Adrenomedullin Enhances Angiogenic Potency of Bone Marrow Transplantation in a Rat Model of Hindlimb Ischemia
Background— Previous studies have shown that adrenomedullin (AM) inhibits vascular endothelial cell apoptosis and induces angiogenesis. We investigated whether AM enhances bone marrow cell–induced angiogenesis.
Methods and Results— Immediately after hindlimb ischemia was created, rats were randomized to receive AM infusion plus bone marrow–derived mononuclear cell (MNC) transplantation (AM+MNC group), AM infusion alone (AM group), MNC transplantation alone (MNC group), or vehicle infusion (control group). The laser Doppler perfusion index was significantly higher in the AM and MNC groups than in the control group (0.74±0.11 and 0.69±0.07 versus 0.59±0.07, respectively, P<0.01), which suggests the angiogenic potency of AM and MNC. Importantly, improvement in blood perfusion was marked in the AM+MNC group (0.84±0.08). Capillary density was highest in the AM+MNC group, followed by the AM and MNC groups. In vitro, AM inhibited MNC apoptosis, promoted MNC adhesiveness to a human umbilical vein endothelial cell monolayer, and increased the number of MNC-derived endothelial progenitor cells. In vivo, AM administration not only enhanced the differentiation of MNC into endothelial cells but also produced mature vessels that included smooth muscle cells.
Conclusions— A combination of AM infusion and MNC transplantation caused significantly greater improvement in hindlimb ischemia than MNC transplantation alone. This effect may be mediated in part by the angiogenic potency of AM itself and the beneficial effects of AM on the survival, adhesion, and differentiation of transplanted MNCs.
Received June 18, 2004; revision received September 9, 2004; accepted November 3, 2004.
Peripheral vascular disease is a crucial health issue that affects an estimated 27 million people.1 Despite recent advances in medical intervention, the symptoms of some patients with critical limb ischemia fail to be controlled. Bone marrow–derived mononuclear cells (MNCs) include a variety of stem and progenitor cells, such as endothelial progenitor cells (EPCs), and contribute to pathological neovascularization.2 MNC transplantation induces therapeutic angiogenesis in ischemic limb3,4; however, some patients fail to respond to this cell therapy. Thus, a novel therapeutic strategy to enhance the angiogenic property of MNCs is desirable.
Adrenomedullin (AM) is a potent vasodilator peptide that was originally isolated from human pheochromocytoma.5 Previous studies have reported that abnormalities of vascular structure are present in homozygous AM knockout mice.6,7 A recent study has demonstrated that blood flow recovery in ischemic limb and tumor angiogenesis are substantially impaired in heterozygous AM knockout mice.8 Furthermore, AM has been shown to inhibit vascular endothelial cell apoptosis and induce angiogenesis through the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway.9,10 These results suggest that AM is indispensable for modulating angiogenesis and vasculogenesis. When these findings are taken together, combination therapy with MNC transplantation and AM infusion may have additional or synergetic effects on therapeutic angiogenesis for the treatment of severe peripheral vascular disease. Thus, the purposes of the present study were (1) to investigate whether local infusion of AM enhances the angiogenic potency of MNC transplantation in a rat model of hindlimb ischemia and (2) to investigate the effects of AM on the survival, adhesion, and differentiation of transplanted MNCs.
Animal Model of Hindlimb Ischemia
Male Lewis rats (weight 250 to 275 g; Japan SLC Inc, Hamamatsu, Japan) were used in the present study. The left common iliac artery of each rat was resected under anesthesia with pentobarbital sodium (50 mg/kg). The distal portion of the saphenous artery and all side branches and veins were dissected free and excised. The right hindlimb was kept intact and used as the nonischemic limb. Transplantation of bone marrow–derived MNCs and infusion of AM were performed in 40 rats immediately after hindlimb ischemia was created. This protocol resulted in the creation of 4 groups: (1) AM infusion plus MNC transplantation (AM+MNC group, n=10), (2) AM infusion plus PBS injection (AM group, n=10), (3) vehicle infusion plus MNC transplantation (MNC group, n=10), and (4) vehicle infusion plus PBS injection (control group, n=10). The Animal Care Committee of the National Cardiovascular Center approved this experimental protocol.
