Elastic Fiber Formation Research Articles

Biaxial Stretch Improves Elastic Fiber Maturation, Collagen Arrangement, and Mechanical Properties in Engineered Arteries.

Tissue-engineered blood vessels (TEVs) are typically produced using the pulsatile, uniaxial circumferential stretch to mechanically condition and strengthen the arterial grafts. Despite improvements in the mechanical integrity of TEVs after uniaxial conditioning, these tissues fail to achieve critical properties of native arteries such as matrix content, collagen fiber orientation, and mechanical strength. As a result, uniaxially loaded TEVs can result in mechanical failure, thrombus, or stenosis on implantation. In planar tissue equivalents such as artificial skin, biaxial loading has been shown to improve matrix production and mechanical properties. To date however, multiaxial loading has not been examined as a means to improve mechanical and biochemical properties of TEVs during culture. Therefore, we developed a novel bioreactor that utilizes both circumferential and axial stretch that more closely simulates loading conditions in native arteries, and we examined the suture strength, matrix production, fiber orientation, and cell proliferation. After 3 months of biaxial loading, TEVs developed a formation of mature elastic fibers that consisted of elastin cores and microfibril sheaths. Furthermore, the distinctive features of collagen undulation and crimp in the biaxial TEVs were absent in both uniaxial and static TEVs. Relative to the uniaxially loaded TEVs, tissues that underwent biaxial loading remodeled and realigned collagen fibers toward a more physiologic, native-like organization. The biaxial TEVs also showed increased mechanical strength (suture retention load of 303 ± 14.53 g, with a wall thickness of 0.76 ± 0.028 mm) and increased compliance. The increase in compliance was due to combinatorial effects of mature elastic fibers, undulated collagen fibers, and collagen matrix orientation. In conclusion, biaxial stretching is a potential means to regenerate TEVs with improved matrix production, collagen organization, and mechanical properties.

New insights into the structure, assembly and biological roles of 10-12 nm connective tissue microfibrils from fibrillin-1 studies.

The 10-12 nm diameter microfibrils of the extracellular matrix (ECM) impart both structural and regulatory properties to load-bearing connective tissues. The main protein component is the calcium-dependent glycoprotein fibrillin, which assembles into microfibrils at the cell surface in a highly regulated process involving specific proteolysis, multimerization and glycosaminoglycan interactions. In higher metazoans, microfibrils act as a framework for elastin deposition and modification, resulting in the formation of elastic fibres, but they can also occur in elastin-free tissues where they perform structural roles. Fibrillin microfibrils are further engaged in a number of cell matrix interactions such as with integrins, bone morphogenetic proteins (BMPs) and the large latent complex of transforming growth factor-β (TGFβ). Fibrillin-1 (FBN1) mutations are associated with a range of heritable connective disorders, including Marfan syndrome (MFS) and the acromelic dysplasias, suggesting that the roles of 10-12 nm diameter microfibrils are pleiotropic. In recent years the use of molecular, cellular and whole-organism studies has revealed that the microfibril is not just a structural component of the ECM, but through its network of cell and matrix interactions it can exert profound regulatory effects on cell function. In this review we assess what is known about the molecular properties of fibrillin that enable it to assemble into the 10-12 nm diameter microfibril and perform such diverse roles.

Contribution of domain 30 of tropoelastin to elastic fiber formation and material elasticity

Elastin is a fibrous structural protein of the extracellular matrix that provides reversible elastic recoil to vertebrate tissues such as arterial vessels, lung, and skin. The elastin monomer, tropoelastin, contains a large proportion of intrinsically disordered and flexible hydrophobic sequences that collectively are responsible for the initial phase separation of monomers during assembly, and are essential for driving elastic recoil. While structural disorder of hydrophobic sequences is controlled by a high proline and glycine residue composition, hydrophobic domain 30 of human tropoelastin is atypically proline-poor, and forms β-sheet amyloid-like fibrils as an individual peptide. We explored the contribution of confined regions of secondary structure at the location of domain 30 in human tropoelastin to fiber assembly and mechanical properties using a set of mutations designed to inhibit or enhance the propensity of β-sheet formation at this location. Our data support a dual role for confined β-sheet secondary structure in domain 30 of tropoelastin in guiding the formation of fibers, and as a determinant of stiffness and viscoelastic properties of cross-linked materials. Together, these results suggest a mechanism for specificity in fiber assembly, and elucidate structure-function relationships for the rational design of elastomeric biomaterials with defined mechanical properties.

