Tight junctions and the modulation of barrier function in disease

Tight junctions create a paracellular barrier in epithelial and endothelial cells protecting them from the external environment. Two different classes of integral membrane proteins constitute the tight junction strands in epithelial cells and endothelial cells, occludin and members of the claudin protein family. In addition, cytoplasmic scaffolding molecules associated with these junctions regulate diverse physiological processes like proliferation, cell polarity and regulated diffusion. In many diseases, disruption of this regulated barrier occurs. This review will briefly describe the molecular composition of the tight junctions and then present evidence of the link between tight junction dysfunction and disease.
Introduction
Tight junctions (TJ) (zonulae occludentes) form a continuous, circumferential, beltlike structure at the boundary between the apical and the basolateral membrane domains in epithelial and endothelial cells. By constituting a regulated diffusion barrier for the paracellular pathway, TJs establish separate compartments in multicellular organisms and are also crucial for the exchange of substances between the internal and external cellular environment by the expression of tissue-specific transport proteins and channels. The transmembrane proteins constituting the TJs are attached to the cytoskeleton, thereby linking cell–cell and cell–substratum adhesion sites. In addition, cytoplasmic plaque proteins constitute scaffolds for TJ assembly or are involved in the regulation of processes like transcription, proliferation and differentiation into a tissue-specific regulated diffusion barrier in physiology and development. Many excellent reviews have summarised data regarding the molecular composition and function of TJs (Ebnet 2008; Gonzalez-Mariscal et al. 2003; Matter and Balda 2003a; Mitic and Anderson 1998; Schneeberger and Lynch 2004; Tsukita and Furuse 1999; Zahraoui et al. 2000), so that the scope of the current review is focused on disturbances of TJ function in human diseases.
Structure of the tight junction
As the apicalmost part of the junctional complex (Farquhar and Palade 1963), the TJ forms a continuous, circumferential belt separating apical and basolateral plasma membrane domains, working as a barrier within the intercellular space and as a fence within the plasma membrane. In recent years, information on the molecular composition of TJs, in particular their transmembrane molecules, has accumulated, forming the basis of our current understanding of the structure and function of TJs in molecular terms (Fig. 1). The morphology of TJs has been intensively analysed by freeze–fracture electron microscopy (Staehelin 1973; Wolburg et al. 2003), where they appear as a set of continuous, anastomosing transmembraneous particle strands on the inner leaflet of the plasma membrane (P-face) with complementary vacant grooves on the outer leaflet (E-face) (Fig. 2). The number and complexity of ramification of the network of TJ strands depends on the cell type, in sum yielding differences in permeability barrier function between different tissues (Staehelin 1973). However, since a direct linear relationship between the complexity of the TJ strand network and the measured transepithelial electrical resistance (TER) could not unambiguousy be established for all cell types analysed, it was predicted that the strands might contain pores that fluctuate between open and closed conformations, suggesting that the TJ strands appear to be remarkably dynamic (Claude 1978).
Fig. 1Molecular composition of tight junctions. The transmembrane proteins occludin, the claudin(s) and junctional adhesion molecule-1 (JAM-1) constitute the barrier formed by TJs sealing the paracellular space. They appear to be interacting in a homophilic manner, and occludin seems to co-polymerase into claudin-based TJ strands. Claudins adhere with each other in a homotypic as well as a heterotypic manner. ZO-1, -2, and -3 bind the cytoplasmic tail of occludin and link the TJ to the actin cytoskeleton. Proteins of the ZO family can shuttle to the nucleus to influence transcriptional processes in cellular proliferation and differentiation. The ZO-proteins have also been shown to interact with claudins and provide molecular scaffolds for TJ assembly. Cingulin is a 140 kDa TJ plaque protein which assoicates with the actomyosin cytoskeleton. Its putative function is transduction of the mechanical force generated by the actomyosin cytoskeleton important for cellular differentiation. The Ras target AF-6 interacts with ZO-1 and serves as a. peripheral component of tight junctions in epithelial cells. Symplekin is a 126 kDa protein that occurs and probably functions in the nucleus as well as in the TJ plaques. Tyrosine phosphorylated Par3 regulates tight junction assembly and promotes cellular polarity by intracellular signalling. Localization of 7H6 TJ-associated antigen along the cell border of vascular endothelial cells has been shown to correlate with paracellular barrier function. Additional proteins have been localised to the TJs but a function has presently not been assigned. Moreover diverse signaling proteins are detected at the apical junctional complex but they are not uniquely confined to the TJFig. 2Freeze–fracture image of the rat intestinal epithelium. The freeze fracture electron micrograph showns the apical brush border, the intramembraneous particle strands of the TJs and the lateral cell surface. The replica shows a continous network of TJ strands. Adapted from (Achler et al. 1989) with permission from D. Drenckhahn, Universität Würzburg. Bar 0.5 μm
Occludin
Recent identification of the TJ-specific integral membrane proteins forwarded our understanding of TJ molecular composition in mammals: occludin (ca. 60 kDa) was identified as the first integral membrane protein localised at TJs in chicken (Furuse et al. 1993) and then also in mammals (Ando-Akatsuka et al. 1996). The occludin transmembrane domain spans the membrane four times with a short cytoplasmic N-terminus and an especially long corboxy-terminal cytoplasmic domain (Fig. 3a). Occludin localizes to the TJs, and its overexpression increases TER in mammalian epithelial cells (Balda et al. 1996). However, transfection of insect cells devoid of endogenous TJs with occludin cDNA surprisingly demonstrated that occludin is not by itself sufficient to form TJ strands but its expression merely produced focal homophilic adhesion sites (Furuse et al. 1996). Moreover, disruption of both occludin alleles in embryonic stem cells did not prevent the formation of an effective diffusion barrier and the polarisation of epithelial cells, as shown by freeze–fracture analysis and TER assessment (Saitou et al. 1998). It was thus concluded that occludin is not required for the formation of TJ strands. However, occludin appears to interact, directly or indirectly, with claudins and is recruited into the long strands formed by coexpression of claudin-1 and claudin-2 (Furuse et al. 1998b, 1999). Balda et al. (1996) however reported that overexpression of both, wild type and COOH-terminally truncated occludin overexpression in cultured MDCK epithelial cells induces not only a modest increase in TER but also significant increase in the flux of the non-charged compound FITC-dextran 4 kDa. Using freeze–fracture electron microscopy and immunocytochemistry, they were able to attribute this increase in paracellular permeability to an altered distribution and membrane topology between neighbouring cells.
Fig. 3Transmembrane proteins of the tight junction. a Murine occludin is a 521 amino acid protein. The first extracellular domain may be involved in cell–cell interaction containing a high tyrosine and glyine domain (triangles), acidic amino acids (orange-red), basic amino acids (green), neutral amino acids (cream). A second functional domain was assigned to the carboxy terminal 150 amino acids which appears to be responsible for the association of occludin with ZO-1. The occludin amino acid sequence is highly conserved between species (Ando-Akatsuka et al. 1996). b Claudins are four transmembrane domain proteins, containing two extracellular and one intracellular loop and a N- and C-terminal cytoplasmic domain. Claudins are a multigene family with so far 24 members identified in diverse species. In the first extracellular loop, claudins have a conserved common motif, GLWxxC(8–10aa)C (blue). Claudins further contain a PDZ-binding motif at the C terminus (orange) capable of bining to TJ plaque proteins like ZO-1. The claudins show a isotype-specific tissue expression pattern. c JAM-1 is an integral membrane protein expressed in endothelial and epithelial cells. Its extracellular domain can dimerize and bind homophilically. The intracellular domain (and in particular a PDZ-binding motif) enable JAM-1 to interact with structural and signaling proteins. JAM-1 is localized at the tight junctions of epithelial and endothelial cells and is involved in the regulation of junctional integrity and permeability. The polypeptide sequence of 299 amino acids has the typical feature observed in a type I integral membrane protein. A putative signal peptide may be cleaved between Leu23 and Val24 (pink), leaving 215 residues in the extracellular domain of the mature protein. A stretch of 17 hydrophobic residues (Ile 239 → Phe 255) presents a potential transmembrane region, and there are 45 residues in the cytoplasmic domain. The extracellular portion contains two domains with intrachain disulfide bonds typical of immunoglobulin-like loops of the V-type. Numerous PKC (blue) and Casein kinase II (yellow) phophorylation sites have been detected involved in platelet activation processes (Sobocka et al. 2000). Cys-43, -111, 146, -214 form dimer containing disulfide bridges and are labeled in black
The claudins
Searching for the proteins forming the structural backbone of the TJ strands, the Tsukita group re-examined the junctional fraction prepared from chicken liver and discovered the first members of the claudin family: two 22-kDa proteins, claudin-1 and claudin-2 (Furuse et al. 1998a).
The claudins also have four transmembrane domains, but do not show any sequence similarity to occludin (Fig. 3b). So far, 24 members of the claudin family have been identified in different species. Claudins appear to be expressed in a tissue-specific manner; some claudins are expressed only in specific cell types, e.g. claudin-5 is expressed primarily by vascular endothelial cells or claudin-11 appears to be expressed solely in oligodendrocytes and Sertoli cells (Morita et al. 1999). Moreover, most cell types express more than two claudin isoforms conferring different size and charge selectivity qualities by the amino acid sequence of their extracellular loops, and the combination of those isoforms results in cell- or tissue-type specific barrier function by “heteropolymerisation” into the resulting TJ strands (see below). As demonstrated by immunogold electron microscopy, there is accumulating evidence now that the claudins do constitute the TJ strands as observed by freeze–fracture electron microscopy (Tsukita et al. 2001). Exogenous expression of claudins in fibroblasts devoid of endogenous TJs resulted in the formation of TJ strands.
Summarizing existing data, there is clear evidence that the claudins constitute the backbone of TJ strands, while occludin, which itself cannot reconstitute such TJ strands, seems to play a permeability regulating role by incorporating itself into the claudin-based strands, a process, the mechanism of which however is not yet understood. The function(s) of occludin thus still remain to be elucidated.
