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Commentary

Does Inhibition of Nuclear Factor Kappa B Explain the Protective Effect of Ticagrelor on Myocardial Ischemia–Reperfusion Injury?

Birnbaum, Yochai MD*; Ye, Yumei MD; Perez-Polo, Jose Regino PhD

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Journal of Cardiovascular Pharmacology: February 2020 - Volume 75 - Issue 2 - p 108-111
doi: 10.1097/FJC.0000000000000787
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We have read with interest the article “Ticagrelor Reduces Ischemia-Reperfusion Injury Through the NFκB–Dependent Pathway in Rats” by Liu et al in the J of Cardiovasc. Pharmacol. July 2019 issue.1

We were puzzled by the minimal number of cytokines tested as there are many multiplex assay systems that measure with great reliability and sensitivity a host of both inflammatory and anti-inflammatory cytokines. This is particularly relevant given that as the authors state “TNFα stimulates the production of anti-inflammatory factors such as IL-10 and IL-13, which could inhibit the transcription and expression of proinflammatory factors” and again “The production of anti-inflammatory factors might somewhat reduce the myocardium damage caused by the excessive increase of inflammatory factors.”1

A major finding of the article has to do with the effects of ticagrelor on the inflammatory activity of the transcription factor nuclear factor kappa B (NFκB). Thus, we were surprised by the reliance on reverse transcription polymerase chain reaction and Western blots for total p65 as indices of NFκB activity. Given that p65 is activated by phosphorylation allowing for its translocation to the nucleus and subsequent binding to DNA promoters, and especially given the authors own reference to: “Han et al2 found that the activation of NFκB aggravated myocardial ischemia reperfusion injury.”

NFκB is a transcription factor that has an important role in induction of genes involved in inflammation and response to injury.3 The family of NFκB transcription factors is typically composed of inactive dimers present in the cytoplasm whose nuclear localization sequence (signal peptide) is inhibited by bound IκBα/β proteins.3,4 Activation of NFκB allows entry into the nucleus of the active phosphorylated dimers. There are 2 pathways for activation. There is a pathway consistent with inflammatory stimulation called the “canonical” pathway and an “alternative” pathway that can play a role in many other processes. In the model described by Liu et al, the relevant pathway is the “canonical” pathway. The NFκB dimers that play a role in inflammatory responses consist of p65(RelA)/p50 or cRel/p50/p52 dimers, although p65/p65 dimers have also been implicated.

In response to cell membrane receptor binding (typically IL-1 or TNFα), the IκBα/β proteins that are bound to cytoplasmic NFκB dimers are phosphorylated and degraded by ubiquination. Signal peptides on the p65/p50 dimer are then exposed and the dimers phosphorylated and translocated to the nucleus where they bind to specific promoter assemblies. NFκB inflammatory activity can be measured in toto by measuring the levels of IκBα/β protein in the cellular cytoplasmic fractions or of phosphorylated p65 in the cellular nuclear fractions by Western blot or more reliably by quantitative immunohistochemical localization to cytoplasm or nuclei by confocal microscopy with appropriate intracellular markers to compensate for the variability of cellular fractionation techniques. Given the complex regulatory outcomes of NFκB activation on both inflammatory and anti-inflammatory cytokines and chemokines as well other signaling proteins, the role of NFκB in inflammation is best ascertained by measuring binding to specific promoter sequences using Electrophoretic Migration Shift Assays (EMSA). Here, labeled DNA promoter sequences in excess are exposed to cellular nuclear fractions and the migration patterns of the protein-DNA complexes measured. NFκB activity specific to a gene is demonstrated by shifts in electrophoretic mobility migration or disappearance of the protein-DNA complex where for competitive controls and supershift analyses, reactions are incubated with a 100‐fold excess of the same unlabeled oligonucleotides or antibodies against the p65 protein, “supershift assays.” More specific assessments can be made by DNase 1 “footprinting analyses” of a specific region of a promoter of interest where binding of the transcription factor to a given DNA promoter sequence protects that region from degradation using DNase 1 enzyme.5

Liu et al1 report that 1 dose of ticagrelor (150 mg/kg), administrated after coronary artery ligation, significantly reduced myocardial infarct size in rats subjected to 45-minute coronary artery ligation followed by reperfusion for 24 hours, 3 days, and 7 days. Their results confirm previous studies showing that ticagrelor, administered either before coronary ligation,6–8 before reperfusion,9,10 or event 24 hours after reperfusion,10 reduces infarct size and improves long-term cardiac function. Liu et al also1 confirmed that ticagrelor attenuated post infarction upregulation of inflammation, reducing the levels of IL-6 and TNF-α.10

It has been shown that ticagrelor, in addition to inhibiting the P2Y12 receptors, blocks the equilibrative nucleotide transporter 1, leading to an increase in the interstitial adenosine levels.11 Previous studies have shown that adenosine receptor activation confers myocardial protection against ischemia–reperfusion injury and reduces experimental infarct size in animals.12 It has been shown that the infarct size limiting effects of ticagrelor is abolished with co-administration of adenosine receptor blockers in the rats [CGS15943 (an A2A/A1 adenosine receptor antagonist)]6 and pigs [8-(p-sulfophenyl) theophylline, an A1/A2-receptor antagonist],8 suggesting that the ticagrelor effect is dependent on the activation of the adenosine receptors. Activation of the adenosine receptors leads to upregulation and activation of cyclooxygenase-2 via increased phosphorylation of Akt, ERK ½, and endothelial nitric oxide synthase (eNOS).10 The infarct-size limiting effect of ticagrelor is dependent on cyclooxygenase-2 activation, and it is blocked by specific cyclooxygenase-2 inhibitors or high-dose aspirin.6

