Margaret T. Armstrong

Regulation of proliferation of the fetal myocardium.

Growth of the myocardium involves the completion of a fixed number of rounds of cell division during the embryonic and fetal stages followed by entry into a postmitotic state and hypertrophy of the postmitotic cardiomyocytes during the later stages of heart growth. It has been suggested that at the time of its determination in the early embryo, the embryonic myocardium is programmed for a fixed and limited number of cell divisions, after which the transition to the postmitotic state occurs autonomously. The proliferative response of cultured myocardium of the fetal chick was explored in four culture settings: monolayer cell culture, collagen lattice culture, organ culture of reaggregated cardiomyocyte tissue (cardiomyocyte spheroid culture), and organ culture of pieces of the ventricle wall. Several growth factors were identified by their ability to stimulate DNA synthesis in cardiomyocytes, identified by the incorporation of 5‐bromodeoxyuridine (BrdU). The serine proteases thrombin and trypsin, fibroblast growth factor‐2 (FGF‐2), transforming growth factor‐α (TGF‐α), and insulin‐like growth factor‐II (IGF‐II) all show growth factor activity but only for cardiomyocytes cultured in three‐dimensional myocardial tissue, and not for cardiomyocytes maintained in monolayer cell culture. Thus, during its proliferation phase, the growth of the fetal myocardium is not controlled solely by internal, cell‐autonomous programs, but is subject to external regulation by a family of peptide growth factors. The unconventional setting of three‐dimensional culture of the myocardium is required to demonstrate its responsiveness to growth factor challenge.

Intercellular invasion and the organizational stability of tissues: a role for fibronectin

Intracellular invasion is the movement of cells of one type into the fabric of other, contiguous tissues. Invasion is a signature behavior of the malignant tumor and also is found as part of the normal behavior of inflammatory blood cells and tissues engaged in the morphogenetic movements of normal embryogenesis and in a number of instances of normal and pathological tissue remodeling in the adult. Informed by the view that the underlying mechanisms of invasion will be similar for tumor cells and invasive blood and embryonic cells, this review adopts a comparative approach to the analysis of invasion. Invasion results in the development of a diffuse interface between contiguous tissues. Its alternative is the maintenance of stable, planar tissue boundaries. This is the more usual condition for contiguous tissues in the animal. This review will focus on the processes that, on the one hand, stabilize planar contact interfaces between tissues, and, on the other, promote the destabilization of tissue integrity by fostering intercellular invasion. Particular attention is devoted to a role for adhesive interactions mediated by the matrix adhesion molecule, fibronectin. In certain instances, fibronectin in the matrix promotes invasion whereas in others, the presence of fibronectin prevents invasion. The distinction appears to depend on whether the invasive tissue is migrating into an acellular extracellular matrix or whether invasion involves densely cellular tissues. In the first instance, fibronectin promotes invasion, whereas in the second, it stabilizes the interface of the contacting tissues and prevents invasion.

Involvement of α2-macroglobulin and C-reactive protein in a complement-like hemolytic system in the arthropod, Limulus polyphemus

Homologues of two plasma proteins of vertebrates, alpha 2-macroglobulin and C-reactive protein, participate in a hemolytic system of the ancient arthropod, Limulus polyphemus. C-reactive protein, which can under the appropriate circumstances activate the classical pathway of the mammalian complement system, is an essential element of the hemolytic system of Limulus. The selective removal of C-reactive protein from the plasma with phosphorylethanolamine-agarose inactivated hemolysis. Addition of affinity-purified C-reactive protein to inactive plasma restored activity. Exposure of plasma to phosphorylethanolamine in solution potentiated hemolysis. alpha 2-Macroglobulin is a member of the same protein family as the complement protein C3 and both require an intact thiol ester for activity. Treatment of Limulus plasma with methylamine under conditions that inactivate thiol-ester-containing proteins reduced the hemolytic activity of some plasma preparations. Addition of purified Limulus alpha 2-macroglobulin to the methylamine-treated plasma restored hemolytic activity. However, alpha 2-macroglobulin is not necessary for hemolysis since the hemolytic activity of some pooled plasma preparations was insensitive to methylamine treatment under conditions that inactivated alpha 2-macroglobulin. Purified C-reactive protein was hemolytic in the absence of alpha 2-macroglobulin. These observations suggest that the proteins in Limulus plasma that participate in hemolysis represent the components of an ancient invertebrate defense system with distant evolutionarily affinities to the vertebrate complement system.

