation of collagen and accumulation of abnormal elastin in the superficial dermis and several matrix metalloproteinases have previously been implicated in this process. Using immunohistochemistry and in situ hybridization, we have studied the localization of two elastolytic matrix metalloproteinases, matrilysin (matrix metalloproteinase-7) and human macrophage metalloelastase (matrix metalloproteinase-12) in solar damage. Human macrophage metalloelastase protein was detected in the superficial dermis in areas of elastotic material. Matrix metalloproteinase-7 was seen in the mid-dermis in regions with less damaged elastic fibers and morphologically better preserved collagen as well as in a band-like pattern below basal keratinocytes in eight of 18 solar elastosis. In samples taken from healthy volunteers 3 d after repeated ultraviolet A or ultraviolet B photoprovocation, occasional immunopositive cells for human macrophage metalloelastase (stromal) or matrix metalloproteinase-7 (sweat gland epithelium) were detected. In samples taken 1 d after ultraviolet B exposure, however, basal keratinocytes were matrix metalloproteinase-7 immunopositive, explaining the linear immunostaining below basal keratinocytes noted particularly in ultraviolet B treated 3 d specimens. Upregulation of metalloelastase was also demonstrated in the skin of hairless mice after repeated ultraviolet exposure. In normal skin, no staining for human macrophage metalloelastase or matrix metalloproteinase-7 was observed in association with elastin. The amount of immunoreactivity for the substrates of matrix metalloproteinase-7, versican, and tenascin, was clearly increased in solar elastosis and photoprovocated skin; versican but not tenascin was detected in the same areas as matrix metalloproteinase-7. Our results suggest that both matrix metalloproteinase-7 and -12 may contribute to remodeling of elastotic areas in sun-damaged skin.

Lung injury and matrix destruction result in the release into the circulation of desmosine, a product of elastin catabolism. To determine whether measurements of desmosine concentrations in the urine can provide important information about the severity of lung injury, we measured the kinetics of lung elastin catabolism following elastase-induced and fibrotic lung injury in mice. Pancreatic elastase instillation in the lung caused immediate elastin destruction, removing as much as 25% of the lung elastin within the first few hours. Peak concentrations of desmosine peptides appeared in the blood within 1-2 hours, and soluble elastin peptides in the lung and urine followed a similar course with peak levels occurring early and then declining rapidly, approaching control levels by 24 hours. More than half of the desmosine peptides removed from the lung following elastase injury were sequestered by the kidney and then slowly released over a period of several days. Lung histology and morphological measurements indicated that bleomycin, TiO2, and SiO2 instillation resulted in severely fibrotic lungs. However, the lung injuries sustained from these agents resulted in minimal elastin damage, representing less than 0.1% of the total lung elastin catabolized as estimated by desmosine analysis. The rapid accumulation of desmosine in renal tissue and its slow release into the urine keep urine concentrations low and minimize the power of urine desmosine assays for quantifying low level tissue destruction.

Although matrix metalloproteinases (MMPs) are expressed in abundance in arterial aneurysms, their contribution to arterial wall degeneration, dilation, and rupture has not been determined. We investigated MMP function in a rat model of aneurysm associated with arterial dilation, elastin loss, medial invasion by mononuclear inflammatory cells, and MMP upregulation. Rupture was correlated with increased gelatinase B (MMP-9) and activated gelatinase A (MMP-2). Syngeneic rat smooth muscle cells retrovirally transfected with tissue inhibitor of matrix metalloproteinases (TIMP)-1 cDNA (LTSN) or with the vector alone as a control (LXSN) were seeded onto the luminal surface of the vessels. The seeding of LTSN cells resulted in TIMP-1 local overexpression. The seeding with LTSN cells, but not LXSN cells, decreased MMP-9, activated MMP-2 and 28-kD caseinase and elastase activity, preserved elastin in the media, and prevented aneurysmal degeneration and rupture. We conclude that MMP overexpression is responsible for aneurysmal degeneration and rupture in this rat model and that local pharmacological blockade might be a reasonable strategy for controlling the formation of aneurysms in humans.