© 2015. Published by The Company of Biologists Ltd | Journal of Cell Science (2015) 128, 1865-1875 doi:10.1242/jcs.162891

COMMENTARY ARTICLE SERIES: CELL BIOLOGY AND DISEASE Mechanobiology of myofibroblast adhesion in fibrotic cardiac disease Alison K. Schroer1 and W. David Merryman1,*

ABSTRACT behavior and phenotype of cardiac cells, which contributes to Fibrotic cardiac disease, a leading cause of death worldwide, manifests maladaptive tissue remodeling (Cuniberti et al., 2006; Merryman as substantial loss of function following maladaptive tissue remodeling. et al., 2006). Therefore, determining how various cardiac cells can affect both the heart valves and the myocardium and respond to changing mechanical environments will aid our is characterized by the activation of and accumulation of understanding of the development of heart disease and potentially . Valvular interstitial cells and cardiac fibroblasts, the uncover new targets for future therapy (Fig. 1A). cell types responsible for maintenance of cardiac extracellular matrix, Valvular interstitial cells (VICs) and cardiac fibroblasts (CFs) are are sensitive to changing mechanical environments, and their ability to primarily responsible for maintaining the ECM, and are sensitive to sense and respond to mechanical forces determines both normal mechanical forces in addition to chemical cues. Mechanically induced development and the progression of disease. Recent studies have signaling promotes myofibroblast (MyoFB) differentiation of VICs uncovered specific adhesion proteins and mechano-sensitive signaling and CFs, resulting in cells that exhibit increased contractility and pathways that contribute to the progression of fibrosis. form increased secretion of growth factors and ECM proteins (Fig. 1B) adhesions with the extracellular matrix, and respond to changes in (Tomasek et al., 2002). MyoFBs interact with the local ECM and sense substrate stiffness and extracellular matrix composition. Cadherins tissue forces through transmembrane ECM receptors called integrins. mechanically link neighboring cells and are likely to contribute to fibrotic Upon adhesion to the ECM, integrins recruit intracellular proteins disease propagation. Finally, transition to the active myofibroblast to form a focal adhesion (FA), which links integrins to the actin phenotype leads to maladaptive tissue remodeling and enhanced and initiates force-dependent signaling. In addition to mechanotransductive signaling, forming a positive feedback loop force transduction from the ECM to the cells, integrins also that contributes to . This Commentary summarizes recent regulate force transmission from intracellular stress fibers to the local findings on the role of through integrins and microenvironment (Baker and Zaman, 2010). MyoFBs are identified α α cadherins to perpetuate mechanically induced differentiation and by their expression of -smooth muscle actin ( -SMA), a specialized fibrosis in the context of cardiac disease. cytoskeletal protein that allows for enhanced generation of cellular force (Hinz et al., 2004). These forces are, in turn, transmitted to the KEY WORDS: Cadherin, , Myofibroblast local microenvironment, altering ECM structure, and substantially affecting tissue mechanics (Petersen et al., 2012). Introduction Intracellular forces are also transmitted to neighboring cells through The heart is a highly dynamic mechanical environment that stretches adherens junctions (AJs), which contain Ca2+-dependent adhesion and contracts over three billion times during the course of the average proteins called cadherins. Classical cadherins form homotypic bonds human life. Proper functioning of cardiac structures is dependent on with cadherins on neighboring cells and mechanically link the their active and passive mechanical properties, and alteration of these cytoskeletal elements of both cells. Different cadherins are found in properties is a hallmark of fibrotic cardiac disease. Given the high various cell types, and major shifts in cell phenotype are often societal cost of heart disease, the mechanisms regulating fibrotic accompanied by a corresponding shift in cadherin expression. disease progression in heart valves and the myocardium have been Neuronal (N)-cadherin is typically expressed in cardiomyocytes extensively studied. In both tissues, inflammation and fibrosis drive (CMs) and quiescent fibroblasts, whereas osteoblast (OB)-cadherin extensive tissue remodeling that significantly impairs cardiac (also known as cadherin-11) is highly expressed in MyoFBs function (Fig. 1A). Inflammation triggers extracellular matrix (Aisagbonhi et al., 2011; Chalpe et al., 2010; Heuberger and (ECM) degradation and the release of profibrotic factors, such as Birchmeier, 2010; Hinz et al., 2004; Li et al., 2012). OB-cadherin angiotensin II (AGT; hereafter referred to as AngII), transforming forms significantly stronger bonds than N-cadherin and is, thus, able growth factor β1 (TGF-β1), and growth factor (FGF), that to transmit increased levels of intercellular tension generated by promote accumulation of ECM and fibrosis of the valves and α-SMA in MyoFBs (Hinz et al., 2004; Hutcheson et al., 2013). Recent myocardium (Chen and Frangogiannis, 2013; Mahler and Butcher, studies have revealed new signaling roles for cadherins in addition to 2011). However, drugs targeting these pathways have had limited their structural function (Leckband and de Rooij, 2014). Signaling success, which has been attributed to their failure to address the effectors downstream of cadherins, including β-catenin, undergo a concurrent mechanical signals that play a crucial role in the initiation substantial crosstalk with cytokine and integrin signaling in the and progression of disease (Frangogiannis, 2014). Conditions that regulation of MyoFB differentiation and function (Caraci et al., 2008; favor increased local tissue strains and stresses increase the risk of Charbonney et al., 2011; Xu et al., 2012). developing fibrotic disease in affected structures by altering the This Commentary focuses on mechanotransduction in VICs and CFs during cardiac fibrotic disease, and its effects on the regulation 1Department of Biomedical Engineering, Vanderbilt University, Nashville, of MyoFB differentiation. Mechanotransduction in endothelial cells TN 37212, USA. is certainly relevant to and has been

