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Flow-induced HDAC1 phosphorylation and nuclear export in angiogenic sprouting

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.


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HDAC1 enables angiogenic invasion under flow.Blocking HDAC1 with the selective HDAC1 inhibitor DHOB (1000 nM) for 24 h (a) under static conditions (without a VEGF gradient) has no effect on angiogenic invasion, while (b) in the presence of interstitial flow and a VEGF gradient sprouting is significantly reduced. The dashed arrow indicates the flow direction. Control devices (treated with DMSO) showed significant invasion under static and flow conditions. (c) Quantification of invasion (displayed as the % of spout area) after one day of continuous DHOB treatment under static and flow conditions. Data represent mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001. (d) DHOB at 1000 nM did not promote EC apoptosis after 1 and 24 h of continuous perfusion as determined by western blot analysis for cleaved caspase 3. (e) Knockdown of HDAC1 was achieved by siRNA transfection. (f) Silencing HDAC1 with siRNA under static (no VEGF gradient) conditions (top row) has a small effect on angiogenic sprouting, while in the presence of interstitial flow and a VEGF gradient (bottom row) sprouting is significantly reduced. Exogenous VEGF (50 ng/ml) was also included in the media. The dashed arrow indicates the flow direction. In contrast, control devices with cells transfected with non-targeting siRNA proceeded with significant invasion under static and flow conditions. (g) Quantification of the % of invasion area after one day of under static and flow conditions for HDAC1 and non-targeting siRNA treated endothelial cells. Data represent mean ± SEM, *p < 0.05, **p < 0.01, ****p < 0.0001.
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f2: HDAC1 enables angiogenic invasion under flow.Blocking HDAC1 with the selective HDAC1 inhibitor DHOB (1000 nM) for 24 h (a) under static conditions (without a VEGF gradient) has no effect on angiogenic invasion, while (b) in the presence of interstitial flow and a VEGF gradient sprouting is significantly reduced. The dashed arrow indicates the flow direction. Control devices (treated with DMSO) showed significant invasion under static and flow conditions. (c) Quantification of invasion (displayed as the % of spout area) after one day of continuous DHOB treatment under static and flow conditions. Data represent mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001. (d) DHOB at 1000 nM did not promote EC apoptosis after 1 and 24 h of continuous perfusion as determined by western blot analysis for cleaved caspase 3. (e) Knockdown of HDAC1 was achieved by siRNA transfection. (f) Silencing HDAC1 with siRNA under static (no VEGF gradient) conditions (top row) has a small effect on angiogenic sprouting, while in the presence of interstitial flow and a VEGF gradient (bottom row) sprouting is significantly reduced. Exogenous VEGF (50 ng/ml) was also included in the media. The dashed arrow indicates the flow direction. In contrast, control devices with cells transfected with non-targeting siRNA proceeded with significant invasion under static and flow conditions. (g) Quantification of the % of invasion area after one day of under static and flow conditions for HDAC1 and non-targeting siRNA treated endothelial cells. Data represent mean ± SEM, *p < 0.05, **p < 0.01, ****p < 0.0001.

Mentions: We next investigated whether HDAC1 plays a role in endothelial cell morphogenesis in response to interstitial flow in our microfluidic device. ECs were continuously treated with an inhibitor of HDAC1, 4-(dimethylamino)-N- [6-(hydroxyamino)-6-oxohexyl]-benzamide (DHOB), for 24 h in the presence of interstitial flow or under static conditions. Exogenous VEGF (50 ng/ml) was also included in the media. ECs in control (CTL DMSO) devices invaded under static and flow conditions (Fig. 2a–c). DHOB inhibited morphogenic invasion under flow conditions, but not in static devices. These results suggest that endothelial morphogenesis is HDAC1-dependent in the presence of interstitial flow but HDAC1-independent under static conditions. In these experiments, DHOB did not induce cell apoptosis: DHOB at the highest concentration employed (1000 nM) did not induce cleaved caspase-3 (CC-3) over 24 h of continuous perfusion (Fig. 2d).


