cGMP protects endothelial cells from hypoxia-mediated leakiness

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European Journal of Cell Biology 87 (2008) 147–161 www.elsevier.de/ejcb

Nitric oxide/cGMP protects endothelial cells from hypoxia-mediated leakiness Gopi Krishna Kollurua, K.P. Tamilarasana, Arun Stephen Rajkumarb, S. Geetha Priyaa, Megha Rajarama, Niyas K. Saleema, Syamantak Majumdera, B.M. Jaffar Alib, G. Illavazaganc, Suvro Chatterjeea, a

Vascular Biology Laboratory, AU-KBC Research Centre, MIT Campus, Anna University, Chennai 600 044, Tamil Nadu, India Optical Nano-Manipulation Group, AU-KBC Research Centre, Anna University, Chennai, Tamil Nadu, India c Defence Institute of Physiology and Allied Sciences, DIPAS, Delhi, India b

Received 14 July 2007; received in revised form 6 October 2007; accepted 8 October 2007

Abstract Leakiness of the endothelial bed is attributed to the over-perfusion of the pulmonary bed, which leads to high altitude pulmonary edema (HAPE). Inhalation of nitric oxide has been successfully employed to treat HAPE patients. We hypothesize that nitric oxide intervenes in the permeability of the pulmonary macrovascular endothelial bed to rectify the leaky bed under hypoxia. Our present work explores the underlying mechanism of ‘hypoxia-mediated’ endothelial malfunction by using human umbilical cord-derived immortalized endothelial cells, ECV-304, and bovine pulmonary artery primary endothelial cells. The leakiness of the endothelial monolayer was increased by two-fold under hypoxia in comparison to cells under normoxia, while optical tweezers-based tethering assays reported a higher membrane tension of endothelial cells under hypoxia. Phalloidin staining demonstrated depolymerization of F-actin stress fibers and highly polarized F-actin patterns in endothelial cells under hypoxia. Nitric oxide, 8-Br-cGMP and sildenafil citrate (phosphodiesterase type 5 inhibitor) led to recovery from hypoxia-induced leakiness of the endothelial monolayers. Results of the present study also suggest that ‘hypoxia-induced’ cytoskeletal rearrangements and membrane leakiness are associated with the low nitric oxide availability under hypoxia. We conclude that nitric oxidebased recovery of hypoxia-induced leakiness of endothelial cells is a cyclic guanosine monophosphate (cGMP)dependent phenomenon. r 2007 Elsevier GmbH. All rights reserved. Keywords: Hypoxia; Endothelial cells; Nitric oxide; Actin filaments; cGMP; Sildenafil

Abbreviations: ARDS, acute respiratory distress syndrome; BPAEC, bovine pulmonary aortic endothelial cells; cGMP, cyclic guanosine monophosphate; DAF-2DA, diaminofluorescein diacetate; DEAN, diethylamine NONOate; DMEM, Dulbecco’s modified Eagle’s medium; EC, endothelial cells; ECV 304, human umbilical vein endothelial cells; eNOS, endothelial nitric oxide synthase; FBS, fetal bovine serum; HAPE, high altitude pulmonary edema; L-NAME, nitro-L-arginine-methyl ester; NO, nitric oxide; NOS, nitric oxide synthase; ODQ, 1H-[1, 2, 4]oxadiazolo[4, 3-a] quinoxalin-1-one; PBS, phosphate-buffered saline; sGC, soluble guanylyl cyclase; SNP, sodium nitroprusside. Corresponding author. Tel.: +91 44 2223 4885x48; fax: +91 44 2223 1034. E-mail address: [email protected] (S. Chatterjee). 0171-9335/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejcb.2007.10.001