*Author for correspondence ([email protected]) described in an excellent recent review (Conway and Schwartz, Journal of Cell Science

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A Fig. 1. Relevance of adhesion mechanobiology to Left atrium fibrotic heart disease. (A) Fibrotic disease affects both heart valves and heart myocardium and is characterized Right atrium by changes in the mechanical properties of the ECM (extracellular matrix) and the cellular phenotypes present in the tissue. Healthy valves primarily have quiescent fibroblast-like cells called valvular interstial cells (VICs) Right ventricle Left ventricle that are embedded in a stable, organized ECM and surrounded by a single layer of valvular endothelial cells (VECs). Similarly, healthy myocardium contains cardiomyocytes (CMs), cardiac fibroblasts (CFs) and lined blood vessels lined with endothelial cells (ECs) that are embedded in stable ECM. Disease can be initiated by Valve leaflet Myocardium acute or chronic cardiac conditions. Inflammation initiates ECM breakdown, accumulation of inflammatory cells and fibroblast-like cells, as well as EndMT, which yields Healthy tissue Healthy endothelial-to-mesenchymal-transformed cells structure (EndMTCs). Inflammation can turn into fibrosis, which is Flexible and characterized by accumulation of stiff ECM and a large functional number of myofibroblasts (MyoFBs) that are derived from VICs, CFs, and EndMTCs. MyoFBs are particularly Disease inititation relevant to disease because they are able to initiate ECM Infllammatiion remodeling and intercellular signaling pathways. (B) The Disease MyoFB phenotype is promoted by combined inputs of progression Fibrosis mechanical and chemical signals that are transduced, Diseased respectively, through integrins and cadherins, and α Fibrotic tissue signaling factor receptors. Increased expression of - pathology SMA in MyoFBs stabilizes, and further activates integrins and cadherins; this increases the generation of intracellular force and actively remodels the ECM. Stiff and MyoFBs also express ECM and growth factors that not dysfunctional only affect themselves but also neighboring cells.

Key Inflammatory B cells Calcified Signaling factors nodule ECM Cellular VIC/CF Intracellular Integrins VEC/EC input force ECM ECM-transmitted CM force EndMTC Cellular MyoFB Release output Integrins of signaling factors and ECM components Cadherins

2013), so this aspect will not be discussed in detail. Likewise, conditions with large clinical impacts are perpetuated by active mechanotransduction in CMs is also important to disease remodeling of the ECM by VICs in valves and by CFs in the progression and has been the subject of another recent review myocardium. (McCain and Parker, 2011). The adhesion molecules of particular interest for this Commentary are integrins and cadherins, which disease are both relevant to mechanically regulated signaling and force Valve disease affects over six million Americans and is associated transmission in the heart. Other important mechanotransducers, with changes in mechanical properties of the leaflets that impair including mechano-sensitive Ca2+-channels and syndecans are normal blood flow through the heart (Go, 2013). The mitral and aortic either not involved in adhesion (Teng et al., 2014), or have already valves on the left side of the heart are most susceptible to disease, been extensively reviewed elsewhere (Frangogiannis, 2010). This which manifests most often as regurgitating flow or stenosis (Go et al., Commentary will describe the interactions between mechanical 2013). Mitral regurgitation is the most common type of heart valve forces and biochemical cues from tissue level down to intracellular disorder (1.7% of adults), but aortic valve disorders are associated signaling, focusing on the particular integrins and cadherins that with higher mortality, especially when calcification and stenosis of the perpetuate these signals and contribute to the progression of fibrotic valve is evident (Go et al., 2013). The initial causes of these conditions cardiac disease. are often linked to altered mechanical loading (e.g. hypertension, ventricular remodeling, bicuspid aortic valve mutation) and, in all Etiology of fibrotic cardiac disease cases, tissue remodeling is perpetuated by inflammation and fibrosis, Fibrotic disease affects both the valves and the myocardium, and is which trigger ECM degradation and accumulation, respectively (de characterized by significant changes in tissue composition and Marchena et al., 2011; Hutcheson et al., 2014). The mitral valve is biochemical signaling that, in turn, affect cell behavior and particularly susceptible to tissue weakening in a process known as accelerate progression of disease (Fig. 1A). Several cardiac myxomatous remodeling, whereas the aortic valve is susceptible to Journal of Cell Science

