Clinical and Experimental Pharmacology and Physiology (2015) 42, 860–864

doi: 10.1111/1440-1681.12427

ORIGINAL ARTICLE

Role of Nox inhibitors plumbagin, ML090 and gp91ds-tat peptide on homocysteine thiolactone induced blood vessel dysfunction Renee M Smith,* Peter Kruzliak,† Zuzana Adamcikova‡ and Anthony Zulli* *Centre for Chronic Disease Prevention and Management, Western CHRE, College of Health and Biomedicine, Victoria University, St Albans, Vic., Australia, †Department of Cardiovascular Diseases, International Clinical Research Center, St Anne’s University Hospital and Masaryk University, Brno, Czech Republic and ‡Department of Public Health, Faculty of Medicine, Pavol Jozef Safarik University, Kosice, Slovak Republic

SUMMARY Antioxidants have not reduced the burden of cardiovascular disease, and current evidence suggests a beneficial role of oxidative stress, via NADPH oxidase (Nox) upregulation, in endothelial function. Homocysteine thiolactone (HcyT) induces blood vessel dysfunction and this correlates with increased vascular oxidative stress. This study aimed to determine if pharmacological inhibition of Nox could impair HcyT induced blood vessel dysfunction. Abdominal aorta were excised from New Zealand White rabbits (n = 6), cut into rings and sequentially mounted in organ baths. Rings were preincubated with 0.55 lmol/L homocysteine thiolactone for 1 h, or combinations of putative Nox inhibitors (plumbagin for Nox4, gp91ds-tat for Nox2, and ML090 for Nox1), 30 min prior to the addition of HcyT, followed by a dose response curve to acetylcholine on phenylephrine preconstricted rings. Plumbagin, ML090 + gp91ds-tat and HcyT reduced responses to acetylcholine, and Plumbagin + Hcyt caused constriction to acetylcholine, which was normalised to plumbagin by ML090. Plumbagin + ML090 or plumbagin + gp91ds-tat completely impaired the effect of acetylcholine. ML090 inhibited the effect of HcyT on reduced response to acetylcholine, whereas gp91ds-tat had no effect. This study concludes that inhibition of Nox1 prevents, whereas inhibition of Nox4 worsens, acetylcholine induced blood vessel relaxation caused by HcyT, while Nox2 inhibition has no effect. However combinations of Nox inhibitors worsen acetylcholine induced blood vessel relaxation. These results suggest that there is cross-talk between Nox isoforms during physiological and pathophysiological processes.

Correspondence: Dr Anthony Zulli, College of Health and Biomedicine, Victoria University, Hoppers Ln, Werribee VIC 3030, Australia. Email: [email protected] Dr Peter Kruzliak, Department of Cardiovascular Diseases, International Clinical Research Center, St Anne’s University Hospital and Masaryk University, Pekarska 53, 656 91 Brno, Czech Republic. Email: [email protected] Received 5 January 2015; revision 29 April 2015; accepted 15 May 2015. © 2015 Wiley Publishing Asia Pty Ltd

Key words: endothelial dysfunction, NADPH oxidase, Nox, Nox inhibitors.

INTRODUCTION A ‘Western diet’ can increase risk factors for cardiovascular disease (CVD) such as high plasma cholesterol and homocysteine. The common underlying mechanism of action for these risk factors is to cause endothelial dysfunction as measured by relaxation to acetylcholine.1 It is well established that impaired relaxation to acetylcholine is due to a decrease in the bio-availability of nitric oxide (NO), as NO bioavailability is believed to be the key factor in the regulation of atherogenesis and hypertension.2–5 NO can react with free radicals6,7 (oxidative radical, O2 , formed by NADPH oxidases8,9) and forms peroxynitrite (ONOO ). The lack of NO stimulates atherogenesis, coupled with increased ONOO can inactivate proteins by nitrating the tyrosine residues of proteins, and we have shown high levels of nitrotyrosine in a rabbit model of coronary artery disease.10 Thus, by inhibiting the enzymes which produce O2 should re-establish normal endothelial function via restoration of nitric oxide (NO) bioavailabilty. However, the disappointing effects of antioxidant treatments in cardiovascular disease (CVD) have lead to the theory that targeted inhibition of the specific enzymes involved in oxidative stress should lead to improvements in CVD outcomes.11,12 In contrast, current published data by other research groups show the exact opposite. As there are different NADPH oxidase enzymes (Nox1, Nox2, Nox3, Nox4, Nox5, DuoX1, DuoX2) which all produce oxidative stress, including free radicals or hydrogen peroxide,13 a recent study by Shafique and colleagues have elegantly shown that overexpression of Nox2, specifically in endothelial cells, improves endothelial function in coronary arterioles of transgenic mice.14 Also, an earlier study showed similar results in mice aorta overexpressing Nox4.15 Taken together, it remains uncertain if targeting Nox enzymes would be beneficial in restoring endothelial dysfunction caused by excess free radical production. In this regard, this study sought to investigate whether inhibition of Nox enzymes with putative inhibitors pumbagin,16,17 ML09018,19 and gp91ds-tat,20–22 and combinations thereof, would affect blood vessel dilation to acetylcholine in both normal and rings incubated with homocysteine thiolactone (the atherogenic

