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From INSERM U541,* Hôpital Lariboisière, IFR Circulation-Paris 7, Université Paris 7, Paris; and the Institut des Vaissseaux et du Sang, Hôpital Lariboisière, Paris, France
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Abstract |
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Diabetics commonly suffer cardiovascular complications, including vascular diseases. In diabetic patients, collateralization is insufficient to overcome the loss of blood flow through occluded arteries leading to ischemia and often nontraumatic limb amputation. However, only few studies focus on the identification of factors that may affect neovascularization in the setting of ischemia in diabetes. It has been suggested that alteration in VEGF expression and signalization or inflammation-related pathway may reduce blood vessels formation in diabetes.16,17 Alternatively, EPC proliferation and adhesion ability were affected in patients with type II diabetes suggesting that BM-derived cells may have altered functions in the setting of diabetes.18 We therefore hypothesized that the impairment of ischemia-induced neovascularization observed in diabetes may be related to a decrease in the pro-angiogenic potential of endogenous BM-MNCs. In the present proposal, we 1) evaluated the pro-angiogenic potential of diabetic BM-MNCs by assessing the effect of administration of BM-MNCs isolated from nondiabetic and diabetic mice on revascularization in the ischemic hindlimb of nondiabetic mice; 2) examined the putative influence of diabetic environment by evaluating the effect of administration of BM-MNCs isolated from nondiabetic and diabetic mice on revascularization in the ischemic hindlimb of diabetic mice; and 3) attempted to analyze the mechanisms involved in diabetes-induced impairment in BM-MNC pro-angiogenic activity. To this aim, we examined the effect of diabetes on BM-MNC differentiation into EPCs in vitro and on the ability of BM-MNCs to participate in vascular-like structure formation; 4) assessed the therapeutic potential of PlGF administration on diabetes-induced BM-MNC dysfunction and impairment of postischemic neovascularization.
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Materials and Methods |
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Induction of Diabetes
To induce moderate diabetes, C57Bl/6 mice (8 weeks old, Iffa Creddo, Lyon, France) were injected intraperitoneally with 40 mg/kg of streptozotocin (STZ; Sigma Chemical Co) in 0.05 mol/L Na citrate, pH 4.5, daily for 5 days. Three days after the fifth injection, blood glucose levels were measured. If serum glucose was less than 9 mmol/L, mice were injected for an additional twice a week at the same dosage. Glucose levels were retested again every week to ensure serum glucose level of greater than 10 mmol/L in mice. Mice with glucose levels less than 10 mmol/L were excluded from further study. In the nondiabetic groups, mice were injected intraperitoneally with 0.05 mol/L Na citrate, pH 4.5.
Administration of BM-MNCs
After 2 months, right femoral artery ligature was performed as previously described.3,4 Using this surgical procedure and using C57Bl6 mice, we did not observe autoamputation in nondiabetic or diabetic animals. At the time of ischemia, nondiabetic and diabetic mice received intravenous injections of 1 x 106 BM-MNCs or PBS. BM-MNCs were also labeled with a green fluorescent marker, PKH2-GL.13 Animals were sacrificed at day 10 after ischemia.
Therapeutic angiogenesis
In a second set of experiments, an osmotic minipump (subcutaneously implanted on the backs of the mice; Alzet, type 2001, Cupertino, CA) delivered for 14 day a daily dose of 1.5 µg of PlGF (R&D Systems, Minneapolis, MN) in nondiabetic and diabetic mice. At day 7, ischemia was induced by right femoral artery ligature and animals were sacrificed at day 14.
Quantification of Angiogenesis
Microangiography
Vessel density was evaluated by high definition microangiography as previously described.3,4 Briefly, mice were anesthetized (isoflurane inhalation) and a contrast medium (barium sulfate, 1 g/ml) was injected through a catheter introduced into the abdominal aorta. Images acquired by a digital X-ray transducer were assembled to obtain a complete view of the hindlimbs. The vessel density was expressed as a percentage of pixels per image occupied by vessels in the quantification area. A quantification zone was delineated by the place of the ligature on the femoral artery, the knee, the edge of the femur and the external limit of the leg.
