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PubMed1999

Influence of microenvironment on mammary epithelial cell survival in primary culture.

Mammary epithelial cells cultured on Engelbreth-Holm-Swarm (EHS) matrix form multicellular structures termed mammospheres, in which cells and matrix become arranged around a central luminal space. In the presence of lactogenic hormones, cells within mammospheres become polarized, form tight intercellular junctions, and secrete milk proteins vectorially into the luminal space. This study examined the mechanism of lumen formation. Histological examination of developing mammospheres showed that cavitation was associated spatially and temporally with the appearance of fragmented nuclear material in apoptotic bodies, and with the presence of cells positively labeled by terminal deoxynucleotide transferase-mediated deoxyuridine nick end-labeling (TUNEL). Analysis of [(32)P]-deoxynucleotide end-labeled genomic DNA by electrophoresis and autoradiography showed DNA laddering indicative of apoptosis. A transient increase in laddering coincided with both lumen formation and the presence of TUNEL-positive cells. Lumen formation, DNA laddering, and detection of TUNEL-positive cells were all accelerated when matrix composition was altered. They were also impaired coordinately when caspase inhibitor was present during the first two days of culture. Therefore, lumen formation in mammosphere cultures is due to selective apoptosis of centrally located cells. Mammosphere cavitation was accompanied by redistribution of matrix constituents to the mammosphere periphery. Western blotting and Western ligand blotting of culture medium showed that lumen formation was also associated with a transient increase in insulin-like growth factor binding protein-5 (IGFBP5), a factor implicated in mammary apoptosis in vivo. We propose that epithelial cell survival during mammosphere development is induced selectively through stabilization by basement membrane constituents, which may act directly on the epithelial cell or confer protection against autocrine apoptotic factors.

Animals↗
PubMed2002

Role of the microenvironment in promoting angiogenesis in acute myeloid leukemia.

Angiogenesis is a crucial event in the survival and progression of solid tumors. To determine whether angiogenesis in acute myeloid leukemia (AML) is an intrinsic property of leukemic cells, the vascularity of bone marrow biopsies was determined. Bone marrow vascularity in newly diagnosed or post-chemotherapy AML patients was increased 4-fold (P < 0.01) and 8.7-fold (P < 0.01), respectively, relative to controls. Vascular endothelial growth factor (VEGF) expression by AML blast cells was assessed by immunohistochemistry, and bone marrow cell supernatants were assayed for secretion of VEGF, fibroblast growth factor-2 (FGF-2), and endostatin by enzyme-linked immunosorbent assay. Diffuse cytoplasmic and strong extracellular VEGF immunoreactivity was seen in bone marrow aspirates from AML patients, but not controls. In contrast, there was no difference in the levels of VEGF, FGF-2, and endostatin secreted by mononuclear cells cultured from bone marrows of AML patients compared to normal controls following two days of culture in vitro. Total angiogenic potential of bone marrow cell supernatants was assessed by endothelial sprouting in vitro and by a chick chorioallantoic membrane assay. No differences were found between 2-day conditioned medium from normal and AML bone marrow mononuclear cells in either assay. Our data show a discrepancy between bone marrow vascularity and VEGF expression in vivo and VEGF expression and angiogenesis from 2-day conditioned medium ex vivo. This suggests that angiogenesis in AML likely represents a response to microenvironmental factors in vivo, rather than being an intrinsic property of leukemic cells.

Acute Disease↗
PubMed1976

Studies of the hemopoietic microenvironment. VIII. Andrenergic and cholinergic innervation of the murine spleen.

Neurohistochemical techniques were used to confirm morphologically the distribution of adrenergic and cholinergic nerves to the splenic microvasculature. The results form the basis of this report. Using these methods, adrenergic innervation was observed only in the adventitia of arteries and arterioles. No cholinergic innervation was found in this site. No adrenergic or cholinergic innervation could be demonstrated to the channels of the red pulp, venules or veins. These data provided morphological evidence that in the murine spleen only splenic arteries and arterioles are innervated; and these have only an adrenergic innervation.

