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Flow-induced HDAC1 phosphorylation and nuclear export in angiogenic sprouting

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ABSTRACT

Angiogenesis requires the coordinated growth and migration of endothelial cells (ECs), with each EC residing in the vessel wall integrating local signals to determine whether to remain quiescent or undergo morphogenesis. These signals include vascular endothelial growth factor (VEGF) and flow-induced mechanical stimuli such as interstitial flow, which are both elevated in the tumor microenvironment. However, it is not clear how VEGF signaling and mechanobiological activation due to interstitial flow cooperate during angiogenesis. Here, we show that endothelial morphogenesis is histone deacetylase-1- (HDAC1) dependent and that interstitial flow increases the phosphorylation of HDAC1, its activity, and its export from the nucleus. Furthermore, we show that HDAC1 inhibition decreases endothelial morphogenesis and matrix metalloproteinase-14 (MMP14) expression. Our results suggest that HDAC1 modulates angiogenesis in response to flow, providing a new target for modulating vascularization in the clinic.

No MeSH data available.


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Interstitial flow-induced HDAC1 phosphorylation and nuclear export in endothelial cells.(a) Representative western blot showing that in the presence of exogenous VEGF (50 ng/ml), interstitial flow leads to increased HDAC1 phosphorylation at Ser421. In the absence of exogenous VEGF and consequently of VEGFR2 phosphorylation, no significant difference could be detected in HDAC1 phosphorylation under flow relative to static. The blot was cropped, and the full-length blot is presented in Supplementary Fig. S1. (b) HDAC1 activity (mU) assay showing a 67% increase in HDAC1 activity under flow relative to static. Data are normalized and shown as % increase in HDAC1 activity under flow conditions relative to static (set as 100%). (c) Western blot analysis (duplicates are shown) and (d) band density quantification showing that in the presence of DHOB (1000 nM) for 1 and 24 h, phosphorylation of HDAC1 at Ser421 is not inhibited. The blot was cropped and the full-length blot is presented in Supplementary Fig. S1. (e) Immunofluorescence staining showing increased p-HDAC1 outside the nuclear (DAPI positive) area under flow (middle row) in relation to static (top row). LMB (20 nM) treatment blocked the increase (bottom row) (Scale bar, 20 μm). (f) Quantification of the area fraction of cytoplasmic t- and p-HDAC1 under static, flow and flow + LMB (20 nM) conditions. Data represent mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
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f3: Interstitial flow-induced HDAC1 phosphorylation and nuclear export in endothelial cells.(a) Representative western blot showing that in the presence of exogenous VEGF (50 ng/ml), interstitial flow leads to increased HDAC1 phosphorylation at Ser421. In the absence of exogenous VEGF and consequently of VEGFR2 phosphorylation, no significant difference could be detected in HDAC1 phosphorylation under flow relative to static. The blot was cropped, and the full-length blot is presented in Supplementary Fig. S1. (b) HDAC1 activity (mU) assay showing a 67% increase in HDAC1 activity under flow relative to static. Data are normalized and shown as % increase in HDAC1 activity under flow conditions relative to static (set as 100%). (c) Western blot analysis (duplicates are shown) and (d) band density quantification showing that in the presence of DHOB (1000 nM) for 1 and 24 h, phosphorylation of HDAC1 at Ser421 is not inhibited. The blot was cropped and the full-length blot is presented in Supplementary Fig. S1. (e) Immunofluorescence staining showing increased p-HDAC1 outside the nuclear (DAPI positive) area under flow (middle row) in relation to static (top row). LMB (20 nM) treatment blocked the increase (bottom row) (Scale bar, 20 μm). (f) Quantification of the area fraction of cytoplasmic t- and p-HDAC1 under static, flow and flow + LMB (20 nM) conditions. Data represent mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Mentions: To examine the mechanism by which HDAC1 modulates angiogenic morphogenesis, we subjected ECs to interstitial flow in the macroscale transwell device. HDAC1 is known to be phosphorylated at Ser421, and this phosphorylation increases its deacetylation activity35. In the presence of VEGF, interstitial flow increased HDAC1 phosphorylation at Ser421 relative to static cultures, suggesting that the phosphorylation of HDAC1 is mechanically regulated (Fig. 3a). In contrast, in the absence of VEGF, and thus in the absence of phosphorylation of VEGFR2, there was no difference in HDAC1 phosphorylation under flow relative to static conditions (Fig. 3a). HDAC1 phosphorylation was accompanied by a 67% increase in its activity (Fig. 3b). These data indicate that phosphorylation, and thus increased activity of HDAC1, plays a role in VEGF- and interstitial flow-induced endothelial morphogenesis.


