Journal of Surgical Research 143, 70–77 (2007)

Phenotypic Heterogeneity in Lung Capillary and Extra-AlveolarEndothelial Cells. Increased Extra-Alveolar Endothelial Permeability

is Sufficient to Decrease Compliance

Kevin Lowe, M.D.,*,†,‡ Diego Alvarez, M.D., Ph.D.,*,† Judy King, M.D., Ph.D.,*,†,§and Troy Stevens, Ph.D.*,†,1

*Center for Lung Biology, †Department of Pharmacology, ‡Department of Surgery, §Department of Pathology,University of South Alabama College of Medicine, Mobile, Alabama

Submitted for publication January 8, 2007

doi:10.1016/j.jss.2007.03.047

Background. In acute respiratory distress syn-drome, pulmonary vascular permeability increases,causing intravascular fluid and protein to move intothe lung’s interstitium. The classic model describingthe formation of pulmonary edema suggests that fluidcrossing the capillary endothelium is drawn by nega-tive interstitial pressure into the potential space sur-rounding extra-alveolar vessels and, as interstitialpressure builds, is forced into the alveolar air space.However, the validity of this model is challenged byanimal models of acute lung injury in which extra-alveolar vessels are more permeable than capillariesunder a variety of conditions. In the current study, wesought to determine whether extravascular fluid ac-cumulation can be produced because of increasedpermeability of either the capillary or extra-alveo-lar endothelium, and whether different pathophysio-logy results from such site-specific increases inpermeability.

Materials and methods. We perfused isolated lungswith either the plant alkaloid thapsigargin, which in-creases extra-alveolar endothelial permeability, orwith 4�-phorbol 12, 13-didecanoate, which increasescapillary endothelial permeability.

Results. Both treatments produced equal increasesin whole lung vascular permeability, but caused fluidaccumulations in separate anatomical compartments.Light microscopy of isolated lungs showed that thapsi-gargin caused fluid cuffing of large vessels, while 4�-phorbol 12, 13-didecanoate caused alveolar flooding.

1 To whom correspondence and reprint requests should be ad-dressed at the Center for Lung Biology, MSB 3340, College of Med-icine, University of South Alabama, Mobile, AL 36688. E-mail:

tstevens@jaguar1.usouthal.edu.

700022-4804/07 $32.00© 2007 Elsevier Inc. All rights reserved.

Dynamic compliance was reduced in lungs with cuff-ing of large vessels, but not in lungs with alveolarflooding.

Conclusions. Phenotypic differences between vascu-lar segments resulted in site-specific increases in per-meability, which have different pathophysiologicaloutcomes. Our findings suggest that insults leading toacute respiratory distress syndrome may increase per-meability in extra-alveolar or capillary vascular seg-ments, resulting in different pathophysiologicalsequela. © 2007 Elsevier Inc. All rights reserved.

Key Words: pulmonary mechanics; vascular perme-ability; endothelial heterogeneity; acute respiratorydistress syndrome; pulmonary edema.

INTRODUCTION

The classic model describing development of pulmo-nary edema suggests that fluid transverses the capil-lary endothelium and flows, according to a gradient ofinterstitial pressure, along the perivascular intersti-tium to accumulate around large pulmonary vessels.Such fluid accumulation is seen, pathologically, asperivascular cuffs, and represents one of two compart-ments in which fluid can collect. The second compart-ment is the alveolar air space which, according to thismodel, fills after perivascular cuffs are formed, as aresult of rising interstitial pressure that forces fluidacross the alveolar epithelium into the air spaces [1].Fluid filling of the alveolar air spaces is thought toresult in alveolar collapse, in turn causing decreasedcompliance and decreased blood oxygenation, eventhough fluid accumulation is present both around largeblood vessels and in the alveoli in acute respiratory

distress syndrome (ARDS). Several lines of evidence

71LOWE ET AL.: ENDOTHELIAL PHENOTYPIC HETEROGENEITY AND PERMEABILITY EDEMA

suggest that this model incompletely describes theedema that forms in ARDS.

