Multiple Laminin Binding Proteins in Human Fetal Heart. (fetal heart/laminin binding proteins/cation dependent protein/cation independen protein/developmental changes) (original) (raw)
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Developmental Dynamics, 1996
The development of the heart from a single heart tube to a four chambered organ with two separated unidirectional flows is a highly complex process. Events like looping, septation, tissue remodelling, and development of valves take place in a time period in which the heart already exerts its pump function. Adhesion of cells to each other and to their extracellular matrix as well as the capability to migrate in such a dynamic environment are extremely important. Integrins and extracellular matrix components have already been implicated in this process. In this report, we describe in detail the differential expression of the a6 integrin subunit during late murine heart development, e.g., in the process from looping to the end of septation. We compare mRNA and protein expression patterns with those of pl and other subunits of laminin-binding integrins, a3 and a7. We show a constant and high expression of 016 in the atrial myocardium and a decrease in expression in the ventricular trabecular myocardium. The compact myocardial wall and the ventricular septum do not express 1x6, except for the myocardium of the distal outflow tract at early stages. Moreover, we describe expression of this integrin subunit in the endocardia1 cushions that contribute to the development of the atrioventricular and semilunar valves. We propose a role for the a6pl laminin receptor in the adhesion of cells to their extracellular matrix at sites of high stress due to cardiac contraction or blood flow induced shear stress. Moreover, site specific endothelial expression within the heart and surrounding extracardiac tissue is discussed. This study suggests a distinct role for a6pl in the heart and provides insight concerning probably important roles of integrins and their extracellular matrix ligands during embryonic develop-ment. o 1996 Wiley-Liss, Inc.
Japanese Heart Journal, 1997
genes in the heart of embryonic, neonatal and adult BALb/c mice. The reverse transcriptase-polymerase chain reaction was employed to determine the mRNA expression of these chains because of the relatively small amount of RNA extracted from rat embryonic hearts. Glyceraldehyde 3-phosphate dehythe hearts of mice at all stages examined. These results demonstrate that the laminin chain gene expression changes in the different developmental stages of the hearts of BALb/c mice.
Identification of an extracellular 130-kDa protein involved in early cardiac morphogenesis
The Journal of biological chemistry, 1993
Previous studies indicate that the transformation of cardiac endothelium into mesenchyme is dependent upon a developmentally regulated signal expressed by its associated myocardium. This process can be mimicked in culture by substituting a non-cytolytic EDTA extract of embryonic heart tissue for the presence of myocardium. Polyclonal antibodies (ES1) generated against the EDTA-extractable proteins both localized to the cardiac extracellular matrix preceding the transformation of endothelium and blocked this process in culture. Based on these observations, we hypothesized that ES1 antigens participate in the formation of cardiac mesenchyme. The present study was undertaken to prepare cDNA and antibody probes for individual ES1 antigens to better characterize their involvement in this important morphogenetic event. An expression library was constructed in Uni-ZAP using poly(A+) RNA from embryonic cardiocyte cultures that had been shown previously to secrete proteins that engender the ...
Clinical & Experimental Immunology, 2008
Cross-reactions between maternally derived autoantibodies and fetal cardiac antigens have been postulated to play a role in the pathogenesis of congenital heart block (CHB). We have explored the cross-reactivity of autoantibodies to the small ribonuclear autoantigens, La/SS-B and Ro/SS-A, with laminin, the major component of cardiac sarcolemmal membrane using affinity-purified antibodies from patients with Sj6gren's syndrome (SS). Anti-La antibodies purified from eight of 10 patients cross-reacted significantly with mouse laminin by ELISA. In contrast, purified antibodies to Ro52 from the same 10 patients showed little or no binding to laminin. Laminin inhibited up to 70% binding of anti-La antibodies to La antigen, and La inhibited up to 65% binding of anti-La antibodies to laminin. The cross-reaction was further examined on cryosections of 10 human fetal hearts aged from 8-7 to 14-9 weeks of gestation, two normal adult hearts, and one pathological adult heart with a diagnosis of dilated cardiomyopathy. Anti-Ro52 antibodies did not bind to the surface of cardiac cells. However, anti-La antibodies from seven of 10 patients tested bound to the surface of fetal myocytes from hearts aged 9 4 to 14-9 weeks of gestation, and also to the myocytes from the pathological adult heart but not to normal adult hearts. Preincubation with La antigen abolished the binding of anti-La antibodies to the surface of adult heart myocytes with dilated cardiomyopathy, and pre-incubation with mouse laminin could partially block this binding. These results suggest that molecular mimicry between laminin and La, but not Ro52, may act as a target for specific maternal autoantibodies, and contribute to the pathogenesis of CHB at a critical stage during fetal cardiac development.
