Introduction
Articular cartilage is a unique connective tissue that physiologically lacks blood vessels. This lack of vessels inevitably coincides with a significantly reduced oxygen level within the tissue, which requires well-adapted mechanisms to ensure survival of the resident cells. The transcription factor hypoxia-inducible factor (HIF)-1 represents one important element in maintaining proper cellular functions under such hypoxic conditions [
1]. As for chondrocytes, HIF-1 is also of great importance by promoting the synthesis of relevant extracellular matrix components [
2]. This synthesis may, at least partly, be mediated by transactivation of Sox9, a key transcription factor for many cartilage-specific genes involving metabolism and chondrogenic differentiation [
3,
4].
The importance of HIF-1 for the formation and maintenance of cartilage tissue has been demonstrated in conditional knockout mice in which deletion of its oxygen-sensitive subunit HIF-1α severely interfered with proper skeletal development and led to massive cell death within the center of the forming cartilaginous elements [
1]. On the contrary, another study in mice with conditional inactivation of the von Hippel–Lindau protein demonstrated that the resulting stabilization of HIF-1α by inhibiting its degradation increased the deposition of extracellular cartilage matrix in the growth plate [
5].
The regulation of HIF-1 activity is complex. Under normoxic conditions, HIF-1α is degraded rapidly. In the presence of molecular oxygen, two prolyl residues within the oxygen-dependent degradation domain of the HIF-1α protein are hydroxylated by HIF-specific oxygen-dependent prolyl-hydroxylases [
6]. This conversion allows capture by the von Hippel–Lindau protein complex followed by ubiquitinylation and rapid degradation by the proteasome [
6,
7]. The synthesis of HIF-1α can be activated by a variety of factors, including reactive oxygen species, glucose metabolites, and a number of growth factors or cytokines involving the phosphatidylinositol 3-kinase or extracellular signal-regulated kinase/mitogen-activated protein kinase pathway [
8‐
10].
In osteoarthritic cartilage, the protein levels of HIF-1 are significantly increased and its activity correlates to the severity of degenerative cartilage changes [
9,
11]. According to the biological functions of HIF-1, it may be assumed that HIF-1 exerts a compensatory protective role in the disease process rather than promoting the progression of the disease.
To further prove this hypothesis, we established two animal models. The first model served to investigate whether inhibition of HIF by 2-methoxyestradiol (2ME2) promotes or initiates osteoarthritis (OA) in the murine knee joint. In contrast to conditional knockout mice, the chemical inhibition allows one to investigate the effects in adult joints in an otherwise healthy organism, and therefore seems better suited referring to studies on OA. Although the exact mechanism of HIF inhibition by 2ME2 has still to be defined, 2ME2 has been shown to reliably decrease the levels of HIF-1α protein in chondrocytes and a number of other cell types – and as a consequence also decreases the expression of a number of HIF-1 target genes including phosphoglycerate kinase 1 (PGK1), vascular endothelial growth factor A, and glucose transporter type 1 [
12‐
14].
The second animal model was used to investigate whether stabilization of HIF-1α by dimethyloxaloylglycine (DMOG) prevents or delays degenerative changes in STR/ORT mice. DMOG is known to efficiently inhibit prolyl-hydroxylases that mediate the oxygen-dependent degradation of HIF [
15]. STR/ORT mice served as an OA model since this strain spontaneously develops degenerative changes in the knee joints [
16‐
19].
Materials and methods
Cell isolation and culture
Primary human chondrocytes were isolated from knee cartilage of six patients undergoing total knee replacement for OA as described previously [
12]. All of the samples were obtained after the patients gave their informed consent. Our institutional ethics committee approved the study protocol.
To minimize the effect of chondrocyte dedifferentiation, only first-passage chondrocytes were used in the present study. After digestion, cells were plated at a cell density of 100,000 cells/cm2 in 24-well plates for RNA isolation or in Flexi-Perm culture chambers (Greiner Bio-One, Solingen, Germany) for immunohistochemistry.
