© 2014. Published by The Company of Biologists Ltd | Disease Models & Mechanisms (2014) 7, 987-996 doi:10.1242/dmm.015255

RESEARCH ARTICLE

TGF-β mediates early angiogenesis and latent in an Emilin1-deficient mouse model of aortic valve disease Charu Munjal1, Amy M. Opoka1, Hanna Osinska2, Jeanne F. James1, Giorgio M. Bressan3 and Robert B. Hinton1,*

ABSTRACT abnormalities that typically manifest as fibrosis then calcification, Aortic valve disease (AVD) is characterized by resulting in stenosis with or without regurgitation. The prevailing view fragmentation (EFF), fibrosis and aberrant angiogenesis. Emilin1 is has been that injury and inflammation result in AVD, but there is an -binding that regulates elastogenesis and increasing evidence that the cell-ECM changes that characterize AVD inhibits TGF-β signaling, but the role of Emilin1 in valve tissue is are mediated by abnormalities in molecular programs that regulate unknown. We tested the hypothesis that Emilin1 deficiency results in cardiac development (Markwald et al., 2010; Hinton and Yutzey, AVD, mediated by non-canonical (MAPK/phosphorylated Erk1 and 2011). Our understanding of the molecular mechanisms underlying Erk2) TGF-β dysregulation. Using , immunohistochemistry, AVD pathogenesis, especially disease progression, has been limited, electron microscopy, quantitative expression analysis, in part, by a lack of animal models that recapitulate the natural history immunoblotting and echocardiography, we examined the effects of of human AVD (Schoen, 2008; Rajamannan et al., 2011). Emilin1 deficiency (Emilin1−/−) in mouse aortic valve tissue. Emilin1 The mature aortic valve comprises three cusps that are hinged to deficiency results in early postnatal cell-matrix defects in aortic valve a crown-shaped annulus within the aortic root. Cusp trilaminar ECM tissue, including EFF, that progress to latent AVD and premature is stratified into fibrosa, spongiosa and ventricularis layers with death. The Emilin1−/− aortic valve displays early aberrant provisional elastic fibers organized as filaments in the ventricularis layer angiogenesis and late neovascularization. In addition, Emilin1−/− (Schoen, 1997; Hinton et al., 2006). Valve interstitial cells (VICs) aortic valves are characterized by early valve interstitial cell activation comprise a heterogeneous population of cells that can be classified and proliferation and late -like cell activation and as quiescent or activated (Rabkin-Aikawa et al., 2004), and some fibrosis. Interestingly, canonical TGF-β signaling (phosphorylated VICs possess characteristics similar to those of smooth muscle cells Smad2 and Smad3) is upregulated constitutively from birth (SMCs) (Bairati and DeBiasi, 1981; Filip et al., 1986). Activated to senescence, whereas non-canonical TGF-β signaling VICs have been implicated in AVD because they are associated with (phosphorylated Erk1 and Erk2) progressively increases over time. pathological cell proliferation and maladaptive ECM remodeling Emilin1 deficiency recapitulates human fibrotic AVD, and advanced (Rabkin-Aikawa et al., 2004). Although healthy adult valve tissue is disease is mediated by non-canonical (MAPK/phosphorylated Erk1 avascular (Duran and Gunning, 1968), neovascularization has been and Erk2) TGF-β activation. The early manifestation of EFF and described in non-rheumatic degenerative AVD and attributed to end- aberrant angiogenesis suggests that these processes are crucial stage inflammatory processes, a secondary result of a wound- intermediate factors involved in disease progression and therefore healing-like response to injury (Rajamannan et al., 2005; Paruchuri might provide new therapeutic targets for human AVD. et al., 2006; Li et al., 2011). Little is known about the potential primary role of aberrant angiogenesis in AVD. KEY WORDS: Elastic fibers, , Aortic valve, Elastic fiber fragmentation (EFF) has long been associated with Fibrosis, Angiogenesis AVD, and the conventional view is that structural degeneration reflects end-stage disease processes (Schoen, 1997). Mouse models INTRODUCTION with elastic fiber assembly defects often have valve abnormalities Aortic valve disease (AVD) affects more than 2% of the general (Hanada et al., 2007; Hinton et al., 2010), and EFF is present in population and typically manifests later in life (Nkomo et al., 2006). pediatric, as well as adult, AVD (Fondard et al., 2005; Hinton et al., Therapeutic intervention remains primarily surgical valve 2006; Wirrig et al., 2011), suggesting that EFF reflects elastic fiber replacement, which is associated with limited durability and assembly defects, in addition to elastase initiated EFF, and therefore significant complications (Keane et al., 1993; Hammermeister et al., might have a role in AVD progression. EFF has been associated with 2000). AVD is characterized by extracellular matrix (ECM) increased elastolytic enzyme activity, which has been identified as a factor contributing to AVD progression in both mouse and human (Fondard et al., 2005; Krishnamurthy et al., 2012). Elastic fibers are 1Division of Cardiology, The Heart Institute, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA. 2Division of Molecular Cardiovascular Biology, The made up of elastin (the core ) and microfibrils ( and Heart Institute, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, associated ), as well as various , such as USA. 3Department of Molecular Medicine, University of Padua, 35121 Padua, emilins and fibulins (Kielty et al., 2002; Wagenseil and Mecham, Italy. 2009). Emilin1 (elastin microfibril interface-located protein) is an *Author for correspondence ([email protected]) elastin- and fibulin-5-binding protein that is necessary for elastogenesis and inhibits transforming growth factor β (TGF-β) This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted signaling (Zanetti et al., 2004; Zacchigna et al., 2006). In addition, use, distribution and reproduction in any medium provided that the original work is properly , keratinocytes and lymphatic endothelial cells in Emilin1- attributed. deficient mice demonstrate increased proliferation due to absent

