Induction of fibulin-5 gene is regulated by tropoelastin gene, and correlated with tropoelastin accumulation in vitro

Fibulin-5 (also known as DANCE) is an elastin-binding protein that is thought to play a role in elastogenesis. We examined the relationship between the gene expression of fibulin-5 and the gene expression and accumulation of tropoelastin by comparing elastin-producing cells (human gingival fibroblasts) with non-elastin-producing cells (human periodontal ligament fibroblasts) by Northern blot analysis. Fibulin-5 gene induction was found only in elastin-producing cells. Induction of the fibulin-5 gene in elastin-producing cells occurred after induction of the tropoelastin gene, and the fibulin-5 level was reduced upon RNA interference-mediated down-regulation of tropoelastin. Fibulin-5 gene induction was also correlated with a rapid increase of tropoelastin accumulation within the cell layer. These results may suggest that the fibulin-5 gene induction is directly or indirectly regulated by tropoelastin gene expression and plays a role in the accumulation of elastic fibers within matrices.     

A novel intracellular fibulin-1D variant binds to the cytoplasmic domain of integrin beta 1 subunit

Fibulin-1 is a member of a growing family of proteins that includes eight members and is involved in cellular functions such as adhesion, migration and differentiation. Fibulin-1 has also been implicated in embryonic development of the heart and neural crest-derived structures. It is an integral part of the extracellular matrix (ECM) and has been shown to bind to a multitude of ECM proteins. However, fibulin-1 was first identified as a protein purified from placental extracts that binds to the cytoplasmic domain of integrin β1. Human fibulin-1 is alternatively spliced into four different isoforms namely A–D. These isoforms share a common N-terminus sequence that contains a secretion sequence but differ in their carboxy-terminal fibulin-1 module. In this report we identify a new splice variant of fibulin-1 that differs from all other fibulin-1 variants in the N-terminus sequence and has a similar carboxy-terminus sequence as fibulin-1D. This variant that we named fibulin-1D prime (fibulin-1D′) lacks a secretion sequence and the anaphlatoxin region of fibulin-1 variants. The protein has an apparent molecular weight of 70.5 kDa. Herein we show that fibulin-1D′ binds to the intracellular domain of integrin β1 as well as to integrin α5β1. The protein was localized intracellularly in CHO cells transfected with a pEF4 plasmid containing full-length coding sequence of fibulin-1D′. We also localized the protein in human placenta. We propose that the fibulin-1D′ variant might play a role in early embryo development as well as in modulating integrin β1 functions including adhesion and motility.     

Effects of fibulin-5 on attachment, adhesion, and proliferation of primary human endothelial cells

BACKGROUND: Fibulin-5 is a novel extracellular protein that is thought to act as a bridging peptide between elastin fibers and cell surface integrins in blood vessel wall. Fibulin-5 binding to endothelial cell (EC) surface integrins may effect cell proliferation and cell attachment to extracellular matrix (ECM) or to artificial surfaces. In this paper, we describe the effects of fibulin-5 on attachment, adhesion, and proliferation of primary human EC. After demonstrating that fibulin-5 over-expression inhibited EC proliferation, we tested the hypothesis that co-expression of fibulin-5 and VEGF165 will lead to unique EC phenotype that will exhibit increased adherence properties and retain its proliferation capacity. METHODS AND RESULTS: Fibulin-5 and VEGF165 gene transfer to primary human saphenous vein endothelial cells was accomplished using retroviral vectors encoding the two genes. Transgene expression was verified using immunohistochemistry, Western blotting, and ELISA. Fibulin 5 over-expression tended to improve immediate EC attachment (30 min after seeding) and improved significantly adhesion (>40%) under shear stress tested 24h after EC seeding. The effects of fibulin-5 and VEGF165 on EC proliferation in the presence or absence of basic FGF were also tested. EC expressing fibulin-5 had reduced proliferation while VEGF165 co-expression ameliorated this effect. CONCLUSION: Fibulin-5 improved EC attachment to artificial surfaces. Dual transfer of fibulin-5 and VEGF165 resulted in EC phenotype with increased adhesion and improved proliferation. This unique EC phenotype can be useful for tissue engineering on endovascular prostheses.     

