Investigational drugs targeting cardiac fibrosis.

Review

Expert Review of Cardiovascular Therapy Downloaded from informahealthcare.com by Chinese University of Hong Kong on 02/13/15 For personal use only.

Investigational drugs targeting cardiac fibrosis Expert Rev. Cardiovasc. Ther. 12(1), 111–125 (2014)

François Roubille*1,2, David Busseuil1, Nolwenn Merlet1, Ekaterini A Kritikou1, Eric Rheáume1,3 and Jean-Claude Tardif1,3 1 Montreal Heart Institute, Universite´ de Montreál, Montreal, QC, Canada 2 Cardiology Department, University Hospital of Montpellier, Montpellier, France 3 Department of Medicine, Universite´ de Montreál, Montreal, QC, Canada *Author for correspondence: Tel.: +1 514 376 3330 +1 514 376 3612 Fax: +1 514 593 2500 [email protected]

Fibrosis is an accumulation of proteins including collagen in the extracellular space, which has previously been considered as irreversible damage in various cardiovascular diseases including heart failure and hypertension. The pathophysiology of fibrosis is currently better understood and can be evaluated by non-invasive methods. Here, the authors present briefly the impact and molecular mechanisms of fibrosis in the myocardium and the promising therapeutic candidates including anti-hypertensive therapies, heart-rate lowering drugs, anti-inflammatory agents, as well as other innovative approaches such as inhibitors of growth factors, miRNA or cell therapy. Surrogate end points allow for larger clinical trials than previously possible with endomyocardial biopsies, and magnetic resonance and molecular imaging should open new fields of research on cardiac fibrosis. Several pre-clinical findings are very promising, and some clinical data support the proofs of concept, mainly those with inhibitors of the renin-angiotensin system. These approaches open the field for regression of fibrosis and include the following: first, some of these drugs are widely used like renin-angiotensin system inhibitors; second, inflammation modulators; third, in near future entirely new approaches targeting the TGF-b pathways, or others like cell therapies or genetic interventions. KEYWORDS: cardiac fibrosis • collagen • extracellular matrix • heart failure • hypertrophy • myocardium • myofibroblast • remodeling • TGF-b

Tissue fibrosis is characterized by an accumulation of proteins, especially collagen, in the extracellular space, which are secreted by profibrotic cells such as fibroflasts. As a result of the excessive accumulation of proteins, nearby cells become hypotrophic and less numerous and their normal activity is hampered. Fibrosis appears as a common pathophysiological phenomenon, especially during the development and progression of cardiovascular diseases. The normal cardiac anatomy is progressively modified by the excessive deposition of extracellular matrix with reduction of microvasculature and disruption of normal myocardial structures. Fibrosis is common to various pathophysiological processes including ischemic cardiomyopathy [1], hypertension [2] (for review, see [3]), heart failure (HF) [4], dilated cardiomyopathy [5,6] and hypertrophic cardiomyopathy [7,8]. It can either be localized (e.g., scarring following myocardial infarction [MI]), or diffuse (e.g., in dilated cardiomyopathy). Myocardial fibrosis can be either reactive (e.g., interstitial fibrosis resulting from increased collagen synthesis by myofibroblasts in the case of chronic inflammatory process or other

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stimuli) or reparative (e.g., as result from replacement of necrotic myocytes by collagen following acute MI), as a result of increases in collagen type I and III synthesis by fibroblasts; in contrast, fibrosis can represent a remodeling process triggered by myocytes loss mainly by apoptosis/necrosis (FIGURE 1). In experimental models, fibrosis leads to an increase of myocardial stiffness and cardiac dysfunction, whereas its regression has beneficial effects on these aspects [9]. Similar findings have been observed in the clinical setting [10], albeit with limitations because of the required endomyocardial biopsies. Taken together, the current literature indicates that targeting fibrosis could alleviate the progression of cardiovascular diseases and improve cardiac function (see TABLE 1 for current ongoing trials). In this review, we discuss the impact of fibrosis in the myocardium (ventricular fibrosis). Atrial fibrosis, involved in arrhythmias, as well as valvular fibrosis are outside the scope of this article (for a recent comprehensive review, see [11]). We discuss the molecular mechanisms involved in this process and the promising candidates that can be used to treat ventricular

2014 Informa UK Ltd

ISSN 1477-9072

111

112

IL-1b

3

4

6

IL-1b

TGF-b

5

Proapoptotic pathway Cellular evolution

Collagen fibrillar

Fibronectin

Putative therapeutic activation

Putative therapeutic inhibition

Myofibroblast precursor

CTGF

BMP-7

1

Fibroblast/ myofibroblast

Collagen I/III

5

TGF-b

4

Extracellular matrix synthesis

Proteoglycans

9

ROS

Fibronectin

Pro-apoptotic pathways

Main effect: induction, activation

Secretion

Biological phenomenon

Cellular type

TGF-b

4

Extracellular matrix degradation

2

NFkB

MMP

8

Main effect: inhibition

Necrosis

Myofibroblast precursors

TNF-a

7

Necrosis

Pro-inflammatory cytokine

via the angiotensinogen-Angiotensin I-Angiotensin II-AT receptor

Autoactivation of TGF-β synthesis

Angiotensin II (Ang 2)

AT1 receptor

Macrophage

PDGF VEGF FGF

Galectin-3

Myocytes


Review áume & Tardif Roubille, Busseuil, Merlet, Kritikou, Rhe

Expert Rev. Cardiovasc. Ther. 12(1), (2014)