Studies of pulmonary edema in animals suggest thatnot all extravascular fluid moves across the capillaryendothelium. In a number of animal models of acutelung injury, extra-alveolar vessels are more permeablethan capillaries, causing fluid to accumulate inperivascular cuffs as the result of extra-alveolar,rather than capillary, permeability [2–5]. These stud-ies emphasize that in focusing on the accumulation offluid in the septal compartment during in ARDS, therole that increased extra-alveolar vascular permeabil-ity plays in the pathophysiology of ARDS may be ig-nored. Similarly, because focus has been on eventsoccurring at the alveolar level, alveolar flooding andinactivation of surfactant is believed to exclusively de-termine mechanical properties of the edematous lung.However, surfactant replacement in ARDS patientsdoes not decrease peak pressure or increase tidal vol-ume [6], suggesting that factors other than surfactantinactivation can decrease compliance in ARDS. An al-ternative hypothesis is that extra-alveolar fluid accu-mulation in perivascular cuffs decreases compliance.This alternative hypothesis is supported by the obser-vation that during experimental hydrostatic edema,compliance decreases prior to alveolar flooding [7].Studies describing increased airway resistance in ani-mal models of pulmonary edema [8–10] and expiratoryflow limitation in ARDS patients [11] suggest that thepathophysiology of ARDS causes decreased flow inextra-alveolar airways. Thus, alveolar flooding alone isnot a sufficient explanation for decreased compliancedue to increased extravascular pulmonary fluid inARDS.

While the classic model of pulmonary edema forma-tion describes capillaries as the source of extra-vascular fluid, extracapillary vessels may be more per-meable than capillaries in a variety of conditions, andextra-alveolar forces may contribute to the changes inpulmonary mechanics associated with ARDS. Our cur-rent study sought to determine whether extravascularfluid accumulates as a result of either increased extra-alveolar vessel or capillary permeability and, if so,whether these two sites of fluid accumulation differen-tially effect pulmonary compliance.

MATERIALS AND METHODS

Lung Isolation and Perfusion

All animal experiments were approved by the Animal Care andUse Committee of the University of South Alabama. Adult maleSprague Dawley rats weighing 250 to 350 g were anesthetized withintraperitoneal pentobarbital (40 mg/kg). The trachea was cannu-lated with p60 tubing connected to a ventilator delivering 10 cc/kgcontaining 5% CO2 enriched room air at 60 breaths per min and 2 cmH2O positive end expiratory pressure. The heart and lungs wereexposed, and the pulmonary artery and left ventricle were cannu-

lated. Lungs were perfused via these cannulae with Earl’s balanced

salt solution containing NaHCO3 and 4% bovine serum albumin.After the cannulae were secured, lungs were suspended from a forcetransducer to measure weight gain. A baseline filtration coefficient(Kf) was measured after an isogravemetric state was achieved [12].Lung volume was derived from integration of flow monitored by aspirometer (AD Instruments, Colorado Springs, CO) connected justproximal to the tracheal cannula. During the experiment lungweight, pulmonary artery pressure, left ventricular pressure, tidalvolume, tracheal pressure, dynamic compliance, and pressure/volume curves were constantly recorded (Power Lab; AD Instru-ments). Preparations with evidence of hemorrhage or edema at thispoint in the experiment were not used. Either 4�-phorbol 12, 13-didecanoate (4�PDD) (3 �M) [13] or thapsigargin (50 nM) [4] wasadded to the perfusate reservoir and allowed to circulate for 20 minbefore a second Kf was measured. In control experiments, the sameprocedure was followed using dimethyl sulfoxide as a vehicle control.The area under the dynamic compliance curve was calculated duringfive breaths at the end of each Kf measurement using Chart 5software (AD Instruments).

Saline Filled Lungs

Heart and lungs were removed in-block and, via the trachealcannula, lungs were filled with normal saline to the level of thetransected trachea. The lungs were then attached to the ventilatorand allowed to float in a saline filled beaker. Compliance data wererecorded after the fifth ventilation.

Tween Rinsed Lungs

Heart and lungs were removed in-block and 3 cc of 0.5% Tween 20were injected into the trachea. The lungs were inflated and deflatedthree times and then as much Tween solution as possible was aspi-rated. The lungs were hung by the tracheal cannula and ventilated.Compliance data were recorded after the fifth ventilation.

