Microfibril-associated Glycoprotein Research Articles

Alternative splicing of MAGP2 generates a non‐glycosylated variant still capable of localizing to extracellular microfibrils

Alterations in microfibril‐associated glycoprotein 2 (MAGP2) have been linked to familial thoracic aortic aneurysm and dissection (FTAAD) and ovarian cancer, suggesting that this protein has an interesting role in normal adult homeostasis. Here, a previously unstudied splice variant of MAGP2 noted in a public database search was characterized. Two mRNA isoforms of MAGP2 were expressed in a human ovarian cancer cell line (CAOV3) by RT‐PCR; sequencing revealed that the longer mRNA was the canonical MAGP2 mRNA that encodes a 173‐amino acid protein (MAGP2‐a), and the shorter variant encoded a 163‐amino acid protein (MAGP2‐b), due to the removal of the seventh exon. Interestingly, the seventh exon contains the conserved asparagine residue (Asp79) thought to be the major site of N‐glycosylation on MAGP2. As expected, MAGP2‐b was both poorly glycosylated and poorly secreted when overexpressed in either 293T cells or a mouse ovarian epithelial cell line, T3, as measured by immunoblotting analysis; this result is consistent with the canonical role of glycosylation modifications of secreted proteins. Since a small fraction of MAGP2‐b protein was secreted, studies were performed to understand what functions MAGP2‐b was capable of, if secreted. To determine if MAGP2‐b could bind and localize to extracellular microfibrils as previously reported for MAGP2‐a, immunofluorescence studies were conducted in T3 cells that generate a striking fibrillin‐2‐rich microfibril matrix in cell culture. Surprisingly, MAGP2‐b could easily be found localized to microfibrils, at levels similar to the MAGP2‐a isoform that is glycosylated and more effectively secreted than MAGP2‐b. To more clearly delineate the functional consequences of glycosylation versus the loss of ten amino acids via alternative splicing, an Asp79Gln (N79Q) mutation was generated in MAGP2‐a to block N‐glycosylation. The N79Q MAGP2‐a protein does not appear to be glycosylated, since the molecular weight of the protein on immunoblots is unchanged by PNGase F treatment. The secretion and microfibril binding properties of the N79Q mutant are very similar to MAGP2‐b; they are both poorly secreted but still able to bind to microfibrils at levels equal to or greater than the fully glycosylated MAGP2‐a variant. Together, these data indicate that glycosylation of MAGP2 is not required for microfibril binding; it also suggests that the non‐glycosylated MAGP2‐b may preferentially bind to microfibrils after cellular export. Why might it be useful for such a MAGP2 variant to exist? In this regard, there is some correlative evidence to suggest that MAGP2 binding of active TGF‐β may negatively regulate TGF‐β activity, based on studies on MAGP2 biochemistry and FTAAD‐associated mutations of MAGP2. Although speculative, the existence of a mechanism that tethers MAGP2 to microfibrils at the expense of free, soluble MAGP2 may make MAGP2 a more effective microfibril tether for active TGF‐β.Support or Funding InformationCSUF Department of Biological Science. Student research assistants funded by CIRM Bridges to Stem Cell Research (Andrew Yale, Cherie Petersen) and HHMI Undergraduate Research Program (Ryan Daniels).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Serum proteome profiling in canine idiopathic dilated cardiomyopathy using TMT-based quantitative proteomics approach

