Disinhibition of SOD-2 Expression to Compensate for a Genetically Determined NO Deficit in Endothelial Cells–Brief Report
Objective— Homozygosity for the −786C-variant of the human nos-3 gene is a risk factor for coronary artery disease (CAD). Interestingly, affected individuals develop CAD more frequently but not earlier than the general population.
Methods and Results— Genotyped primary human umbilical vein endothelial cells (ECs) were exposed to fluid shear stress (FSS) and analyzed for nitric oxide (NO) and superoxide anion (O2−) formation as well as mRNA and protein expression of different antioxidant enzymes. Dysfunctional CC-genotype ECs failed to upregulate NO synthase expression in response to FSS and exhibited a reduced NO synthesis capacity when compared to functionally intact TT-genotype ECs. However, only CC-genotype ECs responded to FSS with an Egr-1–mediated increase in manganese-containing superoxide dismutase (SOD-2) expression, shielding them from endothelin-1–induced oxidative stress in a NO-independent manner.
Conclusions— This FSS-induced rise in SOD-2 expression in CC-genotype ECs effectively stabilizes their antiatherosclerotic phenotype and may explain not only the comparatively slow onset of CAD in homozygous carriers of the C-allele of the nos-3 gene but also define a general strategy for preventing endothelial dysfunction at the outset of atherosclerosis.
Endothelial dysfunction characterized by a decreased bioavailability of nitric oxide (NO) permanently alters the endothelial cell (EC) phenotype from anti to proatherosclerotic.1,2
This reduced bioavailability of NO may be due to insufficient synthesis, ie, low expression or activity of endothelial NO synthase (NOS-3), or enhanced inactivation of NO, eg, by superoxide anions (O2−).
Recently, the −786C-variant of the human nos-3 gene has been shown to be insensitive to fluid shear stress (FSS), the physiologically most important stimulus in vivo for maintaining NOS-3 expression in ECs.3–5 As a consequence, homozygous carriers of the −786C-allele (>12% of all whites, cf 3 to 5) present with endothelial dysfunction typified by a reduced NO-mediated vasodilator response early in life.4 These individuals also have a significantly increased risk for coronary artery disease (CAD) the development of which, however, does not seem to be accelerated compared to patients without this genetic defect3–5 hence arguing for a compensatory mechanism.
In this context, our recently published proteome analysis of genotyped human umbilical vein ECs6 revealed that FSS exclusively upregulates expression of manganese-containing superoxide dismutase (SOD-2) in CC-genotype cells. Here we have investigated the mechanism by which FSS upregulates SOD-2 expression in these cells and its functional implications for the bioavailability of NO.
Full details of the methods used can be found in the supplemental material (available online at http://atvb.ahajournals.org).
In brief, primary ECs isolated from human umbilical cord veins were exposed to FSS (30 dyne/cm2) for the indicated periods by using a cone-and-plate viscometer. Analyses of protein and mRNA expression were performed by real time RT-PCR and Western blot, respectively. Protein detection in cultured and native ECs (umbilical cord veins) was done by immunofluorescence analysis. NO and O2− generation was assessed by using appropriate fluorescent and luminescent dyes. Egr-1–induced expression of the sod2 gene was characterized by electrophoretic mobility shift analysis (EMSA), the use of an appropriate decoy-oligodeoxynucleotide (dODN), and chromatin immuno-precipitation (ChIP). All data are expressed as means±SEM; statistical calculations were performed by paired t test or repeated-measure ANOVA followed by a Tukey-Kramer posthoc analysis as required.
CC-Genotype ECs Produce Less NO but More O2− Than TT-Genotype ECs
Measuring cellular NO formation by 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF) fluorescence revealed a markedly reduced NO synthesis capacity of CC as compared to TT-genotype human umbilical vein ECs (Figure 1A). Both basal and FSS-induced NOS-3 expression in these cells are strongly impaired or even absent,3–5 and this was confirmed by real time RT-PCR analysis (CC-genotype ECs: 57±6 and 61±11 mRNA copies per cell with or without exposure to FSS; TT-genotype ECs: 89±8 and 256±16 mRNA copies per cell; n=6, P<0.05).
To compare the O2− synthesis capacity of these cells both under static conditions and after exposure to FSS, both lucigenin-enhanced chemiluminescence (Figure 1B) and 5-carboxy-2′,7′-dichlorofluorescein (DCF) fluorescence microscopy analysis (cf supplemental Figure I) were used and essentially yielded the same results. Endothelin-1 (ET-1) induced a submaximal acute generation of O2− which was significantly higher in CC as compared to TT-genotype ECs (Figure 1B). Conversely, NOS-3 inhibition augmented ET-1–induced O2− formation 8-fold in TT but only 2.8-fold in CC-genotype ECs after exposure to FSS. This apparent FSS-induced increase in the capacity of CC-genotype ECs to scavenge O2− was further exemplified by the greater reduction of xanthine oxidase-derived O2− with lysates from CC as compared to TT-genotype ECs (Figure 1C).
