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Biomedical subjects

W F Ward

Publications and source records attributed to W F Ward.

At least 73 records · Page 4Linked to original sources
PubMed1986

Cell cycle changes and cytotoxicity in irradiated cultures of bovine aortic endothelial cells.

The purpose of this experiment was to determine the effect of ionizing radiation on cell number, lactate dehydrogenase (LDH) release, cell cycle distribution, [3H]thymidine incorporation, and autoradiographic labeling index in bovine aortic endothelial cells in vitro. Confluent endothelial monolayers were exposed to single doses of 0.5-10 Gy of 60Co gamma rays and were analyzed from 2 to 24 h postirradiation. Irradiated monolayers exhibited a time- and dose-dependent decrease in cell number, increase in LDH release, and redistribution of cells in the cell cycle. Cell cycle redistribution included an increase in the proportion of cells in S phase at 4 h after irradiation and a decrease in S phase at 24 h. The cells also exhibited a decrease in [3H]thymidine incorporation as early as 2 h after 5 Gy. This represented the most rapid radiation response observed in the present study. These data demonstrate that radiation cytotoxicity in confluent, plateau-phase endothelial monolayers is accompanied by changes in the cell cycle distribution of adherent cells, and that reduced [3H]thymidine incorporation is an early marker of radiation injury in this clinically important cell type.

Animals↗
PubMed1985

The relationship between endothelial dysfunction and collagen accumulation in irradiated rat lung.

Male rats were killed 2 months (early fibrosis) or 6 months (peak fibrosis) after a range of single doses of 60Co gamma rays to the right hemithorax. Pulmonary arterial perfusion scans were performed at 2 months on animals scheduled for autopsy at 6 months. Lung angiotensin converting enzyme (ACE) activity was used to monitor endothelial function, and hydroxyproline (HP) concentration served as an index of interstitial collagen accumulation (fibrosis). ACE activity also was measured in right lung bronchoalveolar lavage (BAL) fluid and blood serum, to determine whether information obtained from a minimally invasive procedure might serve as an index or predictor of the severity of lung damage. Linear dose-response curves (r = 0.92-0.99) were obtained for right lung arterial perfusion, ACE activity and HP concentration. At 2 months, perfusion decreased 2.7% per Gy, ACE activity (per lung, per mg wet weight, or per mg protein) decreased 3.0-4.2% per Gy, and HP concentration (per g dry weight) increased 1.7% per Gy. At 6 months, the slopes of the response curves were virtually identical to those at 2 months; the Y intercept of the response curve for ACE activity was unchanged, whereas that for HP concentration was 50% higher at 6 than at 2 months. ACE activity and protein concentration in the BAL increased with increasing dose, but the variation within groups was too large, and the sensitivity was too low to serve as a reliable index of lung status. Serum ACE activity was independent of radiation dose at both autopsy times. Thus in rat lung, arterial perfusion, endothelial dysfunction and interstitial fibrosis exhibit similar but not identical radiosensitivities. The dose-effect curves for these three responses of the lung in situ change less than 5% per Gy over the dose range of 10-30 Gy, a smaller variation than would be predicted from endothelial cell survival data based on clonogenic assays in vitro or in vivo.

Animals↗
PubMed1985

Prostacyclin synthesis in irradiated endothelial cells cultured from bovine aorta.

Confluent monolayers of bovine aortic endothelial cells were examined 2-72 h after exposure to 0.5-5.0 Gy of 60Co gamma-rays. Accumulation of prostacyclin [PGI2, measured as 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha)] in the culture media and PGI2 production stimulated by exogenous arachidonate were correlated with cell detachment and release of lactate dehydrogenase (LDH) activity. Platelet adherence to irradiated and control monolayers also was studied. There were simultaneous time- and dose-dependent increases in cell detachment and in the titers of 6-keto-PGF1 alpha and LDH activity in the culture medium. These changes were evident between 4 and 8 h after 5 Gy or at 24 h after 0.5 Gy. Four hours after 5 Gy, both adherent and detached endothelial cells showed a twofold increase in PGI2 production during a 15-min incubation with arachidonate (10 microM). However, by 72 h this increase was less significant. The accumulation of 6-keto-PGF1 alpha appeared to be related to cell destruction, but radiation also stimulated PGI2 synthesis independent of cell detachment. There was an increased platelet interaction with irradiated monolayers, as a result of platelet adherence to subendothelial matrix exposed after cell detachment. However, irradiation did not alter the nonadherent property of the endothelial cell surface toward platelets.

