Journal: Arthritis Research & Therapy

Article Title: Differential effects of Th1 versus Th2 cytokines in combination with hypoxia on HIFs and angiogenesis in RA

doi: 10.1186/ar3934

Figure Lengend Snippet: Angiogenesis RT² Profiler™ PCR Array data and expression analysis of human rheumatoid arthritis cells . PCR arrays were used with cDNA from rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) from five RA patients and incubated for either (a) 4 hours or (b) 24 hours in 1% oxygen (hypoxia) and (c) from three RA patients exposed to 1 mM dimethyloxalylglycine (DMOG) for 24 hours. Values are fold-change versus normoxia (21% oxygen) or dimethyl sulfoxide (DMSO) control and were analysed against housekeeping gene β-actin, 18S ribosomal RNA, hypoxanthine phosphoribosyltransferase 1 and 60S ribosomal protein L13a. Genes significantly increased or decreased by a factor ≥2 ( P < 0.05, dotted line) in all patients are shown. Data are mean ± standard error of the mean and were analysed by paired t test of ΔCt values, comparing normoxia versus hypoxia (a and b) or DMSO control versus DMOG (c). (d) Western blotting demonstrates induction of hypoxia-inducible factor (HIF)-1α and HIF-2α in total protein lysates from RA FLS in response to 1% oxygen or 1 mM DMOG for 24 hours. An antibody against α-tubulin was used as a loading control. A lane irrelevant to the study was removed as indicated by the line to show lanes of interest adjacent to one another. (e) Freshly dissociated total human RA synovial membrane cells from four patients were analysed for expression of vascular endothelial growth factor (VEGF), leptin, ephrin A3 (EFNA3) and angiopoietin-like (ANGPTL)-4 using quantitative PCR. Changes in mRNA are expressed as fold-change relative to levels under 21% oxygen set as 1.0 (dotted line). * P < 0.05, ** P < 0.01, *** P < 0.001. EFNB2, ephrin B2; FIGF, C-fos-induced growth factor (VEGF D); HAND2, Heart and neural crest derivatives expressed 2; HPSE, heparanase; ID3, inhibitor of DNA-binding 3, dominant negative helix-loop-helix protein; NRP2, neuropillin 2; PLAU, plasminogen activator urokinase.

Article Snippet: FLS and normal human skin fibroblasts (HSF; Lonza, Walkersville, MD, USA) were cultured in DMEM containing 10% foetal bovine serum, 4.5 g/l glucose and L -glutamine, supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin (PAA Laboratories, Coelbe, Germany).

Techniques: Expressing, Incubation, Control, Western Blot, Membrane, Real-time Polymerase Chain Reaction, Binding Assay, Dominant Negative Mutation

Journal: Arthritis Research & Therapy

Article Title: Differential effects of Th1 versus Th2 cytokines in combination with hypoxia on HIFs and angiogenesis in RA

doi: 10.1186/ar3934

Figure Lengend Snippet: Hypoxia-induced hypoxia-inducible factor isoform dependence of angiogenic genes in human rheumatoid arthritis fibroblast-like synoviocytes . Hypoxia-induced hypoxia-inducible factor (HIF) isoform dependence of ephrin A3 (EFNA3), angiopoietin-like (ANGPTL)-4, leptin and vascular endothelial growth factor (VEGF) in human rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). RA FLS were transiently transfected with siRNA oligonucleotides complementary to hypoxia-inducible factor (HIF)-1α (siHIF-1α) or HIF-2α (siHIF-2α) or both simultaneously. An siRNA oligonucleotide complementary to siLuc was used as control. The cell cultures were subsequently exposed to 1% oxygen (hypoxia) for 24 hours. Total RNA was isolated and cDNA generated, and the mRNA level of (a) leptin and (c) ANGPTL-4, (e) VEGF and (g) EFNA3 was determined using quantitative PCR. Changes in mRNA expressed as fold-change relative to levels in the siLuc transfected normoxic controls set as 1.0 (dotted line). The secretion of (b) leptin, (d) ANGPTL-4 and (f) VEGF protein was measured using ELISA. Data expressed as mean ± standard error of the mean of ≥3 independent experiments with sample assayed in triplicate, and were analysed using one-way analysis of variance with Bonferroni's post-hoc test for multiple comparisons versus hypoxic siLuc transfected cells (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: FLS and normal human skin fibroblasts (HSF; Lonza, Walkersville, MD, USA) were cultured in DMEM containing 10% foetal bovine serum, 4.5 g/l glucose and L -glutamine, supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin (PAA Laboratories, Coelbe, Germany).

