cells Review The Role of Lysyl Oxidase Enzymes in Cardiac Function and Remodeling Cristina Rodríguez 1,2,3, * and José Martínez-González 2,3,4, * 1 Institut de Recerca Hospital de la Santa Creu i Sant Pau-Programa ICCC, 08025 Barcelona, Spain 2 Instituto de Investigación Biomédica Sant Pau (IIB-Sant Pau), 08041 Barcelona, Spain 3 CIBER de Enfermedades Cardiovasculares, Instituto de Salud Carlos III, 28029 Madrid, Spain 4 Instituto de Investigaciones Biomédicas de Barcelona (IIBB-CSIC), 08036 Barcelona, Spain * Correspondence: crodriguezs@santpau.cat (C.R.); jose.martinez@iibb.csic.es (J.M.-G.); Tel.: +34-93-556-5897 (C.R.); +34-93-556-5896 (J.M.-G.)  Received: 29 October 2019; Accepted: 20 November 2019; Published: 21 November 2019  Abstract: Lysyl oxidase (LOX) proteins comprise a family of five copper-dependent enzymes (LOX and four LOX-like isoenzymes (LOXL1–4)) critical for extracellular matrix (ECM) homeostasis and remodeling. The primary role of LOX enzymes is to oxidize lysyl and hydroxylysyl residues from collagen and elastin chains into highly reactive aldehydes, which spontaneously react with surrounding amino groups and other aldehydes to form inter- and intra-catenary covalent cross-linkages. Therefore, they are essential for the synthesis of a mature ECM and assure matrix integrity. ECM modulates cellular phenotype and function, and strikingly influences the mechanical properties of tissues. This explains the critical role of these enzymes in tissue homeostasis, and in tissue repair and remodeling. Cardiac ECM is mainly composed of fibrillar collagens which form a complex network that provides structural and biochemical support to cardiac cells and regulates cell signaling pathways. It is now becoming apparent that cardiac performance is affected by the structure and composition of the ECM and that any disturbance of the ECM contributes to cardiac disease progression. This review article compiles the major findings on the contribution of the LOX family to the development and progression of myocardial disorders. Keywords: lysyl oxidases; cardiac function; myocardial hypertrophy; extracellular matrix remodeling 1. Introduction Lysyl oxidase (LOX) proteins comprise a family of copper-dependent enzymes which governs extracellular matrix (ECM) homeostasis and remodeling. Five highly homologous LOX proteins have been identified in mammals: LOX and four LOX-like isoenzymes (LOXL1–4). The primary role of LOX enzymes is to oxidize lysyl and hydroxylysyl residues from collagen and elastin chains into highly reactive aldehydes, which spontaneously react with surrounding amino groups and other aldehydes to form inter- and intra-catenary covalent cross-linkages [1,2]. In the adult mammalian heart, an intricate network of ECM proteins provides a scaffold for the cellular components and participates in the transmission of contractile forces. Studies in both humans and in animal models have evidenced that the abnormal expression/activity of LOX/LOXLs isoenzymes is linked to cardiovascular diseases [1,3,4]. Excessive myocardial collagen deposition and cross-linking (CCL), a process conditioned by LOX enzymes, contributes to cardiac fibrosis and determines left ventricular (LV) stiffness and dysfunction [5–7]. Essentially, fibrosis is characteristic of all forms of heart disease, and most of the pathophysiological stimuli that trigger cardiac fibrosis modulate LOX/LOXLs, playing a critical role in the process [8–10]. Beyond CCL, additional biological functions have been reported for LOX/LOXLs [1,2]. Further, genes encoding LOX enzymes are Cells 2019, 8, 1483; doi:10.3390/cells8121483 www.mdpi.com/journal/cells Cells 2019, 8, 1483 2 of 20 Cells 2019, 8, 1483 2 of 20 functions have been reported for LOX/LOXLs [1,2]. Further, genes encoding LOX enzymes are part of the part mechanoresponsive of the mechanoresponsive genegeneprograms programs responsible responsiblefor forthe the mechanical mechanical regulation regulation of of gene gene expressionin expression incardiac cardiacmyocytes myocytesand andfibroblasts fibroblasts[11].[11].Thus, Thus, LOX/LOXLs LOX/LOXLscontribute contribute toto cardiac cardiac fibrosis, fibrosis, butare but arealso alsoactive activeplayers playersinvolved involvedin inthe thecellular cellularmechanisms mechanismsunderlying underlyingcardiac cardiacdysfunction dysfunctionand and diseaseprogression. disease progression. Inthethe In last last years,years, multiple multiple studies havestudies have contributed contributed to the ofunderstanding to the understanding of the the pathophysiological pathophysiological role of the LOX family in human diseases and, in particular, role of the LOX family in human diseases and, in particular, in the cardiovascular system. in the cardiovascular system. The The involvement involvement of this of this family family on vascular on vascular diseases diseases has has beenbeen recently recently reviewed reviewed [12].Further, [12]. Further, LOX/LOXLs participate in a variety of processes affecting the heart, LOX/LOXLs participate in a variety of processes affecting the heart, from those associated from those associated to post- to myocardial infarction post-myocardial (MI) cardiac infarction remodeling, (MI) cardiac cardiac cardiac remodeling, hypertrophy triggeredtriggered hypertrophy by pressureby or volume pressure overload, or heart failure volume overload, heart(HF) with failure preserved (HF) ejectionejection with preserved fraction (HFPEF) fraction associated (HFPEF) withwith associated obesity and obesity metabolic syndrome or atrial fibrillation. The strong impact of the alteration and metabolic syndrome or atrial fibrillation. The strong impact of the alteration of LOX/LOXLs on of LOX/LOXLs on CCL and and CCL heart function heart supports function supports thethe interest interestof of these thesefamily familyofofenzymes enzymes as as pharmacological targets targets to to improvecardiac improve cardiacremodeling remodelingand andslow slowthe theprogression progressionto toHF. HF.InInthis thisreview reviewarticle articlewewefocus focuson onthose those findingsthat findings thatsupport supportthethefundamental fundamentalinvolvement involvementofofthe theLOX LOXfamily familyin inthe themechanisms mechanismsunderlying underlying cardiacdamage cardiac damageand andrepair. repair. 2.2.The TheLOX LOXFamily Familyof ofECM-Modifying ECM-ModifyingEnzymes Enzymes LOX LOX isis an an extracellular extracellular enzyme enzyme which which governs governs thethe post-translational post-translational modification modification ofof ECM ECM collagen collagenand andelastin. elastin.LOX LOXcatalyzes catalyzesthethe oxidative deamination oxidative deamination of specific ε-amino of specific groups ε-amino of lysine groups and of lysine hydroxylysine residues and hydroxylysine in collagen residues and elastin. in collagen This leads and elastin. toleads This the generation of highlyof to the generation reactive highlypeptidyl reactive α-aminoadipic γ-semialdehydes peptidyl α-aminoadipic and the release γ-semialdehydes of hydrogen and the release of peroxide hydrogenasperoxide by-product [1–3] (Figure as by-product 1). [1–3] This reaction allows the covalent cross-linking of collagen and elastin, which ensures the appropriate (Figure 1). This reaction allows the covalent cross-linking of collagen and elastin, which ensures the tensile strength appropriate and elastic tensile properties strength and elasticof connective tissues. properties of connective tissues. Figure 1. The reaction catalyzed by LOX. LOX activity allows the oxidation of peptidyl lysine residues Figure 1. The reaction catalyzed by LOX. LOX activity allows the oxidation of peptidyl lysine residues in collagen and elastin chains to the corresponding peptidyl aldehydes. in collagen and elastin chains to the corresponding peptidyl aldehydes. LOX is the archetypal member of this family that comprises five closely related copper-dependent LOX is the archetypal member of this family that comprises five closely related copper- enzymes: LOX and four LOX-like isoenzymes (LOXLs: LOXL1, LOXL2, LOXL3, and LOXL4) [13]. dependent enzymes: LOX and four LOX-like isoenzymes (LOXLs: LOXL1, LOXL2, LOXL3, and Although they are product of distinct genes, all of them show a highly conserved catalytic domain LOXL4) [13]. Although they are product of distinct genes, all of them show a highly conserved and diverge in the rest of the sequence. Indeed, this family possesses a conserved carboxyl-terminal catalytic domain and diverge in the rest of the sequence. Indeed, this family possesses a conserved (C-terminal) amine oxidase catalytic domain, which comprises a His-X-His-X-His copper-binding carboxyl-terminal (C-terminal) amine oxidase