Publications by authors named "Dieter P Reinhardt"

88 Publications

The fibrillin-1 RGD motif posttranscriptionally regulates ERK1/2 signaling and fibroblast proliferation via miR-1208.

FASEB J 2021 May;35(5):e21598

Faculty of Medicine and Health Sciences, Department of Anatomy and Cell Biology, McGill University, Montreal, Canada.

Fibrillin-1 is an extracellular matrix protein which contains one conserved RGD integrin-binding motif. It constitutes the backbone of microfibrils in many tissues, and mutations in fibrillin-1 cause various connective tissue disorders. Although it is well established that fibrillin-1 interacts with several RGD-dependent integrins, very little is known about the associated intracellular signaling pathways. Recent published evidence identified a subset of miRNAs regulated by fibrillin-1 RGD-cell adhesion, with miR-1208 among the most downregulated. The present study shows that the downregulated miR-1208 controls fibroblast proliferation. Inhibitor experiments revealed that fibrillin-1 RGD suppressed miR-1208 expression via c-Src kinase and the downstream JNK signaling. Bioinformatic prediction and experimental target sequence validation demonstrated four miR-1208 binding sites on the ERK2 mRNA and one on the MEK1 mRNA. ERK2 and MEK1 are critical proliferation-promoting kinases. Decreased miR-1208 levels elevated the total and phosphorylated ERK1/2 and MEK1/2 protein levels and the phosphorylated to total ERK1/2 ratio. Together, the data demonstrate a novel outside-in signaling mechanism explaining how fibrillin-1 RGD-cell binding regulates fibroblast proliferation.
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http://dx.doi.org/10.1096/fj.202100282RDOI Listing
May 2021

The mgΔ mouse model for Marfan syndrome recapitulates the ocular phenotypes of the disease.

Exp Eye Res 2021 Mar 28;204:108461. Epub 2021 Jan 28.

University of São Paulo, Department of Genetics and Evolutionary Biology, São Paulo, SP, Brazil. Electronic address:

Purpose: Fibrillin-1 and -2 are major components of tissue microfibrils that compose the ciliary zonule and cornea. While mutations in human fibrillin-1 lead to ectopia lentis, a major manifestation of Marfan syndrome (MFS), in mice fibrillin-2 can compensate for reduced/lack of fibrillin-1 and maintain the integrity of ocular structures. Here we examine the consequences of a heterozygous dominant-negative mutation in the Fbn1 gene in the ocular system of the mgΔ mouse model for MFS.

Methods: Eyes from mgΔ and wild-type mice at 3 and 6 months of age were analyzed by histology. The ciliary zonule was analyzed by scanning electron microscopy (SEM) and immunofluorescence.

Results: Mutant mice presented a significantly larger distance of the ciliary body to the lens at 3 and 6 months of age when compared to wild-type, and ectopia lentis. Immunofluorescence and SEM corroborated those findings in MFS mice, revealing a disorganized mesh of microfibrils on the floor of the ciliary body. Moreover, mutant mice also had a larger volume of the anterior chamber, possibly due to excess aqueous humor. Finally, losartan treatment had limited efficacy in improving ocular phenotypes.

Conclusions: In contrast with null or hypomorphic mutations, expression of a dominant-negative form of fibrillin-1 leads to disruption of microfibrils in the zonule of mice. This in turn causes lens dislocation and enlargement of the anterior chamber. Therefore, heterozygous mgΔ mice recapitulate the major ocular phenotypes of MFS and can be instrumental in understanding the development of the disease.
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http://dx.doi.org/10.1016/j.exer.2021.108461DOI Listing
March 2021

Alternative splicing of the metalloprotease ADAMTS17 spacer regulates secretion and modulates autoproteolytic activity.

FASEB J 2021 Feb;35(2):e21310

Icahn School of Medicine at Mount Sinai, New York, NY, USA.

ADAMTS proteases mediate biosynthesis and breakdown of secreted extracellular matrix (ECM) molecules in numerous physiological and disease processes. In addition to their catalytic domains, ADAMTS proteases contain ancillary domains, which mediate substrate recognition and ECM binding and confer distinctive properties and roles to individual ADAMTS proteases. Although alternative splicing can greatly expand the structural and functional diversity of ADAMTS proteases, it has been infrequently reported and functional consequences have been rarely investigated. Here, we characterize the structural and functional impact of alternative splicing of ADAMTS17, mutations in which cause Weill-Marchesani syndrome 4. Two novel ADAMTS17 splice variants, ADAMTS17A and ADAMTS17B, were investigated by structural modeling, mass spectrometry, and biochemical approaches. Our results identify a novel disulfide-bridged insertion in the ADAMTS17A spacer that originates from inclusion of a novel exon. This insertion results in differential autoproteolysis of ADAMTS17, and thus, predicts altered proteolytic activity against other substrates. The second variant, ADAMTS17B, results from an in-frame exon deletion and prevents ADAMTS17B secretion. Thus, alternative splicing of the ADAMTS spacer significantly regulates the physiologically relevant proteolytic activity of ADAMTS17, either by altering proteolytic specificity (ADAMTS17A) or by altering cellular localization (ADAMTS17B).
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http://dx.doi.org/10.1096/fj.202001120RRDOI Listing
February 2021

Slc2a10 knock-out mice deficient in ascorbic acid synthesis recapitulate aspects of arterial tortuosity syndrome and display mitochondrial respiration defects.

Hum Mol Genet 2020 06;29(9):1476-1488

Center for Medical Genetics Ghent, Department of Biomolecular Medicine, Ghent University, 9000 Ghent, Belgium.

