TNF-α Contributes to Endothelial Dysfunction by Upregulating Arginase in Ischemia/Reperfusion Injury
Background— We tested whether tumor necrosis factor (TNF)-α increases arginase expression in endothelial cells as one of the primary mechanisms by which this inflammatory cytokine compromises endothelial function during ischemia-reperfusion (I/R) injury.
Methods and Results— Mouse hearts were subjected to 30 minutes of global ischemia followed by 90 minutes of reperfusion and their vasoactivity before and after I/R was examined in wild-type (WT), tumor necrosis factor knockout (TNF−/−), and TNF 1.6 (TNF++/++) mice. In WT mice, dilation to the endothelium-dependent vasodilator ACh was blunted in I/R compared with sham control. L-arginine or arginase inhibitor NOHA restored NO-mediated coronary arteriolar dilation in WT I/R mice. O2− production was reduced by eNOS inhibitor, L-NAME, or NOHA in WT I/R mice. In TNF−/− mice, I/R did not alter Ach-induced vasodilation and O2− production compared with sham mice. The increase in arginase expression that occurs during I/R in WT mice was absent in TNF−/− mice. Arginase expression was confined largely to the endothelium and independent of inflammatory cell invasion. Arginase activity was markedly lower in TNF−/−, but higher in WT I/R than that in WT sham mice.
Conclusions— Our data demonstrate TNF-α upregulates expression of arginase in endothelial cells, which leads to O2− production then induces endothelial dysfunction in I/R injury.
Endothelial dysfunction has been proposed as one of the mechanisms that contributes to microvascular injury and hypoperfusion after ischemia-reperfusion (I/R).1 Arginase expressed in the endothelium serves as an endogenous competitor of nitric oxidase synthase (NOS) for L-arginine and thus plays a counteracting role in NO-mediated vasodilatory function. A key observation from our previous study1 is that I/R inhibits nitric oxidase (NO)-mediated dilation of coronary arterioles; this occurs by increasing the activity of the enzyme arginase in endothelial cells, which limits the availability of L-arginine to NOS for NO production. Upregulated expression and activity of arginase have been found in corpus cavernosum of diabetic individuals.2 Moreover, an elevated level of arginase activity in cardiac tissue has been shown to be associated with clinical episodes of I/R such as myocardial infarction.2,3 However, the pathophysiological role and the upstream pathway of this upregulated arginase in coronary vasomotor regulation has not been investigated.
TNF-α (TNF-α), a cytokine that is known to be upregulated during I/R,4 can contribute to the increase in arginase activity in bovine pulmonary arterial endothelial cells5 and inhibits endothelium-dependent relaxation of cat coronary arteries to acetylcholine (ACh).6 We hypothesized that TNF-α upregulates endothelial arginase in I/R, which causes a reduction of L-arginine availability to NOS, and leads to superoxide (O2−) production, thus impairing NO-mediated vasodilation. To test this hypothesis, we examined endothelium-dependent NO-mediated dilation in the absence and presence of L-arginine or specific arginase inhibitor, Nω-hydroxy-nor-l-arginine (NOHA) in I/R, the effect of I/R on arginase activity and arginase protein expression in isolated coronary arterioles in wild-type (WT), tumor necrosis factor knockout (TNF−/−), and TNF 1.6 (TNF++/++, a model with cardiac-specific overexpression of TNF-α) mice.
Murine Ischemia/Reperfusion (I/R) Model
The procedures followed were in accordance with approved guidelines set by the Laboratory Animal Care Committee at Texas A&M University. This study used 12- to 15-week-old, 25- to 35-g mice of either sex. We used 2 different strains of WT mice to match the backgrounds of the TNF−/− and TNF++/++ mice. For the TNF−/− mice (Jackson Laboratory; Bar Harbor, Maine), the WT1 is strain B6, 129SF2/J (Jackson Laboratory). Mice, homozygous for TNF++/++ were obtained from coauthor Dr Arthur M. Feldman. The parental strain for this transgenic line is the FVBN, and WT2 controls for this group consist of the FVBN line. The surgical protocol was performed similar to methods described previously7,8 with minor modification. After 30 minutes of the left anterior descending (LAD) coronary artery occlusion and 90 minutes of reperfusion, the heart was harvested and used for functional study or other assays. A sham group was subjected to the same surgical interventions without performing occlusions. We defined WT1 and 2 as WT in this study because the results from WT1 and 2 were identical.
