Unraveling the role of TGFß signaling in thoracic aortic aneurysm and dissection using Fbn1 mutant mouse models.

Although abnormal TGFß signaling is observed in several heritable forms of thoracic aortic aneurysms and dissections including Marfan syndrome, its precise role in aortic disease progression is still disputed. Using a mouse genetic approach and quantitative isobaric labeling proteomics, we sought to elucidate the role of TGFß signaling in three Fbn1 mutant mouse models representing a range of aortic disease from microdissection (without aneurysm) to aneurysm (without rupture) to aneurysm and rupture. Results indicated that reduced TGFß signaling and increased mast cell proteases were associated with microdissection. In contrast, increased abundance of extracellular matrix proteins, which could be reporters for positive TGFß signaling, were associated with aneurysm. Marked reductions in collagens and fibrillins, and increased TGFß signaling, were associated with aortic rupture. Our data indicate that TGFß signaling performs context-dependent roles in the pathogenesis of thoracic aortic disease.

Circulating fibrillin fragment concentrations in patients with and without aortic pathology.

Objective: Fragments of fibrillin-1 and fibrillin-2 will be detectable in the plasma of patients with aortic dissections and aneurysms. We sought to determine whether the plasma fibrillin fragment levels (PFFLs) differ between patients with thoracic aortic pathology and those presenting with nonaortic chest pain. Methods: PFFLs were measured in patients with thoracic aortic aneurysm (n = 27) or dissection (n = 28). For comparison, patients without aortic pathology who had presented to the emergency department with acute chest pain (n = 281) were categorized into three groups according to the cause of the chest pain: ischemic cardiac chest pain; nonischemic cardiac chest pain; and noncardiac chest pain. The PFFLs were measured using a sandwich enzyme-linked immunosorbent assay. Results: Fibrillin-1 fragments were detectable in all patients and were lowest in the ischemic cardiac chest pain group. Age, sex, and the presence of hypertension were associated with differences in fibrillin-1 fragment levels. Fibrillin-2 fragments were detected more often in the thoracic aneurysm and dissection groups than in the emergency department chest pain group (P < .0001). Patients with aortic dissection demonstrated a trend toward increased detectability (P = .051) and concentrations (P = .06) of fibrillin-2 fragments compared with patients with aortic aneurysms. Analysis of specific antibody pairs identified fibrillin-1 B15-HRP26 and fibrillin-2 B205-HRP143 as the most informative in distinguishing between the emergency department and aortic pathology groups. Conclusions: Patients with thoracic aortic dissections demonstrated elevated plasma fibrillin-2 fragment levels (B205-HRP143) compared with patients presenting with ischemic or nonischemic cardiac chest pain and increased fibrillin-1 levels (B15-HRP26) compared with patients with ischemic cardiac chest pain. Investigation of fibrillin-1 and fibrillin-2 fragment generation might lead to diagnostic, therapeutic, and prognostic advances for patients with thoracic aortic dissection.

EMILIN1 deficiency causes arterial tortuosity with osteopenia and connects impaired elastogenesis with defective collagen fibrillogenesis.

EMILIN1 (elastin-microfibril-interface-located-protein-1) is a structural component of the elastic fiber network and localizes to the interface between the fibrillin microfibril scaffold and the elastin core. How EMILIN1 contributes to connective tissue integrity is not fully understood. Here, we report bi-allelic EMILIN1 loss-of-function variants causative for an entity combining cutis laxa, arterial tortuosity, aneurysm formation, and bone fragility, resembling autosomal-recessive cutis laxa type 1B, due to EFEMP2 (FBLN4) deficiency. In both humans and mice, absence of EMILIN1 impairs EFEMP2 extracellular matrix deposition and LOX activity resulting in impaired elastogenesis, reduced collagen crosslinking, and aberrant growth factor signaling. Collagen fiber ultrastructure and histopathology in EMILIN1- or EFEMP2-deficient skin and aorta corroborate these findings and murine Emilin1-/- femora show abnormal trabecular bone formation and strength. Altogether, EMILIN1 connects elastic fiber network with collagen fibril formation, relevant for both bone and vascular tissue homeostasis.

