Growth factor transgenes interactively regulate articular chondrocytes

Adult articular chondrocytes lack an effective repair response to correct damage from injury or osteoarthritis. Polypeptide growth factors that stimulate articular chondrocyte proliferation and cartilage matrix synthesis may augment this response. Gene transfer is a promising approach to delivering such factors. Multiple growth factor genes regulate these cell functions, but multiple growth factor gene transfer remains unexplored. We tested the hypothesis that multiple growth factor gene transfer selectively modulates articular chondrocyte proliferation and matrix synthesis. We tested the hypothesis by delivering combinations of the transgenes encoding insulin‐like growth factor I (IGF‐I), fibroblast growth factor‐2 (FGF‐2), transforming growth factor beta1 (TGF‐β1), bone morphogenetic protein‐2 (BMP‐2), and bone morphogenetic protien‐7 (BMP‐7) to articular chondrocytes and measured changes in the production of DNA, glycosaminoglycan, and collagen. The transgenes differentially regulated all these chondrocyte activities. In concert, the transgenes interacted to generate widely divergent responses from the cells. These interactions ranged from inhibitory to synergistic. The transgene pair encoding IGF‐I and FGF‐2 maximized cell proliferation. The three‐transgene group encoding IGF‐I, BMP‐2, and BMP‐7 maximized matrix production and also optimized the balance between cell proliferation and matrix production. These data demonstrate an approach to articular chondrocyte regulation that may be tailored to stimulate specific cell functions, and suggest that certain growth factor gene combinations have potential value for cell‐based articular cartilage repair. J. Cell. Biochem. 114: 908–919, 2013. © 2012 Wiley Periodicals, Inc.     

Basic fibroblast growth factor accelerates matrix degradation via a neuro-endocrine pathway in human adult articular chondrocytes

Pain-related neuropeptides released from synovial fibroblasts, such as substance P, have been implicated in joint destruction. Substance P-induced inflammatory processes are mediated via signaling through a G-protein-coupled receptor, that is, neurokinin-1 tachykinin receptor (NK(1)-R). We determined the pathophysiological link between substance P and its receptor in human adult articular cartilage homeostasis. We further examined if catabolic growth factors such as basic fibroblast growth factor (bFGF or FGF-2) or IL-1beta accelerate matrix degradation via a neural pathway upregulation of substance P and NK(1)-R. We show here that substance P stimulates the production of cartilage-degrading enzymes, such as matrix metalloproteinase-13 (MMP-13), and suppresses proteoglycan deposition in human adult articular chondrocytes via NK(1)-R. Furthermore, we have demonstrated that substance P negates proteoglycan stimulation promoted by bone morphogenetic protein-7, suggesting the dual role of substance P as both a pro-catabolic and anti-anabolic mediator of cartilage homeostasis. We report that bFGF-mediated stimulation of substance P and its receptor NK(1)-R is, in part, through an IL-1beta-dependent pathway.     

Immunohistochemical localization of osteogenetic protein (OP-1) and its receptors in rabbit articular cartilage.

We assessed the distribution and relative immunohistochemical staining intensity of the bone morphogenetic protein-7, osteogenic protein-1 (OP-1), in its pro- and mature forms, and four of its receptors, type I (ALK-2, ALK-3, and ALK-6) and type II in normal adolescent New Zealand White rabbit articular cartilage. Expression of the protein and its receptors was also examined in cartilage from joints that had been previously subjected to cartilage matrix degradation. Pro-OP-1 was moderately expressed in chondrocytes of the superficial, middle, and deep cartilage zones and in the osteocytes. The expression of mature OP-1 was similar, with the exception of less staining in the superficial zone of cartilage. Expression of these two forms of OP-1 was enhanced in the middle and deep cartilage zones after catabolic challenge. The type I receptor, ALK-6, displayed the strongest staining of the receptors in both cartilage and bone, whereas ALK-2 displayed the weakest staining. No differences were observed in the receptor staining levels after catabolic challenge. This study shows that OP-1 and its receptors have been identified in rabbit articular cartilage and bone, suggesting a possible role for this pathway in cartilage and bone homeostasis.     

