Please wait a minute...
Shopping Cart Email List Chinese
Advanced Search Fig/Tab
Protein & Cell

ISSN 1674-800X

ISSN 1674-8018(Online)

CN 11-5886/Q

Postal Subscription Code 80-984

2018 Impact Factor: 7.575

Featured Articles  |  Most Downloaded  |  Most Reading
Online Submission Subscribe to Our RSS Content Feed
Prot Cell    2011, Vol. 2 Issue (2) : 150-160    https://doi.org/10.1007/s13238-011-1015-4      PMID: 21359602
RESEARCH ARTICLE
Microtubule-associated deacetylase HDAC6 promotes angiogenesis by regulating cell migration in an EB1-dependent manner
Dengwen Li1, Songbo Xie1, Yuan Ren1, Lihong Huo1, Jinmin Gao1, Dandan Cui1, Min Liu2, Jun Zhou1()
1. Department of Genetics and Cell Biology, Tianjin Key Laboratory of Protein Science, College of Life Sciences, Nankai University, Tianjin 300071, China; 2. Department of Biochemistry, Tianjin Key Laboratory of Cellular and Molecular Immunology, Basic Medical College, Tianjin Medical University, Tianjin 300071, China
 Download: PDF(1096 KB)   HTML
 Export: BibTeX | EndNote | Reference Manager | ProCite | RefWorks
Abstract

Angiogenesis, a process by which the preexisting blood vasculature gives rise to new capillary vessels, is associated with a variety of physiologic and pathologic conditions. However, the molecular mechanism underlying this important process remains poorly understood. Here we show that histone deacetylase 6 (HDAC6), a microtubule-associated enzyme critical for cell motility, contributes to angiogenesis by regulating the polarization and migration of vascular endothelial cells. Inhibition of HDAC6 activity impairs the formation of new blood vessels in chick embryos and in angioreactors implanted in mice. The requirement for HDAC6 in angiogenesis is corroborated in vitro by analysis of endothelial tube formation and capillary sprouting. Our data further show that HDAC6 stimulates membrane ruffling at the leading edge to promote cell polarization. In addition, microtubule end binding protein 1 (EB1) is important for HDAC6 to exert its activity towards the migration of endothelial cells and generation of capillary-like structures. These results thus identify HDAC6 as a novel player in the angiogenic process and offer novel insights into the molecular mechanism governing endothelial cell migration and angiogenesis.
Keywords angiogenesis      histone deacetylase 6 (HDAC6)      cell migration      cell polarization      microtubule end binding protein 1 (EB1)     
Corresponding Author(s): Zhou Jun,Email:junzhou@nankai.edu.cn   
Issue Date: 01 February 2011
 Cite this article:   
Dengwen Li,Songbo Xie,Yuan Ren, et al. Microtubule-associated deacetylase HDAC6 promotes angiogenesis by regulating cell migration in an EB1-dependent manner[J]. Prot Cell, 2011, 2(2): 150-160.
 URL:  
https://academic.hep.com.cn/pac/EN/10.1007/s13238-011-1015-4
https://academic.hep.com.cn/pac/EN/Y2011/V2/I2/150
Fig.1  HDAC6 activity is critical for angiogenesis in chick embryos.
(A) Filter paper soaked with the HDAC6 inhibitor tubacin or the vehicle DMSO (control) was placed on the vitelline membrane of a 4-day-old chick embryo cultured . (B) At day 7, capillaries underneath the filter paper were photographed with a stereomicroscope. (C) Experiments were performed as in panels A and B, and the density of blood vessels was quantified (** <0.01). (D) Matrigel with DMSO or tubacin was polymerized around two layers of nylon mesh and placed on the chorioallantoic membrane of a 10-day-old chick embryo. (E) At day 13, blood vessel growth into matrigel/nylon mesh was photographed with a stereomicroscope. (F) Experiments were performed as in panels D and E, and the percentage of positive grids was quantified (* <0.05).
