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Biomedical subjects

D C Zawieja

Publications and source records attributed to D C Zawieja.

At least 19 recordsLinked to original sources
PubMed1999

Role of phospholipase C, protein kinase C, and calcium in VEGF-induced venular hyperpermeability.

We previously demonstrated that vascular endothelial growth factor (VEGF)-elicited increase in the permeability of coronary venules was blocked by the nitric oxide (NO) synthase inhibitor NG-monomethyl-L-arginine (L-NMMA). The aim of this study was to delineate in more detail the signaling pathways upstream from NO production in VEGF-induced venular hyperpermeability. The apparent permeability coefficient of albumin (Pa) and endothelial cytosolic Ca2+ concentration ([Ca2+]i) were measured in intact perfused porcine coronary venules using fluorescence microscopy. VEGF (10(-10) M) induced a two- to threefold increase in Pa, which was blocked by a monoclonal antibody directed against the VEGF receptor Flk-1/KDR, the phospholipase C (PLC) antagonist U-73122, or the protein kinase C (PKC) antagonist bisindolylmaleimide (BIM). In 12 venules that displayed the [Ca2+]i response to bradykinin (10(-6) M) and ionomycin (10(-6) M), only 4 vessels responded to VEGF with a transient increase in [Ca2+]i. Furthermore, Western blot analysis of cultured human umbilical vein endothelial cells showed that VEGF increased tyrosine phosphorylation of PLC-gamma and serine phosphorylation of endothelial constitutive NO synthase (ecNOS). The hyperphosphorylation of PLC-gamma was greatly attenuated by the KDR receptor antibody and U-73122, but not by BIM or L-NMMA. In contrast, U-73122 and BIM were able to inhibit VEGF-elicited serine phosphorylation of ecNOS. The results suggest that VEGF induces venular hyperpermeability through a KDR receptor-mediated activation of PLC. In turn, ecNOS is activated by PLC-mediated PKC and/or cytosolic Ca2+ elevation stimulation.

Animals↗
PubMed1996

Lymphatic microcirculation.

OBJECTIVE: The importance of the lymphatic system during inflammatory response is underscored by the discovery that numerous inflammatory mediators alter the lymph pump. Inhibition of the lymph pump will reduce the lymphatic outflow from the interstitial space and thus initiate the lymphatic generation of edema. We evaluated the effect that reactive oxygen metabolites have on the contractile activity of lymphatic vessels. METHODS: Reactive oxygen metabolites are produced during inflammation and have been implicated in a number of pathologies. Exposure to reactive oxygen metabolites inhibited the lymph pump flow in a time- and concentration-dependent fashion by decreasing contraction frequency, strength, and propagation. RESULTS: Substance P (SP) (1.0 microM) produced the following changes (% control): reductions in diastolic diameter (56%), systolic diameter (47%), and stroke volume of the lymph pump (62%); a large increase in lymphatic contraction frequency (640%); and a modest increase in lymph pump flow (43%). SP also stimulated quiescent vessels to develop typical contraction-relaxation patterns. These effects may serve to minimize the formation of edema in the face of inflammatory edemagenic conditions that are produced by SP. CONCLUSIONS: We concluded that reactive oxygen metabolites significantly inhibit the active lymph pump and that this inhibition could be an important contributing factor in the formation of interstitial edema during inflammation We have also determined the effects of a putative mediator of inflammation (SP) on the lymphatic pumping function.

Animals↗
PubMed1995

Interaction of neutrophils and endothelium in isolated coronary venules and arterioles.

This study reports measurements of porcine neutrophil dynamics in isolated microvessels. Porcine coronary venules and arterioles were isolated, cannulated, and perfused with fluorescently labeled neutrophils at a series of flow velocities. In venules (62.50 +/- 5.41 microns diam) under control conditions, rolling neutrophils were often observed at intraluminal flow velocities ranging from 600 to 6,000 microns/s, and the rolling fraction varied inversely as a function of flow velocity. There was no significant adherence under the control conditions at any of the various flow velocities. Pretreatment of the neutrophils with human recombinant complement 5a (C5a, 10(-8) M) increased adherence at low flow velocities but did not alter the rolling fraction. In contrast to venules, rolling neutrophils were not observed in arterioles (58.80 +/- 5.6 microns diam). Furthermore, neutrophils that were pretreated with C5a did not adhere to the arteriolar endothelium even at low flow velocities. We suggest that 1) isolated microvessels perfused with fluorescently labeled neutrophils are suitable models for the study of the interaction between neutrophils and the microvascular endothelium, 2) shear force plays an important role in neutrophil rolling in coronary venules but is not the major factor that prevents neutrophil rolling and adherence in arterioles, and 3) C5a causes neutrophil adherence in venules but not in arterioles, indicating that different mechanisms underlie the interaction between neutrophils and endothelium in venules and arterioles.