Junction-associated adhesion molecules (JAMs)
A third class of integral membrane proteins comprises members of the immunoglobulin superfamily (Fig. 3c), and can be subdivided into a group consisting of JAM proteins and a group consisting of CAR, CLMP, ESAM and JAM-4 (Ebnet 2008; Ebnet et al. 2004). To date, at least three JAM isoforms have been described, which are expressed differentially in epithelial and endothelial cells, but also in cells devoid of TJ strands like leukocytes (D’Atri and Citi 2002). Except for JAM-1, little is known about the role of these proteins in TJ assembly and function. JAM-1, formerly F11 receptor (Sobocka et al. 2000) (Fig. 3c), may be involved in immune cell transmigration or cell adhesion (Bazzoni et al. 2000) but has not been localized to the junction strands. A putative PDZ binding motif in the cytoplasmic domain of JAM-1 has been identified, which has been able to interact with PDZ domains of ZO-1, AF-6, PAR-3 and MUPP1 (D’Atri and Citi 2002; Itoh et al. 2001). Moreover, JAM-1 has been shown to interact with cingulin. Thus, even though JAM-1 is not part of the TJ strands, it may well be involved in the propagation of signal cascades resulting from homophilic and heterophilic adhesion. A role for JAM-1 in the complex process of adhesion and transmigration of monocytes through endothelial cells has been demonstrated (Martin-Padura et al. 1998). As another function for the JAM proteins, a role in the assembly of TJs and regulation of paracellular permeability could be deciphered by ectopic transfection experiments (Cohen et al. 2001). Interaction of JAM-1 with occludin, so far not determined whether direct or indirect in nature, has been shown (Liu et al. 2000).
TJ plaque proteins and the coordination of signaling at the TJ
In addition to their prime function as a regulated permeability barrier in the paracellular pathway and as fence in the plane of the membrane, TJs play a pivotal role in organizing such diverse processes as morphogenesis, cell polarity, cell proliferation, and differentiation, which require the coordination of signals impinging on and emanating from the plasma membrane. Mounting evidence suggests that the cytosolic TJ plaque is one of the sites in which such signaling is coordinated (Ebnet 2008; Gonzalez-Mariscal et al. 2003; Schneeberger and Lynch 2004). Since identification in 1986 of zonula occludens (ZO)-1 as the first TJ-associated protein (Stevenson et al. 1986), almost 30 additional proteins have been described associated with the cytoplasmic aspect of TJs (Schneeberger and Lynch 2004). They can be grouped into two major categories: The first are the peripherally associated scaffolding proteins like ZO-1 (ZO-2, ZO-3, AF6, and cingulin) that appear to organize the transmembrane proteins and couple them to other cytoplasmic proteins and to actin microfilaments. The second are numerous “signaling” proteins (ZONAB, RhoA, RalA, and Raf-1) proposed to be involved in junction assembly, barrier regulation, gene transcription, and perhaps other, presently undefined pathways.
TJs as permeability barrier
One of the key functions of tight epithelia is the creation of a diffusion barrier by tight control of the transcellular and paracellular pathways. This is acomplished by asymmetrical distribution of transporters and channels for the transcellular route, and on the other side by regulation of diffusion through the paracellular space via integral TJ proteins (Turner 2006). Hereby, certain claudins participate in the formation of ion-selective pores: available data suggest that TJs on the one hand are permeable to small noncharged solutes up to a size of 0.43–0.45 nm in radius as shown by the use of the membrane-impermeant polyethylene glycol as a tracer, but investigations also showed that pores within the TJs are capable of discriminating between ion charge and size, which both are additionally dependent on concentration and pH (Schneeberger and Lynch 2004; van Bree et al. 1988).
The two extracellular loops of occludin consist solely of uncharched amino acid side chains (Fig. 3a) represented mainly by an unusually high content of tyrosine and glycine (Furuse et al. 1993) so that a direct role for occludin in conferring charge selectivity is doubtful. However, occludin could increase electrical resistance by interaction with the extracellular domains of the differently charged residues of the different claudins (see below), since ablation of occludin is not without consequence despite the fact that the protein does not appear to be required for TJ strand formation. Occludin-null mice express a complex phenotype, which is, however, not manifested by structural or functional TJ abnormalities: in occludin −/− mice, TJs were not affected morphologically, and electrophysiological barrier function of intestinal epithelium was normal. However, histopathological abnormalities were detected in several tissues, i.e., chronic inflammation and hyperplasia of the gastric epithelium, calcification in the brain, testicular atrophy, the loss of cytoplasmic granules in striated duct cells of the salivary gland as well as the thinning of the compact bone, pointing to complex functions of the occludin protein (Saitou et al. 2000).
Role of claudin proteins in formation of a TJ permeability barrier
The lack of charged amino acid residues within the extracellular loops of occludin segregates occludin from the candidates contributing directly to the formation of ion-selective pores. The different claudins whose extracellular loops span a wide range of pKion the contrary appear to be excellent candidates to mediate ion selectivity in TJ permeability (Fig. 3b). Major insight into the distinct charge selectivity of the claudins came from the work of Furuse et al. (2001), who could relate the different levels of resistance between MDCK I cells (TER > 10,000 Ωcm2) and MDCK II cells (TER <200 Ωcm2) to a differential claudin isoform expression, while MDCK I cells express claudin-1 and -4 but not claudin-2, MDCK II cells additionally express claudin-2, which seems to mediate higher ion permeability by pore formation/inducing strand breaks. Notably, transfection of the tight MDCK I with claudin-2 cDNA resulted in 20-fold decline in TER but not in the number of TJ strands.
A wide range of further transfection and mutagenesis experiments introducing or ablating different claudins has been performed to date, and been partially helpful to elucidate the conductance conferring properties of the resulting pores (Matter and Balda 2003b). These informative observations must however be viewed with some caution because transfection and site-directed mutagenesis may lead to more than a change in net charge as for example an altered molecular conformation, or altogether the introduction of an unusual claudin protein could lead to disturbance of strand organisation within native strands. By the construction of chimera, Colegio et al. (2003) were moreover able to attribute ion selectivity of the claudins to distinct stretches within the primary amino acid sequence of the extracellular loops leading to alterations in TER and charge selectivity in transfection experiments. Taken together, available data strongly suggest a key role for the extracellular loops of the diverse claudins in the formation of ion selective pores within the TJ strands.
Role of occludin in formation of a TJ permeability barrier
The role of occludin in TJs and a putative contribution to building up the TJ’s ion selectivity (the ion pores) is unclear; it has been demonstrated that overexpression of occludin in MDCK cells reduces ion permeability but in the same time was shown to increase the transepithelial flux of the uncharged compound mannitol (Balda et al. 1996). Further observations then implicated that occludin may be a target for receptor-initiated signalling involving the Rho family of GTPases, and that by an additional not-yet-understood mechanism, occludin phosphorylation may regulate TJ permeability independently of cytoskeletal activity (Hirase et al. 2001).
Some indirect evidence for a role of occludin in strengthening barrier properties arose from heterologous expression studies: when expressed in L-fibroblasts that lack endogenous cadherin-based adherens junctions and ZO-1, little adhesiveness was measured (Kubota et al. 1999). However, when overexpressed in fibroblasts, which do have well developed ZO-1-containing adherens-like junctions (NRK-fibroblasts), increased Ca2+-independent adhesion was observed, indicating that when interacting with ZO-1, occludin mediates adhesive functions (Van Itallie and Anderson 1997). Addition of neutralizing peptides of the peptide sequence of the second extracellular loop elicited competitive interference with cell–cell-adhesion in a dose dependent manner, implicating that this loop directly participates in adhesive interaction (Wong and Gumbiner 1997).
Relating epithelial malignancy to the loss of cell–cell-adhesion, it was demonstrated that overexpression of oncogenic Raf 1 in rat salivary gland epithelial cells (Pa4–Raf1) disrupts TJs and induces an oncogenic phenotype by downregulating the expression of occludin (Wang et al. 2005). Raf1-mediated transformation of Pa4–Raf1 cells was subsequently shown to induce transcriptional downregulation of occludin and forced re-expression of occludin rescued the polarized phenotype of epithelial cells (Wang et al. 2007). Interestingly, forced expression of an occludin mutant lacking the second extracellular loop did not rescue the epithelial phenotype in vitro nor did it prevent tumor growth in vivo. These results demonstrate that the TJ protein occludin has a potent inhibitory effect on the Raf1-mediated tumorigenesis, and the second loop of occludin appears to be required for suppression of Raf1-induced tumor growth, potentially by mediating cell–cell-adhesion as presented above contributing to contact inhibition of cell division.
TJs and human disease
Modulation of barrier function by cytokines
Many cytokines have been shown to influence epithelial and endothelial TJ function and the actin cytoskeleton both in vivo and in vitro. To name the most prominent, the interleukins IL-0, IL-1, IL-3, IL-4, as well as tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ) could be demonstrated to influence TJ barrier function in epithelia and endothelia (Ahdieh et al. 2001; Oshima et al. 2001; Youakim and Ahdieh 1999). This review will emphasise the influence of a few selected cytokines on TJ structure/function and its adjoining actin cytoskeleton in diseases like inflammatory bowel disease and inflammatory disease of the CNS (Table 1).
Table 1Modulation of TJ barrier function in human diseaseClassificationTight junction protein affectedReferenceCancer Breast cancer: invasive ductal cancerClaudin-1, ZO-1Itoh and Bissell (2003), detailed here Prostate cancer: prostatic adenocarcinomasClaudins-1, -3, -4 and claudin-7Sheehan et al. (2007) Thyroid neoplasma, follicular adenomaOccludin and claudin-1, -4 and -7Tzelepi et al. (2007) Gastroesophageal reflux disease: Barrett’s  esophagus (dysplasia)Claudin-18Jovov et al. (2007) Lung cancer: Basaloid squamous carcinomaUltrastructural junctional dedifferentiationGilcrease and Guzman-Paz (1998)Inflammation Inflammatory bowel disease: Morbus CrohnClaudins-2,-3, -5, and -8, ZO-1Zeissig et al. (2007), detailed here  Collagenous collitisOccludin, claudin-2 and -4Burgel et al. (2002) Multiple sclerosisOccludin, claudin-5Förster et al. (2007), detailed hereHereditary diseases Hereditäry deafnessClaudin-14Ben-Yosef et al. (2003), detailed here Familial hypomagnesemiaClaudin-16Simon et al. (1999), detailed here cystic fibrosisOccludin, claudin-1, -4, JAM, ZO-1Coyne et al. (2002)Vision loss Diabetic eye disease: diabetic retinopathyOccludin, ZO-1Felinski and Antonetti (2005), detailed hereViral infection Reoviral infection (hydrocephalus, encephalitis)JAM-1Forrest et al. (2003)Bacterial toxins Clostridium perfringens enterotoxinClaudin-3 and -4McClane (2001), detailed here
Inflammatory bowel disease: Crohn’s disease
Crohn’s disease ranks among the chronic inflammatory bowel diseases (IBD) with diarrhoea as a leading symptom. This is chiefly attributed to epithelial barrier dysfunction, leading to an increased loss of solutes in the form of “leak-flux diarrhoea”. Notably, the integrity of the intestinal barrier is not only impaired in patients with Crohn’s disease but also many first-degree relatives of Crohn’s disease patients present with increased intestinal permeability in the absence of clinical symptoms (Hollander 1993; Peeters et al. 1997; Yacyshyn and Meddings 1995). From this observation it was concluded that barrier dysfunction in Crohn’s disease precedes the inflammatory process in contrast to many other inflammatory diseases, probably arising from genetic defects or driven by environmental factors like intestinal luminal antigens or bacterial toxins.
