Effect of lymphatic cannula outflow height on lung microvascular permeability estimations

Estimates of the pulmonary microvascular membrane reflection coefficient (sigma) and permeability-surface area product (PS) are frequently made with the assumption that a percent change in transmicrovascular fluid flux (Jv) will be represented by an equal percent change in the lymph flow rate (QL) from a single cannulated lung lymph vessel. To test this, we measured QL in seven anesthetized dogs with the outflow end of the lymph cannula set at several heights (H) above and below the lung hilus. The left atrial pressure was then elevated to increase Jv, and QL was again measured at several H's. The percent increase in QL at elevated left atrial pressure depended on H. We used the QL data and lymph and plasma protein concentrations to estimate sigma and PS with a modified form of the Kedem and Katchalsky equations. The calculated values varied considerably with H. Our results indicate that changes in Jv are not represented by equal changes in QL. Therefore, techniques for estimating permeability that depend upon QL as an estimate of Jv may lead to erroneous estimates of sigma and PS.     

Effects of hypoxia on the pulmonary microvascular permeability in adult rabbit lung

Within the last 30 years, researchers have explored what role hypoxia might play in causing permeability changes in the pulmonary microvasculature. Since the data accumulated thus far are unclear, the effects of hypoxia on microvascular transport in the isolated, Ringer's perfused adult rabbit lung was observed and the following parameters were measured or computed for both oxygenated and hypoxic perfusates: pulmonary arterial (r_a) and pulmonary venous (r_v) resistances, pulmonary capillary filtration coefficients (K_f), and pulmonary capillary endothelial reflection coefficients () for NaCl and inulin. Separate reservoir bottles were used to create the desired oxygenated (aeration of solution with 95% O₂-5% CO₂) gas mixture or hypoxic (aeration of solution with 95% N₂-5% CO₂) gas mixture. A higher, but not significant, resistance value was found during the oxygenated state. A significant increase in the pulmonary capillary filtration coefficient during hypoxia (10.72 × 10^–4±0.446 × 10^–4 cm³/s cm H₂O for the hypoxic perfusate and 8.80 × 10^–4±0.384 × 10^–4 cm³/s cm H₂O for the oxygenated perfusate) was found and a significant difference between oxygenated and hypoxic pulmonary capillary reflection coefficients for inulin was computed (oxygenated solution revealed a finding of 0.120±0.003 and the hypoxic solution revealed 0.105±0.002). These findings imply a change in the microvascular permeability during hypoxia. According to the pore theory, a change in pore number, pore size, or both could have occurred. However, from the reflection coefficient data, a change in pore radius seems most likely.     

Losartan attenuates microvascular permeability in mechanical ventilator-induced lung injury in diabetic mice

Mechanical ventilation can cause direct injury to the lungs, a type of injury known as ventilator-induced lung injury (VILI). VILI is associated with up-regulates angiotensinogen and AT1 receptor expression of in the lung. This work explored effects of losartan on VILI in diabetic mice. Ninty-six C57Bl/6 mice were randomly divided into six groups, control group (C group), diabetes group (D group), diabetes mechanical ventilation group (DV group), losartan control group (L + C group), losartan treatment group in diabetic mice (L + D group) and losartan treatment group in mechanical ventilation diabetic mice (L + DV group). Lung W/D, myeloperoxidase (MPO) activity, microvascular permeability, blood–gas analysis, Ang II concentrations and AT-1R protein expression were measured. Compared with D group, DV group increased Ang II concentrations, AT-1R protein expression, W/D ratio, MPO activity, and microvascular permeability. PaO₂ were significantly lower in the DV group than D group or control group. Compared with DV group, L + DV group attenuates ventilator-induced lung injury in diabetic mice and prevented the increase Ang II concentrations, AT-1R protein expression and microvascular permeability caused by ventilation in diabetic mice. This study provides in vivo evidence that losartan attenuates microvascular permeability via down-regulates Ang II and AT-1R expression in mechanical ventilator-induced lung injury in diabetic mice.     

Neutrophil-dependent mediation of microvascular permeability

Macromolecular extravasation induced by the chemoattractants N-formyl-methionylleucylphenylalanine (FMLP), complement fragment C5a, and leukotriene B4 (LTB4) has been reported to be neutrophil dependent. A review of experimental evidence argues against mechanical disruption of the endothelial barrier as a likely mechanism for enhanced microvascular permeability. Other proposed mechanisms attribute macromolecular efflux to extracellular liberation of granule constituents or granule-independent neutrophil release products (e.g., oxygen radicals) that can undermine vascular integrity by direct or indirect actions on endothelial cells or other components of vascular walls (glycocalyx, basement membrane). The cytotoxic potential of neutrophil-release products on endothelial cells prompts consideration of a transcellular pathway for macromolecular transport. Further studies are needed to clarify the precise nature of endothelial injury and its resolution to better understand the physiology behind the transient effects of these mediators on vascular permeability.     

