Recent advances in TGF-β effects on chondrocyte metabolism

Cytokine & Growth Factor Reviews 13 (2002) 241–257

Survey

Recent advances in TGF-␤ effects on chondrocyte metabolism Potential therapeutic roles of TGF-␤ in cartilage disorders Eva Grimaud, Dominique Heymann, Françoise Rédini* Laboratoire de Physiopathologie de la Résorption Osseuse EE 99-01, Faculté de Médecine, University of Nantes, 1 rue Gaston Veil, 44035 Nantes Cedex 01, France

Abstract Novel approaches to treat osteoarthritis are required and progress in understanding the biology of cartilage disorders has led to the use of genes whose products stimulate cartilage repair or inhibit breakdown of the cartilaginous matrix. Among them, transforming growth factor-␤ (TGF-␤) plays a significant role in promoting chondrocyte anabolism in vitro (enhancing matrix production, cell proliferation, osteochondrogenic differentiation) and in vivo (short-term intra-articular injections lead to increased bone formation and subsequent cartilage formation, beneficial effects on osteochondrogenesis). In vivo induction of the expression of TGF-␤ and the use of gene transfer may provide a new approach for treatment of osteoarthritic lesions. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Cartilage disorders; Chondrocyte metabolism; Transforming growth factor-␤

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Activation, expression and regulation of TGF-␤ in healthy and pathological cartilage . . . . . . . . . . . . TGF-␤ signaling in chondrocytes and cartilage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In vivo effects of TGF-␤: intra-articular injections and effects on osteochondrogenesis . . . . . . . . . . . In vitro effects of TGF-␤ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Effect of TGF-␤ on chondrocyte proliferation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Effect of TGF-␤ on matrix metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Potential therapeutic roles of TGF-␤ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. 2. 3. 4. 5.

1. Introduction Osteoarthritis (OA) is a degenerative joint disease characterized by the destruction of articular cartilage during aging and senescence. Homeostasis of normal cartilage in adults represents a delicate balance between degradation and synthesis of the extracellular matrix of the tissue to maintain the functional integrity of the joint. A breakdown of this ∗ Corresponding author. Tel.: +33-2-40-41-28-45; fax: +33-2-40-41-28-60. E-mail address: [email protected] (F. R´edini).

241 242 243 245 246 247 248 250 252 253 253

cartilage matrix leads to the development of fibrillation and fissures and the appearance of ulcerations, together with the disappearance of the full thickness of the joint surface. Moreover, at the clinical stage of the disease, changes caused by OA involve not only cartilage, but also the synovial membrane (where an inflammatory reaction is often observed) and subchondral bone. Cartilage is mainly composed of water and a specific extracellular matrix that makes it resilient and viscoelastic. The main components of this extracellular matrix are type II collagen and high-molecular-weight proteoglycans (PGs) known as aggrecans. Collagen is responsible for the

1359-6101/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 9 - 6 1 0 1 ( 0 2 ) 0 0 0 0 4 - 7

242

E. Grimaud et al. / Cytokine & Growth Factor Reviews 13 (2002) 241–257

tensile strength of the tissue, and the polyanionic constituents of aggrecans (the glycosaminoglycan part) retain water molecules, thus contributing to the viscoelasticity of cartilage. The chondrocyte, the unique cartilage cell type, is found within this extracellular matrix. The pathways likely to account for the loss of cartilage during OA involve expression of proteinases that degrade the major matrix constituents. The specific types of enzymatic activities associated with progressive cartilage removal include those of matrix metalloproteinases (MMPs), i.e. collagenase, gelatinase and aggrecanase, that originate from both synovial cells and chondrocytes. Superficial fibrillation at the articular surface is associated with increased denaturation and loss of type II collagen from collagen fibrils, leading to a decrease of tensile properties. Damage to fibrils leads to a loss of aggrecan, but also of the small proteoglycans decorin and biglycan, which are usually associated with fibrils at the joint surface. The loss of these molecules is associated with increased cleavage of type II collagen by collagenase and of aggrecan by aggrecanase. OA data strongly support the concept that inflammatory cytokines are the major catabolic systems involved in the destruction of joint tissues. It is generally agreed that interleukin-1 (IL-1) may be the pivotal cytokine at early and late stages [1], whereas tumor necrosis factor-␣ (TNF-␣) plays a role in the inflammatory component of OA [2]. Other cytokines released during the inflammatory process in the osteoarthritic joint may be regulatory (IL-6, IL-8) or inhibitory (IL-4, IL-10, IL-13, interferon-␥) [3]. Both IL-1 and TNF-␣ have been found in increased amounts in OA synovial membrane, synovial fluid and cartilage. The imbalance between anabolic growth factors and catabolic cytokine synthesis and/or bioactivity could be the key to cartilage repair failure. However, some evidence indicates that a repair process may take place in degenerative diseases as a result of activation of chondrocyte metabolism. In fact, chondrocytes that are normally non-dividing proliferate in response to damaged extracellular matrix, forming multicellular chondrocyte clones, and produce large amounts of collagen and aggrecans during early stages of the disease. It is therefore possible that systemic or local growth factors play a role in this cartilage repair process. Integrative cartilage repair, which is necessary for durable restoration of cartilage lesions, can be regarded as a wound-healing process. The ability of chondrocytes to increase growth factor expression and restore the rapid decrease of proteoglycan content in the initial phase following acute wounding has been demonstrated [4]. Members of the transforming growth factor-␤ (TGF-␤) superfamily are thought to play key roles in chondrocyte growth and differentiation [5]. This group includes not only the five TGF-␤s (TGF-␤1-5), but also bone morphogenetic proteins [6], activins, müllerian inhibiting substance, inhibins, and growth and differentiation factors [7]. Although TGF-␤s are produced by many different cell types, the concentration of TGF-␤ is approximately 100 times greater in bone than in other tissues, and osteoblasts have a high

concentration of TGF-␤ receptors [8]. TGF-␤ is synthesized as an inactive proform due to its binding to latency-associated peptide (LAP). Activation of TGF-␤ results from proteolytic activity or extreme pH values, among other possible stimuli. TGF-␤ was first described as “cartilage-inducing factor-A” (CIF-A), because it triggered chondrogenic differentiation of embryonic rat muscle cells [9].

2. Activation, expression and regulation of TGF-␤ in healthy and pathological cartilage Articular cartilage itself contains large amounts of latent TGF-␤ [10,11] and this growth factor has been detected immunochemically in chondrocytes. In normal physiological conditions, in which plasminogen and/or plasmin is present in tiny amounts in cartilage, small quantities of active TGF-␤1 are present [10]. On the contrary, in pathological conditions such as fractures, in which chondroprogenitor cells are exposed to high concentrations of plasmin, short-term high concentrations of TGF-␤1 have been observed. Latent TGF-␤ is also present in the matrix of costochondral chondrocytes, and latent TGF-␤ binding protein-1 (LTBP-1) is responsible for storage of this complex in the matrix [11]. In addition, LTBP-2 gene expression in mouse embryos was found to be restricted to cartilage perichondrium and blood vessels, a somewhat surprising result considering that other LTBP genes are widely expressed in rodents [12]. Therefore, LTBP-2 may play a critical role in the assembly of latent TGF-␤ in cartilage. Another recent publication showed that the mechanisms of TGF-␤ activation and activity with regard to collagenase-3 modulation in cartilage appear to be controlled by furin convertase with or without mannose-6 phosphate/insulin-like growth factor-2 receptor [13]. These factors and the small latent TGF-␤ complex are increased in the deep zone of osteoarthritic cartilage, which corresponds to the preferential site of collagenase-3 production. Transglutaminases (TGase) also participate in the activation of latent TGF-␤, and elevated TGase activity in aging articular chondrocytes and may play a role in the activation of TGF-␤ in joint cartilage [14]. In addition, active matrix vesicle-associated MMPs may be involved in the activation of TGF-␤ during late chondrocyte hypertrophy and mineralization of growth plate cartilage [15]. A recent study found that matrix vesicles produced by growth plate chondrocytes contain MMP-3 (stromelysin-1), an enzyme at least partially responsible for activation of small latent TGF-␤1, and that 1.25(OH)2 D3 regulates MMP release from matrix vesicles [16]. The temporal and spatial expression of TGF-␤s and their receptors indicates that TGF-␤1 is present in superficial, transitional and least mature zones, including hypertrophic chondrocytes [17]. TGF-␤ receptor type I (T␤R-I) and T␤R-II are co-expressed with the ligand in these three zones throughout the growth phase. Two independent studies

E. Grimaud et al. / Cytokine & Growth Factor Reviews 13 (2002) 241–257

[18,19] detected the three TGF-␤ isoforms and their receptors at sites of endochondral and intramembranous ossification and found that TGF-␤1 expression was restricted to the proliferative and upper hypertrophic zones. Concerning endochondral ossification, TGF-␤1 and TGF-␤3 were expressed from 6 to 24 weeks in resting, proliferating and maturing zones, while TGF-␤2 was expressed at 6 weeks, but decreased during growth [20]. Unexpectedly, TGF-␤s expression in hypertrophic chondrocytes was weak. T␤R-I was co-expressed with the ligand in resting, proliferating and maturing zones throughout development. When TGF-␤ expression was studied during condylar cartilage development, it appeared that the growth factor was highly expressed in mature and degenerative, as compared to germinal and transitional, layers [21]. During post-natal development of porcine mandibular condylar cartilage, TGF-␤1 showed a significant increase at 24 and 36 months of age, whereas TGF-␤2 increased significantly at 6, 12, 24 and 36 months and TGF-␤3 remained stable at all stages [22]. Changes in TGF-␤1 concentration were investigated in joint fluid of healthy rabbit knees during maturation and following osteochondral injury and spontaneous repair [23]. This study concluded that TGF-␤1 concentration was higher in younger animals, reflecting a better healing capacity, but also a greater susceptibility to osteoarthritic change than for adult animals. During osteoarticular pathologies, synovial fluids of patients with OA contain significant levels of active TGF-␤ [24]. In a recent study, van den Berg reported that TGF-␤ was found in inflamed synovia [25]. Local co-administration of TGF-␤ further enhanced the degree of synovitis, yet prevented cartilage degradation almost completely. These results provide another example of a major lack of correlation between inflammatory mass and destructive potential. Changes in TGF-␤ production and/or expression during osteoarticular diseases have been widely reported. Concerning OA, the mRNAs of TGF-␤ are elevated at an early phase during cartilage repair after moderate proteoglycan depletion, which implies a functional role for these molecules in the repair process [26]. However, the distribution of the three TGF-␤ isoforms differs according to localization in the pathological joint or the experimental model considered. The kinetics of TGF-␤s production in arthritic limbs of mice with collagen-induced arthritis (CIA) indicated the presence of locally produced TGF-␤2 within the lining layer, sublining and pannus at all disease stages, whereas TGF-␤3 was only expressed in scattered cells within the deeper layers of the synovia [27]. The same CIA model showed an abundant expression of all three TGF-␤ isoforms (␤1, ␤2 and ␤3) and their receptors in arthritic synovia, which is of pathogenic importance in the development of CIA [28]. In another experimental model involving STR/ort mice, which develop osteoarthritic lesions of the knee joint, an up-regulation of anabolic growth factors occurred, including TGF-␤1 and catabolic cytokines in the prelesional stages of OA, and anabolic effects predominated [29]. Still another study indicated that TGF-␤1

243

expression in osteophytes of human femoral heads during OA was localized in superficial cells of osteophyte cartilage [30]. Moreover, in a model of experimental osteolathyrism used to study the effect of altered extracellular matrix on the expression of connective tissue components, TGF-␤1 was up-regulated during the cartilagenous stage [31]. Investigations of the potential role of TGF-␤ in fracture healing found that differential expression of TGF-␤2 and TGF-␤3 isoforms occurred, whereas small amounts of TGF-␤1 were present in early callus and increased in expression during chondrogenesis and endochondral ossification [32]. More recently, elevated expression of TGF-␤1 and bone morphogenic protein-2 (BMP-2) was associated with heterotopic endochondral ossification, with mixed tumor formation, in C3(1)/Tag transgenic mice [33]. TGF-␤s expression was also investigated in other osteoarticular pathologies (osteochondrosis and dyschondroplasia) exhibiting cartilage disorders characterized by a failure of chondrocyte differentiation, matrix mineralization and replacement of matrix by bone. A recent study of articular cartilage from affected joints of horses with naturally acquired osteochondrosis and of corresponding joints of clinically normal horses showed increased expression of TGF-␤1, as well as IGF-1 and type I collagen, in cartilage from osteochondrosis lesions [34]. These changes were probably indicative of a healing response to injured tissue rather than a primary alteration. Other studies found deficiencies in TGF-␤ in chondrocytes at sites of porcine osteochondrosis and of TGF-␤3 in avian dyschondroplasia [35,36]. Similar results were reported for alterations of TGF-␤1 expression in dyschondroplastic lesions in the horse [37]. Conversely, in chick embryonic cartilage, the distribution and intensity of TGF-␤ labeling remained unchanged between chondrocytes of the tibial dyschondroplasia (TD) and normal growth plate [38]. Furthermore, TGF-␤1 in growth plates of two lines of broiler chickens with low and high incidences of TD was immunolocalized at low concentrations in the extracellular matrix adjacent to collapsed cartilage canals of the high TD incidence line. This suggests that TGF-␤ is a factor limiting vascular invasion of dyschondroplastic cartilage of TD lesions [39]. Interestingly, a mutation in human cartilage-derived morphogenetic protein-1, a new member of the TGF-␤ superfamily, was demonstrated in association with recessive human chondrodysplasia [40].