Investigational drugs targeting cardiac fibrosis

fibrosis, such as anti-hypertensive therapies, heart-rate (HR) lowering drugs, anti-inflammatory agents, as well as other innovative approaches including inhibitors of growth factors, microRNA and cell therapy. Surrogate end points allow for larger trials overcoming the limitation of endomyocardial biopsies (TABLE 1). We complete this review by highlighting the rapid progress of magnetic resonance imaging (MRI) and molecular imaging that have opened new fields of research on cardiac fibrosis. Molecular mechanisms underlying myocardial fibrosis

In fibrotic processes, the normal cardiac anatomy is progressively modified by the excessive deposition of extracellular matrix with reduction of microvasculature and disruption of normal myocardial structures. The extracellular matrix deposition is largely mediated by fibroblasts (FIGURE 1). Although the origin of these cells remains unclear, it has been proposed that these cells could originate from endothelial cells through endothelial-mesenchymal transition (EMT) [12]. TGF-b1, which is one of the main profibrotic factors capable of enhancing fibrosis through numerous pathways [13], seems to be the main factor inducing EMT [12]. By contrast, bone morphogenetic protein-7 (BMP-7) significantly reduces EMT, and limits the progression of cardiac fibrosis in mouse models [12]. Factors modulating the TGF-b pathway are multiple, including mechanical stress [14], inflammation (mainly through the proinflammatory cytokines IL-1b, IL-6 and TNF-a) and epigenetic modifications [15]. In response to TGF-b, the myofibroblasts (cells that stay in a state between a fibroblast and a smooth muscle cell as regards to differentiation/characteristics and capable of main functions of classical fibroblasts) produce collagen and fibronectin [16], which through a feedback loop enhance the TGF-b-Smad-mitogen-activated-protein-kinase (MAPK)-mediated deposition of collagen [17]. Several other pathways interconnected with the TGF-b network have been described, involving the PDGF and the VEGF (for a recent review see [18]).

Review

Beyond the classical pathway leading to fibrosis schematically presented in FIGURE 1 that involves the TGF-b/Smad pathway, the renin-angiotensin system (RAS, see paragraph 4) and the matrix metalloproteinases (MMP, see paragraph 8), many other factors have recently emerged as important players in fibrosis pathways such as hypoxia-inducible factor 1 [19], nitric oxide synthase (NOS) [20–22], the Nod-like receptor pyrin domain containing 3 (NLRP3) [23], as well as several microRNAs (see paragraph 8). Specific developments are proposed thereafter throughout the review regarding both basic and translational approaches. Surrogate end points

Several fibrosis biomarkers have been proposed. These early biomarkers can be used to identify hypertensive patients without any symptoms who are at risk of developing diastolic heart dysfunction [24]. These surrogate end points provide information on the balance between synthesis and degradation of collagen in clinical settings; an imbalance rather than a strong excess could lead to disease [2,25]. Most of these biomarkers are based on the structure of the cardiac extracellular matrix, which is predominantly composed of collagen type I (75–85%) [26] and III (10–15%). Cardiac fibroblasts are the predominant collagen-producing cells in the heart, responsible for the production of both collagen I and III. Fibrillar collagen is synthesized as a procollagen, which is split by specific proteinases in carboxyl (C)-and amino (N)-terminal propeptides. The N-terminal propeptides of collagen type I or III (PINP and PIIINP) and the C-terminal propeptides (PICP and PIIICP) are used as markers of collagen type I or III synthesis. Importantly, a stoichiometric ratio of 1:1 links the number of collagen type I molecules produced and the number of propeptides of collagen type I released. Regarding the products of degradation of the collagen fibrils, the small telopeptide of collagen type I (ICTP) can be used as marker for the degradation of collagen type I. A stoichiometric ratio of 1:1 links the number of collagen type I molecules degraded and the number

Figure 1. General schema on the main fibrosis actors in cardiac pathogeny and the putative targets for anti-fibrotic strategies. Various physiological stimuli could induce fibrotic process, including cell necrosis or apoptosis, recruiting inflammatory cells, especially macrophages. These cells activate in turn fibroblasts. The main roles played by fibroblasts/myofibroblasts, macrophages and cardiomyocytes are schematically depicted. The fibroblast is responsible for the extracellular matrix production and degradation, under several pathways and regulations as presented. The macrophage is able to activate the fibroblast, and can be dramatically activated by necrotic cells, especially cardiomyocytes. TGF is one of the major molecular actors as explained in the main text. Inflammatory interleukins can trigger apoptotic events as well as enhancing matrix production/degradation. The cross-talk between the various pathways is complex and clinical context-dependent. The central role of the RAS system with the end-effector angiotensin molecule is underlined. Putative targets for anti-fibrotic strategies: All the pathways listed above are theoretically possible targets for therapeutic interventions, as discussed in details in the article. Inhibitors of the indicated pathway are presented in red and the numbers refer to the main molecules presented thereafter, whereas the agonists are represented in green. 1: Agonists of the BMP-7; 2: modulators of MMPs (such as pycnogenol); 3: inhibitors of the macrophages (antibodies under development, cellular targeted therapies); 4: inhibitors of the TGF-b: antibody, antisense oligonucleotide, or receptor antagonist or downstream of the receptor of TGF-b, inhibitors of the Smad transcription factors (such as halofuginone); 5: the RAS inhibitors including ACEs, ARBs, spironolactone and eplerenone (see the main document for details); 6: the modulators of the IL1-b pathway (including antibodies, antagonists of the receptors, see the main document for details); 7: the modulators of the TNF-a pathway (including antibodies, antagonists of the receptors, see the main document for details); 8: the inhibitors of the NFkB (directly or indirectly including the fibrates); 9: inhibitors of the extracellular matrix synthesis. Ang: Angiotensin; BMP-7: Bone morphogenic protein 7; MMP: Metalloproteinase; RAS: Renin-angiotensin system; ROS: Reactive oxygen species.


