recombinant mouse sema3a fc chimera protein  (R&D Systems)

Journal: Bone research

Article Title: Neuronal guidance factor Sema3A inhibits neurite ingrowth and prevents chondrocyte hypertrophy in the degeneration of knee cartilage in mice, monkeys and humans.

doi: 10.1038/s41413-024-00382-0

Figure Lengend Snippet: Fig. 1 Elevated expression of neural factors of human and mice knee cartilage during OA progression. a Left, orthotopic view of 3D reconstruction CT of normal and OA knee joints in humans and mice. Right, Kellgren–Lawrence (KL) analysis of the joint space between normal control (n = 10 patients, n = 8 mice) and OA (n = 10 patients, n = 8 mice) groups. A two-tailed Mann–Whitney U test was used for statistical analysis. Mean with SD. A representative result from five independent experiments is shown. b Left, H&E staining of cartilage in normal control and OA groups. Scale bar, 200 µm (human), 200 µm (mouse). Middle, Safranin O staining of the cartilage in normal control and OA groups. Scale bar, 200 µm(human), 100 µm(mouse). Right, OARSI scores from normal control (n = 10 individuals, n = 8 mice) and OA (n = 10 individuals, n = 8 mice) groups. A two-tailed Mann–Whitney U test was used for statistical analysis. Mean with SD. A representative result from five independent experiments is shown. c Immunofluorescence staining of type X collagen (Col X) in the normal control (n = 10 individuals, n = 8 mice) and OA (n = 10 individuals, n = 8 mice) groups. Quantitative analysis of the cells of positive Col X cells per cartilage was performed among groups. A two-tailed unpaired student’s t-test was used for statistical analysis. Mean with SD. Scale bar, 50 µm. d Immunofluorescence staining of Tuj1 in the normal control (n = 10 individuals, n = 8 mice) and OA (n = 10 individuals, n = 8 mice) groups. Scale bar, 50 µm. Quantitative analysis of the area of positive Tuj1 per cartilage area (mm2) was performed among groups. High magnification of Tuj1 staining in knee cartilage was shown in respective groups (arrowhead). A two- tailed unpaired student’s t-test was used for statistical analysis. Mean with SD. Scale bar, 25 µm. e Immunofluorescence staining of Tau in the normal control (n = 8 individuals, n = 8 mice) and OA (n = 8 individuals, n = 8 mice) groups. Scale bar, 50 µm. Quantitative analysis of the area of positive Tau per cartilage area (mm2) was performed among groups. High magnification of Tau staining in knee cartilage was shown in respective groups (arrowhead). A two-tailed unpaired student’s t-test was used for statistical analysis. Mean with SD. Scale bar, 25 µm. f Immunofluorescence staining of Semaphorin 3A (Sema3A) in the normal control (n = 10 individuals, n = 8 mice), early OA (n = 10 individuals, n = 8 4-weeks OA mice), late OA (n = 10 individuals, n = 8 12-weeks OA mice) groups. Quantitative analysis of the number of positive Sema3a cells in cartilage was performed among groups. Scale bar, 50 µm. A two-tailed unpaired student’s t-test was used for statistical analysis. Mean with SD. A representative result from five independent experiments is shown. OA osteoarthritis. OARSI score Osteoarthritis Research Society International scoring system

Article Snippet: These animals received knee injection under anesthesia following approval from the Ethical Review Board of West China Hospital Sichuan University (No. 2018147A) and were divided into three groups: intra-articular injection of Hyaluronate acid (mean Mol. wt: 0.8~1.2 million, ARTZ@Dispo, JP), Sema3A protein (R&D SYSTEMS, 2150-S3) and lentivirus Rlv-CMV-sema3a-Efla-EGFP (BrainVTA, Wuhan, China) (n= 12, 4 monkeys in each group, 2 knees tested per monkey).

