Microfibril-associated glycoprotein 4 forms octamers that mediate interactions with elastogenic proteins and cells.

Microfibril-associated glycoprotein 4 (MFAP4) is a 36-kDa extracellular matrix glycoprotein with critical roles in organ fibrosis, chronic obstructive pulmonary disease, and cardiovascular disorders, including aortic aneurysms. MFAP4 multimerises and interacts with elastogenic proteins, including fibrillin-1 and tropoelastin, and with cells via integrins. Structural details of MFAP4 and its potential interfaces for these interactions are unknown. Here, we present a cryo-electron microscopy structure of human MFAP4. In the presence of calcium, MFAP4 assembles as an octamer, where two sets of homodimers constitute the top and bottom halves of each octamer. Each homodimer is linked together by an intermolecular disulphide bond. A C34S missense mutation prevents disulphide-bond formation between monomers but does not prevent octamer assembly. The atomic model, built into the 3.55 Å cryo-EM map, suggests that salt-bridge interactions mediate homodimer assembly, while non-polar residues form the interface between octamer halves. In the absence of calcium, an MFAP4 octamer dissociates into two tetramers. Binding studies with fibrillin-1, tropoelastin, LTBP4, and small fibulins show that MFAP4 has multiple surfaces for protein-protein interactions, most of which depend upon MFAP4 octamer assembly. The C34S mutation does not affect these protein interactions or cell interactions. MFAP4 assemblies with fibrillin-1 abrogate MFAP4 interactions with cells.

Vitamin C, lactoferrin and elastin-Advancing the science.

BACKGROUND: This study follows an initial scientific validation linking sodium ascorbate (SAC) with elastin conservation and the clinical trial histology observation that the full formulation tested there stimulated elastin development. In an effort to explain the increased elastin response, a candidate was sought that may provide synergy to SAC during elastin stimulation. Lactoferrin was the constituent chosen to explore in this realm. MATERIALS AND METHODS: Using the previously described ex vivo skin model, freshly collected discarded human skin from 2 donors was used to evaluate the effects of lactoferrin and SAC alone and together, and L-ascorbate CE Ferulic formulation (CEF) on elastogenesis. Four skin explants were topically subjected to the treatments daily for 7 days and one group was left untreated as a negative control. The tissue was fixed and embedded. Sections were evaluated by immunofluorescence using antibodies targeting Tropoelastin and CD44, with DAPI counterstaining to observe nuclei. Images were then analyzed using ImageJ. RESULTS: Treatment with SAC and lactoferrin demonstrated a significant synergistic effect on tropoelastin stimulation compared to the single treatments. In addition, this combination demonstrated intact and increased elastin fibers in contrast to the CEF, which portrayed fragmented elastin fibers. In addition, an additive effect of SAC also contributed to the enhanced CD44, suggesting an increased presence of hyaluronic acid, a new observation for this compound. CONCLUSION: This study complements a series of studies that have been undertaken to validate the efficacy of a novel antioxidant formulation. Aside from its efficacy in ROS management, the SAC constituent is unique in the different forms of Vitamin C for its ability to conserve elastin. Prior clinical studies demonstrated additive elastin stimulation on histology, not just conservation. From this current study, the combination of SAC with lactoferrin may be responsible for this additive stimulatory effect on elastin. This presents a significant advance in topical antioxidant formulations where the Vitamin C component provides antioxidant and collagen stimulation with additional elastin stimulation rather than degradation.

Single Nucleotide Polymorphisms Mutation of Tropoelastin Gene Affects Tropoelastin mRNA and Elastin Expressions in Human Aortic Smooth Muscle Cells.