LPS induces permeability injury in lung microvascular endothelium via AT(1) receptor

Lipopolysaccharide (LPS) is known to stimulate the circulation and local production of angiotensin II (Ang II). To assess whether Ang II plays a role in LPS-induced acute lung injury, rats were injected with LPS, the microvascular endothelial permeability injury was evaluated by histological changes, increased pulmonary wet/dry weight ratio, and pulmonary microvascular protein leak. Besides, increased rat pulmonary microvascular endothelial cell monolayer permeability coefficient (K(f)) was measured after treatment with LPS and/or Ang II, respectively. LPS/Ang II, treatment resulted in a significant increase in K(f). Ang II cooperates with LPS to further increase K(f). Hence, LPS increases pulmonary microvascular endothelial permeability both in vitro and in vivo. Local lung Ang II was increased in response to LPS challenge, and elevated Ang II ulteriorly exacerbates LPS-induced endothelium injury. [Sar(1),Ile(8)]Ang II, a selective block of Ang II type 1 (AT(1)) receptors, eliminated these changes significantly. Our conclusion is that the LPS-induced lung injury may be mediated by the AT(1) receptor.     

Enhanced microvascular permeability of PMA-induced acute lung injury is not mediated by cyclooxygenase products.

Products of cyclooxygenase activity have been proposed to mediate the pulmonary hypertension and increased microvascular permeability associated with phorbol myristate acetate- (PMA) induced acute lung injury. Previously, we reported that thromboxane (Tx) does not mediate PMA-induced pulmonary hypertension in intact anesthetized dogs. In the present study, PMA was administered to isolated canine lungs perfused with autologous blood at constant flow to investigate a possible role for Tx in the PMA-induced increase in microvascular permeability. Changes in permeability were assessed by determining changes in the capillary filtration coefficient (Kfc). In lobes pretreated with papaverine to prevent PMA-induced increases in pulmonary vascular resistance, Kfc increased from a baseline value of 0.2 +/- 0.03 to 1.5 +/- 0.29 ml.min-1.cmH2O-1.100 g wet lobe wt-1 (P < 0.01) 30 min after PMA (5.8 x 10(-8) M, n = 10). Concomitantly, TxB2, the stable metabolite of TxA2, increased from 138 +/- 44 to 1,498 +/- 505 pg/ml (P < 0.05) in the blood. Both the selective Tx synthase inhibitor, OKY-046 (7 x 10(-4) M, n = 6), and the cyclooxygenase inhibitor, indomethacin (10(-4) M, n = 7), prevented the PMA-induced increase in TxB2, but neither compound attenuated the PMA-induced increase in Kfc. ONO-3708 (10(-6) M), a selective prostaglandin (PG) H2/TxA2 receptor antagonist, prevented the vasoconstriction resulting from administration of U-46619, a stable PGH2/TxA2 receptor agonist, but it did not prevent the PMA-induced increases in Kfc (n = 6).(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of amphipathic peptides with an immobilised model membrane

The interaction of melittin and a truncated analogue of melittin with an immobilised phosphatidylcholine monolayer has been studied using dynamic elution techniques. The melittin analogue (21Q analogue) had five amino acids omitted from the C-terminal region of melittin. The influence of temperature and methanol concentration on the binding affinity of the two peptides was determined and compared to the binding behaviour of two control molecules N-acetyltryptophanamide and diphenylalanine. Both peptides exhibited non-linear dependence of affinity on % methanol at different temperatures, while N-acetyltryptophanamide and diphenylalanine exhibited linear behaviour. In addition, both melittin and the 21Q analogue exhibited significant band broadening under a range of experimental conditions, which was not evident for N-acetyltryptophanamide and diphenylalanine. As melittin is known to adopt a significant degree of -helical conformation in the presence of lipids, the results suggest that melittin and the 21Q analogue adopt different conformations and orientations upon binding to the immobilised phosphatidylcholine surface. Overall, the results of this study demonstrate that the immobilised lipid monolayer provides a powerful system to rapidly assess the affinity of peptides for different lipid surfaces.     