Fibulin-5 binds human smooth-muscle cells through alpha5beta1 and alpha4beta1 integrins, but does not support receptor activation

Fibulin-5, an extracellular matrix glycoprotein expressed in elastin-rich tissues, regulates vascular cell behaviour and elastic fibre deposition. Recombinant full-length human fibulin-5 supported primary human aortic SMC (smooth-muscle cell) attachment through alpha5beta1 and alpha4beta1 integrins. Cells on fibulin-5 spread poorly and displayed prominent membrane ruffles but no stress fibres or focal adhesions, unlike cells on fibronectin that also binds these integrins. Cell migration and proliferation were significantly lower on fibulin-5 than on fibronectin. Treatment of cells on fibulin-5 with a beta1 integrin-activating antibody induced stress fibres, increased attachment, migration and proliferation, and stimulated signalling of epidermal growth factor receptor and platelet-derived growth factor receptors alpha and beta. Fibulin-5 also modulated fibronectin-mediated cell spreading and morphology. We have thus identified the beta1 integrins on primary SMCs that fibulin-5 interacts with, and have shown that failure of fibulin-5 to activate these receptors limits cell spreading, migration and proliferation.     

Fibulin-5, an integrin-binding matricellular protein: its function in development and disease

Interactions between the extracellular matrix (ECM) and cells are critical in embryonic development, tissue homeostasis, physiological remodeling, and tumorigenesis. Matricellular proteins, a group of ECM components, mediate cell-ECM interactions. One such molecule, Fibulin-5 is a 66-kDa glycoprotein secreted by various cell types, including vascular smooth muscle cells (SMCs), fibroblasts, and endothelial cells. Fibulin-5 contributes to the formation of elastic fibers by binding to structural components including tropoelastin and fibrillin-1, and to cross-linking enzymes, aiding elastic fiber assembly. Mice deficient in the fibulin-5 gene (Fbln5) exhibit systemic elastic fiber defects with manifestations of loose skin, tortuous aorta, emphysematous lung and genital prolapse. Although Fbln5 expression is down-regulated after birth, following the completion of elastic fiber formation, expression is reactivated upon tissue injury, affecting diverse cellular functions independent of its elastogenic function. Fibulin-5 contains an evolutionally conserved arginine-glycine-aspartic acid (RGD) motif in the N-terminal region, which mediates binding to a subset of integrins, including α5β1, αvβ3, and αvβ5. Fibulin-5 enhances substrate attachment of endothelial cells, while inhibiting migration and proliferation in a cell type- and context-dependent manner. The antagonistic function of fibulin-5 in angiogenesis has been demonstrated in vitro and in vivo; fibulin-5 may block angiogenesis by inducing the anti-angiogenic molecule thrompospondin-1, by antagonizing VEGF₁₆₅-mediated signaling, and/or by antagonizing fibronectin-mediated signaling through directly binding and blocking the α5β1 fibronectin receptor. The overall effect of fibulin-5 on tumor growth depends on the balance between the inhibitory property of fibulin-5 on angiogenesis and the direct effect of fibulin-5 on proliferation and migration of tumor cells. However, the effect of tumor-derived versus host microenvironment-derived fibulin-5 remains to be evaluated.     

The Importance of GLUT3 for De Novo Lipogenesis in Hypoxia-Induced Lipid Loading of Human Macrophages

Atherosclerotic lesions are characterized by lipid-loaded macrophages (foam cells) and hypoxic regions. Although it is well established that foam cells are produced by uptake of cholesterol from oxidized LDL, we previously showed that hypoxia also promotes foam cell formation even in the absence of exogenous lipids. The hypoxia-induced lipid accumulation results from increased triglyceride biosynthesis but the exact mechanism is unknown. Our aim was to investigate the importance of glucose in promoting hypoxia-induced de novo lipid synthesis in human macrophages. In the absence of exogenous lipids, extracellular glucose promoted the accumulation of Oil Red O-stained lipid droplets in human monocyte-derived macrophages in a concentration-dependent manner. Lipid droplet accumulation was higher in macrophages exposed to hypoxia at all assessed concentrations of glucose. Importantly, triglyceride synthesis from glucose was increased in hypoxic macrophages. GLUT3 was highly expressed in macrophage-rich and hypoxic regions of human carotid atherosclerotic plaques and in macrophages isolated from these plaques. In human monocyte-derived macrophages, hypoxia increased expression of both GLUT3 mRNA and protein, and knockdown of GLUT3 with siRNA significantly reduced both glucose uptake and lipid droplet accumulation. In conclusion, we have shown that hypoxia-induced increases in glucose uptake through GLUT3 are important for lipid synthesis in macrophages, and may contribute to foam cell formation in hypoxic regions of atherosclerotic lesions.     