Fig.1  HDAC6 activity is critical for angiogenesis in chick embryos.
(A) Filter paper soaked with the HDAC6 inhibitor tubacin or the vehicle DMSO (control) was placed on the vitelline membrane of a 4-day-old chick embryo cultured . (B) At day 7, capillaries underneath the filter paper were photographed with a stereomicroscope. (C) Experiments were performed as in panels A and B, and the density of blood vessels was quantified (** <0.01). (D) Matrigel with DMSO or tubacin was polymerized around two layers of nylon mesh and placed on the chorioallantoic membrane of a 10-day-old chick embryo. (E) At day 13, blood vessel growth into matrigel/nylon mesh was photographed with a stereomicroscope. (F) Experiments were performed as in panels D and E, and the percentage of positive grids was quantified (* <0.05).
Fig.2  Inhibition of HDAC6 activity impairs blood vessel growth into the angioreactors implanted in mice.
(A) Immunolocalization of HDAC6 in mouse tissues. Frozen sections of liver, small intestine, adrenal gland, and esophagus tissues were stained with antibodies against HDAC6 and CD31. The localization of HDAC6 in the vascular endothelium was then examined with a fluorescence microscope. (B) Diagram of directed angiogenesis assay. Semiclosed angioreactors were implanted subcutaneously into the dorsal flank of athymic nude mice for 11 days, and vascular growth into the angioreactors was photographed (panels C and D). In panel C, angioreactors were filled with matrigel in the absence or presence of heparin/FGF2. In panel D, angioreactors were filled with matrigel in the presence of heparin/FGF2 and DMSO or tubacin. (E) Quantification of the density of blood vessels in the angioreactors (** <0.01). (F) Frozen sections of the vessel-containing matrigel in angioreactors were stained with anti-CD31 antibody (red) and the nuclear dye DAPI (blue). (G) Cell pellets extracted from the vessel-containing angioreactors were stained with FITC-lectin, and the angiogenic response was reflected by the fluorescence intensity (** <0.01). RFU, relative fluorescence unit.
Fig.2  Inhibition of HDAC6 activity impairs blood vessel growth into the angioreactors implanted in mice.
(A) Immunolocalization of HDAC6 in mouse tissues. Frozen sections of liver, small intestine, adrenal gland, and esophagus tissues were stained with antibodies against HDAC6 and CD31. The localization of HDAC6 in the vascular endothelium was then examined with a fluorescence microscope. (B) Diagram of directed angiogenesis assay. Semiclosed angioreactors were implanted subcutaneously into the dorsal flank of athymic nude mice for 11 days, and vascular growth into the angioreactors was photographed (panels C and D). In panel C, angioreactors were filled with matrigel in the absence or presence of heparin/FGF2. In panel D, angioreactors were filled with matrigel in the presence of heparin/FGF2 and DMSO or tubacin. (E) Quantification of the density of blood vessels in the angioreactors (** <0.01). (F) Frozen sections of the vessel-containing matrigel in angioreactors were stained with anti-CD31 antibody (red) and the nuclear dye DAPI (blue). (G) Cell pellets extracted from the vessel-containing angioreactors were stained with FITC-lectin, and the angiogenic response was reflected by the fluorescence intensity (** <0.01). RFU, relative fluorescence unit.
Fig.3  HDAC6 is required for endothelial tube formation and capillary sprouting.
(A) HUVECs were plated onto matrigel and treated with DMSO or tubacin for 4, 24, or 36 h, and the formation of endothelial tubes was examined. (B) Experiments were performed as in panel A, and cumulative tube length was quantified (* <0.05 control). (C) HUVECs were transfected with HDAC6 or control siRNAs, and the expression of HDAC6 and actin was examined by immunoblotting. (D) HUVECs transfected with HDAC6 or control siRNAs were plated onto matrigel, and photographs were taken 24 h later. (E) Experiments were performed as in panel D, and cumulative tube length was quantified (** <0.01 control). (F) Spheroids generated from HUVECs were treated with DMSO or tubacin, and capillary-like sprout formation was then examined. (G) Experiments were performed as in panel F, and cumulative sprout length was quantified (** <0.01 control). (H and I) Capillary sprouting from spheroids generated from HUVECs transfected with HDAC6 or control siRNAs (** <0.01 control).