Techniques: Expressing, Control, Two Tailed Test, MANN-WHITNEY, Staining

Journal: Bone research

Article Title: Neuronal guidance factor Sema3A inhibits neurite ingrowth and prevents chondrocyte hypertrophy in the degeneration of knee cartilage in mice, monkeys and humans.

doi: 10.1038/s41413-024-00382-0

Figure Lengend Snippet: Fig. 2 Conditional knockout of Sema3A in chondrocytes before and after ACLT surgery display different knee phenotypes. a Left, orthotopic view of 3D reconstruction CT images of the Sema3A knockout before ACLT surgery (Sema3AKO Pre), Sema3A knockout after ACLT surgery (Sema3AKO Post) and control ACLT groups. Right, Kellgren–Lawrence analysis of the joint space in Sema3AKO Pre (n = 8), Sema3AKO Post (n = 8), and ACLT (n = 8) groups. A two-tailed unpaired student’s t-test was used for statistical analysis. Mean with SD. A representative X-ray image from five independent experiments is shown. b Top, H&E staining of the cartilage in Sema3AKO Pre, Sema3AKO Post, and ACLT groups. Scale bar, 100 µm. Bottom, Safranin O staining of cartilage in Sema3AKO Pre, Sema3AKO Post, and ACLT groups. Scale bar, 50 µm. Right, The OARSI score of the Sema3AKO Pre (n = 8), Sema3AKO Post (n = 8), and ACLT (n = 8) groups. Safranin O staining of proteoglycan (red) and bone (blue) in sagittal sections of the distal femur compartment. A two-tailed unpaired student’s t-test was used for statistical analysis. Mean with SD. A representative image of each group from five independent experiments is shown. c Immunofluorescence staining of Collagen X in Sema3AKO Pre (n = 8), Sema3AKO Post (n = 8), ACLT (n = 8) groups and the quantitative analysis of the cells of Collagen X positive cells per cartilage. Scale bar, 50 µm. A two-tailed unpaired student’s t-test was used for statistical analysis. Mean with SD. d Immunofluorescence staining of Tuj1 in Sema3AKO Pre (n = 6), Sema3AKO Post (n = 6), ACLT (n = 6) groups and the quantitative analysis of the area of Tuj1 positive area per cartilage area (mm2). Scale bar, 50 µm. A two-tailed unpaired student’s t-test was used for statistical analysis. Mean with SD. A representative result from five independent experiments is shown. e Immunofluorescence staining of Sema3A in Sema3AKO Pre (n = 6), Sema3AKO Post (n = 6), ACLT (n = 6) groups and the quantitative analysis of the number of semaphorin-positive cells per cartilage. Scale bar, 50 µm. A two-tailed unpaired student’s t-test was used for statistical analysis. Mean with SD. A representative result from five independent experiments is shown. f Immunofluorescence staining of F-actin in Sema3AKO Pre (n = 6), Sema3AKO Post (n = 6), ACLT (n = 6) groups and the quantitative analysis of the area of F-actin+ per cartilage area (mm2). Scale bar, 50 µm. A two-tailed unpaired student’s t-test was used for statistical analysis. Mean with SD. A representative result from five independent experiments is shown. g Quantitative analysis of the latency to lick a hind paw following mice from different groups (Sema3AKO Pre (n = 8), Sema3AKO Post (n = 8), ACLT (n = 8)) being placed on a 54 °C plate. A two-tailed unpaired student’s t-test was used for statistical analysis. Mean with SD. ACLT, anterior cruciate ligament transection. OARSI score, Osteoarthritis Research Society International scoring system

Article Snippet: These animals received knee injection under anesthesia following approval from the Ethical Review Board of West China Hospital Sichuan University (No. 2018147A) and were divided into three groups: intra-articular injection of Hyaluronate acid (mean Mol. wt: 0.8~1.2 million, ARTZ@Dispo, JP), Sema3A protein (R&D SYSTEMS, 2150-S3) and lentivirus Rlv-CMV-sema3a-Efla-EGFP (BrainVTA, Wuhan, China) (n= 12, 4 monkeys in each group, 2 knees tested per monkey).

Techniques: Knock-Out, Control, Two Tailed Test, Staining

Journal: Bone research

Article Title: Neuronal guidance factor Sema3A inhibits neurite ingrowth and prevents chondrocyte hypertrophy in the degeneration of knee cartilage in mice, monkeys and humans.

doi: 10.1038/s41413-024-00382-0

Figure Lengend Snippet: Fig. 3 Bidirectional regulation of Sema3A expression in chondrocytes regulates both nerve ingrowth and chondrocyte hypertrophy. a Schematic program of the generation of chondrosphere and dorsal root ganglion (DRG) neurosphere. Sema3A was knocked down in the chondrocytes by siRNA-Sema3A. b Schematic program of the generation of chondrosphere and DRG neurosphere. Sema3A was overexpressed in the chondrosphere by lentivirus CMV-Sema3A-GFP. c The co-culture of chondrospheres and neurospheres, and morphological changes of neuritis growth in the normal control group (left) with normal Sema3A expression in the chondrosphere, Sema3AKD