The Journal of Physiology, 2000
Cardiac muscle contains both âÔ_ and âµ_adrenergic receptor (AR) signalling mechanisms (Steinberg, 1999). In general, the density of âµ_ARs is lower than that of âÔ_ARs (Steinberg, 1999), and higher in atrial than ventricular muscle (Stiles et al. 1983). âµ_ARs are more efficiently coupled to Gs-protein/ adenylate cyclase (AC) than are âÔ_ARs (Green et al. 1992; Levy et al. 1993), resulting in a greater sensitivity to âµ_AR agonist stimulation. In addition, within seconds of agonist exposure âµ_ARs exhibit desensitization resulting from phosphorylation by â-AR kinases (Muntz et al. 1994). â_AR signalling is especially important in the failing heart, which exhibits dramatic and selective remodelling of âÔ_ and âµ_AR signalling (Xiao et al. 1999b). However, the mechanisms responsible for remodelling of â-adrenoceptor signalling are not clear. Laminin is one component of the cardiac extracellular matrix (ECM), whose content and composition undergo profound alterations in response to pathological conditions. Cardiomyocytes attach to laminin and other ECM components via low-affinity, cell surface receptors known as integrins. Integrins comprise a family of heterodimeric transmembrane proteins consisting of various alpha (á) and beta (â) chains which combine to give the integrin complex several different ECM-binding specificities. Adult cardiac myocytes readily attach to laminin via âÔ_integrin receptors (Terracio et al. 1991). Integrins transduce biochemical and mechanical stimuli into the cell to regulate various cellular functions (Cary et al. 1999). Our previous work indicates that laminin binding to âÔ_integrins alters cholinergic regulation of ICa,L by depressing AC activity (Wang et al. 2000). In the present study we report that attachment of atrial myocytes to laminin selectively reduces âÔ_ and enhances âµ_AR signalling mechanisms which regulate ICa,L. These findings may be relevant to the mechanisms underlying â_AR remodelling in the failing heart. METHODS Adult mongrel cats of either sex were anaesthetized with sodium pentobarbital (70 mg kg¢ i.p.). After bilateral thoracotomy, the heart was rapidly excised and mounted on a Langendorff perfusion apparatus for cell isolation, as described (Wu et al. 1991). Experiments were performed on quiescent atrial cells, without
Primary Structure, Developmental Expression, and Immunolocalization of the Murine Laminin α4 Chain
Journal of Biological Chemistry, 1997
The complete primary structure of the mouse laminin ␣4 chain was derived from cDNA clones. The translation product contains a 24-residue signal peptide preceding the mature ␣4 chain of 1,792 residues. Northern analysis on whole mouse embryos revealed that the expression was weak at day 7, but it later increased and peaked at day 15. In adult tissues the strongest expression was observed in lung and cardiac and skeletal muscles. Weak expression was also seen in other adult tissues such as brain, spleen, liver, kidney, and testis. By in situ hybridization of fetal and newborn tissues, expression of the laminin ␣4 chain was mainly localized to mesenchymal cells. Strong expression was seen in the villi and submucosa of the developing intestine, the mesenchymal stroma surrounding the branching lung epithelia, and the external root sheath of vibrissae follicles, as well as in cardiac and skeletal muscle fibers. In the developing kidney, intense but transient expression was associated with the differentiation of epithelial kidney tubules from the nephrogenic mesenchyme. Immunohistologic staining with affinity-purified IgG localized the laminin ␣4 chain primarily to lung septa, heart, and skeletal muscle, capillaries, and perineurium.
