SIRT1 controls endothelial angiogenic functions during vascular growth - PubMed (original) (raw)

. 2007 Oct 15;21(20):2644-58.

doi: 10.1101/gad.435107.

Laleh Ghaeni, Danila Baldessari, Raul Mostoslavsky, Lothar Rossig, Franck Dequiedt, Judith Haendeler, Marina Mione, Elisabetta Dejana, Frederick W Alt, Andreas M Zeiher, Stefanie Dimmeler

Affiliations

SIRT1 controls endothelial angiogenic functions during vascular growth

Michael Potente et al. Genes Dev. 2007.

Abstract

The nicotinamide adenine dinucleotide (NAD(+))-dependent histone deacetylase Sir2 regulates life-span in various species. Mammalian homologs of Sir2 are called sirtuins (SIRT1-SIRT7). In an effort to define the role of sirtuins in vascular homeostasis, we found that among the SIRT family, SIRT1 uniquely regulates angiogenesis signaling. We show that SIRT1 is highly expressed in the vasculature during blood vessel growth, where it controls the angiogenic activity of endothelial cells. Loss of SIRT1 function blocks sprouting angiogenesis and branching morphogenesis of endothelial cells with consequent down-regulation of genes involved in blood vessel development and vascular remodeling. Disruption of SIRT1 gene expression in zebrafish and mice results in defective blood vessel formation and blunts ischemia-induced neovascularization. Through gain- and loss-of-function approaches, we show that SIRT1 associates with and deacetylates the forkhead transcription factor Foxo1, an essential negative regulator of blood vessel development to restrain its anti-angiogenic activity. These findings uncover a novel and unexpected role for SIRT1 as a critical modulator of endothelial gene expression governing postnatal vascular growth.

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Figures

Figure 1.

Figure 1.

SIRT1 regulates the angiogenic activity of endothelial cells. (A) mRNA expression of SIRT1–SIRT7 in HUVECs as assessed by RT–PCR using the indicated primer pairs. GAPDH served as loading control. (B) Statistical summary of the SIRT expression profile as assessed in a microarray analysis of total RNA isolated from HUVECs. (C) Three-dimensional in vitro angiogenesis with collagen gel-embedded spheroids of solvent-treated, nicotinamide-treated (NAM, 5 mM), sirtinol-treated (100 μM), or resveratrol-treated (1 μM) endothelial cells. Cumulative length of all sprouts originating from an individual spheroid was quantified after 24 h. Representative micrographs and a statistical summary are shown. (D) HUVECs were transfected with different siRNAs targeting SIRT1, SIRT2, SIRT3, SIRT5, or a nonrelated scrambled control. A statistical summary of the cumulative sprout length after 24 h originating from individual spheroids in siRNA-transfected endothelial spheroids is shown. The mRNA expression of the SIRTs with deacetylase activity in siRNA-transfected HUVECs as assessed by RT–PCR using the indicated primer pairs is shown in the left panel. (E) The effects of SIRT1 gene silencing on the expression of mRNA transcripts for SIRT1–SIRT7 were assessed by RT–PCR using HUVEC RNA from SIRT1 siRNA-transfected endothelial cells.

Figure 2.

Figure 2.

Effect of SIRT1 on cellular responses in endothelial cells. (A,B) Representative micrographs and statistical summary of three-dimensional in vitro angiogenesis assays with collagen gel-embedded spheroids generated from SIRT1 or scrambled siRNA-transfected endothelial cells. (C,D) Representative micrographs and statistical summary of in vitro Matrigel assays with SIRT1 or scrambled siRNA-transfected endothelial cells. (E) HUVECs were transfected with a SIRT1-specific siRNA or a nonrelated scrambled control. Cell lysates were subjected to Western blotting using antibodies against SIRT1. Antibodies recognizing Foxo1, Akt, or tubulin were used as a loading control. (F–I) Statistical summary of the effects of SIRT1 gene silencing (white bars) on migration, adhesion, proliferation, and apoptosis compared with scrambled siRNA-transfected controls (black bars). The VEGF and bFGF concentrations were 50 ng/mL and 30 ng/mL, respectively. (J) HUVECs were transfected with wild-type (wt) SIRT1, a deacetylation-defective SIRT1 mutant (H363Y), or mock control (pcDNA3). Cells were lysed after 24 h and subjected to Western blot analysis with antibodies against SIRT1 and Myc. (K) Statistical summary of the cumulative sprout length in pcDNA-, SIRT1 wild-type-, or SIRT1 H363Y-transfected endothelial spheroids. (L) Statistical summary of endothelial migration toward the chemoattractant VEGF (50 ng/mL) in pcDNA-, SIRT1 wild-type-, or SIRT1 363Y-transfected cells.

