Microfibril-associated Glycoprotein-1 Research Articles

Fibrillin-1 regulates endothelial sprouting during angiogenesis

Fibrillin-1 is an extracellular matrix protein that assembles into microfibrils which provide critical functions in large blood vessels and other tissues. Mutations in the fibrillin-1 gene are associated with cardiovascular, ocular, and skeletal abnormalities in Marfan syndrome. Here, we reveal that fibrillin-1 is critical for angiogenesis which is compromised by a typical Marfan mutation. In the mouse retina vascularization model, fibrillin-1 is present in the extracellular matrix at the angiogenic front where it colocalizes with microfibril-associated glycoprotein-1, MAGP1. In Fbn1C1041G/+ mice, a model of Marfan syndrome, MAGP1 deposition is reduced, endothelial sprouting is decreased, and tip cell identity is impaired. Cell culture experiments confirmed that fibrillin-1 deficiency alters vascular endothelial growth factor-A/Notch and Smad signaling which regulate the acquisition of endothelial tip cell/stalk cell phenotypes, and we showed that modulation of MAGP1 expression impacts these pathways. Supplying the growing vasculature of Fbn1C1041G/+ mice with a recombinant C-terminal fragment of fibrillin-1 corrects all defects. Mass spectrometry analyses showed that the fibrillin-1 fragment alters the expression of various proteins including ADAMTS1, a tip cell metalloprotease and matrix-modifying enzyme. Our data establish that fibrillin-1 is a dynamic signaling platform in the regulation of cell specification and matrix remodeling at the angiogenic front and that mutant fibrillin-1-induced defects can be rescued pharmacologically using a C-terminal fragment of the protein. These findings, identify fibrillin-1, MAGP1, and ADAMTS1 in the regulation of endothelial sprouting, and contribute to our understanding of how angiogenesis is regulated. This knowledge may have critical implications for people with Marfan syndrome.

MAGP1 maintains tumorigenicity and angiogenesis of laryngeal cancer by activating Wnt/β-catenin/MMP7 pathway.

Microfibril-associated glycoprotein-1 (MAGP1), a crucial extracellular matrix protein, contributes to the initiation and progression of different cancers. However, the role of MAGP1 in laryngeal cancer is not clear. The purpose of this study was to investigate the clinical significance and biological function of MAGP1 in laryngeal cancer. MAGP1 was upregulated in public databases and laryngeal cancer tissues, and high MAGP1 expression led to a poor prognosis and was identified as an independent prognostic marker. Knocking-down MAGP1 inhibited laryngeal cancer cell growth and metastasis. According to gene set enrichment analysis, high MAGP1 expression revealed enrichment in Wnt/β-catenin signaling and knocking-down MAGP1 in laryngeal cancer cells also caused degradation, de-activation, re-location and loss of stability of β-catenin. Additionally, we observed MAGP1 in laryngeal cancer cells inhibits angiogenesis in an MMP7-dependent way. In conclusion, our study suggests a clinical role of MAGP1 in laryngeal cancer, signifying its potential as a therapeutic target in the future.

Identification of the growth factor–binding sequence in the extracellular matrix protein MAGP-1

Microfibril-associated glycoprotein-1 (MAGP-1) is a component of vertebrate extracellular matrix (ECM) microfibrils that, together with the fibrillins, contributes to microfibril function. Many of the phenotypes associated with MAGP-1 gene inactivation are consistent with dysregulation of the transforming growth factor β (TGFβ)/bone morphogenetic protein (BMP) signaling system. We have previously shown that full-length MAGP-1 binds active TGFβ-1 and some BMPs. The work presented here further defines the growth factor-binding domain of MAGP-1. Using recombinant domains and synthetic peptides, along with surface plasmon resonance analysis to measure the kinetics of the MAGP-1-TGFβ-1 interaction, we localized the TGFβ- and BMP-binding site in MAGP-1 to a 19-amino acid-long, highly acidic sequence near the N terminus. This domain was specific for binding active, but not latent, TGFβ-1. Growth factor activity experiments revealed that TGFβ-1 retains signaling activity when complexed with MAGP-1. Furthermore, when bound to fibrillin, MAGP-1 retained the ability to interact with TGFβ-1, and active TGFβ-1 did not bind fibrillin in the absence of MAGP-1. The absence of MAGP was sufficient to raise the amount of total TGFβ stored in the ECM of cultured cells, suggesting that the MAGPs compete with the TGFβ large latent complex for binding to microfibrils. Together, these results indicate that MAGP-1 plays an active role in TGFβ signaling in the ECM.

