Effects of cobalt and chromium ions on lymphocyte migration

A T cell‐mediated hypersensitivity reaction has been reported in some patients with CoCrMo‐based implants. However, the role of cobalt and chromium ions in this reaction remains unclear. The objective of the present study was to analyze the effects of Co²⁺ and Cr³⁺ in culture medium, as well as the effects of culture supernatants of macrophages exposed to Co²⁺ or Cr³⁺, on the migration of lymphocytes. The release of cytokines/chemokines by macrophages exposed to Co²⁺ and Cr³⁺ was also analyzed. The migration of murine lymphocytes was quantified using the Boyden chamber assay and flow cytometry, while cytokine/chemokine release by J774A.1 macrophages was measured by ELISA. Results showed an ion concentration‐dependent increase in TNF‐α and MIP‐1α release and a decrease in MCP‐1 and RANTES release. Migration analysis showed that the presence of Co²⁺ (8 ppm) and Cr³⁺ (100 ppm) in culture medium increased the migration of T lymphocytes, while it had little or no effect on the migration of B lymphocytes, suggesting that Co²⁺ and Cr³⁺ can stimulate the migration of T but not B lymphocytes. Levels of T lymphocyte migration in culture medium containing Co²⁺ or Cr³⁺ were not statistically different from those in culture supernatants of macrophages exposed to Co²⁺ or Cr³⁺, suggesting that the effects of the ions and chemokines were not additive, possibly because of ion interference with the chemokines and/or their cognate receptors. Overall, results suggest that Co²⁺ and Cr³⁺ are capable of stimulating the migration of T (but not B) lymphocytes in the absence of cytokines/chemokines, and could thereby contribute to the accumulation of more T than B lymphocytes in periprosthetic tissues of some patients with CoCrMo‐based implants. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:916–924, 2017.

     

Cobalt and chromium ions reduce human osteoblast-like cell activity in vitro, reduce the OPG to RANKL ratio, and induce oxidative stress

Metal‐on‐metal hip arthroplasty is associated with elevated levels of cobalt and chromium ions. The effects of cobalt and chromium ions on cell number, activity, expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B ligand (RANKL) and oxidative stress on human osteoblast‐like cells were addressed. Saos‐2 cells were supplemented with Co²⁺, Cr³⁺, or Co²⁺ + Cr³⁺ (1:2) at 0, 1, 10, and 100 µg/L and incubated for 24, 48, 72, and 96 h. Cell activity was assessed by MTT‐assay and cell number by Crystal Violet staining. RNA levels of OPG and RANKL were evaluated using real‐time quantitative polymerase chain reaction (qPCR). Compared to controls Co²⁺ reduced cell numbers: at 10 µg/L by 17 ± 8% after 48 h and at 100 µg/L after 24 h by 35 ± 8%. Cr³⁺ decreased cell numbers at 10 µg/L after 48 and 72 h. Co²⁺ + Cr³⁺ combined at 1 µg/L lowered cell numbers after 24 and 96 h (17 ± 13, resp. 13 ± 4%). The 10 and 100 µg/L concentrations reduced cell numbers significantly after 24, 48, and 96 h. Cr³⁺ reduced osteoblast activity at 1, 10, and 100 µg/L at all incubation times. The strongest reduction (11 ± 1%) was seen at 100 µg/L after 96 h. The OPG/RANKL ratio was reduced after 72 h with almost all Co²⁺ and Cr³⁺ concentrations. After 96 h, glutathione, superoxide dismutase, and catalase levels were indicative for an oxidative stress response in all samples. In conclusion, cobalt and chromium ions reduce human osteoblast activity, reduce OPG/RANKL ratio and lead to oxidative stress. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:740–747, 2012     

Understanding the tissue effects of tribo‐corrosion: Uptake,distribution, and speciation of cobalt and chromium in human bone cells

Cobalt and chromium species are released in the local tissues as a result of tribo‐corrosion, and affect bone cell survival and function. However we have little understanding of the mechanisms of cellular entry, intracellular distribution, and speciation of the metals that result in impaired bone health. Here we used synchrotron based X‐ray fluorescence (XRF), X‐ray absorption spectroscopy (XAS), and fluorescent‐probing approaches of candidate receptors P2X7R and divalent metal transporter‐1 (DMT‐1), to better understand the entry, intra‐cellular distribution and speciation of cobalt (Co) and chromium (Cr) in human osteoblasts and primary human osteoclasts. We found that both Co and Cr were most highly localized at nuclear and perinuclear sites in osteoblasts, suggesting uptake through cell membrane transporters, and supported by a finding that P2X7 receptor blockade reduced cellular entry of Co. In contrast, metal species were present at discrete sites corresponding to the basolateral membrane in osteoclasts, suggesting cell entry by endocytosis and trafficking through a functional secretory domain. An intracellular reduction of Cr⁶⁺ to Cr³⁺ was the only redox change observed in cells treated with Co²⁺, Cr³⁺, and Cr⁶⁺. Our data suggest that the cellular uptake and processing of Co and Cr differs between osteoblasts and osteoclasts. © 2014 The Authors. Journal of Orthopaedic Research published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 33:114–121, 2015.     

