Uptake of [18F] EF5 as a Tracer for Hypoxic and Aggressive Phenotype in Experimental Head and Neck Squamous Cell Carcinoma

PURPOSE: This study aims to investigate whether the uptake of 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide ([¹⁸F]EF5) and 2-deoxy-2-[¹⁸F]fluoro-d-glucose ([¹⁸F]FDG) is associated with a hypoxia-driven adverse phenotype in head and neck squamous cell carcinoma cell lines and tumor xenografts. METHODS: Xenografts were imaged in vivo, and tumor sections were stained for hypoxia-inducible factor 1α (Hif-1α), carbonic anhydrase IX (CA IX), and glucose transporter 1 (Glut-1). Tracer uptakes and the expression of Hif-1α were determined in cell lines under 1% hypoxia. RESULTS: High [¹⁸F]EF5 uptake was seen in xenografts expressing high levels of CA IX, Glut-1, and Hif-1α, whereas low [¹⁸F]EF5 uptake was detected in xenografts expressing low amounts of CA IX and Hif-1α. The uptake of [¹⁸F]EF5 between cell lines varied extensively under normoxic conditions. A clear correlation was found between the expression of Hif-1α and the uptake of [¹⁸F]FDG during hypoxia. CONCLUSIONS: The UT-SCC cell lines studied differed with respect to their hypoxic phenotypes, and these variations were detectable with [¹⁸F]EF5. Acute hypoxia increases [¹⁸F]FDG uptake in vitro, whereas a high [¹⁸F]EF5 uptake reflects a more complex phenotype associated with hypoxia and an aggressive growth pattern.     

An In Situ Hybridization Study of Perlecan,DMP1, and MEPE in Developing Condylar Cartilage of the Fetal Mouse Mandible and Limb Bud Cartilage

The main purpose of this in situ hybridization study was to investigate mRNA expression of three bone/cartilage matrix components (perlecan, DMP1, and MEPE) in developing primary (tibial) and secondary (condylar) cartilage. Perlecan mRNA expression was first detected in newly formed chondrocytes in tibial cartilage at E13.0, but this expression decreased in hypertrophic chondrocytes at E14.0. In contrast, at E15.0, perlecan mRNA was first detected in the newly formed chondrocytes of condylar cartilage; these chondrocytes had characteristics of hypertrophic chondrocytes, which confirmed the previous observation that progenitor cells of developing secondary cartilage rapidly differentiate into hypertrophic chondrocytes. DMP1 mRNA was detected in many chondrocytes within the lower hypertrophic cell zone in tibial cartilage at E14.0. In contrast, DMP1 mRNA expression was only transiently detected in a few chondrocytes of condylar cartilage at E15.0. Thus, DMP1 may be less important in the developing condylar cartilage than in the tibial cartilage. Another purpose of this study was to test the hypothesis that MEPE may be a useful marker molecule for cartilage. MEPE mRNA was not detected in any chondrocytes in either tibial or condylar cartilage; however, MEPE immunoreactivity was detected throughout the cartilage matrix. Western immunoblot analysis demonstrated that MEPE antibody recognized two bands, one of 67 kDa and another of 59 kDa, in cartilage-derived samples. Thus MEPE protein may gradually accumulate in the cartilage, even though mRNA expression levels were below the limits of detection of in situ hybridization. Ultimately, we could not designate MEPE as a marker molecule for cartilage, and would modify our original hypothesis.Key words: Mandibular condylar cartilage, perlecan, DMP1; MEPE, in situ hybridization     

Interleukin-1β and tumor necrosis factor-α augment acidosis-induced rat articular chondrocyte apoptosis via nuclear factor-kappaB-dependent upregulation of ASIC1a channel