Idiopathic dilated cardiomyopathy (iDCM) is a primary myocardial disorder with an unknown aetiology, characterized by reduced contractility and ventricular dilation of the left or both ventricles. Naturally occurring canine iDCM was used herein to identify serum proteomic signature of the disease compared to the healthy state, providing an insight into underlying mechanisms and revealing proteins with biomarker potential. To achieve this, we used high-throughput label-based quantitative LC-MS/MS proteomics approach and bioinformatics analysis of the in silico inferred interactome protein network created from the initial list of differential proteins. To complement the proteomic analysis, serum biochemical parameters and levels of know biomarkers of cardiac function were measured. Several proteins with biomarker potential were identified, such as inter-alpha-trypsin inhibitor heavy chain H4, microfibril-associated glycoprotein 4 and apolipoprotein A-IV, which were validated using an independent method (Western blotting) and showed high specificity and sensitivity according to the receiver operating characteristic curve analysis. Bioinformatics analysis revealed involvement of different pathways in iDCM, such as complement cascade activation, lipoprotein particles dynamics, elastic fibre formation, GPCR signalling and respiratory electron transport chain. SignificanceIdiopathic dilated cardiomyopathy is a severe primary myocardial disease of unknown cause, affecting both humans and dogs. This study is a contribution to the canine heart disease research by means of proteomic and bioinformatic state of the art analyses, following similar approach in human iDCM research. Importantly, we used serum as non-invasive and easily accessible biological source of information and contributed to the scarce data on biofluid proteome research on this topic. Bioinformatics analysis revealed biological pathways modulated in canine iDCM with potential of further targeted research. Also, several proteins with biomarker potential have been identified and successfully validated.

Proteomics analysis of pleomorphic adenoma of the human parotid gland.

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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 TGFβ signaling due to an inability to sequester latent or active forms of TGFβ, respectively. Mouse models of excess TGFβ 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 TGFβ 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 TGFβ 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.

Analysis of disease-associated protein expression using quantitative proteomics—fibulin-5 is expressed in association with hepatic fibrosis.

Hepatic fibrosis and cirrhosis are major health problems worldwide. Until now, highly invasive biopsy remains the diagnostic gold standard despite many disadvantages. To develop noninvasive diagnostic assays for the assessment of liver fibrosis, it is urgently necessary to identify molecules that are robustly expressed in association with the disease. We analyzed biopsied tissue samples from 95 patients with HBV/HCV-associated hepatic fibrosis using three different quantification methods. We performed a label-free proteomics discovery study to identify novel disease-associated proteins using a subset of the cohort (n = 27). Subsequently, gene expression data from all available clinical samples were analyzed (n = 77). Finally, we performed a targeted proteomics approach, multiple reaction monitoring (MRM), to verify the disease-associated expression in samples independent from the discovery approach (n = 68). We identified fibulin-5 (FBLN5) as a novel protein expressed in relation to hepatic fibrosis. Furthermore, we confirmed the altered expression of microfibril-associated glycoprotein 4 (MFAP4), lumican (LUM), and collagen alpha-1(XIV) chain (COL14A1) in association to hepatic fibrosis. To our knowledge, no tissue-based quantitative proteomics study for hepatic fibrosis has been performed using a cohort of comparable size. By this means, we add substantial evidence for the disease-related expression of the proteins examined in this study.

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.

MFAP3L activation promotes colorectal cancer cell invasion and metastasis

An abundance of microfibril-associated glycoprotein 3-like (MFAP3L) significantly correlates with distant metastasis in colorectal cancer (CRC), although the mechanism has yet to be explained. In this study, we observed that MFAP3L knock-down resulted in reduced CRC cell invasion and hepatic metastasis. We evaluated the cellular location and biochemical functions of MFAP3L and found that this protein was primarily localized in the nucleus of CRC cells and acted as a protein kinase. When EGFR translocated into the nucleus upon stimulation with EGF, MFAP3L was phosphorylated at Tyr287 within its SH2 motif, and the activated form of MFAP3L phosphorylated ERK2 at Thr185 and Tyr187. Moreover, the metastatic behavior of CRC cells in vitro and in vivo could be partially explained by activation of the nuclear ERK pathway through MFAP3L phosphorylation. Hence, we experimentally demonstrated for the first time that MFAP3L likely participates in the nuclear signaling of EGFR and ERK2 and acts as a novel nuclear kinase that impacts CRC metastasis.