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Introduction Nitric oxide (NO), an endogenously produced autacoid, modulates a variety of biological functions including plasma membrane permeability and paracellular routes (Ignarro, 2002a, b; Moncada, 1997, 1999; Sessa, 2004). NO protects the lungs from increased permeability during injury in a number of species (Poss et al., 1995; Garat et al., 1997; Searles et al., 2004). Benzing et al. (1998) demonstrated that inhaled NO lowers capillary pressure in patients with adult respiratory distress syndrome. It has been suggested that NO serves a potential protective role in pulmonary liquid balance by reducing microvascular pressure, thereby reducing fluid filtration (Mundy and Dorrington, 2000). High altitude pulmonary edema (HAPE) is a hydrostatic edema in the presence of normal left atrial pressure with non-inflammatory high permeability leakage of the alveolocapillary barrier and mild alveolar hemorrhage (Sartori et al., 2007). Uneven hypoxic pulmonary vasoconstriction has been proposed to expose parts of the pulmonary capillary bed to high pressure and vascular injury in HAPE (Hultgren, 1978; Maggiorini et al., 2001). Autopsy findings in patients dying of HAPE also demonstrated a protein-rich alveolar fluid (Arias-Stella and Kruger, 1963). In the rat model, inhaled NO improves survival from HAPE (Omura et al., 2000). Several studies have suggested that inhaled NO improves oxygenation in pulmonary edema (Anand et al., 1998; Kinsella et al., 1997; Perrin et al., 2006; Roberts et al., 1997; Scherrer et al., 1996; Wessel et al., 1997). Work of Mundy and Dorrington (2000) showed that inhibition of NO synthesis augments pulmonary edema in isolated perfused rabbit lung. Scientists from the Defence Research and Development Organization (DRDO), India, successfully used gaseous NO to treat soldiers who suffered from HAPE symptoms in the Himalayas (Himashree et al., 2003). However, the cellular and molecular basis of NO-based recovery from HAPE is not completely understood. Bronchoalveolar lavage studies in subjects with HAPE show a significant elevation of protein content, suggesting that the vascular barrier to protein movement has been decreased (Maggiorini et al., 2001). Work of Kayyali et al. (2002) showed that hypoxia activates MAPK-activated protein kinase MK2 in connection with HSP27 phosphorylation and reorganization of the actin cytoskeleton. Although these observations partially explain the hypoxia ‘downstream’ effects, the relationship between hypoxia-mediated reorganization of the actin network and ‘‘membrane leakiness’’ is not explored yet. In the present work, we show that delivery of NO recovers endothelial cells (ECs) from hypoxia-induced leakiness by reorganizing the actinbased cytoskeleton and by reducing hypoxia-induced membrane tension. Hypoxia-mediated alterations in

ECs were prevented by treatment with sildenafil and cyclic guanosine monophosphate (cGMP) analogs under hypoxic conditions, indicating a major role of the NO/cGMP pathway in the hypoxia-induced alteration of endothelial morphology, permeability and cytoskeletal pattern.

Materials and methods Materials Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Hi-Media, Mumbai, India. Fetal bovine serum (FBS) was from Invitrogen Life technologies. Phalloidin-Alexa Fluor 568 (phalloidin) and diaminofluorescein diacetate (DAF-2DA) from Molecular probe, Oregon, USA. Sodium nitroprusside (SNP), propidium iodide, cytochalasin D, and L-NAME were purchased from Sigma Chemical Co (St. Louis, MO). Polystyrene microspheres were purchased from Polysciences, Inc. (Warrington, PA). Diethylamine NONOate (DEAN) and guanosine-30 50 -monophosphate 8-bromo-sodium salt (8-Br-cGMP) were obtained from EMD Biosciences, Inc., California, USA. Dr. Vijay Shah, GI Research Unit, Mayo Clinic, Rochester kindly provided the eNOS-GFP plasmid. All other chemicals were of reagent grade and were obtained commercially.

Cell culture Human umbilical cord-derived endothelial cells (ECV 304) with additional features of T24 cells were cultured in DMEM supplemented with 10% FBS (v/v) and 1% (w/v) penicillin and streptomycin. Bovine pulmonary aortic endothelial cells (BPAEC) were isolated from the pulmonary artery of freshly killed animals. At the slaughterhouse, large blood vessels (pulmonary artery) were collected aseptically and placed in phosphate-buffered saline (PBS) (pH 7.4). In the laboratory, vessels were washed with PBS. Connective tissue and fat were removed aseptically. Next, ECs were harvested as described elsewhere (Ryan, 1984). Isolated cells were confirmed as ECs by using antibodies against endothelial markers, eNOS and factor VIII, respectively. The cells were used for experiments till passage 6.

Transfection ECV 304 cells were grown in a 12-well plate for at least 18–24 h prior to transfection. The cells were then transfected with plasmid vector encoding eNOS-GFP using the calcium phosphate method as described elsewhere (Chatterjee et al., 2002).

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In vitro hypoxia model ECs in the absence or presence of added compounds were placed into a hypoxia chamber with an inlet and outlet for purging the required percentage of O2. As a control, the cells were incubated under normoxic conditions for the same length of time. For all the hypoxia experiments, a gas mixture (10% O2+90% N2) was purged in the hypoxia chamber for 2 h. The dosage of hypoxia was standardized by varying the O2 concentrations and duration of treatments.

Trypan blue permeability assay We measured the trans-cellular permeability of EC plasma membranes by using a modified trypan bluebased permeability assay (Grankvist et al., 1979). EC overexpressing eNOS-GFP were grown on cover glasses and incubated under normoxia and hypoxia for 30 min, 1 and 2 h at 37 1C/5% CO2. Next, the cells were mounted in a live-cell chamber. Phase-contrast images with 8 s time-lapse were taken at 40  magnification after addition of trypan blue (0.004%). The rate of inclusion of trypan blue dye was calculated from the nuclear color index (color intensity).