Overexpressed MAGP1 Is Associated With a Poor Prognosis and Promotes Cell Migration and Invasion in Gastric Cancer.

Gastric cancer (GC) is a frequently occurring malignancy with high mortality rates. However, the underlying mechanism of GC progression is not very clear. The aim of this study is to reveal the inherent molecular mechanism and develop potential therapeutic targets for advanced GC. The microfibril-associated glycoprotein 1 (MAGP1), identified as a potential oncogene, was found upregulated in GC tissues and high MAGP1 expression was associated with aggressive clinicopathological features. Furthermore, the multivariate Cox regression analysis showed that high MAGP1 expression was an independent predictor of poor prognosis (HR = 2.37, 1.07–5.24; P = 0.033). Mechanistically, MAGP1 promoted the migration and invasiveness of GC cells. In addition, the genes co-expressed with MAGP1 were primarily enriched in focal adhesion and PI3K-Akt pathways. MAGP1 overexpression enhanced the phosphorylation of FAK, AKT, and mTOR, whereas its knockdown also inactivated these factors. Furthermore, the AKT inhibitor suppressed the phosphorylation of AKT, FAK, and mTOR in recMAGP1-treated AGS cells, as well as their migration and invasion capacities. Finally, correlation analysis indicated that MAGP1 is involved in AKT signaling in GC, and is clinically relevant. Taken together, MAGP1 is a promising prognostic marker and potential therapeutic target for advanced GC.

Maladaptive remodeling of pulmonary artery root autografts after Ross procedure: A proteomic study

ObjectivePulmonary autograft root dilatation is the major long-term complication after Ross procedure and the leading cause for reoperation. However, the mechanisms underlying dilatation remain to be elucidated. This study analyzed the proteomic changes seen in the dilated pulmonary autograft compared with normal pulmonary artery and aorta tissues. MethodsPulmonary autograft surgical samples were taken from 9 consecutive patients (mean age 37 ± 14; 15-51 years) with mean diameters of 5.2 ± 0.5 cm (4.6-5.8 cm) reoperated 8 to 16 years after Ross procedure. Control pulmonary artery and aorta samples were from 7 age- and sex-matched cardiac donors. Tunicae mediae from all samples were processed for proteomic analysis via 2-dimensional electrophoresis, matrix-assisted-laser-desorption-ionization-time of flight/mass spectrometry, and bioinformatics. The thus-identified putatively relevant proteins were validated via Western immunoblotting. ResultsPulmonary autograft proteome features differed markedly from control pulmonary arteries, since proteins related to focal adhesions (eg, paxillin), cytoskeleton (eg, vimentin), and metalloprotease-regulating proteoglycans (eg, testican-2) were significantly up-regulated, whereas significant decreases occurred in microfibril-associated glycoprotein1, which controls elastic fiber buildup. Profound changes also occurred in cell-signaling proteins, ie, increases in soluble Jagged-1 fragment and ectodysplasin-2 receptor, and decreases in Notch-1 intracellular domain fragment. Moreover, pulmonary autograft expression levels of Paxillin, Vimentin, Jagged-1 fragment, and Notch1 intracellular domain fragment also differed from those of control aorta. ConclusionsThis study provides the first description of the specific proteomic features of dilated pulmonary autograft tunica media, which separate them sharply not only from those of control pulmonary artery and aorta but also of aortic aneurysms. These findings suggest that dilated pulmonary autografts undergo a unique maladaptive remodeling process deserving further investigation.

Studies on sporadic non-syndromic thoracic aortic aneurysms: II. Alterations of extra-cellular matrix components and focal adhesion proteins.

