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Preventive Medicine
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Age related changes of the extracellular matrix and stem cell maintenance
Andreas Kurtz a,b,⁎,1, Su-Jun Oh a
a College of Veterinary Medicine, Seoul National University, Seoul, Republic of Koreab Berlin-Brandenburg Center for Regenerative Therapies, Berlin, Germany
⁎ Corresponding author at: College of VeterinaryMedi599 Gwanangno, Gwanak-Gu, Seoul 151-742, Republic o
Aging is characterized by reduced tissue and organ function, regenerative capacity, and accompanied by a de-crease in tissue resident stem cell numbers and a loss of potency. The impact of aging on stem cell popula-tions differs between tissues and depends on a number of non cell-intrinsic factors, including systemicchanges associated with immune system alterations, as well as senescence related changes of the localcytoarchitecture. The latter has been studied in the context of environmental niche properties required forstem cell maintenance. Here, we will discuss the impact of the extracellular matrix (ECM) on stem cell main-tenance, its changes during aging and its significance for stem cell therapy. We provide an overview on ECMcomponents and examples of age associated remodeling of the cytoarchitecture. The interaction of stem cellswith the ECM will be described and the importance of an intact and hospitable ECM for stem cell mainte-nance, differentiation and stem cell initiated tissue repair outlined. It is concluded that a remodeled ECMdue to age related inflammation, fibrosis or oxidative stress provides an inadequate environment for endog-enous regeneration or stem cell therapies. Means to provide adequate ECM for stem cell therapies and endog-enous regeneration and the potential of antioxidants to prevent ECM damage and promote its repair andsubsequently support regeneration are discussed.
Organs and tissues undergo distinct physiological and structuralchanges during aging, which can be observed in different organs atvariable times of onset, progression and degree and in tissues withboth, high and low rates of cell renewal. While early signs of senes-cence start in skin and brain at around 20–30 years of age, in kidneyand liver these are usually only seen after about 60–70 years of ageor even later while most tissues start to show symptoms of aging at
cine, Seoul National University,f Korea. Fax: +82 2 8801275.
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about 40 to 50 years, for example the musculoskeletal, cardiovascularand digestive systems. Hallmarks of aging include loss of cell mass,which is most obviously reflected in decreased musculoskeletal mo-bility with loss of muscle and bone mass (sarcopenia and osteoporo-sis, respectively), and thinning and reduced elasticity of skin(wrinkling). In other tissues, such as the brain, liver and kidneys,cell numbers and tissue mass remain rather stable, although a re-duced stem cell and neurogenesis potency are seen, for example inthe brain (Lazarov et al., 2010). Importantly, the thymus and the he-matopoietic system exhibit typical age related changes, reflected inaltered immune-cell profiles including an increased CD8+/CD4+ T-cell ratio, anemia and myeloid cell accumulation in bone marrow(Beerman et al., 2010; Sahin and Depinho, 2010). The age dependentfunctional deterioration of the immune system is associated with an
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increase in autoimmune responses and inflammation in many organs,including neural tissues (multiple sclerosis and presenile dementia),the cardiovascular system (atherosclerosis), synovial tissue (rheuma-toid arthritis), and the pancreas (diabetes mellitus) (Geokas et al.,1990; Klein, 1982; Sell et al., 1996).
Loss of organ mass during aging is accompanied by profoundchanges in the cytoarchitecture of tissues and organs, which havebeen studied most intensively in the bone marrow and skin(Lepperdinger, 2011; Robert et al., 2009a, 2009b), but which arealso present in other tissues. At least partially, changes in thecytoarchitecture are linked to injury and repair processes leading tofibrosis, scar formation, cysts and vascular changes. Cytoarchitecturalchanges and the underlying alterations in extracellular matrix (ECM)composition and structure have profound impact on the stem cellniche and subsequently on stem cell viability, differentiation andplasticity.
