Pressure and stiffness sensing together regulate vascular smooth muscle cell phenotype switching

Fig. 1.. HT stimulates phenotypic switching in VSMC. (A) Overview of the experimental setup and (B) pressure settings. (C and D) Cells were analyzed using CellProfiler before dimensionality reduction and cluster analysis (t-SNE). (E) HT treatment reduced cell area, (F) actin alignment (G), and shape of the cells. (H) The fraction of podosome-forming cells depended on pressure and stiffness (displayed as average per repeat with ~150 to 200 cells each). (I) Podosomes were confirmed by costaining for cortactin, zyxin, or vinculin. (J) Radial profiles are normalized intensities from n = 10 podosomes from five cells each. (K and L) Gelatin degradation (DQ gelatin–positive area) per cell, displayed as average per repeat, from n = 200 to 300 cells each. (M) The large-scale changes to the cellular cytoskeleton were also reflected by a drop in the cellular stiffness as determined by nanoindentation. Top left: Image from tractin (green) and the indenter (bright-field channel). Bottom left: Typical load-indentation curve for the control (green) and HT (red)–treated cells. Right: Young’s moduli [graph from SuperPlotsOfData (78)]. *P < 0.0332; **P < 0.0021; ***P < 0.0002; ****P < 0.0001; ns, not…

Fig. 2.. Quantitative proteomics analysis indicates stiffness- and pressure-dependent changes. (A) Hierarchical clustering of identified proteins indicates primary clustering of HT-treated and control A7r5 VSMCs. Pairwise differential regulation analysis indicates 123 significant changes (FC > 2, P < 0.05; shown in red) between 1-kPa control and HT-treated samples (B), compared to no or only few significant changes for the other comparisons (C to E). (F to I) Literature and gene ontology analysis of the identified 123 differential regulated proteins identified 14 proteins associated with podosome formation (G), 11 other cytoskeletal proteins, 6 extracellular matrix (ECM) components, 10 proteins associated with stress response (H), and 17 proteins associated with atherosclerosis (I) [numbers including overlaps, see (F), outer circle; (G to I) α and β tubulin as well as Myo1e are shown in (G), but are also associated with atherosclerosis; α and β tubulin also with stress response]. From left to right: Gray dots: 1-kPa control, black dots: 1-kPa HT, gray squares: 130-kPa control, black squares: 130-kPa HT.

Fig. 3.. PDBu treatment leads to efficient podosome formation on all stiffnesses, but podosome behavior and matrix degradation are controlled by stiffness. (A and B) PDBu treatment leads to stiffness-dependent podosome formation in A7r5 VSMCs (fraction of podosome-forming cells, displayed as average per repeat, from three independent experiments with ~100 to 200 cells each). (C and D) Podosome lifetime depends on substrate stiffness. (C) Color-coded time projection of A7r5 VSMC treated with PDBu on 1 and 130 kPa shows dynamic podosome formation (see also movies S1 and S2). (D and E) Podosome formation (independent of stiffness) was detected as either single event or cyclically fluctuating podosomes but showed stiffness dependence in their turnover time (measured at half-maximum of the peaks) with an increased lifetime on higher stiffness (E). (F and G) Gelatin degradation was more extensive on compliant surfaces (DQ gelatin–positive area per cell, displayed as average per repeat, from three independent experiments with n = 30 to 100 cells analyzed per repeat and condition). *P < 0.0332; **P < 0.0021; ***P < 0.0002; ****P < 0.0001; P values from ANOVA and Bonferroni test for multiple…

Fig. 4.. Cofilin localization and activity respond to stiffness and regulate podosomal actin turnover. (A) Cortactin phosphorylation is unaffected by stiffness, but cofilin phosphorylation is reduced on soft surfaces, indicating higher activity. Error bars: SD; test for linear trend between stiffness and pCofilin levels: P = 0.0001. (B) Cofilin is enriched at podosomes on soft surfaces. (C) Transfection of phosphomimicking or nonphosphorylatable cofilin regulates the turnover of podosomal actin. (D) A7r5 VSMCs were transfected with mEOS2-actin and iRFP-cofilin WT, S3A (active), or S3E (inactive). (E) Actin turnover was measured after photoswitching of mEOS2-actin, indicating changes in lifetime. (E to G) Fluorescence loss after photoconversion indicates slower podosomal actin turnover in the presence of S3E and faster turnover in the presence of S3A cofilin mutants. Plot of mean and SEM of 30 podosomes from 10 cells per condition (F) and half-time (G). *P < 0.0332; **P < 0.0021; ***P < 0.0002; ****P < 0.0001; P values from ANOVA and Bonferroni test. Box plots are displayed as median, upper and lower quartile (box), and range (whiskers).

