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Biomedical subjects

K E Allen

Publications and source records attributed to K E Allen.

At least 37 records · Page 2Linked to original sources
PubMed1994

Human venous endothelium can promote intimal hyperplasia in a paracrine manner.

PURPOSE: Vein graft stenoses resulting from the development of intimal hyperplasia are the major cause of graft failure in the first postoperative year. This study uses an organ culture of human saphenous vein to model vein graft intimal hyperplasia and assess the involvement of the endothelium in its development. METHODS: Organ cultures of saphenous vein were established comprised of intact vein, vein denuded of endothelium, or cocultures of intact plus denuded vein for 14 days in serum-supplemented medium. At the end of the culture period, veins were processed and sections prepared for immunostaining with monoclonal alpha-smooth muscle actin, Millers elastin, QB END.10, and bromodeoxyuridine. RESULTS: After culture, a cellular neointima developed in the intact veins that was significantly thicker than in those denuded of endothelium (24.5 vs 2.5 microns; p = 0.0001). Denuded veins in coculture with intact veins developed a thicker neointima than did denuded veins alone (12 vs 0 microns; p = 0.01) but less than that of intact veins (12 vs 28 microns; p < 0.01). Proliferation indexes followed the same trend (i.e., intimal smooth muscle cell proliferation was greatest in intact and least in denuded veins). CONCLUSION: The endothelium can promote neointimal formation in cultured human saphenous vein through a paracrine action on the vascular smooth muscle cell.

Endothelium, Vascular↗
PubMed1994

The influence of low molecular weight heparin on neointimal proliferation in cultured human saphenous vein.

OBJECTIVES: To investigate the effect of low molecular weight heparin (LMWH) on neointimal proliferation in cultured human saphenous vein, a model of human vein graft intimal hyperplasia. DESIGN: Dose ranging LMWH concentration study. SETTING: Culture Laboratory, Department of Surgery. MATERIALS: Fifteen segments of human long saphenous vein were incubated at 37 degrees C for 14 days in culture medium with 30% foetal calf serum. LMWH was added to one of the paired segments at 1, 10 and 100 micrograms/ml (five veins each dose). 5-bromo-2-deoxyuridine (Brd-U) was used to label proliferating cells. CHIEF OUTCOME MEASURES: Neointimal thickness (micron and proliferation index (% labelled neointimal cells). MAIN RESULTS: Neointimal thickness and proliferation index were both significantly reduced by LMWH at 100 micrograms/ml [control vs. LMWH, reduction in thickness 21 microns vs. 7 microns (median difference 12 microns, 95% conf. int. 6-18), reduction in proliferation index 33% to 6% (median difference 19%, 95% C.I. 4-32)]. CONCLUSIONS: High dose LMWH reduces neointimal proliferation in cultured human saphenous vein. The practical clinical application of these results may require the use of non anticoagulant heparin-like molecules and/or local drug delivery systems.

Cell Division↗
PubMed1993

Effect of seeding time and density on endothelial cell attachment to damaged vascular surfaces.

An in vitro model to facilitate the study of endothelial cell seeding of damaged vascular surfaces has been developed. This may have applications in the study of endothelial seeding of angioplasty and endarterectomy sites. Using this model, the optimum endothelial seeding time for attachment to damaged vascular surfaces should not exceed 30 min and, to achieve confluent cell attachment, a seeding density > 5 x 10(5) cells/cm2 should be used.

Angioplasty, Balloon↗
PubMed1993

Infrainguinal vein graft stenosis.

Although knowledge of the biological processes involved in the development of intimal hyperplasia has increased markedly in recent years, the precise aetiology of infrainguinal vein graft stenosis remains undetermined. Current therapy is therefore directed at treatment of the established lesion rather than its prevention. There seems little doubt, however, that recent advances in understanding of the vascular biology of normal and pathological saphenous vein will eventually lead to specific targeted therapy that will allow the prevention of vein graft stenosis.

Graft Occlusion, Vascular↗
PubMed1992

Endothelial cell seeding of damaged native vascular surfaces: prostacyclin production.