3. TGF-␤ signaling in chondrocytes and cartilage The TGF-␤ family controls proliferation, extracellular matrix and/or differentiation in articular chondrocytes, and the molecular mechanisms governing ligand binding, receptor oligomerization and signal transduction begin to be elucidated (Fig. 1). The strategic disruption of TGF-␤ types I and II receptor interactions can in fact alter specific cellular responses to TGF-␤ signaling [41]. With respect to TGF-␤ transduction pathways, previous studies have shown that this growth factor stimulates protein kinase C

244

E. Grimaud et al. / Cytokine & Growth Factor Reviews 13 (2002) 241–257

Fig. 1. Main TGF-␤ signaling pathways in chondrocytes and cartilage. AP-1: activation protein-1, ERK: extracellular-signal regulated kinase, LAP: latency-associated-protein, MEK: MAPK ERK kinase, MMPs: matrix metalloproteinases, PKC: protein kinase C, T␤-R: TGF-␤ receptor, TRE: TGF-␤ response element, VDR–RXR: vitamin D receptor/retinoic acid X receptor.

(PKC) via a mechanism independent of phospholipase C or tyrosine kinase. However, a single study reported the involvement of tyrosine kinase in TGF-␤ signaling, showing that H7-sensitive serine/threonine kinase, as well as herbimycin A- and genistein-sensitive protein tyrosine kinases, is implicated in TGF-␤-induced tissue inhibitor of metalloproteinase-3 (TIMP-3) gene expression [42]. The mechanism of TGF-␤1-induced cellular proliferation was examined using cultured rat articular chondrocytes (CRAC). Addition of TGF-␤ caused immediate and transient induction of c-fos, but not myc or jun mRNA, which suggests that TGF-␤1 acts as a primary mitogen in CRAC and that

this mitogenic activity requires PKC activation. Finally, induction of c-fos expression subsequently occurs through an as yet unidentified transactivation mechanism [43]. It was previously reported that TGF-␤ effects on chondrocyte growth regulation and matrix gene transcription involve two different signaling pathways and that inositolphosphate glycan is only implicated in growth regulation [44]. Moreover, TGF-␤ specifically activates mitogen-activated protein kinase 1 (MEK1) and subsequent extracellular signal-regulated kinase (ERK) pathways in CRAC. The activation of this MAPK pathway is involved in mitogenic response to TGF-␤1 [45]. In the same model, TGF-␤

E. Grimaud et al. / Cytokine & Growth Factor Reviews 13 (2002) 241–257

was shown to transduce a predominant signal pathway through MEK–ERK–ELK1, independent of MEK kinase 1 (MEKK1) or TAK1. Moreover, the MEK–ERK pathway activated by TGF-␤ was negatively regulated by a PKA cascade, but transactivated by PKC [46]. In the same model, DNA synthesis and transactivation of type II collagen by TGF-␤ were down-regulated by a specific MEK inhibitor [47]. In addition, dexamethasone, which accelerates cartilage degradation, suppressed TGF-␤-induced chondrocyte proliferation and type II collagen expression, probably through selective inhibition of ERK integrated AP-1 activation. A study using murine ATDC5 chondrocytes showed that the effect of TGF-␤ on inorganic phosphate uptake did not involve PKC or mitogen-activated protein kinases, but possibly a Smad-dependent signaling pathway [48]. Another study indicated that TGF-␤ modulates its effects on matrix production through PKC, but that its effects on alkaline phosphatase and cell proliferation are mediated by PGE2 and protein kinase A production [49]. The effect of TGF-␤ on chondrocyte differentiation was previously shown to be mediated through PKC [50]. The addition of 24,25-dihydroxyvitamin D3 and TGF-␤ produced synergistic effects on resting chondrocyte (RC) alkaline phosphatase-specific activity [51], whereas the addition of 1,25 plus TGF-␤ and 24,25 plus TGF-␤ to growth chondrocytes (GC) and RC cells, respectively, produced a synergistic increase in 35 S-sulfate incorporation and had an additive effect on 3 H-thymidine incorporation. Moreover, the synergistic effect between 24,25 and TGF-␤ was mediated by PKC activation via two parallel pathways: 24,25 through diacylglycerol production and TGF-␤ through G protein activation. Members of the TGF-␤ family transduce signals from the cell membrane to the nucleus via specific types I and II receptors and Smad proteins. More specifically, Smad2 and Smad3 transduce TGF-␤ signaling, Smad4 is a common mediator for BMP and TGF-␤ pathways, and Smad6 and Smad7 inhibit signaling by members of the TGF-␤ superfamily. In cartilage, Smad2 was strongly expressed in proliferating chondrocytes, whereas Smad3 was mainly observed in maturing chondrocytes [52]. Smad4 was broadly expressed in all zones of epiphiseal plate, and the inhibitory Smads (Smad6 and Smad7) were strongly expressed in the cartilage zone containing mature chondrocytes. Moreover, TGF-␤/Smad3 signals inhibit terminal hypertrophic differentiation of chondrocytes and are essential for maintaining articular cartilage, as demonstrated by the use of mutant mice homozygous for a targeted disruption of Smad3 exon 8, which developed degenerative joint disease resembling human OA [53]. The roles of Smad proteins as mediators of TGF-␤ effects on chondrocyte maturation have also been investigated, and Smad2 and Smad3 were found to be key mediators of the inhibitory effect of TGF-␤1 signaling on chondrocyte maturation [54]. The involvement of Smad and MAPK pathways has also been studied in the chondrogenic cell line ATDC5 by means of a TGF-␤-induced effect on aggrecan expression [55]. It ap-

245

peared that TGF-␤ induced rapid, transient phosphorylation of Smad2, and that ERK 1/2 and p38 MAPK activation was also required for TGF-␤-induced aggrecan expression in confluent ATDC5 cells. The molecular mechanisms governing TGF-␤-induced effects on gene transcription in chondrocytes are beginning to be elucidated. The single study to date reported the involvement of a composite element binding vitamin D receptor and retinoic X receptor-␣ that mediates TGF-␤ inhibition of decorin gene expression in articular chondrocytes [56].

4. In vivo effects of TGF-␤: intra-articular injections and effects on osteochondrogenesis The numerous in vivo studies concerning the effect of intra-articular injections TGF-␤ on cartilage metabolism have given quite controversial results, depending on the delay of the treatment. Indeed, the overall results show that short-term treatment leads to an enhancing effect on cartilage metabolism whereas long-term studies demonstrated disturbance of cartilage homeostasis (Fig. 2). Increased bone and subsequent cartilage formation occurred when TGF-␤2 was injected daily into the periosteum of neonatal animals [57]. After five injections, chondrocytes expressing type II collagen mRNA were found around the injection site. In a unilateral model of arthritis, local administrations of TGF-␤1 failed to modify inflammation, but clearly stimulated PG synthesis and restored the PG content of depleted cartilage [58]. TGF-␤2 also prevented the impaired chondrocyte proliferation induced by unloading in growth plates of young rats [59]. Other studies have recently reported a more complex role for TGF-␤ in cartilage formation. TGF-␤ not only modulated the metabolism of articular cartilage in general, but was also targeted to specific subcompartments of the matrix, decreasing collagen volume density pericellularly while increasing it in the intermediate zone [60]. A rapid decrease in the size and number of hypertrophic cells and enhanced subchondral bone formation following intra-articular injections of TGF-␤ into the knee joint of growing rats have also been reported [61]. From day 7 to about 3 weeks, the matrix was stained intensively with safranin O for proteoglycans, but long-term studies found destroyed cartilage in three of six rats, and partial ossification in two rats after 90 and 180 days. A more recent work clearly showed that multiple intra-injections of TGF-␤ induced prolonged disturbances of articular cartilage PG homeostasis, including focal PG depletion and cracks at the level of the tide-mark in several TGF-␤-injected joints, which were quite similar to features of experimental and spontaneous OA in mice [62]. Another property of TGF-␤ concerns its effects on the osteochondrogenic potential of marrow mesenchymal progenitor cells. After exposure to TGF-␤, in vitro and in vivo results are discordant, suggesting that the osteochondrogenic potential is further modulated by the environment in

246

E. Grimaud et al. / Cytokine & Growth Factor Reviews 13 (2002) 241–257

Fig. 2. In vivo effects of intra-articular injections of TGF-␤ on cartilage/chondrocyte metabolism and osteochondrogenesis. MSC: mesenchymal stem cells, coll II: type II collagen.

which the mesenchymal cells are assayed [63]. Another study indicated that TGF-␤1 enhances chondrogenic differentiation of bone-marrow-derived mesenchymal stem cells (MSC) in a dose-dependent manner [64]. Moreover, the distinct chondrogenic pattern of TGF-␤ isoforms depends on the embryonic stage [65], which suggests that TGF-␤ isoforms enhance cartilage differentiation to higher levels in micromass cultures than in situations in which little or no chondrogenic differentiation normally occurs [66]. The ATDC5 cell model was used to investigate the mechanisms underlying the role of TGF-␤ in chondrogenesis, and the results indicate that TGF-␤ stimulates chondrogenesis via initiation of cellular condensation by transition from an initial N-cadherin-contributing stage to a fibronectin-contributing stage during chondrogenetic processes [67]. In fact, chondrogenesis seemed to require the interaction of TGF-␤s and BMPs, while apoptosis was regulated by BMPs alone [68]. Furthermore, TGF-␤s induced the expression of the BMP receptor gene bmpR-1␤ and promoted ectopic chondrogenesis [69]. Another possibility is that the NH2-propeptide of type IIA collagen could act in the extracellular matrix distribution of BMPs and TGF-␤1 in chondrogenic tissue [70]. The results obtained are divergent, in particular with calvarial defects of adult baboons, showing limited chondro-osteogenesis by TGF-␤1 [71]. The same study also reported that rTGF-␤1 induces endochondral bone in the baboon [71] and synergizes with rBMP-7 to initiate rapid bone formation [72]. A contradictory study found TGF-␤1 inhibition of both chondrogenesis and osteogenesis, together with matrix calcification [73]. This effect was greater in cell populations with the highest proliferation rate, possibly because of inhibition of the production of matrix substance rather than of osteoblast proliferation and differentiation.

