Journal: Advanced Science

Article Title: Atrophic Skeletal Muscle‐Derived Extracellular Vesicles Transfer miR‐125a‐5p to Inhibit Bone Formation in Osteoporosis during Aging

doi: 10.1002/advs.202515362

Figure Lengend Snippet: Aged‐SKM‐EVs are increased and taken up by osteoblasts in bone during aging. (A) Representative Hematoxylin and Eosin (H&E) staining of cross‐sections from the mid‐belly of soleus (SOL) muscles and quantification of muscle fiber cross‐sectional area (CSA). n = 6 . Scale bar, 100 µm. (B) Representative tetanic force curve of SOL muscles from 6‐, 12‐, 18‐, and 24‐month‐old mice and quantification of maximal specific force during tetanic stimulation. n = 6 . (C) Representative micro‐CT reconstructed images and quantitative analysis of bone mineral density (BMD), bone volume/total volume (BV/TV), trabecular number (Tb.N), and trabecular separation (Tb.Sp) at the distal femoral metaphysis. Scale bar, 100 µm (upper panels) and 500 µm (lower panels). n = 6 . (D) Representative images of double fluorochrome labeling with calcein green and xylenol orange at the distal femoral metaphysis and quantitative bone histomorphometric analysis of mineral apposition rate (MAR). Scale bar, 25 µm. n = 6 . (E) Representative immunohistochemical staining of osteocalcin (OCN) in distal femoral sections and quantification of osteoblast surface per bone surface (Ob.S/BS). Scale bar, 50 µm. n = 6 . (F) Individual correlations between SOL muscle fiber CSA and BMD, and between CSA and Ob.S/BS. n = 24 . (G) Western blot analysis and quantification of protein expression of RAB27A expression in SOL muscles. n = 3 . (H) Representative immunohistochemical staining and quantification of RAB27A‐positive areas in SOL muscles. Scale bar, 100 µm. n = 6 . (I) Representative transmission electron microscopy (TEM) images of SKM‐EVs. Scale bar, 1 µm. (J) Nanoparticle tracking analysis (NTA) showing particle size distribution and concentration of SKM‐EVs. (K) Western blot analysis of canonical EV markers in SKM‐EVs. (L) Distribution and quantification of eGFP‐labeled EVs in skeletal muscle from HSA Cre ;Cd63 ( loxp − eGFP ) mice. Scale bar, 100 µm. n = 3 . (M) Representative fluorescence images showing circulating eGFP + PKH26 + EVs in plasma from HSA Cre ;Cd63 (loxp‐eGFP) mice, and quantification of the number density of eGFP + PKH26 + EVs (per µm 2 ). Scale bar, 100 nm. n = 6 . (N) Representative immunofluorescence staining of OCN in femoral sections (left), quantitative colocalization analysis (middle), and quantification of the percentage of CD63 GFP + OCN + cells among total OCN + cells (right). Nuclei were co‐stained with DAPI. Scale bar, 100 µm. n = 3 . All data are presented as mean ± SEM. P values were determined by one‐way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test (A–E, G–H), or unpaired two‐tailed Student's t test (L–N). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The primary antibodies used included RAB27A (A23993, ABclonal), CD63 (ab252919, Abcam), OCN (bs‐0470R, Bioss USA), SIRT7 (12994‐1‐AP, Proteintech), and nSMase2 ( C01004 , Signalway Antibody).

Techniques: Staining, Muscles, Micro-CT, Labeling, Immunohistochemical staining, Western Blot, Expressing, Transmission Assay, Electron Microscopy, Concentration Assay, Fluorescence, Clinical Proteomics, Immunofluorescence, Two Tailed Test

Journal: Advanced Science

Article Title: Atrophic Skeletal Muscle‐Derived Extracellular Vesicles Transfer miR‐125a‐5p to Inhibit Bone Formation in Osteoporosis during Aging