First, the cells were cultured under normoxic conditions (21% oxygen) for 3 days in DMEM containing 10% FCS, 2 mM glutamine and 50 U/ml streptomycin/penicillin at 37°C. For gene expression analyses, confluent cells were subsequently treated with different concentrations of DMOG ranging from 0.1 to 1 mM or with 2ME2 in concentrations of 100 or 200 μM, and were cultured either under normoxic (21% oxygen) or hypoxic conditions (1% oxygen) for 24 hours before harvesting. Since 2ME2 is dissolved in dimethyl sulfoxide (DMSO), a portion of the cells was exposed to 1% DMSO alone, which served as an additional control. For hypoxic conditions, the chondrocytes were kept in a sealed incubator (Binder GmbH, Tuttlingen, Germany), and flushed with gas mixture containing 1% oxygen, 5% carbon dioxide and balanced with 94% N2 in a humidified atmosphere. For immunohistochemistry, the cells were treated with 1 mM DMOG and were cultured under normoxic conditions for 5 days.
Animal experiments
The present study investigated the effect of 2ME2 on female Balb/C mice (Charles River, Sulzfeld, Germany) and the effect of DMOG on female STR/ORT mice (Harlan Winkelmann, Borchen, Germany). The animals were fed a standard laboratory diet ad libitum and were allowed to move freely in their cages at any time. For all procedures, the mice were anesthetized by inhalation with 1.5 l/min isofluran (Baxter, Unterschleißheim, Germany) and 1.5 l/min oxygen.
In 14-week-old Balb/C-mice (n = 32), 2ME2 (20 μl) at concentrations of 10 μM (group 1, n = 16) or 100 μM (group 2, n = 16) was carefully injected with a 27G needle into the knee joints through the patella tendon into the intercondylar notch region to avoid contact with the articular surface. The treatment was repeated every other day for a total of six injections during the course of the first 2 weeks of the experiment. The animals received no further treatment for the remaining 1 week or 10 weeks, respectively. A control solution (20 μl) was injected into the right knee joints of all Balb/C mice at the same time. Since 2ME2 was dissolved in a 1% DMSO saline solution, the control solution (0.9% NaCl) was also supplemented with 1% DMSO. Preliminary experiments in vitro excluded any toxic effects of 1% DMSO. The mice were killed either after 3 weeks (n = 8 per group) or 12 weeks (n = 8 per group) by cervical dislocation.
In 8-week-old STR/ORT mice (n = 8), 20 μl of a 1 mM DMOG solution were injected into the left knee joints once per week throughout the whole period of 12 weeks. DMOG was dissolved in 0.9% NaCl. As a control, 20 μl saline solution was injected into the right knee joints at the same time. The STR/ORT mice were sacrificed after 12 weeks.
All knee joints were fixed in 4% paraformaldehyde for 12 hours and were decalcified in 0.5 M ethylenediamine tetraacetic acid/2% paraformaldehyde for 2 weeks. After standard processing, the samples were embedded in paraffin. Serial frontal 5 μm sections of the knee joints were cut and further processed for histological and immunohistological analysis.
All procedures on the animals were approved by the appropriate institutional Review board.
Histological assessment
After deparaffinization, serial sections were stained with toluidine blue or with H&E for further histological investigation. Sections from treated joints and control joints were compared by scoring systems investigating the articular cartilage layer, osteophyte formation and synovial tissue.
The articular cartilage layer was evaluated referring to a mouse-specific scoring system described by Walton [
16] (grade 0 = normal; grade 1 = superficial fibrillation, alterations in proteoglycan staining; grade 2 = deeper fissures, beginning loss of cartilage tissue; grade 3 = substantial loss of uncalcified cartilage tissue, fissures extending to the subchondral bone; grade 4 = complete loss of cartilage tissue, exposure of bone).
Staging of osteophyte formation was analyzed according to a previously described system [
20] (grade 0 = normal; grade 1 = fibrous outgrowths; grade 2 = fibrocartilaginous tissue; grade 3 = mature cartilage tissue; grade 4 = cellular hypertrophy, bone core).
The evaluation of the synovial layer refers to the additive score by Krenn and colleagues [
21], including thickening of the synovial cell lining (grade 0 = single cell layer; grade 1 = two to three cell layers; grade 2 = four to five cell layers; grade 3 = more than five cell layers), synovial inflammation (grade 0 = no inflammation; grade 1 = single lymphocytes; grade 2 = lymphocytic aggregations; grade 3 = formation of lymph follicles) and the cell density of the synovial stroma (grade 0 = normal cell density; grade 1 = slightly increased cellular density; grade 2 = increased cellular density, 3 = high cellular density, presence of multinuclear giant cells).
The sections were analyzed blind, by three independent experts.