1866 COMMENTARY Journal of Cell Science (2015) 128, 1865-1875 doi:10.1242/jcs.162891 tissue stiffening in response to fibrosis and sclerotic remodeling conditions, including valve disease and hypertension, can cause (Geirsson et al., 2012; Merryman and Schoen, 2013). Myxomatous pressure overload in the ventricles that subsequently develops into remodeling is characterized by increased expression of TGF-β1 hypertrophy and fibrosis (Azevedo et al., 2010; Dusenbery et al., (TGFB1), matrix metalloproteinases (MMPs) and several ECM 2014; Huang et al., 2010). Another common initiator of cardiac components, and results in valve billowing and regurgitating blood fibrosis is scar formation after , which affects flow (Aupperle and Disatian, 2012; Geirsson et al., 2012; Hagleret al., over one million Americans annually (Go, 2013). In both chronic 2013). Calcific aortic valve disease (CAVD), a stenotic disease that conditions and myocardial infarction, inflammatory cytokines often necessitates , is characterized by increased induce tissue remodeling and degradation of ECM in the expression of TGF-β1 and – especially collagen-1 – and the myocardium (Gullestad et al., 2012). The loss of ECM can result development of calcified nodules, resulting in a stiff, partially in a temporary decrease in the passive wall stiffness and increase in occluded, stenotic valve (le Polain de Waroux et al., 2007; Mohler diastolic strains in the infarct region, which increase the chance of et al., 2001). These pathologic alterations to the mechanical properties myocardial wall rupture (Banerjee et al., 2006; Gao et al., 2005; Ma of the tissue are primarily mediated by VICs, a heterogenous et al., 2013). Deposition of de novo ECM is necessary to maintain population of fibroblast-like cellsthat differentiate into active MyoFBs structural integrity and requires the switch from inflammatory to in response to fibrotic signaling factors and mechanical cues (Fig. 1) profibrotic signaling factors (Frangogiannis, 2014). In both chronic (Liu et al., 2007). and acute myocardial remodeling, AngII inhibits the degradation of collagen-1 and promotes the expression of FGF-2 and TGF-β1 that, Cardiac fibrosis in turn, promote cell growth and collagen production in the Cardiac fibrosis is a hallmark of heart failure and results in an myocardium (Frangogiannis, 2014; Huang et al., 2010; Porter and increased passive stiffness of the heart wall, diastolic dysfunction Turner, 2009; Schuleri et al., 2012; Virag et al., 2007). TGF-β1 and poor long-term prognosis (Azevedo et al., 2010; Burlew and inhibits inflammation and promotes the differentiation of fibroblasts Weber, 2002; Dusenbery et al., 2014). Many chronic cardiovascular into MyoFBs, and the accumulation of dense ECM in the

A Fig. 2. Mechano-sensitive mechanisms of MyoFB differentiation. (A–C) Myofibroblasts (MyoFBs) play a central role in in the progression of fibrotic disease in the Disrupted basal lamina heart because of their roles in the generation and transmission of cellular force, intercellular signaling and ECM remodeling. One important mechanism yielding MyoFBs is EndMT (A), by which endothelial cells (ECs) lose their endothelial markers (including VE-cadherin) and become migratory and contractile. Valvular EndMT interstitial cells (VICs) and cardiac fibroblasts (CFs) can differentiate into MyoFBs in response to high mechanical MyoFB strain (B) – which is often experienced during inflammation – with the degradation of initial ECM and a corresponding decrease in tissue stiffness. Quiescent VICs and CFs can also differentiate into MyoFBs in response to high mechanical stress (C), caused by both increased tissue stiffness and increased tissue forces. MyoFBs increase the overall stress in the environment by Strain-induced MyoFB Stress-induced MyoFB producing excess ECM and contracting existing ECM differentiation differentiation through increased cellular contractility. MyoFBs also release profibrotic signaling factors, including TGF-β1 B Signaling factors C and Wnt, that promote further MyoFB differentiation and and ECM components tissue stiffening. This forms a positive feedback loop leading to progressively worsening fibrosis. Tissue stiffening also often leads to compensatory increases in ventricular pressure, which increases the applied tissue forces and reinforces this positive feedback loop.

Partially degraded ECM Accumulated ECM Inflammation Fibrosis Disease progression High strain Low strain Tissue strain Low stiffness High stiffness Tissue stiffness Normal pressure High pressure Tissue forces

Key EndMTC VE-cadherin TGF-β1 α-SMA N-cadherin Wnt High contractilitiy EC VIC/CF OB-cadherin FGF ECM component Journal of Cell Science