Animals↗
PubMed1993

Distribution, propagation, and coordination of contractile activity in lymphatics.

The propagation and coordination of lymphatic contractions were studied in the mesentery of the rat small intestine using in situ microscopic observation. Indexes of lymphatic diameter were simultaneously measured at two adjacent lymphangions in spontaneously contracting lymphatics (n = 51). Diameter index, contraction frequency, and the percentage of the intersegmental contractions that were propagated and coordinated (PP) were determined at both sites. The conduction velocity of the contractile activity and the percentage of the coordinated contractions that were propagated both antegrade to the direction of lymph flow and retrograde to the flow stream were determined. The results indicate that 1) 80-90% of the lymphatic contractions in the vessels we evaluated were propagated, 2) the wave of contractile activity propagated both centrally and peripherally, and 3) the conduction velocity of the contractile activity was approximately 4-8 mm/s. We tested the hypothesis that gap junctional communication is responsible for the coordination of the contractile event. To accomplish this, we used the gap junction blockers n-heptanol and oleic acid. PP was 90 +/- 4% under normal conditions and fell to a minimum value of 55 +/- 7% during the gap junction blockade. These results indicate that gap junctional communication played an important role in the propagation and coordination of contractions that occurred in spontaneously active lymphatics.

Alcohols↗
PubMed1993

Histamine increases venular permeability via a phospholipase C-NO synthase-guanylate cyclase cascade.

In this study, we hypothesized that histaminergic increases in venular permeability result from a cascade triggered by activation of phospholipase C (PLC), inducing the synthesis of nitric oxide (NO) and activating guanylate cyclase. The apparent permeability coefficient to albumin (Pa) was measured in isolated porcine coronary venules subjected to constant flow and hydrostatic and oncotic pressures. Histamine (2.5, 5, and 10 microM) transiently and progressively increased Pa. The PLC inhibitor 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate (NCDC; 100 microM) decreased baseline permeability and abolished the effect of histamine. The NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA; 10 microM) and the guanylate cyclase inhibitor 6-anilinoquinoline-5,8-quinone (LY 83583; 10 microM) also blocked the histamine-induced hyperpermeability. L-Arginine (3 mM) reversed the inhibition by L-NMMA. NG-monomethyl-D-arginine did not influence the effect of histamine. Furthermore, sodium nitroprusside (10 microM) augmented Pa by two- to threefold; this effect was blocked in the presence of LY 83583 but not altered in the presence of NCDC. The results suggest that histamine increases coronary venular permeability by a direct action on the venular endothelial cells through a PLC-NO synthase-guanylate cyclase-signaling cascade.

Amino Acid Oxidoreductases↗
PubMed1993

Permeability to albumin in isolated coronary venules.

This study reports measurements of albumin permeability in isolated coronary venules. The isolated microvessel technique allows the quantification of transmural exchange of macromolecules under tightly controlled physical and chemical conditions. Transvenular exchange of albumin was studied in isolated coronary venules during alterations in filtration rate caused by changes in intravascular pressure. The apparent permeability coefficient of albumin (Pa) at an intraluminal pressure of 11 cmH2O was 3.92 +/- 0.43 x 10(-6) cm/s. Elevating intraluminal pressure to 16 and 21 cmH2O increased Pa to 5.13 +/- 0.57 x 10(-6) and 6.78 +/- 0.66 x 10(-6) cm/s, respectively. Calculation of the true diffusive permeability coefficient of albumin (Pd) at zero filtration rate was 1.54 x 10(-6) cm/s. The product of hydraulic conductance (Lp) and (1 - sigma), where sigma is the solute reflection coefficient, was 3.25 x 10(-7) cm.s-1 x cmH2O-1. At a net filtration pressure of 4-5 cmH2O, diffusion accounts for > 60% of total albumin transport across the venular wall. Transmural albumin flux is very sensitive to filtration rate, rising 6.7% for each cmH2O elevation of net filtration pressure. At 11 cmH2O net filtration pressure, convection accounts for nearly 70% of net albumin extravasation from the venular lumen. We suggest that the isolated coronary venule is a suitable preparation for the study of solute exchange in the heart.

