Simulated Hypoxia Research Articles

Chaperoning of Mutant p53 Protein by Wild-type p53 Protein Causes Hypoxic Tumor Regression

Mutant (Mt) p53 abrogates tumor suppression functions of wild-type (WT) p53 through mutant-specific, gain-of-function effects, and patients bearing Mt p53 are chemoresistant. The dominant negative effect of p53 mutants results from their aggregation propensity which causes co-aggregation of WT p53. We explored the mechanism of p53 inactivation in hypoxia and hypothesized whether WT p53 could rescue Mt p53 in hypoxic tumors. WT p53 exists in mutant conformation in hypoxic core of MCF-7 solid tumors, and its conformation is oxygen-dependent. Under simulated hypoxia in cells, WT p53 undergoes conformational change in acquiring mutant conformation. An in vivo chaperone assay shows that WT p53 functions as a molecular chaperone in rescuing conformational and structural p53 mutants in cancer cells both at the transcription and proteome levels. WT p53 chaperone therapy is further shown to cause significant regression of tumor xenografts through reconversion of the mutant phenotype to wild-type p53. The chaperone function of WT p53 is directly linked to the induction of apoptosis in both cancer cells and tumor xenografts. As oncogenic p53 mutants are linked to chemoresistance in hypoxic tumors, p53 chaperone therapy will introduce new dimensions to existing cancer therapeutics. We propose that in cancer cells, WT p53 chaperoning may either exist as a cellular event to potentially reverse the dominant negative effect of its oncogenic mutants or to stabilize yet unidentified factors.

Blood Cardioplegia Serves as a Bicarbonate Donor to the Myocardium during Ischemia: Effects of Anoxia and Hypercapnia on Acid-Base Characteristics of Blood Cardioplegic Solution

Objectives: We investigated the alterations of acid-base characteristics of the blood cardioplegia (BCP) solution during aortic cross-clamping in hearts arrested with BCP and during in vitro-simulated ischemia. Methods: Following aortic cross-clamping, the hearts of 40 patients undergoing cardiac surgery were intermittently infused with an 18°C BCP solution and finally with a 34°C BCP solution prior to aortic cross-clamp release. We measured the pH, partial CO₂ pressure (pCO₂), [HCO₃^–], and [Cl^–] of the coronary sinus effluent in the final BCP solution. The BCP solution was assessed under in vitro gassing at 34°C with 95% N₂ + 5% CO₂ (n = 6), 50% N₂ + 50% CO₂ (n = 3), or 100% CO₂ (n = 6). Results: The coronary sinus effluent, compared with the preinfusion BCP solution, exhibited a significantly lower pH and a greater pCO₂ with no change in the [HCO₃^–] level. In vitro, the 95% N₂ + 5% CO₂ gassing (simulated hypoxia) group exhibited a slight increase in [HCO₃^–] with no change in pCO₂ or pH whereas the 50% N₂ + 50% CO₂ gassing and the 100% CO₂ gassing (simulated hypoxia and hypercapnia) groups exhibited a significant increase in [HCO₃^–] under high pCO₂-induced acidification. Conclusions: Under anoxia and CO₂ retention during aortic cross-clamping, the BCP solution can be a bicarbonate donor to the myocardium.

Effects of hypoxia on epididymal sperm parameters and protective role of ibuprofen and melatonin.

Hypobaric hypoxia is of interest due to an increase of human populations working at high altitude. Testicular damage is related to the physiological response (neoangiogenesis) to increased intrascrotal blood flow as temperature rises. Hypoxia is a stress factor with overproduction of reactive oxygen species (ROS). The effect of hypoxia in mice reproductive parameters is analyzed. Animals were exposed to simulated hypoxia of 4,200 meters above sea level (m.a.s.l.) in a chamber for 33.2 days, both to continuous (HH) or intermittent hypoxia (HI) with an intermittency period of 4 days hypoxia /4 days normoxia (500 m.a.s.l.). The anti-inflammatory drug Ibuprofen was administered to a group of mice to control vasodilation and increased blood flow. Melatonin was administered to another group of mice as a potent ROS scavenger. Animals in both HH and HI exposure were compared to normoxic non-treated controls. There was a hematological response in hypoxia, with an increase in hematocrit and reticulocytosis. There was also increased teratozoospermia. This damage was more pronounced in HH than HI, suggesting that alternating normoxic periods permits compensation for the effects of hypoxia. In both hypoxia systems, the level of lipoperoxidation and the instability of DNA increased. In HH, there was a reduction of teratozoospermia in melatonin-treated mice. Ibuprofen presented a protective effect on the same parameters as melatonin with both HI and HH. The quality of sperm DNA, fragmentation, unpacking and DNA stability diminished. In conclusion, reproductive damage elicited by HH or HI was partially ameliorated by simultaneous treatment with antiflogistic and/or antioxidant agents.

