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Published ahead of print on August 28, 2003, doi:10.1164/rccm.200304-562OC
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American Journal of Respiratory and Critical Care Medicine Vol 168. pp. 1391-1398, (2003)
© 2003 American Thoracic Society

Stretch Activates Nitric Oxide Production in Pulmonary Vascular Endothelial Cells In Situ

Wolfgang M. Kuebler, Ulrike Uhlig, Torsten Goldmann, Gregor Schael, Alexander Kerem, Kay Exner, Christian Martin, Ekkehard Vollmer and Stefan Uhlig

Division of Pulmonary Pharmacology, Research Center Borstel, Borstel; Institute of Physiology, Free University of Berlin; and Institute of Anesthesiology, Deutsches Herzzentrum Berlin, Berlin, Germany

Correspondence and requests for reprints should be addressed to Stefan Uhlig, Ph.D., Division of Pulmonary Pharmacology, Research Center Borstel, Parkallee 22, 23845 Borstel, Germany. E-mail: suhlig{at}fz-borstel.de


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Whereas endothelial responses to shear stress have been studied extensively, the responses to circumferential vascular stretch are yet poorly defined. Circumferential stretch in pulmonary microvessels is largely determined by the transmural pressure gradient, hence by both vascular perfusion and alveolar ventilation pressures. Here, we have studied the production of nitric oxide (NO) by the endothelial nitric oxide synthase (eNOS) in two different models of vascular stretch in the intact lung: In isolated-perfused rat lungs, vascular stretch was induced by elevation of vascular pressure. In situ digital fluorescence microscopy revealed stretch-dependent NO production, which was localized to capillary endothelial cells and inhibited by NOS blockers. In isolated-perfused mouse lungs, vascular stretch was generated by ventilation with elevated negative pressure. Stretch-induced phosphorylation of Akt and eNOS in lung endothelial cells was demonstrated by immunohistochemistry and increased NO production by in situ fluorescence microscopy. Stretch-induced endothelial responses in both models were abrogated by pretreatment with phosphatidylinositol-3-OH kinase inhibitors. These findings demonstrate that circumferential stretch activates NO production in pulmonary endothelial cells by a signaling cascade involving phosphatidylinositol-3-OH kinase, Akt, and eNOS and that this response is independent from the mechanical factors causing vascular distension.

Key Words: overventilation • hydrostatic stress • fluorescence microscopy • endothelial nitric oxide synthase • phosphatidylinositol-3-OH kinase

The free radical nitric oxide (NO) regulates vascular tone, angiogenesis, and cell–cell interactions and is therefore a pivotal determinant of vascular homeostasis (1–3). In addition, it has been implicated with the pathogenesis of vascular and tissue injury due to its potential to yield peroxynitrite in the presence of O2- (4). In endothelial cells, NO is produced by two isoforms of a single heme–containing enzyme, NO synthase (NOS). The inducible NOS is regulated at the level of gene transcription and hence, NO formation requires de novo protein synthesis. In contrast, endothelial NOS (eNOS) is constitutively expressed and regulated not only by calcium-dependent binding of calmodulin (5) but also by serine phosphorylation through the phosphatidylinositol-3-OH kinase (PI3K) and Akt pathway (6).

In addition to tangential shear forces exerted by the viscous drag of streaming blood, vascular endothelial cells are subjected to circumferential stretch forces generated by the transmural pressure gradient. In the pulmonary microcirculation, circumferential stretch may result from both increased hydrostatic pressure and ventilatory movements, but the effects of either form of mechanical stretch on endothelial NO production are not known. We have recently described two models in which the effects of lung microvascular stretch on endothelial cells can be determined in situ, either by vascular pressure elevation in isolated-perfused rat lungs (7, 8) or by overventilation with negative pressure in isolated mouse lungs (9, 10). Here, we applied these models to study in situ the effect of circumferential stretch on lung endothelial NO production and its regulation by the PI3K/Akt pathway.

Some of the results of these studies have been previously reported in the form of an abstract (11).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experiments were performed in isolated-perfused rat and mouse lungs. Details on animal care, drugs, antibodies, and fluorescent probes are provided in the online supplement.