Functional Assessment of Isolated Coronary Arterioles
Please see the supplemental materials, available online at http://atvb.ahajournals.org
mRNA Expression of TNF-α Receptors by Real-Time PCR
Total RNA was extracted from isolated coronary arterioles using Trizol reagent (Life Technologies Inc), and was processed directly to cDNA synthesis using the SuperScript III Reverse Transcriptase (Life Technologies Inc).9 The primers of TNF-α receptor1 (TNF-R1) and 2 (TNF-R2) were designed (primer 3 software) and synthesized (Qiagen). cDNA was amplified using qRT-PCR Kit with SYBR Green (Life Technologies Inc). Data are calculated by 2−ΔΔCT method and are presented as fold change of transcripts for TNF-α receptors’ gene in WT I/R mice normalized to GAPDH, compared with WT sham mice (defined as 1.0-fold).
Electron Paramagnetic Resonance (EPR) Spectroscopy
Please see the supplemental materials.
NOS Activity Assay
NOS detection Kit (Cell Technology Inc) was used for testing NOS activity. Coronary arterioles (5 to 7 vessels per sample, 100 μm inner diameter) in WT-sham, WT-I/R, TNF−/−-sham, TNF−/−-I/R, TNF++/++-sham, and TNF++/++-I/R mice were freshly isolated. NOS activity was measured via fluorescence plate reader at excitation 488 nm and emission at 515 nm. Data are presented as fold change compared with sham-control mice (defined as 1.0-fold).
Arginase Activity Assay
Coronary arterioles (5 to 7 vessels per sample, 100 μm inner diameter) in sham-control and I/R groups from WT, TNF−/−, TNF++/++ mice, and WT mice treated with anti–TNF-α [the neutralizing antibodies to TNF-α (anti-TNF-α, a gift from Dr Gregory Bagby [Department of Physiology, LSUHSC, New Orleans, La], I.P. 0.1 mg/mouse containing 16 mg protein/mL) were administered 3 hours before initiating I/R in WT mice]8 were isolated and prepared in lysis buffer for the arginase activity assay as described previously.1,10 The urea concentration was determined spectrophotometrically by the absorbance at 550 nm measured with a microplate reader (Bio-Tek Instruments). The amount of urea produced, after normalization with protein, was used as an index for arginase activity.
For Western blot analysis,11 isolated coronary arterioles (3 to 4 vessels per sample) before and after I/R were homogenized and sonicated in lysis buffer. Protein concentrations were assessed with use of the BCA kit, and equal amounts of protein (40 μg) were separated by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad). The protein expression of Arginase I or eNOS was tested with the use of rabbit Arginase I polyclonal IgG primary antibody (Santa Cruz Biotechnology) or eNOS primary antibody (Abcam). Horseradish peroxidase-conjugated goat anti-mouse was used as the secondary antibody (Abcam). Signals were visualized by enhanced chemiluminescence (ECL) (Amersham), and quantified by Quantity One (BioRad Versadoc imaging system).
Formalin fixed hearts were embedded in paraffin, sectioned at 7 μm, and mounted on slides. The heart sections8 were incubated with blocking solutions (BSA 3% in Tris buffer) and then with mouse monoclonal anti-arginase antibody (BD Biosciences) and rabbit polyclonal anti-myeloperoxidase (Abcam) or rabbit anti-human Von Willebrand Factor (VWF, Dako Cytomation) and mouse monoclonal anti-arginase antibody overnight at 4°C. Secondary antibodies were added, then sections were mounted in anti fading agent (Slow fade with Dapi, Molecular probe). The slides were observed and analyzed using a fluorescence microscope (Leica microscope with a 63× objective).