Background Sporadic non-syndromic thoracic aortic aneurysms (SNSTAAs) are less well understood than familial non-syndromic or syndromic ones. Here, we focused on morphologic and molecular changes of the extracellular matrix of the tunica media of SNSTAAs. Design Single centre design. Methods Surgical media samples from seven SNSTAAs and seven controls underwent quantitative polymerase chain reaction, proteomics-bioinformatics, immunoblotting, histology and immunohistochemistry analysis. Results A down-regulation of Decorin mRNA with unchanged protein levels associated with a remarkable increase of collagen fibres. A reduced and distorted network of elastic fibres partnered with an attenuated expression of microfibril-associated glycoprotein1 despite the rise of MFAP2 gene-encoded mRNA levels. An increasingly proteolysed paxillin (55 kDa PXN), a focal adhesion protein, combined with an upregulated 62 kDa PXN holoprotein, without changes in amount and phosphorylation of focal adhesion kinase (pp125FAK). The upregulation of SPOCK2-encoded Testican2 proteoglycan and of ectodysplasin (EDA) protein was coupled with a down-regulation of EDA2 receptor (EDA2R). Conclusions Several tunica media extracellular matrix-related changes favour SNSTAA development. A steady level of decorin and a microfibril-associated glycoprotein1 protein shortage cause the assembly of structurally defective collagen and elastic fibres. Up-regulation of PXN holoproteins perturbs PXN/pp125FAK interaction and focal adhesion functioning. Testican2 up-regulation suppresses the membrane-type matrix metalloproteinase inhibiting activities of other SPOCK family members thus enhancing extracellular matrix proteolysis. Finally, the altered EDA•EDA2R signalling would impact on the remodelling of SNSTAA tunica media. Altogether, our results pave the way to a deeper molecular understanding of SNSTAAs necessary to identify their early diagnostic biochemical markers.

Contribution of metabolic disease to bone fragility in MAGP1-deficient mice

Microfibril-associated glycoprotein-1 (MAGP1) is an extracellular matrix protein that interacts with fibrillin and is involved in regulating the bioavailability of signaling molecules such as TGFβ. Mice with germline MAGP1 deficiency (Mfap2−/−) develop increased adiposity, hyperglycemia, insulin resistance, bone marrow adipose tissue expansion, reduced cancellous bone mass, cortical bone thinning and bone fragility. The goal of this study was to assess whether the Mfap2−/− bone phenotypes were due to loss of MAGP1 locally or secondary to a change in whole body physiology (metabolic dysfunction). To do this, mice with conditional deletion of MAGP1 in the limb skeleton were generated by crossing MAGP1-flox mice (Mfap2lox/lox) with Prx1-Cre mice. Mfap2Prx−/− mice did not show any changes in peripheral adiposity, hyperglycemia or insulin sensitivity, but did have increased bone length and cancellous bone loss that was comparable to the germline Mfap2−/− knockout. Unlike the germline knockout, marrow adiposity, cortical bone thickness and bone strength in Mfap2Prx−/− mice were normal. These findings implicate systemic metabolic dysfunction in the development of bone fragility in germline Mfap2−/− mice. An unexpected finding of this study was the detection of MAGP1 protein in the Mfap2Prx−/− hematopoietic bone marrow, despite the absence of MAGP1 protein in osseous bone matrix and absent Mfap2 transcript expression at both sites. This suggests MAGP1 from a secondary site may accumulate in the bone marrow, but not be incorporated into the bone matrix, during times of regional MAGP1 depletion.

Marrow Adipose Tissue Expansion Coincides with Insulin Resistance in MAGP1-Deficient Mice.