1867 COMMENTARY Journal of Cell Science (2015) 128, 1865-1875 doi:10.1242/jcs.162891 myocardial interstitium (Ikeuchi et al., 2004). This increases stress on the remaining contractile myocardium, resulting in further Box 1. A brief overview of EMT during cardiac adverse remodeling and fibrosis largely mediated by CFs that have development differentiated into active MyoFBs (Fig. 1A) (Ersbøll et al., 2014). Development of the heart begins early in embryonic development with a muscular tube lined internally with endocardial cells. Heart valve leaflets are formed by the differentiation of endocardial cells through endothelial- Cardiac cell responses to mechanical stress to-mesenchymal transformation (EndMT) to mesenchymal cells, which In healthy valves and healthy myocardium, quiescent fibroblasts can migrate into a mixture of ECM known as the cardiac jelly. These maintain tissue homeostasis by a controlled and balanced release transformed endocardial cells become the VICs in adult valves and are of ECM proteins and proteases (Davis and Molkentin, 2014). necessary for developing the initial structure of the valve and maintaining However, these fibroblasts transition to an active MyoFB that structure through adulthood (Sewell-Loftin et al., 2014). A similar phenotype during injury or disease in response to the mechanism accounts for the origin of cardiac fibroblasts (CFs) in the synergistic contributions of growth factor signaling (primarily myocardium (muscular tissue). CFs are derived primarily from the β proepicardial organ, a cluster of epithelial-like cells that migrate to cover TGF- 1) and mechanical cues (Fig. 2) (Merryman et al., 2007; the developing heart and form the mature epicardium. A substantial Walker et al., 2004). Decreasing substrate stiffness and treatment proportion of these cells undergo epithelial to mesenchymal transition of MyoFBs with FGF-2 have been shown to reverse MyoFB (EMT) in response to TGF-β1 signaling to gain a more migratory, differentiation and promote the quiescent fibroblast phenotype in fibroblast-like phenotype before they invade the myocardium (Olivey vitro (Greenberg et al., 2006; Kloxin et al., 2010). However, the et al., 2006). These cells are believed to be the origin of vascular smooth increased mechanical stimulation during fibrotic disease may muscle cells, endothelial cells and resident cardiac fibroblasts. During development, CFs differentiate into active MyoFBs – producing fibrullar prevent such a de-differentiation and could be responsible for the collagen and organizing a 3D network for myocardium development and long-term persistence of MyoFBs that are observed in disease. maturation. These MyoFBs express contractile α-SMA, and can apply The three main mechano-sensitive mechanisms of cellular forces to both the developing myocytes and the collagen framework. differentiation that give rise to MyoFBs in the heart during Tissue remodeling during development includes a significant increase in disease initiation are described below (Fig. 2). myocardial stiffness in preparation for the mechanical demands of active Endothelial cells directly contribute to tissue remodeling by circulation. At the end of development, cardiac fibroblasts surround and run parallel to myocytes throughout the myocardium, and are generally differentiating into fibroblast-like cells in response to chemical maintained in a quiescent state; they produce ECM proteins and matrix and mechanical signals through a process known as endothelial-to- metalloproteinases (MMPs) in a tightly regulated balance to maintain mesenchymal transition (EndMT) (Fig. 2A). These cells lose homeostasis (Porter and Turner, 2009). their endothelial adhesions, including vascular-endothelial (VE)- cadherin, and express migratory, mysenchymal cell markers, including N-cadherin, 2 (MMP2) and expression of MMPs and α-SMA increases the ability of CFs to α-SMA (Wylie-Sears et al., 2014). A similar mechanism is migrate through and contract 3D substrates (Wang et al., 2014b). responsible for the origin of both VICs and CFs during High local strains can also signal transition from inflammation development (see Box 1) and is enhanced by active contraction of to fibrosis. In response to increased cyclic strain, aortic VICs the myocardium and surrounding matrix (Sewell-Loftin et al., (AVICs) express reduced levels of inflammatory markers and 2014). Inflammation and TGF-β1 signaling both promote EndMT increased levels of profibrotic factors including TGF-β1 (Smith in valve endothelial cells during the initiation of valve disease et al., 2010). Strain and TGF-β1 together enhance MyoFB (Farrar and Butcher, 2013; von Gise and Pu, 2012; Wylie-Sears differentiation and cell contractility, and increase the formation of et al., 2014). EndMT also accounts for ∼25% of the α-SMA- calcified nodules by AVICs in dynamic culture (Fisher et al., 2013; positive, MyoFB-like cells that are found in the myocardium after Merryman et al., 2007; Santiago et al., 2010). This increase in infarction; this transition is dependent on Wnt, a signaling factor the formation of calcified nodules depends on the establishment that also promotes fibrosis in concert with TGF-β1 (Aisagbonhi and intercellular transmission of cellular tension through α-SMA et al., 2011; Akhmetshina et al., 2012). MyoFB-like cells participate and OB-cadherin, respectively (Fisher et al., 2013; Hutcheson et al., in ECM remodeling during the transition from inflammation to 2013). fibrosis, but future work is needed to completely characterize this A third mechanical trigger for MyoFB differentiation is increased cell population and how it contributes to disease manifestation. mechanical stress (Fig. 2C). In vitro, increased substrate stiffness Another well-established mechanism for MyoFB differentiation promotes MyoFB differentiation in AVICs, leading to increased that is relevant to progressing cardiac disease is the differentiation of formation of calcified nodules, α-SMA expression, and expression of quiescent VICs and CFs in response to high strains (Fig. 2B). TGF-β receptor type 1 (Kloxin et al., 2010; Yip et al., 2009). On stiff Pressure overload increases strains in the valves and myocardium, 2D substrates, CFs exhibit an increase in the expression of α-SMA, and initiation of inflammation causes a breakdown of the ECM that TGF-β1 and collagen-1 (Galie et al., 2011). MyoFBs generate can further increase local strains. Ex vivo aortic valves that were increased internal cellular tension to balance the increase in exposed to pathologic strains (15–20% of original length) expressed extracellular tension they experience on stiffer substrates and, in turn more matrix metalloproteinases – other than MMP2 (i.e. MMP1 and use this tension to remodel their local microenvironment. After cells MMP9) – and collagen-1 than valves that are exposed to have increased their intracellular tension, the surrounding ECM physiologic valve strains (10%) (Balachandran et al., 2009). becomes more taut, which can promote the differentiation of nearby MMPs and other proteases are also expressed by MyoFBs in fibroblasts and perpetuate disease progression (Petersen et al., 2012). infarct regions to break down any damaged ECM, and allow for Besides their active role in ECM modification after differentiation increased fibroblast migration into the infarct region (Aisagbonhi into MyoFBs, CFs can alter myocardial structure by affecting the et al., 2011; Davis and Molkentin, 2014; Schuleri et al., 2012). CFs behavior and function of CMs. Direct intracellular contacts proliferate and express increased levels of α-SMA and MMP2 between CFs and CMs in vitro decrease the contraction velocity in vitro after exposure to – approximately physiological – cyclic of CMs, and increase the expression of inflammatory cytokines strains between 5% and 15% (Dalla Costa et al., 2010). In addition, therein (Banerjee et al., 2006). CF-induced changes in ECM Journal of Cell Science