TGF-␤2 has also been implicated in the inhibition of chondrogenesis caused by Am-80 in limb bud cells [74]. On the contrary, culturing of cells derived from the perichondrium in the presence of both IGF-1 and TGF-␤2 led to increased glycosaminoglycan production and induction of type II collagen, which suggests that these growth factors are highly involved in chondrogenesis [75]. The loss of responsiveness to TGF-␤ through the use of truncated, kinase-defective TGF-␤ type II receptor in mouse tissue promoted terminal chondrocyte differentiation and resulted in the development of joint disease resembling OA [76]. Recent data support the hypothesis that TGF-␤ acts upstream of PTHrP, which has also been shown to regulate chondrocyte differentiation in vivo as well as the rate of hypertrophic differentiation [77]. This suggests that TGF-␤ has both PTHrP-dependent and -independent effects on endochondral bone formation.

5. In vitro effects of TGF-␤ The effects of TGF-␤ on the metabolism of cultured chondrocytes are divergent and depend largely on the experimental conditions used. This section reviews the effects of TGF-␤ on cartilage explants, chondrocytes cultured in monolayers or in three-dimensional conditions, and mesenchymal chondroprogenitor cells (Fig. 3). The increasing effect of TGF-␤ on the synthesis and secretion of cartilage proteoglycans was first reported 10 years ago [78]. Since then, several other studies on cartilage explants have been performed. The temporo-mandibular joint of aged mice is an experimental model commonly used to develop osteoarthritic degenerative lesions. The stimulatory action of TGF-␤ on PG production and cell proliferation in

E. Grimaud et al. / Cytokine & Growth Factor Reviews 13 (2002) 241–257

Fig. 3. Experimental in vitro approaches to study the effects of TGF-␤ on cell proliferation, matrix production and osteogenesis induction.

the temporo-mandibular joint of aged mice suggests that a repair process may exist in osteoarthritic cartilage [79]. Similarly, the association of TGF-␤ with other growth factors or hormones (IGF-1 and GH) increased the size and area of toluidine blue staining and enhanced incorporation of 35 S-sulfate in the same culture explant model [80]. TGF-␤1 also enhanced the incorporation of both 3 H-thymidine and 35 S-sulfate into cartilage cultures of aged mice, indicating that chondrocytes could be stimulated in vitro in OA degenerating cartilage to proliferate and synthesize new matrix through induction of anabolic activity in the tissue [81]. The role of TGF-␤ in protecting against cartilage collagen destruction has been shown in a model of cartilage explants cultured in the presence of IL-1 and oncostatin M [82]. Another parameter that could alter TGF-␤ response is the age of the donor. However, enhanced synthesis of PG induced by TGF-␤1 was demonstrated at all ages [83]. Although TGF-␤1 levels were highest at all ages, the expression of the three isoforms decreased with age. Osteoarthritic cartilage appears to be more sensitive to TGF-␤ than normal cartilage, and TGF-␤ is capable of redifferentiating phenotypically altered chondrocytes in OA [84]. Lastly, TGF-␤ can up-regulate the level of collagenase-3 and cause mimicking in vitro in normal cartilage of the distribution observed in situ in arthritic cartilage [85]. 5.1. Effect of TGF-β on chondrocyte proliferation The effects of TGF-␤ on the proliferation rate of articular chondrocytes have been widely studied, but with controversial results depending on culture conditions, i.e. the presence of growth factors and/or serum in the medium, the type of culture tested (monolayers, three-dimensional, suspension, etc.) and the physiopathological origin of the sample.

247

Overall, most studies have reported an enhancing effect of TGF-␤ on 3 H-thymidine uptake and/or cell number, e.g. in scleral chondrocytes [86], high density primary cultures of rib and ear chondrocytes [87], monolayer cultures of bovine articular chondrocytes [88], monolayers of human intervertebral disk cells transferred into three-dimensional cultures [89], chondrosarcoma chondrocytes [90], monolayer cultures of fetal equine chondrocytes [91], or chondrocytes from the resting and growth zones of costochondral cartilage [92]. However, this stimulating effect was less convincing in some cases, and differed according to the proliferation state of the chondrocyte itself, i.e. inhibiting cell proliferation with quiescent chondrocytes and stimulating synchronized populations in S phase [93]. Another study concerning the effect of TGF-␤ on cell proliferation during subculturing of chondrocytes showed that the growth-stimulatory action of TGF-␤ observed in differentiated chondrocytes decreased during subcultures and that cells in later passages were even growth-inhibited by TGF-␤ [94]. The TGF-␤1 responsiveness of chondrocytes from normal and osteoarthritic patients indicated that the growth pattern was more rapid for the primary osteoarthritic chondrocyte cultures with TGF-␤1 than for normal cells with and without TGF-␤1 and for osteoarthritic chondrocytes without TGF-␤, even though TGF-␤1 increased 3 H-thymidine uptake in each group. Moreover, TGF-␤1 failed to inhibit or delay the dedifferentiation process (loss of phenotype) induced by subculturing the chondrocytes in monolayers [95]. In addition to the differentiation state of the cells, another parameter that influences chondrocyte responsiveness to TGF-␤ is the presence of other growth factors and/or serum in the culture medium. For example, rabbit articular chondrocytes exposed to a combination of TGF-␤ and bFGF underwent a 136-fold increase in cell number [96]. IGF-1 also possesses growth-promoting activity on chondrocyte proliferation, and chondrosarcoma chondrocytes are positively regulated by IGF-1 and TGF-␤1, which interact to augment the mitotic action of chondrocytes [90]. The presence of ascorbic acid was also found to be important. An optimal scheme in which TGF-␤2 was added in primary culture and during the first passage, followed by addition of L-ascorbic acid during the second and third passages, resulted in a seven-fold increase in cell number [88]. Another study showed no remarkable change in DNA synthesis in the presence of TGF-␤1 or TGF-␤2 alone, whereas the combination of either TGF-␤1 or TGF-␤2 with FGF had a synergistic effect on cell proliferation in articular chondrocytes obtained from rabbit knee during the first days after cast immobilization [97]. Concerning the effect of serum, the total DNA content of equine chondrocytes cultured in three-dimensional matrix increased with the addition of TGF-␤1 in FBS-supplemented conditions and decreased in serum-free conditions [98]. In addition to quantitative changes in the proliferative responses of chondrocytes with increasing age, a qualitative change occurred in the pattern of growth factor responses during development. Cells from young donors responded better to

248

E. Grimaud et al. / Cytokine & Growth Factor Reviews 13 (2002) 241–257

PDGF-AA than to TGF-␤, while the opposite pattern was seen in cells from adult donors [99], indicating a profound decline in levels of DNA synthesis and cell replication in response to known chondrocyte growth factors. Most studies have reported an overall stimulatory effect of TGF-␤ on chondrocyte proliferation, but a few found an inhibitory effect. A 48 h treatment of serum-deprived semiconfluent human fetal epiphyseal chondrocytes with TGF-␤1 decreased 3 H-thymidine incorporation and cell number [100]. Similarly, the total number of porcine auricular chondrocytes cultured in vitro in the presence of TGF-␤, decreased after 3 weeks as compared to controls and exhibited fibrous tissue ingrowth when reimplanted in vivo [101]. 5.2. Effect of TGF-β on matrix metabolism The anabolic properties of TGF-␤ on cartilage extracellular matrix metabolism have been well established, but a growing body of evidence now suggests that these effects are not so apparent (Table 1). When considering chondrocyte proteoglycan synthesis or expression, most of the investigaTable 1 In vitro effects of TGF-␤ on chondrocyte matrix metabolism Culture condition and function studied

TGF-␤ effect

Reference

− + + + + − − + + + No change

[103] [103] [104] [105] [89] [89] [56] [87] [106] [107] [97]

Human nasal sceptal chondrocytes (Monolayer/soft agar) 35 S-sulfate

+

[108]

Equine chondrocytes (EC) (3D) serum-free (3D) in presence of serum (Monolayer) 35 S-sulfate

+ − +

[98] [98] [91]

AHAC (Suspension cultures)

Proteoglycan expression and synthesis RAC (monolayer/alginate) decorin expression Biglycan expression Cell-surface PGs synthesis Small PGs synthesis Biglycan synthesis Decorin synthesis (Monolayer) decorin gene regulation (High density primary culture) 35 S-sulfate (Monolayer) presence or not of serum (Monolayer) dedifferentiation process 35 S-sulfate

+

[109]

BAC alginate Aggrecan gene expression PGs synthesis Aggrecan, decorin mRNA 35 S-sulfate

± + + +

[110] [111] [111] [112]

Chondrosarcoma 35 S-sulfate

+

[90]

Rat costochondral (GC and RC) 35 S-sulfate

+

[92,113]

Rat auricular chondrocytes 35 S-sulfate (several exp conditions)

+

[102]

35 S-sulfate

Table 1 (Continued) Culture condition and function studied

Collagen and matrix expression and production Type II collagen (Monolayer RAC) 3 H-pro differentiated Dedifferentiated (EC in 3D) 3 H-pro, 3 H-leu TGF-␤ alone Plus bFGF (AHAC susp culture) + IGF-1 (BAC alginate) + extracellular collagen II (Primary RAC) + bFGF (EC in alginate) mRNA Matrix composition Chondrosarcoma + IGF-1 BAC monolayers ␣1 integrins ␣3 and ␣5 AHAC susp + IGF-1 type I collagen Protease expression and activity Proteases activity (BAC and HAC) mRNA TIMP-3 (HAC) caseinase activities (HAC) adjacent OA lesions mRNA MMPs Distant from OA lesions MMP-13 MMP-1 MMP-8 (OA HAC) collagenase-3 production Equine chondrocytes in alginate mRNA MMP-9 MMP-9 activity

TGF-␤ effect

Reference

− +

[107] [107]

No change + + + − +

[98]

+ + − + +

[115] [90] [116] [116] [109]

− + − − + − − + + +

[115] [117] [118] [119] [119] [119] [119] [120] [114] [114]

[98] [109] [110] [96] [114]

AHAC: adult human articular chondrocytes, BAC: bovine articular chondrocytes, bFGF: basic fibroblast growth factor, EC: equine chondrocytes, exp: experimental, GC: growth chondrocytes, HAC: human articular chondrocytes, IGF: insulin-like growth factor, leu: leucine, MMP: matrix metalloproteinase, OA: osteoarthritic, pro: proline, PG: proteoglycan, RAC: rabbit articular chondrocytes, RC: resting chondrocytes, susp: suspension, TGF-␤: transforming growth factor-␤, TIMP: tissue inhibitor of metalloprotease, 3D: three-dimensional.

tions first studied the effect of TGF-␤ on aggrecan production in terms of total 35 S-sulfate incorporation in various experimental conditions. In all cases reported within the last 5 years, total PG production appears to have been enhanced by the growth factor, regardless of the type of culture (suspension, monolayer, three-dimensional) or cell type used (RAC in monolayers or in alginate, human nasal chondrocytes, equine chondrocytes, BAC in alginate, chondrosarcoma, GC and RC costochondral chondrocytes, rat auricular chondrocytes, etc.) [87,90–92,102,106–113]. Moreover, the influence of the differentiation stage of auricular chondrocytes on the effect of TGF-␤ indicated that the amount of PGs was increased in all chondrocyte populations, i.e. that the effect of TGF-␤ on PG synthesis does not depend on the differentiation stage of the cells [102]. On the other hand, the pericellular matrix was found to be rather important for regulation of the effect of TGF-␤ on proteoglycan synthesis, indicating that, matrix depletion in pathological cartilage might trigger increased matrix synthesis in