Light Microscopy

Lungs were perfusion fixed with 3% glutaraldehyde in 0.1 Mcacodylate buffer under 14 to 15 cm H2O venous pressure and 10mL/kg end inspiratory volume. Lungs were then cut into smallerpieces and immersed in fixative. One mL portions of the lung wererinsed in cacodylate buffer, postfixed for 1 h with 1% osmium tetrox-ide, dehydrated through a graded alcohol series, and embedded inPolyBed 812 resin (Polysciences Inc., Warrington, PA). Thick sec-tions (1 �) were cut with a glass knife and stained with 1% toluidineblue. Thick sections were examined and photographed using a NikonE600 light microscope (Nikon Instruments Inc., Melville, NY).

Transmission Electron Microscopy

Thin sections (80 nm) were cut with a diamond knife and thenstained with uranyl acetate and Reynold’s lead citrate. Cells wereexamined and photographed using a Philips CM 100 transmissionelectron microscope (FEI Company, Hillsboro, OR).

RESULTS

In the current study, we sought to determinewhether equal increases in permeability may beachieved in discrete vascular compartments usingthapsigargin and 4�PDD (Fig. 1) and, if so, whetherdifferent physiological sequelae result. We first identi-fied the thapsigargin and 4�PDD concentrations thatproduce equal increases in permeability by measuring

the Kf. Kf can be determined by monitoring vascular

72 JOURNAL OF SURGICAL RESEARCH: VOL. 143, NO. 1, NOVEMBER 2007

pressures and weight gain in isolated and perfusedlungs [14]. In our experiments, untreated lungs (vehi-cle control dimethyl sulfoxide �0.5%) maintained a Kfof 0.13 mL/min�1cm H2O

�1100g�1, and exhibited no

FIG. 1. Discrete sites of increased vascular permeability. (A)Increased capillary permeability leads to alveolar flooding in 4�PDDtreated lungs. (B) Increased extra-alveolar permeability leads toperivascular cuffs in thapsigargin treated lungs. (Color version offigure is available online.)

FIG. 2. Vascular permeability of untreated and Tg treated lungstreated lungs show cuffing of arteries (A) near a bronchiole (B). Th

approximately 2-fold versus control lungs, *P � 0.01. n � 6 in each gro

increase in extravascular fluid accumulation. Lungsperfused with a 50 nM thapsigargin solution displayedcuffing of large vessels and no accumulation of fluid inthe capillary compartment, and an approximately 2.5-fold increase in Kf versus control lungs (Fig. 2). Incontrast, 4�PDD also increased Kf 2.5-fold, but did notproduce cuffing of extra-alveolar vessels. Rather, inlungs treated with 4�PDD, fluid accumulated in thealveolar air space, but not around large vessels (Fig. 3).Transmission electron microscopy confirmed the pres-ence of fluid within the alveoli in 4�PDD treated lungs.Alveolar flooding and air space collapse were seen inalveoli (Fig. 4A) with a Type 2 pneumocyte at its pe-riphery (Fig. 4B), and floating surfactant (Fig. 4C andD). At higher magnification the endothelium and anendothelial junction appear intact (Fig. 4D). Althoughintact endothelium may seem unlikely in the presenceof obvious alveolar flooding, previous authors have de-scribed an intact endothelial barrier in edematous

) Extra-alveolar vessel cuffing is absent in untreated lungs. (B) Tglveolar spaces (AS) are free of fluid. (C) Tg increased permeability

. (Ae a

up. (Color version of figure is available online.)

73LOWE ET AL.: ENDOTHELIAL PHENOTYPIC HETEROGENEITY AND PERMEABILITY EDEMA

lungs from ARDS patients [15, 16]. This paradox maybe explained by considering that relatively minor ul-trastructural changes in endothelial architecture in-crease permeability [17], and that lung microvascula-ture possesses and extraordinary repair capacity [16].

