1 Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada
http://www.100md.com
糖尿病学杂志 2006年第1期
1 Division of Nephrology, Hypertension, and Transplantation, University of Florida, Gainesville, Florida
2 Division of Pharmacology and Therapeutics, University of Florida, Gainesville, Florida
3 Department of Biomedical Engineering, University of Florida, Gainesville, Florida
4 Department of Mechanical and Aerospace Engineering, University of Florida, Gainesville, Florida
CKD, chronic kidney disease; DETA/NO, diethylenetriaamine NONOate; EPC, endothelial progenitor cell; FITC, fluorescein isothiocyanate; HREC, human retinal endothelial cell; SDF-1, stromal-derived factor-1; VASP, vasodilator-stimulated phosphoprotein; VEGF, vascular endothelial growth factor
ABSTRACT
Stromal-derived factor-1 (SDF-1) is a critical chemokine for endothelial progenitor cell (EPC) recruitment to areas of ischemia, allowing these cells to participate in compensatory angiogenesis. The SDF-1 receptor, CXCR4, is expressed in developing blood vessels as well as on CD34+ EPCs. We describe that picomolar and nanomolar concentrations of SDF-1 differentially influence neovascularization, inducing CD34+ cell migration and EPC tube formation. CD34+ cells isolated from diabetic patients demonstrate a marked defect in migration to SDF-1. This defect is associated, in some but not all patients, with a cell surface activity of CD26/dipeptidyl peptidase IV, an enzyme that inactivates SDF-1. Diabetic CD34+ cells also do not migrate in response to vascular endothelial growth factor and are structurally rigid. However, incubating CD34+ cells with a nitric oxide (NO) donor corrects this migration defect and corrects the cell deformability. In addition, exogenous NO alters vasodilator-stimulated phosphoprotein and mammalian-enabled distribution in EPCs. These data support a common downstream cytoskeletal alteration in diabetic CD34+ cells that is independent of growth factor receptor activation and is correctable with exogenous NO. This inability of diabetic EPCs to respond to SDF-1 may contribute to aberrant tissue vascularization and endothelial repair in diabetic patients.
Stromal derived factor-1 (SDF-1) is a small cytokine belonging to the C-X-C subfamily that was originally isolated from a murine bone marrow stromal cell line (1). SDF-1 and its receptor, CXCR4, are essential for normal ontogeny of hematopoiesis during embryogenesis (2) and play a critical role in lymphopoiesis and myelopoiesis in the adult (3,4). SDF-1 has been shown to be upregulated in the myocardium under ischemia (5) and plays a critical role in homing and chemotaxis of CXCR4-expressing progenitor cells. The ability of SDF-1 to induce chemotaxis has been shown to be regulated by the activity of CD26, a cell-surface dipeptidyl peptidase that degrades SDF-1 (6). Endothelial progenitor cells (EPCs) derived from the bone marrow are modulated by SDF-1.
The stem cell marker CD34 identifies one potential source of EPCs (7). CD34+ mononuclear cells, after 7 days of culture on fibronectin, display an endothelial cell phenotype, are able to incorporate acetylated LDL, produce nitric oxide (NO) when stimulated with vascular endothelial growth factor (VEGF), and express platelet/endothelial cell adhesion molecule-1 and Tie-2 receptor (8).
We have previously demonstrated that SDF-1 levels are increased in the vitreous of diabetic patients with retinopathy and macular edema and that the levels correlate with the severity of diabetic retinopathy (9). In experimental animal models, we have also shown that EPCs play a central role in the development of proliferative retinopathy (10). In these studies, we investigated the ability of diabetic and control CD34+ cells to respond to SDF-1 and the mechanism of the defective response of diabetic CD34+ cells.
RESEARCH DESIGN AND METHODS
Tissue culture and reagents.
All tissue culture reagents were obtained from Invitrogen (Carlsbad, CA) and MediaTech (Herndon, VA). SDF-1 was obtained from R&D Systems (Minneapolis, MN). All other reagents were obtained from Sigma-Aldrich (St. Louis, MO), unless otherwise indicated.
Isolation of CD34+ cells.
The study protocol was approved by the institutional review board at the University of Florida, and written informed consent was obtained from each patient. Blood was collected from 18 patients with stage V chronic kidney disease (CKD) (patients requiring hemodialysis) as a result of type 2 diabetes and 21 diabetic patients with stage I or II CKD (patients with an estimated glomerular filtration rate 60 ml/min). Ten of these patients had type 1 and 11 had type 2 diabetes and 19 were normal control subjects. Blood was collected by routine venipuncture into CPT tubes with heparin (BD Biosciences, Franklin Lakes, NJ). For hemodialysis patients, the blood was collected before hemodialysis. After centrifugation at room temperature in a swinging bucket rotor for 20 min at 1,800g, the mononuclear cells were diluted with PBS supplemented with 2 mmol/l EDTA. The cells were centrifuged for 10 min at 300 RCF and the cell pellet washed; this procedure was repeated once. Each 3.3 x 107 cells peripheral blood mononuclear cells was resuspended in 100 e蘬 PBS supplemented with 2 mmol/l EDTA, to which 33 e蘬 of FcR-blocking reagent (Miltenyi Biotec, Auburn, CA) and 33 e蘬 of magnetic microbeads conjugated with an anti-CD34 antibody were added. After incubation for 30 min at 4°C, the cells were diluted in 10 x volume of PBS supplemented with 2 mmol/l EDTA supplemented with 0.1% BSA. The CD34+ cells were positively selected using an automated magnetic selection autoMACS (Miltenyi Biotec). The selected cells were confirmed to be CD34+ cells by costaining with phycoerythrin-conjugated anti-CD34 (Miltenyi Biotec) and fluorescein isothiocyanate (FITC)-conjugated anti-CD45.
Cell culture.
Primary cultures of human retinal endothelial cells (HRECs) were prepared and maintained as previously described (11,12), and cells in passages 2eC5 were used in these studies. EPCs were cultured from peripheral blood as follows. Peripheral blood mononuclear cells were isolated as above and cultured on fibronectin-coated plates for 6 days in EndoCult Medium (StemCell Technologies) before being treated with 0 or 100 pmol/l or 100 nmol/l of SDF-1 for 24 h. The endothelial nature of the cells was confirmed, after culturing for 6 days, with incorporation of fluorescently labeled acetylated LDL and tetrarhodamine isothiocyanateeCconjugated Ulex europaeus agglutinin-1.
SDF-1eCinduced chemotaxis.
CD34+ cell chemotaxis was done by staining the cells with Calcein-AM (Molecular Probes) before loading them onto the Boyden Chamber. SDF-1 was loaded in the bottom chamber, which was overlaid with a polycarbonate membrane (8-e蘭 pores; Neuro Probe, Gaithersburg, MD) coated with 10% bovine collagen, and the cells were loaded in the top chamber. After 4.5 h at 5% CO2 at 37°C, the percentage of cells that migrated was determined by collecting the media in the lower chamber and determining the relative fluorescence using a Synergy HT (Bio-Tek Instruments, Winooski, VT) with an excitation of 485 ± 20 and an emission of 528 ± 20. Twenty-four-hour pretreatments with 100 e蘭ol/l diethylenetriaamine NONOate (DETA/NO; Cayman Chemicals, Ann Arbor, MI), an NO donor, or 200 e蘭ol/l 2-(4-carboxyphenol)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (Sigma), an NO scavenger, were carried out in the CPT tubes, after centrifugation. Diprotein A, an inhibitor of dipeptidyl peptidase IV, (Alexis, San Diego, CA) was used where indicated at a concentration of 1 mmol/l and was added to the cells just before the migration assay. The 12G5 (R&D), a CXCR4-blocking antibody, was used at a concentration of 1 e蘥/ml.
Endothelial cell tube formation assay.
Endothelial tube formation was assessed on a synthetic basement membrane as per manufacturer’s protocol (Matrigel; BD Biosciences). Briefly, the matrix was thawed overnight at 4°C and polymerized at 37°C for 30 min before use. HRECs were resuspended either in full endothelial cell growth media (for positive control), reduced serum media, or reduced serum media with 100 pmol/l or 100 nmol/l SDF. The cells were then seeded (3 x 103) on the Matrigel and the plates placed in a humidified atmosphere of 5% CO2 at 37°C. Identical fields in each well were photographed every 12 h after plating. The photographs were digitized, and image-analysis software (Image; Scion, Frederick, MD) was used to measure total tube length and branch points. All conditions were tested in duplicate wells in three separate experiments using cells from different donors. EPC tube formation was performed after 6 days of culture.
CD26/dipeptidyl peptidase IV activity.
The activity of CD34+-sorted peripheral blood mononuclear cells was measured in 384-well microplates using the chromogenic substrate Gly-Pro-p-nitroanilide (Gly-Pro-pNA; Sigma-Aldrich) (13). After labeling the CD34+ cells with Calcein-AM as above, a fixed number of isolated CD34+ cells were incubated at 37°C in the presence of 2 mmol/l Gly-Pro-pNA in 100 e蘬 PBS buffer (pH 7.4) containing 10 mg/ml of BSA. The amount of nitroanilide (pNA) formed in the supernatant was determined by readings performed every 5 min at 405 nm using a Synergy HT. The results were plotted as nanomoles of pNA released per 1 x 106 cells released per minute, and the slope was calculated at the linear portion of the curve. Samples were run in triplicate.