Nox inhibitors and endothelial function 40 20

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Fig. 1 Effect of putative Nox inhibitors on endothelial function and after a 1 h incubation with 0.5 lmol/L homocysteine thiolactone. Hcyt, homocystine thiolactone; Plum, Plumbagin; GP, gp91ds-tat. Results are mean  SEM, and one ring per animal was used for n = 6 animals. ***P < 0.001, *P < 0.05 vs control values (compared point to point using two-way ANOVA to control, and HcyT + Plum to Plum; †P < 0.05). , HcyT + Plum; , Plum; , HcyT + GP; , HcyT; , GP; , HcyT + ML090; , ML090; , Con.

form of homocysteine), which is known to reduce acetylcholine mediated vascular relaxation.23 We chose a physiologically relevant dose of homocysteine thiolactone, which is reported as being 5% of total circulating homocysteine,24 and considering clinical studies linking homocysteine to cardiovascular disease show a range between 9.5 and 13 lmol/L, we chose to use a midpoint of 11 lmol/L.25

RESULTS As expected, the addition of HcyT for 1 h reduced acetylcholine mediated vasodilation compared to control (82  5% vs 99  0.9%, P < 0.05). Only the addition of plumbagin severely blunted the affect of acetylcholine (26  6.8% vs 99  0.9%,

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P < 0.001), and this effect was worsened by the addition of HcyT (contraction to 18  6.9% vs 26  6.8%, P < 0.05). The addition of ML090, but not gp91ds-tat, 30 min prior to the addition of HcyT blocked the effect caused by HcyT (98  0.6% vs 82  5%, P < 0.05). There was a trend to a reduced response to acetylcholine by gp91ds-tat (GP) but not ML090 per se (86.2  4.1%, 97.3  0.8% vs 99  0.9%, P = ns; Fig. 1, Table 1). As the addition of HcyT to plumbagin worsened the response to acetylcholine, this effect of HcyT was impaired by ML090 (contraction to 18  6.9% vs 22  5.5%, P < 0.05), and partially by gp91ds-tat (contraction to 18  6.9% vs 3.7  3.1%, P < 0.05), but was not restored by the combination of gp91dstat + ML090 (contraction to 18  6.9% vs contraction to 6.7  6.5%, P = ns; Fig. 2). The combination of gp91ds-tat + ML090 reduced the response to acetylcholine vs control (69  5% vs 99  0.9%, P < 0.001), but not compared to gp91ds-tat or ML090 alone (86.2  4.1%, 97.3  0.8%, respectively, P = ns; ML090 alone shown in Fig. 1). The addition of HcyT worsened the response to acetylcholine vs GP + ML090 only at 3 9 10 6 acetylcholine (83  2.5% vs 72  3.1%, P = 0.042; Fig. 3). Unexpectedly, the combination of either gp91ds-tat or ML090 to plumbagin completely abrogated the response to acetylcholine compared to control (plumbagin + gp91ds-tat, constriction to 5.6  14%, plumbagin + ML090, constriction to 8.2  4.2, plumbagin + gp91ds-tat + ML090, constriction to 6.5  5.6% vs 99  0.9%, P < 0.001) and plumbagin alone (P = 0.043, Fig. 4).

DISCUSSION The major finding of this study is that the putative Nox1 inhibitor, ML090, blocked the effect of HcyT on the response to acetylcholine in the aorta, but this was reversed or worsened when ML090 was used in combination with gp91ds-tat or plumbagin. As well, plumbagin or the addition of ML090 + gp91ds-tat worsened the response to acetylcholine in the aorta.