Permeability assay In this manuscript, ‘‘leakiness’’ denotes to net EC monolayer permeability, which is comprised of paracellular and trans-cellular vascular solute fluxes. Effects of NO, L-NAME, sildenafil citrate, and 8-Br-cGMP on the permeability of endothelial monolayers under normoxia and hypoxia, respectively, were studied. In brief, a half million EC were seeded on the collagencoated polycarbonate membrane in the permeability chamber and incubated for 24 h at 37 1C/5% CO2. The permeability chamber with EC in the absence or presence of added compounds was kept under hypoxia and normoxia, respectively, for 2 h and incubated at 37 1C/5% CO2. Trypan blue (0.004%) was added in the upper half of the permeability chamber, and incubated for 1 h at 37 1C/5% CO2. The solution from the lower chamber was collected to measure the optical density at 580 nm using a Varian Cary 4000 UV–vis spectrophotometer.

Fluorescence microscopy EC were cultured on cover glasses in 12-well plates till they reach 40% confluence before starting the experiments. The cells were then incubated under hypoxia and normoxia with and without 500 mM SNP in 37 1C/5% CO2 for 30 min, 1 and 2 h, respectively. The cover slips were washed gently with PBS, and cells were fixed in 2%

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formaldehyde (freshly prepared from paraformaldehyde) for 7 min, permeabilized with 0.1% Triton X-100 for 2 min and incubated with phalloidin (0.5 mM final concentration) for 1 h. Specimens were viewed under a NIKON TE2000-U fluorescence microscope at 560 nm emission. Photographs were taken with an Andor CCD camera.

Single cell migration assay Cell migration was assessed using the wound healing method. One million ECV 304 cells in 2 ml DMEM/10% FBS were seeded in a 35-mm dish. Twenty-four hours later, when the cells reached confluence, a linear wound was created by scratching the monolayer with a 1 mm wide sterile plastic scraper. Cells were washed with PBS, treated with and without SNP (500 mM) and incubated for a fixed time period (2 h unless otherwise stated) under normoxia and hypoxia. Bright-field images were taken with 4  and 40  magnifications under an inverted bright-field microscope. The rate of wound healing was quantified from the images using Scion Image, Release Alpha 4.0 3.2 and Adobe Photoshop version 6.0.

Measurement of membrane tension by optical tweezers The optical tweezers set-up was built around an inverted microscope (TE-2000U, Nikon Corporation, Japan). A 1.5 W 1064 nm Nd:YAG laser (Laser Quantum, UK) focused through an oil-immersion objective (Plan Apo, N.A. 1.4) was used to form an optical trap. The motion of trapped particles was tracked by backscattering using a 635 nm diode laser (Coherent, USA). Stiffness of the trap (was around 0.008 pN/nm) calibrated by power spectrum analysis. However, for accuracy of measurement, the trap stiffness was calculated each time before the start of an individual experiment. ECV304 ECs treated under normoxia and hypoxia were used for optical tweezers experiments. Prior to experiments, the medium was replaced with PBS containing 2-mm uncoated polystyrene spheres. Using the optical trap, membrane tethers were drawn by pressing the bead against the cell surface for 5 s and pulling out at the rate of 0.5 mm/s and held static at a length of 5 mm. After equilibrium for 20 s, the tether force, F0 (Dai and Sheetz, 1998) and time series of bead fluctuations were measured. The effective stiffness, keff, of the membrane-trap system was computed from power spectral analysis of the time series. keff is a linear combination of the trap stiffness ktrap, and membrane tension Tm given by 1/Tm ¼ 1/ktrap+1/keff (Evans et al., 2005). From this relation, the membrane tension was

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calculated. All membrane tension experiments were carried out at a height of 5 mm above the cover slip. The necessary corrections due to the height of the bead from the surface were made.

from the cells was enhanced with time upon calcium stimulation in normoxia while it remained unchanged in hypoxia-treated EC. A control experiment using L-NAME proved that calcium-induced NO production was L-NAME inhibitable (data not shown). The DAF assay for NO production detected a basal level of L-NAME-inhibitable NO production, which is two-fold higher than the non-specific NO production from cells transfected with empty vector. However, the basal level of DAF-NO fluorescence intensity is four-fold lower than the ionomycin-stimulated one (data not shown). Next, we focused on the sub-cellular localization pattern of eNOS-GFP under hypoxic condition. eNOS-GFP was observed at the plasma membrane and in the perinuclear region of in normoxic EC (Fig. 1B, left panel). These cells were stably transfected with eNOSGFP and not stimulated with calcium ionophore. A treatment of 2 h hypoxia induced relocalization of eNOS-GFP from the plasma membrane to the perinuclear region of the cells (Fig. 1B, right panel). A diffuse fluorescence from cytosol further indicates that eNOS-GFP from the plasma membrane pool leached into the cytosol (Fig. 1B).

NO-imaging with DAF-2DA eNOS-transfected cells cultured on cover glasses in 12-well plates were incubated under hypoxia and normoxia at 37 1C/5% CO2 for 2 h. Cells were washed twice with PBS and then loaded with 200 ml DAF-2DA (10 mM) and incubated for 5 min. After another incubation of 5 min with 1 mM calcium ionophore, cells were stimulated with 0.5 mM calcium chloride and effects were observed under a NIKON TE-2000 inverted fluorescence microscope at 515 nm. Images were taken with an Andor CCD camera. Fluorescence intensity of the cells was calculated by using the image analysis module of Adobe Photoshop 7.0.

