Biochemistry and Biophysics Reports 7 (2016) 180–187

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The effect of diet-induced serum hypercholesterolemia on thesurfactant system and the development of lung injury

Scott Milos a,1, Joshua Qua Hiansen a,1, Brandon Banaschewski a, Yi Y. Zuo b, Li-Juan Yao a,Lynda A. McCaig a, James Lewis a,c, Cory M. Yamashita a,c, Ruud A.W. Veldhuizen a,c,n

a Lawson Health Research Institute, Department of Physiology and Pharmacology, Western University, London, ON, Canadab Department of Mechanical Engineering, University of Hawaii at Manoa, Honolulu, HI, USAc Department of Medicine, Western University, London, ON, Canada

a r t i c l e i n f o

Article history:Received 24 February 2016Received in revised form10 May 2016Accepted 7 June 2016Available online 8 June 2016

Keywords:Pulmonary surfactantCholesterolSerum hypercholesterolemiaARDSSurface tension

x.doi.org/10.1016/j.bbrep.2016.06.00908/& 2016 The Authors. Published by Elsevier

espondence to: Lawson Health Research In4-110, 268 Grosvenor Street, London, ON, Canail address: rveldhui@uwo.ca (R.A.W. Veldhuiand JQH have made equal contribution to t

a b s t r a c t

Acute respiratory distress syndrome (ARDS) is a pulmonary disorder associated with alterations to thepulmonary surfactant system. Recent studies showed that supra-physiological levels of cholesterol insurfactant contribute to impaired function. Since cholesterol is incorporated into surfactant within thealveolar type II cells which derives its cholesterol from serum, it was hypothesized that serum hy-percholesterolemia would predispose the host to the development of lung injury due to alterations ofcholesterol content in the surfactant system.

Wistar rats were randomized to a standard lab diet or a high cholesterol diet for 17–20 days. Animalswere then exposed to one of three models of lung injury: i) acid aspiration ii) ventilation induced lunginjury, and iii) surfactant depletion. Following physiological monitoring, lungs were lavaged to obtainand analyze the surfactant system.

The physiological results showed there was no effect of the high cholesterol diet on the severity oflung injury in any of the three models of injury. There was also no effect of the diet on surfactantcholesterol composition. Rats fed a high cholesterol diet had a significant impairment in surface tensionreducing capabilities of isolated surfactant compared to those fed a standard diet exposed to the sur-factant depletion injury. In addition, only rats that were exposed to ventilation induced lung injury hadelevated levels of surfactant associated cholesterol compared to non-injured rats.

It is concluded that serum hypercholesterolemia does not predispose rats to altered surfactantcholesterol composition or to lung injury. Elevated cholesterol within surfactant may be a marker forventilation induced lung damage.& 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND

license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

The Acute Respiratory Distress Syndrome (ARDS) is a pul-monary disorder caused by one or multiple insults to the lung andis characterized by severe hypoxemia (PaO2:FiO2o200 mmHg)with radiographic bilateral lung infiltrates [1]. ARDS carries amortality rate of approximately 40% and in lieu of effective phar-macological therapies, the mainstay of treatment remains sup-portive through the use of low tidal volume mechanical ventilationto maintain adequate arterial oxygenation [2,3]. The pathophy-siology of ARDS is complex and involves multiple contributingfactors including lung edema formation, maladaptive inflamma-tion, and alterations to the pulmonary surfactant system [4,5].

B.V. This is an open access article u

stitute, Western University,ada N6A 4V2.zen).his manuscript.

Even prior to the development of ARDS, it has been suggested thatcertain individuals may be at an increased risk for the suscept-ibility to disease, and the development of poorer outcomes. Anexample of such susceptibility is chronic alcohol intake, whichmay be related to an increased vulnerability to some of thesepathogenic mechanisms described above [6].