Received 19 December 2013; Accepted 5 June 2014 Emilin1-integrin interactions (Danussi et al., 2011; Danussi et al., Disease Models & Mechanisms

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elastic fiber components to all cusp layers, namely the fibrosa, TRANSLATIONAL IMPACT spongiosa and ventricularis, as well as the annulus, indicating faulty Clinical issue elastic fiber assembly (Fig. 1A,B). EFF and delamination worsens Aortic valve disease (AVD) is a major cause of cardiovascular morbidity over time (Fig. 1B,D,F). Ultra-structural analysis of aged Emilin1−/− and affects more than 2% of the general population of the USA. Aortic aortic valves confirmed marked EFF, delamination and dispersion valve replacement remains the principal treatment strategy for end-stage (Fig. 1H). MMP-9 was examined because it has been shown to play AVD; however, this process is associated with considerable complications. a crucial role in pathological valve remodeling. MMP-9 expression Presently, there are no pharmacologic therapies to directly treat AVD. A −/− better understanding of the cellular and molecular events that underlie was unaltered in adult Emilin1 valves but was increased in aged AVD progression is required to develop novel approaches to treatment. Emilin1-deficient aortic valves in comparison with age-matched Therefore, animal models that mimic the natural history of human AVD controls (Fig. 1J) (Fondard et al., 2005). This observation was are needed to allow researchers to examine the early disease process supported by immunoblot analyses, which demonstrated increased and test new therapies. Emilin1 is an essential elastogenic protein that is expression of active MMP-9, as well as MMP-2, in aged mutant present in both developing and mature aortic valve tissue. Previous valves (Fig. 1K). Importantly, elastase expression, which is not studies have shown that Emilin1 is required for normal elastic fiber expressed in normal healthy valve, was increased in juvenile and assembly and inhibits TGF-β signaling in vasculature. However, the role −/− of Emilin1 dysregulation in AVD pathogenesis is unknown. adult Emilin1 aortic valve tissue and further increased at the aged stage (Fig. 1M,O,Q), demonstrating a role for elastase and Results remodeling enzymes in the progression of AVD. In aged Emilin1−/− In this study, the Emilin1 homozygous knockout mouse (Emilin1–/–) was identified as a new model of latent AVD. Histological and molecular aortic valve tissue, elastin mRNA expression was significantly analyses demonstrated that Emilin1 deficiency is associated with the decreased, whereas that of -1 was unchanged (Fig. 1R). activation of non-canonical (phosphorylated Erk1 and Erk2) and Elastin mRNA expression was also decreased in ascending aorta and canonical (phosphorylated Smad2 and Smad3) signaling in aortic valve left myocardial tissue that had been isolated from Emilin1−/− mice tissue. This results in early elastase-mediated elastic fiber fragmentation (supplementary material Fig. S2O). Taken together, elastic fiber and aberrant angiogenesis in association with early valve interstitial cell assembly defects in Emilin1−/− mice predispose aortic valve tissue (VIC) activation. Interestingly, there is progressive up-regulation of phosphorylated Erk1 and Erk2 signaling over time, which results in the to progressive elastolysis and worsening EFF. activation of a subset of VICs with myofibroblast-like characteristics and marked progression of valve pathology that shows severe fibrosis, Aberrant provisional angiogenesis is an early finding that neovascularization and inflammation. progresses to neovascularization in Emilin1−/− aortic valve Implications and future directions tissue −/− Together, these results demonstrate that the Emilin1-deficient mouse is a Neovascularization (overt angiogenesis) was present in Emilin1 unique model of latent fibrotic AVD that provides important insights into aortic valves at the aged stage only and localized to the annulus early and intermediate disease mechanisms. These findings establish a and proximal cusp, these neovessels also stained positive for CD- central role for elastic fiber dysregulation in early AVD pathogenesis, 31 (Fig. 2B,D). At the earlier adult stage, Emilin1−/− aortic valves suggesting that faulty elastic fiber assembly and consequent elastase- exhibited aberrant provisional angiogenesis, characterized by mediated tissue injury contribute to disease initiation and progression. A better understanding of these early disease processes could contribute to increased interstitial VEGF-A and VEGF-R1 (Flt1) expression, the development of novel treatments for AVD. Because the manifestation which are normally restricted to the valve endothelium, but of AVD is latent in this model, similar to human AVD, preclinical studies unchanged VEGF-R2 (Flk1) expression (Fig. 2F,J,N). In addition, using the Emilin1-deficient mouse model of AVD are warranted. circulatory VEGF-A levels were increased significantly in aged Emilin1−/− mice (data not shown). Interestingly, the angiostatic factors chondromodulin (Chm), endostatin and fibulin-5, which are 2012). Importantly, Emilin1 is expressed in the developing and normally ubiquitously expressed in valve tissue, were markedly mature heart valves (Braghetta et al., 2002; Angel et al., 2011; decreased in aged Emilin1−/− aortic valves (Fig. 2R,T,V), Votteler et al., 2013), and Emilin1 deficiency results in EFF and suggesting that aberrant angiogenesis is the result of both TGF-β activation in the aorta. The role of Emilin1 in AVD increased pro-angiogenic factors and decreased angiostatic factors. pathogenesis is unknown. Quantitative reverse transcription PCR (QRT-PCR) analysis was We tested the hypothesis that Emilin1 deficiency results in AVD, performed in aged aortic valve tissue that showed a significant which is characterized by early EFF and late fibrosis, mediated by increase in both VEGF-A and VEGF-R1, as well as a significant non-canonical TGF-β signaling [through mitogen-activated protein decrease in endostatin. Overall, these results demonstrate that kinases (MAPK) and extracellular-signal-regulated kinases 1 and 2 aberrant angiogenesis is an early disease process that worsens over (Erk1/2)]. We have demonstrated here that Emilin1−/− aortic valves time, identifying a crucial role for angiogenesis in AVD exhibit EFF and early aberrant angiogenesis, as well as late fibrosis progression. and stenosis, and that these pathologic findings are due to complex TGF-β dysregulation, including progressive upregulation of non- Dynamic activation of non-canonical TGF-β (phosphorylated canonical phosphorylated Erk1/2 signaling. This new model of AVD Erk1/2) signaling in Emilin1−/− aortic valve tissue recapitulates the natural history of human AVD, thereby providing To determine the effects of Emilin1 deficiency on TGF-β signaling insights into mechanisms of disease progression that might identify in aortic valve tissue, canonical and non-canonical pathways were new therapeutic targets. examined. In Emilin1−/− aortic valves at the juvenile stage, levels of phosphorylated Smad2 and Smad3 (Smad2/3) were increased when RESULTS compared with age-matched controls (Fig 3A,B). In Emilin1−/− Emilin1−/− aortic valve tissue is characterized by early EFF aortic valves at both the adult and aged stages, there was a and progressive elastolysis significant and similar increase in the absolute number and ratio of Juvenile Emilin1−/− aortic valve tissue exhibits EFF, which is phosphorylated Smad2/3-positive nuclei, as well as protein +/+ characterized by decreased elastic fiber content and dispersion of expression using immunoblotting (Fig. 3D,F-H). In Emilin1 aortic Disease Models & Mechanisms