Coordinate expression of fibulin-5/DANCE and elastin during lung injury repair

Fibulin-5, previously known as DANCE and EVEC, is a secreted extracellular matrix protein that functions as a scaffold for elastin fiber assembly and as a ligand for integrins alphavbeta3, alphavbeta5, and alpha9beta1. Fibulin-5 is developmentally regulated in the lung, and lung air space enlargement develops in mice deficient in fibulin-5. Fibulin-5 is also induced in adult lung following lung injury by hyperoxia. To further examine the role of fibulin-5 during repair of lung injury, we assessed fibulin-5 expression during elastase-induced emphysema in C57/b mice. Mice were treated with either saline or elastase via the trachea, and the lung was examined 20 days after treatment. Fibulin-5 mRNA was induced almost fourfold, whereas elastin mRNA was minimally elevated. Immunohistochemistry studies showed that fibulin-5 was induced in cells within the alveolar wall following elastase treatment. Western analysis demonstrates that fibulin-5 was strongly expressed in isolated primary lung interstitial fibroblasts. Fibulin-5 protein was localized to the fibroblast cell layer in culture, and brief elastase treatment degraded the protein. Intact fibulin-5 did not accumulate in the culture media. Treatment of fibroblasts with the proinflammatory cytokine interleukin-1beta abolished fibulin-5 mRNA expression. Our results indicate that fibulin-5 is coordinately expressed and regulated with elastin in lung fibroblasts and may serve a key role during lung injury and repair.     

Fibulin-5 contributes to colorectal cancer cell apoptosis via the ROS/MAPK and Akt signal pathways by downregulating transient receptor potential cation channel subfamily V member 1

Fibulin-5, a multifunctional extracellular matrix (ECM) protein, is secreted into the ECM, regulating metastasis and invasion in many malignant tumors. However, its role in colorectal cancer (CRC) has not been reported. In this study, we detected the expression of fibulin-5 in 56 CRC patients and eight CRC cell lines, revealing that fibulin-5 was expressed lower in CRC tumor tissues than in peritumor tissues. Furthermore, our study verified that fibulin-5 promoted cell apoptosis and reactive oxygen species (ROS) production by inhibiting transient receptor potential cation channel subfamily V member 1 (TRPV1) in CRC cells. Moreover, NAC (the scavenger of ROS), SB203580 (the inhibitor of p38), PD98059 (the inhibitor of ERK), and SC79 (the activator of Akt) were used to uncover that fibulin-5 induced apoptosis through the ROS/mitogen-activated protein kinase and Akt signal pathways by downregulating TRPV1. Together, these results suggest that fibulin-5 might serve as a novel drug target for the treatment of CRC patients.     

Lysosomal accumulation of oxidized phosphatidylcholine-apolipoprotein B complex in macrophages: intracellular fate of oxidized low density lipoprotein

Oxidized phosphatidylcholine (OxPC) formed in oxidized low density lipoprotein (OxLDL) is thought to be involved in the development of atherosclerosis. OxPC has been found in foam cells in atherosclerotic lesions and suggested to be the epitope for OxLDL recognition by macrophages. OxPC is present as a complex with apolipoprotein B (apoB) in OxLDL, since some OxPC can bind with proteins. In the current study, the intracellular fate of OxPC-apoB complexes after internalization of OxLDL by macrophages was investigated. Murine macrophage cell line J774.1 was incubated with either OxLDL or acetylated LDL for 24 h, then the cells were further incubated for up to 24 h in new medium without lipoprotein. Modified apoB in the cells was quantitated by sandwich ELISA using monoclonal antibodies against OxPC and apoB. Intracellular OxLDL decreased rapidly for the first 4 h to approx. 20% of that before medium change, with the apparent metabolism of OxPC-apoB complex ceasing. OxPC-apoB complexes that remained in the cells after 24 h chasing increased as the period of OxLDL loading in macrophages prolongs. Acetylated LDL in the cells decreased quickly and disappeared after 4 h of chasing. Subcellular fractionation using sucrose density gradient ultracentrifugation of macrophages, which had already accumulated OxPC-apoB complexes by 24 h of incubation with OxLDL and further 24 h chasing, showed that the complex was co-localized with endosomal and lysosomal markers. Immunohistochemical double staining studies demonstrated that OxPC and apoB co-localize in foam cells in early atherosclerotic lesions obtained from human coronary artery. These results suggest that OxPC-apoB complexes originating from OxLDL accumulate in foam cells in human atherosclerotic lesions as well as in macrophages in vitro.     

