Journal: Journal of Advanced Research

Article Title: Discovery and identification of semaphorin 4D as a bioindicator of high fracture incidence in type 2 diabetic mice with glucose control

doi: 10.1016/j.jare.2025.03.014

Figure Lengend Snippet: Exendin-4 reduces Sema4D levels to elevate cortical strength in mice with type 2 diabetes mellitus (T2DM). (A) Schedule of enzyme-linked immunosorbent assay (ELISA) and the corresponding groups. (B) ELISA to determine the levels of ALP, Runx-2, OCN, OPN, and Sema4D in control mice, mice with T2DM, mice with T2DM treated with metformin (Met)/exendin-4 (Ex-4), and GLP-1R knockdown T2DM mice treated with Ex-4. n=6 mice per group. ALP: Alkaline Phosphatase; Runx-2: Runt-related transcription factor 2; OCN: Osteocalcin; OPN: Osteopontin; Sema4D: Semaphorin 4D. (C) Schedule of micro-CT to determine changes in trabecular bone. (D) Micro-CT images representing trabecular formation in control mice, mice with T2DM, mice with T2DM treated with Ex-4, mice with T2DM treated with anti-Sema4D, and mice with T2DM treated with a combination of Ex-4 and anti-Sema4D within a region of interest (ROI). n=6 mice per group. (E) Quantitative analysis of micro-CT images. BV: bone volume; TV: tissue volume; Tb.N: trabecular number; Tb.Th: trabecular thickness; Tb.Sp: trabecular spacing; BS/BV: bone superficial area/bone volume. n=6 mice per group. (F, G) Micro-CT images representing cortical bone formation in control mice, mice with T2DM, mice with T2DM treated with Ex-4, mice with T2DM treated with anti-Sema4D, and mice with T2DM treated with a combination of Ex-4 and anti-Sema4D within an ROI, and quantitative analysis of micro-CT images. n=6 mice per group. (H) Effects of exendin-4 or anti-Sema4D on the biomechanical properties of femoral diaphysis evaluated using the 3-point bending test. Femoral maximum load (N), stiffness (N/mm), maximum stress (MPa), and Young’s modulus (GPa). n=6 mice per group. *p < 0.05; **p < 0.01; ***p < 0.001. Data are presented as mean ± standard deviation.

Article Snippet: To figure out the effect of Sema4D on bone formation, mice were randomly divided into 5 groups (n = 6/group): control group, T2DM group, T2DM + Exendin-4 group (20 mg/kg, MCE,13241–33-3), T2DM + anti-Sema4D group (200 μg/kg, sc-390675), and T2DM + anti-Sema4D + Exendin-4 group.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Knockdown, Micro-CT, Standard Deviation

Journal: Journal of Advanced Research

Article Title: Discovery and identification of semaphorin 4D as a bioindicator of high fracture incidence in type 2 diabetic mice with glucose control

doi: 10.1016/j.jare.2025.03.014

Figure Lengend Snippet: The combination of metformin and exendin-4 can neutralize the effect of Sema4D. (A) Schematic chart of the sample preparation of micro-CT and 3-point bending experiments to determine the effect of the combination of metformin and exendin-4. (B) Micro-CT images representing trabecular formation in control mice and in mice treated with metformin, exendin-4, the combination of metformin and exendin-4, and anti-Sema4D within a region of interest (ROI), and quantitative analysis of micro-CT images. BV: bone volume; TV: tissue volume; Tb.N: trabecular number; Tb.Th: trabecular thickness; Tb.Sp: trabecular spacing; BS/BV: bone superficial area/bone volume. n=6 per group. (C) Micro-CT images representing cortical formation in control mice and in mice treated with metformin, exendin-4, the combination of metformin and exendin-4, and anti-Sema4D within an ROI, and quantitative analysis of micro-CT images. n=6 per group. (D) Effects of metformin, exendin-4, the combination of metformin and exendin-4, or anti-Sema4D on the biomechanical properties of femoral diaphysis evaluated using the 3-point bending test. Femoral maximum load (N), stiffness (N/mm), maximum stress (MPa), and Young’s modulus (GPa). n=6 per group. *p < 0.05; **p < 0.01; ***p < 0.001. Data are presented as mean ± standard deviation.

