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: Profibrotic macrophages increasing fibroblast proliferation via their secreted IGF-1. (a) Dot plots of Igf1 expression in C2 and the other subgroups of macrophages in regenerated aortas 30 and 90 days after graft implantation in WT and Apoe −/− rats. (b) Igf1 expression score in macrophages in regenerated aortas 30 and 90 days after graft implantation in WT and Apoe −/− rats. ∗∗∗∗ indicates p < 0.0001, unpaired t -test. (c) Quantification of IGF-1 concentration in regenerated aortas 30 and 90 days after graft implantation in WT and Apoe −/− rats. ∗∗ 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). UMAP of fibroblasts in regenerated aortas 30 days (d) and 90 days (e) after graft implantation in WT and Apoe −/− rats, heatmap of cell cycle (Ccnd1, Ccnd2, and Ccnd3) scores in UMAP of fibroblasts, and box plots of cell cycle scores in fibroblasts. ∗∗∗∗ indicates p < 0.0001, unpaired t -test. (f) Immunofluorescence staining of Ki67 in regenerated aortas 30 and 90 days after graft implantation in WT and Apoe −/− rats. L indicates lumens. (g) Quantification of Ki67 positive cells in regenerated aortas. ∗∗ indicates p < 0.01, Tukey's post-hoc test. For each time point and each group, five different images from five different animals were analyzed (n = 5). (h) Quantification of IGF-1 in culture mediums of WT and APOE KO macrophages after their culture on PCL scaffolds for 48 h by ELISA. ∗∗ indicates p < 0.01, unpaired t -test. For each group, three different samples were analyzed (n = 3). (i) Immunofluorescence staining of Ki67 in WT and APOE KO fibroblasts after treatment with IGF-1 (10 ng/mL) for 24 h. Cells were counterstained with phalloidin. (j) Quantification of proliferation of WT and APOE KO fibroblasts treated with IGF-1 (10 ng/mL) for 24 h using cell counting kit-8 (CCK-8). ∗∗ indicates p < 0.01, Tukey's post-hoc test. For each group, three different samples were analyzed (n = 3). (k) Quantification of proliferation of WT fibroblasts treated with conditioned medium (CM) with or without IGF-1 blocking antibody (Ab, 1 μg/mL) for 24 h using CCK-8. CM were medium conditioned by WT macrophages cultured on PCL scaffolds for 48 h ∗∗ indicates p < 0.01, Tukey's post-hoc test. For each group, three different samples were analyzed (n = 3).

Article Snippet: Exogenous APOE (0.25 μg/mL, MCE, HY-P701096), TGF-β1 (10 ng/mL, MCE, HY-P7117), IGF-1 (10 ng/mL, MCE), conditioned medium by macrophages, or IGF-1 blocking antibody (1 μg/mL, Invitrogen, MA5-18035) was added into culture medium and incubated with WT or APOE KO fibroblasts for 24 h.

Techniques: Expressing, Concentration Assay, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Cell Counting, CCK-8 Assay, Blocking Assay, Cell Culture

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: Exogenous APOE (0.25 μg/mL, MCE, HY-P701096), TGF-β1 (10 ng/mL, MCE, HY-P7117), IGF-1 (10 ng/mL, MCE), conditioned medium by macrophages, or IGF-1 blocking antibody (1 μg/mL, Invitrogen, MA5-18035) was added into culture medium and incubated with WT or APOE KO fibroblasts for 24 h.

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

Journal: Kidney360

Article Title: Arginase-1 Expressing Macrophages Interact with Myofibroblasts to Regulate Insulin-Like Growth Factor-1-Dependent Tubule Repair