The Journal of Cell Biology, 1988
Affinity chromatography and immunolocalization techniques were used to investigate the mechanism(s) by which endothelial cells interact with the basement membrane component laminin. Bovine aortic endothelial cells (BAEC) membranes were solubilized and incubated with a laminin-Sepharose affinity column. SDS-PAGE analysis of the eluted proteins identified a 69-kD band as the major binding protein, along with minor components migrating at 125, 110, 92, 85, 75, 55, and 30 kD. Polyclonal antibodies directed against a peptide sequence of the 69-kD laminin-binding protein isolated from human tumor cells identified this protein in BAEC lysates. In frozen sections, these polyclonal antibodies and monoclonal antibodies raised against human tumor 69-kD stained the endothelium of bovine aorta and the medial smooth muscle cells, but not surrounding connective tissue or elastin fibers. When nonpermeabilized BAEC were stained in an in vitro migration assay, there appeared to be apical patches of 6...
Regulation of the beta2 subunit chain of laminin in developing rabbit fetal lung tissue
Differentiation, 1996
Laminins are a family of basement membrane-associated heterotrimeric proteins that are important in mediating the growth, migration, and differentiation of a variety of cell types. The β2 subunit chain is a component of several laminin isoforms, e.g., laminin-3, laminin-4, laminin-7, and possibly other, as yet uncharacterized laminin isoforms. Utilizing monoclonal antibodies directed against the β2 subunit chain of laminin, we detected this protein in fetal, neonatal, and adult lung tissues. The relative amount of laminin β2 subunit chain in fetal lung tissue increased as gestation proceeded, reaching its peak around the time of alveolar type II cell differentiation in the rabbit. The laminin β2 subunit chain was localized in early gestational age rabbit fetal lung tissue primarily in basement membranes of prealveolar ducts, airways, and smooth muscle cells of airways and arterial blood vessels. A rabbit laminin β2 cDNA was generated using RT-PCR and utilized as a probe in northern blot analysis to characterize the levels of laminin β2 mRNA in developing rabbit lung tissue. Similar to the pattern of laminin β2 protein induction observed in fetal lung tissue, laminin β2 mRNA levels were maximal late in gestation. Utilizing a laminin β2 chain cRNA probe and in situ hybridization, we detected laminin β2 mRNA in the epithelial cells of prealveolar ducts, the alveolar wall, and airways, as well as in connective tissue cells, and the smooth muscle cells of airways and blood vessels in fetal and adult lung tissues. In addition, using an in vitro explant model, we determined that alveolar type II cells are capable of synthesizing laminin β2 subunit mRNA and depositing this laminin subunit chain in the basement membrane beneath type II cells. The results of this study are suggestive that the laminin β2 chain may be involved in alveolar epithelial cell differentiation.& b d y :
Experimental Cell Research, 2002
Protein levels, mRNA expression, and localization of laminin ␣1 and ␣2 chains in development and in adult mice were examined. Recombinant fragments were used to obtain high-titer-specific polyclonal antibodies for establishing quantitative radioimmunoinhibition assays. This often demonstrated an abundance of ␣2 chain, but also distinct amounts of ␣1 chain for adult tissues. The highest amounts of ␣1 were found in placenta, kidney, testis, and liver and exceeded those of ␣2. All other tissue extracts showed a higher content of ␣2, which was particularly high in heart and muscle when compared to ␣1. Content of ␥1 chain, shared by most laminins, was also analyzed. This demonstrated ␥1 chain levels being equal to or moderately exceeding the sum of ␣1 and ␣2 chains, indicating that these isoforms represent the major known laminin isoforms in most adult mouse tissues so far examined. Moreover, we found good correlation between radioimmuno-inhibition data and mRNA levels of adult tissues as measured by quantitative real-time reverse transcriptase-PCR. Embryonic tissues were also analyzed by radioimmuno-inhibition assays. This demonstrated for day 11 embryos comparable amounts of ␣1 and ␥1 and a more than 25-fold lower content of ␣2. This content increased to about 10% of ␣1 in day 13 embryos. The day 18 embryo showed in heart, kidney, and liver, but not yet in brain and lung, ␣1/␣2 chain ratios comparable to those in adult tissues. Immunostaining demonstrated ␣1 in Reichert's membrane (day 7.5), while ␣2 could not be detected before day 11.5. These data were compared with immunohistochemical localization results on several more embryonic and adult tissue sections. Our results regarding localization are consistent with those of earlier work with some notable exceptions. This was in part due to epitope masking for monoclonal antibodies commonly used in previous studies in esophagus, intestine, stomach, liver, kidney, and spleen.