Negative-Feedback Loop Attenuates Hydrostatic Lung Edema via a cGMP-Dependent Regulation of Transient Receptor Potential Vanilloid 4
Although the formation of hydrostatic lung edema is generally attributed to imbalanced Starling forces, recent data show that lung endothelial cells respond to increased vascular pressure and may thus regulate vascular permeability and edema formation. In combining real-time optical imaging of the endothelial Ca2+ concentration ([Ca2+]i) and NO production with filtration coefficient (Kf) measurements in the isolated perfused lung, we identified a series of endothelial responses that constitute a negative-feedback loop to protect the microvascular barrier. Elevation of lung microvascular pressure was shown to increase endothelial [Ca2+]i via activation of transient receptor potential vanilloid 4 (TRPV4) channels. The endothelial [Ca2+]i transient increased Kf via activation of myosin light-chain kinase and simultaneously stimulated NO synthesis. In TRPV4 deficient mice, pressure-induced increases in endothelial [Ca2+]i, NO synthesis, and lung wet/dry weight ratio were largely blocked. Endothelial NO formation limited the permeability increase by a cGMP-dependent attenuation of the pressure-induced [Ca2+]i response. Inactivation of TRPV4 channels by cGMP was confirmed by whole-cell patch-clamp of pulmonary microvascular endothelial cells and intravital imaging of endothelial [Ca2+]i. Hence, pressure-induced endothelial Ca2+ influx via TRPV4 channels increases lung vascular permeability yet concomitantly activates an NO-mediated negative-feedback loop that protects the vascular barrier by a cGMP-dependent attenuation of the endothelial [Ca2+]i response. The identification of this novel regulatory pathway gives rise to new treatment strategies, as demonstrated in vivo in rats with acute myocardial infarction in which inhibition of cGMP degradation by the phosphodiesterase 5 inhibitor sildenafil reduced hydrostatic lung edema.
- pulmonary edema
- vascular permeability
- vascular endothelium
- phosphodiesterase type 5 inhibitor
- nitric oxide
The pathogenesis of hydrostatic lung edema has been attributed predominantly to an imbalance in Starling forces, ie, fluid extravasation attributable to an increased hydrostatic or reduced oncotic pressure gradient across the microvascular barrier. This classic view has been challenged by the findings of Parker and Ivey in isolated perfused rat lungs, which demonstrated an increase in lung filtration coefficient (Kf) following elevation of left atrial pressure (PLA).1 This increase was attenuated by the β-adrenergic agonist isoproterenol, indicating that the Kf increase was not only caused by an enlarged vascular surface area but also resulted from an increase in vascular permeability that could be counteracted via the cAMP signaling pathway. The latter finding suggests that active endothelial responses may contribute critically to the formation of hydrostatic lung edema.
By use of real-time fluorescence imaging techniques, we recently identified such endothelial responses to an acute elevation in hydrostatic pressure in intact lung microvessels.2 In isolated perfused rat lungs, PLA elevation increases the cytosolic Ca2+ concentration ([Ca2+]i) in lung microvascular endothelial cells by Ca2+ influx via gadolinium-inhibitable cation channels3 and stimulates NO formation by activation of endothelial NO synthase.4 However, the role of these endothelial responses in the pathogenesis of hydrostatic lung edema is yet unclear.
