SPECIAL COMMUNICATION
Free radical production in hypoxic pulmonary artery smooth muscle cells

David W. Killilea1, Raymond Hester2, Ronald Balczon3, Pavel Babal1, and Mark N. Gillespie1

Departments of 1 Pharmacology and 3 Structural and Cellular Biology and 2 Biotechnical Services Laboratory, College of Medicine, University of South Alabama, Mobile, Alabama 36688


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study used an inexpensive and versatile environmental exposure system to test the hypothesis that hypoxia promoted free radical production in primary cultures of rat main pulmonary artery smooth muscle cells (PASMCs). Production of reactive species was detected by fluorescence microscopy with the probe 2',7'-dichlorodihydrofluorescein, which is converted to the fluorescent dichlorofluorescein (DCF) in the presence of various oxidants. Flushing the airspace above the PASMC cultures with normoxic gas (20% O2, 75% N2, and 5% CO2) resulted in stable PO2 values of ~150 Torr, whereas perfusion of the airspace with hypoxic gas (0% O2, 95% N2, and 5% CO2 ) was associated with a reduction in PO2 values to stable levels of ~25 Torr. Hypoxic PASMCs became increasingly fluorescent at ~500% above the normoxic baseline after 60 min. Hypoxia-induced DCF fluorescence was attenuated by the addition of the antioxidants dimethylthiourea and catalase. These findings show that PASMCs acutely exposed to hypoxia exhibit a marked increase in intracellular DCF fluorescence, suggestive of reactive oxygen or nitrogen species production.

fluorescence microscopy; hypoxia; reactive oxygen species; reactive nitrogen species; vascular smooth muscle cells


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

FOR SEVERAL PRACTICAL REASONS, imaging cellular events during hypoxia can be difficult. Removing cells from hypoxia for immediate analysis is usually unacceptable because reoxygenation may induce additional stress on cells and complicate interpretation of results (11, 12). Mimicry of low oxygen conditions by adding cyanide to cell cultures to cause "chemical hypoxia" has been used as an alternative, but there has been persistent concern regarding a lack of correlation of effects of chemical hypoxia to true hypoxia (1, 10). Cell cultures can be made hypoxic by bubbling nitrogen gas through the medium (3, 20), but this approach may compromise sterility of the cultures and may interfere with imaging devices. A different strategy has been the use of chambers that provide a closed environment but are still optically permeable to imaging devices. However, the closed environment itself presents certain problems, including alteration of the environment by cell metabolism and the difficulty in rapidly switching experimental conditions. Therefore, chambers designed to provide a continuous flow of a perfusing gas mixture, such as modified Sykes-Moore chambers, are often preferred, yet these can be expensive and/or technically difficult to fabricate in-house (18). This report describes a simple and economic system for a continuous perfusion of atmosphere above living cell cultures that enables simultaneous microscopic analysis during exposure to the test environment.

To demonstrate the utility of our system, we tested the specific hypothesis that hypoxia caused production of reactive species in cultured rat pulmonary artery smooth muscle cells (PASMCs) using fluorescence of 2',7'-dichlorofluorescein (DCF) as a marker of reactive species generation. Hypoxia-induced intracellular reactive species production has been demonstrated with this approach in other cell types (4, 7), but it is not known whether the PASMC, an important effector cell in the acute and chronic response of the lung to hypoxia, responds similarly.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. PASMCs were isolated from the main pulmonary arteries of male 200- to 250-g Sprague-Dawley rats with an explant technique previously described (2). The cells were maintained in culture medium (1:1 Dulbecco's modified Eagle's medium-Ham's F-12 medium, 10% fetal bovine serum, 100 U/ml of penicillin, and 100 µg/ml of streptomycin) and passaged with trypsin-EDTA treatment. All cell culture reagents were obtained from GIBCO BRL (Life Technologies, Grand Island, NY). The cells were used for experiments within 20 passages and studied when they were at ~80% confluence.