E. Grimaud et al. / Cytokine & Growth Factor Reviews 13 (2002) 241–257

reaction to TGF-␤. Apart from these studies, very few have reported an inhibitory effect of TGF-␤ on PG synthesis or expression. However, the small proteoglycan decorin seems to be inversely regulated by TGF-␤ as compared to the major aggrecan or biglycan [56,89,103]. In fact, decorin exhibits particular properties towards TGF-␤, as it can bind to the growth factor and inhibit its biological properties. Thus, a negative feedback regulation may occur, leading to inhibition of decorin expression by TGF-␤. The presence of serum in the culture medium may also have a direct influence on TGF-␤ action, as reported for equine chondrocytes cultured in solid three-dimensional fibrin matrix [98]. This study showed that total PG accumulation and 35 S-labeled PG synthesis in cultures were increased by addition of exogenous TGF-␤1 in serum-free conditions and decreased by the growth factor in FBS-supplemented conditions. Moreover, the molecular weight of the PGs synthesized was also affected by the presence of serum, leading to production of low-molecular-weight PGs in serum-free conditions and larger-sized PGs in FBS-supplemented conditions. However, the molecular weight of PGs was unchanged by the addition of TGF-␤. The extracellular environment appears to be more critical concerning the effect of TGF-␤ on collagen synthesis and/or expression. The effect of the growth factor varied with the differentiated state of the chondrocytes. An inhibitory effect on collagen synthesis was observed in primary monolayer cultures of RAC, whereas TGF-␤ stimulated production throughout subsequent passages. These effects were correlated with the steady-state levels of mRNA encoding types I, II and III procollagens [107]. In another study, the in vivo role of the extracellular matrix (collagen type II) and the manner in which it interfaces with TGF-␤ were investigated. Extracellular type II collagen was found to augment the TGF-␤1-stimulated increase of ␣1 (II) procollagen gene expression in a dose-dependent manner [110]. Secondary chondroprogenitor cells were obtained by modulating the chondrocyte phenotype with growth factors and stimulating the proliferation of these cells in culture. Rabbit articular chondrocytes exposed to a combination of both TGF-␤ and bFGF ceased production of type II collagen and underwent a 136-fold increase in cell number [96]. The cells were then placed in high-density culture and reexpressed the chondrocyte phenotype in vitro. Other parameters related to matrix composition have been studied in the presence of TGF-␤, and the results have indicated overall stimulation of matrix synthesis in chondrosarcoma [90] and of collagen type I in adult human articular chondrocytes cultured in suspension in the presence of IGF-1 [109]. The effect of TGF-␤1 was also determined on chondrocyte ␤1 integrin expression and integrin-mediated adhesion to extracellular matrix proteins [116]. The results indicate that TGF-␤ decreased the cell surface level of ␣1␤1, whereas ␣3/␣5 ␤1 integrins were increased. TGF-␤ treatment resulted in a decrease in the adhesion of chondrocytes to type IV collagen, while adhesion to type II collagen and fibronectin was stimulated.

249

In addition to its anabolic properties, TGF-␤ modulates protease activities and expression, although its action in vitro is puzzling. TGF-␤ was first described as an inhibitor of metalloproteinase activities, thereby preventing the digestion of extracellular matrix molecules [121]. Further experiments indicated that this growth factor is able to inhibit the IL-1␤-induced increase in neutral proteinase activity [122] and collagenase activity [123]. More recent studies have reported contradictory results. Even though caseinase activity was shown to be inhibited in HAC [118] and TIMP mRNA expression was stimulated in BAC and HAC [117], collagenase-3 production appears to be stimulated by TGF-␤ in human osteoarthritic chondrocytes [120], as well as the expression and activity of MMP-9 in equine chondrocytes cultured in alginate [114]. Another study investigated the ability of TGF-␤ to modulate collagenase expression and synthesis in OA chondrocytes [119]. The results showed non-coordinate regulation after stimulation with TGF-␤, i.e. chondrocytes immediately adjacent to the OA lesion down-regulated collagenase proteins and mRNA upon incubation with TGF-␤1, whereas chondrocytes more distant from the macroscopic lesion increased MMP-13 mRNA, while MMP-1 and MMP-8 decreased after stimulation with TGF-␤1. Other parameters of chondrocyte metabolism have been studied in the presence of TGF-␤, including TGF-␤, which is able to up-regulate IGFBP-3 protein levels and mRNA expression in human fetal epiphyseal chondrocytes [100]. The expression of parathyroid hormone-related protein (PTHrP), a major locally expressed regulator of the proliferation, differentiation, synthetic functions and mineralization of chondrocyte growth cartilage, has been studied in normal knee cartilage samples and cultured articular chondrocytes as well as in cartilage specimens from knees with advanced OA. PTHrP 1–173, one of the three alternatively spliced isoforms, was exclusively expressed and induced by TGF-␤ in cultured chondrocytes [124]. The regulation of PTHrP expression by TGF-␤ has also been studied in epiphyseal and growth plate chondrocytes [125]. The results showed a marked increase in PTHrP mRNA levels by TGF-␤1, TGF-␤2 and TGF-␤3 in epiphyseal chondrocytes. Articular cartilage chondrocytes have the unique ability to elaborate large amounts of extracellular pyrophosphate (PPi), and TGF-␤ appears to be the only cartilage regulatory factor stimulating PPi production. Moreover, TGF-␤ induced comparable increases in the activity of extracellular PPi, intracellular PPi, and cellular PPi-generating enzyme NTP, whose activity is largely attributable to plasma cell membrane glycoprotein-1 (PC-1), which is also stimulated by TGF-␤. Another study found that aging had a differential effect on TGF-␤-induced PPi accumulation versus proliferation in human articular chondrocytes. In fact, TGF-␤ increased PPi levels to a greater extent in chondrocytes from subjects in the older age group than in those obtained from younger subjects [126].

250

E. Grimaud et al. / Cytokine & Growth Factor Reviews 13 (2002) 241–257

The chondrogenic potential of marrow-derived progenitor cells is mediated by numerous factors including TGF-␤1 [127]. The need for TGF-␤1 in this system is not surprising, because this growth factor is found in abundant amounts and plays a role in chondrogenic transformation. Successful in vitro chondrogenesis has been achieved using cells derived not only from bone marrow but also from periosteum and synovium [128]. As there is some evidence that mesenchymal progenitor cells especially (more than differentiated chondrocytes) seem to be targets of TGF-␤-promoted effects [129], the influence of TGF-␤ on the expression and maintenance of the chondrocyte phenotype has been well documented. TGF-␤ influences both the proliferative characteristics and the phenotype of several mesenchymal-derived tissues in vitro and in vivo. One study found that exogenous addition of TGF-␤1 significantly upregulated the expression of type II collagen mRNA in perichondrial cells, suggesting that the chondrocytic phenotype of explant cultures of perichondrium-derived cells is enhanced by exogenous addition of TGF-␤ [130]. In the same experimental model, addition of TGF-␤1 to the culture media of aged perichondrium-derived cells stimulated 3 H-thymidine incorporation and PG synthesis and up-regulated transcriptional expression of the type II collagen gene [131]. Another study found that TGF-␤ associated with a reconstituted basement membrane Matrigel stimulates the chondrogenic phenotype in osteoblastic cells derived from fetal rat calvaria by promoting the appearance of chondroblastic cell aggregates [132]. As TGF-␤1, TGF-␤3 and TGF-␤5 enhanced the expression of N-cadherin, N-CAM, fibronectin and tenascin differentially in precartilage condensations, it was suggested that TGF-␤ isoforms play an important role in the establishment of cell–cell and cell–matrix interactions during precartilage condensations [133]. The authors also showed that TGF-␤1 and TGF-␤5 enhanced PG synthesis in mouse embryonic limb bud mesenchymal cells, while PG catabolism was not affected. Cellular density also had a considerable influence on TGF-␤ actions, as only high-density cultures displayed increased stimulation of PG synthesis, as compared to low and intermediate densities. Secondary chondroprogenitor cells were obtained by modulating the phenotype of articular chondrocytes with growth factors, including TGF-␤, and stimulating the proliferation of these cells in culture [80]. These human secondary chondroprogenitor cells formed only cartilage tissue when assayed in vivo and in tissue bioreactors (an approach essential for autologous repair of degenerated articular cartilage in elderly patients with OA). The composition of the pericellular environment appears to be a critical factor, and the use of specific matrices has influenced chondrocyte response to growth factors such as TGF-␤ in experimental models of cartilage defects. For example, the influence of TGF-␤1 in vitro was determined on transplanted periosteal cells in a fibrin gel in order to repair full-thickness cartilage defects in rabbit knees [134]. A reduction of the cartilage layer was observed under the influence of TGF-␤1, whereas osteochondral integration

and zonal architecture were improved as compared to the control group in which no repair was noted. Lastly, equine mesenchymal stem cells cultures exposed to TGF-␤1 had increased cellular density, with cell layering and nodule formation, that was more marked in cultures treated with TGF-␤1 [135]. The expression of collagen types I and II was up-regulated by TGF-␤1, and treatment of MSC with TGF-␤1 led to dose-related increases in the area and intensity of type II collagen immunoreaction, suggesting that TGF-␤1 enhances chondrogenic differentiation of bone-derived MCS in a dose-dependent manner. Taken together, the majority of the studies concerning TGF-␤ effects on chondrocyte metabolism indicate that this growth factor has a stimulatory effect on cell proliferation and matrix production. However, the cell environment and especially the pericellular matrix composition seem to be critical factors in TGF-␤ response and need to be considered in the development of therapeutic tools involving TGF-␤.

6. Potential therapeutic roles of TGF-␤ Damaged articular cartilage has a limited capacity for repair, partly because of the avascular nature of cartilage and the fact that chondrocytes entrapped in their matrix are insufficiently mobilized to areas of injury. The few cells mobilized to the injury site can set-up a repair matrix, but this neomatrix is morphologically, chemically and mechanically inferior to the original articular cartilage. Therefore, despite numerous surgical options, OA remains incurable, and a better approach to its treatment is imperative. In vivo induction of the expression of growth factors with anabolic properties such as TGF-␤ and the use of gene transfer may provide a new approach for treatment of the disease (Fig. 4). Genes can be transferred into joint cells or MSC by in vivo and ex vivo methods, as reported in several studies. TGF-␤ gene transfer can be considered as gene addition, because its expression in OA is weak and may be enhanced by gene therapy. Both ex vivo delivery using retroviruses and in vivo delivery using adenoviruses, liposomes and naked DNA have proved effective. The indirect ex vivo method is complex and involves several steps including the removal of joint cells (synovium, chondrocytes) or mesenchymal cells from bone marrow, in vitro transfection of the cells by a viral or non-viral gene transfer method, and reintroduction of the transduced cells into the joint. In vitro transfection of joint cells by TGF-␤ has been studied using different joint cells, which in each case led to enhancement of matrix production by the infected cells. A first study investigated the in vitro adenoviral transduction of a human chondrocyte-like cell line, HCS 2/8, with a TGF-␤1 cDNA and found enhanced mRNA expression of type II collagen and PG core protein and a decrease in MMP-3 mRNA expression in transfected cells [136]. Similarly, rabbit articular chondrocytes were transduced with adenoviral vectors carrying TGF-␤1 gene under the transcriptional control of the human

E. Grimaud et al. / Cytokine & Growth Factor Reviews 13 (2002) 241–257

251

Fig. 4. Different strategies using TGF-␤ for cartilage repair: gene therapy, cellular therapy and tissue engineering. Potential therapeutic functions of TGF-␤ are highlighted by boxed text. BMP: bone morphogenetic protein, MSC: mesenchymal stem cells.