Regulation of osteogenic proteins by chondrocytes

The purpose of this review is to summarize the current scientific knowledge of bone morphogenetic proteins (BMPs) in adult articular cartilage. We specifically focus on adult cartilage, since one of the major potential applications of the members of the BMP family may be a repair of adult tissue after trauma and/or disease. After reviewing cartilage physiology and BMPs, we analyze the data on the role of recombinant BMPs as anabolic agents in tissue formation and restoration in different in vitro and in vivo models following with the endogenous expression of BMPs and factors that regulate their expression. We also discuss recent transgenic modifications of BMP genes and subsequent effect on cartilage matrix synthesis. We found that the most studied BMPs in adult articular cartilage are BMP-7 and BMP-2 as well as transforming growth factor-beta (TGF-beta). There are a number of contradicting reports for some of these growth factors, since different models, animals, doses, time points, culture conditions and devices were used. However, regardless of the experimental conditions, only BMP-7 or osteogenic protein-1 (OP-1) exhibits the most convincing effects. It is the only BMP studied thus far in adult cartilage that demonstrates strong anabolic activity in vitro and in vivo with and without serum. OP-1 stimulates the synthesis of the majority of cartilage extracellular matrix proteins in adult articular chondrocytes derived from different species and of different age. OP-1 counteracts the degenerative effect of numerous catabolic mediators; it is also expressed in adult human, bovine, rabbit and goat articular cartilage. This review reveals the importance of the exploration of the BMPs in the cartilage field and highlights their significance for clinical applications in the treatment of cartilage-related diseases.     

CCN Family Member 2/Connective Tissue Growth Factor (CCN2/CTGF) Has Anti-Aging Effects That Protect Articular Cartilage from Age-Related Degenerative Changes

To examine the role of connective tissue growth factor CCN2/CTGF (CCN2) in the maintenance of the articular cartilaginous phenotype, we analyzed knee joints from aging transgenic mice (TG) overexpressing CCN2 driven by the Col2a1 promoter. Knee joints from 3-, 14-, 40-, and 60-day-old and 5-, 12-, 18-, 21-, and 24-month-old littermates were analyzed. Ccn2-LacZ transgene expression in articular cartilage was followed by X-gal staining until 5 months of age. Overexpression of CCN2 protein was confirmed through all ages in TG articular cartilage and in growth plates. Radiographic analysis of knee joints showed a narrowing joint space and other features of osteoarthritis in 50% of WT, but not in any of the TG mice. Transgenic articular cartilage showed enhanced toluidine blue and safranin-O staining as well as chondrocyte proliferation but reduced staining for type X and I collagen and MMP-13 as compared with those parameters for WT cartilage. Staining for aggrecan neoepitope, a marker of aggrecan degradation in WT articular cartilage, increased at 5 and 12 months, but disappeared at 24 months due to loss of cartilage; whereas it was reduced in TG articular cartilage after 12 months. Expression of cartilage genes and MMPs under cyclic tension stress (CTS) was measured by using primary cultures of chondrocytes obtained from wild-type (WT) rib cartilage and TG or WT epiphyseal cartilage. CTS applied to primary cultures of mock-transfected rib chondrocytes from WT cartilage and WT epiphyseal cartilage induced expression of Col1a1, ColXa1, Mmp-13, and Mmp-9 mRNAs; however, their levels were not affected in CCN2-overexpressing chondrocytes and TG epiphyseal cartilage. In conclusion, cartilage-specific overexpression of CCN2 during the developmental and growth periods reduced age-related changes in articular cartilage. Thus CCN2 may play a role as an anti-aging factor by stabilizing articular cartilage.     

Effects of diacerein on biosynthesis activities of chondrocytes in culture

The maintenance of articular cartilage integrity requires a balance between anabolic and catabolic processes which are under the control of chondrocytes. These cells are living in an anaerobic environment and normally do not divide. They are responsible for the continuous maintenance of the cartilage extracellular matrix (ECM). Although multiple factors are involved in the dynamic homeostasis of cartilage, increases in cytokines such as interleukin-1 (IL-1) are associated with a decrease in synthesis and an increase in degradation of the proteoglycans and collagens. Conversely, growth factors such as transforming growth factor-beta (TGF-beta) stimulate chondrocyte synthesis of collagens and proteoglycans, and reduce the activity of IL-1 stimulated metalloproteases, thus opposing the inhibitory and catabolic effects of IL-1. By its capability to reduce IL-1 effects and to stimulate TGF-beta expression in cultured articular chondrocytes, diacerein could favour anabolic processes in the OA cartilage and, hence may contribute to delay the progression of the disease.     