Isolated Blood-perfused Rat Lung
Experimental procedures have been described (7, 12). At baseline, left atrial pressure (PLA) was adjusted to 5 cm H2O, yielding pulmonary artery pressures (PPA) of 10 ± 1 cm H2O. For local delivery of fluorescent probes, a microcatheter was wedged in a pulmonary vein draining a capillary area on the lung surface (7, 8).

Isolated-perfused Mouse Lung
Mouse lungs were placed in a water-jacketed thorax chamber (9, 10, 13) and perfused in a nonrecirculating fashion at constant flow of 1 ml/minute, resulting in PPA of 2 to 3 cm H2O. At baseline, negative pressure ventilation (-3 to -10 cm H2O) with room air at 90 breaths/minute was applied, resulting in tidal volumes (VT) of approximately 200 µl. Additional details are provided in the online supplement.

In Situ Fluorescence Microscopy
In rat and mouse lungs, NO production was determined by digital fluorescence imaging of the NO-sensitive dye 4-amino-5-methylamino-2'-7'-difluorofluorescein (DAF-FM). Because the NO-dependent conversion of DAF-FM into a fluorescent benzotriazole derivative is irreversible, fluorescence intensity (F) relative to baseline (F0) reflects cumulative NO production over time. Its first derivative, determined as {Delta}F/F0 for 5-minute intervals, indicates actual NO production. Details on dye loading and fluorescence imaging are provided in the online supplement.

Western Blot Analysis
Frozen mouse lungs were powdered in presence of liquid nitrogen, lysed, and homogenized. Equal amounts of protein were size-fractionated by gel electrophoresis, transferred to a nitrocellulose membrane, and immunoblotted. Methodological details are given in the online supplement.

Immunohistochemistry
Lung specimens were fixed and paraffin-embedded as described previously (14). Lung sections were stained with primary and secondary antibodies as outlined in the online supplement.

Experimental Design
Lung microvessels were exposed to two different modes of circumferential stretch. In rat lungs, vascular pressure was raised by elevation of PLA from 5 to 15 cm H2O. The PI3K inhibitors Ly294002 (10 µmol/L) or Wortmannin (20 µmol/L), the NOS inhibitor L-NAME (250 µmol/L), or superoxide dismutase (SOD) (2,000 U/ml) were added 10 minutes before PLA elevation. For cell-free perfusion, infusion of Ca2+-rich N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid (HEPES) solution was started 10 minutes before PLA elevation. At the end of the experiments, the exogenous NO-donor S-nitroso-N-acetylpenicillamine (SNAP) (1 mmol/L) was added to test whether endothelial cells still contained unconverted DAF-FM. In mouse lungs, microvessels were stretched by overventilation with negative pressure. Lungs were first ventilated for 60 minutes at an end-inspiratory pressure of -10 cm H2O and end-expiratory pressure of -3 cm H2O. Subsequently, lungs were randomized into three groups. In Group 1 (control), ventilation was continued for 30 minutes with an end-inspiratory pressure of -10 cm H2O. In Group 2 (OV), lungs were overventilated for 30 minutes with an end-inspiratory pressure of -25 cm H2O at constant end-expiratory pressure. In Group 3 (OV/Ly), lungs were pretreated with the PI3K inhibitor Ly294002 (dissolved in 0.05% dimethyl sulfoxide) for 30 minutes before overventilation was introduced as in Group 2.

Statistical Analysis
All data are given as means ± SEM and were statistically analyzed as outlined in the online supplement


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In isolated-perfused lungs, we addressed the effects of circumferential vascular stretch on endothelial NO production in two different models.

Imaging Endothelial NO Production
In rat lungs, the effect of increased vascular pressure on endothelial NO production was determined by fluorescence imaging. Images of DAF-FM fluorescence in subpleural capillaries were essentially similar to those previously reported for endothelial Ca2+-imaging (7, 12), in that the capillary margin was marked by a series of fluorescent spots representing single, dye-loaded cells (Figure 1A) . To identify the phenotype of these cells, we counterstained capillaries with the endothelial marker Alexa Fluor 488 AcLDL (n = 4). As exemplified in Figure 1B, DAF-FM fluorescent cells always stained positive for Alexa Fluor 488 AcLDL, confirming their endothelial phenotype. This is consistent with the notion that lung microvessels of diameter less than 30 µm lack smooth muscle and pericytes (15, 16).



