Vitamin D Modulates PAR-4 Expression in an in Vitro Model of Osteoarthritis

Abstract Osteoarthritis (OA) encompasses degeneration of articular cartilage, subchondral bone erosions and sclerosis. Chondrocyte apoptosis and an oxygen-deprived microenvironment are essential factors in OA pathogenesis. PAR-4 (Prostate apoptosis response-4) is a pro-apoptotic protein implicated in many pathologies as well as in chondrocyte cell death mechanism. Vitamin D supplementation has been identified as a therapeutic tool for a variety of inflammatory pathologies. In the present manuscript, we investigated whether first, PAR-4 expression is influenced by chondrocytes in a model of OA, in vitro, and second, whether vitamin D modulates PAR-4 expression in the same model. To test our hypothesis, we used the primary culture of murine chondrocytes isolated from the femoral and tibial condyles of wistar rats. The expression of the pro-inflammatory effect interleukin IL-1β was evaluated in the presence and absence of vitamin D. Western blot and immunofluorescence analysis confirmed protein expression. In the normoxia condition, the chondrocytes expressed PAR-4 in the cell nucleus, and in the hypoxic condition, PAR-4 was expressed in the cell cytoplasm. We disclosed that the treatment with Vitamin D decreased PAR-4 (p= 0.0137) and caspase-3 (p= 0.0007) expression. Thus, the results suggested that PAR-4 and caspase-3 proteins could be potential targets for OA.However, we believe that research is needed to identify the mechanisms implicated in the regulation of PAR-4 in OA.

Efectos del entrenamiento de intervalos de alta intensidad en altitud simulada. Revisión sistemática / Effects of high-intensity interval training at simulated Altitude. Systematic review

Introducción: en la actualidad, los entrenadores buscan la manera de mejorar las capacidades físicas de los atletas mediante diferentes estrategias de entrenamiento, como la exposición constante o intermitente a la altitud y el entrenamiento de intervalos de alta intensidad. Objetivo: Revisar la literatura actual y describir los efectos sobre el organismo del entrenamiento de intervalos de alta intensidad en altitud simulada en sujetos sedentarios, físicamente activos y entrenados. Resultados: el número de artículos revisados evidencia que, en hipoxia simulada en cámara hipobárica o normobárica (n=13) o máscara de simulación de altitud (n=1), todos utilizaron intensidades altas (n=13) a submáximas (n=1). Los participantes de las investigaciones fueron mujeres con obesidad sedentarias (n=3), hombres y mujeres físicamente activos (n=9) y sujetos entrenados (n=3). El tiempo de intervención de los estudios fue de 3 a 12 semanas, con una altitud simulada de 1824 a 4500 m.s.n.m. Se observaron efectos beneficiosos sobre la composición corporal, aptitud cardiorrespiratoria, aumentos en hemoglobina, eritropoyetina, consumo energético, fuerza máxima concéntrica e isométrica, fuerza absoluta y mejor tolerancia al ejercicio (percepción del esfuerzo). Conclusiones: La combinación de entrenamientos de intervalos de alta intensidad, combinado con una exposición en altitud simulada, puede evidenciar mejoras significativas en el rendimiento cardiorrespiratorio, así como en aspectos de composición corporal, lo que permitiría una mejor predisposición a intensidades más elevadas de actividad y ejercicio físico.

Introduction: Today, coaches are looking for ways to improve athletes' physical abilities through different training strategies, such as constant or intermittent exposu-re to altitude and high intensity interval training. Objective: To review the current literature and describe the effects on the body of simulated high-intensity interval training at altitude in sedentary, physically active, and trained subjects. Results: the number of articles reviewed evidences that, in simulated hypoxia in hypobaric or normobaric chamber (n = 13) or altitude simulation mask (n = 1), all used high intensities (n = 13) to submaximal (n = 1). The research participants were women with sedentary obesity (n = 3), physically active men and women (n = 9), and trained subjects (n = 3). The intervention time of the studies was 3 to 12 weeks, with a simulated altitude of 1824 to 4500 meters. Beneficial effects on body composition were observed, as well as cardiorespiratory fitness, increases in hemoglobin, erythro-poietin, energy consumption, concentric and isometric maximum strength, absolute strength and better exercise tolerance (perception of effort). Conclusions: The combination of high intensity interval training combined with a simulated altitude exposure can show significant improvements in cardiorespiratory performance, as well as in aspects of body composition, which would allow a better predisposition to higher intensities of activity and physical exercise.

