FHL3 Regulates Vascular Smooth Muscle Cell Phenotypic Switching Through the MRTFB-SRF Signaling Axis

Figures

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  Figure 1. FHL3 represses contractile VSMC marker gene expression. (A) A schematic illustration of the pooled CRISPR-Cas9 nuclease knockout screen experimental procedure. Abbreviations: clustered regularly interspaced short palindromic repeats, CRISPR; CRISPR-associated protein 9, Cas9; single-guide RNA, sgRNA. (B) Volcano plot showing results of the pooled CRISPR-Cas9 nuclease knockout screen. Pink symbols indicate genes whose knockout increases MYH11 in VSMCs. (C) VSMCs were transduced with either a lentiviral vector containing a FHL3-targeting shRNA or a non-targeting control shRNA, followed by Western blot analysis of FHL3, MYH11, and TAGLN. Representative Western blot images are presented on the left. Column charts show means (±standard deviation) of MYH11 and TAGLN band intensities standardized against the vinculin band. Each plotted data point represents a technical replicate. p < 0.05 from two-sided Mann–Whitney U test, n = 4 technical replicates per condition. (D) Scatter plot showing an inverse correlation between FHL3 and MYH11 expression levels in VSMCs from different individuals from a VSMC biobank that we recently developed [4]. Each plotted data point represents an…

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  Figure 2. Influence of CAD-associated genetic variant on FHL3 expression in VSMCs. (A) UMAP (Uniform Manifold Approximation and Projection) visualization (left) and bubble plot (right) of FHL3 gene expression in various cell types in atherosclerotic arterial tissues (data from GSE253934 [31]), showing enrichment of FHL3 in vascular smooth muscle cells (SMC). (B) Scatter plot showing colocalization of CAD GWAS with VSMC FHL3 eQTL signals, supporting FHL3 as a candidate effector gene at this CAD locus. n = 1486 biological replicates for FHL3 expression data and n = 1,165,690 biological replicates for CAD GWAS data. (C) Box and whisker plot of FHL3 expression levels in VSMCs by genotype of the CAD-associated genetic variant rs28428561. Each plotted data point represents an independent biological replicate (donor-derived VSMC sample). Each box shows the median, first quartile and third quartile, while each whisker line shows the minimum and maximum values excluding outliers. p value from ANOVA (analysis of variance); n = 1486 biological replicates for both rs28428561 genotype and FHL3 expression data.

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  Figure 3. Influence of CAD-associated genetic variant on FHL3 expression in arterial tissues. (A) Scatter plot showing colocalization of CAD GWAS with arterial tissue FHL3 eQTL signals, supporting FHL3 as a candidate effector gene at this CAD locus. n = 268 (for coronary artery), 472 (for aorta) and 689 (tibial artery) biological replicates for FHL3 expression data and n = 1,165,690 biological replicates for CAD GWAS data. (B) Scatter plot of FHL3 expression levels in arterial tissues by genotype of the CAD-associated genetic variant rs67631072. The numbers of biological replicates are indicated in the figures.

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  Figure 4. FHL3 is associated with MRTFB in VSMCs. (A) A schematic illustration of a reported association between FHL3 and MRTFB based on STRING network analysis. (B). Results of co-immunoprecipitation assay showing that FHL3 and MRTFB are present within the same protein complex in VSMCs. Cell lysates were subjected to immunoprecipitation using either an anti-FHL3 antibody (left) or an anti-MRTFB antibody (right), followed by Western blot analysis using either the anti-FHL3 antibody or the anti-MRTFB antibody, as indicated in the figure. (C). Fluorescent immunocytochemical analysis showing subcellular colocalization of FHL3 (red) and MRTFB (green) in VSMCs. Nuclei are visualized by DAPI staining. Images shown are representative of three independent experiments. Colocalization was quantified using Pearson’s correlation coefficient with Costes automatic thresholding using the Coloc 2 plugin in Fiji.

