David J. Visintine

Propofol attenuates lung endothelial injury induced by ischemia-reperfusion and oxidative stress.

Lung dysfunction after cardiopulmonary bypass and lung transplantation results from oxidant-mediated cellular damage. Previously, we observed the shedding of angiotensin-converting enzyme (ACE) from the endothelial cell surface to be a more sensitive and earlier marker of oxidative lung endothelial injury than lung wet-to-dry weight ratio. The aim of this study was to evaluate the potential of the anesthetic propofol, which has antioxidant properties, to prevent oxidative lung injury by measuring ACE shedding. ACE release from isolated perfused rat lungs increased significantly after ischemia-reperfusion (I/R). Propofol significantly decreased I/R-induced ACE release by 23.4% (P < 0.05). Perfusion with 0.75 mM H2O2 also caused ACE release from the lung microvasculature, which was similarly attenuated by propofol. The protective effect of propofol on H2O2-induced ACE shedding was confirmed in vitro using Chinese Hamster Ovary cells overexpressing human ACE. Thus, propofol can attenuate oxidative injury of the pulmonary endothelium as detected by ACE shedding in I/R and H2O2 models of acute lung injury.

Isoflurane, but not sevoflurane, increases transendothelial albumin permeability in the isolated rat lung: role for enhanced phosphorylation of caveolin-1.

Background: Caveolae mediated transendothelial transport of albumin has recently been shown to be the primary mechanism regulating microvascular endothelial albumin permeability. The authors investigated the effects of isoflurane and sevoflurane on pulmonary endothelial albumin permeability and assessed the potential role of the caveolae scaffold protein, caveolin-1, in these effects. Methods: Isolated rat lungs and cultured rat lung microvessel endothelial cells (RLMVECs) were exposed to 1.0 or 2.0 minimum alveolar concentration (MAC) isoflurane or sevoflurane for 30 min. 125I-albumin permeability–surface area product and capillary filtration coefficient were determined in the isolated lungs. In RLMVECs, uptake and transendothelial transport of 125I-albumin were measured in the absence and presence of pretreatment with 2 mm methyl-β-cyclodextrin, a caveolae-disrupting agent. Uptake of fluorescent-labeled albumin, as well as phosphorylation of Src kinase and caveolin-1, was also determined. In Y14F-caveolin-1 mutant (nonphosphorylatable) expressing RLMVECs, uptake of 125I-albumin and phosphorylation of caveolin-1 were evaluated. Results: In the isolated lungs, 2.0 MAC isoflurane increased 125I-albumin permeability–surface area product by 48% without affecting capillary filtration coefficient. In RLMVECs, isoflurane more than doubled the uptake of 125I-albumin and caused a 54% increase in the transendothelial transport of 125I-albumin. These effects were blocked by pretreatment with methyl-β-cyclodextrin. The isoflurane-induced increase in uptake of 125I-albumin in wild-type RLMVECs was abolished in the Y14F-caveolin-1 mutant expressing cells. Isoflurane also caused a twofold increase in Src and caveolin-1 phosphorylation. Neither 1.0 MAC isoflurane nor 1.0 or 2.0 MAC sevoflurane affected any index of albumin transport or phosphorylation of caveolin-1. Conclusion: Isoflurane, but not sevoflurane, increased lung transendothelial albumin permeability through enhancement of caveolae-mediated albumin uptake and transport in the isolated lung. This effect may involve an enhanced phosphorylation of caveolin-1.

Propofol Attenuates Endotoxin-induced Endothelial Cell Injury, Angiotensin-converting Enzyme Shedding, and Lung Edema

BACKGROUND:Acute lung injury (ALI) is a frequent complication in septic patients. Previously, we found that propofol, a highly lipid-soluble anesthetic, attenuates ischemia-reperfusion and oxidative lung injury in the isolated perfused rat lung. In the present study, we evaluated the effect of propofol on endotoxin-induced ALI and endothelial dysfunction. METHODS:The effect of propofol on endotoxin-induced lung endothelial injury was evaluated by plasma and lung tissue homogenate angiotensin I converting enzyme (ACE) activity, pulmonary vascular anti-ACE monoclonal antibody binding, and lung wet weight to body weight ratio (LW/BW). RESULTS:When injected IV into rats, endotoxin produced endothelial cell injury and lung edema, as indicated by: 1) an increase in plasma ACE activity, 2) a decrease in lung ACE activity and anti-ACE monoclonal antibody binding, and 3) an increase in LW/BW. Monoclonal antibody 1A2 was up to 1.8 times more sensitive than other anti-ACE monoclonal antibodies in detecting the decrease in ACE in lungs of endotoxin-treated rats. Pretreatment of rats with a bolus of propofol before endotoxin injection significantly inhibited the increase in ACE activity in the blood, the decrease in ACE activity in the lung, the decrease in anti-ACE monoclonal antibody binding in the lung, and the increase in LW/BW ratio. Importantly, propofol also significantly increased the survival rate of endotoxin-treated animals. The protective effect of propofol in endotoxin-treated animals in vivo was confirmed in vitro, i.e., propofol decreased endothelial cell injury and ACE shedding from endothelial cells in culture. CONCLUSIONS:These results suggest that propofol offers significant protection against endotoxin-induced pulmonary microvessel endothelial cell injury and that anti-ACE monoclonal antibody 1A2 is a sensitive probe for monitoring endothelial dysfunction and ALI during sepsis.