Apolipoprotein B stimulates formation of monocyte-macrophage surface-connected compartments and mediates uptake of low density lipoprotein-derived liposomes into these compartments

Much of the cholesterol that accumulates in atherosclerotic plaques is found within monocyte-macrophages transforming these cells into "foam cells." Native low density lipoprotein (LDL) does not cause foam cell formation. Treatment of LDL with cholesterol esterase converts LDL into cholesterol-rich liposomes having >90% cholesterol in unesterified form. Similar cholesterol-rich liposomes are found in early developing atherosclerotic plaques surrounding foam cells. We now show that cholesterol-rich liposomes produced from cholesterol esterase-treated LDL can cause human monocyte-macrophage foam cell formation inducing a 3-5-fold increase in macrophage cholesterol content of which >60% is esterified. Although cytochalasin D inhibited LDL liposome-induced macrophage cholesteryl ester accumulation, LDL liposomes did not enter macrophages by phagocytosis. Rather, the LDL liposomes induced and entered surface-connected compartments within the macrophages, a unique endocytic pathway in these cells that we call patocytosis. LDL liposome apoB rather than LDL liposome lipid mediated LDL liposome uptake by macrophages. This was shown by the findings that: 1) protease treatment of the LDL liposomes prevented macrophage cholesterol accumulation; 2) liposomes prepared from LDL lipid extracts did not cause macrophage cholesterol accumulation; and 3) purified apoB induced and accumulated within macrophage surface-connected compartments. Although apoB mediated the macrophage uptake of LDL liposomes, this uptake did not occur through LDL, LDL receptor-related protein, or scavenger receptors. Also, LDL liposome uptake was not sensitive to treatment of macrophages with trypsin or heparinase. Cholesterol esterase-mediated transformation of LDL into cholesterol-rich liposomes is an LDL modification that: 1) stimulates uptake of LDL cholesterol by apoB-dependent endocytosis into surface-connected compartments, and 2) causes human monocyte-macrophage foam cell formation.     

The extracellular matrix proteins fibulin-1 and fibulin-2 in the early human embryo

Summary Fibulin-1 and fibulin-2, two recently identified extracellular matrix proteins with a homologous domain structure, are known to bind various extracellular ligands and calcium. In this study, they have been localized at the light microscopical level in human embryos of gestational weeks 4–10, using polyclonal antibodies. Identical localization patterns were observed for the two fibulins in most of the tissues. In the heart, the endocardial cushion tissue and endocardium, but not the myocardium, were stained, as were the basement membrane zones and adventitia of blood vessels. Staining was also observed in the perichondrium and calcifying regions of developing bones. Moreover, reactions occurred with the gut subepithelium and epithelial basement membranes of the skin. Differences in staining patterns, however, were observed in various neural structures. Fibulin-1 was prominent in the matrix of the leptomeningeal anlage, in basement membranes of the neuroepithelium and the perineurium of peripheral nerves. Fibulin-2 was detected primarily within the neuropithelium, spinal ganglia and peripheral nerves. The early embryonic expression of both fibulins indicates specific roles during organ development and, in particular, involvement in the differentiation of heart, skeletal and neuronal structures.     

