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Biomedical subjects

M B Stemerman

Publications and source records attributed to M B Stemerman.

At least 19 recordsLinked to original sources
PubMed1995

Immediate-early gene expression in human saphenous veins harvested during coronary artery bypass graft operations.

Saphenous vein graft occlusion is a common late complication of coronary bypass grafting. Intimal smooth muscle cell hyperplasia is a component of this pathobiology, but the underlying molecular events are poorly understood. Immediate-early genes are activated shortly after growth stimulation and subserve cellular functions, which may contribute to intimal smooth muscle cell accumulation. In the present study, human saphenous vein grafts were harvested with minimal manipulation during coronary bypass and processed for isolation of total ribonucleic acid to examine change in immediate-early gene expression of messenger ribonucleic acid by Northern blotting techniques. Thirty saphenous vein grafts were incubated at 4 degrees C in Dulbecco's modified Eagle media from 30 minutes to 10 hours. The messenger ribonucleic acids for immediate-early genes c-fos and c-myc were weak or undetectable in controls but were increased (> 10 times controls) within 1 hour (c-fos) and persisted for at least 6 hours (c-myc) after harvest. Our results demonstrate, for the first time in human vascular tissue, incipient immediate-early gene induction. This information may lead to molecular therapies to control saphenous vein graft disease.

Adult↗
PubMed1995

Native low-density lipoprotein increases endothelial cell nitric oxide synthase generation of superoxide anion.

To examine mechanisms by which native low-density lipoprotein (n-LDL) perturbs endothelial cell (EC) release of superoxide anion (O2-) and nitric oxide (NO), ECs were incubated with n-LDL at 240 mg cholesterol per deciliter for 4 days with media changes every 24 hours. n-LDL increases EC release of O2- by more than fourfold and increases nitrite production by 57%. In the conditioned media from day-4 incubations, n-LDL increases total nitrogen oxides 20 times control EC (C-EC) levels. However, n-LDL did not alter EC NO synthase (eNOS) enzyme activity as measured by the [3H]citrulline assay. N omega-Nitro-L-arginine methyl ester, a specific inhibitor of eNOS activity, increases C-EC release of O2- by > 300% but decreases LDL-treated EC (LDL-EC) release by > 95%. L-Arginine inhibits the release of O2- from LDL-ECs by > 95% but did not effect C-EC release of O2-. Indomethacin and SKF 525A partially attenuate LDL-induced increases in O2- production by approximately 50% and 30%, respectively. Thus, n-LDL increases O2- and NO production, which increases the likelihood of the formation of peroxynitrite (ONOO-), a potent oxidant. n-LDL increases the levels of nitrotyrosine, a stable oxidation product of ONOO-, and tyrosine by approximately 50%. In spite of this increase in oxidative metabolism, analysis of thiobarbituric acid substances reveals that no significant changes in the oxidation of n-LDL occur during the 24-hour incubations with ECs.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗
PubMed1994

Induction of cyclooxygenase-2 in rat vascular smooth muscle cells in vitro and in vivo.

Prostaglandins are synthesized from arachidonic acid by the rate-limiting enzyme cyclooxygenase (prostaglandin G/H synthase). Active cyclooxygenase is encoded by two distinct and independently regulated genes, termed cyclooxygenase-1 (cox1) and cyclooxygenase-2 (cox2). In this investigation, we examined the expression of cox1 and cox2 mRNA in rat aorta following balloon deendothelialization (BDE) in vivo and in rat aortic smooth muscle cells (SMC) after serum stimulation in vitro. Two h after BDE, rat aortic cox2 mRNA levels increased greater than 50-fold relative to the lowest detectable levels on days 2 and 14. No message was detectable in non-BDE control rat aortas. Similar to the results found in vivo, cultured SMC exhibited a greater than 45-fold increase in cox2 mRNA levels after a 2-h exposure to serum. This increase was transient because cox2 levels declined at 4 and 8 h. In contrast, minimal changes in cox1 mRNA levels were observed after BDE or serum treatments. Increased levels of cox2 mRNA and corresponding protein synthesis led to an accumulation of total cyclooxygenase protein, which remained elevated 24 h after serum stimulation. Serum-treated SMC also generated greater amounts of cyclooxygenase-dependent metabolites than quiescent SMC as evidenced by marked increases in prostaglandin E2 content in conditioned media. This increase is associated with a 2.5-3.0-fold increased rate of arachidonic acid conversion to prostaglandin E2. Our data indicate that injury and serum stimulation differentially regulate mRNA and protein expression of two distinct cox genes in vascular SMC in vivo and in vitro. The findings suggest that the prostanoid responses after vascular injury are, in part, mediated by acute increases in cox2 mRNA and cyclooxygenase-2 protein.