Article Snippet: To figure out the effect of Sema4D on bone formation, mice were randomly divided into 5 groups (n = 6/group): control group, T2DM group, T2DM + Exendin-4 group (20 mg/kg, MCE,13241–33-3), T2DM + anti-Sema4D group (200 μg/kg, sc-390675), and T2DM + anti-Sema4D + Exendin-4 group.

Techniques: Sample Prep, Micro-CT, Control, Standard Deviation

Journal: Journal of Advanced Research

Article Title: Discovery and identification of semaphorin 4D as a bioindicator of high fracture incidence in type 2 diabetic mice with glucose control

doi: 10.1016/j.jare.2025.03.014

Figure Lengend Snippet: Exendin-4 promotes pseudopodia numbers, leading to the stretching and wider spread of BMSCs via CRMP2. (A) Cell morphology observed using fluorescence microscopy. ImageJ was used to split color channels to observe the pseudopodium in human bone mesenchymal stem cells (hBMSCs). n=5 per group. White arrows indicate the pseudopodium in BMSCs. (B) Immunofluorescence staining to determine F-actin expression in BMSCs. n=5 per group. (C) Western blotting to determine F-actin expression in different groups. n=3 per group. (D) Proteomics analysis to determine the expression of different phosphorylated proteins. (E) Cell morphology observed using fluorescence microscopy. ImageJ was used to split color channels to observe the pseudopodium in BMSCs. n=5 per group. White arrows indicate the pseudopodium in BMSCs. (F) Immunofluorescence staining showing F-actin in different groups. n=5 per group. (G) Immunofluorescence staining indicating CRMP2 expression in BMSCs. n=5 per group. CRMP2: Collapsin Response Mediator Protein 2. (H) Western blotting to determine ALP, Runx-2, and Osterix expression in BMSCs. n=3 per group. (I) Alizarin red staining to analyze mineral deposits in BMSCs with DPYSL2 KD/OE. n=3 per group. DPYSL2 KD/OE: Dihydropyrimidinase like protein 2 knockdown/overexpression. (J) Western blotting to determine pCRMP2 expression in BMSCs with Sema4D or exendin-4. n=3 per group. (K) Immunofluorescence staining to determine pCRMP2 expression in BMSCs. n=5 per group. **p < 0.01; ***p < 0.001. Data are presented as mean ± standard deviation.

Article Snippet: To figure out the effect of Sema4D on bone formation, mice were randomly divided into 5 groups (n = 6/group): control group, T2DM group, T2DM + Exendin-4 group (20 mg/kg, MCE,13241–33-3), T2DM + anti-Sema4D group (200 μg/kg, sc-390675), and T2DM + anti-Sema4D + Exendin-4 group.

Techniques: Fluorescence, Microscopy, Immunofluorescence, Staining, Expressing, Western Blot, Knockdown, Over Expression, Standard Deviation

Journal: Journal of Advanced Research

Article Title: Discovery and identification of semaphorin 4D as a bioindicator of high fracture incidence in type 2 diabetic mice with glucose control