Article Snippet: These animals received knee injection under anesthesia following approval from the Ethical Review Board of West China Hospital Sichuan University (No. 2018147A) and were divided into three groups: intra-articular injection of Hyaluronate acid (mean Mol. wt: 0.8~1.2 million, ARTZ@Dispo, JP), Sema3A protein (R&D SYSTEMS, 2150-S3) and lentivirus Rlv-CMV-sema3a-Efla-EGFP (BrainVTA, Wuhan, China) (n= 12, 4 monkeys in each group, 2 knees tested per monkey).

Techniques: Expressing, Co-Culture Assay, Control

Journal: Bone research

Article Title: Neuronal guidance factor Sema3A inhibits neurite ingrowth and prevents chondrocyte hypertrophy in the degeneration of knee cartilage in mice, monkeys and humans.

doi: 10.1038/s41413-024-00382-0

Figure Lengend Snippet: Fig. 4 Local administration of Sema3A agents in the articular cartilage arrest degeneration in ACLT mice. a, b 3D reconstruction of micro-CT and Kellgren–Lawrence analysis of the joint space in OA (n = 10), Sema3A protein (n = 10), Sema3A overexpressed virus (OS virus) (n = 10), Anti-Sema3A antibody (n = 10), Sema3A low-expressed virus (DS virus) (n = 10) mice groups. Statistical significance was determined by multifactorial ANOVA. All data are reported as the mean ± SD. c, e, f H&E staining, Safranin O staining, and OARSI scores of the OA (n = 10), Sema3A protein (n = 10), OS virus (n = 10), Anti-Sema3A antibody (n = 10), DS virus (n = 10) mice groups. Statistical significance was determined by multifactorial ANOVA. All data are reported as the mean ± SD. Scale bar, 100 µm. A representative result from five independent experiments is shown. d, g Immunofluorescence staining of Collagen X in OA (n = 8), Anti-Sema3A antibody (n = 8), Sema3A protein (n = 8), OS virus (n = 8), Sema3A DS virus (n = 8) mice groups. Statistical significance was determined by multifactorial ANOVA. All data are reported as the mean ± SD. Scale bar, 50 µm. A representative result from five independent experiments is shown. h, k Immunofluorescence staining of Tau in OA (n = 8), Anti-Sema3A antibody (n = 8), Sema3A protein (n = 8), OS virus (n = 8), DS virus (n = 8) mice groups. Statistical significance was determined by multifactorial ANOVA. All data are reported as the mean ± SD. Scale bar, 50 µm. A representative result from five independent experiments is shown. i, l Immunofluorescence staining of Sema3A in OA (n = 8), Anti-Sema3A antibody (n = 8), Sema3A protein (n = 8), OS virus (n = 8), DS virus (n = 8) mice groups. Statistical significance was determined by multifactorial ANOVA. All data are reported as the mean ± SD. Scale bar, 50 µm. A representative result from five independent experiments is shown. j, m Immunofluorescence staining of F-actin in OA (n = 8), Anti-Sema3A antibody (n = 8), Sema3A protein (n = 8), OS virus (n = 8), DS virus (n = 8) mice groups. Statistical significance was determined by multifactorial. ANOVA. All data are reported as the mean ± SD. Scale bar, 50 µm. A representative result from five independent experiments is shown. OARSI score, Osteoarthritis Research Society International scoring system

Article Snippet: These animals received knee injection under anesthesia following approval from the Ethical Review Board of West China Hospital Sichuan University (No. 2018147A) and were divided into three groups: intra-articular injection of Hyaluronate acid (mean Mol. wt: 0.8~1.2 million, ARTZ@Dispo, JP), Sema3A protein (R&D SYSTEMS, 2150-S3) and lentivirus Rlv-CMV-sema3a-Efla-EGFP (BrainVTA, Wuhan, China) (n= 12, 4 monkeys in each group, 2 knees tested per monkey).

