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Biomedical subjects

D Ingber

Publications and source records attributed to D Ingber.

18 recordsLinked to original sources
PubMed1999

How cells (might) sense microgravity.

This article is a summary of a lecture presented at an ESA/NASA Workshop on Cell and Molecular Biology Research in Space that convened in Leuven, Belgium, in June 1998. Recent studies are reviewed which suggest that cells may sense mechanical stresses, including those due to gravity, through changes in the balance of forces that are transmitted across transmembrane adhesion receptors that link the cytoskeleton to the extracellular matrix and to other cells (e.g., integrins, cadherins, selectins). The mechanism by which these mechanical signals are transduced and converted into a biochemical response appears to be based, in part, on the finding that living cells use a tension-dependent form of architecture, known as tensegrity, to organize and stabilize their cytoskeleton. Because of tensegrity, the cellular response to stress differs depending on the level of pre-stress (pre-existing tension) in the cytoskeleton and it involves all three cytoskeletal filament systems as well as nuclear scaffolds. Recent studies confirm that alterations in the cellular force balance can influence intracellular biochemistry within focal adhesion complexes that form at the site of integrin binding as well as gene expression in the nucleus. These results suggest that gravity sensation may not result from direct activation of any single gravioreceptor molecule. Instead, gravitational forces may be experienced by individual cells in the living organism as a result of stress-dependent changes in cell, tissue, or organ structure that, in turn, alter extracellular matrix mechanics, cell shape, cytoskeletal organization, or internal pre-stress in the cell-tissue matrix.--Ingber, D. How cells (might) sense microgravity.

Biomechanical Phenomena↗
PubMed1998

Regulation of cytoskeletal mechanics and cell growth by myosin light chain phosphorylation.

The role of myosin light chain phosphorylation in regulating the mechanical properties of the cytoskeleton was studied in NIH/3T3 fibroblasts expressing a truncated, constitutively active form of smooth muscle myosin light chain kinase (tMK). Cytoskeletal stiffness determined by quantifying the force required to indent the apical surface of adherent cells showed that stiffness was increased twofold in tMK cells compared with control cells expressing the empty plasmid (Neo cells). Cytoskeletal stiffness quantified using magnetic twisting cytometry showed an approximately 1.5-fold increase in stiffness in tMK cells compared with Neo cells. Electronic volume measurements on cells in suspension revealed that tMK cells had a smaller volume and are more resistant to osmotic swelling than Neo cells. tMK cells also have smaller nuclei, and activation of mitogen-activated protein kinase (MAP kinase) and translocation of MAP kinase to the nucleus are slower in tMK cells than in control cells. In tMK cells, there is also less bromodeoxyuridine incorporation, and the doubling time is increased. These data demonstrate that increased myosin light chain phosphorylation correlates with increased cytoskeletal stiffness and suggest that changing the mechanical characteristics of the cytoskeleton alters the intracellular signaling pathways that regulate cell growth and division.

3T3 Cells↗
PubMed1998

Impaired mechanical stability, migration and contractile capacity in vimentin-deficient fibroblasts.

Loss of a vimentin network due to gene disruption created viable mice that did not differ overtly from wild-type littermates. Here, primary fibroblasts derived from vimentin-deficient (-/-) and wild-type (+/+) mouse embryos were cultured, and biological functions were studied in in vitro systems resembling stress situations. Stiffness of -/- fibroblasts was reduced by 40% in comparison to wild-type cells. Vimentin-deficient cells also displayed reduced mechanical stability, motility and directional migration towards different chemo-attractive stimuli. Reorganization of collagen fibrils and contraction of collagen lattices were severely impaired. The spatial organization of focal contact proteins, as well as actin microfilament organization was disturbed. Thus, absence of a vimentin filament network does not impair basic cellular functions needed for growth in culture, but cells are mechanically less stable, and we propose that therefore they are impaired in all functions depending upon mechanical stability.

Actins↗
PubMed1998

In search of cellular control: signal transduction in context.

