Regulation of endothelial CD73 by adenosine: paracrine pathway for enhanced endothelial barrier function.

During episodes of inflammation, multiple cell types release adenine nucleotides in the form of ATP, ADP, 5'-AMP, and adenosine. In particular, following activation, polymorphonuclear leukocytes release larger quantities of 5'-AMP. Extracellular 5'-AMP is metabolized to adenosine by surface-expressed 5'-ectonucleotidase (CD73). Adenosine liberated by this process activates surface adenosine A(2B) receptors, results in endothelial junctional reorganization, and promotes barrier function. We hypothesized that adenosine signaling to endothelia provides a paracrine loop for regulated expression of CD73 and enhanced endothelial barrier function. Using an in vitro microvascular endothelial model, we investigated the influence of 5'-AMP; adenosine; and adenosine analogues on CD73 transcription, surface expression, and function. Initial experiments revealed that adenosine and adenosine analogues induce CD73 mRNA (RT-PCR), surface expression (immunoprecipitation of surface biotinylated CD73), and function (HPLC analysis of etheno-AMP conversion to ethenoadenosine) in a time- and concentration-dependent fashion. Subsequent studies revealed that similar exposure conditions increase surface protein through transcriptional induction of CD73. Analysis of DNA-binding activity by EMSA identified a functional role for CD73 cAMP response element and, moreover, indicated that multiple cAMP agonists induce transcriptional activation of functional CD73. Induced CD73 functioned to enhance 5'-AMP-mediated promotion of endothelial barrier (measured as a paracellular flux of 70-kDa FITC-labeled tracer). These results provide an example of transcriptional induction of enzyme (CD73) by enzymatic product (adenosine) and define a paracrine pathway for the regulated expression of vascular endothelial CD73 and barrier function.

Intraoperative detection of pulmonary thromboemboli with epicardial echocardiography.

We report a novel intraoperative use of epicardial echocardiography in detecting and guiding the removal of pulmonary arterial thromboemboli. We describe a patient with a right atrial thrombus that could not be visualized with intraoperative transesophageal echocardiography. Because we suspected acute pulmonary embolization, epicardial echocardiography was used to visualize the right and left pulmonary arteries. Pulmonary thromboemboli were identified, and pulmonary thromboembolectomy was successfully performed.

Neutrophil-derived 5'-adenosine monophosphate promotes endothelial barrier function via CD73-mediated conversion to adenosine and endothelial A2B receptor activation.

During episodes of inflammation, polymorphonuclear leukocyte (PMN) transendothelial migration has the potential to disturb vascular barrier function and give rise to intravascular fluid extravasation and edema. However, little is known regarding innate mechanisms that dampen fluid loss during PMN-endothelial interactions. Using an in vitro endothelial paracellular permeability model, we observed a PMN-mediated decrease in endothelial paracellular permeability. A similar decrease was elicited by cell-free supernatants from activated PMN (FMLP 10(-6) M), suggesting the presence of a PMN-derived soluble mediator(s). Biophysical and biochemical analysis of PMN supernatants revealed a role for PMN-derived 5'-adenosine monophosphate (AMP) and its metabolite, adenosine, in modulation of endothelial paracellular permeability. Supernatants from activated PMN contained micromolar concentrations of bioactive 5'-AMP and adenosine. Furthermore, exposure of endothelial monolayers to authentic 5'-AMP and adenosine increased endothelial barrier function more than twofold in both human umbilical vein endothelial cells and human microvascular endothelial cells. 5'-AMP bioactivity required endothelial CD73-mediated conversion of 5'-AMP to adenosine via its 5'-ectonucleotidase activity. Decreased endothelial paracellular permeability occurred through adenosine A2B receptor activation and was accompanied by a parallel increase in intracellular cAMP. We conclude that activated PMN release soluble mediators, such as 5'-AMP and adenosine, that promote endothelial barrier function. During inflammation, this pathway may limit potentially deleterious increases in endothelial paracellular permeability and could serve as a basic mechanism of endothelial resealing during PMN transendothelial migration.

Activated endothelial cells elicit paracrine induction of epithelial chloride secretion. 6-Keto-PGF1alpha is an epithelial secretagogue.