Arterial tortuosity syndrome (ATS) is a recessively inherited connective tissue disorder, mainly characterized by tortuosity and aneurysm formation of the major arteries. ATS is caused by loss-of-function mutations in SLC2A10, encoding the facilitative glucose transporter GLUT10. Former studies implicated GLUT10 in the transport of dehydroascorbic acid, the oxidized form of ascorbic acid (AA). Mouse models carrying homozygous Slc2a10 missense mutations did not recapitulate the human phenotype. Since mice, in contrast to humans, are able to intracellularly synthesize AA, we generated a novel ATS mouse model, deficient for Slc2a10 as well as Gulo, which encodes for L-gulonolactone oxidase, an enzyme catalyzing the final step in AA biosynthesis in mouse. Gulo;Slc2a10 double knock-out mice showed mild phenotypic anomalies, which were absent in single knock-out controls. While Gulo;Slc2a10 double knock-out mice did not fully phenocopy human ATS, histological and immunocytochemical analysis revealed compromised extracellular matrix formation. Transforming growth factor beta signaling remained unaltered, while mitochondrial function was compromised in smooth muscle cells derived from Gulo;Slc2a10 double knock-out mice. Altogether, our data add evidence that ATS is an ascorbate compartmentalization disorder, but additional factors underlying the observed phenotype in humans remain to be determined.
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http://dx.doi.org/10.1093/hmg/ddaa071DOI Listing
June 2020

Fibrillin-1 and fibrillin-1-derived asprosin in adipose tissue function and metabolic disorders.

J Cell Commun Signal 2020 Jun 12;14(2):159-173. Epub 2020 Apr 12.

Faculty of Medicine, Department of Anatomy and Cell Biology, McGill University, Montreal, Canada.

The extracellular matrix microenvironment of adipose tissue is of critical importance for the differentiation, remodeling and function of adipocytes. Fibrillin-1 is one of the main components of microfibrils and a key player in this process. Furin processing of profibrillin-1 results in mature fibrillin-1 and releases the C-terminal propeptide as a circulating hunger hormone, asprosin. Mutations in the fibrillin-1 gene lead to adipose tissue dysfunction and causes Marfan syndrome, marfanoid progeroid lipodystrophy syndrome, and neonatal progeroid syndrome. Increased TGF-β signaling, altered mechanical properties and impaired adipogenesis are potential causes of adipose tissue dysfunction, mediated through deficient microfibrils. Circulating asprosin on the other hand is secreted primarily by white adipose tissue under fasting conditions and in obesity. It increases hepatic glucose production and drives insulin secretion and appetite stimulation through inter-organ cross talk. This review discusses the metabolic consequences of fibrillin-1 and fibrillin-1-derived asprosin in pathological conditions. Understanding the dynamic role of fibrillin-1 in the adipose tissue milieu and of circulating asprosin in the body can provide novel mechanistic insights into how fibrillin-1 may contribute to metabolic syndrome. This could lead to new management regimens of patients with metabolic disease.
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http://dx.doi.org/10.1007/s12079-020-00566-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7272526PMC
June 2020

Corrigendum to "The Fibrillin-1 RGD Integrin Binding Site Regulates Gene Expression and Cell Function Through microRNAs" [J. Mol. Biol. 431 (2) (2019) 401-421].

J Mol Biol 2020 Feb 6;432(4):1306. Epub 2020 Jan 6.

Faculty of Medicine, McGill University, Montreal, Canada; Faculty of Dentistry, McGill University, Montreal, Canada. Electronic address:

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http://dx.doi.org/10.1016/j.jmb.2019.12.022DOI Listing
February 2020

Fibulin-4 exerts a dual role in LTBP-4L-mediated matrix assembly and function.

Proc Natl Acad Sci U S A 2019 10 23;116(41):20428-20437. Epub 2019 Sep 23.

Faculty of Medicine, Department of Anatomy and Cell Biology, McGill University, Montreal, QC H3A 0C7, Canada;

Elastogenesis is a hierarchical process by which cells form functional elastic fibers, providing elasticity and the ability to regulate growth factor bioavailability in tissues, including blood vessels, lung, and skin. This process requires accessory proteins, including fibulin-4 and -5, and latent TGF binding protein (LTBP)-4. Our data demonstrate mechanisms in elastogenesis, focusing on the interaction and functional interdependence between fibulin-4 and LTBP-4L and its impact on matrix deposition and function. We show that LTBP-4L is not secreted in the expected extended structure based on its domain composition, but instead adopts a compact conformation. Interaction with fibulin-4 surprisingly induced a conformational switch from the compact to an elongated LTBP-4L structure. This conversion was only induced by fibulin-4 multimers associated with increased avidity for LTBP-4L; fibulin-4 monomers were inactive. The fibulin-4-induced conformational change caused functional consequences in LTBP-4L in terms of binding to other elastogenic proteins, including fibronectin and fibrillin-1, and of LTBP-4L assembly. A transient exposure of LTBP-4L with fibulin-4 was sufficient to stably induce conformational and functional changes; a stable complex was not required. These data define fibulin-4 as a molecular extracellular chaperone for LTBP-4L. The altered LTBP-4L conformation also promoted elastogenesis, but only in the presence of fibulin-4, which is required to escort tropoelastin onto the extended LTBP-4L molecule. Altogether, this study provides a dual mechanism for fibulin-4 in 1) inducing a stable conformational and functional change in LTBP-4L, and 2) promoting deposition of tropoelastin onto the elongated LTBP-4L.
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http://dx.doi.org/10.1073/pnas.1901048116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6789559PMC
October 2019

Quantification of Extracellular Matrix Fiber Systems Related to ADAMTS Proteins.

Methods Mol Biol 2020 ;2043:237-250

Faculty of Medicine, Department of Anatomy and Cell Biology, McGill University, Montreal, QC, Canada.

ADAMTS (a disintegrin-like and metalloprotease domain with thrombospondin type 1 motifs) proteins regulate tissue homeostasis and extracellular matrix (ECM)-related pathogenesis. Some ADAMTS proteins interact with or process multiple ECM proteins, including fibrillins, fibronectin, and collagens. Therefore, characterization and quantification of these ECM fiber systems is essential to understand their functional relationship with ADAMTS proteins. Here we describe unbiased methods to quantify various aspects of ADAMTS-related ECM fiber systems in cell culture and in tissues. We focus on cell counting, overall fiber intensity, fiber length, and focal adhesion analysis in cell culture, and on the quantification of immunohistochemical and immunofluorescent tissue sections. We use ImageJ/Fiji, a widely used Java-based open source software which provides efficient and customizable quantification methods for microscopy images.
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http://dx.doi.org/10.1007/978-1-4939-9698-8_19DOI Listing
April 2020

Biophysical Techniques to Analyze Elastic Tissue Extracellular Matrix Proteins Interacting with ADAMTS Proteins.