Dermal thickening with enhanced collagen contents and epidermal thinning during maturation of dorsal-lateral pig skin.

Event Abstract Back to Event Dermal thickening with enhanced collagen contents and epidermal thinning during maturation of dorsal-lateral pig skin. Ming-Jen Lin1, Ting-Yung Kuo1, Lih-Ren Chen2, 3 and Lynn L. Huang1, 4, 5, 6 1 National Cheng Kung University, Institute of Biotechnology, Taiwan 2 Executive Yuan, Livestock Research Institute, Taiwan 3 Southern Taiwan University, Department of Biotechnology, Taiwan 4 National Cheng Kung University, Institute of Clinical Medicine, Taiwan 5 National Cheng Kung University, Advanced Optoelectronic Technology Center, Taiwan 6 National Cheng Kung University, Research Center of Excellence in Regenerative Medicine, Taiwan Introduction: The global age in this world is growing old fast. According to world health organization, the elderly of 60 and above will constitute two billion people worldwide by 2050.Aging is accompanied with various diseases such as cancer, diabetes, and neurodegenerative diseases. As a result, skin aging study is important in order to understand problems during aging. However, human individuals are not suitable for such skin study; thus, animal model is needed for substitution. Materials and Methods: Animals: For this study seventeen male Lanyu pig in eight groups were used: 1 month-old(n=2), 2.5 month- old(n=6), 9 month-old(n=3), 18 month-old(n=2), 32 month-old(n=1), 41 month-old(n=1), 48 month- old(n=1) and 60 month-old(n=1). The pigs were penned individually to minimize the risk of chamber rupture. The environment was controlled to remain within temperature range of 23 to 28°C, 65% humidity, and the a light cycle of 12 hours on and 12 hours off. The animal were fed 1.25 to 600 to 1000 g of standard swine diet, depending on age, and received an uninterrupted supply of clean, fresh water. All animal procedures were approved by the National Cheng Kung University laboratory animal center. Tissue sample collection and preparation: The tissue sample was collected from different age Lanyu pig by BiopsyPuncher.The cut-out tissue sample was washed with PBS, fixed in 10% formalin solution for 24-48 hours. After fixation, tissues were dehydrated and washed again to be embedded in paraffin. Tissue sections of 5 μm were prepared for histological staining. Histological study: The Lanyu pig skin section prepared from different part of Lanyu pig, immediately fixed in 10% neutral buffered formalin and embedded in paraffin. The paraffin sections (5μm) were made from formalin-fixed samples and stained using hematoxylin and eosin(H&E stain), picro-sirius red staining and Verhoeff's elastin staining. Result and Discussion: The lateral side of pig`s dorsal skin area has been previously studied[1] and Lanyu pig was analyzed in our lab using . Analysis of Lanyu pig showed no significant different in the lateral dorsal skin area. Here, the 1, 9, and 48 month-old Lanyu pigs were studied in which dermal layer was found to increase, and epidermal layer to have decreased in thickness with increasing age. Type Ⅰand Ⅲ collagen were the main collagen in the dermal layer. The Type Ⅲ collagen was shown to be increased in expression level and grew rough in appearance with increasing age. This was further confirmed in the previous research on 0-90 year-old humans, showing that Type Ⅲ collagen increased in the elderly compared to in the youngsters[2]. In consistent, Type Ⅲ collagen increased with increasing age and in overall collagen content in the Lanyu pig. The formation of elastic fibers is due to microfibrils interacting with elastin proteins in the basal layer below epidermis, and further interactions with the epidermis-dermis boundary region. This complex connection formed a network of elastic fibers. Increasing age may lead to increasing proteinase that degrade elastic fibers, forming less elongated elastic fibers, and may exist in the degenerated form due to long half-life of elastic fibers[3]. In consistent, the degenerated form of elastic fibers with increasing age was reported in aging human. Conclusion: In conclusion, the age effect on skin in Lanyu pig is not completely identical to that in human; however, several skin structures were found to be identical between Lanyu pig and human. The work was supported by grants MOST 104-2622-B-006-014 and CZ-20-18-146-103 from the National Science Council, R.O.C.