Endothelial cells play a central role in the coordination of the inflammatory response. In mucosal tissue, such as the lung and intestine, endothelia are anatomically positioned in close proximity to epithelia, providing the potential for cell-cell crosstalk. Thus, in this study endothelial-epithelial biochemical crosstalk pathways were studied using a human intestinal crypt cell line (T84) grown in noncontact coculture with human umbilical vein endothelia. Exposure of such cocultures to endothelial-specific agonists (LPS) resulted in activation of epithelial electrogenic Cl- secretion and vectorial fluid transport. Subsequent experiments revealed that in response to diverse stimuli (LPS, IL-1alpha, TNF-alpha, hypoxia), endothelia produce and secrete a small, stable epithelial secretagogue into conditioned media supernatants. Further experiments identified this secretagogue as 6-keto-PGF1alpha, a stable hydrolysis product of prostacyclin (PGI2). Results obtained with synthetic prostanoids indicated that 6-keto-PGF1alpha (EC50 = 80 nM) and PGI2 stable analogues (EC50 = 280 nM) activate the same basolaterally polarized, Ca2+-coupled epithelial receptor. In summary, these findings reveal a previously unappreciated 6-keto-PGF1alpha receptor on intestinal epithelia, the ligation of which results in activation of electrogenic Cl- secretion. In addition, these data reveal a novel action for the prostacyclin hydrolysis product 6-keto-PGF1alpha and provide a potential endothelial- epithelial crosstalk pathway in mucosal tissue.

Pulmonary vascular effects of isoflurane anesthesia after left lung autotransplantation in chronically instrumented dogs.

BACKGROUND: Single lung transplantation has become a viable therapy for treatment of end-stage pulmonary disease. We previously observed that left lung autotransplantation (LLA) results in a chronic increase in pulmonary vascular resistance and enhanced pulmonary vascular reactivity to sympathetic alpha adrenoreceptor activation. The effects of inhalational anesthetics on the pulmonary circulation after lung transplantation have not been investigated. In the current study, the authors tested the hypothesis that isoflurane anesthesia, known to cause systemic vasodilation, would exert a vasodilator influence on the baseline pulmonary circulation after LLA. In addition, they tested the hypothesis that isoflurane anesthesia, known to attenuate the systemic vasoconstrictor response to sympathetic alpha adrenoreceptor agonists, would reduce the magnitude of the pulmonary vasoconstrictor response to sympathetic alpha adrenoreceptor activation after LLA. METHODS: Left pulmonary vascular pressure-flow (LPQ) plots were generated in chronically instrumented dogs by measuring the pulmonary vascular pressure gradient (pulmonary arterial pressure-left atrial pressure) and left pulmonary blood flow during inflation of a hydraulic occluder implanted around the right main pulmonary artery. Left pulmonary vascular pressure-flow plots were generated in 8 dogs 2-5 weeks after LLA in the conscious and isoflurane-anesthetized states at baseline, after beta adrenoreceptor block with propranolol, and during the cumulative administration of the alpha agonist, phenylephrine. Left pulmonary vascular pressure-flow plots also were generated in eight conscious, sham-operated control dogs at baseline, after beta block, and during phenylephrine administration. RESULTS: Compared with conscious control dogs, LLA resulted in a leftward shift (P < 0.01) in the baseline left pulmonary vascular pressure-flow relation, indicating chronic pulmonary vasoconstriction. Despite the enhanced level of pulmonary vasomotor tone after LLA, isoflurane did not exert a vasodilator influence on the baseline left pulmonary vascular pressure-flow relation. The pulmonary vasoconstrictor response to phenylephrine was enhanced (P < 0.01) after LLA compared with the response measured in conscious control dogs. The magnitude of the pulmonary vasoconstrictor response to phenylephrine after LLA was not attenuated during isoflurane anesthesia. CONCLUSIONS: Isoflurane anesthesia does not exert a vasodilator influence on the pulmonary circulation in the setting of increased pulmonary vascular resistance after LLA. In addition, in contrast to previous studies of the systemic circulation, isoflurane does not attenuate the enhanced pulmonary vasoconstrictor response to sympathetic alpha adrenoreceptor activation after LLA.