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Fig. 1. Elastic fiber assembly defects in Emilin1−/− aortic valve tissue. Sections stained using Hart’s stain show intact elastic fiber filaments localized to the ventricularis layer in Emilin1+/+ mice (A,C,E) and EFF in Emilin1−/− aortic valves that worsened with time (arrowheads, B,D,F). Ultrastructure analyses in aged Emilin1−/− aortic valves show EFF with dispersion (arrowheads, H) when compared with intact elastic fiber filaments in Emilin1+/+ valves (G). MMP-9 and MMP-2 expression increased in the aged aortic valve of Emilin1−/− mice when compared with that of Emilin1+/+ valves using immunohistochemistry (J) and western blotting (K). Western blotting analysis showed increased expression of both the inactive proenzyme form (pro) and the active form of MMP-2 and MMP-9 in Emilin1–/– aortic valves (K). There was increased neutrophil elastase expression in Emilin1−/− aortic valve tissue, especially at the hinge region, at the juvenile, adult and aged stages (arrows, M,O,Q). In aged Emilin1−/− aortic valve tissue, elastin mRNA expression decreased significantly but there was no change in the amount of fibrillin1 mRNA (R). All panels are oriented to show the aortic valve in cross-section with the ventricularis layer situated at the bottom. Mean±s.e.m.; n=12; *P<0.05 Emilin1+/+ versus Emilin1−/−. Scale bars: 10 μm (A-F); 500 nm (G,H); 50 μm (I-P). F, fibrosa; S, spongiosa; V, ventricularis.

valves, phosphorylated Smad2/3 was increased with advanced age (αSMA) were studied in Emilin1−/− aortic valve tissue. SMemb but significantly decreased when compared with that of Emilin1−/− expression was increased at both the adult and aged stages, valves. There was no change in phosphorylated Smad2/3 in indicating VIC activation (Fig. 4B,D). αSMA expression was Emilin1−/− myocardium (supplementary material Fig. S2G). TGF- similarly increased at the adult stage but dramatically increased at βR1 mRNA expression was increased in Emilin1−/− aortic valve the aged stage (Fig. 4F,H). SM22, which is a myofibroblast marker, tissue, as determined by using QRT-PCR (Fig. 3I). The amount of was not expressed in Emilin1−/− aortic valve tissue at the adult stage phosphorylated Erk1/2 was unchanged in juvenile Emilin1−/− aortic but was abundantly expressed at the aged stage (Fig. 4J,L), valves but was modestly increased in adult Emilin1−/− aortic valves identifying a distinct cell activation pattern consistent with latent (Fig. 3J-M) and dramatically increased at the aged stage when myofibroblast-like cell activation. Interestingly, VEGF-A and SM22 compared with the younger mutant (Fig. 3M,O), identifying were colocalized in some VICs (Fig. 4N), primarily in the annulus progressive phosphorylated Erk1/2 activation. There was no region and proximal cusp where neovessels are present, phosphorylated Erk1/2 expression in Emilin1−/− myocardium (data demonstrating that a subset of VICs, rather than endothelial cells, not shown). However, aged Emilin1−/− aorta tissue demonstrated regulate VEGF-A production in the valve interstitium, consistent increased phosphorylated Erk1/2 expression that was localized to with previous observations (Paranya et al., 2001; Paruchuri et al., the aortic root (supplementary material Fig S2I,N). Circulatory 2006; Syväranta et al., 2010). The total number of proliferative cells plasma levels of active TGF-β were significantly increased at the was quantified by proliferation index analysis using phosphorylated adult and aged stages in Emilin1-deficient mice (Fig. 3R). Taken histone H3 (HH3) staining. The results showed an increased together, these findings demonstrate dynamic dysregulation of TGF- proliferative index in Emilin1−/− aortic valve tissue at the juvenile β signaling in Emilin1−/− aortic valve tissue, indicating a possible and adult stages that significantly increased by the aged stage (Fig. role for non-canonical phosphorylated Erk1/2 activation in AVD 4O). Ki67 staining demonstrated similar patterns of proliferation by progression. stage and genotype (supplementary material Fig. S1O). Importantly, activated VICs were also positive for the proliferation marker Early VIC activation and proliferation is maladaptive in phosphorylated HH3, indicated by colocalization of phosphorylated Emilin1−/− aortic valve tissue HH3 and SMemb (supplementary material Fig. S1L). Overall, these In order to examine the VIC phenotype, embryonic smooth muscle results implicate early VIC activation and late myofibroblast-like

myosin heavy chain (SMemb) and alpha smooth muscle actin cell activation in the latent manifestation of AVD. Disease Models & Mechanisms