Marrow adipose tissue (MAT) is an endocrine organ with the potential to influence skeletal remodeling and hematopoiesis. Pathologic MAT expansion has been studied in the context of severe metabolic challenge, including caloric restriction, high fat diet feeding, and leptin deficiency. However, the rapid change in peripheral fat and glucose metabolism associated with these models impedes our ability to examine which metabolic parameters precede or coincide with MAT expansion. Microfibril-associated glycoprotein-1 (MAGP1) is a matricellular protein that influences cellular processes by tethering signaling molecules to extracellular matrix structures. MAGP1-deficient (Mfap2−/−) mice display a progressive excess adiposity phenotype, which precedes insulin resistance and occurs without changes in caloric intake or ambulation. Mfap2−/− mice were, therefore, used as a model to associate parameters of metabolic disease, bone remodeling, and hematopoiesis with MAT expansion. Marrow adiposity was normal in Mfap2−/− mice until 6 months of age; however, by 10 months, marrow fat volume had increased fivefold relative to wild-type control at the same age. Increased gonadal fat pad mass and hyperglycemia were detectable in Mfap2−/− mice by 2 months, but peaked by 6 months. The development of insulin resistance coincided with MAT expansion. Longitudinal characterization of bone mass demonstrated a disconnection in MAT volume and bone volume. Specifically, Mfap2−/− mice had reduced trabecular bone volume by 2 months, but this phenotype did not progress with age or MAT expansion. Interestingly, MAT expansion in the 10-month-old Mfap2−/− mice was associated with modest alterations in basal hematopoiesis, including a shift from granulopoiesis to B lymphopoiesis. Together, these findings indicate MAT expansion is coincident with insulin resistance, but not excess peripheral adiposity or hyperglycemia in Mfap2−/− mice; and substantial MAT accumulation does not necessitate a proportional decrease in either bone mass or bone marrow cellularity.

Losartan and captopril treatment rescue normal thrombus formation in microfibril associated glycoprotein-1 (MAGP1) deficient mice

IntroductionMAGP1 is a glycoprotein present in the elastic fibers and is a part of the microfibrils components. MAGP1 interacts with von Willebrand factor and the active form of TGF-β and BMP. In mice lacking MAGP1, thrombus formation is delayed, increasing the occlusion time of carotid artery despite presenting normal blood coagulation in vitro. MAGP1-containing microfibrils may play a role in hemostasis and thrombosis. In this work, we evaluated the function of MAGP1 and its relation to TGF-β in the arterial thrombosis process. Methods and resultsWe analyzed thrombus formation time in wild type and MAGP1-deficient mice comparing Rose Bengal and Ferric Chloride induced arterial lesion. The potential participation of TGF-β in this process was accessed when we treated both wild type and MAGP1-deficient mice with losartan (an antihypertensive drug that decreases TGF-β activity) or captopril (an angiotensin converting enzyme inhibitor that was used as a control antihypertensive drug). Besides, we evaluated thrombus embolization and the gelatinolytic activity in the arterial walls in vitro and ex vivo. Losartan and captopril were able to recover the thrombus formation time without changing blood pressure, activated partial thromboplastin time (aPTT), PT (prothrombin time), platelet aggregation and adhesion, but decreased gelatinase activity. ConclusionsOur results suggest that both treatments are effective in the prevention of the sub-endothelial ECM degradation, allowing the recovery of normal thrombus formation.

Adamtsl2 deletion results in bronchial fibrillin microfibril accumulation and bronchial epithelial dysplasia--a novel mouse model providing insights into geleophysic dysplasia.

ABSTRACTMutations in the secreted glycoprotein ADAMTSL2 cause recessive geleophysic dysplasia (GD) in humans and Musladin–Lueke syndrome (MLS) in dogs. GD is a severe, often lethal, condition presenting with short stature, brachydactyly, stiff skin, joint contractures, tracheal-bronchial stenosis and cardiac valve anomalies, whereas MLS is non-lethal and characterized by short stature and severe skin fibrosis. Although most mutations in fibrillin-1 (FBN1) cause Marfan syndrome (MFS), a microfibril disorder leading to transforming growth factor-β (TGFβ) dysregulation, domain-specific FBN1 mutations result in dominant GD. ADAMTSL2 has been previously shown to bind FBN1 and latent TGFβ-binding protein-1 (LTBP1). Here, we investigated mice with targeted Adamtsl2 inactivation as a new model for GD (Adamtsl2−/− mice). An intragenic lacZ reporter in these mice showed that ADAMTSL2 was produced exclusively by bronchial smooth muscle cells during embryonic lung development. Adamtsl2−/− mice, which died at birth, had severe bronchial epithelial dysplasia with abnormal glycogen-rich inclusions in bronchial epithelium resembling the cellular anomalies described previously in GD. An increase in microfibrils in the bronchial wall was associated with increased FBN2 and microfibril-associated glycoprotein-1 (MAGP1) staining, whereas LTBP1 staining was increased in bronchial epithelium. ADAMTSL2 was shown to bind directly to FBN2 with an affinity comparable to FBN1. The observed extracellular matrix (ECM) alterations were associated with increased bronchial epithelial TGFβ signaling at 17.5 days of gestation; however, treatment with TGFβ-neutralizing antibody did not correct the epithelial dysplasia. These investigations reveal a new function of ADAMTSL2 in modulating microfibril formation, and a previously unsuspected association with FBN2. Our studies suggest that the bronchial epithelial dysplasia accompanying microfibril dysregulation in Adamtsl2−/− mice cannot be reversed by TGFβ neutralization, and thus might be mediated by other mechanisms.