Animals↗
PubMed1993

Inhibition of smooth muscle cell proliferation and DNA synthesis by Vasoprin--a biologic response modifier.

The effect of Vasoprin, a biologic response modifier (BRM), on smooth muscle cell DNA synthesis and intimal proliferation was studied. In this report, we have measured DNA specific activity (SA) and intimal thickening following balloon injury (balloon de-endothelialization (BDE)) after administration of Vasoprin. Various dose schedules were employed to assess Vasoprin's effect post BDE. Forty-eight hours following vascular injury (the peak of SMC DNA synthesis), Vasoprin administered at various treatment schedules showed between 54% and 72.6% +/- 9% (P < 0.0012) inhibition of DNA SA compared with vehicle controls. Vasoprin also diminished SMC intimal thickening following its administration. Seven days after vascular injury, at which time SMC neointimal proliferation reaches its peak, Vasoprin treated animals showed marked reduction in intimal thickening by 45.5% +/- 6.6% (P < 0.008), compared with the BDE rats receiving an agent vehicle as controls. The results of this report demonstrate that a biologic response modifier (Vasoprin) has a profound effect on SMC proliferation, and that the immune system may be a component in the progression of intimal thickening and hence vascular closure.

Animals↗
PubMed1993

Lipid fluidity modulates platelet aggregation and agglutination in vitro.

To determine the effect of altered membrane fluidity on platelet aggregation/agglutination, fresh, washed human platelets were treated with A2C, a cyclopropyl fatty acid ester which is known to enhance mobility of intrinsic membrane bilayer constituents and increase membrane fluidity. Fluorescence polarization studies demonstrated A2C incubation time- and concentration-dependent increases in platelet membrane fluidity (decreased fluorescence anisotropy). Preincubation with A2C was associated with diminished collagen, thrombin and ristocetin-induced platelet aggregation/agglutination. Aggregation/agglutination was diminished by 93 +/- 5% for collagen (0.2 mg/ml), 53 +/- 3% for thrombin (1.0 U/ml) and 85 +/- 9% for ristocetin (1.1 mg/ml). These data suggest that membrane fluidity is involved in the regulation of platelet function.

Agglutination↗
PubMed1992

Atherogenic levels of low-density lipoprotein increase endocytotic activity in cultured human endothelial cells.

Cultured human umbilical vein endothelial cells (EC) exposed to atherogenic low-density lipoprotein (LDL) levels for protracted periods demonstrated heightened endocytosis. Confluent EC were incubated with LDL 90 to 240 mg/dl cholesterol for 1 to 4 days and endocytosis was measured by 14C-sucrose uptake. Control EC and cells incubated with 90 mg/dl LDL cholesterol showed similar uptakes of 14C-sucrose during all measured time periods. In contrast, EC exposed to 240 mg/dl LDL cholesterol showed an increase in endocytosis beginning at 2 days, whereas 160 mg/dl LDL cholesterol promoted increased uptake by 4 days. The endocytotic activity of LDL-perturbed EC is reduced to levels seen in control cells by cytochalasin B, an actin polymerization inhibitor. This finding suggests a modulatory role for the cytoskeleton in endocytosis changes. Examination of LDL-perturbed EC cytoskeleton reveals structural remodeling resulting in a marked increase in stress fibers. Cytochalasin B exposure causes a loss of stress fibers with the formation of globular filamental aggregates. Such LDL-induced cellular functional changes may contribute mechanistically to endothelial dysfunction, which is widely held to be a major contributing factor in the pathogenesis of atherosclerosis.