Detection of cellular hypoxia
Pimonidazole hydrochloride 60 mg/kg (Hydroxyprobe™-1 Plus Kit; Chemicon, Temecula, CA, USA) was injected intraperitoneally in Balb/C mice (n = 6). The animals were sacrificed after 24 hours. The knee joints and other organs were fixed in 4% paraformaldehyde for 12 hours and were processed as described above. Representative sections were immunostained using a FITC-conjugated monoclonal antibody (Hydroxyprobe™-1 Mab1; Chemicon). Immunostaining was performed according to the manufacturer's recommendations. Antibody binding was detected by a peroxidase-labeled secondary antibody against FITC and was visualized by diaminobenzidine tetrahydrochloride (Chemicon, Temecula, CA, USA). All sections were counterstained with hematoxylin.
Immunohistochemistry
For detection of type II collagen, sections were pretreated with 0.2% hyaluronidase (Roche, Mannheim, Germany) in PBS (pH 5.0) for 60 minutes, and subsequently treated with pronase (2 mg/ml in PBS, pH 7.3; Sigma-Aldrich, Munich, Germany) for 60 minutes at 37°C. No enzymatic pretreatment of the sections was required for detection of CD45. Nonspecific antibody binding was blocked with 5% BSA in PBS.
For signal amplification and visualization of HIF-1α, an amplification system (CSA kit; Dako, Hamburg, Germany) was used according to the manufacturer's instructions. Antigen retrieval was performed for 6 minutes in preheated Dako target retrieval solution, using a pressure cooker.
The slides were incubated overnight at 4°C with polyclonal rabbit antibodies against HIF-1α (Cayman Chemical, Ann Arbor, MI, USA) in a 1:10,000 dilution, with monoclonal mouse anti-human type II collagen antibodies (MP Biomedicals, Irvine, CA, USA) in a 1:500 dilution or with monoclonal rat anti-mouse CD45 antibodies (BD Pharmingen, Heidelberg, Germany) in a 1:200 dilution, followed by washing with Tris-buffered saline. For detection of type II collagen or CD45, the sections were subsequently incubated with a biotinylated donkey anti-mouse secondary antibody (Dianova, Hamburg, Germany) or a biotinylated rabbit anti-rat secondary antibody (Vector Laboratories, Burlingame, CA, USA), followed by treatment with a complex of streptavidin and biotinylated alkaline phosphatase. The sections were developed with fast red and were counterstained with hematoxylin.
For visualization of HIF-1α, a catalyzed signal amplification kit (Dako) based on a streptavidin–biotin–peroxidase reaction was used. 3,3'-Diaminobenzidine served as the chromogen for the peroxidase reaction.
Immunofluorescence
After fixation of the cultured cells with 70% ethanol, the slides were blocked with 5% BSA and were incubated with monoclonal mouse anti-human type II collagen antibodies (MP Biomedicals) in a 1:500 dilution for 50 minutes at 37°C. After washing with PBS, the cells were incubated with a Cy3-conjugated anti-mouse antibody (Dianova) diluted at a ratio of 1:100 for 45 minutes. After washing with Tris-buffered saline, the slides were covered with a mounting medium containing 4',6'-diamidino-2-phenylindole (Vector, Peterborough, UK) and were analyzed by fluorescence microscopy. Control slides were incubated with equivalent concentrations of mouse IgG.
TUNEL staining
For the detection of in situ DNA breaks, the TUNEL reaction was applied using the In Situ Cell Death Detection Kit, AP (Roche, Mannheim, Germany). The proteinase K pretreatment as well as the terminal deoxynucleotidyl transferase concentrations were carefully titrated to allow sensitive and specific detection of apoptotic cell nuclei. After deparaffinization, the sections were washed in PBS and digested with proteinase K (20 μg/ml; Boehringer, Ingelheim, Germany) for 15 minutes at 37°C. After washing with PBS, sections were incubated with terminal deoxynucleotidyl transferase solution in reaction buffer at a volume ratio of 9:1 at 37°C for 1 hour followed by extensive washing. The converter AP antibody was added for 30 minutes at 37°C. After washing, detection was performed by fast red staining and counterstaining with hematoxylin.
RNA isolation and real-time RT-PCR
The expression levels of Sox9, type II collagen α1-chain (Col2A1), type I collagen α2-chain (Col1A2), PGK1, IL-1β, IL-6 and TNFα under the influence of different concentrations of DMOG or 2ME2 were compared for normoxic or hypoxic conditions by real-time RT-PCR using RNA preparations from cultured primary human chondrocytes of three different donors. Total RNA was isolated from the cells by the Nucleo-Spin-RNA-II-Kit (Clontech Laboratories, Mountain View, CA, USA). Quantitative real-time RT-PCR was performed with an ABI Prism 7900 sequence detection system (Applied Biosystems, Foster City, CA, USA) and a QuantiTect Probe RT-PCR Kit (Qiagen, Chatsworth, CA, USA) for one-step RT-PCR.