cytomegalovirus (CMV) early promoter [137]. Transduced chondrocytes responded to elevated endogenous production of TGF-␤1 by increasing their synthesis of PG, type II collagen and non-collagenous proteins. Another experiment was performed by the same group on monolayer cultures of human and canine meniscal cells infected with retroviruses carrying a human TGF-␤ gene [138]. Transduction with the TGF-␤ gene increased the synthesis of PGs and all types of collagen without altering the ratios between them. When a monolayer of rabbit articular chondrocytes infected with recombinant adenovirus carrying gene encoding TGF-␤1 was used, PG synthesis was restored to control levels in the presence of IL-1, indicating that transfer of TGF-␤ to articular chondrocytes can increase matrix synthesis greatly in vitro, even in the presence of the inflammatory cytokine IL-1 [139]. Ex vivo transfer of marker genes to articular cartilage was observed in rabbit knee when retroviral [140] as well as adenoviral [141] vectors were used. In vitro transfection has been performed on mesenchymal cells (MSC) as well as joint cells. MSC were previously shown to be the best candidates for cell therapy to regenerate injured tissue [142]. Complete healing may be achieved through the combination of MSC-mediated therapy with gene transfer of a selective growth factor such as TGF-␤. Ex vivo systemic gene therapy has been approached by using retroviruses to transfer TGF-␤ to splenocytes of DBA mice with collagen-induced arthritis [143]. The modified cells were then introduced into SCID mice, where their ability to produce arthritis was strongly reduced. Adenoviral and retroviral vectors have been used successfully in gene delivery by mesenchymal progenitor cells. Retroviral vectors are one of the preferred modes of transgene transduction into cells

for implantation, because they do not cause the immunologic reaction associated with adenoviral vectors. However, bone-marrow-derived mesenchymal progenitor cells are dependent on actively proliferating cells for successful transduction, which makes their retroviral transduction difficult [144]. Transduction efficiency can be increased if cells are grown in the presence of proliferation-inductive factors such as bFGF and EGF. Adenoviral vectors are considered to be most suitable for in vivo gene transfection of synoviocytes, because these cells normally have a low rate of turnover [145,146]. In a recent study, adenoviral TGF-␤1 was used to transduce human bone-marrow-derived mesenchymal progenitor cells, and effective transduction of these progenitor cells was achieved, with detection of a high concentration of TGF-␤1 in the culture medium [144]. Similarly to results for retrovirus-mediated gene transfer, adenoviral TGF-␤1 transduced progenitor cells used in an in vitro aggregate experiment differentiated into chondrocytes [147], indicating that ex vivo gene transfer methods can be used to produce TGF-␤1 without loss of the chondrogenic potential of these cells. If an effective method can be found for systemic delivery of these cells, then these transduced chondroprogenitor cells may have potential value for treating multiple joint arthrides. Theoretically, chondroprogenitor cells have a particular advantage for solving problems involving chondrogenesis. These cells are readily obtainable, so that their use in gene therapy could be a cost-effective means of delivering chondroinductive proteins to a localized site to induce healing. Moreover, gene transfer to chondroprogenitor cells may be useful in treating familial OA in which mutations in type II collagen gene exist. As normal chondrocytes do not turn over and some of the mutations are dominant nega-

252

E. Grimaud et al. / Cytokine & Growth Factor Reviews 13 (2002) 241–257

tives, expression of a wild-type gene and suppression of the mutant gene can be achieved. In addition to ex vivo approaches, experiments involving in vivo delivery are now beginning to be explored. In vivo gene delivery to chondrocytes has proved to be technically difficult, partly because of extracellular matrix thickness. Recent in vivo studies [148,149] have demonstrated the ability of a non-viral system (plasmid DNA carrying the TGF-␤1 gene driven by a strong mammalian promoter) to transfect autologous primary perichondrial cells and chondrocytes with considerable efficiency. A plasmid DNA encoding human TGF-␤ was administered intramuscularly to rats with streptococcal cell wall-induced arthritis [150]. The results showed that gene transfer of plasmid DNA encoding TGF-␤1 provides a mechanism for delivery of this cytokine that effectively suppresses ongoing inflammatory pathology in arthritis. Successful in vivo liposome-mediated delivery to chondrocytes by a combination of HVJ (Sendai virus) and liposomes has been reported [151]. A study investigated whether the expression of delivered Escherichia coli TGF-␤1 gene is regulated by the stress response of human chondrocyte-like cells (HCS 2/8) when heat-shock protein 70B promoter is inserted into the adenovirus vector [152]. The results show that this promoter could regulate the expression of delivered genes effectively according to the intensity of heat stress. In a study of the localization of gene delivery in joint tissue, control gene (LacZ) expression was observed in almost all synovial tissue samples and in chondrocytes on the surface of degenerated cartilage [153]. Moreover, for 2 weeks following gene delivery TGF-␤ levels in joint fluid were significantly higher than in controls. Another study reported the biological effects of direct, adenovirus-mediated transfer of TGF-␤ gene to the intervertebral disk [154]. Disks injected with Ad/CMV-hTGF-␤1 exhibited extensive, intense positive immunostaining for TGF-␤1, together with a 100% increase in PG synthesis as compared to intact control tissue. These results suggest that the intervertebral disk is an appropriate site for adenovirus-mediated transfer of TGF-␤ gene and subsequent production of growth factors. Transfer of the TGF-␤1 gene to articular chondrocytes in vivo strongly enhanced the synthesis of PGs and collagens [136,155,156], and collagen phenotyping confirmed the continued expression of type II collagen in the transduced cells. Overexpression of active TGF-␤1 through use of an adenoviral vector in the murine knee joint (synovial lining cells) led to hyperplasia of the synovium and chondro-osteophyte formation at the chondro-synovial junctions, suggesting that synovial lining cells contribute to chondro-osteophyte formation [157]. A first study combining the technologies of gene therapy and tissue engineering used a retroviral vector to allow stable introduction of another member of the TGF-␤ superfamily, BMP-7, into periosteal-derived rabbit MSC [158]. Periosteum has chondrogenic potential, making repair or regeneration of cartilage possible in damaged joints [159]. Tissue-engineered cartilage has been investigated in the pres-

ence of growth factors, and the results suggest that bFGF has the potential for clinical applications intended to generate a large volume of tissue-engineered cartilage from a small donor specimen in a short period of time and with a quality similar to that of native human elastic cartilage [160]. Human secondary chondroprogenitor cells, obtained by modulating the phenotype of articular chondrocytes with growth factors and stimulating the proliferation of these cells in culture, formed only cartilage tissue when assayed in vivo and in tissue bioreactors [96]. Another study reported that a novel rhTGF-␤1-F2 fusion protein, containing a von Willebrand’s factor-derived collagen binding domain combined with a collagen type I matrix, is able to capture, amplify and stimulate the differentiation of a population of cells present in rat bone marrow [161]. When these cells are subsequently implanted in inactivated demineralized bone matrix, they produce bone and cartilage.

7. Conclusion The current understanding of the factors involved in OA has evolved greatly during recent years. A better comprehension of the regulating agents will continue to generate new insights into a more accurate identification of effective targets having therapeutic potential in the treatment of such disease. Novel approaches to treat OA are indeed required, because at this time there is no definitive information to indicate which gene is the best to use for the treatment of OA. Progress in understanding the biology of cartilage disorders has led to the use of genes whose products stimulate cartilage repair or inhibit breakdown of the cartilaginous matrix. Of special interest is the prospect of improving the healing of injured tissues by the local delivery of genes which encode anabolic growth factors, and among them TGF-␤, one of the most important factors involved in the control of the repair reaction. The possibility that TGF-␤ may be considered as a therapeutic agent is attractive when considering the overall positive effects exerted by this factor both in vitro and in vivo: augmentation of cell proliferation, matrix production, osteochondrogenic differentiation and maintenance of articular cartilage in the differentiated phenotype, albeit long-term exposure to intra-articular injections of TGF-␤ seems to provoke disturbance of the cartilage extracellular matrix homeostasis. The first experiments using TGF-␤ gene transfer to articular chondrocytes or chondroprogenitor cells are promising, since the growth factor can both induce the synthesis of the specific cartilagenous matrix components (type II collagen and aggrecan) and decrease that of MMP-3, and improve the ability of chondroprogenitor cells to differentiate into cartilage chondrocytes. However, numerous obstacles have to be overcome before gene therapy can be considered for clinical use in humans for the treatment of cartilage disorders. Indeed, undesirable or systemic side effects using a very stable transfection would be very difficult to control. This has been shown in a study

E. Grimaud et al. / Cytokine & Growth Factor Reviews 13 (2002) 241–257

where the induction of the expression of a large amount of TGF-␤1 by the synovial lining cells of rabbit resulted in the death of the animals, whereas smaller amounts caused severe pathologic changes [162]. Despite these obstacles, it already is apparent that gene therapy has the potential of becoming a valuable clinical treatment mode for the cartilage defects.

Acknowledgements Supported by a Contrat de Recherche Stratégique (CreS) INSERM No. 4CR06F. References [1] Eastgate JA, Symons JA, Woods NC, Grinlinton FM, di Giovine FS, Duff GW. Correlation of plasma interleukin-1 levels with disease activity in rheumatoid arthritis. Lancet 1998;2:706–9. [2] Amin AR. Regulation of tumor necrosis factor-␣ and tumor necrosis factor converting enzyme in human osteoarthritis. Osteoarthritis Cartilage 1999;7:392–4. [3] Isomaki P, Punnonen J. Pro- and anti-inflammatory cytokines in rheumatoid arthritis. Ann Med 1997;29:499–507. [4] Bos PK, van Osch GJ, Frenz DA, Verhaar JA, Verwoerd-Verhoef HL. Growth factor expression in cartilage wound healing: temporal and spatial immunolocalization in a rabbit auricular cartilage wound model. Osteoarthritis Cartilage 2001;9:382–9. [5] Frenkel SR, Saadeh PB, Mehrara BJ, Chin GS, Steinbrech DS, Brent B, et al. Transforming growth factor beta superfamily members: role in cartilage modeling. Plast Reconst Surg 2000;105:980–90. [6] Wozney JM, Rosen V. Bone morphogenetic protein and bone morphogenetic protein gene family in bone formation and repair. Clin Orthop 1998;346:26–37. [7] Centrella M, Horowitz MC, Wozney JM, Mc Carthy TL. Transforming growth factor-beta gene family members and bone. Endocr Rev 1994;15:27–39. [8] Robey PG, Young MF, Flanders KC, Roche NS, Kondaiah P, Reddi AH, et al. Osteoblasts synthesize and respond to transforming growth factor-type beta (TGF-beta) in vitro. J Cell Biol 1987;105:457–63. [9] Seyedin SM, Thompson AY, Bentz H, Rosen M, McPherson JM, Conti A, et al. Cartilage-inducing factor-A: apparent identity to TGF-␤. J Biol Chem 1986;261:5693–5. [10] Pedrozo HA, Schwartz Z, Robinson M, Gomes R, Dean DD, Bonewald LF, et al. Potential mechanisms for the plasmin-mediated release and activation of latent transforming growth factor-beta 1 from the extracellular matrix of growth plate chondrocytes. Endocrinology 1999;140:5806–16. [11] Pedrozo HA, Schwartz Z, Gomez R, Ornoy A, Xin-Sheng W, Dallas SL, et al. Growth plate chondrocytes store latent transforming growth factor (TGF)-beta 1 in their matrix through latent TGF-beta 1 binding protein-1. J Cell Physiol 1998;177:343–54. [12] Fang J, Li X, Smiley E, Francke U, Mecham RP, Bonadio J. Mouse latent TGF-beta binding protein-2: molecular cloning and developmental expression. Biochim Biophys Acta 1997;1354:219– 30. [13] Moldovan F, Pelletier J-P, Mineau F, Dupuis M, Cloutier J-M, Martel-Pelletier J. Modulation of collagenase 3 in human osteoarthritis cartilage by activation of extracellular transforming growth factor beta: role of furin convertase. Arthritis Rheum 2000;43: 2100–9. [14] Rosenthal AK, Gohr CM, Henry LA, Le M. Participation of transglutaminase in the activation of latent transforming growth

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25] [26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