Statistical analysis All experiments were performed in triplicate unless otherwise specified. Data are presented as mean+SE. Data was analyzed using t-test, one-way and two-way ANOVA as appropriate. P-values p0.05 were selected as the criterion for a statistically significant difference.

Hypoxia promotes leakiness in ECs Trypan blue was used to determine the viability and permeability of the cells. Healthy cells exclude the blue dye, whereas damaged cells take it up (Lagrange et al., 1999; Boiadjieva et al., 1984). In our experimental setup, trypan blue inclusion experiments were performed well within the physiological range for measuring EC permeability in live cells by restricting the time of experiments to 20 s maximum, and also by minimizing the final concentration of trypan blue, which was 40 mg/ml. This concentration is 100-fold lower than that used in viability assays. Therefore, our trypan blue

Results Hypoxia attenuated NO production from eNOStransfected ECs The NO-sensitive fluorescence dye DAF-2DA was used to monitor NO availability in live eNOS-transfected EC. Fig. 1A demonstrates that DAF fluorescence

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Fig. 2. NO reverses hypoxia-mediated leakiness in EC. (A) Trypan blue inclusion in a single EC under normoxia and hypoxia was monitored using time-lapse microscopy. Results are means7SE from at least five experiments. Total number of cells used for the study was 15. * and @ Po0.05 compared to control and hypoxia, respectively. (B) Trypan blue permeability assay using a monolayer permeability assay chamber. * and # Po0.05 compared to control and hypoxia, respectively. (C) eNOS-GFP overexpressing (eNOS+ve) EC show reduced hypoxia-induced permeability (trypan blue inclusion) when compared to vectortransfected controls (eNOSve). *Po 0.05.

experiment measures mainly the rate of inclusion, not the viability of the cells. Microscopic photometry using single live EC showed that normoxic cells were resistant to the dye, whereas the nuclei of hypoxia-treated cells accumulated trypan blue by a saturable process (saturation level reached within 20 s of trypan blue treatment; Fig. 2A). EC form a monolayer in vivo to create a functional endothelial bed in the lumen of blood vessels. Therefore, a robust cell–cell communication network maintains the physical intactness of the layer (van Nieuw Amerongen and van Hinsbergh, 2002). We re-created the monolayer of EC on a collagen-coated polycarbonate membrane to understand the effects of hypoxia on the permeability of the endothelial monolayer. Accumulation of trypan blue dye in the lower chamber depends on the sensitivity of the EC monolayer to trypan blue. Hypoxia led to an about twofold increase in entry of trypan blue into the lower chamber (Fig. 2B). Phase-contrast images along with propidium iodide staining showed no sign of physical damage and disintegration of the EC monolayer after hypoxia treatments (not shown).

NO protects EC from hypoxia-mediated leakiness NO was delivered via SNP to the hypoxia-treated EC to test the effects of NO on hypoxia-induced leakiness of the cell membrane. A dose of 500 mM SNP completely blocked the hypoxia-dependent inclusion of trypan blue in the EC (Fig. 2A). A similar protective effect of SNP was observed in the experimental set-up, in which trypan blue dye was allowed to pass across the EC monolayer. EC monolayers incubated with 500 mM SNP during hypoxia treatments demonstrated complete recovery from the hypoxia-mediated leakiness of the EC monolayer (Fig. 2B). A parallel set of experiments was carried out with DEAN, another NO donor, to ascertain the specificity of NO effects. Both SNP and DEAN showed similar effects on the permeability without any interference with cell morphology and viability (data not shown). A 2-h hypoxia treatment of eNOS-negative (empty vector) EC reached a saturation level of trypan blue inclusion at the 16th second while the cells overexpressing eNOS resisted trypan blue

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inclusion under hypoxia treatment (Fig. 2C). A 2-h hypoxia treatment introduced changes in the morphology of EC. Phase-contrast microscopy revealed that hypoxia increases the number of rounded cells, which is significantly different from the normal cobblestone-like appearance of EC in monolayer, by 150% in comparison to that of normoxia treatments (Fig. 3A). EC were transiently transfected with eNOS-GFP and subjected to hypoxia (10% oxygen) for 2 h. Varying levels of eNOSGFP expression were evident (Fig. 3B, right panel). The Normal

cell denoted as 1 shows maximum level of expression of eNOS-GFP while 2 and 3 show lower levels of expression, and 4 did not express any eNOS-GFP (Fig. 3B). When these cells were studied for morphology it was observed that the cells with maximum eNOS-GFP expression were morphologically intact with welldefined membrane projections while cell numbers 2 and 3 were tending to be rounded off. Cells without eNOS-GFP showed a smoother surface and long extensions (Fig. 3B, white arrow, number 5, left panel). Round

