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J C Metcalfe

Publications and source records attributed to J C Metcalfe.

At least 55 records · Page 3Linked to original sources
PubMed1995

Transforming growth factor-beta: the key to understanding lipoprotein(a)?

Most large studies of the blood parameters that act as risk factors for myocardial infarction, stroke and atherosclerosis have identified elevated circulating levels of lipoprotein(a) as an important risk factor. Lipoprotein(a) consists of an LDL particle that is covalently bound to the distinguishing protein component apolipoprotein(a). Ever since apolipoprotein(a) was cloned in 1987 and the marked sequence homology to plasminogen was noted, mechanisms for the atherogenic activity of lipoprotein(a), based on the competitive inhibition of plasminogen activity, have been proposed. However, with the availability of transgenic mice expressing both human apolipoprotein(a) and lipoprotein(a), recent studies have demonstrated that lipoprotein(a) acts to inhibit plasminogen activation in vivo. One consequence of this is reduced activation of the cytokine transforming growth factor-beta, an important regulator of vessel wall structure.

Animals↗
PubMed1995

No effect of 60 Hz electromagnetic fields on MYC or beta-actin expression in human leukemic cells.

Epidemiological studies have shown weak correlations between exposure to extremely low-frequency electromagnetic fields (ELF EMFs) and the incidence of several cancers, particularly childhood leukemias, although negative studies have also been reported. These observations have prompted a broad range of in vitro cellular studies in which effects of ELF EMFs have been observed. However, no reported response has been replicated widely in independent laboratories. One potentially important response is the rapid activation of proto-oncogenes and other genes in human leukemic (HL60) cells and a wide variety of other eukaryotic cells, because of the role of these genes in cell proliferation. We describe quantitative Northern analysis of MYC and beta-actin mRNAs from HL60 cells exposed to fields under conditions very similar to those reported previously to activate these genes, namely 60 Hz sinusoidal magnetic fields of 0.57, 5.7 or 57 microT for 20 min. In addition we have used a new design of field-exposure system and introduced a number of other modifications to the protocol to optimize any response. We have also developed a novel method providing enhanced accuracy for the quantitative measurement of mRNA. No significant effect of ELF EMFs on gene expression was observed using any of these systems and analytical methods.

Actins↗
PubMed1995

Proliferation of human aortic vascular smooth muscle cells in culture is modulated by active TGF beta.

OBJECTIVE: Human vascular smooth muscle cells (VSMC) prepared by enzyme dispersal have been reported to proliferate more slowly and remain more differentiated than VSMC derived by explant culture. The aim of this study was to investigate whether these differences are attributable to the production of transforming growth factor beta (TGF beta), a major modulator of VSMC properties in culture, by enzyme dispersal, but not explant, cultures. METHODS: Human aortic VSMC cultures were prepared by enzymatic dissociation of aortic media or by outgrowth of cells from explants derived from the vessel media. Properties, including rate of proliferation and differentiation, of the two different types of culture obtained from 10 donors (age 3-54 years, of either sex) were compared. The role of TGF beta as mediator of these differences was investigated. RESULTS: VSMC from enzyme dispersal cultures proliferated with a long doubling time in 20% fetal calf serum of 68(SEM 2) h, reaching a low saturation density with no "hills and valleys", and the cells were predominantly of stellate morphology. VSMC from explant cultures proliferated with a shorter doubling time of 35(2) h, reached a higher saturation density with "hills and valleys", and showed a spindle shaped morphology. The enzyme dispersed cells, but not the explant cells, released TGF beta into the medium. Addition of exogenous TGF beta to the explant cells extended the doubling time, while addition of a neutralising antibody to the enzyme dispersed cells decreased the doubling time. Tamoxifen, an anti-oestrogenic drug, decreased the rate of proliferation of the explant cells (ED50 = 50 nM; n = 3) but not enzyme dispersed cells by stimulating production of TGF beta. The explant and enzyme dispersal cultures also differed in response to growth factors. The explant cultures stimulated DNA synthesis in response to platelet derived growth factor (PDGF) AA and BB. Enzyme dispersal cultures stimulated DNA synthesis in response to PDGF-BB but response to AA was weaker and also variable. CONCLUSIONS: Human explant derived VSMC systematically differ from enzyme dispersed VSMC obtained from the same donor tissue. Wherever the potential role of TGF beta was investigated, the differences between enzyme dispersal and explant cultures were due to autocrine production of TGF beta by the enzyme dispersal, but not explant cultures.