6-Ketoprostaglandin F1 alpha↗
PubMed1985

Effects of lysosomal inhibitors on 125I-insulin and 125I-asialofetuin degradation by the isolated, perfused rat liver and isolated rat hepatocytes.

To further evaluate the role of the lysosomal system in insulin degradation, we have compared the effects of inhibitors of lysosomal function on the degradation of 125I-insulin with 125I-asialofetuin, a lysosomally targeted molecule, by the intact, perfused rat liver and the isolated rat hepatocyte. The inhibitors employed were chloroquine (125 microM), NH4Cl (10 mM), and leupeptin (50 micrograms/ml). In the intact, perfused liver the observed inhibition of 125I-asialofetuin degradation at 30 min was as follows: chloroquine, 38%; NH4Cl, 32%; and leupeptin, 86%. Chloroquine also inhibited 125I-insulin degradation in the intact, perfused liver (29%), but NH4Cl and leupeptin had no effect. Using the isolated hepatocyte, the observed values for inhibition of 125I-asialofetuin at 60 min were: chloroquine, 85%; NH4Cl, 76%; and leupeptin, 81%. Chloroquine produced a 28% inhibition of 125I-insulin degradation, while NH4Cl and leupeptin had no effect. Chloroquine and NH4Cl decreased cell-associated radioactivity when isolated hepatocytes were incubated with 125I-asialofetuin (leupeptin had no effect), whereas chloroquine caused a 107% increase in cell-associated radioactivity when 125I-insulin was added to the incubation media (NH4Cl and leupeptin had no effect). These results indicate that the effects of chloroquine on insulin degradation are an extralysosomal action and that lysosomes appear not to be involved in the physiologic degradation of the insulin molecule.

Ammonium Chloride↗
PubMed1985

Monocrotaline-induced pulmonary fibrosis in rats: amelioration by captopril and penicillamine.

The purpose of this study was to determine whether Captopril (an angiotensin converting enzyme inhibitor) or D-penicillamine (an inhibitor of collagen crosslinking) can ameliorate pulmonary fibrosis induced by the plant alkaloid monocrotaline. Rats were randomly assigned to one of six treatment groups: (1) control; (2) Captopril, 60 mg/kg/day, p.o.; (3) D-penicillamine, 30 mg/kg/day, p.o.; (4) monocrotaline, 2.4 mg/kg/day, p.o.; (5) monocrotaline plus Captopril, as above; (6) monocrotaline plus penicillamine, as above; and were killed after 6 weeks of continuous drug administration. Monocrotaline-treated rats exhibited several anatomic correlates of pulmonary hypertension, including cardiomegaly, right heart enlargement, and muscularization of the pulmonary arteries and arterioles. These monocrotaline reactions were accompanied by decreased lung activities of angiotensin converting enzyme (ACE) and plasminogen activator (PLA), indicative of endothelial dysfunction; and by increased lung hydroxyproline concentration, indicative of interstitial fibrosis. The presence of interstitial fibrosis was confirmed by electron microscopy. When given concomitantly with monocrotaline, both Captopril and penicillamine partially prevented the cardiomegaly, right heart enlargement, and vascular muscularization. Both agents also diminished the decreased lung PLA activity and increased hydroxyproline concentration observed in monocrotaline-treated animals. Neither modifying agent influenced the monocrotaline-induced decrease in lung ACE activity. Compared with control rats, the rats receiving Captopril alone exhibited decreased heart weight and increased serum ACE activity, and animals receiving penicillamine alone did not differ significantly from control animals for any of the endpoints studied. These data demonstrate that Captopril and penicillamine ameliorate monocrotaline-induced pulmonary fibrosis in rats. Penicillamine, known to inhibit radiation-induced lung injury, thus is shown to be effective in a second model of pulmonary fibrosis. Perhaps more importantly, the hydroxyproline data demonstrate that the ACE inhibitor Captropril exhibits antifibrotic activity in monocrotaline-treated rat lung.