Arteriosclerosis↗
PubMed1991

Effect of low-density lipoprotein on endothelial cell membrane fluidity and mononuclear cell attachment.

To determine the effects of prolonged low-density lipoprotein (LDL) exposure in vitro on cultured endothelial cell (EC) lipid dynamics and cellular function, human umbilical vein ECs were incubated in LDL concentrations [cholesterol (Chol) = 240 mg/dl] associated with the premature development of atherosclerosis. After 4 days of incubation, cells were examined for changes in cellular lipid composition and for membrane fluidity. Results indicate that LDL-EC have increased Chol content (control EC vs. LDL-EC = 22.4 +/- 5.26 vs. 38.9 +/- 0.24 nmol/10(6) cells, P less than 0.05) and cellular Chol-to-phospholipid ratio (0.61 +/- 0.10 vs. 1.21 +/- 0.10 mol/mol, P less than 0.05). Augmentation of EC Chol content was accompanied by a marked decrease in EC cellular membrane fluidity as assessed by fluorescence polarization (anisotropy, r values, 0.172 +/- 0.019 vs. 0.226 +/- 0.014, P less than 0.0001). LDL-induced changes in EC lipid dynamics were associated with enhanced EC binding of monocytes (P less than 0.05) and U937 cells (P less than 0.01). Both LDL-induced decreases in membrane fluidity and enhanced attachment of mononuclear cells were reversed to control levels following a 2-min incubation of LDL-EC with the membrane mobility agent, A2C. These data therefore suggest that LDL-induced modulations in lipid dynamics play an important role in perturbation of EC function.

Cell Adhesion↗
PubMed1990

14, 15-Epoxyeicosatrienoic acid promotes endothelial cell dependent adhesion of human monocytic tumor U937 cells.

Arachidonic acid (AA) can be metabolized in endothelial cells (EC) to a series of epoxides via cytochrome P-450 epoxygenase with 14,15 epoxyeicosatrienoic acid (14,15-EET) as the major product. In this communication we report that 14,15-EET significantly enhances U937 cell attachment to EC with maximal cell attachment at 2.5 to 5 x 10(-7) M 14,15-EET. Thus, 14,15-EET may play a substantial role in inflammation and/or atherogenesis by inducing monocyte attachment to EC.

8,11,14-Eicosatrienoic Acid↗
PubMed1990

Bradykinin induces superoxide anion release from human endothelial cells.

The time-dependent release of superoxide anion (O2-) from bradykinin (Bk)-stimulated human umbilical vein endothelial cells (EC) was measured as the superoxide dismutase-inhibitable reduction of ferricytochrome C employing a novel application of microspectrophotometry. In the absence of Bk, O2- release by EC was not detectable. EC exposure to Bk (10(-6) to 10(-5) M) resulted in a rapid release of O2-. The release of O2- occurred within 5 minutes of exposure. O2- release was partially inhibited by indomethacin (63 +/- 6%), thus suggesting that arachidonic acid metabolism, through cyclooxygenase, contributes to EC O2- production. EC O2- release may be an important component in the pathophysiologic actions of Bk on vascular function.

Bradykinin↗
PubMed1990

Early proto-oncogene expression in rat aortic smooth muscle cells following endothelial removal.

To study the mechanism(s) of vascular smooth muscle cell proliferation in vivo, mRNA levels of c-fos, c-jun, and c-myc were determined by Northern blot analysis following vascular balloon de-endothelialization (BDE). Medial smooth muscle cells (SMC) were separated and studied by enzymatic digestion of the vessel wall. mRNA levels of c-fos and c-jun from aortic smooth muscle cells (SMC) were simultaneously induced within 30 minutes of BDE and declined to baseline by 1.5 hours, c-myc mRNA did not begin to increase until 1 hour after vascular injury. Levels of c-myc peaked at 2 hours and were sustained for an additional 4 hours before gradually declining. Smooth muscle cells derived from enzyme-treated control aortae that did not undergo BDE expressed c-fos and c-jun, but showed no evidence of c-myc message. In contrast, nonenzymatically treated, non-BDE whole aortae (containing both media and adventitia) demonstrated a prominent c-myc signal, but failed to express c-fos and c-jun. Corresponding examination of adventitia derived from enzyme-treated aortae showed this tissue to be a source of all three proto-oncogenes. The results of this study demonstrate the earliest in vivo molecular markers of vascular injury reported to date and implicate SMC proto-oncogene expression in the initiation of SMC proliferation. Furthermore these findings suggest two avenues for proto-oncogene induction, that are due to (1) vessel wall manipulation and (2) humoral stimulation.