A Novel Model of Tobacco Smoke-Mediated Aortic Injury.

OBJECTIVE: Tobacco smoke exposure is a major risk factor for aortic aneurysm development. However, the initial aortic response to tobacco smoke, preceding aneurysm formation, is not well understood. We sought to create a model to determine the effect of solubilized tobacco smoke (STS) on the thoracic and abdominal aorta of mice as well as on cultured human aortic smooth muscle cells (HASMCs). METHODS: Tobacco smoke was solubilized and delivered to mice via implanted osmotic minipumps. Twenty male C57BL/6 mice received STS or vehicle infusion. The descending thoracic, suprarenal abdominal, and infrarenal abdominal segments of the aorta were assessed for elastic lamellar damage, smooth muscle cell phenotype, and infiltration of inflammatory cells. Cultured HASMCs grown in media containing STS were compared to cells grown in standard media in order to verify our in vivo findings. RESULTS: Tobacco smoke solution caused significantly more breaks in the elastic lamellae of the thoracic and abdominal aorta compared to control solution (P< .0001) without inciting an inflammatory infiltrate. Elastin breaks occurred more frequently in the abdominal aorta than the thoracic aorta (P < .01). Exposure to STS-induced aortic microdissections and downregulation of α-smooth muscle actin (α-SMA) by vascular smooth muscle cells (VSMCs). Treatment of cultured HASMCs with STS confirmed the decrease in α-SMA expression. CONCLUSION: Delivery of STS via osmotic minipumps appears to be a promising model for investigating the early aortic response to tobacco smoke exposure. The initial effect of tobacco smoke exposure on the aorta is elastic lamellar damage and downregulation of (α-SMA) expression by VSMCs. Elastic lamellar damage occurs more frequently in the abdominal aorta than the thoracic aorta and does not seem to be mediated by the presence of macrophages or other inflammatory cells.

Pathogenic variants in THSD4, encoding the ADAMTS-like 6 protein, predispose to inherited thoracic aortic aneurysm.

PURPOSE: Thoracic aortic aneurysm and dissection (TAAD) is a life-threatening disease with often unrecognized inherited forms. We sought to identify novel pathogenic variants associated with autosomal dominant inheritance of TAAD. METHODS: We analyzed exome sequencing data from 35 French TAAD families and performed next-generation sequencing capture panel of genes in 1114 unrelated TAAD patients. Functional effects of pathogenic variants identified were validated in cell, tissue, and mouse models. RESULTS: We identified five functional variants in THSD4 of which two heterozygous variants lead to a premature termination codon. THSD4 encodes ADAMTSL6 (member of the ADAMTS/L superfamily), a microfibril-associated protein that promotes fibrillin-1 matrix assembly. The THSD4 variants studied lead to haploinsufficiency or impaired assembly of fibrillin-1 microfibrils. Thsd4+/- mice showed progressive dilation of the thoracic aorta. Histologic examination of aortic samples from a patient carrying a THSD4 variant and from Thsd4+/- mice, revealed typical medial degeneration and diffuse disruption of extracellular matrix. CONCLUSION: These findings highlight the role of ADAMTSL6 in aortic physiology and TAAD pathogenesis. They will improve TAAD management and help develop new targeted therapies.

Fibrillin-1 in the Vasculature: In Vivo Accumulation of eGFP-Tagged Fibrillin-1 in a Knockin Mouse Model.

