Biomedical subjects

S L Diamond

Publications and source records attributed to S L Diamond.

At least 37 records · Page 2Linked to original sources

PubMed1999

Nuclear targeting peptide scaffolds for lipofection of nondividing mammalian cells.

Lipofection of nondividing cells is inefficient because much of the transfected DNA is retained in endosomes, and that which escapes to the cytoplasm enters the nucleus at low rates. To improve the final rate-limiting step of nuclear import, we conjugated a nonclassical nuclear localization signal (NLS) containing the M9 sequence of heterogeneous nuclear ribonucleoprotein (hnRNP) A1, to a cationic peptide scaffold derived from a scrambled sequence of the SV40 T-antigen consensus NLS (ScT). The ScT was added to improve DNA binding of the M9 sequence. Lipofection of confluent endothelium with plasmid complexed with the M9-ScT conjugate resulted in 83% transfection and a 63-fold increase in marker gene expression. The M9-ScT conjugate localized fluorescent plasmid into the nucleus of permeabilized cells, and addition of the nuclear pore blocker wheat germ agglutinin prevented nuclear import. This method of gene transfer may lead to viral- and lipid-free transfection of nondividing cells.

Animals↗

PubMed1999

Engineering design of optimal strategies for blood clot dissolution.

Blood clots form under hemodynamic conditions and can obstruct flow during angina, acute myocardial infarction, stroke, deep vein thrombosis, pulmonary embolism, peripheral thrombosis, or dialysis access graft thrombosis. Therapies to remove these clots through enzymatic and/or mechanical approaches require consideration of the biochemistry and structure of blood clots in conjunction with local transport phenomena. Because blood clots are porous objects exposed to local hemodynamic forces, pressure-driven interstitial permeation often controls drug penetration and the overall lysis rate of an occlusive thrombus. Reaction engineering and transport phenomena provide a framework to relate dosage of a given agent to potential outcomes. The design and testing of thrombolytic agents and the design of therapies must account for (a) the binding, catalytic, and systemic clearance properties of the therapeutic enzyme; (b) the dose and delivery regimen; (c) the biochemical and structural aspects of the thrombotic occlusion; (d) the prevailing hemodynamics and anatomical location of the thrombus; and (e) therapeutic constraints and risks of side effects. These principles also impact the design and analysis of local delivery devices.

Biomedical Engineering↗

PubMed1998

Kinetics of beta2-integrin and L-selectin bonding during neutrophil aggregation in shear flow.

Activated neutrophils aggregate in a shear field via bonding of L-selectin to P-selectin glycoprotein ligand-1 (PSGL-1) followed by a more stable bonding of LFA-1 (CD11a/CD18) to intercellular adhesion molecule 3 (ICAM-3) and Mac-1 (CD11b/CD18) to an unknown counter receptor. Assuming that the Mac-1 counter receptor is ICAM-3-like in strength and number, rate processes were deconvoluted from neutrophil homoaggregation data for shear rates (G) of 100-3000 s-1 with a two-body hydrodynamic collision model (. Biophys. J. 73:2819-2835). For integrin-mediated aggregation (characteristic bond strength of 5 microdynes) in the absence of L-selectin contributions, an average forward rate of kf = 1.57 x 10(-12) cm2/s predicted the measured efficiencies for G = 100-800 s-1. For a selectin bond formation rate constant equal to the integrin bond formation rate constant, the colloidal stability of unactivated neutrophils was satisfied for a reverse rate of the L-selectin-PGSL bond corresponding to an average bond half-life of 10 ms at a characteristic bond strength of 1 microdyne. Colliding neutrophils initially bridged by at least one L-selectin-PSGL-1 bond were calculated to rotate from 8 to 50 times at G = 400 to 3000 s-1, respectively, before obtaining mechanical stability in sheared fluid of either 0.75 or 1.75 cP viscosity. Thus for G > 400 s-1, the interaction time needed for the rotating aggregates to become stable was relatively constant at 52.5 +/- 8.5 ms, largely independent of shear rate or shear stress. Aggregation data and the colloidal stability criterion can provide a consistent set of forward and reverse rate constants and characteristic bond strengths for a known time-dependent stoichiometry of receptors on cells interacting in a shear flow field.