IGF-1 and BMP-7 synergistically stimulate articular cartilage repairing in the rabbit knees by improving chondrogenic differentiation of bone-marrow mesenchymal stem cells

Knee injury is known as a frequently occurred damage related to sports, which may affect the function of cartilage. This study aims to explore whether Insulin-like growth factor 1 (IGF-1) and bone morphogenetic protein-7 (BMP-7)-modified bone-marrow mesenchymal stem cells (BMSCs) affect the repair of cartilage damage found in the knee. Primarily, BMSCs were treated with a series of pEGFP-C1, IGF-1, and BMP-7, followed by determination of IGF-1 and BMP-7 expression. A rabbit cartilage defect model was also established. Afterfward, cell morphology, viability, cartilage damage repair effect, and expression of collagen I and collagen II at the 6th and the 12th week were measured. BMSCs treated with pEGFP-C1/IGF-1, pEGFP-C1/BMP-7, and pEGFP-C1/BMP-7-IGF-1 exhibited elevated expression of BMP-7 and IGF-1. Besides, BMSCs in the P10 generation displayed decreased cell proliferation. Moreover, BMSCs treated with IGF-1, BMP-7, and IGF-1-BMP-7 showed reduced histological score and collagen I expression while elevated collagen II expression, as well as better repair effect, especially in those treated with IGF-1-BMP-7. Collectively, these results demonstrated a synergistic effect of IGF-1 and BMP-7 on the BMSC chondrogenic differentiation on the articular cartilage damage repair in the rabbit knees, highlighting its therapeutic potential for the treatment of articular cartilage damage.     

Regulation of chondrocyte gene expression by osteogenic protein-1

Introduction  

The objective of this study was to investigate which genes are regulated by osteogenic protein-1 (OP-1) in human articular chondrocytes using Affimetrix gene array, in order to understand the role of OP-1 in cartilage homeostasis.     

Aging, osteoarthritis and transforming growth factor-beta signaling in cartilage

Osteoarthritis is a common malady of the musculoskeletal system affecting the articular cartilage. The increased frequency of osteoarthritis with aging indicates the complex etiology of this disease, which includes pathophysiology and joint stability including biomechanics. The balance between anabolic morphogens and growth factors and catabolic cytokines is at the crux of the problem of osteoarthritis. One such signal is transforming growth factor-beta (TGF-beta). The impaired TGF-beta signaling has been identified as a culprit in old mice in a recent article in this journal. This commentary places this discovery in the context of anabolic and catabolic signals and articular cartilage homeostasis in the joint.     

TAK-778, a novel synthetic 3-benzothiepin derivative, promotes chondrogenesis in vitro and in vivo.

TAK-778, a novel synthetic 3-benzothiepin derivative, stimulates the formation of cartilaginous nodules in mouse chondroprogenitor-like ATDC5 cells in vitro in association with upregulation of the gene expression of transforming growth factor-beta(2), but not bone morphogenetic protein-4 and insulin-like growth factor-I. One-shot injection of the TAK-778-containing sustained-release microcapsules accelerated the repair process of the full thickness defects of articular cartilage in rabbit knees. Our in vitro and in vivo results indicate that TAK-778 may be a therapeutically useful synthetic agent for articular cartilage repair.     

Using changes in hydrostatic and osmotic pressure to manipulate metabolic function in chondrocytes