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C Fig. 3. NO protects EC from morphological changes under hypoxia. (A) EC were treated with SNP under normoxia and hypoxia and analyzed for changes in morphology (‘‘normal’’ stellate-shaped vs. ‘‘round’’ cells. * and # Po0.05 compared to normoxia. Y and  Po0.05 compared to hypoxia. (B) Distinct levels of eNOS-GFP expression in eNOS-GFP-transfected EC under hypoxia. EC with normal morphology have a higher level of eNOS-GFP expression and normal cell membrane protrusions (1, black arrows). A change in morphology with anomalous membrane projections was observed with lower eNOS-GFP expression (2 and 3). The cell sans eNOS-GFP expression (4) demonstrated unhealthy characteristics of EC such as stress-associated slender extensions of the cell membrane (white arrow, 5). (C) Distinct morphological changes in L-NAME-treated EC under hypoxia. EC with eNOS expression could withstand the effect of L-NAME in normoxic and hypoxic conditions, while the morphology of the cells deteriorated without eNOS expression. EC without eNOS expression and with L-NAME treatment were the most affected. Data are means7SE (n ¼ 8). At least 200 cells were counted for each experiment. * Po0.05 compared to normoxia/eNOS(+)/L-NAME(+); # Po0.001 compared to normoxia/eNOS()/L-NAME(); @ Po0.05 compared to normoxia/eNOS()/L-NAME(+). (D) EC under normoxia and hypoxia exhibit different morphologies and actin filament (phalloidin staining) patterns.  and # Po0.05 compared to normoxia. y and Y Po0.05 compared to hypoxia. (E) SNP (500 mM) treatment restricts the hypoxia-mediated increase in EC lamellipodia (Lam) formation. Filo: Filopodia. Data are means7SE (n ¼ 8). At least 200 cells were counted for each experiment. * and # Po0.05 compared to normoxia and hypoxia, respectively.

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In a further attempt to strengthen our observation that NO protects EC from hypoxia-induced effects, we treated EC with L-NAME. L-NAME treatment reduced the protective effects of eNOS partly. L-NAME also showed non-specific effects on the morphology of eNOS-negative cells under normoxia. A sharp fall, in the number of morphologically intact cells, was recorded in the eNOS-negative cell population (Fig. 3C).

SNP protects EC from hypoxia-induced alterations in morphology A 500-mM SNP treatment prevented the EC from being rounded off under hypoxia (Fig. 3D). A trypan blue-based viability study showed (data not shown) that rounded off cells are alive. Bright-field observation along with phalloidin staining revealed that the population of EC consists of four sub-populations (Fig. 3D). (A) A normal morphology with stress fibers and

irregular membrane projections. Here, ‘‘normal’’ denotes the usual stellate-shaped EC in a cobblestone-like endothelial monolayer. (B) A ‘‘star’’-shaped morphology with intense phalloidin staining pattern at the broader growth cones. (C) A ‘‘boat’’-like morphology with highly polarized phalloidin-stained areas at the concave side of the boat, and (D) A ‘‘round’’ cell with regularly spaced phalloidin-stained projections in the membrane. Normoxia-treated cells were predominantly (80%) ‘‘normal’’ cells. Hypoxia sharply altered the normal distribution pattern of EC. The number of the cells with normal morphology was reduced to 20% while the number of round and boat cells was increased to 50% and 30%, respectively. No significant change in the number of star-shaped cells was observed across the treatments. When hypoxia-treated cells were incubated with 500 mM SNP, a partial recovery of the morphology was noted (Fig. 3D). A 60% increase in the number of normal cells was observed along with a drop in the number of boat and round cells.

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NO perturbs hypoxia-driven membrane changes A recent work by Vogel et al. (2007) showed that hypoxia/VEGF-induced hyper-permeability can be mediated by activation of Flt-1 independently of the presence of Flk-1, indicating a central role for activation of the PI3-K/Akt pathway, followed by induction of NOS and PKG activity. In turn, activation of the PI3K/Akt pathway mediates NO-induced EC migration and angiogenesis (Kawasaki et al., 2003). Therefore, we planned experiments to understand the connection between hypoxia and migration in relation to NO. A 40% drop in the EC monolayer wound area was observed after 2 h of hypoxia. A 500-mM SNP treatment abolished the hypoxia-induced increase in wound healing (Fig. 5). It is evident that hypoxia perturbs biophysical properties of plasma membrane in EC, e.g. analysis of lipid microdomains by Botto et al. (2006) showed a decrease in caveolin-1, and an increase in CD55 (marker of lipid rafts) in EC in hypoxic intestinal edema. Morphometry showed a significant decrease in EC volume, a marked increase in the cell surface/volume ratio and a decrease in caveolar density (Botto et al.,

