Journal: Bioactive Materials

Article Title: Apolipoprotein E knockout attenuates vascular graft fibrosis by reducing profibrotic macrophage formation through low-density lipoprotein receptor related protein 1

doi: 10.1016/j.bioactmat.2026.01.029

Figure Lengend Snippet: Involvement of profibrotic macrophages in vascular regeneration after graft implantation in vivo . (a) UMAP of macrophages in native aortas and regenerated aortas across different time points after graft implantation in vivo . (b) Dot plots of profibrotic macrophage marker genes (Ctsd, Spp1, Gpnmb, Lgals3, and Fabp5) expressed in different subgroups of macrophages. (c) Percentage of cluster 2 (C2) macrophages in native aortas and regenerated aortas across different time points after graft implantation in vivo . (d) UMAP of expression of Ctsd, Spp1, Gpnmb, Lgals3, and Fabp5 in macrophages in native aortas and regenerated aortas across different time points after graft implantation in vivo . (e) Immunofluorescence staining of CD68 and CTSD in regenerated aortas across different time points after graft implantation in vivo . L indicates lumens. Arrow heads indicate double positively stained cells. (f) WB results of levels of CTSD and SPP1 in native and regenerated aortas across different time points after graft implantation in vivo and quantification of the levels of CTSD and SPP1. ∗∗ indicates p < 0.01, Tukey's post-hoc test. For each time point and each group, five different samples from five different animals were analyzed (n = 5).

Article Snippet: The following primary antibodies were used in this study: APOE (Abcam, ab183597, 1:1000 dilution), COL I (abcam, ab270993, 1:1000 dilution), FN (abcam, ab268020, 1:1000 dilution), CTSD (CST, 74089S, 1:1000 dilution), SPP1 (NeoBiotechnologies, 6696-RBM3-P0, 1:1000 dilution), and LRP1 (Invitrogen, PA5-101013, 1:1000 dilution).

Techniques: In Vivo, Marker, Expressing, Immunofluorescence, Staining

Journal: Bioactive Materials

Article Title: Apolipoprotein E knockout attenuates vascular graft fibrosis by reducing profibrotic macrophage formation through low-density lipoprotein receptor related protein 1

doi: 10.1016/j.bioactmat.2026.01.029

Figure Lengend Snippet: APOE KO reducing profibrotic macrophage formation during vascular regeneration. (a) UMAP of macrophages in native aortas from WT and Apoe −/− rats, heatmap of C2 scores in the UMAP of macrophages in the native aortas, and percentage of C2 cells in macrophages in the native aortas. UMAP of macrophages in regenerated aortas after graft implantation in WT and Apoe −/− rats, heatmap of C2 scores in the UMAP of macrophages in the regenerated aortas, and percentage of C2 cells in macrophages in the regenerated aortas on Day 30 (b) and Day 90 (c). (d) Immunofluorescence staining of CD68 and CTSD in regenerated aortas 30 and 90 days after graft implantation in WT and Apoe−/− rats. (e) Quantification of CD68 and CTSD double positive cells in regenerated aortas on Day 30 and Day 90. ∗∗ indicates p < 0.01, Tukey's post-hoc test. For each time point and each group, five different images from five different samples were analyzed (n = 5). (f) WB results of APOE, CTSD and SPP1 levels in regenerated aortas after graft implantation in WT and Apoe −/− rats for 30 and 90 days. (g) Quantification of levels of APOE, CTSD and SPP1 in regenerated aortas on Day 30 and Day 90. ∗∗ indicates p < 0.01, Tukey's post-hoc test. For each time point and each group, five different samples from five different animals were analyzed (n = 5). (h) WB results of APOE, CTSD and SPP1 levels in WT and APOE KO macrophages after their culture on PCL scaffolds for 48 h. (i) Quantification of levels of APOE, CTSD and SPP1 in WT and APOE KO macrophages after their culture on PCL scaffolds for 48 h ∗ indicates p < 0.05, ∗∗ indicates p < 0.01, unpaired t -test. For each time point and each group, three different samples were analyzed (n = 3). (j) Immunofluorescence staining of APOE and CD68, CTSD and CD68, SPP1 and CD68, respectively, in WT and APOE KO macrophages after their culture on PCL scaffolds for 48 h.