Endothelial cell seeding has been successful in reducing the thrombogenicity of prosthetic vascular grafts in animal and clinical studies. The reduction in thrombogenicity may be attributed to the intrinsic properties of endothelial cells themselves, and their ability to produce anti-thrombogenic mediators such as prostacyclin, and endothelium-derived relaxing factor. Endothelial seeding of damaged vascular surfaces produced during percutaneous transluminal angioplasty and endarterectomy is an attractive possibility due to the excellent attachment characteristics of the sub-endothelial tissue exposed during these procedures. The ability of endothelial seeded damaged vascular surfaces to produce prostacyclin was measured in an in vitro model of vascular injury. Endothelial-seeded damaged surfaces produced significantly higher prostacyclin release than did vessels damaged by balloon dilatation (265.5 pg cm-2 min-1 and 87.5 pg cm-2 min-1 respectively). This study provides evidence that endothelial seeding of damaged native vascular surfaces is technically feasible and that seeding may reduce the thrombogenicity of vascular surfaces following balloon dilatation.

Angioplasty, Balloon↗
PubMed1991

Effects of two methods of endothelial cell seeding on cell retention during blood flow.

The potential benefits of endothelial cell seeding depend not only on effective cell attachment, but also on the ability of the cells to resist the stresses of blood flow. We have investigated the effect of different blood flow rates on immediately seeded grafts (SHORT) and also on preformed confluent monolayers formed by overnight incubation (LONG). Cells were labelled with indium and then exposed to either 100 or 200 ml/min of pulsatile blood flow. Cell retention was measured up to 120 min. At both flow rates the LONG seeding procedure gave significantly better cell retention than the SHORT method (P less than 0.01 at all times). There was no difference in cell retention between the two flow rates in the LONG group, but more cells detached at the higher flow rate in the SHORT group (P less than 0.01). We conclude that the new seeding method, using preformed confluent monolayers, significantly improves cell retention during blood flow.

Blood Flow Velocity↗
PubMed1991

The effect of preformed confluent endothelial cell monolayers on the patency and thrombogenicity of small calibre vascular grafts.

Endothelial cell seeding has been proposed as a method to improve the patency rates in small calibre prosthetic vascular grafts. The seeding methods used at present leave much of the graft luminal surface devoid of endothelial cells and thus still significantly thrombogenic. We have developed a method to preform confluent endothelial cell monolayers, on the grafts prior to implantation, and this study investigates the effect of these monolayers on the early thrombogenicity and patency of polytetrafluoroethylene (PTFE) grafts. Small diameter PTFE grafts were seeded with canine endothelial cells obtained from the external jugular vein. Each of five dogs then received a graft seeded with its own cells and a contralateral, non-seeded control graft. At 1 and 10 weeks after graft implantation graft thrombogenicity was assessed by the use of Indium labelled platelets. The thrombogenicity index (TI) of each graft was determined from counts of gamma activity recorded over a period of 7 days. Grafts were subsequently removed at 12 weeks. At 1 week the mean TI for the seeded grafts was 0.123 (SD 0.019) and that for the controls 0.183 (SD 0.017) (p = 0.005). At 10 weeks only the seeded grafts could be assessed because all of the control grafts had occluded. At this point in time the seeded grafts had a mean TI of 0.159 (SD 0.011) (p = 0.047 vs. seeded at 1 week). By the time of removal at 12 weeks, all control grafts were occluded but only one of the seeded grafts had occluded (p = 0.025). In conclusion, the use of preformed, confluent endothelial cell monolayers for seeding prosthetic grafts significantly reduces the early graft thrombogenicity and improves graft patency. It does not, however, completely halt the increase in thrombogenicity which occurs during the early post-implantation period.

Animals↗
PubMed1990

The effect of varying fibronectin concentration on the attachment of endothelial cells to polytetrafluoroethylene vascular grafts.

Endothelial cell seeding onto untreated polytetrafluoroethylene vascular prostheses is inefficient. In an effort to improve cell attachment, numerous investigators have used fibronectin as a coating material to pretreat the luminal surfaces of these prostheses. The concentrations of fibronectin used have varied enormously, and no one has yet determined the most efficient concentration in terms of cell attachment and cost. Using endothelial cells labeled with indium 111 oxine we have studied the effect of varying fibronectin concentration on the attachment of these cells onto polytetrafluoroethylene vascular grafts. Seeding efficiency was significantly better in all groups of coated grafts, at all times (10, 30, 60, and 120 minutes), compared to uncoated controls (p less than 0.01). Overall, fibronectin at a concentration of 20 micrograms/ml was found to be the most efficient in terms of cell attachment and cost since any further increase in concentration was not accompanied by increased cell attachment. We now routinely use fibronectin at a concentration of 20 micrograms/ml to coat our grafts before endothelial cell seeding.