catalytic domain, which comprises a His-X-His-X-His motif and the lysine tyrosyl quinone (LTQ) cofactor. By contrast, the amino-terminal (N-terminal) copper-binding motif and the lysine tyrosyl quinone (LTQ) cofactor. By contrast, the amino-terminal domains of these enzymes are structurally unrelated and their biological role is largely unknown. (N-terminal) domains of these enzymes are structurally unrelated and their biological role is largely Based on the primary structure of their N-termini, the family can be divided in two subgroups: LOX unknown. Based on the primary structure of their N-termini, the family can be divided in two and LOXL1, which contain a highly basic pro-peptide (PP) sequence, and LOXL2, LOXL3 and LOXL4 subgroups: LOX and LOXL1, which contain a highly basic pro-peptide (PP) sequence, and LOXL2, which possess four tandem repeats of the scavenger receptor cysteine-rich (SRCR) domains that LOXL3 and LOXL4 which possess four tandem repeats of the scavenger receptor cysteine-rich (SRCR) could mediate protein-protein interactions [14] (Figure 2). The high homology found in the catalytic domains that could mediate protein-protein interactions [14] (Figure 2). The high homology found in domain suggests that all of these isoenzymes would have similar substrate requirements and catalytic the catalytic domain suggests that all of these isoenzymes would have similar substrate requirements properties. However, the expression pattern of LOX and LOXLs differ among different tissues, and and catalytic properties. However, the expression pattern of LOX and LOXLs differ among different the dissimilar phenotypes shown by specific knockout animal models indicate that these isoenzymes tissues, and the dissimilar phenotypes shown by specific knockout animal models indicate that these could play different pathophysiological roles. isoenzymes could play different pathophysiological roles. Cells 2019, 8, 1483 3 of 20 Cells 2019, 8, 1483 3 of 20 Figure2. 2. Figure Structure Structure of human of human LOX isoenzymes. LOX isoenzymes. The scheme The scheme evidencesevidences the high of the high homology homology LOX/LOXsof LOX/LOXs isoenzymes in the C-terminal region, formed by the conserved catalytic isoenzymes in the C-terminal region, formed by the conserved catalytic domain which encompasses domain which encompasses the copper binding site and the lysyl tyrosyl quinone (LTQ) cofactor the copper binding site and the lysyl tyrosyl quinone (LTQ) cofactor region. The C-terminal region alsoregion. The C- terminalthe includes region alsoreceptor-like cytokine includes the (CRL) cytokine receptor-like domain. LOX and(CRL) LOXL1domain. LOX sequences propeptide and LOXL1 propeptide (PP) and the sequences site recognized(PP)byand the site BMP1 recognized (symbolized by by BMP1 (symbolized scissors), which allowsby thescissors), cleavagewhich allows of these the cleavage isoenzymes and of release the these isoenzymes of the activeand the are forms, release of the shown. Theactive forms, are shown. N-glycosylation TheLOX-PP sites at the N-glycosylation region aresites at the outlined. LOX-PP LOXLs region are isoenzymes outlined. contain LOXLs isoenzymes four scavenger contain four(SRCR) receptors cysteine-rich scavenger receptors domains probablycysteine-rich involved in(SRCR) domains probably protein–protein interaction. involved in protein–protein interaction. LOX LOXand andLOXL-1 LOXL-1are aresynthesized synthesizedand andsecreted secretedinto intothe theextracellular extracellularenvironment environmentininthe theform formofof inactive inactivezymogens, zymogens,whichwhichare aresubsequently subsequentlycleaved cleavedby bybone bonemorphogenetic morphogeneticprotein-1 protein-1(BMP-1) (BMP-1)and and other procollagen C-proteinases. This proteolytic processing releases the catalytically other procollagen C-proteinases. This proteolytic processing releases the catalytically active forms active forms [1,15] (Figure [1,15] 3). LOX, 3). (Figure the LOX, isoform theforisoform which itsfor proteolytic which itsactivation has been proteolytic more clearly activation has beencharacterized, more clearly is synthesized characterized, as aisprecursor of approximately synthesized as a precursor46ofkDa, which is processed approximately 46 kDa,towhich the enzymatically is processedactive to the form of 32 kDa. active enzymatically The proteolytic form of 32 processing kDa. Theofproteolytic the zymogen is facilitated processing of thebyzymogen fibronectin,is which binds facilitated by LOX and BMP1, fibronectin, whichthereby bindsregulating LOX andthe catalytic BMP1, activity thereby of LOX the regulating [15].catalytic It should be noted activity of that LOXLOX [15].isIt assisted should by beother notedproteins that LOXin the cross-linking is assisted of collagen by other and proteins inelastin. Fibromodulin, the cross-linking a small leucine-rich of collagen and elastin. protein (SLRP), ainteracts Fibromodulin, with LOX protein small leucine-rich and acts as a modulator (SLRP), of itsLOX interacts with activity fostering and acts a site-specific as a modulator of its cross-linking of collagen activity fostering fibrils [16]. a site-specific Similarly, cross-linking of the interaction collagen fibrils between LOX and [16]. Similarly, fibulin-4 seems the interaction betweento promote elastin cross-linking [17]. Overall, LOX activity can be regulated at different LOX and fibulin-4 seems to promote elastin cross-linking [17]. Overall, LOX activity can be regulated levels: regulating LOX transcription, at different modulatingLOX levels: regulating the transcription, extracellular processing modulatingofthe theextracellular pro-enzyme, and stimulating processing or of the pro- inhibiting enzyme, and the catalytic stimulatingactivity of the mature or inhibiting enzyme.activity of the mature enzyme. the catalytic Beyond ECM cross-linking, additional biological functions have been reported for LOX and LOXLs (LOX/LOXLs) [1–3], including the control of cell adhesion migration and proliferation and the modulation of gene transcription and epithelial to-mesenchymal transition. Further, active intracellular forms (both cytoplasmic and nuclear) for LOX/LOXLs have been described [18–20], while the pro-peptide (LOX-PP) released during the proteolytic processing of LOX also exhibits biological activity [2,21] (Figure 3). Therefore, it is likely that LOX/LOXL biology continue revealing novel critical aspects in the near future. Cells 2019, 8, 1483 4 of 20 Cells 2019, 8, 1483 4 of 20 Figure Figure 3. 3. Synthesis Synthesis andand processing processing of LOX.of LOX LOX.is transcribed LOX is transcribed and transduced and transduced leading to theleading to the production of the pre-proLOX form, a pre-proenzyme which is post-translationally modified in endoplasmicin production of the pre-proLOX form, a pre-proenzyme which is post-translationally modified endoplasmic reticulum (ER) andreticulum Golgi to(ER) and Golgi generate the LOX to proenzyme. generate theThis LOX proenzyme. multistep process This multistep involves process (i) cleavage ofinvolves (i) cleavage signal peptide, (ii) of signal peptide, incorporation of (ii) incorporation copper, of copper, (iii) formation of the(iii)lysyl formation tyrosylofquinone the lysyl (LTQ) tyrosyl quinone cofactor and(LTQ) cofactor and of (iv) glycosylation (iv)theglycosylation LOX propeptide of the LOX (LOX-PP). region propeptideThen region this(LOX-PP). Then this inactive precursor isinactive releasedprecursor is released into into the extracellular space,thewhere extracellular space, where it is proteolyzed it is proteolyzed by procollagen by procollagen C-proteinases (mainlyC- proteinases bone (mainly protein morphogenetic bone morphogenetic 1; BMP1) producing protein 1; BMP1) the mature producing catalyticthe LOXmature form,catalytic LOX form, which promotes which promotes extracellular matrixextracellular matrixand (ECM) maturation, (ECM) maturation,which its pro-peptide, and its is pro-peptide, responsible ofwhich is responsible the tumor suppressorof properties the tumorofsuppressor LOX among others effects. properties of LOX Intracellular among others forms of mature effects. LOX have Intracellular formsalso of been maturedetected LOX havein cytosol and nuclei. also been detected In cancer cells,and in cytosol cytosolic nuclei.active LOX forms In cancer cells,control cell adhesion cytosolic active LOX andforms motility through control cell the H O -dependent activation of Src-kinase and the subsequent phosphorylation 2 2 and motility through the H2O2-dependent activation of Src-kinase and the subsequent adhesion of focal adhesion kinase (FAK). Likewise, phosphorylation nuclear of focal LOX modulates adhesion kinase (FAK).chromatin structure Likewise, affecting nuclear LOXgene expression. modulates chromatin structure affecting gene expression. 