MNC Transplantation and AM Infusion
Bone marrow was harvested from the femur and tibia in other male Lewis rats, and MNCs were isolated by Ficoll density gradient centrifugation (Lymphoprep, Nycomed). MNCs (5×106 cells per animal) or PBS was injected into the ischemic thigh muscle with a 26-gauge needle at 5 different points. Human recombinant AM (0.01 μg · kg−1 · min−1) or vehicle was administered for 7 days with a mini-osmotic pump (ALZET, Palo Alto) implanted in the left inguinal region.
Assessment of Blood Perfusion
To measure serial blood flow for 3 weeks, we used a laser Doppler perfusion image (LDPI) analyzer (Moor Instrument). After blood flow was scanned twice, the average flow values of the ischemic and nonischemic limbs were calculated by computer-assisted quantification. The LDPI index was determined as the ratio of ischemic to nonischemic hindlimb blood perfusion.11
Three weeks after MNC transplantation and/or AM infusion, 4 pieces of ischemic tissue from the adductor and semimembranous muscles were obtained and snap-frozen in liquid nitrogen. Frozen tissue sections were stained with alkaline phosphatase by an indoxyl tetrazolium method to detect capillary endothelial cells.3,11 Five fields were randomly selected to count the number of capillaries. The capillary number adjusted per muscle fiber was used to compare the differences in capillary density among the 4 groups.3
Monitoring of Transplanted MNCs in Ischemic Hindlimb Muscle
To examine differentiation of transplanted MNCs, 5×106 MNCs labeled with red fluorescent dye (PKH26-GL, Sigma Chemical Co) were transplanted into the ischemic thigh muscle in rats with (n=3) and without (n=3) AM infusion. Three weeks after transplantation, frozen tissue sections from ischemic muscle were incubated with anti-von Willebrand factor antibody (vWF, DAKO), anti-CD31 antibody (BD Pharmingen), and anti-α-smooth muscle actin antibody (α-SMA, DAKO), followed by incubation with Alexa Fluor 633 IgG antibody (Molecular Probes) and FITC-conjugated IgG antibody (BD Pharmingen), respectively. Five high-power fields (40×) of each section were randomly selected to count the number of transplanted MNCs, vWF-positive cells, and α-SMA–positive cells.
In Situ Detection of MNC Apoptosis
PKH26-labeled MNCs (5×106 cells per animal) were transplanted into the ischemic muscle in rats with (n=2) and without (n=2) AM infusion. Twenty-four hours after transplantation, apoptosis of transplanted MNCs in ischemic tissue was evaluated by terminal dUTP nick-end labeling (TUNEL) assay (ApopTag Fluorescein kit, Serological Corporation), as reported previously.12
In Vitro Apoptosis Assay
The antiapoptotic effect of AM on MNCs was evaluated by TUNEL assay. Human MNCs, isolated from peripheral blood, were plated on 12-well plates (1×106 cells per well) and cultured in serum-free medium for 24 hours with control buffer, AM, or AM plus wortmannin, a PI3K inhibitor (50 nmol/L). TUNEL for detection of apoptotic nuclei was performed according to the manufacturer’s instructions. MNCs were then mounted in medium that contained 4′,6-diamidino-2-phenylindole (DAPI). Randomly selected microscopic fields (n=10) were evaluated to calculate the ratio of TUNEL-positive cells to total cells.
We evaluated whether AM enhances MNC adhesiveness according to a previously reported method.13 In brief, human umbilical vein endothelial cells (HUVECs) were cultured to confluence on 6-well plates with or without pretreatment with tumor necrosis factor-α (1 ng/mL). In the absence or presence of AM (10−7 mol/L), 1×106 MNCs labeled with PKH26 were incubated on an HUVEC monolayer for 24 hours. Nonadherent MNCs were removed, and the number of PKH26-positive cells in each well was counted.
Expression of adhesion molecules in HUVECs was measured by cell ELISA, as reported previously.14 In brief, confluent HUVECs on 96-well plates were treated with AM (10−7 mol/L) or control buffer for 4 hours. HUVECs were then incubated with monoclonal mouse antibodies against intercellular adhesion molecule-1 (ICAM-1, R&D Systems) and vascular adhesion molecule-1 (VCAM-1, R&D Systems). A protein detector ELISA kit (KPL) was used to detect bound monoclonal antibodies.