Among the various pathophysiological processes occurring inARDS, alterations and dysfunction of the pulmonary surfactantsystem has been consistently observed across all causes. Dys-functional surfactant not only contributes directly to altered lungphysiology but also impacts local inflammatory responses [5,7,8].Pulmonary surfactant is a lipoprotein complex synthesized andsecreted by alveolar type II cells into the alveolar space where itreduces the work of breathing by stabilizing the lung throughreducing the surface tension, especially during low lung volumes[9,10]. Obtained from bronchoalveolar lavage material, two ex-tracellular surfactant subtypes can be isolated; the surface activelarge aggregates (LA) and the inactive small aggregates (SA) [11].

nder the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

S. Milos et al. / Biochemistry and Biophysics Reports 7 (2016) 180–187 181

The functional LA are composed of phospholipids (80–90%), neu-tral lipids (3–10%) – the majority of which is cholesterol, and sur-factant associated proteins (5–10%). The precise balance of sur-factant's constituent components is paramount to the material'sbiophysical properties and changes to the composition can impairsurfactant's ability to function [10,12].

In the setting of ARDS, numerous studies have demonstratedprofound alterations to the surfactant phospholipid species com-position, surfactant proteins levels, and decreased levels of the LAsurfactant subtype [5,7,8]. These changes, together with biophy-sical inhibition by serum proteins that have leaked into the lung,have traditionally thought to be the primary contributors to sur-factant dysfunction [12–14]. Interestingly, recent in vitro biophy-sical studies and experiments with animal models of lung injuryhave demonstrated that elevated cholesterol within surfactantrepresents a distinct mechanism of surfactant dysfunction [15–17].For example our group has previously shown that in a rat model ofventilator-induced lung injury (VILI) there was increased surfac-tant associated cholesterol in LA, contributing directly to surfac-tant's biophysical dysfunction. Furthermore, a reduction of cho-lesterol to baseline physiological levels could restore isolatedsurfactants innate biophysical function [17]. The mechanisms bywhich cholesterol content within surfactant increases in the set-ting of ARDS are unknown. It has been shown that under normalphysiological conditions the majority of cholesterol found withinsurfactant is derived by uptake from the circulation [18,19], withsubsequent intracellular regulation of this cholesterol to allow forthe incorporation into surfactant [20,21]. It was therefore hy-pothesized that serum hypercholesterolemia would predisposethe host to the development of severe lung injury due to changesto the cholesterol content within pulmonary surfactant. In order totest this hypothesis rats fed a standard and high cholesterol dietwere subjected to three independent experimental models of lunginjury.

2. Materials and methods

2.1. Animal procedures

Eighty-six 6-week old male Wistar rats (Charles River, St.Constant, Quebec, Canada) were used for these experiments. Allprocedures were approved by the Animal Care Committee atWestern University and are in agreement with the guidelines ofthe Canadian Council of Animal Care. Animals were acclimatizedfor three days and had ad libitum access to water and a standardlaboratory diet. Following acclimatization, animals were rando-mized to either a standard or a high-cholesterol diet (0.5% cholicacid and 1.25% cholesterol by mass; Harlan Teklad, Madison, WI,USA) and allowed food ad libitum. Rats were fed their respective

Table 1Baseline characteristics of non-injured rats fed a standard or high cholesterol diet for 1

Baseline characteristics

Weight (g)Serum analysis Cholesterol (mmol/L)

High density lipoprotein (mmol/L)Triglycerides (mmol/L)

Lavage analysis Total surfactant (mg/kg BW)Large aggregates (LA) (mg/kg BW)Small aggregates (SA) (mg/kg BW)% LA (% of total surfactant)Cholesterol (% of LA)Protein content (mg/kg weight)

* Po0.05 Standard versus high cholesterol diet.

diet for 17–20 days. Animals were subsequently exposed to one ofthree different models of experimental lung injury: 1) acid as-piration, 2) high-tidal volume mechanical ventilation, or 3) whole-lung lavage. In order to obtain non-injured controls, 5–6 animalsfrom each diet group were euthanized by intraperitoneal sodiumpentobarbital overdose (110 mg/kg) for determination of baselineserum cholesterol levels and lung lavage analysis as describedbelow. High density lipoprotein (HDL) and triglycerides levels inserum were determined via enzymatic colorimetric assays byLondon Laboratory Services Group (London On).