The acute-phase proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) demonstrate high-level expression and pleiotropic biological effects, and contribute to the progression and persistence of rheumatoid arthritis (RA). Acid hydrarthrosis is also an important pathological characteristic of RA, and the acid-sensing ion channel 1a (ASIC1a) plays a critical role in acidosis-induced chondrocyte cytotoxicity. However, the roles of IL-1β and TNF-α in acid-induced apoptosis of chondrocytes remain unclear. Rat adjuvant arthritis and primary articular chondrocytes were used as in vivo and in vitro model systems, respectively. ASIC1a expression in articular cartilage was increased and highly colocalized with nuclear factor (NF)-κB expression in vivo. IL-1β and TNF-α could upregulate ASIC1a expression. These cytokines activated mitogen-activated protein kinase and NF-κB pathways in chondrocytes, while the respective inhibitors of these signaling pathways could partially reverse the ASIC1a upregulation induced by IL-1β and TNF-α. Dual luciferase and gel-shift assays and chromatin immunoprecipitation-polymerase chain reaction demonstrated that IL-1β and TNF-α enhanced ASIC1a promoter activity in chondrocytes by increasing NF-κB DNA-binding activities, which was in turn prevented by the NF-κB inhibitor ammonium pyrrolidinedithiocarbamate. IL-1β and TNF-α also decreased cell viability but enhanced LDH release, intracellular Ca²⁺ concentration elevation, loss of mitochondrial membrane potential, cleaved PARP and cleaved caspase-3/9 expression, and apoptosis in acid-stimulated chondrocytes, which effects could be abrogated by the specific ASIC1a inhibitor psalmotoxin-1 (PcTX-1), ASIC1a-short hairpin RNA or calcium chelating agent BAPTA-AM. These results indicate that IL-1β and TNF-α can augment acidosis-induced cytotoxicity through NF-κB-dependent up-regulation of ASIC1a channel expression in primary articular chondrocytes.     

Hypoxia-inducible factor-1alpha regulates autophagy to activate hepatic stellate cells

The role of autophagy in Hif-1α modulated activation of hepatic stellate cells was illustrated in current work. Autophagy markers were determined in livers of Schistosoma japonicum infected mice and hypoxia or LPS treated human hepatic stellate cell, LX-2 cells. The action of Hif-1 to autophagy was defined as increase of autophagy markers was significantly suppressed in Hif-1α siRNA transfected cells upon hypoxia or LPS stimulation. The function of autophagy in activation of LX-2 cells was assessed as increase of activation markers was blocked using autophagy inhibitors under hypoxia and LPS stimulation. Conclusively, Hif-1α regulates activation of hepatic stellate cell by modulating autophagy.     

An in situ hybridization study of MMP-2, -9, -13, -14, TIMP-1, and -2 mRNA in fetal mouse mandibular condylar cartilage as compared with limb bud cartilage

The main purpose of this in situ hybridization study was to investigate MMPs and TIMPs mRNA expression in developing mandibular condylar cartilage and limb bud cartilage. At E14.0, MMP-2, -14, TIMP-1 and -2 mRNAs were expressed in the periosteum of mandibular bone, and in the condylar anlage. At E15.0 MMP-2, -14, TIMP-1 and -2 mRNAs were expressed in the perichondrium of newly formed condylar cartilage and the periosteum of developing bone collar, whereas, expression of MMP-14 and TIMP-1 mRNAs were restricted to the inner layer of the periosteum/perichondrium. This expression patterns continued until E18.0. Further, from E13.0 to 14.0, in the developing tibial cartilage, MMP-2, -14, and TIMP-2 mRNAs were expressed in the periosteum/perichondrium, but weak MMP-14 and no TIMP-1 mRNA expression was recognized in the perichondrium. These results confirmed that the perichondrium of condylar cartilage has characteristics of periosteum, and suggested that MMPs and/or TIMPs are more actively involved in the development of condylar (secondary) cartilage than tibial (primary) cartilage. MMP-9-positive cells were observed in the bone collar of both types of cartilage, and they were consistent with osteoclasts/chondroclasts. MMP-13 mRNA expression was restricted to the chondrocytes of the lower hypertrophic cell zone in tibial cartilage at E14.0, indicating MMP-13 can be used as a marker for lower hypertrophic cell zone. It was also expressed in chondrocytes of newly formed condylar cartilage at E15.0, and continuously expressed in the lower hypertrophic cell zone until E18.0. These results confirmed that progenitor cells of condylar cartilage are rapidly differentiated into hypertrophic chondrocytes, which is a unique structural feature of secondary cartilage different from that of primary cartilage.     