113

114

NCT01437371

NCT00574119

France

USA

Spironolactone, Phase IV

ACE inhibitors, Phase III

Canrenone, Phase III

Molecule, phase

NCT01803828

NCT01516346

Italy

USA

Isosorbide dinitrate + hydralazine, Phase II

Tadalafil, Phase IV

Diltiazem titrated to a target dose of 360 mg daily, Phase II–III

NCT00952627

NCT01640639

NCT01829750

Cardiac progenitor cell infusion, Phase II

Pycnogenol, Phase II

Thalidomide, Phase IV

Only therapeutic trials are mentioned. † Cardiac fibrosis: clinical trials; 272 trials; updated 25 March 2013.

Japan

Cellular therapy

USA

Italy

Anti-inflammatory and anti-oxidative stress agents

NCT00319982

USA

Other antihypertensive therapies and vasodilatators

NCT00403910

NCT

Italy

RAS

Place

In patients with univentricular heart disease primary end point: LV function

Biological markers of fibrosis echocardiography

LVEF

Wave reflection magnitude between baseline and after 24 weeks of randomized therapy LV mass & collagen volume fraction measured by MRI (secondary end point)

LV torsion assessed by MRI

Evolution of diastolic function assessed by echocardiography

Ventricular remodeling by MRI

Optimization of HF treatment in patient older than 80

Changes in echocardiographic left ventricular diastolic volume

Main end point

Secondary end point: fibrosis

Supposed to modulate of MMPs and TIMPs enzyme activities

2018

2011

2014

2013

Fibrosis is a secondary end point, assessed by MRI

Fibrosis is not specifically (in the clinicaltrial.gov site) addressed although thalidomide is supposed to target cardiac inflammation

2018

2013

2011

2012

Was announced 2006

End of the study planned

Secondary end point: fibrosis assessed by MRI

Secondary end point: fibrosis assessed by MRI

Fibrosis is assessed by MRI

Fibrosis is secondary end point

Remarks

Table 1. Clinical trials† currently registered on clinicaltrials.gov including the therapeutic studies.


34

40

100

34

164 patients with diabetic cardiomyopathy

50

20

80

500 patients with mild HF

Number of patients planned

Review áume & Tardif Roubille, Busseuil, Merlet, Kritikou, Rhe



of ICTP, so that ICTP could be considered as a reliable biomarker of collagen type I degradation in conditions of normal renal function (it is cleared by kidneys) [27,28]. In conclusion, these biomarkers provide useful tools to assess the efficacy of various drugs proposed to reduce myocardial fibrosis.


Development of specific end points to assess myocardial fibrosis is crucial

Until recently, only indirect assessment of fibrosis was available, mainly through echocardiography (left ventricle [LV] mass assessment). Endomyocardial biopsies are an invasive evaluation with rare but possible life-threatening complications. They are only representative in diffuse fibrosis and could easily mislead the diagnosis in case of focused lesions. Serial evaluations could target the same myocardial regions but cannot assess exactly the same tissue consecutively twice. Non-invasive methods have, however, been developed to detect cardiac fibrosis. First, cardiovascular magnetic resonance (CMR) imaging now allows serial assessment of global and segmental fibrosis. Second, circulating biomarkers have emerged to evaluate collagen turnover, such as those pertaining to collagen synthesis and degradation. Non-invasive assessment of fibrosis by MRI

Regional myocardial fibrosis can be well visualized by contrastenhanced MRI (late gadolinium enhancement, LGE imaging) and is considered as the gold-standard for quantifying necrosis or scar in MI (FIGURE 2) [29,30] or myocarditis [31,32]. With respect to diffuse fibrosis present, for example, in cardiomyopathies, myocarditis, infiltrative disease and hypertrophy, LGE imaging suffers from inherent limitations as this approach relies on the presence of regions with differences in ‘normal’ signal intensity, typically referred to as ‘normal’ or ‘remote’ myocardium. In diffuse fibrosis, such region may not be present or there may be a diffused gradient between various tissue pathologies. The approach of quantifying the amount of fibrosis or, more recently, extracellular volume (ECV) by T1 mapping allows for global measurements comparing results with reference values and thus may overcome this problem. T1 mapping was utilized for myocardial scarring in patients with acute myocardial infarction (AMI) [33,34] and later in patients with diffuse fibrosis [35,36]. T1, also referred to the longitudinal relaxation time, is modified by the presence of an increased ECV fraction, as it is the case in myocardial fibrosis. Based on signal intensities in images acquired using varying inversion or saturation times, mathematical methods are used to calculate T1 and results are typically presented as colorcoded maps [33,34]. Recent advances in methodology allow for the acquisition of a high-resolution map in a single breath-hold [37]. For imaging fibrosis or measuring ECV, the injection of a contrast agent is considered necessary, which represents a limitation in patients with severe or acute renal dysfunction or known allergy to contrast agents. Calculating the relative T1 mapping indices, especially estimating ECV from the contrast-induced T1 change ratio between myocardium and blood could reduce drastically this bias [38]. ECV has been used www.expert-reviews.com

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ASR

R I

10 0

A

L S P

PIL

Figure 2. Example of fibrotic cardiac disease. Increased signal intensity in a contrast-enhanced CMR image (late gadolinium enhancement), reflecting a non-ischemic scar (arrows) in a patient with myocarditis. Modified with permission from [30].