Micropipette technique setup.
The overall setup to analyze cell deformability was as previously described (14), consisting of a micropipette, a chamber on an inverted interference contrast microscope, two water reservoirs connected for micropipette hydrostatic pressure control, and a video system. Micropipettes were constructed from glass capillary tubes with an outer diameter of 1 mm and an inner diameter of 0.5 mm (A-M Systems, Everett, Washington). The micropipettes used had an average inner diameter of 5.4 e蘭. Resting lymphocytes had diameters ranging from 5 to 17 e蘭. Before use, micropipettes were flushed with 50 e蘬 of plasma to prevent adhesion to the glass surface. Two typical types of experiment, aspiration and recovery when possible, were performed to determine the mechanical properties of individual cells.
Immunohistochemistry.
After fixing the EPCs with 3.7% paraformaldehyde and 0.2% Triton-X in PBS for 30 min at 37°C, the cells were washed with PBS and incubated in blocking solution (1% BSA in PBS) for 30 min at 37°C. The cells were then incubated with 10 e蘥/ml of mouse antivasodilator-stimulated phosphoprotein (VASP) (BD Biosciences Pharmingen) or isotype controls in blocking solution. After an hour at room temperature, the cells were rinsed with PBS and incubated with secondary antibody FITC-conjugated anti-mouse (Southern Biotech) for 30 min; for VASP staining, the secondary was used at a concentration of 10 e蘥/ml, for mammalian-enabled staining, the secondary was used at 5 e蘥/ml. The cells were then rinsed and imaged.
Fluorescence-activated cell sorter analysis of phosphorylated VASP.
CD34+ cells after isolation and DETA/NO treatment were fixed with methanol-free formaldehyde (91.5%) for 5 min before being diluted with PBS and permeabilized for 10 min with Triton X-100 (0.2% final). Portions of the samples were stained at room temperature for 45 min with a FITC-labeled antibody against phosphorylation of Ser239 (16C2 antibody [500 mg/ml]; Nanotools). A second aliquot was labeled with the FITC-labeled antibody after preincubation with a specific blocking phosphopeptide. The fluorescence of these cells was used to determine the parameters for background fluorescence.
Statistical analysis.
Statistical analysis was carried out using Student’s t test and the Mann-Whitney rank-sum test.
RESULTS
CD34+ cells and microvascular endothelial cells respond to physiologic concentrations of SDF-1.
In previous work, we have demonstrated that SDF-1 is elevated in the vitreous fluid of diabetic patients with retinopathy (9), and EPCs participate in pathological retinal angiogenesis (10). However, the concentration of SDF-1 in the vitreous of patients with proliferative diabetic retinopathy was 179 ± 172 pmol/l (means ± SD) (9). This is an SDF-1 concentration that is a full log lower than EPCs have previously been reported to migrate. It is well established that SDF-1 has profound effects at nanomolar concentrations, but the effects of picomolar concentrations have not been well documented. When picomolar concentrations were tested, CD34+ cells had a response to SDF-1 with two peaks of migration, one in the picomolar range and one in the nanomolar range (Fig. 1A). The response to SDF-1 at both the picomolar and nanomolar concentrations were mediated by the SDF-1 receptor, CXCR4, since the response was blocked to a similar extent by the CXCR4 blocking monoclonal antibody 12G5 (Fig. 1B). An SDF-1 bimodal effect on migration was also seen in HRECs as well as Jurkat cells (data not shown).
Picomolar versus nanomolar concentrations of SDF-1 have unique effects on cell behavior.
To examine whether picomolar and nanomolar concentrations had similar efficacy on all properties of SDF-1, we tested its effects on tube formation. HRECs exposed to a 100-pmol/l concentration of SDF-1 demonstrated robust branching without a marked increase in tube length (Fig. 2). However, at 100 nmol/l SDF-1, tube length was statistically increased without increasing the number of branch points. SDF-1 promoted in vitro differentiation of CD34+ cells into EPCs and induced tube formation of cultured EPCs isolated from healthy control subjects at both concentrations of SDF-1 (Fig. 3).
Diabetic patients have defective CD34+ cell migration in response to SDF-1.
If EPC migration to picomolar concentrations of SDF-1 found in the vitreous was the mechanism by which preretinal neovascularization occurred, one might predict that the EPCs isolated from diabetic patients would have enhanced migration at picomolar concentrations of SDF-1. Unexpectedly, CD34+ cells isolated from diabetic patients had markedly diminished migration, with a virtually flat response to SDF-1, over the picomolar and nanomolar concentrations tested (Fig. 4A). This was found in patients with retinopathy and stage V CKD or with stage I or II CKD (Fig. 4B). In addition, the result was the same whether the patients had type 1 or type 2 diabetes (data not shown). Compared with the maximal migration of CD34+ cells in normal control subjects, CD34+ cells from diabetic patients had 30% of the maximal migratory activity (Fig. 4B).
We reasoned that the defect in migration of CD34+ cells could be at the level of the receptor or due to downstream receptor effects. Previously, CD26/dipeptidyl peptidase IV was shown to cleave and inactivate SDF-1, blocking its ability to activate its receptor CXCR4 (13,15,16). To determine whether this was a possible mechanism for the decreased migration observed in CD34+ cells derived from diabetic individuals, we determined the level of CD26/dipeptidyl peptidase IV activity on these cells. We found twice the CD26/dipeptidyl peptidase IV activity present on CD34+ cells derived from diabetic patients versus control subjects (Fig. 4C). However, the increased level of dipeptidyl peptidase IV activity was not limiting migration. Even diabetic patients whose dipeptidyl peptidase IV activity was lower than that of normal control subjects (Fig. 4D) had defective migration. To confirm this, we used an inhibitor of dipeptidyl peptidase IV activity, diprotein A. Incubation of diabetic CD34+ cells with diprotein A, at concentrations that blocked 99% of the dipeptidyl peptidase IV activity (data not shown) increased migration, but the percent increase was still less than that seen in CD34+ cells isolated from normal control subjects (Fig. 4E). Consistent with the defect not being primarily at the receptor level, diabetic CD34+ cells did not migrate to VEGF, suggesting a more general inhibition of migration (Fig. 5).
Cytoskeletal defect in diabetic CD34+ cells.
Classically, the cytoskeletal structure has been investigated by cell deformability either by atomic force microscopy or micropipette technique (17,18). In the latter, micropipettes are made from 1-mm capillary-glass tubing pulled to a fine point by quick fracture to give a flat tip of desired diameter. Two typical types of experiment, aspiration and recovery, are usually performed to determine the mechanical properties of individual cells. Cell deformability can be assessed by aspirating a cell into a micropipette at a constant pressure and then measuring its deformation as a function of time. Using the micropipette technique, it has also been established that the cell rheological properties can be altered by disease and illness. Published data have shown alterations of the mechanical properties of erythrocytes in sickle cell disease (19), white blood cells in diabetes (14,20,21), and a variety of cells in cancer (22eC25). CD34+ cells isolated from patients with diabetes complications were extremely rigid; the deformability could not be determined because they could not be suctioned into the micropipette (Fig. 6). CD34+ cells from normal control subjects entered the micropipette very well (75% of cells completely entered the micropipette, 16.7% partially entered, and 8.3% could not be suctioned into the micropipette; n = 5), while CD34+ cells isolated from diabetic patients were not deformable (100% of the cells could not be suctioned into the micropipette; n = 6). Previously, we have demonstrated a similar disturbance in white cells isolated from diabetic mice (14).
CD34+ cell migration is enhanced by incubation with an NO donor.
Local concentrations of NO dramatically affected hemangioblast behavior and changed vessel phenotype (26). Thus, we investigated whether the migratory defect of CD34+ cells isolated from diabetic patients could be corrected by treatment with NO. Incubation of CD34+ cells in the presence of an NO donor, DETA/NO, for 24 h increased the percentage of cells migrating in response to SDF-1 (Fig. 7). This enhancement was evident at picomolar and nanomolar concentrations of SDF-1. The effect of the NO donor on SDF-1 in stimulating CD34+ cell migration was blocked by coincubating with the NO scavenger 2-(4-carboxyphenol)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (Fig. 7).
NO treatment also corrected the diabetic CD34+ deformability defect (Fig. 6). While 100% of CD34+ cells isolated from diabetic patients were not deformable when treated with vehicle, diabetic CD34+ cells incubated with DETA/NO had a marked increase in deformability (85% of cells could fully enter the micropipette; n = 16). This suggested that NO altered the cytoskeletal structure mediating the correction in the migration defect.
NO causes redistribution of cytoskeletal motors facilitating migration.