Endothelial Ca2+ entry is a potential cause for endothelial retraction by activation of myosin light chain kinase (MLCK) and, thus, may increase endothelial permeability and promote edema formation.5 The mechanosensitive cation channel mediating the endothelial Ca2+ response to hydrostatic pressure remains to be identified. A potential candidate is the transient receptor potential vanilloid 4 (TRPV4), which has been characterized as a cation channel functioning in transduction of membrane stretch, shear stress, and direct mechanical activation (reviewed elsewhere6,7). Of note, TRPV4 is expressed in both micro- and macrovascular lung endothelial cells, and its pharmacological activation was recently shown to cause lung edema.8–10
The role of endothelial NO synthesis in the formation of lung edema has been a matter of controversy. NO may increase or alternatively reduce microvascular permeability, the outcome being determined by the specific conditions and vascular beds under investigation as well as the activity of interrelated signaling pathways and the presence of scavenger molecules.11
To address the regulation of vascular barrier function by endothelial responses in intact lungs, we combined real-time fluorescence imaging techniques with measurements of lung vascular permeability and edema formation. Here, we demonstrate that hydrostatic stress induces Ca2+ influx into endothelial cells via TRPV4 and increases lung vascular permeability by a Ca2+-dependent activation of MLCK. We describe a novel negative-feedback loop in this scenario that effectively limits progressive barrier deterioration via an intrinsic NO/cGMP-signaling pathway that directly regulates TRPV4.
Materials and Methods
Methodological details are provided in the online data supplement at http://circres.ahajournals.org. In brief, isolated lungs of rats or mice were prepared as previously described.3,4 At baseline, lungs were perfused with constant flow of 14 mL/min (rats) or 1 mL/min (mice) at PLA of 5 cmH2O (rats) or 3 cmH2O (mice) and pulmonary arterial pressure (PPA) of 10±1 cmH2O (both), respectively. In situ real-time fluorescence microscopy was performed as previously described.2 In isolated rat or mouse lungs, endothelial [Ca2+]i was determined by fluorescence imaging of the [Ca2+]i-sensitive dye Fura-2. Calcium concentration in endoplasmic stores (ER [Ca2+]) was estimated from the fluorescence intensity of Fura-2FF, which localizes to the endoplasmic reticulum.2 NO production was measured by fluorescence imaging of the NO-sensitive dye DAF-FM. The ratio of fluorescence intensity (F) relative to its individual baseline (F0) reflects cumulative NO production over time, whereas its first derivative ΔF/F0 determined in 5 minutes intervals reflects actual NO production.4 The lung vascular filtration coefficient (Kf) was determined as measure of pulmonary vascular endothelial permeability by dividing the rate of lung weight gain after a PLA increment by the resultant elevation in capillary pressure.12 Conventional whole-cell voltage clamp configuration was performed to measure transmembrane currents in single rat pulmonary arterial endothelial cells as described previously.13
Acute hydrostatic pulmonary edema was induced in vivo in a rat model of myocardial infarction by ligation of the left anterior descending coronary artery (LAD). Phosphodiesterase (PDE)5 inhibition with a single dose of sildenafil (1 mg/kg body weight IV) was initiated on LAD ligation, and lung edema and protein leakage were determined after 90 minutes. All data are presented as means±SEM.
Regulation of Lung Vascular Permeability
Elevation of PLA from 5 to 15 cmH2O increased lung filtration coefficient (Kf), indicating deterioration of the vascular barrier. This effect was attenuated by both Gd3+, which blocks the endothelial Ca2+ influx3 and the MLCK inhibitor ML-7 (Figure 1A). The fact that neither Gd3+ nor ML-7 altered lung perfusion pressures (data not shown) indicates that their effects on Kf did not result from changes in hemodynamics or vascular surface area. To analyze the role of endothelial NO synthesis in the regulation of vascular permeability, we blocked NO synthases by NG-nitro-l-arginine methyl ester (L-NAME) or administered the exogenous NO donor S-nitrosoglutathione (GSNO). GSNO attenuated the pressure-induced increase in Kf, whereas L-NAME amplified it, indicating a barrier-protective role for NO under hydrostatic stress (Figure 1B).