All drugs were obtained from Sigma, except as specifically stated. ACh, SNP, L-NAME, AMT, NOHA,12 L-arginine, and D-arginine were dissolved in PSS for functional studies and in PBS for fluorescence detection. Vehicle control studies indicated that these final concentrations of solvent had no effect on the arteriolar function.
At the end of each experiment, the vessel was relaxed with 100-μmol/L SNP to obtain its maximal diameter at 60 cm H2O intraluminal pressure.8,13 All diameter changes in response to agonists were normalized to the vasodilation in response to 100 μmol/L SNP and expressed as a percentage of maximal dilation. All data are presented as mean±SEM, except as specifically stated (eg, as mean±SD for molecular study). Statistical comparisons of vasomotor responses under various treatments were performed with one-way or two-way ANOVA and intergroup differences were tested with Bonferonni Inequality. Significance was accepted at P<0.05.
Effect of Arginase and TNF-α in ACh-Induced Coronary Arteriolar Dilation in I/R Injury
Please see the supplemental materials (Figure I).
Effect of eNOS, iNOS, Arginase, and TNF-α on Superoxide Production in I/R
Please see the supplemental materials (Figure II).
I/R Increases mRNA Expression of TNF-Rs and the Effect of TNF-α on NOS and Arginase Activity in I/R
mRNA expression of TNF-R1 increased about 2-fold in isolated coronary arterioles in I/R versus sham control in WT mice, but there were no significant differences in mRNA expression of TNF-R2 in I/R versus sham mice (Figure 1A). Administration of arginase inhibitor, NOHA (0.1 mmol/L, 60 minutes prior to initiating I/R), did not affect the mRNA expression of TNF-R1 or TNF-R2 in I/R and sham mice (data not shown). This result confirmed that arginase expression did not affect the level of TNF-R expression.
NOS and arginase activity was assayed in the coronary arteriolar lysate to further elucidate the role of TNF-α in the upregulation of arginase in I/R (Figure 1B). NOS activity was significantly decreased in WT I/R, TNF++/++ sham, and TNF++/++ I/R mice and was not altered in TNF−/− mice before and after I/R compared with WT sham mice (Figure 1B); whereas arginase activity was increased in WT I/R, TNF++/++ sham and I/R mice, but it was not statistically significant from TNF−/− mice; neutralizing antibodies to TNF-α (Ab) blocked induction of arginase activity following I/R in WT mice (Figure 1C). This result further confirmed that TNF-α–induced arginase expression might be the cause of the impairment of NO-mediated vasodilation.
Effect of TNF-α on Arginase I Expression in Coronary Arterioles During I/R
The protein expression of arginase I was lower in TNF−/− mice and higher in TNF++/++ mice than that in WT mice (Figure 2A and 2B), which suggested that arginase titer is inversely linked to TNF-α titer. Arginase expression was significantly increased in WT I/R mice, but was not changed in TNF−/− mice after I/R. I/R did not increase arginase expression in TNF++/++ mice, perhaps because arginase expression mediated by TNF-α was already maximal or saturated in the overexpression (TNF++/++) mice (Figure 2A and 2B). Additionally, anti–TNF-α decreased arginase I expression in WT-I/R versus WT-sham mice (Figure 2C and 2D). These results suggested that TNF-α plays an important role in upregulating arginase in I/R, which may then cause the impairment of NO-mediated vasodilation in I/R.
Effect of TNF-α on eNOS Expression in Coronary Arterioles During I/R
The protein expression of eNOS was decreased in I/R versus sham in WT and TNF++/++ mice (Figure 3). These results suggested that TNF-α plays an important role in downregulating eNOS in I/R, which might be another potential mechanism in the impairment of NO-mediated vasodilation in I/R.