Immunolocalization studies have shown that fibrillin-1 is distributed ubiquitously in the connective tissue space from early embryonic times through old age. When mutated, the gene for fibrillin-1 (FBN1) causes the Marfan syndrome, a common inherited disorder of connective tissue. The multiple manifestations of the Marfan syndrome reflect the known distribution of fibrillin-1 in cardiovascular, musculoskeletal, ocular, and dermal tissues. In this study, a mouse model of Marfan syndrome in which fibrillin-1 is truncated and tagged with green fluorescence was used to estimate the relative abundance of fibrillin-1 in developing tissues. In embryonic tissues, the aorta was the only tissue in which fibrillin-1 green fluorescence was detectable. Other arteries gained detectable fibrillin-1 green fluorescence just after birth. Fibrillin-1 fluorescence was observed at later postnatal times in the lung, skin, perichondrium, tendon, and ocular tissues, while other tissues remained negative. These results indicated that tissues most affected in the Marfan syndrome are the tissues in which fibrillin-1 is most abundant. Focus was placed on the aorta, since aortic disease is life threatening in the Marfan syndrome and fibrillin-1 green fluorescence was most abundant in this tissue. Fibrillin-1 green fluorescence and immunostaining showed that fibrillin-1 is within aortic medial elastic lamellae. Endothelial-specific compared to smooth muscle-specific fibrillin-1 green fluorescence, together with light microscopic analyses of fragmentation of aortic elastic lamellae, demonstrated that smooth muscle cell mutated fibrillin-1 contributed most to progressive aortic fragmentation. However, these studies also indicated that other cells, possibly endothelial cells, also contribute to this aortic pathology. Anat Rec, 2019. © 2019 Wiley Periodicals, Inc.

The acute kidney injury to chronic kidney disease transition in a mouse model of acute cardiorenal syndrome emphasizes the role of inflammation.

Acute cardiorenal syndrome is a common complication of acute cardiovascular disease. Studies of acute kidney injury (AKI) to chronic kidney disease (CKD) transition, including patients suffering acute cardiovascular disease, report high rates of CKD development. Therefore, acute cardiorenal syndrome associates with CKD, but no study has established causation. To define this we used a murine cardiac arrest (CA) and cardiopulmonary resuscitation (CPR) model or sham procedure on male mice. CA was induced with potassium chloride while CPR consisted of chest compressions and epinephrine eight minutes later. Two weeks after AKI was induced by CA/CPR, the measured glomerular filtration rate (GFR) was not different from sham. However, after seven weeks the mice developed CKD, recapitulating clinical observations. One day, and one, two, and seven weeks after CA/CPR, the GFR was measured, and renal tissue sections were evaluated for various indices of injury and inflammation. One day after CA/CPR, acute cardiorenal syndrome was indicated by a significant reduction of the mean GFR (649 in sham, vs. 25 µL/min/100g in CA/CPR animals), KIM-1 positive tubules, and acute tubular necrosis. Renal inflammation developed, with F4/80 positive and CD3-positive cells infiltrating the kidney one day and one week after CA/CPR, respectively. Although there was functional recovery with normalization of GFR two weeks after CA/CPR, deposition of tubulointerstitial matrix proteins α-smooth muscle actin and fibrillin-1 progressed, along with a significantly reduced mean GFR (623 in sham vs. 409 µL/min/100g in CA/CPR animals), proteinuria, increased tissue transforming growth factor-ß, and fibrosis establishing the development of CKD seven weeks after CA/CPR. Thus, murine CA/CPR, a model of acute cardiorenal syndrome, causes an AKI-CKD transition likely due to prolonged renal inflammation.

Fibrillin protein pleiotropy: Acromelic dysplasias.

The fibrillins are large extracellular matrix molecules that polymerize to form microfibrils. Fibrillin microfibrils are distinctive architectural elements that are both ubiquitous in the connective tissue space and also unique, displaying tissue-specific patterns. Mutations in the genes for fibrillin-1 (FBN1) result in multiple distinct pleiotropic disorders. Most of the more than 3000 mutations known today in FBN1 cause the Marfan syndrome. Marfan mutations can occur in any of the 56 domains that compose fibrillin-1. In contrast, rare mutations in FBN1 that are confined to only certain domains cause several different types of acromelic dysplasia. These genetic disorders demonstrate that specific domains of fibrillin-1 perform roles important to musculoskeletal growth. Many of the phenotypes of acromelic dysplasias are the opposite of those found in Marfan syndrome. Knowledge of the functions and structural organization of fibrillin molecules within microfibrils is required to understand how one protein and one gene can be the basis for multiple genetic disorders.