3. LOX Isoenzymes in the Cardiovascular System Beyond Early ECM studies thatcross-linking, associated the additional biological cardiovascular functions phenotype have been of lathyrism reported for by (characterized LOX and aortic LOXLs (LOX/LOXLs) dissection/rupture) with [1–3], including the inhibition theLOX of the control of cell activity enzymatic adhesion migration (reviewed and proliferation in [22]) put the focus on and the modulation of gene transcription and epithelial to-mesenchymal transition. the relevance of the LOX family in the cardiovascular system. Results from genetically modified animal Further, active intracellular models supportforms (both a critical cytoplasmic contribution and nuclear) of these enzymesfor LOX/LOXLs have to cardiovascular been described development, function [18–20], and while the pro-peptide remodeling. LOX knockout (LOX-PP) mice (Loxreleased duringtothe −/− ) develop fullproteolytic term but are processing not viableofand LOX diealso exhibits at the end ofbiological activity the gestation [2,21] or as (Figure neonates 3). These [23]. Therefore, it is show animals likely severe that LOX/LOXL biology continue vascular abnormalities, most revealing likely novel due to acritical aspects defective in the near Tortuous elastogenesis. future. aortas, a high incidence of aortic aneurysms, disruption of elastic fibers and abnormalities in the morphology and adhesive properties of endothelial and 3. LOXmuscle smooth Isoenzymes in thedetected cells were Cardiovascular in Lox−/−System pups. Surprisingly, however, Lox−/− fetal hearts were indistinguishable Early studiesfrom thatthose of wild-type associated animals, while the cardiovascular cardiac LOX phenotype overexpression of lathyrism severely impacts (characterized by aortic ondissection/rupture) cardiac function [24],withastheexplained inhibition inof more detailenzymatic the LOX later in this review. activity Knockout (reviewed mice put in [22]) for LOXL1, the focus the onclosest mammalofparalog the relevance the LOX of family LOX, showin theancardiovascular altered post-partum system. deposition of elastic Results from fibers in genetically pelvic modified organs associated with pelvic organ prolapse, emphysematous lungs and skin animal models support a critical contribution of these enzymes to cardiovascular development,laxity, as well as vascular function and remodeling. LOX knockout mice (Lox−/−) develop to full term but are not viable and die at the end of the gestation or as neonates [23]. These animals show severe vascular abnormalities, Cells 2019, 8, 1483 5 of 20 abnormalities, although less serious than those detected in Lox−/− mice [25]. In turn, transgenic mice with cardiomyocyte-specific expression of LOXL1 developed cardiac hypertrophy [26]. Deletion of LOXL2 in mice increases perinatal death in nearly half of newborn animals, associated with dramatic alterations in the heart (disrupted ventricular septa) [27], while, unexpectedly, animals surviving the perinatal period grow without any major pathological manifestation. Regarding LOXL3 and LOXL4, both have been recently involved in vascular remodeling [28–30], although no apparent cardiovascular alterations have been detected in LOXL3 knockout mice, which exhibit perinatal lethality due to severe craniofacial defects and spinal deformities [31]. Unfortunately, LOXL4-deficient mice are not yet available. LOX and LOXLs isoenzymes have been implicated in the control of vascular homeostasis. The expression of LOX is high in the endothelium of healthy arteries and its inhibition has been linked with the endothelial dysfunction evoked by atherosclerotic risk factors, the instability and rupture of advanced atherosclerotic plaques and aortic aneurysm development [12]. Further, LOX regulates vascular smooth muscle cells (VSMC) proliferation and vascular remodeling, participates in the control of vascular stiffness, oxidative stress and calcification, and modulates platelet activation and thrombosis [12,32–34]. Likewise, LOXL2 controls the age-associated increase in vascular stiffness and it is the main LOX isoenzyme contributing to neovascularization. The mechanisms underlying the LOX/LOXLs-mediated control of vascular homeostasis have been extensively discussed [12] and are beyond the scope of this review. Regarding the heart, LOX and LOXLs are spatiotemporally regulated during embryonic and fetal heart development [35,36], and results in humans and in different experimental models have clearly evidenced that the disturbance of LOX/LOXLs expression/activity is linked to cardiovascular diseases [4–6,37]. Among the five LOX enzymes, LOX is the most abundant in the heart [13], and most studies on cardiac function refer to this isoenzyme, as described below. Less information is available about LOXL1 and cardiac diseases. Nonsynonymous coding single-nucleotide polymorphisms (SNPs) in the LOXL1 gene are the major known genetic risk factor for pseudoexfoliation syndrome (XFS; OMIM#177650) [38], an aging-related systemic disease involving an abnormal ECM deposition, characterized by an increased risk of glaucoma, and a high susceptibility to heart disease among others [39]. In a model that spontaneously develops age-related cardiac-selective fibrosis, the plasminogen activator inhibitor-1 (PAI-1) knockout mice, genome-wide gene expression profiling identified Loxl1 among the most upregulated transcripts involved in profibrotic pathways [40]. Concerning LOXL2, it is highly expressed during the early stages of cardiac development [13], has been recognized as a NOTCH candidate gene potentially involved in valve formation [41], and is a major player in cardiac fibrosis [42]. The contribution of each member of the LOX family to cardiac diseases has been more exhaustively detailed in the next sections. 4. ECM Synthesis and Remodeling in the Heart In the adult mammalian heart, cardiomyocytes are arranged in layers separated by clefts. An intricate network of ECM proteins provides a scaffold for the cellular components and participates in the transmission of the contractile force. Cardiac ECM is mainly composed of fibrillar type I and III collagens (approximately 85% and 11% of total myocardial collagen, respectively), and minor components including elastin, laminin, and fibronectin [43]. Cardiac ECM also contains latent growth factors and proteases whose activation, following cardiac injury, triggers fibrosis, an anomalous matrix remodeling due to an disproportionate deposition of ECM proteins during the wound healing response associated with chemical, mechanical, and immunological stresses. Mature fibrillar collagen is highly stable (half-life 80–120 days), and its turnover is primarily regulated by cardiac fibroblasts. The homeostatic control of cardiac ECM involves a tightly regulated balance between synthesis and degradation of matrix proteins, whose disturbance results in structural and functional abnormalities of the heart. Collagens are synthesized as procollagen chains, with N- and C-terminal propeptide domains, which associate into trimers allowing the folding of the collagen triple helix [44]. Procollagen molecules translocate to the Golgi apparatus, where they are packaged in vesicles Cells 2019, 8, 1483 6 of 20 for their extracellular transport. Once the terminal propeptides are cleaved by zinc (Zn)-dependent metalloproteinases (procollagen N- and C-proteinases), mature collagen molecules spontaneously self-assemble into fibrils [45]. Fibrillar type I and III collagens must be cross-linked to form fibers highly resistant to proteolytic degradation and to acquire their physical properties and tensile strength. Collagen experiences two main types of cross-links: those mediated by LOX enzymes and those derived from the nonenzymatic glycation of lysine and hydroxyllysine residues [46]. Besides cardiomyocytes, fibroblasts represent the largest cardiac cell population. Cardiac fibroblasts, as the main ECM-producing cells, play a key role in preserving the integrity of the ECM network. The cardiac fibroblast population significantly expands during the two first weeks after birth due to the increase of left ventricular pressures [47]. In healthy young adult hearts, cardiac fibroblasts remain quiescent and secrete negligible amounts of ECM proteins [48]. Following cardiac injury, however, a differential population of fibroblasts, namely myofibroblasts, accumulates in sites of injury. Myofibroblasts behave as phenotypically modulated cardiac fibroblasts which combine ultrastructural and phenotypic characteristics of smooth muscle cells (contractile