The epithelial barrier in the intestine is established by a single layer of epithelial cells. However, barrier dysfunction alone does not to seem be sufficient to cause Crohn’s disease, since unaffected relatives of patients with Crohn’s disease comparably present increased permeability, but do not show the abnormal mucosal inflammatory response reported of patients with Crohn’s disease (Weber and Turner 2007). In healthy individuals, the intestinal mucosal barrier is organised through tight junction strands connecting the adjacent epithelial cells (Weber and Turner 2007). These TJs seal the paracellular space and an intact intestinal mucosa. TJ permeability is rate limiting for epithelial permeability and defects thereof are an established source for intestinal barrier defects as observed in Crohn’s disease. The basic understanding of TJ function we developed is that they form an ion-selective and size-selective paracellular gate whose permeability varies throughout the gastrointestinal tract (see above). According to this, it is not surprising that the pattern of claudin isoforms expressed varies alongside the gastrointestinal tract (Nusrat et al. 2000). Recent studies now  show that the expression pattern of characteristic claudins is altered in Crohn’s disease (Weber and Turner 2007) with a concomitant effect on the integrity of TJ strands: Zeissig et al. (2007) showed by freeze–fracture electron microscopy that the overall number of TJ strands sealing the epithelial layer is reduced while concomitantly an increased number of strand breaks was noted in tissue from patiens with Crohn’s disease (Zeissig et al. 2007). The expression of 12 claudin isoforms in the intestinal epithelium was investigated in healthy individuals and patients with Crohn’s disease, leading to the insight that the expression of the claudins-3, -5, and -8 was decreased in active disease. On the contrary, expression of barrier-reducing claudin-2 was increased in histological samples from patients, which was particularly observed in the crypt epithelium. This altered pattern of claudin isoform expression was thus concluded to be partly responsible for the observed mophological and functional disruption of TJs. However, claudin isoform expression patterns were unaltered in patients with inactive Crohn’s disease, rendering this observed change in expression patterns rather a consequence than a cause of active disease.
It is has been acknowledged for a long time that chronic inflammation in Crohn’s disease is associated with the overproduction of proinflammatory cytokines. Searching for the nature of these pro-inflammatory mediators effecting the TJ disruption and dysfunction, investigators (Conyers et al. 1990; Heller et al. 2005; Prasad et al. 2005) had identified IL-13, IFNγ and TNFα as factors in patient’s serum lowering resistance and opening TJs. However, differential effects of the different cytokines crititical to IBD pathogenis have been reported from studies in cell culture models, indicating that the mechanisms by which IFNγ and TNFα inrease paracelluar permeability differ from those used by IL-13 (Prasad et al. 2005). IFNγ and TNFα exposure of model cell lines led to a reduction in claudin-2 expression, while IL-13 exposure induced increased claudin-2 expression. Those observed discrepancies, which might be based on the source of epithelial cell lines, the experimental design or the doses of cytokines applied are the focus of current investigation. Other lines of research are concentrated on gaining more information on the source of pro-inflammatory cytokines in IBD and the mode how those cytokines may work together to modify claudin expression pattern, either in vitro or in vivo (Weber and Turner 2007).
At the cell biological level, recent investigation demonstrated that IFNγ exposure promoted the endocytosis of occludin, JAM-1 and claudin-1 into early and recycling endosomes (Bruewer et al. 2005), an effect which appears to be mediated by RhoA/Rho kinase, which further induced a myosin II-dependent formation of vacuoles in the apical cell moiety (Utech et al. 2005).
At the gene expression level, other research groups could demonstrate that TNFα induced an increase in Caco-2 TJ permeability mediated by activation of the pro-inflammatory transcription factor NFκB. NFκB action leads to a downregulation of specifically ZO-1 gene expression and a redistribution of ZO-1 protein away from the intercellular junctions (Ma et al. 2004). A comparable effect could be described in the nonintestinal model epithelial cell line, MDCK, and in the blood brain barrier model cell line cEND, where also disruption of TJs following exposure of epithelial and endothelial cells to TNFα could be demonstrated (Förster et al. 2007; Poritz et al. 2004).
Blood-brain barrier compromise in neuroinflammation–multiple sclerosis
Homeostasis of the central nervous system (CNS) microenvironment is essential for its normal function. It is maintained by the blood-brain barrier (BBB) which regulates the transport of molecules from blood into brain and backwards. The function of this highly specialised barrier is to (1) protect the brain from blood-borne substances and (2) to provide nutrient supply to the brain parenchyme by specialised transport systems (Rubin and Staddon 1999).
The main structures responsible for this barrier property are the TJ. TJ are strongly developed in endothelial cells of the BBB but only moderately formed between endothelial cells of the peripheral vasculature: leaky blood vessels in the body allow many molecules to cross through to tissue, but the tight construction of the vessels in the CNS guards against brain entry, leading to high electrical resistance in the range of 1,500—2,000 Ωcm2, depending on the species investigated. In BBB-forming brain capillary endothelial cells (BCECs) the TJ proteins occludin and claudin-5, claudin-3, claudin-1 and claudin-12 were demonstrated by immunofluorescence and western blot analysis (Förster et al. 2007; Wolburg and Lippoldt 2002).
Under physiological conditions, brain microvascular endothelial cells form a barrier limiting the extravasation of cells of the immune system like monocytes, lymphocytes and other leukocytes. It has been shown in this context that leukocytes can transmigrate to the BBB without net changes in vascular permeability and cell–cell contacts (Cramer 1992). However, increased leukocyte migration as in multiple sclerosis (MS) has been reported to lead to a re-organisation of the actin cytoskeleton and breakdown of the TJ proteins occludin and ZO-1 (Couraud 1998; Förster et al. 2007). MS is characterised as a chronic and progressive CNS disease with a remitting-relapsing profile. In the course of the disease, both demyelination and microvascular inflammation have been described as central pathophysiological insults. From an animal model for MS, experimental allergic encephalomyelitis (EAE) (Korner and Sedgwick 1996), the inflammatory cytokines TNFα and IL-1 have been delineated to be the key mediators inducing alterations in BBB permeability. Clinically matching data have been collected from MS patients, who show an elevation in TNFα and/or IL-1 in cerebrospinal fluid (CSF) (Weber and Rieckmann 1995). Cytokine-promoted opening of the BBB has been shown to occur due to degradation and decreased synthesis of TJ proteins (Chang and Werb 2001; Harkness et al. 2000; Silwedel and Förster 2006; Yang et al. 2006), resulting in compromised junctional integrity. Particularly, the TJ proteins occludin and ZO-1 have been shown to be negatively affected by the presence of these pro-inflammatory cytokines. Searching for the mechanism of TJ protein degradation, consecutive studies showed, that in many neuroinflammatory conditions, including MS, the matrix metalloproteinases (MMP) play an important role in disrupting the BBB. Amongst these, it has been demonstrated that selectively, MMP-9 (gelatinase B) is increased in CSF levels in MS patients. Tissue inhibitors of metalloproteinases (TIMPs) form complexes with either activated MMPs or with their pro-forms after their secretion, thus regulating MMP activity under physiological conditions (Brew et al. 2000; Yong et al. 2001). Levels of TIMP-1 are however reduced in MS patients relative to control patients, suggesting an imbalance in MMP-9/TIMP-1 ratios in MS (Avolio et al. 2005). MMP-9-mediated opening of the BBB then allows amplification of the inflammation, as demonstrated by radioisotopes (Kermode et al. 1990). Looking for therapeutical agents restoring the unbalaced MMP-9/TIMP-1 ratios in MS, Förster et al. (2007)  showed that glucocorticoid treatment reduces the levels of MMP-9 markedly in a cell culture model of the BBB, an effect which was shown to be based on glucocorticoid-mediated transcriptional activation of the TIMP-1 gene. This study further demonstrated that the administration of the glucocorticoids dexamethasone and hydrocortisone preserved the functional integrity of the TJs and adherens junctions under pro-inflammatory conditions, chiefly by maintaining the levels of the TJ components occludin, claudin-1, claudin-12, ZO-1 and VE-cadherin, while dexamethasone effects on claudin-5 were negligible (Blecharz et al. 2008; Förster et al. 2007).
Another study demonstrated differential susceptibility of cerebral and cerebellar endothelial cell lines to BBB breakdown in response to inflammatory stimuli (Silwedel and Förster 2006): increased fragility of the cerebellar BBB had been reported, as the typical caudal to rostral lesion development of MS and EAE with a preference of the cerebellum indicates (Cross et al. 1993; Juhler et al. 1984; Namer et al. 1993). Matching those studies, the authors could demonstrate a faster and more pronounced increase in permeability in the cerebellar BBB manifested by reduced TER and reduced TJ protein expression using the cerebellar BBB model cell line cerebEND (Silwedel and Förster 2006). The stronger effects of TNFα on endothelial cells of the cerebellum as compared the the cerebrum could be explained on the cellular level, where the TJ protein levels of claudin-5 and occludin were shown to be differently affected in cerebellar and cerebral microvascular endothelial cells: effects on occludin were more pronounced in cerebellar endothelial cells, while cerebral endothelial cells of the BBB showed a stronger decrease in claudin-5. Since it was shown that the main protein buiding up the barrier appears to be occludin, while claudin-5 protein, which is also well detectable in non-BBB endothelial cells, does not seem to influence barrier properties to the same extent (Foerster et al. 2002, 2005, 2006; Wolburg and Lippoldt 2002). One may speculate that the lower overall occludin contents of endothelial cells of the cerebellar BBB compared to the cerebral BBB can partly explain the greater sensitivity of the cerebellar BBB to inflammatory stimuli.