as a surrogate end point to quantify both focused and diffuse fibrosis [39] with prognostic value [40]. Of note, this method has been shown to be reproducible and sensitive, reducing the need for large sample sizes in clinical trials [41], although both intra-operator and inter-operator reproducibilities have to be assessed in large therapeutic studies. To our knowledge, current studies registered on clinicaltrial.gov (TABLE 1) are most often designed choosing biological biomarkers as main end points. Interestingly, T1-mapping could also be useful as a marker for myocardial edema [42], paving the way for both assessment of edema and fibrosis by similar techniques, without contrast agents. Other MRI sequences are currently under study and new tools are intensively evaluated, such as elastography [43–45]. Finally, MRI (as well as other techniques such as nuclear imaging) allows to accurately characterize the ‘quality’ and to some extent the composition of the interstitium. In other words, these imaging modalities can distinguish fibrosis from ‘pseudofibrosis’ where the interstitium is increased not because of collagen deposition, but because of amyloid deposition (amyloidosis [46,47]), accumulation of inflammatory cells (myocarditis [48]) and granuloma (sarcoidosis [49]), among others. New molecular imaging

Complementary to CMR imaging, other promising imaging modalities [50] include positron emission tomography (PET) scanning or single photon emission computed tomography. These molecular imaging approaches could take advantage of targeting various molecules (e.g., integrins, RAS, collagen), cellular processes (i.e., apoptosis) involved in myocardial fibrosis, 115


Review

áume & Tardif Roubille, Busseuil, Merlet, Kritikou, Rhe

and even specific molecules for one particular aspect of fibrosis such as galectin-3 (which could reflect macrophage activity and involvement in cardiac remodeling). This approach seems complementary to CMR as it lacks anatomical evaluation and functional assessment is less accurate than with CMR. In the future, combined imaging techniques (e.g., fusion of CMR and PET images) are likely to provide powerful information, so that clinical trials targeting myocardial fibrosis could be informed through comprehensive imaging simultaneously providing anatomical, functional, cellular and molecular evaluations. Several studies are ongoing to evaluate promising molecular imaging modalities. Among them, the SCAR Study (Technetium-NC100692 scintigraphy to detect avB3 integrin expression as a marker of fibrosis in hypertrophic cardiomyopathy and acute coronary syndrome) will compare 99Technetiumscintigraphy-imaging to MRI in patients with various fibrotic presentations (ClinicalTrials.gov identifier: NCT01230918). Although these imaging improvements open new fields of research, their current applications in translational research remain limited and surrogate end points presently remain necessary. Potential therapies currently available to target fibrosis

Here, we present the available drugs currently under investigation for the prevention or regression of myocardial fibrosis, such as anti-hypertensive therapies, HR-lowering drugs, anti-inflammatory agents and lipid-lowering agents. We also discuss approaches under early development including inhibitors of growth factors, as well as microRNAs and cellular therapy that hold great promise for the future. Anti-hypertensive therapies

Most of the tested antihypertensive therapies have been shown to reduce fibrosis in animal models. In a rat model of aortic banding associated with renal artery ligation, an angiotensin converting enzyme (ACE)-inhibitor, a b-blocker and a calcium-channel blocker all reduced LV fibrosis [51]. However, anti-hypertensive drugs do not all seem to exert similar effects in patients. In hypertensive patients treated with either lisinopril (n = 18) or hydrochlorothiazide (HCTZ) (n = 17), endomyocardial biopsies at baseline and 6 months revealed a decrease of collagen volume fraction (CVF) only in the group treated with the ACE inhibitor [10]. These modifications were consistent with the observed improvement in echocardiographic parameters of diastolic function [10]. A comparison between losartan (n = 21) and amlodipine (n = 16) given for one year revealed that while changes in blood pressure during treatment were similar in the two groups, only losartan resulted in significant decreases in both CVF and PICP [52]. These studies supported the ability of specific anti-hypertensive treatments to regress fibrosis in hypertensive patients independently of their anti-hypertensive efficacy, and RAS inhibitors appear as the most promising agents to achieve this goal. See TABLE 1 for brief presentation of the trials currently ongoing. 116

The RAS is a key regulator of the cardiovascular system and has an important role in fibrotic processes [26], by regulating proliferation and activity of cardiac fibroblasts [53]. When administered in mice, angiotensin II (Ang II), the main effector of the RAS, enhances cardiac fibrosis. In patients, levels of Ang II are elevated in the failing heart [54] and a substantial body of evidence indicates that this peptide contributes to changes in cardiac structure and function, which ultimately lead to progressive worsening in HF. In contrast to Ang II, the heptapeptide angiotensin-(1-7) (Ang-(1-7)) has cardioprotective and anti-remodeling effects [55], including the reduction of collagen production. Various strategies have been proposed to target the Ang II network, including Ang-receptor blockers (ARBs). Targeting other receptors such as those for TNF could potentially also be effective as the Ang II pathway seems to involve one of the TNF receptors [56]. The presence of TNF-a has been shown to be required to induce Ang II-mediated fibroblast maturation from monocytes in an in vitro human monocyteto-fibroblast differentiation model [56]. In vivo, mice deficient in both TNF receptors (TNFR) did not develop cardiac fibrosis in response to Ang II infusion. Furthermore, the Ang IIdependent cardiac fibrosis seemed to involve the signaling through TNFR1, which enhanced the generation of monocytic fibroblast precursors in the heart [56]. Recently, an anti-Ang II vaccine has been proposed and was shown to efficiently decrease myocardial fibrosis in mice [57]. Immunization of mice with conjugated Ang II successfully induced the production of anti-Ang II antibody, which blocked Ang II signaling in human aortic smooth muscle cells [57]. In an Ang II-infused model, the non-immunized mice showed high blood pressure, whereas the immunized mice showed a significant decrease in systolic blood pressure, accompanied by significant reductions in cardiac hypertrophy and fibrosis. Importantly, the anti-Ang II antibody titer was not elevated and there was no accumulation of inflammatory cells, suggesting that vaccines targeting Ang II might be effective to decrease high blood pressure and prevent cardiovascular complications without severe side effects [57]. Angiotensin-converting enzyme inhibitors remain a first-line treatment for hypertension. The ACE inhibitor lisinopril has been shown to induce regression of myocardial fibrosis in rats with genetic hypertension and LV hypertrophy [58]. More importantly, lisinopril was also able to decrease myocardial fibrosis in patients with hypertensive heart disease [10]. In this prospective, randomized, double-blind trial, 35 patients with primary hypertension, LV hypertrophy, and LV diastolic dysfunction were treated with either lisinopril or HCTZ. At baseline and after 6 months, LV catheterization with endomyocardial biopsy and echocardiographic assessment were performed. Myocardial fibrosis, measured by LV CVF, decreased from 6.9 ± 0.6% to 6.3 ± 0.6% (p < 0.05 vs HCTZ). This was also associated with improvement in echocardiographic parameters of diastolic function. ARBs have also been shown to have favorable effects on ventricular fibrosis. Losartan [59] and olmesartan [60] have improved Expert Rev. Cardiovasc. Ther. 12(1), (2014)