The field of molecular cell biology has experienced enormous advances over the last century by reducing the complexity of living cells into simpler molecular components and binding interactions that are amenable to rigorous biochemical analysis. However, as our tools become more powerful, there is a tendency to define mechanisms by what we can measure. The field is currently dominated by efforts to identify the key molecules and sequences that mediate the function of critical receptors, signal transducers, and molecular switches. Unfortunately, these conventional experimental approaches ignore the importance of supramolecular control mechanisms that play a critical role in cellular regulation. Thus, the significance of individual molecular constituents cannot be fully understood when studied in isolation because their function may vary depending on their context within the structural complexity of the living cell. These higher-order regulatory mechanisms are based on the cell's use of a form of solid-state biochemistry in which molecular components that mediate biochemical processing and signal transduction are immobilized on insoluble cytoskeletal scaffolds in the cytoplasm and nucleus. Key to the understanding of this form of cellular regulation is the realization that chemistry is structure and hence, recognition of the the importance of architecture and mechanics for signal integration and biochemical control. Recent work that has unified chemical and mechanical signaling pathways provides a glimpse of how this form of higher-order cellular control may function and where paths may lie in the future.

Animals↗
PubMed1994

Cytostatic inhibition of endothelial cell growth by the angiogenesis inhibitor TNP-470 (AGM-1470).

Recently, we reported the anti-angiogenic action along with anti-tumour activity of TNP-470 (AGM-1470). In this study, the effect of TNP-470 on the growth of human umbilical vein endothelial (HUVE) cells was examined. TNP-470 inhibited the growth of HUVE cells in a biphasic manner. The inhibition was cytostatic in the first phase (complete inhibition at 300 pg ml-1 to 3 micrograms ml-1 with an IC50 of 15 pg ml-1) and cytotoxic in the second phase (> or = 30 micrograms ml-1). The cytostatic inhibition of HUVE cell growth by TNP-470 was durable after washing out TNP-470 in culture. Incorporation of thymidine but not uridine and leucine by HUVE cells was inhibited in the first phase, while that of all three compounds was inhibited in the second phase. Human and rat endothelial cells among various types of cells were the most sensitive to the cytostatic inhibition, while differences in the cytotoxic inhibition were minimal. These results suggest that TNP-470 exerts its specific anti-angiogenic action by inhibiting cytostatically growth of endothelial cells in a relatively specific manner.

Antibiotics, Antineoplastic↗
PubMed1992

Switching from differentiation to growth in hepatocytes: control by extracellular matrix.

Studies were carried out to analyze how different extracellular matrix (ECM) molecules regulate hepatocyte growth and differentiation. Freshly isolated rat hepatocytes were cultured on non-adhesive plastic dishes that were pre-coated with defined densities of either laminin, fibronectin, type I collagen, or type IV collagen. Sparse cell plating densities were used to minimize cell-cell contact formation and all studies were carried out in chemically defined medium that contained a saturating amount of soluble growth factors. Dishes coated with a low ECM density (1 ng/cm2) supported hepatocyte attachment, but did not promote cell spreading or growth. Computerized image analysis confirmed that over 80% of cells remained free of contact with other cells under these conditions. Yet, these round cells maintained high levels of albumin gene expression as well as elevated secretion rates for multiple liver-specific proteins (albumin, transferrin, and fibrinogen), regardless of the type of ECM molecule used for cell attachment. When ECM coating densities were raised from 1 to 1,000 ng/cm2, cell spreading, expression of histone mRNA, DNA synthesis, and cell proliferation all increased in parallel. Activation of growth by high ECM densities was also accompanied by a concomitant down-regulation of differentiated functions and again, dishes coated with all four types of ECM molecules produced similar effects. Thus, the ability to switch hepatocytes from differentiation to growth (i.e., between different genetic programs) is not limited to a single ECM molecule, a distinct three dimensional ECM geometry, or due to alteration of cell-cell interactions. Rather, the regulatory signals conveyed by immobilized ECM molecules depend on the density at which they are presented and thus, on their ability to either prohibit or support cell spreading.