Biophysical Phenomena↗

PubMed1997

Does elevated nitric oxide production enhance the release of prostacyclin from shear stressed aortic endothelial cells?

Nitric oxide (NO) enhances prostacyclin (PGI2) production in agonist-stimulated endothelial cells, while peroxynitrite formed from NO and superoxide anion has been shown to activate cyclooxygenase. Using cultured bovine aortic endothelial cells (BAEC) exposed to arterial levels of laminar shear stress of 25 dynes/ cm2, we tested the hypothesis that NO mediated the elevated synthesis of PGI2 by shear stressed endothelium. Shear stress caused a large and rapid burst and sustained release of NO and PGI2 with the cumulative production at 1 hr enhanced 9.96-fold (n = 4, p < 0.005) and 9.16-fold (n = 3, p < 0.005), respectively, over stationary control production of 0.0257 nmol-NO/cm2-BAEC and 0.0193 ng-PGI2/cm2-BAEC. The NO synthase inhibitors, N(G)-nitro-L-arginine methyl ester (100 microM, LNAME) and N(G)-nitro-L-arginine (10 microM, LNA), caused 87.5 and 65% reductions (n = 3, p < 0.02) of cumulative NO release at 1 hr, respectively, and 45 and 55% reductions (n = 3, p = 0.025) of PGI2 release, respectively. About half of the elevated production of PGI2 in shear stressed cells was due to NO-dependent signaling, indicating that hemodynamic control of these two dilatory molecules is partially coupled.

Animals↗

PubMed1997

Shear stress induction of the endothelial nitric oxide synthase gene is calcium-dependent but not calcium-activated.

Arterial levels of shear stress (25 dynes/cm2) can elevate constitutive endothelial nitric oxide synthase (eNOS) gene expression in cultured endothelial cells (Ranjan et al., 1995). By PhosphorImaging of Northern blots, we report that the eNOS/glyceraldehyde 3-phosphate dehydrogenase (GAPDH) messenger RNA (mRNA) ratio in bovine aortic endothelial cells (BAEC) increased by 4.8- and 7.95-fold after 6-hr shear stress exposure of 4 and 25 dynes/cm2, respectively. Incubation of BAEC with dexamethasone (1 microM) had no effect on shear stress induction of eNOS mRNA. Buffering of intracellular calcium in BAEC with bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)-ester (BAPTA/AM) reduced shear stress induction of eNOS mRNA by 70%. Yet, stimulation of BAEC with ionomycin (0.1-1.0 microM) for 6-24 hr to elevate intracellular calcium had no effect on eNOS mRNA. These studies indicated that the shear stress induction of eNOS mRNA was a calcium-dependent, but not calcium-activated, process. Shear stress was a very potent and rapid inducer of the eNOS mRNA, which could not be mimicked with phorbol myristrate acetate or endotoxin. Inhibition of tyrosine kinases with genistein (10 microM) or tyrphostin B46 (10 microM) or inhibition of G-protein signaling with guanosine 5'-O-(2-thiodiphosphate) (GDP-betaS) (600 microM, 6-hr preincubation) did not block the shear stress elevation of eNOS mRNA.

Animals↗

PubMed1997

Hydrodynamic effects and receptor interactions of platelets and their aggregates in linear shear flow.