Novel Phenazine 5,10-Dioxides Release •OH in Simulated Hypoxia and Induce Reduction of Tumour Volume In Vivo

Phenazine 5,10-dioxides (PDOs) are a new class of bioreductive cytotoxins, which could act towards tumours containing hypoxic regions. The PDOs selective-hypoxic bioreduction was probed in vitro; however, the mechanism of action has not been completely explained. Besides, PDOs in vivo antitumour activities have not been demonstrated hitherto. We study the mechanism of hypoxic/normoxic cytotoxicity of PDO representative members. Electron spin resonance is used to confirm •OH production, alkaline comet assay to determine genotoxicity, and gel electrophoresis and flow cytometry to analyze DNA fragmentation and cell cycle distribution. Chemically induced rat breast tumours are employed to evaluate in vivo activities. For the most selective cytotoxin, 7(8)-bromo-2-hydroxyphenazine 5,10-dioxide (PDO1), exclusive hypoxic •OH production is evidenced, while for the unselective ones, •OH is produced in both conditions (normoxia and simulated hypoxia). In normoxia (Caco-2 cells), PDO1 induces cell-cycle arrest and DNA fragmentation but does not significantly induce apoptosis neither at IC50 nor IC80. No difference in the comet-assay scores are observed in normoxia and simulated hypoxia being the unselective 2-amino-7(8)-bromophenazine 5,10-dioxide (PDO2) the most genotoxic. The in vivo efficacy with the absence of systemic toxicity of PDO1 and PDO2 is checked out. Results from this study highlight the potential of PDOs as new therapeutics for cancer.

The influence of the neuropeptide galanin on the contractility and the effective refractory period of guinea-pig heart papillary muscle under normoxic and hypoxic conditions.

The aim of this study was to discover the effects of galanin, a neuropeptide comprising 29 amino acids capable of activating inward-rectifier K+ channels (IK1) in cardiomyocytes, on the force of contraction (Fc), velocity of contraction (+dF/dt), velocity of relaxation (-dF/dt) and effective refractory period (ERP) of guinea-pig heart. The influence of galanin on the time-course of hypoxia-induced disturbances in contractility and ERP was also examined. Experiments were performed on the isolated right ventricle papillary muscle of guinea-pig heart. In the concentration range 0.001-0.01 microM, galanin had no significant effect on the measured parameters. At 0.03 and 0.1 microM, galanin exerted a positive inotropic action and prolonged ERP. Further increasing the concentration led to a negative inotropic action and significant shortening of ERP. Although simulated hypoxia induced a significant drop in Fc, +dF/dt and -dF/dt, and a significant shortening of ERP, recovery of all the measured parameters was complete after 10 min reperfusion. In the presence of 0.03 and 0.1 microM galanin the effect of hypoxia on the contractility of papillary muscle was more profound and reperfusion did not result in complete recovery. In contrast, addition of 1 microM galanin to the hypoxic solution significantly protected the muscle and recovery of the tissues during reperfusion was rapid and complete (in 5 min). One can conclude that galanin at lower concentrations induced a positive inotropic action and a prolongation of ERP, but increased the sensitivity of heart muscle to hypoxia. At higher concentrations however, galanin exerted a negative inotropic action but protected the muscle against hypoxia-induced disturbances in contractility.