Animals↗
PubMed1976

The hematopoietic microenvironment of the bone marrow: an ultrastructural study of the stroma in rats.

The bone marrow contains branching vascular sinuses lying in a fibroblastic stroma which supports hematopoiesis. This paper describes the stroma and vascular sinuses by scanning and transmission electron microscopy and in freeze-fracture etch replicas in normal fat femoral marrow and in rats made eosinophilic by larvae of trichinella spiralis. The stroma consists primarily of reticular cells which ensheath sinuses as adventitial cells and branch into the surrounding hematopoietic space. They form a spongework on which hematopoietic cells are arranged. Erythroblasts, clustered into islets, and megakaryocytes lie just outside sinuses. Granulocytes, until the metamyelocyte stage, lie in the midst of the hematopoietic cords. Lymphocytes, monocytes and likely stem cells, are clustered about arterial vessels. Macrophages occur throughout the marrow. Fat cells occur adventitial to vascular sinuses and appear to be reticular cells which accumulate fat. Processes of reticular cells closely envelope hematopoietic cells or protrude into them. Reticular cells contain rough ER and are likely fibroblastic. The argyrophilic reticular fibers of the marrow are, however, slender and scanty. Reticular cells are rich in filaments and they may contain many microtubules. They are not phagocytic and possess few lysosomes. The reticular cell cover of a vascular sinus is lifted away as maturing hematopoietic cells approach the sinus, preparatory to crossing the endothelium and entering the circulation. Maturing granulocytes often show microvilli on reaching the basal endothelial surface. The level of eosinophils in the marrow may increase from approximately four to more than 20% after injection of trichinella larvae. Close distinctive association of reticular cells and eosinophils are marked. Reticular cells provide a physical spongwork on which hematopoietic cells are supported. But I postulate that they also trap and induce differentiation of hematopoietic stem cells, and sort the differentiating hematopoietic cells into characteristic locations in their spongework.

Animals↗
PubMed2003

Microenvironment regulation of extracellular signal-regulated kinase activity in chondrocytes: effects of culture configuration, interleukin-1, and compressive stress.

OBJECTIVE: To compare extracellular signal-regulated kinase (ERK) activity in response to interleukin-1 (IL-1) in chondrocytes under various culture configurations designed for the study of cartilage biology and repair, and also in response to dynamic load for chondrocytes in cartilage. METHODS: Isolated bovine articular chondrocytes were maintained in serum-supplemented medium under 4 culture configurations: high-density monolayer, attached to a cut surface of cartilage, within tissue-engineered constructs, or within intact cartilage explants. Samples were subjected to a change of medium with or without IL-1. Cartilage explants were also subjected to dynamic compression. RESULTS: In chondrocyte monolayers, both basal and IL-1-stimulated ERK activities were similarly elevated at 0.5 hours after medium change, diminishing by 74% after 16 hours. In contrast, chondrocytes in other culture configurations exhibited lower basal levels of ERK activity and a moderate activation of ERK in response to IL-1 that was sustained over the 16-hour treatment time. The dynamic component of loading of cartilage explants led to a 5-fold activation of ERK, compared with free-swelling controls, that was indistinguishable from the effects of IL-1. CONCLUSION: ERK signaling in response to IL-1 in chondrocyte monolayers exhibited a pattern that was distinct from that in other culture systems, suggesting that the extracellular matrix plays an important regulatory role in modulating the response to extracellular stimuli. Since IL-1 and dynamic loading have distinct effects on chondrocyte biosynthesis, signaling pathways other than ERK participate in the chondrocyte responses to these stimuli.

Animals↗
PubMed1988

Synovial microenvironment-T cell interactions. Human T cells bind to fibroblast-like synovial cells in vitro.