At the outset of each experimental model of lung injury, sur-gical procedures were utilized for anesthesia and instrumentationas previously reported by Maruscak et. al [22]. Briefly, animalswere anesthetized via intraperitoneal injection of 75 mg/mL ke-tamine and 5 mg/mL xylazine in sterile saline. After appropriatesedation, animals were given a subcutaneous injection of 0.1 mg/kg buprenorphine and a 0.2 mL subcutaneous injection of a 0.5%local anesthetic sensorcaine at the ventral neck area. After ex-posure of the ventral neck area, the left and right jugular veins andthe right carotid artery were exposed and catheterized with PE-50tubing. The left jugular catheter was used to deliver anesthetic/analgesic (0.5–2.5 mg/100 g/h propofol) and the right jugular ca-theter was used to continuously deliver fluid (sterile saline with100 IU heparin/L at 0.5–1.0 mg/100 g/h). The carotid artery cathe-ter was used to measure blood pressure, heart rate, collect bloodfor blood gas measurements (ABL 500 Radiometer, Copenhagen,DK), and continuously deliver fluids (sterile saline with 100 IUheparin/L at 0.5–1.0 mg/100 g/h). All fluids and anesthetics weredelivered via infusion pumps (Harvard Instruments, St. Laurent,QC, Canada). After exposure of the trachea, a 14-gauge en-dotracheal tube was inserted and firmly secured.

Following these surgical preparations, animals were given a0.1 mL IV bolus of 2 mg/mL neuromuscular inhibitor pancuroniumbromide and then immediately connected to a volume-cycled ro-dent ventilator (Harvard Instruments, St. Laurent, Quebec, Canada)via endotracheal tube. The ventilator was set to initially deliver:8 mL/kg tidal volume (Vt), respiratory rate (RR) of 54–58 breathsper minute (bpm), 5 cmH2O positive end-expiratory pressure(PEEP), and a fraction of inspired oxygen (FiO2) of 1.0. Animalswere ventilated for fifteen minutes prior to initial baseline blood-gas measurements. To ensure animals did not have pre-existinglung injury, the baseline (BL) inclusion criteria following thisprocedure was PaO2/FiO2Z400 mmHg.

2.2. Experimental models of lung injury

2.2.1. Acid aspirationA model of acid-aspiration injury was utilized as described pre-

viously [23]. Following the initial surgical procedure, rats meeting theBL inclusion criteria were randomized to receive an intra-tracheal

7–20 days.

Standard diet (n¼6) High cholesterol diet (n¼5)

368.5874.97 362.6374.981.5070.14 7.9771.57*

1.1270.08 1.9470.20*

0.73570.13 2.0570.20*

2.9270.30 2.2870.321.7970.20 1.2670.141.1370.20 1.0270.18

61.4274.93% 56.4872.67%7.3170.40% 9.3471.51%12.1771.62 10.8371.39

Fig. 1. Arterial partial pressure of oxygen (PaO2) to fractional inspired oxygenation(FiO2) ratio during the ventilation of rats fed a standard or high cholesterol dietexposed to acid/air aspiration (A) high-tidal volume mechanical ventilation (B) andwhole lung lavage (C). #¼Po0.05 acid versus air, §¼Po0.05 versus time baselinewithin group. HC-high cholesterol, Vt – Tidal Volume.

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instillation of either 1 M HCl (1 mL/kg) or control air bolus (1 mL/kg).Following instillation, rats were continued to be ventilated (Vt¼8 mL/kg, RR¼54–58 bmp, PEEP¼5 cmH2O and FiO2¼1.0) for 120 min withairway pressure, hemodynamics, and blood gas measurementsmonitored and recorded every 15–30min.

2.2.2. High-tidal volume mechanical ventilationA high-tidal volume mechanical ventilation model of VILI was

used as previously described [15]. After reaching BL inclusioncriteria, standard and high cholesterol diet animals were switchedfrom baseline ventilator settings to a high-stretch ventilationstrategy (Vt¼30 mL/kg, RR¼16–18 bmp, PEEP¼0 cmH2O,FiO2¼1.0) and mechanically ventilated for 180 min [17,22]. Airwaypressure, hemodynamics, and blood gas measurements weremonitored every 15–30 min during ventilation.