Methods Mol Biol 2020 ;2043:213-235

Faculty of Dentistry, McGill University, Montreal, QC, Canada.

Multidomain matrix-associated zinc extracellular proteases ADAMTS and ADAMTS-like proteins have important biological activities in cells and tissues. Beyond their traditional role in procollagen and von Willebrand factor processing and proteoglycan cleavage, ADAMTS/ADAMTSL likely participate in or at least have some role in ECM assembly as some of these proteins bind ECM proteins including fibrillins, fibronectin, and LTBPs. In this chapter, we present four biophysical techniques largely used for the characterization, multimerization, and interaction of proteins: surface plasmon resonance spectroscopy, dynamic light scattering, atomic force microscopy, and circular dichroism spectroscopy.
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http://dx.doi.org/10.1007/978-1-4939-9698-8_18DOI Listing
April 2020

Glycoproteomic Analysis of the Aortic Extracellular Matrix in Marfan Patients.

Arterioscler Thromb Vasc Biol 2019 09 18;39(9):1859-1873. Epub 2019 Jul 18.

From the King's British Heart Foundation Centre, King's College London, United Kingdom (X.Y., A.L.F., J.B.-B., R.L., M.F., F.B., P.S., Q.X., M.M.).

Objective: Marfan syndrome (MFS) is caused by mutations in FBN1 (fibrillin-1), an extracellular matrix (ECM) component, which is modified post-translationally by glycosylation. This study aimed to characterize the glycoproteome of the aortic ECM from patients with MFS and relate it to aortopathy. Approach and Results: ECM extracts of aneurysmal ascending aortic tissue from patients with and without MFS were enriched for glycopeptides. Direct N-glycopeptide analysis by mass spectrometry identified 141 glycoforms from 47 glycosites within 35 glycoproteins in the human aortic ECM. Notably, MFAP4 (microfibril-associated glycoprotein 4) showed increased and more diverse N-glycosylation in patients with MFS compared with control patients. MFAP4 mRNA levels were markedly higher in MFS aortic tissue. MFAP4 protein levels were also increased at the predilection (convexity) site for ascending aorta aneurysm in bicuspid aortic valve patients, preceding aortic dilatation. In human aortic smooth muscle cells, MFAP4 mRNA expression was induced by TGF (transforming growth factor)-β1 whereas siRNA knockdown of MFAP4 decreased FBN1 but increased elastin expression. These ECM changes were accompanied by differential gene expression and protein abundance of proteases from ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family and their proteoglycan substrates, respectively. Finally, high plasma MFAP4 concentrations in patients with MFS were associated with a lower thoracic descending aorta distensibility and greater incidence of type B aortic dissection during 68 months follow-up.

Conclusions: Our glycoproteomics analysis revealed that MFAP4 glycosylation is enhanced, as well as its expression during the advanced, aneurysmal stages of MFS compared with control aneurysms from patients without MFS.
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http://dx.doi.org/10.1161/ATVBAHA.118.312175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6727943PMC
September 2019

Lessons from tracheal tube development for understanding congenital tracheal malformations.

Eur Respir J 2019 03 7;53(3). Epub 2019 Mar 7.

Faculty of Medicine, Dept of Anatomy and Cell Biology, McGill University, Montreal, QC, Canada.

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http://dx.doi.org/10.1183/13993003.00127-2019DOI Listing
March 2019

Fibulin-4 deficiency differentially affects cytoskeleton structure and dynamics as well as TGFβ signaling.

Cell Signal 2019 06 4;58:65-78. Epub 2019 Mar 4.

Department of Molecular Genetics, Erasmus University Medical Center, Rotterdam, the Netherlands; Department of Vascular Surgery, Erasmus University Medical Center, Rotterdam, the Netherlands; Department of Radiation Oncology, Erasmus University Medical Center, Rotterdam, the Netherlands. Electronic address:

Fibulin-4 is an extracellular matrix (ECM) protein essential for elastogenesis and mutations in this protein lead to aneurysm formation. In this study, we isolated vascular smooth muscle cells (VSMCs) from mice with reduced fibulin-4 protein expression (Fibulin-4) and from mice with a smooth muscle cell specific deletion of the Fibulin-4 gene (Fibulin-4/SM22Cre). We subsequently analyzed and compared the molecular consequences of reduced Fibulin-4 expression versus total ablation of Fibulin-4 expression with regard to effects on the SMC specific contractile machinery, cellular migration and TGFβ signaling. Analysis of the cytoskeleton showed that while Fibulin-4/SM22Cre VSMCs lack smooth muscle actin (SMA) fibers, Fibulin-4 VSMCs were able to form SMA fibers. Furthermore, Fibulin-4/SM22Cre VSMCs showed a decreased pCofilin to Cofilin ratio, suggesting increased actin depolymerization, while Fibulin-4 VSMCs did not display this decrease. Yet, both Fibulin-4 mutant VSMCs showed decreased migration. We found increased activation of TGFβ signaling in Fibulin-4 VSMCs. However, TGFβ signaling was not increased in Fibulin-4/SM22Cre VSMCs. From these results we conclude that both reduction and absence of Fibulin-4 leads to structural and functional impairment of the SMA cytoskeleton. However, while reduced levels of Fibulin-4 result in increased TGFβ activation, complete absence of Fibulin-4 does not result in increased TGFβ activation. Since both mouse models show thoracic aortic aneurysm formation, we conclude that not only hampered TGFβ signaling, but also SMA cytoskeleton dynamics play an important role in aortic aneurysmal disease.
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http://dx.doi.org/10.1016/j.cellsig.2019.02.008DOI Listing
June 2019

Novel fibronectin mutations and expansion of the phenotype in spondylometaphyseal dysplasia with "corner fractures".

Bone 2019 04 30;121:163-171. Epub 2018 Dec 30.

