P300 Modulates Endothelial Mechanotransduction of Fluid Shear Stress

Abstract

Purpose

P300 is a lysine acetyltransferase that plays a significant role in regulating transcription and the nuclear acetylome. While P300 has been shown to be required for the transcription of certain early flow responsive genes, relatively little is known about its role in the endothelial response to hemodynamic fluid stress. Here we sought to define the role of P300 in mechanotransduction of fluid shear stress in the vascular endothelium.

Methods

To characterize cellular mechanotransduction and physical properties after perturbation of P300, we performed bulk RNA sequencing, confocal and Brillouin microscopy, and functional assays on HUVEC.

Results

Inhibition of P300 in HUVEC triggers a hyper-alignment phenotype, with cells aligning to flow sooner and more uniformly in the presence of the P300 inhibitor A-485 compared to load controls. Bulk transcriptomics revealed differential expression of genes related to the actin cytoskeleton and migration in cells exposed to A-485. Scratch wound and bead sprouting assays demonstrated that treatment with A-485 increased 2D and 3D migration of HUVEC. Closer examination of filamentous actin revealed the presence of a perinuclear actin cap in both P300 knockdown HUVEC and HUVEC treated with A-485. Interrogation of cell mechanical properties via Brillouin microscopy demonstrated that HUVEC treated with A-485 had lower Brillouin shifts in both the cell body and the nucleus, suggesting that P300 inhibition triggers an increase in cellular and nuclear compliance.

Conclusions

Together, these results point to a novel role of P300 in modulating endothelial cell mechanics and mechanotransduction of hemodynamic shear stress.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12195-024-00805-2.

Introduction

Endothelial cells form the inner lining of the vasculature and act as a barrier between fluids and solutes in circulation and the surrounding tissues [1, 2] and are subjected to various mechanical forces such as shear stress and cyclic stretch, which play an important role in regulating endothelial cell behavior [3]. When exposed to shear stress, endothelial cells undergo drastic cytoskeletal rearrangements that result in cell elongation and alignment parallel to the flow vector, as well as reorganization of cell-cell and cell-matrix adhesions [4]. In addition to triggering cytoskeletal rearrangements, hemodynamic forces also drive transcriptional rewiring and chromatin remodeling in endothelial cells [5–7]. Within 6 h of flow initiation, gene expression profiles of HUVEC change significantly, with classical flow response genes, such as Kruppel-like factors 2 and 4 (KLF2, KLF4), yes-associated protein (YAP), and hairy enhancer of split (HEY1) among others, being upregulated by fluid shear stress [8, 9]. The nucleus plays a central role in mechanotransduction by the endothelium, interacting directly with the surrounding environment via protein complexes, including the linker of nucleus and cytoskeleton (LINC) complex, which bridges the cytoskeleton to the nuclear membrane and underlying chromatin [10]. It is also known that exposure to shear stress drives nuclear stiffening [11] and that the nucleus repositions downstream of the Golgi apparatus in endothelial cells exposed to flow [12].

Within the nucleus, chromatin is organized in a fractal, knotless loop and is differentially packaged into either heterochromatin or euchromatin [13]. Heterochromatin is more condensed and is canonically associated with gene repression while euchromatin is more relaxed and is associated with gene activation [14]. Fluid shear stress has been demonstrated to drive changes in global chromatin compaction in endothelial cells [5]. These observations are further supported by examination of the rheological properties of nuclei in response to flow, where fluorescent puncta in HUVEC nuclei were demonstrated to travel in a magnitude-sensitive and directional manner after initiation of flow [15]. Intriguingly, these magnitude-sensitive and directional movements happened well after the initiation of flow, suggesting that repositioning of subnuclear bodies in response to shear stress may be a functional rather than purely physical response in HUVEC [15]. It is also known chromatin conformation can impact underlying nuclear mechanical properties, with heterochromatin being less compliant than euchromatin [16, 17]. Together, these observations suggest that the chromatin landscape and subsequently the nuclear mechanical environment are altered by fluid shear stress.

P300 is a lysine acetyltransferase with numerous histone and non-histone targets [18] that acts as a key regulator of the nuclear acetylome [19]. It is associated with both enhancer activation and active transcription and is conserved across most multicellular organisms. One of the many acetylation targets of P300 is lysine 27 in the histone H3 tail (H3K27ac), a mark which is associated with active enhancers and transcription. Enhancer activation is also flow-responsive in endothelial cells, as demonstrated by the changes in enrichment of H3K27ac signatures at enhancer motifs before and after flow [8]. In endothelial cells, P300 is required for the activation of early flow response genes such as endothelial nitric oxide synthase (eNOS) [20]. Mice deficient in P300 experience embryonic lethality, with significant defects in heart development and yolk sac vascularization [21], suggesting that P300 has important roles in vascular development. Further, P300 is known to acetylate a variety of transcription factors related to mechanotransduction including KLF4 and YAP [22, 23]. Despite its vital roles in a multitude of biological processes, relatively little is understood about the specific role of P300 in endothelial mechanotransduction.

Here we demonstrate that P300 modulates mechanotransduction of fluid shear stress and the expression of flow-related genes in endothelial cells. Bulk RNA sequencing revealed that P300 inhibition under flow causes an upregulation of genes related to cytoskeletal processes involving the actin cytoskeleton compared to control cells. Disruption of P300 activity drives changes in nuclear morphology and cell shape and rapidly and results in the formation of a perinuclear actin cap under both static and flow conditions. Closer examination of endothelial nuclei after P300 inhibition revealed that perinuclear actin cables on the apical surface of the nucleus appeared to coincide with local nuclear deformations. Examination of mechanical properties via Brilloiun spectroscopy revealed increased compliance in cells treated with P300 inhibitor. These alterations in nuclear morphology and mechanics coincided with several functional alterations in endothelial cells, including decreased time to alignment, increased sprout length as measured by sprouting angiogenesis assay, and decreased migration speed as assessed by scratch wound assay. Together, these results demonstrate that P300 is a key player in regulating the cytoskeletal response to fluid shear stress and that P300-dependent alterations to nuclear and cellular mechanics may modulate sensitivity to fluid shear stress in endothelial cells.

