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Biomedical subjects

D E Ingber

Publications and source records attributed to D E Ingber.

At least 91 records · Page 5Linked to original sources
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↗
PubMed1981

Cell surface properties of normal, differentiating, and neoplastic pancreatic acinar cells.

Acinar, centroacinar, and endocrine cells of the adult rat pancreas each exhibit distinctive cell-surface glycoconjugate patterns as detected by binding of a battery of lectin-ferritin conjugates. Acquisition of these unique glycoconjugate patterns appears to be developmentally regulated, as studies on embryonic rat pancreases at days 15, 17, and 19 of gestation indicate. Further, the three cell types appear to arise from a common stem cell(s) with surface glycoconjugate properties similar to those of the adult centroacinar cell. STudies on the cell-surface properties of a rat acinar cell tumor indicate that the neoplastic acinar cells are likely to be arrested at a developmental stage equivalent to acinar cells of the day 19 embryonic pancreas and are characterized by absence of detectable basal lamina. We hypothesize that pancreatic cancers may arise from the equivalent of the undifferentiated embryonic stem cell(s) and that the morphologic features of the tumor depend on the extent of cell differentiation, including expression of cell-surface glycoproteins and extracellular matrix, prior to neoplastic proliferation.

Animals↗
PubMed1981

Role of basal lamina in neoplastic disorganization of tissue architecture.

We have studied a transplantable carcinoma of the rat pancreas [Reddy, J. K. & Rao, M. S. (1977( Science 198, 78-80] that is composed of cytologically differentiated acinar cells that have lost their epithelial orientation and do not form acini. Light microscopy shows, however, consistent palisading, reorientation, and polarization of these cells in areas of contact with the vasculature. Electron microscopy reveals a normal basal lamina (BL) along the basal portions of repolarized tumor cells that is physically separate from the endothelial BL. We used indirect immunofluorescence to examine the distribution of BL constituents, laminin (Lm) and type IV collagen (type IV), within the different microenvironments of this tumor. In normal pancreas, Lm and type IV are distributed linearly, outlining acini and blood vessels. In the tumor parenchyma, type IV is not detected, whereas Lm appears in a punctate distribution outlining cells. Reorientation of tumor cells is observed only along linearly deposited Lm and type IV bordering vessels. These data indicate that this nonmetastatic tumor has lost the ability to produce or maintain a complete BL within its disorganized parenchyma, while its cells retain the capacity to produce and reorganize along liner BL when in contact with vascular adventitia. We suggest that failure to maintain a complete BL may be involved in the neoplastic disorganization of normal tissue architecture as well as in the breakdown of boundaries during the development of invasive carcinomata.

Animals↗
PubMedDate not supplied

Micropatterned surfaces for control of cell shape, position, and function.

The control of cell position and function is a fundamental focus in the development of applications ranging from cellular biosensors to tissue engineering. Using microcontact printing of self-assembled monolayers (SAMs) of alkanethiolates on gold, we manufactured substrates that contained micrometer-scale islands of extracellular matrix (ECM) separated by nonadhesive regions such that the pattern of islands determined the distribution and position of bovine and human endothelial cells. In addition, the size and geometry of the islands were shown to control cell shape. Traditional approaches to modulate cell shape, either by attaching suspended cells to microbeads of different sizes or by plating cells on substrates coated with different densities of ECM, suggested that cell shape may play an important role in control of apoptosis as well as growth. Data are presented which show how micropatterned substrates were used to definitively test this hypothesis. Progressively restricting bovine and human endothelial cell extension by culturing cells on smaller and smaller micropatterned adhesive islands regulated a transition from growth to apoptosis on a single continuum of cell spreading, thus confirming the central role of cell shape in cell function. The micropatterning technology is therefore essential not only for construction of biosurface devices but also for the investigation of the fundamental biology of cell-ECM interactions.

Animals↗
PubMedDate not supplied

Probing transmembrane mechanical coupling and cytomechanics using magnetic twisting cytometry.

We recently developed a magnetic twisting cytometry technique that allows us to apply controlled mechanical stresses to specific cell surface receptors using ligand-coated ferromagnetic microbeads and to simultaneously measure the mechanical response in living cells. Using this technique, we have previously shown the following: (i) beta 1 integrin receptors mediate mechanical force transfer across the cell surface and to the cytoskeleton, whereas other transmembrane receptors (e.g., scavenger receptors) do not; (ii) cytoskeletal stiffness increases in direct proportion to the level of stress applied to integrins; and (iii) the slope of this linear stiffening response differs depending on the shape of the cell. We now show that different integrins (beta 1, alpha V beta 3, alpha V, alpha 5, alpha 2) and other transmembrane receptors (scavenger receptor, platelet endothelial cell adhesion molecule) differ in their ability to mediate force transfer across the cell surface. In addition, the linear stiffening behavior previously observed in endothelial cells was found to be shared by other cell types. Finally, we demonstrate that dynamic changes in cell shape that occur during both cell spreading and retraction are accompanied by coordinate changes in cytoskeletal stiffness. Taken together, these results suggest that the magnetic twisting cytometry technique may be a powerful and versatile tool for studies analyzing the molecular basis of transmembrane mechanical coupling to the cytoskeleton as well as dynamic relations between changes in cytoskeletal structure and alterations in cell form and function.

Animals↗