1868 COMMENTARY Journal of Cell Science (2015) 128, 1865-1875 doi:10.1242/jcs.162891 stiffness and composition can also promote CM hypertrophy (Ieda stress fibers and ECM fibrils, and initiating downstream et al., 2009). Finally, the release of profibrotic factors, such as TGF- signaling pathways (Baker and Zaman, 2010). This signaling is β1, AngII and FGF by CFs have all been shown to promote CM sensitive to ECM composition, stiffness, and applied strains, and hypertrophy (Rohr, 2011), leading to thickening of the muscular regulates the phenotype of the cell, which in turn affects ECM walls that are then able to generate stronger contractions and synthesis and integrin expression throughout the progression of enhance mechanotransductive signaling throughout the heart. These disease. tissue-level forces are transmitted to the different cardiac cells The β1 subunit is part of most collagen-binding integrins in the through the cardiac ECM and through intercellular adhesions heart and can induce force-dependent cellular responses, between them. In the following sections, we highlight recent including cell growth and MyoFB differentiation through research that provided new insight to the molecular mechanisms by activation of PTK1 (also known as and hereafter referred to as which mechanical signals are transduced from the cellular FAK), MAPK3 and MAPK1 (also known as ERK 1 and ERK2, microenvironment in order to elicit these tissue-level changes. respectively; hereafter referred to as ERKs), MAPK14 (also known as and hereafter referred to as p38), and other mitogen Mechanosensors of stress activated kinases (MAPKs) and their downstream signaling Mechano-sensitive adhesion proteins, including integrins and cascades. The exact functional effects depend on the cell type cadherins, transduce mechanical signals between cells and their and the local microenvironment. For example, CM-specific microenvironment and can stimulate cellular responses including deletion of β1-mediated adhesion results in cardiac fibrosis and cell growth and differentiation. Both integrins and cadherins are heart failure in response to pressure overload by disrupting CM large proteins families, and expression of specific isoforms within membrane integrity and contractile function (Shai et al., 2002). In these families is associated with changes in cellular phenotype and addition, α7β1 integrin, a laminin receptor, has also been shown to progression of disease (Tables 1 and 2). The following sections have a protective effect in CMs that are exposed to ischemic stress summarize those integrin and cadherin isoforms that are (Okada et al., 2013). Removal of the β1-integrin-associated upregulated in fibrotic disease in the heart and discuss the mechanosensitive protein melusin (ITGB1BP2), alters signaling mechanosensitive signaling pathways they initiate. through ERK1/2 and glycogen synthase kinase 3β (GSK-3β), and leads to dilated and fibrosis in response to Integrins sense mechanical signals from the ECM pressure overload in the heart (Brancaccio et al., 2003; Penna Integrins are a diverse class of ECM receptors comprising et al., 2014). These studies highlight that β1 integrin helps to heterodimers of α and β subunits that determine ECM binding protect CMs from adverse effects of the mechanical strains they specificity and intracellular signal transduction. Upon ECM experience. engagement, integrins recruit FA proteins that mechanically link Another β1-containing integrin that protects cardiac cells against the cytoskeleton to the ECM, mediating a force balance between fibrosis is α2β1 integrin, the primary receptor for collagen-1, which

Table 1. Expression of integrin isoforms in normal tissue and during progression of fibrotic disease ECM expression Tissue Integrin type ECM-binding partner Normal tissue Fibrotic disease References Valves α2β1 Collagen-1 Arranged circumferentially, Increased, disorganized expression (Stephens et al., 2010) primarily in the fibrosa layera throughout valve α1β1 IV and VI Basal lamina of endothelium Increased expression throughout (Aupperle and Disatian, and fibrosa valve 2012) α3β1 Collagens IV and VI Basal lamina of endothelium Increased expression throughout (Afek et al., 1999) and fibrosa valve α5β1 Fibronectin Limited expression in basal Increased expression throughout (Gu and Masters, 2010) lamina valve αvβ3 RGD Minimal expression and Primarily exposed and expressed in (Benton et al., 2009) exposure areas of MyoFB differentiation

Myocardium α2β1 Collagen-1 Organized network surrounding Main component of scar after (Burgess et al., 2002) CMs and CF myocardial infarction and general cardiac fibrosis α1β1 Collagen-1 Organized network surrounding Main component of scar after (Burgess et al., 2002) CMs and CF myocardial infarction and general cardiac fibrosis α5β1 Fibronectin Limited expression in basal Increased throughout the myocardium (Burgess et al., 2002) lamina α7β1 Laminin Expressed throughout the Increased throughout the myocardium (Burgess et al., 2002) myocardium β1 Fibronectin and/or Organized network surrounding Increased throughout the myocardium (Okada et al., 2013) collagen CMs and CF α8β1 RGD Organized network surrounding Increased collagen and fibronectin (Bouzeghrane et al., CMs and CF throughout myocardium 2004) αvβ3 RGD Organized network surrounding Increased collagen and fibronectin (Balasubramanian et al., CMs and CF throughout myocardium 2012; Luo et al., 2014) a Each valve leaflet is composed of three layers of tissue, atrialis, fibrosa and spongiosa. Journal of Cell Science