Review

healing after MI in rodents. Furthermore, the ability of ARBs markers of collagen turnover and inflammation including to inhibit the ACE-angiotensin II-TGF-b1 axis has been PINP and PIIINP, MMP type-2, IL-6, IL-8 and TNF-a documented in various extracardiac models [18]. ARBs have also increased with time in the control group. In the treated group, been shown in small clinical studies to reduce fibrosis no impact was evidenced on these biomarkers at 6 months, but biomarkers. For example, candesartan reduced fibrosis bio- PIIINP significantly decreased at 12 months. The treatment markers in 153 patients with atrial fibrillation [61] (this could was also associated with modest effects on diastolic function as reflect either atrial or myocardial fibrosis reduction). assessed by echocardiography, without any impact on clinical A comparison between the ARB losartan (n = 21) and amlodi- variables or brain natriuretic peptide [71]. pine (n = 16) administered for one year revealed that only losSee TABLE 1 for brief presentation of the trials with RAS inhibartan resulted in significant decreases in both CVF and PICP itors currently ongoing. although changes in blood pressure were similar in the two treatment groups [52]. Selective heart rate-reducing treatment: ivabradine Another strategy to reduce the effects of Ang II could be to The If current inhibitor ivabradine provides selective HR promote ACE2. This enzyme is an ACE homolog that hydro- reduction and has recently been introduced in the treatment lyzes Ang II in Ang(1-7), which in turn binds to Mas (a pro- guidelines for HF in patients with LV systolic dysfunction [72]. tein with 7 transmembrane domains and features characteristic In a rabbit model of diastolic dysfunction, ivabradine attenuof class I G-protein-coupled receptors [GPCRs]). This ated LV diastolic dysfunction and also reduced significantly ACE2-Ang(1-7)-Mas counterbalances the Ang II axis, leading both atrial and ventricular fibrosis as well as ventricular collato cardioprotection [62]. This regulatory system appears to be gen type I (FIGURE 3) [73]. Interestingly, ivabradine also decreased insensitive to ACE inhibitors [63] but is up-regulated by ARBs plasma Ang II levels in that study. These data support the hypothesis that selective HR reduction could be a target for [64]. ACE2 inhibition has been shown to increase fibrosis in hypertensive rats [65], whereas its overexpression improves new approaches in diastolic HF, and that ivabradine could be a promising candidate. Furthermore, the beneficial effects could remodeling in diabetic rats [66]. The mineralo-corticoid receptor antagonist spironolactone has involve decreased RAS activity leading to a reduction in cardiac been shown to be a promising drug in animal models [67,68] and fibrosis, although the underlying mechanisms remain to be fully has been recently proposed in clinical conditions associated with understood. In support of this notion, ivabradine was found fibrosis like metabolic syndrome and obeA sity. A comparison between spironolactone Control CD IVA 25 mg/day and placebo for 6 months in 80 patients with metabolic syndrome already treated with Ang II inhibition revealed that only the spironolactonetreated group showed significant improvement of LV diastolic function and parallel decreases in PICP and PIIINP levels [69]. The beneficial effects of spironolactone were not demonstrated in patients with the B 25 Control lower level of myocardial fibrosis or pre20 CD served diastolic function. In a more recent IVA prospective, randomized, double-blind 15 study, the same authors studied 113 patients with obesity, mild LV dia10 *** *** stolic dysfunction and no co-morbidities who were randomly assigned to spironolac5 tone 25 mg/day or placebo for 6 months 0 [70]. Myocardial deformation improved LV with spironolactone when compared with Figure 3. Ivabradine as a putative anti-fibrotic agent in diastolic heart failure in the placebo group, consistent with the a rabbit model. (A) Histology of LV interstitial fibrosis, Masson’s Trichrome staining observed decreases in PICP and (images acquired at 20 magnification). Fibrosis is stained in blue, whereas ventricular PIIINP levels. muscle is stained in pink. (B) Quantification by digital image analysis of interstitial fibrosis Interesting results have also been in the LV sections of control, CD and IVA groups, expressed as the percentage of stained obtained in a small study of 44 patients area in the region of analysis. with diastolic HF who were treated with ***p < 0.001. CD: Cholesterol diet; IVA: Cholesterol-diet+ivabradine. either placebo or the mineralo-corticoid Modified with permission from [73]. receptor antagonist eplerenone [71]. Serum Interstitial fibrosis (%)