We aimed to explore the effects of single nucleotide polymorphisms (SNPs) in tropoelastin gene on tropoelastin mRNA and elastin expressions in human aortic smooth muscle cells (HASMCs). Two SNP loci, rs2071307 (G/A) and rs1785598 (G/C), were selected to construct recombinant lentivirus vectors carrying wild-type and mutant tropoelastin gene. Recombinant plasmids including pWSLV-02-ELN, pWSLV-02-ELN-mut1, and pWSLV-02-ELN-mut2 were constructed, before being amplified by polymerase chain reaction (PCR) and sequenced. The prepared plasmids and the packaging plasmids (pVSV-G and psPAX2) were cotransfected into HEK293T cells to obtain recombinant lentiviruses carrying tropoelastin gene. Afterward, HASMCs were infected with recombinant lentiviruses, and the positive cells sorted by flow cytometry were amplified. Four stable HASMCs cell lines including pWSLV-02-ELN, pWSLV-02-ELN-mut1, pWSLV-02-ELN-mut2, and pWSLV-02 vector were constructed. The expressions of tropoelastin mRNA and elastin in HASMCs were detected by real-time quantitative reverse transcription-PCR and western blot, respectively. Recombinant plasmids including pWSLV-02-ELN-mut1, pWSLV-02-ELN-mut2, and pWSLV-02-ELN were successfully constructed. Recombinant lentiviruses carrying tropoelastin gene were obtained via lentivirus packaging. After infection for 24 h, 3 days and 5 days in HASMCs, tropoelastin mRNA expressions in pWSLV-02-ELN-mut1 and pWSLV-02-ELN-mut2 groups were significantly lower than that of pWSLV-02-ELN group. Besides, after infection for 24 h, 3 days, and 5 days, elastin levels in pWSLV-02-ELN-mut1 and pWSLV-02-ELN-mut2 groups were significantly lower than that in pWSLV-02-ELN group. In conclusion, SNPs mutation of tropoelastin gene affected the expression of tropoelastin mRNA and elastin, suggesting that the polymorphisms of rs2071307 and rs17855988 in tropoelastin gene might be important factors for AD development.

Stochastic simulations of self-organized elastogenesis in the developing lung.

In the normal lung, the dominant cable is an elastic "line element" composed of elastin fibers bound to a protein scaffold. The cable line element maintains alveolar geometry by balancing surface forces within the alveolus and changes in lung volume with exercise. Recent work in the postnatal rat lung has suggested that the process of cable development is self-organized in the extracellular matrix. Early in postnatal development, a blanket of tropoelastin (TE) spheres appear in the primitive lung. Within 7 to 10 days, the TE spheres are incorporated into a distributed protein scaffold creating the mature cable line element. To study the process of extracellular assembly, we used cellular automata (CA) simulations. CA simulations demonstrated that the intermediate step of tropoelastin self-aggregation into TE spheres enhanced the efficiency of cable formation more than 5-fold. Similarly, the rate of tropoelastin production had a direct impact on the efficiency of scaffold binding. The binding affinity of the tropoelastin to the protein scaffold, potentially reflecting heritable traits, also had a significant impact on cable development. In contrast, the spatial distribution of TE monomer production, increased Brownian motion and variations in scaffold geometry did not significantly impact simulations of cable development. We conclude that CA simulations are useful in exploring the impact of concentration, geometry, and movement on the fundamental process of elastogenesis.

Tropoelastin Improves Post-Infarct Cardiac Function.

BACKGROUND: Myocardial infarction (MI) is among the leading causes of death worldwide. Following MI, necrotic cardiomyocytes are replaced by a stiff collagen-rich scar. Compared to collagen, the extracellular matrix protein elastin has high elasticity and may have more favorable properties within the cardiac scar. We sought to improve post-MI healing by introducing tropoelastin, the soluble subunit of elastin, to alter scar mechanics early after MI. METHODS AND RESULTS: We developed an ultrasound-guided direct intramyocardial injection method to administer tropoelastin directly into the left ventricular anterior wall of rats subjected to induced MI. Experimental groups included shams and infarcted rats injected with either PBS vehicle control or tropoelastin. Compared to vehicle treated controls, echocardiography assessments showed tropoelastin significantly improved left ventricular ejection fraction (64.7±4.4% versus 46.0±3.1% control) and reduced left ventricular dyssynchrony (11.4±3.5 ms versus 31.1±5.8 ms control) 28 days post-MI. Additionally, tropoelastin reduced post-MI scar size (8.9±1.5% versus 20.9±2.7% control) and increased scar elastin (22±5.8% versus 6.2±1.5% control) as determined by histological assessments. RNA sequencing (RNAseq) analyses of rat infarcts showed that tropoelastin injection increased genes associated with elastic fiber formation 7 days post-MI and reduced genes associated with immune response 11 days post-MI. To show translational relevance, we performed immunohistochemical analyses on human ischemic heart disease cardiac samples and showed an increase in tropoelastin within fibrotic areas. Using RNA-seq we also demonstrated the tropoelastin gene ELN is upregulated in human ischemic heart disease and during human cardiac fibroblast-myofibroblast differentiation. Furthermore, we showed by immunocytochemistry that human cardiac fibroblast synthesize increased elastin in direct response to tropoelastin treatment. CONCLUSIONS: We demonstrate for the first time that purified human tropoelastin can significantly repair the infarcted heart in a rodent model of MI and that human cardiac fibroblast synthesize elastin. Since human cardiac fibroblasts are primarily responsible for post-MI scar synthesis, our findings suggest exciting future clinical translation options designed to therapeutically manipulate this synthesis.

