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D J Grainger

Publications and source records attributed to D J Grainger.

52 records · Page 3Linked to original sources
PubMed1994

Transforming growth factor beta decreases the rate of proliferation of rat vascular smooth muscle cells by extending the G2 phase of the cell cycle and delays the rise in cyclic AMP before entry into M phase.

Transforming growth factor beta 1 (TGF-beta 1) decreased the rate of proliferation of rat aortic vascular smooth muscle cells (VSMCs) stimulated with serum showing a maximal effect at > 5 ng/ml (200 pM). However, it did not reduce the proportion of cells which passed through S phase (> 90%) and entry into S phase was delayed by less than 3 h. The proportion of cells passing through M phase (> 90%) was also unaffected, but entry into mitosis was delayed by approx. 24 h. This increase in cell cycle time was therefore due mainly to an increase in the G2 to mitotic metaphase period. Addition of TGF-beta 1 late in G1 or late in S phase failed to delay the onset of mitosis, but the presence of TGF-beta 1 between 0 and 12 h after the addition of serum to quiescent cells was sufficient to cause the maximal delay in mitosis of approx. 24 h. The role of cyclic AMP in the mechanism of the TGF-beta 1 effects on the cell cycle was examined. Entry into mitosis was preceded by a transient 2-fold increase in cyclic AMP concentration and TGF-beta 1 delayed both this increase in cyclic AMP and entry into mitosis to the same extent. Addition of forskolin or 8-(4-chlorophenylthio)-cyclic AMP to cells 30 h after stimulation with serum completely reversed the increase in duration of G2 in the presence of TGF-beta 1, suggesting that the rise in cyclic AMP levels which precedes mitosis might trigger entry of the VSMCs into M phase. Addition of forskolin late in S phase (26 h after stimulation with serum) advanced the entry of the cells into M phase and they divided prematurely. This effect was unaffected by the addition of cycloheximide with the forskolin; however, the effect of forskolin on cell division was completely inhibited when cycloheximide was added late in G1. TGF-beta 1 prevented the loss of smooth-muscle-specific myosin heavy chain (SM-MHC), which occurs in primary VSMC cultures in the presence or absence of serum, and the cells proliferated while maintaining a differentiated phenotype. However, TGF-beta 1 did not cause re-differentiation of subcultured VSMCs which contained very low amounts of SM-MHC and the effect of TGF-beta 1 in extending the G2 phase of the cell cycle is exerted independently of its effect on differentiation.

Animals↗
PubMed1994

Local anaesthesia for inguinal herniorrhaphy--a neglected technique.

Ninety-nine inguinal herniorrhaphies were performed under local anaesthetic blockade between 1980 and 1988. This represents 32% of this surgical teams herniorrhaphy operations. A follow-up interview was possible in 82 patients. Satisfaction was expressed by 77 patient (94%) although six of the interviewed patients (7.4%) had moderate/significant discomfort during the procedure. Four of the 99 patients (4%) developed a wound infection.

Adult↗
PubMed1994

Mitogens for adult rat aortic vascular smooth muscle cells in serum-free primary culture.

OBJECTIVE: When vascular smooth muscle cells are dispersed into culture in the presence of serum they modulate their phenotype. The aim of this study was to determine the range of growth factors likely to stimulate replication of medial vascular smooth muscle cells in vivo by examining their responses when cultured in the absence of serum. METHODS: Freshly dispersed vascular smooth muscle cells from the healthy aortic media of adult rats were prepared and plated out in cell culture in the absence of fetal calf serum. DNA synthesis by these cells when plated onto fibronectin in response to various growth factors and vasoconstrictors was analysed by [3H]-thymidine incorporation. RESULTS: Less than 5% of cells plated on culture plastic spread, but 15-25% of cells plated onto fibronectin spread and survived for at least 8 d in culture. These cells responded to platelet derived growth factor BB homodimer (PDGF-BB), basic fibroblast growth factor, and epidermal growth factor by stimulating DNA synthesis at least 10-fold compared with cells in the absence of growth factors. Maximum rate of DNA synthesis occurred 36-42 h after addition of 10% fetal calf serum, whereas maximum rate of DNA synthesis occurred 80-88 h after stimulation with PDGF-BB or basic fibroblast growth factor. By contrast, PDGF-AA homodimer, transforming growth factor type beta, insulin-like growth factor I, angiotensin II, and endothelin I did not stimulate DNA synthesis by more than threefold. CONCLUSIONS: Freshly dispersed vascular smooth muscle cells plated onto fibronectin in the absence of serum proliferate in response to PDGF-BB, basic fibroblast growth factor, and epidermal growth factor by stimulating DNA synthesis. The range of mitogens and the time course of entry into DNA synthesis under these culture conditions suggest that serum-free culture provides a good model for the responses of medial vascular smooth muscle cells in vivo.

