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. 2013 Oct;16(4):759-72.
doi: 10.1007/s10456-013-9353-x. Epub 2013 May 16.

Endothelial FoxO1 is an intrinsic regulator of thrombospondin 1 expression that restrains angiogenesis in ischemic muscle

Emilie Roudier  1 , Malgorzata Milkiewicz, Olivier Birot, Dara Slopack, Andreas Montelius, Thomas Gustafsson, Ji Hye Paik, Ronald A DePinho, George P Casale, Iraklis I Pipinos, Tara L Haas
Affiliations

Endothelial FoxO1 is an intrinsic regulator of thrombospondin 1 expression that restrains angiogenesis in ischemic muscle

Emilie Roudier et al. Angiogenesis. 2013 Oct.

Abstract

Peripheral artery disease (PAD) is characterized by chronic muscle ischemia. Compensatory angiogenesis is minimal within ischemic muscle despite an increase in angiogenic factors. This may occur due to the prevalence of angiostatic factors. Regulatory mechanisms that could evoke an angiostatic environment during ischemia are largely unknown. Forkhead box O (FoxO) transcription factors, known to repress endothelial cell proliferation in vitro, are potential candidates. Our goal was to determine whether FoxO proteins promote an angiostatic phenotype within ischemic muscle. FoxO1 and the angiostatic matrix protein thrombospondin 1 (THBS1) were elevated in ischemic muscle from PAD patients, or from mice post-femoral artery ligation. Mice with conditional endothelial cell-directed deletion of FoxO proteins (Mx1Cre (+), FoxO1,3,4 (L/L) , referred to as FoxOΔ) were used to assess the role of endothelial FoxO proteins within ischemic tissue. FoxO deletion abrogated the elevation of FoxO1 and THBS1 proteins, enhanced hindlimb blood flow recovery and improved neovascularization in murine ischemic muscle. Endothelial cell outgrowth from 3D explant cultures was more robust in muscles derived from FoxOΔ mice. FoxO1 overexpression induced THBS1 production, and a direct interaction of endogenous FoxO1 with the THBS1 promoter was detectable in primary endothelial cells. We provide evidence that FoxO1 directly regulates THBS1 within ischemic muscle. Altogether, these findings bring novel insight into the regulatory mechanisms underlying the repression of angiogenesis within peripheral ischemic tissues.

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Conflict of interest statement

Conflict of interest None.