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Fig. 2. Emilin1 deficiency is associated with aberrant provisional angiogenesis and neovascularization. Overt neovascularization is shown in aged Emilin1−/− aortic valve tissue, restricted to the annulus and proximal cusp, as indicated by Movat’s pentachrome staining (arrowheads, B) and CD-31-positive stained neovessels (arrowheads, D). Emilin1+/+ controls are shown in A and C. VEGF-A (E-H) and VEGF-R1 (I-L) expression was increased at adult (F,J) and aged (H,L) stages in the interstitium of Emilin1−/− aortic valves when compared with that of control Emilin1+/+ tissue, which shows normal expression around the endothelial layer (arrowheads, E,G). Levels of endostatin (Q-R, green; blue, nuclei), chondromodulin (Chm) (S-T) and fibulin-5 (U-V) were lower at the aged stage (R,T,V) when compared with Emilin1+/+ controls (arrowheads, Q,S,U). mRNA expression for VEGF-A, VEGF-R1, VEGF-R2 and endostatin (Endos) in aged Emilin1−/− aortic valve tissue relative to Emilin1+/+ control values (normalized to 1) for each gene (W). Scale bars: 50 μm. Mean±s.e.m.; n=10; *P<0.05 Emilin1+/+ versus Emilin1−/−.

The aged Emilin1−/− aortic valve demonstrates fibrosis and regions of the myocardium and adventitial region of the ascending inflammation, but no calcification aorta (supplementary material Fig. S2D,K). Emilin1−/− myocardium Fibrillar deposition in aortic valve tissue was increased in showed altered sarcomere morphology that was characterized by an aged Emilin1−/− when compared with Emilin1+/+. Specifically, type increased cisternal space due to gaps between the sarcomeres and I and type III collagen were increased significantly (Fig. 5G). There sarcoplasmic reticulum, and contracted sarcomeres with thinner Z- was evidence of mild aortic valve fibrosis at the earlier adult stage discs (supplementary material Fig. S2F); in addition, mitochondrial (data not shown). To determine the potential role of inflammation in number and morphology were unaltered, consistent with a the manifestation of AVD in Emilin1-deficient mice, we examined nonspecific Ca2+ handling abnormality. Periostin, a profibrotic and leukocyte markers. Emilin1−/− aortic valves marker, was increased in the left ventricular myocardium of aged displayed abundant macrophage expression (Mac-3; also known as Emilin1−/− (supplementary material Fig. S2G). Collagen fibers in the Lamp2) at the aged stage (Fig. 5D), but not earlier stages (data not media of Emilin1−/− aorta were randomly arranged and shown). Interestingly, CD-45, a pan-leukocyte marker, remained heterogeneous in size (supplementary material Fig. S2M). Type I, II unchanged compared with controls at all stages (data not shown). and III collagen were not significantly changed in ascending aorta Ultrastructure analysis showed a qualitative abundance of - or myocardium, suggesting that other contribute to the like cells in the hinge region of aged Emilin1−/− aortic valves (Fig. fibrotic response in these tissues. Aged Emilin1−/− mice also 5F,H). The identity of fibroblast-like cells was further established by demonstrated thoracic aortic aneurysm or aortopathy, restricted to the presence of vimentin, an intermediate cytoskeletal protein the aortic root region, and three out of 10 (30%) mice demonstrated present in myofibroblast cells, which was increased in Emilin1−/− aortic dissection. Overall, these findings identify a potential role for aortic valve tissue (supplementary material Fig. S1B). Interestingly, Emilin1 in regulating ECM in cardiovascular tissues. Emilin1-deficient aortic valves did not exhibit calcification at any stage, as shown by the calcification marker Alizarin Red and Runx- Emilin1 deficiency results in AVD in vivo 2 (supplementary material Fig. S1F,H). Overall, these findings In order to evaluate aortic valve function in the Emilin1−/− mouse in identify a role for Emilin1 deficiency in the latent development of vivo, we performed echocardiography in adult and aged mice. The valve fibrosis, similar to the natural history of human AVD. aortic valve peak velocity and corresponding pressure gradient were Latent fibrosis was also present in the ascending aorta and significantly increased in aged Emilin1−/− mice compared with age- myocardium of Emilin1−/− mice, as evidenced by ultrastructural and matched controls (Table 1), consistent with aortic valve stenosis. In morphometric analysis (supplementary material Fig. S2). Excessive addition, 20% demonstrated aortic insufficiency. The aortic root index −/− collagen deposition was largely restricted to the perivasculature was increased in aged Emilin1 mice, consistent with mild aortic Disease Models & Mechanisms