The pressure/volume curve has long been viewed asa sensitive descriptor of pulmonary mechanics in ani-mal models [18] and has been studied extensively as apotential aid in diagnosis and treatment of lung dis-ease [19, 20]. Classic experiments in which pressure/volume relationships were studied in saline filledlungs, in normal air-filled lungs, and in lungs rinsed ofsurfactant describe pressure/volume curves in situa-tion of very low, normal, and very high surface ten-sion, respectively [21]. We generated pressure volumecurves in saline filled lungs (low surface tension),which produce a curve with a near vertical slope. Incontrast, air filled lungs generate curves with an inter-

FIG. 3. Vascular permeability of Tg and 4aPDD treated lungs. (bronchiole (B) Alveolar spaces (AS) are free of fluid. (B) 4�PDD treaflooding (AF). (C) Tg and 4�PDD had equal increases in permeabilitonline.)

mediate slope, and lungs rinsed of surfactant (maximal

surface tension) generate curves with a small slope(Fig. 5). These experiments demonstrate that compli-ance decreases as the result of increasing surface ten-sion within the alveoli. In fact, broncho-alveolar lavagefrom patients with ARDS has increased surface ten-sion [22, 23]. Together, these studies suggest that di-lution or inactivation of surfactant by increased alveo-lar fluid causes decreased compliance in ARDS afflictedlungs. We therefore examined whether lungs treatedwith 4�PDD would be less compliant than controllungs and those treated with thapsigargin. Compliancecan be measured in the actively ventilated lung (dy-namic compliance) or during interrupted ventilation(static compliance). Both increasing airway resistanceand increasing tissue resistance would be expected toincrease tracheal pressure, reduce time for lung infla-tion and decrease dynamic compliance. However, mea-surements of the lung under static conditions would be

Tg treated lungs show extra-alveolar cuffing of an artery (A) near alungs have little or no extra-alveolar cuffing, but do have alveolar

*P � 0.05. n � 6 in each group. (Color version of figure is available

A)tedy,

sensitive only to changes in tissue resistance. To assess

74 JOURNAL OF SURGICAL RESEARCH: VOL. 143, NO. 1, NOVEMBER 2007

both tissue and airway influences on pulmonary me-chanics induced by thapsigargin or 4�PDD, we gener-ated dynamic pressure/volume relationships in iso-lated lungs treated with either thapsigargin or 4�PDD.Lungs treated with thapsigargin had significantly de-creased compliance, while lungs treated with 4�PDDwere not significantly different than control lungs.When we compared groups according to the percentchange in the area under the curve of real-time dy-namic compliance values, 4�PDD treatment did notsignificantly decrease dynamic compliance. In thapsi-gargin treated lungs, however, there was a significantdecrease in dynamic compliance of 17% versus control(Fig. 6). Thus, isolated rat lungs that had increasedextra-alveolar vessel permeability displayed perivas-cular cuffing of large vessels and decreased dynamiccompliance. In contrast, lungs that had equally in-creased permeability in the capillary endotheliumshowed alveolar flooding, but no decrease in dynamic

FIG. 4. SEM of 4�PDD treated lungs. (A) Flooding of alveoli (AF)surfactant filled (S) vesicles is seen in a flooded alveolus. (C) Surfacenlarged view of (C): En � endothelium, Ep � epithelium, EJ �membrane.

compliance.

DISCUSSION

Since the original histological descriptions of pulmo-nary edema, fluid and protein accumulations havebeen seen both in the interstitium surrounding largevessels and in the alveolar air spaces [24]. Fluid accu-mulation around extra-alveolar vessels may result ei-ther as fluid moves across the capillary endotheliumand then distributes into interstitium [1], or may enterthe extra-alveolar interstitium directly from extra-alveolar vessels [2, 4, 5, 25]. The latter situation, inwhich pulmonary edema is caused by fluid movementdirectly across extra-alveolar vessels, can be induced inisolated lungs with the plant alkaloid thapsigargin,which activates store operated calcium entry [4]. Acti-vation of store operated calcium entry through stimu-lation of TRPC1 and TRPC4 [26–29] channels in-creases permeability of extra-alveolar vessels and ofendothelial cell monolayers cultured from those ves-

een among capillaries (C). (B) A Type 2 pneumocyte (TII) containingt granules float in a fluid filled alveolus. (D) Details are seen in an