Partial XBP1 knockdown does not affect viability of oligodendrocyte precursor cells exposed to new models of hypoxia and ischemia in vitro

The endoplasmic reticulum (ER) stress signaling pathway has been implicated in tissue injury in several rodent models of brain ischemia. To understand better the effects of ischemia on white matter in particular, we developed several in vitro models of hypoxia and ischemia in oligodendrocyte precursors. For the first time, we present data showing that exposure of rat oligodendrocyte precursor cells (OPCs) to cobalt chloride (CoCl(2)), antimycin A (AA), or oxygen, glucose and nutrient deprivation (OGND) causes up-regulation of glucose-regulated protein 78/B-cell immunoglobulin-binding protein (Grp78/BiP), C/EBP homologous binding protein (CHOP), and spliced X-box-binding protein 1 (XBP1). To mimic the effects of ischemia, OPCs supplemented with 5% normal growth medium and 95% Hank's balanced salt solution were incubated in a hypoxia chamber set at 0.1% oxygen. Because the toxic effects of AA on OPCs more closely resembled those seen when OPCs were subjected to OGND, we found AA treatment preferable to CoCl(2) as an in vitro model. To investigate the role of XBP1 in survival following an ischemic insult, we generated a stable XBP1 knockdown OPC cell line and subjected it to simulated hypoxia or ischemia. Surprisingly, 65% XBP1 knockdown had no effect on viability following chemical treatment or OGND. These data strengthen the case for targeting the ER stress signalling pathway in an effort to develop new early treatments for ischemic stroke patients but at the same time demonstrate that partial knockdown of XBP1 is not sufficient to protect precursor oligodendrocytes from ischemic damage.

N-n-Butyl Haloperidol Iodide Preserves Cardiomyocyte Calcium Homeostasis during Hypoxia/Ischemia

Aims: N-n-Butyl haloperidol iodide (F₂) is a novel compound derived from haloperidol. In our previous work, F₂ was found to be an L-type calcium channel blocker which played a protective role in rat heart ischemic–reperfusion injury in a dose-dependent manner. In the current study, we aimed to investigate the effects and some possible mechanisms of F₂ on calcium transients in hypoxic/ischemic rat cardiac myocytes. Methods and Results: Calcium transients’ images of rat cardiac myocytes were recorded during simulated hypoxia, using a confocal calcium imaging system. The amplitude, rising time from 25% to 75% (RT25-75), decay time from 75% to 25% (DT75-25) of calcium transients, and resting [Ca²⁺]_i were extracted from the images by self-coding programs. In this study, hypoxia produced a substantial increase in diastolic [Ca²⁺]_i and reduced the amplitude of calcium transients. Both RT25-75 and DT75-25 of Ca²⁺ transients were significantly prolonged. And F₂ could reduce the increase in resting [Ca²⁺]_iand the prolongation of RT25-75 and DT75-25 of Ca²⁺ transients during hypoxia. F₂ also inhibited the reduction in amplitude of calcium transients which was caused by 30-min hypoxia. The activity of SERCA2a (sarcoplasmic reticulum Ca²⁺-ATPase, determined by test kits) decreased after 30-min ischemia, and intravenous F₂ in rats could ameliorate the decreased activity of SERCA2a. The inward and outward currents of NCX (recorded by whole-cell patch-clamp analysis) were reduced during 10-min hypoxia, and F₂ further inhibited the outward currents of NCX during 10-min hypoxia. All these data of SERCA2a and NCX might be responsible for the changes in calcium transients during hypoxia. Conclusion: Our data suggest that F₂ reduced changes in calcium transients that caused by hypoxia/ischemia, which was regarded to be a protective role in calcium homeostasis of ventricular myocytes, probably via changing the function of SERCA2a.

Simulated high-altitude hypobaric hypoxia decreases serum and gastric levels of ghrelin in rats

Simulated high-altitude hypobaric hypoxia decreases serum and gastric levels of ghrelin in rats

Effects of hypoxia on epididymal sperm parameters and protective role of ibuprofen and melatonin

