Journal: Journal of Orthopaedic Translation

Article Title: Targeted knockdown of Piezo1 in synovial macrophages attenuates osteoarthritis development

doi: 10.1016/j.jot.2026.101053

Figure Lengend Snippet: Piezo1 regulates osteoarthritis in mice by modulating macrophage polarization.(A) 3D micro-CT images of the anterior and lateral views of the knee joints of Piezo1 cKO and Piezo1 fl/fl mice following DMM surgery.(B–C) Quantification of osteophyte number and volume in the regions of interest (ROIs), with ROIs marked in green.(D) H&E staining and Safranin O/Fast Green staining of joint cartilage in Piezo1 cKO and Piezo1 fl/fl mice.(E–F) Quantification of synovitis scores and OARSI scores in the synovium and cartilage of Piezo1 cKO and Piezo1 fl/fl mice.(G) Immunostaining for Piezo1 and F4/80 in the synovium of Piezo1 cKO and Piezo1 fl/fl mice with (H) quantitative analysis.(I–L) Immunohistochemical analysis of Aggrecan, ADAMTS4, and IL-6 in the cartilage of Piezo1 cKO and Piezo1 fl/fl mice, with (J–L) quantitative analysis.Data are presented as mean ± SD. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; NS: not significant.

Article Snippet: ADAMTS4 , 82744-2-RR , Proteintech Group (Chicago, IL, USA) , 1:1000 , 1:200.

Techniques: Micro-CT, Staining, Immunostaining, Immunohistochemical staining

Journal: Journal of Orthopaedic Translation

Article Title: Targeted knockdown of Piezo1 in synovial macrophages attenuates osteoarthritis development

doi: 10.1016/j.jot.2026.101053

Figure Lengend Snippet: Targeted knockdown of Piezo1 in synovial macrophages using Man-LNP@Si-Piezo1 alleviates OA symptoms.(A) 3D micro-CT images of the knee joints of different groups.(B–C) Quantification of osteophyte number and volume in ROIs.(D) H&E and Safranin O/Fast Green staining of joint cartilage.(E–F) Quantification of synovitis and OARSI scores.(G–M) Immunohistochemical and immunofluorescence analyses of Aggrecan, ADAMTS4, iNOS, and NLRP3 in cartilage and synovium, with quantification.Data are presented as mean ± SD. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; NS: not significant.

Article Snippet: ADAMTS4 , 82744-2-RR , Proteintech Group (Chicago, IL, USA) , 1:1000 , 1:200.

Techniques: Knockdown, Micro-CT, Staining, Immunohistochemical staining, Immunofluorescence

Journal: Experimental neurology

Article Title: Astrocyte-selective AAV-ADAMTS4 gene therapy combined with hindlimb rehabilitation promotes functional recovery after spinal cord injury.

doi: 10.1016/j.expneurol.2020.113232

Figure Lengend Snippet: Fig. 1. A schematic diagram of the study design. An acute study ending 4 weeks after SCI and another ending 13 weeks after SCI were conducted. To investigate transgene expression and toxicity resulting from the vector, animals were randomly assigned to three treatment groups and tissue was collected 4 weeks following 175 KDyne contusion injuries. To investigate the therapeutic benefit of AAV-ADAMTS4 alone or in combination with hindlimb rehabilitation animals were randomly assigned to experimental groups and on day 0 received a 175 KDyne contusion injury at T10. Those in treatment groups received four injections of AAV-ADAMST4 at each corner of the injury site. Those assigned to the rehabilitation groups were subjected to hindlimb rehabilitation one week after the injury. At week 10, animals received injections of BDA into the hindlimb motor cortex for tracing of hindlimb CST axons. Three weeks later, the animals were euthanised and their spinal cords and brains were collected for histological analyses. This figure was produced using Servier Medical Art, available from www.servier.com/Powerpoint-image-bank. Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.