We have modeled platelet aggregation in a linear shear flow by accounting for two body collision hydrodynamics, platelet activation and receptor biology. Considering platelets and their aggregates as unequal-sized spheres with DLVO interactions (psi(platelet) = -15 mV, Hamaker constant = 10(-19) J), detailed hydrodynamics provided the flow field around the colliding platelets. Trajectory calculations were performed to obtain the far upstream cross-sectional area and the particle flux through this area provided the collision frequency. Only a fraction of platelets brought together by a shearing fluid flow were held together if successfully bound by fibrinogen cross-bridging GPIIb/IIIa receptors on the platelet surfaces. This fraction was calculated by modeling receptor-mediated aggregation using the formalism of Bell (Bell, G. I. 1979. A theoretical model for adhesion between cells mediated by multivalent ligands. Cell Biophys. 1:133-147) where the forward rate of bond formation dictated aggregation during collision and was estimated from the diffusional limited rate of lateral association of receptors multiplied by an effectiveness factor, eta, to give an apparent rate. For a value of eta = 0.0178, we calculated the overall efficiency (including both receptor binding and hydrodynamics effects) for equal-sized platelets with 50,000 receptors/platelet to be 0.206 for G = 41.9 s(-1), 0.05 for G = 335 s(-1), and 0.0086 for G = 1920 s(-1), values which are in agreement with efficiencies determined from initial platelet singlet consumption rates in flow through a tube. From our analysis, we predict that bond formation proceeds at a rate of approximately 0.1925 bonds/microm2 per ms, which is approximately 50-fold slower than the diffusion limited rate of association. This value of eta is also consistent with a colloidal stability of unactivated platelets at low shear rates. Fibrinogen was calculated to mediate aggregation quite efficiently at low shear rates but not at high shear rates. Although secondary collisions (an orbitlike trajectory) form only a small fraction of the total number of collisions, they become important at high shear rates (>750 s(-1)), as these are the only collisions that provide enough time to result in successful aggregate formation mediated by fibrinogen. The overall method provides a hydrodynamic and receptor correction of the Smoluchowski collision kernel and gives a first estimate of eta for the fibrinogen-GPIIb/IIIa cross-bridging of platelets. We also predict that secondary collisions extend the shear rate range at which fibrinogen can mediate successful aggregation.

Adenosine Diphosphate↗

PubMed1996

Computer simulation of systemic circulation and clot lysis dynamics during thrombolytic therapy that accounts for inner clot transport and reaction.

BACKGROUND: We developed a computer model to predict lysis rates of thrombi for intravenous thrombolytic regimens based on the convective/diffusive penetration of reacting and adsorbing fibrinolytic species from the circulation into the proximal face of a dissolving clot. METHODS AND RESULTS: Solution of a one-compartment plasma model provided the dynamic concentrations of fibrinolytic species that served as inlet conditions for stimulation of the one-dimensional spatiodynamics within a dissolving fibrin clot of defined composition. The model predicted the circulating levels of tissue plasminogen activator (TPA) and plasminogen levels found in clinical trials for various intravenous therapies. To test the model predictions under in vitro conditions, plasma clots were perfused with TPA (0.1 mumol/L) and plasminogen (1.0 mumol/L) delivered at constant permeation velocity of 0.1 or 0.2 mm/min. The model provided an accurate prediction of the measured lysis front movement. For TPA administration regimens used clinically, simulations predicted clot dissolution rates that were consistent with observed reperfusion times. For unidirectional permeation, the continual accumulation of adsorbing species at the moving lysis front due to prior rounds of solubilization and rebinding was predicted to provide for a marked concentration of TPA and plasmin and the eventual depletion of antiplasmin and macroglobulin in an advancing (approximately 0.25 mm thick) lysis zone. CONCLUSIONS: Pressure-driven permeation greatly enhances and is a primary determinant of the overall rate of clot lysis and creates a complex local reaction environment at the plasma/clot interface. With simulation of reaction and transport, it becomes possible to quantitatively link the administration regimen, plasminogena activator properties, and thrombolytic outcome.

Clot Retraction↗

PubMed1995

A fluorescence quench and dequench assay of fibrinogen polymerization, fibrinogenolysis, or fibrinolysis.