Regulation of proliferation of embryonic heart mesenchyme: Role of transforming growth factor-β1 and the interstitial matrix

Proliferation of atrioventricular cushion mesenchyme of the embryonic avian heart maintained in three-dimensional aggregate culture is stimulated by interaction with the interstitial matrix. Chicken serum or transforming growth factor-beta 1, which stimulates proliferation, induces matrix deposition in regions of the aggregate showing high labeling indices with tritiated thymidine. Dispersed heart mesenchyme interstitial matrix introduced into serum-free culture is incorporated into the aggregate and stimulates cellular proliferation similar to serum or transforming growth factor-beta 1. Proliferation is reversibly inhibited by the peptide Gly-Arg-Gly-Asp-Ser-Pro. It is suggested that transforming growth factor-beta 1 stimulates the production of interstitial matrix and that a sufficient stimulus for proliferation in this system is the presence of the matrix, which acts as the adhesive support for cellular anchorage.

Fibroblast growth factor-2 stimulates embryonic cardiac mesenchymal cell proliferation

The proliferation response of stage 36 chick atrioventricular valve mesenchymal cells to fibroblast growth factor‐2 (FGF‐2) was studied in the tissue‐like environment of three‐dimensional cell aggregates maintained in organ culture. The mitogenic effects of FGF‐2 on mesenchymal tissue depended on the FGF‐2‐stimulated formation of a fibronectin‐containing extracellular matrix. The matrix was absent in unstimulated aggregates, and co‐localized with regions of actively proliferating cells in stimulated aggregates. Inhibition of fibronectin matrix formation by the inclusion of Arg‐Gly‐Asp‐containing peptides, which compete with fibronectin for binding to the cell surface α5β1 integrin receptors, abolished the proliferation effects of FGF‐2. Inhibition of sulfation of cell surface glycosaminoglycans by treatment with sodium chlorate significantly reduced both the formation of the fibronectin matrix and cell proliferation in response to FGF‐2, suggesting an involvement of the low‐affinity sulfated glycosaminoglycan FGF receptor system. Thus, the FGF‐stimulated growth of embryonic atrioventricular valve mesenchyme in vitro involves the production of a fibronectin matrix. We suggest that the stimulation of the fibronectin matrix represents an essential element in growth factor signaling of mesenchymal tissue, with the matrix serving as an anchorage substratum for the proliferating cells.

Histochemical evidence for lipid A (endotoxin) in eukaryote chloroplasts

Lipopolysaccharide (LPS) (a.k.a., endotoxin) is an essential component of the outer leaflet of the outer membrane of Gram‐negative bacteria and is a potent activator of the innate immune system of animals. Lipid A, the glycolipid core of LPS, is the agent responsible for disease and death from Gram‐negative sepsis, an important cause of human mortality and morbidity. Although it is generally accepted that lipid A is restricted to the prokaryotes, recent efforts to purify molecules from green algae with structural features unique to lipid A have met with success. Furthermore, the vascular plant Arabidopsis thaliana has been found to contain genes that encode all of the enzymes of the biosynthetic pathway for lipid A. It is not known whether vascular plants synthesize lipid A or where lipid A might be located in the tissues. For the present study, we used affinity reagents for lipid A to probe green alga and tissues of the garden pea for a light microscopic localization of lipid A in these eukaryote cells. We find staining for lipid A in free‐living and endosymbiotic green algae and in the chloroplasts of vascular plants, indicating that this molecule is not restricted to prokaryotes, but is found also in select eukaryotes.—Armstrong, M. T., Theg, S. M., Braun, N., Wainwright, N., Pardy, R. L., Armstrong, P. B. Histochemical evidence for lipid A (endotoxin) in eukaryote chloroplasts. FASEB J. 20, E1506 –E1509 (2006)