Adolescent↗
PubMed1994

Activation of transforming growth factor-beta is inhibited in transgenic apolipoprotein(a) mice.

A HIGH concentration of serum lipoprotein(a) is a risk factor for atherosclerosis. Lipoprotein(a) consists of low-density lipoprotein with the additional protein component, apolipoprotein(a), a homologue of plasminogen. Lipoprotein(a) and apolipoprotein(a) enhance proliferation of human vascular smooth muscle cells (hVSMCs) in culture by inhibiting activation of plasminogen to plasmin, thus blocking the proteolytic activation of transforming growth factor-beta (TGF-beta), an autocrine inhibitor of hVSMC proliferation. The hypothesis that this pathway is a key step in atherogenesis is tested on transgenic mice expressing the human apolipoprotein(a) gene. We show here that the activation of TGF-beta is inhibited in the aortic wall and serum of mice expressing apolipoprotein(a), as a consequence of apolipoprotein(a) inhibition of plasminogen activation. These effects are closely correlated with VSMC activation.

Animals↗
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

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↗
PubMed1994

Intracellular pH recovery during respiratory acidosis in perfused hearts.

Na(+)-H+ exchange and Na(+)-dependent HCO3- influx both contribute to recovery of intracellular pH (pHi) after an acidosis induced by using the NH4Cl prepulse technique in mammalian and avian cardiac tissue. We have investigated the relative contributions of these mechanisms to pHi recovery during respiratory acidosis in the Langendorff-perfused ferret heart with and without correction of extracellular pH (pHo). pHi was measured from the chemical shift of the exogenous 31P nuclear magnetic resonance pH indicator 2-deoxy-D-glucose 6-phosphate. Intrinsic intracellular buffering capacity, calculated from the change in intracellular HCO3- concentration after a change in CO2, was reduced from approximately 33 (no inhibitors of acid extrusion present) to 19 +/- 5 mM when H+ extrusion during the acid loading phase was inhibited. During respiratory acidosis (pHo approximately 6.95), the proton efflux rate (JH) calculated at pHi 6.85 was 0.30 +/- 0.04 mmol.l-1.min-1 (n = 9). When pHo was corrected by increasing external HCO3- concentration to 60 mM during respiratory acidosis (pHo approximately 7.33), JH was 1.11 +/- 0.11 mmol.l-1.min-1 (n = 7), and when pHo was partially corrected by the addition of 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid to the perfusion solution (pHo approximately 7.1), JH was 0.64 +/- 0.08 mmol.l-1.min-1 (n = 6). In all three groups Na(+)-H+ exchange and HCO3- influx each contributed approximately 50% to acid-equivalent efflux.(ABSTRACT TRUNCATED AT 250 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1994

High expression of genes for calcification-regulating proteins in human atherosclerotic plaques.

Calcification is common in atheromatous plaques and may contribute to plaque rupture and subsequent thrombosis. However, little is known about the mechanisms which regulate the calcification process. Using in situ hybridization and immunohistochemistry we show that two bone-associated proteins, osteopontin (OP) and matrix Gla protein (MGP), are highly expressed in human atheromatous plaques. High levels of OP mRNA and protein were found in association with necrotic lipid cores and areas of calcification. The predominant cell type in these areas was the macrophage-derived foam cell, although some smooth muscle cells could also be identified. MGP was expressed uniformly by smooth muscle cells in the normal media and at high levels in parts of the atheromatous intima. Highest levels of this matrix-associated protein were found in lipid-rich areas of the plaque. The pattern of expression of these two genes contrasted markedly with that of calponin and SM22 alpha, genes expressed predominantly by differentiated smooth muscle cells and whose expression was generally confined to the media of the vessel. The postulated function of OP and MGP as regulators of calcification in bone and the high levels and colocalization of both in atheromatous plaques suggest they have an important role in plaque pathogenesis and stability.

Arteriosclerosis↗
PubMed1993

Inhibition of PDGF BB stimulated DNA synthesis in rat aortic vascular smooth muscle cells by the expression of a truncated PDGF beta receptor.