Animals↗
PubMed1993

Tamoxifen decreases the rate of proliferation of rat vascular smooth-muscle cells in culture by inducing production of transforming growth factor beta.

Tamoxifen selectively and reversibly decreased the rate of proliferation of adult rat aortic vascular smooth-muscle cells (VSMCs). Half-maximal inhibition of proliferation occurred at 2-5 microM tamoxifen for VSMCs and at > 50 microM for adventitial fibroblasts. The cell cycle time for all the VSMCs in the population was increased from 35 +/- 2 h to 54 +/- 4 h in the presence of 33 microM tamoxifen. Tamoxifen did not affect the time of entry into DNA synthesis, but delayed arrival at mitosis by > 24 h. It therefore extended the duration of the G2-to-M phase of the cell cycle. However, the rate of proliferation of VSMCs was not decreased by tamoxifen (at concentrations up to 50 microM) in the presence of neutralizing antibody to transforming growth factor beta (TGF-beta). The level of mRNA for TGF-beta 1 in VSMCs was strongly induced by 10 microM tamoxifen, and TGF-beta activity in conditioned medium from tamoxifen-treated cells was more than 50-fold higher than from control cells. Tamoxifen therefore extended the G2-to-M phase of the cell cycle in VSMCs by increasing TGF-beta activity in the culture.

Animals↗
PubMed1993

Proliferation of human smooth muscle cells promoted by lipoprotein(a).

Elevated blood concentrations of lipoprotein(a) [Lp(a)] and its constituent, apolipoprotein(a) [apo(a)], constitute a major risk factor for atherosclerosis, but their physiological activities remain obscure. Lp(a) and purified apo(a) stimulated the growth of human smooth muscle cells in culture. This effect resulted from inhibition of plasminogen activation, and consequently the activation by plasmin of latent transforming growth factor-beta, which is an inhibitor of smooth muscle cell growth. Because smooth muscle proliferation is one of the hallmarks of atherosclerotic lesions, these results point to a plausible mechanism for the atherogenic activity of Lp(a).

Animals↗
PubMed1993

Heparin decreases the rate of proliferation of rat vascular smooth muscle cells by releasing transforming growth factor beta-like activity from serum.

OBJECTIVES: Various heparins have been reported to inhibit the proliferation of vascular smooth muscle cells (VSMCs). The effects of eight chemically distinct heparins on the cell cycle and differentiation of primary and passaged cultures of rat aortic VSMCs have been characterised and the mechanism of heparin action investigated. METHODS: VSMCs from adult rat aorta were prepared by enzyme dispersion and stimulated to enter the cell cycle with 10% serum in the presence or absence of heparin. Progressions through S phase and M phase were measured by [3H]-thymidine incorporation and cell counting respectively. Flow cytometry was used to confirm the effects of heparin on VSMC cell cycle progression. The effect of heparin on VSMC differentiation was investigated by analysing smooth muscle specific myosin heavy chain content of the cells after heparin treatment. RESULTS: Eight heparins at concentrations between 5 micrograms.ml-1 and 100 micrograms.ml-1 partially inhibited VSMC proliferation (27% to 76% 96 hours after addition of heparin), but did not affect the entry of the cells into S phase. Flow cytometry confirmed that VSMC populations in the presence of heparin contained significantly (p < 0.005) more cells in the G2/M phase of the cell cycle than control populations. Heparin also blocked the dedifferentiation of primary cultures of VSMCs stimulated by serum. These effects of heparin were completely reversed by the presence of a neutralising antiserum to transforming growth factor beta (TGF beta) and heparin attached to agarose beads was as effective as free heparin as a growth inhibitor of VSMCs. CONCLUSIONS: Heparins of varying molecular weight and anticoagulant properties all partially inhibited VSMC proliferation predominantly by extending the G2/M phase of the cell cycle. Heparin also inhibited dedifferentiation of primary cultures of VSMCs. Heparin (< 100 micrograms.ml-1) acted extracellularly to release TGF beta from serum, which accounted for the effects of heparin on proliferation and differentiation.