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  Figure 5. FHL3 inhibits contractile VSMC marker gene expression by interfering MRTFB-SRF interaction. (A) VSMCs were transduced with either an empty lentiviral vector (LV-vector) to serve as a control, a lentiviral vector to express MRTFB (LV-MRTFB), or lentiviral vectors to express MRTFB (LV-MRTFB) and FHL3 (LV-FHL3), respectively, followed by Western blot analysis of MYH11 and TAGLN. (B) VSMCs were transduced with either a lentiviral vector to express a non-targeting short hairpin RNA (shRNA) to serve as a control, a lentiviral vector to express a shRNA to target MRTFB (MRTFB shRNA), or lentiviral vectors to express shRNAs to target MRTFB (MRTFB shRNA) and FHL3 (FHL3 shRNA), respectively, followed by Western blot analysis of MYH11 and TAGLN. (C) VSMCs transduced to either an empty lentiviral vector (control) or a lentiviral vector to express FHL3, were subjected to immunoprecipitation using an anti-MRTFB antibody, followed by Western blot analysis with antibodies against either MRTFB or SRF, as indicated in the figure. SRF band intensity was normalized to the corresponding SRF input signal. (A–C) Representative Western blot images are presented on the left. Column charts show mean…

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  Figure 6. FHL3 promotes VSMC proliferation and migration via MRTFB. (A) VSMCs were transduced with either a lentiviral vector to express a non-targeting short hairpin RNA (shRNA) to serve as a control or a lentiviral vector to express a shRNA to target FHL3 (FHL3 shRNA), followed by cell proliferation and migration assays. (B) VSMCs were transduced with either an empty lentiviral vector (LV-vector) to serve as a control, a lentiviral vector to express MRTFB (LV-MRTFB), or lentiviral vectors to express MRTFB (LV-MRTFB) and FHL3 (LV-FHL3), respectively, followed by cell proliferation assay and migration assay. (C) VSMCs were transduced with either a lentiviral vector to express a non-targeting short hairpin RNA (shRNA) to serve as a control, a lentiviral vector to express a shRNA to target MRTFB (MRTFB shRNA), or lentiviral vectors to express shRNAs to target MRTFB (MRTFB shRNA) and FHL3 (FHL3 shRNA), respectively, followed by cell proliferation and migration assays. (A–C) Cell proliferation was measured by a colorimetric assay with the use of Cell Counting Kit-8, and cell migration was measured by Transwell assay. Representative images of migrated cells stained with Hoechst 33342 are shown.…

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  Figure 7. Correlation of FHL3 expression with proliferation and migration of VSMCs. (A) A schematic illustration of experimental procedure. (B–E) Scatter plots showing a correlation of FHL3 expression levels with proliferation (B), migration speed (C) and migration distance (D), and no correlation with apoptosis (E) in VSMCs from different individuals from a VSMC biobank that we recently developed [4]. Proliferation, migration, and apoptosis were determined using an Operetta CLS High-Content Analysis System (PerkinElmer), following staining of the cells with 5-ethynyl-2′-deoxyuridine (EdU, in proliferation assay), CellTracker Green CMFDA (in migration assay), and Hoechst 33342 together with propidium iodide (in apoptosis assay), respectively, as described previously [4]. Each plotted data point represents an independent biological replicate (donor-derived VSMC sample). p value from linear correlation analysis; n = 1499 biological replicates for both FHL3 expression and VSMC proliferation/migration data. FPKM: fragments per kilobase of transcript per million mapped reads; r: correlation coefficient.

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  Figure 8. Schematic presentation of the key findings from this study. CAD-associated risk variants reduce expression of FHL3, which inhibits the up-regulation of the VSMC contractile genes MYH11 and TAGLN by interfering with the interaction of MRTFB and SRF. Alterations in the VSMC phenotype influence proliferation, migration, and extracellular matrix production, with context-dependent consequences for plaque development and stability. Abbreviations: coronary artery disease, CAD; myocardin-related transcription factor B, MRTFB; myosin heavy chain 11, MYH11; serum response factor, SRF; transgelin, TAGLN.