Collective cell motion in endothelial monolayers.

Collective cell motility is an important aspect of several developmental and pathophysiological processes. Despite its importance, the mechanisms that allow cells to be both motile and adhere to one another are poorly understood. In this study we establish statistical properties of the random streaming behavior of endothelial monolayer cultures. To understand the reported empirical findings, we expand the widely used cellular Potts model to include active cell motility. For spontaneous directed motility we assume a positive feedback between cell displacements and cell polarity. The resulting model is studied with computer simulations and is shown to exhibit behavior compatible with experimental findings. In particular, in monolayer cultures both the speed and persistence of cell motion decreases, transient cell chains move together as groups and velocity correlations extend over several cell diameters. As active cell motility is ubiquitous both in vitro and in vivo, our model is expected to be a generally applicable representation of cellular behavior.

Fibulin is an extracellular matrix and plasma glycoprotein with repeated domain structure.

We have studied the expression of fibulin in cultured fibroblasts and determined its primary structure by cDNA cloning. Our results show that fibulin is a secreted glycoprotein that becomes incorporated into a fibrillar extracellular matrix when expressed by cultured cells or added exogenously to cell monolayers. In addition, we find that fibulin is present in plasma at a level of 33 +/- 3 micrograms/ml. Sequencing of multiple fibulin cDNAs indicates that a process of alternative splicing results in the expression of three fibulin transcripts. The transcripts encode overlapping polypeptides differing only in carboxy-terminal segments. Common to the three predicted forms of fibulin is a unique 537-amino acid-long cysteine-rich polypeptide and a 29-residue signal peptide. The amino-terminal portion of fibulin contains a repeated element with potential disulfide loop structure resembling that of the complement component anaphylatoxins C3a, C4a, and C5a as well as proteins of the albumin gene family. The bulk of the remaining portion of the molecule is a series of nine EGF-like repeats.

Identification of the low density lipoprotein receptor-related protein (LRP) as an endocytic receptor for thrombospondin-1.

Thrombospondin-1 (TSP1) has potent biological effects on vasculature smooth muscle cells (SMCs) and endothelial cells. The regulation of extracellular accumulation of TSP1 is mediated by a previously obscure process of endocytosis which leads to its lysosomal degradation. Since members of the low density lipoprotein receptor (LDLR) family have been found to mediate endocytosis which leads to degradation of a diverse array of ligands, we evaluated their possible role in the uptake and degradation of TSP1 by vascular SMCs, endothelial-cells and fibroblasts. 125I-TSP1 was found to be internalized and degraded lysosomally by all these cell types. Both the internalization and degradation of 125I-TSP1 could be inhibited by a specific antagonist of the LDLR family, the 39-kD receptor-associated protein (RAP). Antibodies to the LDLR-related protein (LRP) completely blocked the uptake and degradation of 125I-TSP1 in SMCs and fibroblasts but not endothelial cells. Solid-phase binding assays confirmed that LRP bound to TSP1 and that the interaction was of high affinity (Kd = 5 nM). Neither RAP nor LRP antibodies inhibited the binding of 125I-TSP1 to surfaces of SMCs. However, cell surface binding, as well as, endocytosis and degradation could be blocked by heparin or by pre-treatment of the cells with either heparitinase, chondroitinase or beta-D-xyloside. The data indicates that cell surface proteoglycans are involved in the LRP-mediated clearance of TSP1. A model for the clearance of TSP1 by these cells is that TSP1 bound to proteoglycans is presented to LRP for endocytosis. In endothelial cells, however, the internalization of TSP1 was not mediated by LRP but since RAP inhibited TSP1 uptake and degradation, we postulate that another member of the LDLR family is likely to be involved.

Inhibition of in vitro tumor cell invasion by Arg-Gly-Asp-containing synthetic peptides.