Article Snippet: A plasmid containing human ADAMTS4 cDNA (Origene: RC09226; NM_005099) was used as a template and amplified using a ThermoFisher Accuprime PCR kit as per the manufacturer's instructions and the forward primer 5′ TAT TAT ACT AGT GCT GCA GTA CCA GTG CCA TG 3′ and the reverse primer 5′ CTA GTA AAG CTT CGG GAT AGT GAG GTT ATT TC 3′ (Fig. S1a) The ADAMTS4 PCR product was then purified and cloned into an AAV expression plasmid; the ADAMTS4 transgene was followed by a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and bovine growth hormone polyadenylation signal (BGHpA), flanked by AAV2 inverted terminal repeats.

Techniques: Expressing, Plasmid Preparation, Produced

Journal: Experimental neurology

Article Title: Astrocyte-selective AAV-ADAMTS4 gene therapy combined with hindlimb rehabilitation promotes functional recovery after spinal cord injury.

doi: 10.1016/j.expneurol.2020.113232

Figure Lengend Snippet: Fig. 3. AAV-ADAMTS4 transduces primary spinal cord astrocytes and degrades CSPG components. Primary spinal cord astrocytes were transduced with the AAV-ADAMTS4 vector (either 4 × 109 or 8 × 109 vg) and stimulated with 10 ng/mL TGFβ1 two days later. The cells were fixed three days after stimulation and fluorescent immunocytochemistry was then used to detect the ADAMTS4 transgene (ADAMTS4), chondroitin sulphate (CS56) and glycosaminoglycan (WFA). a) 10× magnification images were captured using a Nikon TE-2000-U microscope. Images were thresholded and arbitrary fluorescence units were measured for b) ADAMTS4, c) CS56 and d) WFA. Data represents mean ± SEM (n = 4 independent cultures). A one-way ANOVA followed by a Tukey's multiple comparisons test was used to test statistically significant differences *P < .05, **P < .001, ***P < .0001.

Article Snippet: A plasmid containing human ADAMTS4 cDNA (Origene: RC09226; NM_005099) was used as a template and amplified using a ThermoFisher Accuprime PCR kit as per the manufacturer's instructions and the forward primer 5′ TAT TAT ACT AGT GCT GCA GTA CCA GTG CCA TG 3′ and the reverse primer 5′ CTA GTA AAG CTT CGG GAT AGT GAG GTT ATT TC 3′ (Fig. S1a) The ADAMTS4 PCR product was then purified and cloned into an AAV expression plasmid; the ADAMTS4 transgene was followed by a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and bovine growth hormone polyadenylation signal (BGHpA), flanked by AAV2 inverted terminal repeats.

Techniques: Transduction, Plasmid Preparation, Immunocytochemistry, Microscopy

Journal: Experimental neurology

Article Title: Astrocyte-selective AAV-ADAMTS4 gene therapy combined with hindlimb rehabilitation promotes functional recovery after spinal cord injury.

doi: 10.1016/j.expneurol.2020.113232

Figure Lengend Snippet: Fig. 5. ADAMTS4 gene therapy leads to significantly reduced lesion size. Luxol fast blue-eosin histology was used to investigate the integrity of spinal cord tissue following contusion (a), contusion and AAV-ADAMTS4 treatment (b), contusion with rehabilitation (c), and contusion with AAV-ADAMTS4 treatment and rehabilitation (D). Staining was used to determine the area of the lesion (e), white matter (f), total remaining tissue (g) and grey matter (h). Data represent mean ± SEM (contusion n = 10, contusion + AAV-ADAMTS4 n = 9; contu- sion + rehabilitation n = 12; contusion + AAV -ADAMTS4 + rehabilitation n = 9, sham + AAV-ADAMTS4 n = 4; scale = 1 mm). A Two-Way ANOVA and Bonferroni's multiple comparisons test were used to determine statistically significant differences. Results from the ANOVA tests comparing the mean group dif- ference between the contusion to contusion plus AAV-ADAMTS4 groups are displayed on the figures. *compares contusion to contusion plus AAV-ADAMTS4; #compares contusion plus rehabilitation to contusion plus rehabilitation plus AAV-ADAMTS4. For all figures: NS = Non significant, */#P < .05, **/##P < .001.

