Fibrillin-1 Deposition Research Articles

High fat diet has a protective sex-dependent effect on aortic aneurysm severity in a Marfan syndrome mouse model.

Marfan syndrome (MFS) is a genetic disorder caused by mutations in fibrillin-1 and is characterized by thoracic aortic aneurysms and other complications. Previous studies revealed sexual dimorphisms in aortic aneurysm formation in patients with MFS. The present study aimed to investigate the combined role of a high-fat diet (HFD) and biological sex in aortic disease using the mgR/mgR MFS mouse model. Male and female mgR/mgR mice as well as wild-type (WT) littermate mice were fed a control diet (CD, 10% fat) or HFD (60% fat) from 4 to 12 weeks of age. Key aortic disease parameters analyzed included the diameter of the aortic wall; elastic fiber fragmentation; proteoglycan content; mRNA levels of Mmp12, Col1a1, Col3a1, and Fbn1; and fibrillin-1 deposition in the aortic wall. HFD-fed female mgR/mgR mice had significantly reduced aortic diameters (35%), elastic fiber fragmentation (56%), pathologically enhanced proteoglycans (45%), and expression of Mmp12 (64%), Col1a1 (41%), and Col3a1 (43%) compared to male mgR/mgR mice on HFD. Fibrillin-1 deposition and Fbn1 mRNA levels were unaffected. The data reveal a protective effect of HFD in female mice. In contrast, CD did not exert any protective effects. This study demonstrates a specific sexual dimorphism in MFS mice, with HFD exerting an explicit protective effect on aortic disease severity in female mice. These preclinical data may be useful for developing nutritional recommendations for individuals with MFS in the longer term.

A phenotypic screen of Marfan syndrome iPSC-derived vascular smooth muscle cells uncovers GSK3β as a new target.

Marfan syndrome (MFS) is a rare connective tissue disorder caused by mutations in FBN1. Patients with MFS notably suffer from aortic aneurysm and dissection. Despite considerable effort, animal models have proven to be poorly predictive for therapeutic intervention in human aortic disease. Patient-derived induced pluripotent stem cells can be differentiated into vascular smooth muscle cells (VSMCs) and recapitulate major features of MFS. We have screened 1,022 small molecules in our invitro model, exploiting the highly proteolytic nature of MFS VSMCs, and identified 36 effective compounds. Further analysis identified GSK3β as a recurring target in the compound screen. GSK3β inhibition/knockdown did not ameliorate the proliferation defect in MFS-VSMCs but improved MFS-VSMC proteolysis and apoptosis and partially rescued fibrillin-1 deposition. To conclude, we have identified GSK3β as a novel target for MFS, forming the foundation for future work in MFS and other aortic diseases.

Macromolecular crowding enhances fibrillin-1 deposition in the extracellular matrix.

Biochemical and biophysical factors need consideration when modelling in vivo cellular behaviour using in vitro cell culture systems. One underappreciated factor is the high concentration of macromolecules present in vivo, which is typically not simulated under standard cell culture conditions. This disparity is especially relevant when studying biochemical processes that govern extracellular matrix (ECM) deposition, which may be altered due to dilution of secreted macromolecules by the relatively large volumes of culture medium required for cell maintenance in vitro. Macromolecular crowding (MMC) utilises the addition of inert macromolecules to cell culture medium to mimic such high concentration environments found in vivo. The present study induced MMC using the sucrose polymer Ficoll and examined whether fibrillin-1 deposition by human lung fibroblasts could be augmented. Fibrillin-1 forms extracellular microfibrils, which are versatile scaffolds required for elastic fibre formation, deposition of other ECM proteins and growth factor regulation. Pathogenic variants in the fibrillin-1 gene (FBN1) cause Marfan syndrome, where ECM deposition of fibrillin-1 can be compromised. Using immunocytochemistry, significantly enhanced fibrillin-1 deposition was observed when lung fibroblasts were cultured under MMC conditions. MMC also augmented fibrillin-1 deposition in Marfan syndrome patient-derived skin fibroblasts in a cell line- and likely FBN1 variant-specific manner. The ability of MMC to increase fibrillin-1 deposition suggested potential applications for tissue-engineering approaches, e.g. to generate tendon or vascular tissues, where fibrillin-1 microfibrils and elastic fibres are key determinants of their biomechanical properties. Moreover, it suggested the potency of MMC to better mimic in vivo ECM environments in cell culture studies.