Interaction of amphipathic peptides with an immobilised model membrane

Summary The interaction of melittin and a truncated analogue of melittin with an immobilised phosphatidylcholine monolayer has been studied using dynamic elution techniques. The melittin analogue (21Q analogue) had five amino acids omitted from the C-terminal region of melittin. The influence of temperature and methanol concentration on the binding affinity of the two peptides was determined and compared to the binding behaviour of two control moleculesN-acetyltryptophanamide and diphenylalanine. Both peptides exhibited non-linear dependence of affinity on % methanol at different temperatures, whileN-acetyltryptophanamide and diphenylalanine exhibited linear behaviour. In addition, both melittin and the 21Q analogue exhibited significant band broadening under a range of experimental conditions, which was not evident forN-acetyltryptophanamide and diphenylalanine. As melittin is known to adopt a significant degree of α-helical conformation in the presence of lipids, the results suggest that melittin and the 21Q analogue adopt different conformations and orientations upon binding to the immobilised phosphatidylcholine surface. Overall, the results of this study demonstrate that the immobilised lipid monolayer provides a powerful system to rapidly assess the affinity of peptides for different lipid surfaces.     

Changes during development in the permeability of sclerotia ofSclerotinia minor to an apoplastic tracer

Summary Sclerotia ofSclerotinia minor produced in culture are permeable to the apoplastic tracer sulphorhodamine G (SR) in early stages of their development, but become impermeable as the rind differentiates at the onset of maturation. Reduction in permeability corresponds with deposition of the dark brown pigment in the rind cell walls rather than initiation of the rind as a distinct surface layer. Fluorochrome permeation into cut sclerotia indicates that, while the rind is the primary barrier, the walls and extracellular matrix of the cortex and medulla of mature sclerotia also impede SR movement. Some cells take up fluorochrome into the protoplast. This indicates enhanced proton pumping activity at the cell surface, which suppresses ionisation of the fluorochrome, allowing it to cross the plasma membrane and accumulate in the hyphae. In intact sclerotia such hyphae are very rare and were detected only at one stage of development. However, in cut sclerotia at the two earliest stages of development most of the hyphae near the cut surface accumulated SR and it is possible that this is due to proton pumping activity induced by wounding.Abbreviations ECM extracellular matrix - SR sulphorhodamine G     

Tracing root permeability: comparison of tracer methods

Root epidermis and apoplastic barriers (endodermis and exodermis) are the critical root structures involved in setting up plant-soil interface by regulating free apoplastic movement of solutes within root tissues. Probing root apoplast permeability with “apoplastic tracers” presents one of scarce tools available for detection of “apoplastic leakage” sites and evaluation of their role in overall root uptake of water, nutrients, or pollutants. Although the tracers are used for many decades, there is still not an ideal apoplastic tracer and flawless procedure with straightforward interpretation. In this article, we present our experience with the most frequently used tracers representing various types of chemicals with different characteristics. We examine their behaviour, characteristics, and limitations. Here, we show that results gained with an apoplastic tracer assay technique are reliable but depend on many parameters–chemical properties of a selected tracer, plant species, cell wall properties, exposure time, or sample processing.     

Gallium-transferrin as a macromolecular tracer of vascular permeability

Labeling of plasma transferrin with gallium was investigated to determine whether the gallium-transferrin complex could be effectively used as a macromolecular tracer in studies of capillary permeability using Positron Emission Tomography (PET). Three gallium-plasma preparations were tested and 2 h bio-distribution studies were performed in rats. The three preparations gave similar blood clearance and tissue distribution data, but the methods used for evaluating gallium-transferrin binding were found to be suboptimal. Gallium clearance from blood was biexponential with both components faster than that of ¹²⁵I-albumin. Gallium distribution spaces in all tissues including intracerebral Walker-256 tumors were larger than those of albumin. These results indicate a relative instability of the gallium-transferrin complex in vivo, which appears to preclude its use as an acceptable radiolabeled protein for vascular permeability studies using PET.     

Modulation of solute permeability in microvascular endothelium

Modulation of macromolecular permeability involves creation of venular leaks in response to receptor-operated mechanisms in the endothelial cell membrane elicited by various autacoids (histamine, serotonin, bradykinin). Reversible modulations may occur within seconds in response to specific agents, which indicates receptor-mediated events that act via the endothelial cells' contractile apparatus, leading to subtle changes in junctional microtopography and allowing faster passage of small solutes. This mechanism probably involves activation of the actin-myosin system in endothelial cells. Ca2+ is an important signal substance, as reflected in the permeability-increasing effect of calcium ionophores. The junctional control system may share functional similarities with the contractile system in various types of muscle cells, in particular, smooth muscle. This suggests a function for the extensive vesicular invaginations of the plasmalemmal membrane present in endothelial cells. Rather than being a system to carry macromolecules across the endothelium, its physiological role may be to regulate free cytosolic calcium concentration. It is reminiscent of similar membrane invaginations found in muscle cells. Thus intracellular free calcium may be regulated by a combination of energy-requiring extrusion and passive influx through receptor-operated calcium channels located in the invaginated vesicular membranes, with short diffusion distances to the actin-myosin filaments in the cytoplasm.     