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Fig. 3. Emilin1 deficiency is associated with canonical and non-canonical TGF-β activation in aortic valve tissue. Levels of phosphorylated Smad2/3 (p- Smad2/3) are increased in juvenile (arrowheads, B), adult (arrowheads, D) and aged (arrowheads, F) Emilin1−/− aortic valves compared those in juvenile (A), adult (C) and aged (E) Emilin1+/+ valves. Increased phosphorylated Smad2/3 in adult and aged tissue was confirmed by immunoblotting (G), quantification of Smad2/3-positive nuclei to total nuclei is shown in H. QRT-PCR showed increased mRNA expression of TGF-βR1 in aged Emilin1+/+ (white bar) and Emilin1−/− (black bar) aortic valve (I). MAPK/phosphorylated Erk1/2 signaling progressively increased in Emilin1−/− aortic valves (arrowheads, K,M,O) when compared with age-matched controls (J,L,N). Increased phosphorylated Erk1/2 activation (p-Erk1/2) was confirmed by immunoblotting (P) in both adult and aged Emilin1−/− aortic valve tissue; this increase was significant at the aged stage, as assessed using ImageJ densitometry quantification (Q). Histogram shows circulatory levels of active TGF-β in the plasma samples of Emilin1+/+ and Emilin1−/− mice (R). Scale bars: 50 μm. Mean±s.e.m.; n=14; *P<0.05 Emilin1+/+ versus Emilin1−/−; #P<0.05 adult versus aged.

root dilation, a finding that is commonly associated with AVD (Hahn DISCUSSION et al., 1992). The ascending aorta dimension was normal. In the present study, we demonstrated that the Emilin1 deficient Paradoxically, the aortic valve annulus dimension was increased, mouse is a model of human fibrotic AVD. Importantly this model which together with aortic root dilation is reminiscent of annulo-aortic recapitulates the natural history of human AVD allowing ectasia in human disorders. In 75% of aged investigation of early disease processes. In summary, Emilin1−/− Emilin1−/− mice, there was significant left ventricular dysfunction, a aortic valve tissue demonstrates complex TGF-β dysregulation that common complication of severe AVD. Left ventricular mass and end causes downstream activation of both non-canonical (phosphorylated diastolic dimension were unchanged in Emilin1−/− mice, consistent Erk1/2) and canonical (phosphorylated Smad2/3) TGF-β signaling, with the absence of a primary cardiomyopathy. Interestingly, 25% of and ultimately results in aberrant angiogenesis and mild fibrosis in Emilin1−/− mice die prematurely between 14 and 18 months due to the adult stage due to VIC activation and EFF mediated in-part due unclear causes when compared with Emilin1+/+ mice, which are to increased expression of elastolytic enzymes. Interestingly, over reported to have a longevity of 25 months (Yuan et al., 2009). No time, there is progressive upregulation of phosphorylated Erk1/2 significant changes in aortic valve and ventricular functions were signaling that results in activation of a subset of VICs with observed in adult mutant mice when compared with age-matched myofibroblast-like characteristics, which results in neovascularization control (data not shown). Taken together, these findings identify the and severe fibrosis at the aged stage (Fig. 6). Taken together, the −/− −/−

Emilin1 mouse as a model of latent fibrotic AVD. Emilin1 model of AVD provides unique opportunities to identify Disease Models & Mechanisms

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Fig. 4. Emilin1 deficiency is associated with early VIC and late myofibroblast activation in aortic valve tissue. VIC activation is demonstrated by increased SMemb and αSMA expression at the adult (arrowheads, B,F) and aged stages (arrowheads, D,H) in Emilin1–/–aortic valves when compared with Emilin1+/+ valves (A,C,E,G). Myofibroblast activation is shown by increased SM22 at the aged stage (arrowheads, L) but not the adult stage (J) in Emilin1–/– aortic valves when compared with Emilin1+/+ valves (I,K). Confocal imaging shows colocalization of SM22 (green) and VEGF-A (red) in the endothelium of Emilin1+/+ (M) and the interstitium of Emilin1–/– aortic valve tissue (white arrowheads, N). Emilin1–/– aortic valves demonstrate an increase in the proliferation index at all stages when compared with age-matched Emilin1+/+ valves (O). Aged Emilin1–/– aortic valves also showed significantly increased proliferation when compared with juvenile and adult Emilin1–/– aortic valves. Scale bars: 50 µm (A-L); 10 µm (M,N). Mean±s.e.m.; n=8; *P<0.05 Emilin1+/+ vs Emilin1–/–; #P<0.05 adult Emilin1–/– vs aged Emilin1–/–; ^P<0.05 juvenile Emilin1–/– vs aged Emilin1–/–. predictive biomarkers and to test new therapeutic targets that could valve tissue, Emilin1 is present in the annulus and other cusp represent much needed early intervention strategies to prevent layers, in addition to its presence in the mature elastic fiber advanced AVD and the need for surgery. filaments in the ventricularis layer, suggesting that Emilin1 has Elastic fibers play a crucial role in continuous valve motion by functional roles additional to those associated with elastic fiber contributing to the normal recoil process during the cardiac cycle, assembly (Angel et al., 2011; Votteler et al., 2013). Overall, this and EFF and elastase-mediated disease progression are universal study identifies Emilin1 as an ECM protein that is necessary for findings of AVD that contribute to valve dysfunction (Vesely, mature valve structure and function, and the Emilin1−/− mouse as 1997; Fondard et al., 2005; Schoen, 2008). Emilin1 deficiency and an important model of viable AVD. the resulting elastic fiber assembly defects have been studied in Neovascularization is present in the proximal cusp and annulus tissue of the aorta, showing that Emilin1 inhibits TGF-β signaling regions of aged Emilin1−/− aortic valves in proximity to EFF, and that Emilin1 deficiency causes TGF-β upregulation (Zanetti et consistent with findings in human AVD and the idea that cell-ECM al., 2004; Zacchigna et al., 2006). The findings of the current study homeostasis is disrupted primarily at the hinge region of the valve show that Emilin1−/− aortic valves have intrinsic elastic fiber (Thubrikar et al., 1986; Hinton et al., 2010; Wirrig et al., 2011). In assembly defects, suggesting that EFF might be the result of elastic the present study, we have shown a marked imbalance between pro- fiber assembly defects that trigger further elastase-mediated EFF angiogenic and anti-angiogenic factors that promotes aberrant and a progressive disease process. Several mouse models of angiogenesis in the typically avascular valve tissue. Angiogenic mutated elastic fiber components have valve defects (Ng et al., signaling pathways regulate normal embryonic valve development 2004; Hanada et al., 2007; Hinton et al., 2010; Krishnamurthy et (Combs and Yutzey, 2009), but it is largely unknown whether these al., 2012), suggesting that elastic fiber assembly defects have a pathways contribute to the maintenance of mature adult valve tissue.