Animals↗
PubMed1993

Inhibition of the active lymph pump in rat mesenteric lymphatics by hydrogen peroxide.

The lymphatic system plays an important role in the regulation of fluid and macromolecular exchange. It is a key "safety factor" against the formation of gross edema. Spontaneous contractions in collecting lymphatics of the rat intestine are necessary for the normal transportation of lymph. Hydrogen peroxide is one of the compounds released in inflammation. Therefore, the effects of H2O2 on the pumping activity of spontaneously contracting lymphatics were evaluated in the anesthetized rat (n = 16). Diameter oscillations of the mesenteric collecting lymphatics were monitored before and after the application of H2O2 (4 and 37 microM). The activity of the lymph pump was evaluated using: contraction frequency (F), stroke volume (SV), ejection fraction (EF), and lymph pump flow (LPF). These parameters were determined from the lymphatic diameter tracings. The following changes in lymphatic activity were seen after a 20 minute exposure to 37 microM H2O2: 1) F declined 85%, from 11.6 +/- 1.5 to 1.9 +/- 1.9 cpm. 2) SV fell over 93%. 3) EF decreased 93%, from 0.57 +/- .07 to 0.05 +/- .04. 4) LPF fell dramatically (> 95%) from 41.5 +/- 10.5 to 2.6 +/- 2.5 nl/min. In conclusion, H2O2 produced an intense inhibition of the active lymph pump and it is possible that the inhibition of the active pump contributes to the edema which occurs during inflammation.

Animals↗
PubMed1992

Effects of f-Met-Leu-Phe-induced inflammation on intestinal lymph flow and lymphatic pump behavior.

Previous studies in the literature indicate that intraenteric placement of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe) evokes an intestinal inflammatory response characterized by an accumulation of interstitial fluid and increased lymph flow. Furthermore, it is known that movement of lymph away from the intestine is dependent on the rhythmic pumping of lymph by collecting lymphatics in the mesentery. The purpose of the present study was to determine whether the f-Met-Leu-Phe-induced increase in lymph formation is countered by an increase in lymphatic pump efficiency. Male Sprague-Dawley rats were anesthetized, and a segment of ileum with adjacent mesentery was exteriorized. The mesentery was positioned over an optical window, and a 100-microns collecting lymphatic was selected for study. The preparation was transferred to a video microscope, and the activity of the lymphatic pump was monitored under control conditions and during intraluminal infusion of 1 microM f-Met-Leu-Phe. Lymph propulsion by the lymphatic pump was calculated from the product of stroke volume and contraction frequency. In one group of animals, total lymph flow was determined by cannulating the lymphatic draining the ileal segment. Total lymph flow increased following f-Met-Leu-Phe placement in the intestine. The increased lymph flow was paralleled by a rise in lymphatic pumping. The rise in lymph propulsion by the lymphatic pump resulted exclusively from an increased stroke volume, inasmuch as contraction frequency did not change. The results of the present study suggest that activation of the lymphatic pump during acute inflammation may be important in preventing interstitial edema.

Animals↗
PubMed1992

Oxygen radicals, enzymes, and fluid transport through pericardial interstitium.