Flow-induced HDAC1 phosphorylation and nuclear export in angiogenic sprouting
HDAC1 enables angiogenic invasion under flow.Blocking HDAC1 with the selective HDAC1 inhibitor DHOB (1000 nM) for 24 h (a) under static conditions (without a VEGF gradient) has no effect on angiogenic invasion, while (b) in the presence of interstitial flow and a VEGF gradient sprouting is significantly reduced. The dashed arrow indicates the flow direction. Control devices (treated with DMSO) showed significant invasion under static and flow conditions. (c) Quantification of invasion (displayed as the % of spout area) after one day of continuous DHOB treatment under static and flow conditions. Data represent mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001. (d) DHOB at 1000 nM did not promote EC apoptosis after 1 and 24 h of continuous perfusion as determined by western blot analysis for cleaved caspase 3. (e) Knockdown of HDAC1 was achieved by siRNA transfection. (f) Silencing HDAC1 with siRNA under static (no VEGF gradient) conditions (top row) has a small effect on angiogenic sprouting, while in the presence of interstitial flow and a VEGF gradient (bottom row) sprouting is significantly reduced. Exogenous VEGF (50 ng/ml) was also included in the media. The dashed arrow indicates the flow direction. In contrast, control devices with cells transfected with non-targeting siRNA proceeded with significant invasion under static and flow conditions. (g) Quantification of the % of invasion area after one day of under static and flow conditions for HDAC1 and non-targeting siRNA treated endothelial cells. Data represent mean ± SEM, *p < 0.05, **p < 0.01, ****p < 0.0001.
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f2: HDAC1 enables angiogenic invasion under flow.Blocking HDAC1 with the selective HDAC1 inhibitor DHOB (1000 nM) for 24 h (a) under static conditions (without a VEGF gradient) has no effect on angiogenic invasion, while (b) in the presence of interstitial flow and a VEGF gradient sprouting is significantly reduced. The dashed arrow indicates the flow direction. Control devices (treated with DMSO) showed significant invasion under static and flow conditions. (c) Quantification of invasion (displayed as the % of spout area) after one day of continuous DHOB treatment under static and flow conditions. Data represent mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001. (d) DHOB at 1000 nM did not promote EC apoptosis after 1 and 24 h of continuous perfusion as determined by western blot analysis for cleaved caspase 3. (e) Knockdown of HDAC1 was achieved by siRNA transfection. (f) Silencing HDAC1 with siRNA under static (no VEGF gradient) conditions (top row) has a small effect on angiogenic sprouting, while in the presence of interstitial flow and a VEGF gradient (bottom row) sprouting is significantly reduced. Exogenous VEGF (50 ng/ml) was also included in the media. The dashed arrow indicates the flow direction. In contrast, control devices with cells transfected with non-targeting siRNA proceeded with significant invasion under static and flow conditions. (g) Quantification of the % of invasion area after one day of under static and flow conditions for HDAC1 and non-targeting siRNA treated endothelial cells. Data represent mean ± SEM, *p < 0.05, **p < 0.01, ****p < 0.0001.
Mentions: We next investigated whether HDAC1 plays a role in endothelial cell morphogenesis in response to interstitial flow in our microfluidic device. ECs were continuously treated with an inhibitor of HDAC1, 4-(dimethylamino)-N- [6-(hydroxyamino)-6-oxohexyl]-benzamide (DHOB), for 24 h in the presence of interstitial flow or under static conditions. Exogenous VEGF (50 ng/ml) was also included in the media. ECs in control (CTL DMSO) devices invaded under static and flow conditions (Fig. 2a–c). DHOB inhibited morphogenic invasion under flow conditions, but not in static devices. These results suggest that endothelial morphogenesis is HDAC1-dependent in the presence of interstitial flow but HDAC1-independent under static conditions. In these experiments, DHOB did not induce cell apoptosis: DHOB at the highest concentration employed (1000 nM) did not induce cleaved caspase-3 (CC-3) over 24 h of continuous perfusion (Fig. 2d).

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