Fig.3  HDAC6 is required for endothelial tube formation and capillary sprouting.
(A) HUVECs were plated onto matrigel and treated with DMSO or tubacin for 4, 24, or 36 h, and the formation of endothelial tubes was examined. (B) Experiments were performed as in panel A, and cumulative tube length was quantified (* <0.05 control). (C) HUVECs were transfected with HDAC6 or control siRNAs, and the expression of HDAC6 and actin was examined by immunoblotting. (D) HUVECs transfected with HDAC6 or control siRNAs were plated onto matrigel, and photographs were taken 24 h later. (E) Experiments were performed as in panel D, and cumulative tube length was quantified (** <0.01 control). (F) Spheroids generated from HUVECs were treated with DMSO or tubacin, and capillary-like sprout formation was then examined. (G) Experiments were performed as in panel F, and cumulative sprout length was quantified (** <0.01 control). (H and I) Capillary sprouting from spheroids generated from HUVECs transfected with HDAC6 or control siRNAs (** <0.01 control).
Fig.4  HDAC6 mediates vascular endothelial cell migration.
(A) HUVECs were treated with DMSO or tubacin and scratched, and wound margins were photographed 0 or 24 h later. (B) Experiments were performed as in panel A, and the extent of wound closure was quantified by measuring the wound area compared with the initial wound area (** <0.01 control). (C) HUVECs transfected with control or HDAC6 siRNAs were scratched, and wound margins were photographed 24 h later. (D) Experiments were performed as in panel C, and the extent of wound closure was quantified (** <0.01 control). (E and F) Experiments were performed as in panels A and C, respectively, and the extent of wound closure was examined at different time points.
Fig.4  HDAC6 mediates vascular endothelial cell migration.
(A) HUVECs were treated with DMSO or tubacin and scratched, and wound margins were photographed 0 or 24 h later. (B) Experiments were performed as in panel A, and the extent of wound closure was quantified by measuring the wound area compared with the initial wound area (** <0.01 control). (C) HUVECs transfected with control or HDAC6 siRNAs were scratched, and wound margins were photographed 24 h later. (D) Experiments were performed as in panel C, and the extent of wound closure was quantified (** <0.01 control). (E and F) Experiments were performed as in panels A and C, respectively, and the extent of wound closure was examined at different time points.
Fig.5  HDAC6 stimulates membrane ruffling and cell polarization.
(A) HUVECs transfected with pEGFPC1 were treated with DMSO or tubacin and scratched. The fluorescence of GFP at the leading edge of cells was recorded at 20-second intervals. Rectangular regions were selected as indicated to analyze membrane ruffle dynamics. (B) Experiments were performed as in panel A, and membrane ruffle dynamics were presented as three-dimensional surface plots. (C) HUVECs were treated with DMSO or tubacin and scratched, and cells were fixed 3 h later and stained with anti-α-tubulin antibody, anti-pericentrin antibody, and DAPI to visualize microtubules (green), centrosomes (red), and nuclei (blue), respectively. Broken white lines indicate the wound direction. (D) Experiments were performed as in panel C, and the percentage of polarized cells at the wound margin was quantified (** <0.01). (E and F) HUVECs transfected with control or HDAC6 siRNAs were scratched, and the extent of cell polarization was examined as in panels C and D (* <0.05 control).
Fig.5  HDAC6 stimulates membrane ruffling and cell polarization.