HIF‐1α‐induced HSP70 regulates anabolic responses in articular chondrocytes under hypoxic conditions

We assessed whether heat shock protein 70 (HSP70) is involved in hypoxia inducible factor 1 alpha (HIF‐1α)‐dependent anabolic pathways in articular chondrocytes under hypoxic conditions. Primary rabbit chondrocytes were cultured under normoxia (20% oxygen condition) or hypoxia (1% oxygen condition). Alternatively, cells cultured under normoxia were treated with CoCl₂, which induces HIF‐1α, to simulate hypoxia, or transfected with siRNAs targeting HIF‐1α (si‐HIF‐1α) and HSP70 (si‐HSP70) under hypoxia. HSP70 expression was enhanced by the increased expression of HIF‐1α under hypoxia or simulated hypoxia, but not in the presence of si‐HIF‐1α. Hypoxia‐induced overexpression of ECM genes was significantly suppressed by si‐HIF‐1α or si‐HSP70. Cell viability positively correlated with hypoxia, but transfection with si‐HIF‐1α or si‐HSP70 abrogated the chondroprotective effects of hypoxia. Although LDH release from sodium nitroprusside‐treated cells and the proportion of TUNEL positive cells were decreased under hypoxia, transfection with si‐HIF‐1α or si‐HSP70 almost completely blocked these effects. These findings indicated that HIF‐1α‐induced HSP70 overexpression increased the expression levels of ECM genes and cell viability, and protected chondrocytes from apoptosis. HIF‐1α may regulate the anabolic effects of chondrocytes under hypoxic conditions by regulating HSP70 expression. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:975–980, 2014.     

Combined effects of TNF‐α, IL‐1β, and HIF‐1α on MMP‐2 production in ACL fibroblasts under mechanical stretch: An in vitro study

The dynamics between inflammatory factors, mechanical stress, and healing factors, in an intra‐articular joint, are very complex after injury. Injury to intra‐articular tissue [anterior cruciate ligament (ACL), synovium] results in hypoxia, accumulation of various pro‐inflammatory factors, cytokines, and metalloproteases. Although the presence of increased amounts of matrix‐metalloproteinases (MMP) in the joint fluid after knee injury is considered the key factor for ACL poor healing ability; however, the exact role of collective participants of the joint fluid on MMP‐2 activity and production has not been fully studied yet. To investigate the combined effects of mechanical injury, inflammation and hypoxia induced factor‐1α (HIF‐1α) on induction of MMP‐2; we mimicked the microenvironment of joint cavity after ACL injury. The results show that TNF‐α and IL‐1β elevate the activity of MMP‐2 in a dose‐ and time‐dependent manner. In addition, mechanical stretch further enhances the MMP‐2 protein levels with TNF‐α, IL‐1β, and their mixture. CoCl₂‐induced HIF‐1α (100 and 500 µM) also increases the levels and activity of MMP‐2. Mechanical stretch has a strong additional effect on MMP‐2 production with HIF‐1α. Our results conclude that mechanical injury, HIF‐1α and inflammatory factors collectively induce increased MMP‐2 production in ACL fibroblasts, which was inhibited by NF‐κB pathway inhibitor (Bay‐11‐7082). © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1008–1014, 2011     

In vitro evaluation of human myoblast function after exposure to cobalt and chromium ions

