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Cardiovascular Research 1998 38(1):256-262; doi:10.1016/S0008-6363(98)00003-0
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Expression of a functional neutrophil-type NADPH oxidase in cultured rat coronary microvascular endothelial cells

Ulvi Bayraktutana, Nick Drapera, Derek Langb and Ajay M. Shaha,*

aDepartment of Cardiology, University of Wales College of Medicine, Cardiff CF4 4XN, UK
bDepartment of Pharmacology and Therapeutics, University of Wales College of Medicine, Cardiff CF4 4XN, UK

* Corresponding author. Tel.: +44 (1222) 74 23 38; Fax: +44 (1222) 74 35 00; E-mail: shaham2@cf.ac.uk

Received 20 October 1997; accepted 5 December 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objectives: The production of reactive oxygen species (e.g., superoxide) by endothelial cells is relevant to tissue injury during ischemia-reperfusion, and may also play a role in intracellular signaling pathways. However, the molecular identities of the enzymes responsible for endothelial superoxide production are poorly defined, although xanthine oxidase, NADH/NADPH oxidoreductases and nitric oxide synthase are among proteins suggested to contribute. Recent studies suggest that an NADH/NADPH oxidase similar to that found in neutrophils is an important source of superoxide in vascular smooth muscle. Methods: We investigated whether a phagocyte-type NADH/NADPH oxidase complex is present in rat cultured coronary microvascular endothelial cells. The expression of NADPH oxidase components was studied by RT-PCR and Western blot analyses, while functional activity was assessed by measurement of superoxide production by lucigenin-enhanced chemiluminescence. Results: The major component of the phagocyte-type NADH/NADPH oxidase complex, a cytochrome b558 heterodimer, was shown to be present both at mRNA and protein levels, using oligonucleotide primers designed from published neutrophil and vascular smooth muscle sequences and anti-neutrophil antibodies respectively. Functional activity of the enzyme was also confirmed by NADPH-evoked superoxide production in cell homogenates, which was inhibited either by the superoxide chelator Tiron or by diphenyleneiodonium, an inhibitor of the oxidase. Conclusions: A functional phagocyte-type NADPH oxidase is expressed in coronary microvascular endothelial cells, where it may contribute to the physiological and/or pathophysiological effects of reactive oxygen species. These data, together with reports of the presence of a similar oxidase in other non-phagocytic cell types, suggest that this enzyme complex is widely expressed in many tissues where it may subserve signaling and other functions.

KEYWORDS Experimental; Vasculature; Molecular biology/biochemistry; Endothelial function; Endothelial factors; Free radicals; Gene expression; Signal transduction


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Reactive oxygen species such as superoxide and hydroxyl radical (•OH) are well known to be mediators of cellular injury in many tissues, especially during ischemia-reperfusion [1]. Recent studies indicate that low level production of reactive oxygen species, including H2O2, may also be involved in redox signaling events, for example changes in gene expression [2, 3]. In the vasculature, endothelial cells play an important role in both these processes [4–8]. The molecular identities of the enzymatic sources of endothelial reactive oxygen species are not well defined, although xanthine oxidase, NADH oxidoreductases and nitric oxide synthase are among the enzymes that are suggested to contribute [5–7]. In vascular smooth muscle, an NADH/NADPH oxidase has recently been shown to be an important source of superoxide radicals [9, 10]. Furthermore, this oxidase has been implicated in the pathogenesis of angiotensin II-induced hypertension and vascular smooth muscle hypertrophy, and may also contribute to impaired endothelial-dependent vascular relaxation secondary to inactivation of nitric oxide by superoxide [11, 12].