Results

P300 Inhibition Reduces Time to Alignment in Endothelial Cells Responding to Fluid Shear Stress

To test the hypothesis that P300-mediated acetylation modulates endothelial mechanotransduction of hemodynamic stresses, we treated human umbilical vein endothelial cells (HUVEC) with A-485, a small molecule catalytic inhibitor of the acetyltransferase activity of P300 [24]. In a study comparing the acetylomes of P300 knockout cells and A-485 treated cells, 93% similarity was observed, demonstrating that A-485 effectively reduces the acetyltransferase activity of P300 [19]. Here, we validated A-485 inhibitor activity by measuring levels of H3K27ac, which is deposited by P300 alone [18, 25]. Treatment with A-485 for 24 h caused a significant reduction in H3K27ac as measured by immunofluorescence (Fig. 1A, B) and western blot (Fig. 1C). The effect of A-485 is rapid and specific, with loss of H3K27ac occurring after 1 h of treatment; importantly, this reduction in H3K27ac is not recapitulated by negative control compound, A-486 (Fig. S1) and is specific to this reside, with other histone post translational modifications such as acetylation at lysine 9 (H3K9ac) or trimethylation at lysine 27 (H3K27me3) being unaffected by treatment with A485 (Fig. S2). Orbital shaking has been used previously to modulate shear stress [26], and while it can be difficult to determine the exact shear stress, we cultured cells under conditions estimated by previous groups to be 15 dyn/cm² at the well periphery [27]. When we exposed cells to orbital shaking for 24 h, we observed a slight but statistically significant decrease in nuclear P300 signal compared to static control cells (Fig. S3). Cells treated with A-485 had elevated levels of nuclear P300 under both static and orbital shaking conditions when compared to control cells, but nuclear P300 signal was unaffected by orbital shaking in the context of A-485 treatment (Fig. S3). When we examined samples for signs of adaptation to shear stress, we found that cells treated with A-485 demonstrated more uniform alignment of the major axis of the nucleus to the local flow vector compared to control cells, as measured by nuclear orientation (Fig. 1E). Additionally, cells treated with A-485 under orbital shaking conditions were significantly more elongated compared to DMSO treated cells as demonstrated by comparing cell aspect ratio using F-actin staining (Fig. 1E). We next cultured HUVEC under unidirectional laminar shear stress of 15 dyn/cm² for 72 h using a parallel plate (Hele-Shaw) flow cell. These conditions have been thoroughly characterized and reproducibly induce endothelial cell alignment in HUVEC [28, 29]. To visualize degree of alignment to flow, cells were imaged (Fig. 1F) and the angle of the major axis of the cell body for each condition was plotted in a radial histogram (Fig. 1G). Of n = 2243 cells analyzed per condition, 32.99% of control cells were oriented ± 15 degrees of the flow vector under the control condition whereas 54.11% of cells were oriented ± 15 degrees of the flow vector with A-485 treatment (Fig. 1H). We also observed significant increases to cell aspect ratio after 24 h of flow and A-485 treatment (Fig. 1I). These results suggest that alignment of the cell body to the flow vector is a process that can occur independently of P300 acetyltransferase activity, and that treatment with A-485 enhances the alignment phenotypes displayed by endothelial cells after exposure to flow.

Fig. 1.

P300 inhibition drives reduction in H3K27ac levels in HUVEC and induces hyper-alignment to flow. A Representative images of HUVEC treated with 5 μM A-485 or DMSO under static conditions for 24 h. Cells labeled with phalloidin (white), DAPI (cyan), and H3K27ac (magenta). Scale bars, 50 μm. B Quantification of H3K27ac mean fluorescence intensity from immunofluorescence images. C Western blot and quantification of relative H3K27ac levels in HUVEC treated with DMSO or 5 μM A-485 for 24 h. D Radial histogram of nuclear orientation angle in HUVEC exposed to orbital shaking for 72 h. E Orientation of nuclear major axis and nuclear aspect ratio of cells exposed to orbital shaking for 72 h. F Representative images of HUVEC exposed to flow imparting 15 dyn/cm² for 72 h or 24 h. Cells labeled with DAPI (cyan), VE-cadherin (green), and phalloidin (white). Scale bars, 100 μm. G Radial histogram of cellular orientation angle of HUVEC exposed to 15 dyn/cm² for 72 h. H Percentage of cells with major axis oriented within ± 15 deg. of the flow vector. I Aspect ratio of cells exposed to 15 dyn/cm² for 24 h. Statistical analysis, unpaired parametric T test or Kolmogorov–Smirnov test, ns; not significant, **p < 0.01, ****p < 0.0001. N ≥ 3 experiments. Max. Int. Proj. maximum intensity projection

Inhibition of p300 Modulates Expression of Actin Regulatory Genes

P300 plays a major role in the regulation of transcription [30], and the transcriptome of endothelial cells exposed to fluid shear stress is known to be distinct from the transcriptome of EC cultured under static conditions [8, 31]. Based on the phenotypic features that were present in cells exposed to A-485 in the presence of flow, we sought to interrogate underlying transcriptional differences in an unbiased manner using bulk RNA sequencing. HUVEC were cultured for 24 h under either static or flow conditions (15 dyn/cm²) in the presence of either DMSO or 5 µM A-485 prior to collection of RNA for transcriptomics. Principal component analysis revealed that replicate samples clustered together, with the majority of variance being derived from drug treatment followed by flow condition (Fig. 2A). To visualize differences in gene expression, the top 40 differentially expressed genes were plotted in a heat map using variance-stabilized counts clustered by Euclidean distance (Fig. 2B). Notable differentially expressed genes included thrombospondin, which was significantly down-regulated after treatment with A-485 under both static and flow conditions, as well as tubulin alpha 1B, a key component of microtubules, which was similarly down-regulated compared to control samples under both static and flow conditions. Another notable gene was MMP1, which codes for matrix metalloprotease 1, which was significantly downregulated in A-485-treated cells under static conditions. SERPINE1, which encodes a protein that inhibits fibrinolysis and has been suggested to be a biomarker of cardiovascular pathologies such as cerebral infarction and atherosclerosis [26], was also significantly downregulated by A-485 treatment under static conditions.

Fig. 2.