stress fibers) and synthetically active fibroblasts (extensive endoplasmic reticulum) [48]. Several cues, including an abnormal composition and mechanical characteristics of the ECM, upregulation and release of cytokines and growth factors, and enhanced mechanical stress, govern myofibroblast transdifferentiation. Collagen mainly originates from activated myofibroblasts which play a fundamental role in both reparative and fibrotic processes. Myofibroblast transdifferentiation and enhanced deposition of fibrillar collagen in the cardiac interstitium are characteristic of the cardiac fibrotic response. Fibrillar collagens of the ECM form a complex meshwork that provides structural and biochemical support to cardiac cells and regulates cell signaling. Cardiac function is unequivocally influenced by the composition and structure of the ECM and any disturbance of the ECM contribute to the progression of cardiac diseases. Firstly, in response to injury, collagen deposition and ECM remodeling are adaptive responses that aim to preserve tissue integrity and cardiac function. However, the synergistic response to proinflammatory and profibrotic mediators may overactivate myofibroblasts, leading to an excess of collagen deposition in the matrix. Irrespective of the nature of the injury, and whether it is focal (e.g., MI) or more global (e.g., pressure or volume overload), the common fibrotic response is characterized by collagen synthesis and deposition and upregulation of LOX enzymes, leading to a consequent increase in CCL which makes fibrillar collagen less prone to degradation by matrix metalloproteinases (MMPs). This process alters ECM biomechanical properties and thereby, determines diastolic dysfunction and cardiac stiffness [4,7,37]. Essentially, fibrosis is characteristic of all forms of heart disease, being detrimental for LV function and contributing to the progression to HF. Most of the pathophysiological stimuli that trigger cardiac fibrosis modulate LOX/LOXLs that play a critical role in the process. It is well known the importance of transforming growth factor beta-1 (TGF-β1) in cardiac remodeling and fibrosis [49,50]. LOX/LOXLs are regulated by TGF-β in multiple cells and tissues including the heart. The induction of LOX by TGF-β1 in cardiac fibroblasts requires the activation of PI3K/Akt, Smad3, and MAPK signaling. Concomitantly TGF-β1 increases collagen types I and III, and BMP1 expression [8], thereby contributing to the fibrotic response. Similarly, the induction of LOX by TGF-β1/Smad/AP-1 signaling exacerbates myocardial fibrosis and HF induced by abdominal aortic coarctation in rats [9]. Other profibrotic mediators, including pro-inflammatory cytokines such as TNFα are upregulated in many cardiac diseases. TNFα is a fundamental factor regulating heart function and cardiac fibrosis in a LOX dependent manner. In fact, this cytokine increases LOX expression in cardiac fibroblasts through TGF-β and PI3Kinase signaling pathways [10]. As reviewed below, the increase in LOX/LOXLs expression has been linked with the detrimental cardiac remodeling and dysfunction in several pathological scenarios. 4.1. LOX and LOXLs in Dilated and Hypertrophic Cardiomyopathies and HF The increase in LOX/LOXL expression and/or LOX activity seems to be required by the cardiac fibrotic process to progress. The critical role of the LOX family in CCL determines the close link Cells 2019, 8, 1483 7 of 20 between LOX expression and/or activity and both myocardial stiffness and disturbed LV function. Clinical data and findings from multiple studies in animal models support this relationship. LOX has been involved in the fibrotic process that accounts for end-stage dilated cardiomyopathy (DCM). The increase of LOX mRNA and protein levels in the myocardium in patients with DCM and end-stage HF were associated with a rise in both TGF-β expression and collagen content [51]. Similarly, LOX is increased in areas of intersticial and perivascular fibrosis in the fibrotic myocardium of patients with hypertensive heart disease (HHD) and chronic HF [6]. In these patients a positive correlation between LOX protein levels and CCL, and between CCL and LV stiffness were found. Subsequent studies further support the concept that the degree of CCL (rather than the total amount of collagen) correlates with an impairment of systolic and diastolic function. Patients with HF and normal ejection fraction (EF), displayed an increase in the myocardial content of collagen type I, enhanced CCL, and higher LOX expression, which were associated with a deterioration of diastolic function [7]. Likewise, in patients with hypertension and stage C heart failure, CCL, and not collagen amount, was associated with an elevation of filling pressures [37]. This study concludes that the disproportionate CCL promoted an increase in LV stiffness, which leads to an elevation of filling pressures in these patients [37]. In agreement, in HF patients with normal EF, Kasner et al. found an enhanced myocardial collagen content, but also an increase in LOX expression and CCL, both associated with an abnormal diastolic function [7]. Remarkably, patients with HHD and HF, exhibited an excessive myocardial expression of osteopontin (OPN), that was related to enhanced LOX, higher insoluble collagen, as well as LV stiffness and altered systolic function. Since OPN up-regulates LOX in human fibroblasts in culture, the authors proposed that the OPN-LOX axis might promote an exacerbated deposition of insoluble collagen (stiffer and more resistant to degradation) and the consequent alteration of LV function in these patients [52]. The increase of LOX and OPN levels, as well as those of TGF-β1 and periostin, in cardiac tissues from patients with end-stage HF has been confirmed in subsequent studies [53]. Interestingly, recent research shows the relevance of LOX as a target of miRNAs that regulate cardiac function and fibrosis, in particular of miR-19b [54]. In patients with severe aortic stenosis (AS) and HF, cardiac and circulating miR-19b levels were inversely correlated with LOX protein levels, CCL and LV stiffness [54]. Concerning LOXLs and similar to LOX, cardiac LOXL2 is upregulated in patients with ischemic or idiopathic DCM, in which LOXL2 levels correlate with CCL and with diastolic dysfunction [42]. Furthermore, circulating LOXL2 levels are also elevated in HF patients and correlate with other biomarkers of HF [42]. Therefore, both LOX and LOXL2 contribute to cardiac CCL and condition cardiac performance. Experimental research in different animal models of cardiac hypertrophy has also contributed to build the concept that qualitative rather that quantitative changes in collagen critically impact on cardiac stiffness and remodeling [5]. Certainly, multiple studies have evidenced enhanced expression and/or activity of LOX/LOXLs and CCL and their relationship with adverse myocardial remodeling and dysfunction. Myocardial TGFβ-dependent signaling, LOX expression and CCL were found increased in spontaneously hypertensive rats (SHRs) with LV hypertrophy compared with normotensive animals [55]. Further, in BALB/c mice subjected to experimental hypertension induced by N(G)-nitro-l-arginine methyl ester (l-NAME), the cardiac increase in total fibrillar collagen, CCL, and in the expression and enzymatic activity of LOXL-3 were associated with ventricular stiffness [56]. A similar situation was reported in a model of chronic HF induced by aortic banding, in which the TGF-β1/Smad/AP-1-dependent induction of LOX aggravates myocardial fibrosis and HF [9]. The enhanced LOX-dependent CCL also underlies the pro-hypertrophic role of syndecan 4 in the pressure-overloaded myocardium. The cardiac expression of syndecan 4 rises in mice subjected to aortic banding and impacts on LOX and CCL through a dual mechanism. On one hand, the cytosolic domain of syndecan 4 is responsible for a NFAT-mediated induction of collagen, OPN, and LOX, while its extracellular domain additionally facilitates LOX-dependent CCL by interacting with collagen fibers [57]. In this scenario, disrupting CCL through inhibition of LOX is expected to reverse fibrosis and ameliorate cardiac dysfunction. Indeed, the administration of β-aminopropionitrile (BAPN), Cells 2019, 8, 1483 8 of 20 an irreversible LOX inhibitor, to 38-week-old mice reversed established age-related myocardial fibrosis to a level similar to that of young mice [58]. The inhibition of LOX by BAPN modulated TGF-β signaling and collagen synthesis, but also the degree of macrophage infiltration [58]. In the rat model of aortocaval fistula-induced volume overload (VO), the expression and activity of LOX, as well as collagen and CCL, were progressively upregulated in parallel with