Rat aortic vascular smooth muscle cells (VSMCs) were transfected with a vector encoding a truncated human PDGF beta receptor or the full-length human PDGF beta receptor. Cells stably expressing the truncated human PDGF beta receptor or the full-length human PDGF beta receptor were selected and the effect of PDGF BB on DNA synthesis in these cells was studied. Cells expressing the full-length PDGF beta receptor entered DNA synthesis normally, however, cells expressing the truncated PDGF beta receptor failed to enter DNA synthesis in response to PDGF BB. These data show that the mitogenic response of rat aortic vascular smooth muscle cells to PDGF BB can be inhibited by the expression of a truncated PDGF beta receptor.

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

Calcium measurements with a new high-affinity n.m.r. indicator in the isolated perfused heart.

A new n.m.r. indicator, 1,2-bis-(2-[1-(hydroxycarbony)ethyl- (hydroxycarbonylmethyl)]amino-5-fluorophenoxy)ethane (DiMe-5FBAPTA), with a higher affinity for calcium (apparent Kd 46 nM, pH 7.2, 30 degrees C) than the parent 5FBAPTA chelator (Kd 537 nM, pH 7.1, 30 degrees C) has been used to measure the cardiac intracellular free Ca2+ ([Ca2+]i). DiMe-5FBAPTA was loaded into Langendorff-perfused ferret hearts maintained at 30 degrees C using the acetoxymethyl ester (AM) derivative. The intracellular concentration required to achieve an adequate signal-to-noise (S/N) ratio (> 10:1) for the n.m.r. spectra caused a similar reduction in developed pressure to that obtained using 5FBAPTA-AM. The DiMe-5FBAPTA was used to estimate [Ca2+]i in diastole, through the calcium transient and at rest in the presence of the slow calcium channel blocker diltiazem. At a pacing frequency of 1.0 Hz, end-diastolic [Ca2+]i was 198 +/- 30 nM (n = 9), and reducing the pacing frequency to 0.2 Hz lowered [Ca2+]i to 89 +/- 13 nM (n = 5). Perfusion with diltiazem (100 microM) for 60 min lowered [Ca2+]i to 10 +/- 1 nM (n = 4) in unpaced hearts and to 94 +/- 24 nM (n = 4) in hearts paced at 1.0 Hz. The [Ca2+]i transient measured with DiMe-5FBAPTA was sharper and delayed compared with the transient measured previously with 5FBAPTA. Co-loading the two indicators provided evidence that the indicator with the higher Kd had a dominant effect on the end-diastolic [Ca2+]i. The lower values for end-diastolic [Ca2+]i obtained with DiMe-5FBAPTA are consistent with fluorescent indicator measurements. These observations suggest that perturbations of [Ca2+]i caused by the new indicator are less than those induced by 5FBAPTA. DiMe-5FBAPTA therefore represents a useful step in the development of 19F-n.m.r. calcium indicators.

Animals↗
PubMed1993

Effects of fluorinated inositols on the proliferation of Swiss 3T3 fibroblasts.

The six monodeoxyfluoro-myo-inositols (nFIns) have previously been synthesized as potential inhibitors of signalling pathways mediated by phosphoinositides and their derivatives. Each of the six nFIns isomers was introduced into Swiss 3T3 fibroblasts by the techniques of microinjection or scrape loading at intracellular concentrations of approx. 2-4 mM. Of the six nFIns analogues, only 3FIns and 5FIns inhibited the serum-stimulated proliferation of 3T3 fibroblasts assayed by cell counting. Proliferation was inhibited to a similar extent by 3FIns or 5FIns, irrespective of which technique was used to introduce the nFIns analogues into the cells. Proliferation of cells 35 h after serum stimulation (i.e. when the first cell cycle was completed in control cells) was inhibited by approx. 50% by both 3FIns and 5FIns, and entry into S phase in the first cell cycle was inhibited to the same extent. This indicated that the nFIns analogues were inhibiting proliferation in the G1 phase of the cell cycle. Proliferation during the second cell cycle (35-60 h after stimulation) was inhibited by 75-85%. The inhibitory nFIns analogues were not toxic to the cells, nor did they affect the cellular ATP/ADP ratio. The effectiveness of the nFIns analogues in inhibiting proliferation was directly correlated with their ability to be incorporated into phosphatidylinositol analogues, suggesting that they may act by modulating phosphoinositide signalling pathways or other functions essential for DNA synthesis.