2.2.3. Whole lung lavage model of lung injuryA third experimental model of surfactant depletion was utilized as

previously described [24]. Animals meeting the BL inclusion criteriawere disconnected from the ventilator and subjected to a whole lunglavage using 10 mL saline via syringe attached to the endotrachealtube. The peak inspiratory pressure (PIP) was measured prior to andsubsequently after lavage, and volume of saline administered andrecovered were recorded. After withdrawal of the lavage material, therat was re-attached to the ventilator and given a single breath holdand a blood sample was taken. The lavage protocol was repeated aminimum of four times (to a maximum of 6) until the inclusion cri-teria of PaO2r250 mmHg and PIPr16 cmH2O were achieved. Ratswere then ventilated for an additional 120 min (Vt¼8mL/kg,RR¼54–58 bmp, PEEP¼5 cmH2O and FiO2¼1.0) with monitoring asdescribed above.

2.3. Surfactant isolation, processing, and analysis

At the completion of ventilation, a blood sample was obtained toverify serum cholesterol levels and animals were subsequently eu-thanized with an IV overdose of sodium pentobarbital (110 mg/kg).Animals were exsanguinated and a midline sternectomy was per-formed. Lungs were lavaged previously described [17,22]. The lavagesamples were then centrifuged at 150xg for 10 min and supernatantcontaining the total surfactant was aliquoted and stored at �20 °C.An aliquot of the freshly isolated surfactant was centrifuged at40,000xg for 15 min to separate the supernatant containing the SAsub-fraction and the pellet containing LA. The pellet was re-sus-pended in saline and both sub-fractions were stored at �20 °C. Amodified Duck-Chong phosphorous assay was performed on lipidextracts from the LA and SA surfactant samples to determine thephospholipid content [25,26]. Free cholesterol in LA samples wasdetermined by a Free Cholesterol-E kit according to the manufac-turer's instructions (Wako Chemicals, Richmond, VA, USA).

Total protein in lavage samples were measured via micro BCAprotein assay according to the manufacturer's instructions (PierceBiotechnology, Rockford, IL, USA). IL-6 levels in lavage were mea-sured using an enzyme-linked immunosorbent assay (ELISA) kit(BD Biosciences, San Diego, Calif., USA), according to manufac-turer's instructions.

2.4. Biophysical analysis

For analysis of isolated surfactant's surface tension reducingability, LA samples were re-suspended at a phospholipid con-centration of 2 mg/mL in buffer containing 2.5 mM HEPES, 1.5 mMCaCl2, and 140 mM NaCl, pH¼7.4. Samples were incubated at 37 °Cfor at least 1 h prior to assessing the surface tension reducingproperties using a constrained sessile drop surfactometer (CSD) aspreviously reported [27]. Briefly, the system involved a pedestal,

3 mm in diameter with a 1 mm pinhole, placed within a chamberincubated at 37 °C. Approximately 9–10 mL of surfactant samplewas deposited onto the top of the pre-wet pedestal and allowed toequilibrate for 3 min to allow surfactant adsorption. Followingequilibration, dynamic cycling was performed through repeated

Table 2Physiology of rats that underwent one of three models of experimental lung injury.

Physiology parameter Peak inspiratory pressure (cmH2O) Heart rate (beats per min) Blood pressure (mmHg) PaCO2 in blood (mmHg)

Diet Standard HC Standard HC Standard HC Standard HCAir controlBaseline–air 11.470.4 11.070.2 269713 26877 74.074.5 67.373.8 35.972.6 30.172.7120 min–air 14.270.5§ 14.170.8§ 282713 30477 88.7713 76.5716.9 42.271.8 37.573.4Acid aspirationBaseline–acid 11.270.4 11.770.5 28378 265715 62.774.0 67.372.9 37.371.7 38.371.4120 min–acid 17.470.5#,§ 18.671.6#,§ 283711 274715 63.974.6 65.8710.5 42.871.7 44.972.5High tidal volume mechanical ventilationBaseline–VILI 11.370.4 10.670.5 242711 283712 82.5718.0 79.377.0 34.872.2 38.073.0180 min–VILI 31.872.9§ 31.472.2§ 27979 308721 39.377.0 27.877.8§ 43.2714.9 22.977.3LavageBaseline –lavage 11.070.3 11/070.4 26078.6 25675.3 73.176.8 86.974.9 34.771.2 36.772.2120 min –lavage 19.371.2§ 20.471.6§ 285710.2 26278.2 58.079.0§ 64.077.2§ 53.773.0§ 58.972.8§

PaCO2 – partial pressure carbon dioxide in arterial blood, HC – high cholesterol.§ Po0.05 versus time baseline within treatment group.# Po0.05 acid versus air instillation.