We present a kinetic assay based on the use of fluorescein isothiocyanate (FITC)-labeled fibrinogen as a fluoroactive substrate. The multiple FITCs bound to fibrinogen experienced quenching due to their close proximity. The thrombin-induced polymerization of FITC-fibrinogen led to additional fluorescence quenching due to enhanced neighbor-neighbor interactions in protofibrils and protofibril aggregates. The initial rate of quenching was directly dependent on the thrombin concentration at either low or high ionic strength. The final extent of quenching during polymerization with thrombin could be modulated by prevailing ionic strength and thrombin concentration suggesting that the quenching was due to fibril extension as well as aggregation. The full extent of quenching was greatly reduced by addition to the reaction of unlabeled fibrinogen or Gly-Pro-Arg-Pro, as expected for quenching due to neighbor-neighbor interactions. In contrast to polymerization, cleavage of fibrinogen by plasmin released FITC-labeled fragments free of proximity-based quenching that resulted in a large intensity increase as lysis proceeded--a process termed dequenching. The majority of the dequenching signal during fibrinogenolysis occurred during the generation of fragment X which proceeded as a first-order process with respect to fibrinogen-bound plasmin with kcat = 0.479 s-1. The Kd of active plasmin to fibrinogen was calculated to be 0.42 microM. Addition of epsilon-aminocaproic acid (epsilon ACA)-plasmin complex to FITC-fibrinogen produced little dequenching, demonstrating a requirement for binding in order to initiate lysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Fibrinogen↗

PubMed1995

Constitutive NOS expression in cultured endothelial cells is elevated by fluid shear stress.

The role of chronic fluid shear stress on endothelial constitutive nitric oxide synthase (cNOS) levels may have an important role in vessel diameter control. We subjected primary human umbilical vein endothelial cells (HUVEC) or bovine aortic endothelial cells (BAEC, passages 2-14) to steady laminar shear stress. In both cell types, the intracellular level of cNOS was elevated within 3 h of flow exposure at 25 dyn/cm2 and remained elevated at 6 and 12 h of flow exposure, compared with stationary controls, as indicated by digital immunofluorescence microscopy. Shear stress exposure for 6 h caused a 2.2 +/- 0.3- and 2.8 +/- 0.3-fold elevation of cNOS protein levels in BAEC (n = 3, P < 0.01) and HUVEC (n = 3, P < 0.01), respectively, in the presence or absence of 1 microM dexamethasone. Dexamethasone suppresses induction of the inducible NOS gene, indicating that cNOS was elevated by fluid shear stress. Flow exposure at 4 dyn/cm2 caused no enhancement of cNOS levels in either cell type. The flow induction of the cNOS protein levels was not blocked by preincubation of BAEC with 100-400 microM of NG-nitro-L-arginine methyl ester, indicating that flow-induced NO (or guanosine 3',5'-cyclic monophosphate) was not involved in the elevation of cNOS levels. Protein kinase C inhibitor H-7 (10 microM) had no effect on induction of NOS protein in BAEC exposed to 25 dyn/cm2. The cNOS mRNA levels were found to be elevated by two- to threefold in BAEC after 6 or 12 h of flow exposure at either 4 or 25 dyn/cm2, and this induction of NOS mRNA occurred in the presence of dexamethasone. The elevation of cNOS levels by chronic flow exposure may provide a mechanism for chronic regulation of vessel diameter by endothelial response to prevailing blood flow.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1995

Tissue plasminogen activator (tPA) inhibits plasmin degradation of fibrin. A mechanism that slows tPA-mediated fibrinolysis but does not require alpha 2-antiplasmin or leakage of intrinsic plasminogen.

Thrombolysis is dramatically slower when high concentrations of lytic agent are used. This paradoxical observation, first described as "plasminogen steal," was originally believed to be due to depletion of extrinsic plasminogen and consequent leaching of clot-bound plasminogen. We report that administration of increasing concentrations of recombinant human tissue plasminogen activator (tPA) to fibrin gels resulted in lysis rates that displayed a maximum, with significantly slower rates found at higher tPA, regardless of whether plasminogen was supplied extrinsically or intrinsically. A similar maximum in lysis rates was observed in a system lacking an extrinsic phase when plasminogen was added to fibrin suspensions preincubated with increasing tPA. Thus, intrinsic plasminogen leakage and alpha 2-antiplasmin were not required for the decreased lysis at high tPA. No maximum was observed for increasing concentrations of urokinase. Using fibrin suspensions or gels preincubated with tPA before addition of plasmin, we report that tPA, but not urokinase, caused a dose-dependent inhibition of the fibronolytic action of plasmin. With respect to optimal dosage schemes and the design of novel lytic agents, these findings indicate that (a) there exists a biochemical mechanism against minimizing reperfusion time with increasing tPA dosages and (b) the fibrin affinity of tPA may cause reduced fibrinolysis by plasmin.