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  		Figure 1. In situ 4-amino-5-methylamino-2'-7'-difluorofluorescein (DAF-FM) imaging in venular capillaries of rat lungs. (A) Venular capillary loaded with the nitric oxide (NO)–sensitive dye DAF-FM and imaged at 480-nm excitation. After quenching of DAF-FM fluorescence by brightfield illumination, the capillary was infused with the endothelial marker Alexa Fluor 488 AcLDL and reimaged (B). Note the colocalized fluorescence distributions of DAF-FM and Alexa Fluor 488 AcLDL. Replicated four times. (C) DAF-FM fluorescence response to L-arginine (L-Arg; 250 µmol/L) alone or 10 minutes after NO synthase (NOS) inhibition by L-NAME (250 µmol/L). Fluorescence intensity determined 10 minutes after addition of L-arginine or solvent (HEPES buffer; control group) is expressed relative to its individual baseline (F/F0). n = 5 experiments each, *p value less than 0.05 versus control group, #p value less than 0.05 versus L-Arg. (D) DAF-FM fluorescence response to Ca2+-ionophore 4-bromo A-23187 (5 µmol/L) under Ca2+-free (open bars) or Ca2+-rich (filled bars) conditions in the absence (left) or presence (right) of superoxide dismutase (SOD; 2,000 U/ml). Fluorescence intensity determined 10 minutes after infusion of the ionophore is expressed relative to its individual baseline (F/F0). n = 4 experiments each, *p value less than 0.05 versus Ca2+-free conditions, no significant difference between absence or presence of SOD.

 
When we administered the eNOS substrate L-arginine, endothelial DAF-FM fluorescence increased significantly (Figure 1C). This response was abrogated when vessels were pretreated with the NOS inhibitor L-NAME, verifying the ability of our system to detect endothelial NO production in situ. Administration of the Ca2+-ionophore 4-bromo A-23187 elicited a marked fluorescence increase in lungs perfused under Ca2+-rich but not under Ca2+-free conditions (Figure 1D), reproducing the well described Ca2+-dependence of eNOS activity (5). When lungs were pretreated with the antioxidant enzyme SOD, fluorescence responses to Ca2+-free and Ca2+-rich conditions in the presence of a Ca2+-ionophore were similar to those obtained in the absence of SOD (Figure 1D). Hence, the DAF-FM chromophore reaction does not seem to require O2- generation and, in consequence, peroxynitrite formation, in this setting. Administration of the NO-donor SNAP at the end of the protocol resulted in a marked and rapid (< 1 minute) increase of DAF-FM fluorescence to 4.6- ± 0.2-fold of baseline intensity (F/F0). This response was consistent in all experiments independent of the applied protocol (data not shown). As we discuss later in this article, this finding does not only warrant comparable fluorophore loading to endothelial cells but also confirms that the fluorescence yield was not affected by NO-independent responses.

Vascular Pressure-induced NO Production
We applied this imaging system to analyze the effects of vascular pressure elevation on endothelial NO production. Baseline images obtained at PLA of 5 cm H2O, at which lung capillary pressure is at physiologic levels (17), revealed stable endothelial fluorescence. Accordingly, baseline NO production was below detection limits. Elevation of PLA to 15 cm H2O increased capillary transmural pressure by approximately 132% and dilated capillaries from 17.2 ± 2.1 µm to 19.5 ± 3.2 µm in diameter (p < 0.05), thus stretching the capillary endothelium by approximately 10%. Simultaneously, DAF-FM fluorescence increased in all endothelial cells of the imaged field (Figure 2A) . The fluorescence increase was initiated after a temporal lag of approximately 5 minutes and continued over the 30-minute interval of pressure elevation (Figure 2B). The delayed onset of the fluorescence response was also preserved when endothelial stretch was reinitiated at the end of experiments (n = 3, data not shown). Calculation of NO production revealed a continuous and linear increase during pressure elevation (Figure 2C). Returning PLA to 5 cm H2O terminated the fluorescence increase and reduced NO production to baseline in less than 2 minutes.



