The watchmaker's dilemma: building a tower of microenvironments in vitro.
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Animals↗
Biomedical subjects
M Tavassoli
Publications and source records attributed to M Tavassoli.
Hemopoietic endothelium, incognito.
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Animals↗
Induction of upmodulation of homing receptors in cloned hemopoietic progenitors by growth factors.
Recognition and selective seeding of hemopoietic cells to the marrow after intravenous transplantation is a function of membrane protein known as homing receptor (HR), which is a lectin with galactosyl and mannosyl specificities. We have previously shown that ex vivo incubation of marrow cells with IL-3 or GM-CSF enhances the seeding efficiency and that this enhancement may be the result of upmodulation of homing receptors. In the present work, we have shown that incubation of two cloned progenitor cell lines, FDCP-1 and FDCP-mix, with IL-3 or GM-CSF resulted in a dose-dependent increase in the number of HR per cell. Interferon-gamma did not have such an effect. The upmodulation of receptors was the result of enhanced de novo synthesis and transport of the protein as it was ablated by cyclohexamide, monensin or NH4Cl. On the other hand, degradation of the protein was not affected by the growth factors. The possible mechanisms whereby a brief exposure to growth factor ex vivo could lead to enhancement of homing efficiency are discussed. Induction of upmodulation of HR by ex vivo incubation of donor cells with certain hemopoietic growth factors, could be a useful strategy for enhancing marrow reconstitution after bone marrow transplantation. This could reduce the cost and prevent the side effects of in vivo growth factor therapy.
Ammonium Chloride↗
Enhancement of the grafting efficiency of transplanted marrow cells by preincubation with interleukin-3 and granulocyte-macrophage colony-stimulating factor.
To improve the grafting efficiency of transplanted murine hematopoietic progenitors, we briefly preincubated mouse bone marrow cells with interleukin-3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF) ex vivo before their transplantation into irradiated recipients. This treatment was translated into an increase in the seeding efficiency of colony-forming unit-spleen (CFU-S) and CFU-GM after transplantation. Not only was the concentration of CFU-S in the tibia increased 2 and 24 hours after transplantation, but the total cell number and CFU-S and CFU-GM concentrations were persistently higher in IL-3- and GM-CSF-treated groups 1 to 3 weeks after transplantation. In addition, the survival of animals as a function of transplanted cell number was persistently higher in IL-3- and GM-CSF-treated groups compared with controls. The data indicate that the pretreatment of marrow cells with IL-3 and GM-CSF before transplantation increases the seeding efficiency of hematopoietic stem cells and probably other progenitor cells after transplantation. This increased efficiency may be mediated by upward modulation of homing receptors. Therefore, ex vivo preincubation of donor marrow cells with IL-3 and GM-CSF may be a useful tactic in bone marrow transplantation.
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The effect of ethanol on the uptake, binding, and desialylation of transferrin by rat liver endothelium: implications in the pathogenesis of alcohol-associated hepatic siderosis.
Chronic alcoholism has been reported to be associated with a reduced carbohydrate content of transferrin (TF), particularly, its reduced sialylation state. Low sialylation state of TF now serves as an objective marker of chronic alcohol abuse. To investigate the pathophysiological significance of this finding in relation to hepatic siderosis, also commonly associated with chronic alcoholism, the authors have investigated the effect of ethanol on the uptake, binding, and desialylation of transferrin by isolated rat liver endothelium in vitro. In pulse-chase experiments, transferrin labeled with either 125I (protein-labeled) or 3H (sialic acid-labeled) was incubated with isolated, fractionated liver endothelium with and without ethanol, and the supernates were subjected to column chromatography using RCA120- agarose. Incubation of the endothelium with increasing concentrations of ethanol resulted in a progressive increase in the desialylation rate of transferrin which was maximal when 160 mM concentration of ethanol was used. These data indicate that ethanol significantly promotes the desialylation of transferrin by rat liver endothelium. The implications of these findings in the pathogenesis of hepatic siderosis of the alcoholic are discussed.