Exracellular matrix components and function
The ECM provides a structural framework that is essential for tis-sue growth, remodeling and maintenance, stabilizes tissue structuresand provides needed elasticity. It is a major communication layer be-tween the environment and cells through provision of attachment,migration, survival and functional clues for cells. Hence tissue specificvariability of the ECM determines the cell composition and organ-typical arraignment of cells. The instructive power of the ECM wasimpressively demonstrated in a number of experiments in whichorgans were de-cellularized, leaving only the ECM intact. If theECM-scaffolds from rat heart or lungs were re-populated withisolated heart or lung cells, respectively, functional organs were re-established (Petersen et al., 2010; Ott et al., 2008). The use of decellu-larized ECM as a scaffold for organ specific cells has been expandedto other organs, such as liver, kidney, pancreas and intestine(Baptista et al., 2009; Uygun et al., 2010; Liu et al., 2009; Ross et al.,2009). Moreover, when stem cells are seeded on these natural ECMscaffolds, they are instructed to differentiate into cell types specificfor the source tissue of the ECM and arrange into appropriate func-tional units (Nakayama et al., 2010). This technology has the potentialto offer readily available organ-like units for drug discovery applica-tions and for transplantation.
The capacity to communicate instructive clues to stem cells is di-minished when ECM from injured and senescent tissues is used.Damaging to the ECM are processes that include age-dependent mod-ifications of matrix biosynthesis, postsynthetic modifications of ex-tracellular matrix, and modifications of cell–matrix interactions.Important triggers of ECM-aging include imbalanced proteolytic deg-radation and the release of free radicals (Korpos et al., 2009). Thesecan principally act on any component of the ECM, which is composedof a complex mesh of structural molecules, including elastins, colla-gens, proteoglycans and several glycoproteins that harbor and pre-sent an organ specific array of multiple growth factors, cytokinesand receptor ligands required to mediate cell attachment, migrationand function.
Elastic fibers are a critical component of many tissues, allowingexpansion and contraction and the ECM to function as a shape mem-ory scaffold. Elastic fibers are composite structures with a cross-linked elastin core and an outer layer of fibrillin microfibrils. Whileelastin stores energy, fibrillin microfibrils mediate cell signaling,maintain tissue homeostasis via transforming growth factor beta(TGFβ) sequestration and potentially act to reinforce the elasticfiber (Sprenger et al., 2010; Korpos et al., 2009). The specific mo-lecular structure and elastic fiber stability pre-dispose these ECMcomponents to the accumulation of damage in aging tissues, mostvisible in those with high elastic properties like skin, lung andvasculature. The degradation of elastic fibers is catalyzed by cellularelastases, a prominent process in atherosclerosis, emphysema and
skin aging. Proteolytic fragments of fibronectin and of elastic fiberswere shown to produce noxious effects (Robert, 1998). For example,the presence of saturating concentrations of elastin peptides in thecirculation results in a chronic overstimulation of its receptor withsustained free radical and lytic enzyme production (Kuge et al.,2010). Loss of elasticity is accompanied by a reduced capacity tointeract with cells, which further increases cellular autophagy andsenescence.
Deterioration of ECM in the skin is largely caused by irreversible de-struction of fibrillar collagen, the major structural protein in connectivetissue. Matrix metalloproteinase-1 (MMP-1) expression by dermal fi-broblasts significantly increases with age and causes fragmentationand disorganization of collagen fibrils. Other factors that are regulatedwith age and that modulate the interaction of cells with the ECM-collagens are TGFß, integrins (e.g. alpha2beta1, which itself interactswithMMP1), and reactive oxygen species (Urtasun et al., 2008). Severaldifferentially expressed geneswere identified for skin aging bymicroar-ray analysis, including latent TGFβ-binding protein (LTBP)-2, whichwas up-regulated with age, LTBP3 and the lysyl oxidase-like enzyme(LOXL1), which were both down-regulated with age (Langton et al.,2011).
As a general mechanism, subtle imbalances in local protease andmatrix protein levels can initiate a self-amplifying process leading to agradual weakening of cell-ECM interactions. Communication betweenECM and the cellular genome directly impacts the characteristics andfunctions of tissue resident and tissue infiltrating stem cells (Hynes,2009) through molecular mediators including transmembrane integrinreceptors, focal adhesion kinase, the structural glycoproteins fibronec-tin, laminin and elastonectin and other ECM macromolecules such ascollagen, proteoglycans and elastin and the matricellular componentsthrombospondin 1 (TSP1), osteonectin and the nonsulfated glycosami-noglycan hyaluronuic acid. Many cell surface adhesion-, ECM-, and sig-naling-proteins (e.g. E-cadherin, catenin, CD44, MMP-9 and caveolin-1)are known to be absent or reduced following altered gene promoter-methylation, a process associated with senescence (Patra and Bettuzzi,2007; Sprenger et al., 2010; Fisher et al., 2008).