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  		Figure 2. Capillary NO response to increased vascular pressure in rat lungs. (A) Representative fluorescence images of DAF-FM–loaded lung venular capillaries in situ. Images were obtained at left atrial pressure (PLA) of 5 cm H2O (left) and after 30 minutes of PLA elevation to 15 cm H2O. Vessel margins are depicted by line sketch and blood flow direction by arrow. Note increase of endothelial DAF-FM fluorescence and vasodilation after PLA elevation. (B) Representative profile of DAF-FM fluorescence in a single endothelial cell of lung venular capillary. DAF-FM fluorescence was determined in 10-second intervals at baseline (PLA of 5 cm H2O), during 30 minutes of PLA elevation to 15 cm H2O, and after return to baseline PLA. Fluorescence intensity (F) is expressed relative to its individual baseline (F0). F/F0 reflects NO production integrated over time. (C) Group data of capillary NO production are shown as 5-minute averages at baseline (PLA of 5 cm H2O), during 30 minutes of PLA elevation to 15 cm H2O, and after return to baseline PLA. NO production was quantified as fluorescence increase over 5-minute intervals relative to baseline {Delta} (F/F0). Line derived from linear regression analysis (p < 0.001, rs=0.91), n = 5 experiments, *p value less than 0.05 versus baseline (PLA = 5 cm H2O).

 
Because pretreatment with L-NAME abrogated this response, the fluorescence increase was attributable to NOS activation (Figure 3A) . In contrast, pretreatment with SOD did not attenuate the fluorescence response (Figure 3B). Thus, pressure-induced NO production and chromophore reaction did not seem to depend on endothelial O2- formation. When we perfused microvessels by Ca2+-rich HEPES buffer solution, the fluorescence response was identical to that of blood-perfused capillaries (Figure 3C). Hence, NO production was from the vessel wall, not circulating blood cells. Because vessel distension at constant flow reduces microvascular shear stress, we counteracted this effect by increasing lung perfusion by approximately 43%, i.e., from 14 to 20 ml/minute, during PLA elevation because capillaries dilated by approximately 12.7% and wall shear rate is inversely proportional to the third power of the diameter. Because the fluorescence response was essentially similar to that of lungs at constant flow (Figure 3D), the observed NO production was in response to pressure, not shear stress.



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  		Figure 3. Role of NOS, superoxide, blood cells, and shear rate in NO response of rat lung capillaries. Single experiment tracings show endothelial DAF-FM fluorescence at baseline (PLA of 5 cm H2O), during 30 minutes of PLA elevation to 15 cm H2O, and after return to baseline PLA. (A) The NOS inhibitor L-NAME (250 µmol/L) was added 10 minutes before PLA elevation. (B) The antioxidant enzyme SOD (2,000 U/ml) was added 10 minutes before PLA elevation. (C) Capillary perfusion by cell-free dextran HEPES was initiated 10 minutes before PLA elevation. (D) Total lung perfusion was adjusted from 14 to 20 ml/minutes during the interval of PLA elevation to stabilize estimated shear rate. Replicated four times each.

 
To characterize underlying signaling cascades, we blocked endothelial PI3K by Ly294002 and Wortmannin, respectively. Ly294002 inhibited the pressure-induced increase of endothelial DAF-FM fluorescence (Figure 4) but had no effect on vessel distension (diameter increase by 13.6 ± 2.7%). Similar results were obtained by Wortmannin (n = 5, data not shown). We conclude that increased vascular pressure stimulated NO production by activation of PI3K in lung endothelial cells.



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  		Figure 4. Effect of phosphatidylinositol-3-OH kinase (PI3K) inhibition by Ly294002 (10 µmol/L) on pressure-induced NO production in rat lung capillaries. (A) Representative fluorescence images of DAF-FM–loaded lung venular capillaries in situ. Images were obtained at PLA of 5 cm H2O (left) and after 30 minutes of PLA elevation to 15 cm H2O. Note pressure-induced vasodilation but no fluorescence increase. (B) Representative tracing of DAF-FM fluorescence at baseline (PLA of 5 cm H2O), during 30 minutes of PLA elevation to 15 cm H2O, and after return to baseline PLA. (C) Group data of capillary NO production are shown as 5-minute averages at baseline (PLA of 5 cm H2O), during 30 minutes of PLA elevation to 15 cm H2O, and after return to baseline PLA in the presence of the PI3K-inhibitor Ly294002 (black) and in control subjects (gray). NO production was quantified as fluorescence increase over 5-minute intervals relative to baseline {Delta} (F/F0). n = 5 experiments, *p value less than 0.05 versus control group. The PI3K inhibitor Wortmannin yielded similar results (n = 5, not shown).