The replacement of a native hip joint by a metal-on-metal prosthesis may induce deleterious inflammatory side effects that are associated with the release of wear particles and metal ions. These events are referred to the adverse reaction to metal debris (ARMD) and the adverse local tissue reaction (ALTR). While wear particles seem involved in ARMD, the role of metal ions in ALTR and their impact on myoblasts, located in the prosthesis vicinity, has not been fully identified. To clarify this issue we investigated, using an in vitro culture system, the effect of cobalt and/or chromium ions (Co²⁺ and/or Cr³⁺) on human myoblast proliferation, cellular differentiation, and inflammatory marker expression. Freshly isolated human myoblasts were cultured in media supplemented with graded concentrations of Co²⁺ and/or Cr³⁺. Co²⁺ induced a concentration-dependent decrease of both myoblast viability and myogenic differentiation while Cr³⁺ did not. Co²⁺ or Co²⁺/Cr³⁺ also induced the upregulation of ICAM-1, whereas HLA-DR expression was unaffected. Moreover, allogenic monocytes induced the synergistic increase of Co²⁺-induced ICAM-1 expression. We also found that Co²⁺ stabilized HIF-1α and increased TLR4, tumor necrosis factor-alpha (TNF-α), and interleukin 1β (IL-1β) expression in a dose and time-dependent manner in human myoblasts. This study showed that Co²⁺, but not Cr³⁺, was toxic toward myoblasts and induced, in the surviving cells, expression of inflammatory markers such as ICAM-1, TLR4, TNF-α, and IL-1β. This suggests that Co²⁺, most efficiently in the presence of monocytes, may be a key inducer of ALTR, which may, if severe and long-lasting, eventually result in prosthesis loosening.     

Expression of hypoxia‐inducible factor‐1α and hepatocyte growth factor in development of fibrosis in the transplanted kidney

Late renal graft loss is associated with interstitial fibrosis. Hypoxia‐inducible factor‐1α (HIF‐1α) is thought to facilitate fibrosis through interaction with TGF‐β1, while hepatocyte growth factor (HGF) may act antifibrotic in the kidney allograft. The aim of this study was to investigate the expression of HIF‐1α and HGF in protocol biopsies as possible prognostic biomarkers for renal fibrosis. Thirty‐nine renal transplant recipients were included in the study. Protocol biopsies performed 1 and 2 years after transplantation were used for immunohistochemistry analysis. The correlation between HIF‐1α/HGF and the Banff score was analysed. In addition, progression in renal fibrosis and graft survival among recipients with high or low expression of HIF‐1α/HGF after transplantation was compared. There was no significant correlation between fibrosis and the HIF‐1α expression 1 and 2 years after transplantation, but an inverse significant correlation between the HGF expression and the fibrosis score 1 year after transplantation was shown. Even when adjusting for human leucocyte antigen mismatches, there was a significant relationship between fibrosis and HGF expression. Graft survival was not significantly correlated to HIF‐1α or HGF at 1 year, although the trend was towards better graft survival with high HGF. HGF may have antifibrotic effects in human renal transplants. (Central.Denmark.Region.Committee number: 1‐10‐72‐318‐13)     

Compression‐induced HIF‐1 enhances thrombosis and PAI‐1 expression in mouse skin

Pressure ulcers result from tissue hypoxia caused by external forces. Thrombosis due to external forces is considered important, and hypoxia inducible factor‐1 (HIF‐1) is a master regulator of pressure ulcer development. To date, however, their causal relationship has not been determined. This study therefore investigated the mutual relationship between thrombosis and HIF‐1 activation in compressed mouse skin, based on a hypothesis that HIF‐1 regulation by plasminogen activator inhibitor‐1 (PAI‐1) enhances thrombosis. Compression of mouse skin significantly increased the numbers of thrombi and HIF‐1α‐positive cells compared with control skin. A thrombosis inhibitor significantly reduced the numbers of HIF‐1α‐positive cells and an HIF‐1 inhibitor significantly inhibited thrombosis in compressed skin tissue, suggesting a mutual relationship between thrombosis and HIF‐1 activation. Compression of mouse skin also enhanced the level of Pai‐1 messenger RNA expression, but this increase was significantly reduced by treatment with an HIF‐1 inhibitor, whereas a thrombosis inhibitor had no effect. These results suggested the involvement of PAI‐1 in HIF‐1‐enhanced thrombosis and that an additional factor participates in regulating Pai‐1 expression in compressed skin. These findings may suggest new strategies in pressure ulcer management.     

Constitutive expression of hypoxia‐inducible factor‐1 α in keratinocytes during the repair of skin wounds in horses

As a transient hypoxic state exists within skin wounds in horses and may be important for the healing process, this study sought to identify a molecular hypoxia response occurring in horse limb and body wounds healing by second intention. Hypoxia‐inducible factor 1α (HIF1α) protein expression was studied throughout repair by Western blotting and immunofluorescence. Paradoxically, HIF1α was strongly expressed in intact skin and its expression decreased dramatically following wounding (p<0.01), despite the expected hypoxic state within the wounded tissue. HIF1α levels reincreased in parallel with the epithelialization process, and more rapidly in body wounds than in limb wounds (p<0.01). HIF1α localized predominantly to the keratinocyte layer, in which it was constitutively expressed throughout healing. The HIF1α target gene cyclin‐dependent kinase inhibitor 1A (CDKN1A) showed a pattern of expression similar to HIF1α throughout the healing process and also localized to the keratinocyte layer, suggesting that HIF1α may regulate its constitutive expression. The HIF1α target genes vascular endothelial growth factor A (VEGFA) and solute carrier family 2 (facilitated glucose transporter) member 1 (SLC2A1) however did not have a pattern of expression similar to HIF1α, at the mRNA level. We conclude that HIF1α is expressed in a continuous and hypoxia‐independent manner in equine keratinocytes in both intact and wounded skin, and may regulate the expression of CDKN1A in this cell type.     