Abstract 9386: Marfan Syndrome Mouse Model MgΔ Lpn Presents Alterations in Glomerular Architecture and Increased Renal Vascular Resistance. Losartan Treatment Attenuates These Phenotypes

Introduction: Previously, we have shown that mgΔ lpn murine Marfan syndrome (MFS) model with a fibrillin-1 disruption presents increased Metalloproteinase-9 (MMP-9) expression in the cilliary body in eye. MMP-9 has been related to microfibrils degradation. In this study, we examined the fibrillin-1 and MMP-9 distribution in the kidney and their influence on hemodynamics. Hypothesis: The alteration of fibrillin-1 impacts the glomerular architecture and modify renal hemodynamics. Methods: Female mice at 4 months of age were used: MFS mgΔ lpn mice (n=5); mgΔ lpn treated with losartan for 3 months (n=5); and control wild type mice (n=5). The glomerular structure of the kidney was examined by histology. The distribution and quantification of fibrillin-1 and MMP-9 were analyzed by immunofluorescence assay. Blood flow in the aorta and renal artery was monitored with the probe 2SB-T206 of the Transonic Systems, and the cortical micro-hemodynamic of the kidney was examined by SDF video-microscopy (Sidestream Dark Field Imaging). Results: The mgΔ lpn mice showed reduction of fibrillin-1 deposition and an increase in MMP-9 in the entire kidney tissue, including the glomerular structures. In histologic aspects, the glomerulus showed a reduction of the glomerular area, urinary space, and vascular pole area. The reduced glomerulus dimension possibly squeezes the arteriolar system with a subsequent increase of their vascular resistance. In addition, the cortical microvessels showed a significant reduction in blood flow, and there was an increase in the distribution of microvessels with diameters 6 to 8 μm. Blood flow analysis in the mgΔ lpn showed decreased artery renal flow but did not reveal a difference in aorta blood flow when compared to WT animals. These results indicate vascular resistance in the renal artery. Losartan therapy increased fibrillin-1 and decreased MMP-9 quantifications, improved the glomerulus structures and significantly increased the velocity of the microcirculation blood flow. Conclusions: Alterations in fibrillin-1 modify the glomerular structures and impair kidney hemodynamics. Losartan therapy minimized the renal phenotypes.

Spatiotemporal changes in mechanical matrisome components of the human ovary from prepuberty to menopause.

Spatiotemporal changes in mechanical matrisome components of the human ovary from prepuberty to menopause.

2. Pregnancy and myositis

2. Pregnancy and myositis

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-β, 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.

Nonmyocyte ERK1/2 signaling contributes to load-induced cardiomyopathy in Marfan mice.

Among children with the most severe presentation of Marfan syndrome (MFS), an inherited disorder of connective tissue caused by a deficiency of extracellular fibrillin-1, heart failure is the leading cause of death. Here, we show that, while MFS mice (Fbn1C1039G/+ mice) typically have normal cardiac function, pressure overload (PO) induces an acute and severe dilated cardiomyopathy in association with fibrosis and myocyte enlargement. Failing MFS hearts show high expression of TGF-β ligands, with increased TGF-β signaling in both nonmyocytes and myocytes; pathologic ERK activation is restricted to the nonmyocyte compartment. Informatively, TGF-β, angiotensin II type 1 receptor (AT1R), or ERK antagonism (with neutralizing antibody, losartan, or MEK inhibitor, respectively) prevents load-induced cardiac decompensation in MFS mice, despite persistent PO. In situ analyses revealed an unanticipated axis of activation in nonmyocytes, with AT1R-dependent ERK activation driving TGF-β ligand expression that culminates in both autocrine and paracrine overdrive of TGF-β signaling. The full compensation seen in wild-type mice exposed to mild PO correlates with enhanced deposition of extracellular fibrillin-1. Taken together, these data suggest that fibrillin-1 contributes to cardiac reserve in the face of hemodynamic stress, critically implicate nonmyocytes in disease pathogenesis, and validate ERK as a therapeutic target in MFS-related cardiac decompensation.