Article Snippet: The following primary antibodies were used in this study: APOE (Abcam, ab183597, 1:1000 dilution), COL I (abcam, ab270993, 1:1000 dilution), FN (abcam, ab268020, 1:1000 dilution), CTSD (CST, 74089S, 1:1000 dilution), SPP1 (NeoBiotechnologies, 6696-RBM3-P0, 1:1000 dilution), and LRP1 (Invitrogen, PA5-101013, 1:1000 dilution).

Techniques: Immunofluorescence, Staining

Journal: Bioactive Materials

Article Title: Apolipoprotein E knockout attenuates vascular graft fibrosis by reducing profibrotic macrophage formation through low-density lipoprotein receptor related protein 1

doi: 10.1016/j.bioactmat.2026.01.029

Figure Lengend Snippet: APOE/LRP1 interaction promoting profibrotic macrophage formation during vascular regeneration after graft implantation in vivo . (a) Immunoprecipitation (IP) following mass spectrometry (MS) to screen potential receptors of APOE on surfaces of macrophages. (b) Co-immunoprecipitation (Co-IP) to confirm interaction between APOE and LRP1. (c) Immunofluorescence staining of CD68 and LRP1 in regenerated aortas across different time points. (d) Immunofluorescence staining of APOE and LRP1 in WT macrophages 48 h after their culture on PCL scaffolds. (e) WB results of LRP1, APOE, CTSD and SPP1 levels in WT macrophages cultured on tissue culture plates (negative control, NC) or PCL scaffolds (PCL) for 48 h prior to treatment with shRNA ADV-shRNA(NC) or ADV-shRNA(Lrp1) for 24 h. Quantification of levels of LRP1 (f), APOE (g), CTSD (h) and SPP1 (i) in WT macrophages cultured on tissue culture plates or PCL scaffolds treated with shRNA ADV-shRNA(NC) or ADV-shRNA(Lrp1). ∗ indicates p < 0.05, ∗∗ indicates p < 0.01, N.S. indicates non-significant. Tukey's post-hoc test. For each group, three different samples were analyzed (n = 3). (j) Flow cytometry analysis of CTSD positive cells in WT macrophages cultured on tissue culture plates (negative control, NC) or PCL scaffolds (PCL) for 48 h prior to treatment with ADV-shRNA(NC) or ADV-shRNA(Lrp1) for 24 h and quantification of percentage of CTSD positive cells in WT macrophages in each group. ∗ indicates p < 0.05, Tukey's post-hoc test. For each group, three independent experiments were repeated, and results were analyzed (n = 3).

Article Snippet: The following primary antibodies were used in this study: APOE (Abcam, ab183597, 1:1000 dilution), COL I (abcam, ab270993, 1:1000 dilution), FN (abcam, ab268020, 1:1000 dilution), CTSD (CST, 74089S, 1:1000 dilution), SPP1 (NeoBiotechnologies, 6696-RBM3-P0, 1:1000 dilution), and LRP1 (Invitrogen, PA5-101013, 1:1000 dilution).