253

factor beta 1 in aging articular cartilage. Arthritis Rheum 2000;43:1729–33. D’Angelo M, Billings PC, Pacifici M, Leboy PS, Kirsch T. Authentic matrix vesicles contain active metalloproteases (MMP). A role for matrix vesicle-associated MMP-13 in activation of transforming growth factor-beta. J Biol Chem 2001;276:11347–53. Maeda S, Dean DD, Gay I, Schwartz Z, Boyan BD. Activation of latent transforming growth factor beta 1 by stromelysin in extracts of growth plate chondrocyte-derived matrix vesicles. J Bone Min Res 2001;16:1281–90. Fukumura K, Matsunaga S, Yamamoto T, Nagamine T, Ishidou Y, Sakou T. Immunolocalization of transforming growth factor-betas and type I and type II receptors in rat articular cartilage. Anticancer Res 1998;18:4189–93. Jingushi S, Scully SP, Joyce ME, Sugioka Y, Bolander ME. Transforming growth factor-beta 1 and fibroblast growth factors in rat growth plate. J Orthop Res 1995;13:761–8. Horner A, Kemp P, Summers C, Bord S, Bishop NJ, Kelsall AW, et al. Expression and distribution of transforming growth factor-beta isoforms and their signaling receptors in growing human bone. Bone 1998;23:95–102. Matsunaga S, Yamamoto T, Fukumura K. Temporal and spatial expressions of transforming growth factor-betas and their receptors in epiphyseal growth plate. Int J Oncol 1999;14:1063–7. Li XB, Zhou Z, Luo SJ. Expressions of IGF-I and TGF-beta 1 in the condyle cartilages of rapidly growing rats. Chin J Dent Res 1998;1:52–6. Moroco JR, Hinton R, Buschang P, Milam SB, Iacopino AM. Type II collagen and TGF-betas in developing and aging porcine mandibular condylar cartilage: immunohistochemical studies. Cell Tissue Res 1997;289:119–24. Wei X, Messner K. Age- and injury-dependent concentrations of transforming growth factor-beta 1 and proteoglycan fragments in rabbit knee joint fluid. Osteoarthritis Cartilage 1998;6:10–8. Fava R, Olsen N, Keski-Oja J, Moses H, Pincus T. Active and latent forms of transforming growth factor beta activity in synovial effusions. J Exp Med 1989;169:291–6. van den Berg WB. Joint inflammation and cartilage destruction may occur uncoupled. Springer Semin Immunopathol 1998;20:149–64. van der Kraan PM, Glansbeek HL, Vitters EL, van ven Berg WB. Early elevation of transforming growth factor-beta, decorin, and biglycan mRNA levels during cartilage matrix restoration after mild proteoglycan depletion. J Rheumatol 1997;24:543–9. Mussener A, Litton MJ, Lindroos E, Klareskog L. Cytokine production in synovial tissue of mice with collagen-induced arthritis (CIA). Clin Exp Immunol 1997;107:485–93. Mussener A, Funa K, Kleinau S, Klareskog L. Dynamic expression of transforming growth factor-betas (TGF-beta) and their type I and type II receptors in the synovial tissue of arthritic rats. Clin Exp Immunol 1997;107:112–9. Chambers MG, Bayliss MT, Mason RM. Chondrocyte cytokine and growth factor expression in murine osteoarthritis. Osteoarthritis Cartilage 1997;5:301–8. Uchino M, Izumi T, Tominaga T, Wakita R, Minehara H, Sekiguchi M, et al. Growth factor expression in the osteophytes of the human femoral head in osteoarthritis. Clin Orthop 2000;377:119–25. Ekholm EC, Ravanti L, Kahari V, Paavolainen P, Penttinen RP. Expression of extracellular matrix genes: transforming growth factor (TGF)-beta and ras in tibial fracture healing of lathyritic rats. Bone 2000;27:551–7. Rosier RN, O’Keefe RJ, Hicks DG. The potential role of transforming growth factor beta in fracture healing. Clin Orthop 1998;355(Suppl):S292–300. Maroulakou IG, Shibata MA, Anver M, Jorcyk CL, Liu MI, Roche N, et al. Heterotopic endochondrial ossification with mixed tumor formation in C3(1)/Tag transgenic mice is associated with elevated TGF-beta 1 and BMP-2 expression. Oncogene 1999;18:5435–47.

254

E. Grimaud et al. / Cytokine & Growth Factor Reviews 13 (2002) 241–257

[34] Semevolos SA, Nixon AJ, Brower-Toland BD. Changes in molecular expression of aggrecan and collagen types I, II and X, insulin-like growth factor-I and transforming growth factor-beta 1 in articular cartilage obtained from horses with naturally acquired osteochondrosis. Am J Vet Res 2001;62:1088–94. [35] Thorp BH, Elkman S, Jakowlew SB, Goddard C. Porcine osteochondrosis: deficiencies in transforming growth factor-beta and insulin-like growth factor-I. Calcif Tissue Int 1995;56:376–81. [36] Law AS, Burt DW, Alexander I, Thorp BH. Expression of the gene for transforming growth factor-beta in avian dyschondroplasia. Res Vet Sci 1996;61:120–4. [37] Henson FM, Schofield PN, Jeffcott LB. Expression of transforming growth factor-beta 1 in normal and dyschondroplastic articular growth cartilage of the young horse. Equine Vet 1997;29:434–9. [38] Ren P, Rowland III GN, Halper J. Expression of growth factors in chicken growth plate with special reference to tibial dyschondroplasia. J Comp Pathol 1997;116:303–20. [39] Ling J, Kincaid SA, McDaniel GR, Waegell W. Immunolocalization analysis of transforming growth factor-beta 1 in the growth plates of broiler chickens with high and low incidences of tibial dyschondroplasia. Poult Sci 2000;79:1172–8. [40] Thomas JT, Lin K, Nandedkar M, Camargo M, Cervenka J, Luyten FP. A human chondrodysplasia due to a mutation in a TGF-beta superfamily member. Nat Genet 1996;12:315–7. [41] Hall FL, Benya PD, Padilla SR, Carbonaro-Hall D, Williams R, Buckley S, et al. Transforming growth factor-beta type-II receptor signalling: intrinsic/associated casein kinase activity, receptor interactions and functional effects of blocking antibodies. Biochem J 1996;316:303–10. [42] Su S, DiBattista JA, Sun Y, Li QW, Zafarullah M. Up-regulation of tissue inhibitor of metalloproteinase-3 gene expression by TGF-beta in articular chondrocytes is mediated by serine/threonine and tyrosine kinases. J Cell Biochem 1998;70:517–27. [43] Osaki M, Tsukazaki T, Yonekura A, Miyazaki Y, Iwasaki K, Shindo H, et al. Regulation of c-fos gene induction and mitogenic effect of transforming growth factor-beta 1 in rat articular chondrocyte. Endocr J 1999;46:253–61. [44] Bogdanovicz P, Vivien D, Felisaz N, Léon V, Pujol J-P. An inositolphosphate glycan released by TGF-beta mimics the proliferative but not the transcriptional effects of the factor and requires functional receptors. Cell Signal 1996;8:503–9. [45] Yonekura A, Osaki M, Hirota Y, Tsukazaki T, Miyazaki Y, Matsumoto T, et al. Transforming growth factor-beta stimulates articular chondrocyte cell growth through p44/42 MAP kinase (ERK) activation. Endocr J 1999;46:545–53. [46] Hirota Y, Tsukazaki T, Yonekura A, Miyazaki Y, Osaki M, Shindo H, et al. Activation of specific MEK–ERK cascade is necessary for TGF-␤ signaling and crosstalk with PKA and PKC pathways in cultured articular chondrocytes. Osteoarthritis Cartilage 2000;8:241–8. [47] Miyazaki Y, Tsukazaki T, Hirota Y, Yonekura A, Osaki M, Shindo H, et al. Dexamethasone inhibition of TGF-beta-induced cell growth and type II collagen mRNA expression through ERK-integrated AP-1 activity in cultured rat articular chondrocytes. Osteoarthritis Cartilage 2000;8:378–85. [48] Palmer G, Guicheux J, Bonjour JP, Caverzasio J. Transforming growth factor-beta stimulates inorganic phosphate transport and expression of the type III phosphate transporter Glvr-1 in chondrogenic ATDC5 cells. Endocrinology 2000;141:2236–43. [49] Sylvia VL, Schwartz Z, Dean DD, Boyan BD. Transforming growth factor-beta 1 regulation of resting zone chondrocytes is mediated by two separate but interacting pathways. Biochim Biophys Acta 2000;1496:311–24. [50] Schwartz Z, Sylvia VL, Dean DD, Boyan BD. The synergistic effect of TGF beta and 24,25-(OH)2 D3 on resting zone chondrocytes is metabolite-specific and mediated by PKC. Connect Tissue Res 1996;35:101–6.

[51] Schwartz Z, Sylvia VL, Dean DD, Boyan BD. The synergistic effects of vitamin D metabolites and transforming growth factor-beta on costochondral chondrocytes are mediated by increases in protein kinase C activity involving two separate pathways. Endocrinology 1998;139:534–45. [52] Sakou T, Onishi T, Yamamoto T, Nagamine T, Sampath Tk, ten Dijke P. Localization of Smads, the TGF-beta family intracellular signaling components during endochondral ossification. J Bone Min Res 1999;14:1145–52. [53] Yang X, Chen L, Xu X, Li C, Huang C, Deng CX. TGF-beta/Smad3 signals repress chondrocyte hypertrophic differentiation and are required for maintaining articular cartilage. J Cell Biol 2001;153:35– 46. [54] Ferguson CM, Schwarz EM, Reynolds PR, Puzas JE, Rosier RN, O’Keefe RJ. Smad2 and 3 mediate transforming growth factor-beta 1-induced inhibition of chondrocyte maturation. Endocrinology 2000;141:4728–35. [55] Watanabe H, de Caestecker MP, Yamada Y. Transcriptional cross-talk between Smad, ERK 1/2, and p38 mitogen-activated protein kinase pathways regulates transforming growth factor-betainduced aggrecan gene expression in chondrogenic ATDC5 cells. J Biol Chem 2001;276:14466–73. [56] Demoor-Fossard M, Galéra P, Santra M, Iozzo RV, Pujol J-P, Rédini F. A composite element binding the vitamin D receptor and the retinoic X receptor a mediates the transforming growth factor-␤ inhibition of decorin gene expression in articular chondrocytes. J Biol Chem 2001;276:36983–92. [57] Critchlow MA, Bland YS, Ashhurst DE. The expression of collagen mRNAs in normally developing neonatal rabbit long bones and after treatment of neonatal and adult rabbit tibiae with transforming growth factor beta-2. Histochem J 1995;27:505–15. [58] Glansbeek HL, van Beuningen HM, Vitters EL, van der Kraan PM, van den Berg WB. Stimulation of articular cartilage repair in established arthritis by local administration of transforming growth factor-beta into murine knee joints. Lab Invest 1998;78:133–42. [59] Zerath E, Holy X, Mouillon JM, Farbos B, Machwate M, Andre C, et al. TGF-beta 2 prevents the impaired chondrocyte proliferation induced by unloading in growth plates of young rats. Life Sci 1997;61:2397–406. [60] Itayem R, Mengarelli-Widholm S, Hulth A, Reinholt FP. Ultrastructural studies on the effect of transforming growth factor-beta 1 on rat articular cartilage. APMIS 1997;105:221–8. [61] Hulth A, Johnell O, Miyazono K, Lingberg L, Heinegard D, Heldin CH. Effect of transforming growth factor-beta and platelet-derived growth factor-BB on articular cartilage in rats. J Orthop Res 1996;14:547–53. [62] van Beuningen HM, Glansbeek HL, van der Kraan PM, van den Berg WB. Osteoarthritis-like changes in the murine knee joint resulting from intra-articular transforming growth factor-beta injections. Osteoarthritis Cartilage 2000;8:25–33. [63] Cassiede P, Dennis JE, Ma F, Caplan AI. Osteochondrogenic potential of marrow mesenchymal progenitor cells exposed to TGF-beta 1 or PDGF-BB as assayed in vivo and in vitro. J Bone Min Res 1996;11:1264–73. [64] Worster AA, Nixon AJ, Brower-Toland BD, Williams J. Effect of transforming growth factor beta 1 on chondrogenic differentiation of cultured equine mesenchymal stem cells. Am J Vet Res 2000;61:1003–10. [65] Chimal-Monroy J, Diaz de Leon L. Differential effects of transforming growth factor beta 1, beta 2, beta 3 and beta 5 on chondrogenesis in mouse limb bud mesenchymal cells. Int J Dev Biol 1997;41:91–102. [66] De Bari C, Dell’Accio F, Luyten FP. Human periosteum-derived cells maintain phenotypic stability and chondrogenic potential throughout expansion regardless of donor age. Arthritis Rheum 2001;44:85–95.