Hypobaric hypoxia is of interest due to an increase of human populations working at high altitude. Testicular damage is related to the physiological response (neoangiogenesis) to increased intrascrotal blood flow as temperature rises. Hypoxia is a stress factor with overproduction of reactive oxygen species (ROS). The effect of hypoxia in mice reproductive parameters is analyzed. Animals were exposed to simulated hypoxia of 4,200 meters above sea level (m.a.s.l.) in a chamber for 33.2 days, both to continuous (HH) or intermittent hypoxia (HI) with an intermittency period of 4 days hypoxia /4 days normoxia (500 m.a.s.l.). The anti-inflammatory drug Ibuprofen was administered to a group of mice to control vasodilation and increased blood flow. Melatonin was administered to another group of mice as a potent ROS scavenger. Animals in both HH and HI exposure were compared to normoxic non-treated controls. There was a hematological response in hypoxia, with an increase in hematocrit and reticulocytosis. There was also increased teratozoospermia. This damage was more pronounced in HH than HI, suggesting that alternating normoxic periods permits compensation for the effects of hypoxia. In both hypoxia systems, the level of lipoperoxidation and the instability of DNA increased. In HH, there was a reduction of teratozoospermia in melatonin-treated mice. Ibuprofen presented a protective effect on the same parameters as melatonin with both HI and HH. The quality of sperm DNA, fragmentation, unpacking and DNA stability diminished. In conclusion, reproductive damage elicited by HH or HI was partially ameliorated by simultaneous treatment with antiflogistic and/or antioxidant agents.

Lysyl oxidase expression and inhibition in uveal melanoma

Lysyl oxidase is a marker of poor prognosis in several malignancies and is hypothesized to promote a migratory phenotype in hypoxic breast carcinomas. This study aims to characterize the expression of the lysyl oxidase and lysyl oxidase-like proteins in human uveal melanoma cell lines and archival choroidal melanomas using immunohistochemistry. The transcriptional control of lysyl oxidase will also be investigated under simulated hypoxic conditions using cobalt chloride. Lastly, changes in cellular proliferation and invasion will be assessed after the treatment of cell lines with beta-aminopropionitrile, a lysyl oxidase catalytic inhibitor. Retrospective analysis of lysyl oxidase expression in primary human uveal melanoma showed 82% (27 of 33) of tumors being stained positive. High lysyl oxidase expression correlated with the aggressive epithelioid cell type and was associated with shorter metastasis-free survival. Simulated hypoxia resulted in a significant increase in lysyl oxidase mRNA expression. Inhibiting lysyl oxidase's catalytic activity significantly reduced cellular invasion but had no effect on cell proliferation. Our study is the first to show lysyl oxidase expression in primary choroidal melanomas. This protein may represent a potential therapeutic target that warrants further study in this malignancy.

Intermittent simulated hypoxia for pre-acclimatization

Intermittent simulated hypoxia for pre-acclimatization

Role of hypoxia-inducible factor-1 alpha in the regulation of plasminogen activator activity in rat knee joint chondrocytes

To examine the effects of hypoxia-inducible factor-1alpha (HIF-1alpha) on the plasminogen activator's (PA) activity and on the expression of components of PA system in articular chondrocytes of rats. Chondrocytes from rat knee joint cartilage were cultured under normoxic, hypoxic, CoCl(2) simulated hypoxic, and interleukin-1beta (IL-1beta)-stimulated conditions. siRNA targeting HIF-1alpha was transfected into cells cultured under hypoxic, simulated hypoxic, and IL-1beta-stimulated conditions to silence HIF-1alpha. PA activity was determined by the hydrolysis of the chromogenic substrate H-D-Val-Leu-Lys-pNA (S-2251). The mRNA levels were measured by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). The intracellular/matrix-associate protein levels were detected by Western blot and the soluble protein levels were measured by enzyme linked immunosorbent assay (ELISA). Chromatin immunoprecipitation (CHIP) assay was performed to determine whether HIF-1alpha binds to the hypoxia response element (HRE) of target genes. The enhancement of HIF-1alpha by CoCl(2) resulted in a decrease of PA activity, and the silence of HIF-1alpha by siRNA led to an increase of PA activity. The PA inhibitor-1 (PAI-1) mRNA and protein were increased by hypoxia or simulated hypoxia, which was reversed by the siRNA2-mediated silencing of HIF-1alpha. CHIP assay further confirmed that the induction of PAI-1 involved the binding of HIF-1alpha to the PAI-1 promoter, while the enhancement or silencing of HIF-1alpha did not affect the expression of urokinase type PA (uPA), tissue type PA (tPA) or uPA receptor (uPAR). Additionally, IL-1beta stimulated both HIF-1alpha and PAI-1 in articular chondrocytes, and the IL-1beta-mediated induction of PAI-1 was inhibited partly by HIF-1alpha silencing. HIF-1alpha may inhibit the PA activity through stimulating the expression of PAI-1 in normal articular chondrocytes. The inhibition of HIF-1alpha in the PA activity of articular chondrocytes probably plays an important role in the maintenance of articular cartilage matrix.

