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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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Angiogenic sprouting in a microfluidic device.(a) Schematic of the PDMS microfluidic device. HUVECs are seeded into two channels (red) separated by the polymerized collagen gel (purple). Transendothelial interstitial flow (arrow) is applied across the collagen gel. Each HUVEC channel has independent input and outlet ports allowing strict flow control in both channels. Close - up view of the boxed area shows three apertures that allow invasion and sprouting of the lumenized vessel segments (red) into the central collagen gel (purple). (b) Immunofluorescence staining for Notch-1 (white) and Dll4 (red) expression in GFP transduced HUVECs sprouting in 3D collagen. Nuclei were stained with DAPI (Scale bar, 25 μm). (c) Angiogenic sprouting and invasion into the 3-D collagen matrix under interstitial flow (dashed arrow indicates flow direction) and a VEGF gradient is significantly inhibited in response to 1000 nM of sunitinib. Control devices treated with DMSO proceeded with extensive sprouting (Scale bar, 100 μm). Data represent mean ± SEM, ****p < 0.0001. (d) Schematic of the transwell macroscale device. Collagen and fibronectin were loaded into a 6-well transwell chamber and HUVECs (106/ml) in EGM media were allowed to spread and form a confluent monolayer for 24 h prior to flow initiation. Interstitial flow (0.5 μm/sec) across the HUVEC monolayer was driven using a programmable syringe pump (Harvard Apparatus). Boxed area shows a close-up schematic of interstitial flow imposed across the 6-well transwell. Flow is driven through the EC junctions, collagen gel substrate and the transwell ports analogous to an apical/lumen - basal flow.
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f1: Angiogenic sprouting in a microfluidic device.(a) Schematic of the PDMS microfluidic device. HUVECs are seeded into two channels (red) separated by the polymerized collagen gel (purple). Transendothelial interstitial flow (arrow) is applied across the collagen gel. Each HUVEC channel has independent input and outlet ports allowing strict flow control in both channels. Close - up view of the boxed area shows three apertures that allow invasion and sprouting of the lumenized vessel segments (red) into the central collagen gel (purple). (b) Immunofluorescence staining for Notch-1 (white) and Dll4 (red) expression in GFP transduced HUVECs sprouting in 3D collagen. Nuclei were stained with DAPI (Scale bar, 25 μm). (c) Angiogenic sprouting and invasion into the 3-D collagen matrix under interstitial flow (dashed arrow indicates flow direction) and a VEGF gradient is significantly inhibited in response to 1000 nM of sunitinib. Control devices treated with DMSO proceeded with extensive sprouting (Scale bar, 100 μm). Data represent mean ± SEM, ****p < 0.0001. (d) Schematic of the transwell macroscale device. Collagen and fibronectin were loaded into a 6-well transwell chamber and HUVECs (106/ml) in EGM media were allowed to spread and form a confluent monolayer for 24 h prior to flow initiation. Interstitial flow (0.5 μm/sec) across the HUVEC monolayer was driven using a programmable syringe pump (Harvard Apparatus). Boxed area shows a close-up schematic of interstitial flow imposed across the 6-well transwell. Flow is driven through the EC junctions, collagen gel substrate and the transwell ports analogous to an apical/lumen - basal flow.

Mentions: To determine how interstitial flow modulates angiogenic morphogenesis, we used our previously described microfluidic device that provides control of fluid convection through bio-mimetic vessels17. In this device, human umbilical vein endothelial cells (HUVECs) form parallel vessels separated by a 3-dimensional (3D) collagen I/fibronectin matrix (Fig. 1a). HUVECs stimulated with 50 ng/ml VEGF migrate into the 3D matrix to create new lumenized vessels that eventually connect the two pre-defined vessels (Fig. 1a, schematic), thus mimicking angiogenic sprouting and anastomosis. In this process, ECs extend filopodial extensions analogous to tip cell sprouts seen in vivo3132. The extending sprouts display the ‘typical’ molecular signature of sprouting cells: increased Delta-like ligand-4 (Dll4) staining in tip cells, while the expression of Dll4 is reduced in the trailing stalk cells (Fig. 1b). In contrast, Notch-1 is expressed diffusely in stalk cells (Fig. 1b), consistent with the mechanism of tip-stalk cell communication previously reported4. In addition, these endothelial sprouts respond appropriately to anti-angiogenic treatment. Addition of sunitinib, which blocks VEGFR2 phosphorylation at Tyr951, inhibits invasion into the collagen matrix (Fig. 1c).