doi: 10.1002/advs.202515362

Figure Lengend Snippet: Muscle‐specific inhibition of EV generation alleviates bone loss in aged mice. (A) Schematic diagram illustrating the experimental design. Aged mice were intravenously injected with RGDLTTP peptide‐modified liposome capsulating GW4869 every three days for 6 weeks. (B) Representative immunofluorescent staining for CD63 in TA muscle sections. Nuclei were co‐stained with DAPI. Scale bars, 100 µm. (C) Representative micro‐CT reconstructed images of femora. Scale bar, 100 µm (upper panels) and 500 µm (lower panels). (D) Micro‐CT analysis of BMD, Tb.N, Tb.Th and Tb.Sp at the distal femoral metaphysis. n = 6 . (E) Representative images of double fluorochrome labeling with calcein green and xylenol orange at the distal femoral metaphysis and quantitative bone histomorphometric analysis of MAR. Scale bar, 25 µm. n = 6 . (F) Representative immunohistochemical staining of OCN in distal femoral sections and quantification of Ob.S/BS. Scale bar, 50 µm. n = 6 . (G) Schematic diagram illustrating the experimental design of co‐culture preosteoblasts with control myotubes (CON), atrophic myotubes (ATR) or GW4869 pretreated atrophic myotubes (ATR+GW4869). Myotubes were treated with or without GW4869 (20 µм) before co‐culture. (H) NTA analysis of EVs derived from ATR myotubes treated with DMSO or GW4869. (I) qPCR analysis of osteogenic differentiation‐related genes in preosteoblast after co‐cultured with indicated myotubes. n = 3 . (J,K) Western blot analysis and quantification of osteogenic differentiation‐related protein expression in osteoblasts after co‐cultured with indicated myotubes. n = 3 . (L) Alkaline phosphatase (ALP) staining of osteoblasts after co‐cultured with indicated myotubes for 7 days, respectively. Scale bars, 500um. All data are presented as mean ± SEM. P values were determined by unpaired two‐tailed Student's t test (D‐F), or one‐way ANOVA followed by Tukey's multiple comparisons test (I, K). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The primary antibodies used included RAB27A (A23993, ABclonal), CD63 (ab252919, Abcam), OCN (bs‐0470R, Bioss USA), SIRT7 (12994‐1‐AP, Proteintech), and nSMase2 ( C01004 , Signalway Antibody).

Techniques: Inhibition, Injection, Modification, Staining, Micro-CT, Labeling, Immunohistochemical staining, Co-Culture Assay, Control, Derivative Assay, Cell Culture, Western Blot, Expressing, Two Tailed Test

Journal: Advanced Science

Article Title: Atrophic Skeletal Muscle‐Derived Extracellular Vesicles Transfer miR‐125a‐5p to Inhibit Bone Formation in Osteoporosis during Aging

doi: 10.1002/advs.202515362

Figure Lengend Snippet: Aged‐SKM‐EVs inhibit bone formation and osteogenic differentiation in vivo and in vitro. (A) Schematic illustration of the experimental design. Young mice were intravenously injected with Aged‐SKM‐EVs every three days for 4 weeks. (B) Representative immunofluorescent staining for PKH26 and OCN in Femur sections of young mice 24 h after intramuscular injection with PKH26‐labeled Aged‐SKM‐EVs. Nuclei were co‐stained with DAPI (blue). Scale bars, 50 µm. (C) Representative micro‐CT reconstructed images and quantitative analysis of BMD, Tb.N, Tb.Th and Tb.Sp at the distal femoral metaphysis. Scale bar, 100 µm (upper panels) and 500 µm (lower panels). n = 6 . (D) Representative images of double fluorochrome labeling with calcein green and xylenol orange at the distal femoral metaphysis and quantitative bone histomorphometric analysis of MAR. Scale bar, 25 µm. n = 6 . (E) Representative immunohistochemical staining of OCN in distal femoral sections and quantification of Ob.S/BS. Scale bar, 50 µm. n = 6 . (F) Schematic diagram illustrating the experimental design of preosteoblasts incubated with young SKM‐EVs or Aged‐SKM‐EVs. (G) Representative immunofluorescent staining for PKH26 and phalloidine in preosteoblasts after 24 h of incubation with PKH26‐labeled SKM‐EVs. Nuclei were co‐stained with DAPI (blue). Scale bars, 25 µm. (H) qPCR analysis of osteogenic differentiation‐related genes in preosteoblasts after incubated with young SKM‐EVs or Aged‐SKM‐EVs, respectively. n = 3 . (I,J) Western blot analysis and quantification of osteogenic differentiation‐related protein expression in preosteoblasts after incubated with young SKM‐EVs or Aged‐SKM‐EVs, respectively. n = 3 . (K) ALP staining of osteoblasts after incubated with young SKM‐EVs or Aged‐SKM‐EVs for 7 days, respectively. Scale bars, 500um. All data are presented as mean ± SEM. P values were determined by unpaired two‐tailed Student's t test (C‐E, H, J). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The primary antibodies used included RAB27A (A23993, ABclonal), CD63 (ab252919, Abcam), OCN (bs‐0470R, Bioss USA), SIRT7 (12994‐1‐AP, Proteintech), and nSMase2 ( C01004 , Signalway Antibody).