The interaction of cells with extracellular matrix components such as fibronectin, vitronectin, and type I collagen has been shown to be mediated through a family of cell-surface receptors that specifically recognize an arginine-glycine-aspartic acid (RGD) amino acid sequence within each protein. Synthetic peptides containing the RGD sequence can inhibit these receptor-ligand interactions. Here, we use novel RGD-containing synthetic peptides with different inhibition properties to investigate the role of the various RGD receptors in tumor cell invasion. The RGD-containing peptides used include peptides that inhibit the attachment of cells to fibronectin and vitronectin, a peptide that inhibits attachment to fibronectin but not to vitronectin, a cyclic peptide with the opposite specificity, and a peptide, GRGDTP, that inhibits attachment to type I collagen in addition to inhibiting attachment to fibronectin and vitronectin. The penetration of two human melanoma cell lines and a glioblastoma cell line through the human amniotic basement membrane and its underlying stroma was inhibited by all of the RGD-containing peptides except for the one that inhibits only the vitronectin attachment. Various control peptides lacking RGD showed essentially no inhibition. This inhibitory effect on cell invasion was dose-dependent and nontoxic. A hexapeptide, GRGDTP, that inhibits the attachment of cells to type I collagen in addition to inhibiting fibronectin- and vitronectin-mediated attachment was more inhibitory than those RGD peptides that inhibit only fibronectin and vitronectin attachment. Analysis of the location of these cells that were prevented from invading indicated that they attached to the amniotic basement membrane but did not proceed further into the tissue. These results suggest that interactions between RGD-containing extracellular matrix adhesion proteins and cells are necessary for cell invasion through tissues and that fibronectin and type I collagen are important for this process.

Human osteosarcoma cells resistant to detachment by an Arg-Gly-Asp-containing peptide overproduce the fibronectin receptor.

MG-63 human osteosarcoma cells were selected for attachment and growth in the presence of increasing concentrations of a synthetic peptide containing the cell attachment-promoting Arg-Gly-Asp sequence derived from the cell-binding region of fibronectin. Cells capable of attachment and growth in 5-mM concentrations of a peptide having the sequence Gly-Arg-Gly-Asp-Ser-Pro overproduce the cell surface receptor for fibronectin. In contrast, these cells show no differences in the numbers of vitronectin receptor they express as compared with the parental MG-63 cells. In agreement with the resistance of the selected cells to detachment by the peptide, 25-fold more Arg-Gly-Asp-containing peptide is required to prevent the attachment of these cells to fibronectin-coated surfaces than is needed to inhibit the attachment of MG-63 cells to the same substrate. However, similar concentrations of this peptide inhibit attachment of both cell lines to vitronectin-coated surfaces. The increase in fibronectin receptor is due to an increase in the levels of mRNA encoding the fibronectin receptor. Because of the nature of the selection process, we reasoned that this increase might be due to amplification of the fibronectin receptor gene, but no increase in gene copy number was detected by Southern blot analysis. The peptide-resistant cells display a very different morphology from that of the MG-63 cells, one that has a greater resemblance to that of osteocytes. The resistant cells also grow much more slowly than the MG-63 cells. The increased fibronectin receptor and altered morphology and growth properties were stable for at least 3 mo in the absence of peptide. The enhanced expression of the fibronectin receptor on the resistant cells indicates that cells are capable of altering the amount of fibronectin receptor on their surface in response to environmental factors and that this may in turn affect the phenotypic properties of the cell.

Amino acid sequence of the human fibronectin receptor.

The amino acid sequence deduced from cDNA of the human placental fibronectin receptor is reported. The receptor is composed of two subunits: an alpha subunit of 1,008 amino acids which is processed into two polypeptides disulfide bonded to one another, and a beta subunit of 778 amino acids. Each subunit has near its COOH terminus a hydrophobic segment. This and other sequence features suggest a structure for the receptor in which the hydrophobic segments serve as transmembrane domains anchoring each subunit to the membrane and dividing each into a large ectodomain and a short cytoplasmic domain. The alpha subunit ectodomain has five sequence elements homologous to consensus Ca2+-binding sites of several calcium-binding proteins, and the beta subunit contains a fourfold repeat strikingly rich in cysteine. The alpha subunit sequence is 46% homologous to the alpha subunit of the vitronectin receptor. The beta subunit is 44% homologous to the human platelet adhesion receptor subunit IIIa and 47% homologous to a leukocyte adhesion receptor beta subunit. The high degree of homology (85%) of the beta subunit with one of the polypeptides of a chicken adhesion receptor complex referred to as integrin complex strongly suggests that the latter polypeptide is the chicken homologue of the fibronectin receptor beta subunit. These receptor subunit homologies define a superfamily of adhesion receptors. The availability of the entire protein sequence for the fibronectin receptor will facilitate studies on the functions of these receptors.