Articular cartilage has distinct histological depth zones. In each zone, chondrocytes are subject to different hydrostatic (HP) and osmotic pressure (OP) due to weight-bearing and joint-loading. Previous in vitro studies of regeneration and pathophysiology in cartilage have failed to consider the characteristics of histological heterogeneity and the effects of combinations of changes in HP and OP. Thus, we have constructed molecular, biochemical, and histological profiles of anabolic and catabolic molecules produced by chondrocytes from each depth zone isolated from bovine articular cartilage in response to changes in HP and OP. We cultured the chondrocytes with combinations of loading or off-loading of HP at 0-0.5 MPa, 0.5 Hz, and changes in OP of 300-450 mosM over 1 wk, and evaluated mRNA expression and immunohistology of both anabolic and catabolic molecules and amounts of accumulated sulfated glycosaminoglycan. Any changes in HP and OP upregulated mRNA of anabolic and catabolic molecules in surface-, middle-, and deep-zone cells, in descending order of magnitude. Off-loading HP maintained the anabolic and reduced the catabolic mRNA; high OP retained upregulation of catabolic mRNA. These molecular profiles were consistent with immunohistological and biochemical findings. Changes in HP and OP are essential for simulating chondrocyte physiology and useful for manipulating phenotypes.     

Species-specific biological effects of FGF-2 in articular cartilage: implication for distinct roles within the FGF receptor family

Existing literature demonstrates that fibroblast growth factor-2 (FGF-2) exerts opposing, contradictory biological effects on cartilage homeostasis in different species. In human articular cartilage, FGF-2 plays a catabolic and anti-anabolic role in cartilage homeostasis, driving homeostasis toward degeneration and osteoarthritis (OA). In murine joints, however, FGF-2 has been identified as an anabolic mediator as ablation of the FGF-2 gene demonstrated increased susceptibility to OA. There have been no previous studies specifically addressing species-specific differences in FGF-2-mediated biological effects. In this study, we provide a mechanistic understanding by which FGF-2 exerts contradictory biological effects in human versus murine tissues. Using human articular cartilage (ex vivo) and a medial meniscal destabilization (DMM) animal model (in vivo), species-specific expression patterns of FGFR receptors (FGFRs) are elucidated between human and murine articular cartilage. In the murine OA model followed by intra-articular injection of FGF-2, we further correlate FGFR profiles to changes in behavioral pain perception, proteoglycan content in articular cartilage, and production of inflammatory (CD11b) and angiogenic (VEGF) mediators in synovium lining cells. Our results suggest that the fundamental differences in cellular responses between human and murine tissues may be secondary to distinctive expression patterns of FGFRs that eventually determine biological outcomes in the presence of FGF-2. The complex interplay of FGFRs and the downstream signaling cascades induced by FGF-2 in human cartilage should add caution to the use of this particular growth factor for biological therapy in the future.     

Aging,osteoarthritis and transforming growth factor-β signaling in cartilage

Osteoarthritis is a common malady of the musculoskeletal system affecting the articular cartilage. The increased frequency of osteoarthritis with aging indicates the complex etiology of this disease, which includes pathophysiology and joint stability including biomechanics. The balance between anabolic morphogens and growth factors and catabolic cytokines is at the crux of the problem of osteoarthritis. One such signal is transforming growth factor-β (TGF-β). The impaired TGF-β signaling has been identified as a culprit in old mice in a recent article in this journal. This commentary places this discovery in the context of anabolic and catabolic signals and articular cartilage homeostasis in the joint.     

Adenoviral transduction supports matrix expression of alginate cultured articular chondrocytes

The present study examines the effects of adenoviral (Ad) transduction of human primary chondrocyte on transgene expression and matrix production. Primary chondrocytes were isolated from healthy articular cartilage and from cartilage with mild osteoarthritis (OA), transduced with an Ad vector and either immediately cultured in alginate or expanded in monolayer before alginate culture. Proteoglycan production was measured using dimethylmethylene blue (DMMB) assay and matrix gene expression was quantified by real-time PCR. Viral infection of primary chondrocytes results in a stable long time transgene expression for up to 13 weeks. Ad transduction does not significantly alter gene expression and matrix production if chondrocytes are immediately embedded in alginate. However, if expanded prior to three dimension (3D) culture in alginate, chondrocytes produce not only more proteoglycans compared to non-transduced controls, but also display an increased anabolic and decreased catabolic activity compared to non-transduced controls. We therefore suggest that successful autologous chondrocyte transplantation (ACT) should combine adenoviral transduction of primary chondrocytes with expansion in monolayer followed by 3D culture. Future studies will be needed to investigate whether the subsequent matrix production can be further improved by using Ad vectors bearing genes encoding matrix proteins.     