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compression and expansions of the surfactant drop using a com-puter controlled stepping motor (model LTA-HS actuator, NewportCorporation, Irvine, CA) to control fluid drop volume. Samplesunderwent 20 compression/expansion cycles at a rate of 30 cyclesper minute during which images of the drop were taken at a rateof 10 frames per second. Samples were consistently compressed to72–78% of original area at each cycle. Axisymmetric Drop ShapeAnalysis was used to determine sample area and surface tensionsof each picture taken during the dynamic compression-expansioncycles.

2.5. Statistics

Data are expressed as means7standard error (SE). Differencesbetween diets in experimental groups measured over time wasdone by a two-way repeated measures analysis of variance (AN-OVA) with Bonferroni post-hoc test. An unpaired two-way stu-dent's T-test was used to determine significance for individualtime point comparisons between the two diets in the non-venti-lated rats, the high tidal volume mechanical ventilation rats, and inthe surfactant depletion rats. For comparisons between air in-stilled and acid instilled rats from either diet, a two-way ANOVAfollowed by Bonferroni post-hoc test was used. All statistics wereperformed using statistical analysis program GraphPad Prism(GraphPad Software, La Jolla, CA, USA). Probability (p) values ofless than 0.05 were considered statistically significant.

3. Results

3.1. Baseline characteristics

Baseline characteristics including weights, serum cholesterolprofile, and lavage surfactant characteristics of animals fed stan-dard and high-cholesterol diet groups are shown in Table 1. Asignificant increase in total serum cholesterol, triglycerides, andHDL levels were observed in animals consuming a high-choles-terol diet. At baseline, lavage and surfactant composition includingcholesterol concentration within surfactant was not significantlydifferent between the two diets groups.

3.2. Lung physiology: assessment of lung injury

The oxygenation responses of animals within the different ex-perimental models of lung injury are depicted in Fig. 1. In the firstmodel of injury, rats instilled with acid showed a rapid decrease in

oxygenation that was significantly different from baseline valuesthroughout the course of ventilation (Fig. 1(A)). Furthermore, thePaO2:FiO2 values were significantly lower in acid instilled ratscompared rats receiving air throughout the duration of mechanicalventilation. No significant differences in oxygenation in either acidor air instilled animals were observed between standard or high-cholesterol diet groups. Oxygenation response during 180 min ofhigh tidal volume mechanical ventilation is shown in Fig. 1(B).Significant decreases in blood oxygenation values compared tobaseline were observed from 150 min onwards in both dietgroups; however, there was no significant difference betweenanimals fed a standard or high-cholesterol diet. Finally, in themodel of whole lung lavage, a significant decrease in oxygenationcompared to baseline was observed following the lavage proce-dure with no significant differences observed between standardand high-cholesterol diet groups (Fig. 1(C)).

Physiological measurements of injury from baseline and theend of the ventilation period are shown in Table 2. Althoughseveral significant physiologic effects were observed in each of theexperimental injury groups comparing baseline values to those atthe end of ventilation, no significant differences were observedbetween standard and high-cholesterol diet fed animals in any ofthe experimental models.

3.3. Surfactant analysis

Fig. 2 shows the amount of surfactant recovered from rat lungsby lavage. There was no significant difference in the amount offunctional LA (Fig. 2(A)) or inactive SA (Fig. 2(B)), recovered fromrats that received acid aspiration versus the air control. Moreover,there was no difference observed between standard and high-cholesterol diet animals with respect to each surfactant subtyperecovered in any of the three models of experimental lung injury.Further comparisons of the LA as a percentage of total surfactant(LA/LAþSA) recovered yielded no statistically significant differ-ences between rats that were exposed to either diet (Fig. 2(C)).