Fig. 5.. Rigidity sensing controls cofilin activity through RhoA-ROCK-LIMK signaling. (A) WB analysis of A7r5 VSMCs seeded on different stiffnesses. pCofilin and pLIMK are reduced on compliant surfaces, while other antibodies do not detect stiffness-dependent changes. (B) LIMK2 and ROCK2 knockdown, but not LIMK1 or ROCK1 knockdown, reduces pCofilin levels in A7r5 VSMCs (see fig. S4 for knockdown validation). (C) LIMK2 and ROCK2 knockdown reduces transwell migration of VSMCs. Error bars: SEM. (D) RhoA-GTP pull-down assay shows a reduction in active RhoA on compliant surfaces (quantification from three independent repeats; error bars: SD). (E to G) A7r5 cells were transfected with a RhoA-FRET biosensor and iRFP-Lifeact to analyze the dynamic changes to RhoA activity after PDBu stimulation. Cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP) images were taken every 5 s after donor excitation with a 445-nm laser and with a dual camera setup for simultaneous acquisition (see also fig. S7). iRFP images were taken every 100 s as a reference for the podosome position. (F) Time points of area outlined in white in (E). (G) Kymograph over line marked in yellow in (F). (H) Intensity…

Fig. 6.. Neointima formation is accompanied by reduced extracellular matrix stiffness and changes to LIMK and cofilin phosphorylation. (A to C) Nanoindentation measurements indicate a reduced elastic modulus during neointima formation. (A and B) Bright-field (BF) images before nanoindentation (inset: overlay with measured Young’s modulus) for sham-treated mice (A) or after carotid artery ligation (B: LGT). Quantified in (C), graph from SuperPlotsOfData (78) with P < 0.05 from Welch’s t test for comparison of means between conditions. (D) The increased compliance coincides with increased proteoglycan expression (chondroitin sulfate staining). Inset for LGT shows larger area for overview. (E) Frozen sections from sham-treated mice and animals that underwent carotid artery ligation, stained for pCofilin, pLIMK, and pan-actin together with DAPI. Quantification of the staining intensities of mouse VSMC in sham and ligation (LGT) samples indicates a reduction of both pCofilin and pLIMK, while actin, cofilin, and LIMK intensities are unaffected. Only cell areas were included in the quantification, which was not affected by typical background staining from the elastin layers. Data presented as…

Fig. 7.. Pressure and PDBu stimulation induce calcium influx to regulate slingshot-dependent cofilin phosphorylation. (A) Hydrodynamic pressure stimulation reduces cofilin, but not LIMK phosphorylation. (B) Schematic of pressure application with the nanoindenter (top) and indentation settings (bottom). (C) Example traces of corresponding load and pressure. (D and E) Indentation leads to a consistent increase in Cal520 intensity. Schematic indicates size of bead (in blue) and contact area at 1-μm indentation (in dark blue) in comparison to cell area (in pink). (E) Quantification of n = 15 indented and 10 control cells (located nearby the indented cells) from three independent experiments. Error bars: SD. (F and G) PDBu treatment leads to a prolonged increase in Cal520 intensity, while TG induces a fast release of calcium. (G) Quantification of n = 348, 369, and 562 cells for control, PDBu, and TG treatment, respectively. (H) A23187 reduces cofilin phosphorylation after 1 hour. (I) SSH inhibition by sennoside A (SA) increases cofilin phosphorylation after PDBu treatment, and the reduction in pCofilin after A23187 is counteracted by simultaneous SSH treatment. (J) SSH inhibition [either…

Fig. 8.. Schematic summarizing the pressure- and stiffness-dependent formation of podosomes in VSMCs. (A) Pressure and PDBu signal through Ca2+ and SSH, while rigidity sensing acts through RhoA, ROCK2, and LIMK2 to modulate cofilin phosphorylation. (B) Because cofilin switches between nucleation and severing depending on the concentration (49), fluctuations in cofilin activity result in a cyclic nature of podosome formation: High cofilin activity leads to actin nucleation and actin core assembly of podosomes, which eventually invokes RhoA activation at the podosome ring (through a currently unknown mechanism), thereby increasing ROCK and LIMK activity. This, in turn, reduces the cofilin activity, leading to actin severing and actin core disassembly and a reduction in RhoA activity.