Extracellular Matrix Controls Notch Signaling via Integrin Activation and NICD Ubiquitination. (LB67)

The extracellular Matrix (ECM) has recently been implicated with the regulation of angiogenesis via manipulation of the Notch signaling system however the molecular basis for this has not been explored. We previously showed that the ECM protein microfibril‐associated glycoprotein 2 (MAGP2), suppresses Notch signaling in endothelial cells but promotes Notch signaling in non‐endothelial cells. The goal of this work was to dissect the molecular mechanism by which MAGP2 manipulates Notch signaling. We found that control of Notch by MAGP2 was not dependent on specific Notch receptor‐ligand expression patterns. Interestingly however, we found that mutation of the MAGP2 RGD to a non‐integrin binding RGE caused MAGP2 to promote rather than suppress Notch signaling in endothelial cells. Similarly, soluble integrin blocking RGD peptides and αVβ3 blocking antibodies also increased Notch signaling even in the absence of MAGP2. Further mechanistic dissection revealed that inhibitors of SRC and FAK kinases but not other kinases also increased Notch signaling. Interestingly, in addition to intensifying Notch signaling, RGD peptides and SRC inhibitors also triggered an increase in higher mobility NICD western blot bands and extended NICD half‐life. Our results suggest that these high mobility NICD bands are non‐ubiquitinated forms of NICD fragments. Collectively, we believe these results have revealed a previously undiscovered interaction between integrins and the Notch signaling system that acts to limit Notch signaling via ubiquitination and degradation of active NICD fragments. This novel mechanism may be responsible for ECM control of Notch signaling and angiogenesis.Grant Funding Source: Supported by NIH (NIGMS)

Domain 36 of Tropoelastin in Elastic Fiber Formation

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

Microfibril-associated Glycoprotein 2 (MAGP2) Loss of Function Has Pleiotropic Effects in Vivo

Microfibril-associated glycoprotein (MAGP) 1 and 2 are evolutionarily related but structurally divergent proteins that are components of microfibrils of the extracellular matrix. Using mice with a targeted inactivation of Mfap5, the gene for MAGP2 protein, we demonstrate that MAGPs have shared as well as unique functions in vivo. Mfap5(-/-) mice appear grossly normal, are fertile, and have no reduction in life span. Cardiopulmonary development is typical. The animals are normotensive and have vascular compliance comparable with age-matched wild-type mice, which is indicative of normal, functional elastic fibers. Loss of MAGP2 alone does not significantly alter bone mass or architecture, and loss of MAGP2 in tandem with loss of MAGP1 does not exacerbate MAGP1-dependent osteopenia. MAGP2-deficient mice are neutropenic, which contrasts with monocytopenia described in MAGP1-deficient animals. This suggests that MAGP1 and MAGP2 have discrete functions in hematopoiesis. In the cardiovascular system, MAGP1;MAGP2 double knockout mice (Mfap2(-/-);Mfap5(-/-)) show age-dependent aortic dilation. These findings indicate that MAGPs have shared primary functions in maintaining large vessel integrity. In solid phase binding assays, MAGP2 binds active TGFβ1, TGFβ2, and BMP2. Together, these data demonstrate that loss of MAGP2 expression in vivo has pleiotropic effects potentially related to the ability of MAGP2 to regulate growth factors or participate in cell signaling.

Abstract 3013: Elucidating the functional role of microfibril-associated glycoprotein-2 (MAGP-2) in human ovarian cancer.

Abstract Epithelial ovarian cancer, is the most fatal gynecologic cancer, resulting in approximately 15,000 deaths annually in the US. Our understanding of molecular events relating to the patient survival and overall prognosis is not clear. Using transcriptional profiling technology combined with Cox regression analysis in microdissected serous ovarian cancers, we have identified microfibril-associated glycoprotein 2 (MAGP-2) as a strong prognostic factor toward poor patient outcome. Ectopic overexpression of MAGP-2 was employed to evaluate its functional role in ovarian cancer. MAGP-2 overexpression increased cell migration and resistance to cisplatin in vitro. Moreover, the conditioned medium from MAGP-2 overexpression cells promoted endothelial cell motility. In an orthotopic model using luciferase labeled ovarian cancer cells, MAGP-2 overexpression increased the intraperitoneal tumor growth. In addition, the tumor with high MAGP-2 expression showed increased microvessel density. Our results suggest MAGP2 positively modulates the ovarian cancer tumorigenecity and angiogenesis, which may subsequently lead to poor patient survival. Citation Format: Young-Jeong Na, Vinod Vathipadiekal, Wei Wei, Tim Kwok, Michael Birrer. Elucidating the functional role of microfibril-associated glycoprotein-2 (MAGP-2) in human ovarian cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3013. doi:10.1158/1538-7445.AM2013-3013

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.