To begin to elucidate the possible role that NO has on the cytoskeleton, we examined the distribution of VASP-related proteins in response to exogenous administration of NO. This family of proteins is critical to actin elongation, powering the expansile apparatuses of cells, the filopodia and lamellapodia, (27 and recently rev. in 28) and is regulated by NO (29). Immunohistochemistry of VASP protein was performed on cultured EPCs. Incubation of the EPCs for 24 h with increasing concentration of NO resulted in increased VASP expression (Fig. 8) and a redistribution of this critical protein to filopodia. VASP, in the absence of NO, was distributed evenly throughout the cell. Whereas, with NO stimulation, VASP was upregulated and redistributed to the leading edge of the advancing cell processes. In addition, fluorescence-activated cell sorter analysis demonstrated that NO significantly increased expression of phospho-VASP in EPCs (Fig. 8D).
DISCUSSION
The major finding of this report is that diabetic CD34+ cells have markedly decreased migratory characteristics to multiple different stimuli that can be reversed by exogenous administration of physiologic concentrations of NO. The defect in migration in response to cytokines and growth factors may be responsible for the marked atherosclerosis, peripheral vascular disease, and delayed wound healing typically seen in diabetic patients. The diabetic CD34+ cells were also rigid compared with those cells isolated from healthy control subjects. Like migration, treatment with NO induced deformability of the diabetic CD34+ cells. This suggested that NO was affecting the cytoskeletal structure of the CD34+ cells. This was confirmed by NO causing a redistribution of the cytoskeletal proteins VASP and mammalian-enabled distribution in EPCs.
To fully investigate the nature of the migration defect of CD34+ cells isolated from diabetic patients, we investigated the activity of CD26/dipeptidyl peptidase IV on the surface of these cells. This enzyme cleaves SDF-1 (13,15,16) and could potentially modulate EPC behavior. The CD34+ cell defect in migration was associated with a marked increase in CD26/dipeptidyl peptidase IV activity in some diabetic patients. However, while increased cell surface cleavage of SDF-1 may potentiate the migration defect, our studies suggest that it is not the only mechanism. Diabetic patients with levels of dipeptidyl peptidase IV activity below those of healthy control subjects or whose dipeptidyl peptidase IV activity was inhibited with diprotein A still had diminished cell migration.
Since the diabetic CD34+ cells did not migrate to VEGF or SDF-1 and diabetic white blood cells have been shown to be less deformable than cells from normal control subjects (14), we used the micropipette technique to study the intrinsic cytoskeletal structure. Cells isolated from patients with diabetes complications were so rigid that they could not even be suctioned into the micropipette. We found that 100% of the diabetic cells were not deformable as opposed to only 8.3% of control cells. However, after 24 h exposure with an NO donor, the CD34+ cells from diabetic patients demonstrated the same deformability as normal control subjects.
To determine the mechanism of the increased rigidity of the diabetic CD34+ cells, we studied the localization of VASP by immunohistochemistry; this family of proteins is critical for actin filament elongation powering the advancement of the leading edge of cells (28). In addition, NO has been shown to regulate VASP (29). In response to physiologic concentrations of NO, VASP immunolocalization was altered, VASP protein expression was increased, VASP was concentrated in the filopodia, and VASP phosphorylation was markedly increased. These changes are conducive to cell motility and are consistent with recently published observations concerning the role of NO in inducing long-lasting potentiation of hippocampal synapses by altering neuronal puncta via VASP (30).
There could be additional mechanisms by which NO could correct the diabetic CD34+ cell migration defect. NO is required for proper motility of endothelial cells (31). Diabetes, by affecting Akt phosphorylation, results in diminished NO generation. Exogenous administration of NO would bypass the endogenous need for AKT phosphorylation.
Alternatively, NO may be acting as a reactive oxygen scavenger. Reactive oxygen species, known to be markedly increased in diabetes and associated with complications (32,33), interfere with growth factor signaling (34eC36). Thus, NO, by quenching reactive oxygen species, could possibly enhance VEGF and SDF-1 downstream receptor signaling. Specifically, H2O2 has previously been shown to induce stress fibers in endothelial cells, inhibiting normal motility. NO, by scavenging super oxide and indirectly reducing H2O2 levels, could prevent H2O2 induction of cytoskeletal reorganization via calcium/calmodulin-dependent protein kinase II, Janus kinase 2 and mitogen-activated protein kinase, and extracellular signaleCrelated kinase (37). However, we believe this is less likely since the reactive oxygen scavenger uric acid could not fully correct the migration defect in CD34+ cells (data not shown).
Defects in EPC migration would result in decreased reendothelialization. This defect could extend to the micro- and macrovascular complications seen in diabetic patients. Diabetic eye disease is characterized by aberrant microvascular changes with insufficient intraretinal vascularization and excessive "preretinal" neovascularization. The decreased response of diabetic CD34+ cells to picomolar concentrations of SDF-1 as well as other growth factors may inhibit the ability of these cells to repair early capillary endothelial injury. If this injury is not repaired, acellular capillaries could result. With continued ischemic injury, the retina continues to produce SDF-1 and VEGF that accumulate in the vitreous. With the vitreous acting as a growth factor "sink," increasing growth factor concentrations may ultimately be able to overcome the relatively unresponsive CD34+ cells in the diabetic individual. However, the concentrated SDF-1 and VEGF in the vitreous directs EPC and endothelial cell migration to the surface of the retina, leading to preretinal vascular pathology. Correcting the CD34+ cell migratory defect, before the acellular capillary stage, may result in early capillary repair, preventing the late accumulation of vitreal SDF-1 and classic, preretinal, diabetic proliferation.
Rapid reendothelialization, mediated by CD34+ cells, at the macrovascular level can reduce atherosclerosis, peripheral vascular disease, and restenosis. These macrovascular complications are thought to be the result of endothelial injury (38,39) with possible inadequate endothelial repair and are all accelerated in the diabetic individual.
CD34+ cells, from healthy volunteers, significantly increase revascularization when injected into an ischemic hind limb of hypoinsulinemic diabetic mice (40) and accelerates revascularization and healing of full thickness skin wounds (41). Interestingly, injection of CD34+ cells in an ischemic limb of a normal mouse has no effect (40). This constellation of results suggests that diabetes has a detrimental effect on bone marrow and EPC homeostasis. Other conditions such as ischemia, smoking, and increased cholesterol have also been shown to have injurious effects on EPC number and function (42,43).
Administration of migratory CD34+ cells may represent an easily accessible and safe therapeutic approach to correct the multitude of vascular abnormalities seen in patients with diabetes. However these results should serve as a caution to in vivo studies using CD34+ cells as a therapeutic modality; improvement of CD34+ cell function in diabetic patients without simultaneously decreasing the levels of SDF-1 and other growth factors in the vitreous could result in worsening preretinal neovascularization. This is especially clinically relevant given the development of dipeptidyl peptidase IV inhibitors as an indirect means to stimulate insulin secretion and decrease glucagon secretion (44,45). These inhibitors by inhibiting SDF-1 degradation could potentiate CD34+ cell migration, and while this may slightly improve endothelial repair, it could worsen preretinal neovascularization, especially if given in the absence of decreasing the concentration of SDF-1 within the vitreous.
In conclusion, we have demonstrated that CD34+ cell dysfunction in diabetes is the result of alterations in the EPC cytoskeleton. The defect in SDF-1eCinduced migration seen in diabetic patients with end-organ damage is reversed by physiologic NO concentrations. We demonstrate that NO treatment improves deformability and normalizes the migration of diabetic cells likely by leading to phosphorylation and redistribution of VASP to the advancing edge, directly enhancing cell motility.
ACKNOWLEDGMENTS
This work was supported by National Institutes of Health Grant DK02537, Gatorade Research Funds, a Baxter Renal Discovery Grant to M.S.S., and the Juvenile Diabetes Research Foundation International and National Institutes of Health Grants EY012601, EY007739, and EY14818 to M.B.G.