doi: 10.34067/KID.0000001150

Figure Lengend Snippet: Igf1 is upregulated in an Arg1 -dependent manner in the outer medulla after kidney injury. Arg1 WT and Arg1mko were subjected to IRI/CL-NX and kidneys harvested and microdissected for analysis on day 2. (A) Igf1 mRNA levels in the outer medulla of healthy kidneys and Arg1mko kidneys, n =8 mice per group (B). Immunofluorescence staining of outer medullary sections of Arg1 WT healthy, Arg1 WT injured, and Arg1mko injured kidneys with phospho-IGF1R (red), LTL (green), and DAPI (blue). (C) MFI of phospho-IGF1R signals as shown in (B) were quantified using ImageJ, with five high power fields/kidney section analyzed/group ( n =4 mice). (D) Analysis of mRNA expression of Igf1 in MACS sorted F4/80 + macrophages; ( n =5 mice). (E) Analysis of mRNA expression of Igf1 by naïve, Arg1 low BMDM (transwell coculture of BMDM with BMDM) versus alternatively activated, Arg1 high BMDM (transwell coculture of BMDM with PCRC+GM-CSF, [ n =3]). (F) IGF1 protein concentrations determined by ELISA in the 24 hours conditioned media collected from the conditions described in (E) showing the secreted IGF1 protein levels in MՓ CM1 (naïve, Arg1 low macrophages) and MՓ CM2 (alternatively activated, Arg1 high macrophages) ( n =3/group). P values are shown, and data are presented as mean±SEM. Arg1, arginase-1; Arg1 mko , macrophage-specific Arg1 null; BMDM, bone marrow-derived macrophages; DAPI, 4′,6-diamidino-2-phenylindole; GM-CSF, granulocyte-monocyte colony stimulating factor; IGF1R, IGF1 receptor; IRI, ischemia-reperfusion kidney injury; IRI-CL/NX, ischemia-reperfusion with contralateral nephrectomy; LTL, Lotus tetragonolobus lectin; MACS, magnetic activated cell sorting; MFI, mean fluorescence intensity; MՓ CM, macrophage-derived conditioned medium; PCRC, primary cultured renal cells; pIGF1R, phosphorylated IGF1R; WT, wild type

Article Snippet: IGF1 ELISA assays were performed using Mouse IGF1 ELISA kit (Cat. # ELM-IGF1-1, RayBiotech), and GH ELISA assays were performed by Human GH ELISA Kit (Cat. # ELH-GH-1, RayBiotech).

Techniques: Immunofluorescence, Staining, Expressing, Enzyme-linked Immunosorbent Assay, Derivative Assay, FACS, Fluorescence, Cell Culture

Journal: Kidney360

Article Title: Arginase-1 Expressing Macrophages Interact with Myofibroblasts to Regulate Insulin-Like Growth Factor-1-Dependent Tubule Repair

doi: 10.34067/KID.0000001150

Figure Lengend Snippet: Igf1 is expressed by outer medullary myofibroblasts and endothelial cells. (A) Igf1 expression is greatest in human kidney myofibroblasts based on scRNA-seq of human kidney biopsy samples from publicly available KPMP datasets. (B) scRNA-seq from healthy mouse kidneys confirms that Igf1 expression is highest in myofibroblasts. (C) Igf1 mRNA levels in MACS sorted PDGFR β - and PDGFR β + cell populations from the outer stripe of healthy and injured kidneys, n =5 mice per group. (D) UMAP visualization of identified cell clusters present in freshly isolated PDGFR β + cells from mouse outer stripe 2 days post-IRI. (E) Heatmap showing Igf1 expression across different clusters with highest expression in endothelial cells and myofibroblasts. (F and G) Igf1 levels in FACS-sorted PDGFR β lo and PDGFR β hi cells (F) and PECAM lo and PECAM hi cells (G) isolated from day 2 IRI kidneys, n =4 mice per group. P values are shown, and data are presented as mean±SEM. IRI, ischemia-reperfusion injury; KPMP, Kidney Precision Medicine Project; PDGFR, platelet derived growth factor receptor; PECAM, platelet endothelial cell adhesion molecule-1; scRNA-seq, single-cell RNA sequencing; UMAP, Uniform Manifold Approximation and Projection

Article Snippet: IGF1 ELISA assays were performed using Mouse IGF1 ELISA kit (Cat. # ELM-IGF1-1, RayBiotech), and GH ELISA assays were performed by Human GH ELISA Kit (Cat. # ELH-GH-1, RayBiotech).

Techniques: Expressing, Isolation, Clinical Proteomics, Derivative Assay, Single Cell, RNA Sequencing

Journal: Kidney360

Article Title: Arginase-1 Expressing Macrophages Interact with Myofibroblasts to Regulate Insulin-Like Growth Factor-1-Dependent Tubule Repair

doi: 10.34067/KID.0000001150

Figure Lengend Snippet: Arg1+ macrophage conditioned media induces myofibroblasts and endothelial cells to produce Igf1 in vitro . (A) scRNA-seq analysis of cultured PCRC cells demonstrates that myofibroblasts increase Igf1 mRNA expression in coculture with WT macrophages. (B) PTEC express the highest level of Igf1r after coculture with macrophages. (C) quantitative PCR analysis shows that cultured MACS-enriched PDGFR β + cells express high levels of Igf1 mRNA in response to culture in MՓ CM2 compared with MՓ CM1 and MՓ CM3, n =5. (D) PDGFR β -enriched cells secrete high levels of IGF1 protein in response to culture in MՓ CM2, n =7. (E and F) HUVEC endothelial cells significantly increase Igf1 mRNA expression (E) and IGF1 protein secretion (F) when treated with MՓ CM2 compared with MՓ CM1 and MՓ CM3, n =5. P values are shown, and data are presented as mean±SEM. HUVEC, human umbilical vein endothelial cells; MAC, macrophages; PTEC, proximal tubular epithelial cells; TEC, other tubular epithelial cells.

