Shear stress-induced restoration of pulmonary microvascular endothelial barrier function.

We tested the hypothesis that, in lungs injured by a period of ischaemia and reperfusion (IRI), reduced shear stress contributes to increased pulmonary microvascular endothelial barrier permeability and edema formation. Furthermore, we examined the role of VEGFR2 as a mechanosensor mediating the endothelial response to this altered shear stress.

Methods. Mice. All procedures involving mice were approved by the University College Dublin Animal Ethics Committee (AREC-18-05-McLoughlin) and undertaken following authorisation by the Health Products Regulatory Authority (AE18982.I401). Adult male and female C57 Bl6 mice (10–12 weeks old) were supplied by UCD Biomedical Facility and housed under specific pathogen free conditions with ad libitum access to food and water. Isolated perfused lung. The isolated, ventilated, perfused mouse lung was used to assess vascular function, as previously described (15, 34). Lungs were isolated post-mortem following induction of deep anaesthesia (pentobarbitone sodium, 120 mg/kg I.P.), administration of heparin (1000i.u./kg) and exsanguination. The isolated lungs were placed in a water jacketed chamber maintained at 37°C, ventilated (Rodent Midi-Vent Model 849 Ventilator, product no: 73-4119 (Hugo-Sachs Elektronic-Harvard Apparatus, March, Germany) at constant tidal volume (250 ml, 90 inflations per minute) with 5% CO2 balance air and at a constant positive end expiratory pressure (PEEP) of 2.1 cmH2O (1.6mmHg). To prevent the development of progressive atelectasis, a recruitment manoeuvre was undertaken every five minutes throughout the experimental protocol by increasing PEEP to 15.0 cmH2O (11.5 mmHg) for three inflations. An initial recruitment manoeuvre was performed immediately after tracheal cannulation and then at five-minute intervals throughout the remainder of the protocol. Following cannulation of the pulmonary artery and the left atrium, the lungs were perfused (0.5 - 3.0 ml/minute) while left atrial pressure (LAP) was maintained at 2.0 mmHg in all conditions. Pulmonary arterial pressure (PAP), left atrial pressure (LAP) and airway pressure signals were continuously measured (P75 Blood Pressure Transducers, product no: 73-0020 and Differential Low-Pressure Transducer, product no: 73-3882 respectively, Hugo-Sachs Elektronic-Harvard Apparatus, March, Germany), digitized (220 Hz) and stored for later analysis (AcqKnowledge Data Acquisition 3.8.2 Analysis Software, Biopac Systems Inc, USA). In some protocols, capillary pressure (Pcap) was determined at specific time points using the double-occlusion technique at end expiration as previously described (15, 35, 36). Perfusion solutions. Lungs were perfused with one of two different solutions (Table 1). The first was a low viscosity solution (LVS) with a viscosity relative to water of 1.5 (RV 1.5) i.e. a viscosity close to that of normal plasma (37, 38). The second solution, called the physiological viscosity solution (PVS), had a higher relative viscosity of 2.5. The choice of RV 2.5 as the optimum physiological viscosity solution (PVS) was based on our previous work (15). This viscosity lies within the range of apparent viscosities displayed by normal blood (39, 40). LVS consisted of Dulbeccos’s Modified Eagle’s Medium (DMEM, Sigma, Dublin, Ireland, catalog no. D6046) with Ficoll 70kDa (Sigma, Dublin, catalog no. F2878) added (40 g/l) to provide oncotic pressure. PVS was composed of DMEM (Sigma, Dublin, Ireland, catalog no. D6046) with Ficoll 70 kDa (Sigma, Dublin, catalog no. F2878) (33.8 g/l) and Ficoll 400kDa (Sigma, Dublin, catalog no. F4375) added (32.5g/l) to increase viscosity. The sum of the molar concentrations of Ficoll 70kDa and Ficoll 400kDa in PVS was equal to the molar concentration of Ficoll 70kDa in LVS. Osmolality of the perfusion solutions was measured by determination of solution vapour pressure (VAPRO, model 5520, Wescor, Utah). Colloid osmotic pressures (COP) of the solutions were determined using a polyarylethersulfone (PAES) based hollow fiber dialysis/ultrafiltration membrane (Prismaflex ST150, Baxter, Meyzieu, France, catalogue number. 23E0110CA) as previously described (41); see Supplemental Materials for details. The viscosity of each perfusion solution was measured at 37°C using an Ostwald capillary viscometer as previously described (42). The key properties of the two perfusion solutions are summarised in Table 1 (see also Supplemental File, Table 1). Ischaemia reperfusion injury. After insertion of the pulmonary artery cannula and left atrial cannula, perfusion of the lung was initiated at a flow of 0.5ml/min for one minute. Thereafter, the flow was incrementally increased to 1.0 ml/min, 2.0 ml/min, and 3.0 ml/min at intervals of one minute. Once a flow of 3.0 ml/min was established, the lung was perfused for a minimum of a further five minutes (>10 minutes total) while the venous effluent was discarded; this allowed the clearance of any residual blood from the pulmonary circulation. If during this period the lungs showed no evidence of injury (i.e. showed no evidence of air of vascular leaks) and displayed stable, normal airway and perfusion pressures, they were included in the study groups. No lungs were excluded from the data analysis after meeting these inclusion criteria. Following this initial assessment period, experimental lungs were subjected to ischaemia reperfusion injury (IRI) by stopping perfusion for a period of 20 minutes during which the intravascular pressure was maintained at 2 mmHg. Following this, flow (3.0 ml/min) was re-started and the circuit was closed i.e. the venous effluent was recirculated for the remainder of the protocol. Uninjured control lungs were perfused without interruption at a flow of 3ml/min while the venous effluent was discarded. After 30 minutes, recirculation of perfusate was commenced i.e. at a time corresponding to the end of the warm ischaemia period in injured experimental lung groups. Ventilation of perfusion of all lungs was continued