Flow-induced HDAC1 phosphorylation and nuclear export in angiogenic sprouting
Interstitial flow-induced HDAC1 phosphorylation and nuclear export in endothelial cells.(a) Representative western blot showing that in the presence of exogenous VEGF (50 ng/ml), interstitial flow leads to increased HDAC1 phosphorylation at Ser421. In the absence of exogenous VEGF and consequently of VEGFR2 phosphorylation, no significant difference could be detected in HDAC1 phosphorylation under flow relative to static. The blot was cropped, and the full-length blot is presented in Supplementary Fig. S1. (b) HDAC1 activity (mU) assay showing a 67% increase in HDAC1 activity under flow relative to static. Data are normalized and shown as % increase in HDAC1 activity under flow conditions relative to static (set as 100%). (c) Western blot analysis (duplicates are shown) and (d) band density quantification showing that in the presence of DHOB (1000 nM) for 1 and 24 h, phosphorylation of HDAC1 at Ser421 is not inhibited. The blot was cropped and the full-length blot is presented in Supplementary Fig. S1. (e) Immunofluorescence staining showing increased p-HDAC1 outside the nuclear (DAPI positive) area under flow (middle row) in relation to static (top row). LMB (20 nM) treatment blocked the increase (bottom row) (Scale bar, 20 μm). (f) Quantification of the area fraction of cytoplasmic t- and p-HDAC1 under static, flow and flow + LMB (20 nM) conditions. Data represent mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
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Related In: Results  -  Collection

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f3: Interstitial flow-induced HDAC1 phosphorylation and nuclear export in endothelial cells.(a) Representative western blot showing that in the presence of exogenous VEGF (50 ng/ml), interstitial flow leads to increased HDAC1 phosphorylation at Ser421. In the absence of exogenous VEGF and consequently of VEGFR2 phosphorylation, no significant difference could be detected in HDAC1 phosphorylation under flow relative to static. The blot was cropped, and the full-length blot is presented in Supplementary Fig. S1. (b) HDAC1 activity (mU) assay showing a 67% increase in HDAC1 activity under flow relative to static. Data are normalized and shown as % increase in HDAC1 activity under flow conditions relative to static (set as 100%). (c) Western blot analysis (duplicates are shown) and (d) band density quantification showing that in the presence of DHOB (1000 nM) for 1 and 24 h, phosphorylation of HDAC1 at Ser421 is not inhibited. The blot was cropped and the full-length blot is presented in Supplementary Fig. S1. (e) Immunofluorescence staining showing increased p-HDAC1 outside the nuclear (DAPI positive) area under flow (middle row) in relation to static (top row). LMB (20 nM) treatment blocked the increase (bottom row) (Scale bar, 20 μm). (f) Quantification of the area fraction of cytoplasmic t- and p-HDAC1 under static, flow and flow + LMB (20 nM) conditions. Data represent mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Mentions: To examine the mechanism by which HDAC1 modulates angiogenic morphogenesis, we subjected ECs to interstitial flow in the macroscale transwell device. HDAC1 is known to be phosphorylated at Ser421, and this phosphorylation increases its deacetylation activity35. In the presence of VEGF, interstitial flow increased HDAC1 phosphorylation at Ser421 relative to static cultures, suggesting that the phosphorylation of HDAC1 is mechanically regulated (Fig. 3a). In contrast, in the absence of VEGF, and thus in the absence of phosphorylation of VEGFR2, there was no difference in HDAC1 phosphorylation under flow relative to static conditions (Fig. 3a). HDAC1 phosphorylation was accompanied by a 67% increase in its activity (Fig. 3b). These data indicate that phosphorylation, and thus increased activity of HDAC1, plays a role in VEGF- and interstitial flow-induced endothelial morphogenesis.

View Article: PubMed Central - PubMed

ABSTRACT

Angiogenesis requires the coordinated growth and migration of endothelial cells (ECs), with each EC residing in the vessel wall integrating local signals to determine whether to remain quiescent or undergo morphogenesis. These signals include vascular endothelial growth factor (VEGF) and flow-induced mechanical stimuli such as interstitial flow, which are both elevated in the tumor microenvironment. However, it is not clear how VEGF signaling and mechanobiological activation due to interstitial flow cooperate during angiogenesis. Here, we show that endothelial morphogenesis is histone deacetylase-1- (HDAC1) dependent and that interstitial flow increases the phosphorylation of HDAC1, its activity, and its export from the nucleus. Furthermore, we show that HDAC1 inhibition decreases endothelial morphogenesis and matrix metalloproteinase-14 (MMP14) expression. Our results suggest that HDAC1 modulates angiogenesis in response to flow, providing a new target for modulating vascularization in the clinic.

No MeSH data available.


Related in: MedlinePlus