Senescent cells alter the ECM directly through changes in structur-al proteins, or indirectly through altered growth factor accessibilityand responsiveness (Sprenger et al., 2010). Collagens and lamininsand the enzymes that regulate their turnover, including MMPs andTIMPs, are reciprocally regulated during aging (Zhang et al., 2003;Bavik et al., 2006; Liu and Hornsby, 2007; Sprenger et al., 2008). Forexample, senescent cells often overexpress only specific collagens(e.g. A2) and laminin (e.g. alpha 4) chains and simultaneously over-express MMP activity and degradation processes (Liu and Hornsby,2007; Sprenger et al., 2008). Collagen content is not only regulatedby genetic and epigenetic mechanisms. The reduced levels of TGF-ßcontribute also in lower collagen I synthesis, while at the sametime, elevated MMP1 activity mediates increased collagen I degrada-tion (Ashcroft et al., 1997).
Hyaluronic acid enhances cell migration by influencing ECM po-rosity by binding to collagen I, versican and fibrin and via CD44(selectin)-dependent mechanisms that may in turn modulate the ac-tivities of metalloproteinases (Hayen et al., 1999; Annabi et al., 2004;Ricciardelli et al., 2007). Hyaluronic acid shows aberrant organizationand reduced expression by aged cells, at least in response to injury(Meyer and Stern, 1994; Ghersetich et al., 1994; Robert et al., 2009a,2009b). Like collagen, accumulation of hyaluronic acid can also be ob-served in aged organs and is likely due to longer half life and reducedmetabolism (Robert et al., 2009a, 2009b).
Laminins are glycoproteins composed of homologous a, b and cchains and are the main constituents of basal membranes and impor-tant modulators of cell function, especially for endothelial and epithe-lial cells. Laminin composition and expression may change with ageas has been shown in senescent fibroblasts, which show increased ex-pression of laminin alpha4beta1 (Bavik et al., 2006; Luo et al., 2002).
Table 1Differentially expressed proteins in young (passage 3) and senescent (passage 12) ad-ipose tissue derived mesenchymal stem cells (AdMSC). Proteins related to extracellularmatrix (ECM) remodeling are either up or downregulated. (n=3).
Protein Young>senescent MSC
ECM components
Collagen alpha-3(V) chain
Laminin
Fibronectin
Collagen 1
TGFß
MMP2
LOXL1
Potential cell–ECM interaction related
Keratin, type I cytoskeletal 9
Tropomyosin alpha-1 chain
PRELI domain-containing protein 2
Vimentin
Annexin A11
Cystatin-B
Galectin-3
Syntenin-1
Lamin-A/C
Oxidation associatedElectron transfer flavoproteinsubunit alpha, mitochondrial
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In general, changes in laminin expression and composition are oftenaccompanied by tissue repair and might not be strongly associatedwith cellular aging.
Integrins are transmembrane heterodimers that act as ECM recep-tors for cells. Although some transmembrane proteoglycans alsofunction as co-receptors for matrix components, the principal recep-tors on cells are integrins, which bind at low affinity to ECM proteinssuch as collagens, laminins and fibronectin. Age-related reduction inintegrin alpha1beta1 and alpha2beta1-collagen binding results inless robust cell adhesion and migration and subsequently weakenedattachment and function of the cells, as well as infiltration of othercell types, including endothelial cells (Fisher et al., 2008; Reed et al.,2001). Furthermore, it has been shown that the laminin compositionof the basement membrane modulates infiltration of some immunecells (Wu et al., 2009).
Thrombospondin 1 is one of several members of large matricellu-lar trimeric glycoproteins with aging associated increased expressionlevels. The reduced blood vessel formation during tissue repair inthe elderly is at least partially due to upregulated thrombospondin1 (TSP1), a strong inhibitor of vasculogenesis and angiogenesis(Naumov et al., 2006; Naumov et al., 2006; Kang et al., 2001; Agahet al., 2004; Sadoun and Reed, 2003) in addition to its regulatory ef-fect on metalloproteinase activities.