E. Grimaud et al. / Cytokine & Growth Factor Reviews 13 (2002) 241–257 [67] Kawai J, Akiyama H, Shigeno C, Ito H, Konishi J, Nakamura T. Effects of transforming growth factor-beta signaling on chondrogenesis in mouse chondrogenic EC cells, ATDC5. Eur J Cell Biol 1999;78:707–14. [68] Macias D, Ganan Y, Rodriguez-Leon J, Merino R, Hurle JM. Regulation by members of the transforming growth factor beta superfamily of the digital and interdigital fates of the autopodial limb mesoderm. Cell Tissue Res 1999;296:95–102. [69] Merino R, Ganan Y, Macias D, Economides AN, Sampath KT, Hurle JM. Morphogenesis of digits in the avian limb is controlled by FGFs, TGFbetas, and noggin through BMP signaling. Dev Biol 1998;200:35–45. [70] Zhu Y, Oganesian A, Keene DR, Sandell LJ. Type IIA procollagen containing the cysteine-rich amino propeptide is deposited in the extracellular matrix of prechondrogenic tissue and binds to TGF-beta 1 and BMP-2. J Cell Biol 1999;144:1069–80. [71] Ripamondi U, Bosch C, van den Heever B, Duneas N, Melsen B, Ebner R. Limited chondro-osteogenesis by recombinant human transforming growth factor-beta in calvarial defects of adult baboons (Papio ursinus). J Bone Min Res 1996;11:938–45. [72] Ripamondi U, Duneas N, van den Heever B, Bosch C, Crooks J. Recombinant transforming growth factor-beta 1 induces endochondral bone in the baboon and synergizes with recombinant osteogenic protein-1 (bone morphogenetic protein-7) to initiate rapid bone formation. J Bone Min Res 1997;12:1584–92. [73] Scheijmans CM, Dieudonne SC, Prahl-Andersen B, Burger EH. The influence of transforming growth factor beta 1 on the development of embryonic mouse long bones. Eur J Orthop 1996;18:237–43. [74] Tsuiki H, Fukiishi Y, Kishi K. Relation of TGF-beta 2 to inhibition of limb bud chondrogenesis by retinoid in rats. Teratology 1996;54:191–7. [75] van Osch GJ, van der Veen SW, Burger EH, Verwoerd-Verhoef HL. Chondrogenic potential of in vitro multiplied rabbit perichondrium cells cultured in alginate beads in defined medium. Tissue Eng 2000;6:321–30. [76] Serra R, Johnson M, Filvaroff EH, LaBorde J, Sheehan DM, Derynck R, et al. Expression of a truncated, kinase-defective TGF-beta type II receptor in mouse skeletal tissue promotes terminal chondrocyte differentiation and osteoarthritis. J Cell Biol 1997;139:541–52. [77] Serra R, Karaplis A, Sohn P. Parathyroid hormone-related peptide (PTHrP)-dependent and independent effects of transforming growth factor beta (TGF-beta) on endochondral bone formation. J Cell Biol 1999;145:783–94. [78] Morales TI, Joyce ME, Sobel ME, Danielpour D, Roberts AB. Transforming growth factor-beta in calf articular cartilage organ cultures: synthesis and distribution. Arch Biochem Biophys 1991;288:397–405. [79] Blumenfeld I, Laufer D, Livne E. Effects of transforming growth factor-beta 1 and interleukin-1 alpha on matrix synthesis in osteoarthritic cartilage of the temporo-mandibular joint in aged mice. Mech Ageing Dev 1997;95:101–11. [80] Blumenfeld I, Gaspar R, Laufer D, Livne E. Enhancement of toluidine blue staining by transforming growth factor-beta, insulin-like growth factor and growth hormone in the temporomandibular joint of aged mice. Cells Tissues Organs 2000;167:121–9. [81] Livne E, Laufer D, Blumenfeld I. Osteoarthritis in the temporo-mandibular joint (TMJ) of aged mice and the in vitro effect of TGF-beta on cell proliferation, matrix synthesis, and alkaline phosphatase activity. Microsc Res Tech 1997;37:314–23. [82] Hui W, Rowan AD, Cawston T. Transforming growth factor beta 1 blocks the release of collagen fragments from bovine nasal cartilage stimulated by oncostatin M in combination with IL-1 alpha. Cytokine 2000;12:765–9. [83] Iqbal J, Dudhia J, Bird JL, Bayliss MT. Age-related effects of TGF-beta on proteoglycan synthesis in equine articular cartilage. Biochem Biophys Res Commun 2000;274:467–71.

255

[84] Lafeber FP, van Roy HL, van der Kraan PM, van der Berg WB, Bijlsma JW. Transforming growth factor-beta predominantly stimulates phenotypically changed chondrocytes in osteoarthritic human cartilage. J Rheumatol 1997;24:536–42. [85] Moldovan F, Pelletier JP, Hambor J, Cloutier JM, Martel-Pelletier J. Collagenase-3 (matrix metalloprotease 13) is preferentially localized in the deep layer of human arthritic cartilage in situ: in vitro mimicking effect by transforming growth factor beta. Arthritis Rheum 1997;40:1653–61. [86] Seko Y, Tanaka Y, Tokoro T. Influence of bFGF as a potent growth stimulator and TGF-beta as a growth regulator on scleral chondrocytes and scleral fibroblasts in vitro. Ophthalmic Res 1995;27:144–52. [87] Lee JD, Hwang O, Kim SW, Han S. Primary cultured chondrocytes of different origins respond differently to bFGF and TGF-beta. Life Sci 1997;61:293–9. [88] de Haart WJ, van Osch GJ, Verhaar JA. Optimization of chondrocyte expansion in culture. Effect of TGF beta-2, bFGF and L-ascorbic acid on bovine articular chondrocytes. Acta Orthop Scand 1999;70:55–61. [89] Gruber HE, Fisher Jr EC, Desai B, Stasky AA, Hoelscher G, Hanley Jr EN. Human invertebral disc cells from the annulus: three dimensional culture in agarose or alginate and responsiveness to TGF-beta 1. Exp Cell Res 1997;232:13–21. [90] Matsumura T, Whelan MC, Li XQ, Trippel SB. Regulation by IGF-I and TGF-beta 1 of Swarm-rat chondrosarcoma chondrocytes. J Orthop Res 2000;18:351–5. [91] Nixon AJ, Lillich JT, Burton-Wurster N, Lust G, Mohammed HO. Differential cellular function in fetal chondrocytes cultured with insulin-like growth factor-I and transforming growth factor-beta. J Orthop Res 1998;16:531–41. [92] Nasatzky E, Grinfeld D, Boyan BD, Dean DD, Ornoy A, Schwartz Z. Transforming growth factor-beta 1 modulates chondrocyte responsiveness to 17 beta-estradiol. Endocrine 1999;11:241–9. [93] Boumediene K, Vivien D, Macro M, Bogdanovicz P, Lebrun E, Pujol J-P. Modulation of rabbit articular chondrocyte (RAC) proliferation by TGF-beta isoforms. Cell Prolif 1995;28:221–34. [94] Blanco FJ, Geng Y, Lotz M. Differentiation-dependent effects of IL-1 and TGF-beta on human articular chondrocyte proliferation are related to inducible nitric oxide synthase expression. J Immunol 1995;154:4018–26. [95] Jahng JS, Lee JW, Han CD, Kim SJ, Yoo NC. Transforming growth factor-beta 1 responsiveness of human articular chondrocytes in vitro: normal versus osteoarthritis. Yonsei Med J 1997;38:40–51. [96] Bradham DM, Horton Jr WE. In vivo cartilage formation from growth factor modulated articular chondrocytes. Clin Orthop 1998;352:239–49. [97] Okazaki R, Sakai A, Nakamura T, Kunugita N, Norimura T, Suzuki K. Effects of transforming growth factor betas and basic fibroblast growth factor on articular chondrocytes obtained from immobilized rabbit knees. Ann Rheum Dis 1996;55:181–6. [98] Fortier LA, Nixon AJ, Mohammed HO, Lust G. Altered biological activity of equine chondrocytes cultured in a three-dimensional fibrin matrix and supplemented with transforming growth factor beta-1. Am J Vet Res 1997;58:66–70. [99] Guerne PA, Blanco F, Kaelin A, Desgeorges A, Lotz M. Growth factor responsiveness of human articular chondrocytes in aging and development. Arthritis Rheum 1995;38:960–8. [100] Garcia-Ramirez M, Audi L, Andaluz P, Carrascosa A. Effects of TGF-beta 1 on proliferation and IGFBP-3 production in a primary culture of human fetal epiphyseal chondrocytes (HFEC). J Clin Endocrinol Metab 1999;84:2978–81. [101] Arevalo-Silva CA, Cao Y, Weng Y, Vacanti M, Rodriguez A, Vacanti CA, et al. The effect of fibroblast growth factor and transforming growth factor-beta on porcine chondrocytes and tissue-engineered autologous elastic cartilage. Tissue Eng 2001;7:81–8.

256

E. Grimaud et al. / Cytokine & Growth Factor Reviews 13 (2002) 241–257

[102] van Osch GJ, van der Veen SW, Buma P, Verwoerd-Verhoef HL. Effect of transforming growth factor-beta on proteoglycan synthesis by chondrocytes in relation to differentiation stage and the presence of pericellular matrix. Matrix Biol 1998;17:413–24. [103] Demoor-Fossard M, Rédini F, Boittin M, Pujol J-P. Expression of decorin. Biochim Biophys Acta 1998;179:179–91. [104] Rédini F, Min W, Demoor-Fossard M, Boittin M, Pujol J-P. Differential expression of membrane-anchored proteoglycans in rabbit articular chondrocytes cultured in monolayers and in alginate beads. Biochim Biophys Acta 1997;1355:20–32. [105] Demoor-Fossard M, Boittin M, Rédini F, Pujol J-P. Differential effects of interleukin-1 and transforming growth factor ␤ on the synthesis of small proteoglycans by rabbit articular chondrocytes cultured in alginate beads as compared to monolayers. Mol Cell Biochem 1998;199:69–80. [106] Rédini F, Daireaux M, Mauviel A, Galéra P, Loyau G, Pujol J-P. Characterization of proteoglycans synthesized by rabbit articular chondrocytes in response to transforming growth factor-␤ (TGF-␤). Biochim Biophys Acta 1991;1093:196–206. [107] Galéra P, Vivien D, Pronost S, Bonaventure J, Rédini F, Loyau G, et al. Transforming growth factor-beta 1 (TGF-beta 1) up-regulation of collagen type II in primary cultures of rabbit articular chondrocytes (RAC) involves increased mRNA levels without affecting mRNA stability and procollagen processing. J Cell Physiol 1992;153:596– 606. [108] Bujia J, Pitzke P, Kastenbauer E, Wilmes E, Hammer C. Effect of growth factors on matrix synthesis by human nasal chondrocytes cultured in monolayer and in agar. Eur Arch Otorhinolaryngol 1996;253:336–40. [109] Yaeger PC, Masi TL, de Ortiz JL, Binette F, Tubo R, McPherson JM. Synergistic action of transforming growth factor-beta and insulin-like growth factor-I induces expression of type II collagen and aggrecan genes in adult human articular chondrocytes. Exp Cell Res 1997;237:318–25. [110] Qi WN, Scully SP. Effect of type II collagen in chondrocyte response to TGF-beta 1 regulation. Exp Cell Res 1998;241:142–50. [111] van Susante JL, Buma P, van Beuningen HM, van den Berg WB, Veth RP. Responsiveness of bovine chondrocytes to growth factors in medium with different serum concentrations. J Orthop Res 2000;18:68–77. [112] van Osch GJ, van den Berg WB, Hunziker EB, Hauselmann HJ. Differential effects of IGF-I and TGF beta-2 on the assembly of proteoglycans in pericellular and territorial matrix by cultured bovine articular chondrocytes. Osteoarthritis Cartilage 1998;6:187– 95. [113] Nasatsky E, Azran E, Dean DD, Boyan BD, Schwartz Z. Parathyroid hormone and transforming growth factor-beta 1 coregulate chondrocyte differentiation in vitro. Endocrine 2000;13:305–13. [114] Thompson CC, Clegg PD, Carter SD. Differential regulation of gelatinases by transforming growth factor beta-1 in normal equine chondrocytes. Osteoarthritis Cartilage 2001;9:325–31. [115] Lum ZP, Hakala BE, Mort JS, Recklies AD. Modulation of the catabolic effects of interleukin-1 beta on human articular chondrocytes by transforming growth factor-beta. J Cell Physiol 1996;166:351–9. [116] Loeser RF. Growth factor regulation of chondrocyte integrins. Differential effects of insulin-like growth factor 1 and transforming growth factor beta on alpha 1 beta integrin expression and chondrocyte adhesion to type VI collagen. Arthritis Rheum 1997;40:270–6. [117] Su S, Dehnade F, Zafarullah M. Regulation of tissue inhibitor of metalloproteinases-3 gene expression by transforming growth factor-beta and dexamethasone in bovine and human articular chondrocytes. DNA Cell Biol 1996;15:1039–48. [118] Fawthrop FW, Frazer A, Russell RG, Bunning RA. Effects of transforming growth factor beta on the production of prostaglandin E and caseinase activity of unstimulated and interleukin 1-stimulated

