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. 2009 Dec 1;106(48):20399-404.
doi: 10.1073/pnas.0911921106. Epub 2009 Nov 30.

Synergistic effect of HIF-1alpha gene therapy and HIF-1-activated bone marrow-derived angiogenic cells in a mouse model of limb ischemia

Sergio Rey  1 , KangAe Lee, C Joanne Wang, Kshitiz Gupta, Shaoping Chen, Alexandra McMillan, Nupura Bhise, Andre Levchenko, Gregg L Semenza
Affiliations

Synergistic effect of HIF-1alpha gene therapy and HIF-1-activated bone marrow-derived angiogenic cells in a mouse model of limb ischemia

Sergio Rey et al. Proc Natl Acad Sci U S A. .

Abstract

Ischemia induces the production of angiogenic cytokines and the homing of bone-marrow-derived angiogenic cells (BMDACs), but these adaptive responses become impaired with aging because of reduced expression of hypoxia-inducible factor (HIF)-1alpha. In this study, we analyzed the effect of augmenting HIF-1alpha levels in ischemic limb by intramuscular injection of AdCA5, an adenovirus encoding a constitutively active form of HIF-1alpha, and intravenous administration of BMDACs that were cultured in the presence of the prolyl-4-hydroxylase inhibitor dimethyloxalylglycine (DMOG) to induce HIF-1 expression. The combined therapy increased perfusion, motor function, and limb salvage in old mice subjected to femoral artery ligation. Homing of BMDACs to the ischemic limb was dramatically enhanced by intramuscular AdCA5 administration. DMOG treatment of BMDACs increased cell surface expression of beta(2) integrins, which mediated increased adherence of BMDACs to endothelial cells. The effect of DMOG was abolished by coadministration of the HIF-1 inhibitor digoxin or by preincubation with a beta(2) integrin-blocking antibody. Transduction of BMDACs with lentivirus LvCA5 induced effects similar to DMOG treatment. Thus, HIF-1alpha gene therapy increases homing of BMDACs to ischemic muscle, whereas HIF-1 induction in BMDACs enhances their adhesion to vascular endothelium, leading to synergistic effects of combined therapy on tissue perfusion.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Combined therapy with DMOG-treated BMDACs and AdCA5 improves outcome following femoral artery ligation. (A) Limb perfusion ratio (LPR) in young (3-month) and old (13-month) mice. Mice were treated with saline (black), IM AdCA5 (blue) or IM AdCA5 + IV DMOG-treated BMDACs (green). IM AdCA5 was injected immediately after ligation and IV DMOG-treated BMDACs were delivered 24 h after ligation. Laser Doppler perfusion imaging was performed at the indicated time (in days) after femoral artery ligation and the mean ischemic:non-ischemic LPR was determined. (B) Maximal LPR (maxLPR) was calculated for each treatment group using sigmoidal nonlinear fitting. (C) Motor impairment was scored as follows: 0 = normal response (plantar/toe flexion in response to tail traction); 1 = plantar but not toe flexion; 2 = no plantar or toe flexion; 3 = dragging of foot. (D) The percentage of mice with limb salvage (i.e., no tissue necrosis) or necrosis in each group is indicated. IM, intramuscular adenovirus treatment; IV, intravenous treatment; S, saline; LZ; AdLacZ; CA5, AdCA5; C, BMDACs. Culture of BMDACs in the presence of DMOG (+) or vehicle (−) is indicated. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. saline control. #, P < 0.05; ###, P < 0.001 vs. AdCA5 + saline treatment; n = 4–7 mice per group. Motor impairment and ischemic tissue damage were measured 28 d after surgery.
Fig. 2.
Fig. 2.
Characterization of BMDACs. (A) BMDACs were treated with 0.1 mM DMOG for 4 h and immunoblot assays were performed. (B) BMDACs were cultured under endothelial growth conditions for 4 d in the presence of DMOG or vehicle, and mRNAs were analyzed by reverse transcription and quantitative real-time PCR (RT-qPCR) with fold change (DMOG vs. vehicle) plotted on a log2 scale. (C) BMDACs were cultured in the presence of vehicle (blue) or DMOG (red) for 4 d and cell surface markers were analyzed by flow cytometry using specific antibodies (Table S3). The percentage of positive cells for each marker is indicated. (D) Flow cytometry was performed to determine the percentage of CD31+/VEGFR1+ and CD31+/VEGFR2+ BMDACs after culture in the presence of vehicle (blue) or DMOG (red) for 4 d. *, P < 0.05; **, P < 0.01 DMOG vs. vehicle (n = 4–6).
Fig. 3.
Fig. 3.
BMDAC homing to the ischemic limb. DMOG (+)- or vehicle(−)-treated BMDACs were administered via tail vein injection 24 h after femoral artery ligation and IM injection of adenovirus (AdLacZ or AdCA5) or saline. Gastrocnemius muscles were isolated 8 h later, DNA was extracted, and qPCR performed to detect Sry gene sequences, expressed as the number of copies per 100 ng of genomic DNA. **, P < 0.01; ***, P < 0.001 vs. sham surgery (no ischemia; first bar on Left). ##, P < 0.01 vs. sham surgery, vehicle-treated BMDACs + AdCA5, and DMOG-treated BMDACs + AdCA5 (n = 3–4 mice each).
Fig. 4.
Fig. 4.
BMDAC exposure to DMOG induces integrin mRNA and protein expression. (A) RT-qPCR analysis of CD11a (αL), CD11b (αM), CD11c (αX), and CD18 (β2) mRNA, expressed as fold increase of DMOG- vs. vehicle-treated cells. (B) Flow cytometric analysis of CD11a+/CD18+ (LFA-1+), CD11b+/CD18+ (Mac-1+), and CD11c+/CD18+ (CR4+) BMDACs (n = 3). Antibodies are listed in Table S3.
Fig. 5.
Fig. 5.
Microfluidic BMDAC adhesion assay. Channel surfaces were seeded with HUVECs previously incubated at either 1 or 20% O2 for 24 h. The assay was conducted at a perfusion rate of 96 nL/min (wall shear stress = 0.1 Pa) for 10 min. (A) Sample images from the channels after washing off unattached cells. DMOG-treated (Top channel) and vehicle-treated (Bottom channel) BMDACs were stained with CMFDA and detected with the FITC filter. HUVEC monolayers are visible on phase contrast microscopy. (B) BMDACs were cultured under endothelial growth conditions in the presence of vehicle, 0.1 mM DMOG, or 0.1 mM DMOG + 0.1 mM digoxin for 4 d and subjected to the microfluidic assay. Results were normalized to the mean number of adherent BMDACs under control conditions (Leftmost bar). (C) BMDACs were transduced with control lentivirus encoding enhanced green fluorescent protein (LvEGFP) or lentivirus encoding a constitutively active form of HIF-1α (LvCA5) and analyzed for endothelial adhesion. (D) Flow cytometry was performed to analyze ICAM-1 and E-selectin surface expression on HUVECs incubated at 20% (blue) or 1% (red) O2 for 24 h. The percentage of positive cells is defined by the horizontal bar. (E) BMDACs were preincubated with a blocking CD18 (αCD18) antibody or isotype control and analyzed for endothelial adhesion. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (n = 3–6 independent experiments).
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