The authors thank Dr. G.C. Finlayson for help recruiting patients, Dr. David A. Antonetti for helpful suggestions regarding experimental design and critical review of the manuscript, and Dr. Daniel Purich for discussions concerning VASP.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
Tashiro K, Tada H, Heilker R, Shirozu M, Nakano T, Honjo T: Signal sequence trap: a cloning strategy for secreted proteins and type I membrane proteins. Science 261:600eC603, 1993
Nagasawa T, Hirota S, Tachibana K, Takakura N, Nishikawa S, Kitamura Y, Yoshida N, Kikutani H, Kishimoto T: Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1. Nature 382:635eC638, 1996
Kawabata K, Ujikawa M, Egawa T, Kawamoto H, Tachibana K, Iizasa H, Katsura Y, Kishimoto T, Nagasawa T: A cell-autonomous requirement for CXCR4 in long-term lymphoid and myeloid reconstitution. Proc Natl Acad Sci U S A 96:5663eC5667, 1999
Onai N, Zhang Y, Yoneyama H, Kitamura T, Ishikawa S, Matsushima K: Impairment of lymphopoiesis and myelopoiesis in mice reconstituted with bone marrow-hematopoietic progenitor cells expressing SDF-1-intrakine. Blood 96:2074eC2080, 2000
Ratajczak MZ, Kucia M, Reca R, Majka M, Janowska-Wieczorek A, Ratajczak: Stem cell plasticity revisited: CXCR4-positive cells expressing mRNA for early muscle, liver and neural cells ’hide out’ in the bone marrow. Leukemia 18:29eC40, 2004
Christopherson KW 2nd, Hangoc G, Mantel CR, Broxmeyer HE: Modulation of hematopoietic stem cell homing and engraftment by CD26. Science 305:1000eC1003, 2004
Peichev M, Naiyer AJ, Pereira D, Zhu Z, Lane WJ, Williams M, Oz MC, Hicklin DJ, Witte L, Moore MA, Rafii S: Expression of VEGFR-2 and AC133 by circulating human CD34(+) cells identifies a population of functional endothelial precursors. Blood 95:952eC958, 2000
Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM: Isolation of putative progenitor endothelial cells for angiogenesis. Science 275:964eC967, 1997
Brooks HL Jr, Caballero S Jr, Newell CK, Steinmetz RL, Watson D, Segal MS, Harrison JK, Scott EW, Grant MB: Vitreous levels of vascular endothelial growth factor and stromal-derived factor 1 in patients with diabetic retinopathy and cystoid macular edema before and after intraocular injection of triamcinolone. Arch Ophthalmol 122:1801eC1807, 2004
Grant MB, May WS, Caballero S, Brown GA, Guthrie SM, Mames RN, Byrne BJ, Vaught T, Spoerri PE, Peck AB, Scott EW: Adult hematopoietic stem cells provide functional hemangioblast activity during retinal neovascularization. Nat Med 8:607eC612, 2002
Grant MB, Ellis EA, Caballero S, Mames RN: Plasminogen activator inhibitor-1 overexpression in nonproliferative diabetic retinopathy. Exp Eye Res 63:233eC244, 1996
Grant MB, Tarnuzzer RW, Caballero S, Ozeck MJ, Davis MI, Spoerri PE, Feoktistov I, Biaggioni I, Shryock JC, Belardinelli L: Adenosine receptor activation induces vascular endothelial growth factor in human retinal endothelial cells. Circ Res 85:699eC706, 1999
Christopherson KW 2nd, Hangoc G, Broxmeyer HE: Cell surface peptidase CD26/dipeptidylpeptidase IV regulates CXCL12/stromal cell-derived factor-1 alpha-mediated chemotaxis of human cord blood CD34+ progenitor cells. J Immunol 169:7000eC7008, 2002
Perrault CM, Bray EJ, Didier N, Ozaki CK, Tran-Son-Tay R: Altered rheology of lymphocytes in the diabetic mouse. Diabetologia 47:1722eC1726, 2004
Ohtsuki T, Hosono O, Kobayashi H, Munakata Y, Souta A, Shioda T, Morimoto C: Negative regulation of the anti-human immunodeficiency virus and chemotactic activity of human stromal cell-derived factor 1alpha by CD26/dipeptidyl peptidase IV. FEBS Lett 431:236eC240, 1998
Proost P, Struyf S, Schols D, Durinx C, Wuyts A, Lenaerts JP, De Clercq E, De Meester I, Van Damme: Processing by CD26/dipeptidyl-peptidase IV reduces the chemotactic and anti-HIV-1 activity of stromal-cell-derived factor-1alpha. FEBS Lett 432:73eC76, 1998
Tran-Son-Tay R, Needham D, Yeung A, Hochmuth RM: Time-dependent recovery of passive neutrophils after large deformation. Biophys J 60:856eC866, 1991
Hochmuth RM, Ting-Beall HP, Beaty BB, Needham D, Tran-Son-Tay R: Viscosity of passive human neutrophils undergoing small deformations. Biophys J 64:1596eC1601, 1993
Nash GB, Johnson CS, Meiselman HJ: Mechanical properties of oxygenated red blood cells in sickle cell (HbSS) disease. Blood 63:73eC82, 1984
Athanassiou G, Matsouka P, Kaleridis V, Missirlis Y: Deformability and filterability of white blood cell subpopulations: evaluation of these parameters in the cell line HL-60 and in type II diabetes mellitus. Clin Hemorheol Microcirc 22:35eC43, 2000
Linderkamp O, Ruef P, Zilow EP, Hoffmann GF: Impaired deformability of erythrocytes and neutrophils in children with newly diagnosed insulin-dependent diabetes mellitus. Diabetologia 42:865eC869, 1999
Yao W, Gu L, Sun D, Ka W, Wen Z, Chien S: Wild type p53 gene causes reorganization of cytoskeleton and, therefore, the impaired deformability and difficult migration of murine erythroleukemia cells. Cell Motil Cytoskeleton 56:1eC12, 2003
Glover S, Delaney M, Dematte C, Kornberg L, Frasco M, Tran-Son-Tay R, Benya RV: Phosphorylation of focal adhesion kinase tyrosine 397 critically mediates gastrin-releasing peptide’s morphogenic properties. J Cell Physiol 199:77eC88, 2004
Chen H, Yunpeng W, Shaoxi C, Mian LA: Research on rheological properties of erythrocyte of lung cancer patients. Clin Hemorheol 16:135eC141, 1996
Khan MM, Puniyani RR, Huilgog NG, Hussain A, Ranade GG: Hemorheological profile in cancer patients. Clin Hemorheol 15:37eC44, 1995
Guthrie SM, Curtis LM, Mames RN, Simon GG, Grant MB, Scott EW: The nitric oxide pathway modulates hemangioblast activity of adult hematopoietic stem cells. Blood 105:1916eC1922, 2005
Dickinson RB, Purich DL: Clamped-filament elongation model for actin-based motors. Biophys J 82:605eC617, 2002
Krause M, Dent EW, Bear JE, Loureiro JJ, Gertler FB: Ena/VASP proteins: regulators of the actin cytoskeleton and cell migration. Annu Rev Cell Dev Biol 19:541eC564, 2003
Andre M, Latado H, Felley-Bosco E: Inducible nitric oxide synthase-dependent stimulation of PKGI and phosphorylation of VASP in human embryonic kidney cells. Biochem Pharmacol 69:595eC602, 2005
Wang H-G, Lu F-M, Jin I, Udo H, Kandel ER, de Vente J, Walter U, Lohmann SM, Hawkins RD, Antonova I: Presynaptic and postsynaptic roles of NO, cGK, and RhoA in long-lasting potentiation and aggregation of synaptic proteins. Neuron 45:389eC403, 2005
Ziche M, Morbidelli L, Masini E, Amerini S, Granger HJ, Maggi CA, Geppetti P, Ledda FJ: Nitric oxide mediates angiogenesis in vivo and endothelial cell growth and migration in vitro promoted by substance P. J Clin Invest 94:2036eC2044, 1994
Brownlee MD: Advanced protein glycosylation in diabetes and aging. Annu Rev Med 46:223eC234, 1995
King GL, Brownlee M: The cellular and molecular mechanisms of diabetic complications. Endocrinol Metab Clin North Am 25:255eC270, 1996
Lo YYC, Cruz TF: Involvement of reactive oxygen species in cytokine and growth factor induction of c-fos expression in chondrocytes. J Biol Chem 270:11727eC11730, 1995
Lo YYC, Wong JMS, Cruz TF: Reactive oxygen species mediate cytokine activation of c-Jun NH2-terminal kinases. J Biol Chem 271:15703eC15707, 1996
Nakamura H, Nakamura K, Yodoi J: Redox regulation of cellular activation. Annu Rev Immunol 15:351eC369, 1997
Nguyen A, Chen P, Cai H: Role of CaMKII in hydrogen peroxide activation of ERK1/2, p38 MAPK, HSP27 and actin reorganization in endothelial cells. FEBS Lett 572:307eC313, 2004
Ross R, Glomset J, Harker L: Response to injury and atherogenesis. Am J Pathol 86:675eC684, 1977
Ross R: Atherosclerosis: an inflammatory disease. N Engl J Med 340:115eC126, 1999
Schatteman GC, Hanlon HD, Jiao C, Dodds SG, Christy BA: Atherosclerosis: an inflammatory disease. J Clin Invest 106:571eC578, 2000
Sivan-Loukianova E, Awad OA, Stepanovic V, Bickenbach J, Schatteman GC: CD34+ blood cells accelerate vascularization and healing of diabetic mouse skin wounds. J Vas Res 40:368eC377, 2003
Heeschen C, Lehmann R, Honold J, Assmus B, Aicher A, Walter DH, Martin H, Zeiher AM, Dimmeler S: Profoundly reduced neovascularization capacity of bone marrow mononuclear cells derived from patients with chronic ischemic heart disease. Circulation 109:1615eC1622, 2004
Vasa M, Fichtlscherer S, Aicher A, Adler K, Urbich C, Martin H, Zeiher AM, Dimmeler S: Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Circ Res 89:E1eCE7, 2001
Deacon CF, Ahren B, Holst J: Inhibitors of dipeptidyl peptidase IV: a novel approach for the prevention and treatment of type 2 diabetes Expert Opin Investig Drugs 13:1091eC1102, 2004
Holst JJ, Deacon CF: Glucagon-like peptide 1 and inhibitors of dipeptidyl peptidase IV in the treatment of type 2 diabetes mellitus. Curr Opin Pharmacol 4:589eC596, 2004(Mark S. Segal, Ronak Shah)
2 Division of Pharmacology and Therapeutics, University of Florida, Gainesville, Florida
3 Department of Biomedical Engineering, University of Florida, Gainesville, Florida
4 Department of Mechanical and Aerospace Engineering, University of Florida, Gainesville, Florida
CKD, chronic kidney disease; DETA/NO, diethylenetriaamine NONOate; EPC, endothelial progenitor cell; FITC, fluorescein isothiocyanate; HREC, human retinal endothelial cell; SDF-1, stromal-derived factor-1; VASP, vasodilator-stimulated phosphoprotein; VEGF, vascular endothelial growth factor
ABSTRACT
Stromal-derived factor-1 (SDF-1) is a critical chemokine for endothelial progenitor cell (EPC) recruitment to areas of ischemia, allowing these cells to participate in compensatory angiogenesis. The SDF-1 receptor, CXCR4, is expressed in developing blood vessels as well as on CD34+ EPCs. We describe that picomolar and nanomolar concentrations of SDF-1 differentially influence neovascularization, inducing CD34+ cell migration and EPC tube formation. CD34+ cells isolated from diabetic patients demonstrate a marked defect in migration to SDF-1. This defect is associated, in some but not all patients, with a cell surface activity of CD26/dipeptidyl peptidase IV, an enzyme that inactivates SDF-1. Diabetic CD34+ cells also do not migrate in response to vascular endothelial growth factor and are structurally rigid. However, incubating CD34+ cells with a nitric oxide (NO) donor corrects this migration defect and corrects the cell deformability. In addition, exogenous NO alters vasodilator-stimulated phosphoprotein and mammalian-enabled distribution in EPCs. These data support a common downstream cytoskeletal alteration in diabetic CD34+ cells that is independent of growth factor receptor activation and is correctable with exogenous NO. This inability of diabetic EPCs to respond to SDF-1 may contribute to aberrant tissue vascularization and endothelial repair in diabetic patients.