Animals↗
PubMed1985

Acute radiation effects on the content and release of plasminogen activator activity in cultured aortic endothelial cells.

Confluent monolayers from three lines of bovine aortic endothelial cells were exposed to a single dose of 10 Gy of 60Co gamma rays. Seventy-two hours later, the morphology of the irradiated and sham-irradiated monolayers was examined, and cellular DNA and protein contents were determined. In addition, the release of plasminogen activator (PA) activity into the culture media and PA activity in the cell lysates were assayed. Irradiated monolayers maintained their cobblestone appearance, but individual endothelial cells were enlarged considerably compared to sham-irradiated cells. DNA and protein contents in the irradiated monolayers were reduced to 43-50% and 72-95% of the control levels, respectively. These data indicate that radiation induced cell loss (detachment and/or lysis) from the monolayer, with hypertrophy of surviving (attached) cells to preserve the continuity of the monolayer surface. Total PA activity (lysate plus medium) in the irradiated dishes was reduced to 50-75% of the control level. However, when endothelial PA activity was expressed on the basis of DNA content, the irradiated monolayers from two of the three cell lines contained significantly more PA activity than did sham-irradiated monolayers. Most importantly, the percentage of the total PA activity released into the culture medium by irradiated cells (5-22%) was significantly (P less than 0.001) lower than that released by sham-irradiated cells (23-68%). These data suggest that fibrinolytic defects observed in irradiated tissues in situ may be attributable at least in part to a radiation-induced inhibition of PA release by vascular endothelial cells.

Animals↗
PubMed1985

Plasminogen activator activity in lung and alveolar macrophages of rats exposed to graded single doses of gamma rays to the right hemithorax.

Male rats were sacrificed 2 or 6 months after a single dose of 0-30 Gy of 60Co gamma rays to the right hemithorax. At autopsy, macrophages were lavaged from the right lung, counted, and frozen. The right (irradiated) and the left (shielded) lungs were frozen, then assayed for plasminogen activator (PLA) activity by the fibrin plate lysis method. Freeze-thawed macrophages were assayed for both PLA activity (125I-fibrin clot lysis method) and fibrinolytic inhibitor activity (inhibition of urokinase-induced fibrin lysis). There was a linear, dose-dependent decrease in right lung PLA activity over the dose range of 10-30 Gy at 2 and 6 months postirradiation, reductions of 3.1 and 2.6% per Gy, respectively. PLA activity at all radiation doses was 10-15% higher at 6 months than at 2 months (P less than 0.05), indicative of a partial recovery of this endothelial function in the irradiated lung. There were no significant changes in PLA activity in the shielded left lung at any dose or time. There also was a linear, dose-dependent increase in the number of macrophages lavaged from the right lung at both 2 and 6 months postirradiation, with larger numbers recovered after all doses at 2 months. PLA activity per 10(6) macrophages decreased with increasing radiation dose at both autopsy times, closely paralleling lung PLA activity. This radiation-induced decrease in macrophage PLA activity was not due to increased fibrinolytic inhibitor activity in the irradiated macrophages. These data quantitate the dose response and time course of radiation-induced fibrinolytic defects in rat lung and suggest that information obtained from a minimally invasive procedure such as bronchoalveolar lavage may serve as an index of the degree of pulmonary fibrinolytic dysfunction after irradiation.

Animals↗
PubMed1984

125I-Insulin internalization by perfused rat liver: comparison of its subcellular distribution with that of a lysosomally targeted molecule, 125I-asialofetuin.