Transcriptomics reveal differential expression of genes related to actin cytoskeleton in cells exposed to P300 inhibition under flow. A Principal component analysis plot. Samples cluster primarily by drug and flow condition. B Heatmap of top 40 differentially expressed genes. Values reflect variance-stabilized gene counts. Clustering based on Euclidean distance. C Gene ontology enrichment for biological processes that were differentially enriched between DMSO and 5 μM A-485 under static conditions. D Gene ontology enrichment for biological processes that were differentially enriched between DMSO and A485 under flow conditions. E Expression of several genes related to the cytoskeleton and cell migration under flow conditions. F Expression of several genes related to the endothelial flow response under flow conditions. PC principal component. Padj adjusted P value

To determine how P300 inhibition affected HUVEC under flow or static conditions, gene ontology analysis was performed on treatment pairs within either static or flow conditions. Under static conditions, 8596 genes were differentially expressed between control cells and cells treated with A-485 for 24 h. Gene ontology analysis of genes significantly upregulated in HUVEC treated with A-485 under static conditions revealed that processes related to protein metabolism and macroautophagy were enriched in the A-485 treated cells (Fig. 2C). In comparison, 9236 genes were differentially expressed between control cells and cells treated with A-485 after exposure to 15 dyn/cm² shear stress for 24 h. Gene ontology analysis of significantly upregulated genes on A-485-treated cells compared to DMSO-treated cells under flow conditions revealed biological processes that predominantly related to cell cycle and actin cytoskeleton, including regulation of actin filament-based processes, regulation of actin cytoskeletal organization, and regulation of actin filament organization (Fig. 2D). These results suggest that P300 suppresses these programs. Examination of several genes related to the actin cytoskeleton, including genes that encode actin subunits as well as regulators of actin dynamics such as RhoA [32] and Cdc42 [33] among others, revealed significant upregulation of RhoA in A-485 treated cells under flow conditions along with significant downregulation of several tubulin subunits (Fig. 2E). We also evaluated genes related to known flow response signaling pathways and saw that many flow-related genes are downregulated compared to DMSO treated cells under flow (Fig. 2F). We also examined several genes related to the Notch signaling pathway, which is known to be activated by flow [10]. Interestingly, the majority of genes examined were downregulated under flow in A-485-treated samples relative to DMSO-treated samples after flow (Fig. S4).

P300 Inhibition Modulates Endothelial Migration and Sprouting

To determine whether P300 inhibition and the associated changes in expression of actin regulatory genes modulate cytoskeletal dynamics of ECs, scratch wound assays were performed using HUVEC in the presence or absence of A-485 (Fig. 3A). Control cells had significantly less remaining wound area compared to A-485 treated cells after 24 h, suggesting that inhibiting P300 may impair migration (Fig. 3B). To determine whether the differences in scratch area were due to proliferation or migration, we performed a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay to measure rates of proliferation. At 5 μM A-485, the drug concentration used throughout our studies, we observed a significant decrease in proliferation compared to control conditions (Fig. 3C). We next sought to evaluate the impact of A-485 treatment on EC invasion and 3D migration using a bead sprouting assay [34]. To perform the bead assay, 150 µm beads were coated with HUVEC and embedded in a 2.5 mg/mL collagen hydrogel and cultured with or without A-485. (Fig. 3D). Treatment with A-485 for 72 h led to increased sprout length but did not alter the total number of sprouts per bead (Fig. 3E). Interestingly, A-485 treatment resulted in significantly more cells that migrated as single cells away from beads and into the surrounding collagen hydrogel compared to DMSO controls (Fig. 3F, G). Imaging the collagen hydrogel revealed no difference in pore size adjacent to cells that migrated from the bead surface and that the nucleus was closely surrounded by collagen fibrils, suggesting that A-485-mediated migration was not potentiated by increased proteolysis of the matrix (Fig. S5).

Fig. 3.

Inhibition of P300 slows 2D migration but increases rate of 3D invasion. A Representative images of wound closure over 24-h time course in the presence or absence of 5 μM A-485. Scale bar, 500 μm. B Quantification of wound area as a function of time, normalized by initial wound area. C Cell proliferation as quantified by MTS assay after 24 h of culture as a function of A485 concentration vs. DMSO load control. D Representative images of microbead angiogenesis assay. HUVEC-coated beads are embedded in 2.5 mg/mL collagen I hydrogels. HUVEC express LifeAct-GFP (intensity inverted). Scale bar, 200 μm. E Quantification of multicellular sprout length from bead surface and the number of multicellular sprouts per bead. F Lower magnification images of bead sprouting assay to show single cells migrating from bead surface (red arrows indicate individual cells), and quantification of the number of single cells observed after 24 h. Scale bar, 200 μm. Statistical analysis, unpaired parametric t test, ns; not significant, ***p < 0.001, ****p < 0.0001. N ≥ 3 experiments

Inhibition of p300 Increases Cell and Nuclear Compliance

To migrate in 3D, cell nuclei must deform to pass through pores in the ECM, and previous work has demonstrated that manipulations that increase nuclear compliance increase the rate of migration of cells through collagen hydrogels [35]. It has also been demonstrated that chromatin compaction can modulate the stiffness of nuclei [36]. To determine whether the increased 3D migration of ECs treated with A-485 is due to differential cellular mechanical properties, we fixed HUVECs treated with A-485 and imaged nuclear shape with DAPI and F-actin organization with phalloidin to determine whether P300 inhibition modulates nuclear or cytoskeletal morphology (Fig. 4A). We observed significant increases in nuclear area, eccentricity, and nuclear and cellular aspect ratio after treatment with A-485 (Fig. 4B). No significant differences in cellular area were observed. To further establish that these changes in nuclear and cytoskeletal morphology were due to P300 inhibition and not off-target effects of A-485, we knocked down P300 with siRNA (Fig. 4C). Reduction in H3K27ac signal was proportional to the loss of P300 in EC after P300 knockdown (Fig. 4D), and siP300 cells phenocopied cells treated with A-485, including increased nuclear area, eccentricity, and nuclear aspect ratio compared to cells transfected with nontargeting siRNA (Fig. 4E, F). However, the overall cell area and cell aspect ratio was unchanged between nontargeting and siP300 conditions.

Fig. 4.

