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Biomedical subjects

D E Ingber

Publications and source records attributed to D E Ingber.

17 recordsLinked to original sources
PubMed1992

Altering the cellular mechanical force balance results in integrated changes in cell, cytoskeletal and nuclear shape.

Studies were carried out with capillary endothelial cells cultured on fibronectin (FN)-coated dishes in order to analyze the mechanism of cell and nuclear shape control by extracellular matrix (ECM). To examine the role of the cytoskeleton in shape determination independent of changes in transmembrane osmotic pressure, membranes of adherent cells were permeabilized with saponin (25 micrograms/ml) using a buffer that maintains the functional integrity of contractile microfilaments. Real-time videomicroscopic studies revealed that addition of 250 microM ATP resulted in time-dependent retraction and rounding of permeabilized cells and nuclei in a manner similar to that observed in intact living cells following detachment using trypsin-EDTA. Computerized image analysis confirmed that permeabilized cells remained essentially rigid in the absence of ATP and that retraction was stimulated in a dose-dependent manner as the concentration of ATP was raised from 10 to 250 microM. Maximal rounding occurred by 30 min with projected cell and nuclear areas being reduced by 69 and 41%, respectively. ATP-induced rounding was also accompanied by a redistribution of microfilaments resulting in formation of a dense net of F-actin surrounding retracted nuclei. Importantly, ATP-stimulated changes in cell, cytoskeletal, and nuclear form were prevented in permeabilized cells using a synthetic myosin peptide (IRICRKG) that has been previously shown to inhibit actomyosin filament sliding in muscle. In contrast, both the rate and extent of cell and nuclear rounding were increased in permeabilized cells exposed to ATP when the soluble FN peptide, GRGDSP, was used to dislodge immobilized FN from cell surface integrin receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Actomyosin↗
PubMed1992

Extracellular matrix as a solid-state regulator in angiogenesis: identification of new targets for anti-cancer therapy.

Angiogenesis, the growth of blood capillaries, is regulated by soluble growth factors and insoluble extracellular matrix (ECM) molecules. Soluble angiogenic mitogens act over large distances to initiate capillary growth whereas changes in ECM govern whether individual cells will grow, differentiate, or involute in response to these stimuli in the local tissue microenvironment. Analysis of this local control mechanism has revealed that ECM molecules switch capillary endothelial cells between differentiation and growth by both binding specific transmembrane integrin receptors and physically resisting cell-generated mechanical loads that are applied to these receptors. Control of capillary endothelial cell form and function therefore may be exerted by altering the mechanical properties of the ECM as well as its chemical composition. Understanding of this mechanochemical control mechanism has led to the development of new angiogenesis inhibitors that may be useful for the treatment of cancer.

Animals↗
PubMed1991

Insoluble fibronectin activates the Na/H antiporter by clustering and immobilizing integrin alpha 5 beta 1, independent of cell shape.

Growth of anchorage-dependent cells requires both soluble mitogens and insoluble extracellular matrix molecules such as fibronectin. Soluble growth factors activate chemical signaling pathways and stimulate proliferation by binding to transmembrane receptors. Insoluble fibronectin also binds to cell-surface receptors; however, it is thought to act primarily via effects on the cytoskeleton and cell shape. We recently demonstrated that cell spreading on surface-adsorbed fibronectin activates the Na/H antiporter and that inhibition of this chemical-signaling pathway suppresses growth. We now show that insoluble fibronectin activates the Na/H antiporter by clustering and immobilizing integrin alpha 5 beta 1, independent of effects on cell shape. These results show that an extracellular matrix receptor can behave similarly to a growth factor receptor to activate a signaling pathway implicated in growth control.

Amiloride↗
PubMed1991

Multiple integrins share the ability to induce elevation of intracellular pH.

Previous work has shown that adhesion of anchorage-dependent cells to fibronectin via integrin alpha 5 beta 1 leads to activation of the Na-H antiporter and a rise in intracellular pH (pHi). We now show that adhesion of bovine capillary endothelial cells (BCE) to fibrinogen; collagens type III, IV, and V; laminin; and vitronectin; ligands that bind other members of the integrin family, resulted in significant elevations in pHi. Other ligands (basic fibroblast growth factor, concanavalin A, and thrombin), which bind cells when immobilized on plastic, but that do not bind integrins and do not support cell growth, do not elevate pHi. Adhesion to an antibody against integrin alpha v beta 3 also elevates pHi. Adhesion of peripheral human T lymphocytes to an antibody against the integrin LFA-1 induced a rise in pHi. Antibodies to CD2 or ICAM-2 had only slight effects on pHi, whereas an antibody to the T cell receptor complex that strongly activates T cells induced a large increase in pHi. We conclude that elevation of pHi by integrins is specific and is a property shared by many members of the integrin family.