Dose-Response Relationship, Drug↗

PubMed1995

Fluorogenic fibrinogen and fibrin facilitate macromolecular assembly and dynamic assay of picomolar levels of plasminogen activators under well mixed conditions.

Fibrinogen labeled with fluorescein isothiocyanate (FITC) was tested for its ability to serve as a template for macromolecular assembly as well as to provide a fluorogenic signal to allow continuous monitoring of plasminogen activation and fibrinolysis. As dilute solutions of FITC-fibrinogen or FITC-fibrin fiber suspension were degraded during lysis, release of fluorescent fragments abolished proximity-based quenching and resulted in a 2.0- or 3.6-fold increase in fluorescence intensity, respectively. Addition of plasmin at a final concentration of 10 pM to FITC-fibrinogen (10 nM) produced a detectable level of fluorescence dequenching. The assay had sufficient sensitivity to detect plasmin activity in the presence of excess antiplasmin activity, indicating the dissociation of a reversible antiplasmin-plasmin complex. The detection limit of the reaction assay was 20 pM and 200 pM of recombinant tPA and urokinase, using 10 nM FITC-fibrin and 10 nM and 5 nM plasminogen, respectively. The 10-fold greater sensitivity of the assay for tPA was likely due to the molecular assembly of tPA and plasminogen on the FITC-fibrin. Addition of thrombin (1 U/ml) and plasmin (0.1 nM) to 10 nM FITC-fibrinogen produced fluorescence quenching at first due to fibrinogen polymerization followed by dequenching due to fibrinolysis. Addition of 10 mM epsilon-aminocaproic acid to mixtures of thrombin and plasmin allowed the quenching assay of thrombin activity in the presence of active plasmin. FITC-fibrinogen could be copolymerized with recalcified platelet poor plasma (isolated from citrated whole blood) to yield fibrin that was fluorogenic. Dequenching was observed when plasmin was used to degrade the fibrin formed from the platelet poor plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Mechanically induced calcium mobilization in cultured endothelial cells is dependent on actin and phospholipase.

We sought to evaluate the mechanisms by which mechanical perturbation elevates intracellular calcium in endothelial cells. We report that the transient elevation in intracellular calcium in cultured bovine aortic endothelial cells (BAEC) in response to gentle perturbation with the side of a micropipette was not blocked by depolarization (external K+, 130 mmol/L), nifedipine (10 mumol/L), or Bay K 8644 R(+) (10 mumol/L). Thus, voltage-dependent calcium channels were not involved in the response. Also, amiloride (10 mumol/L) and tetraethylammonium (1 mmol/L) had no effect on calcium mobilization, indicating that Na+ and K+ transporters were not involved. Pretreatment of the cells with the phospholipase C and phospholipase A2 inhibitor manoalide (10 mumol/L) for 10 minutes at 37 degrees C completely abolished the calcium response, as did a 10-minute pretreatment with the inhibitor of actin polymerization, cytochalasin B (1 mumol/L). We observed an inhibitory effect of the phospholipase A2 and phospholipase C inhibitor 4-bromophenacyl bromide (10 mumol/L) on the mechanical response of BAEC that was not as potent as that observed with manoalide. To examine the role of arachidonic acid (AA) and subsequent metabolites that may be released after a putatively mechanical activation of phospholipase A2, we exposed BAEC to exogenous AA. We found that continued exposure of BAEC for 5 minutes to 10 nmol/L to 10 mumol/L AA caused no elevation of intracellular calcium. If mechanical stimulation activates phospholipase A2, the liberated AA and subsequent metabolites do not appear to have much effect on BAEC intracellular calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗

PubMed1994

Transport phenomena and clot dissolving therapy: an experimental investigation of diffusion-controlled and permeation-enhanced fibrinolysis.