Techniques: Micro-CT, Virus, Staining

Journal: Bone research

Article Title: Neuronal guidance factor Sema3A inhibits neurite ingrowth and prevents chondrocyte hypertrophy in the degeneration of knee cartilage in mice, monkeys and humans.

doi: 10.1038/s41413-024-00382-0

Figure Lengend Snippet: Fig. 5 Local administration of Sema3A agents in the articular cartilage arrests the knee joint degeneration in natural OA Rhesus macaques. a, c Representative X-ray images and Kellgren–Lawrence analysis of the joint space in hyaluronate acid (HA) (n = 8), Sema3A protein (n = 8), Sema3A overexpressed virus (OS Virus) (n = 8) Rhesus macaques joint groups. Statistical significance was determined by multifactorial ANOVA. All data are reported as the mean ± SD. b, d OARSI scores for the HA (n = 8), Sema3A protein (n = 8), OS Virus (n = 8) Rhesus macaques groups. Statistical significance was determined by multifactorial ANOVA. All data are reported as the mean ± SD. A representative result from three independent experiments is shown. Scale bar, 50 µm. e–l Immunofluorescence staining of Col X (e, i), Tau (f, j), Sema3A (g, k), and F-actin (h, l) in HA group (n = 8), Sema3A protein group (n = 8), OS Virus group (n = 8) and Rhesus macaques group. All data are reported as the mean ± SD. A representative result from three independent experiments was shown. e, f, h Scale bar, 50 µm. g Scale bar, 25 µm. OARSI score, Osteoarthritis Research Society International scoring system

Article Snippet: These animals received knee injection under anesthesia following approval from the Ethical Review Board of West China Hospital Sichuan University (No. 2018147A) and were divided into three groups: intra-articular injection of Hyaluronate acid (mean Mol. wt: 0.8~1.2 million, ARTZ@Dispo, JP), Sema3A protein (R&D SYSTEMS, 2150-S3) and lentivirus Rlv-CMV-sema3a-Efla-EGFP (BrainVTA, Wuhan, China) (n= 12, 4 monkeys in each group, 2 knees tested per monkey).

Techniques: Virus, Staining

Journal: Bone research

Article Title: Neuronal guidance factor Sema3A inhibits neurite ingrowth and prevents chondrocyte hypertrophy in the degeneration of knee cartilage in mice, monkeys and humans.

doi: 10.1038/s41413-024-00382-0

Figure Lengend Snippet: Fig. 7 Neuronal guidance factor Sema3A plays a dual-functional role in the degeneration of knee cartilage. Schematic summarizing the mechanism of Sema3A exerting a dual-functional role in the knee degeneration process of mouse and Rhesus macaque OA models, and in patients with OA. Sema3A expression in chondrocytes regulates both nerve ingrowth and cartilage hypertrophy. (Created in BioRender)

Article Snippet: These animals received knee injection under anesthesia following approval from the Ethical Review Board of West China Hospital Sichuan University (No. 2018147A) and were divided into three groups: intra-articular injection of Hyaluronate acid (mean Mol. wt: 0.8~1.2 million, ARTZ@Dispo, JP), Sema3A protein (R&D SYSTEMS, 2150-S3) and lentivirus Rlv-CMV-sema3a-Efla-EGFP (BrainVTA, Wuhan, China) (n= 12, 4 monkeys in each group, 2 knees tested per monkey).

Techniques: Functional Assay, Expressing

Journal: In vitro cellular & developmental biology. Animal

Article Title: The role of semaphorin 3A on chondrogenic differentiation.

doi: 10.1007/s11626-024-00909-z

Figure Lengend Snippet: Figure 4. Effects of Sema3A on the ability to synthesize hyaluronan. To evaluate the amount of hyaluronan synthesized during differentia- tion of ATDC5 supplemented with Sema3A, culture supernatants on days 7, 9, 11, and 13 were collected and the amount of hyaluronan was measured. Negative control (n.c); No statistical difference (n.s.) (N = 12).

Article Snippet: Sema3A application To examine the effect of Sema3A on chondrogenic differentiation, ATDC5 cells were treated with Sema3A (1, 10, and 100 ng/mL; recombinant mouse Sema3A Fc chimera; R&D Systems Inc., Minneapolis, MN) at the time of medium change on the seventh day and the cells are cultured for 24 h. Representative data were obtained from four samples from each group.