Animals↗
PubMed1990

Fibronectin controls capillary endothelial cell growth by modulating cell shape.

An in vitro system has been developed to study the mechanism by which fibronectin (FN) regulates capillary endothelial cell growth in the presence of soluble angiogenic mitogens. Endothelial cells were cultured in chemically defined medium containing a constant, saturating amount of basic fibroblast growth factor. Formation of cell-FN contacts was then varied in a controlled fashion by three different techniques: (i) nonadhesive, bacteriological dishes were precoated with increasing densities of FN; (ii) soluble RGD peptides were used to progressively inhibit binding of cell-surface integrin receptors to adsorbed FN; and (iii) FN-coated surfaces were covered with increasingly thick layers of polyhydroxyethylmethacrylate (a nonadhesive polymer) to physically restrict cell access to FN binding sites. Endothelial cells became more extended and proliferated more rapidly as FN coating concentrations were raised from approximately 250 to approximately 10,000 FN molecules per micron 2. Computerized morphometric analysis confirmed that cell shape (projected cell areas) was determined by the density of FN contacts and that DNA synthetic levels were tightly coupled to the extent of cell spreading, regardless of the method used to perturb cell adhesion. In contrast, neither soluble FN nor cell-surface binding of FN-coated microbeads (diameter, 4.5 microns) had any effect on growth when cells were grown in suspension and cell spreading was prohibited. These results suggest that FN controls capillary endothelial cell proliferation based on its ability to support tension-dependent alterations of cell shape--i.e., both by binding to cell-surface integrins and by resisting mechanical loads that are applied to these receptors.

Adrenal Cortex↗
PubMed1990

Control of intracellular pH and growth by fibronectin in capillary endothelial cells.

The aim of this work was to analyze the mechanism by which fibronectin (FN) regulates capillary endothelial cell proliferation. Endothelial cell growth can be controlled in chemically-defined medium by varying the density of FN coated on the substratum (Ingber, D. E., and J. Folkman. J. Cell Biol. 1989. 109:317-330). In this system, DNA synthetic rates are stimulated by FN in direct proportion to its effect on cell extension (projected cell areas) both in the presence and absence of saturating amounts of basic FGF. To investigate direct growth signaling by FN, we carried out microfluorometric measurements of intracellular pH (pHi), a cytoplasmic signal that is commonly influenced by soluble mitogens. pHi increased 0.18 pH units as FN coating densities were raised and cells progressed from round to spread. Intracellular alkalinization induced by attachment to FN was rapid and followed the time course of cell spreading. When measured in the presence and absence of FGF, the effects of FN and FGF on pHi were found to be independent and additive. Furthermore, DNA synthesis correlated with pHi for all combinations of FGF and FN. Ethylisopropylamiloride, a specific inhibitor of the plasma membrane Na+/H+ antiporter, completely suppressed the effects of FN on both pHi and DNA synthesis. However, cytoplasmic pH per se did not appear to be a critical determinant of growth since DNA synthesis was not significantly inhibited when pHi was lowered over the physiological range by varying the pH of the medium. We conclude that FN and FGF exert their growth-modulating effects in part through activation of the Na+/H+ exchanger, although they appear to trigger this system via separate pathways.

Adrenal Cortex↗
PubMed1989

Mechanochemical switching between growth and differentiation during fibroblast growth factor-stimulated angiogenesis in vitro: role of extracellular matrix.

The angiogenic factor, basic fibroblast growth factor (FGF), either stimulates endothelial cell growth or promotes capillary differentiation depending upon the microenvironment in which it acts. Analysis of various in vitro models of spontaneous angiogenesis, in combination with time-lapse cinematography, demonstrated that capillary tube formation was greatly facilitated by promoting multicellular retraction and cell elevation above the surface of the rigid culture dish or by culturing endothelial cells on malleable extracellular matrix (ECM) substrata. These observations suggested to us that mechanical (i.e., tension-dependent) interactions between endothelial cells and ECM may serve to regulate capillary development. To test this hypothesis, FGF-stimulated endothelial cells were grown in chemically defined medium on bacteriological (nonadhesive) dishes that were precoated with different densities of fibronectin. Extensive cell spreading and growth were promoted by fibronectin coating densities that were highly adhesive (greater than 500 ng/cm2), whereas cell rounding, detachment, and loss of viability were observed on dishes coated with low fibronectin concentrations (less than 100 ng/cm2). Intermediate fibronectin coating densities (100-500 ng/cm2) promoted cell extension, but they could not completely resist cell tractional forces. Partial retraction of multicellular aggregates resulted in cell shortening, cessation of growth, and formation of branching tubular networks within 24-48 h. Multicellular retraction and subsequent tube formation also could be elicited on highly adhesive dishes by overcoming the mechanical resistance of the substratum using higher cell plating numbers. Dishes coated with varying concentrations of type IV collagen or gelatin produced similar results. These results suggest that ECM components may act locally to regulate the growth and pattern-regulating actions of soluble FGF based upon their ability to resist cell-generated mechanical loads. Thus, we propose that FGF-stimulated endothelial cells may be "switched" between growth, differentiation, and involution modes during angiogenesis by altering the adhesivity or mechanical integrity of their ECM.