Characterization of Microfibrillar-associated Protein 4 (MFAP4) as a Tropoelastin- and Fibrillin-binding Protein Involved in Elastic Fiber Formation

MFAP4 (microfibrillar-associated protein 4) is an extracellular glycoprotein found in elastic fibers without a clearly defined role in elastic fiber assembly. In the present study, we characterized molecular interactions between MFAP4 and elastic fiber components. We established that MFAP4 primarily assembles into trimeric and hexameric structures of homodimers. Binding analysis revealed that MFAP4 specifically binds tropoelastin and fibrillin-1 and -2, as well as the elastin cross-linking amino acid desmosine, and that it co-localizes with fibrillin-1-positive fibers in vivo. Site-directed mutagenesis disclosed residues Phe(241) and Ser(203) in MFAP4 as being crucial for type I collagen, elastin, and tropoelastin binding. Furthermore, we found that MFAP4 actively promotes tropoelastin self-assembly. In conclusion, our data identify MFAP4 as a new ligand of microfibrils and tropoelastin involved in proper elastic fiber organization.

Identification of Potential Key lncRNAs and Genes Associated with Aging Based on Microarray Data of Adipocytes from Mice.

Objective. This study aimed to screen potential crucial lncRNAs and genes involved in aging. Methods. The data of 9 peripheral white adipocytes, respectively, taken from male C57BL/6J mice (6 months, 14 months, and 18 months of age) in GSE25905 were used in this study. Differentially time series expressed lncRNA genes (DE-lncRNAs) and mRNA genes (DEGs) were identified. After cluster analysis of lncRNAs expression pattern, target genes of DE-lncRNAs were predicted from the DEGs, and functional analysis for target genes was conducted. Results. A total of 8301 time series-related DEGs and 43 time series-related DE-lncRNAs were identified. Among them, 41 DE-lncRNAs targeted 1880 DEGs. The DEGs positively regulated by DE-lncRNAs were mainly related to the development of blood vessel and the pathways of cholesterol biosynthesis and elastic fibre formation. Furthermore, the DEGs negatively regulated by DE-lncRNAs were correlated with protein metabolism. Conclusion. These DE-lncRNAs and DEGs are potentially involved in the process of aging.

Forelimb contractures and abnormal tendon collagen fibrillogenesis in fibulin-4 null mice.

Fibulin-4 is an extracellular matrix glycoprotein essential for elastic fiber formation. Mice deficient in fibulin-4 die perinatally because of severe pulmonary and vascular defects associated with the lack of intact elastic fibers. Patients with fibulin-4 mutations demonstrate similar defects, and a significant number die shortly after birth or in early childhood from cardiopulmonary failure. The patients also demonstrate skeletal and other systemic connective tissue abnormalities, including joint laxity and flexion contractures of the wrist. A fibulin-4 null mouse strain was generated and used to analyze the roles of fibulin-4 in tendon fibrillogenesis. This mouse model displayed bilateral forelimb contractures, in addition to pulmonary and cardiovascular defects. The forelimb and hindlimb tendons exhibited disruption in collagen fibrillogenesis in the absence of fibulin-4 as analyzed by transmission electron microscopy. Fewer fibrils were assembled, and fibrils were disorganized compared with wild-type controls. The organization of developing tenocytes and compartmentalization of the extracellular space was also disrupted. Fibulin-4 was co-localized with fibrillin-1 and fibrillin-2 in limb tendons by using immunofluorescence microscopy. Thus, fibulin-4 seems to play a role in regulating tendon collagen fibrillogenesis, in addition to its essential function in elastogenesis.

Inhibition of versican expression by siRNA facilitates tropoelastin synthesis and elastic fiber formation by human SK-LMS-1 leiomyosarcoma smooth muscle cells in vitro and in vivo

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.

Grass carp Ctenopharyngodon idella Fibulin-4 as a potential interacting partner for grass carp reovirus outer capsid proteins.