an enhanced ventricular mass [59]. Interestingly, in this model, LOX activity negatively correlated with cardiac function, while LOX Cells 2019, 8, 1483 8 of 20 inhibition by BAPN completely circumvented the increase in fibrotic mediators, such as collagens, MMPs aortic and their and banding tissue inhibitors, impacts on LOXandand ameliorated CCL through the cardiac a dual dysfunction mechanism. caused On oneby VO the hand, [60,61]. cytosolic Therefore, domain LOX inhibition of syndecan seems to 4 is responsible forbea cardioprotective, NFAT-mediated induction while the of chronic collagen,increase OPN,ofandcardiac LOX, LOX activity would play a detrimental role favoring the transition from while its extracellular domain additionally facilitates LOX-dependent CCL by interacting with compensated remodeling to decompensated collagen fibersfailure [57]. aggravating HF. Our recent study, using a new transgenic mouse model (TgLOX) that overexpresses In this scenario, human LOX inCCL disrupting the through heart (Figure 4A), of inhibition further LOX evidences is expectedthe to direct reverserelationship fibrosis and between LOX and diastolic function [24]. Myocardial LOX overexpression ameliorate cardiac dysfunction. Indeed, the administration of β-aminopropionitrile causes an age-dependent (BAPN), an diastolic dysfunction irreversible and accelerates LOX inhibitor, cardiacmice to 38-week-old remodeling reversed[24]. More interestingly, established LOX transgenesis age-related myocardial fibrosis exacerbated the cardiac hypertrophy and dysfunction induced by the to a level similar to that of young mice [58]. The inhibition of LOX by BAPN modulated TGF-βchronic infusion of Angiotensin IIsignaling (Ang II) (Figure 5A), triggering and collagen synthesis,abut more alsosevere fibrotic the degree ofprocess macrophagewith higher collagen infiltration [58].deposition and In the rat model CCL (Figure 5B)fistula-induced of aortocaval and an enhanced expression volume overloadof fibrotic (VO), the markers. expression and activity of LOX, as well as Additionally, collagen and CCL, LOX transgenesis were exacerbated progressively the cardiac upregulated inflammatory in parallel with anresponse enhanced induced by Ang ventricular II mass (Figure 6A), enhanced [59]. Interestingly, the expression in this model, LOXofactivity proinflammatory negatively markers correlated such with Il-6, and ascardiac decreased function, thatLOX while of the cardioprotective factor Fgf21 [24]. These animals also exhibit an altered inhibition by BAPN completely circumvented the increase in fibrotic mediators, such as collagens, Ang II-dependent signaling with MMPs increased and theirp38tissue MAPK activation, inhibitors, while and reduced AMP-activated ameliorated protein kinase the cardiac dysfunction caused (AMPK) by VO activation [60,61]. (Figure 6B). Further, Therefore, LOXLOX enhances inhibition cardiac seems to beoxidative stress (Figure cardioprotective, while6C),the an effectincrease chronic due, at least in a of cardiac part, to the attenuation of the induction of SOD1 promoted by Ang II. Whether LOX activity would play a detrimental role favoring the transition from compensated remodeling to the enhanced cardiac oxidative stress exhibited decompensated by TgLOX mice failure aggravating HF.accounts Our recent for the overactivation study, using a new of p38 MAPK, mouse transgenic as described model in(TgLOX) the vascularthat wall in VSMC-specific overexpresses human LOX transgenic in the heart mice [33], 4A), (Figure is an further issue that deservesthe evidences further direct investigation. Therefore, beyond CCL other mechanisms including excessive relationship between LOX and diastolic function [24]. Myocardial LOX overexpression causes an age- inflammation and ROS production dependentcould account diastolic for the cardiac dysfunction phenotype cardiac and accelerates exhibited by TgLOX[24]. remodeling miceMore (Figure 7). Further, this interestingly, LOX study reveals that LOX promotes fibroblast-to-myofibroblast transition. transgenesis exacerbated the cardiac hypertrophy and dysfunction induced by the chronic infusion Indeed, transgenic cardiac fibroblasts expressed of Angiotensin II (Anghigher levels of5A), II) (Figure markers of theaphenotypic triggering more severe switch fibroticof fibroblasts process with to myofibroblasts higher collagen (i.e., α-alphaand deposition smooth CCLmuscle (Figureactin and an 5B) and transgelin enhanced [SM22α]) expressionthanofcontrol fibroticcells (Figure 4B). markers. Figure4.4.Impact Figure ImpactofofLOX LOXtransgenesis transgenesison oncardiac cardiacfibroblast fibroblastphenotype. phenotype.(A) (A)LOX LOXimmunohistochemical immunohistochemical analysisinin analysis heart heart sections sections from from WT WTandand TgLOX TgLOX micemice evidences evidences thethe increased increased cardiac cardiac LOXLOX expression expression in in LOX transgenic mice. Bar: 50 µm. (B) In cardiac fibroblasts from TgLOX mice, which LOX transgenic mice. Bar: 50 µm. (B) In cardiac fibroblasts from TgLOX mice, which exhibits higher LOX exhibits higher LOX expression expression than WTthan cells,WT cells, the expression the expression of markers ofofthe markers of theswitch phenotypic phenotypic switch of fibroblasts to of fibroblasts to myofibroblasts myofibroblasts (α-actin (α-actin and transgelin; and transgelin; SM22α), SM22α), as well as that of typeasI well as that collagen, wereofincreased. type I collagen, were increased. Representative images ofRepresentative Western-blot assaysimages of Western-blot are shown. Glyceraldehydeassays are shown. 3-phosphate Glyceraldehyde dehydrogenase (GAPDH) protein3-phosphate levels dehydrogenase were (GAPDH) analyzed as loading protein control levels were analyzed in Western-blot assays. The as position loading control in Western-blot of the molecular weight assays. The standards position of the molecular weight standards (HyperPAGE prestained (HyperPAGE prestained protein markers, BIOLINE; Ref: BIO-33065) is indicated. protein markers, BIOLINE; Ref: BIO-33065) is indicated. Cells 2019, 8, 1483 9 of 20 Cells 2019, 8, 1483 9 of 20 Figure Figure 5. 5. LOX LOXtransgenesis transgenesisexacerbates exacerbatesAngAngII-induced II-inducedLV LV systolic systolic dysfunction dysfunction andand fibrosis. fibrosis. (A) (A)WTWT (white (white columns) columns) and andTgLOX TgLOX(black (blackcolums) colums)mice micewere wereinfused infusedwith withAngII AngII(1000 (1000ng/kg/min) ng/kg/min)or orsaline saline solution solutionfor for 14 14 days. days. The Theoverexpression overexpressionof ofLOX LOXreduces reducesleft leftventricular ventricular(LV) (LV) ejection ejection fraction fraction (left (left panel) and fractional shortening (right panel) as determined by echocardiography in panel) and fractional shortening (right panel) as determined by echocardiography in Ang II infusedAng II infused mice. mice.Results Resultsareareexpressed expressedasasmean mean ±±SD.SD.**pp <<0.05 0.05vs. vs.WT. WT.(B) (B)Heart Heartsections sectionsfrom from Ang Ang II-infused II-infused mice mice were were stained stained with with Picrosirius Picrosirius red. red. Under Underpolarized polarizedlight, light,cross-linked cross-linkedcollagen collagenappears appearsas as red red refringent refringentfibers. fibers. As Asshown, shown,LOXLOXoverexpression overexpressionenhances enhancesthetheAng AngII-induced II-induceddeposition depositionof ofmature mature collagen(Bar: collagen (Bar: 50 50µm). µm). Similarly, AngLOX Additionally, II-induced cardiacexacerbated transgenesis hypertrophythe was exacerbated cardiac in transgenic inflammatory miceinduced response that overexpress by Ang IILOXL1 (Figurespecifically in cardiomyocytes, 6A), enhanced the expression ofwhich exhibited myocyte proinflammatory markershypertrophy, and decreased such as Il-6, and higher mRNA that levels of the of brain natriureticfactor cardioprotective peptide (Bnp) Fgf21 than [24]. Thesetheiranimals WT littermates. also exhibitEchocardiographic an altered Angdata evidenced II-dependent that LOXL1 signaling transgenesis with increasedcaused p38 MAPKan increase in wall thickness activation, while reducedwith preserved cardiac contraction AMP-activated protein kinase[26]. It shouldactivation (AMPK) that Loxl1 be noted(Figure 6B). expression Further, LOX is enhances induced by hypertrophic cardiac oxidativeagonists in cardiomyocytes stress (Figure 6C), an effect in culture, and, in vivo, in a rat model of hypertrophy induced by abdominal due, at least in a part, to the attenuation of the induction of SOD1 promoted by Ang II. Whether aortic constriction. the enhanced cardiac oxidative stress exhibited by TgLOX mice accounts for the overactivation of