117


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more efficacious to reduce cardiac fibrosis than metoprolol in mice models of HF [74]. Ivabradine could reduce systemic inflammation and pro-inflammatory cytokines [75], as recently demonstrated in a coxsackievirus B3 murine myocarditis model [76]. Both ivabradine and carvedilol similarly attenuated myocardial lesions and fibrosis, inhibited nitric oxide (NO) synthesis by inducible NOS, and decreased the production of TNF-a and IL-6 [76]. Beta-blockers could also be of interest, through the reduction of HR and blood pressure [77], but also by other effects including NO production [78] and anti-oxidative action as has been reported for the beta-blocker carvedilol [79]. Anti-inflammatory agents

Targeting some of the most powerful pro-inflammatory cytokines (TNF-a, IL-1 and IL-6) could have beneficial effects on cardiac fibrosis (see TABLE 1 for brief presentation of the ongoing trials with anti-inflammatory strategies). The TNF-a pathway is clearly involved in cardiac fibrosis in various basic models [56,80,81]. In a mouse model of pulmonary artery banding-induced right ventricle fibrosis, the expression of the Fn14 (TNF receptor superfamily member fibroblast growth factor-inducible molecule 14) was found to be increased [80]. Mice lacking Fn14 had substantially less right ventricle fibrosis and dysfunction compared with wild-type littermates [80]. In support of the role of TNF pathway in fibrosis, transgenic mice with cardiac-restricted overexpression of TNF developed progressive myocardial fibrosis [81]. In this mouse model, cardiac mast cell number increased significantly two- to threefold resulting in a mast cell-cardiac fibroblast cross-talk which seems to be required for the development of myocardial fibrosis in inflammatory cardiomyopathy [81]. Furthermore, cardiac fibroblasts exposed to sustained inflammatory signaling exhibit an increased repertoire of profibrotic phenotypic responses in response to mast cell mediators [81]. As TNFR1 and TNFR2 exert opposing effects on remodeling, hypertrophy, inflammation and apoptosis in HF [82], studying more precisely the role of the different receptor subtypes could help with defining specific effects and proposing tailored therapies [82]. Indeed, TNFR1 exacerbates, whereas TNFR2 ameliorates these phenomena, although signaling through both receptors is required to induce diastolic dysfunction and oxidative stress. [82] The IL-1 signaling system is an obvious target to control cardiac fibrosis [83]. One of the IL-1b receptors has been proposed as a new biomarker for cardiovascular risk assessment [84] in HF patients. Low levels of IL-1 could prevent cardiac fibrosis [85]. A pre-clinical study evaluated the role of the granulocyte colony-stimulating factor (G-CSF) on cardiac hypertrophy and fibrosis in mice with temporary aortic constriction [86]. G-CSF improved both systolic and diastolic functions and decreased cardiac fibrosis possibly through the IL-1b pathway. By contrast, inhibition of IL-1b was beneficial in a murine MI model (knock out for the type I IL-1 receptor) [87]. In this model, the infarct size was unchanged but there was a decreased infiltration of the infarcted myocardium with neutrophils and 118

macrophages and reduced chemokine and cytokine expression. This was followed by an attenuated fibrotic response including a decreased myofibroblast infiltration and reduced collagen deposition in the infarcted and remodeling myocardium. The data on IL-1b remain controversial, and fine-tuned regulation rather than strong inhibition or induction could be considered when targeting cardiac fibrosis. Injection of IL-11 reduced cardiac fibrosis in a mouse model of acute MI [88]. By contrast, infusion of IL-6 seemed to enhance fibrosis in another rat model [89]. Taken together, these findings support the role of inflammatory modulation for the treatment of cardiac fibrosis. Furthermore, new innovative treatments such as galectin inhibitors can be considered. Galectin-3 is a member of the lectin family and has been shown to be involved in inflammation, cell adhesion and activation, including in heart diseases [90]. It was found to be overexpressed in failing hearts in a rat model of HF [91]. Myocardial biopsies revealed that galectin-3 was overexpressed at an early stage specifically in the rats that later developed HF. Galectin-3 was proposed to represent a biomarker for activated macrophages, but could also be involved in activation of cardiac fibroblasts. Importantly, recombinant galectin-3 induced cardiac fibroblast proliferation [91], collagen production and an increase of collagen I over collagen III. Agents currently under study in the field of cardioprotection

Several agents are currently under study in cardioprotection. Despite its disappointing effect on infarct size in clinical trials [92], erythropoietin could have an effect on LVEF, perhaps by reducing fibrosis, as demonstrated in animal models [21,93]. Among promising pharmacological post-conditioning drugs currently under study, cyclosporine has been shown to reduce fibrosis in animal models of AMI [94], chronic aortic banding [95] or induced hypertrophy [96]. By analogy, the flavonoid quercetin has been shown to be cardioprotective in a rat model, reducing fibrosis as efficiently as cyclosporine [97]. These findings have been confirmed in a mouse model [98]. Many drugs are currently under development to achieve pharmacological post-conditioning, and their effect on cardiac fibrosis should also be evaluated. Ischemic post-conditioning inhibits MMP activity, protects extracellular matrix from degradation and down-regulates expression of TGF-b1 in rats. These findings were consistent with a reduction in the population of myofibroblasts within the infarcted myocardium [99]. Vasodilators seem to be also of interest. Sildenafil inhibits cyclic GMP-specific phosphodiesterase type-5A. It has been reported to prevent cardiac hypertrophy and LV dysfunction in various animal models including mice subjected to pressureoverload [100], mice injected with Ang II [101], rats subjected to MI [102] and rats with mitral regurgitation [103]. These preclinical data supported clinical trials on the topic [104]. In the first proof-of concept human study, patients with isolated Expert Rev. Cardiovasc. Ther. 12(1), (2014)



diabetic cardiomyopathy randomly treated 3 months with sildenafil 100 mg per day showed improvement in LV contraction parameters, when compared with controls [105]. Monocyte chemotactic protein-1 (MCP-1) and TGF-b were the only biomarkers decreased by active treatment. Finally, fenofibrate, a powerful peroxisome proliferatoractivated receptor-a (PPAR-a) activator, has been shown to inhibit myocardial fibrosis and diastolic dysfunction in a rat model of hypertensive cardiomyopathy [106]. This could involve inflammatory mechanisms associated with the NF-kB pathway. Emergent therapeutic agents