EPC Culture Assay
Culture of EPCs was performed as described previously.11,15,16 In brief, 2×106 MNCs were plated in Medium-199 supplemented with 20% FCS, heparin, and antibiotics on fibronectin-coated 6-well plates. AM (10−7 mol/L), human recombinant vascular endothelial growth factor (VEGF; 20 ng/mL), or control buffer was added to each plate. After 7 days of culture, nonadherent cells were removed, and adherent cells were incubated with acetylated LDL labeled with DiI (DiI-acLDL, Biomedical Technologies) and FITC-labeled lectin from ulex europaeus (Sigma). Double-positive cells for DiI-acLDL and FITC-labeled lectin were identified as EPCs.16 Randomly selected microscopic fields (n=10) were evaluated to count the number of EPCs.
Fluorescence-Activated Cell Sorting Analysis
Fluorescence-activated cell sorting was performed to identify characteristics of adherent cells after 7 days of culture.16 Cells were incubated for 30 minutes at 4°C with anti-human CD31 antibodies (clone L133.1, Becton Dickinson), anti-human KDR antibodies (clone KDR-1, Sigma), and anti-human VE-cadherin antibodies (clone BV6, Chemicon). Isotype-identical antibodies served as controls. Fluorescence-activated cell sorting analyses were performed with a FACSCalibur flow cytometer and Cell Quest software (BD Biosciences).
Real-Time Polymerase Chain Reaction
Expression of calcitonin receptor-like receptor (CRLR), a receptor for AM, was examined by real-time polymerase chain reaction (PCR). Total RNA was extracted from MNCs, EPCs, and HUVECs with an RNA extraction kit (RNeasy Mini Kit, Qiagen) and converted to cDNA by reverse transcription. Real-time PCR was performed with SYBR green dye (QuantiTect SYBR Green PCR kit, Qiagen) and a Prism 7700 sequence detection system (Applied Biosystems). The PCR primers for CRLR were as follows: sense primer 5′-CATTCAACAAGCAGAAGGCG-3′ and antisense primer 5′-AGCCATCCATCCCAGGTTC-3′. For GAPDH, the primers were as follows: sense primer 5′-CAATGCCTCCTGCA-CCACCAA-3′ and antisense primer 5′-GAGGCAGGGATGATGTTCTGGA-3′. Levels of CRLR mRNA were normalized to that of GAPDH mRNA. PCR-amplified products were also electrophoresed on 2% agarose gels to confirm that single bands were amplified.
In Vitro Matrigel Assay
HUVECs (1×105 cells) were seeded onto 24-well plates coated with Matrigel (Becton Dickinson) in the presence of the combination of control buffer, AM (10−7 mol/L), VEGF (10 ng/mL), or neutralizing antibodies against KDR (2 μg/mL, R&D Systems). After incubation for 18 hours, tube formation area was measured as described previously.17 The control was defined as 100% tube formation, and the percent increase was calculated for each sample.
Measurements of Cytokines
A total of 1×106 MNCs or HUVECs were plated in serum-free medium with or without AM (10−7 mol/L) on 12-well plates. After 24-hour incubation, the conditioned medium was collected, and VEGF, basic fibroblast growth factor, and hepatocyte growth factor were measured with enzyme immunoassay kits (R&D Systems).
Migration assay of smooth muscle cells (SMCs) was performed with Transwell (Coster) 24-well plates composed of a collagen-coated membrane with 8-μm pores. Human aortic SMCs, preincubated with serum-free medium for 24 hours to maintain quiescence, were seeded on the upper chamber at a concentration of 1×106 cells/mL. Serum-free medium containing control buffer, AM (10−7 mol/L), or AM plus wortmannin (50 nmol/L) was placed in the lower chamber. After incubation for 12 hours, the number of migrated cells was counted in the randomly selected fields (n=5).
All values are expressed as mean±SEM. Student’s unpaired t test was used to compare differences between 2 groups. Comparisons of parameters among 3 or 4 groups were made by 1-way ANOVA, followed by Scheffé multiple comparison test. Comparisons of the time course of the LDPI index were made by 2-way ANOVA for repeated measures, followed by Scheffé multiple comparison tests. A probability value <0.05 was considered statistically significant.