The interstitium is the final link in the transportation of nutrients from the bloodstream to the individual cells of an organism. To assess interstitial fluid transport in normal and inflamed tissue, the hydration (H, ml H2O/g dry wt) and hydraulic conductivity (Kp, 10(-8) cm2.s-1.cmH2O-1) of bovine pericardial stroma were determined. The effect of enzymes and neutrophil-derived products of inflammation on the properties of the interstitial model were determined. Samples of the pericardium were exposed separately to trypsin, elastase, hyaluronidase, collagenase, superoxide radicals, and hydrogen peroxide. After exposure, the tissues were washed repeatedly in physiological saline and equilibrated in transport chambers heated to 37 degrees C and pressurized to 50 cmH2O. Fluid flow across the tissues was monitored. A section of tissue was removed and weighed. The tissue section was subsequently dried and reweighed. Tissue thickness, H, and Kp were calculated. H and Kp of the control tissues were 2.82 +/- 0.04 and 1.71 +/- 0.07, respectively. Hydration was significantly increased (22-38%) by exposure to trypsin, elastase, collagenase, and superoxide radicals. Kp increased significantly (30-1055%) in the groups treated with trypsin, hyaluronidase, collagenase, and superoxide radicals. The inflammatory mediators generally increased the hydration and/or the hydraulic conductivity of the model. These results indicate that neutrophil-derived products could be involved in the development of interstitial edema during the inflammatory process.

Animals↗
PubMed1992

Flow modulates coronary venular permeability by a nitric oxide-related mechanism.

This study demonstrates that flow velocity modulates coronary venular permeability to albumin. Apparent permeability coefficients of albumin (Pa) were measured in isolated cannulated coronary venules ranging from 30 to 70 microns in diameter. Hydrostatic and oncotic pressures were controlled while the intraluminal flow velocity was varied. Pa at an intraluminal hydrostatic pressure of 12 cmH2O and a flow velocity of 7 mm/s was 4.01 +/- 0.53 x 10(-6) cm/s. Increasing flow velocity to 10 and 13 mm/s augmented the permeability by 33 +/- 14 and 48 +/- 14%, respectively. The nitric oxide synthase inhibitor, NG-monomethyl-L-arginine (L-NMMA) (10(-5) M), decreased baseline Pa and abolished the flow-induced permeability changes. Administration of L-arginine (3 x 10(-3) M), a physiological precursor of nitric oxide which reverses the effect of L-NMMA, restored the relationship between flow and permeability. From these results we conclude that 1) flow velocity should be considered as a physical force that potentially modulates permeability of venular exchange vessels in the heart and 2) flow modulates coronary venular permeability via the production of nitric oxide.

Animals↗
PubMed1992

Stretch-induced increases in intracellular calcium of isolated vascular smooth muscle cells.

Vascular smooth muscle responds to stretch with an increase in active force development. To investigate the role of Ca2+ in this response, we used the fluorescent dye fura-2 to quantitate changes in cytosolic Ca2+ in single, vascular smooth muscle cells during rapid stretch. Cells were enzymatically dispersed from pig coronary arteries, loaded with fura-2/AM, and studied using a digital-imaging microscope. Stretch of individual cells was accomplished by attachment with suction to two patch-type micropipettes to apply force to the ends of the cell. Stretch induced the release of Ca2+ from intracellular stores as well Ca2+ influx across the plasma membrane. In physiological saline solution containing 1.5 mM Ca2+, intracellular calcium increased with cell stretch in a sigmoidal fashion. This relationship was shifted upward in 10 mM Ca2+ bath solution and abolished after several minutes in Ca(2+)-free solution. The dihydropyridine Ca2+ channel blocker nifedipine, in doses sufficient to completely block inward Ca2+ current, produced only a partial block of the sustained stretch-induced intracellular Ca2+ response. It is concluded that in isolated pig coronary arterial smooth muscle cells, stretch-induced Ca2+ influx occurs in part via a nifedipine-resistant pathway, which may be a stretch-activated cation channel.

Animals↗
PubMed1991

Reactive oxygen metabolites inhibit spontaneous lymphatic contractions.