 
Overventilation-induced NO Production
At elevated vascular pressure, responses attributable to direct compressive effects and those due to circumferential vascular stretch cannot be differentiated. Therefore, we applied a second model, in which circumferential stretch was induced by overventilation with negative pressure. Overventilation at end-inspiratory pressure of -25 cm H2O decreased lung perfusion pressure by 4.7 ± 0.4 cm H2O, whereas capillary transmural pressure increased by approximately 33%, resulting in circumferential vessel distension. Low-magnification fluorescence images of the surface of isolated-perfused mouse lungs were obtained at baseline and after 30 minutes of ventilation according to the randomized protocol. Overventilation increased DAF-FM fluorescence, which was predominantly localized in interalveolar septa where capillaries are abundant (Figures 5A and 5B) . Pretreatment with Ly294002 abrogated this response (Figure 5C). In control lungs ventilated at an end-inspiratory pressure of -10 cm H2O, no increase of DAF-FM fluorescence was detected. Hence, the effects of overventilation were essentially similar to responses to vascular pressure, in that both mechanical stimuli increased microvascular DAF-FM fluorescence and that the response was prevented by PI3K inhibitors.



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  		Figure 5. NO-production in overventilated mouse lungs. Representative fluorescence images of DAF-FM–loaded isolated-perfused mouse lungs at ventilation with end-inspiratory pressure (EIP) of -10 cm H2O (A; baseline) and after 30 minutes of overventilation at EIP of -25 cm H2O (B). (C) Group data of NO production in mouse lungs. Fluorescence intensity (F) after 30 minutes of either ventilation at EIP of -10 cm H2O (Co), overventilation at EIP of 25 cm H2O (OV), or overventilation in the presence of the PI3K-inhibitor Ly294002 (10 µmol/L; Ly/OV) was calculated relative to its individual baseline (F0). n = 6 experiments each, *p value less than 0.05 versus control group.

 
We considered the possibility that PI3K-inhibitors may directly influence lung mechanics. Overventilation more than doubled VT, and this response was not affected by Ly294002 (Figure 6) . Similarly, the overventilation-induced reduction in lung perfusion pressure was independent of the presence of Ly294002 (4.3 ± 1.1 cm H2O).



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  		Figure 6. VT in overventilated mouse lungs. Under baseline conditions, lungs were ventilated with an EIP of 10 cm H2O and an end-expiratory pressure (EEP) of -3 cm H2O. At time point 0, ventilation was continued at EIP of -10 cm H2O in control lungs (open circles, n = 6) or overventilation at EIP of -25 cm H2O was introduced in the presence (filled circles, n = 6) or absence (open squares, n = 6) of the PI3K-inhibitor Ly294002 (10 µmol/L). Differences between control and overventilation groups were statistically significant (p < 0.05).

 
Phosphorylation of Akt and eNOS
PI3K has been reported to stimulate endothelial NO production by phosphorylation of Akt and subsequent Akt-dependent eNOS phosphorylation (6). In mouse lungs, increased Akt phosphorylation was notable 15 minutes after initiation of overventilation (data not shown), and activation was markedly enhanced after 30 and 60 minutes as compared with control lungs (Figures 7A and 7B) . This effect was not attributable to de novo protein synthesis because the expression of nonphosphorylated Akt did not change (data not shown). Ly294002 effectively prevented the Akt phosphorylation (Figure 7C). Because Ly294002 had no effect on the phosphorylation of other kinases such as the mitogen-activated protein kinases Erk 1, Erk 2, and p38 (data not shown), this result was not attributable to a generalized effect of Ly294002 on kinase activities. Akt phosphorylation was localized to capillary endothelium but not to alveolar epithelium or macrophages (Figure 8 , top). In control lungs, phosphorylated Akt was barely detectable in capillaries, airway smooth muscle, alveolar epithelium, or macrophages. In accordance with the notion of a PI3K/Akt-dependent NO synthesis, phosphorylation of the downstream target of Akt, eNOS, was also prominent in capillary endothelial cells of overventilated lungs but not in control lungs (Figure 8, bottom).