2015 AHA Late-Breaking Basic Science Abstracts.

### 23060 ### Validation Of Circulating MicroRnas As Biomarkers In Heart Failure In Two Large Independent Cohorts Antoni Bayes-Genis1, David Lanfear2, Maurice de Ronde3, Josep Lupon1, Joost Leenders4, Elisabet Zamora1, L. Keoki Williams2, De Antonio Marta5, Zhen Liu4, Koos Zwinderman3, Sara-Joan Pinto-Sietsma3, Yigal Pinto3; 1Universitat Autonoma de Barcelona, Barcelona, Spain; 2Henry Ford Hospital, Detroit, MI; 3Academic Med Cntr, Univ of Amsterdam, Amsterdam, Netherlands; 4Mirnext BV, Amsterdam, Netherlands; 5Germans Trias i Pujol Hosp, Badalona, Spain Background: Small studies suggested circulating microRNAs (circmiRs) as biomarkers for Heart Failure (HF). However, standardized approaches and quality assessment are not established, and results have been inconsistent, with little replication between studies. We aimed to implement quality standards to enable comparison between cohorts and assess which circmiRs may add prognostic information in HF. Methods: We measured 15 circmiRs in two independent cohorts totaling >2000 subjects. Cohort I (Barcelona) comprised of n=843 chronic HFrEF patients. Cohort II from Detroit comprised n= 1384 chronic HF patients (892 HFrEF, 492 HFpEF). Each sample was measured in duplicate, and normalized to an abundant and stable circmiR (miR-486-5p). Algorithms were installed to define each circmiR measurement as “valid”, “unmeasurable” or “invalid”. This allowed inclusion of valid low-level circmiR measurements while reducing noise from false amplification signals. Results: In general, between 20–40% of measurements were “invalid”, while miR-499a\_5p and -208a were “unmeasurable” in the majority of patients in both cohorts. Higher levels of circmiRs-133b, -1254, -622, -208a and -499a\_5p were significantly associated with risk of death in both cohorts, with hazard ratios ranging from 1.103 to 1.365 per log increase (p-values 0.001 to 0.05). However, adding these circmiRs to established predictors (age, renal function and NTproBNP) did not further augment the c-stat beyond 0.71 (cohort I) or 0.78 (cohort II). …

Fibrillin-1 microfibrils influence adult bone marrow hematopoiesis

We have recently demonstrated that fibrillin-1 assemblies regulate the fate of skeletal stem cells (aka, mesenchymal stem cells [MSCs]) by modulating TGFβ activity within the microenvironment of adult bone marrow niches. Since MSCs can also influence hematopoietic stem cell (HSC) activities, here we investigated adult hematopoiesis in mice with Cre-mediated inactivation of the fibrillin-1 (Fbn1) gene in the mesenchyme of the forming limbs (Fbn1Prx1−/− mice). Analyses of 3-month-old Fbn1Prx1−/− mice revealed a statistically significant increase of circulating red blood cells, which a differentiation assay correlated with augmented erythropoiesis. This finding, together with evidence of fibrillin-1 deposition in erythroblastic niches, supported the notion that this extracellular matrix protein normally restricts differentiation of erythroid progenitors. Whereas flow cytometry measurements identified a decreased HSC frequency in mutant relative to wild type mice, no appreciable differences were noted with regard to the relative abundance and differentiation potential of myeloid progenitor cells. Together these findings implied that fibrillin-1 normally promotes HSC expansion but does not influence cell lineage commitment. Since local TGFβ hyperactivity has been associated with abnormal osteogenesis in Fbn1Prx1−/− mice, 1-month-old mutant and wild type animals were systemically treated for 8weeks with either a pan-TGF-β-neutralizing antibody or an antibody of the same IgG1 isotype. The distinct outcomes of these pharmacological interventions strongly suggest that fibrillin-1 differentially modulates TGFβ activity in HSC vs. erythroid niches.