Coimmunostaining of Arginase and Myeloperoxidase (MPO) in Coronary Arterioles
We performed immunohistochemistry for arginase and MPO, which is expressed by neutrophil cells, to determine whether arginase was produced by inflammatory cells in coronary microvessels in I/R injury. Our results show that there were no signals for arginase and MPO in sham animals (Figure 4A through 4C), but arginase and MPO were both expressed in coronary vessel wall in I/R mice (red staining with pink arrow for arginase and green with white arrow for MPO, Figure 4D through 4F). Our data show that the increased expression of arginase in I/R was not attributable to an increase of inflammatory cell migration into the vessels because the staining of arginase did not colocalize with the MPO staining (Figure 4, overlay F). Interestingly the inhibition of arginase by NOHA seems to have an effect on neutrophil cell infiltration into the heart in I/R (Figure 4G through 4I). Finally, we used a double immunostaining of arginase and an endothelial cell marker VWF to explore whether arginase was localized in endothelial cells in I/R. As shown in Figure 4 (J through L), the arginase and VWF were almost completely colocalized indicating that the arginase was expressed by endothelial cells in coronary vessels in I/R. The incubation of the preparation with the secondary antibodies did not show nonspecific staining in the coronary microvessels (Figure 4M through 4O, blue arrow). Quantification of MPO-positive cells shows that there were 0 cells per vessel in sham, 3.1 cells per vessel in I/R, and 0.1 cells per vessel in NOHA+I/R in WT mice.
Our results indicate that TNF-α upregulates endothelial arginase in I/R, which causes a reduction of L-arginine availability to NOS, and leads to O2− production, thus impairing NO-mediated vasodilation. Specifically, we found that inhibition of arginase partially restored the impaired vasodilation in I/R in WT and TNF++/++ mice, but did not affect vasodilation in I/R in TNF−/− mice. Importantly, endothelial dysfunction did not occur and O2− production was not increased after I/R in the TNF−/− mice. Arginase expression was elevated after I/R in the WT and TNF++/++ mice, but not affected in the TNF−/− mice, which indicated that TNF-α expression affects expression of arginase in endothelial cells. O2− production was inhibited by arginase inhibitor, NOHA, or NOS inhibitor, L-NAME, which indicates that increased production of O2− in I/R was also partially arginase- and eNOS-dependent. Our immunostaining results showed that arginase was increased in endothelial cells, but not because of the invasion of inflammatory cells in I/R injury. Importantly, our results also indicated that neutralizing antibodies to TNF-α blocks the induction of arginase activity and expression after I/R in WT mice. Taken together, we believe that TNF-α expression compromises coronary endothelial function by increasing arginase expression, leading to O2− generation following myocardial I/R injury.
