by Sergio Rey, Weibo Luo, Larissa A. Shimoda, and Gregg L. Semenza Metabolic reprogramming by HIF-1 promotes the survival of bone marrow–derived angiogenic cells in ischemic tissue by Sergio Rey, Weibo Luo, Larissa A. Shimoda, and Gregg L. Semenza Blood Volume 117(18):4988-4998 May 5, 2011 ©2011 by American Society of Hematology

DMOG increases BMDAC viability by decreasing apoptosis and increasing proliferation. DMOG increases BMDAC viability by decreasing apoptosis and increasing proliferation. (A) Equal numbers of BMDACs were cultured in endothelial growth media in the presence of vehicle or DMOG for 4 days, and viable cells were counted using the Trypan blue exclusion method. *P < .001, DMOG vs vehicle. #P < .001 vs time 0; Bonferroni post hoc test after 2-way ANOVA. The overall effects of time and DMOG treatment on BMDAC survival were significant (P < .001). (B) Apoptosis was detected at day 4 as phycoerythrin-conjugated annexin V (annexin V-PE) positive/7-amino-actinomycin D (7-AAD) negative cells (green rectangle) using flow cytometry. (C) Cell cycle distribution of BMDACs stained with propidium iodide and analyzed by flow cytometry. Data are mean ± SEM (n = 3 or 4). Sergio Rey et al. Blood 2011;117:4988-4998 ©2011 by American Society of Hematology

DMOG elicits transcriptional and metabolic responses in BMDACs DMOG elicits transcriptional and metabolic responses in BMDACs. (A) BMDACs were cultured in the presence of vehicle (V) or DMOG (D). DMOG elicits transcriptional and metabolic responses in BMDACs. (A) BMDACs were cultured in the presence of vehicle (V) or DMOG (D). Mitochondrial mass (top panels), ROS (middle panels), and glucose uptake (bottom panels) were measured in BMDACs by flow cytometry after staining with the fluorescent dyes nonyl acridine orange (NAO), 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), and 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG), respectively (n = 4-6). (B) Mitochondrial DNA content relative to nuclear genomic DNA was determined by quantitative PCR (n = 6). (C) Oxygen consumption (JO2) was analyzed in BMDACs suspended in serum-free EGM2-MV media (n = 6). (D) Quantitative RT-PCR analysis of HIF-1 target genes encoding metabolic regulators in BMDACs cultured in vehicle or DMOG for 4 days (n = 4-6). ETFA and TBP are not HIF-1 targets and were used as negative controls. Gray shaded area indicates ± 2-fold of control. (Inset) Immunoblot assay showing BNIP3, LDHA, and PDK1 protein levels. (E) Extracellular lactate content of BMDAC cultures (n = 9). (F) HIF-1α protein expression in BMDACs was determined by immunofluorescence using flow cytometric analysis (left panel). Gray histogram corresponds to the negative control omitting the primary antibody. Data are mean ± SEM (n = 6; right panel). *P < .05, DMOG vs vehicle, by Student t test in all panels of this figure. Data are mean ± SEM. Sergio Rey et al. Blood 2011;117:4988-4998 ©2011 by American Society of Hematology

Increased survival of DMOG-treated BMDACs under hypoxic and acidic conditions ex vivo. Increased survival of DMOG-treated BMDACs under hypoxic and acidic conditions ex vivo. (A) Effect of pH and hypoxia on BMDAC-D survival at 5.6mM glucose (▴). After culture for 4 days, equal numbers of BMDACs were exposed to low pH (6.4), nonhypoxic (20% O2), and hypoxic (1% O2) conditions. Cell viability was measured using the Trypan blue exclusion method. (B) Effect of glucose concentration on BMDAC-V and BMDAC-D survival under low pH (6.4) and hypoxia (1% O2). After culture for 4 days, BMDAC viability was measured with the LIVE/DEAD Violet fluorescent dye and flow cytometry. Survival curves under 3 glucose concentrations (0.56 [●], 2.8 [▴], and 5.6mM [■]) are shown. *P < .01, BMDAC-V vs BMDAC-D, by Bonferroni post hoc comparisons after 2-way ANOVA. Data are mean ± SEM (n = 3-5). Sergio Rey et al. Blood 2011;117:4988-4998 ©2011 by American Society of Hematology

DMOG induces intracellular alkalinization associated with increased CAR activity and altered expression of CAR isoforms. DMOG induces intracellular alkalinization associated with increased CAR activity and altered expression of CAR isoforms. (A) Intracellular pH of BMDACs. (B) Extracellular pH of BMDACs. (C) Quantitative RT-PCR analysis of Na+/H+ exchangers and active CAR isoforms in BMDAC-D. Gray shaded area represents ± 2-fold difference in BMDAC-D versus BMDAC-V. CAR isoforms: (c) indicates cytosolic; (mt), mitochondrial; (mb), membrane-bound; and (s), secreted. (D) CAR activity of BMDAC homogenates was measured using a colorimetric assay and normalized to the total amount of protein. (E) Effect of CAR inhibition on BMDAC viability under low pH (6.4), glucose (0.56mM), and hypoxia (1% O2). BMDACs were incubated with the CAR inhibitor acetazolamide (1nM to 500μM) for 12 hours; viability was measured with the LIVE/DEAD Violet fluorescent dye and flow cytometry. (A-E) Cells were cultured for 4 days in the presence of vehicle (V) or DMOG (D) before analysis. *P < .05, BMDAC-V vs BMDAC-D, by Student's t test or Bonferroni post hoc comparisons after 1-way ANOVA. Data are mean ± SEM (n = 3). Sergio Rey et al. Blood 2011;117:4988-4998 ©2011 by American Society of Hematology