The effects of oxygen-derived free radicals on the contractile activity of the mesenteric collecting lymphatics were evaluated in the anesthetized rat. Lymphatic contractions were monitored before, during and after the application of oxyradicals. Contraction frequency (F), stroke volume (SV), ejection fraction (EF), contraction propagation (PC), and lymph pump flow (LPF) were determined from the lymphatic diameter tracings. Oxyradicals were generated using hypoxanthine and xanthine oxidase. Exposure to oxyradicals inhibited the lymphatic pumping mechanism: 1) F fell from 15.5 +/- 0.8 to 0.8 +/- 0.7 beats/min; 2) EF went from 0.44 +/- 0.02 to 0.08 +/- 0.04; 3) PC dropped from 92 +/- 2 to 56 +/- 8%; and 4) LPF fell precipitously from 41.0 +/- 5.2 to 0.7 +/- 0.4 nl/min. The effects of the oxyradicals were attenuated by superoxide dismutase, implicating superoxide anion as one of the predominant causative agents. We conclude that oxyradicals significantly inhibit the lymph pump and that this inhibition could be a factor contributing to the formation of interstitial edema during inflammation.

Animals↗
PubMed1991

Calcium measurement in isolated arterioles during myogenic and agonist stimulation.

Vascular smooth muscle calcium was measured during agonist treatment or pressure-induced stimulation of the myogenic response in isolated first-order skeletal muscle arterioles. Arterioles (40-180 microns) with spontaneous tone were isolated from rat cremaster muscle and cannulated. Arterioles were loaded with the calcium-sensitive dye fura-2 and excited at 340 and 380 nm. Images of vessel fluorescence were formed with a fluorescence microscope and digitized using an image processor coupled to a low light level camera. The fluorescent images allowed individual vascular smooth muscle cells to be seen within the arteriolar wall. Fluorescent intensity of the vessel wall, expressed as the ratio of fluorescence at 340 nm/380 nm, was used to estimate changes in vessel wall calcium. Topical application of norepinephrine (10 microM) to the arterioles caused a rapid and sustained constriction of the arterioles (64% of basal diam). The calcium response was biphasic consisting of a transient spike to 271% of basal followed by a decrease to a new steady state at 143% of basal. In comparison, steady-state indolactam (1 microM) produced a similar degree of constriction without an increase in calcium. Adenosine significantly dilated (35%) the arterioles and produced a decrease (24%) in vessel wall calcium. To investigate the myogenic response, intravascular pressure was step increased from 90 to 130 cmH2O. Increasing intravascular pressure caused an initial increase in vessel diameter of approximately 5% followed by active constriction that returned diameter to basal diameter. In association with this diameter change, estimated vessel wall calcium increased rapidly 8 +/- 2% and then continued to increase more slowly and remained elevated at 10-15% above basal levels. This study demonstrates the successful application of calcium-imaging technology in isolated arterioles for study of the role of calcium in arteriolar function. Results indicate that the calcium-contraction relationship differs for different agonists and are further consistent with a role for pressure-induced increases in vascular smooth muscle calcium during the myogenic response.

Adenosine↗
PubMed1990

Thermal effects of MR imaging: worst-case studies on sheep.

The objective of this study was to provide a worst-case estimate of thermal effects of MR imaging by subjecting anesthetized unshorn sheep to power deposition at specific absorption rates (SARs) well above approved standards for periods of time in excess of normal clinical imaging protocols. A control period with no RF power was followed by 20-105 min of RF power application. Afterward, there was a 20-min or longer recovery period with no RF power. Eight sheep were given whole-body RF exposure (1.5- to 4-W/kg SAR) while rectal and skin temperatures were monitored. Four sheep were subjected to 4-W/kg head scans for an average of 75 min while temperatures of the cornea, vitreous humor, head skin, jugular vein, and rectum were measured. In head scanning experiments, skin and eye temperatures increased about 1.5 degrees C. Jugular vein temperature rose a maximum of 0.4 degrees C after an average exposure of 75 min. In whole-body exposures, elevation of rectal temperature was correlated with energy input. Deep-body temperature rises in excess of 2.0 degrees C were attained for 4-W/kg whole-body exposure periods greater than 82 min. Animals exposed for 40 min to 4 W/kg in either body coil (three sheep) or head coil (two sheep) were recovered and observed to be in good health for 10 weeks; no cataracts were found. MR power deposition at SAR levels well above typical clinical imaging protocols caused body temperature to increase. For exposure periods in excess of standard clinical imaging protocols the temperature increase was insufficient to cause adverse thermal effects. Studies in healthy humans are needed to determine whether enhanced heat-loss effector mechanisms are likely to cause deep-body temperatures to plateau at an acceptable level, and to elucidate mechanisms that determine subcutaneous temperature.