The relative quantification of gene expression was performed using the standard curve method. For each sample, the relative amount of the target mRNA was determined and normalized to β2-microglobulin mRNA.
The primer and probe sets for Sox9, Col1A2, Col2A1, IL-1β, IL-6 and TNFα were purchased from Applied Biosystems and from Celera Genomics (Rockville, MD, USA). The primer and probe sets for β
2-microglobulin and PGK1 were designed by the primer express software (PerkinElmer, Emeryville, CA, USA) as described previously [
13].
Statistical analysis
All data are presented as the mean ± standard deviation. The analysis of the morphological scores was performed using the Mann–Whitney test. The results of quantitative gene expression were analyzed using a two-sided Student's t test. P <0.05 was considered significant.
Discussion
The present work documents the importance of the transcription factor HIF-1 for maintaining the integrity of hypoxic articular cartilage. The functional inhibition of HIF-1 coincided with increased apoptosis of articular chondrocytes and led to degenerative changes in murine knee joints. The study therefore supports the results of Schipani and colleagues obtained from conditional HIF-knockout mice in which cell death in the center of cartilaginous elements of the developing skeleton was observed [
1]. Further studies confirmed that its subunit, HIF-1α, plays an important role in chondrogenesis and joint formation – and HIF-1α was recently shown to activate the promotor of SOX9, which is a key regulator of chondrocyte differentiation [
3,
4,
23,
24].
The naturally occurring derivate of estradiol, 2ME2, is considered a substance with anti-tumorigenic properties and is also accepted as a HIF inhibitor. Although its exact mechanism of action is not yet known, the HIF-1-inhibiting properties of 2ME2 have been suggested to be linked to a microtubule depolymerizing effect [
14,
25,
26]. 2ME2 is a substance currently investigated in clinical trials for the treatment of tumors, and its systemic application was shown to be well tolerated with no relevant negative side effects in oxygenated organs [
27].
In vitro studies also confirm that 2ME2 in moderate concentrations has no direct toxic effects on slow-dividing nontumor cells but does lead to a reliable inhibition of HIF-1 [
12,
13].
The present study has shown for the first time that repeated intraarticular administration of 2ME2 decreased the number and the intensity of HIF-1α-positive cells. The relevance of tissue hypoxia even in thin murine articular cartilage was demonstrated by the Hydroxyprobe™ (Chemicon) method, which detected hypoxic cells in deep and superficial layers of avascular cartilage and mensical tissue. This observation indicates the relevance of the transcription factor HIF-1 even in relatively thin murine articular cartilage. One can therefore conclude that 2ME2-exposed cells were no longer able to adapt to a hypoxic environment, resulting in impaired cell functions and cell death – which is presumably the primary pathomechanism for the degeneration of the cartilage tissue in this model. Of course, one has to consider that the molecular mechanism of cartilage degeneration induced by 2ME2 differs to some extent from the pathogenesis of primary OA, in which apoptotic cell death is supposed to play an inferior role [
28]. Nevertheless, the morphological changes strikingly resembled those of osteoarthritic lesions. The lack of synovial inflammation and the absent induction of inflammatory cytokines suggest that cartilage destruction by 2ME2 does not depend on inflammatory reactions, but rather on an impact on the chondrocyte function itself.
The second objective of the present work was to investigate whether stabilization of HIF-1 could prevent the initiation or progression of murine OA in STR/ORT mice, which belong to a family of mouse strains that develop spontaneously degenerative changes [
16,
17,
19]. In preceding
in vitro experiments, DMOG was shown to effectively stabilize HIF-1 by inhibiting its degradation [
12,
15].
Despite the stimulatory effects on SOX9 expression
in vitro, the repeated intra-articular injection of DMOG did not prevent the progression of OA in the knee joints of STR/ORT mice. Neither osteophyte formation nor the score for destruction of the articular cartilage differed between DMOG-treated joints and control joints. Degeneration of the articular cartilage and osteophyte formation were present in all knee compartments. Consistent with previous reports, cartilage degeneration was most prominent in the medial compartment. This phenomenon might be a consequence of mechanical overloading due to the varus deformity of the hind limbs [
17,
19].