Animals↗
PubMed1992

Inhibition of angiogenesis.

The concept of antiangiogenic therapy was first proposed in the early 1970s as a method of restricting tumor growth by inhibiting angiogenesis. In subsequent years sufficient knowledge about the process of angiogenesis itself was obtained so that it is now possible to begin to develop antiangiogenic therapy for clinical use. At least three strategies are feasible: (i) inhibition of release of angiogenic molecules from tumor cells; (ii) neutralization of angiogenic molecules that have already been released; and (iii) inhibition of vascular endothelial cells from responding to angiogenic stimulation. Most of the angiogenic inhibitors that have been discovered at the time of writing, directly interfere with the ability of endothelial cells to form new capillary blood vessels. Antiangiogenic activity is a newly found property of alpha-interferon. Although alpha-interferon is a relatively weak angiogenesis inhibitor in comparison to others, it has been very successful in the treatment of life-threatening hemangiomas in children. Early clinical experience with this first angiogenesis inhibitor to reach clinical trial, indicates that optimal antiangiogenic therapy in the future is likely to be based on the long-term use of inhibitors with low toxicity, and with little chance of inducing drug-resistance. It is apparent that different types of angiogenesis inhibitor may be administered together and that these compounds may also be administered to cancer patients as adjuncts to conventional chemotherapy. It is important to recognize that tumor vasculature has other properties besides angiogenesis, which make it a potential specific target for anti-cancer therapy.

Animals↗
PubMed1991

Potent anti-angiogenic action of AGM-1470: comparison to the fumagillin parent.

The anti-angiogenic activity of AGM-1470, a new synthetic analog of fumagillin isolated from Aspergillus fumigatus, was extensively examined both in vitro and in vivo using four different types of assay and compared to that of the fumagillin parent. Locally administered AGM-1470 inhibited the angiogenesis in the chick embryo chorioallantoic membrane assay and the rat corneal assay. In the rat sponge implantation assay, systemically administered AGM-1470 inhibited angiogenesis induced by basic fibroblast growth factor. Furthermore, in the rat blood vessel organ culture assay, AGM-1470 (1-1,000 ng/ml) was found to selectively inhibit the capillary-like tube formation of endothelial cells with a minimal effect on the non-endothelial cell growth. AGM-1470 showed more potent anti-angiogenic activity and less toxicity than the fumagillin parent. Therefore, AGM-1470 is much better than the fumagillin parent as anti-angiogenic compound.

Animals↗
PubMed1991

Extracellular matrix and cell shape: potential control points for inhibition of angiogenesis.

Capillary endothelial (CE) cells require two extracellular signals in order to switch from quiescence to growth and back to differentiation during angiogenesis: soluble angiogenic factors and insoluble extracellular matrix (ECM) molecules. Soluble endothelial mitogens, such as basic fibroblast growth factor (FGF), act over large distances to trigger capillary growth, whereas ECM molecules act locally to modulate cell responsiveness to these soluble cues. Recent studies reveal that ECM molecules regulate CE cell growth and differentiation by modulating cell shape and by activating intracellular chemical signaling pathways inside the cell. Recognition of the importance of ECM and cell shape during capillary morphogenesis has led to the identification of a series of new angiogenesis inhibitors. Elucidation of the molecular mechanism of capillary regulation may result in development of even more potent angiogenesis modulators in the future.

Animals↗
PubMed1991

Integrins as mechanochemical transducers.

A recent resurgence of interest in mechanical forces and cell shape as biological regulators has revealed extracellular matrix as the site at which forces are transmitted both to and from cells. at the same time, great advances have been made in terms of defining cell-surface integrin receptors as transmembrane molecules that mediate cell attachment and physically interlink extracellular matrix with the intracellular cytoskeleton. Convergence of these two lines of research has begun to elucidate the molecular mechanism by which cells sense physical forces and transduce mechanical signals into a biochemical response.