Abstract 15097: Altered TGFβ Signalling Effector Molecules Contribute to Bicuspid Aortic Valve-associated Thoracic Aortopathy

Upregulated TGFβ signalling plays a key role in mediating congenital thoracic aortic aneurysms (TAA), and has been implicated in bicuspid aortic valve-associated TAA (BAV-TAA). TGFβ is normally stored within the extra-cellular matrix via sequestration proteins bound to the microfibrillar protein, fibrillin. TGFβ is released in response to changes in these sequestration proteins, allowing TGFβ to exert its signalling effects. We hypothesised that in BAV-TAA the upregulation of TGFβ signalling will pathologically alter effector molecule expression, causing matrix degradation and altered TGFβ sequestration. Immunohistochemistry was used to quantify protein expression in aortic tissue from 8 human BAV-TAA and 6 normal controls. In BAV-TAA, TGFβ expression was elevated in all layers (2.5:1; p<0.05), particularly in the adventitia (3.4:1; p<0.001). Microfibril-associated glycoprotein-1 (MAGP-1), which promotes TGFβ release, was elevated (2:1; p<0.03), while latent TGFβ binding protein-1 (LTBP-1), which promotes TGFβ sequestration, was decreased (0.3:1; p=0.03). Matrix metalloproteinase-2 (MMP-2) and MMP-9 expression was elevated (2.6:1; p<0.005, 1.5:1; p<0.05, respectively), and MMP-3 expression was decreased (0.5:1; p<0.05). Fibrillin-1 expression was not altered. The observed changes in TGFβ sequestration proteins will increase unbound TGFβ, leading to upregulation of downstream signalling. The elevated levels of MMP-2 and MMP-9 mediate pathological matrix remodelling, leading to aortic dilatation. The paradoxical decrease in MMP-3 may represent a compensatory mechanism that attempts to reduce overall pathological MMP activity, in order to limit damage to the aortic wall. In conclusion, in BAV-TAA excessive TGFβ signalling is potentiated by reduced TGFβ sequestration, causing extra-cellular matrix degradation by MMPs. This supports the rationale for the use of angiotensin II receptor blockers in BAV-TAA.

MAGP1, the extracellular matrix, and metabolism

Adipose tissue and the extracellular matrix were once considered passive players in regulating physiological processes. Now, both entities are acknowledged for their capacity to engage signal transduction pathways, and for their involvement in maintaining normal tissue homeostasis. We recently published a series of studies that identified a novel mechanism whereby an extracellular matrix molecule, MAGP1 (microfibril associated glycoprotein 1), can regulate energy metabolism in adipose tissue. MAGP1 is a component of extracellular microfibrils and plays a supportive role in maintaining thermoregulation by indirectly regulating expression of the thermogenic uncoupling proteins (UCPs). The focus of this commentary is to draw attention to the role of the extracellular matrix in regulating the bioavailability of signaling molecules, like transforming growth factor β (TGFβ), and exemplify that a better understanding of the extracellular matrix's biological properties could unveil a new source of therapeutic targets for metabolic diseases.