with Thus, LOXL1 also seems to critically contribute to cardiac hypertrophy. Finally, and consistent p38 data from MAPK, HF patients as described in described the vascular above, wall LOXL2 seems to be in VSMC-specific LOXfundamental transgenicfor interstitial mice fibrosis [33], is an and issue that cardiac dysfunction. In mice, pressure overload activates fibroblasts and deserves further investigation. Therefore, beyond CCL other mechanisms including excessive induces the expression of LOXL2, leading inflammation toROS and fibrosis and disturbing production systolicfor could account andthediastolic cardiacfunctions phenotype [42]. Inhibition exhibited of LOXL2 by TgLOX miceby antibody-based (Figure strategies 7). Further, or gene this study knockdown reveals that LOX limits cardiac fibrosis promotes and LV dilatation, ameliorating fibroblast-to-myofibroblast transition. systolic transgenic Indeed, and diastolic dysfunction. cardiac fibroblasts LOXL2 stimulate expressed highermyofibroblast migration levels of markers of theacting downstream phenotypic of switch of TGF-β2 and fibroblasts through a PI3K/AKT to myofibroblasts mediated (i.e., α-alpha smoothmechanism that promotes muscle actin fibroblast-to-myofibroblast and transgelin [SM22α]) than control transformation cells (Figure 4B). [42]. Therefore, both LOX and LOXL2 contribute to the phenotypic switch of fibroblasts emphasizing the importance of ECM stiffness in myofibroblast activation [62]. Cells 2019, 8, 1483 10 of 20 Cells 2019, 8, 1483 10 of 20 Cells 2019, 8, 1483 10 of 20 A Ang II B WT TgLOX Ang II CD3 WT TgLOX Kda -40 P-p38 -40 T-p38 C Ang II P-AMPK -50 WT TgLOX DHE staining T-AMPK -50 -40 GAPDH Figure 6. LOX transgenesis aggravates Ang II-induced cardiac inflammation and oxidative stress and Figure6.6. LOX LOX transgenesis aggravates transgenesissignaling aggravates Ang AngII-induced II-induced cardiac inflammation cardiac inflammation and oxidative stress and Figure disturbs Ang II-dependent pathways. (A) WT and TgLOX mice wereand oxidative infused stress with AngII disturbs and disturbsAng II-dependent Ang II-dependent signaling pathways. signaling pathways.(A) WT and TgLOX mice (A) WT and TgLOX mice were infused were that with infused AngII (1000 ng/kg/min) or saline solution for 14 days. Inmunohistochemistry revealed AngwithII- (1000 (1000 ng/kg/min) or saline solution for for 14 14 days. Inmunohistochemistry revealed that Ang II- challenged TgLOX mice exhibited higher myocardial lymphocyte infiltration (CD3+) than Ang AngII ng/kg/min) or saline solution days. Inmunohistochemistry revealed that WT challenged TgLOX II-challenged TgLOX micemice exhibited higher myocardial exhibited higher myocardial lymphocyte lymphocyte infiltration infiltration (CD3+) (CD3+) thanthanWT WT animals. (B) Representative Western blot assays performed in heart lysates from these animals animals.(B) animals. (B) Representative Representative Western Western blot assays blot assays performed performed in heartinlysates heart from lysates from these theseshowing animals animals showing that LOX transgenesis alters the activation by phosphorylation of both p38 MAPK (p38) and showing that LOX transgenesis alters the activation by phosphorylation of both that LOX transgenesis alters the activation by phosphorylation of both p38 MAPK (p38) and AMPK p38 MAPK (p38) and AMPK (p-p38 and p-AMPK, respectively). Total p38 and AMPK levels are also shown. AMPK (p-p38 and p-AMPK, respectively). Total p38 and AMPK (p-p38 and p-AMPK, respectively). Total p38 and AMPK levels are also shown. Glyceraldehyde levels are also shown. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein levels were analyzed as loading Glyceraldehyde 3-phosphate 3-phosphate dehydrogenase dehydrogenase (GAPDH) (GAPDH) protein levels protein as were analyzed levels were loading analyzed control. as loading The position of control. The position of the molecular weight standards (HyperPAGE prestained protein markers, control. the The weight molecular position of the molecular standards (HyperPAGE weight standards prestained (HyperPAGE protein prestained markers, BIOLINE; protein Ref: markers, BIO-33065) is BIOLINE; Ref: BIO-33065) is indicated. (C) Dihydroethidium (DHE) staining revealed that LOX BIOLINE;(C) indicated. Ref: BIO-33065) is (DHE) Dihydroethidium indicated. (C) Dihydroethidium staining (DHE) stainingexacerbates revealed that LOX overexpression revealed that LOX the Ang overexpression exacerbates the Ang II-mediated increase in cardiac superoxide anion production. overexpression II-mediated exacerbates increase the in cardiac Ang II-mediated superoxide increase in cardiac superoxide anion production. anion production. Cardiac disease LOX ? ECM Cardiac ROS p38 FB crosslinking inflammation production activation differentiation Cardiac dysfunction Figure 7. Figure Theupregulation 7. The upregulationof ofLOX/LOXLs LOX/LOXLs in incardiac cardiacdiseases diseasesaffects affects several several processes processes that that contribute contribute Figure 7. to cardiac The upregulation cardiac dysfunction. dysfunction. ECM:of LOX/LOXLs ECM: extracellular in cardiac extracellular matrix; matrix; FB: diseases affects FB: fibroblast; fibroblast; ROS: several ROS: reactive processes reactive oxygen that contribute oxygen species. species. to to cardiac dysfunction. ECM: extracellular matrix; FB: fibroblast; ROS: reactive oxygen species. Similarly, Ang II-induced cardiac hypertrophy was exacerbated in transgenic mice that Similarly, Ang II-induced cardiac hypertrophy was exacerbated in transgenic mice that overexpress LOXL1 specifically in cardiomyocytes, which exhibited myocyte hypertrophy, and overexpress LOXL1 specifically in cardiomyocytes, which exhibited myocyte hypertrophy, and higher mRNA levels of brain natriuretic peptide (Bnp) than their WT littermates. Echocardiographic higher mRNA levels of brain natriuretic peptide (Bnp) than their WT littermates. Echocardiographic Cells 2019, 8, 1483 11 of 20 4.2. LOX/LOXLs in Post-MI Cardiac Remodeling The human heart has a negligible capacity to cardiomyocyte regeneration after MI. MI is followed by an extensive remodeling of the ECM characterized by an important accumulation of collagen that triggers both replacement and interstitial fibrosis and scar formation. Post-MI fibrosis, developed in both infarcted and non-infarcted cardiac regions, is essential for the recovery of heart function. The cellular response to infarction can be divided into three overlapping phases: the inflammatory phase of infarct healing, the proliferative phase of cardiac repair, and the formation of a mature scar [43]. During the inflammatory phase an enhanced protease activity leads to an extensive degradation of the cardiac ECM, and to the production of a provisional fibrin and fibronectin matrix that acts as a scaffold for inflammatory cells. The proliferative phase is featured by the transdifferentiation of myofibroblasts and the deposition of fibrous tissue. Cardiac fibroblasts play a critical role in LV healing and remodeling after MI. They are reprogrammed into myofibroblasts that are instrumental for the formation of contractile and mature collagen scars that limit the detrimental dilation of infarcted areas. Finally, during the formation of a mature scar, increased LOX promotes the formation and deposition of stiff collagen fibers [63]. Thus, cardiac fibrosis leads to increased collagen deposition and cardiac stiffness derived from an exacerbated CCL. Although the fibrotic response aims to preserve tissue integrity, progressively this pathological fibrosis disturbs LV function linked to poor prognosis associated with a marked increase in the risk of HF, ventricular arrhythmias, and sudden cardiac death. Early studies reported increased collagen content and CCL in the viable free wall of rats subjected to surgery to produce moderate-to-large transmural infarcts [64]. This research suggests that LOX-mediated CCL also determines the adverse remodeling occurring in remote zones to MI, which contributes to HF of ischemic origin. The concomitant post-MI upregulation of LOX and CCL has been demonstrated in multiple animal models. In a rhesus monkey model of MI, ischemic injury generated by ligation of the left anterior descending (LAD) artery promoted an increase in the collagen type I/III ratio in the scar area. Coincidently, LOX expression was upregulated in both the scar area and the border zone, while the expression of MMP1 was downregulated [65]. The degree of LOX upregulation and fibrosis after MI has been related with the extent of LV dysfunction. Indeed, LV fibrosis and higher mRNA levels of collagen I, connective tissue growth factor (Ctgf), Tgfβ and Lox were observed in rats