FSS-Induced SOD-2 Expression Only Occurs in CC-Genotype ECs
Such a protective mechanism may comprise an increased expression of antioxidative enzymes (such as, eg, superoxide dismutase) in response to FSS.6 Whereas SOD-1 expression remained unaffected in either cell type (data not shown), both SOD-2 mRNA (414±60% vs. 107±20% of control) and protein (329±48 versus 122±27% relative intensity) levels were elevated solely in CC-genotype ECs after exposure to FSS (n=5, P<0.05). This finding was corroborated by the significantly greater abundance of SOD-2 protein in the endothelium of freshly isolated human umbilical cord veins from CC as compared to TT-genotype donors (Figure 2A, cf supplemental Figure II).
FSS-Induced SOD-2 Expression in CC-Genotype ECs Is Mediated by Egr-1
Because SOD-2 expression seems to be controlled by Egr-1,7 the role of this transcription factor in FSS-induced SOD-2 expression in CC-genotype ECs was investigated further. EMSA revealed that both basal and FSS-induced Egr-1 levels in the nucleus, indicative of its activation, were much higher in CC as compared to TT-genotyped ECs (Figure 2B). Moreover, by using a dODN neutralizing Egr-1, its critical role in FSS-induced SOD-2 expression in the CC-genotype ECs could be substantiated (Figure 2C). An additional ChIP assay using antibodies directed against Egr-1 and NFκ-B, another transcription factor implicated in SOD-2 expression, revealed that Egr-1 in a FSS-dependent manner binds to a canonical Egr-1 response element close to the transcription start side of the sod2 gene. Though weakly binding to 3 conserved motifs distributed across the sod2 gene, NFκ-B did not reveal any FSS-dependent activity (Figure 2D, cf supplemental Figure III).
As Egr-1 activity is thought to be NO-dependent,8 the observed differences in SOD-2 expression between the 2 genotypes could be related to their NO synthesis capacity. Exposure to the NO donor DETA-NONOate in fact blunted the FSS-induced rise in SOD-2 expression in CC-genotype ECs, whereas their TT-genotype counterparts responded with a robust increase in SOD-2 expression after NOS-3 inhibition (Figure 2E).
FSS-Induced SOD-2 Expression
The insensitivity of the −786C-variant of the nos-3 gene to FSS forms the basis for the increased risk of homozygous carriers of this single nucleotide polymorphism (SNP) to develop CAD. Although endothelial dysfunction can be detected rather early in life,4 manifestation of the disease in affected individuals does not seem to occur prematurely, hence pointing to the existence of (a) compensatory mechanism(s) balancing the insufficient endothelial NO synthesis associated with this genetic defect. Therefore, we compared the NO and reactive oxygen species synthesizing capacity of ECs derived from CC and TT-genotype carriers. Apart from corroborating the reduced ability of CC-genotype ECs to synthesize NO, we discovered that these cells are more resistant to agonist-induced oxidative stress in terms of an increased formation of O2−. As in our recent comparative proteome analysis6 we could confirm that SOD-2 but not SOD-1 expression is upregulated exclusively in CC-genotype ECs in response to FSS, and that SOD-2 protein is clearly more abundant in native ECs derived from CC-genotype individuals. This genotype-dependent difference in SOD-2 expression may account for the greater resistance of CC-genotype ECs to oxidative stress, an increased bioavailability of NO, and thus a temporally inconspicuous atherogenesis.
Egr-1: A Sensor for the Balance Between Oxidative Stress and NO?
In this regard, the NO-sensitive activity of the transcription factor Egr-1,8 which seems to control SOD-2 expression in CC-genotype ECs through binding to a specific sequence motif around position −180 of the sod2 gene, is particularly interesting. Whereas FSS-dependent activation of Egr-1 is inhibited by high levels of NO, low amounts—which may also be the result of an increase in O2− formation—strongly promote Egr-1 translocation to the nucleus in ECs. Because SOD-2 is highly effective in scavenging intracellular O2−, this molecular interplay between NO, Egr-1, SOD-2, and O2− may constitute a self-limiting compensatory control circuit in which Egr-1 acts as a sensor of the endothelial NO/O2− imbalance, not only in individuals homozygous for the −786C-variant of the nos-3 gene but also in endothelial dysfunction in general.
We thank Renate Cattaruzza and Anita Kühner for expert technical assistance.
Sources of Funding
This work was supported by the Deutsche Forschungsgemeinschaft (grant no. HE 1587/9-1).
Received December 19, 2008; revision accepted August 5, 2009.
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