Table 1 Agonist potency (EC50) and maximal effect (Emax) of acetylcholine induced relaxations in rabbit aorta after treatments. EC50 significance is taken at P < 0.05, and significant group inserted into appropriate text box. NS, not significant

Con ML HcyT + ML Plum HcyT + Plum GP HcyT + GP ML + GP HcyT + ML + GP Plum + GP HcyT + Plum + GP Plum + ML HcyT + Plum + ML Plum + ML + GP HcyT + Plum + ML + GP

Emax %

SEM

99.0 97.4 97.8 26.1 17.7 86.3 80.8 68.8 57.6 5.4 3.7 8.2 22.8 6.5 6.7

0.9 0.8 0.6 6.8 6.8 4.1 5.3 4.8 6.9 13.8 3.1 4.2 5.5 5.6 6.5

EC50 Log [M] 7.7 7.7 7.6 6.3 6.2 7.3 7.5 7.2 6.7 6.1 6.3 6.5 6.1 6.8 6.4

SEM

Figure 1

0.1 0.0 0.2 0.6 0.3 0.1 0.1 0.2 0.6 0.3 0.5 0.3 0.3 0.6 0.4

Plum; HcyT + Plum Plum; HcyT + Plum Plum; HcyT + Plum HcyT + GP HcyT + GP

© 2015 Wiley Publishing Asia Pty Ltd

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Figure 4 Plum

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-100 1 -8 10

2 -8 3x10

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Con 10-8

Fig. 2 Effect of Nox inhibition on background Nox4 inhibition with pre-incubation with 0.5 lmol/L homocysteine thiolactone. Hcyt, homocystine thiolactone; Plum, Plumbagin; GP, gp91ds-tat. Results are mean  SEM, and one ring per animal was used for n = 6 animals. *P < 0.05, all values compared point to point using two way ANOVA to ), HcyT + Plum; ( ), HcyT + Plum + HcyT + Plum. ( GP + ML090; ( ), HcyT + Plum + GP; ( ), HcyT + Plum + ML090; ( ), Plum.

Endothelial dysfunction, as measured by a reduction in blood vessel response to acetylcholine, is strongly associated with decreased NO bioavailability due to scavenging by excess oxidative radicals26 caused by excess NADPH oxidase activity,27 yet oxidative stress therapy with antioxidants have clearly shown no benefit in the reduction of CVD mortality.28 To test if excess oxidative stress was the cause of blood vessel dysfunction in our model, we pre-incubated independent aortic rings with the putative blockers ML090 for Nox1, gp91ds-tat for Nox2, and plumbagin for Nox4. Only ML090 inhibited the effect of homocysteine thiolactone in this model, indicating a strong role for

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Fig. 3 Effect of Nox1 and Nox2 inhibition on background pre-incubation with 0.5 lmol/L homocysteine thiolactone. Hcyt, homocystine thiolactone; GP, gp91ds-tat. Results are mean  SEM and one ring per animal was used for n = 6 animals. *P < 0.05. Comparisons made HcyT + GP + ML090 vs GP + ML090; GP + ML090 vs Con. ( ), HcyT + GP + ML090; ( ), GP + ML090; ( ), HcyT; ( ), GP; ( ), Con.

Fig. 4 Effect of Nox inhibition on background Nox4 inhibition. Plum, Plumbagin; GP, gp91ds-tat. Results are mean  SEM and one ring per animal was used for n = 6 animals. *P < 0.05 vs Plum. ( ), Plum + GP + ML090; ( ), Plum + GP; ( ), Plum + ML090; ( ), Plum; ( ), Con.