Complement-induced endothelial dysfunction in rabbits: mechanisms, recovery, and gender differences.

Activation of complement and attenuation of endothelium-dependent relaxation occur in a number of pathophysiological conditions. The aim of this study was to investigate the mechanisms of human complement activation and loss of endothelium-dependent relaxation in rabbit tissue, the duration of this loss, and the effects of gender and serum concentration. In rabbit thoracic aortic rings precontracted with phenylephrine, human serum (HS) concentration dependently induced a loss of endothelium-dependent relaxation to the receptor-dependent vasodilator acetylcholine (ACh) and receptor-independent vasodilator calcium ionophore A23187. Serum-induced loss of ACh-dependent relaxation was decreased when rings were bathed in 1) HS depleted of factor B, C2, or C8, 2) heat-inactivated HS, or 3) HS with complement inhibitor sCR1 or sCR1[desLHR-A]. Superoxide dismutase had no effect on serum-induced loss of ACh-dependent relaxation. Serum-induced loss of ACh-dependent relaxation returned to control values after removal of HS. Serum-induced loss of ACh-dependent relaxation was greater in male than in female aortic rings. These results suggest that 1) complement activation directly attenuates endothelium-dependent relaxation via the classical and alternative pathways independent of superoxide anion formation, 2) this attenuation is concentration dependent, reversible, and dependent on formation of C5b-9, and 3) endothelial tissue from males is more susceptible than that from females to the acute effects of complement activation.

Attenuated hypoxic pulmonary vasoconstriction during isoflurane anesthesia is abolished by cyclooxygenase inhibition in chronically instrumented dogs.

BACKGROUND: Hypoxic pulmonary vasoconstriction (HPV) is a homeostatic mechanism whereby gas exchange is improved through the diversion of blood flow away from poorly oxygenated regions of the lung. The effect of isoflurane anesthesia on HPV is unclear. Using a chronically instrumented canine model, it was hypothesized that isoflurane anesthesia would attenuate HPV compared to the response measured in the same animal in the conscious state. Moreover, because volatile anesthetics increase the production of cyclooxygenase metabolites, it was hypothesized that attenuation of HPV during isoflurane anesthesia would be abolished by cyclooxygenase inhibition. METHODS: Left pulmonary vascular pressure-flow plots were generated in chronically instrumented dose by measuring the pulmonary vascular pressure gradient (pulmonary arterial pressure-left atrial pressure) and left pulmonary blood flow during inflation of a hydraulic occluder implanted around the right main pulmonary artery. In protocol 1 (n - 7), left pulmonary vascular pressure-flow plots were generated during normoxia and hypoxia (systemic arterial PO2 approximately 50 mmHg) in the conscious and isoflurane-anesthetized states. In protocol 2 (n = 7), left pulmonary vascular pressure-flow plots were generated during normoxia and hypoxia (1) in the conscious state, (2) in the conscious state after inhibition of the cyclooxygenase pathway with indomethacin, and (3) during isoflurane anesthesia after cyclooxygenase inhibition. RESULTS: In both the conscious and isoflurane-anesthetized states, the magnitude of HPV was dependent on the level of left pulmonary blood flow. Compared to the response measured in the conscious state, the magnitude of HPV was attenuated during isoflurane anesthesia over the empirically measured range of left pulmonary blood flow. Cyclooxygenase inhibition abolished the isoflurane-induced attenuation of HPV. CONCLUSIONS: This is the first study to demonstrate that isoflurane anesthesia attenuates the magnitude of HPV compared to the response measured in the same animal in the conscious state. Cyclooxygenase inhibition potentiated the magnitude of HPV in both the conscious and isoflurane-anesthetized states, which indicates that vasodilator metabolites of the cyclooxygenase pathways modulate HPV under these conditions. Importantly, the finding that the magnitude of HPV is flow-dependent in both the conscious and isoflurane-anesthetized states may explain conflicting reports in the literature concerning the effects of isoflurane anesthesia on the HPV response.

Computer-assisted mapping of infarcted and viable regions of gross cardiac sections following experimental myocardial infarction.