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  		Figure 7. Activation of Akt-kinase in overventilated mouse lungs. Phosphorylation of the serine/threonine protein kinase Akt was analyzed by immunoblot using antibodies specific for the phosphorylated enzyme, P-Akt. Representative immunoblot (A) and densitometric data (B) from isolated-perfused mouse lungs ventilated for 60 minutes at EIP/EEP of -10/-3 cm H2O, and for another 30 minutes, 60 minutes, or 180 minutes at EIP/EEP of either -10/-3 cm H2O (control group; open circles) or -25/-3 cm H2O (overventilation, filled circles). n = 4 to 6 each, *p value less than 0.05 versus control group. (C) Representative immunoblot from at least three different experiments each, demonstrating inhibition of Akt phosphorylation by the PI3K blocker Ly294002. After baseline ventilation for 60 minutes, isolated mouse lungs were ventilated at EIP/EEP of -10/-3 cm H2O (control group) or -25/-3 cm H2O (overventilation; OV) for another 60 minutes in the presence (Ly) or absence of the PI3K inhibitor Ly294002 (50 µmol/L). Also shown is the solvent control (dimethyl sulfoxide [DMSO]) for overventilated lungs.

 


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  		Figure 8. Immunohistology of Akt and endothelial NOS (eNOS) phosphorylation in overventilated mouse lungs. Representative immunohistochemical stainings of phosphorylated Akt (top) and eNOS (bottom) in lungs ventilated for 60 minutes at EIP/EEP of -10/-3 cm H2O (control group) or -25/-3 cm H2O (overventilation). Note low labeling in alveolar septae of control lungs and higher staining intensities for P-Akt (arrows) and P-eNOS in the endothelia of alveolar capillaries in overventilated lungs.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Circumferential stretch of lung microvessels applied either by elevated vascular pressure or by negative pressure overventilation induced NO production in capillary endothelial cells. In both cases, NO production was prevented by PI3K inhibitors, and PI3K-dependent phosphorylation of Akt and eNOS phosphorylation were evident in overventilated mouse lungs. These findings represent novel evidence that independent of its cause, circumferential vascular stretch evokes PI3K-dependent endothelial NO production, presumably via the Akt signaling pathway.

In Situ Analysis of NO Production
We digitally imaged endothelial DAF-FM fluorescence to monitor NO production in perfused lungs. Diaminofluoresceins (DAFs) have been proposed as novel tools for quantification of NO because their fluorescence intensity increases linearly with the concentration of exogenous NO donors (18), and NO scavengers reduce this reaction in a concentration-dependent manner (19). The detection limit of the DAF-FM assay in cells has been estimated at 3 nmol/L (20), which exceeds the sensitivity of the standard Griess assay for nitrite by approximately 100-fold (21). NO-dependent nitrosation of diaminofluorescein, which itself is only weakly fluorescent, yields strongly fluorescent diaminofluorescein triazoles (20). Previous reports suggest that the nitrosating agent is not NO itself but rather dinitrogen trioxide, the product of NO autoxidation (22). NO reaction with O2-, however, yields peroxynitrite that may oxidize diaminofluoresceins to nonfluorescent, free radical intermediates that directly react with NO to form the fluorescent triazole derivative (23, 24). Thus, the fluorescence yield may be affected by the stoichiometry between O2- and NO (24). In the present study, the fluorescence response to both, a Ca2+-ionophore and vascular pressure elevation was unaffected by SOD, indicating that the chromophore reaction did not depend on the formation of O2- and/or peroxynitrite. However, although exogenous SOD has been demonstrated to enter cells in vitro and scavenge intracellular O2- (25), we cannot exclude the possibility that SOD access to the site of O2- production was insufficient in the isolated lung preparation. In addition, we monitored the fluorescence yield in response to an exogenous NO donor at the end of each experiment. Because the response to SNAP was similar in all experimental groups, we conclude that the sensitivity of the fluorometric in situ assay was not affected by the experimental protocols. The notion that DAF-FM fluorescence is a reliable detector of endothelial NO production is further supported by our findings that the fluorescence response was blocked by L-NAME and coincides with eNOS phosphorylation. Moreover, stable capillary fluorescence in the presence of L-NAME demonstrates that no significant photobleaching occurred within the observation period.