Techniques: In Vivo, Immunoprecipitation, Mass Spectrometry, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Cell Culture, Negative Control, shRNA, Flow Cytometry

Journal: Bioactive Materials

Article Title: Apolipoprotein E knockout attenuates vascular graft fibrosis by reducing profibrotic macrophage formation through low-density lipoprotein receptor related protein 1

doi: 10.1016/j.bioactmat.2026.01.029

Figure Lengend Snippet: Downregulation of APOE by AAV ameliorating fibrosis during vascular regeneration after graft implantation in vivo . (a) Illustration of a strategy of adventitial delivery of AAV-shRNA(Apoe) to inhibit APOE levels in regenerated aortas after graft implantation in vivo . Two weeks after graft implantation in vivo , AAV-shRNA(Apoe) were injected into the adventitia of the regenerated aortas, which were then harvested for analysis three weeks later. (b) M mode images of ultrasound detection of regenerated aortas treated with PBS, AAV-shRNA(NC), and AAV-shRNA(Apoe) for 3 weeks. Arrow heads indicate movement of vascular walls. (c) Tensile tests of regenerated aortas treated with PBS, AAV-shRNA(NC), and AAV-shRNA(Apoe) for 3 weeks. (d) Quantification of RI, PI, and compliance of regenerated aortas treated with PBS, AAV-shRNA(NC), and AAV-shRNA(Apoe) for 3 weeks. ∗∗ indicates p < 0.01, Tukey's post-hoc test. For each group, six different images from six different animals were analyzed (n = 6). (e) Quantification of elastic modulus of regenerated aortas treated with PBS, AAV-shRNA(NC), and AAV-shRNA(Apoe) for 3 weeks. ∗∗ indicates p < 0.01, Tukey's post-hoc test. For each group, six different images from six different animals were analyzed (n = 6). (f) H&E, MTC and EVG staining of regenerated aortas treated with PBS, AAV-shRNA(NC), and AAV-shRNA(Apoe) for 3 weeks. (g) Immunofluorescence staining of COL I, COL III, elastin, αSMA, and eNOS in regenerated aortas treated with PBS, AAV-shRNA(NC), and AAV-shRNA(Apoe) for 3 weeks. L indicates lumens. Arrow heads indicate capillaries. Quantification of adventitia thickness (h), collagen positive areas according to MTC staining (i), elastin positive areas according to EVG staining (j), COL I positive areas (k), COL III positive areas (l), and number of capillaries (m) in adventitial areas of regenerated aortas. (n) Immunofluorescence staining of CTSD and CD68 in regenerated aortas treated with PBS, AAV-shRNA(NC), and AAV-shRNA(Apoe) for 3 weeks. (o) CD68 and CTSD double positive cells in regenerated aortas. ∗∗ indicates p < 0.01, Tukey's post-hoc test. For each group, six different samples from six different animals were analyzed (n = 6). (p) WB results of APOE, CTSD and SPP1 levels in regenerated aortas treated with PBS, AAV-shRNA(NC), and AAV-shRNA(Apoe) for 3 weeks and quantification of levels of APOE, CTSD and SPP1 in regenerated aortas. ∗∗ indicates p < 0.01, Tukey's post-hoc test. For each group, six different samples from six different animals were analyzed (n = 6). (q) Quantification of IGF-1 concentrations in regenerated aortas treated with PBS, AAV-shRNA(NC), and AAV-shRNA(Apoe) for 3 weeks by ELISA. ∗∗ indicates p < 0.01, Tukey's post-hoc test. For each group, six different samples from six different animals were analyzed (n = 3).

Article Snippet: The following primary antibodies were used in this study: APOE (Abcam, ab183597, 1:1000 dilution), COL I (abcam, ab270993, 1:1000 dilution), FN (abcam, ab268020, 1:1000 dilution), CTSD (CST, 74089S, 1:1000 dilution), SPP1 (NeoBiotechnologies, 6696-RBM3-P0, 1:1000 dilution), and LRP1 (Invitrogen, PA5-101013, 1:1000 dilution).