ECM components exert effects beyond their local environment sincethey or their proteolytic fragments are released in the circulation, eitherbound to lipoproteins or as fee peptides, promoting cell migration, an-giogenesis, inflammation and tissue remodeling. Fibronectin and MMPplasma concentrations, for example, increase with age exponentially.Furthermore, ECM bound growth factors are released into the systemby age related changes of binding properties of ECM components(Hayashida, 2010; Chiodoni et al., 2010; Alexi et al., 2011; Kim et al.,2011).
1,3 1
0
3,7
0,80
0,5
2,8
1,8
7,7
5,5
2,6
0,8
2,8
0
1
2
3
4
5
6
7
8
9
Oct4 TGFß Sox2
Rel
ativ
e E
xpre
ssio
n
Nanog LOXL1 Laminin Nestin1
Senescent MSC + ECM Senescent MSC
B C
A
Fig. 1. (A) Expression of potency markers and extracellular matrix (ECM) molecules insenescent adipose tissue derived mesenchymal stem cells (AdMSC) (passage 12) andsenescent AdMSC that have been seeded on ECM derived from young MSC grown inspheres (passage 3). ECM from spheres was obtained by treatment with 1% TritonX100.(B) Mesenchymal stem cell (MSC)-sphere derived extracellular matrix (ECM)and (C) MSC sphere.
Stem cells and the extracellular matrix
Age related changes in ECM not only mediate cell proliferation,differentiation, inflammation and apoptosis, but they also impact onrecruitment, differentiation, and functional integration of stem andtissue specific progenitor cells. We have analyzed the interplay be-tween matrix formation and stem cell senescence in a 3-dimensional(3D) MSC model. When MSC are grown in 3D as spheres, they secretean ECM that is significantly different in protein composition whencompared to adherent cell cultivation. The cells express higher levelsof pluripotency markers and have a higher differentiation capacityand immunomodulatory potency (Fig. 1). Moreover, when we com-pared the 3D-ECM from MSC of young and senescent MSC in this sys-tem, the protein composition was different in numerous proteins thathave been implicated in cell-ECM interaction as determined by prote-omics analysis, including collagens, laminin, TGF-ß and LOXL-1(Rodríguez et al., 2008; Pohlers et al., 2009). When the MSC from se-nescent adherent cultures were seeded on the ECM of young MSCspheres, expression of pluripotency markers increased and differenti-ation potency was improved (Table 1, Fig. 1). Although preliminary,these data strongly indicate that by modifying the ECM, the potencyof stem cells can be modulated and furthermore, that stem cells areable to autocrine establishment of an ECM that supports their viabil-ity. Similar data have been described for restoration of aged, senes-cent fibroblasts to an apparently youthful state by interacting withECM from young fibroblasts. Senescent fibroblasts and MSC expressgenes that are normally induced upon wounding, including genesthat remodel the ECM such as MMP1 and PAI2. Binding of the senes-cence associated zinc finger protein AIA1 to the MMP1-promoterseems also to play a role for this change in gene expression (Choiet al., 2011a, 2011b). The data indicate that gene expression patternsof fibroblasts and MSC can be partially reprogrammed by ECM-
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features. The specific nature of this interaction is still a matter ofresearch.
Mesenchymal to epithelial transition (MET) and the reversed pro-cess, mesenchymal to epithelial transition (EMT), are key cell transi-tion processes during development, tissue remodeling, fibrosis andtumorigenesis. The reprogramming of fibroblasts to induced pluripo-tent stem cells (iPSC) also involves an MET (Samavarchi-Tehraniet al., 2010). Both, EMT andMET are accompanied bymassive ECM re-building. EMT and MET are dependent on the interaction of cells withECM proteins, which trigger intracellular reprogramming events. Inturn, reprogrammed cells express a different ECM protein profile.ECM-resident proteins important for reprogramming include TGFß,bone morphogenic protein (BMP)-4, EGF and IGF binding proteins.BMPs directly stimulate stem cell proliferation and its reductionleads to a reversible decrease of stem cell populations in tissues(Thiery and Sleeman, 2006; Cannito et al., 2010). In MET, matricellu-lar remodeling is to a large degree triggered by cell-intrinsic factors.The role of cell-intrinsic senescence associated factors for tissue reju-venation has been demonstrated in an animal model in which lost tel-omerase activity has been re-established. Organs that showed signs ofsenescence because of experimentally induced lack of telomerase ac-tivity in a telomerase deficient murine model were rejuvenated by re-establishing telomerase expression (Jaskelioff et al., 2011). However,the degree to which cytoarchitectural changes of the ECM occurred inthis model was not investigated although reversal of degenerativeimmune function might have reduced inflammation and subsequentrestoration of ECM structures in this model.
