Fhl1, a new spatially specific protein, regulates vein graft neointimal hyperplasia

Figures

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  FIGURE 1. Time‐course analysis of NIH during the nascent‐NIH phase in vein grafts. (A) Schematic representation of the experimental procedure (n = 5 rats per time point). (B) HE, Masson, Resorcin–Fuchsin (RF), and α‐SMA immunohistochemistry (IHC) staining of vein grafts at sequential time points (control, hour 4, Day 1, Day 3, and Day 7) during the nascent‐NIH phase (n = 5 rats per time point). C‐Med: media tunica of the control external jugular vein; C‐Adv: adventitia of the control external jugular vein; Fb: fibrin; G‐Med: media tunica of the vein graft; G‐Adv: adventitia of the vein graft; L: lumen. Scale bar = 100 µm.

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  FIGURE 2. Time‐course analysis of NIH during the NIH phase in vein grafts. HE, Masson, RF, and α‐SMA IHC staining of vein grafts at consecutive time points (Day 11, Day 14, Day 19, Day 21, Day 28, and Day 35) during the NIH phase (n = 5 rats per time point). G‐Int: neointima of the vein graft; G‐Med: media tunica of the vein graft; G‐Adv: adventitia of the vein graft; L: lumen. Dotted line represents the internal elastic lamina. Scale bar = 100 µm.

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  FIGURE 3. Cellular injury in vein grafts following grafting. (A) Resorcin–Fuchsin staining shows disruption of the internal elastic lamina (IEL) due to vessel distension, indicated by arrows. Scale bar = 100 µm. (B) IHC analysis demonstrates endothelial cell (ERG+) damage and subsequent restoration after grafting, with arrows marking ERG‐stained endothelial cells. Scale bar = 100 µm. (C) IHC analysis reveals inflammation (MPO+), indicated by arrows highlighting neutrophils stained by MPO. Scale bar = 50 µm. Panels A–C, n = 5 rats per time point; L: lumen. (D) Line graph depicting changes in vein graft diameter. (E) Line graph showing the number of endothelial cells over time. (F) Line graph of MPO staining intensity in vein grafts. (G) Representative immunofluorescence (IF) images showing DAPI (blue), α‐SMA (red), and TUNEL (green) staining in vein grafts at control, hour 4, Day 1, and Day 3 (n = 5 rats per time point). The merged image highlights apoptotic smooth muscle cells (arrow). L: lumen. Dotted line indicates the medial layer structure. Scale bar = 50 µm. (H) Quantification of IF staining for medial α‐SMA‐expressing areas in vein grafts. (I) Proportion of TUNEL(+) cells relative to…

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  FIGURE 4. Cellular repair in vein grafts postgrafting. (A) Masson and α‐SMA IHC staining illustrating boundary changes and the presence of repair cells in vein grafts at different time points (control, hour 4, Day 1, Day 3, and Day 7) (n = 5 rats per time point). The dotted line marks the boundary of the control external jugular vein after arterial grafting. C‐Med: media tunica of the control external jugular vein; C‐Adv: adventitia of the control external jugular vein; Fb: fibrin; G‐Med: media tunica of the vein graft; G‐Adv: adventitia of the vein graft; L: lumen. Scale bar = 200 µm. (B) Quantification of α‐SMA IHC staining across different spatial regions at consecutive time points. (C) Schematic representation of neointimal thickness based on Masson staining across time points. Scale bar = 50 µm. Line represents the internal elastic lamina (IEL). (D) Changes in the neointimal area at different time points (top panel). Neointimal cell count at consecutive time points (middle panel). Neointimal area‐to‐lumen area ratio at consecutive time points (bottom panel). n = 5 rats per time point. Statistical analysis indicated a significant difference between Day 19 and Day 14, showing that…

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  FIGURE 5. Spatial transcriptomics atlas and gene expression profiling in control and Day 19 graft veins. (A) Workflow schematic for spatial transcriptomics (ST). (B) The final analysis included nine control veins and nine Day 19 vein grafts. ST spots from unbiased clustering were overlaid on each vessel (biologically independent). Ctrl‐med: media tunica of the control external jugular vein; Ctrl‐adv: adventitia of the control external jugular vein; D19‐int: neointima of the Day 19 vein graft; D19‐med: media tunica of the Day 19 vein graft; D19‐adv: adventitia of the Day 19 vein graft. Scale bar = 100 µm. (C) Principal component analysis revealed significant deviations in gene expression profiles between control veins and Day 19 vein grafts, with PCA descriptors used for statistical measurement. (D) Transcriptome proximity analysis comparing spatial regions between control veins and Day 19 grafts, with the Pearson's correlation coefficient quantifying statistical correlations. (E) Representative pie chart showing cell‐type proximity maps and associated cell populations in spatial RNA‐seq data. (F) Volcano plot highlighting key DEGs (Mylk, Myh11, Tagln, Tpm1, Tpm2, Flna, Myl6, Myl9, Pdlim3,…

