Induction of Hepatocyte Differentiation by the Extracellular Matrix and an RGD-Containing Synthetic Peptide

1991 ◽  
Vol 252 ◽  
Author(s):  
David J. Mooney ◽  
Robert Langer ◽  
Linda K. Hansen ◽  
Joseph P. Vacanti ◽  
Donald E. Ingber
Keyword(s):  
Extracellular Matrix ◽  
Protein Secretion ◽  
Synthetic Peptide ◽  
Cell Spreading ◽  
Specific Protein ◽  
Hepatocyte Differentiation ◽  
Liver Specific Protein ◽  
Extensive Cell ◽  
Adhesive Interactions

ABSTRACTTo design novel biomaterials for hepatocyte transplantation it will be necessary to determine whether specific extracellular matrix (ECM) molecule(s) or the adhesive interactions between the surface and hepatocytes are responsible for regulation of hepatocyte function. Purified ECM molecules (laminin, fibronectin, types I and IV collagen) and a synthetic peptide containing the arginine-glycine-aspartate (RGD) cell-binding sequence were precoated at defined densities to non-adhesive polystyrene dishes. Hepatocytes cultured on dishes coated with a low density of ECM molecules (1 ng/cm2) maintained a round morphology, and high liver-specific protein secretion rates. In contrast, culturing hepatocytes on increasing ECM densities (50–1000 ng/cm2) resulted in extensive cell spreading, a loss of liver-specific protein secretion, and cell growth. Hepatocytes cultured on dishes coated with the RGD-containing peptide did not spread even on a high density of the peptide (10,000 ng/cm2), and albumin secretion remained high for hepatocytes cultured on all peptide densities (1–10,000 ng/cm2). These results suggest that a variety of ECM molecules and synthetic peptides are capable of inducing hepatocyte differentiation in vitro, and these effects depend on their ability to promote cell spreading.

scholarly journals Multiple mechanisms of dissociated epidermal cell spreading.

1983 ◽  
Vol 96 (1) ◽  
pp. 63-67 ◽  
Author(s):  
K S Stenn ◽  
J A Madri ◽  
T Tinghitella ◽  
V P Terranova
Keyword(s):  
Serum Albumin ◽  
Epidermal Cell ◽  
Type Iv Collagen ◽  
Cell Spreading ◽  
Specific Protein ◽  
Epidermal Cells ◽  
Type Iv ◽  
Basement Membrane Protein ◽  
Specific Proteins

To test the possibility that epidermal cells use a common basement membrane protein whenever they spread, in vitro experiments were conducted using trypsin-dissociated guinea pig epidermal cells and the following proteins: human serum, bovine serum albumin, serum fibronectin, Type IV collagen, laminin, and epibolin (a recently described serum glycoprotein which supports epidermal cell spreading; Stenn, K.S., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:6907.). When the cells were added to media containing the specific proteins, all the tested proteins, except for serum albumin, supported cell spreading. Added to protein-coated substrates in defined media, the cells spread on fibronectin, epibolin, and laminin-Type IV collagen, but not on albumin or whole serum. In none of these experiments were the results qualitatively affected by the presence of cycloheximide. Antibodies to a specific protein blocked cell spreading on that protein but not on the other active proteins, e.g. whereas antibodies to epibolin blocked cell spreading on epibolin, they did not affect spreading on fibronectin, collagen, or laminin. In a second assay in which the cells were allowed to adhere to tissue culture plastic before the protein-containing medium was added, the cells spread only if the medium contained epibolin. Moreover, under these conditions the spreading activity of whole serum and plasma was neutralized by antiepibolin antibodies. These results support the conclusion that dissociated epidermal cells possess multiple spreading modes which depend, in part, on the proteins of the substrate, proteins of the medium, and the sequence of cell adhesion and protein exposure.

scholarly journals Basic fibroblast growth factor promotes adhesive interactions of neuroepithelial cells from chick neural tube with extracellular matrix proteins in culture

Development ◽  
1993 ◽  
Vol 119 (3) ◽  
pp. 943-956 ◽  
Author(s):  
Y. Kinoshita ◽  
C. Kinoshita ◽  
J.G. Heuer ◽  
M. Bothwell
Keyword(s):  
Extracellular Matrix ◽  
Neural Tube ◽  
Cell Spreading ◽  
Matrix Proteins ◽  
Fibroblast Growth ◽  
Spreading Effect ◽  
Neuroepithelial Cells ◽  
Beta 1 Integrin ◽  
Adhesive Interactions ◽  
Cell Plating