3T3 Cells↗
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

The uptake of 3H-labelled monodeoxyfluoro-myo-inositols into thymocytes and their incorporation into phospholipid in permeabilized cells.

Monodeoxyfluoro-myo-inositols were applied to electropermeabilized and intact thymocyte preparations to study their metabolism and uptake in order to investigate their suitability as potential inhibitors of phosphoinositide-mediated cellular responses. Only three of the monodeoxyfluoro-myo-inositols were incorporated into the phospholipids of thymocytes: 1D-3-deoxy-3-fluoro-myo-inositol, 5-deoxy-5-fluoro-myo-inositol and 1D-6-deoxy-6-fluoro-myo-inositol, all of which were weaker substrates for phosphatidylinositol synthase than was myo-inositol. The 3-, 5- and 6-fluoro analogues also behaved as competitive inhibitors, with K1 values of 350 +/- 5 microM, 350 +/- 5 microM and 2.9 +/- 2 mM respectively, compared with a Km for myo-inositol of 31 +/- 4 microM. When incubated with electropermeabilized thymocyte preparations, these three analogues of myo-inositol all formed phospholipids with chromatographic properties which corresponded to those of substituted phosphatidylinositol and phosphatidylinositol monophosphate. The uptake of myo-inositol and of the monodeoxyfluoro-myo-inositols into intact thymocytes was studied by a dual-label technique. All the monodeoxyfluoro-myo-inositols were taken up to some extent, but only 2-deoxy-2-fluoro-myo-inositol and 1D-3-deoxy-3-fluoro-myo-inositol were actively concentrated. The monodeoxyfluoro-myo-inositols were also assayed for their ability to inhibit the uptake of myo-inositol into cells. Both 2-deoxy-2-fluoro-myo-inositol and 1D-3-deoxy-3-fluoro-myo-inositol were effective inhibitors of myo-inositol uptake. Furthermore, 1D-1-deoxy-1-fluoro-myo-inositol, which was not taken up actively, was an effective inhibitor of myo-inositol uptake. The three effective inhibitors all showed Ki values of approximately 150 microM, close to the apparent Km for inositol uptake of 180 microM, and the 4-, 5- and 6-fluoro analogues had Ki values in excess of 10 mM.

Animals↗
PubMed1993

Resting and end-diastolic [Ca2+]i measurements in the Langendorff-perfused ferret heart loaded with a 19F NMR indicator.

Intracellular free calcium concentration ([Ca2+]i) was measured in Langendorff-perfused ferret hearts (30 degrees C, pH 7.4) by loading paced hearts with the 19F NMR calcium indicator, the 5,5'-difluoro derivative of 1,2-bis(o-aminophenoxy)ethane-N, N,N',N'-tetraacetic acid (5FBAPTA), to an initial cytosolic concentration of approximately 120 microM. Increasing the pacing frequency raised the end-diastolic [Ca2+]i from 299 +/- 44 nM (mean +/- SEM) at 0.2 Hz to 522 +/- 54 nM at 1.0 Hz and 691 +/- 166 nM at 2.0 Hz. Raising [Ca]o from 1.8 to 7.0 mM at a pacing frequency of 1.0 Hz increased end-diastolic [Ca2+]i to 625 +/- 39 nM. In unpaced hearts perfused with diltiazem (100 microM), [Ca2+]i fell rapidly to a steady-state value of < 100 nM after 60 min. Raising [Ca]o from 1.8 to 7.0 mM had no detectable effect on resting [Ca2+]i. The time course of the [Ca2+]i transient was measured in hearts paced at 1.1 Hz and perfused with 1.8 mM [Ca]o. The peak [Ca2+]i was approximately 2 microM at approximately 150 msec after the pacing pulse, and peak developed LVP occurred at 550 msec compared with 280 msec in control hearts not loaded with 5FBAPTA. Comparisons with data obtained by other techniques, including fluorescent [Ca2+]i indicators, imply that although the end-diastolic [Ca2+]i values obtained with 5FBAPTA in beating hearts are elevated by the concentrations of intracellular 5FBAPTA required for signal detection, the changes in [Ca2+]i observed in response to experimental interventions are qualitatively consistent with previous data.

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↗