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At least 199 records · Page 11Linked to original sources
PubMed1996

Human ryudocan from endothelium-like cells binds basic fibroblast growth factor, midkine, and tissue factor pathway inhibitor.

Ryudocan, a heparan sulfate proteoglycan, was isolated from human endothelium-like EAhy926 cells by a combination of ion-exchange and immunoaffinity chromatography. Purified human ryudocan has biochemical properties similar to those of rat ryudocan isolated from microvascular endothelial cells. Human ryudocan contains only heparan sulfate (HS) glycosaminoglycan chains along with a core protein with an apparent molecular mass of 30 kDa. We evaluated the interactions between purified human ryudocan and several extracellular ligands by using a solid-phase binding assay. We found that basic fibroblast growth factor (bFGF), midkine (MK), and tissue factor pathway inhibitor (TFPI) exhibit significant ryudocan binding. Heparitinase (but not chondroitin ABC lyase) treatment destroyed the ability of ryudocan binding to bFGF, MK, and TFPI. Heparin and HS, but not chondroitin sulfate, inhibited such ryudocan binding. Thus, the HS chains of ryudocan appear to be responsible for its binding to bFGF, MK, and TFPI. The apparent dissociation constants for purified ryudocan were as follows: bFGF, 0.50 nM; MK, 0.30 nM; and TFPI, 0.74 nM. Immunohistochemical analysis revealed that ryudocan was expressed in fibrous connective tissues, peripheral nerve tissues, and placental trophoblasts. These findings suggest that ryudocan may possess multiple biological functions, such as bFGF modulation, neurite growth promotion, and anticoagulation, via HS-binding effectors in the cellular microenvironment.

Amino Acid Sequence↗
PubMed1997

Examining the role of Paneth cells in the small intestine by lineage ablation in transgenic mice.

The Paneth cell lineage is one of four epithelial lineages derived from the adult mouse small intestine's multipotent stem cell. Mature Paneth cells secrete antimicrobial peptides (cryptdins), growth factors, as well as two gene products, a secreted phospholipase A2 and matrilysin, that has been implicated as modifiers of adenoma formation in mice containing a mutation in the tumor suppressor Apc. Immature Paneth cells are located just above and below the cell layer, in intestinal crypts, that has been proposed to contain the multipotent stem cell. Paneth cells differentiate during a downward migration to the crypt base. The location and direction of Paneth cell migration, their high density and long residency time at the crypt base, and the nature of their secreted gene products, suggest that they may influence the structure and/or function of the stem cell niche. Paneth cell ablation can therefore be viewed as an experimental manipulation of the cellular microenvironment that purportedly contains the stem cell and its immediate descendants. Two types of ablation experiments were performed in transgenic mice. Nucleotides -6500 to +34 of the mouse cryptdin-2 gene (CR2) were used to express an attenuated diphtheria toxin A fragment. Light and electron microscopic immunohistochemical analyses of several pedigrees of postnatal day 28 to 180 animals established that ablation of Paneth cells is accompanied by an increase in the proportion of undifferentiated crypt base columnar cells. These cells normally co-exist with Paneth cells. The ablation does not produce a detectable effect on the proliferation or terminal differentiation programs of the other three lineages or on host-microbial interactions. The last conclusion is based on the ability of crypts to remain free of microbes detectable by Gram and Warthin-Starry stains and by retention of the normal crypt-villus distribution of components of the diffuse gut-associated lymphoid tissue. CR2-directed expression of simian virus 40 large T antigen also results in a loss of mature Paneth cells but produces a marked amplification of crypt cells having a morphology intermediate between Paneth and granule goblet cells. EM immunohistochemical analyses suggest that intermediate cells can differentiate to mature goblet cells but not to Paneth cells, as they migrate up the crypt-villus axis. Our findings suggest that (i) stemness in the crypt is not defined by instructive interactions involving the Paneth cell; (ii) expressing a Paneth cell fate may require that precursors migrate to the crypt base; (iii) antimicrobial factors produced by Paneth cells are not required to prevent colonization of small intestinal crypts; and (iv) this lineage does not function to maintain the asymmetric crypt-villus distribution of components of the diffuse gut-associated lymphoid tissue.