Blood Vessel Prosthesis↗
PubMed1989

Deoxyglucose-resistant mutants of Neurospora crassa: isolation, mapping, and biochemical characterization.

Neurospora crassa mutants resistant to 2-deoxyglucose have been isolated, and their mutations have been mapped to four genetic loci. The mutants have the following characteristics: (i) they are resistant to sorbose as well as to 2-deoxyglucose; (ii) they are partially or completely constitutive for glucose transport system II, glucamylase, and invertase, which are usually repressed during growth on glucose; and (iii) they synthesize an invertase with abnormal thermostability and immunological properties, suggesting altered posttranslational modification. All of these characteristics could arise from defects in the regulation of carbon metabolism. In addition, mutants with mutations at three of the loci lack glucose transport system I, which is normally synthesized constitutively by wild-type N. crassa. Although the basis for this change is not yet clear, the mutants provide a way of studying the high-affinity system II uncomplicated by the presence of the low-affinity system I.

3-O-Methylglucose↗
PubMed1986

Characterization of an essential arginine residue in the plasma membrane H+-ATPase of Neurospora crassa.

Treatment of the plasma membrane H+-ATPase of Neurospora crassa with the arginine-specific reagents phenylglyoxal or 2,3-butanedione at 30 degrees C, pH 7.0, leads to a marked inhibition of ATPase activity. MgATP, the physiological substrate of the enzyme, protects against inactivation. MgADP, a competitive inhibitor of ATPase activity with a measured Ki of 0.11 mM, also protects, yielding calculated KD values of 0.125 and 0.115 mM in the presence of phenylglyoxal and 2,3-butanedione, respectively. The excellent agreement between Ki and KD values makes it likely that MgADP exerts its protective effect by binding to the catalytic site of the enzyme. Loss of activity follows pseudo-first order kinetics with respect to phenylglyoxal and 2,3-butanedione concentration, and double log plots of pseudo-first order rate constants versus reagent concentration yield slopes of 0.999 (phenylglyoxal) and 0.885 (2,3-butanedione), suggesting that the modification of one reactive site/mol of H+-ATPase is sufficient for inactivation. This stoichiometry has been confirmed by direct measurements of the incorporation of [14C]phenylglyoxal. Taken together, the results support the notion that one arginine residue, either located at the catalytic site or shielded by a conformational change upon nucleotide binding, plays an essential role in Neurospora H+-ATPase activity.

Arginine↗
PubMed1983

Vanadate-resistant mutants of Neurospora crassa are deficient in a high-affinity phosphate transport system.

Mutant strains of Neurospora crassa have been selected which grow on media containing vanadate, an inhibitor of the plasma membrane ATPase. The mutations all map to a single region (designated van) on the left arm of linkage group VII. The van mutants are unable to take up vanadate from the medium and are also deficient in the uptake of phosphate via a derepressible, high-affinity phosphate transport system. In the van mutants, the K(m) for phosphate transport is elevated as much as 35-fold, indicating that the van locus may code for a structural component of the high-affinity phosphate transport system.

Biological Transport↗
PubMed1981

Geometric isomers of substituted triphenylethylenes and antiestrogen action.

The estrogenic and antiestrogenic activities of tamoxifen ICI 47,699, enclomiphene, zuclomiphene, and the geometric isomers of monohydroxytamoxifen and CI628 were determined in the 3-day immature rat uterine weight test. Tamoxifen, enclomiphene, and the releated geometric isomers of monohydroxytamoxifen and CI628 were partially estrogenic with antiestrogenic properties. ICI 47,699 and zuclomiphene were predominantly estrogenic; however, an antiestrogen effect for zuclomiphene (100 micrograms daily) was demonstrable and large doses of ICI 47,699 (1 or 10 mg daily) inhibited full estrogen action. In contrast, the geometric isomers of monohydroxytamoxifen and CI628 related to ICI 47,699 and zuclomiphene were partially estrogenic with antiestrogenic properties. The estrogenic properties of ICI 47,699 were classified in three ways: elevation of uterine wet weight, increase in whole uterine DNA, and increase in the mitotic activity of luminal epithelial cells. In general, ICI 47,699 was able to initiate estrogenic responses of DNA synthesis or mitosis by translocation of fewer cytoplasmic estrogen receptors to the nuclear compartment than tamoxifen. A model is proposed to explain antiestrogen action in terms of the geometric requirements for receptor binding. It is suggested that the position in space of the alkylaminoethoxyside chain is of fundamental importance. Overall, these data lend support to the view that a structurally specific ligand-estrogen receptor complex can influence the future events within a target tissue to produce either an agonist or an antagonist response.