Regulation of Endothelial [Ca2+]i Response by NO
Because NO attenuated the Ca2+-dependent Kf increase, we tested whether NO may directly interfere with the endothelial [Ca2+]i response. PLA elevation for 30 minutes induced a progressive [Ca2+]i increase in lung capillary endothelial cells as demonstrated by real-time fluorescence imaging (Figure 2A). Inhibition of NO synthase by L-NAME enhanced the endothelial [Ca2+]i response, whereas it was attenuated by addition of GSNO (Figure 2B). The pressure-induced [Ca2+]i increase was likewise intensified by the soluble guanylate cyclase (sGC) inhibitor [1/H/-[1,2,4]oxadiazolo-[4,3-a] quinoxalin-l-one] (ODQ), whereas the sGC stimulator Bay 41-2272 or the cell-permeable cGMP analog 8Br-cGMP largely abrogated it (Figure 2C). These data demonstrate that NO limits the endothelial [Ca2+]i response to hydrostatic stress via activation of its downstream target sGC and subsequent formation of cGMP.
Next, we tested whether cGMP formation also reduces lung vascular permeability and edema formation. Whereas ODQ amplified the Kf increase following PLA elevation, Bay 41-2272 and 8Br-cGMP attenuated the response to an almost similar extent as the Ca2+ influx blocker Gd3+ (Figure 2D). Both 8Br-cGMP and Gd3+ reduced hydrostatic edema formation at PLA of 15 cmH2O, as revealed by lung wet/dry weight ratio analysis (Figure 2E). These findings indicate that NO limits hydrostatic edema formation by a cGMP-mediated attenuation of the endothelial [Ca2+]i response.
The attenuation of the endothelial [Ca2+]i increase by NO may establish a negative-feedback loop which limits not only the increase in Kf but also pressure-induced endothelial NO production itself. Imaging of the NO-sensitive dye DAF-FM in lung endothelial cells in situ revealed enhanced NO production at elevated PLA (Figure 3A), as previously reported.4 This effect was blocked when lungs were perfused with Ca2+ free buffer (Figure 3B) or in the presence of Gd3+ (data not shown). Consistent with the notion that cGMP attenuates the pressure-induced [Ca2+]i response, both Bay 41-2272 and 8Br-cGMP markedly diminished NO production in response to PLA elevation (Figure 3C). Thus, Ca2+-dependent NO production and cGMP-dependent attenuation of the [Ca2+]i response establish a negative regulatory feedback loop in lung vascular endothelial cells.
Regulation of Endothelial TRPV4
To further understand the mechanism by which NO/cGMP limits the endothelial [Ca2+]i increase, we sought to identify the relevant mechanosensitive Ca2+ channel. A putative candidate in this scenario is TRPV4, which is sensitive to both shear and stretch6,7 and on activation induces the formation of lung edema.8–10 Western blot analyses confirmed TRPV4 expression in rat lung homogenates and fresh rat lung endothelial cells (data not shown; findings in keeping with previously published results8), and immunohistochemical analyses localized TRPV4 to lung capillaries in the alveolar septa (data not shown; findings in keeping with previously published results8). Application of ruthenium red (RuR) blocked the increase of endothelial [Ca2+]i and attenuated the increase in Kf following PLA elevation (Figure 4A and 4B). Of note, RuR inhibited the Kf increase to a similar extent as the Ca2+-influx blocker Gd3+, suggesting that the pressure-induced endothelial [Ca2+]i response and the subsequent Ca2+-dependent Kf increase are mediated by TRPV4. Consistent with this notion, pharmacological activation of TRPV4 by 4αPDD increased Kf at baseline PLA of 5 cmH2O. Yet, following stimulation with 4αPDD, PLA elevation did not elicit a further increase in Kf, suggesting that the responsible mechanosensitive channels had already been activated (Figure 4B). To solidify this concept in a genetic loss-of-function model using TRPV4 gene-targeted mice,14 we assessed lung endothelial responses to acute hydrostatic stress in TRPV4-deficient (TRPV4−/−) mice versus wild-type littermates (TRPV4+/+). Pressure-induced endothelial [Ca2+]i increase (data not shown) and NO production (Figure 4C) were preserved in wild-type, but absent in TRPV4−/−, mice, solidifying the functional role of TRPV4 in lung microvascular mechanotransduction. Consistent with this notion, hydrostatic edema formation was drastically reduced in TRPV4−/− mice as compared with wild type (Figure 4D).
