Histone deacetylase activity is essential for the expression of HoxA9 and for endothelial commitment of progenitor cells - PubMed (original) (raw)

. 2005 Jun 6;201(11):1825-35.

doi: 10.1084/jem.20042097. Epub 2005 May 31.

Carmen Urbich, Thomas Brühl, Elisabeth Dernbach, Christopher Heeschen, Emmanouil Chavakis, Ken-ichiro Sasaki, Diana Aicher, Florian Diehl, Florian Seeger, Michael Potente, Alexandra Aicher, Lucia Zanetta, Elisabetta Dejana, Andreas M Zeiher, Stefanie Dimmeler

Affiliations

Histone deacetylase activity is essential for the expression of HoxA9 and for endothelial commitment of progenitor cells

Lothar Rössig et al. J Exp Med. 2005.

Abstract

The regulation of acetylation is central for the epigenetic control of lineage-specific gene expression and determines cell fate decisions. We provide evidence that the inhibition of histone deacetylases (HDACs) blocks the endothelial differentiation of adult progenitor cells. To define the mechanisms by which HDAC inhibition prevents endothelial differentiation, we determined the expression of homeobox transcription factors and demonstrated that HoxA9 expression is down-regulated by HDAC inhibitors. The causal involvement of HoxA9 in the endothelial differentiation of adult progenitor cells is supported by the finding that HoxA9 overexpression partially rescued the endothelial differentiation blockade induced by HDAC inhibitors. Knockdown and overexpression studies revealed that HoxA9 acts as a master switch to regulate the expression of prototypical endothelial-committed genes such as endothelial nitric oxide synthase, VEGF-R2, and VE-cadherin, and mediates the shear stress-induced maturation of endothelial cells. Consistently, HoxA9-deficient mice exhibited lower numbers of endothelial progenitor cells and showed an impaired postnatal neovascularization capacity after the induction of ischemia. Thus, HoxA9 is regulated by HDACs and is critical for postnatal neovascularization.

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Figures

Figure 1.

Figure 1.

HDAC inhibitors abrogate the ex vivo endothelial maturation of circulating mononuclear precursor cells. (a–c) Dose-dependent effect of MS-275 (a), TSA (b), and BuA (c) on the number of EPCs after 72 h (n = 3–7, mean ± SD). (c) The effect of 2 mM BuA on EPC formation under these conditions in the presence or absence of 100 μM of the pan-caspase inhibitor zVAD (n = 4). (d) Relative changes in VEGF-R2+, CD14+, and CD45+ cells in peripheral blood–derived total MNCs after incubation in endothelial growth factor medium for 72 h with or without 2 mM BuA (*, P < 0.05 vs. CD14+, P < 0.005 vs. CD45+; n = 6, mean ± SEM). (e and f) Confocal microscopy of human bone marrow CD34+–derived (e) or murine bone marrow Sca-1+/lin−–derived (f) EPCs stained with DiI-Ac-LDL (red fluorescence) and vWF (green fluorescence), and nuclear TO-PRO-3 staining (blue fluorescence) after exposure toward endothelial differentiation conditions for 72 h in the presence or absence of 2 mM BuA. Representative images out of three to six experiments are shown. (g) Flow cytometric analysis of the expression of integrin subunits α4 (CD49d), α5 (CD49e), or β1 integrin (CD29, fibronectin receptor) in peripheral blood MNCs after exposure toward endothelial differentiation conditions for 72 h with or without 2 mM BuA or 3 μM MS-275 (n = 3–4). (h) Vascular outgrowth from embryonic allantois explants stained with CD31 antibody.

Figure 2.

Figure 2.

HDAC inhibitors decrease expression levels of HoxA9 transcription factor. (a) RT-PCR of HoxA9 (top) and HoxB5 (bottom) after exposure of peripheral blood MNCs to endothelial differentiation conditions for 72 h in the presence or absence of 2 mM BuA, 10 μM MS-275, or 2.5 μM TSA. GAPDH mRNA expression is shown as a control (co; n = 3). (b) Western blot analysis of HoxA9, HoxD9, p21, histone H3 di-acetylation at lysine residues K9 and K14 (Ac-H3), total histone H3, and tubulin from peripheral blood MNCs after incubating in endothelial medium in the presence or absence of 2 mM BuA or 10 μM MS-275 (n = 3–6). (c) Western blot analysis after transfection with siRNA directed against HDAC1 compared with scrambled (scr) in the presence or absence of 1 μM TSA (n = 3).

Figure 3.

Figure 3.

Role of HoxA9 for postnatal neovascularization after ischemia. (a) Incidence of necrotic limbs in wild-type (WT) versus heterozygote HoxA9+/− and homozygote HoxA9−/− mice after hind limb ischemia by ligation of the femoral artery (n ≥ 6/group). (b) For morphological analysis, myocytes were identified by staining for laminin (green) in ischemic tissue of WT (left) and homozygote HoxA9−/− mice (right). (c) Conductant vessels were defined by size (>20 μm) and positive staining for α–smooth muscle actin (red).

Figure 4.

Figure 4.