Roles of L-Arginine and Arginase in Coronary Arteriolar I/R Injury
Episodes of I/R have deleterious effects on endothelial function.14 Endothelial dysfunction is characterized by reduced endothelium-dependent vasodilation, expression of adhesion molecules, adherence of inflammatory cells, transmigration of white blood cells, and production of oxygen free radicals.14 These events will eventually contribute to the development and evolution of myocardial necrosis. Thus endothelial dysfunction may be a key factor in the development and expansion of reperfusion injury. We previously showed that vasodilation to endothelial-dependent vasodilator, ACh, was markedly attenuated in isolated coronary arterioles during I/R in WT mice; but vasodilation to endothelium-independent vasodilator, SNP, was not affected by I/R,8 which suggests that I/R injury in the vasculature is confined to the endothelium. This is consistent with our current findings and the findings by Bohm et al.15
Recent studies16 have implicated a protective effect of NO in the setting of I/R, and it is well accepted that endothelium-derived NO is an important endogenous vasodilator that regulates microvascular tone.10,17 Thus, attenuation of NO production in I/R injury may have devastating consequences. Because NO synthesis requires oxidation of L-arginine via NOS, our findings on the improvement of NO-mediated dilation by L-arginine after I/R in WT and TNF++/++ mice suggest that a reduction in availability of the NOS precursor was involved in the vascular dysfunction and the limited L-arginine might lead to reduced NO and increased O2− anion formation, promoting cardiovascular dysfunction. This observation is in accord with previous evidence where L-arginine supplementation ameliorated endothelium-dependent dilation of large coronary arteries in a feline I/R model.18
We previously demonstrated that myocardial I/R initiated expression of TNF-α, which induces production of O2−, leading to coronary endothelial dysfunction.8 We also found that I/R inhibits NO-mediated dilation of coronary arterioles by increasing arginase activity in endothelial cells, which limits the availability of L-arginine to NOS for NO production.1 However, previous work provides no evidence that the production of O2− is NOS and/or arginase dependent in I/R injury. Vasquez-Vivar et al reported that limitations in arginine availability can switch eNOS from an NO generating NAD(P)H oxidase to a O2− generating enzyme.19 We then used the NOS inhibitor L-NAME to evaluate whether NOS is a source of O2−, because under certain conditions NOS can behave as an NAD(P)H oxidase and produce O2− in amounts greater than NO. The result shows that one of the sources of O2− production is NOS dependent. Our purpose is not to study every cofactor for NOS or the isoforms of NOS, but rather to focus on the role of TNF-α in endothelial injury after I/R, and examine the basis by which TNF-α induces these effects. O2− production in TNF−/− mice was reduced, but increased in TNF++/++ in I/R, which supports the contention that eNOS might be uncoupled from NO production and converted to O2− production. In addition, arginase inhibitor NOHA also prevented O2− production in WT I/R mice, which indicated that arginase is involved in I/R induced O2− production because overexpression of arginase by limiting the arginine pool converted activated eNOS (or any NOS) into an enzyme that produces O2− instead of NO. It is possible that increased expression of iNOS could limit arginine availability and hinder dilation by eNOS. In some other systems, inhibition of arginase has a proinflammatory effect and arginase can protect tissue by generation of polyamines,20 suggesting the level of iNOS in a tissue and how expression of arginase impacts on arginine levels and iNOS activity. However, we do not believe iNOS is involved in the decreased endothelial dependent dilation, because administration of an iNOS inhibitor failed to restore endothelium-dependent dilation, and did not affect superoxide levels.
The Link Between Arginase and TNF-α in Endothelial Dysfunction of Coronary Arterioles in I/R Injury
Endothelial arginase activity in the endothelium mitigates NO production, and thus NO-mediated vasodilatory function, because of consumption of arginine. In the present study, we found that mRNA expression of TNF-R1 increased about 2-fold in WT I/R mice, but arginase inhibitor NOHA did not affect it, which suggests that arginase did not influence the expression of TNF-R. Our studies show that in TNF−/− mice, I/R has no effect on either ACh-induced or SNP-induced vasodilation. This indicates TNF-α plays an important role in I/R impaired vasodilation. To elucidate the relationship between arginase and TNF-α during I/R, we first studied whether endothelial arginase can modulate the overall NO production for dilation in coronary arterioles in WT, TNF−/−, and TNF++/++ mice before and after I/R. Our studies show that arginase inhibitor NOHA improved the NO-mediated vasodilation