Nuclear shape and mechanical properties are altered by P300 inhibition and knockdown under static and flow conditions. A Representative images of nuclear morphology for HUVEC treated with 5 μM A-485 vs. DMSO load control. Scale bars, 50 μm. B Quantification of cell and nuclear morphology. C Representative images of HUVEC transfected with P300 siRNA (siP300) vs. non-targeting control (siNT). Transfected cells immunostained with anti-P300 and anti-H3K27ac antibodies. Scale bar, 50 μm. D Quantification of P300 and H3K27ac mean fluorescence intensity (MFI). E Representative images of nuclear and cell morphology after knockdown of P300 (siP300) vs. non-targeting siRNA (siNT). Scale bars, 50 μm. F Quantification cell and nuclear morphology. G Representative images of HUVEC treated with 5 μM A-485 vs. DMSO load control and imaged by Brilluoin microscopy. Scale bars, 10 μm. H Quantification Brillouin shift within the nucleus and cytoplasm after treatment with 5 μM A-485 vs. DMSO load control. Statistical analysis, unpaired parametric T test (B, D, F) or Welch’s T test, ns; not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. N ≥ 3 experiments

To determine whether the changes in nuclear shape induced by P300 inhibition or knockdown correspond to changes in the mechanical properties of the nucleus, we used Brillouin microscopy to measure the Brillouin shifts in HUVEC treated with A-485 or DMSO for 24 h (Fig. 4G). Brillouin microscopy has been used to measure the mechanical properties of cells and nuclei with subcellular resolution [37], and an increased Brillouin shift has been shown to correspond to an increased Young’s modulus as measured by AFM [38]. We observed significantly lower Brillouin shifts in nuclei and cell bodies of cells treated with A485 compared to controls (Fig. 4H). Based on the log-log linear relationship between Brillouin modulus and Young’s modulus, this suggests that cells and nuclei are more compliant with A-485 treatment as compared to DMSO controls. To determine whether these observed changes in Brillouin shift correspond to differential resistance to deformation through constricted channels, we performed a microfluidic micropipette aspiration assay [39], and consistent with the Brillouin data, we observed increased deformation of cells treated with A-485 into constricted channels as compared to DMSO controls (Fig. S6).

Inhibition and Knockdown of p300 Induces Formation of a Perinuclear Actin Cap

Previous work has demonstrated that a perinuclear actin cap can deform the nucleus and that local actin cap fibers can cause the nucleus to bulge or fold [40, 41]. Informed by RNAseq data demonstrating differential expression of genes coding for actin regulatory proteins (Fig. 2), we sought to determine whether the changes in nuclear morphology driven by P300 inhibition were accompanied by formation of a filamentous actin cap. We imaged actin stress fibers in proximity to the nucleus using confocal microscopy and found that in addition to changes to morphology, filamentous actin was observed at the apical surface of nuclei in cells treated with A-485 for 24 h (Fig. 5A), which was significantly more frequent in cells exposed to A-485 compared to DMSO control (Fig. 5B). Perinuclear actin features were also observed when P300 was knocked down with siRNA (Fig. 5C) and these features were significantly more frequent in P300 knockdown cells compared to cells transfected with nontargeting siRNA (Fig. 5D). These results suggest that P300 directly regulates perinuclear actin assembly in EC.

Fig. 5.

Inhibition and knockdown of P300 results in formation of a perinuclear actin cap. A Representative confocal images of HUVEC treated with DMSO or 5 μM A-485 for 24 h. Cells labeled with phalloidin (white) and DAPI (cyan). Maximum intensity projections of z-stacks of cells and single slices at the apical and basal cell surfaces. Step size, 1um. Scale bars, 20 μm. B Quantification of the number of cells with an actin fibers over the nucleus at the apical plane. C Representative confocal images of HUVEC transfected with either nontargeting or siRNA targeting P300. Maximum intensity projections of z-stacks of cells and single slices at the apical and basal cell surfaces. Step size, 1 μm. Scale bars, 20 μm. D Quantification of cells with apical actin caps. E Representative confocal images of perinuclear actin features in HUVEC exposed to 15 dyn/cm² shear stress for 24 h. Cells labeled with DAPI (cyan), VE-cadherin (green), and phalloidin (white). Maximum intensity projections of z-stacks of cells and single slices at the apical and basal cell surfaces. Step size, 1 μm. Scale bars, 20 μm. F Quantification of cells with apical actin caps. G Representative confocal images of perinuclear actin features in HUVEC exposed to 15 dyn/cm² shear stress for 72 h. Maximum intensity projections of z-stacks of cells and single slices at the apical and basal cell surfaces. Step size, 1 μm. Scale bars, 20 μm. H Quantification of cells with apical actin caps. I Representative images of nuclear deformation in HUVEC treated with 5 μM A485 for 24 h. Orthogonal projection of inset (white dashed box) are displayed along with single confocal planes at the apical cell surface. Yellow arrows indicate locations of nuclear deformation correlated to an apical actin fiber. Scale bar, 5 μm. Statistical analysis, unpaired parametric t test or 2-way ANOVA, ns not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. N ≥ 3 experiments. Max. Int. Proj. maximum intensity projection

When cells exposed to shear stress were imaged to examine perinuclear actin features, we observed that A-485-treated cells had significantly more perinuclear actin features compared to DMSO-treated cells under both static and flow conditions (Fig. 5E, H). No significant difference in perinuclear actin frequency was observed between static and flow conditions in cells treated with A-485 or DMSO for 24 h. (Fig. 5E, F) Interestingly, we observed that nearly 100% of cells exposed to 15 dyn/cm² for 72 h in the presence of A-485 presented perinuclear actin caps (Fig. 5G), and that there were significantly more perinuclear actin features in cells cultured with A-485 during shear stress compared to cells exposed to A-485 under static conditions at this 72 h timepoint (Fig. 5H). These results suggest that fluid shear stress may potentiate the formation of the perinuclear cap over longer time periods in the context of A-485 treatment. Closer examination of perinuclear actin revealed regions in which filaments of the actin cap co-localized with deformations of the nucleus (Fig. 5I). Focal adhesions that anchor actin cap filaments have been shown to have distinct morphology and play differential roles in mechanosensation as compared to basal stress fiber focal adhesions [42], but no significant differences in focal adhesion length were observed between control and A-485-treated cells (Fig. S7). Perinuclear actin frequency in control cells was unaffected by fluid shear stress under either 24 h or 72 h incubation periods (Fig. 5F, H).