Article Snippet: IGF1 ELISA assays were performed using Mouse IGF1 ELISA kit (Cat. # ELM-IGF1-1, RayBiotech), and GH ELISA assays were performed by Human GH ELISA Kit (Cat. # ELH-GH-1, RayBiotech).

Techniques: In Vitro, Cell Culture, Expressing, Real-time Polymerase Chain Reaction

Journal: Kidney360

Article Title: Arginase-1 Expressing Macrophages Interact with Myofibroblasts to Regulate Insulin-Like Growth Factor-1-Dependent Tubule Repair

doi: 10.34067/KID.0000001150

Figure Lengend Snippet: Myofibroblast conditioned media activates the IGF1 receptor and tubular proliferation. (A) Immunofluorescence staining of cultured PCRCs exposed to myofibroblast CM1 (MF CM1, induced by Arg1 low macrophages) or MF CM2 (induced by Arg1 high macrophages) for 15 minutes. 10 ng/ml IGF1 stimulation was used as positive control. The activated IGF1 receptor (green) was detected using anti–phospho-IGF1R antibody and proximal tubule cells identified using AQP1 (red) and nuclei by DAPI staining (blue). (B) Representative densitometry of the images as in (A) showing mean fluorescence intensity, n =4. (C) Representative immunoblot performed on lysates from PCRCs treated with the indicated CM showing pIGF1R, with total IGF1R and GAPDH as loading controls, and (D) corresponding bar graph showing the quantification normalized to total IGF1R, n =3. (E) Ki67 staining of PCRCs exposed to the indicated myofibroblast CM for 24 hours (MF CM3=induced by Arg1 MKO macrophages). 10 ng/ml IGF1 was used as the positive control. (F) Quantification of the percentage of Ki67 positive cells as in (E), n =3 (G) Immunofluorescence costaining of Ki67 and LTL in the outer medulla of WT control kidneys ( Arg1 WT healthy), WT kidneys on day 2 post-IRI ( Arg1 WT Injured), and Arg1 mko kidneys on day 2 post-IRI (Arg1 mko Injured). DAPI staining of nuclei is shown (blue). Arrows indicate Ki67+ nuclei of tubular cells from LTL+proximal tubules. (H) Quantification of numbers of Ki67+ nuclei adjacent to LTL+proximal tubules as in (G), n =5. P values are shown, and data are presented as mean±SEM. GAPDH, glyceraldehyde-3-phopshate dehydrogenase; MF CM, myofibroblast conditioned media.

Article Snippet: IGF1 ELISA assays were performed using Mouse IGF1 ELISA kit (Cat. # ELM-IGF1-1, RayBiotech), and GH ELISA assays were performed by Human GH ELISA Kit (Cat. # ELH-GH-1, RayBiotech).

Techniques: Immunofluorescence, Staining, Cell Culture, Positive Control, Fluorescence, Western Blot, Control

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: Schematic illustration of the senescence-regulatory mechanisms of the sulfated polysaccharide in the glucocorticoid-induced bone marrow microenvironment. Bone marrow senescence plays a critical role in the pathogenesis of osteonecrosis. Glucocorticoids act on bone marrow target cells—adipocytes—to initiate primary bone marrow senescence via triggering a positive feedback loop through the prostaglandin/PPARγ/INK signaling axis. Subsequently, these senescent adipocytes propagate SASP factors to adjacent healthy cells through paracrine signaling or direct cell–cell contact, leading to secondary senescence. Sulfated chitosan (SCS) reprograms the lineage commitment bias of LepR + MSCs by activating the IGF-1/PI3K/Akt/mTOR signaling cascade, suppressing adipogenic differentiation and lipid biosynthesis pathways. SCS attenuates the spread of primary adipocyte senescence into secondary senescence, limiting the progressive amplification of the senescence cascade. Ultimately, this strategy halts the onset of senescence-driven osteonecrosis at an early stage and preserves the functional stability of the bone marrow microenvironment.

Article Snippet: Furthermore, to explore the molecular mechanisms by which SCS regulates MSCs lineage bias, bone marrow supernatant was collected on day 7 following co-treatment with SCS and MPS, and ELISA assays for IGF-1 (R&D Systems, MG100) and BMP-2 (R&D Systems, DBP200) were performed as described above.