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Live-cell microscopy revealed that hypoxia treatment for 2 h increased the number of lamellipodia from 12 to 18 per cell while the number of filopodia remained unaltered. When the cells were kept under hypoxic stress with 500 mM SNP, the number of lamellipodia was 13 per cell (Fig. 3E). It has been shown that interfering with actin polymerization leads to altered permeability of capillaries under hypoxia (Rasio et al., 1989). Another work by Alexander et al. (1988) revealed that blocking actin polymerization causes significant surface area and perimeter increases along with elevated actin stress fibers in cultured ECs. The data suggests that the endothelial junction barrier may be enhanced in part by assembly of actin filaments. These observations led us to investigate the actin polymerization pattern of EC under hypoxia. ‘‘Boat’’-shaped cells are predominantly present in this group. Phalloidin staining revealed that these ‘‘boat’’ cells are highly polarized in terms of actin polymerization. Broader lamellipodia-like structures with robust actin polymerization are visible at the plasma membrane. Hypoxia treatments for 2 h also reduced the number of stress fibers in the cytosol (Fig. 4, upper panels). A 500-mM SNP treatment of the cells partially protected cells from hypoxia-induced changes in the actin polymerization and lamellipodia formation pattern (Fig. 4, lower panels).

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Fig. 4. Actin polymerization pattern of EC under hypoxia. Alexa Fluor-phalloidin staining revealed that SNP (500 mM) treatment prevented the hypoxia-stimulated formation of lamellipodia and sub-cellular central microfilaments as well as the polarization of actin filaments.

2006). Based on these observations and on our actin polymerization pattern studies we hypothesized that hypoxia perturbs membrane stiffness of EC that will in turn modulate the barrier properties of the EC monolayer. In a pioneer effort to measure membrane tension in hypoxia-treated EC by using optically trapped polystyrene spheres adhered to the cell surface (Fig. 6A and B), we demonstrated an increase in the membrane tension of hypoxia-treated cells by 160% in comparison to that of normoxia-treated cells (Fig. 6D). A 500-mM SNP treatment reduced the hypoxia-induced ‘‘stiffness’’ of the membrane specifically in the projected cones of the cells (Fig. 6A, C, and D).

Protecting cGMP from hydrolysis modulates hypoxia-dependent endothelial monolayer permeability It is known that NO effects are mediated by cyclic GMP. Phosphodiesterase (PDE) 5, a major PDE subtype, hydrolyzes cGMP and is more abundant in the lung than in other tissues (Thomas et al., 1990). EC treated with the PDE5 inhibitor sildenafil (1 mM) were also protected from being rounded off under hypoxia (Fig. 7A). A trypan blue-based viability study showed that rounded off cells were alive (data not shown). In monolayer permeability assays, a dose of 1 mM sildenafil had an inhibitory effect on the hypoxia-mediated

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Fig. 6. EC membrane tension under normoxia and hypoxia. (A) Snapshot of ECV cell exhibiting flat and cone-like regions. (B) Schematic representation of the membrane tension measurement using an optical trap. Membrane tether being drawn by a bead confined in an optical trap. Effective tension was computed at equilibrium condition. (C) Snapshot of beads stuck to cells at different regions. (D) Tension values observed over normal, hypoxic and SNP-treated hypoxic cells. Each bar represents the average of 8–10 measurements. * and # Po0.05 compared to normoxia. Y Po0.05 compared to hypoxia.

hyper-permeability of ECs. EC monolayers incubated with 1 mM DEAN during hypoxia treatments failed to demonstrate any recovery from the hypoxia-mediated

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Fig. 7. cGMP levels influence EC (ECV-304) morphology (A, D) and monolayer permeability (B, C) under hypoxia. An increase in the percentage of normal cells was observed in the cell population, which was treated with sildenafil (SF) (A) or with 8-Br-cGMP (D) under hypoxic conditions. In addition, SF protected EC monolayers from hypoxia-induced leakiness (B), whereas theophylline (C) failed. Data are means7SE of 3 (B, C) or 5 (A, D) separate experiments, respectively, assayed in duplicate. * Po0.05, ** Po 0.001 compared to normoxia control, and # Po0.05 compared to hypoxia control.

0.3 0.25 0.2 0.15

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Fig. 8. Blocking sGC with ODQ (10 mM) interferes with hypoxia-mediated changes in EC monolayer permeability. Data are means7SE of three separate experiments assayed in duplicate. * Po0.05 compared to normoxia control and # Po0.05 compared to hypoxia control.

NO and to target soluble guanylyl cyclase (sGC) specifically, the lower concentration of DEAN (i.e. 1 mM) was used to test for the combinatorial effect of sildenafil and DEAN on hypoxia-induced leakiness of EC. EC (ECV-304) monolayers incubated with sildenafil along with DEAN under hypoxia demonstrated a recovery of 30% from the hypoxia-mediated leakiness (Fig. 7B). Theophylline is a non-specific PDE inhibitor, which is not selective for the endothelial-specific PDEs. As expected, theophylline and theophylline along with DEAN (1 mM) did not exert any effect on hypoxiainduced leakiness (Fig. 7C).