Missense mutation in SLIT2 associated with congenital myopia, anisometropia, connective tissue abnormalities, and obesity.

BACKGROUND: SLIT2 is a protein ligand for the Roundabout (ROBO) receptor and was found to play a major role in repulsive midline axon guidance in central nervous system development. Based on studies utilizing knockout models, it has been postulated that SLIT2 is important for preventing inappropriate axonal routing during mammalian optic chiasm development. METHODS: Case report. RESULTS: Here, we report a case of congenital myopia, anisometropia, and obesity in a patient with a SLIT2 point mutation. Examination of the patient's skin biopsy revealed abnormalities in elastin and collagen fibrils that suggest an underlying connective tissue disorder. Structural modeling placed the novel mutation (p.D1407G) in the EGF-like domain 8 and was predicted to affect interactions with SLIT2 binding partners. CONCLUSIONS: To the authors' knowledge, this is the first report of a SLIT2 variant in the context of these ocular findings.

Correction: Sex, pregnancy and aortic disease in Marfan syndrome.

[This corrects the article DOI: 10.1371/journal.pone.0181166.].

Sex, pregnancy and aortic disease in Marfan syndrome.

BACKGROUND: Sex-related differences as well as the adverse effect of pregnancy on aortic disease outcome are well-established phenomena in humans with Marfan syndrome (MFS). The underlying mechanisms of these observations are largely unknown. OBJECTIVES: In an initial (pilot) step we aimed to confirm the differences between male and female MFS patients as well as between females with and without previous pregnancy. We then sought to evaluate whether these findings are recapitulated in a pre-clinical model and performed in-depth cardiovascular phenotyping of mutant male and both nulliparous and multiparous female Marfan mice. The effect of 17ß-estradiol on fibrillin-1 protein synthesis was compared in vitro using human aortic smooth muscle cells and fibroblasts. RESULTS: Our small retrospective study of aortic dimensions in a cohort of 10 men and 20 women with MFS (10 pregnant and 10 non-pregnant) confirmed that aortic root growth was significantly increased in the pregnant group compared to the non-pregnant group (0.64mm/year vs. 0.12mm/year, p = 0.018). Male MFS patients had significantly larger aortic root diameters compared to the non-pregnant and pregnant females at baseline and follow-up (p = 0.002 and p = 0.007, respectively), but no significant increase in aortic root growth was observed compared to the females after follow-up (p = 0.559 and p = 0.352). In the GT-8/+ MFS mouse model, multiparous female Marfan mice showed increased aortic diameters when compared to nulliparous females. Aortic dilatation in multiparous females was comparable to Marfan male mice. Moreover, increased aortic diameters were associated with more severe fragmentation of the elastic lamellae. In addition, 17ß-estradiol was found to promote fibrillin-1 production by human aortic smooth muscle cells. CONCLUSIONS: Pregnancy-related changes influence aortic disease severity in otherwise protected female MFS mice and patients. There may be a role for estrogen in the female sex protective effect.

Codependence of Bone Morphogenetic Protein Receptor 2 and Transforming Growth Factor-ß in Elastic Fiber Assembly and Its Perturbation in Pulmonary Arterial Hypertension.