We have developed a computer-assisted method that creates digital maps of the viable and infarcted regions of tetrazolium-stained gross cardiac sections. Here we describe and test the method, using a canine occlusion-reperfusion infarction model. Quantitative image analysis showed that the method accurately recorded differences between infarcted and viable regions at a spatial resolution of 5-20 pixels per mm2. Microscopic analysis of tissue samples taken from the sections showed that the maps were accurate. In 15 of 15 cases the histology of the samples matched that predicted by the maps. A comparison of infarct area measurements derived from the maps showed that the method was reproducible. The average intraoperator standard deviation was +/- 8% of a slice's infarcted area and the average interoperator standard deviation was +/- 20%. We conclude that the method creates accurate, detailed, and reproducible maps of the infarcted and viable regions of tetrazolium-stained cardiac sections.

Isoflurane and the pulmonary vascular pressure-flow relation at baseline and during sympathetic alpha- and beta-adrenoreceptor activation in chronically instrumented dogs.

BACKGROUND: The extent to which isoflurane anesthesia alters systemic vascular regulation has received considerable attention. In contrast, the pulmonary vascular effects of isoflurane have not been elucidated. Our initial objective was to investigate the net effect of isoflurane on the baseline left pulmonary vascular pressure-flow (LPQ) relation compared with values measured in the conscious state. In addition, we assessed the extent to which isoflurane alters the pulmonary vascular responses to sympathetic alpha- and beta-adrenoreceptor activation. METHODS: Twelve conditioned mongrel dogs were chronically instrumented to measure the LPQ relation. LPQ plots were generated by continuously measuring the pulmonary vascular pressure gradient (pulmonary arterial pressure--left atrial pressure) and left pulmonary blood flow during gradual (approximately 1 min) inflation of a hydraulic occluder implanted around the right main pulmonary artery. LPQ plots were generated at baseline in the conscious and isoflurane-anesthetized states (n = 12). The pulmonary vascular dose-response relation to the sympathetic alpha-adrenoreceptor agonist phenylephrine also was investigated in conscious and isoflurane-anesthetized dogs (n = 6). Finally, after preconstriction with the thromboxane analogue U46619, the dose-response relation to the sympathetic beta-adrenoreceptor agonist isoproterenol was assessed in the conscious and isoflurane-anesthetized states (n = 8). RESULTS: Compared with values measured in the conscious state, isoflurane anesthesia had no net effect on the baseline LPQ relation. The magnitude of the pulmonary vasoconstrictor response to phenylephrine observed in conscious dogs was not altered during isoflurane anesthesia. In contrast, the pulmonary vasodilator response to isoproterenol was markedly potentiated (P < 0.01) during isoflurane anesthesia compared with that in the conscious state. CONCLUSIONS: These results indicate that isoflurane does not exert a net vasodilator influence on the pulmonary circulation at baseline. In contrast to the systemic circulation, the pulmonary vasoconstrictor response to sympathetic alpha-adrenoreceptor activation is maintained during isoflurane anesthesia. Surprisingly, the pulmonary vasodilator response to sympathetic beta-adrenoreceptor activation is actually potentiated during isoflurane. Thus, isoflurane anesthesia has differential effects on the canine pulmonary vascular responses to sympathetic alpha- and beta-adrenoreceptor activation.

Pulmonary vascular resistance in emphysema.