Hereditäry diseases
Hereditäry deafness: nonsyndromic recessive deafness DFNB29
The cochlea of the inner ear is divided into two compartments of different ionic composition. Hereby, the perilymph of the scala vestibuli and scala tympani is high in Na+ but low in K+ concentration, while the endolymph of the scala media resembles the intracellular ionic composition, being high in K+ but low in Na+ (Ferrary and Sterkers 1998). This ion gradient leads to the establishment of an 80–100 mV endocochlear potential, necessary for the depolarisation of sensory hair cells, enhancing the sensitivity of mechano-activated channels at the top of stereocilia (Hudspeth 1989).
Besides, hereditary defects in the gap junction proteins connexin-26 and connexin-43 (Sabag et al. 2005) an association between tight junctions and hearing loss was established. The importance of claudin-14 expression in the cochlea was demonstrated by the identification of two different CLDN14 mutations that cause nonsyndromic recessive deafness DFNB29 in two large consanguineous Pakistani families (Wilcox et al. 2001). By in situ hybridization and immunofluorescence labeling mouse claudin-14 expression in the sensory epithelium of the organ of Corti was observed. By the generation and characterisation of claudin-14 knockout mice as a model for autosomal-recessive deafness DFNB29 it was revealed that deafness occurred due to death of outer hair cells. Searching for the factors that promoted increased death of outer hair cells, morphology of the TJs of claudin-14 knockout mice was evaluated. It was demonstrated that TJ strands between supporting cells and between outer hair cells and supporting cells in the organ of Corti are still present and appear morphologically normal in freeze fracture replicas. The analysed TJ strands which are composed of multiple claudins were thus concluded to have rather a functional than  an ultrastructural defect (Ben-Yosef et al. 2003). Following the reasoning that claudins create charge-selective channels, Ben-Yosef characterised the electrophysiologic properties of claudin 14 by ectopically expressing it in MDCK cells, which revealed a strong discrimination of transfected monolayers against Na+ and K+ ions. Ben-Yosuf and coworkers thus concluded that the absence of claudin 14 from TJs in the organ of Corti might lead to elevated K+ concentration in the space of Nuel, which is normally filled with the K+-poor cortilymph and increased death of outer hair cells. Long-term exposure of the basolateral membranes of outer hair cells to high K+ concentrations had been shown to be toxic and eventually to lead to cell death before (Zenner 1986).
By the identification of two claudin-14 missense mutations that were unable to form TJs after ectopic expression of CLDN14 in L mouse fibroblastes, Wattenhofer et al. (2005) were then able to prove that the basis for the development of deafness is incorrect recruitment and TJ strand formation in cells expressing mutated CLDN14.
Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC)
Inherited and acquired defects in epithelial membrane transport are responsible for many human diseases. In the kidney, tubular claudins play a role in extracellular or paracellular permeability. In this context, it has been shown that mutations in claudin-16 (Paracellin-1) impair the function of TJs in the paracellular resorption of Mg2+ and Ca2+ but not of monovalent cations (Simon et al. 1999), probably founding the molecular basis for the development of familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC), an autosomal recessive renal tubular disorder. In FHHNC patients, the impaired tubular reabsorption of Mg2+ and Ca2+ in the thick ascending limb of the loop of Henle with an eventual progression can lead to end-stage renal disease (Lee et al. 2006). Functional tubular reabsorbtion mechanisms are vitally important, since a normal adult absorbs only 200 mg Ca2+ from the diet each day, but loses 8,000 mg to filtration across the glomerulus. To achieve normal calcium balance, the majority of the filtered load must be reabsorbed by the tubule, to approximately 90%, or 7,000 mg/day and this occurs mainly via the paracellular pathway driven by a lumen positive potential across the tubular wall.
The structural backbone of the paracellular pathway must provide for the significant substrate selectivity and functional heterogeneity between nephron segments and thus also plays a pathophysiologic role in renal diseases. Clinically, the symptoms and gradual kidney damage caused by FHHNC cannot be alleviated by intravenous or oral supplementation with Mg2+, and kidney transplantation is often the only option at later stages of the disease.
Recent studies (Simon et al. 1999) have reported that FHHNC is caused by mutations in a gene, which encodes the TJ protein, paracellin-1. Paracellin-1 (PLCN-1) belongs to the claudin family (claudin-16) that allows the paracellular transport of Mg2+ and Ca2+. As a member of the claudin family of TJ proteins claudin-16 exhibits four transmembrane spans with two extracellular loops and both termini located in the cytoplasm. To date, more than 20 distinct mutations have been identified, which affect either the trafficking of PLCN-1/claudin-16, or its permeability properties (Kausalya et al. 2006).
Interestingly, FHHNC patients do not exhibit the salt wasting or metabolic alkalosis that is found in Bartter’s syndrome (Wong and Goodenough 1999). Based on these findings, it is tempting to hypothesize that PCLN-1 might form a Mg2+- and Ca2+ -conducting pore. It is therefore likely that PCLN-1 selectively mediates the barrier to divalent but not monovalent cations. Thus, not only can the paracellular barrier discriminate between charged and uncharged solutes, but it may also exhibit an even higher level of specificity distinguishing charge and ionic radius and size of the hydrate shell.
As to molecular investigations based on the use of diverse cell culture models for kidney epithelial cells, there is some discrepancy between results obtained upon overexpression of PLCN-1 in two different kidney epithelial cell lines used. In the canine MDCK cell line, another group found PCLN-1 to decrease Na+ permeability, to decrease TER and to enhance transepithelial Mg2+ transport (Ikari et al. 2006). Another recent study performed in the porcine proximal tubule LLC-PK1 cells suggests that paracellin-1 functions to modulate paracellular conductance and not transcellular transport. They hypothesise that PLCN-1 mediates mostly paracellular Na+ permeation to build up the positive lumen potential and that this lumen-positive potential would be the driving force for the reabsorption of Mg2+ (Hou et al. 2005).
As described for other molecules relevant to Mg2+ homeostasis such as ACDP2, (ancient conserved domain protein), a divalent metal transporter, or the Mg2+ transporter MagT1 (Wong and Goodenough 1999), the human PLCN-1 promoter appears to be responsive to external Mg2+ concentrations, since the renal epithelial cell specific PLCN-1 gene expression was shown to positively correlate with the amount of Mg2+ offered in the growth medium (Efrati et al. 2005).
Further, mutations in PLCN-1 can affect claudin-16 protein targeting at the TJs (Kausalya et al. 2006). The mutation T233R for example prevents interaction of claudin-16 with ZO-1 and results in lysosomal targeting of claudin-16 instead. Protein kinase A (PKA) mediates phosphorylation of claudin-16 at Ser217,  failure of which equally leads to dissociation of claudin-16 from ZO-1 and its translocation into the lysosomes (Ikari et al. 2006). In conclusion, although the mechanism of PLCN-1/claudin-16-mediated Mg2+ and Ca2+ reabsorbtion is not yet fully understood, it is clear that these claudins play a crucial role in this process.
Recently, Konrad et al. (2006) additionally unravelled a role for claudin-19 in the development of FHHNC. They had clinically characterized one Swiss and eight Spanish/Hispanic families affected with severe hypomagnesemia due to renal wasting, nephrocalcinosis, and progressive renal failure but had to recognize that the individuals did not have any mutations in CLDN16. They showed that the renal phenotype of the affected patients was virtually undistinguishable from that of patients who presented with FHHNC with proven CLDN16 mutations. However, the affected individuals in these families also have severe visual impairment, characterized by macular colobomata, significant myopia, and horizontal nystagmus (Konrad et al. 2006). Konrad et al. were able to map a new locus for recessive renal magnesium loss on chromosome 1p34.2 and identified mutations in CLDN19, yet another member of the claudin family in patients affected with FHHNC and additionally, with severe ocular abnormalities. CLDN19, which encodes the tight-junction protein claudin-19 was demonstrated to be highy expressed in renal tubules and the retina. In an effort to understand the mechanisms underlying the roles of the two claudins in mediating paracellular ion reabsorption in the kidney, Goodenough and co workers (Hou et al. 2008) showed using pig kidney epithelial cells, that CLDN19 functioned as a Cl(−) blocker, whereas CLDN16 functioned as a Na(+) channel. Mutant forms of CLDN19 were shown to be associated with FHHNC since they were unable to block Cl(−) permeation. Coexpression experiments further elucidated that CLDN16 interacts with CLDN19 and that their association confers cation selectivity, suggesting a role for mutant forms of CLDN16 and CLDN19 in the development of FHHNC.
Vascular system
Diabetic retinopathy
The barrier between the vascular lumen and neural layers in the retina and the brain parenchyme maintains a characteristic microenvironment and is essential for proper neuronal function. This blood-retinal barrier (BRB) consists of two anatomical entities: an inner BRB formed by junctions between endothelial cells of the retinal capillaries, and an outer BRB composed of TJs between retinal pigment epithelial cells. Clinical evidence from fluorescein angiography has demonstrated that the inner BRB appears to be the primary site of vascular leakage leading to macular edema.
The BRB is built up and maintained by TJ complexes that make up the blood vessels of the microvasculature (Mitic and Anderson 1998). The principal proteins found in the retinal endothelial TJs are occludin and claudin-5 (Dejana et al. 2001). Loss of the BRB integrity and vascular permeability leading to macular edema (Vitale et al. 1995) characterise diabetic retinopathy. Macular edema in turn is thought to be directly responsible for vision loss in diabetic retinopathy (Felinski and Antonetti 2005). Vascular endothelial growth factor (VEGF) is thought to be chiefly responsible for this increase in BRB permeability, albeit other permeabilizing factors like histamin or the cytokines IL-1 and TNFα have also been implicated (Felinski and Antonetti 2005). Levels of VEGF, originally identified as an angiongenic factor serving as a mitogen for vascular endothelial cells have been recently correlated with increased vascular permeability (Felinski and Antonetti 2005), compatible with increased VEGF levels detected in the vitreous of diabetic patients for decades. There is enough evidence to show that diabetes and VEGF induce retinal vascular permeability by altering TJ complexes, evidenced for instance by the decrease in occludin content in the retinas of diabetic rats (Felinski and Antonetti 2005). In cultured primary bovine retinal endothelial cells, this observation was mirrored by the observation of decreased occludin contents after VEGF administration (Antonetti et al. 1998). Follow-up investigation underlined this and showed down-regulation of occludin in retinal endothelial cells in culture after VEGF stimulation. VEGF further activates PKC, which leads to increased occludin and ZO-1 phosphorylation (Harhaj et al. 2006). Phosphorylation of occludin and ZO-1 likely contribute to dysregulated endothelial paracellular permeability (Abbott et al. 2006). Regulation of PKC activity and TJ protein modifications may thus have therapeutic implications for treatment of diabetic retinopathy.