Article Snippet: A plasmid containing human ADAMTS4 cDNA (Origene: RC09226; NM_005099) was used as a template and amplified using a ThermoFisher Accuprime PCR kit as per the manufacturer's instructions and the forward primer 5′ TAT TAT ACT AGT GCT GCA GTA CCA GTG CCA TG 3′ and the reverse primer 5′ CTA GTA AAG CTT CGG GAT AGT GAG GTT ATT TC 3′ (Fig. S1a) The ADAMTS4 PCR product was then purified and cloned into an AAV expression plasmid; the ADAMTS4 transgene was followed by a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and bovine growth hormone polyadenylation signal (BGHpA), flanked by AAV2 inverted terminal repeats.

Techniques: Staining

Journal: Experimental neurology

Article Title: Astrocyte-selective AAV-ADAMTS4 gene therapy combined with hindlimb rehabilitation promotes functional recovery after spinal cord injury.

doi: 10.1016/j.expneurol.2020.113232

Figure Lengend Snippet: Fig. 6. AAV-ADAMTS4 increases hindlimb CST collateral sprouting rostral to the lesion and serotonergic innervation caudal to the lesion. BDA was injected into the hindlimb motor cortex of rats 10 weeks after SCI. a) In untransduced animals, loaded axons were mainly confirmed to the CST 600–1200 μm rostral to the lesion edge, whereas treated animals displayed axon sprouting and growth widespread through grey matter (examples indicated by the red arrows; scale bar = 500 μm). ImageJ was used to quantify the total length of traced axons outside of the CST; transduced animals had a significantly greater amount of traced axons outside of the CST. b) Data represents the mean ± SEM. A two-tailed unpaired t-test was used to determine statistical differences between the two groups where *P < .05 (n = 7 for contusion + rehabilitation and contusion + AAV-ADAMTS4 + rehabilitation; n = 4 for contusion and contusion + AAV- ADAMTS4). c) Serotonergic fibers in the ventral horns of sections caudal to the injury (three sections: 600, 1200 and 1800 μm caudal to the injury were used; scale bar = 200 μm) were visualised. d) Images were consistently thresholded and the mean pixel intensity of serotonin immunoreactivity at the ventral horn was measured. Data represent the mean ± SEM (contusion n = 10; contusion + AAV-ADAMTS4 n = 7; contusion + rehabilitation n = 10; contusion + rehabilitation + AAV-ADAMTS4 n = 6). A one-way ANOVA followed by a Tukey's multiple comparisons test was used to test statistically significant differences where *P < .05, **P < .001. e) Protein kinase C gamma immunofluorescence confirmed that abolishment of the CST caudal to the lesion (indicated by *). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: A plasmid containing human ADAMTS4 cDNA (Origene: RC09226; NM_005099) was used as a template and amplified using a ThermoFisher Accuprime PCR kit as per the manufacturer's instructions and the forward primer 5′ TAT TAT ACT AGT GCT GCA GTA CCA GTG CCA TG 3′ and the reverse primer 5′ CTA GTA AAG CTT CGG GAT AGT GAG GTT ATT TC 3′ (Fig. S1a) The ADAMTS4 PCR product was then purified and cloned into an AAV expression plasmid; the ADAMTS4 transgene was followed by a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and bovine growth hormone polyadenylation signal (BGHpA), flanked by AAV2 inverted terminal repeats.

Techniques: Injection, Two Tailed Test

Journal: Experimental neurology

Article Title: Astrocyte-selective AAV-ADAMTS4 gene therapy combined with hindlimb rehabilitation promotes functional recovery after spinal cord injury.