The following three reasons may account for the failure of DMOG to have a beneficial effect. Firstly, endogenous HIF-1 levels may be sufficient in articular chondrocytes, and were even shown to be elevated in OA cartilage [
9,
11,
29]. The data of the present study show that articular chondrocytes strictly depend on sufficient HIF-1 activity to ensure their cellular survival within the hypoxic matrix. Other studies on other cell types, however, describe a contradictory role of HIF-1α in modulating cell viability and apoptosis, which may depend on multitude factors including the cell type or the physico-chemical environment [
30‐
32]. The physiological function of HIF-1α may therefore depend on a well-balanced activity, and surplus levels may not necessarily have further protective effects on the cells or even induce proapoptotic or other detrimental events that could not be detected by the TUNEL method used in the present study.
Secondly, DMOG represents rather a nonselective inhibitor of prolyl-hydroxylases. To our knowledge, agents that selectively inhibit the HIF-degrading prolyl-hydroxylase have not so far been established. DMOG not only inhibits the HIF prolyl-hydroxylase, but also interferes with collagen prolyl-hydroxylases [
33]. The latter are involved in post-transcriptional processing of collagen fibers and formation of the triple helices. The hydroxylation of proline residues is essential for the formation of intramolecular hydrogen bonds and contributes to the stability of the triple-helical conformation [
34]. The intracellular accumulation of type II collagen observed by immunohistochemistry and immunofluorescence can therefore be ascribed to impaired collagen processing, which is known to interfere with proper secretion of fibrillar collagens [
35]. As a consequence, retention and accumulation of collagen molecules may act via a negative feedback loop on their gene expression. Downregulation of the collagen gene expression was more prominent for Col1A2 than for Col2A1. Two mechanisms may counteract specifically the expression of Col2A1. The described putative suppressive effect by intracellular collagen retention may at least partly be neutralized by a stimulatory effect via increased SOX9 expression, which is known to function as an enhancer for Col2A1 [
36].
Thirdly, HIF-1α not only mediates prochondrogenic effects (for example, by enhanced SOX9 expression), but is also involved in catabolic events. HIF-1α was recently shown to be essential for myeloid cell-mediated inflammation [
37]. Elevation of HIF-1α levels by DMOG increased the expression of the catabolic cytokines IL-1β and IL-6. IL-1β has recently been shown to be a HIF-1-target gene since its promotor region carries multiple HIF-1-binding sites resulting in an enhanced expression under hypoxia [
38]. Although moderate doses of IL-1β also support anabolic effects on cartilage metabolism to some degree [
39,
40], it is well proven that this cytokine generally mediates catabolic events by canonical pathways and, therefore, may also account for the negative effect on collagen expression in the present study.
Conclusion
The data from the present work underline the requirement of adequate levels of HIF-1 for the viability of chondrocytes and for the maintenance of the integrity of hypoxic articular cartilage. One has to consider, however, that surplus levels of HIF-1α may also exert catabolic effects – for example, by inducing the cytokines IL-1β and IL-6. HIF-1α may thus exert a paradoxical role in cartilage. On the one hand, HIF-1α ensures energy supply and survival of chondrocytes exposed to a hypoxic environment. Furthermore, HIF-1α stimulates the expression of a number of cartilage-specific genes. On the other hand, HIF-1α also promotes catabolic pathways and may also trigger proapoptotic events. In view of the data of the present study and the data from the literature, HIF-1α may be considered an important element in balancing anabolism and catabolism as well as cellular protection and cell death in cartilage and many other tissues. In this context, the question still remains open if elevated HIF-1α levels in OA exert a compensatory protective role or trigger the disease process.
In view of the detrimental side effect of DMOG on collagen metabolism due to its lack of specificity, one cannot currently rule out that more specific agents might nevertheless have beneficial effects. The future development of strictly selective inhibitors of HIF-degrading prolyl-hydroxylases therefore seems worthwhile, since stabilization of HIF may be of potential therapeutic value at least for cartilage repair approaches by supporting chondrogenic differentiation (for example, via stimulating expression of SOX9).
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
KG carried out the study design, the acquisition of data, the analysis and interpretation of data and drafted the manuscript. DP participated in the study design and drafted the manuscript. SO and KXK were involved in the acquisition of the data. MW participated in the analysis and interpretation of data. FFH and BS conceived the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.