Animals↗
PubMed1990

Atherogenic concentrations of low-density lipoprotein enhance endothelial cell generation of epoxyeicosatrienoic acid products.

To investigate the effects of protracted low-density lipoprotein (LDL) exposure on endothelial cell (EC) epoxyeicosatrienoic acid (EET) generation, human umbilical vein ECs were incubated in atherogenic concentrations of LDL (240 mg cholesterol per deciliter) (LDL-EC). After 4 days' incubation with LDL, EC were stimulated with human thrombin in the presence of 1-[14C]-arachidonic acid. Substantially more EET products were generated by LDL-ECs than by cells not exposed to high levels of LDL (C-EC). Thrombin stimulation caused LDL-EC to produce five- to eightfold more in 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET, with 14,15-EET as the major product. This is the first demonstration, to date, that EETs can be induced in EC. Metapyrone (SKF-525A) markedly inhibited EC EET generation, indicating a role for the cytochrome P-450 enzyme system in human EC arachidonic acid metabolism. One EET product, 14,15-EET, has been found to be chemotactic and to promote adhesion of U937 cells, a human monocytic lymphoma cell line, to EC. Thus, protracted exposure to atherogenic LDL concentrations increases the generation of chemotactic and adhesion factors (ie, 14,15-EET) after thrombin stimulation, possibly through the cytochrome P-450 enzyme system.

8,11,14-Eicosatrienoic Acid↗
PubMed1989

Agonist-dependent generation of lipoxins from rat basophilic leukemia cell (RBL-1).

Incubation of RBL-1 cells in the presence of 15-HPETE and various agonists generated lipoxins and several isomers. Addition of either A23187, fMLP or PMA modulated the number of isomers and amount of lipoxins produced. Administration of A23187 yielded the largest amount of product (5.3 +/- 1.6 micrograms per 10(8) cells) and generated a total of six and three isomers of LXA4 and B4, respectively. This was 2-fold greater than fMLP, which produced a total of two isomers of LXA4 and LXB4. Addition of PMA generated only LXA4 (0.68 +/- 0.26 micrograms). This is similar to the control receiving only 15-HPETE. Biologically derived LXA4 (3 nM) isolated from RBL-1 incubations contracted a rat tail artery preparation to 12% of the maximum induced by phenylephrine (0.125 microM), whereas LXA4 standard (3 nM) elicited 17.6% of the maximum contraction. These results indicate that RBL-1 cells can utilize exogenous 15-HPETE to generate biologically active lipoxins. Further, the yield and isomers of lipoxins can be modified by different agonists.

Animals↗
PubMed1989

Low-density lipoprotein transport in blood vessel walls of squirrel monkeys.

Transmural accumulations of low-density lipoprotein (LDL) were examined in the blood vessel walls of four squirrel monkeys. Vascular wall concentrations of LDL were measured using quantitative autoradiography after 125I-labeled LDL circulation for 30 min. Profiles of relative tissue concentration from different sections in the same region were similar to each other, and there was little animal-to-animal variation. Concentrations were highest near the luminal endothelium, lower near the medial-adventitial border, and lowest within the media. Profiles from different regions fell into three groups: 1) aortic samples had steep intimal concentration gradients and near-zero media concentrations; 2) the iliac, femoral, popliteal, and common carotid arteries had higher intimal concentrations than group 1 but had similar concentrations deep within the media; and 3) the cerebral and coronary arteries, inferior vena cava, and pulmonary artery had intimal concentrations that were similar to group 2, but the concentrations deep within the media were greater than either groups 1 or 2. Arterial bifurcation profiles from the inner wall and the outer walls were similar to each other and to profiles from the upstream and downstream areas. Out of 280 total sites examined, 15 examples of profiles with substantially increased concentrations near the luminal endothelium were found scattered throughout the cardiovascular system, demonstrating that there are focal regions throughout the cardiovascular system which have greatly increased 125I-LDL transendothelial permeability.