central role in AVD pathogenesis. Interestingly, in normal aortic Previous studies have shown that mechanisms of AVD recapitulate Disease Models & Mechanisms

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Table 1. Echocardiography in aged Emilin1−/− mice Emilin1+/+ (n=11) Emilin1−/− (n=12)

Dimensions Annulus (mm) 1.34±0.03 1.58±0.04* Root (mm) 1.76±0.08 1.92±0.06 STJ (mm) 1.61±0.03 1.56±0.03 AAO (mm) 1.66±0.03 1.60±0.03 Root:AAO index 1.08±0.04 1.20±0.03* Valve function Peak velocity (mm/s) 380±70 526±66* Peak gradient (mm Hg) 0.60±0.09 1.20±0.06* Ventricular function Ejection fraction (%) 54.5±1.5 39.5±3.5* Fractional shortening (%) 28.0±0.8 19.5±1.9* LVEDD (mm) 4.60±0.07 4.80±0.13 LV mass (mg) 96.4±4.40 97.4±6.60 Heart rate (bpm) 322±33 383±16

AAO, ascending aorta; LV, left ventricular; LVEDD, left ventricular end diastolic diameter; STJ, sino-tubular junction; *P<0.05, n=10. Results are shown as mean±s.e.m.

The role of canonical TGF-β signaling (phosphorylated Smad2/3) during valve development and disease has been well established (Camenisch et al., 2002; Walker et al., 2004), but little is known about the role of non-canonical TGF-β signaling (phosphorylated Erk1/2). Previous in vitro studies have shown that TGF-β1 signaling activates canonical Smad2/3 and non-canonical MAPK pathways, resulting in VIC activation and fibrosis, suggesting a potential role for phosphorylated Erk1/2 in AVD (Gu and Masters, 2009; Hutcheson et al., 2012). Interestingly, we showed that non-canonical signaling was upregulated and progressively increased over time, indicating the presence of a liability threshold for myofibroblast activation (Fig. 6). We showed that VIC activation occurs early in the disease process and that myofibroblast-like cell activation occurred later in the disease process, associated temporally with the latent findings of marked fibrosis, proliferation and inflammation. Fig. 5. Aged Emilin1−/− aortic valve tissue demonstrates fibrosis and Activated release angiogenic factors in the valve inflammation. Increased collagen deposition (blue staining using Masson’s interstitium, which is typically avascular, providing a putative trichrome) is seen in the Emilin1−/− aortic valve annulus (arrowheads, B) mechanism for the advancement of late neovascularization from when compared with age-matched Emilin1+/+ valves (A). Macrophage early provisional aberrant angiogenesis (Paruchuri et al., 2006). infiltration, as evidenced by Mac-3-positive cells (arrowheads, D), is present Consistently, our findings showed colocalization of activated in aged Emilin1−/− valves only and not Emilin1+/+ controls (C). Electron −/− myofibroblasts with VEGF-A-positive cells. In addition, there was microscopy (EM) of Emilin1 aortic valves showed fibroblast-like cells in all a progressive increase in the proliferation index in Emilin1−/− aortic cusp layers (arrowheads, F,H) along with collagen accumulation. Relative mRNA expression of type I, II and III collagen in the aortic valve tissue of valve tissue, suggesting discrete contributions from each type of aged Emilin1−/− mice to that of Emilin1+/+ mice (I). Scale bars: 50 μm (A-D); VIC activation. Constitutive phosphorylated Smad2/3 upregulation 500 nm (E,F); 100 nm (G,H). Mean±s.e.m.; n=14; *P<0.05 Emilin1+/+ versus might result in early VIC activation, increased elastolytic activity, Emilin1−/−. proliferation and provisional angiogenesis, whereas progressive phosphorylated Erk1/2 upregulation (or a cumulative threshold of developmental programs, suggesting that the dysregulation of both canonical and non-canonical TGF-β signaling) might result in specific structural proteins and signaling pathways incite disease and late myofibroblast activation that results in fibrosis and contribute to faulty homeostasis over time and ultimately valve inflammation, consistent with previous reports (Hutcheson et al., dysfunction later in life (Markwald et al., 2010; Hinton and Yutzey, 2013). Previous reports have shown robust activation of 2011; Mahler and Butcher, 2011). Elastic fiber fragments, or phosphorylated Erk1/2 and proliferation in Emilin1−/− fibroblast degradation products, might have pro-angiogenic, pro-proliferative, cells that is mediated through a PTEN-dependent pathway (Danussi pro-calcific or pro-inflammatory properties (Perrotta et al., 2011; et al., 2011; Danussi et al., 2012). However, in the current study, Pivetta et al., 2014; Parks and Mecham, 2011), in addition to well- PTEN levels were unaltered despite Erk1/2 activation and increased described increases in elastase activity (Robinet et al., 2005), proliferation in Emilin1−/− valves, consistent with a role for activated suggesting that different elastic fiber fragments have different non-canonical TGF-β signaling in aortic valve tissue. A limited maladaptive effects. In this context, Emilin1-deficiency-related EFF number of apoptotic cells were identified as being restricted to the results in aberrant angiogenesis and fibrosis, but not calcification. hinge region of aged Emilin1−/− aortic valves (supplementary The identification of specific mechanisms that regulate different material Fig. S1M), possibly due to increased phosphorylated types of disease progression might provide new potential therapeutic Erk1/2 signaling and high mechanical strain, suggesting that the

targets that prevent AVD progression. biomechanical properties of the Emilin1-deficient valves warrant Disease Models & Mechanisms