Mammalian EGF containing fibulin-like extracellular matrix protein 2 (Fibulin-4/EFEMP2), an extracellular matrix(ECM) protein and a member of the fibulin family, is involved in elastic fiber formation, connective tissue development and some human diseases. In a yeast-two hybrid screening of host proteins interacting with outer capsid protein of grass carp reovirus (GCRV), a grass carp homologue of Fibulin-4 (designated as GcFibulin-4) is suggested to hold the potential to bind VP7, VP56 and VP55, the outer capsid protein encoded by type I, II, III GCRV, respectively. GcFibulin-4 gene of grass carp was cloned and sequenced from the cDNA library constructed for the yeast two-hybrid screening. Full-length cDNA of GcFibulin-4 contains an open reading frame (ORF) of 1323 bp encoding a putative protein of 440 amino acids. Phylogenetic analysis of GcFibulin-4 indicated that it shared a high homology with zebra fish Fibulin-4 protein. Transcriptional distribution analysis of GcFibulin-4 in various tissues of healthy grass carp showed that GcFibulin-4 was highly expressed in muscle, moderately expressed in the intestine and brain, and slightly expressed in other examined tissues; the expression pattern is consistent with tissue tropism of GCRV resulting in hemorrhage symptom in the corresponding tissues. Our results suggested that Fibulin-4 might enable free GCRV particles, the pathogen for grass carp hemorrhagic disease, to target fish tissues more efficiently by interacting with viral outer capsid proteins.

Prostaglandin E-mediated molecular mechanisms driving remodeling of the ductus arteriosus.

The ductus arteriosus (DA), a fetal arterial connection between the pulmonary arteries and aorta, normally closes after birth. Persistent DA patency usually has life-threatening consequences. In certain DA-dependent congenital heart diseases, however, patient survival depends on maintaining DA patency. Complete closure of the DA involves both functional closure, induced by muscle contraction, and anatomical closure, achieved through morphological and molecular remodeling. Anatomical closure of the DA is associated with the formation of intimal thickening, which is characterized by deposition of extracellular matrix in the subendothelial region, sparse elastic fiber formation, and migration of medial smooth muscle cells into the subendothelial space. In addition, fetal molecular remodeling that is suitable for postnatal muscle contraction has been observed in the DA. After the second trimester, high concentration of prostaglandin E2 (PGE2) causes the DA to dilate through the remainder of the fetal period. Emerging evidence from studies using pharmacological approaches and genetically modified mice suggests that, in addition to its vasodilatory effect, this chronic exposure to PGE2 promotes DA-specific anatomical and molecular remodeling through EP4, one of four receptor subtypes for PGE2. Signals that are downstream of PGE2-EP4, such as cyclic AMP (cAMP)-protein kinase A (PKA), exchange protein activated by cAMP (Epac), phospholipase C, and Wnt/β-catenin, may be involved in the regulation of intimal thickening, elastogenesis, and contraction-related genes. Understanding the physiological role of PGE2 in DA remodeling could enable more effective regulation of PDA, both in isolation and in the context of congenital cardiac anomalies.

Fibulin-4 E57K Knock-in Mice Recapitulate Cutaneous, Vascular and Skeletal Defects of Recessive Cutis Laxa 1B with both Elastic Fiber and Collagen Fibril Abnormalities

Fibulin-4 is an extracellular matrix protein essential for elastic fiber formation. Frameshift and missense mutations in the fibulin-4 gene (EFEMP2/FBLN4) cause autosomal recessive cutis laxa (ARCL) 1B, characterized by loose skin, aortic aneurysm, arterial tortuosity, lung emphysema, and skeletal abnormalities. Homozygous missense mutations in FBLN4 are a prevalent cause of ARCL 1B. Here we generated a knock-in mouse strain bearing a recurrent fibulin-4 E57K homozygous missense mutation. The mutant mice survived into adulthood and displayed abnormalities in multiple organ systems, including loose skin, bent forelimb, aortic aneurysm, tortuous artery, and pulmonary emphysema. Biochemical studies of dermal fibroblasts showed that fibulin-4 E57K mutant protein was produced but was prone to dimer formation and inefficiently secreted, thereby triggering an endoplasmic reticulum stress response. Immunohistochemistry detected a low level of fibulin-4 E57K protein in the knock-in skin along with altered expression of selected elastic fiber components. Processing of a precursor to mature lysyl oxidase, an enzyme involved in cross-linking of elastin and collagen, was compromised. The knock-in skin had a reduced level of desmosine, an elastin-specific cross-link compound, and ultrastructurally abnormal elastic fibers. Surprisingly, structurally aberrant collagen fibrils and altered organization into fibers were characteristics of the knock-in dermis and forelimb tendons. Type I collagen extracted from the knock-in skin had decreased amounts of covalent intermolecular cross-links, which could contribute to the collagen fibril abnormalities. Our studies provide the first evidence that fibulin-4 plays a role in regulating collagen fibril assembly and offer a preclinical platform for developing treatments for ARCL 1B.