Animals↗
PubMed1993

Approaches to the development of selective inhibitors of vascular smooth muscle cell proliferation.

Abnormal proliferation of VSMC is a feature of atheromatous plaques and is responsible for obstructive neointimal lesions at the sites of mechanical or immunological intimal damage. We have discussed approaches to the development of specific inhibitors of VSMC proliferation based firstly on studies of the mechanism of action of known inhibitors and secondly on the identification of genes which are unique to and necessary for VSMC proliferation. The effects of angiotensin converting enzyme inhibitors, heparin and hexamethylene bisacetamide on VSMC proliferation have been discussed. Each of these types of agent has been shown to inhibit VSMC proliferation in vitro and/or in vivo. Examination of the mechanisms by which these agents inhibit VSMC proliferation reveals that transforming growth factor beta is an important mediator of their action and that the processes of de-differentiation and proliferation are independently regulated. Studies aimed at identifying genes involved in VSMC proliferation are at an early stage but have already provided strong evidence to support the hypothesis that VSMC in the vessel wall are heterogeneous and contain a subpopulation of cells with an enhanced proliferative capacity. Identification of the genes expressed by these cells may allow specific inhibitors of VSMC proliferation to be developed and may shed light on the pathogenesis of neointimal lesions.

Acetamides↗
PubMed1993

Adult human aortic smooth muscle cells in culture produce active TGF-beta.

Vascular smooth muscle cells (VSMC) from adult human aortas proliferated in culture in response to fetal calf serum (FCS) with a population doubling time of 70-85 h compared with 35 +/- 5 h for VSMC derived from adult rat aortas. Medium conditioned on cultures prepared from aortas from three different donors and mixed 1:1 with fresh Dulbecco's modified Eagle's medium plus 20% FCS [human conditioned medium (HCM)] reduced the rate of proliferation of rat VSMC by 46 +/- 6% (n = 3) after 48 h compared with cells in fresh medium. HCM did not reduce the proportion (> 65%) of rat VSMC that entered DNA synthesis but delayed entry into mitosis by at least 18 h. This effect was similar to previous observations of the action of transforming growth factor-beta (TGF-beta) on rat VSMC (G. K. Owens, A. A. Geisterfer, Y. W. Yang, and A. Komoriya. J. Cell Biol. 107: 771-780, 1988). A TGF-beta assay using DNA synthesis in mink lung epithelial cells confirmed that human, but not rat, VSMC in culture secrete active TGF-beta. Addition of a neutralizing antibody to TGF-beta to human VSMC in the presence of 20% FCS decreased the population doubling time from 74 +/- 3 to 46 +/- 6 h (n = 3). These observations demonstrate that the long population doubling time of human VSMC is due to the production of active TGF-beta and to an inhibitory autocrine loop.

Animals↗
PubMed1992

Hexamethylenebisacetamide selectively inhibits the proliferation of human and rat vascular smooth-muscle cells.

Hexamethylenebisacetamide (HMBA) selectively and reversibly inhibited proliferation of human and rat vascular smooth-muscle cells (VSMCs) compared with endothelial cells, fibroblasts or lymphocytes. Half-maximal inhibition of VSMC proliferation occurred at 2-5 mM-HMBA, and at 30- greater than 50 mM for other cell types. HMBA also prevented de-differentiation, defined by the loss of smooth-muscle-specific myosin heavy chain, of primary rat VSMCs and caused partial re-differentiation of subcultured cells. Other inhibitors of ADP-ribosyltransferase were also selective inhibitors of VSMC proliferation.

Acetamides↗
PubMed1991

A large accumulation of non-muscle myosin occurs at first entry into M phase in rat vascular smooth-muscle cells.