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The findings of the current study identify the Emilin1−/− mouse as a model of latent fibrotic AVD. These findings have substantial clinical implications that emphasize the importance of translational research. AVD progression is a poorly understood process that needs to be defined on a molecular basis in order to test new clinical therapies (Rajamannan et al., 2011). Presently, there are no pharmacological treatment strategies for early-stage AVD; therefore, the elucidation of the natural history of the disease will facilitate preclinical studies aiming to test early intervention strategies, including pharmacological therapies attempting to prevent advanced AVD, potentially encompassing elastase, phosphorylated Erk1/2 or angiogenesis inhibitors. In addition, this model might provide insights into common processes underlying aortopathy associated with AVD. This clinical association is well described and there is some genetic overlap that establishes shared etiologic factors, including TGF-β dysregulation (Hinton, 2012), but this is largely undefined. Mutations in human EMILIN1 have been associated with cardiovascular disease (Shen et al., 2009; Liu and Xi, 2012), but not specifically AVD or aortopathy, and therefore could represent an important candidate gene and/or genetic modifier. Overall, the emilin family of glycoproteins might represent important factors in cardiovascular diseases. Fig. 6. Hypothetical model of complex TGF-β signaling dysregulation in Emilin1-deficient aortic valve tissue. Emilin1 deficiency results in inherent MATERIALS AND METHODS elastic fiber assembly defects and activation of TGF-β signaling. This results Animals in downstream activation of both non-canonical (phosphorylated Erk1/2; p- Emilin1 deficient (Emilin1−/−) and wild-type (Emilin1+/+) littermates were Erk1/2) and canonical (phosphorylated Smad2/3; p-Smad2/3) signaling in studied at juvenile (8-10 days), adult (4-6 months) and aged (12-14 months) Emilin1−/− aortic valve tissue, resulting in early mild fibrosis and aberrant stages. Mice were maintained on a C57Bl6 genetic background, and angiogenesis due to early VIC activation and elastic fiber fragmentation (EFF) mediated, in-part, by increased expression of proteolytic enzymes, genotyping was performed as described previously (Zanetti et al., 2004). All such as elastases, MMP-2 and MMP-9. Over time (red), there is a protocols were approved by the Institutional Animal Care and Use progressive upregulation of phosphorylated Erk1/2 signaling, which results in Committee at Cincinnati Children’s Hospital Medical Center. myofibroblast activation, neovascularization and severe fibrosis at the aged stage. Histology and immunohistochemistry Hearts were isolated and processed as described previously (Hinton et al., 2010). Movat’s pentachrome, Masson trichrome, Hart’s and Alizarin-Red further investigation. This implicates non-canonical MAPK/ stains were used. Immunohistochemistry was performed to assess markers of phosphorylated Erk1/2 activation as a mechanism underlying the VIC activation, myofibroblast differentiation, angiogenesis, TGF-β signaling, progression of AVD and the manifestation of advanced disease, elastolysis and proliferation as described in Table 2. Antibodies were obtained controlling for the important adverse effects of aging. from Cell Signaling (Boston, MA, USA); Abcam (Cambridge, MA, USA); Although non-resident cells have been described in the valve Santa Cruz Biotechnology (Dallas, TX, USA); Sigma-Aldrich (St Louis, MO, interstitium of wild-type mice (Hajdu et al., 2011), the inflammation USA); Invitrogen (Grand Island, NY, USA) and Millipore (Billerica, MA, observed in aged Emilin1−/− aortic valves might be due to other USA) with catalog information and other details given in Table 2. A universal processes. For example, inflammation might be the direct result of streptavidin-biotin and diaminobenzidine detection system (Pierce) was used systemic TGF-β upregulation, given the role of TGF-β signaling for colorimetric detection. Secondary antibodies used for immunofluorescence in inflammation. Alternatively, in light of the abnormalities were goat against mouse IgG Alexa Fluor 568 (Invitrogen, A11004), goat against rabbit IgG Alexa Fluor 568 (Invitrogen, A11011), and goat against appreciated in the adventitia layer of the aorta, inflammation might mouse Alexa Fluor 488 (Invitrogen, A11001). Immunofluorescence was be the result of the migration of non-resident cells from the imaged using a Nikon A1-R confocal microscope. Cell proliferation was adventitia layer into the valve annulus and proximal cusp, studied in histological sections of mouse heart, which were stained using an consistent with the localization of neovascularization. Another antibody against phosphorylated histone H3 (phosphorylated HH3) as alternative is direct infiltration due to a discontinuous endothelial described previously (Hinton et al., 2006). The proliferation index was layer. This results in elastases in the valve interstitium and migration calculated by determining the ratio of positively stained nuclei to the total of circulating hematopoietic (or non-resident) cells from either the number of nuclei in the hinge region of the valve. The phosphorylated newly formed valve neovasculature, which is not present until the Smad2/3 nuclei index was calculated in the same way. Images were analyzed aged stage, or the disrupted endothelium. There were areas of using NIS-Elements software (Nikon). A total of four to six animals were endothelial disruption around the edges of the Emilin1−/− aortic studied per genotype per stage. valve (data not shown), which is consistent with previous findings (Zanetti et al., 2004; Danussi et al., 2012). Previous evidence Electron microscopy Ultrastructure analysis was performed using a Hitachi 7600 transmission suggests that activation of the phosphorylated Erk1/2 pathway is a electron microscope (Hitachi Technologies). Aortic valve, ascending aorta crucial modifier in elastase-mediated diseases (Preston et al., 2002; and left ventricular myocardium were processed and analyzed as described Ghosh et al., 2012). Taken together, complex TGF-β dysregulation previously (Hinton et al., 2010). In order to visualize collagen and elastic −/− mediates early and late pathologic findings in Emilin1 aortic fibers, sections were counter stained with aqueous solutions of 5% tannic valves, and phosphorylated Erk1/2 activation might play a more acid followed by 1% uranyl acetate, and then counterstained with lead

important role than previously appreciated in AVD pathogenesis. citrate. Disease Models & Mechanisms