Recent investigation has further revealed that alterations of the BRB may involve the active proteolytic breakdown of the endothelial cell TJs by MMPs as also evidenced in the case of MS (compare above) (Leppert et al. 2001). Specifically, elevated levels of MMP-9 have been seen when retinal endothelial cells were exposed to high glucose conditions as a cell culture model of diabetes, leading to proteloytic degradation of specifically the TJ protein occludin followed by disruption of the overall TJ complex (Giebel et al. 2005), rendering the regulation of MMP secretion and activity another therapeutical target in diabetic retinopathy.
Multiple sclerosis
See above.
Tight junction alterations in cancer
Decreased expression of claudin-1 correlates with malignant potential in breast cancer
Changes in TJ function have been shown to be an early and key aspect in cancer metastasis (Ehler et al. 2001). Recent studies showed a role for claudin-1 in invasion and metastasis in mammary glandular differentiation (Förster et al. 2002) and carcinogenesis (Morohashi et al. 2007).
Interestingly, there is an absence or significant loss of claudin-1 expression in several established breast cancer cell lines. Claudin-1 expression in primary human mammary epithelial cells, in contrast to low or undetectable levels of expression in a number of breast tumors and breast cancer cell lines, points to claudin-1 as a possible tumor-suppressor gene. In sections from surgically resected breast specimens, a significant loss of claudin-1 protein in breast cancer cells could be shown by immunohistochemistry (Tokes et al. 2005). This finding suggests that claudin-1 may play a role in invasion and metastasis. The expression profile of claudin-1 in non-malignant versus tumor-derived cells has made this gene an interesting candidate for involvement in tumorigenesis, namely by acting as a suppressor of mammary epithelial proliferation.
In order to evaluate the CLDN-1 gene in sporadic and hereditary breast cancer, Krämer et al. (2000) characterized its genomic organization and screened the four coding exons for somatic mutations in 96 sporadic breast carcinomas and for germline mutations in 93 breast cancer patients with a strong family history of breast cancer. In addition, they compared the 5′-upstream sequences of the human and murine CLDN1 genes to identify putative promoter sequences involved in down-regulation of the CLDN1 gene under malignant conditions and examined both the promoter and coding regions of the human gene in various breast cancer cell lines showing decreased claudin-1 expression. However, neither in sporadic or hereditary breast cancers, nor in breast cancer cell lines they found evidence for a role of aberrant claudin-1 in breast tumorigenesis and concluded that other regulatory or epigenetic factors could be involved in the down-regulation of the CLDN1 gene during breast cancer development.
A correlation between TJ abnormalities and neoplasia can be supported by several observations: alterations in the number, appearance, and permeability of TJs have been demonstrated in various cancer types (Cochand-Priollet et al. 1998; Soler et al. 1999; Swift et al. 1983) and in pre-malignant mammary epithelial cells (Förster et al. 2002). At the molecular level, the Drosophila lethal(1) discs-large-1 gene (dlg) product, which localises to TJ-related insect septate junctions has been shown to regulate epithelial cell proliferation. Genetic loss of dlg leads to a neoplastic overgrowth phenotype (Woods and Bryant 1991). The loss of expression of claudin-1 itself has been demonstrated in several mammary carcinoma cell lines (Swisshelm et al. 1999). The expression of the TJ plaque protein ZO-1 has been shown to be reduced in breast carcinomas or breast cancer cells (Hoover et al. 1998). These findings support the hypothesis that claudin-1 might be involved in the development of breast cancer and possibly in other epithelial tumors too.
The molecular pathways leading toward the loss of claudin-1 expression however, remain to be explained.
Bacterial toxins
Clostridium perfringens enterotoxin
Food poisoning by Clostridium perfringens enterotoxin (CPE) leads to diarrhea symptoms classified as “C. perfringens type A food poisoning and antibiotic-associated diarrhea”. The enterotoxin protein has been elucidated to intrude the cells by interacting with epithelial TJ, including certain claudins. Toxin-induced cytolytic pore-formation is a prerequisite for this and involves residues in the N-terminal half of CPE protein, while residues near the C-terminus are required for binding to claudins. In detail, the single 35 kDa CPE polypeptide first causes cellular damage by altering plasma membrane permeability by the formation of an approximately 155 kDa CPE-containing pore complex necessary for the subsequent interaction with TJ proteins via its C-terminal receptor-binding region (McClane 2001). CPE was then perceived to affect TJ structure and function in a way that permeability properties of the epithelial layer are altered leading to diarrhea (McClane 2001).
Further investigation has determined the isoforms of claudins targeted by CPE. Sonoda et al. (1999) were able to show that the COOH-terminal half fragment of CPE (C-CPE) bound effectively to claudin-3 and claudin-4 in MDCK I cells expressing the claudins-1, -2, -3 and -4. They were further able to show that in the presence of C-CPE, reconstituted TJ strands gradually disintegrated and disappeared from their cell surface in C3L cells.
As a consequence, because diverse claudins are overexpressed on some human cancers, the toxin became interesting for targeting chemotherapy in the manner of a Trojan horse (Long et al. 2001; Michl et al. 2001).
Recently, van Itallie et al. (2007) aimed to solve the structure of the CPE claudin-binding domain to advance its therapeutic applications. The structure was shown to be a nine-strand beta sandwich with previously unappreciated similarity to the receptor-binding domains of several other toxins of spore-forming bacteria, giving strong evidence for the presence of a common ancestor for several receptor-binding domains of bacterial toxins (Van Itallie et al. 2007). The very recent elucidation of the structure of a 14-kDa fragment containing residues 194 to the native COOH terminus at position 319 by X-ray diffraction to a resolution below 2 Å showed that the structure is a nine-strand β sandwich with previously unappreciated similarity to the receptor-binding domains of several other toxins of spore-forming bacteria, i.e., the collagen-binding domain of ColG from Clostridium histolyticum and the large Cry family of toxins (including Cry4Ba) of Bacillus thuringiensis (Van Itallie et al. 2008). The authors speculate that the claudin-4 binding site is on a large surface loop between strands β8 and β9 or includes both of these strands. They further were able to clarify that the sequence crystallized binds to purified human claudin-4 with a 1:1 stoichiometry. The binding affinity was determined to be in the submicromolar range similar to that observed for binding of native toxin to cells (Van Itallie et al. 2008). These results could now provide a structural framework to advance therapeutic applications of the toxin.
Conclusion and perspectives
The discovery of the existence of occludin- and claudin-based TJs constituting a permeability barrier in epithelial and endothelial cells has paved the way for the elucidation of the molecular background of diverse human diseases the pathogenesis of which had been unclear before. It became clear that the presence of TJs is indispensable for tissue compartmentalisation and cellular homeostasis. Disturbances in TJ function are reflected in many diseases, but on the other hand, awareness of their involvement has facilitated therapy enormously. For instance, antiinflammatory or antimetastatic drugs could be developed based on their ability to repair broken barrier function. These strategies have become indispensable in the treatment of disease like MS, inflammatory bowel disease or diabetic retinopathy. Following the strategy of pathogenic agents, which use TJ proteins as a docking station to invade cells, even strategies for drug delivery techniques could be implemented. Future attempts will have to address the role of occludin and the exact contribution of the diverse claudins to the formation of ion selective pores, which are still open questions. A more thorough understanding will greatly facilitate diagnosis and the development of specific treatment regimens for diseases originating from impaired TJ function.

tight junction
occludin
claudin
vascular
inflammation
cancer
hereditary
toxin


Cellular and Matrix Mechanics of Bioartificial Tissues During Continuous Cyclic Stretch

Bioartificial tissues are useful model systems for studying cell and extra-cellular matrix mechanics. These tissues provide a 3D environment for cells and allow tissue components to be easily modified and quantified. In this study, we fabricated bioartificial tissue rings from a 1 ml solution containing one million cardiac fibroblasts and 1 mg collagen. After 8 days, rings compacted to <1% of original volume and cell number increased 2.4 fold. We initiated continuous cyclic stretching of the rings after 2, 4, or 8 days of incubation, while monitoring the tissue forces. Peak tissue force during each cycle decreased rapidly after initiating stretch, followed by further slow decline. We added 2 μM Cytochalasin-D to some rings prior to initiation of stretch to determine the force contributed by the matrix. Cell force was estimated by subtracting matrix force from tissue force. After 12 h, matrix force-strain curves were highly nonlinear. Cell force-strain curves were linear during loading and showed hysteresis indicating viscoelastic behavior. Cell stiffness increased with stretching frequency from 0.001–0.25 Hz. Cell stiffness decreased with stretch amplitude (5–25%) at 0.1 Hz. The trends in cell stiffness do not fit simple viscoelastic models previously proposed, and suggest possible strain-amplitude related changes during cyclic stretch.
INTRODUCTION
Mechanical stimuli are important for the health and maintenance of many biological functions.14 This is especially true in the cardiovascular system where the tissues and organs are undergoing constant cyclic loading. It is the cellular response to these mechanical forces that can either maintain healthy tissue or lead to a pathologic state.1
Mechanical stimuli have been used to create more functionally viable engineered tissues.2–5,9,15–17 It has been shown that constant amplitude cyclic stretch (a form of mechanical conditioning) of engineered tissue constructs can cause cells to reorient, increase cell proliferation, and produce more extracellular matrix (ECM).3 However, these studies generally have looked at how the tissue responds to mechanical conditioning over extended time.9,15 In addition, the studies generally quantify the mechanics using only the ultimate strength and modulus4,5,16 along with quantification of such biological factors as cell number, gene expression, and ECM components.2,3,17
Since it is the cells that actively respond to mechanical loads, it is important to understand the effects of mechanical conditioning on the individual components of the tissue (cell and matrix) and how the mechanical properties change from the time that the conditioning is initiated. Our earlier work has shown that tissue forces drop with subsequent stretches during repeated cyclic stretching.19 However, whether this drop occurs in the cells and/or matrix components is not known. Furthermore, it is not known how these drops are affected by different amplitudes and frequencies over many hours of continuous cyclic stretch.
The objective of this study is to measure the short-term (<24 h) mechanical response of collagen gels populated with cardiac fibroblasts to cyclic stretch at different amplitudes and frequencies. Although engineered tissue constructs may be composed of several cell and matrix components, we chose to use a simple system composed of a single cell type and a single defined ECM component to study the mechanics of tissue components during cyclic stretch. This system provides a 3D environment for the cells while allowing the components to be easily modified and quantified. The benefits of using collagen, the main structural component in cardiovascular tissue, are that its initial concentration can be easily modified and it is more stable than other ECM components such as fibrin.4,5,10 By using a single cell type we can avoid complicating effects due to mixed cell populations and clearly understand that cell type’s contribution to the tissue mechanics. Cardiac fibroblasts are one of the two main cell types in cardiac tissue and are responsible for ECM maintenance and remodeling.7 These cells, along with the cardiac myocytes, undergo continuous cyclic stretch with each heartbeat. Thus, it is particularly useful to understand their cellular mechanics during cyclic stretch.