Nox1 in homocysteine thiolactone induced vascular damage. The relationship between Nox1 and homocysteine is controversial.29,30 For example, in cystathionine beta synthase homozygote knockout mice, Nox1 mRNA and activity is increased,29 yet homocysteine did not affect Nox1 in cultured human umbilical vein endothelial cells. Our data support the theory that homocysteine induces Nox1 activity, in that inhibition of Nox1 impairs the blood vessel dysfunction caused by homocysteine thiolactone. Inhibition of Nox1 + Nox2, but not either alone, inhibited the response to acetylcholine in aorta. This data suggests that basal activation of Nox1 and Nox2 are important in the normal blood vessel response to acetylcholine. Indeed, the oxidative radical, O2 , which is produced by Nox1 and Nox2, can activate eNOS.31 In addition, the data presented here supports a role for H2O2, which is produced by Nox4,32 in basal endothelial function, as inhibition of Nox4 by plumbagin severely blunted endothelial response to acetylcholine. It is clear from the literature that H2O2 can activate kinases which activate eNOS31 and genetic upregulation of Nox4 enhances blood vessel dilation in mice,15 and genetic deletion of Nox4 reduces endothelial function.33 Unexpectedly, the combination of Nox4 inhibition with either Nox1 or Nox2 inhibition completely abrogated the vasodilative response to acetylcholine in this model. This suggests a vital and fundamental role for both O2 and H2O2 in eNOS signalling,31 rather than the minor role previously thought. Further studies are warranted to determine the role of both Nox isoforms in normal blood vessel physiology. The data presented in this study show that the addition of homocysteine thiolactone to aorta for 1 h impaired the vasodilative response to acetylcholine if Nox4 or both Nox1 and Nox2 were inhibited, but interestingly, combining Nox4 with either Nox2 or Nox1 inhibition impaired the effect of homocysteine thiolactone on a Nox 4 inhibited background. This data suggests that Nox1 and Nox2 are beneficial in homocysteine induced vascular dysfunction, as this could allow superoxide dismutase and Nox4 to produce H2O2, thus promoting eNOS signalling,31 and this theory is further supported by the addition of a Nox4

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Nox inhibitors and endothelial function inhibitor, showing a marked decrease in vascular dilation from approximately 80–20%. Moreover, this study shows that Nox4 inhibition prior to the addition of HcyT completely blunts the response to acetylcholine in the aorta (Fig. 1), but this effect appears to be inhibited by blocking Nox2, and blunted by blocking Nox1, but not both (Fig. 2). This data suggests that on a background of either control or Nox4 inhibition, the addition of HcyT could stimulate Nox1 to induce blood vessel dysfunction.

STUDY LIMITATIONS Use of putative Nox inhibitors in this study have not been fully characterised in an in vitro model. It remains unknown if ML090, plumbagin or gp91ds-tat have other ‘off target’ effects which can influence acetylcholine induced, NO-dependent vascular relaxation. Nox inhibitors could have ‘off target’ effects on smooth muscle cell function. As endothelial denuded rings cannot be used in this study as that will cause abolishment of acetylcholine induced vascular relaxation, the term ‘response to acetylcholine’ has been used to reflect more accurately the possibility that both endothelia and smooth muscle cells could be affected. In conclusion, this study confirms a role for putative Nox inhibitors in response to acetylcholine in aorta. Whether or not these effects are due to ‘off’ or ‘on’ target effects remains to be validated.

METHODS Male New Zealand White rabbits (n = 6) at 3 months of age were killed by ketamine/xylazine overdose as previously described in our laboratory34,35. The abdominal aorta was removed (from iliac bifurcation to renal artery branch), cleaned of connective tissue and fat, cut into 3 mm rings, and placed in organ baths (OB8, Zultek Engineering, Melbourne, Vic., Australia) for vasoactivity studies. These studies were approved by Victoria University Animal Ethics Committee (Approval #12/019). Aortic reactivity studies The baths were filled with Krebs solution and kept at a constant temperature of 37°C and continuously bubbled with 95% O2/5% CO2 for 1 h. The rings were then stretched to 2.5 g, and after 30 min the rings were re-stretched to their original tension and allowed to stabilise for 2 h. After this, all rings were subjected to a high potassium physiological salt solution (124 mmol/L K+) to induce maximal constriction and after the rings reached plateau (approximately 15 min), the rings were repeatedly flushed with Krebs solution and allowed to rest for 1 h. Separate rings (one ring per animal was used from n = 6 animals per test per drug) were then incubated with (all 1 lmol/L) plumbagin to inhibit Nox4, ML090 to inhibit Nox1, gp91ds-tat to inhibit Nox2, or a combination thereof for 30 min prior to the incubation with 0.55 lmol/L HcyT for 1 h. This dose was chosen as it is 5%21 of 11 lmol/L Hcy.25 Then, rings were precontracted with phenylephrine to 50–70% of maximal constriction to 124 mmol/L K+ and allowed to plateau (30 min),36 followed by a cumulative dose response curve to acetylcholine (10 8 mol/L to all 10 5 mol/L, half log units). After the last dose, a bolus dose of

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sodium nitroprusside was used (10 dilated further.

5

mol/L) to ensure rings

Statistical analysis All groups were compared via two way analysis of variance (ANOVA) using GRAPHPAD PRISM, La Jolla, CA 92037, USA or 1 way ANOVA for Emax, followed by a Bonferroni multiple comparison test. Results are presented as mean  SEM. Significance was taken at P < 0.05 in all cases.

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