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Table 2. Expression of cadherin isoforms in normal tissue and during progression of fibrotic disease Cadherin expression (‘cadherin switching’) Tissue Normal tissue Fibrotic disease Cell type Mechanotransductive effect References Valves N-cadherin OB-cadherin AVIC Increased cell tension, formation of calcified nodules (Hutcheson et al., 2013) VE-cadherin N-cadherin VEC EndMT, migratory, α-SMA-positive cells (Meadows et al., 2009) Myocardium N-cadherin OB-cadherin CF Unknown (Thompson et al., 2014) N-cadherin N-cadherin CM Reduced cell contraction (Thompson et al., 2014) VE-cadherin N-cadherin EC EndMT, migratory, α-SMA-positive cells (Aisagbonhi et al., 2011) forms the largest fraction of healthy ECM in both valves and the (Benton et al., 2009; Gu and Masters, 2010). Furthermore, β3 myocardium (Chen and Simmons, 2011; Gershlak and Black, integrin expression in the heart is significantly increased after 2015). Fibrillar collagen-1 networks are maintained in a tightly myocardial infarction, and expression of β3 integrins in CFs is regulated homeostasis that has been shown to be dependent on necessary for the accumulation of collagen and fibronectin in α2β1-integrin-mediated adhesion in fibroblasts (Fujimura et al., response to pressure overload (Balasubramanian et al., 2012; Luo 2007). Consequently, blocking α2β1 adhesion causes a build-up of et al., 2014). This effect is likely to be mediated by the FA ECM in the skin and in collagen gel lattices, which is prevented by component talin, which links integrins to the cytoskeleton and is α2β1-integrin-induced release of the collagen-1 protease MMP-1 required for αvβ3-integrin-mediated mechanotransduction (Roca- (Fujimura et al., 2007; Liu and Leask, 2013; Riikonen et al., 1995; Cusachs et al., 2009). The talin1 isoform is expressed in the heart Zhang et al., 2014b). during development and disease, and its deletion prevents pressure- However, whereas α2β1 integrin inhibits fibrosis in healthy induced hypertrophy and fibrosis in the myocardium by altering tissues, other β1 integrins promote MyoFB differentiation and signaling through p38, ERK1/2, protein kinase B (Akt) and GSK-3β fibrosis. For instance, α1β1 has been shown to promote both (Manso et al., 2013). All of these kinases are involved in TGF-β1- inflammation and MyoFB differentiation in adult connective tissue induced MyoFB differentiation and promote the expression of (Krieglstein et al., 2002; Rodriguez et al., 2009). In addition, α3β1 α-SMA and increased intracellular tension. Furthermore, β3 integrin promotes EndMT and MyoFB differentiation in fibrotic integrin engagement with ECM proteins enhances TGF-β1 lungs and mediates crosstalk between factors involved in TGF-β1- signaling through Src and p38 to further promote the expression and Wnt-associated signaling (Kim et al., 2009). These integrins are of α-SMA and of β3 integrins, thereby forming another positive expressed in the heart and bind to non-fibrillar collagens IV and VI feedback loop (Pechkovsky et al., 2008). that are upregulated during valve disease and myocardial fibrosis The convergence on TGF-β1 signaling is one example of the (Aragno et al., 2008; Aupperle and Disatian, 2012; Naugle et al., significant crosstalk between integrin- and growth-factor signaling 2006). Another β1 integrin that has been linked to cardiac fibrotic that is involved in the regulation of MyoFB differentiation in the disease is α5β1 integrin, the classic fibronectin receptor. Expression heart (Fig. 3) (Friedland et al., 2009; Schroer, 2014). Non-canonical of α5β1 is increased in fibrotic myocardium and signaling TGF-β1 signaling through Src and p38 promotes the production of downstream of α5β1 promotes the expression of additional α-SMA in AVICs by the transcription factors myocardin related fibronectin in an example of positive feedback (Sarrazy et al., (MRTF) and serum response factor (SRF) 2014; Wang et al., 2010). Secretion of fibronectin contributes to (Elberg et al., 2008; Hutcheson et al., 2012; Watanabe et al., 2009). further MyoFB differentiation by initiating signaling through FAK FGF-2 signaling through FAK and ERK1/2 has been shown to that facilitates the formation of new integrin adhesions and increases prevent MyoFB differentiation in MEFs and to reverse TGF- matrix stiffness (Fig. 2C). Fibronectin-induced signaling promotes β1-mediated expression of α-SMA (Greenberg et al., 2006; MyoFB differentiation, and increased MMP15 expression by and Kawai-Kowase et al., 2004; Schroer, 2014). Src and FAK are increased activity of MMP9 in fibroblasts in 3D in vitro systems, but directly activated by β3 and β1 integrins, respectively, and the these effects are reduced or reversed by the addition of collagen-1 to effects of the adhesions they mediate on MyoFB differentiation is the matrix (Cushing et al., 2005; Zhang et al., 2014a). Another mirrored in this crosstalk (Fig. 3). Specifically, Src promotes α- fibronectin-binding integrin, α8β1, is specifically enhanced in SMA expression, whereas FAK can both inhibit and promote the MyoFBs within fibrotic hearts (Bouzeghrane et al., 2004). Overall, expression of MyoFB markers. Compounding this crosstalk, β1 integrin expression is increased in fibrotic and hypertrophic integrin signaling also regulates the expression and activation of hearts and, moreover, fibroblast-specific deletion of β1 integrin growth factors. For example, β1 integrins regulate the expression of causes insufficient wound healing and reduced MyoFB the angiotensinogen gene (AGT) in CFs through p38 signaling in differentiation in a dermal model (Burgess et al., 2002; Liu and response to mechanical stretch. This effect is mediated by activation Leask, 2013; Liu et al., 2010). Taken together, these data suggest of Rac1 and inhibition of RhoA, intracellular kinases involved in that, despite their protective effect on CMs, β1 integrins exert a pro- cytoskeletal organization and contraction (Verma et al., 2011). fibrotic effect in heart fibroblasts during disease. In addition to such ‘outside-in’ signaling, integrins can also Another important integrin type that has been directly linked to participate in ‘inside-out’ signaling, by which FA proteins can mechanotransduction and MyoFB differentiation are the β3 modify the intracellular domain of integrin subunits to activate them, integrins (Roca-Cusachs et al., 2009). β3 integrins recognize the resulting in stronger adhesions to the ECM (Baker and Zaman, 2010; RGD peptide sequence that is found in fibronectin, collagen and Katsumi et al., 2004; Michael et al., 2009). Vinculin is a force- vitronectin, and are highly expressed during development and sensitive FA protein that is recruited to FAs in a tension-dependent disease (Luo et al., 2014). In accordance with this, AVICs cultured manner, and has been shown to strengthen and stabilize the FAwhen on RGD-coated substrates express increased levels of MyoFB held under high tension, allowing for greater transmission of force markers and of αvβ3 integrins and are more prone to calcification (Carisey et al., 2013; Grashoff et al., 2010; Humphries et al., 2007). Journal of Cell Science