Animals↗
PubMed1998

Dermatan sulfate released after injury is a potent promoter of fibroblast growth factor-2 function.

Proteoglycans have been shown in vitro to bind multiple components of the cellular microenvironment that function during wound healing. To study the composition and function of these molecules when derived from an in vivo source, soluble proteoglycans released into human wound fluid were characterized and evaluated for influence on fibroblast growth factor-2 activity. Immunoblot analysis of wound fluid revealed the presence of syndecan-1, syndecan-4, glypican, decorin, perlecan, and versican. Sulfated glycosaminoglycan concentrations ranged from 15 to 65 microgram/ml, and treatment with chondroitinase B showed that a large proportion of the glycosaminoglycan was dermatan sulfate. The total glycosaminoglycan mixture present in wound fluid supported the ability of fibroblast growth factor-2 to signal cell proliferation. Dermatan sulfate, and not heparan sulfate, was the major contributor to this activity, and dermatan sulfate bound FGF-2 with Kd = 2.48 microM. These data demonstrate that proteoglycans released during wound repair are functionally active and provide the first evidence that dermatan sulfate is a potent mediator of fibroblast growth factor-2 responsiveness.

Body Fluids↗
PubMed2004

Bim is an apoptosis sensor that responds to loss of survival signals delivered by epidermal growth factor but not those provided by integrins.

Anoikis is a rapid apoptosis response that is initiated within a few minutes after inhibition of integrin signaling. In mammary epithelia, anoikis is mediated by subcellular translocation of Bax from the cytosol to mitochondria where it activates the intrinsic apoptosis pathway. The Bcl-2 homology 3 domain-only protein, Bim, has been proposed to have a key role in the apoptosis response of an epithelial cell line with reduced sensitivity to loss of integrin signaling, which undergoes apoptosis over a period of several days in suspension culture. Here we tested the involvement of Bim in the rapid anoikis response of mouse mammary epithelial cells and discovered that Bim does not have a role in detecting integrin-mediated signals. Instead Bim senses the loss of survival cues mediated by epidermal growth factor. Cell lines selected over many passages in culture have lost much of their sensitivity to anoikis signals arising from an altered cellular microenvironment and may undergo apoptosis through acquired mechanisms.

Animals↗
PubMed2003

The low viral production in trophoblastic cells is due to a high endocytic internalization of the human immunodeficiency virus type 1 and can be overcome by the pro-inflammatory cytokines tumor necrosis factor-alpha and interleukin-1.

Maternal-infant transmission of human immunodeficiency virus type-1 (HIV-1) is the primary cause of this retrovirus infection in neonates. Trophoblasts have been proposed to play a critical role in modulating virus spread to the fetus. This paper addresses the mechanism of HIV-1 biology in trophoblastic cells. The trophoblastic cell lines BeWo, JAR, and JEG-3 were infected with reporter HIV-1 particles pseudotyped with envelope glycoproteins from the vesicular stomatitis virus or various strains of HIV-1. We demonstrate that despite a high internalization process of HIV-1 and no block in viral production, HIV-1 established a limited infection of trophoblasts with the production of very few progeny viruses. The factor responsible for this restriction to virus replication in such a cellular microenvironment is that the intracellular p24 is concentrated predominantly in endosomal vesicles following HIV-1 entry. HIV-1 transcription and virus production of infectious particles were both augmented upon treatment of trophoblasts with tumor necrosis factor-alpha and interleukin-1. However, the amount of progeny virions released by trophoblasts infected with native HIV-1 virions was so low even in the presence of pro-inflammatory cytokines that a co-culture step with indicator cells was necessary to detect virus production. Collectively these data illustrate for the first time that the natural low permissiveness of trophoblasts to productive HIV-1 infection is because of a restriction in the mode of entry, and such a limitation can be overcome with physiologic doses of tumor necrosis factor-alpha and interleukin-1, which are both expressed by the placenta, in conjunction with cell-cell contact. Considering that there is a linear correlation between viral load and HIV-1 vertical transmission, the environment may thus contribute to the propagation of HIV-1 across the placenta.

Cell Line↗
PubMed2004

Hyper-responsiveness to stimulation of human immunodeficiency virus-infected CD4+ T cells requires Nef and Tat virus gene products and results from higher NFAT, NF-kappaB, and AP-1 induction.