Next, we determined whether cGMP attenuates the endothelial [Ca2+]i response by preventing Ca2+ influx via TRPV4 or by stimulating Ca2+ uptake into endosomal stores. The cGMP analog 8Br-cGMP failed to increase ER [Ca2+], as determined by Fura-2FF fluorescence at baseline, as well as at elevated PLA (Figure 5A). Yet, 8Br-cGMP blocked the 4αPDD-induced endothelial [Ca2+]i increase to a similar degree as RuR (Figure 5B). The notion that cGMP inhibits endothelial TRPV4 is further supported by whole-cell patch-clamp recordings in pulmonary microvascular endothelial cells. As compared with untreated control cells, 4αPDD activated an inwardly rectifying current that reversed near +20 mV and was blocked by pretreatment with 8Br-cGMP (Figure 5C and 5D).
PDE5 Inhibition Attenuates Hydrostatic Lung Edema
Our finding that cGMP limits endothelial Ca2+ influx via TRPV4 and, thus, attenuates hydrostatic lung edema may give rise to new therapeutic strategies. Because PDE5 which rapidly degrades cGMP to GMP has recently been identified in pulmonary arterial endothelial cells,15 it may present a new target for lung barrier protection. PDE5 expression in lung vascular endothelial cells was confirmed by Western blotting and immunohistochemistry (data not shown). Sildenafil attenuated the increases of both endothelial [Ca2+]i and Kf in response to hydrostatic stress (Figure 6A and 6B), suggesting that PDE5 activity may regulate TRPV4 mediated endothelial Ca2+ influx in the lung. Sildenafil did not alter lung perfusion pressures consistent with the notion that the pulmonary vascular bed is already fully dilated under physiological conditions and lacks a myogenic response.16,17 Thus, sildenafil-induced changes in Kf were not attributable to the vasoactive but exclusively to the barrier-protective properties of the PDE5 inhibitor.
We tested the hypothesis that PDE5 inhibition may counteract lung vascular barrier deterioration and lung edema formation in vivo in a model of acute myocardial infarction. Within 90 minutes, LAD ligation resulted in considerable lung edema formation and protein leakage as evidenced by increased wet/dry lung weight ratio and Evans blue extravasation, respectively (Figure 6C and 6D), and decreased arterial oxygen tension (Figure 6E). Strikingly, administration of intravenous sildenafil at the time of LAD ligation effectively inhibited lung edema formation and protein leakage and increased arterial oxygenation (Figure 6C through 6E).
The present study identifies signaling cascades within endothelial cells that regulate the lung vascular barrier response to hydrostatic stress. Mechanosensitive TRPV4 channels mediate a pressure-induced Ca2+ influx that increases vascular permeability yet, at the same time, activates a negative-feedback mechanism that limits the endothelial [Ca2+]i response and, thus, protects the vascular barrier. This intrinsic feedback loop involves the pressure-induced synthesis of NO and cGMP, which in turn blocks Ca2+ entry via TRPV4. Hence, pharmacological elevation of endothelial cGMP may provide a new strategy in treatment of hydrostatic lung edema.
Identification of this signaling cascade was facilitated by a combination of real-time imaging with Kf measurements in the isolated perfused lung. Real-time imaging has recently developed into a powerful technique to reveal intra- and intercellular signaling pathways at the alveolocapillary barrier.2 Kf determination stands a robust assessment of lung microvascular barrier properties, provided that nonspecific factors that may affect the weight gain can be effectively excluded. In the present study, potential influences of lymphatic drainage, vascular compliance, and epithelial barrier properties were accounted for by the specific setup and protocol for Kf measurements, as outlined in the online data supplement. Similarly, vasoactive responses did not contribute to our findings, because the pulmonary vasculature is fully dilated at rest independent of endogenous NO synthesis and lacks a myogenic response.17,18 Accordingly, none of the applied pharmacological agents caused a change in perfusion pressures in the isolated lung preparation.