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Collagen/ Homotypic GSK-3β by TGF-β1 or Wnt allows for an accumulation of β-catenin Extracellular input RGD TGF-β1 FGF Wnt fibronectin cadherins in the cytoplasm and its subsequent translocation to the nucleus where it activates its target genes (Caraci et al., 2008). In addition to Membrane-bound β3-integrin TGFβR β1-integrin FGFR Frizzled Cadherins β receptor signaling through -catenin, several recent studies have characterized a force-dependent interaction between vinculin and Adhesion-associated α-catenin, another protein linking AJs to the cytoskeleton Src FAK β-catenin protein GSK-3β (Huveneers et al., 2012; le Duc et al., 2010; Yao et al., 2014; Yonemura et al., 2010). Vinculin is recruited to AJs in a force- Intracellular signaling p38 ERK β-catenin α protein dependent manner by -catenin, which results in strengthening of the adhesion and increased cell contractility (Barry et al., 2014; Yao

Myofibroblast marker α-SMA OB-cadherin et al., 2014). This result indicates that integrins and cadherins might share a common mechano-sensitive mechanism, in which vinculin- induced stabilization of either FAs or AJs affects downstream Functional result Generation of Transmission of signaling pathways. p120-catenin is also involved in stabilizing AJs high intracellular force high intercellular force and increases activation of Rac1 when it is bound to either N- or Key Activates/promotes Inhibits Binds to/activates Translocates OB-cadherin, leading to increased expression of mesencymal cadherins (Yanagisawa and Anastasiadis, 2006). Fig. 3. Crosstalk between growth factor and adhesion protein signaling. N-cadherin is the classic cell–cell adhesion protein that is Illustrated here are the intracellular signaling pathways and crosstalk between expressed by quiescent fibroblasts and CMs in the heart. N-cadherin integrin, growth factor and cadherin signaling. β3 integrin signals through the expression and localization at cell–cell contacts is associated with β α same intracelullar pathways as TGF- 1, which increase expression of -SMA increased stability of β-catenin and decreased expression of α-SMA and cellular contractility. β1 integrin shares pathways with both TGF-β1 and FGF, which may in part explain its context-dependent effects on MyoFB (Xu et al., 2012). CFs form N-cadherin-mediated interactions with differentiation. Cadherins regulate the availability of β-catenin to participate in CMs, which influences CM structure and contractility during Wnt signaling, a pathway that promotes cadherin switching and fibrosis. Both development, normal function and disease (Chopra et al., 2011; integrins and cadherins mechanically link the ECM and the actin cytoskeleton, Kudo-Sakamoto et al., 2014; Vreeker et al., 2014). In a recent study, and are sensitive to increases in applied force from extracellular or intracellular N-cadherin bonds between CFs and CMs were shown to α sources; therefore, increased expression of -SMA in MyoFBs forms a positive dynamically deform CM membranes in response to MyoFB feedback loop to further promote adhesion stability and associated signaling pathways. contraction and induce a measurable slowing of CM conduction velocity (Thompson et al., 2014). This study demonstrates a mechanical signal from MyoFBs that affects the electrophysiology Active vinculin can also recruit FA proteins including FAK to of CMs and may contribute to risk of and other potentiate further signaling (Carisey et al., 2013). Intracellular force cardiovascular conditions that are associated with low conduction generated by actin stress fibers can be transmitted through these velocity (King et al., 2013). adhesions to the ECM to alter its organization. Certain growth Whereas quiescent AVICs and CFs express N-cadherins, factors, including TGF-β1, are secreted into the ECM in an inactive during injury and disease these cells differentiate into MyoFBs; form, but αvβ3 integrins can activate such latent TGF-β1 trough this process is characterized by expression of OB-cadherin, which ECM tension, thereby further promoting MyoFB differentiation and has recently garnered much interest as a mechanosensitive fibrosis (Hinz, 2013; Sarrazy et al., 2014; Wipff et al., 2007). MyoFB regulator of inflammation and fibrotic disease (see Box 2). differentiation results in an increase in α-SMA stress fibers, which During differentiation of MyoFBs, TGF-β1 signaling induces an can strengthen and increase the activation of integrins in FAs, increase in OB-cadherin concurrently with a decrease in the forming a positive mechanical feedback loop. The resulting increase expression of N-cadherin (Hinz et al., 2004). Within cell–cell in cellular tension can then be transmitted to neighboring cells adhesions, OB-cadherin is able to withstand significantly higher through cadherin-rich adherens junctions (AJs) as discussed below. forces than N-cadherin, which allows for stronger matrix contraction and the transmission of higher intracellular tension Cadherins sense intercellular forces (Hinz et al., 2004; Hutcheson et al., 2013; Pittet et al., 2008). OB- Cadherins mediate mechanically induced signaling between cells cadherin is highly expressed in diseased heart valves in both VECs through AJs, which link cadherins to the cytoskeleton as described and VICs, and has been implicated in the development of calcified in a recent review (Leckband and de Rooij, 2014). Differential nodules in the aortic valve in CAVD through increased cadherin expression is a main feature of cell differentiation and transmission of intracellular force (Hutcheson et al., 2013; Wang function during disease, and this ‘cadherin switching’ is mediated in et al., 2014a; Zhou et al., 2013). It is also expressed in CFs, but the part by signaling downstream of cadherins (Table 2). β-catenin is functional significance of this expression in the context of cardiac present in AJs, and is intimately involved in crosstalk between fibrosis and wound healing has not been studied. Given the known cadherins, canonical Wnt and TGF-β1 signaling (Fig. 3). Molecular rolesofOB-cadherinininflammationandfibrosisinjoint signaling or mechanical perturbations that disrupt cadherin– connective tissue and lungs, OB-cadherin is likely to have an cadherin interactions between cells have been shown to release important role in myocardial remodeling (Chang et al., 2011; β-catenin into the cytoplasm where it acts in concert with growth Schneider et al., 2012). factor signaling to promote a mesenchymal cell phenotype In addition to functional mechanical roles, OB-cadherin can also (Heuberger and Birchmeier, 2010). For example, exposure to potentiate downstream signals to control cell behavior. Although the oscillatory fluid flow triggers an N-cadherin-mediated release of downstream signaling of OB-cadherin is still relatively β-catenin and a subsequent osteogenic differentiation in stem cells uncharacterized, recent studies have shown significant crosstalk (Arnsdorf et al., 2009). Typically, free cytoplasmic β-catenin is with MyoFB regulatory signals. For instance, it was found that OB- rapidly marked for degradation by GSK-3β; however, inhibition of cadherin engagement promotes differentiation of smooth muscle Journal of Cell Science