subjected to LAD coronary artery ligation presenting signs of HF versus non-failing hearts [66]). Similarly, in mice, MI upregulated the expression of LOX and LOXL1-4 promotes the accumulation of mature collagen fibers in the infarcted area [67]. Both TGFβ and hypoxia-dependent signaling pathways underlie the upregulation of LOX/LOXLs in cardiac fibroblasts, in particular in areas of extensive remodeling. In this model, collagen accumulation and CCL maturation were ameliorated by a post-infarction treatment with BAPN or with a neutralizing antibody against LOX, which also reduced ventricular dilatation and improved cardiac function [67]. It should be noted, however, that a severe impairment of LOX activity and CCL might result in defective scar collagen maturation, compromised scar contractility, and exacerbated adverse remodeling. Indeed, in AMP-activated protein kinase (AMPKα1) knockout (KO) mice subjected to permanent LAD ligation, a reduction in scar maturation and contractility evoked an exaggerated LV dilation. AMPKα1 knockdown was associated with an enhanced proliferation of cardiac fibroblasts in infarcted areas, a reduced expression of α-SMA, a marker of fibroblast transdifferentiation, and a defective maturation of myofibroblasts. These effects might be secondary to the strong down-regulation of the non-canonical TGF-β1/p38 MAPK pathway, and the dramatic reduction of LOX protein levels in the scar of KO hearts [68]. α2β1 integrin has also been involved in the modulation of LOX after MI through its interaction with collagen I [69]. 4.3. LOX Activity in Cardiac Remodeling and Fibrosis Associated With Obesity and Metabolic Syndrome HFPEF is a common clinical syndrome with poor prognosis strongly associated with obesity and the metabolic syndrome. HFPEF is characterized by diastolic dysfunction, or reduced capacity of the heart to fill during diastole, associated with high LV end diastolic blood pressure. The etiology of diastolic dysfunction is multifactorial and includes abnormalities in cardiac ECM remodeling and Cells 2019, 8, 1483 12 of 20 fibrosis. The key role of LOX in cardiac function and remodeling in this pathophysiological scenario is documented in models of diet-induced cardiac dysfunction. In a murine model of diet-induced metabolic syndrome (high-fat high-simple carbohydrate, HFHSC) no changes were observed in the cardiac expression of Lox and Loxl3; however, metabolic syndrome significantly upregulated Bmp1, which increased LOX processing and activity and CCL [70]. The LOX-mediated fibrotic response is concurrent with a marked rise in end-diastolic pressure, higher LV stiffness, and abnormal diastolic filling pattern [70]. Alterations in cardiac ECM CCL seem to be the underlying cause of diastolic dysfunction, and it has been proposed a regulatory role for the adaptive immune system. Indeed, results in mice devoid of functional T lymphocytes fed a HFHSC diet, suggest a role for these inflammatory cells in the modulation of LOX-dependent collagen maturation [71]. Treatment of mice with selective TH1 lymphocyte inducers revealed TH1 immune polarization associated with an increase in cardiac Lox and Loxl3 expression, LOX activity, CCL and ventricular stiffness and a concurrent decrease in cardiac output [72]. Therefore, TH1 lymphocytes are involved in of the development of CCL-mediated diastolic dysfunction, which has potential clinical application for the treatment of diastolic HF [72]. In a recent study we analyzed whether inhibition of LOX activity impacts on the cardiovascular remodeling of ECM triggered by obesity [73]. We found that cardiac LOX was upregulated in rats fed a high-fat diet (HFD), together with cardiac and vascular fibrosis and higher levels of superoxide anion (O2 − ), collagen I and TGF-β. LOX inhibition by BAPN significantly attenuated these changes and improved cardiac hypertrophy. Interestingly, BAPN also ameliorated the HFD-dependent increase in circulating leptin levels. In turn, in cardiac fibroblasts, leptin enhanced collagen I, TGF-β and CTGF levels, Akt phosphorylation and O2 − production, while LOX inhibition attenuated these responses. Therefore, LOX inhibition limited the HFD-mediated fibrosis and oxidative stress in the cardiovascular system. The ability of BAPN to reduce leptin levels in vivo and prevent the leptin-mediated induction of profibrotic mediators and ROS in cardiac cells suggests that the interaction between leptin and LOX regulates downstream signaling pathways underlying the development of myocardial fibrosis in obesity. Finally, LOX has been related to the pro-fibrotic effects and cardiac stiffening caused by the intake of high n-6 polyunsaturated fatty acid (PUFA) diets. In cardiac fibroblasts in culture, high concentrations of linoleic acid (mimicking an excess of dietary n-6 PUFA) upregulated collagen I and LOX [74]. 4.4. LOX and LOXL2 in Atrial Fibrillation Atrial fibrillation (AF) is commonly characterized by increased atrial fibrosis and structural remodeling. Enhanced LOX expression and CCL underlies interstitial fibrosis and remodeling in AF. Indeed, upregulation of LOX has been documented in AF both in humans and in animal experimental models [75–78]. The left atrial appendage from patients with AF displayed an increase in collagen content and cross-linking, as well as enhanced expression of LOX, CTGF and fibronectin and higher Rac1 activity [76]. The mechanism underlying these effects was deciphered using pharmacological and gene knockdown strategies in both cardiac fibroblasts and transgenic mice with cardiac overexpression of constitutively active Rac1 (RacET), which develop atrial fibrosis and spontaneous AF with aging, characterized by increased LOX expression, collagen content, and cross-linking. These studies evidence that Ang II enhances the expression of LOX and fibronectin through the activation of Rac1 GTPase and CTGF. Notably, in RacET mice, torasemide, reduced the aldosterone synthase-mediated profibrotic signaling limiting the induction of CTGF and LOX, preventing atrial fibrosis and reducing the prevalence of AF [77]. Therefore, LOX is involved in a signaling pathway that represents a target for the prevention of fibrotic atrial remodeling. Little information is available about the involvement of other LOX isoenzymes in AF. Recent evidence indicates that serum LOXL2 levels were augmented and correlated with the level of left atrial fibrosis [79]. Indeed, higher LOXL2 levels were found in AF patients with a size of left atrium (LAD) ≥ 40 mm compared with those with LAD < 40 mm, and multivariate regression analysis revealed that they were independent predictors of AF [79]. These data suggest that LOXL2 levels may predict the degree of atrial fibrosis in AF patients and support the contribution of this isoenzyme to the pathogenesis of AF. Cells 2019, 8, 1483 13 of 20 4.5. LOX in Other Pathophysiological Settings Leading to Cardiac Dysfunction In addition to the situations already mentioned, LOX is modulated by a wide array of conditions that impair cardiac performance. This has been evidenced in a variety of animal experimental models. In dystrophin-deficient mice, a model of Duchenne muscular dystrophy (DMD) which develops fibrosis and DCM, an altered profibrotic gene expression profile, in particular, a steep increase in Lox mRNA expression has been documented [80]). The renin–angiotensin–aldosterone system, one of the pathways involved in adverse cardiac remodeling, has been linked to a disturbance in Lox expression. In particular, experiments in null mice evidence that the control of myocardial Lox expression depends on TRAF3 Interacting Protein 2 (TRAF3IP2), a redox-sensitive adaptor molecule and a decisive signaling intermediate in aldosterone/salt-induced cardiac hypertrophy and fibrosis [81]. Likewise, in rats, the hypertrophic and profibrotic cardiac effects of high molecular weight advanced glycation end products (HMW-AGEs) were likely due to the increase of LOX expression and the LOX-mediated CCL, which might be responsible for the higher cardiac stiffness promoted by these compounds [82]. Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a severe disease featured by an extensive fibrotic replacement. In animal models that recapitulate human ARVC, the cardiac fibrotic response was associated with an upregulation of Lox [83,84]. Finally, a strong induction of LOX was identified as the underlying cause of the restrictive cardiomyopathy evoked by a sustained running in SHR rats. The upregulation of LOX results in the deposition of an extensive network of matrix proteins and a substantial elevation of collagen III expression, thereby contributing to the filling disorder in trained SHR [85]. 