Identification of a functional proprotein convertase cleavage site in microfibril-associated glycoprotein 2

Microfibril-associated glycoprotein 2 (MAGP2) is a secreted protein associated with multiple cellular activities including the organization of elastic fibers in the extracellular matrix (ECM), angiogenesis, as well as regulating Notch and integrin signaling. Importantly, increases in MAGP2 positively correlate with poor prognosis for some ovarian cancers. It has been assumed that full-length MAGP2 is responsible for all reported effects; however, here we show MAGP2 is a substrate for the proprotein convertase (PC) family of endoproteases. Proteolytic processing of MAGP2 by PC cleavage could serve to regulate secretion and thus, activity and function as reported for other extracellular and cell-surface proteins. In support of this idea, MAGP2 contains an evolutionarily conserved PC consensus cleavage site, and amino acid sequencing of a newly identified MAGP2 C-terminal cleavage product confirmed functional PC cleavage. Additionally, mutagenesis of the MAGP2 PC consensus cleavage site or treatment with PC inhibitors prevented MAGP2 proteolytic processing. Finally, both cleaved and uncleaved MAGP2 were detected extracellularly and MAGP2 secretion appeared independent of PC cleavage, suggesting that PC processing occurs mainly outside the cell. Our characterization of alternative forms of MAGP2 present in the extracellular space not only enhances diversity of this ECM protein but also provides a previously unrecognized molecular mechanism for regulation of MAGP2 biological activity.

Expression of cytoskeleton and energetic metabolism-related proteins at human abdominal aortic aneurysm sites

The purpose of this study was to evaluate the expression of proteins related to cytoskeleton and energetic metabolism at abdominal aortic aneurysm (AAA) sites using proteomics. Several remodeling-related mechanisms have been associated with AAA formation but less is known about the expression of proteins associated with cytoskeleton and energetic metabolism in AAAs. AAA samples (6.73 ± 0.40 cm size) were obtained from 13 patients during elective aneurysm repair. Control abdominal aortic samples were obtained from 12 organ donors. Proteins were analyzed using two-dimensional electrophoresis and mass spectrometry. The expression of filamin was increased in the AAA site compared to control abdominal aortic samples while microfibril-associated glycoprotein-4 isotype 1, annexin A5 isotype 1, and annexin A2 were reduced compared with control abdominal aortic samples. Reduction in expression level of energetic metabolism-associated proteins such as triosephosphate isomerase, glyceraldehyde 3-phosphate dehydrogenase, and cytosolic aldehyde dehydrogenase was also observed in AAAs compared to controls. Reduction of triosephosphate isomerase expression was also observed by Western blot, which was accompanied by diminished triosephosphate isomerase activity. At the AAA site, pyruvate dehydrogenase expression was reduced and the content of both lactate and pyruvate was increased with respect to controls without changes in lactate dehydrogenase activity. The present results suggest that an anaerobic metabolic state may be favored further to reduce the expression of cytoskeleton-related proteins. The better knowledge of molecular mechanism involved in AAAs may favor development of new clinical strategies.

A thymus-specific noncoding RNA, Thy-ncR1, is a cytoplasmic riboregulator of MFAP4 mRNA in immature T-cell lines