At a molecular level, the smooth muscle NADH/NADPH oxidase is thought to be similar but not identical to an NADPH oxidase found in phagocytic cells such as neutrophils and macrophages [11, 13]. In the latter cell types, NADPH oxidase plays a vital role in non-specific host defence during infection with pathogens by generating large (millimolar) quantities of superoxide during the so-called respiratory burst [14]. The phagocyte NADPH oxidase is a complex multicomponent enzyme which comprises a membrane-associated low-potential cytochrome (cytochrome b558) and at least 4 cytosolic proteins (p47-phox, p67-phox, p40-phox, and a small GTP-binding protein p21-rac 1). The cytochrome b558 heterodimer, comprising a 22 kDa subunit (p22-phox) and a 91 kDa glycosylated subunit (gp91-phox), is responsible for the enzymatic activity of NADPH oxidase, i.e. the one electron reduction of molecular oxygen to superoxide [14]. Activation of NADPH oxidase in phagocytes is tightly regulated and is initiated by translocation of the cytosolic components to the membrane and their association with cytochrome b558 [14]. Interestingly, in vascular smooth muscle although a p22-phox subunit highly homologous to the phagocytic protein has been identified, the gp91-phox subunit could not be demonstrated [13], leading to suggestions that the vascular smooth muscle oxidase may be a different isoform.

In the present study, we investigated whether a phagocyte-type NADPH oxidase is present in endothelial cells. We chose to study rat coronary microvascular endothelial cells (CMVE) since the microvasculature is the major site of production of reactive oxygen species during situations such as ischaemia-reperfusion [1]. We report that both the p22-phox and gp91-phox subunits of cytochrome b558, the major component of the phagocyte-type NADPH oxidase, are expressed at mRNA and protein level in rat CMVE. Furthermore, the oxidase is functionally active as determined by NADPH-evoked generation of superoxide in CMVE homogenates.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
This investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985).

2.1 Endothelial cells
CMVE were isolated from Wistar rat hearts by collagenase digestion, and cultured to passage 2 (14 days) as described previously [15]. Cells were pooled from 4 hearts for each isolation. Briefly, hearts mounted on a Langendorff apparatus were perfused at 37°C with a solution of the following composition (in mM): NaCl 118, KCl 4.7, NaH2PO4 1.2, MgSO4 1.2, NaHCO3 25, glucose 11, pH 7.4 (gassed with 95% O2/5% CO2)-Buffer 1. Epicardial mesothelial cells were devitalised with 70% (v/v) ethanol. After flushing out blood from the coronary circulation, perfusion was changed to Buffer 1 with added CaCl2 0.25 µM and collagenase 0.04% (Sigma type II), which was recirculated for 30 min. Ventricles — excluding any visible large vessels — were then chopped into 15 ml of recirculating solution containing BSA (200 mg, Sigma fraction V), and triturated gently during a 10 min incubation period at 37°C. The suspension was filtered through nylon gauze and centrifuged (150 g, 3 min) to sediment myocytes. The supernatant, including added BSA (100 mg), trypsin 0.01%, and CaCl2 50 µM, was incubated at 37°C for 15 min with stirring. The CMEC pellet was obtained by centrifugation (1000 g, 10 min), washed twice in Buffer 1 with CaCl2 250 µM and 500 µM respectively, and resuspended in 40 ml pre-warmed Medium 199 (Gibco) with added 10% newborn calf serum, 10% fetal calf serum, benzylpenicillin 250 U/ml, streptomycin 250 µg/ml, amphotericin B 12.5 µg/ml, and gentamycin 50 µg/ml. Cell suspensions were plated in 75 cm2 tissue culture flasks and incubated at 37°C. After 1 h, unattached cells and debris were washed off with 0.9% saline. Cultured cells formed confluent monolayers with a ‘cobblestone’ morphology within 5–7 days. Cells were then trypsinised and subcultured to confluence in fresh flasks.

Cultured CMVE were characterised as endothelial by their typical ‘cobblestone’ morphology, and by the uptake of fluorescently labeled acetylated low density lipoprotein by >99% of cells. CMVE stained negatively for smooth muscle {alpha}-actin, and rapidly formed capillary-like tubes on the basement membrane preparation Matrigel.