OBJECTIVE: We determined in patients with pulmonary arterial (PA) hypertension (PAH) whether in addition to increased production of elastase by PA smooth muscle cells previously reported, PA elastic fibers are susceptible to degradation because of their abnormal assembly. APPROACH AND RESULTS: Fibrillin-1 and elastin are the major components of elastic fibers, and fibrillin-1 binds bone morphogenetic proteins (BMPs) and the large latent complex of transforming growth factor-ß1 (TGFß1). Thus, we considered whether BMPs like TGFß1 contribute to elastic fiber assembly and whether this process is perturbed in PAH particularly when the BMP receptor, BMPR2, is mutant. We also assessed whether in mice with Bmpr2/1a compound heterozygosity, elastic fibers are susceptible to degradation. In PA smooth muscle cells and adventitial fibroblasts, TGFß1 increased elastin mRNA, but the elevation in elastin protein was dependent on BMPR2; TGFß1 and BMP4, via BMPR2, increased extracellular accumulation of fibrillin-1. Both BMP4- and TGFß1-stimulated elastic fiber assembly was impaired in idiopathic (I) PAH-PA adventitial fibroblast versus control cells, particularly those with hereditary (H) PAH and a BMPR2 mutation. This was related to profound reductions in elastin and fibrillin-1 mRNA. Elastin protein was increased in IPAH PA adventitial fibroblast by TGFß1 but only minimally so in BMPR2 mutant cells. Fibrillin-1 protein increased only modestly in IPAH or HPAH PA adventitial fibroblasts stimulated with BMP4 or TGFß1. In Bmpr2/1a heterozygote mice, reduced PA fibrillin-1 was associated with elastic fiber susceptibility to degradation and more severe pulmonary hypertension. CONCLUSIONS: Disrupting BMPR2 impairs TGFß1- and BMP4-mediated elastic fiber assembly and is of pathophysiologic significance in PAH.

Intracellular mechanisms of molecular recognition and sorting for transport of large extracellular matrix molecules.

Extracellular matrix (ECM) proteins are biosynthesized in the rough endoplasmic reticulum (rER) and transported via the Golgi apparatus to the extracellular space. The coat protein complex II (COPII) transport vesicles are approximately 60-90 nm in diameter. However, several ECM molecules are much larger, up to several hundreds of nanometers. Therefore, special COPII vesicles are required to coat and transport these molecules. Transmembrane Protein Transport and Golgi Organization 1 (TANGO1) facilitates loading of collagens into special vesicles. The Src homology 3 (SH3) domain of TANGO1 was proposed to recognize collagen molecules, but how the SH3 domain recognizes various types of collagen is not understood. Moreover, how are large noncollagenous ECM molecules transported from the rER to the Golgi? Here we identify heat shock protein (Hsp) 47 as a guide molecule directing collagens to special vesicles by interacting with the SH3 domain of TANGO1. We also consider whether the collagen secretory model applies to other large ECM molecules.

FBN1: The disease-causing gene for Marfan syndrome and other genetic disorders.

FBN1 encodes the gene for fibrillin-1, a structural macromolecule that polymerizes into microfibrils. Fibrillin microfibrils are morphologically distinctive fibrils, present in all connective tissues and assembled into tissue-specific architectural frameworks. FBN1 is the causative gene for Marfan syndrome, an inherited disorder of connective tissue whose major features include tall stature and arachnodactyly, ectopia lentis, and thoracic aortic aneurysm and dissection. More than one thousand individual mutations in FBN1 are associated with Marfan syndrome, making genotype-phenotype correlations difficult. Moreover, mutations in specific regions of FBN1 can result in the opposite features of short stature and brachydactyly characteristic of Weill-Marchesani syndrome and other acromelic dysplasias. How can mutations in one molecule result in disparate clinical syndromes? Current concepts of the fibrillinopathies require an appreciation of tissue-specific fibrillin microfibril microenvironments and the collaborative relationship between the structures of fibrillin microfibril networks and biological functions such as regulation of growth factor signaling.

Functional consequence of fibulin-4 missense mutations associated with vascular and skeletal abnormalities and cutis laxa.

