Journal: Advanced Science

Article Title: Hypertensive Pressure Mechanosensing Alone Triggers Lipid Droplet Accumulation and Transdifferentiation of Vascular Smooth Muscle Cells to Foam Cells

doi: 10.1002/advs.202308686

Figure Lengend Snippet: Piezo1 localises to the VSMC nuclear lamina and controls calcium levels. Immunofluorescent micrographs shows enrichment of Piezo1 at the nuclear membrane of A7r5 VSMCs (A–C). A) Anti‐piezo1 staining shows a dotted appearance around the nuclear membrane (zoom in inset). B) Example line profile (line indicated in A) shows enrichment at the edges. C) Quantification of enrichment against cytoplasm and nuclear intensity. **** P <0.0001 from one‐sample t‐test, compared to a value of 1, i.e., no enrichment. D) The nuclear localisation is further confirmed using a Piezo1‐GFP transfection (zoom in inset); here co‐transfected with Tractin‐Tomato. E–J) Yoda1 treatment leads to rapid Ca 2+ transients in A7r5 (E,F), primary human (G,H) and primary rat (I,J) VSMCs. Data from three independent repeats with n = 30–45 cells.

Article Snippet: The following reagents were used at specified concentrations: Yoda1 (10 µ m , Bio‐Techne, 5586/10), Dooku1 (50 µ m , Bio‐Techne, 6568/10).

Techniques: Membrane, Staining, Transfection

Journal: Advanced Science

Article Title: Hypertensive Pressure Mechanosensing Alone Triggers Lipid Droplet Accumulation and Transdifferentiation of Vascular Smooth Muscle Cells to Foam Cells

doi: 10.1002/advs.202308686

Figure Lengend Snippet: Chronic Yoda1 treatment supports transition to a foam cell phenotype. A‐B) Wound scratch assay shows reduced migration of Yoda1 treated A7r5 cells. C) Nanoindentation indicates a lower Young's Modulus after Yoda1 treatment. D,E) Click‐EdU assay indicates higher cell proliferation following Yoda1 incubation, which is reversed after simultaneous Dooku1 treatment. F,G) TUNEL staining indicates no significant changes to apoptosis after 8 h Yoda1 treatment. H–L). qPCR testing indicate increased CD68 (H), KLF4 (I), LDLR (J) and lower ABCA1 (K) transcription after 8‐hour Yoda1 treatment, while LGALS3 (L) showed no change at this time point. p‐values from unpaired two‐tailed t‐tests (B,C, H–L), or one‐way ANOVA with Tukey correction for multiple comparisons (E,G): * p <0.0332, ** p <0.0021, *** p <0.0002.

Article Snippet: The following reagents were used at specified concentrations: Yoda1 (10 µ m , Bio‐Techne, 5586/10), Dooku1 (50 µ m , Bio‐Techne, 6568/10).

Techniques: Wound Healing Assay, Migration, EdU Assay, Incubation, TUNEL Assay, Staining, Two Tailed Test

Journal: Advanced Science

Article Title: Hypertensive Pressure Mechanosensing Alone Triggers Lipid Droplet Accumulation and Transdifferentiation of Vascular Smooth Muscle Cells to Foam Cells

doi: 10.1002/advs.202308686

Figure Lengend Snippet: Hyperspectral stimulated Raman Scattering (hsSRS) indicates lipid droplet accumulation and lipid metabolic changes after pressure and Yoda stimulation. A,B) Acute Piezo1 activation and HT pressure stimulation leads to lipid accumulation in A7r5 cells. A) Lipid droplet segmentation from phasor blots (bottom row) overlaid in red over 2930cm‐1 image for overall cell outlines (green); B,C) hsSRS shows increase in total lipids after HT pressure and reduction in TAGs after Yoda1 treatment in lipid droplets (B) and cytoplasm (C). D) Quantification of area ratio of lipid droplets; E) Quantification of ratios of Total Lipids compared to Proteins, F) TAGs compared to total Lipids, and G) cholesterol esters (CEs) compared to total lipids. p‐values from one‐way ANOVA with Dunnet correction for multiple comparisons (against ATM): * p <0.0332, ** p <0.0021, **** p <0.0001. Only significant comparisons are shown.

Article Snippet: The following reagents were used at specified concentrations: Yoda1 (10 µ m , Bio‐Techne, 5586/10), Dooku1 (50 µ m , Bio‐Techne, 6568/10).

Techniques: Activation Assay

Journal: Advanced Science

Article Title: Hypertensive Pressure Mechanosensing Alone Triggers Lipid Droplet Accumulation and Transdifferentiation of Vascular Smooth Muscle Cells to Foam Cells

doi: 10.1002/advs.202308686

Figure Lengend Snippet: cPLA2 accumulation and ROS upregulation after Piezo1 activation. A) Immunofluorescent micrographs demonstrating cPLA2 accumulation following Yoda1 treatment. B) example line profiles over nucleus. C) Quantification of enrichment against cytoplasm and nuclear intensity. Pooled data from three independent repeats. *** p <0.0002, **** p <0.0001; p‐values from one‐sample t‐test compared to 1 (black, i.e., 1 = no enrichment) or unpaired two‐tailed t‐test (red). D) Immunofluorescent staining of mouse tissues after artery ligation or sham control. E–H) A7R5 cells incubated with CellROX dye. Acute Piezo1 activation leads to increased ROS production (E, F), which is blocked by simultaneous LOX inhibition through NDGA treatment (G, H). Data from three independent repeats with n = 25–40 cells.

Article Snippet: The following reagents were used at specified concentrations: Yoda1 (10 µ m , Bio‐Techne, 5586/10), Dooku1 (50 µ m , Bio‐Techne, 6568/10).

Techniques: Activation Assay, Two Tailed Test, Staining, Ligation, Incubation, Inhibition