Stretch-induced NO Production
Because mechanical effects of pressure and stretch cannot be easily differentiated in situ, we applied two different models in which circumferential vascular stretch was applied either by elevation of vascular pressure or by negative pressure overventilation. Here, we use the term stretch to qualitatively indicate the consequences of an increase in the transmural pressure gradient. In biophysical terms, this would comprise changes in both circumferential stress and strain, the relation of which is determined by Young's modulus under the assumption that the material obeys Hooke's law (26). In both applied protocols, transmural pressure gradients increased, which will result in passive vessel distension and circumferential stretch because the adult pulmonary vascular bed shows no signs of autoregulation (27). Circumferential stretch was also directly evident as vessel dilation in imaged rat lungs subjected to pressure stress.

In both models, circumferential microvascular stretch increased NO production in isolated lungs. This effect was attributable to stimulation of the constitutively expressed eNOS because NO production was (1) evident in the absence of circulating blood cells, (2) blocked by the NOS-inhibitor L-NAME, and (3) associated with eNOS phosphorylation. At baseline, endothelial NO production was below detection limits of the DAF-FM assay, which is in accordance with the previously reported lack of pulmonary vasoactive effects of NOS inhibitors in healthy rat and mouse lungs (28).

In both models, endothelial responses were essentially similar, in that (1) NO production was not detectable at baseline, but (2) increased significantly when transmural pressure gradients were elevated, and (3) this response was abrogated by PI3K inhibitors. Hence, we conclude that endothelial NO production resulted from circumferential stretch of lung microvessels independent of absolute vascular or alveolar pressures. Of note, stretch-induced endothelial NO production was not only present in different protocols of transmural pressure elevation but also in two different species. This is in accordance with previous articles (elegantly reviewed in Reference 28), that report similar roles of NO in the regulation of lung vascular tone in mice and rats. The fluorescence response, however, was more pronounced in pressurized rats as compared with overventilated mouse lungs, which may be attributable to a stronger increase in the transmural pressure gradient during the protocol for vascular pressure elevation.

The temporal profile of the response was characterized by a lag of approximately 5 minutes in the onset of NO production, which contrasts with previously reported endothelial responses to flow cessation or adenosine triphosphate in which NO production is initiated within seconds (29, 30). Although mechanisms are unclear, this lag was neither attributable to delayed endothelial distortion because microvascular distension is completed in less than 15 seconds (31) nor to slow conversion of DAF-FM because SNAP increased fluorescence in less than 1 minute. The lag was also preserved under conditions of repetitive stretch and may serve as a lowpass filter because lung capillary endothelial cells are physiologically exposed to cyclic stretch by vascular pressure pulsations (32) and the respiratory cycle (33). The lag exceeds the time course of these fluctuations, thereby preventing excessive NO production. For analogous reasons, NO responses in both our models may have been essentially similar irrespective of the fact that vascular pressure induced a continuous stretch, whereas distension due to overventilation was cyclic.

Lung perfusion with HEPES buffer instead of blood ruled out a significant contribution of circulating blood cells as a potential source of NO. Yet, it may seem surprising that the fluorescence response during buffer and blood perfusion was almost identical, although free hemoglobin may act as a vascular sink for NO (34). However, the reaction of NO with intact red blood cells is three orders of magnitude slower than the reaction with equal amounts of free hemoglobin (35). Furthermore, NO scavenging by red blood cells is completely abrogated under flow conditions, presumably due to the additional formation of a skimmed, red blood cell–free plasma layer near the vessel wall (34).

We also considered the possibility that transmural pressure gradients may affect NO production by altered wall shear stress secondary to passive vessel distension. By raising total lung flow proportional to the third power of vessel diameter distension, we stabilized shear rate to the extent possible in an in situ preparation. Although we cannot completely rule out a possible contribution of minute shear alterations, we propose that the endothelial NO response to increased transmural pressure gradients in lung capillaries was predominantly attributable to circumferential stretch effects.

