Kristofer Rubin

High interstitial fluid pressure — an obstacle in cancer therapy

Many solid tumours show an increased interstitial fluid pressure (IFP), which forms a barrier to transcapillary transport. This barrier is an obstacle in tumour treatment, as it results in inefficient uptake of therapeutic agents. There are a number of factors that contribute to increased IFP in the tumour, such as vessel abnormalities, fibrosis and contraction of the interstitial matrix. Lowering the tumour IFP with specific signal-transduction antagonists might be a useful approach to improving anticancer drug efficacy.

PDGF receptors as cancer drug targets.

The benefits of combining PDGF antagonists and VEGF antagonists in a mouse model of pancreatic islet cancer are shown in a report by Bergers et al.: Bergers, G., Song, S., Meyer-Morse, N., Bergsland, E., and Hanahan, D. (2003). Benefits of targeting both pericytes and endothelial cells in the tumor vasculature with kinase inhibitors. J. Clin. Invest. 111, 1287–1295.

Binding of different dimeric forms of PDGF to human fibroblasts: evidence for two separate receptor types

The binding of the three dimeric forms of platelet‐derived growth factor (PDGF), PDGF‐AA, PDGF‐AB and PDGF‐BB, to human fibroblasts was studied. Cross‐competition experiments revealed the existence of two different PDGF receptor classes: the type A PDGF receptor bound all three dimeric forms of PDGF, whereas the type B PDGF receptor bound PDGF‐BB with high affinity and PDGF‐AB with lower affinity, but not PDGF‐AA. The sizes of the two receptors were estimated with affinity labeling techniques; the A type receptor appeared as a major component of 125 kd and a minor of 160 kd, and the B type receptor as two components of 160 and 175 kd. A previously established PDGF receptor monoclonal antibody, PDGFR‐B2, was shown to react with the B type receptor only. The different abilities of the three dimeric forms of PDGF to stimulate incorporation of [3H]TdR into human fibroblasts indicated that the major mitogenic effect of PDGF is mediated via the B type receptor.

Beta 1 integrin-mediated collagen gel contraction is stimulated by PDGF.

The attachment of primary rat hepatocytes and fibroblasts to collagen type I is mediated by non-RGD-dependent beta 1 integrin matrix receptors. In this report we describe a novel 96-well microtiter plate assay for the quantification of fibroblast-mediated contraction of floating collagen type I gels. Fetal calf serum and platelet-derived growth factor (PDGF), but not transforming growth factor-beta 1, stimulated primary rat heart fibroblasts and normal human diploid fibroblasts (AG 1518) to contract collagen gels to less than 10% of the initial gel volume within a 24-h incubation period. Rabbit polyclonal antibodies directed to the rat hepatocyte integrin beta 1-chain inhibited the PDGF-stimulated collagen gel contraction. The inhibitory activity on contraction of the anti-beta 1 integrin IgG could be overcome by adding higher doses of PDGF. The contraction process was not blocked by anti-fibronectin IgG nor by synthetic peptides containing the tripeptide Arg-Gly-Asp (RGD), in concentrations that readily blocked fibroblast attachment to fibronectin-coated planar substrates. Autologous fibronectin or control peptides containing the tripeptide Arg-Gly-Glu were without effect. Immunofluorescence microscopy on fibroblasts grown within collagen gels revealed a punctate distribution of the beta 1 integrin and a lack of detectable levels of endogenously produced fibronectin. Collectively these data suggest a role for integrin collagen receptors with affinity for collagen fibers, distinct from the previously described RGD-dependent fibronectin receptors, in the fibronectin-independent PDGF-stimulated collagen gel contraction process.

T lymphocytes in collagen II-induced arthritis in mice: characterization of arthritogenic collagen II-specific T-cell lines and clones

Collagen type II‐spedfic long‐term cultured T helper cells, derived from the DBA/1 mouse, have been established and characterized. Clones from these T‐cell lines could he shown to recognize either species‐specific or species‐nonspecific determinants on the collagen type II molecule, including determinants on autologous mouse collagen. Induction of arthritis via transfer to both irradiated and normal syngeneic recipient mice was obtained with both collagen type II‐specific T‐cell lines and an autoreactive and collagen type II‐specific T‐cell clone. Fewer cells were needed to evoke arthritis in normal than in irradiated recipients. Cells from lines and the clone used for transfer were by immunocytochemistry shown to have T helper phenotype.

Expression of collagen binding integrins during cardiac development and hypertrophy.

The interaction between components of the extracellular matrix and the cell surface of cardiac myocytes appears to be regulated in part by receptors belonging to the integrin superfamily. The expression of the integrins was investigated at different stages of development of the heart as well as during cardiac hypertrophy. The characterization of the membrane proteins showed that a beta 1-integrin and associated alpha-chains were responsible for the interaction with collagen, laminin, and fibronectin. Immunoprecipitation data indicated that the presence of specific alpha-chains varied with development. These data were correlated with the ability of the isolated myocytes to attach to specific components of the extracellular matrix. The expression of the alpha 1-chain was prominently associated with the recognition of interstitial collagens. The presence of the alpha 1-chain was also associated with stages when collagen synthesis was increased, especially during fetal and neonatal growth and cardiac hypertrophy. Immunohistochemical localization with the antiserum against beta 1-integrin demonstrated its specific localization near the Z lines of cardiac myocytes. The localization both in vitro and in vivo indicated that the beta 1-integrin may play a role in myofibrillogenesis during development. The present immunohistochemical, cell adhesion, and biochemical data clearly indicate that integrins play a major role in the regulation of the interaction between cardiac myocytes and the extracellular matrix during development and disease.