Animals↗
PubMed1989

Inhibition of tumor-cell attachment to extracellular matrix as a method for preventing tumor recurrence in a surgical wound.

Studies with four different transplantable murine tumors demonstrated that surgical instruments contaminated by contact with a tumor mass could produce tumors in a surgical wound. Eighty-seven per cent of mice with wounds made by invisibly contaminated scissors developed tumors. Irrigation with water did not prevent tumor growth. Before spilled tumor cells can invade and grow into a recurrence in the wound site, they must first attach to underlying extracellular matrix. We have devised a simple in vitro assay to identify inhibitors of tumor-cell attachment to develop therapeutic compounds that can prevent tumor-cell reimplantation. Various test compounds, including proteases (trypsin and Dispase), known modulators of matrix metabolism (proline analogues, cycloheximide, heparin, cortisone, cortexolone, and heparin-steroid combinations), large molecular weight polymers (agarose, dextran, polyethylene oxide), and synthetic fibronectin peptides were tested for their ability to inhibit mouse melanoma (B16-F10) cell attachment to gelatinized dishes. Most of these compounds had little or no effect on tumor-cell adhesion when cells were plated in serum-containing medium. However we identified three compounds that inhibited tumor-cell attachment in a reversible fashion: (1) a specific inhibitor of collagen deposition (L-azetidine-2-carboxylic acid); (2) a bacterial neutral protease (Dispase); and (3) synthetic fibronectin peptides that contained the arginine-glycine-asparate (RGD) sequence that is responsible for cell binding. Dispase and the RGD-containing peptides also inhibited cell implantation and prevented tumor formation in a surgical wound. We propose that inhibitors of attachment might be used either alone or with other biologic modifiers to prohibit implantation of free tumor cells at the time of surgery and thus, to prevent local tumor recurrence.

Animals↗
PubMed1987

Endothelial growth factors and extracellular matrix regulate DNA synthesis through modulation of cell and nuclear expansion.

Studies were carried out to analyze the mechanism by which extracellular matrix (ECM) molecules and soluble growth factors interplay to control capillary endothelial cell growth. Bovine adrenal capillary endothelial cells attached to purified matrix components but spread poorly and exhibited low levels of DNA synthesis in the absence of exogenous growth factors or serum. Addition of cationic, heparin-binding growth factor purified from either human hepatoma cells or normal bovine pituitary (fibroblast growth factor) induced extensive cell spreading and up to eight fold increases in DNA synthetic rates relative to levels observed in cells on similar substrata in the absence of mitogen. However, the extent of this response differed depending upon the type of ECM molecule used for cell attachment (fold increase on type III collagen greater than gelatin greater than type IV collagen greater than fibronectin greater than type V collagen much greater than laminin). Computerized morphometry demonstrated that endothelial cell DNA synthetic rates increased in an exponential fashion in direct relation to linear increases in cell and nuclear size (projected areas). Similarly sized cells always displayed the same level of DNA synthesis independent of the type of ECM molecule used for cell attachment or the presence of saturating amounts of growth factor. In all cases, DNA metabolism appeared to be coupled to physical expansion of the cell and nucleus rather than to a specific cell morphology (e.g. polygonal versus bipolar). These findings suggest that ECM may act locally as a "solid state" regulator of angiogenesis through its ability to selectively support or prohibit cell and nuclear extension in response to stimulation by soluble mitogens.

Adrenal Glands↗
PubMed1987

Angiostatic steroids. Method of discovery and mechanism of action.