The subcellular distribution of 125I-insulin in the perfused rat liver was compared with the subcellular distribution of the lysosomally targeted asialoglycoprotein, 125I-asialofetuin. The use of Percoll density gradient medium provided excellent separation of lysosomes from the subcellular membrane fractions. Following perfusion with 125I-asialofetuin, a distinct peak of TCA-precipitable radioactivity could be observed in the lysosomal region of the gradient. In contrast, the gradient distribution of TCA-precipitable radioactivity following perfusion with physiological concentrations of 125I-insulin was unimodal, the observed peak corresponding to the distribution of intracellular membrane marker enzymes. Leupeptin, an inhibitor of lysosomal proteolysis, inhibited the degradation of 125I-asialofetuin but had no effect on 125I-insulin degradation. In addition, leupeptin produced a marked increase in TCA-precipitable radioactivity in the lysosome rich region of gradients prepared from livers perfused with 125I-asialofetuin. No such effect was observed following perfusion with 125I-insulin. These findings are consistent with an initial localization of the internalized insulin molecule with the membraneous system of the liver cell rather than the lysosomal system.

Animals↗
PubMed1984

Monocrotaline-induced pulmonary endothelial dysfunction in rats.

To study the role of endothelial damage in the pathogenesis of lung injury induced by the pyrrolizidine alkaloid monocrotaline, three functions (angiotensin converting enzyme (ACE) activity, plasminogen activator (PLA) activity, and prostacyclin (PGI2) production) associated with the pulmonary endothelium were examined, and were correlated with pulmonary arterial perfusion and ultrastructure in rats receiving monocrotaline in their drinking water (20 mg/liter) for 1-12 weeks. Lung ACE activity increased after 1 week of monocrotaline, then decreased steadily from 1 to 6 weeks, before plateauing at approximately 55% of normal. PLA activity in monocrotaline-treated lungs did not change significantly for the first 2 weeks, then decreased to 59 and 79% of the control value after 6 and 12 weeks, respectively. In contrast, PGI2 production increased progressively, reaching 140 and 270% of the control level after 6 and 12 weeks of monocrotaline treatment, respectively. These endothelial functional changes were not accompanied by significant changes in pulmonary arterial perfusion as visualized by 99mTc lung scans. Electron microscopy of monocrotaline-treated lungs revealed endothelial damage (perivascular and subendothelial edema, degeneration) starting at 1 week, and inflammatory and hemorrhagic reactions starting at 2 weeks. At 6 and 12 weeks, monocrotaline-treated rats also exhibited increased pulmonary arterial wall thickness, right heart enlargement, and cardio- and hepatomegaly. Thus, monocrotaline-induced pulmonary injury is accompanied, and in some cases preceded, by structural and functional abnormalities in the pulmonary endothelium.

Animals↗
PubMed1984

Enzymatic responses to radiation in cultured vascular endothelial and smooth muscle cells.

Confluent monolayers of bovine aortic endothelial and smooth muscle cells were exposed to 0-5.0 Gy of 60Co gamma rays. From 0 to 72 hr after irradiation, the monolayer and culture medium were analyzed for cell (nuclei) number, DNA and protein content, the activities of angiotensin converting enzyme (ACE), lactate dehydrogenase (LDH), and superoxide dismutase (SOD), and LDH isoenzyme profile. Irradiated endothelial cells exhibited a time- and dose-dependent increase in cell detachment, decreased DNA and protein content and reduced ACE active per attached cell, increased LDH and SOD activities per microgram of DNA, and increased LDH activity in the culture medium. The latter was accompanied by a shift from LDH 1 to LDH 4 and 5. The release of LDH activity, observed after 0.5 Gy, was the most sensitive endothelial response, and occurred independent of or preceding cell detachment. Vascular smooth muscle cells contained two to three times more SOD activity than did endothelial cells and exhibited no significant responses to 5.0 Gy.

Animals↗
PubMed1984

Radiation injury in rat lung. IV. Modification by D-penicillamine.