A new method for constructing engineered corneal epithelium based on embryonic stem cells / 中华实验眼科杂志

Objective To construct engineered corneal epithelium from embryonic stem cells (ESCs) using Rock inhibitor combined with hypoxia-normoxia culture condition. Methods Human ESC line H1 was induced to differentiate into epithelial-like cells by addition of retinoic acid (RA) and bone morphogenetic protein 4(BMP4) in the differentiation medium under the adherent culture condition. The ESCs derived epithelial-like cells were expanded in the mixed medium of SHEM and KSFM with the mixture ratio of 1 : 2 with or without Rock inhibitor Y27632. The H1 derived epithelial-like cells were seeded on the denuded ammonic membrane to construct engineered corneal epithelium under hypoxia,normoxia and hypoxia-normoxia culture conditions,respectively. The inducted effect of ESCs into epithelial-like cells,the expansion ability of the epithelial-like cells and the characteristics of the constructed engineered corneal epithelium were evaluated by morphological observation, real-time fluorescent quantitative polymerase chain reaction (RTFQ-PCR) and immunofluorescence technology. Results Compared with the control group,the relative expressions of ESCs marker Oct4 mRNA, Notch signaling pathway related factors Notch1 and Jagged1 mRNA,and Wnt signaling pathways related factors c-myc and Cyclin D1 mRNA were significantly reduced, and the relative expressions of cutaneous ectoderm markers p63 and K18 mRNA were significantly increased at day 8 after induction in the induced group,with significant differences between them (t =14.63,20.15,93.50,11.60, 19.30,18.44,22.63;all at P<0.05). Compared with the without Y27632 group,the relative expressions of p63 and K14 mRNA,Notch signal pathway receptor Notch1 and Jagged1 mRNA were significantly increased,and Wnt signaling pathways downstream targeted gene c-myc and CylinD1 mRNA were significantly decreased at day 8 after induction in the Y27632 group,with significant differences between them (t =20.29,59.22,2.90,39.59,5.32,10.14;all at P<0.05),and the relative expression of K18 mRNA in the two groups was not significantly changed(t=1.38,P>0.05). The ESCs derived epithelium and constructed under hypoxia-normoxia culture condition showed more obvious stratification and tighter cell arrangement in comparison with those cells cultured in consistent hypoxia culture condition or normoxia culture condition. Epithelial markers Pan-CK and K18 as well as epithelial progenitor cell markers p63 and K14 expressed in the whole cell layers of the ESCs derived epithelium constructed under hypoxia-normoxia culture condition. Conclusions The addition of Y27632 enhances the proliferation ability of H1 derived epithelial cells and actives Notch signaling pathway and inhibits Wnt signaling pathway. The culture and construction in the expansion medium with Y27632 under the hypoxia-normoxia culture condition can promote the stratification of H1 derived engineered corneal epithelium.

Hypoxia Enhances Cell Properties of Human Mesenchymal Stem Cells

Atmospheric (in vitro) oxygen pressure is around 150 mm Hg (20% O₂), whereas physiologic (in vivo) oxygen pressure ranges between 5 and 50 mm Hg (0.7–7% O₂). The normoxic environment in cell culture does not refer to a physiological stem cell niche. The aim of this study is to investigate the effect of oxygen concentration on cell properties of human mesenchymal stem cells (MSCs). We analyzed cell proliferation rate, senescence, immunophenotype, stemness gene expression and differentiation potency with human urine stem cells (USCs), dental pulp stem cells (DPSCs), amniotic fluid stem cells (AFSCs), and bone marrow stromal cells (BMSCs). USCs, DPSCs, AFSCs and BMSCs were cultured under either 5% O₂ hypoxic or 20% O₂ normoxic conditions for 5 days. MSCs cultured under hypoxia showed significantly increased proliferation rate and high percentage of S-phase cells, compared to normoxic condition. In real-time PCR assay, the cells cultured under hypoxia expressed higher level of Oct4, C-Myc, Nanog, Nestin and HIF-1α. In immunophenotype analysis, MSCs cultured under hypoxia maintained higher level of the MSC surface markers, and lower hematopoietic markers. Senescence was inhibited under hypoxia. Hypoxia enhances osteogenic differentiation efficiency compared to normoxia. Hypoxia showed enhanced cell proliferation rate, retention of stem cell properties, inhibition of senescence, and increased differentiation ability compared to normoxia.