Acetaldehyde↗
Homing of hemopoietic progenitor cells to the marrow.
The recognition of hemopoietic stem cell after intravenous transplantation of marrow cells occurs initially by a lectin moiety on the surface of marrow sinus endothelium. The cell is then transported across the endothelial cytoplasm much in the way that a soluble ligand, such as transferrin, is transported. In the extravascular compartment, the cell binds to lineage-specific stromal cells. This mechanism, known as homing, is mediated by a lectin-glycoconjugate interaction, the lectin being on the surface of progenitor cell with specificity for galactosyl and mannosyl residues. The binding is subsequently stabilized by membrane-bound proteoglycans, integrin-like receptors, and fibronectin.
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Distribution of homing protein on hemopoietic stromal and progenitor cells.
Homing receptor is a membrane lectin of 110 kd molecular weight that recognizes galactosyl and mannosyl residues of an as yet unknown glycoconjugate. It is responsible for recognition and selective homing of hemopoietic progenitor cells after these cells are transplanted intravenously. Consequently, it is present on the surface of hemopoietic progenitor cells. To determine the distribution of this receptor on other cell types we performed standard binding assays in many cell types using galactosyl and mannosyl residues covalently bound to bovine serum albumin (G-BSA and M-BSA) as an index of homing receptor. BSA moiety was then labeled with 125I. The three cloned hemopoietic cell lines B6Sut, FDCP-1, and FDCP-mix all showed combined binding of G-BSA and M-BSA, whereas the lymphoid cell line L1210 showed only M-BSA, not G-BSA binding and, therefore, was considered to lack homing receptors. Similarly, stromal cell lines D2X and GB1/6 as well as primary marrow stroma (progenitor cell-depleted) did not show homing receptors as evidenced by combined binding of G-BSA and M-BSA. Nor did the nonhemopoietic stromal cell line Swiss 3T3 show the presence of homing receptors by these criteria. We conclude that homing receptors are distributed narrowly and are present on hemopoietic progenitor cells, but absent on hemopoietic stroma.
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Successful stable xenograft of human fetal hemopoietic cells in preimmune fetal sheep.
Human fetal liver hemopoietic stem cells were transplanted into preimmune fetal sheep. Significant numbers of recipient fetuses showed evidence of engraftment of human stem cells which responded in vivo to human-specific hemopoietic growth factors. The chimerism has persisted now for > 2 years without significant graft loss. No evidence of GVHD has been noted. Successful engraftment was associated with the expression of human erythroid, myeloid, and lymphoid differentiation. This xenograft model offers useful possibilities for the study of the biology of human hemopoiesis in vivo.
Animals↗
Response of marrow stroma to ionizing radiation.
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Animals↗
Transendothelial transport of macromolecules: the concept of tissue-blood barriers.
In addition to its many functions in biosynthesis, growth, coagulation and rheology, vascular endothelium is anatomically interposed between the vascular space and the tissue fluid. Recent evidence indicates that it mediates cellular and molecular exchange between these compartments. The exchange can occur through differentiated microdomains of endothelium such as fenestrae. These areas are differentiated with regard to surface charge, protein distribution within the lipid bilayer, membrane fluidity and other features. The exchange is also affected by certain characteristics of the molecule to be transported: molecular size, charge, shape and its carbohydrate content. Proportionately, the largest volume of exchange occurs across the endothelial cytoplasm by vesicular transport systems. Two systems are particularly in evidence; (a) receptor-mediated transcytosis which is specific, and (b) fluid-phase endocytosis. The molecule may become modified in transit and the modification may be of essence in determining its target point and its subsequent metabolism. While most of these modifications involve the carbohydrate moiety of the glycoproteins, glycosylation of non-glycoproteins such as albumin, may also be of physiological significance in transendothelial transport. By virtue of its transport potential, albumin can thus affect the transport of other substances. Recent advances in the molecular transport function of endothelium have been reviewed in the context of its physiological and clinical significance. The basis for the concept of a generalized tissue-blood barrier has been offered.
