Journal: bioRxiv

Article Title: Fibroblasts neurotrophin signaling sustains pathological vascular maturation in rheumatoid arthritis

doi: 10.64898/2026.03.12.711120

Figure Lengend Snippet: (A) High-magnification spatial transcriptomic plots showing arterioles, venules, and capillaries in pre- and post-treatment RA synovial biopsy samples, colored by integrated vascular cell type annotations. The lower panels show high-magnification views of the same regions highlighting expression of NOTCH3 and neurotrophin receptors NGFR and NTRK2 in mural cells surrounding vascular structures. Relevant vascular regions are outlined.(B) RNAscope images showing transcript expression of the VSMC marker MYH11 (magenta) and neurotrophin receptors (B’) NGFR (yellow) and (B’’) NTRK2 (red) in RA synovial tissue sections, overlaid with DAPI (blue). Dotted lines indicate vascular structures defined by MYH11 expression.Scale bars, 150 µm. All images were acquired at 20x magnification and cropped to the same scale.(C) Immunohistochemistry images showing perivascular expression of neurotrophin receptors NGFR, NTRK1, NTRK2, and NTRK3 (brown) in RA synovial tissue. Scale bars, 150 µm. All images were acquired at 20x magnification and cropped to the same scale.

Article Snippet: Following transfer, membranes were blocked for 20 minutes in Everyblot blocking buffer (Bio-Rad # 12010020) and incubated overnight at 4°C with primary antibodies from TrkA and TrkB antibody sampler kit against TrkA, TrkB, p-TrkA/TrkB(Cell Signaling Technology, #4638, 1:500), CNN1 (Proteintech-24855-1-AP), MYH11 (Proteintech-21404-1-AP), NGF (Abcam-ab52918) and GAPDH (Thermo Fisher Scientific, #MA5-15738),or beta-actin (Cell Signaling Technology, #3700).

Techniques: Expressing, RNAscope, Marker, Immunohistochemistry

Journal: bioRxiv

Article Title: Fibroblasts neurotrophin signaling sustains pathological vascular maturation in rheumatoid arthritis

doi: 10.64898/2026.03.12.711120

Figure Lengend Snippet: (A) Schematic illustrating the endothelial cell–ibroblast–mural cell co-culture system.(B) ELISA quantiication of NGF secretion levels (pg/ml) in co-culture.(C) Distribution of RGS5+, MYH11+, and RGS5+/MYH11+ double-positive cells.(D) Proportion of cells in each quadrant, with quadrant boundaries deined by RGS5 and MYH11 expression as described in Methods.(E) Distance of RGS5+ pericytes, MYH11+ VSMCs, and RGS5+/MYH11+ double positive cells from the nearest endothelial cells.(F) Normalized expression of RGS5, MYH11, and PECAM1 relative to distance from the nearest endothelial cells.

Article Snippet: Following transfer, membranes were blocked for 20 minutes in Everyblot blocking buffer (Bio-Rad # 12010020) and incubated overnight at 4°C with primary antibodies from TrkA and TrkB antibody sampler kit against TrkA, TrkB, p-TrkA/TrkB(Cell Signaling Technology, #4638, 1:500), CNN1 (Proteintech-24855-1-AP), MYH11 (Proteintech-21404-1-AP), NGF (Abcam-ab52918) and GAPDH (Thermo Fisher Scientific, #MA5-15738),or beta-actin (Cell Signaling Technology, #3700).

Techniques: Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Expressing

Journal: bioRxiv

Article Title: Fibroblasts neurotrophin signaling sustains pathological vascular maturation in rheumatoid arthritis