Regeneration of elastic fibers by three-dimensional culture on a collagen scaffold and the addition of latent TGF-β binding protein 4 to improve elastic matrix deposition

The objective of this study was to investigate the effects of latent TGF-β binding protein 4 (LTBP-4) on elastic fiber regeneration in three-dimensional cultures of human dermal fibroblasts (HDFs). Appropriate collagen scaffold for elastic fiber regeneration was also examined. Collagen sponges cross-linked at 120 °C and composed of small pores (25 μm on average) was favorable for elastic fiber regeneration by HDFs. Addition of LTBP-4, followed by culture for 21 days, accelerated elastic fiber accumulation within the scaffolds. Conditioned scaffolds containing either HDFs or LTBP-4-built mature elastic fibers were implanted between the dermis and the cutaneous muscle of mice. The combined use of HDFs and LTBP-4 resulted in thicker tissues containing elastic fibers. These results indicate that weakly cross-linked collagen sponges can be used as scaffolds for regenerating elastic fibers both in vitro and in vivo, and that the addition of LTBP-4 accelerates the deposition of both elastin and fibrillin-1, and increases cell proliferation. These techniques may be useful for generating cutaneous or cardiovascular tissue equivalents; furthermore, they may serve as a useful method for the three-dimensional analyses of drugs used to treat skin diseases or to examine the microstructure of elastin networks.

Early fibrillin-1 assembly monitored through a modifiable recombinant cell approach.

Fibrillin proteins constitute the backbone of extra-cellular macromolecular microfibrils. Mutations in fibrillins cause heritable connective tissue disorders, including Marfan syndrome, dominant Weill-Marchesani syndrome, and stiff skin syndrome. Fibronectin provides a critical scaffold for microfibril assembly in cell culture models. Full length recombinant fibrillin-1 was expressed by HEK 293 cells, which deposited the secreted protein in a punctate pattern on the cell surface. Cocultured fibroblasts consistently triggered assembly of recombinant fibrillin-1, which was dependent on a fibronectin network formed by the fibroblasts. Deposition of recombinant fibrillin-1 on fibronectin fibers occurred first in discrete packages that subsequently extended along fibronectin fibers. Mutant fibrillin-1 harboring either a cysteine 204 to serine mutation or a RGD to RGA mutation which prevents integrin binding, did not affect fibrillin-1 assembly. In conclusion, we developed a modifiable recombinant full-length fibrillin-1 assembly system that allows for rapid analysis of critical roles in fibrillin assembly and functionality. This system can be used to study the contributions of specific residues, domains, or regions of fibrillin-1 to the biogenesis and functionality of microfibrils. It provides also a method to evaluate disease-causing mutations, and to produce microfibril-containing matrices for tissue engineering applications, for example, in designing novel vascular grafts or stents.

Microfibril-associated glycoprotein-1 controls human ciliary zonule development in vitro.

The ciliary zonule in the eye, also known as Zinn’s zonule, is composed of oxytalan fibers, which are bundles of microfibrils consisting mainly of fibrillin-1. However, it is still unclear which of the microfibril-associated molecules present in the ciliary zonule controls oxytalan fibers. Microfibril-associated glycoprotein-1 (MAGP-1) is the only microfibril-associated molecule identified in the human ciliary zonule. In the present study, we used siRNA against MAGP-1 in cultures of human non-pigmented ciliary epithelial cells to examine the extracellular deposition and appearance of fibrillin-1 employing Western blotting and immunofluorescence. MAGP-1 suppression led to a reduction of fibrillin-1 deposition. Immunofluorescence also confirmed that RNAi-mediated down-regulation of MAGP-1 led to suppression of fiber development. These results suggest that MAGP-1 plays a crucial role in the extracellular deposition of fibrillin-1 during formation of the human ciliary zonule.

124 PATHOPHYSIOLOGIC CHANGES IN LOXL-1 KNOCKOUT MOUSE WITH PELVIC FLOOR DYSFUNCTION INDUCE A COMPENSATORY AND ABERRANT ELASTIN REGENERATIVE RESPONSE BY VAGINAL SMOOTH MUSCLE CELLS