Capillary Density
Microangiographic analysis was completed by assessment of capillary densities in ischemic and nonischemic muscles at day 10 as previously described.3,4 Frozen tissue sections from gastrocnemius muscle (7 µm) were incubated with rabbit polyclonal antibody directed against total fibronectin (dilution 1:50, TEBU, Yvelines, France) to identify capillaries. Capillaries were revealed with a fluorescent FITC anti-rabbit antibody (dilution 1:10). Capillary density were analyzed in three different sections of gastrocnemius muscle. Five different fields were used in each section (Histolab software, Microvision Instrument, Evry, France). Capillary/myocyte ratios were calculated and the results were then expressed according to ischemic/nonischemic ratios.
Laser Doppler Perfusion Imaging
To provide functional evidence for ischemia-induced changes in vascularization, Laser Doppler perfusion imaging experiments were performed at day 10 as previously described.3,4 Briefly, excess hairs were removed by depilatory cream from the limb, and mice were placed on a heating plate at 37°C to minimize temperature variation. Nevertheless, to account for variables, including ambient light, temperature, and experimental procedures, blood flow was calculated in the foot and expressed as a ratio of ischemic to nonischemic leg. After right femoral artery ligature, mice showing less than 80% of blood flow reduction were excluded from this study.
Isolation of Mouse BM-MNCs
Bone marrow cells were obtained by flushing the tibias and femurs of nondiabetic and diabetic male C57Bl/6 mice. Low-density mononuclear cells were then isolated by density gradient centrifugation with Ficoll as previously described.4
EPC Differentiation Assay
Immediately after isolation, 5 x 106 bone marrow cells were plated on 35-mm cell culture dishes coated with rat plasma vitronectin (Sigma, St Quentin Fallavier, France) and gelatin (0.1%) and maintained in endothelial basal medium (EBM2, BioWhittaker, Paris, France). After 4 days in culture, nonadherent cells were removed and adherent cells underwent immunochemicals analysis as previously described.4
To detect the uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labeled acetylated low-density lipoprotein (AcLDL-Dil), cells were incubated in medium containing AcLDL-Dil (Tebu, Le Perray en Yvelines, France) at 37°C for 1 hour. Cells were then fixed with 2% paraformaldehyde and incubated with FITC-labeled BS-1 lectin (Sigma). Dual-stained cells positive for both AcLDL-Dil and BS-1 lectin were judged to be EPCs, and they were counted per well, as previously described,.4,18 Antibody directed against CD18 (Sigma) was also used to confirm that cultured EPCs did not derive from monocytes/macrophages. Five replicates were done for each experimental conditions. Three independent investigators evaluated the number of EPCs per well by counting three randomly selected high-power fields under fluorescence microscopy. Results are expressed as percentage of total number of cultured cells.
Angiogenesis Assay Using the Matrigel Model
Eight-week-old C57Bl/6 female mice, (Iffa-Creddo) received in their back 0.5 ml subcutaneous injection of either Matrigel + 1 x 106 BM-MNCs isolated from nondiabetic mice, or Matrigel + 1 x 106 BM-MNCs isolated from diabetic mice. In a second set of experiments, BM-MNCs were labeled with a green fluorescent marker, PKH2-GL.13 After the injection, the Matrigel formed rapidly a subcutaneous plug that was left 14 days before removal.
On day 14, the mice were euthanized and the skins were pulled back to expose the Matrigel as previously described.19 Plugs were then removed and fixed with 3.7% formaldehyde at 4°C for 12 hours, embedded in paraffin, sectioned, and stained with Masson Trichrome. Matrigel plugs were sampled systematically, three successive sections of 5 µm, at the top, middle, and bottom of each plug, and then examined (10x, 20x, and 40x magnification; Olympus BH-2, Leica).
Statistical Analysis
Results are expressed as mean ± SEM. One-way analysis of variance was used to compare each parameter. Post hoc Bonferonni’s t-test comparisons were then performed to identify which group differences account for the significant overall analysis of variance. A value of P < 0.05 was considered as statistically significant.
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Results |
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Blood glucose level was 1.9-fold increased in diabetic mice compared to nondiabetic animals (13.1 ± 1.8 mmol/L versus 6.7 ± 1.1 mmol/L, P < 0.05). In addition, body weight was 1.1-fold reduced in diabetic mice when compared to nondiabetic animals (28.3 ± 0.7 g versus 31.2 ± 0.9 g P < 0.05). Treatment with BM-MNCs did not affect these parameters in either group (data not shown).