Techniques: Transfection, Control, Isolation, Generated, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Arthritis Research & Therapy

Article Title: Differential effects of Th1 versus Th2 cytokines in combination with hypoxia on HIFs and angiogenesis in RA

doi: 10.1186/ar3934

Figure Lengend Snippet: Proangiogenic/anti-angiogenic effects of Th1 and Th2 cytokines on rheumatoid arthritis fibroblast-like synoviocyte gene expression . Cell cultures were exposed to 1% oxygen (hypoxia) and/or 10 ng/ml cytokine or left untreated for 24 hours. Total RNA was isolated and cDNA generated, and the mRNA level of (a) vascular endothelial growth factor (VEGF), (c) angiopoietin-like (ANGPTL)-4, (e) leptin and (f) ephrin A3 (EFNA3) was determined using quantitative PCR. Changes in mRNA expressed as fold-change relative to levels in untreated samples set as 1.0 (dotted line). Secretion of (b) VEGF and (d) ANGPTL-4 protein was measured using ELISA. Data expressed as the mean ± standard error of the mean of ≥3 independent experiments with samples assayed in triplicate. Samples analysed using one-way analysis of variance with Bonferroni's post-hoc test for multiple comparisons versus control normoxia († P < 0.05, †† P < 0.01, ††† P < 0.001) or versus hypoxia alone (* P < 0.05, ** P < 0.01,*** P < 0.001).

Article Snippet: FLS and normal human skin fibroblasts (HSF; Lonza, Walkersville, MD, USA) were cultured in DMEM containing 10% foetal bovine serum, 4.5 g/l glucose and L -glutamine, supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin (PAA Laboratories, Coelbe, Germany).

Techniques: Gene Expression, Isolation, Generated, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Control

Journal: Arthritis Research & Therapy

Article Title: Differential effects of Th1 versus Th2 cytokines in combination with hypoxia on HIFs and angiogenesis in RA

doi: 10.1186/ar3934

Figure Lengend Snippet: Th1-induced vascular endothelial growth factor but not angiopoietin-like-4 expression is dependent on hypoxia-inducible factor-1 . Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) were transiently transfected with siRNA oligonucleotides complementary to hypoxia-inducible factor (HIF)-1α (siHIF-1α). An siRNA oligonucleotide complementary to siLuc was used as control. The cell cultures were subsequently exposed to 10 ng/ml cytokines or left untreated for 24 hours. Changes in mRNA in response to IL-1β or TNFα of (a) vascular endothelial growth factor (VEGF) and (b) angiopoietin-like (ANGPTL)-4 expressed as fold-change relative to levels in the siLuc transfected untreated controls set as 1.0 (dotted line). Data expressed as the mean ± standard error of the mean of ≥3 independent experiments with samples assayed in triplicate and analysed versus cytokine-treated siLuc transfected cells. * P < 0.05, ** P < 0.01.

Article Snippet: FLS and normal human skin fibroblasts (HSF; Lonza, Walkersville, MD, USA) were cultured in DMEM containing 10% foetal bovine serum, 4.5 g/l glucose and L -glutamine, supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin (PAA Laboratories, Coelbe, Germany).

Techniques: Expressing, Transfection, Control

Journal: Arthritis Research & Therapy

Article Title: Differential effects of Th1 versus Th2 cytokines in combination with hypoxia on HIFs and angiogenesis in RA

doi: 10.1186/ar3934

Figure Lengend Snippet: Interactions of hypoxia and cytokines modulate induction of angiogenic activity by rheumatoid arthritis fibroblast-like synoviocytes . Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) were cultured in normoxia (21% oxygen), hypoxia (1% oxygen) or with 10 ng/ml IL-4 or TNFα for 24 hours. Alternatively the cells were co-stimulated with cytokines whilst cultured in hypoxia to mimic the environment in RA joints. Supernatants from these cultures were applied to wells of a 96-well plate containing growth factor-reduced matrigel and pre-seeded human microvascular endothelial cell (HMEC)-1 cells and left to form tubules for 4 to 6 hours. (a) A single picture was captured from the centre of each well with a camera (QICAM FAST; QImaging) attached to a microscope (CKX41; Olympus), with a representative example for each condition shown here. Using AngioSys Image Analysis software we analysed several parameters of angiogenesis: (b) percentage of field area covered by HMEC-1 tubules, (c) number of tubules, and (d) tubule junctions formed in the field area. Data expressed as the mean ± standard error of the mean of ≥3 independent experiments with supernatants from each condition assayed in triplicate wells. Samples analysed using one-way analysis of variance with Bonferroni's post-hoc test for multiple comparisons versus control (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: FLS and normal human skin fibroblasts (HSF; Lonza, Walkersville, MD, USA) were cultured in DMEM containing 10% foetal bovine serum, 4.5 g/l glucose and L -glutamine, supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin (PAA Laboratories, Coelbe, Germany).