Stromal derived factor-1 (SDF-1) is a small cytokine belonging to the C-X-C subfamily that was originally isolated from a murine bone marrow stromal cell line (1). SDF-1 and its receptor, CXCR4, are essential for normal ontogeny of hematopoiesis during embryogenesis (2) and play a critical role in lymphopoiesis and myelopoiesis in the adult (3,4). SDF-1 has been shown to be upregulated in the myocardium under ischemia (5) and plays a critical role in homing and chemotaxis of CXCR4-expressing progenitor cells. The ability of SDF-1 to induce chemotaxis has been shown to be regulated by the activity of CD26, a cell-surface dipeptidyl peptidase that degrades SDF-1 (6). Endothelial progenitor cells (EPCs) derived from the bone marrow are modulated by SDF-1.
The stem cell marker CD34 identifies one potential source of EPCs (7). CD34+ mononuclear cells, after 7 days of culture on fibronectin, display an endothelial cell phenotype, are able to incorporate acetylated LDL, produce nitric oxide (NO) when stimulated with vascular endothelial growth factor (VEGF), and express platelet/endothelial cell adhesion molecule-1 and Tie-2 receptor (8).
We have previously demonstrated that SDF-1 levels are increased in the vitreous of diabetic patients with retinopathy and macular edema and that the levels correlate with the severity of diabetic retinopathy (9). In experimental animal models, we have also shown that EPCs play a central role in the development of proliferative retinopathy (10). In these studies, we investigated the ability of diabetic and control CD34+ cells to respond to SDF-1 and the mechanism of the defective response of diabetic CD34+ cells.
RESEARCH DESIGN AND METHODS
Tissue culture and reagents.
All tissue culture reagents were obtained from Invitrogen (Carlsbad, CA) and MediaTech (Herndon, VA). SDF-1 was obtained from R&D Systems (Minneapolis, MN). All other reagents were obtained from Sigma-Aldrich (St. Louis, MO), unless otherwise indicated.
Isolation of CD34+ cells.
The study protocol was approved by the institutional review board at the University of Florida, and written informed consent was obtained from each patient. Blood was collected from 18 patients with stage V chronic kidney disease (CKD) (patients requiring hemodialysis) as a result of type 2 diabetes and 21 diabetic patients with stage I or II CKD (patients with an estimated glomerular filtration rate 60 ml/min). Ten of these patients had type 1 and 11 had type 2 diabetes and 19 were normal control subjects. Blood was collected by routine venipuncture into CPT tubes with heparin (BD Biosciences, Franklin Lakes, NJ). For hemodialysis patients, the blood was collected before hemodialysis. After centrifugation at room temperature in a swinging bucket rotor for 20 min at 1,800g, the mononuclear cells were diluted with PBS supplemented with 2 mmol/l EDTA. The cells were centrifuged for 10 min at 300 RCF and the cell pellet washed; this procedure was repeated once. Each 3.3 x 107 cells peripheral blood mononuclear cells was resuspended in 100 e蘬 PBS supplemented with 2 mmol/l EDTA, to which 33 e蘬 of FcR-blocking reagent (Miltenyi Biotec, Auburn, CA) and 33 e蘬 of magnetic microbeads conjugated with an anti-CD34 antibody were added. After incubation for 30 min at 4°C, the cells were diluted in 10 x volume of PBS supplemented with 2 mmol/l EDTA supplemented with 0.1% BSA. The CD34+ cells were positively selected using an automated magnetic selection autoMACS (Miltenyi Biotec). The selected cells were confirmed to be CD34+ cells by costaining with phycoerythrin-conjugated anti-CD34 (Miltenyi Biotec) and fluorescein isothiocyanate (FITC)-conjugated anti-CD45.
Cell culture.
Primary cultures of human retinal endothelial cells (HRECs) were prepared and maintained as previously described (11,12), and cells in passages 2eC5 were used in these studies. EPCs were cultured from peripheral blood as follows. Peripheral blood mononuclear cells were isolated as above and cultured on fibronectin-coated plates for 6 days in EndoCult Medium (StemCell Technologies) before being treated with 0 or 100 pmol/l or 100 nmol/l of SDF-1 for 24 h. The endothelial nature of the cells was confirmed, after culturing for 6 days, with incorporation of fluorescently labeled acetylated LDL and tetrarhodamine isothiocyanateeCconjugated Ulex europaeus agglutinin-1.
SDF-1eCinduced chemotaxis.
CD34+ cell chemotaxis was done by staining the cells with Calcein-AM (Molecular Probes) before loading them onto the Boyden Chamber. SDF-1 was loaded in the bottom chamber, which was overlaid with a polycarbonate membrane (8-e蘭 pores; Neuro Probe, Gaithersburg, MD) coated with 10% bovine collagen, and the cells were loaded in the top chamber. After 4.5 h at 5% CO2 at 37°C, the percentage of cells that migrated was determined by collecting the media in the lower chamber and determining the relative fluorescence using a Synergy HT (Bio-Tek Instruments, Winooski, VT) with an excitation of 485 ± 20 and an emission of 528 ± 20. Twenty-four-hour pretreatments with 100 e蘭ol/l diethylenetriaamine NONOate (DETA/NO; Cayman Chemicals, Ann Arbor, MI), an NO donor, or 200 e蘭ol/l 2-(4-carboxyphenol)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (Sigma), an NO scavenger, were carried out in the CPT tubes, after centrifugation. Diprotein A, an inhibitor of dipeptidyl peptidase IV, (Alexis, San Diego, CA) was used where indicated at a concentration of 1 mmol/l and was added to the cells just before the migration assay. The 12G5 (R&D), a CXCR4-blocking antibody, was used at a concentration of 1 e蘥/ml.
Endothelial cell tube formation assay.
Endothelial tube formation was assessed on a synthetic basement membrane as per manufacturer’s protocol (Matrigel; BD Biosciences). Briefly, the matrix was thawed overnight at 4°C and polymerized at 37°C for 30 min before use. HRECs were resuspended either in full endothelial cell growth media (for positive control), reduced serum media, or reduced serum media with 100 pmol/l or 100 nmol/l SDF. The cells were then seeded (3 x 103) on the Matrigel and the plates placed in a humidified atmosphere of 5% CO2 at 37°C. Identical fields in each well were photographed every 12 h after plating. The photographs were digitized, and image-analysis software (Image; Scion, Frederick, MD) was used to measure total tube length and branch points. All conditions were tested in duplicate wells in three separate experiments using cells from different donors. EPC tube formation was performed after 6 days of culture.
CD26/dipeptidyl peptidase IV activity.
The activity of CD34+-sorted peripheral blood mononuclear cells was measured in 384-well microplates using the chromogenic substrate Gly-Pro-p-nitroanilide (Gly-Pro-pNA; Sigma-Aldrich) (13). After labeling the CD34+ cells with Calcein-AM as above, a fixed number of isolated CD34+ cells were incubated at 37°C in the presence of 2 mmol/l Gly-Pro-pNA in 100 e蘬 PBS buffer (pH 7.4) containing 10 mg/ml of BSA. The amount of nitroanilide (pNA) formed in the supernatant was determined by readings performed every 5 min at 405 nm using a Synergy HT. The results were plotted as nanomoles of pNA released per 1 x 106 cells released per minute, and the slope was calculated at the linear portion of the curve. Samples were run in triplicate.