Synovitis in rheumatoid arthritis is characterized by infiltration of the synovium by T and B lymphocytes and monocytes, as well as by the proliferation of synovial lining cells, fibroblasts, and endothelial cells. To study synovial cell-T cell interactions in vitro, we established cultures of fibroblast-like synovial cells, and used these cells in a synovial cell-T cell binding assay. Using T cells at various stages of differentiation and activation, we found that human thymocytes and mitogen-activated peripheral blood T cells bound to fibroblast-like synovial cells, whereas fresh peripheral blood T cells did not. Moreover, activated T cells from inflammatory synovial tissue or from synovial fluid also bound to fibroblast-like synovial cells cultured in vivo. Antibodies against certain epitopes of the T cell CD2 (35.1) and synovial cell lymphocyte function-associated antigen-3 (LFA-3) (TS2/9) molecules inhibited synovial cell-thymocyte binding. However, these same anti-CD2 and anti-LFA-3 antibodies only partially inhibited synovial cell binding to activated normal peripheral blood T cells. Moreover, T cells from inflammatory synovium from rheumatoid arthritis and psoriatic arthritis patients also bound to synovial cells in vitro. These findings demonstrate that fibroblast-like synovial cells are capable of binding to human T cells in vitro, and suggest that during the course of inflammatory synovitis, synovial fibroblast-T cell interactions may occur in vivo.

Antigens, Differentiation↗
PubMed1991

Chondrons from articular cartilage. III. Morphologic changes in the cellular microenvironment of chondrons isolated from osteoarthritic cartilage.

Chondrons were isolated from human and canine osteoarthritic cartilage using low-speed homogenization techniques. Changes in chondron morphology were evaluated using differential interference-contrast microscopy, phase-contrast microscopy, and histochemical and ultrastructural methods. Chondrocyte viability was assessed using fluorescein diacetate staining, and chondron metabolism was investigated using autoradiography. The results suggest that initial changes in the collagen and proteoglycan distribution within the chondron are followed by chondrocyte proliferation to form clusters. These techniques offer the potential to study cell matrix interactions in degenerative osteoarthritis.

Aged↗
PubMed2005

TGF-beta1 regulation in hepatocyte-NIH3T3 co-culture is important for the enhanced hepatocyte function in 3D microenvironment.

Co-culture of hepatocytes or hepatocyte spheroids with the supporting NIH3T3 in a 3D microcapsule formed with a hybrid natural/synthetic matrix has led to enhanced hepatocyte functions. We investigated the mechanism of the functional enhancement in co-culture with respect to the contributions of soluble factors and direct cell-cell interactions. The conditioned media from the co-culture induced higher P450 cytochrome oxidase activity (indicated by EROD assay) in the microencapsulated hepatocytes than the conditioned media from the NIH3T3- or the hepatocytes-alone controls. Conditioned media from physically separated co-culture of hepatocytes-NIH3T3 by a membrane insert reduced the functional enhancement. Among the known stimulators of hepatocyte functions, TGF(beta)1 is primarily responsible for the stimulation of hepatocyte functions in this 3D co-culture since the removal of TGF(beta)1 by antibody depletion eliminated the functional enhancement and the reconstitution of TGF(beta)1 restored the functional enhancement. Activation of latent TGF(beta)1 in an extracellular environment were upregulated in co-culture with no observable increase in the TGF(beta)1 expression at transcriptional and translational levels. Our data led to an improved understanding of how co-culture enhances hepatocyte functions in vitro and pave the way for further innovations in liver tissue engineering, drug metabolism studies, and other applications that require functional hepatocytes cultured in vitro.

Animals↗
PubMed2006

Modeling evaluation of the fluid-dynamic microenvironment in tissue-engineered constructs: a micro-CT based model.