5. LOX/LOXLs as Pharmacological Targets in Myocardial Diseases The strong impact of the disturbance of LOX/LOXLs activity on CCL and heart performance [6,7,37] supports the interest of LOX/LOXLs-inhibitory therapies to improve cardiac remodeling and slow HF. Moreover, and considering that tissue drug uptake and distribution could be impaired by an excessive LOX-mediated ECM deposition [86], interfering with cardiac collagen stabilization by LOX/LOXLs inhibition could additionally improve the response to specific pharmacological therapies. Despite the potential interest of LOX inhibitors, LOX-targeting drug discovery has been hampered by the lack of LOX/LOXLs crystallographic structure, an area of active investigation, as indicate recent promising data aiming to clarify the structural–functional relationship of LOXL2 [87]. Furthermore, safety concerns should be considered, since increased expression/activity of these isoenzymes has been associated with cardiac diseases, but a decrease of LOX could underlie the development of several vascular pathologies and promote plaque instability [12]. Because cardiac and vascular pathologies frequently coexist, therapeutic approaches aiming LOX inhibition must be thoroughly evaluated to exclude undesirable side-effects. Moreover, chronic and irreversible cardiac LOX inhibition might cause a severe disruption of the ECM and compromise the structural integrity of the heart. Overall, these considerations evidence that irreversible inhibitors of LOX/LOXLs have critical concerns as therapeutic tools for chronic diseases. In fact, the chronic administration of the broadband LOX inhibitor BAPN provokes neurotoxicity and lathyrism, a disorder characterized by serious connective tissue defects which severely disturbs bone mechanical strength and blood vessel mechanics [22]. Given these constraints, alternative approaches have been assessed (Table 1). Monoclonal antibodies against LOX and LOXL2 have demonstrated their ability to ameliorate cardiac dysfunction and fibrosis in response to pressure overload or MI in mice [42,67]. Simtuzumab, a humanized monoclonal antibody against LOXL2, developed as anti-fibrotic agent, emerges as an interesting therapeutic tool for cardiac diseases. However, and despite promising preclinical data, this drug showed no significant clinical benefit in reducing liver and lung fibrosis [88], and clinical trials testing its efficacy against cardiac fibrosis and dysfunction have not been implemented. Besides monoclonal antibodies, efforts have been made for developing small-molecules that directly inhibit LOXL2 catalytic activity [89]. PXS-5153A, a novel dual LOXL2/LOXL3 inhibitor, dose-dependently decreased LOXL2-mediated collagen oxidation and CCL in vitro, and improved cardiac output after MI in mice [90]. Cells 2019, 8, 1483 14 of 20 Table 1. Therapeutic approaches targeting LOX and LOXLs. Effect on Cardiac Therapeutic Approach Experimental Model Author/Year/Reference # Remodeling and Function Mouse model of age-related BAPN Reduced myocardial fibrosis Rosin et al. 2015 [58] myocardial fibrosis Rat model of cardiac fibrosis Reduced fibrosis and Martínez–Martínez et al. BAPN induced by HFD improved cardiac remodeling 2016 [73] BAPN or Mouse model of MI Reduced ventricular dilatation González–Santamaría et al. Anti-LOX antibody (LAD coronary artery ligation) and improved cardiac function 2016 [67] Reduced cardiac fibrosis and Mouse model of pressure Anti-LOXL2 antibody chamber dilatation, improved Yang et al. 2016 [42] overload (Transaortic constriction) systolic and diastolic functions Reduced myocardial fibrosis Rat model of aortocaval El Hajj et al. 2016 [60] BAPN and improved LV stiffness and fistula-induced volume overload El Hajj et al. 2018 [61] cardiac function PXS-5153A Mouse model of MI Reduced fibrosis and (LOXL2/LOXL3 Schilter et al. 2019 [90] (LAD coronary artery ligation) improved FS and EF inhibitor) Abbreviations: BAPN, β-aminopropionitrile (a pan-LOX inhibitor); EF, ejection fraction; FS, fractional shortening; HFD, high fat diet; LAD, left anterior descending; LV, left ventricular; MI, myocardial infarction. Alternative approaches which could achieve an effective reduction of LOX activity might be derived from the use of metal chelators, which would reduce copper availability and therefore impair LTQ biogenesis, or the blockade of LOX and LOX1 extracellular processing by inhibiting BMP1. It should be highlighted, that the benefit of some drugs widely used for the management of cardiovascular diseases could be derived, at least in part, by their ability to inhibit LOX. This is particularly the case of torasemide, which reduces myocardial LOX expression, CCL and fibrosis and normalizes LV stiffness in patients with hypertensive HF [6]. Similar effects could be ascribed to the losartan metabolite EXP3179 in hypertensive rats [91]). In fact, despite EXP3179 exerted lower antihypertensive effects than EXP3174, a second losartan metabolite that neither modified LOX nor CCL, EXP3179, showed higher anti-fibrotic efficacy than EXP3174. LOX/LOXLs expression and/or activity can also be modulated by molecules interfering mechanisms and signaling pathways involved in cardiac remodeling. This has been illustrated in different experimental models. In SHR, which showed a significant increase cardiac LOX and CCL accompanied by a rise in phosphorylated Smad2, CTGF, and collagen type I, a synthetic peptide from TGF-β1 type III receptor (P144) limits myocardial fibrosis [55]. In rats infused with Ang II, the tetrapeptide Ac-SDKP (N-acetyl-seryl-aspartyl-lysyl-proline), prevented the Ang II-induced increases in LOX and LOXL1 levels, collagen and CCL, TGFβ signaling, NF-κB activation, and the infiltration of CD4+/CD8+ lymphocytes and CD68+ macrophages [92]. In turn, finerenone (a non-steroidal mineralocorticoid receptor antagonist) decreased the expression of CTGF and prevented the aldosterone-induced upregulation of CTGF and LOX. Finerenone attenuated the Rac1 GTPase-mediated upregulation of TGF-β, and in RacET mice, which exhibit increased LV end-diastolic and end-systolic volumes and high TGF-β, CTGF and LOX levels, this drug prevented LV dilatation [93]. Finally, the strong anti-fibrotic potential of the pharmacological inhibition of ROCK is in part due to a decrease in the expression of LOX. Indeed, in a model of engineered connective tissue, composed by cardiac fibroblasts and collagen, Rho-associated kinases (ROCK1 and ROCK2) were identified as key mediators of TGF-β-dependent tissue stiffening of engineered heart muscle tissue, and LOX as a downstream target of this ROCK-actin-MRTF/SRF pro-fibrotic signaling pathway [94]. 6. Conclusions and Therapeutic Opportunities The findings summarized in this review evidence the critical contribution of the LOX family to cardiac function and remodeling. The upregulation of LOX/LOXLs seems to be a feature of several fibrotic processes affecting the heart, from those associated to post-MI cardiac remodeling, cardiac hypertrophy triggered by pressure or volume overload, HFPEF associated with obesity and metabolic Cells 2019, 8, 1483 15 of 20 syndrome or AF. Because LOX/LOXLs play a critical role in CCL, LOX activity emerges as a key biological point to control the quality of ECM, which determines the progression and severity of those clinical entities that course with fibrosis. Concerning therapeutics and although much progress has been made in the search of new antifibrotic agents useful for the treatment of cardiac fibrosis and dysfunction, clinical trials assessing the effectiveness of a priori potentially applicable drugs have failed. Therefore, further efforts will be required to better understand of LOX/LOXLs biology. LOX and LOXLs are also regarded as promising tools for new approaches to grafting and tissue regeneration, but their role in cardiac repair and tissue engineering should also be investigated in more detail. Author Contributions: All the authors have participated in the drafting and revision of the manuscript. Funding: The authors gratefully appreciate the support provided by the Spanish Ministerio de Ciencia, Innovación y Universidades (MICIU), Instituto de Salud Carlos III (ISCIII) [grants RTI2018-094727-B-100, PI18/0919], Fundació La Marató de TV3 (20152330) and Agencia de Gestio d’Ajuts Universitaris i de Recerca (AGAUR; program of Support to Research Groups, Ref. 2017-SGR-00333). Our studies were also co-founded by Fondo Europeo de Desarrollo Regional (FEDER), a way to make Europe. Acknowledgments: We apologize to all those colleagues whose achievements could not be cited due to space limitations. Conflicts of Interest: The authors declare no conflict of interest. References 1. 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