Functional disruption of alpha4 integrin mobilizes bone marrow-derived endothelial progenitors and augments ischemic neovascularization

Abstract

The cell surface receptor alpha4 integrin plays a critical role in the homing, engraftment, and maintenance of hematopoietic progenitor cells (HPCs) in the bone marrow (BM). Down-regulation or functional blockade of alpha4 integrin or its ligand vascular cell adhesion molecule-1 mobilizes long-term HPCs. We investigated the role of alpha4 integrin in the mobilization and homing of BM endothelial progenitor cells (EPCs). EPCs with endothelial colony-forming activity in the BM are exclusively alpha4 integrin-expressing cells. In vivo, a single dose of anti-alpha4 integrin antibody resulted in increased circulating EPC counts for 3 d. In hindlimb ischemia and myocardial infarction, systemically administered anti-alpha4 integrin antibody increased recruitment and incorporation of BM EPCs in newly formed vasculature and improved functional blood flow recovery and tissue preservation. Interestingly, BM EPCs that had been preblocked with anti-alpha4 integrin ex vivo or collected from alpha4 integrin-deficient mice incorporated as well as control cells into the neovasculature in ischemic sites, suggesting that alpha4 integrin may be dispensable or play a redundant role in EPC homing to ischemic tissue. These data indicate that functional disruption of alpha4 integrin may represent a potential angiogenic therapy for ischemic disease by increasing the available circulating supply of EPCs.

Figure 1.

Colony-forming EPCs express α4 integrin in the BM. (A) Flow cytometric analysis of α4 integrin expression in mouse BMMNCs. (B) Colony-forming EPCs were evaluated with a two-step endothelial cell differentiation culture starting with 5 × 10⁶ CD45⁺α4⁺ or CD45⁺α4⁻ FACS-sorted BMMNCs (A, red and blue frame, respectively). The left panel shows a typical colony of EPCs double positive for DiI-acLDL uptake (red) and isolectin B4–FITC binding (green), appearing yellow on merged images. The right panel shows the counts of double-positive colonies grown from CD45⁺α4⁺ and CD45⁺α4⁻ BMMNCs, respectively (n = 3 per group; ***, P < 0.001). (C and D) Flow cytometric analysis of stem cell antigen-1 (Sca-1) and FMS-like kinase (Flk-1) expression in α4⁺ vs. α4-BMMNC or in CD45⁺ vs. CD45⁻ BMMNC, respectively.

Figure 2.

Single dose of anti–α4 integrin Ab significantly increases circEPCs. (A) Flow cytometric analysis for the Sca-1⁺Flk-1⁺ cells in PBMNCs isolated at 24 h after i.v. injection of 200 μg anti–α4 integrin Ab (n = 7 per group; **, P < 0.01). (B) EPC culture assay using PBMNCs isolated at 24 h after i.v. injection of 200 μg anti–α4 integrin Ab. EPCs were cultured for 4 d then identified as adherent cells double positive for DiI-acLDL uptake (red) and isolectin B4–FITC binding (green) (left, original magnification, 200×) (right, ***, P < 0.001). (C) EPC culture assay using PBMNCs isolated at various time points after a single i.v. injection of 200 μg anti–α4 integrin Ab (○, control IgG; ▴, anti–α4 integrin Ab; n = 4 per group at each time point; **, P < 0.01; *, P < 0.05). (D) EPC culture assay using PBMNCs from α4 integrin conditional knockout mice or WT controls (n = 4; ***, P < 0.001).

Figure 3.

Anti–α4 integrin Ab blocks and competes for BM EPC adhesion to VCAM-1 and BM stroma. (A, top) Adhesion of freshly isolated mouse BMMNCs to the immobilized VCAM-1 was quantified with crystal violet microtiter plate. The Ab was added either before (Ab block) or 15 min after (Ab compete) the application of cells. The total incubation time of the Ab with cells was 30 min. (A, bottom) EPC colony assay using the suspension cells that resulted from each treatment in the top (***, P < 0.001 compared with blank control or isotype control; n = 3 per treatment). (B) Similar adhesion assay using different extracellular matrix or antibodies (V, VCAM-1; I, ICAM-1; FN, fibronectin; B/20 or B/200, block with 20 or 200 μg/ml Ab, respectively; C/2, C/20, or C/200 compete with 2, 20, or 200 μg/ml, respectively; n = 3 per treatment; **, P < 0.01; ***, P < 0.001 compared with VCAM-1 coating without Ab group; ^†, P < 0.01 compared with ICAM-1 coating without Ab group. (C) Adhesion of isolated BMMNCs on ex vivo cultured monolayer BM stroma. Quantification was performed by counting the number of adherent cells per square unit and expressed as a percentage (***, P < 0.001 compared with isotype control).

Figure 4.

Anti–α4 integrin Ab augments EPC mobilization after ischemic injury. FVB/NJ mice received surgically induced left HLI and were immediately randomized to receive injections of α4 integrin–blocking Ab or control IgG twice per week. Five mice in each group were killed at each data point (presurgery, 1, 2, or 3 wk post-HLI surgery), and PBMNC EPC culture assays were performed. (○, control IgG; ▴, anti–α4 integrin Ab; **, P < 0.01; ***, P < 0.001).