Micropipette technique setup.
The overall setup to analyze cell deformability was as previously described (14), consisting of a micropipette, a chamber on an inverted interference contrast microscope, two water reservoirs connected for micropipette hydrostatic pressure control, and a video system. Micropipettes were constructed from glass capillary tubes with an outer diameter of 1 mm and an inner diameter of 0.5 mm (A-M Systems, Everett, Washington). The micropipettes used had an average inner diameter of 5.4 e蘭. Resting lymphocytes had diameters ranging from 5 to 17 e蘭. Before use, micropipettes were flushed with 50 e蘬 of plasma to prevent adhesion to the glass surface. Two typical types of experiment, aspiration and recovery when possible, were performed to determine the mechanical properties of individual cells.
Immunohistochemistry.
After fixing the EPCs with 3.7% paraformaldehyde and 0.2% Triton-X in PBS for 30 min at 37°C, the cells were washed with PBS and incubated in blocking solution (1% BSA in PBS) for 30 min at 37°C. The cells were then incubated with 10 e蘥/ml of mouse antivasodilator-stimulated phosphoprotein (VASP) (BD Biosciences Pharmingen) or isotype controls in blocking solution. After an hour at room temperature, the cells were rinsed with PBS and incubated with secondary antibody FITC-conjugated anti-mouse (Southern Biotech) for 30 min; for VASP staining, the secondary was used at a concentration of 10 e蘥/ml, for mammalian-enabled staining, the secondary was used at 5 e蘥/ml. The cells were then rinsed and imaged.
Fluorescence-activated cell sorter analysis of phosphorylated VASP.
CD34+ cells after isolation and DETA/NO treatment were fixed with methanol-free formaldehyde (91.5%) for 5 min before being diluted with PBS and permeabilized for 10 min with Triton X-100 (0.2% final). Portions of the samples were stained at room temperature for 45 min with a FITC-labeled antibody against phosphorylation of Ser239 (16C2 antibody [500 mg/ml]; Nanotools). A second aliquot was labeled with the FITC-labeled antibody after preincubation with a specific blocking phosphopeptide. The fluorescence of these cells was used to determine the parameters for background fluorescence.
Statistical analysis.
Statistical analysis was carried out using Student’s t test and the Mann-Whitney rank-sum test.
RESULTS
CD34+ cells and microvascular endothelial cells respond to physiologic concentrations of SDF-1.
In previous work, we have demonstrated that SDF-1 is elevated in the vitreous fluid of diabetic patients with retinopathy (9), and EPCs participate in pathological retinal angiogenesis (10). However, the concentration of SDF-1 in the vitreous of patients with proliferative diabetic retinopathy was 179 ± 172 pmol/l (means ± SD) (9). This is an SDF-1 concentration that is a full log lower than EPCs have previously been reported to migrate. It is well established that SDF-1 has profound effects at nanomolar concentrations, but the effects of picomolar concentrations have not been well documented. When picomolar concentrations were tested, CD34+ cells had a response to SDF-1 with two peaks of migration, one in the picomolar range and one in the nanomolar range (Fig. 1A). The response to SDF-1 at both the picomolar and nanomolar concentrations were mediated by the SDF-1 receptor, CXCR4, since the response was blocked to a similar extent by the CXCR4 blocking monoclonal antibody 12G5 (Fig. 1B). An SDF-1 bimodal effect on migration was also seen in HRECs as well as Jurkat cells (data not shown).
Picomolar versus nanomolar concentrations of SDF-1 have unique effects on cell behavior.
To examine whether picomolar and nanomolar concentrations had similar efficacy on all properties of SDF-1, we tested its effects on tube formation. HRECs exposed to a 100-pmol/l concentration of SDF-1 demonstrated robust branching without a marked increase in tube length (Fig. 2). However, at 100 nmol/l SDF-1, tube length was statistically increased without increasing the number of branch points. SDF-1 promoted in vitro differentiation of CD34+ cells into EPCs and induced tube formation of cultured EPCs isolated from healthy control subjects at both concentrations of SDF-1 (Fig. 3).
Diabetic patients have defective CD34+ cell migration in response to SDF-1.
If EPC migration to picomolar concentrations of SDF-1 found in the vitreous was the mechanism by which preretinal neovascularization occurred, one might predict that the EPCs isolated from diabetic patients would have enhanced migration at picomolar concentrations of SDF-1. Unexpectedly, CD34+ cells isolated from diabetic patients had markedly diminished migration, with a virtually flat response to SDF-1, over the picomolar and nanomolar concentrations tested (Fig. 4A). This was found in patients with retinopathy and stage V CKD or with stage I or II CKD (Fig. 4B). In addition, the result was the same whether the patients had type 1 or type 2 diabetes (data not shown). Compared with the maximal migration of CD34+ cells in normal control subjects, CD34+ cells from diabetic patients had 30% of the maximal migratory activity (Fig. 4B).
We reasoned that the defect in migration of CD34+ cells could be at the level of the receptor or due to downstream receptor effects. Previously, CD26/dipeptidyl peptidase IV was shown to cleave and inactivate SDF-1, blocking its ability to activate its receptor CXCR4 (13,15,16). To determine whether this was a possible mechanism for the decreased migration observed in CD34+ cells derived from diabetic individuals, we determined the level of CD26/dipeptidyl peptidase IV activity on these cells. We found twice the CD26/dipeptidyl peptidase IV activity present on CD34+ cells derived from diabetic patients versus control subjects (Fig. 4C). However, the increased level of dipeptidyl peptidase IV activity was not limiting migration. Even diabetic patients whose dipeptidyl peptidase IV activity was lower than that of normal control subjects (Fig. 4D) had defective migration. To confirm this, we used an inhibitor of dipeptidyl peptidase IV activity, diprotein A. Incubation of diabetic CD34+ cells with diprotein A, at concentrations that blocked 99% of the dipeptidyl peptidase IV activity (data not shown) increased migration, but the percent increase was still less than that seen in CD34+ cells isolated from normal control subjects (Fig. 4E). Consistent with the defect not being primarily at the receptor level, diabetic CD34+ cells did not migrate to VEGF, suggesting a more general inhibition of migration (Fig. 5).
Cytoskeletal defect in diabetic CD34+ cells.
Classically, the cytoskeletal structure has been investigated by cell deformability either by atomic force microscopy or micropipette technique (17,18). In the latter, micropipettes are made from 1-mm capillary-glass tubing pulled to a fine point by quick fracture to give a flat tip of desired diameter. Two typical types of experiment, aspiration and recovery, are usually performed to determine the mechanical properties of individual cells. Cell deformability can be assessed by aspirating a cell into a micropipette at a constant pressure and then measuring its deformation as a function of time. Using the micropipette technique, it has also been established that the cell rheological properties can be altered by disease and illness. Published data have shown alterations of the mechanical properties of erythrocytes in sickle cell disease (19), white blood cells in diabetes (14,20,21), and a variety of cells in cancer (22eC25). CD34+ cells isolated from patients with diabetes complications were extremely rigid; the deformability could not be determined because they could not be suctioned into the micropipette (Fig. 6). CD34+ cells from normal control subjects entered the micropipette very well (75% of cells completely entered the micropipette, 16.7% partially entered, and 8.3% could not be suctioned into the micropipette; n = 5), while CD34+ cells isolated from diabetic patients were not deformable (100% of the cells could not be suctioned into the micropipette; n = 6). Previously, we have demonstrated a similar disturbance in white cells isolated from diabetic mice (14).
CD34+ cell migration is enhanced by incubation with an NO donor.
Local concentrations of NO dramatically affected hemangioblast behavior and changed vessel phenotype (26). Thus, we investigated whether the migratory defect of CD34+ cells isolated from diabetic patients could be corrected by treatment with NO. Incubation of CD34+ cells in the presence of an NO donor, DETA/NO, for 24 h increased the percentage of cells migrating in response to SDF-1 (Fig. 7). This enhancement was evident at picomolar and nanomolar concentrations of SDF-1. The effect of the NO donor on SDF-1 in stimulating CD34+ cell migration was blocked by coincubating with the NO scavenger 2-(4-carboxyphenol)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (Fig. 7).
NO treatment also corrected the diabetic CD34+ deformability defect (Fig. 6). While 100% of CD34+ cells isolated from diabetic patients were not deformable when treated with vehicle, diabetic CD34+ cells incubated with DETA/NO had a marked increase in deformability (85% of cells could fully enter the micropipette; n = 16). This suggested that NO altered the cytoskeletal structure mediating the correction in the migration defect.
NO causes redistribution of cytoskeletal motors facilitating migration.