Elastofibroma dorsi: a case report with an immunohistochemical and ultrastructural studies

Elastofibroma is a rare, benign, fibrous tumor formed by the proliferation of characteristic elastic fibers that commonly occurs between the lower margin of the scapula and the ribcage. We undertook a histochemical, immunohistochemical, and ultrastructural study of an elastofibroma dorsi beneath the right scapula of a 77-year-old woman. Tumor cells comprised collagen fiber bundles, numerous elastic fibers, and spindle cells resembling fibroblasts. The elastic and collagen fibers in the tumor were stained positively with Elastic van Gieson and Masson trichrome staining, respectively. Immunostaining showed that the fibroblasts were strongly positive for CD34, positive for vimentin, and weakly positive for α-smooth muscle actin. Ultrastructural observations revealed elastin and microfibrils between numerous irregularly arranged collagen fiber bundles. Signs suggestive of elastin deposition were also evident in the tangled collagen fibers themselves. The fibroblasts contained a large amount of rough endoplasmic reticulum and were surrounded on the outside of cells by microfibrils and collagen fibers. Although fibroblasts may produce large quantities of elastin, microfibrils, and collagen, our findings suggested that the deposition of elastin on collagen fibers may be involved in the formation of abnormal elastic fibers.

Elevated expression levels of lysyl oxidases protect against aortic aneurysm progression in Marfan syndrome

Patients with Marfan syndrome (MFS) are at high risk of life-threatening aortic dissections. The condition is caused by mutations in the gene encoding fibrillin-1, an essential component in the formation of elastic fibers. While experimental findings in animal models of the disease have shown the involvement of transforming growth factor-β (TGF-β)- and angiotensin II-dependent pathways, alterations in the vascular extracellular matrix (ECM) may also play a role in the onset and progression of the aortic disease. Lysyl oxidases (LOX) are extracellular enzymes, which initiates the formation of covalent cross-linking of collagens and elastin, thereby contributing to the maturation of the ECM. Here we have explored the role of LOX in the formation of aortic aneurysms in MFS. We show that aortic tissue from MFS patients and MFS mouse model (Fbn1C1039G/+) displayed enhanced expression of the members of the LOX family, LOX and LOX-like 1 (LOXL1), and this is associated with the formation of mature collagen fibers. Administration of a LOX inhibitor for 8weeks blocked collagen accumulation and aggravated elastic fiber impairment, and these effects correlated with the induction of a strong and rapidly progressing aortic dilatation, and with premature death in the more severe MFS mouse model, Fbn1mgR/mgR, without any significant effect on wild type animals. This detrimental effect occurred preferentially in the ascending portion of the aorta, with little or no involvement of the aortic root, and was associated to an overactivation of both canonical and non-canonical TGF-β signaling pathways. The blockade of angiotensin II type I receptor with losartan restored TGF-β signaling activation, normalized elastic fiber impairment and prevented the aortic dilatation induced by LOX inhibition in Fbn1C1039G/+ mice. Our data indicate that LOX enzymes and LOX-mediated collagen accumulation play a critical protective role in aneurysm formation in MFS.

The antidiabetic hormone glucagon-like peptide-1 induces formation of new elastic fibers in human cardiac fibroblasts after cross-activation of IGF-IR.