Ca2+-Dependent Regulation of Lung Vascular Permeability
The endothelial [Ca2+]i response and the increase in lung Kf were inhibited by RuR, whereas the TRPV4 activator 4αPDD mimicked the response to pressure stress, therefore suggesting a role for TRPV4 in the endothelial mechanotransduction and subsequent edema formation. Yet, it has to be considered that RuR blocks several members of the TRPV channel subfamily, as well as L-type Ca2+ channels19 and the mitochondrial Ca2+ uniporter.20 Similarly, 4αPDD is not absolutely specific for TRPV4 because it may, eg, enhance the biological activity of active phorbol esters such as phorbol 12-myristate 13-acetate.21 To rule out these nonspecific effects, we robustly confirmed the functional role of TRPV4 in mechanotransduction and permeability regulation by demonstrating the absence of a pressure-induced endothelial [Ca2+]i response and the attenuation of hydrostatic lung edema in TRPV4-deficient mice. Because TRPV4 activation increases endothelial permeability preferentially in lung capillaries,8 changes in Kf and imaged endothelial [Ca2+]i responses likely occurred in the same vascular compartment. Hydrostatic stress induced a sustained elevation of lung endothelial [Ca2+]i via TRPV4, and inhibition of Ca2+ influx or MLCK attenuated the concomitant Kf increase, consistent with the notion of a Ca2+-dependent contraction of endothelial myofibrils.
NO-Dependent Negative-Feedback Loop
NO has been proposed to either increase or decrease microvascular permeability depending on the specific experimental conditions, species, and vascular bed studied.11 In the lung, the microvascular barrier is more permeable than in systemic vascular beds, as exemplified by the fact that hydraulic conductivity is 10 times smaller and the reflection coefficient is considerably higher in skeletal muscle as compared with lung.22 In the lung hydrostatic stress, both endogenous NO formation and addition of an exogenous NO donor proved barrier protective in that L-NAME amplified the permeability increase, whereas GSNO attenuated it. Real-time fluorescence imaging demonstrated that the barrier-protective effect of NO was attributable to an attenuation of the endothelial [Ca2+]i response and mediated by cGMP. Administration of a cGMP analog blocked the better part of the pressure-induced increase in wet/dry lung weight ratio and, thus, had similar effects as the Ca2+ channel inhibitor Gd3+. Taken together, these findings demonstrate that NO can reduce hydrostatic lung edema via a cGMP-dependent attenuation of the endothelial [Ca2+]i increase.
NO and cGMP may attenuate endothelial [Ca2+]i signaling by inhibition of extracellular Ca2+ entry or by regulation of Ca2+ uptake into and release from endosomal stores.23,24 Measurements of ER [Ca2+] and studies on the regulation of TRPV4 indicate that cGMP impaired the endothelial [Ca2+]i response primarily by inhibition of Ca2+ influx. The obvious interpretation is that endothelial signal transduction cascades established a negative-feedback loop in which hydrostatic pressure triggers TRPV4-mediated endothelial Ca2+ influx, which stimulates NO formation by endothelial NO synthase. NO activates sGC to form cGMP, which in turn limits the endothelial Ca2+ influx. The notion of a closed-loop negative control was confirmed by the fact that sGC stimulation or 8Br-cGMP both attenuated endothelial NO formation.
Negative-feedback control of NO biosynthesis has been reported previously, in that NO and NO-donor agents can directly inhibit endothelial NO synthase in a noncompetitive fashion.25 NO may also downregulate endothelial NO synthase expression via a cGMP-mediated pathway.26 Hence, negative feedback regulation of NO synthesis can occur at the level of direct enzyme inhibition, protein expression, or, as demonstrated in the present study, posttranslational regulation.
