**4. Angiotensin II and TGF-β in cardiac remodeling**

hypertension suggesting that collagen deposition could contribute to decreased myocardial complianceanddisruptedheartelectricalproperties[9].Currently,itiswellknowntherelevance of fibrosis not only to the structure of the cardiac hypertrophy but also to the heart dysfunction [46]. Collagen is the main component of the extracellular matrix in the myocardium, which is synthesized by fibroblasts. However, its deposition in the heart during hypertension also depends of its degradation [3]. Thus, a large body of studies has shown the contribution of the MMP, which is the main proteases to collagen degradation, strongly contributing to the cardiac remodeling after pressure overload or infarct [28, 47]. The imbalances between MMP and endogenous tissue inhibitor (TIMP) are key mechanisms to control the collagen formation and deposition[2,3,48].Indeed, some transcriptionalfactors suchasAP-1 andNF-kBmaymodulate the MMP activity increasing its MMP expression and also TIMP expression [28, 47]. Posttranslationalmechanisms suchasoxidativestress,particularlyperoxynitriteandhydrogenperoxide, may activate MMP and inhibit TIMP activity [35, 49, 50], suggesting a possible mechanism to ROS-induced fibrosis in hypertensive rats [51]. Thus, MMP activity is regulated at three levels: (i) transcriptional level, (ii) endogenous inhibitors and (iii) factor activators (ROS). Interestingly, Ang II may increase MMP activity involving redox-sensitive signaling in fibroblast, thus triggering NF-kB and AP-1 transcriptional factor activation [52]. In fact, antioxidant therapy reverses Ang II-induced cardiac hypertrophy and MMP activity in left ventricle from hypertensive rats [30]. Taken together, Ang II promotes myocyte on the heart and matrix extracellular hypertrophybysimilarmechanismsinvolvingredoxsignaling,whichnotonlyactivatestheRNA expressionofproteinsinthemyocytesorfibroblasts,butalsorisestheactivityofenzymesalready

**Figure 2.** Main pathways concerning Ang II-induced cardiac remodeling. Nox2 is activated by Ang II via AT1 receptor, triggering ROS formation, which activates intracellular pathways related to the cardiac hypertrophy. Nox2: NADPH oxidase isoform 2; ROS: reactive oxygen species; Akt: serine/threonine-specific protein kinase; ERK1/2: extracellular.

Furthermore, it must be recognized that Ang II induces inflammation by triggering cardiac remodeling. The proinflammatory effects of Ang II have been described since 1970 by Finn Olsen [53]. Thenceforward, several studies have supported the contribution of the inflammatory processes associated with Ang II to cardiovascular disorders, including hypertension and

present in the heart, such as MMP.

56 Renin-Angiotensin System - Past, Present and Future

Increased expression of TGF-β was found in the myocardium during cardiac hypertrophy and heart failure [55]. Classically, TGF-β is a multifunctional cytokine recognized as a powerful profibrotic factor. Three isoforms of the TGF-β family have been identified in mammals [56], but TGF-β1 has been constantly associated with several cardiovascular diseases, particularly during the transition from adaptive cardiac hypertrophy into heart failure [56–59]. The overexpression of TGF-β1 induced fibrosis and myocyte hypertrophy in transgenic mice after they were 8 weeks old [58]. Upregulated TGF-β1 mRNA is found in the pressure-overloaded human heart [60], as well as in the dilated cardiomyopathy [57]. The latent form of TGF-β1 is composed of 390-amino acid complexed with the signal peptide and the large amino-terminal prodomains (known as latency-associated proteins, LAPs) which are required for correct folding and dimerization of the carboxyl terminal domain of the growth factor (the mature peptide) [61]. TGF-β1 can be released and activated by the proteolytic cleavage, which disrupts its non-covalent attachment with LAP [62]. The intracellular signaling induced by TGF-β underlies the activation of serine/threonine kinases receptor resulting in Smad phosphorylation, which is responsible to activate target genes [61]. TGF-β may also promote the regulation of the transcription by TGF-β-activated kinase-1 (TAK1) triggering p38 MAPK phosphorylation and activating transcriptional factor (ATF)-2 [56].

Myriad experimental studies reported Ang II-mediated TGF-beta induction, particularly of its expression [63–65]. AT1 receptor seems to be involved with TGF-β upregulation expression at the transcriptional level in as much as losartan treatment inhibited the rise of this cytokine in animals after Ang II infusion [63].

Since AT1 activation produces ROS via NADPH oxidase, Wenzel et al. [63] demonstrated that the induction of TGF-β in cardiomyocytes was diminished in the presence of NADPH oxidase inhibitors. Consistently, antioxidant treatments have shown decreased cardiac TGF-β expression in the experimental model of RAS activation [23, 30]. The redox signaling involved in Ang II-induced TGF-β upregulation seems to be dependent on p38 MAPK and AP-1 pathway, such was observed in ventricular cardiac myocytes [23, 63]. In this regard, the first direct evidence about the causal relation between two important factors for cardiac hypertrophy (Ang II e TGFβ) was observed in TGF-β1-deficient mice. The marked cardiac hypertrophy and the impaired cardiac function induced by chronic suppressor doses of Ang II were not observed in TGFβ1-deficient mice [66]. Thus, cardiac TGF-β is required to hypertrophy signaling induced by Ang II, which in turn activates its AT1 receptor upregulating this cytokine expression.

TGF-β and Ang II are involved in fibroblast differentiation and MMP activity control [3]. In this regard, an imbalance between MMP/TIMP is possibly another common signaling consequently 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 hypertrophy.