Techniques: Activity Assay, Cell Culture, Microscopy, Software, Control

Journal: Frontiers in Neurology

Article Title: Compound Heterozygous COX20 Variants Impair the Function of Mitochondrial Complex IV to Cause a Syndrome Involving Ophthalmoplegia and Visual Failure

doi: 10.3389/fneur.2022.873943

Figure Lengend Snippet: COX20 mRNA and protein expression. (A) mRNA expression of COX20 in the muscle tissues and fibroblasts of the proband and control. GAPDH was used as the loading control. Significant differences in the mRNA levels exist between the groups. (B) Gene and cDNA schematic. c.41A > G and c.222G > T mutations were identified in the affected siblings. According to an NCBI genome analysis, the full length cDNA corresponds to transcript variant 1 (NM_198076.6), and the coding sequence (CDS) is 357 bp in length. Transcript variant 4 (NM_001312873.1) lacks exon 2 and a part (20 bp) of exon 1 of NM_198076.6. The coding region is 222-bp long and shorter than the transcript variant 1 by 135 bp. (C) Sanger sequencing reveals errors in mRNA splicing in fibroblasts of the patient. The results of sequences ①/②/③ were aligned with the reference sequence NM_198076.6. Blanks in the sequence alignment depict deletion or mismatch of nucleotides. Sequences ① were obtained from fibroblasts of control. Alignment of sequence ② shows that the variant c.41A > G led to a 20-bp deletion in exon 1. Alignment of sequence ③ shows that the variant c.222G > T did not affect mRNA splicing. (D–F) Western blot analysis of COX4, COX20, and OXPHOS in muscle tissues and fibroblasts. GAPDH was used as the internal control for muscles, and β-actin for cells. Significant differences are observed in the protein expression of COX20 and CIV but not in the protein expression of CI, CII, CIII, and CV. (G) Blue native-PAGE analysis of mitochondria isolated from fibroblasts reveals lower COX2, COX4, and CIV levels; ** p < 0.01. All data are presented as mean ± SEM and p -values were calculated by the Mann–Whitney U -test.

Article Snippet: Human skin fibroblasts were obtained from iCell (Shanghai, China, h075).

Techniques: Expressing, Control, Variant Assay, Sequencing, Western Blot, Muscles, Blue Native PAGE, Isolation, MANN-WHITNEY

Journal: Frontiers in Neurology

Article Title: Compound Heterozygous COX20 Variants Impair the Function of Mitochondrial Complex IV to Cause a Syndrome Involving Ophthalmoplegia and Visual Failure

doi: 10.3389/fneur.2022.873943

Figure Lengend Snippet: (A,B) OCR of patient (II-1) fibroblasts was measured using a Seahorse XF24 Extracellular Flux Analyzer. Basal respiration, maximal respiration, ATP production, and spare respiratory capacity of fibroblasts from the patient are significantly lower than those of control. (C) To detect the enzymatic activities of complexes, mitochondria were isolated from fibroblasts, and citrate synthase was used as the internal control. Complex IV enzyme activities of the patient fibroblasts were lower than control. (D,E) Functional complementation assays demonstrate that COX20 variants are responsible for the deficiency of complex IV and mitochondria in patient fibroblasts. Fibroblasts transduced with adenovirus vector (ADM-FH) were used as a control group. Western blot analysis shows that compared with fibroblasts transduced with ADM-FH, the expression level of COX20 and CIV in fibroblasts from patients transduced with COX20 increased significantly. (F,G) Seahorse analysis shows that COX20 overexpression promoted maximal respiration and increased mitochondrial spare respiratory capacity. (H) Enzyme activity measurements show that COX20 overexpression can help mitochondria restore the enzymatic activity of complex IV; * p < 0.05, ** p < 0.01 vs. ADM-FH, # p < 0.05 vs. II-1 + ADM-FH and II-1 + AD-COX20. All data are presented as mean ± SEM and p -values were calculated by Kruskal–Wallis tests. ## p < 0.01 versus II-1+ADM-FH and II-1+AD-COX20.

Article Snippet: Human skin fibroblasts were obtained from iCell (Shanghai, China, h075).

Techniques: Control, Isolation, Functional Assay, Transduction, Plasmid Preparation, Western Blot, Expressing, Over Expression, Activity Assay