Effects of BM-MNCs Isolated from Diabetic Mice on Ischemia-Induced Neovascularization
Endogenous BM-MNCs have been shown to secrete angiogenic factors, to incorporate into foci of neovascularization, and thereby to increase new blood vessel formation in ischemic tissue.12,13 Impairment of ischemia-induced neovascularization in diabetes may be related to a decrease in pro-angiogenic potential of endogenous BM-MNCs. Hence, we analyzed the pro-angiogenic potential of diabetic and nondiabetic BM-MNCs in the nondiabetic ischemic leg.
Angiographic Score
Transplantation of BM-MNCs isolated from nondiabetic animals raised the ischemic/nonischemic angiographic score by 1.8-fold over that of nondiabetic animals receiving PBS(P < 0.05) (Figure 1)
. Administration of BM-MNCs isolated from diabetic mice increased by 1.4-fold vessel density when compared to PBS-injected nondiabetic animals (P < 0.05). However, angiographic score was still 1.4-fold lower in mice treated with BM-MNCs isolated from diabetic animals compared to those treated with BM-MNCs isolated from nondiabetic animals (P < 0.05).
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Capillary density analysis after fibronectin staining correlated with microangiographic data (Figure 2)
. Injection of BM-MNCs isolated from nondiabetic animals enhanced ischemic/nonischemic capillary density ratio by 2.7-fold over that of nondiabetic animals receiving PBS (P < 0.01). Administration of BM-MNCs isolated from diabetic mice increased by 1.7-fold capillary number when compared to PBS-injected nondiabetic animals (P < 0.05). However, the capillary number ratio was still 1.6-fold lower from that observed in mice treated with BM-MNCs isolated from nondiabetic animals (P < 0.05).
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Microangiographic and capillary density measurements were associated with changes in ischemic foot blood flow (Figure 3)
. Administration of BM-MNCs isolated from nondiabetic animals increased ischemic/nonischemic blood flow ratio by 2.2-fold in reference to nondiabetic animals receiving PBS (P < 0.05). Injection of BM-MNCs isolated from diabetic mice also increased by 1.4-fold hindlimb blood flow recovery when compared to PBS-injected nondiabetic animals (P < 0.05) but to a lesser extent from that observed in mice treated with BM-MNCs isolated from nondiabetic animals (P < 0.05).
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Vascular endothelial dysfunction induced by diabetes may modulate the ability of BM-derived cells to restore angiogenic function. Therefore, we analyzed pro-angiogenic potential of diabetic and nondiabetic BM-MNCs in diabetic ischemic leg.
Angiographic Score
Transplantation of BM-MNCs isolated from nondiabetic animals raised the angiographic score by twofold over that observed in diabetic animals receiving PBS (P < 0.05) (Figure 1B)
. Administration of BM-MNCs isolated from diabetic animals also increased by 1.4-fold vessel density when compared to PBS-injected diabetic mice (P < 0.05), but to a lower extent than that observed in diabetic mice receiving BM-MNCs isolated from nondiabetic animals (1.5-fold, P < 0.05).
Capillary Density
Transplantation of BM-MNCs isolated from nondiabetic animals raised the ischemic/nonischemic capillary number ratio by 2.4-fold over that of diabetic animals receiving PBS (P < 0.01) (Figure 2B)
. Diabetic mice receiving BM-MNCs isolated from diabetic animals also showed a 1.8-fold increase in capillary number ratio when compared to PBS-injected diabetic mice (P < 0.05). Nevertheless, diabetic BM-MNC-related effects was 1.5-fold lower from those observed with nondiabetic BM-MNC administration (P < 0.05).
Laser Doppler Perfusion Imaging
Transplantation of BM-MNCs isolated from nondiabetic animals raised the blood flow ratio by twofold compared to diabetic animals receiving PBS (P < 0.05) (Figure 3B)
. Diabetic mice receiving BM-MNCs isolated from diabetic animals also showed a 1.4-fold increase in blood flow ratio when compared to PBS-injected diabetic mice (P < 0.05). Nevertheless, diabetic BM-MNC-related effects was 1.4-fold lower from those observed with nondiabetic BM-MNC administration (P < 0.05).