Fibulin-4 is a 60kDa calcium binding glycoprotein that has an important role in development and integrity of extracellular matrices. It interacts with elastin, fibrillin-1 and collagen IV as well as with lysyl oxidases and is involved in elastogenesis and cross-link formation. To date, several mutations in the fibulin-4 gene (FBLN4/EFEMP2) are known in patients whose major symptoms are vascular deformities, aneurysm, cutis laxa, joint laxity, or arachnodactyly. The pathogenetic mechanisms how these mutations translate into the clinical phenotype are, however, poorly understood. In order to elucidate these mechanisms, we expressed fibulin-4 mutants recombinantly in HEK293 cells, purified the proteins in native forms and analyzed alterations in protein synthesis, secretion, matrix assembly, and interaction with other proteins in relation to wild type fibulin-4. Our studies show that different mutations affect these properties in multiple ways, resulting in fibulin-4 deficiency and/or impaired ability to form elastic fibers. The substitutions E126K and C267Y impaired secretion of the protein, but not mRNA synthesis. Furthermore, the E126K mutant showed less resistance to proteases, reduced binding to collagen IV and fibrillin-1, as well as to LTBP1s and LTBP4s. The A397T mutation introduced an extra O-glycosylation site and deleted binding to LTBP1s. We show that fibulin-4 binds stronger than fibulin-3 and -5 to LTBP1s, 3, and 4s, and to the lysyl oxidases LOX and LOXL1; the binding of fibulin-4 to the LOX propeptide was strongly reduced by the mutation E57K. These findings show that different mutations in the fibulin-4 gene result in different molecular defects affecting secretion rates, protein stability, LOX-induced cross-linking, or binding to other ECM components and molecules of the TGF-ß pathway, and thus illustrate the complex role of fibulin-4 in connective tissue assembly.

Characterization of metabolic health in mouse models of fibrillin-1 perturbation.

Mutations in the microfibrillar protein fibrillin-1 or the absence of its binding partner microfibril-associated glycoprotein (MAGP1) lead to increased TGFß signaling due to an inability to sequester latent or active forms of TGFß, respectively. Mouse models of excess TGFß signaling display increased adiposity and predisposition to type-2 diabetes. It is therefore interesting that individuals with Marfan syndrome, a disease in which fibrillin-1 mutation leads to aberrant TGFß signaling, typically present with extreme fat hypoplasia. The goal of this project was to characterize multiple fibrillin-1 mutant mouse strains to understand how fibrillin-1 contributes to metabolic health. The results of this study demonstrate that fibrillin-1 contributes little to lipid storage and metabolic homeostasis, which is in contrast to the obesity and metabolic changes associated with MAGP1 deficiency. MAGP1 but not fibrillin-1 mutant mice had elevated TGFß signaling in their adipose tissue, which is consistent with the difference in obesity phenotypes. However, fibrillin-1 mutant strains and MAGP1-deficient mice all exhibit increased bone length and reduced bone mineralization which are characteristic of Marfan syndrome. Our findings suggest that Marfan-associated adipocyte hypoplasia is likely not due to microfibril-associated changes in adipose tissue, and provide evidence that MAGP1 may function independently of fibrillin in some tissues.

Abnormal Activation of BMP Signaling Causes Myopathy in Fbn2 Null Mice.

Fibrillins are large extracellular macromolecules that polymerize to form the backbone structure of connective tissue microfibrils. Mutations in the gene for fibrillin-1 cause the Marfan syndrome, while mutations in the gene for fibrillin-2 cause Congenital Contractural Arachnodactyly. Both are autosomal dominant disorders, and both disorders affect musculoskeletal tissues. Here we show that Fbn2 null mice (on a 129/Sv background) are born with reduced muscle mass, abnormal muscle histology, and signs of activated BMP signaling in skeletal muscle. A delay in Myosin Heavy Chain 8, a perinatal myosin, was found in Fbn2 null forelimb muscle tissue, consistent with the notion that muscle defects underlie forelimb contractures in these mice. In addition, white fat accumulated in the forelimbs during the early postnatal period. Adult Fbn2 null mice are already known to demonstrate persistent muscle weakness. Here we measured elevated creatine kinase levels in adult Fbn2 null mice, indicating ongoing cycles of muscle injury. On a C57Bl/6 background, Fbn2 null mice showed severe defects in musculature, leading to neonatal death from respiratory failure. These new findings demonstrate that loss of fibrillin-2 results in phenotypes similar to those found in congenital muscular dystrophies and that FBN2 should be considered as a candidate gene for recessive congenital muscular dystrophy. Both in vivo and in vitro evidence associated muscle abnormalities and accumulation of white fat in Fbn2 null mice with abnormally activated BMP signaling. Genetic rescue of reduced muscle mass and accumulation of white fat in Fbn2 null mice was accomplished by deleting a single allele of Bmp7. In contrast to other reports that activated BMP signaling leads to muscle hypertrophy, our findings demonstrate the exquisite sensitivity of BMP signaling to the fibrillin-2 extracellular environment during early postnatal muscle development. New evidence presented here suggests that fibrillin-2 can sequester BMP complexes in a latent state.