Long Hanging Structure of Collagen VII Connects the Elastic Fibers and the Basement Membrane in Young Skin Tissue.

Aging leads to substantial structural changes in the skin. Elastic fibers maintain skin structure, but their degeneration and loss of function with age result in wrinkle formation and loss of skin elasticity. Oxytalan fiber, a type of elastic fiber, extends close to the dermal-epidermal junction (DEJ) from the back of the dermis. Oxytalan fibers are abundant in the papillary layer and contribute to skin elasticity and texture. However, to accurately understand the mechanisms of skin elasticity, the interaction between elastic fibers and DEJ should be elucidated. Here, we investigated elastic fibers and DEJ and their structural alterations with aging. Several basement membrane proteins [collagen (COL) IV, COLVII, and laminin 332], fibrous tropoelastin, and fibrillin-1 in excised human skin tissue were observed using three-dimensional imaging. Age-related alterations in COLVII, elastic fibers, and fibrillin-1 were evaluated. We found that COLVII forms long hanging structures and is co-localized with fibrous tropoelastin in young skin but not aged skin. Fibrillin-1-rich regions were observed at the tips of elastin fibers in young skin tissue, but rarely in aged skin. This co-localization of elastic fiber and COLVII may maintain skin structure, thereby preventing wrinkling and sagging. COLVII is a potential therapeutic target for skin wrinkling.

Liquid to solid transition of elastin condensates.

Liquid-liquid phase separation of tropoelastin has long been considered to be an important early step in the complex process of elastin fiber assembly in the body and has inspired the development of elastin-like peptides with a wide range of industrial and biomedical applications. Despite decades of study, the material state of the condensed liquid phase of elastin and its subsequent maturation remain poorly understood. Here, using a model minielastin that mimics the alternating domain structure of full-length tropoelastin, we examine the elastin liquid phase. We combine differential interference contrast (DIC), fluorescence, and scanning electron microscopy with particle-tracking microrheology to resolve the material transition occurring within elastin liquids over time in the absence of exogenous cross-linking. We find that this transition is accompanied by an intermediate stage marked by the coexistence of insoluble solid and dynamic liquid phases giving rise to significant spatial heterogeneities in material properties. We further demonstrate that varying the length of the terminal hydrophobic domains of minielastins can tune the maturation process. This work not only resolves an important step in the hierarchical assembly process of elastogenesis but further contributes mechanistic insight into the diverse repertoire of protein condensate maturation pathways with emerging importance across biology.

Elastic Fibre Proteins in Elastogenesis and Wound Healing.

As essential components of our connective tissues, elastic fibres give tissues such as major blood vessels, skin and the lungs their elasticity. Their formation is complex and co-ordinately regulated by multiple factors. In this review, we describe key players in elastogenesis: fibrillin-1, tropoelastin, latent TGFß binding protein-4, and fibulin-4 and -5. We summarise their roles in elastogenesis, discuss the effect of their mutations on relevant diseases, and describe their interactions involved in forming the elastic fibre network. Moreover, we look into their roles in wound repair for a better understanding of their potential application in tissue regeneration.

Globule and fiber formation with elastin-like polypeptides: a balance of coacervation and crosslinking.

Elastic fiber assembly is a complex process that requires the coacervation and cross-linking of the protein building block tropoelastin. To date, the order, timing, and interplay of coacervation and crosslinking is not completely understood, despite a great number of advances into understanding the molecular structure and functions of the many proteins involved in elastic fiber assembly. With a simple in vitro model using elastin-like polypeptides and the natural chemical crosslinker genipin, we demonstrate the strong influence of the timing and kinetics of crosslinking reaction on the coacervation, crosslinking extent, and resulting morphology of elastin. We also outline a method for analyzing elastin droplet network formation as a heuristic for measuring the propensity for elastic fiber formation. From this we show that adding crosslinker during peak coacervation dramatically increases the propensity for droplet network formation.