As fibrosis is mainly the consequence of modified activity of specific cell types including myofibroblasts and inflammatory cells, cell therapy appears as a logical and powerful answer (see TABLE 1 for ongoing trials). Indeed, most studies applying stem cell therapy following MI have demonstrated a decrease in fibrosis [107], and recent basic experiments corroborate the feasibility to manipulate fibroblasts in rats [108]. To our knowledge, no clinical trial specifically tailored to evaluate the ability of cell therapy to control myocardial fibrosis is currently ongoing. MicroRNAs have been demonstrated to be powerful antifibrotic agents [109,110]. In a rat model of MI, the expression of miR-101a and miR-101b (miR-101a/b) in the peri-infarct area was decreased, whereas the cardiac performance improved after adenovirus-mediated overexpression of miR-101a as evidenced by echocardiography and hemodynamic measurements [111]. Importantly, interstitial fibrosis was decreased by the treatment in that study. In another mouse model, genetic deletion of miR-214 caused loss of cardiac contractility, increased apoptosis and excessive fibrosis in response to ischemia-reperfusion injury [112]. The roles of some miRNAs in cardiac fibrosis have recently been reported [113,114]. The effects mediated by miRNA could implicate the TGF-b pathway [115] and represent new therapeutic approaches [109,116,117]. Such approaches could target various cells including fibroblasts [118] in various clinical settings, for example, after MI [111,119,120]; however, there are currently many theoretical and practical concerns regarding miRNA use for clinical translation. Inhibitors of NF-kB are currently under active development, especially in cancer research. NF-kB plays a key role in cell survival. The aim of this approach would be to control the apoptosis of myofibroblasts, leading to decreased fibrotic activity [121]. Reduction of collagen fiber turnover through the modulation of NF-kB could be targeted through cannabinoids, nerve and hepatocyte growth factors and the adipocytokines adiponectin and leptin [18]. TGF-b is considered as one of the main regulators of collagen synthesis [122]. This molecule could then be a promising target for inhibition (FIGURES 1 & 4), via antibody, antisense oligonucleotide or receptor antagonist [123]. Downstream of the receptor of TGF-b, the Smad transcription factors could be a more specific target [124], avoiding unwanted complications [125]. Halofuginone, an inhibitor of Smad3, has been shown to exert antifibrotic www.expert-reviews.com

Review

properties [126]. Other pathways independent from the Smad cascade have also been reported to reduce fibrosis in renal [127] or pulmonary [128] fibrotic models, and several clinical trials are ongoing including in cardiac Chagas disease [129]. Because TGF-b is not specific for fibrosis of the heart, local myocardial delivery or better cardiac specificity should be aimed. Targeting directly collagen synthesis and degradation could also be valuable targets. Drugs targeting MMPs are under development and promising results have been presented in animal models [130]. In a MMP-9 null mouse model, different groups of age were evaluated [131]. Although the systolic function was the same, the diastolic function was reduced in old and senescent wild-type mice compared with young controls. This reduction was attenuated in MMP-9 null mice [131]. Concomitantly, the increase in LV collagen content was reduced in MMP-9 null mice. This was consistent with differential expression of different molecules. Periostin expression (which is induced by TGF-b signaling) was reduced, whereas MMP-8 expression, which regulates myocardial collagen turnover and deposition, was increased [131]. The role of MMP-9 on collagen turnover has been confirmed in other models [132,133]. Conclusion

Our knowledge regarding the pathophysiology of fibrosis is growing and the physiological regulating mechanisms are well described. Although various potential therapeutic strategies are now available, careful assessment is necessary to both demonstrate therapeutic benefits on cardiac fibrosis and avoid hypothetical excessive effects (the latter theoretically leading to potential ventricular enlargement (due to loss of the myocardial collagen scaffold), wall thinning and even rupture). Some antifibrotic therapies have been promising in basic models, but only a few of them seem already evaluable in small clinical trials. Whether the regression of fibrosis is correlated with longterm functional improvement and better clinical outcomes remains to be demonstrated in large clinical studies. Such trials are feasible if based on imaging parameters and circulating biomarkers for the accurate but non-invasive assessment of ventricular fibrosis. Expert commentary & five-year view

In this review we showed that cardiac fibrosis is a promising therapeutic target. New tools are available to assess cardiac fibrosis in clinical trials, mainly MRI and biomarkers; but molecular imaging should provide very specific evaluations in a near future. Many basic experiments are very promising, and several clinical data have demonstrated the proof-of-concept, mainly with inhibitors of the RAS. Theoretically it opens the field for reversion of fibrosis, especially in numerous patients with fibrotic chronic diseases such as hypertension (one billion patients worldwide), metabolic syndrome (fast-increasing number) and later to fewer but severe patients suffering from extensive myocardial fibrotic processes such as dilated cardiomyopathy, ischemic disease 119

Review

sol. TβRIII: P144


Anti-TGFβ antibodies: metelimumab, fresolimumab, LY23822770

TGFβ

TGFβ

TβRIII (β-glycan)