Techniques: Amplification, Functional Assay

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS modulates mesenchymal stem cell lineage bias via activation of the IGF-1/PI3K/Akt/mTOR signaling pathway. ( A ) Quantitative analysis of osteocyte morphology in the trabecular bone matrix of the bone marrow at week 6 after MPS treatment with or without SCS, in the presence of various neutralizing antibodies (NAbs) and antagonistic proteins. ( B ) ELISA analysis of IGF-1 and BMP-2 levels in the femoral bone marrow and peripheral serum at day 7 following SCS treatment under MPS conditions. ( C and D ) Western blot analysis of phospho-PI3K, phospho-Akt, and phospho-mTOR (C), as well as phospho-Smad1/5/8, phospho-ERK, and phospho-p38 (D), in CD45 − Ter119 − CD31 − LepR + MSCs after 15-min stimulation with conditioned medium (CM) derived from bone marrow fluid at day 7 following SCS treatment. ( E – G ) Representative flow cytometry plots (E, F) and quantitative analysis (G) of CD45 − CD31 − Sca-1 + CD24 − adipocyte progenitor cells (APCs), CD45 − CD31 − Sca-1 + CD24 + MSCs (E), and CD45 − CD31 − Sca-1 − PDGFRα + (Pα + ) osteoprogenitor cells (OPCs) (F) from femoral bone marrow at day 14 post-MPS induction with or without combined treatment using SCS and IGF-1 NAb or Noggin. ( H and I ) Representative SA-β-Gal staining images (green) of the femur (H), and corresponding quantification (I), at week 4 following MPS treatment with SCS in combination with IGF-1 NAb or DMH1. Insets show magnified views of bone marrow (BM) and trabecular bone matrix (TBM) regions. (Scale bars, 100 μm and 25 μm) ( J ) qPCR analysis of 12 senescence-associated markers in ex vivo femoral bone tissues at week 4 following MPS treatment with SCS in combination with IGF-1 NAb or DMH1. ( K ) Representative Oil Red O staining images of CD45 − Ter119 − CD31 − LepR + MSCs sorted from femurs at day 7 following MPS treatment with SCS in combination with LY294002 or LDN-193189, after in vitro adipogenic induction. (Scale bars, 50 μm and 25 μm) ( L and M ) γ-H2A.X and telomere-associated DNA damage foci (TAFs) co-localization analysis (L), and corresponding quantification (M), in CD45 − Ter119 − CD31 + arteriolar ECs sorted from femurs at day 28 following MPS treatment with SCS in combination with rapamycin or LDN-193189, using immuno-FISH staining. (Scale bars, 7 μm and 1 μm) ( N and O ) Sequential fluorescent labeling using calcein (N) and quantification of mineral apposition rate (O) in femurs treated with SCS and MPS for 4 weeks, with or without LY294002 and/or GW9662. (Scale bars, 50 μm) ( P ) ELISA analysis of five senescence-associated cytokines in femoral bone marrow at day 28 following MPS treatment with SCS in combination with rapamycin and/or T0070907. ( Q and R ) Representative t-distributed stochastic neighbor embedding (t-SNE) plots (Q) from flow cytometric analysis of CD45 − CD31 − Sca-1 + CD24 − APCs, CD45 − CD31 − Sca-1 + CD24 + MSCs, CD45 − CD31 − Sca-1 − Pα + OPCs, CD45 − Ter119 − CD31 + arteriolar ECs, and CD45 − Ter119 − Emcn + sinusoidal ECs at day 14 following MPS treatment with SCS in combination with IGF-1 and/or rosiglitazone, and quantitative analysis of APCs (R) ( S ) Heatmap showing the fluorescent intensity distribution of Lamin-B1 expression across five cellular subpopulations as identified in the t-SNE clustering plot. ∗ P < 0.05 vs. IgG (empty lacunae); # P < 0.05 vs. IgG (filled lacunae). ∗ P < 0.05 vs. SCS; # P < 0.05 vs. SCS + IGF-1 NAb. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using an unpaired two-tailed Student's t -test ( B ), or one-way ANOVA with Tukey's post hoc test ( A, G, I, J, O, P and R ).

Article Snippet: Furthermore, to explore the molecular mechanisms by which SCS regulates MSCs lineage bias, bone marrow supernatant was collected on day 7 following co-treatment with SCS and MPS, and ELISA assays for IGF-1 (R&D Systems, MG100) and BMP-2 (R&D Systems, DBP200) were performed as described above.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay, Flow Cytometry, Staining, Ex Vivo, In Vitro, Labeling, Expressing, Two Tailed Test