Nuclear Shape Remains Disrupted After Dissolution of Perinuclear Actin Cap

The apical actin cap has been shown to be regulated by cytoskeletal tension [40, 42, 43]. To determine the relationship between actin cap structure and nuclear shape, we pulsed A-485-treated HUVEC with latrunculin A, a widely used actin depolymerization agent [44], to prevent actin filament polymerization. After 1 h treatment with 50 nM latrunculin A, we observed dissolution of the apical actin cap (Fig. 6A). We also observed that cells pretreated with A-485 had significantly smaller nuclei after 1 h latrunculin A treatment compared to A-485 treated cells that were not treated with latrunculin A (Fig. 6B). However, nuclear eccentricity and nuclear aspect ratio were unaffected by latrunculin A treatment in cells pretreated with A-485 (Fig. 6B). Control cells exhibited no significant alterations in nuclear area, eccentricity, or aspect ratio after treatment with latrunculin A (Fig. 6A, B). Because we observed total dissolution of the perinuclear actin cap after treatment with latrunculin A, these results suggest that P300 inhibition can modulate nuclear morphology independent of actin cap formation.

Fig. 6.

Nuclear shape changes induced by P300 inhibition persist with actin depolymerization. A Representative images of HUVEC pretreated for 24 h with either DMSO or 5 μM A-485 before 1 h pulse with either DMSO or 50 nM latrunculin A. Scale bar, 50 μm. B Quantification of cells with apical actin caps and corresponding nuclear morphology. Statistical analysis, 2-way ANOVA, ns; not significant, *p < 0.05, ***p < 0.001, ****p < 0.0001. N ≥ 3 experiments

Discussion

Endothelial adaptation to fluid shear stress is essential for maintaining vascular homeostasis [45]. Certain diseases such as atherosclerosis are associated with regions in the vasculature where flow magnitudes are lower or multidirectional, such as near vessel branch points, highlighting the importance of fluid shear stress in maintaining healthy endothelial physiology [46]. Endothelial cells undergo both cytoskeletal rearrangements and transcriptional rewiring in response to flow. Expression of genes such as nitric oxide synthase after exposure to flow has been demonstrated to be P300-dependent [20], and more broadly, the differential enrichment of H3K27ac at enhancers after flow [8] implicates P300 as a regulator of transcriptional rewiring in response to flow. However, the specific role that P300 plays in the endothelial flow response is surprisingly unexplored.

We evaluated the role of P300 in endothelial cell adaptation to flow using a small molecule catalytic inhibitor, A-485 [24], and siRNA-mediated knockdown. Recent studies of endothelial cells exposed to fluid shear stress have revealed significant alterations to H3K27ac signatures at enhancer elements [8]. Because P300 deposits this mark, we hypothesized that inhibiting P300 would disrupt how endothelial cells adapt to flow. Surprisingly, endothelial cells cultured with P300 inhibitor still successfully aligned to the flow vector. In fact, alignment to the flow vector happened more quickly when P300 activity was inhibited (Fig. 1), suggesting that P300 may modulate endothelial sensitivity to fluid shear stress.

Alignment to the flow vector is accompanied by transcriptional changes in endothelial cells from a broad range of vascular beds at different shear magnitudes [47]. Transcriptomic analysis revealed that inhibition of P300 under flow caused significant upregulation of processes related to actin filament organization (Fig. 2). These results are corroborated by the morphological changes observed in EC treated with A-485, such as the increased frequency of actin cap assembly in A-485-treated cells (Fig. 5). RNAseq also demonstrated that cells treated with A-485 express significantly more KLF2 compared to matched control cells under flow conditions, which may explain the accelerated response to fluid shear stress observed in these cells. However, in a human colorectal cancer cell line, P300 has been demonstrated to stabilize a closely related transcription factor, KLF4, via acetylation [23]. Therefore, analysis of KLF2 protein levels after exposure to A-485 would elucidate whether increased KLF2 expression results in elevated KLF2 protein levels despite P300 inhibition. Transcriptomic analysis further identified that cells treated with A-485 and exposed to flow express fewer Notch related genes compared to cells treated with DMSO (Fig. S4). However, when comparing gene expression of cells treated with A-485 and exposed to flow versus static control cells, the HES/HEY family of genes are upregulated under flow, as expected from previous reports describing the Notch family receptors as key mechanosensors of fluid shear stress [48, 49]. These results suggest that P300 could play a role as a rheostat, modulating the threshold shear stress required to activate flow-dependent transcriptional programs.

Nuclear deformation has significant biological consequences, and in endothelial cells computational modeling demonstrates that nuclear morphology adapts to minimize the force applied to the nucleus [50], suggesting an intrinsic relationship between applied fluid stresses and nuclear morphology. Furthermore, disrupted nuclear mechanics are intimately linked with disease phenotypes in diseases such as progeria, where increased resistance to deformation in nuclei of diseased cells has been observed [51, 52]. On the other hand, aberrant compliance can also have detrimental effects, as it makes nuclei more prone to rupture [53]. In cancer, nuclear rupture has been reported to stimulate pathogenic inflammation [16]. In fact, nuclear morphology is often an important factor in characterizing a variety of human malignancies, where irregularity in nuclear shape can be used to differentiate diseased vs. healthy cells [54, 55]. Our observations that inhibition and knockdown of P300 cause significant changes in nuclear shape and compliance, which in turn correlate with functional changes in endothelial cell migration and invasion into 3D ECM, support a role for nuclear mechanics in regulating endothelial behavior. In other words, the functional changes we observe here may not be due to the activity P300 directly, but rather due to P300-mediated changes in nuclear morphology and mechanics which in turn regulate cell behavior.

Having observed significant differences in nuclear shape and enrichment in expression of genes that code for biological processes related to the actin cytoskeleton (Fig. 2), we were motivated to examine perinuclear actin to identify potential relationships between the cytoskeleton and nuclear morphology in our phenotype. Previous imaging studies have identified the perinuclear actin cap as a structure that consists of apical actin fibers which terminate in focal adhesions and have been reported to shape the nucleus [40]. We observed that both P300 knockdown and P300 inhibition via A-485 resulted in significant enrichment in actin caps, which have been reported to exist under significantly higher tension than other filamentous actin structures [42], resulting in compressive forces to the underlying nucleus. Furthermore, in fibroblasts it has been shown that actin cap assembly occurs more rapidly than basal stress fiber assembly in response to shear stress, and that lower shear stress magnitudes are required to trigger apical vs. basal actin assembly [43]. Our data suggest that inhibition of P300 renders HUVEC more sensitive to shear stress magnitude, suggesting a similar coupling of actin cap formation and mechanotransduction of shear stresses. Furthermore, previous work has demonstrated nuclear bulging after depolymerization of actin [40]. To determine whether the actin cap is directly applying compressive stresses to the nucleus to drive changes in morphology, we treated cells with latrunculin A to depolymerize actin, and we observed that dissolution of the perinuclear actin cap in A-485 treated cells does not alter nuclear shape or aspect ratio but does alter nuclear area.