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  FIGURE 6. Differentially expressed genes (DEGs) in the neointima and GO/KEGG analyses. (A) Heatmap showing upregulated DEGs in the neointima (adjusted p ≤ .05; fold change ≥ 2). The red frame marks the 29 upregulated DEGs selected for IHC validation. (B) Volcano plot of DEGs in the neointima. (C) GO analysis of DEGs clustered into biological process (blue), cellular component (green), and molecular function (orange), based on the Cluster Profiler package in R software. (D) KEGG pathway enrichment analysis illustrating the primary biological functions of the DEGs.

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  FIGURE 7. Time course of Fhl1 expression in the neointima of vein grafts. (A) IF staining of Fhl1 protein expression in Day 19 vein grafts. Scale bar = 500 µm. (B) IHC staining of Fhl1 protein expression during the nascent‐NIH phase. Scale bar = 100 µm. (C) IHC staining of Fhl1 protein expression during the NIH phase (left panel). Scale bar = 100 µm. Quantification of Fhl1 expression in the neointima versus the media (right panel). Int: neointima of vein graft; Med: media tunica of vein graft; dotted line indicates the IEL. (D) Quantification of Fhl1 expression in the neointima over consecutive time points. Panels B–D, n = 5 rats per time point. (E) IHC staining of Day 14 vein grafts showing cells migrating from the tunica media to the neointima and beginning to express Fhl1 protein (arrow). (F) Representative IF images of DAPI (blue), Fhl1 (red), α‐SMA (yellow), and Vimentin (green) staining in the neointima of grafts. Scale bar = 50 µm. p‐values correspond to two‐tailed unpaired t‐test for panel C; one‐way ANOVA with Tukey's multiple comparisons test for panel D. ns: not significant; *p < .05, ***p < .001, ****p < .0001.

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  FIGURE 8. Fhl1 deficiency exacerbates neointima formation in vivo. (A) Strategy for CRISPR/Cas9‐mediated genome editing of the Fhl1 gene (left panel). Genotype detection by PCR using rat tail snip (right panel). (B) HE staining of the external jugular vein and Fhl1 expression detected by IHC in Day 28 vein grafts from WT and Fhl1‐KO rats (n = 5 rats per group) (left panel). Quantification of the media area of the external jugular vein and Fhl1 average staining intensity in the neointima of Day 28 grafts (right panel). (C) Representative images (left) and quantification (right) of IF or IHC for PI3K, PCNA, VCAM‐1, CD68 in the grafts, and serum TNF‐α levels in WT and Fhl1‐KO rats at Day 14 postgrafting (n = 6 rats per group). (D) Representative IF images of DAPI (blue), PI3K (red), PCNA (green), PI3K (red) with VCAM‐1 (green), and CD68 (green) with VCAM‐1 (yellow) staining in the neointima of Fhl1 KO grafts. Scale bar = 50 µm. (E) Cross‐sections of HE‐stained vein grafts (left) and quantification of neointima area (right) in WT and Fhl1‐KO rats at Day 14 (n = 6 rats per group) and Day 28 (n = 8 rats per group) postgrafting. Dotted area represents the neointima. L: lumen. Scale bar = 100 µm.…

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  FIGURE 9. FHL1 overexpression inhibits NIH in cultured human saphenous veins (HSV). (A) Representative images (left) and quantification (right) of EGFP fluorescence in HSV rings infected with varying concentrations of virus: 1×1010 PFU/mL, 4×1010 PFU/mL, 7×1010 PFU/mL, and 1×1011 PFU/mL (n = 3 HSV rings per group). (B) IF detection of FHL1 expression in Day 14 HSV rings from the Ad‐NC and Ad‐FHL1 groups (left panel). Quantification of FHL1 regional staining intensity in Day 14 HSV rings (n = 3 HSV rings per group) (right panel). (C) Cross‐sections of HE and RF‐stained HSV rings (left) and quantification of neointima area (right) in Ad‐NC and Ad‐FHL1 groups on Day 14 (n = 3 HSV rings per group). Arrow indicates the internal elastic lamina. Int: neointima of HSV; Med: media tunica of HSV. (D) Representative images (left) and quantification (right) of PCNA immunofluorescence in HSV rings from the Ad‐NC and Ad‐FHL1 groups on Day 14 (n = 3 HSV rings per group). Arrows indicate PCNA(+) cells. Scale bar = 50 µm or 100 µm. L: lumen. ns: not significant, *p < .05, **p < .01, ***p < .001.