(A) HUVECs transfected with pEGFPC1 were treated with DMSO or tubacin and scratched. The fluorescence of GFP at the leading edge of cells was recorded at 20-second intervals. Rectangular regions were selected as indicated to analyze membrane ruffle dynamics. (B) Experiments were performed as in panel A, and membrane ruffle dynamics were presented as three-dimensional surface plots. (C) HUVECs were treated with DMSO or tubacin and scratched, and cells were fixed 3 h later and stained with anti-α-tubulin antibody, anti-pericentrin antibody, and DAPI to visualize microtubules (green), centrosomes (red), and nuclei (blue), respectively. Broken white lines indicate the wound direction. (D) Experiments were performed as in panel C, and the percentage of polarized cells at the wound margin was quantified (** <0.01). (E and F) HUVECs transfected with control or HDAC6 siRNAs were scratched, and the extent of cell polarization was examined as in panels C and D (* <0.05 control).
Fig.6  HDAC6 acts on EB1 to promote cell migration and angiogenesis.
(A) HUVECs were transfected with HDAC6 or EB1 siRNAs, together with pEGFPC1, pEGFPC1-HDAC6 or pEGFPC1-EB1. Cells were scratched, and wound margins were examined with fluorescence microscopy 24 h later. (B) Experiments were performed as in panel A, and the extent of wound closure was quantified. (C) HUVECs were transfected with HDAC6 siRNA together with pEGFPC1, pEGFPC1-HDAC6 or pEGFPC1-EB1, and the formation of endothelial tubes was examined with fluorescence microscopy 6 or 12 h later. (D) Experiments were performed as in panel C, and cumulative tube length was quantified. (E) HUVECs were transfected with HDAC6 siRNA together with pEGFPC1, pEGFPC1-HDAC6 or pEGFPC1-EB1, and capillary-like sprout formation from the endothelial spheroids was examined with fluorescence microscopy. (F) Experiments were performed as in panel E, and cumulative sprout length was quantified.
Fig.6  HDAC6 acts on EB1 to promote cell migration and angiogenesis.
(A) HUVECs were transfected with HDAC6 or EB1 siRNAs, together with pEGFPC1, pEGFPC1-HDAC6 or pEGFPC1-EB1. Cells were scratched, and wound margins were examined with fluorescence microscopy 24 h later. (B) Experiments were performed as in panel A, and the extent of wound closure was quantified. (C) HUVECs were transfected with HDAC6 siRNA together with pEGFPC1, pEGFPC1-HDAC6 or pEGFPC1-EB1, and the formation of endothelial tubes was examined with fluorescence microscopy 6 or 12 h later. (D) Experiments were performed as in panel C, and cumulative tube length was quantified. (E) HUVECs were transfected with HDAC6 siRNA together with pEGFPC1, pEGFPC1-HDAC6 or pEGFPC1-EB1, and capillary-like sprout formation from the endothelial spheroids was examined with fluorescence microscopy. (F) Experiments were performed as in panel E, and cumulative sprout length was quantified.
1 Bertos, N.R., Gilquin, B., Chan, G.K., Yen, T.J., Khochbin, S., and Yang, X.J. (2004). Role of the tetradecapeptide repeat domain of human histone deacetylase 6 in cytoplasmic retention. J Biol Chem 279, 48246–48254 .
pmid:15347674
2 Bornens, M. (2008). Organelle positioning and cell polarity. Nat Rev Mol Cell Biol 9, 874–886 .
pmid:18946476
3 Boyault, C., Sadoul, K., Pabion, M., and Khochbin, S. (2007). HDAC6, at the crossroads between cytoskeleton and cell signaling by acetylation and ubiquitination. Oncogene 26, 5468–5476 .
pmid:17694087
4 Chang, S., Young, B.D., Li, S., Qi, X., Richardson, J.A., and Olson, E.N. (2006). Histone deacetylase 7 maintains vascular integrity by repressing matrix metalloproteinase 10. Cell 126, 321–334 .