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in a context-dependent manner. MyoFB differentiation potentiated Box 2. OB-cadherin – 20 years of insight by integrin and cadherin signaling can contribute to further ECM OB-cadherin (also known as cadherin 11) was first described in 1994 in remodeling and tissue stiffening, which – in turn – enhances the context of osteogenesis and bone development, but recent work has mechanotransductive signaling in nearby cells (Fig. 2). Such a shown its importance in a variety of tissues (Okazaki, 1994). Over 100 system of positive feedback loops can then perpetuate the studies have been published in the past few years that examine the role progression of fibrotic disease. Prolonged inflammatory responses of OB-cadherin in cancer and fibroblast-mediated disease. In the context of cancer, OB-cadherin expression is associated with increased further compound the problem by initiating and propagating ECM migration and metastasis, especially metastasis to bone (Deng et al., remodeling. Both integrin- and OB-cadherin-mediated signaling 2013). As cell differentiation and migration in cancer and inflammatory have been implicated in inflammation, and strategies aimed at disease show some similarities, OB-cadherin has been proposed as a blocking the functions of these adhesion molecules have shown common, possibly therapeutic, target for both types of disease (Assefnia preliminary success in limiting inflammation-triggered maladaptive et al., 2014). OB-cadherin plays an important role in inflammation in remodeling (Chang et al., 2011; Krieglstein et al., 2002; Lee et al., rheumatoid arthritis; it stimulates synovial fibroblasts to release proinflammatory cytokines upon cadherin engagement (Chang et al., 2007; Schneider et al., 2012). The overlapping and integrated 2011; Ding et al., 2014). Celecoxib, a pharmacological inhibitor of Cox-2 networks of chemical and mechanical signals that regulate the that inhibits inflammation during rheumatoid arthritis, has been shown to progression of fibrotic heart disease will continue to challenge the bind to OB-cadherin (Assefnia et al., 2014). Inflammation often leads development of promising therapies. Nevertheless, the crucial role to fibrosis, which is mediated by activated MyoFBs. In this context, OB- of cadherins and integrins in both chemical and mechanical cadherin has been implicated in the progression of dermal and signaling makes them excellent potential targets for therapy and pulmonary fibrosis, and suggested to promote MyoFB differentiation through interaction with β-catenin (Kim et al., 2009; Schneider et al., future study (Agarwal, 2014). 2012; Wu et al., 2014). In accordance with this, injection of a function- blocking antibody against OB-cadherin improves bleomycin-induced Competing interests The authors declare no competing or financial interests. dermal and pulmonary fibrosis, but its effect has not yet been investigated in other organ systems (Schneider et al., 2012; Wu et al., 2014). Taken together, these studies indicate that OB-cadherin is a Funding A.K.S. is supported as a National Science Foundation Graduate Research Fellow. promising target for further research in the field of fibrotic disease. W.D.M. is supported by the National Science Foundation [grant number: 1055384] and National Institutes of Health [grant numbers: HL090407 and HL115103]. Deposited in PMC for release after 12 months. cells through TGFβRII-mediated pathways, and increases the α References expression of -SMA and OB-cadherin through SRF activation Afek, A., Shoenfeld, Y., Manor, R., Goldberg, I., Ziporen, L., George, J., Polak- (Alimperti et al., 2014). Similarly, blocking OB-cadherin or Charcon, S., Amigo, M. C., Garcia-Torres, R., Segal, R. et al. (1999). Increased N-cadherin engagement reduces the expression of MyoFB endothelial cell expression of alpha3beta1 integrin in cardiac valvulopathy in the primary (Hughes) and secondary antiphospholipid syndrome. Lupus 8, 502-507. markers in diseased dermal fibroblasts, which express higher Agarwal, S. K. (2014). Integrins and cadherins as therapeutic targets in fibrosis. basal levels of OB-cadherin and α-SMA compared with healthy Front. Pharmacol. 5, 131. fibroblasts (Verhoekx et al., 2013). Overexpression of OB-cadherin Aisagbonhi, O., Rai, M., Ryzhov, S., Atria, N., Feoktistov, I. and Hatzopoulos, in fibroblasts has also been shown to increase expression of Wnt and A. K. (2011). Experimental myocardial infarction triggers canonical Wnt signaling β and endothelial-to-mesenchymal transition. Dis. Model. 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