The "Elastic Perspective" of SARS-CoV-2 Infection and the Role of Intrinsic and Extrinsic Factors.

Elastin represents the structural component of the extracellular matrix providing elastic recoil to tissues such as skin, blood vessels and lungs. Elastogenic cells secrete soluble tropoelastin monomers into the extracellular space where these monomers associate with other matrix proteins (e.g., microfibrils and glycoproteins) and are crosslinked by lysyl oxidase to form insoluble fibres. Once elastic fibres are formed, they are very stable, highly resistant to degradation and have an almost negligible turnover. However, there are circumstances, mainly related to inflammatory conditions, where increased proteolytic degradation of elastic fibres may lead to consequences of major clinical relevance. In severely affected COVID-19 patients, for instance, the massive recruitment and activation of neutrophils is responsible for the profuse release of elastases and other proteolytic enzymes which cause the irreversible degradation of elastic fibres. Within the lungs, destruction of the elastic network may lead to the permanent impairment of pulmonary function, thus suggesting that elastases can be a promising target to preserve the elastic component in COVID-19 patients. Moreover, intrinsic and extrinsic factors additionally contributing to damaging the elastic component and to increasing the spread and severity of SARS-CoV-2 infection are reviewed.

Elastic tissue disruption is a major pathogenic factor to human vascular disease.

Elastic fibers are essential components of the arterial extracellular matrix. They consist of the protein elastin and an array of microfibrils that support the protein and connect it to the surrounding matrix. The elastin gene encodes tropoelastin, a protein that requires extensive cross-linking to become elastin. Tropoelastin is expressed throughout human life, but its expression levels decrease with age, suggesting that the potential to synthesize elastin persists during lifetime although declines with aging. The initial abnormality documented in human atherosclerosis is fragmentation and loss of the elastic network in the medial layer of the arterial wall, suggesting an imbalance between elastic fiber injury and restoration. Damaged elastic structures are not adequately repaired by synthesis of new elastic elements. Progressive collagen accumulation follows medial elastic fiber disruption and fibrous plaques are formed, but advanced atherosclerosis lesions do not develop in the absence of prior elastic injury. Aging is associated with arterial extracellular matrix anomalies that evoke those present in early atherosclerosis. The reduction of elastic fibers with subsequent collagen accumulation leads to arterial stiffening and intima-media thickening, which are independent predictors of incident hypertension in prospective community-based studies. Arterial stiffening precedes the development of hypertension. The fundamental role of the vascular elastic network to arterial structure and function is emphasized by congenital disorders caused by mutations that disrupt normal elastic fiber production. Molecular changes in the genes coding tropoelastin, lysyl oxidase (tropoelastin cross-linking), and elastin-associated microfibrils, including fibrillin-1, fibulin-4, and fibulin-5 produce severe vascular injury due to absence of functional elastin.

An increase in elastogenic components in the placental villi of women with chronic venous disease during pregnancy is associated with decreased EGFL7 expression level.

Chronic venous disease (CVD) is the response to a series of hemodynamic changes in the venous system and the onset of this disease is often triggered by pregnancy. Placental tissue is particularly sensitive to the characteristic changes which occurs in venous hypertension. In this regard, changes in the extracellular matrix (ECM), that occur to adapt to this situation, are fundamental to controlling elastogenesis. Therefore, the aim of the present study was to analyze the changes that occur in the mRNA and protein expression level of proteins related to elastogenesis in the placental villi of women diagnosed with CVD, in the third trimester of pregnancy. An observational, analytical and prospective cohort study was conducted, in which the placenta from 62 women with CVD were compared with that in placenta from 52 women without a diagnosis of CVD. Gene and protein expression levels were analyzed using reverse transcription­quantitative PCR and immunohistochemistry, respectively. The results showed a significant decrease in the gene and protein expression level of EGFL7 in the placental villi of women with CVD. By contrast, significant increases in the gene and protein expression level of ECM­related proteins, such as tropoelastin, fibulin 4, fibrillin 1 and members of the lysyl oxidase family (LOX and LOXL­1) were also found in the placental villi of women with CVD. To the best of our knowledge, the results from the present study showed for the first time that CVD during pregnancy was associated with changes in the mRNA and protein expression level in essential components of the EGFL7­modulated elastogenesis process in placental villi.