INDUCTION OF B-TYPE RECEPTORS FOR PLATELET-DERIVED GROWTH FACTOR IN VASCULAR INFLAMMATION: POSSIBLE IMPLICATIONS FOR DEVELOPMENT OF VASCULAR PROLIFERATIVE LESIONS

Expression of B-type receptors for platelet-derived growth factor (PDGF) in frozen sections of blood vessels from tissues affected by abnormal vascular cell proliferation was investigated by immunohistochemical techniques and compared with expression of these receptors in blood vessels of normal tissues. Receptors were not expressed, or expressed at low levels, in vessels of normal tissues. In contrast, a pronounced expression of PDGF B-type receptors was seen on vascular smooth muscle cells in atherosclerotic plaques, rejected kidneys, and chronic synovitis. These observations suggest induction of PDGF B-type receptors on vascular smooth muscle cells in inflamed tissues, which would render such cells responsive to growth stimulation by PDGF released from captured platelets, or produced locally (eg, by inflammatory cells or smooth muscle cells). Autocrine or paracrine stimulation of cell growth caused by the effect of PDGF on cells with induced receptors may be important in the formation of the proliferative lesions found in atherosclerosis and in certain forms of chronic inflammation.

The viability of cells grown or centrifuged in a new density gradient medium, Percoll(TM).

A new density gradient medium, Percoll (a modified colloidal silica), has been tested for toxicity in primary cultures of rat liver and calf testicle cells, and in continuous cultures of pig kidney and HeLa cells. The presence of Percoll did not appreciably affect the growth or viability of the cells as judged from cell counts and morphology. The various cells were also centrifugea in gradients of Percoll and subsequently cultured. The in vitro growth of the cells was similar to that of untreated cells. Rat liver cells were labelled in vivo with [125I]asialoceruloplasmin (parenchymal cells) or heat-denatured [125I]albumin (non-parenchymal cells). After dispersion of the cells and iso-pycnic centrifugation in Percoll the non-parenchymal cells banded preferentially at a lower density (1.04−1.05 g/ml) than parenchymal cells (1.07−1.09 g/ml). The two types of cells showed very different morphology in cell culture. The non-parenchymal cells retained their phagocytic properties during culture. Injured cells and cell debris band at the top of the Percoll gradients in contrast to their behaviour in gradients containing low molecular weight substances. Centrifugation in Percoll can be used to enrich viable cells.

Recognition of extracellular matrix components by neonatal and adult cardiac myocytes

Recognition of extracellular matrix (ECM) components by isolated cardiac myocytes from neonatal (4-5 days postpartum) and adult rats was determined by measuring cell attachment to substrates made of ECM components. The substrates were petri dishes coated with either fibronectin, laminin, native monomers of collagen types I, II, III, IV, and V, denatured collagen, or gels containing reconstituted collagen fibers. Adult myocytes attached efficiently to laminin and type IV collagen, weakly to fibronectin, but not at all to the other types of collagen. Neonatal myocytes attached well to all types of collagen and to fibronectin and laminin. Antibodies raised against surface membranes of neonatal myocytes, adult myocytes, or adult hepatocytes were assayed for their ability to inhibit cell attachment to the various ECM substrates. Antibodies against the surface of neonatal myocytes as well as antibodies against the hepatocyte cell surface inhibited the attachment of neonatal myocytes and hepatocytes to collagen but not to fibronectin. Antibodies against the adult myocyte cell surface did not inhibit the attachment of neonatal myocytes or hepatocytes to ECM components. These results indicate the presence of binding molecules on the surface of neonatal myocytes that are involved in the recognition of collagen at a time when collagen is being secreted and formed into a three-dimensional network that attaches to the cell surface of the myocytes. This recognition and adhesion to collagen occurs by a mechanism independent of fibronectin. The binding molecules for collagen could not be detected on normal adult myocytes isolated at a time when the formation of the collagen network has already been completed.

Substrate adhesion of rat hepatocytes: Mechanism of attachment to collagen substrates

Attachment of rat hepatocytes to collagen, which occurs without the aid of fibronectin, was found to be a time-dependent reaction characterized by an initial lag phase of 10-20 min before stable attachment bonds began to form. Increasing the density of molecules in the collagen substrates enhanced the rate of cell attachment. The hepatocytes attached essentially equally well to all the collagen types tested (types I, II, III, IV and V). The initial rate of cell attachment was more rapid to native collagen than to denatured collagen or alpha 1(I) chains, apparently indicating different affinities of the cells for these substrates. However, if cells were incubated for 60 min or more, efficient attachment occurred to the alpha 1(I) chain and to all cyanogen-bromide-treated peptides tested (alpha 1-CB2, alpha 1-CB3, alpha 1-CB4, alpha 1-CB5, alpha 1-CB6A, alpha 1-CB7, alpha 1-CB8, alpha 2-CB2, alpha 2-CB3 and alpha 2-CB4) but not to the aminopropeptide of type I procollagen. A low but significant degree of attachment also took place to substrates made of synthetic peptides with the collagen-like structures (Gly-Ala-Pro)n, (Gly-Pro-Pro)n and (Gly-Pro-Hyp)n, whereas no attachment was observed to polyproline. We suggest that the cell-binding sites in collagen have a simple structure and occur in multiple copies along the collagen molecule. Addition of collagen in solution inhibited initial cell attachment, an effect that persisted longer on substrates made of alpha 1(I) chain than on denatured collagen. The collected data are interpreted in terms of a model for cell-to-collagen adhesion where the formation of stable attachment bonds requires the binding of several low-affinity receptors, clustered at the site of adhesion, to collagen molecules in the substrate.