Techniques: In Vivo, In Vitro, Injection, Staining, Labeling, Micro-CT, Immunohistochemical staining, Incubation, Western Blot, Expressing, Two Tailed Test

Journal: Advanced Science

Article Title: Atrophic Skeletal Muscle‐Derived Extracellular Vesicles Transfer miR‐125a‐5p to Inhibit Bone Formation in Osteoporosis during Aging

doi: 10.1002/advs.202515362

Figure Lengend Snippet: miRNA cargo is responsible for Aged‐SKM‐EVs‐mediated inhibition bone formation in aged mice. (A) Schematic illustration of the experimental design. Aged Dicer floxp/floxp mice were intravenously injected with AAV‐MCK cre for 8 weeks. (B,C) qPCR analysis of Dicer expression and muscle‐specific miRNAs in muscle tissues. n = 6 . (D) Representative micro‐CT reconstructed images and quantitative analysis of BMD, Tb.N,Tb.Th and Tb.Sp at the distal femoral metaphysis. Scale bar, 100 µm (upper panels) and 500 µm (lower panels). n = 6 . (E) Representative images of double fluorochrome labeling with calcein green and xylenol orange at the distal femoral metaphysis and quantitative bone histomorphometric analysis of MAR. Scale bar, 25 µm. n = 6 . (F) Representative immunohistochemical staining of OCN in distal femoral sections and quantification of Ob.S/BS. Scale bar, 50 µm. n = 6 . All data are presented as mean ± SEM. P values were determined by an unpaired two‐tailed Student's t test (B‐F). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The primary antibodies used included RAB27A (A23993, ABclonal), CD63 (ab252919, Abcam), OCN (bs‐0470R, Bioss USA), SIRT7 (12994‐1‐AP, Proteintech), and nSMase2 ( C01004 , Signalway Antibody).

Techniques: Inhibition, Injection, Expressing, Micro-CT, Labeling, Immunohistochemical staining, Staining, Two Tailed Test

Journal: Advanced Science

Article Title: Atrophic Skeletal Muscle‐Derived Extracellular Vesicles Transfer miR‐125a‐5p to Inhibit Bone Formation in Osteoporosis during Aging

doi: 10.1002/advs.202515362

Figure Lengend Snippet: Gain‐ and loss‐of‐function experiments demonstrate that skeletal muscle‐derived miR‐125a‐5p inhibits bone formation during aging. (A) Schematic illustration of the experimental design. Aged mice (20‐month‐old) were intravenously injected with recombinant MyoAAV‐NC, MyoAAV‐miR‐125a‐5p, or MyoAAV‐miR‐125a‐5p‐sponge. (B) Representative fluorescent images of skeletal muscle sections transduced with the indicated MyoAAV vectors. (C) qPCR analysis of miR‐125a‐5p expression in skeletal muscles of mice transduced with the indicated MyoAAV vectors. n = 6 . (D) Representative micro‐CT reconstructed images of femur. Scale bars, 500 µm. (E) Micro‐CT analysis of BMD, Tb.N, Tb.Th and Tb.Sp in the distal femoral metaphysis. n = 6 . (F) Representative double labelling with calcein green and xylenol orange in the distal femoral metaphysis. Scale bars, 25 µm. (G) Bone histomorphometric analysis of MAR in the distal femoral metaphysis. n = 6 . (H) Representative immunohistochemistry staining of OCN in the distal femur. Scale bar, 50 µm. (I) Quantification of Ob.S/BS at the distal femoral metaphysis. n = 6 . All data are presented as mean ± SEM. P values were determined by one‐way ANOVA followed by Tukey's multiple comparisons test (C, E, I). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The primary antibodies used included RAB27A (A23993, ABclonal), CD63 (ab252919, Abcam), OCN (bs‐0470R, Bioss USA), SIRT7 (12994‐1‐AP, Proteintech), and nSMase2 ( C01004 , Signalway Antibody).

Techniques: Derivative Assay, Injection, Recombinant, Transduction, Expressing, Muscles, Micro-CT, Immunohistochemistry, Staining