dothelial junction, S � surfactant filled vesicles, BM � basement

is stanen

sels, but does not increase the permeability of capillar-

75LOWE ET AL.: ENDOTHELIAL PHENOTYPIC HETEROGENEITY AND PERMEABILITY EDEMA

ies or monolayers of capillary endothelial cells [4, 30].Interestingly, attenuation of this permeability re-sponse to thapsigargin is associated with decreasedexpression of TRPC1 and TRPC4 channels in extra-alveolar endothelium in rats with heart failure [31],suggesting that down-regulation of these channels inpulmonary endothelium decreases the permeabilityof vessels under conditions of increased hydrostaticpressure. Conversely, 4�PDD stimulates TRPV4 chan-nels, which are osmo-, mechano-, and temperature-sensitive, and are activated by arachidonic acid metab-olites [32]. These channels are expressed in greaternumbers in capillary endothelium than in extra-alveolar endothelium and, when stimulated, increasepermeability of the capillary compartment withoutsubstantially increasing the permeability of extra-alveolar vessels [13]. These channels may, therefore,represent a mechanism by which circulating inflam-matory mediators increase the permeability of capillar-ies during acute lung injury.

According to the generally accepted model that de-scribes extravascular fluid accumulation within thelung, fluid transverses the capillary endothelium andflows by negative interstitial pressure into the poten-tial space surrounding extra-alveolar vessels. Thisfluid is seen by microscopy as extra-alveolar perivas-cular cuffs [33]. In this mode, pressure builds withinthese cuffs until fluid is forced across the alveolar ep-ithelium into the alveolar air space [1]. Our study

FIG. 5. (A) Dynamic pressure volume surves from saline, un-treated and detergent rinsed lungs. Saline filled lungs were mostcompliant, and tween rinsed lungs were least compliant. (B) Dy-namic pressure/volume curves of representative thapsigargin and4�PDD treated lungs are superimposed on those of Fig. 4(A). Forclarity, only the inspiratory portion of the curves are shown. (Colorversion of figure is available online.)

provides evidence that is not consistent with this clas-

sic model in two respects. First, store operated calciumentry stimulated by thapsigargin causes perivascularcuffs to form due to increased permeability in extra-alveolar vessels, demonstrating that extravascularfluid accumulates without an increase in capillary per-meability. Second, stimulation of TRPV4 channelswith 4�PDD causes fluid to enter the alveolar compart-ment directly from the capillaries without first accu-mulating in the extra-alveolar interstitium. We alsoexplored the physiological effects of this site-specificfluid accumulation, and show that perivascular cuffingdecreases dynamic compliance while an intermediatedegree of alveolar flooding does not. This finding sug-gests that the “stiff lung” associated with ARDS doesnot exclusively result from inactivation of surfactantwithin the alveoli.

The isolated, perfused lung model has been usedextensively to study pulmonary vascular permeability.Kf is a measure that describes changes in permeabilityof the vasculature of isolated lungs based on rate ofweight gain and increases in the static pressure differ-ence across the vascular tree during a period of in-creased outflow pressure [34]. Traditionally, because ofthe much larger surface area of the capillary endothe-lium, Kf was thought to describe the permeability stateof the exchange vessels [14]. However, later studies inisolated lungs subject to ischemia/reperfusion [3], hyp-oxia [5], increased hydrostatic pressure [35], and storeoperated calcium [4] entry establish that Kf also in-creases due to permeability of extra-alveolar vessels.We confirm that increased extra-alveolar vessel perme-ability induced by thapsigargin leads to an increase inKf, and document the presence of perivascular cuffs inthese lungs while, in the same lungs, documenting theabsence of fluid in the alveolar compartment. Impor-tantly, we were able to equal this increase in Kf, whichis due to increased extra-alveolar vessel permeability,by increasing capillary permeability with 4�PDD.These findings are more remarkable if we consider that

FIG. 6. Dynamic compliance in Tg and 4�PDD treated lungs.Dynamic compliance was significantly lower in Tg treated lungs than

in 4�PDD treated lungs. *P � 0.05 versus 4�PDD.

76 JOURNAL OF SURGICAL RESEARCH: VOL. 143, NO. 1, NOVEMBER 2007

equal increases in permeability in these two vascularbeds mean that the permeability increases per surfacearea was necessarily much greater in extra-alveolarvessels. This finding is consistent with previous studiesin uninjured lungs showing that when standardized tosurface area, the arterial endothelium has a 26-foldand the venous endothelium a 58-fold greater perme-ability than capillaries [35]. Indeed, according to recentestimates, this tight capillary endothelial cell barrierfunction represents the most important safety factoragainst formation of pulmonary edema [36].