Protein kinase inhibitors: Cytoplasm

TβRI P GS P (ALK5) G P

Smad 2/3

SB431542 SB505124 GW788388 SD208 LY573636 SM16

TβRIII P

LY2109761 LY2157299

Smad

Antisense oligonucleotide: Trabedersen

2/3 P +

Smad

4

co-TF

P

4 2/3

2/3

P

TGFβ2 mRNA

Sm ad Sm ad Sm ad


TGFβ

TGF-β target gene

SBE Nucleus

Figure 4. Possible ways to target TGF-b. TGF-b signaling is mediated by the binding of its homodimeric form to a receptor complex, triggering the hetero-oligomerization of this receptor complex, followed by the transphosphorylation and the subsequent phosphorylation of Smad-2 and Smad-3 proteins, which induces their association with Smad4 and the translocation of this complex to the nucleus, where it activates the transcription of TGF-b–target genes in co-operation with co-transcriptional factors (co-TF). The therapeutic approaches currently tested to inhibit TGF-b signaling in fibrotic diseases are schematically represented. First, TGF-b signaling can be blocked by sequestering the extracellular ligands using the TGF-b–binding domain of TbRIII (sol.TbRIII) or specific TGF-b neutralizing antibodies. Another strategy is based on the inhibition of the serinethreonine kinase activity of its signaling receptors via small chemical antagonists. Third, antisense nucleotides have also been developed to block TGF-b synthesis. GS: Glycine- and serine-rich box; mRNA: Messenger RNA; SBE: SMAD-binding element. Modified with permission from [129].

and so on. Fibrotic processes are not the same and controversial results are likely to be obtained in various diseases and models. The goal remains to ultimately obtain reversion of pathophysiological features leading to improvement of clinical outcomes.

120

Presently, the first step is the method of how we could accurately evaluate cardiac fibrosis in large populations. MRI is not perfect and not easily available but technical progresses and new sequences are being validated, which should confirm MRI as the best imaging option in this field. New imaging modalities such as molecular imaging are rapidly improving but remain expensive. Biomarkers are well validated and easily available and new candidates could enrich our range. Clearly we are on the edge of clinical trials evaluating the ability of various drugs to decrease fibrotic processes. First family is widely used drugs such as the RAS inhibitors, which could be proposed largely to achieve the goal. Secondly, inflammation modulators already used in clinical routine as well as in new ones are to be tested in near future as they seem promising in the field of atherosclerosis. Third, totally new approach as regard to clinical armory is the TGF-b targeting. Finally new candidates are emerging, such as targeting more specifically cells through cell therapies or genetic interventions (decreasing the profibrotic balance by cardiomyofibroblast or the modulation of putative regulatory cells such as progenitors, inflammatory cells or dendritic cells). In one word, large avenues remain to be explored. First large clinical trials in the field should support or not these enthusiastic perspectives and that is one reason why they appear so crucial. Acknowledgement

We are grateful to Dr. Matthias Friedrich for reviewing the sections pertinent to MRI. Financial & competing interests disclosure

F Roubille has received grants and honoraria from Servier – Abbot – Roche. JC Tardif has received grants from Servier – Roche and honoraria from Servier – Abbot – Roche. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.



Review

Key issues • Fibrosis is an important player in various pathways in cardiovascular pathogeny. • Assessment of cardiac fibrosis is currently possible, through MRI, developing molecular imaging and validated biomarkers. • First trials evaluating cardiac fibrosis and its regression have been completed, validating both the concept and the feasibility. • Promising results have been reported as regards to the renin-angiotensin-aldosterone system antagonists, especially with the mineraloreceptor inhibitors. Expert Review of Cardiovascular Therapy Downloaded from informahealthcare.com by Chinese University of Hong Kong on 02/13/15 For personal use only.

• Other profibrotic processes will be of interest, such as the pro-inflammatory cytokines or bradycardic therapies. If confirmed, their antifibrotic properties could be of interest in clinical settings. • New pathways, more specifically involved in fibrosis, are to be tested including the TGF-b modulation.

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•

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••

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Expanded access to investigational drugs.

Early investigational drugs targeting PPAR-α for the treatment of metabolic disease.

Investigational drugs targeting somatostatin receptors for treatment of acromegaly and neuroendocrine tumors.

Targeting the dopamine receptor in schizophrenia: investigational drugs in Phase III trials.

Investigational drugs for affective disorders. Foreword.

Investigational drugs for treating psoriatic arthritis.

Investigational new drugs for brain cancer.

Emerging and investigational drugs for premature ejaculation.

Investigational drugs in Alzheimer's disease: current progress.

Clinical development success rates for investigational drugs.

Targeting Adenosine Receptors for the Treatment of Cardiac Fibrosis.

MicroRNA-101a inhibits cardiac fibrosis induced by hypoxia via targeting TGFβRI on cardiac fibroblasts.

Synthetic investigational new drugs for the treatment of tuberculosis.

Investigational drugs under development for the treatment of PTSD.

Investigational therapies targeting the ErbB family in oesophagogastric cancer.

Access to 'investigational' cancer drugs: perspective of a trainee.

Dilemmas in the compassionate supply of investigational cancer drugs.

Perspectives on investigational drugs and immunotherapies for glioblastoma.

Successes and setbacks of early investigational drugs for melanoma.

Considerations for Use of Investigational Drugs in Public Health Emergencies.

Investigational drugs for treating anal cancer and future perspectives.

Investigational drugs for anxiety in patients with schizophrenia.

New investigational drugs with single-agent activity in multiple myeloma.

Overview of FDA's Expanded Access Program for Investigational Drugs.