doi: 10.1016/j.jare.2025.03.014

Figure Lengend Snippet: Exendin-4 activates bone formation via the GLP-1R/PI3K/GSK-3β/CRMP2 signaling pathway. (A) Western blotting results of pCRMP2 and F-actin expression in BMSCs with GSK-3β knockdown or overexpression. n=3 per group. GSK-3β: Glycogen Synthase Kinase 3 beta. (B) Immunofluorescence staining of F-actin and pCRMP2 expression in BMSCs. n=5 per group. (C) Western blotting to determine pCRMP2 expression after Sema4D treatment with or without GSK-3β knockdown. n=3 per group. (D) Western blotting to determine pCRMP2 expression after exendin-4 treatment with or without GSK-3β knockdown. n=3 per group. (E) Western blotting to determine the expression of pGSK-3β, pCRMP2, and F-actin after Akt knockdown or overexpression. n=3 per group. Akt: Protein Kinase B/ Ak strain transforming. (F) Western blotting to determine the expression of pGSK-3β, pCRMP2, and F-actin after PI3K knockdown or overexpression. n=3 per group. PI3K: Phosphoinositide 3-Kinase. (G) Western blotting to determine the expression of pGSK-3β after exendin-4 treatment with or without PI3K knockdown and overexpression. n=3 per group. (H) Western blotting to determine P-PI3K expression after exendin-4 treatment with or without GSK-3β knockdown and overexpression. n=3 per group. (I) Western blotting to determine pGSK-3β expression after Sema4D treatment with or without PI3K knockdown and overexpression. n=3 per group. (J) Western blotting to determine pPI3K expression after Sema4D treatment with or without GSK–3β knockdown or overexpression. n=3 per group. *p < 0.05; **p < 0.01; ***p < 0.001. Data are presented as mean ± standard deviation.

Article Snippet: To figure out the effect of Sema4D on bone formation, mice were randomly divided into 5 groups (n = 6/group): control group, T2DM group, T2DM + Exendin-4 group (20 mg/kg, MCE,13241–33-3), T2DM + anti-Sema4D group (200 μg/kg, sc-390675), and T2DM + anti-Sema4D + Exendin-4 group.

Techniques: Western Blot, Expressing, Knockdown, Over Expression, Immunofluorescence, Staining, Standard Deviation

Journal: Journal of Advanced Research

Article Title: Discovery and identification of semaphorin 4D as a bioindicator of high fracture incidence in type 2 diabetic mice with glucose control

doi: 10.1016/j.jare.2025.03.014

Figure Lengend Snippet: Metformin modulates the expression of plexin B1 and GLP-1R in hBMSCs. (A) Differential expression of miRNAs among hBMSCs, hBMSCs+Sema4D, and hBMSCs+Sema4D+Met. MiR-140-3p and miR-3657 showing a significant increase and reduced expression in hBMSCs treated with Met. (B) Differential expression of miR-140-3p, plxnb1, and GLP-1R in different groups. n=3 per group. (C, D) Western blotting for Plexin B1 and GLP-1R expression in BMSCs, BMSCs+Sema4D, and BMSCs+Sema4D+Met. n=3 per group. (E) MiR-3657 expression in BMSCs, BMSCs+Sema4D, and BMSCs+Sema4D+Met. n=3 per group. (F, G) GLP-1R and Plexin B1 levels in BMSCs (con, miR-140-mimics/inhibitor, miR-3657-mimics/inhibitor). n=3 per group. (H) GLP-1R levels in BMSCs (con, miR-140-mimics, miR-140-mimics + miR-3657 inhibitor, miR-140-3p+miR-3657-mimics). n=3 per group.

Article Snippet: To figure out the effect of Sema4D on bone formation, mice were randomly divided into 5 groups (n = 6/group): control group, T2DM group, T2DM + Exendin-4 group (20 mg/kg, MCE,13241–33-3), T2DM + anti-Sema4D group (200 μg/kg, sc-390675), and T2DM + anti-Sema4D + Exendin-4 group.

Techniques: Expressing, Quantitative Proteomics, Western Blot

Journal: Journal of Advanced Research

Article Title: Sympathetic hyperinnervation drives abdominal aortic aneurysm development by promoting vascular smooth muscle cell phenotypic switching