Pharmacokinetic comparison of roxithromycin under normoxic and hypoxic conditions in rats by UPLC/MS/MS / 中国药理学通报

Aim To study and compare the pharmaco-kinetic parameters of roxithromycin under normoxic and hypoxic rats. Methods A highly effective and rapid ultra-performance liquid chromatography with tandem mass spectrometry ( UPLC-MS/MS) method with posi-tive electrospray ionization source was successfully de-veloped and validated for quantification of roxithromy-cin in rat plasma. Sprague-Dawley rats were randomly divided into the hypoxia and normoxic groups. Each rat obtained a single dose of roxithromycin with 10 mg · kg-1 via intragastric administration. The pharmacoki-netic parameter comparison between normoxic and hy-poxic groups was calculated by SPSS software using in-dependent sample t test method. Results The main pharmacokinetic parameters of roxithromycin between the normoxic and hypoxic rats were:the AUC(0-t) 7 576 and 3 761 μg·h·L-1 , MRT(0-t) 5. 6 and 7. 7 h, T1/2 3. 4 h and 3. 9 h, CL 1. 5 and 3. 0 L · h-1 · kg-2 , tmax3. 1 and 3. 4 h, Cmax 1 116 and 372 μg·L-1 , re-spectively. The levels of Cmax and AUC of roxithromy-cin in hypoxic rats were statistically lower than those in normoxic rats. Conclusion The exposure level of rox-ithromycin in hypoxic rats markedly decreased. Our re-sults may provide an important experimental basis to adjust the dosage for roxithromycin in hypoxic clinical practice.

Effect of exogenous PGC -1α on VEGF expression in human retinal vascular endothelial cells / 国际眼科杂志(Guoji Yanke Zazhi)

AlM: To investigate the effect of exogenous peroxisome-proliferator-activated receptor-γcoactivator-1α ( PGC-1α) on vascular endothelial growth factor ( VEGF) expression in human retinal vascular endothelial cells ( HRVEC) .METHODS:Recombinant PGC-1α protein was added to HRVEC, and no recombinant PGC-1α protein was added to HRVEC as control group. After 24h of incubation, two groups of cells were then placed into a normoxic ( 20%O2 ) or hypoxic ( 1% O2 ) environment for another 16h. The expression of VEGF mRNA and protein were detected by real - time PCR, ELlSA and immunofluorescence cytochemistry.RESULTS: VEGF mRNA and protein levels in the cells were significantly increased by recombinant PGC - 1αprotein both under normoxia and hypoxia conditions as compared with control groups (P<0. 01).CONCLUSlON: PGC-1α can upregulate the expression of VEGF in HRVEC under normoxia and hypoxia conditions.

Gene expression promoted by the SV40 DNA targeting sequence and the hypoxia-responsive element under normoxia and hypoxia

The main objective of the present study was to find suitable DNA-targeting sequences (DTS) for the construction of plasmid vectors to be used to treat ischemic diseases. The well-known Simian virus 40 nuclear DTS (SV40-DTS) and hypoxia-responsive element (HRE) sequences were used to construct plasmid vectors to express the human vascular endothelial growth factor gene (hVEGF). The rate of plasmid nuclear transport and consequent gene expression under normoxia (20 percent O2) and hypoxia (less than 5 percent O2) were determined. Plasmids containing the SV40-DTS or HRE sequences were constructed and used to transfect the A293T cell line (a human embryonic kidney cell line) in vitro and mouse skeletal muscle cells in vivo. Plasmid transport to the nucleus was monitored by real-time PCR, and the expression level of the hVEGF gene was measured by ELISA. The in vitro nuclear transport efficiency of the SV40-DTS plasmid was about 50 percent lower under hypoxia, while the HRE plasmid was about 50 percent higher under hypoxia. Quantitation of reporter gene expression in vitro and in vivo, under hypoxia and normoxia, confirmed that the SV40-DTS plasmid functioned better under normoxia, while the HRE plasmid was superior under hypoxia. These results indicate that the efficiency of gene expression by plasmids containing DNA binding sequences is affected by the concentration of oxygen in the medium.

Promotion of proliferation and migration of Müller cells by RPE cells in a co-culture system / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To investigate the role of retinal pigment epithelium (RPE) in the growth of Müller cells using a co-culture system in vitro . METHODS: Müller cells were cocultured with RPE cells under both normoxic and hypoxic conditions in Transwell chamber culture system. Müller cell proliferation was evaluated by MTT assay. The number of cells which migrate through micropores and stay on the outer bottom side of insert systems were observed and counted. RESULTS: The activities of proliferation and migration of Müller cells when cocultured with RPE cells were significantly higher than those of the Müller cells when cultured alone at all time points under both normoxic and hypoxic conditions. However, for both the coculture and control groups, there is no significant difference between the measurements at 3 and 6 hours. CONCLUSION: Evidence suggests that RPE, when co-cultured with Müller cells, can stimulate migration and proliferation of Müller cells under both hypoxic and normoxic conditions in a time-dependent manner; how-ever, there is no evidence to support the synergetic interaction of RPE and Müller cells co-cultured under hypoxic conditions.