A chronic state of immune hyperactivation is a feature of human immunodeficiency virus type-1 (HIV-1) infection. Studies on the molecular mechanisms by which HIV-1 can modulate the activation state of T cells indicate that both Nef and Tat can alter T cell activation. However, the vast majority of data has been obtained from experiments performed with vectors encoding a single virus protein. We demonstrate that infection of human CD4(+) T lymphocytes with fully infectious HIV-1 leads to a hyper-responsiveness of the interleukin-2 promoter. Hypersensitivity in HIV-1-infected T cells was observed upon stimulation with various agents that are engaging different signal transduction pathways. Experiments performed with recombinant heat stable antigen-encoding HIV-1 indicated that the virus-infected cells are the cells with an enhanced response. Both Nef and Tat are involved in this virus-mediated enhancing effect on interleukin-2 promoter activity. Interestingly, whereas Nef seems to be acting mainly through hyperactivation of nuclear factor of activated T cells (NFAT), Tat acts in an NFAT-independent manner. Mobility shift experiments demonstrated that the HIV-1-associated priming of human T cells for stimulation results in a greater induction of transcription factors recognized as essential players in T cell activation, i.e. NFAT, NF-kappaB, and AP-1. A hyper-responsive state was also established upon HIV-1 infection of a more natural cellular reservoir, i.e. primary CD4(+) T lymphocytes. Considering that the HIV-1 life cycle is tightly regulated by the T cell signaling machinery, the priming for activation of a major viral reservoir represents a means by which this retrovirus can create an ideal cellular microenvironment for its propagation and maintenance.

CD4-Positive T-Lymphocytes↗
PubMed2004

Aqueous and micelle-bound structural characterization of the epidermal growth factor receptor juxtamembrane domain containing basolateral sorting motifs.

The EGF receptor is the prototype for four highly related receptors constituting the ErbB family. The EGF receptor is normally targeted to the basolateral membrane in polarized epithelial cells, where it relays information from underlying tissues. Two basolateral sorting signals have been mapped to the cytoplasmic juxtamembrane region of the receptor, a dominant signal comprised of a polyproline core (667-PXXP) and a preceding basic residue (Arg662), and a consensus leucine-based signal (658-LL) responsible for residual sorting when the 667-PXXP signal is absent or defective. The goal of this study was to define the structure of these signals, and gain some insights into how these structures might be regulated by cellular microenvironment. Structural information was acquired for two peptides corresponding to EGF receptor residues Arg645 and Ala674 in aqueous solution or in the presence of membrane-mimicking dodecylphosphocholine micelles, using a variety of NMR and CD spectroscopic methods. Chemical shift data indicate that the 667-PXXP signal does not bind to the micelles and is in random coil state in both aqueous solution and a micellar environment, raising the possibility that 667-PXXP switches to an ordered structure during interaction with the basolateral sorting machinery. In contrast, the adjacent region including 658-LL does bind to micelles mediated by a highly positively charged region located between Arg645 and Arg656. The micelle-bound region also includes Thr654, a known substrate for PKC. This suggests a distinct mode of regulation for this signal involving membrane association and/or phosphorylation.

Amino Acid Sequence↗
PubMed2002

Engineering liver therapies for the future.

Treatment of liver disease has been greatly improved by the advent and evolution of liver transplantation. However, as demand for donor organs continues to increase beyond their availability, the need for alternative liver therapies is clear. Several approaches including extracorporeal devices, cell transplantation, and tissue-engineered constructs have been proposed as potential adjuncts or even replacements for transplantation. Simultaneously, experience from the liver biology community have provided valuable insight into tissue morphogenesis and in vitro stabilization of the hepatocyte phenotype. The next generation of cellular therapies must therefore consider incorporating cell sources and cellular microenvironments that provide both a large population of cells and strategies to maintain liver-specific functions over extended time frames. As cell-based therapies evolve, their success will require contribution from many diverse disciplines including regenerative medicine, developmental biology, and transplant medicine.

Animals↗
PubMed2003

Microfluidic patterning of cells in extracellular matrix biopolymers: effects of channel size, cell type, and matrix composition on pattern integrity.