Loss of Thy-1 inhibits alveolar development in the newborn mouse lung

Transforming growth factor (TGF)-beta mediates hypoxia-induced inhibition of alveolar development in the newborn lung. TGF-beta is regulated primarily at the level of activation of latent TGF-beta. Fibroblasts expressing Thy-1 (CD90) inhibit TGF-beta activation. We hypothesized that loss of Thy-1 due to hypoxia may be a mechanism by which hypoxia increases TGF-beta activation and that animals deficient in Thy-1 will simulate the effects of hypoxia on lung development. To determine if loss of Thy-1 occurred during hypoxia, non-transgenic (C57BL/6) wild-type (WT) mice exposed to hypoxia were evaluated for Thy-1 mRNA and protein. To determine if Thy-1 deficiency simulated hypoxia, WT and Thy-1 null (Thy-1(-/-)) mice were exposed to air or hypoxia from birth to 2 wk, the critical period of lung development, and lung histology, function, parameters related to TGF-beta signaling, and extracellular matrix protein content were measured. To test if the phenotype in Thy-1(-/-) mice was due to excessive TGF-beta signaling, measurements were also performed in Thy-1(-/-) mice administered TGF-beta neutralizing antibody (1D11). We observed that hypoxia reduced Thy-1 mRNA and Thy-1 staining in WT mice. Thy-1(-/-) mice had impaired alveolarization, increased TGF-beta signaling, reduced lung epithelial and endothelial cell proliferation but increased fibroblast proliferation, and increased collagen and elastin. Lung compliance was lower, and tissue but not airway resistance was higher in Thy-1(-/-) mice at 2 wk. Thy-1(-/-) mice given 1D11 had improved alveolar development and lung function. These data support the hypothesis that hypoxia, by reducing Thy-1, increases TGF-beta activation, and thereby inhibits normal alveolar development.

Daxx inhibits stress-induced apoptosis in cardiac myocytes

The role of the death-associated protein Daxx in modulation of apoptosis induced in cardiac myocytes by oxidative stress was studied. Exposure of cultured cardiomyocyte-like cells to oxidative stress or simulated hypoxia increased the level of accumulated ROS and apoptosis. Under conditions of sub-apoptotic stimulation of cardiac myocytes, there was no increase in the level of the Daxx protein, but it translocated from the nucleus to the cytoplasm. Daxx overexpression protected the cells from apoptosis, while they were sensitised to cell death following its down-regulation by siRNA. Moreover, lowering the level of the Daxx protein sensitised cardiac myocytes to spontaneous apoptosis, suggesting that the protein may also have a pro-survival role under physiological conditions. Finally, it was shown that DJ-1, a protein suggested previously to sequester Daxx in the nucleus under conditions of oxidative stress (thereby preventing its cytosolic translocation), was localised solely in the cytoplasm of cardiac myocytes. This indicates that the protein does not modulate the apoptosis regulatory activity of Daxx in cardiac myocytes by its nuclear sequestration. Taken together, Daxx plays a protective role in cultured cardiomyocyte-like cells, at least under the conditions used.

Microcirculation characteristics in apparently healthy subjects during acute hypoxia and intermittent hypoxic training

The characteristics of the microcirculation in the forearm skin of 29 apparently healthy male volunteers were studied in acute hypoxia and during intermittent hypoxic training (IHT) using computer laser Doppler flowmetry. It was shown that short-term exposure of apparently healthy subjects to simulated acute hypoxia optimized the microcirculation owing to sympathetic innervation and concomitant rearrangement of microvascular tone regulation (activation of skin perfusion and reduction of blood flow through arteriovenular shunts when the neurogenic tone component increases). A second (placebo-controlled) series of experiments showed that long-term hypoxic preconditioning (20 IHT sessions) facilitated fixing of the adaptive dynamic rearrangements aimed at microcirculation improvement. The microvasculature response during acute hypoxia and a course of IHT depends on the initial sensitivity of subjects to simulated hypoxia. Significant perfusion enhancement in the tissues studied and microvascular tone normalization were observed in the subjects that were initially sensitive to hypoxia.