Cholesterol and oxysterol metabolism and subcellular distribution in macrophage foam cells. Accumulation of oxidized esters in lysosomes

Cholesterol- and cholesteryl ester-rich macrophage foam cells, characteristic of atherosclerotic lesions, are often generated in vitro using oxidized low density lipoprotein (OxLDL). However, relatively little is known of the nature and extent of sterol deposition in these cells or of its relationship to the foam cells formed in atherosclerotic lesions. The purpose of this study was to examine the content and cellular processing of sterols in OxLDL-loaded macrophages, and to compare this with macrophages loaded with acetylated LDL (AcLDL; cholesteryl ester-loaded cells containing no oxidized lipids) or 7-ketocholesterol-enriched acetylated LDL (7KCAcLDL; cholesteryl ester-loaded cells selectively supplemented with 7-ketocholesterol (7KC), the major oxysterol present in OxLDL). Both cholesterol and 7KC and their esters were measured in macrophages after uptake of these modified lipoproteins. Oxysterols comprised up to 50% of total sterol content of OxLDL-loaded cells. Unesterified 7KC and cholesterol partitioned into cell membranes, with no evidence of retention of either free sterol within lysosomes. The cells also contained cytosolic, ACAT-derived, cholesteryl and 7-ketocholesteryl esters. The proportion of free cholesterol and 7KC esterified by ACAT was 10-fold less in OxLDL-loaded cells than in AcLDL or 7KCAcLDL-loaded cells. This poor esterification rate in OxLDL-loaded cells was partly caused by fatty acid limitation. OxLDL-loaded macrophages also contained large (approximately 40-50% total cell sterol content) pools of oxidized esters, containing cholesterol or 7KC esterified to oxidized fatty acids. These were insensitive to ACAT inhibition, very stable and located in lysosomes, indicating resistance to lysosomal esterases. Macrophages loaded with OxLDL do not accumulate free sterols in their lysosomal compartment, but do accumulate lysosomal deposits of OxLDL-derived cholesterol and 7-ketocholesterol esterified to oxidized fatty acids. The presence of similar deposits in lesion foam cells would represent a pool of sterols that is particularly resistant to removal.     

Fibulin-2 is dispensable for mouse development and elastic fiber formation

Fibulin-2 is an extracellular matrix protein belonging to the five-member fibulin family, of which two members have been shown to play essential roles in elastic fiber formation during development. Fibulin-2 interacts with two major constituents of elastic fibers, tropoelastin and fibrillin-1, in vitro and localizes to elastic fibers in many tissues in vivo. The protein is prominently expressed during morphogenesis of the heart and aortic arch vessels and at early stages of cartilage development. To examine its role in vivo, we generated mice that do not express the fibulin-2 gene (Fbln2) through homologous recombination of embryonic stem cells. Unexpectedly, the fibulin-2-null mice were viable and fertile and did not display gross and anatomical abnormalities. Histological and ultrastructural analyses revealed that elastic fibers assembled normally in the absence of fibulin-2. No compensatory up-regulation of mRNAs for other fibulin members was detected in the aorta and skin tissue. However, in the fibulin-2 null aortae, fibulin-1 immunostaining was increased in the inner elastic lamina, where fibulin-2 preferentially localizes. The results demonstrate that fibulin-2 is not required for mouse development and elastic fiber formation and suggest possible functional redundancy between fibulin-1 and fibulin-2.     

Fibulin-5 expression in the human placenta

Fibulin-5 is a secreted extracellular matrix glycoprotein and displays a diverse panel of biological functions, which can be segregated into elastogenic as well as extra-elastogenic functions. While elastogenic functions of fibulin-5 include essential roles in early steps of elastic fibre assembly, extra-elastogenic functions are widespread. Depending on the cell type used, fibulin-5 mediates cell adherence via a subset of integrins, antagonizes angiogenesis and inhibits migration as well as proliferation of endothelial and smooth muscle cells. In this study, we focused on the spatiotemporal expression of fibulin-5 in the human placenta. With progressing gestation, placental fibulin-5 expression increased from first trimester towards term. At term, placental fibulin-5 mRNA expression is lower when compared with other well-vascularized organs such as lung, kidney, heart, uterus and testis. In first trimester, placenta immunohistochemistry localized fibulin-5 in villous cytotrophoblasts and extravillous cytotrophoblasts of the proximal cell column. In term placenta, fibulin-5 was detected in the endothelial basement membrane and adventitia-like regions of vessels in the chorionic plate and stem villi. Cell culture experiments with the villous trophoblast-derived cell line BeWo showed that fibulin-5 expression was downregulated during functional differentiation and intercellular fusion. Moreover, cultivation of BeWo cells under low oxygen conditions impaired intercellular fusion and upregulated fibulin-5 expression. The spatiotemporal shift from the trophoblast compartment in first trimester to the villous vasculature at term suggests a dual role of fibulin-5 in human placental development.     