The finding that 4�PDD induces fluid accumulationin the alveolar air space without evidence of significantperivascular cuffs indicates that a route of fluid move-ment exists directly from vessels to air spaces, suggest-ing decreased barrier function of both the capillaryendothelium and alveolar epithelium. Indeed, previousstudies using 4�PDD and another TRPV4 agonist,14,15 EET, show that both the endothelium and epi-thelium are damaged [13]. A potential pathway forfluid movement directly from capillaries to alveolarairspace is suggested by descriptions of modeled andclinical ARDS, which document substantial injury tothe epithelial barrier early in the disease process [16,37]. Taken together, these findings suggest that theclassic model in which fluid moves across capillaryendothelium to extra-alveolar interstitium and theninto air spaces after rising interstitial pressure forces abreach of the alveolar epithelium, likely does not de-scribe the only mechanism by which alveolar floodingmay occur in ARDS.

Although perivascular cuffs have often been noted inpathological descriptions of edematous lungs [33, 38],the pathophysiological effects of these cuffs are largelyunknown. Specifically, the effect of extra-alveolar fluidaccumulation on pulmonary mechanics is largely un-known. A decrease in dynamic compliance was associ-ated with perivascular cuffing, which occurred prior toalveolar flooding in isolated dog lungs under conditionsof increased hydrostatic pressure [7]. Investigatorshave also suggested that postmyocardial infarction andcongestive heart failure patients have increased air-way closing pressure due to the presence of extra-alveolar interstitial fluid [39, 40]. In the current study,we provide further support for the idea that extra-alveolar fluid accumulation may negatively affect pul-monary mechanics by documenting that extra-alveolarperivascular cuffs are sufficient to cause decreased dy-namic compliance. The mechanism by which extra-alveolar fluid accumulation decreases dynamic com-pliance remains to be shown. Because dynamic com-pliance depends on airway resistance, it is possiblethat perivascular cuffs compress anatomically relatedbronchi, induce airway narrowing, and increase airwayresistance. Narrowed airways may also cause gas trap-

ping [41], which would shift tidal volume to higher

total lung volumes resulting in a measured decrease indynamic compliance [42]. An alternate explanation isthat perivascular cuffs may interrupt the transfer ofradial tension from the parenchyma to the vascularand bronchial walls and thereby increase tissue resis-tance. Also, the finding that dynamic compliance wasnot decreased in lungs with alveolar flooding warrantsfurther discussion. Studies of the alveolar mechanicssuggest that alveoli expand unequally in edema-tous lungs [43]. Thus, volume delivered by the ventila-tor would likely be directed away from the relativelyfew collapsed alveoli and toward non-flooded alveoliwhich, at the low tidal volumes delivered in our studies(�8 mL/kg), were likely able to accommodate the in-creased volume along the linear portion of the pressurevolume curve. In this case, there would be no decreasein compliance. Questions concerning the mechanismsthat produced our results will be answered by furtherinvestigations of pulmonary mechanics in the setting ofsite-specific increases in permeability.

There is increasing awareness that different insultsmay induce permeability at different sites along thearterial-capillary-venous axis. In the current investi-gation, we exploit phenotypic differences among endo-thelial segments to induce site-specific increases inpermeability. Treating isolated lungs with the calciumagonists thapsigargin or 4�PDD induce fluid accumu-lation in the extra-alveolar interstitium or alveolar airspace, respectively, by increasing the permeability ofthe related vascular compartment. We show that thesetwo compartments do not necessarily communicate assuggested by the traditional model. We also demon-strate that dynamic compliance decreases in lungswith perivascular cuffs, but not in lungs with alveolarflooding, suggesting that mechanisms other than alve-olar flooding and inactivation of surfactant are in-volved in producing the “stiff lungs” seen in ARDS.These findings are not consistent with current para-digms concerning the pathogenesis of pulmonaryedema and suggest the need for further investigationsinto the importance of site-specific increases in perme-ability in the pathophysiology of ARDS.