The Extracellular Matrix Protein MAGP1 Supports Thermogenesis and Protects Against Obesity and Diabetes Through Regulation of TGF-β

Microfibril-associated glycoprotein 1 (MAGP1) is a component of extracellular matrix microfibrils. Here we show that MAGP1 expression is significantly altered in obese humans, and inactivation of the MAGP1 gene (Mfap2−/−) in mice results in adipocyte hypertrophy and predisposition to metabolic dysfunction. Impaired thermoregulation was evident in Mfap2−/− mice prior to changes in adiposity, suggesting a causative role for MAGP1 in the increased adiposity and predisposition to diabetes. By 5 weeks of age, Mfap2−/− mice were maladaptive to cold challenge, uncoupling protein-1 expression was attenuated in the brown adipose tissue, and there was reduced browning of the subcutaneous white adipose tissue. Levels of transforming growth factor-β (TGF-β) activity were elevated in Mfap2−/− adipose tissue, and the treatment of Mfap2−/− mice with a TGF-β–neutralizing antibody improved their body temperature and prevented the increased adiposity phenotype. Together, these findings indicate that the regulation of TGF-β by MAGP1 is protective against the effects of metabolic stress, and its absence predisposes individuals to metabolic dysfunction.

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.

Investigation of age-related decline of microfibril-associated glycoprotein-1 in human skin through immunohistochemistry study

During aging, the reduction of elastic and collagen fibers in dermis can lead to skin atrophy, fragility, and aged appearance, such as increased facial wrinkling and sagging. Microfibril-associated glycoprotein-1 (MAGP-1) is an extracellular matrix protein critical for elastic fiber assembly. It integrates and stabilizes the microfibril and elastin matrix network that helps the skin to endure mechanical stretch and recoil. However, the observation of MAGP-1 during skin aging and its function in the dermis has not been established. To better understand age-related changes in the dermis, we investigated MAGP-1 during skin aging and photoaging, using a combination of in vitro and in vivo studies. Gene expression by microarray was performed using human skin biopsies from young and aged female donors. In addition, immunofluorescence analysis on the MAGP-1 protein was performed in dermal fibroblast cultures and in human skin biopsies. Specific antibodies against MAGP-1 and fibrillin-1 were used to examine protein expression and extracellular matrix structure in the dermis via biopsies from donors of multiple age groups. A reduction of the MAGP-1 gene and protein levels were observed in human skin with increasing age and photoexposure, indicating a loss of the functional MAGP-1 fiber network and a lack of structural support in the dermis. Loss of MAGP-1 around the hair follicle/pore areas was also observed, suggesting a possible correlation between MAGP-1 loss and enlarged pores in aged skin. Our findings demonstrate that a critical “pre-elasticity” component, MAGP-1, declines with aging and photoaging. Such changes may contribute to age-related loss of dermal integrity and perifollicular structural support, which may lead to skin fragility, sagging, and enlarged pores.

Development, Composition, and Structural Arrangements of the Ciliary Zonule of the Mouse

Here, we examined the development, composition, and structural organization of the ciliary zonule of the mouse. Fibrillin 1, a large glycoprotein enriched in force-bearing tissues, is a prominent constituent of the mouse zonule. In humans, mutations in the gene for fibrillin 1 (FBN1) underlie Marfan syndrome (MS), a disorder characterized by lens dislocation and other ocular symptoms. Fibrillin expression was analyzed by in situ hybridization. The organization of the zonule was visualized using antibodies to Fbn1, Fbn2, and microfibril-associated glycoprotein-1 (Magp1) in conjunction with 5-ethynyl-2'-deoxyuridine (EdU), an S-phase marker. Microfibrils, enriched in Fbn2 and Magp1, were prominent components of the temporary vascular tunic of the embryonic lens. Fbn2 expression by nonpigmented ciliary epithelial cells diminished postnatally and there was a concomitant increase in Fbn1 expression, especially in cells located in valleys between the ciliary folds. Zonular fibers projected from the posterior pars plicata to the lens in anterior, equatorial, and posterior groupings. The attachment point of the posterior zonular fibers consisted of a dense meshwork of radially oriented microfibrils that we termed the fibrillar girdle. The fibrillar girdle was located directly above the transition zone, a region of the lens epithelium in which cells commit to terminal differentiation. The development and arrangement of the murine ciliary zonule are similar to those of humans, and consequently the mouse eye may be a useful model in which to study ocular complications of MS.