Quantification of free cholesterol in the surfactant LA fraction isshown in Fig. 3. Within each model of experimental lung injurythere was no difference in surfactant associated cholesterol be-tween rats fed either diet. It was observed that only rats that weresubjected to high-tidal volume mechanical ventilation had ele-vated levels of cholesterol in surfactant compared to baseline noninjured animals.

Table 3 shows values of total protein and IL-6 concentrationsmeasured in lavage fluid isolated from each of the experimentalmodels of lung injury with comparisons made between the two

Fig. 2. Surfactant recovered via lavage from rats fed a standard or high cholesteroldiet and exposed to one of three models of experimental lung injury. Surfactantrecovered as large aggregates (A), small aggregates (B), and percent large ag-gregates of total surfactant (C). No statistical significance, P40.05. HC-high cho-lesterol, Vt-Tidal Volume, LA-large aggregates, BW-body weight.

Fig. 3. Cholesterol content as a percentage of lavage LA phospholipids isolatedfrom rats fed a standard or a high cholesterol diet exposed to one of three modelsof experimental lung injury. No statistical significance, P40.05. HC-High choles-terol, Vt-Tidal Volume.

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diets. Total protein values were used as a marker of alveolar-ca-pillary permeability, while lavage IL-6 levels were utilized as amarker of alveolar inflammation. In general, within each model oflung injury, no differences were observed between standard andhigh cholesterol diet animals with respect to lavage protein or IL-6concentrations.

3.4. Surfactant biophysical function

Minimum surface tension achieved during compression of thesample was used as the primary indicator of biophysical function.Minimum surface tensions (mN/m) achieved at cycles 1, 5, 10, 15,

20 are shown in Fig. 4. Firstly, rats that were fed a high cholesteroldiet trended to have higher minimum surface tensions achievedcompared to standard diet counterparts for respective acid or airtreatments, although this did not reach statistical significance forany cycle (Fig. 4(A)). Secondly, there was no difference in surfacetension reducing capabilities observed for rats exposed to high-tidal volume mechanical ventilation fed either diet at any cycle(Fig. 4(B)). Lastly, rats that were exposed to a whole lung lavagehad significantly impaired surfactant surface tension reductionswhen fed a high cholesterol diet compared to rats fed a standarddiet at later compression cycles (Fig. 4(C)).

4. Discussion/conclusion

Prior studies have implicated an increase in surfactant-asso-ciated cholesterol content as a key contributor to surfactant dys-function in the context of lung injury [17,28]; however, the impactof circulating serum cholesterol levels on surfactant compositionand potential downstream implications for lung injury were un-known. It was therefore hypothesized that serum hypercholes-terolemia would predispose a host to a relatively more severe lunginjury as a result of changes in the pulmonary surfactant system.Our primary findings demonstrate that despite achieving a four-fold increase in serum cholesterol through a high-cholesterol diet,no statistically significant differences were observed between hy-per- and normocholesterolemic rats with respect to conventionalmarkers of lung injury including changes in PIP, PaO2, lavageprotein, and lavage IL-6 concentration across three differentmodels of ARDS. Furthermore, surfactant cholesterol levels andsurfactant pool sizes were not significantly altered in rats fed astandard or high cholesterol diets. Additionally there was nofunctional difference in minimum surface tension achievementobserved in surfactant isolated from rats fed both diets in two ofthe three experimental models of injury. Thus, it is concluded thatdiet-induced serum hypercholesterolemia does not lead to a directincrease in cholesterol content within extracellular pulmonarysurfactant, and does not predispose, nor contribute to the severityof lung injury.

The first observation made in the current study was that therewas no statistical difference between standard and high-choles-terol diet in our baseline control rats with respect to cholesterolcontent within surfactant. This finding was somewhat surprisingin light of an earlier study conducted by McCrae and colleagueswhich examined the surfactant system from adolescent femalemice fed a high-cholesterol diet [29]. These mice had a significant

Table 3Protein and IL-6 concentration in lavage from rats exposed to three models of experimental lung injury.