By measuring the cell forces under various cyclic stretch amplitudes and frequencies, we can better understand the relationship between the applied strains and the cells’ mechanical response. Then we can model this behavior and be able to predict cellular responses to dynamic physiologic and pathologic loading conditions.
METHODS
Tissue Ring Fabrication
Chicken embryonic cardiac fibroblasts (CECF) were isolated from 10 day old chicken embryos (Charles River Laboratories, Wilmington, MA). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 3% fetal bovine serum (FBS), 50 units/ml penicillin and 50 mg/ml streptomycin (P/S). Tissues were fabricated as described previously.20 Briefly, CECF from passage 2–8 were added to a collagen solution containing monomeric rat tail tendon collagen (Upstate Biotechnology Inc., Lake Placid, NY) neutralized with 0.1 N sodium hydroxide (NaOH) and 2× concentrated DMEM. The final solution contained 1 mg/ml collagen and one million cells/ml. Then 1 ml of this solution was poured into cylindrical molds containing an inner mandrel (diameter=9.55 mm). After one hour of incubation, the solution gelled and the molds were filled with incubation medium containing DMEM supplemented with 10% FBS and P/S. After five additional hours, the gels were removed from the molds and transferred to spacers (Fig. 1) placed in incubation medium. The spacers consisted of two fixed rods (2.07 mm diameter) separated by a distance to maintain the internal circumference of the rings at 30 mm. Medium was changed every other day.
FIGURE 1.Cardiac fibroblast populated rings on spacers during static incubation (A) Day 0 [6 h] (B) Day 2 (C) Day 4 (D) Day 8. Mean ring width was 0.64 mm after 8 days incubation (see Fig. 4).FIGURE 2.Illustration of peak force versus time. The gray curve is sample data obtained for 10 stretch cycles. The black line plots the peak force for each cycle vs. time. This peak force line is used for many of the following figures.
Mechanical Tests
After 2, 4, or 8 days of static incubation (Fig. 1) the width of the rings was measured and then the rings were removed from the spacers. The rings were then mounted on a mechanical tester described previously20 that allows independent programmable stretching of four rings while monitoring applied force. The test medium contained DMEM HEPES modification supplemented with 10% FBS and P/S. All rings were placed on the tester at a baseline length that maintained the inner circumference of 30 mm. This was defined as 0% strain. In early tests, stretching began immediately after mounting the rings. This led to increases in peak force after initiation of stretch for low amplitudes at early incubation times. Upon further study, it was determined this was a result of the fresh media and FBS used in the test baths and not a result of a mechanical stretch induced response. In all further studies, the rings were mounted on the tester and held at baseline length for 3 h to allow them to reach equilibrium before testing began.
Effect of Cyclic Stretch Amplitude
Four rings were tested at each of the three days with each ring stretched either 5%, 10%, 20%, or 25% at 0.1 Hz using a triangular displacement waveform (constant strain rate during loading). The force was continuously monitored at 10 Hz over 14 h. For many of the figures, the peak force, defined as the maximum force during each cycle, is plotted vs. time (Fig. 2). We use the term stretch to refer to the amplitude of cyclic stretching, while we use the term strain to refer to the amount of deformation at a particular point in the loading curve.
To separate out the individual tissue component forces (cell and matrix), 2 μM Cytochalasin D (CytoD) was added either 3 h before or 12 h after initiation of stretch. CytoD disrupts actin filaments inside the cell and thus eliminates the majority of the cell-contributed force.21 The remaining force measured 120 min after addition of CytoD is termed the matrix force. Then the estimated cell force is calculated by taking the matrix force and subtracting that from the total tissue force without CytoD.
FIGURE 3.Cell force calculation. Matrix force was measured by adding 2μM CytoD to rings prior to initiation of cyclic stretch. Cell force is calculated by taking the normalized matrix force (see Eq. 1) and subtracting it from the tissue force.
To estimate the cell forces during the initial 12 h of cyclic stretch, it is necessary to use two separate rings subject to identical stretching protocols. This is accomplished by subtracting the forces measured in a ring with CytoD added prior to cyclic stretching from the forces measured in a ring where CytoD was added after 12 h of cyclic stretch. We denote these forces FC0(t) and FC12(t) respectively and similarly, we denote the peak forces PC0(t) and PC12(t). However, the peak forces after 14 h of cyclic stretch, when both rings have been treated with CytoD, can still differ between rings subject to identical cyclic stretch protocols due to variations between rings. To account for this variation, we scale the forces in the ring to which CytoD was added before initiating stretch using the following formula:
We found that the shape of the loading/unloading curve was similar whether CytoD was added before or after initiation of cyclic stretch. We then subtracted this scaled, or normalized, force curve from the force curve for the ring with CytoD added after 12 h to obtain an estimated cell force (Fig. 3):
Single Step Stretch and Hold Effect
To compare the effect of a single stretch to cyclic stretching, a step stretch (stretch to a given stretch magnitude in less than 0.1 s) of 5%, 10%, or 20% was applied and the rings were held at this stretched length for 8 h while the force was monitored. The tissue was then returned to its baseline length and the force was monitored for an additional 6 h. In another set of experiments, CytoD was added 3 h before or 12 h after either a 10% or 20% step stretch.
Effect of Cyclic Stretch Frequency
A similar procedure was followed to test frequency effects. Four rings were tested at each of the three days with each ring stretched at either 0.25 Hz, 0.1 Hz, 0.01 Hz, or 0.001 Hz at 10% stretch amplitude. The current tester’s upper frequency limit is 0.25 Hz and thus unable to stretch at higher frequencies that are closer to physiologic values. CytoD was added either 3 h before or 12 h after initiation of stretch to separate out the cell and matrix components as a result of different frequencies of cyclic stretch.
Cell Number
After the completion of testing, some rings were washed in phosphate buffered saline (PBS) and then placed in 1 ml lysis buffer (0.1% sodium dodecyl sulfate (SDS) in PBS). The samples were sonicated until the rings completely disintegrated to release the DNA from the cells. Finally, 30 μl of sample was placed in 3 ml of Hoechst working solution (30 nM Hoechst 33258 dye (Sigma, St. Louis, MO) in PBS). The fluorescence was then read by a spectrophotometer and cell number determined from a standard curve obtained from samples with known numbers of cells.
Immunohistochemistry
Rings were removed from spacers after 2, 4, or 8 days static incubation and immediately placed in a solution of 4% paraformaldehyde for 30 min, washed with PBS and then permeabilized for 45 min in 0.2% Triton X-100 in PBS. This was followed by 1 h incubation in a blocking solution (2% normal goat serum and 0.02% sodium azide in PBS). After blocking, the rings were incubated overnight at 4°C in blocking solution containing TRITC phalloidin. Finally, the rings were washed again in PBS and mounted for viewing on a confocal microscope (Zeiss Confocor).
RESULTS
Ring Width
Ring width was used as a metric for monitoring the extent of compaction during incubation. The initial width of the rings was approximately 10 mm. This width decreased until day 8 (Fig. 4(b)) when the average width was reduced to 0.64 mm. At this time the cross-sectional area of the ring appeared circular and the volume could be approximately calculated using a circular cross-sectional area and the circumference of the ring. A ring diameter of 0.64 mm corresponds to a 99% reduction in the initial tissue volume.
FIGURE 4.Cell number and ring width during static incubation. (A) Cell number increases from the initial 1 million cells to 2.4 million cells after 8 days of static incubation. (B) Ring width decreased from an initial 10 mm to 0.64 mm after 8 days of static incubation, with a large amount of compaction occurring prior to day 2.
Cell Number
Rings started with a cell density of one million cells/ml (one million cells per ring). This number increased steadily for 4 days to 2.3 million cells then increased only slightly to day 8 with a total cell number of 2.4 million (Fig. 4(a)). When combined with the 99% reduction in tissue volume, this translates into a final cell density of 234 million cells/ml or a 234 fold increase in cell density.
Amplitude Effect
The effect of increasing stretch amplitude on the peak tissue and matrix forces is shown in Fig. 5. At all amplitudes, the peak tissue forces immediately after initiating stretch (Fig. 5(a)) increased with static incubation time while the peak matrix forces were similar (Fig. 5(c)). After 12 h of cyclic stretch, both tissue (Fig. 5(b)) and matrix forces had decreased substantially, with a larger percentage decline in the matrix force (Fig. 5(d)). To examine the relationships between tissue, matrix, and cell forces more closely, figures in the remainder of this paper show data from rings statically incubated for 8 days prior to cyclic stretch unless otherwise noted. Similarities or differences to days 2 and 4 are discussed in the text. In addition, all curves are averages across repeated (n=2–7) experiments. Due to the large number of different conditions tested and the relatively low number of samples (exact numbers shown in Fig. 5(e), 10(e)) rigorous statistical analysis was not possible.
Tissue Force
Higher stretch amplitudes resulted in higher initial peak forces (Figs. 5(a), 6(a), 6(b)). With each subsequent stretch, the peak force decreased with large drops during the first several stretches and sustained decreases during the first two hours, but only small further decreases in peak force over the next 10 h (Fig. 6(a)). In addition, this decrease in peak force was greater at higher stretch amplitudes both in terms of absolute force drop and percentage drop from first stretch. At days 2 and 4, after 12 h of stretch, higher amplitudes still have higher peak forces but the differences were smaller between the amplitudes than the first stretch differences (Fig. 5(a,b)).