ACKNOWLEDGMENTS

This work is supported by NIH grants HL-66299 and HL-60024,the Center for Lung Biology, and the Department of Surgery at theUniversity of South Alabama. The authors thank Freda McDonaldand the histotechnologists for their technical assistance.

REFERENCES

1. Staub NC, Nagano H, Pearce ML. Pulmonary edema in dogs,especially the sequence of fluid accumulation in lungs. J AppPhysiol 1967;22:227.

2. Bohm GM. Vascular permeability changes during experimen-tally produced pulmonary oedema in rats. J Pathol Bacteriol

1966;92:151.

77LOWE ET AL.: ENDOTHELIAL PHENOTYPIC HETEROGENEITY AND PERMEABILITY EDEMA

3. Khimenko PL, Taylor AE. Segmental microvascular permeabil-ity in ischemia-reperfusion injury in rat lung. Am J Physiol1999;276:L958.

4. Chetham PM, Babal P, Bridges JP, et al. Segmental regulationof pulmonary vascular permeability by store-operated Ca2�entry. Am J Physiol 1999;276:L41.

5. Whayne TF, Jr., Severinghaus JW. Experimental hypoxic pul-monary edema in the rat. J Appl Physiol 1968;25:729.

6. Spragg RG, Lewis JF, Walmrath HD, et al. Effect of recombi-nant surfactant protein C-based surfactant on the acute respi-ratory distress syndrome. N Engl J Med 2004;351:884.

7. Noble WH, Kay JC, Obdrzalek J. Lung mechanics in hypervo-lemic pulmonary edema. J Appl Physiol 1975;38:681.

8. Hogg JC, Agarawal JB, Gardiner AJ, et al. Distribution ofairway resistance with developing pulmonary edema in dogs.J Appl Physiol 1972;32:20.

9. Ishii M, Matsumoto N, Fuyuki T, et al. Effects of hemodynamicedema formation on peripheral versus central airway mechan-ics. J Appl Physiol 1985;59:1578.

10. Cook CD, Mead J, Schreiner GL, et al. Pulmonary mechanicsduring induced pulmonary edema in anesthetized dogs. J ApplPhysiol 1959;14:177.

11. Koutsoukou A, Armaganidis A, Stavrakaki-Kallergi C, et al.Expiratory flow limitation and intrinsic positive end-expiratorypressure at zero positive end-expiratory pressure in patientswith adult respiratory distress syndrome. Am J Respir CritCare Med 2000;161:1590.

12. Townsley MI, Korthuis RJ, Rippe B, et al. Validation of doublevascular occlusion method for Pci in lung and skeletal muscle.J Appl Physiol 1986;61:127.

13. Alvarez DF, King JA, Weber D, et al. Transient receptor poten-tial vanilloid 4-mediated disruption of the alveolar septal bar-rier: A novel mechanism of acute lung injury. Circ Res 2006;99:988.

14. Drake R, Gaar KA, Taylor AE. Estimation of the filtrationcoefficient of pulmonary exchange vessels. Am J Physiol 1978;234:H266.

15. Albertine KH. Ultrastructural abnormalities in increased-permeability pulmonary edema. Clin Chest Med 1985;6:345.

16. Bachofen M, Weibel ER. Alterations of the gas exchange appa-ratus in adult respiratory insufficiency associated with septice-mia. Am Rev Respir Dis 1977;116:589.

17. Tomashefski JF, Jr. Pulmonary pathology of acute respiratorydistress syndrome. Clin Chest Med 2000;21:435.

18. Bachofen H, Hildebrandt J. Area analysis of pressure-volumehysteresis in mammalian lungs. J Appl Physiol 1971;30:493.

19. Matamis D, Lemaire F, Harf A, et al. Total respiratorypressure-volume curves in the adult respiratory distress syn-drome. Chest 1984;86:58.

20. Lemaire F. ARDS and PV curves: The inseparable duet? Inten-sive Care Med 2000;26:1.

21. Hoppin FG, Stothert JC, Greaves IA, et al. Lung recoil: Elasticand rheological properties. In: Fishman AP, Ed. Handbook ofPhysiology. Bethesda: American Physiological Society, 1986:195–215.