Total protein (mg/kg BW) IL-6 (pg/mL)

Diet Standard HC Standard HCAir control 14.671.6 16.073.3 457.4748.6 733.27121.0Acid aspiration 71.474.5# 102.9717.1# 1605.57253.1# 1470.67282.3High Vt 150.5741.5 196.6767.4 3149.37787.4 3579.271157Lavage 34.577.9 45.178.3 2600.77283.9 2175.57244.7

HC – High Cholesterol, Vt – Tidal Volume.# Po0.001 acid versus air aspiration.

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increase in both their serum cholesterol levels and the amount ofcholesterol in their surfactant without marked changes to theirlamellar body content or lipid droplets in the lipofibroblast [29].Furthermore, assessment of surfactant biophysical function using acapillary surfactometer demonstrated decreased airway patencyand reduced surface film activity in hypercholesterolemic mice.Potential explanations for the observed differences between ourresults and those of McCrae include species specific differences(rats versus mice), sex differences (male versus female), and dif-ferences in the duration of exposure of animals to a high-choles-terol diet (14–20 days versus 16 weeks). Furthermore these ex-perimental differences have to take the complexity of cholesterolmetabolism within the type II cell into consideration. In this re-gard, future studies examining the consequences of acute andchronic hypercholesterolemia across multiple species of bothsexes, with a more detailed focus on the intracellular cholesterolmetabolism, are warranted.

In order to thoroughly investigate the effects of a high choles-terol diet we utilized three distinct experimental models of lunginjury [30]. Acid aspiration, reflecting gastric aspiration as it occursin patients, provided a clinically relevant model to assess the effectof heightened serum cholesterol on lung injury. Pathophysiologi-cally this injury develops due to a chemical burn and the asso-ciated inflammation [31], and does not typically resolve sponta-neously over the two hour ventilation period. We investigated VILIusing high tidal volume mechanical ventilation since our previousstudies had demonstrated significantly elevated cholesterol levelswithin the surfactant after exposure to this type of injury [17]. Thisinjury develops over a 3 h time period due to repeated overstretching and collapse of the lung alveoli associated with theventilation strategy. Finally, in the whole lung lavage model ofsurfactant depletion, the absence of extracellular surfactant is re-sponsible for the development of lung injury [20] and the im-mediate need for de novo synthesis and secretion of surfactantfollowing surfactant depletion would have provided the mostlikely opportunity to observe diet induced changes in surfactant-associated cholesterol content. Despite the differences in initiatinginsults leading to injury, the various severity of lung injury, andthe physiological changes over time, all three models provideconsistent evidence in support of the conclusion that hypercho-lesterolemia does not impact the cholesterol content of surfactantor the severity of lung injury.

In spite of the absence of differences observed between stan-dard and high-cholesterol fed animals with respect to lung injuryoutcomes, it is notable that differences were observed among thethree different models of lung injury. Specifically, only the VILImodel led to increase in surfactant-associated cholesterol contentin the lavage as compared to the values observed in non-injuredanimals. Given the consistency of this observation across this andother reported studies [17,22,28], it is suggested that this choles-terol effect is related to the damaging result of injurious me-chanical ventilation specifically rather than simply a generic effectof lung injury and associated inflammation. Mechanistically, VILIappears to occur due to repeated collapse and overstretching of

the alveoli and in vitro studies have demonstrated that over-stretching of various lung cells, including the surfactant producingalveolar type II cells, can lead to detrimental intracellular effects[32–34]. The main focus of these studies has been on inflammationbut it is possible that stretch induces effects on cholesterol andsurfactant metabolism that occur within the type II cell. From aclinical perspective, the finding that elevated surfactant choles-terol is, potentially, a ventilation specific phenomenon, makes ittempting to speculate that surfactant-associated cholesterol levelsmay provide a valid biomarker for the damaging effects of me-chanical ventilation.