The fibrillin microfibril scaffold: A niche for growth factors and mechanosensation?

The fibrillins, large extracellular matrix molecules, are polymerized to form "microfibrils." The fibrillin microfibril scaffold is populated by microfibril-associated proteins and by growth factors, which are likely to be latent. The scaffold, associated proteins, and bound growth factors, together with cellular receptors that can sense the microfibril matrix, constitute the fibrillin microenvironment. Activation of TGFß signaling is associated with the Marfan syndrome, which is caused by mutations in fibrillin-1. Today we know that mutations in fibrillin-1 cause the Marfan syndrome as well as Weill-Marchesani syndrome (and other acromelic dysplasias) and result in opposite clinical phenotypes: tall or short stature; arachnodactyly or brachydactyly; joint hypermobility or stiff joints; hypomuscularity or hypermuscularity. We also know that these different syndromes are associated with different structural abnormalities in the fibrillin microfibril scaffold and perhaps with specific cellular receptors (mechanosensors). How does the microenvironment, framed by the microfibril scaffold and populated by latent growth factors, work? We must await future investigations for the molecular and cellular mechanisms that will answer this question. However, today we can appreciate the importance of the fibrillin microfibril niche as a contextual environment for growth factor signaling and potentially for mechanosensation.

Corneal stroma microfibrils.

Elastic tissue was first described well over a hundred years ago and has since been identified in nearly every part of the body. In this review, we examine elastic tissue in the corneal stroma with some mention of other ocular structures which have been more thoroughly described in the past. True elastic fibers consist of an elastin core surrounded by fibrillin microfibrils. However, the presence of elastin fibers is not a requirement and some elastic tissue is comprised of non-elastin-containing bundles of microfibrils. Fibers containing a higher relative amount of elastin are associated with greater elasticity and those without elastin, with structural support. Recently it has been shown that the microfibrils, not only serve mechanical roles, but are also involved in cell signaling through force transduction and the release of TGF-ß. A well characterized example of elastin-free microfibril bundles (EFMBs) is found in the ciliary zonules which suspend the crystalline lens in the eye. Through contraction of the ciliary muscle they exert enough force to reshape the lens and thereby change its focal point. It is believed that the molecules comprising these fibers do not turn-over and yet retain their tensile strength for the life of the animal. The mechanical properties of the cornea (strength, elasticity, resiliency) would suggest that EFMBs are present there as well. However, many authors have reported that, although present during embryonic and early postnatal development, EFMBs are generally not present in adults. Serial-block-face imaging with a scanning electron microscope enabled 3D reconstruction of elements in murine corneas. Among these elements were found fibers that formed an extensive network throughout the cornea. In single sections these fibers appeared as electron dense patches. Transmission electron microscopy provided additional detail of these patches and showed them to be composed of fibrils (∼10 nm diameter). Immunogold evidence clearly identified these fibrils as fibrillin EFMBs and EFMBs were also observed with TEM (without immunogold) in adult mammals of several species. Evidence of the presence of EFMBs in adult corneas will hopefully pique an interest in further studies that will ultimately improve our understanding of the cornea's biomechanical properties and its capacity to repair.