The human alpha 2-macroglobulin receptor: identification of a 420-kD cell surface glycoprotein specific for the activated conformation of alpha 2-macroglobulin.

Ligand affinity chromatography was used to purify a cell surface alpha 2-macroglobulin (alpha 2M) receptor. Detergent extracts of human placenta were applied to an affinity matrix consisting of alpha 2M, previously reacted with methylamine, coupled to Sepharose. Elution with EDTA specifically released polypeptides with apparent molecular masses of 420 and 39 kD. In some preparations, small amounts of a 90-kD polypeptide were observed. The 420- and 39-kD polypeptides appear specific for the forms of alpha 2M activated by reaction with proteinases or methylamine and do not bind to an affinity matrix consisting of native alpha 2M coupled to Sepharose. Separation of these two polypeptides was accomplished by anion exchange chromatography, and binding activity was exclusively associated with the 420-kD polypeptide. The purified 420-kD protein binds to the conformationally altered forms of alpha 2M that are known to specifically interact with alpha 2M receptors and does not bind to native alpha 2M. Binding of the 420-kD polypeptide to immobilized wheat germ agglutinin indicates that this polypeptide is a glycoprotein. The cell surface localization of the 420-kD glycoprotein was confirmed by affinity chromatography of extracts from surface radioiodinated fibroblasts. These properties suggest that the 420-kD polypeptide is a cell surface receptor for the activated forms of alpha 2M.

Suppression of anchorage-independent growth and matrigel invasion and delayed tumor formation by elevated expression of fibulin-1D in human fibrosarcoma-derived cell lines.

Using differential display, we identified an mRNA that is markedly down-regulated in cell line 6A/SB1, derived from a fibrosarcoma formed in an athymic mouse following injection of carcinogen-transformed MSU-1.1 cells. The nontumorigenic parental cell strain, MSU-1.1, expresses high levels of this mRNA. Sequencing of the corresponding cDNA fragment revealed that it corresponded to an expressed sequence tag, which ultimately led to its identification as the fibulin-1D gene. Fibulin-1 is a cysteine-rich, calcium-binding extracellular matrix and plasma protein, which has four isoforms, A-D, derived from alternative splicing. Northern and Western blotting analysis of 16 cell lines established from tumors formed in athymic mice by MSU-1.1-derived cell strains independently transformed in culture showed that 44% exhibited low level or lack of expression of fibulin-1D mRNA and protein. In a similar analysis of 15 malignant cell lines derived from patients, 80% showed low level or no expression. To study the role of fibulin-1D in transformation, we transfected 6A/SB1 cells and a human fibrosarcoma-derived cell line (SHAC) with a fibulin-1D cDNA expression construct. Transfectants displaying high levels of fibulin-1D were isolated and characterized. Elevated expression of fibulin-1D led to reduced ability to form colonies in soft agar and reduced invasive potential as tested in a matrigel in vitro invasion assay. Furthermore, expression of fibulin-1D resulted in a markedly extended latency in tumor formation in athymic mice. These results indicate that low expression of fibulin-1D plays a role in tumor formation and invasion.

The fibulin-1 gene (FBLN1) is disrupted in a t(12;22) associated with a complex type of synpolydactyly.