Modulation of the cGMP level perturbs hypoxiamediated leakiness of ECs To check the role of the cGMP pathway in the recovery of EC from hypoxia effects, EC were treated with 8-Br-cGMP. A 20% increase in the number of normal cells compared to hypoxic conditions was observed. The percentage of rounded up cells was decreased by 40% (Fig. 7D). We have further tested the effect of 8-Br-cGMP on hypoxic permeability, which showed a 30% decrease in BPAEC. Primary EC monolayer incubated with 8-Br-cGMP or sildenafil

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157

Normal

0.4 Monolayer permeability (arb. Unit)

0.35

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@ @

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0.25 0.2 0.15 0.1 0.05 0

A

Ctr l

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Hypoxia @

0.3 *

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@

0.2 @

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8-Br cGMP-DEAN

B Fig. 9. NO (DEAN), sildenafil and 8-Br-cGMP prevent hypoxia-induced increases in EC (BPAEC) monolayer permeability. Data are means7SE of four separate experiments assayed in duplicate. * Po0.05 compared to normoxia control. @ Po0.05 compared to hypoxia control.

along with DEAN demonstrated a recovery of 50% from the hypoxia-mediated leakiness of the monolayer (Fig. 9A and B). In another approach sGC was selectively inhibited with ODQ (10 mM). ODQ treatment of EC abolished the 10-mM DEAN-mediated recovery from hypoxia-induced leakiness (Fig. 8).

Discussion The novel aspect of the present work is the dissection of NO signaling in EC that confers protection of EC against hypoxia-mediated damages. Our study shows that NO protects EC by reversing the hypoxia-driven leakiness (Fig. 2A). Results of the present work specifically imply that NO in part rectifies hypoxiamediated alterations in endothelial barrier functions (Fig. 2A and B). The work of Draijer et al. (1995) indicates that NO/cGMP reduces thrombin-induced endothelial permeability by inhibition of the thrombininduced Ca2+ accumulation and/or by inhibition of cAMP degradation by PDE III. In addition to rectifying the membrane leakiness, NO may protect cell–cell interfaces and maintain the integrity of the EC mono-

layer, thereby ameliorating the leakiness. A recent publication suggests that preconditioning with a low dose of NO donor accelerates repair and maintains endothelial integrity via a mechanism that includes the sGC pathway (Antonova et al., 2007). To verify the cellular and molecular basis of the concept that NO protects HAPE patients by recovering from edema, we studied the effects of hypoxia on permeability of human umbilical cord-derived ECV304 and primary lung macrovascular EC, BPAEC, respectively. Earlier studies showed that permeability of microvascular EC differs from that of macrovascular EC (Zhu et al., 2005). In our experimental setup, we used primary ECs of macrovascular origin (bovine pulmonary artery) to check the NO perturbations in the permeability of hypoxia-treated EC. Lung vasculature shows anatomically heterogeneous functional properties. Work of Kelly et al. (1998) showed that increased [Ca2+]i promotes permeability in macrovascular EC in lung, but not in microvascular EC. A recent work by Irwin et al. (2005) suggested that atrial natriuretic peptide reduced hypoxia-induced pulmonary vascular leak in vivo by acting on microvascular cells but not on macrovscular EC. The results of the present work

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further indicate that NO may target specifically macrovascular EC to modulate permeability under hypoxic conditions. Basal vascular endothelial permeability is normally kept low in part by the restrictiveness of interendothelial junctions. We observed an increase in monolayer permeability under hypoxia that could be the result of trans-cellular hyper-permeability and increasing inter-endothelial gaps (Fig. 2A). Endothelial permeability is believed to be associated with morphology changes of the cells, such as we observed when the endothelial monolayer was subjected to hypoxia (Fig. 3A). Tiruppathi et al. (1992) demonstrated that a-thrombin widens the inter-endothelial junctions because of ‘‘rounding up’’ of the cells and thereby increases the permeability. Hinder et al. (1999) have used an animal model of sepsis to demonstrate that NO inhalation may protect against pulmonary edema associated with endotoxemia and NOS inhibition. It has also been shown that pretreatment of cells with different NO donors increased endothelial cGMP content and blocked hydrogen peroxide-related effects on permeability (Suttorp et al., 1996). The work of Marumo et al. (1999) demonstrated that blocking of NOS enhanced VEGF-induced hyper-permeability, while activation of NOS counteracts the VEGF effects. There is evidence that even basal release of NO helps to maintain normal pulmonary vascular fluid balance (Arkowitz et al., 1997). We critically examined the protective role of NO on permeability and morphology of EC (Fig. 3B and C). We also found that supplemented NO protects EC from hypoxia-mediated damages (Fig. 2A), while NO induces permeability in EC under normoxia (Fig. 2B). It is evident that hypoxia promotes superoxide production in EC that contributes to the enhanced permeability of EC membrane (Gertzberg et al., 2004). NO is known for its high affinity for superoxide radicals (Inoue et al., 2000). Therefore, supplemented NO interacts with superoxide to form peroxynitirite, thereby neutralizing the permeabilityenhancing effects of superoxide and protecting the EC from abnormal barrier functions. The present study reveals that the NO/cGMP pathway perturbs endothelial barrier functions (Figs. 7B, 8, and 9A, B) and also indicates that eNOS-derived NO possibly tunes the integrity of inter-endothelial junctions, and thus serves to maintain the low basal permeability of continuous endothelia (Predescu et al., 2005). The normal function of the endothelium is highly dependent on the endothelial cytoskeleton arrangements. By using a simulation method known as dissipative particle dynamics, in which several atoms united into a single particle. Groot and Rabone (2001) explained that stress and membrane damage are two inter-dependent phenomena. When we measured membrane tension of normoxia- and hypoxia-treated cells by