in WT I/R, TNF++/++ sham, and TNF++/++ I/R mice, but did not affect the NO-mediated vasodilation in WT sham, TNF−/− sham, and TNF−/− I/R mice. Second, we studied the expression of arginase in WT, TNF−/−, and TNF++/++ mice before and after I/R. Endothelial cells can express both isoforms of arginase (arginase I and II), however previous evidence in porcine1,10 and our preliminary experiments in murine coronary arterioles from our laboratory has indicated that arginase I but not arginase II was expressed in coronary arterioles, and this is why we focused on arginase I. Our present results show that arginase expression was higher in TNF++/++ mice and lower in TNF−/− mice. I/R increased arginase expression in WT and TNF++/++ mice, but not in TNF−/− mice. Endothelial function at baseline was diminished in the TNF++/++ versus WT mice and protein expression of arginase was increased at baseline in the TNF++/++ mice. Our results indicate that the I/R increased transcripts for TNF-α in the WT groups, but did not influence those in the TNF++/++ groups. Because TNF-α expression in the TNF++/++ animals is driven by the cardiac myosin promoter, we do not think its expression was sensitive to I/R. Arginase activity was lower in TNF−/− mice than that in WT mice. I/R increased arginase activity in WT mice, but not in TNF−/− mice, as supported by our Western blot analyses. Furthermore, anti–TNF-α decreased arginase I expression and activity in WT I/R mice. Our results indicate that I/R induces TNF-α and TNF-R1 expression, followed by elevation of arginase I, which in turn causes a reduction of L-arginine availability to NOS, and thus impairs NO-mediated vasodilation. Our data demonstrate a physiological role of endothelial arginase in modulating NO-mediated dilation of coronary arterioles and provides a putative pathophysiological route for arginase upregulation in microvascular dysfunction during I/R.
Because TNF-α can block eNOS expression,21,22 it is very important to determine whether eNOS protein levels are also altered in the TNF++/++ mice before and after I/R. This may provide an important mechanism by which endothelial function is adversely affected by TNF-α. Our results show that: (1) I/R decreased NOS activity in WT mice, but I/R did not further decrease NOS activity in TNF++/++ mice and I/R did not affect NOS activity in TNF−/− mice; (2) protein expression of eNOS was decreased in I/R versus sham, in WT and TNF++/++ mice. Although these mechanisms could contribute to the impairment of NO-mediated dilation during I/R, the predominant mechanism in the present model appears to be related to the upregulation of arginase.
Expression of Arginase in Endothelial Cells in I/R Injury
Endothelial function is paramount in maintaining the barrier properties of the endothelium against invasion of inflammatory cells, which many (not all) believe contribute to reperfusion injury. Preservation of endothelial function will both preserve vasomotor control mechanisms and reduce the influx of inflammatory cells. To determine whether the enhanced arginase activity and protein after I/R was attirbutable to its expression in endothelial or inflammatory cells, immunocytochemical determination of their presence in the present study was performed. Our immunostaining results show arginase is expressed in endothelial cells in I/R. Arginase inhibitor NOHA: (1) decreased MPO staining, (2) decreased inflammatory cells migration in I/R, and (3) prevented the invasion of inflammatory cells. These results indicate that the increased levels of arginase in I/R was expressed in endothelial cells in coronary vessels, but was not attributable to an increase of inflammatory cell migration into the vessels because the staining of arginase was independent of MPO. This was further supported by the quantification of MPO-positive cells, which shows that animals treated with NOHA have 0.1 cells/vessel after I/R, and animals without this inhibitor, have 3.1 cells per vessel.
In conclusion, the results of the present study indicate that upregulation of vascular arginase induced by TNF-α inhibits NO-mediated vasodilation during I/R. Arginase is an endogenous competitor of NOS for their common substrate L-arginine, and consequently upregulation of arginase during I/R compromises NO-mediated vasodilatory function through the downregulation of substrate availability. Knowledge gained from this study will provide a better understanding of the mechanism(s) underlying endothelial dysfunction, defined as blunted endothelial dilation of coronary arterioles, after myocardial I/R injury.
Sources of Funding
This study was supported by grants from American Heart Association Scientist Development Grant (110350047A), Pfizer Atorvastatin Research Award (2004-37), NIH grants (RO1-HL077566 and RO1-HL085119) to Dr Cuihua Zhang.
X.G., X.X., and S.B. contributed equally to this study.
Original received November 23, 2006; final version accepted March 21, 2007.
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