Molecular analysis of the reciprocal chromosomal translocation t(12;22)(p11.2;q13.3) cosegregating with a complex type of synpolydactyly showed involvement of an alternatively spliced exon of the fibulin-1 gene (FBLN1 located in 22q13.3) and the C12orf2 (HoJ-1) gene on the short arm of chromosome 12. Investigation of the possible functional involvement of the fibulin-1 protein (FBLN1) in the observed phenotype showed that FBLN1 is expressed in the extracellular matrix (ECM) in association with the digits in the developing limb. Furthermore, fibroblasts derived from patients with the complex type of synpolydactyly displayed alterations in the level of FBLN1-D splice variant incorporated into the ECM and secreted into the conditioned culture medium. By contrast, the expression of the FBLN1-C splice variant was not perturbed in the patient fibroblasts. Based on these findings, we propose that the t(12;22) results in haploinsufficiency of the FBLN1-D variant, which could lead to the observed limb malformations.

Cubilin and megalin: partners in lipoprotein and vitamin metabolism.

Members of the low-density lipoprotein receptor (LDLR) family are unrivalled for their ability to endocytose and target ligands to lysosomes for degradation. Their endocytic and catabolic functions make them essential to homeostatic regulation of the level and activity of their ligands in biological fluids and interstitial spaces. Over the last few years it has become evident that the endocytic function of members of the LDLR family is employed by other kinds of cell surface receptors. Recently, the low-density lipoprotein receptor related protein-2 (megalin) was shown to act in concert with cubilin, a receptor for high-density lipoproteins (HDL)/apolipoprotein A-I (apoA-I), intrinsic factor-vitamin B12 and albumin to mediate ligand endocytosis. In this article, we review the state of knowledge pertaining to cubilin and megalin, emphasizing their joint roles in both lipoprotein and vitamin metabolism.

Low-dose spironolactone reduces plasma fibulin-1 levels in patients with type 2 diabetes and resistant hypertension.

Diabetic patients with hypertension are at particularly high risk of vascular damage and consequently cardiovascular and renal disease. Fibulin-1, an extracellular matrix glycoprotein, is increased in arterial tissue and plasma from individuals with type 2 diabetes. This study aimed to evaluate whether antihypertensive treatment with spironolactone changes plasma fibulin-1 levels. In a multicenter, double-blind, randomized, placebo-controlled study, 119 patients with type 2 diabetes and resistant hypertension were included. A dose of spironolactone 25 mg or matching placebo was added to previous treatment at randomization. Blood pressure (BP) and plasma fibulin-1 were measured at baseline and at 16 weeks follow-up. Overall, 112 patients completed the study. All measures of BP were reduced in the spironolactone group at follow-up. Plasma fibulin-1 was significantly reduced after spironolactone treatment (P=0.009), but increased after placebo (P=0.017). Baseline plasma fibulin-1 correlated with BP and estimated glomerular filtration rate. Increased levels of plasma fibulin-1 (P=0.004) were observed in diabetic participants reporting erectile dysfunction as compared with participants who did not. Treatment with low-dose spironolactone reduced plasma fibulin-1 levels in patients with type 2 diabetes and resistant hypertension. This supports the hypothesis that the antihypertensive effect of the mineralocorticoid receptor blocker in part may be due to regression of vascular remodeling.

A quantitative analysis of the incorporation of fibulin-1 into extracellular matrix indicates that fibronectin assembly is required.

Fibulin-1 is an extracellular matrix glycoprotein found in both loose and dense connective tissues, elastic fibers and some basement membranes. Cultured cells such as fibroblasts assemble endogenously synthesized or exogenously added fibulin-1 into matrix fibrils that also contain fibronectin. Since we have previously shown that fibulin-1 binds to fibronectin (Balbona, K., Tran, H., Godyna, S., Ingham, K. C., Strickland, D. K. and Argraves, W. S. J. Biol. Chem. 267: 20120-20125, 1992), we sought to investigate fibulin-1 incorporation into fibroblast extracellular matrix with an emphasis on evaluating the potential role of fibronectin in the process. In this study, we have used quantitative assays to measure the binding of 125I-fibulin to monolayers of cultured fibroblasts. Our results show that the kinetics of fibulin-1 incorporation into the cell layer and its partitioning into detergent-soluble and -insoluble fractions were similar to those of fibronectin. It was found that antibodies to fibronectin or to the fibulin-1-binding domain of fibronectin-inhibited fibulin-1 incorporation. Cell lines that fail to assemble fibronectin into the matrix, such as HT1080 or PFHR-9, do not incorporate fibulin-1 into their cell layers. However, when HT1080 cells were induced to assemble fibronectin by treatment with dexamethasone, they subsequently acquired the ability to incorporate fibulin-1. Moreover, treatment of cultured fibroblasts with antibodies that inhibit fibronectin assembly significantly inhibit fibulin-1 incorporation into the matrix. When increased amounts of fibronectin were incorporated into cells layers by incubating the cells for varying lengths of time with exogenous fibronectin, a corresponding increase in fibulin-1 incorporation was also observed. Taken together, the data indicate that the incorporation of fibulin-1 requires fibronectin assembly and suggests a dependence on the amount of fibronectin in a matrix. These results highlight the potential of fibronectin to control the deposition of fibulin-1 into those extracellular matrices where both proteins coincide and may have implications in the formation of fibulin-1-containing matrix structures such as basement membranes or elastic fibers.