CHU Sainte Justine Research Centre and Department of Pediatrics, University of Montreal, Montreal, QC H3T 1C5, Canada. Electronic address:

Heterozygous pathogenic variants in the FN1 gene, encoding fibronectin (FN), have recently been shown to be associated with a skeletal disorder in some individuals affected by spondylometaphyseal dysplasia with "corner fractures" (SMD-CF). The most striking feature characterizing SMD-CF is irregularly shaped metaphyses giving the appearance of "corner fractures". An array of secondary features, including developmental coxa vara, ovoid vertebral bodies and severe scoliosis, may also be present. FN is an important extracellular matrix component for bone and cartilage development. Here we report five patients affected by this subtype of SMD-CF caused by five novel FN1 missense mutations: p.Cys123Tyr, p.Cys169Tyr, p.Cys213Tyr, p.Cys231Trp and p.Cys258Tyr. All individuals shared a substitution of a cysteine residue, disrupting disulfide bonds in the FN type-I assembly domains located in the N-terminal assembly region. The abnormal metaphyseal ossification and "corner fracture" appearances were the most remarkable clinical feature in these patients. In addition, generalized skeletal fragility with low-trauma bilateral femoral fractures was identified in one patient. Interestingly, the distal femoral changes in this patient healed with skeletal maturation. Our report expands the phenotypic and genetic spectrum of the FN1-related SMD-CF and emphasizes the importance of FN in bone formation and possibly also in the maintenance of bone strength.
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http://dx.doi.org/10.1016/j.bone.2018.12.020DOI Listing
April 2019

The Fibrillin-1 RGD Integrin Binding Site Regulates Gene Expression and Cell Function through microRNAs.

J Mol Biol 2019 01 28;431(2):401-421. Epub 2018 Nov 28.

Faculty of Medicine, McGill University, Montreal, Canada; Faculty of Dentistry, McGill University, Montreal, Canada. Electronic address:

Fibrillins are the major components of microfibrils in the extracellular matrix of elastic and non-elastic tissues. Fibrillin-1 contains one evolutionarily conserved RGD sequence that mediates cell-matrix interactions through cell-surface integrins. Here, we present a novel paradigm how extracellular fibrillin-1 controls cellular function through integrin-mediated microRNA regulation. Comparative mRNA studies by global microarray analysis identified growth factor activity, actin binding and integrin binding as the most important functional groups that are regulated upon fibrillin-1 binding to dermal fibroblasts. Many of these mRNAs are targets of miRNAs that were identified when RNA from the fibrillin-1-ligated fibroblasts was analyzed by a miRNA microarray. The expression profile was specific to fibrillin-1 since interaction with fibronectin displayed a partially distinct profile. The importance of selected miRNAs for the regulation of the identified mRNAs was suggested by bioinformatics prediction and the interactions between miRNAs and mRNAs were experimentally validated. Functionally, we show that miR-503 controls p-Smad2-dependent TGF-β signaling, and that miR-612 and miR-3185 are involved in the focal adhesion formation regulated by fibrillin-1. In conclusion, we demonstrate that fibrillin-1 interaction with fibroblasts regulates miRNA expression profiles which in turn control critical cell functions.
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http://dx.doi.org/10.1016/j.jmb.2018.11.021DOI Listing
January 2019

Adamts10 inactivation in mice leads to persistence of ocular microfibrils subsequent to reduced fibrillin-2 cleavage.

Matrix Biol 2019 04 7;77:117-128. Epub 2018 Sep 7.

Department of Biomedical Engineering, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA. Electronic address:

Mutations in the secreted metalloproteinase ADAMTS10 cause recessive Weill-Marchesani syndrome (WMS), comprising ectopia lentis, short stature, brachydactyly, thick skin and cardiac valve anomalies. Dominant WMS caused by FBN1 mutations is clinically similar and affects fibrillin-1 microfibrils, which are a major component of the ocular zonule. ADAMTS10 was previously shown to enhance fibrillin-1 assembly in vitro. Here, Adamts10 null mice were analyzed to determine the impact of ADAMTS10 deficiency on fibrillin microfibrils in vivo. An intragenic lacZ reporter identified widespread Adamts10 expression in the eye, musculoskeletal tissues, vasculature, skin and lung. Adamts10 mice had reduced viability on the C57BL/6 background, and although surviving mice were slightly smaller and had stiff skin, they lacked brachydactyly and cardiovascular defects. Ectopia lentis was not observed in Adamts10 mice, similar to Fbn1 mice, most likely because the mouse zonule contains fibrillin-2 in addition to fibrillin-1. Unexpectedly, in contrast to wild-type eyes, Adamts10 zonule fibers were thicker and immunostained strongly with fibrillin-2 antibodies into adulthood, whereas fibrillin-1 staining was reduced. Furthermore, fibrillin-2 staining of hyaloid vasculature remnants persisted post-natally in Adamts10 eyes. ADAMTS10 was found to cleave fibrillin-2, providing an explanation for persistence of fibrillin-2 at these sites. Thus, analysis of Adamts10 mice led to identification of fibrillin-2 as a novel ADAMTS10 substrate and defined a proteolytic mechanism for clearance of ocular fibrillin-2 at the end of the juvenile period.
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http://dx.doi.org/10.1016/j.matbio.2018.09.004DOI Listing
April 2019

A biodegradable synthetic graft for small arteries matches the performance of autologous vein in rat carotid arteries.

Biomaterials 2018 10 26;181:67-80. Epub 2018 Jul 26.

Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA 15261, USA. Electronic address:

Autologous veins are the most widely used grafts for bypassing small arteries in coronary and peripheral arterial occlusive diseases. However, they have limited availability and cause donor-site morbidity. Here, we report a direct comparison of acellular biodegradable synthetic grafts and autologous veins as interposition grafts of rat carotid arteries, which is a good model for clinically relevant small arteries. Notably, extensive but transient infiltration of circulating monocytes at day 14 in synthetic grafts leads to a quickly-resolved inflammation and arterial-like tissue remodeling. The vein graft exhibits a similar inflammation phase except the prolonged presence of inflammatory monocytes. The walls of the remodeled synthetic graft contain many circumferentially aligned contractile non-proliferative smooth muscle cells (SMCs), collagen and elastin. In contrast, the walls of the vein grafts contain disorganized proliferating SMCs and thicken over time, suggesting the onset of stenosis. At 3 months, both grafts have a similar patency, extracellular matrix composition, and mechanical properties. Furthermore, synthetic grafts exhibit recruitment and re-orientation of newly synthesized collagen fibers upon mechanical loading. To our knowledge, this is the first demonstration of a biodegradable synthetic vascular graft with a performance similar to an autologous vein in small artery grafting.
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http://dx.doi.org/10.1016/j.biomaterials.2018.07.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6743079PMC
October 2018

Fibronectin promotes elastin deposition, elasticity and mechanical strength in cellularised collagen-based scaffolds.