Visualization of elastin using cardiac magnetic resonance imaging after myocardial infarction as inflammatory response.

The aim of this study was to investigate the merits of magnetic resonance imaging (MRI) using an elastin-binding contrast agent after myocardial infarction in mouse models with deletions of monocyte populations. Permanent ligation of the left anterior descending (LAD) artery was conducted in 10 wild-type mice and 10 each of three knockout models: CX3CR-/-, CCR2-/-, and MCP-1-/-. At 7 days and 30 days after permanent ligation, cardiac MRI was performed with a 7 T-Bruker horizontal scanner for in vivo detection of elastin with an elastin/tropoelastin-specific contrast agent (ESMA). Histology was performed with staining for elastin, collagen I and III, and F4/80. Real-time PCR was conducted to quantify the expression of genes for collagen I and III, F4/80, and tumor necrosis factor alpha (TNFα). Histological and ESMA-indicated elastin areas were strongly correlated (r = 0.8). 30 days after permanent ligation, CCR2-deficient mice demonstrated higher elastin levels in the scar relative to MCP-1-/- (p < 0.04) and wild-type mice (p < 0.02). The ejection fraction was lower in CCR2-deficient mice. In vivo MRI in mouse models of MI can detect elastin deposition after myocardial infarction, highlighting the pivotal role of elastin in myocardial remodeling in mouse models with deletions of monocyte populations.

Domains 12 to 16 of tropoelastin promote cell attachment and spreading through interactions with glycosaminoglycan and integrins alphaV and alpha5beta1.

Elastin is an extracellular matrix component with key structural and biological roles in elastic tissues. Interactions between resident cells and tropoelastin, the monomer of elastin, underpin elastin's regulation of cellular processes. However, the nature of tropoelastin-cell interactions and the contributions of individual tropoelastin domains to these interactions are only partly elucidated. In this study, we identified and characterized novel cell-adhesive sites in the tropoelastin N-terminal region between domains 12 and 16. We found that this region interacts with αV and α5ß1 integrin receptors, which mediate cell attachment and spreading. A peptide sequence from within this region, spanning domains 14 to mid-domain 16, binds heparan sulfate through electrostatic interactions with peptide lysine residues and induces conformational ordering of the peptide. We propose that domains 14-16 direct initial cell attachment through cell-surface heparan sulfate glycosaminoglycans, followed by αV and α5ß1 integrin-promoted attachment and spreading on domains 12-16 of tropoelastin. These findings advance our mechanistic understanding of elastin matrix biology, with the potential to enhance tissue regenerative outcomes of elastin-based materials.

The evolutionary background and functional consequences of the rs2071307 polymorphism in human tropoelastin.

Elastin is a major polymeric protein of the extracellular matrix, providing critical properties of extensibility and elastic recoil. The rs2071307 genomic polymorphism, resulting in the substitution of a serine for a glycine residue in a VPG motif in tropoelastin, has an unusually high minor allele frequency in humans. A consequence of such allelic heterozygosity would be the presence of a heterogeneous elastin polymer in up to 50% of the population, a situation which appears to be unique to Homo sapiens. VPG motifs are extremely common in hydrophobic domains of tropoelastins and are the sites of transient ß-turns that are essential for maintaining the conformational flexibility required for its function as an entropic elastomer. Earlier data demonstrated that single amino acid substitutions in tropoelastin can have functional consequences for polymeric elastin, particularly when present in mixed polymers. Here, using NMR and molecular dynamics approaches, we show the rs2071307 polymorphism reduces local propensity for ß-turn formation, with a consequent increase in polypeptide hydration and an expansion of the conformational ensemble manifested as an increased hydrodynamic radius, radius of gyration and asphericity. Furthermore, this substitution affects functional properties of polymeric elastin, particularly in heterogeneous polymers mimicking allelic heterozygosity. We discuss whether such effects, together with the unusually high minor allele frequency of the polymorphism, could imply some some evolutionary advantage for the heterozygous state.