doi: 10.1016/j.jare.2024.05.028

Figure Lengend Snippet: Elevated Sema4D expression in AAA. (A-C) Heatmap illustrating expression profiles of nerve guidance factors in aorta samples from Ang II-infused ApoE -/- mice and saline-infused ApoE -/- mice. (D) Dot plots showing expression of nerve guidance factors in macrophage from angiotensin II–induced AAA tissue (AAA) and normal aorta tissues (Normal). (E-F) Western blots and quantification analysis of Sema4D protein levels in human AAA samples and control samples ( n = 6/group) and aortas from Ang II-induced AAA tissues and saline-induced aorta tissues ( n = 6/group). (G-H) Representative immunohistochemical staining and corresponding quantification of Sema4D levels in Ang II-induced AAA models and control ( n = 5/group; scale bars = 200 and 50 µm). (I-J) Flow cytometry analysis of Sema4D + CD68 + cells dissociated from saline-induced aorta tissues and Ang II-induced AAA tissues ( n = 4/group). (K) Representative confocal images of Sema4D (red) colocalized with CD68 (green)–positive macrophages in aorta tissues from Ang II-infused or saline-infused ApoE -/- mice (scale bars = 50 µm). (L) Representative confocal images of Sema4D (red) colocalized with TRAP (green)–positive osteoclast-like cells in aorta tissues from Ang II-infused or saline-infused ApoE -/- mice (scale bars = 50 µm). (M) Secreted protein levels of Sema4D from conditional medium of macrophages treated with vehicle, TNF-α, CaPO 4 and TNF-α + CaPO 4 ( n = 5/group) . Parametric paired t test for Fig. 7F(left), parametric unpaired t test for F(right), 7H, 7J and one-way ANOVA with a post Bonferroni's multiple comparisons test for 7 M. ** P <0.01, *** P <0.001, **** P <0.0001.

Article Snippet: Sema4D knockout (Sema4D -/- ) mice were purchased from Cyagen, maintained in the animal facilities of Southern Medical University on standard rodent chow, and switched to a Western diet containing 0.2 % cholesterol by weight (TD.88137, Harlan Teklad) after AAV-PCSK9 injection.

Techniques: Expressing, Saline, Western Blot, Control, Immunohistochemical staining, Staining, Flow Cytometry

Journal: Journal of Advanced Research

Article Title: Sympathetic hyperinnervation drives abdominal aortic aneurysm development by promoting vascular smooth muscle cell phenotypic switching

doi: 10.1016/j.jare.2024.05.028

Figure Lengend Snippet: Sema4D deletion represses AAA formation by inhibiting sympathetic hyperinnervation. (A) Representative photographs showing macroscopic features of aorta. (B) The AAA incidence in Wildtype group ( n = 21) and Sema4D -/- group ( n = 22). (C) The survival curve from indicated group. (D) The measurements of maximum aortic diameter. (E) Representative macroscopic images of aorta sections stained with H&E, VVG and Masson trichrome (scale bars = 200 and 50 µm). (F) Elastin degradation score in aortas ( n = 10/group). (G) Percentage of collagen area in aortas ( n = 8/group). (H-I) Western blotting and quantification of GAP43 and TH in aortic tissues ( n = 6/group). ( J-K) Representative immunofluorescence images and analysis of β-Tubulin III in PC12 cells treated with monocyte-conditioned medium or osteoclast-conditioned medium, with or without the addition of functional blocking antibodies ( n = 6/group; scale bars = 25 µm). Mono-CM, monocyte-conditioned medium; OC-CM, osteoclast-conditioned medium; ab, antibody. (L-M) Representative immunofluorescence images and analysis of β-Tubulin III in rSema4D-treated PC12 with si-NC or si-Plxnb1 (scale bars = 25 µm; n = 6/group). Parametric unpaired t test for D, 8G, 8I, and 8M, Fisher's exact test for B, log-rank (Mantel-Cox) test for C, nonparametric Mann-Whitney U test for F, and One-way ANOVA with a post Bonferroni's multiple comparisons test for K. * P <0.05, ** P <0.01, **** P <0.0001.

Article Snippet: Sema4D knockout (Sema4D -/- ) mice were purchased from Cyagen, maintained in the animal facilities of Southern Medical University on standard rodent chow, and switched to a Western diet containing 0.2 % cholesterol by weight (TD.88137, Harlan Teklad) after AAV-PCSK9 injection.

Techniques: Staining, Western Blot, Immunofluorescence, Functional Assay, Blocking Assay, MANN-WHITNEY