To determine whether D-penicillamine, known to reduce fibrosis in irradiated rat lung (W. F. Ward, A. Shih - Hoellwarth , and R. D. Tuttle , Radiology 146, 533-537, 1983), also ameliorates radiation injury in the pulmonary endothelium, we measured angiotensin-converting enzyme (ACE) activity, plasminogen activator (PLA) activity, and prostacyclin (PGI2) production in the lungs of penicillamine-treated (10 mg/day, po, continuous after irradiation) and untreated rats from 2 weeks to 6 months after a single dose of 25 Gy of 60Co gamma rays to the right hemithorax. Both ACE and PLA activity in the irradiated right lung of untreated rats decreased dramatically between the 1st and 2nd months after exposure, then reached a plateau through 6 months at approximately 25 and 50% of the normal level, respectively. For the first 2 months after irradiation, penicillamine-treated animals exhibited significantly (P less than 0.05) higher activities of both ACE and PLA than did untreated rats. From 3 to 6 months after irradiation, however, the only significant drug effect on these enzymes was a 25% increase in PLA activity at 6 months. PGI2 production by the irradiated lung of untreated rats increased continuously, and at 6 months was approximately 10 times higher than normal. Penicillamine significantly (P less than 0.05) reduced this hypersecretion, and at 6 months after irradiation, PGI2 production by the lungs of drug-treated rats was only half that of untreated animals. In contrast, the drug had no significant effect on enzyme activities in the lungs of sham-irradiated rats. Thus the antifibrotic agent D-penicillamine delays the onset of radiation-induced enzyme dysfunction in the pulmonary endothelium. In addition at 6 months after irradiation, the lungs of penicillamine-treated rats exhibit 25% more PLA activity and only half as severe a hypersecretion of PGI2 as do the lungs of untreated animals. The drug is most effective in ameliorating endothelial damage during the first 2 months after irradiation, preceding the development of interstitial fibrosis. However, the effect of this penicillamine regimen on pulmonary endothelial function is not as large as its effect on collagen accumulation in irradiated rat lung.

Animals↗
PubMed1983

Collagen accumulation in irradiated rat lung: modification by D-penicillamine.

The collagen antagonist D-penicillamine (10 mg/day, p.o.) significantly ameliorated radiation-induced hydroxyproline (HP) accumulation in the lungs of rats killed 3, 6, 9, or 12 months after a single exposure of 25 Gy of 60Co gamma rays to the right hemithorax. The beneficial effect of penicillamine was observed when HP values were expressed on the basis of wet weight, dry weight, or per whole lung and was not accompanied by significant changes in the size of the soluble (0.5 M citrate, pH 3.6) collagen fraction. This drug regimen had no effect on HP concentration in the shielded left lung and was apparently free of deleterious side effects.

Animals↗
PubMed1983

Effects of hematocrit on oxygenation of the isolated perfused rat liver.

The isolated perfused rat liver is used ubiquitously for metabolic and endocrine studies of hepatic function, yet few data are available regarding the inadequacy of the oxygenation of such preparations. Moreover, the isolated rat liver is usually deprived of its arterial supply and perfused via the hepatic portal vein with low-hematocrit or cell-free solutions. To investigate the efficacy of the oxygen supply, we determined the effect of hematocrit on the relation between oxygen consumption and perfusate flow. We then attempted to define a hematocrit at which hepatic oxygenation was maximal. Livers of male rats anesthesized with pentobarbital sodium were perfused via the portal vein with fresh canine red blood cells suspended in Krebs-Ringer-bicarbonate buffer. Perfusions were carried out at various flow rates, and the relation between perfusate flow and oxygen uptake was determined. At flow rates above 100 ml X min-1 X 100 g liver-1, oxygen uptake was independent of flow but below that value was flow limited, regardless of whether the hematocrit was 10, 20, or 40%. To determine the optimal hematocrit for hepatic oxygen uptake, hepatic portal venous and hepatic venous pressures were held at 10 and 0 mmHg, respectively. The hematocrit was lowered in steps from 80 to 10%. Blood flow increased exponentially as hematocrit fell while oxygen uptake increased to a maximum at approximately 20%. It is concluded that an hematocrit of approximately 20% provides the optimal combination of blood flow and oxygen-carrying capacity while maintaining physiological perfusion pressures, e.g., 10 mmHg.

