Del-1 induces angiogenesis in rabbit model of ischemia. (a) Angiography was performed after unilateral hind-limb ischemia induced in New Zealand white rabbits (3–4 kg body weight) by surgical resection of the femoral artery (19). X’s indicate the ten sites in and surrounding the medial thigh (midthigh) (bracket) in the upper leg, where plasmid was injected (0.5 mg/site). (b) Sustained transgene expression induced by plasmid injection in muscle was detected 1 month after therapy by RT-PCR for hVEGF₁₆₅ and hDel-1. VEGF or Del-1 gene expression was detected in VEGF- or Del-1–plasmid–injected muscle, respectively, but not in control plasmid–injected muscle. MW, molecular weight standards. (c) Angiography was performed before and 1 month after therapy for each treatment. Brackets indicate medial thigh region. (d) Mean number of new vessels (± SD) crossing the medial thigh (area under the brackets in c) for each treatment 1 month after therapy. Asterisks indicate statistically significant result (P < 0.01). (e) Mean CD31 protein expression (± SD) in muscle tissue 1 month after therapy. CD31 levels within injected muscle were assessed by Western blot analysis of lysates of muscle tissue. Asterisks indicate statistically significant result relative to noncoding plasmid control (P < 0.01). Statistical significance for angiography and CD31 analyses was assessed by one-way ANOVA with plasmid treatment as the main effect.

Del-1 interactions with αvβ5 regulate integrin αvβ3 expression. (a and b) Cryosections of CAMs stimulated with Del-1 were incubated with Ab’s against integrin αvβ3 or αvβ5 in combination with Ab’s against vWF. Integrin expression was detected with a rhodamine-conjugated secondary Ab and vWF with an FITC-conjugated secondary Ab. Integrin expression on blood vessels is seen as orange-yellow structures. Representative sections were photographed at ×200. White bar indicates 10 μm. (b) Average integrin expression levels for each integrin in vessels were quantified by determining the average number of red pixels in vessels that was greater than background and dividing by the average number of green pixels greater than background. (c) Integrins αvβ3 and αvβ5 were immunoprecipitated from lysate CAMs stimulated with bFGF or Del-1. Immunoprecipitates were immunoblotted with Ab’s against the cytoplasmic tails of the β3 and β5 subunits. Ratios of αvβ3 to αvβ5 were determined by densitometry. (d) CAMs were stimulated with Del-1 in the presence or absence of saline, anti-αvβ5, or W6/32 control isotype-matched Ab’s. Cryosections of CAMs were then incubated in anti-αvβ3 and anti-vWF Ab’s. Integrin αvβ3 expression was detected with a rhodamine-conjugated secondary Ab and vWF with an FITC-conjugated secondary Ab. Representative sections were photographed at ×200. White bar indicates 10 μm. Integrin αvβ3 expression on blood vessels is seen in yellow. Blood vessels are indicated with arrows. cIgG, control IgG. (e) Expression levels of integrin αvβ3 in vessels were quantified as in b.

Integrin αvβ5 is required for Del-1– but not bFGF-mediated angiogenesis. (a) Angiogenesis was induced by injection of growth factor–reduced Matrigel containing bFGF or Del-1 in homozygous integrin β5-null (β5^–/–) mice and age-matched heterozygous (β5^+/–) sibling mice as well as β5^+/+ mice. Cryosections of Matrigel plugs were immunostained with Ab’s directed against murine CD31. (b) CD31-positive vessels were quantified in five ×200 microscopic fields per plug, and the mean ± SEM for each treatment group was determined. Asterisk indicates statistically significant result relative to wild-type controls (P < 0.001). (c) Angiogenesis was induced by injection of growth factor–reduced Matrigel containing bFGF or Del-1 in homozygous integrin β3-null (β3^–/–) mice, age- and strain-matched C57BL/6 × 129S F₂ hybrid β3-positive mice (F₂), as well as age-matched β3-positive C57BL/6 mice (C57). Cryosections of Matrigel plugs were immunostained with Ab’s directed against murine CD31. (d) CD31-positive vessels were quantified in five ×200 microscopic fields per plug and the mean × SEM for each treatment group was determined. Asterisk indicates statistically significant result relative to wild-type controls (P < 0.001).