Glucagon-like peptide 1 (GLP-1) is a metabolic hormone involved in the stimulation of insulin biosynthesis and secretion. It has been recently reported that GLP-1 also exerts cardioprotective effects and facilitates functional recovery after myocardial infarction through GLP-1 receptor-mediated signaling in cardiomyocytes. GLP-1 treatment has been also demonstrated to produce sustained improvement in cardiac function in long-term studies, suggesting the involvement of mechanisms beyond the acute metabolic and cytoprotective effects. For example, the possible interaction of GLP-1 with the cardiac fibroblasts, which are responsible for the postinfarct remodeling and extracellular matrix production, has not been previously explored. Here, we report that cultures of human cardiac fibroblasts treated with GLP-1 peptides display a selective up-regulation in elastin gene expression and a consequent increase in elastic fibers production, in the absence of the classic GLP-1 receptor. Importantly, we provide experimental evidence that this GLP-1-induced elastogenesis is triggered through the cross-activation of the IGF-I receptor. Because GLP-1 does not stimulate deposition of collagen I, nor promote the proliferation or apoptosis of cultured cardiac fibroblasts, we speculate that its elastogenic effect may also contribute to the beneficial remodeling of the human heart after myocardial infarction.

Lung matrix and vascular remodeling in mechanically ventilated elastin haploinsufficient newborn mice.

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.

Increased upper airway collapsibility in a mouse model of Marfan syndrome

Marfan syndrome (MFS) is a genetic disorder caused by mutations in the FBN1 gene that codifies for fibrilin-1. MFS affects elastic fiber formation and the resulting connective tissue shows abnormal tissue laxity and organization. Although an increased prevalence of obstructive sleep apnea among patients with MFS has been described, the potential effects of this genetic disease on the collapsible properties of the upper airway are unknown. The aim of this study was to assess the collapsible properties of the upper airway in a mouse model of MFS Fbn1(C1039G/+) that is representative of most of the clinical manifestations observed in human patients. The upper airway in wild-type and Marfan mice was cannulated and its critical pressure (Pcrit) was measured in vivo by increasing the negative pressure through a controlled pressure source. Pcrit values from MFS mice were higher (less negative) compared to wild-type mice (−3.1±0.9cmH2O vs. −7.8±2.0cm H2O) suggesting that MFS increases the upper airway collapsibility, which could in turn explain the higher prevalence of OSA in MFS patients.

Pro-elastogenic effects of bone marrow mesenchymal stem cell-derived smooth muscle cells on cultured aneurysmal smooth muscle cells.

Abdominal aortic aneurysms (AAAs) involve slow proteolysis and loss of structural matrix components (collagen and elastin), which lead to wall thinning, weakening and ultimate rupture. At this time, no established non-surgical therapy is available to slow or arrest AAA growth. Inhibiting matrix metalloproteases (MMPs; e.g. MMP2 and -9) overexpressed within AAAs is insufficient to arrest AAA growth, since resident smooth muscle cells (SMCs) are poorly elastogenic and cannot overcome elastolysis to reinstate a healthy elastic matrix. Towards overcoming this limitation, this first study sought to determine the utility of rat bone marrow mesenchymal stem cell (BM-MSC)-derived SMCs to stimulate elastin and elastic matrix synthesis and assembly by aneurysmal SMCs (EaRASMCs). BM-MSCs were successfully differentiated into cells of an SMC lineage (SMLCs). Our study indicates that BM-MSC-derived SMLCs secrete trophic factors, contained in conditioned medium (CM) from their cultures, that, when exposed to EaRASMC cultures in real time, stimulate elastin precursor and matrix deposition and crosslinking by these elastogenically deficient cells, with added benefits in terms of attenuating MMPs, specifically MMP9. The results thus lend support to a proposed cell therapy for AAAs, based on the use of BM-MSC-derived SMLCs. Although we observed no particular improvement in elastic fibre formation, no attenuation of MMP2 activity and increase in amounts of active MMP2 enzyme, we believe that this study justifies follow-up studies to improve upon these outcomes. Future studies will explore the effects of concentrated CM collected from long-term SMLC cultures on EaRASMCs and also investigate the elastogenic output of SMLCs themselves. Copyright © 2014 John Wiley & Sons, Ltd.