Neutral and DEAE dextrans as tracers for assessing lung microvascular barrier permeability and integrity.

Steady-state lymph-to-plasma concentration ratios (L/Ps) of neutral dextrans, cationic DEAE dextrans, and endogenous proteins were determined under normal and increased permeability conditions in six unanesthetized yearling sheep prepared with chronic lung lymph fistulas. Fluorescent dextrans with radii ranging from 1 to 30 nm were intravenously infused, and after 24 h, perilla ketone (PK) was given to alter permeability while the dextran infusion was maintained. Plasma and lymph samples were collected before and after PK administration and analyzed for dextran and protein concentrations after high-performance liquid chromatography size separation. Under both baseline and increased permeability conditions, DEAE dextrans had higher L/Ps than neutral dextrans of similar size but lower L/Ps than proteins of similar size. Comparison of L/Ps before and after PK revealed that the percentage change in permeability for neutral and DEAE dextrans was significantly larger than that for proteins. These results suggest that 1) the pulmonary microvascular barrier behaves as a net negative barrier, 2) some transport mechanisms for proteins and dextrans are different, and 3) neutral and cationic dextrans are more sensitive markers than proteins of the same size for assessing changes in pulmonary capillary permeability.     

Superoxide dismutase as an inhibitor of postischemic microvascular permeability increase in the hamster

The purpose was to elucidate the involvement of superoxide radical (O2-.) in the postischemic increase in the vascular permeability in the hamster cheek pouch. Cheek pouches of anesthetized hamsters were everted, prepared for intravital microscopy, and superfused with a bicarbonate buffered saline solution. Local ischemia for 30 min was obtained using a cuff placed around the proximal part of the cheek pouch. The vascular permeability in the postcapillary venules was quantified as leakage of intravenously injected fluorescein labeled dextran (FITC-dextran, Mw 150,000), using intravital microscopy and fluorimetry. There was a significant and reversible permeability increase after the reperfusion started. In the first series of experiments, combined intravenous infusion and topical application of human recombinant extracellular superoxide dismutase C (EC-SOD C) reduced the postischemic permeability response by 80%. Bovine CuZn-SOD given in exactly the same way reduced the response by 60%. In the second series of experiments, inactivated EC-SOD C was given to the control animals and active EC-SOD C was given to the treated animals. The topical treatment was excluded. Only active EC-SOD C reduced significantly the postischemic permeability increase when present during the ischemic period. Treatment with mannitol (i.v.) did not alter the postischemic response. Since active EC-SOD C and CuZn-SOD but not inactivated EC-SOD C effectively inhibited the response, we suggest that the superoxide anion is involved in the mediation of the postischemic permeability increase in the hamster.     

High microvascular endothelial water permeability in mouse lung measured by a pleural surface fluorescence method.

Transport of water between the capillary and airspace compartments in lung encounters serial barriers: the alveolar epithelium, interstitium, and capillary endothelium. We previously reported a pleural surface fluorescence method to measure net capillary-to-airspace water transport. To measure the osmotic water permeability across the microvascular endothelial barrier in intact lung, the airspace was filled with a water-immiscible fluorocarbon. The capillaries were perfused via the pulmonary artery with solutions of specified osmolalites containing a high-molecular-weight fluorescent dextran. An increase in perfusate osmolality produced a prompt decrease in surface fluorescence due to dye dilution in the capillaries, followed by a slower return to initial fluorescence as capillary and lung interstitial osmolality equilibrate. A mathematical model was developed to determine the osmotic water permeability coefficient (Pf) of lung microvessels from the time course of pleural surface fluorescence. As predicted, the magnitude of the prompt change in surface fluorescence increased with decreased pulmonary artery perfusion rate and increased osmotic gradient size. With raffinose used to induce the osmotic gradient, Pf was 0.03 cm/s at 23 degrees C and was reduced 54% by 0.5 mM HgCl2. Temperature dependence measurements gave an Arrhenius activation energy (Ea) of 5.4 kcal/mol (12-37 degrees C). The apparent Pf induced by the smaller osmolytes mannitol and glycine was 0.021 and 0.011 cm/s (23 degrees C). Immunoblot analysis showed approximately 1.4 x 10(12) aquaporin-1 water channels/cm2 of capillary surface, which accounted quantitatively for the high Pf. These results establish a novel method for measuring osmotically driven water permeability across microvessels in intact lung. The high Pf, low Ea, and mercurial inhibition indicate the involvement of molecular water channels in water transport across the lung endothelium.