Links between nuclear shape, morphology, and underlying nuclear mechanical properties [16], along with our observations of frequent perinuclear actin structures, prompted us to perform Brilluoin microscopy to determine whether nuclei exposed to A-485 were mechanically distinct from nuclei in control cells. To this end, we found that nuclei in cells treated with A-485 were indeed more compliant than control cells (Fig. 4). Nuclear mechanical properties are known to be derived from both properties of the nuclear lamina as well as the chromatin within, with each component contributing differently: chromatin has been reported to behave like a viscoelastic material that resists small deformations within the nucleus while the nuclear lamina acts as a polymeric shell that resists large deformations [36]. Acetylation of histone tails is typically associated with weakened interactions between histones and DNA, which can alter chromatin compaction and therefore can cause changes in nuclear stiffness. In the publication that originally described A-485, the authors observed that inhibition of P300 via A-485 does not drive global reductions in histone acetylation; rather, depletion of acetylation was largely specific to the H3K27 residue [24]. For instance, H3K9 acetylation, another acetylation mark deposited by P300, was relatively stable in the presence of A-485 [24], which we also observed (Fig. S2). Together these data suggest that the reduction in H3K27ac levels we observe with A-485 treatment, while striking, might not be sufficient to drive global changes in chromatin compaction necessary for nuclear stiffening. Another potential explanation for the increase in nuclear compliance we observe might come from alterations in expression of proteins related to nuclear structure and architecture, such as the lamins or vimentin. Our transcriptomic analysis revealed significant downregulation in all three lamin genes (LMNA, LMNB1, LMNB2) as well as vimentin after A-485 treatment under both static and flow conditions. Because these proteins have been linked to nuclear mechanics [56, 57], it is possible that their altered expression drives the mechanical differences we observe and could further underlie the increased rate of invasion of cells into 3D collagen hydrogels with P300 inhibition (Fig. S8).

The nucleus houses the genome and acts as a mechanosensor, placing it in a central role in the endothelial flow response. Our studies revealed that nuclei in cells exposed to P300i were significantly more compliant compared to control nuclei. The nucleus is the largest and stiffest organelle in the cell and is known to polarize downstream of the flow vector due to hemodynamic drag [12]. Increased nuclear compliance coupled with accelerated adaptation to fluid shear stress suggest a potential connection between nuclear mechanical properties and mechanosensitivity in endothelial cells. However, uncoupling the nuclear mechanical properties from underlying changes in chromatin organization, which can subsequently alter transcriptional outputs and cellular behavior, remains a significant challenge.

Methods

Cell Culture

Human umbilical vein endothelial cells (HUVEC) were sourced from LONZA and cultured in complete growth media (EGM-2, Lonza). Experiments using HUVEC were performed between passages 3 and 9. Cells were cultured in 5% CO2 at 37C in a humidified incubator.

Generation of HUVEC LifeAct-GFP

To generate fluorescently-tagged HUVEC, cells were transduced with pLenti.PGK.LifeAct-GFP.W (LifeAct-GFP; AddGene plasmid #51010). Lentivirus was produced by using calcium phosphate transfection (Takara #631312) to transfect HEK-293T cells with pSAX2 (Addgene, plasmid #12260) and pMD2.G (Addgene plasmid #12259). Transfected cells were cultured for 48 h before collecting supernatant. Supernatant containing virus was concentrated using PEG-IT viral precipitator (System Biosciences, Palo Alto, CA). To transduce HUVEC, cells were plated at 75,000 cells per well in a 6-well plate. Virus was added 30 min after plating cells and media was changed after 24 h.

Antibodies and Reagents

Anti-P300 (D8Z4E) and Anti-H3K27ac (D5E4) were acquired from Cell Signaling Technology. Anti-VE-cadherin (F8) was purchased from Santa Cruz Biotechnology. Anti-paxillin (Clone 177) was acquired from BD Biosciences. Rhodamine phalloidin (1 mg/mL), 4′6-Diamidino-2-phenylindole (DAPI), AlexaFluor 488 (1 mg/mL) and AlexaFluor 647 (1 mg/mL) conjugated secondary antibodies were all sourced from Life Technologies. Anti-Paxillin (Clone 177) was purchased from BD Biosciences. NucBlue Live ReadyProbes Reagent (Hoechst 33342) was purchased from Invitrogen. Latrunculin A (ab144290) was purchased from Abcam. A-485 (S8740) was purchased from Selleckchem. Negative control compound A486 was acquired from the Structural Genomics Consortium. CellTiter 96 Aqueous One Solution Cell Proliferation Assay (G3582) was purchased from Promega. EP300 Silencer Pre-designed siRNA (AM16708, ID: 106443) and Silencer Negative Control siRNA #1 (AM4611) were both acquired from Ambion by Life Technologies. Lipofectamine 2000 Transfection Reagent (11668027) was purchased from Invitrogen.

Transfection

HUVEC were transfected with non-targeting siRNA (AM4611) or siRNA targeting P300 (AM16708, ID: 106443) using Lipofectamine 2000 (Invitrogen, 11668027). In brief, HUVEC were plated at 70% confluency in wells coated with rat-tail collagen type I (Corning 354326). To transfect HUVEC, Opti-MEM (Gibco 31985-070) was adjusted to room temperature before being combined with either siRNA or lipofectamine. siRNA and lipofectamine mixtures were prepared separately, with 4 μL 10 uM siRNA being added to 80 μL Opti-MEM and 4 μL lipofectamine 2000 being added to 80uL Opti-MEM for each well. These mixtures were incubated at room temperature for 5 min. After 5 min, siRNA mixture and lipofectamine mixture were combined into one tube and mixed gently at room temperature for 15 min. Immediately before transfection, media was changed on wells. Transfection mixture was added to wells drop-wise and incubated at 37C for 24 h before changing media. After 48 h had passed from the initial transfection, cells were used for experiments.