Role of TRPV4 in the Regulation of Vascular Permeability
TRPV4 channels are expressed in pulmonary arterial and lung microvascular endothelial cells, and TRPV4 activation by 4αPDD has been shown to increase endothelial permeability in the presence of extracellular Ca2+.8 Our data are in agreement with these findings in that 4αPDD induced a marked rise in endothelial [Ca2+]i concomitant with an increase in vascular permeability. After stimulation with 4αPDD, hydrostatic stress had no additional effect on endothelial permeability, suggesting that underlying sensory signaling pathways were already activated.
The present study identified cGMP as a critical regulator of TRPV4 in lung microvascular endothelial cells. As shown by patch-clamp recordings in pulmonary microvascular endothelial cells and by real-time imaging in intact lung microvessels, the 4αPDD-induced inward current and [Ca2+]i increase were both blocked by 8Br-cGMP. Although regulation of the canonical transient receptor potential isoform 3 (TRPC3) by cGMP has been described,27 the present findings are the first to report regulation of a TRPV channels by cyclic nucleotides.
PDE5 Inhibition As Therapeutic Strategy in Hydrostatic Lung Edema
The identification of a cGMP-dependent feedback loop in the regulation of endothelial permeability may give rise to new therapeutic interventions for the prevention or treatment of hydrostatic lung edema. Endogenous activators or pharmacological stimulators of sGC such as NO or Bay 41-2272 may strengthen the vascular barrier by attenuating the endothelial [Ca2+]i response to hydrostatic stress yet, at the same time, may promote lung edema because cGMP blocks fluid absorption by alveolar epithelial cells.28 Inhibition of PDE5 may be a more advantageous concept in this context, because PDE5 is expressed in lung endothelial but not in alveolar epithelial cells.15
In an in vivo model of acute myocardial infarction, LAD occlusion caused hydrostatic lung edema and protein extravasation as previously described.29 PDE5 inhibition by sildenafil reduced lung fluid accumulation by approximately 80% and reconstituted arterial oxygenation to physiological levels. In parallel, PDE5 inhibition decreased protein leakage as determined by Evans blue extravasation, suggesting that the protective effect of sildenafil was primarily attributable to a reduction in endothelial permeability. This notion is supported by our findings in isolated perfused rat lungs in which sildenafil attenuated the pressure-induced increase in endothelial [Ca2+]i and permeability. Vasoactive effects of sildenafil may potentially have been conducive to its antiedematous properties, yet this contribution can be considered small because sildenafil did neither affect mean arterial pressure in vivo nor alter pulmonary hemodynamics in isolated lungs. Similarly, the recently described positive inotropic effects of sildenafil are expected to play a minor role in this setting, because expression of PDE5 in cardiomyocytes is negligible at baseline and only increases in chronic heart failure.30 Sildenafil may also potentially alter lymphatic tone in vivo, yet, again, this would only have a minor impact on hydrostatic edema, because lymphatic drainage from the lung is primarily driven by respiratory mechanics rather than lymphatic contractions.31 PDE5 inhibition may present a promising new strategy to attenuate lung edema in acute hydrostatic stress. This approach is even more attractive because sildenafil has also been demonstrated to ameliorate ischemia/reperfusion injury32 in acute myocardial infarction.
We thank Ursula Hilse for technical assistance.
Sources of Funding
This study received financial support from the Deutsche Forschungsgemeinschaft (Ku1218/4-1); the European Commission under the 6th Framework Programme (contract no. LSHM-CT-2005-018725, PULMOTENSION); Pfizer GmbH, Karlsruhe, Germany; and the Kaiserin-Friedrich Foundation, Berlin, Germany.
Original received July 19, 2007; resubmission received November 28, 2007; revised resubmission received January 28, 2008; accepted February 26, 2008.
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