The Microfibril Hypothesis of Glaucoma: Implications for Treatment of Elevated Intraocular Pressure

Microfibrils are macromolecular aggregates located in the extracellular matrix of both elastic and nonelastic tissues that have essential functions in formation of elastic fibers and control of signaling through the transforming growth factor beta (TGFβ) family of cytokines. Elevation of systemic TGFβ and chronic activation of TGFβ signal transduction are associated with diseases caused by mutations in microfibril-associated genes, including FBN1. A role for microfibrils in glaucoma is suggested by identification of risk alleles in LOXL1 for exfoliation glaucoma and mutations in LTBP2 for primary congenital glaucoma, both of which are microfibril-associated genes. Recent identification of a mutation in another microfibril-associated gene, ADAMTS10, in a dog model of primary open-angle glaucoma led us to form the microfibril hypothesis of glaucoma, which in general states that defective microfibrils may be an underlying cause of glaucoma. Microfibril defects could contribute to glaucoma through alterations in biomechanical properties of tissue and/or through effects on signaling through TGFβ, which is well established to be elevated in the aqueous humor of glaucoma patients. Recent work has shown that diseases caused by microfibril defects are associated with increased concentrations of TGFβ protein and chronic activation of TGFβ-mediated signal transduction. In analogy with other microfibril-related diseases, defective microfibrils could provide a mechanism for the elevation of TGFβ2 in glaucomatous aqueous humor. If glaucoma shares mechanisms with other diseases caused by defective microfibrils, such as Marfan syndrome, therapeutic interventions to inhibit chronic activation of TGFβ signaling used in those diseases may be applied to glaucoma.

Three-dimensional multilayers of smooth muscle cells as a new experimental model for vascular elastic fiber formation studies

ObjectiveElastic fiber formation is disrupted with age and by health conditions including aneurysms and atherosclerosis. Despite considerable progress in the understanding of elastogenesis using the planar culture system and genetically modified animals, it remains difficult to restore elastic fibers in diseased vessels. To further study the molecular mechanisms, in vitro three-dimensional vascular constructs need to be established. We previously fabricated vascular smooth muscle cells (SMCs) into three-dimensional cellular multilayers (3DCMs) using a hierarchical cell manipulation technique, in which cells were coated with fibronectin-gelatin nanofilms to provide adhesive nano-scaffolds. Since fibronectin is known to assemble and activate elastic fiber-related molecules, we further optimized culture conditions. Methods and resultsElastica stain, immunofluorescence, and electron microscopic analysis demonstrated that 3DCMs, which consisted of seven layers of neonatal rat aortic SMCs cultured in 1% fetal bovine serum (FBS) in Dulbecco's modified Eagle's medium, exhibited layered elastic fibers within seven days of being in a static culture condition. In contrast, the application of adult SMCs, 10% FBS, ε-poly(lysine) as an alternative adhesive for fibronectin, or four-layered SMCs, failed to generate layered elastic fiber formation. Radioimmunoassay using [3H]valine further confirmed the greater amount of cross-linked elastic fibers in 3DCMs than in monolayered SMCs. Layered elastic fiber formation in 3DCMs was inhibited by the lysyl oxidase inhibitor β-aminopropionitrile, or prostaglandin E2. Furthermore, infiltration of THP-1-derived macrophages decreased the surrounding elastic fiber formation in 3DCMs. Conclusion3DCMs may offer a new experimental vascular model to explore pharmacological therapeutic strategies for disordered elastic fiber homeostasis.

Domain 36 of Tropoelastin in Elastic Fiber Formation

Elastic fiber assembly is a complex stepwise process involving multiple different proteins and enzymes. Domain 36, encoded by the last exon of the elastin gene, is recognized to be an important domain for deposition onto microfibrils, an essential step in elastic fiber assembly. However, the role of domain 36 in elastic fiber assembly has not been clarified. Here, we utilized our established in vitro assembly model to identify the importance of domain 36 for the assembly process. Our results showed that the lack of domain 36 in bovine tropoelastin results in deficient elastic fiber assembly. A similar result was obtained with the point mutation of two cysteine residues and the deletion of the Lysine-Arginine-Lysine-Arginine (RKRK) sequence in domain 36. Double immunofluorescence of tropoelastin and fibrillin-1, a main component of microfibrils, demonstrated reduced localization of these mutant tropoelastin molecules on fibrillin-1 fibers. Moreover, the binding affinity of these mutants to fibrillin-1 and microfibril-associated glycoprotein (MAGP) was significantly decreased. These data indicate that domain 36 of tropoelastin facilitates elastic fiber assembly by interacting with microfibrils via two cysteine residues and the RKRK sequence.