Immunofluorescence

To examine cellular and nuclear morphology, images of cells were acquired using a laser scanning confocal microscope (FV3000, Olympus) with a 30X U Plan S-Apo N 1.05-numerical aperture silicone oil immersion objective. Images are maximum intensity projections produced from 1 μm steps unless otherwise indicated. Briefly, cells were rinsed with phosphate buffered saline containing calcium and magnesium (PBS++) prior to fixation with 4% paraformaldehyde (Millipore) for 20 min at 37C. Following fixation, cells were washed with PBS++ and then permeabilized with 0.1% (v/v) Triton-X100 (Millipore) in phosphate buffered saline (PBS) for 10 min at room temperature. To prevent nonspecific antibody interactions, cells were then blocked for a minimum of 1 h at room temperature using 2% (w/v) bovine serum albumin (BSA) in PBS. Probing with primary antibody was performed by incubating samples with primary antibodies against VE-cadherin (F-8, v/v, 1:500), P300 (D8Z4E, v/v, 1:800), H3K27ac (D5E4, v/v, 1:800), or Paxillin (clone 177, v/v, 1:200) diluted in 2% BSA in PBS overnight at 4C. After primary antibody incubation, three washes with PBS were performed. Probing with secondary antibody was performed for 1 h at room temperature (goat-anti-mouse AlexaFluor-488 or goat-anti-mouse AlexaFluor-647, v/v, 1:500) in 2% BSA in PBS. Secondary antibody was washed from samples three times with PBS. After immunostaining, samples were counterstained with phalloidin rhodamine (1:2000, v/v) and DAPI (1:1000, v/v) diluted in PBS for 10 min at room temperature to visualize filamentous actin and the nucleus, respectively. Three washes were performed with PBS after counterstaining. Samples were stored in PBS at 4C until imaging. Manual image processing to produce images for figures was performed using ImageJ and computational image processing to measure features such as nuclear or cellular area, shape, and eccentricity was performed using CellProfiler.

Western Blot

To measure protein levels, Western blotting was performed. Briefly, cells were cultured in complete media with or without A485 for 24 h. To harvest cells for Western blot, wells were washed three times with ice cold PBS and lysed on ice with pre-chilled radioimmuoprecipitation assay buffer (Thermo Fisher Scientific) containing 1X HALT protease and phosphatase inhibitor (Thermo Fisher Scientific). Lysates were homogenized by pulse-vortexing and clarified by centrifugation at 16,000×g for 15 min at 4C. Lysates were combined with NuPAGE LDS reducing agent and dithiothreitol and denatured for 5 min at 99C. Gel electrophoresis using NuPAGE 4 to 12% bis-tris gradient gels (Thermo Fisher Scientific, NP0322BOX) was performed before transfer to iBlot2 polyvinylidene difluoride ministack membranes (ThermoFisher Scientific, IB24002) using the iBlot 2 Gel Transfer Device (Invitrogen). Membranes were washed briefly in diH2O followed by tris buffered saline with 0.1% Tween-20 (Millipore Sigma, P1379) (TBS-T) before blocking in SuperBlock Blocking Buffer (Thermo Fisher Scientific, 37537) for 1 h with rocking at room temperature. Membranes were probed with primary antibody overnight with rocking at 4C. Primary antibodies against H3K27ac (D5E4, v/v, 1:2000, Cell Signaling Technology) and GAPDH (2118, 1:5000, v/v, Cell Signaling Technology) were diluted in SuperBlock Blocking Buffer. After probing with primary antibody, membranes were washed 3 times TBS-T. Secondary antibodies conjugated to horseradish peroxidase were diluted in 5% milk (w/v) in TBS-T (1:10,000, v/v) incubated with membranes for 1 h at RT. Bands were visualized via chemiluminescence using SuperSignal West Femto Maximum Sensitivity Substrate reagent (Thermo Scientific, 34094). Images of bands were quantified using ImageJ.

Flow Experiments

Orbital Shaking

HUVEC were exposed to orbital shaking using a VWR Shaker Model 1000. Briefly, HUVEC were seeded in 6 well tissue culture plates coated with type I collagen derived from rat tail (Corning, 354326) at approximately 16,000 cells/cm² and allowed to adhere overnight before initiating orbital shaking. Rotational speed of the orbital shaker was set to 200 rpm, which corresponds to a maximum wall shear stress of 15 dyn/cm² as demonstrated by computational fluid dynamics [27]. Cells were cultured for 72 h in EGM-2 with or without A-485.

Laminar Shear Stress

HUVEC were exposed to laminar shear stress using an Ibidi pump system (Ibidi, 10902). Briefly, ibiTreat u-Slide I Luer (0.6 mm) (Ibidi, 80186) were coated with type I collagen derived from rat tail (Corning, 354326) before seeding. HUVEC were seeded at approximately 24,000 cells/cm² in μ-Slides and allowed to adhere overnight prior to initiating flow experiments. To apply flow to cells, u-Slides were connected to the Ibidi Pump System and cultured for 24 or 72 h under 15dyn/cm² of laminar shear stress. The pump system and slides were housed in a humidified incubator that was maintained at 5% CO₂ and 37 ºC. Static control μ-Slides were seeded and cultured in the same way but were not connected to the Ibidi Pump System. Experiments were performed using EGM-2 cell culture media with or without A485. For bulk RNA sequencing, 3 μ-Slides per condition were serially connected to the Ibidi Pump System and pooled in order to collect enough RNA for library preparation.

Scratch Wound Experiments

HUVEC were seeded in 6 well tissue culture plates coated with type I collagen derived from rat tail (Corning 354326) and allowed to reach confluency. Once cells were 100% confluent, a scratch was created by dragging the tip of a P1000 pipette tip across the well. Immediately after scratching, media in the wells was changed to media with or without A485. Images of wells were captured immediately, 4 h, or 24 h after scratching using an inverted phase contrast Olympus CKX53 microscope using phase contrast with a 4X/NA 0.13 objective. Scratch area was measured using ImageJ.