G protein-dependent basal and evoked endothelial cell vWF secretion

von Willebrand factor (vWF) secretion by endothelial cells (ECs) is essential for hemostasis and thrombosis; however, the molecular mechanisms are poorly understood. Interestingly, we observed increased bleeding in EC-Gα13(-/-);Gα12(-/-) mice that could be normalized by infusion of human vWF. Blood from Gα12(-/-) mice exhibited significantly reduced vWF levels but normal vWF multimers and impaired laser-induced thrombus formation, indicating that Gα12 plays a prominent role in EC vWF secretion required for hemostasis and thrombosis. In isolated buffer-perfused mouse lungs, basal vWF levels were significantly reduced in Gα12(-/-), whereas thrombin-induced vWF secretion was defective in both EC-Gαq(-/-);Gα11(-/-) and Gα12(-/-) mice. Using siRNA in cultured human umbilical vein ECs and human pulmonary artery ECs, depletion of Gα12 and soluble N-ethylmaleimide-sensitive-fusion factor attachment protein α (α-SNAP), but not Gα13, inhibited both basal and thrombin-induced vWF secretion, whereas overexpression of activated Gα12 promoted vWF secretion. In Gαq, p115 RhoGEF, and RhoA-depleted human umbilical vein ECs, thrombin-induced vWF secretion was reduced by 40%, whereas basal secretion was unchanged. Finally, in vitro binding assays revealed that Gα12 N-terminal residues 10-15 mediated the binding of Gα12 to α-SNAP, and an engineered α-SNAP binding-domain minigene peptide blocked basal and evoked vWF secretion. Discovery of obligatory and complementary roles of Gα12 and Gαq/11 in basal vs evoked EC vWF secretion may provide promising new therapeutic strategies for treatment of thrombotic disease.

Cloning and characterization of a single-chain fragment of monoclonal antibody to ACE suitable for lung endothelial targeting

Monoclonal antibody (mAb) 9B9 to angiotensin-converting enzyme (ACE) demonstrates selective accumulation in lung tissue of the rat, hamster, cat, monkey and human after systemic injection. It has also been demonstrated that mAb 9B9 is the useful tool for targeting therapeutic agents or genes to lung endothelium. In this study, we describe the generation and characterization of a single-chain derivative (scFv) of mAb9B9 (scFv 9B9). In vitro, scFv9B9 retains the ability of the parental antibody to recognize human and rat ACE when expressed both on the surface of phage and as a soluble protein in prokaryotic and eukaryotic expression systems. The ability of scFv 9B9 presented by phage or the soluble protein labeled with I(125) to recognize ACE in the pulmonary circulation was also confirmed in an in vivo rat model. Sequence analysis revealed a putative glycosylation site in close proximity to the complementarity determining region 2 (CDR2) of the scFv 9B9 heavy chain. Mutation of Asn68 to Gln in the heavy chain of scFv 9B9 eliminated the glycosylation site and significantly improved the binding affinity of scFv 9B9 to human ACE as determined by cell ELISA and Western Blot. Moreover, Asn68Gln scFv 9B9 showed a greater rate of secretion at 30°C than wild type scFv 9B9, but had a decreased thermal stability at 37°C. The development of a stable and functional single-chain format of mAb 9B9 which specifically recognizes human and rat ACE represents a novel antibody-based reagent suitable for targeted delivery of drugs/genes to the pulmonary circulation.

Effects of anaesthesia, hypercarbia and hypocarbia on regional cerebral blood flow in the goat