Biomaterials 2018 10 12;180:130-142. Epub 2018 Jul 12.

Laboratory for Biomaterials and Bioengineering, Canada Research Chair I in Biomaterials and Bioengineering for the Innovation in Surgery, Department of Min-Met-Materials Engineering, Research Center of CHU de Quebec, Division of Regenerative Medicine, Laval University, Quebec City, QC G1V 0A6, Canada. Electronic address:

One of the tightest bottlenecks in vascular tissue engineering (vTE) is the lack of strength and elasticity of engineered vascular wall models caused by limited elastic fiber deposition. In this study, flat and tubular collagen gel-based scaffolds were cellularised with vascular smooth muscle cells (SMCs) and supplemented with human plasma fibronectin (FN), a known master organizer of several extracellular matrix (ECM) fiber systems. The consequences of FN on construct maturation was investigated in terms of geometrical contraction, viscoelastic mechanical properties and deposition of core elastic fiber proteins. FN was retained in the constructs and promoted deposition of elastin by SMCs as well as of several proteins required for elastogenesis such as fibrillin-1, lysyl oxidase, fibulin-4 and latent TGF-β binding protein-4. Notably, gel contraction, tensile equilibrium elastic modulus and elasticity were strongly improved in tubular engineered tissues, approaching the behaviour of native arteries. In conclusion, this study demonstrates that FN exerts pivotal roles in directing SMC-mediated remodeling of scaffolds toward the production of a physiological-like, elastin-containing ECM with excellent mechanical properties. The developed FN-supplemented systems are promising for tissue engineering applications where the generation of mature elastic tissue is desired and represent valuable advanced in vitro models to investigate elastogenesis.
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http://dx.doi.org/10.1016/j.biomaterials.2018.07.013DOI Listing
October 2018

Roles of fibronectin isoforms in neonatal vascular development and matrix integrity.

PLoS Biol 2018 07 23;16(7):e2004812. Epub 2018 Jul 23.

Faculty of Medicine, Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada.

Fibronectin (FN) exists in two forms-plasma FN (pFN) and cellular FN (cFN). Although the role of FN in embryonic blood vessel development is well established, its function and the contribution of individual isoforms in early postnatal vascular development are poorly understood. Here, we employed a tamoxifen-dependent cFN inducible knockout (cFN iKO) mouse model to study the consequences of postnatal cFN deletion in smooth muscle cells (SMCs), the major cell type in the vascular wall. Deletion of cFN influences collagen deposition but does not affect life span. Unexpectedly, pFN translocated to the aortic wall in the cFN iKO and in control mice, possibly rescuing the loss of cFN. Postnatal pFN deletion did not show a histological aortic phenotype. Double knockout (dKO) mice lacking both, cFN in SMCs and pFN, resulted in postnatal lethality. These data demonstrate a safeguard role of pFN in vascular stability and the dispensability of the individual FN isoforms in postnatal vascular development. Complete absence of FNs in the dKOs resulted in a disorganized tunica media of the aortic wall. Matrix analysis revealed common and differential roles of the FN isoforms in guiding the assembly/deposition of elastogenic extracellular matrix (ECM) proteins in the aortic wall. In addition, we determined with two cell culture models that that the two FN isoforms acted similarly in supporting matrix formation with a greater contribution from cFN. Together, these data show that pFN exerts a critical role in safeguarding vascular organization and health, and that the two FN isoforms function in an overlapping as well as distinct manner to maintain postnatal vascular matrix integrity.
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http://dx.doi.org/10.1371/journal.pbio.2004812DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6072322PMC
July 2018

Increasing Cell Seeding Density Improves Elastin Expression and Mechanical Properties in Collagen Gel-Based Scaffolds Cellularized with Smooth Muscle Cells.

Biotechnol J 2019 Mar 17;14(3):e1700768. Epub 2018 Jun 17.

Laboratory for Biomaterials and Bioengineering, Canada Research Chair I in Biomaterials and Bioengineering for the Innovation in Surgery, Department of Min-Met-Materials Engineering, Research Center of CHU de Québec, Division of Regenerative Medicine, Laval University, Québec, QC G1V 0A6, Canada.

Vascular tissue engineering combines cells with scaffold materials in vitro aiming the development of physiologically relevant vascular models. For natural scaffolds such as collagen gels, where cells can be mixed with the material solution before gelation, cell seeding density is a key parameter that can affect extracellular matrix deposition and remodeling. Nonetheless, this parameter is often overlooked and densities sensitively lower than those of native tissues, are usually employed. Herein, the effect of seeding density on the maturation of tubular collagen gel-based scaffolds cellularized with smooth muscle cells is investigated. The compaction, the expression, and deposition of key vascular proteins and the resulting mechanical properties of the constructs are evaluated up to 1 week of maturation. Results show that increasing cell seeding density accelerates cell-mediated gel compaction, enhances elastin expression (more than sevenfold increase at the highest density, Day 7) and finally improves the overall mechanical properties of constructs. Of note, the tensile equilibrium elastic modulus, evaluated by stress-relaxation tests, reach values comparable to native arteries for the highest cell density, after a 7-day maturation. Altogether, these results show that higher cell seeding densities promote the rapid maturation of collagen gel-based vascular constructs toward structural and mechanical properties better mimicking native arteries.
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http://dx.doi.org/10.1002/biot.201700768DOI Listing
March 2019

Fibrillins.

Methods Cell Biol 2018 8;143:223-246. Epub 2017 Dec 8.