Sprouting Angiogenesis Experiments

Sprouting angiogenesis assay was performed as previously described [58] with modifications. Briefly, Cytodex 3 beads (Cytiva, 17-0485) were incubated with HUVEC expressing LifeAct-GFP for 3 h at 37C with intermittent agitation to prevent clumping. Beads were then incubated overnight to allow for full adhesion of cells to the beads. The following day, beads bearing HUVEC were embedded in collagen hydrogels (2.5 mg/mL). Hydrogels were prepared by titrating collagen I derived from rat tail (Corning, 354326) with reconstitution buffer (0.4 M HEPES, 0.3 M NaHCO3 in diH2O) and 10X DMEM to a final pH of 7.5. Beads were combined with this mixture before gelation to ensure even distribution of beads throughout the hydrogel. After embedding, beads were incubated for 30 min to allow for gelation and then hydrated with media and then incubated overnight at 37C to allow for full gelation. The next day, media was changed to media with either DMSO or 5 uM A485. Media was changed daily on hydrogels. Images of bead-laden hydrogels were captured with an Olympus IX83 inverted widefield microscope using phase contrast with a 4X/NA 0.16 objective.

Brillouin Microscopy

Brillouin microscopy is a spectroscopic technique that utilizes spontaneous Brillouin light scattering and measures the inelastic scattering of light between the incident light and inherent acoustic phonons inside of a material [38, 59]. The result of this interaction introduces a characteristic frequency shift (Brillouin shift), ${\omega }_B$, and is defined as ${\omega }_B=\frac{2n}{\lambda }·\sqrt{\frac{M^{\prime }}{\rho }}·sin\left(\frac{\theta }{2}\right)$, where $n$ is the refractive index of the material, $\lambda$ is the laser wavelength, $M^{\prime }$ is the longitudinal modulus of the probed material, $\rho$ is the density, and $\theta$ is the collection angle of the back scattered light. In a confocal Brillouin setup, the backward scattered light yields $\theta =180$°. In biological samples, an empirical relationship between the Brillouin-derived longitudinal modulus $M^{\prime }$ and the Young’s modulus $E$ has been established: $log\left(M^{\prime }\right)=a·log\left(E\right)+b$, where a and b are material dependent parameters [38]. For this study, we report the value of Brillouin shift in GHz.

An inverted confocal Brillouin microscope was used for all experiments and details of the instrumentation can be found in a previous report [60]. Briefly, we used a 40x/0.95 NA objective (Olympus) and illuminated the sample with light from 660 nm continuous wave laser (20–30 mW). The backward scattered Brillouin signal was coupled into a single mode optical fiber and analyzed using a two-stage VIPA cross-axis spectrometer. The Brillouin spectrum was recorded using an EMCCD (Andor, iXon 897) with an exposure time of 0.05 s. 2D Brillouin images are acquired by scanning the sample using a motorized stage with a step size of 1 μm or 0.5 μm. Cells were imaged at room temperature conditions within 1 h of taking the cells out of the incubator. A vertical slice (YZ) is always mapped first to identify the middle of the cell which then a horizontal slice (XY) is taken at such Z level. Hoechst 33342 (ThermoFisher, NucBlue Live Ready Probes Reagent) was used to stain the nucleus. Bright field and fluorescent images were acquired using the same 40x/0.95 NA objective and a CMOS camera (Andor, Neo).

A home built LabView (National Instruments) program was used to acquire bright field and Brillouin spectra. The calibration materials (water and methanol) with known Brillouin shifts were used to calculate the spectral dispersion (GHz per pixel ratio) and effective free spectral range of the spectrometer. The Brillouin shift of each pixel was obtained by fitting the Brillouin spectrum to a Lorentzian function in MATLAB (MathWorks, R2023b) and 2D Brillouin images were reconstructed from the pixel vector. As cell culture media show a lower shift compared to cells, we identified the shift of the culture media by manually selecting a set of pixels containing the surrounding media (background) and whole cell shifts and masks were identified in Brillouin maps by thresholding to approximately > 3 $\sigma$ from the average value of the Brillouin shift of the medium. Nuclear shifts and masks were identified by co-locating the fluorescence intensity recorded from the fluorescent channel to the Brillouin maps. The cytoplasm shift was calculated subtracting out the nuclear mask from the whole cell mask. All values reported are averaged Brillouin shifts in the selected region.

Bulk RNA Sequencing and Analysis

RNA extraction was performed using Quick-RNA Microprep Kits (Zymo, R1050) according to manufacturer’s instructions. Briefly, u-Slides were disconnected from Ibidi Pump System and rinsed with PBS before lysis buffer was added. Lysis buffer was pulled through the u-Slides multiple times to ensure complete lysis. For each replicate, 3 μ-Slides were pooled in order to generate enough raw material for library preparation. RNA was quantitated using the Qubit RNA High Sensitivity Assay (ThermoFisher Scientific, Q32855). Libraries were prepared using 200 ng input RNA and KAPA Stranded RNA-Seq Library Preparation Kit (Roche, 07962240001) according to manufacturer’s instructions. Libraries were quantitated using the Qubit dsDNA High Sensitivity assay (ThermoFisher Scientific, Q32854). Fragment size and molarity of libraries was assessed using a D50000 ScreenTape (Agilent, 5067-5588). Libraries were pooled and complexity was evaluated using a MiSeq (Illumina) with the MiSeq Reagent Nano Kit v2 (300 cycles) (Illumina, MS-103-1001). Full scale sequencing was performed using a NovaSeq 6000 using an S4 flow cell with the 100 × 8 × 8 × 100 cycle configuration with 8 × 8 indexing (Illumina, 20028313).

Read trimming was performed using bbmap. Reads were aligned to GRCh38.p14 using Salmon [61] and differential expression analysis was performed using DESeq2 [62] in R. Transcripts with less than 10 counts were discarded prior to performing DESeq analysis. Gene ontology enrichment was performed for biological processes using the clusterProfiler package in R [63].

Statistical Analysis

Statistical analyses were performed using GraphPad Prism or R with p < 0.05 indicating significance. Unpaired parametric t test and two-way ANOVA were run as indicated in figure captions. For ANOVAs, a Tukey post-hoc test was run for multiple comparisons between all conditions tested. Data were collected from n ≥ 3 experiments.

Supplementary Information

Below is the link to the electronic supplementary material.

Data Availability

The data that support the findings of this study are available from the corresponding author upon resonable request. Bulk RNA sequencing data has been submitted to the gene expression omnibus (GEO).

Declarations

Conflict of interest

The authors declare no conflicts of interest.