Combined transgene immortalized urothelial cells capable of reprogramming and hepatic differentiation

Human primary cells, including urine-derived cells (UCs), are an excellent source for generation of pluripotent stem cells (iPSCs) to model disease. However, replicative senescence starts early and shortens the time window for generation of iPSCs. We addressed the question whether combinations of transgenes allows efficient immortalization of UCs, iPSC generation, and differentiation into hepatocyte-like cells (HLCs). Retroviral transfer of three gene cassettes HPVE6E7 (H), hTERT/p53DD (T), cyclinD1/CDK4R24C (C) encoding five genes was established in primary UCs. Long-term cell proliferation was observed in cells carrying transgenes H, HT, HC, and HCT, whereas cells carrying transgenes C, T and CT showed early senescence similar to UCs. iPSCs could be exclusively generated from immortalized UCs transduced with transgenes HCT and HC. iPSC colonies appeared however later and in smaller number as compared to UCs. Using an established hepatic differentiation protocol, HLCs were obtained with high efficacy. Of note, a high expression of individual transgenes was observed in immortalized UCs, which was down-regulated after reprogramming in four out of five genes. One transgene was re-expressed in HLCs as compared to iPSCs. Our data suggest that individual transgene combinations result in advanced growth rates of immortalized cells and do not prevent iPSC formation and HLC differentiation. Retroviral transgene expression is mostly silenced in iPSCs but can be rarely re-expressed after hepatic differentiation. An extended time window for iPSC establishment can be proposed that allows straightforward functional analyses of differentiated cells.     

Identification of Arabidopsis histone deacetylase HDA6 mutants that affect transgene expression

A mutant screen was conducted in Arabidopsis that was based on deregulated expression of auxin-responsive transgenes. Two different tightly regulated (i.e., very low expression in the absence of auxin treatment and very high expression after exogenous auxin treatment) auxin-responsive promoters were used to drive the expression of both a beta-glucuronidase (GUS) reporter gene and a hygromycin phosphotransferase (HPH)-selectable marker gene. This screen yielded several mutants, and five of the mutations (axe1-1 to axe1-5) mapped to the same locus on chromosome 5. A map-based cloning approach was used to locate the axe1 mutations in an Arabidopsis RPD3-like histone deacetylase gene, referred to as HDA6. The axe1 mutant plants displayed increased expression of the GUS and HPH transgenes in the absence of auxin treatment and increased auxin-inducible expression of the transgenes compared with nonmutant control plants. None of a variety of endogenous, natural auxin-inducible genes in the mutant plants were upregulated like the transgenes, however. Results of treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine suggest that the axe1 mutations affect transgene silencing; however, histone deacetylase inhibitors had no affect on transgene silencing in mutant or control plants. The specific effect of AtHDA6 mutations on the auxin-responsive transgenes implicates this RPD3-like histone deacetylase as playing a role in transgene silencing. Furthermore, the effect of AtHDA6 on transgene silencing may be independent of its histone deacetylase activity.     

The detrimental effect of iron on OA chondrocytes: Importance of pro-inflammatory cytokines induced iron influx and oxidative stress

Iron overload is common in elderly people which is implicated in the disease progression of osteoarthritis (OA), however, how iron homeostasis is regulated during the onset and progression of OA and how it contributes to the pathological transition of articular chondrocytes remain unknown. In the present study, we developed an in vitro approach to investigate the roles of iron homeostasis and iron overload mediated oxidative stress in chondrocytes under an inflammatory environment. We found that pro-inflammatory cytokines could disrupt chondrocytes iron homeostasis via upregulating iron influx transporter TfR1 and downregulating iron efflux transporter FPN, thus leading to chondrocytes iron overload. Iron overload would promote the expression of chondrocytes catabolic markers, MMP3 and MMP13 expression. In addition, we found that oxidative stress and mitochondrial dysfunction played important roles in iron overload-induced cartilage degeneration, reducing iron concentration using iron chelator or antioxidant drugs could inhibit iron overload-induced OA-related catabolic markers and mitochondrial dysfunction. Our results suggest that pro-inflammatory cytokines could disrupt chondrocytes iron homeostasis and promote iron influx, iron overload-induced oxidative stress and mitochondrial dysfunction play important roles in iron overload-induced cartilage degeneration.