Hypoxia‐inducible factor 3‐alpha expression is associated with the stable chondrocyte phenotype

The hypoxia‐inducible factors HIF‐1α and HIF‐2α are important regulators of the chondrocyte phenotype but little is known about HIF‐3α in cartilage. The objective of this study was to characterize HIF‐3α (HIF3A) expression during chondrocyte differentiation in vitro and in native cartilage tissues. HIF3A, COL10A1, and MMP13 were quantified in mesenchymal stem cells (MSCs) and articular chondrocytes from healthy and osteoarthritic (OA) tissue in three‐dimensional cultures and in human embryonic epiphyses and adult articular cartilage. HIF3A was found to have an inverse association with hypertrophic markers COL10A1 and MMP13 in chondrogenic cells and tissues. In healthy chondrocytes, HIF3A was induced by dexamethasone and increased during redifferentiation. By comparison, HIF3A expression was extremely low in chondrogenically differentiated MSCs expressing high levels of COL10A1 and MMP13. HIF3A was also lower in redifferentiated OA chondrocytes than in healthy chondrocytes. In human embryonic epiphyseal tissue, HIF3A expression was lowest in the hypertrophic zone. Distinct splice patterns were also found in embryonic cartilage when compared with adult articular cartilage and redifferentiated chondrocytes. These in vitro and in vivo findings suggest that HIF3A levels are indicative of the hypertrophic state of chondrogenic cells and one or more splice variants may be important regulators of the chondrocyte phenotype. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1561–1570, 2015.     

Hyperbaric oxygen therapy activates hypoxia‐inducible factor 1 (HIF‐1), which contributes to improved wound healing in diabetic mice

Hyperbaric oxygen (HBO) therapy has been used as an adjunctive therapy for diabetic foot ulcers, although its mechanism of action is not completely understood. Recently, it has been shown that HBO mobilizes the endothelial progenitor cells (EPCs) from bone marrow that eventually will aggregate in the wound. However, the gathering of the EPCs in diabetic wounds is impaired because of the decreased levels of local stromal‐derived factor‐1α (SDF‐1α). Therefore, we investigated the influence of HBO on hypoxia‐inducible factor 1 (HIF‐1), which is a central regulator of SDF‐1α and is down‐regulated in diabetic wounds. The effects of HBO on HIF‐1α function were studied in human dermal fibroblasts, SKRC7 cells, and HIF‐1α knock‐out and wild‐type mouse embryonic fibroblasts using appropriate techniques (Western blot, quantitative polymerase chain reaction, and luciferase hypoxia‐responsive element reporter assay). Cellular proliferation was assessed using H³‐thymidine incorporation assay. The effect of HIF in combination with HBOT was tested by inoculating stable HIF‐1α‐expressing adenovirus (Adv‐HIF) into experimental wounds in db/db mice exposed to HBO. HBO activates HIF‐1α at several levels by increasing both HIF‐1α stability (by a non‐canonical mechanism) and activity (as shown both by induction of relevant target genes and by a specific reporter assay). HIF‐1α induction has important biological relevance because the induction of fibroblast proliferation in HBO disappears when HIF‐1α is knocked down. Moreover, the local transfer of stable HIF‐1α‐expressing adenovirus (Adv‐HIF) into experimental wounds in diabetic (db/db mice) animals has an additive effect on HBO‐mediated improvements in wound healing. In conclusion, HBO stabilizes and activates HIF‐1, which contributes to increased cellular proliferation. In diabetic animals, the local transfer of active HIF further improves the effects of HBO on wound healing.     

Hypoxia‐inducible factor and mammalian target of rapamycin pathway markers in urothelial carcinoma of the bladder: possible therapeutic implications

What’s known on the subject? and What does the study add? The hypoxia‐inducible factor (HIF) and mammalian target of rapamycin (mTOR) pathways are important in tumorigenesis and novel agents targeting these respective pathways have shown promising activity in several malignancies. The current study demonstrates the expression of HIF and mTOR related pathway markers in urothelial carcinoma providing a rationale for clinical trials evaluating agents targeting these pathways.