22. Pison U, Seeger W, Buchhorn R, et al. Surfactant abnormalitiesin patients with respiratory failure after multiple trauma. AmRev Respir Dis 1989;140:1033.

23. Gregory TJ, Longmore WJ, Moxley MA, et al. Surfactant chem-ical composition and biophysical activity in acute respiratory

distress syndrome. J Clin Invest 1991;88:1976.

24. Staub NC. The pathophysiology of pulmonary edema. HumPathol 1970;1:419.

25. Iliff LD. Extra-alveolar vessels and oedema development inexcised dog lungs. J Physiol 1970;207:85P.

26. Brough GH, Wu S, Cioffi D, et al. Contribution of endogenouslyexpressed Trp1 to a Ca2� selective, store-operated Ca2� entrypathway. FASEB J 2001;15:1727.

27. Groschner K, Hingel S, Lintschinger B, et al. Trp proteins formstore-operated cation channels in human vascular endothelialcells. FEBS Lett 1998;437:101.

28. Tiruppathi C, Freichel M, Vogel SM, et al. Impairment of store-operated Ca2� entry in TRPC4(�/�) mice interferes with in-crease in lung microvascular permeability. (See comment). CircRes 2002;91:70.

29. Wu S, Cioffi EA, Alvarez D, et al. Essential role of a Ca2�selective, store-operated current (ISOC) in endothelial cell per-meability: Determinants of the vascular leak site. Circ Res2005;96:856.

30. Kelly JJ, Moore TM, Babal P, et al. Pulmonary microvascularand macrovascular endothelial cells: Differential regulation ofCa2� and permeability. Am J Physiol Lung Cell Mol Physiol1998;274:L810.

31. Alvarez DF, King JA, Townsley MI. Resistance to storedepletion-induced endothelial injury in rat lung after chronicheart failure. Am J Respir Crit Care Med 2005;172:1153.

32. Nilius B, Vriens J, Prenen J, et al. TRPV4 calcium entry chan-nel: A paradigm for gating diversity. Am J Physiol Cell Physiol2004;286:C195.

33. Teplitz C. The core pathobiology and integrated medical scienceof adult acute respiratory insufficiency. Surg Clin N Am 1976;56:1091.

34. Parker JC, Townsley MI. Evaluation of lung injury in rats andmice. Am J Physiol Lung Cell Mol Physiol 2004;286:L231.

35. Parker JC, Yoshikawa S. Vascular segmental permeabilities athigh peak inflation pressure in isolated rat lungs. (See com-ment). Am J Physiol Lung Cell Mol Physiol 2001;283:L1203.

36. Parker JC, Stevens T, Randall J, et al. Hydraulic conductanceof pulmonary microvascular and macrovascular endothelial cellmonolayers. Am J Physiol Lung Cell Mol Physiol 2006;291:L30.

37. Mitsuoka H, Sakurai T, Unno N, et al. Intravital laser confocalmicroscopy of pulmonary edema resulting from intestinalischemia-reperfusion injury in the rat.[see comment]. Crit CareMed 1999;27:1862.

38. Teplitz C. Pathogenesis of pseudomonas vasculitis and septiclegions. Arch Pathol Lab Med 1965;80:297.

39. Interiano B, Hyde RW, Hodges M, et al. Interrelation betweenalterations in pulmonary mechanics and hemodynamics inacute myocardial infarction. J Clin Invest 1973;52:1994.

40. Hales CA, Kazemi H. Small-airways function in myocardialinfarction. New Engl J Med 1974;290:761.

41. Vieillard-Baron A, Prin S, Schmitt JM, et al. Pressure-volumecurves in acute respiratory distress syndrome: Clinical demon-stration of the influence of expiratory flow limitation on theinitial slope. (Erratum appears in Am J Respir Crit Care Med2002;166:1517). Am J Respir Crit Care Med 2002;165:1107.

42. Volgyesi GA, Tremblay LN, Webster P, et al. A new ventilatorfor monitoring lung mechanics in small animals. J Appl Physiol2000;89:413.

43. Schiller HJ, McCann UG, Carney DE, et al. Altered alveolarmechanics in the acutely injured lung. Crit Care Med 2001;29:

1049.