Faculty of Medicine and Faculty of Dentistry, McGill University, Montreal, QC, Canada. Electronic address:

Fibrillins are one of the major components of supramolecular fibrous structures in the extracellular matrix of elastic and nonelastic tissues, termed microfibrils. Microfibrils provide tensile strength in nonelastic tissues and scaffolds for the assembly of tropoelastin in elastic tissues, and act a regulator of growth factor bioavailability and activity in connective tissues. Mutations in fibrillins lead to a variety of connective tissue disorders including Marfan syndrome, stiff skin syndrome, dominant Weill-Marchesani syndrome, and others. Therefore, fibrillins are frequently studied to understand the pathophysiology of these diseases and to identify effective treatment strategies. Extraction of endogenous microfibrils from cells and tissues can aid in obtaining structural insights of microfibrils. Recombinant production of fibrillins is an important tool which can be utilized to study the properties of normal fibrillins and the consequences of disease causing mutations. Other means of studying the role of fibrillins in the context of various physiological settings is by knocking down the mRNA expression and analyzing its downstream consequences. It is also important to study the interactome of fibrillins by protein-protein interactions, which can be derailed in pathological situations. Interacting proteins can affect the assembly of fibrillins in cells and tissues or can affect the levels of growth factors in the matrix. This chapter describes important techniques in the field that facilitate answering relevant questions of fibrillin biology and pathophysiology.
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http://dx.doi.org/10.1016/bs.mcb.2017.08.013DOI Listing
November 2018

Mutations in Fibronectin Cause a Subtype of Spondylometaphyseal Dysplasia with "Corner Fractures".

Am J Hum Genet 2017 Nov;101(5):815-823

Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095, USA.

Fibronectin is a master organizer of extracellular matrices (ECMs) and promotes the assembly of collagens, fibrillin-1, and other proteins. It is also known to play roles in skeletal tissues through its secretion by osteoblasts, chondrocytes, and mesenchymal cells. Spondylometaphyseal dysplasias (SMDs) comprise a diverse group of skeletal dysplasias and often manifest as short stature, growth-plate irregularities, and vertebral anomalies, such as scoliosis. By comparing the exomes of individuals with SMD with the radiographic appearance of "corner fractures" at metaphyses, we identified three individuals with fibronectin (FN1) variants affecting highly conserved residues. Furthermore, using matching tools and the SkelDys emailing list, we identified other individuals with de novo FN1 variants and a similar phenotype. The severe scoliosis in most individuals and rare developmental coxa vara distinguish individuals with FN1 mutations from those with classical Sutcliffe-type SMD. To study functional consequences of these FN1 mutations on the protein level, we introduced three disease-associated missense variants (p.Cys87Phe [c.260G>T], p.Tyr240Asp [c.718T>G], and p.Cys260Gly [c.778T>G]) into a recombinant secreted N-terminal 70 kDa fragment (rF70K) and the full-length fibronectin (rFN). The wild-type rF70K and rFN were secreted into the culture medium, whereas all mutant proteins were either not secreted or secreted at significantly lower amounts. Immunofluorescence analysis demonstrated increased intracellular retention of the mutant proteins. In summary, FN1 mutations that cause defective fibronectin secretion are found in SMD, and we thus provide additional evidence for a critical function of fibronectin in cartilage and bone.
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http://dx.doi.org/10.1016/j.ajhg.2017.09.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5673654PMC
November 2017

Fibrillin-2 and Tenascin-C bridge the age gap in lung epithelial regeneration.

Biomaterials 2017 Sep 22;140:212-219. Epub 2017 Jun 22.

Center for Regenerative Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States.

Organ engineering based on native matrix scaffolds involves combining regenerative cell populations with corresponding biological matrices to form functional grafts on-demand. The extracellular matrix (ECM) that is retained following lung decellularization provides essential structure and biophysical cues for whole organ regeneration after recellularization. The unique ECM composition in the early post-natal lung, during active alveologenesis, may possess distinct signals that aid in driving cell adhesion, survival, and proliferation. We evaluated the behavior of basal epithelial stem cells (BESCs) isolated from adult human lung tissue, when cultured on acellular ECM derived from neonatal (aged < 1 week) or adult lung donors (n = 3 donors per group). A significant difference in cell proliferation and survival was found. We next performed in-depth proteomic analysis of the lung scaffolds to quantify proteins significantly enriched in the neonatal ECM, and identified the glycoproteins Fibrillin-2 (FBN-2) and Tenascin-C (TN-C) as potential mediators of the observed effect. BESCs cultured on Collagen Type IV coated plates, supplemented with FBN-2 and TN-C demonstrated significantly increased proliferation and decreased cellular senescence. No significant increase in epithelial-to-mesenchymal transition was observed. In vitro migration was also increased by FBN-2 and TN-C treatment. Decellularized lung scaffolds treated with FBN-2 and TN-C prior to re-epithelialization supported greater epithelial proliferation and tissue remodeling. BESC distribution, matrix alignment, and overall tissue morphology was improved on treated lung scaffolds, after 3 and 7 days of ex vivo lung culture. These results demonstrate that scaffold re-epithelialization is enhanced on neonatal lung ECM, and that supplementation of FBN-2 and TN-C to the native scaffold may be a valuable tool in lung tissue regeneration.
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http://dx.doi.org/10.1016/j.biomaterials.2017.06.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5553709PMC
September 2017

Unusual life cycle and impact on microfibril assembly of ADAMTS17, a secreted metalloprotease mutated in genetic eye disease.

Sci Rep 2017 02 8;7:41871. Epub 2017 Feb 8.

Department of Biomedical Engineering, Cleveland Clinic Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH, 44195, USA.

Secreted metalloproteases have diverse roles in the formation, remodeling, and the destruction of extracellular matrix. Recessive mutations in the secreted metalloprotease ADAMTS17 cause ectopia lentis and short stature in humans with Weill-Marchesani-like syndrome and primary open angle glaucoma and ectopia lentis in dogs. Little is known about this protease or its connection to fibrillin microfibrils, whose major component, fibrillin-1, is genetically associated with ectopia lentis and alterations in height. Fibrillin microfibrils form the ocular zonule and are present in the drainage apparatus of the eye. We show that recombinant ADAMTS17 has unique characteristics and an unusual life cycle. It undergoes rapid autocatalytic processing in trans after its secretion from cells. Secretion of ADAMTS17 requires O-fucosylation and its autocatalytic activity does not depend on propeptide processing by furin. ADAMTS17 binds recombinant fibrillin-2 but not fibrillin-1 and does not cleave either. It colocalizes to fibrillin-1 containing microfibrils in cultured fibroblasts and suppresses fibrillin-2 (FBN2) incorporation in microfibrils, in part by transcriptional downregulation of Fbn2 mRNA expression. RNA in situ hybridization detected Adamts17 expression in specific structures in the eye, skeleton and other organs, where it may regulate the fibrillin isoform composition of microfibrils.
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http://dx.doi.org/10.1038/srep41871DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5296908PMC
February 2017

The Extracellular Matrix Signature in Vein Graft Disease.