OBJECTIVE

To investigate the rationale for using targeted therapies against hypoxia‐inducible factor (HIF) and mammalian target of rapamycin (mTOR) pathways in urothelial carcinoma of the bladder, by studying the immunohistochemical expression of molecules of these pathways in urothelial carcinoma, as recent pre‐clinical studies and clinical trials have shown the potential utility of such targeted therapies.

PATIENTS AND METHODS

Immunohistochemical stains were performed on a tissue microarray prepared from 92 cases of ≥ pT2 urothelial (transitional cell) carcinoma of bladder, using antibodies against HIF‐1α and VEGF‐R2, and phospho‐S6 and phospho‐4E BP1, molecules of HIF and activated mTOR pathways, respectively. Immunoreactivity was graded from 0 to 3+ (0, 0–5%; 1+, 6–25%; 2+, 26–50%; 3+, > 50% tumour cells positive).

RESULTS

In all, 58, 34, 35 and 17% of the tumours showed grade 2–3+ expression of phospho‐4E BP1, phospho‐S6, HIF‐1α and VEGF‐R2, respectively. Moderate correlation for immunoreactivity was observed between molecules within the same pathway [(phospho‐4E BP1 with phospho‐S6 (rho = 0.411), and HIF‐1α with VEGF‐R2 (rho = 0.265)], but not between molecules across pathways.

CONCLUSIONS

Urothelial carcinomas of the bladder express molecules of the HIF and mTOR pathways, providing a rationale for clinical trials evaluating agents targeting these pathways. Correlation between molecules within the same pathway, and not across pathways, suggests that investigating the usefulness of a specific targeted agent might benefit from pre‐treatment evaluation of pathway marker expression.     

Intra‐articular resveratrol injection prevents osteoarthritis progression in a mouse model by activating SIRT1 and thereby silencing HIF‐2α

We investigated the feasibility of the intra‐articular injection of resveratrol for preventing the progression of existing cartilage degeneration in a mouse model of osteoarthritis (OA). The effects of resveratrol on the expression of silent information regulator 2 type 1 (SIRT1), hypoxia‐inducible factor‐2α (HIF‐2α) and catabolic factors in OA cartilage was explored. OA was induced in the mouse knee via destabilization of the medial meniscus (DMM). Resveratrol was injected weekly into the operated knee beginning 4 weeks after surgery. The OA phenotype was evaluated via histological and immunohistochemical analyses at 8 weeks after DMM. Western blot analysis was performed to identify whether resveratrol modulated the interleukin (IL)‐1β‐induced expression of HIF‐2α in human chondrocytes. Histologically, resveratrol treatment preserved the structural homeostasis of the articular cartilage and the subchondral bone. Following resveratrol injection, the expression of collagen type II was retained, but the expression of inducible nitric oxide synthase and matrix metalloproteinase‐13 was reduced in OA cartilage. Moreover, the administration of resveratrol significantly induced the activation of SIRT1 and the inhibition of HIF‐2α expression in mouse OA cartilage and in IL‐1β‐treated human chondrocytes. These findings indicate that the intra‐articular injection of resveratrol significantly prevents the destruction of OA cartilage by activating SIRT1 and thereby suppressing the expression of HIF‐2α and catabolic factors. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1061–1070, 2015.     

A large‐scale replication study for the association of rs17039192 in HIF‐2α with knee osteoarthritis

Osteoarthritis (OA) is a common disease with a genetic component for its etiology. Recently, a genetic association of a single nucleotide polymorphism (SNP), rs17039192 in HIF‐2α with knee OA has been reported in a Japanese population; however, controversy exits for its replication and a role of HIF‐2α in OA. This study aimed to evaluate the association of the SNP by a large‐scale replication study. A total of 8,457 subjects (3,129 OA cases and 5,328 controls) from seven independent cohorts from six countries (Japan, China, Taiwan, Korea, Greece, and Australia) were recruited and genotyped. The association of rs17039192 with knee OA was evaluated by meta‐analyses. The association of the HIF‐2α SNP was not replicated in any of the populations. Contrary to the previous report, the odds ratios (ORs) of the risk allele frequency were all less than 1. A combined analysis for the seven populations also showed no replication of the association (OR = 0.91, 95% confidence interval = 0.81–1.03). Our large‐scale meta‐analysis showed that the association of rs17039192 in HIF‐2α with knee OA is negative. The significance of HIF‐2α in human OA (idiopathic OA as a common disease) should be further evaluated carefully. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1244–1248, 2012