We have investigated the effects of diffusive and convective transport on fibrinolysis. Using a constant pressure drop (delta P/L) from 0 to 3.7 mmHg/cm-clot to drive fluid permeation, various regimes of lytic agents were delivered into fine and coarse fibrin gels (3 mg/ml) and whole blood clots. Using plasmin (1 microM) delivered into pure fibrin or urokinase (1 microM) delivered into glu-plasminogen (2.2 microM)-laden fibrin, the velocity at which a lysis front moved across fibrin was greatly enhanced by increasing delta P/L. Lysis of fine and coarse fibrin clots by 1 microM plasmin at delta P/L of 3.67 and 1.835 mmHg/cm-clot, respectively, led to a 12-fold and 16-fold enhancement of the lysis front velocity compared to lysis without pressure-driven permeation. For uPA-mediated lysis of coarse fibrin at delta P/L = 3.67 mmHg/cm-clot, the velocity of the lysis front was 25-fold faster than the lysis front velocity measured in the absence of permeation. Similar permeation-enhanced phenomenon was seen for the lysis of whole blood clots. Without permeation, the placement of a lytic agent adjacent to a clot boundary led to a reaction front that moved at a velocity dependent on the concentration of plasmin or uPA used. Overall, these studies suggest that transport phenomena within the clot can play a major role in determining the time needed for reperfusion during fibrinolysis.

Biological Transport↗

PubMed1993

Fluid shear stress induces synthesis and nuclear localization of c-fos in cultured human endothelial cells.

Quiescent cultured primary human umbilical vein endothelial cells were exposed to low levels (4 dynes/cm2) or arterial levels (25 dynes/cm2) of steady laminar shear stress for one hour. Intracellular c-fos protein was measured by immunocytochemistry and quantitative fluorescence video microscopy. The nuclear-localized c-fos protein level was 5.4 +/- 2.0 fold higher (p < 0.01) in the cells exposed to arterial levels of shear stress as compared to the cells maintained in stationary culture. In contrast, the cells exposed to low levels of shear stress showed diffuse but slightly elevated levels of c-fos (2.4 +/- 0.73 times higher than control; p < 0.01) without preferential nuclear localization. The protein kinase C inhibitor, H7 (10 microM) significantly attenuated the induction of c-fos by 50% in cells exposed to arterial shear stress for 1 hour.

Cell Compartmentation↗

PubMed1993

Inner clot diffusion and permeation during fibrinolysis.

A model of fibrinolysis was developed using multicomponent convection-diffusion equations with homogeneous reaction and heterogeneous adsorption and reaction. Fibrin is the dissolving stationary phase and plasminogen, tissue plasminogen activator (tPA), urokinase (uPA), and plasmin are the soluble mobile species. The model is based on an accurate molecular description of the fibrin fiber and protofibril structure and contains no adjustable parameters and one phenomenological parameter estimated from experiment. The model can predict lysis fronts moving across fibrin clots (fine or coarse fibers) of various densities under different administration regimes using uPA and tPA. We predict that pressure-driven permeation is the major mode of transport that allows for kinetically significant thrombolysis during clinical situations. Without permeation, clot lysis would be severely diffusion limited and would require hundreds of minutes. Adsorption of tPA to fibrin under conditions of permeation was a nonequilibrium process that tended to front load clots with tPA. Protein engineering efforts to design optimal thrombolytics will likely be affected by the permeation processes that occur during thrombolysis.

Animals↗

PubMed1993

Mechanical perturbation of cultured human endothelial cells causes rapid increases of intracellular calcium.