Vascular endothelial growth factor plays an important autocrine/paracrine role in the progression of osteoarthritis

Vascular endothelial growth factor (VEGF) plays an essential role in the angiogenesis of growing cartilage. Although VEGF expression in cartilage vanishes in normal adults, VEGF is known to be expressed in chondrocytes of osteoarthritic (OA) cartilage. As little information is available on the VEGF expression in the cartilage of OA-like lesions of the temporomandibular joint (TMJ), VEGF expression in the condylar cartilage of TMJs of rats affected with OA was examined. To evoke OA, mechanical stress was applied by forced jaw opening for 10 or 20 days. After 20 days, marked OA-like lesions were observed in the condyle. VEGF was expressed in the chondrocytes of the mature and hypertrophic cell layers of the intermediate and posterior region of the condyle. The percentage of VEGF immunopositive chondrocytes significantly increased with the period of applied mechanical stress. Furthermore, tartrate-resistant acid phosphatase (TRAP) staining of the condylar cartilage showed significant increment of osteoclasts in the mineralized layer subjacent to the hypertrophic layer where high VEGF expression could be detected. The results suggest that VEGF plays an important role in the progression of OA.Eiji Tanaka and Junko Aoyama contributed equally to this work.     

Age-related changes in the expression of gelatinase and tissue inhibitor of metalloproteinase genes in mandibular condylar,growth plate,and articular cartilage in rats

Summary Mandibular condylar cartilage acts as both articular and growth plate cartilage during growth, and then becomes articular cartilage after growth is complete. Cartilaginous extracellular matrix is remodeled continuously via a combination of production, degradation by matrix metalloproteinases (MMPs), and inhibition of MMP activity by tissue inhibitors of metalloproteinases (TIMPs). This study attempted to clarify the age-related changes in the mRNA expression patterns of MMP-2, MMP-9, TIMP-1, TIMP-2, and TIMP-3 in mandibular condylar cartilage in comparison to tibial growth plate and articular cartilage using an in situ hybridization method in growing and adult rats. MMP-2 and MMP-9 were expressed in a wide range of condylar cartilage cells during growth, and their expression domains became limited to mature chondrocytes in adults. The patterns of TIMP-1 and TIMP-2 expression were similar to those of MMP-2 and MMP-9 during growth, and were maintained until adulthood. TIMP-3 was localized to hypertrophic chondrocytes throughout the growth stage. Therefore, we concluded that TIMP-1 and TIMP-2 were general inhibitors of MMP-2 and MMP-9 in condylar cartilage, while TIMP-3 regulates the collagenolytic degradation of the hypertrophic cartilage matrix.     

Chondroprotection of PPARα activation by WY14643 via autophagy involving Akt and ERK in LPS‐treated mouse chondrocytes and osteoarthritis model

Autophagy maintains cellular homoeostasis. The enhancement of autophagy in chondrocytes could prevent osteoarthritis (OA) progression in articular cartilage. Peroxisome proliferator‐activated receptor α (PPARα) activation may also protect articular chondrocytes against cartilage degradation in OA. However, whether the protective effect of activated PPARα is associated with autophagy induction in chondrocytes is not determined. In this study, we investigated the effect of PPARα activation by its agonist, WY14643, on the protein expression level of Aggrecan and ADAMTS5, and the protein expression level of autophagy biomarkers, including LC3B and P62, using Western blotting analysis in isolated mouse chondrocytes pre‐treated with lipopolysaccharides (LPS, mimicking OA chondrocytes) with or without the autophagy inhibitor chloroquine diphosphate salt. Furthermore, Akt and ERK phosphorylation was detected in LPS‐treated chondrocytes in response to WY14643. In addition, the effect of intra‐articularly injected WY14643 on articular cartilage in a mouse OA model established by the destabilization of the medial meniscus was assessed using the Osteoarthritis Research Society International (OARSI) histopathology assessment system, along with the detection of Aggrecan, ADAMTS5, LC3B and P62 protein levels using immunohistochemistry assay. The results indicated that PPARα activation by WY14643 promoted proteoglycan synthesis by autophagy enhancement in OA chondrocytes in vivo and in vitro concomitant with the elevation of Akt and ERK phosphorylation. Therefore, autophagy could contribute to the chondroprotection of PPARα activation by WY14643, with the implication that PPARα activation by WY14643 may be a potential approach for OA therapy.     