Thrombin stimulation of matrix fibronectin

Trypsin, thrombin, and peptide analogues of the new amino terminus of the proteolyzed thrombin receptor, SFLLRN and SFLLRNPNDKYEPF, stimulated embryonic fibroblasts cultured as 3‐dimensional tissue‐like aggregates to elaborate a fibronectin‐rich extracellular matrix. Enzymatically inactive thrombin and the control peptide FLLRN failed to stimulate matrix production. The induction of cell proliferation correlated with production of the fibronectin matrix. The regions of active cell proliferation in the fibroblast aggregates co‐localized with the matrix and peptide analogues of the RGD cell‐adhesion site of fibronectin reversibly inhibited the accumulation of the fibronectin matrix and the stimulation of cell proliferation by SFLLRN. Two different preparations of the fibronectin matrix stimulated cell proliferation in aggregates cultured in growth factor‐free medium. We suggest that the stimulation of matrix production is a necessary event for mitogenic signaling in mesenchymal tissue. The tight coupling between the matrigenic and mitogenic activities of growth factors was absent in monolayer cultures of chick embryonic fibroblasts since thrombin and trypsin induced proliferation of monolayer‐cultured cells without inducing the production of a fibronectin matrix.

Are cells in solid tissues immobile? Mesonephric mesenchyme studied in vitro

Abstract Fragments of mesonephric mesenchyme isolated from 3-day avian embryos were allowed to round up in vitro. Pairs of tissue fragments were then fused and maintained in suspension culture. For the purpose of identification of cells, fusions were made between chick and quail or tritiated-thymidine labeled chick and unlabeled chick tissue fragments. Mobility of cells was detected by the movement of cells across the interfaces between the individual tissue fragments of the fused tissue masses. Since, after three days in culture, numbers of cells from one fragment were usually present within the depths of its partner fragment, it is concluded that cells within solid pieces of mesonephric mesenchymal tissue show continued mobility. Since large amounts of extracellular material were not found in the intercellular spaces of cultured tissue fragments, it is concluded that cell surfaces serve as the principal substrata for motility. Motility in solid tissue masses is not a reflection of the generalized loss of contact inhibition which is characteristic of invasive cells; contact inhibition of ruffled membrane activity was observed in mesonephric mesenchyme cells cultured in monolayer culture.

Transforming growth factor-β1 localized within the heart of the chick embryo

SummaryTransforming growth factor-β1 is a pleiotropic peptide mediator of growth, differentiation, and extra-cellular matrix synthesis. In the embryonic chick heart prior to the formation of the endocardial cushions, evidence from in vitro experiments suggests that transforming growth factor-β1 may be an inducer of the differentiation of atrioventricular endothelial cells into endocardial cushion mesenchyme. Further in vitro evidence suggests that the factor stimulates mesenchymal cell proliferation, and, thus, growth of the cushions. Using an antibody made against a peptide duplicating the aminoterminal 30 amino acid sequence of transforming growth factor-β1, we stained sections of stage 11, 18, 23, 26, and 36 chick hearts by an in situ immunofluorescence technique. Transforming growth factor-β1 staining localized to the endocardial surface and epicardial surface of the stage 11 heart, but it decreased from these locations in later stages. The cardiac jelly (stage 11), endocardial cushions (stage 18, 23, and 26), and, subsequently, the heart valve leaflets (stage 36) stained intensely for the growth factor.

Capture of Lipopolysaccharide (Endotoxin) by the Blood Clot: A Comparative Study

In vertebrates and arthropods, blood clotting involves the establishment of a plug of aggregated thrombocytes (the cellular clot) and an extracellular fibrillar clot formed by the polymerization of the structural protein of the clot, which is fibrin in mammals, plasma lipoprotein in crustaceans, and coagulin in the horseshoe crab, Limulus polyphemus. Both elements of the clot function to staunch bleeding. Additionally, the extracellular clot functions as an agent of the innate immune system by providing a passive anti-microbial barrier and microbial entrapment device, which functions directly at the site of wounds to the integument. Here we show that, in addition to these passive functions in immunity, the plasma lipoprotein clot of lobster, the coagulin clot of Limulus, and both the platelet thrombus and the fibrin clot of mammals (human, mouse) operate to capture lipopolysaccharide (LPS, endotoxin). The lipid A core of LPS is the principal agent of gram-negative septicemia, which is responsible for more than 100,000 human deaths annually in the United States and is similarly toxic to arthropods. Quantification using the Limulus Amebocyte Lysate (LAL) test shows that clots capture significant quantities of LPS and fluorescent-labeled LPS can be seen by microscopy to decorate the clot fibrils. Thrombi generated in the living mouse accumulate LPS in vivo. It is suggested that capture of LPS released from gram-negative bacteria entrapped by the blood clot operates to protect against the disease that might be caused by its systemic dispersal.