Fibrosis and sclerosis associated ECM rebuilding are major causesfor deteriorating organ function and a barrier to endogenous regener-ation as well as stem cell based therapies. Increased collagen-I depo-sition, which is associated with fibrosis, is often noted in aged heartsas a response to hypertension (Gazoti et al., 2001; Lakatta, 2008). An-other example is the progressive thickening and hardening of arterialwalls as a result of fat deposits on their inner lining. This process iscaused partially by sub-endothelial retention of atherogenic lipopro-teins and associated with excessive accumulation of ECM componentssuch as hyaluronan. Accumulated hyaluran is a substrate for mono-cytes and lymphocytes and may trigger early inflammatory reaction.Although not due to aging per se, these observations show that mul-tiple ECM changes inhibit successful regeneration and underscore theneed to establish appropriate ECM structures for successful endoge-nous regeneration and cell therapy.
Integration of transplanted stem cells for regenerative therapy,their correct tissue specific differentiation, adhesion and migrationare general requirements for stem cell based therapies. Thus to deter-mine whether transplanted stem cells are at all able to integrate intothe changed cytoarchitectural environment of tissues in older pa-tients without losing their regenerative potency, or whether thesestem cells are able to support the provision of endogenous cells to se-nescent tissues, and/or to rebuild the deteriorated cytoarchitectureare clinically highly relevant areas of research. The role of the agedECM and in general of cytoarchitectural changes for vitality and inte-gration of stem cells is not well understood. Clinical experience indi-cates that while adult stem cells do home and remain in damagedtissues, they are usually not able to survive in significant numbers ei-ther as stem cells or as differentiated progeny. Neither do they sup-port rebuilding the damaged tissue microenvironment sufficientlyfor enabling their intrinsic properties of stemness, differentiationand self-renewal. There are notable exceptions, however. Hematopoi-etic stem cells (HSC) repopulate the bone marrow niche, albeit at var-iable age dependent frequencies (Lepperdinger, 2011; Wagner et al.,2008). Mesenchymal stem cells (MSC) have been shown to rebuildmesenchymal tissues such as bone, cartilage and muscle in vivo de-spite damaged cytoarchitectures and inflamed microenvironments(Nöth et al., 2010; Steinert et al., 2008; Roberts et al., 2008). In mostclinical applications, however, MSC appear to act rather indirectly
by modulating the immune system, reducing inflammation and by in-ducing vasculogenesis (Petrie Aronin and Tuan, 2010; Singer andCaplan, 2011; Lasala and Minguell, 2011). Nevertheless, MSC integra-tion and MSC induced tissue repair seem to be possible in mesenchy-mal tissues such as bone, cartilage and adipose tissue, although withvariable efficacy and depending on the hospitality of the stem cellniche (Augello et al., 2010). Despite the in vitro capability of MSC todifferentiate into neurons, cardiomyocytes, kidney cells and hepato-cytes (Puglisi et al., 2011; Choi et al., 2011a, 2011b; Zavan et al.,2010; Choi et al., 2010a, 2010b), MSC and their tissue specific deriva-tives do not usually integrate into the appropriate target tissues anddo not differentiate there into the relevant tissue specific non-mesenchymal cell types. This lack of integration may be due to the in-appropriateness of the tissue specific microenvironment to providethe necessary clues that can be provided in vitro, and the aged anddiseased status of this environment, which is often characterized byinflammation and fibrotic scar formation (Chen, 2010). Engraftmentefficacy may also depend on the mode of cell delivery although pre-clinical and clinical trial experience shows that systemic or local in-jection of MSC does not differ in engraftment rates in most organs(Kurtz, 2008).
Modifying ECM for stem cell therapy
It would be of high importance for regenerative therapies to de-velop methods to adjust the endogenous microenvironment inorder to create a more inhabitable status for stem cells and providethe missing but essential clues for integration, survival and correctdifferentiation. Re-establishment of a proper extracellular matrixmay not only provide an environment for transplanted stem cells,but also promote endogenous stem cell re-population and viability.Another complementing option is the exogenous provision of an ap-propriate matrix structure or scaffold for cells in aged and diseasedtissues before or in parallel with cell transplantation (Carletti et al.,2011; Spadaccio et al., 2010; Lee et al., 2011; Orlando et al., 2011).