doi: 10.64898/2026.03.12.711120

Figure Lengend Snippet: Human fibroblasts were co-cultured with human umbilical vein endothelial cells (HUVECs). Endothelial cells were identified by PECAM1 expression (magenta).(A) RNAscope images showing transcripts of VSMC marker MYH11(red) (A’) and the pericyte marker RGS5 (yellow) (A’’) and the in fibroblast-endothelial co-cultures, overlaid on DAPl(blue). Scale bar, 150 µm. All images were acquired at 20x magnification and cropped to the same scale. (B) RNAscope images showing transcripts of neurotrophin receptors NGFR, NTRK1, NTRK2, and NTRK3 (green) in the co-culture, overlaid on DAPl(blue). Enlarged cropped regions are shown in dotted boxes. Scale bar, 150 µm. All images were acquired at 20x magnification and cropped to the same scale.(C) Fibroblasts were treated with NOTCH3 siRNA and co-cultured with endothelial cells. RNAscope images show transcripts of NGFR (yellow) and NTRK2 (red) in the co-culture, overlaid on DAPl (blue). Enlarged cropped images are shown separately in dotted boxes. Scale bar, 150 µm. All images were acquired at 20x magnification and cropped to the same scale. (D-E) Fibroblasts were treated with siRNAs targeting NGFR, NTRK1, NTRK2, or NTRK3, in the presence or absence of DLL4 (5 µg/mL). (D) RGS5 mRNA expression following knockdown of NGFR or NTRK1. (E) MYH11 mRNA expression following knockdown of NTRK2 or NTRK3, in the presence or absence of DLL4. lndividual data points represent biological replicates; p values are indicated on the graphs. Statistical analysis was performed using a two tailed Student’s t test for comparisons between two groups and one-way ANOVA for comparisons among multiple groups. Post-hoc pairwise comparisons were conducted using t tests with Bonferroni correction to control for multiple comparisons.

Article Snippet: Following transfer, membranes were blocked for 20 minutes in Everyblot blocking buffer (Bio-Rad # 12010020) and incubated overnight at 4°C with primary antibodies from TrkA and TrkB antibody sampler kit against TrkA, TrkB, p-TrkA/TrkB(Cell Signaling Technology, #4638, 1:500), CNN1 (Proteintech-24855-1-AP), MYH11 (Proteintech-21404-1-AP), NGF (Abcam-ab52918) and GAPDH (Thermo Fisher Scientific, #MA5-15738),or beta-actin (Cell Signaling Technology, #3700).

Techniques: Cell Culture, Expressing, RNAscope, Marker, Co-Culture Assay, Knockdown, Two Tailed Test, Control

Journal: bioRxiv

Article Title: Fibroblasts neurotrophin signaling sustains pathological vascular maturation in rheumatoid arthritis

doi: 10.64898/2026.03.12.711120

Figure Lengend Snippet: (A-D) mRNA expression levels of NGFR (A), NTRK1 (B), NTRK2 (C), and NTRK3 (D) in fibroblasts treated with siRNAs targeting the indicated genes, demonstrating knockdown efficiency. (E-H) Fibroblasts were treated with siRNAs targeting NGFR, NTRK1, NTRK2, or NTRK3 in the presence or absence of DLL4 (5 µg/mL), and expression of mural cell markers was assessed. MYH11 mRNA expression following knockdown of NGFR (E) or NTRK1 (F), and RGS5 mRNA expression following knockdown of NTRK2 (G) or NTRK3 (H).lndividual data points represent biological replicates, with P values indicated. Statistical analysis was performed using a two-tailed Student’s t test for comparisons between two groups and one-way ANOVA for comparisons among multiple groups. Post hoc pairwise comparisons were conducted using t tests with Bonferroni correction to control for multiple comparisons.

Article Snippet: Following transfer, membranes were blocked for 20 minutes in Everyblot blocking buffer (Bio-Rad # 12010020) and incubated overnight at 4°C with primary antibodies from TrkA and TrkB antibody sampler kit against TrkA, TrkB, p-TrkA/TrkB(Cell Signaling Technology, #4638, 1:500), CNN1 (Proteintech-24855-1-AP), MYH11 (Proteintech-21404-1-AP), NGF (Abcam-ab52918) and GAPDH (Thermo Fisher Scientific, #MA5-15738),or beta-actin (Cell Signaling Technology, #3700).

Techniques: Expressing, Knockdown, Two Tailed Test, Control

Journal: bioRxiv

Article Title: Fibroblasts neurotrophin signaling sustains pathological vascular maturation in rheumatoid arthritis

