**5. Angiotensin II and TNF-α in cardiac remodeling**

The proinflammatory cytokine TNF-α was first defined as an antimutagenic. Nowadays, amount findings revealed a wide range of pleitropic TNF-α effects including cell proliferation, apoptosis and production of other proinflammatory cytokines [2].

Growing body of evidences evaluated the role of TNF-α in many diseases, particularly in cardiovascular disease. TNF-α has been found upregulated in myocardial from humans and animals with heart failure [70]. A wide variety of cells including macrophages, fibroblast and endothelial cells produce TNF-α. It has been described that cardiomyocytes themselves are capable of synthesizing TNF-α [71]. Bryant et al. [72] have shown that TNF-α synthesized by cardiomyocytes was sufficient to cause severe cardiac remodeling suggesting maladaptive hypertrophy, which may also occur in human heart failure.

The TNF-α is secreted as a cell surface protein (homotrimeric type II transmembrane protein) containing 233-amino-acid, which is activated by proteolytic cleavage to a 76-amino-acid signal peptide [73, 74]. The TNF-α released as a mature protein, which acts as a soluble cytokine through its two receptors: TNF receptor 1 (TNFR1) and TNFR2 [75, 76]. Despite the homology betweenTNFR1 andTNFR2 in extracellulardomains, both intracellulardomains ofTNFR1 and TNFR2 are different. Once activated, TNFR1 leads to recruitment of a protein TRADD (TNFR1 associated death domain protein), which subsequently interacts with three other intracellular proteins forming a complex. When activated, TNFR2 directly recruits TRAF2 and TRAF1 (TNF receptor-associated factor). These differences in TNFR-induced intracellular signaling suggest each receptor has distinct cellular functions. In this sense, dual effects of TNF-α have been

suggested during the progress of cardiac disease. Low concentration of TNF-α has been associated with the protective effects while its high concentrations present deleterious effects [77]. This study did not evaluate the TNF-α receptors contribution. However, other evidences have been shown that the effects of the two receptors on heart failure were opposite, TNFR1 showed proapoptotic and prohypertrophic while TNFR2 developed antiapoptotic and antihypertrophic effects [78]. In addition, other findings have suggested that TNFR1 is responsible forthe major deleterious effects produced by TNF-α in hypertrophic signaling [79, 80]. Moreover, soluble TNFR1 is a predictor of mortality and heart failure in patients with acute myocardial infarct [81]. Preclinical studies demonstrated that TNFR1 plays an important role in Ang II-induced fibrosis in rats while TNFR2 did not affect the increased collagen deposition in response to Ang II infusion [80].

involved in the heart hypertrophy. Ang II-induced increased MMP transcriptional expression has been reported by several studies [30, 42, 44]. Despite AP-1 contribution to the transcription of MMP-2 [47], the NF-kB inhibition attenuated MMP-2 upregulation in both heart and aorta from 2-kidney and 1-clip (2K1C) hypertensive rats [44]. Transgenic mice overexpressing cardiac MMP-2presentedmarkeddecompensatedhypertrophy,includingnotonlycollagendeposition butalsosignificant systolicdysfunction[67].MMP-2seems todegrade some contractileproteins fromheart sarcomeres, suchasmyosinandtroponin[35],whichhaveconstantlybeenassociated with impaired heart capacity to contract in experimental models of heart disease [68]. In this regard, several findings have stated that MMP-2 inhibition ameliorates remodeling and cardiac dysfunction [35, 47, 69]. In addition, Ang II-induced MMP activation may be associated with adaptive remodeling and cardiac dysfunction in 2K1C rats [69]. The Ang II activates MMP-2 by mechanismsinvolvingNADPHoxidaseactivationandROSformation[30,42].Inthissense,TGFβcouldincreaseMMP-2activationsincethiscytokinealsoincreasesROSformation.Indeed,some studies have shown increased TGF-β levels and MMP-2 activity in the left ventricle from hypertensive rats [3, 30]. Hence, the TGF-β-dependent mechanisms to Ang II-induced cardiac remodeling may involve MMP-2 activation by redox signaling. However, future studies are necessary to support the causal relation between MMP-2 activation and TGF-β in Ang II

The proinflammatory cytokine TNF-α was first defined as an antimutagenic. Nowadays, amount findings revealed a wide range of pleitropic TNF-α effects including cell proliferation,