The organization of cells within an extracellular matrix is critical to promote appropriate cellular interactions and tissue function in vivo. The ability to design and create biologically relevant cellular arrangements via microfluidic patterning on surfaces provides new capabilities for tissue engineering and biomimetics. The purpose of this article is to describe techniques using microfluidic patterning of three-dimensional biopolymer matrices to improve cellular pattern integrity and to provide microscale control over cellular microenvironments. Results demonstrated that the incorporation of extracellular matrix biopolymers in cell microfluidic patterning results in a more stable pattern of adherent human endothelial cells than patterning without matrix components after several days in vitro. This may be important for carrying out long-term biological experiments and tissue engineering in vitro. Moreover, chemical components in the patterned biopolymer matrices, such as collagen, chitosan, and fibronectin, influenced the ability of the matrices to control cell migration and pattern stability over time. Thus, microfluidic patterning of cells in extracellular matrix biopolymers was shown to be useful in patterning multiple cell types in well-defined three-dimensional geometries.

Biopolymers↗
PubMed2005

Possible advantages of stem cell transfusion into the peripheral circulation, as opposed to localized transplantation in situ.

Studies to date have overlooked the possible limitations of transplanting stem cells locally (in situ), directly at the site of tissue or organ damage. This approach is contrary to the intrinsic physiological process of tissue and organ regeneration in vivo, which is thought to involve the activation of stem cells resident within the transplanted tissues or their mobilization from ectopic sites, in particular the bone marrow. Signaling pathways and other molecular processes within stem cells transplanted in situ may not be primed to achieve optimal tissue regeneration and may even be confused by the sudden rapid transition in the cellular microenvironment encountered during transplantation. Moreover, there is a risk of the transplanted cells giving rise to undesired lineages at the transplantation site. A novel alternative would be to transfuse stem cells into the peripheral blood circulation, followed by induced homing to the site of tissue damage. This could better replicate the natural physiological process of tissue repair in vivo. Transfusion into the peripheral blood circulation could be a strategy to augment the inadequate mobilization of endogenous adult stem cells from ectopic sites for tissue repair, which may be the case for older patients. The transfused stem cells can then be induced to home in on a damaged tissue or organ, via the controlled release of specific cytokines or chemokines (i.e., stromal cell derived factor-1) emanating from that particular tissue or organ. The gradient of released cytokines/ chemokines within the peripheral blood circulation would then direct the chemotactic migration and homing of the transfused stem cells to the target tissue or organ.

Blood Circulation↗
PubMedDate not supplied

Control of cellular organization in three dimensions using a microfabricated polydimethylsiloxane-collagen composite tissue scaffold.

Parallel channels of various dimensions have been shown to cause a monolayer of cells in culture to align in the direction of the channels. For the engineering of complex organ systems to become a reality, similar control over the cellular microenvironment in three dimensions must be achieved. Using microfabrication, a polydimethylsiloxane (PDMS) scaffold (40 microm wide, 70-microm-deep parallel channels separated by 25-microm-wide walls) was created. A fibroblast-seeded collagen matrix was then molded around this PDMS scaffold. The PDMS scaffold served as an internal skeleton to guide the cells to grow in the prescribed three-dimensional pattern. Organization, aspect ratio, and the z diameter of the cells were analyzed by confocal microscopy. Fibroblasts elongated and organized in the direction of the channels throughout the height of the scaffold. The mean angle of the cells off of the long axis of the channels was 4.3 +/- 0.7 degrees as opposed to 32.6 +/- 2.2 degrees in controls. The morphology of the cells was also affected by the PDMS scaffold. The nuclei were longer (1.25x) and thinner (0.75x) than in control gels; however, no changes in diameter of the cells in the z direction were seen.

Biocompatible Materials↗
PubMed1994

Members of the syndecan family of heparan sulfate proteoglycans are expressed in distinct cell-, tissue-, and development-specific patterns.