Figure 3.

Figure 3.

SIRT1 expression in the vascular endothelium. (A) Cell and tissue lysates were subjected to Western blot analysis with specific antibodies against SIRT1 and eNOS and tubulin. (HUVEC) Human umbilical vein endothelial cells; (HMVEC) human microvascular endothelial cells; (MLEC) murine lung endothelial cells; (Aorta) murine aorta of 129/SV mice. (B) Representative immunostaining is shown for the predominantly nuclear localization of SIRT1 in HUVEC. (C) Immunostainings of SIRT1 with a polyclonal antibody (white) in mouse vascular sections. No signal was detected with a rabbit IgG control antibody (data not shown). Endothelial cells were stained with an antibody recognizing CD31 (PECAM, green). Nuclei were stained in blue (Topro or DAPI).

Figure 4.

Figure 4.

Cardiovascular defects in _SIRT1_-deficient zebrafish embryos. (A) SIRT1 expression at different stages of zebrafish development. (Panels A–F) SIRT1 antisense probe. (Panel A) Dorsal view. (Panels B–F) Lateral views. The embryonic stage is indicated at the bottom left corner. (8-cell stage) maternal expression; (shield) zygotic expression. Bars, 200 μm. (B) RT–PCR analysis performed on total RNA extracted from embryos at the indicated stages using primers specific for SIRT1. β-actin serves as loading control. (C) RT–PCR analysis of SIRT1 mRNA transcripts in control, ATG-MO-injected, and SB-MO-injected embryos. Specific and effective splice-blocking of the SIRT1 transcript in the SB-MO-injected embryos. SB-morpholinos were injected at two different concentrations (SB-MO+ indicates twice the amount of the morpholino as compared with SB-MO). The exon 2–exon 3 spliced form is 249 bp, and 701 bp is the fragment including the intron 2 sequence. (D) Lateral views of the vasculature from either control or SIRT1 knockdown (ATG-MO and SB-MO) zebrafish embryos. Pictures were taken from fixed embryos after immunohistochemistry with an anti-GFP antiserum using a stereomicroscope. (E) Hemorrhages (white arrows) and pericardial swelling (black arrow) in SIRT1 knockdown zebrafish (SB-MO). (F) First and last frame (GFP fluorescence) of time-lapse movies captured from control embryos (24 hpf) and embryos injected at one- to two-cell stage with a SIRT1 splice-blocking morpholino. The embryo’s head is to the left, dorsal is to the top. Arrowheads point to vessels defects.

Figure 5.

Figure 5.

Endothelial cell-specific deletion of SIRT1. (A) Strategy to generate a conditional SIRT1 allele by inserting loxP sites flanking exon 4 of the mouse SIRT1 genomic locus. The structure of the genomic locus, the targeting vector, and the targeted allele are shown. Endothelial-specific deletion was achieved by breeding to transgenic mice harboring a Tie2-Cre transgene. (B) Genotyping of the endothelial-specific SIRT1 mutant mice (Tie2Cretg;SIRT1flox/−). (Cre) Tie2Cretg; (flox) SIRT1flox allele; (+/−) SIRT1+/−. (C) Western blot analysis with a specific antibody against SIRT1 showed that the floxed SIRT1 allele was completely deleted in endothelial cells isolated from the endothelial-restricted SIRT1 mutant mice. Cell lysates were also subjected to Western blot analysis with antibodies targeting eNOS and Tie2 to show the endothelial characteristics of cells; tubulin served as loading control. (D) Mice of the respective genotypes were subjected to hindlimb ischemia, and perfusion was assessed 14 d after onset of ischemia using laser Doppler imaging. Quantitative results are presented. (E) Capillary (CD31, green; laminin, red) and α-smooth muscle actin staining (red) in sections of mice after hindlimb ischemia. A statistical summary of capillary density and the number of α-smooth muscle actin-positive vessels is shown. (F) Mice of the respective genotypes were subcutaneously injected with Matrigel matrix. After 7 d, Matrigel plugs were explanted and the hemoglobin content and invading cell number of the plug were quantified. (G) Histological analysis of H&E stainings from Matrigel plugs isolated from SIRT1flox/+ and Tie2Cretg;SIRT1flox/− mice. (H) Murine endothelial cells of SIRT1flox/+ and Tie2Cretg;SIRT1flox/− mice were isolated by vascular endothelial-cadherin immunopurification. Representative micrographs and statistical summary of the three-dimensional spheroid and Matrigel assay of in vitro angiogenesis with endothelial cells generated from these mice.

Figure 6.

Figure 6.