Hox D3 is required for Del-1–induced angiogenesis. (a) CAMs of 10-day-old chicken embryos stimulated by PBS, bFGF, or Del-1 were transfected with either N1-GFP reporter vector or Hox D3 sense (S), or Hox D3 antisense (AS) DNA expression constructs and N1-GFP. CAMs were fixed prior to excision, and blood vessel branch points (vessel branch pts) were quantified. (b) Expression levels of the chicken (chick) Hox D3, human Hox D3, and chicken GADPH genes in treated CAM tissues were determined by RT-PCR. (c and d) Relative levels of chick Hox D3 (c) and human Hox D3 (d). PCR products were determined by densitometry in comparison with GADPH expression levels for each treatment. (e) Expression levels of integrin β3 and β5, as well as GFP in CAMs stimulated with Del-1 or bFGF and treated with saline (PBS), Hox D3 antisense or sense were determined by Western blot analysis. Average expression levels for each integrin were determined in three separate experiments by densitometry. Asterisks indicate statistical significance relative to bFGF or Del-1 stimulation (P < 0.01).

Del-1 promotes angiogenesis and restores function to murine ischemic muscle in vivo. (a) Expression constructs used to induce hDel-1 and hVEGF expression in vivo contained a CMV enhancer/promoter (enh/pro), 5′ UTR, an optimized intron (IVS-8), cDNA encoding either VEGF₁₆₅ (pVF1164) or full-length human Del-1 (pDL1599), and the hGH 3′ UTR, as well as a kanamycin resistance gene. (b) Expression of Del-1 or VEGF₁₆₅ in hDel-1 (left) and hVEGF₁₆₅ (right) transgene-injected, noncoding plasmid–injected, and untreated muscle. Photographs were taken at ×100 magnification. Arrows denote fibers exhibiting positive staining. Cntl, control. (c) Lysates of muscle injected with hDel-1 (Del-1) or noncoding control plasmid as well as purified Del-1 (pur.Del-1) were immunoblotted for Del-1 expression using rabbit anti–Del-1 Ab’s. (d) Mouse model of hind-limb ischemia induced by ligation of the femoral artery. (e and f) Capillary/myofiber ratio 7 days after treatment with noncoding (control), hDel-1, or hVEGF₁₆₅ expression plasmids. (e) Cryosections of treated muscle immunostained using a rat anti-mouse CD31 Ab. (f) Mean capillary/myofiber ratio ± SEM for three animals per group (*P < 0.01). (g) Hind-limb ischemia was induced by bilateral ligation of the femoral artery of male CD1 mice. Animals were placed on the treadmill for 5 minutes at 5 m/min and then at 10 m/min thereafter. The speed was increased every 2 minutes until fatigue. Results represent the mean ± SEM (n = 7–8 animals/group) treadmill run time for mice treated with formulated noncoding hDel-1 or hVEGF₁₆₅ expression plasmids. Asterisks indicate statistically significant result relative to noncoding plasmid control (P < 0.05).