Bibliometrics↗
Characterization of FDCP-2, a cloned hemopoietic progenitor cell deficient in homing protein.
The progenitor cell clone, FDCP-2, was found to lack the expression of membrane homing lectin that recognizes galactosyl and mannosyl residues of glycoconjugate on the surface of hemopoietic stroma. Adherence of these cells to hemopoietic stroma is significantly less than that of either normal clones B6SUt or FDCP-1, although their adherence to nonhemopoietic stroma 3T3 is preserved. As determined by electron microscopy, the cells lack microvilli, which in their normal counterparts serve to mediate the contact and adherence to hemopoietic stroma. This cell line can be useful as a negative control in elucidating the molecular basis of the homing phenomenon and its function in the regulation of hemopoiesis.
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Embryonic and fetal hemopoiesis: an overview.
Our current knowledge of embryonic and fetal hemopoiesis is critically reviewed in this article. In both murine and human systems, embryonic and fetal development is associated with multiple switching in the sites of hemopoiesis. The phenomenon is first extraembryonic, occurring in blood islands of the yolk sac. Hemopoietic stem cells (HSC) appear to derive from hemangioblasts that are of mesodermal origin. Yolk sac milieu is permissive only for erythropoiesis which proceeds synchronously and may be erythropoietin-insensitive. Yolk sac milieu is not permissive for the development of other cell lines. The final product is nucleated red cells. Yolk sac hemopoiesis is an example par excellence of primitive (as compared to definitive) form of hemopoiesis. HSC then seem to migrate via the bloodstream to the liver and spleen to seed these tissues, which then carry the burden of hemopoiesis until birth and for some time thereafter. Here also erythropoiesis predominates, but some granulopoiesis also occurs. Thus, the milieu is not totally impermissive. Hemopoiesis is in definitive form, lacking synchronicity of cell growth with the end product being anucleated cells and synthesized hemoglobin not limited to embryonic type. The site of hemopoiesis is finally transferred to the bone marrow, which is predominantly granulopoietic. Certain cellular and embryological features of these types of hemopoiesis in the context of more recent molecular understanding of stem cell homing are discussed.
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Cyclophosphamide-induced alterations of bone marrow endothelium: implications in homing of marrow cells after transplantation.
Bone marrow transplantation requires infusion of stem cells i.v. and transport of these cells to the extravascular compartment of the marrow where hemopoiesis takes place. This requires massive cellular traffic across the sinus endothelial barrier. We tested the hypothesis that the conditioning regimen given before transplantation alters the endothelium and permits the traffic of transplanted cells into the hemopoietic compartment. C57B1 mice were given cyclophosphamide i.p. in doses comparable to those used in conditioning regimens. Marrow was perfusion-fixed and studied by electron microscopy at various time after the dose. The salient feature of cyclophosphamide-induced endothelial injury was membrane instability, manifested by sloughing and vesiculation of membrane on the luminal surface. Considerable loss of membrane was associated with a reduction in the surface area of endothelium. There was also an associated loss of integrity of sinus endothelium so that mature circulating red cells, normally confined to the vascular space, could now traverse the vascular wall and appear in the hemopoietic compartment. Moreover, perisinal macrophages, normally confined to extravascular space, were now seen in the lumen. We conclude that the cytotoxic conditioning regimen, given with different objectives, may facilitate the traffic of transplanted cells into the hemopoietic compartment of the marrow.
Animals↗
Molecular basis of homing of intravenously transplanted stem cells to the marrow.
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Bone Marrow↗
Purification and partial characterization of ceruloplasmin receptors from rat liver endothelium.