Flow-through chamber design. Standard 20 × 20-mm glass coverslips were washed sequentially with water and 70% ethanol to remove any factory residue. Holes 15 mm in diameter were punched into standard 60-mm-diameter tissue culture dishes with a chassis punch. The edges of the holes were smoothed with a fine-grain file, and the plates were washed with water to remove debris. A ring of water-resistant glue was applied on the inside of the plates ~2 mm from the edge of the hole. Dry coverslips covering the glue were immediately placed in the plates, taking care not to spread the glue into the chamber of the plate or under the coverslip, and were allowed to dry upside-down. A 4-mm hole was punched off-center into the top of the tissue culture dish lid, which allowed one arm of a stopcock to fit inside (Fig. 1). Silicone sealant (Silastic 732 RTV Adhesive/Sealant, Dow Corning, Midland, MI) was applied around the opening on the outside of the lid to secure the stopcock and prevent airflow around it. The lids and plates were washed sequentially with water to remove residual curing agents and 70% ethanol to sterilize. After they were dry, the plates were ready to be seeded with cells.


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Fig. 1.   Schematic of flow-through hypoxic chamber. Premixed gas flows through a regulator (not shown) into the back of the thermal conducting stage that is heated to 37°C. When the 60-mm2 culture dish is placed inside, a partially closed chamber is formed because the top of the plate rests flush on the inner ring of the stage. A port allows gas to flow under the top of the plate and into the chamber; a second port allows the gas to escape. Flow rate can be adjusted so that backflow of air through the exit port is insignificant. The opening in the bottom of the stage allows the oil objective to come into direct contact with the glass coverslip glued into the bottom of the plate (inset).

The cultures were placed in a model 5000 thermal conducting stage from 20/20 Technology (Whitehouse Station, NJ) as shown in Fig. 1. The apparatus maintained the cultures at 37°C and provided a partially closed environment when a 60-mm-diameter culture dish was fitted inside. Through one opening, certified gas (Air Products, Jacksonville, FL) at a 25 ml/min flow rate was allowed to perfuse the airspace above the medium. Gas exited through the second opening, preventing any pressure buildup or backflow of air. The top of the plate was equipped with a 1-ml syringe attached to the Luer hub of the stopcock. If a drug was to be added during an experiment, the syringe containing the drug would be attached to the stopcock, and the stopcock was placed in the open position before gas perfusion, allowing equilibration of the drug with the desired atmosphere. If no drug was to be added, the stopcock was closed. An additional stopcock added in-line preceding the apparatus allowed rapid switching of environmental conditions if multiple gas mixtures were attached. To verify hypoxia in the medium, PO2 was measured with a blood-gas analyzer (model ABL 30, Radiometer, Cleveland, OH).

Fluorescence microscopy. The production of reactive species as detected by DCF fluorescence during hypoxia was measured with an ACAS 570 confocal laser scanning microscope (Meridian Instruments, Okemos, MI). Nonfluorescent 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA; Molecular Probes, Eugene, OR) was loaded into cell cultures at a final concentration of 5 µM for 20 min. Reactive oxygen species (ROS) can oxidize DCFH-DA to the fluorescent DCF (6, 13, 16, 22). The complete apparatus was mounted on the stage of an Olympus IMT-2 inverted microscope (Olympus, Lake Success, NY). The 488-nm line of an argon laser was used at 100 mW to excite DCF with fluorescence emission collected through a ×100 oil-immersion objective (numerical aperture = 1.3) and a 530/30 band-pass filter in front of the photomultiplier tube.