To begin to elucidate the possible role that NO has on the cytoskeleton, we examined the distribution of VASP-related proteins in response to exogenous administration of NO. This family of proteins is critical to actin elongation, powering the expansile apparatuses of cells, the filopodia and lamellapodia, (27 and recently rev. in 28) and is regulated by NO (29). Immunohistochemistry of VASP protein was performed on cultured EPCs. Incubation of the EPCs for 24 h with increasing concentration of NO resulted in increased VASP expression (Fig. 8) and a redistribution of this critical protein to filopodia. VASP, in the absence of NO, was distributed evenly throughout the cell. Whereas, with NO stimulation, VASP was upregulated and redistributed to the leading edge of the advancing cell processes. In addition, fluorescence-activated cell sorter analysis demonstrated that NO significantly increased expression of phospho-VASP in EPCs (Fig. 8D).
DISCUSSION
The major finding of this report is that diabetic CD34+ cells have markedly decreased migratory characteristics to multiple different stimuli that can be reversed by exogenous administration of physiologic concentrations of NO. The defect in migration in response to cytokines and growth factors may be responsible for the marked atherosclerosis, peripheral vascular disease, and delayed wound healing typically seen in diabetic patients. The diabetic CD34+ cells were also rigid compared with those cells isolated from healthy control subjects. Like migration, treatment with NO induced deformability of the diabetic CD34+ cells. This suggested that NO was affecting the cytoskeletal structure of the CD34+ cells. This was confirmed by NO causing a redistribution of the cytoskeletal proteins VASP and mammalian-enabled distribution in EPCs.
To fully investigate the nature of the migration defect of CD34+ cells isolated from diabetic patients, we investigated the activity of CD26/dipeptidyl peptidase IV on the surface of these cells. This enzyme cleaves SDF-1 (13,15,16) and could potentially modulate EPC behavior. The CD34+ cell defect in migration was associated with a marked increase in CD26/dipeptidyl peptidase IV activity in some diabetic patients. However, while increased cell surface cleavage of SDF-1 may potentiate the migration defect, our studies suggest that it is not the only mechanism. Diabetic patients with levels of dipeptidyl peptidase IV activity below those of healthy control subjects or whose dipeptidyl peptidase IV activity was inhibited with diprotein A still had diminished cell migration.
Since the diabetic CD34+ cells did not migrate to VEGF or SDF-1 and diabetic white blood cells have been shown to be less deformable than cells from normal control subjects (14), we used the micropipette technique to study the intrinsic cytoskeletal structure. Cells isolated from patients with diabetes complications were so rigid that they could not even be suctioned into the micropipette. We found that 100% of the diabetic cells were not deformable as opposed to only 8.3% of control cells. However, after 24 h exposure with an NO donor, the CD34+ cells from diabetic patients demonstrated the same deformability as normal control subjects.
To determine the mechanism of the increased rigidity of the diabetic CD34+ cells, we studied the localization of VASP by immunohistochemistry; this family of proteins is critical for actin filament elongation powering the advancement of the leading edge of cells (28). In addition, NO has been shown to regulate VASP (29). In response to physiologic concentrations of NO, VASP immunolocalization was altered, VASP protein expression was increased, VASP was concentrated in the filopodia, and VASP phosphorylation was markedly increased. These changes are conducive to cell motility and are consistent with recently published observations concerning the role of NO in inducing long-lasting potentiation of hippocampal synapses by altering neuronal puncta via VASP (30).
There could be additional mechanisms by which NO could correct the diabetic CD34+ cell migration defect. NO is required for proper motility of endothelial cells (31). Diabetes, by affecting Akt phosphorylation, results in diminished NO generation. Exogenous administration of NO would bypass the endogenous need for AKT phosphorylation.
Alternatively, NO may be acting as a reactive oxygen scavenger. Reactive oxygen species, known to be markedly increased in diabetes and associated with complications (32,33), interfere with growth factor signaling (34eC36). Thus, NO, by quenching reactive oxygen species, could possibly enhance VEGF and SDF-1 downstream receptor signaling. Specifically, H2O2 has previously been shown to induce stress fibers in endothelial cells, inhibiting normal motility. NO, by scavenging super oxide and indirectly reducing H2O2 levels, could prevent H2O2 induction of cytoskeletal reorganization via calcium/calmodulin-dependent protein kinase II, Janus kinase 2 and mitogen-activated protein kinase, and extracellular signaleCrelated kinase (37). However, we believe this is less likely since the reactive oxygen scavenger uric acid could not fully correct the migration defect in CD34+ cells (data not shown).
Defects in EPC migration would result in decreased reendothelialization. This defect could extend to the micro- and macrovascular complications seen in diabetic patients. Diabetic eye disease is characterized by aberrant microvascular changes with insufficient intraretinal vascularization and excessive "preretinal" neovascularization. The decreased response of diabetic CD34+ cells to picomolar concentrations of SDF-1 as well as other growth factors may inhibit the ability of these cells to repair early capillary endothelial injury. If this injury is not repaired, acellular capillaries could result. With continued ischemic injury, the retina continues to produce SDF-1 and VEGF that accumulate in the vitreous. With the vitreous acting as a growth factor "sink," increasing growth factor concentrations may ultimately be able to overcome the relatively unresponsive CD34+ cells in the diabetic individual. However, the concentrated SDF-1 and VEGF in the vitreous directs EPC and endothelial cell migration to the surface of the retina, leading to preretinal vascular pathology. Correcting the CD34+ cell migratory defect, before the acellular capillary stage, may result in early capillary repair, preventing the late accumulation of vitreal SDF-1 and classic, preretinal, diabetic proliferation.
Rapid reendothelialization, mediated by CD34+ cells, at the macrovascular level can reduce atherosclerosis, peripheral vascular disease, and restenosis. These macrovascular complications are thought to be the result of endothelial injury (38,39) with possible inadequate endothelial repair and are all accelerated in the diabetic individual.
CD34+ cells, from healthy volunteers, significantly increase revascularization when injected into an ischemic hind limb of hypoinsulinemic diabetic mice (40) and accelerates revascularization and healing of full thickness skin wounds (41). Interestingly, injection of CD34+ cells in an ischemic limb of a normal mouse has no effect (40). This constellation of results suggests that diabetes has a detrimental effect on bone marrow and EPC homeostasis. Other conditions such as ischemia, smoking, and increased cholesterol have also been shown to have injurious effects on EPC number and function (42,43).
Administration of migratory CD34+ cells may represent an easily accessible and safe therapeutic approach to correct the multitude of vascular abnormalities seen in patients with diabetes. However these results should serve as a caution to in vivo studies using CD34+ cells as a therapeutic modality; improvement of CD34+ cell function in diabetic patients without simultaneously decreasing the levels of SDF-1 and other growth factors in the vitreous could result in worsening preretinal neovascularization. This is especially clinically relevant given the development of dipeptidyl peptidase IV inhibitors as an indirect means to stimulate insulin secretion and decrease glucagon secretion (44,45). These inhibitors by inhibiting SDF-1 degradation could potentiate CD34+ cell migration, and while this may slightly improve endothelial repair, it could worsen preretinal neovascularization, especially if given in the absence of decreasing the concentration of SDF-1 within the vitreous.
In conclusion, we have demonstrated that CD34+ cell dysfunction in diabetes is the result of alterations in the EPC cytoskeleton. The defect in SDF-1eCinduced migration seen in diabetic patients with end-organ damage is reversed by physiologic NO concentrations. We demonstrate that NO treatment improves deformability and normalizes the migration of diabetic cells likely by leading to phosphorylation and redistribution of VASP to the advancing edge, directly enhancing cell motility.
ACKNOWLEDGMENTS
This work was supported by National Institutes of Health Grant DK02537, Gatorade Research Funds, a Baxter Renal Discovery Grant to M.S.S., and the Juvenile Diabetes Research Foundation International and National Institutes of Health Grants EY012601, EY007739, and EY14818 to M.B.G.