SummaryRegional cerebral blood flow (rCBF) was evaluated by radioactive microsphere indicator technique in unanaesthetized goats and in goats anaesthetized with ketamine, pentobarbitone and halothane. In addition, rCBF was studied in goats anaesthetized with halothane associated with hypercarbia and hypocarbia. Microsphere distribution in unanaesthetized goats demonstrated significant differences in rCBF of the following order and magnitude: thalamus, 151 ± 11 per cent; cerebral gray matter, 111 ± 14 per cent; cerebellum, 92 ± 17 per cent; hypophysia, 75 ± 4 per cent; cerebral white matter, 47 ± 4 per cent (100 per cent = average flow per gm brain). Microsphere distribution following anaesthesia indicated significant changes in relative rCBF to various regions. Ketamine (10 mg/kg) produced a relative decrease in thalamic flow (75 ± 31 per cent ) while increasing gray matter flow (21 ± 10 per cent). Pentobarbitone (35 mg/kg) produced a relative decrease in thalamic (38 ± 19 per cent), gray matter (27 ± 18 per cent), and hypophysis (24 ± 15 per cent) flow, with substantially higher relative flow to white matter (61 ± 23 per cent). Halothane (1.5 per cent) at normocarbia decreased relative thalamic flow (65 ± 28 per cent) but had no effect on other regions. Halothane (1.5 per cent) with hypocarbia decreased relative thalamic flow 65 ± 15 per cent) while increasing relative gray matter flow (42 ± 27 per cent). Halothane (1.5 per cent) with hypercarbia caused a relative increase in thalamic ( 68 ± 28 per cent ) and gray matter (55 ± 34 per cent ) flow.Based on these observations, it was concluded that the goat brain is a heterogeneously perfused organ, and that anaesthesia causes significant shifts in regional cerebral blood flow.RéSUMéLe débit sanguin régional cérébral a été évalué au moyen de microsphères radioactives chez la chèvre à ľétat ďéveil et chez cet animal soumis à une anesthésie à la kétamine, au pentobarbitone et à ľhalothane. On a de plus fait ľétude de ľinfluence de ľhyper et de ľhypocarbie sur le débit cérébral régional de ľanimal anesthésié à ľhalothane.La distribution de microsphères a permis de mettre en évidence des différences significatives dans le débit cérébral régional des animaux à ľétat ďéveil, soit, en ordre décroissant: thalamus, 151 ± 11 pour cent; matière grise, 111 ±14 pour cent; cervelet, 92 ± 17 pour cent; hypophyse, 75 ± 4 pour cent; matière blanche, 47 ± 4 pour cent. Ces pourcentages sont exprimés par rapport au débit sanguin cérébral moyen que ľon dit correspondre à 100 pour cent fin de comparaison.Sous anesthésie, on a observé des modifications profondes des débits régionaux. Par exemple, la kétamine ( 10 mg/kilo) a causé une diminution relative du débit thalamique (75 ± 31 pour cent), associée à une augmentation de débit dans la substance grise ( 21 ± 10 pour cent ).Le pentobarbitone (35 mg/kilo) amenait une diminution relative du débit thalamique (38 ± 19 pour cent), de celui de la substance grise (27 ± 18 pour cent) et du débit hypophysaire ( 24 ± 15 pour cent ) avec une augmentation importante du débit dans la substance blanche (61 ± 21 pour cent).Ľhalothane à 1.5 pour cent en normocarbie causait une diminution relative du débit thalamique (65 ± 15 pour cent) sans affecter le débit aux autres régions. La même concentration ďhalothane, en hypocarbie, diminuait le débit thalamique de 65 ± 15 pour cent tout en augmentant le débit à la substance grise de 42 ± 27 pour cent. En hypercarbie, ľhalothane à 1.5 pour cent augmentait cette fois le débit thalamique de 68 ± 28 pour cent et le débit à la substance grise de 55 ± 34 pour cent.Nous concluons que le cerveau de la chèvre est un organe perfusé de fa÷on hétérogène et que ľanesthésie amène des modifications significatives des débits cérébraux régionaux.

REDUCED EXPRESSION OF ANGIOTENSIN I-CONVERTING ENZYME IN CAVEOLIN-1 KNOCKOUT MOUSE LUNGS

Reduced lung capillary expression of angiotensin I-converting enzyme (ACE), a key enzyme in cardiovascular pathophysiology, and of caveolin-1, an important regulator of endothelial cell signalling, has been demonstrated in various models of pulmonary arterial hypertension (PAH). We addressed the relationship between PAH and ACE expression in caveolin-1 knockout mice (Cav1(-/-)), which have moderate PAH. Tissue ACE activity was reduced by 50% in lungs from 3-month-old Cav1(-/-) mice compared to wild type (WT). A similar reduction in lung endothelial ACE expression was observed by measuring the lung uptake of (125)I-labeled monoclonal anti-ACE antibody and by quantitative immunohistochemistry. These alterations in ACE are limited to capillary segments of the pulmonary circulation. Functionally, the increase in pulmonary artery pressure (PAP) in response to ACE conversion of angiotensin I to angiotensin II in isolated, perfused mouse lungs was reduced significantly in Cav1(-/-) mice compared to WT. Thus, these complementary approaches demonstrate the dependence of lung microvascular endothelial cell ACE protein expression on caveolin-1 expression and underscore the vital role of caveolin-1-regulated pulmonary vascular homeostasis on endothelial ACE expression and activity. In summary, we have revealed a novel role of caveolin-1 in the regulation of ACE expression in pulmonary capillary endothelial cells. Further understanding of the mechanism by which reduced caveolin-1 expression leads altered pulmonary vascular development, PAH, and reduced ACE expression may have important clinical implications in patients with these severe lung diseases.