INTRODUCTION AND OBJECTIVES: Impaired elastic matrix remodeling during pregnancy and after parturition may be linked to pelvic organ prolapse (POP). In lysyl oxidase like-1 (LOXL1) knockout (KO) mice, POP develops after vaginal delivery and correlates with disorganized elastin clusters. The goal of this project was to investigate the alterations in new elastic matrix assembly by vaginal smooth muscle cells (SMC) in age-matched nulliparious (N), multiparous nonprolapsed (MNP) and multiparous prolapsed (MP) LOXL1 KO mice. METHODS: Primary vaginal SMC cultures from all LOXL1 KO mice expressed the SMC markers -actin, SM22, caldesmon, smoothelin and tropomyosin. Passage 1 SMC from each group were cultured for 21 days in DMEM/F12 medium with 10% fetal bovine serum, and then assayed for increase in DNA content, collagen matrix, and alkalisoluble, and insoluble matrix elastin. RT-PCR was used to assess expression of collagen, tropoelastin, fibrillin-1, and lysyl oxidase (LOX). Elastic matrix ultrastructure and fiber integrity was assessed using TEM. RESULTS: SMC from MP mice were smaller, less fusiform, and proliferated more rapidly than SMC from N mice. SMC from MNP mice were larger, more spread, and less proliferative than SMC from MP mice. Elastin, LOX, and collagen mRNA expression were reduced in SMC from MNP mice, and were upregulated in SMC from MP mice compared to N mice. Fibrillin mRNA levels were significantly higher in SMC from MNP mice, while LOX mRNA levels were lower in SMC from multiparous mice. SMC from MNP mice synthesized less elastic matrix per well & per cell than did SMC from MP or N mice. Elastin deposits were sporadic in cultures of SMC from MNP mice. A few aberrant fibers were seen in cultures of SMC from N mice. Such fibers were more numerous in cultures of SMC from MP mice. CONCLUSIONS: New elastic matrix synthesis by LOXL1 KO vaginal SMC diminishes with pregnancy and multi-parity. SMC from MP mice exhibit a more activated, proliferative, and remodeling phenotype than SMC from MNP or N mice. Elastic fiber assembly by MNP LOXL1 KO mice is not compromised by reduced fibrillin deposition, but by poor crosslinking from reduced LOX mRNA and the lack of LOXL1. Reduced elastic matrix regenerative repair due to suppression of elastin mRNA by SMC in MNP mice may lead to POP. Pathophysiologic changes in LOXL1 KO mice induce a compensatory and aberrant elastin regenerative response by vaginal SMC. Therapies to regenerate healthy matrix may have application to POP.

Systemic sclerosis sera affect fibrillin-1 deposition by dermal blood microvascular endothelial cells: therapeutic implications of cyclophosphamide

IntroductionSystemic sclerosis (SSc) is a connective tissue disorder characterized by endothelial cell injury, autoimmunity and fibrosis. The following three fibrillin-1 alterations have been reported in SSc. (1) Fibrillin-1 microfibrils are disorganized in SSc dermis. (2) Fibrillin-1 microfibrils produced by SSc fibroblasts are unstable. (3) Mutations in the FBN1 gene and anti-fibrillin-1 autoantibodies have been reported in SSc. Fibrillin-1 microfibrils, which are abundantly produced by blood and lymphatic microvascular endothelial cells (B-MVECs and Ly-MVECs, respectively), sequester in the extracellular matrix the latent form of the potent profibrotic cytokine transforming growth factor β (TGF-β). In the present study, we evaluated the effects of SSc sera on the deposition of fibrillin-1 and microfibril-associated glycoprotein 1 (MAGP-1) and the expression of focal adhesion molecules by dermal B-MVECs and Ly-MVECs.MethodsDermal B-MVECs and Ly-MVECs were challenged with sera from SSc patients who were treatment-naïve or under cyclophosphamide (CYC) treatment and with sera from healthy controls. Fibrillin-1/MAGP-1 synthesis and deposition and the expression of αvβ3 integrin/phosphorylated focal adhesion kinase and vinculin/actin were evaluated by immunofluorescence and quantified by morphometric analysis.ResultsFibrillin-1 and MAGP-1 colocalized in all experimental conditions, forming a honeycomb pattern in B-MVECs and a dense mesh of short segments in Ly-MVECs. In B-MVECs, fibrillin-1/MAGP-1 production and αvβ3 integrin expression significantly decreased upon challenge with sera from naïve SSc patients compared with healthy controls. Upon challenge of B-MVECs with sera from CYC-treated SSc patients, fibrillin-1/MAGP-1 and αvβ3 integrin levels were comparable to those of cells treated with healthy sera. Ly-MVECs challenged with SSc sera did not differ from those treated with healthy control sera in the expression of any of the molecules assayed.ConclusionsBecause of the critical role of fibrillin-1 in sequestering the latent form of TGF-β in the extracellular matrix, its decreased deposition by B-MVECs challenged with SSc sera might contribute to dermal fibrosis. In SSc, CYC treatment might limit fibrosis through the maintenance of physiologic fibrillin-1 synthesis and deposition by B-MVECs.