BackgroundPostgenomic transcriptome analyses have identified large numbers of noncoding (nc)RNAs in mammalian cells. However, the biological function of long ncRNAs in mammalian cells remains largely unknown. Our recent expression profiling of selected human long ncRNAs revealed that a majority were expressed in an organ-specific manner, suggesting their function was linked to specific physiological phenomena in each organ. We investigated the characteristics and function of ncRNAs that were specifically expressed in the thymus, the site of T-cell selection and maturation.ResultsExpression profiling of 10 thymus-specific ncRNAs in 17 T-cell leukemia cell lines derived from various stages of T-cell maturation revealed that HIT14168 ncRNA, named Thy-ncR1, was specifically expressed in cell lines derived from stage III immature T cells in which the neighbouring CD1 gene cluster is also specifically activated. The Thy-ncR1 precursor exhibited complex alternative splicing patterns and differential usage of the 5' terminus leading to the production of an estimated 24 isoforms, which were predominantly located in the cytoplasm. Selective RNAi knockdown of each Thy-ncR1 isoform demonstrated that microfibril-associated glycoprotein 4 (MFAP4) mRNA was negatively regulated by two major Thy-ncR1 isoforms. Intriguingly, the MFAP4 mRNA level was controlled by a hUPF1-dependent mRNA degradation pathway in the cytoplasm distinct from nonsense-mediated decay.ConclusionsThis study identified Thy-ncR1 ncRNA to be specifically expressed in stage III immature T cells in which the neighbouring CD1 gene cluster was activated. Complex alternative splicing produces multiple Thy-ncR1 isoforms. Two major Thy-ncR1 isoforms are cytoplasmic riboregulators that suppress the expression of MFAP4 mRNA, which is degraded by an uncharacterized hUPF1-dependent pathway.

Human microvascular lymphatic and blood endothelial cells produce fibrillin: deposition patterns and quantitative analysis

Fibrillin microfibrils constitute a scaffold for elastin deposition in the wall of arteries and form the anchoring filaments that connect the lymphatic endothelium to surrounding elastic fibers. We previously reported that fibrillin is deposited in a honeycomb pattern in bovine arterial endothelial cells, which also deposit microfibril-associated glycoprotein (MAGP)-1, whereas thoracic duct endothelial cells form an irregular web. The present immunohistochemical study was designed to verify whether lymphatic and blood human dermal microvascular endothelial cells (HDMECs) isolated from human foreskin by the sequential use of a pan-endothelial marker, CD31, and the lymphatic specific marker, D2-40, deposit fibrillin and MAGP-1. In both cell types, fibrillin and MAGP-1 co-localized and were deposited with different patterns of increasing complexity co-existing in the same culture. Fibrillin microfibrils formed a wide-mesh honeycomb leaving fibrillin-free spaces that were gradually filled. This modality of fibrillin deposition, similar to that of bovine large artery endothelial cells, was basically the same in blood and lymphatic HDMECs. In some lymphatic HDMECs, fibrillin was initially deposited as uniformly scattered short fibrillin strands probably as a result of anchoring filaments carried over from the vessels of origin. Our findings show that blood and lymphatic endothelial cells participate in fibrillin deposition in human skin.

A family of cell-adhering peptides homologous to fibrinogen C-termini

A family of cell-adhesive peptides homologous to sequences on different chains of fibrinogen was investigated. These homologous peptides, termed Haptides, include the peptides Cβ, preCγ, and CαE, corresponding to sequences on the C-termini of fibrinogen chains β, γ, and αE, respectively. Haptides do not affect cell survival and rate of proliferation of the normal cell types tested. The use of new sensitive assays of cell adhesion clearly demonstrated the ability of Haptides, bound to inert matrices, to mediate attachment of different matrix-dependent cell types including normal fibroblasts, endothelial, and smooth muscle cells. Here we present new active Haptides bearing homologous sequences derived from the C-termini of other proteins, such as angiopoietin 1&2, tenascins C&X, and microfibril-associated glycoprotein-4. The cell adhesion properties of all the Haptides were found to be associated mainly with their 11 N-terminal residues. Mutated preCγ peptides revealed that positively charged residues account for their attachment effect. These results suggest a mechanism of direct electrostatic interaction of Haptides with the cell membrane. The extended Haptides family may be applied in modulating adhesion of cells to scaffolds for tissue regeneration and for enhancement of nanoparticulate transfection into cells.