Effect of HIF-1α loss of function on BMDAC survival and gene expression. Effect of HIF-1α loss of function on BMDAC survival and gene expression. (A-C) Bone marrow cells were isolated from Hif1a+/− (HET) mice, which are heterozygous for a null (knockout) allele at the locus encoding HIF-1α, or from their wild-type (WT) littermates and cultured for 4 days under nonhypoxic (N) or hypoxic (H) conditions in endothelial growth medium. The percentage of viable cells (A), number of viable cells (B), and expression of HIF-1 target genes (C) were determined. *P < .05 compared with nonhypoxic WT cells. #P < .01 compared with hypoxic WT cells in panels B and C, Bonferroni post hoc comparisons after 1-way ANOVA. (D) Quantitative RT-PCR analysis of HIF-1 target gene expression and cell viability (E) in BMDAC-V and BMDAC-D cultured for 4 days in the presence (+) or absence (−) of 100nM digoxin (Dig). *P < .05 compared with BMDAC-V without digoxin. #P < .01 compared with BMDAC-D without digoxin, Bonferroni post hoc comparisons after 1-way ANOVA. Data are mean ± SEM (n = 3 or 4). Sergio Rey et al. Blood 2011;117:4988-4998 ©2011 by American Society of Hematology

DMOG increases BMDAC survival in ischemic limbs. DMOG increases BMDAC survival in ischemic limbs. Genomic DNA was isolated from ischemic and nonischemic gastrocnemius muscles of female mice subjected to unilateral femoral artery ligation. All mice were injected with 2 × 108 pfu of AdCA5 distributed equally at 4 intramuscular injection sites in the ischemic limb immediately after surgery. A total of 5 × 105 BMDACs (isolated from male mice and cultured in vehicle or DMOG) were injected intravenously 24 hours after surgery. Muscle was harvested at 8, 16, 34, and 58 hours after injection. DNA was isolated and analyzed using quantitative PCR for a Y-chromosome-specific Sry gene sequence. No signal was detected in gastrocnemius muscles from nonischemic limbs. Decay curves were constructed using a 1-phase exponential decay model with nonlinear regression. BMDAC half-life was calculated from the data, and the difference between vehicle and DMOG was statistically significant (P < .05). Two-way ANOVA showed a statistically significant overall effect of DMOG treatment on BMDAC survival (P < .01 n = 3 or 4 animals per time point). Data are mean ± SEM. Sergio Rey et al. Blood 2011;117:4988-4998 ©2011 by American Society of Hematology

Combined therapy with DMOG-treated BMDACs and AdCA5 improves perfusion and clinical outcome after hindlimb ischemia in 17-month-old mice. Combined therapy with DMOG-treated BMDACs and AdCA5 improves perfusion and clinical outcome after hindlimb ischemia in 17-month-old mice. (A) Laser Doppler perfusion imaging (top and middle panels) and photography (bottom panel) were performed at the indicated time after femoral artery ligation. The top panel shows typical scans of female 17-month-old mice immediately before (Pre) and after (Post) femoral artery ligation in the left hindlimb. Blood flow is quantified using a pseudocolor scale as shown at right (perfusion units [PU]). The middle panel shows representative follow-up images of mice treated with saline, AdCA5, or combined therapy (AdCA5 + BMDAC-D). Because saline- and AdCA5-treated mice had severe tissue necrosis, the last time point (day 14 [D14]) before autoamputation ensued is shown. Bottom panel: Representative photographs of mice at day 28 after surgery (D28). (B) Mean ischemic/nonischemic limb perfusion ratio, as determined by laser Doppler perfusion imaging, was calculated at the indicated time (in days) after hindlimb ischemia. Mice were treated with saline (black), intramuscular AdCA5 (blue), or intramuscular AdCA5 + intravenous BMDAC-D (green). Intramuscular AdCA5 was injected immediately after surgery and intravenous BMDAC-D were delivered 24 hours later. *P < .05 vs saline control by Bonferroni post hoc test after ANOVA (n = 3 mice per group). (C) Tissue damage (left panel) was scored as follows: 0 indicates no tissue damage; 1, cyanosis/discoloration indicative of soft tissue necrosis; 2, loss of 1 or 2 toes; 3, loss of 3 to 5 toes; 4, amputation of entire foot; 5, amputation of entire lower leg; and 6, amputation of entire leg. Motor impairment (right panel) was scored as follows: 0 indicates normal response (plantar/toe flexion in response to tail traction); 1, plantar but not toe flexion; 2, no plantar or toe flexion; and 3, dragging of foot. Motor impairment and ischemic tissue damage were measured 28 days after surgery. *P < .05 vs saline control by Bonferroni post hoc test after ANOVA (n = 3 mice per group). Data are mean ± SEM. Sergio Rey et al. Blood 2011;117:4988-4998 ©2011 by American Society of Hematology