Figures

Fig. 1
Fig. 1
FoxO1, but not FoxO3a, protein level increases in response to PAD and is associated with enhanced p27KIP1 and THBS1 protein expression. Gastrocnemius muscles from control and PAD subjects were assessed by Western blot a for FoxO1 and FoxO3a, p27KIP1 and THBS1 protein levels, and quantified relative to β-actin. Data are mean ± SEM; * indicates significant differences: p = 0.01 for FoxO1; p = 0.047 for p27KIP1; p = 0.01 for THBS1 (Unpaired two tailed Student’s t tests, n = 8–13 per group). b FoxO1 relative protein levels were compared with protein levels of p27KIP1 (top; N = 26) and THBS1 (bottom; N = 16). The Pearson’s correlation coefficient (r) was calculated to assess the relationships between FoxO protein levels and each of the specified parameters
Fig. 2
Fig. 2
FoxO1, but not FoxO3a, protein level increases within ischemic mouse muscle, corresponding with an increase in THBS1 protein expression. FoxO1, FoxO3a and THBS1 (a, b, c) protein levels were assessed in murine gastrocnemius muscle 2, 4 or 7 days following femoral artery ligation and corresponding sham (Sh) operations and quantified relative to αβ-tubulin or β-actin. Data are mean ± SEM; n = 4–6. One-way ANOVA shows a main effect of arterial ligation on the levels of FoxO1 and THBS1 but not on FoxO3a (p = 0.02, p = 0.015 and p > 0.05, respectively, one way ANOVA). * indicates differences versus Sham (Bonferroni post hoc analysis, p < 0.05)
Fig. 3
Fig. 3
FoxO1 and FoxO3a deletion occur in skeletal muscle microvascular endothelial cells of FoxOΔ mice. FoxO1 and FoxO3a levels were measured by Western blotting on protein extracts of gastrocnemius muscles a harvested from 6 week old FoxOL/L and FoxOΔ mice. Protein level relative to β-actin is expressed as mean ± SEM. Differences vs. FoxOL/L are **p = 0.01 (n = 4–7 per group). FoxO1 and FoxO3a levels were assessed by Western blotting on protein extracts of microvascular endothelial cells b isolated from skeletal muscle of FoxOL/L and FoxOΔ mice. ***p < 0.0001 and **p < 0.01 (n = 3 to 4 independent cell isolations from a total of 9 mice per group). Cross-sections of TA/EDL muscles (4 days post femoral artery ligation) were immuostained c for FoxO1 (red) and Isolectin B4 (green). Arrows indicate capillaries positive for FoxO1 staining and arrowheads indicate myocyte/interstitial cell-associated FoxO1 immunoreactivity. Notably, FoxO1 staining did not overlap with Isolectin B4 staining in muscle cross-sections of FoxOΔ mice; asterisks highlight representative capillaries that do not exhibit FoxO1 immunoreactivity. Immunostaining of a FoxOL/L muscle section with normal rabbit IgG as a negative control for the FoxO1 antibody is shown in the bottom left hand panel
Fig. 4
Fig. 4
FoxO deletion promotes endothelial cell outgrowth from ex vivo muscle explants. Muscle explant culture was performed on soleus and plantaris muscle biopsies of non-ischemic FoxOL/L and FoxOΔ mice, and representative images of soleus biopsies are shown (a). The asterisk denotes the original muscle explant; arrows point to endothelial cell outgrowth. The area of migration (upper graph) and the endothelial cell density (lower graph) within the area of migration of explants derived from soleus (white bars) or plantaris (black bars) were calculated (b). Multiple explants from an individual mouse were averaged. Data show mean ± SEM (n = 4–6 mice per condition, *p < 0.05 and **p < 0.01 versus matched FoxOL/L muscle explants, unpaired two-tailed Student’s t test)
Fig. 5
Fig. 5
Ischemia-dependent increases in FoxO1 protein do not occur in FoxOΔ mice. FoxO1 protein levels were assessed at 4 and 10 days post-femoral artery ligation in gastrocnemius muscles of FoxOL/L and FoxOΔ mice. Protein levels were expressed relative to β-actin. Vertical line delineates two different blots. Data are mean ± SEM. Two-way ANOVA indicates a main effect of FoxO deletion (p < 0.0001, n = 4–18 per group). Significant differences between FoxOL/L and FoxOΔ are *p < 0.05 and **p < 0.01 (vs. time matched group), and #p < 0.05 (vs. corresponding sham, Bonferroni post hoc)
Fig. 6
Fig. 6
FoxO deletion promotes blood flow recovery and neovascularization following femoral artery ligation. Hindlimb blood flow of FoxOL/L and FoxOΔ mice a was assessed by Laser Doppler imaging before surgery, and 0, 4, 7, and 10 days after femoral artery ligation. A representative time course of images is shown for each mouse phenotype. White arrows indicate the ischemic limb in post-ligation time-points. Data are mean ± SEM of the ischemic/non-ischemic flow ratio. Two-way ANOVA shows main effects of both time and FoxO deletion (p < 0.0001 and p = 0.0008, respectively; n = 5–12 per group). Post hoc analysis indicated that blood flow ratios in FoxOΔ mice were significantly different at days 4, 7 and 10 compared to day 0 (*p < 0.05 and ***p < 0.001 vs. corresponding day 0). Furthermore, blood flow in FoxOΔ mice was significantly different from that of FoxOL/L mice at days 7 and 10 (###p < 0.001 vs. FoxOL/L mice at the corresponding time point). Capillary to fiber ratio b was assessed in TA/EDL muscles 10 days post-ligation. There was a main effect of FoxO deletion (p < 0.0001, n = 4–8 per group) and significant differences between FoxOL/L and FoxOΔ ***p < 0.001 (Bonferroni post hoc analysis)
Fig. 7
Fig. 7
FoxO deletion prevents the ischemia-induced increase in Thrombospondin 1 within the endothelium of mouse muscle. Microvascular endothelial cells were isolated from skeletal muscle of FoxOL/L and FoxOΔ mice. THBS1 mRNA was assessed by q-PCR (normalized to Hprt1) and THBS1 protein a was assessed by Western blot (normalized to β-actin). **p = 0.004 compared to cells from FoxOL/L mice; *p = 0.01, paired Student’s t test, n = 3 independent isolations, from a total of 8 individual mice per condition. THBS1 mRNA gastrocnemius muscles from FoxOL/L and FoxOΔ mice b was assessed by q-PCR (normalized to Hprt1) at 4 days post-femoral artery ligation or corresponding sham operation. Two-way ANOVA shows main effects of FoxO deletion and arterial ligation (p = 0.04 and p = 0.03, respectively). Significant differences are #p < 0.05 versus Sham FoxOL/L, and *p < 0.05 versus day 4 ligated FoxOL/L (Bonferonni Post hoc, n = 4–7 mice). TA/EDL muscle cross-sections from 4 day ligated or sham operated FoxOL/L or FoxOΔ mice were immunostained c for THBS1 (red) and counterstained with Isolectin B4 (green) to identify capillaries. Arrows point to peri-capillary THBS1 immunoreactivity in the ischemic FoxOL/L tissue. Normal mouse IgG was used as a negative control for the THBS1 antibody
Fig. 8
Fig. 8
FoxO1 is a direct regulator of Thrombospondin-1. Deletion of FoxO gene products in primary skeletal muscle microvascular endothelial cells was induced by adeno-Cre infection (a). FoxO1 protein and THBS1 mRNA were measured after serum starvation. β-actin and Hprt-1 were used as loading control and housekeeping gene, respectively. *p = 0.02 and **p = 0.009 compared to control adeno-β-gal control transduction, paired Student’s t test, n = 3, 4 independent experiments. Overexpression of FoxO1 in microvascular endothelial cells was induced by Adeno-wt-FoxO1 (wt) or Adeno-ca-FoxO1 (ca) infection (b) and confirmed by Western blotting (***p < 0.0001 vs. adeno-β-gal control transduction, one way ANOVA and Bonferroni post hoc analysis, n = 4). THBS1 mRNA was measured by q-PCR (p = 0.005, one way ANOVA; **p < 0.01 compared to the β-gal transduced condition, Bonferroni post hoc analysis, n = 4). Alignment of conserved FoxO consensus elements within the THBS1 promoter (h human, m mouse, c canus) (c). The shaded boxes indicate the core consensus elements within each region. Base mismatches are denoted by italics. The underlined sequence in Region 2 indicates a second consensus site within the murine sequence. Consensus sites and primer locations within the THBS1 locus are illustrated. ChIP was performed using FoxO1 antibody or normal rabbit IgG (d). Primer sets specific for either Regions 1 and 2 within the THBS1 promoter, the Cdkn1b promoter, or the THBS1 coding sequence (exon 3) were used to do PCR on precipitated DNA. In, Input DNA; Fx1, α-FoxO1 antibody; IgG, normal rabbit IgG; L, 100 bp DNA ladder
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