until the lungs became edematous (Pinsp > 7.5mmHg) or until 180 minutes had elapsed. Pilot experiments showed that continuation of ventilation and perfusion after Pinsp exceeded 7.5mmHg was followed shortly by alveolar flooding with the appearance of edema fluid in the main bronchi and trachea. VEGFR2 inhibitors. SU1498 (Sigma Aldrich), a selective inhibitor of the tyrosine kinase activity of KDR (43), was dissolved in dimethyl sulfoxide solution (DMSO) to produce a stock solution, which was added to the perfusate to achieve a final SU1498 concentration of 10 micromol/l and a final DMSO concentration of 0.1% vol/vol. In experimental series where SU1498 was used, vehicle (DMSO) alone was added to the perfusate of the control groups (LVS or PVS perfused) to produce an identical concentration of DMSO. EG00229 (R&D Systems) is an inhibitor of the binding of VEGFA to the NRP1-VEGFR2 complex, thus blocking ligand mediated receptor activation (44-46). EG00229 was dissolved in DMSO to produce a stock solution and added to the experimental perfusate to achieve a final concentration of 30 micromole/l and a final DMSO concentration of 0.3% vol/vol. Control groups received an equal volume of vehicle (DMSO without added EG00229). Edema formation. Wet to dry weight ratios were measured to assess lung fluid content at the end of each protocol (15). Endothelial permeability to albumin. In some experimental series, vascular permeability to albumin was assessed by measurement of extravascular leakage of Evan Blue labelled albumin, as previously described (15). At the end of the period of warm ischaemia when recirculation commenced, the perfusion fluid was switched to a perfusion fluid (LVS or PVS) containing Evans Blue-labelled albumin (0.5g/100 ml, Sigma). At the termination of the experiment, the vasculature was flushed for five minutes with a perfusate (LVS or PVS as appropriate) that it did not contain Evans Blue labelled albumin (3 ml/min) until the draining perfusate was clear, so that only extravasated Evans Blue-labelled albumin remained in the lungs. Wet to dry weight ratios were measured to assess lung fluid content. Formamide (>99.5%, Sigma) was then added to each dried lung and incubated at 70°C for 1 h to extract Evans Blue dye. The lung was then homogenized, the homogenate cleared by centrifugation, and the concentration of Evans Blue determined by absorbance at 620 nm. Tissue content was expressed as micrograms of Evans Blue per milligram of lung dry weight (15, 47). Glycocalyx shedding. The change in heparan sulphate concentration in the perfusate during the period from the end of the warm ischaemia until the end of the protocol was measured by ELISA (LS-Bio Massuchusets, USA, Catalogue No. LS-F39210) according to the manufacturer’s instructions as an index of glycocalyceal breakdown and shedding during that period (48, 49). Analysis of haemodynamic data. After completion of each protocol at pre-selected points throughout the experimental protocols, airway and vascular pressures were determined immediately before a regular recruitment manoeuvre or just prior to the last recruitment manoeuvre before termination of the experiment. Peak inspiratory pressure (Pinsp) was calculated as the average of the peak airway pressure values measured during 10 consecutive inflations and positive end expiratory pressure (PEEP) as the average value of ten consecutive measurements at end expiration during the same respiratory cycles. Pulmonary arterial pressure (PAP) was calculated as the average of 10 values of pulmonary arterial pressure measured at end expiration during the same respiratory cycles. Mean left atrial pressure was calculated using 10 measurements of LAP taken at the same time points. In some experimental series, the rate of change of Pinsp in each lung was calculated as Pinsp at the end of the protocol less Pinsp 10 minutes after perfusion recommenced following the warm ischaemia period (when Pinsp was stable) divided by the interval between those two measurements. Mean pulmonary vascular resistance (PVR) under experimental conditions was calculated using the following conventional formula: PVR = (PAP – LAP)/Q Where PAP is mean pulmonary arterial pressure, LAP is mean left atrial pressure and Q is perfusate flow rate. The resistance to flow in a vascular bed is a function of both the viscosity of the perfusing fluid and geometry of the vascular bed. The contribution of vascular geometry to flow resistance is termed vascular hindrance (15, 50, 51). To assess vascular hindrance we calculated the resistance that would have been measured had the lungs been perfused with solutions whose viscosities equalled that of water as follows: R(H2O) = PVR/RV Where RV is the relative viscosity of the perfusate. Statistical methods. Statistical analysis was performed using GraphPad Prism software version 9.0 (GraphPad Software, San Diego, CA, USA). Randomized number generation was used to assign mice to experimental groups. Values of n give the number of separate lungs in each group. An a priori power calculation based on our previously published data (15) indicated that group sizes of 10 were needed to give a 90% probability of detecting a change of 33% (a reduction of one third) in endothelial permeability (Evans Blue-labelled albumin leak) at an 0.05 level of statistical significance. Data are presented as means (SD). One-way analysis of variance (ANOVA) was used followed by Student Newman Keuls post hoc tests to determine the statistical significance of the difference between means. The duration of survival of lungs in each group (i.e. time until edema had developed or the end of the protocol was reached) was calculated using the Kaplan-Meier product limit estimate and the significance of the difference between these was determined using the log-rank test. When more than two curves were compared in a series of experiments, the Holms-Sidak stepdown correction was used (52). In all experiments, a value of P<0.05 was considered statistically significant; where P > 0.001, the exact P value is shown.