Endothelial Signal Transduction
Whereas eNOS is generally classified as a Ca2+/calmodulin-dependent enzyme (36), various agonists including vascular endothelial growth factor, insulin, insulin-like growth factor-1, or estrogen have recently been reported to stimulate its activity independent of Ca2+ (6, 37–39). In cultured endothelial cells stimulated by shear stress, Dimmeler and coworkers identified an alternative pathway of eNOS activation, which involves PI3K-dependent phosphorylation of the serine/threonine protein kinase Akt. Akt-dependent serine phosphorylation of eNOS increases the Ca2+-sensitivity of the enzyme, rendering its activity maximal at subphysiologic Ca2+ concentrations and facilitating sustained NO production (6). Stretch-induced endothelial signaling events are in accordance with this cascade because NO production was maintained over 30 minutes, inhibited by two different PI3K inhibitors, and associated with PI3K-inhibitable phosphorylation of Akt and eNOS. Although a direct causative relationship between Akt and eNOS phosphorylation or NO production could not be determined in situ due to the lack of suitable blockers, we speculate that eNOS was serine phosphorylated by the protein kinase Akt. Therefore, circumferential stretch may induce an endothelial reaction, which is essentially similar to the response to increased shear stress (6) and hence, constitutes an unspecific endothelial response to mechanical stimuli. Endothelial cell–matrix interactions are potential candidates for the initiation of this signaling cascade because circumferential strain acts on focal adhesion sites via ß1-integrins (40) and induces tyrosine phosphorylation of focal adhesion kinase (41), which in turn associates with its potential substrate, PI3K (42).

The relevance of our findings relates to the controversial question of whether mechanical strain results in deleterious lung vascular effects. Whereas there is growing evidence suggesting that endothelial stretch due to hydrostatic stress or overventilation can increase microvascular permeability (43, 44) and induce the expression of cytokines (10, 45) and adhesion molecules (7), endothelial release of NO has not been implicated with this scenario yet. The potential role of stretch-induced NO production in lung microvessels remains to be elucidated. In both our models, stretch-induced NO production or its inhibition had no apparent effect on vascular tone. This is in accordance with the notion that the healthy adult pulmonary circulation is typically fully dilated and that NOS inhibitors generally have no effect on resting pulmonary vascular tone in mice and rats (28). Of note, adult lungs are characterized by a linear pressure–flow relationship with no signs of autoregulation (27), whereas many systemic vascular beds exhibit a marked myogenic response, i.e., vascular smooth muscle constriction in response to increased vascular pressure (46). Hence, it may be of interest, although beyond the scope of this investigation, to test whether stretch-induced NO production is also a common feature in vascular beds with a strong autoregulatory response.

Due to its antiadhesive and antiaggregatory effects, stretch-induced NO may attenuate the proinflammatory effects of increased pressure (7) or overventilation (9). Furthermore, NO could be involved in strain-induced vascular remodeling, but its role remains obscure because it may both inhibit (47) and amplify (48) vascular smooth muscle proliferation. Finally, the rapid reaction of NO with activated oxygen species may contribute to the pathogenesis of stretch-induced lung injury, but further research is required to illuminate the relevance of endothelial NO production in the pulmonary pathophysiology after mechanical stretch.


   Acknowledgments
 
The authors thank Dörte Karp, Heike Kühl, and Sylvia May for excellent technical assistance.


   FOOTNOTES
 
Supported by grants Uh 88/2-4 and Uh 88/4-1 from the Deutsche Forschungsgemeinschaft.

This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Conflict of Interest Statement: W.M.K. has no declared conflict of interest; U.U. has no declared conflict of interest; T.G. has no declared conflict of interest; G.S. has no declared conflict of interest; A.K. has no declared conflict of interest; K.E. has no declared conflict of interest; C.M. has no declared conflict of interest; E.V. has no declared conflict of interest; S.U. has no declared conflict of interest.

Received in original form April 23, 2003; accepted in final form August 21, 2003


   REFERENCES
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 ABSTRACT
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 RESULTS
 DISCUSSION
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