Other instrument settings included a 3% scan strength and a 1% neutral density filter. Fluorescence intensity was obtained in arbitrary units after background subtraction with Meridian Software version 3.29.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Initial studies determined whether the apparatus described above afforded maintenance of stable normoxic or hypoxic environments. Premixed normoxic (20% O2, 5% CO2, and 75% N2) or hypoxic (5% CO2 and 95% N2) gas was perfused at 25 ml/min through the flow-through chamber, with a headspace of ~20 cm3 over 5 ml of culture medium in a 60-mm-diameter culture dish. Direct sampling of medium from these experiments indicated that although medium PO2 during exposure to normoxic gas remained stable at ~150 Torr, medium PO2 during hypoxia decreased exponentially to ~20 Torr after 30 min of exposure (Fig. 2A). This PO2 level remained stable over the duration of hypoxic exposure. The pH of the medium was also stable during both normoxic and hypoxic exposures, indicating that the changes in fluorescence were not due to pH gradients established by hypoxia (Fig. 2B). Sampling from plates with fluorescent dye had no effect on observed PO2 or pH (data not shown).


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Fig. 2.   PO2 (A) of culture medium decreases exponentially on exposure to hypoxia, whereas pH (B) does not change in the hypoxic environment (n >=  4 cultures/point).

To demonstrate the utility of the above-described perfusion system, we determined whether hypoxia promoted generation of ROS or reactive nitrogen species. PASMC cultures were placed in the apparatus, and 5 µM DCFH-DA was added. The cells were allowed to take up dye for 30 min in a normoxic atmosphere before the initiation of hypoxia. On exposure to the hypoxic gas mixture, the cells became increasingly fluorescent as medium PO2 decreased (Fig. 3). After 60 min, DCF fluorescence was ~500% over normoxic baseline. When the chamber was flushed with normoxic gas, DCF fluorescence returned to baseline values within 20 min (data not shown).


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Fig. 3.   Representative experiment showing increase in dichlorofluorescein (DCF) fluorescence during exposure to hypoxia. A: pulmonary artery smooth muscle cells (PASMCs) were exposed to hypoxia, and images of the same 100-µm2 field were captured at intervals of 0, 5, 10, 15, 20, 30, and 60 min. Fluorescence scale in arbitrary units ranges from low (purple) to high (red) fluorescence. B: summary of hypoxic data. , Hypoxia; triangle , dimethylthiourea (100 µM) was added to the culture medium after 40 min of hypoxic exposure; black-down-triangle , PASMCs were pretreated for 6 h with 300 U/ml of catalase before induction of hypoxia. Values are means ± SE normalized as a percentage of normoxic baseline control for 10 cells in each of at least 3 experiments. * Addition of dimethylthiourea caused significant reductions in DCF fluorescence in hypoxic PASMCs. Catalase prevented hypoxia-induced increases in DCF fluorescence.

To provide confirmatory evidence that hypoxia-induced DCF fluorescence was mediated by reactive species, an antioxidant, dimethylthiourea (DMTU), was used to suppress the production of DCF fluorescence during hypoxia. After 40 min of hypoxia, 100 µM DMTU dissolved in 0.5 ml of culture medium was added to the cells in the chamber through the syringe port. Acute administration of DMTU in this manner reduced DCF fluorescence intensity to ~50% of peak levels within 20 min after introduction into the hypoxic chamber (Fig. 3B). Incubation of PASMCs with catalase (300 U/ml) for 6 h, a condition known to promote substantial elevations in systemic vascular smooth muscle cell catalase activity (17), also prevented hypoxia-induced increases in DCF fluorescence (Fig. 3B).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This report describes the construction of an apparatus to expose living cells to environmental hypoxia while being analyzed by standard microscopy techniques. Along with its simplicity and cost-effectiveness, the apparatus is highly versatile. Many different types of thermal conducting stages are commercially available that could easily be substituted for the 20/20 Technology model to develop similar systems. In addition, the gas exposure system described herein is not limited to analysis of hypoxic atmosphere. A variety of gas mixtures could be used to perfuse the chamber, enabling studies of the cellular effects of hyperoxia, air pollutants, and tobacco and other smokes. A variety of probes also can be used as long as the critical wavelengths are not blocked by the plastic of the culture plate. In this regard, our preliminary studies showed that hypoxia induced a modest increase in fluorescence of the calcium indicator fluo 3-AM (Molecular Probes) in PASMCs (data not shown), thus confirming observations previously reported by others (5, 15). In a similar context, although the present study assessed PO2 in aliquots of the culture medium with a blood gas instrument, an approach that could artifactually elevate measured PO2 values due to contamination of the sample with ambient air, a better estimation of real-time changes in oxygen content could be obtained with a Clark electrode, which could easily be affixed to the apparatus. We believe that the system described herein could permit a more convenient evaluation of a variety of environmental conditions, including hypoxia, on cell physiology without a substantial investment of time and resources compared with other environmental chambers.