Flow-induced HDAC1 phosphorylation and nuclear export in angiogenic sprouting
Angiogenic sprouting in a microfluidic device.(a) Schematic of the PDMS microfluidic device. HUVECs are seeded into two channels (red) separated by the polymerized collagen gel (purple). Transendothelial interstitial flow (arrow) is applied across the collagen gel. Each HUVEC channel has independent input and outlet ports allowing strict flow control in both channels. Close - up view of the boxed area shows three apertures that allow invasion and sprouting of the lumenized vessel segments (red) into the central collagen gel (purple). (b) Immunofluorescence staining for Notch-1 (white) and Dll4 (red) expression in GFP transduced HUVECs sprouting in 3D collagen. Nuclei were stained with DAPI (Scale bar, 25 μm). (c) Angiogenic sprouting and invasion into the 3-D collagen matrix under interstitial flow (dashed arrow indicates flow direction) and a VEGF gradient is significantly inhibited in response to 1000 nM of sunitinib. Control devices treated with DMSO proceeded with extensive sprouting (Scale bar, 100 μm). Data represent mean ± SEM, ****p < 0.0001. (d) Schematic of the transwell macroscale device. Collagen and fibronectin were loaded into a 6-well transwell chamber and HUVECs (106/ml) in EGM media were allowed to spread and form a confluent monolayer for 24 h prior to flow initiation. Interstitial flow (0.5 μm/sec) across the HUVEC monolayer was driven using a programmable syringe pump (Harvard Apparatus). Boxed area shows a close-up schematic of interstitial flow imposed across the 6-well transwell. Flow is driven through the EC junctions, collagen gel substrate and the transwell ports analogous to an apical/lumen - basal flow.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037418&req=5

f1: Angiogenic sprouting in a microfluidic device.(a) Schematic of the PDMS microfluidic device. HUVECs are seeded into two channels (red) separated by the polymerized collagen gel (purple). Transendothelial interstitial flow (arrow) is applied across the collagen gel. Each HUVEC channel has independent input and outlet ports allowing strict flow control in both channels. Close - up view of the boxed area shows three apertures that allow invasion and sprouting of the lumenized vessel segments (red) into the central collagen gel (purple). (b) Immunofluorescence staining for Notch-1 (white) and Dll4 (red) expression in GFP transduced HUVECs sprouting in 3D collagen. Nuclei were stained with DAPI (Scale bar, 25 μm). (c) Angiogenic sprouting and invasion into the 3-D collagen matrix under interstitial flow (dashed arrow indicates flow direction) and a VEGF gradient is significantly inhibited in response to 1000 nM of sunitinib. Control devices treated with DMSO proceeded with extensive sprouting (Scale bar, 100 μm). Data represent mean ± SEM, ****p < 0.0001. (d) Schematic of the transwell macroscale device. Collagen and fibronectin were loaded into a 6-well transwell chamber and HUVECs (106/ml) in EGM media were allowed to spread and form a confluent monolayer for 24 h prior to flow initiation. Interstitial flow (0.5 μm/sec) across the HUVEC monolayer was driven using a programmable syringe pump (Harvard Apparatus). Boxed area shows a close-up schematic of interstitial flow imposed across the 6-well transwell. Flow is driven through the EC junctions, collagen gel substrate and the transwell ports analogous to an apical/lumen - basal flow.
Mentions: To determine how interstitial flow modulates angiogenic morphogenesis, we used our previously described microfluidic device that provides control of fluid convection through bio-mimetic vessels17. In this device, human umbilical vein endothelial cells (HUVECs) form parallel vessels separated by a 3-dimensional (3D) collagen I/fibronectin matrix (Fig. 1a). HUVECs stimulated with 50 ng/ml VEGF migrate into the 3D matrix to create new lumenized vessels that eventually connect the two pre-defined vessels (Fig. 1a, schematic), thus mimicking angiogenic sprouting and anastomosis. In this process, ECs extend filopodial extensions analogous to tip cell sprouts seen in vivo3132. The extending sprouts display the ‘typical’ molecular signature of sprouting cells: increased Delta-like ligand-4 (Dll4) staining in tip cells, while the expression of Dll4 is reduced in the trailing stalk cells (Fig. 1b). In contrast, Notch-1 is expressed diffusely in stalk cells (Fig. 1b), consistent with the mechanism of tip-stalk cell communication previously reported4. In addition, these endothelial sprouts respond appropriately to anti-angiogenic treatment. Addition of sunitinib, which blocks VEGFR2 phosphorylation at Tyr951, inhibits invasion into the collagen matrix (Fig. 1c).

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