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Table 2. Specification of the primary antibodies used for immunohistochemistry Protein Marker Host Dilution Source Catalogue number Antigen retrieval αSMA VIC activation Mouse monoclonal 1:250 Sigma-Aldrich ab5694 Hot citrate SMemb VIC activation Mouse monoclonal 1:250 Abcam ab684 Hot citrate SM22 VIC activation Goat polyclonal 1:250 Abcam ab1035 Hot citrate N elastase Elastase Goat polyclonal 1:50 Santa Cruz Biotech sc9518 Hot citrate MMP-2 Elastase Rabbit polyclonal 1:100 Abcam ab7298 Hot citrate MMP-9 Elastase Rabbit polyclonal 1:200 Abcam ab38898 Hot citrate CD-31 Endothelial cell Rabbit polyclonal 1:50 Abcam ab28364 Hot citrate VEGF-R1 Pro-angiogenesis Rabbit polyclonal 1:100 Santa Cruz Biotech sc316 Hot citrate VEGF-R2 Pro-angiogenesis Rabbit polyclonal 1:100 Cell Signaling 55B11 Proteinase K VEGF-A Pro-angiogenesis Rabbit polyclonal 1:100 Santa Cruz Biotech sc152 Hot citrate Chondromodulin Anti-angiogenesis Rabbit polyclonal 1:75 Lifespan Bioscience LS-C80668 Hot citrate Endostatin Anti-angiogenesis Rabbit polyclonal 1:100 Abcam ab85661 Hot citrate Fibulin-5 Anti-angiogenesis Mouse monoclonal 1:100 Abcam ab66339 Hot citrate Phosphorylated Smad TGF-β Rabbit polyclonal 1:100 Cell Signaling 3101 Hot citrate Phosphorylated Erk1/2 MAPK Rabbit polyclonal 1:100 Invitrogen 700012 EDTA Phosphorylated HH3 Proliferation Rabbit polyclonal 1:300 Millipore 05-806 Proteinase K Ki67 Proliferation Rabbit polyclonal 1:500 Abcam ab15580 Proteinase K Caspase 3 Apoptosis Rabbit polyclonal 1:100 Cell Signaling 9661 Hot citrate Mac-3 Macrophage marker Rabbit 1:50 BD Pharmingen 550292 None

QRT-PCR Acknowledgements Aortic valves were dissected from adult and aged mouse heart from We thank Osniel Gonzales-Ramos, Varun Krishnamurthy and the Cardiac Imaging Emilin1−/− or Emilin1+/+ mice. Aortic valve tissue from three mice was Core Research Laboratory for their assistance. pooled and RNA was isolated using the standard Trizol method. cDNA was generated using ~500 ng RNA followed by QRT-PCR and amplified by PCR Competing interests using gene specific primers (supplementary material Table S1) (Hinton et The authors declare no competing financial interests. al., 2010). Ct values were obtained using Bio-Rad software. The ΔΔCt Author contributions method was used to represent the mRNA fold change. Three experiments C.M. and R.B.H. developed the concepts and approaches, analyzed data and were performed for each stage and genotype. wrote the manuscript. C.M., A.O., H.O., J.J. performed experiments and analyzed data. G.B. provided the mouse model studied and analyzed the data. All authors Immunoblotting approved the final manuscript. Analyses were performed on aortic valves isolated from five stage- and genotype-matched mice, as described previously (Givvimani et al., 2012). Funding A bicinchoninic acid protein assay kit (Thermo Scientific, Rockford, IL, This work was supported by the Cincinnati Children’s Research Foundation USA) was used to estimate total protein, and 30 μg of protein lysate was (R.B.H.); the National Institutes of Health (NIH) [HL085122]; and an Institutional loaded onto 8-12% SDS-PAGE gels and then transferred onto nitrocellulose Clinical and Translational Science Award [NIH/NCRR 8UL1TR000077]. membranes. The membranes were blocked with 3% non-fat dry milk in TBS Tween 20 and incubated with primary antibodies against MMP-2 (1:1000), Supplementary material MMP-9 (1:1000), phosphorylated Smad2/3 (1:300) and phosphorylated Supplementary material available online at http://dmm.biologists.org/lookup/suppl/doi:10.1242/dmm.015255/-/DC1 Erk1/2 (1:1000) overnight at 4°C. Immunoblots were probed with horseradish-peroxidase-conjugated secondary antibodies for 1 hour at room References temperature and developed using chemiluminescence (Amersham Angel, P. M., Nusinow, D., Brown, C. B., Violette, K., Barnett, J. V., Zhang, B., Biosciences). After applying a stripping buffer (Thermo Fisher), the blots Baldwin, H. S. and Caprioli, R. M. (2011). Networked-based characterization of were re-probed with antibodies against GAPDH (Santa Cruz Biotechnology) extracellular matrix proteins from adult mouse pulmonary and aortic valves. J. or total Erk1/2 (Cell Signaling). 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