Animals↗
PubMed1991

Tissue engineering by cell transplantation using degradable polymer substrates.

This paper reviews our research in developing novel matrices for cell transplantation using bioresorbable polymers. We focus on applications to liver and cartilage as paradigms for regeneration of metabolic and structural tissue, but review the approach in the context of cell transplantation as a whole. Important engineering issues in the design of successful devices are the surface chemistry and surface microstructure, which influence the ability of the cells to attach, grow, and function normally; the porosity and macroscopic dimensions, which affect the transport of nutrients to the implanted cells; the shape, which may be necessary for proper function in tissues like cartilage; and the choice of implantation site, which may be dictated by the total mass of the implant and which may influence the dimensions of the device by the available vascularity. Studies show that both liver and cartilage cells can be transplanted in small animals using this approach.

Animals↗
PubMed1990

Synthetic analogues of fumagillin that inhibit angiogenesis and suppress tumour growth.

Neovascularization is critical for the growth of tumours and is a dominant feature in a variety of angiogenic diseases such as diabetic retinopathy, haemangiomas, arthritis and psoriasis. Recognition of the potential therapeutic benefit of controlling unabated capillary growth has led to a search for safe and effective angiogenesis inhibitors. We report here the synthesis of a family of novel inhibitors that are analogues of fumagillin, a naturally secreted antibiotic of Aspergillus fumigatus fresenius. We first isolated this fungus from a contaminated culture of capillary endothelial cells. Purified fumagillin inhibited endothelial cell proliferation in vitro and tumour-induced angiogenesis in vivo; it also inhibited tumour growth in mice, but prolonged administration was limited because it caused severe weight loss. Synthesis of fumagillin analogues yielded potent angiogenesis inhibitors ('angioinhibins') which suppress the growth of a wide variety of tumours with relatively few side-effects.

Animals↗
PubMed1989

Induction of angiogenesis during the transition from hyperplasia to neoplasia.

It is now well established that unrestricted growth of tumours is dependent upon angiogenesis. Previous studies on tumour growth, however, have not revealed when or how the transition to an angiogenic state occurs during early tumour development. The advent of transgenic mice carrying oncogenes that reproducibly elicit tumours of specific cell types is providing a new format for studying multi-step tumorigenesis. In one of these models, transgenic mice expressing an oncogene in the beta-cells of the pancreatic islets heritably recapitulate a progression from normality to hyperplasia to neoplasia. We report here that angiogenic activity first appears in a subset of hyperplastic islets before the onset of tumour formation. A novel in vitro assay confirms that hyperplasia per se does not obligate angiogenesis. Rather, a few hyperplastic islets become angiogenic in vitro at a time when such islets are neovascularized in vivo and at a frequency that correlates closely with subsequent tumour incidence. These findings suggest that induction of angiogenesis is an important step in carcinogenesis.

Adenoma, Islet Cell↗
PubMed1989

Metabolism of tumor regression from angiogenesis inhibition: 31P magnetic resonance spectroscopy.

31P NMR spectroscopy was used to analyzed the in vivo metabolism of reticulum cell sarcoma of mice. The ratio of high- to low-energy phosphates ATPB/(Pi + PME) was measured to reflect the relative metabolic state in the tumor. Of the 34 mice studied, 26 were treated with an antiangiogenesis regimen of heparin and cortisone. Eighty-two percent of the tumors treated eventually decreased in volume (P less than 0.01). Volumes and spectroscopic information of 20 tumors were analyzed. Although the average untreated volume was similar to the volume after 3 days of treatment, the average ATPB/(Pi + PME) ratio rose from 0.34 +/- 0.10 to 0.47 +/- 0.07 (P = 0.02). However, after 6 days of treatment, the volume significantly decreased (P less than 0.0001) but the ratio did not significantly rise further (P = 0.06). The rise in the high-energy phosphate preceded a significant decrease in volume of the tumors. In addition, the replenishment of high-energy stores with tumor regression coincided with the histologic findings of a decrease in the number of tumor cells, a decrease of the mitotic index, and a decrease of the number of necrotic cells present with ongoing treatment. Our data suggest that noninvasive methods of assessing early biochemical response of tumor regression may be possible.