Assembly of fibrillin microfibrils governs extracellular deposition of latent TGFβ

Control of the bioavailability of the growth factor TGFbeta is essential for tissue formation and homeostasis, yet precisely how latent TGFbeta is incorporated into the extracellular matrix is unknown. Here, we show that deposition of a large latent TGFbeta complex (LLC), which contains latent TGFbeta-binding protein 1 (LTBP-1), is directly dependent on the pericellular assembly of fibrillin microfibrils, which interact with fibronectin during higher-order fibrillogenesis. LTBP-1 formed pericellular arrays that colocalized with microfibrils, whereas fibrillin knockdown inhibited fibrillar LTBP-1 and/or LLC deposition. Blocking alpha5beta1 integrin or supplementing cultures with heparin, which both inhibited microfibril assembly, disrupted LTBP-1 deposition and enhanced Smad2 phosphorylation. Full-length LTBP-1 bound only weakly to N-terminal pro-fibrillin-1, but this association was strongly enhanced by heparin. The microfibril-associated glycoprotein MAGP-1 (MFAP-2) inhibited LTBP-1 binding to fibrillin-1 and stimulated Smad2 phosphorylation. By contrast, fibulin-4, which interacted strongly with full-length LTBP-1, did not induce Smad2 phosphorylation. Thus, LTBP-1 and/or LLC deposition is dependent on pericellular microfibril assembly and is governed by complex interactions between LTBP-1, heparan sulfate, fibrillin-1 and microfibril-associated molecules. In this way, microfibrils control TGFbeta bioavailability.

Identification of the Chondrocyte Lineage Using Microfibril-Associated Glycoprotein-2, A Novel Marker That Distinguishes Chondrocytes from Synovial Cells

Methods for the lineage identification of cell or tissue-engineered therapeutics must provide a high degree of performance to confidently distinguish the intended cell type from other lineages that could be present in the finished product. For many applications, these methods also require rapid, high-throughput capability. In this work, methods for the identification of autologous cultured chondrocytes for implantation were investigated. A histological analysis confirmed that fibrous tissue occasionally present in biopsies procured for autologous chondrocyte implantation production comprised synovium. Chondrocyte and synovial cell cultures were then examined using a full transcriptome microarray analysis, which revealed cartilage link protein and microfibril-associated glycoprotein-2 (MAGP2) as the most differentially expressed transcripts between the culture types. Performance characteristics of gene expression assays formed by the analysis of cartilage link protein with normalization to either standard reference genes or to MAGP2 were evaluated. The results demonstrate that the MAGP2-based assay provided superior performance for the purpose of cell culture identification compared to assays using standard reference genes. The selectivity against synovial and heterogeneous samples provided by the novel assay suggests it as an appropriate lineage identification method for cell cultures derived from cartilage.

The elastic fiber protein MAGP‐2 is ectopically expressed in ovarian carcinoma cells

Microfibril‐associated Glycoprotein‐2 (MAGP‐2) is a secreted protein component of elastic fiber microfibril sheaths. Gene expression profiling studies have linked elevated MAGP‐2 expression to advanced stage papillary serous ovarian tumors. These tumors primarily consist of epithelial‐derived tumor cells, but stromal cells are also present, and include endothelial cells from the tumor neovasculature. Since whole tissue assays do not indicate which cells in the tumor expressed increased levels of MAGP‐2, we assayed MAGP‐2 expression by quantitative Reverse Transcriptase PCR (qRT‐PCR) in three ovarian epithelial carcinoma cell lines and normal human ovarian surface epithelial cells. Interestingly, the data revealed that two of the three carcinoma cell lines did express significantly higher levels of MAGP‐2 than the normal cells, suggesting that ectopic expression of MAGP‐2 in the tumor cells themselves may have a role in cancer progression. Our data agree with recently published work on a non‐overlapping set of ovarian epithelial carcinoma cell lines by Mok et al. (Cancer Cell 16:521 2009) showing elevated MAGP‐2 protein levels in approximately 30% of carcinoma cell lines. Studies from our lab and others indicate that ectopic MAGP‐2 can modulate signaling by Notch or integrins, and can induce angiogenesis, suggesting that MAGP‐2 may play multiple roles in ovarian cancer progression. (CSUPERB, MARC)