Can J Cardiol 2016 08 1;32(8):1008.e11-7. Epub 2015 Dec 1.

Department of Dermatology, University of Lübeck, Lübeck, Germany; Institute of Virology and Cell Biology, University of Lübeck, Lübeck, Germany. Electronic address:

Background: Vein graft disease is a major and yet unsolved problem in cardiac revascularization surgery. Although accumulation of extracellular matrix is characteristic for vein graft disease, detailed analysis of the fibrotic material is lacking. Because alterations of collagen cross-links are typical for organ fibrosis, we performed a comprehensive analysis of collagen and elastin in vein graft disease.

Methods: Collagen, elastin, and their respective cross-links were analyzed using histology and amino acid analysis. The expression of collagen-modifying enzymes was analyzed using SYBR Green quantitative real-time polymerase chain reaction. Fibrillin expression was analyzed by immunohistochemistry and quantitative real-time polymerase chain reaction.

Results: Diseased vein grafts showed a marked increase of collagen and of intermediate collagen cross-links, which are markers for newly synthesized collagen. Furthermore, we identified in vein graft disease increased levels of mature hydroxylysine aldehyde-derived cross-links typical for skeletal tissues. This was accompanied by upregulation of lysyl hydroxylase 2 and lysyl oxidase expression. Furthermore, vein graft disease showed a reduction of the elastin/collagen ratio, using elastin cross-links as a marker of elastin content, which was accompanied by an increase of fibrillin-1.

Conclusions: Vein graft disease was accompanied by marked alterations in the composition of the extracellular matrix. The altered collagen cross-link pattern and the reduced elastin/collagen ratio might synergistically increase the stiffness in diseased vein grafts. Furthermore, hydroxylysine aldehyde-derived cross-links can cause a decreased degradability of collagens by matrix-metalloproteinases. Our data suggest collagen cross-links as a therapeutic target in vein graft disease.
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http://dx.doi.org/10.1016/j.cjca.2015.11.014DOI Listing
August 2016

Special issue: Extracellular matrix: Therapeutic tools and targets in cancer treatment.

Adv Drug Deliv Rev 2016 Feb;97:1-3

McGill University, Faculty of Medicine, Department of Anatomy and Cell Biology, and Faculty of Dentistry, Montreal, Canada.

Extracellular matrix (ECM) constituents play not only structural roles during development and tissue homeostasis, but also many biological functions throughout life. Molecular diversity and a vast interactome provide the basis for this multi-functionality. Moreover, native or processed ECM molecules interact with various receptors, thereby activating signaling pathways that control cell differentiation, proliferation, adhesion and migration, all relevant to tumor biology. Thus, there is an emerging field focused on exploiting ECM components as novel therapeutic targets in the treatment of cancer and other diseases, providing potent tools to advance drug delivery and tissue penetration. In this special issue we provide a critical appraisal of this emerging field focusing on: 1) ECM proteins (matricellular proteins, collagen, elastin, fibronectin, proteoglycans), integrins, and protease-facilitated drug delivery; 2) ECM-derived therapeutics (hyaluronan, heparin, heparan sulfate), 3) ECM-like biomaterials, and 4) ECM as critical determinant in drug efficacy, with special emphasis on applications in tumor treatment.
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http://dx.doi.org/10.1016/j.addr.2016.01.001DOI Listing
February 2016

Fibronectin-targeted drug delivery in cancer.

Adv Drug Deliv Rev 2016 Feb 27;97:101-10. Epub 2015 Nov 27.

Faculty of Medicine, Department of Anatomy and Cell Biology, McGill University, Montreal, QC H3A 0C7, Canada; Faculty of Dentistry, McGill University, Montreal, QC H3A 0C7, Canada. Electronic address:

Fibronectin is an extracellular matrix protein with pivotal physiological and pathological functions in development and adulthood. Alternative splicing of the precursor mRNA, produced from the single copy fibronectin gene, occurs at three sites coding for the EDA, EDB and IIICS domains. Fibronectin isoforms comprising the EDA or EDB domains are known as oncofetal forms due to their developmental importance and their re-expression in tumors, contrasting with restricted presence in normal adult tissues. These isoforms are also recognized as important markers of angiogenesis, a crucial physiological process in development and required by tumor cells in cancer progression. Attributed to this feature, EDA and EDB domains have been extensively used for the targeted delivery of cytokines, cytotoxic agents, chemotherapy drugs and radioisotopes to fibronectin-expressing tumors to exert therapeutic effects on primary cancers and metastatic lesions. In addition to drug delivery, the EDA and EDB domains of fibronectin have also been utilized to develop imaging strategies for tumor tissues. Furthermore, EDA and EDB based vaccines seem to be promising for the treatment and prevention of certain cancer types. In this review, we will summarize recent advances in fibronectin EDA and EDB-based therapeutic strategies developed to treat cancer.
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http://dx.doi.org/10.1016/j.addr.2015.11.014DOI Listing
February 2016

Soluble CD109 binds TGF-β and antagonizes TGF-β signalling and responses.

Biochem J 2016 Mar 30;473(5):537-47. Epub 2015 Nov 30.