Insights into the biophysical forces between proteins involved in elastic fiber assembly.

Elastogenesis is a complex process beginning with transcription, translation, and extracellular release of precursor proteins leading to crosslinking, deposition, and assembly of ubiquitous elastic fibers. While the biochemical pathways by which elastic fibers are assembled are known, the biophysical forces mediating the interactions between the constituent proteins are unknown. Using atomic force microscopy, we quantified the adhesive forces among the elastic fiber components, primarily between tropoelastin, elastin binding protein (EBP), fibrillin-1, fibulin-5, and lysyl oxidase-like 2 (LOXL2). The adhesive forces between tropoelastin and other tissue-derived proteins such as insoluble elastin, laminin, and type I collagens were also assessed. The adhesive forces between tropoelastin and laminin were strong (1767 ± 126 pN; p < 10-5vs. all others), followed by forces (≥200 pN) between tropoelastin and human collagen, mature elastin, or tropoelastin. The adhesive forces between tropoelastin and rat collagen, EBP, fibrillin-1, fibulin-5, and LOXL2 coated on fibrillin-1 were in the range of 100-200 pN. The forces between tropoelastin and LOXL2, LOXL2 and fibrillin-1, LOXL2 and fibulin-5, and fibrillin-1 and fibulin-5 were less than 100 pN. Introducing LOXL2 decreased the adhesive forces between the tropoelastin monomers by ∼100 pN. The retraction phase of force-deflection curves was fitted to the worm-like chain model to calculate the rigidity and flexibility of these proteins as they unfolded. The results provided insights into how each constituent's stretching under deformation contributes to structural and mechanical characteristics of these fibers and to elastic fiber assembly.

Transglutaminase-Mediated Cross-Linking of Tropoelastin to Fibrillin Stabilises the Elastin Precursor Prior to Elastic Fibre Assembly.

Elastic fibres are essential components of all mammalian elastic tissues such as blood vessels, lung and skin, and are critically important for the mechanical properties they endow. The main components of elastic fibres are elastin and fibrillin, where correct formation of elastic fibres requires a fibrillin microfibril scaffold for the deposition of elastin. It has been demonstrated previously that the interaction between fibrillin and tropoelastin, the elastin precursor, increases the rate of assembly of tropoelastin. Furthermore, tropoelastin and fibrillin can be cross-linked by transglutaminase-2, but the function of cross-linking on their elastic properties is yet to be elucidated. Here we show that transglutaminase cross-linking supports formation of a 1:1 stoichiometric fibrillin-tropoelastin complex. SAXS data show that the complex retains features of the individual proteins but is elongated supporting end-to-end assembly. Elastic network models were constructed to compare the dynamics of tropoelastin and fibrillin individually as well as in the cross-linked complex. Normal mode analysis was performed to determine the structures' most energetically favourable, biologically accessible motions which show that within the complex, tropoelastin is less mobile and this molecular stabilisation extends along the length of the tropoelastin molecule to regions remote from the cross-linking site. Together, these data suggest a long-range stabilising effect of cross-linking that occurs due to the covalent linkage of fibrillin to tropoelastin. This work provides insight into the interactions of tropoelastin and fibrillin and how cross-link formation stabilises the elastin precursor so it is primed for elastic fibre assembly.

Elastases and elastokines: elastin degradation and its significance in health and disease.

Elastin is an important protein of the extracellular matrix of higher vertebrates, which confers elasticity and resilience to various tissues and organs including lungs, skin, large blood vessels and ligaments. Owing to its unique structure, extensive cross-linking and durability, it does not undergo significant turnover in healthy tissues and has a half-life of more than 70 years. Elastin is not only a structural protein, influencing the architecture and biomechanical properties of the extracellular matrix, but also plays a vital role in various physiological processes. Bioactive elastin peptides termed elastokines - in particular those of the GXXPG motif - occur as a result of proteolytic degradation of elastin and its non-cross-linked precursor tropoelastin and display several biological activities. For instance, they promote angiogenesis or stimulate cell adhesion, chemotaxis, proliferation, protease activation and apoptosis. Elastin-degrading enzymes such as matrix metalloproteinases, serine proteases and cysteine proteases slowly damage elastin over the lifetime of an organism. The destruction of elastin and the biological processes triggered by elastokines favor the development and progression of various pathological conditions including emphysema, chronic obstructive pulmonary disease, atherosclerosis, metabolic syndrome and cancer. This review gives an overview on types of human elastases and their action on human elastin, including the formation, structure and biological activities of elastokines and their role in common biological processes and severe pathological conditions.