Systemic sclerosis sera affect fibrillin-1 deposition and focal adhesion molecule expression by dermal blood microvascular endothelial cells: Therapeutic implications of cyclophosphamide

Systemic sclerosis (SSc, scleroderma) is characterized by fibrosis of the skin and internal organs, autoantibodies and diffuse microangiopathy. In SSc, fibrillin-1 microfibrils are disorganized and unstable. Mutations in FBN1 gene and anti-fibrillin- 1 autoantibodies have also been reported in SSc patients. Fibrillin-1 microfibrils sequester in the extracellular matrix the latent form of the potent pro-fibrotic cytokine transforming growth factor-β (TGF-β). We herein report the effects of SSc sera on the deposition of fibrillin-1 and microfibril-associated glycoprotein-1 (MAGP-1) and the expression of focal adhesion molecules by dermal blood (B-MVECs) and lymphatic (Ly-MVECs) microvascular endothelial cells. Cells were challenged with sera from SSc patients, naive or under cyclophosphamide (CYC) treatment, and healthy controls. Fibrillin-1/MAGP-1 synthesis and deposition and the expression of αvβ3 integrin/ phosphorylated-FAK and vinculin/actin were evaluated by immunofluorescence and quantified by morphometric analysis. Fibrillin-1 and MAGP-1 co-localized in all experimental conditions, forming a honeycomb pattern in B-MVECs and a dense mesh of short segments in Ly-MVECs. In B-MVECs, fibrillin-1/MAGP-1 production and αvβ3 integrin expression significantly decreased upon challenge with sera from naive SSc patients compared with healthy controls. Vinculin was overexpressed in the cells exposed to the serum of naive patients with the diffuse form of SSc. B-MVECs challenged with sera from CYC-treated SSc patients showed fibrillin-1, αvβ3 integrin and vinculin levels comparable to those of controls. Ly-MVECs challenged with SSc sera did not differ from controls in the expression of any of the molecules assayed. Due to the critical role of fibrillin-1 in sequestering TGF-β in the extracellular matrix, its decreased deposition by B-MVECs challenged with SSc sera might contribute to dermal fibrosis. CYC treatment might limit fibrosis through the maintenance of physiologic fibrillin-1 synthesis and deposition by B-MVECs. CYC-induced normalization of αvβ3 integrin expression may restore effective interaction of endothelial cells with fibrillin. Since vinculin is necessary for angiogenesis, its normalization upon CYC treatment may contribute to restore the impaired angiogenesis found in SSc.

ADAMTSL4, a Secreted Glycoprotein Widely Distributed in the Eye, Binds Fibrillin-1 Microfibrils and Accelerates Microfibril Biogenesis

ADAMTSL4 mutations cause autosomal recessive isolated ectopia lentis (IEL) and ectopia lentis et pupillae. Dominant FBN1 mutations cause IEL or syndromic ectopia lentis (Marfan syndrome and Weill-Marchesani syndrome). The authors sought to characterize recombinant ADAMTSL4 and the ocular distribution of ADAMTSL4 and to investigate whether ADAMTSL4 influences the biogenesis of fibrillin-1 microfibrils, which compose the zonule. ADAMTSL4 was expressed by the transfection of HEK293F cells. Protein extracts and paraffin sections from human eyes were analyzed by Western blot analysis and by immunoperoxidase staining, respectively. Immunofluorescence was used to evaluate fibrillin-1 deposition in the ECM of fetal bovine nuchal ligament cells after culture in ADAMTSL4-conditioned medium or control medium. Confocal microscopy was performed to investigate ADAMTSL4 and fibrillin-1 colocalization in these cultures. Western blot analysis identified ADAMTSL4 as a glycoprotein in HEK293F cells and as a major band of 150 kDa in ocular tissues including ciliary body, sclera, cornea, and retina. Immunoperoxidase staining showed a broad ocular distribution of ADAMTSL4, associated with both cells and fibrillar ECM. When cultured in ADAMTSL4-containing medium, fetal bovine nuchal ligament cells showed accelerated fibrillin-1 deposition in ECM. ADAMTSL4 colocalized with fibrillin-1 microfibrils in the ECM of these cells. ADAMTSL4 is a secreted glycoprotein that is widely distributed in the human eye. Enhanced fibrillin-1 deposition in the presence of ADAMTSL4 and colocalization of ADAMTSL4 with fibrillin-1 in the ECM of cultured fibroblasts suggest a potential role for ADAMTSL4 in the formation or maintenance of the zonule.