Animals↗
PubMed1983

Radiation injury in rat lung. II. Angiotensin-converting enzyme activity.

To determine the role of endothelial dysfunction in the pathogenesis of radiation-induced pulmonary injury, lung angiotensin-converting enzyme (ACE) activity, arterial perfusion, and ultrastructure were examined from 1 to 150 days after a single exposure of 25 Gy of 60Co gamma rays to the right hemithorax of rats. Arterial perfusion to the irradiated right lung increased during the first 2 weeks, then decreased to approximately 80% of the left lung value at 30 days postirradiation. Perfusion of the irradiated lung continued to decline, and by 90-150 days was only 40% of that of the shielded lung. ACE activity in the irradiated right lung did not change significantly until 30 days after exposure, when it decreased to 72% of that in the left lung. ACE activity in the right lung declined steadily from 30 to 90 days postirradiation, then reached a plateau through 150 days at less than 20% of normal. Perivascular and interstitial edema was evident at 1 day after irradiation and persisted for 30 days. Endothelial cells exhibited blebbing, fragmentation, and increased basement membrane at 30 days. Mast cells were present in the septa, but interstitial collagen was not increased at that time. From 90 to 150 days postexposure, progressive obliteration of capillaries by fibrotic reactions was observed. Thus decreased ACE activity accompanies radiation-induced hypoperfusion and endothelial ultrastructural changes in rat lung. All of these reactions precede the development of pulmonary fibrosis.

Animals↗
PubMed1983

Radiation injury in rat lung. I. Prostacyclin (PGI2) production, arterial perfusion, and ultrastructure.

Pulmonary prostacyclin (PGI2) production, arterial perfusion, and ultrastructure were correlated in rats sacrificed from 1 day to 6 months after a single exposure of 25 Gy of gamma rays to the right hemithorax. PGI2 production by the irradiated lung decreased to approximately half the normal value 1 day after irradiation (P less than 0.05), then increased steadily throughout the study. By 6 months postirradiation, the right lung produced two to three times as much PGI2 as did either shielded left lung or sham-irradiated lungs (P less than 0.05). Perfusion scans revealed hyperemia of the right lung from 1 to 14 days after irradiation. From its peak at 14 days postirradiation, however, perfusion of the irradiated lung decreased steadily, then reached a plateau from 3 to 6 months at less than half that in the shielded left lung. Electron micrographs of the right lung revealed perivascular edema from 1 to 30 days after irradiation. The right lung then exhibited changes typical of radiation pneumonitis followed by progressive interstitial fibrosis. Platelet aggregates were not observed at any time. Thus, decreased PGI2 production is an immediate but transient response of the lung to radiation injury. Then from 2 to 6 months after irradiation, the fibrotic, hypoperfused lung produces increasing amounts of the potent vasodilator and antithrombotic agent, PGI2. Pulmonary PGI2 production and arterial perfusion are inversely correlated for at least 6 months after hemithoracic irradiation.

Animals↗
PubMed1983

Radiation injury in rat lung. III. Plasminogen activator and fibrinolytic inhibitor activities.

The mechanism of reduced fibrinolysis in lungs of rats whose right hemithorax had been exposed to a single dose of 25 Gy of 60Co gamma rays was determined, and fibrinolytic changes were correlated with perfusion and morphologic alterations. Reduced fibrinolytic activity in the irradiated lung was evident after 1 month, and decreased further at 2 months. From 2 to 6 months postirradiation, right lung fibrinolytic activity reached a plateau at about half of the activity in the shielded left lung or in sham-irradiated control lungs. The reduced fibrinolytic activity was largely due to decreased plasminogen activator activity, rather than to increased inhibitor activity. Changes in fibrinolytic activity of the irradiated lung closely paralleled changes in arterial perfusion. Mild ultrastructural changes in the irradiated lung (endothelial blebbing and interstitial edema) preceded fibrinolytic and perfusion defects. In contrast, marked changes such as fibrin deposition in the alveolar space and interstitial hypercellularity and fibrosis occurred after pulmonary fibrinolytic activity and perfusion were reduced.

Animals↗