doi: 10.64898/2026.03.12.711120

Figure Lengend Snippet: (A-C) Bar plots showing differential regulation of pericyte markers (RGS5, ABCC9), VSMC markers (CNN1, MYH11), and pan-mural cell markers (ACTA2, SMTN, TAGLN) in fibroblasts treated with (A) NGF, (B) BDNF, or (C) NT3 compared with untreated fibroblasts. Bar heights represent log₂ fold change, and bar color indicates the corresponding P values. (D) Schematic of the collagen gel contraction assay in which synovial fibroblasts were embedded in collagen I gels and treatment (E) Representative images of collagen I gel contraction following treatment with NGF, BDNF, or NT3 (F) Quantification of collagen I gel contraction expressed as percent contraction. Values represent mean ± standard deviation (SD). Individual data points indicate biological replicates. Statistical analysis was performed using one-way ANOVA, with P values shown. (G) Schematic illustrating the synovial organoid/explant experimental design. (H) Representative H&E image of synovial explant culture after 3 days of culture. (I) Whole-mount immunoluorescence images showing preservation of endothelial cells after 3 days in culture, marked by CD31 (magenta), and vascular smooth muscle cells marked by MYH11, surrounding CD31 vascular structures. (J) Whole-mount staining showing expression of neurotrophin receptors NGFR, NTRK1, NTRK2, and NTRK3 (green) around vascularature, together with CD31 (magenta) and DAPI (blue). (K) Synovial organoids embedded in Matrigel and treated with NGF, BDNF, or NT3, followed by fixation and staining for a-smooth muscle actin (aSMA) (brown) to visualize mural cells. All 20X images were cropped at similar scale. Scale bar, 150 µm.

Article Snippet: Following transfer, membranes were blocked for 20 minutes in Everyblot blocking buffer (Bio-Rad # 12010020) and incubated overnight at 4°C with primary antibodies from TrkA and TrkB antibody sampler kit against TrkA, TrkB, p-TrkA/TrkB(Cell Signaling Technology, #4638, 1:500), CNN1 (Proteintech-24855-1-AP), MYH11 (Proteintech-21404-1-AP), NGF (Abcam-ab52918) and GAPDH (Thermo Fisher Scientific, #MA5-15738),or beta-actin (Cell Signaling Technology, #3700).

Techniques: Collagen Gel Contraction Assay, Standard Deviation, Preserving, Staining, Expressing

Journal: bioRxiv

Article Title: Fibroblasts neurotrophin signaling sustains pathological vascular maturation in rheumatoid arthritis

doi: 10.64898/2026.03.12.711120

Figure Lengend Snippet: (A) RNAscope images showing transcript expression of pericyte marker RGS5 (yellow) and VSMC marker MYH11 (red) in fibroblasts treated with different neurotrophins, overlaid with DAPI (blue). Scale bar, 150 µm. All images were acquired at 20x magnification and cropped to the same scale.(B) Quantification of total numbers of RGS5+, MYH11+, and RGS5+/MYH11+ double-positive cells following treatment with the indicated neurotrophins.(C) mRNA expression of the pericyte marker RGS5 in fibroblasts transfected with siRNAs targeting NGFR or NTRK1, in the presence or absence of NGF stimulation.(D) mRNA expression of the VSMC marker MYH11 in fibroblasts transfected with siRNAs targeting NTRK2 or NTRK3, in the presence or absence of BDNF or NT3 stimulation, respectively.(E-F) mRNA expression of the mural cell marker CNN1 (calponin) in fibroblasts stimulated with NGF (E) in the presence or absence of a TRKA inhibitor, or stimulated with BDNF (F) in the presence or absence of an NTRK2 inhibitor.

Article Snippet: Following transfer, membranes were blocked for 20 minutes in Everyblot blocking buffer (Bio-Rad # 12010020) and incubated overnight at 4°C with primary antibodies from TrkA and TrkB antibody sampler kit against TrkA, TrkB, p-TrkA/TrkB(Cell Signaling Technology, #4638, 1:500), CNN1 (Proteintech-24855-1-AP), MYH11 (Proteintech-21404-1-AP), NGF (Abcam-ab52918) and GAPDH (Thermo Fisher Scientific, #MA5-15738),or beta-actin (Cell Signaling Technology, #3700).