Blood Perfusion and Capillary Density
Blood perfusion of the ischemic hindlimb increased modestly but gradually in the AM and MNC groups after treatment (Figure 1A). Interestingly, blood perfusion in the AM+MNC group markedly improved within 2 weeks after treatment and showed further improvement thereafter. The LDPI index was significantly higher in the AM, MNC, and AM+MNC groups than in the control group 3 weeks after surgery (Figure 1B). Importantly, the LDPI index was highest in the AM+MNC group among the 4 groups.
Alkaline phosphatase staining of ischemic muscle showed significant augmentation of neovascularization in the AM, MNC, and AM+MNC groups (Figure 2A). The capillary/muscle fiber ratio of ischemic muscle was highest in the AM+MNC group, followed by the MNC group, AM group, and control group (Figure 2B).
Differentiation of Transplanted MNCs
Three weeks after MNC transplantation, PKH26-labeled MNCs were frequently observed in the AM+MNC group, and these transplanted cells were positive for vWF (Figure 3A). Most of these cells were also stained by CD31 (data not shown). The number of PKH26/vWF double-positive cells was significantly higher in the AM+MNC group than in the MNC group (Figure 3B). Although PKH26/α-SMA double-positive cells were not detected in ischemic muscle of each group, newly formed vascular structures in the AM+MNC group included α-SMA–positive cells (Figure 3C). The number of α-SMA–positive cells in the MNC-derived vascular structures was significantly higher in the AM+MNC group than in the MNC group (Figure 3D).
Antiapoptotic Effect of AM on MNCs
In vitro, serum starvation induced MNC apoptosis, as indicated by detection of TUNEL-positive cells (Figure 4A). When incubated in the presence of AM, the percentage of TUNEL-positive cells markedly decreased in a dose-dependent manner (Figure 4B). However, pretreatment with wortmannin, a PI3K inhibitor, diminished the antiapoptotic effect of AM. Similarly, in vivo, local administration of AM decreased TUNEL-positive MNC 24 hours after transplantation (data not shown).
Effect of AM on MNC Adhesiveness
The number of adherent MNCs on an HUVEC monolayer increased significantly in the presence of AM (10−7 mol/L) compared with control (Figures 5A and 5B). With pretreatment using tumor necrosis factor-α, AM also enhanced the adhesiveness of MNCs to HUVECs. AM significantly enhanced expression of ICAM-1 and VCAM-1 in HUVECs (Figure 5C).
Effect of AM on EPC Expansion
After 7-day culture of human MNCs, spindle-shaped or cobblestone-like adherent cells were observed (Figure 6A). Most of the adherent cells were double stained with DiI-acLDL and FITC-labeled lectin. These adherent cells expressed endothelial cell–specific markers: KDR, VE cadherin, and CD31 (Figure 6B). Thus, we identified the major population of the adherent cells as EPCs. Culture of MNCs with AM significantly increased the number of EPCs (Figure 6C). The effect of AM was equivalent to that of VEGF. Real-time PCR revealed that MNCs, EPCs, and HUVECs expressed mRNA of CRLR (Figure 6D). Expression of CRLR mRNA was highest in HUVECs, followed by EPCs and MNCs.
Effects of AM on Tube Formation and SMC Migration
Like VEGF, AM induced tube formation in HUVECs in vitro (Figure 7A). Blocking antibodies against KDR significantly inhibited VEGF-induced tube formation, whereas they did not suppress AM-induced tube formation (Figure 7B). AM did not significantly alter VEGF, basic fibroblast growth factor, or hepatocyte growth factor levels in conditioned medium of cultured MNCs or HUVECs (data not shown). AM significantly increased the number of migrated SMCs compared with control (Figures 7C and 7D). Pretreatment with wortmannin diminished the effect of AM on SMC migration.
In the present study, we demonstrated in vivo that AM infusion or MNC transplantation alone induced angiogenesis in a rat model of hindlimb ischemia, the combination of AM infusion and MNC transplantation enhanced MNC-induced angiogenesis, and AM increased the number of MNC-derived vWF-positive cells and generated α-SMA–positive vascular structures. We also demonstrated in vitro that AM inhibited serum starvation–induced MNC apoptosis, promoted MNC adhesiveness to an HUVEC monolayer, increased the number of MNC-derived EPCs, and stimulated SMC migration.