A new class of steroids has been found that inhibits angiogenesis in the presence of nonanticoagulant heparin. Tetrahydrocortisol is the most potent of the naturally occurring angiostatic steroids. It is a metabolite of cortisone that circulates in the blood, appears in the urine, and was previously believed to be biologically inactive. Both the plasma form and the urinary form are antiangiogenic. The mechanism of action of these steroids depends on their ability to specifically alter basement membrane turnover in growing capillary blood vessels. These steroids represent a prototype of angiogenesis inhibitors that may find potential therapeutic use as adjuncts to anticancer therapy, and in diseases dominated by abnormal neovascularization, i.e., angiogenesis-dependent diseases.

Animals↗
PubMed1986

A possible mechanism for inhibition of angiogenesis by angiostatic steroids: induction of capillary basement membrane dissolution.

A new class of angiostatic steroids acts independently of previously identified steroid functions to inhibit angiogenesis when administered with heparin. Development of angiostatic steroids as therapeutic modulators of blood vessel growth would be greatly facilitated if their mode of action were thoroughly understood. However, the mechanism by which these steroids produce capillary regression is not known. The distributions of fibronectin and laminin were studied in growing and regressing capillaries by immunofluorescence microscopy to determine whether capillary basement membrane (BM) alterations could be involved in the mechanism of antiangiogenesis. In normal 8-day-old chick chorioallantoic membrane, fibronectin and laminin appeared in continuous linear patterns within BM surrounding growing capillaries. In contrast, chorioallantoic membranes treated with combinations of angiostatic steroid and heparin exhibited capillary BM fragmentation and eventually complete loss of fibronectin and laminin from regions of capillary involution. Capillary BM breakdown correlated with capillary retraction, endothelial cell rounding, and associated capillary regression. BM surrounding large vessels, neighboring epithelium, and nongrowing capillaries were not affected. Capillary BM dissolution is the first biochemical action identified for this new class of antiangiogenic steroids.

Allantois↗
PubMed1986

Basement membrane as a spatial organizer of polarized epithelia. Exogenous basement membrane reorients pancreatic epithelial tumor cells in vitro.

The authors have previously described a transplantable pancreatic acinar carcinoma that comprises cells which reorganize and display normal epithelial orientation only when in contact with basement membrane (BM) in vivo. In the present study, they investigated whether exogenous acellular BM or collagenous stroma (prepared from human amnion) was sufficient to reorient pancreatic acinar tumor cells in vitro. Mechanically dispersed tumor cells could not spontaneously attach to standard culture substrata; yet they adhered to exogenous intact BM or to dishes coated with purified laminin or Type IV collagen. Cell contact with amniotic BM resulted in tumor cell shape changes, assembly of intracellular actin into fibrous bundles, and restoration of normal epithelial cell-cell interactions. Computerized morphometry confirmed that tumor cells exhibited a normal polarized distribution of lipid droplets, nuclei, Golgi complexes, and zymogen granules (from base to apex) within 6 hours of culture on BM. Adhering zonules and microvilli were observed only along apical tumor cell surfaces, although full junctional complexes and distinct membrane domains did not reform. Similar attachment, cytoskeletal alterations, and reorientation occurred in the absence of protein synthesis (25 micrograms/ml cycloheximide). In contrast, tumor cells that were maintained on amniotic stroma remained round, displayed circumferential rings of actin, and appeared randomly oriented. Thus, BM may normally serve to integrate and maintain individual cells within a polarized epithelium.

Amnion↗
PubMed1985

Neoplastic disorganization of pancreatic epithelial cell-cell relations. Role of basement membrane.

The authors have analyzed the structural relations of a nonmetastatic rat pancreatic acinar carcinoma and contrasted them with those of normal exocrine pancreas in order to better define the role of basement membrane (BM) in early stages of neoplastic disorganization. These studies showed that normal acinar cells rested on continuous BM (containing laminin, heparan sulfate proteoglycan, and Type IV and V collagens) and displayed a polarized distribution of intracellular organelles, cytoskeletal assemblies (concentration of actin within terminal web), and distinct membrane domains (apical leucine aminopeptidase). In contrast, the parenchyma of the pancreatic acinar carcinoma was free of all BM components except for a discontinuous array of laminin. In these regions, acinar tumor cells appeared randomly oriented, displayed actin in uniform cortical distributions, and lost membrane polarity. However, when tumor cells contacted mesenchymally derived connective tissue along tumor capsule and vascular adventitia, they accumulated intact BM and reoriented in a manner reminiscent of normal pancreas. Tumor cell reorganization was observed in the absence of formation of full junctional complexes or normally polarized membrane domains, although leucine aminopeptidase appeared to be excluded from regions of tumor cell surfaces that were in direct contact with BM. The loss of normal epithelial cell-cell arrangements that is the hallmark of early stages of tumor formation could therefore result from failure to match increases in cell number with commensurate BM extension.

Animals↗