2.2 Reverse transcription (RT) and polymerase chain reaction (PCR)
Total RNA was isolated from confluent cultured CMVE by extraction with phenol-chloroform-guanidinium isothiocyanate [16]. First strand cDNA synthesis was performed using 5 µg total RNA in the presence of 500 ng random hexamers (Promega), 10 mM of each dNTP (Boehringer), 10 mM Tris-HCl pH 8.4, 50 mM KCl, 2.5 mM MgCl2, and 33 U of RNase inhibitor (Promega). The reaction mixture was preincubated at 70°C for 3 min followed by cooling on ice, addition of 200 U Moloney monkey leukemia virus reverse transcriptase (Gibco-BRL) and incubation at 42°C for 90 min. PCR primers for amplification of p22-phox were based on the published rat aortic smooth muscle cell sequence [13]: 5'-GACGCTTCACGCAGTGGTACT-3' (sense) and 5'-CACGACCTCATCTGTCACTGG-3' (antisense) (GenBank accession No. U18729 [GenBank] ). Primers for amplification of gp91-phox were based on the published porcine macrophage cDNA sequence [17]: 5'-GCCTGTGGCTGTGATAAGCAG-3' (sense) and 5'-CTTTTGTTTCAGGCCTGTGA-3' (antisense) (GenBank accession No. U02476 [GenBank] ). PCR reactions were carried out in 100 µl final volumes containing 2 µl of RT reaction, 48 ng each of sense and antisense primers, 200 µmol dNTPs, 1.5 mM Mg2+, and 2.5 U Taq Polymerase (Promega). Amplification was performed for 35 cycles of denaturation at 94°C (1 min), annealing at 65°C (1 min), and extension at 72°C (2 min), followed by a 10 min extension reaction at 72°C. PCR products were separated on a 1.5% agarose gel (Bio-Rad).

2.3 Sequencing reactions
Gel purified PCR products were ligated into a TA cloning vector (Invitrogen) and transformed into E. coli INV{alpha}F' competent cells. Transformed plasmids containing the appropriate inserts were selected, and automated DNA sequencing was carried out on an ABI Prism 377 DNA sequencer (Perkin Elmer). DNA sequencing was repeated using at least 3 different preparations. Sequence comparisons were performed using the Daresbury SEQNET database [18].

2.4 Western blotting
Cultured CMVE were washed twice in ice-cold PBS prior to lysis in 1 ml of boiling lysis solution containing 1% SDS, 10 mM Tris pH 7.4. The amounts of total protein in aliquots were quantified by a micro BCA protein assay kit (Pierce). Equal amounts of protein were run on an 8% SDS-polyacrylamide gel and electroblotted onto nitrocellulose membrane. Equal transfer among lanes was confirmed by reversible staining with Ponceau S (Sigma). Membranes were incubated with monoclonal antibodies (MoAb 449, MoAb 48) raised against human neutrophil NADPH oxidase components (a kind gift of Dr. A. Verhoeven) [19]or a polyclonal anti-human neutrophil p22-phox antibody (a kind gift of Dr. F. Wientjes) [20]. MoAb 449 recognises the human neutrophil p22-phox subunit, while MoAb 48 recognises the gp91-phox subunit. The secondary antibodies were peroxidase-conjugated goat antibodies (Santa Cruz).