Animals↗
PubMed1988

Low-density lipoprotein preparation by combined diafiltration and ultracentrifugation.

A method for isolating low-density lipoprotein by combining diafiltration and ultracentrifugation is described. Diafiltration separates plasma components by use of an ultrafiltration membrane that excludes particles of molecular weight greater than 300,000. The retentate is concentrated three- to fourfold by ultrafiltration, allowing large-scale preparation of low-density lipoprotein. Low-density lipoprotein prepared in this manner is similar in physical, chemical, and biologic properties to low-density lipoprotein isolated by sequential density ultracentrifugation alone. When low-density lipoprotein, prepared by either method, was added to human umbilical vein endothelial cell cultures, no cytotoxicity was observed. The techniques described reduce the demand on multiple rotors and ultracentrifuges for large-scale preparation of low-density lipoprotein suitable and often needed for tissue culture studies.

Arteriosclerosis↗
PubMed1988

Perturbation of cultured human endothelial cells by atherogenic levels of low density lipoprotein.

Cultured human umbilical vein endothelial cells (EC) exposed to atherogenic levels of low density lipoprotein (LDL) for protracted periods demonstrated no measurable evidence of overt cytotoxicity, but were perturbed as indicated by an increase in prostacyclin (PGI2) production. Confluent EC were incubated with high LDL concentrations (240 or 330 mg/dl cholesterol) for 1 to 12 days. LDL was added to culture media containing 25% human lipoprotein-deficient serum to determine the effects of LDL independent of other lipoproteins. LDL did not injure EC as assessed by cell count, vital dye exclusion, 51chromium release, and lactate dehydrogenase release. Although high concentrations of LDL did not cause EC cytotoxicity, such LDL concentrations did results in increased PGI2 generation. PGI2 accumulation in postincubation media was increased two-to-fivefold in otherwise unstimulated cells as measured by radioimmunoassay of the stable PGI2 breakdown product, 6-keto-PGF1-alpha. This elevation persisted for the entire 12-day exposure to high LDL concentrations. These results indicate that prolonged exposure to atherogenic concentrations of LDL does not effect EC viability, but does cause an endothelial perturbation as demonstrated by an increased PGI2 production.

Arteriosclerosis↗
PubMed1988

Reversible changes in stress fiber expression and cell shape in regenerating rat and rabbit aortic endothelium.

The influence of intimal de-endothelialization on stress fiber expression in regenerating rat and rabbit aortic endothelium was studied using immunofluorescence microscopy. Rat thoracic and abdominal aortae were balloon de-endothelialized, and endothelial cell shape and stress fiber expression was studied in both uninjured and de-endothelialized animals. In control animals, the majority of thoracic endothelial cells did not contain stress fibers while the majority of abdominal endothelial cells did. One week after injury, all the endothelial cells distal to the regenerating edge contained very prominent stress fibers. In areas directly adjacent to the still de-endothelialized surface, the endothelial cells had an intense, diffuse cytoplasmic staining without stress fibers. Regenerating endothelium also had a substantially higher length-to-width ratio, but smaller cell areas. Six weeks after injury, the endothelium had completely regenerated, and stress fibers were lost from the majority of the thoracic endothelial cells. Changes in abdominal aorta stress fiber expression were not as marked. In the rabbit, all the control thoracic endothelial cells had stress fibers; however, cells at the leading edge of a narrow region of de-endothelialization had few stress fibers. The results suggest that stress fibers do not play a primary role in cellular migration in situ. The transient increase in stress fiber expression in the rat may result from a temporary demand for greater adhesive capabilities until the subendothelial extracellular matrix is remodeled.

Animals↗