The Identification of CD163 Expressing Phagocytic Chondrocytes in Joint Cartilage and Its Novel Scavenger Role in Cartilage Degradation

Background

Cartilage degradation is a typical characteristic of arthritis. This study examined whether there was a subset of phagocytic chondrocytes that expressed the specific macrophage marker, CD163, and investigated their role in cartilage degradation.

Methods

Cartilage from the knee and temporomandibular joints of Sprague-Dawley rats was harvested. Cartilage degradation was experimentally-induced in rat temporomandibular joints, using published biomechanical dental methods. The expression levels of CD163 and inflammatory factors within cartilage, and the ability of CD163⁺ chondrocytes to conduct phagocytosis were investigated. Cartilage from the knees of patients with osteoarthritis and normal cartilage from knee amputations was also investigated.

Results

In the experimentally-induced degrading cartilage from temporomandibular joints, phagocytes were capable of engulfing neighboring apoptotic and necrotic cells, and the levels of CD163, TNF-α and MMPs were all increased (P<0.05). However, the levels of ACP-1, NO and ROS, which relate to cellular digestion capability were unchanged (P>0.05). CD163⁺ chondrocytes were found in the cartilage mid-zone of temporomandibular joints and knee from healthy, three-week old rats. Furthermore, an increased number of CD163⁺ chondrocytes with enhanced phagocytic activity were present in Col-II⁺ chondrocytes isolated from the degraded cartilage of temporomandibular joints in the eight-week experimental group compared with their age-matched controls. Increased number with enhanced phagocytic activity of CD163⁺ chondrocytes were also found in isolated Col-II⁺ chondrocytes stimulated with TNF-α (P<0.05). Mid-zone distribution of CD163⁺ cells accompanied with increased expression of CD163 and TNF-α were further confirmed in the isolated Col-II⁺ chondrocytes from the knee cartilage of human patients with osteoarthritis, in contrast to the controls (both P<0.05).

Conclusions

An increased number of CD163⁺ chondrocytes with enhanced phagocytic activity were discovered within degraded joint cartilage, indicating a role in eliminating degraded tissues. Targeting these cells provides a new strategy for the treatment of arthritis.     

Melatonin through blockade of Hif-1α signaling mediates the anti-fibrosis under hypoxia in canine Sertoli cells

The hypoxic microenvironment of cryptorchidism is an important factor in the impairment and fibrosis of Sertoli cells which result in blood-testis barrier (BTB) destruction and spermatogenesis loss. Recent studies have shown that melatonin, a well-known pineal hormone exerts beneficial effects against pathological fibrosis in a various of organs. However, it is still unknown whether melatonin can regulate hypoxia-induced fibrosis of Sertoli cells. In this study we evaluate melatonin levels, and its synthesizing enzymes, AANAT and HIOMT expression patterns in canine cryptorchidism and contralateral normal testis. Results show abdominal testes presented low melatonin levels and AANAT and HIOMT expression compared with testes located in the scrotum. Moreover, we established a hypoxia-induced fibrosis model in canine Sertoli cells induced by cobalt chloride (CoCl₂) and found that melatonin inhibited the EMT markers expression and ECM production as well as Hif-1α expression of Sertoli cells in a dose-dependent manner. Furthermore, use of Lificiguat (synonyms YC-1, Hif-1α inhibitor) to interfere with the Hif-1α pathway showed a similar effect with melatonin suppression of the fibrosis in Sertoli cells. The results indicate that melatonin supplementation can alleviate the fibrosis process of Sertoli cells caused by hypoxia, which is associated with regulating the inhibition of Hif-1α signaling.     