Poly(ADP‐ribose) polymerase‐1 mediated glycolytic inhibition as a mechanism of targeted necrosis following renal ischemia

Following ischemic renal injury (IRI), selective damage occurs in the S3 segment of the proximal tubule (PST) due to glycolytic inhibition. However, the exact mechanism by which glycolysis is inhibited is not elucidated. We reported that inhibition of PARP‐1 activity offers protection against necrotic cell death in PSTs following IRI by preserving ATP levels. We hypothesize that discriminating PARP‐1 activation in PSTs following IRI causes poly(ADP‐ribosyl)ation and inhibition of the key glycolytic enzyme GAPDH, leading to inhibition of glycolysis and anaerobic ATP synthesis. We demonstrate using in vitro and in vivo models that GAPDH is poly(ADP‐ribosyl)ated and as a consequence, its activity is inhibited under hypoxic conditions. Inhibition of PARP activity restored GAPDH activity and ATP levels. Supplementation of NAD+ in simulated hypoxia had only a minimal effect on ATP synthesis indicating that glycolytic inhibition is not due to depletion of NAD+ as previously proposed. Inhibition of GAPDH using iodoacetamide, however, exacerbated ATP depletion and necrotic cell death under hypoxic conditions when compared to controls, while inhibition of PARP activity protected the cells. In conclusion, our data indicate that GAPDH‐poly(ADP‐ribosyl)ation and subsequent inhibition of anaerobic respiration exacerbate ATP depletion in PST to induce selective PST cell injury following IRI. AHA and NKF‐Nebraska.

Varying exposure regimes to long term chronic intermittent hypoxia exert different outcomes and morphological effects on Wistar rats at 4600 m

The aim of this study was to compare differences in morphological effects following two regimens of simulated chronic intermittent hypoxia (4600 m) during 12 months, compared to chronic hypoxia and normoxia. Male Wistar rats were randomly assigned to four groups: Chronic intermittent hypoxia (CIH) 2 × 2 (2 days hypoxia, 2 days normoxia, n = 50), CIH4 × 4 (4 days hypoxia, 4 days normoxia, n =50), chronic hypoxia (CH) (permanent, n = 28) and control (NX) (normoxia, n = 24). Hypoxia was simulated in a hypobaric chamber (428 torr). The following parameters were assessed: ventricle wall thickness, presence of congestion, edema, hemorrhage, thrombosis, necrosis, and renal casts. In general, mortality was significantly associated with heart congestion, pulmonary edema, pulmonary hemorrhage, and liver necrosis. Intermittent as well as chronic exposure produced a common pattern of manifestations in all organs, with varying severity and time of occurrence. Right ventricle hypertrophy was observed in CIH2 × 2 and CIH4 × 4 but attained lower values than in CH. Further, the coefficient of right ventricular wall thickness rose markedly dependent upon exposure duration. Renal casts and thrombosis were also shown to be the signs of hypoxic damage. Morphological effects in intermittent hypoxia resembled an intermediate severity position compared to CH and tended to occur later, indicating that a shorter regimen seemed to be less harmful.

Prediction of the susceptibility to AMS in simulated altitude

Acute mountain sickness (AMS) develops when rapidly ascending to high altitudes. However, some mountaineers will suffer from AMS even at 2,000 m and others not until 5,000 m. The awareness of the individual susceptibility for AMS would be helpful for preventive strategies. Thus, the main purpose of this paper is the comparison of existing studies dealing with the prediction of AMS susceptibility and to draw conclusions on presently most valuable tests. A PubMed search has been performed, and preliminary observations from our laboratory have been included. The cautious conclusion derived from the reviewed 16 studies is that values of arterial oxygen saturation (SaO(2)), determined 20-30 min after exposure to simulated hypoxia equivalent to 2,300-4,200 m, seem to be the most useful predictors of AMS susceptibility (>80% correct prediction). Because the sympathetic activation during acute exposure to hypoxia may well contribute to the AMS development, parameters like heart rate variability or blood lactate could even enhance this predictability. The ventilatory response to hypoxia is easily trainable by pre-exposures to hypoxia but considers only part of the complex acclimatization process.