FIGURE 5.Peak tissue and matrix forces for different cyclic stretch amplitudes at 0.1 Hz. (A) Peak tissue force of the first cyclic stretch after 2, 4, or 8 days static incubation. Peak tissue force increases with cyclic stretch amplitude and incubation time. (B) Peak tissue force after 12 h of cyclic stretch after 2, 4, or 8 days static incubation. At day 8 the peak tissue force is similar regardless of cyclic stretch amplitude. (C) Peak matrix force of the first stretch after 2, 4, or 8 days static incubation. Peak matrix force increases with cyclic stretch amplitude. (D) Peak matrix force after 12 h of cyclic stretch after 2, 4, or 8 days static incubation. Higher amplitudes result in higher peak forces. (E) Number of rings averaged for data shown in Figures 5-8 at each amplitude and static incubation day. (F) Individual measurements used in panel B [Peak tissue force after 12 h, 8 days of incubation]. This shows that the trend within an experiment, i.e. rings fabricated on a single day, is consistent but the experiment to experiment variation in forces is large. Y-axis scales are different in each panel to emphasize the trends with amplitude and incubation time.FIGURE 6.Tissue force versus cyclic stretch amplitude after 8 days of static incubation. (A) Peak tissue force over 12 h of cyclic stretch. Peak tissue force dropped quickly after initiation of cyclic stretch followed by a continued slow decline. (B) First 5 tissue stretches after initiation of cyclic stretch. A large decline in tissue force and hysteresis occurred between the first and second stretch. (C) Last 5 tissue stretches after 12 h of cyclic stretch. The peak tissue force is similar at the four amplitudes.
By day 8, similar peak tissue forces were seen after 12 h of cyclic stretch regardless of stretch amplitude between 5% and 25% (Figs. 5(b), 6(a), 6(c)). The rings subject to lower stretch amplitudes (5% or 10%) were able maintain a positive baseline force (i.e. force at the baseline, or zero strain, length). At the highest stretch amplitude the rings had periods of zero force during both the loading and unloading of the ring, meaning that at low strains the rings were slack (Fig. 6(c)). After the first stretch (Fig. 6(b)), the loading curve remained nearly linear at 5% stretch amplitude, but became increasingly nonlinear at higher stretch amplitudes (Fig. 6(c)).
FIGURE 7.Matrix force versus cyclic stretch amplitudes after 8 days of static incubation. (A) Peak matrix force over 12 h of cyclic stretch. Peak matrix force dropped quickly after initiation of cyclic stretch followed by a slow decline for the 12 h duration. (B) First 5 matrix stretches after initiation of cyclic stretch. A large decline in matrix force and hysteresis occurred between the first and second stretch. (C) Last 5 matrix stretches after 12 h of cyclic stretch. Matrix loading curve is highly nonlinear and peak force increased slightly with higher cyclic stretch amplitude.FIGURE 8.Cell force versus cyclic stretch amplitudes after 8 days of static incubation. (A) Peak cell force over 12 h of cyclic stretch. Peak cell force increased for 5% and 10% amplitudes while it decreased for 20% and 25% amplitudes. (B) First 5 cell stretches after initiation of cyclic stretch. (C) Last 5 cell stretches after 12 h of cyclic stretch. The peak cell force and cell stiffness decreased with increased amplitude. The loading curves are nearly linear for all amplitudes.FIGURE 9.Response to step stretch and hold after 8 days of static incubation. The tissue, matrix, and cell forces are shown after a step stretch of either 10% or 20%. The tissue is held at the stretched length for 12 h. The tissue and matrix force for a 20% step and hold is higher than that for a 10% step and hold. The cell force is identical for 10% and 20%.
Matrix Force
Except for the first stretch, the matrix loading curves were nonlinear for all stretch amplitudes tested. Similar to tissue force, higher stretch amplitudes led to higher initial peak forces (Figs. 5(c), 7(b)). For all amplitudes, the peak force decreased with repeated cyclic stretching and this drop in peak force was larger at higher stretch amplitudes (Fig. 7(a)) but higher stretch amplitudes still maintained higher peak forces (Fig. 7(c)). For the first stretch, the ratio of peak forces at day 8 for 25% to 5% was 5:1, but this ratio decreased to 2:1 after 12 h of stretching, due to the peak matrix force dropping by a greater percentage at the higher amplitudes over time. The other distinct feature was the shape of the loading/unloading curves.
After several hours of stretching the shapes of the loading/unloading curves were very similar for all stretch amplitudes, but the strain value at which the matrix force began increasing from zero during each loading cycle was shifted toward larger strain values. The amount of this shift was greater for higher stretch amplitudes (Fig. 7(c)). This meant that the matrix contributed to the tissue force during the loading only when the strain had reached a value from 3 to 7% less than its maximum value. This shape and shift is evident at days 2, 4, and 8. For all stretch amplitudes, the matrix is slack at low strains.
Cell Force
Cell force increased with increased incubation time. The increase in cell force after 12 h of stretch from 2 to 4 days of incubation was due to an increase in cell number (per cell force remained the same). However, the increase in cell force from days 4 to 8 could not be explained by the increase in cell number alone, indicating an increase in per cell force. The shape of the first force-strain curve for the cell differed from all subsequent curves. The initial baseline force was followed by a rise and then a leveling off with further increases in force seen only at the higher amplitudes (Fig. 8(b)). During the second stretch, the cell force loading curve was nearly linear. Unlike the tissue and matrix, the cell force loading curve retained a nearly linear shape throughout the 12 h of cyclic stretch even at 25% amplitude (Fig. 8(b,c)).
At day 2, the peak cell force initially drops for several cycles, but then gradually increases to the end of the test. The size of the initial force drop is dependent on the stretch amplitude. The trend is similar at day 4, except the cell force at 25% stretch remains fairly steady instead of increasing. At day 8, the peak cell force, after the initial drop, increases only for the 5% and 10% stretch, while it decreases for the 20% and 25% stretch (Fig. 8(a)).
Single Step Stretch & Hold Effect
Similar to cyclic stretching at different amplitudes, the larger magnitude step stretches led to higher initial forces (Fig. 9). The tissue force was higher than the matrix force immediately after the step at the same magnitude. However, both tissue and matrix force dropped quickly and to a greater extent for higher magnitudes. The matrix force was lower than the tissue force during the entire 12 h hold period at the same magnitude indicating that the cells contributed to the measured tissue force throughout the hold period (Fig. 9). The rate of decrease during the 12 h hold period is similar for both the tissue and matrix. This is evident in the fact that very quickly after the step stretch the cell force approaches a constant for the 12 h (Fig. 9). The cell force is approximately the same at both 10% and 20% step stretches during the 12 h held at the stretched length. For both step stretch magnitudes, the cell force at the end of the 12 h hold period is similar to the cell force at the baseline length (data not shown) immediately before the step stretch is applied.
Rings that did not have CytoD added before or during the step stretch were returned to their baseline lengths after being held at the stretched length for 8 h. The force dropped to zero but then began to recover with lower magnitude step stretches increasing their forces faster than higher magnitude step stretches. The recovered forces approached their baseline forces prior to stretch. After 6 h of monitoring force at baseline, rings stretched to 5% magnitude and held recovered more of their baseline force (84%) than rings stretched to 20% magnitude (42%).
The force decreases more rapidly in the step stretch and hold than the peak force drops in cyclic stretching at the same amplitude.
Frequency Effect
Figure 10 illustrates the trends in the peak tissue (Fig. 10(a, b)) and matrix force (Fig. 10(c, d)) either at the first stretch or after 12 h of cyclic stretch. It also compares the peak force trends over stretch frequencies and static incubation time. At all frequencies the peak tissue and matrix force increased with static incubation (Fig. 10).
FIGURE 10.Peak tissue and matrix forces for different cyclic stretch frequencies at 10% amplitude. (A) Peak tissue force of the first cyclic stretch after 2, 4, or 8 days static incubation. Peak tissue force increased with cyclic stretch frequency and incubation time. (B) Peak tissue force after 12 h of cyclic stretch after 2, 4, or 8 days static incubation. At day 8 the peak tissue force increased slightly with increased frequency. (C) Peak matrix force of the first stretch after 2, 4, or 8 days static incubation. Peak matrix force increased slightly with increased cyclic stretch frequency. (D) Peak matrix force after 12 h of cyclic stretch after 2, 4, or 8 days static incubation. Peak matrix force similar regardless of cyclic stretch frequency. (E) Number of rings averaged for data shown in Figs. 10–13 at each frequency and static incubation day. (F) Individual measurements used in panel B [Peak tissue force after 12 h and 8 days of static incubation]. This shows that the trend within an experiment (E#) is consistent but the experiment to experiment variation is large. Y-axis scales are different in each panel to emphasize the trends with frequency and incubation time.
Tissue Force
Initially, higher frequencies led to higher peak tissue forces (Figs. 10(a), 11(a), 11(b)). After an initial drop, the peak force remained steady or increased at days 2 and 4. However, at day 8 after the initial drop the peak force continues to decline over the rest of the experiment (Fig. 11(a)). After 12 h, higher frequencies still resulted in higher peak forces (Figs. 10(b), 11(c)). Another difference is in the baseline forces, where lower frequencies had higher baseline forces than high frequencies. Thus, rings cyclically stretched at lower frequencies have lower measured tissue stiffness (slope of the loading curve).
FIGURE 11.Tissue force versus cyclic stretch frequency at day 8. (A) Peak tissue force over 12 h of cyclic stretch. Peak tissue force dropped quickly after initiation of cyclic stretch followed by a continued slow decline. (B) First 5 tissue stretches after initiation of cyclic stretch. A large decline in tissue force occurred between the first and second stretch. (C) Last 5 tissue stretches after 12 h of cyclic stretch. The tissue stiffness increased with increasing cyclic frequency.
Matrix Force
Initially, higher frequencies led to higher peak matrix forces (Figs. 10(c), 12(a), 12(b)). These matrix forces quickly dropped over the first few hours and then continued a steady decline for the remainder of the cyclic stretch (Fig. 12(a)). At day 8, all the peak matrix forces quickly approached the same value about 30 min after the start of stretch (Fig. 12(a)). In addition, unlike the tissue and cell curves, there is little difference between the matrix curves at the different frequencies after 12 h except that lower frequencies contribute over a slightly larger range of strains but the peak force is still the same at all frequencies tested (Fig. 12(c)). As with the matrix force at different amplitudes, the loading curve was nonlinear and the matrix contributed only to the tissue force when the strain was within 4–6% of its maximum value.
FIGURE 12.Matrix force versus cyclic stretch frequency at day 8. (A) Peak matrix force over 12 h of cyclic stretch. Peak matrix force dropped quickly after initiation of cyclic stretch followed by a slow decline for the 12 h duration. (B) First 5 matrix stretches after initiation of cyclic stretch. A large decline in matrix force occurred between the first and second stretch. (C) Last 5 matrix stretches after 12 h of cyclic stretch. Matrix loading curves were highly nonlinear and the peak forces was similar at all frequencies.