In first-passage human umbilical vein endothelial cells (HUVEC) and bovine aortic endothelial cells (passages 13-16), exposure to gentle mechanical perturbation using a micropipette caused a transient rise in intracellular calcium concentration ([Ca2+]i). The increase in calcium concentration ([Ca2+]) occurred each time the cell was nudged. Three responses were evoked in each of 27 cells using 5 independent HUVEC harvests. Increase in [Ca2+] returned to near baseline levels within approximately 30 s. The stimulus did not cause membrane puncture, as indicated by 1) absence of rapid dye leakage, 2) regulated nature of the [Ca2+] response, 3) absence of membrane blebbing, and 4) repeatable nature of the response in the same cell. As an alternative stimulus, we created very narrow fluid streams (1- to 2-microns diam) from a pressurized pipette that generated shear stresses of approximately 0.001-0.1 dyn/cm2 on the cells. However, these low-shear streams had little effect on [Ca2+]i. The poke-induced change in [Ca2+] was not blocked by lowering extracellular [Ca2+] ([Ca2+]o; 10 microM). In the absence of [Ca2+]o, however, HUVEC did not respond to the first poke, indicating a requirement for some [Ca2+]o as a mediator of signaling. After several poke-induced responses, [Ca2+]i could still be released by caffeine (100 microM), indicating the integrity of the intracellular release mechanism(s). These studies indicate that the response of an endothelial cell to a membrane-deforming event involves a priming step utilizing [Ca2+]o, which facilitates the transient increase of [Ca2+]i.

Aniline Compounds↗

PubMed1991

Fluid flow decreases preproendothelin mRNA levels and suppresses endothelin-1 peptide release in cultured human endothelial cells.

Endothelin-1, a 21-amino acid peptide secreted by endothelial cells, has constrictor and mitogenic activity for vascular smooth muscle cells, and its mitogenic activity is synergistic with that of platelet-derived growth factor. Endothelial cell-derived endothelin-1 might therefore contribute to intimal hyperplasia in reendothelialized segments of vascular grafts or of endarterectomy and angioplasty sites. Because intimal hyperplasia occurs most often at sites with disordered flow patterns and lower fluid shear stress, we tested the effects of static culture versus high laminar shear stress (25 dyne/cm2) on endothelin-1 precursor (preproendothelin) gene mRNA transcript levels and endothelin-1 peptide release in cultured human endothelial cells. Primary cultures of human umbilical vein endothelial cells were subjected to controlled levels of shear stress in parallel plate flow chambers for 24 hours. To detect preproendothelin mRNA we applied a linked reverse transcriptase-polymerase chain reaction (RT/PCR) to RNA extracted from cultures. Southern blots of RT/PCR reaction products were hybridized with radioactive phosphorous (32P) labeled probes for the amplified preproendothelin complementary deoxyribonucleic acid (cDNA). Detection by RT/PCR of mRNA for glyceraldehyde 3-phosphate dehydrogenase was used to measure a constitutively expressed control signal. Endothelin-1 release into culture medium was measured by radioimmunoassay. Application of 25 dyne/cm2 of shear stress for 24 hours sharply reduced endothelial cell levels of precursor preproendothelin mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Blotting, Southern↗

PubMed1990

Tissue plasminogen activator messenger RNA levels increase in cultured human endothelial cells exposed to laminar shear stress.

Fluid shear stress can stimulate secretion of tissue plasminogen activator (tPA) by cultured human endothelial cells, while plasminogen activator inhibitor type-1 secretion remains unstimulated. To determine whether hemodynamically induced changes in tPA messenger RNA (mRNA) levels also occur, primary cultures from the same harvest of primary human umbilical vein endothelial cells were either maintained in stationary culture or exposed to arterial levels of shear stress (25 dynes/cm2) for 24 hours. Total cellular RNA was isolated from the shear stressed and stationary cultures and the relative levels of tPA mRNA and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA were determined using a coupled reverse transcriptase/polymerase chain reaction method. As indicated by the amount of amplification product, tPA mRNA levels were many fold higher (greater than 10) in endothelial cells subjected to shear stress for 24 hours than in stationary controls. In contrast, mRNA levels for GAPDH were similar in control and shear stressed cells. The constancy of the measured GAPDH signal indicated that the tPA response was a selective effect of fluid shear stress. When a similar polymerase chain reaction method was used, the mRNA levels of basic fibroblast growth factor (bFGF) were found not to vary in comparison to GAPDH mRNA after 24 hours of shear stress. These results indicate that enhancement of the fibrinolytic potential of endothelial cells in response to hemodynamic forces could involve transcriptional events.

Base Sequence↗