To assess the hemodynamic effects of pulmonary microvasculature disruption in emphysema, we examined resting pulmonary hemodynamics and lung function in 12 carefully identified patients with type A chronic obstructive pulmonary disease. Individuals with respiratory muscle weakness and intercurrent infection were excluded. Standard spirometry, helium dilution lung volumes, and single-breath carbon monoxide diffusing capacity (DCOSB) were obtained within 24 h of right heart catheterization. Resistance to pulmonary blood flow was assessed using the difference between pulmonary arterial (PA) diastolic and mean wedge pressures, and expressed as the pulmonary diastolic gradient (PDG). Mean FEV1/FVC was 51 +/- 8 percent, RV/TLC was 48 +/- 11 percent, DCOSB percent predicted was 62 +/- 29 percent, PaO2 was 72 +/- 11 mm Hg (FIO2, 0.21), and PaCO2 was 39 +/- 5 mm Hg. Mean PDG was 5 +/- 3 mm Hg (normal < or = 3 mm Hg) with normal PA pressures, indicating mildly elevated resistance to pulmonary blood flow. The PDG correlated most closely with DCOSB, rising in curvilinear fashion as DCOSB fell (r = -0.869, p < 0.001). These observations were compared with our previous report of analogous findings in patients with chronic, diffuse interstitial lung disease (ILD). In that group, PDG also increased curvilinearly as DCOSB fell (r = -0.839, p < 0.001). Subjects with FVC greater than 50 percent predicted had elevated PDG with normal pressures, while those with FVC less than 50 percent had pulmonary hypertension. The regression of PDG on DCOSB was strikingly similar to emphysema, although the slope in emphysema was less than that in ILD (p < 0.001). These observations suggest that elevated pulmonary vascular resistance in emphysema stems from disruption of the microcirculation in a fashion similar to that encountered in mild-moderate ILD. However, the magnitude of increase is not sufficient to generate resting pulmonary hypertension in the absence of disturbed gas exchange.

Meclofenamate enhances blood oxygenation in acute oleic acid lung injury.

To assess the role of vasoactive prostanoids in acute lung injury, we studied 16 dogs after intravenous injection of oleic acid (OA; 0.08 ml/kg). Animals were ventilated with 100% O2 and zero end-expiratory pressure. Base-line hemodynamic and blood gas observations were obtained 90-120 min following OA. Observations were repeated 30 min after infusion of meclofenamate (2 mg/kg; n = 10), or after saline (n = 6). Resistance to pulmonary blood flow was assessed using the difference between pulmonary arterial diastolic and left atrial pressures (PDG). Ventilation-perfusion (VA/Q) distributions were derived with the multiple inert gas technique. Prior to infusion, there were no significant differences between the two groups. PDG was elevated mildly above normal levels, and shunt flow was the principal gas exchange disturbance. Saline induced no significant changes in hemodynamics or gas exchange. Meclofenamate enhanced PDG to a small, significant degree and effected a 32% reduction in shunt flow (P less than 0.01). Perfusion was redistributed to normal VA/Q units with little change in low VA/Q perfusion or in overall flow. Arterial PO2 rose from 75 +/- 36 to 184 +/- 143 Torr (P less than 0.05). At autopsy, there were no significant differences in wet to dry lung weights. Prostaglandin inhibition redistributes perfusion from shunt to normal VA/Q units, thereby improving arterial PO2, without altering lung water acutely.

Effect of chronic airways obstruction on measurement of the single-breath carbon-monoxide-diffusing capacity.

We have examined the effect of chronic airways obstruction on the measurement of the single-breath carbon-monoxide-diffusing capacity (DLCLSB). We reviewed the results of 136 consecutive pulmonary function tests (comprising standard spirometry, helium dilution lung volumes and DLCOSB) obtained in patients who had an FEV1/FVC less than 70%. We calculated DLCOSB using two different values for alveolar volume (VA). In the first method (HeDL), VA was measured by single-breath dilution of helium during the test. In the second method (RbDL), VA was measured as the sum of the inspiratory vital capacity, performed during the test, and the residual volume, determined separately by helium rebreathing. The mean HeDL/RbDL, reflecting disparity between computations of DLCOSB in individual subjects was 0.85 +/- 0.13 in patients with moderate obstruction (40 less than or equal to FEV1/FVC% less than 60) and was 0.80 +/- 0.14 in those with severe obstruction (FEV1/FVC% less than 40). The mean HeDL/RbDL was lowest (0.73 +/- 0.12) in those with severe elevation of RV/TLC (RV/TLC% greater than 60). HeDL/RbDL correlated best with RV/TLC (r = -0.71, p less than 0.001). Unexplained variance in HeDL/RbDL was not significantly reduced by including the relationship between HeDL/RbDL and pulmonary function indices commonly used to measure airways resistance. These data suggest (1) the difference between HeDL and RbDL in patients with moderate and severe chronic airways obstruction is greater than previously reported; (2) the disparity between HeDL and RbDL stems from slow space ventilation rather than from increased resistance to air flow, and (3) HeDL underestimates gas transfer in poorly ventilated lung compartments.