The syndecans are a gene family of four transmembrane heparan sulfate proteoglycans that bind, via their HS chains, diverse components of the cellular microenvironment. To evaluate the expression of the individual syndecans, we prepared cDNA probes to compare mRNA levels in various adult mouse tissues and cultured mouse cells representing various epithelial, fibroblastic, endothelial, and neural cell types and B cells at various stages of differentiation. We also prepared antibody probes to assess whether the extracellular domains of the individual syndecans are shed into the conditioned media of cultured cells. Our results show that all cells and tissues studied, except B-stem cells, express at least one syndecan family member; most cells and tissues express multiple syndecans. However, each syndecan family member is expressed selectively in cell-, tissue-, and development-specific patterns. The extracellular domain of all syndecan family members is shed as an intact proteoglycan. Thus, most, if not all, cells acquire a distinctive repertoire of the four syndecan family members as they differentiate, resulting in selective patterns of expression that likely reflect distinct functions.

Animals↗
PubMed2006

Apoptosis: a relevant tool for anticancer therapy.

Apoptosis is a form of cell death that permits the removal of damaged, senescent or unwanted cells in multicellular organisms, without damage to the cellular microenvironment. Defective apoptosis represents a major causative factor in the development and progression of cancer. The majority of chemotherapeutic agents, as well as radiation, utilize the apoptotic pathway to induce cancer cell death. Resistance to standard chemotherapeutic strategies also seems to be due to alterations in the apoptotic pathway of cancer cells. Recent knowledge on apoptosis has provided the basis for novel targeted therapies that exploit apoptosis to treat cancer. These new target include those acting in the extrinsic/intrinsic pathway, proteins that control the apoptosis machinery such as the p53 and proteosome pathway. Most of these forms of therapy are still in preclinical development because of their low specifity and susceptibility to drug resistance, but several of them have shown promising results. In particular, this review specifically aims at providing an update of certain molecular players that are already in use in order to target apoptosis (such as bortezomib) or which are still being clinically evaluated (such ONYX-015, survivin and exisulind/aptosyn) or which, following preclinical studies, might have the necessary requirements for becoming part of the anticancer drug programs (such as TRAIL/Apo2L, apoptin/VP3).

Animals↗
PubMed1998

Modulation of human epidermal cell response to 2,3,7,8-tetrachlorodibenzo-p-dioxin by epidermal growth factor.

Cultured human epidermal cells were treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the presence or absence of epidermal growth factor (EGF). In both normal keratinocytes and a spontaneously immortalized keratinocyte (SIK) line, TCDD treatment in the absence of EGF induced a marked reduction in colony size and cell number, and it perturbed colony morphology. These effects were largely prevented by EGF, indicating that growth factor action in the cellular microenvironment may considerably modify TCDD action in target cells. Both TCDD and EGF substantially reduced expression of the differentiation markers keratin 1 and keratin 10 in the normal and immortalized cells, and did so in an additive fashion. The cells did not display a general loss of differentiated function, since several other markers, including involucrin, were little affected. EGF dramatically stimulated telomerase activity in SIK cultures, and TCDD prevented this action but not by reducing cell growth. However, EGF did not stimulate telomerase activity in normal human epidermal cells despite an evident increase in their growth. The growth factor stimulation of telomerase in the minimally deviated SIK line suggests that derepression of enzyme activity in normal cells may occur in a stepwise fashion during neoplastic progression. TCDD could act as a late stage tumor promoter by selecting for variants in which telomerase is constitutively active.

Cell Differentiation↗
PubMed2002

Hormonal regulation and cell-specific expression of endothelin-converting enzyme 1 isoforms in bovine ovarian endothelial and steroidogenic cells.

Endothelin-converting enzyme 1 (ECE-1) is a key enzyme in the biosynthesis of endothelin 1 (ET-1), a potent regulator of ovarian function. Different ECE-1 isoforms are localized in distinct intracellular compartments. Thus, the spatial and temporal pattern of ECE-1 expression determines the site of big ET-1 activation and the bioavailability of ET-1. This study was undertaken to investigate the hormonal regulation and cell-specific expression of ECE-1 isoforms in endothelial and steroidogenic cells of bovine follicles and corpora lutea (CL). Using enriched follicular and luteal cell subpopulations and in situ hybridization techniques, we showed that the ECE-1 gene is expressed by both endothelial and steroidogenic cells; however, the intracellular ECE-1a isoform was present only in ET-1-expressing endothelial cells. Steroidogenic cells in follicles or in CL, deficient in ET-1, expressed only the plasma membrane ECE-1b isoform. The intensity of antisense ECE-1 labeling in the granulosa cell layer increased with follicular size; insulin-like growth factor I and insulin upregulated ECE-1 expression when cultured with granulosa cells, suggesting that these growth factors may increase ECE-1 in growing follicles. In contrast, ET-1 and LH downregulated ECE-1 in steroidogenic cells. This effect could account for low ECE (and ET-1) levels, which characterize the early luteal phase. These findings suggest that ECE-1 is regulated during different stages of the cycle in a physiologically relevant manner. The hormonal regulation and intracellular localization of bovine ECE-1 isoforms revealed in this study may provide new insights into ET-1 biosynthesis and mode of action in different cellular microenvironments within the ovary.

Animals↗
PubMed2001

Fibrocytes induce an angiogenic phenotype in cultured endothelial cells and promote angiogenesis in vivo.

Angiogenesis is an ordered process requiring the inter-play of numerous cellular and humoral factors. Studies over the past 20 years have identified several growth factors, cytokines, and enzymes that promote blood vessel formation. Most have revealed how individual factors promote an angiogenic phenotype in endothelial cells in vitro or contribute to blood vessel formation in vivo. However, the fundamental question that remains unanswered is how the cellular microenvironment contributes to angiogenesis. Fibrocytes are a recently characterized mesenchymal cell type isolated from peripheral blood that rapidly enter subcutaneously implanted wound chambers and sites of tissue injury. Here we describe the induction of an angiogenic phenotype in microvascular endothelial cells in vitro and promotion of angiogenesis in vivo by cultured fibrocytes. Fibrocytes constitutively secrete extracellular matrix-degrading enzymes, primarily matrix metalloproteinase 9, which promotes endothelial cell invasion. In addition, fibrocytes secrete several proangiogenic factors including VEGF, bFGF, IL-8, PDGF, and hematopoietic growth factors that promote endothelial cell migration, proliferation, and/or tube formation. By contrast, they do not produce representative antiangiogenic factors. Finally, both autologous fibrocytes and fibrocyte-conditioned media were found to induce blood vessel formation in vivo using the Matrigel angiogenesis model.

3T3 Cells↗
PubMed2006

A novel formulation of oxygen-carrying matrix enhances liver-specific function of cultured hepatocytes.

Oxygen is an important component of the cellular microenvironment, mediating cell survival, differentiation, and function. Oxygen supply is a limiting factor during culture of highly metabolic cells such as hepatocytes. Here we present a simple formulation of a fluorocarbon-based oxygen carrier embedded in collagen gel that increases oxygen concentration in culture 6-fold. Rat hepatocytes cultured on oxygen carrier-collagen showed a significant increase in viability and function. Cytochrome P450IA1 activity was increased by 140% in serum-free cultures and by 820% in serum-containing cultures. The significantly higher hepatocellular function on oxygen carrier-collagen matrix persisted and increased during long-term culture. Long-term albumin secretion was increased by 350% in serum-free cultures and by 166% in serum-containing culture. Long-term urea secretion was increased by 79% in serum-free cultures and by 76% in serum-containing cultures. We conclude that oxygen supply may limit hepatocyte function in vitro. This limitation can be overcome by addition of an oxygen carrier to the extracellular matrix. Culture of hepatocytes on oxygen-carrying matrix mimics the oxygen-rich environment of the liver and provides a simple method for enhanced long-term function.

Animals↗
PubMed2002

Secondary parathyroid hyperplasia in tuberous sclerosis: report of a case with large eosinophilic ganglion-like cells similar to those of subependymal giant cell astrocytoma, tubers, and atypical angiomyolipoma.

We report a case of secondary parathyroid hyperplasia in a 49-year-old man with tuberous sclerosis. Two parathyroid glands had collections of large, eosinophilic ganglion-like endocrine cells that to our knowledge have not been previously described at this site. These cells are morphologically similar to those of subependymal giant cell astrocytoma, tubers, and atypical angiomyolipoma, all of which may arise in the setting of tuberous sclerosis. These large, eosinophilic ganglion-like cells found in different affected organs appear to be distinctive of tuberous sclerosis. We suggest these large eosinophilic cells arise from a common stem cell precursor that acquires variable phenotypes according to alterations in the cellular microenvironment.

Angiomyolipoma↗