Dysregulated gene expression in _SIRT1_-silenced endothelial cells. HUVECs were transfected with a SIRT1-specific siRNA or a scrambled oligonucleotide siRNA (each n = 3). Total RNA was isolated after 24 h, and the gene expression profile was assessed with the Affymetrix gene chip expression assay. (A,B) Selected genes that were either down-regulated (A) or up-regulated (B) in _SIRT1_-silenced endothelial cells. (C) RT–PCR analysis of selected genes in scrambled and SIRT1 siRNA-transfected endothelial cells. (D) RT–PCR analysis of selected genes in lung endothelial cells freshly isolated from mice with the respective genotype. (E) Fli1 expression, as revealed by whole-mount in situ hybridization, is reduced in the ISVs (arrowheads) of SIRT1 morphants (SBmo-inj.) compared with that in the control embryos. The GFP signal visualized by fluorescence microscope in live embryos is weaker in SIRT1 morphants. (Panels a,b) Embryos 24 hpf, lateral view; Bar, 50 μm. (Panels c,d) Embryos 4 dpf, lateral view.

Figure 7.

Figure 7.

SIRT1 interacts with Foxo1 in endothelial cells. (A) Interaction of SIRT1 with Foxo1. HUVECs were cotransfected with the Flag-tagged Foxo1 and the Myc-tagged SIRT1. Twenty-four hours after transfection, cells were lysed and Flag-tagged Foxo1 was immunoprecipitated. The immune complexes were assessed for the presence of SIRT1 (Myc) by Western blot analysis with antibodies to Myc. The amounts of Flag-Foxo1 and Myc-SIRT1 present in the cell extracts were analyzed with the respective antibodies. (B) Interaction of endogenous SIRT1 with Foxo1. HUVECs were left untreated or incubated with the SIRT1 inhibitor nicotinamide (NAM) and the class I/II HDAC inhibitor TSA for 2 h. Endogenous Foxo1 was immunoprecipitated with a goat polyclonal antibody to Foxo1 or a control preimmune goat serum (IgG). The immune complexes were assessed for the presence of SIRT1 by Western blot with an antibody to SIRT1. Amounts of endogenous Foxo1 and SIRT1 in the cell extracts were determined with antibodies to SIRT1 and Foxo1. (C) In vitro SIRT1 deacetylation assay. Acetylated Flag-tagged Foxo1 was immunoprecipitated using an antibody targeting the Flag epitope. The acetylated Foxo1 was incubated with immunoprecipitated wild-type SIRT1 (wt) or the deacetylase-defective SIRT1 mutant (H363Y) in the presence or absence of nicotinamide (NAM). Acetylation of Foxo1 was assessed by Western blot with antibodies to acetylated lysine. (D) HUVECs expressing Flag-tagged Foxo1 were incubated for 6 h in the absence or presence of the SIRT1 inhibitor nicotinamide (NAM), the class I/II HDAC inhibitor TSA, or combinations thereof. Flag-tagged Foxo1 was immunoprecipitated, and the acetylation of Foxo1 was assessed by Western blot with antibodies to acetylated lysine. (E) HUVECs were transfected with a SIRT1-specific siRNA or a scrambled control oligonucleotide and 24 h later with pcDNA, Flag-tagged Foxo1, p300, or combinations thereof. Endothelial cells were then incubated with TSA for 2 h. Flag-tagged Foxo1 was immunoprecipitated with antibodies targeting the Flag epitope. The acetylation of Foxo1 was assessed by Western blot analysis. (F) HUVECs were transiently transfected with expression plasmids encoding Foxo1 A3, SIRT1, and a forkhead-responsive element-driven luciferase reporter. Twenty-four hours after transfection, cells were lysed and cell extracts were assayed for luciferase expression. (G) HUVECs were transfected with a SIRT1-specific siRNA or a scrambled control siRNA and 24 h later with expression plasmids encoding pcDNA3, Foxo1 A3, p300, and a forkhead-responsive element-driven luciferase reporter. Twenty-four hours after transfection, cells were lysed and cell extracts were assayed for luciferase expression. (H) ChIP assays were performed with chromatin prepared from HUVECs incubated with nicotinamide (NAM) and TSA for 3 h. Chromatin was immunoprecipitated with mouse IgG or antibodies against SIRT1, Foxo1, or acetylated histone H3, and precipitated genomic DNA was analyzed by PCR using primers flanking conserved Foxo-binding sites in the promoter regions of the Gadd45a, Fli1, and Flt1 genes. (I) Representative images and statistical summary of a three-dimensional in vitro angiogenesis assay with collagen gel-embedded endothelial spheroids transfected with combinations of scrambled, SIRT1, and Foxo1 siRNAs. Cumulative sprout length was quantified after 24 h.

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