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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.

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MMP14 expression is regulated by HDAC1.(a) Representative western blot showing that HDAC1 blockade with DHOB (1000 nM) significantly reduced MMP14 expression after 1 h of treatment under flow but not under static conditions. The blot was cropped and the full-length blot is presented in Supplementary Fig. S1. (b) Representative western blot showing that inhibition of HDAC1 nuclear export with LMB (20 nM) significantly reduced MMP14 expression after 1 h of treatment under flow but not under static conditions. (c) MMP14 selective blockade (100 μg/ml anti-MMP14) resulted in significant reduction of invasion under static (no VEGF gradient) and (d) flow (dashed arrow indicates the direction of flow) and VEGF gradient (bottom row) conditions. Control (CTL-100 μg/ml IgG) devices (both static and flow) proceeded with extensive sprouting. (Scale bar, 100 μm). (e) Quantification of the % area of invasion further supports the microscopic observations. Data represent mean ± SEM, *p < 0.05.
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f5: MMP14 expression is regulated by HDAC1.(a) Representative western blot showing that HDAC1 blockade with DHOB (1000 nM) significantly reduced MMP14 expression after 1 h of treatment under flow but not under static conditions. The blot was cropped and the full-length blot is presented in Supplementary Fig. S1. (b) Representative western blot showing that inhibition of HDAC1 nuclear export with LMB (20 nM) significantly reduced MMP14 expression after 1 h of treatment under flow but not under static conditions. (c) MMP14 selective blockade (100 μg/ml anti-MMP14) resulted in significant reduction of invasion under static (no VEGF gradient) and (d) flow (dashed arrow indicates the direction of flow) and VEGF gradient (bottom row) conditions. Control (CTL-100 μg/ml IgG) devices (both static and flow) proceeded with extensive sprouting. (Scale bar, 100 μm). (e) Quantification of the % area of invasion further supports the microscopic observations. Data represent mean ± SEM, *p < 0.05.

Mentions: We next examined potential downstream effectors involved in sprouting angiogenesis. It is known that MMPs are intimately involved in the angiogenic process, and MMP14 is expressed by endothelial tip cells3839. We found that MMP14 expression increased when HUVECs were subjected to intersitital flow for 1 h (Fig. 5a). We thus assessed whether MMP14 expression in response to flow is dependent on HDAC1. We found that inhibition of HDAC1 with DHOB reduced the increase in MMP14 expression induced by flow (Fig. 5a). Furthermore, inhibition of protein nuclear export with LMB also downregulated MMP14 protein levels after flow (Fig. 5b), suggesting the increase in MMP14 expression after flow may be co-regulated by HDAC1 nuclear export. We then performed our functional assay in the microfluidic channels, further assessing the role of MMP14 in flow-induced angiogenic sprouting. Selective targeting of MMP14 significantly inhibited EC sprouting and invasion under both static (Fig. 5c,e) and flow conditions (Fig. 5d,e).


Flow-induced HDAC1 phosphorylation and nuclear export in angiogenic sprouting
MMP14 expression is regulated by HDAC1.(a) Representative western blot showing that HDAC1 blockade with DHOB (1000 nM) significantly reduced MMP14 expression after 1 h of treatment under flow but not under static conditions. The blot was cropped and the full-length blot is presented in Supplementary Fig. S1. (b) Representative western blot showing that inhibition of HDAC1 nuclear export with LMB (20 nM) significantly reduced MMP14 expression after 1 h of treatment under flow but not under static conditions. (c) MMP14 selective blockade (100 μg/ml anti-MMP14) resulted in significant reduction of invasion under static (no VEGF gradient) and (d) flow (dashed arrow indicates the direction of flow) and VEGF gradient (bottom row) conditions. Control (CTL-100 μg/ml IgG) devices (both static and flow) proceeded with extensive sprouting. (Scale bar, 100 μm). (e) Quantification of the % area of invasion further supports the microscopic observations. Data represent mean ± SEM, *p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5037418&req=5

f5: MMP14 expression is regulated by HDAC1.(a) Representative western blot showing that HDAC1 blockade with DHOB (1000 nM) significantly reduced MMP14 expression after 1 h of treatment under flow but not under static conditions. The blot was cropped and the full-length blot is presented in Supplementary Fig. S1. (b) Representative western blot showing that inhibition of HDAC1 nuclear export with LMB (20 nM) significantly reduced MMP14 expression after 1 h of treatment under flow but not under static conditions. (c) MMP14 selective blockade (100 μg/ml anti-MMP14) resulted in significant reduction of invasion under static (no VEGF gradient) and (d) flow (dashed arrow indicates the direction of flow) and VEGF gradient (bottom row) conditions. Control (CTL-100 μg/ml IgG) devices (both static and flow) proceeded with extensive sprouting. (Scale bar, 100 μm). (e) Quantification of the % area of invasion further supports the microscopic observations. Data represent mean ± SEM, *p < 0.05.
Mentions: We next examined potential downstream effectors involved in sprouting angiogenesis. It is known that MMPs are intimately involved in the angiogenic process, and MMP14 is expressed by endothelial tip cells3839. We found that MMP14 expression increased when HUVECs were subjected to intersitital flow for 1 h (Fig. 5a). We thus assessed whether MMP14 expression in response to flow is dependent on HDAC1. We found that inhibition of HDAC1 with DHOB reduced the increase in MMP14 expression induced by flow (Fig. 5a). Furthermore, inhibition of protein nuclear export with LMB also downregulated MMP14 protein levels after flow (Fig. 5b), suggesting the increase in MMP14 expression after flow may be co-regulated by HDAC1 nuclear export. We then performed our functional assay in the microfluidic channels, further assessing the role of MMP14 in flow-induced angiogenic sprouting. Selective targeting of MMP14 significantly inhibited EC sprouting and invasion under both static (Fig. 5c,e) and flow conditions (Fig. 5d,e).

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