2.5 NADH/NADPH oxidase assay
CMVE homogenates were prepared as described previously for vascular smooth muscle cells [9]. Briefly, cells were lysed on ice in lysis buffer containing 1 mM EGTA, 20 mM monobasic potassium phosphate (pH 7.0), 10 µg/ml aprotinin, 0.5 µg/ml leupeptin, 0.7 µg/ml pepstatin, and 0.5 mM phenylmethylsulfonyl fluoride. Cell homogenates were stored on ice until use. Oxidase activity was measured by lucigenin-enhanced chemiluminescent detection of superoxide in a luminometer [9]. The reaction buffer contained 1 mM EGTA, 150 mM sucrose, 500 µM lucigenin, and 1 mM NADH or NADPH. The reaction was started by addition of 100 µl of homogenate (50–200 µg protein). Luminescence was measured as the rate of photon counts per mg protein, following subtraction of the counts obtained from a buffer blank.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
RT-PCR reactions using oligonucleotide primers designed from the rat aortic smooth muscle cell sequence (for p22-phox) and the porcine macrophage sequence (for gp91-phox) yielded an expected-size 485-bp PCR product for p22-phox and a 423-bp PCR product for gp91-phox in cultured rat CMVE (Fig. 1). Identical-sized PCR products were amplified from the human differentiated monocytoid cell line, U937, which contains NADPH oxidase capable of high-output superoxide production [21]. No PCR products were obtained in samples where RT was not performed, indicating an absence of genomic DNA contamination. Identical results were obtained with several independent CMVE isolates. DNA sequencing revealed 100% homology between the rat CMVE p22-phox partial cDNA sequence and the published rat aortic smooth muscle cell sequence. The rat CMVE gp91-phox PCR product was ~93% homologous to the published porcine macrophage sequence at nucleotide level (Fig. 2).


Figure 1
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  		Fig. 1 (A) 423-bp transcript for gp91-phox; (B) 485-bp PCR transcript for p22-phox. Lane 1, U937 cells (positive control); lane 2, rat CMVE; lane 3, rat CMVE without RT (negative control). Hae III restricted {varphi}X174 DNA was used as a size marker.

 

Figure 2
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  		Fig. 2 Partial cDNA sequence of rat CMVE gp91-phox compared to the porcine macrophage (macro) gp91-phox sequence. Asterisks indicate identical nucleotides.

 
Expression of p22-phox and gp91-phox protein was confirmed by Western analyses. Fig. 3A shows reaction of the anti-neutrophil gp91-phox monoclonal antibody MoAb 48 with a ~90 kDa protein in rat CMVE, with a 75–100 kDa smear obtained with U937 cell protein (as reported previously [19]). MoAb 449 did not react with rat CMVE protein, but a 22–23 kDa band was obtained with U937 cell protein (data not shown). However, a polyclonal anti-p22-phox antibody picked up a 22–23 kDa band both in rat CMVE and U937 cells (Fig. 3B).


Figure 3
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  		Fig. 3 Western blots showing (A) the presence of gp91-phox protein in U937 cells (lane 1) and rat CMVE (lane 2) using an anti-neutrophil gp91-phox monoclonal antibody MoAb 48. The smear from ~75–100 kDa in U937 cells is typical for gp91-phox, and is thought to be due to a mixture of variably glycosylated protein. (B) p22-phox protein detected in U937 cells (lane 1) and rat CMVE (lane 2) using a polyclonal anti-neutrophil p22-phox antibody.

 
No chemiluminescence could be detected in CMVE homogenates in the absence of NADH or NADPH (data not shown). Both NADH and NADPH evoked significant chemiluminescence in the oxidase assay (Fig. 4), with the level of NADPH-evoked activity being 29.2±1.3% that of the NADH-evoked activity (n=10). Tiron (10–100 mM), a chelator of superoxide, completely inhibited both NADH- and NADPH-evoked activity (Fig. 4). Diphenyleneiodonium (DPI, 4–400 µM), an inhibitor of NADH/NADPH oxidases [22], dose-dependently decreased NADH- and NADPH-evoked superoxide production, with a greater effect on NADPH oxidase activity (Fig. 4). Interestingly, DPI was more effective if added at the time of peak superoxide production than if pre-incubated prior to enzyme activation, consistent with previous work indicating that it binds to the activated enzyme [22]. DPI (400 µM) reduced NADH- and NADPH-evoked responses to 55±5% and 17±4% of control values respectively if added at the time of peak chemiluminescence, whereas if pre-incubated it did not reduce NADH-evoked responses (114±66% of control) and only reduced NADPH-evoked responses to 36±15% of control values. There was no significant inhibition of either NADPH- or NADH-evoked activity by inhibitors of xanthine oxidase (oxypurinol 100 µM), cyclooxygenase (indomethacin 10 µM), nitric oxide synthase (L-N-monomethyl arginine 10 µM) or the mitochondrial electron transport chain (rotenone 100 µM) (Fig. 5).