using optical tweezers, hypoxia-treated cells showed region-specific variation in the level of tension. The migration cones with denser actin polymerization developed a 20% higher tension than apparently flatter areas (Fig. 6), and hypoxic conditions with NO donors could protect cells from hypoxia-mediated elevation of membrane tension (Fig. 6). NO has been implicated in the migration of EC. It is evident from the work of Goligorsky et al. (1999) that NO modulates focal adhesions in ECs. The results of our experiments show that hypoxia-associated changes in actin polymerization pattern, permeability and migration of EC are NO dependent (Figs. 2B, 3C, E, 4, and 5). This observation is further supported by earlier work of Baldwin et al. (1998), which confirms that inhibition of NO synthesis increases venular permeability and alters the endothelial actin cytoskeleton. Work of Dai and Sheetz (1995, 1998) furnishes evidence in support to our result that hypoxiainduced polarization of actin in EC overstretches the surface of hypoxia-induced cones and incites higher leakiness in those regions (Fig. 6A and D). We speculate that a drastic rearrangement of cytoskeleton proteins under hypoxia rafts the actin monomers to polymerize in a polarized fashion (Fig. 4). We assume that restricted stiffening of the EC membrane promotes eNOS loosening from the membrane followed by reduced NO production under hypoxia (Fig. 1B). Our assumption is further substantiated by a recent publication indicating that hypoxia produced enlarged EC, with dysfunctional Golgi and loss of eNOS from the plasma membrane (Mukhopadhyay et al., 2007). PDE plays a crucial role in cGMP-dependent signaling by hydrolyzing the cGMP. PDE 5 is largely responsible for cGMP metabolism in the lung endothelium (Matthay, 2002). Sildenafil, a PDE inhibitor, helps in keeping cGMP levels high by inhibiting PDE 5. A recent publication suggests that pulmonary microvascular ECs do not express PDE 5 in culture, and possibly in vivo as well (Zhu et al., 2005). In our experiments, sildenafil led to partial recovery of both types of EC (Fig. 7A and B). The sildenafil effect was also shown to be potentiated by NO donors (Figs. 7A, B and 9A), whereas theophylline, a non-specific PDE inhibitor, failed to confer any protection against hypoxiamediated damages (Fig. 7C). Sildenafil is known to promote vasodilatory effects in acute hypoxia-induced pulmonary hypertension in eNOS KO mice (Preston et al., 2004). Earlier work demonstrated that chronic administration of DA-8159, an inhibitor of PDE5, could attenuate the development of endothelial dysfunction in diabetes, and its effect is associated with an improvement in endothelial function (Ahn et al., 2005). Suttorp et al. (1993) suggested that PDE inhibition is a powerful approach to block the H2O2-induced increase in endothelial permeability. This concept appears especially valuable when endothelial PDE isoenzyme pattern

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and PDE inhibitor profile are matched optimally, as our result shows that inhibition of endothelial-specific PDE5 is more effective than inhibition of PDE4, a nonendothelial subtype (Fig. 7C). The importance of the cGMP-dependent pathway in maintaining the barrier function of the endothelium was further verified when a higher dose (10 mM) of DEAN failed to restore the barrier functions under hypoxia (Fig. 8). Our study demonstrates that cGMP protects endothelial barrier functions under hypoxia. Occurrence of pathologic edema in ARDS, pulmonary hypertension, HAPE, and high altitude cerebral edema is attributed to the leaky endothelial barrier. Dissection of the NO-cGMP pathway, which improves membrane barrier functions in EC, may help identifying a candidate drug from the genre of pharmacological agents such as PDE inhibitors or cGMP analogs. Our findings may be also helpful in further dissecting the mechanism of NO-mediated actin polymerization and polarization in EC under hypoxia. Since vascular permeability in the lung is tightly controlled by an array of physiological variables such as lymph flow, lymph protein concentration, heterogeneity of endothelial behavior, level of histamine and prostaglandins (Brigham et al., 1974; Brigham and Owen, 1975) presently this work does not offer a cure of hypoxia-associated lung diseases. Instead, the work verifies the concept of NO-based recovery of HAPE at the cellular level, and also prepares the springboard to explore the possibilities of NO-based therapy of hypoxia-associated lung diseases in animal models by considering the unique biochemical and biophysical milieu of the lung.

Acknowledgments This study was financially supported by a taskforce grant (Grant# 97(SC-AU)/DIPAS/2005) from Defence Institute of Physiology and Allied Sciences (DIPAS), Defence Research and Development Organization (DRDO), Government of India to S. Chatterjee.

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