Our data, demonstrating a lack of differences between ourstandard and high-cholesterol fed animals has one further im-plication related to the mechanism by which cholesterol increaseswithin surfactant; namely it suggest that the cholesterol does notsimply leak into the alveolar space from the serum. Thus surfac-tant cholesterol metabolism is altered during VILI specifically and,since our data excludes leak and serum cholesterol levels as havinga major influence on this finding, future studies are required toinvestigate which mechanisms are involved. It is possible thatsurfactant cholesterol is increased in VILI due to reduced clearanceby alveolar macrophages from the airspace. However, consideringthat during normal clearance only 20–30% of cholesterol is re-moved in 24 h [35], such a mechanism seems unlikely. A morelikely scenario is an alteration in intracellular metabolism ofcholesterol and surfactant within the alveolar type II cell. Thisintracellular metabolism of cholesterol involves a variety of dif-ferentially regulated proteins associated with import and transportof cholesterol into the lamellar body [20,21] which may be altereddue to VILI. A specific focus for future studies may be proteins likeas NPC1, NPC2, and ABCA1 since these proteins are responsible forcholesterol transport in the alveolar type II cell. Moreover,knockout animals of these proteins produce elevated cholesterolwithin intracellular surfactant and extracellular surfactant [36,37].

Of the various outcomes measured within our study, statisticalsignificance between the two diets was only observed in mea-surements of surface tension reducing properties of LA-surfactantisolated from the lavage model with a similar trend being ob-served in the acid aspiration group. Importantly, these differencesin biophysical function were evidently not of a magnitude thatinduced differences in physiologic outcomes. It is speculated thatthe high cholesterol diet may have had effects on the compositionof other surfactant components, such as relative amounts ofphospholipid species, which may have impacted surfactant func-tion following injury. Previous studies, demonstrating significantchanges to the pulmonary surfactant system following altered diet[38,39], are consistent with such a mechanism.

Although we were successful in the development of hyperch-olesterolemic rats due to a diet high in cholesterol, there is onlylimited information available on the lungs response to such con-dition. It is possible that the lungs respond to such a conditionthrough a compensatory mechanism involving lipid storage inpulmonary lipofibroblasts [29] with potential impact on surfactantonly in a more chronic condition. A limitation of our study

Fig. 4. Minimum surface tension (mN/m) achieved during compression cycles 1, 5,10, 15, and 20 of surfactant isolated from rats by lavage and concentrated 2 mg/mLphospholipid. Rats were fed a standard or high cholesterol diet and were thenexposed to acid/air aspiration (A), high-tidal volume mechanical ventilation (B), orwhole lung lavage (C).*¼Po0.05 high cholesterol versus standard diet. HC – highcholesterol, Vt – Tidal volume.

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therefore, was that the onset of their hypercholesterolemia wasacute and does not represent the human development of thecondition due to chronic ingestion of high-fat and high-cholesterolfoods. It should be noted however, that this short term model wasselected to avoid possible confounding variables such as obesity or

comorbid cardiovascular disease in longer term dietary models. Asecond limitation inherent to the majority of experimental modelsof lung injury is the acute, short term nature of the experimentalprotocols in contrast to the prolonged course of lung injury andmechanical ventilation which may occur over a time from of daysto weeks [4,40]. Therefore, the impact of hypercholesteremiaduring such a prolonged period of ventilation requires furtherinvestigation.

In summary, diet-induced serum hypercholesterolemia did notpredispose rats to the development of more severe lung injury, nordid it affect their surfactant composition. Increases in surfactantcholesterol levels were observed only with the use of high-tidalvolume mechanical ventilation leading to the suggestion that re-peated collapse and over-distension of alveoli may be responsiblefor increased cholesterol within the surfactant. These findingsprovide further insight into the damaging effect of mechanicalventilation and the potential use of surfactant-associated choles-terol levels as a marker of ventilator-induced lung injury. Overall,our data argues against hypercholesteremia as a predisposingfactor affecting surfactant associated cholesterol levels during in-jury and the development of lung injury.

Acknowledgements

The authors thank Dr. Fred Possmayer for helpful discussion.These studies were funded by an operating grant from the Cana-dian Institutes of Health Research (CIHR) (Grant no. FRN 114936)and local funding from the program of experimental medicine(POEM).

Appendix A. Transparency document

Transparency Document associated with this article can befound in the online version at http://dx.doi.org/10.1016/j.bbrep.2016.06.009.

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