pmid:16873063
5 Fischer, D.D., Cai, R., Bhatia, U., Asselbergs, F.A., Song, C., Terry, R., Trogani, N., Widmer, R., Atadja, P., and Cohen, D. (2002). Isolation and characterization of a novel class II histone deacetylase, HDAC10. J Biol Chem 277, 6656–6666 .
pmid:11739383
6 Folkman, J. (2007). Angiogenesis: an organizing principle for drug discovery? Nat Rev Drug Discov 6, 273–286 .
pmid:17396134
7 Gao, J., Sun, L., Huo, L., Liu, M., Li, D., and Zhou, J. (2010). CYLD regulates angiogenesis by mediating vascular endothelial cell migration. Blood 115, 4130–4137 .
pmid:20194890
8 Guardiola, A.R., and Yao, T.P. (2002). Molecular cloning and characterization of a novel histone deacetylase HDAC10. J Biol Chem 277, 3350–3356 .
pmid:11726666
9 Haberland, M., Montgomery, R.L., and Olson, E.N. (2009). The many roles of histone deacetylases in development and physiology: implications for disease and therapy. Nat Rev Genet 10, 32–42 .
pmid:19065135
10 Haggarty, S.J., Koeller, K.M., Wong, J.C., Grozinger, C.M., and Schreiber, S.L. (2003). Domain-selective small-molecule inhibitor of histone deacetylase 6 (HDAC6)-mediated tubulin deacetylation. Proc Natl Acad Sci U S A 100, 4389–4394 .
pmid:12677000
11 Kim, M.S., Kwon, H.J., Lee, Y.M., Baek, J.H., Jang, J.E., Lee, S.W., Moon, E.J., Kim, H.S., Lee, S.K., Chung, H.Y., (2001). Histone deacetylases induce angiogenesis by negative regulation of tumor suppressor genes. Nat Med 7, 437–443 .
pmid:11283670
12 Lamalice, L., Le Boeuf, F., and Huot, J. (2007). Endothelial cell migration during angiogenesis. Circ Res 100, 782–794 .
pmid:17395884
13 Lansbergen, G., and Akhmanova, A. (2006). Microtubule plus end: a hub of cellular activities. Traffic 7, 499–507 .
pmid:16643273
14 Lee, Y.S., Lim, K.H., Guo, X., Kawaguchi, Y., Gao, Y., Barrientos, T., Ordentlich, P., Wang, X.F., Counter, C.M., and Yao, T.P. (2008). The cytoplasmic deacetylase HDAC6 is required for efficient oncogenic tumorigenesis. Cancer Res 68, 7561–7569 .
pmid:18794144
15 Li, R., and Gundersen, G.G. (2008). Beyond polymer polarity: how the cytoskeleton builds a polarized cell. Nat Rev Mol Cell Biol 9, 860–873 .
pmid:18946475
16 Marks, P.A. (2007). Discovery and development of SAHA as an anticancer agent. Oncogene 26, 1351–1356 .
pmid:17322921
17 McKinsey, T.A., Zhang, C.L., Lu, J., and Olson, E.N. (2000). Signal-dependent nuclear export of a histone deacetylase regulates muscle differentiation. Nature 408, 106–111 .
pmid:11081517
18 Rodriguez, O.C., Schaefer, A.W., Mandato, C.A., Forscher, P., Bement, W.M., and Waterman-Storer, C.M. (2003). Conserved microtubule-actin interactions in cell movement and morphogenesis. Nat Cell Biol 5, 599–609 .
pmid:12833063
19 Semenza, G.L. (2007). Vasculogenesis, angiogenesis, and arteriogenesis: mechanisms of blood vessel formation and remodeling. J Cell Biochem 102, 840–847 .
pmid:17891779
20 Urbich, C., R?ssig, L., Kaluza, D., Potente, M., Boeckel, J.N., Knau, A., Diehl, F., Geng, J.G., Hofmann, W.K., Zeiher, A.M., (2009). HDAC5 is a repressor of angiogenesis and determines the angiogenic gene expression pattern of endothelial cells. Blood 113, 5669–5679 .