Animals↗
PubMed1989

Characterization of intact mesenteric lymphatic pump and its responsiveness to acute edemagenic stress.

The contractile properties of the mesenteric collecting lymphatics of the rat were analyzed under control conditions and during periods of enhanced lymph formation using in vivo microscopic techniques. Pressure and diameter were simultaneously monitored in microscopic collecting lymphatics, and lymphatic pump function was analyzed in accordance with basic principles of cardiac mechanics. The lymphatic contractile cycle was divided into two phases of systole and four phases of diastole. Under control conditions, lymphatics contracted with a frequency of 6.4 +/- 0.61 beats/min and ejected approximately 67% of their end-diastolic volume. Ten minutes after the rate of lymph formation was elevated by plasma dilution, end-diastolic diameter, contraction frequency, ejection fraction, and stroke volume increased. Pressure in the lymphatic network became less pulsatile in high lymph flow states. Contractility, an index of inotropic changes in lymphatic pump, was unaltered when lymph flow was increased by plasma dilution. Furthermore, the maximal shortening velocity of lymphatic smooth muscle did not change during periods of enhanced lymph flow. Thus it appears that passive increases in the rate of lymph formation exert few, if any, inotropic effects on the lymphatic pump. The augmented stroke volume and contraction frequency appear to result mainly from intrinsic stretch-dependent mechanisms set in motion by elevated preload. These data represent the first comprehensive characterization of both the flow-generating and muscle characteristics of intact collecting lymphatics and provide a basis for future studies on the physiological regulation of lymphatic contraction.

Animals↗
PubMed1987

Lymph protein concentration in initial and collecting lymphatics of the rat.

Lymph samples were obtained from villus initial and mesenteric prenodal collecting lymphatics of the anesthetized rat using standard micropuncture techniques. The protein concentrations of the lymph samples were determined using a fluorometric microassay. These procedures were performed on preparations either superfused with a modified Krebs solution or covered with paraffin oil. The protein-concentrating ability of the intestinal lymphatics was evaluated to test the osmotic theory of lymph formation. The mean initial lymph protein concentration in superfused preparations was 2.08 g/dl (SE = 0.20). The lymph protein concentration in collecting vessels in superfused preparations was 2.20 g/dl (SE = 0.19). The protein concentration from initial lymphatics in oil-covered preparation was 2.48 g/dl (SE = 0.17). The lymph protein concentration in collecting vessels in oil-covered preparations was 2.32 g/dl (SE = 0.15). The difference between initial and collecting lymph protein concentration found was an order of magnitude lower than that predicted by the osmotic theory. These results cast serious doubts on the effectiveness of the osmotic mechanism of lymph formation as it is presently defined.

Animals↗
PubMed1987

Variations in rat mesenteric tissue thickness due to microvasculature.

Studies of microvascular, tissue and lymphatic transport using microphotometric and microfluorometric techniques are potentially subject to artifact due to variations in tissue specimen thickness. Absorbance techniques utilize the Lambert-Beer law in which A = log I0/I = act, where A is absorbance, I0 is incident light intensity, I is transmitted intensity, a is an absorbance coefficient, c is concentration of substance, and t is path length. If differences in t are known to be present, then inferences of changes in c from changes in A become suspect. In microfluorometry the amount of light gathered is proportional to the number of fluorochromes in the effective cuvette, which is determined by the microscope's numerical aperture and the sample thickness. If variations in thickness are known to occur, the effective cuvette volume may be changing; therefore, inferences of changes in fluorochrome concentration from changes in intensity become suspect. Existing data suggest that rat mesentery is 15-30 microns thick, but variation over a tissue region is unknown. Our goals are to determine thickness variation in avascular, fat-free mesenteric tissue regions; thickness variation near blood vessels; and average tissue thickness. Sprague-Dawley rats were anesthetized with Inactin. Mesenteric tissue from a loop of small intestine was draped over a platform for observation; thickness was measured with an oblique microscope and a microgravimetric technique. The average variation in avascular fat-free tissue was 1.1 micron/100-microns distance, and average thickness was 17.4 microns. There was a significant increase in thickness over the microvasculature.

Animals↗