Animals↗
PubMed1988

Inhibition of angiogenesis through modulation of collagen metabolism.

Combinations of angiostatic steroids and heparin induce basement membrane breakdown, capillary retraction, and endothelial cell rounding as part of their antiangiogenic action (Ingber DE, Madri JA, Folkman J: Endocrinology 119:1768, 1986). Specific modulators of matrix metabolism were examined for their effects and on capillary development in the growing chick chorioallantoic membrane to explore the possibility that structural alterations of extracellular matrix (ECM) could be causally involved in the antiangiogenic mechanism. Regression of growing capillaries was induced by proline analogs (1-azetidine-2-carboxylic acid, cis-hydroxyproline,d,L-3,4-dehydroproline, thioproline) and an inhibitor of prolyl hydroxylase (alpha,alpha-dipyridyl) which interfere with triple helix formation and prevent collagen deposition. Beta-aminopropionitrile, an inhibitor of collagen cross-linking, was also antiangiogenic although an inhibitor of glycosaminoglycan deposition (beta-methyl d-xyloside) was not. Combination of suboptimal doses of active collagen modulators with either angiostatic steroids or heparin resulted in great potentiation of their antiangiogenic effects. Coadministration of proline analogs, angiostatic steroids, and heparin resulted in complete inhibition of angiogenesis (avascular zones in 100% of chorioallantoic membranes) and larger and more extensive avascular zones than previously observed with steroid-heparin combinations. The antiangiogenic effects of these compounds (alone and in combination) were found to be directly related to their ability to inhibit collagen accumulation. Screening of additional compounds that are known to alter ECM turnover resulted in identification of a new angiogenesis inhibitor, all trans-retinoic acid. These findings suggest that growing capillaries may be "switched" into a regressive mode through alterations of collagen metabolism that result in loss of ECM structural integrity. Identification of ECM turnover as a major control point in angiogenesis may also provide a more rational approach for development of new antiangiogenic regimens.

Allantois↗
PubMed1988

A heparin-binding angiogenic protein--basic fibroblast growth factor--is stored within basement membrane.

The basement membranes of bovine cornea are found to contain an angiogenic endothelial cell mitogen, basic fibroblast growth factor (FGF), as determined by heparin-affinity chromatography, immunoblotting, immunofluorescence, and stimulation of capillary endothelial cell proliferation. The growth factor appears to be bound to heparan sulfate and is released from the cornea by treatment with heparin, a hexasaccharide heparin fragment, heparan sulfate, or heparanase, but not by chondroitin sulfate or chondroitinase. These findings indicate that basement membranes of the cornea may serve as physiologic storage depots for an angiogenic molecule. Abnormal release of this growth factor could be responsible for corneal neovascularization in a variety of ocular diseases. Physiologic and pathologic neovascularization in other tissues may also be initiated by release of stored angiogenic factors from the basement membrane. The sequestration of angiogenic endothelial mitogens in the basement membrane may be a general mechanism for regulating their accessibility to vascular endothelium.

Angiogenesis Inducing Agents↗
PubMed1987

Heparin-binding angiogenesis factors: detection by immunological methods.

Immunological methods for the detection of basic fibroblast growth (FGF) are described. Polyclonal antibodies directed against synthetic peptides representing amino-, internal, and carboxy-terminal regions of basic FGF, were raised in rabbits. Five techniques, enzyme-linked immunosorbent assay, immunoblot, electrophoretic transfer (Western) blot, immunoprecipitation and radioimmunoassay, were used to detect basic FGF. These techniques were used to demonstrate that a human hepatoma cell line synthesizes a growth factor structurally related to brain and pituitary basic FGF.

Angiogenesis Inducing Agents↗