Techniques: Synthesized, Negative Control

Journal: International heart journal

Article Title: Intravenous Semaphorin 3A Administration Maintains Cardiac Contractility and Improves Electrical Remodeling in a Mouse Model of Isoproterenol-Induced Heart Failure.

doi: 10.1536/ihj.22-705

Figure Lengend Snippet: Figure 1. Macroscopic and histopathological findings. A: The first line shows representative examples of the macroscopic findings. The second line shows HE staining. The third line shows Masson trichrome-stained sections, and the fourth line shows the tyrosine hydroxylase-stained sec- tions of the myocardium. B: Mean cardiomyocyte diameter. C: The ratio of the fibrous tissue area to the area of the myocardial cross-section. D: Tyrosine hydroxylase-positive area relative to the mean value in the control group, defined as 1. ISP indicates isoproterenol; Sema3A, semaphorin 3A; and HE, hematoxylin-eosin. *P < 0.05 versus control. ‡P < 0.05 versus ISP.

Article Snippet: A subset of mice in the ISP-treated group received 0.5 mg/kg BW of recombinant Sema3A (recombinant mouse Sema3A Fc chimera; R&D Systems, Minneapolis, MN, USA), diluted 10-fold with phosphate-buffered saline, on days 7 and 11 via the tail vein.

Techniques: Staining, Control

Journal: International heart journal

Article Title: Intravenous Semaphorin 3A Administration Maintains Cardiac Contractility and Improves Electrical Remodeling in a Mouse Model of Isoproterenol-Induced Heart Failure.

doi: 10.1536/ihj.22-705

Figure Lengend Snippet: Figure 2. Echocardiographic findings and changes in electrophysiological parameters. A: Echocardiographic findings on day 14. a: Typical M-mode image in the short-axis view at the level of the left ventricular papillary muscle. b–f: Statistical analyses of LVDd (b), LVDs (c), IVSth (d), PWth (e), and FS (f) of the left ventricle in each group. B: Changes in the electrophysiological parameters. a: Representative MAP traces. b–d: Statistical analyses of MAPD20 (b), MAPD90 (c), and ERP (d) values in the experimental groups. ISP indicates isoproterenol; Sema3A, semaphorin 3A; LVDd, left ventricular diastolic diameters; LVDs, left ventricular systolic diameters; IVSth, thickness of the interventricular septum; PWth, thickness of the left ventricular posterior wall; FS, fractional shortening of the left ventricle; MAP, monophasic action potential; MAPD20, mono- phasic action potential duration at 20% repolarization; MAPD90, monophasic action potential duration at 90% repolarization; and ERP, effective refractory period. *P < 0.05 versus control. ‡P < 0.05 versus ISP.

Article Snippet: A subset of mice in the ISP-treated group received 0.5 mg/kg BW of recombinant Sema3A (recombinant mouse Sema3A Fc chimera; R&D Systems, Minneapolis, MN, USA), diluted 10-fold with phosphate-buffered saline, on days 7 and 11 via the tail vein.

Techniques: Control

Journal: International heart journal

Article Title: Intravenous Semaphorin 3A Administration Maintains Cardiac Contractility and Improves Electrical Remodeling in a Mouse Model of Isoproterenol-Induced Heart Failure.

doi: 10.1536/ihj.22-705

Figure Lengend Snippet: Figure 3. Western blot analyses of the expression of Kv4.3, KChIP2, SERCA2, and GAPDH. A: Representative bands corresponding to these proteins in control, ISP, and ISP + Sema3A groups. B-D: Mean densities of bands corresponding to Kv4.3 (B), KChIP2 (C), and SERCA2 (D). Bands were quantified relative to the mean values in each group, with the mean value of the control group set as 1. ISP indi- cates isoproterenol; Sema3A, semaphorin 3A; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KChIP2, K+ channel-interacting pro- tein-2; and SERCA2, sarcoendoplasmic reticulum Ca2+-ATPase 2. *P < 0.05 versus control. ‡P < 0.05 versus ISP.

Article Snippet: A subset of mice in the ISP-treated group received 0.5 mg/kg BW of recombinant Sema3A (recombinant mouse Sema3A Fc chimera; R&D Systems, Minneapolis, MN, USA), diluted 10-fold with phosphate-buffered saline, on days 7 and 11 via the tail vein.

Techniques: Western Blot, Expressing, Control