Natural cartilage remodels both in vivo and in vitro in response to mechanical stresses, hence mechanical stimulation is believed to be a potential tool to modulate extra-cellular matrix synthesis in tissue-engineered cartilage. Fluid-induced shear is known to enhance chondrogenesis in engineered cartilage constructs. The quantification of the hydrodynamic environment is a condition required to study the biochemical response to shear of 3D engineered cell systems. We developed a computational model of culture medium flow through the microstructure of a porous scaffold, during direct- perfused culture. The 3D solid model of the scaffold micro-geometry was reconstructed from 250 micro-computed tomography (micro-CT) images. The results of the fluid dynamic simulations were analyzed at the central portions of the fluid domain, to avoid boundary effects. The average, median and mode shear stress values calculated at the scaffold walls were 3.48, 2.90, and 2.45 mPa respectively, at a flow rate of 0.5 cm(3)/min, perfused through a 15 mm diameter scaffold, at an inlet fluid velocity of 53 microm/s. These results were compared to results estimated using a simplified micro-scale model and to results estimated using an analytical macro-scale porous model. The predictions given by the CT-based model are being used in conjunction with an experimental bioreactor model, in order to quantify the effects of fluid-dynamic shear on the growth modulation of tissue-engineered cartilage constructs, to potentially enhance tissue growth in vitro.

Bioreactors↗
PubMed2001

Formation of highly enantioenriched microenvironments by stochastic sorting of conglomerate crystals: a plausible mechanism for generation of enantioenrichment on the prebiotic earth.

Abiotic generation of local areas of enantioenrichment is to be expected whenever one deals with the 5-10% of organic solids that crystallize as conglomerates. Since an individual crystal of a conglomerate contains only a single enantiomer, simple sorting processes involving winds, waves, or similar forces can act to deposit individual crystals into unique environments. Subsequent dissolution may afford nearly enantiopure solutions. Therefore, in contrast to common perception, enantioenrichment is not a unique signature of living systems, it is simply evidence of a certain degree of chemical complexity.

Chemistry, Organic↗
PubMed1987

Inability of neural crest cells to colonize the presumptive aganglionic bowel of ls/ls mutant mice: requirement for a permissive microenvironment.

The enteric system is formed by cells that migrate to the bowel from the neural crest. In order to gain insight into intraenteric factors that influence this migration, the colonization of the bowel of the ls/ls mouse was investigated. The terminal 2 mm of ls/ls intestine fails to become colonized by crest cells and thus remains aganglionic. The entire bowel of control mice and ls/ls mice was explanted before the appearance in situ of recognizable neurons and grown in organotypic tissue culture. Neurons, detected by the histochemical demonstration of acetylcholinesterase activity, developed throughout the length of the control gut, but, even in vitro, were excluded from the terminal segment of the ls/ls intestine. Co-culture experiments were done, in which primary and secondary sources of crest cells were combined with recipient segments of bowel, to test the ability of the recipient tissue to become colonized by neural precursors. The primary source was murine crest cells migrating away from an explant of the neuraxis. Secondary sources included avian and murine foregut (control and ls/ls) containing migratory crest cells as well as the quail ganglion of Remak. Recipient segments of bowel included control avian and murine hindgut, explanted before the tissue had become colonized by crest cells in situ, as well as the presumptive aganglionic bowel of ls/ls mice. Both primary and secondary sources of crest cells proved to be able to contribute neurons to the control segments of recipient hindgut. Species differences were no barrier to the colonization of the bowel in vitro. Moreover, the ls/ls foregut was as good a source of neural precursors for a normal recipient bowel, as was control avian or murine foregut. In contrast, none of the sources of crest cells that were utilized contributed neurons to the presumptive aganglionic gut of ls/ls mice. Both cells and processes of enteric neurons developing in vitro (detected by demonstrating neurofilament immunoreactivity) tended to be excluded from the presumptive aganglionic tissue. On the other hand, neurites, but not cell bodies, of dorsal root ganglia co-cultured with presumptive aganglionic ls/ls bowel did enter the abnormal zone. These data are consistent with the hypothesis that nonneuronal elements of the wall of the presumptive aganglionic region of the ls/ls gut are abnormal and prevent the colonization of this segment of the gut with viable neural precursors from the neural crest.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