Division of Plastic Surgery, Department of Surgery, McGill University Health Centre, Montreal, QC, Canada, H3G1A4

Transforming growth factor-β (TGF-β) is a multifunctional cytokine implicated in many diseases, including tissue fibrosis and cancer. TGF-β mediates diverse biological responses by signalling through type I and II TGF-β receptors (TβRI and TβRII). We have previously identified CD109, a glycosylphosphatidylinositol (GPI)-anchored protein, as a novel TGF-β co-receptor that negatively regulates TGF-β signalling and responses and demonstrated that membrane-anchored CD109 promotes TGF-β receptor degradation via a SMAD7/Smurf2-mediated mechanism. To determine whether CD109 released from the cell surface (soluble CD109 or sCD109) also acts as a TGF-β antagonist, we determined the efficacy of recombinant sCD109 to interact with TGF-β and inhibit TGF-β signalling and responses. Our results demonstrate that sCD109 binds TGF-β with high affinity as determined by surface plasmon resonance (SPR) and cell-based radioligand binding and affinity labelling competition assays. SPR detected slow dissociation kinetics between sCD109 and TGF-β at low concentrations, indicating a stable and effective interaction. In addition, sCD109 antagonizes TGF-β-induced Smad2/3 phosphorylation, transcription and cell migration. Together, our results suggest that sCD109 can bind TGF-β, inhibit TGF-β binding to its receptors and decrease TGF-β signalling and TGF-β-induced cellular responses.
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http://dx.doi.org/10.1042/BJ20141488DOI Listing
March 2016

Fibrillin-containing microfibrils are key signal relay stations for cell function.

J Cell Commun Signal 2015 Dec 8;9(4):309-25. Epub 2015 Oct 8.

Faculty of Medicine, Department of Anatomy and Cell Biology, and Faculty of Dentistry, McGill University, 3640 University Street, Montreal, Quebec, H3A 0C7, Canada.

Fibrillins constitute the backbone of microfibrils in the extracellular matrix of elastic and non-elastic tissues. Mutations in fibrillins are associated with a wide range of connective tissue disorders, the most common is Marfan syndrome. Microfibrils are on one hand important for structural stability in some tissues. On the other hand, microfibrils are increasingly recognized as critical mediators and drivers of cellular signaling. This review focuses on the signaling mechanisms initiated by fibrillins and microfibrils, which are often dysregulated in fibrillin-associated disorders. Fibrillins regulate the storage and bioavailability of growth factors of the TGF-β superfamily. Cells sense microfibrils through integrins and other receptors. Fibrillins potently regulate pathways of the immune response, inflammation and tissue homeostasis. Emerging evidence show the involvement of microRNAs in disorders caused by fibrillin deficiency. A thorough understanding of fibrillin-mediated cell signaling pathways will provide important new leads for therapeutic approaches of the underlying disorders.
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http://dx.doi.org/10.1007/s12079-015-0307-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4715827PMC
December 2015

Engineered mutations in fibrillin-1 leading to Marfan syndrome act at the protein, cellular and organismal levels.

Mutat Res Rev Mutat Res 2015 Jul-Sep;765:7-18. Epub 2015 May 5.

Faculty of Medicine, McGill University, Montreal, Canada; Faculty of Dentistry, McGill University, Montreal, Canada. Electronic address:

Fibrillins are the major components of microfibrils in the extracellular matrix of elastic and non-elastic tissues. They are multi-domain proteins, containing primarily calcium binding epidermal growth factor-like (cbEGF) domains and 8-cysteine/transforming growth factor-beta binding protein-like (TB) domains. Mutations in the fibrillin-1 gene give rise to Marfan syndrome, a connective tissue disorder with clinical complications in the cardiovascular, skeletal, ocular and other organ systems. Here, we review the consequences of engineered Marfan syndrome mutations in fibrillin-1 at the protein, cellular and organismal levels. Representative point mutations associated with Marfan syndrome in affected individuals have been introduced and analyzed in recombinant fibrillin-1 fragments. Those mutations affect fibrillin-1 on a structural and functional level. Mutations which impair folding of cbEGF domains can affect protein trafficking. Protein folding disrupted by some mutations can lead to defective secretion in mutant fibrillin-1 fragments, whereas fragments with other Marfan mutations are secreted normally. Many Marfan mutations render fibrillin-1 more susceptible to proteolysis. There is also evidence that some mutations affect heparin binding. Few mutations have been further analyzed in mouse models. An extensively studied mouse model of Marfan syndrome expresses mouse fibrillin-1 with a missense mutation (p.C1039G). The mice display similar characteristics to human patients with Marfan syndrome. Overall, the analyses of engineered mutations leading to Marfan syndrome provide important insights into the pathogenic molecular mechanisms exerted by mutated fibrillin-1.
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http://dx.doi.org/10.1016/j.mrrev.2015.04.002DOI Listing
November 2015

In vitro elastogenesis: instructing human vascular smooth muscle cells to generate an elastic fiber-containing extracellular matrix scaffold.

Biomed Mater 2015 Mar 18;10(3):034102. Epub 2015 Mar 18.

Department of Cell and Tissue Engineering, Fraunhofer Institute for Interfacial Engineering and Biotechnology (IGB), 70569 Stuttgart, Germany. Department of Women's Health, Research Institute for Women's Health, Eberhard Karls University Tübingen, 72076 Tübingen, Germany.

Elastic fibers are essential for the proper function of organs including cardiovascular tissues such as heart valves and blood vessels. Although (tropo)elastin production in a tissue-engineered construct has previously been described, the assembly to functional elastic fibers in vitro using human cells has been highly challenging. In the present study, we seeded primary isolated human vascular smooth muscle cells (VSMCs) onto 3D electrospun scaffolds and exposed them to defined laminar shear stress using a customized bioreactor system. Increased elastin expression followed by elastin deposition onto the electrospun scaffolds, as well as on newly formed fibers, was observed after six days. Most interestingly, we identified the successful deposition of elastogenesis-associated proteins, including fibrillin-1 and -2, fibulin-4 and -5, fibronectin, elastin microfibril interface located protein 1 (EMILIN-1) and lysyl oxidase (LOX) within our engineered constructs. Ultrastructural analyses revealed a developing extracellular matrix (ECM) similar to native human fetal tissue, which is composed of collagens, microfibrils and elastin. To conclude, the combination of a novel dynamic flow bioreactor and an electrospun hybrid polymer scaffold allowed the production and assembly of an elastic fiber-containing ECM.
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http://dx.doi.org/10.1088/1748-6041/10/3/034102DOI Listing
March 2015