Integrin αvβ5 is a receptor for Del-1. (a) Biotinylated Del-1 (Del-1), vitronectin (VN), or the 120-kDa cell-binding fragment of fibronectin (FN) immunoblotted with goat–antibiotin HRP. (b–d) Biotinylated Del-1, vitronectin, collagen (COL), or biotinylated 120-kDa cell-binding region of fibronectin incubated with purified integrins αvβ5 (b), αvβ3 (c), or α5β1 (d) were quantified by determining absorbance at 405 nm. (e) FACS profile of HT29 colon carcinoma cells incubated with anti-αvβ5, anti-αvβ3, anti-β1, or no primary Ab (neg) followed by FITC-labeled secondary Ab. Data are plotted as log of fluorescence intensity versus cell number times 10^–5. (f) The adhesion of integrin αvβ5-positive/αvβ3-negative HT29 colon carcinoma cells to Del-1 or vitronectin in the presence of culture medium (med), function-blocking anti-integrin Ab’s anti-αvβ3, anti-αvβ5, or anti-integrin β1. (g) FACS profile of HUVECs incubated with anti-αvβ5, anti-αvβ3, or no primary Ab (neg) followed by FITC-labeled secondary Ab, as in e. (h) The adhesion of αvβ5-positive/αvβ3-positive HUVECs on Del-1 or vitronectin in the presence of culture medium, function-blocking anti-integrin Ab’s anti-αvβ3, anti-αvβ5, or anti-integrin α5β1. (i) FACS profile of human microvascular endothelial cells (HMVECs) incubated with anti-αvβ5, anti-αvβ3, or no primary Ab (neg), followed by FITC-labeled secondary Ab, as in e. (j) The adhesion of quiescent HMVECs on Del-1 or vitronectin in the presence of culture medium, function-blocking anti-integrin Ab’s anti-αvβ3, anti-αvβ5, anti-integrin α5β1, or anti-αvβ3 combined with anti-αvβ5 (anti-β3/β5). HPF, high-power field. Magnification, ×200.

Del-1–mediated angiogenesis requires integrins αvβ3 and αvβ5. (a and b) Del-1–stimulated chick CAMs were treated with saline, anti-αvβ3, anti-αvβ5, or control IgG (W6/32), and blood vessel branch points were counted (a) or photographed at ×10 magnification (b). Ten replicate embryos were tested per treatment group. (c and d) CAMs were treated with 10⁷ PFU of Del-1 or β-galactosidase (LacZ) expressing adenovirus, and blood vessel branch points were counted (c) or photographed at ×10 magnification (d). (e) Del-1–stimulated chick CAMs were treated with saline, anti-αvβ3, anti-αvβ5, or control IgG (W6/32) at 0, 24, and 48 hours. Blood vessel branch points were counted. Ten replicate embryos were tested per treatment group. Asterisks indicate statistically significant result relative to Del-1 stimulation (P < 0.01).

Del-1–αvβ5 interactions induce Hox D3 expression. (a) ECs were plated in monolayers (–BM) or on tissue-culture plates coated with thin layers of basement membrane (+BM) prior to cell lysis and Western blot analysis for cyclin D1, p21^cip, and integrins β3 and β5. (b) RT-PCR was performed for Hox D3, integrins β3 and β5, and GADPH (not shown). Relative levels of each PCR product were determined by densitometry in comparison with GADPH expression levels for each treatment. Asterisks indicate statistical significance relative to monolayer culture (–BM) (P < 0.02). (c) ECs were plated on endothelial monolayers in serum culture, BM + Del-1 (Del), or on BM + Del-1 with anti-αvβ5 function-blocking Ab’s (Del + P1F6), or control Ab’s (Del + cIgG). Integrin β5 and β5 protein expression levels were determined by Western blot analysis. Average expression levels for each integrin were determined in three experiments by densitometry. (d) RT-PCR was performed on RNA isolated from cells treated as in c for Hox D3, integrins β3 and β5, and GADPH. Relative levels of each PCR product were determined by densitometry in comparison with GADPH expression levels for each treatment. Asterisks indicate statistical significance relative to Del-1 stimulation (P < 0.02).

Hox D3 is downstream of αvβ5 but upstream of αvβ3. (a) Q4dh cells transfected with Hox D3 expressing control virus (neg cntl) retroviral constructs were cultured on replicate CAMs in the presence of saline (HOX D3 virus), anti-αvβ3 (Hox D3 + LM609), anti-αvβ5 (Hox D3 + P1F6), or control IgG (W6/32, not shown). CAMs were excised after fixation, and representative CAMs were photographed. Each treatment group contained ten to 12 replicate CAMs. (b) Blood vessel branch points on filter disks were quantified and graphed as mean ± SEM. Asterisks indicate statistical significance relative to Hox D3 virus stimulation (P < 0.01).