Cell Force
Similar to the first force-strain curves at different amplitudes, the cell force initially rose, then leveled off or even decreased at the highest frequency (Fig. 13(b)). At lower frequencies the slope of the loading curve was lower than that for higher frequencies (Fig. 13(c)). This is clear at day 8 when, for low frequency, the baseline cell force was higher and the peak cell force was lower than that of high frequencies (Fig. 13(c)). At the two lowest frequencies the cells were able to maintain a baseline force while at the two highest frequencies the baseline cell force was near zero. This leads to a cell stiffness that was higher at higher cyclic stretch frequencies. This was similar to the trend seen with the tissue stiffness.
FIGURE 13.Cell force versus cyclic stretch frequencies at day 8. (A) Peak cell force over 12 h of cyclic stretch. (B) First 5 cell stretches after initiation of cyclic stretch. (C) Last 5 cell stretches after 12 h of cyclic stretch. Cell stiffness increased with increasing cyclic stretch frequency.
Immunohistochemistry
Rings stained with a dye for actin cytoskeleton showed a distinct morphology that developed during the 8 days of incubation. Initially the cells were sparse with few cells on the outer layer of the rings (Fig. 14(a)). The inner core of the rings initially showed a higher cell concentration that had some alignment along the circumferential direction (Fig. 14(b)). As the rings incubated, this outer layer that was approximately 25 μm thick increased in density but showed little preferential alignment (Fig. 14(c,e)). The inner core of the rings also had some increase in cell density but was noticeably less dense than the outer layer (Fig. 14(d,f)). However, these cells were highly aligned in the circumferential direction (Fig. 14(d,f)).
FIGURE 14.Confocal images at 2, 4, and 8 days of static incubation. F-actin fibers visualized using a TRITC phalloidin stain. (A) Outer surface (projection from 4–14 μm depth) after 2 days static incubation. (B) Inner core (projection from 150–160 μm depth) after 2 days static incubation. (C) Outer surface (projection from 4–14 μm depth) after 4 days static incubation. (D) Inner core (projection from 61–71 μm depth) after 4 days static incubation. (E) Outer surface (projection from 10–15 μm depth) after 8 days static incubation. (F) Inner core (projection from 60–65 μm depth) after 8 days static incubation. Field of View: 230 μm × 230 μm. Circumferential direction is in the vertical direction.
DISCUSSION
We have shown that a few hours after the initiation of stretch, higher cyclic stretch amplitude does not lead to higher cell or tissue forces. Thus, for the majority of the cyclic stretch duration, the peak stress experienced by the cells is independent of the stretch amplitude applied, at least in the range of 5%–25%. This is accomplished by the cells becoming less stiff at higher stretch amplitudes. The exact mechanism for this change in cell stiffness is unknown. We have found that the magnitude of the drop in tissue force after the addition of 2 μm CytoD is similar to the force drop after adding detergent (e.g. deoxycholate). Since the detergent treatment actually removes the cells,18 it is likely the actin cytoskeleton is primarily responsible for the change in cell stiffness. Whether this is a result of cytoskeletal remodeling and/or reduction in active contraction/relaxation of the myosin is not known.
While the initial extracellular matrix consists entirely of collagen it is reasonable to assume that over 8 days of incubation time that additional cell-contributed matrix may be produced. We have incubated rings for 20 days prior to cyclic stretching and found that the matrix force-strain curves are similar to those after 8 days of incubation. Peak forces increase only a small amount at 5% or 10% stretch amplitude after 14 h of cyclic stretch at 0.1 Hz. These results suggest that the matrix mechanics do not change substantially even in the presence of cells that can produce additional matrix.
In addition, we treated some rings with 30 mM ribose after 10 days of incubation, and incubated them for an additional 10 days to non-enzymatically crosslink the collagen.9 This treatment increased the peak matrix force during cyclic stretch with a 3 to 4 fold increase after 14 h compared to the untreated rings incubated for 20 days. The shapes of the matrix force-strain curves changed somewhat, with a shorter period of slack during each force-strain curve. The increased peak force with glycation was similar to Isenberg and Tranquillo9 but comparing the shapes of the force-strain curves is difficult due to differences in experimental methods.
We also found that increased matrix stiffness due to crosslinking had little effect on cell force for 5% or 10% cyclic stretch amplitude at 0.1 Hz. Cell force-strain curves were almost identical for treated and untreated rings after 12 h of cyclic stretch. In 2-D cultures, cell traction depends upon the stiffness of the substrate.6 The cellular mechanism for this response is not clear, but Discher and co-workers suggest that both “outside-in” and “inside-outside-in” pathways may be involved. Whether 3-D culture conditions or cyclic stretching reduce the influence of matrix stiffness on cellular mechanics remains to be investigated.
Since it is the cells that are responding to the mechanical conditioning it is important to realize that while the cells are undergoing a higher strain they are not undergoing a higher stress. So if the cells respond to stress, higher stretch amplitudes will not increase this response. Since both the cells and the tissues at different stretch amplitudes maintain similar stresses, it is reasonable to assume the cells are responding to maintain a certain range of mechanical stresses.
Some limitations to estimating the cell-contributed force should be noted. Unlike the tissue and matrix force we are not able to measure the cell force directly but instead have to estimate it based on the direct measurement of the tissue and matrix. In addition, to estimate the cell force at more than a single time point, we must use the forces from different rings. Due to the ring to ring variations, it is necessary to normalize one ring to another to get quantitative values for the cell force. However, because this normalization occurs at 12 h, it is unknown how well this single point normalization works in predicting the cell force 12 h earlier during the initial stretches. Furthermore, the assumption that the cell force is simply the subtraction of the matrix force from the tissue force is only valid after a critical cell density is reached.11 At days 2 and 4 it is unlikely that this critical cell density is reached but it is surpassed based on cell density by day 8.
Unlike the cells that respond to increased stretch amplitude by decreasing their stiffness, the matrix force-strain curves shift so that the matrix contributes only when the strain is within 3%–7% of the maximum value with the matrix contributing over a slightly larger range of strains at higher stretch amplitudes. This response of the matrix does not appear to depend upon the cells having an actin cytoskeleton, since we found a similar response when cytoD is added either before stretch is initiated or after 12 h of stretch. After 12 h of cyclic stretch, the peak matrix force is only slightly higher with increased stretch amplitude. With different frequencies of cyclic stretch, the peak matrix force is similar but the matrix contributes over a larger range of strains at lower frequencies. For a 10% stretch, the matrix contributes to the last 6% of the stretch at 0.001 Hz while contributing only to the last 4% at 0.25 Hz.
The higher baseline tissue and cell forces seen at lower frequencies (0.001 Hz and 0.01 Hz) are consistent with a simple viscoelastic model because the tissues and cells have more time to recover before being stretched again. The same is seen in the lower amplitudes at 0.1 Hz (5% and 10%) which are able to maintain a baseline force. These correspond to the lowest strain rates and may allow for recovery.
For a linear viscoelastic material undergoing continuous cyclic stretch, both higher stretch amplitudes and higher strain rates will lead to higher peak forces. For cyclic stretch at constant frequency, the highest stretch amplitude also corresponds to the highest strain rate. However, we observed that the peak cell force decreased as the amplitude (and the strain rate) increased. We also note that the decrease in peak cell force is a function of both stretch amplitude and strain rate because similar strain rates (5%/s) at different amplitudes (e.g. 10% @ 0.25 Hz and 25% @ 0.1 Hz) show different tissue, matrix and cell responses In the case of the matrix at 25% @ 0.1 Hz, there is no positive matrix force until the strain increases beyond 10%; whereas there is matrix force below 10% strain during cyclic stretch at 10% @ 0.25 Hz.
To explain this complex behavior a model of cellular mechanics that includes strain-dependent and strain rate-dependent damage may be required. It is reasonable to assume large and/or quick deformations may result in disruption of the actin cytoskeleton8 leading to damage, and thus a reduction in the cell force and stiffness. However, any damage model will be complicated by viscoelastic effects.
One possible mechanism for a strain dependent change in stiffness is an alteration in the amount of cell activation through a stretch-activated pathway. Two of the pathways that exist to activate myosin-light chain kinase and thus force generation in fibroblasts are a calcium dependent pathway and a Rho dependent pathway.13 To test the possible involvement of a calcium dependent pathway, we tested some rings in the presence of 50 μM or 500 μM of gadolinium chloride (GdCl3) to block stretch activated calcium channels.12 In the presence of 50 μM of GdCl3, day 2 rings showed a decrease in peak tissue force at both 5% and 10% cyclic stretch compared to untreated controls. This peak force decrease continued for the first 5–6 h of cyclic stretch then began to increase for the remainder of the 12 h of cyclic stretching but still did not return to the peak tissue force of untreated controls. However, for day 8 rings neither 50 nor 500 μM GdCl3 had an effect on the tissue force compared to untreated controls. Since GdCl3 reduces the tissue and cell force during the first several hours of cyclic stretch at day 2, it suggests that the calcium dependent pathway plays a role in maintenance of cell force at least during early tissue development. It is unclear what causes the cell force to increase after the initial decrease but it could be due to an upregulation of calcium independent pathways or incomplete inhibition of new or existing stretch activated channels. The lack of an effect of GdCl3 at day 8 suggests a reduced role of the calcium dependent pathway.
In addition, these results have important implications for the tissue engineering field, especially for those tissues that undergo continuous deformations such as in the cardiovascular system. By isolating the cell contribution to tissue force, we are able to provide a better understanding of the mechanical environment of the cell and aid in designing mechanical conditioning protocols that improve tissue properties. For example, the peak cell force is not constant during cyclic stretch and thus cell stimulation varies with time. Furthermore, if cell response is dependent on cell stress or force, increasing the amplitude of conditioning will not increase and may decrease the applied cell force.
Due to the large changes in mechanical behavior after initiation of cyclic stretch, it is important to characterize engineered tissues’ response to these loading conditions over longer time periods. Current mechanical characterization relies mostly on a few cycles of preconditioning followed by a test to failure.4,5,9,16 While failure tests are necessary, the response of the tissue and cells under physiologic loading is also vital. We have shown that mechanical properties may change dramatically over hours after initiation of stretch. These changes have implications on tissue function in vivo after implantation and these changes cannot be captured in simple failure tests.
CONCLUSIONS
We have shown that bioartificial tissue constructs consisting of a single cell type and matrix component are a useful model system in which to explore tissue, matrix, and cell mechanics. The cell response to cyclic stretch is complex and depends on both the amplitude and frequency of cyclic stretch. The change in cell stiffness with amplitude behavior does not fit current cellular mechanics models. Future investigations may explore the microstructural basis for this behavior.

cardiac fibroblasts
collagen
tissue engineering
cell mechanics