pmid:19351956
21 Valenzuela-Fernández, A., Cabrero, J.R., Serrador, J.M., and Sánchez-Madrid, F. (2008). HDAC6: a key regulator of cytoskeleton, cell migration and cell-cell interactions. Trends Cell Biol 18, 291–297 .
pmid:18472263
22 Vega, R.B., Harrison, B.C., Meadows, E., Roberts, C.R., Papst, P.J., Olson, E.N., and McKinsey, T.A. (2004). Protein kinases C and D mediate agonist-dependent cardiac hypertrophy through nuclear export of histone deacetylase 5. Mol Cell Biol 24, 8374–8385 .
pmid:15367659
23 Xu, W.S., Parmigiani, R.B., and Marks, P.A. (2007). Histone deacetylase inhibitors: molecular mechanisms of action. Oncogene 26, 5541–5552 .
pmid:17694093
24 Zhang, Y., Kwon, S., Yamaguchi, T., Cubizolles, F., Rousseaux, S., Kneissel, M., Cao, C., Li, N., Cheng, H.L., Chua, K., (2008). Mice lacking histone deacetylase 6 have hyperacetylated tubulin but are viable and develop normally. Mol Cell Biol 28, 1688–1701 .
pmid:18180281
25 Zilberman, Y., Ballestrem, C., Carramusa, L., Mazitschek, R., Khochbin, S., and Bershadsky, A. (2009). Regulation of microtubule dynamics by inhibition of the tubulin deacetylase HDAC6. J Cell Sci 122, 3531–3541 .
pmid:19737819
[1] Qiqun Zeng,Zhenzhen Wu,Hongxia Duan,Xuan Jiang,Tao Tu,Di Lu,Yongting Luo,Ping Wang,Lina Song,Jing Feng,Dongling Yang,Xiyun Yan. Impaired tumor angiogenesis and VEG-Finduced pathway in endothelial CD146 knockout mice[J]. Protein Cell, 2014, 5(6): 445-456.
[2] Dengwen Li,Xiaodong Sun,Linlin Zhang,Bing Yan,Songbo Xie,Ruming Liu,Min Liu,Jun Zhou. Histone deacetylase 6 and cytoplasmic linker protein 170 function together to regulate the motility of pancreatic cancer cells[J]. Protein Cell, 2014, 5(3): 214-223.
[3] Xiaodong Sun, Fang Li, Bin Dong, Shaojun Suo, Min Liu, Dengwen Li, Jun Zhou. Regulation of tumor angiogenesis by the microtubule-binding protein CLIP-170[J]. Prot Cell, 2013, 4(4): 266-276.
[4] Yi Sun, Hua Li. Functional characterization of SAG/RBX2/ROC2/RNF7, an antioxidant protein and an E3 ubiquitin ligase[J]. Prot Cell, 2013, 4(2): 103-116.
[5] Byung-Kwon Choi, Xiumei Cai, Bin Yuan, Zhao Huang, Xuejun Fan, Hui Deng, Ningyan Zhang, Zhiqiang An. HER3 intracellular domains play a crucial role in HER3/HER2 dimerization and activation of downstream signaling pathways[J]. Prot Cell, 2012, 3(10): 781-789.
[6] Kai Gong, Fangfang Zhou, Huizhe Huang, Yandao Gong, Long Zhang. Suppression of GSK3β by ERK mediates lipopolysaccharide induced cell migration in macrophage through β-catenin signaling[J]. Prot Cell, 2012, 3(10): 762-768.
[7] Zhi Huang, Lin Cheng, Olga A. Guryanova, Qiulian Wu, Shideng Bao, . Cancer stem cells in glioblastoma — molecular signaling and therapeutic targeting[J]. Protein Cell, 2010, 1(7): 638-655.
[8] Shenglin Huang, Xianghuo He. microRNAs: tiny RNA molecules, huge driving forces to move the cell[J]. Prot Cell, 2010, 1(10): 916-926.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed