Pressure-dependent NOS activation contributes to endothelial hyperpermeability in a model of acute heart failure

Aims: Acute increases in left ventricular end diastolic pressure (LVEDP) can induce pulmonary edema (PE). The mechanism(s) for this rapid onset edema may involve more than just increased fluid filtration. Lung endothelial cell permeability is regulated by pressure-dependent activation of nitric oxide synthase (NOS). Herein, we demonstrate that pressure-dependent NOS activation contributes to vascular failure and PE in a model of acute heart failure (AHF) caused by hypertension. Methods and results: Male Sprague–Dawley rats were anesthetized and mechanically ventilated. Acute hypertension was induced by norepinephrine (NE) infusion and resulted in an increase in LVEDP and pulmonary artery pressure (Ppa) that were associated with a rapid fall in PaO2, and increases in lung wet/dry ratio and injury scores. Heart failure (HF) lungs showed increased nitrotyrosine content and ROS levels. L-NAME pretreatment mitigated the development of PE and reduced lung ROS concentrations to sham levels. Apocynin (Apo) pretreatment inhibited PE. Addition of tetrahydrobiopterin (BH4) to AHF rats lung lysates and pretreatment of AHF rats with folic acid (FA) prevented ROS production indicating endothelial NOS (eNOS) uncoupling. Conclusion: Pressure-dependent NOS activation leads to acute endothelial hyperpermeability and rapid PE by an increase in NO and ROS in a model of AHF. Acute increases in pulmonary vascular pressure, without NOS activation, was insufficient to cause significant PE. These results suggest a clinically relevant role of endothelial mechanotransduction in the pathogenesis of AHF and further highlights the concept of active barrier failure in AHF. Therapies targetting the prevention or reversal of endothelial hyperpermeability may be a novel therapeutic strategy in AHF.


Acute Heart Failure Model
Rats were anesthetized with isoflurane, a tracheotomy was performed and they were mechanically ventilated with room air at RR = 60/min, PIP = 10 cm H 2 O, PEEP = 3 cm H 2 O. A carotid artery and jugular vein catheter was inserted for recording of blood pressure and drug administration, respectively. Rats received a norepinephrine infusion starting at 7 g/kg/min and titrated to maintain a MAP of 150 mmHg for a period of 2 hours. Shams were submitted to same anesthesia and surgical procedures but received an infusion of lactate ringers instead of norepinephrine. L-NAME was administered as a bolus targeting a plasma concentration of 200 µmol/L before norepinephrine infusion (Supplemental Figure   1)

Rat In-Situ Perfused Lung Preparation
Isolated perfused lung preparation was performed as previously described(1) with minor modifications. Briefly, animals were anesthetized with ketamine-xylazine (90:10 mg/kg) and mechanically ventilated. The chest and pericardial sac were opened and ligatures were placed around the aorta and pulmonary artery. Animals were heparinized (heparin 200U) and exsanguinated.
The left atrium and the pulmonary artery were cannulated and lungs were perfused with Krebs-Ringer-bicarbonate solution supplemented with 3% bovine serum albumin. Pulmonary artery (P PA ) and left atrial pressures (P LA ) were measured continuously via in-line pressure transducers (P-75, Harvard Apparatus, Natick, MA) connected to an analog-to-digital board. Left atrial pressure was set to 3 cm of water pressure for the entire experimental protocol.
An in-line ultrasonic flow probe (Transonic, Ithaca, NY) was placed in the pulmonary artery cannula and vascular pressures data was recorded in real time using a custom-written program (LabVIEW, National Instruments, Austin, TX).
Aliquots of NE were serially added to the perfusate reservoir to produce a dose range from 10 -8 mol/L to 10 -3 mol/L; the concentration of NE was increased every 10 minutes and PA pressure was recorded as described above. Thus, the direct effects of NE on rat pulmonary vasculature pressure were determined.

Arterial Blood Gas Analysis
Whole blood was collected immediately after animal was anesthetized and every 30 minutes after starting specific treatment (Norepinephrine or L-NAME) or lactated ringers infusion. Blood gases, hematocrit (HCT) levels and pH were measured using a GEM Premier 3000 (Instrumentation Laboratory, Orangeburg, NY) according to manufacturer's instructions.

Wet-to-dry ratio
Lungs and bowel were collected at the end of the experiment and wet weights were obtained immediately after organ collection. Samples were placed in an oven at 60°C for 24 hours. Lung and bowel dry weights (LDW and BDW, respectively) were measured and the wet-todry (W/D) ratio was determined

Plasma Volume Determination
During the animal surgery, a constant infusion of fluid (lactate ringers) and saline boluses were given to the rats to maintain MAP and HCT. The amount of fluid required for each animal differed requiring the changes in plasma volume to be corrected for the individual infusion and boluses. We developed a series of calculations to estimate the amount of total plasma loss that occurs during the hypertensive episode and this served as an indicator of systemic fluid escape to the interstitial space. Total blood volume (TBV) was calculated prior to surgery (equation 1).
The plasma volume was derived every 30 minutes based on HCT levels taken from each blood gas. The TBV was corrected for the volume of blood (cTBV) used in each ABG measurement (0.25 mL), by equation 2 The estimated plasma volume (ePV) was calculated by equation 3 ePV= (1-(HCT/100))*cTBV (3) and was then corrected (cPV) for the amount of fluid that was given during that 30 minute period by adding the volume of infused drugs (VDI; norepinephrine and/or L-NAME), fluid (VFI, lactate ringers) and saline boluses (VSI) as specified in equation 4 cPV= ePV+ VDI + VFI + VSI (4) The change in plasma volume (PV) was derived by subtracting the ePV from the cPV (Equation 5) yielding PV.
PV= cPV-ePV (5) The PV from baseline to 120 minutes is presented for all groups.

Lung Injury Score
Lungs were isolated and instilled with10% formalin under a pressure gradient of Membranes were incubated with primary antibody at 4°C overnight, washed and incubated with secondary antibody for 1h at room temperature. Signal was detected by chemiluminescence using the LI-COR system. Band intensities were quantified using Image Studio software (LI-COR).

eNOS uncoupling
To assess whether eNOS is uncoupled during pressure-induced acute heart failure we performed a low temperature gradient gel following standard western blot techniques as previously described (3). Samples were prepared using laemni buffer and b-mercaptoethanol but not boiled and the detection of eNOS monomer/dimer was performed. Protein lysates were resolved using a 4-20% Tris-glycine gradient gel (Biorad). All gels and buffers were pre-equilibrated to 4 °C before electrophoresis, and the buffer tank was placed in an ice bath during electrophoresis and transfer to maintain the gel temperature below 15 °C.
Membranes were incubated with anti eNOS antibody (cell signaling). Signal was detected by chemiluminescence using LiCor system. Band intensities were measured using Image Studio software (LiCor). Fluorescence was read for 30 minutes (exc.485 nm, em.595 nm). Three μL of NADPH 2x10-3 mol/L were then added and a new reading was performed after 30 min. The delta for the fluorescence was calculated (RFU =RFU after NADPH -RFU before NADPH). RFU was normalized by protein content in the sample.

Statistical Analysis
Data are presented as mean ± SD. Groups were compared using one-way ANOVA and comparison of group means was done using Tukey post-test or ttest. P<0.05 was considered statistically significant.

Legends to Figures
Supplemental Figure  1.