Animals↗
PubMed1980

Evidence for the metabolic activation of non-steroidal antioestrogens: a study of structure-activity relationships.

1 The oestrogenic and antioestrogenic activities of tamoxifen and monohydroxytamoxifen have been compared with those of para-methoxy, -methyl, -fluoro, and -chloro tamoxifen in the 3 day immature rat uterine weight test.2 The oestrogenic activity of mestranol, a steroid with low oestrogen receptor binding affinity which is believed to be demethylated to ethinyl oestradiol before exerting its effects, was less potent than ethinyl oestradiol when assayed in the 3 day immature rat uterine weight test. Similarly, para-methoxytamoxifen was less active than monohydroxytamoxifen in oestrogenic and antioestrogenic tests.3 The introduction of a para-methoxy group into tamoxifen did not affect oestrogenic or antioestrogenic activity.4 All the derivatives of tamoxifen were partial oestrogen agonists when compared with oestradiol benzoate in the 3 d immature rat uterine weight test. All test compounds inhibited the uterotrophic activity of oestradiol benzoate (0.16 mug daily) in a dose-related manner. The order of potency was: monohydroxytamoxifen > tamoxifen identical with methoxytamoxifen > p-fluoro identical with p-chloro identical with p-methyltamoxifen.5 Tamoxifen was approximately equiactive with its p-methyl, p-fluoro and p-chloro derivatives in the ability to inhibit [(3)H]-oestradiol binding to rat uterine oestrogen receptors in vitro.6 Tamoxifen was approximately equiactive with its p-methyl and p-fluoro derivatives in the ability to inhibit vaginal cornification of ovariectomized rats upon intravaginal administration with oestradiol (3.2 ng total dose).7 Since tamoxifen in vivo was more active as a partial oestrogen agonist and antagonist than the para substituted fluoro, chloro and methyl derivatives that cannot undergo metabolic hydroxylation to monohydroxytamoxifen, whereas the antioestrogenic activity of the compounds upon local application in the vaginal cornification test was equivalent as was their ability to inhibit [(3)H]-oestradiol-17beta binding to the oestrogen receptor in vitro, it is suggested that at low doses; i.e. over the range of the partial agonist dose-response curve, the biological activity of tamoxifen is the net result of the activities of the parent compound and its metabolites.8 The results demonstrate that metabolic activation of non-steroidal antioestrogens is only an advantage and not a requirement for antioestrogenic activity.

Animals↗
PubMed1978

Effects of inhibitors on the plasma membrane and mitochondrial adenosine triphosphatases of Neurospora crassa.

A comparative study has been made of the effects of a variety of inhibitors on the plasma membrane ATPase and mitochondrial ATPase of Neurospora crassa. The most specific inhibitors proved to be vanadate and diethylstilbestrol for the plasma membrane ATPase and azide, oligomycin, venturicidin, and leucinostatin for mitochondrial ATPase. N,N'-Dicyclohexylcarbodiimide, octylguanidine, triphenylsulfonium chloride, and quercetin and related bioflavonoids inhibited both enzymes, although with different concentration dependences. Other compounds that were tested (phaseolin, fusicoccin, deoxycorticosterone, alachlor, salicyclic acid, N-1-napthylphthalamate, triiodobenzoic acid, cyclic AMP, cyclic GMP, theobromine, theophylline, and histamine) had no significant effect on either enzyme. Overall, the results indicate that the plasma membrane and mitochondrial ATPases are distinct enzymes, in spite of the fact that they may play related roles in H+ transport across their respective membranes.

Adenosine Triphosphatases↗