With regard to active conditioning of the ECM and cytoarchitec-ture, the directed modification of single or multiple matrix compo-nents is currently be pursued. One example is the hydrolysis ofhyaluran in artherosclerotic lesion formation that may activate theprovision of intimal regions by cells and retardation of arterial patho-genesis. A similar approach is the provision of additional and con-trolled matrix materials, with or without cells, directly into thetarget tissue. A relevant example is the injection of fibrinogen hydro-gel or polyethyleneglycol (PEG)ylated fibrinogen biopolymers to-gether with human stem cell derived cardiomyocytes into infractedadult rat hearts, where cell survival increased and overall cardiacfunction significantly improved after 30 days (Williams et al., 2005;Dikovsky et al., 2006). These injectable biomaterials can adjust tothe cytoskeletal geometry and when injected in scar tissue providean adequate contact between ECM and cells.
Most frequently applied experimentally is the ex vivo pre-formation of matricellular complexes. An example is the applicationof hepato-cellularized sheets on an ECM. This technique allows thestacking of several sheets, which engraft efficiently into liver tissue(Yang et al., 2007; Ohashi et al., 2007). Similar approaches are takenwith decellularized organ scaffolds from kidney, pancreas and intes-tine (Baptista et al., 2009). In the kidney, renal branching morpho-genesis and tubulogenesis was achieved by seeding cells from bothuretic bud and metanephric mesenchyme in a three-dimensionalECM gel in the presence of growth factors (Rosines et al., 2010;Steer and Nigam, 2004). Clinically successful was the repair of a tra-cheobronchial defect, which was fixed by implanting an acellularbioartificial airway patch (Macchiarini et al., 2004; Walles et al.,2005). In a further development, a decellularized trachea from adonor was seeded with autologous stem cell-derived chondrocytesand epithelial cells and cultivated in a bioreactor for several days
Fig. 2. Possible interactions of anti-oxidants on extracellular matrix (ECM) related sub-strates. Cells produce and regulate the ECM by modulating secretion of ECM structuralproteins and functional proteins (e.g. elastin, collagens or glucosaminoglycans (GAGs)and laminin or fibronectin), release and expression of ECM-dependent growth factorsand their receptors, such as transforming growth factor-beta (TGFß), fibroblast growthfactors (FGFs) or through the secretion of ECM remodeling proteins such as matrixmetalloproteinases (MMPs) or lysyl oxidase-like (LOXL). Aging causes an imbalanceof the ECM–cell interaction through the production and accumulation of reactive oxy-gen species and oxidative stress. Antioxidant effects are provided by some of the ECMcomponents such as GAGs and other cell-defenses such as LOXL, but their preventivecapacity appears increasingly insufficient with age. The addition of small molecule an-tioxidants such as N-acetyl cystein (NAC) can reverse age related changes in the cellu-lar niche by reducing oxidative stress. The effect of other relevant antioxidants such asergothioneine or flavinoids on ECM protection and maintenance is still not sufficientlyinvestigated.
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before organotypic implantation. This method has been improved byusing intraoperative grafting of a donated cellularized trachea with-out ex vivo cultivation in a patient with congenital stenosis(Orlando et al., 2011; Jungebluth and Macchiarini, 2011). Replace-ment of the esophagus has been achieved in dogs. Here, an engi-neered PGA scaffold consisting of oral keratinocytes and fibroblastson a human amniotic membrane was seeded with smooth muscle tis-sue, rolled around a tube and following intra-abdominal maturationused to replace a segment of esophagus (Nakase et al., 2008). PGAhas astonishing properties as an ECM in tubular structures such asesophagus and intestine (Choi and Vacanti, 1997; Grikscheit et al.,2004), yet more complex and native structures are required forother tissues. In addition, a disadvantage of using allogenic or artifi-cial ECM is that the host's immune system may respond to the mate-rials (Badylak and Gilbert, 2008; Badylak, 2007; Babensee et al.,1998). Modifying the innate ECMmay avoid this complication. For ex-ample, corneal ECM used for corneal endothelium transplantationprovides an advantageous scaffold for corneal epithelial cells for itstransparency, stability in lack of immunogenicity (Grikscheit et al.,2004; Choi et al., 2010a, 2010b; Amano, 2003).