Figure 4
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  		Fig. 4 Left panel: Representative examples of NADH- and NADPH-evoked lucigenin-enhanced chemiluminescence in rat CMVE homogenates, and the effect of addition of DPI. Right panel: Mean data from 7–10 paired experiments showing the effects of Tiron or of DPI on NADH- and NADPH-evoked chemiluminescence.

 

Figure 5
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  		Fig. 5 Mean data showing the effects of oxypurinol (100 µM), indomethacin (10 µM), rotenone (100 µM), and L-N-monomethyl arginine (L-NMMA 10 µM), on NADH- and NADPH-evoked chemiluminescence. N≥3 each.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
Although production of reactive oxygen species by endothelial cells is thought to contribute to reperfusion injury [1, 4–7], and reactive oxygen species are implicated in certain signal transduction pathways [8], the molecular identities of the cellular enzymes responsible for this production are not well defined. Xanthine oxidase is considered to be important during ischemia-reperfusion, while enzymes such as NADH oxidoreductases, nitric oxide synthase and cylcooxygenase have also been implicated [5–7]. A growing body of evidence suggests that in vascular tissues an NADH/NADPH oxidase situated in the medial smooth muscle and adventitia is an important source of reactive oxygen species such as superoxide and H2O2 [9–13]. It appears to be important in angiotensin II-induced vascular smooth muscle hypertrophy and hypertension [11, 12]. Recently, Griendling and co-workers demonstrated that the rat vascular smooth muscle NADH/NADPH oxidase contains a cytochrome b558, and cloned a p22-phox subunit that was highly homologous to the corresponding human neutrophil cDNA coding sequence [13]. However, the presence of the gp91-phox subunit could not be demonstrated, an unexpected finding given that enzymatic activity is thought to reside in this part of the protein.

In the present study, we have demonstrated the presence of both subunits of cytochrome b558 in cultured rat CMVE at mRNA and protein level. The portions of cDNA isolated were highly homologous to the corresponding phagocyte and vascular smooth muscle sequences. Since gp91-phox and p22-phox are essential for the stability of NADPH oxidase and its catalytic activity [14], our data suggest the presence of a functional enzyme in endothelial cells. Indeed, NADPH oxidase activity was confirmed in CMVE homogenates by means of lucigenin-enhanced chemiluminescent detection of superoxide. Furthermore, superoxide production was suppressed by an inhibitor of NADPH oxidase, DPI. Although DPI can inhibit several other flavoproteins (eg, xanthine oxidase, nitric oxide synthase, cyclooxygenase, and components of the mitochondrial electron transport chain), specific inhibitors of these enzymes were ineffective, suggesting that the DPI-sensitive activity was indeed attributable to an NADH/NADPH oxidase. We detected both NADH- and NADPH-evoked superoxide production, but it is not clear whether these activities represent two different enzymes or a single enzyme that utilises both substrates. However, it is notable that DPI was more effective in inhibiting NADPH- than NADH-evoked activity. These data are consistent with previous biochemical studies by Wolin and colleagues [5], who reported that an ‘NADH oxidoreductase’ was a major source of superoxide production in bovine coronary artery endothelial cells, and that this activity could be inhibited by DPI. It remains unclear whether or not this ‘NADH oxidoreductase’ is the same enzyme as ‘NADH/NADPH oxidase’ at a molecular level.