Fibroblast activation protein alpha is expressed by chondrocytes following a pro-inflammatory stimulus and is elevated in osteoarthritis

Arthritis is characterised by the proteolytic degradation of articular cartilage leading to a loss of joint function. Articular cartilage is composed of an extracellular matrix of proteoglycans and collagens. We have previously shown that serine proteinases are involved in the activation cascades leading to cartilage collagen degradation. The aim of this study was to use an active-site probe, biotinylated fluorophosphonate, to identify active serine proteinases present on the chondrocyte membrane after stimulation with the pro-inflammatory cytokines IL-1 and oncostatin M (OSM), agents that promote cartilage resorption. Fibroblast activation protein alpha (FAPα), a type II integral membrane serine proteinase, was identified on chondrocyte membranes stimulated with IL-1 and OSM. Real-time PCR analysis shows that FAPα gene expression is up-regulated by this cytokine combination in both isolated chondrocytes and cartilage explant cultures and is significantly higher in cartilage from OA patients compared to phenotypically normal articular cartilage. Immunohistochemistry analysis shows FAPα expression on chondrocytes in the superficial zone of OA cartilage tissues. This is the first report demonstrating the expression of active FAPα on the chondrocyte membrane and elevated levels in cartilage from OA patients. Its cell surface location and expression profile suggest that it may have an important pathological role in the cartilage turnover prevalent in arthritic diseases.     

Distinct hypoxic regulation of preadipocyte factor-1 (Pref-1) in preadipocytes and mature adipocytes

Preadipocyte factor-1 (Pref-1) is a secretory soluble protein, which exerts pleiotropic effects on maintenance of cancer stem cell characteristics and commitment of mesenchymal stem cell lineages by inhibiting adipogenesis. Observations that obesity renders the microenvironment of adipose tissues hypoxic and that hypoxia inhibits adipogenesis lead us to investigate whether hypoxia increases the expression of anti-adipogenic Pref-1 in preadipocytes, mature adipocytes, and adipose tissues from obese mouse. In 3T3-L1 preadipocytes, hypoxia induces Pref-1 by a hypoxia-inducible factor 1 (HIF-1)-dependent mechanism accompanied by increase in the levels of the active histone mark, acetylated H3K9/14 (H3K9/14Ac). Adipogenesis increased the levels of the heterochromatin histone mark, trimethylated H3K27 (H3K27me3), whereas it decreased the levels of H3K4me3 and H3K9/14Ac euchromatin marks of the mouse Pref-1 promoter. However, differently from preadipocytes, in mature adipocytes hypoxia failed to reverse the repressive epigenetic changes of Pref-1 promoter and to increase its expression. Short term (8 weeks) high fat diet (HFD) increased HIF-1α protein in subcutaneous and epididymal adipose tissues, but did not increase Pref-1 expression. Unlike in 3T3-L1 preadipocytes, HIF-1α did not increase Pref-1 expression in adipose tissues in which mature adipocytes constitute the main population. Interestingly, long term (35 weeks) HFD increased Pref-1 in serum but not in obese adipose tissues. This study suggests that Pref-1 is an endocrine factor which is synergistically increased by obesity and age.     

HMGB1‐induced angiogenesis in perforated disc cells of human temporomandibular joint

High mobility group 1 protein (HMGB1), a highly conserved nuclear DNA‐binding protein and inflammatory mediator, has been recently found to be involved in angiogenesis. Our previous study has demonstrated the elevation of HMGB1 in the tissue of perforated disc of temporomandibular joint (TMJ). Here, we investigated a novel mediator of HMGB1 in regulating hypoxia‐inducible factor‐1α (HIF‐1α) and vascular endothelial growth factor (VEGF) to mediate angiogenesis in perforated disc cells of TMJ. HMGB1 increased the expression of HIF‐1α and VEGF in a dose‐ and time‐dependent manner in these cells. Moreover, immunofluorescence assay exhibits that the HIF‐1α were activated by HMGB1. In addition, HMGB1 activated extracellular signal‐related kinase 1/2 (Erk1/2), Jun N‐terminal kinase (JNK), but not P38 in these cells. Furthermore, both U0126 (ErK inhibitor) and SP600125 (JNK inhibitor) significantly suppressed the enhanced production of HIF‐1α and VEGF induced by HMGB1. Tube formation of human umbilical vein endothelial cells (HUVECs) was significantly increased by exposure to conditioned medium derived from HMGB1‐stimulated perforated disc cells, while attenuated with pre‐treatment of inhibitors for VEGF, HIF‐1α, Erk and JNK, individually. Therefore, abundance of HMGB1 mediates activation of HIF‐1α in disc cells via Erk and JNK pathway and then, initiates VEGF secretion, thereby leading to disc angiogenesis and accelerating degenerative change of the perforated disc.