The use of anti-oxidants and anti-inflammatory agents to reducetwo of the major causes for ECM remodeling is an attractive alterna-tive to direct matrix intervention (Fig. 2). Redox mechanisms notonly control matrix metalloproteinases and thus influence thebalance between matrix deposition and proteolysis, but reactive oxy-gen species and inhibition of mitochondrial antioxidative enzymes
also promote fibrosis, epithelial-mesenchymal transition (EMT) andamyloid deposition (Kar et al., 2010; Kliment and Oury, 2010; Bai etal., 2011; Cabello-Verrugio et al., 2011; Chang et al., 2011). For exam-ple, antioxidant treatment and decreased oxygen tension had a directimpact on improving fitness of MSC when cultivated in ECM (Fan etal., 2011). Inhibition of lysyl oxidase-like 2, a matrix enzyme, resultedin a marked reduction of fibrosis, accompanied by a reduction in acti-vated fibroblasts and decreased TGF-beta signaling (Barry-Hamiltonet al., 2010 Sep). Matrix associated TGF-beta triggers one of themain signals for EMT for fibrosis initiation, and blockage of TGF-betaand angiotensin signaling as well as activation of BMP-7 pathwayscan reverse fibrosis (Strutz and Zeisberg, 2006; Cufí et al., 2010). An-tioxidants are powerful inhibitors of TGF-beta and angiotensin signal-ing and may thus provide means for ECM modeling for cellulartherapy (Kar et al., 2010; Foo et al., 2011; Liu and Gaston Pravia,2010; Rocha et al., 2010). Klotho, a protein has been associated withaging and that interacts with retinoic-acid-inducible gene 1 (RIG1)to inhibit expression of the pro-inflammatory cytokines IL-6 and IL-8. Klotho is a regulator of oxidative stress and thus may be a targetfor the reduction of ECM remodeling and inflammation (Liu et al.,2011 Mar; Tang et al., 2011). Many stem cells use a glycolytic path-way for energy production, providing a protective situation to avoidDNA damage. Upon differentiation, mitochondrial oxidative phos-phorylation becomes the dominant energy production pathway as aresult of a carefully regulated switch. If the oxidative stress-protect-ing forkhead box O (FOXO) or the ataxia–telangiectasia mutated(ATM) genes are knocked out in mice, hematopoetic stem cell num-bers and repopulation potency decreased due to excessive oxidativestress (Yalcin et al., 2008; Tothova et al., 2007; Miyamoto et al.,2007). Treatment with the antioxidant N-acetyl cysteine reducedthe redox microenvironment, which rescued the observed defects.These data further indicate that reduction of oxidative stress providesan environment that protects and regenerates ECM and is crucial forstem cell maintenance and function (Yalcin et al., 2008; Tothovaet al., 2007; Miyamoto et al., 2007; Ito et al., 2004; Mandal et al.,2011).
Conclusion
The interdependency between ECM and cells clearly extends tostem cells, which are highly dependent on ECM signaling for their vi-ability and function. From a cell-therapy point of view, stem cells re-quire a fitting ECM for integration and proper differentiation topromote regeneration and repair of the target tissues. In addition, dif-ferent stem cell types may require different ECM clues to provide theintended benefit. Since such a hospitable ECM is often not readilyavailable in aged and diseased organs, approaches for therapeuticECM modification need to be developed. These approaches will in-clude the direct intervention into the cytoarchitecture by matrixmodulating enzymes, through the establishment of a milieu that pro-motes ECM remodeling and reduces inflammatory events, for exam-ple through anti-oxidant supplementation. Alternatively, stem cellsmay be provided in a pre-established synthetic or biological scaffoldthat is sufficiently functionalized to mimic the appropriate matrix en-vironment and provides necessary instructive clues to stem cells. Fi-nally, improved survival of stem cells beyond a critical time mayprovide these cells with the opportunity to autologously generatean ECM and niche sufficient for viable integration.
Conflict of interest statementThe author declares no conflict of interest.
Acknowledgments
This work was supported by DFG grant GZ KU 851/3-1 LE 1428/3-1to A.K.
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