Dill Extract Induces Elastic Fiber Neosynthesis and Functional Improvement in the Ascending Aorta of Aged Mice with Reversal of Age-Dependent Cardiac Hypertrophy and Involvement of Lysyl Oxidase-Like-1.

Elastic fibers (90% elastin, 10% fibrillin-rich microfibrils) are synthesized only in early life and adolescence mainly by the vascular smooth muscle cells through the cross-linking of its soluble precursor, tropoelastin. Elastic fibers endow the large elastic arteries with resilience and elasticity. Normal vascular aging is associated with arterial remodeling and stiffening, especially due to the end of production and degradation of elastic fibers, leading to altered cardiovascular function. Several pharmacological treatments stimulate the production of elastin and elastic fibers. In particular, dill extract (DE) has been demonstrated to stimulate elastin production in vitro in dermal equivalent models and in skin fibroblasts to increase lysyl oxidase-like-1 (LOXL-1) gene expression, an enzyme contributing to tropoelastin crosslinking and elastin formation. Here, we have investigated the effects of a chronic treatment (three months) of aged male mice with DE (5% or 10% v/v, in drinking water) on the structure and function of the ascending aorta. DE treatment, especially at 10%, of aged mice protected pre-existing elastic lamellae, reactivated tropoelastin and LOXL-1 expressions, induced elastic fiber neo-synthesis, and decreased the stiffness of the aging aortic wall, probably explaining the reversal of the age-related cardiac hypertrophy also observed following the treatment. DE could thus be considered as an anti-aging product for the cardiovascular system.

Tropoelastin: an in vivo imaging marker of dysfunctional matrix turnover during abdominal aortic dilation.

AIMS: Dysfunctional matrix turnover is present at sites of abdominal aortic aneurysm (AAA) and leads to the accumulation of monomeric tropoelastin rather than cross-linked elastin. We used a gadolinium-based tropoelastin-specific magnetic resonance contrast agent (Gd-TESMA) to test whether quantifying regional tropoelastin turnover correlates with aortic expansion in a murine model. The binding of Gd-TESMA to excised human AAA was also assessed. METHODS AND RESULTS: We utilized the angiotensin II (Ang II)-infused apolipoprotein E gene knockout (ApoE-/-) murine model of aortic dilation and performed in vivo imaging of tropoelastin by administering Gd-TESMA followed by late gadolinium enhancement (LGE) magnetic resonance imaging (MRI) and T1 mapping at 3 T, with subsequent ex vivo validation. In a cross-sectional study (n = 66; control = 11, infused = 55) we found that Gd-TESMA enhanced MRI was elevated and confined to dilated aortic segments (control: LGE=0.13 ± 0.04 mm2, control R1= 1.1 ± 0.05 s-1 vs. dilated LGE =1.0 ± 0.4 mm2, dilated R1 =2.4 ± 0.9 s-1) and was greater in segments with medium (8.0 ± 3.8 mm3) and large (10.4 ± 4.1 mm3) compared to small (3.6 ± 2.1 mm3) vessel volume. Furthermore, a proof-of-principle longitudinal study (n = 19) using Gd-TESMA enhanced MRI demonstrated a greater proportion of tropoelastin: elastin expression in dilating compared to non-dilating aortas, which correlated with the rate of aortic expansion. Treatment with pravastatin and aspirin (n = 10) did not reduce tropoelastin turnover (0.87 ± 0.3 mm2 vs. 1.0 ± 0.44 mm2) or aortic dilation (4.86 ± 2.44 mm3 vs. 4.0 ± 3.6 mm3). Importantly, Gd-TESMA-enhanced MRI identified accumulation of tropoelastin in excised human aneurysmal tissue (n = 4), which was confirmed histologically. CONCLUSION: Tropoelastin MRI identifies dysfunctional matrix remodelling that is specifically expressed in regions of aortic aneurysm or dissection and correlates with the development and rate of aortic expansion. Thus, it may provide an additive imaging marker to the serial assessment of luminal diameter for surveillance of patients at risk of or with established aortopathy.