Figure 5.

Tie2/LacZ-BMT+HLI mouse model. Tie2/LacZ BM-transplanted recipient FVB/NJ mice received surgically induced left HLI and were randomized to receive injections of α4 integrin–blocking Ab or control IgG twice per week. On day 14, 10 mice from each group received i.v. injections of BS lectin I–FITC, which identifies vasculature, and were killed. (A) Laser Doppler Perfusion Image showing recovery of blood flow after surgery, expressed as the ratio of perfusion in ischemic limbs to normal limbs (left panel □ control IgG; ▪ anti–α4 integrin Ab. **, P < 0.01; *, P < 0.05). On the right are representative Laser Doppler Perfusion Images at various time points. (B) Representative fluorescent microscope fields of capillaries (BS lectin I–FITC⁺, green) and BM-derived EPCs (anti–β-gal–Rhodamine staining⁺, red) at ischemic area (original magnification, 400×). Arrows indicate BS lectin 1 and β-gal double positive cappillaries. (C) Overall capillary density (BS lectin I–FITC-positive only) (left, n = 6 limbs per group; **, P < 0.01) and BM EPC–derived capillary density (BS lectin I–FITC and β-gal–Rhodamine double positive) (right, n = 6 limbs per group; ***, P < 0.001) in the ischemic area. (D) The rest of the 10 mice in each group were killed on day 60. The wet muscular tissue of the lower limbs was isolated and weighed. Tissue preservation was expressed as the ratio of muscle weight in ischemic limbs to normal limbs (**, P < 0.01). (E) Immunofluorescent double staining for another endothelial marker, CD31 (red) and β-gal (blue) was performed on ischemic limbs at day 14 post-HLI (top) (original magnification, 200×). Arrows indicate CD31 and β-gal double positive capillaries. Quantification of CD31⁺ capillary density (bottom left, n = 6 limbs per group; **, P < 0.01) and CD31 + β-gal + double-positive BM EPC-derived capillary density (pink) (bottom, n = 6 limbs per group; ***, P < 0.001).

Figure 6.

Tie2/GFP−BMT+MI mouse model. Myocardial infarction was induced by ligation of the LAD in Tie2/GFP BM-transplanted mice. Mice were then randomized to receive i.v. injections of anti–α4 integrin Ab or control IgG twice weekly (n = 10 per group). On day 14, the mice received i.v. injections of BS lectin I–Rhodamine and were killed. The infarcted hearts were sectioned in a bread loaf fashion and pathohistological analysis was performed. Shown here are sections obtained at the level of 2 mm below the LAD ligation suture from each animal. (A) Representative fluorescent microscopy of the infarct areas (original magnification, 200×; BM-derived ECs, double positive for GFP [green] and BS lectin I–Rhodamine [red; appear yellow in the merged pictures and are indicated with arrows]). (B) Density of the capillaries with incorporated BM-derived ECs in the ischemic areas (n = 6 per group; ***, P < 0.001). (C) Preexisting survival capillary density in the infarct areas (BS lectin I–Rhodamine⁺ only) (n = 6 per group; ***, P < 0.001). (D) Capillary density in the periinfarct area (n = 6 per group; ***, P < 0.001). (E) Representative Masson's Trichrome staining of hearts after MI. The blue color represents fibrosis or scar which appears reduced in the hearts from anti–α4 Ab treatment group. Bar, 1 mm. (F) Quantification of area of fibrosis (d/c × 100%) confirms a reduction in LV fibrosis after MI in anti-α4 Ab–treated animals (n = 12 per group; **, P < 0.01). (G) Histological dimensions ((a+b)/2) (n = 12 per group; **, P < 0.01).

Figure 7.

Functional disruption of α4 integrin does not alter EPC tissue homing properties. (A) Equal numbers (15 × 10⁶) of BMMNCs were pretreated with either anti–α4 integrin Ab or control IgG, labeled with DiI, and injected into the peripheral circulation of mice with surgically induced HLI and splenectomy. On day 7, the mice were injected with BS lectin I–FITC and killed. Pathological analysis was conducted on the ischemic limb tissues. Capillaries in which ECs derived from injected BM EPCs were incorporated are BS lectin I–FITC and DiI double positive (top, the representative fluorescent microscopy; original magnification, 200×) (bottom, quantification of the densities of double-positive capillaries; n = 6 per group). (B) Background-matched, splenectomized WT recipient mice that had MI induced by LAD ligation received i.v. injections of 10⁶ BMMNCs from either α4 integrin conditional knockout mice or WT littermates. On day 7, the recipients were injected with BS lectin I–FITC and killed. The ischemic cardiac tissues were sectioned. Neovascular tissue containing ECs derived from the injected BM EPCs appear BS lectin I–FITC and DiI double positive (top, representative fluorescent microscopy; original magnification 400×) (bottom, quantification of the densities of double-positive capillaries; n = 6 per group). Arrows indicate double positive capillaries in both panels.