doi: 10.1016/j.expneurol.2020.113232

Figure Lengend Snippet: Fig. 7. ADAMTS4 gene therapy with rehabilitation after thoracic spinal cord injury enhances improvements in behavioural assessments. The measured impaction force did not differ between treatment groups, confirming consistent injuries across all groups (a). The animals were weighed weekly as an indication of health status (b). BBB scores were measured for 10 weeks following the injury (c). Horizontal ladder testing began two weeks after injury where animals were recorded as they crossed a 1-meter horizontal ladder with irregular rungs. Data reported is the sum of the number of foot slips made for three runs. The data represents the mean ± SEM (contusion n = 14, contusion + AAV-ADAMTS4 n = 13; contusion + rehabilitation n = 12; contusion + AAV -ADAMTS4 + rehabilitation n = 9, sham + AAV- ADAMTS4 n = 4). A repeated measures two-way ANOVA was used to determine significant differences between the groups over time and a Bonferroni multiple comparisons test was used to determine differences at each time point where *P < .05, **P < .001, ***P < .0001. Red stars compares the contusion only group to the contusion + AAV-ADAMTS4 group, the yellow stars compares the contusion only group to the contusion + AAV -ADAMTS4 + rehabilitation group, and the purple stars compares the contusion + rehabilitation group to the contusion + AAV-ADAMTS4 + rehabilitation group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: A plasmid containing human ADAMTS4 cDNA (Origene: RC09226; NM_005099) was used as a template and amplified using a ThermoFisher Accuprime PCR kit as per the manufacturer's instructions and the forward primer 5′ TAT TAT ACT AGT GCT GCA GTA CCA GTG CCA TG 3′ and the reverse primer 5′ CTA GTA AAG CTT CGG GAT AGT GAG GTT ATT TC 3′ (Fig. S1a) The ADAMTS4 PCR product was then purified and cloned into an AAV expression plasmid; the ADAMTS4 transgene was followed by a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and bovine growth hormone polyadenylation signal (BGHpA), flanked by AAV2 inverted terminal repeats.

Techniques:

Journal: Experimental and Therapeutic Medicine

Article Title: Oroxylin A attenuates IL-1β-induced inflammatory reaction via inhibiting the activation of the ERK and PI3K/AKT signaling pathways in osteoarthritis chondrocytes

doi: 10.3892/etm.2021.9819

Figure Lengend Snippet: OrA treatment prevents IL-1β-induced degradation of ECM. Chondrocytes were treated with various concentrations of OrA (4, 8 and 16 µM) and stimulated with or without IL-1β (10 ng/ml) for 24 h. (A) Relative mRNA expression levels of MMP-3 and MMP-13 were determined by reverse transcrtiption-quantitative PCR. (B) The protein expression levels of MMP-3, MMP-13, ADAMTS-4 and ADAMTS-5 were determined by western blot analysis. (C) Quantification of MMP-3, MMP-13, ADAMTS-4 and ADAMTS-5 expression. Data are expressed as the mean ± standard deviation from three experimental repeats. # P<0.01 vs. untreated; * P<0.05, * * P<0.01 and * * * P<0.001 vs. IL-1β only. Immunofluorescence analysis of (D) MMP-3 and (E) MMP-13 expression, which were quantified (original magnification, x100). Data are expressed as the mean ± standard deviation from three experimental repeats. * P<0.05, * * P<0.01 and * * * P<0.001. OrA, oroxylin A; ECM, extracellular matrix; IL-1β, interleukin-1β; MMP3, matrix metalloproteinase; ADAMTS, disintegrin and metalloproteinase with thrombospondin motifs.

Article Snippet: Primary antibodies against the unphosphorylated forms of PI3K (cat. no. 4255), AKT (cat. no. 9272), ERK (cat. no. 4695), p38 (cat. no. 14451), JNK (cat. no. 9252), p65 (cat. no. 8242), NF-κB inhibitor α (IκBα; cat. no. 4814), inducible nitric oxide synthase (iNOS; cat. no. 39898), cyclooxygenase 2 (COX-2; cat. no. 12282) and β-actin (cat. no. 3700), and the phosphorylated (p) forms of AKT (cat. no. 13038), ERK (cat. no. 4370), p38 (cat. no. 9216), JNK (cat. no. 9251), and p65 (cat. no. 3039) were obtained from Cell Signaling Technology, Inc. Primary antibodies against MMP3 (cat. no. 17873-1-AP) and MMP13 (cat. no. 18165-1-AP) were purchased from Proteintech Group, Inc., whereas those against aggrecan, disintegrin and metalloproteinase with thrombospondin motifs ADAMTS-4 (cat. no. ab185722), ADAMTS-5 (cat. no. ab41037) and type II collagen (cat. no. ab188570) were from purchased from Abcam.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Standard Deviation, Immunofluorescence