914 POSTER Shedding of GPI-anchored proteins from ovarian cancer cells promotes pericellular plasmin generation and cell invasion

To examine the effects of hypoxia-inducible factor-1α (HIF-1α) on the plasminogen activator's (PA) activity and on the expression of components of PA system in articular chondrocytes of rats.Chondrocytes from rat knee joint cartilage were cultured under normoxic, hypoxic, CoCl2 simulated hypoxic, and interleukin-1β (IL-1β)-stimulated conditions. siRNA targeting HIF-1α was transfected into cells cultured under hypoxic, simulated hypoxic, and IL-1β-stimulated conditions to silence HIF-1α. PA activity was determined by the hydrolysis of the chromogenic substrate H-D-Val-Leu-Lys-pNA (S-2251). The mRNA levels were measured by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). The intracellular/matrix-associate protein levels were detected by Western blot and the soluble protein levels were measured by enzyme linked immunosorbent assay (ELISA). Chromatin immunoprecipitation (CHIP) assay was performed to determine whether HIF-1α binds to the hypoxia response element (HRE) of target genes.The enhancement of HIF-1α by CoCl2 resulted in a decrease of PA activity, and the silence of HIF-1α by siRNA led to an increase of PA activity. The PA inhibitor-1 (PAI-1) mRNA and protein were increased by hypoxia or simulated hypoxia, which was reversed by the siRNA2-mediated silencing of HIF-1α. CHIP assay further confirmed that the induction of PAI-1 involved the binding of HIF-1α to the PAI-1 promoter, while the enhancement or silencing of HIF-1α did not affect the expression of urokinase type PA (uPA), tissue type PA (tPA) or uPA receptor (uPAR). Additionally, IL-1β stimulated both HIF-1α and PAI-1 in articular chondrocytes, and the IL-1β-mediated induction of PAI-1 was inhibited partly by HIF-1α silencing.HIF-1α may inhibit the PA activity through stimulating the expression of PAI-1 in normal articular chondrocytes. The inhibition of HIF-1α in the PA activity of articular chondrocytes probably plays an important role in the maintenance of articular cartilage matrix.

Tonic activation of hypoxia‐inducible factor 1α in avascular articular cartilage and implications for metabolic homeostasis

To determine whether oxygen-dependent activation patterns of hypoxia-inducible factor 1alpha (HIF-1alpha) observed in vascularized tissues are conserved within avascular and hypoxic articular cartilage and whether HIF-1alpha affects cartilage matrix synthesis. Explants of bovine articular cartilage and primary chondrocytes were exposed to normoxia (21% O2), hypoxia (2% O2), and simulated hypoxia (21% O2 plus CoCl2). Western blot and immunofluorescence analyses of HIF-1alpha were performed to determine HIF-1alpha activation patterns. To simulate cartilage loss from disease or injury, the top layers of cartilage were removed from osteochondral explants, and the residual cartilage was assessed for HIF-1alpha immunolocalization and proteoglycan synthesis. We demonstrated continuous nuclear translocation of HIF-1alpha in deeper layers of intact articular cartilage. HIF-1alpha was not completely degraded in chondrocytes exposed to normoxia, but rather, colocalized to the Golgi complex, a finding not previously reported for any cell type. Following alteration of the oxygen gradient by removal of the top layers of cartilage, predominantly perinuclear HIF-1alpha was found in the deeper layers. Restoration of intranuclear HIF-1alpha to these areas was achieved by hypoxia and simulated hypoxia. Under conditions in which HIF-1alpha was inactivated, matrix synthetic activity was altered (P < 0.0001) compared with control cartilage. These findings demonstrate that hypoxia-dependent activation of HIF-1alpha is highly conserved and that changes in oxygen tensions following cartilage loss from injury or disease alter cartilage metabolism in part by changing HIF-1alpha activity. The discovery of tonic activation of HIF-1alpha within intact articular cartilage underscores its potential importance to cartilage homeostasis.