Effects of Diabetes on BM Cell Differentiation into EPCs
BM-MNCs have been shown to differentiate into EPCs in vitro.4,20 Diabetes may reduce differentiation of BM-MNCs into EPCs leading to abrogated postnatal neovascularization.
BM cells from nondiabetic and diabetic mice were isolated, cultured and differentiated into EPCs. A subset of BM-derived cells plated on vitronectin attached and became spindle shaped. EPCs were then characterized as dual-stained cells positive for AcLDL-Dil and BS1-lectin. Only a few positive staining for the monocytic marker CD18 has been observed in our experimental conditions, suggesting that most of the Dil LDL/BS-1 lectin positive cells are not derived from monocytes/macrophages (Figure 4)
. The percentage of cells with Dil LDL/BS-1 lectin positive-staining was low in diabetic and nondiabetic animals without femoral artery ligature (<5%, n = 5 for each group) (data not shown). Ischemia induced by right femoral artery ligature markedly increased the percentage of cells with double-positive staining for AcLDL-Dil and BS-1 lectin in nondiabetic animals (10-fold, P < 0.001 versus nonischemic nondiabetic animals). This effect was hampered by diabetes (55 ± 10% versus 10 ± 3% in nondiabetic mice with femoral artery ligature versus diabetic mice with femoral artery ligature, P < 0.001) (Figure 4)
. In addition, nondiabetic and diabetic BM cells cultured in high glucose conditions did not display changes in their ability to differentiate into EPCs (data not shown).
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BM-MNCs may incorporate into blood vessels in ischemic leg and myocardium.12,13 Hence, diabetes may affect the ability of BM-MNCs to contribute to blood vessel formation and therefore may reduce the revascularization process.
In the Matrigel plug, administration of BM-MNCs isolated from nondiabetic mice formed numerous tube-like structures and the presence of erythrocytes was evidenced in the lumen, demonstrating the existence of a functional vascular structure (Figure 5)
. Injection of fluorescence-labeled nondiabetic BM-MNCs disclosed that fluorescence-positive cells were incorporated into tube-like structures. These results also suggest that new vessels formed in the Matrigel plug are composed from one part of injected labeled BM-derived cells and from the other part of endogenous cells which have migrated in the Matrigel. Conversely, administration of BM-MNCs isolated from diabetic mice induced formation of disorganized cell clusters. In addition, injection of fluorescence-labeled diabetic BM-MNCs showed that fluorescence-positive cells did not markedly contribute to tube-like structure.
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Differentiation into EPCs
We first assessed the effect of PlGF treatment on the ability of BM-MNCs to differentiate into EPCs in the setting of ischemia. PlGF administration increased by 1.4-fold the percentage of cells with double-positive staining for AcLDL-Dil and BS-1 lectin in nondiabetic animals (67.6 ± 7.3%) in reference to untreated nondiabetic mice (48.2 ± 5.4%, P < 0.05). PlGF treatment strongly increased by sixfold the number of cells with double-positive staining in diabetic mice (64.1 ± 7.9% versus 11.2 ± 1.9% in PlGF-treated and untreated diabetic mice, respectively, P < 0.001).
Neovascularization Reaction
We next determined the therapeutic potential of PlGF treatment on diabetes-induced impairment of the neovascularization reaction. In nondiabetic animals, infusion of PlGF increased by 1.5-, 1.4-, and 1.6-fold the angiographic score, capillary number, and limb blood flow, respectively, when compared to untreated nondiabetic animals (P < 0.01) (Figure 6)
. Interestingly, in diabetic mice, PlGF administration enhanced ischemic/nonischemic angiographic score, capillary number, and blood flow recovery by 1.9-, 1.5- and 1.6-fold, respectively, over that of untreated diabetic animals (P < 0.001).