Sequence, recombinant expression and tissue localization of two novel extracellular matrix proteins, fibulin-3 and fibulin-4.

Fibulin-1 and fibulin-2 have previously been identified as basement membrane and microfibrillar proteins with a broad binding repertoire for other extracellular ligands. Here we report on the cloning and sequence analysis of human fibulin-3 (487 residues), also known as protein S1-5, and fibulin-4 (443 residues). These novel members of this protein family are most closely related to fibulin-1C. They consist of a C-terminal globular domain III, also shared by the fibrillins, a central rod-like element composed of five calcium-binding epidermal growth factor-like (EG) modules (domain II) and an N-terminal interrupted EG module (domain I) which replaces the anaphylatoxin-like modules of the other fibulins. This predicted domain structure was supported by electron microscopy of fibulin-4, which demonstrated short rods. Northern blots showed that both novel fibulins are expressed in several human tissues to a variable extent and that they are up-regulated in quiescent fibroblasts. Specific antibodies which were raised against each of the novel fibulins did not cross-react with fibulin-1. Immunohistology of adult mouse tissues showed that fibulin-3, fibulin-4 and fibulin-1 have overlapping but distinct extracellular tissue localizations. A particularly prominent feature was the staining of variable sets of large and small blood vessels.     

Molecular pathways used by platelets to initiate and accelerate atherogenesis

PURPOSE OF REVIEW: The response to injury model in the development of atherosclerosis is broadly accepted by the scientific audience. Platelets are generally not believed to be involved in the initiation of atherosclerosis. New data imply, however, that the response to injury model is too simple for a complete understanding of the inflammatory disease atherosclerosis. The involvement of platelets in the initiation of atherosclerotic lesion formation is critical in directing the atherosclerotic process into regeneration or ongoing vascular injury. RECENT FINDINGS: Platelets internalize oxidized phospholipids and promote foam cell formation. Platelets also recruit circulating blood cells including progenitor cells to the vessel, that are able to differentiate into foam cells or endothelial cells depending on conditions. Platelets express various scavenger receptors that are able to regulate LDL-uptake. LDL-laden platelets are internalized by adherent progenitor cells that in turn differentiate into macrophages and foam cells. SUMMARY: An expanding body of evidence continues to build on the role of platelets as initial actors in the development of atherosclerotic lesions. Platelets bind to leukocytes, endothelial cells, and circulating progenitor cells and initiate monocyte transformation into macrophages. Therefore platelets regulate the initiation, development and total extent of atherosclerotic lesions.     

Fibulin-3 regulates the inhibitory effect of TNF-α on chondrocyte differentiation partially via the TGF-β/Smad3 signaling pathway

Fibulin-3 is an extracellular matrix glycoprotein that is present in elastic tissue and involved in carcinoma development. Previous studies have indicated that fibulin-3 may affect skeletal development, cartilage, and osteoarthritis (OA). This study aims to investigate the function of fibulin-3 on chondrocytes under tumor necrosis factor alpha (TNF-α) stimulation and in murine OA models, and explore the possible mechanism. It was found that fibulin-3 was increased in the cartilage of OA models and in the chondrogenic cells ATDC5 stimulated by TNF-α. Fibulin-3 promoted the proliferation of ATDC5 cells both in the presence and absence of TNF-α. Moreover, overexpression of fibulin-3 suppressed the chondrogenic and hypertrophic differentiation of ATDC5 cells, while knockdown of fibulin-3 caused the opposite effect. Mechanistically, fibulin-3 partially suppressed the activation of TGF-β/Smad3 signaling by inhibiting the phosphorylation of Smad3. SIS3, a Smad3 inhibitor, decreased the chondrogenesis of articular cartilages in OA models, and partially reversed the chondrogenic differentiation of ATDC5 cells caused by knockdown of fibulin-3 in the presence of TNF-α. Furthermore, co-immunoprecipitation (Co-IP) showed that fibulin-3 could only interact with TGF-β type I receptor (TβRI), although overexpression of fibulin-3 reduced the protein levels of both TβRI and TβRII. In conclusion, this study indicates that fibulin-3 modulates the chondrogenic differentiation of ATDC5 cells in inflammation partially via TGF-β/Smad3 signaling pathway.