The authors thank Dr. G.C. Finlayson for help recruiting patients, Dr. David A. Antonetti for helpful suggestions regarding experimental design and critical review of the manuscript, and Dr. Daniel Purich for discussions concerning VASP.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
Tashiro K, Tada H, Heilker R, Shirozu M, Nakano T, Honjo T: Signal sequence trap: a cloning strategy for secreted proteins and type I membrane proteins. Science 261:600eC603, 1993
Nagasawa T, Hirota S, Tachibana K, Takakura N, Nishikawa S, Kitamura Y, Yoshida N, Kikutani H, Kishimoto T: Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1. Nature 382:635eC638, 1996
Kawabata K, Ujikawa M, Egawa T, Kawamoto H, Tachibana K, Iizasa H, Katsura Y, Kishimoto T, Nagasawa T: A cell-autonomous requirement for CXCR4 in long-term lymphoid and myeloid reconstitution. Proc Natl Acad Sci U S A 96:5663eC5667, 1999
Onai N, Zhang Y, Yoneyama H, Kitamura T, Ishikawa S, Matsushima K: Impairment of lymphopoiesis and myelopoiesis in mice reconstituted with bone marrow-hematopoietic progenitor cells expressing SDF-1-intrakine. Blood 96:2074eC2080, 2000
Ratajczak MZ, Kucia M, Reca R, Majka M, Janowska-Wieczorek A, Ratajczak: Stem cell plasticity revisited: CXCR4-positive cells expressing mRNA for early muscle, liver and neural cells ’hide out’ in the bone marrow. Leukemia 18:29eC40, 2004
Christopherson KW 2nd, Hangoc G, Mantel CR, Broxmeyer HE: Modulation of hematopoietic stem cell homing and engraftment by CD26. Science 305:1000eC1003, 2004
Peichev M, Naiyer AJ, Pereira D, Zhu Z, Lane WJ, Williams M, Oz MC, Hicklin DJ, Witte L, Moore MA, Rafii S: Expression of VEGFR-2 and AC133 by circulating human CD34(+) cells identifies a population of functional endothelial precursors. Blood 95:952eC958, 2000
Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM: Isolation of putative progenitor endothelial cells for angiogenesis. Science 275:964eC967, 1997
Brooks HL Jr, Caballero S Jr, Newell CK, Steinmetz RL, Watson D, Segal MS, Harrison JK, Scott EW, Grant MB: Vitreous levels of vascular endothelial growth factor and stromal-derived factor 1 in patients with diabetic retinopathy and cystoid macular edema before and after intraocular injection of triamcinolone. Arch Ophthalmol 122:1801eC1807, 2004
Grant MB, May WS, Caballero S, Brown GA, Guthrie SM, Mames RN, Byrne BJ, Vaught T, Spoerri PE, Peck AB, Scott EW: Adult hematopoietic stem cells provide functional hemangioblast activity during retinal neovascularization. Nat Med 8:607eC612, 2002
Grant MB, Ellis EA, Caballero S, Mames RN: Plasminogen activator inhibitor-1 overexpression in nonproliferative diabetic retinopathy. Exp Eye Res 63:233eC244, 1996
Grant MB, Tarnuzzer RW, Caballero S, Ozeck MJ, Davis MI, Spoerri PE, Feoktistov I, Biaggioni I, Shryock JC, Belardinelli L: Adenosine receptor activation induces vascular endothelial growth factor in human retinal endothelial cells. Circ Res 85:699eC706, 1999
Christopherson KW 2nd, Hangoc G, Broxmeyer HE: Cell surface peptidase CD26/dipeptidylpeptidase IV regulates CXCL12/stromal cell-derived factor-1 alpha-mediated chemotaxis of human cord blood CD34+ progenitor cells. J Immunol 169:7000eC7008, 2002
Perrault CM, Bray EJ, Didier N, Ozaki CK, Tran-Son-Tay R: Altered rheology of lymphocytes in the diabetic mouse. Diabetologia 47:1722eC1726, 2004
Ohtsuki T, Hosono O, Kobayashi H, Munakata Y, Souta A, Shioda T, Morimoto C: Negative regulation of the anti-human immunodeficiency virus and chemotactic activity of human stromal cell-derived factor 1alpha by CD26/dipeptidyl peptidase IV. FEBS Lett 431:236eC240, 1998
Proost P, Struyf S, Schols D, Durinx C, Wuyts A, Lenaerts JP, De Clercq E, De Meester I, Van Damme: Processing by CD26/dipeptidyl-peptidase IV reduces the chemotactic and anti-HIV-1 activity of stromal-cell-derived factor-1alpha. FEBS Lett 432:73eC76, 1998
Tran-Son-Tay R, Needham D, Yeung A, Hochmuth RM: Time-dependent recovery of passive neutrophils after large deformation. Biophys J 60:856eC866, 1991
Hochmuth RM, Ting-Beall HP, Beaty BB, Needham D, Tran-Son-Tay R: Viscosity of passive human neutrophils undergoing small deformations. Biophys J 64:1596eC1601, 1993
Nash GB, Johnson CS, Meiselman HJ: Mechanical properties of oxygenated red blood cells in sickle cell (HbSS) disease. Blood 63:73eC82, 1984
Athanassiou G, Matsouka P, Kaleridis V, Missirlis Y: Deformability and filterability of white blood cell subpopulations: evaluation of these parameters in the cell line HL-60 and in type II diabetes mellitus. Clin Hemorheol Microcirc 22:35eC43, 2000
Linderkamp O, Ruef P, Zilow EP, Hoffmann GF: Impaired deformability of erythrocytes and neutrophils in children with newly diagnosed insulin-dependent diabetes mellitus. Diabetologia 42:865eC869, 1999
Yao W, Gu L, Sun D, Ka W, Wen Z, Chien S: Wild type p53 gene causes reorganization of cytoskeleton and, therefore, the impaired deformability and difficult migration of murine erythroleukemia cells. Cell Motil Cytoskeleton 56:1eC12, 2003
Glover S, Delaney M, Dematte C, Kornberg L, Frasco M, Tran-Son-Tay R, Benya RV: Phosphorylation of focal adhesion kinase tyrosine 397 critically mediates gastrin-releasing peptide’s morphogenic properties. J Cell Physiol 199:77eC88, 2004
Chen H, Yunpeng W, Shaoxi C, Mian LA: Research on rheological properties of erythrocyte of lung cancer patients. Clin Hemorheol 16:135eC141, 1996
Khan MM, Puniyani RR, Huilgog NG, Hussain A, Ranade GG: Hemorheological profile in cancer patients. Clin Hemorheol 15:37eC44, 1995
Guthrie SM, Curtis LM, Mames RN, Simon GG, Grant MB, Scott EW: The nitric oxide pathway modulates hemangioblast activity of adult hematopoietic stem cells. Blood 105:1916eC1922, 2005
Dickinson RB, Purich DL: Clamped-filament elongation model for actin-based motors. Biophys J 82:605eC617, 2002
Krause M, Dent EW, Bear JE, Loureiro JJ, Gertler FB: Ena/VASP proteins: regulators of the actin cytoskeleton and cell migration. Annu Rev Cell Dev Biol 19:541eC564, 2003
Andre M, Latado H, Felley-Bosco E: Inducible nitric oxide synthase-dependent stimulation of PKGI and phosphorylation of VASP in human embryonic kidney cells. Biochem Pharmacol 69:595eC602, 2005
Wang H-G, Lu F-M, Jin I, Udo H, Kandel ER, de Vente J, Walter U, Lohmann SM, Hawkins RD, Antonova I: Presynaptic and postsynaptic roles of NO, cGK, and RhoA in long-lasting potentiation and aggregation of synaptic proteins. Neuron 45:389eC403, 2005
Ziche M, Morbidelli L, Masini E, Amerini S, Granger HJ, Maggi CA, Geppetti P, Ledda FJ: Nitric oxide mediates angiogenesis in vivo and endothelial cell growth and migration in vitro promoted by substance P. J Clin Invest 94:2036eC2044, 1994
Brownlee MD: Advanced protein glycosylation in diabetes and aging. Annu Rev Med 46:223eC234, 1995
King GL, Brownlee M: The cellular and molecular mechanisms of diabetic complications. Endocrinol Metab Clin North Am 25:255eC270, 1996
Lo YYC, Cruz TF: Involvement of reactive oxygen species in cytokine and growth factor induction of c-fos expression in chondrocytes. J Biol Chem 270:11727eC11730, 1995
Lo YYC, Wong JMS, Cruz TF: Reactive oxygen species mediate cytokine activation of c-Jun NH2-terminal kinases. J Biol Chem 271:15703eC15707, 1996
Nakamura H, Nakamura K, Yodoi J: Redox regulation of cellular activation. Annu Rev Immunol 15:351eC369, 1997
Nguyen A, Chen P, Cai H: Role of CaMKII in hydrogen peroxide activation of ERK1/2, p38 MAPK, HSP27 and actin reorganization in endothelial cells. FEBS Lett 572:307eC313, 2004
Ross R, Glomset J, Harker L: Response to injury and atherogenesis. Am J Pathol 86:675eC684, 1977
Ross R: Atherosclerosis: an inflammatory disease. N Engl J Med 340:115eC126, 1999
Schatteman GC, Hanlon HD, Jiao C, Dodds SG, Christy BA: Atherosclerosis: an inflammatory disease. J Clin Invest 106:571eC578, 2000
Sivan-Loukianova E, Awad OA, Stepanovic V, Bickenbach J, Schatteman GC: CD34+ blood cells accelerate vascularization and healing of diabetic mouse skin wounds. J Vas Res 40:368eC377, 2003
Heeschen C, Lehmann R, Honold J, Assmus B, Aicher A, Walter DH, Martin H, Zeiher AM, Dimmeler S: Profoundly reduced neovascularization capacity of bone marrow mononuclear cells derived from patients with chronic ischemic heart disease. Circulation 109:1615eC1622, 2004
Vasa M, Fichtlscherer S, Aicher A, Adler K, Urbich C, Martin H, Zeiher AM, Dimmeler S: Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Circ Res 89:E1eCE7, 2001
Deacon CF, Ahren B, Holst J: Inhibitors of dipeptidyl peptidase IV: a novel approach for the prevention and treatment of type 2 diabetes Expert Opin Investig Drugs 13:1091eC1102, 2004
Holst JJ, Deacon CF: Glucagon-like peptide 1 and inhibitors of dipeptidyl peptidase IV in the treatment of type 2 diabetes mellitus. Curr Opin Pharmacol 4:589eC596, 2004(Mark S. Segal, Ronak Shah)