Ceruloplasmin (CP), a circulating glycoprotein, is known for its copper transport. Recently the spectrum of its activity has been increased to include numerous enzymatic functions. CP binds to the liver endothelium and is transported across the cell via a mechanism involving receptor-mediated endocytosis. To isolate CP receptors, we obtained purified preparations of liver endothelium in rats. The membrane was then isolated by ultracentrifugation and solubilized in Triton X-100. Membrane proteins were labeled with 125I and passed through an affinity column in which CP was covalently linked to Sepharose 4B. Most of the radioactivity was eluted with buffer during the first 5 days. When no more radioactivity was eluted with buffer, elution was done either competitively with cold excess CP or 1 M NaCl. By this technique, a sharp single peak of radioactivity was obtained and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. Under both conditions receptors appeared as a single band with Mr of 35,000 containing 3% carbohydrate and an isoelectric point of 5.2.
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Proteoglycan synthesis by cultured liver endothelium: the role of membrane-associated heparan sulfate in transferrin binding.
Liver endothelium has been reported to possess membrane receptors for the iron-binding protein transferrin (Tf). Similarly, the core protein of proteoglycans (PG) associated with cell membrane in many cell systems can bind Tf. To find out if membrane-associated proteoglycans can explain Tf-binding ability of liver endothelium, we investigated the synthesis and distribution of proteoglycans by isolated, cultured liver capillary endothelium. Cells were isolated and cultured for 48 h in sulfate-free medium and pulse-labeled with 35SO4. The relative distribution of 35SO4-labeled macromolecules, determined in the extracellular (EC), membrane-associated (MA), and intracellular (IC) pools, was respectively 74, 15, and 10%. Membrane-associated proteoglycan (MA-PG) was further purified by ion exchange and gel chromatography. Glycosaminoglycan (GAG) chain characterization indicated about 78% chondroitin sulfate, 7% dermatan sulfate, and about 14% heparan sulfate (HS). Similar GAG chain characterization was made for PG in the EC and IC pools. Transferrin-binding ability of MA-PG was studied by affinity column chromatography, using CNBr-activated sepharose bound to transferrin. About 15% of the labeled MA-PG was specifically bound to Tf-affinity column and could be eluted by excess soluble Tf. This proportion was similar to the proportion of HS in the total membrane-associated pool. Moreover, the eluted labeled material was susceptible to pretreatment with heparitinase, confirming its HS nature. We conclude that the transport capillary endothelium of the liver can synthesize HS proteoglycans which are membrane-associated and this MA-HS pool can bind transferrin. The finding may provide a molecular basis for transferrin binding to liver endothelium and may explain the subsequent transendothelial transport of iron-transferrin complexes into the liver.
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Measurement of homing receptors on the surface of leukemic and nonleukemic cell lines.
Selective homing of hemopoietic progenitor cells to hemopoietic organs is the initial event in hemopoiesis. At a molecular level, it is mediated by a membrane receptor on the surface of hemopoietic progenitor cells. The receptor molecule then binds selectively to a glycoconjugate on the surface of bone marrow stromal cells. The molecular nature of this receptor has been shown previously to be a lectin with specificity for an as yet unknown configuration of galactosyl and mannosyl residues of the glycoconjugate. Normal murine progenitor cell lines, FDCP-1 and B6SUT, were found to possess this homing receptor on their cell membrane, and they also bind well to hemopoietic stroma. However, when three human leukemic cell lines, U937, HL60 and K562, were probed for the presence of this receptor, they were found to be deficient in the homing protein. Further, these cells showed little ability to bind the hemopoietic stromal cell line, GB1/6. These findings suggest that leukemic cells experience a loss, or at least alteration, of homing receptor molecule during leukemic transformation. This can result in a diminished ability to bind to marrow stroma. The lack of homing function may be of pathogenetic significance in the lack of differentiation of leukemic cells and may also contribute to the dissemination of leukemic cells in the circulation.
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