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Discussion |
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In the present study, transplantation of BM-MNCs isolated from nondiabetic mice consistently augmented angiogenesis and collateral vessel formation in ischemic tissue of nondiabetic mice, as previously described.8,12,13 The putative mechanisms for the accelerated angiogenesis induced by transplanted BM-MNCs may include incorporation into vascular structures and local production of angiogenic ligands or cytokines.8,12,13 Interestingly, we demonstrated that administration of diabetic BM-MNCs increased the angiogenic process in the ischemic leg of nondiabetic mice to a lesser extent than that observed with nondiabetic BM-MNC injection. In an effort to elucidate the driving mechanisms that lie behind the decrease in diabetic BM-MNC pro-angiogenic functions, we analyzed the ability of BM-MNCs to differentiate into EPCs in vitro. We showed that diabetes decreased the number of bone marrow-derived differentiated EPCs. In addition, we also demonstrated that diabetic BM-MNCs were unable to participate in vascular-like structure formation in a subcutaneous Matrigel plug. Our results are in line with other findings showing that human EPCs from other types of diabetes (ie, type II) exhibited impaired proliferation, adhesion, and participation in the wound healing process.18 Taken together, these results underscore that the BM-MNC pro-angiogenic potential is affected in diabetes and that the altered function may participate in the abrogated neovascularization process observed in diabetes. BM-MNCs represent a heterogeneous population of cells; therefore, further studies are required to determine whether diabetic BM-MNC dysfunction is specifically related to dysfunction in diabetic BM-EPCs, other cellular populations, or both.
The functional capacity of the BM-derived cells may be governed by the endothelial cells and/or vascular environment in diabetes. However, the pro-angiogenic potential of nondiabetic and diabetic BM-MNC administration was similar in diabetic mice and in nondiabetic animals. Hence, vascular or cellular environment in diabetes does not affect BM-MNC-related effect, suggesting that diabetic environment is unlikely involved in the decrease in endogenous diabetic BM-MNC pro-angiogenic potential. Bone marrow-derived EPCs from young mice restore PDGF pathways and cardiac angiogenic function in the aging host, suggesting a therapeutic role for BM-derived cells administration in reversing the aging associated cardiovascular complications.21 We can also speculate that the role of nondiabetic BM-MNCs is likely not restricted to the restoration of vessel number in ischemic leg of diabetic mice and may have a therapeutic role in reversing the diabetes-induced changes in VEGF production or endothelial function.16,17,22 Endogenous diabetic BM-MNCs may also lack such capacity to reverse vascular/cell dysfunction induced by conditions related to the pathophysiology of diabetes.
Finally, we demonstrated that PlGF infusion increased the neovascularization reaction in ischemic hindlimbs of diabetic mice. Similarly, PlGF administration has been shown to increase postischemic neovascularization in nondiabetic mice.10 In addition, collateral artery growth is more significant in response to treatment with the VEGFR-1-specific ligand PlGF in comparison to the VEGFR-2-specific ligand VEGF-E, highlighting the potential for therapeutic strategy based on PlGF administration.23 Several lines of evidence also suggest that PlGF enhances mobilization and recruitment of BM-derived cells. Hematopoietic stem cells appear to express VEGF-R1 and to be responsive to PlGF.24 In addition, anti-VEGF-R1 suppresses recruitment of myeloid progenitors and transplantation of wild-type bone marrow rescues the impaired collateral growth in PlGF-deficient mice.25 We have extended these previous studies, since we demonstrated that the ability of BM-MNCs to differentiate into EPCs was increased by PlGF administration. Interestingly, PlGF totally restored the diabetes-induced impairment in the ability of BM-MNCs to differentiate into EPCs. Such PlGF-related effects might subsequently participate in the enhanced neovascularization reaction observed in the ischemic leg of PlGF treated-diabetic mice. Taken together, these results offer a foundation for the development of strategies tailored to the treatment of BM-MNC dysfunction and underline the potential for therapeutic strategy based on PlGF administration in restoration of BM-MNC function and postischemic revascularization in the setting of diabetes.
In conclusion, the present study shows that endogenous BM-MNC differentiation into EPCs and pro-angiogenic activity were reduced in type I diabetes in the setting of ischemia. These alterations in BM-derived cell functions might participate in the abrogated neovascularization process observed in diabetes. We also demonstrate that PlGF constitute a potential candidate for therapeutic modulation of postischemic neovascularization in diabetes. Further studies are necessary to confirm these results in type II diabetes and to address the specific mechanisms driving altered BM-derived cell activity and PlGF-related effects.
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Footnotes |
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Supported by grants from Université Paris 7 and from INSERM, AFM, Vaincre la Mucoviscidose ("cellules souches à finalité thérapeutique"). R.T. is a recipient of a fellowship from Fondation pour la Recherche Médicale.
Accepted for publication October 17, 2003.
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T. G. Ebrahimia |