[119]

[120]

[121]

[122]

[123]

[124]

[125]

[126]

[127]

[128]

[129]

[130]

[131]

[132]

[133]

[134]

human articular chondrocytes in culture. Br J Rheumatol 1997;36:729–34. Shlopov BV, Smith GN, Cole AA, Hasty KA. Differential patterns of response to doxycycline and transforming growth factor beta 1 in the down-regulation of collagenases in osteoarthritic and normal human chondrocytes. Arthritis Rheum 1999;42:719–27. Tardif G, Pelletier JP, Dupuis M, Geng C, Cloutier JM, Martel-Pelletier J. Collagenase 3 production by human osteoarthritic chondrocytes in response to growth factors and cytokines is function of the physiologic state of the cells. Arthritis Rheum 1999;46:1147– 58. Edwards DR, Murphy G, Reynolds JJ, Whitham SE, Docherty AJ, Angel P, et al. Transforming growth factor beta modulates the expression of collagenase and metalloproteinase inhibitor. EMBO J 1987;6:1899–904. Harvey AK, Hrubey PS, Chandrasekhar S. Transforming growth factor beta inhibition of interleukin-1 activity involves down-regulation of interleukin-1 receptors on chondrocytes. Exp Cell Res 1991;195:376–85. Rédini F, Mauviel A, Pronost S, Loyau G, Pujol JP. Transforming growth factor-beta exerts opposite effects from interleukin-1 beta on cultured rabbit articular chondrocytes through reduction of interleukin-1 receptor expression. Arthritis Rheum 1993;36:44–50. Terkeltaub R, Lotz M, Johnson K, Deng D, Hashimoto S, Goldring MB, et al. Parathyroid hormone-related proteins is abundant in osteoarthritic cartilage, and the parathyroid hormone related protein 1–173 isoform is selectively induced by transforming growth factor beta in articular chondrocytes and suppresses generation of extracellular inorganic pyrophosphate. Arthritis Rheum 1998;41:2152–64. Pateder DB, Rosier RN, Schwarz EM, Reynolds PR, Puzas JE, D’Souza M, et al. PTHrP expression in chondrocytes, regulation by TGF-beta, and interactions between epiphyseal and growth plate chondrocytes. Exp Cell Res 2000;256:555–62. Rosen F, McCabe G, Quach J, Solan J, Terkeltaub R, Seegmiller JE, et al. Differential effects of aging on human chondrocyte responses to transforming growth factor beta: increased pyrophosphate production and decreased cell proliferation. Arthritis Rheum 1997;40:1275–81. Johnstone B, Hering TM, Caplan AI, Goldberg VM, Yoo JU. In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res 1998;238:265–72. Nishimura K, Solchaga LA, Caplan AI, Yoo JU, Goldberg VM, Johnstone B. Chondroprogenitor cells of synovial tissue. Arthritis Rheum 1999;42:2631–7. Ballock RT, Heydemann A, Izumi T, Reddi AH. Regulation of the expression of the type-II collagen gene in periosteum-derived cells by three members of the transforming growth factor-beta superfamily. J Orthop Res 1997;15:463–7. Dounchis JS, Goomer RS, Harwood FL, Khatod M, Coutts RD, Amiel D. Chondrogenic phenotype of perichondrium-derived chondroprogenitor cells is influenced by transforming growth factor-beta 1. J Orthop Res 1997;15:803–7. Lee MC, Goomer RS, Takahashi K, Harwood FL, Amiel M, Amiel D. Transforming growth factor beta one (TGF-beta 1) enhancement of the chondrocytic phenotype in aged perichondrial cells: an in vitro study. Iowa Orthop J 2000;20:11–6. Basic N, Basic V, Bulic K, Grgic M, Kleinman HK, Luyten FP, et al. TGF-beta and basement membrane matrigel stimulate the chondrogenic phenotype in osteoblastic cells derived from fetal rat calvaria. J Bone Min Res 1996;11:384–91. Chimal-Monroy J, Diaz de Leon L. Expression of N-cadherin, N-CAM, fibronectin and tenascin is stimulated by TGF-beta 1, beta 2, beta 3 and beta 5 during the formation of precartilage condensations. Int J Dev Biol 1999;43:59–67. Perka C, Schultz O, Spitzer RS, Lindenhayn K. The influence of transforming growth factor beta 1 on mesenchymal cell repair of

E. Grimaud et al. / Cytokine & Growth Factor Reviews 13 (2002) 241–257

[135]

[136]

[137]

[138]

[139]

[140]

[141]

[142] [143]

[144] [145]

[146]

[147] [148]

full-thickness cartilage defects. J Biomed Mater Res 2000;52:543– 52. Worster AA, Nixon AJ, Brower-Toland BD, Williams J. Effect of transforming growth factor beta 1 on chondrogenic differentiation of cultured equine mesenchymal stem cells. Am J Vet Res 2000;61:1003–10. Arai Y, Kubo T, Kobayashi K, Takeshita K, Takahashi K, Ikeda T, et al. Adenovirus vector-mediated gene transduction to chondrocytes: in vitro evaluation of therapeutic efficacy of transforming growth factor-beta 1 and heat shock protein 70. J Rheumatol 1997;24:1787– 95. Shuler FD, Georgescu HI, Niyibizi C, Studer RK, Mi Z, Johnstone B, et al. Increased matrix synthesis following adenoviral transfer of a transforming growth factor beta 1 gene into articular chondrocytes. J Orthop Res 2000;18:585–92. Goto H, Shuler FD, Niyibizi C, Fu FH, Robbins PD, Evans CH. Gene therapy for meniscal injury: enhanced synthesis of proteoglycan and collagen by meniscal cells transduced with a TGFbeta(1) gene. Osteoarthritis Cartilage 2000;8:266–71. Smith P, Shuler FD, Georgescu HI, Ghivizzani SC, Johnstone B, Niyibizi C, et al. Genetic enhancement of matrix synthesis by articular chondrocytes: comparison of different growth factor genes in the presence and absence of interleukin-1. Arthritis Rheum 2000;43:1156–64. Kang R, Marui T, Ghivizzani SC, Nita IM, Georgescu HI, Suh J-K, et al. Ex vivo gene transfer to chondrocytes in full-thickness articular cartilage defects: a feasibility study. Osteoarthritis Cartilage 1997;5:139–43. Baragi VM, Renkiewicz RR, Jordan H, Bonadino J, Hartman JW, Roessler BJ. Transplantation of transduced chondrocytes protects articular cartilage from interleukin 1-induced extracellular matrix degradation. J Clin Invest 1995;96:2454–60. Weissman IL. Translating stem and progenitor cell biology to the clinic: barriers and opportunities. Science 2000;287:1442–6. Chernajovsky Y, Adams G, Triantaphyllopoulos K, Ledda MF, Podhajcer OL. Pathogenic lymphoid cells engineered to express TGF-␤1 ameliorate disease in a collagen-induced arthritis model. Gene Ther 1997;4:553–9. Mandell I, Yoo JU, Johnstone B. Gene delivery to mesenchymal cells. Trans Orthop Res Soc 1999;24:112. Nita IM, Ghivizzani SC, Galea-Lauri J, Bandara G, Georgescu HI, Robbins PD, et al. Direct gene delivery to synovium. An evaluation of potential vectors in vitro and in vivo. Arthritis Rheum 1996;39:820–8. Tomita N, Morishita R, Higaki J, Tomita S, Aoki M, Ogihara T, et al. In vivo gene transfer of insulin gene into neonatal rats by the HVJ-liposome method resulted in sustained transgene expression. Gene Ther 1996;3:477–82. Yoo JU, Mandell I, Angele P, Johnstone B. Chondrogenitor cells and gene therapy. Clin Orthop 2000;379(Suppl):S164–70. Goomer RS, Maris TM, Gelberman R, Boyer M, Silva M, Amiel D. Nonviral in vivo gene therapy for tissue engineering of

[149]

[150]

[151]

[152]

[153]

[154]

[155] [156] [157]

[158]

[159] [160]

[161]

[162]

257

articular cartilage and tendon repair. Clin Orthop 2000;379(Suppl): S189–200. Goomer RS, Deftos LJ, Terkeltaub R, Maris T, Lee MC, Harwood FL, et al. High efficiency non-viral transfection of primary chondrocytes and perichondrial cells for ex vivo gene therapy to repair articular cartilage defects. Osteoarthritis Cartilage 2001;9:248–56. Song XY, Gu M, Jin WW, Klinman DM, Wahl SM. Plasmid DNA encoding transforming growth factor-beta 1 suppresses chronic disease in a streptococcal cell wall-induced arthritis model. J Clin Invest 1998;101:2615–21. Tomita T, Hashimoto H, Tomita N, Morishita R, Lee SB, Hayashida K, et al. In vivo direct gene transfer into articular cartilage by intraarticular injection mediated by HVJ (sendai virus) and liposomes. Arthritis Rheum 1997;40:901–6. Arai Y, Kubo T, Kobayashi K, Ikeda T, Takahashi K, Takigawa M, et al. Control of delivered gene expression in chondrocytes using heat shock protein 70B promoter. J Rheumatol 1999;26:1769–74. Ikeda T, Kubo T, Arai Y, Nakanishi T, Kobayashi K, Takahashi K, et al. Adenovirus mediated gene delivery to the joints of guinea pigs. J Rheumatol 1998;25:1666–73. Nishida K, Kang JD, Gilbertson LG, Moon SH, Suh JK, Vogt MT, et al. Modulation of the biologic activity of the rabbit intervertebral disc by gene therapy: an in vivo study of adenovirus-mediated transfer of the human transforming growth factor beta 1 encoding gene. Spine 1999;24:2419–25. Evans CH, Robbins PD. Gene therapy of arthritis. Internal Med 1999;38:233–9. Evans CH, Robbins PD. Potential treatment of osteoarthritis by gene therapy. Rheum Dis Clin N Am 1999;25:333–44. Bakker AC, van de Loo FA, van Beuningen HM, Sime P, van Lent PL, van der Kraan PM, et al. Overexpression of active TGF-beta-1 in the murine knee joint: evidence for synovial-layer-dependent chondro-osteophyte formation. Osteoarthritis Cartilage 2001;9:128– 36. Mason JM, Breitbart AS, Barcia M, Porti D, Pergolizzi RG, Grande DA. Cartilage and bone regeneration using gene-enhanced tissue engineering. Clin Orthop 2000;379(Suppl):S171–8. O’Driscoll W. Articular cartilage regeneration using periosteum. Clin Orthop 1999;379(Suppl):S186–203. Arevalo-Silva CA, Cao Y, Vacanti M, Weng Y, Vacanti CA, Eavey RD. Influence of growth factors on tissue-engineered pediatric elastic cartilage. Arch Otolaryngol Head Neck Surg 2000;126:1234– 8. Andrades JA, Han B, Becerra J, Sorgente N, Hall FL, Nimni ME. A recombinant human TGF-beta 1 fusion protein with collagen-binding domain promotes migration, growth, and differentiation of bone marrow mesenchymal cells. Exp Cell Res 1999;250:485–98. Khang R, Robbins PD, Evans CH. Methods for gene transfer to synovium. In: Robbins PD, editor. Gene therapy protocols. Totowa: Humana Press, 1977. p. 357–68.