Human fibulin-1D: molecular cloning, expression and similarity with S1-5 protein, a new member of the fibulin gene family.

Fibulin-1 is an extracellular matrix (ECM) component of basement membranes and connective tissue elastic fibers, and a blood protein. Multiple forms of fibulin-1 that differ in their C-terminal regions are produced through the process of alternative splicing of their precursor RNA. Two transcripts of 2.4 and 2.7 kb are the predominant fibulin-1 mRNAs expressed in human tissues and cultured cells. While the 2.4 kb transcript had been shown to encode fibulin-1C, the 2.7 kb transcript did not correspond to any of the previously identified human fibulin-1 variants. Herein, we report on the isolation and sequencing of cDNA corresponding to the 2.7 kb fibulin-1 transcript which encodes a novel, alternatively spliced form of human fibulin-1 that we term the D form. The deduced amino acid sequence of the D form is identical in its first 566 residues to the three known fibulin-1 variants (fibulin-1A-C); however, it has a unique 137 amino acid-C-terminal segment encoded by the alternatively spliced portion of its transcript. RNA hybridization analysis showed that the fibulin-1D transcript is coordinately expressed with that of fibulin-1C both in tissues and in cultured cells. Using antibodies specific to the unique C-terminal segment of fibulin-1D and -1C, both proteins were found to be expressed in human placenta. Recombinant fibulin-1D generated in transfected mammalian cells displayed similar ligand-binding properties as placenta-derived fibulin-1 and recombinant fibulin-1C, and it was capable of incorporating into cultured cell ECM in the absence of other fibulin-1 forms. A comparative sequence analysis revealed that the unique C-terminal region of fibulin-1D is similar to the C-terminal regions of fibulin-1C and fibulin-2. Furthermore, the C-terminal regions of fibulin-1C, -1D and -2 are similar to the C-terminal region of a recently described protein termed S1-5. In addition to this C-terminal similarity, S1-5 also contains repeated EGF-like modules and a conserved N-terminal element, thereby leading to the conclusion that S1-5 is a third member of the fibulin gene family.

Identification of chicken and C. elegans fibulin-1 homologs and characterization of the C. elegans fibulin-1 gene.

Fibulin-1, a member of the emerging family of fibulin proteins, is a component of elastic extracellular matrix fibers, basement membranes and blood. Homologs of fibulin-1 have been described in man, mouse and zebrafish. In this study, we describe the isolation and sequencing of chicken fibulin-1C and D cDNA variants. We also describe identification of a C. elegans cDNA encoding fibulin-1D and cosmids containing the C. elegans fibulin-1 gene. Using the cDNA, RT-PCR and computer-based analysis of genomic sequences, the exon/intron organization of the C. elegans fibulin-1 gene was determined. The C. elegans fibulin-1 gene is located on chromosome IV, is approximately 6 kb in length, contains 16 exons and encodes fibulin-1C and D variants. Comparative analysis of the deduced amino acid sequences of nematode and chicken fibulin-1 variants with other known vertebrate fibulin-1 polypeptides showed that the number and organization of structural modules are identical. The results of this study indicate that the structure of the fibulin-1 protein has remained highly conserved over a large period of evolution, suggestive of functional conservation.