Techniques: In Vivo, shRNA, Injection, Staining, Immunofluorescence, Enzyme-linked Immunosorbent Assay

Journal: Journal of Nanobiotechnology

Article Title: Synergistic microenvironment therapy: a dual-function nanogel of supramolecular collagen ll-mimetic matrix and antioxidant MXene for attenuating intervertebral disc degeneration

doi: 10.1186/s12951-026-04211-w

Figure Lengend Snippet: The Composite Hydrogel Substrate Fosters a Pro-Anabolic and Anti-Catabolic Microenvironment for Adherent Nucleus Pulposus Cells. ( A ) Schematic of the in vitro culture model where NPCs are seeded on different hydrogel formulations. ( B ) Histochemical staining for matrix deposition by NPCs cultured on different hydrogels, including Alcian Blue (top row) and Toluidine Blue (bottom row) staining. Scale bar: 200 μm. ( C ) Western blot analysis of anabolic (COL-2, ACAN) and catabolic (MMP-13, ADAMTS-4) protein markers in NPCs cultured on different hydrogel substrates. GAPDH served as a loading control. ( D ) Dot plot visualization of the relative protein expression levels from Fig. 3E The size and color intensity of each dot correspond to the expression level. (E) Western blot analysis of key proteins in the integrin-mediated signaling pathway (p-FAK, p-ERK, p-p38). ( F ) Dot plot visualization of the relative protein expression levels from Fig. 3E. ( G ) Western blot analysis of anabolic and catabolic markers in the presence of an integrin α2β1 blocking antibody (iα2β1). ( H ) Dot plot visualization of the relative protein expression levels from Fig. 3G. ( I ) Representative immunofluorescence images for Collagen II (green), MMP-13 (red), and Aggrecan (green) in NPCs cultured on different hydrogels. Cell nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. ( J ) Quantitative analysis of the relative fluorescence intensity for COL-2, MMP-13, and ACAN from the images in Fig. 3G. Data are presented as mean ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, not significant

Article Snippet: After treatment, cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 5% bovine serum albumin for 1 h. Cells were then incubated overnight at 4 °C with primary antibodies against MMP-13, COL-II, ACAN, GPX4 and SLC7A11 (1:100; ProteinTech, Wuhan, China).

Techniques: In Vitro, Staining, Cell Culture, Western Blot, Control, Expressing, Blocking Assay, Immunofluorescence, Fluorescence

Journal: Journal of Nanobiotechnology

Article Title: Synergistic microenvironment therapy: a dual-function nanogel of supramolecular collagen ll-mimetic matrix and antioxidant MXene for attenuating intervertebral disc degeneration

doi: 10.1186/s12951-026-04211-w

Figure Lengend Snippet: The Mo 2 C@SAPH-R 16 GFOGER Hydrogel Attenuates IVDD Progression and Inhibits Ferroptosis In Vivo. ( A ) Photograph of the intra-discal injection procedure. ( B ) Schematic of the in vivo experimental design. ( C ) Representative X-ray images of rat tail intervertebral discs after 4 weeks and 8 weeks. ( D ) Representative T2-weighted MRI scans of the discs after 4 weeks and 8 weeks. ( E ) Quantitative analysis of the disc height index (DHI). ( F ) Quantitative analysis of the Pfirrmann grade. ( G ) Immunohistochemical staining for ACAN, COL-2, and ACSL4 in disc tissues at 8 weeks. Scale bar: 200 μm. ( H )Immunofluorescence staining for GPX4 in disc tissues at 8 weeks. Scale bar: 200 μm. ( I ) Quantitative analysis of glycosaminoglycan (GAG) content using DMMB assay. ( J ) Photograph of the biomechanical compression testing setup. ( K ) Quantitative analysis of the compressive modulus of the motion segments. Data are presented as mean ± SD( n = 5). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, not significant

Article Snippet: After treatment, cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 5% bovine serum albumin for 1 h. Cells were then incubated overnight at 4 °C with primary antibodies against MMP-13, COL-II, ACAN, GPX4 and SLC7A11 (1:100; ProteinTech, Wuhan, China).

Techniques: In Vivo, Injection, Immunohistochemical staining, Staining, Immunofluorescence, Dimethylmethylene Blue Assay