Vascular smooth-muscle cells (VSMCs) from rat aortae contained very little non-muscle myosin heavy chain (MHC) immediately after dispersal, and the protein did not accumulate if the cells were held in G0/G1 phase by withholding serum or were held in first S phase by the addition of bromodeoxyuridine (BrdU). However, non-muscle MHC accumulated by greater than 20-fold per cell during first M phase, when over 80% of the cells divided between 48 h and 72 h after addition of serum. Delaying the addition of serum caused a delay in the accumulation of the non-muscle MHC until the cells subsequently entered M phase. If the cells were held in M phase at the metaphase/anaphase boundary by nocadazole, the accumulation of non-muscle myosin still occurred, although division was blocked. When the cells were pulse-labelled with [35S]methionine, it was found that non-muscle MHC was one of the major proteins being made and that its synthesis occurred at similar rates throughout the cell cycle. This implied that the rate of degradation of the protein before first M phase was much faster than in M phase, when the protein accumulated rapidly. This was confirmed by direct measurements of the rate at which [35S]methionine-labelled non-muscle MHC disappeared from the cells, which gave a half-life for the protein of about 8 h before M phase but about 5 days in post-mitotic cells, i.e. an increase of approx. 15-fold. These data are consistent with the hypothesis that there is a mechanism in VSMCs which shortens the half-life of the protein before first M phase and that the accumulation of non-muscle MHC which results from the increase in half-life at first M phase may be necessary for division of these cells.

Animals↗
PubMed1984

Reversal of neuromuscular block. Heart rate changes with slow injection of neostigmine and atropine mixtures.

The effect on heart rate of different rates of injection (10 seconds, 1, 3 and 5 minutes) of a fixed dose (neostigmine 2.5 mg and atropine 1.2 mg) and a weight-related dose (neostigmine 50 micrograms/kg and atropine 25 micrograms/kg) of neostigmine-atropine mixture given for the reversal of residual competitive neuromuscular block was studied in 196 healthy adult patients. In both series slow injection lessened and delayed the initial rise in heart rate. The subsequent fall in heart rate was less when the drugs were injected over 3 min compared with the 10 seconds and 1 minute injection groups but when given over 5 minutes there was a steady fall in heart rate, more so with the weight-related dose. It is recommended that a neostigmine and atropine mixture should be administered over 3 min.

Adult↗
PubMed1977

Anidoxime: a clinical trial of an oral analgesic agent.

The new oral analgesic drug anidoxime was compared with dihydrocodeine. There were no side-effects, and no significant differences between the effects of dihydrocodeine 50 mg, anidoxime 75 mg or anidoxime 100 mg.

Administration, Oral↗
PubMedDate not supplied

TGF-beta in blood: a complex problem.

The cytokine transforming growth factor-beta (TGF-beta) was initially purified from human platelets, a rich source of this protein. In addition to platelets, TGF-beta1 is also found in other blood fractions, including plasma and the circulating leukocytes. However, more than 15 years after the initial isolation of TGF-beta1, there remains no consensus on how much TGF-beta1 is present in normal human plasma. Here we review the difficulties associated with measuring TGF-beta concentrations in complex biological fluids, and discuss the current state of knowledge on the distribution of TGF-beta isoforms in various blood fractions as well as the nature of the TGF-beta-containing protein complexes.

Animals↗
PubMedDate not supplied

A common phenotype associated with atherogenesis in diverse mouse models of vascular lipid lesions.

The introduction of a range of different genetic modifications in mice results in altered lipoprotein metabolism and the development of vascular lipid lesions. At present, however, it is unclear to what extent the molecular events underlying lipid lesion formation are similar in these different mouse models of atherosclerosis. The aim of this study was to compare the protein expression pattern of lipid lesions from seven different mouse lines with varying susceptibility to vascular lipid lesion development, to determine to what extent lesions induced by different genetic interventions have a similar composition. The proteins we have measured, using quantitative immunofluorescence, are proteins whose expression is known to be modulated during atherogenesis in humans, including plasminogen activator inhibitor (PAI)-1, transforming growth factor (TGF)-beta 1, osteopontin and the macrophage marker CD11b. In all the mice lines we have investigated, PAI-1 was elevated wherever lesions developed. Active TGF-beta was depressed in the vessel wall of mice which developed lipid lesions, particularly in the intima. In contrast, TGF-beta 1 antigen (active plus latent TGF-beta 1) was increased at lesion sites. Accumulation of osteopontin and, with the marked exception of apolipoprotein(a) transgenic mice, tissue macrophages occurred at sites of lipid deposition in the vessel wall. Each lesion, irrespective of its size and the mouse strain in which it developed, had similar amounts of PAI-1, active TGF-beta and osteopontin per unit area of lesion. These data are consistent with a common phenotype accompanying atherogenesis, irrespective of the genetic basis of susceptibility.

Animals↗