Characterization of the Natural History of Extracellular Matrix Production in Tissue-Engineered Vascular Grafts during Neovessel Formation

Background: The extracellular matrix (ECM) is a critical determinant of neovessel integrity. Materials and Methods: Thirty-six (polyglycolic acid + polycaprolactone and poly lactic acid) tissue-engineered vascular grafts seeded with syngeneic bone marrow mononuclear cells were implanted as inferior vena cava interposition grafts in C57BL/6 mice. Specimens were characterized using immunohistochemical staining and qPCR for representative ECM components in addition to matrix metalloproteinases (MMPs). Total collagen, elastin, and glycosaminoglycan (GAG) contents were determined. MMP activity was measured using zymography. Results: Collagen production on histology demonstrated an initial increase in type III at 1 week followed by type I production at 2 weeks and type IV at 4 weeks. Gene expression of both type I and type III peaked at 2 weeks, whereas type IV continued to increase over the 4-week period. Histology demonstrated fibrillin-1 deposition at 1 week followed by elastin production at 4 weeks. Elastin gene expression significantly increased at 4 weeks, whereas fibrillin-1 decreased at 4 weeks. GAG demonstrated abundant production at each time point on histology. Gene expression of decorin significantly increased at 4 weeks, whereas versican decreased over time. Biochemical analysis showed that total collagen production was greatest at 2 weeks, and there was a significant increase in elastin and GAG production at 4 weeks. Histological characterization of MMPs showed abundant production of MMP-2 at each time point, while MMP-9 decreased over the 4-week period. Gene expression of MMP-2 significantly increased at 4 weeks, whereas MMP-9 significantly decreased at 4 weeks. Conclusions: ECM production during neovessel formation is characterized by early ECM deposition followed by extensive remodeling.

Fibrillin-1 and Fibrillin-2 Are Essential for Formation of Thick Oxytalan Fibers in Human Nonpigmented Ciliary Epithelial Cells In Vitro

The ciliary zonule, also known as Zinn’s zonule, is composed of oxytalan fibers. However, the mechanism by which epithelial cells in the ciliary body form these fibers in not fully understood. We examined human nonpigmented ciliary epithelial cells to determine the appearance and amount of oxytalan fibers in terms of positivity for their major components, fibrillin-1 and fibrillin-2. Examination of fibrillin-1 and fibrillin-2 expression by immunofluorescence revealed that thin fibers positive for fibrillin-1 on Day 2 changed to thick fibers by Day 8. The fibers positive for fibrillin-2 appeared on the thick fibrillin-1-positive fibers after Day 4. Northern blot analysis revealed that the level of fibrillin-1 did not change markedly, while induction of fibrillin-2 gene was evident on Day 5. Western blot analysis showed that fibrillin-1 deposition increased gradually, while that of fibrillin-2 increased markedly from Day 5 to Day 8. Fibrillin-1 suppression did not lead to the formation of fibrillin-2-positive thick fibers, whereas fibrillin-2 suppression led to the formation of fibrillin-1-positive thin fibers, but not thick fibers. These results suggest that both fibrillin-1 and fibrillin-2 are essential for the formation of thick oxytalan fibers in the ciliary zonule and are informative for clarifying the mechanism of homeostasis of the ocular matrix.

A novel defect in fibrillin-1 deposition by dermal fibroblasts clinically presenting as acquired cutis laxa is reversible with dexamethasone and losartan treatment

A novel defect in fibrillin-1 deposition by dermal fibroblasts clinically presenting as acquired cutis laxa is reversible with dexamethasone and losartan treatment