Techniques: RNAscope, Expressing, Marker, Transfection

Journal: bioRxiv

Article Title: Fibroblasts neurotrophin signaling sustains pathological vascular maturation in rheumatoid arthritis

doi: 10.64898/2026.03.12.711120

Figure Lengend Snippet: (A) Synovial tissue explant were embedded in Matrigel and treated with the NOTCH inhibitor DAPT. Organoids were fixed and stained for a-smooth muscle actin (aSMA) to visualize mural cells. (B) Spatial transcriptomic (Xenium) analysis of synovial explants treated with DAPT, showing expression of PECAM1 (red), NOTCH3 (green), and (B’) NGF (blue). (C) Representative images of synovial explants treated with entrectinib or larotrectinib, showing aSMA staining to visualize mural cells. Scale bar, 650 µm. (D) RNAscope images showing expression of RGS5 (yellow), ACTA2 (red), and MYH11 (green), overlaid with DAPI (blue), following entrectinib or larotrectinib treatment. For MYH11 staining either serial section or different sections were used. (E) Quantification of aSMA-positive vascular structures per tissue section following entrectinib or larotrectinib treatment. (F) Quantification of integrated aSMA staining density in synovial explants treated with entrectinib or larotrectinib.(G) Quantification of PECAM1-positive vascular structures per tissue section from 20x images following entrectinib or larotrectinib treatment. Scale bar, 150 µm. All images were acquired at 20x magnification and cropped to the same scale. Values are presented as mean ± standard deviation (SD). Individual data points represent biological replicates. Statistical analysis was performed using a two-tailed Student’s t test for comparisons between two groups and one-way ANOVA for comparisons among multiple groups. Post hoc pairwise comparisons were conducted using t tests with Bonferroni correction to control for multiple comparisons.

Article Snippet: Following transfer, membranes were blocked for 20 minutes in Everyblot blocking buffer (Bio-Rad # 12010020) and incubated overnight at 4°C with primary antibodies from TrkA and TrkB antibody sampler kit against TrkA, TrkB, p-TrkA/TrkB(Cell Signaling Technology, #4638, 1:500), CNN1 (Proteintech-24855-1-AP), MYH11 (Proteintech-21404-1-AP), NGF (Abcam-ab52918) and GAPDH (Thermo Fisher Scientific, #MA5-15738),or beta-actin (Cell Signaling Technology, #3700).

Techniques: Staining, Expressing, RNAscope, Standard Deviation, Two Tailed Test, Control

Journal: JCI Insight

Article Title: Semaglutide reduces murine blood pressure through the vascular smooth muscle GLP-1 receptor

doi: 10.1172/jci.insight.201148

Figure Lengend Snippet: ( A – C ) Annotation maps of the human kidney single-cell transcriptome , showing expression of GLP1R , and vascular smooth muscle marker α1A adrenergic receptor ( ADRA1A ). ( D – F ) An annotation map of the mouse kidney single-cell transcriptome , showing expression of Glp1r and VSMC marker myosin heavy chain 11 ( Myh11 ). ( G – L ) Quantification of Glp1r expression relative to Rpl32 within the ( G ) kidney, ( H ) renal artery, ( I ) lung, ( J ) aortic arch, ( K ) abdominal aorta, and ( L ) carotid artery in control ( Glp1r VSM+/+ ) and Glp1r VSM–/– male mice (n = 5–14). ( M ) Double immunofluorescence staining of α-smooth muscle actin (left panel), GLP-1R (middle panel) and both GLP-1R and α-smooth muscle actin (right panel) in Glp1r VSM+/+ and Glp1r VSM–/– kidney. ( N ) Average food intake per mouse within each cage of group housed mice over 24 hours with or without acute treatment with semaglutide in Glp1r VSM+/+ and Glp1r VSM–/– mice ( n = 4–6). ( O – Q ) Body weight and oral glucose tolerance with calculated area under the curve in Glp1r VSM+/+ and Glp1r VSM–/– mice following acute semaglutide (10 μg/kg) treatment 120 minutes before oral glucose challenge (time 0) ( n = 18–23). Data are presented as mean ± SD. *** P ≤ 0.001 and **** P ≤ 0.0001 by unpaired t test ( G – L and O ), or 2-way ANOVA followed by Tukey post hoc tests ( N and Q ). Sema, semaglutide; Veh, vehicle. Original magnification, ×31.5.

Article Snippet: Deletion of Glp1r in VSMCs of mice with C57BL/6 background ( Glp1r VSM–/– ) were generated by crossing tamoxifen-inducible Myh11- Cre ER T2 mice ( ) (Jackson Laboratory, 019079) with Glp1r fl/fl mice ( , ), provided by R. Seeley, University of Michigan, Ann Arbor, Michigan, USA.

Techniques: Single Cell, Expressing, Marker, Control, Double Immunofluorescence Staining