Members of the low density lipoprotein receptor family control diverse physiological processes.

The low density lipoprotein receptor (LDLR) family is a group of receptors that mediate endocytosis leading to lysosomal degradation of an enormous repertoire of ligands. To date, over 50 distinct macromolecules have been shown to bind members of the family. This wide spectrum of ligand recognition is the basis for an immense diversity in physiological processes in which these receptors participate. In this article, the physiological roles of the LDLR family members are briefly reviewed and a comprehensive list of the ligands with which the receptors interact is presented.

The association of human fibulin-1 with elastic fibers: an immunohistological, ultrastructural, and RNA study.

We examined the pattern of fibulin-1 mRNA and protein expression in human tissues and cell lines. Fibulin-1 transcripts were found in RNA isolated from most tissues and a variety of cultured cells, including fibroblasts, smooth muscle cells, and several epithelial cell lines, but not endothelial cells, lymphomyloid cells, or a number of carcinoma and melanoma lines. Immunohistochemical analysis showed that fibulin-1 is an intercellular component of connective tissues, predominantly associated with matrix fibers in tissues such as the cervix, dermis, intimal and medial layers of blood vessels, heart valves, meningeal tissue of the brain, Wharton's jelly of the umbilical cord, testis, and lung. Most of the fibers that were immunoreactive with fibulin-1 antibodies also stained with antibodies to the elastic fiber proteins elastin and fibrillin, as well as with Verhoeff's elastin stain. Immunoelectron microscopic analysis of elastin fibers of skin and saphenous vein revealed that fibulin-1 was located within the amorphous core of the fibers, similar to elastin, but it was not in the fibrillin-containing, elastin-associated microfibrils. Our finding that fibulin-1 is an elastic fiber component suggests several possible new functions for fibulin-1, e.g., that it is a structural protein that contributes to the elastic properties of connective tissue fibers or that is involved with the process of fibrogenesis.

Low density lipoprotein receptor-related protein-1 expression in the testis: regulated expression in Sertoli cells.

The low density lipoprotein receptor-related protein (LRP-1) is a multiligand receptor capable of mediating endocytosis of a wide array of ligands that relate to both lipoprotein metabolism and proteinase regulation. Many of its ligands are proteinases, proteinase-inhibitor complexes, and lipoproteins known to be contained within the luminal fluid of the seminiferous tubules or in the interstitial spaces of the testis. Immunocytochemical analysis was performed to characterize the pattern of expression of LRP-1 in cells of the rat testis. Immunoperoxidase staining localized LRP-1 to the cytoplasm of Sertoli cells. The distribution and intensity of the Sertoli cell staining was found to vary according to the stages of the cycle of the seminiferous epithelium. Staining was weak in the basal cytoplasm of Sertoli cells during stages II-VIII and strong and granular in the supranuclear cytoplasm during stages XII-XIV and stage I of the cycle. Immunogold labeling showed gold particles associated with the basal and adluminal plasma membranes, with endocytic vesicles, and with endosome membranes. Labeling was also observed on the plasma membrane and membranes of the endocytic apparatus in macrophages and Leydig cells in the interstitial space. Infusion of 125I-Labeled LRP-1 antibody into seminiferous tubules followed by radioautography showed silver grains overlaying the ad-luminal plasma membrane of Sertoli cells at time 0 and in endocytic vesicles and endosomes in the supranuclear region of Sertoli cells 10-minutes postinjection. When the 125I-Labeled LRP-1 antibody was injected into the interstitial space, silver grains overlayed the basal plasma membrane and coated endocytic pits of Sertoli cells at time 0 and, at 10 minutes, the grains labeled endosomes located in the basal pole of Sertoli cells. 125I-Labeled LRP-1 antibody also labeled the plasma membrane and the endocytic apparatus of macrophages and Leydig cells. The absence of immunogold labeling and radioautographic silver grains within lysosomes of Sertoli cells, Leydig cells, and macrophages suggests that internalized LRP-1 is recycled back to the cell surface. The presence of LRP-1 in the endocytic compartment of these cells is consistent with its functioning in the clearance of proteases involved in seminiferous tubule remodeling and/ or the uptake of cholesterol-bound lipoproteins necessary for the biosynthesis of testosterone. In conclusion, the results of these studies demonstrated for the first time the presence of LRP-1 receptor in the endocytic compartments of Sertoli cells and interstitial cells of the rat testis.