Expression of HoxA9 during differentiation of progenitor cells. (a) Western blot analysis of HoxA9 protein expression during ex vivo endothelial differentiation of peripheral blood–derived EPCs for the indicated days. Reprobe with extracellular signal–related kinase (ERK)1/2 indicates equal protein loading (n = 3). (b) RT-PCR analysis of HoxA9, eNOS, VEGF-R2, and GAPDH mRNA expression in ES cells at baseline (day 0) and after exposure toward endothelial differentiation conditions (day 7). (c) Western blot analysis of eNOS and VEGF-R2 protein expression in ES cells at baseline (day 0) and after exposure toward endothelial differentiation conditions (day 7). A representative reprobe with tubulin indicates equal protein loading. (d) Numbers of peripheral blood MNC–derived EPCs after transfection with HoxA9 adenovirus (*, P < 0.05 vs. lacZ control vector; n = 10). Expression control is shown (right). (e and f) Cultivated spleen MNC-derived Dil-ac-LDL+ cells (e) and outgrowing EPC colonies (f) from HoxA9−/−, HoxA9+/−, or WT mice (*, P < 0.05 vs. WT, n ≥ 6).

Figure 5.

Figure 5.

Essential role of HoxA9 for endothelial lineage marker expression in mature endothelial cells. (a) RT-PCR analysis of mRNA expression of the indicated markers in HUVECs transfected with scrambled siRNA or siRNA against HoxA9. A representative agarose gel is shown. (b) Quantitative RT-PCR (Light cycler) of the indicated markers in HUVECs transfected with scrambled siRNA or siRNA against HoxA9 (n ≥ 4). (c) eNOS and HoxA9 protein expression after transfection of HUVECs with scrambled siRNA or siRNA against HoxA9 (*, P < 0.05 vs. scrambled, n = 4). (d) eNOS protein expression in hearts of WT, HoxA9+/−, and HoxA9−/− mice (top, representative blot; bottom, quantification; n = 4/group;*, P < 0.05 vs. WT). (e) eNOS protein expression after transient transfection of a plasmid encoding HoxA9 in HUVECs (*, P < 0.05). (right) Representative Western blot analysis with tubulin as a loading control and staining against c-myc to indicate overexpression of the tagged HoxA9 insert.

Figure 6.

Figure 6.

HoxA9 directly binds to the promoter of eNOS and VEGF-R2. (a) Chromatin immunoprecipitation (ChIP) of endogenous HoxA9 followed by PCR directed against the promoter regions of VEGF-R2, VE- cadherin, eNOS, and, as a positive control, EphB4 (n = 3). Genomic DNA (gDNA) was used as positive control for PCR. (b) Western blot analysis of immunoprecipitated HoxA9 WT or HoxA9 mt in transfected HUVECs. (c) ChIP of DNA-bound myc-tagged HoxA9 from HUVEC lysates after the transient transfection of HoxA9 WT or of a truncated HoxA9 construct deficient in the DNA-binding domain (HoxA9 mt) followed by PCR analysis with promoter-specific primers against sequences of the eNOS promoter (top) or VEGF-R2 promoter (bottom; n = 3). (d–f) Transcriptional activation of the luciferase reporter gene under the regulatory control of the eNOS (d), VEGF-R2 (e), or VE-cadherin promoter (f) in HoxA9 WT or HoxA9 mt cotransfected HUVEC (*, P < 0.05; n = 3–6 vs. mock; RLU, relative light units).

Figure 7.

Figure 7.

Shear stress induction of HoxA9, eNOS, and VEGF-R2 is sensitive to HDAC inhibition. (a) Dose- and time-dependent regulation of HoxA9 expression (Western blot) after exposure of endothelial cells to shear stress. (left) Representative dose-dependency; (right) quantification of time-dependency (15 dynes/cm2; n = 3–6 experiments). (b) FACS analysis of VEGF-R2 expression in peripheral blood–derived EPCs (left), human CD34+ cells (middle), and macrophages (right) after exposure of the respective cell type to shear stress (15 dynes/cm2 for 24 h). (top) Representative traces; (bottom) quantitative analysis. (c) Shear stress (15 dynes/cm2 for 24 h)– stimulated expression of eNOS and VEGF-R2 after HoxA9 siRNA transfection compared with scrambled oligonucleotides. (left) Representative RT-PCR; right, quantification of n = 3 (*, P < 0.05 vs. scrambled + shear stress).

Figure 8.

Figure 8.

Rescue of HDAC inhibitor–mediated reduction of endothelial progenitors and eNOS expression by HoxA9 overexpression. (a) Numbers of peripheral blood MNC–derived EPCs after transfection with HoxA9 adenovirus in the presence or absence of 0.5 mM BuA (*, P < 0.05 vs. lacZ control vector; n = 4). (b) eNOS expression (Western blot analysis) in HUVEC transfected with HoxA9 WT or pcDNA3.1 control plasmid (mock) after incubation with 2 μM TSA for 24 h. (top) Quantification; (bottom) representative Western blot analysis (n = 11; *, P < 0.05 vs. mock- transfected cells; #, P < 0.05 vs. mock + TSA).

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