Fibroblast growth factors have been increasingly assigned mitogenic and trophic roles in embryonic and postnatal development of the nervous system. Little is known, however, of their functional roles in early embryonic neural development at the neural tube stage. We have examined the effect of basic fibroblast growth factor (bFGF) on the adhesive behavior in culture of dissociated brachio-thoracic neural tube cells from 26- to 30-somite stage chick embryos. Cells plated on collagen-coated substratum at a low density attach to the substratum but show poor cell spreading. Addition of bFGF markedly promotes cell spreading, yielding an epithelial morphology. This effect becomes discernible 6–8 hours after cell plating with bFGF and is completed by 24 hours, with half-maximal and maximal effects attained at around 0.4 and 10 ng/ml, respectively. The number of cells remain largely constant up to 24 hours, and then cell survival and/or mitogenic effects of bFGF become apparent. The cell spreading effect is abolished by cycloheximide treatment, inhibited by the anti-beta 1-integrin antibody CSAT, and accompanied by about twofold increases in the expression of beta 1-integrin and vinculin, components of focal adhesion complexes. Cells cultured with bFGF for 24 hours exhibit enhanced cell attachment and cell spreading with little time lag following cell plating. In earlier embryonic stages, developmentally less mature cells depend much more on bFGF for their cell spreading and survival, while in later stages the cell spreading response to bFGF becomes undetectable as neural tube develops to spinal cord. The cell spreading effect of bFGF is realized on specific extracellular matrix proteins including laminin, fibronectin and collagen, but not on vitronectin, arg-gly-asp peptide (PepTite-2000), poly-L-ornithine or others. These results suggest that, in an early stage of neural tube development, bFGF is involved in the developmental regulation of adhesive interactions between neuroepithelial cells and the extracellular matrix, thereby controlling their proliferation, migration and differentiation.

256 ROLE OFTHE BOVINE OOCYTE-SPECIFIC PROTEIN JY-1 IN REGULATION OF MESSENGER RNA ABUNDANCE FOR GENES LINKED TO CUMULUS EXPANSION

2010 ◽  
Vol 22 (1) ◽  
pp. 285 ◽  
Author(s):  
G. Wee ◽  
K. B. Lee ◽  
J. J. Ireland ◽  
G. W. Smith
Keyword(s):  
Extracellular Matrix ◽  
Cumulus Cell ◽  
Germinal Vesicle ◽  
Specific Protein ◽  
Negative Control ◽  
Mrna Abundance ◽  
Cumulus Expansion ◽  
Vitro Fertilization ◽  
Maturation Medium

We have previously demonstrated a requirement of the oocyte-specific protein JY-1 for oocyte and early embryonic development in cattle. Microin- jection of JY-1 siRNA into cumulus-enclosed germinal vesicle-stage oocytes impedes progression to metaphase II and cumulus expansion during in vitro maturation and limits subsequent embryonic development following in vitro fertilization. Negative effects of siRNA-mediated reduction in JY-1 on oocyte maturation, cumulus expansion, and initial cleavage divisions following in vitro fertilization can be rescued bysupplementation with recom- binant JY-1 protein during oocyte culture. However, the mechanisms involved in JY-1 regulation of above developmental endpoints are unknown. The objective of the current study was to determine whether JY-1-induced regulation of cumulus expansion during meiotic maturation is linked to alterations in mRNA abundance for genes that promote formation and stabilization of the mucified extracellular matrix characteristic of an expanded cumulus layer. Cumulus-enclosed germinal vesicle stage-bovine oocytes were microinjected with JY-1 siRNA, subjected to negative control siRNA microinjection or served as uninjected controls (n = 10 oocytes/treatment; n = 5 replicates), and cultured for 48 h in maturation medium containing 50 μM S-roscovitine to block spontaneous germinal vesicle breakdown. Cumulus-oocyte complexes were then washed and in vitro matured for an additional 24 h in maturation medium minus S-roscovitine. Additional JY-1 siRNA injected and uninjected cumulus-enclosed oocytes were cultured as described earlier but in the presence of 1 ng/mL of recombinant JY-1 protein (n = 10 oocytes/treatment; n = 5 replicates). Dose of recombinant JY-1 protein utilized was previously shown to reverse inhibitory effects of JY-1 siRNA injection on cumulus expansion. After in vitro maturation, cumulus cells were harvested and RNA isolated and subjected to reverse transcription. Real-time PCR analysis was then conducted to determine the effect of treatments on cumulus-cell mRNA abundance for HAS2, HAS3, PTX3, and TNFAIP6. Effects of JY-1 supplementation or depletion (siRNA injection) on cumulus-cell mRNA abundance for HAS2 and HAS3 (enzymes involved in hyaluronan synthesis) were not observed. However, abundance of mRNA for TNFAIP6 and PTX3 (molecules implicated in stabilization of the hyaluronan-rich extracellular matrix) was reduced (relative to uninjected and negative control siRNA groups) in response to JY-1 siRNA injection (P < 0.05) and effects of JY-1 siRNA were rescued by JY-1 protein treatment (P < 0.05). Effects of JY-1 protein supplementation on cumulus-cell TNFAIP6 and PTX3 mRNA in uninjected controls were not observed. Results support a requirement of the oocyte-specific protein JY-1 for regulation of expression of genes functionally linked to stabilization of the hyaluronan-rich extracellular matrix and cumulus expansion. This research was supported by USDA 2008-35203-19094 to G. W. Smith.

scholarly journals Reactivity of synthetic peptide analogs of adhesive proteins in regard to the interaction of human endothelial cells with extracellular matrix

Blood ◽  
1991 ◽  
Vol 77 (10) ◽  
pp. 2200-2206 ◽  
Author(s):  
CS Chen ◽  
J Hawiger
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Synthetic Peptide ◽  
Cell Monolayer ◽  
Human Fibrinogen ◽  
Hybrid Peptides ◽  
Peptide Analogs ◽  
Extracellular Matrix Components ◽  
Adhesive Proteins

Abstract Vascular endothelial cells, providing a nonthrombogenic surface to the lumenal aspect of blood vessels, are anchored to matrix adhesion molecules in the subendothelium through their respective receptors belonging to a superfamily of integrins. We analyzed the reactivity of synthetic peptide analogs of adhesive proteins toward human umbilical vein endothelial cells (HUVEC), assaying their detachment from extracellular matrix and attachment to extracellular matrix components in vitro. Synthetic peptide analogs Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), Arg-Gly-Asp-Val (RGDV), Arg-Gly-Asp-Ser (RGDS), and Arg-Gly-Asp-Phe (RGDF), which are analogous to “cell adhesion site” of fibronectin, vitronectin, von Willebrand factor, and alpha-chain of human fibrinogen, respectively, caused significant detachment of HUVEC from the extracellular matrix in vitro at the concentrations ranging from 0.5 to 1.5 mmol/L. They also interfered with attachment of HUVEC to surfaces coated with subendothelial extracellular matrix or its components. The synthetic peptide analog of HHLGGAKQAGDV, which is homologous to the gamma-chain of human fibrinogen sequence 400–411, did not cause any measurable effect on the integrity of HUVEC monolayers (detachment and attachment). “Hybrid” peptides bearing salient features of both sequences, ie, Ala-Lys-Gln-Arg-Gly-Asp-Phe (AKQRGDF) and Lys- Gln-Arg-Gly-Asp-Phe (KQRGDF), had an attenuated effect on the detachment of HUVEC from extracellular matrix. Thus, the integrity of the human endothelial cell monolayer anchored to the extracellular matrix, as measured in detachment and attachment assays, is disturbed by peptides containing RGD sequence whereas the synthetic peptide His- His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val (HHLGGAKQAGDV) is nonreactive.

scholarly journals The Plasmodium liver-specific protein 2 (LISP2) is an early marker of liver stage development

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Devendra Kumar Gupta ◽  
Laurent Dembele ◽  
Annemarie Voorberg-van der Wel ◽  
Guglielmo Roma ◽  
Andy Yip ◽  
...  
Keyword(s):  
Specific Protein ◽  
Liver Stage ◽  
Early Marker ◽  
Liver Specific Protein ◽  
Prophylactic Drugs ◽  
Stage Development ◽  
Molecular Framework ◽  
Relapsing Malaria

Plasmodium vivax hypnozoites persist in the liver, cause malaria relapse and represent a major challenge to malaria elimination. Our previous transcriptomic study provided a novel molecular framework to enhance our understanding of the hypnozoite biology (Voorberg-van der Wel A, et al., 2017). In this dataset, we identified and characterized the Liver-Specific Protein 2 (LISP2) protein as an early molecular marker of liver stage development. Immunofluorescence analysis of hepatocytes infected with relapsing malaria parasites, in vitro (P. cynomolgi) and in vivo (P. vivax), reveals that LISP2 expression discriminates between dormant hypnozoites and early developing parasites. We further demonstrate that prophylactic drugs selectively kill all LISP2-positive parasites, while LISP2-negative hypnozoites are only sensitive to anti-relapse drug tafenoquine. Our results provide novel biological insights in the initiation of liver stage schizogony and an early marker suitable for the development of drug discovery assays predictive of anti-relapse activity.

scholarly journals Reactivity of synthetic peptide analogs of adhesive proteins in regard to the interaction of human endothelial cells with extracellular matrix

Blood ◽  
1991 ◽  
Vol 77 (10) ◽  
pp. 2200-2206
Author(s):  
CS Chen ◽  
J Hawiger
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Synthetic Peptide ◽  
Cell Monolayer ◽  
Human Fibrinogen ◽  
Hybrid Peptides ◽  
Peptide Analogs ◽  
Extracellular Matrix Components ◽  
Adhesive Proteins

Vascular endothelial cells, providing a nonthrombogenic surface to the lumenal aspect of blood vessels, are anchored to matrix adhesion molecules in the subendothelium through their respective receptors belonging to a superfamily of integrins. We analyzed the reactivity of synthetic peptide analogs of adhesive proteins toward human umbilical vein endothelial cells (HUVEC), assaying their detachment from extracellular matrix and attachment to extracellular matrix components in vitro. Synthetic peptide analogs Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), Arg-Gly-Asp-Val (RGDV), Arg-Gly-Asp-Ser (RGDS), and Arg-Gly-Asp-Phe (RGDF), which are analogous to “cell adhesion site” of fibronectin, vitronectin, von Willebrand factor, and alpha-chain of human fibrinogen, respectively, caused significant detachment of HUVEC from the extracellular matrix in vitro at the concentrations ranging from 0.5 to 1.5 mmol/L. They also interfered with attachment of HUVEC to surfaces coated with subendothelial extracellular matrix or its components. The synthetic peptide analog of HHLGGAKQAGDV, which is homologous to the gamma-chain of human fibrinogen sequence 400–411, did not cause any measurable effect on the integrity of HUVEC monolayers (detachment and attachment). “Hybrid” peptides bearing salient features of both sequences, ie, Ala-Lys-Gln-Arg-Gly-Asp-Phe (AKQRGDF) and Lys- Gln-Arg-Gly-Asp-Phe (KQRGDF), had an attenuated effect on the detachment of HUVEC from extracellular matrix. Thus, the integrity of the human endothelial cell monolayer anchored to the extracellular matrix, as measured in detachment and attachment assays, is disturbed by peptides containing RGD sequence whereas the synthetic peptide His- His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val (HHLGGAKQAGDV) is nonreactive.

Specific Labeling of Protein Domains with Antibody Fragments

1981 ◽  
Vol 39 ◽  
pp. 416-417
Author(s):  
U. Aebi ◽  
L.E. Buhle ◽  
W.E. Fowler
Keyword(s):  
Image Processing ◽  
Specific Protein ◽  
Antibody Fragments ◽  
Supramolecular Organization ◽  
Two Dimensional ◽  
Ordered Structures ◽  
Virus Capsids ◽  
Processing Techniques

Many important supramolecular structures such as filaments, microtubules, virus capsids and certain membrane proteins and bacterial cell walls exist as ordered polymers or two-dimensional crystalline arrays in vivo. In several instances it has been possible to induce soluble proteins to form ordered polymers or two-dimensional crystalline arrays in vitro. In both cases a combination of electron microscopy of negatively stained specimens with analog or digital image processing techniques has proven extremely useful for elucidating the molecular and supramolecular organization of the constituent proteins. However from the reconstructed stain exclusion patterns it is often difficult to identify distinct stain excluding regions with specific protein subunits. To this end it has been demonstrated that in some cases this ambiguity can be resolved by a combination of stoichiometric labeling of the ordered structures with subunit-specific antibody fragments (e.g. Fab) and image processing of the electron micrographs recorded from labeled and unlabeled structures.

Cardiac myocytes cultured on a basement membrane extract of the ehs tumor

1986 ◽  
Vol 44 ◽  
pp. 270-271
Author(s):  
L. Terracio ◽  
A. Dewey ◽  
K. Rubin ◽  
T.K. Borg
Keyword(s):  
Extracellular Matrix ◽  
Basement Membrane ◽  
Cardiac Myocytes ◽  
Normal Tissue ◽  
Cell Spreading ◽  
Collagen Iv ◽  
Basement Membrane Extract ◽  
Membrane Extract ◽  
Growth Development ◽  
Multicellular Organisms

The recognition and interaction of cells with the extracellular matrix (ECM) effects the normal physiology as well as the pathology of all multicellular organisms. These interactions have been shown to influence the growth, development, and maintenance of normal tissue function. In previous studies, we have shown that neonatal cardiac myocytes specifically interacts with a variety of ECM components including fibronectin, laminin, and collagens I, III and IV. Culturing neonatal myocytes on laminin and collagen IV induces an increased rate of both cell spreading and sarcomerogenesis.

Neutrophil migration through in vitro interstitial matrix

1992 ◽  
Vol 50 (1) ◽  
pp. 894-895
Author(s):  
J. Roemer ◽  
S.R. Simon
Keyword(s):  
Extracellular Matrix ◽  
Smooth Muscle ◽  
Cell Migration ◽  
Smooth Muscle Cells ◽  
Inflammatory Cell ◽  
Neutrophil Migration ◽  
Culture Conditions ◽  
Aortic Smooth Muscle ◽  
Specific Culture

We are developing an in vitro interstitial extracellular matrix (ECM) system for study of inflammatory cell migration. Falcon brand Cyclopore membrane inserts of various pore sizes are used as a support substrate for production of ECM by R22 rat aortic smooth muscle cells. Under specific culture conditions these cells produce a highly insoluble matrix consisting of typical interstitial ECM components, i.e.: types I and III collagen, elastin, proteoglycans and fibronectin.

Development of an in vitro Model for the Study of the Response of Equine Tendon Fibroblasts to Injury and Medication

1997 ◽  
Vol 10 (01) ◽  
pp. 6-11 ◽  
Author(s):  
R. F. Rosenbusch ◽  
L. C. Booth ◽  
L. A. Dahlgren
Keyword(s):  
Electron Microscopy ◽  
Extracellular Matrix ◽  
Light Microscopy ◽  
Light And Electron Microscopy ◽  
Tendon Healing ◽  
Matrix Proteins ◽  
Isolated Cells ◽  
Superficial Digital Flexor Tendon ◽  
Vitro Model

SummaryEquine tendon fibroblasts were isolated from explants of superficial digital flexor tendon, subcultured and maintained in monolayers. The cells were characterized by light microscopy, electron microscopy and radiolabel studies for proteoglycan production. Two predominant cell morphologies were identified. The cells dedifferentiated toward a more spindle shape with repeated subcultures. Equine tendon fibroblasts were successfully cryopreserved and subsequently subcultured. The ability to produce proteoglycan was preserved.The isolated cells were identified as fibroblasts, based on their characteristic shape by light microscopy and ultrastructure and the active production of extracellular matrix proteins. Abundant rough endoplasmic reticulum and the production of extracellular matrix products demonstrated active protein production and export. Proteoglycans were measurable via liquid scintillation counting in both the cell-associated fraction and free in the supernatant. This model is currently being utilized to study the effects of polysulfated glycosaminoglycan on tendon healing. Future uses include studying the effects of other pharmaceuticals, such as hyaluronic acid, on tendon healing.A model was developed for in vitro investigations into tendon healing. Fibroblasts were isolated from equine superficial digital flexor tendons and maintained in monolayer culture. The tenocytes were characterized via light and electron microscopy. Proteoglycan production was measured, using radio-label techniques. The fibroblasts were cryopreserved and subsequently subcultured. The cells maintained their capacity for proteoglycan production, following repeated subculturing and cryopreservation.

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