Growing body of evidences evaluated the role of TNF-α in many diseases, particularly in cardiovascular disease. TNF-α has been found upregulated in myocardial from humans and animals with heart failure [70]. A wide variety of cells including macrophages, fibroblast and endothelial cells produce TNF-α. It has been described that cardiomyocytes themselves are capable of synthesizing TNF-α [71]. Bryant et al. [72] have shown that TNF-α synthesized by cardiomyocytes was sufficient to cause severe cardiac remodeling suggesting maladaptive

The TNF-α is secreted as a cell surface protein (homotrimeric type II transmembrane protein) containing 233-amino-acid, which is activated by proteolytic cleavage to a 76-amino-acid signal peptide [73, 74]. The TNF-α released as a mature protein, which acts as a soluble cytokine through its two receptors: TNF receptor 1 (TNFR1) and TNFR2 [75, 76]. Despite the homology betweenTNFR1 andTNFR2 in extracellulardomains, both intracellulardomains ofTNFR1 and TNFR2 are different. Once activated, TNFR1 leads to recruitment of a protein TRADD (TNFR1 associated death domain protein), which subsequently interacts with three other intracellular proteins forming a complex. When activated, TNFR2 directly recruits TRAF2 and TRAF1 (TNF receptor-associated factor). These differences in TNFR-induced intracellular signaling suggest each receptor has distinct cellular functions. In this sense, dual effects of TNF-α have been

**5. Angiotensin II and TNF-α in cardiac remodeling**

apoptosis and production of other proinflammatory cytokines [2].

hypertrophy, which may also occur in human heart failure.

hypertrophy.

58 Renin-Angiotensin System - Past, Present and Future

TNF-α-induced intracellular signaling involves canonical NF-kB activation. The complex of intracellular protein is formed when TNFR1 is activated, specific mitogen-activated protein kinase kinases (MAPKKs) are phosphorylated consequently activating c-Jun N terminal kinase (JNK), AP1 and p38 MAPK signaling pathways. Taken together, TNF-α-induced intracellular signaling controls the expression of inflammatory proteins and antiapoptotic genes. Another signaling complex is triggered as a response to the TNFR1 activation resulting in stimulation of the effective caspases, which in turn lead to apoptosis [82].

TNF-α has induced increased ROS formation in endothelial cells by a mechanism dependent of NADPH oxidase subunit: p47 phox subunit [83]. Indeed, cardiomyocytes hypertrophy was induced by recombinant human TNF-α at least in part due to ROS generation [84]. Through experimental models of heart failure, TNF-α inhibition decreased oxidative stress and apoptosis improving cardiac remodeling and dysfunction [85]. Thus, ROS seems to foster a key function in the cardiac hypertrophy induced by TNF-α.

As described above, it is possible to observe common signaling pathways between TNF-α and Ang II. Since Ang II notably has increased TNF-α *in vivo* [86] and *in vitro* studies [87], some evidences have reported a potential role of TNF-α in Ang II-induced cardiac hypertrophy [25, 88–90]. In this context, chronic Ang II infusion promotes cardiac hypertrophy, which was attenuated in TNF-α knockout mice [89]. These findings were further confirmed by the pharmacological inhibition by etanercept, an inhibitor of TNF-α, which blunted cardiac hypertrophy in mice under Ang II infusion [25]. Indeed, the authors showed the involvement of TNF-α in the intracellular signaling in Ang II-induced hypertrophy. Both TNF-α and Ang II induced activation of NF-kB, p38 MAPK and JNK. Accordingly, heart TNF-α knockout mice attenuated the activation of NF-kB, p38 MAPK and JNK signaling in Ang II infusion, suggesting TNF-α is required to induce Ang II cardiac hypertrophy by intracellular signaling pathways [25]. It was observed that the TNFR1 deficient mice did not develop fibrosis under Ang II stimulation, while TNFR2 deficient mice showed increased collagen accumulation in the heart under Ang II infusion [88], which may indicate a promising role of TNF-α in activating TNFR1 as crucial signaling to Ang II inducing cardiac remodeling.

Ang II and TNF-α are involved in increased production of ROS, which in turn activate NF-kB. In this regard, Sriramula et al. [25] also suggest that redox signaling induced by Ang II may be dependent of TNF-α. The authors have found that the increased mRNA, 2 expression and

also the expression of other NADPH oxidase isoforms were blunted in TNF-α knockout mice, which have resulted in lower levels of ROS. Collectively, all findings point out to a causal relation between hypertrophic signaling of Ang II and TNF-α that involve redox pathways on NF-kB, JNK and p38 activation.