Cellular interactions with fibronectin as a model for redundant binding of cells to other extracellular matrix proteins.

Perhaps the most studied property of fibronectin (Fn) is its ability to bind to cells. Interestingly, there are multiple mechanisms by which cells can bind Fn involving as many as ten different cell surface molecules and perhaps six distinct sites within Fn. This apparent redundant binding system is not only restricted to Fn since cells bind other extracellular matrix proteins such as laminin, vitronectin and fibrinogen in a similar manner. The many binding interactions between cells and Fn may serve as a model for understanding redundant binding between cells and other matrix proteins.

Immunological localization of glycoprotein 330, low density lipoprotein receptor related protein and 39 kDa receptor associated protein in embryonic mouse tissues.

In this study we have immunologically examined the expression of the structurally and functionally related receptors, LRP and gp330, and their associated 39 kDa protein (RAP) in tissues of the embryonic mouse. One aim was to determine whether these proteins were coordinantly expressed. The data reveals that gp330 is expressed on the apical surfaces of many specialized absorptive epithelia most prominently, choroid plexus, ependyma, metanephric tubules, ear, thyroid, pericardium, and intestine. LRP was detected in all epithelia expressing gp330 with the exception of the epicardium and metanephric tubule epithelium. However, the subcellular pattern of LRP deposition in polarized epithelium was distinct from the apical pattern of gp330, perhaps indicating that LRP was either sequestered intracellularly or distributed basolaterally. The pattern of LRP expression in tissues of the mouse embryo was however much wider than that of gp330. Prominent expression of LRP was observed in skin, myocardium, mesenchyme, liver, pancreas, and marginal regions of the brain. In the developing liver, LRP was not detected in day 10.5 but was detected in megacaryocyte-like cells of 12.5 day and in hepatocytes of 14.5 day embryonic liver. RAP was observed to be coexpressed with either or both of the receptors but its subcellular pattern of distribution coincided with that of LRP. The coexpression of gp330 and LRP in epithelial cells and the observation that gp330 staining was always localized apically while LRP was distributed basolaterally or sequestered intracellularly suggests that these receptors have distinct functions in polarized epithelial cells.

NT-proBNP is associated with fibulin-1 in Africans: the SAfrEIC study.

OBJECTIVES: The N-terminal prohormone B-type natriuretic peptide (NT-proBNP) is involved in the regulation of volume load and secreted when systemic cardiac overload occurs. Fibulin-1 on the other hand is a component of many extracellular matrix proteins including those present in atherosclerotic lesions, expressed in elastin-containing fibres of blood vessels, and also in the heart. Due to an alarming prevalence of hypertensive heart disease in black South Africans, we investigated the associations of NT-proBNP with fibulin-1 and markers of arterial stiffness in Africans and Caucasians. METHODS: We included 231 Africans and 238 Caucasians from South Africa aged 22-77 years. Serum NT-proBNP and fibulin-1 levels were determined, and arterial compliance and pulse wave velocity were measured. RESULTS: Africans had significantly higher blood pressure and NT-proBNP levels than Caucasians and African men had higher fibulin-1 levels than Caucasian men. In single regression analysis, NT-proBNP was significantly associated with fibulin-1 in African men and Caucasian women. NT-proBNP correlated negatively with arterial compliance in all groups except Caucasian women. After partial adjustments, the association between NT-proBNP and fibulin-1 strengthened in African men only. After full adjustment in multiple regression analysis, the association of NT-proBNP with fibulin-1 was confirmed in African men (R(2)=0.41; ß=0.26; p<0.01) and also in younger women (R(2)=0.34; ß=0.251; p=0.012). CONCLUSIONS: Only Africans indicated a significant independent association between NT-proBNP and fibulin-1, suggesting that cardiovascular alterations are already present in this relatively young African population as opposed to Caucasians.