MNC transplantation causes therapeutic angiogenesis by supplying EPCs and multiple angiogenic cytokines such as VEGF.3,4 The present study showed that local infusion of AM significantly increased blood perfusion and capillary density in ischemic hindlimb muscle. Furthermore, a combination of AM infusion and MNC transplantation significantly increased blood perfusion and capillary density of the ischemic hindlimb compared with MNC transplantation alone. AM has been shown to induce angiogenesis in vitro and in vivo through the PI3K/Akt pathway.10,18 In the present study, AM-induced tube formation was not blocked by neutralizing antibodies against KDR. In addition, AM did not enhance VEGF secretion from MNCs and HUVECs. Thus, beneficial effects of combination therapy with AM and MNCs may be attributable in part to the angiogenic properties of AM itself.
An earlier study has shown that transplanted MNCs disappear from ischemic muscle 7 days after transplantation.19 We demonstrated that apoptosis of MNCs occurred in ischemic muscle 24 hours after MNC transplantation. These results raise the possibility that the angiogenic potency of MNC transplantation is attenuated by MNC apoptosis. In the present study, AM inhibited apoptosis of MNCs in vitro and in vivo, and the antiapoptotic effect of AM was suppressed by wortmannin, a PI3K inhibitor. These findings suggest that AM prolongs MNC survival through the PI3K/Akt pathway and thereby enhances neovascularization in ischemic tissue.
In the present study, AM promoted adhesiveness of MNCs to an HUVEC monolayer. AM significantly enhanced expression of ICAM-1 and VCAM-1 in HUVECs, both of which facilitate adhesion of MNCs to endothelial cells.20 These findings suggest that AM increases MNC adhesiveness to endothelial cells via activation of adhesion molecules. A recent study has shown that MNC adhesiveness to endothelial cells is indispensable for MNC differentiation into endothelial lineage.21 Thus, it is possible that AM infusion enhances the angiogenic potency of MNCs at least in part through promotion of adhesion of MNC to host vascular endothelial cells.
VEGF has been shown to increase the number of EPCs in vitro and in vivo, resulting in angiogenesis and vasculogenesis.13,22 The present study showed that MNCs and EPCs expressed CRLR, a receptor of AM. In vitro, AM increased the number of MNC-derived EPCs that expressed VE cadherin, KDR, and CD31. The effect of AM on EPC expansion was equivalent to that of VEGF. In vivo, AM infusion increased the number of MNC-derived vWF-positive cells, although incorporation of these cells in the capillaries may be due in part to incorporation of hematopoietic cells. These findings suggest that AM may accelerate MNC differentiation into endothelial lineage.
SMC is essential for the generation of functional and mature blood vessels.23 We demonstrated in vivo that local infusion of AM increased the number of α-SMA–positive cells (SMCs) in MNC-derived vascular structures. In vitro, AM enhanced SMC migration, which was inhibited by wortmannin, a PI3K inhibitor. Recent studies using homozygous AM knockout mice have suggested that AM is indispensable for vascular morphogenesis.6,7 When these findings are taken together, it is possible that AM contributes to vessel maturation through enhancement of SMC migration via the PI3K/Akt-dependent pathway.
Currently, a new therapeutic approach to augment the efficacy of MNC transplantation is awaited for the treatment of severe peripheral vascular disease. The present study demonstrated that local infusion of AM enhanced the angiogenic potency of MNC transplantation. In the present study, AM inhibited MNC apoptosis and increased the total number of engrafted cells in ischemic tissue, although this study did not show the effect of AM on specific cell populations of MNCs. In addition, AM promoted cell proliferation, migration, and differentiation. We have already demonstrated the safety of AM infusion in patients with congestive heart failure.24 Thus, combination therapy with AM infusion and MNC transplantation may be a novel and promising therapeutic strategy for the treatment of severe peripheral vascular disease.
A combination of AM infusion and MNC transplantation caused significantly greater improvement in hindlimb ischemia than MNC transplantation alone. This effect may be mediated in part by the angiogenic potency of AM itself and the beneficial effects of AM on the survival, adhesion, and differentiation of transplanted MNCs.
This work was supported by the research grant for cardiovascular disease (16C-6) from the Ministry of Health, Labor and Welfare, Industrial Technology Research Grant Program in ’03 from New Energy and Industrial Technology Development Organization (NEDO) of Japan, Health and Labor Sciences Research Grants-genome 005, the Mochida Memorial Foundation for Medical and Pharmaceutical Research, and the Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research (OPSR) of Japan.
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