Actually, the signaling pathway leading to myocardial protection by ticagrelor is similar to that confirmed by statins.13 Statins activate 5′-nucleotidase that releases adenosine into the interstitial space.14 The infarct size limiting effects of statins also depend on adenosine receptor activation and are blocked with 8-sulfophenyltheophylline or caffeine,14–17 with downstream activation of cyclooxygenase-2.18 Cyclooxygenase-2 inhibition by specific blockers or aspirin attenuates the infarct size limiting effects of statins.19–21 Indeed, a subgroup analysis of The Study of Platelet Inhibition and Patient Outcomes (PLATO) study showed that the effect of ticagrelor on the primary outcome was significantly greater among patients who received lipid-lowering therapy.22 Moreover, rosuvastatin and ticagrelor combination has additive effect on myocardial adenosine levels and hence protection against myocardial ischemia–reperfusion injury.13 Furthermore, chronic treatment with the combination of ticagrelor and rosuvastatin had additive effects on attenuation of inflammation and slowing the progression of atherosclerosis in ApoE(−/−)/db(+)/db(+) double-knockout mice.23

Pretreatment with atorvastatin before ischemia–reperfusion insult has been shown to activate NFκB in the heart,18 although a direct effect involvement in the infarct size limiting effects of statins has not been established. Activation of NFκB by atorvastatin was not seen in eNOS knockout mice but was preserved in inducible nitric oxide synthase knockout mice, suggesting that NFκB activation depends on eNOS activation.

The role of NFκB in protection against ischemia–reperfusion injury, however, is controversial. Liposomal NFκB decoy oligonucleotides administered into the ischemic area at risk reduced myocardial infarct size in pigs subjected to ischemia–reperfusion injury24 and to have anti-inflammatory effects and ameliorate hypoxic brain injury and spinal cord injury in the rat.25–27 Transfection of IκBα attenuates inflammation and accumulation of leukocytes and reduces myocardial infarct size in mice subjected to ischemia–reperfusion injury.28 Genetic blockade of NFκB limits myocardial infarct size in the murine heart after ischemia–reperfusion.29 Onai et al30 showed that inhibition of nuclear translocation of NFκB by an inhibitor of IκB phosphorylation reduces myocardial infarct size. The infarct size limiting effect of tramadol is associated with the attenuation of activation of NFκB.31 Inhibition of NFκB with LncRNA Mirt1 reduces myocardial infarct size.32 On the other hand, NFκB is involved in mediating the delayed cardioprotective effects of ischemic preconditioning33,34 and certain drugs, including the adenosine A3 receptor agonist.35 Thus, most investigators suggest that activation of NFκB before ischemia is involved in mediating protection. By contrast, NFκB is activated after ischemia–reperfusion injury, and acute inhibition of NFκB after initiation of ischemia actually could be protective. Liu et al1 administered ticagrelor after initiation of ischemia and suggested that it attenuated NFκB expression. No other study reported the effect of ticagrelor on NFκB activation. Yet, activation of the adenosine receptors is expected to activate rather than to suppress NFκB.18,35

Dextran sodium sulfate (DDS) is used to induce a model of experimental inflammatory bowel disease and was reported to activate NFκB in the gut.36 DDS can induce inflammation in the heart probably secondary to colitis and systemic inflammatory response with upregulation of IL-1β, TNFα, and TLR4 expression.37 However, (DDS) is not a direct activator of NFκB, and we were not able to find previous studies showing that DDS activates NFκB in the heart. Based on the above-mentioned studies, myocardial NFκB activation before ischemia is expected to induce a state of delayed preconditioning and to limit myocardial infarct size. On the other hand, systemic inflammation could adversely affect protection against ischemia reperfusion and result in bigger infarct. Yet, Liu et al1 found that DDS had no significant effect on infarct size.

As DDS is expected to affect the gut and cause colitis, the baseline condition of the animals can be changed. No data are provided about body weight, body temperature, baseline heart rate, and blood pressure in the different groups. Can it be that the DDS-treated animals were dehydrated? Can it be that absorption of ticagrelor in the gut was reduced in the animals with experimental colitis and therefore the protective effect was attenuated? Data on blood ticagrelor levels or degree of inhibition of platelet aggregation are not provided in the manuscript. However, the fact that ticagrelor was able to reduce hs-CRP levels in the DDS-treated animals (Figure 4b of the article by Liu et al1) suggests that it might not be the case.

The effect of galectin-3 on myocardial ischemia–reperfusion injury is also controversial. In a previous study, Liu et al9 showed that ticagrelor downregulated galectin-3 levels in the infarct zone and suggested that it may mediate the infarct size limiting effect of ticagrelor. Galectin-3 inhibition by pectin or binding peptide G3-C12 limited infarct size in mice.38 Yet, other investigators suggested that knocking down galectin-3 actually increases ischemia–reperfusion injury.39

It could be that ticagrelor ability to limit myocardial infarct size is attenuated in animals with systemic inflammation. Yet, ticagrelor was able to limit infarct size in male obese ZDF rats (ZDF-Leprfa/Crl) with type-2 diabetes13 and to attenuate inflammation and progression of atherosclerosis in ApoE(−/−)/db(+)/db(+) double-knockout mice with type-2 diabetes.23 Moreover, it was shown that ticagrelor is effective in preventing multiorgan failure in patients with sepsis,40 suggesting that this possibility is unlikely.

In conclusion, Liu et al showed that ticagrelor failed to limit infarct size in rats pretreated with DDS. Further studies are needed to confirm whether the primary hypothesis of the authors that the protective effect of ticagrelor involves NFκB activation is right.

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