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.

Oophorectomy‐induced bone loss is attenuated in MAGP1‐deficient mice

Microfibril-associated glycoprotein-1 (MAGP1), together with the fibrillins, are constitutive components of vertebrate microfibrils. Mice deficient in MAGP1 (murine MAGP1 knockout animals (Mfap2(-/-)); MAGP1Δ) is appropriate develop progressive osteopenia and reduced whole bone strength, and have elevated numbers of osteoclasts lining the bone surface. Our previous studies suggested that the increased osteoclast population was associated with elevated levels of receptor activator of NF-κB ligand (RANKL), a positive regulator of osteoclast differentiation. To explore the relationship between RANKL expression and osteoclast differentiation in MAGP1 deficiency, oophorectomy (OVX) was used to stimulate RANKL expression in both WT and MAGP1Δ animals. Bone loss following OVX was monitored using whole body DEXA and in vivo µCT. While WT mice exhibited significant bone loss following OVX, percent bone loss was reduced in MAGP1Δ mice. Further, serum RANKL levels rose significantly in OVX WT mice, whereas, there was only a modest increase in RANKL following OVX in the mutant mice due to already high baseline levels. Elevated RANKL expression was normalized when cultured MAGP1Δ osteoblasts were treated with a neutralizing antibody targeting free TGFβ. These studies provide support for increased RANKL expression associated with MAGP1 deficiency and provide a link to altered TGF-β signaling as a possible causative signaling pathway regulating RANKL expression in MAGP1Δ osteoblasts.

Fibrillin and MAGP-1 production by dermal blood and lymphatic microvascular endothelial cells challenged with systemic sclerosis sera

Systemic sclerosis (SSc) is a connective tissue disorder of unknown ethiology with two different subsets: limited (lSSc) and diffuse cutaneous systemic sclerosis (dSSc) with different patterns of organ involvement, autoantibody profiles and survival (1). The disease is characterized by three major features: vascular endothelial injury, inflammation/autoimmunity and massive deposition of collagen. Many extracellular matrix proteins are altered in SSc, including fibrillin (2). Fibrillin microfibrils form anchoring filaments in lymphatic endothelium. In the wall of arteries they constitute a scaffold for elastin deposition and sequester in the matrix TGFb and other molecules which regulate matrix formation and remodeling (3). The aim of this study was to evaluate the effect of SSc sera on fibrillin and microfibril associated glycoprotein-1 (MAGP-1) deposition by human adult dermal blood (BHMVEC) and lymphatic (LyHMVEC) microvascular endothelial cells. Cells were cultured on gelatine-coated coverslips in EBM containing 15% of serum of lSSc or dSSc patients, naive or under pharmacological therapy, and of controls and fixed at days 2 and 4 of confluence. Double immunofluorescence was performed with a monoclonal antibody to fibrillin-1 and a polyclonal antibody to MFPA2. Fibrillin and MAGP-1 deposition were quantified on 30 random photographic fields/slide. Fibrillin and MAGP-1 were produced by BHMVEC and LyHMVEC in all experimental conditions. They co-localized forming a honeycomb pattern in BHMVEC, as previously described (4), and a dense mesh of short segments in LyHMVEC. Comparative evaluation between LyHMVEC and BHMVEC indicated that, at day 4, LyHMVEC produced more fibrillin than BHMVEC in all experimental conditions. In BHMVEC, Fibrillin and MAGP-1 production significantly increased with time under challenge with naive lSSc sera, and with dSSc sera irrespective of therapy, as compared with control sera. In LyHMVEC, their production significantly increased with time after challenge with both SSc and control sera, without any significant difference. Overall, LyHMVEC produced more but less organized fibrillin and MAGP-1 and were less influenced by SSc sera than BHMVEC. 1) Varga J (2008) Bulletin of the NYU hospital for Joint Diseases 66:198. 2) Wallis DD et al. (2001) Arthritis & Rheumatism 44:1855. 3) Ramirez F, Sakai LY (2010) Cell Tissue Res 339:71. 4) Rossi A et al. (2010) J Anat 217:705.