The phagocyte NADPH oxidase has been very well characterised both at a biochemical and molecular level [14]. It functions to facilitate microbicidal activity via high level superoxide production and its activation is tightly controlled, occurring only in response to specific stimuli triggered during initiation of non-specific host defense pathways. The genes for the two membrane-bound components of cytochrome b558 as well as the cytoplasmic components have been cloned. Molecular defects in these genes result in the syndrome of chronic granulomatous disease (CGD), characterised by recurrent life-threatening infection [14]. By contrast, the vascular smooth muscle NADH/NADPH oxidase appears to be continuously active (even in ‘resting’ cells), and never generates the same high levels of superoxide as the phagocyte enzyme. It clearly has a different function, namely involvement in (patho-)physiological signal transduction pathways. This difference has been speculated to be perhaps the result of a different isoform of (or even the lack of) gp91-phox in smooth muscle [11, 13]. In the current study, we show that both gp91-phox and p22-phox are expressed in endothelial cells, a cell type in which NADPH oxidase activity appears to be functionally more similar to the smooth muscle than the phagocyte enzyme. Although our studies were performed using cultured CMVE, the results are probably applicable to CMVE in situ since we have found that freshly isolated CMVE have a similar level of NADH/NADPH oxidase activity (unpublished observations).

A low level production of superoxide and other reactive oxygen species such as H2O2 in endothelial cells may suggest a role for the NADPH oxidase in cellular signaling pathways rather than in mediating the cellular injury associated with excessive free radical generation. A number of recent studies have implicated reactive oxygen species in redox signaling events in endothelial cells. For example, in human umbilical vein endothelial cells (HUVEC), cytokine-induced expression of vascular cell adhesion molecule-1 (VCAM-1) appears to involve reactive oxygen species-mediated mobilisation of nuclear factor-{kappa}B (NF-{kappa}B), and can be blocked by anti-oxidants [23, 24]. The expression of molecules such as VCAM-1 promotes monocyte adhesion to endothelial cells, and may be an important event in the development of atherosclerosis [24]. Signaling pathways that involve reactive oxygen species have also been implicated in the cyclic strain-induced acute release of plasminogen activator inhibitor-1 [25]and expression of monocyte chemotactic protein-1 [8], and in the tubular morphogenesis of human microvascular endothelial cells [26]. In addition to cytokines and cyclic strain, flow-induced shear stress also appears to be a stimulus for the production of reactive oxygen species by endothelial cells [27]. In many cases, the signal responsible for initiating intracellular signaling events may be H2O2 generated from superoxide rather than superoxide itself; in particular, H2O2 appears to be involved in the activation of NF-{kappa}B and subsequent changes in gene transcription [3, 24, 26]. A pertinent issue is whether superoxide and H2O2 are released inside the cells or outside. Although there appears to be evidence for both intracellular generation [23–26]and extracellular release [27]of reactive oxygen species, where the superoxide generated by NADPH oxidase is released remains to be established. If superoxide is released outside the cell, then other effects could include modulation of nitric oxide-dependent effects on vascular smooth muscle cells and other cell types, since superoxide may reduce the effective levels of nitric oxide as well as form peroxynitrite.

NADPH oxidases similar to the phagocyte enzyme have recently been reported to be present at mRNA/protein level in several other cell types, including fibroblasts [28], hepatoma cell lines [29], carotid body and airway chemoreceptors [30, 31]. The enzyme in these non-phagocytic cells seems to be functionally, and perhaps in some cases even structurally, distinct from the neutrophil oxidase. The major differences between the phagocytic and non-phagocytic enzymes are the level of superoxide generation and the stimuli that trigger or accentuate activation of the enzyme. In some of these cell types, the gp91-phox subunit could not be demonstrated, although whether this reflects its absence or the presence of a different isoform remains to be worked out. These data, together with the findings of the present study, suggest that phagocyte-type NADPH oxidases are widely expressed in many tissues but with probably differing functions in these tissues. The reasons for these differences in enzyme regulation and activity require further study, but possible explanations include minor differences in gp91-phox sequence, in the ‘cytosolic’ components of NADPH oxidase, and/or in the stoichiometry of these subunits. Future studies will define the precise role(s) of the NADPH oxidase complex in endothelial cells.

Time for primary review 28 days.


   Acknowledgements
 
This work was supported by the British Heart Foundation and the UK Medical Research Council.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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