To demonstrate the utility of the above-described system, we tested the hypothesis that hypoxia caused an increase in the generation of reactive species in PASMCs as detected by DCF fluorescence. Our interest in this issue was motivated by reports by Chandel et al. (4) and Duranteau et al. (7). Using a modified Sykes-Moore chamber that is more complex and expensive than the apparatus described in the present report, they (4, 7) showed that hypoxia caused antioxidant-reversible DCF fluorescence in cardiac myocytes and Hep3B cells. These studies also provided evidence that the mitochondrial electron transport chain is a major source of reactive species production (14, 19) and that increased generation of reactive species is required for hypoxia-induced transcriptional events. We reasoned that extending a critical aspect of their findings by demonstrating that PASMCs also responded to hypoxia with increased DCF fluorescence might promote further studies to define the source and molecular targets of reactive species production in this important lung cell population.

We found that acute hypoxia promotes increased DCF fluorescence in cultured PASMCs that is reversed and inhibited, respectively, by the antioxidants DMTU and catalase. Although the utility of DCF to detect reactive species is reasonably well entrenched in the literature (22), there is some controversy regarding exactly which ROS or reactive nitrogen species is responsible (6). DCFH-DA does not react efficiently with superoxide radical or hydrogen peroxide; metals such as those involved in Fenton-type chemistry are likely necessary for oxidation to DCF. Nitric oxide can oxidize DCFH-DA directly but not at physiological levels; however, peroxynitrite can readily oxidize DCFH-DA at low concentrations. Hypochlorous acid can also oxidize DCFH but with low efficiency. The present data show that DMTU, a low molecular weight, thiol-based antioxidant, attenuates hypoxia-induced DCF formation at micromolar concentrations. At such concentrations, DMTU has a weak scavenging affinity for most ROS (9), although it reacts at near diffusion-limited kinetics with the hydroxyl radical (8, 21). Our data also show that catalase inhibits hypoxia-induced DCF fluorescence, thus suggesting involvement of hydrogen peroxide at some point in the free radical reactions evoked by hypoxic exposure. Based on the above considerations, it seems likely that Fenton-active superoxide or hydrogen peroxide plays a significant role in the hypoxia-induced DCF fluorescence. The specific contribution of nitric oxide as well as the potential involvement of mitochondria as a source of reactive species in hypoxic PASMCs should be facilitated with the apparatus described in this report.

In summary, this report describes a simple apparatus for exposing cells to selected gaseous environments while simultaneously examining them on a standard inverted microscope. Using this preparation, we found that hypoxia causes rat PASMCs in culture to generate reactive species as detected by increased DCF fluorescence. The PASMC thus joins a growing list of cells that respond to hypoxia with intracellular free radical generation and provides further support for the concept that ROS or reactive nitrogen species play important roles in hypoxic signal transduction.


    ACKNOWLEDGEMENTS

This investigation was supported by National Heart, Lung, and Blood Institute Grants HL-38495 and HL-58243.


    FOOTNOTES

Address for reprint requests and other correspondence: M. N. Gillespie, Dept. of Pharmacology, College of Medicine, MSB 3130, Univ. of South Alabama, Mobile, AL 36688 (E-mail: mgillesp{at}jaguar1.usouthal.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 24 September 1999; accepted in final form 6 March 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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